硒在农作物生长过程的富集及大豆硒蛋白生物功效研究

发布时间：2018-09-16 21:07

【摘要】：本文针对江西丰城“中国生态硒谷”不同地区含硒土壤种植的水稻(水田、蛋白与淀粉为主)、大豆(旱地、蛋白与油脂为主),从分析其土壤天然硒含量、硒形态研究了不同农作物(水稻、大豆)富硒机理及迁移规律,土壤硒含量对农作物营养成分,加工过程对农作物硒含量和形态变化的影响,对富硒大豆蛋白肽安全性进行了评价,主要研究结论如下。(1)研究了江西丰城“生态硒谷”区域内土壤的性质、水稻和大豆生长过程各组织(根、茎、叶、种子)中硒分布情况以及对其他重金属富集的影响。通过分析丰城不同地区的稻田和旱地土壤中硒形态,发现在董村(C区)稻田和旱地的土壤中水溶性Se含量是蕉坑(A区)的10倍。董村(C区)稻田和旱地土壤中的酸溶态硒、可交换态硒、有机物结合态硒的含量要远高于蕉坑(A区)。这4种形态硒能直接或间接的能被植物吸收利用,导致两个地区所生产出产品中所含硒量有着明显的差异。水稻由秧苗期到幼穗发育期生长过程中,根、茎、叶中硒含量都随着水稻的生长而增加,如茎中硒含量分别为0.104 mg/kg(秧苗期)、0.174 mg/kg(返青期)、0.192 mg/kg(分蘖期)和0.199 mg/kg(幼穗发育期)。但是,从开花结实期到果实成熟期时,根、茎、叶中硒含量都随着水稻的生长而减少,茎中硒含量分别为0.174 mg/kg(开花结实期)、0.156 mg/kg(果实成熟期)。硒在大豆生长过程中的迁移变化情况与水稻类似。天然富硒土壤中硒转移趋势:由高硒含量的土壤先转移到植物(水稻与大豆)的非食用部分组织(根、茎、叶)中,而后逐渐向植物果实(大米、大豆)中迁徙,在硒浓度梯度上,土壤中硒含量根、茎、叶中硒含量果实中硒含量。Cd和Pb在水稻生长期间,叶子中含量与生长时间呈正相关的关系。如Cd和Pb在A区的水稻分蘖期叶中含量分别为2.158 mg/kg和6.897 mg/kg,随着水稻生长到幼穗发育期时,叶子中Cd和Pb含量分别增加到3.787 mg/kg和7.158mg/kg。但是,在水稻的每个生长期内,叶子中Cd和Pb的含量在高硒区(C区)均低于低硒区(A区)中的含量。如,在水稻返青期时,叶子中的Cd在A区和C区的含量分别为1.574 mg/kg和1.412 mg/kg。同时,研究表明在整个生长期内,高硒区的Cd和Pb积累速率比低硒区(A区)的积累速率缓慢。结果表明,在水稻生长过程中,适量的硒浓度对重金属Cd和Pb起到了一定的拮抗作用。(2)不同硒含量(低、中、高硒区)的天然富硒土壤中,随着硒含量的增加,硒含量在精米和米糠中增加的幅度大于硒在稻壳中的积累程度,说明在含硒高的土壤中,硒更多积累在精米和米糠中。不同地区的天然富硒稻谷和大豆的千粒重均有一些变化,但各试验组间没有显著差异;稻谷和大豆籽粒的营养品质发生变化,其中,C区稻谷中粗蛋白质含量(7.23%)比A区蛋白质含量出现显著性提高,蛋白质的含量从6.61%(A区)提高到7.23%,增加了8.5%;C区大豆蛋白质含量(35.08%)比A区蛋白质含量也出现显著性提高,但是,脂肪酸的含量随着硒含量的提高而下降,由A区的18.90%下降到16.52%,下降了12.64%。不同硒含量对大豆蛋白质分子量分布的影响,研究结果表明,硒含量的不同对于蛋白质的种类和分子量没有改变,但是,对于大豆蛋白的四级结构产生了一定影响。根据红外光谱分析可知,在M800cm-1的区域内,低硒区的大豆蛋白质的图谱无明显的吸收峰,而高硒区的富硒大豆(C区)蛋白质在601cm-1和636cm-1处各有一个C=Se的吸收峰,在551cm-1处有C-Se的吸收峰,在830cm-1有Se=O的吸收峰,在2258c m-1处有个Se-H的吸收峰。不同硒含量对大豆蛋白质组成和氨基酸组成的影响,结果表明,不同的硒含量不会改变大豆蛋白质亚基组成,从SDS-PAGE图谱中可以看出,条带没有增加,也没有减少,蛋白质分子量的范围在100-600.0k Da之间。但是,对氨基酸组成有一定的影响,当土壤中硒含量越高,半胱氨酸(Cys)和蛋氨酸(Met)的含量越低,在低硒区(A区)大米蛋白质中的半胱氨酸(Cys)含量为0.682%,而在高硒区(C区)的含量只有0.102%;在低硒区(A区)大米蛋白质中的蛋氨酸(Met)含量为0.523%,而在高硒区(C区)的含量只有0.112%。不同含硒量对大豆脂肪酸组成的影响,结果表明,不同硒含量的大豆油脂中脂肪酸的组成随着不同硒含量而发生了相关的变化,如大豆油脂脂肪酸中的十六酸、十七酸、二十碳烯酸、二十二碳烯酸所占的比例有变化,但是它们之间没有显著差异。大豆油脂中的软脂酸和硬脂酸之间的比例随着硒含量的增加表现为上升趋势关系,但亚油酸和亚麻酸的比例表现为相反的关系。(3)提取剂0.1 mol/LNa OH提取天然富硒大豆硒蛋白效果最好,蛋白质的得率及其硒含量都最高,蛋白质得率为55.75%,硒含量为49.55mg/kg,表明碱法提取富硒大豆蛋白质效率高。正交法提取天然富硒大豆蛋白质最佳工艺参数为:p H10.5、提取温度55℃、提取时间60 min、液料比14∶1,高硒区(C区)大豆质白质的提取率是87.58%,硒含量为53.78mg/kg。正交法制备天然富硒大豆蛋白肽最佳工艺参数:选用风味蛋白酶、酶解时间80min、液固比10:1、反应温度50℃、加酶量0.4%,酶解率达到72.2%,蛋白肽纯度89.8%,硒含量94.65mg/kg。采用分级超滤技术对酶解液进行超滤时,截留分子量5000Da的超滤膜,寡肽得率为78.18%,硒含量125.70 mg/kg。不同硒含量天然富硒大豆蛋白质对·OH、O2-·、DPPH的影响,结果表明,随着浓度的升高(0.2~0.6 mg/m L),不同硒含量的大豆蛋白质对·OH的清除率增加。在浓度为0.6 mg/m L时,高硒区(C区)大豆蛋白质、抗坏血酸、BHT(2,6-二叔丁基-4-甲基苯酚)和低硒区(A区)大豆蛋白质对·OH的清除率分别为48.7%,85.5%,26.5%和8.62%,表明硒对·OH的清除效果起到重要作用。当样品浓度为1.0 mg/m L时,高硒区(C区)和低硒区(A区)天然富硒大豆蛋白质对O2-·的清除率分别是22.52%和5.24%,高硒区(C区)的清除率是低硒区(A区)的3倍,其原因是它们中含硒含量的不同引起的。对DPPH的清除效果方面,在浓度范围为0.2~1.0 mg/m L内,高硒区(C区)天然富硒大豆蛋白质对DPPH·的清除率相应的由24.46%(0.2 mg/m L)增长到49.48%(1.0 mg/m L)。