Effect of Pea Protein and Arabinoxylans Mixtures as Emulsifiers on Vitamin E Absorption in Mice

ABSTRACT

This study aimed to evaluate the impact of pea protein isolate(PPI) and corn arabinoxylans (CAX) as emulsifiers on vitamin E bioavailability. The potential of PPI and CAX as emulsifiers for vitamin E delivery was evaluated by assessing their effect on: (i) vitamin E uptake by cultured human Caco‐2 intestinal cells and (ii) vitamin E postprandial response to 10% oil‐in‐water emulsion enriched in vitamin E in a murine model. Tween 80 (T80) was used as a control emulsifier. A decreased α‐tocopherol uptake by Caco‐2 cells was observed when using PPI as emulsifier compared to the T80 condition. When combining PPI and CAX, especially at low doses of CAX, α‐tocopherol uptake was equivalent to that measured when using T80. In mice, the postprandial response of vitamin E was significantly lower when using PPI + CAX as emulsifiers compared to T80. These results contrast with previous in vitro data, where vitamin E bioaccessibility did not differ when using T80 or PPI + CAX. Models for predicting the bioavailability of lipophilic nutrients when complex plant‐based delivery systems are involved may not always translate to physiological efficacy, supporting the need for validation with in vivo models.

The exclusive use of in vitro models forpredicting the bioavailability of lipophilic nutrients when complex plant‐based delivery systems are involved may not always translate to physiologicalefficacy, supporting the need of validation with in vivo models.

Abbreviations

AX

arabinoxylans

CAX

corn arabinoxylans

FSV

Fat soluble vitamins

PPI

pea protein isolate

O/W emulsion

oil‐in‐water emulsion

T80

Tween 80

1. Introduction

Dietary fat‐soluble vitamins (FSV) regroup 4 families of molecules displaying similar biological activities: vitamin A (retinol, retinyl esters, provitamin A carotenoids), vitamin D (cholecalciferol, ergocalciferol), vitamin E (tocopherols, tocotrienols), and vitamin K (phylloquinone, menaquinones). FSV digestion, transport across the enterocyte, and secretion to the bloodstream can be strongly affected by the food matrix in which they are embedded, as well as by the whole meal composition, as previously reviewed [1, 2]. The bioaccessibility (fraction of the ingested FSV that is biochemically and/or physically released from the food matrix and solubilized within the gastrointestinal fluids in an absorbable form) and bioavailability (fraction of the ingested FSV that reaches the bloodstream and thus its target tissue) of each FSV can be differently modulated by food components. This is likely due to the respective chemical structures of the different FSV [3, 4], which can influence their interactions with food components.

Emulsions are often used to vectorize FSV, and both the physical characteristics of the emulsions and the chemical nature of the emulsifiers used can impact vitamin delivery and absorption [2, 5]. Although in vitro digestions are very useful for screening the potential of different emulsifiers for FSV delivery, results should be validated in in vivo models before bringing promising systems towards applications, as results may differ [6].

We previously reported that among different combinations of legume proteins and arabinoxylans, the combination of pea protein isolate (PPI) and corn arabinoxylans (CAX) as emulsifiers was as efficient as Tween 80 (T80, polysorbate) in promoting the in vitro bioaccessibility of both vitamin E and D₃ when included in 10% oil‐in‐water (O/W) emulsions [7, 8]. For vitamin D₃, the high in vitro bioaccessibility was consistent with its in vivo bioavailability observed in a murine model [8].

The objective of this study was, therefore, to explore whether our in vitro results in terms of vitamin E bioaccessibility could be validated in an in vivo model. To do so, we compared the effects of pea proteins and arabinoxylans and those of T80 on vitamin E uptake by Caco‐2 cells and on vitamin E postprandial response in force‐fed mice.

2. Materials and Methods

2.1. Materials

Pea protein isolate (PPI) (NutralysF85F, min 83% protein) was purchased from Roquette (Lestrem, France). Corn arabinoxylans (CAX, min 79.3% soluble fiber) were kindly donated by AgriFiber (Mundelein, IL, USA). All analytical grade chemicals were from Sigma–Aldrich (Saint–Quentin–Fallavier, France). HPLC‐grade solvents were from Carlo Erba (Peypin, France).

