【作者】 王琦；

【导师】 薛长湖；

【作者基本信息】 中国海洋大学 ， 食品科学， 2008， 博士

【摘要】 目前我国水产品产量已占世界总产量的三分之一左右,位居世界第一位,但加工和综合利用方面与世界水平相比差距还十分明显。近年来,我国水产加工业的产业结构正逐步发生变化,由过去的初步加工、粗加工向精深加工方向发展,且追求多样化、系列化和高附加值的产品。太平洋牡蛎(Crassostrea gigas)产量每年居于我国养殖贝类前列,却尚无好的精深加工利用方式;加工后得到的虾夷扇贝(Patinopecten yessoensis)生殖腺、鸢乌贼( Sthenoteuthis oualaniensis )卵和阿拉斯加狭鳕( Theragra chatcogramma)鱼卵目前也得不到充分的综合利用。如何充分挖掘这类海产动物原料的价值,提高其综合利用率,得到高附加值,高科技含量的产品,一直是亟待解决的问题。海产动物中富含EPA(二十碳五烯酸)、DHA(二十二碳六烯酸)等n-3系列不饱和脂肪酸(n-3 PUFA),贝类和鱼卵又是含丰富磷脂的原料,因而从上述海产动物原料中开发n-3 PUFA磷脂是一个很值得研究的方向。首先本文以太平洋牡蛎、虾夷扇贝生殖腺、鸢乌贼卵和阿拉斯加狭鳕鱼卵等海产动物原料为研究对象,提取其中的总脂肪,分析其中的脂质组成和总胆固醇含量,检测各脂质中的脂肪酸组成,并建立了对于海产动物来源磷脂的灵敏、精确的HPLC-ELSD检测分析方法。发现鸢乌贼卵、鳕鱼卵磷脂中含大量PC,论证了以两者为原料,大量获取以DHA-PC、EPA-PC为主要功能成分的DHA/EPA磷脂的提取工艺的可行性。其次,采用鸢卵粉为原料,用超临界CO2萃取工艺对其中胆固醇进行脱除后用乙醇浸提,并将工艺进行了优化和放大实验,最后得到的提取工艺提取效果好,得到的磷脂产物产量高,纯度好,胆固醇含量低,而且整个工艺过程无任何有毒有害的溶剂残留或污染,绿色环保,有着较高的生产应用价值。对得到的DHA磷脂产物分析,发现其磷脂含量达85%以上,PC含量达60%以上,脂肪酸组成中DHA含量达30%以上。再次,对DHA磷脂产物中的PC的脂肪酸组成情况进行了分析,发现DHA含量达40%,证明其中富含DHA-PC;利用磷脂酶A1对DHA-PC进行酶法降解得到2-DHA-LPC产物,其脂肪酸组成中DHA含量达50%,证明DHA-PC中的DHA以结合在Sn-2上为主,即以2-DHA-PC为主。最后,将DHA磷脂产物制成脂质体,采用灌胃荷S180肉瘤小鼠的方法研究了其抗肿瘤活性及对免疫功能的调节作用。实验结果表明DHA磷脂脂质体具有抑制肿瘤生长的作用,而且能全面调节机体的特异性和非特异性免疫功能。推测其通过增强T细胞、B细胞和巨噬细胞等的功能以抑制肿瘤细胞的生长。分析认为DHA磷脂脂质体提高机体的免疫能力和其抗肿瘤作用主要是通过PC和DHA两种活性物质来实现的,并且克服了DHA单独存在时易被氧化的缺点,制备成脂质体的形式显现更好的生理活性。2-DHA-PC为主要功能成分的DHA磷脂具有广阔的医药和保健品的开发前景。更多还原

【Abstract】 It is well known that marine animals have large amounts of n-3 polyunsaturated fatty acids （PUFA） such as eicosapentaenoic acid （EPA） and docosahexaenoic acid （DHA）. It is also known that shellfish and fish roe contain large proportions of phospholipids in total lipids. In recent years, phospholipids rich in n-3 PUFA （n-3 PUFA-PL） are found to have many novel functions other than the functionalities of PUFA.Oyster Crassostrea gigas and scallop Patinopecten yessoensis, as two kinds of the most important economic bivalves in China, now are widely cultured. The products of oyster and scallop are usually very simple and have low value. Squid Sthenoteuthis oualaniensis is thought to be the most abundant large squid in the tropical and subtropical waters of the Indo-Pacific region and Alaska pollock （ Theragra chatcogramma）is often processed in factories of Qingdao to get the meat product. However during the processing, the gonads from scallop, eggs from squid and roe from Pollock are usually picked out and have no good ways for further use.In this paper meat from oyster Crassostrea gigas, gonads from scallop Patinopecten yessoensis, eggs from squid Sthenoteuthis oualaniensis and roe from Alaska pollock were detected as materials to see if they are good sources of n-3 PUFA-PL.First, total lipids were extracted from the materials of marine animals and separated into neutral lipids, glycolipids and phospholipid. Fatty acid compositions of the lipids were assessed and compared. A high-performance liquid chromatograph equipped with an evaporative light scattering detector （ELSD） （HPLC/ELSD） was also used to assay the phospholipid compositions of the materials. The analysis of the phospholipids by HPLC/ELSD was shown to be an accurate and reproducible analytical method for the determination of cardiolipin （CL）, phosphatidylethanolamine （PE）, phosphatidylinositol （PI）, phosphatidylserine （PS）, phosphatidylcholine （PC）, sphingomyelin （SM） and lysophosphatidylcholine （LPC）. It was found that PC was the most predominant PL in pollack roe and squid egg. Both PC and PE were the main PL in scallop ovary and spermary and oyster meat. It could be seen that the egg/gonad from marine fish and shellfish were rich in PL of different types. As a result, all the materials have large amounts of EPA and DHA in phospholipids and especially the pollock roe and squid eggs contained large amounts of n-3 PUFA-PC.Secondly, squid eggs were processed into powders. The squid-egg powder was extracted first by supercritical carbon dioxide （SC-CO₂） to remove the cholesterol and then ethanol to get the phospholipids product. The technology of using both SC-CO₂ and ethanol for extraction was proved to be practical and effective and had no pollution of poisonous organic solvent. Analysis result showed that the phospholipids product （more than 85% of phospholipids） contained over 60% of PC in weight and over 30 mol% of DHA in fatty acids.Next, the PC of DHA-PL product was analyzed to get the fatty acid composition. More than 40 mol% of DHA was found in PC. Phospholipase A1 was used to act with the PC of DHA-PL product and to hydrolyze the fatty acids at Sn-1 position and get the Sn2- acetylated LPC. The fatty acids composition of the Sn2- acetylated LPC was determined and over 50 mol% of DHA was found.To investigate the effects of the DHA-PC liposomes on antitumor and immune regulation. DHA-PL product was made as unilayer liposomes. Different dosage of DHA-PL liposomes were given to transplanted tumor-bearing mice respectively. The tumor weight、spleen/ B.W、Thymus/B.W、hemolysin content、DTH and the phagocytosis ability of peritoneal macrophage were measured respectively. Results showed that each dosage of DHA-PL liposomes could inhibit the growth of tumor in S180 bearing mice and the average of tumor-inhibitory rate was 36.87%. Furthermore they could enhance spleen/B.W （P<0.05）; increase hemolysin content obviouslly （P<0.05,P<0.01）; heighten delayed allergic level （P<0.05,P<0.01）; promote the phagocytosis ability of peritoneal macrophage （P<0.01）. Then it was concluded that DHA-PL liposomes can regulate both specific and non-specific immune function, inhibit the growth of tumor cells.更多还原