Influence of lamb age and high-oxygen modified atmosphere packaging on protein polymerization of long-term aged lamb loins

Food Chemistry 135 (2012) 122–126

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Influence of lamb age and high-oxygen modified atmosphere packaging on protein polymerization of long-term aged lamb loins Yuan H. Brad Kim ⇑, Sabina Bødker, Katja Rosenvold AgResearch Ltd., Ruakura Research Centre, Hamilton, New Zealand

a r t i c l e

i n f o

Article history: Received 23 December 2011 Received in revised form 20 March 2012 Accepted 19 April 2012 Available online 27 April 2012 Keywords: Lamb Tenderness High oxygen MAP Protein polymerization Oxidation

a b s t r a c t The objective of this study was to determine the influence of lamb age and high-oxygen modified atmosphere (HiOx-MAP) on tenderness of loins during display. Loins from 36 carcasses of two different age groups [4-month-old (New season; NS) and 11-month-old lambs (Old season; OS)] were vacuum-packaged and stored for 8 weeks at 1.5 °C. After storage, the loins were cut into 6-cm thick chops, assigned to either HiOx-MAP or oxygen-permeable overwrap-PVC, and then displayed for 8 days at 3 °C. Initially, packaging methods did not influence shear force (P > 0.05). However, at the end of display, a significant increase in shear force and lipid oxidation was found in OS loins under HiOx-MAP. SDS–PAGE and Western blot results found a greater extent of cross-linked myosin products from OS loins in HiOx-MAP compared to NS loins. These results suggest that HiOx-MAP can adversely influence meat quality of fullytenderized loins, and meat from older lamb may be more susceptible to an oxidizing environment of HiOx-MAP. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Modified atmosphere packaging (MAP) incorporating high oxygen (80%) and carbon dioxide (20%) has been extensively used as a retail-ready packaging method in some countries, because it provides an extension of shelf life of fresh meat by suppressing microbial growth, and prolongs the bright cherry-red ‘‘bloomed’’ colour during display (Eilert, 2005; McMillin, 2008; Phillips, 1996). However, oxidation-related quality defects such as greater extents of discolouration, rancid flavour, and reduced tenderness and juiciness of meat packaged in high-oxygen MAP (HiOx-MAP) compared to low-oxygen utilized packaging methods (e.g. vacuum, oxygen permeable overwrap-PVC and low-oxygen MAP) have been reported in many studies (Aaslyng, Tørngren, & Madsen, 2010; Grobbel, Dikeman, Hunt, & Milliken, 2008; Kim, Frandsen, & Rosenvold, 2011; Kim, Huff-Lonergan, Sebranek, & Lonergan, 2010; Lund, Heinonen, Baron, & Estévez, 2011; Lund, Lametsch, Hviid, Jensen, & Skibsted, 2007). Further, several studies recently reported that decreased tenderness of fresh meat packaged in high-oxygen MAP could be a consequence of a formation of a cross-linked myosin heavy chain (MHC) induced by protein oxidation (Jongberg, Skov, Tørngren, Skibsted, & Lund, 2011; Kim et al., 2010; Lund et al., 2007; Zakrys-Waliwander, O’Sullivan, O’Neill, & Kerry, 2012). For exporting chilled-unfrozen meat cuts, generally, it takes several weeks to ship the meat products to overseas markets. Once arrived, chilled-unfrozen lamb meats are typically repackaged into ⇑ Corresponding author. Tel.: +64 7 838 5152; fax: +64 7 838 5012. E-mail address: [email protected] (Y.H.B. Kim). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.04.109

either HiOx-MAP or a conventional overwrapping with polyvinylchloride (PVC) film for retail display. Due to the extended ageing time of the meat products during shipping at chilling temperature (e.g. 1.5 °C), meat tenderness is well-developed through the myofibrillar protein degradation (Farouk et al., 2007; Olson, Parrish, & Stromer, 1976). However, the long-term aged meat might have an inferior anti-oxidation potential, and thus it could be more susceptible to the oxidative condition associated with HiOx-MAP (Kim et al., 2011; Xiong et al., 2007). In New Zealand, lambs can be generally classified into two different age groups – new season (NS) and old season (OS) lambs. NS lambs are normally born, raised and marketed around August–November (winter–spring in NZ) when 3–4 month old, whereas OS lambs have been kept on farm since the previous January–February (summer in NZ) giving 10– 11 month old lambs when marketed resulting in an approximate seven month difference between lambs from the two seasons. However, as oxidative stability of meat can be adversely influenced by animal age (Xiong et al., 2007), it can be speculated that longterm chilled loins from OS lamb carcasses might be more susceptible to an oxidizing environment (such as HiOx-MAP) and thus result in more oxidation-related quality defects compared to their younger counter parts. Furthermore, although the effect of HiOxMAP on biochemical attributes (particularly protein polymerization with MHC) and tenderness of beef (Kim et al., 2010; ZakrysWaliwander, O’Sullivan, O’Neill, & Kerry, 2012) and pork (Lund et al., 2007) has been determined, its influence on chilled lamb loins for extended periods has not previously been investigated. Therefore, the objective of this study was to determine the influence of lamb age and HiOx-MAP on oxidation stability, protein

