**Development and Characterization of Functional O/W Emulsions with Chia Seed (***Salvia hispanica* **L.) by-Products †**

#### **Luciana M. Julio, Vanesa Y. Ixtaina and Mabel C. Tomás \***

Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CONICET, CICPBA, Universidad Nacional de La Plata (UNLP), 47 y 116, 1900 La Plata, Argentina; luci\_julio86@hotmail.com (L.M.J.); vanesaix@hotmail.com (V.Y.I.)


Published: 1 September 2020

**Abstract:** Physicochemical properties of O/W emulsions containing functional ingredients (high Ν-3 fatty acid content, protein, and soluble fiber) from chia seeds with different protein– carbohydrate combinations (sodium caseinate-lactose, sodium caseinate-maltodextrin, and chia protein-rich fraction-maltodextrin) and chia mucilage were studied. Sodium caseinate with lactose or maltodextrin produced O/W emulsions with small droplet size, high uniformity in droplet size distribution, negatively charged droplets (pH 6.5), pseudoplastic behavior, and high physical stability. Emulsions with chia protein-rich fraction presented wider droplet size distribution and higher D3.2 values than the previous ones, recording a Newtonian behavior. The addition of chia mucilage affected the rheological characteristics of emulsions.

**Keywords:** chia by-products; chia mucilage; O/W emulsions; Ν-3 fatty acids; chia protein-rich fractions

#### **1. Introduction**

The demand for functional foods with multiple health benefits has increased in recent years due to the new trend towards a healthy lifestyle. Functional foods are designed to supply basic nutrients as well as to reduce the risk of some diseases. Advances in food technology resulting in new components, products, processes, and packaging have provided more opportunities for value-added products.

Nowadays, interest in the substitution of synthetic emulsifiers and stabilizers by others of natural origin, such as vegetal polysaccharides and proteins, has grown. The industry generates residual cake with a high fiber and protein content after the extraction of chia oil from the seeds. These by-products are mainly used for animal feed with limited economic and social impacts. Thus, an alternative to adding value to these by-products would be the application of technologies to develop functional food that including them.

A chia protein-rich fraction containing 64.9% of globulins, 20.2% of glutelins, 10.9% of albumins, and 4.0% of prolamins was studied by Sandoval-Oliveros and Paredes-López [1]. This protein-rich fraction presented high contents of glutamic acid, arginine, and aspartic acid, which are important for the proper functioning of the immune system and the prevention of cardiovascular diseases. Besides, chia seed contains between 5 and 6% of mucilage, a tetrapolysaccharide of high molecular weight mainly composed of D-xylose, D-mannose, D-arabinose, D-glucose, and galacturonic and glucuronic acids [2,3]. The intake of chia mucilage as dietary fiber source was associated with numerous health benefits, including the reduction of the risk of coronary heart disease, diabetes, obesity, and different types of cancer [4].

This work deals with the development and characterization of oil-in-water (O/W) emulsions with chia oil, evaluating the influence of different combinations of proteins–carbohydrates and the application of chia by-products (mucilage, protein-rich fraction) on the physicochemical properties of these systems.

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

#### *2.1. Material*

Chia oil (C16:0 9.27%; C18:0 3.41%; C18:1 9.37%; C18:2 17.58%; C18:3 59.02%; C20:0 1.36%) was provided by SDA S.A. (Argentina). Casein sodium from bovine milk was purchased from Sigma Chemical Company (St. Louis, MO), the Maltodextrin DE 13–17% was obtained from Productos de Maíz S.A. (Argentina) and the D-lactose monohydrate from Anedra (Argentina). All reagents used were of analytical grade.

Chia protein-rich fraction with 43.0 of protein, 0.7 of fat, 8.4 of moisture, 14.1 of fiber, 8.4 of ash, and 25.4% of nitrogen-free extract was obtained by a dry processing of defatted chia flour according to Vázquez-Ovando et al. [5]. Chia mucilage was obtained from whole chia seeds according to Segura-Campos et al. [6] method with some modifications. The proximal composition of chia mucilage was 10.7, 8.9, 9.1, 3.9, 13.6, and 53.8% of moisture, ash, protein, fat, fiber, and nitrogen-free extract, respectively.

