Evaluation of Olive Leaf Phenolic Compounds’ Gastrointestinal Stability Based on Co-Administration and Microencapsulation with Non-Digestible Carbohydrates

Citation: Duque-Soto, C.; LeyvaJiménez, F.J.; Quirantes-Piné, R.; López-Bascón, M.A.; LozanoSánchez, J.; Borrás-Linares, I. Evaluation of Olive Leaf Phenolic Compounds’ Gastrointestinal Stability Based on Co-Administration and Microencapsulation with Non-Digestible Carbohydrates. Nutrients 2024,16, 93. https:// doi.org/10.3390/nu16010093 Academic Editors: Sergio Montserrat-de la Paz and Fernando Rivero-Pino Received: 2 November 2023 Revised: 21 December 2023 Accepted: 22 December 2023 Published: 27 December 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nutrients Article Evaluation of Olive Leaf Phenolic Compounds’ Gastrointestinal Stability Based on Co-Administration and Microencapsulation with Non-Digestible Carbohydrates Carmen Duque-Soto 1, Francisco Javier Leyva-Jiménez 2,3 , Rosa Quirantes-Piné4,*, María Asunción López-Bascón5, Jesús Lozano-Sánchez 1,* and Isabel Borrás-Linares 4 1 Department of Food Science and Nutrition, Faculty of Farmacy, University of Granada, Campus Universitario Cartuja s/n, 18071 Granada, Spain; [email protected] 2Area of Food Science and Technology, Faculty of Chemical Sciences and Technologies, University of Castilla-La Mancha, Avda. Camilo JoséCela 10, 13071 Ciudad Real, Spain; javier[email protected] 3Regional Institute for Applied Scientific Research (IRICA), University of Castilla-La Mancha, Avda. Camilo JoséCela 10, 13071 Ciudad Real, Spain 4Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avda Fuentenueva s/n, 18071 Granada, Spain; [email protected] 5 Research and Development Functional Food Centre (CIDAF), Health Science Technological Park, Avenida del Conocimiento 37, Edificio BioRegión, 18016 Granada, Spain; [email protected] *Correspondence: [email protected] (R.Q.-P.); [email protected] (J.L.-S.); Tel.: +34-958240781 (J.L.-S.) Abstract: The large generation of olive by-products has motivated their revalorization into highadded-value products. In this regard, olive leaves pose as an interesting source of bioactive compounds, due to their phenolic content with commonly known antioxidant, anti-inflammatory, and immunomodulatory properties, with potential application in non-communicable diseases. However, their effectiveness and applicability into functional foods is limited by their instability under gastrointestinal conditions. Thus, the development of protective formulations is essential. In this study, the spray-drying encapsulation of a phenolic-rich olive leaf extract with inulin as the encapsulating agent was optimized. Then, the behavior of the free extract under gastrointestinal conditions, its co-administration with the encapsulating agent, and the optimized microencapsulated formulation were studied through an in vitro gastrointestinal digestion process following the INFOGEST protocol. Digestion of the free extract resulted in the degradation of most compounds, whereas this was minimized in the co-administration of the non-encapsulated extract with the encapsulating agent. This protective effect, related to its interaction with inulin, was similar to the microencapsulated formulation. Thus, both approaches, co-administration and microencapsulation with inulin, could be promising strategies for the improvement of the stability of these anti-inflammatory and immunomodulatory compounds under gastrointestinal conditions, enhancing their beneficial effect. Keywords: olive leaf extract; phenolic compounds; microencapsulation; spray-drying; in vitro gastrointestinal digestion; bioaccessibility 1. Introduction Food industry by-products have emerged as promising sources of bioactive compounds and have been proposed for their revalorization into the production of high-addedvalue products, promoting a circular economy approach [ 1 , 2 ]. In this regard, the olive tree is one of the main crops cultivated in Mediterranean countries, which hold 97% of global production, increasing exponentially each year [ 3 , 4 ]. With Spain being the country with the highest production, waste accumulation associated with the production of olive oil is high, implying the production of 1–5 t/ha of pruning residue, including leaves and branches [ 3 – 5 ]. Its abundance and accumulation have been supposed to be an environmental problem, as they have been traditionally ground and burned, increasing contaminant Nutrients 2024,16, 93. https://doi.org/10.3390/nu16010093 https://www.mdpi.com/journal/nutrients Nutrients 2024,16, 93 2 of 20 CO 2 emissions. Thus, a strategy for environmental remediation has emerged through their revalorization, associated with their interesting composition with regards to phytochemicals, especially phenolic compounds, and reducing the production cost through monetizing food waste [6,7]. In this regard, phenolic compounds have gained interest throughout the scientific community for their functional and technological properties, which make them interesting compounds for the development of functional foods. Specifically, phenolic-rich extracts from different products derived from the olive tree have shown great antioxidant [ 8 , 9 ], anti-inflammatory [ 10 – 12 ], immunomodulatory [ 13 ], anti-hypertensive [ 14 ], hypocholesterolemic [ 15 ], hypoglycemic [ 16 ], and cardioprotective effects [ 17 ]. This bioactivity is especially significant for their impact on the treatment and prevention of non-communicable diseases, such as obesity [ 18 ], diabetes [ 19 ], and inflammatory intestinal diseases [ 20 – 22 ], pathologies whose incidence has increased dramatically during the last decades. More specifically, hydroxytyrosol and oleuropein, the most abundant phenolic compounds in olive leaves, have shown an anti-inflammatory effect on intestinal conditions, such as ulcerative colitis [ 23 – 25 ]. Additionally, these health benefits have been related to the impact of these compounds on the colonic microbiota, modulating gut microbial composition, promoting probiotic bacteria while inhibiting pathogenic strains [26,27]. However, their bioactive potential is dependent on their gastrointestinal release from the matrix, intestinal absorption, and pharmacokinetic behavior. In fact, their bioactivity, and consequently health benefits, may be limited by their instability under gastrointestinal conditions. Specifically, phenolic compounds present poor water solubility and stability, being sensitive to unfavorable environments such as high temperatures, light, oxygen, low pH, and enzymatic activity, with most of these occurring under gastrointestinal conditions, which renders them as non-stable substances during digestion [ 28 – 30 ]. In fact, oleuropein, among other phenolic compounds from olive structures, has been shown to be heavily degraded under gastrointestinal conditions, being affected by the low pH values found in the fasting gastric conditions [ 31 – 33 ]. Additionally, they are rapidly metabolized to polar compounds and eliminated from the body, mainly in the urine [ 34 ]. Hence, the nature of their oral administration could compromise their observed effect through a reduction in their bioaccessibility, as the gastrointestinal conditions may enhance this degradation. To solve this problem, the encapsulation of these bioactive compounds has been proposed as a useful strategy to ensure their protection throughout the gastrointestinal tract while allowing for a controlled release in the areas of interest, modulating their access to absorption sites and interaction with colonic microbiota and enhancing their effect on human health. However, the encapsulation technique and conditions applied are limited by the physicochemical stability of phenolic compounds. In this sense, spraydrying has been widely used for bioactive molecules, due to its low cost and industrial scalability [ 35 , 36 ]. This methodology also presents a number of advantages for bioactive compound encapsulation, including a reduced exposure to high temperatures, high quality and stability of the resulting microcapsules, and a low operating cost [ 37 , 38 ]. Among the possible encapsulating agents, non-digestible carbohydrates present an incredible opportunity for colon-targeted delivery of compounds. In this sense, inulin, a fructantype non-digestible polysaccharide, can be catabolized by the colonic microbiota, thus allowing for a transportation of these compounds to their desired sites of action and their interaction with colonic microbiota [ 39 – 41 ]. Additionally, previous studies have established the bioactive potential of this polysaccharide, as it has proved to be an outstanding prebiotic agent, regulating blood sugar and lipids, as well as possessing antioxidant, anticancer, and immune regulation activities, among others [42]. Prior to their implementation, the behavior of the resulting microcapsules under biological conditions needs to be assessed to evaluate their transportation ability. While both in vivo and in vitro models are available, due to the complexity and cost of the former, the latter have emerged as desirable alternatives in the accurate representation of the digestive process. The use of in vitro gastrointestinal digestion protocols supposes an Nutrients 2024,16, 93 3 of 20 approach to a better understanding of the behavior of phenolic compounds under these conditions, as well as allowing consideration of the protective effect that microencapsulation may bring to their phenolic profile. In this sense, the INFOGEST protocol constitutes a harmonized and standardized protocol which allows a correct representation of the gastrointestinal tract as well as a comparison between studies [43,44]. However, although encapsulation of olive leaf extracts has been reported in the literature, the comparison of the phenolic profile resulting from the digestion of these encapsulated formulations with the non-encapsulated phenolic compounds co-administered with inulin is yet to be assessed [ 37 ]. In this sense, with this co-administration, the protective effect due to the interaction of inulin with phenolic compounds on their stability under gastrointestinal conditions could be evaluated. Indeed, this comparative evaluation would be essential for an improvement in the understanding of the digestion of phenolic compounds and their protection from gastrointestinal conditions, as well as to improve the development of functional foods with scalable and effective protective strategies. Therefore, the aim of this study was the evaluation of the differential phenolic profiles, under in vitro gastrointestinal conditions, of a microencapsulated, free co-administered