Natural Additives for Sustainable Meat Preservation: Salicornia ramosissima and Acerola Extract in Mertolenga D.O.P. Meat

Abstract

The search for natural additives from underutilized halophytes and fruit by-products aligns with circular economy principles, addressing consumer demand for healthier and more sustainable alternatives to salt and synthetic antioxidants in foods. Salicornia ramosissima, a halophytic plant rich in minerals, and Malpighia emarginata (acerola), a fruit rich in bioactive compounds, were selected for their potential to enhance meat preservation while reducing reliance on conventional salt and chemical additives. This study evaluated the effects of replacing salt with S. ramosissima powder (1% and 2%) and adding acerola extract (0.3%) in Mertolenga D.O.P. beef hamburgers. Control, 1% salt, acerola, and salicornia formulations were analyzed over 10 days for the following: (1) microbial counts (mesophiles, psychrotrophics, Enterobacteriaceae, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, fungi, Salmonella spp., and E. coli); (2) physicochemical parameters (pH, a_w, and CIE-Lab color); and (3) sensory attributes (odor, color, and freshness). Higher Salicornia concentrations negatively affected color (lower a* values) and sensory perception (darker appearance). Acerola extract improved color stability and delayed the development of off-odors, contributing to higher freshness scores throughout storage. No significant differences in microbial counts were observed between treatments. Overall, acerola and low-dose Salicornia showed potential as natural ingredients for meat preservation, with minimal impact on physicochemical and microbiological quality. These findings support the use of halophytes and fruit extracts in sustainable meat preservation strategies.

1. Introduction

The food industry is undergoing a transformative shift toward sustainable practices, driven by consumer demand for healthier options and regulatory pressures to reduce environmental impacts [1]. Meat is a source of high-quality protein and has been increasingly consumed worldwide, though consumption trends vary regionally, with declines in some European markets due to health and environmental concerns [2]. In fact, meat’s high nutritional value and moisture content render it particularly susceptible to quality deterioration and microbial spoilage, contributing to significant losses across the supply chain—with most of them occurring at the distribution, retail, and consumer levels [3].

Recent trends indicate a growing consumer preference for minimally processed, clean-label products, reflecting broader societal movements toward holistic health and environmental stewardship [4]. This paradigm shift aligns with the United Nations’ 2030 Agenda for Sustainable Development, particularly Goals 3 (Good Health and Well-Being) and 12 (Responsible Consumption and Production), which calls for the sustainable management of natural resources in food systems [5,6]. The meat industry faces a critical challenge in developing innovative preservation methods that extend shelf life, maintain safety and quality, and meet sustainability demands throughout the distribution chain [3,7].

Ground meat products, including hamburgers, represent a significant segment of the global meat market due to their nutritional quality, convenience, and versatility [8]. However, the grinding process dramatically increases the surface area exposed, resulting in a higher risk of contamination and deteriorative processes leading to a shorter shelf life when compared to fresh meat [7]. Studies demonstrate that ground beef undergoes enzymatic and oxidative changes and develops rancidity quicker than intact muscle, with microbial counts often reaching unacceptable levels within 3 days of refrigerated storage [9]. The food industry has traditionally relied on synthetic antioxidants and stringent cold chains to mitigate these effects, but growing consumer resistance to artificial additives has created an urgent need for natural alternatives that can deliver comparable preservation benefits without compromising clean-label status [10,11].

The search for natural preservatives has intensified in recent years, focusing on plant-derived compounds with demonstrated antimicrobial and antioxidant properties [12]. This movement is in response not only to consumer preferences, but also to public health initiatives targeting sodium reduction [13]. The World Health Organization recommends limiting sodium intake to less than 2 g/day, yet processed meat products frequently exceed this threshold, contributing to cardiovascular disease risk [5]. In the European Union, where sodium consumption averages 3.5–5 g/day, regulatory frameworks like Regulation 601/2014 strictly control food additives while paradoxically lacking clear definitions for “natural” alternatives [1,10]. This regulatory gap has spurred research into unconventional ingredients that can fulfil multiple technological functions—including salt reduction, microbial inhibition, and oxidative stability—while meeting clean-label expectations [11,12].

Among the most promising candidates is Salicornia ramosissima, a halophytic plant that thrives in saline coastal environments unsuitable for conventional agriculture [14,15]. This resilient species has evolved sophisticated mechanisms to manage osmotic stress, accumulating mineral ions and bioactive compounds in its succulent stems [16]. Nutritional analyses reveal that Salicornia contains essential minerals (including sodium, potassium, and magnesium), phenolic compounds, and vitamin C at concentrations that vary depending on growth conditions and harvest time [14,17]. Of particular interest is its mineral profile, which confers a salty taste while providing a more balanced sodium-to-potassium ratio than pure NaCl [14]. When processed into powder, Salicornia has shown potential as a partial salt substitute in various food matrices, including bread and sausages, where it can reduce sodium content by 30–50% without compromising preservation efficacy [13,17]. However, its application in meat products, particularly those with Protected Designation of Origin (PDO) status like Mertolenga D.O.P. beef, remains unexplored [17,18]. As part of its food quality policy, the European Union (EU) promotes three types of quality labels for agricultural products and foodstuffs, one of which is Protected Designation of Origin (PDO), which covers agricultural products and foodstuffs that are produced, processed, and prepared in a specific geographical area, using recognized know-how [19]. PDO products present unique formulation challenges, as any additives must preserve the authentic sensory characteristics that define their geographical identity [10].

