Fermentation products for treatment of inflammatory diseases

Abstract

The present invention relates to a fermentation product obtained by a fermentation process of at least one bacterial strain in the presence of at least one plant extract. The fermentation product of the bacteria may comprise a food and / or pharmaceutically acceptable ingredient, or be included in a medical food or nutritional composition. The fermentation product is used for treating and / or preventing inflammatory diseases, in particular inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to a fermentation product obtained by a fermentation process of at least one bacterial strain in the presence of at least one plant extract. The bacterial fermentation product may contain food and / or pharmaceutically acceptable ingredients, or be contained in a medical food or nutritional composition. The fermentation product is used to treat and / or prevent inflammatory diseases, in particular inflammatory bowel disease. Background Art

[0002] The intestinal microbiota is composed of a group of microorganisms present in the human intestine. It is composed of more than one thousand pathogenic bacteria or symbiotic bacteria, of which one hundred and fifty to five hundred are located in the colon. The microbiota establishes a symbiotic and mutually beneficial relationship with the host by digesting carbohydrates, oligosaccharides and producing certain types of vitamins (such as B vitamins, K vitamins and folic acid). In addition, the intestinal microbiota interacts with the innate and adaptive immune systems by producing bacteria-associated molecular patterns (MAMPs) recognized by specific intestinal immune cell receptors (BecattiniS et al., Trends Mol Med, 2016 22: 458-78).

[0003] The change in the quantity and quality of bacterial species compared to the symbiotic conditions of the microbiota of healthy subjects is called dysbiosis. Specifically, bacterial dysbiosis can inhibit the expression of epithelial junction proteins and affect the immune system's recognition of bacteria, leading to abnormal immune responses.

[0004] Various therapeutic strategies have been developed to combat gastrointestinal diseases by modulating the microbiota, such as the use of prebiotics (foods that cannot be digested by humans but have beneficial effects on the microbiota), probiotics (live microorganisms administered orally), postbiotics (probiotic-derived metabolites), or transplantation of fecal microbiota from healthy donors.

[0005] In particular, probiotics are often used as adjuncts to the treatment of inflammatory bowel disease, allergic phenomena, urinary tract infections, colon cancer, hypercholesterolemia and constipation. Lactobacilli are naturally occurring bacteria in the human body that can be ingested in the form of food (especially yogurt and other fermented dairy products) or dietary supplements and have a variety of beneficial effects on human health. For example, they are able to promote food digestion, nutrient absorption, and fight pathogenic microorganisms that can cause various problems in humans and animals. Their intake is recommended for gastrointestinal diseases and for the prevention of gastrointestinal diseases during antibiotic treatment, as well as for the treatment and / or prevention of diarrhea, irritable bowel syndrome, childhood colic, Crohn's disease and necrotizing enterocolitis. In addition, they are recommended for the treatment of Helicobacter pylori infections in the stomach, urinary tract infections and vaginal infections. Finally, Lactobacilli are sometimes used to prevent colds, reduce high cholesterol, Lyme disease and skin diseases.

[0006] Many studies have reported that probiotics can play an important role in maintaining intestinal homeostasis by regulating immunity and increasing epithelial barrier function. Clinical studies and systematic meta-analyses have shown that certain probiotic strains are beneficial for specific patients. Beom J.L. et al. described in J Neurogastroenterol Motil. 2011, 17 (3), 252-266 that the use of probiotics has long been an alternative therapy to conventional drugs for the treatment of many diseases (such as the treatment of IBS). In the context of dysbiosis as a pathogenesis of IBS, probiotic therapy seems reasonable and perhaps ideal because it can restore the intestinal microbiota.

[0007] Guendalini S described the use of Lactobacillus CG (Lactobacillus GG), Lactobacillus reuteri DSM 17938 (Lactobacillus reuteri DSM 17938) and the probiotic mixture VSL#3 in pediatric patients with diarrhea-predominant or post-infectious IBS in Front. Med. on August 28, 2014.

[0008] Liu Yang described in Engineering 2021, Vol. 7, No. 3, 376-38 that the use of Lactobacillus plantarum CCFM8610 can significantly alleviate the clinical symptoms and intestinal microbial dysbiosis in patients with IBS-D. The alleviating effect of Lactobacillus plantarum CCFM8610 on IBS-D may be related to the increase in the relative abundance of butyrate-producing bacterial genera in the intestine.

