Role of a Natural Antioxidant in the Secondary Shelf Life of Ready-to-Use Meat Pâté ^†

Abstract

By-products from the agro-food industry can be natural ingredients for reformulating traditional foods, enhancing quality, extending secondary shelf life (SSL), and reducing food waste. This study evaluates the impact of an olive vegetation water phenolic extract (OVWPE) at two concentrations on ready-to-use meat pâté (MP1 and MP2) under simulated retail storage. We evaluated the phenolic composition, volatile compounds, antioxidant activity, and sensory quality of the OVWPE-added pâté and compared it to the control sample (CTRL; without antioxidant). Results indicated that OVWPE minimized oxidation products, enhancing lipid stability, and also reduced the formation of C₆–C₉ aldehydes linked to rancid off-flavor. Without sensory defects, the OVWPE pâtés showed higher antioxidant activity and α-tocopherol content than the CTRL. OVWPE appears to be a promising antioxidant of natural origin for food formulations, supporting SSL extension and food waste reduction.

1. Introduction

Food is prone to spoilage due to microbial and physicochemical processes, compromising its safety, nutritional value, color, and sensory characteristics [1]. Fat degradation, generally called rancidity, is among the chemical alterations in oil-based products. This causes an accumulation of free radicals, responsible for oxidative stress in the human body and the development of off-flavors [2].

An innovative approach relies on improving food preservation methods, ensuring safety and quality, and extending shelf life. Researchers are exploring using additives of natural origins, recovered from agro-food industry by-products, to meet consumer demand for clean-label products free from conventional additives [3,4]. Virgin olive oil (VOO) stands out among the various supply chains. The mechanical extraction process used to produce olive oil is not very efficient in terms of yield; only 10–20% of the olive fruit produces the VOO. The remaining parts, such as pomace and olive vegetation water (OVW), are typically considered waste [5]. However, in recent years, research attention has shifted not only to improving profitability, a crucial aspect for producers, who face high production costs, but also to recovering the bioactive compounds still present in the waste. These hydrophilic phenols are complex enough to end up in the VOO but remain almost entirely in waste. Only about 2% of the total phenols of the olive are found in VOO [5]. Due to the well-known beneficial effects of phenols contained in OVW, their potential applications as an additive of natural origin in several foods were explored [6].

Recent studies have indicated that grape and tea extracts can enhance liver pâtés, which are high in fat and non-heme iron but low in natural antioxidants [2]. Their high-temperature manufacturing process also makes these pâtés prone to lipid oxidation [2]. This study aimed to assess the effectiveness of OVWPE, derived from OVW, in improving the antioxidant and volatile compounds, antioxidant activity, and sensory quality of ready-to-use meat pâté, thus extending its secondary shelf life (SSL) for 11 days after opening. The SSL refers the duration from the moment the package is opened until the unpackaged food is no longer suitable for consumption [7].

2. Materials and Methods

2.1. Chemicals, Reagents, Materials, and Experimental Set-Up

Pure analytical standards for α-tocopherol, olive phenolic (hydroxytyrosol, tyrosol and verbascoside), and volatile (4-methyl-2-pentanol) compounds were purchased from Merck (Milan, Italy), whereas oleacein and oleocanthal were obtained as described by Taticchi et al. [8]. The 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), and butylated hydroxytoluene (BHT) were purchased from Merck (Milan, Italy). Formic acid, n-hexane, phosphoric acid, hydrochloric acid, methanol, ethanol, 2-propanol, and acetonitrile were supplied by VWR (Milan, Italy). OVWPE was obtained from fresh OVW of olive cultivars blend, harvested in the Umbria region (Italy), and purified according to the protocol reported by Balzan et al. [9]. The OVWPE was the same formulation used for a previous study [10] (Table S1). The ready-to-use meat pâté samples were kindly manufactured by a local food producer (Perugia, Italy), adopting its traditional recipe, as illustrated in Tables S2 and S3. Briefly, all ingredients were mixed, cooked for 30 min, homogenized using a bowl cutter, and added with OVWPE, then filled into 1 kg plastic containers and pasteurized at 90 °C for 15 min before cooling and storage at 4 °C. Three ready-to-use meat pâtés (MPs) were prepared: (i) control sample (CTRL; without antioxidants); (ii) MP1 with the addition of 250 mg phenols from OVWPE/kg of product; and (iii) MP2 with the addition of 500 mg phenols from OVWPE/kg of product. Once opened, each meat pâté sample was stored in a cold room maintained at 4 ± 2 °C for 11 days under 12 h of light exposure (600 lx) and air exposure, simulating the SSL under deli-counter sales conditions, a common sales method in Italian food stores for this kind of foodstuff. An aliquot was removed every 3 h for 11 days, as described previously by Sordini et al. [10].