Complementing halophyte-based salt substitutes, tropical fruits like acerola (Malpighia emarginata) offer potent natural antioxidant solutions [20,21]. Native to Central and South America, acerola cherries contain extraordinary concentrations of vitamin C (ranging from 1000 to 4500 mg/100 g fresh fruit), along with a diverse collection of carotenoids, flavonoids, and phenolic acids [11,20,22]. Unlike synthetic ascorbic acid, acerola’s antioxidant activity derives from synergistic interactions among these compounds, making it particularly effective against both lipid and protein oxidation in complex food systems [7,11]. Though widely used in beverages and dietary supplements, acerola’s application in meat preservation remains limited [7,11]. A few studies have explored its effects on color stability and microbial inhibition, but further research is needed to fully understand its impact, particularly considering the potential risk of masking spoilage through color stabilization. This represents a relevant knowledge gap, especially given acerola’s status as one of nature’s richest sources of antioxidants [11,21].

The intersection of these two natural resources—Salicornia’s mineral-rich profile and acerola’s antioxidant profile—presents an innovative approach to sustainable meat preservation [14,16]. However, critical questions remain about their individual effects on meat products, particularly regarding dose optimization and sensory compatibility [10,17]. Previous research on Salicornia in meat systems has noted potential challenges, including color alterations (notably reduced redness) and flavor deviations at concentrations above 1.5% [17]. Similarly, while acerola extracts effectively inhibit oxidation, their strong acidic profile may require careful balancing to avoid undesirable taste impacts [7,11]. These considerations are especially pertinent for PDO products like Mertolenga D.O.P., where maintaining traditional sensory attributes is paramount [10]. Furthermore, the sustainability credentials of both ingredients warrant examination through the lens of circular bioeconomy principles—Salicornia’s ability to rehabilitate saline soils [15] and acerola’s potential as a by-product valorization opportunity [11] could significantly enhance their appeal to environmentally conscious consumers and producers alike [4,23].

This study addresses these multifaceted challenges through a systematic evaluation of Salicornia ramosissima powder and acerola extract as natural preservatives in Mertolenga D.O.P. beef hamburgers. The research assesses microbial dynamics, physicochemical parameters, and sensory attributes over 10 days of refrigerated storage. By examining these natural additives independently and in combination, the study provides clear guidelines for formulators seeking to reduce sodium content while maintaining oxidative stability in premium meat products [10,13]. From a broader perspective, the findings contribute to sustainable food systems by validating the use of climate-resilient crops (Salicornia) [14,15] and nutrient-dense fruit by-products (acerola) [11] in value-added applications—advancing progress toward multiple Sustainable Development Goals while addressing industry needs for clean-label preservation solutions [1,5,6].

2. Materials and Methods

2.1. Experimental Design

2.1.1. Sampling, Preparation and Processing of Samples

For this study, the semitendinosus and semimembranosus muscles from three male carcasses of Mertolenga D.O.P. beef were collected at 4 days post mortem. These three samples were vacuum-packed and transported under controlled refrigeration (T ≤ 4 °C) and strict hygiene conditions. This ensured optimal meat quality and prevented contamination or deterioration. Within the laboratory, the three batches were physically separated and maintained at 3.0 ± 0.5 °C until further processing.

Meat preparation took place on the same day in the laboratory’s processing room, which was pre-sanitized and maintained at 12.0 ± 0.5 °C. The initial step involved fat extraction, followed by cutting the meat into approximately 4 cm³ pieces using a stainless-steel knife and a cut-resistant stainless-steel glove, both sterilized in water at 82 °C. Samples were taken from each meat batch for microbiological analysis. The remaining meat was then promptly minced using a mincer (PM70, Mainca; Barcelona, Spain) equipped with a 4 mm perforated plate, resulting in a typical fine hamburger consistency. For experimental conditions, each batch was divided into seven equal portions. Each portion was then homogenized with specific ingredients. The following two ingredients were tested independently and in combination: acerola extract (Formulab, LDA, Porto, Portugal) and powdered salicornia (Horta da Ria, LDA, Vagos, Portugal). The concentrations were selected based on previous studies in other food matrices, aiming to explore their novel application in meat products [17,24,25,26].

Table 1. Experimental conditions and ingredient combinations for Mertolenga D.O.P. beef hamburgers.

	Salt	Acerola Extract	Salicornia Powder
Control	-	-	-
Salt	1%	-	-
Salt and Acerola	1%	0.3%	-
Salic. 1%	-	-	1%
Salic. 2%	-	-	2%
Acerola and Salic. 1%	-	0.3%	1%
Acerola and Salic. 2%	-	0.3%	2%

details the experimental conditions and the ingredients used for each treatment.

To achieve proper homogenization of all the components of the hamburger, the ingredients were mixed with 15 mL of distilled water, forming a homogeneous mixture, and added to the minced meat, 5 mL at a time, at 3 random points of the meat, while it was mixed at low speed in a mixer for 30 s. Then, Mertolenga D.O.P. beef miniburgers weighing 20.0 ± 0.5 g, with a 1 cm thickness and 4 cm diameter, were shaped using a Petri dish.

2.1.2. Packaging and Storage of Samples

Mertolenga D.O.P. beef hamburgers were individually packed in co-extruded PA/PE bags (Formulab, LDA, Porto, Portugal), subjected to a modified atmosphere (70% O₂: 30% CO₂; Air Liquide, Lisboa, Portugal), and sealed in a specific packaging machine (V-420 SGA, SAM-MIC, Sabadell, Spain). In total, 315 Mertolenga D.O.P. beef hamburgers were produced, 15 hamburgers for each of the 6 conditions, in triplicate (three batches). For each of the 5 analyzed time points, 3 Mertolenga D.O.P. beef hamburgers from each condition were used. The samples were stored at 1.5 ± 0.5 °C in a refrigeration chamber and analyzed on days 1, 3, 5, 7, and 10. The experimental dynamics consisted of performing microbiological analysis first, followed by instrumental color analysis, and finally sensory analysis on each analysis day.

