The Gliadin Hydrolysis Capacity of B. longum, L. acidophilus, and L. plantarum and Their Protective Effects on Caco-2 Cells against Gliadin-Induced Inflammatory Responses

Background: Non-celiac wheat sensitivity (NCWS) is a poorly understood gluten-related disorder (GRD) and its prominent symptoms can be ameliorated by gluten avoidance. This study aimed to determine the effectiveness of a probiotic mixture in hydrolyzing gliadin peptides (toxic components of gluten) and suppressing gliadin-induced inflammatory responses in Caco-2 cells. Methods: Wheat dough was fermented with a probiotic mix for 0, 2, 4, and 6 h. The effect of the probiotic mix on gliadin degradation was monitored by SDS-PAGE. The expression levels of IL-6, IL-17A, INF-γ, IL-10, and TGF-β were evaluated using ELISA and qRT-PCR methods. Results: According to our findings, fermenting wheat dough with a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was effective in gliadin degradation. This process also reduced levels of IL-6 (p = 0.004), IL-17A (p = 0.004), and IFN-γ (p = 0.01) mRNA, as well as decreased IL-6 (p = 0.006) and IFN-γ (p = 0.0009) protein secretion. 4 h fermentation led to a significant decrease in IL-17A (p = 0.001) and IFN-γ (p = 0.003) mRNA, as well as reduced levels of IL-6 (p = 0.002) and IFN-γ (p < 0.0001) protein secretion. This process was also observed to increase the expression levels of IL-10 (p < 0.0001) and TGF-β (p < 0.0001) mRNA. Conclusions: 4 h fermentation of wheat flour with the proposed probiotic mix might be a good strategy to develop an affordable gluten-free wheat dough for NCWS and probably other GRD patients.


Introduction
Gluten-related disorders (GRDs), an "umbrella" definition encompassing several diseases, including non-celiac wheat sensitivity (NCWS), are becoming increasingly frequent. Gluten is a protein present in wheat, barley, rye, and occasionally in other grains, that is responsible for the elasticity and pliability of dough, making it an essential ingredient in effect of gliadin-derived peptides. It has been extensively utilized in previous GRD-related studies to help determine the pathogenesis of these diseases [48][49][50].
The current study therefore aimed to investigate the enzymatic hydrolysis activity of Bifidobacterium Longum, Lactobacillus acidophilus, and Lactiplantibacillus plantarum on gliadin peptides of wheat flour and their potential effect on gliadin-related inflammatory responses in the Caco-2 cell line.

Materials and Methods
Investigations were carried out at the Research Institute for Gastroenterology and Liver Diseases (RIGLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Data validation was carried out at the Department of Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE), University of Palermo, Palermo, Italy.

Probiotic Preparations
All three lyophilized probiotics, including Bifidobacterium longum (ATCC 15708), Lactobacillus acidophilus (ATCC 4356) and Lactiplantibacillus plantarum (ATCC 8014), were purchased from Takgene Group Company (Tehran, Iran) and used for dough fermentation. The bacteria were cultured in an MRS (De Man, Rogosa and Sharpe) medium supplemented with 0.05% (v/v) L-cysteine at 37 • C for 24-48 h in anaerobic conditions. The strains were kept at −80 • C in MRS broth plus L-cysteine and 50% glycerol.

Sourdough Fermentation
The wheat grain (Mehregan cultivar) with a gluten content of 27.3% was provided by the Agricultural Research, Education, and Extension Organization (AREEO), Karaj, Iran. For the preparation of the sourdough, 80 g of wheat flour was mixed with 190 mL of tap water to obtain 270 g of sourdough. The resulting dough was then divided into two 135 g pieces. The first one was mixed with a combination of the three bacteria, at a cell concentration of ca. 10 8 CFU per g of dough. The second was used without probiotics as a negative control. Both dough pieces were incubated at 37 • C during stirring at 200 rpm. The capacity of B. longum, L. plantarum, and L. acidophilus to hydrolyze gluten was evaluated at 0, 2, 4, and 6 h of incubation.

