【作者】 王磊；

【导师】 王丰俊；

【作者基本信息】 北京林业大学 ， 农业硕士（专业学位）， 2023， 硕士

【摘要】 锌是人体必需的微量元素,日常饮食摄入不足会引发食欲不振、发育不良等疾病。传统补锌剂具有刺激胃肠道,引发不良反应,吸收利用率低等缺点。食源性蛋白肽-锌螯合物结构稳定、吸收利用率高,成为近年来的研究热点。我国核桃产量高,榨油后的核桃粕利用率低。利用核桃粕制备核桃多肽的工艺简单,且核桃多肽营养价值高,是制备肽-锌螯合物的良好选择。本研究以脱脂核桃粕为原料,利用碱溶酸沉法制备核桃蛋白,再利用碱性蛋白酶水解核桃蛋白制备核桃多肽。将核桃多肽与七水硫酸锌螯合制备核桃多肽-锌螯合物,探究核桃多肽-锌螯合物的结构特性和理化特性,为增加核桃多肽-锌螯合物的稳定性和吸收利用度,制备螯合物微胶囊,并研究螯合物及其微胶囊的稳定性。主要研究内容及结果如下:（1）优化核桃多肽-锌螯合物的制备工艺参数。以螯合率为指标,通过单因素-响应面试验确定最佳螯合工艺参数为锌-肽质量比1:3.5,反应p H 8.5,温度50℃,反应时间84 min,此条件下螯合率为84.51±1.86%。利用硫化钠法定性检测制备产物,证明多肽与硫酸锌反应生成螯合物。（2）分析了核桃多肽-锌螯合物的结构特性和螯合机理。核桃多肽和螯合物的氨基酸组成和含量变化结果显示,螯合后的天冬氨酸、谷氨酸、赖氨酸等亲水性氨基酸含量增加,亲水性氨基酸是多肽与锌离子结合的主要氨基酸。紫外全波长扫描光谱、荧光光谱、扫描电镜及其能谱结果表明,螯合反应生成了一种不同于核桃多肽的肽-锌螯合物。粒径-Zeta电位、扫描电镜测定结果表明锌离子的引入使核桃多肽颗粒内部结构发生聚集。傅里叶红外光谱结果显示多肽与锌离子的主要螯合位点为羧基、氨基和肽键。（3）测定核桃多肽-锌螯合物的理化性质。核桃多肽-锌螯合物具有良好的储藏稳定性。7天吸水实验表明,核桃多肽-锌螯合物的最大吸水率为36.35%,明显低于螯合前的吸水率（112.83%）。与核桃多肽（1.20 m L/g）和谷胱甘肽（1.09 m L/g）相比,核桃多肽-锌螯合物的吸油性（0.87 m L/g）较低。核桃多肽-锌螯合物的体外抗氧化活性比螯合前低,螯合物的DPPH自由基清除率IC₅₀为2.91 mg/m L。（4）优化锐孔-固化法制备核桃多肽-锌螯合物微胶囊的工艺参数。利用四因素三水平的单因素-正交试验确定螯合物微胶囊的最佳工艺参数为海藻酸钠浓度为3%,芯壁比为1:2,吐温80添加量为10μL/m L,氯化钙浓度为3%,在此条件下螯合物微胶囊的包埋率为75.67±0.83%。（5）探究核桃多肽-锌螯合物及其微胶囊的稳定性。核桃多肽-锌螯合物在酸性条件下部分分解,核桃多肽-锌螯合物微胶囊在酸性和碱性条件下均具有较好的稳定性。热处理对螯合物及其微胶囊的影响不显著。核桃多肽-锌螯合物及其微胶囊的消化稳定性和生物利用度均高于硫酸锌和葡萄糖酸锌。研究食物共存物对四种锌制剂锌溶解率的影响,发现核桃多肽-锌螯合物及其微胶囊对食物共存物具有一定的抵抗性,能维持较高的锌溶解率。核桃多肽-锌螯合物中的多肽可作为锌离子的吸收载体,实现跨膜运输,降低能耗,有效避免金属离子间的竞争机制,增加吸收效率。含有半胱氨酸、组氨酸、丝氨酸、天冬氨酸和谷氨酸等氨基酸的核桃多肽可与锌离子进行螯合作用,形成可溶性锌螯合物,增加胃肠道对锌元素的吸收利用。对核桃多肽-锌螯合物进行微胶囊化可避免胃酸对螯合物的分解,保证结构的完整,增加锌的生物利用率,所以核桃多肽-锌螯合物是一种理想的补锌剂。更多还原

