Total phenolic content (TPC)

TPC was measured by the Folin-Ciocalteu method (^{Matić and Jakobek, 2021}) with slight modifications. Briefly, 20 µL of CSE (5 mg/mL) were mixed with 160 µL of distilled water, 40 µL of Folin-Ciocalteu’s reagent (2 M), and 60 µL of Na₂CO₃ solution (7 %, w/v). The resultant mixture was incubated at 25 °C for 1 h under dark conditions. The absorbance was measured at 750 nm in a spectrophotometer (Multiskan FC UV-Vis, Thermo Scientific, TYO., Japan), and results were expressed as mg of gallic acid equivalent/g of dried extract (mg GAE/g).

Total flavonoids content (TFC)

TFC was measured by the aluminum chloride-complex formation method (^{Matić and Jakobek, 2021}) with slight modifications. Briefly, 20 μL of CSE (5 mg/mL) were homogenized with 130 μL of methanol and 20 μL of AlCl₃ (5 %, w/v). The resultant mixture was incubated 30 min at 25 °C under dark conditions. The absorbance was measured at 415 nm, and results were expressed as mg of quercetin equivalents/g (mg QE/g).

Caffeoylquinic acid content (CAC)

CAC was evaluated according to ^{Griffiths et al. (1992)} with slight modifications. Briefly, 100 μL of CSE (5 mg/mL) were mixed with 200 μL of urea (0.17 M), 200 μL of glacial acetic acid (0.1 M), and 500 μL of distilled water. The resultant mixture was homogenized with 500 μL of NaNO₂ (0.14 M) and 500 μL of NaOH (1 M), centrifuged at 2250× g for 10 min at 4 °C (Sorvall ST18R, Thermo Fisher Scientific, MA, USA). The absorbance was measured at 510 nm, and results were expressed as mg chlorogenic acid equivalents/g (mg CGA/g).

Free radical scavenging activity

This activity was measured by the DPPH method (^{Ozgen et al., 2006}) with slight modifications. Briefly, 100 μL of CSE (5 mg/mL) were mixed with 100 μL of DPPH ethanol solution (300 μM). The resultant mixture was incubated at 25 °C for 30 min under dark conditions. BHT (0.1 mg/mL) was used as positive control. The absorbance was measured at 517 nm, and the results were expressed as a percentage of inhibition: [(Radical absorbance at 0 min) - (Radical absorbance + antioxidant at 30 min) / (Radical absorbance at 0 min)] x 100.

Radical cation scavenging activity

This activity was determined by the ABTS method (^{Ozgen et al., 2006}) with slight modifications. Briefly, 20 μL of CSE (5 mg/mL) were mixed with 180 μL of ABTS solution (abs 0.7 at 730 nm). The resultant mixture was incubated at 25 °C for 8 min under dark conditions. BHT (0.1 mg/mL) was used as positive control. The absorbance was measured at 730 nm, and the results were expressed as a percentage of inhibition: [(Radical absorbance at 0 min) - (Radical absorbance + antioxidant at 30 min) / (Radical absorbance at 0 min)] x 100.

Reducing power activity

This activity was determined by the ferricyanide/Prussian blue method (^{Berker et al., 2010}) with slight modifications. Briefly, 200 μL of CSE (5 mg/mL) were mixed with 300 µL of phosphate buffer (0.2 M, pH 6.6) and 500 μL of potassium ferrocyanide (1%, w/v) and incubated at 50 °C for 20 min, under dark conditions. The resultant mixture was homogenized with 500 μL of TCA (10%, w/v) and centrifuged at 2300× g at 4 °C for 10 min. Subsequently, 100 μL of the supernatant were homogenized with 100 μL of FeCl₃ (0.1%, w/v). BHT (0.1 mg/mL) was used as positive control. The absorbance was measured at 700 nm, and the results were expressed as absorbance at the same wavelength.

Ferric reducing/antioxidant power

This activity was determined by the FRAP method (^{Berker et al., 2010}) with slight modifications. Briefly, 20 µL of CSE (5 mg/mL) were homogenized with 180 µL of FRAP solution [10:1:1, 300 mM buffer sodium acetate in glacial acetic acid at pH 3.6 and TPTZ (10 mM) in HCl (40 nM) and FeCl₃ (20 mM)]. The reaction mixture was incubated at 25 °C for 8 min under dark conditions. BHT (0.1 mg/mL) was used as positive control. The absorbance was measured at 595 nm, and the results were expressed as mg of Fe²⁺ equivalent/g (mg Fe²⁺/g).

