Skip to Content
MDPIMDPI

Advanced

FoodsFoods
PDF
  • Article
  • Open Access

6 March 2026

12 Pages

Novel Food Supplement Containing a Combination of Postbiotics and Plant-Derived Compounds Regulates Epithelial Barrier Integrity and Immune Response in Human Enterocytes

,
,
,
,
,
,
,
and
1
Department of Translational Medical Science, University Federico II, 80131 Naples, Italy
2
NutriTechLab at CEINGE Advanced Biotechnologies, University Federico II, 80131 Naples, Italy
*
Author to whom correspondence should be addressed.
Foods2026, 15(5), 922;https://doi.org/10.3390/foods15050922
This article belongs to the Section Nutraceuticals, Functional Foods, and Novel Foods
Version Notes
Review Reports
Order Reprints

Abstract

Background: Alterations of epithelial barrier integrity and immune response play a key role in the pathogenesis of allergic diseases and represent promising targets for nutritional interventions. Selected postbiotics and plant-derived compounds have been proposed as potential modulators of epithelial barrier and immune function. Methods: We investigated the effects of a novel food supplement combining heat-inactivated Lacticaseibacillus rhamnosus GG, butyrate, Quercetin, and Perilla frutescens extracts on epithelial barrier function and innate immunity in an experimental model of the human epithelial gut barrier. Results: Exposure to the food supplement resulted in epithelial barrier integrity enhancement, consisting of increased transepithelial electrical resistance, tight-junction protein expression, mucus production, and enterocyte differentiation. Moreover, the formulation markedly stimulated the expression of the innate immunity peptides β-defensin-2 and cathelecidin LL-37. Conclusions: The novel food supplement induces a beneficial modulation of the epithelial gut barrier and immune response. These findings support its potential use as a functional food strategy to restore mucosal homeostasis and to promote immune tolerance in allergic diseases.

1. Introduction

The prevalence, incidence and severity of allergic diseases are increasing worldwide [1,2,3,4]. Recent estimates suggest that food allergies now affect up to 10% of the population in Westernized countries, with a dramatic increase in incidence, particularly among pediatric populations, over the last twenty years [2,3,4]. Alterations of the epithelial barrier and immune tolerance play a pivotal role in the pathogenesis of allergic diseases and could represent targets of intervention to limit the disease burden [5,6]. Selected postbiotics and plant-based dietary compounds have been suggested as potential remedies against allergy [7].
The intestinal barrier represents the largest interface between the external environment and the host’s internal milieu. Its integrity is maintained by a complex system comprising a physical barrier (the mucus layer and tight-junction proteins), a biochemical barrier (antimicrobial peptides), and an immune barrier (organized lymphoid tissue). A breakdown in this delicate balance, often referred to as ‘leaky gut,’ allows for the paracellular translocation of luminal antigens and allergens into the subepithelial space. This process triggers a cascade of pro-inflammatory responses and immune dysregulation, which are fundamental drivers in the development of allergic sensitization [8,9]. The gut microbiome is a key regulator of the mucosal barrier and immune tolerance mechanisms [10,11,12,13]. Many of these effects are mediated by ‘postbiotic’ molecules such gut microbiome-derived metabolites and healthy bacteria cellular components [8,10,11,12,13]. The human microbiome-derived probiotic Lacticaseibacillus rhamnosus GG (LGG) is one of the most studied microorganisms for allergies, with demonstrated ability in modulating epithelial barrier integrity and immune tolerance [9,10,14,15,16,17,18,19]. We have recently demonstrated that the heat-inactivated LGG postbiotic has the potential to exert protective properties against allergies in the same way as living probiotics [7].
The short-chain fatty acid butyrate is one of the best-characterized gut microbiome-derived metabolites with anti-allergic properties, mainly mediated through a regulation of the mucosal barrier and immune tolerance [14]. Specifically, butyrate acts as a critical energy source for enterocytes and promotes the assembly of tight-junction proteins, such as zonula occludens-1 (ZO-1) and occludin, thereby reducing paracellular permeability [14]. Furthermore, it exerts significant immunomodulatory effects by inducing the differentiation of regulatory T cells and enhancing the production of antimicrobial peptides, which are essential for maintaining intestinal homeostasis [14,20].
Quercetin and Perilla frutescens are among the most extensively studied plant-based compounds with anti-allergic and epithelial gut barrier-protective properties [21].
The Postbiotics and Plant-derived compounds against Allergy (PPA) project was conceived to test the hypothesis that a combined formulation could be effective in modulating the epithelial gut barrier and immune function.

2. Materials and Methods

2.1. Study Design and Experimental Workflow

In this preclinical study we investigated the effect of a novel food supplement composed of two postbiotic components (heat-inactivated LGG and butyrate) and two plant-based dietary compounds (Quercetin and Perilla frutescens extracts) on epithelial gut barrier and immune function. These effects may lead to beneficial clinical outcomes for patients with allergic diseases.
The experimental workflow was the following:
For all experiments, we used a well validated model of epithelial gut barrier based on Caco-2 cell monolayers (American Type Culture Collection, Middlesex, UK; accession number: HTB-37) [3,7,14].
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was conducted to evaluate the potential cytotoxic effects of the novel food supplement at various concentrations.
The effects on epithelial gut barrier integrity were evaluated by measuring transepithelial electrical resistance (TEER); tight-junction proteins, Mucin-2 (Muc-2) and lactase expression.
Lastly, the effects on immune function were evaluated assessing the production of two major innate immunity peptides, human-β-defensin-2 (HBD-2) and cathelecidin LL-37 (LL-37), by human enterocytes.

