【摘要】：本文針對江西豐城“中國生態(tài)硒谷”不同地區(qū)含硒土壤種植的水稻(水田、蛋白與淀粉為主)、大豆(旱地、蛋白與油脂為主),從分析其土壤天然硒含量、硒形態(tài)研究了不同農(nóng)作物(水稻、大豆)富硒機理及遷移規(guī)律,土壤硒含量對農(nóng)作物營養(yǎng)成分,加工過程對農(nóng)作物硒含量和形態(tài)變化的影響,對富硒大豆蛋白肽安全性進行了評價,主要研究結(jié)論如下。(1)研究了江西豐城“生態(tài)硒谷”區(qū)域內(nèi)土壤的性質(zhì)、水稻和大豆生長過程各組織(根、莖、葉、種子)中硒分布情況以及對其他重金屬富集的影響。通過分析豐城不同地區(qū)的稻田和旱地土壤中硒形態(tài),發(fā)現(xiàn)在董村(C區(qū))稻田和旱地的土壤中水溶性Se含量是蕉坑(A區(qū))的10倍。董村(C區(qū))稻田和旱地土壤中的酸溶態(tài)硒、可交換態(tài)硒、有機物結(jié)合態(tài)硒的含量要遠(yuǎn)高于蕉坑(A區(qū))。這4種形態(tài)硒能直接或間接的能被植物吸收利用,導(dǎo)致兩個地區(qū)所生產(chǎn)出產(chǎn)品中所含硒量有著明顯的差異。水稻由秧苗期到幼穗發(fā)育期生長過程中,根、莖、葉中硒含量都隨著水稻的生長而增加,如莖中硒含量分別為0.104 mg/kg(秧苗期)、0.174 mg/kg(返青期)、0.192 mg/kg(分蘗期)和0.199 mg/kg(幼穗發(fā)育期)。但是,從開花結(jié)實期到果實成熟期時,根、莖、葉中硒含量都隨著水稻的生長而減少,莖中硒含量分別為0.174 mg/kg(開花結(jié)實期)、0.156 mg/kg(果實成熟期)。硒在大豆生長過程中的遷移變化情況與水稻類似。天然富硒土壤中硒轉(zhuǎn)移趨勢:由高硒含量的土壤先轉(zhuǎn)移到植物(水稻與大豆)的非食用部分組織(根、莖、葉)中,而后逐漸向植物果實(大米、大豆)中遷徙,在硒濃度梯度上,土壤中硒含量根、莖、葉中硒含量果實中硒含量。Cd和Pb在水稻生長期間,葉子中含量與生長時間呈正相關(guān)的關(guān)系。如Cd和Pb在A區(qū)的水稻分蘗期葉中含量分別為2.158 mg/kg和6.897 mg/kg,隨著水稻生長到幼穗發(fā)育期時,葉子中Cd和Pb含量分別增加到3.787 mg/kg和7.158mg/kg。但是,在水稻的每個生長期內(nèi),葉子中Cd和Pb的含量在高硒區(qū)(C區(qū))均低于低硒區(qū)(A區(qū))中的含量。如,在水稻返青期時,葉子中的Cd在A區(qū)和C區(qū)的含量分別為1.574 mg/kg和1.412 mg/kg。同時,研究表明在整個生長期內(nèi),高硒區(qū)的Cd和Pb積累速率比低硒區(qū)(A區(qū))的積累速率緩慢。結(jié)果表明,在水稻生長過程中,適量的硒濃度對重金屬Cd和Pb起到了一定的拮抗作用。(2)不同硒含量(低、中、高硒區(qū))的天然富硒土壤中,隨著硒含量的增加,硒含量在精米和米糠中增加的幅度大于硒在稻殼中的積累程度,說明在含硒高的土壤中,硒更多積累在精米和米糠中。不同地區(qū)的天然富硒稻谷和大豆的千粒重均有一些變化,但各試驗組間沒有顯著差異;稻谷和大豆籽粒的營養(yǎng)品質(zhì)發(fā)生變化,其中,C區(qū)稻谷中粗蛋白質(zhì)含量(7.23%)比A區(qū)蛋白質(zhì)含量出現(xiàn)顯著性提高,蛋白質(zhì)的含量從6.61%(A區(qū))提高到7.23%,增加了8.5%;C區(qū)大豆蛋白質(zhì)含量(35.08%)比A區(qū)蛋白質(zhì)含量也出現(xiàn)顯著性提高,但是,脂肪酸的含量隨著硒含量的提高而下降,由A區(qū)的18.90%下降到16.52%,下降了12.64%。