Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

The potential and capability of the methylotrophic yeast Ogataea methanolica in a "methanol bioeconomy".

Efficient bioconversion of methanol, which can be generated from greenhouse gases, into valuable resources contributes to achieving climate goals and developing a sustainable economy. The methylotrophic yeast Ogataea methanolica is considered to be a suitable host for efficient methanol bioconversion because it has outstanding characteristics for the better adaptive potential to a high methanol environment (i.e., greater than 5%). This capacity represents a huge potential to construct an innovative carbon-neutral production system that converts methanol into value-added chemicals under the control of strong methanol-induced promoters. In this review, we discuss what is known about the regulation of methanol metabolism and adaptation mechanisms for 5% methanol conditions in O. methanolica in detail. We also discuss about the potential to breed "super methylotrophic yeast," which has potent growth characteristics under high methanol conditions.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Maltobionic acid accelerates recovery from iron deficiency-induced anemia in rats.

In experiments 1 and 2, effect of ingestion of maltobionic acid calcium salt (MBCa) on recovery of rats from a latent iron deficiency and from iron deficiency anemia was examined, respectively. After grouping rats into control and iron-deficiency groups, a latent iron deficiency or iron-deficiency anemia was induced in the latter group. And recovery from these states by MBCa containing diets (0%, 3%, and 6% MBCa in diet, classified into MBCa-0, MBCa-3, and MBCa-6 groups) was compared for convalescence period in light of iron sufficient control group. In experiment 1, MBCa ingestion significantly increased the iron concentration in the serum and liver, and promoted recovery from a latent iron deficiency. In experiment 2, hemoglobin and hematocrit levels increased significantly with MBCa intake, and recovery from iron-deficiency anemia was promoted. MBCa effectively promoted the recovery of rats from a subclinical iron deficiency and iron-deficiency anemia.Abbreviations: ANOVA: analysis of variance; DMT1: divalent metal transporter 1; EDTA-2Na: disodium salt of ethylenediaminetetraacetic acid; Fpn: feroportin; Hb: hemoglobin; Ht: hematocrit; ICP-OES: inductivity coupled plasma optical emission spectrometer; MBCa: maltobionic acid calcium salt; nitroso-PSAP: 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol; SE: standard error; SI: serum-iron concentration; TSAT: transferrin saturation; TIBC: total iron-binding capacity; UIBC: unsaturated iron-binding capacity.

On Default Priors for Robust Bayesian Estimation with Divergences.

This paper presents objective priors for robust Bayesian estimation against outliers based on divergences. The minimum γ-divergence estimator is well-known to work well in estimation against heavy contamination. The robust Bayesian methods by using quasi-posterior distributions based on divergences have been also proposed in recent years. In the objective Bayesian framework, the selection of default prior distributions under such quasi-posterior distributions is an important problem. In this study, we provide some properties of reference and moment matching priors under the quasi-posterior distribution based on the γ-divergence. In particular, we show that the proposed priors are approximately robust under the condition on the contamination distribution without assuming any conditions on the contamination ratio. Some simulation studies are also presented.

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats.

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO3, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO3 and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO3. It was concluded that MBCa is a better calcium source than CaCO3 in terms of both calcium retention and absorption. Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.

Patient age as a preoperative factor associated with tunnel enlargement following double-bundle anterior cruciate ligament reconstruction using hamstring tendon autografts.