【Abstract】 Zinc is an essential trace element in the human body.Insufficient daily dietary intake would cause loss of appetite and dysplasia.Traditional zinc supplements have the disadvantages of stimulating the gastrointestinal tract,causing adverse reactions and low absorption and utilization.Foodborne protein peptide-zinc chelates have the stable structure and high absorption utilization,which have become a research hotspot in recent years.The utilization rate of walnut meal is low,the process of preparing walnut polypeptide is simple,and the walnut polypeptide has high nutritional value,which is a good choice for the preparation of peptide-zinc chelate.In this study,walnut protein was prepared from walnut meal by alkaline acid sink method and was hydrolyzed to walnut protein hydrolysate（WPH）.WPH was chelated with Zn SO₄to form walnut protein hydrolysate-zinc chelate（WPH-Zn）.WPH and WPH-Zn were characterized,and WPH-Zn chelate microcapsules were prepared to increase stability and their stabilities in different environments were investigated.The main study contents and results are as follows:（1）Process parameters for the preparation of WPH-Zn chelates were optimized.With the chelation rate index,the optimal chelation process parameters were determined by the one-way response surface test.The optimal chelation process parameters were zinc-peptide mass ratio of 1:3.5,p H of 8.5,temperature of 50℃,and reaction time of 84 min.The chelation rate of WPH-Zn was 84.51±1.86%.Using sodium sulfide precipitation legal test,the product was proved to be a chelate produced by the reaction of the peptide with Zn SO₄.（2）The structural properties and chelation mechanism of WPH-Zn chelate were analyzed.The analysis of the amino acid composition and content of WPH and WPH-Zn chelate showed that the content of hydrophilic amino acids such as aspartic acid,glutamate and lysine increased significantly.This result indicated that hydrophilic amino acids are the main amino acid for binding with zinc ions.Using the comparative analysis of ultraviolet full-wavelength scanning spectrum,fluorescence spectrum,scanning electron microscope（SEM）and its energy spectrum,it was found that the chelation reaction generated a peptide-zinc chelate,which is different from the WPH.The results of particle size-zeta potential and SEM showed that the introduction of zinc ion accumulates the internal structure of WPH particles.Fourier infrared spectroscopy results showed that the main chelation sites of polypeptide and zinc ion were carboxyl group,amino group,and peptide bond.（3）The results of physicochemical properties of WPH-Zn chelate were as follows:WPH-Zn chelate has good storage stability.The maximum water absorption rate after water absorption experiment for 7 days was 36.35%,which was significantly lower than the water absorption rate before chelation（112.83%）.Compared with WPH and glutathione,WPH-Zn chelate had lower oil absorption（0.87 m L/g）.The antioxidant activity in vitro of the WPH-Zn chelate was lower than that before chelation,and the DPPH radical clearance semi-inhibitory concentration was 2.91 mg/m L.（4）Process optimization of WPH-Zn chelate microcapsule prepared by sharp pore-curing method.The optimum reaction process:the concentration of sodium alginate 3%,the nuclear wall ratio 1:2,the amount of Tween 80 10μL/m L,and the concentration of calcium chloride 3%.The optimal encapsulation rate was 75.67±0.83%.（5）The stability of WPH-Zn chelate and chelate microcapsule were explored.The WPH-Zn chelate was partially decomposed under acidic conditions,and the WPH-Zn chelate microcapsules had good stability under acidic and alkaline conditions.The effect of heat treatment on the WPH-Zn chelate and chelate microcapsules were not significant.The digestive stability and bioavailability of WPH-Zn chelate and chelate microcapsule were higher than Zn SO₄and zinc gluconate.The WPH-Zn chelate and chelate microcapsule could resist the influence of food coexistence.The peptides in WPH-Zn chelate can be used as the absorption carrier of zinc ions to realize transmembrane transport,reduce energy consumption,effectively avoid the competition mechanism between metal ions,and increase the absorption efficiency.WPH containing amino acids can chelate with zinc ions to form soluble zinc chelates and increase the absorption and utilization of zinc elements in the gastrointestinal tract.Microencapsulation of WPH-Zn chelate can avoid the decomposition of the chelate by gastric acid,ensure the integrity of the structure,and increase the bioavailability of zinc.Therefore,WPH-Zn chelate is an ideal zinc supplement.更多还原