formulation with inulin and free olive leaf phenolic-rich extract in order to improve protection under gastrointestinal conditions. Thus, the optimization of the microencapsulation of olive leaf extract with inulin using spray-drying was performed using the response surface methodology (RSM). Then, the optimum microencapsulated formulation together with the free formulations (non-encapsulated olive leaf phenolic extract and its combined administration with inulin) were assayed under gastrointestinal conditions to study the potential improvement/enhancement in colon bioaccessibility and the protection of the phytochemicals from the olive leaf extract. The in vitro gastrointestinal digestion was carried out following the INFOGEST protocol for the evaluation of the effect of both co-administration with inulin and the encapsulation strategy by spray-drying on its protection. 2. Materials and Methods 2.1. Chemicals Commercial olive leaf extract was kindly provided by Deretil S.L. (Cuevas del Almanzora, Spain), enriched in hydroxytyrosol and oleuropein. For the encapsulation processes, inulin was purchased from Fagron (Barcelona, Spain). Ethanol and LC-MS-grade methanol and acetonitrile was acquired from Fisher Chemicals (Waltham, MA, USA), whereas acetic and formic acids were from Sigma-Aldrich (Steinheim, Germany). Milli-Q water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Moreover, hydroxytyrosol, oleuropein, luteolin-7-O-glucoside, and loganin standards were acquired from SigmaAldrich or Extrasynthese (Genay Cedex, France). For in vitro digestion, enzymes (pepsin 3412 U/mg and pancreatin 4xUSP) and bovine bile salts (Sigma B-8631) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Chemicals for the preparation of simulated digestive fluids, hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium chloride (NaCl), potassium dihydrogen phosphate (KH 2 PO 4 ), potassium chloride (KCl), sodium hydrogen carbonate (NaHCO 3 ), and ammonium carbonate ([(NH 4 ) 2 CO 3 ]), were purchased from Fisher Chemicals (Waltham, MA, USA). 2.2. Microencapsulation of the Olive Leaf Phenolic-Rich Extract by Spray-Drying The optimization of the microencapsulation of olive leaf extract with inulin by spraydrying was performed using the response surface methodology (RSM) based on a central composite design (CCD) 2 2 model with a star and four central points (Statgraphics Centurion version XVI supported by Statpoint Technologies, Warrenton, VA, USA). The effect of the independent variables, specifically, air temperature (134.75–195.25 ◦ C) and extract:inulin ratio (0.68–4.315), on the response variables, the encapsulation efficiencies (%EE) of hydrox- Nutrients 2024,16, 93 4 of 20 ytyrosol (HT) and oleuropein (OLE), was evaluated. The experimental results were fitted to a second-order polynomial model, as shown in Equation (1): Y=β0+ n ∑ i=1 βiXi+ n ∑ i=1 βii X2 i+ n ∑ i=1 n ∑ j=i+1 βij XiXj(1) where Yrepresents the response variable; β0 is the response constant coefficient fixed at the central point of the experiments; βi , βii , and βj are the regression coefficients of the linear, quadratic, and interaction terms, respectively; and X i and X j represent the values of the independent variables. With the purpose of evaluating the adequacy of the proposed model and the adjustment of the obtained data, three different parameters were assessed (model adequacy, coefficient of determination (R 2 ), and lack-of-fit test). Moreover, optimization was performed using the desirability function on those responses with higher fitting. Moreover, the desirability function is an approach that enables the identification of the simultaneous optimum conditions, estimating the global desirability of the responses (the best condition for both responses) in each run. For that purpose, the desirability function takes values from 0 to 1, where values close to 1 reveal the best conditions to achieve the proposed optimization [ 45 ]. All experimental runs were carried out randomly in a spray-dryer 4M8-TriX instrument (ProCept, Zalzate, Belgium) comprising a process column, an angled T transport tube, a cyclone, and a product manifold. For the encapsulation process, inulin (in a range of 0.69–4.32 g) was previously dissolved in water (in quantities between 48.31 and 44.68 g) at 70 ◦ C until obtaining a homogeneous solution. After that, 1 g of the extract was added and mixed by stirring until complete dilution (50 g), obtaining a total feeding solids from 3.37% to 10.63%. The drying procedures were carried out setting the conditions as follows: inlet air temperature, 135–195 ◦C ; airflow, 0.30 m 3 /min; feeding flow, 2 mL/min; atomization air flow, 13 L/min; nozzle diameter, 0.6 mm; and differential pressure of cyclone, 15–16 mbar. Whereas the outlet air temperature was maintained in the range of 60–90 ◦ C. The attained microparticles after each procedure were kept protected from light and moisture at room temperature. 