Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial.

Objectives: The aims of this study were to select heat-killed lactic acid bacteria (HKL) with antibiotic activity and investigate the efficacy of this bacteria in maintaining periodontal parameters in healthy participants. Materials and methods: An in vitro evaluation was conducted to assess the inhibitory efficacy of lactic acid bacteria against Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum. The effects of HKL administration on various parameters (plaque control record, bleeding on probing, and probing pocket depth) were assessed in a randomized, placebo-controlled trial. Participants in the test and placebo groups (n = 32) consumed oral tablets containing placebo or HKL daily for 8 weeks. Oral bacteria in supra-plaque and saliva were identified using 16S rRNA gene community profiling analysis. Results: Heat-killed Ligilactobacillus salivarius CP3365 significantly (p < 0.05) decreased the viability of oral bacteria and was selected for clinical trials. Administration of HKL CP3365 significantly (p < 0.05) inhibited increases in each parameter. No changes in the relative abundance of P. gingivalis or F. nucleatum subsp. nucleatum were detected by HKL CP3365, but the relative abundance of oral bacteria (genera Porphyromonas, Fusobacterium, and Haemophilus) was significantly (p < 0.05) decreased. Conclusion: HKL CP3365 effectively inhibited oral bacteria growth and was useful for maintaining periodontal health. Clinical Trial Registration: [https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045656].

Effects of Prebiotic Yeast Mannan on Gut Health and Sleep Quality in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Study.

Human gut health is closely related to sleep. We aimed to evaluate the efficacy of yeast mannan (YM) in improving bowel habits and sleep quality, along with metabolomics in fecal samples. A total of 40 healthy adults (age range, 22-64 years) with discomfort in defecation were enrolled and randomly allocated to receive either YM (n = 20; 1.1 g/day) or placebo (n = 20) for four weeks. Participants recorded their defecation habits throughout the test periods. Sleep electroencephalogram (EEG) recording using an EEG device and fecal sampling were performed pre- and post-treatment. The YM group significantly increased defecation frequency and stool volumes compared to the placebo group. After 4 weeks of treatment, the non-REM sleep stage 3 (N3) duration in the YM group was significantly higher than that in the placebo group. YM ingestion significantly lengthened total time in bed (TIB) and significantly shortened N3 latency compared to placebo intake during the trial. The metabolomics analysis found a total of 20 metabolite differences between the YM and placebo groups. As a result of stepwise linear regression, changes in fecal propionate and gamma-aminobutyric acid (GABA) levels were identified as the primary factors explaining changes in TIB and N3 latency, respectively. Our findings suggest that the prebiotic YM could be beneficial to gut health and sleep quality.

Effects of Lactobacillus gasseri CP2305 on Mild Menopausal Symptoms in Middle-Aged Women.

Lactobacillus gasseri CP2305 (CP2305) is a paraprobiotic that exhibits beneficial effects on the intestinal function and microbiota, and increases resistance to psychological stress. The stress response mechanism mainly involves the hypothalamic-pituitary-adrenal axis, which is influenced by the gut-brain axis. Furthermore, the gut-brain axis also communicates bidirectionally with the intestinal microbiota. Additionally, the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes share a common route that affects both mental and health aspects in women. This double-blind, placebo-controlled, parallel-group clinical trial aimed to analyze the influence of the intake of CP2305 on mild symptoms associated with menopause. Eighty women aged 40-60 years ingested CP2305 or placebo tablets for six consecutive menstrual cycles. Assessment was based on the observation of climacteric symptoms with two validated questionnaires-the Simplified Menopausal Index (SMI) and the Greene Climacteric Scale (GCS). The results showed that CP2305 provided significant relief in the SMI total score, SMI vasomotor score, SMI psychological score, GCS total score, GCS somatic score, and GCS vasomotor score compared to the placebo. The percentage of women with symptom relief for the SMI total score was 75.0%, with 30 of 40 women in the CP2305 group, and 55.0%, with 22 of 40 women in the placebo group (p = 0.0594). These findings provide new insights into the function of paraprobiotic CP2305 in relieving mild climacteric symptoms in women.

