高有机硒保存率蔬菜富集和加工机理研究
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摘要
硒是人体的必须营养元素,缺硒可能导致心脏病,甲状腺机能减退,以及免疫能力减弱等症状。近年来,含硒组分具有的抗癌能力受到国际保健科学领域的普遍关注,流行病学研究表明,成年人如果每天补充200μg硒可以使癌症的发病率和死亡率降低50%左右。我国成人的硒摄入量仅为26μg/日,距离中国预防医学中心卫生所推荐的安全进硒量50-240μg/人/日相差甚远。而不同来源和化学形式的含硒组分之间的安全性、生物功能性存在较大差异。植物中硒主要以硒代氨基酸形式存在,生物利用率高,且不易在体内造成蓄积,因此本论文利用蔬菜作为载体,对蔬菜中有机硒的富集、生物转化,以及加工过程对硒的影响进行探讨,对于具有生理功能的含有机微量元素蔬菜型功能食品产业化中的应用提供理论依据和实践指导。
首先选择十字花科云薹属植物甘蓝、菜心、豆科大豆属的毛豆和百合科天门冬属的芦笋作为试验原料,研究蔬菜对于无机硒的吸收和转移规律以及硒的富集对于蔬菜营养成分的影响。研究结果显示:蔬菜中硒的积累量与亚硒酸钠的喷施量线性相关,其中十字花科的甘蓝和菜心属于硒积累植物,蓄积硒的能力较高,转化率高,而毛豆因蛋白含量较高,也可以硒蛋白的形式蓄积较高含量的有机硒。甘蓝和毛豆富硒能力较强,生长周期较长,无机硒残留少,是较适宜的富硒蔬菜品种。采用pH8的浓度为50mg/L的亚硒酸钠溶液,对蔬菜进行2-3次的叶面喷施,是比较简便而且切实可行的农艺富硒方法。适当浓度的硒处理对于蔬菜中蛋白质、叶绿素以及维生素C的合成起促进作用,而可溶性糖的含量呈降低趋势,富硒处理不改变蔬菜中氨基酸的种类和分布,但是分子量分布试验和电泳试验显示,毛豆蛋白中大分子量蛋白(>100kDa)含量降低,而中等分子量和小分子量蛋白含量升高。毛豆中油脂含量随施硒浓度的增加而降低,其中不饱和脂肪酸相对含量降低而饱和脂肪酸的相对含量升高。
其次,进行了含硒组分的分离提取和分析检测。通过HCl缓冲液、蛋白酶和超声波辅助萃取的方法,研究富硒甘蓝和毛豆中硒的提取工艺,结果显示甘蓝中硒主要以游离态形式存在,而毛豆中的硒则以蛋白结合的形式存在。采用pH2.0,超声波处理120sec的方法可以将甘蓝中的绝大部分的硒提取出来,而毛豆中硒的提取则必须采用蛋白酶水解处理,采用超声波处理可以将毛豆蛋白中硒的酶解过程缩短到180s。甘蓝和毛豆中硒代氨基酸的种类和含量的测定采用氯甲酸乙酯衍生化,然后采用气相色谱质谱质谱法进行分析,甘蓝中有机硒主要以甲基硒半胱氨酸和硒代蛋氨酸的形式存在,另有痕量的硒半胱氨酸。毛豆中硒则主要以硒代蛋氨酸的形式存在。
再次,研究了甘蓝和毛豆制汁和脱水过程中硒的损失和降解规律,在4℃贮藏过程中新鲜甘蓝中硒降解率可达30-45%,而毛豆中硒则比较稳定,降解率在10%左右。对蔬菜进行烫漂和调味处理能减少褐变,较好的保持产品的色泽。但烫漂过程会造成甘蓝中硒损失20-30%。脱水过程中毛豆中硒的稳定性高于甘蓝。比较不同脱水工艺对甘蓝和毛豆硒含量的影响,其中冷冻干燥对有机硒的保存率最高,硒蛋氨酸是热不稳定成分,在喷雾干燥过程中损失最大,而甲基硒半胱氨酸即使在冷冻干燥过程中也会有一定的损失。在热风烘干过程中,对于硒的降解的影响,温度和时间均为极显著的影响因素,而且时间的影响要大于温度的影响,拟合方程为Y=1.24321-0.00276X1-0.00075X2 (X1为温度,X2为时间)。硒的降解符合一级动力学模型,Boltzmann方程为:表观活化能Ea为44027 J/mol。
最后,研究了甘蓝含硒组分的体外抗肿瘤细胞活性、体内降血糖能力以及长期摄入富硒产品对小鼠体内抗氧化酶活力以及生理生化指标等的影响。结果显示富硒甘蓝和毛豆提取液对于肿瘤细胞NCI-H446均具有一定的抑制作用。富硒甘蓝提取物对于正常小鼠的血糖没有显著影响,但可降低糖尿病小鼠血糖含量,耐糖量也有一定改善,对糖尿病小鼠的受损胰腺细胞具有一定的修复作用。饲喂富硒甘蓝提取物的小鼠体内过氧化物歧化酶、谷胱甘肽酶含量增加,丙二醛含量降低。灌喂富硒甘蓝汁的对照组和富硒组小鼠血液生理生化指标无显著差异,富硒饲喂组无致畸毒性和遗传毒性。富硒蔬菜产品可作安全可靠的功能补硒食品。
Selenium (Se) is an essential nutrient in the human diet and metabolism. Selenium deficiency may lead to heart disease, hypothyroidism and immune malfunction. In recent years, Se compounds have been internationally recognized to have the ability of anti-cancer. The average total human Se intake in China, as reported by the Chinese Nutrition Association, is only 26μg/d, and this is substantially lower than Chinese Preventive Medicine Center’s recommended intake