Nutritional composition and therapeutic or preventive use thereof

Abstract

The invention relates to a nutritional composition comprising the synergistic combination of a postbiotic mixture comprising inactivated L. rhamnosus LGG and butyric acid or butyrate, a Perilla frutescens dry extract and quercetin, as well as its use in the therapeutic or preventive treatment of allergic disease or condition.

Description

[0001] Nutritional composition and therapeutic or preventive use thereof

[0002] The invention relates to an innovative nutritional composition (which may also be designated as “nutritional supplement”) specifically designed to address the unique nutritional needs of subjects suffering from an allergic condition, with a beneficial impact on the allergic response and nutritional status the aforementioned subjects. The composition of the invention results from advanced research and cutting-edge technology, featuring a unique synergistic combination of postbiotic ingredients, optionally in combination with further ingredients selected from prebiotics and / or micronutrients.

[0003] Background art

[0004] Allergic diseases are rising globally, becoming a global health problem (1). The dramatic increase in prevalence, persistence, and severity of clinical manifestations has led to an increase in hospital admissions, medical visits, worsening of quality of life of affected patients and their parents, burden of care on families and Healthcare System (1). Allergies arise from a breakdown in immune tolerance mechanisms influenced by negative geneenvironment factors interaction with alteration of epithelial barrier and Th2 inflammation (2).

[0005] Allergic subjects are at increased risk of malnutrition, particularly with deficiencies of micronutrients, such as vitamins A, D, E, B12, 'Q-3 polyunsaturated fatty acids, iron, zinc and selenium, as compared to subjects without allergies. These nutrient deficiencies exert a profound impact, triggering nutritional immunity, type 2 inflammation, and restricting dietary uptake of micronutrients at gastrointestinal level (3). Consequently, malnutrition fosters the persistence and the severity of allergic diseases, while an adequate supply of micronutrients promotes immune cells with regulatory and tolerogenic phenotypes (3).

[0006] The impact of malnutrition in the pediatric allergic patient is even more severe leading to risks of body growth and development failure with long-term consequences (3).

[0007] Dysbiosis, which is defined as an alteration of the structure and the function of the gut microbiota, is increasingly recognized as a significant factor in the development of allergies. Dysbiosis resulting from various environmental factors, has been consistently demonstrated in patients affected by allergies and it is characterized by a decrease in butyric-acid- producing bacteria, which leads to a decrease in intestinal butyric acid concentrations (4-6). The decrease in butyric acid concentration suppresses the differentiation of T-cells into regulatory T-cells (Tregs). The reduced number of Tregs impairs the immune system’s ability to control excessive immune responses, thereby contributing to the onset, persistence and severity of allergic diseases (4-6).

[0008] The discovery of the pivotal role of selected dietary factors in influencing immune system development and function has introduced the “immunonutrition” concept. The application of the “immunonutrition” approach in the management of patients affected by allergic diseases may have a beneficial impact on both the nutritional status and the allergic response (7).

[0009] Accordingly, a specifically designed nutritional supplement for allergic patients, able to exert a beneficial effect on the nutritional status and on immune tolerance of said patients is urgently needed.

[0010] Brief description of the invention

[0011] In order to meet these and other needs, the present invention provides a nutritional composition as defined in appended claim 1.

[0012] Further advantageous features of the present invention are defined in the dependent claims.

[0013] The appended claims form an integral part of the present description.

[0014] The synergistic combination of ingredients of the nutritional composition of the invention was shown to be effective in the therapeutic and preventive treatment of an allergic disease or condition, as it was able to improve the immune tolerance mechanisms in patients with allergic diseases or allergic conditions as well as their nutritional status. Detailed Description of the Invention

[0015] The nutritional composition of the invention was specifically designed for targeting the main mechanisms involved in the occurrence, persistence and severity of allergic diseases and conditions, with a beneficial impact on the immune tolerance and nutritional status of patients affected by such diseases or conditions.

