**E**ff**ectiveness of Oat-Hull-Based Ingredient as Fat Replacer to Produce Low Fat Burger with High Beta-Glucans Content**

**Carmine Summo \* , Davide De Angelis , Graziana Difonzo , Francesco Caponio and Antonella Pasqualone**

Department of Soil, Plant and Food Science (DISSPA), University of Bari Aldo Moro, Via Amendola, 165/a, I-70126 Bari, Italy<EMAIL_ADDRESS>(D.D.A<EMAIL_ADDRESS>(G.D.);

<EMAIL_ADDRESS>(F.C<EMAIL_ADDRESS>(A.P.)

**\*** Correspondence<EMAIL_ADDRESS>
Received: 30 June 2020; Accepted: 3 August 2020; Published: 4 August 2020

**Abstract:** Low-fat beef burgers with high beta-glucan content was obtained using a gel made from an oat-hull-based ingredient as fat replacer.  The nutritional composition, cooking yield, textural properties, color characteristics and consumer preference were evaluated, in comparison with a burger without fat replacer (CTRL). After cooking, T2 burger showed a significant increase in the cooking yield and a very low lipid content (3.48 g 100 g−1 ) as well as a level of beta-glucans per single portion (2.96 g 100 g−1 ) near the recommended daily intake. In T1 burger, the decrease of lipid content was mitigated during the cooking process, because the beta-glucans added had a fat-retaining effect. Compared to CTRL, replacing fat led to a softer texture of cooked burgers evaluated by Texture Profile Analysis. The differences in color, significant in raw burgers, were smoothed with cooking. The consumer evaluation, carried out according to the duo-trio test, highlighted significant differences between CTRL and T2 burgers in terms of odor, taste, color and texture. The consumers expressed a higher preference for the T2 burger, probably due to its softer texture and greater juiciness.

**Keywords:** beef burgers; soluble fiber; TPA; consumer evaluation; fatty acid composition

#### **1. Introduction**

Meat and meat products play an important role in human nutrition, constituting a rich source of proteins with high biological value, vitamins (A, B1, B3 and B12), as well as iron, zinc and other micronutrients [1]. The consumption of meat and meat products dates back to antiquity, but these products are still part of the gastronomic tradition of many countries. Therefore, a high number of Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) European brands—which link the quality of food products to a specific geographical area—have been awarded to "Meat products—cooked, salted and smoked" (205 registered products, accounting for 12.95% of the total PDO and PGI products) and "Fresh meat and offal" (180 registered products, i.e., 11.37% of the total PDO and PGI products) [2]. However, the high fat content of meat products (including saturated fatty acids and cholesterol) is related to increased risk of developing coronary heart diseases [3].

In this context, researchers and private companies alike are strongly engaged in trying to improve the nutritional value of meat products by lowering the cholesterol and lipid content, as well as decreasing saturated and increasing polyunsaturated fatty acids. Fats, however, play an important role in meat products, ensuring optimal rheological and textural properties [4] and conferring pleasant sensorial characteristics in terms of flavor and juiciness [5]. Therefore, the reduction of lipid content in meat involves the use of ingredients able to mimic the properties of fat, such as polysaccharides. Several experimental trials have therefore been performed that included various mostly fiber-rich polysaccharide-based fat replacers in the formulation of meat products, such as ground poppy seeds [6], mixtures of wheat fiber and pig skin [7], legume flours [8] and other vegetable sources, as indicated in recent reviews [9]. Dietary fiber can form a compact gel due to the ability to bind water improving the structural characteristics of reduced-fat products [10].

Among dietary fibers, beta-glucans from cereal grains have been recently studied in relation to the health benefits associated with their consumption such as the reduction of cholesterol level and a chemo-preventive effect as reported by Ho et al. [11].

Moreover, beta-glucans show several technologically useful properties (gelling capacity, emulsifying activity, fat/water binding capacity), which make them suitable ingredients in health-promoting functional foods [12]. The major applications of beta-glucans in food formulation are in milk-based products, such as fermented milk products and yogurt [13] and in bakery products [14]. Several beta-glucan sources have also been considered for improving the nutritional quality of meat products, with [15–17] or without [18] fat replacement. However, the level of beta-glucan enrichment reported in previous studies on meat products does not reach the recommended daily intake for beta-glucans, which accounts for 3 g per day [19].

In this frame, the aim of this study was the production of low-fat burgers with a beta-glucan content very close to the recommended daily intake and with good textural and sensorial characteristics.

#### **2. Materials and Methods**

#### *2.1. Preparation of the Fat Replacer*

An oat-hull-based ingredient (Nutraceutica S.R.L., Monterenzio, Italy) containing, as declared by the producer, 55% beta-glucans, <10% proteins, <2% fat, was used to prepare a gel by mixing 27.27 g of flour with 72.73 mL of distilled water for 5 min at 13,500 rpm by means of a T25 Ultraturrax (IKA, Staufen, Germany).

The ratio flour:water was defined in preliminary tests to obtain a gel: (i) able to mimic as much as possible the consistency and homogeneity of the beef fat conventionally used to prepare meat burgers; (ii) having a beta-glucan concentration able to achieve, when added to burgers as total fat replacer, a beta-glucan content as near as possible to the daily intake recommendation (3 g per day) [19].

#### *2.2. Preparation of the Beef Burgers*

Beef meat, purchased at a local butcher's shop, was manually sectioned with a sharp knife to separate the lean meat from the visible adipose and connective tissues.  Adipose tissue and lean meat, both ground, were then mixed manually. During the mixing step, three batches were prepared, according to three different formulations at increasing levels of fat: control (CTRL), with 15% of beef adipose tissue added; T1, with a partial (50%) substitution of beef adipose tissue (i.e., with 7.5% beef adipose tissue and 7.5% oat-hull-based gel added); and T2, with a total substitution of beef adipose tissue (i.e., with 15% oat-hull-based gel added). With the exception of salt, no other spices or ingredients were added. The formulations of the three burgers are reported in Table 1. The burgers, weighing approximately 50 g, were finally shaped (70 mm diameter, 10 mm thickness) using a burger maker mold. The whole experiment was repeated twice.

#### *2.3. Cooking Procedure*

The burgers were cooked according to the American Meat Science Association methodology [20], i.e., were roasted in an electric oven (Delonghi EO 3275, Delonghi Appliances, Treviso, Italy) preheated at 163 ◦C, until their internal temperature, measured by a digital thermometer (LT−101, TFA Dostmann, Reicholzheim, Germany), reached 71 ◦C. Approximatively 10 min was sufficient to cook all the samples perfectly.

Cooked burgers were then submitted to the chemical and textural determinations, as well as consumer test. The colorimetric determinations, instead, were carried out on burgers both before (raw) and after cooking.

**Table 1.** Formulation (g kg−1 ) of three different beef burgers without fat substitution (CTRL) and at 50% (T1) and 100% (T2) fat substitution.

\* Gel as fat replacer formulated with 27.27 g of oat hull ingredient at 55% of beta-glucan concentration emulsified with 72.73 mL of distilled water.

#### *2.4. Chemical Composition of Beef Burgers*

Protein content (total nitrogen × 6.25), ash, and moisture content were determined, according to the AOAC International methods, to be 928.08, 920.153 and 950.46, respectively [21]. The lipid content was determined by Folch method [22] using chloroform and methanol (Sigma Aldrich, Milan, Italy) as extracting solvent. The carbohydrate content was determined as difference. The total beta-glucan concentration was determined by the AOAC International method 995.16 [23] by using the Megazyme mixed-linkage beta-glucan assay kit (Megazyme International, Bray, Ireland). The total energy value for each product was calculated by using the Atwater coefficients as reported in Summo et al. [24]. All determinations were carried out in triplicate.

#### *2.5. Fatty Acid Composition of Beef Burgers*

The fatty acid composition was determined by gas-chromatographic (GC) analysis of fatty acid methyl esters. The lipid fraction was cold-extracted with methanol/chloroform (1:2 *v*/*v*) following the method proposed by Folch et al. [22]. The methylation was carried out according to the AOCS (American Oil Chemists Society) method Ch 1–91 [25]. The GC system and conditions were the same as those reported in a previous paper [26]. The identification of each fatty acid was carried out by comparing the retention time with that of the corresponding methyl ester standard (Sigma Aldrich, Milan, Italy). All determinations were carried out in triplicate.

Atherogenic (AI) and Thrombogenic (TI) indices were calculated according to the following equations [27]:

$$\text{AI} = [\mathbf{C}\_{120} + (\mathbf{4} \times \mathbf{C}\_{140}) + \mathbf{C}\_{160}] / (\mathbf{n} \cdot \mathbf{6} \,\text{PUFA} + \mathbf{n} \cdot \mathbf{3} \,\text{PUFA} + \text{MUFA}) \tag{1}$$

$$\text{TI} = (\text{C}\_{14:0} + \text{C}\_{16:0} + \text{C}\_{18:0}) / [0.5 \times \text{MUFA} + 0.5 \times \text{n-6 PUFA} + 3 \times \text{n-3 PUFA} + (\text{n-3} \times \text{n-3 PUFA} + \text{C}\_{12:0} + \text{C}\_{16:0})] \tag{2}$$

$$\text{PUFA/n-6 PUFA}) \tag{2}$$

where PUFA are polyunsaturated and MUFA monounsaturated fatty acids. C12:0, C14:0, C16:0 andC18:0 are lauric, myristic, palmitic and stearic acids, respectively.

#### *2.6. Cooking Yield*

The cooking yield of beef burgers was determined by measuring the weight (w) of the burgers before and after cooking according to the following equation:

$$\text{Cooking yield} = \text{(w cooledburger/w rawburger)} \times 100. \tag{3}$$

The calculation has been performed on ten burgers.

#### *2.7. Texture Profile Analysis*

Texture profile analysis (TPA) of beef burgers was performed according to Afshari et al. [17] with some modifications, using a texture analyzer model Z1.0 TN (Zwick Roell, Ulm, Germany) equipped with a 3.6 cm cylindrical probe and a 1 kN load cell. The samples were heated in an oven at 60 ◦C in order to simulate the serving conditions. Then, a portion of 2 cm of diameter was cut from the center of the burger. A two-compression cycle was carried out at the speed of 5 mm s−1 , with 5 s of pause between the two compressions, up to 70% of recorded deformation. The following parameters were assessed: hardness (N), indicating the maximum force recorded during the first compression; cohesiveness, measured as the area of work during the second compression divided by the area of work during the first compression; gumminess (N), calculated as hardness × cohesiveness; springiness, measured by the distance of the detected height during the second compression divided by the original compression distance; chewiness (N), calculated as gumminess × springiness. Ten different burgers per formulation were considered, and each burger was subjected to one measurement by TPA.

#### *2.8. Color Determination of Burgers*

Instrumental determination of the surface color of both raw and cooked burgers was carried out by using the CM-600d colorimeter (Konica Minolta, Tokyo, Japan) supported by SpectraMagic NX software (Konica Minolta, Tokyo, Japan). The CIE (International Commition on Illumination) *L*\*, *a*\*, and *b*\* parameters were recorded: lightness (*L*\*), red index (*a*\*) and yellow index (*b*\*), together with ∆E [28].

$$
\Delta \mathbf{E} = \left[ (\Delta L^\*) \mathbf{2} + (\Delta a^\*) \mathbf{2} + (\Delta b^\*) \mathbf{2} \right]^{1/2} \tag{4}
$$

Three samples per formulation were analyzed, and four readings were recorded in different areas of each sample.

#### *2.9. Duo-Trio Consumer Test*

CTRL and T2 burgers were submitted to consumer test according to the duo-trio test methodology [29] to determine if the differences between them could be recognized. Sixty people, regular consumers of meat and neither food-allergic nor intolerant, were recruited among the researchers and students of the Agricultural Faculty of the University of Bari Aldo Moro (Bari, Italy). The study protocol followed the ethical guidelines of the laboratory. Each participant was given information about study aims and individual written informed consent was obtained from each participant. The consumer test was performed at a local restaurant sited in Bari (Italy). Each participant received three samples on the same dish: one as reference (CTRL or T2 randomly, and codified with an alphanumeric code), and the other two were both CTRL and T2 randomly distributed, codified with an alphanumeric code. Each consumer was asked to indicate the sample that was different respect to the reference in terms of color, odor, taste and texture. Moreover, each panelist expressed a judgment indicating which burger preferred. The results were expressed as number of correct answers.

