A novel bifidogenic growth stimulator (BGS) was present in the cell-free filtrate and in methanol extract fraction of the starter (Propionibacterium freudenreichii) cells for the manufacture of Swiss-type cheese. After purification of the BGS isolated from the lyophilized cells, the mass (217.037) was determined by high-resolution electron impact mass spectrometry (MS) and liquid chromatography-MS spectra. Various experimental analyses indicated that the chemical structure of the BGS was 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ). We examined the activity of the P. freudenreichii ET culture containing the BGS on a gut bacterial composition using an anaerobic continuous culture system and found that the BGS seems to enhance the selective utilization of oligosaccharides by bifidobacteria. Furthermore, a placebo-controlled study of the effects of BGS on fecal flora and stool frequency was carried out in healthy human subjects. A drink with the sterilized ET-3 culture was administered once a day for 7 days. Bifidobacterium percentage in the fecal flora and stool frequency were significantly increased by administration of the P. freudenreichii culture. The ACNQ exhibited growth stimulation of bifidobacteria at an extremely low concentration and enhanced the activities of NADH oxidase and NADH peroxidase in bifidobacteria. It was revealed that ACNQ works as a good electron acceptor of NAD (P)H diaphorase. The ACNQred was easily auto-oxidized and also acted as a better electron donor of NAD (P) H peroxidase. These ACNQ-mediated reactions seem to play roles in NAD (P) +-regeneration processes and seem to be responsible for the growth stimulation of bifidobacteria.
As the cause of one disease becomes clear and specific therapies for it are established, other diseases formerly of lesser importance become serious problems. Ulcerative colitis and Crohn's disease, which were unfamiliar diseases in Japan about 20 years ago, are on the increase. The cause remains unclear, but the tendency seems to be closely related to changes in Japanese lifestyle, especially to changes in diet, which has become more similar to the Euro-American diet. Part 1 of this paper outlines the recent development of colonic inflammatory diseases in Japan as seen by the pathologist. The endoscope has enabled remarkable clinical progress, and colonoscopic examinations are performed readily in most patients who complain of intestinal problems. There are many opportunities to detect polyps of the colon, and such polyps rank with inflammatory bowel diseases. The adenoma-carcinoma sequence theory was proposed by Morson in 1968, but most adenomas do not generate cancer over the long term, and the wide utilization of polypectomy has not decreased the colon cancer rate. There is some doubt as to whether all colon cancers develop from adenoma. In recent years, practitioners have discovered very small elevated, flat or depressed colon carcinomas that have no polyp (adenoma) component. There are known as de novo cancers. However, neither the adenoma-carcinoma sequence nor de novo cancer give a complete account for the carcinogenesis of colon cancer yet. Part 2 of this paper explores the pathohistological relation between adenoma and carcinoma.
To evaluate whether intestinal microecology is altered in Crohn's disease, we studied the metabolic activity of faecal flora and its bacteriology. Metabolic activity was analysed by measuring the activities of β-glucuronidase, β-glucosidase and urease in faeces, based on enzyme-substrate reactions, from 24 patients (aged 5-51 years) with Crohn's disease as compared to 16 controls (4-62 years). Faecal bacteriology was determined by analysing bacterial cellular fatty acid profiles by gas-liquid chromatography assay in a subgroup of the study subjects. In Crohn's disease the mean (95% confidence interval) activities of β-glucuronidase: 1.6 (0.9-2.2) nmol/min/mg protein, β-glucosidase: 3.9 (2.4-5.3) and urease: 6.1 (2.9-9.2) were significantly lower than in controls: 3.6 (2.3-4.9), 7.6 (5.6-9.5), 18.1 (11.1-25.0), respectively, p < 0.004. The faecal enzyme activities in Crohn's disease were associated with disease activity: the more active the disease, the lower the enzyme activity. Also faecal bacterial cellular fatty acid profiles in Crohn's disease patients with active disease tended to differ from controls. We suggest that in active Crohn's disease intestinal microecology is altered, in particular, its metabolic activity, which may have a role in the pathogenesis of Crohn's disease.
A gnotobiotic mouse study was investigated to establish a suitable experimental model for evaluating an individual bacterial role for Escherichia coli O157: H7 infection. Germ-free (GF) BALB/c mice (nu/+) were found to die after 12-14 days of E. coli O157: H7 (strain CR 7087) infection, causing rough surface, atrophy, and faded color of the kidney, while specific pathogen-free (SPF) mice survived without any damage after similar infection. Using this model, the effect of Bifidobacterium-monoassociation (BA) and feeding of fructooligosaccharides (FOS) on the lethal activity of enterohemorrhagic E. coli O157: H7 was examined to elucidate if mice inoculated with B. adolescentis strain MBL8321 showed a significantly longer survival time than original GF mice after E. coli O157: H7 (strain CR 7087) infection. An analysis of the biomarkers in the present study provided the following information: 1) B. adolescentis was successfully associated at the 109-1010 colony-forming unit (cfu) level throughout the test period, 2) the numbers of viable E. coli in feces decreased in the order of GF, BA (= BA-FOS) and SPF groups, and the amount of fecal Shiga toxin (Stx), serum creatinine and urea nitrogen also decreased in similar order, 3) the amount of cecal short-chain fatty acids (SCFAs) was found to correlate with the survival time, and 4) the feeding of FOS under this condition afforded no additional increase in either survival time or amount of cecal SCFAs. These results indicate that the study using gnotobiotic mice is suitable for an experimental model of E. coli O157: H7 infection, and suggest tha the intestinal microflora plays an important role for preventing E. coli O157 infection in mice. However, the model is not suitable for evaluating the inhibitory effect related to intestinal fermentation because the parameters for fermentation did not change.