#### *2.2. Methods*

#### 2.2.1. Preparation of Emulsions

Oil-in-water (O/W) emulsions were prepared mixing 10% (*wt*/*wt*) of chia oil and 90% (*wt*/*wt*) of aqueous phase with different compositions (Table 1) using a rotor–stator system Ultraturrax T-25 (Janke and Kunkel GmbH, Staufen, Germany) at 9500 rpm, 1 min. Then, in a second homogenization stage, the samples were passed four times through a high-pressure homogenizer (Panda 2 K, GEA NiroSoavi, Parma, Italy) at 600 bar. Nisine 0.0012% (*wt*/*wt*) and potassium sorbate 0.1% (*wt*/*wt*) were added to the emulsions to prevent microbial growth. Emulsions were stored at 4 ± 1 °C and protected from light for 15 days.

#### 2.2.2. Droplet Size

The droplet size distribution and the De Sauter (D3.2) mean diameter were obtained using a laser diffraction Malvern Mastersizer 2000E particle size analyzer (Malvern Mastersizer 2000E, Malvern Instruments Ltd., Worcestershire, UK) in a range of 0.1–1000 μm.

#### 2.2.3. Ί-potential

The *Ί-*potential was determined using a Zeta Potential Analyzer (Brookhaven 90Plus/Bi-MAS, USA) instrument at room temperature according to Julio et al. [7]. The *Ί-*potential range was set from ƺ100 to 50 mV. For each determination, 50 mg of emulsion was dispersed in 100 mL of milli-Q water.


**Table 1.** Composition of chia oil-in-water (O/W) emulsions.

Chia protein-rich fraction (P), sodium caseinate (C), lactose (L), maltodextrin (M), and chia mucilage (Mg).

#### 2.2.4. Rheological Properties

Rheological measurements were performed using a Haake RS600 controlled stress oscillatory rheometer (Haake, Germany) with a coarse plate–plate sensor system at 25 ± 1 °C. The samples were subjected to a logarithmic increasing of shear rate from 1 to 500 sƺ1 in 2 min, followed by a steady shear at 500 sƺ1 for 1 min, and finally a decreasing shear rate from 500 to 1 sƺ1 in 2 min [8].

#### 2.2.5. Emulsion Stability

Physical stability of emulsions was determined by measurements of dispersed light using a Vertical Scan Analyzer Quick Scan (Coulter Corp., Miami, FL, USA) according to Pan et al. [9]. The emulsions were transferred to cylindrical glass tubes and periodically measured during 15 days.

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

The droplet size distribution (DSD) of chia O/W emulsions with sodium caseinate presented DSD curves with a mono (CL) or bimodal (CM) shape. On the other hand, emulsions prepared with chia protein-rich fraction exhibited wider and trimodal DSD with a shift towards larger droplet sizes. Additionally, systems with chia mucilage addition presented similar DSD curves shape but shifted to lower particle sizes, which was especially noticeable for emulsions with the protein-rich fraction.

The protein–carbohydrate combination had a significant effect (*p* ǂ 0.05) on the D3.2 diameter of emulsions droplets. At the initial time, emulsions with sodium caseinate presented droplet sizes between 0.22 and 0.27 μm and Span values from 1.07 to 1.46, exhibiting a high degree of uniformity. Emulsions containing the protein-rich fraction presented droplet sizes of ~9.86 μm and Span values from 2.23 to 2.65 due to the presence of bigger particle populations. The larger drop size of the emulsions of chia protein-rich fractions could be due to their lower level of protein available under the conditions of the chemical environment. Thus, when the emulsifying agent is not enough to fully stabilize the droplet interface, larger particles may be formed during homogenization [10,11]. Additionally, since sodium caseinate (protein with structural flexibility) is more effective to reduce the interfacial tension at the interface than chia proteins, which are mainly constituted by globular proteins [1], it is expected that it plays a major role as an emulsifier. Besides, a smaller (*p* ǂ 0.05) droplet size for PM + Mg emulsions (7.44 μm) in comparison with PM systems (9.86 μm) was observed. This fact could be due to the increase of viscosity in systems with mucilage, which reduces the movement of the oil droplets, their collision, and coalescence.

