| Tilapia is common fish species, which is mainly used in fish production in aquatic produCS processing. Though a low utilization for Tilapia by-produCS toproduce feed and fertilizer, by-produCS of Tilapia are rich in nutrients, including protein, active peptides, fat and trace elements etc.Extracting active polypeptidefrom Tilapiaby-produCS by bio-enzymatic has a strong application value. Due to the high costs and low re-use rate of free enzyme,it is essential to find appropriate method to improve the repetitive use of bio-enzyme.(1) The optimal enzymatic hydrolysis conditions of Tilapia byproduCSThe response surface method was used to find out the suitable conditions to produce polypeptide fromTilapoa byproduCS with biological enzyme. According to the components of Tilapoa byproduCS amino acidsand commonly used enzyme species in hydrolysis, Elastase, Papain, Proteinase K and Trypsin were chosen to hydrolyze the byproduCS of Tilapia, and optimze hydrolysis conditions under their hydrolytic degree and the yield of polypeptide.When the conditions were:enzyme concentration 20.56 U/g, temperature 38.28℃,3.19 h, pH (natural 7.6), solid-liquid ratio 1:5, hydrolysis degree of Tilapia by-produCS by Elastase reached to 19.89%, the yield of polypeptide was 0.98 mg/mL;when the conditions were:enzyme concentration 50.63 U/g, pH5.14,38.28℃,2 h, solid-liquid ratio 1:5, hydrolysis degree of Tilapia by-produCS by papain reached to 9.99%, the yield of polypeptide was 0.65 mg/mL; when the conditions were:enzyme concentration 31.37 U/g, pH7.06,2.87 h, room temperature (22℃), solid-liquid ratio 1:5, hydrolysis degree of Tilapia by-produCS by proteinase K was 16.83%, polypeptide yield was 0.55 mg/mL; when the conditions were:enzyme concentration 52.37U/g, pH6.92, room temperature (22℃),2.26h, solid-liquid ratio 1:5, hydrolysis degree of Tilapia by-produCS by Trypsin was 12.62%, the yield of polypeptide was 0.42 mg/mL.The best enzyme to hydrolyze Talipia by-product is Elastase.(2)Antioxidant activity of enzymatic hydrolysate from Tilapia by-produCSPreparing polypeptides by using hydrolysis optimum conditions of Elastase and Papain respectively. The influence of different drying procedure (vacuum drying, freeze drying, constant pressure drying, spray drying) to antioxidant activity of polypeptide powder were compared by pyrogallol autoxidation method, DPPH·method, prussianblue method, phosphorus molybdenum complex method,etc.The results showed that polypeptide powder extracted from Tilapia byproduct by Elastase and Papain both have a strong role in antioxidant activity; Elastase and Papain both have a strong reducing power,in the aspect of scavenging capacity of DPPH radicald, superoxide anion radical, hydroxyl r polypeptide powder made by adical, polypeptide powder of Papain was better than Elastase’s; total antioxidant activity and lipid antioxidant activity of polypeptide powder produced by Elastase was larger than the polypeptide powder made by Papain. Differentdryingmethods has a great influence on the antioxidant activity of peptide powder.selecting the appropriate enzyme and drying method to prepare peptide according to the requirements of antioxidant activity. In general, polypeptide powder made byPapain shows greater antioxidant activity.Polypeptide powder were measured by amino acid automatic analyzer and mass spectrometry assays. Molecular weight distribution range of peptide powder produced by Elastase was within 400～1500 kD and concentrated in 500～1400 kD, molecular weight distribution range of peptide powder produced by Papain was from 500 to 1700kD and concentrated in 600～1400 kD, which better illustrated the hydrolysis effect of Tilapia byproduct by Elastase and Papain were pretty well, difference of anti-oxidation effect of polypeptide polypeptide made by papain and made by elastase may be related to mass distribution. Amino acid composition analysis showed that polypeptide powder can be used as a nutritional supplement applied to food produCS because of abundant content of amino acid.(3) Chitosan Halloysites composite microspheres immobilized PapainCS/HNTs composite microspheres were successfully prepared by crosslinking-adsorption method, and used to immobilize Papain.Confirmation of the papain has been fixed by crosslinking adsorption method on CS/HNTs composite microspheres through infrared spectrometer, electron microscopy, fluorescent labeling methods, etc. The immobilized effect found to be best when the ratio of CS and HNTs rate was 1%:2%,the immobilized rate was up to 1.76mg/g; immobilized Papain showed highest relative activity when the concentration of Papain was lmg/mL; relative activity of immobilized Papain grows with time, but after 10h, relative activity of immobilized Papain maintaining a balance trends; The relative activity of immobilized Papain reached maximum at pH 6.8,the relative activity of free enzyme reached maximum at pH 7.2, which realated to support structure; optimum temperature of immobilized Papain was 60℃, the optimum temperature of the free enzyme was 50℃. After 30 days storage, the relative activity of immobilized Papain still holds around 62%, while the free enzyme relative activity decreased to 10%. The relative activity of immobilized enzyme decreased with the increase of frequency, the relative activity of the immobilized enzyme remaining 26.26% after 4 times reusing.Using bio-enzymatic method to enhance the value of tilapia by-produCS, polypeptide by enzymatic preparation has oxidation activity, whichcan be used as additives to food, cosmetics, etc; Papainimmobilized on CS/HNTs microspheres has stong storage ability and operational stability, that greatly enhance the value of papain. |
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