Jinhua Ham Research Articles

Decoding of the saltiness enhancement taste peptides from Jinhua ham and its molecular mechanism of interaction with ENaC/TMC4 receptors

This study focused on unlocking the potential of Jinhua ham-derived peptides (JHP) for enhancing saltiness. JHP (<3 kDa) was obtained through ultrafiltration and desalting, reducing the salt content by 96 %. Four peptide fractions (JHP-P1/P2/P3/P4) were isolated using Sephadex G-25 gel filtration and anion-exchange chromatography. Sensory evaluation and electronic tongue analysis revealed that JHP-P2 (0.5 mg/mL) exhibited the highest saltiness which could replace four-fold NaCl salinity. Three peptides (DL, FMSALF, and HVRRK) identified by UPLC-QTOF-MS/MS were simulated with salty taste receptors ENaC/TMC4. Results indicated that Ser84 and Phe89 of ENaC and Asn404 and Lys567 of TMC4 are crucial for peptide docking related to salty taste. Molecular dynamics simulations showed that the three peptides bind to the TMC4 and ENaC through van der Waals forces, electrostatic interactions, and hydrogen bonds. These findings establish a robust theoretical foundation for salt reduction strategies and provide novel insights into the potential applications of Jinhua ham.

LC-MS-based metabolomics approach to investigate the taste compounds formation of Jinhua ham prepared using Taihu pigs

LC-MS-based metabolomics approach to investigate the taste compounds formation of Jinhua ham prepared using Taihu pigs

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.

Insights into the mechanism of Penicillium inoculation on microbial succession, volatile compound evolutions and sensory development of Jinhua ham

SummaryTo investigate the mechanism of Penicillium inoculation on the flavour improvement of Jinhua ham, the microbial succession, free amino acid contents, volatile compound evolutions, sensory attributes and the relationship between key volatiles and sensory properties were investigated during the processing of Jinhua ham with P. aethiopicum (PA) or P. chrysogenum (PC) inoculation. The total counts of fungal communities in CK, PA and PC groups significantly increased from 2.74 log CFU*g−1 of raw ham to 5.18, 5.39 and 5.50 log CFU*g−1 from day 0 to day 28, and then decreased by 2.06, 1.94 and 1.86 log CFU*g−1 from day 28 to the final products (P &lt; 0.001). The total contents of free amino acids significantly increased from 1584.22 mg/100 g of CK to 2117.20 mg/100 g of PA and 2174.05 mg/100 g of PC (P &lt; 0.01); the contents of alanine, glutamic acid, lysine, valine, leucine and phenylalanine in PC were significantly higher than those of CK and PA (P &lt; 0.05). Sensory scores of meaty aroma, nutty aroma and overall acceptance showed the largest values in PC among three groups. GC–MS results indicated that aromatic aldehydes and pyrazines in PC showed the largest increase, compared with CK and PA groups. PLS‐DA and correlation analysis demonstrated that benzaldehyde, benzeneacetaldehyde and 2,5‐dimethyl pyrazine contributed to the improvement of aroma attributes of Jinhua ham with Penicillium inoculation. The KEGG analysis further revealed that the metabolism of valine and phenylalanine was mainly involved in developing aromatic aldehydes and pyrazines of Jinhua ham with Penicillium inoculation. These results indicated that the inoculation of Penicillium could enhance the flavour quality of Jinhua ham.

Stability and transepithelial transport of oligopeptide (KRQKYD) with hepatocyte-protective activity from Jinhua ham in human intestinal Caco-2 monolayer cells