The surface droplet charge at pH 6.5 was negative for all O/W emulsions, probably due to the ionized groups of the proteins at pH above the isoelectric point (pI) (Figure 1). The electric charge of the emulsion droplets stabilized with sodium caseinate was ƺ35 and ƺ31 mV for CL and CM, respectively, while those coated by the chia protein-rich fraction resulted in ~ƺ23 mV. The more negative charge in droplets of protein-rich fraction systems could be due to the presence of anionic functional groups present in the chia protein structure, mainly related to glutamic and aspartic acids. Besides, the net charge of oil droplets became less (*p* ǂ 0.05) negative when chia mucilage was added into the emulsions, possibly related to the charge suppression caused by electrostatic associations between polypeptide chains and charged groups of the chia mucilage.

**Figure 1.** Ί*-*potential values of chia O/W emulsions at pH 6.5.

Regarding rheological properties, emulsions were affected by the proteins–carbohydrates combination used and the addition of chia mucilage. The experimental data, corresponding to rheological measurements, was fitted to the power-law model, and *n* (flow behavior index) and K (consistency coefficient) parameters were calculated. Differences in the flow behavior of the different O/W emulsions were evidenced (Figure 2). Systems with sodium caseinate recorded values of *n* < 1, exhibiting pseudo-plasticity on different levels. In this sense, emulsions with maltodextrin had greater pseudoplastic behavior than lactose ones. On the other hand, emulsions with the chia protein-rich fraction presented a Newtonian behavior (*n* ~ 1) (Figure 2).

**Figure 2.** Flow curves of chia O/W emulsions. Average values (*n* = 3).

The apparent viscosity of emulsions at 100 sƺ1 (100), typical of food processes such as flow through pipes, agitation, and chewing [12], was also calculated. In this sense, systems with the chia protein-rich fraction had lower 100 values (*p* ǂ 0.05) than emulsions stabilized with sodium caseinate. This fact could be related to a significant amount of nonadsorbed sodium caseinate in the continuous phase, which would lead to the formation of aggregates and a transient network structure with an enhancement in the viscosity [7]. Furthermore, there was an increase (*p* ǂ 0.05) in the viscosity of emulsions with sodium caseinate containing chia mucilage. Similar results were reported by Timilsena et al. [3], who attributed the high viscosity of the chia mucilage solutions to the presence of 4-O-methyl-glucuronic acid and the intermolecular chain networks formation in aqueous media.

The physical stability of each emulsion was examined through its optical characterization during 15 days. The backscattering (BS) profiles evolution, as a function of the sample height (10–20 and 40–50 mm) and the storage time, for the different systems, are presented in Figure 3. Emulsions with sodium caseinate had high physical stability, causing their BS profiles to remain unchanged during the entire storage period (Figure 3a,b). This behavior could be due to the high viscosity level and small particle size of these systems, which reduce the droplet mobility and therefore its upward movement according to Stokes's law. In contrast, emulsions prepared with the chia protein-rich fraction recorded clarification at the bottom of the sample tube at day 5 of storage (Figure 3a,b). This creaming occurrence would be caused by higher mobility and interaction of the oil droplets as a result of weak viscous forces in the aqueous phase of these systems. Emulsions with the chia mucilage addition presented similar BS profiles to those without this by-product.

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**Figure 3.** Backscattering profiles vs. sample tube height: (**a**) 10–20 mm and (**b**) 40–50 mm. Average values (*n* = 2). Std. deviation < 5%.

127

#### **4. Conclusions**

O/W emulsions proved to be suitable systems to deliver and protect chia seed by-products. All protein–carbohydrate combinations used for emulsions preparation led to the improvement of the oxidative stability of chia oil.

Chia mucilage addition had a significant effect on the rheological properties of the emulsions. Systems containing chia mucilage recorded higher viscosity and global stability due to the reduction of the oil droplets movement. Thus, chia mucilage exhibited a potential role as a thickening agent.

The obtained information could be applied to design and develop O/W emulsions as delivery systems of Ν-3 fatty acids and other by-products from chia seed, allowing the revaluation of these novel ingredients.

**Acknowledgments:** This work was supported by grant Ia ValSe-Food-CYTED (119RT0567), Proyecto X756, X907 Universidad Nacional de La Plata and PICT 2016 0323 ANPCyT (Argentina).