2.2. Study Product and Component Sources

The composition of the study product is reported in Table 1. The study product was provided as a powder by AMP Biotech (Benevento, Italy). All components of the study product were food-grade.
Table 1. The composition of the study product.
The LGG postbiotic was obtained as follows. Briefly, LGG (ATCC 53103; 5 × 109 CFU) was cultured anaerobically under sterile conditions to prevent contamination. Bacteria were cultured in 4 L of MRS broth, containing (per liter): 10 g peptone, 8 g meat extract, 4 g yeast extract, 20 g glucose, 1 mL Tween 80, 2 g K_2HPO_4, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g MgSO4 · 7H2O, and 0.05 g MnSO4 · 4H2O. Cells were harvested by centrifugation at 7000 rpm for 15 min at 4 °C, yielding a pellet of 5.43 g. Heat inactivation was performed by suspending the pellet in 110 mL of sterile water and autoclaving at 80 °C for 20 min. The absence of viable bacteria was confirmed by resuspending the pellet in MRS broth, plating serial dilutions (10−1 to 10−7) onto MRS agar, and incubating for 48 h at 37 °C; no colonies were detected. Following inactivation, the bacterial suspension was centrifuged at 8000 rpm for 10 min at 4 °C, and the resulting pellet—corresponding to the heat-inactivated LGG postbiotic—was washed twice with sterile water and stored at −80 °C.

2.3. Human Enterocytes Cell Line

The human enterocyte cell line, the Caco-2 cell, was acquired by American Type Culture Collection, Middlesex, UK (accession number: HTB-37). Experiments were conducting using cells between passage 20 and 30 to ensure phenotypic stability and consistency in barrier function, as it has been reported that late passages (e.g., above passage 60) can lead to alterations in proliferation rates and the expression of differentiation markers [22]. The Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, 4.5 g/L; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Euroclone, Milan, Italy), 1% non-essential amino acids (NEAA; Gibco), 1% sodium pyruvate (Gibco), and 1% penicillin–streptomycin (100 U/mL and 100 μg/mL, respectively; Gibco). Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2, and the medium was refreshed every other day.

2.4. MTT Cytotoxicity Assay

Cell viability in response to different concentrations of the study product was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, a standard colorimetric method that evaluates mitochondrial metabolic activity as a proxy for cell survival. Differentiated Caco-2 cells were seeded into 96-well culture plates at density of 1 × 104 cells/well in 100 µL of complete DMEM medium and allowed to adhere. After 24 h, cells were exposed to the study product at final concentrations of 0.1, 1, 10, 100, or 1000 μg/mL for either 48 h or 4 days at 37 °C. All test solutions were prepared by serial dilution in culture medium to maintain physiological pH and osmotic balance. After the incubation period, 10 μL of MTT solution (5 mg/mL in DMEM) was added to each well and the cells were incubated for an additional 2 h under standard culture conditions to allow for the formation of formazan crystals. The supernatant was then discarded, and the resulting formazan crystals were solubilized in 100 μL of dimethyl sulfoxide (DMSO) per well under gentle agitation. The absorbance was recorded at 490 nm using a microplate spectrophotometer (Thermo Fisher, Multiskan FC microplate photometer). Untreated cells maintained in complete medium were used as the control condition. The data were collected from three independent experiments, each performed with five technical replicates. The results were initially recorded as Optical Density (OD) units and subsequently converted to a percentage of cell viability relative to the control group to facilitate comparison across different experimental runs.

2.5. Transepithelial Electrical Resistance (TEER) Measurement

The integrity of the differentiated enterocyte monolayers was assessed by measuring TEER, a widely accepted non-invasive method to evaluate the formation of tight-junction proteins and barrier function in cell cultures. Caco-2 cells were seeded at a density of 2 × 105 cells per well on polycarbonate Transwell® inserts (6-well format; Corning, Life Science, Kennebunk, ME, USA) and cultured until 21 days post-confluence to allow complete differentiation into a polarized epithelium with mature brush-border features. Before measurement, the cell culture plates were allowed to equilibrate at room temperature (approximately 23–25 °C) for 20 min to minimize temperature-dependent fluctuations in resistance. The TEER was recorded at 24 h intervals over a 72 h period using an epithelial volt–ohm meter (Millicell-ERS-2, Millipore, Billerica, MA, USA) equipped with chopstick electrodes. To ensure accuracy, three independent measurements were taken at different positions within each insert and averaged. The resistance values (Ω) were corrected by subtracting the resistance of the blank inserts and then multipling by the membrane surface area to obtain TEER values in Ω·cm2, calculated as: (measured resistance − blank resistance) × surface area (cm2).

2.6. Stimulation of Human Enterocytes

Caco-2 cells were seeded at a density of 3 × 105 cells per well in six-well plates and allowed to reach full differentiation, which occurred 15 days after confluence, ensuring the development of a stable and polarized epithelial monolayer. The Monolayers were then treated with the study product. The PPA supplement was initially reconstituted in sterile phosphate-buffered saline (PBS) to obtain a concentrated stock solution, which was then vortexed and filtered through a 0.22 μm syringe filter to ensure sterility. This stock was subsequently diluted in complete culture medium to reach a final concentration of 10 μg/mL. The treatment was applied for 48 h at a total volume of 2 mL per well. The chosen concentration and incubation time were established based on preliminary dose–response and time–course experiments. Fully differentiated Caco-2 cell monolayers maintained in complete culture medium under identical experimental conditions but not exposed to the study product served as untreated controls (NT). At the end of the 48 h incubation, the culture supernatants were carefully collected from each well, centrifuged at 1000× g for 10 min at 4 °C to remove any cellular debris, and then stored at −20 °C until further analysis. Each experimental condition was performed in duplicate and repeated in five independent experiments to ensure statistical robustness.

