Journal of Biotechnology 162 (2012) 356 365

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

j ourna l ho me pag e: www.elsev ier .com

Science roin food

K. MakinNestl Research

a r t i c l

Article history:Received 20 JaReceived in reAccepted 13 JuAvailable onlin

Keywords:ProbioticHealth benetHealth claimManufacturingStabilityStorageBiotransforma

ismst of obioticpe. T

microbiotirobio

of pring ants, foity of

1. History of probiotics

Fermentation is one of the oldest methods of preserving foods.By 6000 BCin China. Fedahi were amicroorganPasteur isol1907 that tnikoffs posled to the observationconsumed ltionally lonthe gut suchphenols, ancould be cotained harmof proteolyt1907). Althputed (Chepculminated

CorresponE-mail add

1953, which he dened as active substances that are essential fora healthy development of life (Hamilton-Miller et al., 2003).

0168-1656/$ http://dx.doi.o, cheese was being made from cows and goats milkrmented products such as ker, koumiss, leben, andlso used therapeutically long before the existence ofisms was discovered by Leeuwenhoek in 1683. Louisated lactic acid bacteria from milk in 1857, but it was inhe concept of probiotics was born through Elie Metch-tulation such that consumption of fermented foodsprolongation of life. He based his hypothesis on the

that nomads in Bulgaria and the Russian Steppes whoarge amounts of fermented milk appeared to live excep-g. Metchnikoff hypothesized that proteolytic bacteria in

as Clostridia produced toxic substances such as indoles,d ammonia, which led to autointoxication. This, in turn,untered by consumption of fermented food that con-less lactic acid bacteria, which suppressed the growthic bacteria by lowering the intestinal pH (Metchnikoff,ough Metchnikoffs postulate was subsequently dis-lin and Rettger, 1920), it started a chain of events that

in the coining of the term probiotic by Kollath in

ding author. Tel.: +41 21 7858740; fax: +41 21 7859486.ress: kimmo.makinen@rdls.nestle.com (K. Makinen).

2. The denition of probiotics

Today, no legal denition of probiotics exists, but the deni-tion accepted by most is the denition of the 2001 joint WHO/FAOexpert consultation: probiotics are live microorganisms whichwhen administered in adequate amounts confer a health beneton the host (FAO/WHO, 2002, 2001). We now know that somemicroorganisms that have been inactivated prior to consumptionor extracts of microorganisms can also be health-benecial (Adams,2010), but for the time being, they are disqualied from the genreof probiotics. In contrast, a glance at the market place reveals thatsome strains or combinations of strains are marketed as being pro-biotic without a shred of evidence of their efcacy in humans. Thisis misuse of the term probiotic, which implies a demonstrated(and not only purported) health benet. Such misuse of the termis likely to become less frequent, however, as the regulatory envi-ronment of Europe and elsewhere becomes increasingly stringent,thereby limiting the use of the term probiotic to those strains withappropriate scientic backing. Another misuse of the term probi-otic is typied by a probiotic mattress (available on the market atthe time of this writing) incorporating a layer of microorganisms tocounter dust mite allergens. Because in this case, the microorgan-ism is not administered to the host, but rather, resides in the vicinityof the host, the term probiotic should not be used. Overall, 11 years

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.jbiotec.2012.07.006 and technology for the mastership of p products

en , B. Berger, R. Bel-Rhlid, E. Ananta Center, Nestec Ltd. Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland

e i n f o

nuary 2012vised form 10 July 2012ly 2012e 22 July 2012

tion

a b s t r a c t

Probiotics, dened as live microorganbenet on the host, are a common parthe health benet applications of proof health claim substantiation in Eurootics, introduces the use of non-viableregulatory challenges surrounding propment of food products containing pindustrial production. The descriptionto maintain high viability during dryof probiotic stability in liquid producnutrient bioavailability and digestibil/ locate / jb io tec

biotic applications

which when consumed in adequate amounts confer a healthur daily diet. Since their conception in the early 20th century,s have been expanding, culminating in the recent challengehis paper highlights the different application areas of probi-oorganisms to confer health benets, and explains the recentcs. It then describes in detail the different stages in the devel-tic bacteria starting from the selection of suitable strains for

oduction of probiotic powders with specic focus on strategiesd storage then follows. The paper nishes with a discussionllowed by a description of the use of probiotics to improve

the food products, which they ferment or biotransform. 2012 Elsevier B.V. All rights reserved.

K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365 357

after its conception, the WHO/FAO denition of probiotics remainssufciently accurate, except that in the light of current knowledge,the inclusion of non-live microorganisms in the denition seemsjustied (see below).

3. Health b

To date,reviews haprobiotics. ducted withpopulationsextensively2010) and wthe health This meanshowever clhealth benecase in poinAuthority (the evidencirrelevant ajohnsonii Lapanel, 2011strain showthe same bstrains. Thecontains a wa separate s

The heashare of prothe general2010; Mellelevel of exisconsumer uts. New inFor examplsumed oralin healthy s2008). L. recantly redugastric empbination of shown to retion (Messastimulation2011). Finaprobiotics oet al., 2010)(Jones et albiotics are bacterial strbenet.

4. Identic

Traditionment, probia few simptestinal conin the presepithelial cenow know,benets in strain to be

Bidobacteria sp. are highly sensitive to adult gastric acidity whileLactobacilli are relatively tolerant to it (C. Cavadini, unpublishedobservations). Also, even though it is conceptually attractive thata probiotic strain should be able adhere to intestinal epithelium,

atic uma

ncesenicoundor ex

of, o probs of iited

ate imicatio

uposs, thesignan i

andnism

certaa sty

targexams a csion conjd for

a hurcho

parabharmtic st

use

n thoroorgreasi

healn-via907 umaa su

ter cus, Lambemicrc conand ae puddit

achthawer, id of ed bt of tf atohoulds ca

maue toenet application areas of probiotics

over 900 human intervention studies and countlessve been published on the health benecial effects ofThe studies vary widely in quality and have been con-

a range of probiotic strains, health benets, and target. The health benets of probiotics have been reviewed

elsewhere (Deshpande et al., 2011; Rowland et al.,ill not be detailed here. It is noteworthy, however, that

benecial properties of probiotics are strain-specic. that, for example, two Lactobacillus acidophilus strains,osely related, may not be presumed to have the samecial properties unless so proven in clinical trials. At is the scientic opinion from the European Food SafetyEFSA) on Lactobacillus johnsonii BFE 6128, in which alle provided by the applicant was dismissed for beings it pertained to another related L. johnsonii strain, L.1, rather than the strain under application (EFSA NDA). It is also important not to assume that a probioticn to be health benecial when administered alone, hasenet when administered in combination with otherrefore, any novel mixture of probiotic strains, even if itell-studied probiotic strain, should be substantiated inet of studies.lth benet areas accounting for the biggest marketbiotic sales are immune protection and gut comfort in

healthy adult population (Euromonitor International,ntin, 2008). Their success to some extent reects theting scientic evidence, but is greatly aided by the highnderstanding of, and demand for these health bene-novate applications of probiotics are also emerging.e, some evidence suggests that L. johnsonii La1 con-ly may expedite the recovery of skin immune functionsubjects following UV exposure (Peguet-Navarro et al.,uteri DSM17938 has, in turn, been shown to signi-ce crying time in colicky babies, possibly by improvingtying (Savino et al., 2010, 2007). Interestingly, a com-L. helveticus R0052 and B. longum R0175 has even beenduce anxiety-related symptoms in the general popula-oudi et al., 2011) through a mechanism that may involve

of the parasympathetic nervous system (Bravo et al.,lly, studies are emerging showing a positive effect ofn body weight in healthy overweight adults (Kadooka

and cholesterol levels in hypercholesterolemic adults., 2011). Therefore, the potential applications of pro-many. The major challenge is often to identify whichain will be most likely to be successful for a given health

ation and development of probiotics

ally, and reinforced by the WHO/FAO guidance docu-otic candidate strains have been selected on the basis ofle properties. These are survival in simulated gastroin-ditions (incubation at pH 2.5 followed by incubationence of bile salts), the ability to adhere to intestinallls, and the production of antimicrobial substances. We

however, that these properties neither predict healthhumans, nor are they sine qua non conditions for the

health benecial. For example, experiments show that

systemtic in hsubstapathoghave flines, fInsteaddidatea serienot limmodulan indinduceerthelewell-din humin vitromecha

Forpharmgableit. For oped aexpresBSH detion anbile. Inin hypeis comSuch pprobio

5. The

Eveto micing incconfering nosince 1from hdiarrhewas laand thin a nukilled allergi2005) becom

In acan befreeze-Howevmethoproduccontexment o

It sganismcontainones, dinvestigation of the importance of this characteris-ns is lacking. Finally, the production of antimicrobial

may only be relevant in cases where a specic anti- effect is desired. Nevertheless, such selection criteria

their way to some recent probiotic regulatory guide-ample those adopted in India in 2011 (ICMR-DBT, 2011).r in addition to these basic probiotic properties, can-iotic strains are typically selected via a process involvingn vitro and pre-clinical tests. They can include, but are

to, assays testing the ability of the probiotic strain tomune cell function in vitro or in vivo, which can give

n of what immune prole the probiotic strain mightn interaction with the immune system in humans. Nev-e nal health benet must always be demonstrated ined human trials. To increase the likelihood of successntervention trials, which are costly, better predictive

pre-clinical systems and a better understanding of the of action of probiotics are needed.in health benets, probiotics can be developed using ale approach, which relies on identication of a dru-

et and selection of probiotic strains that can inuenceple, L. reuteri NCIMB 30242 (CardiovivaTM), was devel-holesterol-lowering probiotic based on its high level ofof a class of enzymes called bile salt hydrolazes (BSH).ugate bile acids in the intestine, reducing their reabsorp-cing more cholesterol to be shunted into resynthesis ofman clinical trial, the strain was shown to reduce LDL-Clesterolemic subjects by nearly 9%, over 6 weeks, whichle to levels achieved by plant sterols (Jones et al., 2011).a-style identication and development of candidate

rains is likely to increase.

