advances in food and nutrition research volume 56

ADVISORY BOARDS

KEN BUCKLEUniversity of New South Wales, Australia

MARY ELLEN CAMIREUniversity of Maine, USA

ROGER CLEMENSUniversity of Southern California, USA

HILDEGARDE HEYMANNUniversity of California, Davis, USA

ROBERT HUTKINSUniversity of Nebraska, USA

RONALD JACKSONQuebec, Canada

HUUB LELIEVELDGlobal Harmonization Initiative, The Netherlands

DARYL B. LUNDUniversity of Wisconsin, USA

CONNIE WEAVERPurdue University, USA

RONALD WROLSTADOregon State University, USA

SERIES EDITORS

GEORGE F. STEWART (1
948–1982)
EMIL M. MRAK (1
948–1987)
C. O. CHICHESTER (1
959–1988)
BERNARD S. SCHWEIGERT (1
984–1988)
JOHN E. KINSELLA (1
989–1993)
STEVE L. TAYLOR (1
995– )

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

� Abby K. van den BergProctor Maple Research Center, University of Vermont, Harvey Road,Underhill Center, Vermont, USA (101)

� Sinead C. CorrDepartment of Biochemistry and Immunology, Trinity College Dublin,Ireland (1)

� Susan E. DuncanVirginia Polytechnic Institute and State University (Virginia Tech),Blacksburg, Virginia, USA (17)

� A. DunnGalvinDepartment of Paediatrics and Child Health, Cork University Hospital,Ireland (65)

� Cormac G. M. GahanDepartment of Microbiology, School of Pharmacy and AlimentaryPharmabiotic Centre, University College Cork, Ireland (1)

� Colin HillDepartment of Microbiology, University College Cork, Ireland (1)

� J’ O. B. HourihaneDepartment of Paediatrics and Child Health, Cork University Hospital,Ireland (65)

� Timothy D. PerkinsProctor Maple Research Center, University of Vermont, Harvey Road,Underhill Center, Vermont, USA (101)

� R. T. RileyToxicology & Mycotoxin Research Unit, USDA Agricultural ResearchService, Athens, Georgia, USA (145)

� M. C. SpeerCenter for Human Genetics, Duke University Medical Center, Durham,North Carolina, USA (145)

vii

viii Contributors

� V. L. StevensDepartment of Epidemiology and Surveillance Research, AmericanCancer Society, Atlanta, Georgia, USA (145)

� K. A. VossToxicology & Mycotoxin Research Unit, USDA Agricultural ResearchService, Athens, Georgia, USA (145)

� J. Gelineau-van WaesDepartment of Genetics, Cell Biology & Anatomy, University ofNebraska Medical Center, Omaha, Nebraska, USA (145)

� Janet B. WebsterVirginia Polytechnic Institute and State University (Virginia Tech),Blacksburg, Virginia, USA (17)

CHAPTER 1

Advances in Food and NISSN 1043-4526, DOI: 10

Understanding the Mechanismsby Which Probiotics InhibitGastrointestinal Pathogens

Sinead C. Corr,* Colin Hill,† and Cormac G. M. Gahan‡

Contents I. Introduction 2

II. Evidence for Potential Mechanisms of Action 4

A. Epithelial barrier function and

probiotic signaling 4

B. Production of acid and secretion of

inhibitory substances 7

C. Immunomodulation 8

D. Inhibition of virulence factor expression 10

III. Conclusions 11

Acknowledgment 12

References 12

Abstract In recent years, there has been a growing interest in the use of

probiotic bacteria for the maintenance of general gastrointestinal

health and the prevention or treatment of intestinal infections.

Whilst probiotics are documented to reduce or prevent specific

infectious diseases of the GI tract, the mechanistic basis of this

effect remains unclear. It is likely that diverse modes-of-action

contribute to inhibition of pathogens in the gut environment and

proposed mechanisms include (i) direct antimicrobial activity

through production of bacteriocins or inhibitors of virulence gene

* Department of Biochemistry and Immunology, Trinity College Dublin, Ireland{ Department of Microbiology, University College Cork, Ireland{ Department of Microbiology, School of Pharmacy and Alimentary Pharmabiotic Centre, University CollegeCork, Ireland

utrition Research, Volume 56 # 2009 Elsevier Inc..1016/S1043-4526(08)00601-3 All rights reserved.

1

2 Sinead C. Corr et al.

expression; (ii) competitive exclusion by competition for binding

sites or stimulation of epithelial barrier function; (iii) stimulation of

immune responses via increases of sIgA and anti-inflammatory

cytokines and regulation of proinflammatory cytokines; and

(iv) inhibition of virulence gene or protein expression in gastroin-

testinal pathogens. In this review, we discuss the modes of action

by which probiotic bacteria may reduce gastrointestinal infections,

and highlight some recent research which demonstrates the mech-

anistic basis of probiotic cause and effect.

I. INTRODUCTION

The gastrointestinal tract is a complex ecosystem which can be a reservoirof both beneficial and harmful bacteria. Recently, there has been interestin the role of the gut microbiota in health, and also in the deliberate use ofbacterial supplements to influence this microbial community in a mannerwhich could potentially assist in maintaining health and in diseaseprevention (Holzapfel et al., 1998; Senok et al., 2005). Probiotics (definedas any live microorganism, that when administered to human or animalhosts, has health-promoting benefits) could potentially offer an alterna-tive to conventional therapies such as antibiotics for the prophylaxis ortreatment of intestinal infections (Bourlioux et al., 2003; Rolfe, 2000).From various in vitro and in vivo studies to date, it is clear that probioticsoffer great potential in prevention and treatment of infections (Table 1.1).However, a thorough understanding of their mechanisms of action isrequired to ensure their efficient use. Probiotics are presumed to modu-late the indigenous intestinal flora and improve health via a plethora ofpotential mechanisms of action, such as immunomodulation, directantagonism, or competitive exclusion (summarized in Fig. 1.1)(Gotteland et al., 2006; Sartor, 2004; Venturi et al., 1999). Probiotics caninhibit growth of enteric pathogens by decreasing luminal pH, the secre-tion of bactericidal peptides/proteins, or the stimulation of defensinproduction by epithelial cells (Toure et al., 2003; Zhu et al., 2000). Probio-tics can also block attachment to or invasion of the intestinal epitheliumby pathogens through blocking of epithelial surface receptors or induc-tion of mucins, large carbohydrate molecules which form a barrier alongthe epithelial monolayer (Mack et al., 1999; Mattar et al., 2002).

A number of in vivo studies have been performed which have deter-mined the probiotic capabilities of such strains. While these studies havebeen important in demonstrating probiotic efficacy against various infec-tious diseases, few have specifically identified the mechanistic basis behindthe observed benefits, and many rely on in vitro data to decipher thepossible mechanism of action. However, what has emerged to date is that

TABLE 1.1 A list of potential probiotic strains and their observed beneficial effects

Organism Effect Mechanism Reference

E. coli strain Nissle 1917 Improves epithelial

barrier function

Increases tight junction protein

expression, ZO-2

Zyrek et al. (2007)

L. rhamnosus R0011 Improves epithelial

barrier function

Prevents the pathogen-induced drop in

transepithelial resistance

Sherman et al.

(2005)

L. plantarum 299v Improves epithelial

barrier function

Increases extracellular secretion of mucin,

MUC3

Mack et al. (2003)

VSL#3 probiotic

mixture

Improves epithelial

barrier function

Induces human b-defensin, hBD-2 gene

expression

Schlee et al. (2008)

B. breve Yakult Secretion of inhibitory

substances

Production of acetic acid and thus

lowering of pH

Asahara et al.

(2004)

L. johnsonii NCC533 Secretion of inhibitory

substances

Production of hydrogen peroxide Pridmore et al.

(2008)

L. salivarius UCC118 Secretion of inhibitory

substances

Production of bacteriocin Corr et al. (2007b)

L. casei NCDO1205 Immunomodulation Decrease IL-8 and increase IL-10 response Corr et al. (2007a)

L. rhamnosus GG Immunomodulation Activates NF-kB and regulatesinflammatory response in macrophages

Miettinen et al.

(2000)

B. lactis Bb-12 Immunomodulation Stimulates sIgA Fukushima et al.

(1998)

L. rhamnosus GG Inhibition of virulence

factor expression

Reduces expression of genes encoding

shiga toxin

Carey et al. (2008)

L. plantarum ITM21B Inhibition of virulence

factor expression

Inhibition of urease activity in

Y. enterocolitica

Lavermicocca et al.

(2008)

Transientflora

(including probioticsand pathogens)

Probiotic

E

M

T2DC

4

3

Pathogen1

Mucus

Commensalmicrobiota

Epithelium(including

immune cells)

FIGURE 1.1 Probiotics may protect against infection by pathogens through (1) Direct

antagonism via bacteriocin production. (2) Immunomodulation via immune cell (T-cell,

Dendritic cell) activation. (3) Improvement of epithelial barrier function and competitive

exclusion via induction of mucus and blocking of epithelial binding receptors.

(4) Strengthening of epithelial tight junctions by increased expression of tight junction

proteins, or by a combination of these mechanisms.


the inhibition of pathogens by specific probiotics may represent a highlyspecific commensal–pathogen interaction. It is clear that further under-standing of this phenomenon is required in order to specifically targetgastrointestinal pathogens through the use of appropriate probiotic strains.

II. EVIDENCE FOR POTENTIAL MECHANISMS OF ACTION

A. Epithelial barrier function and probiotic signaling

A key mechanism by which probiotics are thought to exert anti-invasiveactivity is via induction of conformational changes within the epithelialmonolayer (Mack et al., 1999). In a recent study of barrier disruption inT84 epithelial cells by infection with enteropathogenic Escherichia coli,coincubation with the probiotic E. coli strain Nissle 1917 (EcN) or additionof the probiotic after infection abolished this disruption and restoredbarrier integrity (Zyrek et al., 2007). DNA-microarray analysis identifiedmore than 300 genes exhibiting altered expression following incubation ofthe epithelial cells with EcN, including expression and distribution ofzonula occludens-2 (ZO-2), a tight-junction protein.

Further studies have shown that pretreatment of epithelial monolayerswith probiotic bacteria, Lactobacillus acidophilus R0052 and Lactobacillus

Mechanisms of Probiotic Action 5

rhamnosus R0011, reduces epithelial injury following exposure to E. coliO157:H7 and E. coliO127:H6 by preventing the pathogen-induced drop intransepithelial resistance, a measure of barrier integrity (Sherman et al.,2005). These probiotics also reduced the number of foci of rearrangementsof a-actinin, indicative of reduced number of attaching and effacinglesions formed in response to E. coli O157:H7. In this study, viable lacticacid-producing bacteria were necessary to mediate the observed effects. Inanother recent study preincubation of Hep-2 cell monolayers with twostrains of lactobacilli, Lactobacillus delbrueckii subsp. lactis CIDCA 133 andLactobacillus plantarum CIDCA 83114 prior to infection with enterohaemor-rhagicE. coli (EHEC)minimized F-actin rearrangements andmorphologicalalterations in the cell monolayers (Hugo et al., 2008). These studies collec-tively indicate that lactobacilli are capable of directly triggering cellularresponses in host cells that may impede virulence mechanisms of EHEC.

The exact molecular mechanisms by which probiotics stimulate altera-tions in epithelial cell function are currently under investigation. Studieshave shown that probiotic strains such as the VSL#3 probiotic compound(Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, L. aci-dophilus, Lactobacillus casei,L. delbrueckii subsp. bulgaricus, L. plantarum,Strep-tococcus salivarius subsp. thermophilus) can improve epithelial and mucosalbarrier function through production of specific metabolites (Madsen et al.,2001). These include production of short-chain fatty-acids (SCFAs) as by-productofmicrobial fermentation, suchasbutyratewhich induces epithelialcell differentiation and increases barrier integrity (Cook and Sellin, 1998).L. acidophilus has been shown to improve gut barrier function in rats byimproving microflora disturbance, increasing occludin expression, andmaintaining the gut epithelial tight junction (Qin et al., 2005).

Another physiological change potentially induced by probiotics in thehost involves induction or overexpression of mucin. GI tract mucins arelarge, carbohydrate-rich, high-molecular-weight glycoproteins which arethe major components of the mucous layer overlying the intestinal epi-thelium (Mattar et al., 2002). Mucin forms a physicochemical barrierwhich protects epithelial cells from chemical, enzymatic, mechanical,and microbial damage, and limits microbial adherence and subsequentinvasion (Mack et al., 2003). At least 12 mucin genes have been identified,and of these MUC2 and MUC3 are the predominant ileocolonic mucins(Mack et al., 2003). TheMUC2 gene is expressed in goblet cells of the smalland large intestine and is the major secreted mucin of the colon (Macket al., 1999). The membrane-associated mucin MUC3 is not highlyexpressed in the colon but is expressed on both goblet cells and enter-ocytes of the small intestine (Chang et al., 1994). Adherence of selectedLactobacillus strains (L. plantarum 299v, L. rhamnosus GG) to the humanintestinal HT29 epithelial-cell line induces up-regulation of mucin geneexpression, and correlates with increased extracellular secretion of MUC3


(Mack et al., 2003). L. plantarum 299v and L. rhamnosus GG inhibit theadherence of enteropathogenic E. coli to HT29 intestinal epithelial cells viainduction or overexpression of mucin (Mack et al., 1999). In an in vitroCaco-2 cell model, L. casei LGG up-regulates MUC2 expression and has aninhibitory effect on bacterial translocation of the intestinal epithelium(Mattar et al., 2002). Thus, increased expression of intestinal mucin inresponse to lactobacilli mediates inhibition of adherence of pathogensto intestinal cells. However, analysis of this phenomenon using in vivoinfection models has not yet been implemented.

Interestingly, Collado and colleagues (2008) have recently shown thatspecific probiotic strains have the capacity to prevent adhesion of theopportunistic pathogen Enterobacter sakazakii to immobilized humanmucous in vitro. These studies indicate that in addition to inducingupregulation of mucous secretion by the epithelia, specific probioticstrains also have the capacity to competitively exclude or displace patho-gens from human mucous as a mechanism for preventing the transientcolonization of gastrointestinal pathogens.

Potential probiotic strains can also induce the release of defensins fromepithelial cells. These small peptides/proteins are active against bacteria,fungi, and viruses and also stabilize gut barrier function (Furrie et al., 2005).It has been shown that E. coli Nissle 1917 induces human b-defensin-2(hBD-2) gene expression in Caco-2 intestinal epithelial cells (Wehkampet al., 2004). This induction was mediated by NF-kB and AP-1 signalingpathways. Recently, several strains including E. coliNissle 1917, L. acidophi-lus, Lactobacillus fermentum, Lactobacillus paracasei subsp. paracasei, Pediococcuspentosaceus, and the VSL#3 probiotic mixture were found to induce hBD-2gene expression in Caco-2 cells (Schlee et al., 2008). This was also dependenton mitogen-activated protein kinase (MAPK), NF-kB, and AP-1 signalingpathways (Schlee et al., 2008). This induction of hBD-2 may also enhancemucosal barrier function.

The adhesion ability of some probiotic strains affords probiotic bacte-ria the capacity to compete with pathogenic bacteria for receptorsexpressed on epithelial cells, thus blocking contact between epithelialcells and pathogenic bacteria (Sherman et al., 2005; Tsai et al., 2005). In arecent study, BALB/c mice were fed L. acidophilus LAP5 or L. fermentumLF33 originally isolated from swine and poultry for seven consecutivedays before oral challenge with Salmonella enterica serovar Typhimurium(Tsai et al., 2005). Numbers of Salmonella invading livers and spleens ofprobiotic-fed mice were significantly lower than placebo controls, and itwas thought that the adhesiveness of Lactobacillus cells to mouse intestinalepithelium may be an important factor for their antagonistic activityagainst Salmonella invasion in vivo. However, this inference was basedupon in vitro assessment of adherence to intestinal cell lines and was notproven in vivo (Tsai et al., 2005).


B. Production of acid and secretion of inhibitory substances

Lactobacillus and Bifidobacterium spp. are capable of producing organicacids as end products of metabolism. Selected Bifidobacterium species,including B. breve strain Yakult, display anti-infectious activity againstShiga toxin-producing E. coli (STEC) O157:H7 in mice (Asahara et al.,2004). In this study, B. breve Yakult was administered to mice daily forthree consecutive days and mice were infected with STEC on day 3.A dramatic decrease in bodyweight and subsequent death was observedin placebo-fed mice, while bodyweight was maintained and no fatalitieswere observed in B. breve-fed mice. This anti-infective activity wasthought to be due to production of acetic acid by B. breve and loweringof intestinal pH, which had the combined effect of inhibiting Shiga-liketoxin (Stx) production (Asahara et al., 2004).

Lactobacillus and Bifidobacterium spp. have been shown to impedeinfection of human intestinal cells by enterohemorrhagic E. coli O157:H7by the combined action of lactic acid and proteinaceous substances (Gopalet al., 2001). An in vitro study of the ability of L. rhamnosus DR20 andBifidobacterium lactis DR10 to impede infection of differentiated humanintestinal cell-lines by E. coli O157:H7 found that pretreatment of E. coliwith concentrated cell-free culture supernatants from these probioticbacteria significantly reduced numbers of culturable E. coli and the inva-siveness of this strain (Gopal et al., 2001). The probiotic E. coli strain Nissle1917 interferes with S. Typhimurium invasion of human embryonic intes-tinal epithelial INT407 cells via secretion of inhibitory substances,as shown when the probiotic was separated from the bacteria by anonpermeable membrane (Altenhoefer et al., 2004). In a previous study,we utilized a similar transwell chamber system to demonstrate that lacto-bacilli and bifidobacteria (L. casei, L. acidophilus, Lactobacillus salivarius,B. breve, B. infantis, B. longum) are capable of inhibiting Listeria monocyto-genes invasion of C2Bbe1 epithelial cells in the absence of direct contactthrough secretion of proteinaceous molecule(s), active at low pH in thecase of the lactobacilli strains tested (Corr et al., 2007a). However, thenature of the proteinaceous agent needs to be identified.

Recently, Pridmore and co-workers (2008) have examined the produc-tion of hydrogen peroxide by the human gastrointestinal isolate Lactoba-cillus johnsonii NCC533. Through in silico analysis of the genome of thispotential probiotic strain they identified the means by which hydrogenperoxide is synthesized. Furthermore, they demonstrated that the strainactively produced hydrogen peroxide in vitro at levels that were inhibi-tory for S. Typhimurium.

Bacteriocins are compounds with potential anti-microbial activitysynthesized by many bacterial species, including lactic acid bacteria(Cotter et al., 2005; Gotteland et al., 2006). As the ability of bacteriocins to


inhibit or kill pathogens is well documented, these molecules representobvious candidates as mediators of an antipathogen effect. Indeed, bac-teriocins have been shown to be necessary in vivo for long-term oralcolonization by a noncariogenic variant of Streptococcus mutans in a thera-peutic approach known as replacement therapy (Smith et al., 2006). In arecent study, we demonstrated the ability of L. salivarius UCC118 toinhibit L. monocytogenes infection of mice, and directly linked this inhibi-tory effect to production of bacteriocin by L. salivarius (Corr et al., 2007b).We showed that mice orally inoculated with L. salivarius UCC118 wereprotected from subsequent oral infection by L. monocytogenes. However, astable mutant of L. salivarius UCC118 that is unable to produce the bacte-riocin, Abp118, failed to protect mice confirming that bacteriocin produc-tion is the primary mediator of protection against this organism.Furthermore, L. salivarius UCC118 did not offer any protection whenmice were infected with a strain of L. monocytogenes expressing the cog-nate Abp118 bacteriocin immunity protein AbpIM again confirming thatthe observed protective effect was the result of direct antagonism betweenL. salivarius and the pathogen, mediated by the bacteriocin Abp118.

C. Immunomodulation

Probiotic bacteria are capable of tempering the host inflammatoryresponse to infection and are considered to be important mediators ofimmune-regulation in the gastrointestinal environment (Corr et al., 2007a;O’Hara et al., 2006). It is likely that this immunomodulatory role is animportant factor governing the immune clearance of gastrointestinalpathogens and in preventing the establishment of postinfectious inflam-matory conditions (including irritable bowel syndrome, IBS) in the GItract. Furthermore, chronic inflammatory diseases of the GI tract (includ-ing Crohn’s disease) are postulated to be linked to underlying infections(by Mycobacterium avium subsp. paratuberculosis or specific E. coli strains)(Darfefeuille-Michaud et al., 2004; Sechi et al., 2004). Probiotic treatmentraises the possibility that such chronic infections may be amenable tononinvasive intervention in order to limit the cause of the underlyinginflammation.

Probiotic bacteria regulate mucosal immune responses through induc-tion of anti-inflammatory cytokines such as IL-10 and TGF-b, whiledecreasing expression of proinflammatory cytokines, such as TNF andIFN-g (Corr et al., 2007a;Di Giacinto et al., 2005; Silva et al., 2004). B. breveand Streptococcus thermophilus secrete metabolites which inhibit LPS-induced TNF-a secretion from peripheral blood mononuclear cell(PBMC) monolayers (Menard et al., 2004). We demonstrated a significantreduction in interleukin-8 (IL-8) and an increase in IL-10 cytokinessecreted from epithelial cells following pretreatment with probiotics


prior to infection with L. monocytogenes (Corr et al., 2007a). A number ofcommensal strains including L. casei NCDO1205, L. salivarius UCC118,and B. breve UCC2003 were capable of inducing this response. Similarly,both B. infantis 35624 and L. salivarius UCC118 are capable of reducingS. typhimurium-induced proinflammatory responses in vitro (O’Hara et al.,2006). These probiotic commensal strains were capable of blunting IL-8responses and increasing the IL-10 response in an in vitro model ofSalmonella infection.

The mechanistic basis of such responses has been examined by Kellyand co-workers (2004). Bacteroides thetaiotaomicron reduces inflammationdue to Salmonella–TLR5 interactions (Kelly et al., 2004). The mechanismunderpinning this anti-inflammatory response was dependent uponPPAR-g (peroxisome proliferator activated receptor- g)-mediated inhibi-tion of NF-kB and was directly induced by B. thetaiotaomicron. Further-more, L. rhamnosus GG is capable of activating NF-kB and STATs, latentcytoplasmic transcription factors which regulate transcription of genesencoding proteins involved in cytokine signaling and inflammatoryresponses in macrophages (Miettinen et al., 2000).

Some probiotics also stimulate secretory IgA production and activateregulatory T cells (Fukushima et al., 1998). These effects have been seen inhuman studies and demonstrate that anti-Polio sIgA is increased in thoseadministered a probiotic preparation viable B. lactis Bb-12. Similarly, anincrease in IgAþ cells was witnessed in mice administered L. casei(Galdeano and Perdigon, 2006). However, other studies have demon-strated that stimulation of sIgA in humans is stimulated by a prebioticpreparation but not by administration of live probiotic (Bifidobacteriumanimalis) (Bakker-Zierikzee et al., 2006).

Inflammatory conditions of the GI tract may be initiated by a disregu-lated local immune response to the normal microbiota and are hostdependent (Sartor, 2003; Shanahan, 2001). However, a subset of IBSpatients experience symptoms following gastrointestinal infection (post-infectious IBS). In addition, underlying infection has been proposed as apossible trigger in Crohn’s disease and both M. avium subsp. paratubercu-losis or adherent invasive E. coli (AIEC) have been suggested as possiblesources of inflammation (Darfefeuille-Michaud et al., 2004; Sechi et al.,2004). Ingrassia and co-workers have demonstrated that L. casei DN-114001 is capable of inhibiting AIEC strains isolated from Crohn’s diseasepatients in cell culture models of infection, suggesting that probioticintervention may present a future strategy for limiting the pathogenesisof a potential trigger of inflammation in Crohn’s disease (Ingrassia et al.,2005).

Indeed, human studies indicate that specific probiotic strains canreduce symptoms of IBS through immunomodulation (Kajander et al.,2008; O’Mahony et al., 2001) and may have promise for the treatment of


inflammatory bowel disease (IBD) although further research is needed(Hedin et al., 2007). Recently, L. acidophilus has been shown to reduce theinflammatory response in gastric epithelial cells via production of conju-gated linoleic acids (CLA) (Kim et al., 2008). In this study, conditionedmedium containing L. acidophilus-producing CLA interacts with IkBkinase inducing phosphorylation of inhibitory IkBa leading to its dissoci-ation from NF-kB and thus, NF-kB activation. Lactobacillus reuteri hasrecently been shown to secrete factors which potentiate apoptosis bystabilizing IkBa degradation and inhibiting nuclear translocation of p65,thus leading to suppression of NF-kB-dependent gene products thatmediate cell proliferation and cell survival including Cox-2 and Bcl-2,respectively (Iyer et al., 2008). Promotion of cell apoptosis serves as atherapy to prevent colorectal cancer and IBD (Iyer et al., 2008).

The VSL#3 probiotic mix which contains viable lyophilized bifidobac-teria (B. longum, B. infantis, and B. breve), lactobacilli (L. acidophilus, L. casei,L. delbrueckii subsp. bulgaricus, and L. plantarum), and S. salivarius subsp.thermophilus (VSL Pharmaceuticals, Fort Lauderdale, FL), can signifi-cantly modulate the immune response and has been shown to play arole in maintenance of treatment in ulcerative colitis (Venturi et al.,1999). In this study, patients with ulcerative colitis in remission weregiven VSL#3 for 12 months and it was shown that of those taking theprobiotic, the majority remained in remission throughout the studyperiod. Recently, it was shown that culturing human blood dendriticcells with cell-wall components of the probiotic mixture VSL#3 induceddendritic cell maturation and up-regulated production of IL-10 (Hartet al., 2004). Dendritic cells, which play an important role in early bacterialrecognition and in T-cell responses, may be central mediators of theseprobiotic effects. Indeed, administration of VSL#3 is associated with anearly increase in IL-10 production and regulatory CD4þ T cells bearingsurface TGF-b in murine models of colitis, while human studies haveshown increased mucosal regulatory T cells and a reduction in pouchitisdisease activity (Di Giacinto et al., 2005; Pronio et al., 2008). L. acidophilusstrain L-92 has recently been shown to regulate both Th1 and Th2 cyto-kine responses in BALB/c mice possibly through modulation of TGF-b-associated activation of T-regulatory cells, suggesting a potential therapyfor Th1- and Th2-mediated disease including autoimmune disease andinflammatory diseases (Torii et al., 2007).

D. Inhibition of virulence factor expression

A potential mechanism of action by which potential probiotic strains mayimpede pathogens is through the modulation of gene and/or proteinexpression patterns through bacterial signaling mechanisms. Interest-ingly, cell-free supernatants of L. acidophilus have been shown to inhibit


quorum sensing and virulence gene expression in E. coli O157:H7 but didnot affect expression of shiga toxin in this strain (Medellin-Pena et al.,2007). Other researchers have utilized microarray analyses to investigatethe global transcriptional changes in E. coliO157:H7 following coincubationwith L. rhamnosus GG (LGG). Results indicated that LGG coincubationreduces expression of the stx genes encoding shiga toxin production inE. coli O157:H7 (Carey et al., 2008). Subsequently, a variety of Lactobacillus,Pediococcus, and Bifidobacterium strains (L. rhamnosus GG, Lactobacilluscurvatus, L. plantarum, Lactobacillus jensenii, L. acidophilus, L. casei, L. reuteri,Pediococcus acidilactici, Pediococcus cerevisiae, P. pentosaceus, Bifidobacteriumthermophilum, Bifidobacterium boum, Bifidobacterium suis, and B. animalis)were shown to repress stxA expression in this model system, suggestinga globalmechanism bywhich themicrobiota could impede virulence factorexpression in this pathogen (Carey et al., 2008). Similarly, a recent studyexamined the ability of a variety of potential probiotic strains to inhibit theureolytic pathogen Yersinia enterocolitica (Lavermicocca et al., 2008). Theydetermined that one probiotic strain, L. plantarum ITM21B, was capable ofinhibiting urease activity in the pathogen. Overall, it is likely that futurestudies will uncover the regulatory networks that govern signalingmechanisms between pathogens and commensals.

III. CONCLUSIONS

There is mounting evidence to support a role for probiotics as an alterna-tive to conventional methods for prevention and treatment of intestinaldiseases and inflammatory disorders. The introduction of probioticorganisms has been proposed to improve digestive function (Savaianoet al., 1984), reduce chronic inflammation (Di Giacinto et al., 2005; O’Haraet al., 2006), and improve recovery from foodborne disease (Aiba et al.,1998). Previous work using rodent models of disease has demonstrated arole for probiotics in the amelioration of infections caused by Helicobacterpylori (Gotteland et al., 2006), Citrobacter rodentium (a murine model ofEnteropathogenic E. coli (EPEC)) ( Johnson-Henry et al., 2005) andS. Typhimurium (Silva et al., 2004) and clinical trials have shown thatadministration of probiotics can significantly improve eradication ofH. pylori in infected patients (Gotteland et al., 2006). In vitro analyseshave indicated that regulation of mucous production by probiotics canprevent colonization by EPEC (Mack et al., 1999) and there is an apparentcorrelation between immunomodulation by probiotics and elimination offoodborne pathogens (Jijon et al., 2004). Efficient use of probiotic therapieswill require that the precise mechanism(s) by which specific probioticstrains exert their effect is identified. While the molecular details under-pinning probiotic modes of action remain almost entirely unknown,


recently there has been significant progress towards understanding howprobiotics exert their beneficial effects at the molecular level. This sug-gests that the next phase of therapeutic development will represent a‘‘bugs to drugs’’ approach whereby probiotic-based therapeutic agentsare developed as specific pharmabiotics (O’Hara and Shanahan, 2007).

ACKNOWLEDGMENT

The authors wish to acknowledge funding by the Irish Government through the continuedsupport of Science Foundation Ireland for the Alimentary Pharmabiotic Centre, UniversityCollege Cork (http//apc.ucc.ie).

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CHAPTER 2

Advances in Food and NISSN 1043-4526, DOI: 1

Sensory Impacts ofFood–Packaging Interactions

Susan E. Duncan and Janet B. Webster


II. Consumer Perception 20

III. Threshold Concept 21

IV. Sensory Effects 22

V. Methods for Examining Taint and Other Sensory

Effects from Packaging 26

VI. Taints 27

A. Taints from contact materials 27

B. Taints from additives or noncontacting

materials 45

C. Taints from recycled materials 46

VII. Scalping/Sorption 47

VIII. Protection of Sensory Quality by Food Packaging 49

A. Protection against light 50

B. Preventing moisture loss 52

IX. Using Packaging to Improve Sensory Quality 53

A. Sensory impact of novel antimicrobial

ingredients in packaging systems 53

B. Flavor and odor absorbers for improved flavor 54

C. Controlling oxidation through

timed release of antioxidants 55

X. Conclusions 56

Acknowledgment 57

References 57

Virginia Polytechnic Institute and State University (Virginia Tech), Blacksburg, Virginia, USA

utrition Research, Volume 56 # 2009 Elsevier Inc.0.1016/S1043-4526(08)00602-5 All rights reserved.

17

18 Susan E. Duncan and Janet B. Webster

Abstract Sensory changes in food products result from intentional or unin-

tentional interactions with packaging materials and from failure of

materials to protect product integrity or quality. Resolving sensory

issues related to plastic food packaging involves knowledge

provided by sensory scientists, materials scientists, packaging man-

ufacturers, food processors, and consumers. Effective communica-

tion among scientists and engineers from different disciplines and

industries can help scientists understand package–product interac-

tions. Very limited published literature describes sensory percep-

tions associated with food–package interactions. This article

discusses sensory impacts, with emphasis on oxidation reactions,

associated with the interaction of food and materials, including

taints, scalping, changes in food quality as a function of packaging,

and examples of material innovations for smart packaging that can

improve sensory quality of foods and beverages. Sensory evalua-

tion is an important tool for improved package selection and

development of new materials.

I. INTRODUCTION

Packagingmaterials are critical components within packaging systems forimproving product sensory integrity and quality. There is no knownpackaging material, not even glass or metal, which does not interact insome way with the product it protects; these interactions can affect thequality of both the food and the package. Sensory evaluation is an impor-tant tool in the detection of packaging interactions with food. Changes infood or beverage color and appearance, flavor, odor, and textural char-acteristics as affected by packaging material chemistry, processing, andcharacteristics can make or break product success.

Food and beverage packaging functions to contain, protect, communi-cate, and provide convenience in use of the product (Robertson, 2006).A good packaging system is designed with an appropriate barrier toprotect food from external contamination by microorganisms, foreignmaterials, chemical contaminants, or environmental degradation (Kilcast,1996; Robertson, 2006). Printed information on the package provides directcommunicationpertinent to theproduct in the formofwords andgraphics,including colors, to the consumer. Indirect communication, whether inten-tional or unintentional, results when the human sensory response is trig-gered by unexpected or uncharacteristic product changes as a result offood–packaging interactions (Duncan, 2007). Such changesmaynoticeablyaffect product integrity, quality, and shelf-life, resulting in affective(degree of liking or preference) or analytical (recognition of overall orspecific product changes) human responses to the contained product.

Sensory Impacts of Food–Packaging Interactions 19

Flavor, odor, appearance, and texture of foods are not static. Bothpackaging materials and food products may undergo chemical transfor-mations that alter these food characteristics. Reactions such as oxidation,hydrolysis, and vaporization can lead to the production of off-odors andflavors and changes in texture and visual characteristics (Ayhan et al.,2001). These changes may be caused by the packaging materials them-selves, by an interaction between the package and the food, and/orbecause of poor packaging selection (Huber et al., 2002) and may lead toconsumer complaints. New packaging materials or modifications thatimprove product integrity, quality, and shelf-life are valuable if productimprovement is detectable by the consumer or end-user. However, selec-tion of an inappropriate packaging material can result in a negativesensory impression of the contained product by the user, leading todissatisfaction, decreased use, and communication to the food manufac-turer of the product problem. Approximately 50% of all off-odor com-plaints in one company were due to improper packaging, with majorproblem sources ranging from degradation of the packaging materialsto inadequate selection of packaging material (Frank et al., 2001; Huberet al., 2002). Sweets, cakes, and cookies were the primary foods affected bytaints in another food company over a 10-year period, accounting for 50%of the reported cases (Lord, 2003).

Food product manufacturers are at risk of experiencing significantlosses in production, sales, and consumer confidence as a result of detect-able sensory changes from food–package interactions, potentially leadingto a damaged brand image (Huber et al., 2002; Kilcast, 1996; Lord, 2003).Clear communications among technical representatives within the pack-aging supply line are critical (Fig. 2.1). Food manufacturers bear thehighest burden in this line of communication as they have the financialrisk of loss of production from defective products as well as the loss ofproduct, which cannot be reworked, and packages, which cannot bereused (Huber et al., 2002).

The challenge of identifying the source of a sensory problem caused bypackaging is expensive and time consuming. Communications along thepackaging supply line, therefore, should describe the sensory character ofthe material in its virgin state and after package conversion, with foodmanufacturers disclosing processing, packaging, and storage conditionsand expected duration of contact with the foodstuff (Duncan, 2007). Thepackaging supply chain includes the materials or resin manufacturerthrough the packaging supplier/converter to the food processor/manu-facturer, each having a vested interest in material and package/productsuccess. Expensive legal litigation resulting from package/product fail-ure is a potential and serious consequence of poor communication. Mod-ern legislation guards against the spoilage of foods by packagingmaterials by regulating that food packaging materials do not transfer

Retail andfood service

Consumers

Food manufacturers

Packagingmanufacturers

Polymer synthesis/manufacturers

Client interactions Communication Problem resolution

Sensory analyses/flavor and aroma

Instrumental analyses/volatile chemistry

Questions?

Problems...Suppliers

Complaints!

FIGURE 2.1 Clear communications among industry players can help prevent or resolve

sensory impacts for food–package interactions.


constituents to foods in sufficient quantities to endanger human health orcause deterioration of the sensory characteristics of foods (Huber et al.,2002; Soderhjelm and Eskelinen, 1985).

Understanding the sensory impacts related to food or beverage inter-actions with packaging materials may be helpful in preventing or resolv-ing problems as well as in selecting appropriate materials for maintainingproduct integrity, quality, and shelf-life. The source of the change insensory quality associated with a food–packaging interaction may occurat any stage of the food manufacturing or supply chain and from differentsources at each stage. Preventing the problem from occurring is best but,when a problem does occur, good communications and good detectivework are needed to determine the cause of consumer or client complaints(Duncan, 2007).

II. CONSUMER PERCEPTION

Ultimately, the ability of a packaging material to protect food qualitydepends not on whether an interaction has occurred but on whether ornot a consumer can detect that interaction. The repeat purchase of a pack-aged food product is contingent on many factors, with enjoyment andpositive sensory stimulation being among the most important (Kilcast,1996). Consumer expectations associated with sensory characteristics and


quality of a packaged food can be diminished because of unexpected food–packaging interactions. In addition, the perception of contamination causedby unexpected sensory characteristics also raises concerns about safety,even if there is little or no health risk (Anonymous, 1988; Dietrich et al.,2005). However, only a small proportion of consumers purchasing a prod-uct exhibiting a food–packaging interaction may call to comment or com-plain (AFGC, 2007). Therefore, getting sensory observations and qualitativedescriptive information from these contacts is very important.

Consumer communication of a sensory problem in a packaged foodsystem may result in a variety of descriptions from different peopleexperiencing the same problem, suggesting that these reports are unreli-able. The lack of verbal skills or training in analytical descriptive methods,unfamiliarity with chemical species that may be causing the problem, andbias associated with the conditions under which the sensation was expe-rienced make consumer descriptions of sensory changes difficult to actupon (Kilcast, 1996). It is possible that many sensitive people may experi-ence the sensory problem but only a few may report it. This may be whysome incidences of sensory problems associated with food–package inter-actions seem sporadic, limiting perception of the problem scope andmaking detection and resolution of the problem challenging (AFGC,2007). Individuals also perceive flavors at differing concentrations, orthreshold levels, and these levels can vary by a factor of a billion fromone individual to another. A report of a sensory problem by even oneconsumer of known high sensitivity may be reason enough to determinethe cause (Huber et al., 2002; Kilcast, 1996). Consumers are very importantsentinels of quality changes and their comments or complaints mayrepresent the beginning of a major problem.

III. THRESHOLD CONCEPT

The point at which the interaction between the food and package materialcauses a change in the sensory response of an individual is an importantthreshold. At this point, the observer is aware that there is somethingdifferent about the product. Typically, thresholds are discussed in rela-tion to a given chemical compound that contributes a specific odor, flavor,appearance, or textural change. Thresholds for a given compound varywith the medium (food or drink composition) in which it is present, thetemperature at which the product is presented, other stimuli contributingto the sensory character of the product, the methodology used in deter-mining the threshold, and individual sensitivity to the stimulus (Kilcast,1996; Land, 1989; Meilgaard et al., 2007).


If a high proportion of the population is sensitive to the sensorychange and the change is negative in character, there is a high potentialfor consumer complaints about the product. A sensory threshold is thelowest concentration of a compound detectable by a certain proportion,usually 50%, of a given population, indicating that the stimulus is at alevel sufficient to create a sensory perception (AFGC, 2007; Kilcast, 1996;Lawless and Heymann, 1998; Meilgaard et al., 2007). Different types ofthresholds are recognized. The detection (or absolute) threshold is thelowest physical intensity at which a stimulus is perceptible (Kilcast, 1996;Lawless and Heymann, 1998; Meilgaard et al., 2007); the recognitionthreshold is the physical intensity at which a stimulus is correctly identi-fied (Kilcast, 1996; Meilgaard et al., 2007). The range in sensitivity ofindividuals within a population to a given chemical stimulus is typically2000-fold (AFGC, 2007).

IV. SENSORY EFFECTS

Effects of materials on sensory characteristics and quality of foods andbeverages can occur from direct contact, as with a primary packageintended for containment, or by indirect means resulting from the envi-ronmental conditions (relative humidity, temperature, and air quality) aswell as the characteristics of secondary packaging materials. Anymaterialin contact or proximity to a foodstuff may have an effect. This extends tothe materials used for water distribution systems, plumbing, gaskets,adhesives, valves, shipping containers, pallets, and other materials, allof which may contribute to perceivable changes in sensory characteristics.

There is a large body of research on the effects of food packaging onflavor and odor chemistry of various foods. However, the focus of suchresearch is often targeted toward identifying the chemicals and relativeconcentrations that occur as a result of food–package interactions. Therelationship between food–package interactions and human sensoryresponse has received relatively little scientific attention.

There are numerous books, review papers, and research publicationsdescribing chemical interactions of packaging materials with foods thataffect flavor chemistry. Most explain the interactions as a function ofchanges in analytical flavor chemistry, focusing on migration of sub-stances from the package into the food, absorption of flavor componentsfrom the food into the package, and transfer of odors, gases, and lightthrough the polymer package, which then can affect food quality(Ahvenainen, 2000; Barnes et al., 2007; Katan, 1996; Leland, 1997;Piringer and Ruter, 2000; Risch and Ho, 2000). In many research studies,the sensory impact of these reactions is not directly described or is notevaluated, although there may be reference to the impact. Relying only on


analytical evaluation of flavor chemistry can be deceiving because thesecompounds may or may not have an impact on the sensory quality of thefood (Piringer and Ruter, 2000; Torri et al., 2008).