#### *2.10. Statistical Analysis*

Data were subjected to one-way ANOVA followed by the Tukey's HSD test. Significant differences were determined at *p* < 0.05 by the XLStat software (Addinsoft SARL, New York, NY, USA).

The results of duo–trio test were expressed as number of correct answers considering thirty-nine, forty-one and forty-four as minimum correct answers to identify statistically significant differences at *p* < 0.05, *p* < 0.01 and *p* < 0.001, respectively [30].

#### **3. Results and Discussion**

#### *3.1. Chemical Composition*

The addition of the fat replacer significantly influenced the chemical composition of cooked burgers (Table 2). An increase of moisture was observed at increasing content of fat replacer. This is principally due to the high moisture content of the fat replacer. These findings agreed with those of a previous study involving the use of oat beta-glucan as fat replacer [16]. However, in another study, the use of gelled emulsion (based on olive oil, gelatin and 9% inulin) caused an increase of moisture content only in raw patties, whereas a significantly lower moisture of cooked product was observed due to lower cooking yield and water holding capacity of the gel [31]. Therefore, our results could be due also to better moisture retention of fat-substituted burgers during cooking due to the high hydrophilicity of beta-glucans [32], able to increase the water-holding capacity of the product. The total substitution of fat (T2), indeed, caused a significantly higher moisture content than in CTRL and T1.

**Table 2.** Chemical composition, cooking yield and energy value of the cooked beef burger without fat substitution (CTRL) and at 50% (T1) and 100% (T2) fat substitution with an oat-hull-based gel.

Data on the chemical composition were expressed as % on fresh (f.w.) weight. Different letters in the same row indicate significant differences at *p* < 0.05.

On the contrary, the protein content of beef burgers (on fresh matter), showed a progressive and significant decrease when the fat replacement increased. Piñero et al. [15] and Afshari et al. [17] reported that the addition of a beta-glucan-based fat replacer had no significant influence on the protein content. Our findings could be related to a higher level of gel incorporation and a consequently higher moisture content. Moreover, the beef adipose tissue used in CTRL and T2 formulations contained muscular residues, which also contributed to the protein content, in accordance with other authors [33]

Compared to CTRL, the addition of the fat replacer resulted in a slight but significant fat decrease in T1 formulation, whereas the T2 burger showed a more marked decrease. Considering the lipid content of the beef adipose tissue (accounting for 71.5%) used in CTRL and T1 formulations, and the contribution of the residual intramuscular fat of the lean fraction (3.5%), the lipid content of the CTRL raw burger could be estimated at 13.6 g 100 g−1 . After cooking, the CTRL burger showed a lipid content of 8.42% (6.04 g of fat in 71.82 g of cooked burgers); therefore, an estimated fat loss of 56% occurred.  Therefore, even if considering estimated values, cooking induced a more limited fat loss when fat was replaced by the beta-glucan based gel than in the CTRL burger. This phenomenon could be imputable to the ability of the beta-glucans to form a tri-dimensional network which entraps fat and water within the meat protein system [15]. Therefore, it has to be considered that partial fat replacement with beta-glucans lowers fat content in the raw product, but this nutritionally positive effect is mitigated by higher fat retention during the cooking process. As a consequence, a total fat replacement has to be made to achieve a significant nutritional effect on the cooked product.

The use of the fat replacer caused, as expected, a slight but significant increase in the carbohydrate content of T1, even if no significant differences were observed comparing T1 and T2. This was imputable to the presence of carbohydrates in the oat-hull-based ingredient. The addition of vegetable fat replacer in burgers is reported to be influential on the chemical composition of the product [34]. The content of beta-glucans reached a level that made the health claim "beta-glucans contribute to the maintenance of normal blood cholesterol levels" applicable to both T1 and T2 burgers since the concentration of these compounds was always higher than 1 g per recommended portion (in meat products, this quantity corresponds to 100 g). However, the claim regulation specifies that "the beneficial effect is obtained with a daily intake of 3 g of beta-glucans" [19]. In this regard, T2 burger contained 2.96% of beta-glucans. Therefore, the recommended daily intake of beta-glucans, according to the above-mentioned regulation, could be reached by consuming a single portion (100 g) of T2 burger. This result is particularly important because it is possible to achieve a significant improvement in the nutritional characteristics of burgers. Indeed, by combining the total substitution of animal fat with the inclusion of functional macromolecules, a positive effect on cholesterol reduction could be expected. Indeed, it is known that beta-glucan has an active role on the reduction of LDL-cholesterol [11] by modulating the cholesterol metabolism and the gut microbiota [35].

The fat substitution resulted in a significant decrease in energy value, from 203.44 kcal 100 g−1 (CTRL) to 146.24 kcal 100 g−1 (T2). In particular, the T2 formulation allowed the research to obtain a product with lower fat content and, consequently, lower energy value compared to the products proposed by other studies [17–19]. An effective improvement of the nutritional value of meat products was therefore achieved, due to reduced fat content, relatively low energy value and high concentration of beta-glucans.

#### *3.2. Cooking Yield*

The fat replacement caused an increase in cooking yield. The difference, compared with the control burger, became significant in the T2 formulation. These findings agreed with previous studies [17,36] in which higher cooking yield and moisture retention with the increase of beta-glucan content was observed. This behavior can be explained with the already mentioned ability of beta-glucans to form three-dimensional structures with meat proteins, which can easily entrap water and fat, increasing the cooking yield [15].

#### *3.3. Fatty Acid Composition*

Fatty acid composition of burgers is reported in Table 3, as mg 100 g−1 of burger and g 100 g−1 of fatty acids. The nutritional value of beef burgers is also related to the composition of the lipid fraction, which usually is dominated by saturated fatty acids, palmitic and stearic acids in particular, whereas oleic acid was the most abundant unsaturated acid. The fatty acid composition of cooked burgers agreed with other studies carried out on the same category of products [17,37]. Owing to the fat substitution, a significant reduction was observed of the quantity (mg 100 g−1 of burger) of all fatty acids due to the general decrease of lipid content. Moreover, a different level of reduction was observed as a function of the unsaturation rate. In particular, T2 showed a content of palmitic acid 60% lower than the CTRL. The reduction was slightly lower for oleic acid (−57%), whereas linolenic, the most abundant polyunsaturated fatty acid, decreased by 45% comparing T2 with CTRL. This aspect could be better explained considering the composition of fatty acids expressed as percentage. In particular, comparing the T2 with the other formulations, we observed a significantly (*p* < 0.05) lower percentage of saturated fatty acids and a higher percentage of the polyunsaturated fatty acids, whereas the monounsaturated fatty acids remained constant across the formulations. Previous studies report significant differences in the fatty acid composition of subcutaneous and muscular beef fat, with the latter characterized by higher polyunsaturated and lower saturated fatty acids [38,39]. This could explain the differences observed in our samples, because in CTRL and T1 burgers, the fatty fraction added was mainly subcutaneous fat, while in T2 the residual fat was constituted principally by muscular fat.

**Table 3.** Fatty acid composition (g 100 g−1 of burger and g 100 g−1 of fatty acids) and the nutritional index of the beef burger without fat substitution (CTRL) and at 50% (T1) and 100% (T2) of fat substitution with an oat-hull-based gel.

SFA = Saturated fatty acids; MUFA = Monounsaturated fatty acids; PUFA = Polyunsaturated fatty acids; AI = Atherogenic Index; TI = Thrombogenic Index. Different letters in the same row indicate significant differences at *p* < 0.05.

Albeit in low amounts, we detected also some polyunsaturated fatty acids important from a nutritional point of view, such as the arachidonic (C20:4 n-6) eicosapentaenoic (C20:5 n-3) and docosapentaenoic acids (C22:5 n-3), without significant differences among the formulations. The amount of these important fatty acids was lower than that reported in other studies carried out on the raw beef lipid fraction [40]. This difference could be related to the cooking procedure, which causes the loss of these fatty acids [37]. In studies carried out on cooked beef burgers, these fatty acids were indeed not determined [17,41].

As a consequence of the different lipid composition, the nutritional indices linked to the fatty acid composition were also influenced by the fat replacement. In particular, the PUFA/SFA ratio significantly increased in T2 compared to CTRL. Moreover, the atherogenic and thrombogenic indices related to fatty acid composition significantly decreased in T2 burger with 100% fat substitution, although the values were higher than those recommended [42]. The n-6/n-3 ratio was higher in T2 compared to CTRL and T1. It is reported that lowering the n-6/n-3 ratio to less than 4 is desirable to improve the healthiness of the product [43,44]. However, the achievement of this target in meat product is not possible solely with a fat reduction, because fat composition needs to be reformulated by the addition of oils rich in n-3 PUFA [44,45].

Similar improvements were observed by Pintado et al. [45] in fresh sausages obtained using an olive oil in water emulsion containing chia and oat as fat replacer. The authors explained the results with the high level of polyunsaturated fatty acids of chia. The oat-hull-based ingredient used in our study was characterized by a very low lipid content; therefore, its contribution to the fatty acid composition was of relevance. Several studies report that the unsaturated fatty fractions are combined with structural compounds of meat so that their loss during cooking is less influenced than saturated fatty acids [44]. The saturated fatty acids could easily be lost during cooking, and this could explain the observed results.

#### *3.4. Texture Profile Analysis*

Significant differences in the textural properties were observed among burgers with different formulation (Table 4). The incorporation of a fat replacer led to a significant decrease of hardness, cohesiveness, gumminess and chewiness in T1 and T2 burgers compared to CTRL, indicating that these burgers had a softer texture and then required less energy to be compressed. No significant differences, however, were found between T1 and T2, highlighting the fact that the level of fat substitution did not influence the textural properties of beef burgers.

**Table 4.** Texture profile analysis (TPA) of the beef burger without fat substitution (CTRL) and at 50% (T1) and 100% (T2) of fat substitution with an oat-hull-based gel.

Different letters in the same column indicate significant differences at *p* < 0.05.

The trend of moisture and fat as an influence on texture [17] could be explained by a compensation between the differences in moisture and fat contents of T1 and T2 (Table 2), leading to similar textural properties. The effect of the fat substitution level was significant only for springiness, which showed the lowest value in T2 formulation.

Owing to the important structural functions of fat, the influence on the textural properties should be considered when the target of a new food formulation is fat substitution. The use of beta-glucans as fat replacement in beef burger or beef patties was previously studied by other authors with contrasting results, depending on whether beta-glucans were added as powder, gel or emulsion. In particular, Szpicer et al. [16] reported an increase in hardness of meat burgers after the addition of 30% beta-glucan concentrate powder. When the beta-glucans were added as gel [15] or emulsion [36], a significant reduction of hardness and other textural parameters were observed. With the increase of beta-glucans concentration, the amount of water available for proteins decreases and meat products lose springiness [46]. This behavior could be explained by a higher moisture retention of burgers and a consequently lower compactness of protein matrix [36]. Furthermore, beta-glucans have the ability to bind not only water but also fat, allowing the formation of a softer [47] and juicier product [17].

#### *3.5. Color Indices*

Color evaluations on the raw burger were made because the color characteristics of the meat products can influence the consumers' willingness to purchase, with increasing appreciation for bright red products. In raw burgers, a progressive and significant increase of lightness (*L*\*) and yellowness (*b*\*) was observed with fat replacement, while redness (*a*\*) was not significantly influenced (Table 5). The increase of the lightness and yellowness could be related to the presence of yellow pigments such as lutein in oat (the source of beta-glucan enriched gel), as previously reported in [48]. In contrast, *a*\* remained constant, indicating that the fat substitution was not significant on this index. Moreover, in a previous study, the fat substitution with a chia oil emulsion gel caused no significant variations of *a*\* but significant changes of *L*\* and *b*\* [49]. In the same study, *L*\* and *b*\* were slightly higher than ours, probably because of the presence of the oil in the fat replacer.