In this study, characteristics of bacteriocin like inhibitory substances (BLIS) produced by three strains of Lactobacillus acidophilus (BDLA-1, 2409 and MOLA-2), Lactobacillus fermentum (5174) and Lactobacillus plantarum (2903) were studied. The production of BLIS and its stability during 48 hr of growth was monitored in MRS broth adjusted to pH 5.0, pH 6.0 and in MRS broth supplemented with 1.9% sodium-β-glycerophosphate. The BLIS produced by L. acidophilus (BDLA-1, 2409 and MOLA-2), L. fermentum (5174) and L. plantarum (2903) were active against Lactobacillus delbrueckii ssp. bulgaricus (2519), L. casei (2603), L. helveticus (2700), L. jugurti (2819) and L. acidophilus (MJLA-1). The production of BLIS was greatly affected by the pH of MRS broth and the effect was variable for different bacteria. The BLIS produced by all the bacteria showed activity over a wide range of temperature and pH and were sensitive to proteolytic enzymes, but resistant to lipase, phospholipases and amylases. The activity of BLIS in retentates and permeates of cell free MRS-broth was variable with different bacteria during molecular sieving by ultrafiltration. Two stage fractionation with ammonium sulfate was successful in purifying the BLIS and resulted in a single band on silver staining of SDSPAGE gel for two L. acidophilus strains (BDLA-1 and 2409) and L. fermentum (5174) of the 5 bacteria studied. A band of -54 kDa was common for all of the lactobacilli studied which was in line with the acidophilicin LA-1 reported earlier.
We have examined the responses of polymorphonuclear neutrophils (PMNs) and human umbilical vein endothelial cells (HUVEC) to Helicobacter pylori lipopolysaccharides (LPSs). High-antigenicity-polysaccharide-carrying smooth LPS and rough LPS from H. pylori were used for the experiments. PMN activation was determined by chemiluminescence response (CLR). The priming effect of patient's serum or IL-8 on the CLR was also examined. After stimulation of HUVEC with the LPSs, the supernatant was tested for IL-6, IL-8 and RANTES by ELISA. Direct induction of CLR was observed after the stimulation of H. pylori LPSs. Though the response was weak, enhancement occurred in response to high-antigenicity-polysaccharide-carrying smooth LPS by patient's serum against the LPS. IL-8 priming was also effective to enhance CLR, which was induced by H. pylori LPS. Following exposure to H. pylori LPSs, HUVEC secreted IL-6 and IL-8 but not RANTES. The enhanced secretion of both IL-6 and IL-8 occurred in a time-dependent manner. However, the cytokine-inducing ability of H. pylori LPSs is significantly lower than that of other Gram-negative bacteria such as Escherichia coli and Campylobacter jejuni. Structurally similar LPS from Porphyromonas gingivalis also showed weak potency. These results suggest that the ability of H. pylori LPSs is not directly effected to induce inflammation. However, an indirect effect of these LPSs, or continuous stimulation with these LPSs to endothelial cells, may relate to persistent infection.
This study was carried out to evaluate the antagonistic relationship between yogurt and probiotic bacteria and the nature of the inhibitory compound produced by the organisms. Eight strains each of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus and bifidobacteria were isolated from eight commercial AB (L. acidophilus and Bifidobacterium spp.) products containing these four groups of bacteria. The isolates were screened for the production of bacteriocins against each of the 8 isolates of L. acidophilus and Bifidobacterium spp. Twelve strains showed inhibitory activity against all the 8 strains of Bifidobacterium spp. and 5 L. acidophilus isolates with the 'spot on lawn' assay. Of these, only one yogurt bacterium, S. thermophilus was identified to be a bacteriocinproducing organism. The S. thermophilus strain was found to specifically target 2 strains of bifidobacteria. The crude antimicrobial compound was found to be heat stable, resistant over a wide range of pH, and sensitive to proteolytic enzymes, but it retained activity after treatment with lipase. The compound was purified using ultrafiltration, precipitation with ammonium sulfate and dialysis. The bacteriocin-fractionate was also subjected to SDS-PAGE analysis and the molecular weight of the bacteriocin was estimated to be approximately 80 kDa.