Rethinking

tentials of some food components have drawn the attention

styles practices and stress may have a cumulative effecton the immune system increasing the risk for various car-

risk reduction is also well documented (Biziulevicius,Kislukhina, Kazlauskait _e, & Zukait _e, 2006; Gill et al.,

Trends in Food Science & Techincreased significantly in response to the increasing aware-ness of the influence of diet on health. The therapeutic po-

immunomodulatory effects of different bioactive peptidesand their potential for human health promotion and disease* Corresponding author.

0924-2244/$ - see front matter 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2011.08.010sdiovascular diseases, cerebrovascular disorders and cancer(Nakano et al., 2001). On the other hand, research on thee-mail: dominic.agyei@monash.edu)

Bioactive peptides from food proteins have been studied over

the past decade to elucidate their biological potency in the

major systems of the body such as the digestive, cardiovascu-

lar, nervous, and immune systems. Some bioactive peptides

have been established for their antimicrobial roles as well as

humoral and cell-mediated immune functions and thus have

prospects of being incorporated as ingredients in functional

foods, nutraceuticals and pharmaceuticals where their biolog-

ical activities may assist in the control and prevention of

diseases. However, further insightful research on the pharma-

cokinetics of immunomodulatory peptides in vivo and clinical

studies are needed to firmly establish their therapeutic

potency.

BackgroundThe demand for functional foods and nutraceuticals haDominic Agyei* andMichael K. Danquah

Bio Engineering Laboratory, Department of Chemical

Engineering, Monash University, Wellington Road,

Clayton, Victoria 3800, Australia

(Tel.:D61 (0)3 9905 1867; fax:D61 (0)3 9905 5686;food-derived

bioactive peptides for

antimicrobial and

immunomodulatory

activitiesReview

of clinicians, food manufacturers, researchers and con-sumers, thus becoming a major endeavour. It appears thecontemporary world is coming to terms with the ancientquote of Hippocrates Let food be thy medicine and medi-cine be thy food than ever before. The therapeutic poten-tials of functional foods and nutraceuticals areattributable to the presence of specific functional groupsand their molecular derivatives released during food metab-olism. Dietary proteins, aside their ability to supply calo-ries and amino acids, have also been known to offerhealth benefits in vivo and in vitro either in the intactform or as hydrolysates. Food protein hydrolysates that in-duce beneficial biological functionalities are called bioac-tive peptides. Bioactive peptides are produced bymicrobial fermentation, enzyme digestion or proteolysisby enzymes in vitro, and can perform physiological activ-ities in the major body systems (Korhonen & Pihlanto,2006). Such functionalities include antioxidative, antimi-crobial, antihypertensive, cytomodulatory and immuno-modulatory effects (Hartmann & Meisel, 2007; Yanget al., 2009). Fig. 1 shows some physiological effects ofbioactive peptides in the human body.

Bioactive peptides have been produced from awide rangeof food materials including those from animal sources; [milk(Gill, Doull, Rutherfurd, & Cross, 2000), egg, cheese, beef(Jang & Lee, 2005), pork (Jang, Jo, Kang, & Lee, 2008), sea-food (Kim & Wijesekara, 2010), fish (Kim & Wijesekara,2010; Yang et al., 2009), chicken]; plant sources [rice,corn, soy and soy products (Chiang, Tsou, Tsai, & Tsai,2006, Brassica carinata (Pedroche et al., 2007), wheat, broc-coli (Hartmann & Meisel, 2007), pulse crops]; microalgae(Chlorella vulgaris 87/1) (Morris et al., 2009); and fungi[mycoproteins from Fusarium venenatum (Brownsell,Williams, & Andrews, 2001), mushroom (Sun, He, & Xie,2004), Brewers yeast (Kanauchi, Igarashi, Ogata,Mitsuyama, & Andoh, 2005)].

