- Email: [email protected]
Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions
Accepted Manuscript Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions
M. López-Pedrouso, C. Pérez-Santaescolástica, D. Franco, J. Carballo, C. Zapata, J.M. Lorenzo PII: DOI: Reference:
S0963-9969(19)30037-7 https://doi.org/10.1016/j.foodres.2019.01.037 FRIN 8227
To appear in:
Food Research International
Received date: Revised date: Accepted date:
10 December 2018 14 January 2019 15 January 2019
Please cite this article as: M. López-Pedrouso, C. Pérez-Santaescolástica, D. Franco, J. Carballo, C. Zapata, J.M. Lorenzo , Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions. Frin (2019), https://doi.org/10.1016/j.foodres.2019.01.037
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ACCEPTED MANUSCRIPT Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions
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López-Pedrouso, M.1, Pérez-Santaescolástica, C.2, Franco, D.1,
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Carballo, J.3, Zapata, C.2 and Lorenzo, J.M.1*
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Department of Zoology, Genetics and Physical Anthropology, University of
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Santiago de Compostela, Santiago de Compostela -15872, Spain 2
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Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque
Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain 3
Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense,
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Universidad de Vigo, 32004 Ourense, Spain
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Corresponding author. Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract High pressure processing (HPP) is currently being developed to increase the shelf-life of sliced dry-cured ham in convenience package without detrimental effects on texture and sensorial characteristics. This study is focused on protein degradation under pressure conditions and its contribution to
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taste and aroma. Samples of sliced dry-cured ham undergone HPP (600 Pa, 0-
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35ºC) were analyzed from different approaches including proteomic and
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chemical analysis (amino acids and volatile compounds).
Proteomic analysis revealed that high-pressure conditions caused a
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higher level of proteolysis, displaying that actin (ACTC1) was differentially degraded, unlike myosin. Furthermore, main Strecker metabolites-isoleucine
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and leucine-were more abundant at lower temperatures as opposed to 2-methyl butanal and 3-methyl butanal under HPP. Moreover, this study confirmed that
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HPP affected positively linear aldehydes (pentanal, hexanal, heptanal and nonanal) because of produce a decrease of them, which could improve flavor
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and taste of dry-cured ham.
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Keywords: myofibrillar proteins, Strecker metabolites, linear and branched aldehydes, proteolysis and lipid oxidation
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ACCEPTED MANUSCRIPT 1.
Introduction
The traditional manufacturing process of dry-cured ham includes the following steps: salting, post-salting, ripening and drying for a total period between 12 to 24 months (Bermúdez, Franco, Carballo, & Lorenzo, 2014a). All these steps have a strong impact on the meat quality due to complex chemical
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reactions (Harkouss et al., 2015; López-Pedrouso et al., 2018; López-Pedrouso
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et al., 2019; Lorenzo, Bermúdez, Domínguez, Guiotto, Franco, & Purriños,
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2015; Petrova, Tolstorebrov, Mora, Toldrá, & Eikevik, 2016). Nowadays, consumers demand the use of convenience package to increase the shelf life of
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the products as well as to maintain microbiological safety. To overcome the problem of possible food contaminations without the loss of quality, innovative
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and nonthermal techniques are being developed such as high-pressure processing (HPP). However, the introduction of novel technology is another
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important factor, which has a major influence on sensory features. Along with the whole process, the volatile compounds profile (aldehydes, ketones,
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alcohols, hydrocarbons and esters) which defines aroma could be changed due to protein degradation (Martínez-Arellano, Flores & Toldrá, 2016; Pérez-
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Santaescolástica, Carballo, Fulladosa, Garcia-Perez, Benedito, & Lorenzo, 2018a). In addition, other key characteristic associated with proteolysis is the tenderization, which is mainly due to the breakdown of myofibrillar structures (Čandek-Potokar & Škrlep, 2012; Mora, Sentandreu, & Toldrá, 2011). For these reasons HPP is being boosted within food industry, but concomitant effects on odor (rancidity) and texture attributes have been revealed particularly on sliced dry-cured hams (Lorido, Estévez, Ventanas, & Ventanas, 2015; PérezSantaescolástica et al., 2019). It has been reported that HPP modified the
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ACCEPTED MANUSCRIPT protein structure causing alterations in meat color and texture parameters such as pastiness and hardness (Garcıá -Esteban, Ansorena, & Astiasarán, 2004). Moreover, volatile compounds have also been affected by HPP and storage conditions of sliced vacuum-packaged dry-cured ham (Clariana, Guerrero, Sárraga, Diaz, Valero, & García-Regueiro, 2011; Fuentes, Utrera, Estévez,
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Ventanas, & Ventanas, 2014).
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Flavor and aroma are influenced by proteolysis and lipolysis phenomena,
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which release free fatty acids, peptides and free amino acids. These complex processes are attributed to enzymatic and chemical reactions such as Maillard
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reactions and Strecker degradations (Estévez, Ventanas, & Heinonen, 2011; Petrova et al., 2016). In the muscle, lipids and proteins are transformed into
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flavor precursors depending on process conditions (Degnes, Kvitvang, HasleneHox, & Aasen, 2017; Toldrá & Flores; 1998). Specifically, in dry-cured ham
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process, the Maillard reaction is a chemical reaction between a reducing sugar and an amino compound favored by long drying period and low water activity
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(Flores, 2018). On the other hand, the Strecker degradation of amino acids lead to their deamination and decarboxylation turning out aldehydes and ketones.
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These amino acids are intermediates from Maillard reaction which means that both reactions are closely related (Van Boekel, 2006). Therefore, the aroma and flavor are strongly affected by processing techniques, which could modify the kinetics of both reactions. These sensorial attributes could also be studied in detail using the identification and quantification of their volatiles (Bermúdez, Franco, Carballo, Sentandreu, & Lorenzo, 2014b; Pérez-Santaescolástica et al., 2018a). Consequently, the combination of non-volatile and volatile compounds defines a specific taste and smell, which is highly appreciated. Indeed, juiciness
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ACCEPTED MANUSCRIPT and flavor intensity of dry-cured ham are the most important positive characteristics assessed by consumers (Ruiz, Garcıa, Muriel, Andrés, & Ventanas, 2002). To monitor the quality, safety and nutritional requirements during production and storage, foodomics is a very useful tool for the taste, flavor and characterization.
A
proteomic
approach
using
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texture
bidimensional
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electrophoresis (2-DE) coupled with mass spectrometry (MS) could also provide
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biomarkers to optimize food processing (Picard & Gagaoua, 2017; Picard, Gagaoua & Hollung, 2017)
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In fact, it was employed in thermal, microwaves, high pressure, pulsed electric field and ultrasound treatments (Piras, Roncada, Rodrigues, Bonizzi, &
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Soggiu, 2016; López-Pedrouso et al., 2018b). In addition, this approach has been used to study cured and cooked pork ham products (Pioselli, Paredi, &
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Mozzarelli, 2011; Théron, Sayd, Pinguet, Chambon, Robert, & SantéLhoutellier, 2011; Škrlep et al., 2011). From a proteomic point of view, the
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proteolysis of dry-cured ham produced during their manufacturing process has also been studied (López-Pedrouso et al., 2018; Pérez-Santaescolástica et al.,
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2018b; Fabbro, Bencivenni, Piasentier, Sforza, Stecchini, & Lippe, 2016), but only few researches have focused on proteins of dry-cured ham submitted to HPP.
Therefore, the aim of this study was to evaluate the protein alterations of sliced dry-cured ham undergone high-pressure processing. The contribution of protein degradation to the release of volatile compounds and their metabolites should be studied to optimize high-pressure treatment on a final meat product. 2.
MATERIAL AND METHODS
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ACCEPTED MANUSCRIPT 2.1.
Samples
Sixteen dry-cured hams, manufactured according to the traditional system, were sliced (1.5 mm-thick) and vacuum packed in individual plastic bags of polyamide/polyethylene (oxygen permeability of 50 cm3/m2/24h at 23 ºC and water permeability of 2.6 g/m2/24h at 23 ºC and 85% RH, Sacoliva®
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S.L., Spain) and stored in a chamber at 4 ºC ± 2 °C until the treatment
HPP treatments
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2.2.
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application.
The treatment of the packaged slices was applied using a NC Hyperbaric
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WAVE 6000/120 equipment (NC Hyperbaric, Burgos, Spain). Three different treatments were performed at 600 MPa during 6 min, each one accompanied by
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a different temperature: 0 °C, 20 °C and 35 °C. In order to evaluate the effect of HPP treatments, a fourth group of samples were not treated and used as a
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control batch. For proteomic analysis, only samples from control and HPP (0 ºC) groups were analyzed. Samples were stored at room temperature (20 °C)
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for no longer than 4 weeks.
2.3. Proteomic analysis
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2.3.1. Protein extraction and two-dimensional electrophoresis (2-DE) A comparison between control (without HPP treatment) and HPP (0 ºC) batch was carried out using four biological replicas in each case. It is well known that 2-DE maps of porcine muscles were used to detect up to 800 spots (Lametsch & Bendixen, 2001; Morzel et al., 2004). This would provide us enough quantity of data to carry out this experiment between two conditions. Furthermore, the combination of pressure and temperature produce protein denaturation particularly acute in combination of high pressure and temperature
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ACCEPTED MANUSCRIPT (Guyon, Meynier, & De Lamballerie, 2016). In order to avoid interaction between these two variables, the selected temperature for proteomic analysis was 0 ºC with the aim of identify the potential biomarkers related exclusively to high pressure distinguishing them from biomarkers associated with high temperature.
