OECP produced using organic solvents

To obtain OECPs, a mixture of non-polar and polar solvents was used, which allowed the effective extraction of total lipids, following the methodology of Kubiliene et al. (2015). 75 mL of dichloromethane: ethanol: water (1:1:1) were mixed with 25 g of bee propolis, which were shaken for 15 minutes at 4,000 rpm in an Eppendorf® 5427 R centrifuge. Three ultrasound cycles were applied at 60 Kw for 15 min at room temperature while protected from light exposure. Subsequently, the sample was filtered using Whatman® Grade 1:11 µm paper. The filtrate was subjected to funnel decantation to isolate the organic phase from the aqueous phase. The apolar region was subjected to another separation to remove waxes. Finally, the organic phase was rota evaporated at 40 °Cin a Büchi® R-215 Rotavapor System to remove dichloromethane.

Microencapsulation of OECPs and characterization of microencapsulated (MOECP)

The OECPs were microencapsulated using the methodology proposed by Busch et al. (2017) with some modifications. Maltodextrin (30 g) and 0 Gum arabic (0.3 g) were used, dissolved in 150 mL of distilled water, maintaining a constant ratio between the solvent and the encapsulating agent following the formulations proposed in Table 1. Ten milliliters of oily propolis extracts were added, and the resulting mixtures were homogenized using an Ultra-Turrax T25: IKA®, Germany, at 15,000 rpm for 2 min. Then, the emulsion was dried, and the oily propolis extracts were encapsulated using a Büchi® B-191 Mini Spray Dryer. The spray drying operating conditions were suction (%) = 85; pump (%) = 10; air and feed flow: 0.60 m³ min^-1 and 1.3 mm diameter of the nozzle or injector. Yield was calculated according to Equation 1. Samples of microencapsulated OECPs under this methodology were called MOECP.

Where: "Mm" microencapsulated = Mass obtained in the encapsulation, "Me" encapsulant = Mass of the encapsulating agent "Ms" total solids = Mass of the total solids content of the propolis essential oil.

Table 1: Formulation of microencapsulation from two ratios of encapsulating agent and OECP, and parameters of microencapsulation.

The water activity (A_w) of the MOECP was analyzed by placing the samples in a Rotronic® (HygroLab C1 model), and measurements were performed in triplicate at room temperature.

Finally, color analysis of MOECP was performed using a Minolta® colorimeter (CM-3600), the CIE-Lab system (L*, a*, b*), illuminant D65 and an observation angle of 10°. The readings were performed in triplicate according to the methodology proposed by López-Patiño et al. (2021).

Table 1 lists the parameters used to describe the microencapsulation process using the Spray Dryer. Eight mixtures, two extract/encapsulant ratios, four temperatures, and two amounts of oily Colombian propolis extract (OECP) were used.

Total content of Phenols and β-carotene of OECP and MOECP

To evaluate the total content of phenols and β-carotene in the OECP and MOECP, the Folin-Ciocalteu method and the β-carotene test were used, respectively. First, pretreatment was carried out with 1:3 dilutions (extract/solvent) for both OECP and MOECP. For 0.5 mL of OECP and 0.5 mg of MOECP, the samples were diluted in 1.5 mL of dichloromethane.

Folin-Ciocalteu method was used to measure the phenol content. A standard curve was prepared from a stock solution of gallic acid in methanol at a concentration of 1 mg mL^-1. Once the standard curve was constructed, mixtures of 100 µL of control, 100 µL of OECP and 100 µL of diluted MOECP were made, with a 75 µL of Folin-Ciocalteu solution and 825 µL of 2% sodium carbonate solution. The mixtures were vortexed for 1 min, and 250 µL were poured into each well, repeating this process three times for each sample and control. The plate containing the microdilutions was then stored for 90 min in the dark, and, finally, the mixtures were read at 750 nm in a SpectraMax 190 Microplate Reader® plate reader according to the method proposed by Tiveron et al. (2016).

To measure β-carotene, a calibration curve was prepared from a stock solution made with a β-carotene standard (Sigma Aldrich®) whose concentration was 1 mg β-carotene mL^-1 of solvent. Subsequently, 250 µL of the diluted OECP and MOECP samples were read at 470 nm using a SpectraMax 190 Microplate Reader® according to the method proposed by Nair and Meliani (2018).

