【作者】 吴金鸿；

【导师】 王璋；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 来源于蚕丝的丝胶作为一种新型的食品蛋白资源,不但具有食用价值而且具有药用价值,因此对丝胶进行功能性食品的开发利用,不仅提升了丝胶作为丝绸加工副产品的经济价值,而且具有较大的社会效益。本文以丝绸废水中的丝胶为原料,建立了丝胶的提取工艺和酶解工艺;探讨了DA201-C大孔吸附树脂对丝胶酶解产物的精制处理及其分离组分的生物活性;提取丝胶酶解产物中抗氧化性类黑精组分并分析其理化性质和结构特征;以D-半乳糖致衰老动物模型,探讨了丝胶肽抗衰老的效果。本文的研究为开发功能性食品配料――丝胶肽提供了理论基础。本文首先建立了低温乙醇沉淀法提取丝绸废水中丝胶的工艺流程,并使丝胶的提取率达到64%。理化性质研究结果表明醇提丝胶约含有90%以上蛋白质,1%糖和4%灰分;明显地去除了蚕腥恶臭味,但溶解性有所下降;氨基酸组成中含极性侧链的氨基酸达到70%,疏水性值为2.21 KJ.mol-1,表明醇提丝胶为亲水性蛋白质;醇提丝胶相对分子质量分布比较宽,且多数组分分布在10,000～500,000之间。本文进一步通过扫描电镜(SEM)、差式扫描量热法(DSC)、圆二色性(CD)和红外光谱(IR)的测定探讨了醇提工艺对丝胶蛋白质超微结构和分子构象稳定性的影响。SEM结果表明在低温乙醇溶液中蛋白质分子间产生交联聚集形成致密的球状聚集体结构,当脱乙醇并在水溶液中复溶后,这种聚集作用被减弱,分子展开形成三维的网状结构;DSC、CD和IR结果表明蛋白质分子已发生部分变性,而这种变性可能与β-转角构象的出现有关,即部分的分子链通过β-转角这种折叠的构象进行组装,使分子结构从无序的状态向有序的状态转化,从而导致醇提的丝胶蛋白质的溶解性有所下降。此外,GC-MS分析结果表明使丝胶产生蚕腥恶臭味的风味组分主要是杂环类和烃类的挥发性化合物,经过醇提工艺处理后丝胶的蚕腥恶臭味明显被去除,使丝胶适用于食品的开发和利用。本文通过比较五种微生物来源的蛋白酶对丝胶的水解能力,确定最适水解酶为AS1398中性蛋白酶;通过单因素试验和响应面分析确定丝胶最佳酶解条件为:pH 6.1,温度55°C,加酶量([E]/[S])2% (w/w %),该条件下的水解度为23.35%。与醇提丝胶比较,丝胶酶解产物的蛋白质含量略有下降,灰分含量和糖的含量增加;丝胶酶解产物相对分子质量主要分布在200～1000之间,含2～5个氨基酸的短肽比例约占50%左右。本文采用DA201-C大孔吸附树脂对丝胶酶解产物进行精制,即进行初步分离纯化、脱盐和分离褐色组分的处理,结果表明丝胶酶解产物按疏水性的不同利用大孔吸附树脂用乙醇溶液梯度洗脱可分离为四个组分(DK-1,DK-2,DK-3, DK-4);电导率和灰分含量的测定结果表明,利用大孔吸附树脂可以对丝胶蛋白酶解产物中被树脂吸附的组分进行有效地脱盐;通过420 nm特征吸光值的测定和色泽的分析,结果表明DA201-C大孔吸附树脂可以分离提取丝胶酶解产物中黑褐色物质,从而使非褐色的组分(DK-1)达到脱色的效果。经过大孔吸附树脂分离纯化得到含有黑褐色物质的DK-3组分,生物活性分析结果表明DK-3组分具有清除DPPH·的效果,抑制酪氨酸酶活性的作用和螯合Fe2+能力,其EC50分别为2.49 mg/mL,3.13 mg/mL和0.90 mg/mL;将经过纳滤脱盐处理的DK-1组分应用于冻藏鱼糜中,结果表明DK-1组分的添加显著地提高鱼糜肌动球蛋白质的总巯基含量、降低表面疏水性及减缓肌球蛋白质重链(MHC)和肌动蛋白(AC)的降解,说明DK-1组分具有防止鱼糜肌动球蛋白质冷冻变性的作用,而且与浓度存在一定的相关性,在实际应用时,其添加量不宜太高。本文通过SDS-PAGE结合PAS染色法鉴定出丝胶中含有高分子量的糖蛋白;通过β-消去反应判断丝胶中存在O-连接方式的糖肽键;通过GC-MS分析鉴定出嘧啶、2-(5-氯-2-甲氧基亚苄基)-吡咯和吡啶酮等美拉德反应产生的特征性杂环类化合物。以上结果证实丝胶在酶解工艺过程中发生了美拉德反应,从而为丝胶酶解产物中类黑精的产生提供一定的依据。本文将经过大孔吸附树脂分离纯化得到的DK-3组分通过聚酰胺柱层析和RP-HPLC进一步分离纯化,得到在220 nm、280 nm和420 nm均有明显吸收的类黑精组分(RP-M)。抗氧化活性测定结果表明RP-M类黑精组分具有清除DPPH·的效果,其EC50为0.13 mg/mL;可抑制H2O2的氧化损伤,具有明显的神经细胞保护作用,但添加量过高时对细胞反而有毒性作用;RP-M组分氨基酸组成中赖氨酸(Lys)、精氨酸(Arg)和脯氨酸(Pro)及一些疏水性的氨基酸的含量都明显地增加,说明这些氨基酸是组成类黑精肽骨架结构的主要氨基酸;RP-M组分相对分子质量主要分布在1,000～50,000之间,其中大于7,500的成分含量约占50%,而且MALDI-TOF MS测定的质量指纹图谱表明相对分子质量小于1000的短肽为RP-M类黑精组分基本的肽骨架单元,这些结果说明RP-M组分是通过短肽的复合形成聚合度不等的高分子量混合体。ESI-MS-MS质谱分析结果表明RP-M组分含有m/z分别为529.8715、462.3399和398.3169的三个特征的肽段,其氨基酸序列结构分别为:IILLLAAKFG、KVIIKPLI和QPVIAHM。这些肽段的氨基酸序列结构上具备抗氧化肽的特征,说明RP-M类黑精组分的肽骨架结构可能与其抗氧化活性相关。此外,特征肽段氨基酸组成中都含有的Lys和Pro,是形成类黑精最具活性的两种氨基酸残基,对短肽的复合和色素分子的聚合可能起到交联的作用。红外光谱分析结果表明RP-M组分结构中含有芳族胺、芳族羧酸和烷烃结构。紫外-可见光谱分析结果表明,RP-M组分在200 nm~380 nm范围内有强的吸收,在275 nm~280 nm处有一个吸收峰。RP-M组分酸水解样品经RP-HPLC分离纯化得到一个在420 nm处有明显吸收的S-4色素组分。红外光谱、紫外光谱和核磁共振的分析结果表明S-4色素组分是一种α-型吡喃糖类物质,说明类黑精组分的色素可由糖基化的物质形成。本文通过动物试验研究了丝胶肽抗衰老的效果。结果表明丝胶肽可显著提高D-半乳糖致衰老模型老鼠血清中SOD活性和降低MDA含量;可显著增加脾脏的重量;可改善D-半乳糖致衰老模型老鼠学习记忆的能力;减缓D-半乳糖对海马神经细胞的损伤;提高脑组织中抗凋亡基因Bcl-2 mRNA表达水平。结果说明丝胶肽可通过提高体内抗氧化作用,增强机体免疫力功能和延缓脑组织的老化作用起到抗衰老的效果。更多还原