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For each biological replica, a lyophilized sample of dry-cured ham (50
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mg) was mixed with 1 mL of lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS;
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10 mM DTT, and 2% Pharmalyte™ pH 3 -10, GE Healthcare, Uppsala, Sweden) and sonicated (Sonifier 250, Branson, Danbury, CC, USA) under refrigerated
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conditions (0 ºC). Protein purification was performed twice with Clean-Up Kit (GE Healthcare) and subsequently the pellet was dissolved in 200 µL of lysis
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buffer. Protein content in samples was estimated by CB-X protein assay kit (GBiosciences, St. Louis, MO, USA) using a Chromate® microplate reader
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(Awareness Technology, Palm City, FL, USA). The isoelectric focusing (IEF) was carried out in 24 cm pH 4-7 IPG strips (Bio-Rad Laboratories, Hercules,
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CA, USA). In the first step, the strips were rehydrated for 12 h in 450 µL of a solution 0.6% DTT and 1% IPG buffer (Bio-Rad Laboratories) which contained
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250 µg of protein in lysis buffer and rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromophenol blue). Afterward, the
voltage was
sequentially raising until reaching 70,000 Vh, and then the strips were equilibrated with buffer I (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 1% DTT) for 15 minutes and buffer II (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2.5% iodoacetoamide) for another 15 minutes. Finally, the second dimension was carried out in 12% SDS-PAGE using an Ettan DALTsix vertical gel system (GE Healthcare). The resulting 2-DE gels were stained with SYPRO
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ACCEPTED MANUSCRIPT Ruby fluorescent stain (Lonza, Rockland, ME, USA) and the images of gels were obtained by Gel Doc XR+ system (Bio-Rad Laboratories). Image analysis of gels was carried using PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories) and experimental values of isoelectric point (pI) and mass (M r) were estimated.
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2.3.2. Protein Identification by Mass Spectrometry
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The excision and in-gel tryptic digestion of protein gel spots were carried out according to Franco et al. (2015). The digested spots with trypsin were
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ionized using α-cyano-4-hydroxycinnamic acid as matrix and deposited onto a
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384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA). Mass spectrometry analysis was performed on a MALDI-TOF/TOF tandem mass
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spectrometer 4800 MALDI-TOF/TOF (Applied Biosystems, Foster City, CA, USA) using 4000 Series Explorer software v. 3.5 (Applied Biosystems, Foster
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City, CA, USA). The search was carried out using Mascot software v. 2.1 (Matrix Science, Boston, MA, USA) to identify proteins from peptide mass
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fingerprint data employing UniProt/SwissProt database. 2.4. Free
and
isoleucine: extraction,
identification
and
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quantification
leucine
The extraction of free leucine and isoleucine was carried out according to the procedure described by Pérez-Palacios, Ruiz, Barat, Aristoy, & Antequera, (2010) with some modifications. Briefly, five grams of sliced-dry cured ham were homogenized with 25 mL of hydrochloric acid 0.1 N, in an Ika Ultra-Turrax for 8 min under refrigerated conditions by submerging the extract in ice. Afterwards, the solution was centrifuged at 5240g for 20 minutes at 4 ºC and the supernatant layer was filtered through glass wool prior to further analyses. Two
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ACCEPTED MANUSCRIPT hundred ml of this extract was deproteinized by adding 800 mL of acetonitrile and centrifuged for 3 min at 5240g. The derivatization, identification and quantification were carried out according to Franco & Lorenzo (2014). The derivatization and chromatographic analysis conditions were as follows: 10 μL sample was buffered to pH 8.8
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(AccQFluor borate buffer) to yield a total volume of 100 μL. The derivatization
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reaction was initiated by the addition of 20 μL AccQ-Fluor reagent (3 mg/mL in acetonitrile). The chromatographic separation was carried out in a Waters
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AccQ-Tag column (3.9 mm × 150 mm with a 4 μm of particle size) with a flow
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rate of 1.0 mL/min at 37 °C, using a HPLC (Alliance model 2695, Waters, Milford, MA, USA) equipped with a 2475 scanning fluorescence detector. The
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detection of leucine and isoleucine was accomplished by fluorescence with excitation at 250 nm and emission at 395 nm. Both amino acids were identified
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by retention time using an amino acid standard. The Empower 2™ advanced software was employed to control system operation and results management.
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2.5. Aldehydes: extraction, identification and quantification The extraction of aldehydes compounds was performed according to
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methodology proposed by Lorenzo, Gómez, Purriños, & Fonseca (2016). A solid-phase microextraction (Supelco, Bellefonte, PA, USA) composed by fused-silica fiber (10mm length) coated with a 50/30 μm thickness of DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsilox-ane) was used for aldehydes extraction. One gram of sliced dry-cured ham was weighted into a 40 mL vial and then the vial was screw-capped with a laminated teflon-rubber disk. The fiber (SPME) was introduced into the sample vial through the septum and exposed to headspace. Before determination, the fiber was conditioned by
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ACCEPTED MANUSCRIPT heating it into a gas chromatograph injection port at 270 °C for 60 min, according to specifications manufacturer. Aldehyde extraction was carried out in an oven (35 ºC for 30 min) to ensure a homogeneous temperature for the sample and headspace. Prior to extraction samples were maintained for 15 min at the extraction temperature. Once extraction was finished, the fiber was
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withdrawn into the needle and transferred to the injection port of the gas
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chromatograph (6890 N)–mass spectrometer detector (5973 N) system (Agilent Technologies Spain, S.L., Madrid, Spain). A DB-624 capillary column (J&W
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scientific: 30 m, 0.25mm id, 1.4 μm film thickness) was used to aldehydes
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separation. The SPME fiber was desorbed and maintained in the injection port at 260 °C for 5 min. Sample injection was in split-less mode, using Helium as a
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carrier gas with a linear velocity of 40 cm/s. A gradient of temperature was as follow: initially isothermal at 40 °C for 10 min, then raised to 200 °C at a rate of
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5 °C/min, further raised to 250 °C at a rate of 20 °C/min, and finally held at 250 °C for 5 min: total runtime 49.5 min. Injector and detector temperatures were
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both set at 260 °C. The mass spectra were obtained using a mass selective detector working in electronic impact at 70 eV, with a multiplier voltage of 1953
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V and collecting data at a rate of 6.34 scans/s over the range m/z 40–300. Aldehydes were identified using three methods: a) comparing their mass spectra with those contained in the NIST14 library, b) comparing their mass spectra and retention time with authentic standards (Supelco, Bellefonte, PA, USA) and c) calculating the retention index relative to a series of standard alkanes (C 5-C14) (for calculating Kovats indexes, Supelco 44585-U, Bellefonte, PA, USA). Results were expressed as area units (AU)×10 3/g of dry matter. 2.6. Statistical analysis
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ACCEPTED MANUSCRIPT For statistical evaluation of data, the IBM SPSS Statistics 23.0 program (IBM Corporation, Somers, NY, USA) was used. To select spots for mass spectrometry analysis, spot volumes were analyzed by Mann-Whitney test. The fold change (FC) and relative change (RC) were used to assess the changes of the spot volume after high-pressure treatment compared to controls. The fold
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change (FC) is given by FC=VHP/Vcontrol where VHP and Vcontrol were the average
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spot volume in control group and group after a high-pressure treatment,
their
negative
reciprocal.
The
equation
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respectively. If the FC values were less than one, they were represented as used
to
calculate
RC
was
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RC=DV/│DVmax │ where DV= V HP-Vcontrol and DVmax was the maximum observed value of DV.
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To assess the effect of HPP on leucine, isoleucine and branched and linear aldehydes, the four groups of samples were analyzed (control vs. three
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HPP treatments) using an analysis of variance (ANOVA) of one way. When the HPP effect was significant (P<0.05), the least squares means were separated
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using Duncan's t-test at the 95% of confidence level. Results and discussion
3.1.
Proteomic analysis by 2-DE and mass spectrometry
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3.
A comparison between a control batch and HPP batch (600 MPa, 6 min) at 0 ºC was carried out to differentiate the heating caused by HPP effect. In the Figure 1, representative 2-DE proteome images obtained from control and HPP samples are shown. The average number of protein spots detected was 116 and 123 in control and HPP samples, respectively. Each protein spot detected was matched using PDQuest software for the biological replicates. In addition, those gel spots with significant changes in abundance were marked and
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ACCEPTED MANUSCRIPT numbered on the image (Figure 1). In particular, eighteen spots with significantly differential abundance were found, resulting nine spots with significantly qualitative differences (spot no. 2, 3, 4, 12, 13, 14, 16, 17 and 18) and other nine spots with quantitative differences (spot no. 1, 5, 6, 7, 8, 9, 10, 11 and 15). However, it must be emphasized that the majority of these spots
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with qualitative changes were only present in HPP treatment (8 spots of 9
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spots). Consequently, it appears clear to assume that proteomic profiles of
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control and HPP treated samples were highly differentiated. Previous studies of sliced dry-cured ham have shown that a higher number of spots detected on 2-
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DE gel were correlated to a higher proteolysis index (López-Pedrouso et al., 2018). In the present work, a higher number of spots were found in HPP
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samples, indicating more proteolytic degradation. This fact would suggest further proteolysis caused by the effect of pressure matching those observed in
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earlier studies (Rakotondramavo, Rabesona, Brou, de Lamballerie, & Pottier 2018; Ma & Ledward 2004). Overall, the curing step causes proteolysis
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whereas brining and tumbling mainly contribute to denaturation of the proteins. On the other hand, it has been reported that the HPP treatment lead to greater
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protein denaturation and oxidation causing aggregation, gelation or even increasing enzymatic activity. Although both proteomes must include a high level of proteins breakdown, the HPP treatment induced an increase in the proteolysis of dry-cured ham resulting in a higher number of spots. During the HPP process, the non-covalent interactions in the tertiary (hydrophobic and ionic interactions) structure are weakened (Kaur et al., 2016). This change in protein structure could causes a partial unfolding of protein enabling to enable
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ACCEPTED MANUSCRIPT hydrophobic interactions between proteins leading to their aggregation or could give rise to a higher proteolysis as the analysis suggested. The eighteen differentially abundant spots were excised for further identification using MALDI TOF/TOF MS. Fourteen spots were identified with a high Mascot score (> 60) as indicated in Table 1. Among the identified spots,
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ten were actin, which is an important fibrillar protein in meat, representing
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approximately 13% of the total muscle protein in two forms G, and F-actin,
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being G-actin a monomer of F-actin (Appell, Hurst, & Finley, 2018). It is important to highlight that actin spots detected in 2-DE gel had different
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molecular weights ranging from 41.1 to 14.1 KDa (Table 1). However, the actin isoform detected in this study has a theoretical molecular weight of around 42.0
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KDa according to Uniprot database. This difference between theoretical and experimental mass of actin spots can be explained by their different degree of
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fragmentation after a HPP treatment. The total RC value of actins was 2.51 indicating us entire or fragmented actins are most abundant in HPP samples
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(Table 2). On the other hand, the most abundant protein in the animal muscle (around 38%) is myosin. The myosin protein consists of two subunits with very
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different weights, called myosin light chain and heavy chain (Appell et al., 2018). In this comparative proteomic analysis, there was only a single myosin spot found in HPP samples and identified as myosin heavy chain. In addition, the difference between theoretical and experimental mass of myosin spot (223.3 kDa vs. 38.3 kDa) was very high and therefore it reasonable to suppose that spot number 4 is a myosin fragment. However, the presence of actin was more relevant than myosin based on the comparison of their RC (2.51 vs. 0.05) (Table 2).
