Study On The Isolation,purification And Antibacterial Mechanism Of Phenolics From Pecan Husk

Carya illinoinensis(Wangenh.)K.Koch is also known as pecans.Pecan is nutritious,high in unsaturated fatty acids,and also contains other nutrients.Pecan husk is one of the byproducts of pecan processing.About 420,000 tons of pecan husk are produced worldwide each year,and most of them are currently disposed of as waste.It has been reported in the literature that pecan husk is rich in phenolic substances,which have the functions of antioxidant,antibacterial,and anti-inflammatory.Therefore,further development and utilization of pecan husk are of great significance to improve the economic benefits of the pecan industry and reduce resource waste and environmental pollution.In this paper,the effects of different drying methods on the phenols of pecan husk and the enrichment and purification of the phenols in pecan husk were investigated.The phenolic substances of pecan husk were identified,and its inhibitory mechanism against bacteria and its biofilm inhibitory activity were studied.The main research contents are as follows:1.Effects of different drying methods on the phenolic compounds of pecan husk.The pecan husk was dried by freeze-drying,vacuum drying,hot-air drying,and traditional drying,and its phenolic substances were determined.The results showed that freeze-drying could preserve the content of polyphenols and flavonoids in the pecan husk to the greatest extent.The antibacterial experiments were carried out on the phenolic substances obtained by different drying methods,and the results showed that the phenolic substances obtained by freeze-drying had the best antibacterial activity,and the diameters of the inhibition zones were 20.4 ± 0.6 mm(Staphylococcus aureus),18.2 ± 0.3 mm(Streptococcus mutans),15.9 ± 0.6 mm(Pseudomonas aeruginosa),and 12.8 ± 0.4 mm(Escherichia coli).2.Enrichment,purification,and identification of main phenolic compounds in pecan husk.The D101 macroporous resin was preli minarily screened for the enrichment of phenolic substances in pecan husk,and the obtained phenols content was 36.49 ± 1.96%.After secondary purification with polyamide resin,the optimized process is as follows: the loading concentration is 6 mg/m L,the loading flow rate is 1.0 BV/h,and the elution flow rate is 1.0 BV/h,the eluting phase of 40% ethanol was obtained(PPH-FPEEPA),and the phenolic substance content was88.71 ± 2.84%.Compared with the crude extract,the content of phenolic components of PPHFPEEPA increased by 5.6 times.The phenolic substances in it were analyzed by UPLC-Q/TOFMS,and the results showed that nine kinds of phenolic substances were identified from the purified phase of pecan husk,including 5 kinds of flavonols(quercetin-3-O-rhamnoside,kaempferol-3-O-glucoside,quercetin-3-O-sambubioside,hypericin,luteolin-7-O-β-Dglucuronide),One flavonoid(luteolin),one isoflavone(genistein)and two procyanidins(procyanidin dimer type A and procyanidin dimer type B).Among them,quercetin-3-Osambubiglycoside,luteolin-7-O-β-D-glucuronide,and genistein were found in the pecan husk for the first time.3.Study on the inhibitory mechanism of phenolic compounds from pecan husk relative to bacteria.The bacterial structure was observed by scanning electron microscope(SEM),and it was found that PPH-FPEEPA could damage not only the cell wall but also the cell membrane;nucleic acid leakage assay and flow cytometry(FCM)analysis found that the degree of bacterial cell membrane damage increased with increasing concentrations of PPH-FPEEPA.By measuring β-galactosidase,it was shown that PPH-FPEEPA increased the permeability of S.aureus and P.aeruginosa cell inner membrane;the fluorescence intensity of NPN increased significantly,indicating that the permeability of the outer membrane of P.aeruginosa increased.Through the measurement of membrane potential and membrane fluidity,it is shown that PPHFPEEPA can lead to the decrease of membrane fluidity and the phenomenon of membrane hyperpolarization by binding to the cell membrane.In addition,the fluorescence spectrum analysis of membrane protein showed that the fluorescence intensity of Phe,Trp,and Tyr residues decreased significantly,and with the increase of the concentration,the red shift was obvious,indicating that PPH-FPEEPA can lead to changes in the conformation of membrane proteins.4.Study on the inhibitory activity of phenolic compounds from pecan husk on bacterial biofilm.The study found that Sub-inhibitory concentrations of PPH-FPEEPA can effectively inhibit the formation of S.aureus and P.aeruginosa biofilms by reducing the hydrophobicity of the bacterial interface and inhibiting the production of bacterial exopolysaccharides.In addition,through correlation analysis,the concentration of PPH-FPEEPA was positively correlated with its inhibitory rate on biofilms,and negatively correlated with culture time,the observation results of the scanning electron microscope and confocal laser scanning microscope also proved that the antibacterial effect of PPH-FPEEPA increased with the increase of its concentration,indicating that the addition of PPH-FPEEPA can significantly destroy the biofilm,to achieve the effect of inhibiting bacterial biofilm.