# Droplet-based Millifluidic Technique for Encapsulation of Cinnamon Essential Oil: Optimization of the Process and Physicochemical Characterization

Atefeh Farahmand, Bahareh Emadzadeh, Behrouz Ghorani, Denis Poncelet

► To cite this version:

Atefeh Farahmand, Bahareh Emadzadeh, Behrouz Ghorani, Denis Poncelet. Droplet-based Millifluidic Technique for Encapsulation of Cinnamon Essential Oil: Optimization of the Process and Physicochemical Characterization. Food Hydrocolloids, 2022, 129, pp.107609. 10.1016/j.foodhyd.2022.107609. hal-04218594

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# Droplet-based Millifluidic Technique for Encapsulation of Cinnamon Essential Oil: Optimization of the Process and Physicochemical Characterization

Atefeh Farahmand¹, Bahareh Emadzadeh*¹, Behrouz Ghorani ¹& Denis Poncelet*²

¹Department of Food Nanotechnology, Research Institute of Food Science and Technology (RIFST), km 12 Mashhad-Quchan Highway, Mashhad, Iran, PO Box: 91895-157-356

²UMR CNRS 6144 GEPEA, Université de Nantes, F-44000 Nantes, France

*Corresponding authors email: Bahareh Emadzadeh: b.emadzadeh@rifst.ac.ir, Denis Poncelet: denis.poncelet@encapprocess.fr
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**Abstract**

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The "millifluidic" technique could be considered as a novel approach for encapsulation of bioactive compounds and has a promising perspective in the field of food engineering or pharmaceutical science. In this study, we aim to optimize the encapsulation of cinnamon essential oil (CEO) by droplet-based millifluidic technique with four responses: encapsulation efficiency (EE), loading capacity (LC), sphericity factor (SF) and size of calcium-alginate millicapsules.

The effects of alginate concentration (20-30 g/L), flow rate of alginate (1-1.6 mL/min), and flow rate of CEO (0.6-0.8 mL/min) were considered by a Box-Behnken design. The best concentration of chitosan as a coating layer on the optimized samples was selected based on the Young modulus of millicapsules. The regression models showed the significant effect (p<0.05) of the three variables on the characteristics of millicapsules. The optimal millicapsules with 3.58±0.23 mm diameter and 0.96±0.01 SF showed 98.96±1.2% and 70.14±1.8% EE and LC, respectively. SEM images exhibited a rough external surface which changed to a rigid and smooth surface through the chitosan coating. The results of DSC and FTIR tests demonstrated the CEO entrapment in the millicapsules without any chemical interaction with the encapsulant materials. The disarray of crystallinity structure in XRD patterns revealed the successful encapsulation of CEO in the millicapsules. The non-Fickian case II in the mouth and small intestine and anomalous transport in the stomach were the main release mechanisms in the coated millicapsules. The release profile of CEO also fitted well with Ritger-Peppas model.

**Keywords:** Millifluidic, Cinnamon essential oil, Core-shell, Encapsulation, Kinetic release

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49 **1. Introduction**

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The rising demand for consumption of food products with health claims increases the efforts to produce functional foods that help to improve the health conditions, especially in elderly individuals beyond basic nutrition (K. Chen, Chen, Liang, & Xu, 2020; X. Chen, Qiu, Chen, Li, & Liang, 2020; Granato, et al., 2020; Granato, Nunes, & Barba, 2017; Piornos, Burgos-Díaz, Morales, Rubilar, & Acevedo, 2017). This kind of foods is fortified with bioactive compounds such as phytochemicals, minerals, and vitamins, etc., in the battle with a nutrient deficiency (Galanakis, 2021). During the pandemic, the bioactive compounds have also been reported to support the immune system against COVID-19 disease (Galanakis, 2020).

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Plant essential oils consist of a variety of lipophilic bioactive compounds and volatile molecules such as phenol derivatives, sesquiterpene, monoterpenes, and aliphatic components. Metabolism and absorption of phenolic compounds in the gastrointestinal (GI) tract govern their biological characteristics. Only the released compounds in intestine are digested (Adorjan & Buchbauer, 2010). Cinnamon essential oil (CEO) obtained from *Cinnamomum zeylanicum* L., is widely used as a flavoring agent due to its unique organoleptic properties. It has also been proposed as a natural antioxidant and antimicrobial agent due to its phenolic compounds (Gong, Lee, Godec, Zhang, & Abbaspourrad, 2020). Nevertheless, interactions with food components, low solubility in an aqueous phase, high volatility, and negative impact on sensory properties of food products, especially at high concentrations, have faced the direct application of CEO in the food industry with some challenges (Hyldgaard, Mygind, & Meyer, 2012).

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Besides their high volatility, the essential oils are easily decomposed due to sensitivity to environmental conditions. Current researches suggest the entrapment of essential oil into polymeric carriers intending to protect them against adverse environmental conditions (such as oxygen and light), to mask their unwanted intense odor and taste, or to provide a feasible way to
hold or handle the essential oil (Baldim, et al., 2020; Radünz, et al., 2019). Moreover, their encapsulation also helps to overcome their volatility, which leads to a reduction in their loss or uncontrolled release (Sangsuwan, Pongsapakworawat, Bangmo, & Sutthasupa, 2016).

Core-shell structures have been used as a delivery system to control the release mechanism and protect the bioactive compounds of essential oils from chemical reactions and degradation (Akamatsu & Yamaguchi, 2007). Protection of the core is achieved by a solid shell. As a matter of fact, the membrane acts as an obstacle between the capsule's interior and the surrounding environment (Caruso, Schüler, & Kurth, 1999). The size, structure, and morphology of core-shell capsules determine their application. Limitations in controlling the morphology, low reproducibility, and high polydispersity are disadvantages of conventional methods in core-shell production methods such as spray drying, solvent evaporation, self-assembly, and polymerization (W. Li, et al., 2018). Concerning the manufacturing process, there are different state-of-the-art approaches such as micro or millifluidic, co-extrusion, and co-axial electro-hydrodynamic atomization techniques. Besides, the methods are adjustable and convenient for a broad range of core and shell materials (Zhang, Huang, Si, & Xu, 2012).

In the droplet-based millifluidic encapsulation method, the CEO as a dispersed phase is directly extruded via a needle into the co-flowing fluid of alginate (continuous phase) to produce the oil-core droplets through a shear force between two immiscible phases (Martins, Poncelet, Marquis, Davy, & Renard, 2017). For capsule formation, the formed droplet at the tip of a glass tube is dropped into a calcium chloride cross-linking bath, the alginate engulfing the liquid core interacts with Ca²⁺ and produces a calcium-alginate shell. The data in the literatures reflect the interests regarding the encapsulation of oil using the millifluidic set-up to produce highly organized capsules with reproducibility (Pereda, Poncelet, & Renard, 2019; Ponrasu, Yang, Chou, Wu, & Cheng, 2020). Moreover, no toxic chemical is used during the capsule formation, and due to the millimeter size of the capsules, fast production could be observed. (Pendyala, Bithi,
Vanapalli, & Fernandes, 2019; Tamayol, et al., 2013). This is also an inexpensive and versatile technique so that the optimization of the parameters involved in the formation of millicapsules such as concentration and flow rates of phases could be easily adjusted (Martins, et al., 2017).

One of the factors with a vital role in the encapsulation of essential oil is selecting the most appropriate shell material, which is determined based on the core properties and encapsulation method (Purwanti, et al., 2018). Alginate is considered as one of the commonly used biopolymers due to its unique properties such as gel formation in the presence of divalent cations, biodegradability, non-toxicity, and biocompatibility (Kim, Chung, Shin, Yam, & Chung, 2008). Although low stability and leaching of the core through the pores of the alginate shell have also been reported, the problem could be overcome by covering the millicapsules with a cationic polymer like chitosan (Vandenberg, Drolet, Scott, & De la Noüe, 2001). Chitosan has been used as an appropriate coating layer for the alginate millicapsules, so the final millicapsules have a smoother surface with less permeability (S.-M. Koo, Cho, Huh, Baek, & Park, 2001). Good mechanical strength and high stability under acidic and alkaline conditions have been shown for chitosan-coated capsules (Liouni, Drichoutis, & Nerantzis, 2008).

