Development of fermented Atemoya (Annona cherimola × Annona squamosa)-Amazake increased intestinal next-generation probiotics.

Akkermansia muciniphila and Faecalibacterium prausnitzii are next-generation probiotics, which has been reported to protect disease and effectively utilize various carbohydrates (starch and pectin) as nutrients for growth. Atemoya exhibiting fruity flavor, which is suitable for enhancing aroma and attenuating unpleasant taste caused by the koji metabolites. Results indicated that malic acid was increased (from 42.4 to 70.1 mg/100 g) in fermented Atemoya-Amazake. In addition, fermented Atemoya-Amazake elevated growthes in A. muciniphila and F. prausnitzii. Similarly, the populations of Parabacteroides (5.7 fold) and Akkermansia (1.66 fold) were elevated by fermented Atemoya-Amazake treatment in an in vitro simulated gastrointestinal system compared to the control group. Results revealed that fermented Atemoya-Amazake modulated the intestinal microbiota through increasing the production of short-chain fatty acids (exhibiting anti-pathogenic activity) for 2.1, 2.5, 2.6, and 2.1 folds in acetic acid, propionic acid, isobutyric acid, and butyric acid, respectively; suggesting this fermented Atemoya-Amazake could be applied in intestinal protection.

Harnessing Bacillus amyloliquefaciens for Amazake Production: Comparison with Aspergillus oryzae Amazake for Metabolomic Characteristics, Microbial Diversity, and Sensory Profile.

Amazake is a traditional, sweet, non-alcoholic Japanese beverage typically produced through koji fermentation by the fungus Aspergillus oryzae. However, alternative microorganisms such as Bacillus amyloliquefaciens offer potential advantages and novel possibilities for producing similar fermented beverages. This study aimed to replicate the ancestral beverage of amazake by replacing A. oryzae (W-20) with B. amyloliquefaciens (NCIMB 12077) and comparing their fermentation processes and resulting products. Our results show that the production of amazake with B. amyloliquefaciens (ABA) is not only possible but also results in a beverage that is otherwise distinct from traditional amazake (AAO). Saccharification was achievable in ABA at higher temperatures than in AAO, albeit with lower reducing sugar and enzymatic activity values. Amino acids and organic acids were more abundant in AAO, with cysteine being uniquely present in AAO and shikimic acid only being present in ABA. The volatile aroma compound profiles differed between the two beverages, with AAO exhibiting a greater abundance of aldehydes, and ABA a greater abundance of ketones and alcohols. Interestingly, despite these compositional differences, the two beverages showed similar consumer panel acceptance rates. An analysis of their microbial communities revealed pronounced differences between the amazakes, as well as temporal changes in ABA but not in AAO. This study provides promising insights into harnessing the potential of B. amyloliquefaciens as the primary microorganism in the fermentation process of amazake-like beverages, marking an important advancement in the field of fermented low-alcohol beverage production, with possible applications in other fermented foods.

Nutritional and sensory parameters of amazake from the recycling of stale bread.

Stale bread is a waste product with a potential to be recycled. One way to manage this waste material is to process it by fermentation for the purpose of food production. This paper proposes the use of stale wheat and rye bread as ingredients in amazake, a liquid dessert traditionally obtained from rice by fermentation with the koji mould Aspergillus oryzae, followed by liquefaction by the action of fungal enzymes. The stale bread was introduced instead of rice at both the koji stage (wheat bread) and the liquefaction stage (wheat and rye bread). The resulting products had an extended volatile compound profile, from 5 to 15 compounds identified, and modified sensory parameters, compared to the traditional version. Amazake containing bread had an increased protein content, from 1.10 to 6.4 g/100 g, and were more abundant in dietary fibre (up to a maximum of 1.8 g/100 g), additionally enriched with a soluble fraction. The proposed procedure of obtaining of new formula amazake can be directly applied in households to reduce the amount of discarded bread. Due to its simplicity, it also has the potential for further modification in terms of production scale and product parameters.