Recent studies have demonstrated that unhealthy life-

nology 23 (2012) 62e692000; Morris et al., 2009).

ed pe

63D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69Of particular interest are the bioactive peptides of wheyproteins which have been shown to modulate both specificand non-specific immune responses in vitro (Gill et al.,2000) and in mice models (Gauthier, Pouliot, & Saint-Sauveur, 2006). Further, oral administration ofChlorella vul-garis 87/1 hydrolysate produced from pancreatin has beenshown to enhance the stimulation humoral and cell-mediatedimmune functions in undernourished mice (Morris et al.,2009). Antimicrobial and immunomodulatory peptidestherefore have the prospects of use as a new generation ofdrug candidates. However, there exist some limitations tothe full exploitation of immunomodulatory peptides to en-hance human nutrition and health. Review of literature re-veals that the action of immunomodulatory peptides isrelatively nonspecific and this may account for the reasonwhy the exact mechanism of action as well as the in vivofate of these peptides is largely unknown. Further, there areno high throughputmethods for the production of large quan-tities of these peptides. In this commentary, we discuss re-search advances in immunomodulatory and antimicrobial

Fig. 1. Bioactivities of food protein-derivbioactive peptides, production platforms and immunologicalassessment, and some challenges that preclude the large-scale production of bioactive peptides.

Fig. 2. Biological roles of hAntimicrobial and immunological potency ofbioactive peptides

Many peptides, mainly milk-derived, have shown multi-functional properties, where specific peptide sequencesmay possess two or more different biological activities(Pihlanto-Leppala, 2002). Peptides with antimicrobialand/or immunomodulatory activities are ubiquitous in vir-tually all life forms. The mechanism of action of bioactivepeptides in disease control and prevention is by interactingwith microbial host invaders as antimicrobial agents or bysuppressing or stimulating certain immune responses(Hancock & Sahl, 2006) (Fig. 2).

Recent studies have uncovered a wide range of bioactivepeptides obtainable from food proteinmaterials.Mammalianmilk has been shown to contain a number of very potent im-munomodulatory peptides which regulate immune functionsby suppressing or stimulating certain immune factors(Gauthier et al., 2006; Gill et al., 2000; Pihlanto-Leppala,2002). Both immunosuppression and immumostimulationfind use in various areas of disease control. Immunosuppres-

ptides and the body system influenced.sive peptides may be applied during medical conditions suchas graft and transplant rejection as well as inflammations in-duced by autoimmune disorders whilst immunostimulation

ost defense peptide.

processes boost overall immune potency (Gauthier et al.,2006). Some immunomodulatory peptides (such as caseinphosphopeptides) are already being marketed commercially.Casein phosphopeptides (CPP) are casein derived peptidesreleased in the gastrointestinal tracts which, among otherfunctions also stimulate immunoglobulinA (IgA) productionin mice (Ferraretto, Signorile, Gravaghi, Fiorilli, &Tettamanti, 2001; Otani, Nakano, & Kawahara, 2003).

Several studies have also shown microbial or viral inhib-itory peptides. Zeng et al. (2008) obtained some 5e10 kDapeptide hydrolysates from Pacific oysters (Crassostreagigas) and the peptides showed herpes virus growth inhibi-tion properties. In another study, oyster protein extractshave been shown to enhance the proliferation of immuno-cytes in human immunodeficiency virus (HIV-1). Theoyster protein extract enhanced the interleukin (IL-2) de-pendent activation of peripheral blood mononuclear cells(PBMC) and the extract induced effect was marked inHIV seropositive asymptomatic individuals. Thus, oysterprotein extract could prevent or delay the occurrence ofIL-2 dependent immune deficiency including AIDS(Achour et al., 1997).

Further, Yust et al. (2004) identified two fractions of rape-seed protein hydrolysates which inhibited HIV protease inE. coli BL21. The E. coli BL21 cells contained the plasmid,PT5, which carries a cDNA coding for HIV protease. Thesepeptides were detected as they could enter the E. coli cellsand inhibit the HIV protease thus allowing cell growth.

Protein hydrolysates with both antimicrobial and cancercell cytotoxic effects have also been shown from severalstudies (Falciani, Pini, & Bracci, 2009; Jang et al., 2008;Korhonen & Pihlanto, 2006). Table 1 shows the protectiveeffect and immune responses triggered by some bioactivepeptides.

The activity of immunomodulatory peptides is depen-dent on the structure of the peptide molecule. The func-tional property of a bioactive peptide is determined by itsunique three-dimensional (3D) structure which is depen-dent on the type (e.g. cysteine forms disulphide bridges)and the nature (e.g. charge and hydrophobicity) of keyamino acids in the primary sequence. However, bindingcharacteristics of bioactive peptides have been attributedlargely to the secondary structure rather than the tertiarystructure (Kaur, Garg, & Raghava, 2007).

Table 1. Immune activity of some protein fractions and bioactive peptides from different food materials.

