Ataulfo Mango (Mangifera indica L.) Peel Extract as a Potential Natural Antioxidant in Ground Beef

Abstract

Total phenolic content (TPC), total flavonoid content (TFC), antioxidant and antimicrobial in vitro activity of ethanolic (EE) and hydroethanolic (HE) extracts of mango peel Ataulfo were evaluated. The highest TPC, TFC and antioxidant capacity were as shown in EE. Ethanolic extract was incorporated into ground beef at 1000 (BBEA) and 2000 mg/kg meat (BEEB) concentrations and then compared with 250 mg of sodium ascorbate/kg meat (ASC) along with a control (without antioxidant). The effects of adding EE on instrumental colour, metmyoglobin content, thiobarbituric acid reactive substances (TBARS), pH, microbial load and sensorial analysis of ground beef were evaluated for 11 days at 4 °C. BEEB added in raw ground beef improved colour stability while the lipid oxidation in raw and raw-cooked ground beef was inhibited with a greater antioxidant effect than ASC and a similar overall acceptability score (cooked ground beef). However, incorporating BEEB into ground beef did not show significant antimicrobial activity. Therefore, mango peel extracts could potentially be used as a natural antioxidant in ground beef.

1. Introduction

Ground meat is more susceptible to oxidation when compared with meat cuts because of their greater surface area that allows for direct contact between lipids and air [1]. The grinding of meat causes muscle membrane disruption and the exposition of lipid membranes to metal ions, oxygen and temperature [2], making it susceptible to lipid oxidation [3]. Consequently, oxidative processes result in the deterioration of colour pigments (myoglobin) and other proteins in meat and meat products along with lipids which can contribute to detrimental sensory effects and nutritional value [4].

In addition, the oxidation of meat may also have harmful health effects on humans or animals [1]. Multiple toxic compounds are produced which can cause several human illnesses including cancer, atherosclerosis, inflammation and aging processes, among others [5]. Thus, lipid oxidation generates numerous primary and secondary by-products, such as cholesterol oxides, malondialdehyde (MAD) and 4-hidroxynonenal, which are known as carcinogenic potentials [6]. In this regard, Villalobos-Delgado et al. [7] reported that MAD is a carcinogenic and mutagenic agent which can react with DNA, causing deoxyguanosine (M1G), deoxyadenosine (M1A), and deoxycytidine (M1C) adducts, all of which are thought to be mutagenic in mammalian cells and carcinogenic in rat livers [8]. Furthermore, meat can be contaminated with microorganisms which may cause spoilage or foodborne diseases [2]. Other authors reported that minced beef must be kept in storage at 4 °C for a maximum period of 3 days [9], while raw patties have a shelf life of approximately 7 days in refrigeration and aerobiosis [10].

Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hidroxylanisole (BHA) and tertiary butylhydoquinone (THBQ), are employed in order to reduce microbial growth as well as delay lipid and protein oxidation of meat products [11]. However, there are increasing concerns about BHA and BHT because of their possible carcinogenic effects [7]. Sodium ascorbate is one of the most frequently used antioxidants in minced meat [12] because it is effective at reducing oxidation as well as being an efficient scavenger of radical species [13,14]. On the other hand, in recent years, consumer anxiety regarding the safety of synthetic additives in foods have resulted in the use of natural antioxidants and antimicrobials in meat and meat products as alternative compounds for preserving food [12]. Villalobos-Delgado et al. [7] stated that among fruit processing by-products, peel extracts from kinnow citrus hybrid, pomegranate, peanut and grape, among others, have been proposed as natural antioxidants in the elaboration of meat products. In this regard, the use of natural antioxidants in meat products could potentially be a good option considering their high phenolic compound content as well as their health implications and functionality [15].

To this regard, mango peel is reported to have a high bioactive compound content (phenolic acids, flavonoids, xanthones, fibre, carotenoids, vitamins, among others) [16,17,18,19], which have predominantly antioxidant properties [18,20], with acceptable sensory characteristics [17]. Mangiferin (a xanthone), one of the most abundant and potent antioxidants, has been identified in Mango cv Ataulfo [21,22,23].

Furthermore, mango peel extracts from different varieties (Sugar, Tommy Atkins and Badami) have been applied to decrease lipid oxidation in cooked beef homogenates [24] and chicken sausages [25]. To this regard, Mango cv. Ataulfo is a widely used Mexican variety [23] in the juice industry, which generates a high quantity of by-products such as peels, seeds and paste [21] leading to a problem of final disposition and pollution in landfills [26]. Approximately 7–24% of the peel is not commercially used [27,28].

Consequently, as waste, mango peels are susceptible to modification by microorganisms which can lead to problems, including leachates and gas emissions (methane and carbon dioxide) [29,30]. Thus, considering the concerns about improving the sustainability of the food industry and environmental problems related to waste management [31], there is an increasing interest in their valorization as agri-food by-products because they are potentially promising sources of valuable compounds [31,32,33].

Therefore, the aim of this research was to evaluate for the first time the antioxidant and antimicrobial properties of Ataulfo mango peel extracts as well as the effect of their incorporation in raw and raw-cooked ground beef by measuring colour stability, metmyoglobin content, lipid oxidation, pH, microbiological control and sensorial properties during refrigerated storage.

2. Materials and Methods

2.1. Preparation of Mango Peel Extracts

Ataulfo mangoes (Mangifera indica L.) with a weight of 200–300 g (caliber number 16) [34] were purchased at their commercial ripening state in a local market in Huajuapan de León, Oaxaca, Mexico and immediately transported to the laboratory for evaluation. Fruit samples were chosen considering the appearance and yellow-orange peel colouration. All fruit was rinsed under running tap water, disinfected with chlorinated water (100 ppm) and dried with absorbent paper. Selected mangoes showed a pH between (4.0–4.6) and a total soluble solid of 16.9–18.8 °Brix. The fruits were then pre-weighed and manually peeled using a sharp knife with the pulp, seed and peel separated. Yields of pulp, peels and kernels were determined and expressed in percentage (%).