PURPOSE: A few studies have detected associations of post-operative tunnel enlargement with sex, age, and the timing of anterior cruciate ligament reconstruction (ACLR). The aim of the present study was to investigate the correlation between post-operative tunnel enlargement following ACLR using hamstring tendon autografts and preoperative factors. The authors hypothesized that tunnel enlargement is associated with age in patients undergoing ACLR. METHODS: One hundred and six patients (male, n = 57; female, n = 49; mean age, 26.9 years) who underwent double-bundle ACL reconstruction were included in the present study. The time between injury and surgery was 26.3 ± 71.4 weeks. Computed tomographic scans of the operated knee were obtained at 2 weeks and 6 months after surgery. The area of the tunnel aperture was measured for the femoral anteromedial tunnel (FAMT), femoral posterolateral tunnel (FPLT), tibial anteromedial tunnel (TAMT), and tibial posterolateral tunnel. The percentage of tunnel area enlargement was defined as the area at 2 weeks after ACLR subtracted from the area at 6 months after ACLR and then divided by the area at 2 weeks after ACLR. Spearman's correlation coefficient was calculated for each factor. The patients were divided into two groups based on age. Patients aged <40 and ≥40 years were assigned to Groups A and B, respectively. The differences in the outcomes and characteristics of the two groups were evaluated. RESULTS: The percentage of enlargement of the FAMT, FPLT, and TAMT was correlated with patient age (r = 0.31, p = 0.001; r = 0.24, p = 0.012; and r = 0.30, p = 0.002, respectively). In total, 87 and 19 knees were classified into Groups A and B, respectively, based on patient age. The percentage of enlargement of the FAMT was significantly higher in Group B than A (78 vs. 60%, respectively; p = 0.01). The percentage of enlargement of the TAMT was significantly higher in Group B than A (53 vs. 36%, respectively; p = 0.03). CONCLUSION: The percentage of enlargement of the FAMT and TAMT was associated with patient age. These findings suggest the need to consider the possibility of tunnel enlargement when double-bundle ACLR is performed for patients aged >40 years. Age was a preoperative factor associated with tunnel enlargement. LEVEL OF EVIDENCE: III.

The remnant preservation technique reduces the amount of bone tunnel enlargement following anterior cruciate ligament reconstruction.

PURPOSE: The aim of the present study was to investigate the correlation between postoperative tunnel enlargement after ACLR and remnant tissue preservation using the hamstring tendon. METHODS: One hundred and ninety-two subjects (male, n = 101; female, n = 91; mean age 27.1) who had undergone double-bundle ACL reconstruction were included in the present study. The patients were divided into two groups: the remnant tissue preservation group (Group R) and the non-remnant tissue preservation group (Group N). Computed tomographic scans of the operated knee were obtained at 2 weeks and 6 months after surgery. The area of the tunnel aperture for the anteromedial femoral tunnel (FAMT), posterolateral femoral tunnel (FPLT), anteromedial tibial tunnel (TAMT), and posterolateral tibial tunnel (TPLT) was measured. The area at 2 weeks after ACLR was subtracted from the area at 6 months after ACLR and then divided by the area at 2 weeks after ACLR. The differences in the outcomes and characteristics of the two groups were evaluated. RESULTS: Seventy-seven knees were classified into Group R, and 115 knees were classified into Group N. The age, gender, and body mass index did not differ to a statistically significant extent. The percentages of FAMT and TAMT enlargement in Group R were significantly smaller in comparison with Group N (P = 0.003 and P = 0.03, respectively). The percentage of FPLT and TPLT enlargement in the two groups did not differ to a statistically significant extent. CONCLUSION: The remnant-preserving technique reduces the amount of bone tunnel enlargement. The present findings indicate the advantages of the remnant-preserving ACLR technique, and therefore the remnant-preserving technique should be recommended. LEVEL OF EVIDENCE: II.

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats.

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B6 (B6) deficiency in rat liver. Male Wistar rats were fed a control, B6-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B6-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B6-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B6 deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B6 deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.

Resistant starch reduces colonic and urinary p-cresol in rats fed a tyrosine-supplemented diet, whereas konjac mannan does not.

The effect of resistant starch (RS) and konjac mannan (KM) to maintain and improve the large intestinal environment was compared. Wistar SPF rats were fed the following diets for 4 weeks: negative control diet (C diet), tyrosine-supplemented positive control diet (T diet), and luminacoid supplemented diets containing either high-molecular konjac mannan A (KMAT diet), low-molecular konjac mannan B (KMBT diet), high-amylose cornstarch (HAST diet), or heat-moisture-treated starch (HMTST diet). The luminacoid-fed group had an increased content of short-chain fatty acids in the cecum. HAS caused a significant decrease in p-cresol content in the cecum, whereas KM did not. Urinary p-cresol was reduced in the HAST group compared with the T group, but not the KM fed groups. Deterioration in the large intestinal environment was only improved completely in the HAST and HMTST groups, suggesting that RS is considerably more effective than KM in maintaining the large intestinal environment.