Inhibitory Effect of the Glycerophosphate Moiety of Lipoteichoic Acid from Lactic Acid Bacteria on Dexamethasone-Induced Atrogin-1 Expression in C2C12 Myotubes.

Atrogin-1, which is an important regulator of ubiquitin-mediated protein degradation in skeletal muscle, is a major marker of muscle loss and disuse muscle atrophy. To investigate which components of lactic acid bacteria (LAB) suppress dexamethasone (DEX)-induced atrogin-1 expression, mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of components of LAB. Heat-killed cells and lipoteichoic acid (LTA) derived from five LAB strains significantly suppressed DEX-induced atrogin-1 expression. The glycerophosphate (GroP) fraction prepared from chemically-degraded LTA and sn-glycerol-1-phosphate suppressed DEX-induced atrogin-1 expression, whereas the glycolipid anchor fraction of LTA did not. Heat-killed cells obtained by culturing under low-Mn2+ conditions, which generated fewer poly-GroP polymers in LTA, displayed significantly lower inhibitory activity compared to heat-killed cells grown under normal conditions. These results suggested that LTA of LAB contributed to suppressing atrogin-1 expression and that the GroP moiety of LTA was responsible for its inhibitory activity.

D- and l-amino acid concentrations in culture broth of Lactobacillus are highly dependent on the phylogenetic group of Lactobacillus.

d-amino acids produced by Lactobacillus are thought to contribute to the taste quality and health functions; however, no studies have comprehensively evaluated the concentrations of the D- and L-forms of amino acids separately in individual Lactobacillus strains. To gain insight into amino acid concentrations in Lactobacillus, we evaluated amino acid concentrations in culture broth of Lactobacillus separately for the D- and L-forms. Lactobacillus strains were cultured in culture broth, and the amino acid concentrations in supernatant were assessed. The amino acid concentrations obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were subjected to cluster analysis based on Bray-Curtis distance with Ward's minimum variance method. In the analysis of amino acid concentrations under culture with different monosaccharides, the distances among strains cultured with the same monosaccharide were significantly greater than those among cultures of the same strain under different monosaccharides (p < 0.01). The cluster analysis of amino acid concentrations under culture with the same monosaccharide suggested that strains belonging to the same phylogenetic group of Lactobacillus exhibited similar concentrations of amino acids. Data analyses of 70 strains belonging to 17 Lactobacillus taxa indicated that the concentrations of amino acids were highly dependent on the phylogenetic group of Lactobacillus and that the group differences in amino acid concentration were strongly driven by differences in l-serine and d-alanine concentrations. Our results indicate that it is important to evaluate D- and l-amino acids separately when evaluating variations in amino acid concentrations. Because d-alanine has the potential to affect taste quality, the results of this study may provide insight into the taste quality of fermented food produced by Lactobacillus.

Effects of Yeast Mannan Which Promotes Beneficial Bacteroides on the Intestinal Environment and Skin Condition: A Randomized, Double-Blind, Placebo-Controlled Study.

Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall. In vitro fecal fermentation studies showed that YM could exhibit a notable prebiotic effect. The aim of this randomized, double-blind, placebo-controlled study was to assess the efficacy of YM intake on the intestinal environment and skin condition. One hundred and ten healthy female subjects aged 30-49 years were supplemented with YM or placebo for eight weeks. Skin dryness was set as the primary endpoint. No side effects were observed during the study. Microbiota analyses revealed that YM intake selectively increased the relative abundance of Bacteroides thetaiotaomicron and Bacteroides ovatus compared to that by placebo. Feces and urine analyses showed that YM intake lowered the concentration of fecal p-cresol, indole, and skatole, and elevated urinal equol levels compared to those in placebo. Furthermore, YM supplementation ameliorated subjective skin dryness. This study suggests that YM intake could promote beneficial Bacteroides and improve the intestinal environment and skin condition.

The influence of hyperglycemia on neutrophil extracellular trap formation and endothelial glycocalyx damage in a mouse model of type 2 diabetes.

