2. Methodology

#### 2.1 Materials

their antioxidant and antimicrobial properties that have the potential to eliminate free radicals and inhibit the presence of pathogenic microorganisms in food [3]. However, essential oils are chemically unstable when they are exposed to certain environmental conditions such as light, moisture, oxygen, and elevated temperatures, all of which can cause the loss of their antimicrobial and antioxidant properties [4]. In order to protect the essential oils from the environmental conditions and the interaction with other components of the food, these can be encapsulated. Encapsulation is a process of building a barrier between the core and wall material to avoid physicochemical reactions and to maintain the biological, functional, and physicochemical properties of the core materials [5]. It is a good resource in the application of essential oils as antimicrobial and antioxidant agents in food because it helps to mask the characteristic strong odor that can alter the sensory characteristics of the food product in which essential oils are used. Other advantages of encapsulation are as follows: it minimizes the interaction of the active compound with the environment by reducing the rate of evaporation or transfer of core components to the outside, and it permits easy handling of the encapsulated substance and allows a controlled release of the active compound [6]. Among the encapsulation methods of essential oils, encapsulation by complex coacervation implies the electrostatic attraction between two polymers of opposite charges, and coacervate formation occurs over a narrow pH range [5]. Complex coacervation consists of four steps: dissolution, emulsification, coacervate formation, and wall

a. Dissolution: The preparation of aqueous solutions containing two different biopolymers (commonly a protein and a polysaccharide) is achieved by

b. Emulsification: The material to be encapsulated is added to a solution rich in biopolymers, prepared in the previous step, at a temperature above the gelation point and a pH higher than the isoelectric point of the protein used as an encapsulating agent. Agitation is kept constant until obtaining the desired

c. Coacervate formation: The pH is then adjusted below the isoelectric point of the protein to initiate the electrostatic interactions between the polymers with opposite charges: the protein has a positive charge and the polysaccharide a negative charge. As a result, the droplets of the dispersed phase agglomerate

d. Wall formation and drying: The temperature of the system is decreased below the gelation point of the protein until reaching cooling temperatures, which allows the formation of the wall due to the accumulation of the polymer-rich phase around the material of interest. Subsequently, the capsules are dried to

The formation of capsules in complex coacervation is affected by several factors,

such as the composition of the emulsion, which includes the mass ratio of the encapsulating agents, the concentration of the encapsulated material, and the quantity of emulsifiers added [9, 10]. On the other hand, a coacervate can be stabilized by spray drying atomizing the liquid substance forming small drops on a stream of hot gas (air), in which the solvent evaporates producing small particles of

formation and drying [5, 7, 8]:

drop size [5, 7, 8].

obtain a powder [5, 7, 8].

the encapsulated material [11].

84

mixing and sometimes applying heat [7].

Technology, Science and Culture - A Global Vision, Volume II

and the separation of phases takes place [5, 7, 8].

Anise essential oil (AEO) was purchased from Laboratorios Hersol (Mexico City, Mexico). To form the complex coacervates, chia seeds (Salvia hispanica L.) were purchased from Verde Limón Trading Company (Mexico City, Mexico), and gelatin (type B) was purchased from Gelco SA (Bogota, Colombia). Tween 80 (Sigma-Aldrich, USA) was used as an emulsifying agent. Other chemicals used (analytical grade) were purchased from Hycel (Jalisco, Mexico).

#### 2.2 Extraction of chia mucilage

Chia mucilage was extracted using the modified method described in other studies [15, 16]. Chia seeds were hydrated with distilled water in a ratio of 1:20 (w/v) with constant stirring for 4 h at 35 � 1.0°C. The hydrated seeds were freezedried (Triad™ Labconco, USA), and then the mucilage was mechanically separated from the seeds by sieving with a sieve mesh number 35 (500 μm).

#### 2.3 Microencapsulation of anise essential oil by complex coacervation

Complex coacervates were prepared with 5.0 and 7.5% (w/w) of AEO using gelatin and chia mucilage as encapsulating agents and Tween 80 as an emulsifying agent. Coacervates were prepared using an ultrasound homogenizer (Cole-Parmer, CP 505, USA) adjusting the pH by adding HCl 0.1 N dropwise, and the system was cooled down to 25°C to allow the wall formation of the coacervate. The coacervate was spray-dried using a mini spray drier (B-290, BÜCHI Labortechnik, Switzerland). To guarantee a better yield, the coacervates were previously dispersed in aqueous maltodextrin (20% w/w) solution. An inlet air temperature of 160°C and a feeding rate of 5.0 g/min were used. The powders of complex coacervates were collected and stored inside an amber flask at 25 � 1.0°C until further use.

