Encapsulation of propolis extract in ovalbumin protein particles: characterization and in vitro digestion

Abstract The encapsulation of propolis has shown promising results for the protection of bioactive compounds, local and gradual release and masking the astringent taste. Ovoalbumin is a protein of animal origin found in large amounts in egg whites, which has good properties as a wall material for particles.The objective of this study was to microencapsulate propolis by spray drying. The best condition for microencapsulation was achieved with 4% ovalbumin at 120 °C, where there was the greatest encapsulation efficiency (88.20%) and spherical shape. However, the increase of ovalbumin concentration resulted lower yields (< 52%). As for the scanning electron microscopy (SEM), the increase of ovalbumin concentration caused an increase of the size with average diameter and formation of spherical microcapsules. The phenolic compounds were already released in the gastric fluid condition (stomach). Graphical Abstract


Introduction
propolis is a made by bees through resinous and gummy substances.Several studies have reported the beneficial properties of propolis as antimicrobial, antioxidant, anti-inflammatory, antitumor, and with healing and anesthetic activity (pobiega et al. 2019).
in spite of all the beneficial effects already reported on the consumption of propolis and the great interest for the enrichment of foods and beverages, its consumption is still limited due to its bitter taste, which limits its application (Keskin et al. 2019).therefore, alternative ways of introducing propolis into food are sought, without to cause changing of food odor and taste.
the encapsulation technique has known to provide protection for the bioactive compounds, increasing solubility, as well as minimizing changes in taste, odor and color. in addition, microencapsulation has been used to increase nutrient uptake, as it may make possible to release the compounds at the ideal site of absorption, that in the case of bioactive compounds would be in the intestine (Hur et al. 2011).
different substances have been evaluated as wall material in the propolis encapsulation.ovalbumin, the main protein present in egg white (54%), is characterized as an acidic glycoprotein.Recent studies have demonstrated that the use of this protein is effective in the encapsulation of compounds such a curcumin (Visentini et al. 2017), linoleic acid (Sponton et al. 2017), andof propolis (Jansen-alves et al. 2019).
the aim of this study was to evaluate the influence of different concentrations of ovalbumin as encapsulating material, and of the different spray drying temperatures on the encapsulation efficiency, yield, morphology and release profile in gastrointestinal in vitro.

Encapsulation efficiency and yield
according to results of table S1, it can be observed that the drying temperature did not influence (p > 0.05) the encapsulation efficiency of the pE phenolic compounds for the microcapsules made with 1% of ovalbumin.However, when using 4% of ovalbumin, there was a significant difference in the values (p ≤ 0.05) in relation to the increase in the drying temperature.the highest encapsulation efficiency of phenolic compounds was obtained with 4% of ovalbumin at 120 °C by spray drying (88.20%).this result is related to the fact that the higher concentration of ovalbumin allowed a greater number of binding sites with the pE phenolic compounds.
However, a higher inlet temperature also resulted in the higher outlet temperature inside of the product collection vessel.When a higher concentration of the encapsulating material was used, the time for drying and obtaining the powders were longer, that is, the phenolic compounds were exposed for a longer time at the temperatures used in the spray drying process.this leads to the belief that the retention of phenolic compounds in the formulation with 4% of ovalbumin at 160 °C may have been reduced due to a greater exposure to high temperatures.
Furthermore, the drop in the retention of phenolic compounds from the powder with the high concentration of ovalbumin (4%) obtained from a high inlet temperature (160 °C) in the spray drying may have been caused by the breaking of the coating layer of the microcapsules.Excessive evaporation of water in the microcapsules at high temperature can cause cracks in the surface of the coating layer (Chuyen et al. 2018).the data shows that using higher protein concentration for pE encapsulation it was significantly reduced (p ≤ 0.05) the spray drying yield (21.16 to 33.22%).When using 1% of ovalbumin, regardless of the spray drying temperature, higher yields (p ≤ 0.05) were obtained (50.21 to 52.04%).When 4% of ovalbumin was used, the temperature positively affected the yield (p ≤ 0.05).
the use of 4% of ovalbumin caused a reduction in yield due to high adhesion of wall materials to the spray dryer.Formulations with 1% of ovoalbumin led to a lower concentration of microcapsules per mililiter than other treatments, which may explain the low adherence of the powder to the walls of the equipment during drying, contributing to the higher yield of these microcapsules (Jansen-alves et al. 2019).proteins adhere inside the equipment and become rapidly sticky, which hinders their drying and removal (moser et al. 2017).

