3. Results and Discussion
The present study analyzes the quality of meat from lambs fed either a high-cereal concentrate or a concentrate containing corn DDGS, DCP, and EOC. Previously, de Evan et al. [
4] showed that there were no differences between concentrates in growing performance, digestibility of nutrients, and animal health of the lambs. There were also no differences in hot and cold carcass weights, which reached 14.4 and 13.6 kg for the CON and 14.2 and 13.6 kg for the BYP groups, respectively [
4]. The greater content of total soluble polyphenols in the BYP compared with the CON concentrate was attributed to the inclusion of DCP and EOC. The EOC contained 2.03% of total soluble polyphenols (dry matter (DM) basis), which is in agreement with the values ranging from 0.4 to 2.9% reported by others for EOC samples [
7,
25]. Likewise, total soluble polyphenol content of the DCP used in our study (1.33%; dry matter basis) was similar to the values reported by Gorinstein et al. [
26] but slightly greater than the values reported by others [
27,
28]. It has been shown that the content in total soluble polyphenols of DCP is highly variable, as it varies with the type of citrus fruit (lemon, orange, etc.) and the fruit fraction, being greater in the peels than in the pulp of the fruit [
26,
29].
The main differences between concentrates in the FA profile were observed in the contents of lauric (C12:0) and myristic (C14:0) acids, which were lower in the BYP than in the CON concentrate, and in the oleic acid (C18:1 n-9), which was greater in the BYP concentrate. Compared with CON, the BYP concentrate contained less saturated FA (SFA; 36.0 vs. 25.4%, respectively). This is in agreement with previous studies in which diets including DDGS, DCP, or EOC were tested [
8,
9,
10], and it was attributed to the high proportion of unsaturated FA in DDGS, DCP, and EOC [
5,
6,
30].
One of the main factors determining meat quality is its pH, which influences the organoleptic characteristics of the meat [
31]. As shown in
Table 2, no differences between diets were observed in the pH of the longissimus dorsi and semitendinosus muscles, and no interactions of diet × time were detected. As expected, pH decreased (
p < 0.001) at 24 h postmortem in both muscles, reaching values below 6.0 within the range of optimal commercial quality [
32]. The pH values observed in this study are in good agreement with those previously reported for the meat of lambs fed high-concentrate diets and slaughtered at similar body weight [
32,
33,
34] and for lambs receiving diets including the same by-products [
35,
36].
A bright red color in lamb meat is desirable and attractive for consumers [
37]. Although meat color can be strongly affected by the diet, in our study, there were no differences (
p ≥ 0.130) between diets in any color parameter or in the estimated concentrations of pigments (
Table 3) and no diet × time interactions were detected (
p ≥ 0.608), indicating that changes in color over the storage period were not affected by dietary treatment. These results are in accordance with the lack of differences between diets in pH and chemical composition of the meat. In contrast, some authors have reported a discoloration of beef meat when crude olive cake was included in the diet at greater levels than that used in the present study [
38,
39], and Lanza et al. [
40] observed that the inclusion of 10% of orange pulp and 10% carob pulp in the diet of Barbaresca lambs decreased the lightness and intramuscular fat content of the meat.
Refrigerated storage of the meat affected all color parameters and the estimated concentrations of the pigments with the exception of h*, which remained unchanged (
Table 3). Lightness (L*) increased from days 0 to 3, recovering the original values by day 6 of storage, whereas a*, b*, and C* increased by day 3, followed by a stabilization thereafter. Similar increases in b* and C* have been observed by Ripoll et al. [
41] and de la Fuente-Vázquez et al. [
11] during refrigerated storage in atmospheric conditions of lamb meat for 7 days. In contrast, Inserra et al. [
42] reported significant decreases of a* and C* values in lamb meat after 6 days of refrigerated storage in atmospheric conditions. Several factors can influence the stability of the meat color under the same storage conditions, and the diet of the animals and the composition of the meat have been identified as the main ones [
11,
43], helping to explain differences among studies.
The color of the meat depends on many factors, but the concentration of myoglobin and its chemical state is one of the most important factors involved [
43]. The oxymyoglobin is responsible for the bright-red color in meat, but its oxidation to brown-colored metmyoglobin during meat ageing leads to meat discoloration [
11]. In agreement with previous studies [
11,
43], myoglobin decreased (
p < 0.001) and both metmyoglobin and oxymyoglobin increased (
p < 0.001) over the 6 days of storage for both dietary treatments. Every time meat is allowed to bloom, oxymyoglobin is formed [
44], and that is in accordance with the observed increases in a * and b * coordinates at days 3 and 6 compared with day 0. Meat with more than 40% metmyoglobin has been reported to be downgraded by trained consumer panels [
45,
46], but in our study, the estimated proportions were lower than this level in all samples. All these results would indicate high color stability in the meat of lambs from both groups.
