金顶侧耳（Pleurotus citrinopileatus）色艳味鲜，营养丰富，具有降血糖、降血脂、抗肿瘤、抗氧化等生物活性，是一种典型的食药两用型真菌。目前，针对金顶侧耳的研究主要集中在金顶侧耳多糖及其栽培上，而对于金顶侧耳蛋白的研究较少。铁是人体所必需的微量元素，机体缺铁会引发贫血、免疫力低下等多种疾病。传统铁强化剂储存稳定性和生物利用度有限，而通过酶解蛋白获得的多肽与亚铁离子的螯合物可以克服这一缺点。

本文采用超声辅助碱提酸沉法提取金顶侧耳蛋白，并对比研究了超声碱提蛋白与常规碱提蛋白的结构特性与加工特性，接着采用酶解法制备金顶侧耳蛋白多肽，利用超滤与Sephadex G-25凝胶层析对其进一步纯化，将纯化后的多肽与亚铁离子螯合制备肽铁螯合物，并通过紫外扫描、红外扫描、电镜扫描探究其基本表征，最后通过体外模拟消化探究其消化的稳定性。

以金顶侧耳蛋白提取率为考察指标，进行单因素实验和响应面设计试验，最终结合实验实际情况确定超声辅助提取金顶侧耳蛋白的最佳工艺为NaOH浓度0.075 mol/L、液料比43:1、超声时间39 min、超声输入功率350 W（实测声能密度为46.20 W/L），此条件下所得金顶侧耳蛋白提取率为30.50%。

接着进行金顶侧耳蛋白特性的研究，结果表明：金顶侧耳蛋白的分子量主要分布范围在5~15 kDa、30~35 kDa、35~50 kDa区间内以及65 kDa和95 kDa左右，超声辅助提取不会引起蛋白分子量的变化；金顶侧耳蛋白氨基酸含量较为丰富，超声碱提蛋白与常规碱提蛋白中必需氨基酸分别占总氨基酸含量的41.67%、41.82%，必需氨基酸与非必需氨基酸的比值分别为0.7127、0.7187，属于理想蛋白质；红外扫描分析发现超声碱提蛋白与常规碱提蛋白出峰情况大致相同，但有少数特征峰发生了变化。扫描电镜分析发现超声碱提蛋白显示出更多的不规则碎片和无序结构，表面较为粗糙；而常规碱提蛋白组织结构紧密，表面较为光滑。超声辅助提取使得金顶侧耳蛋白具有更高的二硫键含量，更强的疏水性和持水性；碱性条件下超声辅助提取的金顶侧耳蛋白具有更好的溶解性、起泡性及乳化性。

采用酶解法水解金顶侧耳蛋白以制备金顶侧耳多肽，经初步筛选确定碱性蛋白酶为最佳蛋白酶，接着以蛋白水解度和肽铁螯合率为考察指标，通过单因素试验与正交试验确定金顶侧耳蛋白最佳酶解条件为底物浓度3%、酶浓度为6000 U/g、pH 9.0、温度55℃，酶解时间3 h，此条件下蛋白水解度为22.94%，肽铁螯合率为41.82%。

将酶解得到的多肽经超滤分为PCPPI：<3 kDa，PCPPII：3 KDa-10 kDa，PCPPIII：>10 kDa三个组分，发现分子量较小的PCPPI肽铁螯合率最高，可达到51.21%；继续利用Sephadex G-25凝胶层析纯化，得到两个组分PCPPI-1、PCPPII-2，其中PCPPII-2的肽铁螯合率较高，可达到60.07%。

将纯化后的多肽PCPPI-1、PCPPII-2与FeSO4·7H2O按照3:1的比例在pH 6.5、温度35℃的条件下螯合30 min，得到肽铁螯合物PCPPI-1-Fe、PCPPI-2-Fe，并对其进行结构表征。通过氨基酸分析发现多肽中的Asp、Glu、Lys与Arg在肽铁螯合过程中发挥主要作用，通过紫外光谱发现多肽与亚铁离子螯合后产生了新的物质，通过红外光谱发现多肽与亚铁离子的螯合反应主要与肽中的羧基氧与氨基氮有关，通过电镜扫描发现铁离子可以诱导多肽中小颗粒的聚集，导致多肽结构的破坏和较大球形颗粒的形成。最后通过体外模拟消化实验证明了PCPPI-1-Fe、PCPPI-2-Fe比硫酸亚铁、葡萄糖酸亚铁具有更好的消化稳定性。

Pleurotus citrinopileatus is a typical edible and medicinal fungus with brilliant color, fresh taste and rich nutrition. It has the biological activities of reducing blood glucose and blood lipid, anti-tumor, anti-oxidation and anti-fatigue. At present, the studies on Pleurotus citrinopileatus mainly focused on the polysaccharide and its cultivation, while few studies on the protein of Pleurotus citrinopileatus. Iron is an essential trace element for the human body, and deficiency of iron in the body can cause various diseases such as anemia and low immunity. Traditional iron fortifiers have limited storage stability and bioavailability, but peptide iron chelates prepared by chelating peptides obtained by enzymatic protein with ions can overcome this shortcoming.

