Selection Of Feruloyl Esterase-producing Strains And Preliminary Application

Ferulate esterase,also known as cinnamate esterase or cinnamyl esterase,hydrolyzes the polysaccharides ferulate and oligosaccharide ferulate and belongs to a subclass of carboxylate hydrolases.It can hydrolyze ester bonds between ferulic acid or related cinnamic acid and polysaccharides such as arabinoxylan pectin,release free ferulic acid or ferulic acid dimer,and thus degrade the plant cell wall.The release of ferulic acid is widely used in feed industry,papermaking industry,ethanol biosynthesis,textile industry and agriculture.In this paper,the physical and chemical characteristics of feruloesterase strains were screened and identified,and the enzymatic characteristics and preliminary application were studied.The results are as follows.（1）Screening of feruloesterase producing strains.Sieve culture medium with ethyl ferulic acid as the sole carbon source was used to screen the bacteria.109 strains with ferulic acid esterase production were selected from the samples by transparent circle method.The results showed that the five baht bacteria producing the largest transparent circle were strain No.14,No.51,No.93,No.94,No.103,and the radius of the transparent circle was 0.9460 cm,0.9724 cm,0.9513 cm,0.9426 cm,0.8734 cm,among which strain No.51 had the largest transparent circle.The enzyme activity of these strains was tested.The results showed that the enzyme activity of Strain No.51 was the highest,and The enzymatic activity of strain was ranked as follows:No.51>No.14>No.94>No.93>No.103.（2）Bacterial classification.Through morphological observation,physiological and biochemical studies,biological characteristics and 16Sr DNA sequencing,it was determined that Strain No.51 belonged to Kosakonia Cowan II.（3）Enzymatic characteristics:the activity of ferulic acid esterase in this experiment was the highest at 50℃.When the temperature was below 50℃,the enzyme activity showed a gradual increase;when the temperature was above 50℃,the enzyme activity showed a decline with the increase of temperature.Ferulic acid esterase showed good stability in the range of 30-50℃.After being kept at 50℃for 50 min,the enzyme activity remained 58.96%.However,when the temperature was 60℃,the enzyme activity was poor,and the residual enzyme activity was less than 20%after being kept at 50 min.Ferulic acid esterase showed good stability in the range of 30-50℃.After being kept at 50℃for 50 min,the enzyme activity remained 58.96%;however,when the temperature was 60℃,the enzyme activity was poor,and the residual enzyme activity was less than 20%after being kept at 50 min.Optimization of culture medium,culture conditions and enzymatic characteristics.Through uniform experiment and stepwise regression analysis of quadratic polynomial,the culture medium for ferulic acid esterase production was optimized.The results showed that,under the optimal conditions,the values of each factor were:ammonium sulfate 0.1400 g,potassium dihydrogen phosphate 0.0600 g,MgSO₄·7H₂O 0.0480 g,CaCl₂·2H₂O 0.0086 g,Fe Cl₃0.0030 g,yeast powder 0.2290 g,and ethyl ferulic acid 0.8000 m L.After optimization,the enzyme activity increased by16.17%.The optimal fermentation conditions were as follows:90 m L liquid,5.5 p H,4%inoculum,34℃fermentation temperature,20.92%increase of enzyme activity.（3）Preliminary application direction of the strain.When the strain was fermented in bran,the content of ferulic acid was increased with the increase of fermentation time.The fermenting ferulic acid content of the blank group was 13.3740μg/g,and the ferulic acid content of 7d was 16.3715μg/g,which was 22.42%higher than that of the blank group.During fermentation of fermented grains,the content of ferulic acid in the fermented grains fermented 0d was 11.1366μg/g.The content of ferulic acid in the experimental group on 1d was increased compared with that in the fermented grains fermented 0d,and showed a downward trend after 2d.In the control group,ferulic acid in fermented grains was decomposed and its content decreased gradually without increasing.During daqu fermentation,the ferulic acid content of 0d daqu was 4.0548μg/g,and the ferulic acid content in the experimental group showed an increasing trend.The content of ferulic acid in the control group decreased first and then increased.However,compared with the experimental group,the content of ferulic acid in the control group was less,indicating that the addition of strains was beneficial to the release of Ferulic acid in Daqu and could increase the release rate of ferulic acid.The strain can be used in daqu production process and early brewing stage.