Protein oxidation in emulsified cooked burger patties with added fruit extracts: Influence on colour and texture deterioration during chill storage

Meat Science 85 (2010) 402–409

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Protein oxidation in emulsified cooked burger patties with added fruit extracts: Influence on colour and texture deterioration during chill storage Rui Ganhão a, David Morcuende b, Mario Estévez b,* a b

Food Science Department, School of Maritime Technology, Polytechnic Institute of Leiria, Peniche, Portugal Animal Production and Food Science Department, Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain

a r t i c l e

i n f o

Article history: Received 23 September 2009 Received in revised form 3 February 2010 Accepted 8 February 2010

Keywords: Antioxidant Fruit phenolics Functional product Protein oxidation Meat redness Meat hardness Chill storage

a b s t r a c t The influence of protein oxidation, as measured by the dinitrophenylhydrazine (DNPH) method, on colour and texture changes during chill storage (2 °C, 12 days) of cooked burger patties was studied. Extracts from arbutus-berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses (Rosa canina L., RC) and elm-leaf blackberries (Rubus ulmifolius Schott., RU) were prepared, added to burger patties (3% of total weight) and evaluated as inhibitors of protein oxidation and colour and texture changes. Negative (no added extract, C) and positive control (added quercetin; 230 mg/kg, Q) groups were also considered. The significant increase of protein carbonyls during chill storage of control burger patties reflect the intense oxidative degradation of the muscle proteins. Concomitantly, an intense loss of redness and increase of hardness was found to take place in burger patties throughout refrigerated storage. Most fruit extracts as well as Q significantly reduced the formation of protein carbonyls and inhibited colour and texture deterioration during chill storage. Likely mechanisms through which protein oxidation could play a major role on colour and texture changes during chill storage of burger patties are discussed. Amongst the extracts, RC was most suitable for use as a functional ingredient in processed meats since it enhanced oxidative stability, colour and texture properties of burger patties with no apparent drawbacks. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Oxidation of lipids and proteins are major threats to meat quality. The onset of oxidative reactions in muscle foods during handling, processing and storage of leads to undesirable sensory changes and serious health concerns (Gray & Pearson, 1994; Ladikos & Lougovois, 1990; Xiong, 2000). Whereas the influence of lipid oxidation on quality traits such as meat colour and odour is well recognized, the impact of protein oxidation on meat quality requires further attention. In the presence of reactive oxygen species (ROS), protein oxidation is manifested by free radical chain reactions similar to hose of lipid oxidation, including initiation, propagation and termination stages (Gardner, 1979). Amongst other effects, the oxidative degradation of muscle proteins involves modification of the amino acid side chains, with the formation of carbonyl compounds being the most marked change in oxidizing proteins (Stadtman & Levine, 2003; Xiong, 2000). It is generally accepted that the oxidation of myofibrillar proteins affects their technological properties (reviewed by Xiong, 2000) but the consequences of such reactions on particular quality traits is not well understood. Some initial approaches reported the effect of early post-mortem protein oxidation on beef tenderisation through * Corresponding author. Tel.: +34 927257122; fax: +34 927257110. E-mail address: [email protected] (M. Estévez). 0309-1740/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.02.008

inactivation of muscle proteases (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004). Estévez, Ventanas, and Cava (2005) suggested a plausible reason for the effect of protein oxidation on colour and texture of frankfurters during refrigerated storage. Lund, Lametsch, Hviid, Jensen, and Skibsted (2007) reported that fresh pork tenderness is affected by oxidizing proteins during chill storage of meat and Ventanas, Ventanas, Tovar, García, and Estévez (2007) described the likely impact of protein oxidation on colour, texture and flavour of dry-cured products. Some other meat products, such as burger patties, are very susceptible to oxidation as mincing, cooking and the addition of salt, promotes the formation of ROS and hence, the occurrence and intensity of the oxidative reactions (Ladikos & Lougovois, 1990). Nevertheless, the occurrence of protein oxidation during processing and storage of these meat products has not been studied and the impact of these reactions on particular quality traits remains unknown. The commitment to avoiding the unpleasant effects of oxidative reactions in muscle foods and in emphasizing the physiological functions of meat explains the growing interest amongst scientists in the development of functional meat products and the plant kingdom has been recognised as a versatile source of materials with proven functional properties. When added to a particular meat product, plant materials provide dietary fibre and a large variety of minerals, vitamins and phenolic compounds (JiménezColmenero, Carballo, & Cofrades, 2001). Plant phenolics are known

