Recent developments in cheese cultures with protective and ...

Dairy Sci. Technol. 88 (2008) 421–444 Available online at:c© INRA, EDP Sciences, 2008 www.dairy-journal.orgDOI: 10.1051/dst:2008013

Review

Recent developments in cheese cultureswith protective and probiotic functionalities

Franck Grattepanche, Susanne Miescher-Schwenninger,Leo Meile, Christophe Lacroix*

ETH Zurich, Laboratory of Food Biotechnology, Institute of Food Science and Nutrition,Schmelzbergstrasse 7, Zürich, Switzerland

Abstract – Microorganisms play essential roles in the manufacture and ripening of cheese, largelycontributing to the development of organoleptic properties by their metabolism and varied enzy-matic activities, and to microbiological safety through barrier effects of complex microflora andproduction of several low-molecular-weight antimicrobial compounds. Although extensive researchhas been done on bacteriocins of cheese bacteria for controlling pathogens in cheese, until nowonly few applications have emerged. The control of spoilage yeasts and moulds has been tradi-tionally done by chemical additives, but the application of new antifungal protective cultures isvery promising, especially for the cheese industry. It has also been recently shown that naturallyestablished cheese microflora can efficiently prevent the growth of pathogenic or spoilage microor-ganisms. Cheese is also a very suitable but underused carrier for the delivery of probiotic bacteria,conferring health benefits on the host, with specific advantages compared with fermented milks andyoghurts such as high cell viability. This review addresses the latest developments in applications ofprotective cultures (with bacteriocin and antifungal activities) or microflora with barrier effects, andprobiotic cultures for the production of high quality, safe and “healthy” cheese, as well as emphasiz-ing some of the underlying challenges and possible solutions. Furthermore, new safety criteria forfood cultures relating to the presence and transferability of antibiotic resistance genes are discussed.

cheese / bacteriocin / antifungal / probiotics / antibiotic resistance / technology

摘摘摘要要要 –具具具有有有保保保护护护和和和移移移哨哨哨功功功能能能的的的干干干酪酪酪发发发酵酵酵剂剂剂的的的研研研究究究截截截展展展。。。微生物在干酪的制造和成熟中起着关键性的作用,微生物的代谢产物和酶的活性赋予干酪的感官品质,复杂微生物菌群的阻隔效应和产生的一些小分子抗菌化合物是干酪微生物安全的保障。尽管关于细菌素用于控制干酪中病原菌的报道很多,但到目前为止该技术的实际应用还是非常有限。控制干酪中腐败酵母菌和霉菌的传统方法还是添加化学防腐剂,因此,有必要开发一些具有抗真菌作用,特别是适用于干酪生产的保护性发酵剂。最新的研究结果表明,天然干酪中形成的微生物菌群可以有效地防止一些病原微生物和腐败微生物的生长。与富含高活力细菌的发酵乳和酸乳相比,干酪还是一种益生菌很好的载体和传递者,但没引起足够重视,它有利于人体吸收其中有益健康的成分。本文论述了能产生细菌素和抗真菌的保护性发酵剂或具有阻隔效应的微生物菌群;能够生产高质量、安全和有益健康干酪的益生菌发酵剂在干酪生产中应用的最新进展以及在工业化生产中存在的问题。此外,本文还讨论了食品发酵剂中抗生素抗性基因存在和转移的安全标准。

干干干酪酪酪 /细细细菌菌菌素素素 /抗抗抗真真真菌菌菌 /益益益生生生菌菌菌 /抗抗抗生生生素素素的的的抗抗抗性性性 /技技技术术术

* Corresponding author (通讯作者): [email protected]

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422 F. Grattepanche et al.

Résumé – Avancées des connaissances sur les cultures à activité protectrice et probiotiquepour des applications fromagères. Les microorganismes jouent un rôle essentiel dans la prépara-tion et l’affinage des fromages en contribuant de façon importante au développement des propriétésorganoleptiques par leur métabolisme et la grande variété de leurs activités enzymatiques, et à lasécurité microbiologique du produit à travers les effets barrières des microflores complexes et/oula production de composés antimicrobiens de faibles poids moléculaires. De nombreux travaux derecherche ont été réalisés sur les bactériocines produites par des bactéries isolées de fromages pourcontrôler le développement de microorganismes pathogènes. Cependant, très peu d’applicationssont issues de ces travaux. Le développement des levures et moisissures nuisibles à la qualité desfromages est traditionnellement contrôlé par des additifs chimiques mais leur remplacement pardes cultures protectrices antifongiques semble prometteur. Des études ont récemment rapporté lepotentiel de flores naturellement établies sur des fromages pour le contrôle de flores pathogènes etde dégradation. Les fromages constituent également un vecteur adéquat, mais sous-exploité, pourles bactéries probiotiques conférant un bénéfice santé pour le consommateur. Les fromages per-mettent notamment de maintenir un meilleur taux de viabilité de ces bactéries comparativement àd’autres matrices telles que les yoghourts et laits fermentés. Cette revue fait le point sur les derniersdéveloppements sur les cultures protectrices (productrices de bactériocines, à activité antifongiqueou à effets barrières) et probiotiques pour la production de fromages de haute qualité organolep-tique et microbiologique, et ayant un effet bénéfique sur la santé du consommateur. Les contraintestechnologiques associées à l’utilisation de ces cultures sont également abordées et des solutionsproposées. De nouveaux critères pour la sélection de cultures microbiennes destinées à un usagealimentaire et portant sur la présence et le transfert de gènes de résistance aux antibiotiques sontégalement soulignés.

fromage / bactériocine / antifongique / probiotique / antibiorésistance / technologie

1. INTRODUCTION

The many activities of starter and ripen-ing cultures are central for quality and mi-crobiological safety of cheese. Lactic acidbacteria (LAB) are currently almost alwaysadded to cheese milk to produce lacticacid from lactose and contribute to bio-chemical changes during ripening and de-velop the characteristics of cheese. Sev-eral beneficial health effects related to theconsumption of some LAB (called probi-otic culture) have also been demonstrated.This potential has been largely exploited infunctional dairy foods, mainly yoghurt andfermented milks. Although numerous stud-ies have been devoted to incorporation ofprobiotic cultures into cheese, few probi-otic cheeses are commercially produced.

On the other hand, a large varietyof other microorganisms originating frommany sources (milk, equipment, ripeningrooms, human) and growing in the massand on the surface of cheese also partic-ipate in cheese and greatly contribute tothe characteristics and diversity of cheese.

This microbiota, which is relatively diffi-cult to control, may also be contaminatedby spoilage and/or pathogenic microorgan-isms and thereby, be a source of undesiredcontaminants for cheese. In contrast, cul-tures isolated from cheese or milk have aprotective effect against the developmentof contaminants. The implementation bythe cheese industry of such protective cul-tures could contribute with other classicalmeasures (e.g. milk pasteurization, use ofdefined starter) to control the developmentof undesired microorganisms and furtherimprove quality and safety of cheese.

