Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing

The aim of the work was to develop and validate a model of the inactivation of Listeria monocytogenes on dry-cured ham by high hydrostatic pressure (HHP) processing, as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with L. monocytogenes was treated at different HHP conditions (at 347-852 MPa; for 2.3 to 15.75 min; at 7.6 to 24.4 °C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of L. monocytogenes counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. The significance of the quadratic term of pressure and time indicated that little effect was observed below 450 MPa, whereas holding time longer than 10 min did not result in a meaningful reduction of L. monocytogenes counts. Temperature did not show significant influence at the range assayed. The model was validated with results obtained from further experiments and bibliographical data within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, such as prediction of the process criteria to meet the Food Safety Objectives. The results of this work may help food processors to select optimum processing conditions of HHP.     

Volatile compounds in dry-cured Serrano ham subjected to high pressure processing. Effect of the packaging material

The effect of high pressure treatment (400 MPa, 10 min at 12 °C) on the volatile profile of Spanish dry-cured Serrano ham, packaged with or without aluminum foil in a multilayer polymeric bag, was investigated. The analysis of the volatile fraction was carried out by dynamic headspace extraction coupled to gas chromatography–mass spectrometry. Pressure treatment only had a slight effect on the volatile fraction of Serrano ham. Most compounds affected by pressurization, such as alkanes (C₉–C₁₂), 2-undecene, 2-nonanone, 1-octen-3-one, 1-heptanol, 2-hexanol, 2-heptanol, ethyl pentanoate, benzaldehyde and styrene, presumably originated from the metabolism of moulds. A significant effect of pressurization on the migration of compounds from the plastic material was found. Linear and branched chain alkanes, alkenes as well as benzene compounds, were generally less abundant in pressurized samples than in untreated samples. A scalping effect was also observed for compounds such as butanal, pentanal, ethyl esters and pyrazines.     

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.     

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.     

Isomaltulose production by free cells of Serratia plymuthica in a batch process

Free cells of Serratia plymuthica were used to convert sucrose into isomaltulose and trehalulose reducing sugars. The effects of substrate concentration and temperature were observed in a batch process with flasks shaken at 150 rpm. The experimental design and response surface methodology analysis indicated that the conversion parameters had significant influence (p < 0.05) on sucrose conversion, and that a valid model was obtained after an analysis of variance (F_model = 10.48 > F_charted = 5.79) to obtain a response surface and isocurve. Conversion was favoured when a 30% sucrose solution and temperature of 25 °C were used, which resulted in a high conversion into isomaltulose – over 70% – and 7–8% trehalulose. Small amounts of glucose (5–7%) and fructose (5–8%) were formed in the reaction medium.     

Effects of high pressure application (400 and 900 MPa) and refrigerated storage time on the oxidative stability of sliced skin vacuum packed dry-cured ham

The effect of high pressure processing at 400 MPa and 900 MPa on the oxidative stability of sliced and vacuum packaged commercial dry-cured ham was determined by analyzing the antioxidant enzyme activities, TBARS levels (thiobarbituric acid reactive substances), vitamin E content and physicochemical characteristics during refrigerated storage for 50 days in different light conditions. In dry-cured ham pressurized at 400 MPa color changes and sensory analyses were also assessed. The high pressure process at 900 MPa produced a decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities and increased vitamin E content. In contrast, pressurization at 400 MPa, increased SOD activity, and showed no effect on vitamin E content and GSHPx activity. In general the physicochemical parameters determined (fat, moisture and collagen) were unaffected by pressurization. Treatment at 400 MPa increased the instrumental color measurement of lightness (L* values, CIELAB). This level of pressure also modified the hardness, chewiness, saltiness and color intensity. These changes of the sensory attributes in dry-cured ham were significant, but small.     