2.7. Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from stimulated Caco-2 cells using TRIzol reagent (Gibco BRL, Paisley, UK), following a standardized acid-guanidinium thiocyanate-phenol-chloroform extraction protocol. The RNA concentration and purity were assessed with a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA) by evaluating the A260/280 and A260/230 absorbance ratios; only samples with an A260/280 ratio between 1.8 and 2.1 were considered for downstream analysis. Reverse transcription was performed using the High-Capacity RNA-to-cDNA™ Kit (Life Technologies, Waltham, MA, USA) starting from 1 μg of total RNA per sample. The reaction followed the manufacturer’s instructions in a total volume of 20 μL, and the resulting cDNA was stored at −80 °C until further analysis. Gene expression was analyzed by quantitative real-time PCR using a TaqMan Gene Expression Master Mix (Applied Biosystems, Vilnius, Lithuania) to assess the expression of occludin (Hs05465837_g1), ZO-1 (Hs01551871_m1), HBD-2 (Hs00823638_m1) and LL-37 (Hs05006792_s1). Each TaqMan reaction was carried out in a total volume of 10 μL, containing 5 μL of Master Mix, 0.5 μL of the specific probe, and 2 μL of cDNA template. The expression of mucin 2 (Muc2), a key component of the intestinal mucus layer, and lactase, a marker of enterocyte differentiation, was evaluated using SYBR Green Master Mix (Applied Biosystems, Grand Island, NY, USA). For Muc2, the forward primer was 5′-CTCCGCATGAGTGTGAGT-3′ and the reverse primer was 5′-TAGCAGCCACACTTGTCTG-3′. For lactase, the forward primer was 5′-ACACGGTCGATTTCCTCTCT-3′ and the reverse primer was 5′-TGGGTTCTTCATGGTGGAGG-3′. The specificity of the SYBR Green primers was validated by a melting curve analysis performed at the end of each PCR run, showing a single peak for each target gene. Amplification reactions were carried out using a Light Cycler 7900HT (Applied Biosystems) with the following cycling conditions: an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and elongation at 60 °C for 60 s in the Light Cycler 7900HT (Applied Biosystem, Grand Island, NY, USA). All samples were analyzed in triplicate to minimize technical variability. The relative gene expression levels were calculated using the comparative threshold cycle (ΔΔCt) method. β-glucuronidase (GUSB) was used as the housekeeping gene for normalization due to its stable expression in Caco-2 cells under our experimental conditions. GUSB expression was assessed using a TaqMan probe (Hs00939627_m1) and, for SYBR Green analysis, with the forward primer 5′-GAAAATATGTGGTTGGAGAGCTCATT-3′ and the reverse primer 5′-CCGAGTGAAGATCCCCTTTTTA-3′.

2.8. Statistical Analysis

The Kolmogorov–Smirnov test was used to determine whether the continuous variables were normally distributed. Variables following a normal distribution were reported as mean and standard deviation (SD) or standard error of the mean (SEM), depending on the experimental design. According to the data distribution, differences between the two groups were compared using a paired Student’s t-test or the Mann–Whitney U test. For experiments involving multiple comparisons, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test to assess significant differences between specific treatment groups and the control. The level of significance for all statistical tests was two-sided, and a p-value < 0.05 was considered statistically significant (p < 0.05, ** p < 0.01, *** p < 0.001). All analyses were performed using SPSS for Windows (SPSS Inc, version 23.0, Chicago, IL, USA) and GraphPad Prism 10 (La Jolla, CA, USA). Sample sizes and the specific statistical test used for each experiment are detailed in the respective figure legends to ensure transparency in data interpretation.

3. Results

3.1. Modulation of Gut Epithelial Barrier

Preliminary MTT experiments demonstrated that the novel food supplement was well tolerated by human enterocytes. Cells were exposed to a wide range of concentrations (0.1 to 1000 μg/mL) for 48 h and 4 days. The results demonstrated that the PPA formulation was well tolerated, with no significant reduction in cell viability observed at concentrations up to 100 μg/mL (viability > 95% vs. NT control). A significant decrease in metabolic activity was only detected at the highest dose of 1000 μg/mL (p < 0.01). Based on these safety profiles and preliminary dose–response and time–course experiments, the concentration of 10 μg/mL was identified as the optimal dose for investigating the modulatory effects on epithelial barrier and immune function without compromising cellular homeostasis. The effect on the epithelial gut barrier elicited by the novel food supplement was evaluated by measuring the transepithelial electrical resistance (TEER), tight-junction proteins, Mucin-2 (Muc-2) and lactase expression. The TEER measurement, a reliable indicator of the integrity of the paracellular seal, revealed that the exposure of human enterocyte monolayers to the novel food supplement (10 μg/mL) led to a progressive strengthening of the barrier. Specifically, a significant increase in the TEER values was observed starting from 48 h of incubation (p < 0.05 vs. NT), and this reinforcing effect was sustained and further enhanced until the 72 h time point (Figure 1A). The exposure to the novel food supplement resulted in TEER value increases starting from 48 h of incubation; the effect was sustained until 72 h (Figure 1A). This effect occurred in parallel with an upregulation of the tight-junction protein occludin and ZO-1-related gene expression (Figure 1B,C).
Figure 1. Effects of the study product on gut barrier integrity. The novel food supplement exposure (10 μg/mL) resulted in TEER value increases starting from 48 h of incubation; the effect was sustained until 72 h. No TEER value modification was observed in control cells not exposed to the novel food (NT) (A). In parallel, an upregulation of the tight-junction proteins occludin (B) and ZO-1-related gene expression (C) was observed. Data are derived from 5 independent experiments performed in duplicate and are represented as median ± SE (* p < 0.05 vs. NT, *** p < 0.001 vs. NT).
To further assess the effects of the study product on epithelial gut barrier, the expression of Muc2 and lactase genes were also evaluated. After 48 h exposure to the novel food supplement, Muc2 (Figure 2A) and lactase (Figure 2B) expression increased. No modification of Muc2 and/or lactase expression were observed in control cells not exposed to the novel food (NT).
Figure 2. Effects of the study product on Muc2 and lactase expression by human enterocytes. Novel food supplement exposure (10 μg/mL) for 48 h resulted in an Muc2 (A) and lactase (B) gene expression increase. No modifications were observed in control cells not exposed to the novel food (NT). Data are derived from 5 independent experiments performed in duplicate and are represented as median ± SE (*** p < 0.001 vs. NT).

