Effect of pressure and holding time on colour, protein and lipid oxidation of sliced dry-cured Iberian ham and loin during refrigerated storage

Innovative Food Science and Emerging Technologies 10 (2009) 76–81

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Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i f s e t

Effect of pressure and holding time on colour, protein and lipid oxidation of sliced dry-cured Iberian ham and loin during refrigerated storage Ramón Cava a,⁎, Luís Ladero a, Santiago González b, Atanasio Carrasco c, M. Rosario Ramírez d a

Food Technology, Faculty of Veterinary Science, University of Extremadura, Spain Department of Quality Control, Marcos Sotoserrano SLU, Sotoserrano, Salamanca, Spain Department of Engineering, Instituto del Frío, CSIC, Madrid, Spain d Instituto Tecnológico Agroalimentario de Extremadura (INTAEX), Badajoz, Spain b c

a r t i c l e

i n f o

Article history: Received 16 April 2008 Accepted 17 September 2008 Editor proof receive date 20 October 2008 Keywords: Dry-cured Iberian ham Dry-cured Iberian loin High pressure Oxidation Colour Storage

a b s t r a c t The effect of high pressure (HP) treatments (200 MPa 15 min, 200 MPa 30 min, 300 MPa 15 min, 300 MPa 30 min) on colour, lipid and protein oxidation in sliced vacuum-packed dry-cured Iberian ham and loin during refrigerated storage (90 days, + 4 °C) was evaluated. Pressure level and holding time increased the extent of lipid oxidation in both products. Dry-cured ham showed a higher susceptibility to lipid oxidation than dry-cured loin since HP treatment increased TBA-RS values in dry-cured ham samples while HP treatment decreased TBA-RS values in dry-cured loin samples. However, HP treatment did not affect protein oxidation in both meat products. On the other hand, HP treatment affected instrumental colour since nonpressurized dry-cured meat products showed higher redness than pressurized ones. Regarding changes under storage, after 90 days of refrigerated storage lipid and protein oxidation increased while redness decreased in both HP treated and non-treated dry-cured meat products. Changes induced by HP were only noticeable after HP treatment, as storage reduced the initial differences between HP treated and non-treated samples. Therefore, the lack of differences in long stored dry-cured ham and loin HP treated and non-treated indicates that the application of HP (200–300 MPa/15–30 min) could not affect the quality of dry-cured meat products. Industrial relevance: Dry-cured meat products are the meat-based products with the highest sensory quality in Spain and have a high projection in exterior markets. High pressure processing is effective in controlling pathogen and spoilage microorganisms in meat and meat products although it can promote color and oxidation changes that modify sensory characteristics. The study aimed the evaluation of pressure and holding time on color changes and protein and lipid oxidation at vacuum packed slices of Iberian dry-cured ham and loin during subsequent extended chilled storage. High pressure treatment of dry-cured Iberian ham and loin induce changes after treatment although initial differences are not maintained along refrigerated storage. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Dry-cured Iberian ham and loin are meat products with a considerable economical importance as a result of their unique and high sensory quality (Cava, Ventanas, Ruiz, Andrés, & Antequera, 2000). Dry-cured Iberian products are ready-to-eat foods (RTE), usually sold sliced and vacuum packed. Although, low water activity (Aw,) NaCl, nitrite and moisture contents are inhibiting factors of the growth of spoilage and pathogen microorganisms these may be present on the surface of whole hams or on the casings. Final de-boning in dry-cured ⁎ Corresponding author. Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Cáceres 10071, Spain. Tel.: +34 927257169. E-mail address: [email protected] (R. Cava). 1466-8564/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2008.09.005

hams or peeling in dry-cured loins, slicing and packaging operations can lead these to reach the sliced product, constituting a permanent risk of contamination (FSIS, 2006). Sliced dry-cured Iberian meat products, because of the low Aw and high NaCl content, are products with a long shelf-life at refrigeration temperature (more than 3 months); however, the risk of spoilage or food-pathogen growth when contaminated increases in the case of high temperatures for a long storage period. Microbial growth, decolouration and oxidation are important factors for the shelf life and consumer acceptance of packed drycured products from Iberian pigs. Additionally, as a consequence of innovative presentations and new potential markets, industry demands the use of preservation methods that increase the shelflife of treated foods ensuring food safety. Due to the fact that colour and flavour characteristics are the main quality factors in dry-cured

