Protein oxidation affects proteolysis in a meat model system

Meat Science 106 (2015) 78–84 Contents lists available at ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci Protein oxidation affects proteolysis in a meat model system Alberto Berardo a, Erik Claeys a, Els Vossen a, Frédéric Leroy b, Stefaan De Smet a,⁎ a b Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Melle, Belgium Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium a r t i c l e i n f o Article history: Received 4 November 2014 Received in revised form 2 March 2015 Accepted 3 April 2015 Available online 11 April 2015 Keywords: Protein oxidation Dry fermented sausage Proteolysis Carbonyls Thiols Protein electrophoresis Meat model a b s t r a c t The effect of hydrogen peroxide-induced protein oxidation and pH (4.8 and 5.2) on meat proteolysis was investigated in a meat model system for dry fermented sausages. In oxidised samples, increased protein carbonyl contents and decreased thiol concentrations were found. The initial concentration of protein carbonyls was significantly lower in oxidised samples at pH 4.8 than in ones at pH 5.2, but after ten days comparable levels were reached. The inhibition of proteolysis by the addition of a protease inhibitor cocktail did not influence protein oxidation. Yet, proteolysis was negatively affected by low pH values as well as by oxidation, resulting in a reduced release of amino acids during ripening. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Dry fermented sausages are widely consumed, often traditionally important cured meat products, which are characterized by a ripening period in which the desired texture and flavour develop (Leroy, Geyzen, Janssens, De Vuyst, & Scholliers, 2013). Ripening consists of a first fermentation step, usually lasting two to five days, followed by a drying phase that can last several weeks. Although many different types of dry fermented sausages exist in Europe, based on different recipes and processing methods, they can roughly be classified in two main groups, i.e. Northern-type and Southern-type sausages (Hui et al., 2004). The fermentation phase differs markedly between European Northern-type and Southern-type sausages (Ravyts, De Vuyst, & Leroy, 2012). In the former, the pH drops rapidly below 5.0 due to the activity of lactic acid bacteria which convert the added sugars into lactic acid. In the latter, acidification is more moderate due to the lower amounts of fermentable sugars and the lower fermentation temperatures applied. As a result, the pH usually remains above 5.0, which may even increase during maturation due to the metabolic activity of moulds. The pH drop obtained during fermentation provokes protein denaturation and enhances the activity of some important proteolytic enzymes (Astiasaran, Villanueva, & Bello, 1990; Molly et al., 1997). Endogenous exopeptidases and endopeptidases are the main enzymes responsible ⁎ Corresponding author at: Proefhoevestraat 10, 9090 Melle, Belgium. Tel.: +32 9 264 90 01; fax: +32 9 264 90 99. E-mail address: [email protected] (S. De Smet). http://dx.doi.org/10.1016/j.meatsci.2015.04.002 0309-1740/© 2015 Elsevier Ltd. All rights reserved. for proteolysis in dry fermented sausages, while bacterial proteolytic enzymes seem to play a less pronounced role (Hierro, de la Hoz, & Ordonez, 1999; Toldrá, Aristoy, & Flores, 2000). Several authors have indicated cathepsins as the most active endopeptidases involved in proteolysis in cured meat products (Demeyer, Claeys, Ötles, Caron, & Verplaetse, 1992; Molly et al., 1997; Toldrá, Rico, & Flores, 1993; Verplaetse, Demeyer, Gerard, & Buys, 1992). Besides the impact of acidification and proteolysis, fermented sausage production is affected by oxidation processes. Lipid oxidation might impair sensory quality since high levels of malondialdehyde correlate with rancid taste (Wood et al., 2008). The oxidative stability of dry fermented sausages is determined by the balance between prooxidant and antioxidant factors. Additives, like sodium chloride, exert pro-oxidant effects (Ruiz, 2007), whereas sodium ascorbate and nitrite might have either pro-oxidant or antioxidant activities. Myoglobin, abundantly present in meat, also exerts pro-oxidant effects (Carlsen & Skibsted, 2004). Moreover, some lactic acid bacteria used in fermented products produce hydrogen peroxide which is a strong oxidizer (O'Toole & Yuan, 2006). In contrast, meat-associated catalase-positive cocci, which are added as starter cultures or which are naturally present in the sausage batter, may neutralise peroxides (Ravyts et al., 2012). To a certain extent, protein breakdown taking place during the ripening period may improve the oxidative stability since small peptides present higher antioxidant properties than intact proteins (Freitas et al., 2013). Whereas lipid oxidation has been extensively studied during the last decades, the impact of protein oxidation on the quality of dry fermented sausages has still to be elucidated. In meat and meat products, oxidation implies modifications at the protein level resulting in A. Berardo et al. / Meat Science 106 (2015) 78–84 protein carbonylation, breakdown, and aggregation (Lund, Heinonen, Baron, & Estevez, 2011). Those modifications involve changes in protein solubility and functionality, potentially leading to decreased digestibility, disturbance of gelation, emulsification, and water holding capacity, as well