Antiradical activity of OECP and MOECP determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) method

The DPPH method was used to obtain antiradical activity. A mixture of 0.0079 g of DPPH (Sigma Aldrich®) in 2.5 mL of ethanol was prepared and stirred in an amber bottle for 5 min at room temperature. Subsequently, 250 µL of this solution were diluted in 50 mL of 80% ethanol. Next, 300 µL of this mixture were used for a reading at 540 nm in a SpectraMax® 190 Microplate Reader to verify absorbance ranges between 0.5 and 0.6. Finally, 290 µL of the DPPH solution were mixed with 10 µL of diluted OECP or MOECP, respectively. The plate was left in the dark for 30 min, and the sample was read at 540 nm following the method proposed by Ramadan et al. (2012). The antiradical potential was obtained using Equation 2:

Iron Reducing Antioxidant Power (FRAP) assay of OECPs and MOECPs

First, the calibration curve was prepared using a standard of ferrous sulfate diluted in 2 mM ferrous sulfate stock aqueous solution. The OECP and MOECP were diluted in dichloromethane at a 1:4 ratio. Subsequently, 90 µL of each dilution was diluted with 270 µL of distilled water in the dark, and then 2.7 mL of a 10 mM TPTZ Sigma Aldrich® solution was added. The mixtures were heated in a water bath at 37 °C for 30 min and then allowed to cool. Finally, absorbance was measured at 595 nm using a SpectraMax® 190 Microplate Reader. The tests were in triplicate, and the concentration was obtained using the slope and cut-off values, which were expressed in µM of ferrous sulfate, in 1 g of propolis following the method proposed by Thaipong et al. (2006).

Analysis of MOECP with Fourier Transform Infrared Spectroscopy (FT-IR)

To identify the functional groups, present in the MOECP compounds, infrared spectroscopy was used to obtain vibration bands characteristic of one or more specific functional groups. For the test, 0.1 g of each sample was weighed, and ground in an FT/IR-4700 FTIR Spectrometer, JASCO®. The readings were taken in the range of 400-4,000 cm^-1, in triplicate for each sample. Baseline corrections were made to eliminated outlier values and CO₂ and H₂O bands were removed. The spectra were plotted as a function of wavelength and transmittance percentage. To identify the functional groups of the spectra, the band values were compared with the bibliographic information using the method proposed by Fangio et al. (2018).

Microbiological analysis with minimum inhibitory concentration (MIC) of OECP and MOECP

The antimicrobial activity analysis was performed using the well microdilution technique. Plates with 24 wells were used, and 95 µL of Mueller Hinton® culture medium were added to each plate. Then, 0.05 mL of the OECP for the control treatment and dilutions of MOECP in dichloromethane (1:3) were added to achieve a final concentration in each well of 20 mg mL^-1. Once this concentration was reached, 95 µL of Mueller Hinton® medium was added, and serial dilutions were made. Subsequently, a solution of bacteria mix (Escherichia, Staphylococcus aureus, Salmonella enteritidis, Enterobacter aerogenes, Enterobacter agglomerans, Klebsiella sp.) was prepared with 100 µL of saline solution at a concentration of 100 µg mL^-1 and a colony of the bacterial solution with absorbance values between 0.08 to 0.13. Twenty microliters of this solution were added to the wells. Finally, the solutions containing bacteria and OECP or MOECP were incubated for 24 h at 35 °C, and the minimum inhibitory concentration against the microorganisms was determined according to the method proposed by Bonou et al. (2016).

Scanning Electron Microscopy (SEM) of MOECP

The morphology of MOECPs where observed by using scanning electron microscopy (SEM). The tests were performed at the Laboratorio de Microscopía Electrónica de Barrido in the Universidad Nacional de Colombia. The samples were treated with a gold-palladium metallic coating applied with a Q150R ES metallizer (Quorum). Subsequently, they were observed using a Quanta 200 FEI® - USA scanning electron microscope. The operating conditions were at 2×10^-2 torr and 25.0 kV, using secondary electrons. The aim was to determine the presence of agglomerations, the size of the particles, and the shape and presence of porous structures in MOECP according to the method suggested by Sanchez-Reinoso et al (2017).

