苦蕎蛋白的制備、生物活性鑒定及其在豬群中的應(yīng)用
發(fā)布時間：2018-07-26 13:38
【摘要】：蕎麥?zhǔn)且环N糧藥兼用的農(nóng)作物,富含多種營養(yǎng)成分,包括蛋白質(zhì)、膳食纖維、黃酮類物質(zhì)以及礦物元素等。苦蕎蛋白的氨基酸組成比較特別,其營養(yǎng)價值高于大豆、小麥、玉米等作物。已有研究發(fā)現(xiàn)蕎麥蛋白或多肽具有抗菌、抗腫瘤、抗氧化、降膽固醇等多種生理活性,因此在飼料添加劑方面有廣闊的應(yīng)用前景。本研究成功提取了苦蕎蛋白,并對其體內(nèi)、外生物活性和功能進(jìn)行了探討,研究包括以下4個方面:1苦蕎蛋白的提取與分離純化采用硫酸銨鹽析法提取得到苦蕎蛋白粗提物,然后分別采用DEAE-Sepharose離子交換和Sephadex G-75凝膠過濾層析對苦蕎蛋白粗提物進(jìn)行分離純化,SDS-PAGE法分別測定了苦蕎蛋白粗提物和分離峰的分子量。結(jié)果發(fā)現(xiàn)苦蕎蛋白粗提物中的蛋白分子量大小主要介于11KDa-60KDa之間,而活性峰的分子量為38KDa,將其命名為苦蕎鹽溶性球蛋白(WSBWP)。2苦蕎鹽溶性球蛋白(WSBWP)的結(jié)構(gòu)特征研究分別采用Edman測序法和MALDI-TOF質(zhì)譜法分析了苦蕎鹽溶性球蛋白(WSBWP)的一級結(jié)構(gòu),根據(jù)測序結(jié)果和Mascot蛋白質(zhì)數(shù)據(jù)庫搜索結(jié)果發(fā)現(xiàn)其蛋白質(zhì)前體共由393個氨基酸殘基組成,其中信號肽由22個氨基酸殘基組成,成熟肽由371個氛基酸殘基組成。該序列與苦蕎種子貯存蛋白13S球蛋白的α亞基的N末端氨基酸序列完全一致,說明WSBWP很可能就是苦蕎種子貯存蛋白13S球蛋白的酸性亞基。以籽粒莧種子貯存蛋白中的11S球蛋白為模板(同源性為43.65%),在SWISS-MODEL工作平臺上進(jìn)行同源建模,構(gòu)建了WSBWP的三維模型,與大豆11S球蛋白結(jié)構(gòu)相似,因此WSBWP屬于大豆11S球蛋白超家族的一員。3苦蕎鹽溶性球蛋白(WSBWP)及其活性肽的體外活性研究采用體外研究方法分別檢測了苦蕎鹽溶性球蛋白和其酶解活性肽(Pep-1-4)的抗菌活性、膽酸鹽吸附活性、自由基清除能力和抑制腫瘤細(xì)胞增殖的活性�？咕钚詸z測發(fā)現(xiàn)樣品對大腸桿菌和金黃色葡萄球菌有抑制作用;體外膽酸鹽吸附活性研究表明WSBWP、多肽Pep-1和Pep-3對膽酸鈉、�；悄懰徕c和脫氧膽酸鈉三種膽酸鹽都有一定的吸附能力,而WSBWP的膽酸鹽吸附能力不如多肽強(qiáng);抗氧化活性研究檢測了樣品自由基(OH·)的清除能力,發(fā)現(xiàn)WSBWP、多肽Pep-1和Pep-3對OH·都有一定的清除能力;MTT法分別檢測了苦蕎活性肽對人乳腺癌MCF-7細(xì)胞、人胃癌MGC80-3細(xì)胞和人結(jié)腸癌Caco-2細(xì)胞的增殖抑制活性,結(jié)果該活性肽對MCF-7細(xì)胞的增殖抑制作用比較明顯,其抑制效果強(qiáng)于對MGC80-3細(xì)胞和Caco-2細(xì)胞的抑制作用。4苦蕎蛋白在豬群中的應(yīng)用研究初探1)苦蕎蛋白在斷奶仔豬生產(chǎn)中的應(yīng)用研究初探在斷奶仔豬基礎(chǔ)日糧中添加苦蕎蛋白后,發(fā)現(xiàn)斷奶仔豬平均日增重比對照組增加了36.84%,料重比下降了33.33%,腹瀉率下降了41.6%,說明食用苦蕎蛋白提高了斷奶仔豬的生長性能和抗病力。血液生化分析發(fā)現(xiàn)實(shí)驗(yàn)組仔豬血液中的總膽固醇(TC)水平降低了16.75%,而甘油三酯(TG)水平降低了39.73%,高密度脂蛋白膽固醇(HDL-C)水平明顯增加,而且HDL-C與LDL-C的比率增加了32.94%;仔豬血清中的谷丙轉(zhuǎn)氨酶(GPT)水平比對照組高出32.64%,而谷草轉(zhuǎn)氨酶(GOT)水平比對照組高出28.07%,堿性磷酸酶(AKP)水平比對照組略有升高。通過測胃液pH發(fā)現(xiàn)苦蕎蛋白組的仔豬胃液pH由4.28降至3.69。同時,苦蕎蛋白組的仔豬胃的相對重量比對照組增加了20.44%,肝臟相對重量比對照組減少了19.31%。通過Miseq高通量測序得到了斷奶仔豬糞便樣品的微生物組成多樣性,在門水平分析發(fā)現(xiàn)樣品中的優(yōu)勢菌主要是厚壁菌門(Firmicutes)、擬桿菌(Bacteroidetes)和變形菌門(Proteobacteria)。在屬水平分析發(fā)現(xiàn),仔豬食用苦蕎蛋白后,腸道中的乳酸菌(Lactobacillus)、瘤胃菌(Ruminococcus)和經(jīng)粘液真桿菌(Blautia)的含量明顯增加;但一些致病菌如普雷沃菌(Prevotella)、鏈球菌(Streptococcus)的含量明顯下降。結(jié)果認(rèn)為,飼料中給仔豬添加苦蕎蛋白后可提高斷奶仔豬平均日增重、降低料重比,且可改善胃酸分泌,明顯降低腹瀉率。此外,實(shí)驗(yàn)組仔豬的膽固醇、甘油三酯等血液生化指標(biāo)優(yōu)于對照組,腸道菌群組成也發(fā)生了明顯的改善。2)苦蕎蛋白在育肥豬生產(chǎn)中的推廣應(yīng)用苦蕎蛋白在斷奶仔豬取得較好的試驗(yàn)效果后,采用80頭體重在80-90 kg的長白豬、大白豬和太湖黑豬(長×大×太)三元雜交的育肥豬為研究對象進(jìn)行了推廣試驗(yàn)。試驗(yàn)過程將育肥豬隨機(jī)分兩組,對照組和實(shí)驗(yàn)組各40頭豬,試驗(yàn)組日糧中添加5%蕎麥蛋白,對照組添加等量的大豆蛋白,飼喂時間持續(xù)一個月,每天觀察豬只生長狀態(tài)并記錄。試驗(yàn)開始和試驗(yàn)結(jié)束前分別將育肥豬空腹12小時,逐頭稱體重。平時記錄采食量并結(jié)算飼料消耗情況,計(jì)算平均日增重(ADG)、料重比(F/G)以及出欄率。在育肥豬的推廣實(shí)驗(yàn)表明,實(shí)驗(yàn)組育肥豬的平均日增重比對照組增加了22.22%,與對照組相比料重比下降了14.29%(p0.05),提示育肥豬基礎(chǔ)日糧添加苦蕎蛋白后能夠促進(jìn)育肥豬生長,提高飼料轉(zhuǎn)化率。
[Abstract]:Buckwheat is a kind of grain and medicine, rich in a variety of nutrients, including protein, dietary fiber, flavonoids and mineral elements. The amino acid composition of tartary buckwheat protein is very special, its nutritional value is higher than soybean, wheat, corn and other crops. It has been found that buckwheat protein or polypeptide has antibacterial, anti-tumor, antioxidation, Cholesterol reduction and other physiological activities, so it has a broad application prospect in the feed additives. This study successfully extracted the buckwheat protein, and discussed the biological activity and function in the body. The study includes the following 4 aspects: 1 Extraction and purification of Tartary Buckwheat Protein by ammonium sulfate extraction and extraction