Preparation Of Half-fin Anchovy Antibacterial Peptides, Antibacterial Activity And Other Bioactive Capacities Analysis

Half-fin anchovy (Setipinna taty), one kind of small marine fish, used as material, are enzymatic hydrolyzed by proteases to prepare antibacterial peptides, meanwhile, the antibacterial activity and other bioactive capacities of these peptides are investigated, aiming at providing information for the intensitive processing of half-fin anchovy. The contents of this paper include: preparation of half-fin anchovy antibacterial peptides; physicochemical properties of half-fin anchovy antibacterial peptides; isolation and purification of antibacterial peptides from half-fin anchovy antibacterial hydrolysate; antibacterial model of half-fin anchovy antibacterial peptides; antioxidant activities of half-fin anchovy antibacterial peptides and heated products; antiproliferative activities of half-fin anchovy antibacterial peptides and heated products.The antibacterial activities of half-fin anchovy, digested by flavoring proteinase, trypsin, pepsin, alkaline protease and papain, are assayed respectively. Pepsin is selected as the optimal proteinase and response surface methodology is then used to optimize the pepsin hydrolysis parameters using a central composite design (CCD) method. Results shows that pepsin to substrate level of 1100 U/g, pH of 2.0, reaction time of 2.4 h and ratio of water to substrate of 4:1 (v/w) are the optimal conditions to generate antibacterial peptides. The extract rate of soluble peptides and degree hydrolysis (DH) of HAHp are (81.78±0.04)% and (18.12±0.39)%, respectively. Results of gel chromatography indicate that HAHp are composed of small peptides with molecular weight below 3000 Da, furthermore, HAHp exert a broad antibacterial spectrum.The solubility, emulsification and foaming capacity of HAHp are measured in this paper. Results imply that the solubility of HAHp reaches to 80% in the pH range of 2¹2, in addition, HAHp own the emulsification and foaming capacities. Factors that influence the antibacterial activity of HAHp, including heating temperature and time, acidic and alkalic pH values, trypsin andβ-lactamase treatments, metal ions and freeze-melt frequency, are investigated. Results show that HAHp is one kind of heated stable peptides. HAHp keep antibacterial activity in the pH range of 2.4 to 9.4, and the strongest activity is observed in the pH value of 5.4. The antibacterial rate of HAHp after digested by trypsin for 5 h is not decreased. The antibacterial activity of HAHp is not affected byβ-lactamase treatment. HAHp own the antibacterial activity after frozen and melted for six times. Metal inos, such as K⁺, Na⁺, Ca²⁺ and Mg²⁺, do not affect the antibacterial activity of HAHp, whereas, Fe²⁺and Zn²⁺ with concentration above 2.5 mmol/L will decrease the activity. In the present of Fe³⁺, the antibacterial activity of HAHp will be decreased.An antibacterial fraction, HAHp2-3-I, derived from HAHp, is isolated through different chromatographic methods, i.e., Sephadex G-25 and RP-HPLC. Meanwhile, the chromatography of Source 5RPC ST indicates that HAHp2-3-I contains two fractioned peaks. HAHp2-3-I is composed of more than ten small peptides with molecular weight ranging from 1100 to 1700 Da indenfied by LC-MS technique. Five cationic peptides MLTTPPHAKYVLQW, SHAATKAPPKNGNY, PTAGVANALQHA, QLGTHSAQPVPF and VNVDERWRKL, three anionic peptides LATVSVGAVELCY, NPEFLASGDHLDNLQ and PEVVYECLHW, and two neutral peptides LRSKAAAPAEQYE and TPGALLEHPTL are identified. Moreover, the synthesized peptides MLTTPPHAKYVLQW, SHAATKAPPKNGNY and PTAGVANALQHA show the antibacterial activity.The antibacterial activity of HAHp is measured by assaying the growing curve, membrane permeation and scanning electron microscopy of E. coli. Results indicate that HAHp and HAHp2-3-I can inhibit the growing curve of E. coli. HAHp induces the membrane permeation. HAHp and HAHp2-3-I damage the membrane of E. coli. Eight charge peptides of HAHp2-3-I are indentified as novel peptides after sequences compared with antibacterial peptides in UniProtKB/SWISS-PROT and APD data bases. Cationic peptides MLTTPPHAKYVLQW, SHAATKAPPKNGNY and PTAGVANALQHA have high homology with antibacterial peptides deriving from amphibious animal. The second structures of peptides in HAHp2-3-I predicted by Hierarchical Neural Network areα-helix, random coil and extended strand. Heat treatment on HAHp is contributed to improve the in vitro DPPH radical scavenging activity, in addition, DPPH radical scavenging rate of the heated products of HAHp has a good correlation with its browning intensity (y=105.41x+55.036, R²=0.9343). Results of amino acid composition show that the free amino acids content of HAHp-H is significantly lower than that of HAHp. Moreover, csystine is lost completely in HAHp-H, additionally, the content of lysine is decreased. However, the hydrophobic amino acids content of HAHp-H is increased from 45.92% to 48.74%. Results of molecular weight distribution indicate that heat treatment on HAHp results in the higher content of molecular weight of 3,000-5,000 Da and lower molecular weight below 500 Da. HAHp-H exert higher scavenging activity on DPPH and hydroxyl radicals, moreover, the reducing power of HAHp-H is stronger than that of HAHp.The antiproliferative activities of HAHp and HAHp-H on human cell lines DU-145 (prostate cancer), 1299 (lung cancer) and 109 (esophagus cancer) are detected using the MTT method. Results imply that HAHp and HAHp-H have higher inhibitory effects on prostate cancer DU-145 and lung cancer 1299 than on esophagus cancer 109. The IC₅₀ values of HAHp and HAHp-H on prostate cancer DU-145 are 41.14 mg/mL and 13.67 mg/mL, respectively. As for lung cancer 1299, the IC₅₀ values of HAHp and HAHp-H are respectively 40.28 mg/mL and 25.17 mg/mL. Both HAHp and HAHp-H have inhibitory effects at a high concentration of 40 mg/ml on 109 esophagus cancer cells. Moreover, HAHp-H shows stronger antiproliferative activity than HAHp at the same concentration. In the results of cell morphology and HE dye, the morphology changes of prostate cancer DU-145 cells after treated with HAHp and HAHp-H for 48 h are apparent, meanwhile, the characteristic of cell apoptosis is also visualized.