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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2020 Feb 17;57(6):2329–2342. doi: 10.1007/s13197-020-04271-2

Evolution of cranberry juice compounds during in vitro digestion and identification of the organic acid responsible for the disruption of in vitro intestinal cell barrier integrity

Valentine Renaud 1,2, Mélanie Faucher 1,2, Véronique Perreault 1,2, Elodie Serre 1,2, Pascal Dubé 3, Yvan Boutin 4, Laurent Bazinet 1,2,✉
PMCID: PMC7230080  PMID: 32431359

Abstract

Cranberry juice is increasingly consumed for its richness in polyphenols having a positive impact on human health. Unfortunately, when regularly consumed, its high concentration in organic acids may cause some intestinal discomforts. In the present study, its organic acid content was reduced of 41% by electrodialysis with bipolar membrane (EDBM), and the resulted deacidified juice was divided in five different juices readjusted or not with different concentrations of citric and/or malic acid(s) corresponding to the concentration of this/these acid(s) recovered during EDBM or at the titratable acidity (TA) of the non-deacidified cranberry juice. The evolution of the cranberry juice main interesting compounds (organic acids and polyphenols), according to the concentration and nature of the organic acids present, was studied for the first time at each specific stages of the digestion. After digestion, Caco-2 cells were exposed to all digested juices to identify the organic acid(s) responsible for the loss of integrity of the epithelial barrier. It appeared that organic acid contents did not change during the different steps of the digestion while polyphenolic compounds decreased starting from the gastric phase. Whatever the organic acid concentration or nature, the concentration of PACs significantly decreased between the salivary and the gastric steps but was different according to their structure when the concentration of most of anthocyanins significantly decreased at the gastric step. Also, to the best of our knowledge, it was the first time that citric acid was demonstrated as the organic acid responsible for the loss of integrity of Caco-2 cell monolayers.

Keywords: Cranberry juice, Organic acid removal, Electrodialysis with bipolar membrane, Organic acids, Polyphenols, In vitro digestion, Integrity, Epithelial barrier, Caco-2 cells

Introduction

Cranberry is a typical fruit from North America. In Canada, its consumption increased significantly in the last 10 years since the intake per capita tripled from 0.8 kg in 2007 to 2.6 kg in 2016 (MAPAQ 2018). In the province of Quebec, the consumption of cranberries (juice, dried…) is estimated at nearly 21,380 tons in 2016 (MAPAQ 2018). Since a long time, cranberry is well recognized for its benefits on human health due to its high content in polyphenols such as anthocyanins and proanthocyanidins (PACs) (Bazinet et al. 2012). This rich source of polyphenols helps to protect cells against oxidative stress caused by free radicals (Wada and Ou 2002). It has also been proved that cranberry juice consumption prevent the adhesion of Escherichia coli to uroepithelial cells in the urinary tract and gastric ulcers caused by Helicobacter pylori (Sun et al. 2015). This type of juice also inhibits the formation of Streptococcus mutans complexes in saliva and plaque and it reduces the risk of cardiovascular diseases (Gupta et al. 2015; McKay and Blumberg 2007).

Unfortunately, this juice has an acidic pH (about 2.5) and contains high concentrations of citric, malic and quinic acids (Bazinet et al. 2012). These three main organic acids are responsible for the high titratable acidity of cranberry juice and such a high acidity may cause digestive complications. Indeed, these compounds may induce inflammatory intestinal side effects such as diarrhea, bloating or vomiting (Vasileiou et al. 2013). As indicated by Wing et al. (2008), ingestion of 240 mL of cranberry juice three times daily may induce gastrointestinal disorders. Mechanisms of intestinal inflammation have been extensively studied and a dysregulated immune response to commensal flora caused by transient breaks in the mucosal barrier have been reported (Sartor 2006). Caco-2 cells are human epithelial colorectal adenocarcinoma and represent an ideal model to study intestinal system permeability and integrity. Indeed, when cultured under specific conditions, Caco-2 cells differentiate themselves into epithelial cells and create a monolayer which is similar to the enterocytes lining the small intestine (Hidalgo et al. 1989).

Work undertaken by Serre et al. (2016a) showed the effect of global organic acid removal by electrodialysis with bipolar membrane (EDBM) on the protection against disruption of in vitro intestinal Caco-2 cell barrier integrity. EDBM is based on the use of bipolar membranes stacked in an electrodialysis (ED) cell, which are multilayer membranes carrying out water dissociation. These membranes are composed of three parts: an anion-exchange layer, a cation-exchange layer and a hydrophilic interface at their junction. Hence, when a direct current is applied, the water molecules migrating into the hydrophilic layer are splitted into H+ and OH−. EDBM can act in two ways: electroacidification and electrobasification on both sides of the membrane (Bazinet et al. 2004; Mani 1991). EDBM was demonstrated to slightly increase pH of cranberry juice but to decrease drastically and selectively the content of organic acids (Serre et al. 2016b). Furthermore, Serre et al. (2016a) also demonstrated that high concentrations of malic and citric acids in non-deacidified cranberry juice were responsible for the loss of integrity of Caco-2 cell monolayers. However, to the best of our knowledge, the fact that citric or malic acid have similar or different impact on inflammation or loss of integrity of intestinal Caco-2 cell monolayer has never been demonstrated and reported in the literature. Furthermore, the evolution of concentration and structure integrity of polyphenols in cranberry juice during the different specific phases of the digestion has also never been reported.

