In Vitro Digestion of Microcapsule Carriers for Oral Delivery of Bioactive Compounds for Diabetes Treatment and Their Inhibitory Effect on the DPP-4 Enzyme

Abstract

Empty microcapsules, originally designed as carriers of bioactive peptides, were prepared by the combined method of a double-emulsion complex with coacervation spray drying and were subjected to an in-vitro digestion process, producing peptides from the whey protein contained in the microcapsule walls. The inhibitory effect of the enzyme dipeptidyl peptidase-4 (DPP-4) and modulation of the insulin receptor of hydrolyzed microcapsules were evaluated. The hydrolysate of the microcapsules was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, showing the presence of low-molecular-weight peptidic compounds, which apparently were responsible for the DPP-4 inhibitory effect. Fluorescence analysis showed that the effect of the hydrolyzed microcapsules on the insulin receptor was 40% that of insulin. The inhibition of DPP-4 was 54.7%. This work demonstrated that empty microcapsules initially designed as carriers of functional peptides also have the capability to inhibit DPP-4 and modulate insulin receptors by themselves.

1. Introduction

Diabetes is a syndrome that is mainly characterized by an alteration of carbohydrate metabolism caused by either the pancreas not producing enough insulin or organisms not utilizing the insulin efficiently. This produces hyperglycemia, which in turn can result in kidney failure, cardiovascular disease, eye damage, and may even lead to the need for amputation [1]. Patients with diabetes should maintain glucose concentrations within normal limits to prevent complications; thus, a healthy diet and regular physical exercise are initially recommended. However, maintaining a healthy lifestyle is usually extremely difficult [2]. Therefore, this strategy has to be complemented with therapeutic agents that help patients reach the suggested glycemic goals.

To tackle this problem, researchers have been looking into diabetes treatment alternatives that provide adequate glycemic control without the risk of hypoglycemia and weight gain, while reducing the risk of cardiovascular disease [3,4]. Currently, a combination of treatments has been proposed, in which drugs or hypoglycemic components have complementary actions and may facilitate better glycemic control [5].

Similarly, new treatments have been introduced that are based on the incretin effect, which is primarily induced by glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which are released by the intestine cells as a response to food intake. However, the effects of these hormones are very brief, because they are inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4) [6,7,8]. The roles of these drugs increase and extend the effects of incretins by one of two possible mechanisms: producing a GLP1, which is not inactivated by DPP-4 (GLP1 agonist); or by inhibiting the DPP-4 enzyme. Incretins are reported to improve insulin secretion, promote satiety, inhibit glucagon secretion, and delay gastric emptying; moreover, they have been proved to possess cardio- and neuroprotective effects [7].

Multiple efforts, focused mainly on the use of insulin, have been undertaken to develop a strategy for the oral administration of therapeutic proteins. Of the tested vehicles that have been proven the most promising, the one that stands out is the use of nanometric structures that facilitate epithelial penetration, utilizing multi-layered strategies based on mucoadhesive polymers that provide protection from gastric acid. One of the most researched alternatives is the oral administration of peptide drugs, mainly insulin. In this sense, a great variety of vehicles have been designed, which have provided promising results [9]. However, to date there have been no reports that have looked into the system itself, which without the inclusion of any antidiabetic compound can promotes the reduction of glucose in the blood by some unknown mechanism. Thus, the vehicle could, in theory, have synergistic effects with the encapsulated antidiabetic treatment.

Recently, within our research group, the method of microencapsulation of insulin was developed through the combined technique of double emulsion (water in oil in water, W/O/W) with complex coacervation between whey protein isolate (WPI) and sodium carboxymethylcellulose (CMC), or sodium alginate (SA) combined with spray drying. This proved to be a good insulin protector from gastrointestinal pH, maintaining a high biological residual activity; therefore, it appeared to be a good vehicle for therapeutic peptides and proteins [10].