The diet had no influence (
p ≥ 0.166) on chemical composition and texture parameters of the longissimus dorsi (
Table 4). Chemical composition of the meat was within the range reported by others [
11,
34,
47,
48] for lambs from different breeds slaughtered at similar body weight. Our results agree with those from other studies reporting that inclusion of corn DDGS [
10], DCP [
49], or EOC [
50] in the concentrate for fattening lambs had no effects on chemical composition of meat. Some studies reported that the inclusion of corn DDGS in the diet of Rambouillet lambs (20% DDGS [
51]) and Wrzosówka lambs (45% DDGS [
10]) resulted in a more delicate texture. Lanza et al. [
40] observed that feeding 10% of orange pulp and 10% carob pulp to Barbaresca lambs increased the tenderness of their meat. Differences among studies in the composition and inclusion rate of the by-products, in the breed of the animals, and in their weight and age at slaughter can explain the discrepancies with our results.
The lipid oxidation of the meat over a 6-day storage period was assessed by measuring the concentrations of TBARS and CD (
Figure 1 and
Figure 2). The TBARS is one of the most widely used assays for measuring malondialdehyde, an end product of lipid peroxidation, whereas the CDs are primary lipid oxidation products generated in the oxidation of polyunsaturated FA (PUFA [
52]). There was a trend (
p = 0.095) for a significant diet × time interaction for TBARS concentration, but the diet did not affect TBARS levels in the meat (
p = 0.134). Some authors [
53,
54,
55] have reported that the dietary administration of antioxidants to fattening lambs reduced the accumulation of malondialdehyde concentration and improved the resistance of meat to oxidative deterioration. Feeding antioxidant-rich by-products to lambs is a feasible option to increasing the daily intake of antioxidants. In fact, Inserra et al. [
42] and Luciano et al. [
8] fed lambs with concentrates containing high levels of DCP (24 and 35%) and olive cake (25%), respectively, and observed a reduction in TBARS levels in meat compared with those for lambs fed high-cereal concentrates, indicating an improvement in the oxidative stability of the meat. Inserra et al. [
42] attributed this improvement to the greater polyphenol content of the DCP-containing concentrates compared with the control one. In our study, the CON and BYP concentrates contained 1.95 and 6.05 g of gallic equivalents (GAE)/kg DM, respectively, but no differences between groups in the TBARS concentrations were detected when data were analyzed as repeated measures. However, TBARS levels after 6 days of storage were 2.4 times greater for CON than for BYP-fed lambs, and when these data were analyzed independently, differences between groups reached the significance level (
p = 0.026; SEM = 0.0720; n = 12). In agreement with previous studies [
8,
42,
53], the TBARS concentrations increased (
p < 0.001) with storage time, but whereas, in the CON lambs, values increased from day 3 to day 6, TBARS concentrations in BYP lambs remained stable from day 3 thereafter. These results would indicate greater oxidative stability of the meat from the BYP-fed lambs after 6 days of storage. It should be noticed that TBARS concentrations in all samples were below 0.5 mg MDA/kg meat, which is the proposed threshold for the detection of off-flavors (rancidity) by a trained sensory panel [
56].
As shown in
Figure 2, concentrations of CD were not affected either by the diet (
p = 0.219) or the storage time (
p = 0.642), and no diet × time interaction was detected (
p = 0.740). The PUFA are preferential substrates for lipid oxidation in the muscle [
57], and in our study, feeding the BYP concentrate resulted in greater deposition of PUFA in the meat (
p = 0.003;
Table 5) compared to CON lambs. An increase in PUFA concentration can negatively influence the oxidative stability of meat, as the FA susceptibility to oxidation increases with increasing unsaturation degree [
58]. The lack of differences between diets in CD concentrations in our study might be due to the small differences in total PUFA concentration in the meat after slaughter (9.23 and 10.5% of total FA for CON and BYP meat, respectively). In addition, the concentrations of CD in the meat of both groups remained stable over the storage period, indicating that no extensive PUFA oxidation took place. This is in agreement with the lack of reduction in the proportion of any of the PUFA analyzed (
Table 5). In contrast, the proportion of total monounsaturated FA (MUFA) decreased (
p = 0.035) after 6 days of storage, which is in agreement with the increased TBARS concentrations in the meat of both groups at day 6, as malondialdehyde is an end product of both MUFA and PUFA peroxidation [
59]. Our results are in accordance with those of Luciano et al. [
8], who analyzed the effects of feeding lambs with a concentrate containing 25% olive cake to lambs and who observed stronger differences between experimental treatments in TBARS than in CD concentrations in the meat.