(4)随着挤压膨化温度的升高,膨化米粉中的水分损失量呈增大趋势,膨化米粉的沉淀率降低、溶解率和沉淀吸水率增加,且有显著性差异;水溶性糖含量升高,在挤压膨化温度为140-150℃时水溶性糖含量最低,当温度上升到160℃,水溶性糖含量由36.4%上升到38.1%。挤压膨化前、后大米蛋白质的组成发生了变化,未膨化的富硒大米蛋白质中没有分子量为85.0k D的谱带,但是存在25.0k D谱带,大米挤压膨化的各个不同温度段内,膨化米粉的蛋白质组成没有发生显著变化。膨化米粉中蛋白质的吸水性、持水性和起泡性均高于未膨化挤压的大米蛋白质,并随膨化温度升高,呈增大的趋势,但其吸油性呈相反趋势,均低于未挤压膨化的大米蛋白质(E)的吸油性,并随温度升高,蛋白吸油性变小。膨化后米粉的休止角随温度升高而变大,同时膨化后米粉的滑动角与休止角表现相同的结果。SEM微观结构表明,未膨化大米颗粒表面粗糙,结合疏散,大小不均一。挤压膨化后的膨化米粉的表面形成光滑状,其内部空腔与原大米相比,明显增大,整个组织疏松,表现为多孔海绵状结构,但分布均匀。但是,当温度超过170°C后,表面出现裂隙,并随着温度进一步升高,裂隙越明显。X-ray衍射线表明,未膨化的富硒大米X-ray衍射线中高级微晶区明显比膨化后的要多,且变化显著;而在不同温度范围下各膨化米粉X-ray衍射曲线变化不明显。(5)天然富硒大豆酶解物安全性毒理学评价结果显示,富硒大豆肽LD50大于20g/kg·bw,Ames试验和骨髓细胞微核试验结果未见致突变作用,30d喂养试验结果显示试验期间动物未出现拒食现象,动物生长正常,被长浓密、有光泽,表明天然富硒大豆蛋白肽具有良好的食用安全性。对小鼠肿瘤生长的影响结果表明,富硒大豆蛋白肽显著抑制了肿瘤生长(最大抑制率80.2%),可延长染病小鼠寿命3~5 d,对肿瘤辅助治疗有积极作用。正常小鼠补充富硒大豆蛋白肽(202.50μg/kg剂量),血清中谷胱甘肽过氧化物酶(GPX酶)的活性增加了1.2倍,而血清中脂质过氧化产物(LPO)含量表现为下降的情况。对于荷瘤小鼠,随着富硒大豆蛋白肽补充剂量增加,血清GPX酶活最大可增加1.5倍,血清LPO含量则呈降低趋势。在小鼠免疫功能方面,在202.50μg/kg剂量范围,补充富硒大豆蛋白肽能促进免疫器官特别是胸腺发育、增强免疫调节能力。对S180肿瘤细胞形态影响,表明随富硒大豆蛋白肽浓度增加,细胞逐渐收缩变小,胞膜皱缩,胞质颗粒增多,大量细胞碎裂,最终致使其凋亡,表明富硒大豆肽具有明显的抗肿瘤作用。
[Abstract]:In this paper, rice (paddy field, mainly with protein and starch) and soybean (mainly dry land, mainly with protein and oil) were planted in different selenium-containing soils of Fengcheng "China Ecological Selenium Valley" in Jiangxi Province. The selenium-enriched mechanism and migration law of different crops (rice, soybean) were studied by analyzing the natural selenium content in soil, selenium forms, and the effect of selenium content in soil on the nutrition of crops. The main conclusions are as follows: (1) Soil properties, selenium distribution in rice and soybean tissues (roots, stems, leaves, seeds) and other weights during the growing process of rice and soybean in Fengcheng ecological selenium valley, Jiangxi Province were studied. By analyzing the forms of selenium in paddy and upland soils in different areas of Fengcheng, it was found that the content of water soluble Se in paddy and upland soils in Dongcun (area C) was 10 times higher than that in Jiaokeng (area A). The content of acid soluble Se in paddy and upland soils in Dongcun (area C) was much higher than that in Jiaokeng (area A). (3) Selenium content in roots, stems and leaves increased with the growth of rice from seedling stage to young panicle stage. For example, the selenium content in stems was 0.104 mg/kg (seedling stage) and 0.1 mg/kg (seedling stage) respectively. 