Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk

The growth and death or survival of Bacillus cereus in sterile skimmed milk fermented with 18 different lactic acid bacteria (LAB) were investigated. B. cereus alone in milk reached about 10(7)-10(8) colony-forming units (cfu)/ml. When B. cereus was cultivated together with different Lactobacillus or Lactococcus cultures at 30 or 37 degrees C, the B. cereus counts after 72 h of fermentation ranged between < 10 cfu/ml and about 10(6) cfu/ml. The inhibition patterns for the different Lactobacillus and Lactococcus cultures varied. All the Lactococcus cultures (with one exception) reduced pH to 5.3 or lower in 7 h. After 24 h, B. cereus was not detected in any of the fast Lactococcus-fermented milk samples. After 48 h, B. cereus was not detected for 4 of the 12 Lactobacillus cultures. These cultures reduced pH to below 5.0 in 24 h. The other Lactobacillus cultures also inhibited B. cereus, but the counts of B. cereus were still 10(4)-10(6) cfu/ml after 72 h. They also reduced pH at a slower rate. Survival of B. cereus was to a variable extent linked with formation of endospores. Proteinase K did not affect the antimicrobial activity observed. Acid production with decreasing pH, particularly the initial rate of pH decrease, appears to be most important for control of B. cereus with LAB.     

Dry sausage fermented by Lactobacillus rhamnosus strains

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.     

直投式酸奶发酵剂高密度培养工艺的研究

以实验室筛选获得的适于发酵生产用的直投式酸奶发酵剂菌株组合保加利亚乳杆菌Lb-23和嗜热链球菌S.t-14为材料,对其高密度培养工艺进行了研究.结果发现,按球菌:杆菌=1:1的混合培养比例,pH 6.6～6.8,转速85～100r/min,42℃培养18h后,活菌数达到1.27×10~9cfu/mL;菌悬液经1.8%海藻糖、0.25%甘油、5.5%谷氨酸钠和0.5%吐温80保护剂处理,冷冻干燥后获得的发酵剂活菌数达到3.35×10~(11)cfu/g.     

甘蓝泡菜发酵菌种的复配研究

研究了用于蔬菜发酵的3种天然优势乳酸菌菌株——短乳杆菌、干酪乳杆菌、植物乳杆菌单独或复配使用对发酵甘蓝品质的影响。甘蓝接种后,常温条件(约23℃)下发酵3d,监测pH值、总酸、亚硝酸含量、乳酸菌总数的变化情况及感官品质。结果表明短乳杆菌与植物乳杆菌按1∶1复配,总接种量为2%～3%,泡菜的发酵效果最好。当总接种量为2%时,发酵后泡菜的pH为3.35,总酸为0.74%,乳酸菌活菌数为8.93×10~7cfu/mL,感官品质最佳,评分值达到12.1/15,亚硝酸盐含量为14.2mg/kg。     

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.     

传统酸面团中细菌与酵母菌的分离与鉴定

为进一步描述我国传统面食发酵剂的理化性质和菌落组成,收集了北方地区6份发酵剂样品,测定了其酸度和菌落总数,并对分离、纯化、初筛后得到的75株细菌和60株酵母菌进行了测序鉴定。结果显示,样品pH值范围为3.73~5.46,总滴定酸度为8.3~19.8 mL;乳酸菌和酵母菌的计数结果分别为8.35±0.07~9.75±0.12 Log cfu/g,6.31±0.22~8.68±0.04 Log cfu/g。鉴定出包括短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)和旧金山乳杆菌(Lactobacillus sanfranciscensis)在内的乳酸菌8种;酵母菌4种,其中优势菌为酿酒酵母(Saccharomyces cerevisiae);其他细菌6种,主要为解淀粉芽孢杆菌(B.amyloliquefaciens)、地衣芽孢杆菌(B.licheniformis)和醋杆菌。结果表明,我国传统面食发酵剂菌相复杂,以酵母菌和乳酸菌为主,还包括芽孢杆菌、醋酸杆菌在内的多种其他细菌,甚至可能含有致病菌。通过比较细菌和酵母在不同酸面团样品中的分布,发现不同来源样品的微生物种类组成存在差异。     