of non-viable probiotics

ugh the use of the term probiotic is currently limitedanisms alive at the time of consumption, it is becom-ngly evident that even non-viable microorganisms canth benets (Adams, 2010). In fact, products contain-ble microorganisms have been available on the marketwhen Pierre Boucard isolated two strains of Lactobacillin stool, heat-killed them, and marketed them as an anti-pplement called LacteolTM. The anti-diarrhea benetonrmed in clinical studies (Salazar-Lindo et al., 2007)cteolTM is still available as over-the-counter medicationr of countries. In addition to diarrhea treatment, heat-oorganisms have been shown to be effective againstditions in children (Morisset et al., 2011; Peng and Hsu,dults (Ishida et al., 2005), with more research likely to

blished in other health benet areas soon.ion to heat-treatment, inactivation of microorganismsieved through sonication, high pressure treatment,ing, or irradiation (not permitted for food applications).

n the published studies, heat-treatment has been thechoice for strains consumed in foods. Bacterial lysatesy sonication have, in turn, been investigated in theopical applications for skin health benets, such as treat-pic dermatitis (Di et al., 2003; Gueniche et al., 2008).

also be noted that even though non-viable microor-n be produced deliberately, any probiotic product willny non-viable microorganisms in addition to the live

the inevitable loss of viability during manufacture and

358 K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365

storage (see below). It is not clear, however, what, if any, fractionof the health benet is attributable to this inactivated component,and whether these non-viable microorganisms share the behaviorof their deliberately inactivated counterparts.

How cants? The rathat, for exatem (e.g. suin bacteria reside in thnal heat-in

It is unlikprobiotics cIn fact, we klive L. johnspylori in nonteurized doappears to (Michetti ecompound(duction by enzymatic rendering pheat-treatm

The useattention aadvantageslimited shewater activmicroorganroom-tempcould spoil Non-viable hot-reconstufacturing pcreams and

Comparenon-viable dients compto surface sucts. Howevversions of proles, anscientic su

6. EFSA an

Despite effects of papplicationwhere a proated is for panel, 2010standard yoisms Lactothermophilufermentatiomajority (>3assessed byterization onot submitganisms unidentity. Mated with neconsidered

described above in starter cultures) have seen EFSA consider itshealth benecial effects to be sufciently substantiated. Factorsresponsible for this include, studies making up the application notbeing of the required quality (e.g. study design and statistical anal-

the d or ntrap). Invide

demstrats recce fiumduceFSA

xt fet thestake

duct

FAOtic mt theuringiablequiren, thm toic v

two are

in ths (Roch a

cultud mss ane bete orgre noer culed lid mild, recearn

(Rou secoediussense objmenreachletionces.

mebiom

biomep, foae, mep is ehydpsulnd en non-viable microorganisms bring about health bene-tionale is that the key bacterial structural componentsmple, interact with and activate the host immune sys-rface carbohydrates and bacterial DNA) are still presentafter inactivation. The active components could alsoe culture supernatant, provided that it is present in theactivated product in sufcient amounts.ely, however, that all health benets achievable by livean equally be achieved by non-viable microorganisms.now that whilst a fermented milk product containing

onii La1 reduces the activity of the gastric pathogen H.-symptomatic infected subjects, the same product pas-es not (Gotteland et al., 2008). The anti-H. pylori effectbe present in the L. johnsonii La1 culture supernatantt al., 1999) and therefore, the secreted anti-H.-pyloris) can be presumed to be heat-labile or to require pro-the live probiotic strain in situ. It is also likely that mostactivities would not withstand strong heat treatment,robiotics that depend on such activities inactive uponent.

of non-viable microorganisms has attracted muchmong food manufacturers because they have certain

over live probiotics. For example, probiotics have alf life and often, especially in products with highity (aw), require chilled storage conditions. Non-viableisms do not have this limitation, and can be used even inerature ready-to-drink products, which live probioticsdue to unwanted microbe-induced product alterations.microorganisms can also be used in products requiringitution or involving a high temperature during the man-rocess. They are also suitable for topical applications in

shampoos.d to research conducted with probiotics, the study ofmicroorganisms is still in its infancy. Yet, more ingre-rising non-viable benecial microorganisms are likely

oon, due to their ease of application in a range of prod-er, it cannot be assumed that the viable and non-viablethe same microorganism have identical health benetd therefore, each will require its own set of studies forbstantiation.

d probiotic health claims

signicant body of research on the health benecialrobiotics, to date, not a single probiotic health claim

has received a positive ruling from EFSA. The only casebiotic-related health claim was considered substanti-standard yogurt and lactose maldigestion (EFSA NDAb). In this case, the benecial activity was attributed togurt starter cultures (which contain the microorgan-bacillus delbrueckii subsp. bulgaricus and Streptococcuss), which completely deplete the pool of lactose byn, rather than to a probiotic strain per se. The vast/4) of the remaining probiotic health claim applications

EFSA were rejected on the basis of insufcient charac-f the microbial strains, meaning that the applicants did

sufcient molecular data to prove that the microor-der each application were of the claimed taxonomicny of these applications were subsequently resubmit-w characterization data. Out of those probiotic strainssufciently characterized, none (except the mixture

ysis of doubt,(e.g. exulationnot processfuldemon001, haevidenClostridand reotics (Ethe neto meeto the

7. Pro

Theprobioalive avival dthat valso readditiomediueconom

Thecationsgrownprocesmilk sustarterthe foobiomabalancand thotics aof startto chilmenteof drie2000; Kexists

Thetrial mThis leand thing fercount to depsubstagrowthtrated

Thetion stformultion stharsh dof encasizes aata), not showing the claimed effect beyond reasonableot having been done in the correct target populationolation from diseased patients to a healthy target pop-

some cases, the studies making up the application did consistent evidence of the claimed benet, due to suc-onstration of the benet in one study, but a failure toe it in another. So far, only one strain, L. casei DN 114eived the slightly more encouraging ruling insufcientor the cause and effect relationship for the reduction of

difcile toxins in the gut of patients receiving antibioticsd risk of acute diarrhoea in patients receiving antibi-

NDA panel, 2010a) This situation is likely to evolve inw years, however, as new research is being conducted

exact EFSA requirements, which are becoming clearerholders via published EFSA guidelines and opinions.

ion of viable and stable probiotics

/WHO denition of probiotics poses two challenges onanufacturers. The rst is to provide bacteria that are

time of consumption, which necessitates good sur- food production and storage. The second is to ensure

bacteria are present in sufcient quantities, whichs good survival during production and storage, and inat the bacteria grow efciently on food grade growth

high cell counts. Collectively, these factors dictate theiability of the probiotic production process.

major routes of producing probiotics for food appli- illustrated in Fig. 1. In the rst route, probiotics aree nal food matrix, which becomes fermented in theute B). A typical example of such a product is fermenteds yogurt (Route B2), which contains the probiotic strain,re bacteria, and fermentation metabolites secreted into

atrix. Since this product format contains both bacteriald the fermented food matrix, it is critical to maintain aween probiotic growth to achieve sufcient cell countsanoleptic properties of the product. To this end, probi-t grown alone, but together with various combinationsltures (Lourens-Hattingh and Viljoen, 2001).In additionquid yogurt, which represents the most common fer-k product format, an interest to explore the productiononstitutible probiotic yogurt (Bielecka and Majkowska,ey et al., 2009) or probiotic soymilk (Wang et al., 2004),

te B1).nd route to produce probiotics is to grow them in indus-m which in itself is not the nal food matrix (Route A).

the concern for the preservation of sensory attributesective becomes maximizing the yield of biomass dur-tation. The bacterial cells are harvested when the celles a maximum and the growth curve levels off duen of nutrients or accumulation of growth-inhibiting

The cells are harvested by separating them from thedium by centrifugation or ltration, yielding concen-ass.ass can be dried with or without a prior encapsula-

llowed by dry-mixing into end products such as infantilk powders, or cereals (Fig. 1, Route A1). An encapsula-often required in cases where probiotics are exposed toration or storage conditions. There are various methods

ation involving various materials with different particlecapsulating properties (Anal and Singh, 2007). In case

K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365 359

Food grade grow th media Mil k

Fermentati on/

biomassFermen tati on

k

d mil

iotics

of non-fermliquid biomstream withtems such ato Route A2onto cereal2009). Withstep storageRoute A1, tthe end pro

In generduring manditions, andbe discusseproducts.