Differential Scanning Calorimetry (DSC) of MOECP

DSC analysis was used to determine the enthalpy, glass transition temperatures, and endothermic and exothermic events of the MOECP during the useful life of the samples. A DSC 1-500/2722 Mettler Toledo® calorimetric analyzer was used with 2 mg of each sample placed in an aluminum capsule under N₂, which was supplied at a rate of 50 mL min^-1. The temperature range was 30-400 °C, and the heating rate was 10 K min^-1. Finally, thermograms were obtained and analyzed using Mettler Toledo® STARe Thermal Analysis System version 8, as proposed by Busch et al. (2017).

Statistical analysis

For each variable, the means and standard deviations were obtained. To analyze significant differences, ANOVA was performed using the Tukey test (P≤0.05). A principal component analysis (PCA) was also carried out to group the values of the analyses according to similarities and to determinate which variables had a higher incidence in the OECP or MOECP. Matlab® version 7.12.0.635 and Origin® 2018 64 Bit were used (Woźniak et al. 2019).

OECPs extraction

The propolis used in this study were selected because it inhibits coliform microorganisms. Extraction was carried out on eight samples from premontane humid forests, the yield results are presented in Table 2.

Table 2: OECP yields obtained from the propolis collected during the study.

There were no significant differences between the propolis samples, and the yield percentage ranged between 19.06 and 19.85%. This consistency suggests that propolis collected from similar ecological zones under uniform conditions maintains a stable extraction efficiency with apolar organic solvents. Although the yield of the oily extracts was higher than that of essential oils, - had a maximum yield of 1.14%, it was lower than that of the ethanolic extracts, which was between 41 and 60%. Although propolis substances are lipophilic and have a greater affinity with apolar organic solvents, some factors explain the decrease in extraction -relative to that of ethanolic extracts (Sambou et al. 2020). First is that a large proportion of the hydrophobic compounds in propolis are resins (50-70%), whereas oils and waxes comprise 30 and 50% of the total composition (Abdelrazeg et al. 2020). Resins and waxes were removed during the filtration steps; therefore, only oils and substances contained within these matrices will remain (Ahangari and Naseri 2018). The literature reports 41 to 60%, the yields in this study were around 19%. According to Pobiega et al. (2019), water has little affinity to most propolis compounds. Yields with water ranged from 4 to 14%, which affected the results. Although water helped remove waxes and resins, facilitating precipitation, it resulted in a low yield. However, yields increased with ultrasound extraction techniques, which increased the concentration of secondary metabolites and the total mass of the extract. Although OECP yields are modest, the extracted components retain significant biological activity, emphasizing the potential of optimizing extraction techniques, particularly those incorporating ultrasound or alternative solvents, to maximize the recovery of bioactive compounds.

Microencapsulation of OECPs and their characterization (MOECP)

Table 3 presents the results of the yield and aqueous activity of MOECP.

Table 3: Performance and aqueous activity of MOECP using different parameters.

The MOECP exhibited an aqueous activity that ranged from 0.2 to 0.3, which prevented the proliferation of pathogenic fungi and/or bacteria. A significant difference (P<0.05) was observed between the samples according to their concentration. The relationship between encapsulant concentration and the water activity was inversely proportional (the higher the concentration of the encapsulant, the lower the water activity). The yields ranged between 35 and 63%, with a higher value observed in samples that had a high concentration of OECP, such as formulations 1 and 2.

The observed inverse relationship between water activity and the concentration of encapsulating agents, such as maltodextrin and Gum arabic, aligns with findings from other studies. Recently, Iesa et al. (2023) indicated that a higher proportion of encapsulants reduces water activity by limiting free water within the microencapsulate matrix, enhancing both stability and microbial inhibition.

Moreover, the efficiency of microencapsulation can be influenced by the ratio of encapsulating agents and processing conditions, such as temperature, which affects the retention of bioactive compounds and the uniformity of the microcapsules.

