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

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<ul><li><p>noc</p><p>of Sgap</p><p>Fermented milkYoghurt</p><p>surrefrastKluilusGeoes pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus ino 106-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus</p><p>tic acidobioticulatioially at</p><p>International Journal of Food Microbiology 135 (2009) 3438</p><p>Contents lists available at ScienceDirect</p><p>International Journal o</p><p>j ourna l homepage: www.e lsevdistribution, 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-acidication. There is a need in the dairyindustry to enhance the stability of LAB and probiotics in fermentedmilk including yoghurt, especially in developing countries.</p><p>Maintaining the stability of LAB and probiotics frommanufacturingto consumption has been a technological challenge to the foodindustry in developing probiotic dairy foods. Various methods have</p><p>microbial 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 specic mode of interactionis dependent on yeasts as well as bacteria (Jakobsen and Narvhus,1996; Viljoen, 2006).</p><p>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 ofbeen explored to improve the survival of LAsystems under refrigerated conditions, buExamples include addition of prebiotics and</p><p> Part of this work was conducted at the Fonterra Res Corresponding author. Tel.: +65 6516 2687; fax: +</p><p>E-mail address: chmLsq@nus.edu.sg (S.-Q. Liu).</p><p>0168-1605/$ see front matter 2009 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2009.07.017elevated temperaturesd Shah, 2008). Fermen-perature abuses during</p><p>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). The(Champagne and Gardner, 2005; Vasiljevic anted milk products are often subjected to tem1. Introduction</p><p>Dairy products fermented with lacin particular, are popular carriers of prViljoen, 2001). However, the cell popoften declines during storage, especimprove the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermentedmilk. These results provide denitive evidence that yeasts possess stability-enhancing effects on LAB and thatthe specic 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.</p><p> 2009 Elsevier B.V. All rights reserved.</p><p>bacteria (LAB), yoghurts (Lourens-Hattingh andn of LAB and probiotics</p><p>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.ProbioticsStability acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10</p><p>6-fold, but the same yeast failed toLactic acid bacteria nifaciens, Schizosaccharomycfermented milk by ~103 tEnhancement of survival of probiotic andin fermented milk under non-refrigerated</p><p>Shao-Quan Liu a,, Marlene Tsao b</p><p>a Food Science and Technology Programme, Department of Chemistry, National Universityb Food Science Programme, School of Chemical and Life Sciences, Nanyang Polytechnic, Sin</p><p>a b s t r a c ta r t i c l e i n f o</p><p>Article history:Received 7 February 2009Received in revised form 10 July 2009Accepted 17 July 2009</p><p>Keywords:Yeasts</p><p>The effects of yeasts on thefermented milk under non-milk with live LAB. Five yelipolytica, Candida kefyr andnot Streptococcus thermophexamined (Candida krusei,B and probiotics in foodt with limited success.nutrients, stress adapta-</p><p>earch Centre.65 6775 7895.</p><p>ll rights reserved.n-probiotic lactic acid bacteria by yeastsonditions</p><p>ingapore, Singaporeore</p><p>vival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied inigerated conditions (30 C) with a view to develop ambient-stable fermenteds tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowiayveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but) in a mixed yoghurt culture in yoghurt by ~102 to 105-fold. Seven yeaststrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membra-</p><p>f Food Microbiology</p><p>i e r.com/ locate / i j foodmicroyoghurt bacteria for months (Soulides, 1955). A more recent studyshows the biostabilisation of ker 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; Ar et al., 2004; De Freitas</p></li><li><p>lipolytica B9014 were in-house isolates held at the Fonterra ResearchCentre. Lactobacillus rhamnosus DR20 (also known as HN001) is a</p><p>selected yeasts and LAB were conducted and similar trends wereobserved.</p><p>2.2. Experimental protocols</p><p>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.</p><p>2.2.1. Effects of yeasts on survival of yoghurt starter cultures in yoghurtToprepare yoghurt for the experimentpresented in Fig.1,wholemilk</p><p>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.</p><p>whole milk as described above was inoculated with 1% v/vL. rhamnosus DR20 broth culture, with (treatment) and without</p><p>described in Section 2.2.2, except that only one yeast and six LAB wereused, which were described in the legend of Fig. 3.</p><p>culture (total cell count) in yoghurt with 20%w/vmilk solids during incubation at 30 C.</p><p>35S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 3438clinically provenprobiotic and is nowmarketedasHOWARURhamnosusby Danisco (Gill et al., 2000; Gopal et al., 2001).</p><p>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.</p><p>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 4872 h. LAB were plated onMRS agar and incubated at 30 C for 4872 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 24 days.</p><p>Data presented were the averages of duplicate determinationset al., 2009). Nevertheless, the impact of yeasts on the stability ofprobiotics in fermented milk has not been studied in detail.</p><p>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.</p><p>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 nd a solution to the ambientstability of LAB and probiotics in high moisture food systems.</p><p>2. Materials and methods</p><p>2.1. Microorganisms, media, enumeration and culture conditions</p><p>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 Yarrowia(plating) from single experiments. However, replicate experiments ofThe 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 (), K. marxianus ATCC8640 (). The control () had no yeastFig. 1. Effects of yeasts (initial cell count of ~105 cfu/mL) on the stability of yoghurt(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.</p><p>2.2.3. Effects of yeast W. saturnus var. saturnus on survival of differentLAB in fermented milk</p><p>The experiment presented in Fig. 3 was prepared and conducted as2.2.2. Effects of yeasts on survival of probiotic L. rhamnosus DR20 infermented milk</p><p>To prepare fermented milk shown in Fig. 2, the reconstitutedadded.</p></li><li><p>36 S.-Q. Liu, M. Tsao / International Journal of Food Microbiology 135 (2009) 3438Fig. 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. The3. Results</p><p>3.1. Effects of yeasts on survival of yoghurt cultureMY-900(S. thermophilus+L. bulgaricus) in yoghurt</p><p>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.</p><p>As shown in Fig. 1, all the yeasts tested signicantly 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.</p><p>Post acidication was signicant with a change in pH of about0.5 unit (data not shown). The major changes in pH occurred duringthe rst twoweeks of incubation. Lactose fermenting and/or galactosefermenting yeasts grew within the rst 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).</p><p>yoghurt 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.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).</p><p>3.2. Effects of yeasts on survival of probiotic L. rhamnosus DR20...</p></li></ul>

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