【作者】 龚吉军；

【导师】 李忠海； 钟海雁；

【作者基本信息】 中南林业科技大学 ， 林产化学加工工程， 2011， 博士

【摘要】 油茶是我国目前主要的油料植物之一,每年生产油茶籽约100万t、榨油后的副产物——油茶粕约68万t,目前,大多数的油茶粕只是作为有机肥料来使用,造成优质资源的严重浪费。在油茶粕中含有10%-20%的优质蛋白,是一种潜在的植物蛋白资源,而以油茶粕蛋白为原料进行生物活性肽的研究目前尚未见报道。本文对油茶粕蛋白的提取、油茶粕多肽的制备及其生物活性进行了深入的研究,成功制备出具有良好抗氧化、降血压、增强机体免疫力、降血脂和抗菌活性的油茶粕多肽。本研究为拓宽油茶粕的综合利用开辟了新的空间,可为油茶粕多肽的工业化生产提供理论基础与试验依据。在探明油茶粕蛋白组成与等电点(pI=4.6)的基础上,采用碱溶酸沉法,研究了油茶粕蛋白的提取工艺,重点对油茶粕蛋白的碱提工艺进行了响应曲面法优化,最佳的碱提工艺为：料液比1:25、浸提液pH值10.4、浸提时间2.42 h、浸提温度49.2℃,在此条件下,蛋白提取率为65.83±2.45%(n＝4),与模型预测值64.97%无显著差异(P＞0.05)。油茶粕蛋白水解产物的体外抗氧化活性与酶的种类和水解度密切相关。当酶与底物比和水解时间分别为1.50%与6.00 h时,经碱性蛋白酶(Alcalase)水解后所得多肽对O2-·的清除能力、还原能力和抑制亚油酸过氧化的活性均最强,分别为59.93%、0.621和53.19%。当酶与底物比和水解时间分别为1.50%与9.00 h时,经木瓜蛋白酶(Papain)水解后得到的多肽具有最强的清除DPPH的能力(46.89%)。以四氯化碳致毒小白鼠为试验对象,剂量分别为250、500和1000 mg/kg体重,对油茶粕多肽(MW<3 kDa)的体内抗氧化活性进行了研究。结果表明,中、高剂量的油茶粕多肽能使中毒小鼠肝脏的超氧化物歧化酶(SOD)与谷胱甘肽过氧化物酶(GSH-Px)活力显著提高(P<0.05),能使丙二醛(MDA)含量显著降低(P<0.05),说明油茶粕多肽可以显著减轻活性氧对机体的损害,增强机体的抗氧化能力。对制备油茶粕ACE抑制肽的最适酶及其最佳的水解条件分别进行了筛选与优化,在此基础上,将所得多肽(MW<3 kDa)用于自发性高血压大鼠(SHRs)的ACE抑制活性与降压效果研究。蛋白酶的筛选试验中,Alcalase水解所得多肽的IC50是最低的0.96±0.11mg/mL/借助响应曲面法,探明了最佳的水解条件,即pH值8.1、酶与底物比1.57%、水解温度50.5℃、水解时间6.66 h,同时获得了用于ACEI预测的二元回归数学模型。在体内试验中,SHRs在一次灌喂与长期服用100和500 mg/kg·bw的油茶粕多肽后,收缩压显著降低(P<0.05),长期服用油茶粕多肽SHRs的动脉ACE活性受到显著抑制。试验结果证明油茶粕多肽对SHRs具有良好的降压作用。通过建立SD大鼠高血脂模型,采用低、中、高三种剂量(250,500和1000 mg/kg·bw),研究了油茶粕多肽(Alcalase水解)的降血脂效果。结果表明,低、中、高剂量的油茶粕多肽均可显著降低高血脂模型SD大鼠血清中总胆固醇(TC)与甘油三酯(TG)的含量、动脉硬化指数(AI)(P<0.05),还能有效提高高密度脂蛋白胆固醇(HDL-C)的水平(P<0.05)。试验结果提示油茶粕多肽具有良好的降血脂功效,可有效降低动脉粥样硬化的风险。以脾淋巴细胞增殖作用、免疫脏器指数、血清半数溶血值(HC50)、小鼠巨噬细胞的吞噬指数与吞噬率为指标,研究了油茶粕多肽(Alcalase水解)对小鼠的免疫调节功能。试验结果表明,低、中、高剂量(250、500、1000 mg/kg-bw)的油茶粕多肽均能对抗环磷酰胺所致小鼠免疫降低的作用,说明不同剂量的油茶粕多肽均能显著提高免疫低下型小鼠的非特异性和特异性细胞免疫功能(P<0.05)。迟发型超敏反应(DTH)试验结果表明,低、中、高剂量的油茶粕多肽均能显著提高免疫正常小鼠的特异性细胞免疫功能(P＜0.05)。以Pepsin为水解酶,用E. coli为指标菌,通过响应曲面法,对油茶粕蛋白水解制备抗菌肽(MW<10 kDa)的工艺进行了优化,最佳的水解条件为：酶添加量1.70%,pH值1.9,反应温度36.8℃,反应时间60.10 min。在此条件下,对大肠杆菌的抑制率为66.05±0.22%(n=4),与预测值67.88%无显著差异(p>0.05)。对最优条件下所得多肽的抗菌活性进行了研究,结果表明,油茶粕多肽对所测试的细菌、酵母和霉菌均有一定的抑制效果,但差异明显,对细菌的抑菌作用较强,而对酵母菌和霉菌的抑制效果相对较弱。为了探明油茶粕多肽(Alcalase水解)的食用安全性,通过急性毒性试验、遗传试验和亚慢性毒性试验,对其进行了安全性评价。采用一次最大限量法(摄入量为20.00 g/kg·bw)进行急性毒性试验,无小鼠死亡；在遗传试验中,Ames试验、骨髓细胞微核试验和小鼠精子畸形试验结果均为阴性,表明油茶粕多肽对哺乳类动物体细胞染色体及生殖细胞均无损伤作用；在亚慢性毒性试验中,油茶粕多肽对大鼠体重、摄食量、血液学指标、血液生化指标和脏器系数均无明显影响。通过试验结果可以初步判定油茶粕多肽为无毒物质。更多还原

【Abstract】 Camellia oleifera Abel is one of the main oil plants in China. Approximately 100 million tones of oil-tea seed and 68 million tones of oil-tea seed meal (OTSM), a by-product of the production of tea-seed oil, are produced annually in China. At present, however, most of the OTSM is utilized as organic fertilizer. Thus the valuable resources was not under effective utilization. Even though it is the only oil-tea seed residue, OTSM contains about 10%-20% protein (dry basis) which is a potential resource of plant protein. To our knowledge, no data are currently available concerning bioactive peptides derived from OTSM protein. In this paper, the extraction of OTSM protein and the preparation of bioactive peptide derived from the former through proteolytic digestion was studied, and bioactive OTSM peptides (OTSMP) were obtained successfully. The OTSMP obtained through different methods showed good bioactivities, such as