In this context, the objectives of the present study were (1) to study the evolution of organic acids and polyphenolic compounds (anthocyanins and (PACs)) during the different steps (salivary, gastric and intestinal) of the in vitro digestion according to the concentration and nature of the organic acids present in the cranberry juice and (2) to identify the organic acid(s) having the main impact on the integrity of Caco-2 epithelial cells.

Materials and methods

Cranberry juice

A pasteurized and clarified cranberry juice produced by Fruit d’Or (Plessisville, Quebec, Canada) was used for all treatments. This non-deacidified juice was at 8° Brix. The juice was stored at − 20 °C and before each experiment, it was thawed at 4 °C. The Table 1 presents the physicochemical characteristics of the non-deacidified cranberry juice.

Table 1.

Physicochemical characteristics of the non-deacidified and deacidified cranberry juices

Non-deacidified juice Deacidified juice
pH 2.69 2.83
Conductivity (mS/cm) 2.47 2.03
Titratable acidity (mg/L NaOH) 13.85 8.1
Organic acids (g/L)
 Quinic acid 11.62 11.61
 Malic acid 7.37 ± 0.25 4.25 ± 0.14
 Citric acid 13.06 ± 0.41 6.23 ± 0.17
Anthocyanins (mg/L of cyanidin-3-glucoside equivalents)
 Cyanidin-3-arabinoside 14.83 ± 0.59 15.17 ± 0.23
 Cyanidin-3-galactoside 16.42 ± 0.70 16.72 ± 0.36
 Cyanidin-3-glucoside 0.83 ± 0.14 1.00 ± 0.13
 Peonidin-3-arabinoside 10.02 ± 0.37 10.25 ± 0.07
 Peonidin-3-galactoside 20.60 ± 0.77 20.76 ± 0.40
 Peonidin-3-glucoside 2.50 ± 0.08 2.75 ± 0.08
Proanthocyanidins (mg/L of epicatechin equivalents)
 Monomers 25.65 ± 0.95 27.90 ± 0.84
 2–3 mers 89.76 ± 4.54 99.48 ± 3.36
 4–6 mers 28.60 ± 1.11 33.14 ± 1.62
 7–10 mers 3.27 ± 0.63 3.92 ± 0.22
 Polymers 7.58 ± 1.13 6.68 ± 0.14
 Total 154.86 ± 8.13 171.14 ± 6.00
Total polyphenols (mg/L of gallic acid equivalents) 518.98 517.30
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Protocol and sample preparation

In this study, different juices were prepared and tested. First, a non-deacidified juice was taken as a control, and part of this non-deacidified juice was deacidified at 41% (see Sect. Organic acid removal by electrodialysis). After the organic acid removal, this juice was divided in five different juices: the deacidified juice, a deacidified juice where a concentration of malic (3.12 ± 0.11 g/L corresponding to the concentration of malic acid recovered during organic acid removal) and/or citric (6.83 ± 0.17 g/L corresponding to the concentration of citric acid recovered during organic acid removal by electrodialysis) acids was added and a deacidified juice where a concentration of malic acid at the titratable acidity (TA) of the non-deacidified cranberry juice was added. Each organic acid was provided by Sigma Aldrich (St-Louis, MO, USA).

All these juices were in vitro digested, according to the protocol of Versantvoort et al. (2005) and, samples at different stages before and during digestion were collected for further analysis in terms of concentrations of organic acids, total polyphenols, anthocyanins and PACs: initial juices, salivary, gastric and intestinal phases. Subsequently, Caco-2 cells were exposed to the intestinal/final juices (see Sect. Co-culture system) in order to evaluate their impact on the integrity of the intestinal barrier and possibly identify the organic acid(s) responsible for its disruption.

Organic acid removal by electrodialysis

The cell ED configuration and the organic acid removal protocol were similar to those used by Faucher et al. (2018). The electrodialysis cell used during experiments was a semi-industrial EUR-6 cell (Eurodia, Pertuis, France). A 41% deacidified cranberry juice was obtained after 60 min of ED organic acid removal treatment in order to keep quinic acid in cranberry juice as explained by Serre et al. (2016b). The physicochemical characteristics of this deacidified juice are presented in Table 1.

In vitro digestion

The original protocol was taken from Versantvoort et al. (2005) and has been adapted to our conditions. First, four digestive solutions were prepared in order to simulate an in vitro digestion of the samples: saliva juice (pH 6.8 ± 0.2), gastric juice (pH 2.35 ± 0.1), intestinal juice (pH 8.1 ± 0.2) and bile juice (pH 8.1 ± 0.2). Then, samples (6 g) were mixed with saliva juice (3 mL) and agitated at 100 cycles/min for 5 min and at 37 °C. Gastric juice (3 mL) was added and each sample was agitated for 2 h at 37 °C. The pH of each sample was adjusted to 2.4 ± 0.1, using 5 N HCl during all gastric digestion period. Thereafter, intestinal juice (6 mL), bile juice (3 mL) as well as bicarbonate solution (2 mL) were added and agitated during 2 h at 37 °C. Since lipase is already present in porcine pancreatin, it was not added in duodenal juice. At the end of digestions, the pH was adjusted at 7.0 with NaOH in order to inhibit pepsin. This digestion was performed in triplicate and digested samples were frozen at − 20 °C and were thawed at 4 °C before physicochemical analyses (organic acid, anthocyanin and proanthocyanidin).