R. Ganhão et al. / Meat Science 85 (2010) 402–409

to act as effective antioxidants in meat systems through radical scavenging and metal-chelating activities (Rice-evans, Miller, Bolwell, Bramley, & Pridham, 1995). In addition, plant phenolics might enhance the nutritional value of muscle foods as they display antioxidant activity in vivo as well as anti-inflammatory and anti-carcinogenic properties (Bravo, 1998). A large variety of wild fruits and berries are found in the Mediterranean forest such as arbutus-berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses (Rosa canina L., RC) and elm-leaf blackberries (Rubus ulmifolius Schott., RU). These fruits have been found to have large amounts of phenolic compounds and their antioxidant potential has been demonstrated in vitro (Ganhão, Estévez, & Morcuende, 2008). However, the influence of the addition of extracts of these fruits in meat products, in terms of antioxidant effects and impact on meat quality traits is unknown. The protective role played by fruit extracts and phenolic compounds towards food proteins has been seen in model systems (Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008a; Salminen, Jaakkola, & Heinonen, 2008); the effect on real meat products deserves further attention. The present study aimed to evaluate the effect of phenolic-rich extracts of selected Mediterranean wild fruits on texture and colour changes during refrigerated storage of cooked burger patties. The likely impact of protein oxidation on these changes is also addressed. 2. Material and methods 2.1. Chemicals All chemicals and reagents used were purchased from Panreac (Panreac Química, S.A., Barcelona, Spain), Merck (Merk, Darmstadt, Germany) and Sigma Chemicals (Sigma–Aldrich, Steinheim, Germany). 2.2. Fruits Samples of strawberry tree (A. unedo L., AU), common hawthorn (C. monogyna L., CM), dog rose (R. canina L., RC) and elm-leaf blackberry (R. ulmifolius Schott., RU) cultivars were collected at full ripeness in the Cáceres region, Spain (altitude = 450 m) during the summer and autumn of 2007. After hand-harvest, the samples were immediately transferred to the laboratory, cleaned and sorted to eliminate damaged and shrivelled fruits and then frozen at 80 °C. 2.3. Preparation of fruit extracts for burger patties Fruits (30 g), including peel and pulp, were cut into pieces while the seeds were carefully removed. The fruit was ground, dispensed in a falcon tube and homogenized with 10 volumes (w/v) of absolute ethanol. The homogenates were centrifuged at 2600g for 10 min at 6 °C. The supernatants were collected and the residue was re-extracted once more following the procedure previously described. The two supernatants were combined, evaporated using a rotaevaporator and redissolved using 250 g of distilled water. Water solutions from each fruit were prepared and stored under refrigeration until used for the manufacture of porcine burgers (less than 24 h). No insoluble fragments or residues were observed in the water solutions.

403

230 mg/kg, Q). In the basic formulation, the ingredients per kg of patty were as follows: 725 g meat (porcine longissimus dorsi muscle), 250 g distilled water, and 25 g sodium chloride. In the formulation of the treated patties, the 250 g of distilled water were replaced by 250 g of a water solution containing the corresponding fruit extracts or the quercetin. All ingredients were minced in cutter until a homogeneous raw batter was obtained. Sixteen burger patties per batch were prepared in two independent processes (eight patties per batch each time). Burger patties were formed using a conventional burger-maker (100 g/patty), to give average dimensions of 10 cm diameter and 1 cm thickness. Preliminary cooking trials were performed to establish the cooking conditions required to achieve a meat core temperature of 73 °C. Patties were placed on trays and cooked at 170 °C for 18 min in a forced-air oven. The cooking loss was calculated as: Cooking loss = [(Wb Wa)/Wb]  100 where Wb and Wa are the weights of the burger patties before and after cooking, respectively. The cooked burger patties were dispensed in polypropylene trays, wrapped with PVC film and subsequently stored for 12 days at + 2 °C under white fluorescent light (620 lux), simulating retail display conditions. At sampling (days 1, 4, 8 and 12), four burger patties per batch were taken out of the refrigerator and analysed for carbonyl gain and colour and texture. Storage loss was calculated as the weight loss during refrigerated storage of cooked burger patties as follows: storage loss = [(W1 W12)/W1]  100 where W1 and W12 are the weight of the cooked burger patties at days 1 and 12, respectively. 2.5. Proximate composition of burger patties Moisture and total protein contents were determined using official methods (AOAC, 2000a,b). The method of Folch, Lees, and Stanley (1957) was used for determining the fat content of the patties. 2.6. Determination of total carbonyls by the DNPH-method Protein oxidation, as measured by the total carbonyl content, was evaluated by derivatisation with DNPH as described by Oliver et al. (1987) with slight modifications. Burger patties (1 g) were minced and then homogenized 1:10 (w/v) in 20 mM sodium phosphate buffer containing 6 M NaCl (pH 6.5) using an ultraturrax homogenizer for 30 s. Two equal aliquots of 0.2 mL were taken from the homogenates and dispensed in 2 mL eppendorf tubes. Proteins were precipitated by cold 10% TCA (1 mL) and subsequent centrifugation for 5 min at 4200g. One pellet was treated with 1 mL 2 M HCl (protein concentration measurement) and the other with an equal volume of 0.2% (w/v) DNPH in 2 M HCl (carbonyl concentration measurement). Both samples were incubated for 1 h at room temperature. Afterwards, samples were precipitated by 10% TCA (1 mL) and washed three times with 1 mL ethanol:ethyl acetate (1:1, v/v) to remove excess DNPH. The pellets were then dissolved in 1.5 mL of 20 mM sodium phosphate buffer containing 6 M guanidine HCl (pH 6.5), stirred and centrifuged for 2 min at 4200g to remove insoluble fragments. Protein concentration was calculated from the absorption at 280 nm using BSA as standard. The amount of carbonyls was expressed as nmol of carbonyl per mg of protein using an absorption coefficient of 21.0 nM 1 cm 1 at 370 nm for protein hydrazones. The percent inhibition of fruit extracts against carbonyl gain was calculated as follows: [100  (C12 T12)/C12)] where T12 and C12 are the carbonyl contents of the treated and control samples at day 12.