Carbon stable isotopes of glucose during the degradation of rice by the koji fungus Aspergillus oryzae.

Glucose, a key component of traditional Japanese fermented foods, is derived from rice starch via saccharification by hydrolytic enzymes produced by Aspergillus oryzae. The δ 13C value of glucose reflects that of its rice source. However, the influence of saccharification parameters (glucose concentration, degradation temperature, and reaction time) on glucose δ 13C values is unclear. Here, we investigated the influence of saccharification on the δ 13C value of glucose. Our experiments showed a significant difference in the δ 1³C value of glucose (-27.0 ± 0.1 ‰) obtained from saccharification compared to the ingredient rice (-27.1 ± 0.1 ‰) and remaining solid residue (-27.1 ± 0.1 ‰); however, it did not differ significantly from those of rice koji (-27.0 ± 0.1 ‰) and steamed rice (-27.1 ± 0.1 ‰), despite all values being within 0.1 ‰. Notably, glucose concentration, degradation temperature, and reaction time did not significantly affect glucose δ 13C values. These findings demonstrate the remarkable preservation of glucose δ 13C values. The δ 13C values remain aligned with the original δ 13C value of the rice, even with up to 60 % degradation during A. oryzae saccharification. This persistence of the δ 13C value throughout the process offers a potential tool for authenticating the origin of rice-fermented beverages based on the δ 13C value of their glucose component.

Changes in the fecal gut microbiome of home healthcare patients with disabilities through consumption of malted rice amazake.

The aim of the present study was to investigate changes in the gut microbiome both during and after consumption of malted rice amazake (MR-Amazake), a fermented food from Japan, in-home healthcare patients with disabilities, including patients with severe motor and intellectual disabilities. We monitored 12 patients who consumed MR-Amazake for 6 wk and investigated them before and after the intervention as well as 6 wk after the end of intake to compare their physical condition, diet, type of their medication, constipation assessment scale, and analysis of their comprehensive fecal microbiome using 16S rRNA sequencing. Their constipation symptoms were significantly alleviated, and principal coordinate analysis revealed that 30% of patients showed significant changes in the gut microbiome after MR-Amazake ingestion. Furthermore, Bifidobacterium was strongly associated with these changes. These changes were observed only during MR-Amazake intake; the original gut microbiome was restored when MR-Amazake intake was discontinued. These results suggest that 6 wk is a reasonable period of time for MR-Amazake to change the human gut microbiome and that continuous consumption of MR-Amazake is required to sustain such changes.NEW & NOTEWORTHY The consumption of malted rice amazake (MR-Amazake) showed significant changes in the gut microbiome according to principal coordinate analysis in some home healthcare patients with disabilities, including those with severe motor and intellectual disabilities. After discontinuation of intake, the gut microbiome returned to its original state. This is the first pilot study to examine both the changes in the gut microbiome and their sustainability after MR-Amazake intake.

Effects of Amazake Produced with Different Aspergillus on Gut Barrier and Microbiota.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. To explore the preventive effects of dietary foods on IBD, we evaluated the effects of the traditional Japanese fermented beverage "Amazake" on gut barrier function in this study. Black koji Amazake (BA) derived from Aspergillus luchuensis MEM-C strain and yellow koji Amazake (YA) derived from Aspergillus oryzae were made in this study, and their nutrients were analyzed. Mice with mild gut barrier dysfunction induced by Western diet were administered with 10% of each Amazake for two months. Mice gut microbiota were analyzed by 16S rRNA gene sequencing. BA contained a higher amount of isomaltooligosaccharides, citric acid, and ferulic acid than YA. The animal data revealed that BA significantly induced the expressions of antioxidant factors and enzymes such as NF-E2-related factor 2 (Nfr2), heme oxygenase 1 (HO1), and superoxide dismutase-2 (SOD-2). The gut barrier protein, occludin, and fecal immunoglobulin A (IgA) were also significantly enhanced by BA. Furthermore, the levels of serum endotoxin and hepatic monocyte chemotactic protein-1 (MCP-1) were decreased in both the BA and YA groups. In gut microbiota, Lachnospiraceae was increased by BA while Akkermansia muciniphilia was increased by YA. Black koji Amazake contained a higher amount of isomaltooligosaccharides, citric acid, and ferulic acid than yellow koji Amazake and contributed to protecting gut barrier function to reduce endotoxin intrusion and inflammation.