7.1. Intrinsi

Process biotics, but strains. Theabove, straicandidate s

It is welharsh condbiomass. Foknown to piccation (Sidifferent bathe stresseexample, oubetter thanand 1% pandisqualify adevelopmeance have a

Strain rois also a maspontaneouphenotype Margolles eBerger et altolerant mu

ant tioneme

phyres aed bhoulr ste

baseuta

adiat(Laud by

et althe m

Althed ombe

chaner, a

on t thernoloilar Drying Encap sulation

Sterile addit ion

into li quid prod uct

Drying

Biomas s

Dry mix with end

prod uct

Drying

Dried

fermen ted mil

A1 A2 B1

Fermen te

Fig. 1. Schematic routes of production and incorporation of prob

ented liquid products, probiotics can also be added asass concentrate dosed continuously into sterile productin an aseptic environment (Fig. 1, Route A2) using sys-s FlexDos from TetraPak (Prado et al., 2008). Similar, liquid probiotic biomass concentrate can be sprayed

pieces or akes in a uidized bed dryer (Cavadini et al., this approach it is possible to obtain in one process-stable dried cereal coated with probiotics, whereas in

he probiotic has to be dried rst and then mixed withduct.al, three major factors govern the stability of probioticsufacture and storage: strain robustness, process con-

storage conditions. In the following section, they willd in the context of application in dry and liquid food

c stability of probiotic strains

and storage conditions impinge on the stability of pro-stability is also a property that varies between probioticrefore, in addition to the functional properties describedn robustness is a commonly used criterion for selectingtrains to be developed as probiotics.l documented that species respond differently to the

an elegproducimprovwhat isperatuincreastions sanothe

Theusing mUV-irr(EMS) reacheBergerallow strain.preferrited nurisk ofHoweveffectsshouldin tech

Sim

itions of industrial production and storage of probioticr example, compared to B. animalis lactis, B. longum isoorly tolerate temperature increases, oxygen, or des-mpson et al., 2005). Similarly, as mentioned earlier,cterial species and strains show different tolerance tos encountered in the gastro-intestinal tract (GIT). Fort of 35 bidobacteria, one B. breve strain survived far

the other bidobacteria when exposed to 0.5% pepsincreatin (Liu et al., 2007). One should not systematicallyverage non-robust bacterial species from the probioticnt process as within-species variations in stress toler-lso been documented (Liu et al., 2007; Lian et al., 2002).bustness, even though peculiar to each bacterial strain,lleable property. For example, it may increase throughs mutations that provide a stable stress-resistant(Chou and Weimer, 1999; Collado and Sanz, 2007;t al., 2003; Sanchez et al., 2007b,a; Mozzetti et al., 2010;., 2010). Thus, natural selection of spontaneous stress-tants through application of a sublethal stress offers

characterisorgano-lepcient screenstrain is avreduced aband Snche

To bettebacterial ceparental stret al., 2007bidenticatiocesses, whiparametersin additioncross-proteet al., 2004pleiotropic expression Berger et alproduction

Biomas s

stabili zation

Incorpo ration

in prod uctLiqu id

fermen ted mil k

B2

k

into different types of food products.

approach to improve the survival of probiotics during and/or in the GIT following ingestion. The phenotypents obtained by this approach remain in the range ofsiologically compatible. For example, resistance to tem-ltering the structure of ribosomes is unlikely to bey few mutations. It is important that the selected muta-d not confer the strain a physiological disadvantage inp of the production process or in its application in foods.line evolution rate of probiotics can be accelerated by

genic treatments of the original strain, for example withion, or a chemical agent such as ethyl methanesulfonatetier et al., 1988). Alternatively, a similar goal may be

an iterative selective enrichment (Monnet et al., 2003;., 2010) or continuous culture (Mozzetti et al., 2010) thatutant derivatives to quickly outcompete the parental

ough not always possible, the selective enrichment isver the mutagenic treatment, since it generates a lim-r of mutations per genome, which, in turn, reduces theging other important properties of the original strain.ny single mutation may also have unexpected knock-onhe properties of the probiotic strain. A mutated strainefore be considered as a new strain and be re-assessedgical and functional tests.approaches also may be used in order to change other

tics of probiotic strains, such as their effect on thetic properties of the food product, provided that an ef-ing test or a counter-selection method of the originalailable. For example, a B. animalis lactis strain withility to produce acetic acid has been isolated (Margollesz, 2012).r understand the mechanisms of stress response in thells, the robust derivatives can be compared with theain in a top-down systems biology approach (Sanchez; Guillaume et al., 2009; Berger et al., 2010). This allowsn of the most signicantly modied molecular pro-

ch, in turn, may give leads on the main chemico-physical impacting the physiology of the cells. Interestingly,

to protection against the selected stress, cases ofction to other stresses have been documented (Noriega; Desmond et al., 2001). This can be explained by theeffect of some mutations, such as those affecting theof the chaperones GroEL or DnaK (Desmond et al., 2004;., 2010).

360 K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365

7.2. Impact of process conditions on probiotic stability

Strain-intrinsic properties set the baseline for probiotic stabil-ity, which is then acted on by the other major factors governingprobiotic stditions. Thethe survivachanges shstability anprobiotic islower tempproduct hasability of thto identify tstrain and pbasis.

Care alsoof the dryinthe effect ofchanges in ing time, gbeen reportas heat-resistability (BrMuller et alGilliland, 1need to be quences on

7.3. Importapplications

Powder probiotics. dry-mixingmany strating the devacuum-, oPeighambarthe leadingmilder conAlternativehave been gmethods ducapacity at

Out of thlevel of cell agents and tigated (Chaimproving reported toof the powdknowledge universal pall probiotic

An increattempted ents in the this by corr(Santivaranet al., 2007et al., 2012Castro et aSchwab et matrix, such

Ultimately, such investigations will yield a better scientic ratio-nale for protective ingredient selection.

The currently available protective matrices fall into two groups.The rst one is a complex/semi-dened mixture of ingredients, out

ch skshow

and ientsh, 20ducengre004)

secoor ainglees) orangMiaopicaompectlyhelpydra

Dryies balar mn a d Leoessarurtms ont al., 010

20092002ther -stase ruions,le, inr goo). Alste innan

protebiotirage

orage

dryprobill haage. T

covepmeendiviabiringes s

). Lowuctse onois

situaer, tability, namely process (manufacture) and storage con-se conditions often have to be adapted to maximizel of probiotic strains, as will be discussed below. Theould, however, not be such that they compromise thed processability of the whole food matrix in which the

incorporated. For example, even though spray drying ateratures is favorable to probiotic survival, the resulting

too high a moisture content to ensure good process-e powder (Ananta et al., 2005). Therefore, it is importanthe right balance between the survival of the probioticrocessability, and this has to be done on a case by case

has to be taken if a change is implemented upstreamg step (e.g. at fermentation), especially, because often,

these changes is not immediately visible. For example,the composition of the fermentation medium, harvest-rowth temperature and pH during fermentation haveed to modulate technical properties of probiotics, suchstance, freezing- and drying-survival as well as storageashears and Gilliland, 1995; Gilliland and Rich, 1989;., 2011; Palmfeldt and Hahn-Hgerdal, 2000; Reilly and999). Hence, any changes in fermentation conditionscarefully evaluated against potential negative conse-

stability during processing or storage.

ant considerations in the production of probiotics for in dry products

products are among the biggest application elds ofThey are produced by drying the bacteria followed by

with the nal product matrix. The literature detailsegies to achieve good survivability of probiotics dur-hydration step, which can comprise freeze-, spray-,r uidized bed-drying (Santivarangkna et al., 2007;doust et al., 2011). Freeze-drying is still considered as

technology for drying probiotics due to its employingditions that help maintain high level of cell viability.

drying techniques such as spray- or vacuum-dryingaining increased attention as attractive probiotic dryinge to their lower operating costs and higher throughputacceptable level of probiotic survival.e different strategies that can be used to maintain a highviability during drying and storage, the use of protectiveencapsulation of probiotics are the most widely inves-mpagne et al., 1991; Anal and Singh, 2007). Apart fromcell survival during drying, a protective medium was

accelerate water removal and improve the propertieser (Menshutina et al., 2010). However, even though thein the eld of protective agents has been increasing, norotective agent and drying technology that would suits is yet available.asing number of systematic studies, however, haveto decipher the role of individual protective ingredi-maintenance of probiotic dry stability. They have doneelating viability loss with the kinetics of water removalgkna et al., 2005), the occurrence of glassy state (Higl; Kurtmann et al., 2009b; Pehkonen et al., 2008; Ying), the sites of cellular damage (Brennan et al., 1986;l., 1997; Li et al., 2011; Santivarangkna et al., 2009;al., 2007), and the chemical reactions occurring in the

as browning and oxidation (Kurtmann et al., 2009c,a).

of whiit was dryingingredand Pewas reother iet al., 2

Thedients used scharidSantiva2008; turn, tybasic cars dirwhich the deh1995).connmolecuplace iSun anbe necage (Kdepend(Ying eet al., 2Knorr,et al., with ostorage

