| Antrodia cinnamomea(syn.A.camphorata),is a precious medicinal and edible mushroon,which possesses many bioactivties,such as anti-cancer,anti-oxidant,anti-inflammatory,hepatoprotective,and immunomodulation.The wild fruiting bodies of A.cinnamomea are very scarce and cannot fulfill the market demand.Then,submerged fermentation becomes a suitable and widespread way for industrial production of A.cinnamomea due to the advantages of short production period and low cost.However,submerged fermentation of A.cinnamomea(AcSmF)is still faced with some challenges,such as low sporulation and time-and laborconsuming process for seed preparation,which limits the industrial application of AcSmF.In this study,the effectors that promote asexual sporulation in AcSmF were screened from Cinnamomum kanehirae Hay(C.kanehirae),which is the the only natural host for wild A.cinnamomea.Subsequently,the additive concentration of effectors obtained was optimized to further promote the sporulation of AcSmF.Then,the A.cinnamomea mycelium samples cultured in the presence or absence of 1,8-Cineole at different period were used for RNA-seq to reveal the molecular regulatory mechanisms underlying 1,8-Cineole-promoted asexual sporulation of AcSmF.Finally,the antibacterial effect of extracts from the fermentation broth of A.cinnamomea(AcFB)on food-borne pathogens was studied.The contents of this study are listed as follows:(1)The effects of several extracts from C.kanehirae on asexual sporulation of AcSmF were investigated.Then,the water and chloroform extracs of C.kanehirae(CkWE and CkCE)greatly improved the sporulation of AcSmF and increased by 41.22%and 37.34%,respectively.A variety of compounds from CkCE were identified by LC-HR-ESI-MS.The effects of Lhistidinol,baicalein,and 1,8-Cineole on sporulation of AcSmF were further investigated.It was found that 1,8-Cineole dramatically increased the sporulation by 57.25%compared with the control group at the additive concentration of 500 μg/L.(2)The effectors that promote the asexual sporulation of AcSmF from CkWE were screened and its additive concentration was optimized.The CkWE increased the sporulation of AcSmF by 58.00%compared with the control group at the optimum additive concentration of 60 μg/mL.The effectors exist in the supernatant of alcohol precipitation for CkWE.Then,the CkWE was further extracted by normal butanol,ethyl acetate,and chloroform.Among them,the chloroform extracts of CkWE was found markedly promote the sporulation of AcSmF.After LC-HR-ESI-MS analysis and verification,it was found that erythritol is the main effector that promotes sporulation of AcSmF in CkWE with the optimum additive concentration of 1 μg/mL.In addition,1,8-Cineole and erythritol has a synergistic effect in promoting the sporulation of AcSmF.When 500 μg/L of 1,8-Cineole and 1 μg/mL of erythritol were added together,the sporulation of AcSmF reached 7.40 × 107 spore/mL and increased by 69.36%compared with the control group.(3)RNA-seq and bioinformatics analysis were used to reveal the molecular regulatory mechanisms underlying 1,8-Cineole-promoting asexual sporulation of AcSmF.In summary,1,8-Cineole acts on the cell receptors of Wscl and Mid2,then Pkcl receive the signal and send it to the cell wall integrity(CWI)signal pathway to promote the expression of rlml and wetA through a series of cascade reactions,and finally promotes the sporulation of AcSmF.Genes of velB and wetA promote the expression each other and then promote the cell wall synthesis and spore maturation of A.cinnamomea.Moreover,1,8-Cineole increased the membrane fluidity of A.cinnamomea,then heat stress was activated by CWI signal pathway for response.Subsequently,Hsp90 cooperated with the chaperone of Cdc37 to promote the expression of stuA and the relevant genes in CWI pathway.Gene of stuA further promoted the expression of brlA and thus promoting sporulation of AcSmF.In addition,1,8-Cineole promotes the expression of NCP1,a C2H2 zinc finger structural transcription factor,and further promotes the expression of areA,while areA could promote the expression of flbA and flbD through ammonium permease of MEP and inhibit the expression of pkaA.The up-regulated expression of flbD promoted sporulation of AcSmF,while the up-regulated expression of flbA and downregulated expression of pkaA could indirectly promote sporulation of AcSmF by inhibiting PKA pathway.(4)The antibacterial effects of different extracts from AcFB on food-borne pathogenic were investigated.It was found that:1)the chloroform extracts of AcB presents the widest bacteriostatic range by Oxford cup test;2)ethyl acetate and chloroform extracts of AcFB present remarkable inhibitory effect on 7 vibrios and non-vibrios by double gradient dilution method,respectively,and both of the minimum inhibitory concentration(MIC)are 1.565 mg/mL;3)all the chloroform,petroleum ether,and ethyl acetate extracts of AcFB present remarkable inhibitory effect on Salmonella by plate colony count test,and the inhibition rates are 92.7%,91.3%and 83.2%,respectively. |
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