Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Deficiency of ß-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308.

In this study, we investigated a deleterious mutation in the ß-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial ß-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose levels during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

Homozygous gene disruption in diploid yeast through a single transformation.

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

Reduction of the alcohol-stimulative taste of Japanese pot-distilled spirits.

The Japanese traditional pot-distilled spirit shochu has various tastes that are produced by variations in the manufacturing processes. In this study, an alcohol-stimulative taste was organoleptically evaluated using shochu samples, and the chemical components and proton nuclear magnetic resonance (1H NMR) spectra were measured. In some shochu samples, the alcohol-stimulative taste was weaker than that of the standard 15% (v/v) EtOH-H2O mixture, and the water-ethanol hydrogen-bonding structure was stronger compared to a water-ethanol solution. However, some shochu samples filtered with ion-exchange resin had a strong alcohol-stimulative taste comparable to that of the standard 15% (v/v) EtOH-H2O mixture, and the hydrogen-bonding structure was found to be similar to that of the water-ethanol solution. We also investigated the effect of MgCl2 on reducing the alcohol-stimulative taste, and it was observed most strongly with in shochu samples filtered with ion-exchange resin. The change in chemical shift values of the 1H NMR spectra was also the largest in ion-exchange resin filtered shochu samples. The sensory reduction in the alcohol-stimulative taste could be enhanced by the strengthening of the water-ethanol hydrogen-bonding structure. Shochu samples contained many components in larger quantities compared to vodkas. It was found that MgCl2 could reduce the alcohol-stimulative taste of shochu samples. Some salts, such as MgCl2, can be introduced into spirits through the water used to dilute the ethanol content before bottling the products. Our results indicated that some components, such as MgCl2, present in water used can reduce the alcohol-stimulative taste of different spirits produced worldwide.

The effect of Aspergillus luchuensis pectin methylesterase genes pmeA and pmeB on methanol production in sweet potato shochu.

To reduce the methanol content in sweet potato shochu, we studied the pectin methylesterase genes of the shochu-koji mold Aspergillus luchuensis. We found the following three homologs of pectin methyleseterase in the genome of A. luchuensis: pmeA, pmeB, and pmeC. Using pectin as a substrate, the methanol-producing activity of the recombinant of each gene expressed in A. luchuensis was examined and found to be present in recombinant PmeA and PmeB. Additionally, small-scale fermentation of sweet potato shochu using disruptions of pmeA and pmeA-pmeB in A. luchuensis (∆pmeA and ∆pmeApmeB) resulted in significant reduction of the methanol content. Taken together, we revealed that the A. luchuensis pmeA gene was mainly involved in methanol production in sweet potato shochu.

Characterization of aroma profiles of kokuto-shochu prepared from three different cultivars of sugarcane.

Kokuto-shochu is a traditional Japanese spirit prepared from kokuto, obtained by evaporating water from sugarcane (Saccharum officinarum L.) juice. To clarify the effects of sugarcane cultivars on the sensory quality of kokuto-shochu, we investigated the flavor characteristics and composition of volatiles in kokuto-shochu prepared from kokuto using three different sugarcane cultivars, NiF8, Ni15, and RK97-14. Furthermore, experiments were conducted by using the cultivars collected between 2018 and 2020 to observe annual variations in their properties. The amino acid content of the three kokuto varieties did not differ significantly, but the amino acid content of NiF8 was two to five times higher than that of RK97-14, which was the same for all samples collected in the selected years. The browning degrees of kokuto were also higher in NiF8, and they were positively correlated to the amino acid contents of kokuto. The kokuto-like aroma of shochu made from Ni15 was stronger than that of shochu made from RK97-14. The concentration of ethyl lactate in shochu made from Ni15 was higher, however, the concentration of guaiacol was the lowest in the three cultivars' products. Shochu made from NiF8 had the highest levels of Maillard reaction products (MRPs; pyrazines and furans), ß-damascenone, and guaiacol amounts. In contrast, shochu made from RK97-14 tended to have a fruity flavor, and lower MRP levels than those made from NiF8. Thus, it was shown that sugarcane cultivars affect the sensory characteristics and volatiles in kokuto-shochu.

Effect of Koji on Flavor Compounds and Sensory Characteristics of Rice Shochu.