In this review, the latest developments inthe application of protective and probioticcultures for the production of high qual-ity, safe and “healthy” cheese have beenreviewed, with underlying challenges andpossible solutions.

2. PROTECTIVE CULTURESIN CHEESE

The implementation in the nineties ofthe Hazard Analysis Critical Control Point

Protective and probiotic cultures for cheese 423

plan and other preventive measures in thecheese industry have greatly contributedto the reduction of the incidence of food-borne diseases related to cheese consump-tion. For example, during the period 1993–1996, the number of dairy products highlycontaminated by Listeria monocytogenes(i.e. � 100 cfu·g−1) in France decreased by41% [64]. However, a recent study on re-tail cheeses in the UK has shown that 5 and4% of cheeses made from unpasteurized(including thermized milk) and pasteurizedmilk, respectively, were of unsatisfactoryor borderline quality according to EC rec-ommendations 2004/24 and 2005/175 forthe presence of Salmonella, Staphylococ-cus aureus, Escherichia coli and L. mono-cytogenes [82]. A cheese with microbi-ological borderline quality can also berapidly considered unsatisfactory if storedunder inappropriate conditions at home.Yeasts and moulds are also common,sometimes major, spoilage organisms offood products, especially fermented milkproducts and cheeses. These organisms,which cause severe economic losses andsignificantly reduce the shelf life of prod-ucts, may constitute a health hazard due tothe production of mycotoxins [47,48,129].Therefore, there is a real need to enhancethe control of microbiological safety ofcheese from milk to the consumer, andthis can be achieved by microbial cultureswith antimicrobial features and protectiveeffects in cheese.

2.1. Bacteriocins andbacteriocinogenic strains

In the last two decades, several studieshave demonstrated the potential of bacteri-ocins to control growth of pathogenic mi-croorganisms in food products [26]. Bac-teriocins are peptides with antimicrobialactivity produced by a wide diversity ofbacteria. To date, most of the identifiedGram-positive bacteriocins are produced

by LAB, among which there are gener-ally recognized as safe (GRAS) organismsused for milk fermentations [66]. More-over, bacteriocins can be rapidly degradedby proteases in the gastrointestinal tractand therefore they should not interfere withhuman gut microbiota [12]. Finally, bac-teriocins produced in situ by food-gradebacteria do not have to be indicated onthe product label. Most research on bac-teriocins in cheese have targeted the con-trol of L. monocytogenes, or other listeriaspecies used as model for this pathogen,and clostridia spores responsible for lateblowing defects in semi-hard and hardcheeses.

2.1.1. The “classical” bacteriocinsfor cheese

Nisin, a class I bacteriocin (< 5kDapeptides containing unusual lanthionineamino acids) produced by lactococci isthe best documented bacteriocin of LABand remains a model for the develop-ment of other bacteriocins. To date, nisin,as a food additive, is the only bacteri-ocin that has received regulatory approvalfrom the US Food and Drug Administra-tion and European Union (listed as E234)and can be labeled as a “natural preserva-tive”. Its broad activity spectrum includeslisteria, enterococci, staphylococci, strep-tococci, clostridia, Campylobacter jejuni,Helicobacter pylori and antibiotic-resistantstrains of Neisseria gonorrhoeae [94,128].However, the stability of nisin is highlydependent on environmental conditions,in particular pH, and this limits its useto acidic foods [116]. In addition, nisincan be degraded by proteolytic enzymesin cheese, leading to a significant activ-ity decrease during ripening. For exam-ple, Benech et al. [8] showed that only12% of the initial activity produced bya nisinogenic starter remained in Ched-dar cheese after 6 months of ripening.


However, encapsulation of purified nisininto liposomes allowed the retention of90% of initial nisin activity and thereby,resulted in an improved control of Listeriainnocua populations in artificially contam-inated cheese during ripening [8]. How-ever, until now only few applications ofnisin in the cheese industry have emerged,as summarized in recent reviews [21, 55,130].

In contrast to nisin, lacticin 3147, a two-component broad-spectrum antimicrobialpeptide produced by Lactococcus lactissubsp. lactis DPC3147 showed high sta-bility over a wide range of pH [117]. Nodecrease in in situ produced lacticin activ-ity was detected in Cheddar cheese over6 months of ripening [119]. However, theapplication of the lacticin 3147 producerstrain on surfaces of smear-ripened cheeseas a pretreatment for controlling L. mono-cytogenes contamination was not effec-tive [102].

Pediocin AcH (also known as pediocinPA-1 or SJ-1) is another broad-spectrumanti-listerial bacteriocin that belongs toclass II (non-modified heat-stable peptides< 10 kDa). It is produced mainly byPediococcus acidilactici [39], a minor pop-ulation in many cheeses. However, Lac-tobacillus plantarum WHE (commerciallyavailable as ALC 01, anti-listerial culture,Danisco, Germany) isolated from Mun-ster cheese was also shown to produce pe-diocin [40]. Production of pediocin AcHby P. acidilactici H was considerably re-duced when final medium pH exceeded5.0. In contrast, bacteriocin production byLb. plantarum WHE 92 was not affectedfor pH values up to 6.0, which is a clear ad-vantage for cheese application [40]. Spray-ing a cell suspension of Lb. plantarumWHE 92 on surfaces of Munster cheese atthe beginning of ripening inhibited growthof L. monocytogenes for 21 days [41].However, Loessner et al. [83] stressed therisk of development of resistance when pe-diocin AcH or the producer strain is used

to control L. monocytogenes on cheese sur-faces.

2.1.2. Newly developed bacteriocinsfor cheese applications

In recent years, several other bacteri-ocins with potential use in cheese havebeen reported. Streptococcus and Entero-coccus, two genera with particular rele-vance in cheese manufacture, also producea large diversity of bacteriocins [99].

Thermophilin, produced by somestrains of Streptococcus thermophilus, is aclass II bacteriocin with inhibitory activityagainst Streptococcus, Enterococcus,Lactococcus, Bacillus and Listeria [51].Streptococcus macedonicus is a relativelynew species first isolated from Kasseri, aGreek hard-cooked cheese [137]. Mace-docin, produced by Str. macedonicusACA-DC 198, belongs to the lantibi-otic bacteriocins (Class I) and inhibitsseveral LAB as well as Clostridiumtyrobutyricum [59]. C. tyrobutyricum isresponsible for large defects in semi-hardand hard cheeses, such as Swiss-type orGouda, due to high production of butyricand acetic acid, CO2 and H2 from fer-mentation of lactate, leading to significantflavor defects and late cheese blowing [6].Germination of Clostridia spores can beinhibited by additives such as nitrate,lysozyme or nisin, but their uses can berestricted by local regulations [6]. There-fore, the use of the macedocin producerstrain as adjunct culture could be a rele-vant alternative for controlling Clostridiadevelopment. Unlike nisinogenic lacto-cocci, Str. macedonicus is not inhibitedby the cooking step of the manufactureprocess, and macedocin is stable over awide range of pH and upon prolongedfermentation [138]. Recently, Anastasiouet al. [4] evaluated the performance of Str.macedonicus ACA-DC 198 used as solestarter or adjunct culture in the productionof Kasseri cheese from pasteurized milk.