antioxidation, antihypertension, immuno-regulation, antihyperlipemia, and antibacterium. These researches can broad the application area for OTSM, and can provide theoretical foundation and experimental basis for the industral production of OTSMP.After determination the composition and isoelectric point value (pI=4.6) of the OTSM protein, using the methods of dissolving protein under alkaline condition and then settling protein under acidic condition, the extracting technology for protein was studied with the main aspect focused on the optimization of extraction through alkaline solution using response surface methodology (RSM). The optimal conditions of alkaline extracting for protein were as followings:the ratio of material to liquid 1:25, the pH value of extracting solution 10.4, extracting time 2.42 h, extracting temperature 49.2℃. Under these optimum conditions, the extracting rate of protein was 65.83±2.45%(n=4), without significant difference with the predicted value 64.97%(P>0.05).The antioxidant activities of OTSM protein hydrolysates in vitro are highly related to the protease and the degree of hydrolysis (DH). Among all tested samples, alcalase peptides, produced with the E/S ratio and hydrolysis time kept at 1.50% and 6.00 h respectively, possessed the strongest activity (P<0.05) concerning scavenging activities against superoxide anion (59.93%), reducing capacity (0.621) and inhibitory effect on linoleic acid peroxidation (46.89%), while the papain peptides, produced with E/S ratio and hydrolysis time kept at 1.50 % and 9.00 h respectively, had the strongest (P<0.05) scavenging activity against DPPH·(53.19%). Taking rats intoxicated with carbon tetrachloride (CT) as experimental animals, the antioxidative activities of OTSMP (MW<3 kDa) in vivo were studied using three kinds of dose i.e.250,500 and 1000 mg/kg·bw. Results showed that medium and high dose of OTSMP could significantly increase the activities of SOD & GSH-Px and significantly reduce the contents of MDA in liver(P<0.05). These results suggested that OTSMP could obviously enhance the antioxidatve ability of the assayed mices and reduce reactive oxygen species (ROS) damage.The best protease and its enzymatic hydrolysis conditions in the production of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from OTSM was screened and optimized, respectively. The ACE inhibitory activities and hypotensive effects of these peptides in spontaneously hypertensive rats (SHRs) were assayed. In the screening experiment of protease, the peptides obtained by alcalase showed the lowest IC50(0.96±0.11mg/mL). With the aid of response surface methodology (RSM), the optimum hydrolysis conditions(the pH value 8.1, E/S ratio 1.57%, hydrolysis temperature 50.5℃, and hydrolysis time 6.66 h) and a statistical model for experimental validation were determined. A significant decrease (P<0.05)in systolic blood pressure (SBP) was observed in SHRs following one-shot and long-term oral administration of these peptides at a dose of 100 and 500 mg/kg-bw, respectively. A significantly inhibitory effects(P<0.05) on ACE activity in aorta of SHRs after long-term administration of this hydrolysates were also observed. According to the data