The differences observed among raw burgers were smoothed by cooking, after which no significant differences were found for all the color indices, as reported also by Gök et al. [6]. The color of burgers reformulated with fat replacers is influenced by the type of ingredients used for this purpose. In particular, Lucas-González et al. [49] reported a decrease of *L*\* and an increase of *a*\* during cooking of burgers formulated with chestnut flour and chia oil emulsion gels. By contrast, Heck et al. [43] reported an increase of *L*\* and a decrease of *a*\* in cooked burgers produced by the inclusion of linseed or chia oil microparticles. During the cooking process, meat color changes due to the heat-induced denaturation of myoglobin. Our results, assessed on the cooked burgers, were not influenced by fat substitution; however, it is reasonable to say that the primary contribution to color is given by meat. The role of fat in influencing the color of cooked meat is not fully understood [50], but it should have a lower influence on color than other critical parameters, such as pH and storage conditions [50].

**Table 5.** Instrumental color determination of the beef burger without fat substitution (CTRL) and at 50% (T1) and 100% (T2) of fat substitution with an oat-hull-based gel before (Raw) and after (Cooked) cooking.

Different letters in the same row indicate significant differences at *p* < 0.05.

The ∆E of T1 and T2 formulations, calculated by comparing them to the CTRL, was determined in order to improve evaluation of the color differences between samples. The ∆E was higher in raw than in cooked burgers, reaching the maximum of 7.16 in T2 formulation, whereas T1 showed a value of 3.89. ∆E values were between 3.5 and 5.0, meaning that the observer can clearly perceive the difference between samples; thus, T1 raw burgers could be easily distinguished from CTRL. ∆E values higher than 5 indicate the presence of two distinct colors [51]. When considering the cooked burgers, a decrease of ∆E of both T1 and T2 was observed. The changes occurring in T2 burger were particularly interesting due to the drop of ∆E at 2.58. When 2.0 < ∆E < 3.5, even an unexperienced observer can notice the difference in color between products [51].

#### *3.6. Consumer Test*

CTRL and T2 were submitted to a consumer test, according to the duo–trio test methodology [28], which was chosen to determine if the differences between burgers in terms of color, odor, taste and texture were recognizable by consumers. T1 burger was not considered for two main reasons. Firstly, after preliminary sensory analysis, a small group of trained panelists agreed that T1 burger was similar to CTRL. Moreover, considering the nutritional characteristics of T2 burgers, they were noticeably more interesting than T1, therefore we selected only T2 burger, which had no fat added and had a high content of beta-glucans.

As shown in Figure 1, the consumers recognized the difference between CTRL and T2 burgers for all the descriptors. In particular, forty-one people recognized CTRL and T2 for their different color (*p* < 0.01), whereas the number of correct answers increased when considering odor, texture and taste, with highly significant results (*p* < 0.001). The consumer test confirmed the results of textural and colorimetric evaluations (see for example the ∆E parameter). Szpicer et al. [16] also reported that consumers could distinguish products containing fat replacers, based on differences in color, texture, aroma and taste. Moreover, Afshari et al. [17] highlighted that fat substitution was perceived as significantly different by sensory analysis. On the whole, the substitution of fat with the beta-glucan gel changed the textural and sensorial quality of burgers, but the modification did not cause a deterioration of the general appreciation of products. In actual fact, 59.32% of panelists expressed a preference for T2 burger, and 40.68% preferred the CTRL burger. This difference was devoid of statistical significance (*p* > 0.05); therefore, the addition of beta-glucan gel did not cause a significant decrease in the sensorial acceptability of the burgers. Both texture and taste, in fact, are known to influence the acceptability of meat products, especially the juiciness and the tenderness [52]. Moreover, as reported by Desmond et al. [53], a low water binding capacity implicates a negative effect on palatability, due to the lack of juiciness and brittle texture which are both generally unacceptable to the consumers.

unacceptable to the consumers.

consumers could distinguish products containing fat replacers, based on differences in color, texture, aroma and taste. Moreover, Afshari et al. [17] highlighted that fat substitution was perceived as significantly different by sensory analysis. On the whole, the substitution of fat with the beta-glucan gel changed the textural and sensorial quality of burgers, but the modification did not cause a deterioration of the general appreciation of products. In actual fact, 59.32% of panelists expressed a preference for T2 burger, and 40.68% preferred the CTRL burger. (data not shown). This difference was devoid of statistical significance (*p* > 0.05); therefore, the addition of beta-glucan gel did not cause a significant decrease in the sensorial acceptability of the burgers. Both texture and taste, in fact, are known to influence the acceptability of meat products, especially the juiciness and the tenderness [52]. Moreover, as reported by Desmond et al. [53], a low water binding capacity implicates a negative

**Figure 1.**  **Figure 1.**

#### **4. Conclusions 4. Conclusions**

The use of an oat-hull-based gel as fat replacer allowed us to obtain a beef burger with a very low lipid content (3.48 g 100 g−1 in the formulation with a total fat substitution) and with a 2.96 g 100 g−1 content of beta-glucans, almost reaching the recommended daily intake per single portion of burger. With a partial substitution, the decrease of lipid content in the raw product was mitigated during the cooking process (34% and 56% of estimated fat loss in T1 and CTRL respectively). This could be related to the fat-retaining effect of beta-glucans added. Compared to CTRL, replacing fat by the oat-hull-based gel caused a significant decrease in hardness and other textural parameters of cooked burgers. Conversely, the differences in color, significant in raw burgers, were smoothed with cooking. The consumer evaluation, carried out according to the duo–trio test, highlighted significant differences between CTRL and T2 burgers in terms of odor, taste, color and texture. The consumers expressed a higher preference for the T2 burger, probably due to its softer texture and greater The use of an oat-hull-based gel as fat replacer allowed us to obtain a beef burger with a very low lipid content (3.48 g 100 g−1 in the formulation with a total fat substitution) and with a 2.96 g 100 g−1 content of beta-glucans, almost reaching the recommended daily intake per single portion of burger. With a partial substitution, the decrease of lipid content in the raw product was mitigated during the cooking process (34% and 56% of estimated fat loss in T1 and CTRL respectively). This could be related to the fat-retaining effect of beta-glucans added. Compared to CTRL, replacing fat by the oat-hull-based gel caused a significant decrease in hardness and other textural parameters of cooked burgers. Conversely, the differences in color, significant in raw burgers, were smoothed with cooking. The consumer evaluation, carried out according to the duo–trio test, highlighted significant differences between CTRL and T2 burgers in terms of odor, taste, color and texture. The consumers expressed a higher preference for the T2 burger, probably due to its softer texture and greater juiciness.

juiciness. These results are a step forward for the improvement of the nutritional characteristics of meat products and indicate that the use of the oat-hull-based ingredient, rich in beta-glucans, as gel is an These results are a step forward for the improvement of the nutritional characteristics of meat products and indicate that the use of the oat-hull-based ingredient, rich in beta-glucans, as gel is an effective strategy for a complete fat substitution.

effective strategy for a complete fat substitution. **Author Contributions:** Conceptualization, C.S.; methodology, C.S. and A.P.; formal analysis, D.D.A. and G.D.; data curation, D.D.A. and G.D.; Supervision, C.S., A.P. and F.C.; writing—original draft, C.S. and D.D.A.; writing—review and editing, C.S., A.P. and F.C. All authors have revised, read and approved the final **Author Contributions:** Conceptualization, C.S.; methodology, C.S. and A.P.; formal analysis, D.D.A. and G.D.; data curation, D.D.A. and G.D.; Supervision, C.S., A.P. and F.C.; writing—original draft, C.S. and D.D.A.; writing—review and editing, C.S., A.P. and F.C. All authors have revised, read and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

manuscript. **Funding:** This research received no external funding.

**Acknowledgments:** The authors would thank Raffaella Nasti and Isabella Centomani for the technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Quality Characteristics of Healthy Dry Fermented Sausages Formulated with a Mixture of Olive and Chia Oil Structured in Oleogel or Emulsion Gel as Animal Fat Replacer**

## **Tatiana Pintado \* and Susana Cofrades**

Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), José Antonio Novais 10, 28040 Madrid, Spain<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +34-91-5492-300

Received: 8 June 2020; Accepted: 23 June 2020; Published: 24 June 2020

**Abstract:** The present work evaluates the suitability of beeswax oleogels and emulsion gel prepared with a healthy lipid mixture (olive and chia oils) as pork fat replacers for the development of a dry fermented meat product (fuet). Because these systems offer various possibilities, this study has compared their effect on the nutritional quality and sensory acceptability of fuets and their behaviour with regard to technological properties and microbiological and oxidative stability during 30 days of chilled storage. This strategy allowed products with an improved fatty acid profile and a 12-fold decrease of the polyunsaturated fatty acids (PUFA) n-6/n-3 ratio, as compared to the control samples. Irrespective of the structuring method used as animal fat replacer, reformulated samples showed a good oxidative status during chilled storage. In general, no differences that depended on the use of oleogel or emulsion gel were observed in the technological properties and microbiological status, so the choice of one or the other would be conditioned by other factors than the characteristics that the product develops. However, further studies are needed to improve the sensory attributes of the reformulated samples.

**Keywords:** oleogel; emulsion gel; dry fermented sausages; healthier lipid content; chia oil; olive oil

#### **1. Introduction**

Fuet is a type of small-caliber non-acid fermented sausage from northeast Spain made with pork meat, pork fat and various seasonings. Traditionally, fermented sausages were considered safe and healthy foods, but nowadays these products have been associated with health hazards owing to the presence of some components such as saturated fats [1]. In this regard, several options have been assayed to improve lipid content in meat products based on the incorporation of vegetable oils, directly added into the meat matrix, stabilized in oil-in-water emulsion, etc. [2]. However, the interest in alternative technologies has been increasing, and therefore efforts have been made to develop healthy solid fats for foods, attaching importance to their ability to help to promote health and wellbeing [3]. Oleogels and emulsion gels are two different solid oil structured systems that offer interesting characteristics for use as animal fat replacers in the development of healthy meat products [4–8]. In oleogels, liquid oil is transformed into a 'gel-like' structure by using an organogelator, while emulsion gels may be generated from a stable liquid-like emulsion by gelling the continuous phase [3]. Regardless of the type of structured oil system, it is desirable to select an oil or a mixture of oils with a healthy fatty acid profile (reduced saturated fats, rich in unsaturated fats and good Σ polyunsaturated fatty acids (PUFA) n-6/ΣPUFA n-3 ratio, etc.), according to recommendations [9,10]. Accordingly, a mixture of olive oil, which is characterized by its high oleic fatty acid [11], and chia

oil, which is the richest known botanical source of n-3 linolenic acid and does not contain any of the antinutritional compounds (total linamarin, linustatin and neolinustatin) or vitamin B6 antagonist factors that are present in other commercially-available sources of n-3 linolenic acid [12], could provide a way of obtaining new solid lipid materials with healthy fatty acid as animal fat replacers.

Some studies have been carried out to improve the fatty acid profile of cooked (frankfurter sausages) or fresh meat products (patties, longanizas, merguez) by using solid lipid material based on emulsion gels or oleogels [6,13–23]. However, there are very few studies of this kind on fermented meat products. For example, for this purpose linseed emulsion gel [24,25] or oleogel [26] was used to replace animal fat in dry fermented sausages. But we have found no studies that compare the use of emulsion gels and oleogels as animal fat replacers to improve the lipid content in meat products of any kind.

Accordingly, taking into account the particularity of this type of dry fermented meat product owing to the reactions that occur during the ripening process, the present study aimed to evaluate the quality of a functional fermented meat product (fuet) as a function of the olive-chia oil mixture structured as an oleogel or emulsion gel, used as animal fat replacer. The behaviour during one month of chilled storage was also evaluated.

#### **2. Materials and Methods**

#### *2.1. Oleogel and Emulsion Gel Preparation*

Two different animal fat replacers based on solid-structuring oil systems were made: an oleogel (OG) and an emulsion gel (EG). OG consisted mostly of oil (90%), while EG had half that oil content (45%). In both, the lipid phase consisted of a mixture of 80% olive oil (Carbonell Virgen Extra, SOS Cuétara, S.A., Madrid, Spain) and 20% chia oil (Primaria Premium Raw Materials, S.L., Valencia, Spain). The olive oil contained 13% saturated fatty acid (SFA), 75% monounsaturated fatty acid (MUFA) and 8% polyunsaturated fatty acid (PUFA), as reported by Delgado-Pando [27]. According to the information provided by the supplier, the chia oil contained 10% SFA, 5% MUFA and 80% PUFA.