2.2. Microbial Analysis

Microbiological analysis was initially performed on the meat pieces before the production of the Mertolenga D.O.P. beef hamburgers. For this, 10.0 g samples were aseptically collected from each batch for the quantification of total mesophilic and psychrotrophic microorganisms, Enterobacteriaceae, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria (LAB), fungi, and E. coli. Additionally, 25.0 g samples were collected from each batch for the detection of Salmonella spp. and Listeria monocytogenes.

Subsequently, microbiological analysis was conducted on the produced Mertolenga D.O.P. beef hamburgers on days 1, 3, 5, 7, and 10. The Mertolenga D.O.P. beef hamburgers were removed from refrigeration as needed. All conditions under study were analyzed in a laminar flow hood under sterile conditions. For each Mertolenga D.O.P. beef hamburger, 10.0 g was weighed and subsequently diluted in 90 mL of tryptone salt solution (0.3% tryptone and 0.85% NaCl, sterilized at 121 °C for 15 min) within a Stomacher bag. This mixture was then homogenized in a Stomacher (IUL, Barcelona, Spain) for 90 s. Successive decimal dilutions were prepared in test tubes by adding 1 mL of the solution from the Stomacher bag to 9 mL of sterile tryptone salt. Following this, inoculations were performed by surface plating or pour plating onto the respective culture media. The plates were then incubated according to the time and temperature stipulated for each microorganism under study. After incubation, typical colonies were counted, and the results are expressed in log CFU/g. Microbial counts were determined from triplicate analyses per sample, using independent measurements from randomly selected portions to ensure representativeness and accuracy.

2.3. Physical and Chemical Analyses

On days 1, 3, 5, 7, and 10, pH and water activity were measured. The pH value was obtained by the arithmetic mean of three successive measurements taken at random points on each sample, using a pH meter (WTW GmbH, PH 330i, Weilheim, Germany) with a probe placed directly into the sample. To measure water activity, the samples were slowly heated up to 25 °C (optimum temperature for measurement) to prevent evaporation or condensation. An electronic hygrometer (Rotronic, HygroLab C1, Bassersdorf, Switzerland) was used with two attached probes, each with a resolution of ± 0.001 a_w and an accuracy between 0.01 and 0.02 a_w, to obtain the results. The results were obtained by the arithmetic mean of three successive measurements from random points of each sample.

2.4. Instrumental Color Measurement

Color measurements were carried out in the CIE-Lab space, using a Minolta Chromo Meter CR-310 (Konica Minolta, Osaka, Japan) with a standard 2° observer, illuminant D65, and a 4 mm aperture diameter. The Commission Internationale de l’Eclairage (CIE) L* (lightness), a* (redness), and b* values (yellowness) system was used to determine the color of the fresh meat hamburgers after opening the modified atmosphere packaging and before sensory analysis. Color measurements were carried out on days 1, 3, 5, 7, and 10 of storage. The results were obtained by the arithmetic mean of three successive measurements from random points of each sample.

2.5. Sensory Analysis

Sensory analysis was conducted by a trained panel consisting of 8 to 10 members in each evaluation session, all with experience in beef sensory assessment. Samples were evaluated in a dedicated sensory analysis room 60 min after opening the modified atmosphere packaging. All Mertolenga D.O.P beef hamburgers, refrigerated at 3.0 ± 0.5 °C, were presented individually and randomly to each panelist under consistent lighting conditions. Each panelist received a sensory evaluation sheet for each hamburger. The panelists were asked to evaluate sample color, intensity and acceptability of off-flavors, specific odors detected, and overall freshness. These qualities were evaluated with the use of a 9 cm non-structured linear scoring scale, ranging from 0—not intense/unacceptable—to 9—extremely intense/acceptable. Finally, overall freshness was assessed on a 0 to 9 cm line scale and classified as follows: fresh meat from 6 to 9 cm, semi-fresh meat from 3 to 6 cm, and deteriorated meat from 0 to 3 cm.

2.6. Data Analysis

All statistical analyses were primarily performed using SPSS Statistics (Version 33.0, IBM Corp., Armonk, NY, USA). The individual and interactive effects of acerola and salicornia addition were evaluated using ANOVA. When significant differences between groups were identified by the ANOVA test, Tukey’s HSD (Honestly Significantly Different) post hoc test was employed to determine specific group differences. A multivariate analysis was also conducted using Pearson’s correlation coefficient to measure the degree of linear correlation between two variables. For the microbiological counts, one-way analysis of variance (ANOVA) was conducted to test the effect of meat pH for each sampling day. A statistical significance set at a 5% level of probability (p < 0.05) was used.

3. Results

3.1. Microorganisms Quantification

Table 2. Microorganism counts (mean and standard deviation) in fresh vacuum-packed Mertolenga D.O.P. hamburgers.

Microorganism	log ufc/g Sample
Total mesophiles	4.59 ± 0.17
Total psychrotrophs	4.91 ± 0.17
Enterobacteriaceae	3.69 ± 1.43
Pseudomonas spp.	4.19 ± 0.47
Brochothrix thermosphacta	3.61 ± 0.32
Lactic acid bacteria	2.70 ± 0.49
Molds and Yeasts	-
Salmonella spp.	-
E. coli	-
L. monocytogenes	-

shows the microbial counts of the fresh Mertolenga D.O.P beef before the production of Mertolenga D.O.P beef hamburgers and immediately after opening the vacuum package that came directly from the cutting room. The absence of important pathogenic bacteria such as Salmonella spp., E. coli, and L. monocytogenes was noted.

Table 3. Effect of addition of acerola and salicornia on microbial counts (means and standard deviation) of fresh vacuum-packed Mertolenga D.O.P. hamburgers (expressed in log CFU/g⁻¹).