The objective of this work was to monitor the structure of the capsule during the different phases of in vitro digestion to verify that its behavior was in accordance with its design, and to determine its potential hypoglycemic effect, per se, due to the whey protein contained in the capsule. It has been reported that whey protein hydrolysates obtained by digestive enzymes inhibit the in vitro activity of DPP-4, presenting a favorable effect on glycemic control [11,12,13,14,15]. Because the capsule was previously elaborated as a vehicle for insulin and therapeutic proteins, it represents an antidiabetic administration system with two functions: a possible vehicle for a peptide drug; and as a complex with hypoglycemic effects by itself, thus potentiating its effect.

2. Materials and Methods

2.1. Materials

The +90% WPI was purchased from Hilmar Ingredients (Hilmar, CA, USA). Pharmaceutical-grade GELYCEL F1 2000 CMC was obtained from Quimica Amtex S.A. de C.V. (Mexico City, Mexico). Pharmaceutical-grade sodium alginate USP (SA) was purchased from Alquimia Mexicana S. de R. L. (Mexico City, Mexico). GRINDSTED® PGPR 90 polyglycerol polyricinoleate (PGPR) was acquired from Danisco Foods Ingredients. (Grinsted, Denmark). Commercial-grade soybean oil (Nutrioli, Monterrey NL, Mexico), dipeptidyl peptidase-4 (DPP-4) enzyme inhibitory screening kit, pepsin, pancreatin, and the insulin receptor (1011-end) were purchased from Sigma Aldrich (St. Louis, MO, USA). All other reagents and chemicals used were analytical grade.

2.2. Double Emulsion W/O/W Preparation

Double emulsion W/O/W was prepared following the method reported by Cardenas-Bailón et al. [10]. To obtain the empty capsules (capsules with no encapsulated peptide), the internal aqueous phase (IP) consisted of a 5% w/v NaCl solution, which was adjusted to 15% w/v with glycerol because it has been reported that the addition of glycerol to the IP reduces the globule size and increases the stability of water globules in oil emulsions [10,16]. The double emulsion (W/O/W) was prepared using a two-step procedure.

During the first step (Figure 1), a water in oil (W/O) emulsion was prepared by dropping the IP into the oily phase (OP), which was formed by soybean oil and PGPR 8% w/w, and was then homogenized at 10,000 rpm for 5 min using a digital Ultraturrax® T25 (Werke, Unsingen, Germany) at room temperature (25 °C). The W/O emulsion was prepared with 30% IP. The W/O emulsion was emulsified once again (Figure 1) in a 10% w/v WPI solution at 10,000 rpm for 1 min using a digital Ultraturrax® T25 (Werke, Unsingen, Germany) at room temperature (25 °C). The W/O emulsion to WPI dry solid ratio was 1:1.333 (1 g of W/O emulsion emulsified in 13.33 g of a 10% w/v WPI solution).

2.3. Particle Size and Distribution of the Emulsion and Spray-Dried Microcapsules

A 2600 series Malvern IM 026 particle size analyzer (Worcestershire, United Kingdom) was used to measure the particle size and particle size distributions in the double emulsion (W/O/W) and the dried microcapsules. A Zeta Brookhaven Instruments Zeta Plus DR 525 was used for the particle sizing in the first emulsion (W/O). The particle size determination was based on the particle laser diffraction, which was measured by the equipment and translated into a graph of sizes and distribution. Oil was used as a dispersant for the first emulsion (W/O), water for the double emulsion (W/O/W), and hexane for the microcapsules in powder (spray-dried).

2.4. Encapsulation Efficiency

Encapsulation efficiency was determined using a 5% w/v NaCl solution as an indicator, adjusted with glycerol at 15% w/v in the IP. The amount of NaCl in the aqueous external phase released by the double W/O/W emulsion during its preparation was calculated by determining the change in conductivity [10,17]. An Oakton PC700 (Vernon Hills, IL) conductivity meter was used to determine conductivity at room temperature (25 °C). To estimate the amount of NaCl released, a calibration curve was constructed in the following manner: a simple O/W emulsion was prepared (soybean oil in WPI 10% w/v) and the NaCl solution was added in different ratios to simulate the release of NaCl. Then, a conductivity versus encapsulation efficiency curve was plotted, and finally, the emulsion conductivity of the double emulsion W/O/W was measured in triplicate and the encapsulation efficiency was calculated based on the calibration curve.