2.4. Manufacture of burger patties 2.7. Colour measurements Six types of porcine burger patties were prepared with the addition of different fruit extracts (AU, CM, RC, RU) including negative (no added extract, C) and positive controls (added quercetin;

Surface colour measurements of cooked burger patties were performed using a Minolta Chromameter CR-300 (Minolta Camera

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Corp., Meter Division, Ramsey, NJ) which consisted of a measuring head (CR-300), with an 8 mm diameter measuring area and a data processor (DP-301). Before each session the chromameter was calibrated on the CIE colour space system using a white tile. The L* value indicates lightness (L* = 0 darkness, L* = 100 lightness); a* value indicates redness (+60 = red, 60 = green) and b* value indicates yellowness (+60 = yellow, 60 = blue). Colour measurements were made on the surface of each patty in triplicate at three randomly selected locations. Colour measurements were made at room temperature (22 °C) with illuminant D65 and a 0° angle observer at days 1, 4, 8, and 12 of storage. Saturation index and Hue angle (h°) values were obtained by the following equations: saturation index = (a*2 + b*2)0.5; h° = arctg b*/a*. A numerical total colour difference (DE) between burgers at day 1 and day 12 of storage was calculated by: DE1–12 = [(L12 L1)2 + (a12 a1)2 + (b12 b1)2)]1/2. The percent inhibition of fruit extracts against loss of redness was calculated as: 100 [100  (T1 T12)/(C1 C12)] where T and C are the redness values of the treated and control samples, respectively, and subscripts represents freshly cooked (1) and cooked and chilled samples (12). 2.8. Texture measurements Texture profile analysis (TPA) was performed at room temperature with a Texture Analyser TA-XT2i (Stable Micro Systems, Surrey, UK). Three cylindrical samples (2.5 cm diameter) were taken from random locations in each patty and subjected to a two-cycle compression test. The samples were compressed to 40% of their original height with a cylindrical probe of 5 cm diameter and a cross-head speed of 5 mm/s. Texture profile parameters were determined following descriptions by Bourne (1978) and the SMS manual (Stable Micro Systems, Surrey, UK). All analyses were performed in triplicate. The percent inhibition of fruit extracts against hardness increase was calculated as: 100 [100  (T12 T1)/ (C12 C1)] where T and C are the hardness values for the treated and control samples, respectively, and subscripts represents freshly cooked (1) and cooked and chilled samples (12). 2.9. Statistical analysis Four burger patties per batch and per storage day were produced and used as experimental units. All analyses were performed in triplicate in each burger patty (4 burger patties  3 analysis; n = 12 per batch and storage day). Analyses of variance (ANOVA) and Tukey tests by SPSS for Windows (v. 15.0) were carried out to study the effect of the addition of fruit extracts and quercetin on the measured parameters. Differences were considered significant at p 6 0.05. Relationships amongst protein oxidation measurement and instrumental colour and texture parameters were calculated using Pearson’s correlation coefficients.

Table 1 Chemical composition and cooking and storage losses of cooked burger patties with added fruit extracts and quercetin.

B

Moisture FatB ProteinB Cooking lossC Storage lossC

RC

CM

RU

AU

Q

SEMA

70.84 2.19 23.03 22.06 3.23

72.46 1.86 23.38 23.59 2.85

70.63 2.19 23.70 22.71 3.09

70.67 1.97 23.01 21.73 2.95

70.48 1.96 23.50 22.73 2.37

70.93 2.53 22.53 22.24 4.37

0.19 0.06 0.02 0.30 0.18

C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin. Values with a different letter (a–c) within a row are significantly different (P < 0.05). A Standard error of the mean (n = 72; except for storage loss, n = 24). B Expressed as g/100 g burger patty. C Expressed as percentage.

ties (Table 2). This increase was considerably more intense in the control samples (Ddays1–12 = 5.8 nmol carbonyls/mg protein) than in the treated counterparts (Ddays1–12 = 1.1–1.7 nmol carbonyls/ mg protein). The addition of fruit extracts significantly inhibited the formation of protein carbonyls in the burger patties. At all sampling days, control patties had significantly higher amounts of protein carbonyls than the treated ones. No differences in effectiveness between fruit extracts was observed as all treated burgers had similar amounts of protein carbonyls by the end of storage. In fact, all fruit extracts and quercetin showed similar percent inhibitions against the formation of protein carbonyls (Fig. 1). The addition of RU extracts had a significant effect on the colour displayed by cooked burger patties (Table 3). Burger patties with added RU were darker (lower L*-value) and had a redder colour (higher a*-value) than the patties from the other batches. In addition, RU patties displayed a less intense colour (lower saturation index) which was significantly closer to the red axis (lower hue angle value) than the other patties. In general, RC, CM and AU extracts had no effect on colour parameters whereas the addition of Q led to decreased L* values and increased b* and saturation index values. Subsequent refrigerated storage significantly influenced colour parameters with the redness and hue angle values from control and RU patties being the most affected. Redness significantly decreased in control (from 4.16 to 0.54) and in RU patties (from 9.36 to 8.07) but this parameter was unaffected in the remaining patties. In addition, hue angle values significantly increased in control and RU patties while no change was observed in samples from the other batches. Consistently, the total colour difference measured between day 1 and day 12 of refrigerated storage revealed significant differences between samples. Control patties suffered the most intense colour modification followed by RU samples whereas samples with added RC, AU, Q and CM displayed signifi-

Table 2 Protein hydrazonesA gain during refrigeration of cooked burger patties with added fruit extracts and quercetin.

