In Vitro Probiotic Characterization and Safety Assessment of Lactic Acid Bacteria Isolated from Raw Milk of Japanese-Saanen Goat (Capra hircus).

Two novel probiotic strains of lactic acid bacteria were successfully isolated from the raw milk of dairy Japanese-Saanen goats. Selection criteria for positive candidates were grown on de Man-Rogosa-Sharpe or M17 selective medium at 30, 35, or 42 °C anaerobically, and characterized based on Gram reaction, catalase test, and tolerance to low pH and bile salts. Among the 101 isolated positive candidates, two strains, YM2-1 and YM2-3, were selected and identified as Lacticaseibacillus rhamnosus using 16S rDNA sequence similarity. Culture supernatants of the two strains exhibited antipathogenic activity against Salmonella enterica subsp. enterica serovar. Typhimurium, Shigella sonnei, methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157. The antipathogenic activities were retained to some extent after neutralization, indicating the presence of antipathogenic substances other than organic acids in the culture supernatants. The two strains were sensitive with coincidental minimum inhibition concentrations (indicated in the parentheses hereafter) to ampicillin (0.25 µg/mL), chloramphenicol (4 µg/mL), gentamycin (4 µg/mL), kanamycin (64 µg/mL), streptomycin (16 µg/mL), and tetracycline (4 µg/mL). Furthermore, the two strains were resistant to clindamycin (16 µg/mL) and erythromycin (4 µg/mL). In addition, both YM2-1 and YM2-3 strains showed less unfavorable activities, including bile acid bioconversion, carcinogenic-related enzymes, mucin degradation, plasminogen activation, and hemolysis, than the detection limits of in vitro evaluation methods used in this study. In summary, L. rhamnosus YM2-1 and YM2-3 are highly safe and promising probiotic strains applicable in the dairy industry, and were first isolated from the raw milk of Japanese-Saanen goats.

Complete Genome Sequence of Lactobacillus fermentum MTCC 25067 (Formerly TDS030603), a Viscous Exopolysaccharide-Producing Strain Isolated from Indian Fermented Milk.

Lactobacillus fermentum MTCC 25067 (formerly TDS030603) is capable of producing a highly viscous slime exopolysaccharide. We report here the complete genome sequence of the strain, which was deciphered by using PacBio single-molecule real-time sequencing technology.

Anchorless cell surface proteins function as laminin-binding adhesins in Lactobacillus rhamnosus FSMM22.

Anchorless cell surface proteins (CSPs) were extracted with 1 M lithium chloride solution from Lactobacillus rhamnosus FSMM22. Loss of the anchorless CSPs resulted in a 2-fold decrease in FSMM22 cells bound to a constitutive extracellular matrix glycoprotein, laminin, in vitro. DNA-binding protein HU, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and 30S ribosomal protein S19 (RpsS) were identified by mass spectrometry in the extract as laminin-binding adhesins. Among the four proteins, RpsS was immunohistochemically confirmed to exist on the cell surface. Our findings strongly suggest that anchorless CSPs can enhance bacterial adhesion to the host.

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk.

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and ß-glucosidase were detected, but those of α-galactosidase, ß-glucuronidase and N-acetyl-ß-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.

Effects of prebiotic oligosaccharides consumption on the growth and expression profile of cell surface-associated proteins of a potential probiotic Lactobacillus rhamnosus FSMM15.

To investigate carbohydrate preference of a potential probiotic, Lactobacillus rhamnosus FSMM15, six prebiotics, including two milk-derived prebiotics, galactooligosaccharides and lacto-N-biose I, and four plant-origin prebiotics, beet oligosaccharide syrup, difructose anhydride III, fructooligosaccharides, and raffinose, were examined. The strain utilized the milk-derived prebiotics at similar levels to glucose but did not utilize the plant-origin ones in the same manner, reflecting their genetic background, which allows them to adapt to dairy ecological niches. These prebiotics had little influence on the expression pattern of cell surface-associated proteins in the strain; however, an ATP-binding cassette transporter substrate-binding protein and a glyceraldehyde-3-phosphate dehydrogenase were suggested to be upregulated in response to carbon starvation stress.

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Enhancement of Exopolysaccharide Production of Lactobacillus fermentum TDS030603 by Modifying Culture Conditions.

To optimize culture conditions that enhance production of a highly viscous exopolysaccharide of Lactobacillus fermentum TDS030603, a chemically defined medium was examined. The best yield was found to be 199 ± 23 mg/l when 48-hr cultivation was microaerobically performed at 30°C in the chemically defined medium supplemented with 5% glucose and 1% ammonium citrate without pH control. In response to the optimized exopolysaccharide production, the mRNA expression levels of epsB, epsE, and epsG elevated significantly. Our results indicated that the optimal C/N ratio and/or microaerobic condition can alter the expression levels of several exopolysaccharide biosynthesis-related genes promoting the exopolysaccharide production yield.

Isolation of potential probiotic Lactobacillus rhamnosus strains from traditional fermented mare milk produced in Sumbawa Island of Indonesia.

To explore potential probiotics in the traditional foods of Indonesia, fermented mare milk produced in Sumbawa Island was investigated in this study. Gram stain, catalase activity, gas production, cell morphology, carbohydrate utilization pattern, and 16S rDNA sequencing were performed to identify isolated lactic acid bacteria. To assess their probiotic ability, tolerance of low pH, bile salts, artificial gastrointestinal fluids, and adhesion properties to extracellular matrices, were examined. In total 27 strains, 25 Lactobacillus rhamnosus and two Lactobacillus fermentum, were obtained. Among the isolated lactobacilli, three Lb. rhamnosus strains, FSMM15, FSMM22, and FSMM26, were selected as candidates for probiotics, using Lb. rhamnosus GG as index. In vitro binding assay of the three strains against several extracellular matrix proteins revealed that FSMM15 and FSMM26 gave greater binding ratios of mucin/bovine serum albumin (BSA) and significantly higher adhesive abilities to fibronectin than Lb. rhamnosus GG. FSMM22 showed significantly higher adhesion to laminin than Lb. rhamnosus GG.