1. Introduction
In developed countries, and even in developing countries, there is a rising concern on the part of health authorities on how diet can cause numerous diseases [
1]. Therefore, several epidemiological studies have exposed that the consumption of diets with high quantities of fat (>40% of energy from fat) and with a high content in saturated fatty acids induces many health-related disorders [
2]. Thus, one of the most effective behaviors to reduce the risk to develop several diseases is restraining the consumption of saturated fats. In this sense, meat and meat products are one of the principal dietary sources of saturated fats. These fats, which comprised between 30 and 50% of the product, are rich in saturated fatty acids and cholesterol, and they are considered a promoting factor in the development of several diseases including coronary heart disease, metabolic syndrome, obesity and overweight, inflammation, oxidative stress, etc. [
2,
3].
As a result of these undesirable health effects of excessive fat consumption, the meat industry has had to adapt to these consumers’ requirements developing low-fat meat products with healthier lipid profiles. To achieve this objective, besides the reduction of fat content, numerous strategies have been tried including (i) the direct addition of vegetable oils with healthier lipid profiles [
4,
5], (ii) the incorporation of vegetable oils with healthier profile encapsulated in several matrices [
6,
7], (iii) the use of oleogels [
8,
9], and (iv) the use of gelled emulsions [
10,
11]. A gelled emulsion is a colloidal material in which oil-in-water emulsion (O/W) coexists within a gel network. Its formation consists of two stages; in the first, the O/W emulsion is elaborated, and in the second stage, the gelled emulsion is properly obtained with the formation in the aqueous phase of a drop structure of the emulsion inside of the cross-linked structure of biopolymers [
12]. To elaborate these O/W emulsions, several vegetable or marine oils, as well as mixes of them with a healthier fatty acid profile have been utilized, including chia oil, linseed oil, tiger nut oil, and algal oil, among others [
10,
11,
12,
13,
14]. Nevertheless, it should be borne in mind that reducing or replacing the fat content in a meat product is not an easy task. Animal fat is a basic ingredient in the processing of meat products due to its technological (to improve emulsion stability, the impact on rheological and structural capacities, and the adjustment of the drying process in dry-cured meat products, among others) and sensory properties (positive effects on hardness, juiciness, color, tenderness, palatability, and so on) [
15,
16]. Additionally, the addition of vegetables or marine oils with a healthier fatty acid profile may cause an acceleration of lipid oxidation reactions, which can lead to a decrease in the product shelf life as well as a deterioration of their sensorial and nutritional properties [
17].
As mentioned above, several vegetable oils can be used to elaborate gelled emulsion. Chia (
Salvia hispanica L.) oil is a significant oilseed due to its nutritional composition, consisting of up to 65% α-linolenic acid and 20% linoleic acid in the unsaturated fatty acid fraction [
18]. Since 2014, it can be marketed in the European Union. On the contrary, hemp (
Cannabis sativa L.) oil is not widespread on the market, although it is also characterized by an interesting fatty acid composition with a high content of polyunsaturated fatty acids. Thus, in this composition, it is possible to find a high content, up to 75%, of polyunsaturated fatty acids and the unique ratio of 3:1 between omega-6 and omega-3. Hemp oil highly contains linoleic acid and α-linolenic acid in the range of 50–60% and 20–25%, respectively [
19]. In addition, there are high amounts of chlorophyll in the oil due to the harvesting of high amounts of immature seeds [
20].
With the objective to stabilize the O/W emulsion formed, several ingredients (mainly starchy ingredients) have been used. Pseudocereal flours (from quinoa, amaranth, buckwheat, teff, etc.) seem to be excellent candidates for this application [
21,
22]. They contain high-quality proteins, abundant amounts of starch with unique characteristics, large quantities of micronutrients such as minerals, vitamins, and bioactive compounds, and they are gluten-free, which makes them suitable for people suffering from various gluten intolerances. Their main component, starch, has many interesting features such as very small granules ready to form cross-link structures, which made them useful for stabilizing emulsions [
23]. For these reasons, interest in pseudocereals has increased immensely since the turn of the century, and research efforts have been intensified to include them in our diet. Therefore, the objective of this work was (i) to develop gelled emulsions using pseudocereal flours (amaranth, buckwheat, teff, and quinoa) and vegetable oils (chia oil, hemp oil, and their combination at 50%), (ii) to determine their chemical composition, physico-chemical properties, and lipid stability, and (iii) to evaluate their stability during frozen storage.
