金华火腿加工技术起源于唐代，并不断传承创新。由于火腿发酵过程中加入了较多食盐用于发酵和抑制微生物生长，因此金华火腿中的盐含量较高，往往在切片或切块后用于煲汤提鲜。先前的研究已表明金华火腿和宣威火腿中存在丰富的抗氧化肽和抗菌肽，然而关于干腌火腿在煮制过程中生物活性肽的生成和释放的研究还未见报道。本文在利用体外抗氧化和抗菌实验测定金华火腿汤中小分子肽生物活性变化规律的同时，结合分离纯化、分子模拟、肽组学、代谢组学、蛋白质组学等测定和手段，系统探究金华火腿汤中生物活性肽的抗氧化抗菌特性及抗菌机制。主要研究结果如下：

• 不同煮制时间对金华火腿汤中多肽抗氧化、抗菌活性及肽谱影响

本章选用传统加工12个月的金华火腿为原料制作金华火腿汤，并模拟传统煲汤条件，分别煮制1 h、1.5 h、2 h和2.5 h，从汤中提取小分子多肽进行实验。与原料火腿相比，煮制2.5 h后汤中的游离氨基酸含量提高2.47倍，小分子多肽含量提高1.57倍；< 1kDa的肽的比例从生火腿的48.88%，增加到煮制2.5 h后的87.74%；与生火腿相比，火腿汤中肽的DPPH自由基清除活性、羟基自由基清除能力和超氧阴离子自由基清除活力下降，但Fe2+螯合和ABTS+清除能力显著增加；金华火腿汤肽对于四种食源性致病菌（大肠杆菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、单增李斯特菌）均有显著的抑制作用，对细菌的细胞膜有很好的破坏作用，使细菌内部的核酸及蛋白外泄；煮制过程中肽的二级结构发生显著变化，煮制1.5 h组β-折叠占比高于其他组，煮制2 h组α-螺旋占比高于其他组；煮制增加了肽序列和源蛋白的数量，以及从肌球蛋白和肌动蛋白释放的肽的丰度，进一步证明了煮制促进了蛋白水解。

2. 体外模拟胃肠消化对金华火腿汤中多肽抗氧化和抗菌活性的影响

本章利用体外模拟胃肠消化手段处理生火腿及火腿汤，测定火腿及汤中游离氨基酸含量后，提取其中的多肽，来探究体外模拟胃肠消化对金华火腿汤中生物活性肽抗氧化和抗菌活性的影响。胃肠消化使得生火腿和火腿汤中的游离氨基酸含量均显著升高，且汤中的含量均在1650 mg/100 mL肉汤 以上，显著高于生火腿中；模拟胃消化增加了肽的表面疏水性，煮制和肠消化降低了肽的表面疏水性；肽含量进一步增加，且汤中肽含量显著高于生火腿；同时<3 kDa小分子肽的比例进一步升高，经过胃肠模拟消化后，煮制2 h组<3 kDa肽占比超过95%；胃消化可以提高汤肽的DPPH和•OH自由基清除率，降低SOSA值，肠消化则会提高汤肽的SOSA值，降低DPPH和•OH自由基清除率，同时胃肠消化都能提高总抗氧化能力；胃肠消化会降低肽的抗菌性，大肠杆菌和单增李斯特菌核酸及蛋白外泄量减少；在模拟胃肠消化后，肽的螺旋、无规则卷曲比例逐渐减少，折叠比例逐渐升高，肽二级结构变得更有序。

3. 金华火腿汤中抗菌肽的分离纯化鉴定及分子对接

本章首先使用IR120阳离子交换树脂对蒸煮2 h组超滤后<3kDa的样品进行纯化，测定各分离组分的抑菌活性，并选取其中抑菌活性高的组分做反相液相层析纯化。在对各组分抗菌活性测定后，使用LC-MC/MC进一步对高活性组分多肽进行鉴定。在得到氨基酸序列后进行抗菌肽的理化性质及两亲性涡旋轮图的计算机预测，针对其中潜在的高活性肽段，合成肽段后再次进行抑菌实验，并分子对接模拟探究抗菌肽与细菌膜表面蛋白的结合情况。从中共筛选出IKKVVKQASEGP 、LGRVPRGKKKL、LKGGKKQLQKL、MDAIKKKMQMLK四条高抗菌活性肽段，这四条肽段对于两种食源性致病菌的IC50值均低于0.4 mg/mL，均有很强的抑菌活性。其中肽段IKKVVKQASEGP对于两种菌均有最小的IC50值，分别为0.125 mg/mL（S.typhimurium）和0.22 mg/mL（S. aureus）；四条肽段都可以与Omp F稳定结合，且均形成3个以上的氢键。

