Research On The Preparation And Antioxidant Capacity Of Placental Peptide From Tibetan Sheep

The sheep farming is common in China,resulting to that the sheep placenta with great nutritional and medicinal value is abundant.Processing sheep placenta into high-value products and bioactive peptide will pave a way for the full use of sheep placenta.In this study,antioxidant peptide was prepared from Tibetan Sheep placental through ultrasonic-assisted compound enzymolysis technology and determined its antioxidant capacity in vitro by using the many kinds of antioxidant capacity evaluation method.Then Fourier infrared spectrum,scanning electron microscopy?SEM?,X-ray diffraction and circular dichroism method were used to characterization its secondary structure.Sheep placenta peptide was isolated and purified by gel chromatography and the amino acid sequence of the target peptide was determined by Easy nLC1200-Q Eaxtive plus.The main research results are as follows:?1?The protein content was determined to be 85.7%by using Kjeldahl,lipid content of5.23%was messured through Soxhlet extraction method and high performance liquid chromatography was used to analyse amino acids composition.Thus,we can found that Tibetan Sheep placental is a kind of material with high protein,low fat,essential amino acids?26.36%?and antixodative amino acids content?13.25%?.Based on that,the peptide from Tibetan Sheep placental were produced by ultrasound-assisted composite enzyme hydrolysis with the indexes of the degree of hydrolysis?DH?,the yield of peptide?YP?and the antioxidant capacity,based on the screening of protease species and determination of dual-enzyme combination scheme.Furthermore,the one-factor-at-a-time?OFAT?method and response surface methodology?RSM?were used to optimize the condition of ultrasound-assisted enzymolysis process.On the other hand,the amino acid composition and relative molecular weight distribution of the peptide under the optimal technological conditions were analyzed by high performance liquid chromatography?HPLC?.The results showed that the hydrolysis efficiency of simultaneous combined enzymolysis of papain and neutrase was better than that of other proteases strategy,the DH and YP were reached to 37.24%and 13.54%,respectively.The results of RSM optimization showed that the effect of ultrasonic time on DH was the strongest,followed by ultrasonic temperature,enzymolysis time and ultrasonic power.The optimal hydrolysis parameters after optimization was the ultrasonic time of 16.5 min,ultrasonic temperature of 23.5?,ultrasonic power of 438 W,enzymolysis time of 4.9 h.Under the optimized conditions,the DH,PY and the antioxidant capacity were 55.22%,18.52%and 1.08 mM?equivalent to 1.08 mM VE?,respectively.?2?The free radical scavenging ability,metal ion chelation ability and reducing ability of different concentrations of placenta peptide from Tibetan sheep were determined,and the distribution of relative molecular weight,amino acid composition and structural characterization were also investigated to explain the causes of antioxidant capacity.The results showed that the antioxidant capacity of sheep placenta peptide was positively correlated with its concentration,and it had strong radical scavenging ability for DPPH·and·OH with IC50 of 2.83 mg/mL and 0.94 mg/mL,respectively,which were comparable to BHT.In addition,the placenta peptide from Tibetan sheep also has certain O₂^-·scavenging ability,metal ion chelating ability for Fe²⁺and reduction ability for Fe³⁺and Cu²⁺.Moreover,the placenta peptide below 1000 Da of molecular weight accounted for nearly 90%in total peptide,which contained an abundance of Asp,Glu,Gly and so on.Structural characterization results showed that the placenta peptide still remained the proper functional group structures of protein in sheep placental,the micromorphology of dense spherical granular happened,and the rearranged protein molecules aggregated during degradation of proteins to peptide.Based on the results of circular dichroism,the decreased content of?-helices,increased content of?-sheets and random curl were observed.In a word,the placental peptide from the Tibetan sheep have nice antioxidant capacity due to its appropriate content of oligopeptides and amino acids profile,special micromorphology properties and space structure.?3?Tibetan Sheep placental peptide was separated by gel filtration on Sephadex G-15and Easy nLC1200-Q Eaxtive plus was applied to identify the sequence of the active fraction.Sheep placental peptide was isolated to four fractions?F-1,F-2,F-3 and F-4?.The F-2 showed the highest antioxidant capacity.The scavenging capacity of DPPH radical and ABTS radical was 19.61%and 0.56 mM,respectively.The reducing capacity of Fe³⁺was 0.05.Furthermore,27 peptides were identified from F-2.