Influence of high pressure application on the nutritional, sensory and microbiological characteristics of sliced skin vacuum packed dry-cured ham. Effects along the storage period

The effect of high pressure processing at 600 MPa on sliced and vacuum packaged commercial dry-cured ham was determined by analyzing the induced color changes, the physicochemical characteristics, the microbiological count and the changes in sensory attributes and also its effect on lipid oxidation measured through TBARS (thiobarbituric reactive substances), antioxidant enzyme activities and the content of fatty acid from total, free and phospholipid fractions. This effect was also studied during 50 days refrigerated storage with different light conditions. The high pressure processing at 600 MPa modified the color of dry-cured ham producing an increase in lightness L*-parameter. Many sensory attributes were also modified resulting in an increase in hardness, chewiness, brightness, odor intensity and saltiness, while reducing color intensity. High pressure produced a reduction in the aerobic count. The oxidative stability of the pressurized dry-cured ham was not altered as observed from the absence of differences in fatty acid contents and antioxidant enzyme activities. Initially pressurization produced a decrease in TBARS levels, however this effect was not found after 50 days of refrigerated storage.

Industrial relevance

The application of high hydrostatic pressure (HHP) in food technology is a useful tool to combine a minimal processing that increases the shelf-life of the food products, maintaining their safety and nutritional properties and with minimal changes in flavor or taste. This processing technology is very important to increase the safety of the sliced foods which are prone to the microbiological deterioration, as cured meat products and cooked ham. Pressurization of sliced dry-cured ham at 600 MPa has been shown effective reducing spoilage associated microorganisms. The present study was carried out to evaluate the oxidative stability, nutritional quality, safety and sensory attributes of dry-cured ham pressurized at 600 MPa. This is very important because the hypothesis that HHP does not affect to sensorial characteristics needs to be confirmed in different kind of foods. Also, the possible causes of the observed effects should be explained by the study of physic–chemical parameters. In this way the oxidative stability is an important issue because some authors claimed that HHP can increase oxidation in food containing lipids.     

High pressure treatment of dry-cured Iberian ham. Effect on radical formation,lipid oxidation and colour

The effect of high-pressure treatment (200, 400, 600 and 800 MPa) on radical formation in solid samples of dry-cured Iberian ham and in ham slurries was evaluated using the spin-trapping technique and electron spin resonance (ESR) spectroscopy. In addition, hexanal was, as a secondary lipid oxidation product, assessed by gas chromatography. The effect of pressure treatments on ESR signals determined in the spin-trapping assay following pressure treatment in solid samples of dry-cured Iberian ham was not statistically significant. However, the lowest level of pressure applied initiated radical formation and promoted lipid peroxidation, whereas intermediate to high levels of pressure seemed to promote further reaction and disappearance of free radicals. Pressure significantly affected hexanal content in the same way as seen for formation of free radicals. Regarding the slurries prepared from dry-cured ham, pressure significantly increased the tendency of radical formation as seen from the ESR signals and significantly increased the hexanal content. The different pattern between solid ham and ham slurries points toward an initiation mechanism associated with the membrane phospholipids for oxidation. In addition, surface colour (L*, a* and b* values and percent reflectance values) of non-pressurized and pressurized (200–800 MPa) dry-cured Iberian ham samples was assessed. Non-pressurized samples showed a higher lightness than pressurized samples, and redness significantly decreased with pressure treatment.     

Lipid and protein oxidation and sensory properties of vacuum-packaged dry-cured ham subjected to high hydrostatic pressure

The effect of HHP treatment (600 MPa) on the oxidative stability of lipids and proteins of vacuum-packaged Iberian dry-cured ham and the impact on the sensory characteristics of the product was investigated. In order to assess how different commercial presentations are affected by HHP treatment, three different presentations of vacuum-packaged Iberian dry-cured ham were considered, namely, (i) intact format (IF) corresponding to non-sliced vacuum-packaged dry-cured ham, (ii) conventional-sliced format (CSF) corresponding to dry-cured ham slices placed stretched out in the package and (iii) alternative-sliced format (ASF) corresponding to dry-cured ham slices piled up horizontally. The oxidation of dry-cured ham lipids and proteins was enhanced by HHP-treatment with the presentation being highly influential on these oxidative reactions. Pre-slicing dry-cured ham results in a more susceptible product to oxidative reactions during pressurisation and subsequent refrigerated storage. Possible mechanisms, by which HHP-induced oxidative reactions would affect particular sensory traits in vacuum-packaged Iberian dry-cured ham such as colour, texture and flavour attributes, are discussed.     

