【作者】 庄永亮；

【导师】 李八方；

【作者基本信息】 中国海洋大学 ， 食品科学， 2009， 博士

【摘要】 海蜇是一种大型的食用水母,在我国有着广泛的资源分布,具有很高的经济价值、营养价值和药用价值。本论文以黄海海域的新鲜海蜇为原料,采用生化分离技术提取纯化未变性胶原蛋白,研究其理化性质;采用酶工程技术,开发海蜇伞部胶原蛋白梯级肽制品,并以多种体外活性体系确定胶原肽的抗氧化能力。研究不同分子量胶原肽对小鼠B16黑色素瘤细胞黑素合成的影响及作用机制,以及胶原蛋白和胶原肽对紫外线诱导的皮肤光老化模型小鼠的保护作用。本研究对于进一步丰富胶原蛋白的应用领域,开发海蜇新的食(药)用功效,推进海蜇产业链的发展提供科学依据。1.对海蜇化学成分组成测定发现,鲜活海蜇水分达96%以上,干物质含量低于5%。海蜇伞部中粗蛋白、总糖、脂肪、灰分及羟脯氨酸含量分别占干基的82.5%,1.6%、0.3%、15.3%和3.4%;口腕部分别为79.1%,1.8%, 0.4%、17.9%和1.1%。海蜇蛋白质主要由肌原纤维蛋白和碱不溶性蛋白质组成。2.以海蜇伞部为原料,通过采用不同提取方法提取胶原蛋白,并对所提取的胶原蛋白的部分理化性质进行测定。以胶原蛋白的提取率为指标,生物酶法的提取率最高,溶剂预处理化学法和高压辅助物理法提取率相差不大。酶法制备的未变性胶原蛋白鉴定为Ⅰ型胶原。氨基酸分析发现,胶原蛋白中Gly含量最高,约占氨基酸总量的1/3;Ala、Pro、Glu和Asp含量较高,而His、Tyr和Met含量较低,不含有Cys和Trp; SDS-PAGE显示胶原蛋白可能为(α1)3型,且α链类似于脊椎动物I型胶原α1链,分子量为115KDa;胶原蛋白含糖量为2.8%,符合无脊椎动物胶原蛋白特点;热变性温度和热收缩温度分别为28.8℃和51.6℃,均低于牛皮I型胶原;红外光谱说明胶原蛋白保留了大量的三股螺旋结构;溶解性表明海蜇胶原蛋白的等电点为pI6而在pH3时有最大溶解度,酸性条件下盐浓度高于4%时,胶原蛋白的溶解度急剧下降。3.以羟自由基抑制率和水解度为指标,确定海蜇胶原蛋白的水解用酶,并采用响应面方法分析最适酶解条件。结果显示,胰蛋白酶水解条件为pH7.8,水解温度为48.8℃、酶与底物比为3.5%,此条件下,水解产物的羟自由基抑制率为71.87%,水解度为14.69%;碱性蛋白酶properase E水解条件为pH9.1,水解温度为47.3℃、酶与底物比为2.8%,此条件下,水解产物的羟自由基抑制率为79.07%,水解度为17.89%。复合酶水解显著提高水解液清除羟自由基的能力和水解度,复合酶的最佳水解条件为碱性蛋白酶和胰蛋白酶混合水解,温度48.0℃,酶与底物的比值分别为2.8%和3.5%,pH9.1,水解时间3h,此条件下,水解产物的羟自由基抑制率为94.24%,水解度为24.32%。从三种水解产物的分子量大小和氨基酸组成来看,单酶水解的平均分子量主要集中在2000Da左右,复合酶水解的平均分子量在700Da左右;三种水解产物氨基酸组成没有明显不同,富含Gly和Pro,总疏水性氨基酸含量在35%左右。4.以海蜇胶原蛋白复合酶水解液为原料,超滤获取三个不同分子量组分JCH1:Mw>3000Da;JCH2:1000Da<Mw<3000Da;JCH3:Mw<1000Da。应用多指标、多体系评价了不同分子量组分的抗氧化特性。结果显示,JCH3具有强的还原力和脂质氧化抑制能力,并对超氧阴离子具有较高的清除能力,三种体系与浓度均呈正相关性,其中对超氧阴离子的抑制率与浓度呈线性关系;JCH2对羟自由基和过氧化氢活性氧清除效果明显,并体现出较好的铜螯合能力,量效关系明显。水解液中氨基酸的组成和分子量的大小直接影响其抗氧化能力的大小。5.通过体外培养小鼠B16黑素瘤细胞株,探讨了不同分子量胶原肽组分(JCH1,JCH2,JCH3)对细胞内黑色素合成的影响及作用机制。结果显示,三种胶原肽对细胞生长无任何影响(P>0.05),体现了胶原肽无细胞毒性。胶原肽能够有效的降低B16黑素瘤细胞黑素含量(P<0.05,P<0.01),剂量效应关系明显,其中JCH2抑制能力高于JCH1和JCH3,浓度在100μg/ml时,黑色素含量降低了38.8%。从作用机制来看,相对于其它两组分,JCH2表现出较高的抑制酪氨酸酶活性(P<0.01),浓度为50μg/ml时,抑制率为40.5%;同时有效的增加细胞内GSH含量,降低GSSG含量,继而提高两者的比例(P<0.05,P<0.01),保证细胞内的还原能力;另外,JCH2能够显著降低细胞内cAMP含量(P<0.01),从而抑制酪氨酸酶的表达。6.通过紫外线诱发小鼠皮肤光老化模型,研究胶原蛋白和胶原肽对紫外线损伤的调节作用。结果显示,两者能有效地缓解紫外线照射引起的光老化程度,胶原肽表现的生理活性高于胶原蛋白。胶原蛋白和胶原肽显著提高光老化模型的胸腺指数和脾指数,防止免疫器官的萎缩,延缓衰老。胶原蛋白和胶原肽能够提高小鼠皮肤及血清中SOD、GSH-Px、CAT等抗氧化酶活性及GSH的含量,同时降低MDA含量(P<0.05, P<0.01),从而缓解紫外线照射引起的体内氧化压力。胶原蛋白和胶原肽能有效调节皮肤水分、胶原蛋白、脂质、神经酰胺及糖胺聚糖的异常变化。皮肤组织的形态学观察结果进一步表明,胶原蛋白和胶原肽均能有效地改善光老化小鼠皮肤胶原纤维的受损程度,保持I/III型胶原蛋白比例,改善弹性纤维排列与分布,促使皮肤富有弹性。更多还原

【Abstract】 Jellyfish (Rhopilema esculentum) is one of the most important fishery resource in China, which has the high economic, nutritional and medicinal value. In this paper, collagen without telopeptides was prepared from the umbrella of jellyfish Rhopilema esculentum and the phsicochemical characteristics were studied. The appropriate protease and condition of hydrolysis was selected to produce collagen polypeptides. The antioxidant mechanism of polypeptides was investigated by different tests in vitro. Utilizing cultured B16 melanoma cells, inhibition effects of collagen polypeptides with different molecular weight on melanin biosynthesis and their molecular mechanism were researched from the aspects of antioxidation. Further more, protective effects of collagen and collagen polypeptides on mice skin photoaging induced by UV irradiation were discussed. This paper aimed to broaden