PRKAG3 and CAST genetic polymorphisms and quality traits of dry-cured hams-II. Associations in French dry-cured ham Jambon de Bayonne and their dependence on salt reduction

This study aimed to evaluate the effects of PRKAG3 Ile199Val and CAST Arg249Lys and CAST Ser638Arg polymorphisms on the quality traits of the French dry-cured ham Jambon de Bayonne and their interaction with salt reduction. Significant (p<0.05) and suggestive associations (p<0.10) between the polymorphisms and several quality traits of dry-cured ham, mainly related to processing and textural properties, were found. PRKAG3 Ile/Val and CAST 249Lys/638Arg presented the highest scores for sensory and processing properties, whatever the salt content.     

Lipolysis in dry-cured ham: Influence of salt content and processing conditions

Fatty acid composition of neutral lipids (NLs), polar lipids (PLs) and free fatty acids (FFA) from the intramuscular fat of Semimembranosus and Biceps femoris muscles was analysed in 46 Iberian dry-cured hams processed with different amounts of salt (6% high salt batch – HS vs. 3% low salt batch – LS w/w) and different processing systems (traditional – T vs. modified – M).

Total amounts of saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids in NLs decreased in similar proportions during processing of the hams as well as SFA, MUFA and PUFA in the PL fraction, whereas the amounts of SFA, MUFA and PUFA of FFAs significantly increased in Semimembranosus and Biceps femoris muscles. The amount of total fatty acids (TFA), from NLs and PLs, decreased in both muscles throughout the processing. Such a decline was more intense in HS hams than in LS ones, which could be a sign of a promoting effect of sodium chloride on lipolysis. However, the increase in FFA content throughout processing was not more intense in HS hams. Processing conditions studied in this work did not affect the changes in the fatty acid content of each fraction.     

Proteomic analysis of semimembranosus and biceps femoris muscles from Bayonne dry-cured ham

The aim of the study was to delineate and compare the proteomic maps of two muscles of dry-cured ham: the biceps femoris and the semimembranosus. For this purpose, we used two-dimensional electrophoresis on a subcellular muscle fraction: insoluble protein in low ionic strength buffer. After protein identification by MALDI-TOF mass spectrometry and bioinformatic analyses, we found differences in expression levels in the two muscles. Seventy-three proteins or fragments were differentially expressed: 43 were over-represented in semimembranosus and 30 in biceps femoris. Although the study was performed on the insoluble protein fraction in low strength ionic buffer, protein and fragment identifications by mass spectrometry showed that most of the proteins were involved in energy metabolism. The differences observed between the two muscles can be explained by the differences in salt and moisture content in the course of dry-cured ham processing.     

Use of computed tomography to study raw ham properties and predict salt content and distribution during dry-cured ham production

Varying salt content in hams of equal brand is a major challenge for Norwegian dry-cured ham producers. This study was thus undertaken to test existing computed tomography (CT) calibration models for salt on entire hams, regarding predictability of salt content at different processing times including final ham and to study salt distribution during processing of dry-cured ham. Twenty-six hams were scanned by computed tomography (CT) 11 times during dry-curing for this purpose. However, previously established calibration models had to be adjusted as they overestimated salt in dry samples. Prediction of ultimate salt content was more accurate approaching the end of the dry-curing process (RMSEP = 0.351-0.595% salt). Inclusion of remaining weight loss improved the prediction accuracy in un-dried samples by approximately 0.1% NaCl. The prediction errors were sufficiently low to be of practical interest.     