showed in this work, it could be concluded that the alcalase peptides derived from OTSM did possess high ACE inhibitory activity and hypotensive effect in SHRs.Taking model of hyperlipidemia SD rats as experimental animals, lipidemia-lowering effects of OTSMP (digested by alcalase) were studied by means of three kinds of dose, i.e. 250,500 and 1000 mg/kg·bw. Results showed that low, medium and high dose of OTSMP could effectively reducethe levels of TC & TG(P<0.05), also significantly lessen the value of AI(P<0.05). All of three doses of OTSMP could obviously increase the level of HDL-C in the serum of model rats(P<0.05). These results suggested that OTSMP had highly lipidemia-lowering effects and could effectively decrease the risk of atherosclerosis.With spleen lymphocytes proliferation function, index of immune organ, calculation of 50 % hemolysin value (HC50), phagocytic index and phagocytic rate of macrophage as evaluated indices, the immunomodulatory effects of OTSMP (digested by alcalase) on mice were studied with high, medium and low dose (i.e.250,500,1000 mg/kg-bw) of them. Results showed that all of three doses of OTSMP could effectively counteract the immune-lowering-effect of cyclophosphamide on assayed rats (P<0.05). It was suggested that this OTSMP could improve immune function on non-specific and specific immune response of rats with low immune function. Results of delayed type hypersensitivity (DTH) showed that all of three doses of OTSMP could significantly improve immune function on specific immune response of rats with normal immune function (P<0.05).Taking E. coli as evaluated microbe, the hydrolysis conditions of OTSM protein for the production of antimicrobial peptides (MW<10 kDa) was optimized using pepsin through RSM. The best hydrolysis conditions were as followings:the enzyme dosage 1.70%, pH value 1.9, reactional temperature 36.8℃, and reactional time 60.10 min. Under these conditions, the inhibitory rate on E. coli was 66.05±0.22%(n=4), without significant difference with the predicted value 67.88%(P>0.05). Then other antibacterial activities of the obtained OTSMP was assayed. Results showed that OTSMP had inhibitory effects on bacteria, yeast and mould to some degree, but significant difference (P<0.05) was observed. The antibacterial activities of OTSMP was stronger, the inhibitory activities on yeast and fungus were lower.In order to check the edible safty of OTSMP, tests concerning the acute toxicity, genetic toxicity, and subchronic toxicity were undertaken according to specified procedures. Using a maximum limit method (intaking dosage of 20.00 g/kg·bw), no death of rat was observed during the period of acute toxicity tests. In genetic toxicity tests, all results of Ames, micronucleus, and sperm aberrations assay were negative. It was suggested that OTSMP (digested by alcalase) exhibited no damage effects on chromosome of body and reproductive cells of mammals. In subchronic toxicity trials, OTSMP showed no significant influence on body weight, fooder-taking amount, hematologic indices, blood biochemical indices, and viscera indices. Results obtained in this study indicated that the obtained OTSMP was nontoxic substances.更多还原