Co-encapsulation of Paprika and Cinnamon Oleoresins by Spray Drying in a Mayonnaise Model: Bioaccessibility of Carotenoids Using in vitro Digestion

This study aimed to investigate the digestibility and bioaccessibility of spray-dried microparticles co-encapsulating paprika and cinnamon oleoresins using simulated gastrointestinal conditions. It focused on exploring the potential of these co-encapsulated active compounds, which possess diverse technological and functional properties, particularly within a food matrix, in order to enhance their bioavailability. Mayonnaise was selected as the food matrix for its ability to promote the diffusion of carotenoids, as most hydrophobic compounds are better absorbed in the intestine when accompanied by digestible lipids. Model spice mayonnaise, containing 0.5 wt% paprika and cinnamon microparticles content, was formulated in compliance with Brazilian regulations for spices, seasonings, and sauce formulations. Droplet size distribution, optical microscopy and fluorescence microscopy analyses were conducted on the microparticles, model spice mayonnaise, and standard mayonnaise both before and after in vitro gastric and intestinal digestion. Following digestion, all samples demonstrated droplet aggregation and coalescence. Remarkably, dispersed particles (37.40 ± 2.58%) and model spice mayonnaise (17.76 ± 0.07%) showed the highest release rate of free fatty acids (FFAs), indicating efficient lipid digestion. The study found that using mayonnaise as a delivery system significantly increased bioaccessibility (22.7%). This suggests that particles in an aqueous medium have low solubility, while the high lipid composition of mayonnaise facilitates the delivery of active compounds from carotenoids present in paprika and cinnamon oleoresin after digestion.


Introduction
Paprika and cinnamon oleoresins (PCOs) have received attention in the food industry due to their antioxidant and antimicrobial properties, arousing interest in their functional capabilities [1,2].Cinnamon oleoresin, with its distinctive dark and viscous nature, acts as a flavoring agent and it boasts a diverse chemical composition containing ensuring controlled release during gastrointestinal digestion [5,6].Maltodextrin (MD) is commonly employed in spray drying methods for microencapsulation due to its functionalities, including low viscosity, hygroscopicity, high water solubility, and lack of sweetness.Additionally, whey protein isolate (WPI), rich in proteins (β-lactoglobulin, α-lactoglobulin, immunoglobulin, and albumin), exhibits amphiphilic, biological, and emulsifying activity, as well as film-forming ability, making it widely used in encapsulating various bioactive compounds [7].In recent years, several protein-polysaccharide mixtures have been explored as a cost-effective alternative with suitable encapsulating properties for producing particles using the spray drying technique [8][9][10].
Furthermore, recent studies have evaluated the synergistic effect of co-encapsulated compounds.Co-encapsulation involves simultaneously encapsulating two or more active compounds to produce particles rich in different components that are not commonly found together yet can exert pronounced functional effects when incorporated into the same matrix [11,12].Alternatively, co-encapsulation may involve encapsulating a single bioactive using two or more encapsulation techniques.Ferraz et al. [13] previously identified the synergistic effect of paprika and cinnamon oleoresin mixtures by incorporating and characterizing them into emulsions.
Vulić et al. [14] addressed the relevance of encapsulating bioactive compounds from red pepper waste (RPW), a by-product of the vegetable processing industry, using whey protein isolate (WPI) as the encapsulating material.The study evaluated the effects of in vitro gastrointestinal digestion on the release and bioactivity of encapsulated bioactive.The results revealed that RPW encapsulation protected the bioactive compounds against pH changes and enzymatic activities during digestion, increasing their gut bioaccessibility [9].These findings highlight the efficiency of encapsulation for the valorization of bioactive compounds and its potential for nutritional improvements, color enhancement, and bioactive properties in food.
In this context, the current study focuses on examining the in vitro digestibility of co-encapsulated paprika and cinnamon particles.This investigation explores how these particles perform in a physiologically relevant environment, simulating in vivo conditions by incorporating factors such as digestive enzymes, pH variations, digestion duration, and salt concentrations.
Mayonnaise was chosen as the food matrix due to its high lipid concentration, promoting an efficient absorption in the intestine of hydrophobic compounds such as carotenoids and cinnamaldehyde, thereby facilitating their application, enhancing stability, and improving carotenoid bioavailability [10].Mayonnaise, typically comprising 70-80% fat, is one of the most popular sauces or condiments worldwide, consisting of egg yolk, vinegar, oil, and spices [15].The utilization of mayonnaise as a delivery system for the active compounds aims to enhance their bioaccessibility after digestion, offering a potentially effective application of these compounds in food products.In addition to their functional properties, paprika and cinnamon oleoresins can serve as color and flavor agents in food products.
This study presents new research focused on investigating the bioaccessibility of encapsulated compounds when applied to a food matrix.The findings contribute to food science by offering effective encapsulation strategies and potential health-promoting food products enriched with bioactive compounds, expanding our understanding of the functionality of foods.