[0016] The nutritional composition of the invention comprises the synergistic combination of:

[0017] (i) butyric acid or butyrate,

[0018] (ii) inactivated Lactobacillus rhamnosus GG (“LGG”),

[0019] (iii) a Perilla frutescens dry extract, preferably from leaves and seeds, and

[0020] (iv) quercetin.

[0021] In certain preferred embodiments which are illustrated in more detail below, the nutritional composition of the invention further comprises one or more additional ingredients selected from the group consisting of prebiotics and micronutrients.

[0022] The nutritional composition of the invention comprises, inter alia, the combination of ingredients (i) and (ii) mentioned above, which are both postbiotics.

[0023] Butyric acid is a short chain fatty acid (SCFA) produced by the gut microbiome and which is known to exert a pivotal role for human health. Butyric acid is present in the nutritional composition of the invention either as such or in the form of a butyrate. In the present description, the term “butyrate” includes any pharmaceutically or nutritionally acceptable salt of butyric acid, such as sodium butyrate or calcium butyrate, as well as any butyric acid releaser, such as N-(l-carbamoyl-2-phenyl-ethyl) butyramide (FBA) or other known amide derivatives of butyric acid that show the same physicochemical, organoleptic and pharmacokinetic properties as FBA, including e.g. N-(l-butyroyl-carbamoyl-2-phenyl- ethyl)butyramide, 5-benzyl-2-propyl-lH-imidazol-4(5H)-one, N-(l-oxo-3-phenyl-l- (piperidin- 1 -yl)propan-2-yl)butyramide, N-(l -oxo-3 -phenyl- 1 -(pyrrolidin- 1 -yl)propan-2- yl)butyramide, N-(l -(methyl carbamoyl)-2-phenylethyl)butyramide, N-(l - (ethylcarbamoyl)-2-phenylethyl)butyramide, N-(l-(propylcarbamoyl)-2- phenylethyl)butyramide, N-(l-(butylcarbamoyl)-2-phenylethyl)butyramide, N-(l- (pentylcarbamoyl)-2-phenylethyl)butyramide, N-(l-carbamoyl-2-phenylethyl)-N- methylbutyramide, N-(l -carbarn oyl-2-phenylethyl)-N-ethylbutyramide, N-(l -carbarn oyl-2- phenylethyl)-N-propylbutyramide, and pharmaceutically acceptable salts, diastereo-isomers and enantiomers thereof. The aforementioned butyric acid or butyrate in the nutritional composition of the invention is optionally microencapsulated. Microencapsulated forms of butyric acid or butyrate for human consumption are known per se in the prior art.

[0024] Lactobacillus rhamnosus GG (LGG) (ATCC 53103) is the most widely used and characterized postbiotic strain of Lactobacillus rhamnosus, commonly employed in gastroenterology with special regard to the treatment of pediatric diarrhea. The inactivated form of LGG is also known per se in the prior art. Heat or UV inactivation of LGG is also well known per se in the prior art.

[0025] The combination of postbiotic ingredients (i) and (ii) in the nutritional composition of the invention provides the effect of restoring the patient’s main immune tolerance mechanisms with a beneficial impact on gut barrier, gut microbiome, and immune cells.

[0026] As mentioned above, the nutritional composition of the invention further includes (iii) a Perilla frutescens dry extract, preferably from leaves and seeds, and (iv) quercetin, which possess beneficial immunomodulatory and anti-inflammatory properties.

[0027] The Perilla frutescens dry extract is preferably standardized to 2.5% polyphenols.

[0028] In certain preferred embodiments, in addition to the aforementioned active ingredients (i) to (iv), the nutritional composition of the invention further includes one or more prebiotics and / or one or more micronutrients, as well as other further optional active ingredients as illustrated below.

[0029] The one or more prebiotics contribute to restoring a healthy gut microbiome and are preferably selected from the group consisting of 2’-fucosyllactose (2’-FL) (i.e. a human milk oligosaccharide (HMO), resistant starch (i.e. a carbohydrate that resists digestion in the small intestine and ferments in the large intestine), fructo-oligosaccharides (FOSs), inulin and any combination thereof.