Morphology and size distribution
through the analysis of morphology it was observed relatively spherical form of the microcapsules with varied sizes and concavities (Figure S1).However, as can be observed from the micrographs, the microcapsules made with 1% of ovalbumin resulted in smaller sizes, but with irregular shapes and concavities.
Higher concentration of encapsulating material (4% of ovalbumin) caused an increase in the size of the microcapsules, resulting more amount of spherical microcapsules, with an average diameter 6.72 and 6.87 µm at 120 and 160 °C, respectively.this might be explained by a higher solid content used in the present work.it is important to note that the increase in the size of the microcapsules can interfere with the sensory perception of the taste of propolis, since larger microcapsules probably trapped a greater amount of pE.
the formation of roughness or invagination is an undesirable characteristic, due to the possible acceleration of releasing the encapsulated material (lacerda et al. 2016).the observed surface roughness is characteristic of microparticles produced by spray drying at low drying temperatures.in this case, the microparticles surface remains moist and supple during the drying process, so the particle deflates and shrivels as it cools down (tonon et al. 2008).

In vitro digestion study
it can be observed in Figure S2 that a large amount of the encapsulated phenolic compounds were released after 1 h in the gastric fluid, with significant reduction (p ≤ 0.05) of the compounds after 2 h. in the intestinal fluid there was a significant reduction (p ≤ 0.05) of phenolic compounds in all microcapsules.
according to Jimenez-Saiz et al. (2011) depending on the morphology of the microcapsules, a digestion can occur partially or completely in the gastric and intestinal phases.Because a change in protein structure (denaturation and aggregate training) induced by increased heat treatment can alter the accessibility of digestive enzymes (Visentini et al. 2017).
according to Visentini et al. (2017), ovoalbumin with aggregates and with different morphologies is more susceptible to degradation by enzymes compared to native (pure) protein.the ovoalbumin microcapsules that have already undergone heat treatment and rearranged the conformational structure due to high temperatures, suffer greater attack from stomach enzymes.this fact was probably responsible for the rapid release of phenolic compounds from the microcapsules, especially those that were produced with 4% of protein and dried at 160 °C, which ones required longer time inside the spray dryer.
these data refer to the gastrointestinal release of the microparticles, but it does not prevent the application of the microparticles in food to modify or delay the release site of the phenolic compounds, because the presence of other compounds such as fats, sugars, present in most foods can act as protectors in the gastric fluid.

Conclusions
there was influence of ovalbumin concentration on the encapsulation efficiency of phenolic compounds, yield and morphology.the highest encapsulation efficiency was achieved with 4% of ovalbumin at 120 °C.However, the increase in ovalbumin concentration resulted in lower yields.as for morphology, the increase in ovalbumin concentration caused an increase in particle size and also allowed the formation of more spherical microcapsules.none of the microcapsules presented adequate gastrointestinal release behavior, releasing the phenolic compounds in advance, already in the gastric fluid condition (stomach).Future studies with the application of capsules in food are necessary, as the nutrients present can interfere with the release of compounds.

Disclosure statement
no potential conflict of interest was reported by the authors.

Funding
CapES, CnpQ and Fundação de amparo à pesquisa do Estado do Rio Grande do Sul (BR) FapERGS (22/2551-0000840-2) with the cluster 'Supplements and products derived from alternative sources for animal welfare' , for the financial support.