	Treatment	Time					p
		1	3	5	7	10
Mesophiles	Control	5.58 ± 0.43	6.70 ± 0.25	6.81 ± 0.78	7.50 ± 1.29	6.70 ± 1.65	n.s.
	Salt	5.48 ± 0.51	6.61 ± 0.28	6.84 ± 0.95	7.44 ± 0.88	6.46 ± 2.07	n.s.
	Salt and Acerola	5.58 ± 0.66	6.58 ± 0.12	6.99 ± 0.64	6.96 ± 0.38	6.35 ± 1.17	n.s.
	Salic. 1%	6.46 ± 0.05	5.85 ± 1.16	6.96 ± 0.63	6.87 ± 0.02	6.66 ± 0.51	n.s.
	Salic. 2%	6.10 ± 0.63	6.88 ± 0.43	7.36 ± 0.63	6.96 ± 0.50	6.85 ± 0.73	n.s.
	Acerola and Salic. 1%	6.47 ± 0.15	6.43 ± 0.20	7.13 ± 0.46	7.01 ± 0.39	6.11 ± 0.69	n.s.
	Acerola and Salic. 2%	6.52 ± 0.07	7.00 ± 0.63	7.30 ± 0.56	7.22 ± 0.40	6.98 ± 0.81	n.s.
Psychrotrophs	Control	5.92 ± 0.68 b	7.12 ± 0.20 ab	7.48 ± 0.28 a	7.61 ± 0.63 a	7.88 ± 0.33 a	≤0.01
	Salt	5.40 ± 0.67 b	6.99 ± 0.58 ab	7.76 ± 0.35 a	7.79 ± 0.56 a	7.97 ± 0.27 a	≤0.01
	Salt and Acerola	5.58 ± 0.57 b	6.89 ± 0.65 a	7.49 ± 0.31 a	7.75 ± 0.44 a	7.37 ± 0.16 a	≤0.001
	Salic. 1%	5.05 ± 0.52 b	6.88 ± 0.36 a	7.55 ± 0.14 a	7.63 ± 0.90 a	7.86 ± 0.34 a	≤0.001
	Salic. 2%	5.30 ± 0.66 b	7.10 ± 0.51 a	7.98 ± 0.05 a	7.75 ± 0.47 a	7.91 ± 0.03 a	≤0.001
	Acerola and Salic. 1%	5.23 ± 0.48 b	7.15 ± 0.93 a	7.93 ± 0.21 a	7.70 ± 0.64 a	8.09 ± 0.04 a	≤0.001
	Acerola and Salic. 2%	5.18 ± 0.59 b	6.67 ± 0.93 ab	7.72 ± 0.13 a	7.49 ± 0.22 a	7.47 ± 0.57 a	≤0.01
Enterobacteriaceae	Control	2.81 ± 1.12	3.90 ± 0.81	4.66 ± 1.63	4.87 ± 0.42	4.64 ± 0.86	n.s.
	Salt	3.17 ± 0.61	4.06 ± 0.66	4.64 ± 1.26	4.89 ± 0.63	4.26 ± 1.30	n.s.
	Salt and Acerola	3.28 ± 0.20	4.08 ± 0.14	4.71 ± 1.16	4.99 ± 0.94	4.94 ± 0.36	n.s.
	Salic. 1%	3.28 ± 0.91	3.87 ± 0.66	4.71 ± 1.54	4.71 ± 0.54	4.27 ± 1.37	n.s.
	Salic. 2%	2.98 ± 0.53	3.72 ± 0.68	5.14 ± 1.29	4.66 ± 0.56	4.19 ± 1.43	n.s.
	Acerola and Salic. 1%	3.39 ± 0.55 b	4.60 ± 1.37 ab	4.52 ± 0.35 ab	6.18 ± 1.22 a	5.14 ± 0.13 ab	≤0.05
	Acerola and Salic. 2%	3.10 ± 0.35	4.10 ± 0.25	4.95 ± 0.83	5.08 ± 0.78	4.73 ± 1.24	n.s.
LAB	Control	3.27 ± 0.31 c	4.46 ± 0.98 bc	5.96 ± 0.85 ab	7.10 ± 1.16 a	4.89 ± 0.28 abc	≤0.01
	Salt	3.59 ± 1.86 c	4.20 ± 0.46 bc	6.05 ± 1.13 ab	7.50 ± 1.32 a	4.93 ± 0.16 bc	≤0.01
	Salt and Acerola	3.37 ± 0.54 b	4.56 ± 0.72 ab	6.21 ± 0.90 ab	7.05 ± 0.98 b	4.52 ± 0.12 ab	≤0.05