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polymerization and instrumental tenderness of long-term chilled lamb loins during retail display. 2. Materials and methods 2.1. Raw materials and processing A total of 36 lamb carcasses (18 from each season: NS – 3 monthold (17 kg carcass weight) and OS – 10 month-old (16 kg carcass weight) were selected from a commercial abattoir. Lamb carcasses with an ultimate pH24 h of 65.8 measured at between 11th and 12th rib of each carcass were only selected in order to avoid any confounding interactions of a high ultimate pH (P5.8) with other biochemical and physical characteristics. At 24 th post mortem, paired boneless loins (M. longissimus lumborum; New Zealand Meat Specification Code# 3341) from each carcass were excised; yielding 72 loins in total = 36 lambs (n = 18 for NS and OS, respectively)  2 sides, vacuum-packaged and transported (under ice) to AgResearch Ruakura. The loins were then stored for 8 weeks at 1.5 °C to simulate current NZ industry meat export practice. After 8 weeks, the loins were cut into chops (6-cm thick), randomly repackaged into either HiOx-MAP or overwrapped with PVC film and displayed for 8 days at 3 °C under continuous fluorescent natural white light (2800 lx, CRI = 82, Colour temperature = 4000 K; Osram, Auckland, New Zealand). For HiOx-MAP, the loin cuts were put in a shrinkable bag (BB7L, 30 by 39 cm/an oxygen-transmission rate of 20 cc O2/ 1 cm3/m2/24 h at 23 °C and a water-vapour transmission rate of 10 cc/m2/24 h at 23 °C; Cryovac Sealed Air Corporation, Hamilton, New Zealand), and a high-oxygen modified atmosphere (80% O2/ 20% CO2, Certified Standard within ±5%, BOC GASES; Hamilton, New Zealand) was accomplished by using a Securepak 10 Controlled Atmosphere Packaging Machine (Securefresh Pacific, Auckland, New Zealand) by applying vacuum, then flushing the package with the gas mixture, and sealing. For PVC, trays were wrapped with oxygen-permeable polyvinyl chloride film (23,000 cc/O2/m2/24 h at 23 °C). The change of gas composition in HiOx-MAP was monitored by using a headspace oxygen/carbon dioxide analyzer (PBI Dansensor, Glen Rock, NJ) confirming a high-oxygen level (>70% O2) being maintained throughout the entire display period. 2.2. pH pH was measured by inserting a calibrated pH probe (Testo 205 pH meter, Lenzkirch, Germany) directly into the meat. Duplicate readings were taken for each sample at 24 h post mortem and after 8 weeks of ageing at 1.5 °C.

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2-thiobarbituric acid method described by Buege and Aust (1978) with a few modification. In brief, powdered frozen muscle tissues (5.0 g) were homogenized in three volume of distilled water and centrifuged at 2000g for 10 min at 4 °C. The supernatant (2 ml) was mixed with trichloroacetic acid (15%)/thiobarbituric acid (20 mM) solution with BHT (10%), incubated for 15 min in a boiling water bath (around 100 °C) and cooled for 10 min in cold water. After centrifuging the solution one more time, the absorbance of filtered supernatant was measured at 520 nm using a Shimadzu UV-2450 UV–vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan). The absorption found was converted to mg malonaldehyde/kg meat and reported as thiobarbituric acid-reactive substances (TBARS). TBARS level was found using known concentrations of malonaldehyde (MDA) as standard curves. 2.5. SDS–PAGE Whole muscle protein sample preparation for reducing (with bmercaptoethanol; MCE) and non-reducing conditions (with N-ethylmaleimide; NEM) for the SDS–PAGE and Western blot was conducted as described by Kim et al. (2010). The prepared gel samples were loaded at 40 lg of protein per lane on a 7.5% polyacrylamide gel. The gels were silver-stained (Bio-rad Ltd., Albany, New Zealand), and then photographed by using densitometry (GBox: Chemi HR 16; Syngene, USA). 2.6. Western blot Western blots to determine cross-linking of myosin heavy chain (CL-MHC), desmin and troponin-T of the gels samples containing NEM were assayed using a 7.5% (CL-MHC) and 10% gel (desmin and troponin-T) as described by Kim et al. (2010). The blots were incubated at 4 °C with their respective primary antibody (desmin = 1:20,000 dilution with PBS-Tween, monoclonal anti-desmin antibody, D1033; Sigma, St. Louis, MO, USA; troponinT = 1:20,000 dilution with PBS-Tween, monoclonal anti-troponinT antibody, JLT-12; Sigma, St. Louis, MO, USA; MHC = 1:10,000 dilution with PBS-Tween, monoclonal anti-MHC antibody, M8421; Sigma, St. Louis, MO, USA), and their respective secondary antibody (troponin-T, desmin and MHC = 1:20,000 dilution with PBS-Tween, goat anti-mouse-HRP, No. 170-6516; BioRad Laboratories, Auckland, New Zealand). Protein bands were detected using a chemiluminescent detection kit (Pierce ECL Western Blotting Substrate, Thermo Scientific, Rockford, IL, USA), and qualitatively captured the image by using G-Box (Chemi HR 16; Syngene, USA).

