The Effects of a Natural Citrus Phenolic Extract on the Quality Attributes and Oxidative Stability of Pariza-Type Meat Emulsion Product

Abstract

Several synthetic food additives that bear an E-number are used by the meat industry as antioxidants/preservatives of cured meat products, such as pariza-type meat emulsion products. However, these agents have been associated with health problems, namely cardiovascular disease, metabolic syndrome, and potential carcinogenic effects. As a result, natural alternatives are constantly under evaluation with the intention of replacing/minimizing their applications in the meat industry. The aim of the present study was therefore to evaluate the effects of a natural citrus phenolic complex extract on the quality characteristics of pariza-type meat emulsion products. The following three batches of pariza were produced based on the same raw material and recipe: a control group without natural antioxidants and two groups with the addition of the polyphenol complex at the levels of 500 and 1000 ppm. The pH, color, tenderness, and oxidative stability of the meat products were assessed immediately after pariza manufacture (day 0), and 30 and 72 days after the start of its refrigerated storage. As indicated, the oxidative stability of pariza was improved as a result of the natural polyphenol complex addition, since the values of malondialdehyde (MDA), an index of lipid peroxidation, were linearly decreased. Parameters such as pH, lightness (L), and yellowness (b*) were linearly increased, while redness (a*) was linearly decreased, and tenderness was not significantly influenced in the treatment groups compared to the control group. It can be concluded that the natural polyphenol complex under examination can be utilized for the improvement of oxidative stability in pariza.

1. Introduction

Meat products are essential components of the human diet, especially in developed countries, since they are important sources of protein, fat, essential amino acids, minerals, vitamins, and other nutrients. However, the lipids and proteins of meat are easily susceptible to oxidative damage due to the rapid depletion of endogenous antioxidants after slaughter. The oxidation of lipids and free radical production are natural processes which occur in meat processing systems and result in undesirable off-flavors (rancid) and discoloration (fading, browning, or degradation) that can make meat products unpalatable and could cause their rejection [1,2].

Synthetic antioxidant agents therefore play an important role in the meat industry by providing protection against quality deterioration and prolonging the shelf-life of meat products [3]. However, they have been associated with several health problems, such as skin irritation, hypersensitivity, allergies, asthma, metabolic syndrome, gastrointestinal disorders, cardiovascular diseases, and potential carcinogenic effects [4]. In order to limit their applications in meat products, natural agents, such as plant extracts, which are rich in polyphenols could be used as an alternative [5,6]. These substances are secondary plant metabolites that are composed of at least one aromatic ring, to which at least two hydroxyl groups are attached. Polyphenols possess intense antioxidant properties, acting as scavengers of free radicals and reactive oxygen/nitrogen species, inhibitors of enzymatic reactions that are responsible for the formation of free radicals (i.e., lipases split the glycerides and free fatty acids are produced), metal binders, and activators of antioxidant enzymes that lead to the prevention of discoloration and quality deterioration of meat products [7,8].

Citrus fruits are the most produced fruits worldwide since they are easily preserved and transported to any place in the world. At the same time, they possess several health-promoting effects that have been mainly associated with their high levels of vitamin C and phenolic compounds which are the primary constituents of flavonoids (flavanones, flavones, and flavonols existing as glycoside or aglycone forms) [9,10]. A great proportion of their production, when not consumed fresh, is used by the juice industry. As a result, high volumes of citrus by-products are generated that, unfortunately, due to their characteristics, cannot be easily stored and are generally used as feedstuff for livestock or for compost production [11]. However, they contain several compounds, such as phenolic acids, flavonoids, terpenes, and carotenoids with strong antioxidant activities that can be obtained through the appropriate conversion processes and then used in the food industry [12]. The environmental sustainability of the citrus industry is becoming one of the main challenges of this sector, and innovative processes should be performed for the transformation of the biomass obtained from the squeezing of citrus fruits into commercialized compounds with high added value, such as citrus extracts rich in polyphenols [13,14].