[0009] Park JS et al. reported in J Med Food 2018, 21(3), 215-224 that treatment with Lactobacillus acidophilus reduced the severity of dextran sulfate sodium (DSS)-induced colitis. Specifically, Lactobacillus acidophilus inhibits proinflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor-α, IL-1β, and IL-17 produced by helper T cells (Ths) 17 in colonic tissue. In addition, in vitro treatment with Lactobacillus acidophilus directly induced regulatory T cells (Treg) and IL-10 production, while IL-17 production in splenocytes was inhibited.

[0010] Hill C et al. described in Nat. Rev. Gastroenter. Hepatol 2014, 11 (8), 506 that administration of live bacteria brings benefits to the host. Probiotics produce beneficial effects through multiple mechanisms, such as immunomodulation, pain, and changes in nervous system signals. Specifically, probiotics can be integrated into the intestinal microbiota, affecting its composition and activity, including stimulating the existing microbiota through nutritional interactions and inhibiting and / or reducing pathogens by changing the intestinal microbial environment.

[0011] Phytotherapy, especially traditional Chinese medicine, can also help prevent and treat diseases associated with intestinal inflammation. Herbal remedies exert their therapeutic effects through multiple mechanisms, including immunomodulation, antioxidant activity, inhibition of leukotriene B4 and nuclear factor-kB (NF-kB), and antiplatelet activity.

[0012] Triantafyllidi A et al. reported in Ann Gastroenterol. 2015, 28(2): 210-220 that the most important clinical trials conducted so far involve the use of mastic gum, acacia extract, wormwood, aloe, wheat, germinated barley and Boswellia serrata. In ulcerative colitis, aloe vera gel, wheat, andrographis paniculata extract and topical tin powder are superior to placebo in inducing remission or clinical response, while curcumin is superior to placebo in maintaining remission; Boswellia serrata and psyllium seeds in the form of gommoresin are as effective as mesalamine, and evening primrose has a similar relapse rate to ω-3 fatty acids in the treatment of ulcerative colitis. In Crohn's disease, mastic gum, wormwood and tripterygium wilfordii are superior to placebo in inducing remission and preventing postoperative clinical relapse, respectively.

[0013] Lopes Andrade AW et al., Front. Pharmacol., July 29, 2020, 998, report on the beneficial effects of herbal products rich in bioactive compounds with immunomodulatory and antioxidant properties, such as the case of Brugmansia sibiricum (Crassulaceae), a plant used in traditional Brazilian medicine to treat inflammatory diseases.

[0014] Flavonoids are hydroxypolyphenolic molecules abundant in plants, including vegetables, which are the main dietary source of these compounds for humans, as well as wine and tea. Flavonoids have attracted much attention for their beneficial effects on health and disease prevention. Of greatest interest is their antioxidant activity, as they have been shown to have excellent free radical scavenging abilities. However, increasing evidence suggests that flavonoids have many other biological properties, including anti-inflammatory, antiviral, anticancer, and neuroprotective effects through different mechanisms of action.

[0015] In addition to plants, some foods that are routinely consumed in the East, such as legumes, such as mung beans (Phaseolusradiatus or Vigna radiata L.), are also used to treat or prevent certain diseases. Kalim A et al. reported in Pharmacognosy and Phytochemistry 2021; 10(2), 54 that mung beans (Vigna radiata) are one of the most important legume crops and are grown in tropical to subtropical regions around the world. Mung beans are reported to help prevent the risk of hypercholesterolemia, coronary heart disease, and reduce the absorption of toxic substances.

[0016] Tang et al. described the antioxidant, antimicrobial, anti-inflammatory, antidiabetic, antihypertensive, lipid metabolism regulating, antihypertensive and anticancer effects of mung bean, a commonly used medicinal plant, in Chemistry Central Journal 2014, 8:4. Mung bean sprouts contain higher concentrations of nutrients and bioactive compounds than seeds. Mung bean sprouts contain polyphenols, polysaccharides, peptides and isoflavones, which have pharmacological properties such as hypoglycemic, hypolipidemic, antihypertensive, anticancer, hepatoprotective and immunomodulatory effects, which have been determined in vitro and in animal models.

[0017] Toledo O. et al. reported in Food Chemistry 127(3), 1175-1185 that the hydrolyzate of mung bean (common bean) can be used to combat inflammation and oxidation-related diseases, demonstrating that beans significantly inhibited NF-κB transcriptional activation and nuclear translocation of NF-κB p65 subunit.

