Lactobacillus casei Shirota protects from fructose-induced liver steatosis: A mouse model

To test the hypothesis that Lactobacillus casei Shirota (Lcs) protects against the onset of non-alcoholic fatty liver disease (NAFLD) in a mouse model of fructose-induced steatosis, C57BL/6J mice were either fed tap water or 30% fructose solution +/- Lcs for 8 weeks. Chronic consumption of 30% fructose solution led to a significant increase in hepatic steatosis as well as plasma alanine-aminotransferase (ALT) levels, which was attenuated by treatment with Lcs. Protein levels of the tight junction protein occludin were found to be markedly lower in both fructose treated groups in the duodenum, whereas microbiota composition in this part of the intestine was not affected. Lcs treatment markedly attenuated the activation of the Toll-like receptor (TLR) 4 signalling cascade found in the livers of mice only treated with fructose. Moreover, in livers of fructose fed mice treated with Lcs peroxisome proliferator-activated receptor (PPAR)-γ activity was markedly higher than in mice only fed fructose. Taken together, the results of the present study suggest that the dietary intake of Lcs protects against the onset of fructose-induced NAFLD through mechanisms involving an attenuation of the TLR-4-signalling cascade in the liver.     

Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice

Aims: This study aimed at determining whether oral administration of a probiotic strain, Lactobacillus casei strain Shirota (LcS), can improve insulin resistance, which is the underlying cause of obesity‐associated metabolic abnormalities, in diet‐induced obesity (DIO) mice. Methods and Results: DIO mice were fed a high‐fat diet without or with 0·05% LcS for 4 weeks and then subjected to an insulin tolerance test (ITT) or oral glucose tolerance test (OGTT). Oral administration of LcS not only accelerated the reduction in plasma glucose levels during the ITT, but also reduced the elevation of plasma glucose levels during the OGTT. In addition, plasma levels of lipopolysaccharide‐binding protein (LBP), which is a marker of endotoxaemia, were augmented in the murine models of obese DIO, ob/ob, db/db and KK‐A^y and compared to those of lean mice. LcS treatment suppressed the elevation of plasma LBP levels in DIO mice, but did not affect intra‐abdominal fat weight. Conclusions: LcS improves insulin resistance and glucose intolerance in DIO mice. The reduction in endotoxaemia, but not intra‐abdominal fat, may contribute to the beneficial effects of LcS. Significance and Impact of the Study: This study suggests that LcS has the potential to prevent obesity‐associated metabolic abnormalities by improving insulin resistance.     

Survival of Lactobacillus casei strain Shirota in the intestines of healthy Chinese adults

Lactobacillus casei strain Shirota (LcS) is a widely used probiotic strain with health benefits. In this study, the survival of LcS in the intestines of healthy Chinese adults was assessed and the effects of LcS on stool consistency, stool SCFAs and intestinal microbiota evaluated. Subjects consumed 100 mL per day of a probiotic beverage containing 1.0 × 10⁸ CFU/mL of LcS for 14 days. LcS were enumerated using a culture method and the colony identity confirmed by ELISA. Fourteen days after ingestion, the amount of LcS recovered from fecal samples was between 6.86 ± 0.80 and 7.17 ± 0.57 Log₁₀ CFU/g of feces (mean ± SD). The intestinal microbiotas were analyzed by denaturing gradient gel electrophoresis. Principal component analysis showed that consuming LcS significantly changed fecal microbiota profiles. According to redundancy analysis, the amounts of 25 bacterial strains were significantly correlated with LcS intake (P < 0.05), 11 of them positively and fourteen negatively. Concentrations of acetic acid and propionic acid in feces were significantly lower during the ingestion period than during the baseline period (P < 0.05). These results confirm that LcS can survive passage through the gastrointestinal tract of Chinese people; however, they were found to have little ability to persist once their consumption had ceased. Furthermore, consumption of probiotic beverages containing LcS can modulate the composition of the intestinal microbiota on a long‐term basis, resulting in decreased concentrations of SCFAs in the gut.     

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Release studies of Lactobacillus casei strain Shirota from chitosan-coated alginate-starch microcapsules in ex vivo porcine gastrointestinal contents

AIMS: To investigate the release characteristics of Lactobacillus casei strain Shirota (LCS) from chitosan-coated alginate-starch (CCAS) capsule in different regions of ex vivo porcine gastrointestinal (GI) contents. METHODS AND RESULTS: A 0.1 g of CCAS encapsulated bacteria (containing c. 10(8) CFU of LCS) were incubated in different sections of ex vivo porcine GI contents (10 ml) anaerobically at 37 degrees C. Samples were taken from different GI contents at different time intervals (up to 24 h) and estimated for the release of LCS from capsules. There was almost a complete release (1.1 x 10(8) CFU) of LCS from capsules within 8 h of incubation in ileal contents, while it took nearly 12 h to release the completely encapsulated bacteria in colon content under similar conditions. There was only a partial release of encapsulated LCS, incubated in duodenal and jejunal contents, while there was no significant (P > 0.05) release of encapsulated bacteria in gastric contents even after 24 h of incubation. CONCLUSIONS: The capsules were able to release viable probiotic cells completely in ex vivo porcine ileal and colon contents. SIGNIFICANCE AND IMPACT OF THE STUDY: CCAS capsule can be an effective way of protecting probiotic bacteria from adverse gastric conditions and delivering viable bacteria to the host intestinal systems.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Effect of Different Antibiotics and Non-Steroidal Anti-Inflammatory Drugs on the Growth of Lactobacillus casei Shirota