After removing the pulp, two peels per mango were obtained. These peels were cut in half (two pieces per peel, four pieces per mango), dried in a ventilated oven (Pro Smoker, Wisconsin, USA) at 40 °C for 19 h until reaching a moisture content of 12% and then ground in a food processor (KitchenAid, China). The powder was sieved (40 mesh-sieve; MONTINOX, Mexico City, Mexico) to a particle size of 425 μm, packed in bags under vacuum and stored in airtight containers at 4 °C under darkness until producing corresponding extracts.

Three individual batches of each extract were prepared on three different processing days (one per batch) which were used to determine antioxidant and antimicrobial activity in vitro for their subsequent incorporation into ground beef. Two solvents were used for extracting bioactive compounds including 100% ethanol (EE) and hydroethanolic (HE) solution (80:20, water:ethanol v/v) using food grade ethanol following the method described by Adilah et al. [35] with some modifications. Mango peel powder (100 g) was extracted by macerating in 400 mL of solvent under darkness conditions at room temperature. After 24 h, the extracts were filtered (Whatman No. 1) and sample residues were once more reprocessed following the same methodology. The combined extracts of each solvent were concentrated in a rotatory evaporator (Yamato RE300, Tokyo, Japan) at 45 °C and lyophilised using a freeze-dryer (Labconco, Kansas, USA). Finally, one part of the extracts was dissolved in absolute ethanol for the in vitro characterization and the rest was stored in dark containers at 4 °C until the preparation of ground beef.

2.2. Analysis of Extracts

2.2.1. Total Phenolic and Flavonoid Content and In Vitro Antioxidant Capacity

The total phenolic content (TPC) was determined using Folin–Ciocalteu phenol reagent with acid gallic (Sigma-Aldrich, China) as standard according to Lorenzo et al. [4] with some modifications. Aliquots of 50 µL of the extract (200 mg/100 mL) were added to 3 mL of distilled water. Subsequently, 250 µL of Folin–Ciocalteu (0.1 N) reagent (Sigma-Aldrich, Switzerland) was added. The mixture was left to stand for 5 min, after which, 750 µL of Na₂CO₃ (20%) and 950 µL of distilled water were added. The reaction mixture was homogenised with a vortex (IKA vortex 3, Wilmington, USA) and was left to stand for 40 min. The absorbance was determined at 765 nm (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA). The results were expressed as mg of gallic acid equivalents (GAE)/g of sample dry weight (DW) using a calibration curve (0.1–1.0 mg/mL, R² = 0.9997).

Total flavonoid content (TFC) of the extracts was established using quercetin (Sigma-Aldrich, St. Louis, MO, USA) as standard at concentrations between 0.03 and 0.1 mg/mL for the calibration curve (R² = 0.9998). For this, 1 mL of dilution (1:2) from the same extract used to evaluate TPC was mixed with 4 mL of distilled water, 0.3 mL of NaNO₂ (5%) and 0.3 mL AlCl₃ (5%). After being left to stand for 1 min, 2 mL of NaOH (1.0 M) and 2.4 mL of distilled water were added to later homogenize the solution. Then, the absorbance was determined at 330 nm (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA). Results were expressed as mg of quercetin equivalents (EQ)/g of sample dry weight (DW) [36].

The antioxidant capacity in extracts was established through DPPH^• (2,2-diphenyl-1-picry-hydrazyl) assay [36]. A solution with 50 µL of extract (the same extract used in TPC) was used. A total of 2 mL of DPPH^• (0.1 mM) (Sigma-Aldrich, St. Louis, MO, USA) solution in ethanol was added to the solution. It was mixed until homogeneous and the absorbance at 517 nm (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA) was determined at a start time of 0 min to a completion time of 30 min. The results were reported as the percentage of inhibition of the radical Formula (1).

$$\%\mathrm{inhibition}=\frac{\left(A_o-A_a\right)}{A_o}\times 100$$

(1)

where

A_o = Absorbance after 30 min without the antioxidant.

A_a = Absorbance after 30 min with the antioxidant.

For the ABTS^•+, the methodology described by Re et al. [37] was followed. Firstly, the radical ABTS^•+ was generated by the oxidation of 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (14 mM) with potassium persulfate (4.9 mM) to generate a greenish blue radical. The ABTS^•+ solution was diluted to 0.7 ± 0.02 of absorbance with ethanol (≈7 mM). The reaction was carried out with 10 µL of extract added to 990 µL of ABTS^•+ solution at room temperature in darkness. After 7 min, the absorbances were measured with a spectrophotometer (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA) at 734 nm. The results were expressed as µmol Trolox equivalents (TE)/g. Finally, the ferric reducing ability of plasma (FRAP) assay was performed according to the procedure described by Benzie and Strain [38]. The preparation of the working FRAP reagent was carried out by mixing 25 mL of acetate buffer (pH 3.6, 300 mM), 2.5 mL of 2,4,6-Tris(2-piridil)-s-triazina (TPTZ, 10 Mm in 40 mM HCl) solution and 2.5 mL FeCl₃∙H₂O solution (20 mM). The assay was undertaken by mixing 33.2 μL of extract and 1000 μL of reagent at 37 °C in the dark. The absorbance was read at 593 nm (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA) after 7 min. The results were expressed as µmol Trolox Equivalent/g of solid extract.

2.2.2. Antibacterial Activity

The antimicrobial activities of EE and HE were tested against the following strains which were provided by the strain collection at the Microbiology III laboratory of the Autonomous University of Chihuahua: Escherichia coli (ATCC 35218), Staphylococcus aureus (ATCC 25923), Salmonella Typhimurium (ATCC 14028S) and Listeria monocytogenes (ATCC 19114). Pseudomonas fluorescens was isolated from spoiled ground beef obtained from a local supermarket in the city of Chihuahua, Mexico. The sample of ground beef was homogenised in phosphate buffer (dilution 1:10), and the dilution was inoculated into Cetrimide agar and incubated for 24 h at 37 °C. A pure culture strain was characterised by biochemical tests and pigment production.

The Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were both determined using the microdilution method [39]. All strains were grown in nutrient broth (BIOXON, Mexico State, Mexico) to the exponential phase (1.5 × 10⁸ CFU/mL) at 35 °C for 18 h, while 50 µL of the microbial growth was inoculated in the wells. Both extracts were dissolved in ethanol (100%) to 1.0% (w/v) and diluted once more in Tryptic Soy Broth (TSB, BIOXON, Mexico State, Mexico) to achieve finals concentrations of 12.5, 25, 50 and 100 mg/mL. From each concentration of broth containing the extracts, 150 μL was dispensed in the wells of a 96-well round bottom microplate along with the log-phase microbial growth and then incubated at 37 °C for 24 h. Controls for the experiment included a positive control for microbial growth (TSB plus bacterial inoculum, no extract), negative control for the extract (TSB plus extract, no bacterial inoculum) and negative control for the solvent used (TSB plus the highest concentration of alcohol used in the extract dilutions). The MIC value was determined as the concentration of the extract required to inhibit visible growth. From the wells with no visible growth, a loopful of the well content was inoculated into Tryptic Soy Agar (TSA, BIOXON, Mexico State, Mexico) and the Petri plates were incubated at 37 °C for 24 h. The MBC concentration reported no visible colony growth (less than 10 colonies) detected in plates.

2.3. Preparation of Ground Beef

The extract that showed the best response regarding antioxidant activity in vitro (EE) was used for evaluating the lipid oxidation and colour changes in ground beef.

Fresh beef (Biceps femoris muscle) was obtained from a local butcher at 24 h post-mortem. The fat and visible connective tissues were removed from the meat, and the lean meat was then ground in a meat grinder model SM-G73 (Sunmile, China). After mincing, the minced beef was assigned to each the following treatments: Control (without extracts) as negative control, BEEA (1000 mg of EE/kg meat), BEEB (2000 mg of EE/kg meat) and ASC (250 mg of sodium ascorbate/kg meat) as positive control. The antioxidant sources (extracts and ASC) were dissolved in 25 mL of potable water before being added to the formulation. The same quantity of water was added to Control treatment to obtain the same water content for all treatments. No other additives were added to the formulation. All the components were mixed for 3 min with a mixer (KitchenAid., St. Joseph, MI, USA) to homogeneity. In total, fifteen portions of ground beef per treatment (three portions of ground beef per day) were prepared with three independent manufacturing processes (batches) to ensure the experiment’s repeatability.

An 80 g portion of each experimental treatment was packed in polystyrene trays wrapped with air-permeable polyvinyl chloride (PVC) plastic film and subsequently stored at 4 °C for 11 days. At sampling times (1, 3, 6, 9 and 11 days), three portions per treatment were taken and divided into two parts. One part (60 g) was evaluated raw and the pH, TBARS, instrumental colour, metmyoglobin content and microbiological analysis were determined. The other part (20 g) was cooked at 180 °C in a convection oven (Oster, China) until an internal temperature of 72 °C was reached. After cooling at room temperature, the TBARS were quantified. All analyses were determined in triplicate.

2.4. Analysis of Meat Samples

2.4.1. Instrumental and Chemical Analysis of Ground Beef

The pH was determined after being homogenised with an ultraturrax (IKA T18 digital, Germany) with 10 g of sample with 90 mL of distilled water using a pH meter (HANNA instrument, Woonsocket, RI, USA), which was calibrated using pH 7 and pH 4 standard buffer solutions. CIE colour values, L* (lightness), a* (redness) and b* (yellowness) were determined at different positions using an UltraScan Vis 1139 colorimeter (Illuminant D65/10° observer angle and 9.525 mm aperture) (HunterLab, USA). Moreover, a* and b* values were used to calculate the Redness Index (RI) (a*/b*), hue angle (tonality) [tan−1(b*/a*) × (180/π)] and saturation index (vividness) [(a*² + b*²)^½] [40]. The metmyoglobin (MetMb) percentage was determined [41]. The absorbance value was determined by using a spectrophotometer (Genesys 10S UV-Vis, Thermo Scientific, Waltham, MA, USA) at 525, 545, 565 and 572. The phosphate buffer (0.04 M, pH 6.8) was used as a blank. The percentage of metmyoglobin was established using Formula (2) as follows:

$$\%\mathrm{MetMb}=\left[-2.51\left(\frac{A_{572}}{A_{525}}\right)+0.777\left(\frac{A_{565}}{A_{525}}\right)+0.8\left(\frac{A_{545}}{A_{525}}\right)+1.098\right]$$

(2)

The total colour difference (ΔE*) between samples of ground beef on day 1 and day 11 of refrigerated storage was calculated using the following Formula (3):

$${\mathsf{\Delta }\mathrm{E}}_{1-11}={\left(L^{*}{}_{11}-L^{*}{}_1\right)}^2+{\left(a^{*}{}_{11}-a^{*}{}_1\right)}^2+{\left(b^{*}{}_{11}-b^{*}{}_1\right)}^2$$

(3)

The lipid stability in ground beef was determined as malondialdehyde (MDA) content using the thiobarbituric acid reaction (TBARS; thiobarbituric acid reactive substances) [36]. TBARS values were calculated from a standard curve of 1,1,3,3-tetraetoxypropane and expressed as mg MDA/kg meat.

2.4.2. Microbiological Analysis

Ten grams of ground beef were taken aseptically and transferred to 90 mL of sterilised peptone water (0.1 g/100 mL) (DIBICO^®) and homogenised for 2 min. For each treatment, six serial decimal dilutions were prepared. Then, 0.1 mL of each dilution was spread on the surface of agar plates. Total bacterial count (TBC) was established with the use of Plate Count Agar (DIFCO, Sparks, MD, USA) after incubation for 48 h at 35 °C. Microbial colonies were counted and expressed as log₁₀ Colony Forming Units/g ground beef (CFU/g) [42].