Ruminant meat is usually regarded as less healthy for humans than the meat of other species such as poultry and pig, which is mainly due to its high content in SFA [
9]. The differences in the FA profile of the two concentrates used in our study were reflected in the meat FA profile, as lambs fed the BYP concentrate had lower proportions of total SFA (
p = 0.001) and greater of total MUFA (
p = 0.035) and PUFA (
p = 0.021) than those fed the CON one. There were also differences between groups in the individual FA, and the meat from BYP-fed lambs had lower (
p ≤ 0.026) proportions of C15:0, C16:0, and C17:0 and tended to greater proportions of C20:0 (
p = 0.089). Similarly, proportions of C16:1 n-9, C20:1 n-9, C18:2 n-6, C18:3 n-3, and C18:4 n-3 were greater (
p ≤ 0.036) in the BYP compared with CON lambs. Similar results were obtained by others when lambs were fed concentrates containing either corn DDGS [
60] or dried citrus pulp [
9,
61,
62]. The lack of differences (
p = 0.251) between groups in the proportion C18:1 n-9 observed in our study agrees with the results of Kotsampasi et al. [
50], who did not observe differences in this FA in lambs fed destoned EOC, and it was attributed to both the low fat content of the EOC (2.91%, DM basis) and the low level of inclusion in the BYP concentrate (8%). In contrast, several studies have reported increases in the proportion of C18:1 n-9 in the meat of lambs by feeding crude olive cake [
63,
64]. In agreement with previous results in lambs fed high-concentrate diets [
34,
48,
65], for both groups of lambs, the most abundant FA was (C18:1 n-9), followed by C16:0 and C18:0. Although there were no differences (
p = 0.157) between groups in the peroxidability index of the meat, the meat from BYP-fed lambs had lower (
p ≤ 0.003) values in the atherogenic and thrombogenic indexes and a greater (
p < 0.001) hypocholesterolaemic/hypercholesterolaemic ratio (hH), altogether indicating a healthier FA profile compared with CON lambs. Finally, it should be noticed that the column used for the FA analysis did not allow precise identification of
cis and
trans isomers of unsaturated FA [
66], and therefore, differences between groups in the meat FA profile are limited to the FA identified.
The storage of meat for 6 days resulted in subtle changes in the SFA and MUFA profiles, as the proportions of C14:0 tended to decrease (
p = 0.088) and those of C20:1 n-9 increased (
p = 0.009), with no changes detected for other SFAs and MUFAs. In contrast, the proportions of most of the analyzed PUFAs were affected by meat storage. The proportions of C18:2 n-6, C20:3 n-6, C20:4 n-6, C22:4 n-6, and C22:6 n-3 were increased (
p ≤ 0.036) and those of C18:3 n-3 and C18:4 n-3 were reduced (
p = 0.043 and 0.076, respectively) after 6 days of storage, resulting in greater (
p = 0.021) PUFA proportions in the meat compared with day 0. Whereas no diet × time interaction was observed for any SFA and MUFA, significant interactions (
p ≤ 0.049) were detected for C20:3 n-6, C20:5 n-3, and C22:4 and trends (
p ≤ 0.095) were detected for C18:2 n-6, C20:4 n-6, and C22:6 n-3. Majewska et al. [
70] also observed similar changes after refrigerated storage of lamb meat for 1 month, which were attributed to the hydrolytic processes and changes in chemical composition occurring during storage. In contrast, a reduction in the proportions of PUFA as storage time increased has been reported by others [
71,
72], especially when meat contained high proportions of PUFA and was packed in a high-oxygen modified atmosphere. Changes in the FA profiles of meat depend not only on the FA composition and saturation degree but also on the processing methods, storage conditions (temperature, time, atmosphere, etc.), and the concentrations of pro and antioxidants [
73]. The peroxidability index increased (
p = 0.013) over the storage period, but the rest of indexes were not affected by storage.
There were no differences (
p ≥ 0.616) between groups in the color parameters of the subcutaneous fat of the tail root (
Table 6), but the FA profile was markedly affected by diet. The proportion of C16:0 and C17:0 was lower (
p ≤ 0.011) in the BYP-fed lambs than in those fed the CON diet, resulting in a lower proportion (
p = 0.001) of total SFA. C16:1 n-9 and C17:1 were the only MUFAs affected by the diet, and their proportion increased for C16:1 n-9 (
p = 0.008) and tended to decrease for C17:1 (
p = 0.076) by feeding the BYP concentrate. The greatest difference between diets was observed in the PUFA, as all PUFA proportions were greater (
p ≤ 0.019) in the BYP-fed lambs compared with those fed the CON concentrate. As a consequence, the proportion of total PUFA in the subcutaneous fat of the tail root was 1.5 times greater (
p < 0.001) in the BYP group than in the CON one, and the fat from BYP lambs had a greater (
p < 0.001) peroxidability index. As reported in previous studies in lambs [
48,
67,
74], changes in FA profile induced by diet were greater in the subcutaneous than in the intramuscular fat. As discussed by Manso et al. [
48], this can be attributed to the greater deposition rate of the subcutaneous fat compared with the intramuscular fat during the finishing period and to the fact that subcutaneous fat is more responsive to changes in dietary FA supply [
75]. Similarly to that observed for the meat, there were no differences (
p = 0.322) between groups in the peroxidability index of the fat but the fat from BYP-fed lambs had a healthier FA profile as indicated by the lower (
p ≤ 0.012) atherogenic and thrombogenic indexes and the greater (
p = 0.002) hH ratio.