Co-culture system

As described by Serre et al. (2016a), the human leukemia monocytic cell line (THP-1) and the Caco-2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The Caco-2 cells used for this experiment were at passage numbers 32–41 and were seeded at 3 × 105 cells/well onto Transwell insert plates of 12 mm diameter and 0.4 µm pore size (Costar Corp., Cambridge, MA, USA). They were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% non-essential amino acid solution, 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 1 mM sodium pyruvate, and 100 U/mL penicillin (all from Hyclone, Logan, UT, USA) at 37 °C and 5% CO2. Every three days, the media was changed until the cells were completely differentiated at day 21. On this day, transepithelial electrical resistance (TEER) using Millicell®-ERS (Millipore, Bedford, MA, USA) was measured to confirm the integrity of the cell monolayer. Confluent cell monolayers produced TEER values greater than 1000 Ω cm2 after correction for resistance in blank control wells.

THP-1 cells were grown in RPMI supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Hyclone, Logan, UT, USA). The cells were plated at a density of 5 × 105 cells/well in 6-well plates and the plates were incubated for 72 h in the presence of 100 nM phorbol myristate acetate (PMA) (Saint Louis, MO, USA) in order to differentiate them into a macrophage-like phenotype. All media was replaced with Hank’s Balanced Salt Solution (HBSS) (GE, Logan, UT, USA) and the Transwell inserts with cultured Caco-2 cells were added to multiple plate wells previously loaded with THP-1 cells. Afterwards, the HBSS was removed from the apical compartment and it was replaced with 500 µL of in vitro deacidified/digested cranberry juice. The in vitro deacidified/digested cranberry juice was centrifuged at 3250×g for 15 min and then filtered using a 0.22 µm filter prior to use. Solution of Triton (1%) or HBSS were respectively used as positive and negative controls. The inserts were incubated for 3 h at 37 °C and 5% CO2. Before and after addition of cranberry juice, the TEER was measured. Results were expressed as the change in TEER measurement (ΔTEER).

Analyses

Organic acid contents

Concentration of quinic, malic and citric acid was analyzed by HPLC according to the AOAC method 986.13 since they are the three main organic acids in cranberry juice. Before their analysis, organic acids were extracted from non-deacidified cranberry juice and deacidified juices with or without addition of organic acids and digested or not with C18 cartridges (non endcapped 6 mL, 500 mg, Silicycle, Quebec, QC, Canada). First of all, the cartridges were conditioned with methanol (5 mL) and then washed with distilled water (5 mL) and with a solution of acetonitrile/water (10 mL of a 50% V/V). Thereafter, the cartridges were vacuum dried and 10 mL of samples were added. The first 5 mL were discarded and only the last 5 mL were kept for the analysis.

An Agilent 1100 series HPLC system equipped with an UV detector was used to separate the organic acids. Samples (10 µL) were injected on a Synergi Hydro-RP 80A (250 × 4.6 mm, Phenomenex, Torrance, CA, USA) column at room temperature. An isocratic mobile phase was prepared with 0.2 M KH2PO4 solution at pH 2.4. A wavelength of 214 nm was used for the detection of organic acids. Calibration curves and retention times of standards (Sigma Aldrich, Saint-Louis, MO, USA) were used to quantify and identify the three organic acids of interest and their contents were expressed in g/L.

Anthocyanin contents

Prior to anthocyanin analysis by HPLC (Wu and Prior 2005) using an Agilent 1100 series system equipped with a diode array detector, the six different cranberry juices digested or not were filtered using a 0.45 µm nylon filter. Then, 20 µL of samples (20 µL) were injected and analyzed with a Zorbax SB-C18 5 µm (250 × 4.6 mm, Agilent, Santa Clara, CA, USA) column at room temperature. Two mobile phases were prepared for elution at 1 mL/min: solvent A made of water/formic acid (95%/5%) and solvent B, composed of 100% methanol. A wavelength of 520 nm was used for the detection of anthocyanins and these compounds contents were expressed in mg/L cyanidin-3-glucoside equivalents.

Proanthocyanidin contents

For PAC content analysis, samples were filtered with a 0.45 µm nylon filter and a volume of 5 µL were injected onto an Agilent 1260 series HPLC system equipped with a fluorescence detector (Khanal et al. 2009). A Nomura chemical Develosil 100 Diol-5 (250 × 4.6 mm, Phenomenex, Torrance, CA, USA) column was used at 35 °C. According to their polymerization degree, PACs were separated with two solvents at a flow rate of 0.8 mL/min: solvent A, made of acetonitrile and acetic acid (98%/2%) and solvent B, made of methanol, water and acetic acid (95%/3%/2%). Emission wavelength was set at 321 nm while excitation wavelength was set at 230 nm. A calibration curve of epicatechin was used to quantify PACs of different degrees of polymerization. Also, a correction factor was used to convert the different response factors of monomeric to polymeric PACs. Each PAC content was expressed in mg/L of epicatechin equivalents.

Statistical analyses

The data were subjected to an analysis of variance (ANOVA) or a multivariate analysis of variance (MANOVA) and a Tukey test was carried out to evaluate the composition evolution of organic acids and polyphenols of cranberry juice during digestion steps. A Dunnett test was used to measure the effect of organic acids in cranberry juice on Caco-2 cells. For this statistical analytic purpose, SAS software (version 6.1) was used and significant differences were declared at a probability level p < 0.05.

Results and discussion

Evolution of the composition of organic acids and polyphenols in cranberry juice during digestion steps

Organic acid contents

Since citric acid is the most abundant organic acid in cranberry juice, its content in the non-deacidified juice was high (13.06 ± 0.41 g/L) and remained constant all along the digestion steps after taking into account the dilution factor at the different steps (1.50 at salivary, 2.00 at gastric and 3.834 at intestinal steps) (p = 0.74) (Fig. 1a). The initial content of malic acid in the non-deacidified juice was lower than that of citric acid (7.37 ± 0.25 g/L) and remained quite constant along the digestion steps (Fig. 1b). After 60 min of organic acid removal, as expected, the concentration of citric and malic acid was decreased respectively by 52% and 42% and remained constant all along the digestion process.

