Component Analysis And Bioactivity Of Walnut Green Skin Pigment

Food safety caused by foodborne pathogens has attracted extensive attention,because foodborne pathogens can not only change nutritional characteristics,but also harm human body.In order to prevent and control pathogenic microorganisms in food,it is necessary to develop natural non-toxic food preservatives.Walnut green skin,as the main by-product of walnut,is rich in polyphenols and flavonoids,especially walnut quinone,which has good biological activities such as antioxidant and antibacterial properties.Therefore,taking walnut green skin as raw material,this thesis studies the antioxidant and antibacterial properties and antibacterial mechanism of walnut green skin pigment and juglone.1.Study on extraction,separation and purification of walnut green skin pigmentUsing walnut green skin as raw material,the yield of walnut green skin pigment was determined by ultrasonic assisted solvent method under different ethanol concentration,time,solid-liquid ratio and power.The optimal extraction parameters were obtained by response surface optimization.The pigment of walnut green peel was separated and purified by macroporous adsorption resin.Through dynamic adsorption and desorption experiments,three different macroporous adsorption resins D-101,AB-8 and HZ-88 were compared,and the best macroporous adsorption resin was selected for subsequent separation and purification experiments.According to the single factor experimental results,the extraction effect was the best under the conditions of 50%ethanol concentration,1:25 solid-liquid ratio,25 min ultrasonic time and 200 W ultrasonic power.The optimum process conditions obtained by response surface optimization were as follows:ethanol concentration was 44.64%,ultrasonic time was 54.8 min,solid-liquid ratio was 1:27.67,ultrasonic power was 188 W,and the yield was 27.96%.Dynamic adsorption and desorption experiments showed that D-101macroporous adsorption resin had the best effect on the separation and purification of walnut green skin pigment.The optimum conditions for adsorption are initial concentration of 1.5 mg/mL,flow rate of 0.5mL/min,pH=3 and sample volume of 50 mL.The optimum conditions for desorption are elution flow rate of 1.5 mL/min,ethanol concentration of the eluent of 90%,pH=4.2.Antioxidant activity of walnut green skin pigment and its protection against protein oxidative degradation and DNA oxidative damage induced by AAPHThe free radical scavenging ability and reducing ability of walnut green skin pigment extract before and after separation and purification,and the protection experiment of purified walnut green skin pigment against protein oxidative degradation induced by AAPH and plasmid DNA oxidative damage were studied and compared.The results showed that the purified walnut green skin pigment extract had good antioxidant capacity compared with V_C;When the concentration of purified walnut green skin pigment extract reached1.0 mg/mL,it had a significant protective effect on protein degradation induced by AAPH;When the concentration is 0.5 mg/mL,it can significantly protect plasmid DNA from oxidative damage.3.Structural identification of walnut green skin pigment and its antibacterial activityIn this study,the pigment extract of walnut green skin was identified,and its polyphenol content and antibacterial activity were determined.The results showed that the content of total polyphenols in walnut green skin pigment extract was 21.71±0.98 mg/g and the content of total flavonoids was 22.16±0.45 mg/g.Nine main compounds in walnut green skin pigment extract were identified by UPLC-MS/MS and HPLC,which were quercetin,astragaloside,amygdalin,hypericin,isoquercetin,quercetin,caffeic acid,ferulic acid and juglone.Walnut green skin pigment extract has antibacterial activity against E.coli.By increasing the permeability of bacterial cell membrane,it destroys its integrity and leads to the leakage of nucleotides and proteins in cells.SDS-PAGE results showed that walnut green skin pigment extract had a significant effect on the degradation of bacterial whole protein,and could significantly reduce the content of bacteriaβ-galactosidase inhibits the activity of intracellular proteins and eventually leads to the death of E.coli.4.Study on bacteriostatic activity and mechanism of jugloneJuglone,as the main component of walnut green skin pigment extract,has significant antibacterial effect.The growth inhibition curve test showed that juglone had significant antibacterial activity against E.coli,and the minimum inhibitory concentration was 0.0625 mg/mL.The relative conductivity test showed that juglone destroyed the integrity of E.coli membrane and increased the permeability of cell membrane.The results of crystal violet and resin azurol showed that juglone affected the formation and overall activity of E.coli biofilm.SDS-PAGE and PAGE results showed that juglone could reduce the expression of protein,DNA and RNA in E.coli.Juglone interacts with membrane proteins and affects protein expression,resulting in significant changes in content,structure and conformation.Through fluorescence,UV-Vis absorption spectra and molecular docking studies,juglone can bind to the groove of genomic DNA and change its secondary structure and morphology.