Few studies on the millifluidic encapsulation of oil have more focused on the fundamentals and the assessment of the production conditions regardless of the applications of millicapsules and particularly their behavior in the GI tract. Martins, et al. (2017) produced emulsion-loaded microcapsules using droplets millifluidic technique based on the inverse gelation mechanism. They explained the parameters affecting the capsule size, sphericity, and membrane thickness (Martins, et al., 2017). Pereda, et al. (2019) also used millifluidic/inverse gelation to produce w/o emulsion filled millicapsules. They evaluated the effects of curing time and calcium concentration on the oil content, and also the membrane thickness on the mechanical characteristics of the millicapsules (Pereda, et al., 2019). In our previous study, we applied millifluidic/direct gelation technique to develop oil-loaded millicapsules, and optimized the
instrumental, geometrical, and the feed solution properties of the millifluidic device (Farahmand, 123 Emadzadeh, Ghorani, & Poncelet, 2021).

Millifluidic device as a new platform for the encapsulation of the essential oils such as 125 CEO paves the way for the production of millicapsules with highly controlled dimensions and 126 suitable loading capacity. Accordingly, in this study, a double-layered core-shell millicapsules 127 with core of CEO was developed by means of a millifluidic device. A Box-Behnken design was 128 applied to optimize the effects of process parameters (alginate concentration and flow rates of 129 CEO and alginate) on the encapsulation efficiency (EE), loading capacity (LC), size, and 130 sphericity of the produced millicapsules. The success of the encapsulation and some 131 physicochemical characteristics of the chitosan-coated optimal millicapsules were investigated in 132 terms of dimension, morphology, mechanical stability, FTIR spectroscopy, thermal and 133 crystallinity properties. In addition, the kinetics and mechanism of the CEO release from the 134 millicapsules in simulated conditions of GI tract were modeled. 135

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## 2. Material and methods

### 2.1. Materials

Sodium alginate (with a molar mass of $1.57 \times 10^5$ g/mol, low viscosity, and mannuronic 139 (M) to guluronic (G) acid ratio of 1.37), calcium chloride dihydrate ($CaCl_2 \cdot 2H_2O$), and chitosan 140 powders (MW= 50-190 kDa, Degree of deacetylation: 75-85%) were purchased from Sigma- 141 Aldrich (St. Louis, Mo, USA). Tween-20 (HLB=16.7), glacial acetic acid ($CH_3COOH$) (99.8%), 142 tri-sodium citrate ($Na_3C_6H_5O_7$) with mass fraction purity>0.99, and n-hexane were supplied by 143 Merck chemicals Co (Darmstadt, Germany). CEO was obtained from Tabib-daroo Co (Kashan, 144
Iran). All the other chemicals applied in this research were of reagent grade without further
purification.

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148 **2.2. Characterization of CEO**

149 An Agilent 7890B gas chromatography connected with a mass detector (Model 5977A,
150 USA) was used to characterize the aromatic compounds of the CEO. The CEO was diluted by a
151 solvent (acetone) and subsequently injected into the device. In order to prevent damage to the
devices, a 4.5 min delay-time was considered for the evaporation of the solvent/ residual
152 solvent in the sample. The gas chromatography was equipped with a HP-5MS capillary column
153 (phenylmethyl siloxane, 30 m× 0.25 mm, film thickness 0.25µm, Agilent). The injector
154 temperature was 270 °C, and the oven temperature was programmed from 60 °C to 230 °C at a
155 rate of 5 °C/min. The carrier gas was helium at a linear flow rate of 1 mL/min, and the injection
156 volume was 1 µL. The electron impact ionization was performed at 70 eV. Recognition of the
157 essential oil compounds was achieved by matching their mass spectra with those from GC-MS
158 libraries (Purwanti, et al., 2018).

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161 **2.3. Preparation of alginate, chitosan, and calcium solutions**

162 Shell solution was prepared by dissolving suitable amounts of sodium alginate powder
163 (20, 25, and 30 g/L) and Tween-20 (0.6 g/L) in deionized water with a constant stirring (24h) for
164 full hydration. Tween reduces the surface tension of alginate droplet as it exits the millifluidic
tube. Surface tension is known as a holding force exerted on the droplet (Farahmand, et al.,
165 2021). There was no round-shaped millicapsule at the lower concentration of alginate (below 20
g/L), and pumping problems occurred at higher concentrations (more than 30 g/L) due to higher
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viscosity. The cross-linking collector solution was obtained by dissolving an appropriate amount of CaCl₂ (30 g/L) in deionized water. This concentration level was selected based on preliminary tests. Chitosan as a coating solution was also prepared by dissolving required amounts of powder (2.5-15 g/L in 0.1 M acetic acid) to pH= 5.5 and kept stirred on a magnetic stirrer for 1 h (Murata, Maeda, Miyamoto, & Kawashima, 1993).

## 2.4. Encapsulation by double-stage millifluidic method

The core-shell millicapsules were formed using a millifluidic method with the geometry of co-axial flow focusing (Fig.1). The flow rates of dispersed (CEO) and continuous phase (alginate) were adjusted by two syringe pumps (New Era pump systems, Inc, NE-300, USA). The CEO was pumped using a Teflon tube (interior diameter (ID) 0.6 mm and outside diameter (OD) 1.5 mm) at rates ($Q_{CEO}$) of 0.6- 0.8 mL/min. The alginate solution mixed with Tween-20 (0.6 g/L) was also pumped into a Teflon tube (ID= 0.9 mm and OD= 2 mm) at rates ($Q_{Alg}$) of 1-1.6 mL/min. There are two possible flow regimes in the millifluidic method including dripping and getting, which can characterize the dynamics of droplet formation (Farahmand, et al., 2021). The flow rates were chosen according to the formation of dripping region in the glass tube. In this regime, the spherical droplets are formed in the millifluidic tube immediately after the two phases meet (Fig. 1). Both phases were co-flowed into a glass tube ID= 3.2 mm, OD= 5 mm, and length= 20 cm). By shear forces exerted between the two phases, the CEO was extruded in the alginate solution, and oil-core droplets were formed inside the glass tube (Martins, et al., 2017). The core-shell droplets pinched-off from the tip of the glass tube due to the rival forces, and they were collected in a calcium chloride collector stirred at 50 rpm at room temperature. After 30 min gelation time, the millicapsules were rinsed with deionized water, suspended in
chitosan solution for 15 min. The chitosan-coated millicapsules were kept in Ca²⁺ solutions (15 g/L) and stored at 4 °C until use.

Insert figure.1 about here

## 2.5. Experimental design

The impacts of the most important parameters of millifluidic system: alginate concentration (X₁: 20-30 g/L), the flow rate of alginate (X₂: 1-1.6 mL/min), and the flow rate of oil (X₃: 0.6-0.8 mL/min) as independent variables on the EE (Y₁), LC (Y₂), size (Y₃), and SF (Y₄) of the millicapsules were assessed using response surface methodology (RSM). The experiments were planned using Box-Behnken design with 17 experimental runs and 5 central points (Table 1). To elucidate the effect of encapsulation conditions on the responses, a second-order polynomial equation was utilized (Eq. 1):

$$Y=\beta_{0}+\sum_{i=1}^{n} \beta_{i} X_{i}+\sum_{i=1}^{n-1} \sum_{j=i+1}^{n} \beta_{i j} X_{i} X_{j}+\sum_{i=1}^{n} \beta_{ii} X_{i}^{2} \quad (1)$$

Where Y represents the measured responses, β₀ is the constant coefficient, βᵢ is the linear coefficient, βⱼ is the interaction coefficient among variables, βᵢᵢ is the quadratic coefficient, and X is the studied variable. Evaluation of the most suitable model was assessed using various statistical criteria. A coefficient of determination (R²) close to 1, a covariance lower than 10, and an adequate precision higher than 4 were considered for selecting the desired model. The lack of fit test was used to check the adequacy of the fitted model. A non-significant lack of fit confirms well-fitting of the model.

