Optimization of Jinhua Ham Classification Method Based on Volatile Flavor Substances and Determination of Key Odor Biomarkers.

Jinhua ham is a traditional cured meat food in China. For a long time, its grade has mainly been evaluated by the human nose through the three-sticks method, which is highly subjective and is not conducive to establishing evaluation standards through odor markers. In this paper, we analyzed the well-graded Grade I-III hams provided by Jinzi Ham Co., Ltd. (Jinhua, China). Firstly, we used different extraction fibers, extraction temperatures, and extraction time to determine the optimal conditions for headspace solid-phase microextraction (HS-SPME). Then, the aroma components of Jinhua ham were analyzed by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS), and OAV was calculated to screen the key aroma volatiles of three kinds of Jinhua ham. It was found that a total of 56 components were detected in the three types of ham. Among them, there are 21 kinds of key aroma volatiles. Aldehydes, alcohols, and acids are the three main components of Jinhua ham, and the content of aldehydes gradually decreases from Grade I to Grade III ham. The content of acids gradually increased, and we speculated that the increase in acid content was caused by the proliferation of microorganisms in Grade III ham. The key flavor volatiles in Grade I hams was hexanal and 2-methylbutanal. Grade I hams had a strong meat aroma, pleasant fatty, and roasted aroma without any off-flavors. In Grade II ham, the characteristic volatiles (E,E)-2,4-decadienal and ethyl isovalerate were detected. These two volatiles contribute greatly to the flavor of Grade II ham, which makes the flavor of Grade II ham have a special fruity aroma. They also may be prone to sourness and affect the flavor of the ham. Volatiles with low threshold values, such as pyrazines, furans, and sulfur-containing compounds, were relatively high in Grade III hams. This may also contribute to the poorer flavor quality of Grade III hams. This experiment provided a reliable test method and evaluation basis for the rating of Jinhua ham. These results have positive implications for the establishment of odor markers-based grading criteria.

Antioxidant activity of Lactobacillus plantarum NJAU-01 in an animal model of aging.

BACKGROUND: Excessive reactive oxygen species (ROS) can cause serious damage to the human body and may cause various chronic diseases. Studies have found that lactic acid bacteria (LAB) have antioxidant and anti-aging effects, and are important resources for the development of microbial antioxidants. This paper was to explore the potential role of an antioxidant strain, Lactobacillus plantarum NJAU-01 screened from traditional dry-cured meat product Jinhua Ham in regulating D-galactose-induced subacute senescence of mice. A total of 48 specific pathogen free Kun Ming mice (SPF KM mice) were randomly allocated into 6 groups: control group with sterile saline injection, aging group with subcutaneously injection of D-galactose, treatments groups with injection of D-galactose and intragastric administration of 107, 108, and 109 CFU/mL L. plantarum NJAU-01, and positive control group with injection of D-galactose and intragastric administration of 1 mg/mL Vitamin C. RESULTS: The results showed that the treatment group of L. plantarum NJAU-01 at 109 CFU/mL showed higher total antioxidant capacity (T-AOC) and the antioxidant enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) than those of the other groups in serum, heart and liver. In contrast, the content of the oxidative stress marker malondialdehyde (MDA) showed lower levels than the other groups (P < 0.05). The antioxidant capacity was improved with the supplement of the increasing concentration of L. plantarum NJAU-01. CONCLUSIONS: Thus, this study demonstrates that L. plantarum NJAU-01 can alleviate oxidative stress by increasing the activities of enzymes involved in oxidation resistance and decreasing level of lipid oxidation in mice.

The effect of cooking temperature on the aggregation and digestion rate of myofibrillar proteins in Jinhua ham.

BACKGROUND: In order to evaluate the effect of cooking temperature on the nutrition quality of dry-cured hams, 60 biceps femoris samples from 16 Jinhua hams were divided into four groups (control, 70, 100 and 120 °C) and cooked for 30 min. Carbonyl content, sulfhydryl groups, surface hydrophobicity, microstructure, protein aggregation and digestibility of myofibrillar proteins were investigated. RESULTS: Cooking promoted carbonylation and decreased sulfhydryl groups in a temperature-dependent way. Scanning electron microscopy and Nile Red revealed that protein aggregation became a main phenomenon at 120 °C; it coincided with surface hydrophobicity. The increased carbonyl content and decreased sulfhydryl groups contributed to the formation of aggregates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles showed the initial difference in proteolysis rate among four groups. The in vitro digestibility of pepsin and of trypsin and α-chymotrypsin increased from the control to 100 °C and decreased from 100 to 120 °C. CONCLUSION: The increased digestibility could be attributed to the oxidation of proteins and exposing recognition sites of digestive enzymes, while the decreased digestibility was due to the formation of aggregates. Cooking was a main factor that affected the digestibility of Jinhua ham, and cooking at 100 °C could be an ideal way to gain the highest digestibility of Jinhua ham. © 2018 Society of Chemical Industry.