2.3. Encapsulation Efficiency (%EE) Assesment In order to determine the encapsulation efficiency, the phenolic contents of both the non-encapsulated and encapsulated fractions were calculated. First, to recover the nonencapsulated fraction, 150 mg of microparticles was added in 1 mL of MeOH-EtOH, 50:50 (v/v), and dispersed using gentle agitation. The obtained suspension was centrifuged at 90 × gat 4 ◦ C for 1 min, recovering the supernatant which was later centrifuged at 360 × gat 4 ◦ C for 1 min. Finally, the obtained supernatant was filtered through a 0.2 µ m PTFE filter and maintained at − 20 ◦ C and protected from light exposure for later analysis. For the extraction of the encapsulated compounds, 150 mg of microparticles was added to 0.75 mL of MeOH-EtOH, 50:50 (v/v), and vortex-mixed for 1 min. Then, the microparticles were introduced into a refrigerated ultrasound bath for 20 min and centrifuged at 15,000 × g at 4 ◦ C for 10 min. The procedure was repeated on the obtained pellet and both supernatants were combined, centrifuged at 15,000 × gat 4 ◦ C for 10 min, filtered through a 0.2 µ m cellulose filter, and maintained at − 20 ◦ C and protected from light for later analysis. The EE% was determined using Equation (2), described elsewhere: EE% =Totalcompoundcontent −Nonencapsulatedcompoundcontent Totalcompoundcontent ×100 (2) 2.4. INFOGEST Static In Vitro Digestion Olive leaf extract, in the free co-administered formulation with inulin and its microencapsulated form with inulin as the encapsulating agent, was subjected to static in vitro gastrointestinal digestion following the harmonized INFOGEST protocol described in Minekus et al., 2014 with modifications proposed by Brodkorb et al., 2018 regarding phe- Nutrients 2024,16, 93 5 of 20 nolic compounds [ 43 , 44 ]. The digestions were carried out in triplicate for each sample: commercial olive leaf extract (5 g), olive leaf extract combined with inulin (1.3 g of extract with 3.7 g of inulin), and olive leaf extract microencapsulated with inulin as the encapsulating agent (5 g). For the simulation of oral digestion, 5 g of each substrate was dissolved in 5 mL (1:1, w/w) of simulated salivary fluid (SSF) in a 50 mL centrifuge tube, protected from light exposure, and vortexed for 5 min. Then, 7.5 mL of simulated gastric fluid (SGF) containing 2000 U/mL of pepsin, and 5 µ L of 0.3 M CaCl 2 was added to the resulting bolus. The pH was adjusted to 3.0 by addition of 1 M aqueous HCl and milli-Q H 2 O was added until a final volume of 18 mL was achieved. The resulting mixture was homogenized, inertized with N 2 flow to ensure anaerobic conditions, and incubated for 120 min at 37 ◦ C under constant agitation at 150 rpm using a thermostatic incubator (MaxQTM 6000 SHKE6000-8CE, Thermo Scientific, Waltham, MA, USA). Thereafter, samples (1 mL) were taken at the end of the gastric digestion and stored at −80 ◦C for their later analysis. Subsequently, to stop gastric digestion, the pH was increased to 7.0 by adding aqueous 1 M NaOH. For the intestinal phase, 9.8 mL of simulated intestinal fluid (SIF), 100 U/mL of pancreatin, 2.5 mL of bile, 40 µ L of 0.3 M CaCl 2 and, finally, Milli-Q H 2 O was added to achieve a final volume of 40 mL. The resulting mixture was homogenized and inertized with N 2 as previously mentioned. Thus, the intestinal phase was carried out for 2 h, maintaining conditions of temperature and agitation of 37 ◦ C and 150 rpm (MaxQTM 6000 SHKE6000-8CE, Thermo Scientific, Waltham, MA, USA). Then, samples (1 mL) were taken at 30 min intervals and stored at −80 ◦C for their later analysis. Throughout the in vitro digestion process, pH measurements were obtained at intervals of 30 min, adjusting the value to pH 3.0 in the gastric phase and pH 7.0 in the intestinal phase with 1 M aqueous HCl or NaOH when necessary. 2.5. Bioactive Compound Extraction Before phenolic characterization of the digestates by HPLC-MS, the digested samples were defrosted on ice for 2 h for those stored in microcentrifuge tubes or overnight in the refrigerator for those in centrifuge tubes. The samples from all digestive phases were homogenized and centrifuged at 19,500 × g, at 4 ◦ C for 10 min, separating both supernatants (bioaccessible fraction) and pellets (residual fractions). The extraction of phenolic compounds was performed as previously described [ 46 ]. For evaluation of the bioaccessible fraction, 100 µ L of MeOH-EtOH, 50:50 (v/v), was added to 200 µ L of the supernatant, vortexed, and stored at − 20 ◦ C for 30 min for protein precipitation. Then, samples were centrifuged at 19,500 × g, at 4 ◦ C for 10 min and the supernatants evaporated in a vacuum concentrator (Eppendorf Concentrator plus) at ambient temperature for 4–5 h. Dried residues were re-suspended in 100 µ L of MeOH before characterization, homogenized in a refrigerated ultrasound bath, and centrifuged at 19,500 × g, at 4 ◦ C for 10 min, introducing the supernatants in HPLC vials for their analysis. As for the residual fractions, 1 mL of MeOH was added to 100 mg of residue, homogenized, and introduced into a refrigerated ultrasound bath for 15 min. Then, it was agitated at 4 ◦ C and centrifuged at the previous described conditions. The samples were introduced into the vacuum concentrator at ambient temperature for 2–3 h and stored at − 20 ◦ C until analysis. Previous to the analysis, the samples were re-suspended in MeOH to a concentration of 500 µ g/mL. The centrifuged supernatants were also introduced into HPLC vials for their later analysis. 