1. Introduction
There is a growing interest among consumers in natural bioactive compounds that possess the potential to promote health benefits and contribute to the prevention and/or treatment of chronic diseases. Consequently, investigating and researching these natural bioactive compounds is a matter of great importance for numerous laboratories and industries [
1,
2].
Among the bioactive compounds, phytochemicals stand out, due to their ability to inhibit oxidative reactions, thereby protecting the organism from reactive oxygen species, which are implicated in numerous degenerative diseases such as cataracts, atherosclerosis, and cancer [
3].
The Amazon region boasts an extraordinary biodiversity of both native and exotic fruits. Camu camu (Myrciaria dubia (H.B.K) McVaugh) is a fruit that is distributed along Bolivia, Brazil, Colombia, Ecuador, Peru, and Venezuela, and it is emerging as a standout due to its remarkable nutritional prospects [
4,
5]. The composition of camu camu has garnered considerable interest owing to its elevated levels of bioactive compounds such as ascorbic and citric acid and phenolic compounds, including ellagic acid, ellagitannins, and proanthocyanidins [
6,
7,
8], being reported for its purported bioactivities encompassing antihyperglycemic, anti-inflammatory, antihypertensive, and antimicrobial, among others [
9,
10,
11]. In this sense, investigations have demonstrated that camu camu exhibits potent antioxidant and anti-inflammatory properties, surpassing those observed in vitamin C tablets when tested in human subjects [
12]. Moreover, studies have also reported the potential antiobesity effects of camu camu in a rat model of diet-induced obesity [
13]. Additionally, several studies have suggested that camu camu possesses antidiabetic activities, indicating its potential in treating this disease [
7,
14,
15,
16].
To develop functional ingredients based on the active properties of camu camu, it is necessary to extract the bioactive compounds from the fruit or the different parts of the plant [
10,
14], process that can be followed by a drying step to facilitate its industrial handling. However, it is well-known that some bioactive compounds present low solubility in water or are unstable and susceptible to high temperature, light, pH, oxidative stress, and degradative enzymes, which can negatively impact their biological activities. Consequently, developing strategies to protect and preserve them and to enhance their bioaccessibility and bioavailability is crucial [
17,
18]. Among these strategies, encapsulation has emerged as a promising approach to address this challenge. Encapsulation involves entrapping the bioactive components within a protective matrix, which can slow down degradation processes, prevent the loss of their functionality and increase bioavailability [
2,
19]. Therefore, encapsulating the bioactive compounds makes extending their shelf life possible, improving their stability throughout various processing stages and final use at consumption and enhancing their compatibility with different formulations [
1,
20]. Furthermore, the encapsulation strategy could provide controlled release mechanisms and a sustained and targeted delivery of these compounds in the body [
21,
22,
23]. Thus, various encapsulation methods have been explored so far [
24]. The choice of the suitable process varies with the active ingredient's nature, the coating material's properties, and the final product's desired characteristics based on the intended use.
Recent evidence underscores that microencapsulation is a potent process to enhance the stability and bioavailability of the naturally occurring bioactive compounds in camu camu, such as ascorbic acid and anthocyanins. Until now, Fujita et al. have reported the microencapsulation of camu camu juice into Arabic gum and maltodextrin via spray drying [
25]. Figueiredo et al., reported the microencapsulation of the camu camu extract from pulp and peels by spray drying using maltodextrin, inulin, and oligofructose as encapsulating matrixes [
26]. Complementary findings by García-Chacón et al., reveal that spray-drying of camu camu pulp with maltodextrin, whey protein, and a 50:50 mixture of both is a strategy to increase the bioactive compounds stability, modulate the fruit sensory properties, and improve their bioavailability [
27].
However, among the techniques available, electrodynamic processing, including electrospraying or electrospinning, has emerged as a promising and versatile method for encapsulating bioactive agents. Electrospraying offers several advantages over traditional encapsulation techniques [
28]: it enables the production of micro or nano-scale particles; it is simple and versatile in terms of the materials that can be processed; it allows the production of particles at low or ambient temperatures [
28]. However, the main disadvantage of this technology is its low production yield, which has been solved thanks to the integration of electrospraying and gas-driven nebulization [
29]. This technology has been named and patented as electrospraying assisted by pressurized gas (EAPG). The EAPG technology can be used for drying or microencapsulating bioactive compounds at room temperature. It involves atomizing the solution through a pneumatic injector using compressed air that nebulizes within a high electric field. This process enables the formation of fine droplets that get dried at room temperature and are collected as a free-flowing powder.
