Isolation and characterization of the microbial population of different South African kefir grains

Kefir, a slightly acidic fermented milk, is produced by adding lactic acid bacteria and yeasts, in the form of grains, to milk. The bacteria and yeasts present in the kefir grains are known to vary widely. Selective growth media and morphological and biochemical characteristics were used for the isolation and identification of the microbes present in the grains from eight different sources in South Africa. The kefir grains were activated in milk for only 24 h to prevent any changes in the microbial population of the grains. The microbial numbers varied between 6.4 × 10⁴ and 8.5 × 10 ⁸  cfu/g on the media selective for the bacterial species and between 1.5 × 10 ⁵ and 3.7 × 10 ⁸   cfu/g on the media selective for the yeast species. The bacterial genera that were identified included Lactobacillus , Leuconostoc and Lactococcus and the yeast genera included Zygosaccharomyces , Candida and Saccharomyces . The distribution frequencies of the microbes in the different grains were determined and most of the grains were dominated by two microbial species. No pediococci, acetic acid bacteria or propionibacteria were detected.     

Restoration of kefir grains subjected to different treatments

The aim of the study was to find a way to recover the quality of kefir grains that had been subjected to the following treatments: homogenisation, rinsing the grains with water, freeze‐drying and milling, freezing in liquid nitrogen and then frozen storage, and cool storage. The grains were studied in respect of their later replication in milk, their size and their microbiota composition. The daily transfer of treated kefir grains, except freeze‐dried ones, into fresh milk was effective in respect of the recovery of their growth dynamics, size and microbiota balance. The growth dynamics of grains in milk seems to be a very good indicator of their vital and technological functions.     

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.     

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.     

Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain

The physicochemical and gelling properties of kefiran, a water-soluble glucogalactan with probed health-promoting properties, were investigated. Gel permeation chromatograms revealed a single distribution of molecular weight corresponding to 10⁷ Da. Intrinsic viscosity of kefiran determined using Huggins extrapolations was 6.0 dl/g and using Kramer approximations was 5.95 dl/g.Kefiran has a Newtonian behaviour in diluted solutions, which becomes pseudoplastic at higher concentrations. Rheological behaviour of the solution before and after freeze drying was evaluated by small deformation oscillatory rheological measurements. The mechanical spectrum of solution corresponded to an entangled network behaviour. After freeze–thaw treatment of the solution, a rheological behaviour transition from a liquid-like system to a gel was observed. The storage modulus (G′) in cryogels was 35 times higher than the value obtained for the solution. Rheological characteristics of the cryogel were influenced by kefiran concentration. As the polymer concentration increased, higher number of interactions was evident for the increment in both moduli (G′ and G″). The behaviour of kefiran cryogels about 37 °C determines its ability to melt at mouth temperature. These results suggest that kefiran cryogels could be an interesting alternative for its application in food formulations.     

Effect of storage parameters on freeze‐dried kefir grains

The purpose of this study was to use freeze‐drying to preserve microbial activity while extending the shelf life of kefir grains and to determine the best storage temperature. Freeze‐dried kefir grains were lyophilised and were later stored in a multilayer plastic film with a moisture barrier for 90 days at varying temperatures. Microbial activity continued until the 60^th day of storage at 4 °C. PCR analysis was performed to determine Lactobacillus kefiranofaciens as an indicator kefir micro‐organism. It was concluded that the conservation of kefir grains by freeze‐drying protects the natural embedded microbiota; therefore, both the shelf life of kefir grains and the consumption of natural kefir increase.     

Cheese whey fermented with kefir micro‐organisms: Antagonism against Salmonella and immunomodulatory capacity

The antagonistic effect against Salmonella spp. and the immunomodulatory capacity of whey fermented with kefir grains and with three strains isolated from them – Lactobacillus plantarum CIDCA 8327, Lactobacillus kefiri CIDCA 8348 and Kluyveromyces marxianus var. marxianus CIDCA 8154 – were evaluated. Both fermented products interfered with the capacity of Salmonella spp. to associate and invade Caco‐2/TC7 cells and reduced up to the basal level the expression of the CCL20 in response to a pro‐inflammatory stimulus on Caco‐2 CCL20:luc cells. The products with potential application as probiotics obtained by fermentation of whey with kefir micro‐organisms represent an alternative to increase whey economic value.     