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.     

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.     

Accelerated processing of dry-cured ham. Part I. Viability of the use of brine thawing/salting operation

In a previous study, the brine thawing/salting operation using frozen hams as raw material was proposed in order to obtain accelerated processing of dry-cured hams. The time needed to reach the same NaCl concentration on a dry weight basis and the same NaCl concentration in the ham liquid phase for the deeper areas at the end of the post-salting stage were determined.

The aim of this work was to study the influence of the brine thawing/salting operation on the whole dry-cured ham manufacturing process, using the traditional thawing and salting methods as control.

The obtained results indicate that although a strong reduction in the thawing, salting and post-salting stages is obtained by using brine thawing/salting, the time needed in the dry-curing and maturing phases increases compared to those traditionally processed, probably due to the absence of pile salting and thus the reduction in the thickness of the ham piece as a consequence of the ham pressing. From the composition and microbiological point of view, no significant differences were observed among the hams processed by the different treatments.     

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.     

Effects of potassium lactate and high pressure on transglutaminase restructured dry-cured hams with reduced salt content

Ten raw hams (from 5 carcasses) were boned and salted either with salt reduction (15 g/kg NaCl) or salt reduction with addition of potassium lactate (15 g/kg NaCl and 39.74 g/kg of a 60% K-lactate solution). Subsequently, the ham pieces were assembled together with transglutaminase, vacuum packed into water-permeable plastic bags and kept at 3 °C and 85% RH until reaching above 30% weight losses. The effects of K-lactate addition and the high-pressure (HP) treatment at 600 MPa on the physicochemical, instrumental colour and texture (Tensile test) and sensory characteristics of the biceps femoris (BF) muscle were evaluated. The addition of K-lactate did not have a negative effect on colour, flavour or texture of restructured dry-cured hams. The HP treatment increased significantly the pH, L^∗, a^∗ and b^∗ values and the breaking stress, and decreased the water-holding capacity and elasticity (apparent Young’s modulus) of BF muscle. The HP treatment also affected significantly the flavour (increasing saltiness, umami and sweetness) and the sensory texture attributes (increasing muscle binding, hardness, gumminess and fibrousness and decreasing adhesiveness and pastiness), as well as slice appearance (increasing brightness and iridescence and decreasing colour homogeneity).     

Effect of high pressure treatment on microbial populations of sliced vacuum-packed cooked ham

In this study, culture-dependent and culture-independent approaches were used to reveal the microbial diversity and dynamic changes occurring in sliced vacuum-packed cooked ham after high pressure processing (HPP, 400MPa or 600MPa for 10min at 22°C) during refrigerated storage over 90days. Direct extraction of genome DNA and total RNA from meat samples, followed by PCR-denaturing gradient gel electrophoresis (DGGE) and RT-PCR-DGGE on 16S rDNA V3 region, was performed to define the structure of the bacterial populations and active species in pressurized cooked ham. Results showed that HPP affected differently the various species detected. The predominant spoilage organisms of cooked ham, such as Lactobacillus sakei and Lactobacillus curvatus, were found to be very sensitive to pressure as they were unable to be detected in HPP samples at any time during refrigerated storage. Weissella viridescens and Leuconostoc mesenteroides survived HPP at 600MPa for 10min at 22°C and were responsible for the final spoilage. An RNA-based DGGE approach clearly has potential for the analysis of active species that have survived in pressurized cooked ham. High pressure processing at 400 or 600MPa for 10min at room temperature (22°C) has a powerful inhibitory effect on the major spoilage bacteria of sliced vacuum-packed cooked ham. High pressure treatment may lead to reduced microbial diversity and improve the products' safety.     