of Tartary Buckwheat Protein crude protein The crude extract of tartary buckwheat protein was separated and purified by DEAE-Sepharose ion exchange and Sephadex G-75 gel filtration chromatography. The molecular weight of crude buckwheat protein extract and separation peak was measured by SDS-PAGE method respectively. The results showed that the molecular weight of the crude extract of tartary buckwheat protein was mainly between the 11KDa-60KDa and the activity of the buckwheat protein, and the activity of the crude extract of tartary buckwheat protein was found to be between the contents of the crude extract and the activity of the buckwheat protein. The molecular weight of the peak is 38KDa, which is named as the structure characteristics of Tartary Buckwheat salt soluble globulin (WSBWP).2 bitter buckwheat salt soluble globulin (WSBWP), and the first order structure of salt soluble globulin (WSBWP) of Tartary Buckwheat (WSBWP) is analyzed by Edman sequencing and MALDI-TOF mass spectrometry, and its protein is found by the result of sequencing and the search results of Mascot protein database. The precursor was composed of 393 amino acid residues, in which the signal peptide was composed of 22 amino acid residues, and the mature peptide was composed of 371 aromatic acid residues. The sequence was identical with the N terminal amino acid sequence of the alpha subunit of tartary buckwheat seed protein 13S globulin, which indicated that WSBWP was very likely to be an acid subunit of the tartary buckwheat seed storage protein 13S globulin. Based on the 11S globulin of the seed storage protein of grain amaranth as a template (homology 43.65%), the homologous modeling on the SWISS-MODEL working platform was carried out, and a three-dimensional model of WSBWP was constructed, which was similar to the structure of soybean 11S globulin. Therefore, WSBWP belongs to a member of the soy 11S globulin superfamily,.3 bitter buckwheat salt soluble globulin (WSBWP) and its active peptide. In vitro studies, the antibacterial activity of Tartary Buckwheat salt soluble globulin and its enzyme hydrolysable peptide (Pep-1-4), the activity of cholate adsorption, free radical scavenging ability and the activity of inhibiting the proliferation of tumor cells were detected in vitro. The antibacterial activity detection showed that the antibacterial activity was found to inhibit the Escherichia coli and Staphylococcus aureus. The adsorption activity of cholate shows that WSBWP, polypeptide Pep-1 and Pep-3 have a certain adsorption capacity for three kinds of cholate salts, such as sodium cholate, sodium taurocholate and deoxycholate, while the adsorption capacity of WSBWP cholate is not better than that of polypeptides; the antioxidant activity studies the scavenging ability of the free radical (OH) of the sample, and finds WSBWP, polypeptide Pep-1 and Pep-3 to OH. The inhibitory activity of Tartary Buckwheat active peptide on human breast cancer MCF-7 cells, human gastric cancer MGC80-3 cells and human colon cancer Caco-2 cells was detected by MTT method. The inhibitory effect of the active peptide on MCF-7 cell proliferation was more obvious, and its inhibitory effect was stronger than that of MGC80-3 cells and Caco-2 cells. Study on the application of Tartary Buckwheat Protein in pigs 1) study on the application of Tartary Buckwheat Protein in the production of weanling piglets: after adding buckwheat protein to the basal diet of weanling piglets, the average daily gain of weanling piglets increased