Sensory evaluation can help characterize flavors, aromas, appearanceeffects, and textural changes that might not be able to be determinedanalytically. Sensory perception is often more sensitive than analyticalmethods and in some cases is the only way to determine if a change hasoccurred in a food due to an interaction with its packaging. Human sensescan detect changes in volatile chemistry at extremely low concen-trations (10�6–10�12 mol/ml) for some chemicals (Kilcast, 1996; Lawlessand Heymann, 1998; Meilgaard et al., 2007; Reineccius, 2006). However,there are problems with sensory testing that must be kept in mind. Theseproblems include differing perceptions and thresholds for changes inappearance, texture, flavor, and aroma due to the unique physiologicaland psychological makeup of individual human subjects. The rangeof human sensitivity, verbal skills for description of the sensation(s),and affective response to the sensation contribute to the challenges asso-ciated with identifying the source of a sensory problem in a packagedfood system.

The combination of sensory evaluation with analytical approaches isrequired to identify perceptible changes and to identify the potentialchemical changes that may be causing the sensory effect. Low concen-trations of compounds responsible for changes in food characteristicsmay not be detectable by even the most sensitive analytical methodsbut, in combination with appropriately applied sensory methods, theclues provided by both techniques may help identify the problem, pro-vide indications to the cause, and suggest clues for the source of theproblem.

Sensory issues relating to food and packaging interactions may beclassified based on the sensory quality changes that occur. Four broadcategories that infer the direction (positive/negative) of the qualitychange include: (1) taints resulting from the packaging material or as afunctional limitation of the package; (2) scalping of food constituents;(3) packaging function for protection of sensory properties of the food;and (4) improving flavor and odor quality through food–package interac-tion (Fig. 2.2).

Taints are defined as a taste or odor foreign to the product (AFGC,2007; Kilcast, 1996). Taints typically are unpleasant in character and areinitiated by or originate from sources external to the food; they are causedby constituents of the packaging material or the near environment migrat-ing into the product. Common sources of taints related to food packaginginclude packaging materials, inks and dyes, adhesives, and secondarypackaging including pallets, shipping containers, and corrugated card-board materials. Although taints are contaminants of the food

Taint

Scalping

Light

Moisture

Gases/volatiles

Fat

Water

Foodflavorants

Colorant

Materialmolecules

FIGURE 2.2 Negative sensory impacts occur when tainting or scalping occur or

ineffective packaging selections are made.


environment, not all contaminants cause taint (AFGC, 2007). To meetlegal and consumer expectations, the goal should be zero levels of taintingspecies (below sensory threshold) so even the most sensitive members ofthe population cannot detect the taint (Kilcast, 1996). The concentration ofthe penetrating molecules may be very low and still create a significantsensory response. Tainting molecules in the headspace of a package willinfluence the sensory response by the consumer when the package isinitially opened and perhaps motivate a complaint action (Kilcast, 1996).The threshold for detection in the air above the sample is typically lowerthan needed to cause a response in the food medium. The majority ofpublished literature associated with sensory issues of food–packageinteractions is related to taints and flavor scalping.

Scalping (sorption) is also detrimental to consumer perception of foodquality. From a sensory perspective, this type of interaction is different fromtaints. This phenomenon is associatedwith key food constituents absorbinginto a plastic or othermaterial (Brody, 2002). In this situation, desirable foodconstituents, such as aroma compounds, acids, lipids, and pigments, areremoved from the food or beverage by the packagingmaterials. This ismostcommonly described in relation to a decrease in flavor intensity or as analteration of the flavor profile but could also be associatedwith color or odorchanges. Changes or decreases in quality may be perceived by consumers.The exhibition of reduced sensory quality in many shelf-stable foods inplastic packaging is attributed to scalping (Brody, 2002).


Off-flavors and off-odors, in contrast to tainting or scalping, are relatedto changes in food chemistry associated with degradative reactions withinthe foodordeteriorative changes of food components (AFGC, 2007;Kilcast,1996). The character of these changes is typically unpleasant and undesir-able in relation to food quality and consumer satisfaction. In addition,changes in pigment chemistry, moisture loss (or gain), or other reactionswithin the food system may result as a function of improper materialselection. Sensory studies on the protective function of packaging (main-taining/improving) on quality primarily exist as supporting informationto the assessment of chemistry,microbiology, processing, or engineering ofthe food–package system. In many cases, these sensory studies aredesigned to determine if a difference exists because of the experimentalvariables but detailed, descriptive studies of sensory impacts of theprotective function of packaging on food properties is limited.

Novel packaging approaches using active materials or new packagingtechnologies for a desired sensory effect within the food system, or thosethat enhance product sensory quality and improve shelf-life, do not fitinto any of the prior categories. This area of literature is emerging.

The value in understanding the differences in these sources of subtlechanges in sensory quality is associated with distinguishing the cause andpossible solutions associated with the problem. There is a wide array ofpotential contaminants associated with taints and identifying the cause assomething originating from external contamination or from internal prod-uct change helps narrow the search (Kilcast, 1996). Understanding sen-sory impacts from food–package interactions will provide greatercapacity for improving food–package systems. For example, a piney-spruce taint was observed in a routine quality assurance examination ofa packaged ready-to-eat breakfast cereal (Heydanek, 1977). With a carefulanalytical approach that utilized sensory evaluation in the early stages,the source was traced to the resin bonding paper layers in the glassineliner of the package. With the use of gas chromatography–mass spectros-copy (GC–MS), in combination with additional sensory testing of addi-tional glassine materials, the migrant molecules were identified as majorterpenes, fenchyl alcohol, and borneol.

The purpose of this review is to give an overview of the effect that foodpackaging has on the sensory quality of food. The focus will be on humanresponse and sensory terminology used in describing these interactions inrelation to foods and beverages in contact with different materials. Vola-tile chemistry will be described only as it relates to the sensory effects. It isnot the intent of this review to provide detailed information on sensoryevaluation methodology. There are numerous text and reference booksfor developing a basic knowledge of sensory evaluation methods. How-ever, a brief description of nontraditional sensory methods for examiningtaint and other sensory effects from packaging is provided.


V. METHODS FOR EXAMINING TAINT AND OTHER SENSORYEFFECTS FROM PACKAGING

The selection of an appropriate sensory methodology for examiningtaints, scalping, off-flavors, or improved product quality is dependenton the project objectives. No single method can answer all questions, suchas describing the flavor character and intensity as well as consumerresponse, so effective communication of the project goal and objectivesamong the sensory specialist, packaging expert, and project leader isneeded (Duncan, 2007). Appropriate selection and training of participantsin descriptive evaluation is needed to provide accurate and reliable qual-itative and quantitative responses. Descriptive methods, which providemuch more information than discrimination or affective methods, arehelpful in differentiating how a product–package interaction affects sen-sory profiles. However, this is a very time intensive effort because of theinvestment in panel preparation and maintenance. Discrimination testingcan be very effective in determining if a difference occurs because of theproduct–package interaction but these methods do not describe how(change in sensory profile) the product–package interaction affects per-ception. Such methods require little or no training of panelists but morepanelists are needed to increase the power of the test. Untrained panelistsrepresentative of the targeted consumer population should be used forestimating affective consumer responses to taints and off-characteristicsas well as verifying value-added quality maintenance or improvements ofpackaged food products. Since a wide range of individual responsescan occur in consumer testing, a large number of consumers are neededto verify if the product–package interaction is truly having an impact.For all methods, attention to appropriate environmental, sample, andpanelist controls are needed during preparation and presentation.Communication with panelists must be appropriate for instructingpanelists in completing the tasks but restricted in details of samplesand objectives in order to avoid biases that may affect outcomes orinterpretation of the results.

Standardmethods that apply to detecting taint from plastic and paper-boardmaterials are described in US ASTM Std E 462-84 (tests for odor andtaint transfer from packaging film), ASTM E 619-84 (examination of odorsfrom paper packaging), and German Standard DIN 10 955 (testing oftaints transferred by direct contact and also by vapor phase transfer)(Kilcast, 1996). Many of these methods are based on accelerated storageconditions and using food simulants or water or on evaluation of volatilecompounds emitted from packaging material (Kim-Kang, 1990; Torriet al., 2008). Sensory methodology books (Lawless and Heymann, 1998;Meilgaard et al., 2007) provide guidance for appropriate design and


application of sensory methods. Book chapters with specific reference topackaging (Kilcast, 1996, 2003; Lord, 2003) provide some background andgeneral sensory information in the context of the sensory effects of foodpackaging. Recent German references assess difficulties in standardizingmethods for sensory analysis of packaging materials and quality require-ments for colorants and additives used in food and beverage packaging(Anonymous, 2007; Buettner et al., 2007).

VI. TAINTS

Although there are many routes by which taints may occur in foods, oneof the greatest risks is from contact of foods with materials that maycontain potential migrants (Baigrie, 2003; Kilcast, 1996; Reineccius, 2006).Many materials have molecules that can migrate from the package intothe food product. In addition, polymer materials, such as gaskets used inmanufacturing processes, containers used for transport, or packagingmaterials inappropriately used in a process (i.e., irradiation or hot-fill)may cause taints. Water distribution and plumbing materials, flooringmaterials, and disinfectants or other environmental contaminants onfood packaging surfaces also may be sources of tainting molecules.Long-shelf life foods, which are stored in direct contact with packagingmaterials for a long period of time, are at great risk of developing taint.Beverages, extended shelf-life (ESL) or shelf-stable milk products, andother liquids have a high risk for tainting chemical species to transferinto the food matrix. Water and many ESL dairy products have a lowflavor profile so even a low concentration of migrating molecules fromthe polymer may impact sensory characteristics in these products.A higher fat content within the food product, such as chocolate,contained in the packaging material can cause an increased taint withinthe food product. Levels of tainting odors and flavors are highest wherethere is direct contact between the packaging material and foods with ahigh fat content on the surface. Sensory descriptors associated withtainting chemicals from packaging sources have been reported(Ewender et al., 1995; Lord, 2003).

A. Taints from contact materials

Materials may exhibit a tainting odor from the polymers, monomers,additives, adhesives, or process. Packaging related taints, as reportedfrom the Nestle’s Central Packaging Laboratory over a 4-year period(1996–2000) were related to solvents (28%), degraded polyethylene (PE)(24%), styrene (15%), halogenated phenols (15%), degraded paper (3%),


and other unknown sources (15%) (Huber et al., 2002). The virgin materialshould not exhibit an odor; however, that will not guarantee that thematerial will not impact the sensory profile of the food.

Many materials used for food and beverage packaging have charac-teristic odors or sensory active compounds (Torri et al., 2008). The inten-sity and description of the odor may be affected by the number and typeof volatile compounds that are released under environmental conditionsat the time of evaluation. Chemical composition of the material andpolymer morphology may play a role in the sensory characterization.Sensory descriptors do not define a specific chemical compound butmay be related to different compounds, a blend of compounds, and evena limited concentration range of a compound or class of compounds. Forexample, trans-2-nonenal in water changes in sensory (taste) descriptionfrom ‘‘plastic’’ (0.2 mg/l) to ‘‘woody’’ (0.4–2.0 mg/l), ‘‘fatty’’ (8–40 mg/l),and ‘‘cucumber’’ (1000 mg/l) (Piringer and Ruter, 2000). Such terms aredescriptive of the sensation and perception by human response to thechemical stimuli (Table 2.1).

Sensory active components from packaging may influence the percep-tion of product quality (Piringer and Ruter, 2000). Parameters that maycontribute to sensory influence include (Granzer et al., 1986):

� Concentration of the component in the packaging material.� Solubility of the component in the packaging material (partition gasphase/packaging material).

� Solubility of the component in the food (partition gas phase/food).� Sensory threshold level of the component.� Type and intensity of the food aroma.� Diffusion rate of the component in the packaging material.� Diffusion rate of the component in food.� Time and temperature of storage.� Ratio of the amount of packaging material to the amount of food.

Consideration of these parameters is important when tracking thesource of taints and interpreting the relationship between analyticalchemistry and sensory impact of the food–package interaction.

Evaluating odor and flavor taints is frequently done with water, fattyfood simulants (oil, chocolate, unsalted butter), hydrophilic powders(sugar, cornflour), or combined hydrophilic–hydrophobic matrices(milk or cream, biscuits) (Kilcast, 2003). The Robinson test often is usedto evaluate materials for tainting potential. This test places the testmaterial in a sealed container separated from the food simulant or testfood at a relative humidity between 53% and 75%. After about 48 h, thetest food is evaluated for taint compared to a control, using a discrimi-nation method (Lord, 2003). Chocolate is frequently used as the foodsimulant for this test. Intensity of the taint may be evaluated using a

TABLE 2.1 Sensory descriptors associated with taint sources (Baner, 2000; Caul, 1961;

Huber et al., 2002; Piringer and Ruter, 2000; Torri et al., 2008)

Taint sources Material Sensory descriptors

Packaging

materialsaPlastics Acetic acid, acrid, alcohol,

adhesive, burnt, burnt wax,

candle-like, cat urine,

chemical, lube oil, musty,

oxidized polyethylene,paint, paraffins, paste,

phenolic, plastic, pungent,

rancid, soapy, solvent,

stale, styrene, stuffy,

vinyl, waxy

Paper/board Almonds, cardboard, fruity,

green grassy, pine, rancid

Miscellaneous Jute sack, petrolPrinting/

convertingbSolvent based Aromatic, camphorated,

chemical, fruity, solvent,

sweet, toluene

Offset printing Fat, linseed oil, mineral oil,

painty, petrol,

rancid, varnish

External

contaminationcChemical Antiseptic, disinfectant,

herbicide, hospital,insecticide, medical,

metallic, phenolic

Microbiological Moldy, mushrooms, musty

Miscellaneous Catty, cork, naphthalene,

wooden pencil

a Material-intrinsic odorsb Solvent based includes flexo and gravure printingc Absorption of foreign odor from other materials in contact with packaging during production and storageas well as food–package interactions


5-point rating scale (0 ¼ not perceptible; 4 ¼ strong). The use of theRobinson-style test method, using the rating scale, is effective for qualitycontrol of materials intended for food packaging. Understanding thesensory influence of raw and processed materials on food simulantsand complex matrices is helpful in reducing taints in packaged foods.Table 2.2 provides a summary of sensory descriptors associated withfoods and beverages in contact with packaging materials.

TABLE 2.2 Sensory descriptors associated with some packaging materials containing different foods and beverages

Broad

category

Packaging

material Description Product Sensory description References

General Glass Milk Plastic, oxidized, burnt

flavor

Karatapanis et al. (2006)

Metal Beer Musty flavor Council AFaG (1997)

Canned pork products Catty flavor Kim-Kang (1990)

Paper Board stock Cakes, biscuits, chocolate

confectionary

Off-flavor Goldenberg and

Matheson (1975)

Chocolate-coated cakes Off-flavor Goldenberg and

Matheson (1975)

Mushroom odor

Musty, mouldy Caul (1961)

Halogen-like odor Caul (1961)

Papery, cardboardy Caul (1961)

Burnt Anonymous (1988)

Breakfast cereal Piney flavor Heydanek (1977)

Milk Stale, fruity flavor Karatapanis et al. (2006)

Painty, chalky, fruity,

rancid, sulfide, and

resinous odor

Anonymous (1988)

Packing sacks Cocoa powder Mouldy odor and

disinfectant flavor

Whitfield et al. (1984)

Cocoa beans Mouldy flavor Anonymous (1988),

Whitfield et al. (1984)

Waxed parchment

paper

Butter Refrigerator/stale Lozano et al. (2007)

Adhesives Coatings Waxy, oily, rancid Caul (1961)

Spoiled protein (sulphide) Caul (1961)

Resinous Caul (1961)

Printing films Cakes, biscuits, chocolate

confectionary, sugar

confectionary

Off-flavor Goldenberg and

Matheson (1975)

Plastics Polyamide/ionomer

laminate

Retort pouch Ham products Cat urine odor Piringer and Ruter (2000)

Polyester, aluminum

foil, polyethylene

laminate

Retort pouch Fruit-flavored soft drinks Off-flavor Passy (1983)

Glassine Liner Ready-to-eat breakfast

cereals

Pine or spruce-like odor Heydanek (1977)

Candy wraps Bitter, burnt, old rubber-

like flavor

Kim-Kang (1990)

Polypropylene Oats Stearic, paint, bitter, hay,

rancid odor, and flavor

grainy

Larsen et al. (2005)

Heydanek (1978)

Boiled sweets Jasmine-like, herbal and

floral

Lord (2003)

‘‘Lube’’ oil, burnt, phenolic Caul (1961)

Orange juice Musty odor Feigenbaum et al. (1998)

Polyolefins Low density

polyethylene

(LDPE)

Candle-grease, musty,

rancid, soapy, pungent,

acrid, sickly, astringent,

synthetic, metallic, and

dry flavor

Linssen and Roozen

(1994)

Water Musty Linssen and Roozen

(1994)

High density

polyethylene

(HDPE)

Milk Stale, fruity flavor Karatapanis et al. (2006),

Moyssiadi et al. (2004)

Corn chips (snack foods) Plastic odor Sander et al. (2005)

Water Tastes: sweet, metallic,

stony, pungent, dusty,

Villberg et al. (1997)

(continued)

TABLE 2.2 (continued )

Broad

category

Packaging


stale, plastic, foul, stink

bug, candle grease

Odors: Sweet, chemical,

stale, dirty, foul

HDPE water pipes Water Earthy-musty flavor Council AFaG (1997)

Waxy, plastic, citrus Heim and Dietrich

(2007b), Dietrich

(2007)

Polyethylene (PE) Wine Pungent, musty odors

Coffee Rio, medicinal, phenolic,

or iodine-like flavor

Council AFaG (1997)

Wine Musty cork flavor Council AFaG (1997)

Prawns and ocean fish Iodoform taint Council AFaG (1997)

Oxidized oil, waxy,

rubbery

Caul (1961)

Candle-like, stale, stuffy,

musty, soapy, rancid

odor

Piringer and Ruter (2000)

PE-coated

paperboard

Milk, water, fruit juices Plastic flavor

Wax-like odor

Leong et al. (1992)

Piringer and Ruter (2000)

Cross-linked PE

Water pipes

Water Alcohol, sweet chemical,

plastic, bitter,

mechanical, glue,

burning, spicy, fruity,

almond,rotten swampy,

burning plastic pipe

Durand and Dietrich

(2007)

PE þ rubber net Water Yellowish color, plastic

taste/odor

Kontominas et al. (2006)

Polyethylene

terephthalate

(PETE)

Papery, scorched cloth Caul (1961)

Milk Plastic, oxidized, burnt

flavor

Karatapanis et al. (2006),

Moyssiadi et al. (2004)

Colas Turpentine-like odor Kim-Kang (1990)

PETE þ rubber net Water Yellowish or opaque color,

taste/odor

Kontominas et al. (2006)

Polystyrene Oat products Insecticide or plastic flavor Heydanek (1978)

Woody, sweet Caul (1961)

Plastic-like chemical odor

and flavor

Heydanek (1978)

Coffee creamer and

condensed milk

Astringent chemical plastic

flavor

Baner (2000)

Chocolate and lemon

cream cookies

Off-flavor and odor Passy (1983)

PVC Fruit drinks Catty flavor Kim-Kang (1990)

Orange and lemon

drinks

Off-flavor Kim-Kang (1990)

Packed cheese (with PE/

PETE lid)

Pungent, chemical pine

odor and flavor

Lord (2003)

Epoxy Drinking water Plastic, glue, putty,

adhesive, chemical,

musty

Heim and Dietrich

(2007a), Dietrich

(2007)

Cellophane Sweet, woody, rubbery Caul (1961)

Cellulose film Sandwiches Off-flavor Goldenberg and

Matheson (1975)

Other Environmental

odorants

Floral Caul (1961)

Woody Caul (1961)

(continued)


Broad

category

Packaging


Musty, cardboardy, burnt,

floral, painty, chalky,

fruity, rancid, sulfide,

resinous, woody odors

Caul (1961), Anonymous

(1988)

Printing Inks Off-odor Caul (1961)

Musty odor Anonymous (1988)

Musty, cardboardy, burnt,

painty, chalky, fruity,

rancid, sulfide, and

resinous odors

Anonymous (1988)

Oils Oily, fatty, buttery Caul (1961)

Varnishy, painty Caul (1961)

Inky, rancid Caul (1961)

Garbagey Caul (1961)

Rubber

hydrochloride

(pilofilm)

Sour milk (Casein) Caul (1961)

Halogen-like odor Caul (1961)

Vinyl Saran Aromatic sweet, chlorine,

oxidized oil, or solventy

Caul (1961)

Vinyl chloride Alcohol, soapy Caul (1961)

Glued seams Cocoa powder Moldy odor and

disinfectant flavor

Anonymous (1988)

Mixture Maple syrup Off-odor Kim-Kang (1990)

Granular gelatin Fish odor


1. Polystyrene (PS)Residual styrene monomer from polystyrene production has been asso-ciated with tainting problems in different food products (Baner, 2000;Heydanek, 1978; Huber et al., 2002; Piringer and Ruter, 2000). Sensorydescriptors for styrene monomer include chemical, insecticide, and plas-tic (Baner, 2000; Caul, 1961; Heydanek, 1978). Thresholds for styrenemonomer are very low in water (taste threshold: 0.022–0.37 mg/kg) andair (odor threshold: 0.050 mg/kg). Thresholds in complex foods andbeverages range from 0.2 to 0.3 mg/kg for orange fruit juice drink, a 3%oil-in-water emulsion, and skim milk (0% fat), 1–3 mg/kg for whole milk,oil-in-water emulsions of 15–30% oil, cocoa powder (10–20% fat), andgreater than 3 mg/kg for condensed milk (10% fat), butter, and cream(33% fat) (Baner, 2000). PS starts to decompose at very low levels afterseveral hours and at temperatures greater than 240 �C. Ethylbenzene,which is commonly used to dilute solvents during PS polymerization, isanother source of these taints (Baner, 2000).

Styrene monomer concentration in foods packaged in 31 different PS-containing food packages and contact materials averaged 224 mg/kgwith two products having concentrations between 800 and 1500 mg/kg,well above the sensory threshold limits (Baner, 2000). Strict specificationsfor styrene monomers as well as for residual solvents, toluene, and odorand taint transfer for supplier materials should be set (Huber et al., 2002).

Polystyrene used for plastic cups has been a source of off-flavors(Huber et al., 2002). Oat products stored in polystyrene containers devel-oped an ‘‘insecticide or plastic’’ off-flavor after 6 weeks of storage(Heydanek, 1978). Corn products stored under the same conditions didnot develop these taints. Compounds with odors similar to the off-flavorin oats were identified by GC–MS and gas chromatography–olfactometry(GC–O). A similar pattern of volatile peaks were observed in both the PSpackage and the oat products. The taint was identified as being related tothe styrene monomer, which was probably present in the PS feedstock.The level of styrene residual was much greater in oats (�146 mg/kg), at20 times the styrene odor threshold in air (0.73 mg/kg), than in corn(�1.5 mg/kg). Styrene levels in corn were at twice the odor thresholdbut were not high enough to cause a taint in the corn product. It is possiblethe higher fat content of the oats, 11% as compared to 2% for the corn,increased product affinity for the styrene monomer.

Coffee creamers and condensedmilk packaged in thermoformed poly-styrene single serve (5–10 g product) portion pack containers havedemonstrated styrene taint problems (Baner, 2000). These products aretypically packaged at ultra-high temperatures (UHT) and, if packagedaseptically, may be stored without refrigeration. The higher storagetemperature increases the potential for styrene taint in the productand substantially decreases the potential product shelf-life because of


tainting issues. The use of laminate materials, placing PE and ethylenevinyl alcohol (EVOH) on the food contact surface, reduces the migrationof styrene monomers from PS to the food. However, even with theselaminate barrier materials there still have been styrene taints resultingfrom the styrene monomer migrating from the PS layer to the inner PElayer when the material was shipped and stored in roll form prior toforming (Baner, 2000). The estimated styrene concentration migratingfrom the PS into creamers was 23–31 mg/kg, far exceeding the sensorythreshold of 0.1–3 mg/kg in cream and potentially creating a sensoryimpact when the cream is diluted into a food or beverage, such as tea orcoffee. The large ratio of package surface to cream volume in these singleserve portion packs contributes to this high concentration. The high fatcontent of cream also increases the transfer rate whereas nonfat milkwould have much lower, probably undetectable, styrene taint under thesame conditions (Baner, 2000). However, other research suggests thatmilkfat masks low levels of taint from other materials in whole milkcompared to nonfat milk (Leong et al., 1992; van Aardt et al., 2001a).

Storage temperature and changes in product sensory profile withstorage also affect the perception of taint from styrene monomer andother tainting compounds. Styrene monomer migration was suggestedas a possible contributor to flavor and odor changes of butter packaged inwaxed parchment paper during refrigerated storage (Lozano et al., 2007).Butter wrapped in common commercial wrapping foil or wax parchmentpaper demonstrated different flavor profiles at 6 and 12 months of storageat refrigeration (4 �C) and freezer (�20 �C) temperatures (Lozano et al.,2007). A trained sensory panel (n ¼ 8) evaluated intensity (0 (absence);15 (high)) of selected sensory characteristics (cooked/nutty, milkfat/lactone, refrigerator/stale, and salty taste) representing characteristics offresh and stored butters. Butters stored for 6 months at refrigerationtemperature in waxed parchment paper had detectable ‘‘refrigerator/storage’’ flavor (intensity of 1.0–1.1) compared to fresh and foil-wrappedbutters but products in both packaging had lower intensities of positiveflavor attributes (‘‘cooked/nutty,’’ ‘‘milkfat/lactone’’). At 12 months,mean intensity values for both parchment and foil wrapped refrigeratedproducts were between 1.6 and 1.9, indicating that this flavor problemhad increased and the intensity of ‘‘cooked/nutty’’ notes continued todecrease. Frozen products wrapped in parchment exhibited a very low(0.5) intensity of refrigerator/stale flavor after 12 months of storage ascompared to no perception of this characteristic in foil-wrapped butter.Frozen storage helped to maintain levels of milkfat/lactone notes and,while cooked/nutty notes decreased slightly more in parchment-wrapped butter than in foil-wrapped, this characteristic still wasobserved at higher levels in frozen products than in refrigerated productsat 12 months.


Styrene monomer migration from the package stored at refrigerationtemperatures was detected by GCO but frozen storage can decrease themigration rate (Lozano et al., 2007). Odor threshold levels of styrene,ethylbenzene, and toluene in oil were reported as 3.4, 4.1, and94.7 mg/kg, respectively, and were higher than the level of these com-pounds found in all butter samples. Lozano et al. (2007) suggested theadditive effect of multiple benzene derivative compounds at subthresh-old levels, in combination with declining fresh flavor compounds, alteredthe perception of the refrigerator/stale flavor intensity as the parchment-wrapped product aged. The authors also suggested that differences inmatrix between oil (100% lipid) and butter (80% lipid, 20%water) must beconsidered when relating thresholds from oil to butter. They hypothe-sized that styrene monomer thresholds in butter would be lower thanthose reported in oil. This is an important consideration when applyingthreshold data of compounds in food simulants to the sensory detectionof these compounds in complex food products.

Although the common method for assessing thresholds is a 3-samplealternative forced choice method with increasing concentrations, there aresome product characteristics that make presentation of more than onesample at a time unsuitable (Lawless and Heymann, 1998). Linssen et al.(1991) evaluated PS packaging material taint in chocolate ingredients(chocolate flakes (15% fat), cocoa power (10% and 20% fat), intended forbeverages. Chocolate ingredients were mixed with or without (control) PSsheet pieces (0.5 dm2, 2.0 dm2), and stored in glass jars for 7 days at 30 �C.Drinks were prepared from chocolate ingredients, water, and sugar forsensory testing. Untrained panelists (n ¼ 48–50) evaluated the drinkscompared to a standard (no PS) for a difference comparison as well asfor recognition of styrene. A signal detection discrimination method wasselected because product stickiness and lingering taste character limitedthe number of samples that could be presented simultaneously.

Using the signal detection method, panelists responded to each sam-ple with one of four category choices (Linssen et al., 1991). The categoriesfor the difference test and the styrene recognition test were ‘‘(same as the)standard, perhaps standard, perhaps not the standard, not standard’’ and‘‘styrene recognized, perhaps styrene recognized, perhaps styrene notrecognized, styrene not recognized.’’ R-indices, used to express theresults of signal detection over the characteristic background, representthe probability of correctly distinguishing (chance ¼ 50%; 100% indicatesperfect discrimination) between products or correct recognition of styreneby the panelists. There was no difference in taste, based on R-indices closeto 50%, between test samples and the standard for cocoa drinks andchocolate flakes in contact with low styrene exposure (0.5 dm2). Higherstyrene exposure (2.0 dm2) in contact with chocolate flakes increased theprobability of identifying the difference, with R-indices of 64% and 72%


for the plain and milk chocolate flakes, respectively. Milk chocolate has alower bitter taste intensity than plain chocolate and is more susceptible tostyrene taint. Comparisons to a 200 ppb styrene standard in waterincreased the probability of identifying the styrene taint in chocolateflakes (R-indices of 62–88%). Providing a recognition standard increasesthe probability of discriminating the difference. Increasing the contactarea and polymer thickness increased the migration concentration ofstyrene and increased the chance of taint.

Because the potential for styrene taint to occur is high, any packagingmaterials containing PS should be evaluated under accelerated migrationtesting conditions with the intended product (Baner, 2000). The productshould be tested using a sensory discrimination test, such as a triangletest, in comparison with a reference product stored under the same con-ditions but not in contact with the packaging. Accelerated testing condi-tions must consider food product quality to avoid significant chemical ormicrobiological spoilage.

2. Polyamides (PA)Good oxygen barrier properties of a material do not guarantee a goodflavor barrier. Polyamides have good oxygen barrier properties but do notprovide a good flavor barrier because of their hydrophilic properties(Brody, 2002). Two ham products, cooked and packaged in PA/ionomerlaminate films from different film manufacturers, were identified, byconsumer complaints, as smelling like cat urine (Piringer and Ruter,2000). The PA source was different for eachmanufacturer but the ionomerwas traced to one source. Linkage to a specific printing ink on the filmwasevident and the source was traced to diacetone alcohol (DAA), a chemicalprecursor of mesityloxide. The cat urine aroma resulted from a reactionby sulfur-containing proteins in the ham that had migrated into theionomer film with DAA, converting it to mesityloxide (Piringer andRuter, 2000). Printing inks must be free of mesityloxide and its precursorswhen used on ionomer-containing laminates or films intended for pack-aging foods with sulfur-containing proteins (Piringer and Ruter, 2000).

3. PolyolefinsThis class of materials includes the family of plastics based on ethyleneand propylene (Robertson, 2006). Low, linear, and high density PE andpolypropylene (PP) materials are common food packaging materials. Theuse of polyolefins, such as PE and PE terephthalate (PETE) in contact withfoods and beverages is common. However, contact with foods, especiallyunder conditions of heat or long duration, can potentially impact sensorycharacteristics of the contained product. Packaging should be carefullyselected, especially for applications that involve heat treatment at hightemperatures while in contact with foods.


Residual solvents from processing of polyolefins and other materialscan affect sensory quality of packaged foods. Entrapment of solvents,inks, or other tainting chemicals within layers of newly processed mate-rial on a reel increases the risk of taint into food. Frank et al. (2001)reported that chocolate wrappers (paper/aluminum low density PE(LDPE)/Surlynj) with a high odor score (3.0; 4.0 ¼ very strong differencefrom reference), based on a trained descriptive panel, demonstrateddecreasing odor intensity, from the initial odor score 1.3 when aeratedfor up to 48 h. However, the score (2.6) at 24 h was still above the maxi-mum odor score for acceptable use (2.5 out of 4) as food packaging.Packaging materials are rolled on large reels for shipping and, if notappropriately aerated prior to use, solvents remaining on the inner layersof the roll can cause taint in wrapped chocolate and other foods (Baner,2000; Frank et al., 2001). Aeration (gassing-off ) of unrolled materialsenables the packaging supplier to reduce the odor through gassing offof solvents, thus, improving the product quality. Food manufacturersshould inquire if adequate aeration time was provided prior to packageconversion and require such specifications be met.

The intended use of materials in contact with foods can be a criticalparameter in determining material selection. Heating of materials incontact with foods can increase the risk of taint in some circumstances.Plastic nettings, made of plastic-based threads from PE and PETE, areoften used for containing raw meats and vegetables. Vegetables, such aspotatoes or carrots, are typically washed and often peeled after removalfrom the package before raw consumption or cooking, thus, reducing therisk of taints. However, some meat products are cooked in these nettingmaterials at temperatures exceeding 100 �C for an hour or more.Migration of molecules from these packaging materials into fatty andaqueous-based food simulants was detected at ranges below and abovethe European Union established migration limits (60 mg/l) (Kontominaset al., 2006). The impact of migrating molecules was measured by heatingdifferent PE or PETE net or threads in potable water for 4 h at 100 �C.A panel of five judges evaluated the taste, odor, and color of the aqueoussimulant based on a 5-point intensity scale (0 ¼ no difference betweenexperimental and control sample (water); 4 ¼ very large difference).A preset designation established that a score greater than 1 indicatedan unacceptable sensory score and evidence of sensory impact fromcontact of the material with the aqueous simulant. Only 7 of the 15materials tested received a score lower than or equal to 1, the sensoryacceptability limit. Sensory characteristics that developed in most watersamples included a yellowish discoloration or opaque appearance andoff-odor and off-taste described as plastic at slight to moderate intensities.Netting materials (PE or PETE) containing rubber and rubber plus cottonresulted in higher sensory impacting characteristics compared to


materials consisting of PE or PETE only. Only one sample of PETE thread(no rubber or cotton) produced objectionable plastic taste (score ¼ 2) andslight odor as well as a moderate difference in color (yellowish, dispersiondevelopment) compared to the water control sample.

There was no direct relationship between sensory characteristicsfound in the water migration behavior of molecules in the various nettingmaterials tested (Kontominas et al., 2006). Some samples that exhibitedacceptable levels of migration had a moderate or greater intensity of off-flavor, off-odor, or color impact. Not all materials with high migrationlevels demonstrated sensory impacts in the water. There were samplesthat had values below the upper limit for migration as well as no realchange (less than or equal to 1) in sensory characteristics compared to thecontrol water sample.

Oxidation products of plastics materials may be responsible foroff-odor development in heated plastic materials. Relevant sensory com-pounds are not the alkanes and alkenes, which have high sensorythresholds, but the less concentrated oxygenated compounds, which havelow sensory thresholds (Piringer and Ruter, 2000). One-heptene-3-oneand 2-nonenal were identified as important tainting compounds fromPE-containing packaging materials (Piringer and Ruter, 2000).Kontominas et al. (2006) indicated 2-ethyl hexanal, heptanal, octanal, and2,6-di-tert butyl quinone might have been responsible for tainting from thePE and PETE net and threads. PE odor is characterized by a number ofsensory descriptors such as candle-like, stale, musty, stuffy, rancid, andsoapy (Piringer and Ruter, 2000). Many of these sensory descriptors areused in describing oxidation of foods components.

Heat during polymer processing (polycondensation and melt proces-sing) and package formation can cause formation of low molecularweight acetaldehyde (Robertson, 2006). Dabrowska et al. (2003) andEwender et al. (2003) reported that the concentration of acetaldehyde,due to the degradation of the PETE polymer during bottle formation,reaches �4.5–5.5 ppm when the process is continuous and stable. How-ever, concentrations of more than 50 ppm have been observed even whenthere was only a short standstill in the bottle production line (Dabrowskaet al., 2003). The production of acetaldehyde appears to be initiated whenPETE bottles are exposed to ozonated water used for the disinfection andwashing of bottles during their manufacture (Dabrowska et al., 2003).Several authors have reported that aldehydes are major oxidation pro-ducts during ozone disinfection (Weinberg et al., 1993; Richardson, 1998)and Mehta and Bassette (1978) reported high amounts of acetaldehydeproduction after milk cartons were exposed to ethylene oxide steriliza-tion. However, Song et al. (2003) reported that no new compounds wereformed in PETE upon exposure to ozonated water. Acetaldehydemigration from PETE can alter flavor profiles of foods and beverages.


Acetaldehyde imparts a fruity, green apple flavor and is found inmany food products, including fruits, beverages, and yogurt. The sensoryimpact of the acetaldehyde taint from packaging is highly dependent onthe food system. van Aardt et al. (2001a) determined human thresholdlevels for acetaldehyde in spring water (167 ppb), milk, and chocolatemilk using a three-sample alternate forced choice test series with a panelof 25 people. Threshold values for acetaldehyde in milk (whole milk,4040 ppb; low-fat milk, 4020 ppb; nonfat milk, 3939 ppb) were notaffected by fat content. Chocolate milk had a threshold value of10,048 ppb, which compared well with the results of Bills et al. (1972)who looked at the threshold level of acetaldehyde in strawberry milk(11,700 ppb). The higher threshold for chocolate milk, as compared towhole milk, is likely due to the masking effect that chocolate flavoringagents had on acetaldehyde flavor. Several authors have reported anincreased acetaldehyde concentration due to exposure to light(Cadwallader and Howard, 1998; Cladman et al., 1998; Jenq et al., 1988;van Aardt et al., 2001b). The combination of these two sources—migrationfrom PETE packaging and exposure to light—could potentially raise thelevel of acetaldehyde in a food product above threshold levels.

van Aardt et al. (2001b) studied the sensory impact of acetaldehydemigration from PETE bottles into milk stored under light. Acetaldehydeconcentration in light-exposed milk (3.25% milkfat, 18 days at 4 �C) pack-aged in clear PETE, clear PETE with UV blocker, amber PETE and highdensity PE (HDPE) was about the same as milk packaged in glass storedat the same conditions (range for all packaging: 1265–2930 mg/kg).A significant difference in the concentration of acetaldehyde in light-exposed milk as compared to light-protected milk was found. A trainedsensory panel (n ¼ 8) used a 9-point verbal category scale (1 ¼ notdetectable; 9 ¼ very strong) and rated the acetaldehyde intensity around2 (‘‘trace, not sure’’) for both light-exposed and light-protected milk fromall packaging. No significant difference in sensory perception of acetalde-hyde off-flavor due to either light exposure or bottle type was observed.This lack of a difference, even when significant differences in acetaldehydeconcentration were found in light-exposed milk as compared to light-protected milk, is likely due to acetaldehyde concentrations below sensorythresholds for milk. Setting maximum specifications for acetaldehyde con-centrations in PETE would protect against sensory impacts in bottledwater, which has a very low sensory threshold for this compound.

Studying the sensory impact of materials in contact with water isvaluable in understanding the impact of materials use in water distribu-tion and food packaging. The trend for replacing copper piping withpolymer-based plumbing materials has created a host of taste and odorproblems associated with potable water (AwwaRF, 2002; Dietrich et al.,2005; Rigal 1992, 1995; Rigal and Danjou, 1999; Tomboulian et al., 2004).


Cross-linked polyethylene (PEX) used as home or retail plumbingmaterials can cause perceptible changes in the odor of tap water(Durand and Dietrich, 2007). A water industry standard flavor profileanalysis (Standard Methods 2170B) was used by 10 trained panelists todetermine the odor profile of synthetic tap water stored in PEX pipe,manufactured by a silane cross linking procedure. Water, with and with-out disinfectants (chlorine, chloramine), was stored in PEX pipes for 3–4days and for three consecutive periods. Odor descriptions generally weresummarized as ‘‘burning-solvent, plastic’’ odor at the weak to moderatelevel. A variety of descriptors were used with ‘‘alcohol, plastic, and sweetchemical’’ terms common in the first flush period. The addition of disin-fectants provided odor characteristics described as ‘‘glue.’’ Subsequentflushes changed the odor profile with terms such as fruity, spicy, bitter,almond, rotten swampy, and burning plastic suggesting that the odor wasbecoming more distinct and perhaps even more objectionable. The chem-ical 2-ethyoxy-2-methylpropane (ETBE) was identified as a contributor tothe odor of PEX pipe.

Marchsan and Morran (2002) found that flavor descriptions variedbetween chlorinated and nonchlorinated water in contact with PE andPP with stronger tastes frequently found in chlorinated samples. ‘‘Plas-tic/rubber’’ terms were used for chlorinated and nonchlorinated watersstored in PP and PE as well as in nonchlorinated waters from acryloni-trile/butadience/styrene (ABS). ‘‘Plastic/chemical’’ descriptors wereused for chlorinated and nonchlorinated waters in PP and PE and poly-urea materials, and in ABS materials for chlorinated waters only.Polyurethane materials contributed chemical tastes to chlorinated watersand medicinal flavors to nonchlorinated water. The ‘‘chemical’’ term alsowas applied to chlorinated water stored in PP, PE, and ABS and non-chlorinated water stored in ABS. ‘‘Medicinal’’ also was used to describeboth nonchlorinated and chlorinated waters stored in PP.

Off-flavors in water and milk packaged in PE-coated paperboardcartons have been described by consumers as ‘‘unpleasant plastic’’(Berg, 1980). Leong et al. (1992) examined the sensory impact of milk(nonfat (0.05%), lowfat (2%), and whole (3.25%)) packaged in gable-topPE-coated paperboard cartons (half-pint (236 ml), quart (946 ml), andhalf-gallon (1890 ml). A 10-member panel was selected based on discrim-inating ability for milk off-flavors and a paired comparison test or apairwise ranking test was used to evaluate packaging flavor in the sam-ples on days 1, 3, and 6 of storage at 2.2 �C. Control samples initially werepackaged in HDPE at the manufacturer but transferred to glass containerswithin a few hours after transport to the laboratory. Half-pint milk sam-ples (all fat levels) packaged in PE-coated paperboard were clearly distin-guishable from milk packaged in glass. The ability to discriminatebetween milk packaged in PE-coated paperboard and glass increased


with decreasing fat content. Packaging flavor seemed to develop mostwithin the first three days of storage. More packaging flavor was found inhalf-pint packaged milk than in larger containers, probably because of thehigher contact surface:volume ratio. Leong et al. (1992) documented thatheat sealing of the cartons was not the source of the taint. Packaging flavorwas found in water samples on day 1 similar to the results for low fatmilk. Packaged flavor in milk stored in PE-coated paperboard developswithin 24 h and is more easily detected with decreasing fat content,possibly because milk fat appears to mask or dilute this flavor defect(Leong et al., 1992). This conflicts with most evidence that as fat contentin a food product increases, so does that of the migration of volatilecompounds from the packaging material to the food product and sodoes the presence of off-odors and flavors. However, nonfat milk haseven less flavor character than lowfat and whole milk, which may be onereason that the package flavor is more evident. The most common pack-aging material for milk is now HDPE.

