Study On The Fermentation Regulation Of Shiraia Bambusicola For Hypocrellin A And Its Antimicrobial Properties

Hypocrellins（HYPs）are hydrophobic perylene quinones isolated from the stroma of medicinal Hypocrella bambusae（Berk.&Broome）Sacc.or Shiraia bambusicola Henn.Studies on the anticancer,antiviral,and antibacterial activities of HYPs have been reported over the last 40 years.With outstanding photoinducible activities,HYPs can be adopted for photodynamic therapy（PDT）.It is based on the photocatalytic production of singlet oxygen and reactive oxygen（ROS）,which have an untargeted impact on cell membranes,cell walls,proteins,lipids,and nucleic acids and leads to cell death.Compared to the widespread reuse of traditional antimicrobial drugs,which have heightened the resistance of numerous microorganisms,resulting in traditional antimicrobial drugs becoming challenging to fight microbiological infections,the non-targeting mechanism of PDT reduces the probability of bacteria developing resistance.Despite the good phototherapeutic activity of HYPs,there are still some pressing challenges in its application.On one hand,HA is a lipophilic compound with poor water solubility,which cannot conveniently be converted into pharmaceutical preparations,especially intravenous injections;On the other hand,the origins of natural HYPs products are limited,and microbial fermentation technology is currently a feasible method for large-scale production of HYPs.In order to gain a more thorough insight into HYPs antimicrobial activity against multiple pathogenic microorganisms and to be able to exert HYPs antimicrobial activity with maximum efficiency,this work systematically investigated the inhibitory or killing effectiveness of HA,a relatively high-yielding HYPs,against Aspergillus niger,Candida albicans,Staphylococcus aureus,and Escherichia coli.Furthermore,in order to fully enhance the antimicrobial rate of HYPs,the effect pattern of light（wavelength,intensity,duration,etc.）,HA concentration,p H,and other factors on the antimicrobial activity of HA were investigated.In addition,morphological observation and transcriptome analysis further investigated the molecular mechanism of HA antimicrobial activity.Research results are as follows:The S.bambusicola GDMCC 60438 strain had the highest HYPs production efficiency at p H=6.4 and 32°C.As a result,the yield of HA（the major component of the HYP product is HA,which is 95%）was 154.54 mg/L.By adding 1 mg/L of V_B1 at the 12th h of fermentation,the HA yield could be increased by 32.41%compared to the control group;by adding 200m M of KCl at the 12th h of fermentation,the HA yield increased by 68.61%compared to the control group,reaching 243.06 mg/L.The HA samples demonstrated stronger antifungal activity under 465-470 nm light irradiation.A1-10 ppm HA inhibits the growth of A.niger CMCC（F）98003 and A.aculeatus AA-01.This inhibition correlated significantly with light dose and light duration.The kinetic inhibitory activity of HA against light in mycobacteria under aerobic conditions was correlated with the amount of ROS catalytically produced.Scanning electron microscopy and transcriptome studies revealed a disrupted cell wall/membrane structure of A.niger,which could be one of the important reasons for the photodynamic inhibition of mycorrhizal growth by HA.Low HA concentrations（<2.5 ppm）also had inhibiting activity on the strain growth and biofilm formation of C.albicans CMCC（F）98001,which could also have the biofilm removed.The activity of anti-C.albicans of HA could be significantly improved in conjunction with H2O2（<50 m M）.The mechanism of action increased cell membrane permeability.In addition,it increased intracellular ROS,which affected the increase in the C.albicans,while inhibiting mycelium formation,thereby reducing the infectivity and virulence of C.albicans.HA showed significant inhibition efficiency against S.aureus CMCC（B）26003.In a p H=7.5 solution environment under light irradiation at 465-470 nm,a 2.5 ppm HA could completely be inhibiting S.aureus.The activity depended on the light dose and HA concentration.More prolonged exposure（>15 min）at lower light doses was more conducive to its anti-bactericidal effects.In this study,HA production was enhanced by regulating the liquid fermentation process of S.bambusicola.A.niger,A.aculeatus,C.albicans,S.aureus and E.coli were selected as the targets of HA,and the appropriate conditions of HA use such as concentration,light intensity,light wavelength and irradiation duration were explored.The results of the above study has certain significance to promote the application of HA and solve the problem of microbial drug resistance.