2.6. Bioactive Compound Bioaccessibility The bioaccessibility was calculated as previously described [ 46 ] using Equation (3) [ 47 ], corresponding to the phenolic compound fraction freed from the studied formulation into the gastrointestinal tract at the end of the simulation (240 min of the intestinal phase) and, thus, accessible for intestinal absorption. Additionally, the accumulative presence through- Nutrients 2024,16, 93 6 of 20 out the digestion process of the bioaccessible fraction was calculated using Equation (4), as a percentage of the initial composition of the extract [ 48 ]. The initial phenolic content in the olive leaf extract for the bioaccessibility evaluation was assessed by re-suspending 5 g of extract in the final volume of the intestinal phase (18 mL) prior to being submitted to the extraction procedure described in the previous section. Bioaccesibility(%)=PC content inIP4 (mg) Initial PCcontent (mg)×100% (3) Recovery(%)=PC content inDS (mg) Initial PCcontent (mg)×100% (4) where PC is phenolic compounds; IP4 is the final sample from the intestinal phase (time 240 min); DS is the digested samples for each phase; and initial PC content is the phenolic content present in the olive leaf extract. 2.7. Bioactive Compound Characterization Using HPLC-MS Analyses were performed using an Agilent 1200 liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with a micro vacuum degasser, binary pump, autosampler, thermostatic column compartment, and diode array detector. The HPLC column used for separation was an Agilent Zorbax Eclipse Plus C18 (1.8 µ m, 4.6 ×150 mm ). The mobile phases consisted of water plus 0.1% formic acid (A) and acetonitrile (B). The multi-step linear gradient applied was the following: 0 min, 5% B; 2 min, 30% B; 25 min, 95% B; 30 min, 95% B; 32 min, 5% B and then, the initial conditions were maintained for 3 min. The flow was 0.5 mL/min, the temperature was maintained fixed at 25 ◦C, and the injection volume in the HPLC system was 5 µL. The HPLC system was coupled to a microTOF mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with an ESI interface (Agilent Technologies, Palo Alto, CA, USA) operating in negative-ion mode, in a mass range of 50–1000 m/z. Nitrogen was used as a nebulizing/ionizing and drying gas at conditions of 2 bar and 10 L/min. The drying temperature was set at 190 ◦ C, capillary voltage of +4 kV, and end-plate offset at − 500 V. Other optimum values for the ion-transfer parameters were output voltage, 120 V; skimmer 1, 40 V; hexapole 1, 23 V; hexapole RF, 100 Vpp; skimmer 2, 22.5 V; lens 1 transfer, 50 µ s; and lens 1 pre-pulse storage, 3 µs. In order to recalibrate mass spectra obtained during analysis to achieve a mass precision of 5 ppm, 5 mM sodium formate was use as calibration agent at the beginning of each analysis. 2.8. Data Processing The chemical characterization of the phenolic compounds in the free extract, the microencapsulated formulation, and the digested samples (bioaccessible and residual fractions) was carried out using the software DataAnalysis 4.0 (Bruker Daltoniks, Bremen, Germany). For identification purposes, the mass analyzer data (exact mass and isotopic pattern) were processed for obtaining a molecular formulae list of the analyzed compounds with a 5 ppm tolerance error. The elucidation was achieved by comparing the obtained putative molecular formulae with the previous literature and personal databases of phenolic compounds present in this plant matrix. In addition, for the quantification of the olive leaf extract, microparticles and the different digestate samples, the injection into the HPLC-TOF-MS instrument was carried out in triplicate, and the peak area of each tentative compound was measured in the obtained chromatogram for each replicate. After the selection of an adequate commercial standard, based on structural similarity with the target analyte, the concentrations of the identified phenolic compounds were calculated by the interpolation of the peak area detected in the replicate analysis of each sample in the corresponding surrogate standard Nutrients 2024,16, 93 7 of 20 calibration curve. The phenolic content was expressed as mean concentration ± standard deviation for each sample. 2.9. Statistical Analyses The experiments were performed in triplicate and comparisons were made using the SPSS statistical software (SPSS version 28; SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Tukey’s post hoc tests with α at 0.05 were applied to determine statistical differences among conditions and digestive phases at a 95% confidence level. 3. Results and Discussion 3.1. Characterization of Olive Leaf Extract In the present study, a commercial olive leaf extract was used to assess its stability along the gastrointestinal digestion process and the influence of its co-administration with inulin and microencapsulation. The extract contains, according to the manufacturer’s specifications, oleuropein (OLE) and hydroxytyrosol (HT) in contents around 48% and 1%. This extract was comprehensively characterized using HPLC-MS by using the method previously described and the obtained chromatogram is shown in Figure 1. Major and minor compounds detected were tentatively identified by the interpretation of their MS spectra obtained using a TOF-MS combined with the data provided by databases and the literature. These compounds were also quantified by using surrogate standard approximation, as described in Section 2.8. The composition of the commercial extract is summarized in Table 1. Nutrients 2024, 16, x FOR PEER REVIEW 7 of 21 in triplicate, and the peak area of each tentative compound was measured in the obtained chromatogram for each replicate. After the selection of an adequate commercial standard, based on structural similarity with the target analyte, the concentrations of the identified phenolic compounds were calculated by the interpolation of the peak area detected in the replicate analysis of each sample in the corresponding surrogate standard calibration curve. The phenolic content was expressed as mean concentration ± standard deviation for each sample. 