Proteomics and Meat Quality Aspects of Chilled Goat Meat

Chevon (goat meat) is most preferred and widely consumed meat in the country. Generally, goat meat is marketed as fresh meat immediately after slaughter. Several times, consumers complain about the toughness of goat meat. This is mainly attributed to improper development of rigor mortis and hindering the process of conversion of muscle to meat. Hence, post-mortem chilling and ageing of carcasses are required to ensure food safety, maximize shelf-life, reduce shrinkage and improve the tenderness of meat. It is well established that meat tenderizes during post mortem storage. The biochemical and physiochemical mechanisms during the tenderization processes are reported (Lametsch et al. 2003). Several muscle proteins play important role in the meat tenderization process (Hopkins and Thompson 2002).
       
Previous studies on effect of chilling and ageing on expression individual skeletal muscles were focused on beef and lamb. In India, meat quality aspects in various breeds have been studied (Jayanthi et al. 2021; Raman et al. 2021; Prasad et al. 2022). However, no studies in goat meat were focussed on proteomics and ageing effects. With the objective to identify reliable proteins involved in ageing and tenderness, the present study was undertaken to compare meat quality characteristics and expression of different muscle proteins between aged and non-aged goat meat.
 

This work was carried out in ICAR-Central Institute of Postharvest Engineering and Technology during the year 2014. Final data analysis was carried out in 2016 at ICAR-National Research Centre on Meat, Hyderabad. Adult goat carcasses (Beetel breed; 12 months age) from animals slaughtered in government approved local municipality abattoir were used.  A total of 12 goat carcasses (6 for each treatment) were randomly assigned to two post mortem treatments: 1. Control/non-aged (4-6 h post mortem) and 2. Chilled and aged meat. Chilling was initiated at (16±1°C) within 2 h of post-mortem and continued for 6-8 h. For ageing fresh muscle samples of semitendinosus muscle obtained from chilled goat carcass were vacuum packaged in polyamide packaging films (Nylon-6, 100 µm) and stored in the chiller (6±2°C) for 72 h. Thus collected semitendinosus muscles were used to measure pH, colour, and shear force value, free and bound water, cooking loss, moisture and protein identification studies. 
       
Carcass pH and temperature were measured using a portable pH-meter with a penetration pH electrode (Hanna Instruments, Woonsocket, RI, USA). Hunter L (lightness), a (redness) and b (yellowness) values were measured after the newly cut surface was exposed to ambient air (Hunter and Harold 1987). Hue (H*) and Chroma (C*) were calculated using the equations H* = tan ^-1 (b/a) and chroma C* = (a²+b²)^0.5. The cooking loss values were calculated based on the difference in weight before and after cooking at 80°C, for 25 min and the same samples were used for the determination of shear force with a Warner-Bratzler shear blade attached to TA-XTplus Texture analyser (Stable Micro Systems, Godalming, Surrey, UK). The crosshead speed was 5 mm/s. The water lost, termed “free water” or centrifuged free water, was the percentage difference in weight before and after centrifugation. (Kristensen and Purslow 2001).
       
The procedure described by Kang and Rice (1970) was used with slight modifications for extraction of salt soluble proteins (SSP) Proteomic service facilities of Centre for Cellular and Molecular Platforms (C-CAMP), Bangalore, India were utilized for the identifications of proteins using LC-MS/MS. Protein extracts were precipitated using ethanol and acetone (1:1), precipitated protein pellet was trypsin digested before subjected to MS analysis. Digested peptides were dried by speed vacuum and reconstituted in 100 µL of 2% acetonitrile with 0.1% formic acid and 1 µL of the same were injected on to the column. Digested peptides from in-sol samples were subjected to 70 LC run followed by acquisition of data on LTQ-Orbitrap-MS. Generated data was searched for the identity on MASCOT as search engine using Swiss-prot, TrEMBL and Capra Hircus databases. List of proteins and sequence generated through LC-MS/MS was used for the analysis. Spectra were processed and analysed by the Global Protein server Explorer 3.6 software (Applied Biosystems, Carlsbad USA). This software uses an internal MASCOT (Matrix Science, Marylebone, UK) program for matching MS and MS/MS data against database information. The data obtained were screened against mammalian database downloaded from Swiss-Prot/TrEMBL homepage (http://www.expasy.ch.sprot).
       
The meat quality data obtained from six goat carcasses were subjected to a one-way analysis Ageing/chilling treatment was considered as a major source of variation. When statistical differences were detected, differences were considered significant at the P=0.05 level.

