Bioavailability of acetate from two vinegar supplements: capsule and drink.

The bioavailability of acetate in various vinegar supplements, e.g. as capsules and drinks, remains unclear. Thus, we conducted a cross-over clinical study in 30 healthy subjects. After an overnight fast, subjects received each test sample in a randomised sequence: 9 vinegar capsules (containing 750 mg acetic acid in total) with 150 mL of water, 100 mL of vinegar drink (containing 750 mg acetic acid), and 150 mL of water as reference. Blood samples were collected before (defined as 0 min), at 15, 30, 45, 60, 90, 120 and 180 min after each test sample intake. In the vinegar drink group, serum acetate concentration increased immediately after intake, peaked at 15 min and returned to baseline at 90 min. That in the vinegar capsule group rose slowly, peaked at 30 min and returned to baseline at 120 min. The peak values in both groups exceeded 200 µmol/L, the physiologically active concentration confirmed by in vitro experiment. In the reference group, levels remained constant throughout the 180-min period. The amount of absorbed acetate from the vinegar capsule group and the drink group was evaluated by the difference value of the area under the serum acetate concentration-time curve (AUC) between in each vinegar group and in the reference group (expressed as AUC(capsule-ref) and AUC(drink-ref ), respectively). AUC(capsule-ref) was about 80% of AUC(drink-ref ), but there was no significant difference between them.

Preparation of (13)C-labeled ceramide by acetic acid bacteria and its incorporation in mice.

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with (13)C-labeled acetic acid. The GC/MS spectrum of the trimethylsilyl derivative of (13)C-labeled dihydroceramide gave molecular ions with an increased mass of 12-17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, (13)C-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [(13)C]dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to (13)C-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [(13)C]sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [(13)C]sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.

Effect of continuous ingestion of acetic Acid bacteria on memory retention and the synaptic function in aged rats.

We administered Acetobacter malorum NCI1683 (S24), containing a high concentration of dihydroceramide (7.2 mg/g of dry cell weight), consecutively to aged rats (male Crlj:Wistar rats, 22 months old). The ingestion of Acetobacter malorum for 89 d significantly extended the memory retention in passive avoidance tests, increased the release of acetylcholine with depolarization of brain synaptosomes and decreased the causative agents of neurodegenerative diseases in the cerebral cortices.

Cloning and characterization of clpB in Acetobacter pasteurianus NBRC 3283.

The clpB gene in Acetobacter pasteurianus was cloned and characterized. Although the clpB gene was transcribed in response to a temperature shift and exposure to ethanol, the clpB disruption mutant was only affected by high temperature, suggesting that the ClpB protein is closely associated with heat resistance in A. pasteurianus.

Vinegar intake enhances flow-mediated vasodilatation via upregulation of endothelial nitric oxide synthase activity.

This study examined the effect of acetate on endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) by immunoblotting assay and the ability of acetic acid to upregulate flow-mediated vasodilatation in humans. In HUVECs, acetate induced a biphasic increase in the phosphorylated form of eNOS. The amount of phosphorylated eNOS was significantly increased by exposure to 200 mumol/l acetate for 20 min (early phase) and for 4 h (late phase). The inhibitors of cAMP-dependent protein kinase (PKA) and AMP-activated protein kinase (AMPK) blocked acetate-induced eNOS phosphorylation in the early and the late phase respectively. Furthermore, in postmenopausal women, maximum forearm blood flow (FBF) in response to shear stress increased in the vinegar (acetic acid) administered group compared to the placebo group. These results suggest that acetic acid-induced eNOS phosphorylation contributes to upregulation of flow-mediated vasodilatation in humans.

Acetic acid bacterial lipids improve cognitive function in dementia model rats.

