To search for enzymes that improve the efficiency of protoplast production in Tricholoma matsutake, Paenibacillus sp. MU1, which harbors degradation activity for the dried fruit body of T. matsutake, was isolated. The bacterial strain has the potential to degrade the cell wall of T. matsutake, which was isolated from a soil sample using medium including insolubilized fraction of T. matsutake fruit body as a sole nutrient source. The draft genome sequence of the bacterium comprised 36 contigs, totaling 6.6 Mbp, with an average length of 184,698 bp. Whole genome sequence data identified the strain as Paenibacillus sp. MU1. Among its degrading activities, the bacterial α-1,3-glucanase (Mu1Agl) was purified to homogeneity, and its enzymatic properties were characterized. Mu1Agl acts on mutan (α-glucan with α-1,3 and α-1,6 linkages), but other polysaccharides and oligosaccharides are not substrates. Mu1Agl exhibited maximum activity at 40ｰC at around pH 6.0 in phosphate buffer. The Mu1Agl gene was cloned with reference to mass spectrometry and whole genome analysis information. The deduced amino acid sequence showed high homology with discoidin domain-containing proteins or α-1,3-glucanase of P. glycanilyticus and other Paenibacillus species. These results indicated that Mu1Agl belongs to the minor group (Subgroup 3) of GH family 87.

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Mushrooms harbor diverse bacterial communities that can enhance their growth. Investigation on the role of these bacteria as growth-promoting agents offers a promising approach for improving mushroom production. In vitro co-culturing of fungi and bacteria is a useful method for screening these growth-promoting effects. Previous methods using a circular design and dual compartments have shown that Paraburkholderia fungorum GIB024 is a growth promoter of Rhizopogon roseolus TUFC 10010, but challenges were encountered in clarifying the specific influence of the bacterium or the medium with the control. To address this limitation, we developed a tri-compartment screening design in a single Petri dish. This design allowed for direct observation of mycelial growth towards the bacterial compartment (BC) or control compartments (CC). Using this tri-compartment method, medium alone did induce any directional bias in the growth of mycelium on the control plate, as evidenced by the similar growth towards BC, CC, and perpendicular directions. Additionally, applying P. fungorum GIB024 to the BC, but not to the mushroom compartment (distal location) resulted in a positive effect on the mushroom. Specifically, the mycelial growth occured more towards the BC rather than the CC or perpendicularly. This new method provides a useful tool for investigating the role of bacteria with growth-promoting effects on mushrooms.

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Tofuyo (traditional fermented food of Okinawa) was produced using mushrooms instead of Monascus sp. and Aspergillus oryzae. To screen mushrooms that produce alcohol-tolerant protease, 76 strains of edible mushroom were cultivated on rice medium and total protease activity was measured in the presence of 20% ethanol. Ten strains of mushroom, such as Auricularia sp. SKB074 (4.46 U / L) and Auricularia americana SKB073 (4.03 U / L), showed higher activity than that of commercial rice-koji (1.46 U / L). Culture conditions and preliminary fermentation tests were investigated, and Auricularia auricula-judae NBRC100150 and Lepista sordida SKB010 were selected to produce tofuyo based on the taste and aroma of the prototype. Tofu was fermented using mushroom-koji of these strains in the presence of alcohol, and the texture of the produced tofuyo was evaluated using the texture profile method. Tofuyo fermented for 14 or 28 days using L. sordida SKB010 showed texture parameters comparable to those of commercial tofuyo. Tastes and aromas of tofuyo fermented by L. sordida SKB010 showed less distinctiveness, and depending on the alcohol and seasoning used, commercialization as tofuyo with new characteristics might be possible.

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During the cultivation of Lentinula edodes, brown film was formed; however, the microbiome associated with brown film has not been examined. We investigated bacterial communities and attempted to find potential bacterial biomarkers on the brown film for distinguishing different flushes of L. edodes cultivation. In bacterial composition, Firmicutes was a dominant bacterium in the first flush, and Proteobacteria was a dominant bacterium in the third flush (last flush). The first flush group showed a significant difference in the structure of bacterial communities compared to the third flush group. In biomarker screening, Bacillus was selected in the first flush group, and Burkholderia-Caballeronia-Paraburkholderia and Sphingomonas were selected in the third flush group by linear discriminant analysis. These findings suggested that Bacillus, and Burkholderia-Caballeronia-Paraburkholderia and Sphingomona are potential bacterial biomarkers for determining early stage and late stage of L. edodes cultivation, respectively.

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We produced shio-koji (Japanese fermented seasoning)-like seasoning using mushrooms instead of Aspergillus oryzae. Shio-koji was fermented on a small scale using 50 mushroom strains and the resulting reducing sugar content of each sample was measured. Shio-koji produced by fermentation with Ganoderma lucidum NBRC31863 and Grifola frondosa SKB003 had high reducing sugar contents of 234.3 g / L and 219.4 g / L, respectively. The formol nitrogen content was highest in shio-koji produced by fermentation with Neolentinus lepideus SKB008 (0.147%). We selected six strains of mushrooms and fermented shio-koji at a medium scale using these strains. The findings showed that G. frondosa SKB003 had the highest reducing sugar content (143.2 g / L) and that Wolfiporia cocos NBRC10024 had the highest protease activity (7.37 U / L). We therefore selected G. frondosa SKB003 and W. cocos NBRC10024 for testing, and subjected beef soaked in shio-koji produced using these strains and controls (commercial shio-koji and 7% saline solution) to sensory evaluation testing. In terms of ratings, no significant differences were detected among samples. However, noticeable differences were observed among the samples using a three-point discrimination preference method, which showed that the shio-koji produced by fermentation with G. frondosa SKB003 was rated most highly.

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