3.2. Modulation of Immune System

The effect on immune system modulation by the novel food supplement was evaluated by measuring the gene expression of two major components of innate immunity produced by human enterocytes: human beta defensin 2 (HBD-2) and cathelecidin LL-37.
The exposure to the novel food supplement resulted in an upregulation of both innate immunity peptides gene expression by human enterocytes (Figure 3A,B). No modifications of HBD-2 and LL-37 gene expression were observed in control cells not exposed to the novel food (NT).
Figure 3. Effects of the study product on β-defensin-2 (HBD-2) and cathelicidin LL-37 gene expression by human enterocytes. The novel food supplement exposure (10 μg/mL) for 48 h resulted in β-defensin-2 (HBD-2) (A) and cathelicidin LL-37 (B) gene expression increase in human enterocytes. No modifications were observed in control cells not exposed to the novel food (NT). Data derived from 5 independent experiments performed in duplicate and are represented as median ± SE (* p < 0.05 vs. NT).

4. Discussion

The Postbiotics and Plant-derived compounds against Allergy (PPA) project was designed to investigate the effect of a novel food supplement based on the combination of four bioactive components with well demonstrated efficacy against allergy. In this paper, we provided data on the preclinical phase of the project aimed at evaluating the effect of this novel food supplement, composed of heat-inactivated Lacticaseibacillus rhamnosus GG (LGG) and butyrate with Quercetin and Perilla frutescens on epithelial gut barrier and immune function. We found that this novel food supplement exerts multiple beneficial regulatory actions on the human enterocytes involved in epithelial gut barrier and immune function.
The two postbiotic components of the novel food supplement were heat-inactivated LGG and butyrate. We have recently demonstrated that heat-inactivated LGG postbiotic could regulate several mechanisms involved in the immune tolerance network, increasing the activation of Treg cells and IL-10 production and reducing major Th2 cytokine (i.e., IL-4, IL-5, and IL-13) production in children affected by food allergy. The upstream mechanism of these effects involved the activation of major regulatory molecules of immune function Tgfb1, Ifna2, Ptgs2 and Csf2 [7]. Furthermore, positive effects on gut barrier integrity with an upregulation of TEER value, tight-junction proteins, Muc2 and lactase expression, with a subsequent reduction in FITC dextran permeability, were also observed [7]. The other postbiotic included in the novel food supplement is butyrate, a major short-chain fatty acid (SCFA) produced by the human gut microbiome [23]. Butyrate has emerged as a key mediator of immune tolerance, with documented anti-allergic properties. Experimental and clinical evidence indicates that butyrate enhances epithelial barrier integrity, promotes regulatory T cell differentiation, and suppresses type-2 inflammatory responses [23]. Moreover, many of these effects are mediated by epigenetic mechanisms, thereby fostering a tolerogenic mucosal environment [24,25]. Specifically, butyrate acts as a critical energy source for enterocytes and promotes the assembly of tight-junction proteins, thereby reducing paracellular permeability. These pleiotropic effects are largely mediated by its interaction with specific G protein-coupled receptors (GPCRs), such as GPR41, GPR43, and GPR109A. The activation of these receptors leads to the inhibition of histone deacetylases (HDACs), a crucial mechanism that facilitates the transcription of genes involved in mucosal repair and anti-inflammatory signaling [26,27]. Importantly, reduced intestinal levels of butyrate-producing bacteria have been consistently associated with increased risk of food allergy and impaired immune tolerance, particularly in early life. These findings support the concept that restoring butyrate availability—either through microbiome modulation or direct supplementation—may represent a promising nutritional strategy for preventing and managing allergic diseases [28].
The two plant-based dietary compounds included in the novel food supplement were Quercetin and Perilla frutescens extracts. Quercetin represents one of the most extensively studied dietary flavonoids with anti-allergic and epithelial gut barrier-protective properties. Evidence suggest that regular intake of this dietary flavonoid may contribute to the prevention and management of allergic diseases through the stabilization of the epithelial gut barrier, the inhibition of mast cell activation, the attenuation of pro-inflammatory signaling and the modulation of gut microbial composition in ways that promote SCFA-producing taxa [29,30]. These effects are mediated by multiple pathways, including RhoA/ROCK, Ca2+/JNK/Src, and Nrf2-dependent mechanisms [29,30].
Perilla frutescens, a traditional edible plant widely used in Asian diets that is rich in polyphenols, has been proposed as a functional food ingredient against allergy with epithelial gut barrier-protective effects and modulatory action against oxidative and inflammatory stress [31,32]. These effects involve the activation of the Nrf2 pathway and the transcription of tight junction proteins [33]. Interestingly, the addition of Quercetin extracts has been proposed to increase the anti-inflammatory and anti-histamine effects of Perilla frutescens [31].
Allergy derives from a breakdown of the immune tolerance network [11,34]. To explore the potential efficacy of this novel food supplement against allergy we focused on several components of the immune tolerance network. Mature enterocytes form the epithelial gut barrier, via tight-junction proteins, and prevent abnormal exposure to environmental antigens. Increased intestinal permeability allows allergens to pass through, activating the immune system and causing allergic reactions [35]. We found that stimulating human enterocyte monolayers with the novel food supplement resulted in intestinal permeability reduction, as suggested by the increase in TEER values. This effect was mediated, at least in part, by the modulation of human enterocyte maturation, as demonstrated by increased expression of the brush-border enzyme lactase, and the two major tight-junction proteins, occludin and ZO-1. Interestingly, a reduced expression of all of these three compounds has been observed in patients affected by allergy [35]. We also found that the novel food supplement was able to increase the expression of Muc2 in human enterocytes. Muc2 is a key mucin protein forming the protective mucus layer in the intestine essential for epithelial gut barrier function. The mucus layer plays a central role in preventing local as well as systemic allergy [36]. When the Muc2-based mucus barrier is compromised (e.g., due to poor diet or gut microbiota imbalance), food allergens can cross the epithelial gut barrier, increasing antigen exposure and worsening allergic responses, with a negative impact on immune tolerance [37]. Muc2-deficient mice display lower Treg cell counts in the small intestine lamina propria. Interestingly, when exposed orally to Muc2 protein from wild-type (WT) mice, deficient mice were able to shift their immune cell profiles towards increased Tregs and reduced inflammatory cell subsets. Muc2 glycoproteins induce the production of IL-10, TGF-β, retinoic acid (RA), and TSLP by DCs and epithelial cells, and thus promoting immune function [37].
Beyond their structural and metabolic functions, intestinal epithelial cells actively contribute to mucosal immunity through the production of antimicrobial peptides such as HBD-2 and LL-37. These molecules constitute a crucial component of the innate defense system, providing protection against microbial invasion while promoting epithelial repair and immune tolerance. Impaired expression of HBD-2 and LL-37 have been associated with increased intestinal permeability and allergic sensitization [38,39]. We found that the novel food supplement was able to significantly increase the expression of both HBD-2 and LL-37 genes by human enterocytes, suggesting another potential mechanism of action in protecting against allergic diseases. These host defense peptides (HDPs) do not only possess direct antimicrobial activity, but also function as immunomodulatory molecules or ‘alarmins’ that facilitate the transition from a Th2-skewed allergic environment toward mucosal homeostasis. The cooperative potential of the supplement’s ingredients may enhance the biochemical defense of the mucosa by providing the necessary structural and chemical signals to the epithelial cells. These findings are consistent with previous evidence showing that bioactive postbiotic molecules such as butyrate and heat-inactivated LGG postbiotic can induce HBD-2 and LL-37 synthesis via TLR2–NF-κB signaling pathways [39]. Moreover, polyphenolic compounds including Quercetin and Perilla extracts may further enhance epithelial resilience by modulating redox-sensitive transcription factors such as Nrf2, which also influence the gene expression of innate immunity peptides [40].
The major limitations of our study are related to the lack of pharmacological synergy assessment, including component-specific controls and a synergy-modeling approach. Future studies incorporating single-component controls and dedicated synergy assessment models will be required to quantitatively dissect the contribution of each component. In addition, the epithelial gut barrier model adopted in the study, while widely accepted as a model of the intestinal barrier [7,41], does not replicate the complexity of the in vivo intestinal environment, which includes immune cell interactions, microbial communities, and mucus-secreting goblet cells [42,43]. Another limitation of the present study is the evaluation of the modulatory effects elicited by the novel food supplement on tight-junction proteins, lactase, Muc-2 and innate immunity peptides based on the assessment of mRNA level without direct evaluation of protein abundance or subcellular localization. While transcriptional changes do not always translate into protein-level effects, the observed upregulation of these protein-related genes were consistently accompanied by functional improvements in epithelial gut barrier integrity, as demonstrated by increased transepithelial electrical resistance and reduced paracellular permeability [44,45]. Nevertheless, future studies are required to confirm these findings at the protein level and to investigate tight-junction protein organization using immunoblotting and immunofluorescence approaches.
In conclusion, although further preclinical and clinical studies are necessary, these findings provide a solid preclinical rationale for the development of functional food strategies aimed at supporting mucosal homeostasis in allergic diseases.

Author Contributions

Conceptualization, L.C., S.C. and E.C.; methodology, F.O., A.C., R.D.S., V.M. and M.M.; validation, L.C., S.C. and E.C.; formal analysis, R.F.I.; investigation, F.O., S.C. and L.C.; resources, R.F.I.; data curation, F.O. and L.C.; writing—original draft preparation, F.O., S.C. and L.C.; writing—review and editing, L.C.; visualization, L.C.; supervision, L.C. All authors have read and agreed to the published version of the manuscript.