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ACCEPTED MANUSCRIPT It has been reported that the actomyosin complex suffers a greater degradation dry-cured ham during processing. Particularly, it has been described as an extensive degradation of myosin heavy chain while actin is remarkably more stable at this high ionic condition (Fabbro et al., 2016; Wang, Zhang, Li, Shen, & Zhang, 2017). Furthermore, a heating process causes the
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rupture of hydrogen bonds of proteins, in contrast to HPP treatment, which
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affects the hydrophobic and electrostatic interactions (Duranton, Simonin,
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Chéret, Guillou, & de Lamballerie, 2012). For this reason, the proteomic analysis was carried out at 0 ºC to avoid the temperature effect. In addition, the
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high ionic strength conditions during dry-cured ham process could change the myosin light chain conformation affecting the binding of myosin with actin
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through to their phosphorylation and dephosphorylation. In saline conditions, actin shows a high degree of phosphorylation improving its stability against µ-
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calpain (Wang et al., 2017). This suggests that salt curing of dry-cured ham produced an intense degradation of myosin that at the same time was
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unaffected by HPP. Regarding actin, the salty conditions should make this protein more stable, but the HPP induces their fragmentation, confirmed by the
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presence of some actin fragments on the gel. According to Kęska & Stadnik (2017), myofibrillar proteins, specifically myosin-2, are precursors of peptides and amino acids, which have a strong impact on the taste of dry-cured meat, while sarcoplasmic proteins had not incidence on taste-active components generation. On the other hand, two spots were identified as triosephosphate isomerase with a total RC of approximately 0.31. Triosephosphate isomerase is an enzyme, which increases the glycolytic metabolism and produces NADH and
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ACCEPTED MANUSCRIPT ATP, consequently it is strongly correlated with meat quality (Gagaoua, Monteils, Couvreur, & Picard, 2017; Kim & Dang, 2005; Schilling et al., 2017). In this proteomic study, spot number 1 identified as triosephosphate isomerase has an experimental mass similar to theoretical mass which would indicate that the protein is entire. On the contrary, the spot number 15 could suggest that
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there were protein aggregates even under reducing conditions due to the
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theoretical mass was lower than experimental mass as in other treatments
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occurred previously (Di Luccia, Gatta, Nicastro, Petrella, Lamacchia, & Picariello, 2015; López-Pedrouso et al., 2018b).
Levels of leucine and isoleucine affected by HPP treatment
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3.2.
As hydrolysis of proteins by enzyme action and chemical reaction
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release free amino acids, their quantification can be used as an indirect way to quantify proteolysis. Among free amino acids, leucine and isoleucine have a
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great importance because of their high concentration and are classified as lipophilic amino acids. Both are related to meat taste and flavor directly or
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indirectly via for being metabolites of Strecker aldehydes. As shown in the Figure 2, relative leucine content was higher than isoleucine in all treatments.
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The average relative content of leucine ranged from 102.62 to 130.50 mg/100 g dry cured ham (d.b.) whereas isoleucine varied from 62.18 to 77.43 mg/100 g dry cured ham (d.b.). Indeed, leucine was the most abundant free ami no acid in dry-cured ham according to Pérez-Santaescolástica et al. (2018a) and both are considered as predominant free amino acids like in the other types of hams such as Jinhua, Xuanwei, Parma and Bama (Zhang et al., 2018). Both amino acids have a specific tastes and high concentrations consequently, it can be supposed that both directly influence the development of aroma and taste. It is
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ACCEPTED MANUSCRIPT known that leucine together with glutamate, lysine, alanine and lactate are the highest contributor of taste in dry-cured hams (Zhang et al., 2018). Specifically, leucine and isoleucine were strongly associated with bitter taste of dry-cured meat products (Kęska & Stadnik, 2017). However, it has also indicated that the spatial arrangement of amino acids can be changed with the food process,
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resulting different tastes from the same amino acid (Solms, 1969). On the other
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hand, it has recently reported that high levels of leucine and isoleucine were
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correlated with proteolysis index contributing to a high adhesiveness in drycured hams (Pérez-Santaescolástica et al., 2018a).
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There was a significant difference (P<0.05) between control samples and samples after HPP treatment. As shown in the Figure 2, it can be observed that
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there was significant (P<0.05) differences between samples after HPP treatment at low temperatures (0 ºC and 20 ºC) and samples at 35 ºC. In
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particular, the relative leucine and isoleucine content increased under HPP at low temperatures (0 ºC and 20 ºC) in comparison with control samples unlike 35
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ºC with the lowest values for both amino acids. Therefore, these findings suggest that the effect of HPP treatment on relative isoleucine and leucine
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content is highly dependent on temperature. According to Guyon et al. (2016), the high-pressure process greatly contributes more to the hydrolysis of proteins into amino acids such as leucine and isoleucine. This suggests that all HPP treatments should increase both concentrations in dry-cured ham due to protein degradation. On the other hand, Kim, Kemp, & Samuelsson (2016) have described that leucine and isoleucine are metabolites involved in chemical reactions forming volatiles compounds as aldehydes. This means an indirect way of influence in taste and aroma as mentioned above. It is possible to
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ACCEPTED MANUSCRIPT hypothesize that the higher temperatures facilitate the Strecker reaction between leucine and isoleucine and α-carbonyl compounds and this fact could explain the lower content of both amino acids after HPP treatment at 35 ºC. 3.3.
Aldehyde volatile compounds affected by HPP treatment
Wide varieties of volatile compounds were detected using SPME
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followed by GC-MS (Pérez-Santaescolástica et al., 2018b). Volatile compounds
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are classified in chemical families, but aldehydes are a key group in dry-cured
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ham for their abundance and important contribution to taste and aroma. Their sensorial notes are fruity, toasted, oily, fatty and rancid, commonly associated
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with cured meats (Sánchez-Peña, Luna, García-González, & Aparicio, 2005). As shown in table 3, the HPP treatment significantly (P<0.001) affected the total
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aldehydes concentration resulting in the highest values in control samples (23430.18 AU×103/g of dry-cured ham). Again, the temperature fixed in each
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HPP treatment resulted in significant (P<0.05) differences in total aldehydes between samples at low temperature (average value of 12331.38 AU×103/g of
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dry-cured ham) and at 35 ºC (19687.56 AU×103 /g of dry-cured ham). Among the aldehydes determined with statistical significance, the most abundant were
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two branched aldehydes as 2-methyl-butanal and 3-methyl-butanal and four linear aldehydes as pentanal, hexanal, heptanal and nonanal. All linear aldehydes showed a significant reduction (P<0.05) due to the HPP effect which means that its contribution in overall aroma was decreased. The reductions ranged from 37.43% to 62.54% for nonanal and heptanal, respectively. The most likely explanation for these results may be the fact that aldehydes are considered highly reactive molecules especially α- and βunsaturated (alkenals, alkadienals, and hydroxyalkenals) which could react with
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ACCEPTED MANUSCRIPT proteins (Guyon et al., 2016). The results suggest that linear aldehydes could be reacting to a great extent in HPP conditions; therefore, their concentration decreases in comparison with samples untreated. In the case of the most abundant aldehyde, the hexanal concentration diminished more than 2.5 times by high-pressure effect. This result is consistent with Fuentes et al. (2014) that
PT
indicated a decrease of hexanal level in dry-cured ham slices treated with high
RI
pressure (600 MP). In addition, hexanal plays a key role in dry-cured products
SC
and at high concentration lead to the development of an unpleasant rancid odor (Lorenzo, Carballo, & Franco, 2013; Shahidi & Pegg; 1994; Carrapiso, Martín,
NU
Jurado, & García, 2010). Indeed, like other linear aldehydes, hexanal come from lipid oxidation by breakdown products of unsaturated lipids (Petričević,
MA
Radovčić, Lukić, Listeš, & Medić, 2018). Specifically, hexanal could be formed from the breakdown of linoleic, gamma-linolenic and arachidonic acids (Shahidi,
that HPP prevents
temperatures.