FoodProduct

Protein Identified Peptide Enzyme treatmentused

Immune effect Ref.

Bovine milk as1-casein Pancreatin Y lymphocytes proliferation (Gill et al., 2000)Isracidin N e terminal

sequence 1e23Chymosin Protect mice against infection by

Staphylococcus aureus.[ phagocytic response in miceinfected with Candida albicans.Protect cows and sheepagainst mastitis

(Gill et al., 2000)

b-casein Pancreatin Y lymphocytes proliferation (Gill et al., 2000)

ic

ing etht ofbiom

64 D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69b-casomorphin Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu (60e70)

Trypsin

Para-k-casein Phe-Phe-Ser-Asp-Lys(17e21)

Trypsin

a-Lactalbumin Hydrolyseda-lactalbumin

Mushroom Auriculariapolytrichahemoagglutinativeprotein (APP)

uncharacterizedpeptides ofmolecular weight13.4 kDa

Soy beans Unidentified soyproteins

Alcalase

Egg Ovalbumin Peptides notspecified

e

Wheat Gluten Immunopeptides Enzymatdigestion

Chlorellavulgarian87/1

Unidentified algaeproteins

uncharacterizedpeptides ofmolecular weight2e5 kDa

Pancreat(followintreatmenChlorella

[, increase; Y, decrease; yHuman studies.Y lymphocytes proliferation, [ miceresistance to Klebsiella pneumoniainfection

(Gill et al., 2000)

[ antibody formation and[ phagocytosis in vitro

(Gill et al., 2000)

[ mice immune response to SRBCsand HRBCs

(Gill et al., 2000)

activates murine splenocytes,increasing their proliferation andgamma-interferon (IFNg) secretionin vitro

(Sheu et al., 2004)

Murine splenic lymphocyteproliferation, [ phagocytic effectof peritoneal macrophages

(Kong, Guo, Hua,Cao, & Zhang, 2008)

Antimicrobial activities,immunomodulatory, anti-cancer,and anti-hypertensive activities

(Mine & Kovacs-Nolan, 2006)

[ Natural killer cell activityy (Horiguchi,Horiguchi, &Suzuki, 2005)

anol

ass)

stimulation of humoral immunefunctions, haemopoiesis,monocyte-macrophage systemactivation,

(Morris et al., 2009)

65D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69The structural characteristics, hydrophobicity, basicity aswell as the composition and sequence of amino acids playa crucial role in determining the biological activities trig-gered by bioactive peptides (Hancock & Sahl, 2006;Korhonen & Pihlanto, 2006). Most peptides with antimicro-bial properties are short, hydrophobic and cationic in nature(Hancock & Sahl, 2006). Further, the presence of key aminoacids has been known to impart specific functionalities. Forexample, all the most potent angiotensin-I-converting en-zyme (ACE) inhibitory peptides, namely, valine-proline-proline and isoleucine-proline-proline, contain at least oneproline residue. Also, glycine is a key amino acid in the syn-thetic peptides (Tyr-Gly and Tyr-Gly-Gly) which have beenshown to enhance the proliferation of human peripheralblood lymphocytes (Pihlanto-Leppala, 2002).

Bioactive peptides as therapeuticsThe conventional use of drugs such as antibiotics and

vaccines as a means to control diseases suffers several de-merits leading to consequences on both manufacturers andconsumers. Factors such as high cost of manufacture ofvaccine, bureaucratic procedures for certification, changesin government and government regulations, and require-ments for adjuvants and ancillary medical supplies oftenlead to periodic production interruptions and shortage oftherapeutics.

Within the first half of the last decade there were nation-wide shortages of six recommended childhood vaccines tar-geting nine diseases, and the supply of adult influenzavaccine was interrupted three times in the US alone(Coleman, Sangrujee, Zhou, & Chu, 2005; Obaro &Palmer, 2003). It is therefore pertinent that measures areimplemented for the sustainability of pharmaceutical prod-ucts by developing alternative sources of these biologics.Immunomodulatory peptides, due to their hormonal or neu-ronal functions can be used as therapeutic agents. However,unlike bioactive peptides, the effects of vaccines on the im-mune system are highly specific inducing protective adap-tive immune responses and immunological memory.Thus, bioactive peptides cannot be used as effective substi-tutes for vaccines. Nevertheless, due to the physiologicaleffects they trigger in the body system, immunomodulatorypeptides could act as safe substitutes for antibiotics andother pharmacological agents.