After some changes in the process (i.e.time to reach a pH of 5.2 in the drainedcurd and temperature/duration of ripen-ing), no major effects were observedon microbiological, physicochemical orsensorial characteristics of cheeses madewith Str. thermophilus ACA-DC 198 aloneor mixed with a commercial starter culturecompared with control cheese with onlycommercial starter [4]. Furthermore, Str.macedonicus ACA-DC 198 was used asstarter or adjunct culture and macedocinwas detected until the end of cheese ripen-ing (90 days) [4]. However, the efficiencyof macedocin inhibiting Clostridia in situhas not yet been tested in cheese.

Enterococci are used as starter or non-starter lactic acid bacteria (NSLAB) inmany traditional Mediterranean cheeses,where they greatly contribute to the de-velopment of organoleptic properties dur-ing ripening through proteolysis, lipoly-sis and breakdown of citrate into aromacompounds [60]. Although some mem-bers of the Enterococcus genus are alsopathogenic and involved in the transfer ofantibiotic resistance and/or virulence fac-tors, others are considered safe and evenused as probiotics [52]. Many enterococcican produce bacteriocins belonging exclu-sively to the heat-stable, non-lantibioticclass II, with the exception of cytolysin,a two-peptide lantibiotic (Class I) associ-ated with virulence of some Enterococcusfaecalis strains due to its hemolytic activ-ity [99]. Nuñez et al. [101] have inves-tigated the inhibition of two L. monocy-togenes strains by enterocin 4, producedin situ by E. faecalis INIA 4 during themanufacture and ripening of Manchegocheese. The population of L. monocyto-genes Ohio, initially at 105 cells per mLin raw milk, decreased to less than 102

cells per gram of curd 8 h after the startof manufacture when E. faecalis INIA 4was used as sole starter or adjunct culture.From ripening days 2 to 60, L. monocyto-genes Ohio was only recovered in cheeses

manufactured with E. faecalis INIA 4, withor without commercial culture, after an en-richment procedure of 25 g of cheese sam-ple [101]. In contrast, the population ofL. monocytogenes Ohio in control cheesesremained stable at 3.9 × 104 cells per gram.However, E. faecalis INIA 4 as sole starterfailed to inhibit growth of L. monocyto-genes Scott A under the same conditions.

Bactericidal synergism between heatpretreatment (65 ◦C, 5 min) and the en-terocin AS-48 produced by E. faecalisA-48-32 was shown on inhibition of S.aureus in skimmed milk [97]. Comparedwith acidified skimmed milk a lower effi-cacy was measured in cheese when E. fae-calis A-48-32 and Enterococcus faeciumUJA32-81, both enterocin AS-38 produc-ers, were used as starter or adjunct cul-ture, to inhibit growth of S. aureus [97].This result was tentatively explained bya lower production of enterocin AS-38 incheese and/or difference in bacteriocin ac-tivity due to cheese matrix and temper-ature conditions (i.e. 28 ◦C for acidifiedskimmed milk and 4 ◦C for cheese ripen-ing) [97]. Another important factor, pH,may have also influenced bacteriocin activ-ity but was not reported in the study.

To conclude this section, the relevanceof bacteriocinogenic strains as “protectivecultures” varies among the different stud-ies. Indeed, the efficacy of a bacteriocinproducer or bacteriocin with high poten-tial in simple experimental set-up condi-tions (e.g. synthetic medium, controlledtemperature and pH) to control growthof pathogens can be very different whenused in cheese due to several factors: com-petition with the starter culture, complexinteractions between the bacteriocin andcheese matrix, presence of inhibitors (e.g.proteases), and/or non-optimal conditions(e.g. pH, temperature) for growth and bac-teriocin synthesis. In addition, cheese com-position may also greatly differ from coreto surface in terms of microflora diversity,environmental conditions, particularly salt


and water contents, and pH. Thus, bac-teriocin production and efficiency cangreatly vary within cheese. Bacteriocino-genic strains used as starter or adjunct cul-ture must also show good growth in milkand/or cheese since bacteriocin produc-tion is usually directly correlated with finalbiomass.

Safety considerations using bacteriocinsor their producer strains is an important is-sue since cross-resistances between differ-ent classes of bacteriocins and/or antibi-otics have already been reported [74, 98].Moreover, some bacteriocinogenic strainsalso belong to genera or species (e.g. ente-rococci) which include pathogenic strains.Applications of such strains at high levelsin cheese should be carefully assessed forthe risk to benefit ratio. Solutions proposedto overcome these major drawbacks will bediscussed below (Sect. 5). Finally, the bal-ance of the microflora of cheese, a key pri-ority for proper ripening and final quality,can be affected by bacteriocins or bacteri-ocinogenic strains; this problem should becarefully assessed for each cheese type asdiscussed below.

2.1.3. Impact of bacteriocins andbacteriocinogenic strains oncheese ripening

The microflora of cheese, through itsdiverse metabolic activities and release ofenzymes, plays an important role in the de-velopment of typical texture, flavor and ap-pearance of ripened cheeses. Only GRASbacteria for specific food uses can be incor-porated in dairy products. For in situ bac-teriocin production in cheese, the strainsmust also be able to grow at high lev-els and/or be metabolically active in thecheese environment. Therefore, only bac-teriocins from LAB, generally isolatedfrom cheese, have been used. Bacteriocins,most of them with a narrow, and some likenisin and pediocin with a broader activ-ity spectrum, are active on bacteria closely

related to the producer strain or confined tothe same ecological niche [75]. Thus, thepresence of bacteriocin, produced in situ oradded, can disrupt the natural balance anddynamics of the cheese microflora, includ-ing starter cultures.