2.2. Vitamin E‐Loaded Emulsion Preparation

An appropriate volume of vitamin E (α‐tocopherol or γ‐tocopherol) stock solution in HPLC‐grade ethanol was transferred to a glass tube. Ethanol was carefully evaporated under nitrogen, and the dried residue was solubilized in olive oil to achieve a tocopherol concentration of 0.18 mg α‐tocopherol/mL olive oil for cell uptake or 15 mg γ‐tocopherol/mL olive oil for in vivo experiments. Then, 10% (w/w) O/W emulsions were prepared by emulsifying the olive oil and different PPI and CAX aqueous solutions elaborated with Milli‐Q water (4% PPI (w/w), 4% PPI + 0.9% CAX (w/w), 4% PPI + 0.15% CAX (w/w) solutions) following the indications of Bravo‐Núñez et al. [7]. An emulsion stabilized with 2% T80, prepared using the same procedure, was used as a control. Emulsions had a final vitamin concentration of 0.018 mg α‐tocopherol/mL emulsion for cell experiments and 1.5 mg γ‐tocopherol/mL emulsion for in vivo experiments.

2.3. Cell Culture and α‐Tocopherol Absorption Experiments

The Caco‐2 cell line was routinely cultured in DMEM containing 8% FBS, 1% penicillin‐streptomycin, and 1% non‐essential amino acids at 37°C in a 10% CO2 atmosphere. Cell lines were grown on 12‐well plates with transwell inserts for 21 days before experiments.

Micellar fractions of in vitro digestions of α‐tocopherol‐loaded emulsions were prepared for cell absorption experiments. Digestion of emulsions was carried out with a test meal as previously described [7, 9]. All digestions were run in quadruplicate (a different freshly elaborated emulsion was used for each replicate), and micelles were frozen at −80°C until use.

Cytotoxicity of micellar fractions diluted in serum‐free complete medium (serial dilutions up to 1/16) was determined using an MTT assay as described elsewhere [10].

Twenty‐four hours before each experiment, the media were replaced with a serum‐free medium at both the apical and basolateral sides. At the beginning of each experiment, media were harvested, and the apical side of the cell monolayers was exposed to 1 mL of the α‐tocopherol‐rich micelles properly diluted (1/8) with serum‐free complete medium, whereas 2 mL of serum‐free complete medium were added to the basolateral side. Cells were then incubated for 4 h at 37°C. At the end of the experiment, media from the apical side of the cell monolayer were collected. Cells were washed with 1 mL cold PBS to eliminate adsorbed micelles, then scraped and collected in 0.5 mL PBS. Scraped cell, apical and basolateral media samples were stored at –80°C before tocopherol assays. Each experiment was repeated in quadruplicate. Vitamin E absorption by the enterocytes was calculated as follows:

$$\begin{array}{ccc}& & \mathrm{Vita}\min E\mathrm{upta}\mathrm{ke}\mathrm{by}\mathrm{Caco}2\mathrm{cells}\left(\%\right)\hfill \\ & =& \frac{\begin{array}{c}\mathrm{Vita}\min E\mathrm{amou}\mathrm{nt}\mathrm{in}\mathrm{cells}\hfill \\ +\mathrm{in}\mathrm{baso}\mathrm{late}\mathrm{ral}\mathrm{media}\hfill \end{array}}{\begin{array}{c}\mathrm{Vita}\min E\mathrm{amou}\mathrm{nt}\mathrm{in}\mathrm{dilu}\mathrm{ted}\mathrm{mice}\mathrm{lles}\hfill \\ \mathrm{at}\mathrm{the}\mathrm{begi}\mathrm{nning}\mathrm{of}\mathrm{the}\mathrm{expe}\mathrm{rime}\mathrm{nt}\hfill \end{array}}\times 100\hfill \end{array}$$