3. Results The proximate composition of cooked burger patties with added fruit extracts and Q is shown in Table 1. Burger patties were analysed for moisture (70.5–72.5 g/100 g), fat (1.9–2.5 g/100 g) and protein (22.5–23.7 g/100 g) contents. The addition of fruit extracts did not affect the chemical composition as no significant differences were found amongst batches. The cooking and storage losses ranged from 21.7 to 23.6 g/100 g and from 2.4 to 4.4 g/100 g, respectively. The loss of weight after cooking and chill storage of burger patties was not affected by added fruit extracts. Chill storage had a significant effect on protein oxidation as the amount of carbonyl compounds increased significantly in all pat-

C

C Day 1 4 8 12 SEMC

RC a,z

3.68 5.70a,y 6.30a,y 9.52a,x 0.51

CM b,y

2.58 3.33b,xy 4.03b,x 4.21b,x 0.17

RU b,y

2.56 3.52b,xy 3.51bc,xy 3.97b,x 0.17

AU c,y

2.14 2.48b,xy 2.58c,xy 3.23b,x 0.16

SEMB

Q b,y

2.60 3.27b,xy 3.92b,x 4.29b,x 0.17

c,y

2.17 3.05b,xy 3.18bc,xy 3.73b,x 0.12

0.13 0.18 0.23 0.36 –

C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin. Values with a different letter (a–c) within a row of the same storage day are significantly different (P < 0.05). Values with a different letter (x–z) within a column of the same batch are significantly different (P < 0.05). A Expressed as nmol hydrazones/mg protein. B Standard error of the mean within the same storage day (n = 72). C Standard error of the mean within the same batch (n = 48).

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Fig. 1. Percentage inhibition of fruit extracts against the formation of protein hydrazones, loss of redness and increase of hardness during refrigerated storage of cooked burgers. a,b,c Different letters between groups denote significant differences (p < 0.05); ns = non significant.

Table 3 Instrumental colour measured during refrigerated storage of cooked burger patties with added fruit extracts. C

RC

CM

RU

AU

Q

SEMA

68.11a 69.19a 67.69a 67.91a 0.26

67.45ab 68.67a 68.07a 67.47a 0.33

66.76ab 67.63ab 66.48a 66.17a 0.28

59.25c,x 60.30c,x 60.04b,x 57.34b,y 0.24

66.91ab,xy 67.73ab,x 68.04a,x 65.24a,y 0.27

66.23b 66.82b 66.34a 66.52a 0.26

0.38 0.39 0.48 0.80 –

4.46b 4.37b 4.38b 4.32b 0.10

4.31bc 4.07b 4.04b 3.88b 0.09

4.25bc 4.15b 3.90b 4.07b 0.09

3.95c 3.95b 3.77b 3.98b 0.07

0.23 0.25 0.31 0.46 –

L* Day 1 4 8 12 SEMB a* Day 1 4 8 12 SEM

4.16bc,x 2.23c,y 1.13c,z 0.54c,z 0.24

9.36a,x 8.74a,y 8.42a,yz 8.07a,z 0.10

b* Day 1 4 8 12 SEM Saturation index Day 1 4 8 12 SEM

13.07b 13.21a 13.18a 13.43a 0.11

13.59ab 13.38a 13.03a 13.24a 0.25

14.48a 13.68a 13.59a 14.15a 0.19

8.28c 9.34b 9.54b 9.62b 0.21

13.23b 13.04a 13.38a 13.32a 0.17

14.22a 14.15a 13.74a 14.40a 0.15

0.26 0.24 0.26 0.39 –

13.73c 13.41ab 13.23 13.44 0.11

14.31abc 14.07ab 13.75 13.93 0.27

15.11a 14.28ab 14.18 14.68 0.20

12.54d 12.85b 12.75 12.57 0.12

13.90bc 13.69ab 13.94 13.93 0.18

14.76ab 14.70a 14.25 14.94 0.15

0.13 0.16 0.17 0.27 –

72.44ab,z 80.40a,y 85.08a,x 87.74a,x 1.01

71.80b 72.04b 71.50c 71.99b 0.19

73.43ab 73.53b 73.47bc 74.67b 0.27

41.32c,y 46.52c,xy 48.42d,xy 49.97c,x 0.90

72.21ab 72.46b 73.81bc 73.05b 0.27

74.46a 74.46b 74.70b 74.58b 0.24

1.39 1.33 1.61 2.35 –

4.20a

2.16c

1.02d

2.95b

1.73c

2.14c

0.13

Hue angle Day 1 4 8 12 SEM

DE *

C: Control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin. Values with a different letter (a–c) within a row of the same storage day are significantly different (P < 0.05). Values with a different letter (x–z) within a column of the same batch are significantly different (P < 0.05). A Standard error of the mean within the same storage day (n = 72). B Standard error of the mean within the same batch (n = 48).

cantly smaller changes. Once again, all fruit extracts and Q were similarly efficient at inhibiting the loss of redness during refrigerated storage of cooked burger patties. Most colour parameters significantly correlated with protein oxidation measurements (Table 4).

