Oxidation Status and Antioxidant Activity of Analogue Meat Products in Modified Atmosphere Packaging

Abstract

The study aims to assess the impact of modified atmosphere packaging (MAP) on the oxidation status of five types of analogue meat products, crucial for extending shelf life and maintaining quality, and seeks to optimize packaging strategies to mitigate oxidation and provide possible solutions for enhancing the overall quality of analogue meat products. Gas ratios in MAP, as well as thiobarbituric acid reactive substances (TBARS), free fatty acids (FFA), total polyphenol content, and antioxidant capacity were assessed through four different assays (2,2-diphenyl-1-picryl-hydrazyl: DPPH, Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid): ABTS, Ferric Reducing Antioxidant Power: FRAP, Cupric reducing antioxidant capacity: CUPRAC) for analogue meat products (steak, noodles, filet, burger, and mince) on the last day of their shelf life. O₂ ratios in the MAP for all the products did not differ significantly (p > 0.05), but CO₂ concentrations significantly differed (p > 0.05) in the MAP of the evaluated products. The minced product exhibited higher oxidative stability with the lowest TBARS (3.20 mg MDA·kg⁻¹) and FFA (1.12% total fat as oleic acid), along with a high antioxidant capacity (DPPH: 32.26, ABTS: 4.49% inhibition, CUPRAC: 11.48 Trolox mmol/kg). The filet product was more susceptible to the oxidation process, as evidenced by the significantly (p > 0.05) higher TBARS value (9.71 mg MDA·kg⁻¹), lower polyphenol content (1.01 mg gallic acid/g), and antioxidant capacity (FRAP: 4.75 mmol/g, CPRAC: 5.57 Trolox mmol/kg).

1. Introduction

Meat analogue products represent an initial step in dietary shifts toward reducing meat consumption. The objective of creating meat analogue products is to replace meat by replicating its functionality in terms of sensory properties (such as taste, odor, and texture) and nutritional values [1,2]. Meat analogues are primarily categorized into the following two groups: those pre-cooked by the producer and those produced raw (heated by the consumer). Pre-heated products typically include comminuted or ground meat analogues such as sausages, ham, spreads, as well as pre-cooked burgers, meatballs, and nuggets. Raw-produced meat analogues, displayed in the market in a “raw” form, mainly consist of burgers, minced meat, and meatballs [3].

In today’s markets, an increasingly diverse array of meat analogue products is readily available, each crafted through unique methodologies and ingredient combinations to mimic the taste and texture of conventional meat. These products are rich in essential nutritional components, featuring proteins sourced from soy, wheat gluten, and mushrooms, along with lipids derived from coconut, palm, or various oils like sunflower, canola, soy, and corn. Furthermore, polysaccharides such as starches and fibers contribute to the structural integrity and dietary fiber content of these analogues, while additional ingredients like hydrocolloids, gums, colorants, and flavor enhancers play pivotal roles in refining their sensory profile and overall palatability, catering to diverse consumer preferences and dietary needs [4,5,6]. Nevertheless, meat analogues employ a broad spectrum of additive ingredients meticulously selected to emulate not only the color and texture but also the sensory attributes and nutritional qualities inherent in conventional meat products. These additives are intricately formulated to mimic the nuanced characteristics of meat, encompassing its visual appeal, mouthfeel, aroma, and nutritional composition. Through the strategic incorporation of these diverse additives, meat analogues endeavor to offer consumers a compelling alternative that closely resembles the sensory experience and nutritional profile of traditional meat-based dishes, thereby catering to evolving dietary preferences and culinary expectations [7].

Modified atmosphere technology used for food packaging maintains the quality of fresh produce for a long time and extends its shelf life [8]. In this preservation technique (MAP), the natural air surrounding the food in the package is replaced with a specific gas or gas mixture, usually CO₂, O₂, and N₂. MAP gases are suitable for extending the shelf life of perishable foodstuffs such as various kinds of meat, fish, vegetables, fruits, and other products [9]. Each product type must be packaged in an optimized modified atmosphere since the metabolic processes in agricultural products (plant and animal origins) differ from kind to kind, and the respiration, transpiration, and gas permeation (via the packaging) processes that occur simultaneously must be balanced by the MAP system [10]. MAP with N₂ or CO₂ can restrict the O₂ amount in the package thus slowing down the oxidation process of the food products [11]. Carbon dioxide is a colorless, odorless, and tasteless gas and is the most used gas in MAP for food packaging. Its role primarily involves limiting oxidation and preventing the growth of aerobic bacteria and mold thus extending shelf life [12]. It has been found that the length of shelf life is directly proportional to the ratio of CO₂ inside the MAP. However, a high level of CO₂ in the MAP has disadvantages such as souring (change in product taste) and the collapse of packages (resulting from CO₂ reaction with the film and/or product) and, for this reason, it is usually used with other gases like N₂ [13]. The main role of nitrogen is to substitute for oxygen contained in the atmospheric air in the package. The level of nitrogen in the packaging remains higher for a longer period due to its slow dispersion [8].