Beeswax (Manuel Riesgo, S.A., Madrid, Spain), which was used as an organogelator in the OG formulation, was prepared as previously described by Gomez-Estaca [6]. Briefly, the oil mixture (90%) and beeswax (10%) were heated (65 ◦C) under constant stirring (500 rpm) in a food processor (Vorwerk Thermomix TM 31, Wuppertal, Germany) until complete melting and mixing. The resulting solution was then immediately poured into metal containers under pressure to compact it and prevent air bubbles, and it was stored at 3 ± 1 ◦C after standing for 60 min at room temperature in darkness.

EG was prepared as described by Pintado [7]. Briefly, soy protein isolate (10%) (Manuel Riesgo, S.A., Madrid, Spain) was mixed with water in a Thermomix TM 31 (Wuppertal, Germany) food processor (30 s, approx. 5600 rpm). Then, as a gelling agent, gelatin (3%) (type B, 200–220 bloom) from Manuel Riesgo, S.A. (Madrid, Spain) was added and combined (15 s, approx. 5600 rpm). The final mixture was mixed at approx. 5600 rpm with gradual addition of the appropriate amount (45%) of the oil mixture described previously. Finally, it was placed in metal containers under pressure to compact it and prevent air bubbles, and stored in a chilled room at 3 ± 1 ◦C for 20 h until use.

#### *2.2. Fuet Design and Preparation*

Sufficient fresh post-rigor pork meat (a mixture of biceps femoris, semimembranosus, semitendinosus, gracilis and adductor muscles) and pork backfat were obtained from a local market. Both the pork meat and the backfat were vacuum packed in batches of approximately 1000 and 500 g respectively, to be frozen and stored at −20 ◦C until used (less than one month).

Four different fuet-type dry fermented sausages were formulated (Table 1) in a pilot plant. Two formulations without replacement of pork backfat were prepared as references: one with normal fat content (NF/C) and the other with reduced fat content (RF/C). Additionally, two reduced-fat fuets were formulated, in which pork backfat was partially replaced by oleogel (RF/OG) or emulsion gel (RF/EG). Although the level of fat replacement was the same, different amounts of OG and EG had to be added to obtain a similar lipid content.

**Table 1.** Formulation (g/100 g) of different fuets.

 Reduced-fat fuets reformulated by partially replacing (80%) pork backfat with oleogel (RF/OG) or emulsion gel (RF/EG). All samples contain 5.5% of special commercial seasoning preparation for fuet.

Previously thawed pork meat and pork backfat (~18 h at 2 ± 2 ◦C) and the new lipid materials (OG in RF/OG and EG in RF/EG) were minced to a particle size of 6 mm (Van Dall S.r.l., model FTSIII, Treviglio, Italy). The ingredients for each formulation (Table 1) were placed in a mixer (MAINCA, Barcelona, Spain) and homogenized for 1 min. Half of the water and a commercial seasoning preparation for fuet (COMPLET FUETIB CU-425, Pilarica, Valencia, Spain) were added to the mixture and it was mixed for 1 min. Then the other half of the water and seasoning were added and the result was mixed again for 2 min. The mixture was stuffed (manual stuffer, MAINCA, Barcelona, Spain) into 34/36 mm-diameter natural pork casings (Julio Criado Gómez, S.A., Madrid, Spain), resulting in sausages weighing about 200 g. The sausages were dipped in a meat surface starter suspension of *Penicillium nalgiovense* and *Penicillium candidum* (TEXEL NEO 1 Danisco, DuPont™, Madrid, Spain) prepared according to the manufacturer's instructions. The sausages were placed in a ripening cabinet (BINDER model KBF 240, Tuttlingen, Germany) under the following conditions: 2 days at 19 ◦C and 80–85% relative humidity (RH) and 15 days at 13 ◦C and 75–80% RH. These conditions were set for all the products in order to have no other variables, despite the fact that the water content conditions the ripening process of fermented products [28]. The fuets were packed in plastic bags under aerobic conditions and kept in chilled storage (2 ± 2 ◦C) for 30 days.

Samples from each formulation were taken at 0 (the end of the ripening process and the beginning of storage), 15 and 30 days of chilled storage for analysis.

#### *2.3. Processing Losses*

Losses were calculated by weight difference during the fuet ripening period and expressed as a percentage of the initial weight.

#### *2.4. Chemical Composition and Energy Value of Fuets*

The chemical composition of the fuets was analyzed at the end of the ripening period. Each analysis was performed three times. Moisture and ash content were determined using official methods [29]. A LECO FP-2000 Nitrogen Determinator (Leco Corporation, St Joseph, MI, USA) was used to evaluate protein content and fat level was measured in accordance with Bligh and Dyer [30]. The energy value was calculated on the basis of 9 kcal/g for fat and 4 kcal/g for protein.

The fatty acid content was evaluated in triplicate by saponification and bimethylation according to Lee [31] in samples previously freeze-dried (Lyophilizer Telstar Cryodos Equipment, Tarrasa, Spain). The analysis of fatty acid methyl ester (FAME) was carried out on an Agilent gas chromatograph (Model 7820A, Santa Clara, CA, USA) fitted with a GC-7 Agilent HP-88 capillary column (60 m × 250 µm × 0.2 µm) using a flame ionization detector The temperature of the injector and the detector was 250 and 260 ◦C respectively. On the other hand, the temperature profile of the oven was 125 ◦C, increasing by 8 ◦C/min to 145 ◦C (held for 26 min) and 2 ◦C/min to 220 ◦C (held for 5 min). C13:0 was used as internal patron and for the identification of fatty acids, that was carried out by

comparison of the retention times, it was used the standard 47015-U Supelco PUFA No.2 Animal Source (Sigma-Aldrich Co., St. Louis, MO, USA). Fatty acids were expressed as g of fatty acid/100 g product.

#### *2.5. Technological Properties*

Technological properties were evaluated during the chilled storage of the fuets, at 0, 15 and 30 days.

The pH was determined (in quadruplicate) at room temperature in water in a ratio of 1:10 (w/v) using a 827 Metrohm pH-meter (Metrohm AG, Zofingen, Switzerland).

Water activity (Aw) was measured (in triplicate) at 25 ◦C, after removing the casing, in a LabMaster-aw instrument (model 1119977, Novasina AG, Lachen SZ, Switzerland).

Colour was measured (ten times) in fuet cross-sections using a Konica Minolta CM-3500 D spectrophotometer (Konica Minolta Business Technologies, Tokyo, Japan) set to D65 illuminant/10◦ observer. The CIELAB colour space was used to obtain the colour coordinates L\* (black (0) to white (100)), a\* (green (–) to red (+)), and b\* (blue (–) to yellow (+)).

Texture profile analysis (TPA), as described by Bourne [32], was carried out using a TA-XTplus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) equipped with a 30 kg load cell. Six cores (diameter = 12 mm, height = 20 mm) per sample were axially compressed to 50% of their original height at a crosshead speed of 0.8 mm/s to calculate hardness (N). The tests were performed on the samples at room temperature immediately after refrigeration at 3 ◦C.

#### *2.6. Lipid Oxidation*

The fuets were assessed for oxidative stability by measuring secondary oxidation products, based on changes in concentrations of thiobarbituric acid-reactive substances (TBARs) and the main volatile aldehyde compounds formed by lipid oxidation [33].

TBARs, which were expressed as mg malonaldehyde (MDA)/kg fuet based on a standard curve prepared from 1,1,3,3-tetraethoxypropane in advance, were determined according to Delgado-Pando [34],. Volatile compounds of the fuet samples were extracted by solid phase micro-extraction and determined according to Alejandre [24]. The gas chromatograph (Agilent, model 6890N, Santa Clara, CA, USA) was equipped with a 5973 Mass Selective Detector and it used a DB-WAXetr polyethylene glycol capillary column (60 m × 320 µm × 0.25 µm). For the analysis the oven temperature was set initially at 40 ◦C (4 min hold), increased to 110 ◦C at 4 ◦C/min, to 180 ◦C at 6 ◦C/min, and to 240 ◦C at 8 ◦C/min (15 min hold). Helium was used as a carrier gas at 1.3 mL/min; injector and detector temperatures were held at 250 and 240 ◦C, respectively. Identification of the peaks was based on comparison of their mass spectra with the spectra of a commercial library (Wiley 7th edition and NIST/EPA/NIH 02 mass spectral library) and by comparison of their retention times with those of standard compounds. For semi-quantitative purposes, peak area was measured by integration of the total ion current of the spectra. Results were expressed as area/sample weight (g) × 103 .

Determinations for each sample, volatile compounds, and TBARs were performed in triplicate at day 0 and after 30 days of chilled storage.

#### *2.7. Microbiological Analysis*

Total viable counts (TVC) and lactic acid bacteria (LAB) were evaluated as described Pintado et al. [19]. For results exposure, all microbial counts were converted to logarithms of colony-forming units per gram (Log cfu/g).

#### *2.8. Sensory Analysis*

The sensory analysis was carried out with a panel of 30 assessors selected from the Institute of Food Science, Technology and Nutrition (ICTAN-CSIC) staff. These people were chosen because they are acquainted with meat products and the terminology used for the analysis. For samples preparation, the fuets were cut into 3-mm-thick slices. Two slices per sample were presented to the panellists, who were instructed to rinse their mouth with bread and water between samples. The sensory attributes (general appearance, odour, flavour, texture and overall acceptability) were evaluated on a 10-point scale, 0 being considered as "dislike strongly" and 10 as "like strongly". The panellists were also asked to make any comments that they considered relevant about their sensory perception of the samples.

#### *2.9. Statistical Analysis*

The whole experiment was performed twice. Statistical tests were made employing the SPSS computer program (v24 SPSS Statistical Software, Inc., Chicago, IL, USA). One-way and/or two-way analyses of variance (ANOVA) were performed. Differences between pairs of means were assessed on the basis of confidence intervals using Tukey's Honestly-significant-difference (HSD) test. The level of significance was *p* ≤ 0.05.

#### **3. Results and Discussion**

#### *3.1. Processing Losses*

At the end of the ripening process, the losses that products suffered were calculated to evaluate the yield for the various products as a consequence of the reformulation strategy (Figure 1).  Several authors [28,35,36] have observed higher losses in reduced-fat fermented sausages than in sausages with normal fat. In the present study, the strategy of reducing fat and improving the lipid profile by using oleogel (OG) and emulsion gel (EG) led to products with better binding properties than when only the fat content was reduced (RF/C) (Figure 1). No differences were observed between samples with OG or EG despite the higher quantity of water added directly during the preparation of RF/OG than in the case of RF/EG, in which water was stabilized or entrapped in an emulsion (Table 1).

**Figure 1.** Weight losses and the processing yield of the fuets as a consequence of the ripening process.  Reduced-fat fuets reformulated by partially replacing (80%) pork backfat with oleogel (RF/OG) or emulsion gel (RF/EG). Different letters indicate significant differences by formulation in weight losses and processing yield (*p* < 0.05).

#### *3.2. Chemical Composition and Energy Value*

The fuet composition (Table 2) was mainly influenced by the formulation (Table 1). However, for this type of meat product, the ripening process should be taken into account because during this

period there is a high water loss (Figure 1), which is one of the characteristics that determine the final composition of the product because it results in concentration of the components.

**Table 2.** Chemical compositions and nutritional significance ratios of different fuets after the ripening process.

SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. For sample denominations, see Table 1. Different letters in the same row indicate significant differences (*p* < 0.05) between formulations. Means ± standard deviation.

As expected on the basis of the fuet formulations (Table 1), products with two different fat levels were obtained (Table 2). The two strategies used in this work, the replacement of animal fat by water alone or by the use of structured oils, OG (RF/OG) and EG (RF/EG), led to products with similar (*p* > 0.05) fat content (Table 2). Moisture content increased significantly as a result of the reduction in fat level, as other authors have found in dry fermented sausages [24,37]. Despite the differences in water losses (Figure 1), no significant differences were found in moisture content that depended on the type of fat used as the lipid source (all-animal-fat, OG, or EG). Reduced all-animal-fat fuet (RF/C) had the highest (*p* < 0.05) ash content, probably because it had the highest losses (Figure 1) during processing. The protein levels of the fuets were between 31.17% and 37.74% (Table 2). The use of oleogel and emulsion gel as fat replacers resulted in samples with different (*p* < 0.05) protein contents, probably because of the use of soy protein isolate as emulsifier in the preparation of the emulsion gel.