2.9. Statistical Analyses The experiments were performed in triplicate and comparisons were made using the SPSS statistical software (SPSS version 28; SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Tukey’s post hoc tests with α at 0.05 were applied to determine statistical differences among conditions and digestive phases at a 95% confidence level. 3. Results and Discussion 3.1. Characterization of Olive Leaf Extract In the present study, a commercial olive leaf extract was used to assess its stability along the gastrointestinal digestion process and the influence of its co-administration with inulin and microencapsulation. The extract contains, according to the manufacturer’s specifications, oleuropein (OLE) and hydroxytyrosol (HT) in contents around 48% and 1%. This extract was comprehensively characterized using HPLC-MS by using the method previously described and the obtained chromatogram is shown in Figure 1. Major and minor compounds detected were tentatively identified by the interpretation of their MS spectra obtained using a TOF-MS combined with the data provided by databases and the literature. These compounds were also quantified by using surrogate standard approximation, as described in Section 2.8. The composition of the commercial extract is summarized in Table 1. Figure 1. Base peak chromatogram of olive leaf extract at a concentration of 500 mg/mL, where the peaks have been numbered according to their elution order. Table 1. Composition of the commercial olive leaf extract. Peak RT (min) Proposed Compound Molecular Formula m/z Concentration (%) 1 9.08 Hydroxytyrosol glucoside C14H20O8 315.1108 0.107 ± 0.008 2 10.01 Hydroxytyrosol C8H10O3 153.059 1.0 ± 0.1 3 11.61 Oleoside/Secologanoside isomer 1 C16H22O11 389.1135 0.26 ± 0.03 4 15.51 Oleoside/Secologanoside isomer 2 C16H22O11 389.1135 0.10 ± 0.02 Figure 1. Base peak chromatogram of olive leaf extract at a concentration of 500 mg/mL, where the peaks have been numbered according to their elution order. Table 1. Composition of the commercial olive leaf extract. Peak RT (min) Proposed Compound Molecular Formula m/zConcentration (%) 1 9.08 Hydroxytyrosol glucoside C14H20O8315.1108 0.107 ±0.008 2 10.01 Hydroxytyrosol C8H10O3153.059 1.0 ±0.1 3 11.61 Oleoside/Secologanoside isomer 1 C16H22O11 389.1135 0.26 ±0.03 4 15.51 Oleoside/Secologanoside isomer 2 C16H22O11 389.1135 0.10 ±0.02 5 18.00 Verbascoside C29H36O15 623.199 0.67 ±0.06 6 19.37 Luteolin-7-O-glucoside C21H20O11 447.095 1.5 ±0.1 7 19.81 Oleuropein diglucoside isomer 1 C31H42O18 701.231 0.38 ±0.04 8 21.68 Oleuropein diglucoside isomer 2 C31H42O18 701.231 0.78 ±0.01 9 23.19 Luteolin glucoside isomer C21H20O11 447.095 0.28 ±0.03 10 25.72 Oleuropein C25H32O13 539.181 48 ±1 11 27.24 Oleuropein isomer C25H32O13 539.181 4.9 ±0.2 12 29.57 Ligstroside C25H32O12 523.182 0.89 ±0.04 13 31.75 Oleuropein aglycone C19H22O8377.1223 0.24 ±0.02 Nutrients 2024,16, 93 8 of 20 3.2. Microencapsulation of Olive Leaf Extract by Spray-Drying With the purpose of achieving an optimization of the encapsulation process for HT and OLE contained in the commercial olive leaf extract, several statistical analyses were conducted. For all evaluated responses, model adequacy, lack-of-fit, R 2 , and ANOVA were performed to determine the fitting of the proposed experimental model. In this sense, model adequacy was used to indicate the best-choice mathematical model; the lack-of-fit test revealed the fitting quality of the model applied; R 2 revealed the ability to predict the behavior of the response variables and, finally, ANOVA indicated the statistically significant effect of temperature (X 1 ) and the extract:encapsulating agent (E:EA) ratio (X 2 ) on the response variables [45]. The experimental conditions of the different runs of the design performed to optimize the encapsulation together with the results of the encapsulation degree of HT and OLE are shown in Table 2. The analysis of variance (ANOVA) of the proposed experimental model for each response variable (%EE of HT and OLE) is summarized in Table 3. Table 2. Experimental design conditions with experimental and fitted results for the response variables (encapsulation efficiency of main compounds). Run T (◦C) E:EA %EE HT %EE OLE Exp. Pred. Exp. Pred. 1 195.25 2.50 66.68 62.17 64.96 64.33 2 134.75 2.50 69.82 65.77 67.62 61.99 3 165.00 2.50 60.73 65.49 72.27 60.49 4 165.00 0.69 8.15 5.28 13.73 9.31 5 190.00 4.00 80.35 81.11 70.23 70.22 6 140.00 1.00 10.77 16.27 13.72 18.31 7 165.00 2.50 67.12 65.49 49.10 60.49 8 165.00 2.50 65.21 65.49 53.08 60.49 9 190.00 1.00 5.24 9.02 26.18 27.74 10 165.00 4.32 81.89 81.77 71.62 69.78 11 140.00 4.00 81.33 81.81 72.75 75.77 12 165.00 2.50 64.32 65.49 64.14 60.49 T: inlet air temperature; E:EA: extract:inulin