Theexceptexampsary fo2009btransla2007; Aon the on proand sto

7.4. St

Thewhich they stof storucts. Itdevelo

Depbiotic loss duimprov2009bof prodcally thinto a m

Thehowevimmed milk has been investigated most. For instance,n that bacteria dried in the presence of milk survived

storage better than when dried in the presence of single or a dened mixture of ingredients (Li et al., 2011; Pyar11; Lian et al., 2002). Interestingly, the protective effectd when a part of skimmed milk solids was replaced bydients, such as prebiotics (Ananta et al., 2005; Corcoran.nd group of protective matrices comprises single ingre-

dened mixture of ingredients. The most commonly ingredient is sugars such as mono-, di-, or oligosac-r sugar derivatives such as sorbitol (Li et al., 2011;kna et al., 2005; Schwab et al., 2007; Pehkonen et al.,

et al., 2008). The dened mixtures of ingredients, inlly have low- and high-molecular weight sugars as theironent (Ying et al., 2011). Low molecular weight sug-

interact with bacterial proteins and the lipid bilayer,s maintain them in a close-to-native conformation inted state and upon subsequent rehydration (Leslie et al.,ng in the presence of high-molecular weight sugars alsocteria in a glassy state, which considerably reduces theobility responsible for deteriorative reactions that take

dry state (Andersen et al., 1999; Crowe et al., 1998;pold, 1997). In addition to sugars, an antioxidant mayy to reduce oxidative damage during drying and stor-ann et al., 2009a). Which antioxidant should be used,

the composition of the protective matrix, however2011). Proteins (Chavez and Ledeboer, 2007; Heidebach), amino acids (Martos et al., 2007; Sunny-Roberts and; Pyar and Peh, 2011) and prebiotics/bers (Desmond; Schwab et al., 2007), used individually or mixed

ingredients, may also positively impact the drying- andbility of lactic acid bacteria.les for selecting the optimal protective matrix have

however, and need to be interpreted with caution. For some cases, glassy state was shown to be unneces-d bacterial stability (Ying et al., 2012; Kurtmann et al.,o, good survival during drying does not necessarilyto good survival during storage (Chavez and Ledeboer,ta et al., 2005). Hence, to provide a comprehensive viewctive effects of single ingredients or their combinations

c stability, any investigation should assess both drying--stability.

stability of probiotics in dry food products

ing step represents a relatively short period duringiotics are exposed to stressful conditions. Afterwards,ve to remain viable in the nal product for up to 2 yearsable 1 illustrates the water activity (aw) of dried prod-rs a broad range and should be considered during the

nt of probiotic products.ng on the storage conditions and duration, loss of pro-lity during storage may be magnitudes higher than

drying. In general, maintaining probiotics at low awtorage stability (Abe et al., 2009c; Kurtmann et al.,

aw, however, can only be achieved in a limited number such as food supplements, in which probiotics are typi-ly active ingredient, dried to a very low aw, and enclosedture-tight capsule.tion pertinent to probiotic supplements does not apply,

o the majority of dried food products. As shown in

K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365 361

Table 1Typical water activity and storage temperatures of various dehydrated products.

Dry/dehydrated products Water activity Length of shelf life Temperatures

Cereals, biscuits, snacks, confectionery, pet food Aw 0.20.5 Mild climate1824 months*

Hot climate1218 months*

During distributionInfant formula, milk powder, cereals Aw < 0.2Supplements Aw < 0.1

* Length of shelf life will also depend on distribution time and conditions.

Table 1, the aw of dried food products ranges from 0.1 to 0.5. Pro-biotic bacteria are normally added into them at very low doses,typically 0.011% of product weight, depending on the concentra-tion of viable cells in the probiotic powder. Hence, at equilibrium,the aw of the product dominates over the aw of the probiotic pow-der. In principle, the product could be dried further to reduce its awto values more favorable to probiotic stability. However, this pro-cedure is energy-intensive and might have a detrimental impact onthe sensory attributes of the product. Therefore, probiotic stabilitymust be achieved within the connes of the product aw. This rep-resents a signicant challenge to manufacturers of probiotic foods,especially when the aw of the food product is high.

The literature contains a vast set of data on how to improvethe storage stability of probiotics. The interpretation of this data ishampered, however, by certain factors, as described below. Regard-less of the probiotic process conditions prior to storage, each studyshould contage. This, uMany studiably at variinter-laboraprobiotic suthe variouslated experimprove prcontext, forcounts thatically 1061loss during probiotic cean economi

0.001

0.01

0.1

1

10

0

log

loss

/mon

th

Fig. 2. Overvieliterature datamost stable pr

Fig. 2 sulog loss per different putext of Tabl(the strain types of dry

Whilst aassumed tootic preparin one monAbe et al. (product awat 25 C). ItBidobacteto oxygen tduring stor

timelosets shle 1 achalls pro

arky co

port

st pts. Cnt, wwn ik wi, 200robiore m

cont cont, in w

with stabely wtic ceed (H05).

led ps of Brmorice arol and report the temperature and the aw during stor-nfortunately, was not done in many published papers.es also lack kinetic data. Kinetic data, acquired prefer-ous storage temperatures and aw, would allow bettertory comparison of results as well as an estimation ofrvival in alternative storage conditions. Furthermore,

published strategies tended to focus only on the iso-imental case and not consider how the strategies mightobiotic stability in product-realistic conditions. In this

example, it is not enough to merely secure viable cells meet the minimal requirement at end of shelf life (typ-07 cells/g) but also to limit the magnitude of viabilityshelf life. This reduces the need for initial overdosing oflls in the product and keeps the cost of production atcally acceptable level.

B. longum - Abe et al, 2009

B. infans - Abe et al, 2009B. breve - Abe et al, 2009

B. longum - Abe et al, 2009B. lacs - Heidebach et al, 2010L. rhamnosus - Ananta et al, 2005

L. rhamnosus - Pehkonen et al, 2008L. rhamnosus - Miao et al, 2008

L. rhamnosus - Ying et al, 2011L. rhamnosus - Ying et al, 2012

L. paracasei - Miao et al, 2008L. paracasei - Heidebach et al, 2010

Theet al. cproduc

Tabunder varioubenchmstrateg

7.5. Im

MoproducsegmeAs shoing milViljoenwith pThey aasepticproducmarkemixed

TherelativprobioreportRoy, 20in chilspecieFurthefruit ju0.60.50.40.30.20.1aw

w of storage inactivation rate of probiotic bacteria at 25 C based on. In papers where various protective formulations were used, only theeparation was included in this graph.

of probiotic(Saarela et

The majproducts ation tempethe producet al., 20113040 C (up to 50 C)On shelfRT and higher

mmarizes the inactivation rate of various probiotics (inmonth) stored at 25 C as a function of aw (data sets fromblications). It also illustrates, when viewed in the con-e 1, the suitability of the various probiotic preparationsand the protective ingredients) to be used in different

food matrices. log loss of 1 over a period of one year of shelf life is

be economically acceptable, the majority of the probi-ations shown in Fig. 1 reached this level of loss alreadyth. In contrast, the probiotic preparations reported byAbe et al., 2009c,a) fullled this requirement up to aof 0.3, (maximal log loss of 0.08 per month when stored

is also interesting to note, as shown in the gure, thatria, which are generally considered as more sensitivehan Lactobacilli, were the most stable bacterial genusage.-aw- and shelf life-conditions used in the papers of Abely approximate the conditions relevant to dried foodown innd demonstrate that it is possible to stabilize probioticsenging storage conditions. Plotting the stability data ofbiotic preparations onto a graph such as Fig. 2 will help

whether the improvement achieved with a particularrresponds to stability in a product-realistic scenario.

ant considerations in liquid products

robiotic-containing liquid products are refrigeratedhilled dairy products are the most popular ones in thisith probiotic yogurt as the dominant product format.n Fig. 1, probiotic yogurt is manufactured by ferment-th probiotics and starter cultures (Lourens-Hattingh and1). Another increasingly popular chilled liquid producttics is non-fermented dairy- or fruit-based beverages.ade by adding probiotics into the food product underditions and packaging the resulting mix into the naltainer. An oat-based fermented drink also exists in thehich oats are rst fermented with probiotics and then

fruit juice (e.g. the ProvivaTM juice concept).ility of probiotic-containing chilled dairy products isell mastered. Products that retain up to 106107 viablells/mL after 46 weeks of refrigerated storage have beenolzapfel and Schillinger, 2002; Champagne et al., 2005;

Nevertheless, further work on the stability of probioticsroducts is needed. For example, it is known that someidobacteria do not survive well in yogurt (Roy, 2005).e, low pH and the presence of various organic acids innd fruit preparations may make achieving good stability

s in fruit juice or in fruit-containing yogurt challengingal., 2011; Santo et al., 2011).or factors that impact probiotic stability in chilled liquidre the type of starter culture, the pH, the fermenta-rature, the presence of oxygen, and the composition oft (Klijn, 2005; Lourens-Hattingh and Viljoen, 2001; Ng; Talwalkar and Kailasapathy, 2004; Abe et al., 2009b).