[0030] The one or more micronutrients contribute to restoring the nutritional status and immune function of the patient and are preferably selected from vitamins, Q-3 polyunsaturated fatty acids, and trace elements (“oligoelements”). Preferred vitamins are selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin B12 and any combination thereof. Preferred 'Q-3 polyunsaturated fatty acids are selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A) and the combination thereof. Preferred trace elements are selected from the group consisting of iron, zinc, selenium and any combination thereof. The term “iron” in the present description includes any pharmaceutically or dietary acceptable forms of iron, such as ferrous and ferric iron salts, for example ferrous sulfate, ferrous gluconate, ferric citrate, and ferric sulfate. The term “zinc” in the present description includes any pharmaceutically or dietary acceptable forms of zinc, such as e.g. zinc sulfate, zinc acetate, and zinc gluconate. The term “selenium” in the present description includes any pharmaceutically or dietary acceptable forms of selenium, such as selenium cystine, selenium methionine, sodium selenite, etc.

[0031] An additional optional active ingredient which may be present in the composition of the invention is the digestive enzyme diamine oxidase (DAO), which supports the degradation of food-derived histamine. Conventional but not limiting sources of DAO are pork kidney protein extract with 7% DAO, or legume extract (e.g. Pisum sativum and Lens Culinalis extract).

[0032] Additional optional active ingredients which may be present in the composition of the invention are Ribes nigrum extract or macerate (preferably standardized at 4% anthocyanosides); Zingiber officinalis (Ginger) rhizome dry extract (preferably standardized at 10% gingeroles); Scutellaria baicalensis dry extract.

[0033] The nutritional composition of the invention is formulated for oral or enteral administration, for example as a nutritional supplement, a beverage, a food product or a pharmaceutical product. A pharmaceutical product is prepared, for example, in the form of a tablet, a sachet, a capsule, a vial or a syrup. A food product or nutritional supplement includes for example food products or nutritional supplements designed for subjects affected by allergies, including hypoallergenic nutritional supplements and formulas for children with food allergies.

[0034] The active ingredients concentration in the nutritional composition of the invention may vary depending on the age of the patient (i.e., pediatric or adult patients), the route of administration, the pharmaceutical or dietary form.

[0035] Table 1 below provides the preferred daily dose ranges for each of the essential active ingredients of the nutritional composition of the invention, which are in line with the respective dietary reference values and tolerable upper intake levels known in the art.

[0036] Table 1

[0037] *The daily dose range of butyric acid refers to the net amount of butyric acid which is present in the butyric acid salt and / or butyric acid derivate and / or releaser actually employed.

[0038] In addition to the essential active ingredients listed in Table 1, the nutritional composition of the invention optionally further includes one or more additional active ingredients, which are illustrated in Table 2 below along with their preferred daily dose ranges. Such ranges are in line with the respective dietary reference values and tolerable upper intake levels known in the art. Table 2

[0039] **Any type of iron, selenium, zinc salts

[0040] *The daily dose range of butyric acid refers to the net amount of butyric acid which is present in the butyric acid salt and / or butyric acid derivate and / or releaser actually employed.

[0041] The scope of the invention therefore includes a nutritional composition as disclosed herein, which is in a unit dosage form and comprises:

[0042] - butyric acid: 150-1000 mg per unit dosage form;

[0043] - inactivated L. rhamnosus GG (“LGG”): 108- 1011CFU per unit dosage form; - Perilla frutescens dry extract (preferably standardized at 2.5% polyphenols): 50-500 mg per unit dosage form; and

[0044] - quercetin: 20-200 mg per unit dosage form. Exemplary amounts of the essential active ingredients:

[0045] Butyric acid: 150 mg, 160 mg, 170, mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg,

[0046] 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg,

[0047] 850 mg, 900 mg, 950 mg, 1000 mg per unit dosage form.

[0048] Inactivated LGG: IxlO8CFU, 5xl08CFU, IxlO9CFU, 5xl09CFU, IxlO10CFU, 5xl010CFU, IxlO11CFU, 5x1011CFU per unit dosage form.