#### 2.4 Characterization of spray-dried powders of AEO

Particle size: The granulometric distribution of the powders was determined by a dynamic light scattering particle analyzer (Bluewave, Microtrac Inc., USA), and the instrument was previously calibrated. Span, expressing the polydispersity of the powder [17], was calculated using the following equation:

$$\text{Span} = \frac{D\_{\90} - D\_{10}}{D\_{\S 0}} \tag{1}$$

The moisture content of powders: Moisture content was determined using the AOAC method 926.12 [18].

Solid yield: It was calculated as the ratio of the powder weight collected after the spray drying process and the initial weight of solid components in the liquid coacervate before drying [19].

Flow properties: To determine the flow properties of the powders, bulk density and tapped density were obtained [20]. Bulk density (ρbulk) of the powders was determined by introducing a known weight of powder into a graduated cylinder and then measuring its volume. Tapped density (ρtap) was determined in a similar way applying manual mechanical force to compact the powder particles until no difference in the volume was observed. Compressibility index (CI) and Hausner ratio (HR) are flow properties that measure the propensity of a powder to be compressed [20]. To calculate the CI and HR, the following equations were used:

$$\text{CI} = \frac{\rho\_{tap} - \rho\_{bulk}}{\rho\_{tap}} \times \mathbf{100} \tag{2}$$

$$HR = \frac{\rho\_{tap}}{\rho\_{bulk}}\tag{3}$$

7.5% of AEO (D50 = 12.43 μm). Even if the particle size distribution of the samples looks similar (Figure 1), a significant difference (P < 0.05) in the median diameter (D50) between the samples was determined, which may be due to the variation of the essential oil concentration in the sample and the low standard deviation of the measurements. According to the literature, particle size of the microcapsules produced depends on the material properties, the solid concentration, the viscosity of the encapsulating material, and the operating conditions of the spray drying process [23]. The obtained values agree with the size range for various powders dried by

Effect of Oil Content in the Physicochemical Characteristics of Spray-Dried Powders of Anise…

The moisture content may change the physicochemical characteristics of powders during storage, becoming an important variable of stability during the shelf life of the product [25]. The values observed in this study are among the range of moisture content accepted for food powders (1–5%) [26]. As shown in Table 1, the sample with 5.0% of AEO had the largest moisture content. This result is expected since this sample has fewer solids compared to the sample with 7.5% of AEO. A significant difference between samples (P < 0.05) was obtained, which suggests that the oil content affected the final moisture content of the dried coacervates.

% moisture (w.b.) 2.07 0.02<sup>a</sup> 1.25 0.01<sup>b</sup> Solid yield (%) 80.0\* 79.7\*

Compressibility index <sup>50</sup> 0.01<sup>a</sup> <sup>50</sup> 0.01<sup>a</sup> Hausner ratio 2.00 0.01<sup>a</sup> 2.00 0.01<sup>a</sup> EE (%) 93.0 0.3<sup>a</sup> 96.6 0.2b

Different letters in row show significant differences (P < 0.05) between samples.

\*All properties were calculated by triplicate except for solid yield.

Characterization of spray-dried powders of AEO.

) 0.233 0.001a 0.227 0.001b

) 0.466 0.001a 0.454 0.001b

5.0% AEO 7.5% AEO

atomization, which is commonly between 10 and 50 μm [24].

Particle size distribution of spray-dried powders with 5.0 and 7.5% of AEO.

DOI: http://dx.doi.org/10.5772/intechopen.90099

3.2 Moisture content

Bulk density (g/cm3

Table 1.

87

Tapped density (g/cm<sup>3</sup>

Figure 1.

Encapsulation efficiency (EE): Surface oil (SO) and total oil (TO) of the dried coacervates were determined to calculate the encapsulation efficiency. SO was determined by the modified method described by other authors [21]. In 20 mL of n-hexane, a sample of 0.5 � 0.01 g of dried coacervates was dispersed, with constant stirring for 15 s, then filtered (Whatman #41), and dried with a vacuum oven (G0553–10, Cole-Parmer, USA) at 70°C � 1.0°C to evaporate the excess of n-hexane. TO content in the dried coacervates was determined by the modified method using acid digestion as suggested by other authors [22]. A sample of 0.5 � 0.01 g of dried coacervates was dissolved in 3 mL of HCl 4 N with constant stirring until total dilution, and then 20 mL of n-hexane was added maintaining constant stirring for 1 h. Using a separation funnel, the HCl was separated, and the oil phase with n-hexane was recovered in a flask and was placed in a vacuum oven (G0553-10, Cole-Parmer, USA) to evaporate the n-hexane at 70°C � 1.0°C for 30 min. SO and TO content were determined gravimetrically. The percent of EE was calculated using the following equation:

$$\text{EE} \left( \% \right) = \frac{\text{TO} - \text{SO}}{\text{TO}} \times 100 \tag{4}$$

#### 2.5 Statistical analysis

Analysis of variance (ANOVA) and Tukey's comparison tests were performed to statistically analyze the obtained data, using Minitab (v.17, LEAD Technologies Inc., USA) with a confidence level of 95%.

