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The growth and interaction of yeasts and lactic acid bacteria isolated from Zimbabwean naturally fermented milk in UHT milk

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The growth and interaction of yeasts and lactic acid bacteria isolated from Zimbabwean naturally fermented milk in UHT milk
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  Ž . International Journal of Food Microbiology 68 2001 21–32www.elsevier.com r locate r ijfoodmicro The growth and interaction of yeasts and lactic acid bacteriaisolated from Zimbabwean naturally fermented milk in UHT milk  Tendekayi H. Gadaga a,b, ) , Anthony N. Mutukumira a , Judith A. Narvhus b a  Institute of Food, Nutrition and Family Sciences, Uni Õ ersity of Zimbabwe, P.O. Box MP 167, Mt. Pleasant, Harare, Zimbabwe b ˚  Department of Food Science, Agricultural Uni Õ ersity of Norway, P.O. Box 5036, N-1432, As, Norway Received 7 August 2000; received in revised form 17 December 2000; accepted 6 January 2001 Abstract Nine yeast and four lactic acid bacterial strains, previously isolated from Zimbabwean traditionally fermented milk, were Ž . inoculated into ultra-high temperature treated UHT milk in both single and yeast–lactic acid bacteria co-culture. The lactic Ž . acid bacteria LAB strains consisted of   Lactococcus lactis  subsp.  lactis  biovar.  diacetylactis  C1,  L. lactis  subsp.  lactis Lc39,  L. lactis  subsp.  lactis  Lc261 and  Lactobacillus paracasei  subsp.  paracasei  Lb11. The yeast strains used were Candida kefyr   23,  C. lipolytica  57,  C. lusitaniae  63,  C. lusitaniae  68,  C. tropicalis  78,  Saccharomyces cere Õ isiae  71,  S.dairenensis  32,  C. colliculosa  41 and  Dekkera bruxellensis  43. After 48-h fermentation at 25 8 C, the samples were analysed Ž . for pH, viable yeast and bacterial counts, organic acids, volatile organic compounds VOC and carbon dioxide. The  Lactococcus  strains reduced the pH from about 6.6 to between 4.0 and 4.2, while  Lb. paracasei  subsp.  paracasei  Lb11reduced the pH to about 5.4. Most of the yeasts, however, did not affect the final pH of the milk except for  C  .  kefyr   23,which reduced the pH from 6.6 to 5.8. All the  Lactococcus  strains grew two log cycles during the 48-h fermentation period,while  Lb. paracasei  subsp.  paracasei  Lb11 grew about one log cycle.  S. cere Õ isiae  71,  C. colliculosa  41 and  D.bruxellensis  43 showed poor growth in the milk in both single and co-culture. The other species of yeast grew about two logcycles.  Candida colliculosa  41,  S  .  dairenensis  32 and  D. bruxellensis  43 showed reduced viability when in co-culture with  Lb .  paracasei  subsp.  paracasei  Lb11. The samples in which  C  .  kefyr   23 was used were distinct and characterised by largeamounts of acetaldehyde, carbon dioxide and ethanol. However, in the samples where  S  .  dairenensis ,  C. colliculosa, D.bruxellensis, C  .  lusitaniae ,  C  .  tropicalis ,  C  .  lipolytica  and  S  .  cere Õ isiae  were used in co-culture, the final pH and metabolitecontent were mainly determined by the corresponding LAB strain. From the observations, it was concluded that  C  .  kefyr   23grows well in UHT milk and produces VOC that may be important to the flavour profile of the fermented milk.Enhancement of production of some flavour compounds such as acetaldehyde and malty compounds in some yeast–LABco-cultures was presumed to be indicative of interaction between the microorganisms.  