Gliadin Extract
For gliadin extraction, approximatley 10 g of each dough sample was incubated with 50 mM Tris-HCl buffer (pH = 8.8) for 60 min at 4 • C followed by vortexing and centrifuging at 20,000× g for 20 min. After removing the supernatant that contained albumin and globulin, the pellet was extracted twice with Tris-HCl buffer (pH = 8.8), and the supernatant was discarded. The pellet was then mixed with 40 mL of 70% v/v ethanol at 25 • C for 2 h. The supernatant containing gliadin was stored at −80 • C. The concentration of the extracted gliadin was determined using a bicinchoninic acid (BCA) protein assay kit (DNAbiotech, Tehran, Iran).

SDS-PAGE Electrophoresis of Gliadins
Gliadin degradation after 0, 2, 4, and 6 h of incubation with a mix of B. longum, L. acidophilous, and L. plantarum was assessed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A mini SDS-PAGE (Bio-Rad Laboratories GmbH, Munich, Germany) was used, separation was performed using the classical Laemmli method [51], and the bands were stained with Coomassie Blue.

In Vitro Digestion of Gliadins
A pepsin/trypsin (PT) digest of extracted gliadins (to mimic normal intestinal degradation) was prepared to be exposed to the human colon carcinoma Caco-2 cell line, as follows: 120 mg of extracted gliadin was dissolved in 20 mL of sodium acetate buffer (50 mM, pH  4). The sample was incubated at 37 • C, with agitation for 2 h after the addition of pepsin (3200 U/mg, P-6887, Sigma-Aldrich, St. Louis, MO, USA) to the mixture. After incubation, 142 mg of sodium phosphate was added, and the pH was brought to 7.0 using a 1 M NaOH solution. The mixture was treated with trypsin (2500 U/mg, T-7418, Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 • C with agitation for another 2 h. The obtained solution was heated at a temperature greater than 95 • C for 10 min to stop the enzymatic reaction. Finally, the digested solution was lyophilized and stored at −20 • C.
Untreated Caco-2 cells were used as negative controls to assess the base levels of cytokines secretion. LPS-treated Caco-2 cells were used as positive control to assess the levels of cytokines secretion under inflammatory conditions. 2.7. Evaluating B. longum, L. acidophilous, and L. plantarum Effects on the Inflammatory Effects of Gliadin Peptides in Intestinal Epithelial (Caco-2) Cells 24 h after seeding, cells were incubated with 1 mg/mL of PT-digest of gliadins (extracted from 0, 2, 4, and 6 h probiotic fermented and unfermented doughs) [52], 1 µg/mL of LPS (Lipopolysaccharides, as a pro-inflammatory response triggering control) [53], and B. longum, L. acidophilous, and L. plantarum (alone in a mix) (Multiplicity of Infection (MOI) = 100), and the incubation was continued for 24 h. Following incubation, cells and supernatants were collected and analyzed for the production of pro-and anti-inflammatory cytokines.

Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)
To evaluate IL-6 and INF-γ production as proinflammatory cytokines, the supernatant was obtained, and protein concentrations were determined using the enzyme-linked immunosorbent assay (ELISA) technique (Karmania Pars Gene, Rafsanjan, Iran) according to the manufacturer's instructions. Triplicate results were read at 450 nm by an ELISA reader (TECAN, Salzburg, Austria).

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
A YTA Total RNA Purification mini kit for the Blood/Cultured Cell/Tissue kit (Yekta Tajhiz Azma, Tehran, Iran) was used to extract the total RNA from Caco-2 cells. The NanoDrop-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA) was used to measure the concentration and purity of the extracted RNA. Reverse transcription was performed using the 2 step 2X qRT-PCR Premix (Taq) kit (BioFact™, Daejeon, Republic of Korea), and to investigate the expression levels of IL-6, INF-γ, IL-17, IL-10, and TGF-β as pro-and anti-inflammatory cytokines, a SYBR Green-based quantitative real-time PCR assay was performed using the Rotor-Gene Q MDx. Gene Runner software version 6.0 was used to design the primer sequences and their specificity was checked by NCBI Primer-BLAST and performing PCR experiments followed by 1.5% agarose gel electrophoresis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference housekeeping gene and relative quantitation (RQ) for each gene expression calculated by the 2 −∆∆Ct method.

