The Research On The Preparation And Bioactivities Of Peptide Derived From Camellia Oleifera Abel Seed Meal

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.