Koji is an important starter for rice shochu brewing and influences the rice shochu quality. Consequently, we studied the impacts of koji on the flavor compounds and sensory characteristics of rice shochu using molds Aspergillus kawachii SICC 3.917 (A-K), Aspergillus oryzae SICC 3.79(A-O), Aspergillus Niger CICC 2372 (A-N), Rhizopus oryzae CICC 40260 (R-O), and the traditional starter Qu (control). The effects of koji on the aroma components, free amino acids (FAAs), and overall sensory aspects of rice shochu were studied. These findings indicated that koji significantly affected the rice shochu's quality. The content of total FAAs in rice shochu A-K (30.586 ± 0.944 mg/L) and A-O (29.919 ± 0.278 mg/L) was higher than others. The content of flavor compounds revealed that the aroma of rice shochu with various koji varied greatly from the smells of alcohols and esters. Shochu A-O had a higher concentration of aroma compounds and it exhibited a strong aroma and harmonious taste compared with the others. This research using taste compounds, FAAs, flavor intensity, and partial least squares regression (PLSR) showed that shochu A-O appeared to possess the best sensory qualities, with elevated concentrations of alcohols and sweet FAAs and lesser concentrations of sour FAAs. Therefore, the A-O mold is promising for the manufacture of rice shochu with excellent flavor and sensory characteristics.

Ameliorative effect of the oil components derived from sweet potato shochu on impaired short-term memory in mice after amyloid ß25-35 injection.

Sweet potato shochu oil is a by-product of shochu production and usually discarded although some physiological functions are considered. In this study, we investigated the effects of shochu oil on short-term memory using a murine model of spontaneous alternating behavior induced by the intracerebroventricular (ICV) administration of amyloid ß25-35 (Aß25-35). Mice were orally administered shochu oil for 15 days. Experiments with a Y-maze model revealed that the Aß25-35 caused a significant decrease in spontaneous alternation behavior, and supplementation with shochu oil significantly improved this behavior. DNA microarray analysis revealed that the administration of shochu oil downregulated the expression of S100a9 and Ptgs2, which reportedly exacerbate amyloid ß deposition in Alzheimer's disease. The administration of shochu oil upregulated the expression of Dnaja1 and PP2A, which is typically downregulated in Alzheimer's disease. These data suggest that shochu oil possible ameliorates on impaired short-term memory in mice after amyloid ß25-35 injection, as indicated by its effects on improving spontaneous alternation behavior and modulating the expressions of related genes.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

Effects of feeding wood kraft pulp silage containing sweet-potato shochu distillery by-product on feed intake, feed digestibility, rumen fermentation, blood components, and growth performance in Japanese Black fattening steers during early period.

The effects of long-term feeding of wood kraft pulp (KP) silage containing sweet-potato shochu distillery by-product (SDP) on feed intake, feed digestibility, rumen fermentation, and growth performance of Japanese Black steers were investigated during the early fattening period. Ten Japanese Black steers (9.8 ± 0.6 months of age) were used in this study. Five steers (KP group) were fed KP silage as a replacement for 10% timothy hay (dry matter bases), in contrast to the other five (control group). KP silage consisted of 92.9% KP and 7.1% SDP (dry matter bases). The experiment was conducted for 18 weeks. No significant differences were observed in terms of feed intake, feed digestibility, or daily body weight gain between the groups. In addition, diurnal changes in the rumen pH and ruminal lipopolysaccharide activity did not differ between the groups. However, the plasma concentration of aspartate transaminase in the KP group was slightly lower (P = 0.078) than that in the control group. Thus, our study suggested that feeding KP silage does not reduce feed intake or affect the rumen fermentation or growth performance of Japanese Black fattening steer.

Factors that cause replacement of shochu yeast by wild yeast upon Sashi-moto repetition.

In shochu-making, a small amount of fermenting moromi is added to a koji/water mixture instead of yeast culture to initiate fermentation. This is a characteristic process called Sashi-moto. It is known that shochu yeast is replaced by wild yeast upon repetition of Sashi-moto. The shochu yeast strains Kagoshima No. 2 (K2), Kagoshima No. 4 (C4), and Kagoshima No. 5 (H5), but not Kagoshima No. 6 (A6), were replaced by wild yeast (strain No. S5-g). K2 and C4 were easily replaced compared to H5, and the specific growth rates of K2 and C4 were lower than that of S5-g under higher osmotic pressure. Although the specific growth rate of H5 was higher than that of S5-g, its yeast population at the stationary phase was smaller than S5-g. On the other hand, both the specific growth rate and yeast population of A6 were higher than those of S5-g. The specific growth rate of yeast would be affected by osmotic tolerance and specific characters of the yeast strain.

