Microbial Species Diversity,Community Dynamics,and Metabolite Kinetics of Water Kefir Fermentation

Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.     

The microbial flora of sugary kefir grain (the gingerbeer plant): biosynthesis of the grain fromLactobacillus hilgardii producing a polysaccharide gel

Summary The microflora of sugary kefir grains was principally mesophilic and consisted chiefly of lactic acid bacteria [Lactobacillus casei, Lactobacillus hilgardii (=brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis] and a small proportion of yeasts (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica andC. valida). Few coliforms and faecal streptococci were observed. Observation by scanning electron microscopy revealed that the filamentous yeasts adhered to the bacteria on the periphery of the grain.Lactobacillus hilgardii, the single microorganism isolated which was able to produce a gelling polysaccharide, was important in the biosynthesis of the grain. Pieces of gel produced by this strain, and transferred in a yeast extract-sucrose solution, grew and resembled the household kefir grains. This represents a new, cheap way of producing immobilized cells by self-embedding in a neutral polysaccharide.

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Flore microbienne du grain de kefir sucré (plant de la bière de gingembre): biosynthèse du grain par Lactobacillus hilgardii produisant un gel de polysaccharide

Résumé La microflore des grains de kefir sucré est essentiellement mésophile et comprend principalement des bactéries lactiquesLactobacillus casei, Lactobacillus hilgardii (= brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis ainsi qu'une petite proportion de levures (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica etC. valida). On a observé peu de coliformes et de streptocoques fécaux. L'observation au microscope électronique à balayage a révélé que les levures filamenteuses adhèrent aux bactéries sur la périphérie du grain.Lactobacillus hilgardii, le seul microorganisme isolé susceptible de produire un polysaccharide gélifiant, est important dans la biosynthèse du grain. Des morceaux de gel produit par cette souche et transférés dans une solution d'extrait de levure et de saccharose, croissent et ressemblent aux grains de kefir domestique. Ceci représente une manière nouvelle, peu coûteuse de produire des cellules immobilisées par auto-piégeage dans un polysaccharide neutre.

     

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     

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.     

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir

The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24 h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80 mg/ml, and that of acetic acid increased from 0.08 to 1.12 mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24 mg/ml.     

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.     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

Identification and probiotic properties of lactobacilli isolated from two different fermented beverages

Purpose

Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals.

Methods

Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential.

Results

Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract.

Conclusion

Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

     

Detection of interactions between yeasts and lactic acid bacteria isolated from sugary kefir grains

Different techniques were tested for studying the synergism between the micro-organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter-relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.     

Growth performances ofLactobacillus hilgardii immobilized in dextran gel and in continuous fermentation

A variant ofLactobacillus hilgardii was immobilized by its own production of dextran gel, forming grains. The best rate of weight increase of the gel in continuous fermentation was 16.3±3.3%/h, at pH 4.8±0.1 and with a dilution rate of 0.22 to 0.26/h. Observation by scanning electron microscopy located most of the bacteria as microcolonies on the surface. A similar arrangement appeared in calcium alginate beads. The best population density (10¹⁰ cells/g) was obtained in grains at pH 5.8, after 30h. At a similar pH value, 4.8, the growth rate was higher in alginate beads than in dextran gel but the final population density was approximately the same. Acidification rate increased faster with mixed gel at pH 5.2 than with dextran at pH 5.8.     

Interaction between <Emphasis Type="Italic">Oenococcus oeni</Emphasis> and <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> isolated from wine. Modification of available nitrogen and biogenic amine production

During the mixed culture of Lactobacillus hilgardii 5w, a common spoilage wine bacteria and Oenococcus oeni X₂L, an amensalistic growth response of the malolactic bacteria was produced due to a competition for nitrogenous nutrients, mainly peptides. Arginine was fully consumed and peptide concentration diminished 60% with respect to both pure cultures at the end of exponential growth. Histamine release increased 34% with respect to L. hilgardii single culture. Under the poor nutritional conditions present during winemaking, L. hilgardii could increase histamine production and adversely affect malolactic fermentation conducted by O. oeni and hence the quality of the final product.     

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry

Aims: The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid‐state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation. Methods and Results: Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power–time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD–time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long‐run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O₂ protective effect of rye sourdough. Conclusions: Measurements of the power–time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. Significance and Impact of the Study: Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.     

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.     

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.     

Functional Compounds in Fermented Buckwheat Sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48–54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei ). The two variants of a heterofermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4–8 and 30°C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81–8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40°C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.     

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.     

Milk Kefir: Ultrastructure,Antimicrobial Activity and Efficacy on Aflatoxin B1 Production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

The association of kefir microbiota was observed by electron microscopic examination. Scanning electron microscopic (SEM) observations revealed that kefir grain surface is very rough and the inner portions had scattered irregular holes on its surface. The interior of the grain comprised fibrillar materials which were interpreted as protein, lipid and a soluble polysaccharide, the kefiran complex that surrounds yeast and bacteria in the grain. Yeast was observed more clearly than bacteria on the outer portion of the grain. Transmission electron microscopic (TEM) observations of kefir revealed that the grain comprised a mixed culture of yeast and bacteria growing in close association with each other. Microbiota is dominated by budded and long-flattened yeast cells growing together with lactobacilli and lactococci bacteria. Bacterial cells with rounded ends were also observed in this mixed culture. Kefir grains, kefir suspensions, and kefiran were tested for antimicrobial activities against several bacterial and fungal species. The highest activity was obtained against Streptococcus faecalis KR6 and Fusarium graminearum CZ1. Growth of Aspergillus flavus AH3 producing for aflatoxin B1 for 10 days in broth medium supplemented with varying concentrations of kefir filtrate (%, v/v) showed that sporulation was completely inhibited at the higher concentrations of kefir filtrate (7–10%, v/v). The average values of both mycelial dry weights and aflatoxin B1 were completely inhibited at 10% (v/v). This is the first in vitro study about the antifungal characteristics of kefir against filamentous fungi which was manifested by applying its inhibitory effect on the productivity of aflatoxin B1 by A. flavus AH3.     

Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48-54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei). The two variants of a hetero-fermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4.8 and 30 degrees C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81.8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40 degrees C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.     

Microbiology of sayur asin fermentation

Summary Sayur asin is a fermented mustard cabbage product of Indonesia. The cabbage is fermented naturally in the presence of brined water taken from boiled rice. Fermentation was characterized by a sequential growth of the lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus confusus, Lactobacillus curvatus, Pediococcus pentosaceus, and Lactobacillus plantarum. Starch degrading species of Bacillus, Staphylococcus and Corynebacterium exhibited limited growth during the first day of fermentation. The yeasts, Candida sake and Candida guilliermondii contributed to the fermentation. Lactic acid, acetic acid, succinic acid, ethanol and glycerol were products of fermentation. Glucose, generated by the degradation of rice starch and maltose, was metabolized by the species that grew.