of 50-240μg/d. Epidemiological studies indicate if adults are supplemented with 200μg/d selenium, the cancer morbidity and mortality may reduce about 50%. Different sources and the chemical form of selenium compounds have different safety and biological functionality levels. Se of plant origin is mainly in the form of selenium amino acids that have higher bioavailability and will not easily cause toxic accumulation in the human body. Therefore, the use of vegetable as a vector of organic selenium enrichment is a appropriate choice. This thesis studied the Selenium bioconversion in vegetables, the formation mechanism of organic selenium, and the processing effects of selenium enriched. The results can provide the basic knowledge for application and the guidance in the industrialization of functional food containing this trace elements.
First, flowering Chinese cabbage (Brassica campestris L. ssp. Chinensis (L.), cabbage (Brassica oleracea var. capitata L.) edamame(Glycine Max (L.) Merrill.) and asparagus (Asparagus officinalis L.) were selected as the selenium enrichment test materials for studying inorganic selenium uptake and transfer pattern in vegetables and effects of selenium treatment on nutritional ingredients for the vegetables. The results show that selenium was a transportable and recyclable element in vegetables. Within certain ranges of spraying concentration, selenium content enriched was linearly correlated with the concentration of NaSeO32-. Twice spraying with lower concentration of NaSeO32- and adequate adjustment of pH improved selenium absorption in vegetables. Spraying of selenium on cabbage at a later growth phase led to higher selenium accumulation in vegetables. Selenium treatment with appropriate concentrations increased protein, vitamin C and chlorophyll synthesis, whereas decreased soluble sugar content. Selenium enrichment did not change the amino acid type and distribution, However, the molecular weight distribution tests and electrophoresis showed that high molecular weight protein in edamame (100kDa) decreased, whereas medium and small molecular weight protein increased. Selenium enriched edamame had a decreased oil content, in which unsaturated fatty acids rate reduced and saturated fatty acids rate increased.