the research field of collagen, produce high-valued functional food and medicine from jellyfish and provide a valuable scientific basis for the industrialization development of jellyfish. The main results are as follows:1. The main compositions of jellyfish were determined. Moisture content of fresh jellyfish was above 96%. Contents of crude protein, mucopolysaccharides, fat and ash in dry samples of jellyfish umbrella were 82.5%,1.6%, 0.3% and 15.3%, respectively and contents in jellyfish oral-arm were 79.1%, 1.8%, 0.4% and 17.9%, respectively. Proteins of jellyfish were mainly composed of myofibrillar proteins and alkali-insoluble proteins.2. The studies of the extractions and the biochemical characterizations of collagen from jellyfish umbrella were undertaken. The rates of enzymic extraction were highest among the extraction methods and the rates of extractions by solvent pretreatment and pressure pretreatment were similar. Pepsin-soluble collagen was extracted from jellyfish umbrella and classified as type I collagen. The compositional features of the amino acids were with the highest contents of Gly (300 residues/1000) but with low contents of His, Tyr and Met. Combined with the results of SDS-PAGE, collagen was (α1)3 trimers whileαchain (about 115KDa) resembeledα1 chain of type I collagen of vertebrate. The content of total sugar was 2.8%. The denaturation temperature (Td) and shrinkage temperature (Ts) were 28.8℃and 51.6℃respectively, both lower than those of bovine skin collagen. FTIR investigations showed the existence of helical arrangements of collagen. Furthermore, collagen had the highest solubility at pH3 and the pI was at about 6. No significant changes in solubility were observed in the present of NaCl up to 4% (w/v) and a sharp decrease was found with NaCl above 4% (w/v).3. Jellyfish collagen was hydrolyzed by different protease treatments to obtain antioxidative polypeptides. The collagen hydrolysates by trypsin and properase E exhibited the high drgree of hydrolysis (DH) and hydroxyl radical scavenging activities. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions (including enzyme to substrate ratio, pH and temperature) to prepare protein hydrolysates from jellyfish umbrella collagen. Trypsin was selected to hydrolyze collagen at pH 7.8, 48.8℃, enzyme-to-substrate ratio ([E]/[S]) 3.5%; Properase E was pH9.1, 47.3℃, [E]/[S] 2.8%. The R2 value of radical scavenging activity and DH indicated that the designed models could correctly explain the data variation and significantly represent the actual relationships between the reaction parameters. Double enzymes hydrolysis could improve DH and hydroxyl radical scavenging activities. The optimum parameters of the bienzyme hydrolysis were : pH 9.1, 48.0℃, [E]/[S] 3.5% and 2.8% respectively. Molecular weight (MW) of single enzymic hydrolysis was concentrated to 2,000Da, and that of bienzyme hydrolysis was 700Da. Amino acids compositins of three hydrolysis conditions were similar. They were rich in Gly and Pro and the total hydrophobic amino acids (THAA) contents were above 35%.4. The bienzymic hydrolysates were fractionated into three ranges of molecular weight (JCH1>3,000Da, 