[0049] Perilla frutescens dry extract (preferably standardized to 2.5% polyphenols): 50 mg, 60 mg, 70, mg, 80 mg, 90 mg, 100 mg, 120, mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg per unit dosage form.

[0050] If the butyric acid is present in composition of the invention the form of a butyric acid salt, a butyric acid derivative or butyric acid releaser, the above-mentioned ranges and exemplary amounts refer to the net amount of butyric acid present in the salt, derivative or releaser actually employed.

[0051] Optionally, the above-illustrated unit dosage form further comprises:

[0052] - vitamin A: 250-750 pg per unit dosage form, and / or

[0053] - vitamin D: 15-35 pg per unit dosage form, and / or

[0054] - vitamin E: 5-60 mg per unit dosage form, and / or

[0055] - vitamin B12: 1.5-1000 pg per unit dosage form, and / or

[0056] - iron: - 7-20.5 mg per unit dosage form, and / or

[0057] - zinc: 2.9-13 mg per unit dosage form, and / or

[0058] - selenium: 15-70 pg per unit dosage form, and / or

[0059] - -inulin: 1-12 g per unit dosage form, and / or

[0060] - FOSs: 2- 10 g per unit dosage form, and / or

[0061] - 2FL’ : 2.9-4 g per unit dosage form, and / or

[0062] - resistant starch: 1-10 g per unit dosage form, and / or

[0063] - ' -3 polyunsatured fatty acids: 1-5 g per unit dosage form, and / or

[0064] - diamine oxidase (DAO) derived from protein extract from pork kidney with 7% DAO, or legumes extract (Pisum sativum and Lens Culinalis): 0.3 - 0.9 mg per unit dosage form, and / or

[0065] - Ribes Nigrum (4% anthocyanosides): 50-250 mg per unit dosage form, and / or

[0066] - Zingiber Officinalis (Ginger) rhizome, dry extract (10% gingeroles): 10-50 g per unit dosage form, and / or

[0067] - Scutellaria Baicalensis, dry extract: 50-200 g per unit dosage form.

[0068] Exemplary amounts of the optional active ingredients:

[0069] Vitamin A: 250 pg, 300 pg, 310 pg, 320 pg, 330 pg, 340 pg, 350 pg, 360 pg, 370 pg, 380 pg, 390 pg, 400 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg per unit dosage form.

[0070] Vitamin D: 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg per unit dosage form.

[0071] Vitamin E: 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg per unit dosage form.

[0072] Vitamin B12: 1.5 pg, 1.6 pg, 1.7 pg, 1.8 pg, 1.9 pg, 2.0 pg, 2.1 pg, 2.2 pg, 2.3 pg, 2.4 pg, 2.5 pg, 2.6 pg, 2.7 pg, 2.8 pg, 2.9 pg, 3.0 pg, 5 pg, 10 pg, 50 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg, 950 pg, 1000 pg per unit dosage form.

[0073] Iron: 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 20.1 mg, 20.2 mg, 20.3 mg, 20.4 mg, 20.5 mg per unit dosage form.

[0074] Zinc: 2.9 mg, 3.0 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg per unit dosage form. Selenium: 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg per unit dosage form.

[0075] Inulin: 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g per unit dosage form.

[0076] FOSs: 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g per unit dosage form.

[0077] 2FL’ : 2.9 g, 3.0 g, 3.1 g, 3.2 g, 3.3 g, 3.4 g, 3.5 g, 3.6 g, 3.7 g, 3.8 g, 3.9 g, 4.0 g per unit dosage form.

[0078] Resistant starch: 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g per unit dosage form.

[0079] ' -3 polyunsatured fatty acids: 1 g, 2 g, 3 g, 4 g, 5 g per unit dosage form.

[0080] Diamine oxidase: 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg per unit dosage form. Ribes nigrum'. 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg per unit dosage form.

[0081] Zingiber officinalis'. 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg per unit dosage form.

