Study On The Physicochemical Properties Of Pepper Seed AMP And The Inhibition Effect Of Against Aspergillus Flavus

As a natural immune barrier,antimicrobial peptides（AMP）are widely found in various organisms in nature because their unique antibacterial mechanisms and safety are increasingly concerned by related fields.In our Pre-laboratory work shows pepper seed antimicrobial peptides had a strong inhibitory activity against Aspergillus flavus growth.On this basis,the subject further optimized the extraction conditions and separation and purification methods of pepper seed AMP,clarified its physicochemical properties and range of antibacterial activity,studied its antagonistic effect on Aspergillus flavus and its mechanism,and based on the“safety of food poisoning”.“Rational Evaluation Procedures and Methods”evaluates the safety level of pepper seed AMP and examines their effect on the actual anti-mildew application of corn.It provides reference for the development and utilization of pepper by-product pepper seeds and the application of plant-derived antimicrobial peptides as a fungicide in feed storage.The main research contents and results are as follows:（1）Separation and purification of pepper seed AMPAspergillus flavus was used as an indicator to Compared with 6 varieties of chili peppers with a large amount of usage in southwest China.The antibacterial effect of Guizhou bell pepper was the best.The pepper seed was used as a follow-up test material,and its extraction conditions were optimized to obtain the best extraction conditions:The EDTA buffer（pH 5.6）was used as the extract,the ratio of material to liquid was 1:5,the extraction time was 16 h,and the concentration of ammonium sulfate was 80%.At this time,the AMP bacteriostasis diameter can reach 18.55mm,which was 50.32%higher than that before optimization（12.34mm for sterile water extraction）.The pepper seed AMP were separated and purified by desalting of dialysis bags,ultrafiltration of 10 kDa ultrafiltration tubes and Sephadex gel chromatography.Thepurifiedantibacterialpeptideswereelectrophoreticallypureby Tricine-SDS-PAGE,and the molecular weight was around 7.8 kDa.（2）Characteristics of pepper seed AMPBy measuring the concentration of pepper seed AMP,temperature,acid and alkali conditions,UV irradiation,metal ions,urea concentration,the antibacterial effect of pepper seed AMP,the results show that the antibacterial activity of pepper seed AMP’s with the increase in concentration.However,when the concentration was0.625 mg/mL,the AMP still has a significant antibacterial effect;the antibacterial peptide has the ability to resist temperature,and can maintain the original antibacterial activity even if it acts for 15 minutes at 121°C.At the pH value of 2-12,it can form a significant inhibition zone,and the activity is highest when the pH value was 12.The effect of UV irradiation for 24 h,0-6 mol of urea and 4 kinds of protease treatment on antimicrobial peptides is not effective.Influencing,the metal ions(Na⁺,K⁺,Ca²⁺,Mg²⁺,Mn²⁺,Cu²⁺)at the test concentration of 10^-2 mol/L have no effect on the activity of the antibacterial substances,but the presence of Fe²⁺on the antibacterial activity of AMPwill be inhibited.By measuring the antibacterial range of pepper seed’s AMP,the results showed that the pepper seed antibacterial peptide has a wider range of inhibition.In particular,it has a strong inhibitory effect on spoilage bacteria（A.flavus,Rhizopus nigricans）,food-borne pathogens（Bacillus cereus,Staphylococcus aureus）,and plant pathogens（F.oxysporum）caused by feedstuffs,indicating its development for the preservation of preservatives and biological pesticide potential.（3）Inhibition effect of against Aspergillus flavusThe pepper seed AMP can strongly inhibit the growth of Aspergillus flavus.The minimum inhibitory concentration（MIC）was 156.25μg/mL.The different folds of MICs had different degrees of inhibition on the Aspergillus flavus spores.Incubation of the pepper seed AMP for 18 h under 4MIC conditions completely inhibited the spore germination of Aspergillus flavus.The addition of 1MIC AMP at different stages of Aspergillus flavus can inhibit its continued growth and inhibit its spore production.The addition of pepper seed AMP at different stages of the growth of Aspergillus flavus inhibits the use of nutrient substances in the medium.Sytox Green（SG）combined with a fluorescent dye to study the permeability of the pepper seed AMP to Aspergillus flavus Sex,found that AMP treatment of Aspergillus flavus mycelium,cell membrane permeability has changed.It showed that the pepper seed AMP could inhibit spore germination at the spore stage of Aspergillus flavus,and the mycelia stage could destroy the cell membrane of Aspergillus flavus,which reduced its ability to absorb nutrients,limited its growth,and inhibited the production of spores.（4）Evaluation of the safety of pepper seed AMPThe safety of pepper seed’s AMP as additives was studied to provide a toxicological safety basis for their application.Acute toxicity tests and subchronic toxicity tests were conducted according to"Toxicological Evaluation Procedures and Methods for Food Safety".The results showed that once the mice were fed with 5 g/kg·BW of the pepper seed AMP,no obvious poisoning and feeding were observed after one week of feeding.According to the standard of acute toxicity,the antibacterial peptide belongs to the actual non-toxic level.The results of the 30-day feeding experiment showed that the pepper seed antibacterial peptide had no significant effect on the feed utilization rate,organ index,blood routine,and blood biochemical indexes of the rats at the daily dose of 300 mg/kg·BW（P>0.05）.No pathological changes of the pepper seed antibacterial peptides were found in rats,indicating that the pepper seed antibacterial peptides are safe and do not cause accumulation toxicity in vivo.（5）Application of pepper seed AMP as antifungal agents in storage of cornComparing different doses of pepper seed AMP,natamycin,calcium propionate,and capsaicin antimicrobial peptides,and natamycin and calcium propionate compound antibacterial agents to treat corn,and stored at 28°C and 75%relative humidity for 20 days.The results showed that low dose（0.075mg/kg）of pepper seed antibacterial peptide can inhibit mold contamination,0.3g/kg of pepper seed AMP antibacterial effect of calcium propionate and natamycin standard addition.At the same time,it can be found that the simultaneous application of the pepper seed antibacterial peptide with calcium propionate or natamycin is better than the equivalent concentration of the two alone.It shows that the pepper seed antibacterial peptide itself can be used as a fungicide,and it works better with other antifungal agents.In summary,the pepper seed AMP has strong tolerance to the environment and has a wide range of application;it has an antagonistic effect on Aspergillus flavus and can effectively inhibit the growth of Aspergillus flavus at different growth stages of Aspergillus flavus;a single high-dose intake Both（5g/kg·BW）and long-term cumulative ingestion（300mg/kg·BW）will not produce toxic effects on the body and have good safety.Both alone and in combination with other antibacterial agents had a good anti-mold effects.It shows that the pepper seed AMP has the potential to be used as a feed additive in feed storage.