In general, the addition of fruit extracts significantly increased the hardness of cooked burger patties and other related parameters such as chewiness (Table 5). It is particularly remarkable the intense texture modifications suffered by the control and CM burger patties during the 12 days of storage. Hardness and chewiness sig-

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Table 4 Pearson’s correlation coefficients (R2)a between protein oxidation (Pox) as measured by protein hydrazones and the instrumentally measured colour and texture parameters. Pox L* a* b* Saturation index Hue angle Hardness Adhesiveness Springiness Cohesiveness Chewiness Resilience

p-value

0.33 0.60 0.22 0.21 0.50 0.01 0.18 0.48 0.44 0.12 0.31

<0.001 <0.001 0.048 0.284 <0.001 0.988 0.230 <0.001 <0.001 0.375 <0.001

a n = 48 Cooked burger patties for correlation coefficients calculated from measurements on day 1 and day 12 of refrigerated storage.

nificantly increased in the aforementioned groups whereas these parameters were unaffected in burger patties with added Q and RC, AU and RU extracts. At day 12, burger patties with added CM displayed significantly higher values for hardness and chewiness. Fruit extracts and Q displayed significantly different percent inhi-

bitions against hardness increase during chill storage. RC was the most effective fruit extract at inhibiting the increase of hardness, followed by RU, AU, Q and finally, CM. Various other texture parameters, namely, springiness, cohesiveness and resilience showed significant correlations with protein oxidation measurements.

4. Discussion 4.1. Protein oxidation The protein carbonyl content was used as a measure of the extent of oxidative reactions affecting muscle proteins during refrigerated storage of burger patties. The onset of lipid oxidation in cooked and refrigerated meats and the impact of these reactions on meat quality (i.e. warmed-over flavour) have been extensively studied (reviewed by Ladikos & Lougovois (1990) and Gray & Pearson (1994)) whereas research on the onset and extent of protein oxidation in processed meat products is limited. The increase of protein carbonyls show that muscle proteins in cooked patties are susceptible to oxidative reactions leading to carbonyl gain. Carbonyl compounds are formed as a result of the oxidative degradation of side chains of lysine, proline, arginine and histidine residues

Table 5 Texture parametersA measured during refrigerated storage of cooked burger patties with added fruit extracts and quercetin. C

RC

CM

RU

AU

Q

SEMB

Day 1 4 8 12 SEMC

23.28c,y 28.89ab,xy 29.39ab,xy 34.08bc,x 1.04

31.45ab 34.58a 33.99a 33.45bc 0.98

29.00ab,z 32.83ab,yz 36.17a,xy 42.14a,x 1.16

32.65a 35.81a 36.08a 36.29b 0.99

28.48ab 30.99ab 32.55ab 32.39bc 0.92

24.36bc 25.62b 25.64b 27.11c 0.89

0.81 0.88 0.89 0.91 –

Day 1 4 8 12 SEM

-0.02 0.10 0.21 0.19 0.06

-0.61 -0.08 -0.27 -0.09 0.09

-0.35 -0.21 0.09 0.02 0.09

-0.26 0.04 -0.14 -0.22 0.08

-0.43 -0.24 -0.33 -0.18 0.10

-0.17 0.15 0.14 0.12 0.10

0.07 0.07 0.07 0.08 –

Day 1 4 8 12 SEM

0.96y 0.96y 0.97y 1.02a,x <0.01

0.94y 0.95xy 0.96xy 0.98ab,x <0.01

0.95 0.95 0.95 0.98ab <0.01

0.94y 0.95xy 0.95xy 0.97ab,x <0.01

0.96 0.97 0.95 0.95b <0.01

0.96 0.97 0.95 0.99ab <0.01

<0.01 <0.01 <0.01 <0.01 –

Day 1 4 8 12 SEM

0.66a 0.66 0.67 0.67 <0.01

0.64b 0.65 0.65 0.65 <0.01

0.65ab 0.66 0.66 0.65 <0.01

0.65ab 0.65 0.65 0.66 <0.01

0.65ab 0.66 0.65 0.66 <0.01

0.66a 0.65 0.66 0.66 <0.01

<0.01 <0.01 <0.01 <0.01 –

Day 1 4 8 12 SEM

14.58b,y 18.16ab,xy 19.87x 22.30ab,x 0.77

18.94ab 21.17ab 21.15 22.76ab 0.67

18.82ab,y 20.61ab,y 22.48y 27.65a,x 0.81

20.27a 21.96a 22.32 23.00ab 0.56

17.74ab 22.07a 20.16 20.43b 0.71

15.37b 16.23b 17.49 17.83b 0.59

0.50 0.59 0.52 0.67 –

Day 1 4 8 12 SEM

0.46a 0.45a 0.46a 0.48 <0.01

0.42b,y 0.41b,y 0.41c,y 0.45x <0.01

0.43ab 0.43ab 0.42bc 0.44 <0.01

0.43ab 0.42b 0.42bc 0.45 <0.01

0.44ab 0.45a 0.43abc 0.46 <0.01

0.46a 0.45a 0.45ab 0.48 <0.01

<0.01 <0.01 <0.01 <0.01 –

Hardness

Adhesiveness

Springiness

Cohesiveness

Chewiness

Resilience

C: control; RC: Rosa canina; CM: Crataegus monogyna; RU: Rubus ulmifolius; AU: Arbutus unedo; Q: quercetin. Values with a different letter (a–c) within a row of the same storage day are significantly different (P < 0.05). Values with a different letter (x–z) within a column of the same batch are significantly different (P < 0.05). A Units for texture parameters in Section 2. B Standard error of the mean within the same storage day (n = 72). C Standard error of the mean within the same batch (n = 48).