Citrus by-products have already been added to meat products (cooked and dry-cured sausages, cooked turkey meat, mortadella, etc.) with positive effects on their quality characteristics, such as pH, color parameters, and lipid oxidation indices [15,16,17,18]. These results are promising and could lead to the minimization of synthetic agent use and a reduction in meat residual nitrite content, contributing to sustainable practices that can extend meat shelf life, improve its sensory properties, and fortify human health [19]. At the same time, meat and meat products with functional characteristics can be developed to promote health and prevent the risk of Western diseases, such as type 2 diabetes, hypertension, heart disease, stroke, obesity, and several types of cancer [20,21].

Pariza is a type of pasteurized salami, also known as bologna sausage, primarily made from pork. It is considered one of the most popular cooked meat products in Greece [22]. In this study, pariza was selected due to its susceptibility to lipid oxidation as this product usually contains around 9–20% of fat. Similar studies have been conducted by Varga-Visi et al. [23] and Colmenero et al. [24] in order to examine the rancidity, color, and other sensory properties of pariza products under chilling storage conditions. Furthermore, Shin et al. [25] measured lipid oxidation in irradiated meat using TBARS (Thiobarbituric Acid Reactive Substances) to evaluate the extent of oxidation, and it was shown that irradiation, which is similar to sunlight or artificial light exposure, increased lipid oxidation. Considering the above, the aim of this study was to evaluate the effects of a natural citrus phenolic (CP) complex extract on the quality characteristics of a pariza-type meat emulsion product.

2. Materials and Methods

2.1. Pariza Samples Preparation

Each batch contained pork meat, lard, and water as the main ingredients and was prepared as follows: The frozen pork was weighed, cut into small pieces, i.e., particle size around 3–4 mm, and then added to the cutter (GEA CMV 500L type: Cut Master V500L, s/n:288/0668, GEA, Düsseldorf, Germany). There, it was comminuted to even smaller pieces for a short time. Then, the appropriate amounts of spices and stabilizers were added. At this stage, the addition of the examined proprietary citrus phenolic complex extract (Flavomix AX200, Polypan Group, Moschato, Greece) occurred at the levels of 500 and 1000 ppm in the CP500 and CP1000 groups, respectively. The producer describes Flavomix AX200 powder as an extract from Citrus simensis and Citrus aurantium with the total phenolics at 200 g/kg of the product (Figure 1). Once emulsification had occurred, the RPM (rotations per minute) was set to high thus creating significant friction. Therefore, all the raw materials must be stored at low temperatures. During the initial mixing, the temperature of the ingredients would be ±0–1 °C, and the grinding process would continue until the mixture was reduced to a paste. At the stage of emulsification, the temperature would reach 9 ± 1 °C, while the room’s temperature would be 8–10 °C. However, the final meat mass should not exceed 10 °C. Thus, while mixing for 8–10 min, part of the required quantity of water and ice was added in portions. Then, the starch and lard trimmings were added and mixed for 1–2 min. When the paste was ready, it was filled (Vemag filling Machine, model: HP25E Vemag, Verden/Aller, Germany) into synthetic casings to the required volume, that is, out of 450 kg of the total emulsion mass, each pariza piece was filled to 300 g. The formed salamis were then cooked using steam in a specially designed chamber under controlled conditions (Schroter steam cooking chamber, Borgholzhausen, Germany). The cooking program applied was as follows: the chamber temperature was initially set at 55 ± 2 °C for 4 h, and then it was increased to 76 °C until the internal temperature of the samples at the center reached 72 ± 2 °C. Finally, the samples were cooled down and refrigerated at 4 ± 0.5 °C. The pariza flow chart is presented in Figure 2. A total of 24 replicates were prepared for each group, with 8 per sampling day.

