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The aim of this study was to investigate the modifications of the proteome and flavor-related proteins in unsmoked bacon resulting from ultrasound treatment with the application of label-free quantitation technology together with bioinformatics analysis. Results showed that the expression levels of 137 proteins were markedly affected by ultrasound with most of them being significantly upregulated. The proteins distributed in the cytoplasm and the cytosol, the mitochondrion, and the nucleus were more susceptible to ultrasound treatment. Meanwhile, 20 flavor-related proteins, mostly myofibrillar proteins and metabolic enzymes mainly involved in the metabolic pathways of signaling and cellular processes and environmental information processing, were screened out. In addition, the differential expressions of flavor-related proteins induced by ultrasound were verified by western blotting. This study displayed insightful information from the proteomics perspective for a better understanding of the influential effect of ultrasound treatment on meat flavor., This work was financially supported by the China Agriculture Research System of MOF and MARA (CARS-35), the earmarked fund for the Jiangsu Agricultural Industry Technology System (JATS(2021)476), and the Kekedala City of the 4th Division Science and Technology Plan (2022GY03).

The effect of the partial NaCl replacement by other salts (potassium, calcium, and magnesium chloride) on the formation of volatile compounds through the processing of dry-cured ham was studied using solid-phase microextraction (SPME). Three salt formulations were considered, namely, I (100% NaCl), II (50% NaCl and 50% KCl), and III (55% NaCl, 25% KCl, 15% CaCl(2), and 5% MgCl(2)). There was an intense formation of volatile compounds throughout the processing of dry-cured hams, particularly during the "hot-cellar" stage. The differences between treatments were found to be more remarkable at the end of the curing process. Hams from formulations I and II had significantly higher amounts of lipid-derived volatiles such as hexanal than hams from formulation III, whereas the latter had significantly higher amounts of Strecker aldehydes and alcohols. Plausible mechanisms by which salt replacement may affect the generation of volatile compounds include the influence of such replacement on lipid oxidation and proteolysis phenomena. The potential influence of the volatiles profile on the aroma of the products is also addressed in the present paper.

The main objective of this work was to identify and characterize the peptides generated by simulated gastrointestinal digestion of pork meat (longissimus dorsi) by the sequential action of pepsin and pancreatin. The obtained hydrolysate was analyzed by liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization source (nano LC-ESI-MS/MS). Using this technique 51 different peptides were identified in the hydrolysate, corresponding to fragments of the main structural muscle proteins and some well-known sarcoplasmic proteins. To the best of our knowledge, this constitutes the highest number of peptides identified in pork meat digests. Peptide fragment size ranged from six to sixteen amino acids, being rich in proline residues and thus making them more resistant to further degradation by digestive enzymes. The present study constitutes a clear evidence of the extensive degradation that pork muscle proteins would undergo after gastrointestinal digestion, giving rise to a wide variety of short peptides. So, the use of in vitro digestion contributes to a better knowledge about the generation of peptides from diets with high protein quality.

A proteomic approach has been carried out to investigate the extensive proteolysis occurring in the processing of Serrano ham. In this study, a total of 14 peptide fragments derived from myosin light chain I and titin have been identified for the first time. Nine of these peptides originated from myosin light chain I protein, with the loss of dipeptides at the N-terminal position observed in some of them. This suggests that dipeptidyl peptidases are involved in the generation of dipeptides, which contribute to the generation of the characteristic taste associated with Serrano ham. The other five peptides came from the PEVK region of the titin protein. This region is believed to confer elasticity to the sarcomere as well as the ability to bind calpains. The hypothetical action of mu-calpain and calpain 3 enzymes over this region would make these enzymes potentially responsible for protein breakdown during the early dry-curing stage.

The binding of sarcoplasmic and myofibrillar proteins extracted from postrigor pork muscle and from 7 and 12 months dry-cured hams with volatile compounds such as 3-methyl-butanal, 2-methyl-butanal, 2-pentanone, hexanal, methional, and octanal was studied using solid phase microextraction and gas chromatography analysis. The binding ability of sarcoplasmic proteins from pork muscle was higher than the ability shown by 7 and 12 months dry-cured ham sarcoplasmic homogenates and also higher than the binding ability of myofibrillar homogenates. The effect of the ionic strength on the binding was also studied. This effect was more important on myofibrillar proteins due to its ability to produce changes on the protein conformation that affect their binding ability. However, the sarcoplasmic protein binding ability was more related to the small compounds present in this homogenate than with the aqueous phase ionic strength.

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.

Lactobacillus casei CRL 705 was screened, among other meat isolates, for its proteinase and aminopeptidase activities toward synthetic substrates and, according to that, selected for specific assays on muscle proteins. The hydrolytic effects of whole cells, cell free extracts (CFE), and the combination of both on muscle sarcoplasmic and myofibrillar protein extracts was evaluated by SDS-PAGE and reverse phase HPLC analyses. The proteinase activity of whole cells caused the degradation of a great number of sarcoplasmic protein bands. A partial hydrolysis was also associated with CFE that when combined with whole cells showed an important additional degradation. Peptide profiles from sarcoplasmic protein extracts were greatly modified regardless of the addition of whole cells or CFE, although their combination intensified these changes. The generation of free amino acids was remarkable when whole cells and CFE were incorporated together to sarcoplasmic protein extracts.