2.2. Analytical Determination

During the SSL, each sample (CTRL, MP1, and MP2) was analyzed in duplicate at various intervals: 0, 1, 2, 3, 4, 7, 8, 9, 10, and 11 days after the packaging was opened.

2.2.1. Extraction and Evaluation of Phenolic Compounds from OVWPE and α-Tocopherol Content

The extraction and analysis procedures for phenolic compounds from OVWPE and α-tocopherol were the same as already described in previous works [9,10]. The total phenolic content was determined by summing the concentrations of the individual phenolic compounds identified and quantified (hydroxytyrosol, tyrosol, verbascoside, oleacein, and oleocanthal). Each extraction and analysis was performed in duplicate.

2.2.2. Evaluation of Antioxidant Activity

The antioxidant activity of samples was evaluated by antiradical activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) assay according to the method described by [11] and modified [10]. Each measurement was performed in duplicate.

2.2.3. Evaluation of Volatile Compounds

Evaluation of volatile compounds in ready-to-use meat pâté was performed via headspace solid-phase microextraction, followed by gas chromatography–mass spectrometry (HS-SPME/GC-MS), according to the procedures of Sordini et al. [10]. The concentration of C₆–C₉ aldehydes was calculated by summing the concentration of hexanal, (E)-2-heptanal, and nonanal.

2.3. Sensory Analysis

The ready-to-use MPs (0-day of storage after opening) were subjected to a quantitative descriptive sensory analysis by a panel of 10 experienced judges previously trained for sensory analysis, according to the ISO 8586: 2012 standard [12]. Different attributes were evaluated, including appearance, odor, taste, overall pleasantness, and off-flavors. Their intensity was rated using 9 cm unstructured line scales.

2.4. Statistical Analysis

The chemical data were the mean values of two extractions evaluated two times ± standard deviation, while the sensory data were the mean of the sensory evaluation performed by 10 trained panelists. SigmaPlot software v15.0 (Systat Software Inc., San Jose, CA, USA) was used to perform a priori one-way analysis of variance (ANOVA) with Tukey’s test (p < 0.05).

3. Results and Discussion

3.1. Evaluation of Phenolic Compounds from OVW and α-Tocopherol Concentration of Ready-to-Use Meat Pâté During SSL

Table 1. Evolution of total phenols from OVW (mg/kg), α-tocopherol (mg/kg), DPPH˙ (µmol TE/g f.w.), and C₆–C₉ aldehydes (µg/kg) of meat pâté during SSL (0–11 days).

