麻疯树籽粕脱毒及其抗菌肽的细胞膜色谱制备法和抗菌机理的研究
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摘要
麻疯树是目前世界上公认的最具有开发制备生物柴油潜力的树种。据统计，到2015年我国麻疯树栽培面积将达到9000万亩，每年将约有650多万吨的麻疯树籽粕产出，到时将能产出约200多万吨的麻疯树粗蛋白，是一种亟待开发的天然植物蛋白质资源。由于毒素和抗营养因子的存在，长期以来，麻疯树籽粕主要被用作价值较低的肥料，限制了其应用范围。因此，合适脱毒方法的建立，是提高麻疯树籽粕资源利用价值首要解决的问题。由于天然抗菌肽来源有限、分离纯化步骤繁琐，生产效率较低，限制了抗菌肽行业的发展。因此，开发寻找新型天然抗菌肽的蛋白来源，探索绿色、高效、高通量的抗菌肽分离纯化方法迫在眉睫。
本论文以麻疯树籽粕为原料，在为麻疯树籽粕探索出一种合适的脱毒工艺，制备安全无毒麻疯树籽粕的同时，关注脱毒麻疯树籽粕蛋白的深度开发利用。通过建立一步细胞膜色谱技术及改进的两步细胞膜色谱技术，快速、准确地从脱毒麻疯树籽粕及其蛋白水解物中筛选和制备麻疯树源抗菌肽，并通过对抗菌机理研究手段的创新和对现有的研究手段进行优化组合及设计，实现在活体环境下探索麻疯树源抗菌肽的抗菌特征和机理，为麻疯树籽粕蛋白的增值开发利用提供理论依据和技术支持。主要研究结果如下：
（1）采用3种不同脱脂方法（溶剂提取法、挤压膨化预处理联合溶剂提取法和高温压榨法）对麻疯树籽进行脱脂处理。结果表明：不同的脱脂过程能显著影响胰蛋白酶抑制剂和植物凝集素的含量及活性（p<0.05），其中挤压膨化粕（SEEPM）和高温压榨粕（SPM）中的胰蛋白酶抑制剂和植物凝集素的含量（3.15mg/g和3.43mg/g，2.67mg/g和1.53mg/g）均显著低于溶剂萃取粕（SEM）（27.28mg/g和38.78mg/g）。但不同脱脂处理并不会对麻疯树籽粕中的佛波酯、植酸、皂苷有良好的脱除效果，仍需进一步采取其他脱毒方式。由于挤压膨化粕中低的残油率和毒性物质含量，因此挤压膨化预处理联合溶剂浸提法为麻疯树籽粕最适脱脂方法。
（2）以在最适脱脂方法下得到的麻疯树籽粕为研究对象，采用5种不同处理（酶处理：纤维素酶和果胶酶；酶处理+60%甲醇或65%乙醇处理；90%甲醇或90%乙醇处理）对其进行脱毒。结果表明：不同的脱毒方法对麻疯树籽粕中的毒素及抗营养因子含量有显著影响（p<0.05）。其中，经过酶解联合65%乙醇洗脱处理后，粕中佛波酯被全部脱除，胰蛋白酶抑制剂、植物凝集素、植酸和皂苷的含量均显著下降（p<0.05）。且该法得到的脱毒粕中蛋白质含量最高（74.86%），蛋白质体外消化率高达92.37%。此外，该粕中氨基酸总量为66.87克/100克蛋白，必需氨基酸齐全，配比合理，营养指标（EAAI、BV、NI和PDCAAS）均高于其他脱毒粕。可见，酶解处理联合65%乙醇洗脱的方法为麻疯树籽粕最适脱毒方法。