Identification of antioxidant peptides of Jinhua ham generated in the products and through the simulated gastrointestinal digestion system.

BACKGROUND: The objective of this study was to purify and identify antioxidant peptides present in the extract of Chinese dry-cured Jinhua ham. Jinhua ham extracts were separated into five fractions (A-E) by size-exclusion chromatography. Each fraction was subjected to a simulated gastrointestinal (GI) digestion system and fractions showing strong antioxidant activities were collected and subjected to liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for further purification and identification. RESULTS: Using MS/MS analysis, 33 peptides were identified in these fractions. Several key peptides were selected for synthesis and their antioxidant activity determined. The peptide showing strongest DPPH radical scavenging activity was GKFNV, which showed 92.7% antioxidant activity at a concentration of 1 mg mL(-1); the peptide LPGGGHGDL showed the highest hydroxyl radical scavenging activity; and LPGGGT and HA showed strong inhibition activity against erythrocyte hemolysis (about 45%) before digestion. On the other hand, KEER may contribute to Fe(2+) chelating ability in fraction C after GI digestion. CONCLUSION: Jinhua dry-cured ham seems to be a potential source of antioxidant peptides generated in the products and in GI digestion.

Insights into the identification of bitter peptides from Jinhua ham and its taste mechanism by molecular docking and transcriptomics analysis.

In order to identify the peptides responsible for bitter defects and to understand the mechanism of bitterness in dry-cured ham, the peptides were identified by LC-MS/MS, and the interaction between bitter peptides and receptor proteins were evaluated by molecular docking and molecular dynamics simulation; the signal transduction mechanism of bitter peptides was investigated using the model of HEK-293T cells by calcium imaging and transcriptomics analysis. The results of LC-MS/MS showed that 11 peptides were identified from the high bitterness fraction of defective ham; peptides PKAPPAK, VTDTTR and YIIEK derived from titin showed the highest bitterness values compared with other peptides. The results of molecular docking showed that lower CDOCKER energy was observed in the interaction between these peptides and hT2R16 in comparison with these receptors of hT2R1, hT2R4, hT2R5, hT2R8 and hT2R14, and the interaction of hT2R16 and peptides was stabilized by hydrophobic interaction and hydrogen bond. The average RMSF values of VTDTTR were higher than that of YIIEK and PKAPPAK, while EC50 values of VTDTTR were lower compared with PKAPPAK and YIIEK. Transcriptomics analysis showed that 529 differentially expressed genes were identified in HEK-293T cells during the stimulating by VTDTTR and were mainly enriched into neuroactive ligand-receptor interaction, MAPK pathway, cAMP pathway and calcium signaling pathway, which were mainly responsible for the bitter signal transduction of VTDTTR. These results could provide evidence for understanding the bitter defects of dry-cured ham and the taste mechanism of bitter peptide.

Insight into Antioxidant Activity and Peptide Profile of Jinhua Ham Broth Peptides at Different Cooking Times.

