*3.3. Antioxidant Compounds, Oxidation and Color Stability of LTL under Storage Conditions*

The content of antioxidant compounds observed in LTL samples stored at two retail display conditions is shown in Table 4. No significant interactions were observed between the main effects.


**Table 4.** Content of antioxidant vitamins and antioxidant stability in LTL muscle at the end of each storage and retail display condition.

<sup>1</sup> D, Dietary treatment: 0DG, control; 15DG, 15% DG; 30DG, 30% DG and 45DG, 45% DG (%DM basis); R, retail treatment: R1, aerobic exposure for seven days; R2, vacuum-packed conditions for 25 days, plus aerobic exposure for three days. <sup>2</sup> SEM: standard error of the mean. <sup>3</sup> D\*R, dietary treatment x retail treatment interaction. Means in the same row having different letters are significant at the *p* ≤ 0.05 level. NS: no significant.

The content of α-tocopherol was not affected either by the feeding diet or the storage conditions, and the average concentration was 0.58 μg/g meat. Regarding γ-tocopherol, a higher content was observed in meat from steers fed with DG diets, while it was not affected by the storage conditions. However, in all cases, the values of α- and γ-tocopherol observed were lower than those observed at 72 h post-mortem. Previously, it has been reported that vitamin E affects lipid and color stability after refrigerated storage [13]. In the present study, during storage, α-tocopherol levels dropped around 34% in meat from all diets, except in those from 45DG, in which it decreased by 64%, whereas γ-tocopherol levels dropped around 20% in meat from all diets, except in those from 45DG, in which it decreased by 48%. Indeed, the FRAP assay showed that the total reducing activity in meat was greater for R1 than for R2, but also that it was not affected by the feeding diets. Other authors have reported that, after meat aging and aerobic exposure, both the FRAP values and the content of antioxidant vitamins decreased [34]. In our study, the effect of storage on FRAP was not observed on vitamin content.

Regarding retinol content, the meat from animals fed 30DG had greater content than that from animals fed 15DG and 45DG, while the meat from 0DG had the lowest content (Table 4). Retinol content was not affected by the retail display conditions.

Together with α-tocopherol, other compounds like the minor forms of vitamin E, carotenoids, and their retinoid derivatives, protect tissues against free radicals and prevent oxidation reactions [13]. All these compounds could be administered in the feeding diets, as stated before. However, pro-oxidant compounds, like unsaturated fatty acids, could also be administered in the diets. It has been reported that, during refrigerated storage, oxidative processes increase exponentially [13], since factors like temperature, the presence of light, and oxygen act as catalysts [6,9]. In our study, the values of TBARS measured at the end of the storage period in LTL samples showed a tendency (*p* = 0.08) to interact between dietary treatment and retail display conditions (Figure 2). Meat from DG diets showed similar TBARS values, independently of the storage condition. Interestingly, the meat from 0DG showed the highest TBARS values when exposed to R2 conditions. In this sense, greater TBARS values have been reported in meat samples from 0% DG diets, compared to meat samples from 50% DG diets, after 21 days of aging [38]. However, de Mello et al. [17] observed greater TBARS values in meat from animals fed 30% DG than in meat from animals fed 0 and 15% DG and aged for 42 days. These authors observed meat surface discoloration after aging followed by a retail display and related these events with a higher content of PUFAs in meat when feeding cattle with 30% DG.

**Figure 2.** Lipid oxidation (mean ± SEM) measured as thiobarbituric acid reactive substances (TBARS) in LTL at the end of storage and retail display conditions for each dietary treatment (0, 0%DG control; 15, 15% DG; 30, 30%DG; 45, 45% DG, DM basis).

The TBARS assay is used as a marker of lipid oxidation and as a predictor of the perception of rancidity in meat. In different studies, values of 2–2.5 mg MDA/kg have been established as the accepted limit at which meat shows no rancidity [6]. In our study, all the TBARS values observed were below that threshold.

As stated above, anti- and pro-oxidants can be provided in the feeding diets, and their balance would be a determinant for the oxidative stability of meat. In our study, the feeding diets supplied liposoluble vitamins to meat, mostly γ-tocopherol. However, α-tocopherol is the isomer with the greatest antioxidant action and at least 3.0 to 3.5 μg/g of meat is needed to preserve tissues from oxidation and/or to maintain color stability [7,23,39]. Interestingly, it has been proposed that a dosage level of γ-tocopherol and a similar dosage level of α-tocopherol are approximately equally effective in preventing oxidation in red meat [39]. In our study, the sum of the content of antioxidant vitamins (1.39 μg/g meat on average) was below the optimal levels proposed to preserve tissues from oxidation, but the extent of oxidative damage was far below the threshold for rancidity. Therefore, it could be assumed that the antioxidant compounds present in meat samples were exerting a cooperative antioxidant activity.

