Isolation Of A Highly Pathogenic Beauveria Bassiana Infected With Silkworm And Study On Its Protease

Beauveria bassiana,a major entomopathogenic fungus,infects silkworm larvae,producing the white muscardine silkworm Bombyx Batryticatus.The bioactive substances in Bombyx Batryticatus are widely used in medical,healthcare,and cosmetic industries.Many active ingredients and bioactive substances in the silkworm are also beneficial to human health.However,Beauveria bassiana is easily influenced by the environment during the growth process.After multiple passages,it is found that the virulence and growth state of Beauveria bassiana are extremely unstable,and it is prone to obvious virulence degradation.The spores produced in fermentation process have phenomena of short storage time and easy inactivation,which are not suitable for large-scale production.In this study,we isolated 7 B.bassiana strains（SDJC-1,SDJC-2,SDJC-3,GXHC-1,SDND-BB,AT-3006,and SQJC-1）from different samples of Bombyx Batryticatus.Based on the results of biological characteristics（colony morphology,sporulation characteristics,vegetative growth）,protease and chitinase activities,and the lethality in silkworm,a strain SDJC-3 with high pathogenicity and excellent traits was screened out.The strain SDJC-3 had excellent biological characteristics,high sporulation,the highest protease and chitinase activities,and the lethal rate of 75%to silkworm.The Bombyx Batryticatus prepared with SDJC-3 was large and plump and had clear ring of silk gland in the cross-section,exhibiting good quality.Therefore,the strain SDJC-3 was selected as the starting strain to carry out the following studies.The fermentation conditions of the screened strain SDJC-3 were optimized by response surface optimization technology.Firstly,through the optimization of single-factor fermentation conditions,the optimal growth temperature,shaking speed and volume ratio of fermentation broth were 27℃,180 r/min and 1/5,respectively.Secondly,taking the biomass of Beauveria bassiana after fermentation（dry weight of mycelium）as the response value,the three-factor（temperature,shaking speed,volume）three-level response surface test was carried out on the strain SDJC-3 using the software Design of Experiments,the results showed that the volume had the greatest effect on the fermentation of Beauveria bassiana,followed by the shaking speed,and the temperature had a lesser effect.The optimal fermentation conditions of strain SDJC-3were as follows:the temperature,shaking speed and volume ratio of fermentation broth were27.4326℃,165.393 r/min and 23.0066%,respectively.Under these conditions,the maximum fermentation biomass could be 15.481 g/L,increased to 116.19%of the previous fermented biomass.The enzymatic properties of the protease produced by the strain SDJC-3 were studied,and it was found that the optimum temperature of the protease was 25℃,the optimum p H was 8.0,and metal ions(K^+,Mg²⁺,Mn²⁺,Na⁺)could significantly activate the protease.The silkworm was hydrolyzed with protease,and the antioxidant activity of the hydrolyzed product was determined.within the range of certain density,the scavenging ability of the silkworm hydrolyzate to superoxide anion free radicals and DPPH free radicals was 75.0%and 72.6%,respectively,while the scavenging ability on hydroxyl radical was 59.9%.We isolated 7 B.bassiana strains from different samples of Bombyx Batryticatus.Based on the results of biological characteristics,protease and chitinase activities,and the lethality in silkworm,strain SDJC-3 with high pathogenicity and excellent traits was screened out.The optimization of fermentation conditions,the study of enzymatic properties and the study of antioxidant active substances of hydrolyzate were carried out.The research results provide a theoretical basis for the research on the selection and breeding of highly pathogenic Beauveria bassiana strains,the quantitative preparation of the silkworm and the antioxidant active substances of the silkworm,and the further exploration of the application potential of the silkworm.