Turkish kefir and kefir grains: microbial enumeration and electron microscobic observation†

Microbial populations in kefir and kefir grains were enumerated by plating. Total lactic acid bacteria, lactoccocci, lactobacilli and yeast populations increased during fermentation and increased slightly during cold storage. Kefir grains had a lactic acid bacteria : yeast ratio of 10 ⁹  : 10 ⁶ . In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods, which indicated yeast colonization on the surface and middle part of the kefir grain. Three types of lactobacilli (short, long and curved) were noted throughout the grain. Lactococci were not observed under SEM; preparation of kefir grains for SEM may have caused removal of lactococci from the grains.     

Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods

Abstract: In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture‐independent and culture‐dependent methods. In the culture‐independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture‐dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence‐based PCR (rep‐PCR) fingerprinting, and the results were validated by 16S rDNA full‐length sequencing. According to the results of culture‐dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture‐dependent and culture‐independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Practical Application: Due to their artisan‐ and region‐dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from different regions of Turkey were studied using culture‐dependent and culture‐independent molecular methods.     

Quality characteristics of kefir as a carrier for probiotic Lactobacillus rhamnosus GG

Kefir was prepared using kefir grains of different origins (US, Australia and India) and starter culture. The sensory scores for kefir made using starter culture and US kefir grains were significantly higher than for the other samples (P < 0.05). The index of viscosity was significantly higher (P < 0.05) in kefir prepared using Indian kefir grains. Shear stress–shear rate plots indicated yield stress in all kefirs. All samples exhibited shear‐thinning behaviour. The average count of Lactobacillus rhamnosus GG in kefir after incubation was 7.09 log CFU/g. No significant difference (P > 0.05) was observed in quality characteristics in kefir after the addition of LGG, indicating that it could be a potential probiotic carrier.     

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)₅-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.     

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284

The aim of this study was to analyze the β-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. β-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The β-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.     

Microbial diversity and stability during primary cultivation and subcultivation processes of Tibetan kefir

In this study, we have investigated the microbial diversity and stability in the Tibetan kefir during the primary cultivation and subcultivation processes via a combination of culture‐independent and culture‐dependent methods. According to the culture‐independent methods, the profiles of PCR‐DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis) indicated that nine microbial species were predominant at different cultivation stages; seventy four isolates of seven predominant species were obtained and identified via the culture‐dependent methods. PCR‐DGGE further confirmed that Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus paracasei, Streptococcus thermophilus, Kazachstania unispora and Saccharomyces cerevisiae were the most dominant species in the Tibetan kefir during the process of primary cultivation, among which Lb. kefiri, Lb. paracasei and K. unispora showed relative strong stability during both the processes of primary cultivation and subcultivation. These findings suggested that some isolates of the three species possessed the potentiality of being used in the development of direct vat set (DVS) starters for the production of Tibetan kefir and the related products.     

Effects of cow's and goat's milk as fermentation media on the microbial ecology of sugary kefir grains

In the present study, we have investigated the importance of fermentation media on grain formation and the microbial characteristics of sugary kefir. The sugary kefir grains were fermented in brown sugar, cow's milk or goat's milk. Using culture-dependent and culture-independent methods, we identified the microorganisms present in both the grains and filtrate and then evaluated their distribution. The structure of the grains was also observed by scanning electronic microscopy (SEM). The identification results indicated that there were remarkable changes in microbial ecological profiles of the sugary kefir grains and their filtrates when brown sugar and milk were compared as fermentation media. Three lactic acid bacteria (LAB) species (Leuconostoc mesenteroides, Lactobacillus mali and Lactobacillus hordei) were found in the grains fermented using brown sugar. However, four species, named Leu. mesenteroides, Lactococcus lactis, Bifidobacterium psychraerophilum and Enterococcus faecalis, were identified in the grains fermented using either cow's or goat's milk. The size and structure of the kefir grains were also significantly influenced by the culture medium. We hypothesize that the grains originally may contain many different microorganisms and the identified changes are an adaption to each specific medium during grain formation and growth. The distribution of strains thus may vary depending on the carbon and energy sources available for grain fermentation and these microbial changes will further affect the granulation and growth of the grains. This study is important to our understanding of the mechanism of kefir grain formation and growth because it explores the relationship between fermentation media and kefir microorganisms.     

Identification of microbial flora in kefir grains produced in Turkey using PCR

Kefir grains might have different ratios and/or content of microflora according to their origin. The purpose of this study was to determine microbial flora of kefir grains produced in three regional universities in Turkey using polymerase chain reaction (PCR) and consensus sequence primers. According to the results of PCR products with the specific primers, the following were identified as the natural inhabitants of the kefir grains: Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Bifidobacterium bifidum and Kluyveromyces marxianus kefir. The results of this study revealed that traditional kefir produced by using kefir grains as natural starter cultures contains lactic acid bacteria especially Lactobacillus spp. One of the sources also contained B. bifidum. This is the first record on the presence of B. bifidum in kefir grains.     