The study evaluated the stability of an oligopeptide (Lys-Arg-Gln-Lys-Tyr-Asp, KRQKYD) and its transport mechanism by simulating gastrointestinal digestion and a model of human intestinal Caco-2 monolayer cells in vitro. In this study, the effects of environmental factors (temperature, pH and NaCl concentration) and simulated gastrointestinal digestion on the stability of KRQKYD were evaluated by indicators of the levels of alanine transaminase (ALT), aspartate transaminase (AST) and malondialdehyde (MDA) in an alcohol-induced hepatocyte injury model. The results showed that KRQKYD still maintained satisfactory hepatocyte-protective activity after treatment with different temperatures (20−80 °C), pH (3.0−9.0), NaCl concentration (1 %−7 %) and simulated gastrointestinal digestion, which indicated that KRQKYD showed good stability to environmental factors and simulated gastrointestinal digestion. Furthermore, the intact KRQKYD could be absorbed in a model of Caco-2 monolayer cells with a Papp value of (9.70 ± 0.53) × 10−7 cm/s. Pretreatment with an energy inhibitor (sodium azide), a competitive peptide transporter inhibitor (Gly-Pro) and a transcytosis inhibitor wortmannin did not decrease the level of transepithelial KRQKYD transport, indicating that the transport mechanism of KRQKYD was not associated with energy dependent, vector mediated and endocytosis. The tight junction disruptor cytochalasin D significantly increased the level of transepithelial KRQKYD transport (P < 0.05), suggesting that intact KRQKYD was absorbed by paracellular transport.

Investigation of conformational changes of cathepsin B as a feasibility assessment for NaCl reduction in Jinhua ham

Investigation of conformational changes of cathepsin B as a feasibility assessment for NaCl reduction in Jinhua ham

The Contribution of Microorganisms to the Quality and Flavor Formation of Chinese Traditional Fermented Meat and Fish Products.

Guizhou sour meat and sour fish, Chaoshan fish sauce, Sichuan sausage and bacon, Cantonese sausage, Jinhua ham, and Xinjiang air-dried beef are eight representatives of Chinese traditional fermented meat and fish products (FMFPs), which are favored by Chinese consumers due to their high nutritional value and quality. The quality of the spontaneously fermented Chinese traditional FMFP is closely correlated with microorganisms. Moreover, the dominant microorganisms are significantly different due to regional differences. The effects of microorganisms on the texture, color, flavor, nutrition, functional properties, and safety of Chinese traditional FMFPs have not been not fully described. Additionally, metabolic pathways for flavor formation of Chinese traditional FMFPs have not well been summarized. This article describes the seven characteristic Chinese traditional FMFPs and correlated dominant microorganisms in different regions of China. The effects of microorganisms on the texture, color, and flavor of Chinese traditional FMFPs are discussed. Furthermore, the metabolic pathways of microbial regulation of flavor formation in Chinese traditional FMFPs are proposed. This work provides a theoretical basis for improvement of Chinese traditional FMFPs by inoculating functional microorganisms isolated from Chinese traditional fermented foods.

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.

Production of branched-chain flavor compounds from hydrolysis of pork myofibrillar protein by optimizing the inoculation of Limosilactobacillus fermentum YZU-06 and Staphylococcus saprophyticus CGMCC 3475

This study aimed to develop a mixed starter of Limosilactobacillus fermentum YZU-06 and Staphylococcus saprophyticus CGMCC 3475 isolated from Jinhua ham for enhancing the production of branched-chain flavor compounds from pork myofibrillar protein. Different ratios of mixed strains (3:1, 2:1, 1:2, and 1:3) and single strains were employed to incubate with myofibrillar protein extract to detect viable counts, pH, proteolysis, and branched-chain volatiles at 24, 48 and 72 h of incubation. The result showed that the inoculated strains had acid-producing characteristics and bacterial suitability. The mixed starter of 2:1, 1:2, and 1:3 treatments exhibited a higher degree of protein degradation, as indicated by a 90% similarity through cluster analysis of myofibrillar protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compared to the other treatment groups, 2:1 group showed a significantly higher peptide content of 123.58 μg/mL and a greater concentration of free amino acids (236.67 mg/L). Furthermore, 2:1 group obtained the highest levels of 3-methylbutanal (56.06 μmol/L), 2-methylpropanal (1.76 μmol/L), and 2-methylbutanal (0.48 μmol/L). Our findings emphasize the importance of appropriate proportions of L. fermentum YZU-06 and S. saprophyticus CGMCC 3475 for the starter to produce branched-chain flavor compounds of meat products.