The oxidation of unsaturated fatty acids in soy or peas (mostly used in analogue meat products) and the presence of glycosides such as phenols and saponins lead to the formation of unpleasant flavors that can negatively affect the acceptability of such products by consumers [11]. The fats and proteins undergo intensive processing in meat analogue products, leading to an increased risk of rancidity and oxidation. For this reason, antioxidants are added to such products, serving the same purpose as their addition to meat products. Antioxidants derived from plants are a diverse group of chemical compounds. The primary bioactive components sourced from plants and utilized in the meat industry include polyphenols (such as flavonols, anthocyanins, and tannins) and terpenes (which are the primary constituents of essential oils) [14,15]. The process of replacing animal fat with vegetable oils (consisting mainly of unsaturated fatty acids that undergo more rapid oxidation processes) coupled with strong processing operations predisposes meat analogue products to lipid and protein oxidation. Therefore, it is essential to investigate the effects of storage conditions (including duration, temperature, and packaging type) on the oxidation status of such products [3].

Research on meat analogues primarily focuses on formulation and process development. There is limited information available on their safety, shelf life, and long-term nutritional and health impacts [16]. However, recently, some studies have begun to address the shelf life of plant-based meat analogues by analyzing the microbial community composition, such as Hai et al. [17]. The research aims to assess the oxidation status and antioxidant activity of five types of analogue meat products when packaged in a modified atmosphere on the final day of their shelf life. In other words, this investigation seeks to understand the effect of different formulations (five plant-based products) stored under the same packaging conditions on several lipid oxidation parameters, offering insights for improving the shelf life and quality of analogue meat products. The findings will contribute to enhancing the stability and quality of analogue meat products, guiding future developments in food preservation and formulation.

2. Materials and Methods

2.1. Materials

Five types of analogue meat products are used in the study as follows: steak, noodles, filet, burger, and mince. The main compositions of the analogue meat products are explained in

Table 1. Main compositions (in g/100 g) of analogue meat products.

Types	Ingredients	Nutritional Values per 100 g
		Protein	Fat	Saccharides	Fibers	Salt
steak	Water; breadcrumbs (16.4%): water, wheat flour, rapeseed oil, yeast, salt, spice extracts (sweet pepper, turmeric); vegetable oils: rapeseed and sunflower in different proportions, proteins: wheat (5.8%); soy (5.5%) and dried egg; mayonnaise: sunflower oil, dried egg yolks, fermented alcohol vinegar, mustard, iodized table salt (table salt, potassium iodate, sugar); citrus fiber; corn starch; fermented alcohol vinegar; wheat flour; dried yeast extract; spice mix (chili pepper, sweet pepper, cumin, oregano); tomato concentrate; onion powder; garlic; garlic powder; salt	16.0	12.0 including saturated fatty acids 1.3	16.0 including sugars 2.4	5.5	1.3
noodles	Water; soy protein (31.7%); rapeseed oil (4%); fermented alcoholic vinegar; dried yeast extract; table salt; aroma	22.5	4.7 including saturated fatty acids 0.4	1.2 including sugars 0.1	9.8	1.4
filet	Water; soy protein (10.4%); vegetable oils: rapeseed and sunflower in different proportions; flour (wheat, rice), onion (4.4%); dried egg whites; starch (corn, potato); wheat protein (3.1%); apple puree; citrus fibers; spice mixture (white pepper, sweet paprika, turmeric); onion and garlic powder; fermented alcohol vinegar; table salt; dried onion; garlic; dried yeast extract; yeast; flavorings (contains celery); hydrolysed proteins (soybean, corn) black pepper extract	15.5	7.0 including saturated fatty acids 0.5	10.0 including sugars 1.8	6.8	1.3
burger	Water; soy protein (16%); wheat protein (5%); vegetable oils: rapeseed and sunflower in different proportions; onion; corn starch, stabilizers E461—methylcellulose, E407—carrageenan); edible salt; fermented alcoholic vinegar; dried yeast extract; flavouring; onion powder; garlic powder; barley malt extract; caramelized sugar; maltodextrin; mixture of spices (roman cumin, black pepper, coriander, allspice); E330—citric acid	16.0	6.0 including saturated fatty acids 0.7	8.0 including sugars 1.0	4.0	1.5
minced	Water; soy protein (22.9%); vegetable oils: rapeseed and coconut; stabilizer (methylcellulose); fermented alcohol vinegar; natural aromas; garlic and onion powder; fruit and vegetable concentrates (blackcurrants, beets, peppers, carrots); black pepper; malted barley extract	17.3	7.9 including saturated fatty acids 2.9	3.6 including sugars 1.0	5.0	1.0