Many dairy processors blow mold HDPE containers for milk, juice,and water in the processing plant. Without proper specifications forsensory quality of the granules, taints may be readily noticeable in theseproducts. HDPE pellets were reported to have low concentrations ofodor-producing compounds but the sensory impact of these compounds,as determined by GCO was strong (Villberg et al., 1997). Leachate waterfrom high quality HDPE was described as having a sweet, metallic, stony,and pungent taste and sweet, chemical, stale, dirty, and foul odor. SomeHDPE pellets contributed negative taste (dusty, stale, plastic, foul, stinkbug, and candle grease descriptors) and odors (stale, dirty, foul). Themajority of odor compounds were carbonyl compounds. 2-octenal,which gives a mushroom odor, and butylacrylate, which gives a glue-like odor, were the strongest, while moderate odors were imparted by2-propanal (glue-like odor) and methyl hexanal (green, pungent) andwere found only in the poor quality pellets. Other compounds found toleach into water from poor quality HDPE were 2,4-heptadienal, nonanal,and undecadienal. Ethyl propanate leached out in extremely smallamounts but gave a very strong odor, which smelled like glue (Villberget al., 1997).

HDPE and epoxy, frequently used in home plumbing, were impli-cated in having the greatest impact on odors in tap water (Dietrich, 2007).A trained sensory panel, using the water industry standard flavor profileanalysis method, evaluated six different plumbing materials for odorintensity. In order of increased odors in tap water (simulated) as afunction of material indicated chlorinated polyvinyl chloride (cPVC) ashaving the least increase in odors, with cross-linked PE (PEX)-a, copper,and PEX-b having increasing levels of odors. Increased odors can causesensory annoyance. Water stored in HDPE was characterized as having


a ‘‘waxy, plastic, citrus’’ odor at moderate levels (Heim and Dietrich,2007b). Odor intensity increased in the presence of chlorine and ‘‘chemi-cal, plastic’’ descriptors were used; chloramine disinfectants also causedan increase in odor intensity described as ‘‘waxy-crayon, plastic.’’

Piagentini et al. (2002) studied the effects of citric acid, ascorbic acid,and type of packaging film on the sensory characteristics, chlorophyllretention, and weight loss of fresh cut spinach in refrigerated storage.Spinach was packaged in either mono-oriented PP bags or in LDPE bagsand stored at refrigeration temperatures for 14 days. A trained sensorypanel evaluated off-odor, appearance, wilting, and color using a 10 mmunstructured line scale. Storage time significantly (< 0.001) affected sen-sory attributes, while the type of packaging film only influenced off-odordevelopment (p < 0.001). Off-odor development was greater for orientedPP than for LDPE. The type of packaging film had no effect (p > 0.05) onvisual sensory characteristics. The intensity of off-odor packaged in LDPEreached an average of 7.3 after 14 days (9 ¼ none, 0 ¼ severe). Theoriented PP had an average sensory value of 5 after 14 days. Di Pentimaet al. (1995) found similar results with broccoli, whole spinach, and aspar-agus packaged in different plastic films stored at 4 �C.

4. Polyvinyl chloride (PVC) and chlorinated PVC (cPVC)PVC and cPVC can have a metallic odor and taste due to accidentalcontamination by antimony (Tamboulian et al., 2002). Phenolic andacetone odors are attributed to m-chlorophenol and cyclohexanone,respectively. Threshold levels for these compounds in water are0.005 ppm for m-chlorophenol and 0.12 ppm for cyclohexanone. PVCpipes and polymer coatings have been found to have an organosulfurodor that is attributed to ethyl-2-mercaptoacetate. This compound arisesfrom the interaction between low molecular weight alcohols with syn-thetic organic compounds added to PVC as a heat stabilizing agent (Sideset al., 2001). Heim and Dietrich (2007b) did not find a significant odor insynthetic tap water stored in cPVC pipes compared to water stored inglass control pipes.

5. EpoxyEpoxy is used as a lining for water reservoirs, water mains, and homeplumbing systems (Heim and Dietrich, 2007a). These applications canimpact sensory quality of tap water in food manufacturing, food serviceoperations, and residential homes. This effect may be most noticeable inwater but residual aroma and flavor compounds may cause a taint infoods prepared with these water sources. An odor assessment, using awater industry standard flavor profile analysis method, identified astrong relationship between water (simulated tap water, pH 7.7–7.9)stored in epoxy-lined copper pipes for 3–4 days and an odor described


as ‘‘plastic, adhesive, putty.’’ In addition, a noticeable decrease in chlorineand chloramine disinfectant odors were identified (Heim and Dietrich,2007a).

B. Taints from additives or noncontacting materials

The primary chemicals associated with taints are solvents or inks,aliphatic aldehydes and ketones, phenols or halogenated phenols, andanisoles (Lord, 2003).

1. Printing inks, varnishesColor, graphics and labels on primary and secondary packages are usedto inform, advertise, attract attention, and promote the product within.Printing (or packaging) inks and varnishes contain colorants, binders,solvents, and additives. Most of the off-odor related consumer complaintslinked to packaging at Nestle were attributed to residual solvents frominadequate printing or converting processes or the use of low-qualitypromotional items (Huber et al., 2002). These systems may be character-ized as solvent-based, water-based, oleo-resinous, or UV- or electronbeam (energy) curing (Aurela and Soderhjelm, 2007). Although thesematerials are applied to the external surface of the packaging material,low molecular weight compounds will easily migrate through the pack-aging material, with the exception of glass and aluminum foils, into thefood. Some of these compounds have a noticeable smell and can contrib-ute to taint of the food product. Oxidized aromas in the foods may bepartially related to oxidation of vegetable oils used in offset printing oralkyd resins, used as binders in inks. Aldehydes and ketones resultingfrom the oxidation process can unexpectedly modify the flavor and odorof food within the package, creating a negative, even repulsive, sensoryresponse (Soderhjelm and Eskelinen, 1985). Mineral oils may containaromatic compounds, which can diffuse readily through fibrous or plasticpackaging materials. Toluene and xylene, which are aromatic com-pounds, should be avoided in printing of food packages (Aurela andSoderhjelm, 2007). Hydrocarbon compounds in lithographic inks havebeen sources of taints (Kilcast, 1996). Low odor inks and varnishes shouldbe chosen. At the minimum, adequate time for airing for solvent or UV-cured systems, which may produce taints from trace residues of acrylatemonomers and from benzophene photoinitiators, before packagingshould be allowed (Aurela and Soderhjelm, 2007; Kilcast, 1996). Printedand varnished paperboards containing residual solvents may be one ofthe main sources of taints in foods (Soderhjelm and Eskelinen, 1985).

Placing printed premiums (coupons) within a food package is com-mon but these materials also may be sources of taints. Premiums intendedfor packaged dry mix beverages were tested for their contribution of


odors prior to inclusion (Apostolopoulos, 1998). Overwrapped (PE) andunwrapped premiums were placed in Mason jars, sealed, and heated at49 �C for 1 h and cooled. An odor evaluation panel (n ¼ 4), familiar withsolvent odors associated with the packaging industry, rated odor inten-sity on a scale of 0–10 (0 ¼ no odor; 8–10 ¼ excessive odor), described theodor, and also indicated if the odor was objectionable or not. The solventodor was attributed to the PE resin or the paint used with the premiums.Cyclohexane concentration, as determined by GC/MS was 16 times higherin unwrapped premiums than attributed to the overwrapped premiums;toluene, 2-methyl heptane, and 3-methyl hexane also were detected. Thesensory panel identified no odor associated with the overwrapped pre-miums but identified a very strong (rating of 9), solvent-like, objectionableodor for the unwrapped premiums. The PE overwrap contributed nodistinct odor and effectively contained the premium odor. The paint usedin the manufacturing and printing of the premiums was implicated. How-ever, unsealed or punctured overwrap would not provide appropriateprotection, potentially leading to taints in the beverage powders.

2. Coloring agentsColoring agents are often used in materials to provide protection fromvisible and ultraviolet light, and should be considered as a potentialsource of taints. Heinio and Ahvenainen (2002) studied the odor of pack-aging materials as a function of different coloring agents. However, therewas no direct indication, based on odor, that the coloring agent was thesource of taint in the packaged food. They recommended that odor testingshould only be regarded as an indicator.

3. AntioxidantsTomboulian et al. (2002) has reported that butylated hydroxytoluene(BHT) can impart a ‘‘burnt plastic’’ odor and is an additive in HDPEpipes. Quinone may be derived from BHT due to interactions with resid-ual chlorine in pipes (Anselme et al., 1985). Yam et al. (1996) reported thatantioxidants, such as vitamin E, Irganox 1010, and BHT, contributed tooff-flavors in water. Vitamin E yielded less off-flavor, possibly due tolower aldehyde and ketone concentrations. Extrusion temperatures over280 �C and exposure time for melt contributed to more oxidation of LDPEfilms and higher intensities of off-flavors in water in contact with LDPEwith different antioxidants (Andersson et al., 2005).

C. Taints from recycled materials

Recycled materials may contain absorbed odorous or flavorful moleculesfrom earlier use that, when introduced into a new packaging material,may cause taint (Franz andWelle, 2003; Kilcast, 1996). Analytical detection


limits of instrumentation may be higher than sensory thresholds fromsome flavor compounds so, while the recycled material may appear tohave low or nondetectable concentrations of volatile contaminants, theremay be sufficient levels for sensory detection (Franz and Welle, 2003).

Limonene, for example, is readily absorbed from citrus juices intopackaging materials. Analysis of recycled PETE after exposure to modelcompounds showed average and maximum values of 18.6 and 86 ppb foracetaldehyde and 2.8 and 20 ppb for limonene. Analysis of contaminantssuch as solvents in recycled plastics showed extremely low levels rangingfrom 1.4 to 2.7 ppb and resulted from only 0.03% to 0.04% of the recol-lected PETE bottles (Franz et al., 2004). Recycled HDPE, PP, PS, and PETEpolymers demonstrated sensory properties characteristic of the virginpolymers but also additional odor notes (Huber and Franz, 1997).A sensory panel readily identified the recycled polymer from virginpolymers based on these additional odor notes. Recycled HDPE wasmost different from the virgin material whereas recycled PETE had thelowest odor deviation. Recycled PS and PP had more odor notes than thematched virgin material but did not have as great a difference as wasfound for HDPE. Use of recycledmaterials should be considered on a caseby case basis, using appropriate sensory testing to verify sensory intert-ness of recycled PETE or any other materials (Franz and Welle, 2003).If the contained product is bland in odor and flavor, the impact of thesemolecules may be even more evident (Kilcast, 1996). Recycled materialswould not be appropriate for water or milk packaging.

With the continuing desire to recycle and reuse plastic packagingmaterials to reduce their environmental impact, the absorption of com-pounds into the packaging material will become increasingly important.It has been reported that the recycling process used for plastics are notcompletely efficient in their ability to eliminate absorbed compounds.These compounds can then desorb into the new product upon reuse ofthe plastic (Safa and Bourelle, 1999). Deep cleaning, or supercleaning,technologies for recycled polymers are safe and produce bottles withvery low levels of contamination, positively influencing sensory propertiesof the recycled materials (Franz and Welle, 2003; Franz et al., 2004).

VII. SCALPING/SORPTION

Sorption of flavor compounds, or more colloquially ‘‘scalping,’’ is consid-ered a major factor in the degradation of food quality (Arora et al., 1991;Ayhan et al., 2001; Charara et al., 1992; Fukamachi et al., 1996). The termsorption encompasses the properties of absorption, adsorption, and clus-ter formation and describes the penetration and movement of a chemicalcompound into a polymer (Robertson, 2006). Aroma and flavor


perception often involves the interplay of many compounds in a specificproportion. Therefore, any disturbance of this balance due to sorption canchange the sensory characteristics of the product and reduce its accept-ability (Arora et al., 1991; Sajilata et al., 2007). All plastic materials havesome sorption capacity for flavor molecules (Gremli, 1996), which canresult in a sensory impact. Higher storage temperatures will accelerate thesorption of volatiles. Volatiles associated with flavor of a given foodproduct may be decreased by as much as 20% by sorption (Gremli, 1996).

Absorption of flavor molecules into the package may be affected by anumber of parameters associated with the material and the food (vanWillige, 2002). Crystallinity, morphology, and polarity of polymers caninfluence the rate of absorption. Size, concentration, copermeants, andpolarity of flavor molecules within the food system also affect absorption.Storage temperature and time exposed to the food matrix affect polymerand food matrices, creating additional challenges in determining effectsof materials in contacts with foods.

Not only can absorption alter the aroma and flavor of a product, it canalso change the mechanical properties of the polymer. Swelling and gaspermeability are factors that effect the physical properties of a polymer(Robertson, 2006; Sadler and Braddock, 1991; Safa and Bourelle, 1999).Swellingoccurswhencompoundsare absorbed into thepolymeranddistortthe shape of the package. As absorption increases there is also a subsequentincrease in gas permeability. This increase in gas permeability can affect theshelf-life and sensoryquality of a foodby, for example, increasingoxidation.In very severe cases, absorption can affect package integrity.

Color and appearance of a food are important quality aspects onwhich consumers base many initial purchase and consumption decisions.Nylon-6 polyamides may scalp dye materials from foods, altering thefood color intensity. Liquids and moist foods (high water content) indirect contact with the polymer have the greatest potential for changesor loss of color. Migration of food colorants into packaging material canalter the package and product appearance or cause staining of foodcontact surfaces on household items. Such problems increase consumercomplaints and lead to decreased sales (Oehrl et al., 1991). However, thereis very little published research that directly considers color and appear-ance changes as a function of interaction of materials and colorants.

There are a number of studies that look at the absorption of flavorcompounds into different polymer packaging materials (Arora et al., 1991;Ayhan et al., 2001; Charara et al., 1992; Fukamachi et al., 1996; Hernandez-Munoz et al., 2001; Imai et al., 1990; Konczal et al., 1992; Letinski andHalek,1992; Moshonas and Shaw, 1989; Nielsen et al., 1992; Sadler and Braddock,1991; Safa and Bourelle, 1999; van Willige et al., 2000, 2002, 2003).There are fewer studies looking at the effect that this absorption has onthe sensory quality of the food. The few sensory studies that have been


done to date are contradictory and more research into this area is impera-tive (Ayhan et al., 2001; Durr et al., 1981; Kwapong and Hotchkiss, 1987;Mannheim et al., 1987; Moshonas and Shaw, 1989; Pieper et al., 1992;Sadler et al, 1995; Sharma et al., 1990).

Several studies found that scalping of flavor volatiles by polymers didnot affect the sensory quality of juices. Sharma et al. (1990) found that fruitsquashes and tropical fruit beverages stored with PP and PE had nodifferences detected using triangle testing. Pieper et al. (1992) reportedthat a 50% decrease in limonene concentration, along with a smalldecrease in alcohol and aldehyde concentration, did not affect the sensoryquality of orange juice. Sadler et al. (1995) tested the effect of volatilecompound absorption from orange juice into LDPE, PETE, and EVOHstored at 4.5 �C for 3 weeks on the sensory characteristics of the juice andobserved no change. van Willage (2002) reported that a sensory panel(n ¼ 27) could not find a significant difference in flavor of reconstitutedorange juice packaged in LDPE, PET, or polycarbonate (PC) althoughanalytical flavor chemistry documented a large decrease in flavorconstituents.

Other investigators, however, found that the absorption of flavor com-pounds by polymer packaging material did affect the perception of odorand flavor (Ayhan et al., 2001; Kwapong and Hotchkiss, 1987; Mannheimet al., 1987; Moshonas and Shaw, 1989). A sensory panel found no signifi-cant difference in color in orange juice processed by pulsed electric fieldstored in glass, PETE, HDPE, and LDPE, even though there were signifi-cant analytical differences (Ayhan et al., 2001). A significant difference inflavor was found in juice stored in LDPE after 56 days compared to theother packaging materials but no significant differences were found inflavor after 112 days in glass, PETE, and HDPE. Overall, the retention ofall flavor compounds was significantly higher in glass and PETE thanHDPE and LDPE. An increase in storage temperature had adverse effectson flavor and color. There was more loss of aldehydes and esters in allpackages after 2 weeks than hydrocarbons, and flavor loss was moreadvanced in HDPE and LDPE than in PETE and glass. These results canbe explained by both the absorption of flavor compounds and by theacceleration of the production of degradation products due to oxygenpermeability and increased storage temperature (Ayhan et al., 2001).

VIII. PROTECTION OF SENSORY QUALITYBY FOOD PACKAGING

Packaging materials can significantly increase the shelf-life of a food byreducing or slowing the degradation of the food. Package characteristicssuch as decreased oxygen and light permeability, for example, are


responsible for the increased shelf-life. The proper choice of packaging,while at times difficult to do, is extremely important for protecting andmaintaining sensory quality.

Saint-Eve et al. (2008) identified that packaging choice affected thesensory quality, specifically aroma, of flavored stirred yogurts with 0%or 4% fat content. A trained sensory panel (n ¼ 8–15) evaluated yogurtpackaged in glass, PS, or PP over a 28-day refrigerated storage period.Evaluations, using an unstructured line scale with ‘‘weak’’ and ‘‘veryintense’’ as the anchors, reflected 10 odor, 15 aroma, 2 taste, and 3texture-in-mouth characteristics. While aroma intensity and profilechanged for yogurts packaged in all materials over time, glass providedthe best protection for aroma and flavor intensity as it had the best barrierproperties. A time effect was evident with relation to sensory perceptionand scalping of aroma compounds. Loss of some aroma compounds wasgreater in yogurts stored in PP than in PS but the flavor chemistrystabilized before the 28th day of storage for yogurt packaged in PP. Thekinetics of aroma compound sorption were slower in PS than in PP,perhaps because of the differences in crystallinity between the two mate-rials at 4 �C. Nonfat yogurts packaged in glass and PS developed similarsensory odor and aroma changes over the 28-day storage. Fruity noteswere better retained in PS-packaged products compared to PP-packagingbut more acids were also noted. The authors also suggested that higher fatcontent (4%) product may lose less volatiles into packaging by absorptionbecause of the lipophilic nature of many aroma compounds, therebyreducing the interaction with packaging materials. Fewer or less intenseflavor and odor defect characteristics were identified in 4% yogurts pack-aged in PS at the end of shelf-life than for yogurt packaged in glass or PP.

A. Protection against light

One major reason for nutrient loss and off-flavor development today isdue to extended exposure to fluorescent light in food retail display cases.Many foods and beverages are susceptible to light-induced reactions,especially those with photo-sensitizers. Natural pigments found in foodsthat commonly act as photochemical initiators are flavonoids, riboflavin(vitamin B2), chlorophyll, heme, and vitamin K.

The chemical effects of photo-oxidation on food components resultsin off-flavor development or changes in pigmentation or appearance.Dairy products, which are very susceptible to photo-oxidation, developa distinct, unpleasant flavor described as ‘‘cardboard’’ or ‘‘burnt feathers’’(Bodyfelt et al., 1988). Sunstruck flavor of beer is also a noteworthy flavordefect caused by light. Fruits, vegetables, pigmented beverages, candies,and other colored food systemsmay demonstrate color change from photo-oxidation of pigments. Ingredients, such as powderedmilk, flavorings, and


colorants, will develop off-flavors, off-odors, or color changes as a functionof light exposure. The use of packaging materials to protect food systemsfrom the effects of light is common. However, the most effective solutionsfor protection of product and ingredient sensory quality is to provide acomplete light block, which is not always the most effective method formarketing a product to consumers.

The primary plastic packaging materials used for refrigerated milkproducts are HDPE and PETE (Anonymous, 2002). Clear glass, olefin-coated fiberboard, blow molded PE, and high-density PC or PE also areused. Glass allows 91% light transmission, HDPE allows 57%, while fiber-board allows 4% light transmission. Fiberboard containers were found toprotect milk from light oxidation for up to 48 h whereas milk in plastic orglass containers developed light oxidized flavorwithin 12 h of exposure to100–200 ft-c fluorescent light. Milk in clear PE pouches showed off-flavordevelopment in 6 h after exposure to 100 ft-c and 3 h at 200 ft-c.

Many investigators have found that packaging materials that protectmilk and dairy products from photo-oxidation are important to sensoryquality (Christy et al., 1981; Cladman et al., 1998; Dimick, 1973; Gorgern,2003; Papachristou et al., 2006a,b; Rysstad et al., 1998; Simon and Hansen,2001; van Aardt et al., 2001b; Zygoura et al., 2004). Zygoura et al. (2004)found that both clear and pigmented (2% TiO2) PETE had significantlyhigher lipid oxidation than paperboard and 3-layer pigmented coex-truded HDPE and monolayer pigmented HDPE between days 3 and 7(end of test). Oxygen permeability of the packaging material did not affectoxidation, but this could have been due to the large headspace of thepackages used. Sensory evaluation, using a trained panel and a flavor andintensity rating scale (0 ¼ unfit for consumption, 5 ¼ very good), showedthat milk packaged in clear bottles had a much lower acceptabilityscore than milk packaged in pigmented bottles.

Simon and Hansen (2001), using an untrained panel and a ‘‘differencefrom control’’ test, found that milk packaged in oxygen barrier board(EVOH and foil) deteriorated much more slowly than milk packaged instandard or juice boards. The foil-lined board had the added benefit of alight block. Inhibition of oxygen permeation into a package does notsolely protect against degradation. Milk packaged in HDPE with a carbonblack layer (light barrier) and no oxygen barrier was shown to have betterprotection against light oxidation than HDPE with an EVOH oxygenbarrier but no light barrier (Gorgern 2003; Moyssiadi et al., 2004).

PETE has an advantage over high-density poly(ethylene) (HDPE), thepolymer currently used for the larger sizes of milk packaging, since theoxygen transmission rate at 4 �C, 50% relative humidity, and 21% oxygenof a commercial one-pint PETE bottle is 19 ml/day compared to 390–460 ml/day for a commercial one-pint HDPE bottle (van Aardt et al.,2001b). However, translucent HDPE has an advantage over clear PETE


in that it blocks approximately 40% light between 300 and 700 nm,whereas clear PETE only blocks �20% light in the same range (vanAardt et al., 2001b).

The efficacy of film over-wraps, made from single and multilayers ofiridescent film, to reduce the production of light oxidation in milk and foreffectiveness in controlling light oxidized flavor in milk was tested(Webster, 2006; Webster et al., 2007). A balanced incomplete block multi-sample difference test using a ranking system and a trained panel wasused for the evaluation of light oxidation flavor intensity. Packaging over-wraps limited the production of light oxidation flavor in milk over timebut not to the same degree as the complete light block. Blocking all visibleriboflavin excitation wavelengths was better at reducing light oxidationflavor than blocking only a single visible excitation wavelength. However,blocking transmission of all riboflavin excitation wavelengths at the levelssuggested by the International Dairy Federation (IDF) was not sufficientto completely protect against the production of light oxidation flavor,suggesting the presence of a photosensitizer other than riboflavin inthe milk.

Bray et al. (1977) found that 73% of 2000 consumers preferred nonex-posed milk to light exposed milk.

B. Preventing moisture loss

Protection of color and texture, as well as odor and flavor, of foodproducts also is an important consideration when choosing the appropri-ate food packaging material. Nylon-LDPE plastics helped protectvacuum-packaged burnt coconut meat and coconut water from colorand texture changes over a 28-day storage compared to a PVC film( Jangchud et al., 2007). Burnt aromatic coconut water and meat werestored at 5 �C and 80–90% relative humidity in two package treatmentswith an unwrapped treatment as the control and evaluated over a 28-dayshelf-life. Packaging treatments included: (1) PVC film (thickness: 11 mm;water vapor transmission rate (WVTR): 210 g/(m2d); OGTR: 8133 cc/(m2d bar)); and (2) under conditions of vacuum in a Nylon: linear LDPEplastic bag (15 mm,120 mmthickness each, respectively;WVTR: 5.1 g/(m2d);OGTR: 73 cc/(m2d bar)). Sensory evaluation of color (water: yellow,transparency; meat: white), texture (hardness), as well as several odorand flavor characteristics were assessed on a 15-cm unstructured linescale (0 ¼ ‘‘weak’’; 15 ¼ ‘‘strong’’) by 12 experienced/trained panelists.Vacuum packaging effectively ESL of the burnt coconut by limiting coloralteration in the coconut water and meat and maintaining hardness of thecoconut meat. Panelists rated the coconut packaged in Nylon: linearLDPE with high acceptability after 28 days whereas the PVC-filmwrapped treatment had declined in quality, partially as a result of


increased microbial growth, by 14–18 days. The barrier properties of theNylon: linear LDPE film maintained the desired vacuum storage of theproduct, which contributed to the improved product quality.

IX. USING PACKAGING TO IMPROVE SENSORY QUALITY

Smart packaging, referring to value-added packaging features thatenhance the functionality of a product, encompasses active packaging aswell as other developments that improve product safety and efficiency(Robertson, 2006). Examples of active packaging systems include oxygenand ethylene scavengers, carbon dioxide scavengers and emitters, preser-vative releasers, ethanol emitters, moisture absorbers, and flavor/odoradsorbers. Innovative technologies for these systems may have intendedimpact on sensory characteristics of foods through control of microbialgrowth, oxidation reactions, moisture migration, and absorbance of unde-sirable odor-impacting molecules. While there is a large body of literaturein the area of smart (active, intelligent, other) packaging materials andsystems, the relationship to sensory impact on humans is secondary to thechemical or microbiological impacts, including analytical flavor chemis-try, that are primarily studied. We have illustrated the potential forsensory impacts of some packaging innovations with a few examples.

A. Sensory impact of novel antimicrobial ingredientsin packaging systems

Controlling microbial degradation of food systems is paramount in main-taining sensory quality. Inclusion of antimicrobial agents from naturallyderived sources within the polymer reduces the risk from a consumerperspective (Suppakul et al., 2008). Extracts of clove, grapefruit seeds,huanglian, rhubarb, and basil have been embedded in LDPE to test forantimicrobial effects (Suppakul et al., 2008). Linalool and methylchavicol,antimicrobial compounds from basil were embedded in LDPE anddemonstrated inhibitory effects on microbial growth in naturally con-taminated cheddar cheese samples. These materials (control LDPE film,linalool-LDPE, and methylchavicol-LDPE) also were tested for sensoryimpacts on wrapped, cubed cheddar cheese over 6 weeks of storage at4 �C. The materials and the cheese were both sterilized by ultraviolet lightprior to contact. Using a triangle test method, a panel of 10 membersevaluated the cheese for basil taint. Linalool-embedded LDPE did notimpact flavor of the cheddar cheese over the 6-week storage as panelistscould not detect the difference between cheese stored in LDPE or linalool-embedded LDPE. However, methylchavicol-embedded film caused ataint in the cheese as early as 7 days. This sensory impact was detectable


again only in the fourth week of the study. Methylchavicol was reportedto have a more distinct flavor than linalool and was more evident in thecheese. While the microbial shelf-life of cheese was improved with use ofmethylchavicol-embedded LDPE as compared to linalool-LDPE andLDPE control materials, sensory effects were noticeable with use of thiscompound, illustrating the potential for reducing the commercial successof the antimicrobial material (Suppakul et al., 2008). Selection of naturallyoccurring antimicrobial agents or other active additives should be basedon both intended efficacy as well as potential for protecting the sensoryintegrity of the product.

B. Flavor and odor absorbers for improved flavor

Some food processing methods impose negative sensory quality para-meters on food systems. UHT processing of fluid milk and citrus juices,for example, increases the perception of cooked or stale flavor and odornotes because of thermal degradation of macro- and micronutrientswithin the system. An increase in low molecular weight aldehydes andketones has been identified as primary contributors to these negativesensory characteristics in heat processed beverages (Suloff, 2001). Milk,soymilk, and water are bland, having low flavor and odor profiles, andlow concentrations of migrating molecules from PETE or other materialscan influence sensory quality (Norton, 2003; van Aardt et al., 2001a,b).Odor-adsorbingmaterials were synthesized from cyclodextrin, d-sorbitol,and nylon MXD6 blended with PETE for removal of aroma compoundsassociated with lipid oxidation (Suloff et al., 2003) and identified thatselective adsorption of low molecular weight aldehydes, compared tolarger aldehydes, occurred with partition coefficients of three to sixtimes higher magnitude. Cooperatively, Norton (2003) demonstrated thehuman sensory impact by evaluating the sensory threshold for targetedcompounds and the efficacy of the odor-absorbing compounds in springwater, milk, and UHT procesed ESL soymilk.

Norton (2003) initially established the sensory impact of selectedmolecules to establish if absorption by the scavenging compoundswould be at efficacious levels. Low molecular weight aldehydes andketones (hexanal, 2-heptenal, 2-pentanone, and 2,4-nonadienal) in springwater, pasteurized milk (2% milkfat), and ESL soymilk were selected asrepresentative compounds of oxidation and high temperature processingnotes. Human thresholds, based on 12 panelists, were determined, inascending order of concentration using a series of 3-sample alternateforced choice tests, by both logistic regression and the geometric meanapproach. Testing was done under controlled conditions in individualsensory booths. Untrained panelists initially were provided a referencesample of the selected compound at supratheshold concentration in the


testing medium. Thresholds calculated using logistic regression wereconsistently higher than those calculated using the geometric meanapproach, but both methods found thresholds of hexanal, 2-heptenal,2-pentanone, and 2,4-nonadienal to vary significantly when comparingthe three media. Hexanal and 2,4-nonadienal had lower thresholds than2-heptenal and 2-pentanone. Odor detection thresholds of 2-heptenal,2-pentanone, and 2,4-nonadienal were lowest in water, followed bymilk, then soymilk at product temperatures of 4 �C.

Triangle tests (n¼ 36 panelists, a 0.05) verified that five combinations ofabsorber addition in volatile-spiked springwater, milk and soymilk alteredthe aroma of the product. Addition of b-cyclodextrin in both hexanal-spiked spring water and milk significantly influenced odor. Panelists alsofound a significant difference (p < 0.05) in 2-pentanone spiked milk withthe addition of both b-cyclodextrin and d-sorbitol and in 2-heptenal spikedsoymilk with the addition of d-sorbitol. Because of its ability to adsorbodors caused by lipid oxidation, b-cyclodextrinmay be a good scavenger inpackaging systems for milk and soymilk. However, since b-cyclodextrin isvery reactive with low molecular weight compounds, there is a possibilitythat desirable aromas could also be scavenged. d-sorbitol also was some-what effective as a scavenger for aroma compounds, particularly in milk.The authors did not report on efficacy of these active components inabsorbing volatiles when imbedded into the material.

C. Controlling oxidation through timed release of antioxidants

Active packages can be designed that contain compounds, such as anti-oxidants, which can migrate into the food and improve sensory quality byreducing oxidation, increasing shelf-life, and increasing nutritive quality.Additives are commonly used in polymer processing to inhibit oxidativereactions (Robertson, 2006). Excess addition of antioxidants, such asbutylated hydroxyanisole (BHA) or BHT, has been effective in controllingoxidized flavor in dry breakfast cereals and crackers and other dryproducts (Hoojjat et al., 1987; Jadhav et al., 1996; Miltz et al., 1995).

vanAardt et al. (2005a,b, 2007) studied the effect of antioxidant additioninto milk and poly(lactide-co-glycolide) (PLGA) films to determine iftimed release of antioxidant addition from packaging might be effectivein limiting photo-oxidation of fluid milk and milk powders. Similaritytesting, using triangle test methods, identified that levels of added anti-oxidants (0.05% a-tocopherol or 0.025% a-tocopherol or 0.025% ascorbicacid) to reduced fat milk (2%milkfat) were not significantly perceptible bypanelists (n ¼ 30). Subsequently, milk was spiked with and without anti-oxidants and exposed to light (1100–1300 lx) for 10 h at 4 �C. A sensorypanel (n¼ 24) compared the samples for difference, using the triangle testmethod. Milk with tocopherol/ascorbic acid addition was different from


the light-exposed control and had a fresher milk flavor, indicating thatantioxidant addition had controlled photo-oxidation. However, tocoph-erol alone did not provide any benefit. A timed addition of antioxidantsfrom packaging was studied by the direct addition of antioxidants to milkover a 6-week period under lighted refrigeration conditions (van Aardtet al., 2005b). The timed effect did reduce concentrations of some odor-active compounds associatedwith light-induced oxidation, as determinedby GCO evaluations by a trained panel (n¼ 3). A combination of BHA andBHT significantly reduced the concentrations of heptanal and 1-octene-3-ol, which are common light oxidation off-flavor compounds in light-exposed milk (van Aardt et al., 2005b). The single, initial addition of acombination of a-tocopherol and ascorbyl palmitate significantly reducedhexanal production for the first 4weeks of the study but not thereafter. In aseparate study, antioxidants were incorporated into PLGA films; filmswere stored in milk powders and water and oil food simulants in thepresence and absence of light (van Aardt et al., 2007). Milkfat was stabi-lized to a degree, based on GC analysis of volatiles, against photo-oxidation of milk powders in the presence of antioxidant-loaded PLGAbut sensory analysis was not used as confirmation. Although the sensorystudies completed (van Aardt et al., 2005a,b) document the potentialbenefit for timed-release of antioxidants, there is no direct evidence thatthe change in flavor chemistry from the PLGA films resulted in a sensoryimpact of the milk powders or food simulants.

The potential for smart packaging for improvement of food qualityand safety is very high. Although sensory evaluation is essential in theearly stages of development, there is an important role for sensory evalu-ation simultaneously with analytical assessments of new materials incontact with food systems. New materials or novel applications of mate-rials may deliver value in improved sensory quality or create unexpectedsensory impact that may not be interpreted from analytical chemical orphysical methods of assessment.

X. CONCLUSIONS

Sensory impacts from food–packaging interactions are probably moreprevalent than acknowledged. Studies that have included controlled sen-sory analysis concurrently with analytical methods for studying the effectsofmaterials on foods have demonstrated that human assessment is needed.All sectors of the food packaging supply line are important in controllingthe risk of taints and avoiding quality degradation from scalping of valu-able volatiles and pigments. Commitment to understanding sensoryimpacts from interaction of food and packaging materials can lead toinnovations for improved quality and shelf-life of food systems.


ACKNOWLEDGMENT

This research was supported in part by the NSF-IGERT grant under Agreement No.DGE-0333378 Macromolecular Interfaces with Life Sciences (MILES).

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CHAPTER 3

Advances in Food and NutISSN 1043-4526, DOI: 10.1

Department of Paediatrics

Developmental Trajectoriesin Food Allergy: A Review

A. DunnGalvin and J’ O. B. Hourihane

Contents I. Background 66

rition R016/S1

and Ch

esearch, Volume 56 # 2009043-4526(08)00603-7 All righ

ild Health, Cork University Hospital, Ireland

Elsts

A
. T ransition points: neurocognitive development 67
B. R
esearch with children 68
II. P
revalence, Mechanisms, and Clinical
Manifestations of Food Allergy
68
III. T
he Impact of Food Allergy on HRQL 73
A
. R esearch using generic HRQL measures 73
B. H
RQL Research using disease-specific measures 74
IV. T
he Psychological Burden of Food Allergy 76
V. T
he Influence of Parents on Child Adjustment 78
VI. S
ocial Support 79
VII. T
he Impact of Stress on Biopsychosocial
Development
80
VIII. T
he Impact of Sex and Gender in Food Allergy 81
A
. T he influence of sex 82
B. T
he influence of gender 83
IX. R
isk Behavior in Food Allergy 84
X. D
evelopmental Pathways in Food Allergy 86
A
. T ransition points in development for food
allergic children
90
B. P
arental views and management of food allergy 91
XI. D
iscussion and Implications for Future Research 92
Referen
ces 95
evier Inc.reserved.

65

66 A. DunnGalvin and J’ O. B. Hourihane

Abstract Increasing recognition of the importance of the relationships

between perceptions, emotions, behaviors and health has changed

the way health and disease are portrayed and researched. A chronic

condition may affect and/or interact with already existing norma-

tive demands and changes in socialization. Although the prevalence

of food allergy and anaphylaxis have been reportedly increasing,

the emotional and social impact of growing up with food allergy

has received little emphasis.

In this paper, we present current findings on the biopsychosocial

impact of food allergy on children in order to gain insight into the

food allergy experience, from the perspective of the child, teen, and

parent living with food allergy, with particular attention to develop-

mental aspects. Due to the scarcity of publications on the psycho-

social dimensions of food allergy, we also draw on selected

literature on children’s and parent’s experience of, and coping with

chronic disease that may inform research into food allergy. To this

end, we review some general developmental mechanisms that may

underpin and explain normative age-graded shifts in patterns of

coping across childhood and adolescence. We also highlight gaps

in the literature and assess implications of current research in food

allergy and other chronic diseases for intervention and prevention of

negative short and long term outcomes.

I. BACKGROUND

The growing prevalence of allergic diseases present an increasing chal-lenge for populations and health care systems around the world and foodallergies constitute a notable part of this increase (Sicherer, 2002). Theemotional and social aspects of food allergy have not received muchattention to date although an association between allergy and anxietydisorders in children and adolescents have been found to persist intoadulthood (Katon et al., 2004a,b), and evidence linking psychologicalstress to the expression of asthma and atopy continues to grow (Wright,2005). In child/adolescent populations with asthma, up to one third maymeet criteria for comorbid anxiety disorders (Bender-Berz et al., 2005;Ortega et al., 2002).

Thoughts, feelings, and behaviors affect our health and well-being.Recognition of the importance of these influences on health and disease isconsistent with evolving conceptions of mind and body and represents asignificant change in medicine and the life sciences. Recent developmentsinclude the idea that emotional processes, such as stress, moderate activ-ity in nearly all systems of the body and can directly influence thepathophysiology of disease. Discovery of these and other relationships

Developmental Trajectories in Food Allergy 67

between behavior and health has changed the way health and disease areportrayed. This interest is also reflected in the rapid development ofhealth psychology and its more multidisciplinary cousin, behavioralmedicine.

These fields grew rapidly in the 1980s and now constitute majorendeavors in most university and medical center settings (Barlow et al.,2002a). The movement coincides with the growing recognition of theimportance of children’s views of their experiences that has begun topermeate many areas of research (Hill, 2006). Methods for research withchildren are, however, relatively underdeveloped, emphasizing the needto develop and document methods for research with children (Hennessyand Heary, 2004).

Within the basic and clinical scientific community, there is increasingrecognition that developmental trajectory frameworks offer a conceptualmodel for health development and a more powerful approach to under-standing diseases. A developmental trajectory or pathway may be under-stood as a lifelong process of developmental integration that involvescomplex interactions between biological and environmental factors thatinfluence the phenotypic expression of physiology, psychology, andbehavior (Halfon and Hochstein, 2002).

The health of children is a product of complex, dynamic processesproduced by the interaction of external influences, such as children’sfamily, social, and physical environments, and their genes, biology,and behaviors. Because children are rapidly changing and developingin response to these interactions, the developmental process plays animportant role in shaping and determining their health.

A. Transition points: neurocognitive development

Developmental pathway models can take account of the cumulative andinteractive contribution of physiological and environmental variables.They may also delineate sensitive or transition points in developmentwhen physiological or environmental variables associated with a chroniccondition may have a relatively greater impact and/or interact withalready existing normative demands and changes in socialization(Halfon and Hochstein, 2002). One key transition point occurs at between4 and 7 years in most cultures and involves entry into formal education.Children’s social networks start to change from networks in which chil-dren primarily interact with adults to networks in which children primar-ily interact with other children, with consequent exposure to socialcomparison and competition in school classrooms and peer groups(Dixon and Stein, 2006). The clinical literature suggests that children’spatterns of social functioning in middle childhood is predictive ofrelationship patterns at later points in development (Gifford-Smith


and Browning, 2003). Furthermore, there is evidence of shifts in cognitivedevelopment in which enhanced memory, new reasoning abilities, andnew strategies for recall emerge (Dixon and Stein, 2006). Adolescencemarks a similar transition point. Growth of primarily physiologicallybased processes, such as attention, perception, and information proces-sing, provide the foundation for important social and emotional changesthat occur during these years that contribute to children’s growing senseof identity and self-esteem (Harter, 1997).

Research in chronic disease in childhoodmay be particularly apposite,as children who grow upwith a chronic illness not only have to meet theirage-related developmental tasks, but they also have to manage theirdisease, which leads to a heightened risk of maladaptation (Hill, 2006).A chronic condition may affect and/or interact with already existingnormative demands and changes in socialization (Schmidt, 2003). Thus,although most children follow normative developmental pathways andencounter predictable transition points, disease-specific pathways maybe embedded within these trajectories and influence the phenotypicexpression of physiology, psychology, and behavior.

B. Research with children

Children are increasingly acknowledged to have rights in the determina-tion of medical decisions that affect them. This has encouraged research tobe undertaken with children themselves to understand their own viewson the impact of a disease on their experiences and relationships. It hasbecome increasingly important for researchers and healthcare profes-sionals to understand how the perceptions, experience, and impact of achronic disease might influence a patient’s interpretation and response toit, so that we, in turn can respond more appropriately.