[0018] Chao WW et al. reported in Journal of Medicinal Food Vol. 18, No. 7 that red bean has anti-inflammatory effects and has the potential to be a beneficial ingredient, and described that at concentrations of 50 μg / mL to 200 μg / mL, it can significantly inhibit the inflammatory response of LPS-stimulated macrophages in a dose-dependent manner by reducing cellular NO and downregulating iNOS, COX-2, TNF-α and IL-6 gene expression. In addition, it can also reduce H2O2-induced oxidative damage in macrophage RAW 264.7.

[0019] The activity of plants or plant extracts may be affected by bacteria. CG Rizzello et al., Microbial Cell Factories 2013, 12:44 reported that plant extracts fermented with lactobacilli have beneficial effects on organisms. For example, fermentation of Echinacea and Lactobacillus plantarum was reported to have a better antimicrobial effect than Echinacea alone.

[0020] Oh NS et al., J. Dairy Sci., 2020, 103: 2947-2955 reported that milk fermented with extracts of Cudrania tricuspidata and Lactobacillus 505 had stronger antioxidant properties than Cudrania tricuspidata alone, and the resulting product had a hepatoprotective effect against colorectal cancer and induced liver metastasis.

[0021] WO 2020 / 0070087 describes the use of a combination of Bifidobacterium longum and Bifidobacterium lactis for the treatment of Crohn's disease, wherein the probiotic composition increases the concentration of anti-inflammatory cytokines.

[0022] US 2007 / 298019 describes a composition for treating inflammatory bowel disease, comprising Lactobacillus delbrueckii ssp. Bulgaricus and a plant component.

[0023] KR 2021-0128947 describes the use of Lactobacillus sachsii that is able to convert soybean roots or extracts into useful isoflavone metabolites, exerting their antioxidant properties.

[0024] However, there is still a need for alternative compositions with anti-inflammatory activity that are acceptable to patients, in particular compositions with intestinal anti-inflammatory activity that are easily accepted by patients for use in humans and / or animals.

[0025] There is also a need for effective, especially naturally derived, compositions for use as alternatives to and / or in combination with antibiotics to reduce the amount of antibiotics used, without the development of antibiotic resistance, and that are well tolerated by patients and do not develop resistance. The nutritional compositions are also suitable for patients receiving other therapies, where additional antibiotic treatment may produce adverse reactions, as well as for immunocompromised patients and pediatric patients.

[0026] Surprisingly, it has been found and forms the subject of the present invention, a nutritional composition comprising a fermentation product obtained by a fermentation process of at least one bacterial species in the presence of at least one plant extract. The composition comprising the fermentation product of the present invention combines the efficacy of lactobacilli with the efficacy of plant matrix metabolites obtained for their action.

[0027] The composition of the present invention is used for treating and / or preventing inflammatory diseases, such as inflammatory bowel disease. Summary of the invention

[0028] The object of the present invention is to provide a fermentation product obtained by a fermentation process with a strain of Lactobacillus brevis deposited at the German Collection of Microorganisms and Cell Cultures of the Leibniz Institute under the DSM number DSM 33682 in the presence of at least one plant extract selected from the group consisting of Scutellaria baicalensis Georgi, Boswellia serrata, Phaseolus, Curcuma longa, Camellia sinensis, broccoli sprouts and mixtures thereof.

[0029] The object of the present invention is to provide a composition comprising a fermentation product obtained by fermentation of the bacterial strain Lactobacillus brevis deposited at the German Collection of Microorganisms and Cell Cultures of the Leibniz Institute under the number DSM 33682 in the presence of at least one plant extract selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Phaseolus, Curcuma longa, Tea, Broccoli sprouts and mixtures thereof.

[0030] The object of the present invention is to provide a method for obtaining a fermentation product by fermenting a Lactobacillus brevis strain deposited at the German Collection of Microorganisms and Cell Cultures of the Leibniz Institute under the DSM number DSM 33682 in the presence of at least one plant extract selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Phaseolus, Curcuma longa, Tea, Broccoli sprouts and mixtures thereof.

