Aristotelia chilensis Leaf Powder as a Sustainable Alternative to Synthetic Antioxidants in Fresh Sausages: Advancing Toward More Natural and Ecological Meat Production

Abstract

The development of sustainable food systems requires natural solutions that reduce dependence on synthetic additives while ensuring instrumental quality, sensory acceptability, and consumer safety. This study evaluated Aristotelia chilensis leaf powder, a Chilean native plant rich in polyphenols, as a natural and sustainable alternative to synthetic antioxidants in the production of fresh sausages. The leaf powder was incorporated at 500, 1000, and 1500 mg/kg, and effects on proximal composition, pH, color, fatty acid profile, volatile organic compounds and sensory attributes were assessed. No changes were found in proximal composition or pH. Treatments with 500 and 1000 mg/kg increased oleic and eicosapentaenoic acids and reduced trans fatty acids and lower concentrations were observed of aldehydes linked to lipid oxidation. Overall, 500 mg/kg of maqui leaf powder (ML) was identified as the optimal treatment, as it reduced oxidation indicators, improved the lipid profile, and maintained sensory acceptability. In addition to its technological functionality and effect on sensory acceptability, the use of Aristotelia chilensis as a natural ingredient in sausages could reinforce food sustainability by reducing dependence on synthetic petroleum-derived additives, revaluing local biodiversity within a circular economy framework, and meeting consumer demand for healthier products with clean labels.

1. Introduction

Fresh sausages are one of the most widely consumed meat products [1]. Their ease of preparation and characteristic flavor have made them a valued food in various cultures. However, the proteins and fats responsible for their flavor constitute, at the same time, a challenge for their preservation, since these nutrients favor the development of biochemical reactions that lead to loss of quality and significantly reduce the shelf life of these products [2]. Oxidation is the most frequent degradation process affecting the quality of fresh sausages [3]. Synthetic antioxidants have been widely used in the meat industry as a barrier technology to delay oxidative processes in meat products due to their stability, efficacy and economic advantage [4]. For example, sodium nitrite is one of the most widely used antioxidants and preservatives in meat products [5]. However, its use in fresh meat products is very restricted and subject to various regulations [6]. Instead of sodium nitrite, other synthetic additives petroleum derivatives are used, such as BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), PG (propyl gallate), TBHQ (tertbutyl hydroquinone) [7]. In meat products, these additives can stabilize color parameters, reduce the formation of metmyoglobin, and provide oxidative stability of lipids and proteins, avoiding the generation of volatile compounds derived from lipids, without affecting sensory attributes. However, synthetic antioxidants are unsustainable ingredients that are hazardous to human health, as studies have concluded that regular consumption of these synthetic compounds could have adverse health effects and has been linked to mutational damage to DNA [8]. From an environmental perspective, synthetic antioxidants such as BHT are of concern. Their high stability and the technological advantages they offer have led to widespread use across various industries, resulting in the accumulation of toxic residues in numerous biological and environmental matrices [9]. Simultaneously, consumers are increasingly prioritizing their health and are more demanding about food quality. They demand healthier meat products free from antioxidants, preservatives and artificial colors [10]. Consequently, the markets for functional foods formulated without synthetic additives is gaining more and more ground [11,12].

Therefore, it is important to find alternative solutions to synthetic antioxidants and preservatives to produce meat products. In this context, the functionality of polyphenolic compounds derived from fruits, vegetables and herbs as antioxidants and antimicrobials has been studied in meat products and their functionality has been demonstrated in vitro [12,13,14,15,16,17]. In addition, studies have shown that polyphenolic extracts of maqui leaves exhibit excellent antioxidant and/or antimicrobial capacity in formulations of fresh meat products such as beef patties with very promising results, which makes it a good candidate to replace synthetic additives in the meat industry [18]. Maqui (Aristotelia chilensis mol. Stuntz) is a plant native to southern Chile, known for its fruit’s rich in anthocyanins, which give it an intense purple color. Maqui fruits are known as superfoods owing to their high concentration of antioxidants. However, maqui leaves have an approximate concentration of bioactive compounds 3.2 times higher than their fruits (148.76 mg GAE/g and 45.7 mg GAE/g, respectively) [18,19]. The main compounds present in the maqui leaves include flavonoids, flavonols, phenolic acids, and stilbenes [20]. Despite the natural qualities of both fruits and leaves of maqui there are limited studies on the effect of its incorporation into the formulation of popular meat products such as sausages. Therefore, the study aimed to evaluate the quality of pork/beef sausages prepared with maqui leaf powder (MLP).

2. Materials and Methods

2.1. Plant Material

This study and the procedures developed were approved by the Scientific Ethical Committee of the University of La Frontera (Approval No. 125/21, Temuco, Chile). Maqui (Aristotelia chilensis (Mol.) Stuntz) leaves sampled in summer at the Maquehue Experimental Farm of the Universidad de La Frontera, Chile (38°50′16.01″ S, 72°41′39.98″ W) were used. The samples were washed with distilled water and dried in an oven at 35 °C (Binder ED25, Binder, Tuttlingen, Germany) until they reached constant weight. Then, they were ground in an ultracentrifuge mill (ZM 200, Retsch GmbH and Co. KG, Haan, Germany) passing them through an 80 µm sieve. The resulting maqui leaf powder (MLP) was stored in polyethylene bags at −21 °C until further use.

2.2. Preparation of Sausages with Maqui Leaves Powders

The study was conducted using a completely randomized design to evaluate the effect of maqui leaf powder concentrations on sausage quality parameters. Five treatments were produced: (a) Control without antioxidants (CON); (b) 500 mg/kg of sodium erythorbate (SA); (c) MLP at Ma500 mg/kg (Ma500); (d) MLP at Ma1000 mg/kg (Ma1000); (e) MLP at Ma1500 mg/kg (Ma1500). Pork loin (50%), beef (20%), (20%), and pork bacon were ground into 8 mm and 6 mm plates, respectively, using a refrigerated grinder (La Minerva, Bologna, Italy). Additionally, 5% water, 1.5% salt, and spices (sweet paprika (2.5%), garlic (0.25%), hot paprika (0.25%), cumin (0.25%), and pepper (0.25%) were added to the formulation. The concentrations of SA and MLP (500–1500 mg kg⁻¹) were adjusted based on 100% of the total mass to maintain a constant proportion in all experimental treatments, ensuring consistency in the final product.

The mixture was homogenized, then stuffed into pork casings (32–34 mm diameter) to achieve a final weight of 50 g. The sausages were aired for 24 h at 2 °C and 85% RH in the absence of light to eliminate surface moisture. Then, twenty sausages of 50 g each were produced per treatment and portioned (n = 5 per day and per treatment) for physicochemical analyses (days 1, 6, 13, and 21). Subsequently, the sausages were conditioned for 24 h at 2 °C in a chamber and placed on trays protected by a modified atmosphere (60% CO₂/40% N₂) for 21 days at 2 °C with 1076 lux of warm white, fluorescent light. This experiment was repeated twice with the same ingredients and production phases.

2.3. Proximal Composition Analysis

The characterization of the proximal composition of the developed prototypes was carried out following the procedures described in the standards for fat [21], protein [22], ash [23], and moisture analysis [24]. In MLP prototypes, the procedure for the determination of total fat was carried out following the procedure described by AOCS Am 5-04 [21] for which an automatic extractor (Ankom ^XT10 ANKOM Technology Corp., Macedon, NY, USA) was used. The determination of the protein content was conducted by the Kjeldahl procedure using Vapodest®, C. Gerhardt GmbH & Co. KG, Königswinter, Germany for hydrolysis and extraction of proteins. Moisture content was determined gravimetrically by weighing 5 g of samples and maintaining them at 105 °C in a heating and drying oven (Memmert UFP 600, Memmert, Schwabach, Germany) until constant weight. Ash content was also determined by a gravimetric method, for which 3 g of samples were weighed into porcelain capsules and calcined at 600 °C in a muffle furnace (Carbolite^® RWF 12-13, Hope Valley, UK) until constant weight.