Funding

Italian Ministry of Health–Health Operational Plan Trajectory 5-Line of action “Creation of an action program for the fight against malnutrition in all its forms and for the dissemination of the principles of the Mediterranean diet” (Mediterranean Diet for Human Health Lab, “MeDiHealthLab”, code T5-AN-07, CUP E63C22002570006).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The study was supported by the Department of Translational Medical Science of the University of Naples ‘Federico II,’ Naples, Italy, which received funding from the National Recovery and Resilience Plan, European Union–Next Generation EU (On Foods–Research and Innovation Network on Food and Nutrition Sustainability, Safety and Security—Working on Foods; code PE0000003) and from the Italian Ministry of Health—Health Operational Plan Trajectory 5—Line of action ‘Creation of an action program for the fight against malnutrition in all its forms and for the dissemination of the principles of the diet Mediterranean’ (Mediterranean Diet for Human Health Lab ‘MeDiHealthLab’; code T5-AN-07).

Conflicts of Interest

Authors declare that they have no competing interests.

Abbreviations

The following abbreviations are used in this manuscript:
PPAPostbiotic and plant-derived compounds against allergy
TEERTransepithelial electrical resistance
ZO-1Zonula occludens-1
Muc-2Mucin-2
HBD-2Β-defensin-2
LGGLacticaseibacillus rhamnosus GG
LL-37Cathelicidin LL-37
MRSMan, Rogosa and Sharpe
DMEMDulbecco’s modified eagle medium
NEAANon-essential amino acids
DMSODimethyl sulfoxide
NTUntreated control
GUSBΒ-gluconidase

