Preparation and evaluation of a functional effervescent powder based on inclusion complexes of orange oil and β-cyclodextrin derivatives

Abstract Flavoured functional effervescent powders are becoming increasingly popular by consumers due to their health benefits and easy dissolution. In present study, orange flavoured effervescent powders having functional properties were prepared. Orange oil (O) was blended with different essential oils (EOs) that have high antioxidant activity. The orange oil and the O-EOs blends were subjected to odour sensory evaluation, gas chromatography-mass spectrometry (GC-MS) analysis and evaluation of radical scavenging activity. Combinations of two water soluble β-cyclodextrin polymers, 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) and epichlorohydrin-β-cyclodextrin (EPI-β-CD), were prepared at different molar ratios (3:1 and 1:3, F1 and F2, respectively). The O-EOs blends that showed the highest antioxidant activities and best odour qualities were encapsulated with F1 and F2, separately. The orange flavoured inclusion complexes were prepared by freeze drying method. The particle sizes of the inclusion complex powders were in the nanoscale. Characterization of the inclusion complexes nanoparticles was performed by scanning electron microscopy (SEM), and fourier transform infrared microscopy (FT-IR). The results confirmed the successful formation of the inclusion complexes. However, inclusion complex of O-EOs (300 mg) blend with F1 (OF1cIC) showed the smallest particle size (113.90±15.90 nm), the more negative zeta potential (−27.10±1.27 mV), the highest encapsulation efficiency (95.51%) and best odour quality. Therefore, it was mixed with an effervescent powder having high acceptable characteristics. The orange flavoured effervescent powder showed superior flowability.

Preparation and evaluation of a functional effervescent powder based on inclusion complexes of orange oil and β-cyclodextrin derivatives 1

. Introduction
In the upcoming time, the global market for effervescent products will be grown signi cantly due to their enjoyable sensation, ease in swallowing and health bene ts [1]. Global effervescent production market is expected to increase from USD 4.217 million in 2022 to USD 64.08 million in 2029 [2]. Effervescent products contain a mixture of acids such as citric acid (CA) and tartaric acid (TA) with sodium bicarbonate (NaHCO 3 ) that can react quickly after adding water and produce carbon dioxide (CO 2 ) gas causing a refreshing sparkle effect [1,3]. Chemical preservatives are used to improve the microbiological and oxidative stability of carbonated beverages, however, rising health consciousness in uenced people to shift towards healthy products. The beverages industries are capitalizing on this trend by producing effervescent products having functional properties to improve consumer health and lifestyle [4]. The use of essential oils (EOs) as functional ingredients in food and beverages industries is gaining momentum, both for their relatively safe status, their wide acceptance by consumers and their exploitation for potential multi-purpose functional use [5]. However, the use of EOs as natural preservatives and avoures is greatly limited by their volatility and low water solubility. In addition to being aromatic compounds, EOs are sensitive to light and oxidation and thus lose their quality. Encapsulation is the main option to protect and preserve avour in food products [6. In last years, encapsulation technology has been developed signi cantly for the design and formulation procedures for stabilization, solubilization and delivery of the active compounds in food and pharmaceutical industries [7]. Encapsulation with cyclodextrins (CDs) can improve the stability of sensitive ingredients such as EOs [6], extend their shelf-life, protect them from oxidation and control release of their components [8]. Cyclodextrins have a relatively hydrophilic external surface and a hydrophobic interior cavity [9]. This unique structure allows them to form inclusion complexes. Inclusion complex is a unique form of chemical structure in which guest molecule is enclosed within the cavity of the host. Among CDs, β-cyclodextrin (β-CD) is the most widely used due to its suitable cavity size for common guests with molecular weight between 200 and 800 Da, availability and reasonable price [10]. However, due to its low aqueous solubility, the use of β-CD is limited in formulations [11]. To overcome this issue, hydrophilic derivatives of β-CD have been investigated. 2-Hydroxypropyl-β-cyclodextrin (2-HP-β-CD) derivative is relatively high aqueous soluble (above 60 g in 100 mL water at 25°C) with low toxicity and satisfactory inclusion ability [12]. Since few years, studies have been carried out toward the use of epichlorohydrin-βcyclodextrin (EPI-β-CD) polymer [13,14]. The polymerized form of β-cyclodextrin viz epichloro-βcyclodextrin remains within the cavity structure of β-CD providing capability of forming inclusion complexes with a variety of guest molecules [15]. Furthermore, it is proved to be more effective compared to its parent β-CD [16]. Orange avoured effervescent is very popular to consumers. The main objective of the present study was to prepare orange avoured effervescent powder having promising antioxidant activity and high avour quality. To achieve this aim the antioxidant activity of orange oil was improved by blending with essential oils (EOs) having high antioxidant activity. A mixture of two soluble derivatives of β-cyclodextrin (2-HP-β-CD and EPI-β-CD) at different molar ratios were used to encapsulate the orange -essential oils (O-EOs) blend that showed the highest antioxidant activity and avour acceptability. The produced inclusion complexes were freeze dried and those showed the highest entrapping e ciency were subjected to different physicochemical analysis. The effervescent powders of the orange avoured powder was prepared and subjected to further physical analysis.
All other chemicals were of analytical grade and the solvents were puri ed and distilled before use.