R. Cava et al. / Innovative Food Science and Emerging Technologies 10 (2009) 76–81

Iberian meat products (Cava et al., 2000), conventional technologies such as thermal processing, which could induce undesirable changes on sensory properties of these types of dry-cured meat products, cannot be used to reduce the risk associated to spoilage microorganisms and food-borne pathogens, and so other mild technologies must be used. High pressure processing of foods offers several advantages over existing alternatives, for example there is little effect on flavour, colour and vitamins (Kimura et al., 1994). High pressure (HP) treatment has been used for the preservation of chicken, pork, meat products, surimi gels as well as salmon products, with considerable positive effects on protease activity, textural properties, taste and flavour (Knorr, 1993; Garriga, Aymerich, & Hugas, 2002; O'Brien & Marshall, 1996; Ohshima, Ushio, & Koizumi, 1993). The use of HP for the microbial decontamination has been extensively reviewed but complete microbial inactivation is currently not possible (Knorr, 1995; Smelt, 1998). Nevertheless, HP treatment induces undesirable changes on meat and meat products characteristics, modifying texture and colour of meat and increasing lipid oxidative reactions, which decline the acceptability, especially in some rich protein foods treated at pressures higher than 400 MPa (Cheftel & Culioli, 1997). Industry is greatly interested in applying high pressure to dry-cured products (Andrés, Adamsen, Moller, Ruiz, & Skibsted, 2006; Garriga et al., 2002; Morales, Calzada, & Nunez, 2006; Serra et al., 2007) but there is a need to establish optimum processing conditions to ensure their microbiological safety, especially if shelf-life is extended, minimizing the negative effects of pressure on oxidation processes in relation to discolouration and to development of rancidity that could impair sensory quality. A few reports studying the effect of high pressure effects on drycured products under refrigerated storage have been published (Andres, Moller, Adamsen, & Skibsted, 2004; Andrés et al., 2006; Cava, Mingoarranz, & Carrasco, 2002; Hugas, Garriga, & Monfort, 2002; Núñez et al., 2003) and to our knowledge this is the first report investigating the changes on lipid and protein oxidation and colour of dry-cured Iberian ham and loin during storage for 90 days at 4 °C. The aim of this study was to evaluate the combined effect of two pressure levels (200 MPa and 300 MPa) and two holding times (15 min and 30 min) on lipid and protein oxidation and on colour on the surface of vacuum-packed slices of dry-cured Iberian hams and loins and the implications of high pressure treatment on these traits during refrigerated storage. 2. Materials and methods 2.1. Dry-cured ham and loin samples Dry-cured Iberian hams and loins from pigs fed on concentrate were used for the experiment. Dry-cured loins and hams were manufactured following a traditional process. Loins were seasoned by rubbing in a mixture of salt, nitrite, olive oil and spices such as Spanish paprika (Capsicum annuum, L.), oregano (Origanum vulgare L.) and garlic (Allium sativum, L.). Loins were kept at 4 °C for 4 days to allow the seasoning mixture to penetrate. Then, the loins were stuffed into collagen casings and held at 4 °C at a relative humidity of 80% for 30 days. Finally, the loins were ripened at 12 °C and 70% relative humidity for 90 days. Loins were processed for a total dry-curing time of 4 months. For the manufactured of dry-cured hams, ham surface was rubbed with sodium chloride containing nitrate/nitrite and placed on salt piles to complete the salting process at 3–4 °C and 95% relative humidity. After salting, hams were kept at 0–10 °C and 75–90% relative humidity for ~6 months. Then, the hams were ripened for ~ 18 months in at 10–25 °C and 60–80% relative humidity. Hams were processed for a total dry-curing time of 24 months In order to slice both meat products, m. Biceps femoris were dissected from the hams and the casings of the dry-cured loins were removed.