The results of the colorimetry analysis for MOECP are presented in Table 4. The samples had a similar tone (h), without differences (P>0.05), but varied in terms of chroma (C) (P<0.05). This result was due to changes in luminosity and the blue-yellow ratio when the OECP concentration and temperature changed. As the temperature decreased and the concentration of the encapsulating material increased, the intensity of yellow in the samples increased, and the microencapsulation became less luminous. In the eight formulations, there was a significant difference between to the amount of OECP used and the temperature.

Table 4: OECP and MOECP color parameters.

The concentrations of bioactive compounds, such as polyphenols and β-carotene, as well as antioxidant and antiradical activity, were reduced in samples 1 to 8 (MOECP), compared with OECP. This phenomenon can be attributed to increased temperature. The most affected metabolites were β-carotene, where the concentration was reduced by up to nine-fold. This can be attributed to the breaking of the double bonds between carotenoids and xanthophylls with increasing temperature, which generated chemical changes in the carotenoids resulting in the appearance of low molecular weight substances, short-chain isoprene derivatives, and oxygenated substances.

Similarly, a decrease in polyphenols was observed with increasing temperature, which could change the original structure and functionality. Phenols react in the presence of substances such as organic acids, lipids, and nitrogenous compounds, giving rise to the appearance of substances such as benzocaine, ethers, glycolipids, aldehydes, and ketones, which have a lower antioxidant capacity than polyphenolic substances or compounds with - OH (Islam et al. 2014; Kalušević et al. 2017).

Total content of Phenols, β-carotene and Antiradical activity of OECP and MOECP

The antioxidant activity, and bioactive compounds content, such as polyphenols and carotenoids in OECP and MOECP are presented in Table 5. Antioxidant activity decreases with increase temperature, leading to the degradation of labile compounds.

Table 5: Antioxidant activity and polyphenol and carotenoid content of OECP and MOECP.

Formulations 1 and 2 maintained greater reducing capacity and antiradical activity, indicating less degradation of labile compounds, because they were prepared at lower temperatures. As the temperature increases, Maillard and Strecker degradation reactions generate substances with a lower antioxidant potential than bioactive compounds in OECP. The affected compounds, apart from polyphenols, are low molecular weight volatiles and nitrogenous substances, such as proteins, amino acids, and sugars that result in aldehydes, ketones, pyrazines, and furans. In this study, the values of antioxidant activity and bioactive compounds such as carotenoids were still present in the microencapsulated extracts (MOECP) although at a lower concentration than in the non-encapsulated extract (OECP). On the contrary, previous studies that evaluated the ethanolic extracts of propolis (Baysan et al. 2018) and encapsulated fruit extracts reported better antioxidant activity in encapsulated forms than in non-encapsulated extracts (Tamanna et al. 2015).

Iron Reduction Antioxidant Power Assay (FRAP) for OECP and MOECP

When analyzing the FRAP values for the OECP and MOECP (Table 5), the reducing capacity of the MOECP decrease with increasing temperature and decreasing OECP concentration. According to Rodrigues et al. (2021), microencapsulation reduces the levels of agents in propolis, such as polyphenols, via oxidative processes. Previous studies have shown that microencapsulating has a maximum reducing capacity at temperatures between 100 - 120 °C and that these encapsulates have a greater reducing capacity than encapsulates that only contain maltodextrin and Gum arabic (Pratami et al. 2020). Both polysaccharides and polyphenols have cyclic structures that reduce metal ions such as Fe⁺³, which is transformed into Fe⁺². There are more phytochemical compounds in propolis extracts that contribute to the reducing activity, but they have not been correlated with the reducing power (Pratami et al. 2020).

Analysis of MOECP using FT-IR

Figure 1 shows the results of the infrared spectra reading with the MOECP treatments, which resulted from breaking the double bonds of the carotenoids and xanthophylls as the temperature increased, causing chemical changes in the carotenoids that resulted in the appearance of low molecular weight substances, short-chain isoprene derivatives, and oxygenated substances.

Figure 1: FT-IR spectra of MOECPs. (a) Sample 1 (100 °C), (b) Sample 2 (120 °C), (c) Sample 3 (140 °C), (d) Sample 4 (160 °C), (e) Sample 5 (100 °C), (f) Sample 6 (120 °C), (g) Sample 7 (140 °C), (h) Sample 8 (160 °C). MOECP: Microencapsulated oily extract of Colombian propolis.