OBJECTIVES: Hyperglycemia induces vascular dysfunction that is thought to be initiated by neutrophils. Neutrophil activation produces endothelial injury by pathways that include NETosis, a type of specific cell death. In this study, we investigated the effects of hyperglycemia on neutrophil activation, cell death, NETosis, and endothelial glycocalyx damage using a mouse diabetes model. METHODS: We used db/db mice as a type 2 diabetes model, and C57BL/6 mice were the controls. At 5, 8, and 12 weeks of age, the proportion of CD11b+ granulocytes/monocytes, neutrophil extracellular trap (NET)-forming granulocytes/monocytes, and damaged and nonviable granulocytes/monocytes was analyzed. In addition, serum levels of high mobility group box 1, histone H3, and glycocalyx components that included syndecan-1 and hyaluronan were measured. RESULTS: In diabetic mice, we observed an increased proportion of CD11b+ granulocytes/monocytes. The proportion of NET-forming granulocytes/monocytes increased from the early stages of the experiments. The proportions of damaged and nonviable granulocytes/monocytes increased over time. In the 12-week-old diabetic mice, serum histone H3 levels increased. Circulating levels of syndecan-1 and hyaluronan decreased over time and were lower in diabetic mice. CONCLUSION: Neutrophil activation and cell death induce endothelial glycocalyx damage, and NET formation also participates in the mechanisms of vascular injury in type 2 diabetes.

Daily Intake of Paraprobiotic Lactobacillus amylovorus CP1563 Improves Pre-Obese Conditions and Affects the Gut Microbial Community in Healthy Pre-Obese Subjects: A Double-Blind, Randomized, Placebo-Controlled Study.

Despite the fact that gut microbiota is closely associated with obesity, few studies have focused on the influences of paraprobiotics as food ingredients on both obesity prevention and the gut microbial community. In this study, we evaluated the effects of fragmented Lactobacillus amylovorus CP1563 (CP1563) as a paraprobiotic for obesity prevention and investigated its effects on the gut microbial community in pre-obese subjects. One hundred sixty-nine healthy subjects with a body mass index from 25.0 to 29.9 kg/m2 ingested beverages with or without the fragmented CP1563 containing 10-hydroxyoctadecanoic acid (10-HOA) for 12 weeks. The changes in abdominal, total, visceral, and subcutaneous fatty areas were significantly lower in the CP1563-10-HOA group than in the placebo group at 12 weeks. Furthermore, 16S rRNA gene sequencing of fecal DNA revealed that the changes in the abundances of the genera Roseburia and Lachnospiraceae;g were significantly greater in the CP1563-10-HOA group than in the placebo group, and the changes in the abundances of the genus Collinsella was significantly smaller in the CP1563-10HOA group than in the placebo group. Our results showed that continuous ingestion of the fragmented CP1563 containing 10-HOA reduced abdominal body fat and affected the gut microbial community in pre-obese healthy subjects. Our findings may contribute to the understanding of the relationship between the anti-obesity effect of paraprobiotics and gut microbiota.

Additive effect of Lactobacillus acidophilus L-92 on children with atopic dermatitis concomitant with food allergy.

BACKGROUND: Atopic dermatitis (AD) in infants is often related to food allergies (FA). The beneficial effects of lactic acid bacteria towards allergic diseases have been reported, but there are few reports on their effect and preferable dosages on AD in young children with concomitant FA. OBJECTIVE: To examine additional effects of two different dose of paraprobiotic Lactobacillus acidophilus L-92 (L-92) on the clinical treatment in young children afflicted by AD with diagnosed or suspected FA. METHODS: Fifty-nine AD young children from 10 months to 3 years old, with FA or who had not started to ingest specific food(s) because of high specific IgE levels, were recruited and randomly allocated into L-92 group (daily intake of 20 mg L-92/day) and placebo group. Participants were given test sample with conventional treatment for AD over a 24-week period. The severity of eczema was evaluated using SCORing Atopic Dermatitis (SCORAD) index before intervention, and at 4, 12, and 24 weeks after intervention. RESULTS: After 24 weeks of intervention, a significant decrease in SCORAD was observed only in the L-92 group when compared with the baseline values. Significant decreases in thymus and activation-regulated chemokine (TARC) and total IgE were also detected 24 weeks after intake in the L-92 group compared with the placebo group. CONCLUSION: It was suggested that intake of sufficient amounts of L-92 works as an adjunctive treatment of young children afflicted by AD with diagnosed or suspected FA.

