水飞蓟籽仁蛋白和油的主要特性与生物活性研究
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摘要
水飞蓟(Silybum marianum)为菊科草本植物,其籽主要用来提取分离其有效成分水飞蓟素。由于水飞蓟素主要存在于籽壳中,采用脱壳工艺不仅可使其壳仁分离,便于高纯度水飞蓟素的高效提取,同时也为籽仁的综合利用创造了条件。由于籽仁中油和蛋白质的含量均较高,因此,有必要深入研究水飞蓟油和水飞蓟蛋白的主要特性与生物活性,为其作为新资源食品的开发提供理论依据,也为实现水飞蓟资源的综合利用、提高其附加值提供技术支持。
本论文以水飞蓟籽仁为原料,在研究水飞蓟油和水飞蓟蛋白的基本营养成分和主要特性的基础上,进一步研究了水飞蓟油和水飞蓟蛋白酶解物的抗氧化、抗衰老活性,主要研究内容与结果如下：
1.分析了水飞蓟籽仁的主要营养成分及其含量。籽仁脱脂粉中蛋白质含量为47.23%,其氨基酸种类齐全,谷氨酸和天冬氨酸含量丰富；脱脂粉中矿物质元素含量丰富,钙、锌、铜等元素的含量高于一般谷物；籽仁中含油量为34.12%,其中不饱和脂肪酸含量高达77.21%,且主要以亚油酸为主(48.20%),油中α-VE含量为339.42mg/kg。
2.初步研究了水飞蓟籽仁脱脂粉的食用安全性和水飞蓟蛋白的消化性能。水飞蓟籽仁脱脂粉对小鼠的经口LD50>15g/kg,属于无毒级别；利用胃蛋白酶-胰蛋白酶体外模拟消化水飞蓟蛋白结果表明,其中的高分子量蛋白比大豆分离蛋白中的更容易被降解,消化性能优良。
3.采用响应面分析法优化了制备水飞蓟蛋白的工艺条件。在pH11、液料比16：1、温度50℃的条件下,蛋白的提取率为54.52%。
4.采用Osbome蛋白分类法对水飞蓟蛋白进行分级分离,并对其主要特性进行了研究。结果表明,水飞蓟蛋白以清蛋白为主(42.94%),其次是球蛋白(10.1%)；水飞蓟总蛋白及其四种蛋白组分都含有丰富的谷氨酸、精氨酸、天冬氨酸和亮氨酸,必需氨基酸构成较平衡；SDS-PAGE结果显示：水飞蓟蛋白主要以中、低分子质量蛋白为主,主要集中在32.4-44.5kD和14.5-24.8kD两个区域,清蛋白和球蛋白的主要电泳条带分布与总蛋白一致；表面疏水性测定结果显示：球蛋白的表面疏水指数最高,清蛋白的最低；对总蛋白、清蛋白和球蛋白的主要加工特性的研究结果表明,总蛋白的加工特性优于清蛋白和球蛋白。
5.从5种蛋白酶中筛选出水飞蓟蛋白的最佳水解酶为中性蛋白酶,其酶解水飞蓟蛋白的最适条件为：底物浓度2%,反应温度55℃,加酶量14000U/g,pH值7.0,酶解时间120min。在此条件下,蛋白酶解物对·OH清除率达到了88.57%。水飞蓟蛋白酶解物中约有65%是分子量小于1000Da的寡肽。
6.研究了水飞蓟蛋白酶解物抗氧化活性的稳定性。结果表明,在试验范围内,水飞蓟蛋白酶解物的耐热性较好,在酸、碱条件下,抗氧化稳定性良好；NaC1、蔗糖和葡萄糖对酶解物的抗氧化稳定性影响不明显；苯甲酸和山梨酸会降低酶解物的抗氧化活性；不同金属离子对酶解物的抗氧化稳定性影响程度不同；酶解物对胃肠道的酶系有较好的耐受性。
7.对水飞蓟蛋白酶解物进行超滤分级,研究了不同分子量肽段的体外抗氧化活性。结果表明,分子量<1kD的水飞蓟肽的体外抗氧化活性最强,其次为1-3kD肽,3-10kD肽的体外抗氧化活性最弱；分子量<1kD的肽可以显著抑制H202诱导的小鼠红细胞的氧化溶血,抑制小鼠肝细胞线粒体的肿胀和MDA的生成,提高肝细胞线粒体Na+-K+-ATPase、Ca2+-Mg2+-ATPase和SDH的活性,维持肝细胞线粒体的膜电位和膜流动性。
8.研究了水飞蓟油和水飞蓟蛋白酶解物的抗衰老活性。结果表明,水飞蓟油和水飞蓟蛋白酶解物可显著降低D-半乳糖诱导的衰老小鼠的血脂水平,提高小鼠肝、脑组织的抗氧化能力和小鼠肝细胞线粒体膜的流动性与膜电位及其线粒体中SDH、Na+-K+ATPase和Ca2+-Mg2+ATPase的活性；显著提高小鼠肝、脑组织中Bc12的含量,降低caspase-3的活性；水飞蓟蛋白酶解物可显著降低小鼠肝、脑组织中8-OHdG的水平；水飞蓟油可显著降低肝组织中8-OHdG的水平,但脑中8-OHdG的含量与模型组没有显著差异；水飞蓟油和水飞蓟蛋白酶解物均可保护小鼠肝、脑组织细胞形态的完整性。在试验剂量范围内,水飞蓟蛋白酶解物以中剂量组效果最好,水飞蓟蛋白酶解物的抗衰老效果优于水飞蓟油。
Silybum Marianum is compositae herbaceous plant. Its seed is mainly used for extraction of silymarin. Silymarin is the effective component of Silybum Marianum, which mainly exists in the seed shell. Separation of the shell and kernel can not only facilitate efficient extraction of silymarin with high purity, but also facilitate the comprehensive utilization of seed kernel. Seed kernel contains abundant oil and protein therefore it is necessary to study the main properties and biological activities of Silybum marianum oil and protein. The research can not only provide the theoretical basis for the new resources of food development of Silybum marianum seed kernel protein and oil, but also provide the technical support for comprehensive utilization and improve added value of Silybum Marianum.