		Days of Storage After Opening
	Samples	0	1	2	3	4	7	8	9	10	11
Total phenols from OVW (mg/kg)	CTRL	-	-	-	-	-	-	-	-	-	-
	MP1	154.8 ± 1 Ba	123.3 ± 6.5 Bb	111.2 ± 4.5 Bc	99.9 ± 3.8 Bcd	95.7 ± 4 Bd	85.1 ± 4.3 Be	70.2 ± 2.3 Bf	62.3 ± 4.1 Bg	23.9 ± 0.4 Bh	15 ± 0.3 Bi
	MP2	301.1 ± 1.2 Aa	290.6 ± 2.2 Ab	286.2 ± 1.2 Ab	246.2 ± 1.4 Ac	244 ± 4 Ac	233 ± 3.2 Ad	202.6 ± 1.8 Ae	181.5 ± 0.6 Af	168.9 ± 4.1 Ag	120.6 ± 2.2 Ah
α-tocopherol (mg/kg)	CTRL	333.0 ± 0.9 Aa	323 ± 1.8 Bb	316.6 ± 0.8 Cc	315.7 ± 0.4 Bc	313.5 ± 2.7 Bc	299 ± 1.9 Cd	291.6 ± 1.6 Ce	290.5 ± 0.1 Ce	288.8 ± 0.8 Ce	273.6 ± 1.2 Cf
	MP1	331.4 ± 2 Aa	331.2 ± 0.4 Aa	321.3 ± 0.4 Bb	318.9 ± 2.4 Bcd	319.3 ± 0.8 Bbd	314.6 ± 0.7 Bcd	307.1 ± 3.1 Be	305.7 ± 0.7 Be	298.5 ± 0.6 Af	280.9 ± 2 Bg
	MP2	332.7 ± 1.6 Aa	332 ± 1.8 Aa	330 ± 4.2 Aa	328.5 ± 1.6 Aab	326.5 ± 0.8 Aabc	321.7 ± 2 Abd	320.4 ± 0.7 Acd	316.4 ± 1 Ade	312.4 ± 1.2 Aef	308 ± 2.7 Af
DPPH˙ (µmol TE/g f.w.)	CTRL	0.51 ± 0.01 Ca	0.47 ± 0.03 Cab	0.44 ± 0.04 Bab	0.43 ± 0.03 Cab	0.42 ± 0.04 Bab	0.40 ± 0.03 Bb	0.20 ± 0.02 Cc	0.16 ± 0.01 Ccd	0.17 ± 0.02 Ccd	0.13 ± 0.01 Cd
	MP1	0.70 ± 0.06 Ba	0.69 ± 0.02 Ba	0.66 ± 0.05 Aa	0.61 ± 0.03 Bac	0.52 ± 0.01 Bbc	0.46 ± 0.03 Bb	0.43 ± 0.01 Bb	0.41 ± 0.03 Bbe	0.31 ± 0.01 Bde	0.23 ± 0.01 Bd
	MP2	0.83 ± 0.06 Aa	0.78 ± 0.04 Aab	0.77 ± 0.07 Aab	0.75 ± 0.06 Aab	0.70 ± 0.02 Abc	0.60 ± 0.05 Abcd	0.62 ± 0.04 Abcd	0.56 ± 0.03 Abd	0.52 ± 0.02 Abd	0.45 ± 0.03 Ad
C6–C9 aldehydes (µg/kg)	CTRL	193 ± 17 Aa	269 ± 13 Ab	267 ± 10 Ab	292 ± 12 Abc	308 ± 17 Abc	330 ± 17 Acd	339 ± 9 Acd	370 ± 8 Ade	399 ± 2 Aef	436 ± 11 Af
	MP1	191 ± 9 Aa	202 ± 6 Ba	207 ± 7 Ba	219 ± 5 Bb	225 ± 5 Bb	231 ± 5 Bbc	265 ± 3 Bc	293 ± 6 Bd	311 ± 6 Be	397 ± 6 Bf
	MP2	206 ± 14 Aa	210 ± 11 Ba	215 ± 1 Ba	221 ± 2 Bb	221 ± 15 Bb	234 ± 4 Bb	248 ± 6 Cb	277 ± 4 Cc	297 ± 4 Cd	341 ± 13 Ce

