Dissertation
Dissertation > Industrial Technology > Light industry,handicrafts > Food Industry > Aquatic products processing industry > Fisheries by-product processing and utilization of

Study on Preparation of Antioxidative Peptides from Shrimp Processing Byproducts Hydrolysate

Author ZhaoJing
Tutor JiangJiaXin; HuangGuangRong
School
Course Biochemistry and Molecular Biology
Keywords Shrimp Processing Byproducts antioxidative peptide Isolation andPurification antioxidative activity stability
CLC TS254.9
Type Master's thesis
Year 2012
Downloads 112
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In this paper, taked the shrimp processing byproducts (SPB) as a rawmaterial, pretreatment of material, choice of hydrolase, the optimal conditions forenzymatic hydrolysis were studied to produce antioxidative hydrolysate adoptingenzymolysis technique. DPPH radical scavenging activity (DSA) was detected as theevaluation guide. Amino acid composition, molecular weight distribution, antioxidantability in different assay systems and the stability of antioxidative activity wereanalized to understand the properties of shrimp processing byproducts hydrolysate(SPBH). The hydrolysate was further isolated and purified by several steps includingmethanol extraction, ion-exchange chromatography, gel filtration chromatographyand a two-step reverse-phase high-performance liquid chromatography (RP-HPLC) inorder to purify a peptide with potent antioxidant property. The purified antioxidantpeptide was identified by electrospray ionization tandem mass spectrometry(ESI/MS/MS).The main findings are as follows:(1) SPB has a fat content of1.5%. The hydrolysate has a lower content of solubleprotein and degree of hydrolysate (DH), but a higher DSA after being defatted.(2) SPB was independently hydrolyzed by Trypsin, Pepsin, Protamex,Flavourzyme, Neutrase, and Alcalase. Results showed that the Alcalase hydrolysatetook on the highest DSA and also gived the highest DH of all. The molecular weightdistribution of each hydrolysate was not that big of a difference.(3) A central composite design (CCD) part of response surface analysis (RSM) wasused as an experimental design based on the single-factor to product peptides withhigh DPPH and ABTS radical scavenging activity (DSA and ASA). The results forDSA showed that the optimal enzymatic hydrolysis conditions were temperature50.5℃, time duration120min, pH7.2, enzyme concentration415U/g. DH, DSA, andthe average length of peptides were21.8%,66.9%, and4.6, respectively. The resultsfor ASA showed that the optimal enzymatic hydrolysis conditions were temperature59.6℃, time duration120min, pH8.4, enzyme concentration597.3U/g. DH, ASA,and the average length of peptides were40.2%,38.3%, and2.5, respectively. There isno direct relationship between the degree of hydrolysis and antioxidative activity.(4) The hydrolysate produced under optimized enzymatic hydrolysis parameterswith high DSA was choosed for properties research. The results were as follows: (a) The hydrolysate had an extremely high content of favor amino acids (Glu,Asp,and Gly), which make up44.0%of the total amino acids. The essential aminoacids of SPBH made up32.1%of all amino acids. The content of hydrophobic aminoacids (Pro, Gly, Ala, Val, Ile, Leu and Phe) was40.3%.(b) Most peptides of this hydrolysate were smaller than6.5kDa. The fraction withlower (molecular weight) MW than1kDa exhibited the highest activity of all.(c) DSA, ASA, the reducing power and the metal chelating activity were detectedto evaluate the antioxidative activity of the hydrolysate. The IC50values of BHT andSPB hydrolysates for DSA were0.29and0.50mg/mL; The IC50values of BHT andSPB hydrolysates for ASA were7.2and7.4μg/mL; At the absorbance of0.5for thereducing power, the concentration of BHT was0.22mg/mL, while the concentrationof hydrolysates protein was4.8mg/mL. The metal chelating activity was38.9%at0.12mg/mL concentration while the chelating activity of EDTA reached73.4%at0.1mg/mL.(5) The antioxidant stability experiments showed that the hydrolysate was stablewhen it was heated up to100℃and the relative antioxidative activity could bemaintained nearly70%at very low pH of2.0. Glucose and sucrose had a negativeeffect on the antioxidative activity, in which the relative activity of about80%wasretained. Sodium chloride and sodium benzoate had little or no effects on theantioxidative activity of the hydrolysate. The effects of Zn2+and Cu2+on theantioxidative activity were significant and dependent on metal concentration.Therefore, the hydrolysate should be avoided to contact with the substance includingZn2+and Cu2+during processing.(6) The hydrolysate was isolated and purified by several steps and DSA was usedto evaluate the antioxidant activity of fractions. The purified antioxidant peptide wasidentified as Ser-Val-Ala-Met-Leu-Phe-His (804.4Da), and the DSA was65.7%at50μg/mL, which was a3.18-fold higher compared with the first step separation byion-exchange chromatography. The high antioxidant activity may be due to thepresence of Phe-His segment at the C-terminus of the peptide.

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