Related to this, the role of psycho-educational interventions in facilitat-ing adaptation to chronic disease has received growing recognition andis in keeping with policy developments advocating greater involvementof patients in their own care (Barlow et al., 2002a)

II. PREVALENCE, MECHANISMS, AND CLINICALMANIFESTATIONS OF FOOD ALLERGY

Atopy may be defined as a genetically and environmentally determinedpredisposition to clinically expressed disorders, including allergic rhini-tis, atopic dermatitis or eczema, food allergy, and allergic asthma, regu-lated through immune phenomena in which many cells (i.e., mast cells,eosinophils, and T lymphoctytes) and associated cytokines, chemokines,and neuropeptides play a role.


Atopy identifies allergic diseases such as atopic dermatitis (eczema),bronchial asthma, and hayfever, which tend to cluster in families and areassociated with the production of specific IgE antibodies to commonenvironmental allergens (Sicherer, 2002). The ‘‘atopic march’ refers tothe natural progression of allergic conditions, characterized by a typicalpattern of sensitization and manifestation of symptoms appearing atcertain ages, persisting over years or decades, but often spontaneouslyresolving with age (Sampson, 2003). Epidemiological studies haveattempted to disentangle the various phenotypes, focusing on singlemanifestations at certain age windows, since different specific pheno-types may be induced or modulated by different genetic, environmental,or lifestyle factors. Most investigators seem to agree that a complexinteraction between genetic and environmental factors regulates develop-ment of different atopic features, however much of the natural history ofatopic diseases and its determinants are still not well understood( Johannson et al., 2004). Food allergy is defined as an adverse immuneresponse to food allergens.

The growing prevalence of allergic diseases present an increasingchallenge for populations and health care systems around the worldand food allergies constitute a notable part of this increase (Sicherer 2002;Sicherer et al., 2001; von Berg et al., 2003). The most common food aller-gens are peanuts, tree nuts, seafood, eggs, and milk, however, the list isconstantly growing (Sampson, 2003). Several studies have confirmed theincrease in prevalence of food allergies, especially peanut allergy and as adisease burden throughout the world; however, they seem to increasinglyaffect countries with a formerly low prevalence and is becoming a grow-ing public health problem (Hourihane, Smith and Strobez, 2002). Thesefindings were substantiated by Al-Muhsen and colleagues (2003) suggest-ing that peanut allergy now accounts for the majority of severe food-related allergic reactions. There appears to be a correlation between theincreased consumption of a novel food and the risk of allergic reactions.Examples of foods introduced into the North American diet which conse-quently began to provoke allergic reactions as consumption increasedinclude kiwi, mango, avocado, and other exotic fruits (Burks, 2006;Sampson, 2003).

Food allergy affects �6–8% of young children and 3–4% of youngadults in the UK, US, and Europe (Eggesbo et al., 2001; Sampson, 2005;Sicherer and Sampson, 2006). In contrast, food intolerance describes anabnormal physiological response to an agent which is nonimmune-mediated ( Johansson et al., 2004). The prevalence of food allergies varybetween countries. The only comprehensive permanent nationwidereporting system and register for severe allergic reactions to food wasinstituted in Norway in 2000 (Lovik et al., 2003), and so more quantitativeestimates are difficult to provide. In Ireland, Hourihane (1998) estimates


that there are 16,000 children with peanut allergy; however, there hasbeen no formal prevalence study undertaken to date. In addition, currentdata on severe and fatal reactions may be misleading because anaphylac-tic reactions are often mislabeled as asthma deaths, because of a lack ofantecedent history or information. In effect, inadequate diagnosis of foodallergy may, in part, reflect the lack of adequate provision of relevanthealth services.

The European Academy of Allergy and Clinical Immunology hasproposed a revised nomenclature for allergic and related reactions( Johansson et al., 2004). According to this proposal adverse reactions tofood should be termed ‘‘food hypersensitivity.’’ The term food allergyshould be used when immunological mechanisms have been demon-strated, and includes both IgE- and non-IgE-mediated reactions. Allother reactions, which have sometimes been referred to as ‘‘food intoler-ance,’’ should be termed nonallergic food hypersensitivity (Fig. 3.1).

Adverse reactions to foods were first described over 2000 years ago byHippocrates, who is credited with the observation that cow’s milk couldcause urticaria and gastric upset, and, 500 years later Galen also describeda case of allergy to cow’s milk (Kimber and Dearman, 2002). Adversereactions to foods were published intermittently during the twentiethcentury, but it is only during the last 20–30 years that an increasingawareness of food allergy has emerged in western industrializedsocieties.

Food allergy occurs when the body’s immune system mounts anexaggerated response against the offending food, which acts as anallergen. It is a type of hypersensitivity reaction. It can be either:

� A type I, IgE-mediated reaction: This is the usual cause of food allergy.After initial sensitization, the release of mediators such as histamine aretriggered each time a person is exposed to the food. It is these mediatorsthat cause the symptoms.

Non-IgE-mediated food allergy IgE-mediated food allergy

Food allergy

Food hypersensitivity

Non-allergic hypersensitivity

FIGURE 3.1 Continued

Typical developmental pathway Food allergy developmental pathway

Infancy and early childhood Parental protection

Entry to formal schooling Transition point Precipitating events (stressful events in the children’s lives caused by developmental & food allergy related factors)

Cognitive appraisal and emotional effects

Biology

• Sex• FAMechanisms

Environment

Distal factors (socio-econom ic, education, culture)

Proximal factors

• Gender • Age• Context• Prior experience • Parent attitude • Social support (parent, teacher, peers,

friends)• General awareness

Integration of food allergic identity Rejection of food allergic identity

Functions (Interpers onal & Intrapersonal)

• Risk & safety • Eating & food • Control • Self-esteem • Identity • Interaction with peers • Emotional regulation

• Principal strategies Maximisation/avoidance

• Supporting strategies

• Principal strategies Minimisation/risk

• Supporting strategies

Adolescence Transition point

Precipitating events (stressful events in the teens lives caused by developmental & food allergy related factors)

Functions (Interpers onal & Intrapersonal)

• Risk &safety • Eating & food • Control • Self-esteem • Reinforce identity • New places • New people • Autonomy • Assert independence • Validation of beliefs • Emotional regulation

Habitual cognitive/emotional appraisal mechanisms

Habitual response patterns (physiological, emotional, behavioural)

Longterm psychological impact

Living with uncertainty

Search for normality

FIGURE 3.1 Nomenclature proposed by the European Academy of Allergology and

Clinical Immunology (Johansson et al., 2004). The developmental pathway in food

allergy. Our developmental model outlines the content of stressors (e.g., social events,

restaurants, food, allergic reactions, concern for risk and safety, concern for identity);

modifying variables on appraisal (e.g., attitudes of others, low general awareness, poor

labeling, age, sex, context; parental attitude); specific resources (e.g., types of social

support; restaurants; labelling); emotional impact (uncertainty, difference); psychologi-

cal impact (e.g., generalized anxiety, low self-efficacy); functions (e.g., reducing

uncertainty; ‘‘fitting-in’’) and consequences for behavior and participation (e.g., principal,

supporting).


� A delayed, type IV-mediated reaction: These reactions are mediatedmainly by T cells. They typically affect the gastrointestinal tract or skin,for example, exacerbation of eczema in children after milk ingestion.


In an IgE-mediated reaction, symptoms involving the oropharynx andgastrointestinal tract may occur within minutes of ingesting a food aller-gen. Itching and swelling of the lips, tongue, and soft palate as well asnausea, abdominal pain, vomiting, and diarrhea have all been demon-strated secondary to food allergy (Sampson, 2003).

Anaphylaxis refers to a sudden, severe, potentially fatal, systemicallergic reaction that can involve skin, respiratory tract, gastrointestinaltract, and cardiovascular system (Sampson, 2003). The term anaphylaxisis derived from the Greek words meaning ‘‘without’’ (ana) and ‘‘protec-tion’’ (phylaxis). The most dangerous symptoms include breathing diffi-culties and a drop in blood pressure, or shock, which are potentially fatal.Symptoms of anaphylaxis may develop within seconds or a few hoursafter ingestion of a food allergen, with the vast majority of reactionsdeveloping in the first hour. Symptoms can include swelling (especiallylips, tongue, or throat), difficulty breathing, abdominal cramps, vomiting,diarrhea, circulatory collapse, coma, and death. Typical allergy medica-tions such as antihistamines work too slowly and cannot reverse theeffects of chemical mediators. Adrenaline or epinephrine, therefore isthe treatment of choice and must be administered by injection promptly.

Food allergy, particularly to peanuts, is the most common cause ofanaphylaxis outside hospital (Bock et al., 2001), yet there are other com-mon food causes such as shellfish, fish, milk, soy, wheat, and eggs (Aseroet al., 2007). These foods may not only cause fatal or near-fatal reactions,and they also tend to induce ‘‘persistent sensitivity’’ in most patients, incontrast to other foods such as milk, eggs, and soybeans, which arefrequently associated with milder reactions and are usually ‘‘outgrown.’’

The life-threatening nature of anaphylaxis ‘‘makes prevention thecornerstone of therapy,’’ (Sampson, 2003). Avoidance of the responsiblefood allergen and emergency management in the form of injectable epi-nephrine (Epipen, Anapen, or Twinjet) in case food allergen is acciden-tally ingested is the only reliable therapy offered to those living with foodallergy.

Anticipatory guidance measures form the cornerstone of advice,including; reading food ingredient labels, concern for cross-contamination, vigilance in a variety of social activities, and immediateaccess to the Epipen (Munoz-Furlong, 2003). However, avoidance is com-plicated by the fact that peanuts, nuts, soy, can be found inmany foods (e.g.,breads, muffins, pastries, biscuits, cereals, soups, icecreams, seasoning,sauces) and in different forms as an emulsifier or thickening agent.

A growing number of families must live and cope with food allergy ona day to day basis, socio-emotional impact of food allergy on children,and adolescents has been little researched to date (DunnGalvin et al.,2007). Although researchers in the field of food allergy have stated thatthe scarcity in psychological and social literature on the experience of


living with food allergy demands attention, the majority of research infood allergy has been biomedical in orientation, focusing on issues suchas the molecular structure of allergens, or methods of diagnosis (seeDunnGalvin et al., 2007).

More recently, there has a growing interest in the development ofquestionnaires to measure the impact of food allergy on health-relatedquality of life (De Blok et al., 2007). Health related quality of life (HRQL) isa multidimensional construct, consisting of physical, psychological, andsocial components (Eiser and Morse, 2001).

III. THE IMPACT OF FOOD ALLERGY ON HRQL

Most studies to date used generic HRQL questionnaires to investigate theimpact of food allergy on quality of life. However, a disease-specificquestionnaire is necessary because generic measures although useful forcomparison across diseases, do not incorporate issues directly related tothe patients condition and therefore lack the sensitivity necessary todetect changes as a result of treatment interventions (Fayers andMachin, 2000). Further generic measures lack the detail that is requiredto assess how different subgroups are impacted by food allergy (De Bloket al., 2007).

A. Research using generic HRQL measures

Primeau and colleagues (2001) studied a sample of 301 patients andevaluated the quality of life and family relations of children and adultswith peanut allergy, compared to that of children and adults with rheu-matological disease. Their study was the first on the subject and theycompared HRQL in rheumatology and food allergy patients. It wasshown that the parents of allergic children believed that their childrenhad difficulties in many areas. Remarkably, their children had moreimpairment, especially in daily activities and in their familial social inter-actions, compared to children with significant rheumatological disease.The authors found that families with peanut allergic children experiencesignificantly more disruption in their familial and social interactions/activities than families with a child with CRD and suggested that thismay be due to the constant risk of sudden death in the peanut allergygroup leading to greater parent restriction of activities.

The comparison was criticized by Avery et al. (2003) because childrenwith rheumatological diseases have other symptoms to include severepain and chronic physical disabilities, which exceeds experiences ofchildren with peanut allergy. Primeau et al. (2000), at the time of theirstudy, recognized and acknowledged this comparison as a potential


compromise. They admitted that their choice was influenced by theaccessibility to available data.

Sicherer et al. (2001) measured the parental perception of physical andpsychological functioning. The authors randomly selected 400 membersof the food and anaphylaxis network, with families of children aged 5–18years old and had 253 responses. Results indicated that peanut allergyimpacted significantly on general health, parental distress, and familyactivities. Those with two or more food allergies scored significantlylower, depending on how many foods they were avoiding. There wasalso evidence to suggest that the educational and emotional supportneeds of these families are not being met.

Avery et al. (2003) assessed the effect of peanut allergy on the quality oflife in children aged 7–12 years old and contrasted this with experiencesof children with insulin-dependant diabetes mellitus (IDDM). Theyrecruited 20 children with peanut allergy and 20 children with IDDM(ages ranging from 7 to 12 years). Their results indicated that childrenwith IDDM have similar problems as children with peanut allergy. Thisincludes, food choices, social restriction, issues relating to school, thecarriage and use of a syringe and the chronic nature of the condition.The results also suggested that children with peanut allergy are moreanxious and parents feel that their needs are not taken into account.Results showed that children with peanut allergy had a poorer qualityof life and are more anxious concerning accidental ingestion of peanutthan children with diabetes are of having a hypoglycemic reaction.

Another generic questionnaire-based study, among 1451 adolescents,indicated that 19% of the participants reported that they had a perceivedadverse reaction to food, however their condition was not physician-diagnosed (Marklund et al., 2004). When compared to adolescents with-out such conditions, this kind of allergy-like condition, regardless of theunderlying mechanisms, was associated with lower HRQL.

B. HRQL Research using disease-specific measures

The first validated HRQL food allergy specific measure; the Food AllergyQuality of Life–Parental Burden (FAQL-PB) questionnaire (Cohen et al.,2004) measures the parental burden associated with having a child withfood allergy. Scores in the food-allergic cohort were significantly lower forgeneral health perception, parental distress and worry, and interruptionsand limitations in usual family activities, than in healthy controls. Scaleswere also lower in subjects with multiple food-allergies.

More recently, several measures have been developed to assess qualityof life in parents, children and teens, propelled by Europrevall. Europre-vall has initiated/propelled a leap in research.


Europrevall is Europrevall is an EU project which aims to improvequality of life for parents, children, teenagers and adults with food allergy(De Blok et al., 2007; Dunn Galvin et al., 2007). Europrevall multidisciplin-ary integrated project (IP) involving 17 European member-states, Switzer-land, Iceland, and Ghana. Of the 63 partners, there are 15 clinicalorganisations and six small-medium sized enterprises (SMEs) as well asthe leading allergy research organisations in Europe. Since the projectbegan in 2005, new partners have also joined from New Zealand,Australia, Russia, India, and China.

By integrating information and developing tools for the use of Euro-pean food allergy scientists, health professionals, food and biotech indus-tries, and consumers it is Europrevall’s hope that causes of food allergycan become better understood, diagnosis of food allergy can becomeswifter and the quality of life of food allergy sufferers improved.

Two food allergy specific questionnaires have been developed andpublished under the auspices of Europrevall. The first measures HRQL inchildren aged 0–12 years and is parent administered; ‘‘Food AllergyQuality of Life’’ questionnaire (FAQLQ-PF; DunnGalvin et al., 2008) andthe second measures quality of life in teens (FAQLQ-TF; Flokstra-DeBloket al., 2008).

The FAQLQ-PF and -TF were developed and validated in four stages:(1) item generation using focus groups with both children and parents,expert opinion, and literature review; (2) item reduction, using clinicalimpact and factor analysis; (3) internal and test-retest reliability andconstruct validity were evaluated; and (4) cross-cultural and contentvalidity was examined by administering the questionnaire in a US sample(FAQLQ-PF, only).

Both studies found a severe impact of food allergy on HRQL inrelation to psychosocial aspects of children’s and teens everyday lives.For example, in the initial focus groups put in place to generate items forthe FAQLQ-PF, parents suggested that the anxiety associated with therisk of a potential reaction has more profound effects on emotional andsocial aspects of a child’s everyday life, than clinical reactivity induced byfood intake. The importance of a subscale assessing this aspect of anxietywas subsequently confirmed using clinical impact and factor analyticmethodologies.

In addition, multivariate analysis showed an interaction between sexand age group for impact of general emotional impact on HRQL scale, ineffect, parents of boys reported higher mean scores up to the age of6 years; parents of girls reported higher mean scores in the 6–12 yearsage group.

Studies using these measures have thrown light on the everydayburden of living with food allergy.


It has therefore been well established that food allergy can have aprofoundly negative impact on quality of life, extending well beyondthe immediate clinical effects of the individuals condition. However,very little is known about the attitudes, beliefs, and coping strategies offood allergic children about food allergy generally and their conditionspecifically and little attention has been paid to the most effective meth-ods of communication with this group (Miles et al., 2006).

IV. THE PSYCHOLOGICAL BURDEN OF FOOD ALLERGY

Although it appears that food allergy leads to intrapersonal (e.g., anxiety)and interpersonal (e.g., social restrictions) problems in adaptation, therehas been little research into the socio-emotional impact of food allergy onpsychological and social functioning. Indeed, in most studies on chronicdiseases, usually psychological maladjustment factors such as behaviorproblems or depression are studied, but social maladjustment factors,such as social anxiety or social skills, have rarely been included (Meijeret al., 2002).

The literature also lacks well-designed studies describing the psycho-logical burden of food allergy. Studies are scarce and are often carried outin a mixture of diagnoses, reflecting the difficult field of adverse reactionsto food. Many of the studies that have been undertaken are limitedbecause they rely only on questionnaires, on the particular questionnaireselected, and on prestudy or preexisting hypotheses which limits novelfindings. Questionnaire studies produce findings on ‘‘how often’’ or‘‘how many’’ but are very limited in answering ‘‘why.’’

For example, patients attending allergy clinics were found to havehigher levels of depression when compared with the general population,(e.g., Kova’cs et al., 2003). In a community sample, Knibb et al. (1999)found that women with a perceived food intolerance or allergy havesignificantly higher scores for somatic symptoms, anxiety and insomnia,and severe depression than women with no reported food allergy orintolerance. Vatn (1997), found that patients identifying themselves assensitive to food or chemicals had high scores for depression, anxiety,shyness, and defensiveness.

Coping has been defined as ‘‘action regulation under stress’’ andrefers to how individuals ‘‘mobilize, guide, manage, energize, directbehavior, emotion, and attention and how they fail to do to’’ (Skinnerand Wellborn, 1994, p. 113) under stressful conditions.

However, it appears that literature on adjustment or coping in chronicillness focuses mainly on coping in illness-specific situations. However,general coping styles may be more predictive for the psychosocial devel-opment of chronically ill children and teens than illness-related coping


because these styles reflect how children cope with developmental tasks(Meijer et al., 2002). General coping styles (e.g., in social situations) may beparticularly relevant in food allergy because it is the anxiety associatedwith the risk of a potential reaction that has more profound effects onemotional and social aspects of a child’s everyday life, than clinicalreactivity induced by food intake (DunnGalvin et al., 2008). In addition,food allergy, once diagnosed and after restrictions are put in place, maybe primarily asymptomatic. Living with peanut allergy places increasedstress on the child and the child’s parents and siblings (King et al., 2008). Italso causes differing levels of anxiety throughout the family. The authorsfound that mothers of children with peanut allergy feel that they havesignificantly poorer HRQL and suffer more anxiety and stress than thefather.

However, clinicians assume that ‘‘appropriate’’ levels of anxiety isadaptive in children and parents living with food allergy. Primeau et al.,2000 and Avery et al., 2003 suggest that the high level of stress in familieswith a peanut allergic child may have beneficial effects on coping strate-gies. Only one study, using a study-specific questionnaire, suggested thatdeprivation due to restrictions in lifestyle may lead to social anxiety infood allergy (Bollinger et al., 2006).

Adaptational processes of children and adolescents with chronic con-ditions are of utmost importance because of the long-term consequencesof childhood conditions. For example, in child/adolescent populationswith asthma, up to one third may meet criteria for comorbid anxietydisorders (Bender-Berz et al., 2005). In adult populations with asthma,the estimated rate of panic disorder ranges from 6.5% to 24% (Katon et al.,2004a,b). Children with any chronic condition have twice the risk ofdeveloping mental health disorders of healthy children without anaccompanying physical disability (Schmidt, 2003). A recent paper(Knibb et al., 2008) may provide another clue to the impact of livingwith food allergy on long term adjustment. The authors used the IllnessPerception Questionnaire (Moss-Morris et al., 2002) to measure the extentto which illness perceptions and coping strategies are associated withlevels of psychological distress among 156 adults with food allergy.Results showed that a strong illness identity and emotional representa-tions of food allergy in adults were associated with higher levels ofpsychological distress; as were less adaptive coping strategies such asfocusing on and venting of emotions. Strong personal control beliefs wereassociated with the lower levels of distress, as were adaptive copingstrategies such as positive reinterpretation and growth. Coping skillspartially mediated the link between the illness perceptions and the out-come; however, illness identity, emotional representations, and personalcontrol retained an independent significant association with psychologi-cal distress.


V. THE INFLUENCE OF PARENTS ON CHILD ADJUSTMENT

Parental perceptions can have a profound impact on the way that childrenthemselves perceive their own health and illness and on how they inter-pret risk associated the disease (Noojin and Wallander, 1997). Althoughthe impact of any condition on a child’s life varies according to the specificcharacteristics of the disease, the chronically ill child is often more depen-dent on parents with adolescents complaining more often a delay inindependent life-styles (Wallander and Varni, 1998).

An important developmental issue relates to the fact that children andadolescents are dependants, largely reliant on adults for significantaspects of illness behavior. This behavior includes the way in whichsymptoms are responded to, including the extent to which medical con-sultation and lifestyle alterations are undertaken. Parents and carers arepowerful in responding to (or ignoring) children’s physical complaints,attributing significance (or reassurance) to these complaints, facilitating(or otherwise) the children’s use of health care facilities and their involve-ment in (or withdrawal from) normal life activities (Noojin andWallander, 1997). Thus, parental distress, response to diagnosis and con-sequent coping strategies will have implications for how children them-selves cope and manage food allergy along developmental trajectories.

We could only find one published study that looked at the impact ofparent influence in food allergy. Recent findings by Bollinger et al. (2006,p. 419) suggests that ‘‘children with food allergy may be at an increasedrisk of social-emotional developmental difficulties.’’ Many parents admit-ted to overprotecting their children through an understandable desire toensure their children’s safety. However, such restrictions can stunt chil-dren’s social and emotional development and increase children’s percep-tion of ‘‘illness intrusiveness.’’ Keating and Miller (2005) discuss researchfindings on infant distress which suggests two socioemotional roots ofcompetence, with possible interactions between them: age-specific emo-tion regulation style; and parental response and facilitation. They use theterm ‘‘habits of mind’’ to describe the increasing coordination and inte-gration of the competence and regulatory system trajectories over thecourse of development.

Our research group have carried out a study (submitted) on parentstress triggers using a specially designed Implicit Association Test (IAT).The IAT measures associative links between environmental triggers andpsychological states. Explicit or ‘‘self-report’’ questionnaires have givenrise to concerns related to the influence of conscious self-presentationalbiases which often result in inaccurate answers (Greenwald et al., 1998;Nosek, 2007). In addition, the very act of completing the self-reportmeasure may change emotions attitudes and beliefs about the construct


under investigation (Greenwald et al., 1998). Therefore, the IAT was usedto measure the differential association of a target category (environmentalstimuli) with an attribute dimension (anxious vs. relaxed) in parents offood allergic children (N ¼ 60) and matched controls (N ¼ 30). A studyspecific questionnaire (SSQ) was used to measure explicit attitudes. TheIAT targets the specific concerns/attitudes/beliefs parents of food aller-gic children while minimizing confounders. In contrast, the perceivedthreat of social situations where food may or may not be present (a centralconcern of parents of food allergic children) is incomprehensible to par-ents of nonfood allergic children. Results showed that parents are signifi-cantly more anxious when they believe they are not in control of theirchild’s environment. In contrast, there were no significant results withregard to parents of nonfood allergic children. It is clear therefore thatparents face a difficult balancing act between encouraging growing chil-dren’s independence and ensuring their safety that may have implica-tions for children’s own perception of control in living and coping withfood allergy.

VI. SOCIAL SUPPORT

Not only interpersonal functioning, but also interpersonal needs of chil-dren with chronic conditions differ from those of healthy children. Socialsupport is understood as a ‘‘resistance’’ or ‘‘buffering’’ factor in chronicdiseases. Studies with children have shown that higher stress increasesrisk for adjustment problems, such as greater anxiety and depressivesymptoms, while higher social support reduces the risk for adjustmentproblems (e.g., Wallander and Varni, 1998). Although many studiesaggregate social support variables, the sources and types of available tochildren may differentially predict psychosocial adjustment (Schreursand de Ridder, 1997). In addition, classmate support (contrasted withparent, friend, and teacher support) was found to be the strongest predic-tor of adjustment in children with limb deficiencies (Varni et al., 1991),cancer (Varni et al., 1994a,b,c), and pediatric rheumatic disease (Von Reisset al., 2002). However, other studies have found that family social support,but not peer support, was a significant predictor of adjustment in childrenjuvenile rheumatoid arthritis (Varni et al., 1988).

The diverse findings may occur because social support needs differ asa function of the particular disease. However, given the sparse literatureon support in chronic disease in children in general, and food allergy inparticular, further research is needed to examine the relative importanceof different sources and types of support on the adjustment of children.For example, in a recent qualitative study into children’s, teens, andparents perceptions of living with food allergy (DunnGalvin et al., 2009),


we found that social support, per se, does not necessarily protect againstemotional distress, but rather how the individual perceives and interpretshis or her social network. In children with generalized avoidance strate-gies, friends or parents may actually reinforce children’s beliefs of agenerally unfriendly world. Our findings show that parental distress,threat perception, and coping strategies reflect how children themselvesrespond to and manage food allergy. Parental influence and beliefs mayhave greatest impact during sensitive (psychological) or critical (physio-logical) periods of development when children and adolescents are mostvulnerable, for example, during middle childhood and/or adolescence.This type of specificity permits precise empirical guidance in the devel-opment of our educational interventions for children, teens, and parentswith food allergy.

In addition, we found that different dimensions of social support mayreflect different dimensions of adjustment, for example, children whospoke about classmate support or teacher support appeared more incontrol and much less anxious about food allergy, compared to thosewho referred only to support from parents and close friends. Those whodid not seek (or receive) instrumental or emotional support from friendsor teachers appeared to engage in much riskier behavior. These differen-tial relationships underline what Varni (1994b, p. 35) refers to as the‘‘specificity of the relationship between resistance factors and outcomes.’’

VII. THE IMPACT OF STRESS ON BIOPSYCHOSOCIALDEVELOPMENT

From a physiological viewpoint on development, we find that environ-mental stress may be important in perinatal programming (Wright, 2005).Studies in rodents and primates have shown that environmental manip-ulations that increase maternal stress result in elevated cortisol levels anddysfunctional behaviors in offspring that are mediated, in part, througheffects on gene expression (Meaney and Szyf, 2005). Wright and collea-gues (2005) found that higher caregiver stress in the first 6 months afterbirth was associated with increases in the children’s allergen-specificproliferative response (a marker of the allergic immune response) highertotal IgE levels, and increased production of TNF-a and reduced IFN-g ina birth cohort of children predisposed to atopy. Therefore, during T cellmaturation when the atopic phenotype is being determined by exposureto allergens, stress may be an additional factor.

During an immune response, the brain and the immune system com-municate with each other in order to maintain homeostasis (Knonfol andRemick, 2000). Two major pathways, the HPA-axis and the CNS areinvolved in this bi-directional interaction (Elenkov et al., 2000). The effects


of stress on neuroimmuregulation in turn may modulate the hypersensi-tivity response in developing children. Cytokines play a crucial role in thepathogenesis of allergic diseases. In addition to acting as chemical mes-sengers between immune cells, cytokines can serve as mediators betweenthe immune system and the brain (Dantzer, 2001).

Catecholamines, glucocorticoids, and proinflammatory cytokines(TNF-a) are considered to be the principal messengers between theimmune system and the nervous system in the stress response. For exam-ple, chronic stress enhances the production of TNF-a, in turn increasedTNFa levels can activate the HPA axis. For example, increased numbers ofregulatory T cells in peripheral blood were observed in both atopic andnonatopic students under exam stress, as well as skewed Th1/Th2 ratioand reduced NK cell numbers that were unique to atopic students (Kangand Fox, 2000). Although some studies (e.g., Wright, 2005) have examinedthe impact that the activity of the stress system may have on immuneactivation and symptoms, very few studies have considered whetherimmune activation and the experience of having an atopic disease, partic-ularly during childhood, influences the long-term responsiveness of theHPA axis. For example, Rosencrantz et al., 2005 used fMRI during antigenchallenge to examine regional brain activation in adults with mild allergicasthma and identified activity in the anterior cingulated cortex (ACC) inresponse to asthma associated emotion words (e.g., wheeze). After anti-gen challenge, increased levels of IL-1 and IL-6 have been noted (Marshallet al., 2002). Depression has also shown to be associated with excessivesecretion of proinflammatory cytokines such as IL-1 and IL-6 (Maddoxand Pariante, 2001).

VIII. THE IMPACT OF SEX AND GENDER IN FOOD ALLERGY

The relationship of sex and gender to health and disease is complex, andvaries across an individual’s lifespan, and between cultures and differentsocial contexts. Attention to sex and gender in biomedical and healthsciences research is being actively promoted by the European UnionCommission under their research policy of ‘‘mainstreaming genderequality’’(Klinge and Bosch, 2005). Sex denotes the differences attributedto biological origins alone, while gender refers to the social and culturalinfluences that lead to differences between women andmen (DunnGalvinet al., 2007). One consequence of variables related to both sex and genderis that potentially differing patterns of disease prevalence, differentdegrees of severity, and different patterns of mortality and morbiditymay be identifiable between men and women (e.g., Wizeman andPardue, 2001).


A. The influence of sex

In population based studies sex differences in atopy (assessed as skin testreactivity to one or more of a panel of allergens) have been reportedthroughout childhood and into early adulthood, such that rates in girlsare lower than in boys up to at least 15 years of age, in most studies up to25 years of age, but are not consistently observed thereafter (Forde et al.,2003). In contrast to the sex differences in atopy assessed as skin testpositivity which vary and change direction across the human lifespan,sex differences in atopy assessed as total serum IgE levels are consistentacross the lifespan, with levels in females being lower than those in males(Burney et al., 1997). With reference to asthma and food allergy, preva-lence is higher in boys before puberty, while this sex ratio is reportedlyreversed after puberty (Becklake and Kauffman, 1999). Physiologicalpathways for these sex differences have been discussed with referenceto ‘‘immune dimorphism,’’ the term given to differences in immuneresponses and regulation between the sexes.

The mechanistic involvement of sex hormones in immune reactionshas increasingly been acknowledged in recent years (Osman, 2003). Tes-tosterone and oestrogen affect diverse cellular processes including pro-tein synthesis, cell division and migration, neuronal growth and axonalbranching, and synaptic remodeling. Receptors for sex steroids have beenidentified on lymphocytes, monocytes, and mast cells (e.g., Balzano et al.,2001). Lymphocytes are known to express both testosterone and estrogenreceptors (Osman, 2003) whereas androgens enhance CD8þ lymphocyteactivity and are correlated with the activation of IFN-g-secreting cells inhealthy adults (Balzano et al., 2001). In allergy, sex hormone receptors onlymphocytes and leukocytes may modulate the type of immune reactionand regulate inflammation. For example, estrogens have a receptor-mediated effect on the releasibility of mast cells influencing the thresholdlevels in the effector phase of allergy (Da Silva, 1999).

Different patterns of cytokine responses between males and femalesmay be implicated in gender specific effects. Mechanisms linking psycho-logical stress, personality, and emotion to neuroimnoregulation as well asincreased risk of atopy have been increasingly elucidated (Wright, 2005).We already know that components of stress and the stress response differbetween men and women. The tend-and-befriend response, mediated byoxytocin and endogenous opioids, may be more applicable to womenthan the fight-or-flight response, which was based largely on studies ofmen (Wright, 2005). Even within the flight-or-flight response patternthere are sex-based differences. The HPA axis interacts with reproductivefunction, such as menstruation.

For example, when challenged by psychosocial stressors, males havebeen found to show a significant increase in glucocorteroid sensitivity


but decreased proinflammatory plasma cytokine production, whereasfemales show a significant decrease in glucocorticoid sensitivity butunchanged proinflammatory plasma cytokine production (Rohlederet al., 2001). Furthermore, these sex differences were found only underactive mental stress, not under passive cold stress. This raises the possi-bility that sex differences in self-reporting (see DunnGalvin et al., 2006)and even prevalence in some diseases could at least in part be explainedby sex differences in the nature of the physiological response to stress,and, further that the nature of stressors may also influence sex differencesin immune reactivity to stress (Kang et al., 2004) involving a complexinteraction between biology and environment. For example, there aregender differences in the types of stressors to which an individual is likelyto be exposed.

The complexity of these sex and gender based interactions may explainthe more adverse effects of food allergy on female over male generalemotional well-being. For example, females with food allergy were foundto be at increased risk of negative socio-emotional outcomes (Bollinger et al.,2006). Patients attending allergy clinics reported higher levels of depressioncompared to the general population (Kova’cs et al., 2003). A birth cohortstudy in Finland (Timonen, 2003) revealed that, at epidemiological level,skin prick test positive females exhibited up to an 1.8-fold greater risk ofdeveloping lifetime depression when compared with skin prick test nega-tive subjects. In addition, the corresponding risk increased up to 2.7-foldamong females, who had a positive skin prick test together with self-reported allergic symptoms. Maternal atopy alone almost doubled therisk of lifetime depression in female probands when compared withfamilies in which no maternal atopy existed. In contrast, parental atopydid not predict any type of depression in male probands. However, aspreviously discussed, social adjustment or social anxiety was not investi-gated; therefore, we only have results on more extreme or clinical psycho-logical or behavioral disorders, such as those described in the DSM-IV.

B. The influence of gender

There has been little psycho-social research on the influence of gender inthe context of food allergy (see DunnGalvin et al., 2007). Marklund andcolleagues (2004) investigated the extent to which females and maleadolescents experience perceived allergy-like conditions and the impactof these on everyday life. They found that adolescent females reportedallergy-like conditions more frequently than adolescent males. Althoughall adolescents with allergy-like conditions reported significantly lowerHRQL in seven of eight health scales that measured bio-psycho-socialfunctioning, however, females reported more severe HRQL-deteriorationcompared with males. This is consistent with research that shows an


excess of psychological vulnerability in adolescent girls with chronicconditions when compared to boys suffering from the same conditions,including epilepsy and asthma (Austin et al., 2000), insulin dependentdiabetes mellitus (La Greca et al., 1995a,b), and cerebral palsy (MaGill andHurlbut, 1986). Of the allergy conditions reported by Marklund, morethan 50% of the adolescents stated they had food hypersensitivity withpositive allergy tests. However, a sex and/or gender breakdown forconfirmed food hypersensitivity or method of diagnosis was notincluded. Thus, it is not possible to determine if there is a gender differ-ence in perceived versus actual food allergy for these individuals. How-ever, work by Knibb and colleagues (1999) has demonstrated a genderbias in reporting self-diagnosed food allergy and intolerance, with signif-icantly more females self-reporting than males.

Differences in self-reports of ill-health and psychological distress havealso been observed in adolescents in the general population. For example,Sweeting and West (2003) found that self-reported general ill-health andphysical symptoms, as well as psychological distress was significantlyhigher and increasing from age 11 for females compared to males. Thisincreased with age and by age 15, there was a female excess in general ill-health, including psychological distress and ‘‘malaise,’’ limiting illness,poor self-rated health, headaches, stomach problems, and dizziness. Thismay also explain possible gender differences in self-assessed health in thecontext of perceived food allergy.

This has been explained by the concept of ‘‘illness centrality’’ (Wiebeet al., 2002), or the level at which particular illness has been integrated intothe self-concept. In a recent study (DunnGalvin et al., 2009), we found thatas the child develops, the level of integration of food allergy into the selfconcept also develops in a mostly gender-specific manner and has con-sequences for the child’s understanding of food allergy and everydaymanagement of the condition. We found that girls tend to incorporatefood allergy into their self-concept, making it a defining part of who theyare, whereas males ‘‘contain’’ the illness by minimizing its importance.Interestingly, although boys evinced lower anxiety levels because of thistendency, they were more prone to ‘‘risky’’ behavior (e.g., not botheringto read labels), whereas girls were more anxious, but also demonstratedmore self-care behaviors.

IX. RISK BEHAVIOR IN FOOD ALLERGY

Risk is considered as the probability of a negative event occurring and canbe quantified. However, this form of risk can be understood as ‘‘danger.’’The perception of risk is a socially constructed phenomenon, and is moredifficult to measure. Psychological risk is based on perception rather than


fact, and is therefore based on qualitative, not quantitative characteristicsof the hazard being considered. For example, when one individual feels asharp pain, he/she may interpret it as a possible ‘‘heart-attack’’ andphone for an ambulance, whereas another individual might just reachfor an indigestion remedy. As already discussed, food allergy has animpact on quality of life, due in part to the constant vigilance requiredon the part of the allergic individual, or their caregiver, to ensure acciden-tal ingestion of food allergens does not occur. However, limited researchexists in risk perception in food allergy and there is no research to date onprocess variables and/or causal pathways involved in the initiation,treatment, and cessation of health risk behaviors in food allergy.

Research looking at the incidence of severe allergic reaction has sug-gested that adolescents and young adults are more at risk. In a Norwegianstudy on severe allergic reactions to food, the main risk group wascomprised of young adults aged 20–35 (Lovik et al., 2003). Teenagersrepresent a high-risk group for anaphylactic fatalities caused by foodallergy, accounting for 53% of a group of UK fatalities (Pumphrey, 2000).

A recent paper by Sampson and colleagues (2006) found that adoles-cents and young adults appear to be at an increased risk for fatal foodallergic reactions, and suggested that they may adopt more risk-takingbehaviors with regard to their food allergy; however, gender differencesor possible causal mechanisms were not explored. The study populationincluded persons with a high degree of severity of food-induced allergicdisease, with numerous food allergies, and frequent and severe reactions,however, 37%with severe symptoms did not receive epinephrine, and 38%did not have it with them during severe reactions. The authors suggestionsfor intervention was to encourage clinicians to emphasize to patients thatfood is often a part of all group activities, and an accidental exposure couldoccur, making it necessary and safest to always have an epipen available.They also suggested teaching parents to remind teenagers about carryingepipens to social events. It is clear, therefore, that research is needed toestablish underlying emotional risk factors associated with risk taking.

Perception of risk in adults is usually described, or interpreted, withreference to health belief models (HBMs) (Ajzen and Fishbein, 1980; Janzand Becker, 1984). Individuals are more likely to engage in health beha-viors if they perceive: vulnerability to health threats; that the conse-quences are severe; that treatment or preventive measures will besuccessful. Although there are variants to the framework, the differentmodels share many of the same elements. In effect, theories assume thatindividuals rationally weigh benefits and costs and act according to theoutcome of this analysis. Subsequent modifications to the models includethe addition of perceived social or monetary barriers to the adaptiveresponse. A cue to action which can be internal (e.g., symptoms) orexternal (e.g., health communication) is hypothesized to trigger these


cognitive processes. Of course, demographic and sociopsychological vari-ables may influence perceptions and indirectly affect the likelihood of theresponse (van der Pligt, 1994, 2002). Although the HBM have been foundto predict children’s expectations to use medicines to treat illnesses (Bushand Iannotti, 1990), there is limited support for the model and for compo-nents of the model predicting children’s health behaviors. This is espe-cially evident for perceived vulnerability to health threats. For example,early research revealed mostly negative relations between children’shealth behaviors and perceived vulnerability to risk (e.g., Gochman andSaucier, 1982) and initial findings were later extended to adolescents(Greening and Stoppelbeim, 2000), thus challenging the hypothesispostulated by HBM that greater perceived risk is related to adaptivebehaviors in children and adolescents.

Such findings suggest that risk perception is a complex process thatwarrants a deeper understanding from both health educators andresearchers. For example, in light of the incongruence between knowl-edge and action in terms of compliance and risk, there is an increasingrecognition of the need to qualitatively explore people’s experiences,perceptions, and understandings of what it is like to live with a chroniccondition, including its management, in order to better understand thedecisions people make about managing their condition.

Developmental factors are also important when considering risk per-ception in food allergy. For example, in middle childhood, as processesrelating to the impact of social comparison develop, children may betempted to reject safety rules in order to ‘‘fit-in.’’ Adolescents andyoung adults frequently eat away from home, they face growing peerpressure, and alcohol consumption may be high. The latter may bothimpair their ability to assess risk and augment the physiological effectsof allergen encounter such as vasodilation (Sampson, 2004). For manyadolescents, the social pressure for psychosexual autonomy directlyclashes with the prolonged dependence on family, which may be particu-larly pronounced in chronic diseases such as food allergy. Researchers inthe field of allergy suggest that teenagers should be a priority group forthe development and evaluation of interventions to improve their adher-ence to management plans. However, interventions will not prove suc-cessful unless we know the disease-specific developmental trajectory bywhich some teenagers become high risk and others do not.

X. DEVELOPMENTAL PATHWAYS IN FOOD ALLERGY

All these studies represent outcomes, but, there has been little, if any,research in allergic diseases in general and food allergy in particular, intothe developmental pathways that lead to these observable consequences.


