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.     

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.     

Characterization of interactions between Lactobacillus hilgardii and Saccharomyces florentinus isolated from sugary kefir grains

Stimulation of the growth of Lactobacillus hilgardii and production of lactic acid in the presence of Saccharomyces florentinus have already been observed in a poor glucose medium. In this study we showed that CO₂, pyruvate, propionate, acetate and succinate excreted by the yeast were responsible for this phenomenon, whereas ethanol, fumarate and a cocktail of vitamins had no effect. Fermentation by the yeast did not enrich the total nitrogen or free amino acid content of the medium. The use of proteases confirmed that it was improbable that any nitrogenous compound could be responsible for the bacterial activation. Between values of 0.1 and 5%, the initial yeast: bacteria ratio had no effect on the stimulation of Lact. hilgardii, even though yeast fermentation was drastically affected by a low initial value. The interactions differed greatly according to the sugar source. With monosaccharides, stimulation began after 48 h of culture, and was higher on glucose than on fructose. With sucrose, stimulation appeared at the beginning of fermentation. Different mechanisms seemed to be involved.     

Characterization of the polysaccharides from aLactobacillus brevis and from sugary kefir grains

Summary The gel-forming polysaccharide of the sugary kefir grains (11.5% of dry matter) or one taken from aLactobacillus brevis culture were identified as dextrans with some 1)-Gp-(3 links in the main chain, with a ratio (branched/total units) of 0.19 and 0.14 respectively, instead of 0.07 for the non-gelling polysaccharide.     

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.     

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.     

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.     

Influence of pH, temperature and initial yeast: bacteria ratio on the stimulation of Lactobacillus hilgardii by Saccharomyces florentinus isolated from sugary kefir grains

The effects of pH, temperature and initial yeast: bacteria ratio on Lactobacillus hilgardii and Saccharomyces florentinus cultivated either in pure or mixed culture were studied. Quadratic polynomial as a function of factors was proposed to express the lactic acid production at different sampling times, and the percentage increase in lactic acid production by Lact. hilgardii in mixed culture compared with pure culture. Temperature was the factor which had the main influence on lactic acid production in mixed culture, whereas stimulation of bacteria depended greatly on pH value. In the range 0·1-20%, the initial yeast: bacteria ratio had no effect on these responses, but presence of the yeast was absolutely necessary to obtain high production of lactic acid. Optimum culture conditions were determined to maximize these characteristics.     

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.     

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     

Properties of β-galactosidase of Lactobacillus kefiranofaciens K-1 isolated from kefir grains

β-Galactosidase from Lactobacillus kefiranofaciens K-1 was isolated and characterized. Optimal temperature and pH for the enzyme reaction were 50^°C and pH 6.5, respectively. Molecular weight was estimated to be approximately 311000. Glucose and galactose inhibited the activity, but the inhibition by galactose was rather weaker than observed in other β-galactosidases. MnCl₂ and MgCl₂ had no effect on the activity. FeSO₄, AgNO₃ and HgCl₂ acted as the inhibitor. β-Mercaptoethanol and L-cysteine activated the enzyme, while iodoacetamide inhibited the activity. The K _m values were 4.92 mmol/1 for ONPG and 1.27 mmol/1 for lactose.     

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.     

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.     

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.

     

多项分类方法鉴定西藏地区藏灵菇中的乳酸球菌

【目的】采用多项分类法对16株分离自藏灵菇中的乳酸球菌进行准确鉴定。【方法】首先应用传统的生理生化试验,之后采用16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)进行了鉴定,最后,通过16S rRNA基因序列分析进行验证。【结果】将16株菌株初步鉴定为3个菌群:片球菌群、乳球菌群和肠球菌群,进一步鉴定为14株耐久肠球菌,1株乳酸片球菌,1株乳酸乳球菌乳酸亚种,16S rRNA基因序列分析验证的结果与前3种试验方法的结果相一致。【结论】试验结果表明传统的生理生化鉴定和16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)相结合的多项分类方法有利于乳酸球菌种间的准确鉴定。     

Lactobacilli isolated from the stomach of conventional mice.

Twenty strains of lactobacilli isolated from the stomach of conventional mice were tested for their ability to ferment or hydrolyze substrates that may be present in the stomach habitat. The lactobacilli could be placed in four groups (A to D) depending on their ability to ferment N-acetylglucosamine, dextrin, cellobiose, gum arabic, and xylan. The majority of the isolates belonged to groups A and D. Group A strains did not resemble previously described Lactobacillus species, but group D strains were identified as L. leichmannii. A representative group A isolate colonized the surface of the nonsecretory epithelium of the stomach of gnotobiotic mice; a group D isolate did not.     

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.     

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.     

Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage

Aims:  To investigate Klebsiella oxytoca strain BAS-10 growth on ferric citrate under anaerobic conditions for exopolysaccharide (EPS) production and localization on cell followed by the purification and the EPS determination of the iron-binding stability constant to EPS or biotechnological applications.
Methods and Results:  Klebsiella oxytoca ferments ferric citrate under anaerobic conditions and produces a ferric hydrogel, whereas ferrous ions were formed in solution. During growth, cells precipitate and a hydrogel formation was observed: the organic material was constituted of an EPS bound to Fe(III) ions, this was found by chemical analyses of the iron species and transmission electron microscopy of the cell cultures. Iron binding to EPS was studied by cyclic voltammetric measurements, either directly on the hydrogel or in an aqueous solutions containing Fe(III)-citrate and purified Fe(III)-EPS. From the voltammetric data, the stability constant for the Fe(III)-EPS complex can be assumed to have values of approx. 10¹²–10¹³. It was estimated that this is higher than for the Fe(III)-citrate complex.
Conclusions:  The production of Fe(III)-EPS under anaerobic conditions is a strategy for the strain to survive in mine drainages and other acidic conditions. This physiological feature can be used to produce large amounts of valuable Fe(III)-EPS, starting from a low cost substrate such as Fe(III)-citrate.
Significant and Impact of the Study:  The data herein demonstrates that an interesting metal-binding molecule can be produced as a novel catalyst for a variety of potential applications and the EPS itself is a valuable source for rhamnose purification.