Effect of α-tocopherol, rosemary extract and their combination on lipid and protein oxidation in beef sausages

This study focuses on the oxidative changes in lipids and proteins of beef sausages with incorporated a-tocopherol, rosemary extract or their combination during 3 months’ storage at 4°C. For this purpose, sausages were formulated with no antioxidant (Control, C), 200 ppm a-tocopherol (T), 200 ppm rosemary extract (R), and 100 ppm a-tocopherol + 100 ppm rosemary extract (TR). To observe oxidative changes in lipids; peroxide value, thiobarbituric acid reactive substances (TBARS), and total oxidation value (TOTOX), in proteins; sulfhydryl and carbonyl contents were measured. Use of antioxidants and storage time significantly affected oxidative stability of sausages (P<0.05). Antioxidants, individually or in combination, retarded lipid peroxidation and improve the oxidative stability of sausage during storage. The antioxidant combination showed synergistic effect on protein oxidation, as the lowest carbonyl contents were found in TR samples. As a result, a combination of antioxidants with different effect mechanisms could be the better option to prevent oxidative changes in meat products.


Introduction
Oxidation is the main non-microbial phenomenon in meat and meat products which creates undesirable changes in product quality, limits the shelf life and produces adverse health effects due to the development of toxic secondary oxidation products. Lipid and protein oxidations occur through free radical reactions, and due to their high amount of lipids and high concentration of prooxidants, meat products are highly susceptible to oxidative changes [1][2][3].
Use of antioxidants is one of the main strategies for preventing oxidation in meat products. Apart from synthetic antioxidants, natural compounds with antioxidative effect are proposed since they are safe, cheaper and consumers prefer natural sources to synthetic compounds [4]. Antioxidative effects can be achieved by different mechanisms, such as chelation of metals, scavenging free radicals, breaking chain reactions, and inhibition of lipid peroxidation [5].
α-tocopherol, an oil-soluble natural antioxidant, is widely used in the meat industry due to this compound acting as an electron donor and breaking the chain reactions. Previous reports showed the high antioxidative effect of α-tocopherol in meat products [6][7][8]. Rosemary (Rosmarinus officinalis L.) extract is an effective natural antioxidant for meat products due to its high amount of phenolic diterpenes and phenolic acids [9,10], and due to phenolic hydroxyl groups, rosemary extract can scavenge free  [11]. Previous studies have demonstrated the effects of using antioxidants individually in meat product formulations. However, the use of a combination of antioxidants with different action mechanisms could be a better way prevent lipid and protein oxidation.
To the best of our knowledge the use of α-tocopherol and rosemary extract in combination in beef sausages has not been studied yet. Therefore, the aim of this study was to investigate the effects of individually or combinative use of α-tocopherol and rosemary extract on lipid and protein oxidation in beef sausages.

Material and methods
Four different beef sausage formulations were prepared (Table 1). Minced beef and beef fat were purchased from a local butcher, α-tocopherol, and rosemary extract were obtained from Kimbiotek (İstanbul, Turkey). Minced beef, curing ingredients, and half of the ice were homogenized and ground for 1 min in a cutter (Alpina, Switzerland). Beef fat, α-tocopherol, and/or rosemary extract (depending on formulation), other ingredients, and the remaining of the ice were added to the meat mixture, and batters were homogenized for 3 minutes to obtained sausage emulsion. Emulsions were stuffed into casings and smoked at 40°C for 2 hours (Afos, England) then heat treated in a boiling vessel until the core temperature reached 70°C. Once cooking was completed, sausages were cooled, vacuum packaged, and stored at 4°C for 3 months. Oxidative changes of lipids were determined in terms of peroxide values [12], thiobarbituric acid reactive substances (TBARS) [13], and total oxidation value (TOTOX) [14]. Protein oxidation was investigated by the determination of sulfhydryl [15] and carbonyl groups [16]. The effects of antioxidants and storage period were investigated by using two-way ANOVA analysis. Means were compared by using Duncan's Post-Hoc tests in the SPSS 23 software.