[0082] Scutellaria baicalensis'. 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg per unit dosage form.

[0083] The nutritional composition of the invention may further include carriers and / or excipients suitable for dietary or pharmaceutical use, the selection and use of which falls within the ability of the skilled formulator.

[0084] As disclosed in the examples, in vitro evaluation using immune cells collected from patients treated with a nutritional composition according to the invention showed reduced Th2 cytokines response and increased IL-10 and regulatory T cells response, indicating an immune shift toward tolerance. Additionally, the nutritional composition according to the invention enhanced gut barrier integrity and mucus production in an in vitro model of gut barrier, further supporting gut health.

[0085] Additionally, in a pilot clinical trial involving 30 pediatric patients affected by cow's milk protein allergy (CMP A) the inventor observed that a nutritional composition according to the invention was able to normalize body growth, micronutrients serum levels and to stimulate immune tolerance.

[0086] The in vitro and in vivo experimental results described in the examples below show the effectiveness of the nutritional supplement of the invention in the therapeutic and preventive treatment of an allergic disease or condition, such as cow's milk protein allergy (CMP A) or an allergic reaction.

[0087] As part of a broader therapeutic strategy, the nutritional supplement of the invention may be co-administered with an active pharmaceutical ingredient known per se to manage allergic diseases or conditions in order to enhance its effects. Said active pharmaceutical ingredient known per se to manage allergic diseases or conditions is preferably selected from the group consisting of antihistamines, antileukotrienes corticosteroids, bronchodilators, biologies, immunotherapy and immune-modulating agents.

[0088] Accordingly, the scope of the invention further comprises a combined preparation comprising the nutritional composition of the invention and one or more pharmaceutical active ingredients effective in the management of allergic diseases or conditions, preferably selected from the group consisting of antihistamines, antileukotrienes corticosteroids, bronchodilators, biologies, immunotherapy and immune-modulating agents, for the simultaneous, separate or successive administration in the therapeutic or preventive treatment of an allergic disease or condition in a subject, wherein the subject is preferably a pediatric subject and the allergic disease or condition is preferably CMPA or an allergic reaction.

[0089] The following examples, which are provided by way of illustration only, highlight the benefits of the nutritional composition according to the invention, such as immune tolerance enhancement (increased regulatory T cells), gut barrier function improvement, and nutritional status improvement (normalizing growth, vitamin D and DHA levels) in pediatric patients with CMPA.

[0090] The experimental results mentioned in the examples are further illustrated in figures 1-8, concerning the effects of the nutritional composition (also called “nutritional supplement”) according to the invention on immune and gut health. The “intervention” group in Figures 1-3 is the group that received the nutritional supplement according to the invention, which in Figures 4-8 is designated as “BLG+LGGp” for brevity. The complete composition of the nutritional supplement administered is provided in Table 4.

[0091] Drawings description:

[0092] Figure 1 shows the mean body weight, length / height, and head circumference at baseline and after 6 months for both the intervention and placebo groups. The “intervention” group experienced significant growth in all parameters compared to the placebo group, with a notable difference in body weight after 6 months (p<0.05). Figure 2 shows the mean serum Vitamin D levels at baseline and after 6 months. A statistically significant increase in Vitamin D levels was observed in the “intervention” group compared to the placebo group after 6 months (p<0.05), indicating the supplement's effectiveness in improving Vitamin D status.

[0093] Figure 3 shows the mean serum DHA concentrations as a percentage of total fatty acids at baseline and after 6 months. The “intervention” group demonstrated a significant increase in DHA levels compared to the placebo group (p<0.05), suggesting the supplement effectively enhanced DHA status during the study period.

[0094] Figure 4 shows that the administered nutritional supplement (BLG+LGGp) significantly reduced the production of pro-inflammatory Th2 cytokines (IL-4, IL-5, IL-13) in response to beta-lactoglobulin (BLG) stimulation, indicating its ability to suppress allergic cytokine responses (*p < 0.05 vs BLG).

