Study On Preparation,Properties And Antibacteria Activity Of Potato Trypsin Inhibitor

Trypsin inhibitors have a variety of physiological functions and have a broad application prospect in agriculture,medicine and other fields.Potato trypsin inhibitor?PTI?,which was often directly discharged as a by-product of potato starch processing,can't exert its maximum value,The recovery of trypsin inhibitor in potato starch wastewater will be beneficial to the high value utilization of potato by-products and promote the comprehensive utilization of potato crops.In this study,two methods for recovering trypsin inhibitors were optimized and compared using simulated potato starch processing wastewater as raw material.And the physicochemical properties such as solubility,foam property,emulsification property and stability of PTI prepared under optimal conditions as well as its interaction with trypsin were further studied.At the same time,the antibacterial activity of PTI was measured and its antibacterial mechanism was discussed in order to provide theoretical guidance for the application of PTI in antibacterial properties.The extraction conditions of macroporous resin adsorption chromatography and acid precipitation combined with salting out were optimized by investigating the activity,yield,purity and so on.Macroporous resin adsorption chromatography was used to separate potato protease inhibitors from other proteins.After optimization,the protein content of the product was 88.32%,and the sugar content was 2.70%.The yield was 462 mg/kg,the inhibition ratio was 2672 TIU/mg and the purification factor was 15.7 times,showing a light yellow powder.After the single factor experiment to explore the optimal process conditions for the preparation of PTI by acid precipitation combined with salting out,the protein content of the final product was 81.81%,the sugar content was 2.8%,the yield was 600 mg/kg,the inhibition ratio was 3275 TIU/mg and the purification ratio was 19.3 times,showing a pure white powder.Compared with the two methods,the product of acid precipitation combined with salting out method has higher activity,higher purification times and higher yield.Therefore,the PTI prepared by acid precipitation combined with salting out method was studied in the following experiments,and the purity of PTI was determined by SDS-PAGE electrophoresis and MALDI-TOF MS.The results showed that the electrophoretic pure grade PTI with higher activity was obtained by this method.PTI was taken as the research object to investigate its functional property,stability inhibition type and its interaction with trypsin.At room temperature,PTI showed good solubility in a wide p H range and solubility varied with temperature and p H.Compared with the lyophilized powder of potato simulation wastewater and soybean trypsin inhibitor?STI?,PTI showed favorable foam property,gas-water interface property and emulsification property.The effects of p H and temperature on the stability of the PTI were explored by determining residual trypsin inhibitory activity and analyzing conformation of the PTI before and after different treatments.,PTI,a protein rich in?-sheet conformation,has certain heat resistance and good p H stability.The abrupt temperature of inhibitory activity and secondary structure is70?.The changes of secondary structure were mainly manifested as the decrease of?-sheet and?-turn contents,and the corresponding increase of?-helix and random coil,while the relative trypsin inhibitory activity of PTI in the range of p H 2-10 remained above 90%and the secondary structure of PTI was not sensitive to p H changes.In vitro simulated digestion stability experiments showed that PTI had a certain resistance to gastrointestinal digestion.At the end of gastrointestinal digestion,its final inhibitory activity loss rate was about 29.28%and the release rate of-NH₂group was about 24.39%.PTI was a non-competitive inhibitor with the low IC₅₀of 6.861±0.107 mg/L and was bound to the necessary group outside the enzyme activity center by non-covalent bond.The further isothermal titration experiment indicated that the binding of PTI and trypsin was a spontaneous exothermic process with a single binding site.At 25?,it was a spontaneous enthalpy-driven reaction caused by the electrostatic force.When the temperature reached 37?and 50?,it is an enthalpy-entropy compensation-driven process dominated by an enthalpy-driven process and the force was mainly hydrogen bond or Van der Waals'force.To evaluate the bacteriostatic activity of PTI,the inhibition zone diameter?DIZ?was determined through filter paper diffusion method and the minimum bacteriostatic concentration?MIC?and minimum bactericidal concentration?MBC?were determined through micro double dilution method.Furthermore,the antibacterial mechanism of PTI was explored through scanning electron microscopy,cell membrane permeability and integrity,oxidative damage as well as inhibition of bacterial protease activity.PTI showed significant inhibition against Bacillus subtilis,Bacillus cereus and Pseudomonas aerimonas with the diameter of bacteriostatic zone values of 12.56 mm,12.35 mm and 11.76 mm,respectively.The MIC ranged from 1.88 to 3.75 mg/m L,and the MBC?3.75 mg/m L?were the same for three bacteria.PTI destroyed the morphology of bacterial cells and led to the increase of relative conductivity and the leakage of nucleic acid in bacterial suspension,indicating that PTI affected the membrane permeability of bacteria.Fluorescence microscopy images showed that the integrity of cell membrane of bacteria was destroyed and the production of intracellular ROS was promoted by PTI at the concentration of MIC.PTI was able to inhibit the activity of bacterial protease.In conclusion,PTI probably play a bacteriostatic role through the multi-target synergistic effect.