74 mg / kg (turning green), 0.192 mg / kg (tillering) and 0.199 mg / kg (young panicle development). However, selenium content in roots, stems and leaves decreased with the growth of rice from flowering and fruiting stage to fruit ripening stage, and selenium content in stems was 0.174 mg / kg (flowering and fruiting stage) and 0.156 mg / kg (fruit ripening stage) respectively. Selenium transfer trend in natural Se-enriched soils is similar to that in rice. Selenium transfer trend in natural Se-enriched soils is from soil with high Se content to non-edible tissues (roots, stems, leaves) of plants (rice and soybeans), and then gradually to plant fruits (rice, soybeans). On the Se concentration gradient, the Se content in soil is the Se content in roots, stems, leaves and fruits. For example, the contents of Cd and Pb in rice leaves at tillering stage in A region were 2.158 mg/kg and 6.897 mg/kg, respectively. As the rice grew to the young panicle stage, the contents of Cd and Pb in leaves increased to 3.787 mg/kg and 7.158 mg/kg, respectively. The contents of Cd and Pb in leaves in high-selenium area (C area) were lower than those in low-selenium area (A area). For example, the contents of Cd in A area and C area were 1.574 mg/kg and 1.412 mg/kg respectively during rice green-back period. Meanwhile, the accumulation rate of Cd and Pb in high-selenium area was slower than that in low-selenium area (A area). The results showed that proper selenium concentration played an antagonistic role on Cd and Pb during rice growth. (2) Selenium content increased more in milled rice and rice bran than in rice husk in natural selenium-rich soils with different selenium content (low, medium and high selenium area). Selenium was more accumulated in milled rice and rice bran. The 1000-grain weight of natural Se-enriched rice and soybean had some changes in different regions, but there was no significant difference among the experimental groups. The nutritional quality of rice and soybean had changed. The crude protein content (7.23%) in C region was significantly higher than that in A region. The content of protein in C region (35.08%) was significantly higher than that in A region. However, the content of fatty acids decreased with the increase of selenium content, from 18.90% in A region to 16.52% and 12.64% respectively. The results showed that the different selenium content had no effect on the type and molecular weight of protein, but had some effect on the fourth-order structure of soybean protein. There was a C=Se absorption peak at 601cm-1 and 636cm-1 in white matter, a C-Se absorption peak at 551cm-1, a Se=O absorption peak at 830cm-1 and a Se-H absorption peak at 2258cm-1. The results of SDS-PAGE showed that the bands did not increase or decrease, and the protein molecular weight ranged from 100 kDa to 600.0 kDa. However, the amino acid composition was affected to a certain extent. The higher the selenium content in soil, the lower the contents of cysteine (Cys) and methionine (Met) and the lower the content of cysteine (Cys) in the