the
lipid oxidation, particularly at low
EP T
concluded
ED
2001), hence is widely used as oxidation level indicator. It may, therefore, be
On the other hand, branched aldehydes, which are 2-methyl-butanal and
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3-methyl-butanal, are related to proteolysis and amino acids degradation (Toldrá, 1998). From a sensorial point of view, both aldehydes are potent odorants associated with the nutty, cheesy and salty sensory notes (SánchezPeña et al., 2005). Both aldehydes come from isoleucine and leucine, via Strecker degradation, which is considered as a step within Maillard reaction. This reaction involves oxidative deamination followed by decarboxylation resulting aldehydes with one carbon atom less the corresponding amino acid (Estévez et al., 2011; Resconi, Escudero, & Campo, 2013). The results show
18
ACCEPTED MANUSCRIPT that HPP treatment at 35 ºC had an impact on both aldehydes, because of their concentration significantly increased (P<0.05) with increments of 35.30% and 41.05% for 2-methyl-butanal and 3-methyl-butanal, respectively. However, these findings do not support previous research of Martínez-Onandi, RivasCañedo, Picon, & Nuñez (2016) who found that HPP treatment (600 MPa, 6
PT
min) produced a reduction in the amount of 2-methyl-butanal. Although these
RI
results must be interpreted with caution because they are influenced by water
SC
activity and intramuscular fat content, as noted above. Furthermore, it is necessary to highlight that it has been reported that the ability of 2-methyl-
NU
butanal to bind peptide extracts of dry-cured ham was too weak to be detected (Martínez-Arellano et al., 2016). It seems that Strecker aldehydes are less
MA
reactive than linear aldehydes and consequently more stable. Based on these reasons, it can be concluded that Strecker aldehydes can be associated with
4.
ED
greater proteolysis degree of dry-cured ham after HPP treatment. Conclusions
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High pressure conditions caused a significant impact on proteome of sliced dry-cured ham. The effect of HPP treatment was clearly demonstrated on
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myofibrillar proteins such as actin which was likely given rise to isoleucine and leucine. In addition, 2-methyl-butanal and 3-methyl-butanal coming from both Strecker metabolites increased their concentration due to HPP effect. Therefore, protein degradation by HPP effect can trigger a series of chain reactions depending on conditions (pressure and temperature) which cause a strong impact on volatile compound profile. A further research needs to be done to establish whether all changes in the volatile profile induced by high-pressure
19
ACCEPTED MANUSCRIPT process will be positive for the improvement of dry-cured ham sensorial attributes. Acknowledgements This research was supported by Grant RTA 2013-00030-CO3-03 from INIA
(Spain). Acknowledgements
to
INIA
for granting
Cristina Pérez
PT
Santaescolástica with a predoctoral scholarship. José M. Lorenzo is member of
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the MARCARNE network, funded by CYTED (ref. 116RT0503).
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ACCEPTED MANUSCRIPT CAPTION TO FIGURES
Figure 1. 2-DE gel images obtained from sliced dry-cured ham samples after a standard process (left) and a HPP treatment at 0ºC (right). Spots with significant
These spots were analyzed by MALDI-TOF/TOF MS.
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differences (P<0.05) by effect of pressure treatment are indicated and numbered.
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Figure 2. Relative leucine and isoleucine content in dry-cured ham under
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HPP treatment at different temperatures. Mean ± standard deviation expressed in mg FAA/ mg total FAA per 100 g dry matter. Differences in letter: a to c per
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each FAA represent significant differences (P<0.05) among treatments.
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ACCEPTED MANUSCRIPT Table 1. Identification of selected protein spots by MALDI-TOF/TOF Protein Descriptionb
Protein Accessionb
Mascot Score
1
Triosephosphate isomerase
TPI1
87
Sequence coverage (%) 29
2
Alpha actin 1
ACTC1
93
9
3
3
Alpha actin 1
ACTC1
108
17
5
4
Myosin-7
MYH7
131
7
13
5
Alpha actin 1
ACTC1
340
6
Alpha actin 1
ACTC1
166
7
Hemoglobin subunit beta
HBB
91
9
Alpha actin 1
ACTC1
378
10
Alpha actin 1
ACTC1
11
Alpha actin 1
ACTC1
12
Alpha actin 1
ACTC1
14
Alpha actin 1
15
Triosephosphate isomerase
17
Alpha actin
5
15
2
24
8
119
SC
30
11
154
9
3
90
23
8
ACTC1
172
18
4
TPI1
79
30
5
ACTC1
85
17
4
MA
NU
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16
ED
Sus scrofa (pig) was employed as taxonomy filter in Mascot search
Theoretical mass was provided by UniProt database
d
Experimental mass was estimated on 2-DE gels using molecular weight standards
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c
6
9
The spot numbers are shown in Figure 1
b
Number of matched peptides
24
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a
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Spot no.a
26
Theo (Uni
ACCEPTED MANUSCRIPT Table 2. Spot volumes with significant differences by effect of a high-pressure treatment
in sliced dry-cured ham. Fold change (FC) and relative change (RC) of the selected spots Control
HPP
Mean ± SE
Mean ± SE 249.53 ± 45.25 337.46 ± 152.42 134.77 ± 38.14 43.11 ± 15.00 897.29 ± 162.25 191.86 ± 47.54
Spot no.
Protein (gene name)
1
Triosephosphate isomerase (TPI1)
2
Alpha actin 1 (ACTC1)
-
3
Alpha actin 1 (ACTC1)
-
4
Myosin-7 (MYH7)
-
5
Alpha actin 1 (ACTC1)
486.98 ± 80.85
6
Alpha actin 1 (ACTC1)
340.11 ± 52.40
7
Hemoglobin subunit beta (HBB)
109.81 ± 30.37
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124.48 ± 38.76
35.04 ± 10.43
FC
RC
+ 2.00
+ 0.25
+∞
+ 0.68
+∞
+ 0.27
+∞
+ 0.09
+ 1.84
+ 0.83
- 1.77
- 0.30
- 3.13
- 0.15
227.48 ± + 1.27 + 0.10 18.14 574.66 ± 9 Alpha actin 1 (ACTC1) 151.05 ± 20.66 + 3.80 + 0.85 147.24 473.80 ± 10 Alpha actin 1 (ACTC1) 132.30 ± 27.02 + 3.58 + 0.69 110.71 11 Alpha actin 1 (ACTC1) 99.09 ± 23.00 56.07 ± 14.76 - 1.77 -0.09 411.04 ± 12 Alpha actin 1 (ACTC1) +∞ + 0.83 143.85 496.40 ± 13 Uncharacterized protein +∞ + 1.00 129.27 175.91 ± 14 Alpha actin 1 (ACTC1) +∞ + 0.35 82.73 Triosephosphate isomerase 288.90 ± 15 137.34 ± 4.72 + 2.10 + 0.31 (TPI1) 25.18 142.94 ± 16 Uncharacterized protein +∞ + 0.29 46.31 269.59 ± 17 Alpha actin 1 (ACTC1) +∞ + 0.54 81.93 18 Uncharacterized protein 153.73 ± 16.46 -∞ - 0.31 Average ( ± SE, Standard error) volumes were assessed from 2-DE gels using the PDQuest software 179.16 ± 11.03
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Uncharacterized protein
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8
27
ACCEPTED MANUSCRIPT Table 3. Effect of HPP at different temperatures on main branched and linear aldehydes of drycured ham expressed as AU×103 /g of dry-cured ham (mean ± standard deviation) Treatment m/z
LRI
R
CO
HPP-0
HPP-20
57
671
ms, lri
863.96± 326.20
3-methyl butanal
58
659
ms, lri
1736.34±521.48
a
pentanal
57
728
ms, lri, s
1215.3± 547.46 b
hexanal
56
865
ms, lri
15717.34 ±4194.15 c
heptanal
70
974
ms, lri, s
1025.45 ±324.52 c
384.07± 105.33 a
450.05± 163
nonanal
57
1148
ms, lri, s
553.39± 170. 73 b
346.82 ±74.69 a
338.68± 91
RI
23430.18±4949.22
SC
Total aldheydes
PT
2-methyl butanal
a
a-c
c
879.99± 427.70
a
1547.91±340.04
a
1878.31±791
541.18 ± 203.79 a
594.13± 192
6040.25±1821.88
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ED
EP T AC C
a
11065.57± 2337.37 a
Mean values in the same row (corresponding to the same compound) not followed by a common letter differ significantly (P<0.05). m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30m×0.25mm id, 1.4μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo, Montes, Purriños, & Franco, 2012; Lorenzo, Bedia, & Bañon, 2013b; Lorenzo, 2014; Lorenzo & Domínguez, 2014; Lorenzo & Carballo, 2015; Pateiro, Franco, Carril, & Lorenzo, 2015; Pérez-Santaescolástica et al., 2018a; Pérez-Santaescolástica et al., 2018b; Purriños, Franco, Bermudez, Carballo, & Lorenzo, 2011a; Purriños, Franco, Bermúdez, Temperan, Carballo, & Lorenzo, 2011; Purriños, Franco, Carballo, & Lorenzo, 2012, Purriños, Carballo, & Lorenzo, 2013); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Treatments: CO= control (without treatment); HPP-0=High pressure treatment at 0 ºC; HPP20=High pressure treatment at 20 ºC; HPP-35=High pressure treatment at 35 ºC.
