Preparation,purification And Functional Characterization Of Protein Hydrolysate From Chinese Pond Turtle (Chinemys Reevesii)

Chinese pond turtle（Chinemys reevesii）,also known as Chinese grass turtle,is mainly distributed in China,South Korea,Japan,and other parts of Asia.Chinese traditional medicine believes that the Chinese turtle have the effect of prolonging life,and modern research also shows that the Chinese pond turtle has high nutritional value.With the rise of turtle breeding industry,how to improve the comprehensive utilization rate and commercial value of turtle resources through deep processing is an urgent problem to be solved in turtle processing industry.In this paper,Chinese Pond Turtle was used as raw material.The research showed that the functional properties of protein peptides obtained from enzymatic hydrolysis of Chinese pond turtle meat were improved,showing great potential in inhibiting antioxidant and antidiabetes activities,which laid a scientific foundation for the deep processing and utilization of Chinese pond turtle.The main research contents and results are as follows:Firstly,the basic nutritional components of muscle,liver,and hard shell of the Chinese pond turtle was evaluated and compared with the recommended daily nutrient intake（RDI）.The functional properties performance of muscle,liver,and hard shell were also evaluated.The results showed that the protein content in muscle was the highest（74.97%dry weight）,the ash content in hard shell was the highest（49.66%dry weight）,and the fat content in liver was the highest（9.68%dry weight）.31 kinds of mineral elements（4 macroscopic and 27microscopic,namely sodium,potassium,calcium,magnesium,iron,aluminum,copper,zinc,chromium,manganese,boron,lead,selenium,molybdenum,titanium,vanadium,cobalt,nickel,barium,etc.）were analyzed.Sodium was the highest in hard shell（25.86 g/100 g dry weight）,while cobalt was the lowest in muscle and liver（0.01 mg/100 g dry basis）.The contents of total essential amino acids,free essential amino acids,and vitamin B6 in muscle were higher,which were 28.36 g/100 g dry weight,266.29 mg/100 g dry weight,and 14.47mg/100 g wet weight,respectively.The contents of unsaturated fatty acids and vitamin K in liver were high,reaching 7.49 g/100 g dry weight and 1.59 g/100 g wet weight,respectively.The contents of total minerals,hydroxyproline,and vitamin B3 in the hard shell were the highest（48.10 g/100 g dry weight,2.18 g/100 g dry weight,and 17.52 mg/100 g wet weight,respectively）.There were significant differences in protein molecular weight distribution among samples.The maximum percentages in liver,muscle,and hard shell were 87.34%（≤1000 Da）,85.00%（≤1000 Da）,and 46.50%（≥10000 Da）,respectively.Under different p H conditions,the functional properties of Chinese pond turtle freeze dried powder muscle,liver,and hard shell changed significantly.The hard shell achieved the highest solubility（45.89%）followed by muscle（21.29%）at acidic condition（p H 2.0）;the higher emulsification activity index was obtained in hard shell(90.22 m²g^-1;p H 4.0),whereas the highest emulsion stability index（89.93%,p H 6.0）in muscle.The water holding capacity and oil holding capacity of muscle were higher,which were 7.97 and 2.19 m L/g dry weight,respectively.The results also showed that 100 g Chinese pond turtle had a significant contribution to RDI.Secondly,through the results of the first part,it is found that the protein content in muscle is the highest and has good antioxidant activity.Literature studies have proved that papain can produce high-quality protein hydrolysates from aquatic proteins,but it has not been reported for turtle muscle hydrolysis.Therefore,in this chapter,papain system is selected to study its hydrolysis effect on Chinese turtle muscle.Under the optimum conditions（the ratio of substrate to buffer is 1:1 w/v,the ratio of enzyme to substrate is 5%w/w,p H value is 6.0,temperature is 60℃,enzymatic hydrolysis time is 6 h）,the maximum degree of hydrolysis（DH）is 19.52%.When DH was 19.52%,the highest yield of protein hydrolysate was 17.26%and the protein content was 