（3）利用抗菌肽与菌体细胞膜间存在着特异性亲和力，建立了一步细胞膜色谱技术及改进的两步细胞膜色谱技术（细胞膜固相亲和萃取联合HPLC指纹图谱技术），成功地从粕及其水解物中制备2种麻疯树源抗菌肽（JCpep7和JCpep8）。通过质谱技术（MALDI-TOF-MS/MS）鉴定其氨基酸残基序列分别为Lys-Val-Phe-leu-Gly-leu-Lys和Cys-Ala-Ile-Leu-Thr-His-Lys-Arg。抗菌实验表明利用细胞膜亲和色谱技术分离麻疯树源抗菌肽是可行的。所得2种抗菌肽对大肠杆菌、铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、枯草芽孢杆菌与肺炎链球菌均有抗菌活性，最适pH为中性，均对热稳定，不耐受高离子强度，对胃蛋白酶、胰蛋白酶和木瓜蛋白酶具有一定的抵抗力，但不能抵抗蛋白酶K的酶解。在低浓度下均对兔红细胞无溶血和凝集作用。
（4）首次采用毛细管电泳技术，将活体金黄色葡萄球菌作为“假固定相”，依据毛细管电动色谱原理，用相对淌度代替淌度，描述活性菌体对抗菌肽（JCpep7和JCpep8）的迁移行为的影响，建立了一种新的毛细管电泳法测定抗菌肽与活性菌体结合常数的方法。结果表明：抗菌肽JCpep7和JCpep8与活性金黄色葡萄球菌（LSACs）之间的结合过程是自发进行的，而且结合效果均很显著。随着温度的增加，2种抗菌肽与LSACs的结合常数也随着增大。其中JCpep7以疏水相互作用力为主要作用，而JCpep8则以静电吸引力和疏水相互作用为主要作用。
（5）利用毛细管电动色谱相对淌度法（初始结合常数实验）及对现有研究抗菌肽抗菌机理的手段（生长曲线抑制实验、内外膜破坏实验及菌体微观形态学变化）进行优化组合及设计，实现了在活菌体环境下探索抗菌肽（JCpep7和JCpep8）的抗菌机理。结果表明：抗菌肽JCpep7和JCpep8能快速结合到金黄色葡萄球菌细胞壁上，并对细胞壁造成损伤，进而扩散到细胞膜。当抗菌肽浓度在细胞膜上超过一定的阈值，就会破坏细胞膜的完整性，使得胞内物质泄漏，导致菌体死亡。可见，细胞壁膜是抗菌肽（JCpep7和JCpep8）发挥抗菌作用的主要靶点。其中JCpep7是以“毯式”模式的膜损伤抗菌特征，而JCpep8是以“桶状”模式的膜损伤抗菌特征。
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.
本论文以麻疯树籽粕为原料，在为麻疯树籽粕探索出一种合适的脱毒工艺，制备安全无毒麻疯树籽粕的同时，关注脱毒麻疯树籽粕蛋白的深度开发利用。通过建立一步细胞膜色谱技术及改进的两步细胞膜色谱技术，快速、准确地从脱毒麻疯树籽粕及其蛋白水解物中筛选和制备麻疯树源抗菌肽，并通过对抗菌机理研究手段的创新和对现有的研究手段进行优化组合及设计，实现在活体环境下探索麻疯树源抗菌肽的抗菌特征和机理，为麻疯树籽粕蛋白的增值开发利用提供理论依据和技术支持。主要研究结果如下：
（1）采用3种不同脱脂方法（溶剂提取法、挤压膨化预处理联合溶剂提取法和高温压榨法）对麻疯树籽进行脱脂处理。结果表明：不同的脱脂过程能显著影响胰蛋白酶抑制剂和植物凝集素的含量及活性（p<0.05），其中挤压膨化粕（SEEPM）和高温压榨粕（SPM）中的胰蛋白酶抑制剂和植物凝集素的含量（3.15mg/g和3.43mg/g，2.67mg/g和1.53mg/g）均显著低于溶剂萃取粕（SEM）（27.28mg/g和38.78mg/g）。但不同脱脂处理并不会对麻疯树籽粕中的佛波酯、植酸、皂苷有良好的脱除效果，仍需进一步采取其他脱毒方式。由于挤压膨化粕中低的残油率和毒性物质含量，因此挤压膨化预处理联合溶剂浸提法为麻疯树籽粕最适脱脂方法。