The use of antibiotics may be accompanied by side ef-fects which could compromise consumer health throughdrug-to-drug interactions, fever, and allergenic reactions.Development of resistance by microorganisms is anotherimportant issue that hinders the use of many small moleculedrugs. Immunomodulatory peptides have the potential toaddress these setbacks. As peptides, they accumulate lessin body tissues and thus their use may be accompaniedby little or no side effects. Further, resistance by microor-ganism has not been known to accompany the use of immu-nomodulatory peptides, yet still this is not anticipatedbecause these peptides do not interact directly with themicroorganism but rather through diverse mechanisms ininnate immune system responses (Hancock & Sahl, 2006;Kim & Wijesekara, 2010).

Moreover, the use of bioactive peptides to generate ther-apeutic products for disease control is economically moreattractive compared with the use of and antibiotics or vac-cines. Many of the diseases (such as HIV, malaria and mea-sles) for which vaccines are needed, exist principally inthird world countries with low standards of living. Thiscoupled with the high cost of biological raw materials re-quired makes development of such vaccines cost-ineffective which discourages most drug manufacturingcompanies, being profit driven firms (Obaro & Palmer,2003). Bioactive peptides, on the other hand, can be pro-duced from by-products of food protein bio-processing.Fish skin, bones, cakes from pulse crops and residualminced meat are relatively cheap and the utilization ofsuch by-products may reduce production cost with anadded advantage of an efficient waste disposal (Agyei &Danquah, 2011; Yang et al., 2009).

Moreover, immunomodulatory bioactive peptides areencrypted in a wide range of food materials proteins(Table 1) and most of these foods are abundant in nature.Some commercial food products containing bioactive pep-tides have already found their place in Foods for SpecifiedHealth Use (FOSHU) products (Hartmann & Meisel, 2007).Additionally, a host of bioactive peptides can be producedfrom a single food source by the use of a combination ofenzymes. Gill et al. reported about 20 immunomodulatorypeptides of casein and whey proteins in milk alone(Gill et al., 2000). Many of the milk-derived peptides alsoshow multifunctional properties. Thus a single peptidemay perform more than one function in the body systems.Conclusively, peptide based therapeutics offer several ad-vantages over conventional drugs as a results of theirhigh bioactivity and biospecificity to targets, and the widespectrum of their therapeutic action (Marx, 2005).

Bioactive peptides as therapeutics - hindrances andsuggested solutions

Although immunomodulatory, antihypertensive and an-timicrobial peptides have good prospects of use in the alliedhealth and food industries some potential drawbacks needto be addressed before the bioactive peptides can be opti-mally exploited to that end. Some of the major drawbacksand suggested strategies to overcome them have been high-lighted in Table 2.

The very potent antimicrobial peptides have been knownto be cationic with hydrophobic amino acid residues(Hancock & Sahl, 2006). These properties, together withtheir large hydrodynamic size therefore lead to a compromisein their solubility and membrane permeability. This setbackcan be overcome through specialized delivery strategies suchas peptide encapsulation with micro- and nano-sized coordi-nation polymers or particles such as dendrimers, liposomes,and polyectrolyte microspheres.

66 D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69Further, the susceptibility of peptides to protease degra-dation is another major setback which can shorten thein vivo half-life of the peptide drug and may also lead to un-desirable pharmacological reactions and potentially toxicend-products. To this, interventions such as chemical mod-ification of peptides and the incorporation of unnaturalamino acids (including D-amino acids) have been knownto enhance the stability of peptides. The presence of certainkey biomolecules enhances the stability of certain peptides.For example, the presence of a proline residue at the car-boxyl terminal end of a peptide has been shown to increasethe peptides resistance to degradation by digestive en-zymes (Yamamoto, Ejiri, & Mizuno, 2003).

Table 2. The cons of peptide based therapeutics and strategies toaddress them.