The acidifying capacity of nisin-tolerant lactococci was not significantlyaffected by the presence of a nisinogenicstrain, whereas a significant decrease wasobserved for a commercial culture andnisin-sensitive lactococci during growthin milk [8]. Benech et al. [7] studied thetextural, physicochemical and sensoryattributes of Cheddar cheese made witha mixed culture containing Lactococcuslactis subsp. lactis biovar. diacetylactisUL719 and two nisin-tolerant lactococci.After 6 months of ripening, rheologicalproperties were not significantly differentwhile proteolysis, lipolysis and formationof hydrophilic and hydrophobic pep-tides increased in cheese containing thenisin Z producer compared with controlcheese [7]. Enzymatic capabilities of Lc.diacetylactis UL719 cells or autolysisof a nisin-sensitive subpopulation of thestarter may be responsible for proteolysisin cheese containing the bacteriocino-genic strain [7]. In this study, the twonisin-tolerant lactococci were also selectedfor their high acidifying capacity in milk.However, for cheeses produced using thesetwo strains, in combination or not withLc. diacetylactis UL719, a bitter off-flavorand an acidic taste were observed [7],probably due to a low amino-peptidasicactivity of these strains compared withcommercial starter cultures. Nevertheless,cheese bitterness could be corrected andflavor improved by incorporating a highproteolytic strain, Lactobacillus caseisubsp. casei L2A, in the mixed lactococcistarter containing the nisin Z producer [7].Similar results were recently reported forHispanico cheese manufactured using alacticin 481 producing strain, Lc. lactisINIA 639, a non-bacteriocinogenic strain,


Lc. lactis INIA 437, and Lactobacillus hel-veticus with high amino-peptidase activityand sensitive to lacticin 481 [57]. Analysisof volatile compounds also showed anenhanced production of 2-methylpropanal,2-methylbutanal, ethanol, 1-propanol,ethyl acetate, ethyl butanoate and ethylhexanoate in lacticin cheeses, whichreached higher scores for aroma qualityand intensity compared with controlcheese made with both starter and adjunctculture [56].

Bacteriocins have been shown to inducelysis of sensitive starter cultures, leadingto a fast release of intracellular enzymeswhich enhance cheese flavor develop-ment [86,88,95]. Bacteriocins may also fa-cilitate diffusion in the cells of molecules,such as amino acids, through membranepermeabilization. The increased amino-acid pool may lead to an increased rateof transamination and formation of α-ketoacids which can be further degraded enzy-matically into flavor compounds [87].

2.2. Protective cultures producingnon-proteinaceous low-molecular-weight antimicrobialcompounds

Recently, antifungal cultures havegained importance for cheese applicationsalthough limited work has been done inthis field. The antifungal properties of LABhave been recently reviewed by Schnürerand Magnusson [125]. The assessment ofcheese spoilage by yeasts and moulds iscomplicated because fungal activity duringripening can be either needed or detrimen-tal to product quality, depending on thetype of cheese and microorganisms [48].Spoilage of cheese due to fungal growthis caused by the production of volatilecompounds, leading to off-flavors, andalso mycotoxin accumulation, which maypromote allergies [47, 129]. Penicilliumspp. and Aspergillus spp. are important

spoilage moulds in preservative-free hard,semi-hard and semi-soft cheeses, whereasCandida spp., Kluyveromyces marxianusand Pichia spp. are main contaminants inunripened soft cheeses [47, 48].

The control of spoilage yeasts andmoulds is traditionally done by chemicaladditives such as sorbic and benzoic acids,which both have a broad activity spectrum.Furthermore, the antibiotic natamycin pro-duced by Streptomyces natalensis is effec-tive against fungi and is used as a commonpreservative on hard cheese surfaces [22,34]. In contrast, consumers are increas-ingly demanding high quality, safe andmildly processed foods with long shelf lifeand low or no addition of chemical preser-vatives. Furthermore, fungi also showedan increased resistance to antibiotics andchemical preservatives such as sorbic andbenzoic acids [18]. Thus, the applicationof antifungal protective food cultures has ahigh potential, especially in dairy productssuch as cheeses or fermented milks.

So far, only a few antifungal pro-tective cultures are commercialized andtheir applications are still emerging, es-pecially for dairy products but also forother foods and feeds (Tab. I). Two pro-tective cultures are currently marketed byDanisco (Danisco, Copenhagen, Denmark)as the HOLDBACTM range, combiningtwo strains of Propionibacterium freuden-reichii subsp. shermanii JS and Lactobacil-lus rhamnosus LC705 [133] or Lactobacil-lus paracasei SM20 [91,92]. Both cultureshave been successfully tested in yoghurtand on the surface of hard cheese to pre-vent spoilage by yeasts and moulds [91].

In contrast to antibacterial peptides,only little is known about antifungalmechanisms. So far, research has mainlybeen directed towards identifying dif-ferent antifungal metabolites producedin simple in vitro fermentations, butdetected antifungal metabolites were notsolely responsible for antifungal featuresof a particular strain or culture. Due


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]


to complex and synergistic interactionsbetween different low-molecular-weightcompounds and likely cell-to-cell interac-tions, the overall mechanisms are difficultto elucidate [125]. Recently, we identifiedthe production of several antimicrobials,including 2-pyrrolidone-5-carboxylic acid,3-phenyllactic acid, hydroxyphenyl-lactic acid, and succinic acid besidespropionic and acetic acids, during fer-mentations of Lb. paracasei SM20and Propionibacterium jensenii SM11in whey-based medium (unpublisheddata). Similarly, Lb. rhamnosus LC705was shown to produce 2-pyrrolidone-5-carboxylic acid [140]. Furthermore,mixtures including acetic, caproic, formic,propionic, butyric, n-valeric, and benzoicacids, as well as 3-hydroxy fatty acids,proteinaceous compounds and cyclicdipeptides were described as antifungalcompounds of LAB [29,84,100,127,132].One common characteristic was that allthese compounds had to be present at lowconcentrations in contrast to high mini-mal inhibitory concentrations for fungi,confirming the complexity of antifungalmechanisms [132, 140].

The application of antifungal protectivecultures is very promising, especially forthe cheese industry. Several strains havebeen recently developed for cheese appli-cations but further research is required toelucidate their mechanisms, reduce costsand for implementation by the industry.High effectiveness of protective culturesis often related to high inoculation levels,which can affect food quality and increasecosts compared with traditional chemicalpreservation [91]. Clearly, careful opti-mization of antifungal systems and theirapplications are required.

2.3. Barrier effects of complex cheesemicrobiota

Cheese surface consortia are known tobe complex ecosystems, exhibiting high

species and strain diversity of bacteria andeukaryotes. Recently developed molecu-lar tools such as pulsed field gel elec-trophoresis (PFGE), which is a culture-dependent method, temporal temperaturegel electrophoresis (TTGE), denaturinggradient gel electrophoresis (DGGE), sin-gle strand conformation polymorphism(SSCP), and terminal restriction fragmentlength polymorphism- (TRFLP-) or lengthheterogeneity- (LH-) PCR have been suc-cessfully used to explore diversity and dy-namics of cheese microflora [30, 35, 49,80,103,113,114]. Throughout cheese man-ufacturing and ripening, the developmentof the microflora is influenced by negativeinteractions between the different micro-bial populations, such as competition fornutrients, as well as stimulating interac-tions, such as pH increase and productionof growth factors [96]. Both bacterial andeukaryotic populations evolve, with somespecies disappearing and others becomingpredominant.