Insert table.1 about here

## 2.6. Characterization of millicapsules
### 2.6.1. Dimension and sphericity factor

After the gelation procedure, the shape and size of the millicapsules were calculated through Image J software (National Institutes of Health, USA). At least 20 millicapsules were captured using a digital microscope (Dino-lite Pro, Taiwan) in each experimental run. The photographs of samples were captured with 1280×1024 resolutions. 30× magnifications were used for all experimental runs. The diameter of each millicapsules was calculated assuming it is a circular shape. The Sphericity factor (SF) was measured as explained by (Davarcı, Turan, Ozcelik, & Poncelet, 2017) (Eq.2):

$$SF= (2 d_{min})/(d_{max}+d_{min}) \quad (2)$$

Where d is the millicapsule diameter. The SF> 0.95 was considered as spherical millicapsule and 0.9 <SF< 0.95 indicates oval and pear millicapsule. Lower values denoted shape deformation.

### 2.6.2. Morphology

The surface morphology of the uncoated and coated millicapsule was captured under a field emission scanning electron microscope (FESEM) (MIRA3, TESCAN, Czech Republic) at room temperature and acceleration voltage of 15 kV. Coating of the CEO-loaded millicapsules with gold was performed by sputter coater for 150s at 20 mA. SEM images with different magnifications from a range of 80-500 were taken at room temperature.

### 2.6.3. Encapsulation efficiency (EE) and loading capacity (LC)

Quantification of loaded CEO in millicapsule was achieved according to Parris, Cooke, and Hicks (2005) with some modifications. 0.5 g of the oil-loaded millicapsule was dispersed in
5 mL of sodium citrate (0.055 M) (as a chelating agent for calcium ions) to de-crosslink the millicapsules. Then 5 mL n-hexane was added and the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was gathered, and the absorbance of the extracted CEO was measured using a UV-Vis spectrophotometer (DR 5000, HACH) at a wavelength of 280 nm. The absorbance results of the extracted CEO were compared to the standard calibration curve of Cinnamaldehyde standard solutions (R²= 0.99). The EE and LC of CEO were determined using to Eq. (3) and (4), respectively:

$$EE (\%) = (\text{amount of loaded CEO}/ \text{initial amount of CEO}) \times 100 \quad (3)$$

$$LC (\%) = \text{amount of loaded CEO}/ \text{weight of collected millicapsules} \times 100 \quad (4)$$

### 2.6.4. Mechanical strength

A texture analyzer (TA.XT-Plus, Stable Micro Systems, UK) equipped with a 20 mm cylindrical probe was used to measure the millicapsule's strength. A compression test was applied to assess the millicapsule deformation up to a distance of 2 mm, with a linear compression speed of 0.6 mm/min, pre-test speed of 1 mm/min, and a post-test speed of 2 mm/min. The normal force and the distance were calculated simultaneously at the upper plate (Fery & Weinkamer, 2007; Pereda, et al., 2019). The optimal millicapsules were coated using the chitosan solution (0, 0.25, 0.5, 1, 1.5% w/v prepared in 0.1 M acetic acid), and their strengths also were compared to the uncoated millicapsule. The maximum mechanical strength was the criterion to select the best concentration of chitosan solution for further studies. Each concentration of the chitosan was tested by five replications. All the experiments were measured at ambient temperature. Young modulus E (Pa), was estimated using to Eq. 5:
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$$E = (3(1-\nu^2) \cdot F) / \sqrt{d \cdot H^3} \qquad (5)$$

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### 2.6.5. FTIR spectroscopy

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FTIR spectra for pure sodium alginate, CEO, chitosan powder, unloaded millicapsule, CEO-loaded millicapsule, and coated millicapsules (loaded or unloaded with CEO) were plotted from wavenumber 400-4000 cm⁻¹ by a spectrophotometer (Bruker Alpha FTIR, US). Samples were prepared by grinding the millicapsule with KBr and pressed into a disk. A few CEO droplets were also placed on the KBr pellet (Hosseini, Zandi, Rezaei, & Farahmandghavi, 2013).

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### 2.6.6. Thermal properties of millicapsule

Thermal transition characteristics of the CEO, sodium alginate and chitosan powders, unloaded, CEO-loaded millicapsules, and coated millicapsules were measured by a differential scanning calorimeter (DSC, Mettler, Toledo, Switzerland). Sample powder (about 15 mg) was sealed in an aluminum pan. DSC curve was recorded from 25 to 400 °C at a heating flux of 10 °C/min under 30 mL/min nitrogen flow (J. Li, Kim, Chen, & Park, 2016).
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### 2.6.7. XRD

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The structural changes were recorded by X-ray diffraction (XRD) (Siemens, D5000) over a 2θ range of 5-50 ° with a step angle of 0.04 °/min (Hosseini, et al., 2013). The results obtained were then used to plot the graphs using Origin 2019b (Origin lab Inc., USA) software.

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### 2.6.8. In vitro digestion and release kinetics

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The in vitro essential oil release was investigated in three phases of mouth, stomach and intestinal tract. Digestion protocols for simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared as follows:

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#### 2.6.8.1. Simulated oral digestion

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Specific ratios of salts and α-Amylase concentration in SSF was prepared according to (Minekus, et al., 2014). For the analysis, ~25 g of millicapsules were suspended in 20 mL prewarmed SSF, which the pH was adjusted (Metrohm, pH meter model 780) to 6.8 by standard solutions of 1 M (NaOH/HCl). The samples were placed in an incubator shaker (Memmert, Germany) at 37°C and 95 rpm for 2 min (Bastos, de Sá Costa, Siqueira, & Garcia-Rojas, 2020). Interval time of sampling was 20 second.

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#### 2.6.8.2. Simulated gastric digestion

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500 mL SGF was prepared by mixing 2g/100g NaCl and 3600 U/mL pepsin enzyme and pH adjusted to 2.5 with 1 mmol/L HCl. 25 grams of millicapsules treated in the oral phase were added to 500 mL of prewarmed (37°C) SGF and was incubated for 2 h continuously shaking at
303 100 rpm. Sampling was performed at specific interval times (2, 15, 30, 45, 60, 75, 90, 105 and
304 120 min) (Gorbunova, Evteev, Evdokimov, & BANNIKOVA, 2016).

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**2.6.8.3. Simulated intestinal digestion**
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After gastric digestion, treated millicapsules were added to 400 mL SIF and they were
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incubated at 37°C for 2 h at 100 rpm. SIF contained 0.7g/100g monobasic potassium phosphate,
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0.1g/100g bile salts and 0.4g/100g pancreatin. The SIF solution pH was set to 7 utilizing 0.5
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mmol/L NaOH. 20 min interval times were applied (Gorbunova, et al., 2016).

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The mechanisms of release process were evaluated by various mathematical models such
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as Higuchi (Eq. 6), Ritger-Peppas (Eq. 7), Korsmeyer-Peppas (Eq. 8), and Kopcha (Eq. 9):

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$M_t/M_\infty = kt^{1/2}$ Eq. 6 (Higuchi, 1963)

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$M_t/M_\infty = kt^n$ Eq. 7 (Ritger & Peppas, 1987)

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$M_t/M_\infty = (k.t^n)/100$ Eq. 8 (Korsmeyer, Gurny, Doelker, Buri, & Peppas, 1983)

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$M_t/M_\infty = A \times t^{1/2} + B \times t$ Eq. 9 (Kopcha, Tojo, & Lordi, 1990)

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In these models, M<sub>t</sub>, M<sub>∞</sub>, k, and t represent the value of released CEO at time t, the released
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CEO at infinite time, release constant, and time of release in simulated media, respectively.
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Furthermore, the n value in Ritger-Peppas and Korsmeyer-Peppas equations corresponds the
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release mechanism (for spherical capsule, n≤ 0.43 Fickian diffusion, 0.43< n <0.85 non-Fickian
diffusion (Anomalous transport), n= 0.85 case II transport, and above 0.85 shows a super case II
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mechanism. The A value in Kopcha model indicates the Fickian constant, but B value represents
the erosion constants.
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## 2.7. Statistical analysis

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The model analysis in response surface methodology was performed by Design expert software version 10 (Stat-Ease Inc., Minneapolis, USA). Data obtained in the mechanical stability of optimal millicapsules were investigated by analysis of variance (ANOVA) and compared by Duncan test using Minitab software (version 16, Statsoft, USA).