（2）以在最适脱脂方法下得到的麻疯树籽粕为研究对象，采用5种不同处理（酶处理：纤维素酶和果胶酶；酶处理+60%甲醇或65%乙醇处理；90%甲醇或90%乙醇处理）对其进行脱毒。结果表明：不同的脱毒方法对麻疯树籽粕中的毒素及抗营养因子含量有显著影响（p<0.05）。其中，经过酶解联合65%乙醇洗脱处理后，粕中佛波酯被全部脱除，胰蛋白酶抑制剂、植物凝集素、植酸和皂苷的含量均显著下降（p<0.05）。且该法得到的脱毒粕中蛋白质含量最高（74.86%），蛋白质体外消化率高达92.37%。此外，该粕中氨基酸总量为66.87克/100克蛋白，必需氨基酸齐全，配比合理，营养指标（EAAI、BV、NI和PDCAAS）均高于其他脱毒粕。可见，酶解处理联合65%乙醇洗脱的方法为麻疯树籽粕最适脱毒方法。
（3）利用抗菌肽与菌体细胞膜间存在着特异性亲和力，建立了一步细胞膜色谱技术及改进的两步细胞膜色谱技术（细胞膜固相亲和萃取联合HPLC指纹图谱技术），成功地从粕及其水解物中制备2种麻疯树源抗菌肽（JCpep7和JCpep8）。通过质谱技术（MALDI-TOF-MS/MS）鉴定其氨基酸残基序列分别为Lys-Val-Phe-leu-Gly-leu-Lys和Cys-Ala-Ile-Leu-Thr-His-Lys-Arg。抗菌实验表明利用细胞膜亲和色谱技术分离麻疯树源抗菌肽是可行的。所得2种抗菌肽对大肠杆菌、铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、枯草芽孢杆菌与肺炎链球菌均有抗菌活性，最适pH为中性，均对热稳定，不耐受高离子强度，对胃蛋白酶、胰蛋白酶和木瓜蛋白酶具有一定的抵抗力，但不能抵抗蛋白酶K的酶解。在低浓度下均对兔红细胞无溶血和凝集作用。
（4）首次采用毛细管电泳技术，将活体金黄色葡萄球菌作为“假固定相”，依据毛细管电动色谱原理，用相对淌度代替淌度，描述活性菌体对抗菌肽（JCpep7和JCpep8）的迁移行为的影响，建立了一种新的毛细管电泳法测定抗菌肽与活性菌体结合常数的方法。结果表明：抗菌肽JCpep7和JCpep8与活性金黄色葡萄球菌（LSACs）之间的结合过程是自发进行的，而且结合效果均很显著。随着温度的增加，2种抗菌肽与LSACs的结合常数也随着增大。其中JCpep7以疏水相互作用力为主要作用，而JCpep8则以静电吸引力和疏水相互作用为主要作用。
（5）利用毛细管电动色谱相对淌度法（初始结合常数实验）及对现有研究抗菌肽抗菌机理的手段（生长曲线抑制实验、内外膜破坏实验及菌体微观形态学变化）进行优化组合及设计，实现了在活菌体环境下探索抗菌肽（JCpep7和JCpep8）的抗菌机理。结果表明：抗菌肽JCpep7和JCpep8能快速结合到金黄色葡萄球菌细胞壁上，并对细胞壁造成损伤，进而扩散到细胞膜。当抗菌肽浓度在细胞膜上超过一定的阈值，就会破坏细胞膜的完整性，使得胞内物质泄漏，导致菌体死亡。可见，细胞壁膜是抗菌肽（JCpep7和JCpep8）发挥抗菌作用的主要靶点。其中JCpep7是以“毯式”模式的膜损伤抗菌特征，而JCpep8是以“桶状”模式的膜损伤抗菌特征。
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.
引文
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