2.5. Coacervation of W/O/W Double Emulsion

Coacervation of the W/O/W double emulsion was conducted based on the report of Cardenas-Bailón et al. [10]. CMC or SA were selected as polysaccharides for coacervation of the W/O/W double emulsions, since they form a stable coacervate with whey protein at pH 2.5, which is the gastric pH. Even though both polysaccharides are mucoadhesive, the complexes with SA are more viscous, making the coacervation process slower.

The W/O/W double emulsion was mixed with a CMC or a SA solution at 1% w/v with a 1:2 ratio of W/O/wall material (WPI + CMC or WPI + SA). The pH of the mixture was gradually adjusted with HCl 0.1 N to approximately 3.5 and then with HCl 1 N until the coacervation pH (pH = 2.5) was reached under constant stirring (Figure 1).

2.6. Spray Drying of Coacervated Microcapsules

The coacervated microcapsules were spray-dried using a Mobile Minor Niro Atomizer spray dryer (Niro, Soeborg, Denmark) equipped with a spinning wheel spraying system. The inlet temperature used was 180 °C and the outlet temperature used was 70 °C [10]. The coacervated microcapsules were maintained under a constant slow stirring during feeding into the dryer to avoid the conglomeration of the coacervate. The powder obtained was maintained at 2–5 °C until further analysis.

2.7. In Vitro Digestion Design of “Empty” Spray-Dried Capsules

Several methods of digestion have been proposed in the literature, however a consensus has not been reached for a standardized model. In this work, the model of in vitro digestion reported by Minekus et al. [18] was used. A 0.5 g sample of powder was weighed in a Falcon tube and suspended in 40 mL of simulated gastric fluid (SGF) at 2.5 pH. It was incubated for 2.0 h at 37 °C with gentle stirring in a dual-action shaker (Lab-line 3508, USA) set at position 2, in the presence of pepsin (2000 units/mL in HCl 0.1 M).

At the end of the gastric digestion, the sample was centrifuged (5 min, 1000 g) and transferred into a solution of simulated intestinal fluid (SIF) at pH 7. It was then incubated under gentle stirring (position 2) in a dual-action shaker for 2 hours at 37 °C with pancreatin added, which was based on the specific trypsin enzymatic activity of 100 units/ml. At the end of the intestinal digestion, the hydrolysate solution was heated in boiling water for 10 min to inactivate the enzymes. Hydrolysate samples were taken at 0 min (beginning) and 120 min (end) of the gastric phase, and for the intestinal phase samples were taken at 0, 60, and 120 min, in order to quantify total protein and determine the degree of hydrolysis at different digestion times.

2.8. Determination of Protein Concentration

Protein concentration was determined using the Bradford reagent (Sigma Aldrich B6916). The assay consisted of mixing 1 part of the hydrolyzed sample from each step in the digestion process (previously centrifuged at 5000 g for 20 min) with 30 parts of Bradford reagent. The absorbance was registered at 595 nm and the protein concentration was determined by comparison with a standard curve using bovine serum albumin (BSA) as the standard protein.

2.9. Determination of Degree of Hydrolysis during Simulated Digestion

The degree of hydrolysis of the protein was determined using the o-phtaldialdehyde (OPA) method described by Nielsen et al. [19] to estimate the percentage of cleaved peptide bonds with regards to the original protein. A serine standard was prepared as follows: 5 mg serine was diluted in 50 mL deionized water (0.9516 meq/ml), which was placed in contact with the OPA reagent, and the absorbance at 340 nm was determined using a UV-visible spectrophotometer (VICTOR Nivo, Perkin Elmer, USA). A 1:100 dilution of hydrolysates in distilled water was prepared. The assays were conducted in microplates; 20 µL of the sample and 150 µL OPA reagent were placed in each well. The OPA reagent was prepared according to the methods of Nielsen et al. [19]. The plate was incubated for 2 min at 37 °C and the absorbance was determined.