as having a potential impact on flavour due to the formation of certain carbonyls and Schiff bases (Lund et al., 2011). Contradictory effects of protein oxidation on proteolysis were reported. On the one hand, the increased hydrophobicity due to oxidation favours the recognition and the subsequent degradation of oxidised proteins by proteases (Davies, 2001; Pacifici, Kono, & Davies, 1993). This occurs in mild oxidative conditions, in which the proteolytic susceptibility of myosin heavy chain increases by the action of oxygen radicals (Xue, Huang, Huang, & Zhou, 2012). On the other hand, intense oxidative conditions generate cross-links between proteins so that the resulting aggregates are poor substrates for proteases (Pacifici et al., 1993). Moreover, the direct oxidation of proteolytic enzymes impairs their activity (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004), with cysteine proteases being highly susceptible (Lametsch, Lonergan, & Huff-Lonergan, 2008). To the best of our knowledge, the influence of oxidation on proteolysis in dry fermented sausages has not been studied before. Sausage preparation processes, like meat grinding and the consequent exposure to oxygen as well as the addition of sodium chloride, might trigger protein oxidation and affect proteolysis. Yet, the understanding of how physico-chemical changes occurring in dry fermented sausages, including pH drop and proteolysis, interact with protein oxidation may enable strategies to control its negative effects. Therefore, the aim of this study was to investigate protein oxidation in a meat model system for dry fermented sausages and to assess its effect on proteolysis, and conversely the effects of pH and proteolysis on protein oxidation. 2. Materials and methods 79 2.2. Sarcoplasmic protein solubility Sarcoplasmic protein solubility was measured in a low ionic strength solution (150 mM NaCl), as described previously (Claeys, De Vos, & De Smet, 2002), and was expressed in mg soluble protein/g of meat. Three grams of meat was homogenised in 30 mL of 150 mM NaCl and 0.01 mM iodo-acetic acid. The samples were centrifuged and filtered. The protein concentration of the supernatant, assumed to contain the soluble sarcoplasmic protein fraction, was determined using the biuret method. 2.3. Protein carbonyl content The protein carbonyl content was determined by derivatization with DNPH (2,4-dinitrophenyl hydrazine) as described by Levine, Williams, Stadtman, and Shacter (1994) with some modifications. Three grams of meat with 30 mL of phosphate buffer (20 mM, pH 6.5 containing 0.6 M NaCl) was homogenised and four aliquots of 0.2 mL were treated with 1 mL ice-cold TCA (10%) to precipitate the proteins. After centrifugation the supernatant was discarded and two aliquots were treated with 0.5 mL of 10 mM DNPH dissolved in 2.0 M HCl and two aliquots were treated with 0.5 mL of 2.0 M HCl (blank). After 1 h of reaction, 0.5 mL of ice cold 20% TCA was added. The samples were then centrifuged and supernatant was discarded. Excess DNPH was removed by washing three times with 1 mL of ethanol:ethylacetate (1:1, v/v). The pellets were dissolved in 1 mL of 6.0 M guanidine hydrochloride in 20 mM phosphate buffer (pH 6.5). The carbonyl concentration (nmol/mg protein) was calculated from the absorbance at 280 nm and 370 nm of the samples using the following equation (Levine et al., 1994): Chydrazone A370 6 ¼  10 Cprotein εhydrazone;370  ðA280 −A370  0:43Þ 2.1. Dry fermented sausage preparation The experimental set-up was a 2 × 2 × 2 full factorial design with two pH values installed, induction or not of oxidation, and the addition or not of a protease inhibitor cocktail. The experimental set-up was repeated twice at two different days and each time a batch of sausage models was prepared. Lean pork from shoulder muscles, which contained 4.5% fat upon analysis by the ISO 1444-1973 method, was ground through a 3-mm plate and mixed with the curing agents sodium chloride (2.5%, m/m), sodium nitrite (0.015%, m/m), and sodium ascorbate (0.05%, m/m). The batch was subsequently divided into subbatches for the different treatments. The batch was first divided into two equal parts and the pH was set at 5.20 ± 0.10 in the first batch and at 4.80 ± 0.10 in the second batch, in both cases by adding lactic acid. The pH remained in the ranges of 5.20 ± 0.10 and 4.80 ± 0.10 during ripening. The batches were further split into sub-batches for the oxidation treatment, control (C) versus oxidised (O), and for the addition or not of a protease inhibitor cocktail. Oxidation was induced by adding hydrogen peroxide before the stuffing (12 μL/g meat of a 6% hydrogen peroxide solution). Based on a preliminary test, the concentration of hydrogen peroxide was chosen to ensure an increase of about 4 nmol carbonyl/mg of protein. The cocktail of protease inhibitors was made by mixing pepstatin A (60 μM) and E-64 (1.4 mM). Pepstatin A was dissolved in ethanol and subsequently mixed with E-64 which had been previously dissolved in water (1:1, v/v). The protease inhibitor cocktail was added at 0.02 mL per gram of meat. A solution containing ethanol and water (1:1, v/v) replaced the protease inhibitor cocktail in batches where it was not added. The meat mixtures were stuffed into falcon tubes of 50 mL. Samples were taken after 0, 5 and 10 days of incubation at 22 °C. The length was chosen to allow sufficient proteolysis in order to mimic the first days of ripening. where εhydrazone,370 is 22,000 M−1 cm−1 and the carbonyl concentrations obtained from the blanks were subtracted from the corresponding treated sample. 