362 K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365

OO

OH OO

OH

OH1 35

3

Lcin

OH

C

inic A

ids (b

Among theplays a pivogrowth andcation rate the yogurt Hence, impculture typebenecial inwell documof viable of plow hydrogability to de2011) and lpost-acidi

Anotherity in fermoligosacchaotic growthMartnez-Vpanied by proteolytic plementatioprobiotics f2010). Furthimproved tto the juicepositive effe

Ambientmat that cochallengingpost-acidiet al., 2005product forIt has been viable for sethat they caproduct (Toprobiotics asensory attrate the ba

pointGaia

all tics i

biotes

biotirable

dataioca

fermood

examat imlic acOHO

O

OH

OHOH

4La1

1

iuQ)LAQC-3(enotcaLciniuQlyoeffaC

OH

OH

OH O

COOH

O

OH

OH

OH

OH

Chloro genic Acid

La1

Qu

Fig. 3. Hydrolysis of coffee chlorogenic lactones (top) and chlorogenic ac

se factors, the selection of the yogurt starter culturetal role since it dictates not only the degree of probiotic

survival but also the process characteristics (e.g. acidi-and growth temperature) and the sensory properties of(Lourens-Hattingh and Viljoen, 2001; De Vuyst, 2000).rovement of probiotic stability by optimizing starters and composition has received a lot of attention. Theteractions between starter cultures and probiotics areented in the literature. To ensure a high concentrationrobiotics in yogurt, a starter culture should ideally haveen peroxide producing capacity (Ng et al., 2011), highgrade various reactive oxygen species (Odamaki et al.,ow H+-ATPase activity, which directly correlates withcation capacity (Ongol et al., 2007)

feasible route to the improvement of probiotic stabil-ented dairy products is the addition of prebiotics orrides to yogurts. This was reported to enhance probi-

and stability (Donkor et al., 2007; Vasiljevic et al., 2007;

at the by Bioinner wprobio

8. Promatric

ProcompaRecentact as brials byin the f

Forities thphenoillaluenga et al., 2012), and was sometimes accom-increased production of organic acids and increasedactivity of probiotics. It has also been shown that sup-n of a model fruit juice with green tea extract stabilizesor up to 6 weeks of refrigerated storage (Shah et al.,ermore, the addition of oat or barley bers to fruit juicehe survival of B. breve added as a frozen concentrate

(Saarela et al., 2011). The mechanism underlying thect of these additions is still unknown.

liquid products are another interesting product for-uld be considered for probiotics. However, it is very

to keep probiotics stable (Klijn, 2005) and to preventcation of the product by starter cultures (Jankowska) at ambient temperatures. Therefore, at present, thismat remains a white spot for probiotic applications.shown, though, that some probiotic strains can remainveral months of storage at room temperature, providednnot metabolize the sugars present in the fermentedurnade et al., 2005). It is also challenging to stabilizet ambient temperatures whilst minimally impacting

ributes. One way to achieve this is to physically sepa-cteria from the liquid food matrix and only mix them

some probesterase, ferferulate, ferand Dmowsto improvincontent of Lactobacilluwhich reduand therebet al., 2004,reducing th2008), thusdiet. To disstarch or syfor the desiet al., 2011)

Regardin(La1) couldgenic lactoresponsibletrials were strate as weOH

O

OH

OH

4

5 +

dicAcieffaCenotca

OH

OOH

OH

O

OH

OH+

cid Caff eic Acid

ottom) catalyzed by L. johnsonii La1 esterase.

of consumption. The Probiotic Straw as manufacturedin which dried probiotic cells are immobilized in the

of the straw seems to be a good vehicle for deliveringn this fashion.

ics and their biocatalytic activities in food

cs are typically added into foods to perform a function to dietary supplements such as vitamins or minerals., however, illustrate the potential of probiotic strains totalysts, thereby improving the properties of raw mate-entation or by performing specic biotransformations

production chain or during the passage through the GIT.ple, some probiotic bacteria have -glucosidase activ-prove the bioavailability of isoavones, avonols andids (Cho et al., 2011; Ding and Shah, 2010). Similarly,

iotic bacteria also produce esterases (e.g. cinnamoyluloyl esterase) and have been used to transform methylulic acid and cinnamic acids (Lai et al., 2009; Szwajgierka, 2010; Szwajgier and Jackubczyk, 2010). In additiong bioavailability, probiotics can be used to reduce theundesired compounds in food materials. For instance,s fermentum CRL722 has high -galactosidase activity,ces the content of atulence-causing sugars in soymilky enhances the digestibility of this product (Leblanc

2005). Some Bidobacteria strains, in turn, are potent ate phytate content of whole wheat dough (Palacios et al.,

improving the bioavailability of iron from whole-graincover new strains with superior capacity to degradenthesize vitamins, genetic screening of food microbiotared functional properties could be considered (Turpin.g sensory attributes, it was reported that L. johnsonii

reduce the bitterness of coffee by hydrolyzing choloro-nes (Fig. 3 top) formed during roasting and partially

for the bitter taste of coffee (Frank et al., 2011). Theconducted in model systems with 3-CQAL as pure sub-ll as in coffee brew (Bel-Rhlid et al., 2006). The enzyme

K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365 363

responsible for this hydrolysis was identied and characterized asan esterase. La1 esterase can also catalyse the hydrolysis of thecoffee antioxidantchlorogenic acids into quinic and caffeic acids(Figure 3 bottom), whose biovailability is higher than that of chloro-genic acids

L. johnsostudied comcated in a metabolizein vitro andsonii La1 delactic acid the probiota two-fold pounds fromto L. johnson

These extive biocatacontext depprobiotic emerging kecial effecsee new prfood matrixfuture studiof stability

9. Conclus

Probiotihas expandcomfort anious, sometresearch cothese benetory enviroof focused evident thabenets tobe alive at non-viable likely to retaken to seimproved vare robust mconditions various straing the drymatrix throstorage temresearchersstability of tions. In adinteraction obtained vistrains leadity of food iis the possiuct.

Acknowled

We thanfor critically

References

Abe, F., Miyauchi, H., Uchijima, A., Yaesima, T., Iwatsuki, K., 2009a. Stability ofbidobacteria in powdered formula. International Journal of Food Science &Technology 44, 718724.

omitahe start. Lechijim

peraturnatioC.A., 2iers.., Singstrial 18, 24E., Vold Lactn, A.B.ze-dri

of fre547.

d, R., rolysisllus johFood Cd, R., K

20061B., Mo

selectment, M., Mhe sururt p

rs, M.M storanal of .A., Foenstoal behe. Prorica 1, M., Wobacill.P., Te

llus bu4.

i, C., B6286gne, Cc acid125.gne, Cres to

B.E., Lproce

H.A., R specrimened Sta., Le

hytocng chllus s410.., Wetes fr1.

M.C., m for lied M, B.Miotic lpplied, D., Mchara. Journ.H., Cais. Annt, L., 20res. Fde, Gent Od, C., FroESL-C 338.(Couteau et al., 2001)nii La1 can also hydrolyse rosmarinic acid (RA), a widelypound from rosemary extracts, which has been impli-

number of health benets. RA is hypothesized to bed by gut microbiota prior to absorption. Experiments

in the GIT model (TIM-1) system showed that L. john-graded RA into caffeic acid and 3.4-dihydroxyphenyl

(Bel-Rhlid et al., 2009). Therefore, the combination ofic L. johnsonii La1 and rosemary extract could result ineffect: improved bioavailability of the bioactive com-

RA, together with the probiotic properties attributedii La1.amples show that bacterial strains can be used as effec-lysts. Whether they should be called probiotic in thisends on whether the criteria for the use of the termdescribed above are met. Nevertheless, in the light ofnowledge on bacterial biocatalysis and the health ben-ts of various natural compounds, the market is likely tooducts emerge incorporating mixtures of bacteria and, modied by means of microbial enzymes. Therefore,es on probiotic stability should also include assessmentin the bio-transformed food matrices.

ion

cs represent a fast evolving eld which in past yearsed from the traditional health benet areas of digestived immune protection to diverse applications in var-imes unexpected, health benet areas. The probioticmmunity is now facing the challenge of substantiatingts to the standard required by the stringent regula-nment in Europe, which will necessitate the generationhealth claim dossiers. It is also becoming increasinglyt non-viable microorganisms can also confer health

the host. However, a probiotic, by denition mustthe time of consumption, and despite the efcacy ofmicroorganisms for certain health benet, others arequire live microorganisms. Therefore, care must belect intrinsically robust strains, which may be furtheria accelerated evolution strategies. Even strains thatust be protected from viability loss during the harsh

encountered during probiotic powder production, andtegies have been developed for this purpose. Follow-ing step, probiotics must remain viable in the foodughout shelf-life, a property that largely depends on theperature and the water activity of the product. Many

have investigated strategies to improve the storageprobiotics, but not always at product-realistic condi-dition to the health benets derived from the directof the microorganisms with the host, some might bea biocatalytic properties exhibited by some probioticing to improvement of bioavailability and digestibil-ngredients. An interesting side benet of this strategybility to improve sensory attributes of the food prod-

gment

k, Diane Zimmermann, Eric Emond, and Jeroen Muller reviewing the manuscript.

Abe, F., Ton tyogu

Abe, F., UtemInte

Adams, mod

Anal, A.Kinduogy

Ananta, drie

Andersefreestate540

Bel-RhliHydbaciand

Bel-RhliWO

Berger, rallytrea

Bieleckaon tyogh

BrasheaatedJour

Bravo, JBientionnervAme

BrennanLact

Castro, Hbaci879

CavadinEP08

Champalacti118

Champacultu

Chavez, and

Cheplin,withexpeUnit

Cho, K.Mof pduriBaci402

Chou, L.Sisola233

Collado,anisApp

Corcoranprobof A

Couteauand acid

Crowe, Jbios

De Vuyscultu

DeshpanCurr

Desmonof GNFB, S., Yaeshima, T., Iwatsuki, K., 2009b. Effect of production conditionsbility of a human bidobacterial species Bidobacterium longum intters in Applied Microbiology 49, 715720.a, A., Miyauchi, H., Yaesima, T., Iwatsuki, K., 2009c. Effects of storage

re and water activity on the survival of bidobacteria in powder form.nal Journal of Dairy Technology 62, 234239.010. The probiotic paradox: live and dead cells are biological response

Nutrition Research Reviews 23, 3746.h, H., 2007. Recent advances in microencapsulation of probiotics forapplications and targeted delivery. Trends in Food Science & Technol-0251.kert, M., Knorr, D., 2005. Cellular injuries and storage stability of sprayobacillus rhamnosus GG. International Dairy Journal 15, 399409., Fog-Petersen, M.S., Larsen, H., Skibsted, L.H., 1999. Storage stability ofed starter cultures (Streptococcus thermophilus) as related to physicalezing matrix. Lebensmittel-Wissenschaft und-Technologie (LWT) 32,