	Salic. 1%	3.21 ± 0.62 c	4.58 ± 0.47 bc	6.18 ± 1.05 ab	6.83 ± 0.83 a	5.11 ± 0.66 abc	≤0.01
	Salic. 2%	3.02 ± 0.33 c	3.91 ± 0.37 bc	6.10 ± 0.81 a	6.45 ± 0.95 a	5.06 ± 0.24 ab	≤0.01
	Acerola and Salic. 1%	4.13 ± 1.93	3.97 ± 0.66	5.59 ± 0.88	6.93 ± 0.91	5.16 ± 0.55	n.s.
	Acerola and Salic. 2%	4.03 ± 1.89	4.40 ± 0.62	5.52 ± 0.60	6.71 ± 0.17	6.29 ± 1.37	n.s.
B. thermosphacta	Control	3.55 ± 0.87	5.81 ± 0.14	5.84 ± 0.91	5.56 ± 1.02	5.83 ± 1.30	n.s.
	Salt	3.74 ± 0.73	5.29 ± 0.43	5.79 ± 1.26	6.38 ± 0.91	5.18 ± 2.65	n.s.
	Salt and Acerola	3.95 ± 0.85	5.32 ± 0.53	5.71 ± 0.83	5.67 ± 0.74	5.29 ± 1.31	n.s.
	Salic. 1%	3.95 ± 0.74	5.35 ± 0.10	5.45 ± 1.26	5.13 ± 0.89	4.93 ± 1.74	n.s.
	Salic. 2%	3.73 ± 1.22	4.91 ± 0.53	6.20 ± 1.65	4.68 ± 1.65	4.75 ± 2.48	n.s.
	Acerola and Salic. 1%	3.25 ± 0.70	4.93 ± 0.44	5.07 ± 1.52	4.35 ± 1.85	4.85 ± 0.61	n.s.
	Acerola and Salic. 2%	3.50 ± 0.58	4.92 ± 0.23	5.77 ± 1.22	5.36 ± 0.83	4.66 ± 2.34	n.s.
Molds and Yeasts	Control	2.60 ± 0.52	2.89 ± 0.77	3.50 ± 0.74	3.84 ± 0.08	3.89 ± 0.36	n.s.
	Salt	2.90 ± 0.26	3.00 ± 0.35	3.45 ± 0.40	3.79 ± 0.38	3.39 ± 0.23	n.s.
	Salt and Acerola	2.68 ± 0.14	3.07 ± 0.22	3.32 ± 0.34	3.50 ± 1.11	3.19 ± 1.01	n.s.
	Salic. 1%	2.85 ± 0.24	3.34 ± 0.39	3.09 ± 0.36	3.13 ± 0.98	3.57 ± 0.74	n.s.
	Salic. 2%	2.59 ± 0.03	2.63 ± 0.57	3.31 ± 0.62	3.25 ± 0.51	3.44 ± 0.41	n.s.
	Acerola and Salic. 1%	3.05 ± 0.36	3.00 ± 0.18	2.97 ± 0.25	3.56 ± 0.32	3.76 ± 0.97	n.s.
	Acerola and Salic. 2%	3.19 ± 0.81	2.97 ± 0.36	3.69 ± 0.44	3.44 ± 0.69	3.20 ± 0.59	n.s.
Pseudomonas spp.	Control	3.66 ± 0.42	4.43 ± 0.32	4.53 ± 0.52	4.91 ± 0.41	4.49 ± 0.70	n.s.
	Salt	4.13 ± 0.96	4.38 ± 0.17	4.41 ± 0.74	5.19 ± 0.66	4.86 ± 0.61	n.s.
	Salt and Acerola	3.84 ± 0.57	4.14 ± 0.17	4.41 ± 0.45	4.86 ± 0.93	4.11 ± 0.16	n.s.
	Salic. 1%	3.84 ± 0.38	3.78 ± 0.20	4.76 ± 0.52	4.71 ± 0.58	4.30 ± 0.43	n.s.
	Salic. 2%	3.32 ± 0.24	4.08 ± 0.18	4.37 ± 0.60	4.43 ± 0.37	4.55 ± 1.21	n.s.
	Acerola and Salic. 1%	3.69 ± 0.41	3.95 ± 0.21	4.31 ± 0.67	4.91 ± 0.54	5.00 ± 0.06	n.s.
	Acerola and Salic. 2%	4.46 ± 0.93	4.10 ± 0.34	4.82 ± 0.78	4.76 ± 0.72	4.24 ± 0.34	n.s.