Inactivation of Salmonella and Escherichia coli O157:H7 on artificially contaminated alfalfa seeds using high hydrostatic pressure

Alfalfa sprouts contaminated with Salmonella and Escherichia coli O157:H7 have been implicated in several outbreaks of foodborne illnesses in recent years. The seed used for sprouting appears to be the primary source of pathogens. Seed decontamination prior to sprouting presents a unique challenge for the sprouting industry since cells of the pathogenic survivors although undetectable after sanitizing treatments, can potentially multiply back to hazardous levels. The focus of this study was to therefore test the efficacy of high hydrostatic pressure to eliminate a ∼5 log CFU/g load of Salmonella and E. coli O157:H7 on alfalfa seeds. Pressure treatment of 600 MPa for up to 25 min at 20 °C could not result in complete inactivation of Salmonella. High-pressure treatment was then carried out either at sub-ambient (4 °C) or elevated (40, 45 and 50 °C) temperatures to test the ability of high pressure to eliminate Salmonella. Pressure treatment at 4 and 20 °C did not deliver any satisfactory inactivation of Salmonella while high pressure at elevated temperatures achieved complete kill. Pre-soaking seeds prior to high-pressure treatment also enhanced pressure inactivation of Salmonella but at the expense of seed viability. High-pressure treatment of 500 MPa for 2 min at 45 °C was able to eliminate wild-type Salmonella and E. coli O157:H7 strains without bringing about any appreciable decrease in the seed viability.     

Validation of high pressure processing for inactivating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study identified and validated high hydrostatic pressure processing (HPP) for achieving greater than 3.52-log reductions of Vibrio parahaemolyticus in the Pacific oysters (Crassostrea gigas) and determined shelf life of processed oysters stored at 5 °C or in ice. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10^4-5 cells per gram and processed at 293 MPa (43 K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). A HPP of 293 MPa for 120 s at groundwater temperature (8 ± 1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. Oysters processed at 293 MPa for 120 s had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This HPP can be adopted by the shellfish industry as a post harvest process to eliminate V. parahaemolyticus in raw oysters.     

Accelerated processing of dry-cured ham. Part 2. Influence of brine thawing/salting operation on proteolysis and sensory acceptability

In a previous study, the brine thawing/salting operation using frozen hams as raw material was studied as a valid alternative for the accelerated processing of dry-cured hams. But no information was available on how this treatment could affect some important biochemical mechanisms and the sensory quality of hams. The aim of this work was to study the influence of the simultaneous brine thawing/salting operation on proteolysis and sensory acceptability of the produced dry-cured hams. The results confirm that dry-cured hams can be produced by using brine thawing/salting with a substantial reduction in the thawing and salting time needed. This accelerated process resulted in similar or even better sensory preferences than hams produced through the traditional method. However, the preference of consumers based on the appearance was lower for most of the hams than when using the traditional method, probably due to a wider slice section of the brined hams that can be corrected by adequate pressure during the salting. Thus, this treatment can be used without affecting the quality of dry-cured hams.     

The effect of NaCl-free processing and high pressure on the fate of Listeria monocytogenes and Salmonella on sliced smoked dry-cured ham

NaCl is an important multifunctional ingredient applied in dry-cured ham elaboration. However, its excessive intake has been linked to serious cardiovascular diseases causing a recent increase in the development of reduced salt products. In the present study Listeria monocytogenes and Salmonella, food-borne pathogens which can cross-contaminate post processed products, were spiked with < 100 CFU/g on slices of both standard (S) and NaCl-free processed (F) (elaborated with KCl + potassium lactate instead of NaCl) smoked dry-cured ham. Although L. monocytogenes and Salmonella counts decreased faster in S ham, pathogens were present in both types of non-pressure treated ham during the entire refrigerated storage period (112 days). Pressurisation at 600 MPa for 5 min caused the elimination of both pathogens in S ham after 14 days. In contrast, Salmonella and L. monocytogenes were present in F ham until days 28 and 56, respectively, indicating that the NaCl-free processed dry-cured ham had lower stability than standard smoked dry-cured ham.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.