Production Process Optimization And Performance Of Phospholipase A₂ And Enzymatically Hydrolyzed Phospholipids

Phospholipid is a kind of natural emulsifier,which has been widely used in food,feed,health care products,medical treatment and light industry.However,the hydrophilicity of natural phospholipids is not good enough,and its application in some fields is far from the desired goal and effect.In order to improve the properties of phospholipids,natural phospholipids have been modified by physical,chemical and biological methods.In the above method,the biological method（enzymatic modification）is used to change a hydrophobic fatty acid ester in the phospholipid molecule to a hydroxyl group,and the method of obtaining lysophospholipids is simple,environmentally friendly,and has fewer by-products.The hydrophilicity and emulsification of the product lysophospholipids are greatly improved.Phospholipase A₂is the most important enzyme used for phospholipase modification.In addition,phospholipase A₂can also be used in many fields such as enzymatic deguming of vegetable oils,preparation of decellularized matrix and cosmetics.In this study,the preservation,fermentation,isolation and purification of phospholipase A₂were explored and optimized,which provided a basis for the development of suitable production process and scheme.In rich and poor water system water phospholipase A₂catalytic phospholipid the influence factors of the preparation of lysophospholipids and process conditions are optimized and exploration;The relationship between the emulsification index and the degree of enzymatic hydrolysis was also studied.The main research contents and results are as follows:1.Study on bacterial culture and solid culture:The effect of antibiotic concentration on bacterial culture and spore preservation was investigated.When the concentration of antibiotics in solid medium was 150μg/m L,the screening effect was the best,and after the spores were stored in 20%glycerol tubes for 2 years,the enzyme activity could recover to the maximum after reactivation.This study provided a method basis for the screening of recombinant plasmids for antibiotics and the preservation of spores in glycerol tubes.2.The study of phospholipase A₂liquid culture:it was found that the enzyme activity reached 10100 U/m L when the liquid medium with 100μg/m L antibiotic concentration was transferred to the seventh passage.When the seventh passage strains were cultured in liquid medium with the concentration of 100μg/m L antibiotics,the enzyme activity reached 10100 U/m L when the third passage strains were cultured.This study provided theoretical and technical guidance for the large-scale production of phospholipase A₂using fermenter in factories in the future.3.The technological conditions for the separation and purification of phospholipase A₂were as follows:first,the enzyme protein was precipitated with 30%（w/w）ammonium sulfate,and then phospholipase A₂was purified by gel filtration chromatography,and then powder phospholipase A₂could be prepared by vacuum freeze-drying technology.4.The emulsification index was found to be positively correlated with the degree of enzymatic hydrolysis.Therefore,emulsification index could be used to reflect the content and conversion rate of enzymatic hydrolysis phospholipid.Finally,the relationship between emulsification index and lysophospholipid content in products was determined as follows:Q（Lysophospholipid content,%）=（Emulsification index （U,U/mmol）-1389）/12.418orQ（Lysophospholipid content,%）=（Emulsification index （U,U/g）-1664）/14.8825.Preparation of lysophospholipids catalyzed by phospholipase A₂:In water-rich enzymatic hydrolysis system,enzyme amount 725 U/g（phospholipase A₂/concentrated phospholipid,0.65 g phospholipid/g concentrated phospholipid）,Na H₂PO₄-Na₂HPO₄buffer（0.10 mol/L,p H=7.5）,substrate ratio 30%（concentrated phospholipid/total reaction system,W/W）,reaction state stirring,The reaction time was 18 hours.The content of lysophospholipids increased by 55.50%,and the total content of lysophospholipids in the product reached 75.50%.In the water-poor enzymatic hydrolysis system,97%phospholipid concentration（phospholipid content 65%,lipid content 35%）,enzyme amount 0.2%（phospholipase A₂activity 250,000 U/g,w/w,）,1%5 mol/L Na OH solution,1.8%water,and the reaction state was still for 24 h.The total content of lysophospholipid in the product reached more than 52.20%.