Fig. 1.

Fig. 1

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Measurement of a citric and b malic acid contents in non-deacidified cranberry juice and deacidified juices with or without extra content of organic acids during the three digestion steps and in comparison with the initial juice. Columns with different capital letters are significantly different at a probability level of 0.05 (MANOVA, Tukey test). Columns with different lowercase letters are significantly different at a probability level of 0.05 (ANOVA) (n = 3)

When malic acid was added in deacidified cranberry juice, and that whatever the level of malic acid added, no impact was observed on citric acid concentration, the concentration of citric acid was the same as the one in deacidified juice. As for the concentration of malic acid of the deacidified juice, it was quite similar to the one of the non-deacidified cranberry juice when malic acid was added. On the other hand, when malic acid was added in deacidified cranberry juice but at the same titratable acidity of the non-deacidified juice, the concentration of malic acid was not the same as that of the initial non-deacidified cranberry juice (p < 0.0001) and increased by 77%, meaning that a high quantity of malic acid was added in order to obtain the same pH of the initial non-deacidified cranberry juice. For both organic acid concentrations, the addition of malic acid in deacidified cranberry juice did not change significantly their concentrations all along the digestion steps.

When citric acid was added in the deacidified juice to compensate for its organic acid removal, the concentration in citric acid was quite similar to the one in the non-deacidified cranberry juice while no impact was observed on malic acid concentration: the concentration of malic acid was the same as the one in deacidified juice. Statistical analyses showed that when citric acid was added to the deacidified cranberry juice, the concentration of citric acid did not change all along the digestion steps.

These results were in accordance with those of Serre et al. (2016a) who showed that the final contents of organic acids after the global digestion process did not significantly change in non-deacidified and deacidified cranberry juice in comparison with non-digested juices. They also showed that the concentration of organic acid was only affected by the organic acid removal treatment duration. Furthermore, in the present study, when citric and/or malic acid was added to the deacidified cranberry juice, its content was similar to, respectively, the concentration in citric or malic acid of the non-deacidified cranberry juice. These results were in accordance with the concentrations of organic acids expected since organic acids were added to the deacidified cranberry juice after the organic acid removal process.

Anthocyanin contents

Statistical analyses of anthocyanin concentrations revealed that there were statistically significant differences between the digestion steps and initial juices, and also according to the nature of the anthocyanins (Table 2). Indeed, whatever the concentration and nature of the organic acids present in the cranberry juice, the general trend for five of the six anthocyanins measured (Cyanidin-3-arabinoside, C-3-galactoside, C-3-glucoside, Peonidin-3-arabinoside, P-3-galactoside) indicated that there was no difference between the initial juice and the salivary phase while a decrease in the concentration of anthocyanins appeared during digestion between the salivary and gastric phase. Furthermore, according to the nature of the anthocyanin, and that whatever the concentration and nature of the organic acids present, the decrease in their concentrations was different: some anthocyanins presented a decrease in their concentration between 34% and 56% (C-3-arabinoside, C-3-galactoside, C-3-glucoside, P-3-arabinoside, P-3-galactoside) while P-3-glucoside had the same concentration after the gastric phase. Interestingly, after the gastric phase, there was a disappearance of anthocyanins in the intestinal phase. For the sixth anthocyanin (C-3-glucoside), surprisingly, its concentration was the same between the initial and salivary step, and in contrary to the five previous anthocyanins, seemed to disappear completely during the gastric phase.

Table 2.

Evolution during digestion of anthocyanin contents in non-deacidified cranberry juice and deacidified juices with or without extra content in organic acids and in comparison with their respective initial juice