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Both m. Biceps femoris and loins were sliced in 3 mm thickness slices using a slicing machine. 2.2. Vacuum packaging, high pressure treatment and refrigerated storage Dry-cured ham and loin slices were vacuum-packaged in nylon/ polyethylene bags (9.3 ml O2/m2/24 h at 0 °C) with 5 slices/package and day of chill storage. Packages were randomly assigned to each treatment. Packages (5packs/treatment/day of storage) were pressurized at two pressure levels (200 MPa and 300 MPa) and two holding times (15 min and 30 min) giving four experimental treatments (200 MPa/15 min, 200 MPa/30 min, 300 MPa/15 min and 300 MPa/30 min), as well as a non-pressurized control group. High pressure treatments were performed in a batch isostatic press from ACB Gec-Alsthom, mod AGIP n.665 (Nantes, France) capable of operating at 500 MPa with a pressure vessel measuring 0.10 m diameter and 0.30 m in length (capacity 2.35 l). Two high pressure pneumatic pumps DSXHF202 and DSXHF903 (Haskel. Ltd., California, USA) pumped the pressure medium into the vessel from a reservoir. The pressure in the vessel was measured using a pressure-transducer HP28 (Intersonde Ltd., Watford, England) connected near to the inlet of the vessel. Pressure vessel and transmitting medium (water) were maintained at temperature b14 °C by a constant flow of water through an external jacket. A thermocouple type K was fitted through the lower plug to measure the inner temperature of the vessel during the pressure treatment. After pressure treatment, both bags containing pressurized and non-pressurized samples were stored under refrigeration at +4 °C in the darkness for 0, 60 and 90 days. 2.3. Chemical analysis 2.3.1. Lipid oxidation Lipid oxidation was measured using a complete dry-cured loin or ham slice homogenised using a kitchen blender. The extent of lipid oxidation was estimated as TBA-RS following the method described by Salih, Smith, Price, and Dawson (1987). TBA-RS were measured on two slices of each pack and were expressed as mg malondialdehyde (MDA)/kg meat. 2.3.2. Protein oxidation Protein oxidation was measured by estimation of carbonyl groups formed during incubation with 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl following the method described by Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987). Carbonyl concentration was measured on the treated sample by measuring DNPH incorporated on the basis of absorption of 21.0 mM− 1 cm− 1 at 370 nm for protein hydrazones. Results were expressed as nmol of DNPH fixed per milligram of protein. Protein oxidation was measured on two slices from each pack and was expressed as nmol carbonyls/mg protein. Protein concentration was calculated spectrophotometrically at 280 nm using bovine serum albumin (BSA) as standard. 2.3.3. Instrumental colour measurements Colour measurements were taken immediately after opening the package (to prevent colour degradation because of light and oxygen) in accordance with the recommendations on colour determination of the American Meat Science Association (Hunt et al., 1991). The following CIELAB colour coordinates were determined: lightness (L⁎), redness (a⁎, red± green) and yellowness (b⁎, yellow ± blue). The colour parameters were determined using a Minolta CR-300 colorimeter (Minolta Camera Co., Osaka, Japan) (Illuminant D65/0° standard observer and a 0.8 cm port/viewing area). Before use, the colorimeter was standardized using a white tile (mod CR-A43). In addition, total colour differences (ΔE) were calculated. The measurements were repeated on eight randomly selected locations on each slice and averaged for statistical analysis.

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Table 1 Effect of pressure and holding time on TBA-RS (mg MDA/Kg) of sliced dry-cured Iberian ham during refrigerated storage

Table 3 Effect of pressure and holding time on protein oxidation (nmol carbonyls/mg protein) of sliced dry-cured Iberian ham during refrigerated storage

Pressure

Pressure

200 MPa

300 MPa

SEM

Holding time

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

Day 0 Day 60 Day 90 SEM P

2.47 b y 3.42 b y 6.58 x 0.53 ⁎⁎⁎

4.39 a 5.09 ab 7.13 0.50 ns

5.53 a 5.57 a 5.62 0.24 ns

4.86 a 5.06 ab 5.85 0.38 ns

4.32 a y 5.13 ab y 7.31 x 0.40 ⁎⁎⁎

0.25 0.24 0.35

P

⁎⁎⁎ ⁎ ns

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 25); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 15); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