益生菌发酵绿豆乳的菌种筛选

本文以分离自益生菌乳制品、微生态制剂、保健品的13株国际公认的益生乳酸菌为试验菌株,接种于纯绿豆乳培养基中进行发酵。通过检测其凝乳与否、凝乳时间、以及凝乳时pH、活菌数、感官评价等指标,筛选适宜发酵绿豆乳的繁殖力强、发酵活性高的益生菌菌株。结果表明：经过层层筛选得到的干酪乳杆菌07-211、植物乳杆菌08．193、瑞士乳杆菌05-29等三株益生菌发酵6h均可均-凝乳,有绿豆香,且凝乳时,其活菌数分别可达到3．9×10^8、1．6×10^8、1．4×10^8cfu/mL凝乳时pH分别为4．16、4．30、4．32,滴定酸度分别为83．5、81．0、75．2oT,可作为新型绿豆乳制品的发酵菌株。     

Microbiological attributes and biogenic amine content of probiotic Turkish fermented sausage

Microbiological attributes and biogenic amine content of Turkish fermented sausage manufactured by using probiotic starter cultures (Lactobacillus casei, L. acidophilus or their combination) were investigated before and after fermentation-drying period and during refrigerated storage at 4?±?1?°C for 8?months at 2?month intervals. As results of the study, during fermentation and storage biogenic amine content (histamine, putrescine, cadaverine and tyramine) of the samples were increased significantly. Salmonella, Staphylococcus aureus, coliform and fecal coliform microorganisms were not detected during fermentation and storage. Probiotic microorganism counts of all samples were higher than the lower limit of 6.0 log cfu/g which is requested for probiotic foods.     

The occurrence and growth of microorganisms during the fermentation of fish sausage

Minced fish (mullet) sausage mixes containing added sugar, salt, nitrate, nitrite and spices were fermented (48 h, 30 degrees C) by indigenous flora or by a starter culture (Pediococcus acidilactici) and the microbial ecology and behaviour of various bacteria was monitored. Pediococcus pentosaceus and Lactobacillus plantarum dominated the indigenous fermentation, achieving populations of 10(7)-10(8) cfu/g by 48 h, and decreasing the pH of the mix to 4.5-4.7. Significant growth (10(5)-10(7) cfu/g) of Staphylococcus warneri, Staphylococcus aureus, Staphylococcus epidermidis. Micrococcus varians and Micrococcus luteus also occurred during this fermentation. Less growth was exhibited by Bacillus megaterium and yeasts. Pediococcus acidilactici dominated the fermentation when it was inoculated as a starter culture, but indigenous lactic acid bacteria (P. pentosaceus and L. plantarum) also grew to 10(7)-10(8) cfu/g. The growth of other bacteria and yeasts was restricted during fermentation with starter culture. Inoculated Escherichia coli, Salmonella typhimurium, Salmonella sofia, and Staphylococcus aureus grew to 10(6)-10(7) cfu/g in the sausage mix during indigenous fermentation. Lesser growth occurred for Bacillus cereus, Clostridium perfringens and Vibrio parahaemolyticus. Growth of these bacteria was significantly inhibited in sausage mix fermented with P. acidilactici.     

人工发酵剂对湘西腊肠微生物菌群和理化特性的影响

本文从动态角度探讨自然发酵、戊糖片球菌发酵、木糖葡萄球菌发酵和混合菌发酵腊肠在发酵过程中五种菌相(细菌总数、乳酸菌、葡萄球菌、酵母菌和肠细菌)和理化特性的阶段性变化。微生物数据显示,四组腊肠初始肠细菌数量约5.30 logCFU/g,而发酵结束,添加了戊糖片球菌腊肠组的肠细菌数量约1.00 logCFU/g,没有添加该菌腊肠组的肠细菌数量约3.30 logCFU/g,说明戊糖片球菌能有效抑制肠细菌的生长,保证了产品的安全性和稳定性;发酵初期,腊肠p H值迅速降5.30以下,到发酵中期,腊肠p H开始回升;在发酵过程中,戊糖片球菌腊肠组的TBARS(硫代巴比妥酸)值显著(p0.05)高于其他三组腊肠,而其它理化特性无显著差异。从数据分析可知,腊肠的发酵剂能有效改善其卫生质量,而对腊肠的p H值、AW和亚硝酸盐残留量没有显著影响。     

