The Mechanism of Antimicrobial Activity of Conjugated Bile Acids against Lactic Acid Bacilli.

The mechanism underlying antimicrobial activity of conjugated bile acids against strains of lactic acid bacilli is not well understood. The purpose of this study was to investigate two typical conjugated bile acids (glycochenodeoxycholic acid and taurochenodeoxycholic acid) for their mechanisms of antimicrobial activity against four strains of different species of lactic acid bacilli at the physiological pH of the small intestine of humans. The bacterial cell membrane integrity, transmembrane potential, and transmembrane pH gradient were examined using the fluorescence probes SYTO 9 plus propidium iodide, 3,3'-dipropylthiadicarbocyanine iodide, and 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, respectively. The intracellular ATP levels were measured by the firefly luciferase-based bioluminescence method. It was found that the antimicrobial activity of conjugated bile acids against the strains of lactic acid bacilli is strain-specific, and glycochenodeoxycholic acid showed significantly greater antimicrobial activity than taurochenodeoxycholic acid against the strains of lactic acid bacilli. The conjugated bile acids inhibited the growth of strains of lactic acid bacilli by disrupting membrane integrity, dissipating transmembrane potential, reducing the transmembrane pH gradient, and depleting intracellular ATP. In conclusion, the antimicrobial activity of conjugated bile acids against lactic acid bacilli is a multifactorial phenomenon. This study will provide valuable information for developing strategies to improve the ability of lactic acid bacilli to tolerate bile in vivo.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Bile salt hydrolase activity is present in nonintestinal lactic acid bacteria at an intermediate level.

It is generally considered that bile salt hydrolase (BSH) activity is hardly detected in nonintestinal lactic acid bacteria (LAB). The aim of this study was to investigate the distribution and intensity of BSH activity in LAB isolated from naturally fermented vegetables and milk. A total of 624 lactic acid bacterial strains classified into 6 genera and 50 species were isolated from 144 naturally fermented vegetable samples and 103 naturally fermented milk samples, and their BSH activity was screened by gas chromatography with electron capture detection. The BSH-positive strains were further analyzed quantitatively for their deconjugation ability against six human-conjugated bile salts by HPLC based on the disappearance of the conjugated bile salts from the reaction mixture. The results showed that 39% of the strains possessed BSH activity distributed in 24 lactic acid bacterial species. The strains of the fermented vegetable origin showed a 0.5-fold higher incidence of BSH-positive strains than those of the fermented milk origin, and the lactic acid bacilli exhibited 2.5-fold higher incidence of BSH-positive strains than the lactic acid cocci in general. The strains of the fermented vegetable origin generally had greater bile salt deconjugation ability than those of the fermented milk origin. More than 97% and 93% of the BSH-positive strains exhibited a greater substrate preference for glycoconjugated bile salts than tauroconjugated bile salts and for dihydroxy bile salts than trihydroxy bile salts, respectively. This study demonstrated that BSH activity was also present in nonintestinal LAB.

A comparative analysis of derivatization strategies for the determination of biogenic amines in sausage and cheese by HPLC.

The six biogenic amines in sausage and cheese were analyzed by HPLC with UV detection after off-line derivatization with dansyl chloride, 9-fluorenylmethoxycarbonyl chloride, benzoyl chloride and dabsyl chloride, respectively. The results showed that both the off-line 9-fluorenylmethoxycarbonyl and dabsyl derivatization were not suitable for HPLC analysis of biogenic amines when batch injection was used because the derivatives were instable, whereas both the off-line dansyl and benzoyl derivatization were suitable for HPLC analysis of biogenic amines when batch injection was used, but the latter needed to maintain the derivatives at 4 °C to ensure that benzoylated tyramine was not degraded when waiting for the analysis. The off-line dansyl derivatization had an obvious advantage in the analysis of biogenic amines in sausage and cheese samples by HPLC combined with batch injection because the method has a wider linear range and higher sensitivity, accuracy, precision and stability of the derivatives.

Bile Salt Hydrolase and S-Layer Protein are the Key Factors Affecting the Hypocholesterolemic Activity of Lactobacillus casei-Fermented Milk in Hamsters.

SCOPE: Lactobacillus casei F0822-fermented milk has exhibited significant hypocholesterolemic activity in hamsters in the previous study. Under this premise, the objective of this study is to further explore whether bile salt hydrolase (BSH) and S-layer protein (SLP) of the strain have a significant influence on hypocholesterolemic activity of the fermented milk. METHODS AND RESULTS: Independent and double interposon mutants of BSH and SLP genes are constructed from wild-type L. casei F0822 via chromosomal insertion of chloramphenicol or/and erythromycin resistance genes based on double-crossover homologous recombination. The mutants- and the wild-type strain-fermented milk is prepared (viable counts of approximately 8.0 × 108 colony-forming units mL-1 each) and intragastrically administered to high-cholesterol-fed hamsters once daily at a dose of 1.25 mL d-1 for 28 d, respectively. Both the BSH-deficient mutant- and the SLP-deficient mutant-fermented milk significantly (p < 0.05) increase serum total and LDL-cholesterol levels in hamsters compared with the wild-type strain-fermented milk. However, only the BSH-deficient mutant-fermented milk could significantly (p < 0.05) increase hepatic total and esterified cholesterol levels in hamsters. CONCLUSION: Both BSH and SLP have a significant influence on the hypocholesterolemic activity of L. casei F0822-fermented milk in hamsters. Nevertheless, the BSH is greater than the SLP in this regard.

Hamsters Are a Better Model System than Rats for Evaluating the Hypocholesterolemic Efficacy of Potential Probiotic Strains.

SCOPE: Rats and hamsters are the most commonly used animal models for evaluating the hypocholesterolemic activity of potential probiotic strains, whereas little or no information has been reported on whether the animal models would affect the experimental conclusions regarding the hypocholesterolemic efficacy of the strains. METHODS AND RESULTS: Both high-cholesterol-fed rats and hamsters were intragastrically administered viable cells of bile salt hydrolase-active Lactobacillus acidophilus K16 once daily (1 × 1010 CFU per kg body weight) for 28 d. It was found that the strain did not significantly (p > 0.05) affect the serum and hepatic cholesterol levels in rats, whereas it significantly decreased (p < 0.01 or p < 0.001) the serum total and non-HDL-cholesterol as well as hepatic-free, esterified, and total cholesterol levels in hamsters by 29.6%, 38.8%, 15.8%, 36.2%, and 34.0%, respectively. CONCLUSION: These data suggest that the hypocholesterolemic efficacy of L. acidophilus K16 is substantially different between high-cholesterol-fed hamsters and rats and that hamsters are a better model system than rats for evaluating the hypocholesterolemic efficacy of potential probiotic strains due to their similarity to humans in biliary bile acid composition, including types of bile acids and their conjugation form.

Bile acid patterns in commercially available oxgall powders used for the evaluation of the bile tolerance ability of potential probiotics.

This study aimed to analyze the bile acid patterns in commercially available oxgall powders used for evaluation of the bile tolerance ability of probiotic bacteria. Qxgall powders purchased from Sigma-Aldrich, Oxoid and BD Difco were dissolved in distilled water, and analyzed. Conjugated bile acids were profiled by ion-pair high-performance liquid chromatography (HPLC), free bile acids were detected as their p-bromophenacyl ester derivatives using reversed-phase HPLC after extraction with acetic ether, and total bile acids were analyzed by enzymatic-colorimetric assay. The results showed that 9 individual bile acids (i.e., taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid) were present in each of the oxgall powders tested. The content of total bile acid among the three oxgall powders was similar; however, the relative contents of the individual bile acids among these oxgall powders were significantly different (P < 0.001). The oxgall powder from Sigma-Aldrich was closer to human bile in the ratios of glycine-conjugated bile acids to taurine-conjugated bile acids, dihydroxy bile acids to trihydroxy bile acids, and free bile acids to conjugated bile acids than the other powders were. It was concluded that the oxgall powder from Sigma-Aldrich should be used instead of those from Oxoid and BD Difco to evaluate the bile tolerance ability of probiotic bacteria as human bile model.

A new method for the in vitro determination of the bile tolerance of potentially probiotic lactobacilli.

A new in vitro method was developed to determine the bile tolerance of potentially probiotic lactobacilli. The overnight culture of various lactobacilli strains was inoculated into sterile, half-strength MRS broth supplemented with and without 0.3% (wt/vol) oxgall, buffered with 0.1 M sodium phosphate buffer at a final pH of 7.3, and incubated at 37 °C for 12 h under anaerobic conditions. The bile tolerance ability of the lactobacilli strains was expressed as the percentage of the propagation generations of the bacterial cells in the presence of oxgall to those in the absence of oxgall. The bile tolerance ability of 11 strains of 8 Lactobacillus species, including 3 bile salt hydrolase (BSH)-negative strains and 8 BSH-positive strains, was analyzed using the newly developed method and two traditional methods. The results showed that bile tolerance ability of the strains was considerably different depending on the analysis method used. The newly developed method mimics the physiological environment of the human small intestine, and avoids changes in pH and bile salt composition during the incubation period, which are drawbacks of the traditional bile tolerance test methods. Therefore, the analysis method developed in this study is more suitable to screen or compare the bile tolerance ability of lactobacilli strains.

Over-expression of prolyl isomerase Pin1 promotes cervical tumorigenesis and metastasis.