2.4. pH and Color

pH of the prototypes was measured using a digital pH meter (Hanna Instruments Inc., Cluj, Romania) previously calibrated. Color was measured using a portable colorimeter (Konica Minolta Sensing, model CR-10, Tokyo, Japan) using the CIELab system, where the redness index or a* (red–green intensity), yellowness or b* (yellow–blue intensity), color saturation or C* (chromaticity) and lightness or L* (dark to light) were determined [18].

2.5. Fatty Acid Profile

For fatty acid analysis, sausages were minced with a domestic mincer to homogenize samples. Lipids were extracted according to the methodology proposed by Folch et al. [25]. Fatty acid methyl esters (FAME) were formed using 800 µL of n–hexane (Merck, Darmstadt, Germany, 100795, SupraSolv^® grade, ≥99.0% purity) and 1.3 mL of 2 N potassium hydroxide in methanol (Merck, Germany, 111787, Titripur^®), added to each sample and then magnetically stirred for 30 min. The supernatant was filtered with 0.5 g anhydrous sodium sulfate (Merck, Darmstadt, Germany, 106647, Suprapur^® grade, 99.99% purity) and centrifuged at 2000× g at room temperature for 5 min. FAME was analyzed using a gas chromatograph (Clarus 500, Perkin Elmer, Boston, MA, USA) coupled with a flame ionization detector (FID), split injection mode, and autosampler. FAME separation was performed with an SP^TM 2380 fused silica capillary column (60 m × 0.25 mm × 0.2 µm film thickness) (Supelco, Bellefonte, PA, USA) by injecting one microliter of FAME extract. A gradient program was used for column temperature: initial temperature was set at 150 °C, after 1 min temperature was increased at a rate of 1 °C min⁻¹ to 168 °C, maintained for 11 min, then increased at 6 °C/min to 230 °C, and this temperature was maintained for 8 min. The detector and injection port temperature were 250 °C and nitrogen was used as carrier gas. Individual FAMEs were identified by retention time using a standard 37–component FAME Mix C4-C24 (Supelco, Bellefonte, PA, USA).

2.6. Volatile Compound Profile

The extraction, separation, identification and determination of volatile compounds were carried out following the procedure and conditions described by Cutillas et al. [26]. A solid-phase microextraction (SPME) system with a Pal RTC–120 autosampler (CTC Analytics AG, Zwingen, Switzerland)) was used for the separation of volatile compounds, while a 7890B GC System gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to a 5977B MSD mass selective detector (Agilent Technologies) was used for the separation of volatile compounds. The SPME tool was loaded with a fused silica fiber (10 mm length) coated with 50/30 mm thick DVB/CAR/PDMS (divinylbenzene/carboxene/polydimethylsiloxane) (Supelco, Bellefonte, PA, USA). Before analysis, the fiber was conditioned by heating in a SPME fiber conditioning station at 270 °C for 30 min. Meanwhile, 1 ± 0.02 g of sausages sample was weighed into a 20 mL vial (Agilent Technologies, Santa Clara, CA, USA) and immediately capped with a laminated Teflon rubber disk for headspace SPME (HS-SPME) volatile compound extraction. Samples were equilibrated for 15 min at 37 °C, and successively the extraction process was conducted for 30 min at the same. After extraction, the fiber was desorbed and kept at 260 °C for 8 min in the injection port of the gas chromatograph-mass spectrometer (GC-MS) system. Helium was used as carrier gas with a constant flow rate of 1.2 mL/min. For volatile separation, a DB-624 capillary column (30 m, 0.25 mm i.d., film thickness 1.4 μm; J and W Scientific, Folsom, CA, USA) was employed. The oven temperature program was isothermal for 10 min at 40 °C, raised to 200 °C at a rate of 5 °C/min, successively raised/raised to 250 °C at a rate of 20 °C/min, and held for 5 min (total run time for analysis was 49.5 min). Injector and detector temperatures were adjusted and maintained at 260 °C. Mass spectra were acquired using the 5977B selective detector operating at electron energy at 70 eV, with an electron multiplier voltage of approximately 900 V, and collecting data at 2.9 scans/s in the range m/z 40–550 in scan acquisition mode. Mass source was maintained at 230 °C, while the mass was set at 150 °C. After chromatographic analysis, data processing and identification were performed with Mass Hunter Quantitative Analysis B.07.01 software (Agilent Technologies), where integration was conducted with the Agile2 algorithm, while peak detection was performed by deconvolution. Compounds were identified by comparing their mass spectra with those included in the NIST14 (National Institute of Standards and Technology, Gaithersburg, MD, USA) library (2014, version 2.2). Volatile compounds were considered when the spectra matched more than 85% and appeared in more than 50% of the sample group. Finally, after integration, peak detection, and identification of each compound, the extracted ion chromatogram (EIC) of the quantifier ion was obtained from each peak. The results were expressed as area units (AU × 10⁴/g sample).

2.7. Sensory Evaluation

The effect of the inclusion of MLP on the organoleptic attributes of sausages at the beginning of storage was evaluated (day 1). A trained sensory panel composed of six people (three women and three men, aged 34 to 50 years) from the Center for Technology and Innovation in Meat Quality of the Universidad de La Frontera conducted the sensory investigations focusing on the following attributes: appearance, color, juiciness, odor, flavor, texture and overall acceptability. In addition, a sensory evaluation based on visual descriptions was performed on days 6, 13, and 21. The sausages’ color attributes, percentage of discoloration, and overall acceptability were evaluated using a 5-point scale. For color and overall acceptability, the scale was: 1 = excellent, 2 = good, 3 = acceptable, 4 = poor, and 5 = unacceptable. For surface discoloration, the following scale was used: 1 = none, 2 = 0–10%, 3 = 11–20%, 4 = 21–60%; 5 = 61–100%. The analyses were performed under controlled conditions in a white–light room, according to the procedures described in ISO 6658 [27]. Each sausage wrapped in aluminum foil was cooked on a S + S/S + S/S + S double large contact electric grill (Milan Toast, Sulbiate, MB, Italy) preheated at 150 °C until a core temperature of 70 °C determined using a manipulated probe was reached. A 9–point hedonic scale was used, where nine corresponded to liking very much and one to disliking very much.

2.8. Statistical Analysis

Statistical analysis was performed with IBM SPSS Statistics 23 software (IBM Corporation, Somerset, NY, USA). A completely randomized design was applied: five treatments × twenty sausages per treatment × two batches. Results were expressed as the mean ± standard deviation of the mean. Normality and homogeneity of variance of the variables were previously confirmed by Shapiro–Wilk and Levene’s tests, respectively. A two-factor Analysis of Variance (ANOVA) was performed to evaluate the index of pH, color index (a*, b*, L), sensory attributes (color, percentage of discoloration and general acceptability) and volatile organic compound profile. Treatment and storage time were considered as fixed effects. Sausage proximal composition, fatty acid profile and sensory analysis were evaluated by one-factor ANOVA. When significant differences were detected, Tukey’s test was performed, with a significance level of (p < 0.05). Additionally, a Principal Component Analysis (PCA) was performed to evaluate the relationship between the physicochemical variables using the R.4.0.5 statistical program.

3. Results and Discussion

3.1. Changes in Proximal Composition, pH and Color

Moisture (58.75–59.19%), fat (19.72–20.71%), protein (15.97–16.45) and ash (1.90–1.98%) concentrations did not show statistically significant differences between treatments (p > 0.05) (

Table 1. Proximal composition of sausages reformulated with natural antioxidants from maqui (Aristotelia chilensis Mol. Stuntz) leaves determined at the beginning of storage (day 1).