Naturally established cheese microflorawere found to be more or less permissivefor the growth of pathogenic or spoilagemicroorganisms [13, 85, 93, 124]. The in-hibitory activity was related to biodiversityand/or dynamics of the microflora becauseno antimicrobial activity was detected withpure cultures of the isolated strains.

Different hypotheses can be formu-lated to explain the antimicrobial activ-ity of complex consortia. Certain factorsassociated with their metabolism (e.g. or-ganic acids and H2O2 production, nutrientdepletion) together with cheese manufac-ture parameters (e.g. temperature, salt con-centration) might exert synergic actions,inhibiting pathogenic or spoilage microor-ganisms. It is also well known that bacte-riocin synthesis can be regulated by quo-rum sensing systems [62]. In this case,signaling molecules produced by one pop-ulation of cheese consortia are believed totrigger bacteriocin production by another


population, thus explaining the absence ofanti-microbial activity of pure cultures.

The selection of complex surface mi-croflora to control the growth of spoilageand pathogenic microorganisms in cheeseappears to be a promising avenue becauseno modification of process is required.However, further studies are needed to un-derstand better mechanisms contributingto antimicrobial activity. The controlledpropagation-production of such microflorafor large-scale applications is a technolog-ical challenge, as presented below.

3. PROBIOTIC CULTURESIN CHEESE

Probiotics are defined by theFAO/WHO [45] as “live microorgan-isms which when administered in adequateamounts confer a health benefit on thehost”. The beneficial effects in treatmentand prevention of various diseases or gutdisorders such as inflammatory boweldisease or lactose intolerance are greatlydebated in the literature [106]. Probioticmicroorganisms include several genera ofbacteria and yeasts [68], and among those,strains of enterococci, lactobacilli andpropionibacteria are important for manu-facturing some cheeses [52, 90, 108], andwell adapted to the cheese environment.On the other hand, bifidobacteria andLactobacillus acidophilus are most usedin functional dairy foods containing probi-otics, especially milk, yoghurt, ice creamand desserts [20]. However, conditionsencountered in these products are very dif-ferent from those of their natural habitat,the gastrointestinal tract of humans and an-imals. Thus, product composition can havedeleterious effects on their viability, whichis one of the most important prerequisitesfor beneficial health effects [106]. Due toits limited acidity, low oxygen level, highlipid content and low storage temperature,

cheese is a suitable carrier for deliveringlive probiotic bacteria [15].

3.1. Characteristics of probioticbifidobacteria and lactobacillirelated to their viability incheeses

Viability of probiotics is a key param-eter for efficacy of probiotic products. Al-though the amount of cells required to pro-duce beneficial health effects is not known,some authors have suggested a daily in-take of at least 108–109 viable cells as theminimum intake to provide a therapeuticeffect [118]. A minimum level of 106 cfuof probiotic bacteria per gram of productat the time of consumption is generallyaccepted and selected to provide bacterialconcentrations that are technologically at-tainable and cost-effective [79, 123]. Ac-cording to this criterion, most cheesesseem to be suitable carriers for probioticbacteria, as indicated by the high stabil-ity of viable cell counts reported for manycheeses during storage (Tab. II).

Probiotic bifidobacteria and lactobacilliof gut origin are generally classifiedas strictly anaerobic or microaerophilicbut their resistance to oxygen is strain-and species-dependent [134]. They usu-ally show poor resistance under prolongedacidic conditions, with lactobacilli be-ing less affected than bifidobacteria [20].These characteristics explain the highersurvival of probiotic cultures in cheesecompared with other dairy products, in par-ticular yoghurts and fermented milks [72,126]. Indeed, yoghurt has an initial pHof about 4.1–4.4 that decreases to 3.8–4.2 during refrigerated storage due to lac-tic acid production by Lactobacillus del-brueckii subsp. bulgaricus. On the otherhand, the curd of soft cheeses has apH below 5.0 after production (about4.5 for Camembert cheese) but it in-creases rapidly during ripening. In 35-day


ripened Camembert cheese, pH in thecore and the rind reached high values,around 6.8 and 7.5, respectively [2]. ThepH of hard and semi-hard cheeses, gener-ally above 5.0 after production, remainedrelatively constant throughout the ripen-ing period [23]. Furthermore, the cheesecore can be considered as an anaerobicenvironment with very low redox poten-tial (Eh) of about –250 mV [9]. The re-dox potential of Camembert cheese dur-ing ripening has been shown to increasefrom +330 to +360 mV on the surfaceand decrease from –300 to –360 mV inthe core [2]. In contrast, Eh is positive inyoghurt and increases with storage timeabove +120 mV [32,33]. Other parametershave been reported to influence probioticviability in cheese, such as temperature andduration of ripening, presence of NSLABwith antagonistic activities, milk pasteur-ization and salt content [63, 104, 105, 112,141].

Several bifidobacteria strains have beensuccessfully incorporated into cheeseswith no major changes in the process(Tab. II). This is different from yoghurt,where the incorporation of bifidobacte-ria is generally limited to Bifidobacteriumanimalis, a bacteria isolated from animals,due to its high acid and oxygen tolerancecompared with human bifidobacteria [20,72]. Because the health benefits of animalbifidobacteria have not been researched tothe same extent as human species [72],cheese is a promising carrier for enlargingthe range of bifidobacteria added to func-tional foods. Furthermore, cheese is alsoa suitable protective carrier against harshstomach conditions. Vinderola et al. [139]have reported high survival for Bifidobac-terium bifidum, Lb. acidophilus and Lb.casei added to Argentinian Fresco cheeseand subjected to a pH of 3.0 for 3 h at37 ◦C. In an in vitro test, Cheddar cheesecontaining a probiotic strain of E. faeciumgave greater protection to the strain ex-posed to porcine gastric juice at pH 2.0

than yoghurt [58]. Cellular response ofP. freudenreichii to technological stressesduring Swiss-type cheese manufacture hasalso been reported to increase resistance ofcells to acidic conditions [71].

However, although cheese is likely oneof the best carriers for probiotics, theaddition of high numbers of viable andmetabolically active cells can affect prod-uct quality, especially organoleptic proper-ties, as discussed below.

3.2. Effects of probioticbifidobacteria and lactobacillion cheese quality

The gross chemical composition ofcheese (i.e. salt, protein, fat and moisture)and pH are generally not influenced byadded probiotic bacteria [10, 11, 19, 28, 31,37, 61, 104, 105, 141]. However, in Ched-dar cheese containing Bifidobacterium lac-tis Bb-12, Mc Brearty et al. [89] measureda higher moisture level of 40%, exceed-ing the legal limit, compared with con-trol cheese containing about 38% moisture.This effect, leading to low body/texturescores in sensory analysis, was explainedby a rapid acidification during cheese man-ufacture with the starter added togetherwith B. lactis Bb-12 [89].

