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PROTEOMICS OF PARMA DRY-CURED HAM: ANALYSIS OF SALTING EXUDATES Gianluca Paredi, Roberto Benoni, Giovanni Pighini, Luca Ronda, Adam Dowle, David Ashford, Jerry Thomas, Giovanna Saccani, Roberta Virgili, and Andrea Mozzarelli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01293 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 18, 2017
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Journal of Agricultural and Food Chemistry
PROTEOMICS OF PARMA DRY-CURED HAM: ANALYSIS OF SALTING EXUDATES
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Gianluca Paredi^°, Roberto Benoni^, Giovanni Pighini^, Luca Ronda§, Adam Dowle@, David
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Ashford@, Jerry Thomas @, Giovanna Saccani#, Roberta Virgili# and Andrea Mozzarelli^°#*
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^Department of Food and Drug, §Department of Medicine and Surgery, °Interdepartmental Center
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SITEIA.PARMA, University of Parma, Parma, Italy
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@
Bioscience Technology Facility, Department of Biology, and University of York, York, UK #
Stazione Sperimentale per l’Industria delle Conserve Alimentari (SSICA), Parma, Italy #
National Research Council, Institute of Biophysics, Pisa, Italy
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*Corresponding author: Andrea Mozzarelli, Department of Food and Drug, University of Parma,
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Viale delle Scienze 23/A, 43124, Parma, Italy. Email
[email protected] 18 19
Andrea Mozzarelli orcid.org/0000-0003-3762-0062
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Abstract
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The production of Parma dry-cured ham involves steps of salting, drying and ripening. Although
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sea salt is the only preserving agent, strategies are pursuing aimed at reducing salt content for
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decreasing its negative impact on consumer health. A 24-hour pressure treatment was applied
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before salting for reducing thickness and inequalities in shape. To evaluate the potential impact of
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the pressure step on process outcome, differential proteomic analyses by complementarity 2D-
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PAGE and LC-MS/MS were carried out on exudates collected at day 1, 5 and 18 of the salting
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phase for hams treated or untreated with pressure. Specific proteins were found differentially
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abundant in exudates from pressed vs unpressed hams and as a function of time. These changes
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include glycolytic enzymes and several myofibrillar proteins. These findings indicate that pressure
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causes a faster loosening of myofibrillar structure with release of specific groups of proteins.
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Keywords: dry-cured ham, salting exudates, proteomics, 2D-PAGE, LC-MS/MS
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INTRODUCTION
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Parma dry-cured ham is a traditional high quality Italian food product, classified as “Protected
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Designation of Origin” (P.D.O). In 2015, over 8.000.000 thighs were processed according to the
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tutelary rules of Parma Ham Consortium that, with the dossier CEE N°2081/921, regulate raw
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matter requirements and production process. Briefly, domestic heavy pigs bred for Parma ham are
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fed according to an established dietary regimen until slaughtering, occurring when pigs are at least
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nine months old and 160 Kg of average live weight. Trimmed fresh hams should meet specific
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requirements of weight, covering fat thickness, absence of pathological conditions and visual
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defects. The traditional processing of dry-cured ham consists of two salting steps, drying phases at
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low and room temperature and an aging step for at least 12 months overall processing time. On
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average, at the end of the whole production process, the ham weight decreases by about 30%. The
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protected designation of origin “Parma Ham” is deserved to those dry-cured hams meeting P.D.O.
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requirements. Quality criteria aim to mark with the brand only dry-cured hams with limited quantity
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of salt and moisture, and with an adequate proteolysis degree, in order to achieve the typical flavor
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without affecting texture and taste. According to the P.D.O. specifications, the allowed ranges for
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salt and moisture vary from 4.2 to 6.2 g/100g, and from 59 to 63.5 g/100g, respectively. Proteolysis
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degree, expressed as percent ratio between nitrogen soluble in 5% trichloroacetic acid and total
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nitrogen, is established to be from 24 to 31. Higher variations are permitted for salt and proteolysis
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since more affected by meat quality and processing.