n.s.: not significant. Different superscript letters within the same row indicate statistically significant differences between treatments (p < 0.05), according to Tukey’s post hoc test.

presents the microbiological evolution of the hamburgers during storage. All treatments showed consistent increases in microbial counts throughout the storage period. Significant time-dependent growth (p ≤ 0.05) was observed specifically for psychrotrophics, Enterobacteriaceae, lactic acid bacteria (LAB), B. thermosphacta, and Pseudomonas spp. in certain treatments. Notably, the natural additives (acerola extract, Salicornia powder, and their combination) showed no significant differences compared to the salt and the control treatments (p > 0.05) for any microbial group. Most importantly, these results confirm that the natural alternatives performed at least as well as conventional salt in maintaining microbiological quality.

3.2. Physical–Chemical Parameters

Table 4. Effect of addition of acerola and salicornia on physical–chemical parameters (pH, water activity (a_w), and CIELab color coordinates) (means and standard deviations) of fresh vacuum-packed Mertolenga D.O.P. hamburgers.

	Treatment	Time					p
		1	3	5	7	10
pH	Control	5.77 ± 0.19	5.74 ± 0.26	5.78 ± 0.14	5.69 ± 0.26	5.64 ± 0.23	n.s.
	Salt	5.66 ± 0.14	5.59 ± 0.08	5.54 ± 0.04	5.49 ± 0.03	5.44 ± 0.15	n.s.
	Salt and Acerola	5.69 ± 0.18	5.67 ± 0.11	5.62 ± 0.13	5.66 ± 0.16	5.50 ± 0.13	n.s.
	Salic. 1%	5.65 ± 0.15	5.53 ± 0.12	5.50 ± 0.08	5.39 ± 0.07	5.34 ± 0.15	n.s.
	Salic. 2%	5.62 ± 0.20	5.58 ± 0.21	5.42 ± 0.18	5.38 ± 0.13	5.25 ± 0.01	n.s.
	Acerola and Salic. 1%	5.70 ± 0.19	5.62 ± 0.17	5.52 ± 0.10	5.49 ± 0.10	5.62 ± 0.26	n.s.
	Acerola and Salic. 2%	5.70 ± 0.18	5.70 ± 0.25	5.60 ± 0.13	5.59 ± 0.20	5.51 ± 0.19	n.s.
	p	n.s.	n.s.	n.s.	n.s.	n.s.
aw	Control	1.00 ± 0.00	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	1.00 ± 0.00	n.s.
	Salt	0.98 ± 0.01	0.98 ± 0.01	0.98 ± 0.01	0.99 ± 0.00	0.99 ± 0.01	n.s.
	Salt and Acerola	0.99 ± 0.01	0.99 ± 0.00	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	n.s.
	Salic. 1%	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	n.s.
	Salic. 2%	0.99 ± 0.01	0.98 ± 0.00	0.98 ± 0.01	0.98 ± 0.00	0.98 ± 0.01	n.s.
	Acerola and Salic. 1%	0.99 ± 0.01	0.99 ± 0.00	0.99 ± 0.01	0.99 ± 0.01	0.99 ± 0.01	n.s.
	Acerola and Salic. 2%	0.99 ± 0.01	0.98 ± 0.00	0.98 ± 0.00	0.98 ± 0.00	0.98 ± 0.00	n.s.
	p	n.s.	n.s.	n.s.	n.s.	n.s.
L*	Control	39.03 ± 0.96 a	44.38 ± 2.58 a	41.04 ± 0.43 a	41.79 ± 1.36 a	41.16 ± 2.32	≤0.05
	Salt	37.04 ± 1.85 ab	40.08 ± 0.59 ab	40.25 ± 0.70 ab	38.99 ± 1.72 ab	41.30 ± 3.17	n.s.
	Salt and Acerola	38.40 ± 2.43 a	39.06 ± 2.94 ab	37.62 ± 2.27 abc	38.70 ± 1.32 abc	40.33 ± 1.54	n.s.
	Salic. 1%	33.85 ± 0.68 bc	37.66 ± 0.77 b	37.86 ± 0.61 abc	38.06 ± 0.70 bc	39.92 ± 0.54	≤0.001
	Salic. 2%	33.61 ± 0.91 bc	37.67 ± 3.99 b	37.46 ± 1.73 bc	36.90 ± 1.30 bc	37.12 ± 0.90	n.s.
	Acerola and Salic. 1%	36.13 ± 2.28 abc	37.66 ± 1.49 b	38.81 ± 1.31 ab	38.99 ± 0.74 ab	37.67 ± 0.40	n.s.
	Acerola and Salic. 2%	32.61 ± 0.61 c	36.67 ± 2.47 b	34.54 ± 0.66 c	35.53 ± 1.22 c	37.05 ± 1.92	≤0.05
	p	≤0.001	≤0.05	≤0.001	≤0.001	≤0.05
a*	Control	21.86 ± 0.84 a	23.30 ± 3.99	21.17 ± 2.48 a	21.38 ± 1.51 a	16.75 ± 0.60	≤0.05
	Salt	19.56 ± 0.77 ab	21.46 ± 0.71	22.35 ± 1.35 a	20.97 ± 1.70 ab	18.22 ± 1.53	≤0.05
	Salt and Acerola	18.85 ± 0.25 ab	22.22 ± 1.59	19.45 ± 1.82 ab	18.78 ± 0.92 abc	16.07 ± 3.23	≤0.05
	Salic. 1%	17.63 ± 1.96 b	16.23 ± 4.10	17.37 ± 0.77 ab	17.61 ± 0.18 bc	15.18 ± 3.85	n.s.
	Salic. 2%	14.43 ± 1.62 cd	15.23 ± 2.77	17.00 ± 5.35 ab	14.01 ± 1.23 de	12.96 ± 2.49	n.s.
	Acerola and Salic. 1%	16.53 ± 0.31 bc	15.57 ± 2.53	17.00 ± 0.44 ab	16.69 ± 1.47 cd	15.96 ± 3.76	n.s.
	Acerola and Salic. 2%	12.85 ± 1.01 d	17.27 ± 5.52	14.05 ± 1.82 b	12.60 ± 0.75 e	12.55 ± 1.99	n.s.
	p	≤0.001	≤0.05	≤0.05	≤0.001	n.s.
b*	Control	12.51 ± 0.95	11.96 ± 1.18	12.27 ± 1.55	11.28 ± 2.69	8.17 ± 2.92 b	n.s.
	Salt	11.05 ± 0.72	12.71 ± 1.01	13.36 ± 0.41	12.23 ± 0.93	11.51 ± 0.40 ab	≤0.05
	Salt and Acerola	10.99 ± 1.41	12.68 ± 1.21	11.46 ± 1.25	10.82 ± 1.23	8.87 ± 1.30 ab	n.s.
	Salic. 1%	12.76 ± 0.95	12.84 ± 1.30	12.58 ± 0.66	12.41 ± 0.74	11.79 ± 0.11 a	n.s.
	Salic. 2%	12.16 ± 0.20	12.14 ± 2.06	12.95 ± 1.46	11.80 ± 0.66	11.02 ± 0.40 ab	n.s.
	Acerola and Salic. 1%	12.99 ± 0.77	12.34 ± 0.27	12.75 ± 0.40	11.83 ± 1.96	10.51 ± 0.86 ab	n.s.
	Acerola and Salic. 2%	11.40 ± 0.57	13.42 ± 1.46	11.99 ± 0.62	11.29 ± 0.71	10.32 ± 0.67 ab	≤0.05
	p	n.s.	n.s.	n.s.	n.s.	≤0.05

n.s.: not significant. Different superscript letters within the same column (^a, ^b, ^c, etc.) indicate statistically significant differences between treatments (p < 0.05), according to Tukey’s post hoc test. Color coordinates are expressed in the CIE L* (lightness), a* (redness), and b* (yellowness) system.

summarizes the physical and chemical results obtained throughout storage. The physicochemical analysis revealed consistent patterns across all treatments: pH values showed a gradual decrease during storage, while water activity (a_w) remained stable. Statistical analysis confirmed no significant differences between treatments for either parameter (p > 0.05), demonstrating that neither acerola extract nor Salicornia powder negatively affected these critical quality indicators compared to the control.

