International Journal of Food Microbiology 135 (2009) 34–38

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeastsin fermented milk under non-refrigerated conditions☆

Shao-Quan Liu a,⁎, Marlene Tsao b

a Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Singaporeb Food Science Programme, School of Chemical and Life Sciences, Nanyang Polytechnic, Singapore

☆ Part of this work was conducted at the Fonterra Res⁎ Corresponding author. Tel.: +65 6516 2687; fax: +

E-mail address: chmLsq@nus.edu.sg (S.-Q. Liu).

0168-1605/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2009.07.017

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 February 2009Received in revised form 10 July 2009Accepted 17 July 2009

Keywords:YeastsLactic acid bacteriaProbioticsStabilityFermented milkYoghurt

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied infermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermentedmilk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowialipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (butnot Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~102 to 105-fold. Seven yeastsexamined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membra-nifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus infermented milk by ~103 to 106-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillusacidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 106-fold, but the same yeast failed toimprove the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermentedmilk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and thatthe specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecularmechanism of such interaction of yeasts with LAB remains to be found.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Dairy products fermented with lactic acid bacteria (LAB), yoghurtin particular, are popular carriers of probiotics (Lourens-Hattingh andViljoen, 2001). However, the cell population of LAB and probioticsoften declines during storage, especially at elevated temperatures(Champagne and Gardner, 2005; Vasiljevic and Shah, 2008). Fermen-ted milk products are often subjected to temperature abuses duringdistribution, storage and retailing, until consumption. This tempera-ture abuse is aggravated in developing countries where chilled-chaindistribution is inadequate. This results in a reduction of the nutritionalvalue and shortens the shelf life of the fermentedmilk product largely,if not solely, due to over-acidification. There is a need in the dairyindustry to enhance the stability of LAB and probiotics in fermentedmilk including yoghurt, especially in developing countries.

Maintaining the stability of LAB and probiotics frommanufacturingto consumption has been a technological challenge to the foodindustry in developing probiotic dairy foods. Various methods havebeen explored to improve the survival of LAB and probiotics in foodsystems under refrigerated conditions, but with limited success.Examples include addition of prebiotics and nutrients, stress adapta-

earch Centre.65 6775 7895.

ll rights reserved.

tion, use of protectants, and microencapsulation (Shah, 2000;Champagne and Gardner, 2005; Doleyres and Lacroix, 2005; Rosset al., 2005; Roy, 2005; Vasiljevic and Shah, 2008). It remains to beseen whether these methods can enhance the stability of LAB andprobiotics under non-refrigerated conditions.

Yeasts have a long history of proven safe use in the fermentation offoods and beverages. On the other hand, yeasts can cause spoilage offoods and beverages (Fleet, 1990; Jakobsen and Narvhus, 1996). Themicrobial ecosystem of fermented foods and beverages is complex,consisting of a range of microbes such as bacteria, yeasts andsometimes moulds. The interaction between yeasts and bacteriainvolves stimulation or inhibition and the specific mode of interactionis dependent on yeasts as well as bacteria (Jakobsen and Narvhus,1996; Viljoen, 2006).

An early study indicates that the viability of milk cultures of aLactobacillus bulgaricus strain is maintained for months when culturedwith certain yeasts (Graham,1943). Another early research shows thatyeast Torulopsis sp. (now Candida sp.) can extend the viability ofyoghurt bacteria for months (Soulides, 1955). A more recent studyshows the biostabilisation of kefir with Saccharomyces cerevisiae withregard to ethanol formation and sugar utilisation, but there is nomention of stability of LAB (Kwak et al., 1996). None of these studiesreport the cell count of LAB, storage temperature and biochemicalproperties of the yeasts involved. Some evidence indicates that yeastscan help in the stabilisation of lactic acid bacterial population incheese and yoghurt (Viljoen et al., 2003; Arfi et al., 2004; De Freitas

35S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 34–38

et al., 2009). Nevertheless, the impact of yeasts on the stability ofprobiotics in fermented milk has not been studied in detail.

USpatent 6,294,166 (Hsia, 2001) describes amethod formaintainingthe stability of dried viable probiotics and LAB stored under ambientconditions using dried non-viable yeasts and protein. It is hypothesisedthat the dried non-viable yeasts act like yeast extracts, providingnutrients such as vitamins to the probiotics and LAB. Clearly, the matrixof this dried mixture differs substantially from that of high moisturefoods and beverages. It is questionable and remains to be exploredwhether this method can be directly applied to highmoisture foods andbeverages to maintain LAB stability.

