*3.5. Protein Oxidation*

There were no differences between breed and finishing feed treatments on total carbonyl and free thiol content in either *Semimembranosus* or *Vastus lateralis* (Table 3). On the

other hand, protein oxidation significantly differed between the three packaging methods in both muscle types. Total carbonyl was significantly lower in VSP compared to HioxMAP and TrigasMAP. There was a small but significant difference between total carbonyl of HioxMAP and TrigasMAP with TrigasMAP inducing a lower carbonyl generation. The free thiol content values were significantly lower in VSP compared to either HioxMAP or TrigasMAP for both muscle types, suggesting that minimising protein oxidation in sheep meat can be achieved by the use of oxygen at a level below 50%. Within SPD treatment, Merino *Semimembranosus* or *Vastus lateralis* had lower free thiol contents compared to equivalent muscles from Composite sheep, indicating the importance of cameline in sheep finishing diets for Merino sheep to reduce protein oxidation. This difference was not observed for total carbonyl.

#### **4. Discussion**

#### *4.1. Colour and pH*

Differences in Instrumental CIELab parameters due to breed was more apparent in *Vastus lateralis* compared to *Semimembranosus*. Merino *Vastus lateralis* had higher L\*, and lower a\*, b\*, hue, and chroma than the same muscle from Composite sheep. These results suggest that the meat from Merino sheep was less colour stable, which coincided with a substantially higher pH compared to meat from Composite sheep. This agrees with a previous study which found the fastest drop in oxy-/met-myoglobin ratio in meat from Merino, compared to other crossbreeds [14]. The lower colour stability in Merino sheep meat was also reported in other studies [9,10]. Furthermore, significant differences in fatty acid composition and vitamin E concentration were found in the *Longissimus* of Merino and crossbred sheep [7,10]. These differences are likely to result in variation in the oxidation of myoglobin, thus affecting the colour stability of meat.

Breed and pH have been shown to be among the most important predictors in sheep meat colour stability. Meat from Merino often has a different ultimate pH to meat from crossbreed sheep [10,14]. A study on lamb from the Australian Cooperative Research Centre for Sheep Industry Innovation showed that Merino *Longissimus* with a higher pH had the least colour stability in overwrap [14]. The link between breed, ultimate pH, and colour stability is complex. Meat pH has been linked to myoglobin autooxidation, changes in enzymatic activities, iron molecule oxidation, and light scattering, all of which affect the appearance of the meat [15]. It is worth noting that most studies on sheep meat have focused mainly on the *Longissimus*, which is known to differ from *Semimembranosus* and *Vastus lateralis* in muscle fibre type, contributing to differences in colour [15].

Myoglobin oxidation and oxygenation status is affected by the level of oxygen during retail packaging. This study shows that the storage of lamb under high (80%) and moderate (50%) oxygen environments for 10 days significantly reduces the colour stability of both *Semimembranosus* and *Vastus lateralis*, when compared to lamb stored in VSP. The significant decrease in L\*, a\*, b\*, hue and chroma in HioxMAP, compared to VSP, are consistent with the results of previous studies [3,16,17]. Lower chroma and higher hue values are undesirable in red meat as it represents paler and duller meat [18]. The colour results from TrigasMAP in the present study suggest that after 10 days of storage, TrigasMAP does not offer enhanced colour stability, similar to results of Resconi et al. [16] in which beef were displayed in different O2 levels for up to 8 days. However, it is possible that meat in TrigasMAP with a shorter retail display time may have better colour than in HioxMAP. Zakrys et al. [19] suggested that 50% O2; 30% N2; 20% CO2 may provide opportunity for improved shelf life by enhancing the a\* value of beef, compared to HioxMAP after a 3-day storage. Meat surface colour has been shown to deteriorate after three days of storage in HioxMAP [20]. This was attributed to the reduction of metmyoglobin reducing activity during prolonged storage, thereby favouring the oxidative process of oxymyoglobin to metmyoglobin. The study of Khliji et al. [21] indicated that consumers discriminate against red meat with a\* values below 14.5. The a\* values for both muscles in HioxMAP and TrigasMAP were well below this threshold for both muscles in this study. Thus, retail

displaying of sheep meat in HioxMAP or TrigasMAP for 10 days is not recommended for colour enhancement.