Incorporation of probiotic cultures incheese does not generally affect primaryproteolysis which in many cheeses resultsfrom activity of the coagulant agent (ex-cept for high cook cheeses) and, to alesser extent, of plasmin and subsequently,residual coagulant and enzymes from thestarter microflora [131]. However, changesin secondary proteolysis and increases infree amino acid content have often beenreported when probiotics were added tocheese [11, 61, 89, 104, 105]. Peptides andamino acids directly contribute to cheeseflavor (such as sweet, bitter or malty) andcan be precursors for the synthesis of other


TableII.V

aria

bilit

yof

prob

iotic

cultu

res

inch

eese

expe

rim

ents

.

Che

ese

Type

Rip

enin

g /

Stor

age

cond

itio

ns

Stra

ins

Cel

l cou

nts

(log

cfu

·g–1

)C

hang

es in

pro

cess

or

rem

arks

Ref

.

Star

t1E

nd1

Che

ddar

6–7

°C,

24 w

ksB

. bifi

dum

6.

07.

0A

dditi

on o

f bi

fido

bact

eria

pri

or to

mix

ing

and

pack

ing

into

hoo

psC

omm

erci

al c

ultu

re o

r im

mob

ilize

d fr

eeze

-dri

ed

cultu

re

[37]

4 °C

,12

wks

B. i

nfan

tis

AT

CC

279

20G

6.7

6.59

Che

ese

milk

sta

ndar

dize

d w

ith

crea

m f

erm

ente

d w

ithB

. inf

anti

s A

TC

C 2

7920

G[3

1]

8 °C

, 28

wks

B. l

acti

s B

b12

B. l

ongu

m B

B53

6 8.

96.

48.

25.

2A

ddit

ion

of b

ifid

obac

teri

a w

ith s

tart

er c

ultu

re[8

9]

9–10

°C

, 32

wks

B. l

acti

s B

94B

. lac

tis

Bb1

2B

ifido

bact

eriu

m s

p.D

R10

Lb.

aci

doph

ilus

L10

L

b. a

cido

phil

us L

a5L

b. p

arac

asei

L26

Lb.

cas

ei L

c1L

b. r

ham

nosu

s D

R20

8.0

8.0

8.7

7.6

8.3

8.4

7.8

8.2

7.6

8.2

8.7

3.7

3.6

7.4

7.2

8.0

Add

ition

of

mix

ed p

robi

otic

cul

ture

s w

ith

star

ter

cult

ure

B94

, Bb1

2 an

d D

R10

wer

e m

ixed

toge

ther

L10

and

La5

wer

e m

ixed

toge

ther

L26

, Lc1

and

DR

20 w

ere

mix

ed to

geth

er

[112

]

4 °C

,24

wks

Lb.

aci

doph

ilus

496

2 L

b. c

asei

279

B. l

ongu

m 1

941

Lb.

aci

doph

ilus

L10

Lb.

par

acas

ei L

26B

. lac

tis

B94

8.3

8.5

8.0

8.4

8.5

7.5

8.3

8.6

8.3

8.5

8.5

7.5

Add

ition

of

mix

ed p

robi

otic

cul

ture

s w

ith

star

ter

cultu

res

LA

4962

, LC

279

and

BL

1941

wer

e m

ixed

toge

ther

L10

, L26

and

B94

wer

e m

ixed

toge

ther

[104

, 10

5]

Whi

te-b

rine

d ch

eese

4 °C

, 90

day

sB

. bifi

dum

Bb0

2L

b. a

cido

phil

us L

a59.

09.

07.

07.

0A

dditi

on o

f pr

obio

tic c

ultu

res

with

sta

rter

cul

ture

[141

]


TableII.C

onti

nued

.

Che

ese

Type

Rip

enin

g /

Stor

age

cond

itio

ns

Stra

ins

Cel

l cou

nts

(log

cfu

·g–1

)C

hang

es in

pro

cess

or

rem

arks

Ref

.

Star

t1E

nd1

Sem

i-ha

rd c

hees

e12

°C

,4

wks

Lb.

aci

doph

ilus

Lb.

par

acas

ei6.

97.

397.

89.

1A

dditi

on o

f la

ctob

acill

i with

sta

rter

cul

ture

[10,

11]

Sem

i-ha

rd g

oat’s

m

ilk

chee

se6

°C,

70 d

ays

B. l

acti

sL

b. a

cido

phil

us8.

3–8.

67.

8–8.

56.

9–8.

06.

8–7.

5Pr

obio

tic b

acte

ria

used

as

star

ter

cultu

res

Mod

ific

atio

n of

pro

cess

: add

ition

of

milk

pro

tein

hy

drol

yzat

e, a

mou

nt o

f st

arte

r in

ocul

um,

tem

pera

ture

of

milk

and

cur

d, c

urd

cutti

ng,

conc

entr

atio

n of

sal

t, ri

peni

ng ti

me

[63]

Can

estr

ato

Pugl

iese

Har

d ch

eese

12 °

C,

86 d

ays

B. b

ifidu

m B

b02

B. l

ongu

m B

b46

7.1

7.0

6.0

5.0

Add

itio

n of

bif

idob

acte

ria

with

sta

rter

cul

ture

Mod

ific

atio

n in

the

proc

ess:

hea

ting

of th

e cu

rd[2

8]

Min

as F

resh

che

ese

5°C

, 21

day

sL

b. a

cido

phil

us L

a56.

36.

7A

dditi

on o

f la

ctob

acill

i with

sta

rter

cul

ture

[19]

Cre

scen

za S

oft,

rind

less

che

ese

4 °C

, 14

day

sB

. bifi

dum

B. l

ongu

mB

. inf

anti

sM

ixed

cul

ture

FC

Mix

ed c

ultu

re I

C

6.02

6.02

6.02

6.02

6.02

8.0

7.1

5.2

5.0

5.2

Add

itio

n of

bif

idob

acte

ria

with

sta

rter

cul

ture

Bif

idob

acte

ria

wer

e te

sted

in m

ixed

cul

ture

or

indi

vidu

ally

Mix

ed c

ultu

re w

as im

mob

iliz

ed o

r no

t

[61]

Arg

entin

ian

Fre

sco

chee

se,

Soft

che

ese

4 °C

,60

day

sB

. lon

gum

(2

stra

ins)

B. b

ifidu

m (

2 st

rain

s)B

ifido

bact

eriu

m s

p (1

str

ain)

Lb.

aci

doph

ilus

(2

stra

ins)

Lb.

cas

ei (

2 st

rain

s)

6.4–

7.6

7.7–

8.7

7.0–

7.6

6.0–

8.7

7.0–

8.4

5.9–

7.3

6.9–

8.6

6.6–

7.6

5.6–

8.6

7.0–

8.7

Add

ition

of

prob

iotic

cul

ture

with

sta

rter

cul

ture

Prob

iotic

cul

ture

s w

ere

mix

ed in

dif

fere

nt

com

bina

tion

s

[139

]

1 Cel

l con

cent

rati

ons

at th

e be

ginn

ing

and

the

end

of th

e ri

peni

ng p

erio

d.2 F

or C

resc

enza

che

ese,

sta

rtin

g co

ncen

trat

ions

cor

resp

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to lo

g of

cfu

per

mL

of

mil

k us

ed f

or c

hees

e-m

akin

g.


flavors or volatile aroma, resulting in off-flavors [5, 128].