Challenge Strategies

Low oral bioavailability Use of parenteral delivery routesLow stability Chemical modification of peptides

(with retention of activity)Use of peptidomimeticsUse or incorporation of unnatural orD-amino acids

Toxicity and effect onbody systems

Toxicological studies in animal models

Difficulty in delivery Encapsulation in coordination polymersor particles (such as dendrimers, andpolyectrolyte microspheres)Use of micro- or nano-sized liquidmarbles

Difficulty in transportacross membranes

Encapsulation in liposomes

High cost of synthesis Use of cheap food protein sources andbi-products of protein processing assubstratesUse of natural sources such asbacteriocinsAlso, the sequence, ser(P)-ser(P)-ser(P)-glu-glu (P beingphosphate) present in casein phosphopeptides constitutesa motif unto which cationic minerals can bind. The result-ing complex is resistant to proteolysis en route the gastro-intestinal system (Ferraretto et al., 2001).

Other approaches include the use of peptidomimetics, orcyclic peptides and research in this area is receiving atten-tion (Hancock & Sahl, 2006). Shen et al., have developeda new chemical method of peptide synthesis which canbe used to incorporate unnatural amino acids into a peptidechain as well as custom design the chirality of the end prod-uct (Shen, Makley, & Johnston, 2010). However, such inter-ventions will involve a trade-off between enhanced stabilitywithout significant loss in activity.

Moreover, in peptide therapeutics manufacture, the sin-gle largest setback is the high cost of manufacturing pep-tides (Hancock & Sahl, 2006). This is because theconventional manufacturing approaches such as transgenic,recombinant, or synthetic methods are relatively expensiveand thus prohibitive for scale-up operations (Marx, 2005).However, the plausibility of manufacturing physiologicallypotent peptides from food proteins can be used to overcomethe high cost setback in peptide manufacture. Break-throughs in this area include the use of bi-products fromfood protein processing operations as substrates for enzy-matic and/or microbial fermentation. Also, naturally pro-duced peptides sources such as bacteriocins which do notrequire synthetic manufacture can also be used.

Bioactive peptides e bioprocessingBioactive peptides are produced from proteins by the use

of one or more of the following methods: (1) enzymatic hy-drolysis with digestive enzymes, (2) fermentation of foodproteins with proteolysis starter cultures and (3) proteolysisby enzymes derived from plants or microorganisms(Korhonen & Pihlanto, 2006). However, some intact proteinshave been known to possess bioactive properties. Lysozymeand a-lactalbumin have been shown to possess antibacterialproperties (Expositoa & Recio, 2006). However, enzymatichydrolysis of whole molecules is known to be the most com-monly used method for bioactive peptides. The enzymesused could be of gastrointestinal origin, or from bacterial,fungal and plant sources. The use of proteolytic enzymesor their precursors from recombinant DNA technology hasalso been reported (Korhonen & Pihlanto, 2006). Commonlyused enzymes are pepsin, trypsin, alcalase, chymotrypsin,papain and pancreatin, which are either used alone or in com-bination with other enzymes. Production of bioactive pep-tides by enzymatic hydrolysis is performed at the optimalconditions of enzyme(s), and the type of peptides generatedis dependent on the hydrolytic specificity of the enzyme(s).

Moreover with this method, specific enzyme combina-tions could be used to simulate the fate of a protein moleculewhen it passes through the digestive system. For examplePedroche et al. (2007) sequentially digested B. carinata pro-tein isolates in vitrowith trypsin, chymotrypsin and carboxy-peptidase A and obtained peptides with antioxidant andangiotensin converting enzyme (ACE) inhibitory activities.Bioactive peptides generatedwith this order of enzymes com-binations is believed to be mimic those produced by the hu-man digestive system (Pedroche et al., 2007). Moreover,a combination of different enzyme treatments usually leadsto the production of many novel peptides. The use of immo-bilized enzymes over the conventional soluble enzymes forthe production of bioactive peptides is gaining momentum.In a study by Pedroche et al., bioactive peptides were pro-duced from B. carinata hydrolysed by immobilized enzymeson a glioxyl-agarose support (Pedroche et al., 2007).

Fermentation of food proteins with proteolytic startercultures is another method of bioactive peptide production.The proteolytic system of lactic acid bacteria (LAB), forexample, involves a plethora of proteases that offer variedenzymatic specificities. LAB possess cell envelope associ-ated proteinases (CEP) as well as intracellular peptidasessuch as endopeptidases, aminopeptidases, tripeptidasesand dipeptidases for possible application in the productionof bioactive peptides. Probiotics bacteria of the genera

