Study On The Enzymatic Properties Of Recombinant Human Lysozyme And Effect On The Intestinal Flora Of Mice

Forage and food need corresponding additives due to requirements inprocess technique, unique taste, and anti-corrosion, due to easy operations, apparenteffect, and low cost, many kinds of forages and food additives are widely used.However, with deeply related research, deficiencies of traditional additives are knownto us. It’s important to find a new additive with safety, high efficiency, and no harm.Human lysozyme is one member of lysozyme family and widely exists in body fluidand tissue. The lysozyme produced by microbes fermentation after engineeringbacteria is induced by related human lysozyme code genes via gene engineeringtechnique is named as recombinant human lysozyme. The recombinant humanlysozyme has common properties as common lysozyme, such as broad-spectrumantibiosis, retaining and anticorrosion, and inflammation reducing and anti-virus. Butits overall performance is better than egg white lysozyme and can replace thetraditional antiseptic and could be added in forage and food as function element, so ithas popular application potential. Moreover, recombinant human lysozyme isnon-specific immune factor in human body and is good model for researching onmolecular evolution and molecule immune relationship, which has great academicvalue.This research is aimed at discovering practical effect of recombinant human lysozymeas forage and food additives and its effects on body intestine bacterium and serum bioand chemical index via researching enzyme properties of recombinant humanlysozyme, real prevention and retaining effects, and influence on rats’ intestinebacterium and serum bio and chemical index.In this paper, first of all, based on the turbidimetry value of micrococcus,lysozyme activity was determined under different reaction conditions （temperature,pH value, artificial gastric and bowel liquid, and common metal ions）; Then, soakpieces of Lctalurus punctatus in different conceration of recombinant humanlysozyme solution then the organoleptic quality, bacterial colony number, pH value,TVB-N value, TBA value, acid value was determined respectively in different times;At last, mice were orally administered with the lysozymes through drinking waterincluding the egg white lysozyme and recombinant human lysozyme of2500and 5000U/ml. The serum were collected to determinate the concentration of total protein,urea nitrogen, triglyceride and cholesterol through picking eye ball of the mice at theexperimental time of1,7,14,21days, then the mice were slaughtered for theintestinal contents taken out in sterile environment to culture the Escherichia coli.,Staphylococcus aureus, Enterococcus faecalis, Bifidobacterium and Lactobacillus.The enzyme properties experimental results showed that egg white lysozymeexhibited the optimal temperature was40℃, and the optimal pH value was6.0;Recombinant human lysozyme exhibited the optimal temperature was45℃, and theoptimal pH value was6.0. Both lysozyme activities would decline rapidly above50℃, but keep high in the different pH6.0-9.0conditions. Their activities woulddecline rapidly when both lysozyme were kept in artificial gastric and bowel liquid,only recombinant human lysozyme would contain44.85%activity in artificial bowelliquid after120min; The Fe³⁺, Ca²⁺, K+, Mn2+, Zn2+, Mg2+, Al3+, Cu2+ions couldrestrain the lysozyme activity which had no significant difference between twolysozymes （P>0.05）. The retaining test showed that different concentration ofrecombinant human lysozyme solution can process various degrees after dipped onreducing the decreasing speed of fish organoleptic quality, TVB-N value, increasingspeed of TBA value, bacterium number and acidity. It can slow down the process ofcorrosion of fish and exerts a function as extending retaining time and using itsedibility and merchandise value to maximum degree. In practice,0.25%ofrecombinant human lysozyme can get good retaining performance and extends theretaining time of Lctalurus punctatus from4days to12days. The animal experimentshowed that compared with control group, both lysozyme can restrain the growth ofintestinal Staphylococcus aureus and Enterococcus faecalis, but promote the growthof Bifidobacterium and Lactobacillus in the later experimental time; Onlyrecombinant human lysozyme had obvious inhibitory effect against Escherichiacoli（P<0.05）. Two lysozymes can increase total protein concentration and reduce ureanitrogen and cholesterol concentration for the mice orally administered with twolysozymes, but only above three serum biochemical indicators of recombinant humanlysozyme group（5000U/ml） showed significant difference（P<0.05） at theexperimental time of21day.The result displays that recombinant human lysozyme and egg white lysozymecould regulate the intestinal flora and affect serum biochemical indicators, and theeffect of recombinant human lysozyme was more obvious than that of egg white lysozyme for the mice. It should be considered how to control the conditions oftemperature, pH value, artificial gastric and bowel liquid, and metal ions when twolysozyme were added into the foods as an additive. Besides, recombinant humanlysozyme could significantly prolong storage period of aquatic product, so its verysuitable to use as forage and food additives.