2.4. Statistical analysis Samples from dry-cured Iberian hams and loins were completely and randomly assigned to each treatment. There were five replicates per treatment and day of storage. The effects of treatment and time of storage for each type of dry-cured product were analyzed using the Analysis of Variance procedure of SPSS, version 10.0. Mean was used to compare differences between treatments. Tukey's test was used to compare the mean values of the treatments and storage time. Mean values and standard errors of the means (SEM) were reported. 3. Results 3.1. Effect of pressure and holding time on lipid and protein oxidation The effect of high pressure treatment — pressure level and holding time on TBA-RS values of sliced vacuum-packed dry-cured Iberian ham during refrigerated storage is shown in Table 1. High pressure treatment significantly increased (p;;0.05) TBA-RS values at Day 0 when compared with non-pressurized samples. Neither pressure level nor holding time affected TBA-RS values in treated samples. In relation to the time of storage, TBA-RS values tended to increase in all treatments throughout refrigerated storage, although differences were only significant (p b 0.05) in non-pressurized and 300 MPa/30 min treated samples. At Day 60, significant differences in lipid oxidation were found among treatments (p b 0.05) belonging to non-pressurized samples the lowest TBA-RS values. At the end of refrigerated storage (Day 90), no differences in lipid oxidation were found between pressurized and non-pressurized control samples. Table 2 shows the effect of pressure level and holding time on the TBA-RS values of sliced vacuum-packed dry-cured Iberian loin pressurized and stored at +4 °C. Contrarily to the effect of pressurization described for sliced dry-cured Iberian ham, pressurization of vacuum packed slices of dry-cured loins did not increase the extent of lipid oxidation at Day 0, but resulted in an unexpected reduction in TBA-RS

200 MPa

300 MPa

Holding time

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

Day 0 Day 60 Day 90 SEM P

7.6 xy 5.5 b x 10.9 y 0.87 ⁎

7.0 7.4 ab 8.3 0.66 ns

5.5 x 6.2 ab xy 9.6 y 0.75 ⁎

5.1 6.3 ab 6.8 0.38 ns

6.8 8.6 a 6.8 0.51 ns

SEM

P

0.50 0.35 0.58

ns ⁎ ns

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 25); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 15); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

(p b 0.05) when compared with non-pressurized samples. In pressurized samples, pressure level affected lipid oxidation, being TBA-RS values higher in samples pressurized at 200 MPa than those treated at 300 MPa. After 90 days of refrigerated storage TBA-RS significantly increased in all samples (p b 0.05), except in 200 MPa/15 min pressurized samples and non-treated samples in which TBA-RS decreased in comparison with Day 0. At Day 90, samples pressurized at 200 MPa/30 min, 300 MPa/15 min and 300 MPa/30 min showed the highest (p b 0.05) TBA-RS, whereas samples pressurized at 200 MPa/15 min and nonpressurized samples had the lowest TBA-RS (p b 0.05). The effect of high pressure treatment — pressure level and holding time — on carbonyl contents, as an indicator of protein oxidation, of vacuum-packed sliced dry-cured Iberian ham and loin during refrigerated storage is shown in Tables 3 and 4, respectively. In general, no differences in carbonyl contents were found between pressurized and non-pressurized samples neither in dry-cured ham nor in dry-cured loin after pressurization. Only at day 60 dry-cured ham pressurized samples had higher values of protein oxidation than non pressurized samples and in dry-cured loin samples treated at 300 MPa/30 min had the highest values. Refrigerated storage increased the amounts of carbonyls in dry-cured ham, although the increases were only statistically significant (p b 0.05) in non-pressurized and in 200 MPa/30 min pressurized samples. In dry-cured loin the same trend was found, storage increased protein oxidation although not statistically different. So, initial carbonyl content kept constant or tended to increase slightly throughout storage. In both dry-cured products, no differences in carbonyl content were found between pressurized and non-pressurized control samples at Day 90 of storage. 3.2. Effect of pressure and holding time on colour parameters Changes in instrumental colour parameters (CIE L⁎a⁎b⁎) of vacuumpacked sliced dry-cured ham is shown in Table 5. No differences in

Table 2 Effect of pressure and holding time on TBA-RS (mg MDA/Kg) of sliced dry-cured Iberian loin during refrigerated storage

Table 4 Effect of pressure and holding time on protein oxidation (nmol carbonyls/mg protein) of sliced dry-cured Iberian loin during refrigerated storage

Pressure

Pressure

200 MPa

300 MPa

SEM

Holding time

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

Day 0 Day 60 Day 90 SEM P

0.69 a x 0.27 y 0.26 b y 0.06 ⁎⁎⁎

0.37 b,c x 0.29 x,y 0.23 b y 0.02 ⁎⁎⁎

0.38 b,c y 0.26 y 0.53 a x 0.04 ⁎⁎⁎

0.43 b x,y 0.31 x 0.54 a y 0.03 ⁎

0.26 c y 0.30 y 0.69 a x 0.06 ⁎⁎⁎

0.03 0.02 0.04

P

⁎⁎⁎ ns ⁎⁎⁎

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 25); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 15); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