Improving γ-Oryzanol and γ-Aminobutyric Acid Contents in Rice Beverage Amazake Produced with Brown, Milled and Germinated Rices.

Rice is an important source of γ-oryzanol (GO) and γ-aminobutyric acid (GABA), which are bioactive compounds that may benefit blood lipid and pressure control. Both GO and GABA can be improved by germination and fermentation. Fermentation with A. oryzae produces Koji, a rice-based starter for Amazake, a naturally sweet beverage. Germinated rice (brown and milled rice), were tested to improve those bioactive compounds during the fermentation process. The resulting Koji was optimised to GO and GABA through a response surface methodology; α-amylase activity and starch content were also assessed. The different rice matrix resulting from the germination largely impacted the biosynthesis of GABA, α-amylase and starch contents. Amazake, obtained by germinated rice, has increased GO and GABA contents when compared to the one obtained from milled rice (from a non-detectable value to 27.65 ± 0.23 mg/100 g for GO and from 163.95 ± 24.7 to 271.53 ± 5.7 mg/100 g for GABA). A panel of 136 Portuguese consumers tasted the beverage in a blind overall tasting test followed by an informed test, using 9-point scales. The consumer scores had a mean value of 4.67 ± 1.9 and 4.9 ± 1.8, meaning that cultural differences may play an important role with regard to liking and accepting Amazake.

Effects of Milk Amazake on Skin Elasticity, Hydration, and Transepidermal Water Loss: An 8-Week Double-Blind, Randomized, Controlled Trial.

Amazakes made from rice and koji mold are rich in nutrients, such as groups of vitamin B, minerals, essential amino acids, and oligosaccharides, and can improve skin moisturization. However, there are few reports on milk amazake, made from milk and koji mold. Therefore, in this double-blind, randomized controlled trial, we investigate the effect of milk amazake on skin function. Healthy women and men (n = 40) were randomly allocated to the milk amazake or placebo group. The test beverage was consumed once daily for 8 weeks. Skin elasticity, hydration, and transepidermal water loss (TEWL) were measured at baseline and at weeks 4 and 8, and all subjects completed the trial. Skin elasticity (R2 and R5) at 8 weeks was significantly increased in the milk amazake group compared with baseline. In addition, changes in R5 in the milk amazake group were significantly higher than those in the placebo group. Conversely, TEWL, an evaluation item of skin moisturizing function at 8 weeks, was significantly decreased in the active group compared with baseline. In conclusion, milk amazake may be useful as a functional food for improving skin function.

Improving chestnut physicochemical properties through fermentation - Development of chestnut Amazake.

The increased awareness of population regarding the impact of consumption habits is leading to interest in new, innovative, diversified and health promoting foods. In this work, two new amazake fermented products were developed with chestnut (Castanea sativa Mill.), using rice or chestnut koji as source of glycolytic enzymes. The analysis of the amazakes evolution showed improvements in chestnuts physicochemical characteristics. The fermented products presented higher values of soluble protein, sugars, starches, antioxidant capacity, and similar values of ascorbic acid for chestnut koji amazake. The adhesiveness increased, which is related to the enhanced concentrations of sugars and starches. The evolution into less structured products was observed in the firmness followed by a consistent decrease of the viscoelastic moduli. The developed chestnut amazakes can represent a suitable alternative to traditional amazake, creating an opportunity for valorisation of chestnut industrial by-products, as new, tasty, and nutritive fermented products with potential functional characteristics.

Koji amazake Maintains Water Content in the Left Cheek Skin of Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group, Comparative Trial.