Results and discussion
As seen in Table 2, antioxidant and storage period showed a significant effect on peroxide values (PV), TBARS, and TOTOX of beef sausages (P<0.05). PVs of sausages were between 4.03 -5.21 meqO2/kg at the beginning of storage, but 8.11 -14.11 meqO2/kg at the end of storage (Table 3). Although some fluctuations were recorded during storage, in general, the impact of the storage period on PVs was significant (P<0.05). During storage, PV of C treatment increased continuously, PV of T sausages rose until month 2, and then significant decrement was observed (P<0.05). At month 3, the highest PV was recorded in C treatment (P<0.05). Antioxidant addition, whether individual or in combination, had a significant effect on peroxide content (P<0.05). Similar results were observed by Georgantelis et al. [17] wherein both α-tocopherol and rosemary extract were used in fresh pork sausages. In all cases, PVs of sausages were lower than 25 meqO2/kg, which is described as the limit for fatty foods [18].  Table 3. Initial TBARS values ranged between 0.15 -0.42 mg MA/kg; the highest TBARS values were found in control treatment and R sausages, while T and TR sausages showed similar TBARS values (P>0.05). All samples showed an increased in TBARS value until month 3. C treatment had the highest TBARS value at the end of storage time. TBARS values of R, T and TR sausages increased up to month 2, then a significant reduction was observed, probably due to the decomposition of aldehydes (P<0.05). The final values in month 3 ranged between 0.35 -0.75 mg MA/kg, with TR sausages having the lowest oxidation rate (P<0.05). Throughout the storage, TBARS values of all sausages were lower than 2.0 mg MA/kg, which is described as the limit of TBARS values in meat and meat products [13]. Similar to our results, Azizkhani and Tooryan [19] reported that using tocopherols and rosemary extract as a combination showed the greatest antioxidative effect in beef sausage storage during 3-month storage. Georgantelis [17] reported that TBARS values of fresh pork sausages formulated with chitosan and its combinations with either α-tocopherol or rosemary also showed the most intense antioxidative effect.
TOTOX, total oxidation value, is described as 2 x peroxide value + TBARS value [14]. Initial TOTOX values of sausage were between 8.24 and 10.61, and significantly increased during storage (Table 3). TOTOX values of sausages showed a similar trend as PV. The highest TOTOX value was observed for T sausage in the month 2. However, at the end of storage, C sausage had significantly higher TOTOX values compared to sausages with antioxidants. According to Decker et al. [20], the TOTOX value for food should be lower than 26; in this case, all of our sausages except C sausage in month 3 (28.98) were within the limit. Similar to lipid oxidation, oxidative change in proteins is believed to proceed via free radical chain reaction [21]. The use of antioxidant and storage time had significant effects (P<0.05) on protein oxidation of sausages in terms of sulfhydryl and carbonyl contents ( Table 2). Initial sulfhydryl contents of sausages were between 1.39 -2.25 nmol/mg protein, while sulfhydryl contents of sausages were between 1.77 -2.76 nmol/mg protein at the end of storage (Table 4). Sulfhydryl contents of sausages increased in month 1, then decreased significantly during storage (P<0.05). The sulfhydryl reduction in sausages could be explain by the formation of disulphide-crosslinks [22].
Carbonyl contents of samples were between 1.16 -1.81 nmol/mg protein at the beginning of storage ( Table 4). The highest carbonyl content was found in C sausage while the lowest carbonyl content was observed in TR sausage (P<0.05). During storage, significant increments were observed in all sausages (P<0.05). Control sausages showed the highest formation rate of carbonyl groups of all the treatments after 3 months' storage. Using α-tocopherol and rosemary extract combination showed a synergistic effect on carbonyl contents of sausages, and TR sausages had the lowest carbonyl content among the treatments at the end of storage time (P<0.05).

Conclusion
Use of antioxidants individually in meat products is one of the effective ways to inhibit oxidative changes. However, using antioxidant combinations which have different effect mechanisms to limit oxidation reactions could be a better option. The present study indicates that using α-tocopherol, rosemary extract or their combination prevents oxidative changes in beef sausages during 3-month cold storage. At the end of storage, sausages with antioxidants had significantly lower TBARS and TOTOX values. TOTOX value of C sausage in month 3 was higher than the limit value. Moreover, the αtocopherol and rosemary extract combination showed a synergistic effect on carbonyl contents of sausages. In conclusion, the use of antioxidant combinations could be a novel approach to delay oxidative changes in meat products.