Treatment	Moisture (%)	Fat (%)	Protein (%)	Ash (%)
CON	58.76 ± 0.94 *	20.42 ± 1.42	16.45 ± 0.40	1.98 ± 0.06
SA	59.20 ± 0.23	20.91 ± 0.72	15.98 ± 0.67	1.90 ± 0.08
Ma500	58.38 ± 0.73	20.14 ± 1.27	16.45 ± 0.24	1.94 ± 0.09
Ma1000	58.67 ± 0.69	20.64 ± 0.22	16.08 ± 0.34	1.94 ± 0.05
Ma1500	59.71 ± 0.38	19.72 ± 0.35	15.99 ± 0.40	1.97 ± 0.02
Sig	n.s.	n.s.	n.s.	n.s.

* Mean ± standard deviation of three replicates per treatment. CON (non-antioxidant control); SA (synthetic antioxidant Sodium Erythorbate); MLP at 500 mg/kg (Ma500); MLP at 1000 mg/kg (Ma1000) and MLP 1500 mg/kg (Ma1500). Sig (Significance): n.s. (not significant).

). This suggests that MLP does not modify the proximal composition of the sausages. Table S1, shown as Supplementary Materials, shows the proximal composition of MLP. These results are consistent with those previously reported for pork sausages reformulated with olive leaf extracts [28]. Changes in pH during storage for all five treatments are shown in

Table 2. Evolution of pH and color (a*; b*; L) during the 21 days of storage of sausages reformulated with maqui (Aristotelia chilensis Mol. Stuntz) leaves powders (MLP).

		Storage Time at 2 °C
Parameter	Treatment	Day 1	Day 6	Day 13	Day 21	p-Value
pH	CON †	5.82 ± 0.01 1	5.40 ± 0.02 2	5.52 ± 0.07 2	5.53 ± 0.06 2	0.000
	SA	5.86 ± 0.08 1	5.39 ± 0.05 2	5.46 ± 0.05 2	5.44 ± 0.03 2	0.000
	Ma500	5.76 ± 0.05 1	5.38 ± 0.04 2	5.58 ± 0.07 2	5.51 ± 0.05 2	0.000
	Ma1000	5.78 ± 0.09 1	5.37 ± 0.06 2	5.44 ± 0.08 2	5.53 ± 0.03 2	0.000
	Ma1500	5.79 ± 0.08 1	5.36 ± 0.03 2	5.49 ± 0.05 2	5.52 ± 0.03 2	0.000
	p-value	n.s.	n.s.	n.s.	n.s.
Color
a*	CON	26.16 ± 1.99	26.95 ± 2.19	27.13 ± 2.3	25.35 ± 0.60	n.s.
	SA	26.23 ± 2.00	28.51 ± 0.21	29.09 ± 0.27 a	27.45 ± 1.18 a	n.s.
	Ma500	26.16 ± 2.06	27.08 ± 0.74	26.50 ± 0.50	24.77 ± 0.57	n.s.
	Ma1000	26.40 ± 1.95	26.29 ± 1.57	26.81 ± 0.69	24.82 ± 1.07	n.s.
	Ma1500	25.25 ± 1.23	25.48 ± 0.38	24.49 ± 0.83 b	23.54 ± 1.43 b	n.s.
	p-value	n.s.	n.s.	0.010	0.011
b*	CON	32.64 ± 3.10 1	34.25 ± 2.17	32.96 ± 4.08	31.75 ± 1.63	n.s.
	SA	35.16 ± 3.18	35.64 ± 2.3 1	37.29 ± 0.75 a2	34.33 ± 1.75	0.41
	Ma500	33.49 ± 3.2 1	34.68 ± 1.971	34.08 ± 1.92	30.83 ± 2.20	n.s.
	Ma1000	36.53 ± 0.58 1	33.32 ± 2.12	33.68 ± 1.45	31.81 ± 1.92 2	n.s.
	Ma1500	34.09 ± 1.04	34.47 ± 1.42	30.92 ± 1.55 b	30.60 ± 2.20 b	0.020
	p-value	n.s.	n.s.	0.045	n.s.
L*	CON	46.71 ± 1.52	50.80 ± 2.00	49.05 ± 3.71	45.08 ± 3.49	n.s.
	SA	51.04 ± 3.78	48.57 ± 3.59	51.73 ± 1.08	46.89 ± 1.67	n.s.
	Ma500	47.51 ± 1.65	46.99 ± 3.28	48.57 ± 0.72	44.49 ± 3.05	n.s.
	Ma1000	51.00 ± 3.10	47.69 ± 1.86	47.83 ± 3.54	46.89 ± 0.81	n.s.
	Ma1500	48.66 ± 0.09	48.56 ± 2.01	46.70 ± 1.48	46.95 ± 0.65	n.s.
	p-value	n.s.	n.s.	n.s.	n.s.

† CON: antioxidant-free sausages; SA: sausages with synthetic antioxidant Sodium Erythorbate; MLP at 500 mg/kg (Ma500); MLP at1000 mg/kg (Ma1000) and MLP 1500 mg/kg (Ma1500). ^a,b Different superscripts in the same column and for the same parameter indicate significant differences (treatment effect; p < 0.05; Tukey’s Test); ^1,2 Different superscripts in the same row (treatment) and for the same parameter indicate significant differences between days (effect of storage time; p < 0.05; ANOVA). The p-value (same column) indicates significant differences between days. p-values (same row) indicate significant differences between the treatments.

. pH measurements on day one showed no differences between treatments. Over the course of storage, a decrease in pH of approximately 0.4 units was observed for all treatments. However, no significant differences were noted between treatments on any of the sampling days. Carballo et al. [29] reported similar results for lamb sausages prepared with natural hops extract and packaged under a modified atmosphere of 80% NO₂/20% CO₂. According to the authors, under these conditions, lactic acid bacteria (LAB) are dominant in the medium. In addition, microorganisms such as Brochothrix thermosphacta and Enterobacteriaceae, as well as other heterofermentative bacteria, can dominate the medium, causing an increase in lactic acid and acetic acid content.

Combined effect of storage time and the inclusion of MLP as a natural antioxidant on the color of pork and beef sausages stored for 21 days was evaluated (Table 2). Measurement of fresh meat color was conducted using the CIELab system. This system involves the analysis of the total myoglobin concentration and the relative proportions of one or more redox forms of myoglobin (i.e., deoxy-, oxy-, met-). None of the color index (a*, b*, L) were significantly altered by the inclusion of MLP until day 13 of storage. However, from day 13 onwards, a significant interaction effect (p < 0.05) between time and treatment was observed. Specifically, the treatment with the highest concentration of MLP experienced a reduction of 1.94 units in the redness value (a*) compared to the SA treatment (synthetic antioxidant). This same trend remained constant until the end of storage. Similarly, no significant differences were observed in the yellowness index (b*) at the beginning of storage. However, by day 13, the AS treatment showed an increase in this indicator reaching a value of 37.29 (p < 0.05), significantly higher than the rest of the treatments. Sodium erythorbate is used in meat formulations as an antioxidant and preservative but can also act as a color accelerator and stabilizer [30]. In contrast, MLPs are rich in pigment chlorophyll. Nonetheless, sausages made with MLP showed adequate redness index (24.77, 24.82, and 23.53 for Ma500, Ma1000, and Ma1500, respectively). By the end of the storage period, color index (lightness) showed no differences compared to the control and synthetic antioxidants.