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# 3. Results and discussions

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## 3.1. Identification of CEO constituents

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Analysis of GC-MS chromatogram of CEO identified 10 compounds representing 99.97% (Table 2). CEO spectrum consisted of one major peak indicating trans-Cinnamaldehyde (84.04%). Hu, Zhang, Xiao, and Wang (2018) also reported that the major compound in the CEO spectrum was Cinnamaldehyde (64.94%) and this essential oil consisted of 89.78% aromatic compounds. Fadel, Hassan, Ibraheim, Abd El Mageed, and Saad (2019) reported that the major identified constituent of cinnamon bark oil spectrum was Cinnamaldehyde (90.2%), followed by eugenol (2.20%), and β-Caryophyllen (2.18%). The Different values of Cinnamaldehyde being reported in the previous researches might be due to the use of various extraction methods. In this study, the concentration of (E)-Cinnamaldehyde was used as a marker to measure the encapsulated CEO.

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## 3.2. Millifluidic encapsulation process

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### 3.2.1. Effect of independent variables on EE and LC
In this experimental design, the effects of independent variables on the responses were analyzed using ANOVA (see **supplementary, Tables 1& 2**). The ANOVA for predicted models showed a statistical dependency (p<0.05) on the variables. The fitted equations for each response are reported in **Table 3**.

Insert table.3 about here

The EE values varied from 97.34 to 99.40% (Fig. 2, A-C). These values were significantly higher than the results obtained by other CEO encapsulation methods (Muhammad, et al., 2020; Noghabi & Molaveisi, 2020; Paris, Ramirez-Corona, Palou, & López-Malo, 2020). Oil contents of millicapsules were in the range of 45.11-70.16% (Fig. 2, D-F).

Fig. 2 exhibits the three-dimensional response surface plots of EE and LC as a function of independent variables at different levels. A higher EE could be obtained by augmenting the alginate concentration ([Alg]: 20 to 30 g/L) as a continuous phase (Fig. 2, A). Increasing the alginate concentration and consequently, its viscosity inside the tube could forms capsules with more efficiency and a stiffer shell (Gholamian, Nourani, & Bakhshi, 2020; Phawaphuthanon, Behnam, Koo, Pan, & Chung, 2014). The EE of the oil is highly dependent on the cross-linking degree of the alginate shell. Higher concentrations of alginate would result in a thick and dense shell and therefore, prevent the CEO leakage from the millicapsules. This shell has more active calcium-binding sites (G-blocks) with higher degree of cross-linking. (Piornos, et al., 2017). A similar trend could be observed for LC (Fig. 2, D). Surface tension and interfacial tension are considered as holding forces, while gravitational and drag forces are assumed as detaching forces of the core-shell droplet at the tip of millifluidic tube. Furthermore, surface tension and gravitational forces are exerted on the continuous phase droplets (shell), but interfacial tension
and drag forces affected the dispersed phase droplets (core) (Chan, Lee, Ravindra, & Poncelet, 2009; Farahmand, et al., 2021; X. Wang, Zhu, Shao, Luo, & Zhang, 2019). The drag force (as a detaching force) is proportional to the continuous phase viscosity. Augmentation of alginate concentration, and also its viscosity exerted higher drag force on the dispersed phase (Martins, et al., 2017). Hence, the small CEO-loaded droplets detached more quickly from the tube. Subsequently, the small millicapsules in gelling bath contained high CEO content compared to their total weight. This is because of the different transferring speed of CEO and viscous alginate, so the LC of millicapsule improved.

When the alginate flow rate ($Q_{Alg}$) was maintained fixed at the central value, increasing the flow rate of CEO ($Q_{CEO}$) up to 0.7 mL/min resulted in improved EE (Fig. 2, B). However, the higher values of 0.7 mL/min exhibited a reverse effect. Higher density of CEO than alginate solution (1035 and 1001 kg/m³, respectively) led to faster transferring of CEO in tube. In addition to density, shear stress of the tube wall could reduce the velocity of the alginate solution (Akbari, Rahimi, & Fattahi, 2017) and therefore, CEO flow rates higher than 0.7 mL/min led to a failure in oil entrapping. The LC is the millicapsule ability in CEO loading based on the amount of polymer used. The amount of entrapped CEO in the millicapsules was directly related to the flow rate. The higher flow rates, the more injection of CEO into the droplet compared to the shell solution and therefore, the higher LC (Fig. 2, E, F).

As shown in Fig. 2 (C), high flow rates of alginate were desirable for obtaining high EE values. As mentioned above, the CEO droplets moved faster than alginate in the tube. By increasing the alginate flow rate, formation speed of the alginate droplets also enhanced. Consequently, the gravitational force became dominant, which led to successful encapsulation of CEO droplets. An inverse trend was observed concerning to LC as a function of alginate flow rate (Fig. 2, F). Increasing the flow rate of the continuous phase affects the gravitational force on the droplets, which helps faster detaching of small droplets from the tube (Cramer, Fischer,
& Windhab, 2004). It seems that the high flow rates of alginate caused the higher shell/oil ratio (w/w) in the produced millicapsule. The reduction of required volume of oil loading occurred via more injection of alginate solution, which led to a decrease in LC. (Fig. 2, F). As reported by Chan (2011), oil content of calcium alginate beads produced by extrusion-dripping method was reduced at higher ratios (w/w) of shell to oil.

Insert figure.2 about here

### 3.2.2. Effect of independent variables on size

The diameter of the millicapsules varied from 3.46 to 3.99 mm (Fig. 3 A-C). A converse effect of alginate flow rate on the millicapsules diameter was shown in Fig. 3. A. The higher flow rate of alginate induces higher pressure on the interface of two phases, and also a high velocity gradient. As a result, a high drag force acting on the dispersed phase resulted in its breakup into the smaller droplets (T. Fu, Wu, Ma, & Li, 2012). Similar results also were observed by Akbari, et al. (2017)

Increasing the alginate concentration from 20 to 30 g/L caused a reduction in millicapsule diameter (from 3.99 to 3.59 mm) (Fig. 3, B). This may be due to viscosity increasing of the continuous phase surrounding the CEO droplets inside the tube. Hence, high viscosity of alginate could restrict the fast transfer speed of the CEO and also reduces the interfacial tension as the holding force of oil droplets (Chan, 2011). The reduction of interfacial tension resulted in dominating the detached forces (drag and gravitational), so smaller droplets separated more quickly. As a result, smaller millicapsules produced in the gelling bath.

As depicted in Fig.3, C, increasing the CEO flow rate from 0.6 to 0.8 mL/min had a positive effect on the millicapsules diameter. Higher flow rate of the inner fluid compared to the continuous phase results in a larger droplet by reducing the shear stress imposed on the inner
phase (Ren, Ju, Xie, & Chu, 2010). The higher values of density and CEO flow rate resulted in more core material enters into the droplet compared to the shell. Inducing more resistance to the continuous phase as a result of the injection inertia of the dispersed phase at high flow rate produces larger droplets (T. Fu, et al., 2012). Similar results were reported by W. Wang, Waterhouse, and Sun-Waterhouse (2013) for canola oil.