2.2. Meat Quality Evaluation

2.2.1. pH, Color and Shear Force Values

A portable pH meter (HI 99163 model, Hanna Instruments, Cluj, Romania) was used for the recording of acidity values in the pariza samples on day 0, but also on days 30 and 72 after storage at 4 °C. Standardization of the pH meter was carried out at room temperature (20 ± 2 °C) using buffers with pH values of 4.0 and 7.0 (Merck, Darmstadt, Germany) [26]. Color attributes (lightness—L, redness—a*, and yellowness—b*) were also measured in the pariza samples on day 0, and 30 and 72 days after storage at 4 ± 0.5 °C, by a Miniscan XE (HunterLab, Reston, VA, USA) chromameter that was standardized with the use of a white and black tile and assigned on the CIE-LAB system [27]. Shear force value was also measured in the samples, with a cross-section of 1 cm², by a Warner–Bratzler (WB) shear blade fitted to a Zwick Testing Machine Model Z2.5/TN1S (Zwick GmbH & Co., Ulm, Germany) (in triplicate). Peak force values were calculated in Newton. Warner–Bratzler shear force value measurement is a typical procedure for the evaluation of meat tenderness [28]. As previously pointed out, 8 replicates were prepared for each group per sampling day. The selection of sampling days was based on the shelf life of the product (72 days) and the selection of an intermediate time frame (30 days).

2.2.2. Lipid Oxidation Status

Malondialdehyde (MDA) levels served as an indicator of lipid oxidation and were measured on day 0, and on days 30 and 72 after storage at 4 ± 0.5 °C, by a selective third-order derivative (3D) spectrophotometric method [29]. In brief, 5 mL of butylated hydroxytoluene (BHT) in hexane (8 g/L) and 8 mL of aqueous trichloroacetic acid (TCA) (50 g/L) were mixed with 2 g each of the raw or cooked meat sample (in duplicate) and then homogenized (Unidrive x 1000, CAT, M. Zipperer GmbH, Ballrechten-Dottingen, Germany). The mixture was then centrifuged for 5 min at 5000× g. After hexane was discarded, 2.5 mL of the bottom layer was mixed with 1.5 mL of aqueous 2-thiobarbituric acid (TBA) (8 g/L). The obtained solution was further incubated at 70 ± 1 °C for 30 min. It was then left to cool under running water, equilibrated, and was finally subjected to 3D spectrophotometry (Hitachi U3010 Spectrophotometer, Hitachi High-Tech Corporation, Tokyo, Japan) in the range of 500–550 nm. MDA content (ng/g wet tissue) was measured on the basis of the height of the third-order derivative peak at 521.5 nm, by comparing with the slope and intercept data of the standard calibration curve created using the MDA precursor 1,1,3,3-tetraethoxypropane (TEP).

2.3. Statistical Analysis

Data were analyzed by a mixed model procedure appropriate for the repeated measurements per subject, with the addition of citrus polyphenols as a fixed effect and storage duration as repeated factor by Sas/Stat [30]. The linear dose response was examined with orthogonal contrasts using the CONTRAST statement. Differences in the means were detected at 0.05 with Bonferroni adjustment, and the results were displayed as least squares (LS) means ± standard error of mean (S.E.M.).

3. Results and Discussion

As indicated in

Table 1. Effect of citrus polyphenol (CP) complex on the quality characteristics of pariza-type meat emulsion product on day 0, 30 and 72 days after refrigerated storage.

Parameter	Sampling Day	CONTROL 1	CP500	CP1000	SEM	p-Value	p-Linear
pH	0	6.33 aB	6.34 aB	6.33 aB	0.01	NS	NS
	30	6.36 aA	6.37 aA	6.36 aA	0.01	NS	NS
	72	6.26 bC	6.29 aC	6.31 aB	0.01	<0.05	<0.01
	p-value	<0.05	<0.01	<0.01
Lightness (L)	0	52.47 bB	54.08 aA	53.96 aB	0.28	<0.001	<0.01
	30	55.37 aA	53.82 bA	54.81 aA	0.28	<0.05	NS
	72	52.79 aB	53.10 aB	53.50 aB	0.28	NS	<0.001
	p-value	<0.001	<0.05	<0.05
Redness (a*)	0	27.96 aA	25.19 bB	24.52 bB	0.39	<0.001	<0.001
	30	23.99 bB	26.04 aAB	23.07 bC	0.39	<0.001	NS
	72	27.43 aA	27.15 aA	26.08 bA	0.39	<0.05	<0.001
	p-value	<0.001	<0.05	<0.01
Yellowness (b*)	0	6.67 aA	6.15 bA	6.96 aA	0.12	<0.001	NS
	30	5.39 cC	5.63 bB	6.20 aB	0.12	<0.001	<0.001
	72	6.09 bB	5.94 bAB	6.77 aA	0.12	<0.001	<0.001
	p-value	<0.001	<0.01	<0.001
Shear Force (N)	0	4.24 aA	3.88 aAB	4.09 aA	0.15	NS	NS
	30	3.98 aA	3.67 aB	3.98 aA	0.15	NS	NS
	72	4.25 aA	4.18 aA	4.09 aA	0.15	NS	NS
	p-value	NS	<0.05	NS
MDA 2 (ng/kg)	0	20.60 aC	17.36 bC	18.03 bC	0.72	<0.05	<0.05
	30	23.54 aB	21.42 bB	20.20 bB	0.72	<0.05	<0.001
	72	30.98 aA	27.42 bA	25.34 cA	0.72	<0.01	<0.001
	p-value	<0.001	<0.001	<0.05

