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BACKGROUND: Patients with chronic kidney disease (CKD) reportedly show dysbiosis, which is the imbalance of gut microbiome. Dysbiosis increases the uremic toxin level in the intestine, and uremic toxins transfer into the blood, causing CKD progression. Sake lees, a traditional Japanese fermented food, may help reduce uremic toxins by altering the gut microbiome. Additionally, D-alanine, which is present in sake lees, may have a renoprotective effect. The present pilot study aims to evaluate the effect of adding sake lees to the standard CKD dietary therapy in reducing blood uremic toxins. METHODS: This pilot study is a single-center, open-label, randomized controlled trial. Twenty-four patients with CKD will be enrolled and allocated 1:1 to the intervention and control groups. The intervention group will receive standard CKD dietary therapy with an additional intake of 50 g of sake lees per day for 8 weeks, whereas the control group will only receive standard CKD dietary therapy. The primary endpoint is the change in serum indoxyl sulfate after 8 weeks. The secondary endpoint is the plasma D-alanine and fecal microbiome changes. CONCLUSION: This pilot study provides insight into the development of a new diet focused on gut microbiome and D-amino acids in patients with CKD. CLINICAL TRIAL REGISTRATION: This protocol was approved by the Clinical Trial Review Board of Kanazawa University Hospital on October 27, 2022 (2022-001 [6139]) and available to the public on the website of the Japan Registry of Clinical Trials on November 22, 2022 (jRCT1040220095).

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Sake lees are a byproduct of Japanese rice wine and used as ruminant feed with high protein and ethanol contents. This study aimed to explore the effects of ensiled total mixed ration (TMR) containing sake lees on rumen fermentation, digestion, nitrogen (N) use, and plasma metabolites in sheep. Four mature wethers were assigned to a 4 × 4 Latin square design with a factorial arrangement of ensiling treatment (non-ensiled or ensiled TMR) and types of sake lees (traditional or liquefied). Although dietary ethanol consumption was higher in sheep fed ensiled TMR, ruminal and plasma concentrations of methanol and ethanol increased after feeding in all dietary treatments, which was accompanied by the increased plasma concentrations of formate. The intake and digestion of dry matter and N were lower in sheep fed ensiled TMR than in those fed non-ensiled TMR. The plasma concentrations of most amino acids decreased in sheep fed ensiled TMR. Although plasma methionine, serine, and glycine concentrations were not altered by diet, homocysteine concentration was the highest in sheep fed ensiled TMR containing traditional sake lees. The negative impacts of ensiling treatment on N digestion and amino acid utilization should be considered for formulating ensiled TMR containing sake lees.

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Increasing food loss and waste (FLW) is a global problem, and efforts are being made to use waste food as potential livestock feed material. The amount of self-supplied feed is lower in Japan than in other countries, and the government recommends FLW use for animal feed. Sake (Japanese rice wine) is a traditional alcoholic beverage. During the sake manufacturing process, large amounts of squeezed solids or "lees" (sake lees) are generated. Sake lees are nutritious and functional, but are prone to spoilage. In this study, we investigated whether sake lees should be mixed with animal feed immediately or after drying. To assess the usefulness of sake lees as a poultry feed ingredient and determine the effect of sake lees on intestinal immunity, we performed a feeding trial with three treatments: a raw sake lees (RSL) diet, dried sake lees (DSL) diet, and control diet. Three-week-old broilers were fed these diets (n=8 per group) for two weeks. We then calculated feed efficiency and performed RT-qPCR to assess the effects of diet on intestinal immunity. The growth performance in the RSL diet group was equivalent to that in the control diet group. The DSL diet became difficult for broilers to eat, resulting in decreased growth performance. In the ileum of RSL-diet broilers, the mRNA expression levels of TGF-ß1 and avian ß-defensin (AvBD)12 were significantly increased compared to those of control diet broilers (p<0.05), and a significant correlation was observed between the two genes (p<0.05). Our results indicated that sake lees should not be dried and should be mixed immediately with feed, and this sake lees when fed to chicken activates the intestinal immunity. However, sake lees have a lower fat content than corn, and it is thus important to combine sake lees with high-energy feed.

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More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

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We have previously reported that dietary glucosylceramide (GlcCer) and rice extracts containing GlcCer reduce the formation of aberrant crypt foci (ACF) in the colons of 1,2-dimethylhydrazine (DMH)-treated mice, as a precursor model of colon cancer. This study investigated the impact of alkali-stable neutral lipids (NLs) containing free ceramides (Cer) and sterols on the formation of ACF in mice for the purpose of searching for functional components, irrespective of GlcCer, in rice extracts. The fraction was prepared from sake lees as a rice fermentation byproduct. Dietary NLs suppressed the marked increase in colon ACF treated with DMH.