[0095] Figure 5 shows that the administered nutritional supplement (BLG+LGGp) significantly increased the number of regulatory T cells (CD4+ / CD25+ / FoxP3+) and enhanced IL-10 production in peripheral blood mononuclear cells (PBMCs). Flow cytometry confirmed the rise in Tregs numbers, suggesting an enhanced immune tolerance (*p < 0.05 vs BLG).

[0096] Figure 6 shows the upregulation of regulatory dendritic cell markers (Tgfbl, Ifna2, Ptgs2, Csf2) in PBMCs treated with the nutritional supplement (BLG+LGGp); Treg induction is mediated through a dendritic cell-dependent mechanism.

[0097] Figure 7 shows that the administered nutritional supplement (BLG+LGGp) significantly increased TEER values, indicating enhanced epithelial barrier function. Additionally, RT- PCR analysis showed that the nutritional supplement (BLG+LGGp) upregulated the expression of tight junction proteins occludin and ZO-1, essential for maintaining gut barrier integrity (*p < 0.05 vs control).

[0098] Figure 8 shows that the administered nutritional supplement (BLG+LGGp) promoted increased expression of mucin 2 (Muc2), crucial for mucus production, and lactase, indicating enhanced enterocyte differentiation. These effects contribute to a healthier intestinal barrier (*p < 0.05 vs control).

[0099] Examples

[0100] 1. In vitro experiments on PBMCs

[0101] In vitro experiments on PBMCs from pediatric patients with IgE-mediated CMPA were conducted to evaluate the immunomodulatory effects of a nutritional supplement according to the invention comprising the ingredients illustrated in Table 3 below. Blood samples from 10 children were used to isolate PBMCs, which were stimulated with BLG in the presence or absence of the product. The study measured cytokine production and regulatory T cells (Tregs) levels.

[0102] The results showed that the nutritional supplement of the invention significantly reduced pro-inflammatory Th2 cytokines (IL-4, IL-5, IL- 13) and increased IL- 10, an antiinflammatory cytokine crucial for immune tolerance. Flow cytometry revealed an increase in Tregs (CD4+ / CD25+ / FoxP3+), and RT-PCR analysis indicated upregulation of regulatory dendritic cell markers, suggesting that Treg expansion was mediated by a dendritic cell-dependent mechanism. These findings suggest that the nutritional supplement modulates immune responses by reducing pro-allergic cytokines and promoting regulatory pathways, enhancing immune tolerance in CMPA patients.

[0103] 2. Human enterocytes cell line study

[0104] An in vitro study using Caco-2 cells, a model of the intestinal barrier, investigated the effects of the nutritional supplement according to the invention (Table 3) on gut barrier integrity. Cells were treated with the nutritional supplement of the invention for 48 hours, and the barrier function was assessed through Trans-Epithelial Electric Resistance (TEER) and the expression of tight junction proteins (occludin and ZO-1), mucus production genes (Muc2), and enterocyte differentiation markers (lactase). Treatment with the nutritional supplement of the invention significantly increased TEER values, indicating improved gut barrier integrity. It also upregulated the expression of tight junction proteins occludin and ZO-1, which are crucial for maintaining epithelial integrity. Additionally, the nutritional supplement of the invention boosted the expression of Muc2, a key component of the mucus layer, and lactase, suggesting enhanced enterocyte differentiation.

[0105] These findings demonstrate that the nutritional supplement of the invention strengthens gut barrier function, promotes epithelial differentiation, and supports mucus production, potentially helping manage CMPA by reinforcing gut integrity and promoting immune tolerance.

[0106] Table 3 3. Pilot randomized controlled trial

[0107] A pilot randomized clinical trial was carried out with a nutritional supplement according to the invention comprising the essential ingredients illustrated identified in Table 3 above as well as additional prebiotics and micronutrients. The total active ingredients composition of the nutritional supplement employed in the pilot clinical study is provided in Table 4 below.