Controlling cathepsin B activity in Jinhua ham through multifactorial analysis and modeling of temperature, pH and salt content

The activity of cathepsin B in Jinhua ham was affected by various processing parameters, which were investigated and quantified using a multi-layer artificial neural network (ANN). The key factors that influence ham quality are processing temperature, pH, and sodium chloride content. The study discussed the univariate analysis and multivariate interactions between two or three factors. The established ANN model was used for further investigation, and the actual enzyme activity of cathepsin B was characterized. The results showed that high temperature (greater than 60 °C) and an alkaline environment or the addition of Na + cation significantly reduced the enzyme activity (p < 0.05). The optimal conditions for protease activity were found to be at 40 °C and pH 6.0. The enzyme was found to be unstable at a temperature of 20 °C or pH 6.5–7.0 in the presence of NaCl, indicating significant interactions between temperature, pH value and NaCl content (p < 0.01). Furthermore, the ANN model showed good match between true and predicted values. The correlation coefficient was 0.98468, and the model provided supplementary information about the effect of NaCl on cathepsin B activity within 0.4M–0.6M. The findings could be useful in Jinhua ham production and the study of the structure-activity relationship of cathepsin B.

Quality Control of Jinhua Ham from the Influence between Proteases Activities and Processing Parameters: A Review

Endogenous proteases are significant for Jinhua ham quality. Protein degradation affects the chemical traits, texture and the formation of flavor substances. Protease activities are affected by different process parameters, such as processing temperature, maturation time, salt content and the drying rate. They affect ham quality, which can be controlled by process parameters. The influences of key factors on Jinhua ham quality are briefly summarized, which can provide a theoretical basis for the selection of specific parameters in dry-cured ham processing. Furthermore, some suggestions are proposed for correcting and improving the flavor and textural defects of ham, yet the effectiveness depends on the operating conditions. The determination of enzyme activity is not real-time and unsupervised at the moment. Future research will focus on the determination of the actual endogenous protease activity and the quantitative relationship between the enzyme activity and main processing parameters.

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.

Insight into the impacts of Jinhua ham processing conditions on cathepsin B activity and conformation changes based on molecular simulation

To produce the high quality products and improve the stability of Jinhua hams, the activities of cathepsins and their correlation with processing parameters were explored. The effect of heat treatment on the structure of cathepsin B, together with potential of hydrogen and NaCl concentration, were investigated. At 45 °C, the activity of cathepsin B was maximum and it decreased rapidly at high temperature. The decrease on sulfhydryl group content indicated that the enzyme activity varied due to the breaking of disulfide bond in molecule. Infrared spectroscopy indicated that the secondary structure of cathepsin B had altered. Meanwhile, the root mean squared deviation and the root mean square fluctuation curves reflected that the cathepsin B vitality was in a decline under high temperature. In addition, in the presence of 0.6 mol ⋅ L−1 NaCl, lots of conformational changes were evidenced. Under acidic conditions (pH = 6), there were more formed hydrogen bonds, and the hydrogen bonding interactions became stronger. Simulation results contributed to providing an important theoretical basis for understanding the roles of processing parameters on the quality control of Jinhua ham.

Peptidomic insights on structural protein hydrolysis by exopeptidase activities and its effects on bitterness defects of dry‐cured ham

SummaryExcessive bitterness is a common problem and is related to the hydrolysis of structural proteins degradation in the dry‐cured ham industry. To better understand the effect of structural proteins (myosin, titin, troponin) degradation on sensory development of Jinhua ham, sensory attributes, exopeptidase activities, peptide fragments and cleavage sites of structural proteins were investigated in both premium and defective hams. The results of sensory evaluation and electronic tongue revealed that more than 1.5‐fold values in bitterness intensities and less than 0.67‐fold values in richness intensities were observed in defective ham, compared with premium ham. The activities of exopeptidase were determined, and the results indicated that the residual activities of TPP I in defective ham were higher, while the residual activities of LAP and AAP were lower, compared with premium ham. The large numbers of unique peptide fragments derived from myosin, titin and troponin were identified in defective ham. Several unique cleavage sites including L‐399, L‐1047 and K‐1053 for MYH 1 and MYH 4, K‐257, R‐694, K‐1374 and K‐1651 for MYH 7, and K‐92 and K‐103 for TNN C1 and TNN C2 were only presented in defective ham, which were intensely response to the excessive accumulation of peptides and the development of excessive bitterness in defective ham. This study could provide a theoretical basis for the regulation of bitterness defects in dry‐cured ham in the future.

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.

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.