Protection of the endothelial glycocalyx by antithrombin in an endotoxin-induced rat model of sepsis.

INTRODUCTION: Injury and loss of the endothelial glycocalyx occur during the early phase of sepsis. We previously showed that antithrombin has a protective effect on this structure in vitro. Here, we investigated the possible protective effects of antithrombin in an animal model of sepsis. METHODS: Wistar rats were injected with endotoxin, and circulating levels of syndecan-1, hyaluronan, albumin, lactate and other biomarkers were measured in an antithrombin-treated group and an untreated control group (n = 6 in each group). Intravital microscopy was used to observe leukocyte adhesion, microcirculation, and syndecan-1 staining. RESULTS: The circulating levels of syndecan-1 and hyaluronan were significantly reduced in the antithrombin-treated group, compared with the untreated controls. Lactate levels and albumin reduction were significantly attenuated in the antithrombin-treated group. Intravital microscopic observation revealed that both leukocyte adhesion and blood flow were better maintained in the treatment group. The syndecan-1 lining was disrupted after endotoxin treatment, and this derangement was attenuated by treatment with antithrombin. CONCLUSION: Antithrombin effectively maintained microcirculation and vascular integrity by protecting the glycocalyx in a rat sepsis model.

Protective effect of a newly developed fucose-deficient recombinant antithrombin against histone-induced endothelial damage.

Antithrombin is expected to modulate both prothrombotic and proinflammatory reactions in sepsis; vascular endothelium is the primary target. In the present study, we sought to evaluate the protective effects of a newly developed fucose-deficient recombinant antithrombin. Endothelial cells were treated in vitro with histone H4 to induce cellular damage. Low to high doses of either plasma-derived antithrombin or recombinant thrombomodulin were used as treatment interventions. Morphological change, apoptotic rate, cell viability, cell injury, and syndecan-4 level in the medium were evaluated. Immunofluorescent staining with anti-syndecan-4 was also performed. Both types of antithrombin reduced cellular damage and apoptotic cell death. Both plasma-derived and recombinant antithrombin improved cell viability and reduced cellular injury when administered at a physiological concentration or higher. Syndecan-4 staining became evident after treatment with histone H4, and both antithrombins suppressed the staining intensity at similar levels. The syndecan-4 level in the medium was significantly decreased by both antithrombins. None of the indicators showed a significant difference between plasma-derived and recombinant antithrombin. In conclusion, both recombinant and plasma-derived antithrombin can protect vascular endothelial cells. Recombinant antithrombin may represent a useful new therapeutic agent for sepsis-associated vascular damage.

Efficacy of prolonged ingestion of Lactobacillus acidophilus L-92 in adult patients with atopic dermatitis.

To evaluate the safety and efficacy of prolonged ingestion of Lactobacillus acidophilus L-92 (L-92) on skin symptoms in adult atopic dermatitis (AD) patients, a placebo-controlled double-blinded parallel-group comparison study was performed. This included daily administration of heat-killed and dried L-92 or placebo for 24wk in 50 AD patients who were 16yr old or older. The severity of skin symptoms was evaluated at baseline and at 4, 8, 12, 16, 20, and 24wk during the intervention using the investigator global assessment, eczema area and severity index, and scoring atopic dermatitis. Serum cytokine and blood marker levels were also measured at baseline and at 4, 8, 16, and 24wk during the intervention. No adverse events were reported during the study period. Compared with the placebo group, the L-92 group showed significant decreases in investigator global assessment, eczema area and severity index, and scoring atopic dermatitis. Subjective symptoms in adult AD patients were reduced by intake of L-92. Furthermore, it was suggested that sustained ingestion of L-92 resulted in suppression of scratching behavior and maintenance of remission status of skin symptoms. Sixteen weeks after the study commenced, a significant decrease in lactate dehydrogenase and a significant increase in transforming growth factor-ß were observed in the L-92 group compared with the placebo group. In the L-92 group, a significant elevation of IL-12 (p70) level at the end of treatment period compared with before the treatment was observed. This study suggested that L-92 suppresses type-2-helper-T-cell-dominant inflammation by activating regulatory T cells and type 1 helper T cells.