#### 3. Results

#### 3.1 Particle size

Samples showed an asymmetrical distribution with a positive skew (to the right) for the particle size (Figure 1). Span values for the samples with 5.0 and 7.5% of AEO were 1.60 and 1.06, respectively; these small span values indicate a narrow size distribution of the AEO powders [17]. Small particle size was observed in the evaluated microcapsules; the smaller particle size obtained was for the sample with

Effect of Oil Content in the Physicochemical Characteristics of Spray-Dried Powders of Anise… DOI: http://dx.doi.org/10.5772/intechopen.90099

Figure 1. Particle size distribution of spray-dried powders with 5.0 and 7.5% of AEO.

7.5% of AEO (D50 = 12.43 μm). Even if the particle size distribution of the samples looks similar (Figure 1), a significant difference (P < 0.05) in the median diameter (D50) between the samples was determined, which may be due to the variation of the essential oil concentration in the sample and the low standard deviation of the measurements. According to the literature, particle size of the microcapsules produced depends on the material properties, the solid concentration, the viscosity of the encapsulating material, and the operating conditions of the spray drying process [23]. The obtained values agree with the size range for various powders dried by atomization, which is commonly between 10 and 50 μm [24].

#### 3.2 Moisture content

The moisture content of powders: Moisture content was determined using the

Flow properties: To determine the flow properties of the powders, bulk density and tapped density were obtained [20]. Bulk density (ρbulk) of the powders was determined by introducing a known weight of powder into a graduated cylinder and then measuring its volume. Tapped density (ρtap) was determined in a similar way applying manual mechanical force to compact the powder particles until no difference in the volume was observed. Compressibility index (CI) and Hausner ratio (HR) are flow properties that measure the propensity of a powder to be compressed [20]. To calculate the CI and HR, the following equations were used:

> CI <sup>¼</sup> <sup>ρ</sup>tap � <sup>ρ</sup>bulk ρtap

EE %ð Þ¼ TO � SO

statistically analyze the obtained data, using Minitab (v.17, LEAD Technologies

TO

Analysis of variance (ANOVA) and Tukey's comparison tests were performed to

Samples showed an asymmetrical distribution with a positive skew (to the right) for the particle size (Figure 1). Span values for the samples with 5.0 and 7.5% of AEO were 1.60 and 1.06, respectively; these small span values indicate a narrow size distribution of the AEO powders [17]. Small particle size was observed in the evaluated microcapsules; the smaller particle size obtained was for the sample with

HR <sup>¼</sup> <sup>ρ</sup>tap ρbulk

Encapsulation efficiency (EE): Surface oil (SO) and total oil (TO) of the dried coacervates were determined to calculate the encapsulation efficiency. SO was determined by the modified method described by other authors [21]. In 20 mL of n-hexane, a sample of 0.5 � 0.01 g of dried coacervates was dispersed, with constant stirring for 15 s, then filtered (Whatman #41), and dried with a vacuum oven (G0553–10, Cole-Parmer, USA) at 70°C � 1.0°C to evaporate the excess of n-hexane. TO content in the dried coacervates was determined by the modified method using acid digestion as suggested by other authors [22]. A sample of 0.5 � 0.01 g of dried coacervates was dissolved in 3 mL of HCl 4 N with constant stirring until total dilution, and then 20 mL of n-hexane was added maintaining constant stirring for 1 h. Using a separation funnel, the HCl was separated, and the oil phase with n-hexane was recovered in a flask and was placed in a vacuum oven (G0553-10, Cole-Parmer, USA) to evaporate the n-hexane at 70°C � 1.0°C for 30 min. SO and TO content were determined gravimetrically. The percent of EE was calculated using the following equation:

� 100 (2)

� 100 (4)

(3)

Solid yield: It was calculated as the ratio of the powder weight collected after the spray drying process and the initial weight of solid components in the liquid

AOAC method 926.12 [18].

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coacervate before drying [19].

2.5 Statistical analysis

3. Results

86

3.1 Particle size

Inc., USA) with a confidence level of 95%.

The moisture content may change the physicochemical characteristics of powders during storage, becoming an important variable of stability during the shelf life of the product [25]. The values observed in this study are among the range of moisture content accepted for food powders (1–5%) [26]. As shown in Table 1, the sample with 5.0% of AEO had the largest moisture content. This result is expected since this sample has fewer solids compared to the sample with 7.5% of AEO. A significant difference between samples (P < 0.05) was obtained, which suggests that the oil content affected the final moisture content of the dried coacervates.


Different letters in row show significant differences (P < 0.05) between samples. \*All properties were calculated by triplicate except for solid yield.

#### Table 1.

Characterization of spray-dried powders of AEO.