Distribution of microorganisms with particular reference to encapsulated bacteria in kefir grains

Propagable and non-propagable kefir grains in a form resembling cauliflower florets were observed by scanning electron microscopy (SEM) and light microscopy. In propagable grains short and long rod-shaped bacteria and yeasts formed separate colonies on the outside surface and inside. Internally, filaments which derived from capsules around the cells extended radially from a population of long rod-shaped bacteria. In non-propagable grains long rod-shaped bacteria with filamentous appendages were not observed, but only short rod-shaped bacteria and yeasts. Indian ink preparations showed presence of encapsulated bacteria in propagable grains and absence of these in non-propagable ones. The above results suggest that encapsulated bacteria are responsible for propagation of kefir 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.     

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.     

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.     

Films based on kefiran,an exopolysaccharide obtained from kefir grain: Development and characterization

Kefiran, an exopolysaccharide produced by microorganisms present in the kefir grains, is a glucogalactan that has several health promoting properties. In the present work, the ability of kefiran to form films and the effect of glycerol addition at different concentrations on film properties was evaluated. Kefiran was able to form films at concentrations ranging from 5 to 10 g/kg. The concentration 10 g/kg was selected because the films were easily removed from the plate. All film-forming solutions exhibited a pseudoplastic behavior; glycerol addition did not modify the solution rheological properties.     

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem.     

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.     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25 份标示含双歧杆菌样品进行检测。结果表明：该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到104 CFU/mL。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25 份市售实际样品进行测试,有5 份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

浓香型纯粮白酒鉴别方法的研究

根据纯粮白酒在碱性加热条件下在波长363 nm处存在吸光度值差异的原理,可以通过纯粮白酒标准曲线来确定该酒样中纯粮白酒的比例。通过盲样酒的实际检测比对,4个样品的准确度均＞90%,相对标准偏差RSD均＜1%,证实了碱性实验在鉴别纯粮白酒及其在酒精勾兑白酒中的比例含量上的可行性。     

Identification of Saccharomyces cerevisiae in bakery products by PCR amplification of the ITS region of ribosomal DNA

 A non-conventional sensitive and specific method for the detection of Saccharomyces cerevisiae in food is proposed. Polymerase chain reaction (PCR) led to the identification of this yeast in some commercial bakery products. The comparative use of two methods of clean-up of DNA from Triticum spp., S. cerevisiae and some foods shows an improvement in extraction yield for the phenol-free method. Analysis of some sequences of ribosomal genes shows different amplified products for Triticum and S. cerevisiae. Internal transcribed spacer amplification (ITS₁/ITS₄) produced a single band of about 650 and 800 bp for Triticum and S. cerevisiae, respectively. ITS₁/ITS₂ universal fungal primers gave a single band of about 300 and 450 bp for Triticum and S. cerevisiae, respectively. Both amplification products are able to distinguish between the yeast and the DNA from T. aestivum and T. durum, showing polymorphism in the ITS₁ region. Amplification on two primers designed using the sequence of the ITS₁ region of S. cerevisiae rDNA (SC₁/SC₂) led to a single band of 300 bp, species-specific for the yeast. These primers enabled an unambiguous distinction between fermented foods containing S. cerevisiae and those leavened by baking powders to be made. We suggest that this PCR assay could be used for quality control and for the trace detection of S. cerevisiae as a potential food allergen. Received: 7 January 1999     

Viability of microencapsulated Bifidobacterium animalis ssp. lactis BB12 in kefir during refrigerated storage

The viability of free and immobilized cells of Bifidobacterium animalis ssp. lactis BB12 incorporated into kefir was studied for 28 days during refrigerated storage. The immobilized bifidobacteria were added directly to previously prepared kefir. Titratable acidity, pH, ethanol, fat, protein and lactose were evaluated in the kefir with bifidobacteria after the storage. The survival of the free and microencapsulated bifidobacteria was evaluated during the storage period and in simulated gastric juice. The pH of kefirs ranged from 4.3 to 4.6. Encapsulation improved significantly the survival of bifidobacteria during exposure to nisin, during the storage period and in simulated gastric juice.     

双歧绿豆乳果冻的研制

实验以绿豆、魔芋粉、卡拉胶、脱脂奶粉为主要原料,采用正交试验方法探讨双歧绿豆乳果冻的最佳配方。结果表明:双歧绿豆乳果冻的最佳配方为:奶粉质量分数为2%,凝胶剂质量分数为0.8%(魔芋粉与卡拉胶两者的配比为7∶3),总糖质量分数为14%,柠檬酸质量分数为0.12%。利用该配方生产的双歧绿豆乳果冻,具有双歧乳独有的功效和香味,口感爽滑,酸甜适度,富有弹性,冻体完整,是一种新型发酵型天然营养果冻。