Effect Of Extracellular Protease Secreted By Pseudomonas Fluorescens W3 On UHT Milk Quality

Under low temperature conditions,the growth of mesophilic microorganisms,mainly lactic acid bacteria,in raw milk is inhibited,but contaminated psychrophilic bacteria can continue to multiply and gradually become the main microorganisms in milk.At present,the most common psychrotrophic bacteria isolated from raw milk is Pseudomonas fluorescens.Although its bacteria can be killed by UHT(Ultra High Temperature)treatment,the protease produced by them at low temperature have strong heat resistance after UHT treatment and it can still retain a certain amount of activity and reduce the quality of UHT milk during storage,thereby shortening the shelf life.This thesis takes the dominant psychrophilic bacteria in raw milk-Pseudomonas fluorescens W3 as the research object,uses ammonium sulfate precipitation to extract the extracellular protease secreted by W3,and then studies the enzymatic properties,and finds that the enzyme is the most suitable The temperature and pH are 40? and 8.5 respectively;Ca2+ and Mn2+ promote enzyme activity,and Cu2+,EDTA and DTT inhibit enzyme activity,which indicates that the protease contains disulfide bonds and the active center may contain Ca2+.Determination of enzyme kinetic parameters found that the Vmax value of the protease was 13.947 U/mL and the Km value was 13.647 mg/mL,indicating that the enzyme is easier to bind to the substrate.Analysis of the heat resistance of protease found that after heating at 65? for 30 min,the residual enzyme activity in whole milk and skimmed milk were 64.25%and 58.20%,respectively;after heating at 137? for 5 s,the residual enzyme activity in whole milk and skimmed milk The enzyme activity was 51.53%and 41.13%,respectively,indicating that the milk component has a certain protective effect on the protease activity.The protease extracted by ammonium sulfate precipitation was added to UHT milk and observed at 40? for 30 h.It was found by SDS-PAGE that the protease hydrolyzed?-casein first,followed by ?-casein,and finally ?-casein.Lactoglobulin and ?-whey protein were hardly hydrolyzed,indicating that the protease mainly hydrolyzes casein.The study of the physical and chemical properties of UHT milk found that with the increase of time,the color of UHT milk gradually deepened,the particle size became larger,the absolute value of the zeta potential decreased,the viscosity increased,the acidity increased,the sediment content increased,and the hydration value decreased.The texture of UHT milk was measured and found that the hardness,cohesion and adhesion index of UHT milk increased with time,indicating that protease had a negative impact on the quality of UHT milk.The shelf life prediction model of UHT skimmed milk and whole milk at 37?,25? and 4? was established by using the Arrhenius equation.The degree of proteolysis obtained by the screening was used as the limiting index to determine the psychrophilic bacteria contaminated in the raw milk.When the number reaches 103 CFU/mL,the corresponding protease activity is about 0.50 U/mL.Under this condition,the shelf life prediction equation of UHT skim milk is:(?),UHT whole milk is:(?).Using this equation,the shelf life of UHT skim milk when stored at 25? is 165 d;UHT whole milk is 150 d,And the reliability of the prediction model was preliminarily verified at 37?.