Microstructure is closely related to techno-functional properties in microencapsulated powders intended to protect bioactive compounds. Soursop leaves provide phytochemicals that need to be protected to ensure their functionality. This investigation aimed to study the microstructure of microcapsules containing soursop leaves extract and its linkage with physical and chemical characteristics of the resulting powder. Microcapsules were prepared by spray drying using gum Arabic and maltodextrin as encapsulating agents at 5 and 10%. Powders were characterized by scanning electron microscopy, particle size analysis, solubility, infrared spectroscopy and encapsulation efficiency. Microphotographs showed spherical shape particles ranging from 0.25 to 13.87 μm, where the particles morphology depended on the concentration and the type of the encapsulant used. At higher concentration of encapsulant, there was an increase in the sphericity, integrity, size, and surface smoothness of particles. This relationship was inverse for solubility in treatments with gum Arabic. The extract encapsulation was confirmed by Fourier Transform infrared spectroscopy and encapsulation efficiency index, revealing that the treatment with maltodextrin at 10% showed a better capability for entrapment (72.12%). The results evidence that microstructure of microcapsules is closely linked to the type and concentration of encapsulant, which in turn determine the physical and chemical characteristics of powders intended for instant drinks solubility and entrapping soursop bioactive compounds.
Soursop (Annona muricata L.) is traditionally used in ethnomedicine by different populations around the world [
Bioactive phytochemicals are susceptible to fast inactivation or degradation once they are extracted from its original source [
Microencapsulation, which is one of the most used techniques for bioactive compounds conservation, is defined as a process where small particles or drops are surrounded by a cover or embedded in a homogeneous or heterogeneous matrix to generate microscopic capsules with advantageous properties [
Depending on the microencapsulation technique and the encapsulants used, it is possible to obtain particles with heterogeneous superficial structure, size, and shape, which lead to a deficient conservation of the compounds of interest [
There is no former information about studies on morphology of soursop leaves extract microencapsulated by spray drying. The aim of this investigation was to study the microstructure of microcapsules of hydroalcoholic extract from soursop leaves (Annona muricata L.) microencapsulated via spray drying using gum Arabic and maltodextrin as encapsulants at two different concentrations, and its linkage with physical and chemical properties.
The herbal Augusto Weberbauer (MOL) of the Universidad Nacional Agraria La Molina, certified the authenticity of the species under study. The leaves were collected in a farm in Pisco, Ica, Peru (Latitude 13˚44.817'S, Longitude: 76˚9.897'W) during December 2016. Ethanol 99.9% (Merck, Germany) was used for extraction. The encapsulants used were gum Arabic (Nexira food, France) and maltodextrin DE = 15 - 20 (Lihua Starch, China), which were selected in former trials due to its tolerance to ethanolic solutions. Other reagents used were Acetic acid (Merck, Germany), Gallic acid (Sigma-Aldrich, USA), Folin-Ciocalteu reagent (Merck, Germany), and sodium carbonate (Merck, Germany).
The collected fresh leaves were selected, washed with drinking water and dried in absence of sunlight for 10 days (Final humidity = 12%). They were previously oven-dried (UM400 Memmert) at 50˚C until reaching 10% humidity before grinding. Then, the leaves were ground in a leaf mill (3383-L30 Thomas Scientific) applying a N˚ 20 sieve. The leaves powder was hermetically stored in polyethylene bags until being used.
The previously powdered leaves were weighed in Erlenmeyer flasks, mixed with a hydroalcoholic solution (20% ethanol) at 1:36 rate (mass:volume), and placed in a water bath (AL 25 LAUDA Aqualine) at 70˚C for 30 minutes. Then, the extraction was stopped by immersing the flasks in a bath with ice. The extract was filtered using a nylon fiber. The filtrate was centrifuged at 2,500 rpm (IEC HN-SII Damon) for 30 minutes.
The resulting supernatant from the extraction was mixed with gum Arabic and maltodextrin to obtain solutions at 5% and 10% (w/w). The mixture was homogenized (IKA® T18 Ultra-Turrax®) at 10,000 and 15,000 rpm for 5 minutes each turn and kept in refrigeration until being used. Mixtures were spray dried in a Mini Spray Dryer B-2 90 (Büchi, Switzerland) with a 1 mm nozzle at 140˚C, an air flow rate of 32.5 m3/h, a feeding flow rate of 10 mL/min and pump at 10%.