2.4.3. Sensory Analysis

The sensory analysis was carried out according to the procedure described by do Prado at al. [43] with some modifications. The sensorial attributes of the cooked ground beef were evaluated by a panel of 100 different untrained panellists (consumers) comprising students and staff from the Technological University of the Mixteca. The sensory evaluation was performed in three separate tasting sessions: the first and the second were carried out with 35 panellists while the third was carried out with 30 panellists. On day 0, 4 portions of ground beef (50 g) from each treatment were first stored raw under refrigeration (4 °C) for 48 h. After this period had elapsed, each treatment was cooked until reaching an internal temperature of 72 °C. The sensory analysis was carried out on the same day as cooking, with the samples from each treatment preheated (65 °C) in an 800 W microwave oven and then served warm (~45 °C) to panellists. The samples (5 g) were randomly coded using three-digit numbers and then evaluated by each consumer in a set of four different samples, with one from each of the experimental treatments (Control, BEEA, BEEB and ASC). The panellists evaluated odour, colour, flavour and overall acceptability using a 9-point hedonic scale: 1-dislike extremely; 2-dislike very much; 3-dislike moderately: 4-dislike slightly; 5-neither like nor dislike; 6-like slightly; 7-like moderately; 8-like very much; and 9-like extremely. During evaluation, the panellists were provided with water and salt-free bread crackers to cleanse their palate between tasting of samples. Scores of between 6 and 9 were considered acceptable.

2.5. Statistical Analysis

For the TPC, TFC and antioxidant activity variables (DPPH^•, ABTS^•+ and FRAP as-says), a t-Student test was carried out in function of the effect of type of solvent (ethanol and hydroethanolic solution). The antioxidant effect (treatments; Control, BBEA, BEEB and ASC) on total colour difference and sensorial analysis in ground beef was analysed by analysis of variance (ANOVA), including batch as a random effect. Moreover, the data obtained from the ground beef analysis (pH, instrumental colour, metmyoglobin content, total bacteria count and TBARS) were analysed using a one-way Analysis of Variance (ANOVA) to evaluate the effect of antioxidant (treatments; Control, BBEA, BEEB and ASC) within each time period and to evaluate the effect of time (1, 3, 6, 9 and 11) within treatments. Then, when the ANOVA for each statistical analysis showed significance (P < 0.05), the ANOVA was followed by the Duncan’s test (P < 0.05). All data were expressed as mean values ± standard error (SEM). Statistical analyses were carried out using Statistica software (V. 10).

3. Results

The yields of pulp, kernel and peel from Ataulfo mango fruits were 75.6 ± 1.48%, 11.5 ± 1.29% and 12.4 ± 0.73%, respectively (data not shown). Jahurul et al. [20] reported that the peel is 7-24% of the total fruit weight, depending on the mango variety. In this study, the yield of peels (12.4%) was higher than other report with same that reported by Vega-Vega et al. [44] for the Ataulfo variety (8.90%) and similar to Tommy Atkins, Sensation, Kensington and Hadden varieties (11.6–13%) [44,45], while it was lower than that Manila variety (17.8%) [46].

3.1. Analysis of Extracts

3.1.1. Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and In Vitro Antioxidant Activity

Table 1. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity by three different test systems (DPPH^•, ABTS^•+ and FRAP) of EE and HE.

Treatments	TPC (mg GAE/g DW)	TFC (mg EQ/g DW)	DPPH• (% Inhibition)	ABTS•+ (µmol TE/g DW)	FRAP (µmol TE/g DW)
EE	344.0 a	153.7 a	77.6 a	1.3 a	11.6 a
HE	132.5 b	59.7 b	59.4 b	0.9 b	5.6 b
SEM	5.69	4.11	2.25	0.13	1.34
P-level	***	***	***	**	***

EE: ethanolic extract of Ataulfo mango peel; HE: hydroethanolic extract of Ataulfo mango peel. ^a,b Means values within the same column with different letters are significantly different (P < 0.05). SEM: standard error of the mean. P-level, Significance: ** (P < 0.01), *** (P < 0.001).

shows the results of TPC, TFC and the antioxidant activity (DPPH, ABTS^•+, FRAP) of EE and HE. Between the two extracts, the ethanolic extract had highest (P < 0.05) amount of phenolic content. These results could be explained by the polarities of solvents and the type of phenolic compounds present. Ethanol has been classified as a polar-protic solvent, leading to preferential extraction of low molecular weight compounds, such as glycoside and non-glycoside phenolic compounds [47]. In addition, different polyphenolic compounds have been identified in mango peel extracts [17]. In particular, Masibo and He [48] showed that phenolic constituents of mango peel include mangiferin (xanthone-C-glucoside; main phenolic compound), quercetin, rhamnetin, ellagic acid, kaempferol, with their related conjugates.

Rojas et al. [28] reported a TPC of 137 to 509 mg GAE/g from Ataulfo mango peels similar to those found in the current investigation for EE (344.0 mg GAE/g DW). However, the results from this study show a higher TPC than the values found by other authors [19,26,49] with 6.50–68.13 mg GAE/g in extracts from Ataulfo mango peels. In contrast, Alañón et al. [16] and Marçal and Pintado [17] reported values ranging from 14.85 to 127.6 mg GAE/g for other varieties were observed. This variability might be due to the extraction technique, solvent used or environmental conditions as well as pre- and postharvest conditions [49].

Furthermore, EE exhibited considerably higher TFC than HE (153.7 vs. 59.7 mg EQ/g extract, respectively). Values of 502–795 mg catequin equivalents/100 g from Tommy Atkins, Haden and Kent extracts freeze-dried mango peel were reported [50]. Additionally, TFC in both extracts were higher than the value of 1.75 mg QE/g in extracts from Kesington Pride mango peels [29]. The extraction method influences the flavonoid extracted profile. Therefore, fewer polar flavonoid structures such as isoflavones, flavonones and flavones are extracted with chloroform, acetone, methylene chloride and diethyl ether while polar flavonoid fractions such as flavonoid glycosides (e.g., quercetin and kaempferol) are extracted with alcohol–water mixture or alcohol [51,52,53].