when not considering vitamin E degradation; or

$$\begin{array}{ccc}& & \mathrm{Vita}\min E\mathrm{upta}\mathrm{ke}\mathrm{by}\mathrm{Caco}2\mathrm{cells}\left(\%\right)\hfill \\ & & =\frac{\begin{array}{c}\mathrm{Vita}\min E\mathrm{amou}\mathrm{nt}\mathrm{in}\mathrm{cells}+\mathrm{in}\mathrm{baso}\mathrm{late}\mathrm{ral}\mathrm{media}\hfill \end{array}}{\begin{array}{c}\mathrm{Vita}\min E\mathrm{amou}\mathrm{nt}\mathrm{in}\mathrm{cells}+\mathrm{in}\mathrm{baso}\mathrm{late}\mathrm{ral}\mathrm{media}\hfill \\ +\mathrm{in}\mathrm{apic}\mathrm{al}\mathrm{media}\mathrm{at}\mathrm{the}\mathrm{end}\mathrm{of}\mathrm{the}\mathrm{expe}\mathrm{rime}\mathrm{nt}\hfill \end{array}}\times 100\hfill \end{array}$$

when considering vitamin E degradation.

2.4. Mouse Experiments

2.4.1. Animals

6‐week‐old C57BL/6 male mice were purchased from Janvier laboratory (Le Genest St Isle, France). Animals were housed in temperature‐, humidity‐, and light‐controlled room in cages (2‐3 animals/cage). They had free access to tap water and a standard chow diet (R03‐25 irradiated, Safe, Augy, France). Mice were allowed to acclimatize for 1 week before the start of the experiments. The protocol was approved by the local Ethics committee of Marseille and the Ministry of National Education, Higher Education and Research of France (approval number APAFIS#13473‐2018020918403330v3).

2.4.2. Postprandial Experiments

Experiments were carried out on 7‐week‐old mice (weight between 20 and 30 g). The number of mice per group was 5 to ensure statistical sensitivity [8, 11] while adhering to the ethical principle of minimizing the number of animals. Three days before the experiment, a blood sample was obtained at fast (zero baseline sample) by submandibular puncture. Mice were fasted overnight before each experiment. On the day of the experiment, mice were force‐fed 300 µL of the emulsions previously described, receiving 450 µg of γ‐tocopherol. Blood samples were then taken at t = 1.5, 3, and 4.5 h after force‐feeding. A final blood sample was taken at 6 h by intracardiac puncture after euthanasia. Blood samples were collected in heparinized tubes and immediately centrifuged according to manufacturer's instructions, and the resulting plasma samples were immediately frozen at −80°C. Liver and intestine samples were promptly harvested after euthanasia. Intestines were carefully rinsed with ice‐cold PBS and cut into five 6‐cm‐long segments corresponding to: duodenum, jejunum proximal, jejunum medium, jejunum distal, and ileum. All samples were immediately snap‐frozen in liquid nitrogen and stored at −80°C until analysis. For analyses, 100 mg of intestinal/ liver samples were suspended in 1 mL of PBS and homogenized with two 3 mm‐diameter stainless steel balls in 2 mL Eppendorf tubes using a MM301 ball mill (Retsch, Eragny sur Oise, France) before vitamin E and lipid extraction.

2.5. Vitamin E and Lipid Extraction

To evaluate vitamin E content from different samples (diluted micelles, apical content, basolateral content, cells in PBS, plasma and homogenized liver ), vitamin E was extracted from 500 µL samples. When needed, distilled water was added to the samples to reach a final volume of 500 µL. The same volume of internal standard solution (retinyl acetate in ethanol) and 2 mL of hexane were added to glass tubes, and samples were vigorously mixed for 10 min with a vortex at ambient temperature. Then, samples were centrifuged (10 min, 4°C, 1300 g) and the upper phases were collected in hemolysis tubes and evaporated until dryness under nitrogen. Another 2 mL of hexane was added to the glass tubes, and the process was repeated. The upper phases were collected and evaporated in the same hemolysis tubes. The dried extracts were dissolved in 200 µL of pure methanol.