Both strategies, the pork backfat reduction and the partial pork backfat replacement by oleogel and emulsion gel systems, improved the fatty lipid profile, with decreased SFA and increased PUFA (*p* < 0.05). With regard to SFA, the use of OG and EG as animal fat replacers significantly reduced the myristic, palmitic and stearic acid contents in the fuets by more than half compared to the control (NF/C) (Table 2). The highest (*p* < 0.05) MUFA content was in the control samples (NF/C). However, MUFA represented 54% of total fat in NF/C, whereas in RF/OG and RF/EG MUFA content was approximately 60% of total fat. Oleic acid was the main fatty acid in all samples (Table 2), which is consistent with reports for fatty acid composition in pork fat [38] and in olive oil [27], which was the main oil used in the development of OG and EG. The RF/OG and RF/EG products showed the highest (*p* < 0.05) PUFA content, with a notable increase in α-linolenic fatty acid (ALA) in both samples owing to the presence of chia oil, which is the richest known botanical source of n-3 linolenic acid [12]. Consequently, owing to the technological advantages that chia seed and chia flour offer and their high lipid content (30–35%), both products have also been used (added directly or in emulsion or emulsion gel) to improve the fatty acid profile of various meat products, such as frankfurters, burgers, longanizas, etc. [19,39–41]. The PUFA/SFA ratio is one of the main parameters currently used to assess the nutritional quality of the lipid fraction of foods, and a PUFA/SFA ratio above 0.4 is recommended [38]. The PUFA/SFA ratio in the all-animal-fat samples (N/FC and R/FC) was around 0.2 (Table 2), which is consistent with reports by other authors concerning conventional meat products [20,42], whereas replacement of pork fat by the new healthy lipid materials (OG and EG) increased this ratio (*p* < 0.05) to 0.6 (Table 2), thus complying with the recommendations. The PUFA n-6/n-3 ratio is also of great interest, because diets with high PUFA n-6/n-3 ratios promote the pathogenesis of many diseases (cardiovascular diseases, cancer, etc.), whereas increased n-3 PUFA content exerts a suppressive effect [43]. The nutritional recommendation for this ratio is that it should be lower than 4, and the strategy based on the replacement of animal fat by OG or EG produced a drastic decrease to values close to 1 in the PUFA n-6/n-3 ratio in the RF/OG and RF/EG fuets, complying with the recommendations. Increasing the PUFA/SFA ratio as well as reducing the PUFA n-6 / n-3 ratio to get closer to the reference values, has been tested using new lipid materials such as EG or oleogels elaborated with oils that have a healthy profile of fatty acids (olive, flax, chia, etc.). This strategy, which has been tried on other types of meat products (fermented, cooked or fresh), has given similar results to those obtained in the present study [6,8,17,18,24].

According to the composition specified, the energy value of the normal-fat fuets (NF/C) was approximately 392 kcal/100 g. As a consequence of the reformulation strategies based on lipid content improvement, the energy value decreased to values between 328 kcal/100 g (RF/OG and RF/C samples) and 338 kcal/100 g in fuet with emulsion gel (RF/EG). These changes represent an energy reduction of around 14–16% in the reformulated products. Similar or lower energy reductions have been observed in other reduced-fat fermented sausages [24,37].

#### *3.3. Nutritional and Health Claims*

According to the composition presented in Table 2 and Regulation (European Commission) no 1924/2006 and Regulation (EU) no 432/2012 [44,45], all the fuets could be labelled with the nutritional claim "high protein content" and the corresponding health claims presented in Table 3. On the other hand, the sample with reduced all-animal-fat content (RF/C) showed a fat reduction of more than 30% with respect to the control and could therefore labelled with a "reduced fat content" claim.

**Table 3.** Nutrition and health claims authorised in fuets according to Regulation (EC) No 1924/2006 and Commission Regulation No 432/2012.

Furthermore, the strategy based on partial replacement of animal fat by healthy structured oil systems (OG and EG) allows other nutritional and health claims for these fuets according to European regulations [44,45]. With regard to nutritional claims, the RF/OG and RF/EG fuets could be labelled with "high unsaturated fat" and "high omega-3 fatty acids" claims (Table 3). With regard to health claims, the labelling of these samples could include the claim that "ALA contributes to the maintenance of normal blood cholesterol levels" (Information shall be given to the consumer that the beneficial effect is obtained with a daily intake of 2 g of ALA). Taking into account that it is recommended to limit the consumption of processed meat to 50 g per day [46], this amount of the RF/OG and RF/EG samples covers more than 50% of ALA needs. Accordingly, the presence of chia and olive oil oleogel or emulsion gel in the fuets reflected healthier nutritional properties when compared with the control samples.

#### *3.4. Technological Properties*

In order to know the consequences of the different composition of the fuets as well as the phenomena that occurred during the ripening process, the technological properties were evaluated during the storage period, after ripening, which is when the product is consumed. The water activity (Aw) of the fuets was affected by the formulation (Table 4), with values ranging between 0.87 and 0.90 just after the ripening period (day 0 of storage). The use of OG or EG as fat replacement in the fuets did not significantly condition the initial Aw, but their values were higher (*p* < 0.05) than those observed in the samples with all-animal fat and than those expected for this kind of product [47]. However, Triki [48] observed decreased Aw values in chorizo (a Spanish fermented sausage) fermented sausages. In the present work, what may have happened is that the use of the mixture of structured olive and chia oils in the development of the fuets conditioned the ripening process, requiring a longer time to produce a reduction in water activity levels. In general, chilled storage had hardly any effect on water activity (Table 4). Similar behavior has been observed in fermented sausages during storage [48].

**Table 4.** Technological properties of fuets during chilled storage: pH and water activity (Aw) values, colour parameters (L\* lightness, a\* redness and b\* yellowness) and texture profile analysis (TPA) (Hardness, N).

For sample denominations, see Table 1. Different letters in the same column and different number in the same row indicate significant differences (*p*<0.05) between formulations or chilled storage process. Means±standard deviation.

The sausage formulations and chilled storage conditioned (*p* < 0.05) the pH values of the fuets (Table 4). However, all the pH values were within the normal range reported for similar commercial products [47] or products in which animal fat was replaced by n-3 long-chain PUFA in konjac glucomannan matrix or linseed EG [25,49].  Similar behavior has been described for fuets in which animal fat was replaced by sunflower oil [28] and in higher caliber (50 mm) dry fermented sausages made with linseed oil EG as animal fat replacer [25]. On the other hand, [24] did not observe an effect on pH values as a consequence of fat replacement (26.3%, 32.8% and 39.5%) by linseed oil gelled emulsion in dry fermented sausages. During chilled storage a significant increase in pH values was observed. Similar results have been found in dry fermented sausage produced using different lactobacilli as starter culture [50]. These authors found that the pH started to increase after 28th day of ripening and the increase continued during storage at refrigeration (8◦ C). An increase of pH could be related to the breakdown of lactic acid following the depletion of the added sugar [50].

Table 4 shows the values obtained for lightness (L\*), redness (a\*) and yellowness (b\*) in the control and reformulated fuets. As a result of reducing animal-fat content (comparison between NF/C and RF/C), increases (*p* < 0.05) in lightness and yellowness were observed, while no effect on redness values was found. However, as a result of the replacement of pork backfat by structured chia and olive oil systems (RF/OG and RF/EG samples), in comparison with the control (NF/C), only yellowness increased (*p* < 0.05) (Table 4). This means that the strategy of reducing and replacing animal fat with a mixture of structured olive and chia oils gives rise to products that maintain the characteristic redness of this type of product, unlike what happens when there is only a reduction in fat content (RF/C), which causes greater changes in colour. It is important to note that the healthier fuets (RF/OG and RF/EG samples) were more stable, with smaller changes in colour parameters after 30 days of chilled storage, than the products made with only animal fat (Table 4).

The hardness of the fuets varied as a result of the modifications that were assayed (Table 4). Initially it was noted that there was a significant increase in hardness in the fuets with reduced animal fat, probably owing to greater water losses in the RF/C samples (Figure 1). These results are in agreement with those found by several authors [28,51,52], who reported higher hardness in low-fat dry fermented sausages than in high-fat ones. The type of structured oil system used as the animal fat replacer conditioned the hardness of the fuets. Thus, fuets made with EG as animal-fat replacer (RF/EG) showed similar (*p* > 0.05) hardness to the control (NF/C), whereas those with oleogel (RF/OG) had the lowest (*p* < 0.05) hardness values. Hardness has a negative relation with moisture content in dry fermented meat products, as other authors have observed [51,53]. Accordingly, given that the RF/OG and RF/EG samples had similar moisture values (Table 2) and processing losses, the differences in hardness could be attributed to how the water was added during the preparation of the products, directly to the meat matrix (RF/OG) or stabilized in EG (RF/EG).  Similar behavior was observed by other authors when they used linseed oil EG or OG as an animal fat replacer in dry fermented sausages [25,26]. Conversely, in salchichón (a Spanish fermented sausage) and fuet, the replacement of various animal-fat levels by fish oil encapsulated in konjac gel (salchichón) or by sunflower oil added directly (fuet) resulted in harder samples [26–28]. As expected, during chilled storage all the samples experienced an increase (*p* < 0.05) in hardness (Table 4), probably because all the samples lost water during that period. However, it should be noted that, as with color, the changes in the texture of the OG and EG fuets during chilled storage were smaller than those in the control samples made with animal fat.

#### *3.5. Lipid Oxidation*

Lipid oxidation is the main non-microbial cause of quality deterioration in meat products and one of the most important reactions of fermented meat products that generates volatile compounds [33]. Accordingly, the effect of the partial replacement of pork backfat by structured chia and olive oil

systems (oleogel or emulsion gel) on lipid oxidation, measured as volatile compounds and MDA levels, is shown in Table 5.

**Table 5.** Parameters related to lipid oxidation of fuets during chilled storage: thiobarbituric acid-reactive substances (TBARs) values (mg malonaldehyde (MDA)/kg sample) and volatile compounds (area/sample weight (g) × 103 ).

For sample denominations, see Table 1. Different letters in the same row indicate significant differences by formulation and different number in the same column indicate differences by chilled storage (*p* < 0.05). Means ± standard deviation.

TBARs values were significantly higher in the RF/OG and RF/EG samples, reflecting increased lipid oxidation in the fuets owing to the higher level of unsaturated fat, although their oxidation levels remained well below the rancidity threshold which is usually when the MDA concentration is above 1 mg per kg of sample [33]. Chilled storage did not have a significant effect on TBARs values, probably because of the stability provided by the structured systems in which the oil mixture was located, unlike what occurs when the oil is incorporated directly [55]. Similar results have been found in various meat products with an improved lipid profile based on plant and marine oils stabilized in different ways [48].

Aldehydes are the most abundant volatile compounds produced by lipid oxidation, and hexanal is the aldehyde that has been considered to be the best indicator [33]. As expected, higher (*p* < 0.05) levels of all volatile compounds were observed after the ripening process (day 0) in samples with OG (RF/OG) or EG (RF/EG) used as animal fat replacer (Table 5). These results are in agreement with those obtained in the determination of TBARs and those found by some other authors. Thus, Alejandre [24] and Glisic [25] observed higher levels for aldehydes in dry fermented sausages in which the lipid content was improved by using linseed emulsion gel as an animal fat replacer. On the other hand, although RF/EG showed higher (*p* < 0.05) hexanal levels than RF/OG, non-significant differences were observed in heptanal, octanal and nonanal levels depending on the structured oil system used as healthier lipid material (Table 5). Josquin [56] assayed the replacement of pork backfat with pure, pre-emulsified or encapsulated fish oil in fermented sausages and observed differences in volatile levels, depending on the strategy used to incorporate the oil. The sausages in which encapsulated oil was incorporated had lower volatile compound levels than the others.