Materials and methods
The material and methods section is presented as supplementary material.

Results and Discussion
Structural characterization before and after in vitro digestion.
Droplet size distribution of the dispersed particles, model spice mayonnaise, and standard mayonnaise obtained before and after gastric and intestinal digestion were provided in supplementary files (see Supplementary Material, Fig. S1, and Table S1).Mean droplet size and polydispersity was calculated from droplet size distribution.
Before digestion (initially), the dispersed particles showed a monomodal size distribution.Model spice mayonnaise and standard mayonnaise showed a bimodal size distribution (Fig. S1), with span values varying between 1.79 ± 0.00 and 1.88 ± 0.01 and mean droplet diameter (D 4,3 ) ranging between 2.15 ± 0.02 and 11.92 ± 0.05 μm (Table S1).These differences in particle size distribution in the initial phase are affected by homogenization, emulsion composition and viscosity of the different samples [16].
Particle size of dispersed particles increased after the gastric step, which was attributed to the presence of pepsin protease.This protease caused the hydrolysis of WPI on the surface of the particles, allowing particles coalescence.Also, due to the acidic pH of the stomach, which is close to the isoelectric point of WPI, there was a decrease in the surface charge of the particles and, as a consequence, the repulsion between them [17,18] and increase of coalescence.Similar behavior was observed for model spice mayonnaise and standard mayonnaise but with a lower intensity probably due to the lack of a significant influence on the system from the high oil content, since attraction and coalescence act on the protein part and cause destabilization.In addition, the high viscosity of mayonnaise can delay the mobility of particles and hinder coalescence/flocculation, since the particles need to get close to generate destabilizing effects [17,19].
After digestion (in the intestine), all samples tended to have a multimodal size distribution and presented high span and D4.3 values (p < 0.05), suggesting an extensive aggregation of droplets.Goldin et al. [20] and Quian et al. [21] reported that changes in aggregation were caused by several physicochemical mechanisms.Generally, bile salts can remove and replace compounds at the droplet/particle interface, allowing pancreatin (lipase) adsorption.Pancreatin hydrolyzes lipids and releases fatty acids into the digestive fluid.Pancreatin also acts as a protease and contributes to the hydrolysis of WPI at the interface.With protein breakdown, particles destabilize and coalesce.Emulsified digestible triacylglycerols (i.e., long chain triacylglycerols and medium chain triglycerides) are converted to free fatty acids (FFAs) and monoacylglycerols in the presence of adsorbed lipases, thereby altering the internal composition, structure, and properties of the lipid droplets that may also have decreased their coalescence stability.Due to the low concentration (0.5%, w/w) of particles in the model spice mayonnaise, there were many irregularly shaped clumps of small particles present, as indicated by the arrow in Fig. 1.
The presence of carotenoids at droplet surfaces may have protected the emulsified oil from oxidation [22], which would be more beneficial to model spice mayonnaise than to standard mayonnaise during digestion.These results corroborate fluorescence microscopy images (Fig. 1).Moreover, when triacylglycerols are digested into FFAs (Fig. 3) by lipase, the composition of the lipid droplets is altered, leading to changes in droplet morphology and aggregation stability [21].