2.4. Thiol concentration The thiol concentration was determined after derivatization by Ellman's reagent, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) adopted from Jongberg, Torngren, Gunvig, Skibsted, and Lund (2013). Two grams of frozen meat was homogenised in 50 mL of 5% SDS in TRIS buffer (pH 8.0) and incubated for 30 min in a water bath at 80 °C. The homogenate was centrifuged to eliminate insoluble particles. Two millilitres of 0.1 M TRIS buffer (pH 8) and 0.5 mL of 10 mM DTNB dissolved in TRIS buffer were added to 0.5 mL of supernatant. For each sample, a blank was included containing 0.5 mL of supernatant and 2.5 mL of TRIS buffer. A solution containing 0.5 mL of 5.0% SDS in TRIS buffer, 0.5 mL of 10 mM DTNB, and 2.0 mL of TRIS buffer was used as a reagent blank. All mixtures were protected against light and allowed to react for exactly 30 min. The absorbance was measured spectrophotometrically at 412 nm and the thiol concentration was calculated using the formula of Lambert–Beer (ε412 = 14,000 M− 1 cm−1) and expressed in nmol thiols/mg protein. The protein concentration of the blank was determined spectrophotometrically at 280 nm using a BSA standard curve. 2.5. Electrophoresis About 2.5 g of meat was homogenised in 50 mL of 0.01 M imidazole buffer (pH 7.0), containing 2% SDS, and kept at 95 °C for 10 min to dissolve proteins. After cooling to room temperature, protein solutions were centrifuged and filtered. After determination of protein concentration (Kjeldahl), solutions were diluted to obtain 3 mg of crude protein 80 A. Berardo et al. / Meat Science 106 (2015) 78–84 per mL and were divided into two equal aliquots. In one aliquot, 2% of 2mercaptoethanol was added to investigate proteolysis. In the aliquot without 2-mercaptoethanol, disulphide bonds remained intact, which was used to follow protein oxidation. Protein solutions were frozen and preserved at −18 °C until electrophoresis. Gels were prepared according to Greaser, Yates, Krywicki, and Roelke (1983). The separating gel contained 8.0% of total (T) acrylamide plus N,N′-methylene-bis-acrylamide, with a crosslinking of 0.5%, containing 0.1% of SDS at pH 8.8. The stacking gel contained 3.0% of total (T) acrylamide plus N,N′-methylene-bisacrylamide, with a cross-linking of 4.76%, containing 0.1% of SDS at pH 6.8. Per well, 10 μL (30 μg protein) was loaded. The gels were mounted in a vertical slab gel apparatus (Hoefer Scientific Instruments, SE 600 series), using constant power supply ECPS 2000/300 (Pharmacia, Sweden) for electrophoresis. Electrophoresis was performed at constant 20 mA and continued until the bromo-phenol blue front reached the bottom of the gel. The staining of the proteins was carried out by immersing the gels overnight in a solution containing 0.1% of Coomassie brilliant blue R250 dissolved in a 20% methanol and 2% phosphoric acid (v/v) solution at room temperature. After destaining in fresh fixing solution, the gels were scanned using a Bio-Rad computing densitometer model CDS-100, and the peak intensity was recorded. 2.6. Free and peptide-bound α-NH2-N Free α-NH2-N and peptide-bound α-NH2-N were determined according to the method described by Oddy (1974). Briefly, 1 g of frozen meat was homogenised in 20 mL of 0.6 M perchloric acid (PCA). The homogenate was centrifuged and filtered. For the total α-NH2-N, 2 mL of PCA extract was mixed with 5 mL of HCl (8.4 M) and incubated at 100 °C for 24 h for hydrolysis. After hydrolysis, solutions were neutralised and diluted to 50 mL. One millilitre of a buffer solution, containing 0.5% of ninhydrin (pH 5.8) and 100 μl of 50% ascorbic acid solution, was added to either 0.2 mL of the PCA extract + 0.8 mL of water (for free α-NH2-N) or 1 mL of the neutralised hydrolysed extract (for total α-NH2-N). A solution containing 1 mL of distilled water, 1 mL of 0.5% ninhydrin buffer solution (pH 5.8) and 100 μl of 50% ascorbic acid solution was used as a blank. All mixtures were left in boiling water for 20 min and, after cooling to room temperature, 5 mL of a 60% ethanol solution was added. The absorbance was measured spectrophotometrically at 570 nm. Results were calculated as mg α-NH2-N/100 g of dry matter (ISO 1442-1973), by comparing with solutions of L-leucine of known concentration, used as a standard and treated the same way. The peptide-bound α-NH2-N contents were calculated by subtracting the free α-NH2-N from the total α-NH2-N. 2.7. Cathepsin B + L and cathepsin D activities Cathepsin B + L activity was determined fluorometrically (Fluoroscan Ascent FL, Thermo Scientific) with the common substrate N-CBZ-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin at 37 °C as previously described (Uytterhaegen, Claeys, & Demeyer, 1992) with some modifications. Briefly, 3 g of meat was homogenised in 25 mL cold (2 °C) 0.1 M citrate buffer pH 5.0, containing 0.2% Triton X100, and centrifuged (4000 g; 15 min). Twenty-five microlitres of the filtered supernatant was mixed with 225 μl of 30 μM N-CBZ-L-phenylalanyl-Larginine-7-amido-4-methylcoumarin in 0.1 M phosphate buffer (pH 6) containing 0.07% Brij 35 and 1.29 mg/mL cysteine-HCl. The excitation wavelength was set at 335 nm and emission was recorded at 460 nm. Data were collected every minute for 20 