q 2001 Elsevier Science B.V. Allrights reserved. Keywords:  Yeasts; Fermented milk; Flavour compounds; Organic acids ) Corresponding author. Institute of Food, Nutrition and FamilySciences, University of Zimbabwe, P.O. Box MP 167, Mt. Pleas-ant, Harare, Zimbabwe. Fax: q 47-64-94-37-89. Ž .  E-mail address:  henry.gadaga@inf.nlh.no T.H. Gadaga . 1. Introduction Ž . Yeasts, together with lactic acid bacteria LAB ,are part of the microbial flora of Zimbabwean natu- 0168-1605 r 01 r $ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S0168-1605 01 00466-4  ( )T.H. Gadaga et al. r  International Journal of Food Microbiology 68 2001 21–32 22 Ž rally fermented milk Feresu and Muzondo, 1990;Mutukumira, 1995; Mutukumira, 1996; Gadaga et . al., 2000 . Earlier work showed that  Saccharomycescere Õ isiae ,  Candida lusitaniae ,  C  .  colliculosa  and  S  . dairenensis  are some of the predominant yeast Ž . species isolated from amasi Gadaga et al., 2000 .Yeasts affect the quality of fermented milk throughthe production of flavour compounds and other Ž . metabolites Jakobsen and Narvhus, 1996 . Lactosefermentation and assimilation, lactate assimilation,lipolysis and proteolysis are the important reactionsof yeasts which are responsible for the diverse flavour Ž compounds reported in many dairy products Roostita . and Fleet, 1996 .Several microbial interactions involving yeastshave been suggested in fermented products such asblue cheese, white mould cheese, bacterial surface Ž ripened cheeses, kefir and koumiss Subramanianand Shankar, 1985; Fleet, 1990; Jakobsen and . Ž . Narvhus, 1996 . Marshall 1987 reviewed possibleinteraction between yeasts and lactic acid bacteria infermented milk, but the mechanisms of these interac-tions have not been elucidated. Interaction between  Lactobacillus hilgardii  and  S  .  florentinus  isolatedfrom sugary kefir grains has also been reported,where the yeast stimulated the LAB through produc-tion of carbon dioxide, pyruvate, propionate and Ž . succinate Leroi and Pidoux, 1993a . In addition,some LAB release galactose into the medium as a Ž by-product of lactose metabolism Marshall, 1987;Davidson and Hillier, 1995; Marshall and Tamime, . 1997 , which may be used by galactose-assimilating,but lactose-negative, yeasts. Proteolytic yeasts suchas  Yarrowia lipolytica  and  C. catenulata  grow inmilk and produce free amino acids such as leucine,phenylalanine, lysine, arginine, glutamic acid and Ž . valine Roostita and Fleet, 1996 , which can be asource of metabolisable substrate for other microor-ganisms, resulting in the production of secondarymetabolites including flavour compounds. Release of free amino acids could also promote the growth of LAB with a poor proteolytic system.The high yeast counts in Zimbabwean traditional Ž  y 1 . fermented milk ca. 6.9 log cfu g suggest that 10 the yeasts have a mechanism for growth in the milk  Ž . Mutukumira, 1996; Gadaga et al., 2000 . The aim of this study was to assess the ability of yeasts isolatedfrom Zimbabwean naturally fermented milk to grow Ž . in ultra-high temperature UHT treated milk. Possi-ble interactions between the yeasts and LAB werealso investigated by studying final populations in themilk and by comparing their ability to producevolatile compounds, organic acids and carbon diox-ide in pure and co-culture. 