On the other hand, with regard to the DPPH^• assay, the highest antioxidant activity (expressed as percentage of inhibition) was observed for EE (77.6%), followed by HE (59.4%). Kothalawala and Yatiwella [54] reported similar values between 80% and 85% in peel mango extracts from Sri Lankan cultivars. Similarly, EE showed higher ABTS^•+ scavenging ability (1.3 µmol TE/g) than HE (0.9 µmol TE/g). These results suggest that compounds in EE react quickly with ABTS^•+ or DPPH^• radicals through the donation of hydrogen atoms compared to HE. In addition, EE showed the highest FRAP reducing power with 11.6 µmol TE/g, followed by HE (5.6 µmol TE/g). The FRAP value for EE was similar to 12.76 µmol TE/g in the same variety [26]. Thus, these authors reported that peel from the Mexican Ataulfo variety showed antioxidant activity which could be attributed to the high mangiferin content which is classified as a “superantioxidant” [18,48].

Therefore, the antioxidant capacity in EE could be related to their bioactive compound contents such as mangiferin, gallates (methyl gallate) and gallotannins (from 5 to 13 units), chlorogenic and vanillic acids as well as gallic, hydroxycinnamic, hydroxybenzoic, caffeic, coumaric, ellagic, caffeic and ferulic acids, as well as carotenoids (all-trans-β-carotene, 9-cis-β-carotene, all-trans-violaxanthin, 9-cis-violaxanthin, all-trans-lutein as well as 13-cis-β-criptoxanthin) which have been identified in mango cv. Ataulfo peels [21,22,55,56]. Therefore, the mentioned phenolic compounds’ antioxidant activity has been closely related to their free radical scavenging capabilities, their potential chelation of pro-oxidant metals, their role as reducing agents and quenchers of singlet oxygen [57].

3.1.2. In Vitro Antimicrobial Activity of EE and HE

The results in

Table 2. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of EE and HE against Gram-positive and Gram-negative bacteria.

Microbial Strains	Minimal Inhibitory Concentration (mg/mL)		Minimal Bactericidal Concentration (mg/mL)
	EE	HE	EE	HE
S. Typhimurium	50	100	100	100
E. coli	>100	>100	>100	>100
Ps. fluorescens	>100	>100	>100	25
S. aureus	25	12.5	25	100
L. monocytogenes	50	25	100	50

EE: ethanolic extract of Ataulfo mango peel. HE: hydroethanolic extract of Ataulfo mango peel.

show that EE and HE exhibited antimicrobial activity against S. aureus with a MIC of 25 and 12.5 mg/mL, respectively. Moreover, the MBC for EE and HE was 25 mg/mL for S. aureus and Ps. fluorescens. These values correspond with the activity reported by Vega-Vega et al. [44], who found that the most sensitive strains of Ataulfo mango peel extracts at 25 mg/mL were L. monocytogenes and S. aureus with 100% inhibition. Thus, this study’s results indicate that Ataulfo mango peel extracts have mild antimicrobial activity against Gram-negative and Gram-positive bacteria but are more effective against S. aureus.

It can be observed that HE showed greater antimicrobial activity (MIC) towards Gram-positive bacteria (S. aureus and L. monocytogenes) compared to EE. The combined use of water and organic solvent could facilitate the extraction of chemicals that are soluble in water and/or organic solvent [58]. Ayala-Zavala et al. [59] indicated that solvents of higher polarity (ethanol or ethanol–water mixtures) can extract flavonoid glycosides and higher molecular weight phenolics, leading to higher yields of total extracted polyphenols. Thus, the varying degrees of effectiveness of the extracts could refer to the action and nature of phenolic compounds which are considered to cause inhibitory action and possess great structural variations. Therefore, the extracts’ antibacterial activity might be due to the hydroxyl (-OH) groups in phenolic compounds which can interact with the cell membranes of bacteria causing membrane structural disruption as well as the leakage of cellular components, thereby preventing cell division and the growth of microorganisms [60]. Moreover, polyphenols can denature enzymes and bind to substrates such as minerals, vitamins and carbohydrates, making them unavailable for microorganisms [61]. Finally, Tirado-Kulieva et al. [62] indicated that the antibacterial effect of phenolic compounds in mango by-products is more efficient against Gram-positive bacteria, while Gram-negative bacteria are more resistant because of the presence of lipopolysaccharides in their membrane, which make them repel or slow down the interaction with polyphenols.

3.2. Analysis of Ground Beef with EE

3.2.1. Colour Characteristics

Table 3. Colour characteristics and metmyoglobin (MetMb) content (%) of raw ground beef formulated with mango peel ethanolic extracts (EE) stored under refrigeration for 11 days.