Crespy, V., Page-Zoerkler, N., Nagy, K., Raab, T., Hansen, C.E., 2009. of rosmarinic acid from rosemary extract with esterases and Lacto-nsonii in vitro and in a gastrointestinal model. Journal of Agriculturalhemistry 57, 77007705.raehenbuehl, K., Aesbach, R., Lerch, K., 2006. Soluble coffee product.25505.ine, D., Mansourian, R., Arigoni, F., 2010. HspR mutations are natu-ted in Bidobacterium longum when applying successive heat-shocks. Journal of Bacteriology 192, 256263.ajkowska, A., 2000. Effect of spray drying temperature of yoghurt

vival of starter cultures, moisture content and sensoric properties ofowder. Nahrung/Food 44, 257260.., Gilliland, S.E., 1995. Survival during frozen and subsequent refriger-

ge of Lactobacillus acidophilus cells as inuenced by the growth phase.Dairy Science 78, 23262335.rsythe, P., Chew, M.V., Escaravage, E., Savignac, H.M., Dinan, T.G.,ck, J., Cryan, J.F., 2011. Ingestion of Lactobacillus strain regulates emo-avior and central GABA receptor expression in a mouse via the vagusceedings of the National Academy of Sciences of the United States of08, 1605016055.anismail, B., Johnson, M.C., Ray, B., 1986. Cellular damage in dried

us acidophilus. Journal of Food Protection 49, 4753.ixeira, P.M., Kirby, R., 1997. Evidence of membrane damage in Lacto-lgaricus following freeze drying. Journal of Applied Microbiology 82,

allevre, O., Gaier, W., 2009. Cereal products containing probiotics.3B9..P., Gardner, N., Brochu, E., Beaulieu, Y., 1991. The freeze drying of

bacteria. A review. Canadian Institute of Science and Technology 24,

.P., Gardner, N.J., Roy, D., 2005. Challenges in the addition of probiotic foods. Critical Reviews in Food Science and Nutrition 45, 6184.

edeboer, A.M., 2007. Drying of probiotics, optimization of formulationss to enhance storage survival. Drying Technology 25, 11931201.ettger, L.F., 1920. Studies on the transformation of the intestinal ora,

ial reference to the implantation of bacillus acidophilus: II. Feedingts on man. Proceedings of the National Academy of Sciences of thetes of America 6, 704705.e, J.H., Yun, H.D., Ahn, B.Y., Kim, H., Seo, W.T., 2011. Changeshemical constituents (isoavones avanols, and phenolic acids)eonggukjang soybeans fermentation using potential probioticsubtilis CS90. Journal of Food Composition and Analysis 24,

imer, B., 1999. Isolation and characterization of acid- and bile-tolerantom strains of Lactobacillus acidophilus. Journal of Dairy Science 82,

Sanz, Y., 2007. Induction of acid resistance in Bidobacterium: a mech-improving desirable traits of potentially probiotic strains. Journal oficrobiology 103, 11471157.., Ross, R.P., Fitzgerald, G.F., Stanton, C., 2004. Comparative survival ofactobacilli spray-dried in the presence of prebiotic substances. Journal

Microbiology 96, 10241039.cCartney, A.L., Gibson, G.R., Williamson, G., Faulds, C.B., 2001. Isolationcterization of human colonic bacteria able to hydrolyse chlorogenical of Applied Microbiology 90, 873881.rpenter, J.P., Crowe, L.M., 1998. The role of vitrication in anhydro-ual Review of Psychology 60, 73103.00. Technology aspects related to the application of functional starterood Technology and Biotechnology 38, 105112.., Rao, S., Patole, S., 2011. Progress in the eld of probiotics: year 2011.pinion in Gastroenterology 27, 1318.itzgerald, G.F., Stanton, C., Ross, R.P., 2004. Improved stress toleranceoverproducing Lactococcus lactis and probiotic Lactobacillus paracasei

Applied and Environment Microbiology 70, 59295936.

364 K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365

Desmond, C., Ross, R.P., OCallaghan, E., Fitzgerald, G., Stanton, C., 2002. Improvedsurvival of Lactobacillus paracasei NFCB 338 in spray-dried powders containinggum acacia. Journal of Applied Microbiology 93, 10031011.

Desmond, C., Stanton, C., Fitzgerald, G.F., Collins, K., Ross, R.P., 2001. Environmentaladaptation of probiotic lactobacilli towards improvement of performance duringspray dryi

Di, M.L., Centi, M.G., 2003stratum copatients. E

Ding, W.K., Shsoymilk sufermentat

Donkor, O.N., activity ofInternatio

EFSA NDA panrelated toyoghurt sythe gut ofin patients1924/2006

EFSA NDA panrelated to pursuant t

Efsa NDA panrelated to tem (ID 99(EC) No. 19

Euromonitor Itive Health

FAO/WHO, 20powder mthe United

FAO/WHO, 200culture OrOntario, C

Frank, O., Zeheroast coffeFood Rese

Gilliland, S.E., storage ofence 73, 1

Gotteland, M.,E., Speiskywith cranb421426.

Gueniche, A., Kton, L., BiedVitreoscilldouble-bli159, 1357

Guillaume, E., proteomic771784.

Hamilton-Milltion and e

Heidebach, T., lation on fr98, 30931

Higl, B., KurtmRisbo, J., 2storage stamatrix. Bio

Holzapfel, W.HResearch I

ICMR-DBT, 20Journal of

Ishida, Y., NakaO., Fujiwarperennial Dairy Scie

Jankowska, A.,microoraNutrition S

Jones, M.L., Maof a micro30242 yogNutrition,

Kadooka, Y., SKagoshimabiotics (Larandomize

Kearney, N., Mment of a s338. Intern

Klijn, A., 2005. Physiological and Molecular Characterisation of Stress Response inBidobacterium longum NCC2705. University College Cork, Cork, Ireland.

Kurtmann, L., Carlsen, C.U., Risbo, J., Skibsted, L.H., 2009a. Storage stability of freeze-dried Lactobacillus acidophilus (La-5) in relation to water activity and presenceof oxygen and ascorbate. Cryobiology 58, 175180.

n, L.peratuence ress 2n, L., bactecultur, Lorcarases p

of di502M., Gisis inagene

J.G., G, 2004llus fernal of

J.G., Pis ablets. FEM., Israeect boEnviroan, F.s on vechnoC., Hsirnatioiang, Z

acqu-Hattial Dairs, A.,. Chaate 198.s, A., S

in withicrob

z-Villation o

durinG.I., Membrrs in n, J., 2

Nutritina, Nof pro117di, M

geot, Chotro2 and

nal of koff, En, W.183.

Mills,ides onologi, P., Da, N., Rre suri infe, C., Beruecki048

t, M., . A n

tory eition i, V., Gacroixobactobial .A., Roe techhe groobiolong. International Dairy Journal 11, 801808.C., Cinque, B., Masci, S., Giuliani, M., Arcieri, A., Zicari, L., De, S.C., Cifone,. Effect of the lactic acid bacterium Streptococcus thermophilus onrneum ceramide levels and signs and symptoms of atopic dermatitisxperimental Dermatology 12, 615620.ah, N.P., 2010. Enhancing the biotransformation of isoavones inpplemented with lactose using probiotic bacteria during extended

ion. Journal of Food Science 75, M140M149.Nilmini, S.L.I., Stolic, P., Vasiljevic, T., Shah, N.P., 2007. Survival and

selected probiotc organisms in set-type yoghurt during cold storage.nal Dairy Journal 17, 657665.el, 2010a. Scientic opinion on the substantiation of a health claim

fermented milk containing Lactobacillus casei DN-114 001 plusmbiosis (Actimel), and reduction of Clostridium difcile toxins in

patients receiving antibiotics and reduced risk of acute diarrhoea receiving antibiotics pursuant to Article 14 of Regulation (EC) No.. EFSA Journal 8, 1903.el, 2010b. Scientic Opinion on the substantiation of health claimslive yoghurt cultures and improved lactose digestion (ID 1143, 2976)o Article 13(1) of Regulation (EC) No. 1924/2006. EFSA Journal 8, 1763.el, 2011. Scientic Opinion on the substantiation of health claimsLactobacillus johnsonii BFE 6128 and natural defences/immune sys-0) and skin health (ID 991) pursuant to Article 13(1) of Regulation24/2006. EFSA Journal 9, 2024.

nternational, 2010. Probioticsand Prebiotics: Moving Beyond Diges- Opportunities and Challenges.

01. Health and nutritional properties of probiotics in food includingilk with live lactic acid bacteria. Food and Agriculture Organization of

Nations and World Health Organization, Cordoba, Argentina.2. Guidelines for the evaluation of probiotics in food. Food and Agri-ganization of the United Nations and World Health Organization,anada.ntbauer, G., Hofmann, T., 2011. Bioresponse-guided decomposition ofe beverage and identication of key bitter taste compounds. Europeanarch and Technology 222, 492508.Rich, C.N., 1989. Stability during frozen and subsequent refrigerated

Lactobacillus acidophilus grown at different pH. Journal of Dairy Sci-1871192.