Purpose: Improvement in water content and skin barrier function on human skin is believed to be induced by koji amazake, a non-alcoholic beverage derived from rice fermented by Aspergillus oryzae (A. oryzae). In order to scientifically identify the effects of koji amazake on human skin, we performed a randomized, double-blind, placebo-controlled, parallel-group comparative trial and quantified the content of glucosylceramide (GlcCer) which would be responsible for the effects. Participants and Methods: Healthy adults concerned with their skin dryness were divided into koji amazake (N = 30) or placebo group (N = 30). During this test, the test beverages were ingested at 118 g/day. Their water content and trans-epidermal water loss (TEWL) were measured at 0 week (baseline) and 8 weeks. The content of GlcCer in test beverages was quantified by HPLC-ELSD. Results: In comparison with the placebo group, the water content in the left cheek of individuals in the koji amazake group was maintained for 8 weeks. In addition, changes in water content from the baseline to 8 weeks differed significantly between the koji amazake (0.19) and placebo groups (-3.98). Unexpectedly, there was no significant difference in the TEWL between koji amazake and placebo group. We analyzed GlcCer in both koji amazake and placebo beverages, which were found to contain 1.35 ± 0.11 and 0.30 ± 0.07 mg/118 g, respectively. The amount of GlcCer in koji amazake was approximately equal to the dosage of plant-derived GlcCer which has the ability to improve water content and TEWL in humans. Conclusion: Present study has shown that intake of koji amazake contributes to maintain the water content only on the left cheek. The content of GlcCer derived from koji amazake was adequate for maintenance of the water content compared to previous reports. Therefore, it was concluded that GlcCer in koji amazake acts as a functional ingredient.

Effects of Malted Rice Amazake on Constipation Symptoms and Gut Microbiota in Children and Adults with Severe Motor and Intellectual Disabilities: A Pilot Study.

Constipation is a frequent complication in patients with severe motor and intellectual disabilities (SMID). The aim of this study was to investigate changes in constipation symptoms and gut microbiota associated with the intake of malted rice amazake, a fermented food in Japan, in patients with SMID. Ten patients consumed the test food for six weeks, and their physical condition, dietary and medication status, and constipation assessment scale (CAS) were investigated. Comprehensive fecal microbiome analysis using the 16S rRNA sequence method was performed. The results showed a significant decrease in CAS, and a significant increase in Lactobacillales and decrease in Escherichia-Shigella after consuming malted rice amazake. To investigate the difference in the effects of malted rice amazake consumption, based on the characteristics of the original gut microbiota, the patients were grouped according to the similarity of their gut microbiota before the intervention; Firmicutes-rich Group 1 (n = 5), Actinobacteria-rich Group 2 (n = 4), and Proteobacteria-rich Group 3 (n = 1). The CAS decreased in Groups 1 and 2. The relative abundance of Bifidobacterium showed an increasing tendency both overall and in Group 1, but it was originally higher in Group 2. Our results suggest that malted rice amazake consumption reduces constipation symptoms and simultaneously changes the gut microbiota, but the changes may vary depending on the original composition of the gut microbiota.

Intake of Koji Amazake Improves Defecation Frequency in Healthy Adults.

Reportedly, the intake of koji amazake, a beverage made from steamed rice fermented by Aspergillus oryzae, improves defecation frequency. However, its functional ingredients and mechanism of action remain unclear. To compare the effects of koji amazake and a placebo beverage on defecation frequency and to identify the functional ingredients and mechanism of action, a randomized, placebo-controlled, double-blind parallel-group comparative trial was performed on two groups. The koji amazake had 302 ± 15.5 mg/118 g of A. oryzae cells, which was not in the placebo. Compared with the placebo group, the koji amazake group showed a significant increase in weekly defecation frequency at 2 weeks (5.09 days vs. 4.14 days), 3 weeks (5.41 days vs. 4.18 days), and 4 weeks (5.09 days vs. 3.95 days), along with an increase in the weekly fecal weight at 4 weeks (724 g vs. 501 g). The intake of koji amazake did not induce significant intergroup differences in the fecal SCFA concentration, whereas it significantly decreased the relative abundance of Blautia and significantly increased that of Bacteroides at 3 weeks. Therefore, koji amazake intake improved defecation frequency, and A. oryzae cells played potentially important roles as functional ingredients.