There remains a need to develop a method for maintaining thestability of LAB and probiotics in high moisture foods and beveragesfor an extended period of time under non-refrigerated conditions. Theobjectives of this research were to examine the effects of variousyeasts on the stability of probiotic and non-probiotic LAB in fermentedmilk and yoghurt systemswith a view to find a solution to the ambientstability of LAB and probiotics in high moisture food systems.

Fig. 1. Effects of yeasts (initial cell count of ~105 cfu/mL) on the stability of yoghurtculture (total cell count) in yoghurt with 20%w/vmilk solids during incubation at 30 °C.The yoghurt culture MY-900 was a blend of S. thermophilus and L. bulgaricus. The yeastswere: S. bayanus CVC-NF74 (♦),W. saturnus var. saturnus CBS254 (⁎), Y. lipolytica B9014(○), C. kefyr NCYC143 (Δ), K. marxianus ATCC8640 (×). The control (●) had no yeastadded.

2. Materials and methods

2.1. Microorganisms, media, enumeration and culture conditions

All the microorganisms used were obtained from the culturecollection held at the Fonterra Research Centre, Palmerston North,New Zealand. These microorganisms were either obtained fromelsewhere or were in-house isolates, as indicated below. The followingyeasts were originally from various culture collections: Williopsissaturnus var. saturnus CBS254 (Centraalbureau voor Schimmelcultures,Utrecht, the Netherlands); Kluyveromyces marxianus ATCC8640 andLactobacillus acidophilus ATCC4356 (American Type Culture Collection,VA, USA); Candida kefyrNCYC143 (National Collection of Yeast Cultures,Institute of Food Research, Norwich, England); Geotrichum candidumCMICC335426 [Commonwealth (now CAB International) MycologicalInstitute Culture Collection, Richmond, UK]; Lactobacillus reuteriDSM20016 (Deutsche Sammlung von Mikroorganismen und Zellkultu-ren, Braunschweig, Germany); Lactobacillus johnsonii LA1 (NCC533)(Nestle Culture Collection, Lausanne, Switzerland). Saccharomycesbayanus CVC-NF74 was a commercial wine yeast from Lallemand Inc.,Ontario, Canada. Schizosaccharomyces pombe 972, Pichia subpelliculosa,Pichia membranifaciens SR-55 and Candida kruseii MUY-14 were fromthe Culture Collection of Institute of Molecular Biosciences, MasseyUniversity, New Zealand (collection of Dr. G.J. Pilone, now retired).Streptococcus thermophilus B2513, L. bulgaricus B3118 and Yarrowialipolytica B9014 were in-house isolates held at the Fonterra ResearchCentre. Lactobacillus rhamnosus DR20 (also known as HN001) is aclinically provenprobiotic and is nowmarketedasHOWARURhamnosusby Danisco (Gill et al., 2000; Gopal et al., 2001).

Standard microbiological media were used to cultivate yoghurtcultures, LAB andyeasts. Yoghurt starter cultureswere grown in10%w/vreconstituted skimmilk at30 °C for 24h. LABwere cultured inMRSbroth(Gibco) at 30 °C for 24 h. Yeasts were grown, with or without aeration(150 rpm), at 30 °C for 24 to 48 h in a medium (pH 5.0) of 2% w/vglucose, 0.25%w/veachof yeast extract,malt extract andBacto-peptone.Broth cultures were used in the experiments described in Section 2.2.

Selective media were used to enumerate LAB and yeasts. Yoghurtcultureswere plated on bothM17 agar (BDDifco) andMRS (Gibco) agarplates, which were incubated at 37 °C for 48–72 h. LAB were plated onMRS agar and incubated at 30 °C for 48–72 h. Both M17 agar and MRSagar contained an appropriate amount of natamycin (Danisco) to inhibityeasts, as per manufacturer's instruction. Yeasts were plated onoxytetracycline-glucose yeast extract (Oxoid) agar containing 0.1 g/Lof chloramphenicol and plates were incubated at 25 °C for 2–4 days.

Data presented were the averages of duplicate determinations(plating) from single experiments. However, replicate experiments of

selected yeasts and LAB were conducted and similar trends wereobserved.

2.2. Experimental protocols

In the context of this study, yoghurt refers to milk productsfermented with standard yoghurt cultures (S. thermophilus andL. bulgaricus). Fermented milk refers to milk products fermented withLAB other than standard yoghurt cultures.