Highly sensitive detection of influenza virus in saliva by real-time PCR method using sugar chain-immobilized gold nanoparticles; application to clinical studies.

A highly sensitive and convenient method for detecting influenza virus was developed using modified end-point melt curve analysis of a RT-qPCR SYBR Green method and influenza virus-binding sugar chain-immobilized gold-nanoparticles (SGNP). Because SGNPs capture influenza viruses, the virus-SGNP complex was separated easily by centrifugation. Viral RNA was detected at very low concentrations, suggesting that SGNP increased sensitivity compared with standard methods. This method was applied to clinical studies. Influenza viruses were detected in saliva of patients or inpatients who had been considered influenza-free by a rapid diagnostic assay of nasal swabs. Furthermore, the method was applied to a human trial of prophylactic anti-influenza properties of yogurt containing Lactobacillus acidophilus L-92. The incidence of influenza viruses in saliva of the L-92 group was found to be significantly lower compared to the control group. Thus, this method was useful for monitoring the course of anti-influenza treatment or preventive measures against nosocomial infection.

Lactobacillus acidophilus L-92 Cells Activate Expression of Immunomodulatory Genes in THP-1 Cells.

To understand the immunomodulatory effects of Lactobacillus acidophilus L-92 cells suggested from our previous study of in vivo anti-allergy and anti-virus effects, host immune responses in macrophage-like THP-1 cells after 4 h (the early phase) and 24 h (the late phase) of cocultivation with L-92 cells were investigated by transcriptome analysis. In the early phase of L-92 treatment, various transcription regulator genes, such as, NFkB1, NFkB2, JUN, HIVEP2 and RELB, and genes encoding chemokines and cytokines, such as CCL4, CXCL11, CCL3 and TNF, were upregulated. Two transmembrane receptor genes, TLR7 and ICAM1, were also upregulated in the early phase of treatment. In contrast, many transmembrane receptor genes, such as IL7R, CD80, CRLF2, CD86, CD5, HLA-DQA1, IL2RA, IL15RA and CSF2RA, and some cytokine genes, including IL6, IL23A and CCL22, were significantly upregulated in the late phase after L-92 exposure. Some genes encoding cytokines, such as IL1A, IL1B and IL8, and the enzyme IDO1 were upregulated at both the early and the late phases of treatment. These results suggest that probiotic L-92 might promote Th1 and regulatory T-cell responses by activation of the MAPK signaling pathway, followed by the NOD-like receptor signaling pathway in THP-1 cells.

The milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro attenuate arterial dysfunction in L-NAME-treated rats.

Both endothelial dysfunction and arterial stiffness are surrogate markers of atherosclerosis and thus cardiovascular (CV) events. The milk-derived peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) inhibit angiotensin-converting enzyme, dilate blood vessels ex vivo and stimulate nitric oxide (NO) production in cells. In this study, we investigated the effects of either VPP or IPP on arterial function and on target organ damage in vivo. Male Wistar rats were treated with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 g l(-1)), L-NAME+VPP (0.3 g l(-1)) or L-NAME+IPP (0.3 g l(-1)) in their drinking water for 8 weeks. Plasma nitrite and nitrate (NOx) levels were significantly increased in normal Wistar rats after supplementation with either VPP or IPP but not in rats that were chronically treated with L-NAME. Acetylcholine-induced vasorelaxation in the thoracic aorta ring was impaired by L-NAME, whereas vasorelaxation was significantly greater in mice treated with L-NAME+VPP for 1 or 4 weeks or L-NAME+IPP for 4 weeks than in mice treated with L-NAME alone. Four weeks of treatment with either VPP or IPP attenuated the increase in pulse wave velocity (PWV) that was induced by L-NAME. Cardiac and renal damage were observed after 8 weeks of treatment with L-NAME, and either VPP or IPP attenuated this damage. These results show that VPP or IPP attenuates arterial dysfunction and suggest that milk-derived peptides might prevent CV damage.

Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate atherosclerosis development in apolipoprotein e-deficient mice: a preliminary study.

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1ß, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.

Anti-influenza virus effects of both live and non-live Lactobacillus acidophilus L-92 accompanied by the activation of innate immunity.