28
1168.02±483
7535.70 ±221
13597.19± 30
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Proteolysis under pressure conditions and its possible sensory consequences were assessed Proteomic analysis showed that high-pressure conditions led to a higher level of proteolysis Actin (ACTC1) is a candidate biomarker to control high pressure processing High-pressure conditions at low temperatures produced an increase of Strecker metabolites Branched and linear aldehydes affected in different ways by high pressure processing
29
Figure 1
Figure 2
M. López-Pedrouso, C. Pérez-Santaescolástica, D. Franco, J. Carballo, C. Zapata, J.M. Lorenzo PII: DOI: Reference:
S0963-9969(19)30037-7 https://doi.org/10.1016/j.foodres.2019.01.037 FRIN 8227
To appear in:
Food Research International
Received date: Revised date: Accepted date:
10 December 2018 14 January 2019 15 January 2019
Please cite this article as: M. López-Pedrouso, C. Pérez-Santaescolástica, D. Franco, J. Carballo, C. Zapata, J.M. Lorenzo , Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions. Frin (2019), https://doi.org/10.1016/j.foodres.2019.01.037
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ACCEPTED MANUSCRIPT Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions
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López-Pedrouso, M.1, Pérez-Santaescolástica, C.2, Franco, D.1,
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Carballo, J.3, Zapata, C.2 and Lorenzo, J.M.1*
1
Department of Zoology, Genetics and Physical Anthropology, University of
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Santiago de Compostela, Santiago de Compostela -15872, Spain 2
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Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque
Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain 3
Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense,
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Universidad de Vigo, 32004 Ourense, Spain
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Corresponding author. Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract High pressure processing (HPP) is currently being developed to increase the shelf-life of sliced dry-cured ham in convenience package without detrimental effects on texture and sensorial characteristics. This study is focused on protein degradation under pressure conditions and its contribution to
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taste and aroma. Samples of sliced dry-cured ham undergone HPP (600 Pa, 0-
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35ºC) were analyzed from different approaches including proteomic and
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chemical analysis (amino acids and volatile compounds).
Proteomic analysis revealed that high-pressure conditions caused a
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higher level of proteolysis, displaying that actin (ACTC1) was differentially degraded, unlike myosin. Furthermore, main Strecker metabolites-isoleucine
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and leucine-were more abundant at lower temperatures as opposed to 2-methyl butanal and 3-methyl butanal under HPP. Moreover, this study confirmed that
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HPP affected positively linear aldehydes (pentanal, hexanal, heptanal and nonanal) because of produce a decrease of them, which could improve flavor
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and taste of dry-cured ham.
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Keywords: myofibrillar proteins, Strecker metabolites, linear and branched aldehydes, proteolysis and lipid oxidation
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ACCEPTED MANUSCRIPT 1.
Introduction
The traditional manufacturing process of dry-cured ham includes the following steps: salting, post-salting, ripening and drying for a total period between 12 to 24 months (Bermúdez, Franco, Carballo, & Lorenzo, 2014a). All these steps have a strong impact on the meat quality due to complex chemical
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reactions (Harkouss et al., 2015; López-Pedrouso et al., 2018; López-Pedrouso
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et al., 2019; Lorenzo, Bermúdez, Domínguez, Guiotto, Franco, & Purriños,
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2015; Petrova, Tolstorebrov, Mora, Toldrá, & Eikevik, 2016). Nowadays, consumers demand the use of convenience package to increase the shelf life of
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the products as well as to maintain microbiological safety. To overcome the problem of possible food contaminations without the loss of quality, innovative
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and nonthermal techniques are being developed such as high-pressure processing (HPP). However, the introduction of novel technology is another
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important factor, which has a major influence on sensory features. Along with the whole process, the volatile compounds profile (aldehydes, ketones,
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alcohols, hydrocarbons and esters) which defines aroma could be changed due to protein degradation (Martínez-Arellano, Flores & Toldrá, 2016; Pérez-
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Santaescolástica, Carballo, Fulladosa, Garcia-Perez, Benedito, & Lorenzo, 2018a). In addition, other key characteristic associated with proteolysis is the tenderization, which is mainly due to the breakdown of myofibrillar structures (Čandek-Potokar & Škrlep, 2012; Mora, Sentandreu, & Toldrá, 2011). For these reasons HPP is being boosted within food industry, but concomitant effects on odor (rancidity) and texture attributes have been revealed particularly on sliced dry-cured hams (Lorido, Estévez, Ventanas, & Ventanas, 2015; PérezSantaescolástica et al., 2019). It has been reported that HPP modified the
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ACCEPTED MANUSCRIPT protein structure causing alterations in meat color and texture parameters such as pastiness and hardness (Garcıá -Esteban, Ansorena, & Astiasarán, 2004). Moreover, volatile compounds have also been affected by HPP and storage conditions of sliced vacuum-packaged dry-cured ham (Clariana, Guerrero, Sárraga, Diaz, Valero, & García-Regueiro, 2011; Fuentes, Utrera, Estévez,
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Ventanas, & Ventanas, 2014).
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Flavor and aroma are influenced by proteolysis and lipolysis phenomena,
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which release free fatty acids, peptides and free amino acids. These complex processes are attributed to enzymatic and chemical reactions such as Maillard
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reactions and Strecker degradations (Estévez, Ventanas, & Heinonen, 2011; Petrova et al., 2016). In the muscle, lipids and proteins are transformed into
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flavor precursors depending on process conditions (Degnes, Kvitvang, HasleneHox, & Aasen, 2017; Toldrá & Flores; 1998). Specifically, in dry-cured ham
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process, the Maillard reaction is a chemical reaction between a reducing sugar and an amino compound favored by long drying period and low water activity
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(Flores, 2018). On the other hand, the Strecker degradation of amino acids lead to their deamination and decarboxylation turning out aldehydes and ketones.
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These amino acids are intermediates from Maillard reaction which means that both reactions are closely related (Van Boekel, 2006). Therefore, the aroma and flavor are strongly affected by processing techniques, which could modify the kinetics of both reactions. These sensorial attributes could also be studied in detail using the identification and quantification of their volatiles (Bermúdez, Franco, Carballo, Sentandreu, & Lorenzo, 2014b; Pérez-Santaescolástica et al., 2018a). Consequently, the combination of non-volatile and volatile compounds defines a specific taste and smell, which is highly appreciated. Indeed, juiciness
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ACCEPTED MANUSCRIPT and flavor intensity of dry-cured ham are the most important positive characteristics assessed by consumers (Ruiz, Garcıa, Muriel, Andrés, & Ventanas, 2002). To monitor the quality, safety and nutritional requirements during production and storage, foodomics is a very useful tool for the taste, flavor and characterization.
A
proteomic
approach
using
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texture
bidimensional
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electrophoresis (2-DE) coupled with mass spectrometry (MS) could also provide
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biomarkers to optimize food processing (Picard & Gagaoua, 2017; Picard, Gagaoua & Hollung, 2017)
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In fact, it was employed in thermal, microwaves, high pressure, pulsed electric field and ultrasound treatments (Piras, Roncada, Rodrigues, Bonizzi, &
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Soggiu, 2016; López-Pedrouso et al., 2018b). In addition, this approach has been used to study cured and cooked pork ham products (Pioselli, Paredi, &
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Mozzarelli, 2011; Théron, Sayd, Pinguet, Chambon, Robert, & SantéLhoutellier, 2011; Škrlep et al., 2011). From a proteomic point of view, the
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proteolysis of dry-cured ham produced during their manufacturing process has also been studied (López-Pedrouso et al., 2018; Pérez-Santaescolástica et al.,
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2018b; Fabbro, Bencivenni, Piasentier, Sforza, Stecchini, & Lippe, 2016), but only few researches have focused on proteins of dry-cured ham submitted to HPP.
Therefore, the aim of this study was to evaluate the protein alterations of sliced dry-cured ham undergone high-pressure processing. The contribution of protein degradation to the release of volatile compounds and their metabolites should be studied to optimize high-pressure treatment on a final meat product. 2.
MATERIAL AND METHODS
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ACCEPTED MANUSCRIPT 2.1.
Samples
Sixteen dry-cured hams, manufactured according to the traditional system, were sliced (1.5 mm-thick) and vacuum packed in individual plastic bags of polyamide/polyethylene (oxygen permeability of 50 cm3/m2/24h at 23 ºC and water permeability of 2.6 g/m2/24h at 23 ºC and 85% RH, Sacoliva®
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S.L., Spain) and stored in a chamber at 4 ºC ± 2 °C until the treatment
HPP treatments
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2.2.
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application.
The treatment of the packaged slices was applied using a NC Hyperbaric
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WAVE 6000/120 equipment (NC Hyperbaric, Burgos, Spain). Three different treatments were performed at 600 MPa during 6 min, each one accompanied by
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a different temperature: 0 °C, 20 °C and 35 °C. In order to evaluate the effect of HPP treatments, a fourth group of samples were not treated and used as a
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control batch. For proteomic analysis, only samples from control and HPP (0 ºC) groups were analyzed. Samples were stored at room temperature (20 °C)
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for no longer than 4 weeks.
2.3. Proteomic analysis
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2.3.1. Protein extraction and two-dimensional electrophoresis (2-DE) A comparison between control (without HPP treatment) and HPP (0 ºC) batch was carried out using four biological replicas in each case. It is well known that 2-DE maps of porcine muscles were used to detect up to 800 spots (Lametsch & Bendixen, 2001; Morzel et al., 2004). This would provide us enough quantity of data to carry out this experiment between two conditions. Furthermore, the combination of pressure and temperature produce protein denaturation particularly acute in combination of high pressure and temperature
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ACCEPTED MANUSCRIPT (Guyon, Meynier, & De Lamballerie, 2016). In order to avoid interaction between these two variables, the selected temperature for proteomic analysis was 0 ºC with the aim of identify the potential biomarkers related exclusively to high pressure distinguishing them from biomarkers associated with high temperature.