[0031] The object of the present invention is to provide a process for obtaining a pharmaceutical or nutritional composition comprising a fermentation product according to the invention obtained in the presence of at least one plant extract, and a pharmaceutical or food excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: Colonic injury scores and disease activity index in mice treated with Prob1. T-test was first performed for the vehicle group versus the TNBS group, with p values ​​< 0.001 (●●●) and p < 0.0001 (●●●●). One-way ANOVA (or equivalent) was applied to compare all other groups with TNBS, with p values ​​< 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

[0033] Figure 2: MPO levels measured in the colon of mice treated with TNBS-induced colitis. T-test was first performed for the vehicle group versus the TNBS group, with p-values ​​< 0.0001 (●●●●). One-way ANOVA (or equivalent) was applied to compare all other groups with TNBS, with p-values ​​< 0.01 (**), p< 0.001 (***), and p< 0.0001 (****).

[0034] Figure 3: mRNA of pro-inflammatory cytokines measured in the colon of mice treated with TNBS-induced colitis. T-test was first performed to compare the vehicle group with the TNBS group, with p value < 0.01 ( ●● ). All other groups were compared with TNBS using one-way ANOVA (or equivalent method) with p values ​​< 0.05 (*), p < 0.01 (**), and p < 0.0001 (****).

[0035] Figure 4: mRNA of immune cells measured in the colon of mice treated with TNBS-induced colitis. T-test was first performed for the vehicle group versus the TNBS group, with p-values ​​< 0.01 (●●). One-way ANOVA (or equivalent) was applied to compare all other groups with TNBS, with p-values ​​< 0.05 (*), p < 0.01 (**), and p < 0.0001 (****).

[0036] Figure 5: Zonulaoccludens 1 protein levels measured in the colon of TNBS-induced colitis treated mice. T-test was first performed for vehicle vs. TNBS, p value < 0.0001 (●●●●). All other groups were compared to TNBS using one-way ANOVA (or equivalent method), p value < 0.05 (*) and p < 0.01 (**).

[0037] Figure 6: Protein levels of immune cells measured in the colon of TNBS-induced colitis treated mice. T-test was first performed for the vehicle group versus the TNBS group, with p values ​​< 0.001 (●●●) and p < 0.0001 (●●●●). One-way ANOVA (or equivalent) was applied to compare all other groups with TNBS, with p values ​​< 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

[0038] Figure 7: β-diversity measured in feces of mice treated with TNBS-induced colitis - (a) dominant phyla; (b) dominant families. T-test was first performed to compare the vehicle group with the TNBS group: p value < 0.05 ( ●● ). All other groups were compared with TNBS using one-way ANOVA (or equivalent method) with p values ​​< 0.05 (*) and p < 0.01 (**). DETAILED DESCRIPTION

[0039] The present invention describes a fermentation product obtained by a fermentation process of a Lactobacillus strain in the presence of at least one plant extract.

[0040] The present invention describes a fermentation product obtained by a fermentation process of a Lactobacillus brevis strain (deposited at the German Collection of Microorganisms and Cell Cultures of the Leibniz Institute, DSM No. 33682) in the presence of at least one plant extract selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Phaseolus, Mung bean, Mung bean sprouts, Turmeric, tea, broccoli sprouts and mixtures thereof.

[0041] The fermentation product may comprise the probiotic Lactobacillus brevis DSM No. 33682, in the presence of a supernatant produced by the same fermentation process in an amount of 0% to 30% (p / p) of the fermentation mass weight.

[0042] The amount of supernatant is regulated by the steps of centrifugation, freeze drying or spray drying process.

[0043] The plant extract used in the present invention is characterized by having antioxidant properties and may be in the form of a solid extract.

[0044] The probiotic Lactobacillus brevis DSM 33682 was fermented in the culture medium, and the characteristics of the obtained product were 1x10 8 CFU / ml to 1x10 10 CFU / ml was fermented in the presence of a plant substrate selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Mung bean sprouts, Turmeric, tea, broccoli sprouts and mixtures thereof.

[0045] The probiotic Lactobacillus brevis DSM 33682 was added at 1x10 8 CFU / ml to 1x10 10 The amount of CFU / ml is inoculated into a culture medium, and the culture medium is supplemented with a plant matrix selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Phaseolus, Mung bean, Mung bean sprouts, Turmeric, tea, broccoli sprouts and a mixture thereof.