Correlation of characteristic flavor and microbial community in Jinhua ham during the post-ripening stage

In this work, the changes in volatile flavor compounds and microbial communities in post-ripening Jinhua ham were analyzed. Hexanal and nonanal were the main flavor substances in Jinhua ham stored for 1-3 years, and the content of which were increasing during the post-ripening stage and finally reached 261.80 μg/100g and 445.33 ± 15.29 μg/100g, respectively. High-throughput sequencing showed that Aspergillus, Saccharomyces, Staphylococcus, and Cobetia were the dominant microorganisms in post-ripening Jinhua ham. The dominant fungi were Aspergillus (post-ripened for 1-2 years) and Saccharomyces (post-ripened for 3 years). Staphylococcus accounted for the largest proportion of the bacteria in Jinhua ham, and the proportion gradually increased with the passage of time, from 50.21% to 64.66% during the post-ripening. Spearman correlation analysis showed that Saccharomyces (P < 0.001), Aspergillus (P < 0.01), and Staphylococcus (P < 0.05) were positively correlated with the flavor of Jinhua ham in the post-ripening stage. The revealed correlation of microorganisms and the flavor in post-ripening Jinhua ham can be a foundation for the quality improvement of dry-cured ham and the development of fermented meat starter.

Evolution of antioxidant peptides and their proteomic homology during processing of Jinhua ham

This study aimed to clarify the evolution of antioxidant peptides and their proteomic homology during a continuous period of Jinhua ham processing. The peptide content increased during the process, reaching a value of 1.92% (weight %, based on meat weight) in ham at the end of dry-ripening, which was approximately 8-fold higher than that prior to salting. From scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and hydroxyl radical (•OH) and chelating ability on Fe2+, it was suggested that peptides generated at the end of dry-ripening had the highest level of antioxidant activity. Size-exclusion chromatography indicated that the antioxidation of peptides might be related to their MWs, where more fractions with small MWs result in higher antioxidant activity. The results of endogenous protease activities showed 22.33% of the initial activities of Cathepsin B were retained, suggesting that Cathepsin B plays a key role in the formation of antioxidant peptides. Homology-based proteomics showed that peptides primarily arose from myosin (32.37%), followed by actin (16.91%), troponin T (12.08%) and others.

Charactering the spoilage mechanism of “three sticks” of Jinhua ham

To investigate the spoilage characteristics of Jinhua ham, sensory scores, volatile compounds, biogenic amine, physicochemical parameters and microbial counts were evaluated between normal and spoiled hams. The results showed that off-odors of spoiled hams were dominated by rancid, sour, sulfide and ammonia odors derived from these compounds including butanoic acid, methanethiol and dimethyl disulfide. Total content of biogenic amine in spoiled hams was significantly higher (more than 10-fold) compared with normal hams, and putrescine, cadaverine and histamine were the key components of biogenic amine of spoiled hams. Lower salt content, and higher moisture, TVB-N and thiobarbituric acid reactive substances (TBARS) values were observed in spoiled hams compared with normal hams. The populations of Enterobacteriaceae and Enterococcus of spoiled hams were obviously higher than that of normal hams. High moisture and low salt content caused the abnormal growth of Enterobacteriaceae and Enterococcus in spoiled hams, which contributed to the spoilage of Jinhua ham.

Characterization of a lactic acid bacteria using branched-chain amino acid transaminase and protease from Jinhua Ham and application in myofibrillar protein model

This study was to screen a strain with both branched-chain amino acid transaminase (BCAT) and protease activities for producing methyl-branched flavor compounds in a myofibrillar protein extract model. Lactic acid bacteria (LAB) was isolated from Jinhua ham and screened with BCAT activity by an electrochemical sensor and protease activity by the agar plate method. In the culture medium, strain L6 was selected with high utilization rate and characteristic metabolites content of branched-chain amino acids (BCAAs), and identified as Lactobacillus fermentum YZU-06 (L. fermentum). Compared with the previously reported L. plantarum, L. fermentum exhibited an excellent capacity of hydrolyzing myofibrillar protein with the higher contents of free amino acids, peptides and small molecular weight proteins. Moreover, L. fermentum group presented more BCAAs metabolites of 2-methylbutanal and 3-methylbutanal than that of L. plantarum group. In conclusion, L. fermentum YZU-06 is a promising starter culture to improve the flavor of fermented meat products.