lowed by ultrafiltration in membrane reactors, and ion ex-

67D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69change membrane chromatography have also been used(Chabeaud et al., 2009; Li, Chen, Wang, Ji, & Wu, 2007;Pedroche et al., 2007; Sheu, Chien, Chien, Chen, & Chin,2004). The use of a combination of methods for the produc-tion, separation, purification and identification of bioactivepeptides is also becoming popular. Such methods lead toproducts of high purity and/or yield. Electro-membrane fil-tration is a method used for the isolation and enrichment ofbioactive peptides (Bargeman et al., 2002). This methodcombines electrophoresis and membrane filtration andthus very efficient l for strongly charged biomoleculessuch as bioactive peptides. Additionally, key operating pa-rameters such as the type of membrane, electrical fieldstrength, salination of hydrolysate and hydrolysate concen-tration can be manipulated to improve the product transferand separation rates (Bargeman et al., 2002).

Bottlenecks to advancementsEven though bioactive peptides have numerous poten-

tials, there exist some limitations to the production of largequantities to satisfy the growing market demands. Pres-ently, the production and utilization of bioactive peptideshas not realized its full potential due to two major chal-lenges namely scientific and relating techno-economic set-backs and regulatory issues.

Scientific and techno-economic challengesThere is a dearth of reports that address the production

of bioactive peptides from a bioprocess engineering andeconomics perspective in order to assess commercial via-bility. Laboratory-scale preparations are not optimisedand routinely results in low volumetric titres. The lack ofviable bioprocesses, transferable to industrial scale, is a ma-jor hindrance to the rapid percolation of bioactive peptidesinto the therapeutic consumer markets.nologies used in isolating and purifying biomoleculescoupled with chemical engineering convective mass trans-fer principles could offer a cost-effective and scalable pro-duction platform for immunomodulatory peptides. To date,the methods that have been used for peptide fractionationand enrichment are those that enable recovery of bioactivepeptides with minimal destruction. Briefly, they involve ul-trafiltration, ion exchange and gel filtration technologies.Coupled methodologies such as enzymatic hydrolysis fol-Lactobacillus and Bifidobacterium have been most widelystudied for the production of probiotics and bioactive pep-tides (Alhaj, Kanekanian, Peters, & Tatham, 2010). The ac-tivities of peptidases are affected by growth conditions ofthe producing microorganism hence the formation of pep-tides by this method can be manipulated to some extent(Korhonen & Pihlanto, 2006).

The development of optimized production methods forthe commercial production of bioactive peptides is an on-going research endeavour. Advances in bio-separation tech-The scaling up of production methods for bioactive pep-tide requires optimization of certain key steps in the raw ma-terial conditions and process operations. Novel separationand enrichment technologies to enrich bioactive peptide frac-tions are needed (Pihlanto-Leppala, 2002). Some advanceshave been made in the use of continuous reactor and mem-brane ultrafiltration processes for the continuous productionand separation of peptides (Korhonen & Pihlanto, 2006)however, most of these technologies are not scalable. Theissue of fouling is also a major technical challenge that ac-companies the use of membrane-ultrafiltration for peptides.

The lack of scalable purification and enrichment method-ologies can result in an overall increase in the cost of peptidemanufacture. It has been estimated that the separation andpurification stages in industrial biotechnology processescan account for up to 70% of the capital and operating costs(Brady, Woonton, Gee, & OConnor, 2008). The achieve-ment of high-yield bioactive peptides manufacturing hingeson detailed optimisation raw materials and process opera-tions essential for the generation of specific peptides of dis-tinct bioactivities under economic conditions.

Another bottleneck is that in vivo scientific data fromtoxicological studies of bioactive peptides is lacking. Thisis a major conclusion drawn from all the studies made onimmunomodulatory peptide in the last decade. Due to theirrelatively small size, bioactive peptides, unlike their parentproteins, may react with other food components especiallyduring processing conditions producing reaction productsthat may be detrimental to health (Korhonen & Pihlanto,2006). Unlike the ACE inhibitory peptides which oftenhave very small peptide sequences (usually di- and tri-peptides) the antimicrobial and immunomodulatory pep-tides generally have longer sequences with larger molecularweights which raise safety and allergenic concerns. Thus,to be safely used as therapeutic products clinical testshave to be conducted to ascertain the safety of bioactivepeptides, their mode of delivery, and dosage.