2.3. Shear force

2.7. Data analyses

Shear force was measured for the loins displayed for 1 and 8 days under either HiOx-MAP or PVC. The loins were placed into plastic bags and were cooked in a water bath set at 99 °C to an internal temperature of 75 °C, which was monitored by a DigiSense scanning temperature logger (Eutech Instruments Pte Ltd., Singapore). After cooling, 10  10 mm cross section samples were cut and sheared perpendicular to the fibre direction using MIRINZ Tenderometer (MIRINZ Inc., Hamilton, New Zealand) to assess instrumental tenderness values by determining the amount force to shear (Chrystall & Devine, 1991; MacFarlane & Marer, 1966). Ten replicates were measured for each sample. The results were expressed as shear force (kg F).

The design was a split–split plot where the whole plot was the age classes (NS/OS), the sub-plot was the packaging method with a random assignment (HiOx-MAP/PVC), and the sub–sub-plot was the retail display day. The data were analysed using the ANOVA directives of GenStat (2008) with animals and loin sides within animals as the block. Treatment effects were determined using Wald statistics. Least squares means for each attribute were separated (F test, P < 0.05) by using least significant differences.

2.4. Lipid oxidation

A slight increase in pH was observed after 8 weeks of storage (increased from 5.7 to 5.8 from 1 day to 8 weeks post mortem; P < 0.05; Table 1). A similar result of a subtle increase in pH after the long-term chills-storage of lamb loins under vacuum was

Lipid oxidation of the loins displayed for 1 and 8 days under the different retail packaging conditions was measured using the

3. Results and discussion 3.1. pH

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reported (Kim et al., 2011). However, there was no difference in pH between NS and OS loins either at 1 day post mortem or after 8 weeks of vacuum storage at 1.5 °C (P > 0.05; Table 1). 3.2. Shear force The long term chill-storage of loins resulted in very low shear force values overall (below 4 kg F), which could be considered very tender by consumers (Bickerstaffe, Bekhit, Robertson, Roberts, & Geesink, 2001). However, interestingly, a significant packaging  display day  age class interaction in shear force values was found during retail display (Table 1). The PVC packaged loins had no change in shear force values regardless of the age class and retail display period (P > 0.05), whilst a significant increase was observed in the OS loins packaged under HiOx-MAP at the end of display period. In contrast, the NS loins in HiOx-MAP had no change in shear force during display (P > 0.05). Tenderness change of meat packaged in HiOx-MAP has been reported in beef (Kim et al., 2010; Tørngren, 2003) and pork (Lund et al., 2007) studies, where increased instrumental tenderness values and decreased sensory tenderness and juiciness of fresh meat in HiOx-MAP were found after storage. However, the post mortem storage period of the meat tested in these studies was less than 2 weeks, whereas the current study determined well-aged long-term chilled meat (8 weeks at 1.5 °C) prior to retail packaging into HiOx-MAP. Thus, the current shear force data indicate that meat tenderness can be still negatively influenced by HiOx-MAP during retail display even after the long-term chill-storage period. Further, it is suggested that the extent of an increase in toughness of meat under HiOx-MAP may be influenced by different animal age classes. 3.3. Lipid oxidation There were significant packaging  age class and packaging  display day interactions in lipid oxidation (Table 1). The loins packaged in HiOx-MAP resulted in an increase (P < 0.05) in TBARS values at the end of display, whereas the loins in PVC appeared to have no significant change over display period (although there was a general trend toward an increase in lipid oxidation with display time). Particularly, the OS loin in HiOx-MAP resulted in the highest TBARS value at day 8 indicating that the largest extent of oxidation had occurred in the OS loin in HiOx-MAP; there was a

