Study On The Preparation Of Sericin Peptides, Their Biological Activities And Structure

Sericin, derived from silk cocoon, is a new food protein source, which can be used as food and medicine, so the exploration and utilization of sericin for the functional food manufacture will not only raise the economic value of sericin which used to be as a by-product of silk industry, but also reveal the significant social benefit.In this dissertation, the extraction technology as well as the enzymatic hydrolysis technology for sericin derived from silk wastewater were developed; the refinement of sericin hydrolysates using the macroporous adsorption resin DA201-C was researched and the biological activities of the obtained fractions were further investigated; the antioxidant melanoidin derived from sericin hydrolysates was purified and their physiochemical properties and structure were determined; the anti-aging effect of sericin peptides on the D-galactose induced aging model rats was assessed. The study of this dissertation would give the theoretical foundations for utilization of sericin hydrolysates as functional food additives.Initially, chilled alcohol precipitation method was used to extract sericin, and the suitable extraction process with the yield of 64% was set up. Results from physiochemical analysis show that sericin extrated with alcohol contained about 90% of protein, 1% of sugar and 4% of ash; the solubility was decreased, but silk special bad smell was removed significantly; the hydrophilic amino acid amount was up to 70 % in the total amino acids and the hydrophobicity value was 2.21 KJ.mol-1, which suggest that sericin was a kind of hydrophilic protein; the relative molecular weight of sericin was mainly in the range of 10,000～500,000.Further, the effects of the extraction process with alcohol on the mircro- molecular structure and the stabilization of molecular conformation of sericin were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), circular dichroism (CD), and infrared (IR) spectroscopy. SEM result shows that protein molecules were aggregated together to form a compact and spherical structure, but it changed to loose and netty structure after ridding of the alcohol and resolving in the water; the results obtained from the DSC,CD and IR suggested that the conformation of protein molecules was partially changed, which was possibly relative to the appearance ofβ-Turn conformation, which could facilitate molecule folding by reversing the direction of the polypeptide chain, and make the molecule conformation changed from random structure to order structure, thus lead to the decrease of solubility. In addition, GC-MS analytic result reveals that the flavor components resulted in the silk bad smell were mainly the heterocyclic and alkyl volatile components, but they could be removed by using alcohol, thus sericin extrated with alcohol was suitable for the application in food.The effects of five kinds of proteases on the hydrolysis of sericin were compared by the degree of hydrolysis (DH). AS 1398 was found to be the most effective enzyme. By the single-factor tests and the response surface analysis, the optimum hydrolysis conditions were estimated as pH 6.1, temperature 55°C, and E/S (%) of 2.00, respectively. Accordingly, the highest DH value was estimated as 23.35%. Furthermore, compared to the sericin, the protein content of the hydrolysates was decreased, but increased in the contents of sugar and ash. The relative molecular weight distribution of the hydrolysates was among 200～1000, and the component with relative molecular weight under 500 was of 50% or so.Further, the macroporous adsorption resin DA201-C was used to refine sericin hydrolysates by fractionation, desalting and decoloring treatments. Results show that four fractions (DK-1,DK-2,DK-3, DK-4) were separated by DA201-C resin eluted with alcohol in step wash, the adsorptive fractions had been desalted effectively according to the analysis of the conductivity value and the ash content, and dark-brown components could be isolated from sericin hydrolysates according to the analysis of the optical density at 420 nm and the chroma parameters of the fractions. Moreover, the DK-3 fraction shows the DPPH free-radical scavenging activity, the inhibitory effect on mushroom tyrosinase and ferrous ion-chelating activity in vitro, and the corresponding value of EC50 was 2.49 mg/mL, 3.13 mg/mL and 0.90 mg/mL, respectively. The DK-1 fraction after being desalted by nanofiltration