Moreover, the exact mechanism of action of immuno-modulatory and antimicrobial peptides is still unclearalthough they have been studied extensively. Transmem-brane pore formation, inhibition of cell wall and/ornucleic-acid synthesis, and activation of the host autolyticenzyme system are some of the proposed mechanisms(Biziulevicius, 2004). Biziulevicius et al. (2006) also indi-cated that the immunostimulatory activity of food proteinhydrolysates is a consequence of their antimicrobial activ-ity. According to their hypothesis, some food-borne pep-tides act as antimicrobials by activating the autolyticprocesses of the intestinal microflora in situ and thus causethe release of microbial lysis products. These lysis productswhich are immunoenhancers account for the immunostimu-latory effects resulting from the consumption of certainfood proteins. In vivo and human interventional studieswith bioactive peptides are expensive and thus in vitro ex-periments are often performed to identify potential bioac-tive peptides before committing to complete human trials.

68 D. Agyei, M.K. Danquah / Trends in Food Science & Technology 23 (2012) 62e69Foltz et al., however, have argued in their review that it isinappropriate to use the in vitro stimulatory or inhibitory ef-fects as the justification to test the in vivo effects of bioac-tive peptides because the approach largely neglects the lowbioavailability of peptides resulting from poor absorption,distribution, metabolism, and excretion (ADME). In theirview, it is only valid to propose in vivo efficacy for bioac-tive peptides when the peptide exhibits reasonable proteo-lytic stability and physiologically relevant ADME profiles(Foltz, van der Pijl, & Duchateau, 2010).

Moreover, major conclusions drawn from most studieson immunomodulatory peptides have been contradictory.Such differences have in part been attributed to variationsin methodologies, raw materials, and models used however,they need to be addressed. Further, possible systemic sideeffects have to be considered through in vivo studies(Gauthier et al., 2006). These technological issues thereforehave to be settled before bioactive peptides can be ex-ploited optimally for human nutrition and health. The effi-cacy and safety of bioactive peptides should be investigatedfurther by carrying out animal studies to verify beneficialeffects and to clarify adverse affects.

Another area requiring intensive research is elucidatingthe fate of bioactive peptides during gastro intestinal transitas well as the evaluation of peptide stability in the livingbody systems. Walsh et al. (2004) have demonstrated thatthe antihypertensive peptide obtainable from b-lactoglobulin[b-LG f(142e148)] is not sufficiently stable to gastrointesti-nal and serum proteases. Thus, this peptide may be helplessto act as a hypotensive agent in humans following oral inges-tion. Findings from studies on the fate of peptides en-routethe body systems will provide useful information needed inthe design of technologies aimed at formulating peptidesinto forms capable of surviving the harsh conditions createdby the stomach and pancreatic juices.

Regulatory related challengesIt is highly prognostic that in the next few decades, the

bioactive peptides market will become a self-standing in-dustry with its own professional associations promotingits interests due to the recent expansion of the areas of ap-plication of bioactive peptides. Government and consumerprotection bodies must therefore come out with regulationsthat govern the production, marketing and utilization ofsuch products. Information needed for design of regulatorymeasures will depend largely on the advances made in sci-entific studies on the toxicity, mode of action in the bodyand nutritional intake recommendations.

ConclusionsFood proteins have been shown to offer health benefits be-

yond serving the nutritional needs of the body. Such nutri-tional intervention can be of immense benefit in enhancingthe health of immune compromised as well as healthy indi-viduals. In humans, studies have established the effect of cer-tain lifestyle related practices on some parameters of theimmune system. By implication, peptides with antihyperten-sive, antimicrobial and immunomodulatory properties maybe used as preventive or control aids against lifestylerelated-diseases such as hypertension, cancers, cardiovascu-lar diseases, diabetes, osteoporosis, stress and obesity.

Immunomodulatory peptides may also be incorporatedinto food and drug formulations that can be consumed assupplements. However, to realize the full therapeutic poten-tial of these peptides in vivo models and clinical studies areneeded to clarify concerns such as the mode of action, tox-icity, dosage, mode of delivery, and possible systemic ef-fects that these peptides might have on the body. Also,completely optimized bioprocesses which are transferableto large-scale operations need to be developed for the pro-duction, and purification of these important biopeptides.

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Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activitiesBackgroundAntimicrobial and immunological potency of bioactive peptidesBioactive peptides as therapeuticsBioactive peptides as therapeutics - hindrances and suggested solutionsBioactive peptides bioprocessing

Bottlenecks to advancementsScientific and techno-economic challengesRegulatory related challenges

ConclusionsReferences