3.2. Volatile Compound Profile

Fresh meat, in its original state, has a mild odor with little aroma and a blood-like taste. The oxidation of lipids and proteins, together with the development of microbial communities during storage, produce unpleasant odors characteristic of spoiled meat products (rancid, sour, spicy, fermented meat, old cheese, sulfurous and putrid odors) [31,32,33,34]. In this study, a total of 121 VOCs (volatile organic compounds) were identified, which are segmented by chemical families.

Table 3. Profile of volatile and identified organic compounds in fresh sausages reformulated with maqui (Aristotelia chilensis Mol. Stuntz) leaf powder (MLP) stored for 21 days at 2 °C (expressed as % area).

		Day 1					Day 21
VOCs (%)	RT	Match Factor (%)	m/z	CON	AS	Ma500	Ma1000	Ma1500	CON	AS	Ma500	Ma1000	Ma1500
n- hexane	4.88	98.94	56.0	0.84	0.82	0.42 1	0.15 a1	0.90 1	0.60	0.68	4.79 a2	2.56 b2	2.88 b2
Trichloromethane	7.37	98.28	83.0	0.13	0.16	0.07 1	0.03 1	0.17 1	0.08	0.11	1.12 a2	0.72 a2	0.70 a2
Octane	16.98	96.56	85.0	0.27	0.21	0.09	0.08	0.17	0.29	0.12	0.09	0.11	0.08
Cyclopentane	18.38	88.85	70.0	0.02	0.02	0.01	0.02	0.02	0.11	0.03	0.07	0.07	0.07
Ethylbenzene	21.43	98.02	91.0	0.63	0.44	0.51	0.37	0.46	0.52	0.41	0.40	0.39	0.41
Benzene, 1,3-dimethyl-	21.84	99.50	91.0	2.36	1.70	1.97	1.46	1.82	1.93	1.58	1.49	1.50	1.58
o-Xylene	23.08	97.74	91.0	0.53	0.39	0.46	0.34	0.43	0.49	0.39	0.38	0.39	0.40
Bicyclo [3,1,0] hex-2-ene, 2-methyl-5-(1-methylethyl)-	23.83	97.36	91.0	0.27	0.27	0.21	0.21	0.28	0.29	0.29	0.26	0.25	0.26
(1R)-2,6,6-Trimethylbicyclo[3,1,1]hept-2-ene	24.17	98.40	93.0	1.52	1.28	1.22	1.40	1.55	1.62	1.47	1.49	1.43	1.5
Bicyclo [3,1,1] heptane, 6,6-dimethyl-2-methylene-, (1S)-	26.26	98.00	77.0	2.39	2.20	1.90	1.94	2.40	2.18	2.14	1.97	1.92	1.94
Heptane, 2,2,4,6,6-pentamethyl-	26.39	96.42	56.0	0.21	0.20	0.21	0.22	0.23	0.23	0.25	0.25	0.22	0.21
Nonane, 3,7-dimethyl-	30.33	88.93	71.0	0.00	0.01	0.00	0.00	0.00	0.08	0.15	0.25	0.26	0.23
Dodecane	33.79	95.56	71.0	0.02	0.05	0.02	0.02	0.03	0.36	0.64	1.01	0.96	0.84
Decane, 2,9-dimethyl-	33.80	87.53	57.0	0.03 1	0.07 1	0.02 1	0.02 1	0.04 1	0.53 2	0.93 2	1.47 2	1.39 2	1.20 2
Butane, 2,2-dimethyl-	36.98	94.31	71.0	0.01 1	0.03 1	0.01 1	0.00 1	0.01 1	0.36 a2	0.55 2	0.79 2	0.70 2	0.73 2
Octane, 2,7-dimethyl-	36.99	90.92	57.0	0.01 1	0.051	0.01 1	0.01 1	0.01 1	0.49 2	0.75 2	1.07 2	0.94	0.99 2
HDROCARBONS				8.22	6.821	6.6	6.06	7.39	8.1	7.47	7.66 1	8.44 1	8.25 1,2
1-Butanol, 3-methyl-	16.61	99.36	70.0	1.18 1	2.07 1	2.59 1	3.32 1	3.88 1	0.24 2	0.19 2	0.44 2	0.94 2	2.01 2
1-Butanol, 2-methyl-	16.79	95.94	56.0	0.07	0.15	0.16	0.23	0.28	0.04	0.03	0.07	0.16	0.41
Silanediol, dimethyl-	18.32	98.93	45.0	0.55	0.22	0.42	0.50	0.02	0.21	0.07	0.06	0.06	0.37
1-Pentanol	18.37	95.43	55.0	0.02	0.02	0.02	0.02	0.02	0.14	0.04	0.09	0.09	0.09
2,3-Butanediol	21.30	95.58	45.0	0.43 1	0.43	0.66	0.49	0.22	1.88 2	0.68	0.51	0.67	0.44
1-Hexanol	23.24	97.43	56.0	0.05	0.06	0.06	0.07	0.11	0.82	0.24	0.48	0.56	0.48
2-Ethyl-1-hexanol	29.61	96.75	57.0	0.09	0.05	0.06	0.03	0.06	0.06	0.07	0.07	0.09	0.05
1-Hexanol, 2-ethyl-	29.61	95.82	83.0	0.03	0.01	0.02	0.01	0.02	0.02	0.02	0.02	0.02	0.01
Benzyl alcohol	31.12	95.59	79.0	0.13	0.16	0.16	0.33	0.74 1	0.29	0.30	0.61	0.99	1.61 2
Cyclohexanol, 2,6-dimethyl-	33.17	86.89	71.0	0.07	0.08	0.06	0.08	0.10	0.06	0.06	0.07	0.07	0.07
Phenylethyl Alcohol	33.77	99.11	91.0	0.11 1	0.19 1	0.36 1	0.71 1	0.97 1	3.38 2	4.94 2	8.46 a2	13.36 b2	17.77 c2
3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)-	34.86	90.65	111.0	0.02	0.02	0.02	0.02	0.02	0.04	0.04	0.04	0.05	0.05
p-Menth-2-en-7-ol, trans-	38.12	87.98	93.0	0.02	0.03	0.03	0.03	0.04	0.05	0.05	0.03	0.05	0.05
p-Cymen-7-ol	39.34	98.38	135.0	0.23	0.27	0.35	0.59	1.29	0.55	0.58	0.60	0.90	1.27
ALCOHOLS				3.00	3.76	4.95 1	6.43 a1	7.78 b1	7.78 2	7.32 2	11.55 a2	18.56 b2	24.68 c2
Butanal, 3-methyl-	9.80	99.48	58.0	0.47	0.52	0.29	0.13	0.06	0.10	0.09	0.12	0.10	0.10
Butanal, 2-methyl-	10.41	97.57	57.0	0.19	0.25	0.14	0.07	0.02	0.02	0.03	0.07	0.09	0.09
Hexanal	19.17	98.42	56.0	0.01	0.01	0.00	0.00	0.00	0.68 1	0.08	0.17 2	0.13 2	0.07
Benzeneacetaldehyde	30.86	91.81	91.0	0.17 1	0.26 1	0.22 1	0.22 1	0.13 1	21.58 2	21.15 2	11.65 2	8.05 2	5.23 2