Preparation of orange oil-essential oil blends
Orange oil (100 µL) was blended with different amounts (0.25 µL, 0.50 µL and 0.75 µL) of the essential oils of camphor, basil and marjoram to improve its antioxidant activity, while preserving the characteristic avour. The blends showed the best results were blended with 0.1 µL of oregano essential oil.

Odour acceptability evaluation
The odour acceptability of each orange oil-essential oil (O-EO) blend was conducted by 10 well trained panelists drawn from Food Technology and Nutrition Institute. The panelists sniffed and scored the perceived orange aroma of each blend on a category scale 0.0 (not acceptable) to 10.0 strongly acceptable.
Results were expressed as percentage of DPPH or ABTS radical inhibition according to Eq. 1. Where: A 0 is the absorbance of the control sample and A s is the absorbance of sample. All preparation and assays were performed in triplicate 2.2.1.3. Gas chromatography-mass spectrometry (GC-MS) analysis Orange oil, EOs and their blends were subjected to GC-MS analysis using a gas chromatography (Agilent 8890 GC System) coupled to a mass spectrometer (Agilent 5977B GC/MSD) and equipped with a HP-5MS fused silica capillary column (30 m, 0.25 mm i.d., 0.25 mm lm thickness) according to Fadel et al. [20].
Qualitative analysis of the separated peaks was carried out by matching with data from the library of mass spectra (National Institute of Standard and Technology, NIST) and comparison with those of authentic compounds and published data [21]. While the quantitative analysis was performed by the normalization method.

Preparation of orange avoured inclusion complexes (O-F-IC)
The inclusion complexes were prepared via freeze-drying method [22]. Two water soluble β-cyclodextrine derivatives, 2-HP-β-CD and EPI-β-CD, were mixed at 3: 1 and 1: 3 (F1 and F2) molar ratios, respectively. Each mixture (2g) was dissolved separately in 15 mL distilled water and stirred at 600 rpm for 30 min. The O-EOs blend showed the best odour acceptability and highest antioxidant activity was selected and added at different contents (200, 250 and 300 mg) to each aqueous solution of the β-CDs mixtures (Table 1S). The solutions were agitated using the vortex till disappearance of any oil droplets then stirred at 150 rpm for 48h to ensure complete entrapment of the O-EOs blend. The obtained inclusion complexes solutions were lyophilized at − 80°C, under pressure of 0.01 m bar (Alpha-1-4 LSC model, CHRIST, freeze drier, Harz-Germany). The lyophilized inclusion complexes were stored in sealed containers inside a desiccator at 20°C until use. For each loaded sample EE were determined in triplicate.
The antioxidant activity of each entrapped O-EOs blend was determined as mention above.