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This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

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Sake lees (Sake-kasu) are the sediments of Japanese sake brewing process from fermented rice with Aspergillus oryzae and yeasts. Sake lees contain various enzymes and metabolites derived from the Sake starter culture, and expected to add aroma, flavor and softness to sausages. We investigated the effects of Sake lees supplementation on fermented dry sausage characteristics over an aging period of 35 days. Sake lees supplementation significantly accelerated sarcoplasmic and myofibrillar protein decomposition and increased peptide and free amino acid content compared to untreated sausage meat. Sake lees significantly acidified the sausages, enhanced their sour taste, and influenced their acceptability. Sake lees supplementation also significantly improved the hardness of the final product and conferred a preferable flavor to it. These results suggest that the various enzymes and compounds in Sake lees improve the flavor and texture of fermented dry sausages.

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Objectives: We studied the effects of fish oil and sake lees combined with a high fat diet in rats, and assessed serum lipids concentrations and anxiety behavior. Methods: Male Sprague-Dawley rats aged 5 weeks were fed a diet containing the Soybean oil (Control), Soybean oil + Sake lees (S), Fish oil (FO) or FO + Sake lees (FO+S) for 18days. The control diet contained a soybean oil component. It was measured the concentrations of serum lipids and anxiety behavior. Results: The concentration of serum cholesterol were significantly lower in the FO+S group than in the Control group. The concentration of serum triglyceride and free fatty acids were significantly lower in the FO group and the FO+S group than in the Control group. In anxiety behavior, the stay time in the zone of open arm were significantly higher in the S group than in the Control group. Conclusion: These results suggested that the combination of fish oil and sake lees in the diet improved the concentration of total cholesterol in the serum, which should assist in the prevention and improvement of dyslipidemia. In addition, intake of sake lees is expected to be effective in suppressing anxiety behavior.

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In this study, we investigated the alterations in the purine composition of swordfish prepared using a traditional Japanese processing method of soaking in sake lees. These alterations are the byproducts of the yeast fermentation of rice-koji and are renowned for enhancing the umami nature of food. Using a conventional assay method for hydrolyzing all of the purines into four bases and our developed method for simultaneously analyzing purines, we observed the alterations in four purine bases in the soaked sake lees and swordfish. The findings showed that the total purine content, and hypoxanthine-related and guanine-related purines in swordfish decreased after soaking in sake lees. We also analyzed the free purine composition and showed that the ratio of IMP in swordfish was decreased by soaking, while that of inosine in sake lees was increased by soaking swordfish in it.

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Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of sake lees hydrolysate against acetaminophen-induced hepatotoxicity in mice. Sake lees hydrolysate was administered orally to ICR mice for seven days. Six hours after acetaminophen treatment, the mice were sacrificed, and blood and liver samples were collected for analysis. Treatment with acetaminophen markedly increased the levels of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase. Pretreatment with sake lees hydrolysate significantly prevented the increases in the serum levels of these enzymes and inhibited acetaminophen-mediated glutathione depletion. In addition, histopathological evaluation of the livers also revealed that sake lees hydrolysate prevented acetaminophen-induced centrilobular necrosis. The expression of γ-glutamylcysteine synthetase (γ-GCS), hemeoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the liver were decreased after acetaminophen treatment, whereas pretreatment with sake lees hydrolysate led to an increased expression of all three proteins. Furthermore, sake lees hydrolysate induced the expression of these proteins in HepG2. These results suggested that sake lees hydrolysate could induces HO-1 and γ-GCS expression via activation of the Nrf2 antioxidant pathway, and protects against acetaminophen-induced hepatotoxicity in mice.

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We evaluated the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay. Sake cake, a raw material used to make amazake, had high innate immunity-stimulating activity, whereas rice malt, another raw material used to make amazake, did not, even after fermentation. These results suggest that the silkworm muscle contraction assay is a useful tool to screen foods with high innate immune-stimulating activity and that amazake made from sake cake has immunomodulatory potential.

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BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasing worldwide as one of the leading causes of chronic liver disease. Sake lees (SL) are secondary products of sake manufacturing and are considered to have beneficial effects on human health. To investigate these effects, we used high fat diet (HFD)-fed mice treated with or without the SL extract. METHOD: Mice were the HFD ad libitum for 8 weeks and were administered 500 µL of distilled water with or without the SL extract (350 mg/mL) by a feeding needle daily for the last 4 weeks. Food intake, body weight, and liver weight were measured. Triacylglycerol content and the mRNA and protein expression levels of various lipid and glucose metabolism-related genes were determined in liver tissues. The levels of triglyceride, free fatty acids, glucose, insulin, and liver cell damage markers were determined in serum. Fatty acid-induced lipid accumulation in HepG2 cells was assessed in the presence or absence of the SL extract. RESULTS: Mice fed a HFD and treated with the SL extract demonstrated a significant reduction in hepatic lipid accumulation and mRNA and protein levels of peroxidome proliferator-activated receptor γ (PPARγ), PPARα, CD36, and phosphoenolpyruvate carboxykinase 1 in the liver, while the SL extract did not affect body weight and food intake. Moreover, insulin resistance and hepatic inflammation in HFD-fed mice improved after administration of the SL extract. In HepG2 cells, the SL extract suppressed fatty acid-induced intracellular lipid accumulation. CONCLUSIONS: These findings suggest that treatment with the SL extract could potentially reduce the risk of NAFLD development, and that the SL extract may be clinically useful for the treatment of NAFLD.

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