rice protein in the low selenium (A) region. The content of methionine (Met) in low selenium area (A area) and high selenium area (C area) was 0.523% and 0.112% respectively. The effect of different selenium content on the fatty acid composition of soybean oil showed that the fatty acid composition of soybean oil with different selenium content varied with the selenium content. The proportion of hexadecanoic acid, hexadecanoic acid, eicosanoic acid and docosanoic acid in fatty acids of soybean oil changed, but there was no significant difference between them. The ratio of linolenic acid was opposite. (3) The extractant 0.1 mol/LNa OH had the best effect on extracting selenium-rich soybean protein. The protein yield and selenium content were the highest. The protein yield was 55.75% and the selenium content was 49.55 mg/kg. This indicated that the alkaline method was efficient in extracting selenium-rich soybean protein. The optimum technological parameters were as follows: P H10.5, extraction temperature 55, extraction time 60 min, ratio of liquid to material 14 The yield of oligopeptides was 78.18% and the content of selenium was 125.70 mg/kg. The effects of natural selenium-rich soybean protein with different selenium contents on OH, O2 -, DPPH were studied. The results showed that the yield of oligopeptides was 78.18% and the content of selenium was 125.70 mg/kg when the enzyme hydrolysate was ultrafiltered by staged ultrafiltration. When the concentration was 0.6 mg/ml, the scavenging rate of soybean protein with high selenium content (region C), ascorbic acid (region C), BHT (2,6-di-tert-butyl-4-methylphenol) and low selenium content (region A) was 48.7%, 85.5%, 26.5% and 8.62%, respectively. When the concentration of sample was 1.0 mg/ml, the scavenging rate of natural selenium-rich soybean protein in high-selenium area (C area) and low-selenium area (A area) was 22.52% and 5.24%, respectively. The scavenging rate of high-selenium area (C area) was 3 times that of low-selenium area (A area), which was caused by the different selenium content in them. Within the concentration range of 0.2-1.0 mg/ml, the DPPH scavenging rate of natural selenium-rich soybean protein in high-selenium region (C region) increased from 24.46% (0.2 mg/ml) to 49.48% (1.0 mg/ml). (4) With the increase of extrusion temperature, the water loss in extruded rice flour increased, the precipitation rate of extruded rice flour decreased, the solubility and precipitation water absorption of extruded rice flour increased. The content of water soluble sugar was the lowest when extrusion temperature was 140-150 C. When extrusion temperature was 160 C, the content of water soluble sugar increased from 36.4% to 38.1%. The protein composition of the extruded