During ripening, lipolysis also playsan important role in the development ofcheese characteristics. The addition of pro-biotic cultures does not seem to affect thefree fatty acid profile of cheese, likely dueto a higher lipolytic activity of startersand some NSLAB compared with probi-otic cultures [28, 61, 63].

Most cheeses containing probiotic lac-tobacilli and bifidobacteria have highacetic acid content due to heterofermenta-tion [28,31,61,63,89,104,105]. Bifidobac-teria produce mainly acetic and lactic acid,in a molar ratio 2:3, from lactose fermen-tation via the fructose-6-phosphate shuntpathway. Some lactobacilli can also pro-duce acetic acid, but to a lesser extent com-pared with bifidobacteria [36]. Acetic acidcontributes to the typical flavor of differentcheeses, but excessive concentrations canalso result in off-flavors [53, 120].

Lactose maldigestion can be alleviatedby β-galactosidase activities of bifidobac-teria [67]. A complete lactose hydroly-sis was observed in Crescenza, Canes-trato Pugliese and Cheddar-like cheeses towhich bifidobacteria were added, eventu-ally leading to a small galactose accumula-tion [28, 31, 61].

As already reported by several au-thors [15, 118], cheese is a promising foodmatrix for probiotics. However, only a fewprobiotic cheeses have been successfullydeveloped for the market compared withyoghurts or fermented milks. Furthermore,strain selection and possible process ad-justments should be carefully evaluated tomaximize probiotic cell viability duringcheese manufacture and storage, as well asto limit possible changes in organolepticproperties. Finally, dairy propionibacteriawhich have been recently proposed as pro-biotics [107] could be further researchedfor applications as probiotic and ripeningcultures, especially for Swiss-type cheeses.

4. ANTIBIOTIC RESISTANCE

Until recently, safety criteria for foodcultures were mainly based on a long his-tory of safe use [54, 121]. These crite-ria were often considered sufficient foruse of any strains belonging to a par-ticular “safe” genus or species in cheesetechnology. However, it is now increas-ingly accepted that strains of almost anybacterial species can acquire plasmids ortransposons containing antibiotic (AB) re-sistance genes or virulence factors andtherefore, safety considerations have tobe placed on particular strains. Increasingconcerns over the AB resistance situationin food-related bacteria are partly due toexcessive use of antibiotics in both humanand veterinary medicine and agriculture [1,81, 109] and to the high transferability po-tential of acquired AB resistance genes [3,110]. During the last decade, the hypothe-sis that non-pathogenic microorganisms infermented food, e.g. LAB in cheese, canalso function as reservoirs for transferableAB resistance determinants has been ver-ified [122, 135]. As a consequence, hori-zontal gene transfer (HGT) of such deter-minants to other bacteria can occur in thecheese matrix and surface and once cheeseis consumed, between cheese organismsand commensal microbiota in the gastroin-testinal tract. HGT by conjugation betweenthe cheese isolate Lb. plantarum harboringthe plasmid pLFE1 with the erythromycinresistance gene erm(B) and E. faecalis wasmeasured at high frequency in gnotobioticrats even without selective pressure [46].

The presence of AB resistance genes inboth isolated LAB from fermented foods,such as cheese, and LAB of human ori-gin is the first evidence for such AB re-sistance gene transfer, as recently reviewedby Ammor et al. [3]. The best docu-mented examples are tetracycline and ery-thromycin resistance genes found in ente-rococci in European cheeses [70, 135]. ABresistance genes were also characterized in


cheese LAB isolates belonging to the gen-era Lactobacillus, Lactococcus and Leu-conostoc [3]. Furthermore, transferabilityof AB resistance genes derived from plas-mids and transposons of cheese LAB iso-lates was demonstrated in vitro betweendifferent species of LAB by conjugation onagar plates [109].

A second indication of HGT is theincreasing prevalence of AB-resistantnon-Enterococcus LAB isolates fromcheeses [3,27,50,69,109,136]. Phenotypicanalysis of AB resistance among LABwas always limited by different method-ological approaches used to determineAB susceptibility-resistance breakpointsof dairy strains and bifidobacteria. Theuse of the micro-dilution method fordetermining minimal inhibitory concen-trations of antibiotics in non-clinical,non-Enterococcus LAB and bifidobacteriawas highly recommended [38, 76]. Re-sults for phenotypic resistance are thencompared with the breakpoints establishedfor the species [50]. Now available arenew molecular screening tools based onmicroarray hybridization allowing rapidscreening of AB resistance genes [111].With a microarray targeting ninety dif-ferent AB resistance determinants mainlyfrom Gram-positive bacteria, Kastneret al. [73] showed that among thirty LABstarter strains used for cheese production,none contained acquired AB resistancegenes, in contrast to several meat LABstarters and two probiotic cultures: Lac-tobacillus reuteri SD 2112 with theplasmid-encoded tetracycline resistancegene tet(W) and the lincosamide resistancegene lnu(A); and Bifidobacterium animalissubsp. lactis DSM 10140 with tet(W).

To conclude, AB resistance screening ofindustrial cultures and novel strains shouldbe done systematically. Strain identifica-tion by phenotypic and genotypic methodsand the absence of transferable AB resis-tance genes and virulence factors are im-portant criteria which will be implemented

in European food legislation [42, 43]. Ac-cording to criteria set by the EuropeanFood Safety Authority [44], intrinsic ABresistance or AB resistance due to muta-tion of chromosomal genes represent ac-ceptable risks of dissemination by HGT,but acquired AB resistances are risk factorsfor public health.

5. TECHNOLOGICALCHALLENGES ANDPROSPECTS FOR INNOVATIVETECHNOLOGIES

The incorporation of functional culturesin cheese must have no deleterious ef-fects on the cheese manufacturing pro-cess and product quality. This implies thatmetabolic activities (e.g. lactic acid, aromaand flavor productions) of cultures withadded functionalities must be similar tothose of the original starter culture. On theother hand, a functional culture should notbe impaired to provide the desired effect.Technological challenges and possible so-lutions for application of protective andprobiotic cultures in cheese are discussedbelow.