2.2.1. Effects of yeasts on survival of yoghurt starter cultures in yoghurtToprepare yoghurt for the experimentpresented in Fig.1,wholemilk

powder (20% w/v) was reconstituted in 1.8 L of water at 50 °C. Thereconstitutedwholemilkwas thenheat-treatedat90 °C for10min.Aftercooling to 30 °C, the reconstitutedwholemilkwas inoculatedwith 1%v/v mixed yoghurt starter culture MY-900 (S. thermophilus andL. bulgaricus) from Danisco, with (treatment) and without (control)the addition of 1% v/v yeast broth culture (initial yeast cell count in theinoculated milk was approximately 105 cfu/mL). 50-mL aliquots of theinoculated milk were then dispensed into 70-mL sterile plastic screw-capped containers before incubation at 30 °C for several weeks.

2.2.2. Effects of yeasts on survival of probiotic L. rhamnosus DR20 infermented milk

To prepare fermented milk shown in Fig. 2, the reconstitutedwhole milk as described above was inoculated with 1% v/vL. rhamnosus DR20 broth culture, with (treatment) and without(control) the addition of 1% v/v yeast broth culture (initial yeast cellcount in the inoculated milk was approximately 105 cfu/mL). This wasfollowed by dispensing 50-mL aliquots of the inoculated milk into 70-mL sterile plastic screw-capped containers before incubation at 30 °Cfor up to 11 weeks.

2.2.3. Effects of yeast W. saturnus var. saturnus on survival of differentLAB in fermented milk

The experiment presented in Fig. 3 was prepared and conducted asdescribed in Section 2.2.2, except that only one yeast and six LAB wereused, which were described in the legend of Fig. 3.

Fig. 2. Effects of yeasts (initial cell count of ~105 cfu/mL) on the stability of L. rhamnosusDR20 in fermented milk with 20% w/v milk solids during incubation at 30 °C. Theyoghurt culture MY-900 was not added. The yeasts were: (A) G. candidumCMICC335426 (▲), S. pombe 972 (×), P. subpelliculosa B9049 (⁎); (B) P. membranifaciensSR-55 (○), Y. lipolytica B9014 (■), K. apiculata B9050 (×), C. kruseii MUY-14 (♦). Thecontrol (●) had no yeast added.

36 S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 34–38

3. Results

3.1. Effects of yeasts on survival of yoghurt cultureMY-900(S. thermophilus+L. bulgaricus) in yoghurt

Five yeasts were tested for their effects on survival of yoghurtcultures in yoghurt at 30 °C and the results are shown in Fig. 1. Theseyeasts represent lactose fermenting and galactose fermenting C. kefyrNCYC143 and K. marxianus ATCC8640, lactose non-fermenting butgalactose fermenting S. bayanus CVC-NF74, lactose non-fermentingand galactose non-fermenting W. saturnus var. saturnus CBS254, andnon-fermentative Y. lipolytica B9014.

As shown in Fig. 1, all the yeasts tested significantly enhanced thesurvival of yoghurt cultures by 102 to 105-fold during incubation at30 °C compared with the control (no added yeast). Microscopicexamination of colonies picked fromM17 andMRS agar plates showedthat it was the L. bulgaricus that survived in the presence of addedyeasts (i.e. S. thermophilus died). Different yeasts had different positiveeffects on yoghurt culture stability, although some yeasts showedsimilar effects. C. kefyr NCYC143, K. marxianus ATCC8640 andY. lipolytica B9014 were less effective. Most effective yeasts wereS. bayanus CVC-NF74 and W. saturnus var. saturnus CBS254.

Post acidification was significant with a change in pH of about0.5 unit (data not shown). The major changes in pH occurred duringthe first twoweeks of incubation. Lactose fermenting and/or galactosefermenting yeasts grew within the first week to ~106 cfu/mL. Lactosenon-fermenting and galactose non-fermenting yeast did not grow butstayed viable. The cell counts of most yeasts remained at ~105 cfu/mLthroughout the incubation period, except for Y. lipolytica B9014, whichdeclined markedly during incubation (data not presented).