4. 基于代谢组和蛋白质组联合组学的抗菌肽IKKVVKQASEGP抑菌机制

本章通过非靶向代谢组学和非标记定量蛋白质组学联用，对于抗菌肽处理后鼠伤寒沙门氏菌的胞内代谢变化和蛋白表达上下调进行分析，确定IKKVVKQASEGP主要影响的代谢通路情况，来探究抗菌肽IKKVVKQASEGP的抑菌机理。代谢组学检测出抗菌肽处理后沙门氏菌显著差异代谢物516种，其中上调241种，下调275种，涉及辅因子的生物合成、嘌呤代谢、ABC转运体、谷胱甘肽代谢、嘧啶代谢等通路；蛋白质组学检测出抗菌肽处理后沙门氏菌735个显著差异表达蛋白中共有558个蛋白表达下调，仅有176个蛋白的表达上调，涉及丙酮酸代谢、氨基酸的生物合成、嘌呤代谢、碳代谢、糖酵解/糖异生等通路；蛋白质组学和代谢组学联合分析后，可以推测抗菌肽通过干扰细菌细胞膜相关的蛋白合成，显著下调能量代谢、氨基酸合成代谢、核苷酸合成代谢相关酶蛋白的含量，影响相关代谢物的合成代谢，从而破坏细胞膜功能、影响细菌正常的能量代谢和呼吸作用、干扰正常转录和翻译，以此发挥抗菌作用。

Jinhua ham processing technology is originated in the Tang Dynasty and has been continuously inherited and innovated. Due to the addition of plenty salt for fermentation to inhibit microbial growth during ham fermentation, the salt content in Jinhua ham is high. For this reason, it is often sliced or diced to increase the flavor of broths. Previous studies have shown the presence of large amounts of antioxidant and antimicrobial peptides in both Jinhua ham and Xuanwei ham, while the production and release of bioactive peptides from dry-cured ham during cooking has not been reported. This article ystematically investigated the antioxidant and antibacterial activity and antibacterial mechanisms of bioactive peptides in Jinhua ham broth using in vitro antioxidant and antibacterial assays, combined with isolation and purification, molecular simulation, peptidomics, metabolomics, and proteomics. The main results of this study are as follows：

1. Effects of different cooking times on antioxidant and antibacterial activities and peptide profiles of peptides in Jinhua ham broth

In this chapter, Jinhua ham processed traditionally for 12 months was used as raw material to make Jinhua ham broth, and traditional broth-making conditions was simulated to stew Jinhua ham broth for 1 h, 1.5 h, 2 h and 2.5 h, respectively. Small molecule peptides were extracted from the broth for experiments. After 2.5 h of cooking, the free amino acid content of the broth increased 2.47-fold and the small molecule peptide content increased 1.57-fold. The proportion of peptides < 1 kDa increased from 48.88% in raw ham to 87.74% after 2.5 h of cooking. Compared with raw ham, the DPPH radical scavenging activity, hydroxyl radical scavenging ability and superoxide anion radical scavenging activity of peptides in ham broth decreased, but Fe2+ chelation and ABTS+ scavenging ability were significantly increased. Jinhua ham broth peptides had significant inhibitory effects on all four food-borne pathogenic bacteria(Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes), and had strong destructive effects on the cell membrane of bacteria, causing nucleic acid and protein leakage from the inside of bacteria. The secondary structure of peptides changed significantly during cooking, with higher percentage of β-folding in the 1.5 h cooking group and higher percentage of α-helix in the 2 h cooking group than the other groups. Cooking increased the number of peptide sequences and source proteins, as well as the abundance of peptides released from myosin and actin, thus further demonstrating that cooking promoted proteolysis.