Attribute	Storage Day	Treatments				SEM	P-Level
L*		Control	BEEA	BEEB	ASC
	1	52.1 a,A	51.1 a,A	51.3 a,A	52.1 a,A	0.43	n.s
	3	51.0 a,A	50.7 a,A	51.5 a,A	51.2 a,A	0.36	n.s
	6	48.1 b,B	50.2 a,A	50.7 a,A	51.2 a,A	0.39	***
	9	45.4 b,C	49.7 a,A	49.6 a,A	50.8 a,A	0.62	***
	11	51.2 a,A	49.1 a,A	49.7 a,A	50.5 a,A	0.60	n.s.
	SEM	0.50	0.44	0.47	0.33
	P-level	***	n.s	n.s	n.s
a*	1	7.0 b,A	8.8 a,A	9.6 a,A	8.1 a,b,A	0.30	*
	3	6.6 b,A	8.4 a,B	9.0 a,A	8.0 a,A	0.32	**
	6	5.0 b,A	6.1 a,BC	6.4 a,B	7.8 a,A	0.32	**
	9	5.0 b,A	5.9 a,C	6.0 a,B	6.9 a,A,B	0.20	**
	11	3.2 b,B	5.2 a,C	5.3 a,B	5.4 a,B	0.24	***
	SEM	0.27	0.31	0.28	0.32
	P-level	***	***	***	*
b*	1	12.5 a,A	12.8 a,A	13.7 a,A	12.6 a,A	0.29	n.s.
	3	11.4 b,A,B	11.7 b,A,B	13.0 a,A,B	12.4 a,b,A	0.24	*
	6	10.4 b,B,C	11.1 a,b,B	11.2 a,b,C	12.7 a,A	0.30	*
	9	9.5 b,C	9.9 b,C	11.7 a,C	12.1 a,A	0.27	***
	11	11.8 a,A,B	10.0 a,C	11.0 a,C	11.0 a,A	0.30	n.s.
	SEM	0.30	0.23	0.26	0.26
	P-level	***	***	***	n.s.
a/b*	1	0.6 c,A	0.8 a,A	0.7 b,A	0.7 b,A	0.02	***
	3	0.5 b,A	0.7 a,A	0.7 a,A	0.6 a,A	0.02	***
	6	0.6 a,A	0.6 a,B	0.6 a,B	0.6 a,A	0.02	n.s.
	9	0.7 a,A	0.5 b,B	0.5 b,B	0.6 a,b,A	0.02	*
	11	0.3 b,B	0.5 a,B	0.5 a,B	0.5 a,B	0.03	***
	SEM	0.03	0.02	0.02	0.02
	P-level	***	***	***	*
Chroma	1	14.3 a,A	16.0 a,A	16.4 a,A	15.0 a,A	0.35	n.s.
	3	12.9 b,A,B	14.4 a,b,B	15.9 a,A	14.9 a,A	0.35	*
	6	12.4 b,A,B	12.7 b,C	12.9 a,b,B	15.0 a,A	0.39	*
	9	11.3 b,B	11.6 b,D	12.9 a,B	13.9 a,A,B	0.29	***
	11	11.1 b,B	11.5 b,D	12.3 a,B	12.3 a,B	0.29	**
	SEM	0.32	0.33	0.34	0.35
	P-level	*	***	***	*
Hue	1	61.1 a,B	53.2 c,B	57.2 b,B	57.2 b,B	0.71	***
	3	62.2 a,B	54.4 c,B	55.3 b,c,B	58.3 b,A,B	0.78	***
	6	57.9 b,B	61.5 a,A	60.7 a,A	59.3 b,A,B	0.85	**
	9	57.7 b,B	63.4 a,A	62.4 a,b,A	60.2 a,b,A,B	0.86	*
	11	74.3 a,A	62.5 b,A	63.8 b,A	63.8 b,A	1.18	***
	SEM	1.12	0.85	0.73	0.84
	P-level	***	***	***	*
MetMb (%)	1	25.5 a,B	20.2 a,C	23.1 a,A	21.0 a,B	0.97	n.s
	3	35.1 a,B	19.3 b,C	14.2 b,B	26.1 ab,AB	2.94	**
	6	38.3 a,B	24.1 bc,B	19.0 c,A,B	26.2 ab,AB	1.81	**
	9	43.6 a,A	26.3 bc,B	21.5 c,A,B	31.7 b,A	3.15	**
	11	45.3 a,A	30.9 bc,A	20.6 c,A,B	35.4 b,A	3.20	**
	SEM	2.83	1.14	1.13	1.71
	P-level	**	***	**	*

Control: ground beef without extracts; BEEA: ground beef added with 1000 mg of EE/kg meat; BEEB: ground beef added with 2000 mg of EE/kg meat; ASC: ground beef added with 250 mg of sodium ascorbate/kg meat. ^a–c: Means values in the same row with different letters are significantly different (P < 0.05). ^A–D: Means values in the same column with different letters are significantly different (P < 0.05). SEM: standard error of the mean. P-level, Significance; n.s.: not significant, * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

shows the effects of EE on the colour characteristics of raw ground beef. Lightness (L*) values were constant (P > 0.05) even with the use of antioxidants. In the Control, however, the values tended to decrease until day 9, while there was a slight increase on the last day of storage, which could be attributed to the oxidation processes. Pateiro et al. [11] reported that higher levels of protein oxidation during storage leads to a lower water-retention capacity in the product and, consequently, increased light dispersion. Thus, an increase in L* values occurs when there is less myoglobin on the surface as well as increased light scattering [63]. Regarding a*, b*, a*/b* and chroma values, most treatments showed a decrease during the period storage (P < 0.05), but an increase in yellowness (b*) was observed for the Control group between days 9 and 11. The increase in b* (yellowness) may be attributed to the accumulation of Schiff pigments from lipid to protein complexes because of oxidative stress [15]. Furthermore, MacDougall [64] reported that the gradual oxidation of myoglobin and the accumulation of MetMb result in a loss of a* and a gain in b*, which can be interpreted as an increase in the hue angle in the direction of yellow with a loss in chroma. The change in direction towards yellow is perceived as being browner (MetMb). This resulted in higher hue values for the Control compared with the other treatments (P < 0.05) on day 11. Thus, at the end of the storage period, BEEA, BEEB and ASC showed higher a* and a*/b* values and a lower hue angle compared to the Control (P < 0.05), indicating that mango peel extracts inhibited the discolouration of ground beef during cold storage.

On the other hand, the maximum amount of MetMb was observed in the Control (45.3%) while the raw ground beef at 2000 EE mg/kg meat showed the lowest percentages (20.6%) on day 11 of storage. MetMb formation is usually a consequence of the action of free radicals, generated during lipid oxidation on the heme group of myoglobin, which triggers oxidation of the molecule and leads to colour loss [3]. Furthermore, Motoyama et al. [65] reported that bacterial contamination increases MetMb percentages in meat via increased oxygen consumption. The high oxygen demand of aerobic bacteria in their logarithmic growth phase leads to the oxidation of myoglobin to MetMb [66]. Sánchez-Escalante et al. [67] mentioned that when the proportion of MetMb reaches 40%, the product is rejected by consumers. According to them, the oxidised pigment would be perceived after 6 days of storage in Control.

Therefore, the results revealed that the presence of antioxidant compounds in EE protected colour by retarding the formation of MetMb. The inhibition of MetMb in ground beef could be due to the ability of polyphenols to scavenge the radical generated [68] and could also act as metal-chelating agents which form stable complexes with haeme and non-haem ions released from disrupted cell membranes due to the grinding of the meat [1]. Different authors have documented the effects of natural antioxidants from agroindustrial by-products such as guarana seed [11], avocado peel and seed [15], peanut skin [1] and red grape pomace [12] on colour stability in different meat products such as raw pork patties, ground beef and raw lamb patties.

Moreover, total colour differences (E1–11) were 7.8, 5.2, 4.4 and 4.5 for Control, BEEA, BEEB and ASC, respectively (

Table 4. Total colour difference (ΔE_1–11) measured on the surface of raw ground beef stored under refrigeration for 11 days at 4 °C for 11 days.