Insert figure.3 about here

### 3.2.3. Effect of independent variables on SF

As demonstrated in Fig.3, D, increasing the alginate pump speed helped to produce a more spherical millicapsule. There are two stages in droplet detaching from the tip of tube: squeezing stage with slight decrease in the thinning rate of the neck (reason of droplet formation with irregular shape) and pinching-off stage with rapid thinning rate. The squeezing stage is function of continuous flow rate. It was mentioned that the rate of neck thinning in the squeezing stage increases as the continuous flow rate enhances. This is due to improving the drag force, which detaches the droplet neck (Pan, Chen, Zhang, & Li, 2020). The impinging velocity of the droplet with the surface of the CaCl₂ bath is determining the shape of the produced capsule. In other words, irregular-shaped capsules are formed at a low velocity of collision (Gao, He, Fu, Qiu, & Jin, 2016). Increasing the alginate flow rates and also the gravitational force enabled the droplet to faster detaching. As a result, collision dynamics did not affect the final shape of produced millicapsule.

By increasing the alginate concentration from 20 to 30 g/L the more spherical millicapsule were produced, and the SF increased from 0.94 to 0.97 (Fig. 3, E). It seems the core-shell droplets from dilute alginate solution are mostly unable to retain sphericity during
cross-linking stage by restoring forces (interfacial tension and hydrostatic pressure) or against
the drag force upon collision with the surface of calcium chloride bath (Lee, Ravindra, & Chan,
2013). Hence, irregular-shaped millicapsules were observed at lower concentrations of alginate.

As illustrated in Fig. 3, F, increasing the CEO flow rate resulted in a decrease in the sphericity. The shape evolution of millicapsules depends on the competition between the cross-linking process and the fluid drag force. Deformation of the droplet is highly affected by the drag enforced by the gelling bath (Haldar & Chakraborty, 2018). The entrance of the dispersed phase (CEO) in a large volume into the droplet would lead to a thin shell with less cross-linking degree. This droplet could not tolerate the fluid drag force; therefore, irregular-shaped millicapsules would produce.

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### 3.2.4. Optimization of variables and verification of design

Since the encapsulation efficiency and sphericity values were appropriate in all experimental runs, the optimized encapsulation process was selected based on achieving the EE and SF values in range, the maximum LC, and the minimum diameter of millicapsules using the numerical point prediction method. The optimized conditions included the alginate concentration of 30 g/L, alginate flow rate of 1.09 mL/min, and CEO flow rate of 0.8 mL/min. Desirability as accuracy criterion in the optimized conditions was 0.91, which is quite close to 1. The predicted EE, LC, SF, and size values were 98.11%, 71.46%, 0.97, and 3.52 mm, respectively, while the values for the optimized conditions were measured as 98.96± 1.2%, 70.14± 1.8%, 0.96± 0.01, and 3.58± 0.23 mm. These results corresponded well with the predicted data approving that the regression model was adequate for prediction of the millifluidic encapsulation of CEO.

The literature review showed that the millifluidic method compared to other oil-core encapsulation techniques exhibited higher efficiency and ability to produce spherical millicapsules (Chew & Nyam, 2016; S. Y. Koo, Cha, Song, Chung, & Pan, 2014; Morales, et

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al., 2017; Sittipummongkol, Chuysinuan, Techasakul, Pisitsak, & Pechyen, 2019; Volić, et al., 2018).

The dripping flow pattern of the droplet formation in the tube was shown in **Fig. 4**.
Droplet formation occurs directly after the two phases meet in the dripping process. **Fig. 4 (C)**, demonstrated the optimal millicapsules at the time of falling into the bath and after 30 min gelation (**Fig. 4, D**).

Insert figure.4 about here

3.3. Characterization of optimal millicapsules

3.3.1. Mechanical stability

The alginate millicapsules undergo mechanical stress caused by compression, osmotic pressure, and shear forces during food processing. The diameter of millicapsules, amount of core loading, cross-linking time, strength, and the degree of the cross-linking exhibit a considerable effect on the mechanical stiffness. Alginate millicapsules are sensitive to deformation, rupture, and even undesirable early release of the core. Hence, the optimal millicapsules was coated with a chitosan layer for further analysis. The Young modulus values of the coated millicapsules as a function of chitosan concentration is plotted in **Fig. 5**. The millicapsules coated with chitosan concentrations higher than 0.5% w/v were stronger than the millicapsules without coating. There was not a significant difference (p<0.05) between the Young moduli of uncoated samples and the millicapsules coated with 0.25% w/v chitosan concentration (Fig. 5). The highest value of Young modulus and the mechanical stability was achieved at 1.5% w/v chitosan (16×10⁵ Pa), which was approximately 1.76 times higher than uncoated millicapsules (9.1×10⁵ Pa) (Fig.5).
This can be translated into the strong electrostatic interactions between amino groups of chitosan and carboxyl groups of alginate due to the reticulation of GG and MG blocks of alginate with
Ca²⁺ and the formation of cooperative ionic bonds between the M residues with the protonated amino groups of chitosan (Dupuy, Arien, & Minnot, 1994). Similar results were also reported by Bedade, Sutar, and Singhal (2019). Consequently, the optimal millicapsules were coated with 1.5% w/v chitosan concentration.

Insert figure.5 about here

### 3.3.2. Surface morphology

The surface morphology of coated and uncoated millicapsules containing CEO was assessed using scanning electron microscopy (Fig. 6, a-f). The micrographs (Fig6, c, d) exhibited small dents and rough external surface with many wrinkles on the matrix of the uncoated millicapsules. However, coating the millicapsules with chitosan drastically reduced the surface fissures and holes, making it rigid, smooth and dense (Fig. 6, e, f). Compared to the uncoated millicapsules, this structure provides a higher degree of core protection via the permeability reduction. Improving the mechanical strength of the chitosan-coated capsule was also reported by Sun, et al. (2020).

Insert figure.6 about here

### 3.3.3. Thermal properties

DSC curves of the pure CEO, shell materials, the unloaded, the CEO-loaded, and the chitosan-coated optimal millicapsules are depicted in Fig. 7. A broad endothermic peak at 78 °C associated with water loss and a sharp exothermic peak at 245.32 °C attributed to polymer chains decomposition were demonstrated in DSC curve of alginate powder (Fig. 7, a). The thermal
characteristics of pure CEO were also investigated by DSC thermograms (Fig. 7, b). Two endothermic peaks at 48.1 °C and 195.4 °C were detected, which assigned the evaporation of the residual solvent in essential oil and CEO degradation, respectively. It is worth mentioning that the residual solvent was not detected in the GC-MS chromatogram due to the delay-time of the dilution step with acetone (See section 2.2). According to Süfer and Bozok (2020), endothermic pattern of essential oil thermogram is strongly associated with double bonds and an indicator of decomposition. Shao, Yu, Chen, and Gao (2021) and Yang, et al. (2021) reported the degradation temperature of CEO as 159 °C and 166.26 °C, respectively. Evaluation of thermal properties approved the heat sensitivity and volatile nature of CEO. Elimination of absorbed water in chitosan powder was evident at 77 °C endothermic peak and a sharp exothermic peak at 295.21 °C revealed the decomposition onset of chitosan chains. The "egg-box" structure formation with calcium ions resulted in disappearing of the exothermic peak in the DSC thermogram of calcium-alginate (unloaded) millicapsules (Fig. 7, d) (Lupo, Maestro, Gutiérrez, & González, 2015).