In this paper, the ultrasonic-assisted alkali extraction and acid precipitation method was used to extract Pleurotus citrinopileatus protein, and the structural characteristics and processing characteristics were compared between the ultrasonic-assisted alkali extraction and conventional alkali extraction. Subsequently, the enzyme hydrolysis method was used to prepare the Pleurotus citrinopileatus protein polypeptide, which was further purified by ultrafiltration and Sephadex G-25 gel chromatography. The purified polypeptide was chelated with iron to prepare peptide-iron chelate. Then its basic characteristics were explored by UV spectrum, IR spectrum and electron microscopy scanning. Finally, the digestion stability was explored by in vitro simulated digestion.

The single factor experiments and response surface design experiments were performed with the protein extraction rate of Pleurotus citrinopileatus as the inspection index, and then combined with the actual situation, the optimal process of ultrasonic-assisted extraction of Pleurotus citrinopileatus protein was determined as follows: NaOH concentration is 0.075 mol/L, liquid-to-material ratio is 43:1, ultrasonic time is 39 min, and ultrasonic input power is 350 W (measured sound energy density is 46.20 W/L). Under these conditions, the protein extraction rate of Pleurotus citrinopileatus was 30.50%.

Then, the study on the characteristics of Pleurotus citrinopileatus protein showed that the molecular weight of Pleurotus citrinopileatus protein mainly distributed in 5-15 kDa, 30-35 kDa, 35-50 kDa, 65 kDa and 95 kDa, and ultrasonic-assisted extraction did not cause the change of molecular weight of protein. The amino acid content of Pleurotus ostreatus protein was abundant. The essential amino acids of ultrasonic alkaline extraction protein and conventional alkaline extraction protein account for 41.67% and 41.82% of the total amino acid content, and the ratio of essential amino acids to non-essential amino acids was 0.7127 and 0.7187, belong to ideal proteins. FTIR spectrum analysis showed that the ultrasonic-assisted alkali extraction protein peaks were almost the same as the conventional alkali extraction protein peaks, but there were changes in a few characteristic peaks. The analysis by SEM showed that the ultrasonic-assisted alkali extraction protein showed more irregular fragments and disordered structure, and the surface was relatively rough. The protein structure extracted by conventional alkali was compact, and the surface was relatively smooth. Ultrasound-assisted extraction enabled the Pleurotus citrinopileatus protein to have higher disulfide bond content, better hydrophobicity and water holding capacity. The protein extracted by ultrasound-assisted extraction under the alkaline condition showed better solubility, foaming ability and emulsifying ability.

The pleurotus citrinopileatus protein was hydrolyzed by enzymolysis to prepare pleurotus citrinopileatus polypeptide, and alkaline protease was determined as the optimal protease through preliminary screening. With protein hydrolysis degree and chelating rate of peptide and iron as indicators, the optimal enzymolysis conditions of Pleurotus citrinopileatus protein were determined by single factor and orthogonal test as substrate concentration 3%, enzyme concentration 6000 U/g, pH 9.0, temperature 55℃ and enzymolysis time 3 h. Under these conditions, protein hydrolysis degree was 22.94% and chelating rate of peptide and iron was 41.82%.

The polypeptide obtained by enzymolysis was divided into three components by ultrafiltration: PCPPI(<3 kDa), PCPPII(3 KDa-10 kDa), PCPPIII(>10 kDa). It was found that the iron chelation rate of PCPPI with smaller molecular weight was the highest, reaching 51.21%. Continue to be purified by Sephadex G-25 gel chromatography, two components PCPPI-1 and PCPPII-2 were obtained. Within them, PCPII-2 had a high chelation rate of peptide iron, which can reach 60.07%.

Then, chelating the purified polypeptides PCPPI-1, PCPPII-2 and FeSO4·7H2O in a ratio of 3:1 under the condition of pH 6.5 and temperature of 35℃ for 30 min to obtain peptide iron chelates: PCPPI-1-Fe, PCPPI-2-Fe, and their structures were characterized. It was showed by amino acid analysis that Asp, Glu, Lys and Arg in peptides played a major role in the process of peptide iron chelation. It was showed by UV spectrum that new substances were produced before and after chelation. It was showed by FTIR spectrum that the chelation reaction between the polypeptide and iron was mainly related to carboxyl oxygen and amino nitrogen in the peptide. It was found by SEM that iron could induce the aggregation of small particles in the polypeptide, leading to the destruction of the polypeptide structure. Finally, the simulated digestion experiment in vitro proved that PCPPI-1-Fe and PCPPI-2-Fe had better digestion stability than ferrous sulfate and ferrous gluconate.