Hitherto, camu camu has been encapsulated mainly into polysaccharides [
25,
27]. However, based on our encapsulation expertise [
30,
31], food-grade proteins can provide increased stability to sensitive bioactive ingredients [
32]. Therefore, this work aimed to study the potential of the room temperature EAPG technology to dry and microencapsulate a camu camu extract (CCX). Whey protein concentrate (WPC) and zein (ZN) were selected as encapsulating agents. The microcapsules were characterized by morphology, total soluble polyphenols (TSP) content, and antioxidant activity. Additionally, this study aimed to assess the storage stability of CCX EAPG-derived microcapsules under different humidity and temperature conditions and against photooxidation
2. Materials and Methods
2.1. Materials
Camu camu powder (CCP) was provided by Qomer BioActive Ingredients (Valencia, Spain). Whey protein concentrate (WPC) 80% was purchased from Beurrespa S.L. (Madrid, Spain). According to the distributor, WPC was claimed to contain 81.6% protein (on dry basis) and 7.5 % fat. Maize zein (ZN), gallic acid, sodium carbonate, Folin-Ciocalteau reagent, magnesium nitrate, potassium acetate, and silica gel were purchased from Sigma-Aldrich (Saint Louis, USA). Methanol (reagent grade) was purchased from Labbox (Premià de Dalt, Spain). Ethanol 96 vol.% from Panreac Química SLU (Barcelona, Spain). Deionized water was used throughout the study.
2.2. Preparation of Camu Camu Extract (CCX)
The CCP was extracted following a standardized adapted method as described by Fracassetti et al. [
8]. A mixture of alcohol and water (85%) was used as the extraction solvent. The extraction process was carried out at a concentration of 5% (w/v), where 50 g of CCP was mixed with 1000 mL of the extraction solvent. The mixture was homogenized using a magnetic stirrer (H20 series, LBX Instruments, Premia de Dalt, Spain) at room temperature until complete dissolution of CCP. Subsequently, the mixture was sonicated in a SONOPULS HD 2200.2 (Bandelin electronic GMbH & Co. KG, Berlin, Germany) for an additional 30 min. After sonication, the solution was centrifuged at 3000 rpm for 15 minutes at 4°C using an Avanti J-26 XPI Beckman Coulter centrifuge (Brea, CA, USA). The resulting supernatant was then filtered using Whatman No. 1 filter paper under vacuum filtration. The filtered supernatant was collected and used for further encapsulation processes.
2.3. Preparation of the Polymer Solution
To prepare the encapsulating solutions, a 20% (w/v) solution of whey protein concentrate (WPC) in water was prepared, and a 4% (w/v) solution of zein (ZN) in an 85% (v/v) ethanol aqueous solution. Then, the encapsulating solutions were created by slowly adding the pre-prepared camu camu extract (CCX) to the respective encapsulating matrix solutions at ratios of 1:1 w/w and 2:1 w/w (encapsulant to dry weight CCX ratio). The solutions were continuously stirred using a magnetic stirrer (H20 series, LBX Instruments, Premia de Dalt, Spain) at room temperature overnight until a homogenous mixture of polymer solutions was obtained.
2.4. EAPG Process
The EAPG process was used to dry the CCX solution and to encapsulate the CCX into WPC and ZN, using the CapsultekTM pilot plant from Bioinicia S.L. (Valencia, Spain). The pilot plant consists of a nebulizer that generates aerosol droplets, which are subjected to a high electric field, a drying chamber, and a cyclonic collector. The process was conducted under controlled ambient conditions of 25 °C and 30% relative humidity (RH), as detailed in the study by Busolo et al. [
29]. The solution was pumped to the injection unit at a flow rate of 10 mL/min, with an air pressure of 10 L/min and a voltage of 15 kV. The resulting particles were collected from the cyclone as a free-flowing powder, stored in flasks under vacuum at -20 °C, and protected from light to prevent oxidation until further analysis.
2.5. Microscopy
Scanning electron microscopy (SEM) analysis of the particle morphology was conducted using a Hitachi S-4800 field-emission scanning electron microscope (Hitachi High Technologies Corp., Tokyo, Japan). The microscope was operated at an electron beam acceleration of 5 kV. Before analysis, approximately 5 mg of the capsules from each sample were coated with a thin layer of gold/palladium to enhance conductivity and imaging quality during SEM observations. The SEM images were captured, and the average diameters of the particles were determined using Image J Launcher v1.41 software developed by the National Institutes of Health (Bethesda, USA).
2.6. Assessment of the Stability of CCX Formulations
The stability of CCX formulations was evaluated under different storage conditions of relative humidity (RH) and temperatures and accelerated oxidation conditions under ultraviolet (UV) radiation. To simulate different humidity levels, samples of CCX formulations were placed in desiccators containing either silica gel (0% RH), potassium acetate oversaturated solution (23% RH), or magnesium nitrate oversaturated solution (56% RH). The desiccators were kept at a cooling temperature (4°C) or ambient temperature (~21°C) accordingly. The samples were stored under the above conditions and monitored for 40 days. Weekly samples were collected and analyzed.