ratio; %EE: encapsulation efficiency; results are expressed as %; HT: hydroxytyrosol; OLE: oleuropein; Exp.: experimental; Pred.: predicted. Table 3. Analysis of variance (ANOVA) of the proposed experimental model for each response variable. Source Encapsulation Efficiency HT OLE p-Value p-Value Model 0.000 a0.002 a X1: Temperature 0.2398 0.824 X2: E:EA ratio 0.000 a0.008 a X1X20.482 0.744 X2 10.458 0.529 X2 20.00 a0.068 a Lack-of-fit 0.058 0.844 R20.98 0.93 E:EA: extract:encapsulating agent; R 2 : quadratic correlation coefficient; HT: hydroxytyrosol; OLE: oleuropein; asignificant (p< 0.050). As can be observed in Table 2, the encapsulation rate of HT ranged from 5.24% (run 9) to 82% (run 10). Concerning the fitting parameters (Table 3), the model adequacy was revealed to be satisfactory (p ≤ 0.05), lack-of-fit was p> 0.05, and R 2 was 0.98, indicating the good fitting and predictive capabilities of the proposed model. Moreover, the ANOVA results were used to discern the effect of each factor on the encapsulation of HT. In this Nutrients 2024,16, 93 9 of 20 sense, the linear and quadratic effects of the E:EA ratio ( X2 ) were the most influential factors on the encapsulation degree of HT. Thus, a simplified equation that explains the behavior of this response is displayed in Equation (5): %EE HT =−38.57 +60.16X2−7.51X2 2(5) A graphical explanation of the proposed equation can be observed in Figure 2A, which helps to explain the results obtained for HT after performing the experimental design. It is possible to observe the relevant positive effect of the E:EA ratio, reaching a higher degree of encapsulation with higher inulin concentration independently of the effect of temperature, since it did not significantly influence this response. The higher availability of the encapsulating agent when a higher inulin concentration was applied, facilitated the entrapment of HT, increasing the encapsulation efficiency of this compound [ 49 ]. Therefore, the optimum conditions provided by the model for maximum HT encapsulation were 160 ◦ C and an E:EA ratio of 4.00. When these conditions were applied, the predicted theoretical value for the response variable and the experimental result were quite similar (82.50% and 84.50%, respectively). Nutrients 2024, 16, x FOR PEER REVIEW 10 of 21 higher total OLE content (111 mg OLE/g of microparticles vs. 24.5 mg OLE/g of microparticles) [37]. At this point, it was decided to simultaneously optimize both response variables. For this purpose, the desirability function was used to find those conditions that provided predictable and reliable information. Therefore, the variable responses %EE of HT and OLE were maximized simultaneously and are plotted in Figure 2C considering their performance as a function of the two factors assessed. Figure 2. Response surface plots of (A) %EE of HT, (B) %EE of OLE, and (C) %EE of HT and OLE maximized simultaneously. The highest desirability index (value DI = 1) was found at 145 °C and an E:EA ratio of 3.87, obtaining theoretical values of 82.04% and 74.56% for %EE HT and %EE OLE, respectively. After performing experiments with the proposed optimum conditions identified through the desirability function, the experimental results (%EE HT 80.44% and %EE OLE 79.45%) were similar to those predicted (Table 4). Moreover, the total amounts of HT (2.3 mg/g of microparticles) and OLE (93 mg/g of microparticles) were obtained using HPLC-MS in order to evaluate the final concentration after the spray-drying process. Finally, in order to obtain complete information on the degree of encapsulation of minor compounds contained in the optimized microencapsulated powder, an exhaustive study of the degree of encapsulation of the remaining compounds was carried out. In addition, these results will help to understand in more detail the protection and controlled release of other olive bioactive compounds after in vitro digestive processes carried out in the subsequent trials. Overall, the results displayed in Table 4 reveal a high encapsulation degree of phenolic alcohols, secoiridoids, and flavonoids, with hydroxytyrosol and its glycoside being the most encapsulated compounds (80.44% and 79.91%, respectively). On the other hand, secoiridoids presented encapsulation degrees from 58.70% to 79.45%, with oleuropein being the most encapsulated in this group, followed by oleoside/secologanoside isomer 1 (76.96%). It should be noted that the differences in the degree of encapsulation of the different isomers may be due to conformational differences of the compounds, causing steric hindrance due to the prevented exposure of the functional groups that interact with the encapsulating agent, and consequently, reducing the interaction capacity [51,52]. Flavonoids also presented a great encapsulation degree (above 70%). This results may be associated with the interaction between the hydroxyl groups of the flavoFigure 2. Response surface plots of ( A ) %EE of HT, ( B ) %EE of OLE, and ( C ) %EE of HT and OLE maximized simultaneously. On the other hand, after performing the proposed experimental design, the encapsulation efficiency reached for OLE ranged from 13.72% (run 6) to 72.75% (run 11) (Table 2). Regarding the statistical analysis (Table 3), the model adequacy was p ≤ 0.05, the lack-of fit test was p> 0.05, and R 2 was 0.93, revealing