The lactose fermenting and galactose fermenting yeasts C. kefyrNCYC143 andK.marxianusATCC8640 grewandproduced large amountsof gas and a strong alcohol aroma, causing spoilage. The lactose non-fermenting but galactose fermenting yeast S. bayanus CVC-NF74 alsogrew but produced lesser amounts of gas and alcohol, still causingspoilage. The yeast Y. lipolytica B9014 was strongly lipolytic, imparting astrong cheesy and butyric off-odour to the yoghurt. The formation ofhigh levels of alcohol by the yeasts C. kefyr NCYC143 and K. marxianusATCC8640 and strong lipolysis by theyeast Y. lipolyticaB9014 (anddeathof this yeast) may explain their relative ineffectiveness in stabilisingyoghurt cultures. The lactose non-fermenting and galactose non-fermenting yeast W. saturnus var. saturnus CBS254 caused no defects(no formation of gas and alcohol).

3.2. Effects of yeasts on survival of probiotic L. rhamnosus DR20 infermented milk

L. rhamnosus DR20 ferments lactose but does not producegalactose from lactose, unlike standard yoghurt cultures. Therefore,galactose utilisation by yeasts was not considered an issue in thisparticular experiment. Seven lactose non-fermenting yeasts wereexamined for their effects on the stability of L. rhamnosus DR20 infermented milk at 30 °C and the results are presented in Fig. 2.

As shown in Fig. 2, L. rhamnosusDR20 grew initially to ~109 cfu/mLregardless of yeast addition, followed by a decline in its cellpopulation. The rate of L. rhamnosus DR20 population decline wasimpacted by the presence or absence of yeast and yeast type. All theyeasts examined significantly improved the survival of L. rhamnosusDR20 by 103 to 106-fold during incubation at 30 °C compared with thecontrol (no added yeast). Most yeasts showed similar positive effectson L. rhamnosus DR20 stability. The yeasts Y. lipolytica B9014 andS. pombe 972 were relatively less effective.

Post acidification was drastic with a final pH of about 3.7 (data notshown). The major changes in pH occurred during the first two weeksof incubation. Most yeasts stayed viable and their cell counts remainedrelatively constant at ~105 cfu/mL, except for the cell count of yeastsY. lipolytica B9014 and S. pombe 972, which showed a significantdecline during incubation (data not presented). The gradual loss ofyeast viability appears to correlatewith their declining effectiveness instabilising L. rhamnosus DR20.

3.3. Effects of yeastW. saturnus var. saturnus B9043 on survival of probioticand non-probiotic LAB in fermented milk

The experiments described above demonstrate that the stability-enhancing effect of yeasts on yoghurt culture MY-900 and L. rhamnosusDR20 appears to be characteristic of yeasts. It is both academicallyinteresting and practically useful to find out whether this stability-enhancingpropertyof yeasts is applicable to different LAB. In this regard,W. saturnus var. saturnus CBS254 was chosen as the model yeast in thisstudy due to its lactose non-fermenting and galactose non-fermentingattribute. The effects of the yeastW. saturnus var. saturnus CBS254 on thestability of six LAB are presented in Fig. 3.

As indicated in Fig. 3, not all of the six LAB tested responded to thepresence of the yeast W. saturnus var. saturnus CBS254 with regard totheir stability. The yeastW. saturnus var. saturnus CBS254 significantlyimproved the stability of L. rhamnosus DR20, L. reuteri DSM20016 andL. acidophilus ATCC4356 by up to 106-fold. However, this yeast did notenhance the stability of S. thermophilus B2513 and L. bulgaricus B3118.The data on the impact of this yeast on the survival of L. johnsonii LA1were not conclusive and further research is required. The apparentsecondary growth of L. reuteri DSM20016 and L. johnsonii LA1 wasobserved in the absence of the added yeast (control) and could be dueto nutrients released from cellular autolysis, although the exact causeis unknown.

Fig. 3. Effects of yeast W. saturnus var. saturnus CBS254 (▲) (initial cell count of ~105 cfu/mL) on the stability of L. rhamnosus DR20, S. thermophilus B2513, L. reuteri DSM20016,L. bulgaricus B3118, L. acidophilus ATCC4356 and L. johnsonii LA1 in fermented milk with 20% w/v milk solids during incubation at 30 °C. The control (●) had no yeast added.

37S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 34–38

The changes in the pH of fermented milk varied with LAB butgenerally fell below pH 4.0. The major changes in pH occurred duringthe first two weeks of incubation (data not shown). The cell count ofthe yeast W. saturnus var. saturnus CBS254 stayed relativelyunchanged at about 104 to 105 cfu/mL during incubation (data notshown).