min. The slope of the resulting line, describing the relationship between the time and the fluorescence readings, is a measure for the activity. Results are expressed as pmol 7-amino-4-methylcoumarin (NHMec) released per min and per g of meat. Cathepsin D was extracted and its activity was assayed as previously described by Claeys, De Smet, Demeyer, Geers, and Buys (2001). Results are expressed as μg haemoglobin hydrolysed per min and per g of meat. 2.8. Statistical analysis Data were analysed using the general linear model ANOVA procedure with the fixed effects of treatment (C and O), protease inhibitors (addition or not), and pH (4.8 and 5.2). Free and peptide-bound αNH2-N data and cathepsin B + L and cathepsin D activities data were analysed using the general linear model ANOVA procedure with the fixed effects of treatment (C and O) and pH (4.8 and 5.2). In this case data from samples containing inhibitors were not considered. The 2way interaction terms were only included in the model when significant (P b 0.05). The data at days 0, 5, and 10 were analysed separately. Tukeyadjusted post hoc tests were performed for all pairwise comparisons. Pvalues b 0.05 were considered significant. All the statistical analyses were carried out by SAS Enterprise guide 6. 3. Results 3.1. Sarcoplasmic protein solubility The solubility of sarcoplasmic proteins was assessed in a low ionic strength solution. The main effects of pH, oxidation treatment and use of inhibitors on protein solubility were all significant, whereas the interaction terms were not (Table 1). The sarcoplasmic protein solubility was almost 20% lower in sausages at pH 4.8 than in ones at pH 5.2. The oxidation treatment resulted in an approximately 10% lower protein solubility in comparison with the control treatment. In addition, samples treated with protease inhibitors displayed lower protein solubility. However, whereas the pH and oxidation treatments immediately exerted an effect on protein solubility, the addition of protease inhibitors revealed its effect only from day 5 on of ripening. 3.2. Protein oxidation Protein oxidation was quantified by estimation of the protein carbonyl content (Fig. 1) and thiol concentrations (Fig. 2), as well as by protein electrophoresis (Fig. 3). The pH and oxidation treatments significantly affected carbonyl and thiol concentrations, whereas the addition of the protease inhibitor cocktail did not significantly influence protein oxidation. Table 1 Effect of pH, induced oxidation and protease inhibitors on sarcoplasmic protein solubility (mg soluble protein/g meat) during ripening in a sausage model. pH Day 0 Day 5 Day 10 a Oxidation treatment (O) Protease inhibitors (PI) 4.8 5.2 Control Oxidation No Yes 21.3 18.5 19.1 26.4 22.7 22.9 25.0 21.8 22.5 22.7 19.4 19.6 23.9 22.9 23.9 23.8 18.3 18.1 Root mean square error. RMSEa 1.291 0.764 1.652 P-value pH O PI b0.001 b0.001 b0.001 0.003 b0.001 0.004 0.785 b0.001 b0.001 A. Berardo et al. / Meat Science 106 (2015) 78–84 81 Fig. 1. Effect of treatment on carbonyls in sausage models during ripening at pH 4.8 (A) and 5.2 (B). In the control treatment, carbonyl contents were restricted to values between 2.0 and 3.5 nm DNPH/mg protein, while the oxidation treatment always had significantly higher values, except for the sausages at day 0 and pH 4.8 (Fig. 1), reflected in a significant pH × oxidation interaction term for that particular sampling moment. The control treatment displayed a slight but not significant increase in carbonyl content during ripening whereas the oxidation treatment resulted in a significant increase in carbonyl contents at day 5 of ripening compared to day 0. The initial thiol concentration (day 0) was significantly lower in the oxidation treatment than in the control treatment (Fig. 2). Later on, after five days of ripening, all treatments displayed a significant loss of thiol groups. At pH 5.2, samples of the oxidation and control treatments converged to similar values, both at days 5 and 10. For pH 4.8, however, samples from the oxidation treatment displayed considerably lower amounts of thiol groups after 5 and 10 days, than samples from the control treatment (significant pH × oxidation interaction term). Finally, the electrophoresis patterns of proteins not treated with 2-mercaptoethanol displayed differences with respect to the oxidation treatment (Fig. 3). The electrophoresis patterns were comparable for both pH values; therefore, only the ones obtained at pH 5.2 are shown. At day 0, oxidised samples (D band) showed a thicker band at the top of the gel (protein aggregates) in comparison with samples from the non-oxidised sausages. The peak intensities of protein aggregates and myosin heavy chain in control and oxidised samples at day 0 were measured and are shown in Fig. 4. Protein aggregates were significantly higher in oxidised samples than in the control samples. On the contrary, the latter presented a higher amount of myosin heavy chain. During ripening, proteins displayed a decreased intensity of all bands and the appearance of a broader band at the top of the gel. When oxidised proteins were treated with 2-mercaptoethanol (G, H, I bands), a disulphide bond breaker, the intensity of the upper band (aggregates) clearly decreased and the lower bands were recovered. 3.3. Proteolysis Sausage models prepared with the addition of inhibitors did not show any significant increase of free α-NH2-N and peptide-bound α-NH2-N Fig. 2. Effect of treatment on thiol concentration in sausage models during ripening at pH 4.8 (A) and 5.2 (B). 