For intestine samples, vitamin E extraction was done following the Bligh and Dyer method, to also extract triglycerides (TG). Briefly, vitamin E and lipids were extracted from 200 µL of intestine solution. 300 µL of distilled water, 150 µL of internal standard (retinyl acetate in ethanol), 1000 µL of methanol, and 500 µL of chloroform were added to glass tubes, and samples were vigorously mixed for 2 min with a vortex at ambient temperature. Then, an extra 500 µL of distilled water and 500 µL of chloroform were added, and samples were again vigorously mixed for 10 min with a vortex at ambient temperature. Samples were centrifuged (10 min 4°C, 1300 g) and the inferior phases wre collected in hemolysis tubes and evaporated under nitrogen. 500 µL of chloroform were added to the glass tubes and the process of mixing plus centrifugation was repeated. The upper phases were collected and evaporated in the same hemolysis tubes. The dried extracts were dissolved in 400 µL of pure methanol. 200 µL of the resulting methanol solution were transferred to HPLC vials for vitamin E analysis. The remaining volume was evaporated until dryness under nitrogen and subsequently dissolved in 200 µL of isopropanol. Samples were then collected in HPLC vials and kept at −20°C until lipid measurements.

2.6. Vitamin E HPLC Analysis

A volume of 180 µL was used for HPLC analysis with a 250 × 4.6 mm RP C18, 5 µm Zorbax column (Agilent Technologies, Montpellier, France) maintained at a constant temperature (35°C) and a guard column with a mobile phase of 100% methanol (flow rate = 1.5 mL/min). For the calibration curves, the injected volume ranged from 5 to 180 µL. The HPLC system comprised a Dionex separation module (P680 HPLC Pump and ASI‐100 Automated Sample Injector, Dionex, Aix‐en‐Provence, France), a Dionex UVD340U photodiode array detector (vitamin E detection at 292 nm and retinyl acetate detection at 325 nm), and a JASCO fluorimetric detector (JASCO, Nantes, France). The software used was Chromeleon software (v6.50 SP4 Build 1000, Dionex). For fluorimetric analysis, α‐tocopherol and γ‐tocopherol were detected at 325 nm after light excitation at 292 nm. α‐tocopherol and γ‐tocopherol calibration curves allowed quantification of vitamin E by comparing the sample peak area with standard reference curves.

2.7. Triglyceride Analysis

TG from plasmas and intestines were measured with a kit for Total triglycerides—GPO Method from Biolabo (Les Hautes Rives, Maizy, France), following the instructions of the manufacturer. For plasma, analyses were directly performed with the plasma. For intestine samples, TG were extracted as described in Section 2.5, and resuspended in isopropanol.

2.8. Statistical Analysis

Data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism software, version 8.4.3 (GraphPad Software, San Diego, California, U.S.A.). Data were tested for normality (Shapiro–Wilk test). The distribution of all dependent variables was normal. One‐way ANOVA (cells) or t‐test (in vivo) was used for data analysis. For one‐way ANOVA, Tukey's test was used as a post‐hoc test for pairwise comparisons. Values of p ≤ 0.05 were considered significant.

3. Results

3.1. α‐Tocopherol Uptake by Caco‐2 Cells

The total α‐tocopherol content recovered in the apical and cellular compartments at the end of the experiment was not significantly affected by emulsifiers, meaning that α‐tocopherol was not highly degraded during the experiment. α‐tocopherol was not detected in the basolateral compartments. The percentage of recovered α‐tocopherol was 96 ± 2%, 93 ± 5%, 98 ± 4%, and 97 ± 3% for 2% T80, 4% PPI, 4% PPI + 0.15% CAX, and 4% PPI + 0.9% CAX, respectively.

The α‐tocopherol uptake by enterocytes after a 4 h exposure is presented in Figure 1A,B. When not considering vitamin degradation (Figure 1A), α‐tocopherol uptake was significantly higher when using 2% T80 than when using 4% PPI (p = 0.01) or 4% PPI + 0.9% CAX (p = 0.03) as emulsifiers. No significant differences were found between T80 and 4% PPI + 0.15% CAX, although the p‐value was close to significance (p = 0.0526). When considering α‐tocopherol degradation (Figure 1B), significant differences were only found between 2% T80 and 4% PPI (p = 0.03). p values for 4% PPI + 0.15% CAX and 4% PPI + 0.9% CAX were 0.06 and 0.07, respectively.

FIGURE 1.

Caco‐2 α‐tocopherol uptake after 4 h exposure to micelles containing 422 ng α‐tocopherol /mL micelles. (A) Uptake not considering α‐tocopherol degradation and (B) Uptake considering α‐tocopherol degradation. Data are expressed as mean ± standard error of the mean (SEM). Sample size was n = 2–4 of technical replicates (variance due to technical issues). Data were tested for normality (Shapiro–Wilk test). Data were analyzed by One‐way ANOVA. Tukey's test was used as a post‐hoc test. Significant differences are expressed as: ^* = p ≤ 0.05 and ^** = p ≤ 0.01.