Characterization of the orange avoured inclusion complexes.
The following methods were selected to evaluate the physical properties, the particle morphology, group interaction of the O-EOs blend with the mixture of soluble polymers β-CDs (2-HP-β-CD + EPI-β-CD) of the inclusion complexes that showed the highest EE, best odour acceptability and highest antioxidant activity.

Scanning electron microscopy (SEM)
The surface morphology of the selected orange avoured inclusion complexes was examined by scanning electron microscopy (QUANTA FEG 250, Oregon, USA) under 15 Kv accelerating voltage. Each sample was coated with a ne layer of gold/ palladium before viewing under 500 times magni cation.

Fourier transform infrared spectroscopy (FT-IR) analysis
The possible interaction between the selected O-EOs blend and the mixture of β-cyclodextrine derivatives was detected by FT-IR analysis. The samples were scanned in infrared range of 400-4000 cm -1 (JASCO 6100, Tokyo, Japan). FT-IR samples were prepared in the form of potassium bromide pellets.

Preparation of effervescent powder
Different amount of citric acid (CA), tartaric acid (TA) and sodium bicarbonate (NaHCO 3 ) in the ratio of 1: 2: 3.5 were used for the formulation of the effervescent powder [24,25]. Each formula was dissolved in 50 mL distilled water and investigated regarding their effect on effervescent time, clarity and pH of the obtained solutions. Effervescent time, the moment at which a clear solution was obtained after effervescent completion, was determined using stop watch [1]. Clarity of the obtained solutions was assessed visually, while pH was determined using a digital pH meter (Vstar 92, Orion Versa Star Tm , Thermo Fisher Scienti c INC, USA) at 25°C. The formula of the effervescent components that showed the acceptable results was selected and mixed with 5 g fructose, as a sweetener, and then mixed well with the selected orange avoured inclusion complex (O-CDs-IC).

Evaluation of the physical properties of orange avoured effervescent powder 2.2.5.1. Powder density
The bulk and tapped densities of the powder were determined according to the following equations 3 and 4 [25].
Bulk density (g/mL) = Weight of sample/Bulk volume Eq. 3 Tapped density (g/mL) = Weight of sample/Tapped volume Eq. 4 Bulk volume: is the volume of certain weight of sample measured by a graduated cylinder.
Tapped volume: is the minimum volume of sample reached by tapping the cylinder

Determination of compressibility Carr's index
Compressibility is the ability of a powder to decrease in volume under pressure. It is a simple, fast, and accurate method for predicting the powder ow characteristics [25]. Carr's index of the effervescent powder was calculated from Eq. 5.

Determination of Hausner ratio
The Hausner ratio for the effervescent powder was calculated by the following Eq. 6 [25].
Hausner ratio = Tapped density/ Bulk density Eq. 6 It is related to inter-particle friction and can be used to assess powder ow properties.

Measurement of angle of repose
The owability of the selected orange avoured effervescent powder was assessed by measuring the angle of repose by a xed funnel method according to Hussain and Al-khedairy [25]. The data were statistically processed to estimate the average ± standard deviation (SD) of triplicates.

Radical scavenging activity of the essential oils
In the present study DPPH and ABTS radicals assays were used to determine the free radical scavenging activity of orange oil (O) and Ca, Ma, Ba and Or EOs. The radical scavenging activity of both method showed a similar decreasing order such as Ba > Ca > Or > Ma > O (Fig. 1a, b). The highest inhibition of the free radicals DPPH and ABTS by basil EO may be attributed to the presence of linalool and eugenol in considerable concentrations (Table 2S). It was reported that the basil EO of linalool-eugenol chemo type showed high AOA [29]. The synergistic interaction between the antioxidant compounds found in the basil  [31], in addition to the synergistic interaction among other antioxidant compounds such as linalool, α-terpeneol, ρ-cymene and 1, 8-cineole (Table 2S)