Results are the mean values of two extractions evaluated two times ± standard deviation. Uppercase letters (A–C) in the column indicate significant differences (Tukey’s test; p ≤ 0.05) among different formulations at the same storage time. Lowercase letters (a–i) in the row indicate significant differences (Tukey’s test; p ≤ 0.05) within the same formulation during storage. Total phenols from OVW were expressed as sum hydroxytyrosol, tyrosol, oleacein, and verbascoside; C₆–C₉ saturated aldehydes were expressed as the sum of hexanal, (E)-2-heptanal, and nonanal. Legend: control sample (CTRL; without antioxidants); MP1 with the addition of 250 mg phenols from OVWPE/kg of product; and MP2 with the addition of 500 mg phenols from OVWPE/kg of product.

shows the evolution of the total phenols from OVW and α-tocopherol concentration (mg/kg) in MPs during the SSL. As expected, phenolic compounds derived from OVWPE were absent in the CTRL sample, which did not contain the extract. Conversely, these compounds were detected in the enriched samples (MP1 and MP2) at 60% and 66% of their originally added levels, respectively (Table 1). This reduction is likely attributable to their early involvement in oxidative reactions during the mixing phase or as a result of the heat treatment applied for product stabilization at the industrial scale. Balzan et al. [9] reported similar losses during the preparation and cooking of fresh pork sausages that were enriched with a purified phenolic extract from OVW. Over time, the total phenols content showed a significant (p ≤ 0.05) decrease corresponding to 90% for MP1 and 60% for MP2, respectively. This evolution is also because the oxidative phenomena occur more quickly in these conditions (air and light exposure). In OVWPE, the oleuropein derivatives (oleacein and hydroxytyrosol, in particular) accounted for 96% of the total phenols; they are well documented for their potent antioxidant activity, primarily through direct inhibition of lipid peroxidation [6,9,10]. Their strong radical-scavenging capacity is attributed to the presence of ortho-dihydroxy (catechol) groups on the aromatic ring, which enable efficient hydrogen atom donation to neutralize lipid radicals and stabilize the resulting species via resonance [13]. Previous studies have suggested this mechanism [6,9]. Eleven days after opening the package, residual bioactive phenols were still detectable in MP1 and MP2, though at lower levels.

The health benefits of α-tocopherol, which include its antioxidant properties and its role as a vitamin E source, are well established [14].

Supplementing the human diet with α-tocopherol is recommended to prevent oxidative damage [15]. As shown in Table 1, the α-tocopherol content, naturally high in this gastronomy preparation, appeared to be well preserved from oxidation due to the antioxidant action of olive phenols present in the OVWPE, particularly at the highest supplementation level. In the CTRL sample, a significant (p ≤ 0.05) decrease after just one day of storage was observed, and MP1 also showed a decline after two days; no significant variation was detected in MP2 until the 7th day. This result demonstrates that as long as the phenol levels in the MP1 and MP2 samples remain high, α-tocopherol is shielded from oxidation and can retain its higher concentrations for consumers. After 11 days of SSL, total reductions in the α-tocopherol levels ranged from 18% in the CTRL sample to 7% in the MP2.

3.2. Evaluation of Antioxidant Activity of Ready-to-Use Meat Pâté During SSL

The results of antioxidant activity measured by DPPH˙ assay were directly related to the phenol concentration in the OVWPE, with the highest activities observed in the MP2 sample (Table 1). A similar trend was observed in all samples, decreasing significantly (p ≤ 0.05) with increasing storage time. At the end of SSL, the DPPH˙ values decreased by 75%, 67%, and 46% in CTRL, MP1, and MP2, respectively, compared to the initial values. On the other hand, Gueboudji et al. [16] reported that the phenolic extract from olive mill wastewater (OMW) exhibits higher antioxidant activity compared to ascorbic acid and BHT, as demonstrated through various assays (DPPH, ABTS, and FRAP).

