Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria.

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.

Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains.

Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi. However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi. Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi. To develop a highly efficient gene-targeting system for L. starkeyi, we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆l sku70∆lslig4 and the triple mutant ∆lsku70∆lsku80∆lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast.

Multicopy integration and expression of heterologous genes in the oleaginous yeast, Lipomyces starkeyi.

The oleaginous yeast, Lipomyces starkeyi, is an excellent lipid producer with great industrial potential. However, methods for molecular breeding have not been established for L. starkeyi. We describe the development of a system for targeted rDNA integration of multiple copies of a gene into L. starkeyi genome by spheroplast-polyethylene glycol transformation.

Antifungal activity of plant defensin AFP1 in Brassica juncea involves the recognition of the methyl residue in glucosylceramide of target pathogen Candida albicans.

An antifungal defensin, AFP1, of Brassica juncea inhibits the growth of various microorganisms. The molecular details of this inhibition remain largely unknown. Herein, we reveal that a specific structure of fungal sphingolipid glucosylceramide (GlcCer) is critical for the sensitivity of Candida albicans cells to AFP1. Our results revealed that AFP1 induces plasma membrane permeabilization and the production of reactive oxygen species (ROS) in wild-type C. albicans cells, but not in cells lacking the ninth methyl residue of the GlcCer sphingoid base moiety, which is a characteristic feature of fungi. AFP1-induced ROS production is responsible for its antifungal activity, with a consequent loss of yeast cell viability. These findings suggest that AFP1 specifically recognizes the structural difference of GlcCer for targeting of the fungal pathogens.

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.

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.

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.

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.