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Studies On Tilapia Skin Collagen Degradation Behavior And Preparation Of Peptide Calcium Chelate

Posted on:2021-05-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:L ZhangFull Text:PDF
GTID:1361330611467067Subject:Sugar works
Abstract/Summary:PDF Full Text Request
A large amount of protein-rich tilapia skin is produced in frozen fish fillets processing industry,which has high utilization value.Many studies had been carried out in this paper.Firstly,collagen was extracted from tilapia fresh skin and degraded by hydrochloric acid and heat treatment to form gelatinized collagen,and the degradation process was studied.Secondly,in order to solve the problems of collagen peptide content determination in the fish gelatin system,the Biuret method was improved and evaluated.On the other hand,an alkaline collagenase from Bacillus subtilis was isolated,purified,and studied of the enzyme properties,tructure and immobilized with sulfonated polystyrene?SPS?nanoparticles.Based on the knowledge of protein structure and Lumping 3-D model,the degradation behavior was analyzed in enzymatic reaction system for the preparation of collagen peptide by hydrolysis of Tilapia collagen by the immobilized alkaline protease from Bacillus subtilis.The final study was to use collagen peptide as raw material to prepare the peptide calcium chelate,optimize the process,evaluate the stability of the chelate,and use the Caco-2 cell model to evaluate the calcium absorption promoting effect of the chelate in vitro.The main research contents and results are as follows:?1?Observation of fiber tissue showed that type I collagen was the main collagen in fish skin.Fresh tilapia skin and scales were used as raw materials,acid-soluble collagen?ASC?and pepsin-soluble collagen?PSC?were extracted by acid method and acid-enzyme combination method.The characterization and properties of the four collagen products were compared,and the results showed that the purities of ASC and PSC from tilapia skin were85.69%and 72.88%respectively,the purities of ASC and PSC from tilapia scales were70.35%and 73.92%,respectively.Glycine was the most abundant amino acid in the products,with 20.85%,21.01%,21.16%and 19.21%,respectively,which was in accordance with the primary structural characteristics of collagen.The UV absorption maximum wavelengths of ASC and PSC from tilapia skin and scales were 234 nm,222 nm,236 nm,and 226 nm,respectively,which also accorded with the characteristics of collagen.The Ts temperatures of fish skin and scale ASC,PSC are about 89.0?,81.3?,74.0?and 70.7?,respectively.FT-IR showed that the four products retained the natural triple helix structure;Results of high performance gel exclusion chromatography?GPC?showed that the weight-average molecular weights of ASC and PSC from tilapia skin and scales were 139570 Da,20891 Da,131909 Da and 20428 Da.According to the molecular weight and distribution,fish skin ASC is closest to natural collagen and has better application value in medical and health food fields.?2?Using acid-soluble collagen?ASC?from tilapia fresh skin as material,gelatinization was induced by hydrochloric acid,and gelatin was obtained by hot water extraction.The gelatinization process was studied by infrared spectroscopy,circular dichroism,SDS-PAGE electrophoresis and DSC thermal stability analysis.The infrared spectra showed that the tri-helical structure of collagen gradually unwound with the increase of acid treatment time,because the sharpness of absorption peak near 1460?1230 cm-1 decreased obviously.The gelatinization degree of collagen was higher at 4 h of acid treatment,and the gelatin yield reached 77.41%by hot water extraction.The results of DSC and SDS-PAGE electrophoresis also showed that acid treatment caused deprotation of collagen tri-helical structure and degradation of macromolecules subunits,and the two degradation processes were in equilibrium stage in the first 4 h of acid treatment.As a result,it can be determined that the acid treatment time is less than 4 h to obtain higher quality gelatin.?3?Taking tilapia skin collagen peptide as the reference,a modified Biuret colorimetric method for the determination of peptide content was developed.The maximum absorption wavelength of color rendering complex,the range of concentration of peptide,trichloroacetic acid?TCA?and p H with high linearity were investigated.The repeatability,precision evaluation and recovery rate of the standard curve were tested under optimized conditions.The applicability of this method to the detection of collagen peptides from the tilapia skin and scales with different molecular weight was also studied.The results showed that when the mass