Daily Intake of D-ß-Hydroxybutyric Acid (D-BHB) Reduces Body Fat in Japanese Adult Participants: A Randomized, Double-Blind, Placebo-Controlled Study.

Currently, there is considerable interest in ketone metabolism owing to the benefits for human health. Conventionally, strict dietary restrictions on carbohydrates are required to increase plasma ketone levels, while supplementation with D-ß-hydroxybutyric acid (D-BHB) can easily increase plasma ketone levels. We hypothesized that a daily intake of D-BHB could promote weight loss, especially through fat reduction. Herein, D-BHB (OKETOATM) was produced via a proprietary fermentation process from sugar. In this randomized, double-blind, placebo-controlled study, we assessed the safety and fat-reduction effects after 12 wk of daily ingestion of D-BHB (2.9 g) in 22 healthy Japanese adults and 22 control participants. Blood samples were collected pre- and post-treatment. Blood chemistry, anthropometric variables, and the body composition of the participants were investigated. Data analysis revealed that visceral fat at 12 wk significantly decreased by 9.0 cm2 (p=0.037), as evidenced by analysis of covariance. Blood parameters and body condition showed no significant differences between the two groups, and the participants reported no adverse effects or discomfort. Furthermore, data were analyzed by regrouping the participants. After removing one suspicious diabetes participant, all others showed significant decreases in visceral fat, body weight, BMI, and fat weight. Additionally, those aged under 50 y old had significantly decreased abdominal circumference and body fat percentage, in addition to visceral fat, body weight, BMI, and fat weight. Overall, our findings indicate that daily D-BHB intake may reduce body fat without dieting or exercise intervention. This study was registered with the UMIN Clinical Trials Registry as UMIN000045322.

Effect of Exogenous Acute ß-Hydroxybutyrate Administration on Different Modalities of Exercise Performance in Healthy Rats.

PURPOSE: A ketone body (ß-hydroxybutyrate [ß-HB]) is used as an energy source in the peripheral tissues. However, the effects of acute ß-HB supplementation on different modalities of exercise performance remain unclear. This study aimed to assess the effects of acute ß-HB administration on the exercise performance of rats. METHODS: In study 1, Sprague-Dawley rats were randomly divided into six groups: endurance exercise (EE + PL and EE + KE), resistance exercise (RE + PL and RE + KE), and high-intensity intermittent exercise (HIIE + PL and HIIE + KE) with placebo (PL) or ß-HB salt (KE) administration. In study 2, metabolome analysis using capillary electrophoresis mass spectrometry was performed to profile the effects of ß-HB salt administration on HIIE-induced metabolic responses in the skeletal and heart muscles. RESULTS: The maximal carrying capacity (rest for 3 min after each ladder climb, while carrying heavy weights until the rats could not climb) in the RE + KE group was higher than that in the RE + PL group. The maximum number of HIIE sessions (a 20-s swimming session with a 10-s rest between sessions, while bearing a weight equivalent to 16% of body weight) in the HIIE + KE group was higher than that in the HIIE + PL group. However, there was no significant difference in the time to exhaustion at 30 m·min -1 between the EE + PL and the EE + KE groups. Metabolome analysis showed that the overall tricarboxylic acid cycle and creatine phosphate levels in the skeletal muscle were higher in the HIIE + KE group than those in the HIIE + PL group. CONCLUSIONS: These results indicate that acute ß-HB salt administration may accelerate HIIE and RE performance, and the changes in metabolic responses in the skeletal muscle after ß-HB salt administration may be involved in the enhancement of HIIE performance.

Stress increases blood beta-hydroxybutyrate levels and prefrontal cortex NLRP3 activity jointly in a rodent model.

AIM: This study aimed to assess the response of endogenous beta-hydroxybutyrate to psychological stress, and its association with nucleotide-binding domain, leucine-rich repeat, pyrin domain-containing 3 (NLRP3) inflammasome, and stress-induced behavior. METHODS: Male C57BL/6J mice were subjected to 1-hour restraint stress to examine changes in the endogenous beta-hydroxybutyrate and active NLRP3 levels in the prefrontal cortex. Subsequently, we created a depression model applying 10-day social defeat stress to the male C57BL/6J mice. RESULTS: One-hour restraint stress rapidly increased beta-hydroxybutyrate levels in the blood. The active NLRP3 levels in the prefrontal cortex also increased significantly. A correlation was found between the increased beta-hydroxybutyrate levels in the blood and the active NLRP3 levels in the prefrontal cortex. The mice exposed to social defeat stress exhibited depression- and anxiety-like behavioral changes in the open field, social interaction, and forced swim tests. There was a correlation between these behavioral changes and endogenous beta-hydroxybutyrate levels. Among the social defeat model mice, those with high beta-hydroxybutyrate levels tended to have more depression- and anxiety-like behavior. CONCLUSIONS: The increased blood beta-hydroxybutyrate levels due to psychological stress correlate with the active NLRP3 levels in the prefrontal cortex, suggesting that the increased beta-hydroxybutyrate levels due to stress may reflect a reaction to brain inflammation. In addition, mice with higher blood beta-hydroxybutyrate levels tend to exhibit increased depression- and anxiety-like behaviors; thus, an increase in blood beta-hydroxybutyrate levels due to stress may indicate stress vulnerability.