References

  1. Abrams, E.M.; Greenhawt, M.; Shaker, M.; Alqurashi, W. Separating Fact from Fiction in the Diagnosis and Management of Food Allergy. J. Pediatr. 2022, 241, 221–228. [Google Scholar] [CrossRef] [Scilit]
  2. Nocerino, R.; Carucci, L.; Coppola, S.; Cecere, G.; Micillo, M.; Castaldo, T.; Russo, S.; Sandomenico, M.; Marino, A.; Gualano, R.; et al. Epidemiology of Paediatric Italian Food Allergy: Results of the EPIFA study. J. Allergy Clin. Immunol. Glob. 2024, 3, 100246. [Google Scholar] [CrossRef] [Scilit]
  3. Tanno, L.K.; Demoly, P. Food allergy in the World Health Organization’s International Classification of Diseases (ICD)-11. Pediatr. Allergy Immunol. 2022, 33, e13882. [Google Scholar] [CrossRef] [Scilit]
  4. Turner, P.J.; Conrado, A.B.; Kallis, C.; O’Rourke, E.; Haider, S.; Ullah, A.; Custovic, D.; Custovic, A.; Quint, J.K. Time trends in the epidemiology of food allergy in England: An observational analysis of Clinical Practice Research Datalink data. Lancet Public Health 2024, 9, e664–e673. [Google Scholar] [CrossRef] [Scilit]
  5. Lu, H.-F.; Zhou, Y.-C.; Yang, L.-T.; Zhou, Q.; Wang, X.-J.; Qiu, S.-Q.; Cheng, B.-H.; Zeng, X.-H. Involvement and repair of epithelial barrier dysfunction in allergic diseases. Front. Immunol. 2024, 15, 1348272. [Google Scholar] [CrossRef] [Scilit]
  6. Ogulur, I.; Mitamura, Y.; Yazici, D.; Pat, Y.; Ardicli, S.; Li, M.; D’aVino, P.; Beha, C.; Babayev, H.; Zhao, B.; et al. Type 2 immunity in allergic diseases. Cell. Mol. Immunol. 2025, 22, 211–242. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  7. Oglio, F.; Paparo, L.; Carucci, L.; Gaeta, A.; Armiento, S.; Coppola, S.; Molinaro, A.; De Castro, C.; Masino, A.; Mauriello, V.; et al. Postbiotic effects elicited by heat-inactivated Lacticaseibacillus rhamnosus GG against cow’s milk allergy in human cells. Front. Immunol. 2026, 16, 1671729. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  8. Farnetano, M.; Carucci, L.; Coppola, S.; Oglio, F.; Masino, A.; Cozzolino, M.; Nocerino, R.; Canani, R.B. Gut microbiome features in pediatric food allergy: A scoping review. Front. Allergy 2024, 5, 1438252. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  9. Berni Canani, R.; Paparo, L.; Nocerino, R.; Di Scala, C.; Della Gatta, G.; Maddalena, Y.; Buono, A.; Bruno, C.; Voto, L.; Ercolini, D. Gut Microbiome as Target for Innovative Strategies Against Food Allergy. Front. Immunol. 2019, 10, 191. [Google Scholar] [CrossRef] [Scilit]
  10. Aitoro, R.; Paparo, L.; Amoroso, A.; Di Costanzo, M.; Cosenza, L.; Granata, V.; Di Scala, C.; Nocerino, R.; Trinchese, G.; Montella, M.; et al. Gut Microbiota as a Target for Preventive and Therapeutic Intervention against Food Allergy. Nutrients 2017, 9, 672. [Google Scholar] [CrossRef] [Scilit]
  11. Canani, R.B.; Gilbert, J.A.; Nagler, C.R. The role of the commensal microbiota in the regulation of tolerance to dietary allergens. Curr. Opin. Allergy Clin. Immunol. 2015, 15, 243–249. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  12. Salminen, S.; Collado, M.C.; Endo, A.; Hill, C.; Lebeer, S.; Quigley, E.M.M.; Sanders, M.E.; Shamir, R.; Swann, J.R.; Szajewska, H.; et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 2021, 18, 649–667. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  13. Berni Canani, R.; Sangwan, N.; Stefka, A.; Nocerino, R.; Paparo, L.; Aitoro, R.; Calignano, A.; Khan, A.A.; Gilbert, J.A.; Nagler, C.R. Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants. ISME J. 2016, 10, 742–750. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  14. Berni Canani, R.; Nocerino, R.; Terrin, G.; Coruzzo, A.; Cosenza, L.; Leone, L.; Troncone, R. Effect of Lactobacillus GG on tolerance acquisition in infants with cow’s milk allergy: A randomized trial. J. Allergy Clin. Immunol. 2012, 129, 580–582.e5. [Google Scholar] [CrossRef] [Scilit]
  15. Berni Canani, R.; Nocerino, R.; Terrin, G.; Frediani, T.; Lucarelli, S.; Cosenza, L.; Passariello, A.; Leone, L.; Granata, V.; Di Costanzo, M.; et al. Formula selection for management of children with cow’s milk allergy influences the rate of acquisition of tolerance: A prospective multicenter study. J. Pediatr. 2013, 163, 771–777.e1. [Google Scholar] [CrossRef] [Scilit]
