【作者】 王彩霞；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2014， 博士

【摘要】 我国是淡水鱼生产及加工大国，淡水鱼加工产生大量下脚料，若不进行有效处理，不仅造成资源浪费，而且污染环境。在中国的传统饮食文化中，吃脑可以补脑，鱼脑更被视为幼儿及青少年补脑佳品。药食同源的理念贯穿于中国的饮食文化中，除作为食材，中国民间常以鱼脑入药，医治头部病症。鱼脑营养丰富，富含多不饱和脂肪酸(PUFA)、磷脂、蛋白等物质，其中被称为“脑黄金”的二十碳五烯酸(EPA，C20:5n3)和二十二碳六烯酸(DHA，C22:6n3)含量较高。目前对鱼下脚料的利用中，鱼脑未经分离直接和鱼头被作为饲料处理或简单加工为低值产品，对其中有价值的功能性成分尚未提取和利用。本文对鲢鱼脑的营养价值进行了全面分析，并与鳙鱼，草鱼和青鱼脑比较；对鲢鱼脑脂肪的理化特性、挥发性物质及脂类组成进行了分析；对鲢鱼脑不同脂类组分在抗菌、抗血小板活化、抗氧化及抗肿瘤等方面的生理功效进行了进一步研究，探讨了鱼脑益智及保健的机理。本研究对于对阐明鱼脑的营养价值、揭示鱼脑的生理功能具有学术意义，对相关产业开发具有实用价值。首先对鲢鱼脑进行营养评价，并与鳙鱼、草鱼、青鱼对比，通过基本营养成分测定、氨基酸组成分析、氨基酸营养评价、矿物元素分析及脂肪酸组成分析等对鱼脑的营养价值进行了研究。结果表明：鱼脑脂肪含量高，鲢鱼、鳙鱼、草鱼、青鱼脑的脂肪含量分别为20.53g/100g、13.72g/100g、53.75g/100g、73.81g/100g；四种鱼脑蛋白质必需氨基酸组成合理，无限制性氨基酸；Na、Ca和P是四种鱼脑中含量最高的三种矿物质元素；脂肪酸分析表明四种淡水鱼脑PUFA含量丰富，二十碳四烯酸(ARA，C20:4n6)、EPA和DHA含量较高；四种淡水鱼脑中鲢鱼脑蛋白质(1.92g/100g)、必需氨基酸(7.96mg/g)及总氨基酸(16.64mg/g)含量最高，鲢鱼脑ARA(2.54%)、EPA(5.42%)、DHA(7.28%)、ω-3PUFA总量(20.56%)及ω-3/ω-6(2.77)远高于其余三种淡水鱼脑，草鱼脑的ω-6/ω-3为7.62，高于安全推荐限量4.0。鱼脑的营养价值主要来源于鱼脑中高含量及高质量的PUFA。在营养评价的基础上对鲢鱼脑脂肪的理化指标，挥发性物质，脂类组成及含量，尤其是磷脂的组成、含量及脂肪酸进行了分析。鲢鱼脑总脂肪(TL)具有较好的理化性质，其中碘价为124.26g/100g、过氧化值为2.42meq/kg、酸价为0.58mg/g，达到精制鱼油一级标准；鲢鱼脑TL共检出40种挥发性物质，烃类含量最高；极性脂肪(PL)和磷脂分别占鲢鱼脑TL的的22.73%和10.95%，磷脂中的磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)和磷酯酰丝氨酸(PS)之和占磷脂总量的83.9%；脂肪酸分析表明PL中PUFA含量为35.17%，高于中性脂肪(NL)，PL中DHA含量是NL的6.3倍；鲢鱼脑PC、PE和PS的PUFA含量分别为33.19%、39.34%、36.27%，PC、PE和PS中EPA及DHA的含量均较高。丰富的磷脂含量及磷脂中结合的大量PUFA对于增强记忆，防治心血管疾病及癌症的预防和治疗具有积极意义。研究了鲢鱼脑TL、NL、PL抑菌，抗血小板活化因子(PAF)及抗氧化活性。结果表明：鲢鱼脑TL、NL及PL具有一定的抑菌能力，PL由于PUFA含量高，抑菌活性大于NL；鲢鱼脑TL、NL及PL对PAF诱导的血小板聚集表现出抑制作用，IC50分别为104.13μg、2.35μg、87.47μg，因此NL抗PAF活性的效价最高，这可能是因为NL中PAF拮抗剂的含量更高；氧化损伤是心脑血管疾病及癌症的重要诱因之一，在癌症的发生和发展中起重要作用。体外抗氧化研究发现鲢鱼脑脂肪可有效清除DPPH自由基，细胞抗氧化表明鲢鱼脑脂肪对H2O2氧化损伤的HEK-293细胞具有一定的保护作用，可提高H2O2氧化损伤HEK-293细胞的存活率，改善受损细胞的形态，提高抗氧化关键酶超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶的活性，PL富含磷脂，PUFA含量高，因此抗氧化活性高于NL。细菌除引起食品的腐败外，许多细菌活动也会产生PAF，而心脑血管疾病的发生与血小板的活化密切相关，此外，氧化损伤也是心脑血管疾病的重要诱因。鲢鱼脑脂肪抗菌、抗PAF及抗氧化的研究和发现对于揭示鱼脑的保健机理具有重要意义，表明鲢鱼脑脂肪具有预防或作为辅助因子用于治疗心脑血管疾病的潜能。研究了鲢鱼脑TL、NL及PL的体外抗肿瘤活性。结果表明：鲢鱼脑TL可抑制MCF-7，HeLa，CACO-2，HepG2细胞的增殖；TL、NL及PL对MCF-7细胞和HeLa细胞增殖的抑制活性表现为一定的时间效应和剂量效应；鲢鱼脑TL、NL及PL作用于MCF-7细胞和HeLa细胞后细胞形态发生明显变化，细胞数减少，细胞贴壁异常，染色质聚集，细胞表现为浓密致染；细胞周期分析发现，鲢鱼脑TL、NL及PL作用后，MCF-7细胞和HeLa细胞被阻滞在S期，其中处理48h后HeLa细胞出现较大凋亡峰，TL、NL及PL的Sub-G1比例分别为5.62%、3.06%、9.98%，因此鲢鱼脑脂肪通过阻滞S期及诱导细胞的凋亡而抑制MCF-7细胞和HeLa细胞的增殖；细胞凋亡率检测证实了鲢鱼脑脂肪对MCF-7细胞及HeLa细胞的作用方式是诱导凋亡而非坏死；对鲢鱼脑脂肪诱导肿瘤细胞凋亡机制的研究发现，鲢鱼脑脂肪作用后，MCF-7细胞和HeLa细胞细胞内活性氧(ROS)升高，TL、NL及PL处理MCF-7细胞24h后，ROS较对照分别上升了23.12%、17.69%、32.76%；JC-1染色，流式细胞仪对线粒体膜电位(Ψ)的分析指出鲢鱼脑TL、NL及PL处理会导致MCF-7细胞和HeLa细胞Ψ的降低，因此引起MCF-7细胞和HeLa细胞ROS的升高及的Ψ降低是鲢鱼脑脂肪诱导细胞凋亡的重要原因；PL富含磷脂，PUFA含量高，因此在鲢鱼脑三个脂肪组分中表现出最强的HeLa细胞和MCF-7细胞抑制活性。更多还原

【Abstract】 China is rich in freshwater fishes and they are abundantly processed into diverse products.However, with the rapid increase of processing, large amounts of by-products are producedevery year. If the by-prducts are not exploited scientifically and rationally, it not only wastesthe resources, but also pollutes the environment. In China, fish brain is considered to benutritious and known to contribute to health and intelligence. Besides, fish brain has beenused in traditional Chinese medicine for the treatment of head diseases. Fish brain is rich inpolyunsaturated fatty acid (PUFA), phospholipids, protein et al. Eicosapentaenoic acid (EPA,C20:5n3) and docosahexaenoic acid (DHA, C22:6n3) which have been proved nutritious aswell as beneficial to physical and intelligent development are abundantly contained in fishbrain. However, brain, with other by-products is discarded or or used as material for feed.Lots of functional substances are not extracted. In the