#### **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/).

#### *Proceedings*

### **A Comparative Study of the Physical Changes of Two Soluble Fibers during In Vitro Digestion †**

**Natalia Vera 1,2, Laura Laguna 3, Liliana Zura 2 and Loreto A. Muñoz 2,\*** 


Published: 1 September 2020

**Abstract:** This research aimed to compare the apparent viscosity and the degree of fragmentation/aggregation produced in dispersions of xanthan gum and chia mucilage during the gastrointestinal tract by using an in vitro digestion. Both soluble fibers exhibited pseudoplastic behavior, independent of the concentration and stage of digestion (oral, gastric or intestinal). The viscosity decreased from the oral to intestinal stage in all the concentrations, produced mainly by the "dilution effect" by the addition of digestive fluids. The particle size of xanthan gum increased drastically in the gastric stage mainly due to the decrease in pH, but at intestinal level returned to its original pattern, while particle size and pattern of mucilage during all the stages of digestion remained unchanged, maintaining its integrity. In general terms, since chia mucilage and xanthan gum maintain their viscosity and integrity through the gastrointestinal tract, they could be used as functional ingredients improving the functionality of foods.

**Keywords:** chia seed; in vitro digestion; mucilage; soluble fiber; xanthan gum

#### **1. Introduction**

According to several authors and health organizations, diets high in dietary fiber have a higher incidence in the prevention of many major non-communicable diseases compared to diets lower in this component [1–5]. Many of the beneficial effects have been explained by their behavior at gastrointestinal level. According to EFSA (European Food Safety Authority), dietary fiber corresponds to non-digestible carbohydrates plus lignin, including all carbohydrate components occurring in foods that are non-digestible in the human small intestine and pass into the large intestine. Based on their solubility, dietary fiber can be classified into water soluble (pectins, gums, mucilages, etc.) and insoluble fractions (cellulose, lignin, etc.); both types have different molecular characteristics and physiological effects on the gastrointestinal tract [6]. Soluble dietary fiber (DSF) intake is generally associated with slow transit through the stomach and increasing of the small intestine transit time; this behavior is related with its ability to form viscous solutions [7]. In addition, soluble fibers are fermented by the colonic microbiota, releasing different levels of short chain fatty acids (SCFAs) and play a critical role in the composition and metabolic activity of the microbiome, which affects the intestinal health and ultimately the immune system and the body's ability to resist some chronic diseases [8].

Chia seed has been described as a source of soluble fiber, its mucilage has the ability to retain large amounts of water and produce viscous dispersions, even at low concentrations [9]. On the other hand, xanthan gum is a commercial soluble fiber obtained by fermentation of *Xanthomonas campestris*, is soluble in cold water and in solution exhibits high pseudoplastic flow [10].

The objective of this study was to evaluate comparatively the viscosity and degree of aggregation/fragmentation changes produced during the in vitro digestion of mucilage from chia seeds and xanthan gum.

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

#### *2.1. Materials*

Chia seeds were provided by Benexia (Functional Products Trending S.A., Santiago, Chile); the crude mucilage was obtained from chia seeds by using the method proposed by Muñoz et al. (2012) and xanthan gum was purchased from Sigma-Aldrich [11]. To perform the in vitro digestion, the enzymes and reagents were purchased from Sigma-Aldrich and Merck. The comparisons among means were performed using one-way ANOVA (Analysis of Variance) and the significant differences were determined by the Tukey test (*p* < 0.05). All these tests were performed using the software, Statgraphics Centurion XV.I.

#### *2.2. In Vitro Digestion*

Suspensions of the two DSF at low, medium and high concentrations (0.3, 0.5 and 1.0% *w*/*w*) were subjected to in vitro digestion simulating the gastrointestinal conditions (oral, gastric and intestinal). To perform the experiments, the standardized static in vitro digestion protocol proposed by Minekus et al. (2014) was used [12]. The simulated gastrointestinal fluids such as salivary (SSF), gastric (SGF) and intestinal (SIF) and the enzymes were prepared according to the same protocol.

#### *2.3. Steady Shear Flow Behavior*

The apparent viscosity to each soluble fiber dispersion was determined without digestion as control and before and after each digestion stage by applying an increasing shear rate from 0.1 to 100 sƺ1 in triplicate using a Rotational Rheometer, RheolabQC (Anton Paar GmbH, Austria-Europe). The rheometer was equipped with a double gap concentric cylinder and a Peltier temperature plate set at 37 °C, simulating body temperature. The flow behavior index (*n*) and consistency index (*k*) values were obtained by fitting to the Power Law model (Equation (1)):

$$\mathbf{M} = \mathbf{k} \mathbf{\hat{\mathbf{y}}}^{\prime} \tag{1}$$

where is the shear viscosity (Pa s), k is the consistency index (Pa sƺ1), · is the shear rate (sƺ1) and n is the fluid behavior index (dimensionless).

