Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic

Abstract

Background: The Japanese traditional cuisine, Washoku, considered to be responsible for increased longevity among the Japanese, comprises various foods fermented with the non-pathogenic fungus Aspergillus oryzae (koji). We have recently revealed that koji contains an abundant amount of glycosylceramide. Intestinal microbes have significant effect on health. However, the effects of koji glycosylceramide on intestinal microbes have not been studied.

Materials and methods: Glycosylceramide was extracted and purified from koji. C57BL/6N mice were fed a diet containing 1 % purified koji glycosylceramide for 1 week. Nutritional parameters and faecal lipid constituents were analyzed. The intestinal microbial flora of mice on this diet was investigated.

Results: Ingested koji glycosylceramide was neither digested by intestinal enzymes nor was it detected in the faeces, suggesting that koji glycosylceramide was digested by the intestinal microbial flora. Intestinal microbial flora that digested koji glycosylceramide had an increased ratio of Blautia coccoides. Stimulation of B. coccoides growth by pure koji glycosylceramide was confirmed in vitro.

Conclusions: Koji functions as a prebiotic for B. coccoides through glycosylceramide. Since there are many reports of the effects of B. coccoides on health, an increase in intestinal B. coccoides by koji glycosylceramide might be the connection between Japanese cuisine, intestinal microbial flora, and longevity.

Keywords: Aspergillus; Blautia; Glycosylceramide; Intestinal microbial flora; Japanese cuisine; Koji; Prebiotic.

Fig. 1

Extraction and purification of glycosylceramide from koji. Lipids were extracted with chloroform–methanol from 1.8 kg of pregelatinized koji. The ester-bond containing lipids were degraded with mild alkaline treatment, and the lipid phase was extracted using Bligh and Dyer fractionation. The chloroform-soluble fraction was recovered (a), and the acetone-insoluble fraction was recovered (b). GlcCer indicates glycosylceramide, a and b indicate hydroxylated and nonhydroxylated cerebroside respectively

Fig. 2

Metabolic fate of koji glycosylceramide in the intestine. a Total lipid profile of feces from mice fed with or without koji glycosylceramide. Nor indicates the feces of non-added mice and Kgc indicates those of koji glycosylceramide-fed mice. The number indicates the replicate number of experiments. b Total lipid profile of koji glycosylceramide incubated with intestinal extracts. c NBD-TLC of C12-ceramide incubated with intestinal extracts. Intestinal extract was recovered from mice, mixed with purified koji glycosylceramide or NBD-C12-ceramide, incubated at 37 °C for 16–30 h, developed, and visualized by orcinol–H₂SO₄ reagent or fluorescence. SG indicates Sterylglucoside and GlcCer indicates glycosylceramide, a and b indicate hydroxylated and nonhydroxylated cerebroside respectively

Fig. 3

Analysis of intestinal microbial flora of mice fed with koji glycosylceramide. a Cluster tree of microbes increased or decreased in the feces of mice fed with koji glycosylceramide. The underline indicates microbes whose read values were significantly increased (p < 0.05). The broken underline indicates microbes whose percentage values were significantly increased (p < 0.05). The square indicates microbes whose read values and percentage values were significantly increased. Stars indicate microbes which were larger in Kgc than in Nor. b Box plot representing the relative abundance of the genera (Blautia coccoides) enriched in the feces of koji glycosylceramide-fed mice. Mice were fed with koji glycosylceramide for 1 week. The feces were recovered, and freeze-dried. Genomic DNA was extracted and purified from the feces. The V3–V4 variable region of 16S rRNA was amplified using PCR and sequenced using Miseq, Illumina. Data were analyzed by MiSeq Reported 16S metagenomics system. p value indicates one-tailed unpaired Student’s t-test under symmetry conditions (n = 3)

Fig. 4

In vitro analysis of the bacterial growth-stimulating effect of koji glycosylceramide. Purified glycosylceramide was added to bacterial cultures and growth (OD₆₀₀) was measured after 24 h of culture. The results are the mean of triplicate independent experiments with standard errors. The statistical significance of differences between averages was assessed by the unpaired one-tailed Student’s t-test (***p < 0.001). a B. coccoides incubated with or without 4 μg/μL koji glycosyleeramide. b B. coccoides incubated with or without 4 μg/μL soybean glucosylceramide. c L. casei incubated with or without 4 μg/μL soybean glucosylceramide. d E. coli incubated with or without 4 μg/μL soybean glucosylceramide. Detailed materials and methods are described in the text