Living cells of probiotic Bifidobacterium bifidum YIT 10347 detected on gastric mucosa in humans.

The probiotic strain Bifidobacterium bifidum YIT 10347 has been demonstrated to inhibit Helicobacter pylori activity, prevent injury to the gastric mucosa, and improve general gastric malaise symptoms in H. pylori positive patients. This study aimed to investigate the adhering activity and localisation of B. bifidum YIT 10347 to gastric cells and tissue in vitro, and in human in vivo to clarify the mechanism of its beneficial effects on the stomach. The in vitro study found the adhesion rate of B. bifidum YIT 10347 to human gastric epithelial cells was about 10 times higher than that of lactic acid bacteria and other bifidobacteria. In the human study, 5 H. pylori negative and 12 H. pylori positive subjects ingested milk fermented with B. bifidum YIT 10347. B. bifidum YIT 10347 cells were measured by RT-qPCR for in gastric biopsy samples. Living B. bifidum YIT 10347 cells were detected in the biopsy samples in H. pylori negative subjects (105 cells/g and 104 cells/g at 1 h and 2 h after ingestion, respectively) and H. pylori positive subjects (104 cells/g at 1 h after the ingestion). Moreover, immunostaining analysis of tissue sections found that B. bifidum YIT 10347 cells were located at the interstitial mucin layer of the stomach. These results suggest that cells of probiotic B. bifidum YIT 10347 adhered to the human gastric mucosa in a live state, and that the higher adhering activity of B. bifidum YIT 10347 to the gastric mucosa may be involved in its beneficial effects on the human stomach.

Effect of Bifidobacterium bifidum BF-1 on gastric protection and mucin production in an acute gastric injury rat model.

Homeostasis in the stomach environment is maintained by the balance of protective factors such as gastric mucus and aggressive factors such as gastric acid, stress, alcohol, and drugs. An overload of aggressive factors that upsets this balance can induce gastric injury. Fermented milk that contains Bifidobacterium bifidum BF-1 (BF-1), a probiotic strain, and Streptococcus thermophilus YIT 2021 (ST) is known to improve Helicobacter pylori-associated gastritis in humans. Here, we investigated the gastroprotective potential of BF-1 in a rat model of acid-ethanol-induced acute gastric injury to fully elucidate its potential compared with ST. Living BF-1, ST, or vehicle was orally administrated to rats, and acid-ethanol gastric injury was induced 2h later. The gastric injury rate was determined and shown to be significantly lower in the BF-1 group than in the vehicle group, which showed a similar level to the ST group. The production of gastric mucin and the expression of several target genes associated with protection and inflammation were examined before and after induction of gastric injury. Interestingly, mucin 5ac (muc5ac) gene expression in gastric corpus samples and gastric mucin production in stomach samples from the BF-1 group, but not the ST group, were significantly higher than those in the respective samples from the vehicle group. These findings indicate that BF-1 has the potential to provide gastroprotection, alleviating acute gastric injury by enhancing the production of gastric mucin in a rat model.

Bifidobacterium bifidum BF-1 suppresses Helicobacter pylori-induced genes in human epithelial cells.

Helicobacter pylori infection alters gene expression in host cells. Specifically, inflammatory chemokines such as IL-8 are upregulated in the gastric mucosa during H. pylori infection. Although the mechanism by which H. pylori causes inflammation of the gastric mucosa is not yet understood, many studies have suggested that nuclear factor kappa B (NF-κB) plays a key regulatory role in host cells. We have shown that preincubation with Bifidobacterium bifidum strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses induction of IL-8 by the pathogen. To investigate how how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been preincubated with BF-1 and infected with H. pylori. We found that preincubation with BF-1 suppresses the expression of H. pylori-induced genes in human cells and that most of the affected genes are related to the NF-κB signaling pathways. These results suggest that BF-1 can affect the regulatory mechanism of the NF-κB signaling pathways.

Effect of Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels in humans.

Helicobacter pylori infection is an important risk factor for gastric diseases. Some probiotics are useful for suppressing H. pylori infection. Bifidobacterium bifidum YIT 4007 can improve the experimental gastric injury in rats and the disease stages on the gastric mucosa in peptic ulcer patients. We evaluated the fermented milk using a clone (BF-1) having the stronger ability to survive in the product than this parent strain to clarify the in vitro suppressive effect of BF-1 on H. pylori and the in vivo efficacy of BF-1 fermented milk on H. pylori and gastric health. In the mixed culture assay of BF-1 and H. pylori, the number of pathogens was decreased such that it was not detected after 48 h in the Brucella broth with a decrease in pH values. In the cell culture experiment with human gastric cells, the H. pylori infection-induced IL-8 secretion was suppressed by the preincubation of BF-1. In a human study of 12-wk ingestion (BF-1 group, n = 40; placebo group, n = 39) with a randomized double-blind placebo-control design, the H. pylori urease activity and gastric situation were evaluated using a urea breath test (UBT) and the serum pepsinogen (PG) levels as biomarkers for inflammation or atrophy, respectively. In the H. pylori-positive subjects, the difference (DeltaUBT) of the UBT value from the baseline value in the BF-1 group (n = 34) was lower than that in the placebo group (n = 35) at 8 wk. The baseline UBT values showed a negative correlation with DeltaUBT values at 8 and 12 wk in the BF-1 group but not in the placebo. In the PG-positive subjects classified by the PG test method, the BF-1 group was lower in DeltaUBT values than the placebo group at 8 and 12 wk. In the active gastritis class by PG levels, the BF-1 group was lower in their DeltaUBT values than the placebo at 8 and 12 wk. The PG I levels in the BF-1 group were lower than the placebo at 12 wk. The PG II levels in the BF-1 group did not change during the ingestion period, but the placebo was increased. The PG I/II ratios slightly decreased from baseline at 12 and 20 wk in the BF-1 and placebo groups. These patterns were also observed in the H. pylori-positive subjects. The improving rates of upper gastrointestinal symptomatic subjects and total symptom numbers in the BF-1 group were higher than those in the placebo. These results indicate that BF-1 fermented milk may affect H. pylori infection or its activity, gastric mucosal situation, and the emergence of upper gastrointestinal symptoms.

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene.

The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.

Transit of radical scavenging activity of milk products prepared by Maillard reaction and Lactobacillus casei strain Shirota fermentation through the hamster intestine.

Oxidative stress in the colon is associated with the incidence of colon cancer. In situ, the suppression of oxidative stress in the colon would be an effective form of prevention of the cancer. In this study we investigated the transit of the radical scavenging activity of milk products through the hamster intestinal tract. Two types of skim milk products were prepared by Maillard reaction and then lactic acid fermentation. Heat treatment enhanced the radical scavenging activity for 2,2-diphenyl-1-picrylhydrazyl radical of skim milk. The activity was further increased by fermentation with Lactobacillus casei strain Shirota. Normal hamsters were fed these milk products for 14 d. For potential radical scavenging activity per unit dry weight of feces and cecal content, the groups ranked in the order of fermented product-fed hamsters > heated product-fed hamsters > control hamsters, reflecting the order of the potential of the corresponding diets. Approximately 12% of the activity of the heated and the fermented product diets passed through the gastrointestinal tract. These results suggest that some of the radical scavenging activity generated by food processing reached the colon in nonabsorbable products.

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.

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.