不同硒含量對大豆蛋白質(zhì)分子量分布的影響,研究結(jié)果表明,硒含量的不同對于蛋白質(zhì)的種類和分子量沒有改變,但是,對于大豆蛋白的四級結(jié)構(gòu)產(chǎn)生了一定影響。根據(jù)紅外光譜分析可知,在M800cm-1的區(qū)域內(nèi),低硒區(qū)的大豆蛋白質(zhì)的圖譜無明顯的吸收峰,而高硒區(qū)的富硒大豆(C區(qū))蛋白質(zhì)在601cm-1和636cm-1處各有一個C=Se的吸收峰,在551cm-1處有C-Se的吸收峰,在830cm-1有Se=O的吸收峰,在2258c m-1處有個Se-H的吸收峰。不同硒含量對大豆蛋白質(zhì)組成和氨基酸組成的影響,結(jié)果表明,不同的硒含量不會改變大豆蛋白質(zhì)亞基組成,從SDS-PAGE圖譜中可以看出,條帶沒有增加,也沒有減少,蛋白質(zhì)分子量的范圍在100-600.0k Da之間。但是,對氨基酸組成有一定的影響,當(dāng)土壤中硒含量越高,半胱氨酸(Cys)和蛋氨酸(Met)的含量越低,在低硒區(qū)(A區(qū))大米蛋白質(zhì)中的半胱氨酸(Cys)含量為0.682%,而在高硒區(qū)(C區(qū))的含量只有0.102%;在低硒區(qū)(A區(qū))大米蛋白質(zhì)中的蛋氨酸(Met)含量為0.523%,而在高硒區(qū)(C區(qū))的含量只有0.112%。不同含硒量對大豆脂肪酸組成的影響,結(jié)果表明,不同硒含量的大豆油脂中脂肪酸的組成隨著不同硒含量而發(fā)生了相關(guān)的變化,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變化,但是它們之間沒有顯著差異。大豆油脂中的軟脂酸和硬脂酸之間的比例隨著硒含量的增加表現(xiàn)為上升趨勢關(guān)系,但亞油酸和亞麻酸的比例表現(xiàn)為相反的關(guān)系。(3)提取劑0.1 mol/LNa OH提取天然富硒大豆硒蛋白效果最好,蛋白質(zhì)的得率及其硒含量都最高,蛋白質(zhì)得率為55.75%,硒含量為49.55mg/kg,表明堿法提取富硒大豆蛋白質(zhì)效率高。正交法提取天然富硒大豆蛋白質(zhì)最佳工藝參數(shù)為:p H10.5、提取溫度55℃、提取時間60 min、液料比14∶1,高硒區(qū)(C區(qū))大豆質(zhì)白質(zhì)的提取率是87.58%,硒含量為53.78mg/kg。正交法制備天然富硒大豆蛋白肽最佳工藝參數(shù):選用風(fēng)味蛋白酶、酶解時間80min、液固比10:1、反應(yīng)溫度50℃、加酶量0.4%,酶解率達(dá)到72.2%,蛋白肽純度89.8%,硒含量94.65mg/kg。采用分級超濾技術(shù)對酶解液進行超濾時,截留分子量5000Da的超濾膜,寡肽得率為78.18%,硒含量125.70 mg/kg。不同硒含量天然富硒大豆蛋白質(zhì)對·OH、O2-·、DPPH的影響,結(jié)果表明,隨著濃度的升高(0.2~0.6 mg/m L),不同硒含量的大豆蛋白質(zhì)對·OH的清除率增加。在濃度為0.6 mg/m L時,高硒區(qū)(C區(qū))大豆蛋白質(zhì)、抗壞血酸、BHT(2,6-二叔丁基-4-甲基苯酚)和低硒區(qū)(A區(qū))大豆蛋白質(zhì)對·OH的清除率分別為48.7%,85.5%,26.5%和8.62%,表明硒對·OH的清除效果起到重要作用。