After chilled storage, a significant decrease was observed in the volatiles studied, except for nonanal in the samples made with OG or EG, whereas the samples with all-animal fat generally showed values (Table 5) similar to those at the beginning of storage.

#### *3.6. Microbiological Analysis*

Microbiological factors during chilled storage are known to affect the stability and shelf life of meat products. Figure 2 shows changes in total viable count (TVC) and lactic acid bacteria (LAB). All samples presented high initial microbial counts (>8 log cfu/g) of TVC and LAB, which in general were maintained during chilled storage. However, fuet formulated with emulsion gel (RF/EG) experienced a significant increase in TVC and LAB counts after 30 days in refrigeration, reaching levels close to log 9 cfu/g (Figure 2). These results are in accordance with others observed in dry fermented sausages in which various animal-fat levels were replaced [35,48].

**Figure 2.** Microorganism (**a**: total viable count; **b**: lactic acid bacteria) counts (log cfu/g) of fuets during 30 days of chilled storage. For sample denominations see Table 1.

#### *3.7. Sensory Analysis*

The external appearance of the fuets was similar regardless of the formulation strategy used (Figure 3). However, some differences were observed in their cross-sectional appearance, depending on the lipid source that was used. Thus, while the animal fat was perfectly differentiated in the meat matrix, the oleogel or EG in R/OG and R/EG, respectively, could not be seen (Figure 3).

**Figure 3.** Effect of formulation strategies on the external and cross-sectional appearance of the fuets after the ripening process. For sample denominations see Table 1.

The results of the hedonic analysis for the attributes evaluated are shown in Figure 4. In general, for all of them, the samples made with all-animal fat received higher scores than the others. With regard to RF/OG and RF/EG, the panelists evaluated them with similar scores for all attributes. The lower scores that the reformulated samples received could be attributed to the high aldehyde content as compared to the control (Table 5), as other authors have reported for this type of meat product [57]. On the other hand, the differences observed between their appearances (Figure 3) may have conditioned how the panelists evaluated other sensory attributes [58]. Furthermore, after 30 days of storage, when they showed lower aldehyde contents (Table 4), RF/OG and RF/EG received higher scores for flavor or general acceptability. Alejandre et al. [24] did not observe differences in taste and juiciness but found differences in odor between control dry fermented sausages and others made with linseed emulsion gel as animal-fat replacer. However, the sensory attributes could be further improved by slight modifications to the product, including modifications to the conditions associated with the ripening process.

**Figure 4.** Sensory analysis scores for general appearance, odor, flavor, texture and general acceptability of the fuets: **a)** after ripening process; **b)** after 30 days of chilled storage. For sample denominations see Table 1.

#### **4. Conclusions**

The healthy oil mixture based on chia and olive oil, structured into an oleogel or emulsion gel, was proved to be an interesting option for the development of functional dry fermented sausages. These products could be labelled with certain nutritional and health claims according to European legislation, mainly because of the high α-linolenic fatty acid content. The strategy of reducing and replacing animal fat with a mixture of structured olive and chia oils gives rise to products that maintain the color characteristic of this type of product and a good oxidative and microbiological status during chilled storage. Fuets made with EG as animal-fat replacer had similar hardness to the control whereas those with oleogel were softer. Nevertheless, further studies are necessary to improve sensory attributes of the reformulated fuets with this type of lipid material but no great differences resulting from the use of one or the other were observed. Moreover, the strategy based on reduction and improvement of the lipid fraction yielded products that were stable during chilled storage.

**Author Contributions:** T.P. and S.C. contributed equally to this work. Both, designed this study, performed the experiments and collaborated in the statistical analysis and drafted the main manuscript. In addition, Paloma González and María Solano participated in the experimental phase of this study during their academic practices. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Economy and Competitiveness, AGL2014-53207-C2-1-R and PID2019-103872RB-I00, and the Intramural 202070E177 was funded by The Spanish National Research Council (CSIC)

**Acknowledgments:** We are grateful to the Analysis Service Unit facilities of Institute of Food Science, Technology and Nutrition (ICTAN) for the analysis of chromatography and mass spectrometry. Furthermore, the authors wish to thank Paloma González and María Solano for their support during the experimental work.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Clean Label Alternatives in Meat Products**

**Gonzalo Delgado-Pando 1 , Sotirios I. Ekonomou 2 , Alexandros C. Stratakos 2 and Tatiana Pintado 1,\***

**Abstract:** Food authorities have not yet provided a definition for the term "clean label". However, food producers and consumers frequently use this terminology for food products with few and recognisable ingredients. The meat industry faces important challenges in the development of clean-label meat products, as these contain an important number of functional additives. Nitrites are an essential additive that acts as an antimicrobial and antioxidant in several meat products, making it difficult to find a clean-label alternative with all functionalities. Another important additive not complying with the clean-label requirements are phosphates. Phosphates are essential for the correct development of texture and sensory properties in several meat products. In this review, we address the potential clean-label alternatives to the most common additives in meat products, including antimicrobials, antioxidants, texturisers and colours. Some novel technologies applied for the development of clean label meat products are also covered.

**Keywords:** clean label; meat products; nitrites alternatives; phosphates alternatives

#### **1. Introduction**

Over the last few years, food producers have identified the term "clean label" as an important market trend. Nevertheless, what does "clean label" mean? So far there, is no official nor clear definition of the term [1,2]. Asioli et al. [3] proposed two ways the consumers can interpret a product as being clean label. In a broad sense, by looking at the front of pack, consumers might assume a product is clean label if related visual claims appear, such as "free from . . . ", "organic", "no additives", etc. In a strict sense, the authors conclude that, on the back of the pack, consumers associate clean-label products with those that have a short list of ingredients, are non-synthetic, are common for the consumers, etc. Therefore, a definition of clean label should relate to the number and type of additives (synthetic or not) a product has as well as its wholesomeness. An attempt of a definition was released in the official blog of the Institute of Food Technologists: "clean label means making a product using as few ingredients as possible, and making sure those ingredients are items that consumers recognize and think of as wholesome" [4]. We believe that this is a very accurate definition of the term. It relates to all the three important aspects of the clean-label trend: short list of ingredients, trust in the ingredients and perceived healthiness. In line with this, Aschemann-Witzel et al. [5] found that consumers perceived ingredients as belonging to one of these two opposing categories: known-"natural"-good or unknownsynthetic-bad. The former being the one related with the clean-label option. It is important to remark the following finding: there is a correlation for an additive of being perceived as potentially unsafe, unhealthy or of low quality if the name is not common or difficult to pronounce [6,7]. A survey in the USA showed that, depending on the ingredient name, the perceived naturalness differs. When asked about added salt, 65.6% of the respondents considered it natural. However, when they were asked about added sodium chloride, only 32% considered it natural [8]. As with the term "clean label", the term "natural" does not

**Citation:** Delgado-Pando, G.; Ekonomou, S.I.; Stratakos, A.C.; Pintado, T. Clean Label Alternatives in Meat Products. *Foods* **2021**, *10*, 1615. https://doi.org/10.3390/ foods10071615

Academic Editor: Rubén Domínguez

Received: 23 June 2021 Accepted: 9 July 2021 Published: 13 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have a proper definition given by the regulators. Although consumer might understand it as a synonym of non-chemical, good and healthy, this is far from the reality where sodium chloride is the same as common salt or nitrites from synthetic origin are the same as the ones extracted from the Swiss chard. Nonetheless, consumer perception must be taken into account for product success and we do not need to forget that safety plays an essential role for the consumer, along with health, being a top-ten consumer trend in 2021 [9].

The meat industry faces important challenges, and as part of the food industry conglomerate, it needs to address changes towards clean-label options. Meat products, per definition, need to utilise an important amount of additives during their processing, so that the typical technological and organoleptic characteristics are met. In addition, many of the additives also employed during meat processing are essential to preserve the safety and shelf life of the products. Many synthetic-sounding ingredients offer functionalities that are paramount for meat quality. For this reason, nowhere else are these challenges greater than in meat production.

Additives are one of the most researched substances in the world, as they are constantly monitored by food-safety agencies, such as The European Food Safety Authority (EFSA) in the EU and (Food and Drug Administration) FDA in the USA. Within the EU, there is a list of permitted additives and their maximum level of use depending on the type of product [10]. For meat products, the list is long, including antimicrobials, antioxidants and texturisers as the most used ones, but also some other additives (such as colours, stabilisers and acidity regulators) are allowed to be used in some of the European meat products (Table 1). Consumers might perceive these additives as unhealthy or unnecessary due to their chemical-sounding name. However, all the additives used in meat processing are considered safe within the established limits by the food safety authorities.

In this article, we present a thorough review of the clean-label options in the form of ingredients or novel technologies that can offer a real clean-label alternative to the most common additives used in meat processing.

**Table 1.** Additives permitted in the EU for meat product according to Reference [10].

**Table 1.** *Cont.*

#### **2. Clean-Label Ingredients in Meat Products**

#### *2.1. Antimicrobial*

Consumers' demand for safe and high-quality meat and meat products is more dynamic and diversified nowadays than in the past. They want minimally processed, easily prepared, all-natural ready-to-eat (RTE) meat products [11]. To date, the trend in consumers' food demands, clean labelling has rapidly increased, particularly for meat products containing many food additives [12]. Researchers in parallel with producers and manufacturers have been challenged to develop healthy meat products with high quality and safety criteria. The microorganisms associated with the spoilage of meat and meat products are bacteria such as *Pseudomonas*, *Acinetobacter*, *Brochothrix thermosphacta*, *lactobacillus* spp., *Enterobacter*, as well as yeasts and moulds that can affect the organoleptic characteristics of food [13].

The extended use of nitrites led to growing awareness and concern about the healthiness of meat products. Numerous safety issues about nitrite have been raised because it can be converted into N-nitroso with amines in meat products, known as carcinogenic compounds to humans [14,15]. Therefore, several studies counter this challenge and help

produce meat products with low or no-nitrite salts using potential alternatives with similar antimicrobial effects without causing any health hazards [16,17]. Additionally, nitrite play a major role in inhibiting the growth of foodborne pathogens such as *Listeria monocytogenes*, *salmonella* spp., *Campylobacter jejuni*, *Escherichia coli O157:H7*, *Flavobacterium*, *micrococcus* spp. and *clostridium* spp. that can cause important public health problems with million cases of foodborne diseases occurring each year [15,18].

Another additive used as preservative in meat products is sulphites. Sulphites or SO2 are antibacterial agents more powerful against gram-negative bacteria [19]. These additives are considered allergens as certain people have adverse reactions to their consumption, especially those sensitive to asthma, including triggering of anaphylactic reactions, hypotension, abdominal pain, dermatitis, etc. [20]. In addition to be declared as allergen content, sulphites and sulphiting agents are controlled and, in the EU, sulphites and SO2 are the only ones permitted at a maximum dose of 450 mg/kg and only for the following meat products: breakfast sausage, longaniza fresca, butifarra fresca and burger meat when it has 4% of cereal or vegetable.

In the meat processing industry, several traditional thermal and novel non-thermal preservation techniques are being used to increase the products' shelf life and enhance the sensory properties. To achieve this, meat curing is a well-developed processing stage that includes the addition of salt, nitrite and nitrate even on fresh-cut meat imparting several distinctive properties to the meat products [21,22]. The main synthetic nitrites used in the meat industry are sodium nitrite (NaNO2) and potassium nitrite (KNO2) because they are cost-effective, stable, and easy to prepare and use [23]. Before using compounds of natural origin as a replacement for nitrite, their antimicrobial efficacy should be examined, and this review provides a comparison of the published data. Foodborne pathogens can easily contaminate raw meat or meat products, and during prolonged periods of storage, spoilage microorganisms may produce an unwanted visual appearance and diminish their organoleptic properties. Research for additives of natural origin with antimicrobial activities, especially of plant origin, has notably increased in recent years [23]. Numerous natural extracts have been applied to meat and meat products, with herbs and spices being the most used as clean-label alternatives to nitrites and sulphites [24]. Among these, some plant extracts can serve as natural nitrate sources, as nitrate naturally occurs in the environment (plants, soils, water, etc.) [25]. However, nitrites of natural origin do not offer any healthier advantage towards synthetic nitrites, and they only provide a cleanlabel option for the consumer. Table 2 presents some potential antimicrobial alternatives from natural origin for nitrite and sulphites that can be used effectively in clean-label meat products.