Morphology study by scanning electron microscopy (SEM)
The morphology of the dry extract, the pure encapsulants, and the microcapsules was evaluated with a JSM-6380LV (JEOL, Japan) microscope. The powdered samples were placed on the upper surface of metallic stubs covered with Scotch double sided tape and vacuum immobilized using gold (S150, Edwards). Microphotographs were taken at magnifications ranging from 1200 to 20000x.
Particle Size analysis
The size was expressed in terms of the volume-weighted mean diameter (D[4,3]) which was determined by the estimate of the diameters of more than 650 particles using the software Axio Vision Rel. 4.8 from panoramic photos obtained with SEM. In accordance with Jinapong et al. [
D [ 4 , 3 ] = ∑ n i d i 4 ∑ n i d i 3 (1)
where ni is the number of particles of di diameter.
To verify the encapsulation process, IR spectrums for the microencapsulated extracts (04 treatments), the sole spray dried extract, and the pure encapsulants were obtained using a portable spectrometer TruDefender FT (Ahura Scientific, USA) diamond point with a resolution of 3 cm−1 from 4000 to 650 cm−1. Before placing the samples (powdered), a correction of air background was made. The peaks were analyzed with the software Resolutions pro 4.0 Varian, Inc.
Total phenolic compounds were used as a referential indicator of the amount of phytochemicals in the powders. Encapsulation efficiency for each treatment was determined by quantifying the content of phenolic compounds inside “core” (CPC) and on the surface (SPC) of the microparticles using the Folin-Ciocalteu method adapted from Simon-Brown et al. [
A = 31.401 × C − 0.0001 (2)
where A is the absorbance at 726 nm and C is the concentration in mg/mL.
The encapsulation efficiency was calculated using the Equation (3):
E E ( % ) = ( C P C − S P C C P C ) × 100 (3)
The solubility of the microencapsulated extracts was determined according to the adapted method from De Souza et al. [
S B T = ( W f − W i W m ) × 10 , 000 (4)
where Wf is the weight of flask plus solids (g), Wi is the weight of empty flask (g) and Wm is the weight of the 25 mL aliquot (g).
EE and SBT data were expressed as the average ± standard deviation (S.D.) from at least three replicas. The significance of differences between encapsulants and concentrations was evaluated by analysis of variance (ANOVA) using a Randomized Complete Block design considering encapsulants as blocks and concentrations as treatments. The statistical analysis was performed using Microsoft Excel.
Morphological characteristics of microparticles such as sphericity, shape and surface (smoothness/roughness), and particle size were examined by analyzing SEM images (
In this study, the microstructure of the spray dried soursop extract without encapsulant and the original structure of commercial encapsulants in powder were used as controls. Each component showed a characteristic microstructure inherent to its chemical nature and as a consequence of the technological process experienced during its elaboration (Figures 1(a)-(c)). The drying operation is a complex process where energy and matter transfer mechanisms take place, causing physical, chemical and structural changes [
reference patterns for purity evaluation of the encapsulants, the optimization for obtaining powdered plant extracts and their sub-products might take much more studies.
Visual analysis allowed to understand how the morphology of the supplies gets altered because of the spray drying process. Figures 1(1)-(4) reveals that the entrapment process of the extract into the encapsulants was effective.
In general, the shape of the microparticles was spherical and had a heterogeneous size. The sphericity and the heterogeneity in size is characteristic of the spray drying process [
The particles with MD (
With both encapsulants there is a greater proportion of spherical particles when a concentration of 10% is used. This verifies that a greater concentration of encapsulant increases the number of spherical particles mainly with MD; similarly, Simon-Brown et al. [
From the techno-functional point of view, the particles with a rough surface are more sensitive to oxidation reactions compared with those with a smooth surface due to their greater superficial areas [
According to
With both encapsulants, there was an increment of the particle diameter when the encapsulant concentration was increased. Additionally, the particles with GA tended to show a slightly larger size in comparison to those prepared using MD, as has been reported in similar studies [
The drying temperature is determinant since significantly influences the conservation of thermosensitive bioactive compounds [
Drop formation into microcapsule and proposed mechanisms to explain in a graphical way how some defective particles were formed during spray drying are
Treatment | D [4,3] (µm) | Size range (µm) |
---|---|---|
EXT GA 5% | 6.97 | 0.28 - 13.87 |
EXT GA 10% | 7.51 | 0.25 - 11.21 |
EXT MD 5% | 7.05 | 0.29 - 12.86 |
EXT MD 10% | 7.36 | 0.30 - 13.43 |
presented in
During the formation of the microparticle a given thickness of the solid surface (Shell) is needed to give it mechanical resistance to avoid disruption [
The fragmentation of particles (
The microparticles with GA at 5% showed to have experienced a “ballooning” phase tending to breakage after having overreached their extension; in spite of this phenomenon was visually lower at highest encapsulant concentrations, it was also shown the presence of post rupture particle remainders. The incidence of breaking off is an indicator of the microencapsulation process efficiency [
being both inversely proportional.