a good adjustment of the model as well as a good predictive capacity. Similar to the results found for HT, the linear and quadratic effects of the E:EA ratio ( X2 ) were shown to be also determinant in the OLE encapsulation efficiency, having positive effects on the encapsulation degree (Equation (6)). %EE OLE =−19.84 +48.44X2−6.36X2 2(6) The behavior of this response throughout the experimental design is shown in Figure 2B . The response surface plot for OLE encapsulation displays the positive effect of the concentration of inulin in the mixture introduced in the spray-drying to achieve a higher encapsulation of this compound. In spite of not having statistically significant effects, the temperature seemed to cause a slight decrease in the encapsulation efficiency of OLE. This result can be associated with the degradation of OLE at higher temperatures and, also, with the glass transition temperature of inulin. Thus, when the temperature is below this point the physical properties of the polymer change to those characteristic of the glassy or crystalline state. Hence, temperatures above the glass transition temperature cause a sticky behavior of the Nutrients 2024,16, 93 16 of 20 healthy volunteers was increased when compared to the control group [ 79 ]. Additionally, the presence of pectin has been reported to increase the bioavailability of quercetin and isorhamnetin in mice compared to other formulations [80]. The presented in vitro results are in line with this preliminary in vivo evidence, and will further improve the development and evaluation of innovative approaches of the administration of phenolic compounds in protective formulations, enhancing their implementation into functional foods and nutraceutical formulations. This will provide the foundation for the future in vivo evaluation of promising formulations and new products for improving and maximizing their health benefits. 4. Conclusions The presence of inulin and the microencapsulation of the extract during in vitro gastrointestinal digestion of the different formulations showed an alteration in the phenolic degradation profile. Indeed, the presence of inulin proved to have a beneficial effect on the phenolic stability, as interactions between inulin and the present phenolic compounds improved their protection under digestive conditions. Thus, the presence of inulin improves the gastric and intestinal stability of OLE and modulates the degradation of HT precursors, favoring the stability of its glucoside form. However, the combined effect with the encapsulation process could be a more attractive alternative as it would allow for an increased protection of the encapsulated compounds, although a slight degradation could be observed under gastric conditions, while maintaining the stability of the superficial (non-encapsulated) or liberated content. These presented results could contribute to the development of innovative formulations for obtaining functional foods and neutraceutical products with improved stability compared to the free extracts, thus maximizing the potential health benefits observed in their administration. Nevertheless, further research into inulin–phenol interactions during digestion is needed in order to fully elucidate its digestion dynamic. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu16010093/s1, Figure S1: Evolution of the residual fraction of oleuropein diglucoside isomers, oleuropein, oleuropein aglycone, hydroxytyrosol and its oxidized and glucoside forms under gastric (120 min) and intestinal (150, 180, 210 and 240 min) stages of in vitro gastrointestinal digestion; Figure S2: Evolution of the residual fraction of verbascoside, oleoside/secologanoside, ligstroside and luteolin-7-glucoside and its isomer under gastric (120 min) and intestinal (150, 180, 210 and 240 min) stages of in vitro gastrointestinal digestion. Author Contributions: Conceptualization, R.Q.-P., J.L.-S. and I.B.-L.; methodology, R.Q.-P., F.J.L.-J. and J.L.-S.; software, C.D.-S., F.J.L.-J. and M.A.L.-B.; validation, I.B.-L., J.L.-S. and R.Q.-P.; formal analysis, I.B.-L., J.L.-S. and R.Q.-P.; investigation, C.D.-S., F.J.L.-J. and R.Q.-P.; resources, R.Q.-P. and I.B.-L.; data curation, C.D.-S., F.J.L.-J. and M.A.L.-B.; writing—original draft preparation, C.D.-S. and F.J.L.-J.; writing—review and editing, R.Q.-P., J.L.-S. and I.B.-L.; visualization, J.L.-S.; supervision, I.B.-L.; project administration, R.Q.-P. and I.B.-L.; funding acquisition, R.Q.-P., J.L.-S. and I.B.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by “Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía” (INTESOLIVE project, PY18-RE-0033) and Grant TED2021-132489A-I00 funded by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR. C. Duque-Soto is grateful to the Regional Government of Andalucía for a predoctoral contract (PREDOC_00110) and to the Doctoral Program in Nutrition and Food Sciences, University of Granada. M.A. López-Bascón is grateful to “Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía” for a postdoctoral researcher contract (POSTDOC_21_00031). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All the data generated by this research have been included in the article. For any assistance, it is possible to contact with the corresponding authors. 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