Statistical Analysis
Data were analyzed using Prism 6.04 for Windows software (GraphPad, La Jolla, CA, USA). Data were expressed as mean ± standard deviation (SD), and one-way ANOVA were used to evaluate differences in means between groups. p values below 0.05 were deemed statistically significant.

Ethical Approval
This article does not contain any studies with human or animal subjects and was approved by the ethics committee of the Research Institute for Gastroenterology and Liver Diseases Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.RIGLD.REC.1397.010).

Gliadin Hydrolysis by Selected Probiotics
According to the SDS-PAGE profiles of the gliadin polypeptides, a 6 h fermentation of dough with a mix of B. longum, L. acidophilous, and L. plantarum showed the highest gliadin hydrolysis capacity ( Figure 1).

Statistical Analysis
Data were analyzed using Prism 6.04 for Windows software (GraphPad, La Jolla, CA, USA). Data were expressed as mean ± standard deviation (SD), and one-way ANOVA were used to evaluate differences in means between groups. p values below 0.05 were deemed statistically significant.

Ethical Approval
This article does not contain any studies with human or animal subjects and was approved by the ethics committee of the Research Institute for Gastroenterology and Liver Diseases Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.RIGLD.REC.1397.010).

Gliadin Hydrolysis by Selected Probiotics
According to the SDS-PAGE profiles of the gliadin polypeptides, a 6 h fermentation of dough with a mix of B. longum, L. acidophilous, and L. plantarum showed the highest gliadin hydrolysis capacity ( Figure 1).

IL-6 and INF-γ Protein Expression
Compared to the untreated Caco-2 cells (negative controls), the Caco-2 cells that were treated for 24 h with LPS (positive controls) and non-fermented-dough-extracted PT-gliadin exhibited significantly higher levels of IL-6 (p < 0.0001 in both cases) and IFN-γ (p = 0.003 and 0.002, respectively). No significant difference in the secretion of the two proinflammatory cytokines was observed between LPS and non-fermented dough-extracted PT-gliadin treated Caco-2 cells, as well as between untreated Caco-2 cells and those treated with B. longum, L. acidophilus, and L. plantarum, alone or in a mix (p > 0.05) ( Figure  2).

IL-6 and INF-γ Protein Expression
Compared to the untreated Caco-2 cells (negative controls), the Caco-2 cells that were treated for 24 h with LPS (positive controls) and non-fermented-dough-extracted PT-gliadin exhibited significantly higher levels of IL-6 (p < 0.0001 in both cases) and IFN-γ (p = 0.003 and 0.002, respectively). No significant difference in the secretion of the two proinflammatory cytokines was observed between LPS and non-fermented dough-extracted PT-gliadin treated Caco-2 cells, as well as between untreated Caco-2 cells and those treated with B. longum, L. acidophilus, and L. plantarum, alone or in a mix (p > 0.05) (Figure 2).

Discussion
Sourdough fermentation is a traditional process for bread leavening that may markedly reduce wheat allergens and break down gluten, but it does not eliminate gluten proteins completely [43,54]. Therefore, researchers are trying to find novel biotechnological strategies based on using probiotics to make almost totally gluten-free products more accessible [55]. Probiotics have been shown to be an excellent source of endopeptidases, which can reduce gluten content in wheat foods and seem to be the strongest candidates for improving the quality of gluten-free foods [42]. In this regard, it has been reported that "ancient" wheats, which partially differ in composition from "modern" wheat species, seem to be better tolerated (lower rate of symptom onset, better palatability, and lower cost than gluten-free products) by a subset of NCWS patients [56,57]. As Bifidobacteria and Lactobacilli strains are known to be the most common probiotics used in foods and supplements, we evaluated the effect of a probiotic mix containing B. longum, L. acidophilous, A 4 h fermentation with these probiotics considerably decreased IL-17A (p = 0.001) and IFN-γ (p = 0.003), increased IL-10 (p < 0.0001) and TGF-β (p < 0.0001), and reduced (not statistically significant) IL-6 (p > 0.05) mRNA levels compared to the non-fermented dough-extracted PT-gliadin-incubated Caco-2 cells.