Microparticles with GA showed a predominant rough surface (
Mushroom hat type microparticles can be observed in the microphotographs of extract with 10% GA (
It is not evident the existence of an independent nucleus (
Factors that influence the formation of hollow particles are a rapid evaporation rate [
One of the primordial properties of encapsulated powder intended for direct human consumption is its easy reconstitution which will depend on the size, density, porosity, superficial charge, superficial area of the particle, and the presence of amphipathic substances [
Therefore, it is necessary to optimize the spray drying conditions (type and concentration of the encapsulant, type of nozzle, entrance temperature, air speed, among others), depending on the expected use of the product, taking into account the greatest number of variables with significant effect, which is achieved through a previous screening.
IR spectrums
The infrared spectrum of the encapsulant materials, the pure spray-dried extract, and the microencapsulated extracts are presented in
All the spectra showed a common band of absorption at the wavelength of 3300 cm−1 (
Both encapsulants reduced the band intensity characteristic of the extract (peaks at 1585 and 1507 cm−1); however, unlike MD, the gum Arabic spectrum shows a peak (at around 1600 cm−1) very closely located to the characteristics peaks of extract, which has also been identified by Bouaziz et al. [
Encapsulation efficiency of phenolic compounds
Significant differences were found by analyzing the four treatments (
In this study, percentages of EE varied from 53.46% to 72.12%; the values of efficiency increase when the concentration of the encapsulant increases, and it is higher in samples with MD, trend observed earlier by Saikia et al. [
Samples | EE (%) | Solubility |
---|---|---|
EXT GA 5% | 53.46 ± 1.931Aa | 93.23 ± 0.991Aa |
EXT GA 10% | 64.76 ± 1.461Ab | 88.49 ± 0.731Ab |
EXT MD 5% | 54.10 ± 0.992Ba | 98.15 ± 0.632Bc |
EXT MD 10% | 72.12 ± 1.102Bc | 97.38 ± 0.992Bc |
Averages with different capital letters and low case letters in each column indicate a significant difference (p ≤ 0.05) between blocks and treatments, respectively.
reported values from 63% to 79% using MD. Additionally, it was observed that the value for treatment with GA at 5% becomes equivalent to the treatment with MD at 5%, but EE increases significantly with MD at 10%.
Considering that the extract is a mixture of compounds with unique properties this variability between values of EE is partially explained to be due to the nature of the metabolite and its affinity with the matrix [
Solubility of microencapsulated powders ranged from 88.5% to 98.2% (
The encapsulation of soursop dry leaves extract by spray drying was achieved, getting microparticles of spherical shape and heterogeneous size (0.25 - 13.43 μm). The morphology of the microparticles depended on the concentration (5 and 10%) and type of the encapsulant (GA and MD), giving a positive correlation between the encapsulant concentration and the microparticles sphericity, mainly for MD. Also, the powder solubility was greater using MD, but inversely proportional to the concentration of the encapsulant, which is associated with the particle size. The extract encapsulation was qualitative confirmed via FTIR spectroscopy. Encapsulation efficiency essays showed that the treatment with MD at 10% presented a greater performance for encapsulating phenolic compounds originally existing in the extract. The characterization of the encapsulated extract is a starting point in the search of its application in food matrixes.
The authors thank the partial financing provided by the Ministry of Education of Peru (MINEDU): Project for the strengthening of doctoral programs at UNALM “Food Science” CONV-000179-2015-FONDECYT-DE, to the Pacific Alliance scholarship program that allowed the use of facilities provided by the Center of Spectroscopy and Electron Microscopy of University of Concepcion (CESM). We also thank Dr. Luis Rodríguez-Saona for the support given for the use of a portable FTIR spectrometer.
The authors declare no conflicts of interest regarding the publication of this paper.
Jordán-Suárez, O., Glorio-Paulet, P. and Vidal, L. (2018) Microstructure of Annona muricata L. Leaves Extract Microcapsules Linked to Physical and Chemical Characteristics. Journal of Encapsulation and Adsorption Sciences, 8, 178-193. https://doi.org/10.4236/jeas.2018.83009