苦荞蛋白的制备、生物活性鉴定及其在猪群中的应用
发布时间：2018-07-26 13:38
【摘要】：荞麦是一种粮药兼用的农作物,富含多种营养成分,包括蛋白质、膳食纤维、黄酮类物质以及矿物元素等。苦荞蛋白的氨基酸组成比较特别,其营养价值高于大豆、小麦、玉米等作物。已有研究发现荞麦蛋白或多肽具有抗菌、抗肿瘤、抗氧化、降胆固醇等多种生理活性,因此在饲料添加剂方面有广阔的应用前景。本研究成功提取了苦荞蛋白,并对其体内、外生物活性和功能进行了探讨,研究包括以下4个方面:1苦荞蛋白的提取与分离纯化采用硫酸铵盐析法提取得到苦荞蛋白粗提物,然后分别采用DEAE-Sepharose离子交换和Sephadex G-75凝胶过滤层析对苦荞蛋白粗提物进行分离纯化,SDS-PAGE法分别测定了苦荞蛋白粗提物和分离峰的分子量。结果发现苦荞蛋白粗提物中的蛋白分子量大小主要介于11KDa-60KDa之间,而活性峰的分子量为38KDa,将其命名为苦荞盐溶性球蛋白(WSBWP)。2苦荞盐溶性球蛋白(WSBWP)的结构特征研究分别采用Edman测序法和MALDI-TOF质谱法分析了苦荞盐溶性球蛋白(WSBWP)的一级结构,根据测序结果和Mascot蛋白质数据库搜索结果发现其蛋白质前体共由393个氨基酸残基组成,其中信号肽由22个氨基酸残基组成,成熟肽由371个氛基酸残基组成。该序列与苦荞种子贮存蛋白13S球蛋白的α亚基的N末端氨基酸序列完全一致,说明WSBWP很可能就是苦荞种子贮存蛋白13S球蛋白的酸性亚基。以籽粒苋种子贮存蛋白中的11S球蛋白为模板(同源性为43.65%),在SWISS-MODEL工作平台上进行同源建模,构建了WSBWP的三维模型,与大豆11S球蛋白结构相似,因此WSBWP属于大豆11S球蛋白超家族的一员。3苦荞盐溶性球蛋白(WSBWP)及其活性肽的体外活性研究采用体外研究方法分别检测了苦荞盐溶性球蛋白和其酶解活性肽(Pep-1-4)的抗菌活性、胆酸盐吸附活性、自由基清除能力和抑制肿瘤细胞增殖的活性。抗菌活性检测发现样品对大肠杆菌和金黄色葡萄球菌有抑制作用;体外胆酸盐吸附活性研究表明WSBWP、多肽Pep-1和Pep-3对胆酸钠、牛磺胆酸钠和脱氧胆酸钠三种胆酸盐都有一定的吸附能力,而WSBWP的胆酸盐吸附能力不如多肽强;抗氧化活性研究检测了样品自由基(OH·)的清除能力,发现WSBWP、多肽Pep-1和Pep-3对OH·都有一定的清除能力;MTT法分别检测了苦荞活性肽对人乳腺癌MCF-7细胞、人胃癌MGC80-3细胞和人结肠癌Caco-2细胞的增殖抑制活性,结果该活性肽对MCF-7细胞的增殖抑制作用比较明显,其抑制效果强于对MGC80-3细胞和Caco-2细胞的抑制作用。4苦荞蛋白在猪群中的应用研究初探1)苦荞蛋白在断奶仔猪生产中的应用研究初探在断奶仔猪基础日粮中添加苦荞蛋白后,发现断奶仔猪平均日增重比对照组增加了36.84%,料重比下降了33.33%,腹泻率下降了41.6%,说明食用苦荞蛋白提高了断奶仔猪的生长性能和抗病力。血液生化分析发现实验组仔猪血液中的总胆固醇(TC)水平降低了16.75%,而甘油三酯(TG)水平降低了39.73%,高密度脂蛋白胆固醇(HDL-C)水平明显增加,而且HDL-C与LDL-C的比率增加了32.94%;仔猪血清中的谷丙转氨酶(GPT)水平比对照组高出32.64%,而谷草转氨酶(GOT)水平比对照组高出28.07%,碱性磷酸酶(AKP)水平比对照组略有升高。通过测胃液pH发现苦荞蛋白组的仔猪胃液pH由4.28降至3.69。同时,苦荞蛋白组的仔猪胃的相对重量比对照组增加了20.44%,肝脏相对重量比对照组减少了19.31%。通过Miseq高通量测序得到了断奶仔猪粪便样品的微生物组成多样性,在门水平分析发现样品中的优势菌主要是厚壁菌门(Firmicutes)、拟杆菌(Bacteroidetes)和变形菌门(Proteobacteria)。在属水平分析发现,仔猪食用苦荞蛋白后,肠道中的乳酸菌(Lactobacillus)、瘤胃菌(Ruminococcus)和经粘液真杆菌(Blautia)的含量明显增加;但一些致病菌如普雷沃菌(Prevotella)、链球菌(Streptococcus)的含量明显下降。结果认为,饲料中给仔猪添加苦荞蛋白后可提高断奶仔猪平均日增重、降低料重比,且可改善胃酸分泌,明显降低腹泻率。此外,实验组仔猪的胆固醇、甘油三酯等血液生化指标优于对照组,肠道菌群组成也发生了明显的改善。2)苦荞蛋白在育肥猪生产中的推广应用苦荞蛋白在断奶仔猪取得较好的试验效果后,采用80头体重在80-90 kg的长白猪、大白猪和太湖黑猪(长×大×太)三元杂交的育肥猪为研究对象进行了推广试验。试验过程将育肥猪随机分两组,对照组和实验组各40头猪,试验组日粮中添加5%荞麦蛋白,对照组添加等量的大豆蛋白,饲喂时间持续一个月,每天观察猪只生长状态并记录。试验开始和试验结束前分别将育肥猪空腹12小时,逐头称体重。平时记录采食量并结算饲料消耗情况,计算平均日增重(ADG)、料重比(F/G)以及出栏率。在育肥猪的推广实验表明,实验组育肥猪的平均日增重比对照组增加了22.22%,与对照组相比料重比下降了14.29%(p0.05),提示育肥猪基础日粮添加苦荞蛋白后能够促进育肥猪生长,提高饲料转化率。
[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.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828.5
本文编号：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.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828.5
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