Initial Salivary Gastric Intestinal
Cyanidin-3-arabinoside (mg/L)
 Non-deacidified 14.83 ± 0.59aA 13.99 ± 0.19aA 6.10 ± 1.13bA 0 ± 0cA
 Deacidified 15.17 ± 0.23aA 14.23 ± 0.08aA 5.95 ± 1.29bA 0 ± 0cA
 Deacidified + malic acid 14.99 ± 0.04aA 14.03 ± 0.06bA 5.50 ± 0.23cA 0 ± 0dA
 Deacidified + citric acid 14.76 ± 0.02aA 14.21 ± 0.26bA 6.44 ± 0.21cA 0 ± 0dA
 Deacidified + malic and citric acid 14.86 ± 0.26aA 14.21 ± 0.21aA 5.65 ± 0.53bA 0 ± 0cA
 Deacidified + TA malic acid 14.83 ± 0.11aA 14.09 ± 0.10bA 5.37 ± 0.17cA 0 ± 0dA
Cyanidin-3-galactoside (mg/L)
 Non-deacidified 16.42 ± 0.70aA 15.48 ± 0.28aA 9.10 ± 1.12bA 0 ± 0cA
 Deacidified 16.72 ± 0.36aA 15.90 ± 0.20aA 8.81 ± 1.35bA 0 ± 0cA
 Deacidified + malic acid 16.73 ± 0.20aA 15.84 ± 0.20bA 8.23 ± 0.27cA 0 ± 0dA
 Deacidified + citric acid 16.31 ± 0.07aA 15.99 ± 0.19aA 9.44 ± 0.26bA 0 ± 0cA
 Deacidified + malic and citric acid 16.35 ± 0.28aA 15.91 ± 0.17aA 8.58 ± 0.74bA 0 ± 0cA
 Deacidified + TA malic acid 16.51 ± 0.29aA 15.69 ± 0.22bA 8.57 ± 0.30cA 0 ± 0dA
Cyanidin-3-glucoside (mg/L)
 Non-deacidified 0.83 ± 0.14aAB 0.78 ± 0.07aA 0.44 ± 0.45abA 0 ± 0bA
 Deacidified 1.00 ± 0.13aA 0.91 ± 0.10aAB 0 ± 0bA 0 ± 0bA
 Deacidified + malic acid 0.90 ± 0.10aAB 0.98 ± 0.05aAB 0 ± 0bA 0 ± 0bA
 Deacidified + citric acid 0.78 ± 0.05aAB 0.85 ± 0.03aAB 0 ± 0bA 0 ± 0bA
 Deacidified + malic and citric acid 0.71 ± 0.04aAB 0.96 ± 0.12bAB 0 ± 0cA 0 ± 0cA
 Deacidified + TA malic acid 0.79 ± 0.04aB 1.03 ± 0.05bB 0 ± 0cA 0 ± 0cA
Peonidin-3-arabinoside (mg/L)
 Non-deacidified 10.02 ± 0.37aA 9.42 ± 0.14aA 5.33 ± 0.98bA 0 ± 0cA
 Deacidified 10.25 ± 0.07aA 9.94 ± 0.04aB 5.06 ± 1.26bA 0 ± 0cA
 Deacidified + malic acid 10.06 ± 0.11aA 9.54 ± 0.23bAB 5.01 ± 0.19cA 0 ± 0dA
 Deacidified + citric acid 9.99 ± 0.08aA 9.91 ± 0.22aAB 5.47 ± 0.05bA 0 ± 0cA
 Deacidified + malic and citric acid 10.13 ± 0.29aA 9.89 ± 0.26aAB 4.97 ± 0.42bA 0 ± 0cA
 Deacidified + TA malic acid 10.07 ± 1.10aA 9.69 ± 0.15aAB 4.86 ± 0.35bA 0 ± 0cA
Peonidin-3-galactoside (mg/L)
 Non-deacidified 20.60 ± 0.77aA 19.03 ± 0.30aA 12.56 ± 1.23bA 0 ± 0cA
 Deacidified 20.76 ± 0.40aA 19.61 ± 0.14aAB 12.37 ± 1.68bA 0 ± 0cA
 Deacidified + malic acid 20.39 ± 0.05aA 19.39 ± 0.10bAB 11.67 ± 0.37cA 0 ± 0dA
 Deacidified + citric acid 20.17 ± 0.06aA 19.59 ± 0.28bAB 13.04 ± 0.21cA 0 ± 0dA
 Deacidified + malic and citric acid 20.37 ± 0.41aA 19.82 ± 0.29aAB 11.91 ± 0.60bA 0 ± 0cA
 Deacidified + TA malic acid 20.23 ± 0.17aA 19.28 ± 0.34bB 12.14 ± 0.56bA 0 ± 0cA
Peonidin-3-glucoside (mg/L)
 Non-deacidified 2.50 ± 0.08aA 2.70 ± 0.08aA 2.40 ± 0.26aA 0 ± 0bA
 Deacidified 2.75 ± 0.08aA 2.83 ± 0.24aA 2.37 ± 0.48aA 0 ± 0bA
 Deacidified + malic acid 2.54 ± 0.12aA 2.67 ± 0.23aA 2.47 ± 0.14aA 0 ± 0bA
 Deacidified + citric acid 2.38 ± 0.05aA 2.65 ± 0.23aA 2.53 ± 0.62aA 0 ± 0bA
 Deacidified + malic and citric acid 2.50 ± 0.10aA 2.93 ± 0.12bA 2.23 ± 0.20aA 0 ± 0cA
 Deacidified + TA malic acid 2.46 ± 0.31aA 2.93 ± 0.24aA 2.40 ± 0.18aA 0 ± 0bA
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The results on the same column, for each individual anthocyanin, with a different capital letter for the same digestion step/phase are significantly different at a probability level of 0.05. The results on the same line with a different lowercase letter for the same juice are significantly different at a probability level of 0.05 (n = 3)

Studies conducted on the stability of anthocyanins and their degradation under simulated gastrointestinal digestion, showed similar results. Serre et al. (2016a) showed that the global digestion process of cranberry juices appeared to degrade anthocyanin contents present in cranberry juice but they did not study the degradation at the different phases. It has also been showed that anthocyanin molecules possess an equilibrium under different pH conditions and four molecular species may exist; the red basic flavylium cation (pH 1–3) and three secondary structures; the colourless carbinol pseudobase (pH 4–5), the blue quinoidal base (pH 6–8) and the yellow chalcone pseudobase (pH 8–9) forms (Fig. 2) (Zhao and Temelli 2017). The flavylium cation conformation has been confirmed to be the dominant structure for monomeric anthocyanins under the acidic condition, and this conformation can benefit the stability of anthocyanins (Março and Scarminio 2007). Kamonpatana et al. (2012) suggested that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota. Bermúdez-Soto et al. (2007) showed that anthocyanins were sensitive to alkaline conditions and digestive enzymes. Yang et al. (2018) explained that, under simulated intestinal condition, the decrease of the total anthocyanin concentration mainly resulted from the degradation of anthocyanins via cleaving their aromatic ring as suggested from previous studies. Furthermore, it has been shown that the concentration of P-3-glucoside significantly decreased under simulated intestinal tract in chokeberry, wild blueberry, pomegranate and red wine (Bermúdez-Soto et al. 2007; Mosele et al. 2015; Yang et al. 2018). Concerning C-3-glucoside, He et al. (2009), suggested that acidic conditions might cause the release of the sugar moiety from its aglycone due to hydrolysis, whereas flavylium cation conformation could stabilize anthocyanins under acidic conditions for the digestion of black raspberry in rat tissues. Yang et al. (2018) speculated that hydrolysis and/or polymeric reaction might take place more rapidly on C-3-glucoside than the formation of its flavylium ion structure, which caused its concentration decrease under the simulated tract at the gastric step.