2.10. Laser Scanning Confocal Microscopy

The capsule microstructure at the end of the gastric and intestinal phases was observed using a Zeiss LSM 710 NLO Multiphoton Confocal Microscope (Carl Zeiss, Germany). Fluorescein isothiocyanate (FITC) was used to stain the protein, Oil Red O dye was used to stain lipids, and calcofluor white dye was used to stain the polysaccharide polymers. A 50 μL sample was transferred and 5 μL of each dye at 1% (w/v) was added, and they were placed on a concave confocal microscope slide. Finally, the images were examined and registered.

2.11. DPP-4 Inhibition Test

A DPP-4 Inhibitor Screening Kit (Sigma Aldrich, St. Louis, MO, USA) was used to validate the inhibiting effect of the “empty” microcapsules after digestion using a Flex Station 3 multi-mode microplate reader (Molecular Devices, LLC, San José, CA, USA). Fluorescence emissions were collected in the kinetic mode in 96-well clear plates for 30 min. The relative percent inhibition was calculated using Equation (1), where ΔF/ΔT indicated the change in fluorescence during the chosen time interval.

$$\%\mathrm{Inhibition}=\frac{\left(\frac{\Delta F}{\Delta T}\right)enzyme - \left(\frac{\Delta F}{\Delta T}\right)Enzyme inhibited substrate }{ \left(\frac{\Delta F}{\Delta T}\right)\mathit{e}\mathit{n}\mathit{z}\mathit{y}\mathit{m}\mathit{e}}$$

(1)

2.12. Qualitative Analysis of the Peptides Formed by the In Vitro Digestion Process

The dried droplet method was used to prepare matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) samples. A 1 μL sample was mixed with 1 μL of the matrix (7:3 v/v α-ciane-4-hydroxycinnamic acid (α-CHCA) and formic acid) and the mixture was placed on MALDI plates and subsequently air dried. The mass spectra were acquired in the positive mode using a MALDI-TOF autoflex spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) and were processed using the Flex Analysis (Bruker Daltonics Inc., Billerica, MA, USA) software.

2.13. Interaction of the Microcapsule Hydrolysates on the Insulin Receptor

The insulin receptor fraction InsR 1011-end (Sigma Aldrich, St. Louis, MO, USA) was used for the determination. Measurements were conducted at room temperature (25 °C) using a spectrofluorometer (Thermo Scientific Varioskan LUX, Multi-mode Microplate Reader) to reveal the folding or the structural loss of the insulin receptor. To do this, scanning from 250 to 410 nm was conducted using a UV-visible fluorescence spectrophotometer (FlexStation 3 multi-mode microplate reader, Molecular Devices, LLC, San José, CA, USA). The obtained absorption spectra included the insulin receptor, the combination of the insulin receptor with insulin, and the insulin receptor combined with the supernatant obtained from in vitro digestion. The resulting spectra were compared as a function of their fluorescent intensity and the fluorescence change was calculated at the maximum emission (300 nm).

2.14. Statistical Analysis

Analysis of variance (ANOVA, p < 0.05), Tukey´s comparison test (p < 0.05), and Dunnett´s method were carried out using the statistical software Minitab® 17.1.0 to assess differences between the mean of each factor level and the mean of a control group.

3. Results and Discussion

3.1. Particle Size Distribution in the W/O and W/O/W Emulsions

The particle size of the first emulsion (W/O) was determined to verify its stability. As shown in Figure 2, the particle size obtained had a mean diameter of 720 ± 32 nm, which was adequate because it has been reported that particle sizes lower than 1 µm result in better stability, preventing coalescence and avoiding phase separation [17,20].