¹ The treatment groups were control (CONTROL), without CP addition, and two groups with the addition of the polyphenol complex at the levels of 500 and 1000 mg/kg (CP500 and CP1000, respectively). ² Malondialdehyde. a,b,c Values sharing dissimilar superscripts within a row and parameter are significantly different. A,B,C Values sharing dissimilar superscripts within a column and parameter are significantly different.

, the pariza pH values were not affected by CP addition on day 0 and 30. Fernández-Ginés et al. [31] also found no significant differences in pH values of bologna sausages after the addition of citrus fiber at 0.5–2%, as well as the refrigerated storage of 28 days, indicating no significant effect of citrus polyphenols on pariza meat acidity. However, on day 72, increased pH values were observed in CP500 and CP1000 when compared with the control group (p < 0.05). In contrast with the results of the present study, the pH values were decreased from 5.7 to 5.5 in the ground beef patties treated with 450 μg/g of orange (Citrus reticulata) pomace after refrigerated storage for 9 days [32]. A decrease in pH values were also observed in Spanish-type dry-cured “Fuet” sausages from 5.44 to 5.34 after the application of a citrus extract at 200 ppm during ripening at 14 °C for 21 days [33]. Discrepancies may be attributed to the difference in meat product and storage period, which was much shorter in the aforementioned studies. The pH values in the three experimental groups were higher on day 30 compared to day 0, and this finding could be attributed to the production of proteases by psychotropic bacteria [34] that were not affected by CP addition. Finally, the pH values were also decreased in all experimental groups, possibly as an effect of the growth of lactic acid bacteria [35].

The color of meat and meat products dramatically affects consumer purchasing decisions, since it is perceived as a significant attribute associated with quality and freshness [36]. Consumers generally consider bright red as a color indicating a fresh and high-quality meat product, while a pale, discolored, or darker color is related with poor preservation and low quality [37]. Meat can have a bright red color as an effect of deoxymyoglobin conversion into oxymyoglobin. However, the continued exposure of the meat surface to oxygen results in increased oxidation rates and the production of free radicals that further induce the transformation of myoglobin into the brown pigment, metmyoglobin [38]. Thus, it can be concluded that instrumental meat color evaluation is very crucial as it provides objective measurements [39]. Instrumental measures of L* (brightness) and a* (redness) are straightforward and can easily be applied to muscle color. On the other hand, the colors represented by b* (yellowness) are not typical or intuitively related to meat, since they are more correlated to brown than yellow [40].

