【作者】 张靖；

【导师】 靳烨；

【作者基本信息】 内蒙古农业大学 ， 食品科学， 2021， 硕士

【摘要】 羊骨作为羊肉加工副产物具有很高的营养价值,但其生物活性物质还未被深入研究,因此本研究以羊骨为原料,通过优化酶解工艺制备羊骨抗氧化肽,对其抗氧化能力和稳定性进行分析,并表征其二级结构。进一步纯化多肽,对抗氧化能力最强的肽段进行氨基酸序列鉴定,通过功能注释、生物信息学预测筛选潜在的功能多肽。将筛选出的多肽进行化学合成,验证抗氧化活性,结果如下:羊骨氨基酸总含量为15.75 g/100g,其中疏水氨基酸占28.25%,必需氨基酸占20.44%,是抗氧化肽提取的理想原料。羊骨抗氧化肽响应面优化结果为:使用碱性蛋白酶,酶解p H 8.9、酶添加量6400 U/g、酶解时间3.25h、底物质量分数8.8%、酶解温度50℃,该条件下,水解度为（20.78±0.83）%,羊骨抗氧化肽的·OH清除能力、DPPH·清除能力、ABTS⁺自由基清除能力的IC₅₀值分别为6.07 mg/m L、23.45 mg/m L、1.18 mg/m L,酶解物浓度为2 mg/m L时,测得还原力为0.15±0.003。羊骨抗氧化肽（SBAP）对高温不耐受,耐酸、碱性较弱;添加Na Cl可提高SBAP的还原力;还原糖使SBAP保持原有活性,或活性略有升高;而金属离子降低SBAP的抗氧化活性;SBAP活性对苯甲酸钠和山梨酸钾稳定;体外模拟消化表明,SBAP对人工胃肠液具有一定的惰性。结构表征显示,SBAP保留源蛋白的特征官能团,微观形貌呈疏松多孔的不规则大孔海绵状结构,α-螺旋结构和无规卷曲含量降低,β-折叠和β-转角含量升高。将最优工艺参数下制备的酶解液进行超滤分离,得到P-I（M_W<3 k Da）、P-II（3 k Da<M_W<10 k Da）和P-III（M_W>10 k Da）。氨基酸测定结果表明:除谷氨酰胺/谷氨酸（Gln/Glu）,SBAP中其他氨基酸含量均高于羊骨粉,且必需氨基酸含量占总氨基酸的比值（EAA/TAA）为27.03%,疏水性氨基酸多达39.96%。P-I疏水性氨基酸所占比例最高,为43.13%,含有对疏水性组分有很强结合力的疏水性氨基酸（Pro、Phe、Met等）和非极性脂肪族氨基酸（Val）,且相同浓度条件下,P-I的·OH清除能力、DPPH·清除能力、ABTS⁺自由基清除能力和还原力活性最高。电镜图中直观地看出,P-I较其他肽段更加疏松,结构似絮状网络;体外模拟消化表明P-I表现出更高活性。对P-I进行LC-MS/MS质谱鉴定,Peptide Ranker评分大于0.85的肽段有17条,经GO和KEGG蛋白功能注释结果表明,酶解制备得到的多肽参与到生物系统、免疫系统、消化系统等多种生物过程中。结合文献报道以及功能预测网站对鉴定到的多肽进行筛选,确定合成VYPFPGPIPN、LGFPL和SLVYPFPGPIPN三条多肽。它们均具有抗氧化活性,但抗氧化活性存在差异,同时,合成肽间既存在正协同作用也存在负协同作用。更多还原

【Abstract】 Sheep bone has high nutritional value as a by-product.But its bioactive substances have not been studied in depth.In this study,antioxidant peptide was prepared from sheep bone through enzymolysis technology.The antioxidant ability,stability and secondary structure of the antioxidant peptides were analyzed.The peptides were purified,and the amino acid sequences were identified.Potential functional peptides were screened by functional annotation and bio-informatics prediction.Chemical synthesis of the selected peptides to verify the antioxidant activity.The results of this topic are as follows:The total content of amino acids in sheep bone is 15.75 g/100g,hydrophobic amino acids and essential amino acids accounted for 28.25%and20.44%,respectively.Sheep bone is an ideal raw material for the extraction of antioxidant peptides.The results of response surface methodology were as follows:alkaline protease,p H was 8.9,the amount of enzyme added was 6400U/g,hydrolysis time was 3.25 h,substance mass fraction was 8.8%and the hydrolysis temperature was 50℃.Under these conditions,the degree of hydrolysis was（20.78±0.83）%,The IC₅₀values of·OH scavenging activity,DPPH·scavenging activity and ABTS⁺free radical scavenging capacity of antioxidant peptides from sheep bone were 6.07 mg/m L,23.45 mg/m L and1.18 mg/m L,respectively.When the concentration of hydrolysate was 2mg/m L,the reducing power was 0.15±0.003.The stability of sheep bone antioxidant peptide（SBAP）was studied.The results showed that SBAP was not tolerant to high temperature,acid and alkali.With the increase of Na Cl content,the reducing power of SBAP improved.Reducing sugar can keep the original activity of SBAP antioxidant peptide,or slightly increase the activity while metal ions decreased the antioxidant activity of SBAP.The activity of SBAP is stable to sodium benzoate and potassium sorbate.The simulated digestion in vitro shows that SBAP is inert to artificial gastrointestinal fluid.The structure characterization showed that SBAP retained the characteristic functional groups of the source protein,the micro morphology of SBAP was irregular macroporous spongy structure with loose pores.The decreased content ofα-helices and random,increased content ofβ-sheets andβ-turns were observed.Three components with different molecular weight were obtained:P-I（M_W<3 k Da）,P-II（3 k Da<M_W<10 k Da）and P-III（M_W>10 k Da）.The results of amino acids of ultrafiltration components showed that except（Gln/Glu）,the content of other amino acids in SBAP was higher than that in sheep bone,and the ratio of essential amino acids to total amino acids（EAA/TAA）was 27.03%,and the hydrophobic amino acids were 39.96%.P-I had the highest proportion of hydrophobic amino acids 43.13%,including hydrophobic amino acids（Pro,Phe,Met,etc.）and non-polar aliphatic amino acids（Val）.Under the same concentration,P-I had the highest activity of·OH scavenging activity,DPPH·scavenging activity,ABTS⁺scavenging activity and reducing power.It can be seen directly from the electron microscope that component P-I is more loose than other peptides,and its structure is like flocculent network.Simulated digestion in vitro shows that component P-I has higher activity.The results of LC-MS/MS showed that there were 17 peptides with peptide ranker score greater than 0.85 in P-I.GO and KEGG protein function annotation showed that the peptides prepared by enzymatic hydrolysis were involved in many biological processes,such as biological system,immune system,digestive system and so on.According to the literature reports and the function prediction website,the identified peptides were screened to synthesize VYPFPGPIPN,LGFPL and SLVYPFPGPIPN.The three peptides have antioxidant activity,but there are differences in antioxidant activity.At the same time,there are both positive and negative synergistic effects among synthetic peptides.更多还原