The purpose of this study was to investigate whether some non-steroidal anti-inflammatory drugs (NSAIDs) could cause inhibition of the growth of Lactobacillus casei Shirota (LcS) or whether this microorganism is able to use some of them as the sole carbon source, considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance or improve the healing of gastric ulcers. Acetylsalicylic acid (ASA), sodium acetylsalicylate (SAS), acetaminophen, sodium naproxen, and sodium ibuprofen were added as the sole carbon source to a basal medium and tested for biodegradation by LcS. The same NSAIDs were added in different concentrations to disks and plated on MRS Agar to test the possible inhibitory effect of these compounds on LcS. Also, the resistance of LcS to 12 different antibiotics was studied on MRS agar. None of the NSAIDs tested could be used by LcS as the sole carbon source at the assayed concentrations. In the case of the disk diffusion method, sodium naproxen showed inhibition zones for the 500-μg disks and sodium ibuprofen was inhibitory for the 250- and 500-μg disks. However, when the macrobroth dilution method was used, the growth of LcS was inhibited by ASA, SAS, acetaminophen, and sodium ibuprofen. This strain showed resistance to the antibiotics sulfamethoxazole with trimethoprim, pefloxacin, and gentamicin. This is the first study on the effect of NSAIDs on probiotic bacteria. The results of the biodegradation test indicate that the simultaneous consumption of NSAIDs and a dairy beverage with LcS is not likely to change the bioavailability of the drugs.     

Effect of an oral administration of Lactobacillus casei strain Shirota on the natural killer activity of blood mononuclear cells in aged mice

The natural killer (NK) activity of blood mononuclear cells and splenocytes in aged mice fed on a diet containing Lactobacillus casei strain Shirota (LcS group) was significantly (P < 0.01) higher than that in mice fed on a diet without LcS. In the LcS group, there was a significant positive correlation (r = 0.63, P < 0.01) between the NK activity of blood mononuclear cells and the NK activity of splenocytes.     

Lipoxygenase inhibitor from Lactobacillus casei

An inhibitor of plant lipoxygenase from culture filtrates of Lactobacillus casei was purified by column chromatography and shown to be benzoic acid. The isolated benzoic acid had an IC₅₀ of 350 M against purified soybean lipoxygenase at pH 9. L. casei therefore may have the potential to be used as a preservative against the oxidation of unsaturated fatty acids, thereby preventing undesirable flavours in foods.     

Modulation of Lactobacillus casei in ileal and fecal samples from healthy volunteers after consumption of a fermented milk containing Lactobacillus casei DN-114 001Rif

Lactobacillus casei DN-114 001 is a probiotic strain able to interact with the immune system and to interfere with gastrointestinal pathogens. The derived strain DN-114 001Rif was studied during its transit through the upper and distal intestine of human volunteers. Seven volunteers participated in the study, which involved intestinal intubation to sample ileal contents and collection of fecal samples, with a wash-out period of 8 days between the 2 steps. The retrieval of the probiotic was analyzed in the ileum every 2 h for 8 h following the ingestion of one dose of the test product and in the feces prior to, during, and after daily consumption of the test product for 8 days. Persistence of the probiotic amplifiable DNA was assessed using temporal temperature gradient gel electrophoresis and real-time PCR. Fluorescent in situ hybridization allowed analysis of the composition of the dominant digestive microbiota. The ingestion of L. casei DN-114 001Rif led to a significant and transient increase of its amplifiable DNA in ileal and fecal samples. This is related to a high stability in the composition of dominant groups of the gut microbiota. Data from ileal samples are scarce and our study confirms the potentiality for interaction between probiotics and the human immune system.     

Lower plasma triglyceride level in Syrian hamsters fed on skim milk fermented with Lactobacillus casei strain Shirota

The effect of fermented skim milk (FSM) by Lactobacillus casei strain Shirota on plasma lipids in hamsters was examined. Hamsters fed on cholesterol-free and -enriched diets containing 30% FSM had lower levels of plasma triglyceride than those fed on the control diet. In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10% to 30%. Unfermented milk tended to lower the level of triglyceride, but not significantly. The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet. L. casei strain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth. These results indicate that FSM lowered the plasma triglyceride level in hamsters.     

The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C

1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall.