New Inhibitory Effect of Latilactobacillus sakei UONUMA on the Cholesterol Biosynthesis Pathway in Human HepG2 Cells.

Many pharmaceuticals and dietary foods have been reported to inhibit cholesterol biosynthesis, mainly by inhibiting the presqualene enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase rather than a postsqualene enzyme. In this study, we examined the inhibitory effects of Latilactobacillus sakei UONUMA on cholesterol biosynthesis, especially postsqualene, in human HepG2 hepatoma cells. We quantified cholesterol and its precursors, and the mRNA and protein levels of enzymes involved in cholesterol biosynthesis. Three L. sakei UONUMA strains exhibited new inhibitory effects on cholesterol biosynthesis and inhibited the mRNA level of sterol-delta24-reductase (DHCR24), which is involved in the postsqualene cholesterol biosynthesis pathway. These strains will be useful for the prevention and treatment of hyperlipidemia.

Regulatory effect of paraprobiotic Lactobacillus gasseri CP2305 on gut environment and function.

BACKGROUND: Lactobacillus gasseri CP2305 (CP2305) is a strain of Lactobacillus isolated from a stool sample from a healthy adult that showed beneficial effects on health as a paraprobiotic. In a previous study, we demonstrated that CP2305-fermented heat-treated milk modified gut functions more than artificially acidified sour milk. Thus, the regulatory activity of the former beverage was attributed to the inactivated CP2305 cells. OBJECTIVE: The aim of this study was to elucidate the contribution of non-viable paraprobiotic CP2305 cells to regulating human gut functions. We thus conducted a randomized, placebo-controlled, double-blinded parallel group trial. DESIGN: The trial included 118 healthy participants with relatively low or high stool frequencies. The test beverage was prepared by adding 1×10(10) washed, heat-treated, and dried CP2305 cells directly to the placebo beverage. The participants ingested a bottle of the assigned beverage daily for 3 weeks and answered daily questionnaires about defecation and quality of life. Fecal samples were collected and the fecal characteristics, microbial metabolite contents of the feces and composition of fecal microbiota were evaluated. RESULTS: The number of evacuations and the scores for fecal odors were significantly improved in the group that consumed the CP2305-containing beverage compared with those of the group that consumed the placebo (p=0.035 and p=0.040, respectively). Regarding the fecal contents of microbial metabolites, the level of fecal p-cresol was significantly decreased in the CP2305 group relative to that of the placebo group (p=0.013). The Bifidobacterium content of the intestinal microbiota was significantly increased in the CP2305 group relative to that of the placebo group (p<0.008), whereas the content of Clostridium cluster IV was significantly decreased (p<0.003). The parasympathetic nerve activity of the autonomic nervous system became dominant and the total power of autonomic activity was elevated in the CP2305 group (p=0.0401 and p=0.011, respectively). CONCLUSIONS: The continuous ingestion of heat-treated CP2305 cells clearly affected intestinal functionality. This is the first report of sterilized Lactobacillus cells having a significant impact on the environment and functions of the intestinal tract. The observed effects might be due, at least in part, to the brain-gut interaction.

Oral administration of the milk casein-derived tripeptide Val-Pro-Pro attenuates high-fat diet-induced adipose tissue inflammation in mice.

Inflammation of adipose tissue triggers the metabolic syndrome, atherosclerosis and CHD. In the present study, we investigated whether the milk casein-derived tripeptide valine-proline-proline (VPP) has an anti-inflammatory effect on the adipose tissue of high-fat diet (HFD)-fed mice. Male C57BL/6J mice (7 weeks of age) were fed ad libitum with either a HFD and plain tap water (HFD group) or a HFD and water containing 0·3 mg VPP/ml (HFD+VPP group) for 10 weeks. The results showed that the expression level of CD18 in the peripheral blood monocytes of the HFD+VPP group was significantly decreased compared with the level observed in those of the HFD group. Activated monocytes and pro-inflammatory macrophages were accumulated in the stromal vascular fractions of the adipose tissue from HFD-fed mice, which were significantly decreased in those supplemented with VPP. The formation of crown-like structures rich in pro-inflammatory macrophages was also significantly reduced in the adipose tissue of mice administered with VPP. Real-time PCR analysis revealed that the expression of monocyte chemoattractant protein-1 and that of the pro-inflammatory cytokine IL-6 in adipose tissue tend to be lower in the HFD+VPP group than in the HFD group. These observations indicate that oral administration of VPP exerts an anti-inflammatory effect on the adipose tissue of HFD-fed mice, which may eventually lead to the primary prevention of chronic inflammation-related diseases.

Preventive Roles of Rice-koji Extracts and Ergothioneine on Anxiety- and Pain-like Responses under Psychophysical Stress Conditions in Male Mice.

This study determined the effect of daily administration of Rice-koji on anxiety and nociception in mice subjected to repeated forced swim stress (FST). In a parallel experiment, it was determined whether ergothioneine (EGT) contained in Rice-koji displayed similar effects. Anxiety and nociception were assessed behaviorally using multiple procedures. c-Fos and FosB immunoreactivities were quantified to assess the effect of both treatments on neural responses in the paraventricular nucleus of the hypothalamus (PVN), nucleus raphe magnus (NRM), and lumbar spinal dorsal horn (DH). FST increased anxiety- and pain-like behaviors in the hindpaw. Rice-koji or EGT significantly prevented these behaviors after FST. In the absence of formalin, both treatments prevented decreased FosB expressions in the PVN after FST, while no effect was seen in the NRM and DH. In the presence of formalin, both treatments prevented changes in c-Fos and FosB expressions in all areas in FST mice. Further, in vitro experiments using SH-SY5Y cells were conducted. Rice-koji and EGT did not affect cell viability but changed the level of brain-derived neurotrophic factor. In conclusion, Rice-koji could reduce anxiety and pain associated with psychophysical stress, possibly mediated by the modulatory effects of EGT on neural functions in the brain.

Effect of casein hydrolysate, prepared with protease derived from Aspergillus oryzae, on subjects with high-normal blood pressure or mild hypertension.

Casein hydrolysate, prepared with Aspergillus oryzae protease, contains angiotensin I-converting enzyme inhibitory peptides, such as Val-Pro-Pro and Ile-Pro-Pro. We conducted a randomized, double-blind, placebo-controlled study to evaluate the effect of casein hydrolysate on the blood pressure of 144 subjects with high-normal blood pressure (n = 104) and mild hypertension (n = 40). Subjects were randomly assigned to two groups for a 12-week intake period. In the test group, both systolic (SBP) and diastolic (DBP) blood pressure decreased significantly compared with the placebo group: SBP/DBP significantly decreased from 138.2 +/- 6.5/84.4 +/- 5.3 mm Hg at week 0 to 132.3 +/- 7.3 (P < .001)/81.2 +/- 4.8 mm Hg (P < .001) at week 12. In the stratified analysis, the test product showed an antihypertensive effect in both the subject group with high-normal blood pressure and that with mild hypertension. No side effect was observed in any subjects in this study. These results demonstrate that the casein hydrolysate, prepared with A. oryzae protease, produced a significant reduction in blood pressure in a population of subjects with high-normal blood pressure or mild hypertension without an adverse event.

Casein-derived tripeptide, Val-Pro-Pro (VPP), modulates monocyte adhesion to vascular endothelium.

AIM: A food-derived bioactive tripeptide, Val-Pro-Pro (VPP), has been shown to possess angiotensin I-converting enzyme (ACE) inhibitory activity and foods containing such peptides exhibit an anti-hypertensive effect in clinical settings. METHODS: The present study focused on the effect of VPP on monocyte adhesion to endothelium under flow conditions using phorbol 12-myristate 13-acetate (PMA)-stimulated monocytic THP-1 cells. RESULTS: Pre-incubation of THP-1 cells with VPP (1 mM, 24 hours) significantly decreased the PMA-induced adhesion of THP-1 cells (p<0.05) to human umbilical vein endothelial cells (HUVECs). PMA-induced up-regulation of beta1 and beta2 integrin activation in THP-1 cells was downregulated by VPP, which significantly suppressed only the PMA-induced phosphorylation of JNK (p<0.05) in THP-1 cells. In addition, preincubation of THP-1 with SP600125, a specific inhibitor of JNK, resulted in significant reduction of the PMA-induced adhesion of THP-1. Interestingly, another tripeptide with comparable ACE inhibitory activity, Leu-Gly-Pro (LGP), failed to reduce the PMA-induced adhesion of THP-1, suggesting a distinct anti-inflammatory effect of VPP on THP-1 adhesion. CONCLUSION: These observations suggest that VPP moderates monocyte adhesion to inflamed endothelia via attenuation of the JNK pathway in monocytes, which might contribute to the primary prevention of atherosclerosis.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): IX. Evaluation of the mutagenic potential of synthesized L-valyl-L-prolyl-L-proline in the Salmonella-Escherichia coli/microsome, incorporation assay.

The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential.

Effect of powdered fermented milk with Lactobacillus helveticus on subjects with high-normal blood pressure or mild hypertension.

OBJECTIVE: Two tripeptides (Val-Pro-Pro and Ile-Pro-Pro) that have inhibitory activities for angiotensin I-converting enzyme are produced in milk fermented with Lactobacillus helveticus. In this study we evaluated the effect and safety of powdered fermented milk with L. helveticus CM4 on subjects with high-normal blood pressure or mild hypertension. METHODS: A randomized, placebo-controlled, double-blind study was conducted using 40 subjects with high-normal blood pressure (HN group) and 40 subjects with mild hypertension (MH group). Each subject ingested 6 test tablets (12 g) containing powdered fermented milk with L. helveticus CM4 daily for 4 weeks (test group) or the same amount of placebo tablets for 4 weeks (placebo group). RESULTS: During treatment, the decrease in systolic blood pressure (SBP) and diastolic blood pressure (DBP) in the test group tended to be greater than in the placebo group for both blood pressure groups. At the end of treatment (week 4), a significant decrease in DBP in the HN group was observed (i.e. 5.0 mm Hg (0.1, 9.9; p = 0.04) compared with the placebo group). There was no significant change in SBP (3.2 mm Hg (95% CI -2.6, 8.9; p = 0.27). In the MH group, SBP decreased by 11.2 mm Hg (4.0, 18.4; p = 0.003) and there was a statistically non-significant decrease in DBP of 6.5 mm Hg (-0.1, 13.0; p = 0.055) compared with the placebo group. No marked changes were observed in other indexes, including pulse rate, body weight and blood serum variables, and no adverse effects attributed to the treatment was found in each group. CONCLUSIONS: Daily ingestion of the tablets containing powdered fermented milk with L. helveticus CM4 in subjects with high-normal blood pressure or mild hypertension reduces elevated blood pressure without any adverse effects.