Screening,Immobilization And Application Of Microbial Lipase-producing Strains

Lipase?Lipase,EC 3.1.1.3?belongs to carboxyl ester hydrolase,it can catalyze the hydrolysis of natural substrate oil and produce fatty acids,glycerol and glycerol monolipids,mainly distributed in animal,plant and microbial tissues.In this paper,a microbial lipase producing strain was screened from soil,and the conditions of enzyme production and immobilization were analyzed for its application in food.The results were as follows:1.A lipase producing strain Y3 was screened from 6 soil samples by initial screening of rhodamine B plate and fermentation re-screening on initial fermentation medium.The initial enzyme activity of Y3 strain was 7.6 U/mL.The strain was identified by morphological identification and 26SrDNA sequence analysis.The strain belongs to Fusarium of the genus?Fusarium tricinctum?;The fermentation conditions of Y3strain enzyme production were optimized by response surface analysis.The optimum medium conditions were as follows:5%sucrose,4%tryptone and 0.5%MgSO₄,the enzyme activity reached 12.74 U/mL,which was 1.68 times that of the initial enzyme activity.The optimum fermentation conditions are:when the culture time was 2 days,culture temperature was 30°C,the pH value was 5,and the enzyme activity reached 12.546 U/mL,which was 1.65 times of the initial enzyme activity.2.Using sodium alginate-polyvinyl alcohol as immobilized carrier and glutaraldehyde as crosslinking agent to immobilized microbial lipase.The optimum conditions of immobilized enzyme and compared the enzymatic properties of immobilized enzyme and free enzyme were discussed.Final determination the optimal conditions for immobilized enzyme were sodium alginate concentration 2%,glutaraldehyde concentration 2%,polyvinyl alcohol concentration 8%,CaCl₂ concentration 1.5%,immobilized cross-linking time 10 h,under this condition,the relative enzyme activity of immobilized enzyme reached71%.Compared with the free enzyme,the immobilized lipase had higher thermal stability and pH stability,and had good reusability and storage stability.3.Headspace solid-phase microextraction?SPME?was used to extract volatile components from meat and dairy products before and after microbiological lipase treatment,and was analyzed and identified by electronic nose and gas chromatography-mass spectrometry?GC-MS?.The results of electronic nose test showed that the addition of microbial lipase could effectively improve the flavor of pork,shellfish and yoghurt.The main volatile components in pork,milk scallops and yoghurt were aldehydes,alcohols,ketones,acids,aromatic hydrocarbons and so on,which were consistent with the results of electronic nose detection.Taking starch and chitosan as examples,the effect of microbial lipase on polysaccharide was investigated.The amount of lipase was 2.5 mL;The concentration of the substrate was 6%;The temperature is 60?;The optimum time of starch and chitosan were 45 min and 75 min,respectively.The optimum pH value of starch is 4 and chitosan is 7.This indicates that microbial lipase plays an important role in the flavor of meat,dairy and polysaccharides.4.Using strain Y3 as a template,the lipase gene was amplified by PCR to obtain the target gene fragment,which was 90%or more homologous to the sequence of Fusarium sp.lipase gene published by NCBI,indicating that the gene fragment was the same size as the target fragment.