當(dāng)樣品濃度為1.0 mg/m L時,高硒區(qū)(C區(qū))和低硒區(qū)(A區(qū))天然富硒大豆蛋白質(zhì)對O2-·的清除率分別是22.52%和5.24%,高硒區(qū)(C區(qū))的清除率是低硒區(qū)(A區(qū))的3倍,其原因是它們中含硒含量的不同引起的。對DPPH的清除效果方面,在濃度范圍為0.2~1.0 mg/m L內(nèi),高硒區(qū)(C區(qū))天然富硒大豆蛋白質(zhì)對DPPH·的清除率相應(yīng)的由24.46%(0.2 mg/m L)增長到49.48%(1.0 mg/m L)。(4)隨著擠壓膨化溫度的升高,膨化米粉中的水分損失量呈增大趨勢,膨化米粉的沉淀率降低、溶解率和沉淀吸水率增加,且有顯著性差異;水溶性糖含量升高,在擠壓膨化溫度為140-150℃時水溶性糖含量最低,當(dāng)溫度上升到160℃,水溶性糖含量由36.4%上升到38.1%。擠壓膨化前、后大米蛋白質(zhì)的組成發(fā)生了變化,未膨化的富硒大米蛋白質(zhì)中沒有分子量為85.0k D的譜帶,但是存在25.0k D譜帶,大米擠壓膨化的各個不同溫度段內(nèi),膨化米粉的蛋白質(zhì)組成沒有發(fā)生顯著變化。膨化米粉中蛋白質(zhì)的吸水性、持水性和起泡性均高于未膨化擠壓的大米蛋白質(zhì),并隨膨化溫度升高,呈增大的趨勢,但其吸油性呈相反趨勢,均低于未擠壓膨化的大米蛋白質(zhì)(E)的吸油性,并隨溫度升高,蛋白吸油性變小。膨化后米粉的休止角隨溫度升高而變大,同時膨化后米粉的滑動角與休止角表現(xiàn)相同的結(jié)果。SEM微觀結(jié)構(gòu)表明,未膨化大米顆粒表面粗糙,結(jié)合疏散,大小不均一。擠壓膨化后的膨化米粉的表面形成光滑狀,其內(nèi)部空腔與原大米相比,明顯增大,整個組織疏松,表現(xiàn)為多孔海綿狀結(jié)構(gòu),但分布均勻。但是,當(dāng)溫度超過170°C后,表面出現(xiàn)裂隙,并隨著溫度進一步升高,裂隙越明顯。X-ray衍射線表明,未膨化的富硒大米X-ray衍射線中高級微晶區(qū)明顯比膨化后的要多,且變化顯著;而在不同溫度范圍下各膨化米粉X-ray衍射曲線變化不明顯。(5)天然富硒大豆酶解物安全性毒理學(xué)評價結(jié)果顯示,富硒大豆肽LD50大于20g/kg·bw,Ames試驗和骨髓細(xì)胞微核試驗結(jié)果未見致突變作用,30d喂養(yǎng)試驗結(jié)果顯示試驗期間動物未出現(xiàn)拒食現(xiàn)象,動物生長正常,被長濃密、有光澤,表明天然富硒大豆蛋白肽具有良好的食用安全性。對小鼠腫瘤生長的影響結(jié)果表明,富硒大豆蛋白肽顯著抑制了腫瘤生長(最大抑制率80.2%),可延長染病小鼠壽命3~5 d,對腫瘤輔助治療有積極作用。正常小鼠補充富硒大豆蛋白肽(202.50μg/kg劑量),血清中谷胱甘肽過氧化物酶(GPX酶)的活性增加了1.2倍,而血清中脂質(zhì)過氧化產(chǎn)物(LPO)含量表現(xiàn)為下降的情況。對于荷瘤小鼠,隨著富硒大豆蛋白肽補充劑量增加,血清GPX酶活最大可增加1.5倍,血清LPO含量則呈降低趨勢。在小鼠免疫功能方面,在202.50μg/kg劑量范圍,補充富硒大豆蛋白肽能促進免疫器官特別是胸腺發(fā)育、增強免疫調(diào)節(jié)能力。對S180腫瘤細(xì)胞形態(tài)影響,表明隨富硒大豆蛋白肽濃度增加,細(xì)胞逐漸收縮變小,胞膜皺縮,胞質(zhì)顆粒增多,大量細(xì)胞碎裂,最終致使其凋亡,表明富硒大豆肽具有明顯的抗腫瘤作用。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S511;S565.1

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