76.63%.The total amino acid content of protein peptides with different degrees of hydrolysis was more than 96.77%.When DH was 19.52%and p H value was 6.0,excellent solubility（95.56%）and emulsifying activity index(108.76m²g^-1)were obtained;when the p H value is 2.0,the foaming capacity of DH is the highest,which is 100.0%.The water holding capacity is the highest at 13.53%in DH,which is 4.38g/g.The antioxidant activity showed the strongest hydroxyl radical scavenging activity（95.25%）,ABTS（84.88%）,DPPH（75.89%）,iron chelation（63.25%）,and copper chelation（66.90%）at 11.96%of DH,while the reduction capacity（0.88）was the highest at 19.52%of DH.Thirdly,the hydrolysis of muscle by alkaline protease,flavor protease,trypsin,and bromelain was studied.The effects of enzyme type,enzyme/substrate ratio and enzymolysis time were studied,and the functional properties,antioxidant,cytotoxicity,and antidiabetic activity（α-amylase andα-glucosidase inhibition）of the hydrolyzed protein were evaluated.The yield of papain hydrolysate reached the highest level,19.52%,but it was found that the antioxidant activity of papain inhibited IC₅₀ value and functional characteristics（water and oil holding capacity）were not very good.The amino acid composition of the hydrolysate showed that the contents of glutamate,aspartic acid,lysine,hydroxyproline,and hydrophobic amino acids were high.The antioxidant activity of flavor protease hydrolysate was the strongest,the inhibition rates of DPPH and ABTS were 68.32%and 74.12%,respectively,and the FRAP value was 0.30.Flavor protease hydrolysate has the strongest effect on the inhibition rate ofα-glucosidase was 61.80%,and the strongest inhibitory effect on cell HT-29 cytotoxicity（82.26%）.The hydroxyl radical scavenging rate of trypsin hydrolysate was the best（92.70%）,and the chelating activity of bromelain to Fe²⁺was the strongest（63.29%）（p<0.05）.When bromelain was used,the highest inhibition rate 76.89%toα-amylase was obtained.Enzymatic hydrolysis can significantly increase the low molecular weight peptide and enhance the functional properties of the hydrolysate.The protein solubility of trypsin hydrolysate was98.72%at p H 1.0,and the emulsifying activity index was 182.81 m²g^-1 at p H 7.0.Bromelain hydrolysate had high emulsifying stability（97.58%）at p H 10.The water holding capacity of alkaline protease hydrolysate is the best,which is 5.08 g/g,and the oil holding capacity of bromelain hydrolysate is the highest,which is 6.28 g/g.In addition,scanning electron microscopy（SEM）showed that the spherical structure treated with flavor protease was better than that treated with alkaline protease.The results of X-ray diffraction showed that the relative crystallinity of the hydrolysate of flavor protease was the highest（37.53%）,indicating the best storage resistance.The zeta potential of alkaline protease hydrolysate was the highest（-28.23 mv）.Also,the UV absorption value of flavor enzyme hydrolysate was the lowest,which showed that the storage resistance was the highest.Finally,the flavor enzyme hydrolysate was selected for further separation and purification,and the peptide component with high purity was obtained,and its antioxidant activity was studied.The flavour protease hydrolysates were separated by gel column chromatography on Sephadex G-10,and three fractions（FH-1,FH-2 and FH-3）were obtained.The percentages of peptides with molecular weight less than 150 Da in fractions FH-3,FH-2,and FH-1 were 93.25%,85.22%,and 76.76%,respectively.The antioxidant activity of different fractions was evaluated by the free radical scavenging ability of DPPH and ABTS.The results showed that the IC₅₀ values for ABTS were FH-3:0.42±0.01 mg/m L;FH-2:0.51±0.03 mg/m L,and FH-1:0.64±0.02 mg/m L,had stronger activity than flavor enzyme protein hydrolysate(IC₅₀ is 0.91±0.01 mg/m L).The IC₅₀values for DPPH were FH-2:5.04±0.13 mg/m L;FH-3:5.43±0.11 mg/m L and FH-1:5.77±0.20 mg/m L,which were better than flavor enzyme hydrolysate(IC₅₀ was 9.36±0.21 mg/m L).The results showed that the purified peptide fraction had higher antioxidant activity than the protein hydrolysate.