Identification of 2-furanmethanethiol contributing to roast aroma in honkaku shochu and awamori.

Honkaku shochu and awamori are traditional Japanese spirits. 2-Furanmethanethiol (2FM), a volatile thiol, was identified as a roast aroma compound in honkaku shochu and awamori. The detection threshold of 2FM in 25% (v/v) ethanol water solutions was determined as 1.6 ng/L. The odor activity values, calculated using the detection threshold suggested that 2FM affects the quality of honkaku shochu and awamori. The odor activity values of 2FM were higher in barley shochu distilled at atmospheric pressure than in sweet potato shochu, rice shochu and awamori; therefore, 2FM is considered to contribute to the characteristics of barley shochu.

Performance of stacked microbial fuel cells with barley-shochu waste.

The treatment of barley-shochu waste combined with electricity generation was examined using stacked microbial fuel cells (SMFCs). The maximum chemical oxygen demand (CODCr) removal efficiency and maximum power density were achieved at 36.7 ± 1.1% and 4.3 ± 0.2 W m⁻³ (15.7 ± 0.9 mW m-2). The acetic acid concentration in effluent increased, whereas the citric acid, ethanol and sugar concentrations decreased during the operation. Microbial community analysis of the anode cell suspension and raw barley-shochu waste revealed that Clostridiaceae, Acetobacteraceae, and Enterobacteriaceae became predominant after the operation, implying that microorganisms belonging to these families might be involved in organic waste decomposition and electricity generation in the SMFCs.

The white koji fungus Aspergillus luchuensis mut. kawachii.

The white koji fungus, Aspergillus luchuensis mut. kawachii, is used in the production of shochu, a traditional Japanese distilled spirit. White koji fungus plays an important role in the shochu production process by supplying amylolytic enzymes such as α-amylase and glucoamylase. These enzymes convert starch contained in primary ingredients such as rice, barley, buckwheat, and sweet potato into glucose, which is subsequently utilized by the yeast Saccharomyces cerevisiae to produce ethanol. White koji fungus also secretes large amounts of citric acid, which lowers the pH of the shochu mash, thereby preventing the growth of undesired microbes and enabling stable production of shochu in relatively warm regions of Japan. This review describes the historical background, research tools, and recent advances in studies of the mechanism of citric acid production by white koji fungus.

Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308.

Aspergillus luchuensis mut. kawachii is used primarily in the production of shochu, a traditional Japanese distilled alcoholic beverage. Here, we report the chromosome-level genome sequence of A. luchuensis mut. kawachii IFO 4308 (NBRC 4308) and a comparison of the sequence with that of A. luchuensis RIB2601. The genome of strain IFO 4308 was assembled into nine contigs consisting of eight chromosomes and one mitochondrial DNA segment. The nearly complete genome of strain IFO 4308 comprises 37,287,730 bp with a GC content of 48.85% and 12,664 predicted coding sequences and 267 tRNAs. Comparison of the IFO 4308 and RIB2601 genomes revealed a highly conserved structure; however, the IFO 4308 genome is larger than that of RIB2601, which is primarily attributed to chromosome 5. The genome sequence of IFO 4308 was deposited in DDBJ/ENA/GenBank under accession numbers AP024425-AP024433.

Making Traditional Japanese Distilled Liquor, Shochu and Awamori, and the Contribution of White and Black Koji Fungi.

The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called "honkaku shochu." It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a unique temperature control method to ensure that these three important elements, which greatly affect the taste of the produced liquor, are balanced without any excess or deficiency. This review describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan and whose market is expected to expand worldwide, with special attention paid to the koji fungi cultivation step. Furthermore, we describe the history of the koji fungi used today in the production of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also report the latest research progress on this topic.

The Escherichia coli CitT transporter can be used as a succinate exporter for succinate production.

