Abstract
Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg−1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light–dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacterium adolescentis, a “psychobiotic”, increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.
Highlights
We investigated potential changes in the expression sion of selected genes involved in serotonin signaling and metabolism (tph1a, tph1b, tph2,of sion of selected genes involved in serotonin signaling and metabolism tph2, selected genes involved in serotonin signaling and metabolism (tph1a,(tph1a, tph1b,tph1b, tph2, htr1aa, htr1aa, slc6a4a, and mao) in the gut and in the brain of zebrafish fed with one of the three htr1aa, slc6a4a, and mao) in the gut and in the brain of zebrafish fed with one of the three slc6a4a, and mao) in the gut and in the brain of zebrafish fed with one of the three kinds kinds of tempeh (Figure 6B)
We found that tempeh fermented with Rhizopus oryzae MHU 002 had the highest GABA content (9.712 ± 0.404 g kg−1, Figure 1D); MHU 002-fed fish spent less time in the light zone than the controls (109.67 ± 7.15 s, Figure 2E); the MHU 002 group had the lowest increase in BDNF in the brain (Figure 6A)
Proteobacteria (e.g., E. coli) were reduced from 84.97% to 51.90% in zebrafish fed tempeh fermented with MHU001 for 2 weeks, while Actinobacteria (e.g., Bifidobacterium) increased more than two times from 0.79% to 1.80%
Summary
Tempeh is a traditional fermented soybean food product consumed mainly in Southeast Asia, especially in Indonesia. It is prepared through fermenting dehulled and cooked soybeans with Rhizopus fungi [1]. The fungal mycelium binds the soybeans together into a compact cake [2]. While R. oligisporous, R. oryzae, and R. stolonifera have been identified as soybean tempeh starters, only R. oligosporous [3] and R. oryzae [4]
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