  16. Berni Canani, R.; Di Costanzo, M.; Bedogni, G.; Amoroso, A.; Cosenza, L.; Di Scala, C.; Granata, V.; Nocerino, R. Extensively hydrolyzed casein formula containing Lactobacillus rhamnosus GG reduces the occurrence of other allergic manifestations in children with cow’s milk allergy: 3-year randomized controlled trial. J. Allergy Clin. Immunol. 2017, 139, 1906–1913.e4. [Google Scholar] [CrossRef] [Scilit]
  17. Nocerino, R.; Di Costanzo, M.; Bedogni, G.; Cosenza, L.; Maddalena, Y.; Di Scala, C.; Della Gatta, G.; Carucci, L.; Voto, L.; Coppola, S.; et al. Dietary Treatment with Extensively Hydrolyzed Casein Formula Containing the Probiotic Lactobacillus rhamnosus GG Prevents the Occurrence of Functional Gastrointestinal Disorders in Children with Cow’s Milk Allergy. J. Pediatr. 2019, 213, 137–142.e2. [Google Scholar] [CrossRef] [Scilit]
  18. Nocerino, R.; Coppola, S.; Carucci, L.; de Giovanni di Santa Severina, A.F.; Oglio, F.; de Michele, R.; di Sessa, I.; Masino, A.; Bedogni, G.; Canani, R.B. The step-down approach in children with cow’s milk allergy: Results of a randomized controlled trial. Allergy 2023, 78, 2477–2486. [Google Scholar] [CrossRef] [Scilit]
  19. Nocerino, R.; Bedogni, G.; Carucci, L.; Aquilone, G.; Oglio, F.; Coppola, S.; Masino, A.; Canani, R.B. Long term impact of formula choice in children with cow milk protein allergy: 6-year follow-up of the Atopic March Cohort Study. Clin. Nutr. 2025, 48, 134–143. [Google Scholar] [CrossRef] [Scilit]
  20. Makino, T.; Furuta, Y.; Wakushima, H.; Fujii, H.; Saito, K.; Kano, Y. Anti-allergic effect of Perilla frutescens and its active constituents. Phytother. Res. 2003, 17, 240–243. [Google Scholar] [CrossRef] [Scilit]
  21. Paparo, L.; Nocerino, R.; Ciaglia, E.; Di Scala, C.; De Caro, C.; Russo, R.; Trinchese, G.; Aitoro, R.; Amoroso, A.; Bruno, C.; et al. Butyrate as a bioactive human milk protective component against food allergy. Allergy 2021, 76, 1398–1415. [Google Scholar] [CrossRef] [Scilit]
  22. Coppola, S.; Avagliano, C.; Sacchi, A.; Laneri, S.; Calignano, A.; Voto, L.; Luzzetti, A.; Canani, R.B. Potential Clinical Applications of the Postbiotic Butyrate in Human Skin Diseases. Molecules 2022, 27, 1849. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  23. Folkerts, J.; Redegeld, F.; Folkerts, G.; Blokhuis, B.; van den Berg, M.P.M.; de Bruijn, M.J.W.; van Ijcken, W.F.J.; Junt, T.; Tam, S.; Galli, S.J.; et al. Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling. Allergy 2020, 75, 1962–1974. [Google Scholar] [CrossRef] [Scilit]
  24. Di Costanzo, M.; De Paulis, N.; Biasucci, G. Butyrate: A Link between Early Life Nutrition and Gut Microbiome in the Development of Food Allergy. Life 2021, 11, 384. [Google Scholar] [CrossRef] [Scilit]
  25. Chayalak, W.; Thammacharoen, S.; Deachapunya, C.; Poonyachoti, S. Preventive effects of quercetin on the leaky gut by suppression of IL-1beta-induced transcription factors involved in an inflammatory pathway in colonic epithelial cells. Thai J. Vet. Med. 2024, 53, 3. [Google Scholar] [CrossRef] [Scilit]
  26. Yuan, M.; Sun, T.; Zhang, Y.; Guo, C.; Wang, F.; Yao, Z.; Yu, L. Quercetin Alleviates Insulin Resistance and Repairs Intestinal Barrier in db/db Mice by Modulating Gut Microbiota. Nutrients 2024, 16, 1870. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  27. Mayangsari, Y.; Sugimachi, N.; Xu, W.; Mano, C.; Tanaka, Y.; Ueda, O.; Sakuta, T.; Suzuki, Y.; Yamamoto, Y.; Suzuki, T. 3,5,7,3′,4′-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells. J. Agric. Food Chem. 2021, 69, 10174–10183. [Google Scholar] [CrossRef] [Scilit]
  28. Cho, S.-Y.; Park, S.-J.; Kwon, M.-J.; Jeong, T.-S.; Bok, S.-H.; Choi, W.-Y.; Jeong, W.-I.; Ryu, S.-Y.; Do, S.-H.; Lee, C.-S.; et al. Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage. Mol. Cell. Biochem. 2003, 243, 153–160. [Google Scholar] [CrossRef] [Scilit]
  29. Wu, X.; Dong, S.; Chen, H.; Guo, M.; Sun, Z.; Luo, H. Perilla frutescens: A traditional medicine and food homologous plant. Chin. Herb. Med. 2023, 15, 369–375. [Google Scholar] [CrossRef] [Scilit]
