## 2 Materials and Methods
### Oxidation Shelf-Life Study  
The pH was measured on raw burgers on days 0, 1, and 3 of storage, and on cooked burgers on day 0. Measurements were made in triplicate (Shen et al., 2018) using sensION+ PH31 pH equipment (Hach Lange SLU, Barcelona, Spain).  
Color was measured using a Konica Minolta CR 400 chromatometer (Minolta Camera Co., Osaka, Japan), standardized using a calibration plate. Lightness (L*), a* chromaticity (red-green), and b* chromaticity (blue-yellow) were measured according to the CIELab system. Measurements were taken in triplicate on raw burgers on days 0, 1, and 3 of storage. Additionally, color was also measured on the cooked burgers on day 0, considering both sides of the burger.  
The total color differences (\(\upDelta\)E) of each sample with respect to the control formulation and in relation to day 0 of storage were calculated as follows:  
\[\upDelta\text{E}=\sqrt{\left(\text{L}_{\text{s}}^{*}-\text{L}_{\text{c}}^{*} \right)^{2}+\left(\text{a}_{\text{s}}^{*}-\text{a}_{\text{c}}^{*}\right)^{2}+ \left(\text{b}_{\text{s}}^{*}-\text{b}_{\text{c}}^{*}\right)^{2}} \tag{1}\]  
Thiobarbituric acid reactive substances (TBARS) content, directly related to lipid oxidation, was measured following the method described by Ohkawa et al. (2019) with some modifications (Mohkawa et al., 2019). The TBARS values, expressed as mg of malonaldehyde (MDA) per kg of beef burger sample, were calculated using the following formula:  
\[\text{TBARS}\left(\text{mg MDA}/\text{kg}\right)=\left(\text{Abs}_{532}/\text {W}\right)\times 9.48 \tag{2}\]  
\(\text{Abs}_{532}\) was the absorbance at 532 nm, and W was the weight of the sample (g). The analyses were repeated in triplicate at 0, 1, and 3 days of refrigerated (4 \({}^{\circ}\)C) storage.  
Protein oxidation was measured in relation to the thiol concentration. The concentration of the thiol groups was determined after derivatization by 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (Mohkawa et al., 2019), following the method described by Nieto et al. (2019). The absorbance corresponding to the thiol concentration in the samples was correlated with \(\text{Abs}_{412-\text{afterDTNB}}-\text{Abs}_{412-\text{beforeDINE}}-\text{Abs}_{412- \text{blank}}\). The analyses were carried out in triplicate at 0, 1, and 3 days of refrigerated storage.  
Lipid oxidation was also evaluated with the concentration of volatile compounds, determined according to the method described by Martinez-Zamora et al. (2019). A total of 5 g of burger sample was placed in glass vials, and volatile compounds were extracted using solid-phase microextraction (SPME) (Supelco, Bellefonte, PA, USA). Analyses were performed on a Hewlett-Packard 6890 N Series GC gas chromatograph fitted with an HP 5973 mass spectrometer and MSD Chemstation (Hewlett-Packard, Palo Alto, CA, USA). Compounds were identified by comparing their mass spectra with those contained in the NIST05 (National Institute of Standards and Technology, Gaithersburg, MD, USA) library. Analyses were performed on the four burger formulations on days 0 and 3 of refrigerated storage. Results were expressed as area units (AU) \(\times\) 10\({}^{5}\)/g of sample.