Treatments	ΔE1–11	SEM
Control	7.8 a	0.90
BEEA	5.2 b	0.65
BEEB	4.4 b	0.78
ASC	4.5 b	0.56

Control: ground beef without extracts; BEEA: ground beef added with 1000 mg of EE/kg meat; BEEB: ground beef added with 2000 mg of EE/kg meat; ASC: ground beef added with 250 mg of sodium ascorbate/kg meat. SEM: standard error of the mean. ^a,b: Means values in the same column with different letters are significantly different (P < 0.05).

) with differences observed between Control and the rest of the treatments (P < 0.05), while BEEA, BEEB and ASC were similar (P > 0.05). Several authors have reported a classification scale for visual perception of ΔE as follows [69]: 0.5-1.5 ‘small difference’, 1.5–3.0 distinct difference’, 3.0–6.0 very distinct, 6.0–12.0 ‘great difference’ and ≥12 ‘very much’. According to this scale, the changes in colour in this study from BEEA, BBEB and ASC could be considered as very distinct, while Control would be considered as a ‘great difference’. Pateiro et al. [11] reported that colour differences might be a result of the product’s discolouration due to oxidation processes during the storage period. Thus, BEEB, BEEA and ASC ground beef showed lower colour differences than Control during storage, demonstrating a better oxidative stability. This finding corresponds with the study by Rodríguez-Carpena et al. [15], who noticed that control raw pork patties underwent a more intense colour deterioration than patties with the incorporation of avocado by-products such as peels.

3.2.2. Lipid Oxidation

During the raw ground beef storage (Figure 1a), most of the treatments (except BEEB) increased (P < 0.05) the production of TBARS indicating the formation of aldehydes. On day 1, TBARS values for BEEB were significantly (P < 0.05) lower than any other treatment. Moreover, after 3 days of storage, the concentrations of TBARS in raw ground beef treated with EE were considerably lower than the Control (P < 0.05), showing that EE effectively protected against lipid oxidation.

On the other hand, the cooking process moderately promoted lipid oxidation in BEEA and BEEB. Cooking disrupts cellular organization and causes protein denaturation leading to the loss of antioxidant enzyme activity and the release of protein-bound iron [70]. Iron, in its various forms, could oxidise unsaturated fatty acids in meat [71]. Therefore, in the cooked product (Figure 1b), treatments with EE showed significantly (P < 0.05) lower TBARS values compared to Control and ASC (commercial antioxidant). After 11 days of storage, these last treatments showed a significant (P < 0.05) increase in lipid oxidation compared to the treatments with EE. Thus, the use of sodium ascorbate (ASC) in ground beef increased lipid oxidation to values even higher than control treatment. Sodium ascorbate is effective at reducing oxidation as ascorbate, which is known to be an efficient scavenger of radical species [13,14]. However, it is also responsible for pro-oxidant behavior by reducing transition metals such as iron perpetuating the formation of hydroxyl radicals (OH·) through the Fenton reaction [14]. In this regard, Bañón et al. [10] reported that ascorbate can reduce haematic Fe³⁺, react with O₂ and inhibit free radical formation, but may also form the pro-oxidative species (Fe²⁺ and Cu⁺).

Regarding the effect of the extract addition (BEEA and BEEB), TBARS values remained almost constant during all 11 days of storage. Thus, at the end of the storage period, BEEA and BEEB showed MDA values below 0.3 mg of malondialdehyde/kg of meat, indicating that EE was more efficient against lipid oxidation in raw and raw-cooked ground beef. These treatments showed values that were below the threshold for rancid flavor perception of 0.5 to 1.0 mg MDA/kg meat [40,72]. Thus, the ability of EE to inhibit MAD formation derives from their high phenolic compound content and high antioxidant capacity. Masibo and Hen [48] reported that mango peels contain two main polyphenols which are mangiferin and quercetin 3-0-galactoside. Mangiferin has been considered as a more potent antioxidant than vitamin C and E with iron-complexing abilities. Consequently, it can be used to reduce iron-induced oxidative change [73]. Considering the great variety of compounds in mango peels, the antioxidant capacity of EE cannot be attributed to only one compound, suggesting a synergistic effect of all compounds.

The effectiveness of vegetable and fruit by-products in controlling lipid oxidation by reducing MDA formation has been reported in raw and cooked meat products [10,11,15,25,74,75].

3.2.3. pH and Microbiological Analysis

Table 5. pH and total bacteria counts (TBC) of raw ground beef formulated with mango peel ethanolic extracts (EE) stored under refrigeration for 11 days.

	Storage Day	Treatments				SEM	P-Level
		Control	BEEA	BEEB	ASC
pH	1	5.63 a,D	5.55 a,C	5.22 b,C	5.26 b,C	0.15	**
	3	5.43 ab,D	5.39 b,C	5.25 c,C	5.68 a,C	0.16	***
	6	6.23 a,C	6.01 b,B	6.02 b,B	6.05 b,B	0.16	**
	9	6.65 a,B	6.38 b,B	6.10 b,AB	6.27 b,A	0.17	***
	11	7.10 a,A	6.44 b,A	6.35 b,A	6.49 b,A	0.17	**
	SEM	0.16	0.15	0.15	0.17
	P-level	***	***	***	***
TBC	1	3.2 a,C	2.9 a,C	3.1 a,C	3.1 a,C	0.25	n.s.
	3	4.0 a,C	3.2 a,C	3.6 a,C	3.4 a,C	0.23	n.s.
	6	6.7 a,B	5.1 b,B	5.6 b,B	6.0 a,b,B	0.25	*
	9	7.5 a,A,B	7.6 a,A	7.4 a,A	7.7 a,A	0.13	n.s.
	11	8.2 a,A	8.0 a,A	7.6 a,A	8.0 a,A	0.15	n.s.
	SEM	0.34	0.40	0.35	0.37
	P-level	***	***	***	***