Nonanal	32.08	99.23	98.0	0.12	0.08	0.02	0.01	0.01	0.14	0.12	0.08	0.08	0.04
3-p-Menthen-7-al	35.57	96.99	109.0	0.07	0.11	0.04	0.03	0.02	0.05	0.09	0.05	0.05	0.04
Benzaldehyde, 4-(1-methylethyl)	37.62	98.65	133.0	5.98 1	8.92	4.87	3.87 1	4.03	7.06 2	9.85	6.79	7.44 2	6.04
4-Isopropylcyclohexa-1,3-dienecarbaldehyde	39.15	97.46	79.0	0.89	1.25	0.74	0.67	1.05	1.12	1.41	1.12	1.15	1.03
ALDEHYDES				7.91 a1	11.40 b1	6.33 ac1	5.00 ac1	5.35 ac1	30.75 a2	32.82 a2	20.03 b2	17.09 b2	12.65 c2
2,3-Butanedione	6.36	96.95	86.0	0.10	0.25	0.43	0.31	1.08 1	0.07	0.18	0.09	0.03	0.03 2
2-Butanone	6.60	92.04	72.0	0.00 1	0.00 1	0.00 1	0.02 1	0.08 1	0.44 2	0.19 2	0.49 2	0.58 2	0.53 2
Acetoin	15.78	96.15	45.0	31.64 a1	31.51 a1	40.35 b1	38.00 b1	19.31 c1	2.50 2	2.65 2	2.78 2	1.67 2	1.89 2
Ethanone. 1-(1H-pyrrol-2-yl)	32.35	97.86	94.0	0.07	0.10	0.08	0.09	0.11	0.13	0.14	0.14	0.14	0.13
6-Methyl-3,5-heptadiene-2-one	32.97	87.13	109.0	0.07	0.09	0.07	0.07	0.09	0.12	0.13	0.11	0.12	0.09
KETONES				31.88 a1	31.95 b1	40.93 c1	38.49 c1	30.67 d1	3.25 a2	3.29 2	3.60 2	2.54 2	2.67 b
Thiirane, methyl-	5.47	97.21	74.0	3.71 1	2.29 1	3.85 1	3.34 1	3.89 1	0.03 2	0.02 2	0.01 2	0.01 2	0.01 2
Sulfide, allyl methyl	11.58	98.21	88.0	1.02	0.78	0.64	0.66	0.85	0.74	0.62	0.46	0.54	0.45
Disulfide, methyl 2-propenyl	24.48	96.51	120.0	0.23	0.18	0.26	0.20	0.27	1.23	1.20	1.46	1.10	0.95
Diallyl disulphide	31.22	99.44	113.0	1.04	1.03	0.71	0.79	0.75	0.94	1.05	0.69	0.74	0.60
SULFUR				6.01 1	4.28 1	5.46 1	5.00 1	5.76 1	2.95 2	2.89 2	2.62 2	2.39 2	2.01 2
Ethylenimine	4.88	87.76	41.0	0.75 1	0.74 1	0.36 1	0.14 1	0.80 1	0.54 2	0.60 2	2.44 2	2.24 2	2.54 2
1H-Pyrrole, 3-methyl-	15.98	94.29	81.0	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Propane. 2-nitro-	24.17	92.77	41.0	0.14	0.10	0.08	0.07	0.06	0.14	0.10	0.11	0.10	0.11
3-Methylpyridazine	30.65	88.55	94.0	0.01	0.01	0.01	0.01	0.02	0.02	0.02	0.02	0.03	0.03
NITROGEN				0.92	0.87	0.47 1	0.24 1	0.9 1	0.72 a2	0.74 a2	2.59 b2	2.39 b2	2.7 b2
alpha-Pinene	24.17	98.34	93.0	1.53	1.28	1.23	1.41	1.55	1.62	1.47	1.49	1.43	1.50
beta-Pinene	26.26	97.18	91.0	3.30	2.97	2.58	2.64	3.27	3.02	2.96	2.70	2.67	2.69
beta-Myrcene	26.66	97.81	93.0	0.96	0.97	0.81	0.93	1.16	1.28	1.43	1.14	1.19	1.25
3-Carene	27.48	98.80	93.0	2.84	2.23	2.40	2.93	3.00	3.45	2.95	3.08	3.43	3.45
α-Terpinene	27.88	93.12	121.0	0.09	0.10	0.08	0.09	0.12	0.12	0.15	0.11	0.13	0.13
D-Limonene	28.29	98.64	68.0	1.32	1.19	1.08	1.43	1.62	1.68	1.61	1.58	1.69	1.69
o-Cymene	28.49	98.47	119.0	20.12 a	20.46 a	16.94 b	18.11 ab	23.77 c	22.14	20.17	19.92	20.84	19.35
Eucalyptol	28.77	97.47	154.0	0.03	0.04	0.03	0.03	0.04	0.04	0.04	0.04	0.04	0.04
γ-Terpinene	29.42	99.11	91.0	8.36	8.48	6.98	7.40	10.03 a1	7.47	8.35	7.02	7.23	7.15
α-Terpinolene	30.54	93.79	136.0	0.03	0.04	0.03	0.03	0.04	0.05	0.06	0.05	0.06	0.06
Terpinen-4-ol	34.85	92.34	93.0	0.02	0.03	0.02	0.02	0.04	0.04	0.05	0.04	0.05	0.05
Caryophyllene	42.04	99.62	91.0	0.44	0.48	0.43	0.44	0.65	0.71	0.78	0.73	0.80	0.79
Humulene	42.92	94.42	93.0	0.04	0.04	0.04	0.04	0.05	0.08	0.09	0.08	0.09	0.09
TERPENES				39.08 1	38.30 1	32.65 1	35.49 1	45.35 1	41.69 2	40.12 2	37.97 2	39.65 2	38.23 2
Ethyl Acetate	6.79	88.20	43.0	0.05	0.01	0.06	0.04	0.05	0.48	0.42	0.46	0.64	0.64
Acetic acid ethenyl ester	9.80	86.04	43.0	0.46	0.44	0.25	0.11	0.05	0.08	0.08	0.09	0.09	0.08
Methyl isovalerate	17.30	90.51	74.0	0.00	0.01	0.01	0.02	0.05	0.01	0.01	0.01	0.01	0.01
Butanoic acid, 2-methyl- ethyl ester	21.04	91.41	102.0	0.00	0.00	0.01	0.02	0.04	0.03	0.03	0.03	0.04	0.05
Butanoic acid, 3-methyl- ethyl ester	21.26	95.47	85.0	0.02	0.00	0.01	0.03	0.07	0.03	0.03	0.04	0.05	0.06
1-Butanol, 3-methyl- acetate	22.50	99.02	70.0	0.02	0.02	0.04	0.11	0.37	0.02	0.01	0.02	0.02	0.04
1,5-Dimethyl-1-vinyl-4-hexenyl butyrate	32.00	87.89	93.0	0.04	0.05	0.04	0.04	0.07	0.07	0.08	0.07	0.08	0.07
Linalyl acetate	32.01	86.47	71.0	0.03	0.04	0.03	0.04	0.05	0.06	0.06	0.06	0.06	0.06
Methyl salicylate	35.74	96.09	120.0	0.02	0.02	0.02	0.02	0.04	0.06	0.07	0.06	0.07	0.06
ESTER				0.64 1	0.59 1	0.48 1	0.44 1	0.78 1	0.83 2	0.77 2	0.84 2	1.06 2	1.09 2
Dimethyl ether	2.92	92.89	46.0	0.08	0.07	0.09	0.13 1	0.08	0.91	0.71	0.85	1.53 2	0.97
ETHER				0.08 1	0.07 1	0.09 1	0.13 a1	0.08 1	0.91 2	0.71 2	0.85 2	1.53 2	0.97 2