Second, selenium component extraction method and speciation were studied. Through HCl buffer and ultrasound-assisted extraction method, selenium component in cabbage and soybean can be extracted. Results show that the main selenium form in cabbage is in free state, and main selenium form in edamame is protein binding. Using 0.01 HCl buffer, assisted with 120 sec ultrasonic probe, most selenium in cabbage can be extracted. For edamame selenium extraction, it is necessary to adopt proteolytic processing. Ultrasound can be used to deal with selenium soybean protein to shorten the enzyme process to 180s. Derivative of selenium amino acid in cabbage and edamame was done with ethyl chloride formate. Then, they were detected with gas chromatography mass mass spectrometry. Chemical analysis of selenium showed that main selenium species in cabbage was Se-methyl-selenocysteine although selenomethionine and selenocystein were also detected. For edamame, the main selenium species was selenomethionine.
Further, effect of the cabbage and soybean juice process and the dehydrate process on selenium content were studied. When fresh cabbage and edamame were stored under 4℃for 4 weeks,the selenium degradation rate in cabbage was up to 30-45% and soybean selenium was relatively stable, with a degradation rate at about 10%. Blanching and seasoning could reduce the browning degree, and maintain the green color. But blanching process caused the loss of selenium in cabbage at about 20-30%. Edamame selenium was more stable than cabbage during the dehydrated processing. Comparing different dehydration process, freeze-drying had the highest preservation rate for organic selenium. Selenomethionine is a heat-labile component, and the spray drying process resulted in the highest loss of selenomethionine. Met-Secysteine had a certain extent loss even in the freeze-drying process. In the drying process, temperature and time were the most significant factors for the degradation of selenium but the latter had even greater impact . Fitting equation is Y=1.24321-0.00276X1-0.00075X2 (X1 is temperature , X2 is time). Se degradation agreed with a dynamic model of the Boltzmann equation: with the apparent activation energy (Ea) of 44,027 J/mol.
Finally, the extrac of selenium cabbage was used to study in-vitro antitumor activity and the antidiabetic mechanisms in type II diabetic mice. A long-term intake of selenium enriched products for mice was done to study the antioxidant enzymes and physiological and biochemical indicators. Cytotoxic bioactivity study on H446 cell line showed that selenium extract had stronger inhibition in vitro. The cell morphology sticked to the bottle wall showed bigger change and the sum of cells was decreased distinctly than the control treated under microscope. The higher concentration the samples had, the more obvious phenomenon of cell apoptosis was. Selenium cabbage extract had no significant effect on normal mice, but could reduce blood glucose levels in diabetic mice. After gastric perfusion of selenium, mice index of SOD, GSH-Px increased and MDA reduced, blood physiological and biochemical indicators of the control group of mice and selenium-enriched group had no significant difference. Teratogenic toxicity and genetic toxicity of selenium enriched juice showed that selenium vegetable products were safe and reliable to be used as functional food.