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(Stadtman & Levine, 2003). The formation of specific protein carbonyls, a-amino adipic semialdehyde (AAS) and c-glutamic semialdehyde (GGS), during in vitro oxidation of myofibrillar proteins has been confirmed (Estévez, Ollilainen, & Heinonen, 2009). These semialdehydes could have contributed to the carbonyl gain found as they account for around 70% of total protein carbonyls in oxidized proteins (Requena, Chao, Levine, & Stadtman, 2001). The large amount of protein carbonyls in the present study denotes more intense oxidative reactions than described in previous studies on raw pork and beef subjected to chill storage (Lund, Lametsch, et al. (2007); Lund, Hviid, & Skibsted, 2007; Martinaud et al., 1997). The initiation of muscle proteins oxidation requires further clarification although it is generally accepted that heme and non-heme iron as well as oxidizing lipids play major roles (Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008b; Estévez et al., 2008a; Kroger-Ohlsen, Ostdal, & Andersen, 2003; Salminen et al., 2008). In the present experiments, mincing and the high temperatures applied during cooking could have enhanced protein oxidation in the patties. Disruption of the tissues leads to the release of pro-oxidants naturally present in the muscle and enhance the incorporation of oxygen in the system. Heating degrades myoglobin causing the release of iron which is believed to increase its pro-oxidant potential in cooked meats (Kristensen & Purslow, 2001; Schrickler & Miller, 1983). In fact, it is probable that nonheme iron is a main initiator of protein oxidation in cooked meat systems as the formation of the major carbonyls, AAS and GGS, requires the presence of metals (Requena et al., 2001) and iron has been shown to be an effective promoter of carbonyl formation in myofibrillar proteins (Estévez et al., 2009). The results confirm the ability of the selected fruit extracts to act as efficient inhibitors of muscle protein oxidation. The protecting effect of added fruit extracts against protein oxidation can be attributed to the phenolic compounds naturally present in RC (11.8 mg gallic acid equivalents (GAE)/g fruit), CM (20.7 mg GAE/ g fruit), RU (5.0 mg GAE/g fruit) and AU (4.3 mg GAE/g fruit) (Ganhão et al., 2008). These fruits were found to contain high amounts of phenolic compounds and display intense antioxidant activities in vitro against DPPH and ABTS radicals (Ganhão et al., 2008). Specifically, RC and CM contains high amounts of proanthocyanidins, RU is particularly rich in ellagic acid, ellagetannins and anthocyanins and AU has high contents of catechins and benzoic acids (Bahorun, Luximon-Ramma, Crozier, & Aruoma, 2004; Dall’Acqua, Cervellati, Loi, & Innocenti, 2008; Hellström, Törrönen, & Mattila, 2009). Previous studies have reported antioxidant activities of phenolic compounds and diverse plant materials against protein carbonyl formation in various meat products such as beef patties (Lund, Hviid, et al., 2007), frankfurters (Estévez et al., 2005) and liver pâtés (Estévez, Ventanas, & Cava, 2006). Plant phenolics also display antioxidant potential against other protein oxidations such as tryptophan depletion in myofibrillar proteins (Estévez et al., 2008a; Estévez et al., 2008b) and loss of thiol groups in turkey meat (Mercier, Gatellier, Viau, Remignon, & Renerre, 1998). As redox-active compounds, plant phenolics also display pro-oxidant actions towards myofibrillar and other food proteins (Viljanen, Kivikari, & Heinonen, 2004; Estévez et al., 2008a). This effect, however, was not observed in the present study. In fact, fruit extracts displayed higher percent inhibitions against carbonyl gain than selected plant phenolics added at similar GAE concentrations (Estévez et al., 2008a). The precise mechanisms involved in the antioxidant actions of plant phenolics on myofibrillar proteins are not well understood. Phenolics from the tested fruits are known to act as efficient radical scavengers and could have blocked the pro-oxidant action of ROS on proteins. Additionally, some phenolics such as cyanidin-3-glucoside, which is present in RC and RU, display metal-chelating activities (Rice-Evans et al., 1995). This might be particularly rele-