The L value was increased in CP-treated groups on day 0, decreased in CP500 on day 30, while no significant differences were observed on day 72. However, a significant linear dose response effect was shown on day 0 and 72 (p-linear < 0.01 and <0.001, respectively), indicating that lightness was increased as the CP level in pariza increased (Table 1). On the other hand, the value of lightness was increased on day 30, as already shown in previous studies with pork sausages during the first week of storage (an increase at the level of 6%) [41], due to changes in the structure of meat proteins [42], but it was finally decreased on day 72, possibly as a result of higher oxidation rates and metmyoglobin formation [38]. Yellowness was also linearly increased on day 30 and 72 (p-linear < 0.001); b* values were higher in CP1000 compared to the other groups on day 30 and 72 after storage at 4 °C (Table 1). On the other hand, redness was linearly reduced in the pariza treated with citrus polyphenols on days 0 and 72 (p-linear < 0.001), indicating an opposite trend, with both CP groups showing lower values on day 0, whereas only CP1000 on day 72 was reduced compared to the controls (p < 0.05), possibly as an effect of nitrosopigment degradation [17]. In a previous study, lightness was also increased by 5–7%, and redness was decreased by 15% in bologna sausages after the application of lemon albedo at 2.5–10%; however, the opposite results were observed in dry-cured sausages, i.e., an increase in the a* value at the level of 11–13% [17]. Addition of citrus peel extracts (1%) reduced L from 45.25 to 39.70 but increased the a* and b* values from 5.57 to 7.65 and 10.10 to 10.67, respectively, in meatballs stored at −20 °C for 6 months [43]. Lightness was not affected, while redness and yellowness were increased from 11.9 to 13.4 and 13.9 to 15.1, respectively, in ground beef patties treated with 450 μg/g of orange (Citrus reticulata) pomace [32]. Increased values of a* in cooked beef (by 20%) and cooked chicken (by 10%) were also observed as a result of citrus peel addition at 0.1% [44].

Shear force value, which is a very important sensory attribute, was assessed to indicate any possible side effects of the addition of citrus polyphenols in pariza that would increase the risk of a poor eating experience for the consumer [45]. No effect of CP complex on the tenderness of pariza-type meat emulsion product was pointed out, since the shear force values were not significantly different among the groups throughout the experimental period (Table 1). Hardness, which was one of the four measures of sensory tenderness (the others were cohesiveness, toughness, and chewiness) [46], was also not affected by the addition of orange pomace (450 μg/g) in ground beef patties [32].

Bioactive compounds contained in citrus by-products generally display intense antioxidant properties against free radicals, molecules with an unpaired electron in their atomic structure [47]. The improved oxidative stability of pariza was observed as an effect of CP addition, since MDA values were lower in treated groups than controls, showing a dose-dependent effect (p-linear < 0.001; Table 1). This effect could be attributed to the antioxidant properties of citrus polyphenols that protect pariza against the negative effects of oxidation processes [8]. Decreased TBA values by 13–25% were also observed in Bologna sausages after the addition of orange fiber at 0.5–2%, respectively [17]. Mahmoud et al. [48] evaluated the lipid oxidative stability of beef burger treated with 2.5–10.0% of orange peel and indicated reduced TBA values by 11–46%, respectively. Orange peel extract at 0.4% also improved lipid stability of beef muscle stored at −20 °C for 60 days, since TBARS values were decreased by 25% [49]. The addition of orange or lemon peel flour into beef patties stored at 4 °C for 15 days resulted in reduced TBA values [50]. Furthermore, the application of grapefruit peel extracts in meatballs during frozen storage led to an improvement of oxidative stability [43]. Sayari et al. [51] reported that the addition of grapefruit peel extracts in turkey sausage reduced lipid oxidation rates and that the TBA values were lower by 73.45% after 13 days. Klangpetch et al. [52] also found lower malondialdehyde (MDA) values in raw chicken wings drumettes stored at 4 °C for 14 days as an effect of Kaffir lime peels extract addition. Lipid oxidation decreases in raw beef and pork and cooked pork and chicken were also found after the addition of citrus peel at 0.1% [44]. Citrus extract-coated trays delayed lipid oxidation in cooked turkey meat, as indicated by the significantly lower TBARS values [53].

4. Conclusions

The presence of antioxidants in citrus by-products allows for their application in food processing to obtain healthy products and fortify the human organism against the development of cancer and other diseases. As far as the authors are aware, this research is the first one that examines the effect of a citrus extract on the quality characteristics and the oxidative stability of pariza. As indicated by our preliminary results, CP extracts improved oxidative stability by reducing the formation of malondialdehyde and can be used for the extension of shelf life in pariza-type meat emulsion products. However, further research is warranted before any definitive recommendations can be made.