4. Discussion

In this study, we report that yeasts enhanced the survival of certainLAB by up to 106-fold in fermented milk and yoghurt during storage at30 °C. The extended time of incubation of up to 11 weeks was chosenin order to obtain information on the long-term survival of LAB in thepresence of added yeasts. The aim was to develop ambient-stablefermented milk products with viable LAB that can match the shelf lifeof ambient-stable pasteurised fermented milk such as heat-treatedyoghurt and yoghurt drink (i.e. no live cultures).

On the other hand, the outcome of this research would enable theextension of shelf life of fermented milk by enhancing survival of LAB,

especially when temperature abuse occurs, or ultimately reduce theneed for or even eliminate the costly chilled-chain distribution system.

The findings from this study provide the first definitive evidencethat yeasts can indeed enhance viability of LAB in fermented milksystems, which is in agreement with previous observations (Graham,1943; Soulides, 1955; Viljoen et al., 2003; Arfi et al., 2004; De Freitaset al., 2009). The LAB stability-enhancing effects of yeasts could beattributed to the interactions between yeasts and LAB (Viljoen, 2006).However, these studies provide no information on the cell count ofLAB and biochemical properties of the yeasts involved; neither dothese studies provide any insight into the mechanism of yeasts andLAB interactions.

It is generally believed that the yeasts excrete nutrients that benefitthe LAB. This is indeed the case with some LAB and yeasts (Gobbettiet al., 1994a; Viljoen, 2006). If this is the mechanism for all yeast andLAB interactions, yeast extract is expected to improve the stability ofLAB. Our preliminary study showed that yeast extract had no orlimited effect on the survival of LAB (yoghurt cultures andL. rhamnosus DR20). This suggests that nutrient excretion by yeasts

38 S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 34–38

might not be themechanism for our findings. Furthermore, not all LABresponded to the presence of yeasts with respect to their stability asshown in Fig. 3, suggesting that there may be different mechanismsfor different yeast–LAB combinations. There are numerous reports onthe metabolic interactions between yeasts and LAB in co-cultures(Leroi and Pidoux, 1993a,b; Leroi and Courcoux, 1996; Gobbetti et al.,1994a,b; Cheirslip et al., 2003a,b; Alexandre et al., 2004; Guerzoniet al., 2007). However, these studies do not shed any light on themechanism of enhancement of LAB survival by yeasts under acidic andnon-refrigerated conditions.

All the yeasts tested showed stability-enhancing effects on LAB,although only one strain per yeast species was presented. We hadevidence that different strains of the same species showed similareffects; for example, S. cerevisiae strains (data not shown, for brevity).On the other hand, there were LAB strain differences in their responsesto the presence of added yeast. This was demonstrated in Figs. 1 and 3,which show that the survival of L. bulgaricus (not S. thermophilus) in themixed yoghurt cultureMY-900 respondedwell to the presence of addedyeast, yet L. bulgaricus B3118 did not respond to the presence of addedyeast. While this could be due to strain variations, further studies at thebiochemical and molecular level are warranted.

In this study we found that W. saturnus var. saturnus CBS254 wasthe choice of yeast, as it imparted a fruity flavour attribute and it doesnot ferment lactose and galactose (Kurtzman, 1998). Williopsis yeasts,including W. saturnus and W. californica, are part of the naturalmicroflora of cheeses and olive (Wyder and Puhan,1999; Seiller, 2002;Ciafardini et al., 2006). Several Williopsis yeasts (mainly strains of W.saturnus var. saturnus and var. mrakii) are mycocinogenic and areantagonistic toward other yeasts (Nomoto et al., 1984; Ohta et al.,1984; Michalčáková et al., 1993; Vital et al., 2002), suggesting thatthese yeasts can act as protective cultures against potential spoilage.These properties make W. saturnus yeasts desirable choices forincorporation into fermented milk.

The sensory properties of the fermented milk were not system-atically evaluated in this study, because the focus of this researchwas onthe survival of LAB. However, as mentioned above, as long as the addedyeast did not ferment lactose and galactose, and there were no addedfermentable sugars, the sensory characteristics of the fermented milkwere not expected to be adversely affected. As demonstrated in thisinvestigation, gas and alcohol formations were inevitable if the yeastfermented lactose and/or galactose such as K. marxianus ATCC8640,C. kefyr NCYC13 and S. bayanus CVC-NF74. Furthermore, lipolytic off-odour would occur if the yeast was lipolytic as in the case of Y. lipolyticaB9014. The specific impact of yeasts or yeast–LAB combinations on thesensory attributes of fermented milk must be assessed individually.Clearly, further research is warranted in this aspect.