Dipeptidyl peptidase III (EC 3.4.14.4) was purified from porcine skeletal muscle by ammonium sulfate fractionation and by chromatography and rechromatography on a Biosept-DEAE HPLC column. The enzy...

A tripeptidase was purified to homogeneity from the cell extract of Lactobacillus sake by ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration chromatography, and two steps of anion exchange chromatography. After SDS-PAGE a single band of protein was detected of approximately 55 kDa. A similar molecular mass was estimated by gel filtration. The tripeptidase activity was optimal at pH 7.0 and at 40 degrees C. The enzyme was strongly inhibited by metal chelators, reducing agents, and bestatin while thiol group reagents, serine proteinase inhibitors, and aspartic proteinase inhibitors had no effect on the activity. The enzyme was activated by Mn(2+) and almost totally inhibited by Zn(2+) and to a lesser extent by Sn(2+). The enzyme only exhibited activity against tripeptides, and those hydrolyzed at higher rates were Ala-Ala-Ala, Ser-Ser-Ser, and Leu-Gly-Gly.

Spanish “Serrano” dry-cured hams were processed under traditional practices which included two different length of the ripening−drying stage dry-curing methods. Dry-cured hams typically have high production cost because of the length of the ripening−drying stage which makes the product less competitive. In order to study the generation of dry-cured ham flavor the volatile components were investigated. Sensory properties were analyzed by GC/olfactometry and descriptive sensory techniques. The relationship of the volatile components with sensory descriptors was examined by factor analysis and resulted in a solution composed of four factors defined as “pork”, “cured”, “pleasant”, and “off-flavor”. The short ripening process was characterized by aldehydes, such as hexanal and 3-methyl butanal, alcohol (1-penten-3-ol), and dimethyl disulfide, that gave an olfactory sensation of fresh-cured pork flavor. The “pleasant” aroma in the short process had already been developed and was defined by ketones, esters, pyra...

The effect of myoglobin content on the activity of different proteolytic systems, lysosomal enzymes, calcium dependent neutral proteinases, and aminopeptidases was examined concretely. Increasing myoglobin content inhibited cathepsin activities (B, B + L, and H). A similar effect was obtained with μ-calpain, which showed about 10% of its initial activity in the presence of 1 mg/mL of myoglobin. In contrast, m-calpain and calpastatin were almost insensitive to different levels of myoglobin in the medium. Exopeptidases, aminopeptidase B and alanyl aminopeptidase, were inhibited by myoglobin to approximately the same extent as the cathepsins. These results suggest that myoglobin might be considered an endogenous inhibitor, which could affect the endogenous proteolytic activity differently in different types of muscles. Consequently, the rate of both meat tenderization and generation of free amino acids may also be regulated by myoglobin. Keywords: Myoglobin; inhibition; muscle proteases; cathepsins; calpains...

[EN] One of the main biochemical changes that take place during the processing of dry-cured ham is the degradation of the muscle protein fraction, mainly due to the action of muscle enzymes. In the present study, the isolation and tentative identification of 137 fragments from myosin light chain 1 (MLC 1), together with 88 fragments originated from myosin light chain 2 (MLC 2), have been achieved for the first time in Spanish dry-cured ham, proving the intense proteolysis experienced by myofibrillar proteins after dry-cured processing. This study was carried out by use of proteomic technology for peptide identification, and the possible enzymes contributing to the degradation of these proteins were also further discussed., Project AGL2010-16305 from MICINN (Spain) and FEDER funds are acknowledged.

An attempt to decrease the NaCl content in dry-cured products through the use of three different salting treatments (II: 55% NaCl, 25% KCl, 15% CaCl(2), 5% MgCl(2); III: 45% NaCl, 25% KCl, 20% CaCl(2), 10% MgCl(2); and IV: 30% NaCl, 50% KCl, 15% CaCl(2), 5% MgCl(2)) in comparison to a control (I: 100% NaCl) was assayed to evaluate the biochemical and sensory characteristics in the final product. Most proteolytic enzyme activities from the loins submitted to the experimental salting treatments, especially treatments II and IV, remained higher than those salted traditionally (control). The higher aminopeptidase activity was also reflected in a larger release of free amino acids. Finally, a sensory paired comparison test revealed that those loins salted with the treatment II were not significantly different from the loins salted traditionally (100% NaCl), so that this treatment could be successfully used for sodium reduction.