With regards to the second emulsion (W/O/W), Figure 3 shows a bimodal distribution with two peaks located between 32–37 µm and 66.8–77.5 µm; the particle mean diameter was 35.52 µm for the double emulsion.

With respect to the encapsulation efficiency, as mentioned in Section 2.4, a calibration curve was constructed with a NaCl solution, and a linear relationship between the encapsulation efficiency and the system conductivity was observed. The encapsulation efficiency for the double emulsion (W/O/W) was 89.5 ± 1.10%. The particle size bimodal distribution and 89.5 ± 1.10% encapsulation efficiency obtained were very similar to those previously reported [10].

3.2. Particle Size after Spray Drying and during In Vitro Digestion

The particle sizes of the coacervated microcapsules after spray drying for WPI–SA and WPI–CMC systems resuspended in 2.5 pH buffer were 21.67 ± 0.18 µm and 23.20 ± 0.66 µm for equivalent sphere diameters (D4,3), respectively. On the other hand, the Sauter mean diameter (D3,2) for the same microcapsules prepared with the WPI–SA system was 14.08 ± 0.13 µm and for the WPI–CMC system was 15.28 ± 0.12 µm.

Particle size and particle size distribution were determined at the beginning and end of the digestion process both for the gastric and intestinal phase. As shown in

Table 1. Particle size for whey protein isolate with sodium alginate (WPI–SA) and whey protein isolate with sodium carboxymethylcellulose (WPI–CMC) systems at the beginning (0 min) and end (120 min) of gastric and intestinal digestion (pH 2.5).

Gastric phase	Time	WPI–SA	WPI–CMC
	D3,2 (0 min)	15.21 µm ± 0.554D	18.74 µm ± 0.436C
	D3,2 (120 min)	20.75 µm ± 0.566B	22.47 µm ± 0.535A
Intestinal phase	Time	WPI–SA	WPI–CMC
	D3,2 (0 min)	12.85 µm ± 0.281B	13.35 µm ± 0.125A
	D3,2 (120 min)	2.82 µm ± 0.107D	4.24 µm ± 0.325C

Means with different letters differ significantly (p < 0.05).

, a small increase in the Sauter mean diameter (D3,2) was observed for both coacervation systems at the end of the gastric phase; nevertheless, this increase was significantly different (p < 0.05). Because the coacervate is stable at gastric pH, the small increase in particle size can be attributed to the particle expansion because of the polysaccharide hydration in contact with the gastric digestion solution [21,22,23].

Figure 4 shows the particle size distribution for both coacervation systems at gastric pH (2.5 pH). It can be seen that in both cases, after 120 min in the gastric phase there was a change in the distribution towards particles of greater size and an increase in their frequency. This was possibly caused by the fact that coacervates tend to group together because of the absence of repulsion charges on the surface [24], in addition to the aforementioned polysaccharide hydration.

With regards to the action of pepsin present on the capsules in the gastric digestion solution, results suggest no action of the enzyme in the gastric phase. The coacervation pH was 2.5; therefore, the particle was stable at the gastric pH, and whey protein remained protected by the polysaccharide.

Based on the capsule design, the microcapsules were expected to remain stable in the acidic conditions of the stomach and to disintegrate at the intestinal pH (7.0 pH). As is shown in Table 1, the particle size during intestinal digestion showed a considerable reduction in terms of Sauter mean diameter (D3,2). This was because complex coacervation is a reversible process, dissociating the complex coacervate when conditions (especially pH and temperature) are unfavorable [10,25]. At the same time, as the coacervate disintegrates, the protein layer was exposed to hydrolysis by trypsin and α-chymotrypsin present in pancreatin of the SIF. When this protein layer was hydrolyzed, the particle size was reduced to the point of releasing the W/O first emulsion [23].