Table 1 Effects of different lamb age classes (New Season; NS or Old season; OS) and retail packaging types (high-oxygen modified atmosphere package and MAP or overwrap PVC) on pH, shear force values and lipid oxidation (TBARS) of lamb M. longissimus during retail display, which were vacuum-chilled stored for 8 weeks at 1.5 °C prior to retail display for 8 days at 3 °C. Trait

MAP

Shear forceB C

TBARS

NS OS NS OS

Day 1

Day 8

Day 1

Day 8

3.2a 3.4a 0.9a 0.4a

3.1a 3.8b 2.7bc 3.9c

3.2a 3.5a 1.6ab 0.4a

2.9a 3.3a 2.0ab 1.2a

1 day D

pH A

NS OS

SEDA

PVC

a

5.67 5.70a

0.19 0.66

8 weeks 5.79b 5.82b

0.02

Standard errors of difference between means. Shear force values were expressed as kg F. C TBARS (mg malondialdehyde/kg meat) was measured to determine lipid oxidation. D pH measured at 1 day post mortem and after 8 weeks of vacuum chilled storage at 1.5 °C. abc Pooled means with different superscript letters within each trait are different (P < 0.05). B

trend (P = 0.08) of lower lipid oxidation in the NS loin compared to the OS loin in HiOx-MAP at day 8. This observation is consistent with previous studies (Jongberg et al., 2011; Kim et al., 2010; Zakrys, Hogan, O’Sullivan, Allen, & Kerry, 2008), where high oxygen atmospheres induced greater extent of lipid oxidation of fresh meat during storage compared to lower oxygen or anaerobic packaging conditions. An increase in lipid oxidation is associated with rancid odour or undesirable flavour of meat products (Jayasingh, Cornforth, Brennand, Carpenter, & Whittier, 2002; Tarladgis, Watts, Younathan, & Dugan, 1960; Zakrys, Hogan, O’Sullivan, Allen, & Kerry, 2008). The OS loin in HiOx-MAP appeared to be more susceptible to the oxidizing environment compared to the NS loin, which could indicate different oxidative stability based on the age of the animals. Xiong et al. (2007) found that the rate of lipid oxidation increased with an increase in animal age concluding that the oxidative stability of lipids decreased with age. Thus, the OS loin might have inferior oxidative stability against a high oxygen condition compared to the NS loin during retail display that might affect overall lipid oxidation stability after the long-term vacuum chill-storage. 3.4. Protein polymerization of MHC and proteolysis SDS–PAGE results revealed a clear presence of a high-molecular weight protein polymerization product in the loins packaged in HiOx-MAP after 8 days of retail display period (Fig. 1). Westernblot assay further determined that the high molecular weight protein aggregation was cross-linked MHC, which was more prevalent in the loins packaged in HiOx-MAP compared to the loins in PVC (Fig. 2). It is noteworthy that a greater extent of intermolecular cross-linked MHC was observed in the OS loin compared to the NS loins at day 8. This phenomenon clearly corresponded to the results from lipid oxidation discussed above, where greater TBARS values were found in the loins under HiOx-MAP and particularly in the OS loin rather than in the NS loin. The formation of crosslinked MHC due to protein oxidation of fresh meat stored in HiOx-MAP has been reported by several studies (Jongberg et al., 2011; Kim et al., 2010; Lund et al., 2007; Zakrys-Waliwander, O’Sullivan, O’Neill, & Kerry, 2012). Lund et al. (2007) determined that fresh pork loins packaged in HiOx-MAP revealed the formation of cross-linked MHC through disulphide bonding due to protein oxidation during storage, which subsequently resulted in reduced tenderness and juiciness. Zakrys-Waliwander, O’Sullivan, O’Neill, and Kerry (2012) also found the occurrence of protein oxidation (based on a decrease in free thiol groups and an increase in carbonyl contents) in beef steaks during HiOx-MAP storage and subsequent high molecular (500 kDa) cross-linked MHC formation, which negatively influenced instrumental beef tenderness and drip loss. An oxidative cross-linking/aggregation of myosin and possibly with titin was reported by Kim et al. (2010). Based on Western blot and diagonal-SDS–PAGE assays with MALDI-TOF MS/MS identification for detected disulphide bonds, they concluded the crosslinked MHC formation could be a causative factor for reduced tenderness of beef steaks packaged in HiOx-MAP (Kim et al., 2010). Therefore, in the present study, the increased shear force values of the OS loins in HiOx-MAP during display storage could be due to the formation of cross-linked MHC by the occurrence of protein oxidation under the high-oxygen atmosphere condition. The current study did not measure protein oxidation; however, the close interaction of lipid and protein oxidation in the meat systems has been reported, and lipid-derived radicals and hydroperoxides might promote protein oxidation of meat proteins as well (Lund et al., 2011). Interestingly, the OS loin under HiOx-MAP appeared to be more susceptible to oxidative environment resulting in greater magnitude of protein aggregation compared to the NS loin under the same packaging condition and subsequently affecting meat

