Preparation And Characteristics Of Antimicrobial Peptides From Prickly Ash(Zanthoxylum Bungeanum Maxim.)Seed Protein

Prickly ash(Zanthoxylum bungeanum Maxim.) seed is main by-product in the production of prickly ash pericarp, rich in protein, is a high quality protein source. In-depth study of the prickly ash seed protein can enhance its economic value and has important implications for Zanthoxylum bungeanum Maxim, industry value added efficiency by using modern food biotechnology. Antimicrobial peptides are a class of polypeptide that has many purposes such as anti-bacteria, fungi, viruses, parasites and inhibit or kill Tumor cells. Antimicrobial peptides can not only be degraded and digested by enzyme system of the human body, but also can maintain the maximum nutritional value of food. Therefor they are one of the widely studied natural preservatives. In this study, the protease enzyme was selected to prepare antimicrobial peptides from prickly ash seed protein. The research studied the optimization of hydrolysis conditions, purification of hydrosates and the properties of antimicrobial peptides. The main results as follows:1. Pepsin and acid protease were selected to prepare antimicrobial peptides from prickly ash seed protein after screening tests of protease. One-factor-at-a-time method and response surface analysis based on Box-Behnken design, using response surface methodology to analyze each factor. The inhibitory rate on Escherichia coli in response value was established a mathematical model. The optimal hydrolysis conditions of pepsin were that substrate concentration 49mg/mL, enzyme/substrate ratio (w/w) 0.9%, pH2.0, hydrolysis temperature 32℃ and hydrolysis time 3h. Under these conditions, the degree of hydrolysis (DH) was 9.22%, the inhibitory rate on E. coli was 60.69%. The best hydrolysis conditions of acid protease were as followings:substrate concentration 30mg/mL, enzyme/substrate ratio (w/w) 3.0%, pH4.0, hydrolysis temperature 51.2℃ and hydrolysis time 4.7h. Under these conditions, the degree of DH was 9.05%, the inhibitory rate on E. coli was 56.98%.2. The separation of peptide A and B were followed by ultrafiltration, Sephadex G-50 gel chromatography respectively, than the purified products were measured relative molecular mass. The results of ultrafiltration showed that the peptide A and B of the relative molecular mass in the range of 5kD and lOkDa (Called peptide A-b and peptide B-b, respectively) had antibacterial activity significantly, the inhibitory rate were 62.79% and 66.94%. Then by Sephadex G-50 gel chromatography, peptide A-b was separated four peaks, in which the peak Ⅳ (peptide A-b-Ⅳ) had the best antibacterial activity, the inhibitory rate on E. coli was 100%; Peptide B-b was separated four peaks too, in which the peak III (peptide B-b-Ⅲ) had the optimal antibacterial activity, the inhibitory rate on E. coli was 69.99%. The Tricine-SDS-PAGE analysis showed that the relative molecular mass of peptide A-b-IV and peptide B-b-III were about 8kDa and 10kDa respectively.3. The research used colony forming unit technique to study the stability of peptide A-b and their antimicrobial performance. The results showed that the peptide A-b can inhibit the Escherichia coli, salmonella, Bacillus subtilis and Staphylococcus aureus. The antimicrobial activity of peptide A-b on Escherichia coli increased with rising concentration. When treated with temperature and heat-time, the antibacterial activity of peptide A-b displayed no remarkable differences with control (p>0.05). The peptide A-b remained excellent antibacterial activities after pH2.0-12.0, being lowest at pH 2.0(58.13%) and highest at pH 12.0(79.17%). With the concentration of metal ions increasing, the antibacterial activity of peptide A-b reduces, increases and changes little respectively after treatment of K+, Ca2+ and Fe3+, and the antibacterial activity of antimicrobial peptides would increase obviously when treating with 0.1mol/L K+. The antibacterial activity decreased slightly after treatment with organic solvents. The antibacterial activity increased significantly after treatment with Tween 20 and Tween 80 (p<0.0l), respectively. While insignificant of antibacterial activity after treatment with SDS was dedected. Therefore, the peptide A-b had better stability of temperature, pH, metal ions and organic solvents, and the surfactants (Tween 20 and Tween 80) could improve the antibacterial activity of antimicrobial peptides significantly.4. The solubility, foaming capacity and emulsifying property of peptide A-b were investigated with respect to pH and temperature. The results showed that the peptide A-b had a better solubility (NSI>80%) at a wide range of pH≥6. temperature 30℃ to 50℃. The peptide A-b had better foaming capacity in acidic and alkaline conditions than in neutral condition, and it had good foam stability (≥82%) within 10min and pH≥6. The peptide A-b had some foaming capacity (≥40%) at a certain range of temperature 35 ℃ to 55℃, maintaining good foam stability within 10min (≥73%). The emulsifying property of peptide A-b decreased with rising concentration, while the emulsifying stability increased with rising concentration. The emulsifying property and emulsifying stability of peptide A-b were worst at pH4.0, emulsifying property was 2.44m2/g and emulsifying stability was less than 22 min. The peptide A-b had a highly hydrophilic and its hydrophilic lipophilic balance (HLB) value was greater than 16.7.