3.2. Triglycerides and γ‐tocopherol Postprandial Response in Mice

Plasma postprandial TG levels were decreased at t = 3 h when using 4% PPI + 0.9% CAX as emulsifiers compared to 2% T80 (p = 0.03, Figure 2A), although no significant difference was observed for the area under the curve (AUC). TG content of the intestinal mucosa was only significantly decreased when using 4% PPI + 0.9% CAX as emulsifiers compared to 2% T80 in the ileum (p = 0.04) (Figure 2B).

FIGURE 2.

(A) Postprandial triglyceride response, (B) Small intestine triglyceride content, (C) Postprandial γ‐tocopherol response, (D) Small intestine γ‐tocopherol content, and (E) Liver γ‐tocopherol content of mice (male, n = 5) 6 h after force‐feeding 300 µL of 10% oil‐in‐water emulsions providing 450 µg γ‐tocopherol. Data are expressed as mean ± standard error of the mean (SEM). Data were tested for normality (Shapiro–Wilk test). Data were analyzed by t‐test. Significant differences are expressed as: ^* = p ≤ 0.05 and ^** = p ≤ 0.01.

Plasma γ‐tocopherol levels from t = 1.5 h were significantly higher when using 2% T80 as emulsifier than when using 4% PPI + 0.9% CAX for all times (for t = 3, 4.5, and 6 h, p‐values of 0.001, 0.003, and 0.004, respectively) (Figure 2C). γ‐tocopherol content of the duodenum (p = 0.02) and median jejunum (p = 0.03) (Figure 2D), and γ‐tocopherol uptake by the liver (p = 0.02, Figure 2E) were also increased when γ‐tocopherol was delivered with emulsions stabilized with 2% T80 compared to when delivered with emulsions stabilized with 4% PPI + 0.9% CAX.

4. Discussion

Previous in vitro results highlighted that PPI + CAX could constitute an efficient alternative to T80 to deliver vitamin E and D₃ [7, 8], with these promising results being confirmed for vitamin D₃ in a murine model with different vitamin D₃ levels [8]. The previous in vitro results for vitamin E only considered the capacity of PPI + CAX to stabilize emulsions and promote vitamin E release during digestion [7]. In this study, we first explored the uptake by Caco‐2 cells of vitamin E from digested emulsions to see how these emulsifiers could affect vitamin E transport across the enterocyte. It is known that food components can influence the rate of uptake of FSV at the enterocyte level [8]. The improved uptake of α‐tocopherol when using 2% T80 instead of 4% PPI (Figure 1A,B) can be related to how emulsifiers regulate the interaction of vitamins with the enterocytes. The efficiency of T80 for bioactive delivery to enterocytes has been extensively reported (e.g., [12]), likely via membrane fluidization, leading to increased permeabilization and ultimately to a loss of integrity depending on the level and chronicity of exposure [13]. This mechanism of action is supported by the detrimental effect of chronic exposure to T80 at high doses on gut health [14]. Moreover, decreased α‐tocopherol uptake with 4% PPI, and to a lesser extent with the combination of PPI and CAX, is probably related to the presence of antinutrients in the emulsifiers. We previously reported that the used PPI and CAX were associated with saponins, tannins, and phytates (7.4 ± 1.15 and 5.4 ± 0.76 mg saponins/g, 0.44 ± 0.05 and 1.76 ± 0.36 mg tannins/g, 24.8 ± 2.7 and 33.38 ± 4.48 mg phytates/g, for PPI and CAX, respectively) [7]. It was also previously shown that antinutrients could decrease vitamin K uptake by enterocytes [4], and vitamins E and K show similarities in their chemical structure.

While not statistically significant, the observed trend of reduced vitamin E degradation when combined with PPI + CAX may account for the differences in vitamin uptake by enterocytes. This protective effect of CAX against vitamin degradation in a cell model agrees with previous experiments in which soy proteins were combined with arabinoxylans for vitamin delivery [15]. It is also consistent with our previous in vitro results [7]. We hypothesize that this effect is related to the antioxidant properties of arabinoxylans [16], which may partially compensate for the negative effect of associated antinutrients. The potential interactions between antinutrients from PPI and CAX were not specifically assessed in this study.