Fig. 2.

Fig. 2

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Flavylium ion conversion at increasing pH.

Adapted from Zhao and Temelli (2017)

Proanthocyanidin contents

Statistical analyses of the PAC content evolution revealed that as for anthocyanins, there were statistically significant differences between juices at the three digestion steps and with the initial juice and also according to the nature of the PACs (Table 3). Indeed, whatever the concentration and nature of the organic acids present in the cranberry juice, the general trend for monomers, 2–3 mers and 7–10 mers, indicated that there was no real difference between the initial juices and the salivary step. Contrary to 4–6 mers and polymers which indicated significant differences between these two steps with an averaged (all juice averaged) decrease of 16% for the 4–6 mers and 18% for the polymers. However, for all PACs measured, the statistical analysis indicated that there were significant differences between the salivary step and the gastric step. Indeed, whatever the concentration and nature of the organic acids present in the cranberry juice, monomer and 2–3 mer concentrations slightly decreased of about 22% while 4–6 mer, 7–10 mer and polymer concentrations disappeared totally from the gastric step until the intestinal step. Interestingly, the concentration of 2–3 mers increased drastically and significantly at the intestinal phase when the concentration of the four other PACs did not change.

Table 3.

Evolution during digestion of the proanthocyanidin (PAC) contents in non-deacidified cranberry juice and deacidified juices with or without extra content in organic acids and in comparison with their respective initial juice

Initial Salivary Gastric Intestinal
Monomers (mg/L)
 Non-deacidified 25.65 ± 0.95aA 24.06 ± 0.28aA 18.73 ± 0.55bA 19.58 ± 0.88bA
 Deacidified 27.90 ± 0.84aB 25.72 ± 0.51aB 19.09 ± 1.12bA 18.45 ± 1.87bA
 Deacidified + malic acid 26.96 ± 0.61aAB 24.84 ± 0.38bAB 20.01 ± 0.35cA 18.48 ± 1.13cA
 Deacidified + citric acid 27.34 ± 0.94aAB 24.54 ± 0.64bAB 19.70 ± 0.98cA 18.73 ± 0.18cA
 Deacidified + malic and citric acid 27.44 ± 0.73aAB 25.03 ± 0.55aAB 20.19 ± 0.58bA 18.53 ± 2.35bA
 Deacidified + TA malic acid 26.98 ± 0.55aAB 23.77 ± 0.55bAB 19.99 ± 0.45cA 19.89 ± 0.48cA
2–3 mers (mg/L)
 Non-deacidified 89.76 ± 4.54aA 76.37 ± 1.02aA 59.59 ± 6.67bA 416.33 ± 7.76cA
 Deacidified 99.48 ± 3.36aB 82.87 ± 1.29bB 73.58 ± 3.94cB 366.33 ± 2.60dB
 Deacidified + malic acid 96.72 ± 3.30aAB 82.22 ± 0.34bB 80.97 ± 1.61bBC 391.44 ± 9.17cBC
 Deacidified + citric acid 94.81 ± 1.00aAB 83.03 ± 0.91bB 83.56 ± 1.42bC 411.40 ± 2.23cC
 Deacidified + malic and citric acid 95.43 ± 2.47aAB 83.36 ± 1.30bB 82.36 ± 0.50bBC 427.44 ± 4.47cBC
 Deacidified + TA malic acid 91.94 ± 1.77aAB 80.77 ± 1.07bB 81.84 ± 0.62bBC 416.74 ± 3.93cBC
4–6 mers (mg/L)
 Non-deacidified 28.60 ± 1.11aA 23.91 ± 0.34bA 0 ± 0cA 0 ± 0cA
 Deacidified 33.14 ± 1.62aB 28.50 ± 0.53bB 0 ± 0cA 0 ± 0cA
 Deacidified + malic acid 31.91 ± 1.03aBC 28.66 ± 0.25bB 0 ± 0cA 0 ± 0cA
 Deacidified + citric acid 30.75 ± 0.63aABC 28.63 ± 0.48bB 0 ± 0cA 0 ± 0cA
 Deacidified + malic and citric acid 30.84 ± 0.95aABC 28.49 ± 0.58bB 0 ± 0cA 0 ± 0cA
 Deacidified + TA malic acid 29.71 ± 0.32aA 27.57 ± 0.27bB 0 ± 0cA 0 ± 0cA
7–10 mers (mg/L)
 Non-deacidified 3.27 ± 0.63aA 2.54 ± 0.12aA 0 ± 0bA 0 ± 0bA
 Deacidified 3.92 ± 0.22aA 2.99 ± 0.13bB 0 ± 0cA 0 ± 0cA
 Deacidified + malic acid 3.71 ± 0.13aA 2.86 ± 0.15bAB 0 ± 0cA 0 ± 0cA
 Deacidified + citric acid 2.92 ± 0.51aA 2.77 ± 0.07aAB 0 ± 0bA 0 ± 0bA
 Deacidified + malic and citric acid 2.94 ± 0.73aA 2.86 ± 0.22aAB 0 ± 0bA 0 ± 0bA
 Deacidified + TA malic acid 2.96 ± 0.61aA 2.84 ± 0.07aAB 0 ± 0bA 0 ± 0bA
Polymers (mg/L)
 Non-deacidified 7.58 ± 1.13aA 6.22 ± 1.79aA 0 ± 0bA 0 ± 0bA
 Deacidified 6.68 ± 0.14aA 5.07 ± 0.36bA 0 ± 0cA 0 ± 0cA
 Deacidified + malic acid 6.67 ± 0.38aA 5.25 ± 0.32bA 0 ± 0cA 0 ± 0cA
 Deacidified + citric acid 6.52 ± 0.32aA 5.25 ± 0.20bA 0 ± 0cA 0 ± 0cA
 Deacidified + malic and citric acid 6.81 ± 0.15aA 5.26 ± 0.36bA 0 ± 0cA 0 ± 0cA
 Deacidified + TA malic acid 6.35 ± 0.38aA 5.18 ± 0.60bA 0 ± 0cA 0 ± 0cA
Total (mg/L)
 Non-deacidified 154.86 ± 8.13aA 133.10 ± 0.97bA 78.32 ± 6.13cA 435.91 ± 8.63dA
 Deacidified 171.14 ± 6.00aB 145.15 ± 2.15bB 92.67 ± 2.99cB 384.78 ± 3.77dB
 Deacidified + malic acid 165.97 ± 4.16aAB 143.84 ± 0.62bBC 100.98 ± 1.26cC 409.92 ± 9.78dC
 Deacidified + citric acid 162.34 ± 2.35aAB 144.22 ± 1.58bBC 103.26 ± 1.38cC 430.13 ± 2.20dC
 Deacidified + malic and citric acid 163.47 ± 4.76aAB 145.01 ± 2.10bB 102.55 ± 1.08cB 445.97 ± 6.80dC
 Deacidified + TA malic acid 157.95 ± 3.44aAB 140.12 ± 1.31bC 101.83 ± 0.97cC 436.63 ± 4.35dC
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The results on the same column, for each individual PAC, with a different capital letter for the same digestion phase are significantly different at a probability level 0.05. The results on the same line with a different lowercase letter for the same juice are significantly different at a probability level 0.05 (ANOVA) (n = 3)