【Abstract】 Sericin, derived from silk cocoon, is a new food protein source, which can be used as food and medicine, so the exploration and utilization of sericin for the functional food manufacture will not only raise the economic value of sericin which used to be as a by-product of silk industry, but also reveal the significant social benefit.In this dissertation, the extraction technology as well as the enzymatic hydrolysis technology for sericin derived from silk wastewater were developed; the refinement of sericin hydrolysates using the macroporous adsorption resin DA201-C was researched and the biological activities of the obtained fractions were further investigated; the antioxidant melanoidin derived from sericin hydrolysates was purified and their physiochemical properties and structure were determined; the anti-aging effect of sericin peptides on the D-galactose induced aging model rats was assessed. The study of this dissertation would give the theoretical foundations for utilization of sericin hydrolysates as functional food additives.Initially, chilled alcohol precipitation method was used to extract sericin, and the suitable extraction process with the yield of 64% was set up. Results from physiochemical analysis show that sericin extrated with alcohol contained about 90% of protein, 1% of sugar and 4% of ash; the solubility was decreased, but silk special bad smell was removed significantly; the hydrophilic amino acid amount was up to 70 % in the total amino acids and the hydrophobicity value was 2.21 KJ.mol-1, which suggest that sericin was a kind of hydrophilic protein; the relative molecular weight of sericin was mainly in the range of 10,000～500,000.Further, the effects of the extraction process with alcohol on the mircro- molecular structure and the stabilization of molecular conformation of sericin were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), circular dichroism (CD), and infrared (IR) spectroscopy. SEM result shows that protein molecules were aggregated together to form a compact and spherical structure, but it changed to loose and netty structure after ridding of the alcohol and resolving in the water; the results obtained from the DSC、CD and IR suggested that the conformation of protein molecules was partially changed, which was possibly relative to the appearance ofβ-Turn conformation, which could facilitate molecule folding by reversing the direction of the polypeptide chain, and make the molecule conformation changed from random structure to order structure, thus lead to the decrease of solubility. In addition, GC-MS analytic result reveals that the flavor components resulted in the silk bad smell were mainly the heterocyclic and alkyl volatile components, but they could be removed by using alcohol, thus sericin extrated with alcohol was suitable for the application in food.The effects of five kinds of proteases on the hydrolysis of sericin were compared by the degree of hydrolysis (DH). AS 1398 was found to be the most effective enzyme. By the single-factor tests and the response