CON: antioxidant-free sausages; SA: sausages with synthetic antioxidant Sodium Erythorbate; MLP at 500 mg/kg (Ma500); MLP at 1000 mg/kg (Ma1000) and MLP 1500 mg/kg (Ma1500). ^a–c Different superscripts in the same column and for the same parameter indicate significant differences (treatment effect; p < 0.05; Tukey’s Test); ^1,2 Different superscripts in the same row (treatment) and for the same parameter indicate significant differences between days (effect of storage time; p < 0.05; ANOVA).

shows the identified VOCs grouped according to their chemical families. On the first day, a predominance of terpenes and ketones was observed for all treatments. The main terpenes found were o-cymene 16.94–23.77% and γ-terpinene 6.98–10%. Domínguez et al. [32] also observed a high concentration of terpenes in salami, accounting for 63.2% of total VOCs. The origin of terpenes and terpenoids in this type of product is mainly related to the addition of spices (black and white pepper) or can be found in meat due to its presence in the animal diet [33,34]. In fact, significant differences were observed in the concentrations of o-cymene and γ-terpinene (p < 0.05) that were higher in the Ma1500 treatments. In this sense, plant species such as maqui are rich in terpenes and the incorporation of MLP resulted in an increase in the content of these VOCs [35,36,37]. A high concentration of ketones was also found, which was defined by acetoin (30.97–40.93%). Acetoin is the main volatile compound present in fresh beef and is produced due to the breakdown of carbohydrates and plays a key role in terms of taste, providing a buttery, creamy and sweet smell [37]. During storage, the concentration of acetoin decreased in all treatments, and on day 21 it was between 1.67 and 2.78%. Liu et al. [38] reported a significant decrease in acetoin content during shelf-aging in salami sausages inoculated with different yeast strains and Lactobacillus rhamnosus. In this study, acetoin decrease was associated with microbial fermentation of carbohydrates and formation of butter-buttery flavor and odor of the sausages. In addition, Stanborough et al. [39] reported that acetoin concentration had decreased between days 14 and 21 of storage from 9.4 to 5.8. Facultative anaerobic microorganisms such as yeast, Lactobacillus spp. Lactobacillus sakei, Brochothrix thermosphacta and Enterobacteriaceae can generate acetoin and its derivatives from pyruvate [40,41,42]. In fact, acetoin biosynthesis during storage is often an indicator of fermentative degradation [42]. In yeast fermentation this biosynthesis occurs in the initial phase, where there is a vigorous increase in the production and excretion of acetoin to the medium. However, it has been reported that during the later stages of fermentation the level of this hydroxyketone may decrease considerably as it is taken up by yeast cells and reduced to 2-3-butanediol. That is, at these stages, the elimination is greater than the excretion of acetoin [43]. Likewise, it has also been reported that some bacteria, such as those of genus Bacillus (Bacillus subtilis and Bacillus pumilus), use acetoin as a carbon source, producing a fermentative breakdown through the enzyme acetoin dehydrogenase and generating intermediate metabolites such as acetyl butanediol. 2,3-butanediol was found in the VOC profile, but the concentration significantly increased only in the control treatment. Therefore, this compound can be used as an initial substrate to be transformed into other end products of the alcohol series.

In this study, the growth of microbial communities present during storage was not evaluated and further study of the type of microorganisms they dominate, how they are affected by the inclusion of maqui extracts, and their effect on VOCs formation should be considered.

The compounds associated with the spoilage of meats and their derivatives have different chemical natures, including esters, ketones, aldehydes, sulfur compounds, amines, and volatile fatty acids [44]. During storage, a significant increase (p < 0.05) in the content of aldehydes (12.65–32.82%), alcohols (7.32–24.68%), hydrocarbons (10.28–17.07%), and nitrogenous compounds (0.58–4.47%) was observed. In this sense, the analysis of intersubject effects showed a significant interaction (p = 0.002) between the day factor and the treatment factor. At the end of storage, the total aldehyde concentration was significantly higher in the SA treatments and in the sausages CON than in the treatments with natural antioxidants from maqui leaves. In other words, on day 21 of storage, aldehyde formation in the treatments with maqui leaf antioxidants was reduced by 34; 44 and 58% compared to the CON. In meat products, aldehyde formation can come from triglyceride hydrolysis and fatty acid metabolism, such as b-oxidation of unsaturated fatty acids or autooxidation of lipids [31]. In fact, in meat and meat products, the formation of aldehydes is the main indicator of lipid oxidation [44]. Therefore, this could indicate a significant inhibition of lipid oxidation in natural antioxidant treatments of maqui leaves. In addition, a proportional relationship was observed between the increase in the concentration of antioxidants in maqui leaves and the decrease in lipid oxidation (p < 0.05). Velázquez et al. [18] reported the antioxidant effect on lipid oxidation of MLP in beef burgers. The high formation of aldehydes during storage was due to the formation of benzeneacetaldehyde. Benzeneacetaldehyde reached a concentration of 21% in the CON and SA lots. In contrast, for maqui treatments, the concentration was 11.65, 8.05 and 5.23%, respectively. Benzeneacetaldehyde is a compound resulting from the oxidation of lipids and is associated with acorn-fed, rancid, and spicy aroma and has been associated in previous studies with the deterioration of vacuum-packed meat due to lipid and protein oxidation and the growth of Pseudomonas spp., C. maltaromaticum, C. divergens at the expense of oxidative deamination of L-phenylalanine [45,46,47,48]. Most straight-chain aldehydes are produced by the oxidation of unsaturated fatty acids. For example, hexanal is one of the main volatile aldehyde indicators of lipid oxidation, as it is generated as a primary product of polyunsaturated fatty acids, such as ω-6 [48]. In this study, we observed a significant increase in hexanal concentrations during storage (p < 0.05). Hexanal content was higher in CON treatment. All antioxidant treatments (natural and synthetic) showed lower hexanal concentrations than the CON treatment. This suggests the inhibition of lipid oxidation by antioxidants. This was especially true for higher MLP concentrations. Nevertheless, the concentrations reached were well below the detectable odor threshold (5.87 ppm) [49].

On the other hand, hexane was the hydrocarbon that was present in the highest proportion at the end of storage (0.60–4.79%). Long-chain hydrocarbons, such as hexane, have been reported to have less influence on flavor due to their high odor threshold [50].

On the other hand, the formation of alcohols also increased significantly during storage. A significant interaction (p < 0.001) between time and treatment was observed for the family of volatile alcoholic compounds. The MLP treatments showed statistically significant differences compared to the CON (7.78%) and SA (7.32%) treatments. Interestingly, the increase in the concentration of alcohols in the sausages with MLP was proportional to the increase in the concentration of Ma500 (11.55%), Ma1000 (18.56%) and Ma1500 (24.68%) powders. The main alcohol found in all batches was phenylethyl alcohol. At the beginning of storage, concentration was not higher than 1%, but by day 21 its concentration had increased considerably reaching concentrations of 8, 13 and 17% in the Ma500, Ma1000 and Ma1500 lots, respectively; while the CON and SA lots reaching concentrations of 3 and 4%, respectively. Unlike lipid oxidation byproducts, phenylethyl alcohol is known and used as a flavoring agent for its pleasant rose aroma and is found naturally in many plant tissues and essential oils. It can also originate from the fermentative metabolism of yeast during storage in a modified atmosphere. Therefore, it is recommended that future research address the VOC profile of maqui leaves and evaluate their dynamic changes in more detail. However, we believe that the increase in this volatile compound is attributed to its presence in maqui leaves or to the fermentative metabolism of yeasts.

In addition, some alcohols that are indicators of meat degradation were identified, such as 2,3-butanediol and 3-methyl-1-butanol. These compounds are considered indicators, as they are synthesized from the degradation of amino acids such as leucine, valine and isoleucine, so they are closely linked to protein oxidation and have a fermented, fuselated, alcoholic, pungent and ethereal odor [32,51]. In addition, formation of 3-methyl-1-butanol during storage has been reported to be positively correlated with the development of Pseudomonas [51]. Volatile flavoring compounds containing nitrogen originate from the breakdown of proteins, free amino acids and nucleic acids [48]. Our results show an increase in the concentration of ethylenimine (aziridine) in all treatments, going from 0.80% on day 1 to 4.44% on day 21. However, its presence has been reported in chicken meat, in the muscles of the cooked beef shoulder [48].