Considering the particle size and distribution of the microcapsules during the intestinal digestion, Figure 5a,b show a significant reduction in the particle size distribution. This confirms that the coacervate disintegrates at intestinal pH and the whey protein is attacked by the proteases present in pancreatin of the SIF, thus reducing the size of the particle. This confirms the correct behavior of the microcapsule as a vehicle capable of withstanding gastric digestion and disintegrating at intestinal digestion pH to deliver peptides within the first emulsion in the intestine.

3.3. Protein Concentration and Degree of Hydrolysis of Microcapsules during In Vitro Digestion

Figure 6 shows the results for protein concentration and degree of hydrolysis for the gastric and intestinal digestion phases for the WPI–CMC and WPI–SA systems.

Regarding the results for protein concentration shown in Figure 6, a decrease was observed over hydrolysis time for both the gastric and intestinal phases. The protein concentration found at the beginning of the gastric phase could be caused by the presence of free non-coacervated protein, because the coacervation efficiency for both systems was approximately 90% and 78% for WPI–CMC and WPI–SA, respectively [10]. This indicates that a small amount or protein and polysaccharide did not interact during coacervation, leaving some protein in the solution, which during the drying process was deposited on the surface of the coacervated dry capsules and was then suspended into the SGF during digestion. Figure 6a shows that the concentration of protein at the beginning of the gastric phase is significantly higher for the WPI–SA system, which is consistent with the lower coacervation efficiency for this system (higher amount of protein that did not coacervate with SA).

The decrease in protein concentration during in vitro digestion has also been reported in a soy milk bioaccessibility study and was attributed to the enzymatic hydrolysis of the protein-structure-forming peptides [26], which could not be detected by the Bradford protein assay. This is because the blue dye Coomassie Brilliant Blue G-250 (CBBG) has a low affinity for low-molecular-weight peptides (<3 kDa) [27,28,29,30].

With respect to the degree of hydrolysis of the protein during the gastric phase, at the initial time (0 min), there were no detectable values for hydrolysis in either system of coacervation. At the end of this phase (120 min), a slight increase in the degree of hydrolysis was observed—up to 1.85% for the capsules prepared with WPI–CMC and 4.83% for the WPI–SA system, which indicated a limited action of pepsin. This slight increase may be explained by the hydrolysis of the free protein that remained on the surface of the capsule after coacervation, as was previously mentioned. Statistical analysis show that the degree of hydrolysis is significantly higher for the WPI–SA system, which is consistent with the higher protein concentration of the beginning of the gastric phase.

For the intestinal digestion phase, the protein concentration and the hydrolysis percentage were determined at 0, 90, and 120 min. As shown in Figure 6b, for both systems of coacervation the protein concentration decreased with the digestion time, and at the same time there was an increase in the degree of hydrolysis, which was 1.85%, 16.69%, and 23.87% for the WPI–CMC system, and 4.83%, 16.91%, and 21.79% for the WPI–SA system, respectively. This indicated an effective action of the digestive enzymes on the exposed protein of the microcapsules. These results are in accordance with the reports of Spellman et al. [31] and Mulcahy et al. [32], who obtained a degree of hydrolysis from 5.5% to 24% for whey protein with different enzymes and hydrolysis conditions [31,32,33,34]. Based on the degree of hydrolysis obtained in this work, these hydrolysates could be classified as extensive hydrolysates because they present a degree of hydrolysis higher than 10% [35].

From the above results, it can be concluded that the “empty” microcapsules, coacervated with both polysaccharides (CMC and SA), remained stable at 2.5 pH, because they maintained a low free protein concentration, very low degree of hydrolysis, and a particle size that was very similar to the coacervated powder from the beginning to the end in the gastric digestion. However, at pH 7 the microcapsules suffered a decrease in the concentration of free protein, a considerable increase in the degree of hydrolysis, and a reduction in particle size during the intestinal digestion phase. Therefore, the developed microcapsules are capable of protecting any peptide compound or any compound sensitive to gastric digestion, allowing them to reach the intestine in a biologically active form inside the internal W/O emulsion, which is released during intestinal digestion.