Andrews, M., Toledo, M., Munoz, L., Caceres, P., Anziani, A., Wittig,, H., Salazar, G., 2008. Modulation of Helicobacter pylori colonizationerry juice and Lactobacillus johnsonii La1 in children. Nutrition 24,

naudt, B., Schuck, E., Volz, T., Bastien, P., Martin, R., Rocken, M., Bre-ermann, T., 2008. Effects of nonpathogenic gram-negative bacterium

a liformis lysate on atopic dermatitis: a prospective, randomized,nd, placebo-controlled clinical study. British Journal of Dermatology1363.Berger, B., Affolter, M., Kussmann, M., 2009. Label-free quantitatives of two Bidobacterium longum strains. Journal of Proteomics 72,

er, J.M., Gibson, G.R., Bruck, W., 2003. Some insights into the deriva-arly uses of the word probiotic. British Journal of Nutrition 90, 845.Foerst, P., Kulozik, U., 2010. Inuence of casein-based microencapsu-eeze-drying and storage of probiotic cells. Journal of Food Engineering6.ann, L., Carlsen, C.U., Ratjen, J., Forst, P., Skibsted, L.H., Kulozik, U.,

007. Impact of water activity, temperature, and physical state on thebility of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactosetechnology Progress 23, 794800.., Schillinger, U., 2002. Introduction to pre- and probiotics. Food

nternational 35, 109116.11. ICMR-DBT guidelines for evaluation of probiotics in food. IndianMedical Research 134, 2225.mura, F., Kanzato, H., Sawada, D., Hirata, H., Nishimura, A., Kajimoto,a, S., 2005. Clinical effects of Lactobacillus acidophilus strain L-92 onallergic rhinitis: a double-blind, placebo-controlled study. Journal ofnce 88, 527533.

Reps, A., Proszek, A., Krasowska, M., 2005. Effect of high pressure on and sensory characteristics of yoghurt. Polish Journal of Food andciences 14 (55), 7984.rtoni, C.J., Parent, M., Prakash, S., 2011. Cholesterol-lowering efcacyencapsulated bile salt hydrolase-active Lactobacillus reuteri NCIMBhurt formulation in hypercholesterolaemic adults. British Journal of19.ato, M., Imaizumi, K., Ogawa, A., Ikuyama, K., Akai, Y., Okano, M.,, M., Tsuchida, T., 2010. Regulation of abdominal adiposity by pro-ctobacillus gasseri SBT2055) in adults with obese tendencies in ad controlled trial. European Journal of Clinical Nutrition 64, 636643.eng, X.C., Stanton, C., Kelly, J., Fitzgerald, G., Ross, R.P., 2009. Develop-pray dried probiotic yoghurt containing Lactobacillus paracasei NFBCational Dairy Journal 19, 684689.

KurtmanteminuProg

Kurtmanotic Agri

Lai, K.K.esteples5018

Lautier, geneMut

Leblanc,G.G.baciJour

Leblanc,722 of ra

Leslie, Sprotand

Li, B., TitantBiot

Lian, W.Inte

Liu, Z., Jwith

Lourenstion

Margolle2003chol191

MargolleStratal M

Martneaddimilk

Martos, on mLette

MellentiNew

Menshuing 1170

MessaouRoupsycR005Jour

Metchniman161

Miao, S.,charTech

MichettPortcultupylo

Monnetdelb86, 3

Morisse2011piraNutr

MozzettF., LbidMicr

Muller, Jof thing tMicr, Carlsen, C.U., Skibsted, L.H., Risbo, J., 2009b. Water activity-re state diagrams of freeze-dried Lactobacillus acidophilus (La-5):of physical state on bacterial survival during storage. Biotechnology5, 265270.Skibsted, L.H., Carlsen, C.U., 2009c. Browning of freeze-dried probi-ria cultures in relation to loss of viability during storage. Journal ofal and Food Chemistry 57, 67366741., G.L., Gonzalez, C.F., 2009. Biochemical properties of two cinnamoyluried from a Lactobacillus johnsonii strain isolated from stool sam-abetes-resistant rats. Applied and Environment Microbiology 75,4.llot, B., Auffray, Y., Castillo-Sanchez, X., Boutibonnes, P., 1988. Muta-

Streptococcus lactis exposed to UV irradiation and alkylating agents.sis 3, 245247.arro, M.S., Silvestroni, A., Connes, C., Piard, J.C., Sesma, F., Savoy de,. Reduction of alpha-galactooligosaccharides in soyamilk by Lacto-mentum CRL 722 in vitro and in vivo evaluation of fermented soyamilk.Applied Microbiology 97, 876881.iard, J.C., Sesma, F., de Giori, G.S., 2005. Lactobacillus fermentum CRL

to deliver active alpha-galactosidase activity in the small intestineS Microbiology Letters 248, 177182.

li, E., Lighthart, B., Crowe, J., Crowe, L., 1995. Trehalose and sucroseth membranes and proteins in intact bacteria during drying. Appliednment Microbiology 61, 35923597.

, Liu, X., Zhao, J., Zhang, H., Chen, W., 2011. Effects of cryoprotec-iability of Lactobacillus reuteri CICC6226. Applied Microbiology andlogy 92, 609616.ao, H.C., Chou, C.C., 2002. Survival of bidobacteria after spray-drying.nal Journal of Food Microbiology 74, 7986.., Zhou, K., Li, P., Liu, G., Zhang, B., 2007. Screening of bidobacteriaired tolerance to human gastrointestinal tract. Anaerobe 13, 215219.ngh, A., Viljoen, B.C., 2001. Yogurt as probiotic carrier food. Interna-y Journal 11, 117.

Garcia, L., Sanchez, B., Gueimonde, M., los Reyes-Gavilan, C.G.,racterisation of a Bidobacterium strain with acquired resistance toa preliminary study. International Journal of Food Microbiology 82,

nchez, B., 2012. Selection of a Bidobacterium animalis subsp. lactis a Decreased Ability To Produce Acetic Acid. Applied and Environmen-iology 78, 33383342.luenga, C., Frias, J., Gomez, R., Vidal-Valverde, C., 2012. Inuence off rafnose family oligosaccharides on probiotic survival in fermentedg refrigerated storage. International Dairy Journal 16, 768774.inahk, C.J., De Valdez, G.F., Morero, R., 2007. Effects of protective agentsane uidity of freeze-dried Lactobacillus delbrueckii ssp, bulgaricus.Applied Microbiology 45, 282288.008. Probiotics: successfull strategies from the global market place.tion Business..V., Gordienko, M.G., Voinovskiy, A.A., Zbicinski, I., 2010. Spray dry-biotics, process development and scale-up. Drying Technology 28,7.., Lalonde, R., Violle, N., Javelot, H., Desor, D., Nejdi, A., Bisson, J.F.,., Pichelin, M., Cazaubiel, M., Cazaubiel, J.M., 2011. Assessment of

pic-like properties of a probiotic formulation (Lactobacillus helveticus Bidobacterium longum R0175) in rats and human subjects. BritishNutrition 105, 755764.., 1907. Lactic acid as inhibiting intestinal putrefaction. In: Heine-

(Ed.), The Prolongation of Life: Optimistic Studies London. , pp.

S., Stanton, C., Fitzgerald, G., Roos, Y., Ross, R.P., 2008. Effect of disac-n survival during storage of freeze dried probiotics. Dairy Science &y 88, 1930.orta, G., Wiesel, P.H., Brassart, D., Verdu, E., Herranz, M., Felley, C.,ouvet, M., Blum, A.L., Corthesy-Theulaz, I., 1999. Effect of whey-basedpernatant of Lactobacillus acidophilus (johnsonii) La1 on Helicobacterction in humans. Digestion 60, 203209.al, C., Corrieu, G., 2003. Improvement of the resistance of Lactobacillusi ssp bulgaricus to freezing by natural selection. Journal of Dairy Science3053.Aubert-Jacquin, C., Soulaines, P., Moneret-Vautrin, D.A., Dupont, C.,on-hydrolyzed, fermented milk formula reduces digestive and res-vents in infants at high risk of allergy. European Journal of Clinical65, 175183.rattepanche, F., Moine, D., Berger, B., Rezzonico, E., Meile, L., Arigoni,, C., 2010. New method for selection of hydrogen peroxide adaptederia cells using continuous culture and immobilized cell technology.Cell Factories 9, 60.ss, R.P., Sybesma, W.F., Fitzgerald, G.F., Stanton, C., 2011. Modicationnical properties of Lactobacillus johnsonii NCC 533 by supplement-wth medium with unsaturated fatty acids. Applied and Environmentgy 77, 68896898.

K. Makinen et al. / Journal of Biotechnology 162 (2012) 356 365 365

Ng, E.W., Yeung, M., Tong, P.S., 2011. Effects of yogurt starter cultures on the sur-vival of Lactobacillus acidophilus. International Journal of Food Microbiology 145,169175.

Noriega, L., Gueimonde, M., Sanchez, B., Margolles, A., los Reyes-Gavilan, C.G., 2004.Effect of the adaptation to high bile salts concentrations on glycosidic activ-ity, survival at low pH and cross-resistance to bile salts in Bidobacterium.International Journal of Food Microbiology 94, 7986.

Odamaki, T., Xiao, J.Z., Yonezawa, S., Yaeshima, T., Iwatsuki, K., 2011. Improved via-bility of bidobacteria in fermented milk by cocultivation with Lactococcus lactissubspecies lactis. Journal of Dairy Science 94, 11121121.

Ongol, M.P., Sawatari, Y., Ebina, Y., Sone, T., Tanaka, M., Tomita, F., Yokota, A., Asano,K., 2007. Yoghurt fermented by Lactobacillus delbrueckii subsp, bulgaricus H+-ATPase-defective mutants exhibits enhanced viability of Bidobacterium breveduring storage. International Journal of Food Microbiology 116, 358366.