Escherichia coli strain, whose gene is one of the subunits of succinate dehydrogenase (sdhA), and gene of the transcriptional repressor of isocitrate lyase (iclR) were disrupted, accumulated 6.6 times as much intracellular succinate as the wild-type MG1655 strain in aerobic growth, but succinate was not found in the culture medium. E. coli citT gene that encodes a citrate transporter was cloned under the control of the lacI promoter in pBR322-based plasmid and the above strain was transformed. This transformant, grown under aerobic condition in M9-tryptone medium with citrate, accumulated succinate in the medium while no succinate was found in the medium without citrate. CitT was active as a succinate transporter for 168 h by changing the culture medium or for 24 h in fed-batch culture. This study suggests that the CitT transporter functions as a succinate exporter in E. coli for succinate production in the presence of citrate.

Hair growth-promoting activity of components derived from sweet potato shochu.

Sweet potato shochu oil is one of the by-products of sweet potato shochu production. We investigated the functionality and industrial use of shochu oil as a food-derived raw material. Because of the increased incidence of self-consciousness in people owing to thinning hair, in this study, we examined the hair growth-inducing effects of shochu oil. Minoxidil, the only topical medication approved for hair growth treatment in Japan, was used as a control for the evaluation of hair growth-promoting activity of shochu oil. Human follicle dermal papilla cells treated with shochu oil showed upregulated expression of vascular endothelial growth factor in a concentration-dependent manner, indicating that shochu oil induced the activation of the hair growth cycle. In vivo, epidermal treatment with shochu oil also promoted hair growth in C3H mice. More than 35 components were detected in shochu oil via gas chromatography-mass spectrometry. The main components, accounting for 98.5% of shochu oil, were as follows, in order of decreasing concentration: ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl caprate, ethyl laurate, ethyl myristate, and ethyl α-linolenate. Among these, ethyl palmitate, ethyl linoleate, and ethyl α-linolenate promoted hair growth in C3H mice. These results indicate that shochu oil can be used as a hair restorer. To the best of our knowledge, this study is the first to demonstrate the hair growth-promoting activity of shochu oil.

Reduction of alkalinity supplementation for acid-based wastewater treatment using a thermophilic multi-feed upflow anaerobic sludge blanket reactor.

Generally, Shochu distillery wastewater treatment is required the addition of alkalinity agents for an increase of pH in the UASB reactor. However, to reduce the cost of alkalinity supplementation, cost-effective reactor operation method has been desired. This study aimed to reduce the alkalinity supplementation for a thermophilic (55°C) multi-feed up-flow anaerobic sludge blanket (MF UASB) reactor for the low-cost treatment of the wastewater from the production of the Japanese distilled alcohol called shochu. Shochu distillery wastewater contains high concentrations of organics (46,500-57,600 mgCOD L-1; COD: chemical oxygen demand) and volatile fatty acids (16,200-25,000 mgCOD L-1), and low pH (4.1-4.5). With alkalinity supplementation of 0.045 mgCaCO3 mgCOD-1 using 24% NaOH, the MF UASB reactor achieved an 87 ± 2% COD removal rate with an organic loading rate of 24 kgCOD m-3 day-1 for 554 days reactor operation (hydraulic retention time of 10 h and influent concentration of 10,000 mgCOD L-1). The organic removal rate decreased to 19 ± 3% in the MF UASB reactor when alkalinity supplementation was reduced to 0.015 mgCaCO3·mgCOD-1. In this study , the minimum alkalinity supplementation was 0.045 mgCaCO3 mgCOD-1 at an organic loading rate of 24 kgCOD m-3 day-1.

Effects of solutes on the alcohol-stimulative taste of vodkas.

Quantitative analyses of chemical components and sensory tests were carried out on vodka samples to test for stimulative taste of ethanol. Proton nuclear magnetic resonance (1H NMR) spectra of the vodkas were measured with 600 MHz NMR. The effects of salts on the alcohol-stimulative taste were investigated for 15% (v/v) EtOH-H2O solution. 1H NMR spectroscopy results showed that a magnesium salt could reduce the stimulative taste of ethanol organoleptically and, at the same time, strengthen the hydrogen-bonding structure of water-ethanol, although the effect of the magnesium could not be clarified quantitatively in the products of vodka. It was also suggested that a change in the water-ethanol hydrogen-bonding structure could lead to a reduction in the stimulative taste of ethanol in vodka.