[0108] The pilot clinical study was aimed at assessing the effects of the nutritional supplement on the nutritional status and growth of children affected by CMPA. Thirty children aged 1-3 years were divided into two groups: one received the supplement (n=15), and the other received a placebo (n=15) for 6 months. The primary outcomes measured were serum vitamin D and DHA levels, along with growth parameters such as weight and height. After 6 months, the intervention group showed a significant improvement in growth, with higher body weight (13.3 kg vs. 12.2 kg in the placebo group, p<0.05). Serum vitamin D levels also increased significantly in the intervention group (22.5 ng / ml vs. 18.7 ng / ml in the placebo group, p<0.05), reaching sufficient levels. Similarly, DHA levels significantly increased in the intervention group (3.05% vs. 2.1% of total fatty acids, p<0.05).

[0109] These findings indicate that the nutritional supplement positively affected growth and improved vitamin D and DHA levels, addressing key nutrient deficiencies in children with CMPA.

[0110] Statistical analysis

[0111] The statistical analysis was conducted on anonymized data entered into a cleaned and locked database. The Kolmogorov-Smirnov test was used to assess the normality of continuous variables, which were reported as mean (SD) if normally distributed, or as median and interquartile range (IQR) if not. Categorical variables were presented as counts and percentages. Depending on the data distribution, paired Student’s t-tests or Mann-Whitney U tests were used to compare values at baseline and after 6 months. A p-value of <0.05 was considered statistically significant. Analyses were performed using SPSS (version 23.0) and GraphPad Prism 7.

[0112] Table 4

[0113] References

[0114] 1) Shin YH, Hwang J, Kwon R, Lee SW, Kim MS; GBD 2019 Allergic Disorders Collaborators; Shin JI, Yon DK. Global, regional, and national burden of allergic disorders and their risk factors in 204 countries and territories, from 1990 to 2019: A systematic analysis for the Global Burden of Disease Study 2019. Allergy. 2023 Aug;78(8):2232-2254. doi: 10.1111 / all.15807. Epub 2023 Jul l l. PMID: 37431853; PMCID: PMC10529296.

[0115] 2) Wang J, Zhou Y, Zhang H, Hu L, Liu J, Wang L, Wang T, Zhang H, Cong L, Wang Q. Pathogenesis of allergic diseases and implications for therapeutic interventions. Signal Transduct Target Ther. 2023 Mar 24;8(1):138. doi: 10.1038 / s41392-023-01344-4. PMID: 36964157; PMCID: PMC10039055.

[0116] 3) Vassilopoulou E, Venter C, Roth-Walter F. Malnutrition and Allergies: Tipping the Immune Balance towards Health. J Clin Med. 2024 Aug 11;13(16):4713. doi: 10.3390 / jcml3164713. PMID: 39200855; PMCID: PMC11355500. 4) Berni Canani R, Gilber JA, Nagi er CR (2015) The role of the commensal microbioma in the regulation of tolerance to dietary allergens. Curr Opin Allergy Clin Immunol 15:243- 249; 5) Pantazi AC, Mihai CM, Balasa AL, Chisnoiu T, Lupu A, Frecus CE, Mihai L, Ungureanu A, Kassim MAK, Andrusca A, Nicolae M, Cuzic V, Lupu VV, Cambrea SC. Relationship between Gut Microbiota and Allergies in Children: A Literature Review. Nutrients. 2023 May 29; 15(11):2529. doi: 10.3390 / nul5112529. PMID: 37299492; PMCID: PMC10255222.

[0117] 6) De Filippis F, Paparo L, Nocerino R, Della Gatta G, Carucci L, Russo R, Pasolli E, Ercolini D, Berni Canani R. Specific gut microbiome signatures and the associated pro- inflamatory functions are linked to pediatric allergy and acquisition of immune tolerance. Nat Commun. 2021 Oct 13; 12(1):5958. doi: 10.1038 / s41467-021-26266-z. PMID: 34645820; PMCID: PMC8514477.

[0118] 7) Venter C. Immunonutrition: Diet Diversity, Gut Microbiome and Prevention of Allergic Diseases. Allergy Asthma Immunol Res. 2023 Sep;15(5):545-561. doi: 10.4168 / aair.2023.15.5.545. PMID: 37827976; PMCID: PMC10570780.

