Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.

[1]  Ying Huang,et al.  Characterization of Lactobacillus plantarum Lp27 isolated from Tibetan kefir grains: a potential probiotic bacterium with cholesterol-lowering effects. , 2013, Journal of dairy science.

[2]  Xia Chen,et al.  Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet. , 2012, Journal of dairy science.

[3]  P. Horvatovich,et al.  Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance , 2011, BMC Microbiology.

[4]  P. Deedwania,et al.  Lipid-lowering therapy with statins for the primary and secondary prevention of cardiovascular disease. , 2011, Cardiology clinics.

[5]  R. Kumar,et al.  Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague–Dawley rats , 2010, British Journal of Nutrition.

[6]  Heping Zhang,et al.  Screening of potential probiotic properties of Lactobacillus fermentum isolated from traditional dairy products , 2010 .

[7]  P. Bork,et al.  A human gut microbial gene catalogue established by metagenomic sequencing , 2010, Nature.

[8]  Mingsheng Dong,et al.  Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. , 2009, Food microbiology.

[9]  M. Zago,et al.  A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters. , 2009, International journal of food microbiology.

[10]  Á. Lima,et al.  Antimicrobial Activity of Broth Fermented with Kefir Grains , 2009, Applied biochemistry and biotechnology.

[11]  W. Holzapfel,et al.  Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya. , 2008, International journal of food microbiology.

[12]  Technical guidance - Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. , 2008, EFSA journal. European Food Safety Authority.

[13]  M. Kleerebezem,et al.  Functional Analysis of Four Bile Salt Hydrolase and Penicillin Acylase Family Members in Lactobacillus plantarum WCFS1 , 2008, Applied and Environmental Microbiology.

[14]  E. Urdaneta,et al.  Intestinal beneficial effects of kefir-supplemented diet in rats , 2007 .

[15]  G. Giraffa,et al.  Incidence of antibiotic resistance and virulence determinants among Enterococcus italicus isolates from dairy products. , 2007, Systematic and applied microbiology.

[16]  M. Liong,et al.  Effects of a Lactobacillus casei synbiotic on serum lipoprotein, intestinal microflora, and organic acids in rats. , 2006, Journal of dairy science.

[17]  Ming-Ju Chen,et al.  Hypocholesterolaemic effects of milk-kefir and soyamilk-kefir in cholesterol-fed hamsters , 2006, British Journal of Nutrition.

[18]  Colin Hill,et al.  Bile Salt Hydrolase Activity in Probiotics , 2006, Applied and Environmental Microbiology.

[19]  E. Purdom,et al.  Diversity of the Human Intestinal Microbial Flora , 2005, Science.

[20]  E. Farnworth,et al.  Immunomodulating capacity of kefir. , 2005, The Journal of dairy research.

[21]  A. Benini,et al.  Assessment of novel probiotic Lactobacillus casei strains for the production of functional dairy foods , 2004 .

[22]  S. Sarkar.  Potential of acidophilus milk to lower cholesterol , 2003 .

[23]  D. Gevers,et al.  Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausage , 2003, Applied and Environmental Microbiology.

[24]  P. Cullen,et al.  The impact of dietary mono‐ and poly‐unsaturated fatty acids on risk factors for atherosclerosis in humans , 2002 .

[25]  G. Antoni,et al.  Chemical and microbiological characterisation of kefir grains , 2001, Journal of Dairy Research.

[26]  S. Salminen,et al.  Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. , 2001, International journal of food microbiology.

[27]  F. Shanahan,et al.  In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. , 2001, The American journal of clinical nutrition.

[28]  G. Klein,et al.  Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods , 2000, Journal of applied microbiology.

[29]  K. Michaelsen,et al.  Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans , 1999, Applied and Environmental Microbiology.

[30]  A. Hosono,et al.  Hypocholesterolemic Effects of Lactobacillus casei subsp. casei TMC 0409 Strain Observed in Rats Fed Cholesterol Contained Diets , 1999 .

[31]  R. Satokari,et al.  Persistence of Colonization of Human Colonic Mucosa by a Probiotic Strain, Lactobacillus rhamnosusGG, after Oral Consumption , 1999, Applied and Environmental Microbiology.

[32]  M. Médici,et al.  Evidence for hypocholesterolemic effect of Lactobacillus reuteri in hypercholesterolemic mice. , 1998, Journal of dairy science.

[33]  G. Kociubinski,et al.  Isolation and characterization of Bifidobacterium strains for probiotic formulation. , 1998, Journal of food protection.

[34]  L. Morelli,et al.  Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract , 1998, Journal of applied microbiology.

[35]  G. Klein,et al.  Taxonomy and physiology of probiotic lactic acid bacteria. , 1998, International journal of food microbiology.

[36]  D. Brassart,et al.  The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo , 1997, Applied and environmental microbiology.

[37]  J. Grill,et al.  Adhesion of different bifidobacteria strains to human enterocyte‐like Caco‐2 cells and comparison with in vivo study , 1995, Letters in applied microbiology.

[38]  B. Richelsen,et al.  Hypocholesterolaemic effect of a new fermented milk product in healthy middle-aged men. , 1995, European journal of clinical nutrition.

[39]  E. Schiffrin,et al.  Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. , 1995, Journal of dairy science.

[40]  A. Servin,et al.  Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. , 1993, FEMS microbiology letters.

[41]  L. Angulo,et al.  Microflora present in kefir grains of the Galician region (North-West of Spain) , 1993, Journal of Dairy Research.

[42]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[43]  D. Walker,et al.  Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. , 1990, Journal of dairy science.

[44]  Takayuki Ezaki,et al.  Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains , 1989 .

[45]  S. Feighner,et al.  Development of a differential medium for bile salt hydrolase-active Lactobacillus spp , 1989, Applied and environmental microbiology.

[46]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[47]  Yen-Po Chen,et al.  Lactobacillus kefiranofaciens M1 isolated from milk kefir grains ameliorates experimental colitis in vitro and in vivo. , 2012, Journal of dairy science.

[48]  Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance , 2005 .

[49]  A. Hosono,et al.  Hypocholesterolemic effects of viable and heat-sterilized cells of Lactobacillus GG in rats fed a high-cholesterol diet , 2004 .

[50]  J. C. Carvalho,et al.  Study of anti-inflammatory activity of Tibetan mushroom, a symbiotic culture of bacteria and fungi encapsulated into a polysaccharide matrix. , 2003, Pharmacological research.