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Probiotics and carcinogens both have a significant effect on the microfloral composition of the human intestine. The objective of this study was to investigate the impact of an important carcinogen, 4-Nitroquinoline-1-Oxide on colonic microflora and the efficacy of the probiotic Lactobacillus salivarius REN as an agent of counteracting these effects. Using denaturing gradient gel electrophoresis (DGGE) combined with redundancy analysis, we demonstrated that both 4-Nitroquinoline-1-Oxide and L. salivarius REN significantly altered the bacterial communities of rat colons. A total of 27 bacterial strains were identified as being affected by treatment with 4-Nitroquinoline-1-Oxide or L. salivarius REN using a t-value biplot combined with band sequencing. 4-Nitroquinoline-1-Oxide treatment increased the abundance of two potential pathogens (one Helicobacter strain and one Desulfovibrio strain), as well as reducing the abundance of two potentially beneficial strains (one Ruminococcaceae strain and one Rumen bacteria). The Helicobacter strain was initally detected in carcinogen-treated rat intestinal microflora, but L. salivarius REN treatment effectively suppressed the growth of the Helicobacter strain. These results suggested that L. salivarius REN may be a potential probiotic, efficiently acting against the initial infection with, and the growth of pathogenic bacteria.

Despite its ubiquity in fermented dairy products, the safety of lactic acid enterococcal bacteria remains controversial. In this study, five Enterococcus durans strains - A1, A2, B1, B2, and C1 - were isolated from traditional fermented yak milk from Tibet. To evaluate the strains' safety, biogenic amine production, antibiotic resistance and presence of known virulence determinants were investigated. Strain A1 can produce biogenic amines for histamine, spermine, and spermidine (mean values: 8.64, 8.31, and 0.30 mg/L, respectively). Polymerase chain reaction amplification for Strain A1 found genes involved in expression of gelatinase (gleE), cytolysin (cylA, cylB, and cylM), sex pheromones (ccf and cpd) and cell wall adhesion (efaA). Strain A2 showed sensitivity or intermediate resistance to all tested antibiotics, and no virulence determinants except gelE and ccf, but did produce tyramine at a relatively high level (912.02 mg/L). Both strains B1 and B2 could produce histamine (10.43 and 10.56 mg/L, respectively), and showed vancomycin resistance; B1 also produced tyramine (504.02 mg/L). Strain C1 could produce all five biogenic amines tested in the study -putrescine, histamine, tyramine, spermine, and spermidine; concentrations were 6.51, 9.59, 205.85, 5.55, and 5.39 mg/L, respectively. All E. durans strains found in Tibetan traditional fermented yak milk thus offer potential risk.

Bama County (Guangxi, China) is famous for its longevous population. In this study, intestinal microflora of 17 healthy elderly subjects of different ages and from different regions (rural and urban) in Bama, were analyzed by denaturing gradient gel electrophoresis (DGGE). Significant effects of age and living region on the whole intestinal bacterial communities were observed by redundancy analysis (RDA). A total of 11 bacterial strains that were correlated with age and living region were identified using a t-value biplot combined with band sequencing. Four bacterial strains were correlated with both age and living region of the elderly in Bama. Two Bacteroides strains and one Ruminococcaceae strain were abundant in the rural, younger elderly; conversely, one Desulfovibrio strain was high in the urban, older elderly. Another Bacteroidetes strain was only correlated with the participant's age, and its abundance increased with the age of the elderly. The richness of one Clostridium sordellii strain, which was only correlated with the elderly living region, was high in the urban elderly. The study also found five other novel bacterial strains that were correlated with the age or living region of the elderly in Bama. These results expand our understanding of age- and region-effects on the intestinal microflora of the elderly and raise the possibility of developing probiotics originating from centenarians.

The acid adaption is commonly used as a strategy to enhance the acid tolerance of bifidobacteria. However, the acid tolerance response (ATR) mechanism elicited by this method is unclear. Real-time relative-quantitative PCR was applied to analyze the changes in the expressions of ffh, uvrA, groES, and dnaK involved in the ATR after acid-adaptation in Bifidobacterium longum BBMN68 in different growth phases. BBMN68 was cultured at a constant neutral pH during the whole growth phase. Without acid-adaptation, the survival ratios at the lethal pH 3.0 were 0.25% and 17% in the exponential and stationary phases, respectively. The genes ffh, uvrA, groES, and dnaK were significantly higher in the stationary phase than in the exponential phase. The results indicated that although there was no acid stress, the acid tolerance of cells was elevated from the exponential phase into stationary phase. After acid-adaptation at pH 5.0 for 120 min, the survival ratios of BBMN68 in the exponential and stationary phases were increased to 2.5 and 31%, respectively. In the exponential phase, ffh, uvrA groES, and dnaK were significantly decreased after acid-adaptation. In the stationary phase, after acid-adaptation for 15, 60, and 120 min, the genes uvrA, groES, and dnaK were significantly decreased, whereas, ffh was significantly up-regulated at 15 min, and then suppressed at 60 and 120 min after acid-adaptation. The results represented that the ATR in B. longum was different from other bacteria, and ffh may be the transient acid gene.

Intestinal microflora analysis was performed on 52 healthy elderly subjects of different ages and in different regions in Bama County, Guangxi, China. The participants were assigned to three groups depending on their age and location: longevous (group M; mean age = 98 years; n = 21); rural younger elderly (group S; mean age = 70 years; n = 18); and urban elderly (group C; mean age = 82 years; n = 13). Ten groups of bacteria were quantified using real-time PCR. Age-related differences were observed in the number of Clostridium coccoides-Eubacterium rectale--there were more in longevous participants. Region affected the numbers of Bacteroides--Prevotella and Clostridium perfringens subgroup, and longevous participants had significantly more of the two bacterial groups than urban elderly participants. Region-related effects were also observed for the relative abundance of E. coli, and rural elderly participants had a lower proportion. Both age and regional effects were observed in the amount of total bacteria, and longevous participants had higher numbers than urban elderly participants. A significantly higher proportion of lactobacilli was observed in rural younger elderly participants than urban elderly participants, but independent age or regional effects did not contribute to this difference. This study suggests that age and region can affect the intestinal microflora of elderly people.

We studied the effects of two bifidobacteria strains isolated from healthy centenarians on intestinal function in mice. Bifidobacterium adolescentis BBMN23 and Bifidobacterium longum BBMN68 were orally administrated to specific pathogen-free BALB/c mice at different doses (2 x 10(11), 2 x 10(9), or 2 x 10(7) CFU/kg body weight) each day for 4 weeks. Villus height, crypt depth, villus width, and villus/crypt ratio (V/C) were determined. The content of duodenal secreted immunoglobulin A (sIgA) was also evaluated. There were clear increases in height and width of duodenal villi in both treated groups. Crypt depths were deeper in animals treated with BBMN23 than in controls, while depths were reduced in animals receiving BBMN68. The V/C ratio was increased after feeding with BBMN68, while BBMN23 had no significant effect. Both strains improved the sIgA content of the duodenum. These results suggest that BBMN23 and BBMN68 may improve intestinal digestion and ability and enhance immune barrier function in the intestine.

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.