In this dissertation, the basic nutrition composition and physicochemical properties of Silybum marianum seed kernel protein and oil were studied. The researches concering antioxidant and anti-aging activities of Silybum marianum oil and protein hydrolysates were also investigated. The main results were as follows:
1. The protein content of Silybum marianum seed kernel defatted powder was47.23%and amino acid composition was reasonable, especially it was rich in glutamic acid and aspartate. Its mineral element content was high, the content of calcium, zinc, copper and other elements were higher than other grain. The oil content in the seed kernel was34.12%, of which unsaturated fatty acid content was77.21%. Linoleic acid was the main unsaturated fatty acid (48.20%). α-VE content in oil was339.42mg/kg.
2. The edible security of the Silybum marianum seed kernel defatted powder and the digestion performance of Silybum marianum protein were preliminarily studied. The oral LD50on mice about Silybum marianum seed kernel defatted powde was more than15g/kg. So it belongs to the non-toxic level. In vitro digestibility of Silybum marianum protein using pepsin trypsin and pepsin was studied. High molecular weight proteins in Silybum marianum protein were easier to be hydrolysed than SPI. So Silybum marianum protein had good digestion performance.
3. The optimum processing conditions of Silybum marianum protein was determined through response surface methodology. Under the optimized processing conditions as pH11, liquid-solid ratio16:1and50℃, the maximum extraction rate was54.52%.
4. Silybum marianum protein was fractionated by the osborne protein classification method and its main properties were studied. Albumin was the main constituent (42.94%), followed by globulin (10.1%). Both total protein and four protein components of Silybum marianum contained abundant glutamic acid, arginine, aspartic acid and leucine. The composition of essential amino acid was balanced. The SDS-PAGE result showed that Silybum marianum protein was mainly composed of protein of middle and low molecular weight. The main bands were in the range of32.4～44.5kD and14.5～4.8kD. Globulin had the highest surface hydrophobic index, albumin was just the reverse of globulin. The processing performance of total protein was better than that of albumin and globulin.