The results of the FT-IR spectra suggest that as the temperature increased, there was a noticeable degradation of carotenoids, leading to the formation of low molecular weight substances, short-chain isoprene derivatives, and oxygenated compounds. This thermal degradation is characterized by the disappearance of characteristic absorption bands associated with carotenoid double bonds and the emergence of new peaks corresponding to these degradation products. Such findings are consistent with studies that have observed similar thermal-induced changes in carotenoid structures, which are known to contribute to the aroma profiles of various foods and natural products (Stutz et al. 2015). Additionally, the appearance of oxygenated compounds in the FT-IR spectra suggests oxidative processes occurring during encapsulation, which can influence the stability and bioactivity of the encapsulated propolis extract.

Microbiological analysis of OECP and MOECP

Table 6 presents the results of the microbiological analysis of the MOECP samples.

Table 6: Minimum inhibitory concentration (MIC) of propolis OECP- according to various parameters used in the Spray Dryer.

The MOECP activity against Salmonella enteritidis and Klebsiella sp. was null because the bioactive compounds that inhibit these bacterial strains were subjected to spray-drying and volatilization processes or other chemical reactions that altered their functional groups. In addition, the substances in the extract that do not undergo changes during encapsulation lack sufficient activity. This phenomenon was also evidenced -by MOECP activities against gram-positive and coliform organisms such as Escherichia coli and microorganisms of the Enterobacter genus, whose minimum inhibitory concentrations were higher than in OECP. In this study, the activity against gram-positive microorganisms such as Staphylococcus aureus was higher than that of other microparticles obtained from other propolis derivatives, such as ethanolic extracts (Da Cruz Almeida et al. 2017).

Previous studies have investigated microencapsulated with maltodextrin as the wall material, where the encapsulated bioactive compounds were plant extracts such as guava (Psidium guajava L) (Fernandes R et al. 2014), cinnamon (Cinnamomum zeylanicum) (Ostroschi et al. 2018), soybean (Glycine max) (Kalušević et al. 2017) and walnut (Juglans regia) (Cheraghali et al. 2018). The results of these studies indicated that none of the bioactive compounds exhibited activity against coliforms or gram-positive bacteria. Therefore, the results presented in Table 6 mean that MOECP had better in vitro antimicrobial activity than other plant extracts because of the high content of bioactives with a bactericidal capacity.

Figure 2 presents the results of the multivariate analysis performed on MOECP for antimicrobial activity.

Figure 2: Principal Component Analysis. A) Score plot of microencapsulated propolis oily extracts and B) Loading plots of variables evaluated in the analysis of propolis oily extracts for antimicrobial activity.

One of the most important factors was a clear differentiation of samples according to their biological activity. Only formulations 1 and 2 shared similarities in terms of their bioactivity. Additionally, MOECP activity against Staphylococcus aureus and Escherichia coli differentiated the samples analyzed better. The activity against microorganisms of the genera Enterobacter, Klebsiella sp., and Salmonella was similar among the eight formulations, indicating that the drying conditions did not affect the activity against these pathogens. There were only two variables that effectively differentiated the samples, and the multivariate protocol allowed the samples to be differentiated.

Microscopy of the MOECP

Figure 3 shows the morphological characterization of MOECP samples using SEM. It is possible to identify that the microparticles from the oily propolis extract and the mixture of maltodextrin and Gum arabic were mostly spherical although there were amorphous particles with smaller particles were grouped. These clusters were larger in samples with higher concentrations of the oily propolis extract, such as formulations 1 and 2. Lower concentrations and higher temperatures resulted in larger particles with larger contact surfaces and lower densities of particles, possibly because of the higher evaporation rate in these treatments. The particles should be as smooth as possible to facilitates the retention of material. The results showed that temperature was the variable that most affected the micro-encapsulation process and that the samples subjected to higher temperature values had a rougher surface as determined by the microscopy analysis, meaning these particles presented higher bioactive losses.