3.4. Confocal Laser Scanning Microscopy

The images obtained by confocal microscopy (Figure 7) show the behavior of the microcapsule in different phases of in vitro digestion. At the end of the gastric phase (Figure 7a,b), the polysaccharide (blue color) is shown surrounding the surface of the particle, which is consistent with the coacervation process performed at 2.5 pH and remains stable during gastric digestion. The stable coacervate forms insoluble complexes that block some of the places where the enzyme would perform proteolysis [36].

At the beginning of the intestinal phase (Figure 7c,d), changes in the structure of the microcapsule are observed—there is a lower amount of polysaccharides and the stained protein (green color) with FITC is exposed. This change can be explained by the change in pH in the intestinal phase, during which the coacervate dissociates, exposing the whey protein layer of the double emulsion, making it available for hydrolysis by the proteases present in the SIF. As the protein layer is hydrolyzed, the particle size is reduced to the point of releasing the W/O first emulsion (Figure 7e,f), which results in the coalescence of the oil droplets (red colors), as can be seen in the figure [23].

3.5. DPP-4 Inhibitory Activity by the Hydrolysate Obtained from Microcapsule Digestion

It has been reported that whey protein hydrolysates produced with digestive enzymes such as pepsin or trypsin in short hydrolysis times inhibit in vitro DPP-4 activity [37]. To test the effect of the microcapsule hydrolysates obtained during the intestinal phase and after four hours of digestion, simulating the digestion in human, the DPP-4 test was performed. The DPP-4 enzyme inhibition test was conducted for hydrolysates in both the gastric and intestinal phases at different times. In the gastric phase, negative results were obtained at the initial and final times for the WPI–SA and WPI–CMC systems (data not shown). The same results were obtained at 0 min of the intestinal phase (Figure 8).

The results obtained in the intestinal phase (Figure 8) were 44.9% and 43.8% inhibition for 25 µL hydrolysates from empty microcapsules coacervated with SA or with CMC, respectively, at 60 min of hydrolysis. Increasing the hydrolysis time to 120 min, which corresponds to the end of the intestinal phase, the inhibition percentages increased to 54.7% and 53.1% for the capsule hydrolysates with SA and CMC, respectively. On the other hand, for sitagliptin (the reference standard), 51.2% inhibition was obtained at a concentration of 19 nM and no significant difference was observed between hydrolysates for either system at 60 or 120 min hydrolysis. This is a promising result that demonstrates the potential hypoglycemic capacity of the empty capsules after the hydrolysis of the whey protein contained in the wall of the capsules. Consequently, these empty capsules may have a synergistic effect with the peptides or hypoglycemic compounds that can be encapsulated within them.

Several researchers have reported the inhibition of DPP-4 by whey protein digestion; however, few have determined this phenomenon in vitro. Of these, Lacroix and Li-Chan [14,38] reported that individual peptides derived from whey protein generally showed a lower potential than the hydrolysates or the fractions from which they were isolated, suggesting that these peptides act synergistically with each other. For this reason, the fractionation of the hydrolysates obtained in this work was not performed.

It is important to highlight that the present work has shown that during digestion of the empty capsules, peptides with good inhibitory activity of the DPP-4 enzyme were generated in a simulated digestion that included both the gastric and intestinal phase, unlike the previously reported works that used short time periods and a single enzyme of either gastric or plant origin [15]. Based on the above results, the microcapsules coacervated with CMC were select for the following assays due to an easier coacervation procedure because of the lower viscosity compared with SA.

3.6. Qualitative Analysis of the Peptides Formed by the In Vitro Digestion Process.

To obtain the approximate molecular weight of the peptides in the resulting hydrolysate from the intestinal digestion, samples were subjected to a matrix-assisted laser ionization test with a time-of-flight detector (MALDI-TOF), using the alpha-cyano-4-hydroxycinnamic acid matrix (CHCA) for peptide analysis. Because of the hypoglycemic effect of the hydrolysate, commercial insulin was used as the control.