In vitro human colonic microbiota utilises D-ß-hydroxybutyrate to increase butyrogenesis.

The ketone body D-ß-hydroxybutyrate (DBHB) has gained attention owing to its cellular signalling function; however, its effect on the human colonic microbiota remains unclear. Here, DBHB dynamics in the human colon were investigated using an in vitro colonic microbiota model, which maintained most of the operational taxonomic units detected in the original faeces. Over 54% of 0.41% (w/v) DBHB was metabolised by microbiota models originating from seven faecal samples after 30 h of fermentation (regarded as DBHB utilisers); however, <19% of DBHB was metabolised by microbiota models from five faecal samples (regarded as non-utilisers of DBHB). In utilisers, DBHB administration increased the relative abundance of the genus Coprococcus, correlated with increased butyrogenesis. Increased butyrogenesis was not observed in DBHB non-utilisers. Based on PICRUSt analysis, the relative abundance of ß-hydroxybutyrate dehydrogenase was maintained in microbiota models from DBHB utilisers following DBHB administration; however, it decreased in microbiota models from non-utilisers. After 21 h of fermentation, the intracellular glutamate concentration, which is indicative of growth, showed a positive correlation with DBHB utilisation (R2 = 0.70). Human colonic microbiotas with high growth activity demonstrate efficient utilisation of DBHB for increased butyrate production, which affords health benefits.

Efficient production and secretion of oxaloacetate from Halomonas sp. KM-1 under aerobic conditions.

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize glucose for the intracellular storage of the bioplastic poly-(R)-3-hydroxybutyric acid (PHB) and extracellular secretion of pyruvate under aerobic conditions. In this study, we investigated the effects of sodium chloride concentration on PHB accumulation and pyruvate secretion in the KM-1 strain and, unexpectedly, observed that oxaloacetate, an important intermediate chemical in the TCA cycle, glycogenesis, and aspartic acid biosynthesis, was secreted. We then further analyzed oxaloacetate productivity after changing the sodium chloride additive concentration, additive time-shift, and culture temperature. In 42-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 39.0 g/L oxaloacetate at a rate of 0.93 g/(L h). The halophilic bacteria Halomonas has already gained attention for industrial chemical-production processes owing to its unique properties, such as contamination-free culture conditions and a tolerance for high substrate concentrations. Moreover, no commercial scale oxaloacetate production was previously reported to result from bacterial fermentation. Oxaloacetate is an important intermediate chemical in biosynthesis and is used as a health food based on its role in energy synthesis. Thus, these data provided important insights into the production of oxaloacetate and other derivative chemicals using this strain.

Efficient production and secretion of pyruvate from Halomonas sp. KM-1 under aerobic conditions.

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain.

Greenhouse gas emissions from the treatment of household plastic containers and packaging: replacement with biomass-based materials.

The purpose of this study was to quantify the life-cycle greenhouse gas (GHG) emissions reduction that could be achieved by replacement of fossil-derived materials with biodegradable, biomass-based materials for household plastic containers and packaging, considering a variety of their treatment options. The biomass-based materials were 100% polylactide or a combination of polybutylene succinate adipate and polylactide. A scenario analysis was conducted considering alternative recycling methods. Five scenarios were considered: two for existing fossil-derived materials (the current approach in Japan) and the three for biomass-based materials. Production and waste disposal of 1 m(3) of plastic containers and packaging from households was defined as the functional unit. The results showed that replacement of fossil-derived materials with biomass-based materials could reduce life-cycle GHG emissions by 14-20%. Source separation and recycling should be promoted. When the separate collection ratio reached 100%, replacement with biomass-based materials could potentially reduce GHG emissions by 31.9%. Food containers are a priority for replacement, because they alone could reduce GHG emissions by 10%. A recycling system for biomass-based plastics must be carefully designed, considering aspects such as the transition period from fossil-derived plastics to biomass-based plastics.

Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 by nitrate fed-batch cultivation with glucose under microaerobic conditions.