by 36.84%, the ratio of weight to weight decreased by 33.33%, and the diarrhea rate decreased by 41.6%, indicating that the protein of edible buckwheat was improved. The growth performance and resistance of weanling piglets. The blood biochemical analysis showed that the total cholesterol (TC) level in the blood of the experimental group decreased by 16.75%, the triglyceride (TG) level decreased by 39.73%, the high density lipoprotein cholesterol (HDL-C) level increased significantly, and the ratio of HDL-C to LDL-C increased by 32.94%. The level of enzyme (GPT) was 32.64% higher than that of the control group, while the level of GOT was 28.07% higher than that of the control group, and the alkaline phosphatase (AKP) level was slightly higher than that of the control group. The pH of the piglet gastric juice of the buckwheat protein group was reduced to 3.69. from 4.28, and the relative weight of the stomach in the buckwheat protein group was 20.44% higher than that of the control group. The relative weight of the dirty relative weight was reduced by 19.31%. by Miseq high throughput sequencing to obtain the microbial diversity of the weaned piglets' fecal samples. In the gate level analysis, the dominant bacteria in the sample were mainly Firmicutes, Bacteroidetes and Proteobacteria. After the use of buckwheat protein, the content of lactic acid bacteria (Lactobacillus) in the intestinal tract, Ruminococcus and Blautia increased obviously, but some pathogenic bacteria, such as Prevotella and Streptococcus, decreased obviously. The results showed that the weanling piglets could be improved by adding buckwheat protein in the feed. In addition, the blood biochemical indexes such as cholesterol and triglyceride in the experimental group were better than those of the control group, and the composition of the intestinal flora was obviously improved by.2). The buckwheat protein in the production of fattening pigs was popularized and applied to the weanling piglets. After the experimental results, the breeding pigs with 80 weight of 80-90 kg long white pig, big white pig and Taihu black pig (long x Tai x too) were used for the study. The finishing pigs were randomly divided into two groups, 40 pigs in the control group and the experimental group, 5% buckwheat protein in the experimental group and the same amount of soybean in the control group. Protein, the feeding time lasted for one month, and the growth state of the pig was observed every day and recorded. The fattening pig's empty stomach was weighed for 12 hours before and before the end of the experiment. The average daily gain (ADG), the weight ratio (F/G) and the column rate were recorded and the feed consumption was recorded. The average daily gain of the fattening pig was 22.22% higher than that of the control group, and the ratio of feed weight to the control group was 14.29% (P0.05). It suggested that the fattening pig's basal diet supplemented with buckwheat protein could promote the growth of fattening pigs and raise the feed conversion rate.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S828.5
本文編號：2146184
