【作者】 王英丽；

【导师】 吉日木图；

【作者基本信息】 内蒙古农业大学 ， 食品科学， 2021， 博士

【摘要】 随着人口老龄化加快,胶原蛋白系列产品受到消费者的广泛关注,其市场发展前景广阔。胶原蛋白产品受自身家族庞大、原料开发不足、功能活性评价不够全面、科学依据不充分及工业化生产不成熟等因素的制约,为此寻找开发一些新型的胶原蛋白生物原料是必要的措施。本研究以双峰驼驼掌为研究对象,基于蛋白质组和多肽组学研究了驼掌三个组织部位,即驼掌掌筋(ZJC)、驼掌掌垫(ZDC)和驼掌筋膜(JMC)的营养特性、差异肽段及差异蛋白表达规律,并进一步研究了驼掌源胶原蛋白提取工艺及特征、以及不同酶组合制备胶原蛋白肽的生物学功能。(1)常规成分、胶原蛋白和各氨基酸含量分析结果表明,驼掌三个组织存在组织差异性,共同营养特性为P/W值约0.45;驼掌掌垫(ZDC)拥有高L/P的营养特性,且其氨基酸组成更接近于理想蛋白的标准。驼掌三个组织氨基酸构成差异不显著,并与胶原蛋白的氨基酸构成相似。(2)经蛋白质组学鉴定驼掌三个组织均保留了大量保守性蛋白,包括软骨寡聚基质蛋白、金属结合蛋白和胶原蛋白等。与ZJC和JMC相比,ZDC的差异蛋白表达数量比较大(占鉴定蛋白总数目的17.8%)。从蛋白质的分子量和等电点分析获得ZDC的大分子量蛋白质的种类和相对含量较多,驼掌三个组织的蛋白质均呈现弱酸性(PI=6.80)。驼掌组织共有18个胶原蛋白亚基类型,且以Ⅰ型、Ⅳ型为主,其中Ⅵ型胶原蛋白为其特征胶原蛋白,并发现了16个特征肽段。KEGG通路分析结果表明驼掌三个组织在六大类生物代谢通路中均得到特有的注释信息,且信号通路信息具有一定的相似度;PPI结果表明驼掌三个组织在糖代谢通路较多的蛋白相互作用,尤其在ZDC,说明驼掌胼胝状肉垫具有特有热量代谢机制。(3)基于多肽组学驼掌组织质谱鉴定结果表明,ZDC与ZJC、JMC间存在较大差异性,其特有肽段数量远高于驼掌其他组织,并占总鉴定数量的46.2%。GO分析结果显示驼掌组织间差异蛋白主要涉及绑定结合和细胞组成方面;KEGG通路分析显示ZDC主要涉及在遗传信息处理的核糖体,而JMC与ZJC则主要在细胞过程,说明驼掌三个组织在蛋白质代谢上存在组间差异。PPI结果显示在糖酵解/糖异生通路蛋白交互作用多,其中GAPDH连接度高。内源性肽活性分析表明驼掌中有71.45%的内源性肽具有生物学活性,主要包括ACE抑制作用、DPP-IV抑制作用、抗氧化功能等14个方面的生物学活性,其中ZDC活性肽段数最多(1871个),占活性肽段总数的87.6%;鉴定到驼掌胶原蛋白活性肽段37个,主要来自I型、V型、VI型胶原蛋白。(4)RSM优化提取驼掌源胶原蛋白(CAC)结果表明,提取时间为48h、酶用量为6.0%、料液比1:14、乙酸浓度0.5 mol/L,可获得较高的CAC得率。驼掌胶原蛋白具有胶原蛋白的所有特征吸收峰和完整的三螺旋结构。经蛋白组学鉴定显示它有7个胶原蛋白亚基类型和一个前胶原增强因子。(5)不同酶组合制备CAC肽的试验结果表明,双酶组合酶解有利于得到更多的CAC肽。营养特性分析显示CAC肽的各项氨基酸测定结果均高于CAC原料。FTIR红外光谱分析表明不同酶组合CAC肽中都发现有更多的脯氨酸被暴露出来。多肽组学鉴定分析,双酶CAC肽(<3KDa)鉴定肽段数量和长度分布较为集中,数量明显减少。与双酶CAC肽(<3KDa)相比,单酶CAC肽(<3KDa)中活性肽段对应的生物学活性相对比较广谱,进一步暗示了双酶CAC肽(<3KDa)具有较强的生物学活性。更多还原