surface analysis, the optimum hydrolysis conditions were estimated as pH 6.1, temperature 55°C, and E/S (%) of 2.00, respectively. Accordingly, the highest DH value was estimated as 23.35%. Furthermore, compared to the sericin, the protein content of the hydrolysates was decreased, but increased in the contents of sugar and ash. The relative molecular weight distribution of the hydrolysates was among 200～1000, and the component with relative molecular weight under 500 was of 50% or so.Further, the macroporous adsorption resin DA201-C was used to refine sericin hydrolysates by fractionation, desalting and decoloring treatments. Results show that four fractions (DK-1,DK-2,DK-3, DK-4) were separated by DA201-C resin eluted with alcohol in step wash, the adsorptive fractions had been desalted effectively according to the analysis of the conductivity value and the ash content, and dark-brown components could be isolated from sericin hydrolysates according to the analysis of the optical density at 420 nm and the chroma parameters of the fractions. Moreover, the DK-3 fraction shows the DPPH free-radical scavenging activity, the inhibitory effect on mushroom tyrosinase and ferrous ion-chelating activity in vitro, and the corresponding value of EC50 was 2.49 mg/mL, 3.13 mg/mL and 0.90 mg/mL, respectively. The DK-1 fraction after being desalted by nanofiltration was added into the frozen fish surimi, and results show that the addition of the DK-1 fraction could significantly increase the total sulfhydryl content of actomyosin, decrease surface hydrophobicity of actomyosin and inhibit the decompose of myosin heavy chain (MHC) and actin (AC), suggesting the DK-1 fraction had the cryoprotective effect on fish surimi proteins. But this effect was relative to the dosage, and the data denote that the dosage of application could not be too high.In this dissertation, glycoproteins with high molecular weight from sericin were identified by SDS-PAGE stained with PAS, O-glycosidic linkage was found byβ-elimination reaction, and characteristic heterocyclic volatile components derived from the maillard reaction, such as pyridine, 2-piperidone and 2-[5-chloro-2-methoxyphenyl] pyrrole, were identified from sericin hydrolysates by GC-MS analysis. These data approve that the maillard reaction occurred in the process of sericin enzymatic hydrolysis, and give the experimental foundations for the produce of melanoidins in sericin hydrolysates. Further, melanoidins (RP-M) with antioxidant activities and adsorptive features at 220 nm, 280 nm and 420 nm, was isolated from the DK-3 fraction by polyamide resins and RP-HPLC. The results from the analysis of antioxidant activity showed that RP-M had the DPPH free-radical scavenging activity, and IC50 of RP-M was 0.13 mg/ml. Moreover, RP-M exhibited the neuroprotective effect on hydrogen peroxide (H2O2) induced oxidative damage in PC12 cells, but the highest dosage in this study shows