5. Neutral protease was the optimal hydrolases for Silybum marianum protein among5proteases which were investigated. The optimum conditions of enzymatic hydrolysis were determined as substrate concentration [S]2%, temperature55℃,[E]/[S]14000U/g, pH7.0, time120min. Under these conditions, the·OH scavenging rate was88.57%. The hydrolysates contained around65%oligopeptide whose molecular weight were smaller than1000Da.
6. The stability of antioxidant activity of protein hydrolysates of Silybum marianum was studied. Protein hydrolysates of Silybum marianum had good stabilities in acid and alkaline conditions. NaCl, sucrose and glucose have little effects on the antioxidation activity, while benzoic acid and sorbic acid could reduce the activity. Different metal ions had the different effects on the antioxidation activity. The hydrolysates could keep high activity in vitro digestive system.
7. Protein hydrolysates of Silybum marianum were ultrafiltrated consecutively by the ultrafiltration membrane. The antioxidant activities of peptides with different molecular weight were studied.<1kD peptide had the strongest antioxidant activity in vitro, followed by1-3kD peptide and3-10KD peptide in turn.<1kD peptide could inhibit the hemolysis of red blood cells induced by H2O2, the swelling of liver mitochondria and the generation of MDA in mice. It also could improve the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and SDH, and maintain liver mitochondria membrane potential and membrane fluidity.
8. Results of anti-aging activity showed that Silybum marianum oil and protein hydrolysates could significantly decrease the blood lipid level and increase the antioxidant capacity of liver and brain in D-galactose-induced aging mice. Both of them could improve liver mitochondria membrane fluidity, membrane potential and the activities of SDH, Na+-K+-ATPase, Ca2+-Mg2+-ATPase, decrease the concentration of caspase-3and improve the activity of Bcl2in the liver and brain of aging mice. Protein hydrolysates of Silybum marianum could significantly reduce the levels of8-OHdG in the liver and brain of aging mice. While Silybum marianum oil could significantly reduce the levels of8-OHdG only in liver. Compared with the model group, there were no significant differences in the levels of8-OHdG in brain. Silybum marianum oil and protein hydrolysates could protect the integrity of the cell morphology of liver and brain of mice. In the test dose range, the middle dose group of protein hydrolysates of Silybum marianum had the best effect. The effect of anti-aging of the hydrolysates was better than Silybum marianum oil.