Odour sensory evaluation and radical scavenging activity of orange-EOs blends (O-EOs)
Blends of orange oil with Ca, Ba or Ma EOs at different concentrations, 0.25, 0.5 and 0.75 µL /100 µL orange oil, were subjected to odour sensory analysis. As shown in Table 1, the blends containing orange oil and each EO at concentration of 0.25 µL /100 µL orange oil showed the highest scores. Therefore, these blends were selected and subjected to GC-MS analysis and determination of their AOA (Table 2 and Fig. 1c,   d). As shown in Fig. 1c, addition of 0.25 µL of each EO (basil, camphor and marjoram) to orange oil increased the inhibition of the free radical DPPH from 19.31% to 57.02, 24.33 and 22.90%, respectively. The scavenging ability of the three blends on ABTS radical showed the same trend as DPPH (Fig. 1d). This nding is correlated to the presence of the antioxidant compounds in the three blends ( Table 2). As shown in Fig. 1c, (Table 2 and Fig. 1c). As shown from Fig. 1d, the results of ABTS assay showed the same trend. From Fig. 1c, d and Table 1, O + Ba + Or blend showed the highest scavenging ability (60.79%) and highest odour sensory score, therefore it was encapsulated at different quantities in β-CDs derivatives (F1 and F2) (Table 1S) 3.4. Odour acceptability and encapsulation e ciency of the orange avoured inclusion complexes As shown in Table 3 .8% [32]. As shown in Table 5, O-F1-IC showed higher EE than O-F2-IC. This nding may be correlated to the fact that increasing the concentration of CDs resulted in increasing the spaces occupied by CDs polymers and causing a decrease of the free volume within the polymer matrix (a compact structure with smaller pore sizes) and subsequently the amount of the oil that can be encapsulated within those pores will be decreased [33]. Previous studies revealed that the physicochemical properties of both the CDs polymers (cavity diameter, derivative nature) and the encapsulated guest (geometry, volume and hydrophobicity) play a crucial role in the formation of inclusion complexes [6,34]. The higher EE in the present study may be attributed to the freeze drying process used in preparation of the inclusion complexes, which prevent the degradation and evaporation of the volatile compounds and thus minimizes their loss [35].  For each column, same letters means insigni cant difference at p < 0.05

Antioxidant activity of the encapsulated O-EOs blend
The antioxidant activity of the encapsulated O-EOs in the inclusion complexes (Table 3) was lower than that of the free O-EOs blend (Fig. 1c). This decrease could be because the cyclodextrine polymers block the functional groups of the active compounds that react with DPPH radical [36], bearing in mind that the cyclodextrine polymers didn't have antioxidant activity. A positive correlation was found between the DPPH scavenging activity and encapsulation e ciency [11]. This result is in agreement with previous studies. The guava life oil encapsulated in 2-HP-β-CD showed slightly lower DPPH scavenging activity than that of the free guava oil [37]. Similar results have been reported by Rakmai et al. [11] who correlated the antioxidant activity of black pepper oil encapsulated in HP-β-CD to the presence of the antioxidant compounds limonene and α-pinene.

Characterization of the orange avoured inclusion complexes
As shown in Table 3 inclusion complexes O-F1C-IC, O-F2B-IC and O-F2C-IC showed the best results regarding, odour acceptability, EE and DPPH scavenging activity, therefore were selected to be characterized.
3.6.1. Particle size, polydispersity and zeta potential As shown in Table 4, the particle sizes of three investigated inclusion complexes were in the nanoparticle scale [38]. Among these samples, O-F1C-IC showed the smallest particle size and highest EE (Table 3 and  Table 4). This result is consistent with previous study of Hadian et al. [34] who reported an opposite relationship between EE and size of nanoparticle of β-cyclodextrin containing geraniol. The higher particle sizes of O-F2B-IC and O-F2C-IC than O-F1C-IC may be correlated to their higher content of EPI-β-CD which has a higher molecular weight than 2-HP-β-CD. It has been reported that increases in β-CD concentration induce aggregation and results in increasing the particle size [38][39][40]. Polydispersity index (PDI) of the inclusion complex nanoparticles was < 0.5. This indicates their stability and homogeneity [34].   For each column, same letters means insigni cant difference at p < 0.05