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For each biological replica, a lyophilized sample of dry-cured ham (50
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mg) was mixed with 1 mL of lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS;
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10 mM DTT, and 2% Pharmalyte™ pH 3 -10, GE Healthcare, Uppsala, Sweden) and sonicated (Sonifier 250, Branson, Danbury, CC, USA) under refrigerated
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conditions (0 ºC). Protein purification was performed twice with Clean-Up Kit (GE Healthcare) and subsequently the pellet was dissolved in 200 µL of lysis
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buffer. Protein content in samples was estimated by CB-X protein assay kit (GBiosciences, St. Louis, MO, USA) using a Chromate® microplate reader
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(Awareness Technology, Palm City, FL, USA). The isoelectric focusing (IEF) was carried out in 24 cm pH 4-7 IPG strips (Bio-Rad Laboratories, Hercules,
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CA, USA). In the first step, the strips were rehydrated for 12 h in 450 µL of a solution 0.6% DTT and 1% IPG buffer (Bio-Rad Laboratories) which contained
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250 µg of protein in lysis buffer and rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromophenol blue). Afterward, the
voltage was
sequentially raising until reaching 70,000 Vh, and then the strips were equilibrated with buffer I (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 1% DTT) for 15 minutes and buffer II (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2.5% iodoacetoamide) for another 15 minutes. Finally, the second dimension was carried out in 12% SDS-PAGE using an Ettan DALTsix vertical gel system (GE Healthcare). The resulting 2-DE gels were stained with SYPRO
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ACCEPTED MANUSCRIPT Ruby fluorescent stain (Lonza, Rockland, ME, USA) and the images of gels were obtained by Gel Doc XR+ system (Bio-Rad Laboratories). Image analysis of gels was carried using PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories) and experimental values of isoelectric point (pI) and mass (M r) were estimated.
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2.3.2. Protein Identification by Mass Spectrometry
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The excision and in-gel tryptic digestion of protein gel spots were carried out according to Franco et al. (2015). The digested spots with trypsin were
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ionized using α-cyano-4-hydroxycinnamic acid as matrix and deposited onto a
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384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA). Mass spectrometry analysis was performed on a MALDI-TOF/TOF tandem mass
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spectrometer 4800 MALDI-TOF/TOF (Applied Biosystems, Foster City, CA, USA) using 4000 Series Explorer software v. 3.5 (Applied Biosystems, Foster
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City, CA, USA). The search was carried out using Mascot software v. 2.1 (Matrix Science, Boston, MA, USA) to identify proteins from peptide mass
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fingerprint data employing UniProt/SwissProt database. 2.4. Free
and
isoleucine: extraction,
identification
and
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quantification
leucine
The extraction of free leucine and isoleucine was carried out according to the procedure described by Pérez-Palacios, Ruiz, Barat, Aristoy, & Antequera, (2010) with some modifications. Briefly, five grams of sliced-dry cured ham were homogenized with 25 mL of hydrochloric acid 0.1 N, in an Ika Ultra-Turrax for 8 min under refrigerated conditions by submerging the extract in ice. Afterwards, the solution was centrifuged at 5240g for 20 minutes at 4 ºC and the supernatant layer was filtered through glass wool prior to further analyses. Two
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ACCEPTED MANUSCRIPT hundred ml of this extract was deproteinized by adding 800 mL of acetonitrile and centrifuged for 3 min at 5240g. The derivatization, identification and quantification were carried out according to Franco & Lorenzo (2014). The derivatization and chromatographic analysis conditions were as follows: 10 μL sample was buffered to pH 8.8
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(AccQFluor borate buffer) to yield a total volume of 100 μL. The derivatization
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reaction was initiated by the addition of 20 μL AccQ-Fluor reagent (3 mg/mL in acetonitrile). The chromatographic separation was carried out in a Waters
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AccQ-Tag column (3.9 mm × 150 mm with a 4 μm of particle size) with a flow
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rate of 1.0 mL/min at 37 °C, using a HPLC (Alliance model 2695, Waters, Milford, MA, USA) equipped with a 2475 scanning fluorescence detector. The
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detection of leucine and isoleucine was accomplished by fluorescence with excitation at 250 nm and emission at 395 nm. Both amino acids were identified
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by retention time using an amino acid standard. The Empower 2™ advanced software was employed to control system operation and results management.
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2.5. Aldehydes: extraction, identification and quantification The extraction of aldehydes compounds was performed according to
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methodology proposed by Lorenzo, Gómez, Purriños, & Fonseca (2016). A solid-phase microextraction (Supelco, Bellefonte, PA, USA) composed by fused-silica fiber (10mm length) coated with a 50/30 μm thickness of DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsilox-ane) was used for aldehydes extraction. One gram of sliced dry-cured ham was weighted into a 40 mL vial and then the vial was screw-capped with a laminated teflon-rubber disk. The fiber (SPME) was introduced into the sample vial through the septum and exposed to headspace. Before determination, the fiber was conditioned by
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ACCEPTED MANUSCRIPT heating it into a gas chromatograph injection port at 270 °C for 60 min, according to specifications manufacturer. Aldehyde extraction was carried out in an oven (35 ºC for 30 min) to ensure a homogeneous temperature for the sample and headspace. Prior to extraction samples were maintained for 15 min at the extraction temperature. Once extraction was finished, the fiber was
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withdrawn into the needle and transferred to the injection port of the gas
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chromatograph (6890 N)–mass spectrometer detector (5973 N) system (Agilent Technologies Spain, S.L., Madrid, Spain). A DB-624 capillary column (J&W
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scientific: 30 m, 0.25mm id, 1.4 μm film thickness) was used to aldehydes
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separation. The SPME fiber was desorbed and maintained in the injection port at 260 °C for 5 min. Sample injection was in split-less mode, using Helium as a
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carrier gas with a linear velocity of 40 cm/s. A gradient of temperature was as follow: initially isothermal at 40 °C for 10 min, then raised to 200 °C at a rate of
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5 °C/min, further raised to 250 °C at a rate of 20 °C/min, and finally held at 250 °C for 5 min: total runtime 49.5 min. Injector and detector temperatures were
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both set at 260 °C. The mass spectra were obtained using a mass selective detector working in electronic impact at 70 eV, with a multiplier voltage of 1953
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V and collecting data at a rate of 6.34 scans/s over the range m/z 40–300. Aldehydes were identified using three methods: a) comparing their mass spectra with those contained in the NIST14 library, b) comparing their mass spectra and retention time with authentic standards (Supelco, Bellefonte, PA, USA) and c) calculating the retention index relative to a series of standard alkanes (C 5-C14) (for calculating Kovats indexes, Supelco 44585-U, Bellefonte, PA, USA). Results were expressed as area units (AU)×10 3/g of dry matter. 2.6. Statistical analysis
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ACCEPTED MANUSCRIPT For statistical evaluation of data, the IBM SPSS Statistics 23.0 program (IBM Corporation, Somers, NY, USA) was used. To select spots for mass spectrometry analysis, spot volumes were analyzed by Mann-Whitney test. The fold change (FC) and relative change (RC) were used to assess the changes of the spot volume after high-pressure treatment compared to controls. The fold
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change (FC) is given by FC=VHP/Vcontrol where VHP and Vcontrol were the average
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spot volume in control group and group after a high-pressure treatment,
their
negative
reciprocal.
The
equation
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respectively. If the FC values were less than one, they were represented as used
to
calculate
RC
was
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RC=DV/│DVmax │ where DV= V HP-Vcontrol and DVmax was the maximum observed value of DV.
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To assess the effect of HPP on leucine, isoleucine and branched and linear aldehydes, the four groups of samples were analyzed (control vs. three
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HPP treatments) using an analysis of variance (ANOVA) of one way. When the HPP effect was significant (P<0.05), the least squares means were separated
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using Duncan's t-test at the 95% of confidence level. Results and discussion
3.1.
Proteomic analysis by 2-DE and mass spectrometry
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3.
A comparison between a control batch and HPP batch (600 MPa, 6 min) at 0 ºC was carried out to differentiate the heating caused by HPP effect. In the Figure 1, representative 2-DE proteome images obtained from control and HPP samples are shown. The average number of protein spots detected was 116 and 123 in control and HPP samples, respectively. Each protein spot detected was matched using PDQuest software for the biological replicates. In addition, those gel spots with significant changes in abundance were marked and
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ACCEPTED MANUSCRIPT numbered on the image (Figure 1). In particular, eighteen spots with significantly differential abundance were found, resulting nine spots with significantly qualitative differences (spot no. 2, 3, 4, 12, 13, 14, 16, 17 and 18) and other nine spots with quantitative differences (spot no. 1, 5, 6, 7, 8, 9, 10, 11 and 15). However, it must be emphasized that the majority of these spots
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with qualitative changes were only present in HPP treatment (8 spots of 9
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spots). Consequently, it appears clear to assume that proteomic profiles of
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control and HPP treated samples were highly differentiated. Previous studies of sliced dry-cured ham have shown that a higher number of spots detected on 2-
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DE gel were correlated to a higher proteolysis index (López-Pedrouso et al., 2018). In the present work, a higher number of spots were found in HPP
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samples, indicating more proteolytic degradation. This fact would suggest further proteolysis caused by the effect of pressure matching those observed in
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earlier studies (Rakotondramavo, Rabesona, Brou, de Lamballerie, & Pottier 2018; Ma & Ledward 2004). Overall, the curing step causes proteolysis
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whereas brining and tumbling mainly contribute to denaturation of the proteins. On the other hand, it has been reported that the HPP treatment lead to greater
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protein denaturation and oxidation causing aggregation, gelation or even increasing enzymatic activity. Although both proteomes must include a high level of proteins breakdown, the HPP treatment induced an increase in the proteolysis of dry-cured ham resulting in a higher number of spots. During the HPP process, the non-covalent interactions in the tertiary (hydrophobic and ionic interactions) structure are weakened (Kaur et al., 2016). This change in protein structure could causes a partial unfolding of protein enabling to enable
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ACCEPTED MANUSCRIPT hydrophobic interactions between proteins leading to their aggregation or could give rise to a higher proteolysis as the analysis suggested. The eighteen differentially abundant spots were excised for further identification using MALDI TOF/TOF MS. Fourteen spots were identified with a high Mascot score (> 60) as indicated in Table 1. Among the identified spots,
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ten were actin, which is an important fibrillar protein in meat, representing
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approximately 13% of the total muscle protein in two forms G, and F-actin,
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being G-actin a monomer of F-actin (Appell, Hurst, & Finley, 2018). It is important to highlight that actin spots detected in 2-DE gel had different
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molecular weights ranging from 41.1 to 14.1 KDa (Table 1). However, the actin isoform detected in this study has a theoretical molecular weight of around 42.0
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KDa according to Uniprot database. This difference between theoretical and experimental mass of actin spots can be explained by their different degree of
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fragmentation after a HPP treatment. The total RC value of actins was 2.51 indicating us entire or fragmented actins are most abundant in HPP samples
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(Table 2). On the other hand, the most abundant protein in the animal muscle (around 38%) is myosin. The myosin protein consists of two subunits with very
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different weights, called myosin light chain and heavy chain (Appell et al., 2018). In this comparative proteomic analysis, there was only a single myosin spot found in HPP samples and identified as myosin heavy chain. In addition, the difference between theoretical and experimental mass of myosin spot (223.3 kDa vs. 38.3 kDa) was very high and therefore it reasonable to suppose that spot number 4 is a myosin fragment. However, the presence of actin was more relevant than myosin based on the comparison of their RC (2.51 vs. 0.05) (Table 2).