200 MPa

300 MPa

Holding time

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

Day 0 Day 60 Day 90 SEM P

3.8 ab 5.0 5.3 0.3 ns

2.7 b 4.9 4.8 0.41 ⁎

3.2 b 3.7 4.6 0.25 ns

3.2 b x 5.9 y 4.7 xy 0.43 ⁎

4.6 a 4.9 4.5 0.44 ns

SEM

P

0.18 0.26 0.33

⁎⁎⁎ ns ns

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 25); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 15); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

R. Cava et al. / Innovative Food Science and Emerging Technologies 10 (2009) 76–81

lightness (CIE L⁎-value) were found in sliced dry-cured ham as a result of the HP treatment and in addition, the time of storage did not significantly change lightness in packed samples. Yellowness (CIE b⁎value) followed a similar trend as that described for L⁎-value, neither pressurization nor the time of storage affected these parameters. Pressurization at 200 MPa/30 min, 300 MPa/15 min and 300 MPa/ 30 min significantly reduced a⁎-value (p b 0.05) (∼15%) when comparing with non-treated samples. Redness significantly decreased during the storage in all groups (p b 0.05), even though this diminution in a⁎-value did not progress between 60 and 90 days of storage. The effects of HP on the colour dry-cured loins were similar than in dry-cured ham (Table 6). In this sense, pressure treatment did not affect lightness (L⁎-value). Similarly, lightness did not change in vacuumpacked dry-cured loin during storage, with the exception of nonpressurized control samples in which a significant reduction of initial L⁎value was found after 90 days of refrigerated storage (p b 0.05). However, HP treatment caused a reduction in redness (a⁎-value) (p b 0.05) when compared with non-pressurized control samples. Redness did not significantly change during storage at +4 °C in pressurized and in nonpressurized samples, and at Day 60 and Day 90 no redness differences were found between treatments. Yellowness was not affected by pressure treatment, however, during refrigerated storage yellowness tended to rise in all samples, with significant increases in those pressurized during long holding periods (30 min). At Day 0, total colour changes (ΔE) did not follow a clear pattern to describe changes of colour as a result of pressure level or holding time (Table 7). However, ΔE-values were higher in dry-cured Iberian hams than in dry-cured Iberian loins, being in agreement with changes in colour coordinates previously described. In general, ΔE tended to decrease with time of storage, indicating a smaller difference in the colour between non pressurized control samples and high pressure treated. The increase in ΔE values at Day 90 was unexpected in 200 MPa/ 15 min and 300 MPa/15 min treated dry-cured hams as well as 200 MPa/ 30 min and 300 MPa/30 min in dry-cured loin. 4. Discussion Pressure treatment induced modifications in colour and lipid oxidation in sliced dry-cured ham and loin agreeing with findings previously reported in these type of meat products (Andres et al., 2004; Cava et al., 2002; Núñez et al., 2003). This pro-oxidant effect has been previously described in different pressurized meats and muscle foods.

Table 5 Effect of pressure and holding time on instrumental colour (CIE L⁎a⁎b⁎) of sliced drycured Iberian ham during refrigerated storage Pressure

200 MPa

300 MPa

SEM P

Holding time

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

Lightness (CIE L⁎)

46.39 42.58 43.52 0.92 ns 35.23 a x 27.66 y 27.04 y 1.06 ⁎⁎⁎ 25.22 20.01 20.67 1.16 ns

42.18 43.45 42.28 0.87 ns 32.63 a,b x 25.42 y 25.08 y 1.01 ⁎⁎⁎ 20.38 18.37 19.16 1.13 ns

43.08 40.28 43.94 0.90 ns 31.40 b x 26.32 y 26.73 y 0.77 ⁎⁎⁎ 20.63 16.45 21.84 1.38 ns

42.90 42.71 42.57 0.92 ns 30.81 b x 26.33 y 25.67 y 0.76 ⁎⁎ 19.52 19.07 18.29 0.95 ns

46.76 43.13 44.43 1.26 ns 29.20 b x 25.55 y 26.52 y 0.53 ⁎⁎ 23.26 19.97 21.71 1.44 ns

Day 0 Day 60 Day 90 SEM P Redness Day 0 (CIE a⁎) Day 60 Day 90 SEM P Yellowness Day 0 (CIE b⁎) Day 60 Day 90 SEM P