本论文以水飞蓟籽仁为原料,在研究水飞蓟油和水飞蓟蛋白的基本营养成分和主要特性的基础上,进一步研究了水飞蓟油和水飞蓟蛋白酶解物的抗氧化、抗衰老活性,主要研究内容与结果如下：
1.分析了水飞蓟籽仁的主要营养成分及其含量。籽仁脱脂粉中蛋白质含量为47.23%,其氨基酸种类齐全,谷氨酸和天冬氨酸含量丰富；脱脂粉中矿物质元素含量丰富,钙、锌、铜等元素的含量高于一般谷物；籽仁中含油量为34.12%,其中不饱和脂肪酸含量高达77.21%,且主要以亚油酸为主(48.20%),油中α-VE含量为339.42mg/kg。
2.初步研究了水飞蓟籽仁脱脂粉的食用安全性和水飞蓟蛋白的消化性能。水飞蓟籽仁脱脂粉对小鼠的经口LD50>15g/kg,属于无毒级别；利用胃蛋白酶-胰蛋白酶体外模拟消化水飞蓟蛋白结果表明,其中的高分子量蛋白比大豆分离蛋白中的更容易被降解,消化性能优良。
3.采用响应面分析法优化了制备水飞蓟蛋白的工艺条件。在pH11、液料比16：1、温度50℃的条件下,蛋白的提取率为54.52%。
4.采用Osbome蛋白分类法对水飞蓟蛋白进行分级分离,并对其主要特性进行了研究。结果表明,水飞蓟蛋白以清蛋白为主(42.94%),其次是球蛋白(10.1%)；水飞蓟总蛋白及其四种蛋白组分都含有丰富的谷氨酸、精氨酸、天冬氨酸和亮氨酸,必需氨基酸构成较平衡；SDS-PAGE结果显示：水飞蓟蛋白主要以中、低分子质量蛋白为主,主要集中在32.4-44.5kD和14.5-24.8kD两个区域,清蛋白和球蛋白的主要电泳条带分布与总蛋白一致；表面疏水性测定结果显示：球蛋白的表面疏水指数最高,清蛋白的最低；对总蛋白、清蛋白和球蛋白的主要加工特性的研究结果表明,总蛋白的加工特性优于清蛋白和球蛋白。
5.从5种蛋白酶中筛选出水飞蓟蛋白的最佳水解酶为中性蛋白酶,其酶解水飞蓟蛋白的最适条件为：底物浓度2%,反应温度55℃,加酶量14000U/g,pH值7.0,酶解时间120min。在此条件下,蛋白酶解物对·OH清除率达到了88.57%。水飞蓟蛋白酶解物中约有65%是分子量小于1000Da的寡肽。
6.研究了水飞蓟蛋白酶解物抗氧化活性的稳定性。结果表明,在试验范围内,水飞蓟蛋白酶解物的耐热性较好,在酸、碱条件下,抗氧化稳定性良好；NaC1、蔗糖和葡萄糖对酶解物的抗氧化稳定性影响不明显；苯甲酸和山梨酸会降低酶解物的抗氧化活性；不同金属离子对酶解物的抗氧化稳定性影响程度不同；酶解物对胃肠道的酶系有较好的耐受性。
7.对水飞蓟蛋白酶解物进行超滤分级,研究了不同分子量肽段的体外抗氧化活性。结果表明,分子量<1kD的水飞蓟肽的体外抗氧化活性最强,其次为1-3kD肽,3-10kD肽的体外抗氧化活性最弱；分子量<1kD的肽可以显著抑制H202诱导的小鼠红细胞的氧化溶血,抑制小鼠肝细胞线粒体的肿胀和MDA的生成,提高肝细胞线粒体Na+-K+-ATPase、Ca2+-Mg2+-ATPase和SDH的活性,维持肝细胞线粒体的膜电位和膜流动性。
8.研究了水飞蓟油和水飞蓟蛋白酶解物的抗衰老活性。结果表明,水飞蓟油和水飞蓟蛋白酶解物可显著降低D-半乳糖诱导的衰老小鼠的血脂水平,提高小鼠肝、脑组织的抗氧化能力和小鼠肝细胞线粒体膜的流动性与膜电位及其线粒体中SDH、Na+-K+ATPase和Ca2+-Mg2+ATPase的活性；显著提高小鼠肝、脑组织中Bc12的含量,降低caspase-3的活性；水飞蓟蛋白酶解物可显著降低小鼠肝、脑组织中8-OHdG的水平；水飞蓟油可显著降低肝组织中8-OHdG的水平,但脑中8-OHdG的含量与模型组没有显著差异；水飞蓟油和水飞蓟蛋白酶解物均可保护小鼠肝、脑组织细胞形态的完整性。在试验剂量范围内,水飞蓟蛋白酶解物以中剂量组效果最好,水飞蓟蛋白酶解物的抗衰老效果优于水飞蓟油。
Silybum Marianum is compositae herbaceous plant. Its seed is mainly used for extraction of silymarin. Silymarin is the effective component of Silybum Marianum, which mainly exists in the seed shell. Separation of the shell and kernel can not only facilitate efficient extraction of silymarin with high purity, but also facilitate the comprehensive utilization of seed kernel. Seed kernel contains abundant oil and protein therefore it is necessary to study the main properties and biological activities of Silybum marianum oil and protein. The research can not only provide the theoretical basis for the new resources of food development of Silybum marianum seed kernel protein and oil, but also provide the technical support for comprehensive utilization and improve added value of Silybum Marianum.