This present study aimed to investigate the effects of various cooking times (1 h, 1.5 h, 2 h, 2.5 h, named as JHBP-1, JHBP-1.5, JHBP-2, JHBP-2.5) on the antioxidant activity and peptide profile of Jinhua ham broth peptides (JHBP). The peptides extracted from uncooked ham were used as an uncooked group with the name of JHBP-0. The results revealed that the antioxidant efficacy in the four cooked groups changed dramatically compared to JHBP-0. After cooking, the DPPH radical scavenging activity, hydroxyl radical scavenging activity and superoxide anion radical scavenging activity decreased, except for the Fe2+ chelation and ABTS+ scavenging capacity which increased significantly. However, the cooked groups still showed a strong antioxidant capacity. In particular, the superoxide anion radical scavenging ability and the Fe2+ chelation action were significantly stronger compared to glutathione (GSH) and butylated hydroxytoluene (BHT) (p < 0.05). JHBP-1.5 also displayed stronger antioxidant capacity than the other three cooked groups, and its secondary structure and mass distribution changed significantly after cooking, specifically with an increased proportion of helix and <1 kDa peptides. Moreover, the constitution of free amino acids (FAAs) and the types of peptides released in the broth increased significantly with a longer cooking time. In total, 1306 (JHBP-0), 1352 (JHBP-1), 1431 (JHBP-1.5), 1500 (JHBP-2), and 1556 (JHBP-2.5) peptide sequences were detected using LC-MC/MC. The proportion of <1 kDa peptides also gradually increased as the cooking time extended, which is consistent with the molecular weight distribution measurements.

Insights into the Protein Differentiation Mechanism between Jinhua Fatty Ham and Lean Ham through Label-Free Proteomics.

Jinhua lean ham (LH), a dry-cured ham made from the defatted hind legs of pigs, has become increasingly popular among consumers with health concerns. However, the influence of fat removal on the quality of Jinhua ham is still not fully understood. Therefore, a label-free proteomics strategy was used to explore the protein differential profile between Jinhua fatty ham (FH) and lean ham (LH). Results showed that 179 differential proteins (DPs) were detected, including 82 up-regulated and 97 down-regulated DPs in LH vs. FH, among which actin, myosin, tropomyosin, aspartate aminotransferase, pyruvate carboxylase, and glucose-6-phosphate isomerase were considered the key DPs. GO analysis suggested that DPs were mainly involved in binding, catalytic activity, cellular process, and metabolic process, among which catalytic activity was significantly up-regulated in LH. Moreover, the main KEGG-enriched pathways of FH focused on glycogen metabolism, mainly including the TCA cycle, pyruvate metabolism, and glycolysis/gluconeogenesis. However, amino acid metabolism and oxidative phosphorylation were the main metabolic pathways in LH. From the protein differentiation perspective, fat removal significantly promoted protein degradation, amino acid metabolism, and the oxidative phosphorylation process. These findings could help us to understand the effects of fat removal on the nutritional metabolism of Jinhua hams and provide theoretical supports for developing healthier low-fat meat products.

Physicochemical property, volatile flavor quality, and microbial community composition of Jinhua fatty ham and lean ham: A comparative study.

The physicochemical property, volatile flavor compounds, and microbial community structure of Jinhua fatty ham (FH) and lean ham (LH) were investigated and compared by high-throughput sequencing and HS-GC-IMS. Results showed that FH had higher pH and slightly lighter and yellower color than LH. Meanwhile, 33 volatile flavor compounds were identified from FH and LH, among which LH showed higher abundance of total alcohols and acids, but FH had generally richer aldehydes, ketones, esters, heterocyclic, and sulfur-containing compounds. Moreover, FH and LH did not have significant difference in α-diversity of bacterial community, but LH presented a much lower α-diversity of fungal community than FH. Besides, the dominant microorganisms (relative abundance >2%) in FH were Ruminococcaceae UCG-005, Staphylococcus, Ruminococcaceae UCG-014, Meyerozyma, and Aspergillus at the genus level, while in LH were Staphylococcus, Psychrobacter, Halomonas, Propionicicella, Ruminococcaceae UCG-005, Meyerozyma, Yamadazyma, and Aspergillus. Furthermore, the analysis of Pearson's correlation and metabolic network confirmed that the discriminative flavor compounds of FH were mainly ß-oxidation and degradation products of fatty acids, while those of LH were mostly derived from the Strecker reaction or microbial metabolism of amino acids. The present study could help understand the potential pathway of characteristic microorganisms affecting flavor formation of fat-deficient dry-cured hams and provide theoretical supports for developing healthier fermented meat products.

Identification and Mechanism Elucidation of Anti-Inflammatory Peptides in Jinhua Ham: An Integrative In Silico and In Vitro Study.