This limits the ability of clinicians, researchers, and policy makers topredict and evaluate cognitive and emotional development in the foodallergic child, with implications for prevention, treatment, interventionand health policy. For example, allergists assume, but have never con-firmed, that high levels of vigilance in children performs an adaptiveprotective function and psycho-social outcomes have not been investi-gated to date. Furthermore, a developmental perspective has the potentialto provide an explanatory framework for previous disparate findings ofthe impact of food allergy on children and adolescents.

The way in which children and adolescents cope with chronic healthconditions is considered as an increasingly important predictor of healthin clinical and psychosocial research (Schmidt, 2003). Consideration of thedevelopmental perspective is not only useful when studying children, butalso helps to explain cognitive processes in response to stressors in adult-hood. However, coping has been studied less frequently in children andadolescents than in adults. Researchers contend that this may be due tothe difficulty in assessing developmental processes that are occurringsimultaneously in children and teens (Schmidt, 2003). The inter-relatedness of coping and development implies that coping is a processthat is shaped by developmental organization and, likewise, developmentis shaped by coping processes (Schmidt, 2003; 214).

However, early adopted strategies of coping with chronic disease mayserve as a buffer against these disease-related consequences, even if acertain stability of coping strategies across situations and developmentalstages cannot be assumed. Coping has not only been shown to be relatedto patient well-being, but mediates health behavior as well as health careutilization (Barlow et al., 2002b). For health care providers, there is socio-economical interest to support the development of adaptive and activecoping strategies in children with chronic conditions as early as possible.

A recent study (DunnGalvin et al., 2009), under the aegis of Europre-vall, represents a first attempt to provide an integrated developmentalframework to explain the onset, development, and maintenance of foodallergy related cognitions, emotions and behavior. 62 children/teenagersaged 6–15 years took part in 15 age appropriate focus groups, 52% ofchildren were female. Parents were also interviewed. All children werephysician diagnosed with IgE-mediated food allergy and had been issuedwith an anapen/epipen.

Through qualitative enquiry, a framework for evaluating childrenwith food allergy was developed. Developmentally appropriate techni-ques were designed to stimulate discussion, maintain interest, and mini-mize threat to the child’s self-esteem. Six main themes emerged from theanalysis that encompassed precipitating events (stressful events in thechildren’s lives caused by food allergy related factors); psychologicalimpact (cognitive appraisal and emotional effects); and behavioral


consequences or coping strategies. Findings indicate that coping in foodallergy is more than simply a strategy, it is a cumulative history ofinteractive processes (both age, gender, and disease specific) that areembedded in a child’s developmental organization.

Our findings show that food allergy is a central ‘‘lens’’ in children’slives through which they interpret experiences. When children and teensare confronted with a stressful event, such as a birthday party, a novelsituation, an allergic reaction, or making new friends, the way in whichthey appraise the event, and its attendant emotional impact are viewedthrough this lens. How this lens is constructed and its psychological impact(uncertainty, anxiety, confusion, difference) on individual children ismodified by age, gender, context, prior experience, attitudes of parents,attitude of peers, and level of general awareness.

Age is an important factor in determining the type of event childrenare likely to encounter from structured events such as birthday parties inyounger children to more unstructured and unplanned events as childrenbecome more independent.

Children live within the context of their families, which have interac-tion patterns, rules, organizing principles, and general belief systems, aswell as those specifically regarding health and disorder. Parental stressand perceptions of level of threat and consequent anxiety has a profoundimpact on the way that children themselves perceive risk and control.In focus groups with parents, many admitted to overprotecting theirchildren through an understandable desire to ensure their children’ssafety. Teens used very similar language and phrases when talkingabout aspects risk and control that younger children ascribed to theirparents—and that parents themselves used in focus groups.

In most children under the age of 8 years, there is a certainty ofparental and adult knowledge and a consequent sense of control of eventsrelating to food allergy. Children in the 6–8 years age group described thefood they eat as ‘‘special’’ and also described themselves in this manner.In addition, younger children are more confident in social situationsbecause of the protective presence of the parent. However, a transitionpoint occurs around>8 years when children begin to describe themselvesas different and the term special is ascribed to parents and takes on anegative connotation. At this time, children also begin to learn or feel thatthey cannot conclusively prevent an allergic reaction from occurring,giving rise to a state of uncertainty, and impacting on children’s percep-tion of autonomy and self-belief in their ability to control events intheir lives.

Older children have to learn to live with constant uncertainty, oftenreinforced by parent’s own anxiety about safety. This sense of uncertaintycontinues to grow as children develop and when children were asked‘‘when do you have a reaction,’’ many recounted stories of accidental


ingestion of allergic foods when visiting relatives, at school, or at socialevents ‘‘even though I was careful.’’

This state of uncertainty is also reinforced as children and teensencounter a widespread lack of awareness that encompasses teachers,schoolfriends, classmates, shops, restaurants, coffeeshops, afterschoolactivities, social events, and other parents. Therefore, children becomeaware, both through parents or through direct experience that a cleardichotomy exists between safe places and people and risky places orpeople, perceived by participants as ‘‘those who understand and thosewho don’t.’’ This directly impacts on perception of control and illnessintrusiveness. In addition, difficulties in adapting concrete rules oftenresults in a perception of external control over a particular event, particu-larly in the transition period between parental and ‘‘self’’ control.

A food allergic child’s identity appears to be closely tied to the dietaryand social restrictions that come with their condition. In middle child-hood as peer comparison begins to appear, children with food allergybegin to make causal connections between experiences in the world atlarge and inner feelings, a strong negative association can developbetween appraisal in terms of an objective health threat and appraisal interms of the emotional response to a health threat. This perception of‘‘difference’’ is isolating and has consequences for how children perceivethemselves and how they feel they are perceived by others. Indeed, teensspeak about social events mostly in terms of their restrictions.

Events are appraised in the context in which they occur and an aware-ness of expected behavior. For example, whereas an allergic reactionwhich takes place in the home may be regarded as a low-anxiety event,one which takes place in school can be appraised a highly stressfulbecause it impacts on the child’s goal to ‘‘fit-in.’’ This goal confrontschildren with the difficult balancing act of protecting their ego and man-aging risk. He or she learns to appraise (and weight) threats to personalsafety with threats to social identity. The stress appraisal process mayresult in children ‘‘just chancing it will be ok’’ and deliberately eating anallergic food when in the company of others, whereas others protect theirself-esteem by avoiding novelty as much as possible. With regard to riskybehavior, another important factor emerged as a motivation for deliber-ately eating an allergic food. It appears that some older children attemptto determine their own risk thresholds; ‘‘I would have a little bit. . . andsee what happens. . . you might get a bit sick only.’’ This may be a way togain some control over feelings of uncertainty that are integral to growingup with food allergy.

Levels of anxiety appear to range from mild anxiety in a situationwhere the allergic food is present to more severe ‘‘trait-like’’ anxiety.About 20% of the participants mentioned being anxious ‘‘a lot of thetime,’’ the majority of which were girls. Coping strategies were found to


lie on an maximization/avoidance to minimization/risk continuum. Themajority of girls were found to use ‘‘avoidant’’ strategies to cope withliving with food allergy. Many clinicians assume that these strategies arenecessary and adaptive, if they are proportionate. However, resultsshowed high levels of anxiety, vigilance, and generalized avoidance ofsituations and people not directly related to food consumption are asso-ciated with maladaptive avoidant strategies. A surprising finding wasthat anxious children and teens are not necessarily those who experiencethe most or recent reactions. For example, many of the children whodescribed themselves as anxious or worried about food allergy couldnot remember ever having had a serious reaction. ‘‘Minimizing’’ strate-gies, such as not reading labels, not telling others that you are food allergicin risky situations and deliberately eating an allergic food (mostly foundin boys), are also maladaptive in that children who use them are morevulnerable to experiencing an allergic reaction. Opportunities to engagein risky behavior increase as children enter their teens. Teenagers repre-sent a high-risk group for anaphylactic fatalities caused by food allergy,accounting for 53% of a group of UK fatalities (Pumphrey, 2000).

A. Transition points in development for food allergic children

With reference to transition points in development, a negative represen-tation of food allergy can result in psychological distress, which mayaccompany, or follow, transition (or sensitive) points in the developmen-tal pathway, for example, when children learn that they (or their parents)cannot conclusively prevent an allergic reaction.

In the middle childhood years, children must begin to gain autonomyand self-belief in their ability to control events in their lives. Appraisalprocesses outlined above may result in increasing attention being given toprocessing information relating to food allergy, accompanied by emo-tional arousal and consequent increased detection of threat, whethersocial (self-concept) or personal (safety). However, recent experience ofa reaction is not a necessary precondition for this.

Because food allergy places particular limitations on children’s livesand frequently leads to restrictions in a variety of activities, there is thepotential for these perceived illness-induced limitations to generalize todisease- unrelated events as children progress along the developmentpathway. For example, we have evidence of an over-interpretation ofambiguous information in terms of processing of potential threat notdirectly related to food; ‘‘you’d be worried if you were somewherenew. . . that you weren’t before; ‘when other kids see an advert for acircus, they think fun, I think danger.’’

In adolescence we encounter another transition point, when teensencounter unstructured ‘‘novel situations’’ and peers and their automatic


response patterns are challenged resulting, as before, in increased atten-tion to threat. This appears to apply particularly to those with avoidantcoping strategies at the middle to high points of the avoidance/riskcontinuum and has an impact on cognitions, emotions and ultimatelybehavior. Although many teens appear to accept an ongoing conflictbetween personal safety and social self-concept, others resolve this byintensifying previous behaviors, whether minimization/risk or maximi-zation/avoidant, moving them up or down the continuum. Finally,increasing coordination and integration of regulatory systems (e.g., infor-mation processing, and appraisal processes) over the course of develop-ment means that by the time children reach their teens, self-perception,emotional reactions, and cognitive appraisal mechanisms have becomerelatively stable and consistent.

B. Parental views and management of food allergy

Food allergy adds an extra element to the ordinary challenges of parent-ing. There was to considerable overlap between parent and children/teenfocus group findings. Both parents and children spoke of feelings ofanxiety, frustration, anger, difference, embarrassment, and uncertaintyin response to situations stemming from food allergy. In addition, parentsspoke about the difficulty of balancing the needs of their nonallergicchildren against those of the child with food allergy, and negotiating theprecarious line between supporting children’s independence and keepingthem safe. These challenges sometimes led to conflict between parentsand children and between father and mother which, in turn, impacted onthe quality of family life. Furthermore, the sense of huge responsibility,over and above that felt by the parent of a ‘‘normal’’ child, and guilt,means that levels of stress often continue for significant periods of time.

In order to keep their children safe, and cope with their own anxiety,a substantial proportion of mothers, in particular, appear to micro-manage their young children’s lives, which may lead to dependenceand low self-efficacy in some teens and, in contrast, to rebellion andrisky behavior in others. On this point, as in the child/teen transcripts,there appears to be gender differences, with parents reporting thatboys tend to hide their food allergy from others and girls rely largely onparents, and longstanding friends as they enter adolescence, to managesocial situations involving food. There are also gender differencesbetween fathers and mothers, although this difference may pertain tolanguage style and role expectations, in that mothers were more likelyto talk about feelings of anxiety and helplessness.

Parents had different concerns at different developmental points, forexample, when children were young parents keenly felt the burden ofresponsibility in managing risk in their children’s lives, but were less


anxious about children’s safety because of higher perceived control. Par-ents were particularly anxious in the transition period from primary tosecondary school. In primary school, classes are generally smaller andparents are familiar with friends, however, in secondary school childrenencounter new social and peer pressures, and hence an increased risk.Parents worried that children would not carry their ‘‘pens,’’ would takerisks with food in response to peer pressure, or would not inform othersof their food allergy.

XI. DISCUSSION AND IMPLICATIONS FORFUTURE RESEARCH

Because food allergy is only sporadically symptomatic, the anxiety anduncertainty associated with a potential reaction has more profound effectson a child’s everyday life than physiological symptoms; therefore, chil-dren with food allergy may be at increased risk of negative short- andlong-term socioemotional outcomes. Low general awareness encounteredin the social world appears also to contribute to the later development ofpsychological distress and/or to risky behavior. Additionally, variables,such as personality traits and gender are likely to moderate individualimpact. Findings also demonstrate the need for health professionals towork closely with the understandings individuals have about their foodallergy, about ‘compliance’ and about risks associated with the condition.

Studies to date show that many of the issues around food allergyreported by participants and their parents are operative at an emotionallevel. As such, they fall outside the responsibility or indeed the capacity offamily doctors or allergists to address in a direct way, entailing the needfor an educational intervention specific to food allergy. Many existinginterventions in chronic diseases focus only on disease management andinformation and fail to address the wider psychosocial consequences ofliving with chronic disease (Barlow and Ellard, 2004; Gage et al., 2004).Although it is very important that children and their families understandthe condition and its treatment, Gibson and colleagues (2004) maintainthat asthma education limited to information transfer alone is ineffective.Further, research suggests that the goal of an intervention should not onlyto be change the mean level of a particular coping strategy but also tocreate a stable growth dynamic by including issues that are food allergyspecific, as well as age and context specific. Research in food allergysuggests that greater support and clear information is important at timeof diagnosis and at the different transition points along the developmentpathway. Greater emphasis is needed on the social and emotional aspectsof food allergy, on knowing ‘‘what to expect,’’ and on enhancing the self-management skills of children and their families. In addition, public


information on food allergy is needed in general, and in health, education,catering, and retailing, in particular.

Food allergic children appear to experience disease-specific stressorsand use particular coping strategies that evolve in response to age-,gender-, and context-specific stressors. Particular issues revealed by theliterature are summarized below:

� For young children particular issues include: how to feel part of socialoccasions such as birthday parties while being and feeling safe; how tocommunicate with friends, restaurant staff, and other children andadults in novel situations; how to deal with difficult people and situa-tion (e.g., teasing and bullying); how to create a positive self-image thatincludes food allergy.

� For older children and young teens particular issues include: how tobalance peer pressure and positive self-perception while staying safe;how to communicate in novel situations such eating out and makingnew friends; how to manage feelings such as embarrassment, self-consciousness, difference, managing the removal of the parental safetynet, and development of effective self-care.

� For older teens particular issues include: how to deal with going outindependently with friends, starting to drink alcohol, the first indepen-dent holiday, romantic relationships, and attending clubs or discos.

� For parents particular issues include: anxiety; teaching children to beindependent and safe, what to expect as children grow, managing theremoval of the parental safety net and helping children developeffective self-care.

� Gender specific issues include: how to deal with gender roles (e.g., boysdo not seek social support; girls find it difficult to be assertive).

Over the last 20 years, new lifecourse frameworks have been devel-oped which offer a conceptual model for health development and a morepowerful approach to understanding diseases (Halfon and Hochstein,2002). Such models are germane in the field of food allergy sincebiological hypersensitivity to environmental stimuli is a central featureof atopic disorders. Although it is recognized that allergen contact canelicit symptoms at higher and lower dose at different time points, render-ing different thresholds in allergen provocation tests within the sameindividual, there is only a limited understanding of the mechanismsinvolved in the developmental course of food allergies.

A developmental framework has the potential to link formerly dispa-rate concepts such as health-related quality of life, the maturation of theimmune system, and delineate mechanisms linking psychological stress,personality and emotion to neuroimmunoregulation as well as toincreased risk of negative impact. Such a model may be used to explainboth physiological and psychological phenomena, and their interaction,


and consequently provide a shared language as a basis for multidisciplin-ary studies in food allergy.

A growing appreciation of the interactions between behavioral, neu-ral, endocrine, and immune processes have underlined the need for amultidisciplinary approach. For example, it is possible that psychologicaldistress and atopy have overlapping biological contributors, increasingthe likelihood of ‘‘trait’’ anxiety. Mechanisms linking psychological stressand emotion to neuroimnoregulation as well as increased risk of atopyhave been increasingly elucidated (e.g.,Wright, 2005). Stress early in lifecan, for example, result in long-term alterations of the function of theHPA-axis (Helm et al., 2002). In addition, an association between TNF-aand the cognitive affective subscale of the Beck Depression Inventory,which measures depressed mood independent of physical symptoms,demonstrated a negative affect-specific activation of proinflammatorycytokines that may actually promote disease progression (Knonfol andRemick, 2000).

A biopsychosocial framework may reveal new links between physio-logical and psychological systems that, in turn, may provide new insightsto guide future explorations that result in novel clinical or therapeutictreatments that relieve the burden of food allergy. Such a frameworkentails the adoption of methodologies that illuminate pathways in devel-opment such as qualitative methods and structural equation modeling.

One of our aims in ongoing research is to move beyond quantitativereports of HRQL impact to an examination of the underlying mechan-isms. A developmental framework, such as that illustrated (Fig. 3.1), hasthe potential to link formerly disparate concepts such as health-relatedquality of life, the maturation of the immune system, cytokine secretion,to the influence of sex and gender variables, as well as to increased risk ofnegative physiological or psychological impact. Such a model may beused to explain both physiological and psychological phenomena, andtheir interaction, and consequently provide a shared language as a basisfor multidisciplinary studies in food allergy. Longitudinal studies arenecessary when the goal is to investigate cause and effect. At presentEuroprevall is conducting a birth cohort study in food allergy, thatincludes clinical and psychological measures. Such studies may alsolead to novel treatment options in the future.

For clinicians, the early recognition and incorporation of a develop-mental framework into a treatment plan is essential and sets the stagefor an effective medical care and the eventual transition from paediatricto adult care. For health care providers, there is socio-economical interestto support the development of adaptive and active coping strategies inchildren with food allergy as early as possible, targeted at specific transi-tion points, and with age and gender relevant content.


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CHAPTER 4

Advances in Food and NutISSN 1043-4526, DOI: 10.1

Proctor Maple Research Ce

Maple Syrup—Production,Composition, Chemistry,and Sensory Characteristics

Timothy D. Perkins and Abby K. van den Berg


rition R016/S1

nter, U

esearch, Volume 56 # 2009043-4526(08)00604-9 All righ

niversity of Vermont, Harvey Road, Underhill Center, Vermon

Elsts

t, U

II. H
istory 102
III. M
aple Sap Flow 104
IV. S
ap Collection 106
V. S
ap Processing: Evaporation 107
VI. A
nnual Syrup Production 110
VII. S
ap Chemistry 111
A
. T ransformation during storage 115
B
. T ransformations during reverse
osmosis/nanofiltration
116
C
. T ransformations during evaporation by heating 117
VIII. S
cale/sugar Sand Formation During Sap Processing 121
IX. S
yrup Standards 124
X. S
yrup Chemistry 126
A
. D ensity 126
B
. C arbohydrates 126
C
. p H 126
D
. C onductivity 127
E
. C olor 127
F
. R heology 127
G
. In organic composition 128
H
. O rganic acids 131
I. F
lavor compounds 131
J. S
ensory evaluation of flavor 133
evier Inc.reserved.

SA

101

102 Timothy D. Perkins and Abby K. van den Berg

K
. O ff-flavors in maple syrup 134
L
. N utritional aspects of maple syrup 135
XI. O
ther Maple Products 136
XII. C
ontamination 137
XIII. A
dulteration 137
XIV. S
ummary 140
Referen
ces 140
Abstract Maple syrup is made from sap exuded from stems of the genus Acer

during the springtime. Sap is a dilute solution of primarily water and

sucrose, with varying amounts of amino and organic acids and

phenolic substances. When concentrated, usually by heating, a

series of complex reactions produce a wide variety of flavor com-

pounds that vary due to processing and other management factors,

seasonal changes in sap chemistry, and microbial contamination.

Color also forms during thermal evaporation. Flavor and color

together are the primary factors determining maple syrup grade,

and syrup can range from very light-colored and delicate-flavored

to very dark-colored and strong-flavored.

I. INTRODUCTION

Maple syrup is produced from the sap of several species of maple (Acer),chiefly through the concentration of sap via thermal evaporation.Although the chemistry of maple syrup is dominated by sucrose, a widevariety of sap collection and processing factors, microbiological interac-tions in sap, environmental influences, as well as the packing and storageof the finished product, combine to produce a range of chemistry andflavor profiles in maple syrup. Because of the large concentration factor(�40 gal of sap are required to produce 1 gal of syrup) and often delicateflavor profiles involved, several off-flavors are commonly found. Finally,the large price differential between maple syrup and other sweetenersprovides incentive for adulteration.

II. HISTORY

Several different legends describe how Native Americans discovered thatthe sap of maple trees was sweet and could be boiled down to formmaplesugar (Heiligmann et al., 2006). The most likely explanation is that theyobserved birds and animals cutting holes or gashes into the twigs of trees,or drops of sap falling after branch breakage by snow or wind. Thesesmall wounds ooze sap in the spring, forming small drops of sap that are

Maple Syrup—Production, Composition, Chemistry, and Sensory Characteristics 103

concentrated by the sun andwind, or form sweet icicles of sap. The NativeAmericans undoubtedly recognized this and collected sap by cuttingslashes in the trunks of maple trees and used skins, hollowed woodenvessels or clay pots to collect the sap, which was then concentrated byboiling to form a very dark and strong-tasting sugar. At certain times ofthe year, maple sugar could comprise a significant portion of their totalcaloric intake.

Early colonists throughout New England took up the practice ofmaking maple sugar due to the high cost of imported sugars, and becausethe practice occurs at a time of year when other agricultural endeavors arenot possible. Spouts made of hollowed out stem sections of elder or sumactwigs were inserted into holes cut in the trunks of maple trees withchisels. Later, metal spouts were produced and used in holes drilledwith augers. Sap was first collected into hollowed-out tree trunks, thenlater wooden or metal buckets. In most cases, the final product was maplesugar (a solid), and only a relatively small amount of maple syrup (liquid)was produced.

Over the next few hundred years, the practice of tapping trees andcollecting sap changed considerably. While some maple producers con-tinue to use metal spouts and metal buckets to collect sap, plastic spoutsand tubing are now relatively common. Some experimentation withmetal tubing began in the late-1800s and early-1900s, however, the firstsuccessful commercial tubing systems arose in the 1950s and early-1960swith the introduction of plastic (PVC) tubing and associated plastic(Nylon) spouts and fittings to collect and transport sap to a centrallocation, greatly reducing the labor required to collect sap. Initiallytubing systems were run across the ground and vented, but continuedexperimentation and use resulted in tubing lines being suspended,unvented, and a drop line introduced to reduce reabsorption of sap bytrees further down the line in the collection system. Shortly after tubingcame into use, some researchers and producers began attempting toaugment sap yield by applying vacuum to the tubing systems.The early results were encouraging, but maple sap yield was greatlybolstered with the advent of a new generation of tubing composed ofPolyethylene, along with associated changes in spouts and fittings, andincreased use of vacuum pumps designed for maple applications in themid-1990s. The end result is that current high-yield production methodscan achieve sometimes double or more the standard yield from bucketsor gravity (nonvacuum) tubing installations.

The processing of sap into maple syrup has also changed greatly fromcolonial days. Early settlers used a batch method to boil sap in largekettles over open fires. This required a very long time and huge quantitiesof wood to produce a very dark and strong-tasting maple sugar with amoderate to substantial load of impurities. Modern maple evaporators


provide relatively continuous processing of sap, are very energy efficient,and generally produce a much lighter-colored and lighter-flavored maplesyrup fairly quickly.

In addition to changes in collection and processing, the product itselfhas also changed. Presently, most maple is made into and marketed assyrup (liquid), with much smaller sales of maple sugar, cream, or candythan was historically the case.

III. MAPLE SAP FLOW

Under the appropriate conditions, a sweet sap can be collected from mostmaple (Acer) species. In general, however, only the sap of sugar, black,and red maple is commonly used to make maple syrup. Where mapletrees are found in abundance and weather conditions are appropriate,commercial maple production can occur. This ranges from Nova Scotia toMinnesota from east to west, and from southern Ontario and Quebecin the north to areas of West Virginia in the south (Heiligmann et al.,2006). Boxelder is sometimes tapped in areas of Manitoba and thePacific northwest.

The physiological process responsible for sap flow in maple treesprobably results from a combination of physical and osmotic forces(Cirelli et al., 2008; Milburn and O’Mally, 1984; Tyree, 1983). In thephysical model, fluctuations in wood temperature that span the freezingpoint during the leafless period (fall or spring) create alternating negativeand positive pressures within the trunk and branches. When wood tem-perature falls below freezing, the water vapor within the billions of air-filled lumen of fiber cells freezes, forming a frost-like layer on the inside ofthe cell wall. Since the vapor pressure is much lower over ice than liquidwater, this, and to a much lesser degree the contraction of the air bubble,create a vapor pressure gradient, causing water to move apoplastically(along cell walls) into the lumen, where it continues to freeze. Due tostrong cohesion, and the vapor pressure gradient, water is pulled upthrough vessel elements towards the lumen. Eventually, the entirewood (fibers and vessel elements) and sap freezes. Freezing occurs firstin the fine branches in the crown of the tree, then progressively down-ward. The amount of water uptake is dictated by soil water availabilityand the rate of freezing. A slow freeze ensures maximum uptake, whereasin a rapid freeze vessel elements in the stem of the tree may freeze beforewater uptake is complete.

During the warming phase, as the wood increases in temperatureabove the freezing point, the frost layer thaws, and the gas bubbleexpands. Sap pressure at the stem level increases very rapidly to a peakpressure, which is largely caused by gravitational potential, and


somewhat also by gas bubble expansion. This pressure may reach up to40 psi (275 kPa). Over time, the pressure slowly recedes as sap is forcedout of small wounds or into other areas of the tree, until the pressurewithin the tree equals the air pressure outside the tree, at which point flowceases. The flow rate and total yield from tapholes is proportional to treesize and to the pressure gradient, thus sap flows faster earlier in a ‘‘run’’than later. Root pressure is not a significant factor in maple sap exudation.

Recent evidence (Cirelli et al., 2008) clearly demonstrates that there isalso a considerable osmotic component to the development of sap pres-sure in maple due to anatomical barriers to sucrose between the vesselsystem and fibers. Further work is necessary to determine the precisecontribution of physical and osmotic factors on sap exudation.

Maple producers exploit the sap flow phenomenon during the time ofyear when temperatures are expected to fluctuate around the freezingpoint by drilling small holes into the stem, inserting spouts, and collectingthe sap in some fashion. Only trees that have reached a certain diameter(10–12 in. at breast height) are generally tapped. This ensures that the treewill be able to withstand the stress of tapping and regrow sufficient woodduring the growing season to compensate for the loss due to tapping andthe accompanying zone of discoloration (walling off, a normal woundresponse in trees to limit microbial infection). Sap will typically onlyflow for 1–2 months before microbial contamination of the taphole, orthe lack of proper flow conditions (freeze–thaw) cause the flow to cease.In general, each taphole will produce about 10–20 gal of sap duringthe season, depending upon the collection technology employed, theenvironmental conditions during the season, and the size and sap sugarcontent of the tree.

Although sap will flow in both the fall and spring of the year, the vastmajority of maple production occurs in the spring for several reasons. Sapin the spring is sweeter than in the fall, and decreasing temperatures asthe season progresses from fall to winter can cause damage to equipment(split bags and buckets due to frozen sap) and frost-heaving of spouts outof tapholes. Trees should also not be tapped more than once per year.

Sap sugar content is not high at all times of the year. There is a strongseasonal pattern of production, accumulation, and utilization of non-structural carbohydrate forms in maple. Starch, the dominant form ofreserve carbohydrate in sugar maple, tends to be quite low during thephotosynthetic period, and accumulates in the stem and twig woodtowards the end of the growing season. Soluble sugars tend to increaseduring the winter and early spring as a function of temperature(Cortes and Sinclair, 1965). Sucrose is clearly the dominant solublesugar in the xylem, with only minor amounts of glucose and fructose.Still lesser amounts of stachyose, raffinose, and xylose are also present(Wong et al., 2003).


IV. SAP COLLECTION

Sap may be collected into galvanized or aluminum buckets or plasticbags, all of which require periodic (up to several time daily during highflow periods) emptying. When buckets or bags are used, maple producersmust collect the sap into a larger, generally mobile container and bring itto the site where it is processed.

Sap may also be collected with a network of plastic tubing (Fig. 4.1).Collection with tubing generally does not require visiting each tree duringthe season after the intial tapping, as the sap flows through progressivelylarger tubing into a holding tank, often located at the site of sap proces-sing into syrup. When using tubing, proper design, layout, and mainte-nance of the tubing system must be observed in order to maximize sapyield. The general rule for installing a tubing system is ‘‘tight, straight,and downhill.’’ Other factors that affect collection with tubing include thesize of tubing, the number of taps on a lateral line, tubing layout, andseveral other considerations. Sap yields on gravity (buckets, bags, ortubing without vacuum) average about 8–10 gal of sap (�0.20–0.25 galof syrup equivalent) per tap over a season.

Vacuum is used in many modern tubing installations, with a pumpevacuating the tubing system to a level of 20–25 in. mercury (in Hg). Byapplying vacuum to the tubing network, the pressure gradient betweenthe inside of the stem and the ambient air inside the tubing increases,resulting in a much higher sap flow rate and sap yield. This practice does

FIGURE 4.1 Maple tubing system. Spout is inserted into a hole drilled into the tree

(on upper right), a dropline extends downward from the spout, and connects to the

lateral 5/16 in. tubing line. The lateral line runs from tree to tree, and connects to

the mainline (1 in. diameter pipeline in this case) which is suspended on tensioned

steel wire.


not significantly impact sap sugar content, chemistry, or the amount ofinternal tree damage (Wilmot et al., 2007a,b). The primary purpose is toaugment sap yield, and to encourage sap flow during time periods whenflows might otherwise be marginal. Sap yields on a well-designed andoperated vacuum tubing system can reach 25 gal per tap (0.6 gal of syrupequivalent) each season.

Tapholes are drilled into tree trunks at a height of 3–7 in. above the soilwithin an area that is free of visible damage or older tapholes (whichbecome progressively harder to see as the tree grows). Tapholes arenormally 7/16 in. in diameter, but 5/16 in. diameter spouts introducedin the mid-1990s produce similar yields under vacuum with less treedamage (Wilmot et al., 2007a) are gaining widespread acceptance. Tap-holes are drilled to�1.5–2.5 in. deep into the wood at a slight uphill angleto allow sap to naturally flow out of the taphole. A metal spout (if usingbuckets or bags) or a plastic spout is placed in the hole and lightlyhammered in place. Spouts are generally removed at the end of themaple sugaring season to allow the tree to heal.

Several attempts have been made to increase the length of time sapflows from the taphole. Paraformaldehyde (PFA) was used for a fewdecades as a microbicide to reduce taphole ‘‘drying.’’ PFA use wasphased out when it was determined to be harmful to the tree, and theuse of any microbicide is currently illegal in all maple production areas.More recently, a denatured ethanol has been promoted to ‘‘sterilize’’tapholes in some areas, but the lack of any lasting impact on microbialpopulations has limited its effectiveness in producing higher sap yields;in fact, some research has shown the practice to significantly reduce sapflow from such ‘‘sterilized’’ tapholes. There is considerable ongoingresearch into how to maximize sap yield in maple production, particularlyin vacuum tubing operations.

Because sap is a perishable product, it is generally processed relativelysoon after collection to minimize microbial contamination and the accom-panying reduction in syrup quality. Sap is often filtered and UV-sterilizedafter collection to reduce microbial loads in order to maintain sap quality.Ozone treatment, although useful in water treatment, does not appear tobe effective in maple applications, presumably due to the strong protec-tive effect of sugars on microbial populations (Labbe et al., 2001).

V. SAP PROCESSING: EVAPORATION

After collection, maple sap must be transformed through some means ofconcentration into maple syrup. The two major processes utilized areevaporation by heating and reverse osmosis followed by heating. Themodern maple evaporator (Fig. 4.2) is typically composed of several

12

3

4

5

6

Sap inlet

Syrup drawoff

Floatbox

FIGURE 4.2 A contemporary maple syrup evaporator. Sap follows a winding path from

the sap inlet, sequentially through the pan sections to the syrup drawoff.


parts: a heat source, an arch to contain and concentrate the heat, and pans,which contain the liquid and allow it to become concentrated.

Most evaporators utilize wood or oil as fuel, and commercial evapora-tors (as opposed to hobby-sized units) are insulated and efficient.Although wood was a common fuel source historically, it is being largelysupplanted by oil due to the high cost of labor and convenience. Oil iseasy to move around (as opposed to wood) and is easy to control (nearlyinstant on and off, with high and low settings). Most commercialevaporators are oil-fired, although some very large operations use highpressure steam to achieve a very rapid processing rate and to avoidscorching of evaporator pans.

The arch is made of cast iron or steel and sits under the pans to containthe heat. Oil-fired units have the burner located at the front, slantingupward, and with the fire pointing towards the rear (stack). The insideof an oil-fired arch is lined with ceramic blanket insulation. Wood-fired


units are usually lined with fire-brick and contain metal grates, some-times with forced ventilation. A wood-fired evaporator has doors in thefront through which wood is added. The hot gases flow back towards therear of the unit and stack. Pans, normally composed of stainless steel, siton top of the arch. Today, nearly all evaporator pans are Tungsten InertGas (TIG) or Metal Inert Gas (MIG) welded, and conform to widespreadfood industry construction practices. Formerly, most pans were soldered,and constructed of stainless steel, although sometimes older units werecomposed of English tin, or less frequently, copper. Prior to 1994, mostsolder contained lead; after 1994 lead-free silver solder (tin-silver) wasused for pan fabrication. Considerable attention has been recently focusedon developing and promulgating standards for maple equipmentmanufacturing (LMEA, 2001).

Most evaporators have two distinct types of pans, the back pan (alsocalled the flue pan or sap pan) and the front pan (or syrup pan) (Figs. 4.2and 4.3). The back pan, where themajority of the water is evaporated fromthe sap, has deep flues that are designed to maximize heat exchange andevaporation. Back pans come in two configurations, drop-flue and raised-flue, based upon whether the flues in the pan extend above or are evenwith the position of the pan on the arch rails. Neither type is clearlydominant in the industry. Sap enters into the back pan; generally viagravity feed through a pipe connected to the sap storage tank and isregulated by a mechanical float or an electronic valve. The back pan isinternally divided into two or more sections which result in a semichan-neled flow from the sap inlet to an outlet near the front pan.

Frontpan section

66–67%

62% 47% 29% 19%

11%

15%

Sap inlet2%

Exhaust flue

Syrupdrawoff

Oil burner

Backpan section

FIGURE 4.3 Schematic diagram of a maple evaporator. Numbers and shading density

show the density (�Bx) of liquid within different partitions of the evaporator during

evaporation. Sap enters the back pan section of the evaporator at the lower right and

flows through a feed-pipe into the partition at the upper right. It then flows around the

back, and then forward. Two front pans contain two partitions each. Sap flows into

the front pan section through a pipe connecting the back pan to the front pan at the lower

middle. Sap/syrup then flows through each front pan partition, increasing in density and

developing color and flavor during the process. Syrup exits the evaporator at the lower

left via an automatic drawoff, which openswhen the density of syrup close to the drawoff

area is correct, then closes again when the density drops below that of finished syrup.


Sap flows from the back pan into the front pan, sometimes usinganother float or valve to regulate the entry of sap. The bottom of thefront pan is flat, and the front ‘‘pan’’ may actually be made up of one ormore pans, with each separate pan itself divided into several partitions.Front pans are made in one of two configurations: those in which the sapruns parallel to the main axis of the evaporator, termed reverse-flowevaporators, and pans in which the sap flows from side-to-side, termedcross-flow evaporators. In both cases, syrup flows out the last partition ofthe front pan via a manual or automatic draw-off valve into a pail ordirectly into a pipeline feeding a filtration system.

Modern maple evaporators are designed to have a relatively continu-ous or semicontinuous flow. In theory, an evaporator can be envisioned asa long continuous stainless steel ‘‘gutter,’’ with heat applied under theentire surface. Sap flows in one end of the gutter (the back pan), and syrupflows out of the other (draw-off of the front pan). Because it is impracticalto have an evaporator that is tens of feet long, the gutter is bent intosections to reduce the overall size, and to allow a single heat source to belocated under the evaporator pans. Commercial evaporators arecommonly sized to fit the number of taps in the sugaring operation, andrange in size from 3 ft.� 10 ft. (width� length) up to 6 ft.� 18 ft., with theback pan section typically occupying about two-thirds of the overallevaporator surface area. The depth of sap in the pans is generally keptquite low, only 1.5–2.0 in., to maximize boiling rate.

Ancillary equipment is oftenused in conjunctionwith evaporators.Hoodsare employed to channel steam out of the processing facility (termed a sugarhouse, sugar shack, maple house, etc.). Several types of devices are manu-factured which sit over the evaporator pans to take advantage of the steamenergy to preheat or preconcentrate the sap before it enters the back pan.

Often syrup is drawn off at a density slightly lower than of finishedmaple syrup, and is brought to final density in a separate finishing pan.Finished syrup is filtered to remove solids and generate a clear product.Filtration through a wool or synthetic cone filter is sometimes used insmaller operations; most modern commercial production scale operationsemploy a pressure filter utilizing diatomaceous earth as the filteringmedia. Bulk syrup is hot-packed in 30–50 gal drums (stainless or galvanizedsteel, epoxy lined steel, or plastic) for storage before being reheated andpackaged into retail containers as needed.

VI. ANNUAL SYRUP PRODUCTION

The yearly worldwide production of maple syrup is roughly 8–9 milliongal (�45,000 metric tons), of which�15% is produced in the United States,and 85% produced in Canada. The New England/New York region


cumulatively produce about 75% of the total domestic U.S. crop. Quebecis the largest Canadian producer, with about 90% of the total Canadianproduction originating there. The largest markets of maple are in the U.S.,Canada, Europe, and, increasingly, Asia.

VII. SAP CHEMISTRY

Maple sap is a dilute solution of mainly water and sugar, along with traceamounts of other substances, including organic acids, free amino acids,protein, minerals, and phenolic compounds. Although the proportionsare somewhat variable, sap is normally composed of 95–99% water and1–5% sugar.

The primary sugar found in uncontaminated sap is sucrose, whichranges from �96–99% of the total sugar present (Table 4.1). As microor-ganisms contaminate sap, particularly later in the sap flow season, invert(reducing) sugar levels increase, reaching up to about 0.20–0.25% of thetotal sugar concentration in sap (Dumont, 1994; Heiligmann et al., 2006).At times, several other sugar forms, including mono-, di-, tri-, and higheroligosaccharides, may also be found in maple sap (Dumont, 1994; Haqand Adams, 1961; Stinson et al., 1967), although typically in very lowconcentrations.

Sap pH ranges from 3.9 to 7.9, but in most cases is only slightly acidic,with a typical range of 6.5–7.0. There is a slight trend for sap to becomemore acidic as the sap flow season progresses (probably due to microbialaction). Conductivity normally ranges from 320 to 520 mS/cm.

The total concentration of sugars in sap varies due to several factors.Variation between individual trees may be quite large due to differencesin genetics, growth rate, and crown density. However, the ranking of anyone tree relative to its neighbors tends to remain relatively constant bothwithin a season and from season to season (Taylor, 1956).

TABLE 4.1 Sugar composition (% dry weight) of the

solid fraction of maple sap (from Perkins et al., 2006,

used with permission)

Sucrose 96–99

Polysaccharides nd— 0.5

Oligosaccharides nd—0.02

Glucose nd—0.17Fructose nd—0.10

Quebrachitol nd—0.15

Unidentified nd—0.67


Sap sugar content within an individual tree may vary from one seasonto another to a relatively high degree, probably as a result of photosyn-thetic carbohydrate gain in the prior growing season. Sap sugar contentalso varies within a season, typically decreasing throughout the season.

Although at one time, it was believed that the use of tubing systemswith vacuummight result in a dilution of sap sugar content and increasedinternal wounding in trees, recent research has shown that this is not thecase.Wilmot et al. (2007b) demonstrated that sap collected at high vacuumlevels (>18 in. Hg) did not result in diluted sugar content, did not containsignificantly different levels of minerals, and did not cause larger zones ofdiscoloration in maple stems.

The average sugar content of a forest stand utilized for maple produc-tion has a large impact on the economics of maple production, as asugarbush containing a higher level of sugar in the sap will requireconsiderably less energy to produce 1 gal of syrup compared to a sugar-bush with a lower sugar content. Maple producers can increase sap sugarcontent by selecting individual trees with higher sap sugar content duringthinning, and by encouraging good crown and stem growth throughcrop tree management techniques. Fertilization of trees, although not acommon practice in maple operations for a number of reasons, can be usedto correct nutritional deficiencies and stimulate growth (Perkins et al.,2004a), and may increase sugar yield from a site (Perkins et al., 2004b).

The inorganic composition of maple sap is highly variable (Table 4.2).Potassium and calcium make up the bulk of the inorganic fraction of sap,with substantial amounts of magnesium and manganese, and only traceamounts of sulfur, phosphorus, zinc, and copper, followed by aluminum,sodium, boron, and iron. In contrast to what is observed in sugar concen-tration, most of these elements show a slight or strong tendency toincrease in concentration during the sap flow season (Marvin andGreene, 1959). Lead is generally below detection limits unless contami-nated by lead-containing equipment.

The gases expressed from maple stems during sap exudation showhigher carbon dioxide and correspondingly lower oxygen concentrationscompared to ambient air, indicating substantial respiratory activity inmaple wood during the leafless period of late-winter to early-spring(Marvin and Greene, 1959).