**Table 2.** *Cont.*

Removing nitrite from meat products could be problematic because of its high antimicrobial efficacy. Hence, McDonnell et al. [33] evaluated several compounds for their antimicrobial efficacy against *L. monocytogenes* to uncured and alternative cured RTE processed meat and poultry products. The addition of vinegar, lemon and cherry powder blend (1.5%) delayed the growth of *L. monocytogenes* inoculated on the surface of cured ham and deli-style turkey breast. They suggested using the three antimicrobials on uncured roast beef as no growth of *L. monocytogenes* was observed after 12 weeks of storage at 4 ◦C. Moreover, *L. monocytogenes* effectively inhibited and decreased by 4 and 3 Log on RTE bologna type turkey meat coated with Nisaplin and Guardian (antimicrobial gelatin) films, respectively, after 56 days of refrigeration (4 ◦C) storage [34]. The efficacy of chitosan coating as an alternative to chemical protective additives demonstrated by Bostan and Mahan [35] on sausages. All sausages were dipped into 0.25, 0.50 and 1.00% chitosan solutions prepared with 1.00% acetic acid. The authors observed that the shelf life of the products increased and that 0.25% chitosan concentration was enough to inhibit the growth of aerobic bacteria, whereas higher concentrations were needed to inhibit the lactic acid

bacteria (LAB). Soultos et al. [36] observed a positive effect of chitosan (0.50 and 1.00%) against the total viable count, LAB, *pseudomonas* spp., *B. thermosphacta*, *Enterobacteriaceae*, yeasts and moulds on Greek-style fresh pork sausages. Golden et al. [37] evaluated the efficacy of antimicrobial blends containing dried vinegar (DV), together with fruit and spice extracts with salt, against *C. perfringens* in uncured ham compared to traditionally cured ham. They manifested that combining the clean-label antimicrobials used had similar inhibition effects against *C. perfringens* in uncured compared to traditionally cured ham.

Additionally, a broad range of essential oils (EOs) with antimicrobial effects is widely used on meat products to prevent the growth of foodborne pathogens and spoilage microorganisms and extend the shelf life. EOs are secondary metabolites obtained from plants [38], are composed of a complex mixture of volatile compounds of low molecular weight and are characterised by being mainly liquid at room temperature [39]. Oregano oil has been extensively used on meat with positive results against common spoilage microbiota [40–42] and pathogens such as S. Enteritidis [43], S. typhimurium [28,41], *S. aureus* and *L. monocytogenes* [44]. Interestingly, Hernández-Hernández et al. [45] used a novel method to encapsulate Mexican oregano (*Lippia graveolens Kunth*) EO and found that it was efficient to control the naturally occurring microbiota of fresh pork meat during cold storage. Although it is challenging to replace nitrite with a single antimicrobial compound owing to its broad-spectrum activity [46], especially against inactivation of *C. botulinum* spores in cured meat products [21], a combination of nitrite and different antimicrobial agents may be successful. In this way, De Oliveira et al. [47] reported that different levels of winter savoury with 100 ppm of sodium nitrite allowed them to control the growth of *C. perfringens* on mortadella sausages. The authors attributed the antimicrobial activity of the EOs to the presence of carvacrol, ρ-cymene, linalool and thymol. The study by Bellés et al. [48] showed that the use of carvacrol in lamb burgers could be an option as an alternative to sulphites, as it showed a delay on microbial growth. Cui et al. [49] evaluated the antimicrobial efficacy of nutmeg, sage and clove plant extracts in a model meat food. They observed a synergistic effect of the natural extracts with 10 ppm NaNO2 against *C. botulinum*, showing a potential combination in the control of botulism in minimally processed meat. Furthermore, Xi et al. [50] reported that lemon and lime powders and grape seed extract are less effective against *L. monocytogenes*. Still, cranberry powder together with nitrite (150 ppm) reduced the growth of *L. monocytogenes* by 2–4 Log CFU/g in cured cooked meat. Cranberry powder, long recognised as a source of natural antimicrobials, combined with nitrite (150 ppm) and grape seed extract, also offers a potential combination to inhibit *L. monocytogenes* growth in natural and organic processed meats [50]. The antimicrobial activity of the EOs is commonly attributed to the presence of the phenolic compounds [12,44,51] that can disturb the phospholipid bilayer of the cytoplasmic membrane and damage the membrane proteins leading to increased permeability of the cell membrane. However, there are several other mechanisms leading to the inactivation of the target microorganism, such as the disruption of a variety of enzyme systems [52] and destruction of genetic material [53].

The application of EOs is partially limited due to their intense aroma, which may cause adverse organoleptic effects and limited consumer's acceptance. To overcome this problem, novel thermal and non-thermal techniques [53,54] and the use of EOs as part of the hurdle technology together with other compounds and other processing technologies, such as the encapsulation of EOs in nanostructures, are essential to improve the shelf life and the sensory attributes of meat products.

#### *2.2. Antioxidants*

Antioxidants are added to meat and meat products to extend their shelf life through the deactivation of free radicals, and thus slowing down the rancidity. Various factors can promote lipid oxidation in meat products. Based on their mode of action, primary antioxidants prevent lipid peroxidation by preventing a chain reaction, reacting directly with lipid radicals and converting them into relatively stable products; and secondary antioxidants

act by donating a hydrogen atom (H·) and binding to catalysts such as metal ions [55,56]. The list of approved antioxidants is small within the EU but larger for the USA. The only synthetic "pure" antioxidants approved in the EU list are gallates, tert-Butylhydroquinone (TBHQ) and butylated hydroxyanisole (BHA), which are allowed for only one specific meat product: dried meat. Other additives that provide antioxidant capacity but also have other functions are nitrites, ascorbates, erythorbates and citrates. Even though the safety of synthetic antioxidants has been questioned, the safety of antioxidants of natural origin is not much different [57], as the chemical compounds are the same irrespective of their origin. However, consumers relate the word "natural" to "good", as we mentioned before. For this reason, there has been an increase of the research and use of antioxidants of natural origin.

Antioxidants of natural origin have been identified in spices, herbs, fruits or vegetables and applied on meat and meat products primarily for their flavours and aroma. However, several natural extracts have been proven to offer the same functionality as their synthetic alternatives, with the advantage of being label-friendly and process compatible. Phenolic compounds are well known as a major group of natural antioxidants [28,58,59]. A growing list of clean-label natural extracts with antioxidant activity Generally Recognised as Safe (GRAS) by the FDA in the last years (USFDA, 2018) can be used in the meat industry. To name some of the commercially available antioxidants used throughout the meat industry, these are coffee, grape seed, green tea, oregano, sage (Greek and Spanish), lavender, lime, dill, parsley and rosemary extract between them being the most used in the meat industry [60,61]. Conversely, the EU has only approved rosemary extract as antioxidant additives for meat products [10], but the spices can be used as ingredients in the formulation following all the safety controls.

One of the most important natural antioxidants is 3,4-dihydroxyphenylethanol or hydroxytyrosol (HXT), showing interesting antioxidant characteristics and having beneficial effects on health [62]. Martinez-Zamora et al. [63] tested both natural (HXTo) and synthetic (HXTs) antioxidants on lamb meat burgers. Natural HXTo consisted of organic hydroxytyrosol (HXTo, sample 7% purity from olive tree leaves, 200 ppm) showed higher preservative activity in maintaining the nutritional value than the control synthetic HTX (HXTs, 99% purity, 200 ppm) made with sulphites. Rosemary, orange and lemon extracts were investigated in cooked Swedish-style meatballs, with the citrus extracts showing a 50% control of rancidity. The rosemary (water and oil soluble) extracts presented a complete elimination of rancidity after 12 days of storage at 8 ◦C [64]. In the same way, Kim et al. [65] also observed that rosemary extract had high antioxidant properties that could delay the onset of rancidity in meat fats. In this context, to explore for alternatives to synthetic additives, numerous industrial by-products of chestnuts (wood, flowers, leaves, shells, etc.) [66–69] and various fruits [32,70–74] have been used for their antioxidant activity on meat and meat products. The use of industrial by-products agrees with the circular economy concept [67]. It reduces the environmental impact of food processing and waste production while bringing benefits for the meat industry that avoids significant losses by protecting the meat products from oxidation, increasing their quality and shelf life.

As we mentioned earlier, many natural extracts can negatively affect the aroma of meat products. However, there are several plants, such as spinach, radishes and celery, that contain more than 2500 mg nitrate/kg [25,75], and their extracts can be used as natural sources of nitrate in meat products. Celery has been extensively studied and used commercially because it does not affect the sensory attributes of meat products [76]. The addition of celery powder in cooked sausages significantly inhibited the quality deterioration during cold storage for four weeks [77]. Sausages containing celery powder (0.8%) showed comparable pH, thiobarbituric acid reactive substances (TBARS) and volatile basic nitrogen (VBN) values to the control samples containing sodium nitrite (0.01%). These results manifested that celery powder effectively protected sausages from quality deterioration and can be used as nitrite source from natural origin. Similarly, added celery juice powder and starter culture in emulsified sausages presented good quality characteristics without significant differences with the control samples containing sodium nitrite [78]. Nitrate obtained from

plant sources can be used directly in the brine solution or the product together with a starter culture (to form nitrate into nitrite) or as a "cultured", "prefermented" or "pre-converted" nitrate-containing plant source. The meat industry mainly applies the second method because they can control the specific natural pre-converted nitrites they use and their concentrations [76,79].

When evaluating natural antioxidant compounds that may prevent or retard protein and lipid oxidation, it is essential to consider the compound's fat solubility, effective dose, optimum temperature, pH and thermal stability, as well as cost, availability and regulatory status. The meat industry has an excellent opportunity to utilise antioxidants of natural origin in their products, following the consumers' demands for clean-label meat products.

#### *2.3. Texturisers*

Phosphates are the most widely used additive in processed-meat products because of their functional effects. Phosphates possess a certain antimicrobial effect and inhibit lipid oxidation, which condition the colour and the flavour of the products; but the main reason for their use is that they increase the water-holding capacity (WHC) affecting texture and sensory qualities [80]. Based on this, their replacement can lead to several technological limitations; therefore, it is essential to find alternatives that will not compromise the functions phosphates provide. Fibres, seaweeds and vegetable powders are ingredients with similar capacities to phosphates and could offer an opportunity towards clean-label meat products [80]. Phosphates are of concern for people with chronic kidney disease, as their excess in blood is associated with cardiovascular risk [81]. For the healthy individuals, even though phosphates present no concern with respect to genotoxicity or carcinogenicity and their acute oral toxicity is low, the EFSA found that the exposure was higher than the acceptable daily intake for some population groups in their re-evaluation of these additives in 2019 [82]. This is another reason for trying to find alternatives to phosphates in meat products.

In general, strategies based on the reduction or elimination of phosphates have been studied in emulsion-type sausages (Table 3); however, they have been used in others, such as ham, bacon, delicatessen meats, breaded chicken products or injected poultry pieces [80].

Fibres present potential as functional alternatives to phosphate due to their technological advantages (high water- and fat-holding capacity, improved emulsion stability, and texture enhancement) and their positive effect on health [95]. In that sense, several richfibres components (whole seeds, fibre extracts, etc.) have been used to improve the texture and sensory attributes of meat products, mainly in those with reduced fat or reduced salt content [95]. However, in the development of free-phosphates meat products, the use of fibres as replacers is not so widespread.

Chia seed presents several functional advantages but can also affect consumers' health positively due to its high content of soluble dietary fibre [96]. In that sense, chia mucilage (formed after soaking chia seeds in water) has been used in powder and gelled form in two concentrations (2 and 4%) as sodium tripolyphosphate replacer in the development of bologna sausages [87]. New healthier products showed similar yield than controls, with both concentrations of mucilage, and in the two forms (powder and gel). Other alternative could be the use of mushrooms due to their high levels of nutrients (protein, polysaccharides, fibre and vitamins) and several biological benefits. Lyophilized and pulverized winter mushrooms were used in different concentrations (0, 0.5, 1.0, 1.5 and 2.0%) as sodium pyrophosphate (0.3%) replacer in emulsion-type sausages to evaluate their technological properties [89]. Over 1% of mushrooms powder, the exudation of fat from sausages was inhibited and an increase of pH was noted. Moreover, lipid oxidation of sausages was inhibited. However, it was observed that free-phosphates samples were softer [89] (Table 3).