2. Materials and Methods
2.1. Plant Material
Chia oil (CH) was obtained from Herbolarios Navarro, (Alicante, Spain), while hemp oil (H) was purchased from Laboratorios Almond, S.L. (Librilla, Spain). Amaranth flour (A) was obtained from Tentorium Energy S.L. (Tarragona, Spain); buckwheat flour (BW) and white quinoa flour (WQ) were purchased from Biogran S.L. (Madrid, Spain), and whole teff flour (T) was obtained from El granero integral, S.L. (Madrid, Spain). The gelling agent was gellan gum (an extracellular polysaccharide excreted by microorganism Pseudomonas elodea). It is a water-soluble linear structure with a repeating unit of tetrasaccharide) and instant gel (gelatin of animal origin (pork) with 180 bloom), which was obtained from Sosa Ingredients S.L. (Barcelona, Spain).
2.2. Lipid Profile of Vegetable Oils
The identification of fatty acids was carried out according to the method 969.33 [
24]. For that, fatty acids of all samples were transmethylated producing fatty acid methyl esters (FAME). The FAMEs were analyzed on HP 6890 chromatography equipment with a flame ionizer detector and a Suprewax-280 capillary column (30 m, 0.25 μm of film, 0.25 mm internal diameter; Tecknokroma Barcelona, Spain). The injector and detector temperatures were 250 and 270 °C respectively. The temperature program was as follows: the initial temperature was 60 °C, and this was maintained for 1 min after the injection; subsequently, it was raised at a rate of 10 °C/min until reaching 170 °C and was kept at this temperature for 2 min. After these 2 min, it was raised at a speed of 3 °C/min until reaching 230 °C, and it was kept at this temperature for 10 min, and finally, it was raised at a speed of 2 °C/min until reaching 260 °C and maintained for 1 min at this temperature. The carrier gas was helium with an internal column pressure of 11 psi. The injector volume was 0.2 μL in splitless. The response factors were calculated using fatty acid standards, and their identification was made by comparison with the retention times of these FAME standards (Supelco 37 component FAME Mix, Bellefonte, PA, USA). With the data obtained from the chromatograms, the following parameters were calculated: total saturated fatty acids (SFAs), total unsaturated fatty acids (UFAs), total monounsaturated fatty acids (MUFAs), total polyunsaturated fatty acids (PUFAs), the ratio between saturated and unsaturated fatty acids (SFAs/UFAs), and the ratio between omega-3 and omega-6 fatty acids (ω-3/ω-6). All analyses were carried out in triplicate (three independent batches), and the results were expressed as g fatty acid/100 g oil.
2.3. Gelled Emulsions Preparation
Gelled emulsion (GE) preparation essentially involves producing a protein-stabilized emulsion using emulsifying agents and incorporating a gelling agent such as a hydrocolloid or other ingredients with the gelling capacity to convert the emulsion into a GE. Twelve different types of oil-in-water (O/W) GE samples were formulated, as shown in
Table 1. Eight GE samples were made combining each of the flours with each of the oils: amaranth flour with chia oil or hemp oil (ACH and AH, respectively); buckwheat flour with chia oil or hemp oil (BWCH and BWH, respectively); whole teff flour with chia oil or hemp oil (TCH and TH, respectively) and finally, white quinoa flour with chia oil or hemp oil (WQCH and WQH, respectively). For the other four GE, a blend of chia oil and hemp oil (50:50
v/
v) was made, and each flour was combined with this oil blend. The other four GEs elaborated were amaranth flour with chia and hemp oils blend (AM); buckwheat flour with chia and hemp oils blend (BWM); whole teff flour and with chia and hemp oils blend (TM), and white quinoa flour with chia and hemp oil blends (WQM).
The O/W GE samples were prepared as follows. For each type of GE, first the gelling agent “instant gel” was mixed in a homogenizer (Thermomix 31, Vorwerk-España M.S.L., S.C., Spain) with water for 2 min at 60 °C at high speed. Then, the flour was added and mixed for 1 min at medium speed. In the next step, the temperature was turned down to 37 °C and gellan gum was added and mixed for 2.5 min at 250 rpm. In the last step, the mixture was mixed with the gradual addition of the appropriate amount of oils or their blends for 5 min, at 37 °C and 1100 rpm. The elaborated GEs were placed in metal containers and stored at 4 °C for 20 h until use. The whole process was replicated three times (three independent batches).
2.4. Gelled Emulsion Analysis
2.4.1. Proximate Composition
Protein, fat, ash, and moisture content were determined on GE samples using the appropriate methodology from the Association of Official Analytical Chemist [
24]. Protein content was determined by the Kjeldahl method with a factor of nitrogen of 6.25. The Soxhlet method was used for fat content determination, with petroleum ether as the extractant. Ash content was determined by incinerating the samples at 525 °C, while moisture was determined by heating the samples in an oven until constant weight.
2.4.2. Physicochemical Properties
The pH of GE samples was measured using a Crison combination electrode connected to a pH-meter Crison model 510, (Barcelona, Spain). These measures were made directly into the emulsion.