the cytoxicity on cells. Compared to sericin, the content of amino acid residues in RP-M, including Lys, Arg, Pro and other hydrophobic amino acids, was increased significantly, suggesting they were the dominant amino acid residues in the RP-M peptide skeletons. The relative molecular weight of RP-M was mainly in the range of 1,000～50,000, and the components with relative molecular weight above 7,500 was of 50% or so. Moreover, the mass fingerprinting of RP-M determined by MALDI-TOF MS denotes that the peptide skeletons of RP-M consisted of small peptides with relative molecular weight under 1,000, so the above results imply that the melanoidin component-- RP-M, was a mixture with high molecular weight, which was formed by polymerizing between peptide mixture with pigments.The primary structure of the three peptide fragments at m/z 529.8715、462.3399 and 398.3169, were identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS), and the amino acid sequence was IILLLAAKFG、KVIIKPLI and QPVIAHM, respectively. The primary structures of peptide fragments had the functional structure feature similar with other antioxidant peptides, which denotes that the antioxidant activity of RP-M might be related to their peptide skeletons structures. In addition, these peptide fragments all consisted of the characteristic amino acids, such as Lys and Pro, which are the most active amino acids that facilitate the produce of melanoidin. The IR spectrum of RP-M denotes it possessed the structures of aromatic amine, aromatic carboxylic acid and alkane. The shape of the UV/VIS spectrum of RP-M had strong absorption in the range of 200 nm~380 nm, and with maxima in the range of 275 nm~280 nm. The acid hydrolysates of RP-M was prepared and purified by RP-HPLC. A pigment component (S-4) with strong absorption in the wavelength of 420 nm was obtained. The analysis results from infrared (IR) spectroscopy, UV/VIS spectroscopy and nuclear magnetic resonance (NMR) show that the S-4 pigment component was a kind of alpha-pyranoside saccharides. This result implies that the pigment part of melanoidin was consisted of glycosylation products.In this dissertation, the anti-aging effects of sericin peptides on the D-gal induced aging rats were measured. Results show that the consumption of sericin peptides could obviously increase superoxide dismutase (SOD) activities and decrease malondialdehyde (MDA) levels in the rat serum; markedly increase the weight of rat spleen; enhance the ability of learning and memory in aging model rats; notably suppressed neuronal loss in hippocampal CA1 region and prevent the morphological damage of pyramidal cells; enhance the expression levels of the anti-apoptotic gene Bcl-2. These data suggest that the anti-aging effects of sericin peptides in vivo were relative to the function of antioxidant activities, the improving function of immunity, and the role of delaying the aging change of the brain.更多还原