study, the nutritional value of brainfrom silver carp was evaluated and compared with big head carp, grass carp and black carp.The physicochemical properties, volatile compounds and lipid classes of silver carp brainlipids were determined. Furthermore, anti-platelet-activating factor (PAF), antibacterial,antioxidant and anticancer activities of lipids extracted from silver carp brain were studied.The mechanisms of fish brain in contributing to health and intelligence were discussed. Thisstudy was of theoretical significance and of great practical significance in exploiting fishbrain as functional products.Proximate composition, amino acid, essential amino acid score, mineral elements and fattyacid (FA) profiles of brain from silver carp were investigated and compared with big headcarp, grass carp and black carp. The results indicated that brains from the four fish specieswere rich in lipids. The lipids content of silver carp, bighead carp, grass carp and black carpwere20.53g/100g,13.72g/100g,53.75g/100g and73.81g/100g, respectively. No limitingamino acid existed in brains. Na, Ca, and Mg were the predominant minerals in brains of thefour fish species. FA analysis revealed that brains from the four freshwater fish speciescontained abundant PUFA, especially arachidonic acid (ARA, C20:4n6), EPA and DHA.Among the four fish species, silver carp brain had the highest protein content (1.92g/100g),essential amino acid (7.96mg/g), total amino acid (16.64mg/g), ARA (2.54%), EPA (5.42%)and DHA (7.28%) contents, total ω-3PUFA (20.56%) and ω-3/ω-6ratios (2.77). However,ω-6/ω-3ratio in brain of grass carp (7.62) was higher than safe range (4.0).The advantages of fish brain stem from the high content and quality of lipids. Thus, on thebasis of nutritional value evaluation, the physicochemical properties, volatile vompounds,lipid classes, especially phospholipids of silver carp brain lipids were analyzed. The lipidsextracted from brain of silver carp exhibited good physicochemical properties, of which theiodine value (124.26g/100g), peroxide value (2.42meq/kg) and acid value (0.58mg/g) reached to the first-grade criterion of refined fish oil. Forty volatile compounds wereidentified, of which hydrocarbons predominated. The ratios of polar lipid (PL) andphospholipids to total lipids were22.73%and10.95%respectively. Phosphatidylcholine (PC),phosphatidylethanolamine (PE) and phosphatidylserine (PS) accounted for83.9%of thephospholipids. The PUFA level in PL (35.17%) is higher than that in neutral lipid (NL), ofwhich the level of DHA in PL was6.3times of that in NL. The PUFA levels of PC, PE and PSwere33.19%,39.34%and36.27%, respectively. PC, PE and PS were rich in EPA and DHA.The abundant phospholipids and PUFA content may assist to explain the intelligencebeneficial activities as well as pharmacological activities of fish lipids against variousdiseases including cardiovascular diseases, liver disease, cancer and so onThe capabilities of lipids (TL, NL, PL) from silver carp brain in antibacterial, anti-PAFand antioxidant activities were studied. TL, NL and PL from silver carp brain exhibitedantibacterial activity. Because of higher PUFA content in PL, it showed stronger antibacterialactivity than NL. The silver carp brain lipids exhibited strong anti-PAF activity. The IC50value of TL, NL and PL was104.13μg,2.35μg and87.47μg, respectively. Thus, theanti-PAF activity of TL was mainly attributed to NL because NL may contain more PAFantagonists. The scavenging effects for1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assayindicated TL, NL and PL possessed strong antiradical