A wide range of free amino acids are found in sterile maple sap(Heiligmann et al., 2006), including glycine, alanine, asparagines, threo-nine, leucine, isoleucine, valine, and methionine. Morselli and Whalen(1986) examined the change in the distribution of various amino acidsover two maple sap seasons. Their results indicated that initially, only asmall number (6–7) of amino acids were found in sap, all in relatively lowconcentration. As the season progressed, the diversity of amino acidsincreased to 12–15. In addition, the concentration of amino acids present

TABLE 4.2 Inorganic (minerals and metals) composition of maple sap

Element

Rangea

(ppm) Rangeb (ppm)

Meanb

(ppm)

Meanc

(ppm)

Potassium 27–95 50–81 65 25

Calcium 21–77 35–75 50 40

Magnesium 2.6–9.0 3.9–8.1 5.6 3

Manganese 2.7–9.7 1.7–5.5 3.5

Sulfur 0.18–1.80 0.77Phosphorus 2.5–8.0 0.2–1.1 0.65

Zinc 0.24–1.47 0.55

Copper 0.08–1.56 0.50

Aluminum 0.04–0.20 0.10

Sodium nd—4.0 0.02–0.17 0.08

Boron 0.02–0.16 0.08

Iron nd—3.7 0.01–0.07 0.04

Lead nd nd

a Marvin and Greene (1959).b van den Berg and Perkins (unpublished).c Dumont (1994).


in late season sap may rise to over 10 ppm (Dumont, 1994), probablyreflecting the breaking of winter dormancy and an acceleration of meta-bolic activity in the trees. These changes could have a significant impacton the characteristics of syrup produced, as the nitrogen content of sap,primarily amino-N (Pollard and Sproston, 1954), has a large influence onthe development of flavor and off-flavor compounds in maple syrup.

Uncontaminatedmaple sap has the same appearance as water in that itis nearly colorless, with very high light transmission through the visiblerange (Fig. 4.4). Some absorption is found in sections of the UV and nearinfrared (NIR) ranges.

Several organic acids are also found in maple sap (Table 4.3). In general,the total quantity of organic acids starts out low, and rises throughout thesap flow season. Malic acid (concentration 800–45,000 ppb) is by far themost common organic acid, ranging from just over 50–99% of the total acidpresent. Succinic acid and oxalic acid are also fairly dominant forms. Otheracids occur sporadically in low concentration (Dumont, 1994; Mollica andMorselli, 1984).

A wide range of phenolic compounds, varying in type and concentra-tion, can also be found in maple sap (Dumont, 1994). Most of these appearto be derived from lignin (Kermasha et al., 1995). They range in concen-tration in sap up to 0.1 ppm. The most dominant phenols present tend to

100

90

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40

30

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10

190

240

290

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390

440

490

Wavelength (nm)

Ligh

t tr

ansm

ittan

ce (

%)

540

590

640

690

740

790

840

890

940

990

1040

1090

0

FIGURE 4.4 Transmission profile of maple sap at 2 �Bx from 190–1100 nm. Sap was

collected on March 29, 2006, then stored frozen until analyzed.

TABLE 4.3 Organic acids in maple sap (Dumont, 1994).

Organic Acid Mean (ppb)

Malic 14,940

Fumaric 1677

Succinic 910

Oxalic 380

Aconitic 193

Citric 109

Tartaric 59

Total 18,033


be: sinapic acid, coumaric acid, syringaldehyde, and coniferaldehyde,with lesser amounts of vanillic acid, syringic acid, homovanillic acid,and ferulic acid (Table 4.4). No clear temporal trends in total phenol levelsare apparent (Dumont, 1994), although some work does suggest thatvariation in vanillin glycoside concentrations are correlated with seasonalchanges during the sap flow period (Belford et al., 2006). A variety offlavonoids, including catechin, flavanols, and dihydroflavonols, are alsofound in maple sap (Deslauriers, 2000). Some of these exhibit strongseasonal tendencies.

TABLE 4.4 Phenolic compounds in maple sap (after Dumont, 1994)

Compound Mean (ng/ml)a

Sinapic acid 31.8

Coumaric acid 15.0

Syringaldehyde 14.9Coniferaldehyde 13.4

Vanillin 9.7

Vanillic acid 9.5

Homovanillic acid 8.4

Syringic acid 3.9

Ferulic acid 2.4

Coniferol 1.6

Total 110.7

a Normalized to 1 �Bx.


Many of these compounds are likely to be flavor precursors, althoughthe influence of each on the final flavor profile of the resulting maplesyrup is greatly affected by storage, as well as the type and length ofprocessing. Because there are a number of transformations during proces-sing, and many of these compounds are volatile when heated, theconcentration is not necessarily increased in maple syrup.

There is an increasing interest in the quantity and composition ofphenols in maple sap and syrup, due to the antioxidant, antiradical, andantimutagenic activities of these compounds (Theriault et al., 2006).

A. Transformation during storage

While in the xylem, sap generally is considered to be sterile. Immediatelyupon being exposed to taphole conditions, it is acted upon by a widevariety of microorganisms, including bacteria, fungi, and yeasts. Themajor impact of this contamination on the syrup making process is theconversion of a small quantity of sucrose by invertase.

Because it is colder and all the equipment is clean, sap collected earlyin the maple sap flow season tends to be very low in microbial load, andthus low in invert sugar concentration. As the season progresses, dailytemperature tends to increase. This results in the sap collecting systembecoming colonized with microorganisms, increasing the invert level ofthe sap. Some producers use intermediate season tubing washes or rinsesto reduce microbial contamination, although the efficacy of mid-seasoncleaning is most likely relatively minor and short-lived.


Proper filtering of sap, cool storage before processing, and rapidprocessing are commonly used to reduce microbial growth in sap.Growth of microorganisms may slightly or significantly alter the form ofseveral elements in sap, especially by causing the forms of nitrogen andphosphorus to be changed, by increasing the protein component in sap,and by altering the turbidity of sap. Sap that has been extensively colo-nized by yeasts may undergo fermentation. Extremely high levels ofmicroorganisms, especially at the end of the season, may cause syrup tobecome ‘‘ropey,’’ a sticky, stringy, gelatinous texture that is almost impos-sible to remove, thus rendering it essentially unmarketable.

B. Transformations during reverse osmosis/nanofiltration

Due to the high cost of fuel, and to reduce the time required to process sapinto maple syrup, an increasing number of maple producers are usingreverse osmosis/nanofiltration (hereafter collectively termed RO) toincrease the sugar concentration of sap prior to boiling. Sap processedvia RO is called ‘‘concentrate,’’ and tends to be slightly yellow in color.Although early units were adapted from water desalination units,commercial RO machines specialized for the maple industry are nowavailable from a variety of manufacturers. There is also a strong trendtowards increasing levels of concentration of sap with RO. In the past,most maple producers were content to concentrate sap to 8 �Bx.Currently, many producers are striving to concentrate to the highestdegree possible (�25 �Bx). The major effect of preconcentration of sapvia RO is the removal of water. Going from 2 to 8 �Bx achieves a 75%reduction in the amount of evaporation necessary (from about 43 gal ofsap to about 11 gal to produce a gallon of maple syrup). A furtherconcentration of sap to 16 �Bx would require only 5.5 gal of sap toproduce 1 gal of maple syrup (Fig. 4.5).

Concentrate is extremely perishable. Most maple operations utilize ROmachines that are capable of providing only slightly more concentrate perhour than can be utilized by the evaporator. In practice, most producerswill concentrate a small amount of sap, start the evaporator, and continueto run the RO during boiling so that the concentrate is not allowed tobuild up and spoil.

The by-product of sap concentration by an RO, permeate, is also usedin maple operations as a source of very clean water. Due to the lowmineral concentration of permeate water, it is used for cleaning tubingand evaporator equipment, as well as the RO membrane itself, whichshould be run through a wash and rinse cycle after each use. Thechemicals and dosage to be used are specified by the membrane and ROmanufacturers, and should be carefully followed to avoid damaging themembrane or contaminating the sap concentrate.

Density (�brix)

00

5

10

15

20

25

30

35

40

45

50

55

60Sap SyrupConcentrate

5 10 15 20 25 30 35 40 45 50 55 60 65 70

Sap

(ga

llons

)

FIGURE 4.5 The relationship between the density and the number of gallons of sap

required to produce 1 gal of maple syrup. The normal range of raw sap, sap concentrated

by reverse osmosis (concentrate), and finished syrup are shown.


Obviously, the use of RO has tremendous consequences to the amountof evaporation, and thus heating time necessary to make syrup. Becausemuch of the color and flavor development occur during the heating phaseof processing, RO use should strongly affect the resultant color and flavorof maple syrup produced, although there has been little research clearlydemonstrating the effects. Ongoing efforts (van den Berg and Perkins,unpublished data) do appear to indicate some significant changes insyrup compositional attributes.

Depending upon the membrane and RO used, the chemistry of theconcentrated sapmay be somewhat different thanwhatmight be expectedby a simple concentration factor alone. Some membranes sacrifice a slightamount of sugar and mineral passage for high flow rates, while othersprovide very high sugar retention, with correspondingly low flow rates.Although it has not been extensively investigated, limited research hasdetermined that phenols are concentrated, but aldehydes and alcohols insap are reduced during RO use (Kermasha et al., 1995). Prefilters andmembranes may also serve as a source of microbial contamination of sap.

C. Transformations during evaporation by heating

Although the single greatest influence of the process of transforming sapinto syrup is the removal of water, the effect on the chemistry and flavor isnot simply due to concentration. Rather, a number of complex reactions


are involved which result in the chemistry and flavor profile of maplesyrup. Given the huge number of permutations in storage, processingconditions and rates, and temporal variations in sap chemistry, onlygeneralizations are possible.

The first and most obvious transformation that occurs during evapo-ration is the change in sugar content (Fig. 4.6). Sap enters the process at anaverage of around 2 �Bx, and the finished product, maple syrup is around66–67 �Bx (Figs. 4.3 and 4.5). The process inside the evaporator is theoreti-cally one in which a semicontinuous gradient of sugar is formed. Initially,as an evaporator is first started with sap only, the inflow of sap into theback pan will gradually cause a gradient to form. This gradient eventuallyperpetuates throughout the entire evaporator, with a density near that ofsyrup near the draw-off, and near that of sap at the inlet of the back pan.In reality, the evaporator is more or less a series of interconnected pans,each with their own sugar content, but certainly influenced by the sugarconcentration in the pan immediately before and after it. The semicontin-uous flow of sap in, and regular draw-off of syrup, maintains the sugargradient within the entire evaporator system. Sometimes, after a shortperiod of boiling, maple producers will ‘‘sweeten’’ the partition nearestthe draw-off with syrup to hasten the development of a proper gradient,although this is not necessary for the gradient to form.

As the density gradient along the evaporator develops, a concomitantincrease in boiling temperature also is found (Isselhardt et al., 2007). Rawsap boils at 212 �F, and syrup at 66.5 �Bx boils at 219.3 �F (at standardatmospheric pressure). Most producers use a hydrometer, calibrated inBrix or Baume and corrected for temperature, to determine the finishedsyrup density.

The second most obvious transformation during heating is a change incolor (Fig. 4.6). As sap progresses through the evaporator, it darkens dueto nonenzymatic browning, a complex suite of chemical changes arisingfrom nonenzymatic activity acting on sugar solutions. The longer the timeand intensity of heating, the greater the effect on color and flavor devel-opment (Willits et al., 1952). These same processes are also intimatelyinvolved in flavor formation. The rate of nonenzymatic browning reac-tions varies greatly depending upon sap chemistry (particularly the invertsugar and amino acid concentration) and on processing rate and condi-tions. In general, syrups that are low in invert sugar concentration (typicalearly season sap) produce light-colored and light-flavored maple syrup,whereas those that have high invert levels produce darker colored andstronger tasting syrups due to increased substrate availability for nonen-zymatic browning reactions (Naghski and Willits, 1957).

Maple producers attempt to control the color and flavor profiles ofthe syrup they produce primarily though rapid processing of sap.

0

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ar c

onte

nt (

�Bx)

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duct

ivity

(µS

·cm

)

B

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C

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Sap ta

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nk

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an 1

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pan

3

Front

pan

4

Syrup

Tem

pera

ture

(�F

)A

40 �F

10

0

20

30

40

50

60

70

80

90

100

Ligh

t tra

nsm

ittan

ce (

%)

E

FIGURE 4.6 Changes in temperature (A), sugar content (B), pH (C), conductivity

(D) and light transmittance (E) at different stages of thermal processing from maple

sap to syrup.


Light-colored syrups are generally more difficult to produce, and are veryuseful in blending of maple syrup to achieve a good color and flavorbalance. Lighter syrups therefore generally command a higher price thandarker syrups.

During heating, sap pH initially increases rapidly as the solutionbecomes more concentrated, generally transitioning from neutral orslightly acidic to slightly to moderately alkaline (Fig. 4.6). Akochi et al.(1997) demonstrated that this change is most likely the result of chemicalreactions occurring during the heating process rather than the loss of


organic acids. By the time sap reaches a concentration of 8–12 �Bx, sap pHbegins to decline and continues to steadily drop until reaching the pointwhen it is the density of syrup. During this time, hexoses undergo alka-line degradation to form triose sugars, which readily decompose duringheating to produce color bodies. Alkaline degradation and color forma-tion do not occur exactly simultaneously, as the conversion of hexoses totrioses appears to occur early in the boiling process, but most of the colorformation occurs in the latter stages of heating, primarily in the last fewpartitions of the evaporator. Cyclotene, furaneol, isomaltol, and otherthermal sugar degradation products are probably formed at this stage ofprocessing (Potter and Fagerson, 1992).

A large number of lignin-derived flavors have been identified inmaple syrup (Filipic et al., 1969; Potter and Fagerson, 1992). Duringboiling, there are large increases in phenol-related flavor compoundssuch as furaldehydes, vanillin, and syringyl aldehyde (Kermasha et al.,1995). Furfural and hydroxymethylfurfural color precursors form as aresult of carmelization and Maillard reactions between amino acids andreducing sugars, as well as via oxidative polymerization of phenoliccompounds. These are further reduced to caramels and melanoidins,and eventually to colored polymer bodies. Due to the variable concentra-tion of precursors in sap, the range of color and of flavor compounds inmaple syrup is very broad. Like the case for sap, there is a tendency forsyrup to decrease in pH during the production season.

Because one of the dominant color formation pathways involves thereactions among amino acids and reducing sugars, controlling invertsugar levels has historically been the key to producing light maplesyrup. RO usage and highly efficient evaporators that result in rapidprocessing and low sap residence time can also affect color and flavordevelopment. The recent innovation of injecting air through small pipesinto boiling sap has proven to also produce light-colored syrup. Recentwork has shown that significant improvement in syrup light transmissioncan result from air injection (Fig. 4.7, van den Berg et al., 2009a), with fewother significant alterations to bulk syrup chemistry. The way in which airinjection achieves this is still somewhat unclear. Air injection does resultin an overall lowering of temperature of the boiling sap (Isselhardt et al.,2007; UVM Proctor Maple Research Center, unpublished data), suggest-ing the lightening effect may be due simply to reduced thermal-inducedbrowning. Research at Centre Acer in Quebec, Canada, has shown that airinjection may increase the formation of oxidative species which affectcolor and flavor formation during evaporation. These projects areexpected to elucidate in more detail the effects of air injection and othernew maple processing technologies on the chemistry and flavor profilesof maple syrup.

Control

Air injection

3/15 3/25 3/29 3/31 4/3 4/5 4/7

FIGURE 4.7 Maple syrup produced in paired evaporators boiling sap from the same

source on the same dates. The evaporator that produced the syrup on the top was the

standard, control evaporator. The syrup on the bottom was produced in an identical

evaporator equipped with air injection.


VIII. SCALE/SUGAR SAND FORMATION DURINGSAP PROCESSING

As the sap is boiled in an evaporator, the concentration of other sub-stances also changes. Dissolved minerals and metals go through a satura-tion phase, and eventually precipitate as a scale-like substance on thesurfaces of the evaporator. This scale, also termed ‘‘niter’’ or ‘‘sugarsand,’’ can take many forms, and is quite variable in composition(Table 4.5, Fig. 4.8). The type and amount of scale changes throughoutthe season, and is highly variable from one season to the next.

Nearest the sap inlet of the back pan it is composed of a protein-rich,sticky substrate that is rich in calcium malate and is probably largelycaused by the denaturing of organic substances and microbes.

In general, sugar sand is amixture of calciummalate and sugar. Gener-ally, the higher the levels of calcium and malic acid present in the sap, thegreater the amount of scale formation (Davis et al., 1963). Further into theevaporator the scale deposited is denser and adheres strongly to pansurfaces. In the front pans, as the density of the solution becomes quitehigh, scale forms very rapidly on evaporator surfaces, and small particlesof scale become suspended in the syrup. This suspended material, alsooften referred to as ‘‘niter’’ or ‘‘sugar sand,’’ must be filtered from syrupprior to packing in drums or into retail containers, as it will impart a grittytexture to syrup, and can cause an undesirable off-flavor in the syrup.

TABLE 4.5 Composition of maple sugar sand (from Perkins et al.,

2006, used with permission)

Sugar sand (in run) 0.05–1.42% dw

pH 6.30–7.20

Calcium 0.61–10.91%

Potassium 0.146–0.380%

Magnesium 0.011–0.190%

Manganese 0.06–0.29%Phosphorus 0.03–1.18%

Iron 38–1,250 ppm

Copper 7–143 ppm

Boron 3.4–23 ppm

Molybdenum 0.17–2.46 ppm

Free Acid 0.07–0.37%

Total malic acid 0.76–38.87%

Acids other than malic 0.08–2.62%Undetermined material 6.94–34.16%

Calcium malate 1.30–49.41%

Sugars in dried samples 33.90–85.74%

Sugar sand in dried samples 14.26–66.09%

FIGURE 4.8 Variation in maple scale (sugar sand/nitre) appearance and form.



Scale on evaporator surfaces, especially in the front pans, is a nui-sance to the maple producer. If allowed to build up excessively, itreduces heat transfer to the liquid, can cause off-flavors, and may resultin scorching of the pans. Maple sugar makers deal with scale accumula-tion in their evaporators in several ways. The most apparent is to shutdown the process, drain the partially processed sap, and clean the pans.Often a food-approved acid solution is used to hasten the process (greatcare must be taken in handling and disposal of acids and in ensuringthat all acid residues are adequately rinsed from the pans prior to usingthem again). An alternative process is used with reverse-flow pans inwhich the flow of sap in the front pans is changed by switching thelocation of the draw off. By alternating sides on which syrup is removed,the incoming partially processed sap will redissolve a portion of thescale from the pans, and thus delay the necessity to shut down andclean the pans. Producers using cross-flow pans will often remove thepan nearest the draw-off, move the second front pan forward, and inserta spare clean pan in that position, thereby reducing the amount of timein which the evaporator is shut down. When boiling sap concentrate(8–20 �Bx), producers obviously experience much more rapid build-up ofscale in evaporators than when using sap (2 �Bx), and the scale can alsobegin to form further back in the evaporator system. Given the increas-ing quantity of syrup being produced using reverse osmosis, thisproblem (and the use of acid to clean pans) is increasing rapidly.Research is ongoing in several locations to better characterize the scale,and to find easier and more environmentally safe methods to deal withthe problem. One recent interesting approach is the use of electrodialysisto demineralize sap prior to evaporation by heating (Bazinet et al., 2007).Although the process did reduce calcium and malic acid levels inconcentrated sap, thus presumably reducing scale formation, it may beconsidered illegal under most existing maple purity laws.

A similar issue in evaporator pans is foam. Foam develops in allportions of evaporator pans during boiling, and must be controlled tomaximize heating and to prevent scorching of pans. Historically, milk,cream, or animal fats were used to cut the foam. Present practice is to usevegetable oils or other commercially available defoamers. The primarydeterminant of which defoamer to use is often based upon whether or notthe maple producer is ‘‘organic’’ certified, as certification requires the useof certified organic vegetable oils. Although there has been relatively littlescientific study of the composition of foam, an analysis of foam skim-mings has shown foam to be a sink for lead (Stilwell and Musante, 1996),and occasionally foam has been suggested as a source of off-flavors inmaple syrup.


IX. SYRUP STANDARDS

Pure maple syrup generally must meet strict standards for density,clarity, color, and flavor. In general, there is agreement between thevarious grades of maple syrup produced in the U.S. and Canada;however, the specific names may vary somewhat (Table 4.6). Some U.S.states have their own grade and color designations. A complete descrip-tion of maple syrup grade and color descriptors is available in the NorthAmerican Maple Syrup Producers Manual (Heiligmann et al., 2006,p. 172). The Canadian standards are mandatory, whereas U.S.D.A.standards are voluntary.

The minimum solids content to meet U.S.D.A and Canadian regula-tions is 66% total solids (at 20 �C in Canada, unspecified in the U.S.Standards). Individual states in the U.S. are able to set their own stan-dards. Vermont andNewHampshire set a minimum of 66.9 �Bx (at 60 �F).Some jurisdictions set an upper density limit (typically 68.9% solids),whereas others do not. Density is generally measured with a hydrometer,hydrotherm, or, increasingly with refractometers, although regulationsoften specify the ‘‘legal’’ method in each area.

In all cases, syrup must be clear of suspended crystals or particulatesthat might cause cloudiness and impart a gritty texture to the product.

Syrup color is determined by spectrophotometric light transmittanceat 560 nm (Fig. 4.9). Syrup must meet (or exceed) a certain cut-off level in

TABLE 4.6 Grades of maple syrup in Canada and the U.S.

Light

transmittancea

Canada U.S.

�66.0% Solids �66.0% Solids

Grade Color Grade Color

�75.0% Canada

No. 1

Extra

light (AA)

U.S. Grade A Light

amber

60.5–74.9% Canada

No. 1

Light (A) U.S. Grade A Medium

amber44.0–60.4% Canada

No. 1

Medium (B) U.S. Grade A Dark

amber

27.0–43.9% Canada

No. 2

Amber (B) U.S. Grade

B for

reprocessing

<27.0% Canada

No. 3

Dark (C) Substandard

a U.S. Standards are based upon the transmittance of permanent glass standards with photometricequivalents. Canadian Standards are based upon photometric light transmission at 560 nm. Some statesmay have different names for the grade classes or slightly different density requirements.


order to be placed within a grade. In practice, producers generally use avisual comparator based upon ‘‘permanent’’ glass or plastic filters, ormore commonly, with a temporary standard composed of burnt sugarsuspended in glycerol. Such temporary grading kits are useful as guides,but care should be taken in their use. At least one company has recentlyintroduced a digital maple syrup transmittance meter, however, forseveral reasons such devices are not currently in widespread use withinthe industry.

Syrup is also graded based upon its flavor. Although this is somewhatsubjective, experienced producers are able to distinguish the generalcategory and intensity of maple flavors, as well as detect gross off-flavorsthat cause syrup to be down-graded to a commercial or substandard level.

Because of maple purity laws in both the U.S. and Canada, there arerelatively few processing techniques which can be employed by mapleproducers. In general, processes may neither add, nor remove substancesfrom the product, other than what is accomplished through evaporation.Certain exceptions are allowed, such as the use of diatomaceous earth forfiltering, the use of reverse osmosis machines, and various defoamingagents. Addition of other sugars, decolorizing agents (activated carbon,anion exchange decolorizing resins, bone char, flavor modifying agents,pHmodifiers), preservatives, flavorings, colorants, and other foodadditivesare not allowed.

100%

90%

80%

Grade

A lig

ht am

ber

Medium

ambe

r

Dark a

mber

Substa

ndar

d/com

mercia

l

Grade

B

70%

60%

50%

40%

Ligh

t tra

nsm

ittan

ce

30%

20%

10%

0%190 290 390 490 590

Wavelength (nm)690 790 890 990 1090

FIGURE 4.9 Transmission profiles of maple syrup. Profile for each grade was derived

from 10–40 scans of individual syrups. Grades delineations are based upon U.S.D.A.

standards. Color grade is determined by the transmittance of light at 560 nm.


X. SYRUP CHEMISTRY

A. Density

Maple syrup is, by definition, �66–68% total dissolved solids (at 20 �C).Since the major solid constituent (98% or more) of syrup is sugar, % totalsolids and �Bx are generally used interchangeably. Syrup that is lessdense than 66 �Bx is subject to fermentation if contaminated by microbes.Syrup denser than 67 �Bx may develop crystals inside the container.Therefore, a fairly narrow range of acceptable density is required toensure that quality issues are not found. Water makes up the bulk of theremaining solution (�32–34%), but due to the high concentration ofdissolved solids, the water activity of maple syrup is fairly low, averagingaround 0.87–0.88, thereby restricting the development of pathogenicmicroorganisms.

B. Carbohydrates

Most of the total carbohydrate fraction of syrup (88–99 þ%) is in the formof sucrose, with varying amounts of invert sugars present, primarily as afunction of microbial contamination of sap during storage prior to proces-sing (Edson, 1910; Edson and Jones, 1912). In some cases, conversion ofsucrose to invert sugars may continue after packing into retail containers,especially if syrup is low in density and fermentation occurs. A slightamount of sucrose hydrolysis may also occur during thermal processing.Total sucrose composition in syrup may range from �47–74%, typicallywith a mean of 65–68%. Glucose and fructose may range from nondetec-tible up to over 9% (Stuckel and Low, 1996), but typically are both foundin the 0.4–0.5% range in finished syrup. A recent study by van den Berget al. (2006) revealed no consistent relationships among maple syrupgrades and carbohydrate profiles (sucrose, glucose, fructose). However,lighter-colored syrups are generally thought to contain lower amounts ofinvert sugars than darker syrups, and are thus selected for use in makingmaple candy and cream, where crystallization properties are important.

C. pH

The published range of pH of maple syrup varies from somewhat acidic(4.7) to slightly basic (8.7), but typically is near neutral, with reportedmean values around pH 6.3–6.8 (Chenard and Gagnon, 1997; Perkinset al., 2006). The pH of the syrup is related to several factors, includingcollection and storage of sap, the methods employed to process the sapinto syrup, time of season, and microbial contamination.


D. Conductivity

Due to dissolved minerals in maple syrup, the conductivity can rangefrom about 90 to slightly above 230 mS/cm, with a trend towardsincreasing conductivity as light transmittance of syrup decreases.Conductivity of syrup is sometimes used as a rapid screening test foradulteration of syrup, especially in Canada.

E. Color

Maple syrup ranges greatly in color and opacity. The light transmittancefrom the UV through the near IR regions is shown in Fig. 4.9. UV light isnot well transmitted through maple syrup. Transmittance increases rap-idly in the visible spectrum, reaching a gradually upward sloping pla-teau at around 650 nm before reaching a peak in the near IR range ataround 920 nm. The color of maple syrup ranges from a very lightyellow-amber to near black. Generally, most syrups have a light yellowto a dark red-brown color, due mostly to color bodies originating fromnonenzymatic browning during evaporation. Because invert sugarbrowns at a lower temperature than sucrose, the level of glucose andfructose in sap, as well as rate of processing are the primary determi-nants of syrup color and light transmission. Maple syrup produced byvacuum or microwave processing is nearly colorless as a result of therebeing no or low thermal degradation of the sugars (Favreau et al., 2001;Willits et al., 1952). Chen et al. (2001) analyzed the colorimetric propertiesof maple syrup and found that total color value and ‘‘redness’’ increasedstrongly with increasing color grade, but that color was not an adequatetool to discern compositional differences between authentic maple syrupand table syrups composed of other sugars. Most likely, this is becausetable syrups utilize burnt caramel as a color agent in the formulation ofthese products.

F. Rheology

The rheological properties of Canadian maple syrup have beendescribed by Ngadi and Yu (2004). Although maple syrup is primarilyNewtonian in its flow characteristics, grade, temperature, and densitycan independently and interactively influence the apparent viscosity,with a range of 0.138–0.160 Pa s at 25 �C. Colder, more dense, and darkergrade syrups have higher viscosity than warmer, thinner, and lightergrades. Chen et al. (2001) found a slightly higher range of 0.128–0.247Pa s at 25 �C, with a mean of 0.164 Pa s at 25 �C, probably as a result of ahigher variation in syrup density and geographic origin. They did notfind a relationship between syrup grade and viscosity. Corn syrup


containing cellulose gum as a thickening agent had a vastly differentrheological signature, making structural analysis a possible method todetect some forms of maple syrup adulteration.

G. Inorganic composition

A number of studies have described the range of minerals and metalsfound in maple syrup (Dumont, 1996; Morselli, 1975; Stuckel and Low,1996). These studies and other unpublished works are summarized inChenard and Gagnon (1997), and by Perkins et al. (2006) (Table 4.7). Whilemany of these constituents originate in the composition of the sap as itcomes directly from the tree (which can vary widely), other elements maybe introduced during the collection and evaporation process.

Potassium is by far the mineral found in highest concentration inmaple syrup, with a range from various studies of 541–4031 ppm.Typically, the average values are around 2000–3300 ppm. Calcium isalso found in substantial quantities, with a mean of 630–1470 ppm.The mean magnesium concentration is 130–375 ppm. Lesser amounts ofmanganese, sodium, and phosphorus are usually found.

The content of various metals in maple syrup fluctuates widely, oftenas a result of contact with sap collection and processing equipment. Iron,zinc, copper, and tin are generally present to varying degrees. Tin oftenoriginates from uncoated metal containers sometimes used to marketmaple syrup. Upon opening a metal container, oxidation can cause tinlevels to rise to the point where syrup begins to appear slightly greenish,and acquire a distinct ‘‘metallic’’ off-flavor over several months time.

Little research has focused on the differences in syrup composition bygrade. While light transmittance is different (by grade definition), thetrends in other aspects of syrup chemistry are not as simple. Two unpub-lished studies at the University of Vermont Proctor Maple ResearchCenter show that there are no consistently predictable trends in bulksyrup chemistry related to grade (color class).

Maple sap collection and processing equipment produced prior to1994 was frequently made with lead solder, terneplate, or galvanizingmaterial containing lead. Lead is not detectable in sap collected directlyfrom the tree, but fairly rapidly dissolves in sap. The composition of thesap collection and processing materials, surface area, and length ofcontact time are important factors in determining the degree of leadcontamination of sap. Upon boiling, lead is concentrated; however, agreat deal of lead is sequestered in scale and sugar sand, and is thusfiltered out. The dissolved lead fraction cannot be filtered, and thus maycontaminate the syrup. The U.S., Canada, and some states have set maxi-mum permissible lead levels ranging from 250 to 500 ppb, and the vastmajority of syrup produced falls well under those limits. As equipment

TABLE 4.7 Reported values and ranges for composition of maple syrup (adapted from Perkins et al., 2006, used with permission)

Morsellia Stuckel and Lowb Dumontc Perkinsdvan den Berg, Perkins

and IsselhardteTypical

mean

General

Total Solids (%) 66.5 63.2–69.5 – – 62.0–68.0 65Moisture (%) 33.5 26.5–39.4 – – – –

pH – 5.6–7.9 5.7–8.5 – 5.5–7.3 6.4

Conductivity

mS/cm2– – – – 96–318 189

Carbohydrates

Sucrose (%) 58.5–65.8 51.7–75.6 42.3–74.0

– 59.4–73.8 66

Glucose (%) 0–7.3 0.0–9.6 0.0–7.2 – 0.0–1.6 0.4

Fructose (%) Trace 0.0–4.0 0.0–6.8 – 0.0–1.1 0.5

Minerals and

Metals

Nitrogen (%) – – – – 0.001–0.077 0.03

Potassium (ppm) 1300–

3900

1055–2990 541–

4031

1600–

2590

963–3319 2283

Calcium (ppm) 400–2800 266–1707 183–

1943

600–1250 278–2494 911

Magnesium (ppm) 12–360 10–380 11–575 0–198 25–543 177

Manganese (ppm) 2–220 – <1–252 0–117 0.01–223 39.8

Sodium (ppm) 0–6 – <1–261 0–27 0.01–492 36

(continued)


Morsellia Stuckel and Lowb Dumontc Perkinsdvan den Berg, Perkins

and IsselhardteTypical

mean

Phosphorus (ppm) 79–183 – <2–2335 20–113 0.01–91 37Iron (ppm) 0–36 – 0–18 0–18 0.01–61 10

Zinc (ppm) 0–90 – 2–43 0–96 0.01–527 22

Aluminum (ppm) – – – – 0.01–18 2

Boron (ppm) – – – – 0.01–3 0.2

Sulfur (ppm) – – – – 0.01–100 18.2

Copper (ppm) 0–2 – 0–8 0–6 – 1.2

Tin (ppm) 0–33 – – 0–24 – 8

Lead (ppm) 0–0.25 – 0–0.49 0–0.35 – 0.20Cadmium (ppm) – – 0–0.09 0–0.07 – 0.02

Chloride (ppm) 31–191 20–400 93

Organic Acids

Malic Acid (%) 0.141 0.06–0.66 0.32–

0.90

– –

Fumaric Acid (%) 0.006 0.001–0.012 0.0–0.13 – –Citric Acid (%) 0.015 – – – –

Succinic Acid (%) 0.012 – 0.0–0.26 – –

a Morselli (1975)b Stuckel and Low (1996)c Dumont (1996)d Perkins (1998, unpublished data)e van den Berg, Perkins and Isselhardt (unpublished data).


containing lead reaches the end of its service lifetime, the overall level oflead in maple syrup should decrease.

Only a few studies have examined the minor inorganic constituents ofmaple syrup. Both sodium and chloride are sometimes found in maplesyrup, occasionally at fairly high levels. Morselli and Whalen (1987)found a mean chloride concentration of 93 ppm in syrup, with a rangeof 31–191 ppm. Trees growing in areas of heavy road-salt use had higherconcentrations of salt in sap, and eventually in syrup. Sodium rangesfrom 0.1 to 492 ppm, with a typical mean of 2–36 ppm. Sodium can beelevated due to roadside salt, or more commonly, to the use of sanitizers(sodium hypochlorite) in maple tubing. More recently, the ratio of sodiumto chloride has been investigated as an indicator of artificial decoloriza-tion of syrup (an illegal activity) with anion exchange resins (Perkins andvan den Berg, unpublished data).

H. Organic acids

Several organic acids are typically present inmaple syrup (Table 4.7).Malicacid is the predominant form, comprising on average about 0.5% of thetotal weight of syrup. Various studies have yielded a range of 0.06–0.90%,but typically average about 0.5 ppm.Citric acid, succinic acid, and fumaricacid are also present in trace amounts in syrup (Stuckel andLow, 1996), buttypically are found in far lower concentrations than malic acid. Malic acidis a dominant constituent of sugar sand, and malic and citric acids aresometimes used to detect different forms of adulteration, as the signaturesof these compounds are quite different in some other types of sugars.

I. Flavor compounds

Over 130 volatile flavor compounds have been identified in maple syrup,although in widely varying levels and combinations depending upon thegrade of syrup as influenced by the type and intensity of processing, thetime of season (probably as a result of microbial influence on sap chemis-try), and possibly by geographic location. Many of the flavor compoundsare phenol derivatives. Several published studies describe some of these(Akochi et al., 1994, 1997; Alli et al., 1992; Belford et al., 1991; Deslauriers,2000; Filipic et al., 1969; Kermasha et al., 1995; Potter et al., 1995;Underwood, 1971). The dominant types of flavor compounds include:phenolics, pyrazines, carbonyl-based molecules, and others. Analysis ofthe primary flavor contributors tomaple syrup is complicated by the largenumber, combinations, and levels of flavor compounds present, as well asthe detection threshold and saturation nature of the taste reaction of thesecompounds.


In general, lighter-color (higher grade) syrups tend to contain lowerlevels of flavor compounds than darker (lower grade) syrups. Someextremely light syrups have very little flavor (other than sweet). In themaple industry, these are sometimes termed ‘‘techno-syrup,’’ as they areconsidered to be more prevalent in operations using a higher level ofprocessing technology (reverse osmosis, steam- or vapor compressionevaporators). The veracity of this connection has not been established,however, it is enough of a concern that researchers are beginning toinvestigate the effects of processing equipment and intensity on maplesyrup chemistry and flavor.

Phenolic flavor compounds present in syrup are largely the degrada-tion products of lignin components of sap. These compounds include:vanillin, syringaldehyde, dehydroconiferyl alcohol, syringoyl methylketone, and 2,6-dimethoxyphenol. Vanillin is generally a strongcontributor to the flavor of light-colored, early-season maple syrup(Potter et al., 1995), whereas a variety of furanones, cyclotene, and othercaramelization-derived compounds appear to dominate in darker syrups(Belford et al., 1991; Potter et al., 1995).

Pyrazines are generally products of Maillard reactions, and tend toincrease fairly steadily during boiling (Akochi et al., 1997). The presenceand concentration of several forms of pyrazine in maple syrup are quitevariable, and they contribute both to the flavor and off-flavor attributes ofmaple syrup depending upon the form and concentration of both thepyrazine and other flavor compounds present which may mask anyoff-flavor. In particular, moderately high levels of 2,5-dimethylpyrazineare thought to result in the objectionable ‘‘metabolism’’ off-flavor foundoccasionally in maple syrup (van den Berg et al., 2009b). In some years,‘‘metabolism’’ (also called ‘‘woody’’) can affect up to 25% of the totalannual maple syrup crop. At lower concentrations, especially when com-bined with strong carmelization flavors, 2,5-dimethylpyrazine likely con-tributes acceptable flavors to maple syrup. Research is ongoing toinvestigate processes to reduce or mitigate ‘‘metabolism’’ off-flavor inmaple syrup.

Reverse osmosis of sap increases the relative proportion of phenoliccompounds, while decreasing aldehydes and alcohols (Kermasha et al.,1995). Most maple producers concentrate sap to around 8–10%, reducingthe amount of water to be evaporated by 75%. Maple syrup producedfrom concentrated sap tends to be lighter in flavor and color, due to the farreduced boiling time required to achieve the proper density. Given theincreasing cost of fuel, the tendency among some producers in recentyears has been to increase the level of concentration. Sap concentration byRO up to 15–25% is not unheard of at the present time. Many producersand packers in the maple industry feel that high concentration (> 10%) ofsap by RO produces a ‘‘flat’’ syrup, lacking in flavor. This topic is also thefocus of ongoing research.


Overall, dozens of compounds are involved in the development of thecharacteristic flavors found in maple syrup. The number of compounds,the different sap to syrup processing involved, the wide range of maplesyrup flavor and intensity, and somewhat frequent off-flavors all compli-cate the understanding of the flavor of maple syrup.

J. Sensory evaluation of flavor

Although the dominant taste is usually sweet, maple syrup can rangefrom a very light, sweet, but otherwise nearly tasteless substance, to avery dark, bitter, burnt taste. Most syrups used for table use on pancakesor in cooking are intermediate in color and have a moderate to strongmaple-caramel flavor, without objectionable off-flavors. Light colored,low-flavor syrups are generally used for blending with darker syrups toachieve the correct color and flavor profile. Moderate syrups can be usedwithout blending. Dark syrups usually have strong flavors (and some-times slight-moderate off-flavors that are masked by strong ‘‘good’’flavors) and are used for blending, or as ingredients in recipes requiringa maple flavor. Very dark, commercial syrups (which are not sold fortable use) range from very strong tasting syrups with good flavor, to verystrongly bitter syrups with mild to moderate off-flavors. These syrups areused in commercial food manufacturing processes where a strong mapleflavor is required. The degree of off-flavor deemed acceptable isdetermined by the user and the application.

For most producers and consumers, the description of syrup flavor isgenerally fairly straightforward, focusing on a simple scale of light flavorto strong (dark) flavor. The lightest syrups (Canada No. 1 Extra Light andU.S.D.A. Grade A Light Amber) are generally sweet, with only a slighthint of maple flavor. As one tastes progressively darker syrups, the tastebecomes dominated less by sweet alone, and more by a maple-caramelflavor. The strongest table grades (Canada No. 2 Amber and VermontGrade B) are dominated by an intense caramel flavor (although notenough to cause a bite or bitter taste), generally with a mix of severalother flavors in lesser amounts. Often the darker color and strong flavorscause people to believe that these syrups are thicker, although all syrupsmust legally meet the same density requirements.

A relatively recent addition to the maple industry is the ‘‘FlavorWheelfor Maple Products’’ (Agriculture and AgriFood Canada, 2004), patternedafter similar classification systems in wines and other food products. TheFlavor Wheel for Maple Products (hereafter FWMP) contains 13 flavorfamilies, including: maple, confectionary, vanilla, milky, empyreumatic,floral, fruity, spicy, foreign (deterioration/fermentation), foreign (envi-ronment), plant—herbaceous, plant—humus/forest/cereals, and plant—ligneous. Within each family are one or more subfamilies of flavors,


which on the outer ring are further divided into individual flavors. TheFWMP is a useful tool in the maple industry, and although it is fairly new,and requires substantial training to be used correctly, is likely to becomemore widespread in maple research and large-scale marketing as it pro-vides a common lexicon for those in the industry to describe the sensorycharacteristics of maple in a very detailed way.

Typically, there is a progression from lighter-colored and flavoredsyrups (Canadian No. 1 AA or U.S.D.A. Grade A Light Amber) early inthe maple production season, dominated by delicate maple and vanillaflavors, to moderately colored and tasting syrups (Canadian No. 1 A andU.S.D.A. Grade A Medium Amber) in the middle of the season, withlight-moderate confectionary (light brown sugar) and empyreumatic(light caramel) flavors, to dark-colored and strong flavored syrups(Canadian No. 2 B or U.S.D.A. Grade B), characterized by heavy confec-tionary (molasses) and empyreumatic (burnt-sugar) notes.

There has been some recent research investigating the ‘‘Terroir’’ ofmaple. Preliminary work has shown that the underlying bedrock compo-sition may influence the chemistry of the sap and resulting syrup (Corbettand Munroe, 2006), but the effects on maple syrup flavor are not as clear(Costanza-Robinson et al., 2007).

