1. Normalization of intestinal flora
1.1 C. butyricum MIYAIRI antagonistic effects on toxinogenic Escherichia coli and 20 E. coli strains isolated from live stocks (cows, pigs and chickens) were evaluated by the plating method. C. butyricum MIYAIRI inhibited the growth of all E. coli strains tested.
1.2 C. butyricum MIYAIRI antagonistic effects on Campylobacter jejuni, Salmonella typhimurium and Staphylococcus aureus strains isolated from chicken (meet) were evaluated by the plating method and the contact culture method and using C. butyricum MIYAIRI culture fluid. The growth of all these bacteria was inhibited regardless of the method used. Effects on Campylobacter were thought to be bactericidal.
1.3 Bifidobacterium sp., Lactobacillus acidophilus, and Streptococcus faecalis were inoculated to basic media containing C. butyricum MIYAIRI culture fluid and cultured. The growth of all these bacteria was promoted.
1.4 C. butyricum MIYAIRI antagonistic and symbiotic effects on Clostridium difficile and Bifidobacterium sp. were evaluated using the same plate. C. butyricum MIYAIRI showed antagonistic effects on the former and symbiotic effects on the latter.
1.5 C. butyricum MIYAIRI was simultaneously cultured with Clostridium difficile by the contact culture method. The former inhibited the growth of the latter.
1.6 C. butyricum MIYAIRI inhibited the growth of the intestinal pathogens Vibrio cholerae 01, V. cholerae non-01, Aeromonas hydrophila, and Shigella flexneri under an anaerobic environment in mixed culture using GAM broth and BHI broth.
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2. Reduction in substances with harmful effects in the intestine
2.1 C. butyricum MIYAIRI was cultured with amine- and ammonia-producing Clostridium putrificum and Escherichia coli. Production of amine and ammonia was inhibited in the presence of C. butyricum MIYAIRI compared to the production observed in its absence.
2.2 Whether C. butyricum MIYAIRI is able to consume ammonia was studied in an in vitro study. About 60% of ammonia was consumed by C. butyricum MIYAIRI.
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3. Stability in gastric juice
3.1 C. butyricum MIYAIRI, Lactobacillus acidophilus and Streptococcus faecalis were kept in contact with human gastric juice (pH 1.0-5.4) with shaking for 2 hours (normal length of stay of foods in the stomach) in order to evaluate the resistance of these bacteria to human gastric juice. Live C. butyricum MIYAIRI cell counts did not decrease over a wide pH range (1.0-5.4), while live L. acidophilus and S. faecalis cell counts decreased markedly at pH 1.0-3.5.
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B. In vivo studies
1. Normalization of intestinal flora
1.1 An antibiotic product (chloramphenicol) was administered for 1 week in order to disturb intestinal flora in 4-week-old ICR mice (2 animals in the control group and 3 animals in the test group), and changes in fecal intestinal flora were evaluated. Enterobacteriaceae and Lactobacillus showed a tendency to decrease in the control group, but these bacteria showed no changes in the group which received C. butyricum MIYAIRI at the same time, indicating that C. butyricum MIYAIRI prevents disturbance of intestinal flora due to the antibiotic. Fecal C. butyricum MIYAIRI counts remained between 103 and 106 per gram of feces during the treatment period.
1.2 Effects of C. butyricum MIYAIRI on the incidence of diarrhea and mortality were evaluated in 4 to 8-week-old Golden hamsters in which diarrhea was caused by administering an antibiotic product (cefatrizine). C. butyricum MIYAIRI reduced incidence of diarrhea and prevented deaths due to diarrhea caused by Clostridium difficile, indicating that it inhibits the growth of C. difficile and contributes to the normalization of intestinal flora.
1.3 Incidence of Clostridium difficile and Toxin A produced by it in feces samples obtained from adult inpatients under antibiotic therapies were compared in the C. butyricum MIYAIRI group and the control group. Incidence of both C. difficile and Toxin A was lower in the C. butyricum MIYAIRI group.