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ACCEPTED MANUSCRIPT It has been reported that the actomyosin complex suffers a greater degradation dry-cured ham during processing. Particularly, it has been described as an extensive degradation of myosin heavy chain while actin is remarkably more stable at this high ionic condition (Fabbro et al., 2016; Wang, Zhang, Li, Shen, & Zhang, 2017). Furthermore, a heating process causes the
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rupture of hydrogen bonds of proteins, in contrast to HPP treatment, which
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affects the hydrophobic and electrostatic interactions (Duranton, Simonin,
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Chéret, Guillou, & de Lamballerie, 2012). For this reason, the proteomic analysis was carried out at 0 ºC to avoid the temperature effect. In addition, the
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high ionic strength conditions during dry-cured ham process could change the myosin light chain conformation affecting the binding of myosin with actin
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through to their phosphorylation and dephosphorylation. In saline conditions, actin shows a high degree of phosphorylation improving its stability against µ-
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calpain (Wang et al., 2017). This suggests that salt curing of dry-cured ham produced an intense degradation of myosin that at the same time was
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unaffected by HPP. Regarding actin, the salty conditions should make this protein more stable, but the HPP induces their fragmentation, confirmed by the
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presence of some actin fragments on the gel. According to Kęska & Stadnik (2017), myofibrillar proteins, specifically myosin-2, are precursors of peptides and amino acids, which have a strong impact on the taste of dry-cured meat, while sarcoplasmic proteins had not incidence on taste-active components generation. On the other hand, two spots were identified as triosephosphate isomerase with a total RC of approximately 0.31. Triosephosphate isomerase is an enzyme, which increases the glycolytic metabolism and produces NADH and
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ACCEPTED MANUSCRIPT ATP, consequently it is strongly correlated with meat quality (Gagaoua, Monteils, Couvreur, & Picard, 2017; Kim & Dang, 2005; Schilling et al., 2017). In this proteomic study, spot number 1 identified as triosephosphate isomerase has an experimental mass similar to theoretical mass which would indicate that the protein is entire. On the contrary, the spot number 15 could suggest that
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there were protein aggregates even under reducing conditions due to the
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theoretical mass was lower than experimental mass as in other treatments
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occurred previously (Di Luccia, Gatta, Nicastro, Petrella, Lamacchia, & Picariello, 2015; López-Pedrouso et al., 2018b).
Levels of leucine and isoleucine affected by HPP treatment
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3.2.
As hydrolysis of proteins by enzyme action and chemical reaction
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release free amino acids, their quantification can be used as an indirect way to quantify proteolysis. Among free amino acids, leucine and isoleucine have a
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great importance because of their high concentration and are classified as lipophilic amino acids. Both are related to meat taste and flavor directly or
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indirectly via for being metabolites of Strecker aldehydes. As shown in the Figure 2, relative leucine content was higher than isoleucine in all treatments.
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The average relative content of leucine ranged from 102.62 to 130.50 mg/100 g dry cured ham (d.b.) whereas isoleucine varied from 62.18 to 77.43 mg/100 g dry cured ham (d.b.). Indeed, leucine was the most abundant free ami no acid in dry-cured ham according to Pérez-Santaescolástica et al. (2018a) and both are considered as predominant free amino acids like in the other types of hams such as Jinhua, Xuanwei, Parma and Bama (Zhang et al., 2018). Both amino acids have a specific tastes and high concentrations consequently, it can be supposed that both directly influence the development of aroma and taste. It is
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ACCEPTED MANUSCRIPT known that leucine together with glutamate, lysine, alanine and lactate are the highest contributor of taste in dry-cured hams (Zhang et al., 2018). Specifically, leucine and isoleucine were strongly associated with bitter taste of dry-cured meat products (Kęska & Stadnik, 2017). However, it has also indicated that the spatial arrangement of amino acids can be changed with the food process,
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resulting different tastes from the same amino acid (Solms, 1969). On the other
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hand, it has recently reported that high levels of leucine and isoleucine were
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correlated with proteolysis index contributing to a high adhesiveness in drycured hams (Pérez-Santaescolástica et al., 2018a).
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There was a significant difference (P<0.05) between control samples and samples after HPP treatment. As shown in the Figure 2, it can be observed that
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there was significant (P<0.05) differences between samples after HPP treatment at low temperatures (0 ºC and 20 ºC) and samples at 35 ºC. In
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particular, the relative leucine and isoleucine content increased under HPP at low temperatures (0 ºC and 20 ºC) in comparison with control samples unlike 35
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ºC with the lowest values for both amino acids. Therefore, these findings suggest that the effect of HPP treatment on relative isoleucine and leucine
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content is highly dependent on temperature. According to Guyon et al. (2016), the high-pressure process greatly contributes more to the hydrolysis of proteins into amino acids such as leucine and isoleucine. This suggests that all HPP treatments should increase both concentrations in dry-cured ham due to protein degradation. On the other hand, Kim, Kemp, & Samuelsson (2016) have described that leucine and isoleucine are metabolites involved in chemical reactions forming volatiles compounds as aldehydes. This means an indirect way of influence in taste and aroma as mentioned above. It is possible to
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ACCEPTED MANUSCRIPT hypothesize that the higher temperatures facilitate the Strecker reaction between leucine and isoleucine and α-carbonyl compounds and this fact could explain the lower content of both amino acids after HPP treatment at 35 ºC. 3.3.
Aldehyde volatile compounds affected by HPP treatment
Wide varieties of volatile compounds were detected using SPME
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followed by GC-MS (Pérez-Santaescolástica et al., 2018b). Volatile compounds
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are classified in chemical families, but aldehydes are a key group in dry-cured
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ham for their abundance and important contribution to taste and aroma. Their sensorial notes are fruity, toasted, oily, fatty and rancid, commonly associated
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with cured meats (Sánchez-Peña, Luna, García-González, & Aparicio, 2005). As shown in table 3, the HPP treatment significantly (P<0.001) affected the total
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aldehydes concentration resulting in the highest values in control samples (23430.18 AU×103/g of dry-cured ham). Again, the temperature fixed in each
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HPP treatment resulted in significant (P<0.05) differences in total aldehydes between samples at low temperature (average value of 12331.38 AU×103/g of
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dry-cured ham) and at 35 ºC (19687.56 AU×103 /g of dry-cured ham). Among the aldehydes determined with statistical significance, the most abundant were
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two branched aldehydes as 2-methyl-butanal and 3-methyl-butanal and four linear aldehydes as pentanal, hexanal, heptanal and nonanal. All linear aldehydes showed a significant reduction (P<0.05) due to the HPP effect which means that its contribution in overall aroma was decreased. The reductions ranged from 37.43% to 62.54% for nonanal and heptanal, respectively. The most likely explanation for these results may be the fact that aldehydes are considered highly reactive molecules especially α- and βunsaturated (alkenals, alkadienals, and hydroxyalkenals) which could react with
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ACCEPTED MANUSCRIPT proteins (Guyon et al., 2016). The results suggest that linear aldehydes could be reacting to a great extent in HPP conditions; therefore, their concentration decreases in comparison with samples untreated. In the case of the most abundant aldehyde, the hexanal concentration diminished more than 2.5 times by high-pressure effect. This result is consistent with Fuentes et al. (2014) that
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indicated a decrease of hexanal level in dry-cured ham slices treated with high
RI
pressure (600 MP). In addition, hexanal plays a key role in dry-cured products
SC
and at high concentration lead to the development of an unpleasant rancid odor (Lorenzo, Carballo, & Franco, 2013; Shahidi & Pegg; 1994; Carrapiso, Martín,
NU
Jurado, & García, 2010). Indeed, like other linear aldehydes, hexanal come from lipid oxidation by breakdown products of unsaturated lipids (Petričević,
MA
Radovčić, Lukić, Listeš, & Medić, 2018). Specifically, hexanal could be formed from the breakdown of linoleic, gamma-linolenic and arachidonic acids (Shahidi,
that HPP prevents
temperatures.