0.79 0.78 0.71

ns ns ns

0.55 0.33 0.31

⁎⁎ ns ns

0.91 0.96 0.94

ns ns ns

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 25); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 15); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

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Table 6 Effect of pressure and holding time on instrumental colour (CIE L⁎a⁎b⁎) of sliced drycured Iberian loin during refrigerated storage Pressure

200 MPa

Holding time Lightness (CIE L⁎)

Redness (CIE a⁎)

Yellowness (CIE b⁎)

Day 0 Day 60 Day 90 SEM P Day 0 Day 60 Day 90 SEM P Day 0 Day 60 Day 90 SEM P

300 MPa

Control (n = 5)

15 min (n = 5)

30 min (n = 5)

15 min (n = 5)

30 min (n = 5)

50.08 x 47.75 x,y 47.33 y 0.49 ⁎ 25.12 a 26.25 25.75 0.39 ns 20.65 21.51 21.74 0.39 ns

49.74 48.65 47.97 0.61 ns 22.22 b y 25.24 x 25.33 x 0.53 ⁎⁎ 19.72 21.67 21.27 0.50 ns

50.11 49.11 50.75 0.62 ns 21.55 b 24.00 24.23 0.58 ns 20.55 y 21.82 x,y 23.82 x 0.52 ⁎⁎

50.05 49.21 48.47 0.60 ns 22.28 b 24.12 25.39 0.78 ns 19.57 21.42 21.50 0.47 ns

49.97 49.77 51.22 0.59 ns 23.45 a,b 24.53 23.37 0.74 ns 20.38 y 23.14 x,y 24.14 x 0.67 ⁎

SEM

P

0.43 0.37 0.56

ns ns ns

0.36 0.48 0.54

⁎⁎ ns ns

0.32 0.31 0.45

ns ns ns

a,b,c: Means within the same row with different letters differ significantly (Tukey's test, p b 0.05) (n = 50); x,y,z: Means within a column with no common letter differ significantly (Tukey's test, p b 0.05) (n = 30); SEM: standard errors of the mean; P: ANOVA p-value. * p b 0.05, **p b 0.01, ***p b 0.001, ns: non-significant differences.

Cheftel and Culioli (1997) revised the effect of high pressure on lipid oxidation in meat and fish, describing that pressure treatments lead to catalysis of lipid oxidation and thus increasing TBA-RS numbers. In a study in dry-cured Iberian ham, Andres et al. (2004) described that pressurized samples showed a lower lightness and a higher lipid oxidation derived products than non-pressurized samples, and that the redness of the samples significantly decreased with pressure treatment. Similarly, Cava et al. (2002) and Núñez et al. (2003) using surface response methodology (RSM) modelized the changes induced by pressure level (range 24–400 MPa) and holding time (range 7–28 min) on colour and lipid oxidation of vacuum-packed slices of dry-cured Iberian ham and loin, describing that the higher pressure level and holding time were, the higher the TBA-RS and discolouration were. Although, there are few references about the oxidation of proteins in processed meats, the formation of carbonyl groups in proteins has been widely used as a measure of oxidation (Srinivasan & Hultin, 1995). The relationship between protein and lipid oxidation has been described via peroxy radical formation which reacts with proteins and/or lipids (Davies, 1986; Srinivasan & Hultin, 1995). Protein oxidation may occur more rapidly than lipid oxidation in biological systems such as muscle (Davies & Golberg, 1987; Srinivasan & Hultin, 1995), since proteins are within the aqueous phase where many radicals are formed (Soyer & Hultin, 2000). Contrarily to the previously described for muscle systems, in dry-cured products in which the amount of water is low, the oxidation processes as a result of pressurization and/or storage are not much relevant. Differences in the extent of the carbonyls formation between dry-cured loins and hams could be related to the length of the maturation process

Table 7 Effect of pressure treatment and refrigerated storage on total colour changes (ΔE) of sliced dry-cured Iberian ham and loin during refrigerated storage Dry-cured Iberian ham

Dry-cured Iberian loin

Pressure

200 MPa

200 MPa

Holding time

15 min 30 min 15 min 30 min 15 min 30 min 15 min 30 min

Day 0 Day 60 Day 90

6.9 2.9 2.8

300 MPa

6.8 4.4 1.3

8.0 1.6 2.9

6.3 2.2 1.5

3.1 1.4 0.9

300 MPa

3.6 2.6 4.3

3.0 2.6 1.2

1.7 3.1 5.2

ΔE = [(ΔL⁎) 2 + (Δa⁎) 2 + (Δb⁎) 2] 0.5 where ΔL⁎, Δa⁎ and Δb⁎ are the differences in L⁎, a⁎ and b⁎ between treated and non-treated samples.