82 A. Berardo et al. / Meat Science 106 (2015) 78–84 A B C D E F G H I Protein aggregates Table 2 Effect of induction of oxidation and pH on free α-NH2-N (mg α-NH2-N/100 g dry matter) during ripening in a sausage model (samples in absence of inhibitors). Oxidation treatment (O) Myosin Heavy Chain Day 0 Day 5 Day 10 a Actin Fig. 3. Electrophoresis patterns of protein solutions. A, B and C: control treatments at days 0, 5, and 10, respectively (not treated with 2-mercaptoethanol). D, E and F: oxidation treatments at days 0, 5, and 10, respectively (not treated with 2-mercaptoethanol). G, H and I: oxidation treatments at days 0, 5, and 10, respectively (treated with 2mercaptoethanol). All depicted samples originated from the experiment at pH 5.2 without added inhibitors. during ripening (data not shown). On the contrary, free α-NH2-N and peptide-bound α-NH2-N significantly increased in samples in the absence of inhibitors during ripening, both in the control and oxidation treatments (Table 2). At day 0, both treatments had similar values for free α-NH2-N, whereas at days 5 and 10 the oxidation treatment had significantly lower values compared to the control treatment. In contrast, peptide-bound αNH2-N did not show significant differences between the two treatments (data not shown). The samples at pH 4.8 had significantly lower values of free α-NH2-N than the ones at pH 5.2, at all time points. The electrophoresis patterns of proteins treated with 2mercaptoethanol are reported in Fig. 5. The electrophoresis patterns were comparable for both pH values; therefore, only the ones obtained at pH 5.2 are shown. In fermented sausage models with active endogenous proteases, the formation of new bands during ripening was found below the position of myosin heavy chain. When using protease inhibitors, no changes in the patterns were found during ripening. Fig. 4. Peak intensities of protein aggregates and myosin heavy chain in control and oxidised samples at day 0. pH Control Oxidation 4.8 5.2 269 390 520 269 333 381 250 337 403 288 386 497 RMSEa P-value O pH 38 24 51 0.989 0.027 0.009 0.266 0.033 0.048 Root mean square error. Results of cathepsin D activity are reported in Fig. 6. At day 0 control and oxidised samples at pH 5.2 presented, although not significantly, lower cathepsin D activities than the counterparts at pH 4.8. At days 5 and 10 of ripening significantly lower cathepsin D activity was recorded in oxidised samples at pH 5.2 compared to control samples at both pH values, illustrating the significant pH × treatment interaction term at these sampling days. Cathepsin B + L activity is shown in Fig. 7. The oxidised samples showed a strongly reduced cathepsin B + L activity at day 0 and almost no activity at days 5 and 10. At days 0 and 10, control samples at pH 4.8 reported a significantly lower cathepsin B + L activity than control samples at pH 5.2. 4. Discussion Proteolysis is an important process characteristic of meat fermentation, in particular during the first two weeks of ripening (Beriain, Lizaso, & Chasco, 2000; Casaburi et al., 2008). The electrophoresis patterns obtained in the present study underline this aspect, matching earlier observations in dry fermented pork sausages (Defernando & Fox, 1991). The occurrence of proteolysis was evident from the increase in both free α-NH2-N and peptide-bound α-NH2-N, reflecting the activity of amino peptidases and endopeptidases, respectively. Increases of free and peptide-bound α-NH2-N were also found by Dierick, Vandekerckhove, and Demeyer (1974) during the first nine days of dry fermented sausage ripening. Little is known about the potential effects of proteolysis on protein oxidation in fermented meats. Small peptides may present antioxidant activity, which depends on their composition, structure and hydrophobicity (Sarmadi & Ismail, 2010). The antioxidant activity is exerted in different pathways: inactivation of reactive oxygen species (ROS), scavenging of free radicals, and chelating of transition metals (Broncano, Timón, Parra, Andrés, & Petrón, 2011; Sarmadi & Ismail, 2010). The properties of peptides are in turn determined by protease specificity. In the present study, the addition of a protease inhibitor cocktail lowered protein solubility as a measure of proteolysis, but this did not result in differences in oxidative stability, even though proteolysis was successfully blocked at the level of both endopeptidases and exopeptidases. We therefore hypothesize that the active endogenous proteases either generated peptides with no antioxidant activity or that the antioxidant peptide concentration may not have been high enough to exert detectable effects. On the other hand, radicals may have easier access to susceptible amino acids in meat peptides so that an effect of proteolysis on the susceptibility to oxidation cannot be excluded. However, this should result in an increased content of oxidation products, which was not observed. Additional experiments and analyses are required to independently check and quantify the antioxidant activity of muscle peptides and their susceptibility to oxidation in this type of product. Vice versa, another objective of the present study was to elucidate if protein oxidation can affect proteolysis during meat fermentation. In meat, protein oxidation