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Nowadays, due to the high consumption of salted processed food, the sodium chloride intake is
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approximately 9-12 g/day2. High salt intake increases blood pressure and might cause
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cardiovascular and renal diseases3. Furthermore, the European code against Cancer 4th edition4,5
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recommends to “limit red meat and food high in salt” in order to reduce cancer risk and
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hypertension. However, in dry-cured ham production process, sodium chloride is crucial because it
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acts as a bacteriostatic agent6, reduces water activity, and plays an active role in the development of
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sensory and quality parameters of the final product7. Differently from the processing of dry-cured 3 ACS Paragon Plus Environment
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hams in other countries, such as Spain and France where nitrite and nitrate are used, no other
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ingredient than sea salt is allowed in Parma dry-cured ham. Therefore, the reduction of sodium
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chloride in the production of dry-cured ham is not an easy task to accomplish. Furthermore, low salt
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levels are associated with higher moisture and proteolysis degree than standard salt levels,
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triggering defects such as softness and pastiness in dry-cured ham8.
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Proteomic analyses provide an insight of the molecular modifications that muscles undergo
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throughout the entire phases of meat transformation, from muscle to meat and from meat to food
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product9,10. In a previous study, we investigated by two-dimensional electrophoresis (2D-PAGE)
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the qualitative/quantitative variations in the proteome of exudates as a consequence of the
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application of different technological treatments in the production of cooked ham11. Proteomics
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studies have been also carried out aimed at the characterization of the modifications that dry-cured
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ham undergoes under different conditions. Particularly, changes of myofibrillar and water soluble
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protein components during ripening were investigated12. Furthermore, the protein pattern of
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semimembranosus and biceps femoris muscles from Bayonne dry-cured hams was compared with
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2D-PAGE13. Recently, a label free mass spectrometry approach was applied to determine the
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changes in water soluble proteome at different processing times14 and to characterize the proteolytic
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activity of endogenous endopeptidases on muscle proteins15.
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In this study, the effect of the application of a pressure treatment to fresh hams before the salting
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phase was investigated by proteomic tools. The pressure treatment is not usually applied to pork
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thighs to be processed into dry-cured hams. However, it has been attempted as a tool to reduce ham
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thickness and shape variability, thus shortening and equalizing the time required for salt penetration
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inside ham and moisture flow outside16. The expected effect is to counteract the increase moisture
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and proteolysis usually occurring in the reduced-salt dry-cured ham. A similar approach was
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previously adopted16 resulting in an increased salt concentration and a water activity reduction in
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dry-cured hams during the intermediate processing stages, critical for product safety. No
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modification of weight losses and final salt content with respect to non-pressed hams was observed. 4 ACS Paragon Plus Environment
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The proteomics analysis was pursed via the characterization of the exudate collected from the brine
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losses of hams salted as such or after pressure application, at different times. Exudates are protein-
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rich media obtained during the salting steps in the production of both cooked and dry-cured ham,
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generated from the salt-mediated rupture of muscle cells over the time-dependent salt penetration
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within meat. Thus, exudate reports on the overall effect of salting on muscle cells. Proteomic
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analysis was carried out by 2D-PAGE with peptide mass fingerprinting to identify protein spots.
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Furthermore, a LC-MS/MS approach was exploited to overcome the drawbacks of 2D-PAGE, such
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as the difficulty in the separation of proteins characterized by extreme basic pI, low or high
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molecular weight, high hydrophobicity, insolubility and lower abundance17. To our knowledge, this
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is the first investigation that exploits ham exudates recovered from the salting stage for the
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proteomic characterization of the early processing steps from pork to dry-cured ham.
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MATERIAL AND METHODS
Sample preparation. Eight fresh legs from Landrace
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Large White domestic heavy pigs were selected in a local slaughterhouse according to the
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requirements of Parma Ham Consortium reported in CEE N°2081/921. Ham selection was carried
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out in compliance with post mortem time, weight, shape, trimming way, fat thickness, meat quality
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and visual appearance provided for by abovementioned tutelary regulations. Next, before salting,
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four hams were randomly assigned to pressure treatment and four hams to standard storage. Weight
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was 13.26 ± 0.30 kg and 13.15 ± 0.14 kg, fat thickness 2.78 ± 0.86 cm and 2.75 ± 0.50 cm, pH 5.62
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± 0.03 and 5.69 ± 0.14 for unpressed and pressed hams, respectively. Fresh hams were conditioned
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at low temperature (1-3°C) and 80-90% RH for 24 h prior to salting, placed horizontally on a shelf,
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side by side, and arranged with the large thick bottom of each unit sided by the narrow shank of the
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adjacent one, to form a continuous ham layer. In the case of the hams to be pressed, a plank was put
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on four subsequent hams and eight specifically made concrete blocks, 6 kg each, were put on the
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plank, perpendicularly to the hams for 24 hours, exerting a pressure corresponding to 12 kg/ham. At
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the end of the pressure treatment, the height was measured in the thickest part of the ham when 5 ACS Paragon Plus Environment
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placed horizontally, close to the trimming line between the uncovered muscular mask and the part
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covered by the rind, using a gauge designed for this measure. Unpressed hams had height of 18.51 ±
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0.79 cm, whereas pressed hams had height of 18.21 ± 0.66 cm and 15.58 ± 0.23 cm before and after
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pressure application, respectively.