K. Off-flavors in maple syrup

Within the maple industry, more attention is sometimes given to recog-nizing off-flavors, as these can result in syrups being down-graded, orplaced into the commercial or substandard class. The origin of off-flavorsmay be either intrinsic to the sap, or arise from external factors. Off-flavors regularly found in maple syrup include: metabolism (a poorlyunderstood woody-cardboard flavor arising from environmental condi-tions), buddy (made from late season sap, characterized by a strong, bitterchocolate flavor), salty (inadequate rinsing of equipment), chemical(inadequate cleaning/rinsing procedures), scorch/burnt (overheatingdamage), moldy or fermentation (microbial contamination), and severalothers (Heiligmann et al., 2006). Producers, packers, and agriculturalinspectors generally learn to recognize these and are able to diagnoseproduction and/or storage issues causing the off-flavor. Although typi-cally color and flavor development progress hand-in-hand during evapo-ration, those in the maple industry must also assess whether the flavor ofthe syrup is typical of the color. A syrup found to have a flavor notcharacteristic of the grade is usually down-graded (placed into the nextlowest syrup grade). Although primarily designed as a tool to describepositive sensory attributes of maple flavor, the FWMP does includesome off-flavors as well. Some off-flavors are dealt with by blending, anattempt to reduce the off-flavor to undetectable or unobjectionable levels.


Other off-flavors (metabolism, buddy, chemical) are often too severe toallow the syrup to be used in this manner, and are thus relegated tocommercial status and used for industrial flavoring, or to make sugar(during which the process reduces or eliminates most of the volatile off-flavor compounds).

Due to improper packing or storage conditions, more frequently foundin syrups packed at too low a density, microbial contamination cansometimes cause off-flavors. Because of the low water activity of maplesyrup, few organisms are generally able to successfully colonize maplesyrup. When contamination does occur, it will result in a moldy off-flavor. Bulk storage of syrup low in density will result in a fermentationoff-flavor (fruity). Generally these barrels are easy to spot, as the top andbottom of the drum will bulge outward due to the buildup of carbondioxide. Both moldy and fermented syrup, unless severely damaged, canbe reheated, filtered, and blended with a good-flavored syrup to achievean acceptable result. Bulk storage of syrup in barrels in good condition,especially stainless steel barrels, will maintain syrup color and flavor for along period of time (1–2 years).

Syrup is packed into retail containers at a temperature of �180–185 �F.Exceeding this temperature may promote the development of nitre,whereas packing too cool does not result in complete kill of any micro-organisms that may be in the container. After packing, containers areusually turned on their sides to sterilize the container top as well. Ifcontainers are not cooled rapidly (especially when large orders arepacked tightly together before cooling), they are subject to ‘‘stack burn,’’wherein the syrup darkens rapidly. Maple syrup is commonly packagedin tin, glass, and plastic containers. Glass retains syrup color and flavorwell for 6þmonths, especially if kept cold. Syrup packed into plastic maybegin to darken rapidly, although flavor is generally unaffected. For thisreason, maple producers and packers will often pack a syrup that issomewhat lighter than the grade to account for darkening on the retailshelf. Alternatively, packing is done frequently with only a small amountof syrup in plastic kept in storage or on retail shelves. Tin, although lesscommon presently, is particularly good for short-term maple syrupstorage. Tin retains syrup color very well. Prolonged storage of syrup intin, especially after the container has been opened, may result in a ‘‘tin’’ or‘‘metallic’’ off-flavor. With long-term storage in tin containers a slightlygreen off-color may develop.

L. Nutritional aspects of maple syrup

Although predominantly sugar, maple syrup does contain some amountof several minerals and vitamins (Tables 4.7–4.8). A one-fourth cupserving contains 217 calories, but supplies 100% of the Canadian

TABLE 4.8 Vitamins in maple syrup (adapted from Perkins et al., 2006, used with

permission)

Morsellia CANDIb

Thiamin (ppm) – 1.3

Niacin (ppm) 0.16 1

Riboflavin (ppm) 0.046 0.6

Folic Acid Trace –

Biotin Trace –Pyridoxine (B6) Trace –

A Trace –

a Morselli (1975).b CANDI (1997).


and U.S. F.D.A. daily recommended value of manganese, 34% riboflavin(vitamin B2), 11% zinc, and lesser amounts of magnesium, calcium, andpotassium. Frequently it is said that darker syrups contain higher levels ofminerals and vitamins, and while some limited studies have demon-strated this to some degree, the rather high variability in syrup chemistryoften overwhelms this trend. Of more interest, recent work has shownthat the phenolic compounds in maple sap and syrup confer some degreeof antioxidant, antiradical, and antimutagenic properties to mapleproducts (Theriault et al., 2006).

Consumer preference of maple syrup appears to be trending towardsdarker (stronger tasting) grades.

XI. OTHER MAPLE PRODUCTS

A variety of other ‘‘value-added’’ maple products are made from maplesyrup. These include maple cream (also known as maple butter), whichdespite the name, does not contain any dairy product. It is made by heatingsyrup to 12–13 �C above the temperature of boiling water, then cooling itrapidly (usually in awater bath), and then stirring it slowly, eithermechani-cally or by hand, to encourage the formation of fine crystals. To preventseparation, the enzyme invertase isused in someareas (it isnot legal in everyU.S. state) to encourage the conversion of a portion of the sucrose to glucoseand fructose. Similarly, a small amount of potassium sorbate is used in someareas to inhibit mold formation to lengthen shelf-life of the product.

Maple candy, maple block or cake sugar, loose maple sugar (Indiansugar), and other types of maple products are made by boiling syrup todifferent densities followed by pouring into molds, pans, or grinding to a


fine texture. Specific procedures to follow can be found in Heiligmann et al.(2006).

A new product consisting of maple syrup in which a portion of thesucrose has been converted to invert sugar (using invertase) was intro-duced recently. The advantages are that higher density syrup can be soldwith a greatly reduced tendency to crystallize. In some areas this productis not allowed to be sold as ‘‘maple syrup,’’ due to the addition ofinvertase and because some areas have upper limits on the density ofmaple syrup (which this product exceeds). Due to the high level of invertsugar in this syrup, this product has a somewhat different flavor profile aswell, with a more pronounced caramel-like flavor.

Maple syrup and maple sugar are often used as flavoring agents incommercial and retail cooking. When added as an ingredient, typically adark and strong tasting syrup will be used. Maple can add a distinctiveflavor to foods, and is also often used as a humectant in some recipes.

XII. CONTAMINATION

Due to the large concentration factor involved in converting sap to syrup(typically 40:1), any slight contamination of sap can result in off-flavors insyrup. All sap collection and processing materials must be kept very cleanand free from cleaning or other residues, as these may impact the flavor ofthe resultant syrup. Soap is used very sparingly, if at all, in maplesugaring operations, as most detergents leave a residue that is almostimpossible to entirely remove. Most cleaning is done with copiousamounts of hot water. Chlorine or other special-purpose agents are usedfor sanitizing tubing and processing equipment, with repeated rinsingnecessary to remove all residues. Given the increasing use of ROmachines and the use of strong acids to clean evaporators, strict attentionto chemical safety, particularly in using proper procedures to eliminatethe risk of inadvertent contamination, is very important.

XIII. ADULTERATION

Due to the wide differences in the price of maple and sugars from otherorigins, there is a considerable incentive for adulteration. The simplest isthe addition of cane, corn, or beet sugar to maple syrup. A great deal ofresearch has been conducted on this problem.

As a general screening method for addition of nonmaple sugars, theconductivity of maple syrup can be checked. Syrups with conductivityreadings above 1200 mS/cm are suspect, and thus subjected to additionaltesting.


Determination of the carbohydrate profile in suspect syrups may alsobe used. One historical method was to examine the specific rotationinduced in linearly polarized light by the syrup using a polarimeter.Sucrose has an optical rotation of þ66.5�, glucose þ52.7�, and fructose�92 �. Addition of sugars with a proportion of sucrose to invert sugarsdifferent frommaple is readily detectible. Currently, HPLC can be used toreadily determine the sucrose/invert ratios. These methods are only ableto detect gross adulteration of maple syrup, due to the large naturalvariation in invert sugars found in maple syrup (0–12%).

More recently, the carbon stable isotope ratio test (SIRA) has become aneasy method to detect adulteration with cane and corn syrup (Carro et al.,1980). Because maple trees are C3 plants with a somewhat differentphotosynthetic pathway for carbon fixation, the ratio of 13C/12C in thesugar produced is different than cane or corn.Maple has a d13C of approxi-mately�24.5,whereas corn and cane are closer to a d13Cof�8 to�12. Thus,even a small addition of cane or corn syrup is readily detectable. Becausebeets are also C3 plants, the SIRA test is not able to detect adulterationwith beet sugar. Improvement of the SIRA method is possible usingmalic acid as an internal standard (Tremblay and Paquin, 2007).

Due to the inability to reliably detect beet sugar additions, the site-specific natural isotope fractionation nuclear magnetic resonance (SNIF–NMR) method used widely in the wine industry was adapted for themaple industry (Martin et al., 1996). This method determines the sitespecific isotope concentrations of organic compounds by nuclear mag-netic resonance of ethanol fermented from the suspect sample.

Other methods to detect adulteration include anion-exchange liquidchromatography in combination with pulsed amperometric detection(Stuckel and Low, 1995) and FTIR, FT-Ramen, and NIR Spectroscopy(Paradkar et al., 2002, 2003).

At times, a large price differential between light and dark grades ofmaple syrup has given rise to another type of adulteration in the form ofbleachingof syrup.Suchapractice is illegal throughout theU.S. andCanada.Avariety ofmethods canbe employed (Fig. 4.10).Hydrogenperoxide servesas a direct bleaching agent which acts by oxidation of color bodies in syrup.Use of hydrogen peroxide also causes serious flavor defects, the effectwears off somewhat over time, and residual hydrogen peroxide is readilydetectible using simple test strips. Acid, which is added to the backpanduring evaporation, can also result in syrup lightening, probably by lower-ing the pH enough to reduce the rate of alkaline degradation, and thus colorbody precursor formation. Carbon filtration is another method of syruplightening. Formerly somewhat difficult to do, as it required charcoal orbone char, lightening via carbon filtration is somewhat easier now giventhe prevalence of a wide selection of resins that are designed specificallyfor sugar decolorization. Although adulteration via decolorization with

FIGURE 4.10 Effect of treatment of syrup with decolorizing resin (Top: from left to

right, control syrup, decolorized, further decolorized) and hydrogen peroxide

(Bottom: control syrup, treated syrup).


resins can be difficult to detect, there have been somemethods developed toscreen suspect syrup. One promising method is to examine syrup forincreased level of anions substituted for the highly positively chargedsyrup color bodies (Perkins and van den Berg, unpublished data).


XIV. SUMMARY

Although pure maple sap is predominately a dilute solution of sucroseand water, and maple syrup is mostly concentrated sap, the mixture ofminor amounts of minerals, amino acids, phenolic compounds, andorganic acids found in sap in varying amounts during the short sapexudation season, along with substances added, formed, or sequesteredduring the collection and processing phases of maple syrupmanufacturing, and the interactions of sap and syrup with microorgan-isms at many stages of the process, make the chemistry of maple syruprather complicated. In all cases, regardless of how sap is collected orprocessed, the end result is usually a pure product—maple syrup—witha unique and distinctive flavor.

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CHAPTER 5

Maternal Fumonisin Exposureas a Risk Factor for NeuralTube Defects

J. Gelineau-van Waes,* K. A. Voss,† V. L. Stevens,‡

M. C. Speer,§ and R. T. Riley†


II. Neural Tube Defects 148

A. Neural tube defects: Overview 148

B. Environmental risk factors for neural tube

defects: Fumonisins 148

C. Nutritional risk factors for neural tube defects:

Folic acid 149

D. Genetic risk factors for neural tube defects 151

III. Fumonisin Exposures 153

A. Fumonisins and regulatory policy 153

B. Measurements of fumonisin exposure 154

IV. Reproductive Toxicology of Fumonisins 155

A. Animal studies: Overview 155

B. Mouse models of fumonisin-induced neural tube

defects 156

V. Mechanisms of Fumonisin Toxicity 158

A. Structural considerations 158

B. Role of sphingolipids in fumonisin toxicity 160

C. Fumonisin inhibition of de novo sphingolipid

metabolism 162

* Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center,Omaha, Nebraska, USA

{ Toxicology & Mycotoxin Research Unit, USDA Agricultural Research Service, Athens, Georgia, USA{ Department of Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia, USA} Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, USA

Advances in Food and Nutrition Research, Volume 56 # 2009 Elsevier Inc.ISSN 1043-4526, DOI: 10.1016/S1043-4526(08)00605-0 All rights reserved.

145

146 J. Gelineau-van Waes et al.

D. Fumonisin and accumulation of bioactive

sphingoid base-1-phosphates 164

E. Fumonisin depletion of glycosphingolipids and

disruption of folate transport 166

VI. Conclusions 170

References 171

Abstract Fumonisins are mycotoxins produced by the fungus F. verticil-

lioides, a common contaminant of maize (corn) worldwide. Maternal

consumption of fumonisin B1-contaminated maize during early

pregnancy has recently been associated with increased risk for

neural tube defects (NTDs) in human populations that rely heavily

on maize as a dietary staple. Experimental administration of

purified fumonisin to mice early in gestation also results in an

increased incidence of NTDs in exposed offspring. Fumonisin

inhibits the enzyme ceramide synthase in de novo sphingolipid

biosynthesis, resulting in an elevation of free sphingoid bases and

depletion of downstream glycosphingolipids. Increased sphingoid

base metabolites (i.e., sphinganine-1-phosphate) may perturb signal-

ing cascades involved in embryonic morphogenesis by functioning

as ligands for sphingosine-1-P (S1P) receptors, a family of G-protein-

coupled receptors that regulate key biological processes such as

cell survival/proliferation, differentiation and migration. Fumonisin-

induced depletion of glycosphingolipids impairs expression and

function of the GPI-anchored folate receptor (Folr1), which may

also contribute to adverse pregnancy outcomes. NTDs appear

to be multifactorial in origin, involving complex gene-nutrient-

environment interactions. Vitamin supplements containing folic

acid have been shown to reduce the occurrence of NTDs, and

may help protect the developing fetus from environmental terato-

gens. Fumonisins appear to be an environmental risk factor for

birth defects, although other aspects of maternal nutrition and

genetics play interactive roles in determining pregnancy outcome.

Minimizing exposures to mycotoxins through enhanced agricultural

practices, identifying biomarkers of exposure, characterizing

mechanisms of toxicity, and improving maternal nutrition are all

important strategies for reducing the NTD burden in susceptible

human populations.

I. INTRODUCTION

Fumonisins are mycotoxins produced by the fungus Fusarium verticil-lioides, F. proliferatum, and more rarely, other Fusarium species (Bolgeret al., 2001; Gelderblom et al., 1988) (Fig. 5.1A). They are found invariable amounts in maize (corn) and maize-based foods worldwide

Fusarium verticillioides Fumonisin

COOH

HOOC

Me Me

O

O

O

OCOOH

HOOC

OH

OH OH

NH2

1

FIGURE 5.1 The photograph on the left shows an ear of maize infected with the

Fusarium verticillioides fungus. The mold is white to pinkish in color, and produces the

fumonisin toxin. Decay often begins with insect-damaged kernels, however, levels of

fumonisins capable of causing toxicity in rodents can occur in asymptomatic maize. The

chemical structure shown on the right is that of the mycotoxin fumonisin B1. Fumonisin

was first isolated in 1988 by investigators at PROMEC (program on mycotoxins and

experimental carcinogenesis) in Tygerberg, South Africa.

Fumonisin and Neural Tube Defects 147

(Bolger et al., 2001; Humpf and Voss, 2004) and their presence in othercommodities has occasionally been reported (Castoria et al., 2005;da Silva et al., 2000; Kritzinger et al., 2003). Consumption of fumonisinB1-contaminated maize has been associated with a variety of differentdiseases in animals, including leukoencephalomalacia in horses (Smithet al., 2002) and pulmonary edema in swine (Constable et al., 2000, 2003;Haschek et al., 2001). Independent studies have also established a causalrelationship between fumonisin B1 exposure and liver and kidney toxic-ity (Bolger et al., 2001; Voss et al., 2001), and/or liver and kidney carci-nogenicity in rodents (Gelderblom et al., 1991; Howard et al., 2001a,b).The International Agency for Research on Cancer has evaluated fumo-nisin B1 as a possible human carcinogen (Group 2B) (IARC, 2002).Although the human health effects of fumonisins are not proved, theconsumption of foods made from F. verticillioides- or fumonisin B1-contaminated maize has been associated with high rates of esophagealcancer in parts of southern Africa, China, and northeastern Italy (Bolgeret al., 2001, Shephard et al., 2007). Elevated levels of fumonisin have alsobeen found in maize from Mazandaran Province in Iran, another regionwith a high incidence of esophageal cancer (Shephard et al., 2002;Yazdanapanah et al., 2006). However, fumonisin exposures in this andother areas of Iran in which esophageal cancer is less frequent were low(� 0.22 mg/kg body weight/day) due to the relatively small amount ofmaize consumed (Yazdanpanah et al., 2006). In terms of human healthrisks, maternal consumption of fumonisin B1-contaminated maize ormaize-based food products during early gestation has recently been asso-ciated with increased risk for birth defects, specifically, neural tube


defects (NTDs) (Hendricks, 1999; Kromberg and Jenkins, 1982; Marasaset al., 2004; Melnick and Marazita, 1998; Missmer et al., 2006; Moore et al.,1997; Ncayiyana, 1986; Xiao et al., 1990).

II. NEURAL TUBE DEFECTS

A. Neural tube defects: Overview

Approximately 3–4% of all newborns have a significant abnormality inbody structure or function. Birth defects (congenital anomalies) are theleading cause of death in children under 1 year of age. NTDs, which occurwith a frequency of approximately 1/1000 live births (Nakano, 1973), areamong the most common of all human birth defects, yet their etiologicbasis and embryology remain poorly understood. NTDs are commoncongenital malformations that occur when the embryonic neural tube,which ultimately forms the brain and spinal cord, fails to properly closeduring the first few weeks of development. Anencephaly, which is essen-tially the absence of the brain, is invariably fatal, and results from failureof the anterior neural folds to properly elevate and fuse along the dorsalmidline during early embryogenesis. Spina bifida refers to incompletedevelopment of the posterior neural tube, and failure of fusion of one ormore vertebral arches, often accompanied by protrusion of the spinal cordand its associated membranes. Patients with spina bifida can have avariety of associated conditions, the severity of which is often determinedby the level of the lesion. Most commonly, patients with spina bifida havedifficulty walking and require either braces or a wheelchair, have hydro-cephalus requiring shunting, and/or have difficulty with bowel andbladder control. Empirical risk figures, along with numerous clinicaland experimental studies, suggest that NTDs are multifactorial in origin,having genetic, environmental, and nutritional components that contributeto their prevalence (Campbell et al., 1986).

B. Environmental risk factors for neural tubedefects: Fumonisins

Maternal fumonisin exposure has been proposed as a potential riskfactor for human NTDs among populations consuming large amounts offumonisin-contaminated maize or maize-based products (Marasas et al.,2004). Globally, NTDs present a tremendous burden to human populationsin rural areas of the world where maize is a dietary staple, and fumonisincontamination is common (Hendricks, 1999; Kromberg and Jenkins, 1982;Marasas et al., 2004; Melnick and Marazita, 1998; Moore et al., 1997;


Ncayiyana, 1986; Torres et al., 2007; Xiao et al., 1990). The incidence of NTDsin these regions, including Guatemala, northern China, and the Transkei ofSouth Africa, is often 6–10 times higher than the average global neural tubedefect rate (Hendricks, 1999; Kromberg and Jenkins, 1982; Marasas et al.,2004; Melnick and Marazita, 1998; Moore et al., 1997; Ncayiyana, 1986; Xiaoet al., 1990). Since fumonisins are ubiquitous, and maize-based meals areoften the primary food commodity available, human exposures can besignificant (Marasas, 1996; Meredith et al., 1999; Miller, 2001; Shephardet al., 1996, 2007). In Guatemala, a recent analysis of fumonisin B1 inmaize samples, coupled with data on daily maize intake, demonstratedthat human consumption of maize products could frequently result infumonisin exposures exceeding the recommended World Health Organi-zation (WHO) provisional maximal tolerable daily intake (Torres et al.,2007). In the United States, the Texas Department of Health reported aneural tube defect cluster among Mexican-American women along thesouth Texas border in 1990–1991 (Hendricks, 1999). Corn crops in theLower Rio Grande Valley registered unusually high levels of fumonisinB1 during this period, and epidemiological studies revealed that CameronCounty women who conceived during 1990–1991 had a substantiallyhigher neural tube defect rate (2.7/1000 live births) than those who con-ceived during the period from 1986 to 1989 (1.5/1000 live births). Mexican-Americans in Texas consume large quantities of maize, primarily in theform of tortillas, and thereforemay be exposed to high levels of fumonisins.Results of a follow-up population-based case-control study conductedamong the south Texas Mexican-American population suggested thatfumonisin exposure increased the risk of NTDs, proportionate to dose,up to a threshold level, at which point fetal death was postulated to bemore likely to occur (Missmer et al., 2006).

C. Nutritional risk factors for neural tube defects: Folic acid

The etiology of NTDs is largely unknown, but appears to be multifactorialin origin, involving complex gene-nutrient-environment interactions.Consumption of large quantities of fumonisin-contaminated maize ormaize-based food products during early gestation may represent anenvironmental risk factor for having a child with a neural tube defect.However, other aspects ofmaternal nutrition are also important in ensuringa positive pregnancy outcome, and adequate maternal folate appears toplay a critical role in protecting the developing fetus from the harmfuleffects of environmental teratogens. Diets heavily dependent onmaize arelikely to be folate deficient since maize contains only low levels of thisvitamin (Burton et al., 2008). In Mexico and Central America, maize-based foods are typically prepared using an alkaline process known as


nixtamalization (Palencia et al., 2003) which has been shown to furtherreduce folate, riboflavin, and other vitamins (Burton et al., 2008; Cardenaset al., 2001).

Folate is an essential vitamin derived from plant sources that plays animportant role in DNA biosynthesis and amino acid metabolism.Multipletransport systems play a role in mediating internalization of folatesthrough the plasma membrane into the cell for utilization in a varietyof critical housekeeping functions. The primary mechanisms for folatedelivery into the cell are through (1) carrier-mediated (reduced folatecarrier; RFC1) or (2) receptor-mediated (folate receptor a, b, d, g) pro-cesses. These two transport systems are distinguished by their uniquepatterns of tissue expression, divergent specificities for oxidized vs.reduced folates, and differing protein structures and mechanisms forthe transmembrane transport of folates. As mammalian cells are notcapable of synthesizing folates de novo, these two transport systems playa critical role in mediating folate uptake for the biosynthesis of purines,pyrimidines, and some amino acids that are necessary for cell survivaland proliferation.

The murine folate binding protein 1 (Folr1) is homologous to thehuman folate receptor-alpha (FRa), a membrane bound protein withhigh binding affinity for folic acid. During early development, Folr1 ishighly expressed in the placenta, yolk sac membrane, and dorsal neuraltube, and is later observed in the choroid plexus, ependymal cells, andperipheral edge of the retina (Saitsu et al., 2003). The glycosylphosphati-dylinositol (GPI)-anchored Folr1 internalizes folate via endocytosis, and islocalized in unique sphingolipid-enriched plasma membrane microdo-mains known as lipid rafts (Elortza et al., 2003; Foster et al., 2003). Lipidrafts function as specialized platforms for coordinating protein–proteininteractions involved in the initiation of signal transduction cascades andintracellular signaling (Brown and London, 1998). Since most embryoniccells do not express Folr1 (Page et al., 1993), the presence of these receptorsin neuroepithelial cells (Barber et al., 1999; Saitsu et al., 2003) suggests acritical role for Folr1-mediated folate transport in the normal morphoge-netic events involved in neural tube closure (Piedrahita et al., 1999;Spiegelstein et al., 2004).

Human clinical and epidemiological studies have demonstrated thatmaternal use of folic acid in early pregnancy can significantly reduce boththe occurrence (Czeizel and Dudas, 1992), as well as the recurrence(MRC, 1991) of neural tube defect-affected pregnancies. These findingshave been further validated by observational studies of women takingdaily periconceptional multivitamin supplements containing folic acid(Shaw et al., 1995; Werler et al., 1993). However, while the epidemiologicand experimental data support the hypothesis that this apparent


reduction in neural tube defect risk may be specifically attributable tofolic acid, the mechanisms underlying the protective effects of folic acidare not fully understood.

The incidence of NTDs is elevated in regions of the world (Guatemala,South Africa, China) where maize consumption with fumonisin is docu-mented or plausible and where diets are also likely to be deficient in folate(Chu and Li, 1994; Cifuentes, 2002; Marasas, 2001; Marasas et al., 2004;Shannon and Fenichel, 1990; Yoshizawa et al., 1994). The high incidenceof NTDs among Mexican-American women living in the U.S.–Mexicoborder region in Texas is likely related to maternal fumonisin exposurecoupled with folate deficiency (Hendricks, 1999; Marasas et al., 2004;Missmer et al., 2006). Interestingly, fumonisin content in Guatemalanand U.S. maize samples has been shown to be similar in some years(Marasas et al., 2004); however, Guatemalans typically consume moremaize than Americans, resulting in significantly higher fumonisin expo-sures. Finally, other studies found a 50% decrease in the incidence ofNTDs with folic acid supplementation in Mexico (Martinez de Villarrealet al., 2002), suggesting that folic acid supplementation may offer someprotection against the adverse effects of fumonisin exposure.

D. Genetic risk factors for neural tube defects

In addition to environmental and nutritional risk factors, there is clearly agenetic susceptibility component that contributes to the incidence ofcongenital malformations. Neural tube defect risk has been shown tovary across ethnic groups, suggesting that genetic variation (polymorph-isms) may predispose particular individuals, or groups of individuals toNTDs. Several lines of evidence suggest a genetic component to this typeof birth defect. First, NTDs are associated with known genetic syndromes,including Meckel syndrome, anterior sacral meningomyelocele, and analstenosis. Secondly, in NTDs occurring without other syndromes, therecurrence risk for siblings is approximately 2–5%, which is up to a50-fold increase over that observed in the general population. Khouryet al. (1988) have shown that for a recurrence risk to be this high, anenvironmental teratogen would have to increase the risk at least100-fold to exhibit the same degree of familial aggregation, making agenetic component essentially required. Additionally, a variety of muta-tions leading to NTDs in mice have been documented, providing furtherevidence in support of a genetic factor in humans. To date, more than 80different murine loci predisposing to NTDs have been identified (Coppet al., 2003; Juriloff and Harris, 2000). The complex, early embryologicaldevelopment of NTDs presents fascinating challenges to the geneticist.One traditional approach to identifying disease genes in families has been


through genomic screening of multiplex pedigrees. Advances in geneticmarker availability, including the development of highly polymorphicmicrosatellite repeat markers (STRPs) and single nucleotide repeat poly-morphisms (SNPs), the dense genetic maps of microsatellites (Bromanet al., 1998) and of SNPs (Matise et al., 2003), the development of high-throughput genotyping methods (Ben Othmane et al., 1998), and innova-tions in statistical genetic linkage analysis (Gudbjartsson et al., 2000;Kruglyak et al., 1996; Kong and Cox, 1997; Martin et al., 2000; O’Connelland Weeks, 1995) have paved the way for large-scale investigations ofcomplex human diseases. Recently, a genomic screen of 44 multiplexpedigrees (those with at least two sampled affected individuals) wasperformed using highly polymorphic microsatellite repeat markers andidentified regions of interest on chromosomes 7 and 10 (Rampersaudet al., 2005). Occasionally, large multiplex pedigrees—those with threeor more affected individuals—are identified and such families can beinvaluable for identifying a gene associated with NTDs (Stamm et al.,2006). Presumably, the affected individuals, since related to one another,all share the same genetic variant. Once a putative causative geneticvariant is identified in such a family, its relevance in other families canbe quickly assessed.

Another common approach to characterizing the genetic influence of acondition is to assess biologically plausible candidate genes—geneswhose involvement in a process is suggested by the known function ofa gene. In NTDs, biologically plausible candidate genes might be thoseknown to be involved in neural tube closure in other animal systems, areknown to be important generally in early development, or are involvedwith folate metabolism. Folate metabolism pathway genes have oftenbeen examined for association with NTDs due to the well-establishedability of folic acid to reduce the risk of this debilitating birth defect by50–70%. Some studies have shown that the protective effect of folic acidmay be decreased in populations of Hispanic descent (Shaw et al., 1995;Suarez et al., 2000). Blood folate levels have a strong genetic componentwith an estimated heritability of 46% (Morrison et al., 1998), yet maternalfolate supplementation can only prevent 50–70% of NTDs (Chatkupt et al.,1994). Folic acid supplementation does not entirely eliminate recurrencerisk (Chatkupt et al., 1994; Milunsky et al., 1989), suggesting that additional,genetic factors are responsible for the development of this type of birthdefect. These non-folate responsive cases may represent highly geneticcases of NTDs (Scriver, 1985). The ability to identify those women geneti-cally predisposed to having a child with a neural tube defect, and whoserisk could be minimized by the use of folic acid supplements, would allowgenotype-directed pharmacogenetic interventions.

The mechanism by which dietary folate supplementation preventsNTDs is not understood (Prevention of Neural Tube Defects: Results of


the Medical Research Council Vitamin Study, MRC Vitamin StudyResearchGroup, 1991). Folic acid derivatives are essential for the synthesisof DNA, as well as for cell division, tissue growth, and DNA methylation(Morrison et al., 1998), and may also protect cells against oxidative dam-age. DNA methylation enables proper gene expression and chromosomestructure maintenance, both of which are critical in the developingembryo (Razin and Kantor, 2005). Low dietary folate levels could directlylimit folate availability to cells or indirectly disrupt methionine metabo-lism, thereby increasing homocysteine in the maternal serum (Rosenquistand Finnell, 2001). Either mechanism implicates folate receptor andmethionine–homocysteine regulatory genes. Thus, the study of variationin genes involved in folate metabolism makes good biologic sense. NTDsin humans result from the combined effects of genetic, nutritional,and environmental influences, and as such are a classic example of amultifactorial disorder. Identifying the genetic factors will be critical forcharacterizing the interactions between genes and the environment, andunderstanding these interactions will provide the basis for identifyingindividuals at greatest risk for having a child with a birth defect, as well asfacilitating the design of novel preventive strategies.

III. FUMONISIN EXPOSURES

A. Fumonisins and regulatory policy

Based on the adverse health effects observed in animals, the Center forFood Safety and Nutrition, U.S. Food and Drug Administration (FDA)issued industry guidance levels for fumonisins in maize in 2001(‘‘Guidance for Industry on Fumonisin Levels in Human Foods andAnimal Feeds’’; http://cfsan.fda.gov/�dms/fumongu2.html) statingthat human health risks associated with exposure to fumonisins werepossible. Recommended levels for foods range from 2 to 4 ppm totalfumonisins (fumonisin B1 plus fumonisin B2 plus fumonisin B3) depend-ing on the intended use of the maize and whether or not the kernels arewhole or degermed. Proposed limits (fumonisin B1 plus fumonisin B2) inEurope, (effective October 2007), range from 0.2 to 2 ppm dependingupon the commodity and the targeted consumer (i.e., adults or infants)(Commission of European Communities, 2005). The International Agencyfor Research on Cancer (IARC, 2002) classified fumonisin B1 as ‘‘possiblycarcinogenic to humans (class 2B)’’ and in 2001 the Joint FAO/WHOExpert Committee of Food Additives (JECFA) recommended a provi-sional maximum tolerated daily intake (PMTDI) of 2 mg/kg body weightfor fumonisin B1, fumonisin B2 and fumonisin B3, ‘‘alone or in

http://www.cfsan.fda.gov/dms/fumongu2.html


combination’’ (Bolger et al., 2001), and suggested that further research wasneeded to examine the teratogenic potential of this compound.

B. Measurements of fumonisin exposure

One of the difficulties in studying the environmental component to birthdefects is that the time of ‘‘injury’’ precedes the occurrence of the condi-tion by weeks or months. In humans, neural tube closure is complete byapproximately 28 days after conception, frequently before a woman evenknows she is pregnant. Prospective studies of exposure would requireextensive population screening to identify the occasional affected caseand would require years to develop a large enough sample size for theanalysis of gene–environment interactions. Thus, use of biomarkers attime points after the event of interest (e.g., pregnancy) may be a usefulapproach for characterizing environmental influences (Krapels et al., 2004;Leck, 1983; Murray et al., 1997; Prakash et al., 2002; Weis et al., 2005).Fumonisin B1 is rapidly cleared from the system and can only be detectedin human urine and feces for up to 3–5 days after ingestion (Sewram et al.,2003; Riley, unpublished data). In the absence of modern technologyfor prenatal monitoring (i.e., ultrasound), fetal malformations may notbe detected until the child is born. At this point, it is too late to accurat-ely determine the level of maternal fumonisin exposure that occurredduring the critical gestational window spanning neural tube closure(i.e., 3–4 weeks post-conception). A possible surrogate for direct fumoni-sin exposure is a rise in the sphinganine/sphingosine ratio, but thismeasurement is also limited to short-term detection (Sewram et al.,2003) unless exposure is fairly constant. Several studies in rodents haveshown that once elevated by a high dose, the levels of sphinganine ineither urine or kidney will remain significantly elevated by exposure to alow dose of fumonisin that would not ordinarily elicit an increase insphinganine (Enongene et al., 2002; Wang et al., 1999). For example, ratswere fed diets containing 10 ppm of fumonisin B1 for 10 days and thenchanged to either diets containing 0 or 1 ppm fumonisin B1 (Wang et al.,1999). In the rats consuming the 0 ppm diet, the urinary sphinganinereturned to control values (�0.2 nmol/ml) by day 20, whereas in theanimals changed to the 1 ppm fumonisin B1 diet, sphinganine was stillsignificantly elevated on day 20 (2.5 nmol/ml).

Chemical analysis of hair samples may also provide a method forexamining chronic mycotoxin exposures. In 2003, Sewram et al. (2003)reported that human hair testing could be used to detect fumonisins.After extraction and clean up, high performance liquid chromatographycoupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS)was able to detect fumonisin B1, fumonisin B2, and fumonisin B3 fromhuman hair samples (Sewram et al., 2003). However, these were


composite samples taken from barbershops in various regions ofSouth Africa. Thus, hair testing has not yet been employed to measureindividual fumonisin exposure level. Hair analysis has many potentialadvantages. Compared to urine and blood testing, which are only able todetect exposures from �2 to 4 days past, hair analysis provides a largerwindow of xenobiotic detection. Exposure detection ranges fromweeks tomonths depending upon the length of the hair (Kintz, 2004) and assess-ment of the hair in segments can be performed so that the segment mostclosely reflecting time-at-exposure is selected for study. Nonetheless, themethod for measuring fumonisin in hair has not been validated so thatlevels in hair can be used to predict past or ongoing exposure. Morerecently, a simple method for measuring fumonisin in urine samplesfrom women in a cohort recruited in Morelos County, Mexico hascorrelated urinary fumonisin B1 with tortilla consumption and suggeststhat urinary fumonisin B1 could be a valuable tool in investigating theassociated health effects of exposure (Gong et al., 2008).

As part of the evaluations of the cluster of NTDs along the Texas–Mexico border in 1990–1991 (Missmer et al., 2006), three different metricsto evaluate fumonisin exposure were considered. These metrics included(1) number of tortillas consumed during the first trimester (collected viaquestionnaire); (2) post-partum sphinganine/sphingosine ratio to estimatefumonisin exposure from maternal blood; and (3) nanograms of fumoni-sin ingested per day during the periconceptional period, estimated fromgrouped 6-month averages for sampled tortillas from homes andmarkets.The outcome of this study suggested that ingestion of maize-basedproducts in the amounts observed in Mexican-American diets were asso-ciated with increased risk for NTDs. All of the experimental findingswere robust across exposure metrics, suggesting that the questionnairedata were a realistic measure of fumonisin exposure, particularly whencoupled with estimates of seasonal variation in fumonisin levels.

IV. REPRODUCTIVE TOXICOLOGY OF FUMONISINS

A. Animal studies: Overview

Previous reproductive toxicology studies in laboratory animals examiningthe effects of prenatal exposure to fumonisin demonstrated a potentialrisk to the developing fetus. Studies using an aqueous extract of contami-nated maize-culture material of F. verticillioides reported that fumonisinwas developmentally toxic in hamsters (Floss et al., 1994; Penner et al.,1998). In addition, purified fumonisin B1 was shown experimentally tocause fetal toxicity in rats and mice (Collins et al., 1998; Reddy et al., 1996).In another study, pregnant CD1 mice treated with a semipurified extract


of F. verticillioides culture material exhibited a dose-dependent increase inthe number of resorptions and fetal abnormalities (Gross et al., 1994).However, in several of the studies, developmental abnormalities alsooccurred in the presence of maternal toxicity. In animal model systems,cultured rat embryos exhibited an increase in the incidence of NTDs(Flynn et al., 1997) following exposure to the hydrolyzed fumonisin B1,also known as aminopentol 1 (AP1), which is produced during the nixta-malization process used to make maize flour for masa and tortillas(Dombrink-Kurtzman et al., 2000). Sadler et al. (2002) reported an associa-tion between fumonisin B1 exposure and NTDs in murine embryo cultureand showed that folinic acid supplementation was able to ameliorate theteratogenic effects of fumonisin. Early gestational exposure to the myco-toxin also induces a high incidence of NTDs in offspring of LM/Bc mice(Gelineau-van Waes et al., 2005), and a somewhat lower, but biologicallysignificant incidence of NTDs in embryos of CD1mice followingmaternalintraperitoneal injections of purified fumonisin B1 (Voss et al., 2006). In theinbred LM/Bc mouse strain, maternal administration of 20 mg/kg/dayfumonisin B1 on gestational days 7.5 and 8.5 (intraperitoneal injection)resulted in NTDs (exencephaly, which is the mouse equivalent of anen-cephaly in humans) in all litters examined (n ¼ 10 litters), and in 79% ofthe exposed embryos. Similar to the results obtained in murine embryoculture (Sadler et al., 2002), maternal supplementation with folic acid wasable to reduce the in vivo incidence of fumonisin-induced NTDs in LM/Bcmice by approximately 50% (Gelineau-van Waes et al., 2005). Preliminaryresults using the LM/Bc mouse model (n¼ 3 l) indicate that the oral routeof fumonisin exposure (20 mg/kg of fumonisin B1 on E7.5 and E8.5;equivalent to 80–120 mg of fumonisin B1/kg diet) also results in NTDs(20%) in exposed embryos.

B. Mouse models of fumonisin-induced neural tube defects

1. Fumonisin B1-induced neural tube defects in culturedmouse embryos

Sadler et al. (2002) assessed the effect of fumonisin B1 treatment ondevelopment of neurulating mouse embryos in culture. Fumonisin B1

treatment resulted in a dose-dependent increase in the percentage ofembryos exhibiting NTDs (Sadler et al., 2002). Concurrent alterations inlevels of sphingoid bases were also found. Supplementation of theembryos with high levels of folate during the fumonisin B1 treatmentreduced the incidence of NTDs without correcting the sphingolipidabnormalities, suggesting that the mycotoxin influenced neural tubematuration by affecting the function of the folate receptor. Cumulatively,these findings provided a conceptual framework to account for how


exposure to fumonisin B1 could increase the risk of NTDs by disruptinglipid rafts through sphingolipid depletion and, consequently, impairedfolate receptor function.

2. Maternal fumonisin exposure and neural tube defectsFurther evidence of a causal relationship between gestational fumonisinexposure and altered embryonic morphogenesis was provided by theestablishment of an in vivo mouse model (Gelineau-van Waes et al.,2005). Early gestational administration of the purified fumonisin B1 myco-toxin to pregnant mice of the inbred SWV and LM/Bc mouse strainsresulted in an increased incidence of fetal resorptions and/or malforma-tions (Gelineau-van Waes et al., 2005). Although reproductive toxicologystudies in which purified fumonisin B1 was administered to hamsters(Penner et al., 1998) or fumonisin-contaminated culture material wasadministered to mink (Powell et al., 1996) reported a decrease in birthweight and/or an increase in fetal death as the primary finding(s), veryfew LM/Bc embryos/fetuses were resorbed, even at the highest dose offumonisin administered. Rather, a significant proportion of fumonisin-exposed LM/Bc embryos failed to complete neurulation. The number ofaffected fetuses per litter increased as the dose of fumonisin wasincreased, and at the highest dose administered (20 mg/kg/day on E7.5and E8.5), the litters of all treated dams were positive for NTDs, and 79%of the exposed LM/Bc fetuses were exencephalic (Gelineau-van Waeset al., 2005) (Fig. 5.2).

BA

ExencephalicControl

FIGURE 5.2 (A) Image of a normal (vehicle control) E10.5 embryo; (B) image of an E10.5

exencephalic embryo collected from an LM/Bc dam that was treated with purified

fumonisin B1 early in gestation (20 mg/kg i.p. on E7.5 and E8.5).