can be triggered by hydrogen peroxide, iron, or myoglobin. The reaction between iron and hydrogen peroxide forms highly reactive hydroxyl radicals. Yet, myoglobin can also react with hydrogen peroxide generating hypervalent myoglobin species 83 A. Berardo et al. / Meat Science 106 (2015) 78–84 A B C D E F Protein aggregates Myosin Heavy Chain Actin Fig. 7. Cathepsin B + L activity. Fig. 5. Electrophoresis patterns of protein solutions treated with 2-mercaptoethanol. A, B, and C: samples treated with inhibitors at days 0, 5, and 10, respectively. D, E, and F: samples not treated with inhibitors at days 0, 5, and 10, respectively. All depicted samples originated from the experiment at pH 5.2 without oxidation treatment. (Lund et al., 2011). Metal-catalysed oxidation was reported to provoke increases of carbonyls, although formation of cross-links can also occur (Lund et al., 2011). Similarly, myoglobin-oxidising systems generate protein carbonyls and cross-links (Estevez, 2011; Lund et al., 2011). Moreover, nitrite and hydrogen peroxide form peroxynitrite which is a strong oxidant. Consistently, the peroxide-induced samples in the present study indeed showed higher carbonyl contents and a loss of thiol groups, which was also accompanied by decreased protein solubility. To the best of our knowledge, this is the first study indicating that the induction of protein oxidation reduces proteolysis in a model for fermented sausages. It has been suggested that a certain degree of oxidation may favour proteolysis, since increased protein hydrophobicity might facilitate the degradation by proteases (Davies, 2001; Pacifici et al., 1993) and carbonylated proteins seem to be more susceptible to proteasome-driven proteolysis (Nyström, 2005). However, the present study indicates the opposite effect. Indeed, the induced oxidation decreased the activity of proteases. In particular, the activity of cathepsin B + L was completely stopped. These results show that these cysteine cathepsins are highly susceptible to oxidation. Similarly, Lametsch et al. Fig. 6. Cathepsin D activity. (2008) reported that oxidation forms a disulphide bond within the μcalpain active site, which inhibits the activity of that cysteine protease. On the contrary, the activity of cathepsin D was only slightly decreased suggesting a high resistance of this aspartic protease towards oxidation. Moreover, protein oxidation generated protein aggregates through the formation of disulphide bonds, which were clearly visible in the upper part of the electrophoresis patterns of the oxidised samples. Similarly, Park, Xiong, Alderton, and Ooizumi (2006) observed formation of protein aggregates on top of the electrophoresis patterns of oxidised myofibrillar proteins. In the same study, a decreased intensity of myosin heavy chain following oxidation was reported, which is confirmed in the present study. Lund, Lametsch, Hviid, Jensen, and Skibsted (2007) identified the high molecular weight on top of the gel as cross-linked myosin heavy chain. Consequently, protein aggregates, which have low proteolytic susceptibility, might also undergo limited proteolysis. Indeed, severe oxidation favours the formation of protein aggregates impeding the recognition of proteins by proteolytic enzymes (Pacifici et al., 1993). In particular, the formation of covalent bonds, e.g., disulphide and dityrosine bonds, normally occurring during oxidation, induces proteins to cluster and precipitate (Chao, Ma, & Stadtman, 1997; Davies & Delsignore, 1987; Meucci, Mordente, & Martorana, 1991). As a result of diminished proteolysis, the release of amino acids during ripening decreased in the oxidised samples. This may be of importance as amino acids can be converted by meat-associated catalasepositive cocci into aroma compounds (Ravyts et al., 2012), or even serve as alternative energy substrates for both the lactic acid bacteria and catalase-positive cocci (Janssens et al., 2014; Rimaux et al., 2010). Yet, amino acids can also be converted into aldehydes through the Strecker degradation. Villaverde, Ventanas, and Estévez (2014) hypothesized that protein carbonyls might also play a role in the formation of Strecker aldehydes. As proteolysis is also affected by the overall fermentation and thus acidification process, the effect of pH also needs consideration. Therefore, two different pH levels were chemically imposed (pH 5.2 and 4.8) on the sausage batter, corresponding to typical pH values reached after fermentation in Southern-type and Northern-type fermented sausages, respectively. At pH 4.8, lower sarcoplasmic protein solubility was found, reflecting a higher protein denaturation. Indeed, a decrease of protein solubility usually occurs in dry fermented sausages during the fermentation step (Astiasaran et al., 1990; Klement, Cassens, & Fennema, 1974). Therefore, it may be assumed that more residues were exposed to oxidation at low pH, leading to, among others, a higher protein carbonylation. Surprisingly, protein carbonylation in the oxidised samples was retarded during ripening at pH 4.8. Possibly, the lactic acid used to drop the pH might have played a role due to its potential antioxidant activity (Groussard et al., 2000). The higher amount of lactic acid needed for the samples at pH 4.8 might explain the lower carbonylation at the start of the fermentation in these samples. Despite this 84 A. Berardo et al. / Meat Science 106 (2015) 78–84 delayed protein oxidation, proteolysis was indifferently reduced in samples at pH 5.2 and pH 4.8, following induced oxidation. A pH of 5.2 also implied a higher release of amino acids during ripening. Verplaetse et al. (1992) reported similar results in sausages were the pH was dropped by using glucono-delta-lactone since the optimum activity of the major aminopeptidases in meat is at neutral pH (Toldrá et al., 1992). Moreover, the cathepsin B + L activity was higher at pH 5.2. Indeed, the optimum pH values for cathepsins B and L are 5.5–6.5 and approximately 5, respectively (Geesink & Veiseth, 2009). Since cathepsin B can also act as an exopeptidase (Geesink & Veiseth, 2009), it might have increased the amount of free amino acids in those samples. 