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vant in cooked meats as phenolics could have hindered the prooxidant action of non-heme iron by chelation. Both antioxidant mechanisms would explain the results obtained since certain ROS such as hydroxyl radicals and iron are directly involved in the formation of protein carbonyls from myofibrillar proteins (Estévez et al., 2009). By inhibiting protein carbonyl formation, fruit extracts enhance the nutritional value of porcine burgers. Besides the loss of essential amino acids, the formation of carbonyl compounds from oxidizing protein might have additional repercussions on particular quality traits, as discussed below. 4.2. Colour deterioration The red-brownish colour of cooked meats is mainly determined by the presence of denatured-globin hemochromes formed as a result of high temperatures (Fox, 1994). During cooking, the heme protein is denatured and the iron is oxidized into its ferric form and the hematin pigment remains intact (Lawrie, 1998). Additionally, the formation of coloured Maillard products on heating, the physico-chemical state of proteins and other meat components and the addition of colorants are also influential (Fox, 1994; Lawrie, 1998). Amongst the fruit extracts, RU was the only one to significantly influence the colour of freshly cooked burger patties (day 1). Anthocyanins, which are major pigment constituents of RU, literally dyed burger patties with a distinct purplish colour. The impact of RU pigments on burger patties led to an intense increase of redness and a decrease of the other colour parameters compared to burger patties from the other batches. In contrast to RU, the other fruits contain small quantities of anthocyanins which explain their lack of influence on the colour of cooked patties. An intense redbrownish colour is expected and generally preferred by cooked meat consumers (Aaslyng et al., 2007; Berry, 1998). In the absence of sensory evaluation of the samples, the impact of the peculiar colour displayed by patties with added RU extracts on consumer’s acceptance remains unknown. Control burger patties underwent intense discolouration during chill storage. According to Francis and Clydesdale (1975), the colour modifications instrumentally measured can be considered as noticeable visual changes when the total colour difference (DE1– 12) values are higher than 2. Thus, colour changes in the control patties during chill storage would be very noticeable to consumers whereas the addition of fruit extracts and Q preserved the colour of freshly made patties. The loss of redness and increase of hue angle values have been described in raw and cooked meats subjected to refrigerated and frozen storage (Estévez, Morcuende, & Cava, 2003; Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007; Hassaballa, Mohamed, Ibrahim, & Abdelmageed, 2009; Ramírez, Morcuende, Estévez, & Cava, 2004). The discolouration of raw burger patties is generally attributed to the oxidation of ferrous heme– iron (Fe2+) into its ferric form (Fe3+) induced by lipid oxidation products (Yin & Faustman, 1993). Under these circumstances, oxymyoglobin is transformed into metmyoglobin and the colour shifts from a pleasant bright red to an undesirable brownish colour. The specific reason for the discolouration of cooked meats is not clear (Fox, 1994). Ramírez et al. (2004) adapted the classical theory for raw meat discoloration to cooked meats by claiming that the colour deterioration in cooked pork during refrigerated storage was caused by lipid oxidation products. Unfortunately, these authors provide no valid arguments or further details about the mechanism. Fernández-Ginés, Fernández-López, Sayas-Barberá, Sendra, and Pérez-Alvarez (2003) suggested that colour deterioration during refrigerated storage of cured and cooked meats is explained by the oxidative degradation of certain nitrosopigments. Estévez and Cava (2004) and Estévez et al. (2005) reported significant correlations between protein oxidation and discolouration of cooked liver pâtés and frankfurters subjected to chill storage. According to these

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authors, the oxidative degradation of the denatured-globin and the oxidative cleavage of the hematin pigment would lead to the release of iron from the heme molecule causing the eventual discolouration of the cooked meat. It is reasonable that the colour changes reported in the present study are caused by oxidative reactions since the addition of substances with proven antioxidant activity inhibited the discoloration of cooked, chilled patties. Although lipid-derived products could have induced pigment oxidation and meat discolouration, the impact of protein oxidation might be considerably higher as the present patties had 10-fold more protein than lipid (20% protein vs. 2% lipid). Protein radicals and other protein oxidation products are known to induce lipid oxidation (Viljanen, Kivikari, & Heinonen, 2004) and hence, heme pigments in cooked meats could also be affected by their pro-oxidant action. The significant negative correlation between redness and protein oxidation measurements ( 0.60; p < 0.001) support this hypothesis. The discoloration suffered by patties with added RU might not be due to oxidative changes in muscle pigments and proteins as this fruit extract was highly effective against protein oxidation. The oxidative degradation of anthocyanins, which are mainly responsible for the colour of RU patties, has been well documented (Rein & Heinonen, 2004) and would explain the slight discolouration recorded for these samples during chill storage. Besides the effect of oxidation on meat and fruit pigments, physico-chemical changes affecting other meat components could have enhanced the colour changes. In fact, protein carbonyls, assessed in this study, as a measure of protein oxidation, are involved in the formation of protein cross links through further oxidative reactions (Requena et al., 2001; Xiong, 2000). The formation of cross links and protein aggregates as a result of protein oxidation during chill storage of burger patties could have affected light reflection and hence, could have contributed to the discolouration. Hassaballa et al. (2009) have recently ascribed colour changes in cooked tuna muscle to the aggregation of myofibrillar proteins during frozen storage. 4.3. Texture deterioration The increase of hardness and other related texture parameters such as chewiness in chilled control burgers is highly undesirable as this could have a great impact on consumer acceptability. Hardness increases during refrigerated storage of burger patties and other cooked meat products has been reported (Estévez et al., 2005; Fernández-Ginés et al., 2003; Fernández-López, Sayas-Barberá, Sendra, & Pérez-Alvarez, 2004; Hassaballa et al., 2009). In agreement with the effect of added fruit extracts on colour changes, the addition of most fruit extracts and Q inhibited the texture deterioration seen in the control samples. Although loss of moisture during storage could explain the increase of hardness in burger, this is not applicable in the present study since all samples underwent similar weight losses during storage. In addition, the proximate composition of control and treated patties was similar at day 1 and at day 12 (data not shown). On the other hand, oxidative damage to proteins has an impact on protein solubility, leading to the aggregation and complex formation due to cross link formation (Karel, Schaich, & Roy, 1975). It is plausible that protein oxidation led to an increase of hardness in burger patties through the formation of protein carbonyls, the loss of protein functionality and the formation of cross links between proteins. Phenolics from fruit extracts would have reduced hardness increases in patties through the inhibition of protein oxidation during refrigerated storage. The lack of significance for the correlation between protein oxidation measurements and instrumental hardness is likely due to the contradictory effect on CM. This fruit extract was efficient against protein oxidation and particularly effective at inhibiting