Thermal and photochemical degradation of myoglobin pigments in relation to colour stability of sliced dry-cured Parma ham and sliced dry-cured ham produced with nitrite salt

Lipophilic and hydrophilic extracts of the red pigments from Parma ham and nitrosylated pigment of dry-cured ham produced with nitrite salt were prepared with acetone/water (75/25 v/v %) solution and aqueous phosphate buffer, respectively. The spectral characteristics differed for both the lipophilic and the hydrophilic Parma ham pigment compared with the dry-cured ham produced with nitrite salt. The red lipophilic pigment(s) extractable from Parma ham was(were) found to be very stable towards thermal degradation in acetone/water (75/25 v/v %) solution for temperatures up to 70 °C in contrast to the lipophilic pigment(s) extractable from dry-cured ham produced with nitrite salt, which was(were) found to have an energy of activation of 99 kJ/mol for thermal degradation. In contrast, quantum yields for photodegradation of the lipophilic ham pigments exposed to 366 nm (420 nm) monochromatic light were larger for Parma ham than for nitrite-cured ham [1.6×10^–5 (6.9×10^–6) versus 1.6×10^–6 (2×10^–6) mol einstein^–1] as determined for acetone/water (75/25 v/v %) solution. In agreement with these findings for the extracted lipophilic pigments, sliced Parma ham showed better colour stability than sliced dry-cured ham produced with nitrite salt, when stored in the dark at low oxygen concentration, in contrast to a faster initial discolouration for Parma ham when exposed to light, as shown for chilled storage for 35 days under retail conditions for the two products each packed at two oxygen levels (0.4 and 21%).     

Accuracy of near infrared spectroscopy for prediction of chemical composition, salt content and free amino acids in dry-cured ham

The capability of near infrared (NIR) spectroscopy was examined for the purposes of quality control of the traditional Slovenian dry-cured ham “Kraški pršut.” Predictive models were developed for moisture, salt, protein, non-protein nitrogen, intramuscular fat and free amino acids in biceps femoris muscle (n = 135). The models' quality was assessed using statistical parameters: coefficient of determination (R²) and standard error (se) of cross-validation (CV) and external validation (EV). Residual predictive deviation (RPD) was also assessed. Best results were obtained for salt content and salt percentage in moisture/dry matter (R_CV² > 0.90, RPD > 3.0), it was satisfactory for moisture, non-protein nitrogen, intramuscular fat and total free amino acids (R_CV² = 0.75–0.90, RPD = 2.0–3.0), while not so for protein content and proteolysis index (R_CV² = 0.65–0.75, RPD < 2.0). Calibrations for individual free amino acids yielded R_CV² from 0.40 to 0.90 and RPD from 1.3 to 2.9. Additional external validation of models on independent samples yielded comparable results. Based on the results, NIR spectroscopy can replace chemical methods in quality control of dry-cured ham.     

Consumers’ acceptance of innovations in dry-cured ham: Impact of reduced salt content, prolonged aging time and new origin

The objective of this study was to evaluate effects of information about reduced salt content, prolonged aging time and new origin on the acceptance of dry-cured ham. The study was carried out in Norway and origins of tested hams were Norway and Spain. Consumers’ acceptance of hams was investigated in blind, expected and informed conditions. Results showed that ratings in the informed condition changed in the direction of the expectations and significant assimilation effects occurred for two products. Two consumer clusters were identified. Consumers in the first cluster were more open to trying new kinds of food and this attitude was exemplified by a relatively high expected score for dry-cured ham with reduced salt level, long aging and Spanish origin. Consumers in the second cluster were more sceptical to new food and new dishes. This was reflected in a relatively high expected score for the traditional Norwegian ham with high salt level and short aging time.     

Nucleotides and their degradation products during processing of dry-cured ham, measured by HPLC and an enzyme sensor

The aim of this work was to study how nucleotide degradation during the processing of dry-cured ham is affected when using three types of salting (100% NaCl; 50% NaCl and 50% KCl; 55% NaCl, 25% KCl, 15% CaCl₂ and 5% MgCl₂). Divalent salts in the salting mixture depressed the breakdown rate from the beginning of the process (salting and post-salting) up to the ripening stage (7 months) when the inosine (Ino), hypoxanthine (Hx) and xanthine (X) concentrations matched for the three treatments. The evolution of Hx and Hx + X were analysed by HPLC and an enzyme sensor, respectively, during processing. Time and temperature conditions during the curing time did not affect Hx stability. The usefulness of the enzyme sensor was confirmed and it is a practical tool to determine Hx + X in dry-cured ham, as an index of minimum curing time. A good correlation between enzyme sensor and HPLC data was observed.     

