Preparation Of IgY Against Polyphenol Oxidase (PPO) And The Effect Of Inhibiting Shrimp Melanosis

Shrimp melanosis caused by polyphenol oxidase (PPO) is the key factor in theprocess of shrimp products. It affects the sensory quality and commercial value ofshrimp deeply. Inhibiting activity of the polyphenol oxidase and development ofsecurity, efficient biological materials in controlling shrimp melanosis are the hotresearch area at preasent. Egg yolk antibody (IgY) is only one type of yolk formedfrom IgG which transfer into immunoglobulin in poultry serum. A very importantproperty of IgY is its remarkable inhibitory effect on the corresponding antigenbacteria. It can not only affect the antigen activity significantly, but also be used as apotential novel biological anti-staling agent.In this research,4℃fresh Penaeus vannmei is chosen as research object. Thepurpose is to inhibit activiry of PPO in Penaeus vannamei. The PPO obtained byextracting and purifying from Penaeus vannmei, is used to immunize laying hens asantigen to prepare the specific IgY. Then these IgY immune activity is explored atdifferent temperatures and pH, On which basis, the effect of inhibiting activity in vitrois determined. At last, the consolidated sensory evaluation, chemical indicators(TVB-N, pH) and microbiological indicators is taken to evaluate the performance ofPPO specific IgY in vivo sample preservation. Main achievements are listed asfollowing:The crude enzyme is extracted from Penaeus vannmei. It was isolated andpurified by acetone precipitation purification, Sephadex G-100gel chromatographyand DEAE Sephadex A-50Ion chromatography. Then, pure PPO purity was identifiedby SDS-PAGE gel electrophoresis. The crude enzyme activity is0.45U/mg. The totalamount of protein is10940.2mg. After purification, the activity of pure PPO is14.4U/mg. The total multiples of purification is32, and protein recovery rate is1.19%.The activity recovery rate is38%. Determined by SDS-PAGE, pure enzyme molecularweight is about45kD, and the purity has been reached electrophoresis level. The purified PPO was taken as antigen to immunize hens. IgY extracted fromegg yolk. Its immune titer was measured by ELISA. After three times injections, titerof IgY turn to be about1:2000. Then, the immunological activity of the tenth weekIgY was determined at different temperatures and pH. The parameters showed that itsactivity was very stable under60℃，and it maintained above50%activity betweenpH5and pH9.In vitro, the effect of IgY to inhibit activity of PPO was positively correlatedwith concentration. The greater concentration is, the more obvious effect is. At0℃and37℃, the0.5mg/mL of specific IgY can exhibit a certain inhibitory effectcompletely. After a period of reaction, the residual enzymatic activity is maintained atabout70%. With the increasing of concentration, the inhibiting effect is also graduallylifting. When the concentration is5mg/ml, its inhibitory effect climbed to the top, theremaining activity was maintained at about30%.By indices such as pH, TVBN, total colonies and sensory evaluation at4℃, vivosamples were analyzed. The results showed that the performance of specific IgY inPenaeus vannmei preservation is very significant. Therefore, the melanosis could bedelay from24h to72h after pretreated by5mg/mL IgY solution. The shelf life ofPenaeus vannmei chilled at4℃also extend from48h to96h.