Figure 3: MOECP microparticles at 10 µm magnification. A) Sample 1 (100 °C), B) Sample 2 (120 °C), C) Sample 3 (140 °C), D) Sample 4 (160 °C), E) Sample 5 (100 °C), F) Sample 6 (120 °C), G) Sample 7 (140 °C), H) Sample 8 (160 °C). MOECP: Microencapsulated oily extract of Colombian propolis.

The results of three-dimensional microscopy of MOECP particles are related to their antioxidant activity. It has been reported that, as temperature increases, the presence of defects and cracks increases, which reduces the effectiveness of the wall material because they can degrade through oxidation and hydrolysis processes (Andrade et al. 2017). In terms of morphology, the macroparticles, regardless of their temperature and concentration, retained the same shape, as reported by Ramakrishnan et al. (2018), who used the same wall materials to encapsulate tamarind juice. Maltodextrin was responsible for circular morphology because the proportion of this polysaccharide with respect to Gum arabic was constant. Changes in the shape in the encapsulates were minimal. Microscopy revealed that in the particles with a higher concentration of the extract had more clusters because the spray drying process had a greater mass, which led to microparticles with greater thickness, higher molecular weight, and possibly more clusters (Insang et al. 2022). Samples with more clusters and greater thickness are expected to have a greater extract and, therefore, greater action against pro-oxidant substances and pathogenic bacteria.

DSC analysis of MOECP

Figure 4 and Table 7 show the results of the DSC analysis, which indicate the endothermic and exothermic changes in the MOECP.

Figure 4: DSC spectra of the MOECPs. a) Light Blue: Sample 1 (100 °C), b) Brown: Sample 2 (120 °C), c) Purple: Sample 3 (140 °C), d) Light Green: Sample 4 (160 °C), e) Yellow Ochre: Sample 5 (100 °C), f) Black: Sample 6 (120 °C), g) Dark Green: Sample 7 (140 °C), h) Dark Blue: Sample 8 (160 °C). MOECP: Microencapsulated oily extract of Colombian propolis.

Table 7: Endothermic and exothermic changes in MOECP based on DSC thermal analysis.

The thermograms showed two endothermic events between 50 and 180 °C, while one exothermic event can be observed between 290 and 320 °C. The samples with a higher concentration of propolis (treatments four to eight) had endothermic peaks with increased height and thickness, while those with a lower concentration of propolis had narrower and weaker peaks. The two melting peaks correspond to Gum arabic and maltodextrin; they presented a difference in intensities, where the peak for Gum arabic (located between 50 and 65 °C) was fainter than that of maltodextrin (located between 150 and 180 °C). In turn, the samples with a higher concentration of the oily propolis extract (1, 2, 3, and 4) had less degradation than those with an extract: encapsulant ratio of (1:3) (5, 6, 7 and 8). It was concluded that, at a higher temperature and lower concentration, the intensity of the endothermic peaks increased, and, consequently, the amount of encapsulated extract decreased. It is recommended to maintain the encapsulation process conditions proposed in formulation two since it had the highest stability as represented by the higher endothermic peak.

The endothermic peak at 50 °C signaled volatilization of water, while the peaks at 150 °C corresponded to fusion processes where there were fusions between low molecular weight compounds, such as volatiles and polyphenols, and the wall material. The exothermic peak between 290 - 320 °C for all extracts indicated the loss of compound and rupture of some microencapsulated particles. This peak was more intense for samples with a higher oily extract concentration because more bioactive compound molecules underwent chemical changes, such as depolymerization and decomposition processes. The higher the intensity of the peaks, the greater the stability of the samples were, therefore, the formulations with higher OECP concentration were more stable. The heat values were higher for formulations 1 and 2, which had a higher concentration of OECP and a lower drying temperature, indicating that the samples were more resistant to high temperatures, pathogens, mechanical forces, degenerative reactions, and other decomposition processes (Ballesteros et al. 2017).

In the future, the addition of a protein system to the elaboration of microparticles, such as ovalbumin and pea protein, can be considered because encapsulates made with various bioactives from these biopolymers have presented more intense endothermic peaks, and exothermic events were not observed that signal the release of secondary metabolites from the particles and, therefore, the restoration of the structural polysaccharides to their original state (Jansen-Alves et al. 2018; Jansen-Alves et al. 2024).