Discussion
Sourdough fermentation is a traditional process for bread leavening that may markedly reduce wheat allergens and break down gluten, but it does not eliminate gluten proteins completely [43,54]. Therefore, researchers are trying to find novel biotechnological strategies based on using probiotics to make almost totally gluten-free products more accessible [55]. Probiotics have been shown to be an excellent source of endopeptidases, which can reduce gluten content in wheat foods and seem to be the strongest candidates for improving the quality of gluten-free foods [42]. In this regard, it has been reported that "ancient" wheats, which partially differ in composition from "modern" wheat species, seem to be better tolerated (lower rate of symptom onset, better palatability, and lower cost than gluten-free products) by a subset of NCWS patients [56,57]. As Bifidobacteria and Lactobacilli strains are known to be the most common probiotics used in foods and supplements, we evaluated the effect of a probiotic mix containing B. longum, L. acidophilous, and L. plantarum on degrading gliadin peptides of wheat flour dough and reducing their inflammatory effects in a Caco-2 cell line. Similar to previous in vitro studies, we observed the inflammatory effect of peptic-tryptic digested gliadin peptides (extracted from non-fermented dough) on the Caco-2 intestinal cells [58,59]. Our results also showed the capacity of B. longum, L. acidophilous, and L. plantarum to reduce pro-inflammatory and increase anti-inflammatory cytokine expression by these cells. Álvarez-Mercado et al., in a recent study, observed how B. longum subsp. Infantis reduced the secretion of pro-inflammatory cytokines by Caco-2 cells [59]. Another study, conducted by Zhao et al., reported that B. longum subsp. longum K5 produced a decrease in the levels of pro-inflammatory cytokines and inflammatory mediators in RAW 264.7 and Caco-2 cells stimulated with LPS [60]. The inflammatory regulation role of L. acidophilus has also been observed in human intestinal epithelial cells [61] and human MKN45 cells [62]. An attenuating effect of L. plantarum MYL26 on inflammation in Caco-2 cells was also reported by Chiu et al. [63].
Scricciolo et al. [64], in a recent randomized, double-blind, placebo-controlled monocentric study, did not observe any effectiveness of using a proline-specific endopeptidase (P1016) to improve NCWS patients' symptoms following gluten reintroduction. On the contrary, in a study conducted by Ido et al. [65], an enzyme mix sourced from microorganisms and papaya was found to effectively reduce symptoms in NCWS patients undergoing a gluten challenge. In detail, the authors used a mixture of peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively. A group of thirty patients diagnosed with NCWS were randomly assigned to a two-week single-blind challenge with gluten plus either the enzyme mix or placebo. The patients then underwent a crossover phase after a GFD wash-out period. The authors observed a significant difference in the overall symptom score between post-washout and post-gluten challenge stages for the enzyme mixture and placebo groups (p < 0.05). However, there were no noticeable differences in serum levels of IL-8, TNF-α, or RANTES after the consumption of gluten, gluten with the enzyme mix, or gluten with a placebo [65].
In our study, we demonstrated the desirable effects of probiotics for producing easily available gluten-free wheat flour. The fermentation of wheat dough using a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was found to be effective in gliadin degradation and decreased IL-6, IL-17A, and IFN-γ mRNA levels, as well as IL-6 and IFN-γ protein secretion. However, a 4 h fermentation with this mix might be the optimal protocol for making glutenfree wheat flour dough suitable for patients with GRD. This is due to the strong ability of the 4 h fermentation to decrease pro-inflammatory cytokine mRNA expression (IL-17A and IFN-γ) or protein secretion (IL-6 and IFN-γ), and at the same time, increase antiinflammatory cytokine mRNA levels (IL-10 and TGF-β). These results agree with those of previous studies reporting protective effects of certain probiotic strains against some gliadinmediated inflammatory responses. In a previous study, Enterococcus mundtii QAUSD01 and Wickerhamomyces anomalus QAUWA03 were reported to be able to degrade gliadin in various wheat varieties [66]. Probiotic VSL#3 (Ferring Pharamaceuticals, Milan, Italy), containing Streptococcus thermophilus, L. plantarum, L. acidophilus, L. casei, L. delbrueckii spp. bulgaricus, B. breve, B. longum, and B. infantis, has also been reported to decrease the toxicity of wheat flour following a 24 h fermentation [67]. In addition, Leszczyn'ska et al. reported that a 24 h fermentation of wheat flour with L. plantarum, L. brevis, L. sanfranciscencis, and bakery yeasts (Saccharomyces cerevisiae) lowered the allergenicity of wheat flour [68].