**Table 3.** Ingredients used as phosphates alternatives in the development of clean-label meat products.

Fructo-oligosaccharides (FOSs) are soluble prebiotic fibres that have been used as an alternative clean-label ingredient to phosphates in the production of restructured chicken steaks and cooked hams [83,93]. For phosphates-free restructured steaks' development, inulin was added in gel and powder form (4.5%). In the case of hams, FOSs were employed in different concentrations as substitutes for phosphates and dextrose, using response surface methodology. In general, the behaviour of these healthier products was similar when comparing with samples with phosphates. However, authors indicated the need to tolerate some processing compromises, such as a reduction in yield [83,93]. Other type of fibres used to avoid the use of phosphates was bamboo fibre. Its use in Bologna sausages (2.5 and 5%) resulted in being similar to others cited. Although some technological properties were conditioned with bamboo fibres, sausages maintained emulsion stability and yields [85].

By-products of the food industry that have a high fibre content could be a phosphate replacement that would allow for the industry to obtain healthier meat products while improving sustainability (many of them would otherwise go unutilised) (Table 3). Citrus fibre, a by-product of the fruit-juice industry, has been used in different concentrations (0.50, 0.75 and 1.00%) instead of tripolyphosphate with optimal results for some functional properties, such as adequate emulsion stability and yield [84]. However, authors considered that citrus-fibre levels must be assayed more critically depending on the content and type of protein present in the products. Aside from applying phosphates replacement strategies directly in the reformulation of the product, others have tried it in marinades for chicken products. Plum ingredients, dried plum powder and dried plum fibre (0.06%), and a blend of them (0.06%) were used to replace sodium tripolyphosphate in chicken breast fillets marinade [97]. A hedonic analysis and a 5-point just-about-right (JAR) demonstrated that

the marinade of the blend of plum fibre and powder was not distinguishable from the control. Moreover, no differences were observed in cooking and thawing losses. Mango peel is another by-product that has been evaluated as a phosphate substitute to marinade chicken breast. Samples treated with mango peel showed similar cooking and thawing yield than those with marinate solution containing tripolyphosphate [86].

By-products obtained from the meat industry, such as porcine blood plasma or dehydrated beef proteins, could be used as phosphates alternatives and have been studied added directly to meat products (frankfurters) or through brines (for beef strip loins) [91,92]. The use of both meat industry by-products as phosphate replacers resulted in being positive regarding their yield; however, sensory quality was affected, as it increased animal taste and odour in frankfurters [91] and decreased tenderness in beef steaks [92].

Sea tangle (*Lamina japonica*) is a type of brown algae with water retention and binding ability that has been added to totally replace the sodium pyrophosphate (0.2%) in an emulsion-type sausage. Both 1.5 and 3% of sea tangle offered similar cooking loss to sausages without negative effects on sensory acceptability [88]. Natural calcium powders obtained from eggs and oyster shells were used individually or in combination as phosphate alternatives to formulate pork meat products [94]. It was observed that the combination of oyster (0.2%) and egg (0.3%) shell powder would enable the replacement of synthetic phosphate with desirable qualities in the reformulated products.

Based on some of the ingredients mentioned, commercial alternatives to phosphates have been patented. An example that has been evaluated in marinade chicken-meat products is SavorPhos (Formtech Solutions Inc., College Station, TX, USA), a proprietary blend labelled as citrus flour, all natural flavourings and less than 2% of sodium carbonate [90]. The use of SavorPhos blend as replacer of a commercial phosphate blend, both in water and oil-based marinades, resulted in an optimal option in rotisserie chickens and chicken breasts. Similar yields were obtained with water-based marinades; however, the use of SavorPhos improved the yield with oil-based marinades. Moreover, texture values of breast were improved with the use of SavorPhos and without negatively affecting colour or sensory acceptability [90].

#### *2.4. Colours*

Food colours are used to help improve the appearance of food products that could be affected by exposure to light, moisture, air and temperature variations, as well as to enhance the naturally occurring colours or give colour to otherwise colourless products. This type of additives comes from natural and synthetic origin and according to EU legislation [10] only a few are accepted and most of them limited to some dosage and specific products. From the additives of synthetic origin, only two are permitted for meat products within the EU: Allura Red AG and Ponceau 4R. The former can be applied for luncheon meat, breakfast sausages and burger meat, whereas Ponceau 4R can only be applied in three specific products: chorizo, salchichón and sobrasada. The clean-label alternatives for these colours are the food colours from natural origin, such as cochineal and carminic acids, as well as caramels, carotenes, paprika extracts or beetroot red. However, not all of these colours are permitted in the aforementioned products (Table 1). In addition, some of the food colours might present poor stability to light and time (such as beetroot red or paprika extracts), are not soluble in fat (such as cochineal) or are not soluble in water (such as carotenes) [19]. A problematic with food colours is the consumer perception of their use. Some might have a negative perception as food colours can mask other colours in the food product [98] and also for the relationship of some of them with attention-deficit/hyperactivity disorder in children [99]. Consumers might perceive a meat product as clean label if it has food colours of natural origin in it, but even these food colours can dissuade the consumer if the food colour is not a recognizable ingredient in that product, e.g., caramel in sausages. For this reason, the use of food colours in clean-label meat products should be limited to the few already accepted in the traditional recipes.

#### **3. Novel Technologies for the Development of Clean-Label Meat Products**

Thermal processing in addition to the use of additives, have been the only generally recognized methods for reducing food spoilage. However, the high temperatures used during these processes induce changes in the structure of food and losses of consistency and, in addition, lipid oxidation, which is the main cause of rancidity. These negative effects on the nutritional and sensory properties and the probable health risks have given rise to new technologies called non-thermal processing/mild processing/hurdle techniques [100]. High-pressure processing (HPP), ultrasound and packaging—mainly modified atmospheric packaging (MAP)—are non-thermal techniques that currently are gaining interest in the development of minimally processed food products. However, these techniques also need of an optimisation step to maintain the product quality while also extending or maintaining its shelf life.

High-pressure processing (HPP) is a treatment based on the application of high pressure (100–800 MPa), at mild temperatures (<45 ◦C), that is uniformly distributed through the product by a liquid transmitter. The utilization of HPP allows us to inactivate microorganisms and enzymes for a longer period without the need of chemical additives. Nonetheless, to assure food safety and to extend shelf life, the applied pressure and the temperature must be chosen according to the characteristics of the product [101]. In general, the treatment involves a minimal impact on sensory quality and nutritional value, but the noticeable differences in thermal and aggregative behaviour of proteins can condition the products' colour and texture [102]. In beef patties, the texture and cooking loss increased with higher pressure levels [103], but a contrary effect was observed in beef gels in which HPP treatment improved the yield and texture parameters [104]. Furthermore, Maksimenko et al. [104] observed a decrease in colour values of beef gels under HHP treatment. On the other hand, as a consequence of the aggregation that HPP caused on proteins, the digestion of the meat can be improved [105]. However, highpressure treatment may also induce lipid oxidation depending on the processing time and the pressure level applied [101]. However, this negative effect could be solved by using antioxidants of natural origin, thus maintaining the condition of clean label. For example, the use of sage powder on beef burgers pressurized at 600 MPa retarded the lipid oxidation of products over 60 days of chilled storage [106].

The introduction of the ultrasonic treatment promotes the production of pro-health, minimally processed food, which is currently very popular among consumers. Power ultrasound is a non-thermal processing technology that uses sound energy at frequencies higher than human audible range (>20 kHz) and lower than microwave frequencies (10 MHz) with many applications on muscle products, included meat tenderization, acceleration of maturation and mass transfer, and shelf-life extension [107]. Moreover, is a treatment characterized with a low impact on the organoleptic properties and the nutritional value of meat products. The use of ultrasound reduces microbial contamination due to its capacity to cause damage on biological cells, especially microbial cell membranes [108]. In addition, the use of ultrasound may allow us to reduce the use of additives, such as phosphates, due to its ability to improve the emulsification and gelling properties of proteins [109,110]. The characteristics of this technology make it attractive to reduce or even eliminate the use of additives and obtain clean-label meat products [108,111].

In addition, PEF (Pulsating Electric Field) or Pulse Light are non-thermal technologies that are receiving increased attention. Both technologies, in comparison with conventional thermal sterilization make it possible to achieve effective inactivation of microorganisms in a much shorter processing time and using less energy [108]. Moreover, the impact on nutritional and sensory characteristics of the final products is, in general, minimal.

Food packaging is an indispensable element that serves as the protection from contamination, external environment and mechanical damage. Currently, a new generation of packaging is emerging with several functions that, among others, extend the shelf life of meat products. For example, it has been observed that the combination of vacuumpackaging technology and shrinking largely extends shelf-life in comparison with tradi-

tional packaging [108]. In addition, this packaging is growing as an eco-friendly technology due to the use of biodegradable films. The new packaging materials are developed by considering not only the sustainability of their materials but also to extend shelf life, in a healthier and convenient way. The packaging that not only acts as a barrier from the outside environment but also has some active functions towards improving the shelf life is called active packaging. There are four classes of active packaging depending on the function: scavenging or absorbing, emitting, creating barriers and regulating [112]. The first class comprises mainly gas or liquid absorbers and is barely used in meat products; however, in fresh meat, they are more popular (e.g., sachets that absorb losses from fresh meat). Within the active packaging, emitting antioxidants and creating antimicrobial barriers are the most popular functions for meat products in order to prevent oxidation and microbial spoilage, and thus improving shelf life. The use of edible coatings with antioxidants and/or bioactive compounds (as the ones mentioned in Section 2.1) are being tested in different meat products. Zhao et al. [113] found that chitosan and carvacrol starch packaging films delayed microbial spoilage by up to 25 days in ham. A novel edible film made up of calcium alginate was developed by Noor et al. [114] that included *Asparagus racemosus* as bioactive ingredient. The use of this film prevented the lipid oxidation and improved the storage quality of a model meat product. A recent and thorough review of edible coatings as active packaging in meat products can be found in [115]. Consumers might perceive some risks associated with this new active packaging (technology acceptance, toxicity of new materials, economic risk, malfunction, etc.) and, thus, reject it. Although most of the attitudes towards active packaging are neutral to mildly positive, there is low familiarity with it, and if educational communication is not provided of the information of its value (i.e., extending shelf life), consumers might reject this technology [116].

#### **4. Conclusions and Future Trends**

The use of some additive is so extended in the manufacturing of meat products that the meat industry did not worry about finding alternatives until very recently. Consumers are demanding safe, nutritious and healthier meat products and have put the focus on the additives they contain. A clean-label meat product should only contain the ingredients from the traditional recipes easily recognised by the consumers. Some additives, such as texturisers or colours, are being replaced with alternative options. However, avoiding the use of some additives can create situations where food safety is at risk. Some alternatives rely on the origin of the additive: natural vs. synthetic (e.g., nitrites from green vegetables vs. synthetic nitrites), as natural is perceived as a good trait for most of the consumers. This would be enough for the industry, as products with "natural" alternatives will be perceived as being clean label. Nonetheless, the health problems associated with some additives do not distinguish if the substance is extracted from the nature or synthesised in a laboratory, the chemical component remains the same. We believe that future research should focus on the application of synergistic alternatives, such as a combination of novel technologies and the use of preservatives with no health implications. There is a surge in different antioxidants and antimicrobials from natural sources, but these would need to be thoroughly evaluated before being utilised as alternatives just for being "natural". Innovations in the packaging industry are yet to be widely applied in the meat industry. Once they are fully developed, they will make an important impact on the products' shelf life in a sustainable manner. The meat industry and meat scientists should explore further the clean-label alternatives to develop safer, nutritious and healthier meat products.

**Author Contributions:** Conceptualization, G.D.-P. and T.P.; investigation, G.D.-P., S.I.E., A.C.S. and T.P.; writing—original draft preparation, G.D.-P., S.I.E., A.C.S. and T.P.; writing—review and editing, G.D.-P., S.I.E., A.C.S. and T.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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