Isolation Of High-yield Esterase And Non-citrinin Monascus Strain And Its Esterase Production Condition Optimation

Microbial esterase plays an important role in increasing the components of ester aroma in fermented foods,like liquor.The main esterase-producing microbes include Monascus spp.,yeast,Rhizopus spp.and some bacteria,among which Monascus spp.possess the relatively strong ability to produce esterase.In this research,Shanxi aged vinegar Daqu and Zhijiang liquor Daqu were taken as the objects,from which high-esterase-producing Monascus spp.may be isolated.Those isolated strains were identified through morphological observation,combinated with ITS(Internal transcribed spacer)sequence analysis and ISSR(Inter-simple sequence repeats)analysis methods,then a Monascus strain with high-yield esterase was achieved.The production of citrinin in this strain was also detected,and its fermentation conditions for esterase-production were optimized by the orthogonal method.The main results are as follow:1.Isolation and identification of strains41 Monascus strains were isolated from Shanxi aged vinegar Daqu and 2 Monascus strains were isolated from Zhijiang liquor Daqu.Among those strains,M51411 is identified as M.anka and the others are identified as M.pilosus strains through morphological identification,combinated with ITS sequence analysis and ISSR analysis.There are 8 strains of Monascus isolated from the commercial Hongqu products,and 4 of them,as well as the43 strains from Daqu and 2 strains from our lab were used to determine the esterase activity in next experiments.2.Esterase activity determination and citrinin analysisThe esterase activities of 49 Monascus strains in�1�were detected,the results show that the esterase activities of 49 strains range from 3.000 mg/g�100 h to 10.000 mg/g�100 h.One of the strains from Hongqu products has the highest enzyme activity of 10.266 mg/g�100 h while the enzyme activity of M.pilosus M76113 which from Daqu is 8.194 mg/g�100 h in the third.Considering the intellectual property rights of strains,we choose M.pilosus M76113 as the object in next experiments.The production of citrinin in M.pilosus M76113 was detected by UPLC,and the sequences of citrinin related gene were amplified.The results show that there is no citrinin detected and no stripes amplified,in combination with the both results,M.pilosus M76113may not produce citrinin can be concluded.3.Optimization of producing-esterase condotions by M.pilosus M76113In order to explore the effects of different fermentation conditions on esterase accumulation in M76113 solid-state fermentation,the single factor experiments were carried out,and the preliminary optimal fermentation conditions in 250 m L Erlenmeyer flask are:20g substrate weight,20 m L tap water,5%inoculum size(Inoculating 10%M76113 spores suspension of which concentration was 10⁶m L^-1 into PDB and shaking cultivating for 24 h with the speed of 170 r/min.),30 ^oC incubation temperature,11 d incubation time.Based on the single factor experiments results,the orthogonal experiments were designed to optimize the conditions of fermentation.The results show that,the optimal fermentation conditions in 250 m L Erlenmeyer flask are:20 g substrate weight,20 m L tap water,5%inoculum size(Inoculating 10%M76113 spores suspension of which concentration was 10⁶m L^-1into PDB and shaking cultivation for 24 h with the speed of 170 r/min.),32 ^oC incubation temperature,12 d incubation time.The optimized esterase activity reach to 10.904mg/g�100 h that increased 33.07%compared with the initial enzyme activity of M.pilosus M76113,and was similar to the enzyme activity of esterase commercial Hongqu products.