. A total of 10 samples from each product (Garden Gourmet, Nestlé Czechia s.r.o., Praha, Czech Republic) were obtained from retail markets. All the samples were in the same modified atmosphere packaging (35% CO₂ and 65% N₂). A high-barrier polypropylene (green plastics) tray, which was hermetically sealed with a barrier lidding film, was used for the packaging of the analogue meat products in the modified atmosphere. The samples were stored at 2 ± 2 °C until the last day of their shelf life. On the last day of their shelf life, the samples were analyzed in order to determine their oxidation status and antioxidant activity.

2.2. Methods

2.2.1. Gas Concentration Measurement in Modified Atmosphere Packaging (MAP)

The measurement of gas concentrations in the MAP was conducted by using gas analyzer Check Point II (PBI Dansensor AS, Ringsted, Denmark), through the insertion of a probe inside the packaging atmosphere. For each sample, two measurements were recorded.

2.2.2. Thiobarbituric Acid Reactive Substances (TBARS)

Each sample (10 g), mixed with distilled water (95.7 mL) and HCl (2.5 mL 4 N), were left to homogenize for 2 min. The samples were distilled and around 50 mL of distillate was obtained. Next, 5 mL of the distillate with 5 mL of trichloroacetic acid (15%, 0.375% thiobarbituric acid reagent) was heated for 35 min in a boiling water bath and then left to cool. The absorbances of the samples were measured by spectrophotometer against an appropriate blank at 532 nm. Calculation of the results were conducted by multiplication of the absorbance value with 7.8. The TBARS values were expressed as mg malondialdehyde per kg of sample (MDA mg·kg⁻¹ sample) [18].

2.2.3. Free Fatty Acids (FFA)

FFA were estimated by the titration method, where 5 g of sample with 50 mL of ethanol/diethyl ether (1:1) and 5 drops of phenolphthalein were heated (20 °C) in a water bath and then titrated with an alcoholic solution of potassium hydroxide until a red coloration was achieved. FFA is the amount of potassium hydroxide (mg) which is required to neutralize the FFA in one gram of sample [19]. FFA were represented by total fat (%) as oleic acid.

2.2.4. Total Polyphenol Content Determination

First, 0.1 g of the sample was homogenized in 20 mL of ethanol and water (1:1) in order to prepare the sample for extraction. The extraction was filtered (after 30 min in an ultrasound bath), and the filtered extract (1 mL) was mixed with (5 mL) Folin–Ciocalteu/water solution (1:10) and Na₂CO₃ (4 mL, 75 g/L), before being incubated in darkness (for 30 min). The created solution was measured by spectrophotometer (at 765 nm). Gallic acid was used to get the calibration curve, the results of which were expressed in mg/g of the gallic acid equivalent [20]. The calibration curve of gallic acid (r2 > 0.99) was used for the calculation of results.

2.2.5. Antioxidant Activity Estimation

• 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method

The sample extract was prepared according to Jung et al. [21]. First, 3 g of the sample was homogenized with 15 mL of trichloroacetic acid (5%), then 10 mL of chloroform was added. DPPH was dissolved in methanol (0.025 g/L) in order to prepare a fresh solution of radical stock. This solution was measured by spectrophotometer against a blank (at 515 nm) and the absorbance was detected as A0. The prepared sample extract (0.2 mL) was added to the DPPH solution (3.8 mL) and left to react for 10 min. Then, the absorbance was measured and indicated as A10. Measurements were performed in duplicate [22].