首先选择十字花科云薹属植物甘蓝、菜心、豆科大豆属的毛豆和百合科天门冬属的芦笋作为试验原料,研究蔬菜对于无机硒的吸收和转移规律以及硒的富集对于蔬菜营养成分的影响。研究结果显示:蔬菜中硒的积累量与亚硒酸钠的喷施量线性相关,其中十字花科的甘蓝和菜心属于硒积累植物,蓄积硒的能力较高,转化率高,而毛豆因蛋白含量较高,也可以硒蛋白的形式蓄积较高含量的有机硒。甘蓝和毛豆富硒能力较强,生长周期较长,无机硒残留少,是较适宜的富硒蔬菜品种。采用pH8的浓度为50mg/L的亚硒酸钠溶液,对蔬菜进行2-3次的叶面喷施,是比较简便而且切实可行的农艺富硒方法。适当浓度的硒处理对于蔬菜中蛋白质、叶绿素以及维生素C的合成起促进作用,而可溶性糖的含量呈降低趋势,富硒处理不改变蔬菜中氨基酸的种类和分布,但是分子量分布试验和电泳试验显示,毛豆蛋白中大分子量蛋白(>100kDa)含量降低,而中等分子量和小分子量蛋白含量升高。毛豆中油脂含量随施硒浓度的增加而降低,其中不饱和脂肪酸相对含量降低而饱和脂肪酸的相对含量升高。
其次,进行了含硒组分的分离提取和分析检测。通过HCl缓冲液、蛋白酶和超声波辅助萃取的方法,研究富硒甘蓝和毛豆中硒的提取工艺,结果显示甘蓝中硒主要以游离态形式存在,而毛豆中的硒则以蛋白结合的形式存在。采用pH2.0,超声波处理120sec的方法可以将甘蓝中的绝大部分的硒提取出来,而毛豆中硒的提取则必须采用蛋白酶水解处理,采用超声波处理可以将毛豆蛋白中硒的酶解过程缩短到180s。甘蓝和毛豆中硒代氨基酸的种类和含量的测定采用氯甲酸乙酯衍生化,然后采用气相色谱质谱质谱法进行分析,甘蓝中有机硒主要以甲基硒半胱氨酸和硒代蛋氨酸的形式存在,另有痕量的硒半胱氨酸。毛豆中硒则主要以硒代蛋氨酸的形式存在。
再次,研究了甘蓝和毛豆制汁和脱水过程中硒的损失和降解规律,在4℃贮藏过程中新鲜甘蓝中硒降解率可达30-45%,而毛豆中硒则比较稳定,降解率在10%左右。对蔬菜进行烫漂和调味处理能减少褐变,较好的保持产品的色泽。但烫漂过程会造成甘蓝中硒损失20-30%。脱水过程中毛豆中硒的稳定性高于甘蓝。比较不同脱水工艺对甘蓝和毛豆硒含量的影响,其中冷冻干燥对有机硒的保存率最高,硒蛋氨酸是热不稳定成分,在喷雾干燥过程中损失最大,而甲基硒半胱氨酸即使在冷冻干燥过程中也会有一定的损失。在热风烘干过程中,对于硒的降解的影响,温度和时间均为极显著的影响因素,而且时间的影响要大于温度的影响,拟合方程为Y=1.24321-0.00276X1-0.00075X2 (X1为温度,X2为时间)。硒的降解符合一级动力学模型,Boltzmann方程为:表观活化能Ea为44027 J/mol。
最后,研究了甘蓝含硒组分的体外抗肿瘤细胞活性、体内降血糖能力以及长期摄入富硒产品对小鼠体内抗氧化酶活力以及生理生化指标等的影响。结果显示富硒甘蓝和毛豆提取液对于肿瘤细胞NCI-H446均具有一定的抑制作用。富硒甘蓝提取物对于正常小鼠的血糖没有显著影响,但可降低糖尿病小鼠血糖含量,耐糖量也有一定改善,对糖尿病小鼠的受损胰腺细胞具有一定的修复作用。饲喂富硒甘蓝提取物的小鼠体内过氧化物歧化酶、谷胱甘肽酶含量增加,丙二醛含量降低。灌喂富硒甘蓝汁的对照组和富硒组小鼠血液生理生化指标无显著差异,富硒饲喂组无致畸毒性和遗传毒性。富硒蔬菜产品可作安全可靠的功能补硒食品。
Selenium (Se) is an essential nutrient in the human diet and metabolism. Selenium deficiency may lead to heart disease, hypothyroidism and immune malfunction. In recent years, Se compounds have been internationally recognized to have the ability of anti-cancer. The average total human Se intake in China, as reported by the Chinese Nutrition Association, is only 26μg/d, and this is substantially lower than Chinese Preventive Medicine Center’s recommended intake of 50-240μg/d. Epidemiological studies indicate if adults are supplemented with 200μg/d selenium, the cancer morbidity and mortality may reduce about 50%. Different sources and the chemical form of selenium compounds have different safety and biological functionality levels. Se of plant origin is mainly in the form of selenium amino acids that have higher bioavailability and will not easily cause toxic accumulation in the human body. Therefore, the use of vegetable as a vector of organic selenium enrichment is a appropriate choice. This thesis studied the Selenium bioconversion in vegetables, the formation mechanism of organic selenium, and the processing effects of selenium enriched. The results can provide the basic knowledge for application and the guidance in the industrialization of functional food containing this trace elements.