  30. Adam, G.; Robu, S.; Flutur, M.-M.; Cioanca, O.; Vasilache, I.-A.; Adam, A.-M.; Mircea, C.; Nechita, A.; Harabor, V.; Harabor, A.; et al. Applications of Perilla frutescens Extracts in Clinical Practice. Antioxidants 2023, 12, 727. [Google Scholar] [CrossRef] [Scilit]
  31. Cazalla, E.; Cuadrado, A.; García-Yagüe, Á.J. Role of the transcription factor NRF2 in maintaining the integrity of the Blood-Brain Barrier. Fluids Barriers CNS 2024, 21, 93. [Google Scholar] [CrossRef] [Scilit]
  32. Carucci, L.; Shah, N.; Canani, R.B. Editorial: New insights in pediatric gastrointestinal food allergies. Front. Allergy 2024, 5, 1531494. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  33. Canani, R.B.; Caminati, M.; Carucci, L.; Eguiluz-Gracia, I. Skin, gut, and lung barrier: Physiological interface and target of intervention for preventing and treating allergic diseases. Allergy 2024, 79, 1485–1500. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  34. Shan, M.; Gentile, M.; Yeiser, J.R.; Walland, A.C.; Bornstein, V.U.; Chen, K.; He, B.; Cassis, L.; Bigas, A.; Cols, M.; et al. Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals. Science 2013, 342, 447–453. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  35. Parrish, A.; Boudaud, M.; Kuehn, A.; Ollert, M.; Desai, M.S. Intestinal mucus barrier: A missing piece of the puzzle in food allergy. Trends Mol. Med. 2022, 28, 36–50. [Google Scholar] [CrossRef] [Scilit]
  36. Wehkamp, J.; Schmid, M.; Stange, E.F. Defensins and other antimicrobial peptides in inflammatory bowel disease. Curr. Opin. Gastroenterol. 2007, 23, 370–378. [Google Scholar] [CrossRef] [Scilit]
  37. Paparo, L.; Aitoro, R.; Nocerino, R.; Fierro, C.; Bruno, C.; Canani, R.B. Direct effects of fermented cow’s milk product with Lactobacillus paracasei CBA L74 on human enterocytes. Benef. Microbes 2018, 9, 165–172. [Google Scholar] [CrossRef] [Scilit]
  38. Lia, F.; Baron, B. Analysis of Polyphenolic Composition, Antioxidant Power and Stress-Response Effects of Fractionated Perilla Leaf Extract on Cells In Vitro. Biologics 2025, 5, 2. [Google Scholar] [CrossRef] [Scilit]
  39. Paparo, L.; Coppola, S.; Nocerino, R.; Pisapia, L.; Picariello, G.; Cortese, M.; Voto, L.; Maglio, M.; Miele, E.; Carucci, L.; et al. How dietary advanced glycation end products could facilitate the occurrence of food allergy. J. Allergy Clin. Immunol. 2024, 153, 742–758. [Google Scholar] [CrossRef] [Scilit]
  40. Hooper, L.V.; Littman, D.R.; MacPherson, A.J. Interactions between the microbiota and the immune system. Science 2012, 336, 1268–1273. [Google Scholar] [CrossRef] [Scilit]
  41. Anjum, M.; Laitila, A.; Ouwehand, A.C.; Forssten, S.D. Current Perspectives on Gastrointestinal Models to Assess Probiotic-Pathogen Interactions. Front. Microbiol. 2022, 13, 831455. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  42. Wang, H.-B.; Wang, P.-Y.; Wang, X.; Wan, Y.-L.; Liu, Y.-C. Butyrate enhances intestinal epithelial barrier function via up-regulation of tight junction protein Claudin-1 transcription. Dig. Dis. Sci. 2012, 57, 3126–3135. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  43. Ghosh, S.; Whitley, C.S.; Haribabu, B.; Jala, V.R. Regulation of Intestinal Barrier Function by Microbial Metabolites. Cell. Mol. Gastroenterol. Hepatol. 2021, 11, 1463–1482. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  44. Ferris, M.M.; Subitoni Antonio, L.; Al-Sadi, R. Probiotics and the intestinal tight junction barrier function. Front. Cell Dev. Biol. 2025, 13, 1671152. [Google Scholar] [CrossRef] [Scilit]
  45. Wang, J.Y.; Xiao, L.; Wang, J.Y. Posttranscriptional regulation of intestinal epithelial integrity by noncoding RNAs. Wiley Interdiscip. Rev. RNA 2017, 8, e1399. [Google Scholar] [CrossRef] [Scilit]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Heat Treatment Modulates Structure, Functionality, and Digestion-Related Antioxidant Activity of Xanthoceras sorbifolium Seed Meal Protein
Previous
Systematic Identification of Characteristic Metabolites and Analysis of Quality and Metabolomic Profiles of Yunnan Kucha White Tea
Next