3.3. Fatty Acid Profile

The fatty acid profile was characterized by a predominant distribution of monounsaturated fatty acids (MUFA) (44.2–44.8%), saturated fatty acids (SFA) (38.5–38.9%) and polyunsaturated fatty acids (PUFA) (18.1–18.7%) in all treatments (

Table 4. Fatty acid profile of sausages reformulated with natural antioxidants from maqui (Aristotelia chilensis Mol. Stuntz) leaves (MLP) determined at the beginning of storage (day 1).

Fatty Acid (%)	CON	SA	Ma500	Ma1000	Ma1500
C10:0	0.088 ± 0.02	0.082 ± 0.02	0.082 ± 0.08	0.096 ± 0.01	0.079 ± 0.01
C12:0	0.149 ± 0.01	0.111 ± 0.03	0.123 ± 0.06	0.128 ± 0.01	0.080 ± 0.03 a
C14:0	1.28 ± 0.05	1.20 ± 0.06 a	1.28 ± 0.36	1.22 ± 0.06 a	1.27 ± 0.03
C16:0	22.6 ± 0.145	22.6 ± 0.34	22.4 ± 0.77	22.6 ± 0.08	22.18 ± 0.20
C16:1n-7	1.92 ± 0.07	1.92 ± 0.12	1.80 ± 0.08	1.91 ± 0.08	1.66 ± 0.12 a
C17:0	0.443 ± 0.05	0.373 ± 0.08	0.356 ± 0.87	0.482 ± 0.06	0.569 ± 0.11
C17:1n-7	0.404 ± 0.08 a	0.323 ± 0.06 b	0.268± 0.23 c	0.352 ± 0.08 ab	0.460 ± 0.10 c
C18:0	13.0 ± 0.10	13.1 ± 0.12	13.2 ± 0.51	13.3 ± 0.08	13.1 ± 0.07
C18:1n-9	39.1 ± 0.38 a	39.9 ± 0.39 bc	40.2 ± 0.20 c	39.6 ± 0.51 b	39.7 ± 0.33 bc
9t,12t-C18:2	1.82 ± 0.12 a	1.55 ± 0.15 b	1.55 ± 0.07 b	1.45 ± 0.01 c	1.396 ± 0.08 c
C18:2n-6	15.4 ± 0.11	15.4 ± 0.45	15.2 ± 0.03	15.5 ± 0.09	15.4 ± 0.19
C20:0	0.304 ± 0.01 a	0.224 ± 0.06	0.2155 ± 0.06	0.224 ± 0.09	0.295 ± 0.04 a
C18:3n-3	0.800 ± 0.01	0.731 ± 0.07 a	0.762 ± 0.06 a	0.737 ± 0.07 a	0.840 ± 0.07
C20:1n-9	0.906 ± 0.03	0.870 ± 0.09	0.924 ± 0.043	0.806 ± 0.00 a	0.913 ± 0.09
C21:0	0.785 ± 0.08	0.717 ± 0.00 a	0.764 ± 0.00	0.734 ± 0.00 a	0.793 ± 0.00
C20:4n-6	0.369 ± 0.01	0.383 ± 0.02	0.396 ± 0.02 a	0.377 ± 0.01	0.380 ± 0.06 a
C20:5n-3	0.225 ± 0.04 a	0.177 ± 0.09 b	0.154 ± 0.06 b	0.371 ± 0.04 c	0.223 ± 0.07 a
C24:0	0.125 ± 0.01 a	0.208 ± 0.05	0.186 ± 0.04	0.117 ± 0.02 a	0.179 ± 0.00
C22:6n-3	0.099 ± 0.01	0.072 ± 0.05 a	0.061 ± 0.018 a	0.127 ± 0.02	0.072 ± 0.07
SFA	38.9 ± 0.50	38.5 ± 0.84	38.5 ± 1.97	38.9 ± 0.55	38.5 ± 0.60
MUFA	44.1 ± 0.57	44.5 ± 0.67	44.8 ± 1.34	44.1 ± 0.66	44.2 ± 0.63
PUFA	18.7 ± 0.34 a	18.3 ± 0.86	18.1 ± 0.26	18.3 ± 0.38	18.5 ± 0.57 a
n-3	1.12 ± 0.07	0.98 ± 0.21	0.98 ± 0.13	1.24 ± 0.1	1.14 ± 0.20
n-6	17.2 ± 0.24	17.0 ± 0.62	16.8 ± 0.11	17.0 ± 0.20	17.0 ± 0.33
n-9	40.0 ± 0.411	40.8 ± 0.481	41.2 ± 0.2 a	40.4 ± 0.51	40.7 ± 0.41
Trans	1.82 ± 0.12	1.55 ± 0.15 a	1.55 ± 0.07 a	1.45 ± 0.10 c	1.39 ± 0.08 c
PUFA/MUFA	0.424	0.411	0.404	0.4315	0.420

SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids. Results are shown as the mean ± standard deviation. ^a–c Different superscripts in the same day. and for the same fatty acid indicate significant differences (treatment effect; p < 0.05; Tukey’s test). This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

). Among the most abundant fatty acids, palmitic acid (C16:0) was identified as the main SFA; oleic acid (C18:1n-9) and linoleic acid (C18:2n-6) as the most predominant MUFA and PUFA, respectively. A reduction in trans fatty acid (9t,12t-C18:2) concentration was observed in both SA and MLP (Ma500; Ma1000 and Ma1500) antioxidant treatments compared to CON treatment. In addition, an inverse relationship was observed between the concentration of MLP and the levels of this trans fatty acid. These results suggest a possible protective effect of natural antioxidants against trans-fat formation. Additionally, an increase in the concentration of arachidonic acid (C20:4n-6) was observed in the treatments (Ma500 and Ma1500) (p < 0.05).

Fatty acids such as alpha-linolenic acid (ALA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) are the most important for human health because of their role in the regulation of the immune system, hormone synthesis and cognitive functions, among many others. Since humans cannot synthesize these fatty acids endogenously due to the lack of delta 15 and delta 22 desaturase enzymes capable of introducing double bonds beyond carbon 9 into the alkylated chain of fatty acids [51], their incorporation through diet is essential. In our study, batch Ma500 showed a higher concentration (1.1 units) of oleic acid (C18:1n-9) and batch Ma1000 showed an increase in EPA levels (0.15 units) (p < 0.05) with respect to the CON treatment. Overall, these results align with previous research that has reported increases in the concentration of MUFA and PUFA fatty acids, such as linoleic acid (C18:2n-6) in beef patties treated with MLP [18]. Although the lipid profile of maqui leaves has not yet been fully characterized it is known that these types of polyunsaturated fatty acids are mainly found in the chloroplasts of green leafy vegetables [52]. In addition, it has been previously reported that the lipid fraction of maqui fruit is highly unsaturated, especially due to the presence of oleic acid (33%) and linoleic acid (46%) [53].

3.4. Organoleptic Analysis

The effect of the inclusion of MLP on the color, odor, texture, flavor, and general acceptability of sausages was evaluated. Appearance, color, juiciness, odor, and texture were all affected by the inclusion of MLP (

Table 5. Organoleptic results of sausages reformulated with natural antioxidants from maqui leaves MLP (Aristotelia chilensis Mol. Stuntz) at the beginning of storage (day 1).

Treatment	Appearance	Color	Juiciness	Odor	Flavor	Texture	General Acceptability
CON	7.0	7.2 a	7.0	6.6 a	6.6 a	6.4 a	6.2 a
AS	7.6	7.8 b	7.8	7.8	8.0	7.6	7.6
Ma500	7.8	7.8 b	7.0	8.2	8.4	8.0	7.8
Ma1000	6.8	6.2 a	8.0	7.4	7.6	8.0	7.6
Ma1500	7.2	6.4 c	7.2	7.2	7.4	7.2	7.2
p-value	0.100	0.028	0.084	0.045	0.050	0.041	0.037

Results are shown as the mean. ^a–c Different superscripts in the same day. and for the same fatty acid indicate significant differences (treatment effect; p < 0.05; Tukey’s test).