The following formula was used for the calculation of results:

DPPH in % inhibition = (Absorbance 0 − Absorbance 10/Absorbance 0) × 100

where Absorbance 0 is at time 0 min (control) and Absorbance 10 is at time 10 min (after sample reaction).

• 2,20-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) Method

First, 0.1 g of the sample was homogenized in dark vials with 20 mL of ethanol and water (1:1). The sample was extracted (in an ultrasound water bath for 30 min) and then filtrated. Next, 0.007 M ABTS solution (10 mL) was mixed with 0.00245 M potassium persulphate solution (10 mL) and left to react for around 12–16 h. This solution was diluted till its final absorbance (at 735 nm) became 0.7. The prepared sample extract (20 µL) was mixed with the ABTS reaction mixture (1980 µL) and left to react in darkness for 5 min. Then, it was measured by spectrophotometer (at 735 nm) to determine the absorbance [23].

Calculation of the results were performed according to the following formula:

ABTS [in %] = [(ABTS Absorbance − sample Absorbance)/ABTS Absorbance] × 100

where ABTS Absorbance is the absorbance of ABTS solution.

• Ferric Reducing Antioxidant Power (FRAP) method

First, 0.1 g of the sample was homogenized in 20 mL of ethanol and water (1:1) in order to prepare for the sample extraction. The extraction was filtered (after 30 min in an ultrasound bath). The filtered extract (180 µL) was incubated with distilled water (300 µL) and 3.6 mL of working solution (acetate buffer + TPTZ + FeCl₃ × 6H₂O in a ratio of 10:1:1) in the dark (for 8 min). Then, the absorbance of the solution was measured by spectrophotometer (CE7210, UK) at 593 nm. Torlox was used as the standard; the results were expressed as the amount of Trolox (μmol) in one g of sample [24]. Calibration curve of Trolox (r² > 0.99) was used for the calculation of results.

• Cupric reducing antioxidant capacity (CUPRAC) method

The CUPRAC method was conducted according to Apak et al. [25]. First, 0.1 g of the sample was sonicated with 20 mL of an ethanol–water mixture (1:1) for 30 min in dark glass vials and then filtered. Next, 1 mL of the extract was mixed with each of the following: 1 mL of 0.01 M Copper (II), 1 mL of 0.0075 M Neocuproin, and 1 mL of NH4Ac buffer pH = 7.0. After incubation in darkness (for one hour), the absorbance of the samples was measured against a blind sample by spectrophotometer (at 450 nm). Trolox was used for the preparation of a calibration curve and the results were expressed as μmol of Trolox per kilogram of sample.

2.3. Statistical Analysis

Mean and standard deviation were computed for data using Microsoft Office Excel 2016. Correlation analysis was conducted by Pearson’s correlation analysis. Significance in the differences (p < 0.05) among the analogue meat products was determined by the analysis of variance (ANOVA), with post hoc Tukey’s test using the SPSS 20 statistical software (IBM Corporation, Armonk, NY, USA).

3. Results

The results of the gas concentrations in the Modified Atmosphere Packaging (MAP) are presented in

Table 2. Gas concentrations (35% CO₂ and 65% N₂) (ratio in %, mean ± SD) in modified atmosphere packaging of analogue meat products.

Samples	Gases Concentrations
	O2	CO2	Rest Gases (N2)
steak	0.30 ± 0.19	35.27 ± 1.06 a	64.43 ± 0.94 b
noodles	0.07 ± 0.01	37.70 ± 0.26 ab	62.24 ± 0.25 c
filet	0.45 ± 0.19	35.23 ± 1.36 a	64.34 ± 1.22 b
burger	0.22 ± 0.07	34.63 ± 0.63 ac	65.14 ± 0.59 b
minced	0.08 ± 0.06	33.97 ± 0.74 c	65.97 ± 0.70 a

Different superscripts a, b, c indicate significant differences (p < 0.05) among analogue meat products.

. On the final day of their shelf life, the percentage of O₂ concentration in the MAP for all the investigated products was below 0.5, with no significant differences observed among them (p > 0.05). The CO₂ concentration in the MAP of the minced products was significantly lower (p < 0.05) compared to the other evaluated products. The highest nitrogen ratio (p < 0.05) was found in the MAP of the minced products, while the lowest was observed in the MAP of the noodle products.

The results of the oxidation status, including TBARS, free fatty acid, polyphenols, and antioxidant capacity, are presented in

Table 3. TBARS, free fatty acid, polyphenols and antioxidant capacity (mean ± SD) of analogue meat products.