First, flowering Chinese cabbage (Brassica campestris L. ssp. Chinensis (L.), cabbage (Brassica oleracea var. capitata L.) edamame(Glycine Max (L.) Merrill.) and asparagus (Asparagus officinalis L.) were selected as the selenium enrichment test materials for studying inorganic selenium uptake and transfer pattern in vegetables and effects of selenium treatment on nutritional ingredients for the vegetables. The results show that selenium was a transportable and recyclable element in vegetables. Within certain ranges of spraying concentration, selenium content enriched was linearly correlated with the concentration of NaSeO32-. Twice spraying with lower concentration of NaSeO32- and adequate adjustment of pH improved selenium absorption in vegetables. Spraying of selenium on cabbage at a later growth phase led to higher selenium accumulation in vegetables. Selenium treatment with appropriate concentrations increased protein, vitamin C and chlorophyll synthesis, whereas decreased soluble sugar content. Selenium enrichment did not change the amino acid type and distribution, However, the molecular weight distribution tests and electrophoresis showed that high molecular weight protein in edamame (100kDa) decreased, whereas medium and small molecular weight protein increased. Selenium enriched edamame had a decreased oil content, in which unsaturated fatty acids rate reduced and saturated fatty acids rate increased.
Second, selenium component extraction method and speciation were studied. Through HCl buffer and ultrasound-assisted extraction method, selenium component in cabbage and soybean can be extracted. Results show that the main selenium form in cabbage is in free state, and main selenium form in edamame is protein binding. Using 0.01 HCl buffer, assisted with 120 sec ultrasonic probe, most selenium in cabbage can be extracted. For edamame selenium extraction, it is necessary to adopt proteolytic processing. Ultrasound can be used to deal with selenium soybean protein to shorten the enzyme process to 180s. Derivative of selenium amino acid in cabbage and edamame was done with ethyl chloride formate. Then, they were detected with gas chromatography mass mass spectrometry. Chemical analysis of selenium showed that main selenium species in cabbage was Se-methyl-selenocysteine although selenomethionine and selenocystein were also detected. For edamame, the main selenium species was selenomethionine.
Further, effect of the cabbage and soybean juice process and the dehydrate process on selenium content were studied. When fresh cabbage and edamame were stored under 4℃for 4 weeks,the selenium degradation rate in cabbage was up to 30-45% and soybean selenium was relatively stable, with a degradation rate at about 10%. Blanching and seasoning could reduce the browning degree, and maintain the green color. But blanching process caused the loss of selenium in cabbage at about 20-30%. Edamame selenium was more stable than cabbage during the dehydrated processing. Comparing different dehydration process, freeze-drying had the highest preservation rate for organic selenium. Selenomethionine is a heat-labile component, and the spray drying process resulted in the highest loss of selenomethionine. Met-Secysteine had a certain extent loss even in the freeze-drying process. In the drying process, temperature and time were the most significant factors for the degradation of selenium but the latter had even greater impact . Fitting equation is Y=1.24321-0.00276X1-0.00075X2 (X1 is temperature , X2 is time). Se degradation agreed with a dynamic model of the Boltzmann equation: with the apparent activation energy (Ea) of 44,027 J/mol.
Finally, the extrac of selenium cabbage was used to study in-vitro antitumor activity and the antidiabetic mechanisms in type II diabetic mice. A long-term intake of selenium enriched products for mice was done to study the antioxidant enzymes and physiological and biochemical indicators. Cytotoxic bioactivity study on H446 cell line showed that selenium extract had stronger inhibition in vitro. The cell morphology sticked to the bottle wall showed bigger change and the sum of cells was decreased distinctly than the control treated under microscope. The higher concentration the samples had, the more obvious phenomenon of cell apoptosis was. Selenium cabbage extract had no significant effect on normal mice, but could reduce blood glucose levels in diabetic mice. After gastric perfusion of selenium, mice index of SOD, GSH-Px increased and MDA reduced, blood physiological and biochemical indicators of the control group of mice and selenium-enriched group had no significant difference. Teratogenic toxicity and genetic toxicity of selenium enriched juice showed that selenium vegetable products were safe and reliable to be used as functional food.
引文
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