colour changes but patties with added CM suffered marked texture changes. Unlike the other fruits, Crataegus spp. contains large amounts of pectins with highly efficient gelling capacities (Kuliev & Poletaeva, 1982). The texture changes in patties with added CM are probably induced by physico-chemical modifications of CM polysaccharides during chill storage. The retrogradation of these gums during storage probably led to the increase of hardness in the food matrixes. Regarding the influence of fruit extracts on texture changes, RC had the most intense effect against hardness increase compared to the other fruit extracts. 5. Conclusions The wild Mediterranean fruits tested displayed intense antioxidant activity against protein oxidation and could play an important role as functional ingredients in burger patties by improving their oxidative stability and quality. The addition of R. ulmifolius would affect the colour displayed by burger patties whereas the addition of C. monogyna would supply some negative texture properties to these products. Some other fruits such as R. canina improve the oxidative stability, colour and texture of cooked burger patties with no apparent drawbacks. Protein oxidation might play a major role in the nutritional and sensory quality of meat products. Further investigations should confirm some of the likely hypothesis and mechanisms reported. The development of more sensitive and specific methods for assessing protein oxidation are required to shed light on the chemical nature of these complex reactions. References Aaslyng, M. D., Oksama, M., Olsen, E. V., Bejerholm, C., Baltzer, M., Andersen, G., et al. (2007). The impact of sensory quality of pork on consumer preference. Meat Science, 76, 61–73. AOAC. (2000a). Moisture content 950.46. In Official methods of analysis (17th ed.). Gaithersburgh, Maryland: Association of Official Analytical Chemists. AOAC. (2000b). Protein content in meat 928.08. In Official methods of analysis (17th ed.). Gaithersburgh, Maryland: Association of Official Analytical Chemists. Bahorun, B., Luximon-Ramma, T., Crozier, A., & Aruoma, A. (2004). Total phenol, flavonoid, proanthocyanidins and vitamin C levels and antioxidant activities of Mauritian vegetables. Journal of the Science of Food and Agriculture, 84, 1553–1561. Berry, B. W. (1998). Cooked color in high pH beef patties as related to fat content and cooking from the frozen or thawed state. Journal of Food Science, 63, 1–4. Bourne, M. C. (1978). Texture profile analysis. Food Technology, 33, 62–66. Bravo, L. (1998). Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56, 317–333. Dall’Acqua, S., Cervellati, R., Loi, M. C., & Innocenti, G. (2008). Evaluation of in vitro antioxidant properties of some traditional Sardinian medicinal plants: Investigation of the high antioxidant capacity of Rubus ulmifolius. Food Chemistry, 106, 745–749. Estévez, M., & Cava, R. (2004). Lipid and protein oxidation, release of iron from heme molecule and colour deterioration during refrigerated storage of live pâté. Meat Science, 68, 551–558. Estévez, M., Kylli, P., Puolanne, E., Kivikari, R., & Heinonen, M. (2008a). Oxidation of skeletal muscle myofibrillar proteins in oil-in-water emulsions: interaction with lipids and effect of selected phenolic compounds. Journal of Agricultural and Food Chemistry, 56, 10933–10940. Estévez, M., Kylli, P., Puolanne, E., Kivikari, R., & Heinonen, M. (2008b). Fluorescence spectroscopy as a novel approach for the assessment of myofibrillar protein oxidation in oil-in-water emulsions. Meat Science, 80(4), 1290–1296. Estévez, M., Morcuende, D., & Cava, R. (2003). Oxidative and colour changes in meat from three lines of free-range reared Iberian pigs slaughtered at 90 kg live weight and from industrial pig during refrigerated storage. Meat Science, 65, 1139–1146. Estévez, M., Ollilainen, V., & Heinonen, M. (2009). Analysis of protein oxidation markers – a-Aminoadipic and c-glutamic semialdehydes – In food proteins by using LC-ESI-multi-stage tandem MS. Journal of Agricultural and Food Chemistry, 57, 3901–3910. Estévez, M., Ventanas, S., & Cava, R. (2005). Protein oxidation in frankfurters with increasing levels of added rosemary essential oil: Effect on colour and texture deterioration. Journal of Food Science, 70, 427–432. Estévez, M., Ventanas, S., & Cava, R. (2006). Effect of natural and synthetic antioxidants on protein oxidation and colour and texture changes in refrigerated stored porcine liver pâté. Meat Science, 74, 396–403. Fernández-Ginés, J. M., Fernández-López, J., Sayas-Barberá, E., Sendra, E., & PérezAlvarez, J. A. (2003). Effects of storage conditions on quality characteristics of bologna sausages made with citrus fiber. Journal of Food Science, 68, 710–715.

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