山西地方自然发酵蔬菜发酵过程中细菌多样性分析

为探究山西地方自然发酵蔬菜发酵过程中细菌菌群结构,采用Illumina HiSeq对不同发酵时期的发酵蔬菜样品中细菌进行测序,且对地方自然发酵蔬菜制作过程中细菌总数及乳酸菌数、基本理化指标进行检测与分析。结果表明,地方自然发酵蔬菜的发酵前期细菌多样性丰富,随着发酵时间的延长细菌种群结构趋于稳定,发酵中后期样品主要菌属为乳杆菌属（Lactobacillus）;pH值随发酵时间延长呈下降趋势,总酸反之,成品发酵蔬菜中细菌总数和乳酸菌数分别为1.2×107 CFU/g和2.5×106 CFU/g,亚硝酸盐含量为0.15 mg/kg,pH值为3.22,总酸含量为1.98 g/kg,乳酸菌数与亚硝酸盐呈彼消此长趋势。本研究揭示了山西地方发酵蔬菜中细菌群落结构及差异,为地方自然发酵蔬菜的商业化生产提供理论依据。     

Sensory Acceptability and Stability of Probiotic Microorganisms and Vitamin C in Fermented Acerola (Malpighia emarginata DC.) Ice Cream

ABSTRACT:  Six fermented acerola ice creams were produced, containing different starter cultures ( Bifidobacterium longum , Bi.lactis , and traditional yogurt starter culture— Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus ) and final pH (5 and 4.5). The ice creams were evaluated for probiotic culture viability, vitamin C stability, and sensory acceptance. Mix fermentations were stopped when pH 5.0 and 5.5 were attained. However, after the addition of acerola pulp the determined pH were 4.5 and 5, respectively. Mixes were frozen and stored for 15 wk at −18 °C. The viable counts for probiotic cultures remained above the recommended minimum limit of 10⁶ cfu/g during 15 wk storage even in products with pH 4.5. Vitamin C concentration remained around 140 mg/100 g of product. The attributes of aroma, taste, texture, and overall acceptance obtained scores in the range of 5.15 to 7.22. The fermented acerola ice cream was a suitable food for the delivery of vitamin C and Bifidobacterium strains with excellent viability and acceptable sensory characteristics.     

Characterization and Technological Properties of Lactic Acid Bacteria in the Production of “Sorghurt,” a Cereal-Based Product

A total of 57 predominant LAB strains isolated from cassava and maize grains fermentation processes for fufu and ogi were identified using phenotypic and genomic fingerprinting methods such as rep-PCR and ARDRA. They were divided into facultatively heterofermentative rods (26.3%), obligately heterofermentative rods (31.6%), and tetrad forming homofermentative cocci (42.1%). Selected strains were further identified by sequencing the 16S rDNA gene. Technological studies such as acidification, hydrogen peroxide production, starch hydrolysis, enzymatic activities, degradation of oligosaccharides, and in vitro adherence properties were carried out. Lactobacillus plantarum strains demonstrated better and rapid acid production capability, followed by the Pediococcus strains, while L. fermentum strains exhibited slower acid production. Hydrogen peroxide production was observed among the LAB groups. The test strains utilized the indigestible sugars raffinose and stachyose. Only L. pentosus demonstrated high amylase activity comparable to that of L. amylovorus DSM 20531. Lactobacillus plantarum and L. fermentum strains showed high β-glucosidase activity. Six strains were selected as starter cultures. The strains were tolerant to acidic pH levels and bile salt. The yoghurt-like “sorghurt” produced using the selected starter cultures had a final pH of less than pH 4.0, and a viable count of less than 5.5 Log₁₀cfu/mL at the end of a 24 h fermentation period. The samples were generally acceptable to the taste panelists. The starter organisms demonstrated varying degree of adherence to HT29 MTX cell line. Therefore, employing functionally defined LAB strains may be one practical approach for incorporating health-promoting features into appropriate food products suitable for targeted population.     