【Abstract】 With the rapid aging of the population,collagen series products have been widely concerned by consumers and their market development prospects were broaden.The large collagen family,insufficient raw material development,incomplete functional activity evaluation,insufficient scientific basis and immature industrial production,etc,have been restricting its development.Therefore,it is necessary to seek some new collagen biological raw materials.In this study,camel hoof of Camelus bactrianus as the research object,their nutritional characteristics,differential peptide segments and differential protein expression in three parts of camel hoof,namely,ZJC,ZDC and JMC,were studied based on proteomics and polypeptidomics analysis.The extraction technology and characteristics of camel-derived collagen were analyzed.And the biological functions of collagen peptides which prepared by different enzyme combinations were further studied.(1)The results of conventional ingredients,collagen and amino acids content analy-sis showed that there were tissue differences in camel hoof,and its common nutritional characteristic was that the P/W value was about 0.45.ZDC has high L/P nutritional property,and its amino acid composition was closer to the standard of ideal protein.There was no significant difference in the amino acid composition of the three tissues of camel hoof,and the amino acid composition was similar to that of collagen.Camel hoof could be used as a food protein source of collagen.(2)The identification results of proteomics were that a large number of conserved proteins were retained in the three tissues of camel hoof,including cartilage oligomeric matrix protein,metal binding protein and collagen,etc.Compared with ZJC and JMC,the number of differentially expressed proteins in ZDC was larger,17.8% of total identified proteins.According to the molecular weight and isoelectric point analysis of proteins,it showed that ZDC had more types and contents of large molecular weight proteins.Camel hoof tissues were present weak acidity(PI=6.80).The collagen types in camel hoof tissue were identified to 18 types,mainly type I and type IV,among which type IV collagen was the characteristic collagen,and 16 characteristic peptides were found.KEGG pathway analysis showed that the three tissues of camel hoof obtained unique annotation information from six kinds of biological metabolic pathways,and the signal pathway information had certain similarity.PPI results showed that there were more protein interactions in the glucose metabolism pathway in camel hoof tissues,especially in ZDC,and its indicated that callose pads of camel hoof had special mechanism of heat metabolism.(3)The results of polypeptideomics identification showed that there was a great difference between ZDC,ZJC and JMC,and the number of specific peptides in ZDC was much higher than that in other tissues of camel hoof,accounting for 46.2% of the total number of identified peptides.GO analysis showed that the differential proteins in camel hoof tissues were mainly involved in binding and cell composition.KEGG pathway analysis showed that ZDC was mainly involved in ribosomes of genetic information processing,while JMC and ZJC were mainly involved in cellular processes.It showed that there were differences in protein metabolism among three tissues of camel hoof.PPI results showed that there were many protein interactions in the glycolysis/ gluconeogenesis pathway,among which the protein with high connectivity was GAPDH.And the results were that 71.45% endogenous biological activite peptides in camel hoof,which mainly included ACE inhibitory,DPP-IV inhibitory,antioxidant function and other 14 aspects.The number of active peptides in ZDC was the largest(1871),accounting for 87.6% of the total number of active peptides.And thirty-seven collagen active peptides were identified,mainly from type I,V and VI collagen.(4)Optimization of the response surface was achieved at extraction time of 48 hrs,enzyme dosage of 6.0%,solid-to-liquid ratio of 1:14 and acetic acid concentration of 0.5mol/L.Moreover,the yield of collagen from Bactrian camel hoof achieved good stability and reliability under the above optimal parameter conditions.CAC had all the characteristic absorption peaks of collagen and complete triple helix structure.Proteomic identification revealed seven collagen types and one procollagen enhancer.(5)The results of the preparation of CAC peptides by different enzyme combinations showed that the double enzyme preparation was beneficial to get more CAC peptides.But there was no significant correlation between the yields of CAC peptide and ACE inhibitory ability under different enzymatic hydrolysis methods.The analysis of nutritional characteristics showed that the determination of various amino acids of CAC peptides were higher than that of CAC.The FTIR showed that more proline was exposed in CAC peptides with different enzyme combinations.Identification of polypeptideomics showed that the number and length of the double-enzyme CAC peptide(< 3KDa)were more concentrated,but there had been a marked decrease in numbers.Compared with double-enzyme CAC peptide(< 3KDa),the single-enzyme CAC peptide(< 3KDa)had a wide range of biological activities.It was further suggested that the double-enzyme CAC peptides(< 3KDa)have strong biological activities.更多还原