activities. HEK-293cells were used asan oxidative stress model to investigate the protective effects of silver carp brain lipids againstH2O2-induced cytotoxicity and apoptosis. TL, NL and PL showed protective effects onH2O2-induced HEK-293cells, including increasing cell viability, improving injured cellmorphology as well as increasing the activities of superoxide dismutase, Glutathioneperoxidase and catalase which are regarded as the first line of the antioxidant defense systemduring oxidative stress. Therefore, lipids from silver carp brain possessed strong antioxidantactivty, and because of abundant phospholipids and higher level of PUFA contained in PL, PLplayed more important in antioxidant acitivity. Many foods primarily deteriorate because ofmicrobes that give rise to the loss of quality and safety. In addition, numerous bacteria areable to produce PAF. PAF, which is a key in atherosclerosis development, is also an extremelypotent aggregating agent participating in the inflammatory development of plaque and theblockage of blood vessels that finally lead to coronary heart disease. Besides, oxidativedamages are engaged in the development of cardiovascular disease and cancer. The presentstudy may help to explain the protective role of fish lipids against diseases and may beresponsible for the effectiveness of fish brain in benefiting health. Furthermore, the resultsmay shed light on the utilization of silver carp brain lipids as eligible bioactive ones forprevention and treatement of cardiovascular disease.The anticancer activity of silver carp brain lipids (TL, NL, PL) were investigaged. Theproliferations of MCF-7cells，HeLa cells，CACO-2cells and HepG2cells were suppressed by TL from silver carp brain. TL, NL and PL suppressed the proliferations of MCF-7cells andHeLa cells in time-and dose-dependent manner. Exposure MCF-7cells and HeLa cells to TL,NL and PL resulted in loss of adhesion and the cells rounding up, indicating the possibility ofapoptosis occurrence. The results observed from fluorescence microscope showed thatincubated MCF-7cells and HeLa cells with sliver carp brain lipids resulted in nucleifragmentation as indicated in condensed chromatin and bright staining in morphology,indicating apoptosis. Cell cycle progressions were determined by flow cytometry. The resultsindicated that after treatment with TL, NL and PL, MCF-7cells and HeLa cells were arrestedat S stage. The obvious apoptotic peaks were observed after HeLa cells were incubated withlipids for48h. Compared to control, the Sub-G1proportions of TL, NL and PL were increased5.62%,3.06%and9.98%, respectively. Therefore, lipids from silver carp brain inhibited theproliferations of MCF-7cells and Hela cells through promoting apoptosis and blocking Sphase. The detecting of apoptosis proved that the effects of silver carp brain lipids onMCF-7cells and HeLa cells were primarily apoptosis rather than necrosis. The production ofROS was significantly increased in lipids treated MCF-7cells and Hela cells in comparison ofcontrol. For instances, after treated by TL, NL and PL for24h, compared to control, the ROSlevel of MCF-7cells increased23.12%,17.69%and32.76%, respectively. Further analysisindicated that TL, NL and PL could cause the collapse of mitochondrial membrane potential(Ψ) of MCF-7cells and HeLa cells. Thus, the apoptotic effects of silver carp brain lipids onMCF-7cells and HeLa cells were associated with the elevated level of ROS accumulation andthe loss of Ψ. The anticancer role of PL surpassed that of NL which may attributed toabundant phspholipids and higher PUFA level in PL.更多还原