Overexpression of the prolyl isomerase PIN1 is involved in tumorigenesis, but the role of PIN1 in cervical cancer is unclear. In this study, we examined PIN1 protein expression by immunohistochemistry in 221 paraffin-embedded samples from cervical cancer patients, cervical intraepithelial neoplasia patients, and control tissues, and found that high expression of PIN1 was significantly associated with lymph node metastasis (P=0.002), advanced stage according to the International Federation of Gynecology and Obstetrics guidelines (P=0.026). When endogenous PIN1 expression was knocked down using siRNA, cell proliferation, colony formation, migration, and invasion were inhibited in the SiHa cervical cancer cell line. Additionally, PIN1 knockdown increased E-cadherin and ß-catenin expression, and decreased expression of N-cadherin and Vimentin, suggesting that PIN1 can promote epithelial-mesenchymal transition (EMT). These results indicate that the Overexpression of the prolyl isomerase PIN1 in cervical cancer indicates tumor-Promotive properties of PIN1 that may be a marker of poor prognosis in cervical cancer patients, and the molecular determinants of epithelial polarity which have tumorigenesis enhancing impact, might through EMT.

Screening for cholesterol-lowering probiotic based on deoxycholic acid removal pathway and studying its functional mechanisms in vitro.

Elevated serum cholesterol in humans is generally a risk factor correlated with the development of coronary heart disease (CHD). Reducing deoxycholic acid (DCA) content in the intestine can reduce serum cholesterol levels, which reduce the incidence of CHD. A total of 150 strains of lactic acid bacteria and bifidobacteria were isolated from human fecal samples. The DCA removal ability of these strains was evaluated. Results showed that 9 strains displayed above 10% DCA removal rate. The probiotic potentials of the 9 strains were evaluated. The strain Lactobacillus casei F0822 was screened out due to the stronger adhesion to HT-29 cells and tolerance to bile and acid. DCA removal for this strain resulted from that the S-layer protein locating the cell surface bound DCA. The FTIR spectra showed that the carboxyl group in DCA was the principal group by which DCA was bound to the S-layer protein of L. casei F0822. These findings suggested that L. casei F0822 is a better candidate probiotic strain, which has the potential to reduce human serum cholesterol levels.

Effect of tween series on growth and Cis-9, trans-11 conjugated linoleic acid production of Lactobacillus acidophilus F0221 in the presence of bile salts.

Cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) producing bacteria have attracted much attention as novel probiotics which have shown beneficial effects on host health. However, bile salts are able to inhibit bacterial growth and c9, t11 CLA production. For recovering growth and c9, t11 CLA production of Lactobacillus acidophilus F0221 in the presence of bile salts, Tween series (Tween 20, Tween 40, Tween 60 and Tween 80) were added in growth culture containing 0.3% oxgall. Results showed that the viable counts were significantly (P < 0.05) recovered to 8.58-8.75 log CFU/mL in the presence of all Tween treatments. However, recovery of c9, t11 CLA production was only demonstrated in the presence of Tween 80 (72.89 µg/mL). Stepwise increasing oxgall in a concentrations range from 0.1% to 0.9% according to human intestinal physiological environments, Tween 80 still showed significant (P < 0.05) recovery ability on growth (8.91-8.04 log CFU/mL) and c9, t11 CLA (69.22-34.27 µg/mL) production. The effect of Tween 80 on growth and production was also investigated in the presence of different types of bile salts (sodium salts of cholic acid (CA), deoxycholic acid (DCA), chendeoxycholic acid (CDCA), glycocholic acid (GCA) and taurocholic acid (TCA)). Results showed that Tween 80 could significantly (P < 0.05) recover c9, t11 CLA production in the presence of all types of bile salts, but the Tween 80 could only significantly (P < 0.05) recover viable counts of the strain in the presence of CA, DCA and CDCA. This recovery ability could be attributed to the protection of leakage of intracellular material. Additionally, although bile salts inhibited growth and c9, t11 CLA production by the growing cell, it promoted the c9, t11 CLA production by the resting cell.

Inhibition of Shigella sonnei adherence to HT-29 cells by lactobacilli from Chinese fermented food and preliminary characterization of S-layer protein involvement.

In this study, seven lactobacilli with a high degree of antagonistic activity against three pathogens and good adherence to HT-29 cells were selected. The ability of these seven lactobacilli to inhibit adhesion of Shigella sonnei to intestinal mucosa was studied on cultured HT-29 cells. Lactobacilli were added simultaneously with, before or after S. sonnei to test for their effectiveness in exclusion, competition and displacement assays, respectively. Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L and Lactobacillus casei Q8-L all exhibited significant inhibitory activity. In order to elucidate the inhibitory functions of S-layer proteins, the S-layer proteins were removed with 5 M LiCl from the M5-L, J10-L and Q8-L strains. Under such conditions, inhibition activity was decreased in all three strains, as revealed in exclusion, competition and displacement assays. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 45 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 40.5%, 41.5% and 43.8% of the total amino acid for the M5-L, J10-L and Q8-L strains, respectively. These findings suggest that the M5-L, J10-L and Q8-L strains possess the ability to inhibit S. sonnei adherence to HT-29 cells, and S-layer proteins are involved in this adhesion inhibition.