The main purpose of this work was to study the generation of Angiotensin I-converting enzyme inhibitory (ACEI) peptides after gastrointestinal digestion of pork meat by the action of pepsin and pancreatin at simulated gut conditions. The hydrolysate was further subjected to reverse phase chromatography in order to separate the fractions with ACEI activity. Using MALDI-TOF/TOF mass spectrometry, 12 peptides were identified in these fractions. It is worth highlighting the novel peptides ER, KLP, and RPR with IC(50) values of 667 microM, 500 microM, and 382 microM, respectively. Results obtained by MALDI-TOF/TOF mass spectrometry were complemented by a second approach consisting of the analysis of the hydrolysate directly by nanoLC-ESI-MS/MS followed by a study of the obtained sequences and comparison with known ACEI peptide sequences. By using these two approaches, a total of 22 peptides were selected for its synthesis and further in vitro assay of ACEI activity. The strongest ACE inhibition was observed for peptide KAPVA (IC(50) = 46.56 microM) followed by the sequence PTPVP (IC(50) = 256.41 microM). Sequence similarity searches revealed that these two peptides derive from muscle titin, constituting the first identified ACEI peptides coming from this protein. This is also the first time that ACEI sequences MYPGIA and VIPEL have been reported. Other identified and synthesized sequences showed less ACEI activity. The obtained results evidence the potential of pork meat proteins as a source of antihypertensive peptides after gastrointestinal digestion.

The aim of this study was to evaluate the effect of different cooking procedures on the concentrations of creatine and creatinine and the ratio of creatinine/creatine in cooked ham. Two cooking methods (constant temperature and increasing temperature, constant T and DeltaT, respectively) were tested on different locations in porcine longissimus dorsi muscle and ham (semimembranosus, biceps femoris, and gluteus muscles). The results showed larger creatine conversion into creatinine in the surface layer than in the core as well as higher creatinine/creatine ratio values when applying the DeltaT in comparison to the constant T method. A correlation between the creatinine/creatine ratio and the heat treatment was established, and 15 samples of commercial cooked hams were analyzed to support these results. This creatinine/creatine ratio analyzed in the surface of the ham could be used as a rapid and nondestructive indicator to determine the effectiveness of the heat treatment in cooked ham processes.

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.

Three levels (0%, 1%, and 2%) of conjugated linoleic acid (CLA) were combined with two levels (low and high) of monounsaturated fatty acids (MUFA) for pig feeding. The activity of neutral lipase (NL), acid lipase (AL), phospholipase (PL), neutral esterase (NE), and acid esterase (AE) was measured in extracts from muscle and subcutaneous adipose tissues. The addition of CLA in the diet only affected the lipolytic activity in muscle, whereas differences in MUFA content of pig diets were mainly responsible for the lipolytic enzyme modifications observed in adipose tissue. Nevertheless, a significant effect of the interaction CLA x MUFA on the activity of several lipolytic enzymes was observed in both tissues. The effect of either linoleic acid (LA) or CLA on the activity of muscle and adipose lipolytic enzymes was determined by in vitro assays. Remarkable inhibitory or activation effects were detected depending on the enzyme and kind of tissue.

The effect of curing agents (salt, glucose, nitrate, nitrite, and ascorbic acid) on the binding of skeletal peptides (carnosine and anserine) and a sarcoplasmic protein (myoglobin) with key flavor compounds (hexanal, octanal, 2-pentanone, 2-methylbutanal, and 3-methylbutanal) has been studied by solid-phase microextraction (SPME). Curing agents had an effect on the interaction process between carnosine and volatile compounds, which was higher than the interactions observed with anserine and myoglobin. Sodium chloride decreased the interaction of volatiles with carnosine except for octanal, which was increased, and 2-pentanone, which was unaltered. Ascorbic acid exerted the highest effect by decreasing the interaction of carnosine with all of the volatile compounds except for octanal and 2-pentanone. The interaction with anserine was affected by sodium chloride, nitrate, and nitrite, producing a decrease in the interaction with hexanal, octanal, and methional. Finally, sodium chloride, glucose, and nitrite increased the interaction of myoglobin with hexanal, octanal, and methional. The effect of simulated stages of the curing process on the binding was also studied. A combined effect of the curing agents resulted in a change in the relative proportions of volatile compounds that can lead to different flavor perceptions of dry-cured meat products.

The ability of skeletal dipeptides (carnosine and anserine) and a sarcoplasmic protein (myoglobin) to interact with key flavor compounds (hexanal, octanal, methional, 2-pentanone, 2-methylbutanal, and 3-methylbutanal) has been studied using the solid phase microextraction (SPME) technique. Conditions for SPME analysis (fiber coating, sampling time, and linearity of detection) were optimized. The effect of pH on the binding was also investigated. Thermodynamic models were applied to evaluate the binding parameters n (number of binding sites), K (affinity constant), and DeltaG (Gibb's free energy) to all of the flavor compounds studied, and they showed an absence of cooperative effect. Carnosine was the peptide with the highest affinity for all of the volatile compounds except 2-pentanone. Its interaction with hexanal and methional was significantly affected by pH. Anserine showed a lower level of interactions with hexanal, methional, 2-methylbutanal, and 3-methylbutanal, whereas myoglobin interacted with only hexanal and 2-methylbutanal. Differences in aroma retention can thus result in different sensory perceptions of muscle foods.