Although vitamin absorption by the enterocyte brings valuable information, in a previous work, we observed with vitamin D₃ that although PPI and CAX reduced vitamin uptake in a Caco‐2 model, no significant differences were observed in vivo. This suggests that, for vitamin D₃, emulsifiers may delay uptake without affecting overall absorption, since the postprandial vitamin D response was similar whether T80 or PPI + CAX were used as emulsifiers [8]. We therefore investigated one of the most promising conditions—4% PPI + 0.9% CAX—by assessing its impact on vitamin E bioavailability using an in vivo model. This approach allows the integration of all physiological processes involved. The other conditions were not evaluated to reduce the number of animals used.

For this set of experiments, γ‐tocopherol was chosen because, unlike α‐tocopherol, it is not the form added in the diet administered to mice, and it is not re‐secreted into the bloodstream by the liver. α‐ and γ‐tocopherol only differ by the position of one methyl group on the chromanol ring [17], and major changes in emulsion interfacial behavior have not been observed [18]. This makes γ‐tocopherol a better in vivo tracer of the vitamin E fraction absorbed by the gut, given that intestinal absorption of α‐ and γ‐tocopherol forms is similar [19].

For both emulsifiers and in agreement with previous results, vitamin E accumulation in intestinal mucosa was mainly concentrated in the distal jejunum and the ileum [20], as vitamin E absorption mostly happens at the distal small intestine. TG accumulation in the intestinal mucosa showed the same trend. The lower postprandial triglyceridemia when using 4% PPI + 0.9% CAX agrees with previous studies that reported reduced postprandial lipemia after consumption of legumes and fibers [21]. Significantly lower plasma and hepatic levels of γ‐tocopherol were measured when using 4% PPI + 0.9% CAX instead of 2% T80. Vitamin E also accumulated in the intestinal mucosa when using 2% T80, which suggests a lower absorption at the enterocyte level for 4% PPI + 0.9% CAX, in agreement with the results obtained in Caco‐2 cells (Figure 1A). This suggests a detrimental effect of fibers (CAX), which could be related to their viscosity‐enhancing properties in foods [22], thus hindering the diffusion of mixed micelles to the brush border of intestinal enterocytes. However, previous data reported no effect of fibers on vitamin E absorption [23, 24], and in this study, CAX levels in the emulsions were relatively low (0.9%). We thus hypothesize that this negative effect is more likely due to the antinutrients present in the emulsifiers, as discussed above. The divergence between the results of this study for vitamin E and those of our previous study with vitamin D₃ [8] could be related to the specificity of epithelial transporters for each vitamin [25], together with the potential interactions antinutrients–transporters–vitamins. These interactions were not addressed in this work and should be further confirmed.

5. Conclusion

This study highlights the importance of confirming in vitro results with in vivo models before bringing promising systems towards applications, as results can differ. The successful application of pea protein isolates and arabinoxylans as emulsifiers to deliver vitamin E may only be possible when reducing the antinutrient content of these emulsifiers, which could be achieved by fermentation or enzymatic hydrolysis processes, keeping in mind that these processes could impact their emulsifying capacity.

Author Contributions

Angélique Berthomé: Investigation, Formal analysis; Donato Vairo: Methodology, Investigation; Charlotte Sabran: Methodology, Investigation; Katherine Alvarado‐Ramos: Investigation; Ángela Bravo‐Núñez: Conceptualization, Methodology, Validation, Investigation, Formal analysis, writing – original draft, Writing – review & editing, Funding acquisition; Emmanuelle Reboul: Conceptualization, Methodology, Validation, Writing – review & editing, Funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

Ángela Bravo‐Núñez's competitive postdoctoral contract “Margarita Salas” was funded by the government of Spain (Ministerio de Universidades) and by the European Union (NextGeneration‐EU). Bench fees were covered by the “Centre de Recherche en CardioVasculaire et Nutrition” (Marseille, France).

Data Availability Statement

Data available under request. All authors read and agreed with the manuscript.