Detoxification Of Jatropha Meal And Study On Cell Membrane Chromatographic Screening Method And Antimicrobial Mechanism Of Jatropha’s Antimicrobial Peptides

Currently, Jatropha curcas is recognized as the most economical of preparation biodiesel. Up to2015,the output of Jatropha curcas seed meal protein will be more than200million tons, which is a potentialprotein resource. Due to the toxins and anti-nutritional factors, Jatropha curcas seed meal was used as thelower-value fertilizer, limiting its applications. Therefore, the most suitable detoxification methodestablished is the key point. Futhermore, low yield hinders antimicrobial peptides (AMPs) application. As aconsequence, developing new protein sources for AMPs and a promising approach for high-throughputscreening of AMPs are urgent.The purpose of this study is not only to establish the most suitable methods of detoxification forJatropha curcas seed meal, but also pay attention to the use of the detoxification protein of Jatropha curcasseed meal. And then cell membrane affinity chromatography (CMAE) and the improved CMAE (cellmembrane affinity chromatography-offline-liquid chromatography fingerprint) were developed to isolateand purify AMPs from Jatropha curcas. Furthermore, the antimicrobial mechanism of antimicrobialpeptides (JCpep7and JCpep8) in the living cell environment of Staphylococcus aureus (S. aureus) wasinvestigated. The main results and conclusions obtained were as follows.(1) In order to establish the most suitable methods of defatting for Jatropha curcas seed meals, theJatropha curcas L. seed meals were defatted by three methods (screw extruding-expanding pretreatmentfollowed by petroleum ether extraction, petroleum ether extraction and screw-press). It was shown thatdifferent methods of defatting had great impact on trypsin inhibitors activity (TIA) and lectin in the oilseedmeals (p<0.05). And TIA and lectin in the SEEPM (3.15mg/g and3.43mg/g) and in the SPM (2.67mg/gand1.53mg/g) were significantly lower than that in the SEM (27.28mg/g and38.78mg/g). But thephorbolesters, phytates and saponins were not removed by the different methods of defatting, indicatingthat there must be a detoxification way in the future. Due to the low content of residual oil, toxins andanti-nutritional factors in the SEEPM, the SEEPM obtained by the defatting method (screwextruding-expanding pretreatment followed by petroleum ether extraction) is better for detoxification.(2) The Jatropha curcas seed meal obtained by the most suitable methods of defatting was detoxifiedby five different treatments (enzymatic treatment; enzymatic treatment60%methanol and65%ethanoltreatments, respectively;90%methanol and90%ethanol treatments, respectively). It was shown thatdifferent detoxification treatments had significant (p<0.05) effect on the toxin and antinutritionalcomponents (p<0.05). The treatment that hydrolysis of enzymes (cellulase plus pectinase) followed bywashing with ethanol (65%) was the best detoxification method. After this treatment, the phorbolesterswere decreased by100%. The antinutritional components (phytates, tannins, saponins, TIA and lectin) weredecreased to a tolerable level. In this detoxified meal, the crude protein content (74.86%) was highest, andthe in vitro protein digestibility was high to92.37%. In addition, the total amino acid was66.87g/100gprotein and the nutritional indices (EAAI, BV, NI and PDCAAS) were higher than other detoxificationmeal. It can be concluded that the process of hydrolysis of enzymes (cellulase plus pectinase) followed bywashing with ethanol (65%) was a promising way to detoxify Jatropha curcas meal.(3) Based on the membrane-binding activity of AMPs, novel methods named CMAE and the improvedCMAE were developed to isolate and purify AMPs from Jatropha curcas. Combining withMALDI-TOF-MS/MS, two novel antimicrobial peptides JCpep7(Lys-Val-Phe-leu-Gly-leu-Lys) andJCpep8(Cys-Ala-Ile-Leu-Thr-His-Lys-Arg) were successfully isolated and identified. The antibacterialtests showed that the novel methods were feasible. These two antimicrobial peptides were active against thetested microorganisms and thermal stability. In addition, the two antimicrobial peptides, which had nohemolytic activities and cytotoxicity in the low concentrations, can resist the enzymolysis of pepsin,trypsase and papain, but could not resist the enzymolysis of proteinase K.(4) The characterization of initially binding process between antimicrobial peptides (JCpep7andJCpep8) and the living Staphylococcus aureus cells (LSACs) used as pseudo-stationary phase in Capillary Electrochromatogrpahy (CEC) was firstly investigated. It was shown that the binding process betweenantimicrobial peptides (JCpep7and JCpep8) with LSACs is spontaneous and significant. Hydrophobicinteraction plays a major role in the binding process of JCpep7. However, the electrostatic attraction andhydrophobic interaction play a major role in the binding process of JCpep8.(5) The antimicrobial mechanism of antimicrobial peptides (JCpep7and JCpep8) in the living cellenvironment of S. aureus was investigated by time-dependent experimental settings, such as thecharacterization of initially binding process, antibacterial activity kinetics, outer membrane and innermembrane disruption assays, as well as bacterial ultrastructure. Data showed that JCpep7and JCpep8canfirstly and rapidly bind with the cell wall. Once binding to the cell wall, JCpep7and JCpep8can break thecell wall, then continues to move forward to reach the cell membrane and gathers. When the concentrationof JCpep7and JCpep8was beyond a certain threshold, it led the bacteria died caused by forming a stablemembrane holes in the cell membrane. JCpep7killed the bacteria according to the model of “carpet” model,however, JCpep8killed the bacteria according to the model of “barrel-stave” model.