Regarding UV radiation, a commercially available OSRAM Ultra-Vitalux lamp (OSRAM, Garching, Germany) irradiated the samples with UV light. This lamp consists of a quartz discharge tube and a tungsten filament, producing a blend of radiation like natural sunlight. The lamp was operated at a power of 300 W, and only wavelengths like those present in daylight passed through the special glass bulb. After 1 h of exposure, the radiation between 315-400 nm was measured to be 13.6 W, and the radiation between 280 and 315 nm was measured to be 3 W. Approximately 20 g of capsules were evenly spread on Petri dishes and positioned at 20 cm under the UV lamp. The powder in the Petri dish was stirred daily to maintain a uniform treatment. The thickness of the powder layer was maintained below 5 mm to ensure consistent exposure to UV light. The capsules were subjected to the specified storage conditions, and their stability was evaluated over 40 days. Weekly samples were taken from the capsules for analysis.
The analysis of the samples was specifically made to focus on variations in total soluble polyphenols and antioxidant capacity, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy was utilized.
2.7. Total Soluble Polyphenols
The total soluble polyphenols (TSP) content in the CCX formulations was determined using the Folin-Ciocalteu method described by Singleton and Rossi [
33]. For the analysis, 10 mg of the CCX formulations were diluted in ethanol at a concentration of 50% (v/v). Similarly, an equivalent amount of WPC-CCX capsules was diluted in ethanol at 33% (v/v), and an equivalent amount of ZN-CCX capsules was diluted in ethanol at 75% (v/v). After dissolution, 20 μL of the capsule's solution was mixed with 1.2 mL of water and 300 μL of sodium carbonate solution at a concentration of 7.5% (w/v) in Eppendorf tubes. The mixture was then homogenized for 1 minute using a vortex shaker and left to stand for 5 minutes.
Next, 380 μL of water was added to the mixture, followed by 100 μL of a 10% (v/v) Folin reagent solution. The reaction proceeded for 15 min under dark conditions and at room temperature. Subsequently, the absorbance of the resulting solution was measured in triplicate at a wavelength of 765 nm using a UV/Vis spectrophotometer (UV4000 Dinko Instruments, Barcelona, Spain). The absorbance values obtained were then used to calculate the total soluble polyphenols content of the samples, expressed in gallic acid equivalents (mg GAE/g of dried CCX).
2.8. Antioxidant Activity
The antioxidant capacity of the obtained CCX formulations was determined using the DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) free radical scavenging method. This method is commonly employed to assess the ability of samples to scavenge free radicals and indicate their antioxidant activity. To assess the antioxidant capacity, 10 mg of the dried CCX (or the equivalent amount of WPC-CCX or ZN-CCX capsules) was dissolved in ethanol at specific concentrations (50% v/v for the dried CCX, 33% v/v for WPC-CCX, and 75% v/v for ZN-CCX). Then, 50 μL of the prepared solutions were mixed with 950 μL of methanol.
An aliquot of 0.1 mL of the methanol solutions was then added to 1.9 mL of a DPPH methanolic solution (0.094 mM concentration). The mixture was vortexed and kept in dark conditions for 30 min at room temperature to allow the reaction between the DPPH radical and the samples.
A methanol solution without the sample was used as a blank for comparison. After the incubation period, the absorbance of the reaction mixture was measured in triplicate at a specific wavelength (e.g. 517 nm) using a UV/Vis spectrophotometer (UV4000 Dinko Instruments, Barcelona, Spain). The absorbance values obtained from the samples were compared to the blank to calculate the radical scavenging activity. The radical scavenging activity can be calculated using the following equation:
DPPH Inhibition (%) is the inhibition percentage of DPPH, Ac is the absorbance of pure DPPH solution, and As is the absorbance of the incubated sample after reacting with DPPH.
By employing the DPPH free radical scavenging method and calculating the radical scavenging activity, the antioxidant capacity of the CCX formulations could be assessed, providing information about their ability to neutralize free radicals and potentially protect against oxidative stress.
2.9. Attenuated Total Reflection- Fourier Transform Infrared (ATR-FTIR)
The ATR-FTIR spectra of CCX formulations were measured using a Bruker Tensor 37 FT-IR Spectrometer equipped with an ATR sampling accessory called the low-temperature Golden Gate from Specac Ltd. This accessory ensures proper contact between the diamond crystal and the encapsulated samples, enhancing the quality of the spectra. Approximately 50 mg of the capsules were used for each measurement.