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The salting phase was performed in two steps using a mixture of 2- and 3-mm grain size salt. Wet
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salt (nearly 15% added water) was rubbed on ham rind, whereas dry salt was added to unskinned
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ham surface. In the first salting phase, the legs were covered with ≈3.0% salt (% ham weight) and
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placed at 1-3°C, 80-90% RH for six days. Subsequently, hams were desalted and underwent a
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second salting step with ≈2.0% added salt, for 12 days, under the same environmental conditions.
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After each salting treatment, hams were desalted by scrubbing off the unabsorbed salt with brushes.
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At the end of the salting step, average weight losses corresponding to 2.72% and 3.34% were
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recorded for unpressed and pressed hams, respectively. During salting, exudates dripped from hams
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were collected from each ham placed individually into a large vessel, after 1, 5 and 18 days. Before
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processing, exudates were stored at -80°C. For processing, exudates were thawed and centrifuged at
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16000 g for 15 minutes to remove insoluble material. Due to the high salt concentration, 1 mL of
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each sample solution was dialyzed against 40 mM Tris, 0.5% SDS, pH 7.4, for 24 hours. Protein
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concentration was determined using the Bradford assay with bovine serum albumin for calibration.
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Two-dimensional gel electrophoresis (2D-PAGE). Protein precipitation with acetone was
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carried out for 500 µl of dialyzed sample and the pellet solubilized in 7 M urea, 2 M thiourea and
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4% CHAPS buffer. Protein samples from the four pressed hams were pooled prior subsequent
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analysis, thus obtaining a single sample for each day (day 1, day 5 and day 18). The same pooling
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procedure was carried out for the four unpressed hams. IEF strips (11 cm pH 4-7) were rehydrated
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for 16 hours at 20 °C with 200 µg of protein solubilized in 7 M urea, 2 M thiourea, 4% w/v
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CHAPS, 65 mM DTE and 0.2 % v/v ampholites. Protein focusing was achieved with a multi-phase
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programme: 1) 600 V for 1 hour 2) 1000 V for 1 hour 3) 4000 V for 1 hour 4) 8000 V for 1 hour 5)
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8000 V until 26000 V/H in the pH range 4-7. Then, strips were equilibrated for 15 minutes in 6 ACS Paragon Plus Environment
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reducing buffer containing 6 M urea, 2% w/v SDS, 50 mM Tris–HCl pH 8.8, 30% v/v glycerol, 1%
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w/v DTT, and for 15 minutes in alkylating buffer containing 6 M urea, 2% w/v SDS, 50 mM Tris–
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HCl pH 8.8, 30% v/v glycerol, 4% w/v iodoacetamide. The second dimension was performed with
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Criterion TGX any kD resolving gel (Bio-Rad®) applying 200 V for 50 minutes. Finally, gels were
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stained with Bio-Safe Coomassie (Bio-Rad®).
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Image analysis. Two replicate 2D gels were run for each pooled sample and analyzed with
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PDQuest 8.0 software (BioRad®). The average variation coefficient for replicate gels was found to
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be 32%, that is comparable to CV values obtained in previous investigations18-20. Detection and
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matching steps were carried out automatically using the same parameters for each replicate group.
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Moreover, a manual control step was performed to build a more accurate master gel for each
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replicate group. The generated higher level match-set maps were compared to detect qualitative and
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quantitatively differences between the 2D reference maps for pressed and unpressed hams at
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different sampling times, and for either pressed or unpressed hams as a function of time. A 2-fold
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change threshold in spot averaged and normalized abundance was considered relevant for the
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analysis, according to common practice in 2D-PAGE data analysis 21-24.