Instrumental color analysis (CIE-Lab) revealed significant treatment and time effects on the hamburgers’ appearance. For lightness (L*), values increased until day 3 and then slightly decreased, yet remained higher than the initial measurements, indicating an overall lightening of the samples during storage. For redness (a*), values increased until day 3, indicating enhanced red coloration, but then progressively declined until day 10, ultimately falling below the initial values. This trend is consistent with the oxidation of myoglobin and the accumulation of metmyoglobin during storage [10]. Treatment effects were particularly evident, with control samples maintaining the highest values, followed by salt and acerola treatments, which were statistically similar. Salicornia-containing samples showed concentration-dependent darkening, with the 2% addition resulting in a significantly lower lightness compared to the other treatments. Yellowness (b*) remained stable throughout storage (p > 0.05), except on day 10, when significant differences emerged between the control (lowest b* value) and 1% salicornia (highest b* value) (p ≤ 0.05). These results demonstrate that while salicornia significantly influenced color parameters (particularly at a 2% concentration), acerola maintained color characteristics comparable to conventional salt treatment.

3.3. Sensory Analysis

The trained sensory panel documented significant changes in product characteristics throughout the storage period. Color evaluation revealed that all samples underwent progressive darkening, with initial bright red hues gradually transitioning to darker red and brown tones. Notably, burgers containing acerola extract exhibited slower browning development compared to other treatments. In contrast, Salicornia-supplemented samples were immediately identified as darker (Day 1), displaying significantly deeper red tones and more pronounced browning—an effect that intensified with an increasing Salicornia concentration. The 2% Salicornia treatment showed the most affected color alterations, remaining statistically distinct from both the control and salt-only samples at all evaluation points.

Time had a highly significant effect on off-odor development (p < 0.001), confirming the expected progression of spoilage throughout the storage period. In contrast, the treatment itself did not significantly affect the overall off-odor scores (p > 0.05), indicating that the addition of natural ingredients such as acerola extract and Salicornia powder did not worsen the sensory perception of spoilage.

The overall freshness evaluation (OFE) provided crucial shelf-life insights (Figure 1). Initial OFE scores averaged 6.99 cm (>6 cm = “fresh”), except for treatments containing 2% Salicornia (5.8 cm = “semi-fresh”). While all treatments showed a declining freshness, acerola maintained higher OFE scores through Day 6—exceeding the control and salt samples by 1–2 days before reaching the “deteriorated” threshold (Day 7 vs. Days 5–6). Importantly, the Acerola + 1% Salicornia combination significantly improved OFE when compared to Salicornia-only treatments, while 2% Salicornia mixtures showed the most rapid quality decline, being classified as “spoiled” by Day 4.

4. Discussion

Regarding microbiological parameters, it is possible to verify that the behavior of the microbiota present in the meat was similar between the different conditions analyzed (p > 0.05). The most dominant microorganisms of the deteriorative microbiota analyzed were lactic acid bacteria and B. thermosphacta, as described by other authors who concluded that these microorganisms were the most prevalent in refrigerated meat in a modified atmosphere with less than 50% CO₂ and O₂ [27]. Although the initial contamination of lactic acid bacteria was relatively low, their growth was exponential and reached values greater than 7 log cfu/g, which led to acid odors and discoloration, typical sensory alterations involved in the deterioration of meat by lactic acid bacteria [28]. This was essentially due to the accumulation of end products of their metabolism, such as lactic acid, acetic acid, and butyric acid, which were also responsible for the appreciable reduction in pH. The increase in LAB until day 7 reflects their metabolic activity and ability to dominate under refrigerated conditions and modified atmosphere packaging. However, the significant decrease observed by day 10 may be associated with the progressive accumulation of inhibitory metabolites, such as organic acids, the depletion of essential nutrients, and the proliferation of other spoilage microorganisms. These changes, along with physicochemical alterations—such as reduced pH, increased oxidative stress, and intensified microbial competition—may have created unfavorable conditions for LAB survival and growth, contributing to their decline [29,30]. B. thermosphacta was one of the prevalent microorganisms in the meat samples. This microorganism produces diacetyl and a range of fatty acids due to the aerobic metabolism of glucose, which give rise to sour, acidic, or musty odors. These sensory defects can be observed once this organism reaches values of 5 log cfu/g [31]. Several scientists have concluded that total mesophilic bacterial counts between 10⁷ and 10⁸ cfu/g per sample are sufficient to start causing off-odors and slime [32]; these values were reached, on average, by day 7. Tremonte et al., 2016, evaluated the antimicrobial effect of acerola on red meat’s shelf life [33]. The results reported in their study highlight that acerola extract is able to produce a strong antagonistic effect against Pseudomonas species and B. thermosphacta, which are involved in the spoilage of fresh meat products. They described a strong antimicrobial activity of acerola extract, probably due to the presence of several compounds belonging to different bioactive groups, which represent a series of hurdles for microbial growth.

According to Leygonie et al. (2012) [34], the freezing and subsequent thawing of fresh meat can lead to a decrease in pH. This is explained by the fact that pH is a measure of the amount of free hydrogen ions (H⁺), so it is possible that freezing leads to the subsequent production of exudate, which could cause the denaturation of buffer proteins, the release of hydrogen ions, and a consequent decrease in the measured pH. Alternatively, fluid loss from meat can cause an increase in solute concentration, which also results in a decrease in pH. Regardless, all samples were frozen and thawed following the same procedure, and it can be considered that the deviation of results due to the freezing/thawing process was identical in all samples and, therefore, the differences found between different conditions can be considered plausible. In all treatments, the average initial pH obtained at time 1 showed a value of 5.68 ± 0.15, which is in line with other authors who have demonstrated that this value should be between 5.70 and 5.90 for fresh beef [35,36]. Samples treated with acerola had the lowest pH throughout the analysis period. This evidence suggests that the addition of acerola, due to its high content of ascorbic acid, could make it possible to lower the pH in beef [37]. However, in the control and salt samples, the pH also decreased. This drop in pH can be explained by the high prevalence of lactic acid bacteria in the meat throughout the period analyzed, due to the production of lactic, acetic, and butyric acid, among others, as exposed by other authors [29]. The use of Salicornia ramosissima at different concentrations led to a slight reduction in water activity (a_w), which is consistent with the literature, as ionic compounds typically lower a_w by binding free water molecules [38].