rice flour did not change significantly at different temperatures. The water absorption, water holding capacity and foaming capacity of the protein in the extruded rice flour were higher than those of the non-extruded rice protein, and showed an increasing trend with the increase of extruding temperature. The oil absorption of rice protein (E) was lower than that of non-extruded rice protein (E), and the oil absorption of rice protein decreased with the increase of temperature. Comparing with the original rice, the inner cavity of the extruded rice flour is obviously enlarged, and the whole structure is loose, showing a porous spongy structure, but the distribution is uniform. However, when the temperature exceeds 170 degrees C, the surface cracks appear, and the cracks become more obvious with the temperature rising further. The results of X-ray diffraction showed that the high-grade microcrystalline region in the non-expanded Se-enriched rice X-ray diffraction line was obviously more than that in the expanded rice, and the change was significant, but the X-ray diffraction curves of the expanded rice flour were not obvious in different temperature ranges. (5) The toxicological evaluation of the enzymatic hydrolysate of natural Se-enriched soybean showed that the Se-enriched soybean peptide LD50 was more than 20g/kg.bw, Ames. No mutagenicity was found in the experiment and the micronucleus test of bone marrow cells. The results of 30-day feeding test showed that the animals did not show antifeeding. The animals grew normally, were dense and glossy, indicating that the natural selenium-rich soybean protein peptide had good food safety. It significantly inhibited tumor growth (the maximum inhibition rate was 80.2%), prolonged the life span of infected mice for 3-5 days, and had a positive effect on adjuvant treatment of tumor. As for the tumor-bearing mice, with the increase of Se-enriched soybean protein peptide supplementation dosage, the serum GPX enzyme activity increased by 1.5 times, while the serum LPO content decreased. In the immune function of mice, supplementation of Se-enriched soybean protein peptide could promote the development of immune organs, especially thymus, and enhance the immune regulation. The effect of Se-enriched soybean protein peptide on the morphology of S180 tumor cells showed that with the increase of Se-enriched soybean protein peptide concentration, the cells gradually contracted, membrane shrank, cytoplasmic granules increased, a large number of cells fragmented, and ultimately led to apoptosis, indicating that Se-enriched soybean protein peptide has obvious anti-tumor effect.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511;S565.1

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