Effect of chilling on quality characteristics of meat
 
The average carcasses temperature in chilled carcas dropped from 36.2±1°C to 14.4±1°C in 4 h.  The pH of meat decreased from 6.65±0.2°C to 5.72±0.3°C during this period. Color values were not significantly different (Table 1). The chilled and aged meat samples showed higher release of moisture during cooking and thus higher cooking losses as compared to non-aged meat samples. Bendall (1978) suggested that carcasses should be chilled slowly so that the internal temperature does not drop to less than 10°C within first 10 h.


 
Shear force value
 
Chilled and aged meat had significantly (p<0.05) lower shear force value as compared to non-chilled fresh meat (Table 1). Difference in shear force values between aged and non-aged carcasses was nearly 30%. Several studies indicated that tenderization of meat during ageing can be attributed to various factors like sarcomere length, breakdown of proteins into smaller units, (Devine 1996). They further, supported that tenderization is dependent on the degree of sarcomere shortening and the activation of proteolytic enzymes and that these two factors act synergistically to give the tenderness.
 
Free and bound water and total moisture
 
The free water was significantly (P<0.05) higher in chilled/aged meat whereas bound water was significantly higher in non-aged/fresh meat (Table 1). Increase in free water content is also related to protein breakdown during ageing. Devine et al. (2004) observed that meat that entered rigor at 15°C and further aged was having higher tenderness and free water content.
 
Proteomic analysis of different proteins in un-aged and aged/chilled meat
 
LC-MS/MS identification and information related to the proteins are shown in Tables 2 and 3. Myofibrillar, sarcoplasmic, mitochondria and ribosomal proteins were dominant in both non-aged and aged meat. Four different isoforms of myosin were identified and myosin-1 was having highest scores and coverage and myosin-3 was having lowest score and coverage. Tropomyosin which provides physical integrity to muscle cells, was having higher score and coverage in aged than non-aged meat. Myosin light chains are distinct from the heavy chains and have their own properties. MLC found in aged meat had higher score and coverage compared to MLC observed in non-aged meat. Most of the sarcoplasmic proteins found are related to oxidative metabolism including citric acid cycle (e.g. creatine kinase), glycolysis (e.g. glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase) oxidative stress, transport, protein repair etc. Lactate dehydrogenase is generally released during tissue damage and was observed in aged and non-aged meat. Endoplasmic reticulum calcium ATPase- 1 was also observed in both the samples. Higher score and coverage for cytochrome c was observed in non-aged meat, but, the HSP 70 identified in aged meat showed higher protein score and coverage than HSP identified in non-aged meat. Calsequestrin, a calcium-binding protein of the reticulum; calmodulin, a small, highly conserved protein; cytochrome complex, or cyt c, a small heme protein  were also identified. Other sarcoplasmic proteins identified were sarco/endoplasmic Calcium ATPase (SERCA). These identified metabolic enzymes are all associated with ATP-generating pathways, either the glycolytic pathway or energy metabolism.




       
During the conversion of muscle to meat, major changes in muscle protein architecture are primarily noticeable at the expression levels of major myofibrillar proteins like myosin, actin, titin, nebulin, troponin-T, desmin and filamin (Lonergan and Lonergan 2005). In a study by Jia et al. (2007) on protein changes in two different bovine muscles (M. longissimus dorsi and semitendinosus) after 24 h storage, five proteins (cofilin, lactoylglutathionelyase, substrate protein of mitochondrial ATP-dependent proteinase SP-22, HSP27 and HSP20) changed with a similar pattern in both muscles, while 15 proteins showed altered expression pattern specific for the two different muscle types. Stefania et al. (2010) observed changes in the insoluble protein fraction of bovine longissimus thoracics muscle from eight Norwegian Red (NRF) dual-purpose young bulls during the first 48 h post-mortem. They found significant changes in a total of 35 proteins identified two metabolic enzymes (2, 3-bisphosphoglycerate mutase and NADH dehydrogenase) and one protein involved in the stress responses/apoptosis of the cell (Hsp70).
 
 

In comparison with non-aged meat, aged meat showed improved meat quality characteristics. In aged meat, higher expression myofibrillar proteins involved in tenderization was observed.  Further, Myosin light chain/MLC-3 and MLC-1, myosin-I, myosin-II and myosin-III, troponin-C, are probable protein markers associated with tenderization during ageing of goat meat.
 

This research was funded by the Ministry of Food Processing Industries, Government of India under the scheme “Research and Development in food processing sector” (No: 7/MFPI/RandD/2011).
 

The authors declare that there is no conflicts of interest.