This decrease could be explained by a rearrangement of the molecules or a change in their conformation into small chains and thus explaining the increase of the content of 2–3 mers in the intestinal phase. Similar results were obtained by Serre et al. (2016a), who showed that 4–6 and 7–10 degree of polymerization groups of PACs disappeared at the end of the digestion but no information was given for the different digestion phases. Results from in vitro and in vivo models demonstrated that PAC oligomers with a degree of polymerization lower than five were absorbable (Déprez et al. 2000). Spencer et al. (2000) showed that oligomers were unstable under conditions of acidic environment of the gastric medium and were decomposed essentially into epicatechin monomeric and dimeric units. Furthermore, they showed that the higher the polymerization index of monomer, the more readily the components were cleaved.

Effect of organic acids of cranberry juice on Caco-2 cells

Transepithelial/transendothelial electrical resistance (TEER) is a very sensitive quantitative technique to measure the integrity and permeability of tight junction dynamics in cell culture models of endothelial and epithelial monolayers (Srinivasan et al. 2015). This measurement method was used to assess the impact of deacidified cranberry juice with and without extra content in organic acids and in comparison with non-deacidified cranberry juice on the Caco-2 epithelial cell monolayer integrity (Fig. 3).

Fig. 3.

Fig. 3

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Effect of different deacidified cranberry juices with or without extra content of organic acids in comparison with a non-deacidified cranberry juice on the variation in TransEpithelial Electrical Resistance (ΔTEER) (Ω cm2) of Caco-2 monolayers (*p < 0.05). HBSS is Hank’s Balanced Salt Solution, non-deacidified juice is the digested non-deacidified cranberry juice, deacidified juice is the digested non-deacidified cranberry juice after organic acid removal by electrodialysis treatment, deacidified + malic acid, deacidified + citric acid and deacidified + malic and citric acid correspond to the deacidified/digested cranberry juice where the concentration lost during organic acid removal treatment of these organic acids is added and deacidified + TA malic acid is the deacidified/digested cranberry juice where the concentration of malic acid at the titratable acidity of the non-deacidified cranberry juice is added. S.E.M corresponds to Standard Error of the Mean (n = 3)

According to the ANOVA results, there were statistically significant differences between the non-deacidified juice and some deacidified juices with or without addition of organic acid, and that, depending on the type of acid. First of all, to ensure the consistency of the trial and as expected for a negative control, incubation with HBSS for 3 h did not affect the ΔTEER (− 32 ± 13 Ω cm2). However, 1% Triton (positive control) severely impacted the integrity of the Caco-2 cells with a ΔTEER of − 861 ± 24 Ω cm2.

Amongst juices, the non-deacidified cranberry juice was taken as a control and was compared to each sample of deacidified cranberry juice with or without extra content of organic acid. ΔTEER of 1% Triton and non-deacidified cranberry juice were not significantly different, which meant that non-deacidified cranberry juice severely modified cell integrity. In contrast, exposure to deacidified cranberry juice implied a 36% decrease of the impact on the Caco-2 cell monolayer integrity. Furthermore, addition of malic acid in the deacidified cranberry juice, and that whatever the level of malic acid added did not impact the cell monolayer integrity as the non-deacidified cranberry juice (p < 0.05). But, addition of citric acid and the combination of malic and citric acids in the deacidified cranberry juice, presented a ΔTEER not significantly different from the non-deacidified cranberry juice, meaning that citric acid might be the organic acid responsible for the loss of integrity of the intestinal cells.