5.1. Challenges related tobacteriocinogenic culturesin cheese

As discussed previously, bacteriocino-genic strains can affect the growth of startercultures, and milk and curd acidification.Conversely, good growth of the producerstrains in the presence of the starter cul-ture is also needed because bacteriocinproduction is directly related to cell con-centration. To overcome this major lim-itation, bacterial strains for starter cul-tures must be carefully selected. Bouksaimet al. [14] highlighted the importance of theinoculum ratio of bacteriocinogenic strainsand starter cultures for controlled acidi-fication and production in Gouda cheese.


A very small change in the proportion ofmixed culture inoculum from 0.4/1.4% to0.6/1.4% between the nisinogenic strain(Lc. diacetylactis UL719) and Flora Dan-ica starter led to a large increase in num-bers of UL719 accompanied by changes innisin Z activity in cheese, i.e. from non-detectable to 256 international units (cor-responding to 6.4 μg pure nisin) per gramof cheese, although composition of cheeseswas similar to control cheese made with1.4% Flora Danica only. This study clearlyshowed the great challenge of controllingbacteriocinogenic strain activity in cheese.The use of bacteriocin-resistant strains hasalso been proposed by several authors toovercome the deleterious effects of bac-teriocin on acidifying capacity of startercultures [8,95]. However, the selection pro-cess can be difficult, particularly in the caseof cheese with complex microflora. Fur-thermore, other technological parameters(e.g. bacteriophage resistance of starter andbacteriocin cultures) are also important forcheese applications.

Technological properties (e.g. ability togrow and acidify in milk) and/or safety sta-tus (e.g. bacteriocinogenic strains carryingAB resistance and/or belonging to genus orspecies which includes pathogenic strains)of bacteriocinogenic strains can also limittheir applications in cheese. As a possi-ble solution, the heterologous productionof bacteriocin in GRAS strains with rele-vant technological properties was success-fully attempted [16, 115].

Despite extensive research done onbacteriocins and bacteriocinogenic strains,few products are commercially avail-able, reflecting the difficulties of imple-mentation of such protective cultures incheese manufacturing. Although heterolo-gous production of bacteriocins has goodpotential to facilitate applications of bac-teriocinogenic cultures in cheese, regula-tory considerations and general consumeropposition to use of genetically modified

organisms in food will likely block this de-velopment.

5.2. Innovative technologies forproduction of cheese cultures

Industrial processes for food cultureproduction, including cheese cultures andprobiotics, almost exclusively use con-ventional batch fermentation with sus-pended cells cultivated separately. Sep-arately produced cultures are eventuallymixed after production to formulate com-mercial preparations. For bacterial surface-ripened cheeses, cheeses after salting areeither inoculated with commercial definedmixed cultures containing different strainsof Brevibacterium linens, Debaromyceshansenii and/or Geotrichum candidum, orin some countries are washed with smearsfrom older cheeses (“old-young” smearingmethod) [17].

However, both approaches have disad-vantages, such as limited biodiversity forcontrolled inoculation and contaminationrisks by undesirable contaminants for the“old-young” smearing method. The devel-opment of technologies enabling a con-trolled propagation of complex microfloracould be very useful for controlling con-tamination and developing cheese surfaceflora, leading to high product quality andsafety.

Cell immobilization has been used toperform high cell density fermentationsfor both cell and metabolite productionswith several advantages over free-cell fer-mentations, including: high cell densities,reuse of biocatalysts, improved resistanceto contamination and bacteriophage attack,enhancement of plasmid stability, preven-tion from washing-out during continu-ous cultures, and the physical and chem-ical protection of cells [78, 79]. Recently,we developed and validated new contin-uous fermentation systems with immobi-lized human fecal microbiota to closely


model intestinal fermentation and stablycultivate complex microbiota [24,25]. Sim-ilar approaches could be used to stabilizeand propagate complex cheese surface mi-croflora, and eventually develop dynamicfermentor models to study cheese surfaceecosystems.

Current data also suggest that cell im-mobilization combined with continuousculture might also be used to efficientlyproduce, in a one-step process, functionalcultures and probiotic cells with enhancedtolerance to environmental stresses and im-proved technological and functional char-acteristics [78, 79]. Recent studies withimmobilized lactobacilli and continuousmixed strain cultures have reported signifi-cant physiological changes, such as a largeincrease in cell tolerance to nisin and lowpH, and enhanced acidification capacity ofcheese cultures [65, 77].

6. CONCLUSION

Despite a great deal of basic and appliedresearch carried out on identification, char-acterization and development of bacterio-cinogenic strains in cheese over the pastthirty years or even more, very few ap-plications have reached the market. Thissituation is largely explained by the diffi-culty of applying bacteriocinogenic strainsin the context of industrial cheese pro-duction where needs for robust and re-producible processes are high. Selectionof competitive bacteriocinogenic strains incheese environments and suitable starterswith low sensitivity to the bacteriocin is aprerequisite for successful large-scale ap-plications. Development of culture rota-tion to respond to bacteriophage attacksis also needed. Furthermore, strict pro-cess control is required to achieve propergrowth of starter, secondary and adjunctcultures and production of uniform, highquality and safe cheese. If this can beachieved, in situ bacteriocin productioncould be used both to enhance microbio-

logical safety of cheese and to acceleratecheese ripening. One of the most promis-ing applications of protective cultures ison cheese surfaces where post-processingcontamination is more likely to occur andno interference with starter cultures is ex-pected. The selection and developmentof complex natural cheese surface mi-croflora with protective properties againstspoilage and pathogenic contaminants isespecially attractive but will require devel-opment of suitable technologies for con-trolled propagation of complex cultures.Furthermore, the development of antifun-gal cultures with protective effects associ-ated with minimal metabolism in cheeseis rapidly evolving and similar conceptsrelying on synergistic mixtures of non-protein low-molecular-weight metabolitescould be further developed to target bacte-rial contaminants.

Cheese is also a very suitable but under-used carrier for the delivery of probotics,with specific advantages compared to fer-mented milks and yoghurts for maintain-ing high cell viability and with potential toenlarge the range of probiotic strains usedin functional dairy products. It is there-fore expected that new functional cheeseswill be developed on the market, eventu-ally harboring resident cultures with probi-otic features not yet characterized and ex-ploited.

Finally, safety of food cultures willbecome increasingly important with theemergence and dissemination of AB resis-tance genes. In the near future cheese andprobiotic cultures, like any other food cul-tures, will have to fulfill strict testing toguarantee the absence of acquired AB re-sistance genes with transfer potential.

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