It is known that the nutritional strategies that modify the fatty acid profile of meat could increase the susceptibility of meat to lipid oxidation by increasing n-3 PUFAs [7,13]. We showed that the content of PUFAs and n-6 PUFAs did not change with the DG level, while that of n-3 PUFAs decreased numerically [40]. This effect was statistically seen in the n-6/n-3 PUFA ratio, which increased as the DG level increased due to the decrease in n-3 PUFAs. In the present study, we observed that the inclusion of DG in the feeding diets supplied both more antioxidant compounds and fewer pro-oxidant compounds.

In this sense, it has been reported that the supplementation of DG diets with vitamin E does not improve the effect against lipid oxidation, since the meat from 30DG-diets, with and without vitamin E supplementation, had similar values of TBARS after 7 days of retail display [41].

The meat color parameters found at the end of the retail display conditions are presented in Table 5. In the case of the R2 storage condition, an extra measurement was done at the end of the vacuum-packed storage, prior to display conditions (R2a). The color parameters analyzed showed no interaction between the main effects.


**Table 5.** Color parameters (CIELab) in LTL muscle at the end of each storage and retail display condition.

<sup>1</sup> D, Dietary treatment: 0DG, control; 15DG, 15% DG; 30DG, 30% DG and 45DG, 45% DG (%DM basis); R, retail treatment: R1, aerobic exposure; R2a, end of vacuum-packed storage (25 days); R2b, end of aerobic exposure of R2. <sup>2</sup> SEM: standard error of the mean. <sup>3</sup> D\*R, dietary treatment x retail treatment interaction. Means in the same row having different letters are significant at the *p* ≤ 0.05 level. NS: no significant.

Meat color did not differ statistically with the inclusion of increasing levels of DG in the diets. These results are in agreement with those presented by de Mello et al. [17], who compared beef from DG diets with control diets (0% only corn, 15% and 30% DG). On the other hand, Depenbusch et al. [19] reported a linear decrease in redness (a\* value), after 7 days of retail display conditions, in steaks from heifers fed increasing levels of DG (0% to 75%). Moreover, to analyze differences in beef color, delta color change (ΔE) was calculated, following AMSA procedures, between steaks from DG diets and steaks from the control diet. The values of ΔE obtained for 15DG, 30DG, and 45 DG were 1.58, 1.69, and 2.13, respectively. Some authors reported that values of ΔE from 2 to 10 indicate that differences in color are perceptible by the human eye at a quick look [42] while other authors reported that values between 1.5 to 3.0 in beef could be evident to consumers [43].

Meat color stability is the result of the balance between anti- and pro-oxidant compounds [44]. It could be assumed that meat with an increase in PUFA content, due to the inclusion of DG in the diet, would be more susceptible to lipid oxidation and discoloration. Thus, as previously reviewed, it is difficult to define a general effect on meat color due to the inclusion of DG in the diet [3].

During storage, the formation of metmyoglobin (MMb) modifies meat color with a decrease in both a\* and C\* parameters [45]. Besides, the rate at which MMb develops could be affected by the conditions of packaging and storage. In the present study, storage conditions influenced color parameters. Samples from R2 conditions were brighter and had higher values of a\* and C\* than steaks from R1 conditions. Indeed, samples from R1 and R2 showed a value of ΔE of 2.93, which indicated that differences could be evident to the human eye [42,43]. Additionally, a decrease in a\* and C\* values was observed in R2b with respect to R2a.

Meat from R1 had lower values of color parameter than meat from R2b (Table 5). These results are in accordance with those of Jose and McGilchrist [46], who proposed that in low pH beef, aging increased bloom depth and made meat appear brighter and redder in color. However, other authors reported that aging time before retail display did not affect the redness (a\* parameter) of meat samples from DG diets, while retail display conditions decreased the values of a\* [41]. Similarly, in our study, a decrease in a\* and C\* values were observed when changing from vacuum to aerobic packaging in R2 (R2b with respect to R2a). This decrease could be associated with an increase in MMb content when samples were exposed to aerobic conditions.