The antiviral effects of both a live and non-live Lactobacillus acidophilus strain L-92 (L-92) were investigated by oral administration (10 mg/mouse per d) daily for 21 d in a mouse model infected intranasally with influenza virus (H1N1). Virus titres in the lung of mice administered either live or non-live L-92 cells daily for 15 d were repressed 6 d after virus infection compared with the control group. Natural killer (NK) activity in the orally administered non-live L-92 group was higher compared with that of the control group before virus infection and on day 6. In contrast, NK activity in the live L-92 group compared with the control group was not significantly changed on both days, but was significantly higher on day 1. In contrast, live L-92 showed a greater repression of virus proliferation compared with non-live L-92, 6 d after the infection. Live L-92 decreased the number of neutrophils in the lung and suppressed lung weight, leading to the consequent deterioration of consolidation scores of the lung. These results indicated that pretreatment of live or non-live L-92 cells had protective effects against influenza virus infection. Among the measured cytokines and chemokines, eotaxin, macrophage colony-stimulating factor, IL-1b, RANTES (regulated on activation, normal T cell expressed and secreted) and interferon-a were significantly increased in the lung: IL-17 was significantly increased in Peyer's patch of the live L-92 group compared with the control group. A mechanistic study suggested that the enhancement of NK activity in the lung caused by stimulating various antiviral cytokines and chemokines after the oral administration of L-92 cells might be important in protecting against virus infection.

Transcriptional response of Lactobacillus acidophilus L-92 after attachment to epithelial Caco-2 cells.

Transcriptome analysis showed that Lactobacillus acidophilus L-92 cells having anti-allergy effects on human up-regulated 41 genes involved permease, ABC transporter, proteinase and transcriptional regulator after attached to epithelial Caco-2 cells. Inversely, 37 genes were down-regulated, including ATP synthases, ABC transporters and transcriptional regulators.

Milk casein-derived tripeptides, VPP and IPP induced NO production in cultured endothelial cells and endothelium-dependent relaxation of isolated aortic rings.

Milk casein-derived tripeptides, valyl prolyl proline (VPP), and isoleucyl prolyl proline (IPP) inhibit angiotensin-converting enzyme (ACE) and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans. On the top of these results, we have recently reported that the hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, enforcing us to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides. For this purpose, we examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO(x) (NO(2) and NO(3)) concentration in the medium was significantly higher than that of the control. Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists. These lines of results suggested that both VPP and IPP induced production of vasodilative substances including NO.

Casein hydrolysate containing the antihypertensive tripeptides Val-Pro-Pro and Ile-Pro-Pro improves vascular endothelial function independent of blood pressure-lowering effects: contribution of the inhibitory action of angiotensin-converting enzyme.

Accumulating evidence shows that deterioration of vascular endothelial function underlies the pathophysiology of cardiovascular diseases following lifestyle-related diseases. Both Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which are tripeptides derived from proteolytic hydrolysate of milk casein, inhibit angiotensin-converting enzyme (ACE), suggesting that both VPP and IPP may improve vascular endothelial function, because many ACE inhibitors are known to improve endothelial function. We investigated the effects of ACE-inhibitory food component in humans with mild hypertension, since there has been no report on such effects. The study was conducted by the placebo-controlled, double-blind crossover method in 25 male subjects with mild hypertension. After casein hydrolysate containing both VPP and IPP were administered for 1 week, reactive hyperemia of the left upper forearm was measured using plethysmography as an index of vascular endothelial function. Since one subject dropped out, we analyzed the data of 24 subjects. The reactive hyperemia of the left upper forearm was produced by a 5 min occlusion using inflation of a cuff. The maximum blood flow during reactive hyperemia was 20.8+/-6.7 mL/min/100 mL tissue in the placebo group, whereas it increased remarkably to 30.0+/-10.4 mL/min/100 mL tissue in the group administered casein hydrolysate containing both VPP and IPP (p<0.001). There was no change in systemic blood pressure, indicating that the improvement of the vascular endothelial function attributable to VPP and IPP is independent of hemodynamic changes. We conclude that casein hydrolysate containing VPP and IPP improves the vascular endothelial dysfunction in subjects with mild hypertension. The continuous intake of VPP and IPP could help to prevent cardiovascular diseases in hypertensive subjects.