Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.     

开菲尔活菌型酸乳饮料的研制

以鲜牛奶、蔗糖为主要原料,Kefir M为发酵剂,羧甲基纤维素钠和高甲氧基果胶为稳定剂,通过对不同配方及工艺条件进行正交试验,确定了开菲尔酸乳饮料的最佳工艺和配方。结果表明,当发酵温度为28 ℃,羧甲基纤维素钠为0.20%,高甲氧基果胶为0.20%,蔗糖为8%,制作的开菲尔活菌型酸乳饮料口感和稳定性较好。     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78–84% similarity, and yeasts 80–92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.     

Effect of different growth conditions on biomass increase in kefir grains

Kefir is a functional dairy product and the effects of kefir consumption on health have been well documented. Kefir grains have naturally high numbers of lactic acid bacteria and yeasts and are used in manufacturing kefir. The biomass of kefir grains slowly increases after successive fermentations. The effects of adding whey protein isolate, modified whey protein (MWP, fat replacer; Carbery Inc., Cork, Ireland), or inulin to milk and different atmospheric conditions (ambient or 6% CO₂) during fermentation on the increase in biomass of kefir grains were investigated. Reconstituted milks (10% milk powder) enriched with whey protein isolate (2%), MWP (2%), and inulin (2%) were inoculated with kefir grains and fermented in ambient and 6% CO₂ incubators at 25°C until a final pH of 4.6 was reached. Biomass increments of kefir grains were determined weekly over 30 d. Lactic acid bacteria and yeast contents of kefir grains were also determined. The highest biomass increase (392%) was found in kefir grains grown in milk supplemented with whey protein isolate under ambient atmospheric conditions. Application of CO₂ did not provide a significant supporting effect on the biomass of kefir grains. Addition of MWP significantly accelerated the formation of kefir grain biomass (223%). The use of whey protein isolate, MWP, or inulin in milk did not cause any adverse effects on the microbial flora of kefir grains.     

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

BACKGROUND: Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species‐specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. RESULTS: The microbiological composition of foods was analysed by molecular methods. Using species‐specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. CONCLUSION: A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species. Copyright © 2012 Society of Chemical Industry     

Chemical composition and sensory analysis of cheese whey‐based beverages using kefir grains as starter culture

The aim of the present work was to evaluate the use of the kefir grains as a starter culture for tradicional milk kefir beverage and for cheese whey‐based beverages production. Fermentation was performed by inoculating kefir grains in milk (ML), cheese whey (CW) and deproteinised cheese whey (DCW). Erlenmeyers containing kefir grains and different substrates were statically incubated for 72 h at 25 °C. Lactose, ethanol, lactic acid, acetic acid, acetaldehyde, ethyl acetate, isoamyl alcohol, isobutanol, 1‐propanol, isopentyl alcohol and 1‐hexanol were identified and quantified by high‐performance liquid chromatography and GC‐FID. The results showed that kefir grains were able to utilise lactose in 60 h from ML and 72 h from CW and DCW and produce similar amounts of ethanol (～12 g L^?1), lactic acid (～6 g L^?1) and acetic acid (～1.5 g L^?1) to those obtained during milk fermentation. Based on the chemical characteristics and acceptance in the sensory analysis, the kefir grains showed potential to be used for developing cheese whey‐based beverages.     

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.     

Diversity of bacteria present in milk kefir grains using culture-dependent and culture-independent methods

In the present work bacteria associated with milk kefir grains from several Brazilian States, Canada and the United States of America under traditional conditions have, for the first time, been studied using a combination of pheno-and genotypic methods. Conventional culturing was performed and a total of 270 isolates were obtained from all samples. Isolates were identified using biochemical tests and partial sequence analysis of 16S rDNA. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant bacterium was Lactobacillus kefiri. PCR-DGGE revealed the presence of Gluconobacter japonicus and Lactobacillus uvarum which were no isolated. Conventional isolation revealed the presence of L. helveticus, L. kefiri and Acetobacter syzygii not identified among the sequenced DGGE bands. This study is the first to report the presence of Lactobacillus satsumensis and Acetobacter syzygii in milk kefir grains.     

分离自藏灵菇的乳酸菌的益生特性

从藏灵菇中分离纯化5株乳酸菌,初步鉴定2株为嗜酸乳杆菌,3株为乳酸乳球菌。选取其中2株菌研究其益生特性。结果表明,从藏灵菇中分离出的乳酸菌具有良好的益生特性,2株乳酸菌在pH值为4～6可生长良好;耐热范围为30～60℃;胆盐耐受性为0.1%～0.5%;发酵液对金黄色葡萄球菌、沙门氏菌、大肠杆菌等肠道病原菌有抑制作用;对抗生素有不同程度的耐药性。     

玉米乳酸菌饮料的工艺研究

以玉米为原料,采用全酶法糖化工艺与多株乳酸菌互生发酵相结合的方法,在37℃条件下对玉米酶解液发酵48h。发酵液经过滤后加适量的砂糖、柠檬酸及复合稳定剂,可调制成酸甜可口、富含多种氨基酸的乳酸发酵饮料。     