In this sense, it has been reported that during refrigeration storage, lipid oxidation occurs more slowly than discoloration [14]. McKenna et al. [47] reported that color-stable muscles had lower TBARS than less color-stable ones. These authors found values up to 0.35 mg/kg for *M. longissimus lumborum* and *M. longissimus thoracis* after 5 days of a retail display while preserving color traits. In agreement, in our study, the values of TBARS observed were lower than 0.35 mg/kg, except for meat from the 0DG treatment at R2 storage conditions. Furthermore, the values of a\* measured for all samples in our study were above 14.5, the value considered as a threshold for beef color acceptability [48].

Moreover, a PCA was performed to depict the relationship between the oxidant/antioxidant status and the color parameters, at each storage condition (Figure 3). The new components identify where the maximum variance of the data occurs in a multidimensional space. For the R1 condition, the first component (PC1) accounted for 29.5% of the variance, the second component (PC2) accounted for 21.0% of the variance, and the third component (PC3) accounted for 19.3% of the variance. For R2 conditions, the principal components accounted for 36.3% (PC1), 28.2% (PC2), and 16.7% (PC3) of the variance. Interestingly, it was observed that the relationship between the variables differed with the type of storage. In R1 storage, retinol and FRAP showed a positive correlation with a\* and C\* and a negative correlation with TBARS. In R2 storage, γ-tocopherol and FRAP showed a positive correlation with a\* and C\* and a negative correlation with TBARS. For both storage conditions, a\* and C\* parameters showed a negative correlation with TBARS.

**Figure 3.** Principal component analysis (PCA) depicting the relationship between the oxidant/antioxidant status and the color parameters, at (**A**) R1 conditions (aerobic exposure for seven days) and at (**B**) R2 conditions (vacuum-packed storage for 25 days, plus aerobic exposure for three days).

#### **4. Conclusions**

The inclusion of DG in the feeding diets of beef steers increased the content of antioxidant compounds in the diets. This was reflected in a greater content of γ-tocopherol in beef from DG diets, and a numerical increase in retinol content. Although the values of α-tocopherol were below those indicated as optimal for oxidation delay, the values of TBARS observed were far lower than the ones associated with meat rancidity. Therefore, it could be assumed that the antioxidant compounds present in the meat from animals fed with DG exerted a cooperative antioxidant activity.

The meat exposed to refrigerated storage was affected by the conditions of retail display since FRAP and color parameters decreased. The inclusion of DG affected the extent of lipid oxidation. In this regard, the samples from DG-diets showed similar values of TBARS for both storage conditions, and these values were lower than those from 0DG and extended storage. Thus, DG inclusion in finishing diets positively impacts the balance between anti- and pro-oxidant compounds and meat oxidative stability during the display conditions evaluated.

**Author Contributions:** Conceptualization, M.M., D.P. and G.G.; methodology, M.M., S.A.R., L.R., D.P. and G.G.; software, M.M., S.A.R. and L.R.; validation, M.M., S.A.R., L.R., D.P. and G.G.; formal analysis, M.M., L.R. and G.G.; investigation, M.M., S.A.R., L.R., D.P. and G.G.; resources, L.R., D.P. and G.G.; data curation, G.G. and M.M.; writing—original draft preparation, M.M., D.P. and G.G.; writing—review and editing, M.M., S.A.R., L.R., D.P. and G.G.; visualization, M.M.; supervision, D.P. and G.G.; project administration, M.M.; funding acquisition, M.M., L.R., D.P. and G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Instituto Nacional de Tecnología Agropecuaria (INTA PEI#517), by Instituto Promoción Carne Vacuna Argentina (Convenio de Cooperación Técnica IPCVA- INTA, 2017) and by Consejo Nacional de Investigaciones Científicas y Técnicas, Beca Doctoral Temas Estratégicos 2016–2021 (Res DN#5040).

**Institutional Review Board Statement:** Experimental procedures were approved by the Ethics Committee of the School of Veterinary Sciences of the University of Buenos Aires (Comité Institucional de Cuidado y Uso de Animales de Laboratorio, Facultad de Ciencias Veterinarias, protocol number 2016/42), Argentina. The assay was carried out as part of common zootechnical procedures and the animals did not suffer any intervention beyond those typical in such farming conditions. Muscle samples were collected for the analysis during the routine carcass cutting procedure at the meat processing plant.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from corresponding author.

**Acknowledgments:** The authors express their gratitude to Karina Moreno, Marta Signorelli and Cecilia Barreto for their technical assistance. The authors are members of the Healthy Meat network funded by CYTED (119RT0568).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