Diversity and functional characteristics of lactic acid bacteria from traditional kefir grains

Traditional kefir grains were collected from distinct parts of Turkey, and their microbial profile was determined. A wide bacterial biota was observed formed by distinct lactic acid bacteria (LAB) in which Lactococcus lactis strains appeared to be dominant. Yeast species were also identified in kefir grains. Significant levels of antifungal and antibacterial activities were monitored in kefir isolates. All tested LAB produced an exopolysaccharide (EPS) containing glucose and galactose, and some strains formed a fructan‐type EPS. Importantly, low levels of antibiotic resistance were observed among the kefir isolates.     

EFFECT OF NATIVE AND LYOPHILIZED KEFIR GRAINS ON SENSORY AND PHYSICAL ATTRIBUTES OF WHEAT BREAD

Native and lyophilized kefir grains were added directly to bread dough or as a starter in sourdough. Because of inadequate leavening activity of kefir, yeast‐leavened breads were prepared. Kefir addition influenced the quality attributes and the shelf life of wheat bread. The pH of bread containing kefir decreased as compared to that in the control bread. The acidity of bread with kefir increased as compared to that in the control bread. An addition of kefir grains, regardless of the form, lowered the bread volume and increased the mold‐free shelf life from 4 (control) to 5–7 days. The addition of kefir grains in bread dough contributed to a milder taste, a more delicate yogurt‐like or dairy aroma. The breads made with sourdoughs containing native or lyophilized kefir grains scored higher for crumb quality number than breads made by directly adding kefir grains.     

A review on traditional Turkish fermented non-alcoholic beverages: Microbiota,fermentation process and quality characteristics

Shalgam juice, hardaliye, boza, ayran (yoghurt drink) and kefir are the most known traditional Turkish fermented non-alcoholic beverages. The first three are obtained from vegetables, fruits and cereals, and the last two ones are made of milk. Shalgam juice, hardaliye and ayran are produced by lactic acid fermentation. Their microbiota is mainly composed of lactic acid bacteria (LAB). Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus paracasei subsp. paracasei in shalgam fermentation and L. paracasei subsp. paracasei and Lactobacillus casei subsp. pseudoplantarum in hardaliye fermentation are predominant. Ayran is traditionally prepared by mixing yoghurt with water and salt. Yoghurt starter cultures are used in industrial ayran production. On the other hand, both alcohol and lactic acid fermentation occur in boza and kefir. Boza is prepared by using a mixture of maize, wheat and rice or their flours and water. Generally previously produced boza or sourdough/yoghurt are used as starter culture which is rich in Lactobacillus spp. and yeasts. Kefir is prepared by inoculation of raw milk with kefir grains which consists of different species of yeasts, LAB, acetic acid bacteria in a protein and polysaccharide matrix. The microbiota of boza and kefir is affected from raw materials, the origin and the production methods.     

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.     

重组开菲尔菌粒发酵性能及菌相组成研究

为了简化开菲尔混菌发酵体系的菌种复杂程度,并保留开菲尔特有的发酵风味,对来源于拉萨、成都、乌鲁木齐、西宁4个地区的开菲尔菌粒进行了重组培养,得到重组菌粒TK-ZJUJ06。高通量测序分析检测到其细菌菌相组成为8个属,分别为乳杆菌属(39.46%)、明串珠菌属(0.62%)、乳球菌属(43.28%)、链球菌属(0.54%)、醋酸菌属(11.53%)、希瓦氏菌属(0.67%)、不动杆菌属(0.05%)以及假单胞菌属(0.14%);生理生化培养方法及26S rDNA序列分析测得其酵母菌相为酿酒酵母(Saccharomyces cerevisiae)。发酵性能及挥发性成分分析结果表明,该重组菌粒发酵性能优良,酸度117 °T,挥发性脂肪酸含量65%。与对照相比,菌相组成简单,并能够保留开菲尔特有的发酵风味。     

A modelling study on kefir grain biomass growth: Influence of various minerals

In this study, the effect of initial kefir grain concentration and various minerals (MgO, MgSO₄.7H₂O, ZnSO₄.7H₂O, CaSO₄.1/2H₂O and Fe₂O₃) on the propagation of kefir grains and the pH evolution of kefir were investigated. The activated kefir grains were inoculated into very low fat (0.1%) cows’ milk under constant temperature (25°C) and impeller speed (100 rpm) for a propagation time of 24 h. Several mathematical models proposed to predict the pH value and kefir grain biomass increase vs propagation time were evaluated by the experimental results for all mineral additions; the Richards equation was found to be the most appropriate one. After evaluation of the experimental data, the most influential mineral addition providing 46.3% increase in kefir grain biomass was found to be MgO with a concentration of 0.30 g Mg²⁺/L. At this optimal condition, a model was derived for the kefir grain biomass concentration representing pH dependence.     