These results confirmed that organic acid removal from cranberry juice reduced negative effects on the Caco-2 cell monolayer and showed for the first time that an addition of malic acid did not impact the integrity of the barrier while citric acid drastically impacted the cell monolayer integrity. Furthermore, studies conducted on tight junctions of epithelial cells have shown a decrease in their TEER when exposed to organic acids such as citric acid (Cho et al. 1989; Daugherty and Mrsny 1999). These results also explained in details and succeeded to identify the organic acid responsible for the global observations reported by Serre et al. (2016a) concerning the fact that a non-deacidified cranberry juice seriously damaged the Caco-2 cell monolayer integrity while a deacidified cranberry juice less impacted it.

Figure 4 is a proposed mechanism of the action of citric acid present in cranberry juice on the opening of tight junction and resulting in chronic inflammatory response. The intestinal barrier is composed of epithelial cells interconnected by tight junction proteins (claudin and occludin) and adherens proteins (cadherin). It has been shown that adherens proteins bind with other cadherin proteins of a neighboring epithelial cell via calcium ions (Ca2+) (Hayashi et al. 1999). Concerning the tight junction proteins, they ensure the tightness of the epithelial, thanks to the presence of Ca2+ which allows the formation of the actin-myosin complex, itself, linked to the ZO-1 and ZO-2 proteins by spectrin (Fig. 4 Step 1). Therefore, calcium is essential for the formation and the maintenance of cellular epithelial junctions and for the integrity and sealing of the epithelial barrier (Noach et al. 1993; Tomita et al. 1996). In addition, at the pH of the intestine (8.1), the organic acids present in the cranberry functional juice are in their anionic form (Serre et al. 2016b) and the anionic form of citric acid is known to be a calcium chelator. Indeed, the structure and conformation of the molecule provide three potential binding sites for Ca2+ and form a stable complex (Cho et al. 1989). In contrast, the ability of malate to bind Ca2+, although its structure presents two binding sites, has never been reported in the literature. Furthermore, Caco-2 cells have organic anion transporters (OATs) inserted into their apical plasma membrane responsible of the transcellular transport. Citrate transport occurs by a combination of Na+-independent pathways, possibly mediated by an OAT, and Na+-dependent mechanisms (Weerachayaphorn and Pajor 2008). Unfortunately, malate transport by OATs has also never been reported in the literature. This would confirm the fact that even at a high concentration (at the titratable acidity of the non-deacidified juice), deacidified cranberry juice with malic acid added did not have a significant effect on the intestinal barrier. Therefore, transcellular transport of citrate into epithelial cells would bring to a decrease of intracellular Ca2+; by chelating Ca2+, citric acid would deprive adherens proteins and tight junction proteins of calcium, increasing paracellular uptake by opening the tight junctions of the epithelium (Fig. 4 Step 2) (Cho et al. 1989; Froment et al. 1989; Nolan et al. 1990). The opening of epithelial cells would result in the transport of microorganisms through the paracellular pathway, thus leading to a chronic inflammatory response and also a decrease in the integrity of the intestinal barrier (Fig. 4 Step 3).

Fig. 4.

Fig. 4

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Proposed mechanism of citric acid present in cranberry juice on the opening of tight junctions and resulting chronic inflammatory response. Step 1: intestinal cell barrier with a normal integrity, step 2: disruption of tight junction complexes, via depletion of intracellular calcium following the transport of citric acid and step 3: paracellular absorption in the epithelium through opened tight junctions and resulting chronic inflammatory response

Conclusion

It appeared from these results that the main organic acid, in cranberry juice, responsible for the loss of integrity of Caco-2 epithelial cells was citric acid. Indeed, the anionic structure and conformation of the molecule, at the pH of the intestine, provide three potential binding sites for Ca2+ to form a stable complex. In contrast, the ability of malate to bind Ca2+, although its structure presents two binding sites, was not demonstrated. This would confirm the fact that even at a high concentration (at the titratable acidity of the non-deacidified juice), deacidified cranberry juice with malic acid added did not have a significant effect on the intestinal barrier. However, no effect of the digestion process was observed on the concentration of organic acids present in cranberry juice while a decrease in anthocyanins and PACs (with a degree of polymerization higher than three) contents appeared mainly at the gastric step and that whatever the concentration and nature of the organic acids initially present in the cranberry juice. This decrease observed at this stage is necessary to ensure the bioavailability of these phenolic compounds during the consumption of cranberry juice.

To our knowledge, this is the first time that the evolution of organic acids and polyphenolic compounds present in a non-deacidified cranberry juice have been studied during the different steps of the digestion and this according to the concentration and nature of the organic acids present in the cranberry juice. Furthermore, it is also the first time that citric acid was identified for having the main impact on the loss of integrity of Caco-2 epithelial cells when consuming such a juice with high titratable acidity. The market for such a new functional juice deacidified by electrodialysis with bipolar membrane is estimated at more than 1.0 million dollars per year. Based on these convincing results, the next step actually underway in our team, is an in vivo study to demonstrate a decrease of the loss of integrity of the enterocytes lining the small intestine when deacidified cranberry juice is consumed.

Acknowledgements

The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) is acknowledged. This work was supported by the NSERC Industrial Research Chair on Electromembrane processes aiming the ecoefficiency improvement of biofood production lines (Grant IRCPJ 492889-15 to Laurent Bazinet). The authors thank Mrs. Véronique Richard (Institute of Nutrition and Functional Foods (INAF)) for technical assistance with HPLC analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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