The same fumonisin treatment regime performed in mice from thehighly inbred SWV strain, however, yielded very different results. At thehighest fumonisin B1 dose administered (20 mg/kg/day on E7.5 andE8.5), approximately 15% of the implants were resorbed, and only oneexencephalic fetus was observed in the 10 SWV litters examined. Sphin-ganine levels were elevated in the placentas and embryos of both strains,although to a much greater extent in the LM/Bc mice, suggesting thatgenetics plays a role in the observed differences in sphingolipid metabo-lites and pregnancy outcome after fumonisin exposure. Observationsfrom preliminary feeding studies (summarized in Voss et al., 2006) areconsistent with the concept of strain-dependent differences. In thesestudies, LM/Bc and CD1 mice were fed fumonisin-contaminated dietsbeginning 5 weeks before mating and continuing throughout gestation.At a dietary fumonisin B1 concentration of 150 ppm, late fetal deathsoccurred in two of the nine (22%) CD1 litters, and the incidence of latefetal deaths in affected litters ranged from 40 to 64%. No NTDs werefound in the CD1 mice. In contrast, no late fetal deaths were found inthe five LM/Bc litters, but one LM/Bc litter was positive for NTDs.The results from the studies in LM/Bc and SWV mice, in conjunctionwith the preliminary results of the feeding studies comparing the LM/Bcand CD1 strains clearly point to an as yet unknown genetic susceptibilitycomponent with respect to risk for NTDs following maternal fumonisinB1 exposure.

In the LM/Bc mouse strain, maternal fumonisin exposure resulted indecreased expression of folate receptor (Folr1) protein in the yolk sacmembrane and neuroepithelial cells of developing embryos. In addition,reduced levels of 3H-folate uptake by neurulating embryos demonstratedthat fumonisin depletion of glycosphingolipids (gangliosides) had animpact on both expression and function of theGPI-anchored folate receptor.Moreover, similar to the results observed in the embryo culture model offumonisin exposure, supplementing pregnant dams with high levels offolic acid (50 mg/kg/day on E0.5–E9.5) afforded some protection againstfumonisin teratogenicity, and reduced the incidence of NTDs from 79 to50% in exposed LM/Bc fetuses (Gelineau-van Waes et al., 2005).

V. MECHANISMS OF FUMONISIN TOXICITY

A. Structural considerations

The chemical structure of fumonisin (Fig. 5.1B) is remarkably similar tothat of the sphingoid bases sphinganine (Sa) and sphingosine (So), andfumonisin has been shown to inhibit the enzyme ceramide synthase inde novo sphingolipid metabolism (Wang et al., 1991). Ceramide synthase


catalyzes the formation of ceramide from the condensation of fatty-acylCoA and sphinganine (Sa) or sphingosine (So). Inhibition of ceramidesynthase by fumonisins is competitive (Merrill et al., 2001; Riley and Voss,2006; Riley et al., 2001, 2006) and likely involves the tricaballylic acid andamine groups, which occupy the enzyme’s respective fatty-acyl CoA andthe sphingoid base binding sites (Merrill et al., 2001). Experimentalevidence indicates that the primary amine is essential for the fumonisinmolecule’s ceramide synthase inhibitory activity (Lemke et al., 2001;Norred et al., 2001), which is believed to be the mechanistic ‘‘trigger’’ fortoxicity. Inhibition of ceramide synthase results in an accumulation ofupstream sphingoid bases and sphingoid base-1-phosphates, and a deple-tion of downstream complex glycosphingolipids (Fig. 5.3). To date, atleast 28 analogues of fumonisin have been identified (Reeder et al.,2002). Fumonisin B1 (FB1) is the most common and, from a toxicologicalstandpoint, the most thoroughly studied. Structures of the less common

Palmitoyl CoA + serine

Sphinganine

Glucosylceramide

Lactosylceramide

GangliosidesGangliosides

Sphinganine-1-P

Fumonisin Ceramide synthase

Ceramide

Fumonisin inhibits de novo sphingolipid biosynthesis Fumonisin inhibits de novo sphingolipid biosynthesis

Sphingosine Sphingosine-1-P

GPI-anchored folatereceptor (folr1)

S1P receptors

Lipid raft

FIGURE 5.3 Simplified schematic of the de novo sphingolipid biosynthetic pathway.

Fumonisin inhibits the enzyme ceramide synthase, resulting in (1) an accumulation of

sphinganine, which is subsequently phosphorylated to form bioactive sphinganine-1-

phosphate, a ligand for the G protein-coupled S1P receptors; and (2) a depletion of

downstream glycosphingolipids (gangliosides), important components of ‘‘lipid rafts’’ in

which GPI-anchored receptors such as the folate receptor (Folr1) are found.


fumonisin B2, and fumonisin B3, differ from that of fumonisin B1 by thenumber and position of hydroxyl groups on the backbone.

Base hydrolysis removes the tricarballylic acid groups yielding thecorresponding hydrolyzed fumonisins (HFB1, HFB2) (Dombrink-Kurtzman et al., 2000; Merrill et al., 2001). Hydrolyzed fumonisins formduring the traditional corn cooking (in alkaline water) method known asnixtamalization that is used to make masa for tortillas (Dombrink-Kurtzman et al., 2000; Palencia et al., 2003). Hydrolyzed fumonisin B1

inhibits ceramide synthase less effectively in vitro (Merrill et al., 2001)and, in contrast to fumonisin B1, was not toxic when fed to female micefor 28 days (Howard et al., 2002). Likewise, N-(acetyl)-fumonisin B1 andN-(carboxymethyl)-fumonisin B1 which, like N-(1-deoxy-D-fructose-1-yl)fumonisin B1, lack a free primary amine group, were not toxic and didaffect sphingolipid metabolism when fed to female mice (Howard et al.,2002). Heating fumonisin B1 with reducing sugars such as glucose yieldsbrowning reaction products such as N-(1-deoxy-D-fructose-1-yl) fumonisinB1 and N-(carboxymethyl)-fumonisin B1 (Lu et al., 2002). FumonisinB1 also forms conjugates when heated with model starch or proteincompounds (Humpf and Voss, 2004). In this case, however, binding likelyoccurs through the tricarballylic group. The extent to which fumonisinbinds to foodmatrices and the bioavailability of matrix-bound fumonisinsis unknown. However, the presence of matrix bound (or ‘‘hidden’’)fumonisins in maize-based cereal (Kim et al., 2003), and alkali-processedfood (Park et al., 2004) has been demonstrated.

B. Role of sphingolipids in fumonisin toxicity

Several biochemical modes of action have been postulated to explainfumonisin-induced toxicity and carcinogenicity in animals, but the pri-mary hypothesis involves perturbation of sphingolipid metabolism(Bolger et al., 2001; IARC, 2002; Wang et al., 1991; WHO, 2000). Sphingo-lipids are important structural components of the plasma membrane andalso function as intra- and intercellular signaling molecules (Chalfant andSpiegel, 2005; Merrill et al., 2001). The toxic effects of fumonisin appear todepend on disruption of various aspects of lipid metabolism, membranestructure, and signal transduction pathways mediated by lipid secondmessengers. The demonstrated effects include altered rates of cellproliferation vs. apoptosis, altered cell–cell communication and cell adhe-sion, oxidative stress, and modulation of gene expression (Abel andGelderblom, 1998; Bhandari et al., 2002; Seefelder et al., 2003). The biomo-lecular events linking ceramide synthase inhibition, disruption of sphin-golipid metabolism, and toxicity have not been elucidated andmechanistic roles for oxidative damage (Abel and Gelderblom, 1998;Kouadio et al., 2005; Lemmer et al., 1999; Sahu et al., 1998), critical changes


in cell fatty acid composition (Gelderblom et al., 2001), up- or down-regulation of transcription or post-transcriptional stabilization of cyto-kines and other signaling molecules (Bhandari et al., 2002; Bondy et al.,2000; Gelderblom et al., 2001; Merrill et al., 2001; Sharma et al., 2003) havealso been proposed. The extent to which such events occur independentlyor secondary to sphingolipid metabolism disruption is unknown. Asidefrom influencing apoptosis and mitosis (Dragan et al., 2001; Howard et al.,2001a), fumonisin interferes with cell–cell or cell–matrix adhesion andmembrane permeability, perhaps through sphingolipid dependentmechanisms (Gon et al., 2005; Pelagalli et al., 1999; Ramasamy et al.,1995). It is noteworthy that detachment and sloughing of tubule epithelialcells is a feature of fumonisin-induced renal disease (Hard et al., 2001).In addition, complex sphingolipids are important for the uptake andtrafficking of folate (Stevens and Tang, 1997) via the GPI-anchored folatereceptor.

Apoptosis and mitosis in liver (hepatocytes) and kidney (proximaltubule epithelium of the outer medulla) are early consequences of fumo-nisin exposure (Bolger et al., 2001; Dragan et al., 2001; Howard et al., 2001a;Voss et al., 2001). They occur simultaneously and it is proposed that animbalance between cell death and increased regenerative pressurethrough continuous mitosis is a critical component of fumonisin’s carci-nogenic mode of action, especially in the kidney (Dragan et al., 2001;Howard et al., 2001a). In this regard, the survival and replication ofdamaged cells escaping apoptosis is likely critical for renal carcinogene-sis. Sphingoid bases and other sphingolipids are signaling molecules forapoptosis and mitosis (Merrill et al., 2001; Taha et al., 2006). Specifically,sphingosine and ceramide are pro-apoptotic, growth inhibitory and cyto-toxic whereas sphingosine-1-phosphate (S1P) exerts pro-growth andanti-apoptotic effects. Sphinganine causes apoptosis in LLC PK1 kidneycells in vitro (Kim et al., 2001; Riley et al., 1999; Yu et al., 2001), possiblythrough a calmodulin-dependent mechanism (Kim et al., 2001). Theimportance of elevated levels of free sphinganine in fumonisin-inducedapoptosis has been demonstrated using myriocin, a potent inhibitor ofserine palmitoyltransferase that abolishes the fumonisin-induced increasein free sphinganine and the increased apoptosis (Riley et al., 1999) Additionof exogenous sphinganine to the incubation medium also enhanced thetoxicity of fumonisin to Chinese hamster ovary (CHO) cells, which arerelatively resistant to the sphingolipid and pro-apoptotic effects of fumo-nisin (Yu et al., 2001). Thus, the overall metabolic balance of the varioussphingolipids is likely a critical determinant of whether cells undergoapoptosis, remain quiescent, or replicate and, in this regard, the ratio ofS1P to ceramide is likely to be important (Spiegel and Milstien, 2002).

Sphingolipid metabolism is also affected by cytokines and othersignaling molecules. Tumor necrosis factor a (TNFa) is a cytokine that


exerts both pro- or anti-apoptotic effects and has been shown to modifythe extent of fumonisin-induced liver injury in mice (Sharma et al., 2002),perhaps through a mechanism that involves NF-kB (Gopee and Sharma,2004). Enhancement of liver apoptosis (compared to wild-type mice) inTNFa knockout mice has been observed and was likely affected throughover-expression of Fas ligand, indicating that Fas-dependent pathwaysalso mediate fumonisin-induced apoptosis (Sharma et al., 2003). Activa-tion of neutral sphingomyelinase by TNFa could also enhance apoptosisby increasing cell ceramide or sphingosine levels or, alternatively, pro-mote cell survival in the event that significant amounts of sphingosineand, ultimately, S1P were formed from the sphingomyelinase-generatedceramide pool. Like TNFa and possibly Fas ligand, other cell surfacereceptor agonists influence cell survival and replication by activatingsphingomyelinase, ceramidase or sphingosine kinases, thereby increasingthe production of ceramide, sphingosine, and S1P, respectively (Merrillet al., 2001; Spiegel and Milstien, 2002).

C. Fumonisin inhibition of de novo sphingolipid metabolism

Fumonisin exposure has been shown to result in subsequent alterations inthe major pools of sphingolipids, including increased concentrations offree sphingoid bases and their 1-phosphate metabolites, and decreasedbiosynthesis of ceramide and downstream glycosphingolipids (Merrillet al., 2001; Riley et al., 1996) (Fig. 5.3). Sphinganine levels accumulaterapidly following inhibition of ceramide synthase, providing a biomarkerfor fumonisin exposure that has been validated in vitro, as well as intissue, serum, and urine (Norred et al., 1997; Riley et al., 1993; Wanget al., 1991). The significantly elevated levels of sphinganine observed inLM/Bc maternal and fetal tissues following fumonisin exposure areindicative of fumonisin inhibition of the enzyme ceramide synthase.The marked increase in sphinganine in embryonic tissue following earlygestational exposure to fumonisin (E7.5 and E8.5) suggests that the toxincrosses the placenta and that there is an effect at the level of the embryothat cannot simply be attributed to maternal toxicity. At this early timepoint, the placenta is not well developed, leaving the embryo potentiallyvulnerable to teratogenic insult.

Fumonisin inhibition of ceramide synthase leads to the accumulationof free sphinganine and sphingosine in tissues, serum and urine in a widerange of species including catfish, trout, poultry cattle, horses, rabbits,and mice (Bolger et al., 2001; Merrill et al., 2001; Riley et al., 2001). Freesphingosine or sphinganine are subsequently phosphorylated by sphingo-sine kinase to form the corresponding sphingoid base-1-phosphates(S1P or sphinganine-1-P), which may then be dephosphorylated byeither sphingosine phosphate phosphatases (Sgpp1, Sgpp2) or lipid phosphate


phosphatases (Ppap2a, Ppap2b), or alternatively, irreversibly degraded bysphingosine phosphate lyase (Sgpl) to form phosphoethanolamine andhexadecenal (Fig. 5.4). Phosphoethanolamine is a precursor for thebiosynthesis of phosphatidylethanolamine, a compound that has alsobeen shown to accumulate following fumonisin exposure both in vitroand in vivo. Accumulation of bioactive S1P or sphinganine-1-P in serum ortissues of horse (Constable et al., 2005), swine (Piva et al., 2005), rats (Rileyand Voss, 2006), adult mice (Suzuki et al., 2007), ducks (Tardieu et al.,2006), and livers of mouse embryos (Riley et al., 2006) has been reportedafter fumonisin exposure.

The in vivo sphingolipid effects of fumonisins are reversible (Merrillet al., 2001). Within 3 weeks after replacing fumonisin-contaminated dietwith control feed, the elevated tissue sphinganine (Sa) and sphingosine(So) concentrations and Sa/So ratios (used as a biomarker of fumonisinexposure) of rats decreasedmarkedly, almost reaching pretest values, andthe histological appearance of the liver and kidney returned (loss offumonisin-linked lesions) to normal (Voss et al., 1998). In ponies, serumsphingoid base concentrations and serum chemistry indicators of hepaticinjury (alanine and aspartate transaminase activities) rose and felltogether as the fumonisin contaminated feed was given to and withdrawnfrom the animals (Wang et al., 1992).

Sphingosine kinase 1 (sphk1)Sphingosine kinase 2 (sphk2)

Sphingosine phosphate phosphatases (Sgpp1, Sgpp2)Lipid phosphate phosphatases (Ppap2a, Ppap2b)

Sphingosinephosphate lyase

TNFa, IFNgPDGF, VEGFEstrogen

Sphingosine(sphinganine) Sphingosine-1-P

(sphinganine-1-P)Ethanolamine-P

S1P establishes chemokine gradients important for cell migration

Stored in RBCs, platelets

FIGURE 5.4 Biochemical pathway illustrating (1) phosphorylation of sphingosine or

sphinganine by the enzymes sphingosine kinase 1 (sphk1) and/or sphingosine kinase

2 (sphk2); (2) dephosphorylation of sphingosine-1-phosphate (S1P) or sphinganine-1-

phosphate by sphingosine phosphate phosphatases (sgpp1, sgpp2) and/or lipid phos-

phate phosphatases (ppap2a, ppap2b); and (3) irreversible degradation of S1P or sphin-

ganine-1-phosphate by sphingosine phosphate lyase (sgpl) to form

phosphoethanolamine. Both S1P and sphinganine-1-phosphate are stored in red blood

cells and platelets, and function as ligands for the G protein-coupled S1P receptors.


D. Fumonisin and accumulation of bioactive sphingoidbase-1-phosphates

Although sphingolipids are important structural components of the exo-plasmic leaflet of the plasma membrane, their role in mediating cellsignaling processes has also recently been recognized. Sphingoid basemetabolites, such as sphingosine-1-phosphate (S1P) are involved in signaltransduction cascades that regulate key biological processes (Futermanand Hannun, 2004; Spiegel and Milstien, 2002, 2003; Stunff et al., 2004;Watterson et al., 2003). S1P turnover is mediated either by reversibledephosphorylation to sphingosine or by irreversible cleavage to ethanol-amine phosphate and fatty aldehyde (Zhou and Saba, 1998). The dynamicbalance between S1P (pro-survival) and ceramide/sphingosine (pro-apoptosis) has been proposed to form a ‘‘cellular rheostat’’ that deter-mines cell survival vs. cell death (Spiegel andMilstien, 2002). S1P has bothextracellular and intracellular signaling functions, and functions as aligand on cell membrane S1P receptors, a family of five G-protein-coupledreceptors (S1P1–5; previously endothelial differentiation gene (Edg) recep-tors) that play important roles in such diverse functions as cell survival/proliferation, differentiation and migration, as well as angiogenesis andendothelial barrier integrity (Brinkmann, 2007; Dev et al., 2008; Takabeet al., 2008) (Fig. 5.5). SIP gradients and S1P receptors also play an impor-tant role in mediating immune cell migration to sites of inflammation(Brinkmann, 2007; Cyster, 2005). The phenotypes observed in S1P recep-tor knockout mice indicate a certain degree of functional redundancybetween the various receptor isoforms, but also establish a critical rolefor S1P receptors in angiogenesis and neurogenesis during embryonicdevelopment (Kono et al., 2004). Inactivation of S1P1 in mouse modelsresults in embryolethality, and the offspring die between E12.5 andE13.5 due to severe hemorrhage. S1P1,2,3 are expressed in neural tissue(especially the telencephalon), S1P5 is expressed in CNS white matter(oligodendrocytes), and both sphingosine kinases (Sphk1, Sphk2) areexpressed in the developing brain and spinal cord (Dev et al., 2008;Kono et al., 2004; Mizugishi et al., 2005), suggesting a role for S1Preceptor-mediated signaling in neural tube closure. Although there is noobvious phenotype when either Sphk1 or Sphk2 alone is inactivated in aknockout mouse model, Sphk1/Sphk2 double knockout embryos die byE12.5 with severe vascular and neural abnormalities, including failure ofneural tube closure (Mizugishi et al., 2005). Interestingly, Sphk1-/-/Sphk2þ/�mutant mice are infertile, and follow-up experiments suggest a criticalrole for early gestational activation of de novo sphingolipid metabolism inthe maintenance of pregnancy (Mizugishi et al., 2007).

Sphinganine-1-P also functions as a ligand for S1P receptors, althoughthe binding affinity for the different S1P receptor isoforms differs from

AC c-Src

RaccAMP ERK

Gq

PLCCa2+

PKC

Cell proliferation

Cdc 42

S1P1 S1P2 S1P3 S1P4 S1P5

Rho

Rock, PLDcadherin

Detachment, retractioncell Migration

G12/13

Gi/o

Pi3K

Akt

Adhesion, extensioncell migration

Cell cycle regulation

eNOS

Cell shape changes

FIGURE 5.5 There are five isoforms of S1P receptors (S1P1–5), a family of G protein-

coupled membrane receptors previously known as ‘‘Edg’’ receptors (endothelial differ-

entiation gene). Ligand binding and activation of S1P receptors initiates multiple intra-

cellular signaling cascades involved in cellular proliferation, differentiation, survival and

migration.


that of S1P (Im et al., 2001). The high levels of sphinganine and sphinga-nine-1-P that accumulate after fumonisin exposure may therefore elicitdifferent downstream signaling cascades than S1P due to differentialbinding and activation of the various S1P receptor isoforms. Moreover,high concentrations of ligand have been shown to cause internalizationand downregulation of S1P cell surface receptors, thereby acting as a‘‘functional antagonist’’ rather than as an agonist (Brinkmann, 2007).Further differences in downstream signaling effects include a role forS1P in intracellular signaling, which apparently is not observed forsphinganine-1-phosphate (Taha et al., 2006).

The activation of S1P receptors reduces renal and mesenteric bloodflow in rats (Bischoff et al., 2001), and it can be speculated that decreasedblood flow and hypoxia contribute to the extreme sensitivity of ratkidneys to fumonisins. Fumonisin exposure has also been shown todecrease cardiac output, heart rate, and mean arterial pressure as wellas increase pulmonary arterial pressure and pulmonary resistance in pigs(Constable et al., 2000, 2003), findings which suggest that left-sided cardiacinsufficiency underlies porcine pulmonary edema. Plasma sphingoid base


concentrations (Constable et al., 2003) and serum S1P (and sphinganine-1-P)(Piva et al., 2005) increase in fumonisin-exposed pigs. Sphingosine blocksL-type calcium channels and ryanodine receptors in rabbits (Sabbadiniet al., 1992). Therefore, one possible mechanism for fumonisin-inducedporcine pulmonary edema involves interference with calcium ion trans-port, which in turn is critical for cardiac function and vascular tone(Constable et al., 2003). Both sphinganine and sphingosine relaxedphenylephrin-contracted and uncontracted thoracic aortic and pulmo-nary arterial rings in vitro (Hsiao et al., 2005) and, while S1P had littleeffect on the aortic rings, it increased the tension of uncontracted pulmo-nary arterial rings. From these observations, it can be further speculatedthat plasma S1P also contributes to pulmonary edema by increasingpulmonary arterial tension, likely through a mechanism involving S1Preceptors. S1P1,2,3 receptors are known to mediate endothelial barrierintegrity and vascular permeability (Brinkmann, 2007; Gon et al., 2005;Sanchez et al., 2007; Singleton et al., 2005), while agonism of S1P3 receptorsresults in sinus bradycardia (Sanna et al., 2004).

The process of neural tube closure requires the integration of multiplesignaling cascades involved in cell proliferation, migration, and differen-tiation. It is therefore likely that fumonisin inhibition of ceramidesynthase, resulting in elevated levels of sphinganine-1-phosphate(as opposed to tightly regulated levels of S1P) may alter the dynamics ofS1P receptor-mediated signaling pathways involved in coordinatingthese events, thereby contributing to the failure of neural tube closure.

E. Fumonisin depletion of glycosphingolipids and disruptionof folate transport

In addition to an accumulation of upstream sphingoid bases and sphin-goid base-1-phosphates, depletion of the downstream, complex sphingo-lipid metabolites of ceramide (i.e., gangliosides) has also been shown tooccur after fumonisin exposure (Fig. 5.3). Stevens and Tang (1997)reported in an in vitro study that fumonisin-induced depletion of glyco-sphingolipids inhibited vitamin uptake via the GPI-anchored folatereceptor and suggested that dietary exposure to fumonisin could there-fore adversely affect folate uptake, and potentially compromise cellularprocesses dependent on this vitamin. The high-affinity folate receptor(murine Folr1; human FRa) is a GPI-anchored protein found associatedwith detergent-insoluble membranemicrodomains rich in cholesterol andsphingolipids known as ‘‘lipid rafts’’ (Elortza et al., 2003). These raftscreate domains of reduced fluidity in membranes and arise from theshared biophysical properties of sphingolipids and cholesterol (Brownand London, 2000; Rietveld and Simons, 1998). They are hypothesized tofunction in membrane and endocytic sorting of proteins in polarized


epithelial cells, as foci for recruiting signaling molecules to the plasmamembrane, and in cell adhesion (Ilangumaran et al., 1999; Lin et al., 1998;Simons and Ikonen, 1997). A role for membrane rafts in regulating thefolate receptor was suggested by studies in which the depletion of cellularcholesterol was found to impair the uptake of 5-methyltetrahydrofolateinto cells (Chang et al., 1992).

Gangliosides stabilize the association of GPI-anchored proteins withthe outer leaflet of cell membranes (Watanabe et al., 2002) and are impor-tant components of specialized membrane microdomains, or ‘‘lipid rafts’’in which GPI-anchored proteins cluster. Ganglioside GM1 has beenshown to be highly expressed in the developing brain and spinal cord(Silani et al., 1993), and decreased levels of ganglioside GM1a and GD1ahave previously been reported in anencephalic human fetal brain (Cacic,1995). In the LM/Bc in vivomousemodel of fumonisin exposure, maternalsupplementation with ganglioside GM1 was more effective than folate inpreventing NTDs. Expression of ganglioside GM1 in plasma membranemicrodomains of the developing neuroepithelium may be necessary forcoordinating/orchestrating protein–lipid interactions and signal trans-duction events involved in normal neural tube closure. GPI-anchoredproteins such as the folate receptor associate with gangliosides in thetrans-golgi network, prior to sorting and transport to the plasmamembrane.Association of Folr1 with ganglioside GM1 may be important for deliveryof the GPI-anchored folate receptor to the apical plasma membrane inneuroepithelial cells. In addition, co-localization of Folr1 with GM1 inlipid rafts appears to facilitate receptor function, and promote normalneural tube closure. Different gangliosides associate with different pro-teins in ‘‘lipid rafts’’ in a cell-type specific manner, and the rafts functionas specialized platforms for the initiation of signal transduction (Brownand London, 1998; Hoessli et al., 2000). Although the specific interactionsbetween ganglioside GM1 and Folr1 and their precise role in neural tubeclosure are unknown at this time, several hypotheses are currently underinvestigation. Since the GPI-anchored folate receptor has previously beenisolated from lipid raft microdomains enriched in signaling molecules,including kinases/phosphatases, heterotrimeric G proteins, and smallGTP-binding proteins (Elortza et al., 2003; Foster et al., 2003; Miotti et al.,2000), it seems reasonable to hypothesize that Folr1 may play a criticalrole in signal transduction pathways necessary for cell survival, prolifer-ation, and activation of the actin cytoskeleton necessary for normal neuraltube closure.

Fumonisin inhibition of de novo sphingolipid biosynthesis (Merrillet al., 2001; Riley et al., 1996), resulting in depletion of complex glyco-sphingolipids, disruption of lipid rafts, and compromised folate transportvia Folr1, which may have significant implications with respect todevelopmental anomalies such as NTDs (Hansen et al., 2003; Piedrahita


et al., 1999; Rothenberg et al., 2004; Saitsu et al., 2003). The inhibition offolate uptake by cells is expected to have the same consequences as adietary deficiency in this vitamin. Therefore, the possibility that inhibitionof folate receptor-mediated vitamin uptake contributes to the develop-ment of NTDs has been investigated. Mice in which expression of thegene encoding the folate receptor (Folr1) was knocked out were found tohave defects in neural tube development and die before birth (Piedrahitaet al., 1999). This phenotype could be reversed by supplementing thepregnant knockout mice with excess folate, suggesting that the loss ofvitamin uptake mediated by the folate receptor was responsible for thedevelopmental defects (Piedrahita et al., 1999). Reduction of folate receptorexpression in mouse embryos with antisense technology (Hansen et al.,2003) or blocking Folr1 activity in pregnant rats with antibodies to thistransporter (da Costa et al., 2003) both resulted in impaired embryogenesisthat could be reversed with excess folate supplementation. Rothenberget al. (2004) found autoantibodies against this transporter in serum from asubset of womenwith neural tube defect-affected pregnancies, suggestingthat antibody-induced inactivation of the folate receptormay be responsiblefor some NTDs in humans. Cumulatively, these findings indicate thatimpairment of folate receptor function could result in NTDs.

Fumonisin B1-induced depletion of the major sphingolipids to40–65% of their normal levels in Caco-2 cells (which express high levelsof the folate receptor) was found to significantly impair 5-methyltetrahy-drofolate uptake (Stevens and Tang, 1997). While this result demonstratesthat sphingolipid depletion inhibits vitamin uptake, it does not indicate aspecific effect on the folate receptor because 5-methyltetrahydrofolate canbe taken up by both the RFC1 and the GPI-anchored folate receptor (Folr1)(Corona et al., 1998; Miotti et al., 1997). Therefore, to specifically determinethe effects of fumonisin B1 treatment on the GPI-anchored folate receptor,uptake was measured with folic acid. This synthetic folate is bound by thefolate receptor (Folr1) with an affinity that is approximately 100,000 timesgreater than that of the RFC1, and folic acid is transported almost exclu-sively by the former (Goldman, 1971; Kamen and Capdevila, 1986). Folatereceptor function was found to be inhibited by 20–35% in cells derivedfrom a human placental choriocarcinoma ( JAR), monkey kidney epithe-lium (MA-104), and a human adenocarcinoma (Caco-2) that had beendepleted of roughly 40% of their sphingolipids by fumonisin B1 treatment.Other uptake processes, including receptor-mediated and facilitativetransport, were not affected by this treatment (Stevens and Tang, 1997).Thus, depletion of cellular sphingolipids specifically inhibits folatereceptor-mediated vitamin uptake.

Folate receptor function can be altered by changing the level of thisprotein, its activity, or both. To determine if folate receptor levels were


affected by sphingolipid depletion, the amount of this transporter wasmeasured in fumonisin B1-treated cells. Total cellular folate receptorwas measured by quantifying specific folic acid binding in detergentsolubilized cells while the amount of receptor active in endocytosisand available for folate uptake was measured in intact cells under condi-tions where endocytosis was maintained. Fumonisin B1-induced sphin-golipid depletion did not change total cellular levels of folate receptorsignificantly in Caco-2, JAR and MA-104 cells. However, this treatmentdid decrease the amount of receptor active in endocytosis by between25 and 40%. This decrease in the amount of folate receptor available forvitamin uptake could contribute to the fumonisin B1-induced inhibition ofthis transporter.

The effect of fumonisin B1 treatment on folate receptor function wasinvestigated by determining if sphingolipid depletion altered the endo-cytic kinetics of this transporter. These experiments were done usingFRaTb-1 cells, which were generated by stably transfecting CHO cellswith the GPI-anchored folate receptor and the transferrin receptor(Mayor et al., 1998). The movement of the folate receptor through theendocytic pathway was followed by using a fluorescent folic acid analogthat remained tightly bound to this protein. Fumonisin B1-treatment didnot significantly alter the rate at which the folate receptor wasinternalized. However, the recycling of this receptor to the cell surfacewas accelerated approximately threefold by sphingolipid depletion(Chatterjee et al., 2001). The recycling rate was also faster in cells depletedof cholesterol through treatment with the HMG-CoA reductase inhibitorcompactin (Chatterjee et al., 2001). The fact that reduction of either of themajor lipid species found in membrane rafts specifically perturbedthe recycling of the folate receptor suggests that association withthese domains regulates the intracellular sorting and trafficking of thisGPI-anchored protein. Thus, the inhibition of folate receptor-mediatedvitamin uptake caused by fumonisin B1-induced sphingolipid depletionappears to result from a combination of altered endocytic traffickingand reduced levels of this receptor available for folate uptake. The molec-ular mechanisms by which sphingolipid depletion causes these changesare unknown.

In summary, fumonisin mycotoxins exert a wide range of diverse andspecies-specific toxicological effects. The weight of evidence suggeststhat inhibition of ceramide synthase and disruption of sphingolipidmetabolism is the initial mechanistic event. The ensuing disruption ofthe metabolism of the sphingoid bases, sphingoid base 1-phosphates, andcomplex glycosphingolipids and the physiological processes modulatedby these molecules are critical for fumonisin toxicity.


VI. CONCLUSIONS

NTDs present a tremendous burden to human populations in rural areasof the world where maize is a dietary staple (Hendricks, 1999; Krombergand Jenkins, 1982; Marasas et al., 2004; Melnick andMarazita, 1998; Mooreet al., 1997; Ncayiyana, 1986; Xiao et al., 1990). The observed associationbetween consumption of fumonisin-contaminated maize and increasedincidence of NTDs in several world communities, coupledwith the existingexperimental evidence in animal models strongly supports the need forfurther investigation into the teratogenic potential of this compound.Epidemiologic data on dietary fumonisin exposures, including levels infoods, and daily food consumption, coupled with measurements of reli-able biomarkers of exposure are needed. These exposure assessments,along with information on maternal genetics and nutrition (i.e., maternalfolate status) as they relate to pregnancy outcome will help to establish acomprehensive understanding of the impact of fumonisin as a humanhealth hazard, and risk factor for birth defects. Animal feeding trialsincorporating dietary administration of the mycotoxin will help to estab-lish relevant ‘‘no observable adverse effect levels’’ (NOAEL), and mecha-nistic studies in animal models will help to identify and validatebiomarkers of exposure, and establish the complex biochemical, genetic,immune, and nutritional factors that contribute to increased susceptibilityto fumonisin-induced NTDs. Establishing the basic mechanisms underly-ing fumonisin toxicity and identifying polymorphisms in candidate genesthat contribute to susceptibility will facilitate our understanding of neuraltube defect risk in women exposed to this mycotoxin during early preg-nancy. Collectively, this information will also help in developing strate-gies for prevention. The experimental evidence suggests that maternalfolate supplementation is effective in ameliorating the teratogenic effectsof fumonisin. Maternal diet and nutritional status play a key role in fetaldevelopment, and strategies such as folate supplementation or folatefortification of foods may prove effective in reducing the incidence ofNTDs in communities likely to be consuming high levels of fumonisin-contaminated maize. Fumonisins are present at low levels in maizethroughout the world, but high levels of the toxin may occur, dependingon the environmental conditions and genetics of the host plant. Strategiesfor reducing the risk of fumonisin contamination in maize supplied to themarket include improved crop management practices, and improvedbreeding strategies, as well as transgenic approaches for increased ear-mold resistance (Betz et al., 2000; Duvick, 2001). Minimizing exposures tomycotoxins through enhanced agricultural practices and biotechnology,characterizing potential health risks and mechanisms of toxicity, andimproving maternal nutrition are all important strategies or considera-tions for reducing the neural tube defect burden in high risk humanpopulations.


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INDEX

A

Adherent invasive E. coli (AIEC), 9Anterior cingulated cortex (ACC), 81Anxiety disorders, 66

C

Carbon stable isotope ratio test (SIRA), 138C2Bbe1 epithelial cells, 7Chinese hamster ovary (CHO) cells, 161chlorinated PVC (cPVC) food-packaging, 38Conjugated linoleic acids (CLA), 10Contemporary maple syrup evaporator

ancillary equipment, 110arch, 108–109fuel source, 108pans, 109–110

Coping, 76

D

De novo sphingolipid metabolismbiochemical pathway, 164sphinganine levels, 163

DNA-microarray analysis, 4

E

Enterohaemorrhagic E. coli (EHEC), 4Extended shelf-life (ESL), 27, 52, 54

F

Folate binding protein 1 (Folr1), 150Folate receptor-alpha (FRa), 150Food allergy

biopsychosocial development, stressimpact, 80–81

comorbid anxiety disorders, 66developmental pathways

avoidant and minimizing strategies, 90awareness, 89emotional development, 86–87event control, 88parental views andmanagement, 91–92

precipitating events, 87transition points, 90–91

gender impactinfluence, 83–84vs. sex impact, 81

HRQL impact, researchdisease-specific measures, 74–75generic measures, 73–74

neurocognitive development, 67–68parental perceptions

developmental issues, 78IAT measures, 78–79

prevalence, mechanisms and clinicalmanifestations

allergic diseases, 69anaphylaxis, 72atopy, 68–69food hypersensitivity, 70–72

psychological burdenadaptational processes, 77coping, 76–77

researchbiopsychosocial framework, 94children, 68literature issues, 93

risk behaviorfactors, 85health belief models (HBMs), 86perception, 85

sex impactvs. gender impact, 81influence, 82–83

social support, 79–80Food allergy quality of life-parental burden

(FAQL-PB), 74–76Food-packaging interactions

consumer perception
interactions, 20–21sensory problem, 21
evaluation, 18packaging materials, 19quality improvement

antimicrobial ingredients, 53–54flavor and odor absorbers, 54–55

183

184 Index

Food-packaging interactions (cont.)
oxidation control, 55–56
quality protectionlight, 50–52moisture loss prevention, 52–53package characteristics, 49–50

yogurt package, 50scalping/sorption

color and appearance, 48polymers and, 49properties, 47–48

sensory effectsdiscrimination testing, 26evaluation, 23flavor chemistry, 22–23

gas chromatography–massspectroscopy (GC–MS), 25

negative sensory impacts, 24packaging materials, 22scalping/sorption, 24–25sensory methodology books, 26–27taints, 23–24

taintscontact materials, 27–45potential migrants, 27

technical representatives communication,20

threshold conceptdetection/absolute, 22sensory response, 21

Fumonisin exposure and toxicityanimal studies, 155–156bioactive sphingoid base-1-phosphates

fumonisin and accumulation, 164–166neural tube closure, 166

porcine pulmonary edema, 165–166signaling functions, 164SIP gradients and S1P receptors,

164–165chemical structural considerations

ceramide synthase, 159

corn cooking, 160de novo sphingolipid biosynthetic

pathway, 159–160de novo sphingolipid metabolism

biochemical pathway, 163sphinganine levels, 162in vitro and in vivo, 162–163

folate transport disruptionfolate uptake, 168receptor levels, 168–169

glycosphingolipids fumonisin depletion

GPI-anchored protein, 166LM/Bc in vivo mouse model, 167treatments, 168–169vitamin uptake, 169

measurements, 154–155and regulatory policy, 153–154reproductive toxicology

animal studies, 155–156mouse models, 156–158

sphingolipids, 160–161

G

GI tract mucins, 5Glycosylphosphatidylinositol (GPI), 150

H

Health belief models (HBMs), 85Health related quality of life (HRQL)

disease-specific measures, 74–76generic measures, 73–74

Human b-defensin-2 (hBD-2) geneexpression, 6

I

IBD. See Inflammatory bowel diseaseImmunomodulation, 8–10Implicit association test (IAT), 78Inflammatory bowel disease (IBD), 10Insulin-dependant diabetes mellitus

(IDDM), 74

L

Lactobacillus acidophilus R0052, 4–5Lactobacillus rhamnosus GG (LGG), 11Lactobacillus rhamnosus R0011, 5

M

Maple candy, 136Maple cream, 136Maple syrup

adulteration
bleaching treatment, 137–139carbon stable isotope ratio test (SIRA),
138screening method, 137–139

annual production, 110–111chemistry

composition values and ranges,129–130

conductivity and color, 127

Index 185

density and carbohydrates, 126flavor compounds, 131–133flavor sensory evaluation, 133–134inorganic composition, 128, 131nutritional aspects, 135–136off-flavors, 134–135organic acids, 131pH, 126rheology, 127–128vitamins, 136

contamination, 137cream and candy, 136–137and decolorizing resin, 139history

maple trees sap, 102–103sap processing, 103–104

maple sap flowphenomenon, 105physiological process, 104–105

sap chemistryevaporation by heating and

transformation, 117–121inorganic composition, 112–113organic acids, 113–115phenolic compounds, 115pH range, 111reverse osmosis/nanofiltration

transformation, 116–117storage transformation, 115–116sugar composition, 111–112transmission profile, 114

sap collectionplastic tubing, 106–107tapholes, 107

sap processing, evaporation, 107–110scale/sugar sand formation

composition and variation, 122evaporator surfaces, 123niter, 121

standardscolor, flavor and purity, 125grades, 124

Maple tubing system, 106–107

N

Neural tube defects (NTDs)environmental risk factors, fumonisins,

148–149fumonisin-induced mouse models

in cultured mouse embryos, 156–157maternal fumonisin exposure, 157–158

genetic risk factors

DNA methylation, 153folic acid, 152–153genes—genes candidate, 152syndromes, 151

nutritional risk factors, folic acidfolate mechanisms, 150maize consumption, 151

overview, 148spina bifida, 148

P

Paraformaldehyde (PFA), 107Peripheral blood mononuclear cell (PBMC),

8Polyamides (PA) food-packaging, 38Polyethylene terephthalate (PETE)

food-packaging, 33–34, 38–39, 49, 54bottles

contaminant analysis, 47light-exposed and protected milk, 41ozonated water, 40

high-density poly ethylene (HDPE),51–52

Polyolefins food-packaging, 38acetaldehyde, 41aeration, 39chlorinated and nonchlorinated water, 42HDPE and epoxy, 43–44off-odor development, 44oxidation products, 40packaging flavor, 43sensory characteristics, 39–40water distribution, 41–42

Polystyrene (PS) food-packagingmonomer thresholds, 37refrigerated storage, 36signal detection method, 37–38storage temperature, 35–36styrene monomer concentration, 35styrene taint, 38

Polyvinyl chloride (PVC) food-packaging,38

Probiotics, gastrointestinal pathogensmechanisms, 2, 4

acid production and inhibitorysubstances secretion, 7–8

epithelial barrier function andprobiotic signaling, 4–6

immunomodulation, 8–10virulence factor expression, 10–11

strainsadhesion ability, 6

186 Index

Probiotics, gastrointestinal pathogens (cont.)
and beneficial effects, 3immunomodulation, 9–10potential mechanism of action, 10–11VSL#3, 5
Provisional maximum tolerated daily intake(PMTDI), 153

Pyrazines, 132

R

Reverse osmosis (RO), 116–117Robinson test, taints, 28–29

S

Shiga toxin-producing E. coli (STEC), 7Short-chain fatty-acids (SCFAs), 5Single nucleotide repeat polymorphisms

(SNPs), 152SIRA. See Stable isotope ratio testSmall-medium sized enterprises (SMEs), 75Sphingosine-1-phosphate (S1P), 161, 164Stable isotope ratio test (SIRA), 138Study specific questionnaire (SSQ), 79

T

Taints, food-packagingadditives/noncontacting materials

coloring agents and antioxidants, 46printing inks, varnishes, 45–46

contact materials, 27epoxy, 44–45polyamides (PA), 38polyolefins, 34–44polystyrene (PS), 35–38PVC and cPVC, 44Robinson test, 28–29sensory active components, 28sensory descriptors, 29–34

definition, 23–24recycled materials

absorbed odorous, 46PETE, 47

T84 epithelial cells, 4Triangle test, food-packaging

antioxidant level, 55basil taint, cheese, 53–54squashes and fruit beverages, 49

Tumor necrosis factor a (TNFa), 161

advances in food and nutrition research volume 56

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