5. Conclusions In summary, oxidation of proteins may reduce protein breakdown during dry fermented sausage ripening. Besides the negative effects of protein oxidation in terms of reduced oxidative stability, the lower amounts of free amino acids may negatively affect the sensorial qualities, as they are important for flavour development upon conversion by the meat microbiota and as a source of Strecker aldehydes. Further research is required to evaluate and quantify the antioxidant properties of the peptides generated by proteolysis and their susceptibility to oxidation, as well as the actual impact of the decreased proteolysis due to oxidation on quality deterioration. Acknowledgements This work was financially supported by the Fund for Scientific Research — Flanders (FWO-Vlaanderen) Project G.0327.12. References Astiasaran, I., Villanueva, R., & Bello, J. (1990). Analysis of proteolysis and protein insolubility during the manufacture of some varieties of dry sausage. Meat Science, 28(2), 111–117. Beriain, M. J., Lizaso, G., & Chasco, J. (2000). Free amino acids and proteolysis involved in ‘salchichon’ processing. Food Control, 11(1), 41–47. Broncano, J. M., Timón, M. L., Parra, V., Andrés, A. I., & Petrón, M. J. (2011). Use of proteases to improve oxidative stability of fermented sausages by increasing low molecular weight compounds with antioxidant activity. Food Research International, 44(9), 2655–2659. Carlsen, C. U., & Skibsted, L. H. (2004). Myoglobin species with enhanced prooxidative activity is formed during mild proteolysis by pepsin. Journal of Agricultural and Food Chemistry, 52(6), 1675–1681. Casaburi, A., Di Monaco, R., Cavella, S., Toldrá, F., Ercolini, D., & Villani, F. (2008). Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits. Food Microbiology, 25(2), 335–347. Chao, C. C., Ma, Y. S., & Stadtman, E. R. (1997). Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems. Proceedings of the National Academy of Sciences of the United States of America, 94(7), 2969–2974. Claeys, E., De Smet, S., Demeyer, D., Geers, R., & Buys, N. (2001). Effect of rate of pH decline on muscle enzyme activities in two pig lines. Meat Science, 57(3), 257–263. Claeys, E., De Vos, E., & De Smet, S. (2002). Protein solubility of longissimus from stress sensitive and stress resistant pigs. Proceedings 48th International Congress of Meat Science and Technology, Rome, Italy (pp. 616–617). Davies, K. J. A. (2001). Degradation of oxidized proteins by the 20S proteasome. Biochimie, 83(3–4), 301–310. Davies, K. J., & Delsignore, M. E. (1987). Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure. Journal of Biological Chemistry, 262(20), 9908–9913. Defernando, G. D. G., & Fox, P. F. (1991). Study of proteolysis during the processing of a dry fermented pork sausage. Meat Science, 30(4), 367–383. Demeyer, D., Claeys, E., Ötles, S., Caron, L., & Verplaetse, A. (1992). Effect of meat species on proteolysis during dry sausage fermentation. Proceedings of the 38th International Conference on Meat Science and Technology, Clermont-Ferrand, France (pp. 775–778). Dierick, N., Vandekerckhove, P., & Demeyer, D. (1974). Changes in non-protein nitrogencompounds during dry sausage ripening. Journal of Food Science, 39(2), 301–304. Estevez, M. (2011). Protein carbonyls in meat systems: A review. Meat Science, 89(3), 259–279. Freitas, A. C., Andrade, J. C., Silva, F. M., Rocha-Santos, T. A. P., Duarte, A. C., & Gomes, A. M. (2013). Antioxidative peptides: Trends and perspectives for future research. Current Medicinal Chemistry, 20(36), 4575–4594. Geesink, G. H., & Veiseth, E. (2009). Muscle enzymes: Proteinases. In L. M. L. Nollet, & F. Toldrá (Eds.), Handbook of muscle foods analysis (pp. 91–110). CRC Press. Greaser, M. L., Yates, L. D., Krywicki, K., & Roelke, D. L. (1983). Electrophoretic methods for the separation and identification of muscle proteins. Reciprocal Meat Conference, 36, 87–91. Groussard, C., Morel, I., Chevanne, M., Monnier, M., Cillard, J., & Delamarche, A. (2000). Free radical scavenging and antioxidant effects of lactate ion: An in vitro study. Journal of Applied Physiology, 89(1), 169–175. Hierro, E., de la Hoz, L., & Ordonez, J. A. (1999). Contribution of the microbial and meat endogenous enzymes to the free amino acid and amine contents of dry fermented sausages. Journal of Agricultural and Food Chemistry, 47(3), 1156–1161. Hui, Y. H., Goddik, L. M., Hansen, A. S., Nip, W. K., Stanfield, P. S., & Toldrá, F. (2004). Handbook of food and beverage fermentation technology. New York: Marcel Dekker Incorporated. Janssens, M., Van der Mijnsbrugge, A., Sánchez Mainar, M., Balzarini, T., De