Meat homogenates preparation

Fresh pork meat (Semimembranosus muscle, at 48 h postmortem) was purchased from a local processor (Norson®, Hermosillo, Mexico). Any visible extra-muscular fat was trimmed, and then the meat minced using a 4.5 mm-hole plate (meat grinder 4152, 4 Hobart Dayton, OH, USA). The minced pork meat was mixed with salt (1.5 %, w/w) and pork back fat (10 % in the final formulation, w/w). Subsequently, the obtained minced meat (1 g) was homogenized with 10 mL of distilled water at 6000 rpm at 5 °C for 1 min (Ultraturrax T25, IKA, Germany), and 100 µL of the respective antioxidants: CN, without antioxidants; T1 and T2, CSE at 250 and 500 ppm, respectively; BHT, synthetic antioxidant at 500 ppm. The mixture was oxidized with 50 µL potassium ferrocyanide at 0, 0.5, and 1.0 % (w/v). After that, meat homogenates were stored at 4 °C for 1 h and subjected to oxidative stability evaluation.

pH values

The pH values of meat homogenates were determined using a potentiometer with automatic temperature control (pH211, Hanna Instruments Inc., RI, USA) following the procedure 981.12 (^{AOAC, 2020}).

Thiobarbituric acid reactive substances (TBARS)

Lipid oxidation of meat homogenates was determined by the TBARS method (^{Pfalzgraf et al., 1995}). Meat homogenates (10 g) were homogenized with 20 mL of TCA (10%, w/v) (4500 rpm at 5 °C for 1 min) and centrifuged (2500× g at 5 °C for 20 min). Subsequently, 2 mL of the filtered supernatant were mixed with 2 mL of TBA solution (20 mM) and incubated at 98 °C for 20 min. After that, the absorbance was measured at 531 nm. The results were expressed as mg of malondialdehyde/kg of pork meat (mg MDA/kg).

Color parameters values

The color parameters values of meat homogenates were evaluated as described previously (^{Sánchez et al., 2001}). The color parameters were performed using a spectrophotometer (CM 508d, Konica Minolta Inc., TYO, Japan) with a D65 illuminant and a 10° observer calibrated with a white calibration cap (CM-A70). Color parameters consisted of lightness (L*), redness (a*), yellowness (b*), Chroma (C*), and hue (h*).

Metmyoglobin content (MMb)

MMb of meat homogenates was measured using a spectrophotometer as described previously (^{Sánchez et al., 2001}). The maximum value of the quotient K/S525 and K/S572 at the beginning of the experiment (day 0) was fixed as 0 % MMb, while 100 % MMb was obtained after oxidizing the meat sample in potassium ferricyanide (1 %, w/v).

Statistical analysis

Data obtained from all measurements were expressed as mean ± standard deviation (SD) of at least three independent trials. Polyphenols content and antioxidant activity data were subjected to a t-test. In addition, data from meat quality assays were subjected to a two-way analysis of variance, using the treatments (CN, T1, T2, and BHT) and level of oxidized agent (0, 0.5, and 1.0 %) as the fixed effect, including the interaction. A Tukey-Kramer test was conducted for means comparison (p < 0.05). Also, a principal components analysis was performed to determine the relationship between the analyzed parameters and treatments (SPSS, version 21).

pH values

Figure 1 illustrates the results of the treatment and the induced pro-oxidation effect, using potassium ferrocyanide in pH changes of pork meat homogenates, which were incorporated with CSE and BHT. The results showed an effect of treatment and pro-oxidation interaction (p < 0.05) on this parameter. At 0% of oxidizing, BHT samples showed the higher (p < 0.05) pH values for meat homogenates (6.01) respect to CN, T1 and T2 whose values ranged between 5.90-5.97. At 1.0 % of the oxidizing agent, T1 and T2 showed lower (p < 0.05) pH values than other treatments.

Figure 1 Effect of treatment and pro-oxidation on pork meat homogenates pH values. Lowercase letters indicate significant differences between treatments; capital letters indicate significant differences in each treatment at different pro-oxidation levels (p < 0.05).  

Figura 1 Efecto del tratamiento y pro-oxidación sobre los valores de pH de homogenizados de carne de cerdo. Las letras minúsculas indican diferencias significativas entre tratamientos; las letras mayúsculas indican diferencias significativas en cada tratamiento a diferentes niveles de pro-oxidación (p < 0.05). 

A previous investigation evaluated the influence of the anticaking agent potassium ferrocyanide (80, 2500, and 17500 ppm) on the oxidative stability of frozen minced pork meat, and results showed that pH values were not affected by pro-oxidant incorporation (^{Hansen et al., 1996}). In addition, it has been reported that including coffee husk aqueous-ethanol extract (100 and 200 ppm) in raw frozen chicken patties did not affect initial pH values for CN samples (^{De Farias Marques et al., 2022}). However, regardless of the antioxidant and pro-oxidant agent addition, in our study, pH values remained within the acceptable range (> 5.5 - 6.1) reported for pork meat (^{Kim et al., 2016}a). In agreement with our study, it has been evidenced that initial pH values of raw and cooked chicken patties were not affected by 1.5 and 3.0 % of coffee silverskin powder addition (^{Martuscelli et al., 2021}a). Also, initial pH values of raw and cooked pork patties were not affected by non-compliant green coffee beans extract at 0.15, 0.30, and 0.60 % (^{Bergamaschi et al., 2023}).

TBARS values

The incorporation of extracts obtained from agro-industrial residues into meat products is a promissory strategy to improve oxidative stability (^{Hadidi et al., 2022}). pH is an intrinsic meat quality parameter involved in lipid oxidation chain reactions (^{Chaijan, 2008}). Oxidative stability decrease has been associated with several oxidant products, including transition metals and salt, which affect their physicochemical properties and promote lipid and protein oxidation (^{Min et al., 2010}). In this context, potassium ferrocyanide, a non-physiological iron compound used as an anti-caking agent in salt for human consumption, has also been related to pro-oxidant effects in meat systems (^{Hansen et al., 1996}; ^{Van Nguyen et al., 2012}).

Figure 2 reports the results of the treatment and pro-oxidation effect on lipid oxidation of pork meat homogenates incorporated with CSE and BHT. The results showed an effect of treatment and pro-oxidation interaction (p < 0.05) on this parameter. At 0 % of oxidizing, no differences (p > 0.05) were found between treatments on TBARS values for meat homogenates; however, these values increased (p < 0.05) by the pro-oxidation effect. At 1.0 % of oxidizing agents, T1, T2, and BHT showed the lowest (p < 0.05) TBARS values compared to samples from CN.

Figure 2 Effect of treatment and pro-oxidation on pork meat homogenates lipid oxidation values. Lowercase letters indicate significant differences between treatments; capital letters indicate significant differences in each treatment at different pro-oxidation levels (p < 0.05).  

Figura 2 Efecto del tratamiento y pro-oxidación sobre los valores de oxidación de lípidos de homogenizados de carne de cerdo. Las letras minúsculas indican diferencias significativas entre tratamientos; las letras mayúsculas indican diferencias significativas en cada tratamiento a diferentes niveles de pro-oxidación (p < 0.05). 

In a previous study, the influence of potassium ferrocyanide at concentrations of 80, 2500, and 17500 ppm on lipid oxidation in frozen minced pork meat was evaluated. The results indicated that TBARS values remained unaffected at 0 d of storage. However, these values exhibited an increased at 56 d of storage, with a concentration-dependent effect (^{Hansen et al., 1996}). In another work, it was evidenced that the incorporation of Fe²⁺ and Fe²⁺ + NaCl increased MDA formation in raw beef patties, 45.1 and 51.2% of inhibition respect CN samples (^{Min et al., 2010}). Also, it has been demonstrated that incorporation of potassium ferrocyanide (2.5, 7.5, and 100 ppm) to cod fillets (Gadus morhua) accelerated in concentration-dependent lipid oxidation during the salting process, as well as during storage for 6 months (^{Van Nguyen et al., 2012}).

In agreement with our study, the incorporation of spent coffee ground aqueous extract (500 and 1000 ppm) to raw pork meat homogenates reduces MDA formation, approx. 39.5 and 46.9% of inhibition respect to CN, after being subjected to thermal treatment at 37 °C for 12 h (^{Kim et al., 2016b}). Also, it has been reported that incorporation of coffee husk aqueous-ethanol extract (100 and 200 ppm) to raw and cooked frozen chicken patties during storage (- 18 °C for 45 d), enhances antioxidant stability through MDA reduction, approx. 90 and 66 % of inhibition, respectively, respect to CN (^{de Farias Marques et al., 2022}). In another study, it was demonstrated that incorporation of CSE (500 ppm) into raw pork meat homogenates reduces MDA formation, approx. 55 % of inhibition respect to CN, after being subjected to thermal treatment at 37 °C for 8 h (^{Vargas et al., 2023}). Additionally, although the oxidation of meat products can begin from their processing and storage, it has been reported that meat products treated with antioxidants, and whose values did not exceed 0.6 mg MDA/kg, do not present rancid aroma (^{Georgentelis et al., 2007}).