Optimization Of Hydrolysis Conditions For Peptides From Grass Carp Fish Skin:Promotion For Streptococcus Thermophiles Proliferation And Characterization Of Antioxidant Activity

As a multifunctional probiotic,Streptococcus thermophilus is widely used in daily life.However,the growth condition of streptococcus thermophilus is very harsh,and its growth relies on the environment to provide a variety of peptides,free amino acids,vitamins and other growth substances.Therefore,finding a reasonable and effective nitrogen source to promote the high-density growth of Streptococcus thermophilus has been a hot topic in China.Grass carp,one of the four major fishes in freshwater aquaculture in China,is characterized by rapid growth,wide feed sources and rich nutritional value.At present,grass carp processing in China is mainly in the traditional way,focusing on fresh sales,fishmeal feed and other primary processing products,with low added value.In the process of processing aquatic products,a large number of by-products are produced,of which fish skin accounts for more than 8%.If these by-products can not be effectively used,they will not only cause waste,but also cause environmental pollution.Fish skin contains a large amount of protein,among which type I collagen is the most abundant,much higher than other parts of the fish body,and its amino acid sequence is rich in bioactive sequences.If collagen peptide is extracted from the by-product of aquatic products,not only polypeptide and amino acid can be obtained,but also the price and the cost are low.Therefore,in this work,grass carp fish skin was chosen to prepare peptides by enzymatic hydrolysis and the peptides so produced were used to make growth medium for S.t,and the promotion effect on the S.t proliferation was also studied.On this basis,the enzymatic hydrolysate was purified by macroporous resin and the antioxidant activity in vitro was analyzed.The main research results are as follows:1.Taking the degree of hydrolysis as the index,the effects of different proteases?pepsin,flavor neutral protease,papain,trypsin,alkaline enzyme?on the degree of hydrolysis of grass fish skin were studied,and the protease with the highest degree of hydrolysis was selected.The results showed that flavor neutral protease had the highest degree of hydrolysis.The hydrolysis time,the ratio of water to raw materia and enzyme concentration were selected for the single-factor experiment,and the Box-Benhnken response surface combination design experiment was carried out on the basis of the single-factor experiment.The results showed that the optimum enzymatic hydrolysis process was:the hydrolysis time was 6.87h,the ratio of water to raw material of 1:9.60?w/v?,and enzyme concentration was 5.51%.2.The free amino acid of the enzymatic hydrolysis product of flavor neutral protease contains the essential amino acid to promote the growth of streptococcus thermophilus.The hydrolysis time,the ratio of water to raw materia and enzyme concentration were selected for the single-factor experiment,and the Box-Benhnken response surface combination design experiment was carried out on the basis the single-factor experiment.The results showed that the optimal enzymatic hydrolysis process was as follows:the h hydrolysis time was 4.13h,the ratio of water to raw materia was 1:9.89,and the enzyme concentration was 3.1%.Therefore,the optimal enzymatic hydrolysis condition with the degree of hydrolysis as the index was not related to the enzymatic hydrolysis condition with the promotion of streptococcus thermophilus as the index.Considering the effect of the enzymatic hydrolysis product of herbaceous fish skin on the proliferation of probiotics in this study,the enzymatic hydrolysis process for the preparation of polypeptides to promote the proliferation of streptococcus thermophilus was selected for subsequent experimental study.3.Study on the purification process of macroporous resin:screening of macroporous resin type,single factor test and orthogonal test were used to obtain the best deodorization and decolorization effect of DA201-C macroporous resin on grass fish skin peptides.All other things being equal,when the temperature is between 15?to 35?,resin content is between 2.5%to 7.5%,pH is between 6 to 8,the processing time is between 1.0 h to 1.5 h,trends decoloring effect is best.Orthogonal experiment results showed that DA201-C macroporous resin decolorizing optimum process is:temperature 25?,resin content 5%,pH of 7,the processing time of 1.5 h.The results of amino acid analysis showed that the loss rate of free amino acid in the grass fish skin peptides was higher than that of total amino acid,and the loss rate of bitter amino acid in free amino acid was higher.GC-MS results showed that compared with the pre-deodorization,the total amino acid in the grass fish skin peptides after deodorization decreased,the proportion of bitter amino acid decreased,DA201-C macroporous resin adsorbed more bitter amino acids.The total volatile substances in the grass fish skin peptides decreased by 12 species after adsorption by DA201-C macroporous resin.There was no significant difference in the proliferation of Streptococcus thermophilus by using grass fish skin peptides before and after deodorization and decolorization.4.The Grass Carp fish skin hydrolysate prepared under the optimum conditions was fractionated to four fragments according to molecular weight sizes:GFSPH??>10000 Da?;GFSPH??5000?10000 Da?;GFSPH??3000?5000 Da?;GFSPH??<3000 Da?.The results showed that the effect of different molecular weight herbacterin fish skin peptides on the proliferation of streptococcus thermophilus was the most significant when the molecular weight was less than 3000 Da.The results of antioxidant test in vitro showed that the polypeptides of grass carp fish skin peptides had strong antioxidant capacity,GFSPH?of DPPH radical scavenging IC₅₀ was 5.99mg/mL,the hydroxyl radical scavenging free radicals of IC₅₀ was 10.44 mg/mL;GFSPH?of radical scavenging ability as follows:DPPH>hydroxyl free radicals>super oxygen anion.