From the practical point of view, several hurdles need to beovercome before commercialising this finding to develop ambient-stable fermented milk and yoghurt with high cell counts of livecultures: post acidification, aroma stability and potential spoilage.Selection of lactose-negative and galactose-negative yeasts is critical.However, it has been reported that lactose-negative and galactose-negative yeasts may still grow in milk (Roostita and Fleet, 1996),although we observed no growth of this type of yeasts in our study atthe inoculated level of ~105 cfu/mL. Moreover, to minimise potentialspoilage and to ensure product safety, good manufacturing practiceand hygiene are essential.

References

Alexandre, H., Costello, P.J., Remize, F., Guzzo, J., Guilloux-Benatier,M., 2004. Saccharomycescerevisiae–Oenococcus oeni interactions inwine: current knowledge and perspectives.International Journal of Food Microbiology 93, 141–154.

Arfi, K., Leclercq-Perlat, M.-N., Baucher, A., Tache, R., Delettre, J., Bonnarme, P., 2004.Contribution of several cheese-ripening microbial associations to aroma compoundproduction. Lait 84, 435–447.

Champagne, C.P., Gardner, N.J., 2005. Challenges in the addition of probiotic cultures tofoods. Critical Reviews in Food Science and Nutrition 45, 61–84.

Cheirslip, B., Shimizu, H., Shioya, S., 2003a. Enhanced kefiran production by mixedculture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae. Journal ofBiotechnology 100, 43–53.

Cheirslip, B., Shoji, H., Shimizu, H., Shioya, S., 2003b. Interactions between Lactobacilluskefiranofaciens and Saccharomyces cerevisiae in mixed culture for kefiran produc-tion. Journal of Bioscience and Bioengineering 96, 279–284.

Ciafardini, G., Zullo, B.A., Cioccia, G., Iride, A., 2006. Lipolytic activity ofWilliopsis californicaand Saccharomyces serevisiae in extra virgin olive oil. International Journal of FoodMicrobiology 107, 27–32.

DeFreitas, I., Pinon,N.,Maubois, J.-L., Lortal, S., Thierry, A., 2009. The additionof a cocktail ofyeasts species to Cantalet cheese changes bacterial survival and enhances aromacompound formation. International Journal of Food Microbiology 129, 37–42.

Doleyres, Y., Lacroix, C., 2005. Review: technologies with free and immobilised cells forprobiotic bifidobacteria production and protection. International Dairy Journal 15,973–988.

Fleet, G.H., 1990. Yeasts in dairy products. Journal of Applied Bacteriology 68, 199–211.Gill, H.S., Rutherfurd, K.J., Prasad, J., Gopal, P.K., 2000. Enhancement of natural and acquired

immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) andBifidobacterium lactis (HN019). British Journal of Nutrition 83, 167–176.

Gobbetti, M., Corsetti, A., Rossi, J., 1994a. The sourdough microflora. Interactionsbetween lactic acid bacteria and yeasts: metabolism of amino acids. World Journalof Microbiology and Biotechnology 10, 275–279.

Gobbetti, M., Corsetti, A., Rossi, J., 1994b. The sourdough microflora. Interactionsbetween lactic acid bacteria and yeasts: metabolism of carbohydrates. AppliedMicrobiology and Biotechnology 41, 456–460.

Gopal, P.K., Prasad, J., Smart, J., Gill, H.S., 2001. In vitro adherence properties of LactobacillusrhamnosusDR20 and Bifidobacterium lactisDR10 strains and their antagonistic activityagainst an enterotoxigenic Escherichia coli. International Journal of Food Microbiology67, 207–216.

Graham, V.E., 1943. The maintenance of viability in Lactobacillus bulgaricus culture bygrowth in association with certain yeasts. Journal of Bacteriology 45, 51.

Guerzoni,M.E., Vernocchi, P.,Ndagijimana,M., Gianotti, A., Lanciotti, R., 2007.Generationofaroma compounds in sourdough: effects of stress exposure and lactobacilli–yeastsinteractions. Food Microbiology 24, 139–148.

Hsia, H.S., 2001. Nutrition supplement containing Lactobacillus acidophilus, yeast andsoy protein. Patent. US 6,294,166.