The optimal therapeutic approach for patients with NCWS remains a topic of ongoing debate among researchers [28][29][30][31]. This debate is attributable to the lack of a univocal understanding of the etiopathogenesis of NCWS. Research groups hold conflicting views, often citing gluten, WGAs, ATIs, or FODMAPs as the sole cause behind clinical manifestations in NCWS patients [26,[29][30][31]69]. On the basis of the current understanding of this GRD, NCWS should be viewed as a complex entity, with several subtypes of patients. Multiple studies have shown that some individuals with NCWS experience symptom improvement or remission by adhering to a GFD, and their symptoms worsen during double-blind placebo-controlled (DBPC) challenges with gluten [14,29,[70][71][72]. Contrary to this, some studies suggest that simply reintroducing gluten is not enough to re-trigger symptoms. Instead, it is believed that other components present in wheat could also play a role [30,31,69]. To date, no study has defined whether there is a tolerability threshold for these components of wheat. Moreover, the limited availability of comprehensive long-term research studies does not permit any definite conclusions as to whether NCWS is a persistent condition. Consequently, it remains uncertain if patients can regain wheat tolerance after years of avoiding the triggering stimuli.
Our study allowed us to identify a mix of probiotics easily available on the market, which, by digesting gliadin peptides in a relatively short time (4 h), are able to reduce the inflammatory and increase the anti-inflammatory response in an in vitro model with Caco-2 cells. Its most promising use could be as a starter to ferment the dough of wheat, creating a tolerable, palatable, easily accessible, and low-cost product, which could be potentially tolerated by a subgroup of NCWS subjects, as well as other GRD patients and at the same time prevent the onset of nutritional deficiencies.
The primary limitation of our study is its exclusive reliance on in vitro data. The potential tolerance of wheat dough fermented using our probiotic mix needs to be verified in appropriately designed in vivo studies. Another limitation is that we assessed the effectiveness of the probiotic mix solely on the Mehregan cultivar of wheat. As a result, we cannot rule out potential variations in the efficacy of the mix when applied to other types of wheat, with different percentages of gliadin peptides and compositions. To address these limitations, we have initiated plans for a multicenter study. This new study aims to test the effectiveness of the probiotic mixture in gliadin degradation and tolerance in NCWS patients using wheat dough obtained from various Italian and Iranian cultivars, including both modern and ancient varieties, and through both in vitro and in vivo assessments.

Conclusions
Adhering to a GFD can be extremely challenging, particularly due to the limited availability of gluten-free food options. Therefore, finding a probiotic mixture that can help make gluten-free wheat flour available in a short time could represent a significant advancement towards enhancing the quality of life for the countless individuals worldwide grappling with gluten-related disorders. According to our findings, a 4 h fermentation of wheat flour with a probiotic mixture containing B. longum, L. acidophilus, and L. plantarum (ca. 10 8 CFU per g of dough) might be a good strategy for developing an affordable glutenfree wheat dough, which can be used to prepare different food items for these patients. However, our understanding of the exact molecular mechanisms underlying the action of these probiotics is currently limited and requires further investigation. Funding: The study was funded by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran (Grant no: 24221) and also the Italian Health Ministry, Grant PE-2016-02363692 "Non-celiac gluten sensitivity (NCGS): is gluten the true culprit? A clinical and immunological study on the tolerability of different wheat grains in NCGS patients". The study sponsor had no role in the study design, in the collection, analysis, and interpretation of the data, in writing the manuscript, nor in the decision to submit the paper for publication.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.