[Abstract]:Buckwheat is a kind of grain and medicine, rich in a variety of nutrients, including protein, dietary fiber, flavonoids and mineral elements. The amino acid composition of tartary buckwheat protein is very special, its nutritional value is higher than soybean, wheat, corn and other crops. It has been found that buckwheat protein or polypeptide has antibacterial, anti-tumor, antioxidation, Cholesterol reduction and other physiological activities, so it has a broad application prospect in the feed additives. This study successfully extracted the buckwheat protein, and discussed the biological activity and function in the body. The study includes the following 4 aspects: 1 Extraction and purification of Tartary Buckwheat Protein by ammonium sulfate extraction and extraction of Tartary Buckwheat Protein crude protein The crude extract of tartary buckwheat protein was separated and purified by DEAE-Sepharose ion exchange and Sephadex G-75 gel filtration chromatography. The molecular weight of crude buckwheat protein extract and separation peak was measured by SDS-PAGE method respectively. The results showed that the molecular weight of the crude extract of tartary buckwheat protein was mainly between the 11KDa-60KDa and the activity of the buckwheat protein, and the activity of the crude extract of tartary buckwheat protein was found to be between the contents of the crude extract and the activity of the buckwheat protein. The molecular weight of the peak is 38KDa, which is named as the structure characteristics of Tartary Buckwheat salt soluble globulin (WSBWP).2 bitter buckwheat salt soluble globulin (WSBWP), and the first order structure of salt soluble globulin (WSBWP) of Tartary Buckwheat (WSBWP) is analyzed by Edman sequencing and MALDI-TOF mass spectrometry, and its protein is found by the result of sequencing and the search results of Mascot protein database. The precursor was composed of 393 amino acid residues, in which the signal peptide was composed of 22 amino acid residues, and the mature peptide was composed of 371 aromatic acid residues. The sequence was identical with the N terminal amino acid sequence of the alpha subunit of tartary buckwheat seed protein 13S globulin, which indicated that WSBWP was very likely to be an acid subunit of the tartary buckwheat seed storage protein 13S globulin. Based on the 11S globulin of the seed storage protein of grain amaranth as a template (homology 43.65%), the homologous modeling on the SWISS-MODEL working platform was carried out, and a three-dimensional model of WSBWP was constructed, which was similar to the structure of soybean 11S globulin. Therefore, WSBWP belongs to a member of the soy 11S globulin superfamily,.3 bitter buckwheat salt soluble globulin (WSBWP) and its active peptide. In vitro studies, the antibacterial activity of Tartary Buckwheat salt soluble globulin and its enzyme hydrolysable peptide (Pep-1-4), the activity of cholate adsorption, free radical scavenging ability and the activity of inhibiting the proliferation of tumor cells were detected in vitro. The antibacterial activity detection showed that the antibacterial activity was found to inhibit the Escherichia coli and Staphylococcus aureus. The adsorption activity of cholate shows that WSBWP, polypeptide Pep-1 and Pep-3 have a certain adsorption capacity for three kinds of cholate salts, such as sodium cholate, sodium taurocholate and deoxycholate, while the adsorption capacity of WSBWP cholate is not better than that of polypeptides; the antioxidant activity studies the scavenging ability of the free radical (OH) of the sample, and finds WSBWP, polypeptide Pep-1 and Pep-3 to OH. The inhibitory activity of Tartary Buckwheat active peptide on human breast cancer MCF-7 cells, human gastric cancer MGC80-3 cells and human colon cancer Caco-2 cells was detected by MTT method. The inhibitory effect of the active peptide on MCF-7 cell proliferation was more