A case of intra-articular ganglion cysts of the knee joint: correlation between arthroscopic and magnetic resonance imaging.

BACKGROUND: Intra-articular ganglion cysts of the knee are rare. Here we report a case of an arthroscopically confirmed ganglion cyst arising from the posterior cruciate ligament (PCL) along with preoperative magnetic resonance imaging (MRI) findings. CASE PRESENTATION: A 39-year-old female admitted a hospital with left knee pain with flexion and extension. MRI revealed a cystic lesion along the PCL. The lesion exhibited slight but homogeneous hyperintensity on T1 weighted images. Thin septals were visible within the lesion. Arthroscopic examination revealed a mass lesion with a white fibrous capsule, located near the PCL. A gel-like liquid spurted from the mass upon puncture. The lesion was completely resected. Histological examination revealed loose connective tissue and fibroblasts with collagen, thus confirming the diagnosis of a ganglion cyst. CONCLUSION: Many reports have suggested intra-articular ganglion cysts of the knee are rare. In our study, a cystic lesion may have been impinged between the PCL and intercondylar notch, resulting in flexion and extension difficulty in the left knee. Arthroscopic resection is the major treatment of intra-articular ganglion cyst, and preoperative MRI findings can predict the correct arthroscopic approach. We have reported a case in which an intra-articular ganglion attached to the PCL.

Comparison of the effects of treatment with celecoxib, loxoprofen, and acetaminophen on postoperative acute pain after arthroscopic knee surgery: A randomized, parallel-group trial.

BACKGROUND: Selective cyclooxygenase-2 (COX-2) inhibitors, conventional non-selective nonsteroidal anti-inflammatory drugs (NSAIDs), and acetaminophen have been adopted for the relief of mild to moderate acute and chronic pain. However, it remains unclarified whether the therapeutic differences in pain sensation exist among these agents. The aim of this study was to compare the efficacy of different types of analgesic agents for postoperative acute pain management. METHODS: A single-center, randomized, controlled study was performed in consecutive patients who underwent the second-look procedure with removal of internal fixation after anterior cruciate ligament reconstruction or arthroscopic meniscal repair/meniscectomy. Celecoxib (400 mg for the first dose and then 200 mg), loxoprofen (60 mg), or acetaminophen (600 mg) was orally administered from postoperative 3 h. The pain intensity on a 100-mm VAS scale and subjective assessment of therapeutic pain-relief were compared among these three treatment groups until postoperative 2 days. The acquired data were analyzed according to the per-protocol analysis principle. RESULTS: A total of 432 patients were screened, and 160 were enrolled. The VAS score tended to decrease over time in all groups. There was a significant improvement in the pain score both at rest and on movement, and subjective impression in the celecoxib-treated group compared with acetaminophen at postoperative 2 days. On the other hand, loxoprofen resulted in the benefit only in the pain score at rest in comparison with acetaminophen. Any comparisons between celecoxib and loxoprofen showed insignificant differences throughout observations. No adverse effects were confirmed in each group. CONCLUSIONS: These obtained findings in our dose setting conditions suggest that celecoxib and loxoprofen treatments were superior to acetaminophen in pain-relief, though the superiority of loxoprofen over acetaminophen was modest. Overall, selective COX-2 inhibitors including conventional NSAIDs seem to have a possible advantage in acute pain management of relatively less invasive surgery.

Accumulation of lipid in rat liver was induced by vitamin B6 deficiency and was ameliorated by supplemental phosphatidylcholine in the diet.

We investigated the efficacy of supplementing the diet with pteroylmonoglutamic acid (PGA), choline, or phosphatidylcholine (PC) in ameliorating the lipid accumulation in rat liver that is induced by vitamin B6 (B6) deficiency. In Experiment 1, male Wistar rats were fed a control, B6-deficient, or PGA-, choline-, or PC-supplemented (10 mg, 4 g, and 6.3 g/kg of diet, respectively) B6-deficient diet containing l-methionine at 9 g/kg of diet for 35 days. In Experiment 2, rats were fed a control, B6-deficient, or PC-supplemented (at 3.15, 6.3, or 12.6 g PC/kg of diet) B6-deficient diet for 35 days. Choline or PC supplementation ameliorated liver lipid deposition and returned plasma lipids to normal. Judging from these results, it appeared that B6 deficiency decreased the synthesis of PC in the liver, thereby decreasing the secretion of very low-density lipoproteins, and in consequence producing lipid accumulation in the liver and reductions of plasma lipids.

Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeast Candida boidinii.

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.

Novel physiological roles for glutathione in sequestering acetaldehyde to confer acetaldehyde tolerance in Saccharomyces cerevisiae.

In this work, we identified novel physiological functions of glutathione in acetaldehyde tolerance in Saccharomyces cerevisiae. Strains deleted in the genes encoding the enzymes involved in glutathione synthesis and reduction, GSH1, GSH2 and GLR1, exhibited severe growth defects compared to wild-type under acetaldehyde stress, although strains deleted in the genes encoding glutathione peroxidases or glutathione transferases did not show any growth defects. On the other hand, intracellular levels of reduced glutathione decreased in the presence of acetaldehyde in response to acetaldehyde concentration. Moreover, we show that glutathione can trap a maximum of four acetaldehyde molecules within its molecule in a non-enzymatic manner. Taken together, these findings suggest that glutathione has an important role in acetaldehyde tolerance, as a direct scavenger of acetaldehyde in the cell.

Involvement of 5-methyltetrahydrofolate in the amelioration of hyperhomocysteinemia caused by vitamin B(6) deficiency and L-methionine supplementation.

We attempted to clarify the reason why folate fortification ameliorates hyperhomocysteinemia induced by vitamin B(6) deficiency. Hyperhomocysteinemia caused by vitamin B(6) deficiency significantly decreased the rat liver 5-methyltetrahydrofolate level which was significantly improved by folate fortification. This result suggests that the amelioration of hyperhomocysteinemia in response to folate supplementation had enhanced the removal of homocysteine via methionine synthase.

Genome Sequence of the Lanthanide-Responsive Methylotrophic Bacterium Methylorubrum extorquens Strain GM97.

We present the complete genome sequence of Methylorubrum extorquens strain GM97, which formed large colonies on a 1/100 nutrient plate with samarium (Sm3+). The genome for strain GM97 was estimated to be 7,608,996 bp, which suggests that the strain is closely related to Methylorubrum extorquens strains.

A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter.

Introduction: In Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and lactic acid bacteria such as Leuconostoc can proliferate because of their tolerance toward low temperature and their low nutritional requirements. Later, Lactobacillus becomes the dominant genus, leading to weakly acidic conditions that contribute to control yeasts and undesired bacterial contaminants. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary. Methods: In this study, we performed 16S rRNA amplicon sequencing analysis after sampling from floor, equipment surfaces, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan. Results: Amplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, equipment surfaces, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes present in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the taste and flavor. Discussion: These findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.

Treatment with Interleukin-25 Suppresses Short-Term High-Fructose Diet-Induced Hepatic Gene Expression and Activities of Fatty Acid Synthesis Enzymes in Rats.

This study aimed to investigate the effects of interleukin-25, which belongs to the interleukin-17 family, on short-term high-fructose diet-induced hepatic triacylglycerol accumulation. Rats were fed a high-starch (control) or high-fructose diet for 7 d, with or without intraperitoneal administration of recombinant interleukin-25 from days 3-7. Treatment with interleukin-25 significantly reduced the mRNA levels and activity of fatty acid synthesis enzymes and caused a nominal reduction in hepatic triacylglycerol levels in rats fed a high-fructose diet but not in those fed a control diet. Interleukin-25 treatment did not affect the mRNA levels of ß-oxidation enzymes in either the control or fructose-fed rats. These results suggest that treatment with interleukin-25 suppresses short-term high-fructose diet-induced fatty acid synthesis and leads to the alleviation of triacylglycerol accumulation in the liver.

A unique case in which Kimoto-style fermentation was completed with Leuconostoc as the dominant genus without transitioning to Lactobacillus.

The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation in which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.