This study aimed to effectively identify anti-inflammatory peptides in Jinhua ham, a dry-cured meat product made from the hind legs of pigs by curing and fermenting processes, and elucidate their anti-inflammatory mechanism. The investigation involved a combination of chromatographic purification, in silico screening, and in vitro validation. The first peak of JHP (JHP-P1) was purified using two-part exchange chromatography, in which 3350 peptides were identified by nano-HPLC-MS/MS, among which QLEELKR and EAEERADIAESQVNKLR showed significant anti-inflammatory potential (prediction scores: 0.759 and 0.841). In molecular docking and in vitro RAW264.7 cell experiments, these peptides displayed a strong affinity for Toll-like receptor 4-myeloid differentiation-2 (TLR4-MD-2), specifically binding around Arg 380, Lys 475, His 401, Gln 423, Asp 426, etc. This binding inhibited TLR4 expression and prevented trimer formation about TLR4-MD-2 and lipopolysaccharide (LPS), strongly inhibiting the inflammatory cascade. JHP suppressed LPS-induced cytokine overproduction and partially inhibited the phosphorylation of proteins in the MAPK/NF-κB pathway. These results demonstrated that combining in silico methods (activity prediction and molecular docking) is an effective strategy for screening anti-inflammatory peptides. This study provided a theoretical basis for identifying more anti-inflammatory peptides and applying them in functional foods.

Analysis of Volatile Compounds in Jinhua ham Using Three Extraction Methods Combined with Gas Chromatography-Time-of-Flight Mass Spectrometry.

The volatile compounds in Jinhua ham samples after different aging times were characterized using solvent-assisted flavor evaporation (SAFE), solid-phase microextraction (SPME), and needle trap (NT) extraction methods combined with gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS). Hundreds of aroma compounds were identified, including aldehydes, alcohols, ketones, furans, esters, acids, pyrazines, and sulfides. The results showed that NT extracted the greatest number of volatile compounds, whereas the extraction efficiency of SPME headspace adsorption was highest among the three sample preparation methods. Principal component analysis of SPME effectively distinguished the variation in the aroma of the Jinhua hams specific to aging time. Butyrolactone, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, phenylacetaldehyde, and acetic acid were considered as the main volatile compounds in the Jinhua ham samples at three years of aging. The results showed that SPME-GC-TOF/MS effectively discriminated among samples by age. By comparing the three extraction methods, this study provides a theoretical basis for the selection of extraction methods of volatile aroma compounds in Jinhua ham.

Insights into ultrasonic treatment on the mechanism of proteolysis and taste improvement of defective dry-cured ham.

To investigate the effects of ultrasonic treatment on proteolysis and taste development of defective dry-cured ham, sensory attributes, enzyme activities, protein degradation and free amino acids were evaluated after different ultrasonic treatments. The ultrasonic treatment of 1000 W & 50 °C significantly increased the intensities of overall taste, umami, sweetness and richness, and decreased bitterness values compared with other groups. The residual activities of DPP I and cathepsin B + L in 1000 W & 50 °C maintained 48.71% and 24.94% of control group, respectively; the intense degradation of structural proteins was observed by label-free proteomics, accordingly. The contents of total free amino acids from 4522.64 mg/100 g muscles in control group increased to 5838.75 mg/100 g muscles in 1000 W & 50 °C; the largest increase of sweet and umami amino acids observed in 1000 W & 50 °C was responsible for the improvement of taste quality of defective dry-cured ham.

Stearic acid prevent alcohol-induced liver damage by regulating the gut microbiota.

The pathological characteristics of alcohol-associated liver damage (ALD) mainly include liver lipid accumulation, which subsequently leads to alcohol-associated steatohepatitis, fibrosis and cirrhosis. Dietary factors such as alcohol and fat may contribute to the development of ALD. A chronic alcohol-fed mouse model was used to investigate the effect of fatty acids in Jinhua ham on ALD. The fatty acids in Jinhua ham could prevent the occurrence of ALD from chronic alcohol consumption. In addition, the fatty acids in Jinhua ham with liver protective activity were long-chain saturated fatty acids (LCSFAs), including palmitic acid and stearic acid. In contrast, long-chain polyunsaturated fatty acids aggravated the pathogenesis of ALD. Furthermore, the mechanism underlying the prevention of ALD by fatty acids in Jinhua ham was ascribed to increasing relative abundances of Akkermansia muciniphila and Lactobacillus in the gut, which were beneficial to regulating intestinal homeostasis, ameliorating intestinal barrier dysfunction and reducing alcohol-associated hepatitis and oxidative stress damage. This study demonstrated that dietary supplementation with saturated fatty acids could prevent or mitigate ALD by regulating the gut microbiota (GM) and improving the intestinal barrier, while provided a more affordable dietary intervention strategy for the prevention of ALD.