3.3. Evaluation of Volatile Compounds of Ready-to-Use Meat Pâté During SSL

Table 1 shows the evolution of the concentration of C₆–C₉ saturated aldehydes (hexanal, (E)-2-heptanal, nonanal) (µg/kg) in ready-to-use meat pâté during the SSL. The concentration of C₆–C₉ aldehydes is frequently used as a marker of lipid oxidation due to its high sensitivity [17,18]. In fact, Aparicio-Ruiz et al. [19] identified hexanal and nonanal as key indicators of lipid oxidation and rancidity in fatty foods, with perception thresholds of 80 and 150 µg/kg, respectively. In all samples, the concentration of C₆–C₉ saturated aldehydes increased significantly (p ≤ 0.05) during the SSL. However, the OVWPE slowed the formation of new C₆–C₉ aldehydes, while in the CTRL sample, these compounds increased immediately and reached systematically higher values of MP1 and MP2 over the 11 days. As in the present study, many authors have observed that the effects of different extracts were concentration-dependent [10], decreasing the production of C₆–C₉ aldehydes at increasing levels of antioxidant addition. Our results are in line with those reported by Pateiro et al. [18], who observed that the concentration of hexanal in liver pâté was lower in the samples enriched with green tea, chestnut, and grape extracts compared to the control group without antioxidants. Interesting, Munekata et al. [6] reviewed olive processing by-products as sources of polyphenols that enhance meat preservation. These phenolic compounds protect against oxidation, inhibit spoilage and pathogens, maintain sensory qualities, and minimally affect other quality indicators across meat types, products, and storage conditions.

3.4. Sensory Profile of Ready-to-Use Meat Pâté at 0 Days

The impact of OVWPE addition on the sensory characteristics of ready-to-use meat pâté was evaluated (Figure 1). As the microbiological analysis results were not available in real-time and the safety of the samples for panelists could not be assured, sensory evaluations were exclusively conducted on day 0 to establish baseline characteristics, with no evaluations performed at subsequent time points. None of the three tested samples exhibited any sensory defects and were therefore considered acceptable for human consumption. Notably, the sensory profile of the samples at the time of package opening (0 days) revealed that MP2 exhibited significantly (p < 0.05) more pronounced aromatic herbal odor, spiciness, and overall pleasantness compared to the control (CTRL) sample. Our results can be explained by the fact that the C₆ aldehydes, such as hexanal, contribute to the characteristic herbal odor (odor threshold value 75 µg/kg) [20], and is also recognized as a marker of lipid oxidation and may lead to rancid off-flavors when present at higher concentrations [19,21]. The taste attribute of bitterness and the trigeminal sensation of spiciness were also significantly enhanced in the phenol-enriched samples and are associated with the presence of OVW phenols [10]. Compared to the CTRL sample, several sensory attributes improved with the addition of phenols. These findings are consistent with those reported by Difonzo et al. [22], who studied the sensory properties of non-thermally stabilized olive-based pâté enriched with olive leaf extract during refrigerated storage (4 ± 1 °C) for 120 days under modified atmosphere packaging (MAP) conditions.

4. Conclusions

This study demonstrates that OVWPE is an effective antioxidant of natural origin for ready-to-use meat pâté. The OVWPE addition effectively preserved bioactive compounds, delayed oxidative degradation by slowing lipid oxidation, and reduced the formation of volatile compounds that contribute to unpleasant off-flavors. Our outcomes support the dose-dependent efficacy of OVWPE and its potential for integration alongside or in place of synthetic antioxidants in clean-label formulations. These preliminary findings lay the groundwork for future research, which will include consumer sensory acceptability studies, microbiological analysis, and comparative tests with other common natural antioxidants used in the food industry, thus facilitating a comprehensive evaluation of OVWPE’s efficacy. In this context, owing to its ability to enhance food quality, prolong shelf life, and reduce the food waste, OVWPE represents a promising strategy for the valorization of EVOO by-products. Indeed, the EVOO supply chain is experiencing significant transition towards sustainability, with an emphasis on enhanced management of by-products during the ex-traction process. This encompasses the large-scale implementation of technological solutions aimed at recovering the phenolic fraction from OVW.