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Fig. 3. Representative Western blot depicting desmin (A) and troponin-T (B) degradation products of lamb M. longissimus lumborum from two different age classes [4-month-old (New season) and 11-month-old lambs (Old season)] under different retail packaging conditions (high-oxygen modified atmosphere package and MAP or overwrap PVC) run on 10% gels. The loins were vacuum-stored for 8 weeks at 1.5 °C prior to the retail package and displayed for 8 days at 3 °C under light. REF = a lamb loin (longissimus lumborum) samples at 48 h post mortem was used as a reference; INT = intact protein; DEG = degraded protein. D1 = display day 1 and D8 = display day 8.

Fig. 1. Representative silver stained SDS–PAGE (7.5% gel) of whole muscle protein extractions of M. longissimus lumborum from Old Season lamb carcasses under a non-reducing condition. The loins were vacuum-stored for 8 weeks at 1.5 °C prior to the retail package (high-oxygen modified atmosphere package and MAP or overwrap PVC) and display for 8 days at 3 °C under light. CL-MHC = cross-linked myosin heavy chain; MHC = myosin heavy chain; D1 = display day 1 and D8 = display day 8.

qualitative analysis as shown in Fig. 3. These observations indicate that the proteolysis of myofibrillar proteins during the 8 weeks of ageing had been fully-processed, and thus no further degradation was occurred during the additional retail display period. Further, it confirmed the suspect that the occurrence of protein polymerization of cross-linked MHC during retail display was the likely factor inducing the significant increase in the shear force values of the OS loins in HiOx-MAP (based on no impacts of packaging types and age classes on desmin and troponin-T degradation). Kim et al. (2010) reported similar results that HiOx-MAP did not influence the extents of proteolysis and l-calpain autolysis of beef steaks, and concluded that an oxidative cross-linking of MHC with a high molecular weight protein could be a possible explanation for decreased tenderness of beef steaks packaged in HiOx-MAP. 4. Conclusions

Fig. 2. Representative Western blot depicting cross-linked myosin heavy chain of lamb M. longissimus lumborum from two different age classes [4-month-old (New season) and 11-month-old lambs (Old season)] under different retail packaging conditions (high-oxygen modified atmosphere package and MAP or overwrap PVC) run on 7.5% gels. The loins were vacuum-stored for 8 weeks at 1.5 °C prior to the retail package and displayed for 8 days at 3 °C under light. CL-MHC = cross-linked myosin heavy chain; MHC = myosin heavy chain. D1 = display day 1 and D8 = display day 8.

tenderness. This could suggest that antioxidant properties of proteins besides of lipid oxidation stability might be negatively associated with a physiological age of animals (Xiong et al., 2007). There were no impacts of packaging treatments and lamb age classes on desmin and troponin-T degradation based on the

The results from the present study determined that meat quality of long term chill-stored lamb loins could be adversely influenced by HiOx-MAP through elevated oxidative conditions during retail display. Particularly, a formation of oxidative crosslinked MHC and a concomitant increase in shear force values of the OS loin in HiOx-MAP indicate a negative impact of a high-oxygen atmosphere on tenderness of fully-tenderized lamb loins during retail display. Furthermore, the findings from the current study suggest that oxidative stability of lipids and protein in post mortem lamb muscle could decrease with an increase in lamb age, and thus meat from older lamb could undergo a greater extent and faster rate of oxidizing process under the HiOx-MAP retail packaging condition. Therefore, a proper care should be made for selecting a retail display packaging type for meat from older age animals. Further studies regarding a possible antioxidant mechanism/system against protein oxidation and concomitant protein polymerization of meat under HiOx-MAP would be warranted to minimize oxidative meat quality defects. Acknowledgements This project was supported by the AgResearch Research & Capability Fund. The appreciation is extended to Dr. Martin Upsdell for

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