Selection of antifungal protein-producing molds from dry-cured meat products

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillus niger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicillium chrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37 kDa for AS51D and 9 kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.     

PRKAG3 and CAST genetic polymorphisms and quality traits of dry-cured hams - I. Associations in Spanish dry-cured ham Jamón Serrano

The functional single polymorphisms identified in the calpastatin (CAST) gene have been related to the rate of meat tenderization and the protein turnover after slaughter, and the Ile199Val polymorphism identified in the coding region of the protein kinase AMP-activated (PRKAG3) gene has been proven to affect ultimate pH in muscle. The aim of the present study was to show the effects of these genetic polymorphisms on the quality traits of Spanish dry-cured ham Jamón Serrano. A tissue sample from 665 crossbreed pigs were genotyped for PRKAG3 Ile199Val, CAST Arg249Lys and CAST Ser638Arg polymorphisms, and a subsample of 120 dry cured hams was selected to perform physico-chemical, rheological, instrumental colour and sensory analyses. Associations between the polymorphisms and several quality traits of dry-cured ham, mainly related to flavour and texture, were found. The genotypes PRKAG3 Ile/Ile, CAST249 Arg/Arg and CAST638 Arg/Arg, and the haplotype CAST 249Arg-638Arg were the most favourable for Jamón Serrano production.     

PRKAG3 and CAST genetic polymorphisms and quality traits of dry-cured hams-III. Associations in Slovenian dry-cured ham Kraški pršut and their dependence on processing

The aim of the present study, the third in a series of three papers, is to show the effects of PRKAG3 and CAST gene polymorphisms on the quality traits of the Slovenian dry-cured ham “Kraški pršut” and their interaction with ham producers. Significant interaction of polymorphisms with producer in the case of salt content, lipid oxidation (PRKAG3 Ile199Val), proteolysis index (CAST Arg249Lys) and pastiness (CAST Ser638Arg) indicated that genotype manifestation was reliant on the manufacturing practice. PRKAG3 Ile199Val polymorphism affected several physico-chemical, rheological and sensory traits. The Ile/Ile genotype yielded less salty and softer hams, indicating beneficial effects on dry-cured ham quality. The effect of CAST polymorphisms was less pronounced, although the observed associations with pastiness, proteolysis index and several free amino acid concentrations indicate its possible influence on proteolysis, with haplotype CAST 249Arg/638Ser being associated with a higher degree of proteolysis.     

Dynamics and characterization of yeasts during ripening of typical Italian dry-cured ham

The evolution of the yeast population during manufacturing and ripening of dry-cured Parma ham was investigated. Contamination levels ranged from 10(5) to 10(7) cfu/g on muscle surface, 10(4) to 10(6) cfu/g on covering fat and exceeded 10(7) cfu/g on spreadable fat mince ("sugna"). Two hundred and sixty one yeast isolates underwent identification test, showing that the predominant species of yeast population during the whole maturing process were Debaryomyces hansenii, Candida zeylanoides, Debaryomyces maramus, and to a lesser extent, Candida famata and Hyphopichia burtonii. The species Candida catenulata, Candida guilliermondii, Candida edax and other genera like Cryptococcus and Wingea were occasionally found. The yeast counts and species distribution changed according to the stage of processing and to the ham sampling location. At the end of the cold phase, the washing procedure was effective in lowering the yeast count in muscle and fat surface layers, but during the next ageing stages, yeast colonization of unskinned ham muscle increased again, though species distribution changed if compared to previous manufacturing phases. The ripening steps taken into account from the end of the cold phase to the final outcome, were always characterized by more than one yeast species, suggesting that yeasts other than Debaryomyces spp. could play a remarkable role on the sensory and safety properties of typical Italian dry-cured ham.     

Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing

To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347–852 MPa), time (2.3–15.8 min) and temperature (7.6–24.4 °C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process.     

Influence of partial replacement of NaCl with KCl, CaCl2 and MgCl2 on lipolysis and lipid oxidation in dry-cured ham

Sodium intake above nutritional recommendations may involve harmful consequences to health such as the increased risk of cardiovascular diseases. Dry-cured ham constitutes a product with a relatively large amount of sodium. Thus, to obtain a healthier product for consumers with reduced sodium content, two formulations containing KCl alone (formulation II) or mixed with CaCl2 and MgCl2 (formulation III) have been proposed to partially replace NaCl. Lipolysis and lipid oxidation occurring in hams processed with these formulations have been studied since they have direct influence on the final flavor. No significant differences in acid lipase activity or lipid oxidation were found at the end of the process between the alternative formulations and formulation I (control with 100% NaCl). Differences in some free fatty acids, generated along the processing, were detected among treatments and at the end of dry-curing. Data suggests a slight trend towards a major lipolysis during treatment III.     

High pressure treatments on the inactivation of Salmonella Enteritidis and the physicochemical, rheological and color characteristics of sliced vacuum-packaged dry-cured ham

The effect of high pressure (HP) on Salmonella Enteritidis in sliced dry-cured ham stored under temperature abuse (8°C) during 60d was investigated. After treatment, reductions of S. Enteritidis were 1.06, 2.54 and 4.32 log units in ham treated at 400, 500 and 600MPa for 5min at 12°C, compared to non-pressurized samples. After 60d, counts of S. Enteritidis in ham treated at 400 and 500MPa were 2.56 and 2.66 log units lower than in non-treated ham, whereas the pathogen was only detected after enrichment in ham treated at 600MPa. Lipid oxidation increased with storage and pressurization, whereas total free amino acid contents were similar in HP and control samples after 60d. Dry-cured ham treated at the highest pressures exhibited lower shear resistance, whereas the maximum force to compress the sample was slightly changed. Color (L*, a* and b*) varied with pressurization and storage. Changes induced by HP in dry-cured ham were attenuated during storage.     

Effect of the relative humidity of drying air during the resting period on the composition and appearance of dry-cured ham surface

This study evaluates the effect of three different relative humidities (RH) during the resting period on the composition of the external part of Semimembranosus muscle (2 mm thick) and the appearance of the surface of the dry-cured ham after resting and ageing. Forty-eight hams stored after salting for 40 days at 4±1 °C and three different RH (52±3, 78±3 and 85±3%) were studied. Half of the hams in each treatment were sampled after the resting period and the rest were aged for 8 additional months at the same temperature and RH. Storage of hams at 52% RH during resting produced a white appearance in some parts of the rind and increased the surface covered with oil drip. Hams stored at 78% showed a similar composition to those stored at 52%, except for the higher moisture content and lower Na/moisture ratio at the end of the resting period, and potassium content (on dry matter, dm) at the end of the process. At the end of resting no white rind was observed, and at the end of the process the surface covered with oil drip was slightly lower than at 52%. Storage of hams at 85% RH increased the surface pH, Na2HPO4.12H2O crystallisation, Mg (dm), Mg/moisture, K (dm) and NPN/NT at the end of the resting period, and increased the pH and Mg (dm), Mg/moisture, Na/moisture, K/moisture, the ash (dm) and ash/moisture contents and decreased the a_w and the surface covered with oil drip at the end of the process.     