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(24 months in dry-cured Iberian ham and 4 months in dry-cured Iberian loin) and the different implication of high pressure treatment and storage might be related to initial carbonyl contents, higher in dry-cured hams than in dry-cured loins. Changes on colour and lipid oxidation induced by high pressure at Day 0 were not detected at Day 60 and 90 of refrigerated storage, indicating that storage diminished initial differences. As a result, evolution of lipid oxidation and discolouration during refrigerated storage was similar in pressurized and non pressurized samples. The lack of effect of pressurization on colour changes and lipid oxidation is not in agreement with previous studies in meat, which described a more rapidly increase in TBA-RS and reduction in a⁎-value with processing time (Cheah & Ledward, 1996). Additionally, the discolouration through pressure processing appears as a result of oxidation of ferrous myoglobin to ferric myoglobin with a decrease of a⁎-values and an increase in the proportion of metmyoglobin at the expense of oxymyoglobin. This change could be prevented by the formation of nitrosylmyoglobin, more resistant to oxidation (Carlez, VecianaNogues, & Cheftel, 1995; Goutenfongea, Rampon, Nicolas, & Dumont, 1995). More recently, a Zn-porphyrin complex has been reported to contribute to the red colour of Parma ham and Iberian ham when nitrate and nitrite are absent in this cured meats products (Møller, Adamsen, Catharino, Skibsted, & Eberlin, 2007; Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004). The contribution of Zn-porphyrin complex to the color of the analyzed samples and the effect of high pressure treatments on Zn-porphyrin cannot be evaluated in the present study and need more studies to elucidate both aspects. However, from findings, dry-cured Iberian ham seemed to be more sensitive than dry-cured loin to pressure treatment and storage in terms of lipid oxidation and discolouration. These results indicate that mechanisms involved in lipid oxidation in pressurized dry-cured hams and dry-cured loins could be different, and different chemical characteristics such as: i. fat content (Cava et al., 2000), ii. salt and nitrite contents (Bruun-Jensen & Skibsted, 1996), iii. use of paprika and garlic in dry-cured loin manufacturing (Aguirrezábal, Mateo, Domínguez, & Zumalacárregui, 2000; Gómez, Alvarez-Orti, & Pardo, 2000) and/or iv. initial oxidation level (Carrasco, Tárrega, Ramírez, Mingoarranz, & Cava, 2005; Cava, Tárrega, Ramirez, Mingoarranz, & Carrasco, 2005) could play an important role on the discolouration and oxidative stability of lipids mediated by pressure treatment and/or storage. Initial oxidation values seem to be critical in terms of the effect of pressurization on colour and TBA-RS and further changes during refrigerated storage and could explain the different behaviour of dry-cured ham and loin. This dissimilar response of dry-cured Iberian hams and loins towards lipid oxidation and colour changes induced by the technological treatment has been previously reported by Cava et al. (2005) and Carrasco et al. (2005) in irradiated at 5 and 10 kGy. In both studies, a similar behaviour was found for irradiated samples, being changes induced by irradiation greater in dry-cured hams than in dry-cured loins. Discolouration processes and lipid oxidation phenomena are closely related indicating that both participate for oxidative processes of lipids and myoglobin due to the fact that the oxidation of muscle pigments can catalyze the oxidation of lipids (Akamittath, Brekke, & Schanus, 1990). Discolouration took place during the first period of study (0–60 days) whereas the greater changes in lipid oxidation were detected at the end of storage (60–90 days), indicating that discolouration processes were initiated before lipid oxidation changes. Zhao, Wells, and McMillin (1994) hold that lipid oxidation is normally not considered a limiting factor for shelf life as lipid oxidation occurs at a slower rate than discolouration or microbial growth. The rate of formation of TBA-RS in the period 0–90 days (Δnon-pressurized: +0.05 mg MDA/kg/day vs Δ pressurized: +0. 01/+0.03 mg MDA/kg/day) and colour changes (a⁎-value: Δnon-pressurized: −0.09/day vs Δpressurized: −0.03/ −0.08/day) were higher in non treated dry-cured ham samples than in pressurized ones. This fact could be due to the protective effect of high