concentration of collagen peptide?labeled molecular weight of 3 KDa?was within 0.3?1.5mg/m L,13%TCA was used,adjusted the p H 12.5 of the system,at 545 nm,the absorbance was linearly fitted with the mass concentration of peptide?R2=0.999?.RSD from the repeatability and precision test were 1.86%and 1.84%,recovery were 113.9%and 109.7%,RSD was 1.39%and 1.00%.It showed that the method had wide linear range,good repeatability and high accuracy.By detecting different kinds of tilapia peptides and comparing the deviation of recovery and concentration,the improved Biuret method had been proved to be a simple,rapid and suitable method for the determination of peptides from tilapia.?4?In order to analyze a collagenase zymin from Bacillus subtilis,the enzyme was isolated and purified,the protein structure was identified and the enzymatic properties were studied.The results showed that the specific activity of enzyme could be increased to 608.17U/?g by purification.The molecular weight of the enzyme measured by SDS-PAGE electrophoresis was approximately 31.0 KDa.The amino acid sequence of the enzyme was identified by mass spectrometry,and the three-dimensional structure of the protein was simulated.It is speculated that this enzyme may belong to the subtilis protease family.The optimum reaction temperature and p H of enzyme were 60?and 7.4 respectively in the hydrolysis system of tilapia skin collagen.The enzyme has good thermal stability under40?and acid-base stability in the range of p H 5.0-7.0.The Michaelis constant was 88.22 and ethylenediamine tetra acetic acid?EDTA?could reduce enzyme activity to about 60%,and ethylene glycol double?2-amino ethyl ether?four acetic acid?EGTA?could completely deactivate the enzyme.The optimal immobilization conditions were as follows:Volume ratio of sulfonated polystyrene?SPS?nanoparticles carrier to enzyme was 3:50?m L:m L?,the immobilized temperature was 25?,and the p H was 4.5.Under this condition,the immobilization ratio was 73.48%and the specific activity was 274.05 U/?g,and the specific activity of immobilized enzyme was about 53.74%of that of free enzyme.?5?To show the enzymatic hydrolysis behavior and results of tilapia skin gelatinized collagen degraded by immobilized alkaline protease,based on high performance gel exclusion chromatography?GPC?and liquid chromatography-mass spectrometry?HPLC-MS/MS?,combined with bioinformatics knowledge,computer simulation and image processing techniques were used to draw 3-D diagrams which can characterize the dynamic characteristics of enzymatic hydrolysis reaction process.Using antioxidant capacity as the evaluation basis,the enzymatic hydrolysates of 180 min were sequenced by mass spectrometry,10 fragments were identified,and the molecular structure of peptide fragments was predicted by Chem Draw19.0-Chem3D software.?6?Using tilapia skin collagen peptide powder with nominal molecular weights of 1 KDa,3 KDa and 5 KDa and anhydrous calcium chloride as raw materials,the preparation process of collagen peptide calcium chelate was optimized.Response surface method was used to optimize the chelating conditions with the response value of calcium chelating rate.The results showed that the optimum chelating conditions were p H 7.00,temperature 40.00?,ratio of peptide to calcium mass 7:1 and time 30.00 min,and the predicted chelating rate was39.5%±0.5%.The peptide calcium chelate was characterized by FTIR,scanning electron microscopy?SEM?and energy dispersive spectrometry?EDS?,and the stability was determined.The results showed that Ca2+was chelated successfully.The peptide calcium chelate was not stable at high temperature,and it was easy to dissociate in the acid environment,and the influence was greater than that of alkaline environment.It was stable in the environment co-existing with lactose and sodium chloride.Pepsin and trypsin could decompose the chelate,and the effect of trypsin was greater than that of pepsin;Caco-2monolayer cell model was used to evaluate the effect of peptide calcium chelate on calcium transport in vitro.It was found that the chelate with concentration above 3 mg/L had a good effect on calcium transport.At 3 h,the peptide calcium chelate of 7 mg/L promoted calcium transport to 2.52?g/mg and the calcium absorption rate to 41.04%.Compared with other studies,its effect on promoting calcium transport and absorption is better than that of tilapia skin protein peptide?Val-Gly-Leu-Pro-Asn-Ser-Arg?calcium chelate,and weaker than that of cod skin collagen peptide?Ala-Gly-Pro-Ala-Gly-Pro-Arg?calcium chelate.
Keywords/Search Tags:Tilapia skin, Collagen, Collagen peptide, Gelatinization, Biuret, Collagenase, Peptide calcium chelate
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