In this dissertation, the basic nutrition composition and physicochemical properties of Silybum marianum seed kernel protein and oil were studied. The researches concering antioxidant and anti-aging activities of Silybum marianum oil and protein hydrolysates were also investigated. The main results were as follows:
1. The protein content of Silybum marianum seed kernel defatted powder was47.23%and amino acid composition was reasonable, especially it was rich in glutamic acid and aspartate. Its mineral element content was high, the content of calcium, zinc, copper and other elements were higher than other grain. The oil content in the seed kernel was34.12%, of which unsaturated fatty acid content was77.21%. Linoleic acid was the main unsaturated fatty acid (48.20%). α-VE content in oil was339.42mg/kg.
2. The edible security of the Silybum marianum seed kernel defatted powder and the digestion performance of Silybum marianum protein were preliminarily studied. The oral LD50on mice about Silybum marianum seed kernel defatted powde was more than15g/kg. So it belongs to the non-toxic level. In vitro digestibility of Silybum marianum protein using pepsin trypsin and pepsin was studied. High molecular weight proteins in Silybum marianum protein were easier to be hydrolysed than SPI. So Silybum marianum protein had good digestion performance.
3. The optimum processing conditions of Silybum marianum protein was determined through response surface methodology. Under the optimized processing conditions as pH11, liquid-solid ratio16:1and50℃, the maximum extraction rate was54.52%.
4. Silybum marianum protein was fractionated by the osborne protein classification method and its main properties were studied. Albumin was the main constituent (42.94%), followed by globulin (10.1%). Both total protein and four protein components of Silybum marianum contained abundant glutamic acid, arginine, aspartic acid and leucine. The composition of essential amino acid was balanced. The SDS-PAGE result showed that Silybum marianum protein was mainly composed of protein of middle and low molecular weight. The main bands were in the range of32.4～44.5kD and14.5～4.8kD. Globulin had the highest surface hydrophobic index, albumin was just the reverse of globulin. The processing performance of total protein was better than that of albumin and globulin.
5. Neutral protease was the optimal hydrolases for Silybum marianum protein among5proteases which were investigated. The optimum conditions of enzymatic hydrolysis were determined as substrate concentration [S]2%, temperature55℃,[E]/[S]14000U/g, pH7.0, time120min. Under these conditions, the·OH scavenging rate was88.57%. The hydrolysates contained around65%oligopeptide whose molecular weight were smaller than1000Da.
6. The stability of antioxidant activity of protein hydrolysates of Silybum marianum was studied. Protein hydrolysates of Silybum marianum had good stabilities in acid and alkaline conditions. NaCl, sucrose and glucose have little effects on the antioxidation activity, while benzoic acid and sorbic acid could reduce the activity. Different metal ions had the different effects on the antioxidation activity. The hydrolysates could keep high activity in vitro digestive system.
7. Protein hydrolysates of Silybum marianum were ultrafiltrated consecutively by the ultrafiltration membrane. The antioxidant activities of peptides with different molecular weight were studied.<1kD peptide had the strongest antioxidant activity in vitro, followed by1-3kD peptide and3-10KD peptide in turn.<1kD peptide could inhibit the hemolysis of red blood cells induced by H2O2, the swelling of liver mitochondria and the generation of MDA in mice. It also could improve the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and SDH, and maintain liver mitochondria membrane potential and membrane fluidity.
8. Results of anti-aging activity showed that Silybum marianum oil and protein hydrolysates could significantly decrease the blood lipid level and increase the antioxidant capacity of liver and brain in D-galactose-induced aging mice. Both of them could improve liver mitochondria membrane fluidity, membrane potential and the activities of SDH, Na+-K+-ATPase, Ca2+-Mg2+-ATPase, decrease the concentration of caspase-3and improve the activity of Bcl2in the liver and brain of aging mice. Protein hydrolysates of Silybum marianum could significantly reduce the levels of8-OHdG in the liver and brain of aging mice. While Silybum marianum oil could significantly reduce the levels of8-OHdG only in liver. Compared with the model group, there were no significant differences in the levels of8-OHdG in brain. Silybum marianum oil and protein hydrolysates could protect the integrity of the cell morphology of liver and brain of mice. In the test dose range, the middle dose group of protein hydrolysates of Silybum marianum had the best effect. The effect of anti-aging of the hydrolysates was better than Silybum marianum oil.
引文
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