1,000Da<JCH2<3,000Da, JCH3<1,000Da) using an ultrafiltration membrane bioreactor system. The antioxidant activities of JCH1, JCH2, and JCH3 were investigated using different in vitro tests. JCH3 showed the highest reducing power, antioxidant activity in linoleic acid emulsion system and superoxide anion radical scavenging activity. JCH2 had the strongest hydroxyl radical and hydrogen peroxide scavenging activities and metal chelating ability. The antioxidant activities of three collagen polypeptides increased with increasing concentrations used and the correlations of superoxide anion radical scavenging activities were linear. The results suggested that the antioxidant activities of jellyfish collagen hydrolysates were related to their molecular weights and amino acids contents.5. Utilizing cultured B16 melanoma cells, inhibition effects of collagen polypeptides (JCH1, JCH2, JCH3) on melanin biosynthesis and their molecular mechanism were researched from the aspects of antioxidation. Effects of collagen polypeptides on cell growth were no significant (P>0.05) which indicated collagen polypeptides had no cytotoxic activity. Collagen polypeptides exerted obvious inhibition on productions of melanin (P<0.05, P<0.01) in concentration dependent manners. Inhibitory activity of JCH2 was higher than JCH1 and JCH2. Afer incubation with JCH2 (100μg/ml) for 48h, melann content was decreased by 38.8%. Compared the other factions, JCH2 showed the highest tyrosinase inhibition (40.5% of control at dose of 50μg/ml). Intracellular GSH levels in B16 melanoma cells were enhanced by JCH2, while GSSG levels were suppressed. Consequently, the GSH/GSSG ratio was increased. It could ensure intracellular reduce capacity. Fourthermore, JCH2 could decrease intracellular levels of cAMP significantly (P<0.01), and then supress the expression of tyrosinase mRNA. The results showed JCH2 was a novel anti-melanogenic peptide from natural resources and exerted actions via its antioxidative properties and copper-chelating ability6. Effects of collagen and collagen polypeptides on chronic ultraviolet radiation-induced skin photoaging in hairless mice were studied in this chapter. Collagen and collagen polypeptides could protect immune system by increasing the indexs of thymus and the indexes of spleen. Collagen and collagen polypeptides coulde increase the activities of SOD, CAT, and GSH-PX (P<0.05) and the content of GSH (P<0.01) while decrease the content of MDA (P<0.05), which alleviated the oxidant press UV-induced. Meanwhile, collagen and collagen polypeptides could recover the abnormal changes of skin collagen, lipid, total ceramide and glycosaminolycan. Fourthremore, histological studies showed collagen and collagen polypeptides could improve the breakdown of dermal interstitial collagen and cross-linking of inter- and intra-collagen molecules, maintain the ratio of type I/III collagen, and ameliorate the order and distribution of elastic fiber. The results showed collagen and collagen polypeptides could relieve the damages of UV treatment, and activities of collagen polypeptides were higher than collagen. Therefore, collagen polypeptides might have a good anti-photoaging action.更多还原