Parameters	Steak	Noodles	Filet	Burger	Minced
TBARS (mg MDA·kg−1)	6.67 ± 1.08 b	5.22 ± 0.58 c	9.71 ± 1.73 a	4.81 ± 1.27 c	3.20 ± 0.73 d
FFA (% total fat as oleic acid)	2.31 ± 0.17 c	3.77 ± 0.30 a	3.52 ± 0.30 b	1.48 ± 0.31 d	1.12 ± 0.20 e
polyphenols (mg gallic acid/g)	2.63 ± 0.03 a	1.08 ± 0.03 d	1.01 ± 0.01 e	1.90 ± 0.03 b	1.15 ± 0.03 c
DPPH (% inhibition)	18.78 ± 1.82 b	18.91 ± 3.30 b	11.53 ± 2.49 c	13.83 ± 3.71 c	32.26 ± 10.09 a
ABTS (% inhibition)	0.82 ± 0.32 c	1.25 ± 0.25 c	1.86 ± 0.20 b	2.26 ± 0.10 b	4.49 ± 0.37 a
FRAP (mmol/g)	11.28 ± 1.07 a	5.34 ± 0.36 c	4.75 ± 0.47 c	6.96 ± 0.26 b	7.36 ± 0.37 b
CUPRAC (Trolox mmol/kg)	11.77 ± 0.95 a	8.96 ± 0.12 b	5.57 ± 0.03 d	8.05 ± 0.23 c	11.48 ± 0.26 a

Different superscripts a, b, c, d indicate significant differences (p < 0.05) among analogue meat products within row.

. The TBARS values of the minced products were significantly lower (p < 0.05) compared to the other evaluated products, while the highest values (p < 0.05) were observed in filet samples. No significant differences (p > 0.05) in TBARS values were observed between the noodles and burgers. The free fatty acid (FFA) content of the minced samples was significantly lower (p < 0.05) than the other evaluated analogue meat products, followed by burgers, steaks, filets, and noodles, respectively. The polyphenol content in the steaks was twice as high (2.63 mg gallic acid/g) compared to the filets (1.01 mg gallic acid/g), noodles (1.08 mg gallic acid/g), and minced products (1.15 mg gallic acid/g). The highest antioxidant capacity, as determined by the DPPH and ABTS methods, was observed in the minced products and was significantly higher (p < 0.05) than other evaluated analogue meat products. The steaks and noodles exhibited higher DPPH (% inhibition) than the filets and burgers, while showing lower ABTS (% inhibition). According to the FRAP and CUPRAC assays, the highest antioxidant capacity was observed in the steak samples, while the filet products contained the lowest capacity. The CUPRAC method of antioxidant assessment indicated that the steak and minced samples had a higher antioxidant capacity (with 11.77 and 11.48 Trolox mmol/kg, respectively), followed by the burgers (6.96 Trolox mmol/kg), noodles (5.34 Trolox mmol/kg), and filets (4.75 Trolox mmol/kg), respectively.

Table 4. Correlation (coefficient of determinations r) of gas composition in MAP with measured oxidative–hydrolytic stability and antioxidant properties.

Samples	MAP	TBARS	FFA	Polyphenols	DPPH	ABTS	FRAP	CUPRAC
steak	O2	0.1457	0.2604	0.6313 *	0.0677	0.4749	0.5224	0.7347 *
	CO2	0.4077	−0.1018	−0.6656	−0.4555	−0.7386	0.6474	−0.6485
noodles	O2	0.6716	0.8408	0.9286 *	0.6357 *	0.6763	0.7975	0.7215
	CO2	−0.1855	0.0238	−0.4748	0.6268	0.2912	0.5163	−0.1904
filet	O2	0.3498	0.4547	0.6899	0.2586	0.6803	0.8518 *	0.9452 *
	CO2	−0.1109	0.515	−0.0775	0.173	0.5805	−0.1263	0.0283
burger	O2	−0.3942	0.0934	0.5522	0.746	0.9221 *	0.2679	0.5994
	CO2	0.0723	0.2039	0.1127	0.0135	0.0434	0.0282	−0.054
minced	O2	0.0916	0.8866 *	0.3217	−0.689 *	0.5126	0.4112	0.1931
	CO2	−0.1338	−0.8738 *	−0.2709	0.7345 *	−0.3037	−0.2249	−0.2671

* Statistically significant (p < 0.05) correlation.

shows the correlation analysis (R-regression, done by Pearson’s correlation) of the gas composition in the Modified Atmosphere Packaging (MAP) with measured oxidative–hydrolytic stability and antioxidant properties.