Insights into the evolution of myosin light chain isoforms and its effect on sensory defects of dry-cured ham.

To better understand the contribution of myosin light chain (MLC) isoforms to sensory defects in Jinhua ham, dipeptidyl peptidase (DPP) activities, peptide fragments, cleavage sites and the potential of DPP to develop sensory defects of dry-cured ham were evaluated and discussed in normal and defective hams. Higher residual activities of DPP I were found in defective ham compared with normal ham; approximate 3-fold peptide fragments were identified in defective ham than in normal ham. These regions of positions 11-35 and 116-141 in MLC 1, 13-53 and 139-156 in MLC 2, and 18-50 in MLC 3 contributed to the intense generation of peptide fragments in defective ham. PLS-DA further revealed DPP I showing intense response to degrade peptides. Cleavage sites including Glu-128, Tyr-132 and Glu-133 were responsible for the intense release of dipeptides in defective ham. These cleavages could play key role in discriminating taste attributes between defective and normal hams.

Characterization of Jinhua ham aroma profiles in specific to aging time by gas chromatography-ion mobility spectrometry (GC-IMS).

A rapid method for analyzing of Jinhua ham samples in different aging time was created based on gas chromatography-ion mobility spectrometry (GC-IMS). The GC-IMS chromatograph provided information regarding the identities and intensities of 37 volatile flavor compounds, including both monomers and dimers. Principal component analysis (PCA) effectively distinguished the variation in the aroma of the Jinhua hams specific to aging time. Alcohol (octanol, 2-methylbutanol), ketones (2-butanone, 2-hexanone, 2-heptanone, acetoin, gamma-butyrolactone), aldehydes (butanal, 3-methylbutanal), ester (propyl acetate) and carboxylic acids (3-methylbutanoic acid) were considered as the main volatile compounds in the Jinhua ham samples. This GC-IMS method, then, proved to be feasible for the rapid and comprehensive detection of volatile compounds in Jinhua hams, and multivariance analysis (i.e.: PCA) was able to provide information related to aging time.

Evaluating the effect of protein modifications and water distribution on bitterness and adhesiveness of Jinhua ham.

Defects with textures and flavors are a common problem, causing many economic losses in the dry-cured ham industry. To obtain a better understanding of the defects of dry-cured ham, texture, protein denaturation, protein structure, and water distribution of normal and defective hams were investigated. Compared with normal ham, more than 1.5-fold values in adhesiveness and bitterness, and less than 0.8-fold values in hardness were found in defective ham. The intense denaturation of sarcoplasmic proteins and actin, and the dramatic transformation of α-helix to ß-sheet were the key modification of proteins; a high proportion (92.39%) of immobile water contributed to the excessive softness and adhesiveness of defective hams. Furthermore, high denaturation of proteins could accelerate the degradation of proteins, which further developed the bitterness and adhesiveness of defective hams. Partial least squares regression demonstrated that the discrepancies in protein denaturation, protein structure and water distribution were related with bitterness and adhesiveness of Jinhua ham.

The Effect of Coating Incorporated with Black Pepper Essential Oil on the Taste Quality of Jinhua Ham After Storage for Four Months.

In this study, 1 H NMR and multivariate data analysis were used to investigate the effect of coating incorporated with black pepper essential oil (CIBPEO) on the taste of Jinhua ham after 4 months of storage; four treatments of control check (CK), base formula coating (BC), BC + 0.05% BPEO, and BC + 0.1% BPEO were used for the coating of hams. Results showed that the metabonome was dominated by 23 metabolites, including amino acids, sugar, organic acids, alkaloids, nucleic aides and their derivatives, and others. BPEO decreased the intensity of sourness, sweetness, bitterness, aftertaste, and the relative nonvolatile taste metabolites compared to CK and BC; the decrease of intensity was not dependent on the BPEO contents. These findings demonstrated that CIBPEO could give a new taste balance to Jinhua ham and be beneficial to a group of people with a particular sensory preference, who are sensitive to undesirable sourness and bitterness, and prefer a light overall taste. PRACTICAL APPLICATION: The coating incorporated with black pepper essential oil during storage could give a new taste balance to Jinhua ham and be beneficial to a group of people with a particular sensory preference, who are sensitive to undesirable sourness and bitterness, and prefer a light overall taste.