was added into the frozen fish surimi, and results show that the addition of the DK-1 fraction could significantly increase the total sulfhydryl content of actomyosin, decrease surface hydrophobicity of actomyosin and inhibit the decompose of myosin heavy chain (MHC) and actin (AC), suggesting the DK-1 fraction had the cryoprotective effect on fish surimi proteins. But this effect was relative to the dosage, and the data denote that the dosage of application could not be too high.In this dissertation, glycoproteins with high molecular weight from sericin were identified by SDS-PAGE stained with PAS, O-glycosidic linkage was found byβ-elimination reaction, and characteristic heterocyclic volatile components derived from the maillard reaction, such as pyridine, 2-piperidone and 2-[5-chloro-2-methoxyphenyl] pyrrole, were identified from sericin hydrolysates by GC-MS analysis. These data approve that the maillard reaction occurred in the process of sericin enzymatic hydrolysis, and give the experimental foundations for the produce of melanoidins in sericin hydrolysates. Further, melanoidins (RP-M) with antioxidant activities and adsorptive features at 220 nm, 280 nm and 420 nm, was isolated from the DK-3 fraction by polyamide resins and RP-HPLC. The results from the analysis of antioxidant activity showed that RP-M had the DPPH free-radical scavenging activity, and IC50 of RP-M was 0.13 mg/ml. Moreover, RP-M exhibited the neuroprotective effect on hydrogen peroxide (H2O2) induced oxidative damage in PC12 cells, but the highest dosage in this study shows the cytoxicity on cells. Compared to sericin, the content of amino acid residues in RP-M, including Lys, Arg, Pro and other hydrophobic amino acids, was increased significantly, suggesting they were the dominant amino acid residues in the RP-M peptide skeletons. The relative molecular weight of RP-M was mainly in the range of 1,000～50,000, and the components with relative molecular weight above 7,500 was of 50% or so. Moreover, the mass fingerprinting of RP-M determined by MALDI-TOF MS denotes that the peptide skeletons of RP-M consisted of small peptides with relative molecular weight under 1,000, so the above results imply that the melanoidin component-- RP-M, was a mixture with high molecular weight, which was formed by polymerizing between peptide mixture with pigments.The primary structure of the three peptide fragments at m/z 529.8715,462.3399 and 398.3169, were identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS), and the amino acid sequence was IILLLAAKFG,KVIIKPLI and QPVIAHM, respectively. The primary structures of peptide fragments had the functional structure feature similar with other antioxidant peptides, which denotes that the antioxidant activity of RP-M might be related to their peptide skeletons structures. In addition, these peptide fragments all consisted of the characteristic amino acids, such as Lys and Pro, which are the most active amino acids that facilitate the produce of melanoidin. The IR spectrum of RP-M denotes it possessed the structures of aromatic amine, aromatic carboxylic acid and alkane. The shape of the UV/VIS spectrum of RP-M had strong absorption in the range of 200 nm~380 nm, and with maxima in the range of 275 nm~280 nm. The acid hydrolysates of RP-M was prepared and purified by RP-HPLC. A pigment component (S-4) with strong absorption in the wavelength of 420 nm was obtained. The analysis results from infrared (IR) spectroscopy, UV/VIS spectroscopy and nuclear magnetic resonance (NMR) show that the S-4 pigment component was a kind of alpha-pyranoside saccharides. This result implies that the pigment part of melanoidin was consisted of glycosylation products.In this dissertation, the anti-aging effects of sericin peptides on the D-gal induced aging rats were measured. Results show that the consumption of sericin peptides could obviously increase superoxide dismutase (SOD) activities and decrease malondialdehyde (MDA) levels in the rat serum; markedly increase the weight of rat spleen; enhance the ability of learning and memory in aging model rats; notably suppressed neuronal loss in hippocampal CA1 region and prevent the morphological damage of pyramidal cells; enhance the expression levels of the anti-apoptotic gene Bcl-2. These data suggest that the anti-aging effects of sericin peptides in vivo were relative to the function of antioxidant activities, the improving function of immunity, and the role of delaying the aging change of the brain.