1.4 Fecal bacterial flora and C. butyricum MIYAIRI counts were compared before and after administration of C. butyricum MIYAIRI to adult inpatients who underwent chest surgery. In patients without abdominal symptoms, C. butyricum MIYAIRI increased E. coli, Lactobacillus, and Enterococcus and decreased Klebsiella and Candida. Inpatients with abdominal symptoms, C. butyricum MIYAIRI increased E. coli and Lactobacillus, decreased Klebsiella, Candida, and Pseudomonas Proteus, and resolved symptoms. C. butyricum MIYAIRI (106-107 per gram) were detected in fecal samples a few days after starting the treatment.
1.5 Cows (3 per block; body weight, about 700 kg) were given feed rich in soy bean-curd refuse for 80 days, during which time Enterobacteriaceae and Lactobacillus in the first stomach were regularly monitored. In the block given C. butyricum MIYAIRI, Lactobacillus increased, while Enterobacteriaceae decreased compared to the control block. In addition, the ratio of contents of volatile fatty acid and lactic acid in the first stomach remained constant in the C. butyricum MIYAIRI block, indicating that it normalizes bacterial flora in the first stomach. 103-104 cells per 1 ml of C. butyricum MIYAIRI were detected from the first stomach during the treatment in the C. butyricum MIYAIRI block.
1.6 C. butyricum MIYAIRI was administered to 2 cows weighing about 250 kg, and changes in bacterial flora in the first stomach and feces were monitored. C. butyricum MIYAIRI increased Lactobacillus and decreased Enterobacteriaceae in both the first stomach and feces.
1.7 Suckling piglets (4 per block; 31 and 36 days after birth) treated with C. butyricum MIYAIRI were raised for 4 weeks in a hot and crowded environment, and the effects of C. butyricum MIYAIRI on their growth and bacterial flora in different segments of the GI tract (duodenum, ileum and cecum) were evaluated. The growth of Enterobacteriaceae and Staphylococcus was inhibited and bacterial flora were normalized in the C. butyricum MIYAIRI block compared to the control. 103-104 cells per gram of C. butyricum MIYAIRI were detected from each segment. Body weight gain increased by about 17% in the C. butyricum MIYAIRI block compared to the control.
1.8 Broilers (20 per block; 7 days after hatching) treated with C. butyricum MIYAIRI were raised for 3 weeks in the rainy season and summer, and the effects of C. butyricum MIYAIRI on their growth and bacterial flora in different segments of the GI tract (duodenum, ileum and cecum) were evaluated. The growth of Enterobacteriaceae and Streptococcus was inhibited and bacterial flora were normalized in the C. butyricum MIYAIRI block compared to the control. 102-104 cells per gram of C. butyricum MIYAIRI were detected from each segment. Body weight gain increased by 7% to 11% in the C. butyricum MIYAIRI block compared to the control.
1.9 Broilers (10 per block; 14 days after hatching) treated with C. butyricum MIYAIRI were raised for 4 weeks at intermittently high temperature, and the effects of C. butyricum MIYAIRI on their growth and bacterial flora in different segments of the GI tract (duodenum, ileum and cecum) were evaluated. The growth of Enterobacteriaceae and Staphylococcus was inhibited and bacterial flora were normalized in the C. butyricum MIYAIRI block compared to the control. 102-103 cells per gram of C. butyricum MIYAIRI were detected from each segment. Body weight gain increased by 14% in the C. butyricum MIYAIRI block compared to the control.
1.10 Broilers (7,300 per block; 32 days after hatching) treated with C. butyricum MIYAIRI were raised for 29 days, and bacterial flora in the ileum and cecum were evaluated. The growth of Enterobacteriaceae, Campylobacter, and Clostridium perfringens was inhibited and bacterial flora were normalized in the C. butyricum MIYAIRI block compared to the control. 102-103 cells per gram of C. butyricum MIYAIRI were detected from each segment. Body weight gain increased by about 2% in the C. butyricum MIYAIRI block compared to the control.