Control: raw ground beef without extracts; BEEA: raw ground beef added with 1000 mg of EE/kg meat; BEEB: raw ground beef added with 2000 mg of EE/kg meat; ASC: raw ground beef added with 250 mg of sodium ascorbate/kg meat. SEM: standard error of the mean. ^a,b: Means values in the same row with different letters are significantly different (P < 0.05). ^A–D: Means values in the same column with different letters are significantly different (P < 0.05). P-level, Significance; n.s.: not significant, * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

shows the effects of EE on pH and the evolution (log₁₀ CFU/g) of total bacteria counts in raw ground beef during refrigerated storage. The pH values for all treatments increased gradually over the storage period (P < 0.05). An increase in pH may be attributed to the accumulation of ammonia, which is then attributable to the degradation of proteins and amino acids by bacteria [76]. Bekhit et al. [77] reported that microbial and endogenous enzymes act on amino acids at a high pH and decompose alkaline ammonia compounds, thereby increasing the pH of meat. However, once the storage period had elapsed, BEEA, BEEB and ASC showed pH values within the limits established by the Mexican Legislation (pH < 6.8) in fresh ground beef [78], with BEEB showing lower pH values than Control (P < 0.05).

On the other hand, total bacteria count of all treatments increased (P > 0.05) as storage progressed, especially in the Control. An increase in the pH has an impact on the rate of biochemical processes and provides a better environment for the growth of the microbial population in meat [77]. No differences (P > 0.05) were observed between treatments on days 1, 3, 9 and 11. However, the total bacterial count was lower in BBEA (5.1 log₁₀ CFU/g) and BEEB (5.6 log₁₀ CFU/g) than the control (6.7 log₁₀ CFU/g) after 6 days of storage. These treatments remained below the limit for fresh ground beef, which is 5 × 10⁶ CFU/g (6.7 log₁₀ CFU/g) established by Mexican Legislation [78]; and below 7 log₁₀ CFU/g, a value considered by the International Commission for Microbial Specifications for Foods (ICMSF) as the limit which determines the end of fresh meat shelf-life [79].

The slight inhibitory effects achieved with EE on the microbial population might be attributable to their phenolic compounds such as polyphenols, which include flavonoids and other bioactive components that have demonstrated antimicrobial activity [60]. Ayala-Zavala et al. [59] reported that the site(s) and number of hydroxyl groups in the phenol group are associated with their antimicrobial capacity and relative toxicity to microorganisms, with evidence showing that increased hydroxylation leads to increased microbial toxicity. In this regard, flavonoids present three phenolic rings with various hydroxyl groups. Furthermore, Guil-Guerrero et al. [33] and Villalobos-Delgado et al. [80] mentioned that the antimicrobial capability of polyphenols has showed different mechanisms of action such as cytoplasmic membrane destabilization, cell membrane permeabilization, deprivation of essential minerals (iron and zinc) by means of chelating and inhibition of extracellular microbial enzymes, along with direct interference in microbial metabolisms.

Consequently, the antimicrobial activity of fruit seeds and peels is well documented. Yu et al. [1] observed that the incorporation of peanut skin extract (PSE) to raw ground beef promoted a decrease in bacterial growth (on day 6 and after), while control (0% PSE) increased steadily over the storage period. Pateiro et al. [11] also reported that guarana seed extracts showed a reduced microbiological population on day 7 of storage.

3.2.4. Sensorial Analysis

Finally, regarding the sensory analysis, the highest odour, flavour and overall acceptance scores of cooked ground beef were obtained for BEEB and ASC (positive control), with no differences (P > 0.05) among them and scores above six (

Table 6. Sensory analysis of cooked ground beef formulated with mango peel ethanolic extracts (EE).

Treatments	Odour	Colour	Flavour	Overall Acceptability
Control	5.2 b	6.2 a	5.1 b	5.6 b
BEEA	5.3 b	6.2 a	5.7 b	5.7 b
BEEB	6.0 a	6.0 a	6.3 a	6.3 a
ASC	6.1 a	6.2 a	6.4 a	6.4 a
SEM	0.10	0.09	0.10	0.10
P-level	**	n.s	**	*

Control: ground beef without extracts; BEEA: ground beef added with 1000 mg of EE/kg meat; BEEB: ground beef added with 2000 mg of EE/kg meat; ASC: ground beef added with 250 mg of sodium ascorbate/kg meat. SEM: standard error of the mean. ^a,b: Means values in the same column with different letters are significantly different (P < 0.05). P-level, Significance; n.s.: not significant, * (P < 0.05), ** (P < 0.01).

). Regarding colour, there were no differences (P > 0.05) between treatments. Despite this, consumers noticed grey discolouration in the Control, which may be indicative of alterations due to lipid oxidation.

Thus, these results indicate that the incorporation of EE did not produce a perceptibly bad colour and flavour in cooked ground beef, despite the fact that extract could contribute an astringent flavour and a more yellow-brown colour. He et al. [81] reported that astringency in fruits is primarily derived from tannins and other polyphenolic compounds. To this regard, the Ataulfo variety mango peel is a rich source of hydrolysable tannins [56]. In contrast, the greater acceptability of BEEB could be related to the fresher flavour and more intense odour perceived by the panellists.

4. Conclusions

Among the two extracts evaluated, the ethanolic extract (EE) showed the highest phenolic and flavonoid content while also exhibiting good antioxidant activity (reducing and scavenging activity). Furthermore, the antimicrobial activity in vitro showed its inhibitory effect against Staphylococcus aureus (foodborne pathogen). Additionally, the incorporation of EE into raw ground beef maintained colour stability by reducing the loss of redness with a redder and more vivid colour. Furthermore, this extract had an antioxidant effect on lipid oxidation in raw and raw-cooked ground beef, especially at a high concentration (2000 mg/kg), making them as effective or more effective than ASC during 11 days of storage. Additionally, for the sensory analysis of cooked ground beef, EE at a high concentration showed similar scores to ASC for odour, flavour and overall acceptability. However, although EE showed mild antimicrobial activity in vitro, it only showed microbiological stability in raw ground beef until day 6 of storage.

Therefore, the extraction of antioxidants from mango peel waste and their incorporation into meat products could mitigate the formation of malondialdehyde (toxic and mutagenic metabolite).