There were two sharp endothermic peaks corresponded to loss of bound water in the thermograms of uncoated millicapsules (Fig. 7, d, e), which were disappeared in the chitosan-coated millicapsules due to complete coverage (Fig 7, f). Chitosan-alginate reaction can be confirmed by disappearing the exothermic peaks of pure chitosan and alginate (Fig 7, a, c, f) (Sankalia, Mashru, Sankalia, & Sutariya, 2007). As demonstrated in Fig. 7, f, there was only an endothermic characteristic peak at 225.7 °C, which was shifted to higher temperatures than the CEO thermogram. Furthermore, the evaporation peak of the residual solvent in CEO at 48.1 °C (Fig. 7, b) has disappeared in the thermograms of chitosan-coated and CEO-loaded millicapsules (Fig. 7, e, f). This revealed the successful incorporation of essential oil in encapsulating polymer and also higher thermal stability of encapsulated CEO. Besides, the absence of additional peaks for millicapsules compared to the pure samples pointed no interactions among CEO/ alginate/chitosan (Hadidi, Pouramin, Adinepour, Haghani, & Jafari, 2020).
Insert figure.7 about here

**3.3.4. FTIR**

The FTIR analysis confirmed the presence of core, shell component, and the interaction between the chitosan and shell layer, as highlighted in **Fig. 8**. As shown in **Fig.8, a**, characteristic absorption bands of alginate powder at 3433 cm⁻¹ (stretching vibration of OH), 2924 cm⁻¹ (CH), 1629 cm⁻¹ (COO⁻, asymmetric), and 1029 cm⁻¹ (C-O-C) were similar to other researches (Angadi, Manjeshwar, & Aminabhavi, 2012; Daemi & Barikani, 2012). CH2-stretching vibration of the pure CEO was observed at 2814 cm⁻¹ (**Fig.8, b**). Characteristic absorption peaks at 1677 and 1625 cm⁻¹ were associated with aldehyde carbonyl (C=O stretching vibration) and also the presence of the cinnamaldehydes and aldehydes in the essential oil. Other peaks around 1575 and 1124 cm⁻¹ were related to the aromatic ring (C=O skeleton vibration) and C-O deformation vibration, respectively (de Oliveira, Paula, & de Paula, 2014). The bands such as those at 1450 and 1294 cm⁻¹ originated from various organic compounds verified in the CEO by GC/MS analysis (see **Table 2**). The FTIR spectrum of chitosan powder (**Fig.8, c**) demonstrated the characteristic bands at 3436, 2873, 1660, 1580, 1422, 1381, 1159, and 895 cm⁻¹ that corresponded to OH stretching, C-H stretching, C=O stretching of amide I, bending vibrations of N-H in N-acetylated residues, N-H stretching of amide II, N-H stretching of amide III, C-O-C stretching, and pyranose ring, respectively (Hasheminejad, Khodaiyan, & Safari, 2019).

Incorporation of the CEO in the calcium-alginate millicapsules (**Fig. 8, e**) led to a higher intensity for the CH stretching peak at 2855 cm⁻¹, which was associated with the CEO ester groups (Hosseini, et al., 2013). Furthermore, similar peaks of the CEO and CEO-loaded spectra with minor differences revealed the successful entrapment of the CEO in the millicapsules
without interaction with the alginate matrix. The spectra of CEO-loaded and chitosan-CEO-loaded millicapsules (Fig. 8, e, f) showed many similarities with the CEO spectrum (Fig. 8, b). However, due to the millicapsules and CEO bands overlapping, some small variations in the intensities and the shapes of the bands were observed. Sharpening of the peak at 1625 cm⁻¹ due to the alginate carboxyl groups and disappearance of the bands at 1580 cm⁻¹ revealed the formation of electrostatic interactions between chitosan and alginate (Lai, Lin, Mo, Xu, & Lin, 2017).

Insert figure.8 about here

### 3.3.5. Crystallinity

Products of microencapsulation process with crystalline structure tend to dissolve slowly in solution due to the dissolution of surface crystals which exposed to solvent (de Barros Fernandes, et al., 2016). Hence, XRD analysis was performed to confirm the successful encapsulation of CEO in the chitosan-loaded millicapsules and its impact on the physical state of shell materials. The alginate powder (Fig. 9, a) exhibited some characteristic peaks at 13.5 °, 23.17 °, 24.19 °, 26.67 °, 30.19 °, and 36.75 ° (2θ), which showed the semi-crystallinity structure of alginate (Lakouraj, Mojerlou, & Zare, 2014). The diffraction spectrum of the chitosan powder showed a weak and a strong peak at 2θ of 11 ° and 20.14 °, respectively (Fig. 9, b). These peaks represented that chitosan had two different forms of crystal morphologies. The first peak is related to hydrated forms and the next one corresponds to a more regular anhydrous form (Ogawa, Yui, & Miya, 1992).

No peak was shown in the diffractogram of unloaded and chitosan-unloaded millicapsules (Fig. 9, c, d), which is implied destruction of the crystalline structure of the alginate and chitosan during ionic gelation and electrostatic binding, respectively. In other words, complexion of the
chitosan and alginate resulted in slowing down the formation of the hydrogen bonds among amino
groups and hydroxyl groups, so a sharp peak of the chitosan powder disappeared. Furthermore,
the chitosan-unloaded millicapsules showed an amorphous morphology (Liang, Liu, & Wu, 2007;
Lim & Ahmad, 2017). The disappearance of the exothermic peaks of chitosan and alginate proved
the successful interaction between the two biopolymers. This result implies the transformation
possibility of the crystal to amorphous structure (Crcarevska, Dodov, & Goracinova, 2008). As
compared with chitosan-unloaded millicapsule, the characteristic peak at 2θ of 23 ° in the X-ray
diffraction pattern of CEO-loaded millicapsule confirmed the CEO entrapping in the chitosan-
millicapsules (Fig. 9, f). Accordingly, it is obvious that the incorporation of CEO might result in
the disarray of the chitosan-coated millicapsules packing structure. A similar transition from
crystalline form to amorphous has already been observed recently (Bagheri, Ariaii, &
Motamedzadegan, 2020; Hosseini, et al., 2013; Matshetshe, Parani, Manki, & Oluwafemi, 2018).

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**3.3.6. In vitro digestion and release kinetic in the simulated gastrointestinal tract (GIT)**

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The release behavior of the CEO from the optimal coated and uncoated (control)
millicapsules in the mouth (2 min), stomach (120 min), and small intestine (120 min) is depicted
in **Fig. 10**. As shown in the figure, only a small amount of CEO (2.06 %) was released from
coated millicapsules in the SSF. This was attributed to the short-time of exposure to simulated
saliva system and coating matrix. However, unlike the oral phase, rate of the CEO release from
coated millicapsule reached to 27.38 % in SGF (**Fig. 10**). The intense acidic condition (pH= 2.5)
and existence of pepsin enzyme in the SGF could promote the release from millicapsules, but it
was obvious that chitosan coating retarded the CEO release rate. The electrostatic interaction
between amino groups of chitosan and carboxylic groups of alginate play an important role in retarding the release rate (Luo & Wang, 2014). At the end of this stage, 72.62 % of the CEO was preserved in the coated millicapsules. The higher and faster release of CEO from coated millicapsules was observed in the small intestine (Fig. 10). The CEO release was increased during exposure time in SIF, which coated millicapsules released 77.96 % of its loaded oil during passing 240 min in GI tract phases. The fast release of CEO was associated with the high number of digested millicapsules. As a result, entrapped CEO in the partially digested millicapsules was released further in the SIF (pH= 7) due to the effect of pancreatin enzyme on wall material (J. Fu, et al., 2020). The CEO-loaded millicapsules with chitosan coating can assuredly be applied as intestinal site-specific delivery system, because of its pH-dependent behavior in targeted release.

According to Fig. 10, 7.06 % of CEO was released from the control sample into SSF (during the first 2 min of digestion). However, about 66.8 % of CEO was leached from the control sample into SGF, which was 2.5 times higher than the coated millicapsules leakage. In the intestine stage, the CEO loaded in the control sample was completely released during 40 min and the millicapsules were digested. Lack of chitosan coating led to the fast release of CEO in the acidic conditions of stomach (Fig. 10).

Insert figure.10 about here

The model-fitting kinetics of the CEO release from coated and uncoated millicapsules with various models (Higuchi, Ritger-Peppas, Korsmeyer-Peppas, and Kopcha) revealed that the Ritger-Peppas model was regarded as the most suitable model for describing the mechanism of CEO release in coated millicapsules. The mentioned model showed the highest R² and the lowest RMSE values compared to the other models in all digestion phases of coated millicapsules (Table 4). In Ritger-Peppas model, the n value presents the release mechanism of essential oil. According
to **Table 4**, for coated millicapsules the n parameter in mouth and small intestine phases were higher than 0.85, indicating that the dominant mechanism was non-Fickian diffusion (super case II) as an extreme type of transfer. In the stomach phase, the n value of millicapsule was between 0.43 and 0.85 (Table 4), denoting non-Fickian diffusion (anomalous transport) (Ritger, et al., 1987).