. Morphology of the orange avoured inclusion complexes
The particle morphology of the three orange effervescent inclusion complexes was observed under a scanning electron microscope (SEM). As shown in Fig. 2, all samples showed irregular shaped particles.
The highly porous nature of the sample con rms their high encapsulation e ciency (Table 3)  assigned to CH2 and CH3 bending vibrations [42,43]. The similarity between the spectra of 2-HP-β-CD and EPI-β-CD indicates that the basic structural units of cyclodextrin polymer are preserved in both of them [44,45]. As shown in Fig. 3 bands. This result indicates the successful entrapment of the O-EOs blend and formation of the inclusion complexes [11,37].

Evaluation of effervescent powder
The results revealed that increasing the amount of effervescent components resulted in increasing the effervescent time (Table 5). Formula 5, showed the long effervescent time (215s), however it was excluded according to the pharmacopeia standard which stated that effervescent time should be < 180s. Moreover, the presence of high amount of citric acid (CA) in the effervescent powder may displace the encapsulated compound from the cyclodextrin cavity and consequently decrease its solubility [25,48] and hence its amount in the end product. As shown in Table 5, all formula showed a slight acidic, and thus have better palatability [49]. As shown in Table 5, formula 4 showed the best results and thus was selected for the preparation of the orange avoured effervescent powder.

Physical properties of the orange avoured effervescent powder
The owability of the effervescent powder is mainly evaluated by different parameters such as bulk density, tapped density, Carr's compressibility index, Hausner ratio and angle of repose [50,51].The bulk density and tapped density of the prepared orange avoured effervescent powder were 0.29 ± 0.01 and 0.35 ± 0.01 g/mL, respectively. It has been reported that a powder with Carr's compressibility index and Hausner ratio lower than 20% and 1.25, respectively is considered to be free owable [52]. Accordingly, the values of Carr's compressibility index and Hausner ratio in the present study (16.53 ± 0.75 and 1.20 ± 0.01, respectively) indicated good ow properties for the orange avoured effervescent powder (Table 5). Concerning the angle of repose, the measured value, 34° ± 0.63, also re ected good powder owability according to the previous study where free owing powder was imparted when angle of repose ranged between 30-38° [51]. Thus, the evaluated parameters (Table 3S) indicated better packing ability and free owability of the effervescent powder.

Conclusion
Currently, there is an increasing interest to impart the carbonated beverages functional properties. Orange is one of the most popular beverages. In present study, the antioxidant activity of orange oil was improved by blending with different essential oils having high antioxidant activity, while keeping the desired avour.
Combinations of two soluble β-cyclodextrine derivatives, 2-hydroxylpropyl-β-cyclodextrine and epihydrin-βcyclodextrine, at 3: 1 and 1: 3 molar ratios, respectively, were used to encapsulate the orange essential oils blend (O-EOs) that showed the highest odour quality and antioxidant activity. The physicochemical analysis, scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FT-IR) con rmed the successful formation of the orange avoured inclusion complexes nanoparticles. The slight decrease in the antioxidant activity of the encapsulated O-EOs blend compared to the free blend may be correlated to blocking of the functional groups of the active compounds that react with DPPH radical.
Among the investigated inclusion complexes that produced by encapsulation of O-EOs with the mixture of 2-HP-β-CD and EPI-β-CD (at molar ratio 3:1) showed the smallest particle size, the best odour quality, the highest antioxidant activity, the highest encapsulation e ciency and the more negative zeta potential.
Therefore, it was used in preparation of the orange avoured effervescent powder, the physical analysis proved its high quality.

Declarations
Appendix A. Supplementary data Supplementary data to this article can be found at online