The texture of each sample was evaluated using a TA-XT2i texturometer (Stable Micro Systems, Surrey, England). The “Measure Force in Compression” Test was selected, and the accessory TTC spreadability rig (HDP/SR, Stable Micro Systems) was used. It is composed of a 90° male cone probe and five cone-shaped product holders that were precisely matched females. Both cones were 25 mm apart, and the sample was placed into the female cone and pressed down to eliminate air pockets. Any excess sample was scraped off with a knife to leave a flat test area. GE samples were stabilized at 5 °C for 30 min before testing and were forced to flow out at 45° with a test speed of 3 mm/s. During compression, the force increases up until the point of maximum penetration depth. This force value was taken as the “firmness (N)” at this specified depth. The “work of shear (N.s)” represents the total amount of force required to perform the shearing process [
25,
26].
2.5. Stability of Gelled Emulsion during Frozen Storage
Since GE samples should be kept frozen until their application to avoid quick oxidation (high unsaturated fat content), it has been decided to assess the influence of freezing time on several properties related to their stability (resistance of emulsion characteristics to changes over time) such as emulsion stability (retention of fluids in the system at maximum levels), color, and lipid oxidation. For that, each emulsion was placed into Petri dishes that were covered, sealed with parafilm, and frozen at −23 °C in an air freezer W7 8210 0X (Whirlpool, MI, USA) for 15 days. After that, samples were thawed in refrigeration conditions (1 h), and color parameters, emulsion stability, and lipid oxidation were assessed as described below.
2.5.1. Emulsion Stability
The emulsion stability was determined following the procedure from [
27] with slight modifications. Samples were introduced into centrifuge tubes of 15 mL and centrifuged at 3000 rpm for 1 min. Then, they were heated in a water bath for 30 min at 70 °C and cooled at room temperature; after that, they were centrifuged again at 3000 rpm for 3 min. The samples were left standing upside down to release the separated fat and water onto filter paper. The results are expressed in g of total fluid expelled/100 g of sample and were calculated using the following expression:
2.5.2. Instrumental Color Analysis
The instrumental color parameters of GE samples were measured in the CIEL*a*b* color space using a Minolta CM-700 (Minolta Camera Co., Osaka, Japan), with illuminant D65, SCI mode, and an observer angle of 10°. Low reflectance glass (Minolta CR-A51/1829-752) was placed between the samples and the equipment. The CIEL*a*b* coordinates determined were
L* (lightness),
a* (red/green), and
b* (yellow/blue). The magnitudes
h°* (hue) and
C* (chrome) were calculated with Equations (2) and (3), respectively.
2.5.3. Oxidative Stability
The oxidative stability of emulsions was evaluated by measuring changes in thiobarbituric acid reactive substances (TBARs). TBARs determination for each sample was performed in triplicate by the method described by Rosmini et al. [
28]. TBARs values were calculated from a malonaldehyde (MA) standard curve and were expressed as mg MA/kg sample.
2.6. Statistical Assay
The whole process was replicated three times (three independent batches). Each replication was performed on a different production day, and each batch was analyzed in triplicate. Means and standard deviations of data obtained from the analysis of GE samples are shown in corresponding tables. A one-way ANOVA test and the Tukey-b post hoc test were used to determine significant differences in both the different types of GE samples and the different times of frozen storage. SPSS version 24.0 was used (SPSS Inc., Chicago, IL, USA) for the evaluations at a significance level of p < 0.05.
4. Conclusions
The use of pseudocereal flours (amaranth, buckwheat, teff, and white quinoa) and vegetable oils (hemp oil, chia oil, and a blend of both) results in a technologically viable option to elaborate gelled emulsions with a healthier lipid profile (>70% PUFA). The only combination that is not suitable for further application is the use of teff flour with the blend of both oils. Several combinations of all these ingredients allow the elaboration of GE with different firmness that will be useful for their application in different types of foods. If the oxidative stability of the GE is taken as a quality criterion, AM (amaranth flour + blend oils) and WQCH (white quinoa flour + chia oil) samples are the most suitable for the substitution of fat in the development of new foods low in fat or with a healthier lipid profile. On the other hand, TH (teff flour + hemp oil), and WQH (white quinoa flour + hemp oil) show better behavior (emulsion stability) under the frozen and thawing process, which made them suitable for frozen foods. In any case, more studies are needed to improve the stability of the emulsion. Possible alternatives to improve this stability could be to (i) increase the concentration of the emulsifying agent (pseudocereal flours) and reduce the water content, (ii) increase the concentration of the gelling agents and reduce the water content, or (iii) increase the concentration of the emulsifying agent and the gelling agents and reduce the water content. To sum up, the use of these gelled emulsions in foods development brings a new strategy to produce healthy foods.