To establish a sustainable society, commodity chemicals need to be developed from biomass resources. Recently, (R)-3-hydroxybutyric acid ((R)-3-HB), a monomer of bioplastic poly-(R)-3-hydroxybutyric acid (PHB), has attracted attention for its possible use in the chemical industry. Halophilic bacteria have been considered for bioprocess applications due to certain characteristics such as the ability to grow in media containing high levels of the starting carbon source and the ability to be rarely contaminated. A halophilic bacterium Halomonas sp. KM-1 stores PHB intracellularly under aerobic conditions and secretes (R)-3-HB under microaerobic conditions. In this study, we optimized culture conditions to maximize (R)-3-HB secretion by KM-1 cells. By a simple nitrate fed-batch cultivation, Halomonas sp. KM-1 secreted 40.3g/L (R)-3-HB with a productivity of 0.48g L(-1)h(-1) with 20% (w/v) glucose. This level is one of the highest recorded productivity of (R)-3-HB to date.

Co-digestion of polylactide and kitchen garbage in hyperthermophilic and thermophilic continuous anaerobic process.

Two series of two-phase anaerobic systems, consisting of a hyperthermophilic (80°C) reactor and a thermophilic (55°C) reactor, fed with a mixture of kitchen garbage (KG) and polylactide (PLA), was compared with a single-phase thermophilic reactor for the overall performance. The result indicated that ammonia addition under hyperthermophilic condition promoted the transformation of PLA particles to lactic acid. The systems with hyperthermophilic treatment had advantages on PLA transformation and methane conversion ratio to the control system. Under the organic loading rate (OLR) of 10.3 g COD/(L day), the PLA transformation ratios of the two-phase systems were 82.0% and 85.2%, respectively, higher than that of the control system (63.5%). The methane conversion ratios of the two-phase systems were 82.9% and 80.8%, respectively, higher than 70.1% of the control system. The microbial community analysis indicated that hyperthermophilic treatment is easily installed to traditional thermophilic anaerobic digestion plants without inoculation of special bacteria.

Promotion of polylactide degradation by ammonia under hyperthermophilic anaerobic conditions.

The objective of this study was to evaluate the promotion effect of ammonia on the biodegradation of polylactide (PLA) under hyperthermophilic (80°C) and thermophilic (55°C) anaerobic condition. The results showed that PLA was transformed to lactic acid under hyperthermophilic conditions, but that the transformation ratio was negligible under thermophilic conditions. The hydrolysis process can be markedly increased with ammonia addition and microorganism activity. The maximum transformation ratios of the two kinds of PLA used in this study were 65.2% and 51.8%, respectively, with ammonia addition of 4 g N/L over 3 days treatment of anaerobic sludge. After the hyperthermophilic pretreatment, the hydrolysis products were converted to methane by methanogens under the thermophilic and anaerobic conditions. The final methane conversion ratios of the two kinds of PLA after 22 days treatment were 81.8% and 77.0%, respectively.

Methylothermus thermalis gen. nov., sp. nov., a novel moderately thermophilic obligate methanotroph from a hot spring in Japan.

A novel moderately thermophilic methanotroph, strain MYHT(T), was isolated from a hot spring in Japan. The isolate grew on methane or methanol at 37-67 degrees C, and optimally at 57-59 degrees C. It was found to be a Gram-negative aerobe, with colourless colonies of non-motile coccoid cells, possessing type I intracytoplasmic membranes and regularly arranged surface layers of linear (p2) symmetry. Strain MYHT(T) expressed only the particulate methane monooxygenase and employed the ribulose monophosphate pathway for formaldehyde assimilation. It is a neutrophilic and halotolerant organism capable of growth at pH 6.5-7.5 (optimum pH 6.8) and in up to 3% NaCl (optimum 0.5-1% NaCl). Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain MYHT(T) is most closely related to the thermophilic undescribed methanotroph 'Methylothermus' HB (91% identity) and the novel halophilic methanotroph Methylohalobius crimeensis 10Ki(T) (90% identity). Comparative sequence analysis of particulate methane monooxygenase (pmoA) genes also confirmed the clustering of strain MYHT(T) with 'Methylothermus' HB and Methylohalobius crimeensis 10Ki(T) (98 and 92% derived amino acid sequence identity, respectively). The DNA G+C content was 62.5 mol%. The major cellular fatty acids were C(16:0) (37.2%) and C(18:1)omega9c (35.2%) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. On the basis of comparative phenotypic and genotypic characteristics, a new genus and species, Methylothermus thermalis gen. nov., sp. nov., is proposed, with MYHT(T) as the type strain (=VKM B-2345(T)=IPOD FERM P-19714(T)).