Chemical composition and microbiological changes during spontaneous and starter culture fermentation of Enam Ne–Setaakye, a West African fermented fish-carbohydrate product

Enam Ne-Setaakye, a fermented fish-carbohydrate meal was made from minced fish flesh (75%), yam (20.5%), onions(1%), ginger(1%) and salt(2.5%). A total of 113 isolates belonging to the genus Lactobacillus, Pediococcus, Bacillus, Pseudomonas, Klebsiella, Staphylococcus, Debaromyces and Candida were obtained from the natural fermentation process of the product. Lactobacillus brevis and Pediococcus pentosaceus were the dominant species with 24% and 19% occurrence respectively, while Candida species least occurred (3%).Changes in the viable count of the microflora showed the dominance of lactic acid bacteria from the 48 h of fermentation period (5.8᎒ cfu/g) to the end (2.3᎒ cfu/g). Enterobacteriaceae which was detected at a level of 10 cfu/g at 0 h, were not detectable after 72 h. Moulds were not isolated, while the yeast population became detectable from 72 h reaching 10 cfu/g at the end of fermentation. On the other hand, a shorter fermentation period of 72 h with a LAB count of 10 cfu/g, non-detection of enterobacteriaceae, moulds and a yeast population of 10 cfu/g were obtained when the fermentation was carried out using L. brevis and P. pentosaceus as starter cultures. A pH level of 4.3, 3.1% titratable acidity and 2.1% (^w/_wp) salt content were obtained in the starter culture fermented Enam Ne-Setaakye. There were significant differences in the moisture and free fatty acid contents of both naturally and starter culture fermented samples, but the protein content (16%) was not significantly different. Sensory evaluation of the products showed consumer preference for the steamed samples.     

Microbiological attributes and biogenic amine content of probiotic Turkish fermented sausage

Microbiological attributes and biogenic amine content of Turkish fermented sausage manufactured by using probiotic starter cultures (Lactobacillus casei, L. acidophilus or their combination) were investigated before and after fermentation-drying period and during refrigerated storage at 4 ± 1 °C for 8 months at 2 month intervals. As results of the study, during fermentation and storage biogenic amine content (histamine, putrescine, cadaverine and tyramine) of the samples were increased significantly. Salmonella, Staphylococcus aureus, coliform and fecal coliform microorganisms were not detected during fermentation and storage. Probiotic microorganism counts of all samples were higher than the lower limit of 6.0 log cfu/g which is requested for probiotic foods.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

Inhibition of Listeria innocua growth by antimicrobial-producing lactic acid cultures in vacuum-packed cold-smoked salmon

The biopreservative potential of three antimicrobial-producing lactic acid bacteria strains was evaluated on cold-smoked salmon. Lactobacillus casei, Lactobacillus plantarum and Carnobacterium piscicola were added singly or in association to cold-smoked salmon, artificially contaminated with Listeria innocua and stored under vacuum for 30 days at 4 degrees C. All the lactic cultures were able to inhibit Listeria innocua growth, showing a bacteriostatic or bactericidal effect, without affecting negatively the sensory quality of the product. Lactobacillus casei was bacteriostatic when inoculated at 6 log cfu/g, but bactericidal at 8 log cfu/g, reducing Listeria innocua of 3.3 log cfu/g in comparison with the test at the end of storage. Lactobacillus plantarum and C. piscicola strains, inoculated singly at 6 log cfu/g reduced Listeria innocua counts of 2.8 and 2.7 log cfu/g, respectively, compared with the test. The association Lactobacillus casei-Lactobacillus plantarum was the most effective among the treatments with 6 log cfu/g inoculum, as Listeria innocua counts decreased of 3.2 log cfu/g compared with the test. The treatment with Lactobacillus casei-C. piscicola association was less effective than C. piscicola alone.     