Basically, three important phenomena happen during the contact of medium with the millicapsules: diffusion, swelling and erosion. From the mathematical point of view, these processes could be described using two models: Fickian (case I) and non-Fickian (case II, Anomalous Case, and Super Case II) (Malekjani & Jafari, 2021). In this study, swelling and polymeric chain relaxation (disentanglement of the matrix) were the main release mechanisms during the contact of millicapsules with SGF (anomalous transport), so that, SGF penetrated rapidly into the structure of the millicapsules, while the matrix slowly dissolved into the SGF (pH=2.5). The thickness of the matrix enhanced due to the SGF uptake and consequently, disentanglement of the polymeric chain occurred.

Furthermore, $|A/B|<1$ in the Kopcha model of coated millicapsules in the three phases indicated the matrix erosion (Kopcha, et al., 1990). In this case, the degradation rate of the matrix was much faster than the penetration of the medium into the polymeric structure (Malekjani & Jafari, 2021).

Noppakundilograt, Piboon, Graisuwan, Nuisin, and Kiatkamjornwong (2015) also proposed Ritger-Peppas model with non-Fickian transport as the best model of release mechanism of eucalyptus oil from alginate microcapsule. Ritger-Peppas model was selected as the most suitable model in mouth and small intestine for the control sample. However, Kopcha was the best model to describe the release of CEO from the uncoated millicapsules in the stomach. The n values of the Ritger-Peppas model in mouth and small intestine stages denoted non-Fickian
diffusion (anomalous transport in mouth and super case II in small intestine). |A/B|>1 of Kopcha model in the stomach stage of the control sample represented Fickian diffusion (Kopcha, et al., 1990).

Insert table.4 about here

4. Conclusion

In this work, the CEO was encapsulated using millifluidic device and RSM was applied for assessing the effects of the independent variables on the EE, LC, size, and SF of the produced millicapsules. The results showed that all three variables had significant effects (p< 0.05) on the responses. The optimal millimeter-scale capsule (3.58± 0.23 mm) had the highest EE (98.96± 1.2%) and LC (70.14± 1.8%) values and it could be considered as a completely round millicapsules (SF= 0.96± 0.01). The chitosan-coating layer (at 1.5% w/v concentration) created the highest mechanical stability in the millicapsules. Surface fissures, pleats, and holes were disappeared in the SEM image of the coated millicapsules. Endothermic peak in the chitosan-CEO- loaded millicapsules was shifted to the higher temperatures, which highlighted the higher thermal stability of the encapsulated CEO. Similar peaks with minor differences in the FTIR spectrums of free CEO and its encapsulated form also confirmed the successful entrapment without any chemical interaction with the shell matrix. Transition of the crystalline form of pure materials to the amorphous form of millicapsules also proved the proper incorporation of the CEO. Release kinetics of cinnamon essential oil in the simulated conditions of GIT followed a non-Fickian mechanism. Ritger-Peppas model was selected as the most suitable model to predict the essential oil release from coated millicapsules. However, Fickian diffusion was observed for uncoated millicapsules in stomach. Concerning to the results, the designed millicapsules could
701 be considered as promising delivery system for well-controlling the CEO release and would be
702 a good choice for intestinal site-specific delivery system.

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964
Figures:

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Double-stage millifluidic method for preparing the oil-core millicapsules: CEO as dispersed phase is extruded through the co-axial flow into the alginate solution (continuous phase).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Three-dimensional plots of the response surfaces for EE (A-C) and LC (D-F) as a function of concentration ([Alg]) and flow rate of alginate (QAlg) (A, D), alginate concentration ([Alg]) and flow rate of CEO (QCEO) (B, E), and flow rates of CEO (QCEO) and alginate (QAlg) (C, F).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Three-dimensional plots of the response surfaces for the size (A-C) and the SF (D-F) as a function of the concentration and the flow rate of alginate (A, D), the alginate concentration and the flow rate of CEO (B, E), and the flow rates of CEO and alginate (C, F).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Droplet formation pattern (A) and droplet detachment (B). Formation of optimal millicapsules at the moment of gelling (C) and after 30 min curing-time (D)</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 5. Young modulus versus chitosan concentration for the optimal millicapsules</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Digital microscopy (a and b, ×30) and FESEM (c-f, ×80-500) images of CEO-loaded millicapsules (stage 1) and chitosan-coated millicapsules with CEO core (stage 2) prepared at the optimized conditions</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** DSC thermograms of sodium alginate (a), pure CEO (b), chitosan (c), unloaded millicapsule (d), CEO-loaded millicapsule (e), and chitosan-CEO-loaded millicapsule (f).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 8.** FTIR spectra of sodium alginate (a), pure CEO (b), chitosan (c), unloaded millicapsules (d), CEO-loaded millicapsules (e), and chitosan-CEO-loaded millicapsules (f).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 9.** XRD patterns of sodium alginate (a), chitosan (b), unloaded millicapsules (c), chitosan-unloaded millicapsules (d), CEO-loaded millicapsules (e), and chitosan-CEO-loaded millicapsules (f).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 10.** The cumulative release profile of the cinnamon essential oil from the coated and uncoated millicapsules during in vitro digestion</figcaption></figure>
Tables:

<table><caption>**Table 1.** Coded and uncoded factors for RSM ascertained from 17 experimental runs of Box-Behnken design</caption><thead><tr><th rowspan="2">Run</th><th colspan="3">Uncoded variables</th><th colspan="3">Coded variables</th></tr><tr><th>[Alg],<br>(g/L)</th><th>Q<sub>Alg</sub>,<br>(mL/min)</th><th>Q<sub>CEO</sub>,<br>(mL/min)</th><th>X<sub>1</sub></th><th>X<sub>2</sub></th><th>X<sub>3</sub></th></tr></thead><tbody><tr><td>1</td><td>25</td><td>1.6</td><td>0.8</td><td>0</td><td>+1</td><td>+1</td></tr><tr><td>2</td><td>25</td><td>1.3</td><td>0.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>3</td><td>25</td><td>1.3</td><td>0.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>4</td><td>20</td><td>1.3</td><td>0.6</td><td>-1</td><td>0</td><td>-1</td></tr><tr><td>5</td><td>25</td><td>1.0</td><td>0.6</td><td>0</td><td>-1</td><td>-1</td></tr><tr><td>6</td><td>25</td><td>1.3</td><td>0.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>7</td><td>25</td><td>1.3</td><td>0.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>8</td><td>30</td><td>1.3</td><td>0.6</td><td>+1</td><td>0</td><td>-1</td></tr><tr><td>9</td><td>30</td><td>1.6</td><td>0.7</td><td>+1</td><td>+1</td><td>0</td></tr><tr><td>10</td><td>25</td><td>1.3</td><td>0.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>11</td><td>20</td><td>1.0</td><td>0.7</td><td>-1</td><td>-1</td><td>0</td></tr><tr><td>12</td><td>20</td><td>1.6</td><td>0.7</td><td>-1</td><td>+1</td><td>0</td></tr><tr><td>13</td><td>20</td><td>1.3</td><td>0.8</td><td>-1</td><td>0</td><td>+1</td></tr><tr><td>14</td><td>30</td><td>1.3</td><td>0.8</td><td>+1</td><td>0</td><td>+1</td></tr><tr><td>15</td><td>30</td><td>1.0</td><td>0.7</td><td>+1</td><td>-1</td><td>0</td></tr><tr><td>16</td><td>25</td><td>1.0</td><td>0.8</td><td>0</td><td>-1</td><td>+1</td></tr><tr><td>17</td><td>25</td><td>1.6</td><td>0.6</td><td>0</td><td>+1</td><td>-1</td></tr></tbody></table>
<table><caption>**Table 2.** Chemical compositions of the CEO identified by GC-MS</caption><thead><tr><td>No</td><td>Compounds</td><td>Concentration (%)</td></tr></thead><tbody><tr><td>1</td><td>Hexadecanol-2</td><td>0.70</td></tr><tr><td>2</td><td>Decane</td><td>6.27</td></tr><tr><td>3</td><td>M- Pyrrol</td><td>3.38</td></tr><tr><td>4</td><td>Linalool</td><td>1.14</td></tr><tr><td>5</td><td>Cis-Cinnamaldehyde</td><td>0.58</td></tr><tr><td>6</td><td>Trans-Cinnamaldehyde</td><td>84.04</td></tr><tr><td>7</td><td>Eugenol</td><td>1.90</td></tr><tr><td>8</td><td>Alfa-Copaene</td><td>0.63</td></tr><tr><td>9</td><td>Caryophyllene</td><td>0.84</td></tr><tr><td>10</td><td>Cadina-1 (10),4-diene</td><td>0.49</td></tr><tr><td>11</td><td>Un-identified traces</td><td>0.03</td></tr></tbody></table>
<table><caption>**Table 3.** Second-ordered quadratic models developed for EE, LC, size and SF</caption><thead><tr><th>Responses</th><th>Predicted models</th><th>R<sup>2</sup></th><th>R<sup>2</sup><sub>adj</sub></th><th>Covariance</th><th>Adequate<br>precision</th></tr></thead><tbody><tr><td>EE</td><td>Y<sub>1</sub> (EE) = 98.58+ 0.84 X<sub>1</sub> + 0.17 X<sub>2</sub> - 0.05 X<sub>3</sub> - 0.08 X<sub>1</sub>X<sub>2</sub> - 0.10 X<sub>1</sub>X<sub>3</sub> - 0.01 X<sub>2</sub>X<sub>3</sub> - 0.16 X<sub>1</sub><sup>2</sup> + 0.03 X<sub>2</sub><sup>2</sup> - 0.09 X<sub>3</sub><sup>2</sup></td><td>0.99</td><td>0.99</td><td>0.01</td><td>207.97</td></tr><tr><td>LC</td><td>Y<sub>2</sub> (LC) = 55.35+ 8.82 X<sub>1</sub> - 3.77 X<sub>2</sub> + 0.65 X<sub>3</sub> - 0.33 X<sub>1</sub>X<sub>2</sub> - 0.47 X<sub>1</sub>X<sub>3</sub> - 1.28 X<sub>2</sub>X<sub>3</sub> + 0.73 X<sub>1</sub><sup>2</sup> + 1.21 X<sub>2</sub><sup>2</sup> + 0.71 X<sub>3</sub><sup>2</sup></td><td>0.99</td><td>0.99</td><td>0.1</td><td>568.80</td></tr><tr><td>Size</td><td>Y<sub>3</sub> (size) = 3.63- 0.18 X<sub>1</sub> - 0.08 X<sub>2</sub> + 0.02 X<sub>3</sub> + 0.01 X<sub>1</sub>X<sub>2</sub> + (5×10<sup>-3</sup>) X<sub>1</sub>X<sub>3</sub> + (5×10<sup>-3</sup>) X<sub>2</sub>X<sub>3</sub> + 0.05 X<sub>1</sub><sup>2</sup> + 0.02 X<sub>2</sub><sup>2</sup> + (10<sup>-3</sup>) X<sub>3</sub><sup>2</sup></td><td>0.99</td><td>0.99</td><td>0.19</td><td>98.74</td></tr><tr><td>SF</td><td>Y<sub>4</sub> (SF) = 0.95+ 0.02 X<sub>1</sub> + 0.01 X<sub>2</sub> - (3.75×10<sup>-3</sup>) X<sub>3</sub> + (5×10<sup>-3</sup>) X<sub>1</sub>X<sub>3</sub> + (7.5×10<sup>-3</sup>) X<sub>2</sub>X<sub>3</sub></td><td>0.96</td><td>0.95</td><td>0.41</td><td>25.14</td></tr></tbody></table>
<table><caption>**Table 4.** Kinetics constant of cinnamon essential oil release profile in the simulated GIT (mouth, stomach, and small intestine)</caption><thead><tr><th rowspan="2">Models</th><th rowspan="2">Parameters</th><th colspan="2">Mouth</th><th colspan="2">Stomach</th><th colspan="2">Small intestine</th></tr><tr><th>coated</th><th>uncoated</th><th>Coated</th><th>uncoated</th><th>coated</th><th>Uncoated</th></tr></thead><tbody><tr><td rowspan="3">Higuchi</td><td>K</td><td>0.03±0.04</td><td>7.97±0.11</td><td>2.61±0.06</td><td>1.24±0.09</td><td>0.51±0.01</td><td>1.22±0.19</td></tr><tr><td>R<sup>2</sup></td><td>0.96</td><td>0.91</td><td>0.98</td><td>0.64</td><td>0.93</td><td>0.97</td></tr><tr><td>RMSE</td><td>5.31</td><td>0.46</td><td>6.03</td><td>2.66</td><td>1.27</td><td>0.01</td></tr><tr><td rowspan="4">Ritger-Peppas</td><td>K</td><td>0.02±0.01</td><td>4.64±1.02</td><td>2.51±0.17</td><td>5.86±0.77</td><td>0.05±0.01</td><td>0.25±0.04</td></tr><tr><td>n</td><td>1.02±0.18</td><td>0.63±0.03</td><td>0.51±0.07</td><td>0.49±0.31</td><td>1.33±0.04</td><td>1.17±0.08</td></tr><tr><td>R<sup>2</sup></td><td>0.96</td><td>0.99</td><td>0.99</td><td>0.93</td><td>0.99</td><td>0.99</td></tr><tr><td>RMSE</td><td>1.19</td><td>0.07</td><td>0.3</td><td>1.33</td><td>0.7</td><td>0.08</td></tr><tr><td rowspan="4">Korsmeyer-Peppas</td><td>K</td><td>104.85±1.17</td><td>464.18±2.43</td><td>272.35±3.44</td><td>58.61±2.41</td><td>8.41±0.59</td><td>25.27±1.76</td></tr><tr><td>n</td><td>1.01±0.03</td><td>0.63±0.03</td><td>0.49±0.01</td><td>0.49±0.66</td><td>1.21±0.06</td><td>1.17±0.06</td></tr><tr><td>R<sup>2</sup></td><td>0.96</td><td>0.99</td><td>0.99</td><td>0.94</td><td>0.98</td><td>0.99</td></tr><tr><td>RMSE</td><td>2.31</td><td>7.05</td><td>0.67</td><td>0.52</td><td>6.43</td><td>8.25</td></tr><tr><td rowspan="4">Kopcha</td><td>A</td><td>0.56±0.04</td><td>3.44±0.19</td><td>-0.22±0.13</td><td>5.43±0.72</td><td>-0.23±0.3</td><td>-2.62±0.55</td></tr><tr><td>B</td><td>0.77±0.02</td><td>1.17±0.05</td><td>0.38±0.06</td><td>0.04±0.01</td><td>0.41±0.02</td><td>0.83±0.04</td></tr><tr><td>R<sup>2</sup></td><td>0.95</td><td>0.99</td><td>0.82</td><td>0.96</td><td>0.96</td><td>0.98</td></tr><tr><td>RMSE</td><td>2.79</td><td>0.35</td><td>9.02</td><td>0.08</td><td>8.63</td><td>0.93</td></tr></tbody></table>
<figure><img src="image_12.png" /></figure>

Cinnamon essential oil

<figure><img src="image_13.png" /></figure>

Alginate+ Tween

<figure><img src="image_14.png" /></figure>

Inlet of shell
solution

<figure><img src="image_15.png" /></figure>

Inlet of core material

<figure><img src="image_16.png" /></figure>

Calcium chloride bath

Chitosan layer

<figure><img src="image_17.png" /></figure>

CEO

<figure><img src="image_18.png" /></figure>

CLSM

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

Optimal millicapsule

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>