2. Effect of in vitro simulated gastrointestinal digestion on antioxidant and antibacterial activities of peptides in Jinhua ham broth

In this chapter, we investigated the effects of in vitro simulated gastrointestinal digestion on the antioxidant and antimicrobial activities of bioactive peptides in Jinhua ham broth by dealing with raw ham and ham broth, and extracting the peptides from the ham and broth after measuring the free amino acid content. Gastrointestinal digestion significantly increased the free amino acid content in both raw ham and ham broth, and the content in broth was all above 1650 mg/100 mL broth which was significantly higher than that in raw ham. Simulated gastric digestion increased the surface hydrophobicity of peptides, and intestinal digestion decreased the surface hydrophobicity of peptides. The peptide content further increased, and the peptide content in broth was significantly higher than that in raw ham. Meanwhile, the proportion of small molecule peptides(<3 kDa) further increased. After simulated gastrointestinal digestion, the proportion of <3 kDa peptides in the 3-h cooking group exceeded 95%. Gastric digestion increased the DPPH and •OH radical scavenging and decreased the SOSA value of broth peptides, while intestinal digestion increased the SOSA value and decreased the DPPH and •OH radical scavenging of broth peptides.However both gastrointestinal digestion increased the total antioxidant capacity. Gastrointestinal digestion decreased the antimicrobial property of peptide, and the amount of nucleic acid and protein  from E. coli and L. monocytogenes excretion decreased. After gastrointestinal digestion, the proportion of helical and irregular curl gradually decreased, the proportion of fold gradually increasesd, and the peptide secondary structure became more orderly.

3. Isolation and purification of antibacterial peptides from Jinhua ham broth for identification and molecular docking

In this chapter, the 2h-group with <3 kDa after ultrafiltration was first purified using IR120 cation exchange resin, the antibacterial activity of each isolated fraction was determined, and the fraction with a high antibacterial activity was selected for reverse liquid chromatography purification. After further antimicrobial activity study of each fraction, LC-MC/MC identified peptides with high active fractions was carried out. After obtaining the amino acid sequences, computer prediction of physicochemical characteristics and amphiphilic vortex wheel diagram of the antimicrobial peptides was performed. For the potentially highly active peptides among them, peptides were synthesized and then subjected to inhibition experiments again, and molecular docking simulations were performed to probe the binding of the antimicrobial peptides to bacterial membrane surface proteins. A total of four highly active peptides, IKVVKQASEGP, LGRVPRGKKKL, LKGGKKQLQKL and MDAIKKKMQMLK were screened, and all of the four peptides showed strong antibacterial activities with IC50 values lower than 0.4 mg/mL against two foodborne pathogenic bacteria. Among them, peptide IKKVVKQASEGP had the smallest IC50 values of 0.125 mg/mL (S. typhimurium) and 0.22 mg/mL (S. aureus), respectively. All of the four peptides could bind to Omp F stably, and form more than three hydrogen bonds.

4. Mechanism of bacterial inhibition by the antibacterial peptide IKKVVKQASEGP based on combined metabolomic and proteomic

In this chapter, the intracellular metabolic changes and up- and down-regulation of protein expression in Salmonella typhimurium after antibacterial peptide treatment were analyzed using a combination of untargeted metabolomics and unlabeled quantitative proteomics to determine the metabolic pathways mainly affected by IKKVVKQASEGP and to investigate the mechanism of bacterial inhibition by the antibacterial peptide IKKVKQASEGP. Metabolomics detected 516 significantly differentially expressed metabolites in Salmonella after antibacterial peptide treatment, of which 241 were up-regulated and 275 were down-regulated, involving cofactor biosynthesis, purine metabolism, ABC transporter, glutathione metabolism, pyrimidine metabolism etc. Proteomics detected 735 significantly differentially expressed proteins in Salmonella after antibacterial peptide treatment, with a total of 558 proteins being down-regulated in expression and only 176 proteins being up-regulated, involving pyruvate metabolism, amino acid biosynthesis, purine metabolism, carbon metabolism, glycolysis/gluconeogenesis  etc. After combined proteomics and metabolomics analysis, it can be speculated that the antibacterial peptide significantly down-regulated the content of enzyme proteins related to energy metabolism, amino acid anabolism and nucleotide anabolism by interfering with bacterial cell membrane-related protein synthesis, affecting related metabolites. The antibacterial effect was achieved by disrupting the cell membrane function, affecting the normal energy metabolism and respiration of bacteria, and interfering with normal transcription and translation.