Palacios, M.C., Haros, M., Rosell, C.M., Sanz, Y., 2008. Selection of phytate-degradinghuman bidobacteria and application in whole wheat dough fermentation. FoodMicrobiology 25, 169176.

Palmfeldt, J., Hahn-Hgerdal, B., 2000. Inuence of culture pH on survival ofLactobacillus reuteri subjected to freeze-drying. International Journal of FoodMicrobiology 55, 235238.

Peguet-Navarro, J., Dezutter-Dambuyant, C., Buetler, T., Leclaire, J., Smola, H., Blum,S., Bastien, P., Breton, L., Gueniche, A., 2008. Supplementation with oral probioticbacteria protects human cutaneous immune homeostasis after UV exposure-double blind, randomized, placebo controlled clinical trial. European Journal ofDermatology 18, 504511.

Pehkonen, K.S., Roos, Y.H., Miao, S., Ross, R.P., Stanton, C., 2008. State transitions andphysicochof Lactoba1732174

Peighambardofor preserv& Technol

Peng, G.C., Hsucasei for tPediatric A

Prado, F.C., Parbeverages

Pyar, H., Peh, KLactobacillof Dairy Te

Reilly, S.S., Gilrefrigerate714718.

Rowland, I., CaF., Marteau report o436439.

Roy, D., 2005.products.

Saarela, M., AlImprovingInternatio

Salazar-Lindo,G., Colin, Pof mild acNutrition 4

Sanchez, B., ChY., de los Rtion and thApplied an

Sanchez, B., CAnglade, PMargolles,lactis to biMicrobiolo

Santivarangkna, C., Foerst, P., Kulozik, U., 2007. Alternative drying processes for theindustrial preservation of lactic acid starter cultures. Biotechnology Progress 23,302315.

Santivarangkna, C., Kulozik, U., Foerst, P., 2005. Effect of carbohydrates on thesurvival of Lactobacillus helveticus during vacuum drying. Letters in AppliedMicrobiology 42, 271276.

Santivarangkna, C., Kulozik, U., Kienberger, H., Foerst, P., 2009. Changes in membranefatty acids of Lactobacillus helveticus during vacuum drying with sorbitol. Lettersin Applied Microbiology 49, 516521.

Santo, A.P., Perego, P., Converti, A., Oliveira, M.N., 2011. Inuence of food matriceson probiotic viability a review focusing on the fruity bases. Trends in FoodScience & Technology 22, 377385.

Savino, F., Cordisco, L., Tarasco, V., Palumeri, E., Calabrese, R., Oggero, R.,Roos, S., Matteuzzi, D., 2010. Lactobacillus reuteri DSM 17938 in infantilecolic: a randomized, double-blind, placebo-controlled trial. Pediatrics 126,e526e533.

Savino, F., Pelle, E., Palumeri, E., Oggero, R., Miniero, R., 2007. Lactobacillus reuteri(American type culture collection strain 55730) versus simethicone in thetreatment of infantile colic: a prospective randomized study. Pediatrics 119,e124e130.

Schwab, C., Vogel, R., Ganzle, M.G., 2007. Inuence of oligosaccharides on theviability and membrane properties of Lactobacillus reuteri TMW1.106 duringfreeze-drying. Cryobiology 55, 108114.

Shah, N.P., Ding, W.K., Fallourd, M.J., Leyer, G., 2010. Improving the stability of pro-biotic bacteria in model fruit juices using vitamins and antioxidants. Journal ofFood Science 75, M278M282.

, P.J., Stanton, C., Fitzgerald, G.F., Ross, R.P., 2005. Intrinsic tolerance of Bi-cteriuage. Jo., Leonismsoberturviva0) strarnatior, D., roducnorumr, D.,

ophilu. Techar, A., Kence ensive, S., nismsW., Hs of lagenoy 77, 8ic, T., KcontaY.-C., and dratio217.

, Sangarchits on

gricult., SuancedGG i602.emical aspects of cryoprotection and stabilization in freeze-dryingcillus rhamnosus GG (LGG). Journal of Applied Microbiology 104,3.ust, S.H., Golshan Tafti, A., Hesari, J., 2011. Application of spray dryingation of lactic acid starter cultures: a review. Trends in Food Science

ogy 22, 215224., C.H., 2005. The efcacy and safety of heat-killed Lactobacillus para-reatment of perennial allergic rhinitis induced by house-dust mite.llergy and Immunology 16, 433438.ada, J.L., Pandey, A., Soccol, C.R., 2008. Trends in non-dairy probiotic. Food Research International 41, 111123..-K., 2011. Effect of cryoprotective agents on survival and stability ofus acidophilus cultured in food-grade medium. International Journalchnology 64, 578584.liland, S.E., 1999. Bidobacterium longum survival during frozen andd storage as related to pH during growth. Journal of Food Science 64,

purso, L., Collins, K., Cummings, J., Delzenne, N., Goulet, O., Guarner,, P., Meier, R., 2010. Current level of consensus on probiotic science

f an expert meeting London, 23 November 2009. Gut Microbes 1,

Technological aspects related to the use of bidobacteria in dairyLait 85, 3956.akomi, H.L., Matto, J., Ahonen, A.M., Puhakka, A., Tynkkynen, S., 2011.

the storage stability of Bidobacterium breve in low pH fruit juice.nal Journal of Food Microbiology 149, 106110.

E., Figueroa-Quintanilla, D., Caciano, M.I., Reto-Valiente, V., Chauviere,., 2007. Effectiveness and safety of Lactobacillus LB in the treatmentute diarrhea in children. Journal of Pediatric Gastroenterology and4, 571576.ampomier-Verges, M.C., Collado, M.C., Anglade, P., Baraige, F., Sanz,eyes-Gavilan, C.G., Margolles, A., Zagorec, M., 2007a. Low-pH adapta-e acid tolerance response of Bidobacterium longum biotype longum.d Environment Microbiology 73, 64506459.hampomier-Verges, M.C., Stuer-Lauridsen, B., Ruas-Madiedo, P.,., Baraige, F., de los Reyes-Gavilan, C.G., Johansen, E., Zagorec, M.,

A., 2007b. Adaptation and response of Bidobacterium animalis subsp.le: a proteomic and physiological approach. Applied and Environmentgy 73, 67576767.

Simpsondobastor

Sun, W.Qorga

Sunny-Ron s(E80Inte

Szwajgiesp. pPolo

Szwajgieacidrum

Talwalkinupreh

Tournadorga

Turpin, ertiemetolog

Vasiljevotic

Wang, ria rehy209

Ying, D.Demeffecof A

Ying, DEnhsus 597m species to heat and oxygen and survival following spray drying andurnal of Applied Microbiology 99, 493501.pold, C., 1997. Cytoplasmic vitrication and survival of anhydrobiotic. Comparative Biochemistry and Physiology 117A, 327333.s, E.O., Knorr, D., 2009. The protective effect of monosodium glutamatel of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800ins during spray-drying and storage in trehalose-containing powders.nal Dairy Journal 19, 209214.Dmowska, K., 2010. Novel ferulic acid esterases from Bidobacteriumed on selected synthetic and natural carbon sources. Acta Scientiarum. Technologia Alimentaria 0, 305318.

Jackubczyk, A., 2010. Biotransformation of ferulic acid by Lactobacilluss K1 and selected Bidobacterium strains. Acta Scientiarum Polono-nologia Alimentaria 9, 4559.ailasapathy, K., 2004. A review of oxygen toxicity in probiotic yogurts:

on the survival of probiotic bacteria and protective techniques. Com-e Reviews in Food Science and Food Safety 3, 117124.Aymes, F., Reniero, R., 2005. Shelf stable product with living micro-. WO2005/089560.umblot, C., Guyot, J.P., 2011. Genetic screening of functional prop-actic Acid bacteria in a fermented pearl millet slurry and in theme of fermented starchy foods. Applied and Environment Microbi-7228734.ealy, T., Mishra, V.K., 2007. Effects of beta-glucan addition to a probi-

ining yogurt. Journal of Food Science 72, C405C411.Yu, R.-C., Chou, C.-C., 2004. Viability of lactic acid bacte-

bidobacteria in fermented soymilk after drying, subsequentn and storage. International Journal of Food Microbiology 93,

uansri, L., Weerakkody, R., Singh, T.K., Leischtfeld, S.F., Gantenbein-, C., Augustin, M.A., 2011. Tocopherol and ascorbate have contrastingthe viability of microencapsulated Lactobacillus rhamnosus GG. Journalural and Food Chemistry 59, 1055610563.n, J., Sanguansri, L., Weerakkody, R., Augustin, M.A., 2012.

survival of spray-dried microencapsulated Lactobacillus rhamno-n the presence of glucose. Journal of Food Engineering 109,

Science and technology for the mastership of probiotic applications in food products1 History of probiotics2 The definition of probiotics3 Health benefit application areas of probiotics4 Identification and development of probiotics5 The use of non-viable probiotics6 EFSA and probiotic health claims7 Production of viable and stable probiotics7.1 Intrinsic stability of probiotic strains7.2 Impact of process conditions on probiotic stability7.3 Important considerations in the production of probiotics for applications in dry products7.4 Storage stability of probiotics in dry food products7.5 Important considerations in liquid products

8 Probiotics and their biocatalytic activities in food matrices9 ConclusionAcknowledgmentReferences