Use of isolated kefir starter cultures in kefir production

Kefir is a beverage produced by lactic-alcoholic fermentation of milk using kefir grain. For the first time in Iran, the microbial flora of kefir grain was isolated and identified (Motaghi et al. 1997). In this paper various ratios of starter cultures of kefir grains were investigated. Various ratios of lactic acid bacteria, yeasts and acetic acid bacteria were tested and the quality (colour, smell, flavour, acidity, effervescence and viscosity) of the product was assessed. At constant incubation time and temperature (24 h, 25 °C using homogenised milk with 2.5% fat), samples with various ratios of starter culture (3–5% w/v) were examined and analysed for protein, fat, sugar, alcohol, carbon dioxide, acidity, density, and riboflavin content. Samples produced with 3% (v/v) bacterial mixed culture and 2% (v/v) yeast (K3 procedure) culture were considered as best with respect to quality and organoleptic quality. The comparison of the results with the organoleptic tests of previous studies showed that the kefir produced with kefir grain is more desirable as compared with kefir produced with starter cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain

Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior.     

Lactic acid bacteria and yeasts in kefir grains and kefir made from them

In an investigation of the changes in the microflora along the pathway: kefir grains (A)→kefir made from kefir grains (B)→kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83–90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and Lactobacillus brevis. Yeasts (10–17%) identified were Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52–65% and 79–86%, respectively) predominated. Within the group of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus (70–87% of the isolated bacilli) predominated. Along the pathway A→B→C, the streptococcal proportion in the total kefir microflora increased by 26–30% whereas the lactobacilli decreased by 13–23%. K. marxianus var. lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 1–6 DOI: 10.1038/sj/jim/7000186 Received 02 August 2000/ Accepted in revised form 15 July 2001     

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it''s necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.     

Evaluation of freeze-dried kefir coculture as starter in feta-type cheese production

The use of freeze-dried kefir coculture as a starter in the production of feta-type cheese was investigated. Maturation of the produced cheese at 4 degrees C was monitored for up to 70 days, and the effects of the starter culture, the salting method, and the ripening process on quality characteristics were studied. The use of kefir coculture as a starter led to increased lactic acid concentrations and decreased pH values in the final product associated with significantly higher conversion rates compared to salted rennet cheese. Determination of bacterial diversity at the end of the ripening process in salted kefir and rennet cheeses by denaturing gradient gel electrophoresis technology, based on both DNA and RNA analyses, suggested a potential species-specific inhibition of members of the genera Staphylococcus and Psychrobacter by kefir coculture. The main active microbial associations in salted kefir cheese appeared to be members of the genera Pseudomonas and Lactococcus, while in salted rennet cheese, Oxalobacteraceae, Janthinobacterium, Psychrobacter, and Pseudomonas species were noted. The effect of the starter culture on the production of aroma-related compounds responsible for cheese flavor was also studied by the solid-phase microextraction-gas chromatography-mass spectrometry technique. Kefir coculture also appeared to extend the shelf life of unsalted cheese. Spoilage of kefir cheese was observed on the 9th and 20th days of preservation at 10 and 5 degrees C, respectively, while spoilage in the corresponding rennet cheese was detected on the 7th and 16th days. Microbial counts during preservation of both types of unsalted cheese increased steadily and reached similar levels, with the exception of staphylococci, which were significantly lower in unsalted kefir cheese. All types of cheese produced with kefir as a starter were approved and accepted by the panel during the preliminary sensory evaluation compared to commercial feta-type cheese.     

Water kefir: Factors affecting grain growth and health-promoting properties of the fermented beverage

Nowadays, the interest in the consumption of healthy foods has increased as well as the homemade preparation of artisanal fermented product. Water kefir is an ancient drink of uncertain origin, which has been passed down from generation to generation and is currently consumed practically all over the world. Considering the recent and extensive updates published on sugary kefir, this work aims to shed light on the scientific works that have been published so far in relation to this complex ecosystem. We focused our review evaluating the factors that affect the beverage microbial and chemical composition that are responsible for the health attribute of water kefir as well as the grain growth. The microbial ecosystem that constitutes the grains and the fermented consumed beverage can vary according to the fermentation conditions (time and temperature) and especially with the use of different substrates (source of sugars, additives as fruits and molasses). In this sense, the populations of microorganisms in the beverage as well as the metabolites that they produce varies and in consequence their health properties. Otherwise, the knowledge of the variables affecting grain growth are also discussed for its relevance in maintenance of the starter biomass as well as the use of dextran for technological application.     

Profile of microbial communities present in tibico (sugary kefir) grains from different Brazilian States

The microorganisms associated with Brazilian tibico (sugary kefir) grains from eight different Brazilian States were investigated using a combination of culture-dependent and culture-independent methods. The bacterial genera included Lactobacilllus, Acetobacter, Gluconobacter, Bacillus and yeast genera included Pichia, Saccharomyces, Kazachstania, Candida, Zygosaccharomyces and Yarrowia. Some bacteria and yeast detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at some Brazilian tibico grains by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria’s Gluconobacter liquefaciens and Bacillus cereus and the yeast Pichia cecembensis, Pichia caribbica and Zygosaccharomyces fermentati are described for the first time in tibico grains. Our findings are relevant to the knowledge of tibico grains used as starter culture for fermented beverages consumed by the Brazilian population.     

Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production

In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.     

Investigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

     

A note on the heterofermentative Lactobacillus isolated from kefir grains

M arshall , V.M., C ole , W.M. & F arrow , J.A.E. 1984. A note on the heterofermentative Lactobacillus isolated from kefir grains. Journal of Applied Bacteriology 56 , 503–505.
Heterofermentative lactobacilli have been isolated from kefir grains obtained from four different sources. A number of these isolates ferment only L-arabinose and gluconate and are similar to the species 'Lactobacillus desidiosus' . The DNA of these isolates, however, have 85–109% homology with 'L. caucasicus' NCDO 190 which is now regarded as L. kefir . The relationship between these strains is discussed.     

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.
Methods and Results:  Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk ( D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica . The three micro-organisms studied varied considerably in their ability to survive to spray drying processes . Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased.
Conclusions:  Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures
Significance and Impact of Study:  It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.     

Whey-cheese production using freeze-dried kefir culture as a starter

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.     

A note on the heterofermentative Lactobacillus isolated from kefir grains

Heterofermentative lactobacilli have been isolated from kefir grains obtained from four different sources. A number of these isolates ferment only L-arabinose and gluconate and are similar to the species 'Lactobacillus desidiosus'. The DNA of these isolates, however, have 85-109% homology with 'L. caucasicus' NCDO 190 which is now regarded as L. kefir. The relationship between these strains is discussed.     

Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities

Kefir is a fermented milk beverage. The milk fermentation is achieved by the of kefir grains, a cluster of microorganisms held together by a polysaccharide matrix named kefiran. Kefir grains are an example of symbiosis between yeast and bacteria. They have been used over years to produce kefir, a fermented beverage that is consumed all over the world, although its origin is Caucasian. A vast variety of different species of organisms forming the kefir grains, comprising yeast and bacteria, have been isolated and identified. Kefir is a probiotic food. Probiotics have shown to be beneficial to health, being presently of great interest to the food industry. Kefir has been accredited with antibacterial, antifungal and antitumoural activities among other beneficial attributes. This review includes a critical revision of the microbiological composition of kefir along with its beneficial properties to human health.     

Taxonomical classification of yeasts isolated from kefir based on the sequence of their ribosomal RNA genes

Yeast strains present in 10 samples of kefir of different commercial and domestic origins have been isolated and classified taxonomically on the basis of the internal transcribed sequences (ITS) of their ribosomal RNA genes. A total of 18 yeast strains representing 10 different species have been characterized. Of the three commercial kefir samples analyed, two contained the well characterized yeast Kluyveromyces lactis while no yeast was found in the other one. A broader spectrum of yeast species was found among the home-made kefir samples, of which Issatchenkia orientalis, Saccharomyces unisporus, Saccharomyces exiguus and Saccharomyces humaticus were the most representative species.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3 in different yogurt and kefir combinations as prefermentation contaminant

AIMS: To compare microbiological safety of yogurt, kefir and different combinations of yogurt and kefir samples by using three foodborne pathogenic strains (Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3) as indicators. METHODS AND RESULTS: Fresh yogurt and kefir drinks were added to pasteurized milk at a 5% rate either separately or together, and then incubated at different temperatures (43 degrees C for yogurt and 30 degrees C for kefir), depending on appropriate growth temperature of their starter microflora. While traditional yogurt was found to be the least suppressive on the three pathogenic micro-organisms, samples obtained from two subsequent fermentation process (samples fermented at 43 degrees C for 3 h and at 30 degrees C for 21 h) were more suppressive than that of traditional kefir. There was no significant survival difference between E. coli O157:H7 and L. monocytogenes 4b in samples tested (P > 0.05), but Y. enterocolitica O3 was more susceptible than other two test strains (P < 0.05). CONCLUSIONS: The microbiological safety of the dairy product fermented at two consecutive periods was superior than that of traditional yogurt or kefir alone. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when yogurt and kefir starter micro-organisms are combined in a milk fermentation process with different time and temperature periods.     

Yeasts from kefir grains: isolation, identification, and probiotic characterization

Kefir—a traditional beverage whose consumption has been associated with health benefits—is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment–length polymorphisms). We identified 4 species belonging to 3 genera—Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)—and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Kefir micro-organisms: their role in grain assembly and health properties of fermented milk

Kefir is a homemade viscous and slightly effervescent beverage obtained by milk fermentation with kefir grains, which are built up by a complex community of lactic acid and acetic acid bacteria and yeasts confined in a matrix of proteins and polysaccharides. The present review summarizes the role of kefir micro-organisms in grain assembly and in the beneficial properties attributed to kefir. The use of both culture-dependent and independent methods has made possible to determine the micro-organisms that constitute this ecosystem. Kefir consumption has been associated with a wide range of functional and probiotic properties that could be attributed to the micro-organisms present in kefir and/or to the metabolites synthetized by them during milk fermentation. In this context, the role of micro-organisms in kefir health promoting properties is discussed with particular attention to the contribution of yeast as well as bioactive metabolites such as lactic and acetic acid, exopolysaccharides and bioactive peptides. Even though many advances on the knowledge of this ancient fermented milk have been made, further studies are necessary to elucidate the complex nature of the kefir ecosystem.