The proteomics homology of antioxidant peptides extracted from dry-cured Xuanwei and Jinhua ham.

The objective of this study was to investigate the antioxidant activity and the homology of peptides extracted from dry-cured Xuanwei and Jinhua ham. The antioxidant activity of crude peptides was assessed by ORAC and ABTS assays and the scavenging effects on DPPH and O2- free radicals. LC-ESI-Q-TOF-MS/MS was used to analyze the peptide composition. Based on the identified peptides, homologous proteins were matched by the Peaks software. Overall, 243 and 213 peptides were identified with their parent proteins in Xuanwei and Jinhua dry-cured hams. Based on the ORAC and TEAC values, XHP showed higher antioxidant ability than JHP (P < 0.05). After further purification by G-15 chromatography, the results indicate that the oligopeptides with less than 1000 Da had greater antioxidant activity than the other two fractions. Homology-based proteomics showed that the majority of peptides originated from myosin which accounted for 26% in XHP and 32% in JHP respectively.

Contribution of Histidine and Lysine to the Generation of Volatile Compounds in Jinhua Ham Exposed to Ripening Conditions Via Maillard Reaction.

To evaluate the role of Maillard reactions in the generation of flavor compounds in Jinhua ham, the reactions of glucose and ethanal with histidine and lysine, respectively, were studied by simulating the ripening conditions of Jinhua ham. The volatile products produced were analyzed using solid phase microextraction-gas chromatography/mass spectrometry. The results showed that 8 volatile compounds were generated by the reaction of glucose and histidine and 10 volatile compounds were generated by the reaction of glucose and lysine. Reactions of ethanal with lysine and with histidine both generated 31 volatile compounds that contributed to the flavor of Jinhua ham. This indicates that histidine and lysine related to Maillard reactions possibly play important roles in the generation of the unique flavor compounds in Jinhua ham. This research demonstrates that free amino acids participate in the generation of volatile compounds from Jinhua ham via the Maillard reaction and provides a basic mechanism to explain flavor formation in Jinhua ham. PRACTICAL APPLICATION: Jinhua ham is a well-known traditional Chinese dry-cured meat product. However, the formation of the compounds comprising its special flavor is not well understood. Our results indicate that Maillard reactions occur in Jinhua ham under ripening conditions. This work illustrates the contribution of Maillard reactions to the flavor of Jinhua ham.

Comparison of microbial communities from different Jinhua ham factories.

Microbes in different aged workshops play important roles in the flavor formation of Jinhua ham. However, microbial diversity, community structure and age related changes in workshops are poorly understood. The microbial community structure and diversity in Jinhua ham produced in factories that have 5, 15, and 30 years of history in processing hams were compared using the pyrosequencing technique. Results showed that 571,703 high-quality sequences were obtained and located in 242 genera belonging to 18 phyla. Bacterial diversity and microbial community structure were significantly different with the years of workshops. Three-phase model to characterize the changes of ham microbial communities was proposed. Gas chromatography-mass spectrometry assays indicated that the hams produced in different aged workshops have great differences in number and relative contents of volatiles compounds. These results suggest that different aged factories could form special and well-balanced microbial diversity, which may contribute to the unique flavor characteristics in Jinhua ham.

Stability of an antioxidant peptide extracted from Jinhua ham.

The effects of NaCl, temperature, pH, light intensity and a simulated gastro-intestinal digestion were assessed to determine the stability of antioxidant peptides extracted from Chinese Jinhua ham. Antioxidant peptides showed good stabilities when NaCl content was less than 6%, temperature was lower than 60 °C and they were not exposed to light directly. However, the antioxidant peptides lost antioxidant activities rapidly under alkaline condition. The results of a simulated two-stage digestion system showed that antioxidant activity increased with pepsin treatment but declined with further hydrolysis with trypsin. Pepsin was effective to hydrolyze peptides into smaller fractions leading to the increased exposure of internal hydrophobic amino acids, but trypsin could hydrolyze peptides into more free amino acids resulting in the decline in surface hydrophobicity which affected antioxidant activity of peptides. These suggest that antioxidant peptides extracted from Jinhua ham could maintain their antioxidant activity under the proper conditions.