Non-destructive analysis of aw, salt and water in dry-cured hams during drying process by means of computed tomography

Computed tomography (CT) has been previously used as a non-destructive technology to accurately determine salt and water content in dry-cured ham at the initial stages of the elaboration process. However, since the accuracy of predictive models has been affected by the dry level of the sample, in this study, prediction models for salt and water contents and also for a_w to be applied during drying process were developed. Results showed that the number of independent variables needed to obtain regression models with a similar accuracy to the models previously developed is higher in drier samples. Furthermore, an important effect of fat content is also observed since prediction of a_w, salt content and water content were significantly improved when the fattiest samples were removed from the calibration or when fat content was included in the predictive models. Because of the different physicochemical characteristics of muscles, models developed specifically for Semimembranosus (SM), Biceps femoris (BF) or Semitendinosus (ST) muscles were more accurate. It can be concluded that models for prediction during the drying process were accurate enough to consider CT as a useful tool applicable for controlling and optimizing the dry-cured ham elaboration processes.     

Time course of peptide fingerprints in semimembranosus and biceps femoris muscles during Bayonne ham processing

The aim of this work was to define reliable markers of muscle and processing time in dry-cured ham using a rapid, precise semi quantitative method for the protein fraction soluble in low ionic strength buffer. For this purpose protein labchip Agilent was used to separate proteins and peptides and accurately determine their molecular weights and concentrations electrophoretically. In this way the protein fingerprinting of dry-cured ham at different process times was characterised, together with targets and products of proteolysis. In addition, the comparison of all the electrophoregrams indicated muscle and dry-curing process markers.     

Study of the counts,species and characteristics of the yeast population during the manufacture of dry-cured “lacón”. Effect of salt level

The aim of this work was to study the yeast population during the manufacture of dry-cured “lacón” (a Spanish traditional meat product) and the effect of the salting time. For this study, six batches of “lacón” were manufactured with three different salting times (LS (3 days of salting), MS (4 days of salting) and HS (5 days of salting)). Yeast counts increased significantly (P < 0.001) during the whole process from 2.60 to 6.37 log cfu/g. An increased length of salting time did not affect yeast counts throughout the manufacture of dry-cured “lacón”, although the highest yeast counts were obtained from LS batches. A total of 226 isolates were obtained from dry-cured “lacón” during drying-ripening stage, of which 151 were yeasts and were identified at the species level using molecular techniques. The total of 151 identified yeasts belonged to 4 different genera: Debaryomyces, Candida, Cryptococcus and Rhodotorula. Debaryomyces hansenii was the most abundant species isolated throughout the whole process as much in the interior as in the exterior of the pieces of three salt levels of “lacón” studied, while Candida zeylanoides was only isolated from the interior of MS and HS batches and from the exterior of LS and HS groups, but at lesser proportion than D. hansenii.     

Use of recA gene sequence analysis for the identification of Staphylococcus equorum strains predominant on dry-cured hams

Spanish dry-cured ham is an uncooked meat product highly appreciated due to its characteristics flavour. In this study, we examined the accuracy of biochemical tests and 16S rDNA sequencing in the identification of 56 staphylococcal strains isolated during industrial Spanish dry-cured ham processes. Important differences were observed comparing genotypic and phenotypic data. Staphylococcus xylosus was the prevalent species identified by biochemical methods (87.5%), however, sequencing of the 16S rDNA resulted in an unambiguous identification of Staphylococcus equorum (73.2%) and Staphylococcus vitulinus (8.9%) strains. Reliable identification of meat staphylococci, mainly among S. xylosus and S. equorum strains could be also achieved by means of recA gene sequence comparison. Two degenerate primers previously described for lactic acid bacteria were used to amplify an internal fragment of the recA gene. This fragment was amplified from twelve staphylococcal type strains representing frequent meat species. The results indicated that recA sequencing is an adequate method to discriminate among meat staphylococci. In addition, S. xylosus and S. equorum strains could be more accurately discriminated by recA sequencing than 16S rDNA or sodA sequencing. The S. equorum sequence diversity showed at the intra-species level by recA gene sequencing confirmed the high heterogeneity described among S. equorum strains.