Correlations between O₂ (in the steak samples) and two of the measured chemical parameters, the polyphenol content (R² = 0.6313) and CUPRAC (R² = 0.7347), were found to be statistically significant (p < 0.05); in the burger samples, a significant (p < 0.05) correlation was found with ABTS (R² = 0.9221); in the filet samples, with FRAP (R² = 0.8518) and CUPRAC (R² = 0.9452; the highest found positive correlation); in the noodles samples, with polyphenols (R² = 0.9286) and DPPH (R² = 0.6357); and in the minced samples, with FFA (R² = 0.8866) and DPPH (R² = −0.689). Correlations between CO₂ (in minced samples) and two of the measured chemical parameters, FFA (R² = −0.8738; the highest found negative correlations) and DPPH (R² = 0.7345), were found to be statistically significant (p < 0.05).

4. Discussion

An oxygen-free modified atmosphere is typically employed for packaging analogue meat products to keep the package free from oxygen until the last day of shelf life. However, this is not always guaranteed, and oxygen permeation may occur through the packaging barriers [26,27]. In our study, although the packages of analogue meat products are supposed to be free from oxygen, a very small ratio of O₂ was observed. Nevertheless, this oxygen level in the MAP of all five products was less than 0.5%. The decrease in CO₂ concentration in the MAP of the minced products could be attributed to its higher moisture content. The reaction of carbon dioxide with moisture in the products leads to CO₂ reduction and the formation of carbonic acid [27]. Nitrogen is primarily used in MAP to displace oxygen, functioning as an inert filler [28]. However, changes in the atmosphere within the MAP are attributed to the natural interaction of the gas mixture with the packaged products [27].

Lipid oxidation is a process with several stages, in which different compounds are formed. The TBARS value is used to quantify levels of secondary products of lipid oxidation. These secondary products mainly include aldehydes and ketones [29]. The low TBARS values in minced samples could be attributed to the high content of saturated fatty acids from coconut oil used in this product (Table 1). Coconut oil is more resistant to oxidation and polymerization due to its composition of high saturated fatty acids compared to other plant oils [30]. In many plant-based products resembling ground animal-based meat products, such as minced products, burger patties, and nuggets, a combination of liquid fats (like sunflower oil) and solid fats (such as coconut oil) is used to achieve the appropriate balance. In such products, it is preferable that the fats are solid at room temperature and turn liquid after heat treatment to mimic the properties of the corresponding meat products [5]. Although no legislative limit for malondialdehyde (MDA) in meat has been officially published, MDA values above 0.5 mg/kg indicate some oxidation, and levels over 1.0 mg/kg are considered unacceptable in many studies [31]. Jeong et al. [32] found that the TBARS values of meat analogues with different fat replacers stored under freezing conditions for six months did not exceed 1.0 mg/kg. The TBARs of all meat analogues in this study exceeded 1.0 mg/kg and at a high rate (3.20–9.71 mg/kg). Such high levels of MDA could be attributed to several processing procedures during the analogue meat production, as well as its content of oxidant susceptible oils, such as sunflower, soybean, olive, and peanut oils [33]. The incorporation of vegetable oils in different reformulated meat products has been the focus of several studies. In this regard, Pintado and Cofrades [34] indicated that sausages with reduced levels of animal fat and the incorporation of vegetable oils in different forms, such as oleogel and emulsion gel, usually showed lower oxidative stability, which is consistent with the results of our study.

The observed minimal levels of free fatty acids (FFA) found in minced products, in contrast to the other products examined in our study, corroborate the findings of the TBARS analysis and suggest a more favorable oxidation status for this specific product category. The emergence of free fatty acids in oils predominantly arises from the process of lipolysis, wherein hydrolytic enzymes catalyze the breakdown of triglycerides into constituent fatty acids and glycerol [35]. Lipolysis occurring within legumes results in the liberation of free fatty acids, including notable compounds such as linoleic and alpha-linolenic acids. These liberated fatty acids play a significant role in imparting bitterness to oat and pea protein isolates, thereby serving as potential contributors to the development of off-flavors within these food products [36,37]. Lipid oxidation stability in complex food products, such as meat analogues, which consist of a number of different ingredients, depends on the quality of the ingredients used in their production. For example, oils and other ingredients should be as fresh as possible with minimal levels of lipid oxidation thus extending the shelf life of the final product.