Claims

CLAIMS1. A nutritional composition comprising butyric acid or butyrate, inactivated Lactobacillus rhamnosus GG, Perilla frutescens dry extract, and quercetin.

2. The nutritional composition according to claim 1, further comprising one or more prebiotic substances and / or one or more micronutrients.

3. The nutritional composition according to claim 2, wherein the one or more prebiotic substances are selected from the group consisting of 2’-fucosyllactose, resistant starch, inulin and fructo-oligosaccharides (FOSs), and wherein the one or more micronutrients are selected from the group consisting of vitamins, Q-3 polyunsaturated fatty acids and trace elements.

4. The nutritional composition according to claim 3, wherein the 'Q-3 polyunsaturated fatty acids are selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A) and combinations thereof.

5. The nutritional composition according to claim 3 or 4, wherein the vitamins are selected from the group consisting of vitamin A, vitamin D, vitamin E and vitamin B 12 and combinations thereof.

6. The nutritional composition according to any of claims 3 to 5, wherein the trace elements are selected from the group consisting of iron, zinc, selenium, salts thereof and any combination thereof.

7. The nutritional composition according to any of claims 1 to 6, further comprising diamine oxidase (DAO).

8. The nutritional composition according to any of claims 1 to 7, further comprising Ribes nigrum extract or macerate.

9. The nutritional composition according to any of claims 1 to 8, further comprisingScutellaria baicalensis dry extract.

10. The nutritional composition according to any of claims 1 to 9, further comprising Zingiber officinalis rhizome dry extract.

11. The nutritional composition according to any of claims 1 to 10, which is in a unit dosage form.

12. The nutritional composition according to claim 11 , which comprises, per unit dosage form, 108-10nCFU of inactivated L.rhamnosus GG, 50-500 mg oiVerilla frutescens dry extract, 20- 200 mg of quercetin, 150-1000 mg of butyric acid or butyrate.

13. The nutritional composition according to claim 12, which further comprises, per unit dosage form, 2.9-4 g of 2’-fucosyllactose, and / or 1-10 g of resistant starch, and / or 1-12 g of inulin, and / or 2-10 g of FOSs, and / or 250-750 pg of vitamin A, and / or 15-35 pg of vitamin D, and / or 5-60 mg of vitamin E, and / or 1.5-1000 pg of vitamin B12, and / or 1-5 g of ' -3 polyunsaturated fatty acids, and / or 7-20.5 mg of iron in any pharmaceutically acceptable form, and / or 2.9-13 mg of zinc in any pharmaceutically acceptable form, and / or 15-70 pg of selenium in any pharmaceutically acceptable form, and / or 0.3-0.9 mg of diamine oxidase (DAO), and / or 50-250 mg of Ribes nigrum, and / or 10-50 mg of Zingiber officinalis rhizome dry extract, and / or 50-200 mg of Scutellaria baicalensis dry extract.

14. The nutritional composition according to any of claims 1 to 13, for use in the therapeutic or preventive treatment of an allergic disease or condition in a subject, preferably a pediatric subject.

15. The nutritional composition for use according to claim 14, wherein the allergic disease or condition is cow’s milk protein allergy (CMP A) or an allergic reaction.

16. The nutritional composition for use according to claim 14 or 15, wherein the nutritional composition improves immune tolerance, and / or enhances gut barrier integrity, and / or reduces pro-allergic cytokines in said subject.

17. A combined preparation for the simultaneous, separate or successive administration in the therapeutic or preventive treatment of an allergic disease or condition in a subject, wherein the combined preparation comprises a nutritional composition according to any one of claims 1 to 13 and one or more further anti-allergic pharmaceutical active ingredients preferably selected from the group consisting of anti -histamines, antileukotrienes corticosteroids, bronchodilators, biologies, immunotherapy agents and immune-modulating agents, wherein the subject is preferably a pediatric subject and the allergic disease or condition is preferably CMPA or an allergic reaction.

Images