the
lipid oxidation, particularly at low
EP T
concluded
ED
2001), hence is widely used as oxidation level indicator. It may, therefore, be
On the other hand, branched aldehydes, which are 2-methyl-butanal and
AC C
3-methyl-butanal, are related to proteolysis and amino acids degradation (Toldrá, 1998). From a sensorial point of view, both aldehydes are potent odorants associated with the nutty, cheesy and salty sensory notes (SánchezPeña et al., 2005). Both aldehydes come from isoleucine and leucine, via Strecker degradation, which is considered as a step within Maillard reaction. This reaction involves oxidative deamination followed by decarboxylation resulting aldehydes with one carbon atom less the corresponding amino acid (Estévez et al., 2011; Resconi, Escudero, & Campo, 2013). The results show
18
ACCEPTED MANUSCRIPT that HPP treatment at 35 ºC had an impact on both aldehydes, because of their concentration significantly increased (P<0.05) with increments of 35.30% and 41.05% for 2-methyl-butanal and 3-methyl-butanal, respectively. However, these findings do not support previous research of Martínez-Onandi, RivasCañedo, Picon, & Nuñez (2016) who found that HPP treatment (600 MPa, 6
PT
min) produced a reduction in the amount of 2-methyl-butanal. Although these
RI
results must be interpreted with caution because they are influenced by water
SC
activity and intramuscular fat content, as noted above. Furthermore, it is necessary to highlight that it has been reported that the ability of 2-methyl-
NU
butanal to bind peptide extracts of dry-cured ham was too weak to be detected (Martínez-Arellano et al., 2016). It seems that Strecker aldehydes are less
MA
reactive than linear aldehydes and consequently more stable. Based on these reasons, it can be concluded that Strecker aldehydes can be associated with
4.
ED
greater proteolysis degree of dry-cured ham after HPP treatment. Conclusions
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High pressure conditions caused a significant impact on proteome of sliced dry-cured ham. The effect of HPP treatment was clearly demonstrated on
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myofibrillar proteins such as actin which was likely given rise to isoleucine and leucine. In addition, 2-methyl-butanal and 3-methyl-butanal coming from both Strecker metabolites increased their concentration due to HPP effect. Therefore, protein degradation by HPP effect can trigger a series of chain reactions depending on conditions (pressure and temperature) which cause a strong impact on volatile compound profile. A further research needs to be done to establish whether all changes in the volatile profile induced by high-pressure
19
ACCEPTED MANUSCRIPT process will be positive for the improvement of dry-cured ham sensorial attributes. Acknowledgements This research was supported by Grant RTA 2013-00030-CO3-03 from INIA
(Spain). Acknowledgements
to
INIA
for granting
Cristina Pérez
PT
Santaescolástica with a predoctoral scholarship. José M. Lorenzo is member of
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the MARCARNE network, funded by CYTED (ref. 116RT0503).
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ACCEPTED MANUSCRIPT CAPTION TO FIGURES
Figure 1. 2-DE gel images obtained from sliced dry-cured ham samples after a standard process (left) and a HPP treatment at 0ºC (right). Spots with significant
These spots were analyzed by MALDI-TOF/TOF MS.
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differences (P<0.05) by effect of pressure treatment are indicated and numbered.
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Figure 2. Relative leucine and isoleucine content in dry-cured ham under
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HPP treatment at different temperatures. Mean ± standard deviation expressed in mg FAA/ mg total FAA per 100 g dry matter. Differences in letter: a to c per
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each FAA represent significant differences (P<0.05) among treatments.
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ACCEPTED MANUSCRIPT Table 1. Identification of selected protein spots by MALDI-TOF/TOF Protein Descriptionb
Protein Accessionb
Mascot Score
1
Triosephosphate isomerase
TPI1
87
Sequence coverage (%) 29
2
Alpha actin 1
ACTC1
93
9
3
3
Alpha actin 1
ACTC1
108
17
5
4
Myosin-7
MYH7
131
7
13
5
Alpha actin 1
ACTC1
340
6
Alpha actin 1
ACTC1
166
7
Hemoglobin subunit beta
HBB
91
9
Alpha actin 1
ACTC1
378
10
Alpha actin 1
ACTC1
11
Alpha actin 1
ACTC1
12
Alpha actin 1
ACTC1
14
Alpha actin 1
15
Triosephosphate isomerase
17
Alpha actin
5
15
2
24
8
119
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30
11
154
9
3
90
23
8
ACTC1
172
18
4
TPI1
79
30
5
ACTC1
85
17
4
MA
NU
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16
ED
Sus scrofa (pig) was employed as taxonomy filter in Mascot search
Theoretical mass was provided by UniProt database
d
Experimental mass was estimated on 2-DE gels using molecular weight standards
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c
6
9
The spot numbers are shown in Figure 1
b
Number of matched peptides
24
AC C
a
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Spot no.a
26
Theo (Uni
ACCEPTED MANUSCRIPT Table 2. Spot volumes with significant differences by effect of a high-pressure treatment
in sliced dry-cured ham. Fold change (FC) and relative change (RC) of the selected spots Control
HPP
Mean ± SE
Mean ± SE 249.53 ± 45.25 337.46 ± 152.42 134.77 ± 38.14 43.11 ± 15.00 897.29 ± 162.25 191.86 ± 47.54
Spot no.
Protein (gene name)
1
Triosephosphate isomerase (TPI1)
2
Alpha actin 1 (ACTC1)
-
3
Alpha actin 1 (ACTC1)
-
4
Myosin-7 (MYH7)
-
5
Alpha actin 1 (ACTC1)
486.98 ± 80.85
6
Alpha actin 1 (ACTC1)
340.11 ± 52.40
7
Hemoglobin subunit beta (HBB)
109.81 ± 30.37
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124.48 ± 38.76
35.04 ± 10.43
FC
RC
+ 2.00
+ 0.25
+∞
+ 0.68
+∞
+ 0.27
+∞
+ 0.09
+ 1.84
+ 0.83
- 1.77
- 0.30
- 3.13
- 0.15
227.48 ± + 1.27 + 0.10 18.14 574.66 ± 9 Alpha actin 1 (ACTC1) 151.05 ± 20.66 + 3.80 + 0.85 147.24 473.80 ± 10 Alpha actin 1 (ACTC1) 132.30 ± 27.02 + 3.58 + 0.69 110.71 11 Alpha actin 1 (ACTC1) 99.09 ± 23.00 56.07 ± 14.76 - 1.77 -0.09 411.04 ± 12 Alpha actin 1 (ACTC1) +∞ + 0.83 143.85 496.40 ± 13 Uncharacterized protein +∞ + 1.00 129.27 175.91 ± 14 Alpha actin 1 (ACTC1) +∞ + 0.35 82.73 Triosephosphate isomerase 288.90 ± 15 137.34 ± 4.72 + 2.10 + 0.31 (TPI1) 25.18 142.94 ± 16 Uncharacterized protein +∞ + 0.29 46.31 269.59 ± 17 Alpha actin 1 (ACTC1) +∞ + 0.54 81.93 18 Uncharacterized protein 153.73 ± 16.46 -∞ - 0.31 Average ( ± SE, Standard error) volumes were assessed from 2-DE gels using the PDQuest software 179.16 ± 11.03
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Uncharacterized protein
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8
27
ACCEPTED MANUSCRIPT Table 3. Effect of HPP at different temperatures on main branched and linear aldehydes of drycured ham expressed as AU×103 /g of dry-cured ham (mean ± standard deviation) Treatment m/z
LRI
R
CO
HPP-0
HPP-20
57
671
ms, lri
863.96± 326.20
3-methyl butanal
58
659
ms, lri
1736.34±521.48
a
pentanal
57
728
ms, lri, s
1215.3± 547.46 b
hexanal
56
865
ms, lri
15717.34 ±4194.15 c
heptanal
70
974
ms, lri, s
1025.45 ±324.52 c
384.07± 105.33 a
450.05± 163
nonanal
57
1148
ms, lri, s
553.39± 170. 73 b
346.82 ±74.69 a
338.68± 91
RI
23430.18±4949.22
SC
Total aldheydes
PT
2-methyl butanal
a
a-c
c
879.99± 427.70
a
1547.91±340.04
a
1878.31±791
541.18 ± 203.79 a
594.13± 192
6040.25±1821.88
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ED
EP T AC C
a
11065.57± 2337.37 a
Mean values in the same row (corresponding to the same compound) not followed by a common letter differ significantly (P<0.05). m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30m×0.25mm id, 1.4μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo, Montes, Purriños, & Franco, 2012; Lorenzo, Bedia, & Bañon, 2013b; Lorenzo, 2014; Lorenzo & Domínguez, 2014; Lorenzo & Carballo, 2015; Pateiro, Franco, Carril, & Lorenzo, 2015; Pérez-Santaescolástica et al., 2018a; Pérez-Santaescolástica et al., 2018b; Purriños, Franco, Bermudez, Carballo, & Lorenzo, 2011a; Purriños, Franco, Bermúdez, Temperan, Carballo, & Lorenzo, 2011; Purriños, Franco, Carballo, & Lorenzo, 2012, Purriños, Carballo, & Lorenzo, 2013); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Treatments: CO= control (without treatment); HPP-0=High pressure treatment at 0 ºC; HPP20=High pressure treatment at 20 ºC; HPP-35=High pressure treatment at 35 ºC.
28
1168.02±483
7535.70 ±221
13597.19± 30
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Proteolysis under pressure conditions and its possible sensory consequences were assessed Proteomic analysis showed that high-pressure conditions led to a higher level of proteolysis Actin (ACTC1) is a candidate biomarker to control high pressure processing High-pressure conditions at low temperatures produced an increase of Strecker metabolites Branched and linear aldehydes affected in different ways by high pressure processing
29
Figure 1
Figure 2