The spectra were precisely collected using ATR-FTIR spectroscopy within the 4000-600 cm-1 wavenumber range, capturing a comprehensive infrared frequency spectrum crucial for molecular bond characterization. The protocol averaged 10 scans per spectrum to enhance the signal-to-noise ratio, a standard analytical chemistry practice for data quality improvement. Spectral resolution was set at 4 cm-1, allowing proper resolution for molecular vibration interpretation.
The OPUS 4.0 data collection software program, developed by Bruker, was used to analyze the spectral data. This software enables the processing, analysis, and interpretation of the ATR-FTIR spectra, allowing for the identification and characterization of the encapsulated samples. Measurements were performed in triplicate to ensure the reproducibility and reliability of the results.
2.10. Statistical Analysis
The data were expressed as mean ± standard deviation. To determine the significance of the differences observed among the different samples, an analysis of variance (ANOVA) was conducted. Following ANOVA, a Tukey test was performed to compare the means of multiple groups. In this analysis, differences were considered significant when the p-value was less than 0.05. For this analysis, the software used was Statgraphics Centurion Version 17.2.04, developed by Statistical Graphics Corp.
Figure 1.
SEM micrographs of dried CCX structures. (A) The neat CCX dried using the EAPG process. (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w. The scale bar corresponds to 30 µm.
Figure 1.
SEM micrographs of dried CCX structures. (A) The neat CCX dried using the EAPG process. (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w. The scale bar corresponds to 30 µm.
Figure 2.
Evolution of the TSP Decay of CCX EAPG-derived microcapsules over 40 days under diverse storage conditions. (A) EAPG dried CCX; (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w.
Figure 2.
Evolution of the TSP Decay of CCX EAPG-derived microcapsules over 40 days under diverse storage conditions. (A) EAPG dried CCX; (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w.
Figure 3.
Evolution of the DPPH inhibition Decay of CCX EAPG-derived microcapsules over 40 days under diverse storage conditions. (A) Non-encapsulated CCX; (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w.
Figure 3.
Evolution of the DPPH inhibition Decay of CCX EAPG-derived microcapsules over 40 days under diverse storage conditions. (A) Non-encapsulated CCX; (B) WPC—CCX 1:1 w/w; (C) WPC—CCX 2:1 w/w; (D) ZN—CCX 1:1 w/w; (E) ZN—CCX 2:1 w/w.
Figure 4.
Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) of EAPG dried CCX.
Figure 4.
Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) of EAPG dried CCX.
Figure 5.
Spectral evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for the EAPG dried CCX stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 5.
Spectral evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for the EAPG dried CCX stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 6.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for WPC-CCX 1:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 6.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for WPC-CCX 1:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 7.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for WPC-CCX 2:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 7.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for WPC-CCX 2:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 8.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for ZN-CCX 1:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 8.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for ZN-CCX 1:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 9.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for ZN-CCX 2:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Figure 9.
Spectral Evolution of Attenuated total reflection—Fourier transform infrared spectroscopy (ATR- FTIR) for ZN-CCX 2:1 w/w EAPG-derived microcapsules stored under various conditions (A) 0 days; (B) 40 days, 0% RH, 4°C; (C) 40 days, 0% RH, 21°C; (D) 40 days, 23% RH, 21°C; (E) 40 days, 56% RH, 21°C; (F) 40 days, UV light exposure. The spectra were maximized to the band with the highest intensity in the wavelength range between 1800 and 800 cm−1.
Table 1.
Particle size, total soluble polyphenol content, and antioxidant activity for CCX dried by EAPG (CCX-EAPG) and encapsulated CCX into whey protein concentrate (WPC) and zein (ZN).
Table 1.
Particle size, total soluble polyphenol content, and antioxidant activity for CCX dried by EAPG (CCX-EAPG) and encapsulated CCX into whey protein concentrate (WPC) and zein (ZN).
Sample |
Particle size (μm) |
DPPH inhibition (%) |
TSP (mg GAE/g dried CCX) |
CCX |
|
89.06 ± 0.02 b
|
1.13 ± 0.05 a
|
CCX – EAPG |
10.01 ± 1.84 a
|
94.03 ± 0.02 a
|
1.14 ± 0.07 a
|
WPC - CCX 1:1 |
6.74 ± 2.57 ab
|
91.60 ± 0.06 ab
|
1.15 ± 0.04 a
|
WPC - CCX 2:1 |
7.24 ± 2.49 ab
|
94.07 ± 0.04 a
|
1.11 ± 0.05 a
|
ZN - CCX 1:1 |
6.24 ± 2.33 ab
|
85.32 ± 0.02 b
|
1.21 ± 0.06 a
|
ZN - CCX 2:1 |
5.85 ± 1.45 b
|
93.22 ± 0.04 a
|
1.15 ± 0.05 a
|