obvious, and its inhibitory effect was stronger than that of MGC80-3 cells and Caco-2 cells. Study on the application of Tartary Buckwheat Protein in pigs 1) study on the application of Tartary Buckwheat Protein in the production of weanling piglets: after adding buckwheat protein to the basal diet of weanling piglets, the average daily gain of weanling piglets increased by 36.84%, the ratio of weight to weight decreased by 33.33%, and the diarrhea rate decreased by 41.6%, indicating that the protein of edible buckwheat was improved. The growth performance and resistance of weanling piglets. The blood biochemical analysis showed that the total cholesterol (TC) level in the blood of the experimental group decreased by 16.75%, the triglyceride (TG) level decreased by 39.73%, the high density lipoprotein cholesterol (HDL-C) level increased significantly, and the ratio of HDL-C to LDL-C increased by 32.94%. The level of enzyme (GPT) was 32.64% higher than that of the control group, while the level of GOT was 28.07% higher than that of the control group, and the alkaline phosphatase (AKP) level was slightly higher than that of the control group. The pH of the piglet gastric juice of the buckwheat protein group was reduced to 3.69. from 4.28, and the relative weight of the stomach in the buckwheat protein group was 20.44% higher than that of the control group. The relative weight of the dirty relative weight was reduced by 19.31%. by Miseq high throughput sequencing to obtain the microbial diversity of the weaned piglets' fecal samples. In the gate level analysis, the dominant bacteria in the sample were mainly Firmicutes, Bacteroidetes and Proteobacteria. After the use of buckwheat protein, the content of lactic acid bacteria (Lactobacillus) in the intestinal tract, Ruminococcus and Blautia increased obviously, but some pathogenic bacteria, such as Prevotella and Streptococcus, decreased obviously. The results showed that the weanling piglets could be improved by adding buckwheat protein in the feed. In addition, the blood biochemical indexes such as cholesterol and triglyceride in the experimental group were better than those of the control group, and the composition of the intestinal flora was obviously improved by.2). The buckwheat protein in the production of fattening pigs was popularized and applied to the weanling piglets. After the experimental results, the breeding pigs with 80 weight of 80-90 kg long white pig, big white pig and Taihu black pig (long x Tai x too) were used for the study. The finishing pigs were randomly divided into two groups, 40 pigs in the control group and the experimental group, 5% buckwheat protein in the experimental group and the same amount of soybean in the control group. Protein, the feeding time lasted for one month, and the growth state of the pig was observed every day and recorded. The fattening pig's empty stomach was weighed for 12 hours before and before the end of the experiment. The average daily gain (ADG), the weight ratio (F/G) and the column rate were recorded and the feed consumption was recorded. The average daily gain of the fattening pig was 22.22% higher than that of the control group, and the ratio of feed weight to the control group was 14.29% (P0.05). It suggested that the fattening pig's basal diet supplemented with buckwheat protein could promote the growth of fattening pigs and raise the feed conversion rate.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S828.5
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