Vuyst, L., & Leroy, F. (2014). The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation. Food Microbiology, 39, 53–60. Jongberg, S., Torngren, M. A., Gunvig, A., Skibsted, L. H., & Lund, M. N. (2013). Effect of green tea or rosemary extract on protein oxidation in Bologna type sausages prepared from oxidatively stressed pork. Meat Science, 93(3), 538–546. Klement, J. T., Cassens, R. G., & Fennema, O. R. (1974). Effect of bacterial fermentation on protein solubility in a sausage model system. Journal of Food Science, 39(4), 833–835. Lametsch, R., Lonergan, S., & Huff-Lonergan, E. (2008). Disulfide bond within mu-calpain active site inhibits activity and autolysis. Biochimica et Biophysica Acta, Proteins and Proteomics, 1784(9), 1215–1221. Leroy, F., Geyzen, A., Janssens, M., De Vuyst, L., & Scholliers, P. (2013). Meat fermentation at the crossroads of innovation and tradition: A historical outlook. Trends in Food Science and Technology, 31(2), 130–137. Levine, R. L., Williams, J. A., Stadtman, E. P., & Shacter, E. (1994). Carbonyl assays for determination of oxidatively modified proteins. In P. Lester (Ed.), Methods in enzymology oxygen: Radicals in biological systems part C, Vol. 233. (pp. 346–357). Academic Press. Lund, M. N., Heinonen, M., Baron, C. P., & Estevez, M. (2011). Protein oxidation in muscle foods: A review. Molecular Nutrition & Food Research, 55(1), 83–95. Lund, M. N., Lametsch, R., Hviid, M. S., Jensen, O. N., & Skibsted, L. H. (2007). High-oxygen packaging atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during chill storage. Meat Science, 77(3), 295–303. Meucci, E., Mordente, A., & Martorana, G. E. (1991). Metal-catalyzed oxidation of human serum-albumin — Conformational and functional-changes — Implications in protein aging. Journal of Biological Chemistry, 266(8), 4692–4699. Molly, K., Demeyer, D., Johansson, G., Raemaekers, M., Ghistelinck, M., & Geenen, I. (1997). The importance of meat enzymes in ripening and flavour generation in dry fermented sausages. First results of a European project. Food Chemistry, 59(4), 539–545. Nyström, T. (2005). Role of oxidative carbonylation in protein quality control and senescence. The EMBO Journal, 24(7), 1311–1317. O'Toole, Desmond K., & Yuan, Kun Lee (2006). Fermented foods. In Kun Lee Yuan (Ed.), Microbial biotechnology: Principles and applications (pp. 227–292). World Scientific Publishing Co. Pte. Ltd. Oddy, V. H. (1974). A semiautomated method for the determination of plasma alpha amino nitrogen. Clinica Chimica Acta, 51(2), 151–156. Pacifici, R. E., Kono, Y., & Davies, K. J. A. (1993). Hydrophobicity as the signal for selective degradation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex, proteasome. Journal of Biological Chemistry, 268(21), 15405–15411. Park, D., Xiong, Y. L., Alderton, A. L., & Ooizumi, T. (2006). Biochemical changes in myofibrillar protein isolates exposed to three oxidizing systems. Journal of Agricultural and Food Chemistry, 54(12), 4445–4451. Ravyts, F., De Vuyst, L., & Leroy, F. (2012). Bacterial diversity and functionalities in food fermentations. Engineering in Life Sciences, 12(4), 356–367. Rimaux, T., Vrancken, G., Pothakos, V., Maes, D., De Vuyst, L., & Leroy, F. (2010). The kinetics of the arginine deiminase pathway in the meat starter culture Lactobacillus sakei CTC 494 are pH-dependent. Food Microbiology, 28(3), 597–604. Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004). Oxidative environments decrease tenderization of beef steaks through inactivation of mu-calpain. Journal of Animal Science, 82(11), 3254–3266. Ruiz, J. (2007). Ingredients. In F. Toldrá (Ed.), Handbook of fermented meat and poultry (pp. 59–76). Oxford: Blackwell Publishing. Sarmadi, B. H., & Ismail, A. (2010). Antioxidative peptides from food proteins: A review. Peptides, 31(10), 1949–1956. Toldrá, F., Aristoy, M. C., & Flores, M. (2000). Contribution of muscle aminopeptidases to flavor development in dry-cured ham. Food Research International, 33(3–4), 181–185. Toldrá, F., Aristoy, M. C., Part, C., Cervero, C., Rico, E., Motilva, M. J., & Flores, J. (1992). Muscle and adipose-tissue aminopeptidase activities in raw and dry-cured ham. Journal of Food Science, 57(4), 816–818. Toldrá, F., Rico, E., & Flores, J. (1993). Cathepsin-B, cathepsin-D, cathepsin-H and cathepsin-L activities in the processing of dry-cured ham. Journal of the Science of Food and Agriculture, 62(2), 157–161. Uytterhaegen, L., Claeys, E., & Demeyer, D. (1992). The effect of electrical stimulation on beef tenderness, protease activity and myofibrillar protein fragmentation. Biochimie, 74, 275–281. Verplaetse, A., Demeyer, D., Gerard, S., & Buys, E. (1992). Endogenous and bacterial proteolysis in dry sausage fermentation. Proceedings of the 38th International Conference on Meat Science and Technology, Clermont-Ferrand, France (pp. 851–854). Villaverde, A., Ventanas, J., & Estévez, M. (2014). Nitrite promotes protein carbonylation and Strecker aldehyde formation in experimental fermented sausages: Are both events connected? Meat Science, 98, 665–672. Wood, J. D., Enser, M., Fisher, A. V., Nute, G. R., Sheard, P. R., Richardson, R. I., Hughes, S. I., & Whittington, F. M. (2008). Fat deposition, fatty acid composition and meat quality: A review. Meat Science, 78(4), 343–358. Xue, M., Huang, F., Huang, M., & Zhou, G. (2012). Influence of oxidation on myofibrillar proteins degradation from bovine via mu-calpain. Food Chemistry, 134(1), 106–112.