#### *4.2. Water Holding Capacity and Texture*

Contradictory results have been reported for the purge loss of meat in different packaging treatments, while others reported an increase in the purge of meat under vacuum [22]. Taylor et al. [17] showed that the weight loss of vacuum-packed beef and pork was less than MAP (75%O2/25%CO2)-packed samples after storage. Similar results were reported in other studies [23,24]. However, it was suggested in a review by McMillin [25] that the purge loss of meat displayed under vacuum packaging is higher than MAP, partly attributed to the negative pressure. In the current study, the two muscles responded differently to the effect of packaging. HioxMAP led to a higher purge loss for *Semimembranosus* but lower purge loss for *Vastus lateralis* compared to VSP, emphasising that future purge loss investigations should consider muscle differences.

Unlike purge loss, similar cooking losses were observed for both muscle types with a higher cooking loss found for both HioxMAP and TrigasMAP compared to VSP, in agreement with previous studies [26]. On the other hand, cooking loss due to breed differences appear to be muscle-specific, with *Semimembranosus* from Merino having a lower cooking loss compared to the same muscle from Composite. While the underlying mechanisms of water holding capacity remains an ongoing research area, previous studies showed that variations in muscle fibre type and connective tissue composition play a role in cooking loss differences [27–29].

Packaging appeared to affect the two muscles differently. WBSF of *Semimembranosus* in HioxMAP and TrigasMAP were higher than VSP. Similar results have been reported for beef topside and beef round, where the beef topside is a more likely response to ageing than beef round muscle after storage [30]. Numerous studies have shown the negative effect of HioxMAP on sheep meat eating quality. Frank et al. [1] showed a significantly lower sensory tenderness of lamb in HioxMAP compared to VSP. Similar results on various texture measurements were also found for meat from other species [11,13,31]. Previous studies found while the WBSF of beef *Longissimus* did not differ between oxygen levels from 40–80%, sensory panellists preferred beef in lower O2 environments 40–50% [19,32,33]. Furthermore, various studies have established that the toughening of meat in HioxMAP is caused by increased protein oxidation resulting in more disulfide bond formation between actomyosin complexes, less degradation of structural proteins, e.g., desmin and troponin T, and deactivation of calpain [13,32,34–36]. It is worth noting that the exact mechanisms appeared to be muscle- and species-specific [2,3,34]. Our results on texture are consistent with the protein oxidation results, which showed that significant differences were only found when VSP was compared to HioxMAP and TrigasMAP. Together, these results indicate VSP is the preferred packaging method for lamb regardless of breed and finishing feed treatments.

#### *4.3. Lipid Oxidation*

Lipid oxidation is a key quality determinant in meat, as it causes the development of off-flavours and rancidity in meat. Free radical formation from lipid oxidation has also been linked to increased myoglobin oxidation and thus discolouration [37]. Feeding strategy of livestock can play a significant role in manipulating lipid oxidation of meat. The present study found a reduction of lipid oxidation of *Semimembranosus* and *Vastus lateralis* from sheep finished on diets supplemented with camelina forge or camelina meal. These findings compliment previous studies which found significant decreases in TBARS for forage fed animals when compared to grain-fed animals [5,38,39]. Lamb muscles finished on diets supplemented with camelina cake has been shown to have a different fatty acid composition compared to those on the standard pelleted diet without camelina supplementation [40]. Furthermore, we have reported in a separate study that both camelina hay- and camelia meal-supplemented diets reduced (*p* < 0.001) arachidonic acid concentration of *Longissimus*

from these animals compared with the SPD diet [7]. In addition, the SCM diet significantly increased alpha linolenic acid (ALA) concentration of the *Longissimus* compared to SPD and SCF, resulting in an increase in total omega-3 concentration and the decrease in the ratio of *n* − 6/*n* − 3 in meat [7].

The packaging results in this study show that TrigasMAP is an effective method to reduce lipid oxidation in packaging, regardless of breed and finishing feed treatments, consistent with previous studies on the effect of varying oxygen content on lipid oxidation [19,34,41]. Reducing oxygen content in retail packaging is even more important when sheep is not finished on supplemented diet.

Consumers discriminate against the off-flavour of beef when TBARS reaches the 2.28 mg MDA/kg meat threshold [42]. While similar investigations are needed for sheep meat, the present findings suggest that retail display of sheep meat in HioxMAP for 10 days leads to unacceptable flavour regardless of breed, feed or muscles. Supplementation of finishing feeds with camelina forage and TrigasMAP offers the potential to reduce TBARS values to below this threshold, thus reducing the economic loss for the industry. It should be noted that VSP provided consistently minimal lipid oxidation regardless of breed, feed or muscle treatments.