2. Materials and methods 2.1. Storage and transportation of yeast isolates Yeasts strains isolated from Zimbabwean fer- Ž . mented milk Gadaga et al., 2000 were stored on Ž . Ž malt extract agar MEA Merck, Damstadt, Ger- . many slants at 4 8 C and were transported to Norwayin an insulated cooler bag with ice packs. The yeastswere further stored on MEA slants at 4 8 C at theAgricultural University of Norway. 2.2. Selection of yeast strains Nine yeast strains were selected from 44 isolates,based on their ability to produce relatively high Ž . levels of volatile organic compounds VOC and Ž carbon dioxide in UHT milk Tine Norske Meierier, . Oslo, Norway , after fermentation for 48 h at 25 8 C Ž . results not shown . The following yeast strains wereselected:  C. kefyr   23,  S. cere Õ isiae  71,  C  .  lipolytica 57,  C  .  lusitaniae  68,  C  .  tropicalis  78,  C  .  lusitaniae 63,  C. colliculosa  41,  S. dairenensis  32 and  Dekkerabruxellensis  43. All the cultures are at present storedin the culture collection of the Department of FoodScience, Agricultural University of Norway. Concen-trated cultures of the yeasts were then prepared. 2.3. Preparation of concentrated yeast cultures Each actively growing culture of the selectedyeasts was inoculated separately into malt extract Ž . Ž . broth Merck 200 ml in 250-ml screw cappedbottles and incubated for 72 h at 25 8 C in a waterbath. The cultures were then centrifuged at 5000 = g Ž . Ž in a refrigerated 4–10 8 C centrifuge Sorval 5RB, . Du Pont Instruments, DE, USA . The pellet was Ž . re-suspended in sterile reconstituted 10%, w r v Ž . Ž . skimmed milk 20 ml containing 10% v r v glyc-erol and then stored at y 80 8 C.  ( )T.H. Gadaga et al. r  International Journal of Food Microbiology 68 2001 21–32  23 2.4. Preparation of concentrated lactic acid bacteriacultures Concentrated pure cultures of   Lactococcus lactis subsp.  lactis  biovar.  diacetylactis  C1,  L .  lactis subsp.  lactis  Lc39,  L .  lactis  subsp.  lactis  Lc261 and  Lb. paracasei  subsp.  paracasei  Lb11, previouslyisolated from Zimbabwean traditional fermentedmilk, were prepared. The  Lactococcus  and the  Lac - tobacillus  strains were inoculated into M17 broth Ž and MRS broth Oxoid Unipath, Hampshire, Eng- . Ž . land 250 ml , respectively, and incubated at 30 8 Cfor 24 h. The cultures were centrifuged in a similarway to the yeast cultures and then re-suspended in Ž . 25 ml of reconstituted 10%, w r v skimmed milk.The cultures were stored at y 80 8 C and later used as Ž . direct vat set DVS cultures for inoculating UHTmilk. 2.5. Inoculation of UHT milk with DVS cultures The thawed concentrated cultures had viable LABand yeast counts ranging between 9.81–10.3 and7.6–8.3 log cfu ml y 1 , respectively. A volume of  10 the individual concentrated cultures was added to the Ž . UHT milk 40 ml , calculated to obtain a standardinitial inoculum of about 7.0 log cfu ml y 1 LAB 10 y 1 Ž . and 5.0 log cfu ml yeast Tables 2 and 3 . A 10 Ž . portion of each inoculated milk sample 10 ml wasaseptically pipetted into a headspace vial for carbondioxide analysis and incubated together with the restof the milk at 25 8 C for 48 h. The viable microbialcounts, VOC, organic acids, carbon dioxide and pHwere determined in the incubated milk. 2.5.1. Viable microorganisms Lactococcus  and  Lactobacillus  strains were enu- Ž . Ž . merated pour plate on M17 and MRS agar Oxoid ,respectively, and incubating at 30 8 C for 48 h. Yeasts Ž . were enumerated spread plate on yeast extract glu- Ž . Ž . cose chloramphenicol agar YGCA IDF, 1990 andincubated at 25 8 C for 3 days. 2.5.2. Chemical analyses Ž pH was determined using a Radiometer