Acetic acid bacteria, fermentative microorganisms of traditional foods, have unique alkali-stable lipids (ASL), such as dihydroceramide which is a precursor of sphingolipids. Sphingolipids are important components of the brain tissue. We examined the effect of oral administration of ASL in a rat model of dementia (7-week-old, male) with a basal forebrain lesion. In a water maze test, the dementia model rats demonstrated poor spatial orientation. The administration of ASL (165 or 1650 mg/kg of body weight per day, for 14 days) produced a significant improvement in learning ability in the dementia model rats. In vitro experiments showed ASL had the ability to promote neurite outgrowth in pheochromocytoma (PC12) cells. Among the ASL components, dihydroceramide has the most potent effect on the differentiation of PC12 cells. It is highly possible that oral administration of dihydroceramide-containing ASL reverses the decline in cognitive function in dementia.

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources.

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h(-1). The 13C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO2 production rate and the biomass concentration decreased, where the 13C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.

Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283.

The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.

Elevation of ceramide in Acetobacter malorum S24 by low pH stress and high temperature stress.

Acetic acid bacteria have unique and highly pure membrane lipid components, such as 2-hydroxypalmitoyl-sphinganine (dihydroceramide) and can grow and produce acetic acid at around pH 3.0, suggesting that ceramide in cell membranes may be involved in the tolerance to acidic pH. Acetobacter malorum S24 was selected for the production of ceramide and grown in YPG medium containing 0.8% ethanol. Ceramide biosynthesis was induced at pH 4 and below, suggesting that ceramide biosynthesis is induced by low pH stress. Elevation of ceramide was also induced by high temperature stress (40-70 degrees C). After the strain was cultured in an optimal growth medium, the cells were collected and treated at pH 3 and 40 degrees C for 4 days, resulting in a 30-fold elevation of both the yield and content of ceramide.

Vinegar intake reduces body weight, body fat mass, and serum triglyceride levels in obese Japanese subjects.

Acetic acid (AcOH), a main component of vinegar, recently was found to suppress body fat accumulation in animal studies. Hence we investigated the effects of vinegar intake on the reduction of body fat mass in obese Japanese in a double-blind trial. The subjects were randomly assigned to three groups of similar body weight, body mass index (BMI), and waist circumference. During the 12-week treatment period, the subjects in each group ingested 500 ml daily of a beverage containing either 15 ml of vinegar (750 mg AcOH), 30 ml of vinegar (1,500 mg AcOH), or 0 ml of vinegar (0 mg AcOH, placebo). Body weight, BMI, visceral fat area, waist circumference, and serum triglyceride levels were significantly lower in both vinegar intake groups than in the placebo group. In conclusion, daily intake of vinegar might be useful in the prevention of metabolic syndrome by reducing obesity.

Acetic acid upregulates the expression of genes for fatty acid oxidation enzymes in liver to suppress body fat accumulation.

We investigated the effect of acetic acid (AcOH) on the prevention of obesity in high-fat-fed mice. The mice were intragastrically administrated with water or 0.3 or 1.5% AcOH for 6 weeks. AcOH administration inhibited the accumulation of body fat and hepatic lipids without changing food consumption or skeletal muscle weight. Significant increases were observed in the expressions of genes for peroxisome-proliferator-activated receptor alpha (PPARalpha) and for fatty-acid-oxidation- and thermogenesis-related proteins: acetyl-CoA oxidase (ACO), carnitine palmitoyl transferase-1 (CPT-1), and uncoupling protein-2 (UCP-2), in the liver of the AcOH-treatment groups. PPARalpha, ACO, CPT-1, and UCP-2 gene expressions were increased in vitro by acetate addition to HepG2 cells. However, the effects were not observed in cells depleted of alpha2 5'-AMP-activated protein kinase (AMPK) by siRNA. In conclusion, AcOH suppresses accumulation of body fat and liver lipids by upregulation of genes for PPARalpha and fatty-acid-oxidation-related proteins by alpha2 AMPK mediation in the liver.

Acetic acid activates PKD1L3-PKD2L1 channel--a candidate sour taste receptor.

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca2+ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.

Hydrogen peroxide resistance of Acetobacter pasteurianus NBRC3283 and its relationship to acetic acid fermentation.

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.

Identification of two major ammonia-releasing reactions involved in secondary natto fermentation.

Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.