Ingredients, Functionality, and Safety of the Japanese Traditional Sweet Drink Amazake.

The sweet drink amazake is a fermented food made from Aspergillus oryzae and related koji molds in Japan. There are two types of drinks called amazake, one made from koji (koji amazake) and the other made from sake lees, a by-product of sake (sakekasu amazake). The sweetness of koji amazake is from glucose, derived from starch broken down by A. oryzae amylase. The other, sakekasu amazake, depends on added sugar. The main components are glucose and sucrose, but they also contain more than 300 other ingredients. Koji amazake contains oligosaccharides and ergothioneine, and sakekasu amazake has a resistant protein and α-ethyl glucoside, which are characteristic ingredients of each amazake. However, there are also common ingredients such as glycosylceramide. Functionality is known to include anti-fatigue, bowel movement, skin barrier, and other effects on human health. In particular, the bowel movement-improving effects have been well studied for both amazakes. These functions result from ingesting approximately 100 mL per day, but human clinical trials have clarified that this amount has no effect on blood glucose levels and weight gain. In the future, the identification of substances associated with each function is required.

Amazake made from sake cake and rice koji suppresses sebum content in differentiated hamster sebocytes and improves skin properties in humans.

Amazake is a traditional Japanese health drink. Here, we examined the effects of amazake on skin in cells and humans. Treatment with sake cake or rice koji suppressed intracellular lipid accumulation in differentiated hamster sebocytes, likely through the reduced expression of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA. In double-blind, placebo-controlled trial, seventeen Japanese women ingested either amazake or placebo for 4 weeks. Ingestion of the amazake decreased the sebum content compared to the placebo. The questionnaires showed improvements in "face color," "dark circles under the eyes," "glossy hair," and "waking up well", only in the amazake. In accordance with the questionnaires, additional analysis revealed the change in the L* values under the eyes was statistically increased in the amazake compared to the placebo. These results indicate that amazake may decrease sebum content in cells and humans and increase the L* values under the eyes, with some additional beneficial effects in humans.

Effect of temperature on saccharification and oligosaccharide production efficiency in koji amazake.

Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C-60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C-60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.

Evaluation of the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay.

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.

Metabolite profile of koji amazake and its lactic acid fermentation product by Lactobacillus sakei UONUMA.

The koji amazake is a traditional sweet Japanese beverage. It has been consumed for over a thousand years in Japan; nonetheless, little is yet known of the ingredients in koji amazake. Therefore, this study aimed to analyze the metabolites of koji amazake using a metabolomics approach. Additionally, we reformed the flavor of koji amazake by lactic acid fermentation (LAF-amazake) using Lactobacillus sakei UONUMA, which was isolated from snow caverns. The purpose of this article is to identify the ingredients in these beverages. In LAF-amazake and koji amazake, sugars, amino acids, organic acids, and vitamin B complex were determined in the two beverages, and over 300 compounds were detected in total. Thirteen saccharides were identified including two unknown trisaccharides, and there were no differences in these between the two beverages. In LAF-amazake, lactic acid, vitamin B2 (riboflavin), B3 (nicotinic acid and nicotinamide), and B6 (pyridoxine) were significantly increased as compared to koji amazake, whereas malate and glutamine decreased. These results suggested that LAF, malolactic fermentation, and glutamine deamidation occurred simultaneously in LAF-amazake. L. sakei UONUMA strains produced these vitamins. Moreover, it was surprising that acetylcholine, a well-known neurotransmitter, was newly generated in LAF-amazake. Here, we have succeeded in reforming the flavor of koji amazake and obtained these metabolic data on the two beverages. The present study could provide useful basic information for promoting functional analyses of koji amazake and LAF-amazake for human health.