model . PHM92 pH meter with a combination glass elec- Ž trode and temperature probe Radiometer, Copen- . hagen after calibrating with commercial buffers Ž . Ž . Merck pH 4 and 7 . Volatile organic compounds Ž . VOC were determined by headspace gas chro- Ž . matography HS-GC and the organic acids weredetermined using high performance liquid chro- Ž . matography HPLC according to the method of  Ž . Narvhus et al. 1990; 1998 . The VOC determinedwere acetaldehyde, ethanol, acetone, diacetyl, ace-toin, 3-methyl-butanal, 3-methyl-butanol, 2-methyl-butanal and 2-methyl-butanol. The following organicacids were determined: citric, orotic, pyruvic, suc-cinic, lactic, formic, uric, and propionic acids. Car-bon dioxide was determined using the method of  Ž . Narvhus et al. 1991 . All the determinations weredone in duplicate. 2.6. Statistical analysis Average values of VOC, organic acids and carbondioxide data were analysed using principal compo- Ž . Ž . nent analysis PCA with cross-validation using the w Ž  w Unscrambler Programme Unscrambler 7.01, . Camo, Trondheim, Norway . Each variable wasweighted by dividing with the standard deviation of that variable. Interpretation of the data was made byinspection of the scores and loadings plots. Singleand co-cultured samples were compared by perform-ing one-way analysis of variance using Minitab v Ž . 12.02 Minitab programme. 3. Results The results are presented according to changes inmicrobial cell counts, pH, organic acids, VOC andproduction of carbon dioxide after 48-h fermentation.The values given are the mean from two independentexperimental runs. 3.1. Viable microorganisms The  Lactococcus  strains grew approximately twolog cycles both in single and co-cultures. The finalnumbers of the  Lactococcus  strains in the fermentedmilk were in the range 9.04–9.51 log cfu ml y 1 . 10  Lb. paracasei  subsp.  paracasei  Lb11 grew approxi-mately one log cycle and the viable counts ranged y 1 Ž . between 8.43 and 8.84 log cfu ml Table 1 in 10 both single and co-culture. The viable counts of thethree  Lactococcus  strains were not changed by co-inoculation with yeast, except for lower viable counts   (   )  T  .H . G a d  a  g a e t   a l   .   r I   n t   er  n a t   i   o n a l   J   o ur  n a l   o  f  F  o o d M i   cr  o b  i   o l   o  g  y 6  8 2  0  0 1 2 1 – 3 2  2  4   Table 1The viable LAB counts in UHT milk after 48-h fermentation with selected combinations of yeasts and LAB strains y 1 Ž . LAB strain Initial inoculum Final LAB count Lactic acid bacteria counts after growth with yeasts log cfu ml 10 y 1  y 1 Ž . Ž . log cfu ml log cfu ml 10 10  C. kefyr C. lipolytica S. cere Õ isiae C. lusitaniae C. tropicalis C. lusitaniae C. colliculosa S. dairenensis D. bruxellensis 23 57 71 68 78 63 41 32 43  L. lactis  subsp. 7.45 9.3 9.30 9.42 9.40 9.39 9.29 9.51 9.42 9.35 9.44 lactis  biovar. diacetylactis  C1  L. lactis  subsp. 7.98 9.35 9.31 9.04 9.26 9.34 9.21 9.41 9.33 9.24 9.19 lactis  Lc39 a a a a a  L. lactis  subsp. 7.19 9.06 9.38 9.22 9.39 9.31 9.25 9.25 9.19 9.34 9.22 lactis  Lc261 a a a  Lb. paracasei  7.93 8.68 8.81 8.64 8.68 8.84 8.70 8.53 8.59 8.74 8.45subsp.  paracasei Lb11 a Ž . Final LAB counts in co-cultures were significantly  P F 0.01 higher than the single culture. Results are from two replicate trials.  ( )T.H. Gadaga et al. r  International Journal of Food Microbiology 68 2001 21–32  25Table 2The viable yeast counts in UHT milk after 48-h fermentation with selected combinations of yeast and LAB strains y 1 Yeast strain Initial yeast Final yeast counts in Final yeast counts log cfu ml with LAB y 1  y 1 Ž . Ž . inoculum cfu ml single culture cfu ml  L. lactis  subsp . L. lactis L. lactis Lb. paracaseilactis  biovar. subsp . lactis  subsp . lactis  subsp.  paracaseidiacetylactis  C1 Lc39 Lc261 Lb11 C  .  kefyr   23 5.90 7.45 7.27 7.32 7.86 7.19 C  .  lipolytica  57 5.30 6.90 6.41 7.03 6.33 6.66 a S  .  cere Õ isiae  71 5.60 6.45 6.08 6.52 5.78 6.66 C  .  lusitaniae  68 5.00 7.52 7.16 7.55 7.94 6.90 C  .  tropicalis  78 5.48 7.21 7.19 7.20 6.58 7.02 C  .  lusitaniae  63 5.85 7.65 7.32 7.28 7.29 6.83 a C  .  colliculosa  41 5.78 6.86 6.56 6.74 6.65 6.00 a S  .  dairenensis  32 5.70 7.21 6.48 6.40 6.78 5.78 a  D. bruxellensis  43 6.42 6.30 6.57 6.45 6.00 5.62 a Ž . The final yeast counts were significantly  P F 0.01 lower in co-culture than in single culture. The results are from two replicate trials. of   L. lactis  subsp.  lactis  Lc39 when in co-culturewith  C. lipolytica  57. The final viable counts forLc261 were comparatively higher in co-culture with C. kefyr   23,  S. cere Õ isiae  71, and  S. dairenensis  32than in single culture.  Lb. paracasei  subsp.  paraca - sei  Lb11 had higher viable counts in co-culture with C  .  kefyr   23,  C. lusitaniae  68 and  S. dairenensis  32 Ž . Table 1 .The ability of the selected yeast cultures to growin milk was variable. The final populations were inthe range of 5.45–7.65 log cfu ml y 1 .  S. cere Õ isiae 10 71,  C. colliculosa  41 and  D. bruxellensis  43 insingle culture showed poor ability to grow in the Ž . UHT milk Table 2 .  C. kefyr   23 grew to finalpopulations of 7.19–7.86 log cfu ml y 1 .  C. collicu - 10 losa  41,  S  .  dairenensis  32 and  D. bruxellensis  43when grown in co-culture with Lb11 had a lowerfinal population than in the srcinal inoculum, sug-gesting that their viability may have been reduced at Ž . the end of the fermentation Table 2 . 3.2. pH  L. lactis  subsp.  lactis  biovar.  diacetylactis  C1,  L.lactis  subsp.  lactis  Lc39 and  L. lactis  subsp.  lactis Lc261, in single and co-culture, reduced the pH from6.6 to levels ranging between 4.0 and 4.2.  Lb. paracasei  subsp.  paracasei  Lb11 lowered the pH to Ž about 5.4 after the 48-h fermentation period Table . 3 , and consequently, did not coagulate the milk.However, the yeast strains in single culture did notcause any notable changes in the pH of the UHTmilk except  C  .  kefyr   23, which reduced the pH to Ž . 5.8 Table 3 . 3.3. Organic acids, VOC and carbon dioxide Data for organic acids, VOC and carbon dioxideproduced by the different lactic acid bacteria and Ž . yeast cultures, were analysed using PCA Fig. 1 .The first two principal components accounted for Table 3The pH of UHT milk after 48-h fermentation with selected combinations of yeast and LAB strains a LAB strain pH in pure LAB culture pH in co-culture with yeasts  L. lactis  subsp.  lactis  biovar.  diacetylactis  C1 4.2 " 0.09 4.18–4.23  L. lactis  subsp.  lactis  Lc39 4.1 " 0.08 4.05–4.16  L. lactis  subsp.  lactis  Lc261 4.2 " 0.03 4.04–4.13  Lb. paracasei  subsp.  paracasei  Lb11 5.4 " 0.04 5.08–5.43 a The range of pH of samples fermented with the LAB strains in co-culture with  C. kefyr   23,  C. lipolytica  57,  C. lusitaniae  63,  C.lusitaniae  68,  C. tropicalis  78,  S. cere Õ isiae  71,  S. dairenensis  32,  C. colliculosa  41, and  D. bruxellensis  43. The results are from tworeplicate trials.
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