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Peptide mass fingerprinting. Protein spots excised from gels were digested with trypsin.
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Briefly, spots were de-stained using a mixture of 50% v/v ethanol and 15% acetic acid. Two
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washing steps of the spots were performed in 20 mM ammonium bicarbonate and 50% v/v
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acetonitrile for 20 minutes. A further washing step was carried out in acetonitrile before drying in a
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vacuum centrifuge. Rehydration of spots was performed for 30 minutes with 20 µL of 20 mg/ml
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Proteomics Grade trypsin in 20 mM ammonium bicarbonate, 9% v/v acetonitrile and 0.1 mM
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hydrochloric acid. After rehydration, 50 µL of 20 mM ammonium bicarbonate was added and the
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spots were incubated at 37°C for 16 hours. The supernatants containing peptides were recovered
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and combined with peptides that were further obtained from gels upon two washes with 50% v/v
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acetonitrile/0.1% v/v TFA. The supernatant was dried and resolubilised in 20 µL 50% v/v 7 ACS Paragon Plus Environment
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acetonitrile/0.1% v/v TFA. For MALDI TOF/TOF analysis, 0.5 µL of sample were mixed with 0.5
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µL of 10 mg/mL α-cyano-4-hydroxycinnamic acid (HCCA) in 30% v/v acetonitrile 0.07% v/v TFA
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before spotting onto the plate. MALDI spectra were acquired on the 4800 Plus MALDI
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TOF/TOF™ (Ab Sciex) in positive ion reflectron mode combining 500 shots in the mass range 700-
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3500 Da. External calibration was performed using Sigma peptide standards: bradykinin fragment
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1–7 (m/z 757.3997), angiotensin II (human) (m/z 1046.5423), P14R (m/z 1533.8582), ACTH
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fragment 18–39 (human) (m/z 2465.1989) and insulin chain B oxidized (m/z 3,494.6513). Resulting
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MS spectra were submitted to database searching using the Mascot online search engine against the
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SwissProt database. The search was restricted to mammalian proteins. Carbamidomethyl
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modification was set as a fixed modification whereas methionine oxidation was set as a variable
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modification and two missed cleavages were tolerated. Peptide mass tolerance was set at 100 ppm.
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LC-ESI-MS/MS. Protein samples were digested according to FASP protocol25 and LC-
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MS/MS analysis was carried out using a nanoAcquity UPLC system (Waters) for peptide
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separation, coupled to a maXis LC-MS/MS System (Bruker Daltonics) with a nano-electrospray
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source. Chromatographic separation was performed with a nanoAcquity Symmetry C18, 5 µm trap
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(180 µm x 20 mm Waters) and a nanoAcquity BEH130 1.7 µm C18 capillary column (75 µm x 250
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mm, Waters) using a flow rate of 300 nL/min, at 60°C. A gradient of two solvents (solvent A: 0.1%
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(v/v) formic acid and solvent B: acetonitrile containing 0.1% (v/v) formic acid) was applied for the
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separation. The initial condition was 5% solvent B, followed by a linear gradient to 30% solvent B
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over 125 min, then a linear gradient to 50% solvent B over 5 min, and finally a wash with 95%
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solvent B for 10 min. Positive ESI-MS and MS/MS spectra were acquired using AutoMSMS mode.
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Instrument settings were: ion spray voltage: 1,400 V, dry gas: 4 L/min, dry gas temperature 160 °C,
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ion acquisition range: m/z 50-2,200. Resulting tandem mass spectra were submitted to the SwissProt
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database using the Mascot search engine. The search was restricted to porcine proteins and two
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missed cleavages were allowed. Carbamidomethyl modification was set as a fixed modification, 8 ACS Paragon Plus Environment
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whereas methionine oxidation was set as a variable modification. A value of 10 ppm was used for
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peptide tolerance and a 0.1 Da for the fragment tolerance. Peptide matches were filtered to require a
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minimum expected score of 0.05 at 0.05 significance. The Mascot calculated emPAI value was used
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to estimate the relative abundance of identified proteins. Molar percentage was calculated as
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reported using the formula: ∑
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the number of observed peptides and Nobservable is the number of calculated observable peptides26.
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Only differences in molar fraction percentage of at least 1.5-times were considered relevant,
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according to common practice in the analysis of LC-MS/MS experiments21-24. The resulting
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proteins were submitted to Mann–Whitney U test with p