In determining the objective color of the meat samples according to the CIE-Lab color space, it was found that the initial mean value of the parameter L* for acerola corresponded to 38.16, which is in agreement with other authors who analyzed the color parameters and defined the L* coordinate as having a value of 37.61 ± 0.31 on day 1 [39]. In the study with salicornia, the mean initial value of the L* parameter corresponded to 35.9, which is not in accordance with the parameter defined by [39], which can be explained by the addition of salicornia, which, due to its strong color, significantly altered the color pattern of the Mertolenga D.O.P beef hamburgers. As expected, it was found that at the initial time, there was an increase in the a* coordinate due to the passage of the deoxymyoglobin form into oxymyoglobin in the oxygen-rich modified atmosphere package [32]. The initial average value of the parameter a* in the acerola study corresponded to 21.86 ± 0.84, similar to the results obtained by other authors in minced meat packed in modified-atmosphere packaging with a high oxygen content [39]. Over time, the a* coordinate declined due to the gradual oxidation of myoglobin and the accumulation of metmyoglobin [32]. The initial mean value of the parameter a* in the study with salicornia samples corresponded to 17.70, which is not in line with that observed by the previously mentioned authors [39], which can be explained by the fact that the samples with salicornia acquired a browner color from the salicornia properties. These color changes can be attributed to the presence of chlorophyl and other pigments naturally found in salicornia [17]. There were no significant differences regarding the b* parameter either in the sample with acerola or in the samples with salicornia, considering the time effect. These results are in line with those demonstrated by other authors, in which no significant differences were found in the parameter b* of beef stored in modified-atmosphere packaging over time [39].

Among other factors able to influence consumer choices, color represents one of the most important visual clues for red fresh meat [40]. Regarding color, as one would expect, the bright red color starts to become dark red and the brown color intensifies over time due to chemical and microbial deterioration causing the oxidation of myoglobin and the consequent accumulation of methemoglobin [32]. In our study, Mertolenga D.O.P. beef hamburgers containing acerola extract demonstrated significantly attenuated browning development compared to control samples throughout the evaluation period. This protective effect on color stability aligns with acerola’s known antioxidant properties, where its phenolic compounds interfere with myoglobin oxidation—a process where lipid oxidation products convert oxymyoglobin’s Fe²⁺ to metmyoglobin’s Fe³⁺, causing discoloration. By potentially neutralizing hydroperoxides and free radicals, acerola’s antioxidants appear to slow this oxidative cascade, thereby preserving redness longer than control samples [33]. This study clearly demonstrated a concentration-dependent relationship between Salicornia content and color perception, with higher concentrations progressively intensifying both dark red and brown coloration at all time points. Importantly, the initially observed darker pigmentation in Salicornia-treated samples—distinct from the control and salt samples at early stages—should be attributed to the plant’s natural pigments rather than product deterioration. This interpretation is supported by the parallel color evolution patterns observed across all treatments during storage, where the gradual darkening of red tones and increased browning developed consistently due to metmyoglobin accumulation (as previously discussed). These findings suggest that while Salicornia’s inherent pigmentation significantly impacts initial product color, it does not accelerate the fundamental oxidative processes responsible for meat discoloration during storage. Samples with 2% salicornia acquired a darker color due to the plant’s own pigments, thus reducing the red color of the meat and, consequently, the overall freshness value. The use of acerola (Malpighia emarginata) showed satisfactory results regarding a reduction in the rot smell, improved the maintenance of the typical red color of the meat, and obtained positive results in the overall evaluation of freshness. As would be expected, the effect of time on rot odor was noticeable in all samples. However, samples with the addition of acerola significantly differed from the control and salt samples. This fact may have been due to the lower microbial contamination that the samples with acerola showed in relation to Pseudomonas, a microorganism known for its great proteolytic and consequently deteriorative activity [27]. This lower contamination may have allowed for a lesser presence of off-odors, since this microorganism is directly linked to their presence. The samples with acerola obtained better results in the overall freshness assessment during all periods under analysis, increasing the shelf life of the hamburger by at least 1 day. These results are in line with another study that concluded that the use of acerola at 0.15% improved the sensory qualities of Mertolenga D.O.P beef hamburgers [7].

5. Conclusions

Our findings demonstrate that acerola (Malpighia emarginata) effectively preserves meat quality, significantly reducing rotten odor development, maintaining typical red coloration, and improving overall freshness scores—extending shelf life by approximately 24 h compared to controls. Salicornia (Salicornia ramosissima) exhibited concentration-dependent effects: while 1% supplementation delayed off-odor perception, higher concentrations (2%) introduced plant-derived pigmentation that darkened meat color, adversely affecting consumer appeal.

From a sustainability perspective, both ingredients offer compelling advantages. Acerola represents a valorization opportunity for tropical fruit byproducts, converting nutrient-rich waste into functional food additives. Salicornia—a halophyte requiring no freshwater irrigation—could help salt reduction strategies in processed meats.

We conclude that acerola serves as a potent natural additive for premium meat products like Mertolenga D.O.P. burgers. For Salicornia, while concentrations of >1% impair sensory quality, further research should explore (a) optimal dosing to leverage its mineral profile and salt replacement potential; (b) synergistic applications with other natural preservatives; and (c) environmental impact assessments of large-scale cultivation.