#### *2.4. Determination of Degree of Aggregation/Fragmentation*

The degree of aggregation/fragmentation was determined at each stage of in vitro digestion in terms of particle size distribution in six-fold, by laser light scattering with a Malvern Mastersizer 2000 (Malvern Instruments, Worcestershire, UK) software version 5.6, using water at 25 °C as solvent.

#### **3. Results**

#### *3.1. Steady Shear Flow Behaviour*

The steady flow behavior, consistency (*k*), and flow index (*n*) behavior of the mucilage and xanthan gum at low, medium, and high concentration, before and during each stage of in vitro digestion can be seen in Figure 1 and Table 1, respectively. All the dispersions show non-Newtonian behavior, with decreasing viscosity with increasing shear rate, also known as pseudoplasticity or shear-thinning behavior (Figure 1). The apparent viscosity to the samples without digestion (Figure 1a–c) show a directly proportional relationship with the concentration and *k* increase as concentration increased. Similar behavior was reported by Timilsena et al., (2015) where the rheological properties of the purified chia seed polysaccharide were evaluated [13]. Moreover, the apparent viscosity of mucilage and xanthan gum decreased from the oral to intestinal stage in all the concentrations (Figure 1d–l), caused mainly by the addition of digestive fluids (SSF, SGF and SIF) and was less affected by the pH changes and ionic strength. Similar behavior was previously observed by Lazaro et al. (2018) and Fabek et al. (2014) when different soluble fibers were subjected to in vitro digestion [14,15]. According to Vuksan et al. (2011), many of the beneficial physiological effects produced by the dietary fiber intake are associated with their capacity to hydrate and increase the viscosity of the human digesta. In this study, both DSF provided viscosity at gastrointestinal level, therefore, their use in food matrices would help to improve functionality [16].

**Figure 1.** Apparent viscosity during in vitro digestion.

#### *3.2. Degree of Aggregation/Fragmentation*

Figure 2 shows the distribution of the particle size of the mucilage and xanthan gum during the in vitro digestion. Figure 2a–c shows the changes in particle size of xanthan gum at the three concentrations. In this case, xanthan gum did not show differences in the bimodal distribution between control and oral stage for the three concentrations, but at gastric level the particle size distribution changed; the curve moves to the right which indicates increasing size.


*Proceedings* **2020**, *53*, 21

This behavior has been previously explained as the decrease of the intermolecular electrostatic repulsion at low pH [10], which allows the expansion of the fiber chains. Finally, at intestinal level, the particle size of xanthan gum returned to the original size mainly due to the increase in pH, at the same time as the concentration increases the conformation becomes monomodal. On the other hand, the particle size and pattern at the three concentrations of mucilage during all the stages of digestion remained unchanged (Figure 2d–f). This behavior has not been previously reported and could indicate a better physiological response when the mucilage from chia seed is ingested. Furthermore, the gastric empty will be slower by using mucilage than xanthan gum and possibly could reduce the nutrient absorption through the intestinal mucosa [17].

**Figure 2.** Degree of aggregation/fragmentation during in vitro digestion, (**a**–**c**) correspond to xanthan gum and (**d**–**f**) correspond to mucilage from chia seed.

#### **4. Conclusions**

In this study both soluble fibers, mucilage and xanthan gum, had the ability to retain viscosity through the gastrointestinal tract, which could indicate their ability to modulate certain physiological responses enhancing functionality when they are added into the food matrix. In addition, the aptitude of these fibers to maintain their structure suggests that they can be used to develop structured foods as a strategy to modulate the digestive process, delaying the gastric emptying.

**Acknowledgments:** This work was supported by the following grant: Ia ValSe-Food-CYTED (119RT0567); the National Fund for Scientific Development and Technological, Project FONDECYT 11150307, Chile and the Fundación para la Innovación Agraria PYT-2018-0261 FIA.

#### **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/).