Figure 9 shows the spectra obtained for commercial insulin (Figure 9a), as well as the hydrolysate obtained at the end of the digestion process for the empty capsules (Figure 9b) for the WPI–CMC system. As can be seen in this figure, the commercial insulin shows a signal at 5773.61, very close to its molecular weight, which is 5.8 kDa. This signal does not appear in the spectrum of the sample that corresponds to the supernatant of the in vitro digestion of the empty capsules (Figure 9b), but there are other peaks in the spectrum corresponding to low-molecular-weight compounds (0–1000 m/z). Figure 9b shows with more detail the signals obtained for samples of in vitro digestion of the empty capsules in the region of 0–700 m/z.

Figure 10a shows two well-defined signals at 524 and 568 m/z in the commercial insulin mass spectrum. These signals are also present in the spectrum of the hydrolysate product of the in vitro digestion of the empty capsules (Figure 10b), but with an increase in intensity. Additionally, a set of new and more intense signals appears mainly at 481, 496, 506, 524, 544, 568, 615, and 650 m/z. This study proves the appearance of other low-molecular-weight peptide masses result from the digestion of the microcapsule, differing from the appearance of commercial insulin. These peptides may be responsible for the physiological response in glucose control of the hydrolysate of the WPI that has been demonstrated in previous works [11,12,13,14,15,37,38,39].

3.7. Interactions of Hydrolysates of Empty Capsules with Insulin Receptor

Fluorescence spectroscopy tests were performed to determine if the peptides resulting from the digestion of the empty capsules interact with the insulin receptor, for which the InsR insulin receptor domain 1101-end was used. Fluorescence was used as a tool to monitor the folding of proteins. Lee et al. [40] reported that the interaction of insulin with the insulin receptor can be observed by the increase in fluorescence intensity in the range of 250–410 wave lengths. Figure 11 shows the change in fluorescence intensity for the insulin receptor, insulin receptor–insulin complex, and insulin receptor–hydrolysate complex.

Figure 11 shows, on the one hand, the fluorescence of the insulin receptor caused by the presence of tryptophan and tyrosine residues, and on the other, the increase in fluorescence intensity when the interaction of the insulin receptor and insulin occurs, which coincides with a report by Lee et al. [40,41].

Previous studies propose changes in the secondary structure of the receptor upon binding to insulin [40,42,43], which possibly implies the formation of more rigid helices and tertiary and quaternary structural changes that occur when two protomers come into contact.

Regarding the response of the insulin receptor upon contact with the hydrolysate of in vitro digestion of the empty capsules, an increase in fluorescence intensity was also observed, which suggested that the hydrolysate also produces a conformational change on the insulin receptor, but of lower intensity than that obtained with insulin (40% at 300 nm). Judging from the florescence intensity response shown in Figure 11, it can be concluded that the peptides in the hydrolysate of the empty capsules are interacting with the insulin receptor in a somehow similar manner to insulin.

4. Conclusions

In vitro digestion of the empty capsules showed that the integrity of the microcapsule remained stable at gastric pH (2.5 pH), and that at pH 7, the coacervate disintegrates in the intestinal phase, allowing digestion of the protein-producing peptides capable of inhibiting the DPP-4 enzyme. It is important to highlight that this study demonstrated that the whey protein present in the wall of the empty microcapsules generates peptides with good DPP-4 inhibition activity in simulated digestion, which takes into account physiological conditions in both gastric and intestinal phases, unlike previously reported works that used short time periods and a single enzyme.

Furthermore, there was an interaction of these peptides with the insulin receptor in a similar manner to insulin but of lower intensity (40%) than that obtained with insulin.

It could be concluded that the designed microcapsule, aside from being a vehicle for oral delivery of bioactive peptides, is a complex with inhibitory effect on DPP-4 with potential hypoglycemic activity in itself, which can be enhanced by encapsulating functional peptides or antidiabetic compounds that can help to develop oral formulations with complementary actions for diabetes treatment.