Research On Process Of Preparation And Activity For Collagen Peptides Of Tilapia Skin

In the processing of tilapia,a large amount of collagen-rich fish skin waste is produced.At present,only a small part is used for the production of feed or fertilizer,and most of them are discarded as waste to pollute the environment.The tilapia skin was extracted and hydrolyzed into collagen peptide,which not only improved its added value,but also reduced resource waste and environmental problems.However,at present,the domestic collagen peptide products have the disadvantages of heavy fishy smell,deep color,uneven distribution of polypeptide molecular weight and insufficient output.In view of this,the fish skin was used as the raw material in this paper.Fish skin was degreased and deodorization,and the most suitable hydrolyzed protease was selected.Enzymatic hydrolysis was performed to determine the optimal hydrolysis process through single-factor response surface experiments.It was decolorized with activated carbon.The related activities of hydrolysates were investigated.The hydrolysates were isolated and purified.The main results are as follows.1.The moisture content of tilapia skin was 10.25%,protein content was 76.98%,fat content was 11.10%,ash content was 1.22% and collagen content was 62.05%.Three factors: extraction time,extraction temperature and extraction pressure were were selected to degrease fish skin.The fish oil yield was the experimental index.The single factor analysis of the extraction conditions showed that the optimal combination of factors was the extraction time 50 min,extraction temperature 50 ?,extraction pressure 0.5 MPa,analytical temperature 70 ? and material moisture 10.25%.At this condition the yield was10.47% and the degreasing rate was up to 94.32%.Therefore,the fish skin was defatted and deodorant using this optimal process condition,and it laid the foundation for preparing high-quality collagen peptides from tilapia skin in the later stage.The fish oil which extracted by continuous phase transition from low temperature was yellow to reddish-brown,light fishy smell and no odor,moisture and volatiles were 0.26%,the iodinevalue was 123.47 gI/100 g,the acid value was 7.83 mgKOH/g,the peroxide value was 1.25mmol/kg,and the insoluble impurities were 0.23%,which complied with top standard for crude fish oil of SC/T 3502-2000.There was no residual solvent in the fish oil,indicating that the skin oil could also be used for other research.A total of 27 fatty acids were identified from fish skin oil,saturated fatty acids accounted for 28.35% of all fatty acids,monounsaturated fatty acids accounted for 34.75% of all fatty acids,and polyunsaturated fatty acids accounted for 28.40% of all fatty acids.2.Compared with the indexes of the degree of hydrolysis,molecular weight distribution,sensory evaluation and hydroxyproline content,alkaline protease had the best sensory evaluation,higher degree of hydrolysis,more molecular weight of 180-1000 Da,less molecular weight of <180 Da(free amino acid)and higher content of hydroxyproline,so alkaline protease was determined as the best enzyme for preparing collagen peptide from tilapia skin.3.The degree of hydrolysis,molecular weight distribution and hydroxyproline content were used as indexes to investigate the effects of different enzyme hydrolysis time,pH,temperature,enzyme amount and liquid ratio on the enzymatic hydrolysis of alkaline protease.On the basis of single factor test,according to the requirement of Box-Behnken response surface method,four factors affecting the enzyme hydrolysate were selected: time,amount of enzyme,temperature,pH as independent variables,and the degree of hydrolysis(DH)as the response value.Four factor and three level response surface methodology was used to optimize the extraction process parameters of collagen peptide.The optimal conditions for alkaline protease hydrolysis were: time 7 h,enzyme addition 7%,temperature 60 °C,pH 10.38.Under this condition,the degree of hydrolysis of tilapia skin hydrolyzed by alkaline protease was 23.01%.4.Activated carbon addition,decolorization temperature,decolorization time and decolorization of pH were selected to treat collagen peptide.The decolorization rate and protein recovery rate were used as the experimental indicators.The decolorization conditions were analyzed by single factor.The optimum process were activated carbon addition 1.00%,decolorizing temperature 50 °C,decolorizing time 45 min and decolorizing pH 5.0.Under this condition,the decolorization rate was 69.32% and the protein recoveryrate was 87.43%.The decolorization effect of the collagen peptide was best,and the loss of protein was also less.5.The collagen peptide after decolorization would be studied for its related activity.Tyrosinase activity was inhibited by collagen peptide,but the inhibition effect was slightly lower than that of kojic acid.The result could preliminarily prove that the collagen peptide can inhibit the activity of tyrosinase.The result of peptide content detected by BCA was the same as that detected by GFC method.The transdermal absorption rate of collagen peptide reached 49.73% and 53.75% respectively in 24 h,indicating that it had good transdermal absorption performance.The results of HPLC-C18 qualitative detection showed that three components of collagen peptide were the most overexposed.6.The collagen peptide was used to promote HSF cells proliferation and purification.Proliferation of HSF cells was promoted by collagen peptide.HSF cells proliferation rate was maximum for 71.25%.The collagen peptide was isolated and purified using proliferation of HSF cells as an index.The results showed that the four fractions(A,B,C,and D)separated by the glass column had different degrees of promoting the proliferation of HSF cells.Among them,the proliferative ability of A component was the strongest,and the maximum proliferation rate reached 32.11%.Therefore,the A component was selected for further purification.Then four components A1,A2,A3 and A4 were isolated and each component had different degrees of promoting effect on the proliferation of HSF cells.Overall,the proliferative capacity of A1 component was the strongest,with a maximum proliferation rate of 55.12%.The purity of the separated components was determined by HPLC,and the purity was judged according to the peak type.The results showed that the purity of collagen peptide after separation and purification was increasing.