开菲尔粒中乳酸菌的分离与鉴定

从4个不同来源的开菲尔粒中分离得到40株乳酸菌，并对这40株乳酸菌进行了鉴定．结果为德氏乳杆菌8株，德氏乳杆菌保加力亚种6株，高加索奶乳杆菌2株，嗜酸乳杆菌1株，粪肠球菌7株，屎肠球菌2株，嗜热链球菌1株，乳脂链球菌3株。     

发酵型核桃乳饮料的研制

研究了发酵型核桃乳饮料的生产工艺。对核桃乳原浆和发酵型核桃乳饮料的理化性质进行了测定 ,并对发酵型核桃乳饮料生产工艺中的关键步骤(前配料、发酵剂、均质压力)进行了研究。     

16S rDNA PCR-RFLP分析鉴定开菲尔发酵剂中的乳酸菌

对传统乳制品开菲尔发酵剂通过菌种分离和纯化得到67 株乳酸菌,经过传统形态学分类区分成6 种形态类型。在此基础上进行16S rDNA限制性片段长度多态性聚合酶链反应（polymerase chain reaction-restriction fragmentlength polymorphism,PCR-RFLP）分析法鉴定为4 种分子类型,分别为：乳明串株菌（L. lactis）、坚强肠球菌（E. durans）、意大利肠球菌（E. italicus）、嗜热链球菌（S. thermophilus）。其中坚强肠球菌菌数超过50%,占所分离菌株的62.7%,为优势菌。     

活性乳酸菌饮料生产工艺研究

研究了活性乳酸菌饮料的生产工艺，对菌种、稳定剂和酸味剂进行了筛选，确定了适合工业化的生产工艺。实验表明，嗜热链球菌和保加利亚乳杆菌比例为1：1，羧甲基纤维素：藻酸丙二醇酯：还原胶=4：1：1，添加量为0.3%；柠檬酸：乳酸：酒石酸=3：3：2，添加量为0.25%；加糖量为12%。解决了乳酸菌饮料的稳定性问题，所得产品品味较好。     

蛋黄保健营养乳酸饮料的研究

以蛋黄和牛乳为主要原料,研制出一种营养丰富、均衡、有蛋黄风味的乳酸饮料。在研究中确定了主要工艺流程,并通过发酵菌种选育驯化、发酵条件试验、物料配比确定、稳定剂筛选和正交实验,优选出最佳配方以及对影响制品质量的其他因素进行了探讨。     

Diversity and screening of lactic acid bacteria in la-baicai made by Korean-Chinese in northeastern China

This study investigated the lactic acid bacteria (LAB) population in la-baicai (spicy cabbage), a traditional fermented food made by the Korean-Chinese community in northeastern China and screened for functional LAB. LAB diversity was analyzed using denaturing gradient gel electrophoresis (DGGE), and 81 LAB strains were isolated and identified based on 16S rDNA sequencing analysis. Polymerase chain reaction DGGE detected 21 LAB species, belonging to the genera Lactobacillus (Lb.), Leuconostoc (Leu.), Pediococcus and Weissella, in 45 la-baicai samples. Lb. plantarum and Lb. sakei were considered to be dominant in the bacterial community. Among 81 LAB isolated by traditional pure culture methods were Lb. plantarum (25 strains), Lb. brevis (two strains), Lb. casei, (four strains), Lb. pentosus (three strains), Enterococcus faecium (45 strains), and E. durans (two strains). The tolerances of these LAB were investigated. Six LAB with high salt (NaCl)-tolerance were screened from the isolates, and 16% (w/v) was the highest salt-tolerance reached. Among the isolates, strain N1, identified as Lb. pentosus, survived well in a simulated digestive environment. Traditional fermented la-baicai from northeastern China is a rich LAB resource. Further study is needed and would be worthwhile to advance the benefits of these LAB.     

发酵蓝莓乳饮料生产工艺的探讨

以蓝莓、鲜奶为主要原料制成发酵乳饮料,通过试验对稳定剂及饮料配方进行了研究,结果表明:复合稳定剂的组成为果胶(0.20%)、CMC(0.15%)、PGA(0.10%);最佳配方为蓝莓汁的添加量8%,发酵乳的添加量30%,糖的添加量8%,pH值4.0。     

猕猴桃乳酸菌饮料的研制

以猕猴桃为原料,以pH值和感官评价作为检测指标,依次经过菌种筛选、单因素试验及正交试验确定猕猴桃乳酸菌饮料的最佳工艺条件,并分析该饮料在保藏期期间总酚酸、维生素C以及乳酸菌含量的变化情况。结果表明,适用于猕猴桃乳酸菌饮料的最佳菌种为植物乳杆菌（Lactobacillus plantarum）,最佳发酵工艺条件为初始pH值5.0,加糖量10%,料液比1∶1（g∶mL）,接种量106 CFU/g,发酵温度37 ℃,发酵时间28 h。在此最佳工艺条件下,产品总酚酸含量174.89 mg/100 g,维生素C 46.84 mg/100 g,pH值4.27,可溶性固形物18%,活菌数对数值9.96,微生物指标符合相关标准。     

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.