). The sensory panel results revealed that the inclusion of 1000 and 1500 mg/kg (Ma1000 and Ma1500) of MLP slightly affected the color of the sausages, producing a darker shade with greenish hues due to the chlorophyll pigments in the maqui leaves. Compared to the SA treatment, which achieved the highest color score, Ma1000 and Ma1500 obtained scores 1.6 and 1.4 units lower, respectively. However, the Ma500 treatment (500 mg/kg) showed no significant differences in color acceptability compared to SA and outperformed the CON. Regarding the other sensory attributes (appearance, juiciness, odor, flavor, texture, and overall acceptability), Ma500 obtained scores equal to or higher than those of the CON and SA treatments. These results suggest that MLP at concentrations of 500 mg/kg can be used as functional ingredients in meat formulations without affecting the organoleptic perception by the consumer. Furthermore, the results showed that other attributes such as odor, flavor, texture, and overall acceptability were enhanced by the inclusion of MLP at the beginning of storage. Comparable results have been reported by other authors who have used leaf powders in meat formulations. For example, in a study by Boruzi et al. [54] the inclusion of walnut leaf powders had positive effects on the appearance and flavor of pork hamburgers, and the treatments with walnut leaf powders showed overall acceptability scores like those of the synthetic antioxidant BHT.

The evolution of sensory attributes throughout storage (days 1, 6, 13, and 21) showed that the inclusion of maqui leaf powder (MLP) had a time- and concentration-dependent effect (Figure 1). At the beginning of storage (days 1 and 6), the color scores were similar across all treatments, indicating a homogeneous appearance immediately after processing. However, from day 13 onward, a tendency toward increasing scores was evident, reflecting a loss of the characteristic red color, especially in treatments with higher MLP concentrations (Ma1000 and Ma1500). On day 21, Ma1500 presented the highest values, indicating greater visual alteration, likely associated with the presence of chlorophyll pigments that intensified greenish tones and the possible oxidation of meat pigments. In contrast, Ma500 and AS maintained low and stable scores, demonstrating better color retention during storage, comparable to that of the synthetic antioxidant.

The discoloration behavior followed a trend similar to that observed for the color scores. During the first six days, no significant differences were observed between the treatments. However, from day 13 onward, discoloration progressively increased, reaching its maximum value on day 21. Treatments Ma1000 and Ma1500 showed the highest percentages (≈25–30%), demonstrating a more rapid loss of surface color characteristics of sausages. In contrast, Ma500 and AS maintained low levels of discoloration (<15%), confirming that moderate concentrations of MLP exert a protective effect against chromatic oxidation.

Likewise, overall acceptability showed a gradual decrease throughout the storage period, which was more pronounced in treatments with higher doses of MLP. Until day 13, the Ma500 and AS values remained stable, indicating that the addition of 500 mg/kg MLP did not negatively affect sensory perception. However, by day 21, acceptability decreased markedly at Ma1500, coinciding with increased discoloration and darkening. This suggests that the visual changes generated by the high MLP content negatively influenced the panel’s overall evaluation.

3.5. Principal Component Analysis

Figure 2 shows the Pearson correlation results, highlighting the relationships between pH, color (a*, b*, and L*), SFA, MUFA, PUFA, and COVs. The pH showed a high degree of negative and positive correlation with the COVs acetoin (r = −0.71) and 1-hexanol (r = 0.72), respectively. In addition, moderate correlations were observed with benzaldehyde, 4-(1-methylethyl) (cuminaldehyde) (r = −0.58), and the a* color index (r = −0.53). The interrelationship between pH, lipid oxidation, and their effects on meat color has been extensively studied. Although the interaction between pH, lipid oxidation, and meat color is complex, lipid oxidation and oxymyoglobin reactions are favored at lower pH values. Similarly, the primary and secondary products of lipid oxidation can oxidize myoglobin, causing a loss of redness [55,56]. In fact, this study observed a negative correlation between the redness index and benzaldehyde formation (r = −0.71). Redness a* also showed a moderate negative correlation with the volatile alcohols benzyl alcohol (r = −0.58) and phenylethyl alcohol (r = −0.66). Among the correlated VOCs, a strong correlation was found, as expected, between benzeneacetaldehyde and benzyl alcohol and phenylethyl alcohol (r = −0.867), (r = 0.895).

Principal component analysis (PCA) was performed to determine the relationship between physicochemical parameters and to evaluate the grouping of treatments according to their quality characteristics. To explore the changes in volatile compounds, acetoin, n-hexane, benzaldehyde, 4-(1-methylethyl)-, disulfide, methyl 2-propenyl, 1-hexanol, benzyl alcohol, phenyl ethyl alcohol, and ethyleneimine were used as active variables. pH, L*, a*, b*, SFA, MUFA, and PUFA were used as complementary variables. The PCA results indicated that the first two components of the analysis explained 68.63% of the data. PC1, representing 46.6% of the variance, was mainly associated with pH, redness index (a*), and the VOCs 1-hexanol, benzyl alcohol, phenylethyl alcohol, disulfide, methyl 2-propenyl, acetoin, benzaldehyde, 4-(1-methylethyl), and benzoic aldehyde.

PC2 accounted for 21.99% of this variance. The variables with the highest participation in this dimension were SFA, MUFA, and PUFA fatty acids; lightness (L*); and the COVs ethylene and n-hexane.

The distribution of treatments in the profile graph (Figure 3) allowed three associations to be identified based on the quality characteristics of the sausages at the end of storage. The association between the CON and SA treatments was marked by a similarity between the redness (a*) and yellowness (b*) indices, which were higher at the end of storage in the samples with natural antioxidants, especially Ma1000 and Ma1500 treated samples. In addition, these treatments also showed similarities in terms of volatile aldehyde content (benzeneacetaldehyde and benzaldehyde, 4-(1-methylethyl)-), which could suggest greater lipid and protein oxidation in these treatments. In the second group, an association was found between the Ma1000 and Ma1500 treatments owing to the similarity in pH and alcoholic VOCs (benzyl alcohol and phenylethyl alcohol). Although there is little information regarding the VOC profile of maqui leaves, the presence of aromatic alcohols, such as benzyl alcohol, in its fruits has been previously reported. Therefore, it is likely that this compound was present in the treatments with higher concentrations of MLP (1000 and 1500 mg/kg) [57]. The third group included the Ma500 treatment, which was differentiated by its content of n-hexane, ethylene, acetoin, disulfide, and methyl 2-propenyl. The presence of n-hexane in raw fermented sausages has been previously reported [58].

4. Conclusions

This study demonstrated that Aristotelia chilensis leaf powder, used at a concentration of 500 mg/kg, is a natural and effective ingredient capable of improving the oxidative stability and fatty acid profiles of fresh sausages without affecting proximal composition or other important quality indicators, such as pH, odor, flavor, and overall acceptability. Importantly, however, at higher concentrations, sensory indicators such as sausage color and appearance may be affected. Still, these results indicate that maqui leaves may be an ingredient that can promote sustainability in the meat industry. Furthermore, from a sustainability perspective, the use of native plant resources in the meat industry contributes to more environmentally friendly production processes by replacing petroleum-derived synthetic additives, reducing environmental impact, and promoting biodiversity. This approach aligns with consumer demand for a new generation of healthier, clean-label meat products. Overall, maqui leaf powder represents a sustainable strategy for the development of healthier and more environmentally friendly meat products.