Lipolysis
Lipid digestion of dispersed particles, model spice mayonnaise, and standard mayonnaise in the intestinal phase is presented in terms of FFA release (Fig. 2).In all delivery systems, the volume of NaOH increased rapidly in the

Impact of Model Spice Mayonnaise on Carotenoid Bioaccessibility
Carotenoids in paprika oleoresin are primarily responsible for the color of microparticles incorporated into mayonnaise [27,28].All carotenoid pigments in paprika have chromophoric properties that allow their grouping into two different isochromatic fractions: red and yellow.The red fraction contains pigments exclusive to the capsicum genus (capsanthin, capsorubin, and capsanthin-5, 6-epoxide).In contrast, the yellow fraction comprises the other pigments (zeaxanthin, violaxanthin, antheraxanthin, β-cryptoxanthin, β-carotene, etc.) that act as precursors of the first [20,29].The results obtained for the content of each isochromatic fraction (red and yellow) and the total carotenoid content of the dispersed particles, model spice mayonnaise, and standard mayonnaise before and after in vitro digestion are shown in Table 1.
The bioaccessibility and simulated bioavailability of total carotenoid content were assessed after in vitro digestion of dispersed particles and model spice mayonnaise, as shown in Fig. 3.The standard mayonnaise was also evaluated, and the carotenoids value was subtracted in order to avoid a false-positive result for the tests.Bioaccessibility and simulated bioavailability in the dispersed particles were 19.8 ± 1.45 and 9.6 ± 1.02%, respectively.In the model spice mayonnaise, bioaccessibility was 22.7 ± 1.65% and simulated bioavailability 15.4 ± 1.39% (Fig. 3).The bioaccessibility of the encapsulated carotenoids is highly dependent on their first 10 min of digestion and then slowed down.Therefore, there was a rapid increase in FFA release during the first minutes of digestion followed by a gradual increase over longer times, suggesting that lipase molecules were rapidly absorbed on lipid droplet surfaces and started to hydrolyze the triacylglycerol molecules.
During digestion, the rate of lipolysis might be regulated by the mobility of lipases interacting with the dispersed oil phase [23].Our calculation shows that the lipids in the dispersed particles were 37.40 ± 2.58% digested but only 17.76 ± 0.07 and 17.29 ± 0.01% digested in spice and standard mayonnaise, respectively.Similar results were obtained by Xia et al. [24] in high-fat samples when studying the influence of lipid content in a corn oil preparation on the bioaccessibility of β-carotene.Fluorescence microscopy (Fig. 2) indicated the presence of a great number of nondigested lipid droplets after digestion.Incomplete digestion of the lipid phase in a high-fat system, such as mayonnaise, may result from insufficient lipase to digest all droplets due to an inadequate volume of bile salts to solubilize lipids and remove them from the surface of the droplets.An emulsifier can also disrupt lipase access to lipids, and some FFAs can stay on the surface of the droplets and block lipase digestion [25,26].high bioaccessibility [30,31].Similar results were found by Roman et al. [32], who evaluated the digestibility of mayonnaise enriched with sea buckthorn carotenoids.

Conclusion
This study successfully explored the digestibility and bioaccessibility of co-encapsulated paprika and cinnamon oleoresins through spray-dried microparticles under in vitro simulated gastrointestinal conditions.The application of coencapsulated oleoresins showed potential, revealing higher bioaccessibility within a mayonnaise delivery system than in dispersed particles.Pre-and post-digestion structural analysis showed altered droplet size distribution and aggregation.Mayonnaise's incorporation enhanced carotenoid stability and diffusion, indicating effective post-digestion solubility in the digesta [24]; this solubility is influenced by many factors, including the physical composition of carotenoids and wall material composition; molecular weight; surface charge; and dosage of stabilizers and emulsifiers [30].These results suggest mayonnaise is an efficient delivery system for paprika and cinnamon microparticles since it has a high lipid content that contributes for the diffusion of active compounds from carotenoids after digestion.
Vulić et al. [14] reported that encapsulating carotenoid and phenolic phytochemicals from red pepper waste using WPI protected these phytochemicals against pH changes and enzymatic activities during digestion and contributed to an increase in the bioaccessibility of both carotenoids and phenolics in the gut.The presence of a mayonnaise delivery system helps in the formation of micelles in the intestinal phase and increases the solubilization capacity of total carotenoid content in these micelles, leading to a

Fig. 1
Fig. 1 Optical and confocal images of dispersed particles, model spice mayonnaise, and standard mayonnaise in different digestion phases (initial, gastric, and intestinal).Nile red stained the lipid phase of all samples.White arrows indicate the location of paprika and cinnamon

Fig. 3
Fig. 3 Bioaccessibility and simulated bioavailability values of total carotenoids for dispersed particles and model spice mayonnaise.Different lowercase letters between dispersed particles and model spice mayonnaise indicate a significant difference (p ≤ 0.05) was revealed by the Tukey test

Table 1
Carotenoid content before and after in vitro digestion