pressure at 200–300 MPa on heme pigments, controlling metmyoglobin formation and thus reducing lipid oxidation. Although colour of meat is affected by pressure treatment as a result of globin denaturation and oxidation of myoglobin to metmyoglobin (Carlez et al., 1995), nitrite was found to prevent such pressure-induced discolouration of minced meat (Goutenfongea et al., 1995). In the same way, Bruun-Jensen and Skibsted (1996) found in nitrosylmyoglobin solutions that the rate of oxidation of MbFe(II)NO to MbFe(III) decreased with increasing hydrostatic pressure in the pressure range 50–350 MPa, indicating a protective effect of high pressure against MbFe(II)NO oxidation. MbFe(II)NO is believed to be an important antioxidant in cured meats due to its fast radical exchange reactions (Andersen, Johansen, Shek, & Skibsted, 1990). 5. Conclusions Although the reason for the observed catalysis of lipid oxidation is not clear, the present work does give further insight into some of the pressure induced lipid, protein and colour changes in dry-cured meat products. The conflicting results obtained (i.e. dry-cured products pressurized at different treatments exhibited both increased and reduced lipid oxidation) are a manifestation of the complexity of the oxidative processes in dry-cured meats. The effect of HP and storage on the oxidative stability and colour changes in the studied dry-cured meat products is sensitive to a high number of extrinsic and intrinsic factors. Among them are the conditions of pressurization (pressure and holding time) as well as the time of storage, the raw material composition (moisture and fat contents, fatty acid composition, pickling mixture consisted of paprika, olive oil and garlic, which are rich in natural antioxidants) and the initial level of oxidation. HP induced changes are only important after treatment thus changes are similar in HP treated and non-treated during storage, being changes on colour and lipid and protein oxidation during refrigerated storage of a greater importance that those induced by HP. The lack of differences in stored products treated with HP with respect to non-treated products shows that the application of HP at level 200–300 MPa for 15–30 min could not compromise colour and oxidative stability of dry-cured meat products. Acknowledgements Authors thank Marcos Sotoserrano S.L.U. for providing dry-cured Iberian ham and loin samples. References Aguirrezábal, M. M., Mateo, J., Domínguez, M. C., & Zumalacárregui, J. M. (2000). The effect of paprika, garlic and salt on rancidity in dry sausages. Meat Science, 54, 77−81. Akamittath, J. G., Brekke, C. J., & Schanus, E. G. (1990). Lipid oxidation and colour stability in restructured meat systems during frozen storage. Journal of Food Science, 55, 1513−1517. Andersen, H. J., Johansen, H. S., Shek, C. K., & Skibsted, L. H. (1990). Nitric oxide exchange in nitrosylmyoglobin. Zeitschrift fuÉr Lebensmittel-Untersuchung und -Forschung, 191, 293−298. Andrés, A. I., Adamsen, C. E., Moller, J. K. S., Ruiz, J., & Skibsted, L. H. (2006). Highpressure treatment of dry-cured Iberian ham. Effect on colour and oxidative stability during chill storage packed in modified atmosphere. European Food Research and Technology, 222, 486−491. Andres, A. I., Moller, J. K. S., Adamsen, C. E., & Skibsted, L. H. (2004). High pressure treatment of dry-cured Iberian ham. Effect on radical formation, lipid oxidation and colour. European Food Research and Technology, 219, 205−210. Bruun-Jensen, L., & Skibsted, L. H. (1996). High-pressure effects on oxidation of nitrosylmyoglobin. Meat Science, 44, 145−149. Carlez, A., Veciana-Nogues, T., & Cheftel, J. C. (1995). Changes in colour and myoglobin of minced beef meat due to high pressure processing. Lebensmittel-Wissenschaft undTechnologie, 28, 528−538. Carrasco, A., Tárrega, R., Ramírez, M. R., Mingoarranz, F. J., & Cava, R. (2005). Colour and lipid oxidation changes in dry-cured loins from free-range reared and intensively reared pigs as affected by ionizing radiation dose level. Meat Science, 69, 609−615. Cava, R., Mingoarranz, F.J. & Carrasco, J.A. (2002). Colour and lipid oxidation changes in dry-cured loins treated with high hydrostatic pressure: development of a response surface model to optimize the combination of pressure and time of treatment 48th Annual International Congress of Meat Science and Technology, Proceedings Vol I. Roma (Italy).

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