Comparative analyses of microbial community diversities of Tibetan kefir grains from three geographic regions

Tibetan kefir grains are the traditional starter for fermented milk products in Tibet, China. To gain a deeper understanding of their bacterial and fungal community diversities, we compared kefir grains from three different regions, Nyingchi, Lhasa and Yangbajing, using high‐throughput sequencing. The results of sequence analysis grouped the reads into four bacterial and four fungal phyla, comprising 34 bacterial and 139 fungal genera, and 44 bacterial and 195 fungal species. Firmicutes and Proteobacteria were two dominant bacterial phyla, and Lactobacillus and Acetobacter were the dominant bacterial genera. Basidiomycota and Ascomycota were the dominant fungal phyla. There was significant difference in the Basidiomycota and Ascomycota among three regions (P < 0.05). A significant difference in genera was observed in the percentage of Saccharomyces, Trichosporon, Aspergillus and Candida between the samples from Nyingchi, Lhasa and Yangbajing (P < 0.05). Based on principal component analysis (PCA), the Tibetan kefir grain samples from Nyingchi could be distinctly differentiated from the other two regions in term of fungal community, but no obvious difference existed in the bacterial population of samples from the three regions.     

Characterisation of the exopolysaccharide kefiran produced by lactic acid bacteria entrapped within natural kefir grains

The main goal of this work was to characterise and quantify the exopolysaccharide kefiran and to discover an effective procedure for its isolation from kefir grains, originating from the Caucasian Mountains. Capillary electrophoresis was used for the characterisation and quantification of the d ‐glucose and d ‐galactose in our samples at a mass ratio of 1:0.7. The effect of fermentation time on growth of kefir grains and the content of kefiran within the grains were determined. The pH profiles were monitored dynamically. In addition, the influence of fermentation temperature on kefir grains mass concentration (γ_KG) and the content of kefiran within the grains (w_KEF/KG) were studied. The highest values for both were obtained at 30 °C.     

Inferring the role of microorganisms in water kefir fermentations

Water kefir is a slightly alcoholic, lactic and acetic beverage fermented by yeasts, lactic acid bacteria and acetic acid bacteria that are associated with the polysaccharide of the water kefir grains. In this study, the three main metabolic products of microorganisms were evaluated during a traditional 192‐h water kefir fermentation and also after inoculating the microorganisms in fresh medium or sterilised broth from different fermentation stages. The first process to occur was alcoholic fermentation, carried out in particular by Saccharomyces cerevisiae. After 24 h, lactic and acetic acid accumulation was generated by Lactobacillus hilgardii and Acetobacter tropicalis. By the end of fermentation, ethanol had been almost entirely consumed and oxidised to acetic acid, possibly by a dissimilatory route of Acetobacter species. An original hypothetical diagram is proposed for the carbon flux from sucrose, and the metabolic role of the main yeasts and bacteria is assigned for the distinct stages of water kefir fermentation.     

Chemical and microbiological characterisation of kefir grains

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.     

Populations and potential association of Saccharomyces cerevisiae with lactic acid bacteria in naturally fermented Korean rice wine

The microbial populations focused on predominant yeast species and lactic acid bacteria (LAB) in 15 commercial makgeolli brands, where a fungal starter nuruk was used were examined. Viable yeast counts were obtained on yeast potato dextrose (YPD) and MRS agar containing sodium azide. MRS-C (0.1% cycloheximide supplemented) was used for selective counts of LAB. Saccharomyces cerevisiae was found to be predominant in the 15 samples tested, with an average count of 4.6×10⁷ CFU/mL. Contrary to the earlier studies, Lactobacillus plantarum and Weissella cibaria were shown as predominant LAB species with an average count of 1.7×10⁷ CFU/mL. Surprisingly, as many as 7 log viable cells/mL were present at the ethyl alcohol concentration of 6–7%. The data from real-time PCR also indicated that the yeast populations remains almost constant during the refrigerated storage of 12 days, while that of LAB decrease slightly first 9 days and increase after then, despite the overall increase in acidity. Data from the differential microbial counts suggest that yeast S. cerevisiae might be associated with 2 LAB species, L. plantarum and W. cibaria, under ethyl alcohol stress during the turbid rice wine fermentation.     

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.