Jakobsen, M., Narvhus, J., 1996. Yeasts and their possible beneficial and negative effectson the quality of dairy products. International Dairy Journal 6, 755–768.

Kurtzman, C.P., 1998. Williopsis Zender, In: Kurtzman, C.P., Fell, J.W. (Eds.), The Yeasts, aTaxonomic Study, 4th ed. Elsevier, Amsterdam, pp. 413–419.

Kwak, H.S., Park, S.K., Kim, D.S., 1996. Biostabilisation of kefir with a nonlactose-fermenting yeast. Journal of Dairy Science 79, 937–942.

Leroi, F., Courcoux, P., 1996. Influence of pH, temperature and initial yeast: bacteria ratioon the stimulation of Lactobacillus hilgardii by Saccharomyces florentinus isolatedfrom sugar kefir grains. Journal of Applied Bacteriology 80, 138–146.

Leroi, F., Pidoux,M.,1993a. Detection of interactions betweenyeasts and lactic acidbacteriaisolated from sugary kefir grains. Journal of Applied Bacteriology 74, 48–53.

Leroi, F., Pidoux, M., 1993b. Characterization of interactions between Lactobacillus hilgardiiand Saccharomyces florentinus isolated from sugary kefir grains. Journal of AppliedBacteriology 74, 54–60.

Lourens-Hattingh, A., Viljoen, B.C., 2001. Review: yoghurt as probiotic carrier food.International Dairy Journal 11, 1–17.

Michalčáková, S., Sulo, P., Sláviková, E., 1993. Killer yeasts of Kluyveromyces and Hansenulagenerawith potential application in fermentation and therapy. Acta Biotechnology 13,341–350.

Nomoto, H., Kitano, K., Shimazaki, T., Kodama, K., Hara, S., 1984. Distribution of killeryeasts in the genera Hansenula. Agricultural and Biological Chemistry 48, 807–809.

Ohta, Y., Tsukada, Y., Sugimori, T.,1984. Production, purificationand characterizationofHYI,an anti-yeast substance, produced by Hansenula saturnus. Agricultural and BiologicalChemistry 48, 903–908.

Roostita, R., Fleet, G.H., 1996. Growth of yeasts in milk and associated changes to milkcomposition. International Journal of Food Microbiology 31, 205–219.

Ross, R.P., Desmond, C., Fitzgerald, G.F., Stanton, C., 2005. A review: overcoming thetechnological hurdles in the development of probiotic foods. Journal of AppliedMicrobiology 98, 1410–1417.

Roy, D., 2005. Review: technological aspects related to the use of bifidobacteria in dairyproducts. Lait 85, 39–56.

Seiller, H., 2002. Yeasts in milk and dairy products. In: Roginski, H., Fuquay, J.W., Fox, P.F.(Eds.), EncyclopediaofDairy Science, vol. 4. Academic Press,NewYork, pp. 2761–2769.

Shah, N.P., 2000. Probiotic bacteria: selective enumeration and survival in dairy foods.Journal of Dairy Science 83, 894–907.

Soulides, D.A., 1955. A synergism between yoghurt bacteria and yeasts and the effect oftheir association upon the viability of the bacteria. Applied Microbiology 3, 129–131.

Vasiljevic, T., Shah, N.P., 2008. Review: probiotics — from Metchnikoff to bioactives.International Dairy Journal 18, 714–728.

Viljoen, B.C., 2006. Yeast ecological interactions. Yeast–yeast, yeast–bacteria, yeast–fungiinteractions and yeasts as biocontrol agents (Chapter 4). In: Querol, A., Fleet, G.H.(Eds.), Yeasts in Food and Beverages. Springer, Berlin, pp. 83–109.

Viljoen, B.C., Lourens-Hattingh, A., Ikalafen, B., Peter, G., 2003. Temperature abuseinitiating yeast growth in yoghurt. Food Research International 36, 193–197.

Vital, M.J.S., Abranches, J., Hagler, A.N., Mendoca-Hagler, L.C., 2002. Mycocinogenic yeastsisolated fromAmazon soils of theMaraca Ecological Station, Rorainma-Brazil. BrazilianJournal of Microbiology 33, 230–235.

Wyder, M.-T., Puhan, Z., 1999. Role of selected yeasts in cheese ripening: an evaluationin aseptic cheese curd slurries. International Dairy Journal 9, 117–124.