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2. Reduction in substances with harmful effects in the intestine
2.1 14CO2 in expired air was determined in Wistar rats (3 per group; 6 weeks of age) treated with 14C-urea in order to evaluate the effects of C. butyricum MIYAIRI on the decomposition of urea by urease. 14CO2 in expired air was smaller by up to about 17% in the C. butyricum MIYAIRI group than in the control, indicating that C. butyricum MIYAIRI either inhibits the activity of urease produced by intestinal bacteria or decreases urease production by reducing the number of urease-producing bacteria.
2.2 Ten maternal pigs were raised for 48 days, and fecal ammonia concentrations and Enterobacteriaceae and Lactobacillus counts were determined before and after administration of C. butyricum MIYAIRI. Fecal ammonia concentrations decreased by about 30% after administration of C. butyricum MIYAIRI. It increased Lactobacillus and decreased Enterobacteriaceae.
2.3 Pigs (3 per block; 30 days after birth) were raised for 64 days, and changes in fecal ammonia concentrations were monitored. Fecal ammonia concentrations decreased by about 50% in the C. butyricum MIYAIRI block compared to the control.
2.4 C. butyricum MIYAIRI was administered to 15 pigs for 1 month, during which time amounts of ammonia in the pig house and feces were monitored. Amounts of ammonia in the pig house decreased 40% to 60% after treatment compared to the baseline before treatment. Odor also decreased. Fecal ammonia amounts decreased 61%.
2.5 Broilers (3 per block; body weight, about 1.3 kg) were raised for 5 days, during which time ammonia concentrations in the cecum and portal blood were monitored. Ammonia contents in the cecum and portal blood decreased by about 39% and 12%, respectively, in the C. butyricum MIYAIRI block compared to the control.
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3. Acceleration of vitamin E absorption
3.1 Wistar rats (6 per group; 6 weeks of age) were given vitamin E-deficient diet, standard diet, or vitamin E-rich diet for 5 weeks, after which plasma vitamin E concentrations were determined. Plasma vitamin E concentrations were higher in the C. butyricum MIYAIRI group than in the control group regardless of the type of diet given, and differences were statistically significant in the blocks given vitamin E-deficient and rich diet. This finding indicates that C. butyricum MIYAIRI is effective in increasing the vitamin E absorption rate.
3.2 C. butyricum MIYAIRI (as formula feed containing vitamins A and D3 and brewer's yeast as well) was administered to dairy calves (10 per block; 10 days after birth) for 80 days, after which plasma vitamin A and E concentrations were determined. Plasma concentrations of both vitamins A and E were higher in the C. butyricum MIYAIRI block than in the control, indicating that C. butyricum MIYAIRI increases the vitamin A and E absorption rate.
3.3 Vitamin E-added feed was given to fattening cattle (6 per block; 8 months after birth) for 10 weeks, after which vitamin E concentrations in portal blood were determined. Serum vitamin E concentrations showed a tendency to be higher in the C. butyricum MIYAIRI block than in the control block, suggesting that C. butyricum MIYAIRI enhances the absorption of vitamin E from the GI tract.
3.4 Vitamin E-added feed was given to broilers (5 per block; 2 days after hatching) for 4 weeks, after which serum vitamin E concentrations were determined. Serum vitamin E concentrations were significantly higher in the C. butyricum MIYAIRI block than in the control block, suggesting that C. butyricum MIYAIRI enhances the absorption of vitamin E from the GI tract.
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4. Other studies
4.1 C. butyricum MIYAIRI (as formula feed containing vitamins A and D3 and brewer's yeast as well) was administered to dairy bull calves during the nursing period. The accident rate and treatment frequency decreased in the C. butyricum MIYAIRI block compared to the control. Body weight gain per day increased by about 30%.
4.2 Effects of C. butyricum MIYAIRI on the rate of chick raising were evaluated using White Leghorns S, D and H (8,074-15,073 per group). The chick mortality in the C. butyricum MIYAIRI block was 15% to 17% of that in the control block. Enterobacteriaceae and Staphylococcus detection rates determined in different organs decreased in the C. butyricum MIYAIRI block compared to the control.