To effectively mitigate chemical lipid oxidation reactions in food materials, a range of strategies can be employed, encompassing the reduction in oxygen levels, prevention of exposure to ultraviolet light, storage at lower temperatures, and the incorporation of antioxidants [38]. The synergy between plant endogenous antioxidants and those added during processing plays a pivotal role in regulating lipid oxidation through a multitude of mechanisms, thereby enhancing the stability and shelf life of food products [39,40]. Due to the intensive processing of proteins and fats in meat analogues, antioxidants are added to prevent fat rancidity and protein oxidation. The production of meat and meat analogues often involves the use of several spices and aromatic plants to enhance their antioxidant properties [41]. The higher polyphenol content in steak and the lowest in filet samples were reflected in the FRAP and CUPRAC values. High correlation coefficients be-tween the polyphenols and antioxidant activities (of cereal and pseudocereal) and FRAP and CUPRAC have been reported. Both assays (FRAP and CUPRAC) are based on similar mechanisms, involving electron transfer reactions with metal ions for oxidation and analyzing the ability to reduce the radical cation [42]. The high antioxidant capacity observed in the minced product via DPPH, ABTS, and CUPRAC assays may help avoid excessive secondary oxidation in such products, as indicated by its lower content of TBARS and FFA. Bolanho and Beléia [43] reported a high correlation between DPPH and ABTS in soybean-based products, as both methods detect free radical scavenging capability. DPPH and ABTS assays are typically based on mechanisms involving both hydrogen atom and electron transfers [44]. The lower polyphenol content and antioxidant capacity (FRAP and CUPRAC values) of the filet products could be the reason for the higher susceptibility of this product to secondary oxidation, as evidenced by the significantly high TBARS values.

The observed polyphenol content and antioxidant capacity, as indicated by the FRAP and CUPRAC values, in the filet products compared to the other samples may underlie the increased susceptibility of the former to secondary oxidation, a hypothesis supported by the markedly elevated TBARS values. This correlation raises further inquiry into the interplays among polyphenols, antioxidant capacity, and lipid oxidation kinetics within filet products. A comprehensive exploration of these factors could illuminate underlying mechanisms and inform targeted interventions aimed at bolstering the oxidative stability of filet products, thereby enhancing their shelf life and overall quality. Adopting a multidimensional approach that affects biochemical analysis, sensory evaluation, and lipidomics may reveal novel insights into the dynamics governing lipid oxidation in filet products, paving the way for more effective preservation strategies and product formulations in the food industry.

5. Conclusions

Analogue meat products are manufactured from a relatively high number of ingredients, some of which often include vegetable oils. In addition, these products undergo extensive processing during manufacturing, which together may have an impact on making them susceptible to oxidation and fat rancidity. Consequently, the packaging method for such products plays a crucial role in determining their shelf life. The aim of our study was to evaluate the oxidation status of five analogue meat products that utilized the same Modified Atmosphere Packaging on the last day of their shelf life. The study found that the minced product exhibited greater oxidative stability, showing the lowest levels of TBARS and FFA, along with a high antioxidant capacity (DPPH, ABTS, and CUPRAC). In contrast, the filet product was more susceptible to the oxidation process, displaying the highest TBARS content and the lowest levels of polyphenols, antioxidant capacity (FRAP and CUPRAC). There are the following potential limitations in this study: (1) The assessment of the oxidation status of the analogue meat products was limited to the last day of their shelf life, without monitoring the progression of the oxidation process from the first day of packaging. However, the expiry date is more relevant for consumers than the production date. (2) There is no established legislative limit for the oxidation parameters used to assess the status of samples and their acceptability for human consumption. (3) Evaluating the microbiological status and sensory analysis of such products is also necessary, as it can contribute to determining their safety for consumption. Further comprehensive investigation is warranted to thoroughly explore and address the limitations highlighted earlier. By conducting more extensive and rigorous studies, researchers can elucidate complex relationships, identify potential confounding factors, and offer nuanced solutions to enhance the understanding and applicability of the findings. This extended inquiry will not only contribute to advancing scientific knowledge but also pave the way for the development of more effective strategies and interventions in the field of vegetarian food commodities.