发酵剂对发酵香肠微生物及理化特性的影响

采用3种复合型发酵剂(VBL-97、VBM-60、SHI-59)和1种单一型发酵剂(BOM-13)生产发酵香肠,研究不同发酵剂对发酵香肠微生物及理化性质的影响。结果表明4组发酵香肠在发酵和成熟过程中,乳酸菌始终保持为优势菌,当达到成熟时,乳酸菌数在7.21~8.13 lgcfu/g范围内,葡萄球菌(除BOM-13外)高于5.50 lgcfu/g,肠杆菌科菌数低于3.00 lgcfu/g。BOM-13型发酵剂由单一清酒乳杆菌组成,发酵和成熟时间长于其他三组,所以成品的硫代巴比妥酸值(TBARS值,0.84 mg/100 g)和非蛋白氮值(NPN,0.46%)高于其它3组产品。由此可知由于发酵剂组成不同,相应地发酵条件(温度和时间)和成熟时间亦不同,但最终四种发酵剂都可生产出高品质产品(p H值5.3;AW0.82;NPN值高;TBARS值1.0 mg/kg;乳酸菌为优势菌且7.0 lgcfu/g;肠杆菌科菌3.0lgcfu/g)。VBL-97型发酵剂产酸能力和抗氧化能力强,而BOM-13型发酵香肠蛋白分解程度最高。     

乳杆菌发酵剂对酸肉挥发性风味成分的影响

分别采用植物乳杆菌、清酒乳杆菌和类植物乳杆菌作为发酵剂对酸肉发酵过程中挥发性风味成分的影响。 电子鼻分析结果表明,酸肉的整体风味随发酵时间的增加差异显著;利用固相微萃取-气相色谱-质谱技术对酸肉发 酵过程中的挥发性风味物质进行检测,结果表明,酯类物质的相对含量显著增加,且增加的主要是乙酯类物质,发 酵结束时,添加植物乳杆菌、类植物乳杆菌和清酒乳杆菌组酸肉的乙酯类物质相对含量（56%、53%和60%）均显 著高于对照组（47%）,说明乳杆菌发酵剂的添加有利于酸肉中乙酯类物质的形成;通过Heatmap聚类分析发现, 发酵结束时,添加乳杆菌发酵剂的3 组酸肉整体风味比较接近,但与对照组差异较大,说明乳杆菌发酵剂的添加影 响了酸肉的风味品质。此外,发酵过程中添加发酵剂的酸肉中乳酸菌数量增加较快,肠杆菌数量显著降低,这保证 了酸肉的食用安全性。     

Investigation on culturable microflora in tibetan kefir grains from different areas of china

Four samples of Tibetan kefir grains (TK-ZJUJ 01-04) from Tibet and surrounding areas were investigated via phenotypic and genotypic methods to compare and analyze the diversity of culturable microflora among different origins. As a result, 4 genera of microorganisms from TK-ZJUJ01: Bacillus subtilis (2.9 × 10(7) cfu/mL), Lactococcus lactis (8.2 × 10(7) cfu/mL), Kluyveromyces marxianus (3.0 × 10(6) cfu/mL), Saccharomyces cerevisiae (9.0 × 10(6) cfu/mL); 4 genera from TK-ZJUJ02: Lactobacillus kefiri (1.0 × 10(8) cfu/mL), Pichia kudriavzevii (5.0 × 10(6) cfu/mL), K. marxianus (1.9 × 10(7) cfu/mL), Kazachstania unispora (6.2 × 10(7) cfu/mL); 6 genera from TK-ZJUJ03: Leuconostoc lactis (4.6 × 10(7) cfu/mL), L. lactis (3.0 × 10(7) cfu/mL), Lactobacillus plantarum (3.0 × 10(7) cfu/mL), K. unispora (3.0 × 10(6) cfu/mL), K. marxianus (2.0 × 10(6) cfu/mL), (1.7 × 10(7) cfu/mL); and 4 genera from TK-ZJUJ04: L. plantarum (1.8 × 10(7) cfu/mL), Acetobacter fabarum (5.0 × 10(6) cfu/mL), K. unispora (6.2 × 10(7) cfu/mL), Pichia guilliermondii (6.2 × 10(7) cfu/mL) were identified. Yeasts like P. kudriavzevii and P. guilliermondii isolated in this study were the first time reported in Tibetan kefir grains. For TK-ZJUJ 01-03, lactic acid bacteria were the major microorganisms, which accounted for more than 50% of all the microbial population, while for TK-ZJUJ04, the largest microbial group was yeasts which accounted for more than 50%. In a word, study of diversity and composition of microflora provided us theoretical foundation for further investigation and application of Tibetan kefir grains. Practical Application: This is the basic research in order to develop and industrialize a new kind of yogurt starter which is naturally formed microbiota with both lactic acid bacteria and yeasts in it.