[0046] One aspect of the present invention is a product obtained by fermentation of Lactobacillus brevis DSM 33682 in the presence of Phaseolus extract. Preferably, the product is obtained by fermentation of Lactobacillus brevis DSM 33682 in the presence of mung bean sprout extract. More preferably, the mung bean sprout extract is in lyophilized form.

[0047] The product of the present invention is prepared by inoculating Lactobacillus brevis DSM 33682 at 1×10 8 CFU / ml to 1x10 10CFU / ml is obtained by inoculating into a medium to which a mung bean extract of 5 g / L to 15 g / L higher than the fermentation broth concentration is added. Preferably, the product is obtained by fermentation of Lactobacillus brevis DSM 33682 in the presence of a mung bean sprout extract of 5 g / L to 15 g / L higher than the fermentation broth concentration.

[0048] The fermentation product of the present invention is characterized in that it is obtained by a process comprising the following steps:

[0049] (a) Add 1x10 8 CFU / ml to 1x10 10 CFU / ml amount of bacterial strain Lactobacillus brevis DSM33682;

[0050] (b) adding Phaseolus extract, Mung bean extract or Mung bean sprout extract to the culture medium at a concentration of 5 g / L to 15 g / L based on the volume of the fermentation product;

[0051] (c) maintaining mild agitation at a temperature between 30°C and 40°C for 8 hours to 24 hours under anaerobic or aerobic conditions;

[0052] (d) centrifuging at a speed of less than 4000 rpm for 5 minutes to 40 minutes;

[0053] (e) Freeze-drying or freezing.

[0054] The culture medium may be any commercial culture medium known to those skilled in the art that is suitable for growing lactobacilli.

[0055] These media contain nutrients, buffers, and are sterile to prevent any contamination. The soil may contain:

[0056] (a) Peptones: A group of water-soluble compounds obtained by hydrolysis (acid or enzyme) of proteins (casein, soy protein, etc.);

[0057] (b) NaCl: Add at appropriate concentration according to osmotic pressure requirement.

[0058] (c) Sugar: Glucose, lactose, mannitol are added to specific soils for specific purposes;

[0059] (d) Yeast, meat, and organ extracts: provide growth factors and inorganic salts;

[0060] (e) Concentrates: lysed blood, hemoglobin, dehydrated milk, gelatin vitamins.

[0061] Some media may contain selective supplements such as antibiotics, bile salts, and colorimetric indicators to follow the fermentation metabolism of the bacteria under study by a color change of the medium at a critical pH.

[0062] The Phaseolus extract or Mung bean extract or Mung bean sprout extract used to prepare the fermentation product of the present invention is characterized by an oxidation-reduction potential of 100 mV to 400 mV when in a phosphate buffer at a concentration of 0.5% (w / v) to 20% (w / v).

[0063] In the presence of Phaseolus extract or mung bean extract or mung bean sprout extract, the fermentation product of Lactobacillus brevis DSM 33682 can be obtained by fermenting for 8 hours to 24 hours at a temperature of 30°C to 40°C under anaerobic or aerobic conditions (preferably anaerobic). At the end of the fermentation, the solution is centrifuged at a speed lower than 4000rpm, preferably between 200rpm to 3000rpm, for 5 minutes to 40 minutes. Preferably, the solution is centrifuged at a speed of 3000rpm for 10 minutes. The concentration multiple of the fermentation product obtained from the fermentation broth is 6 to 10 times that of the fermentation broth. The fermentation product can be frozen, lyophilized or dried by spray drying.

[0064] The process for obtaining a fermentation product of Lactobacillus brevis DSM33682 in the presence of a Phaseolus extract, a Mung bean extract or a Mung bean sprout extract comprises the following steps:

[0065] (a) Lactobacillus brevis DSM 33682 was added at 1×10 8 CFU / ml to 1x10 10 The amount of CFU / ml was placed in the culture medium;

[0066] (b) adding a Phaseolus extract, a Mung bean extract, or a Mung bean sprout extract to the medium at a concentration 5 g / L to 15 g / L higher than that of the fermentation broth.

[0067] (c) under anaerobic or aerobic conditions at a temperature of 30°C to 40°C with mild agitation for 8 hours to 24 hours;

[0068] (d) centrifuging at a speed of less than 4000 rpm for 5 minutes to 40 minutes;

[0069] (e) Freeze drying or freezing.

[0070] A fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of a Phaseolus extract or a Mung bean extract or a Mung bean sprout extract under anaerobic conditions is characterized by having anti-inflammatory activity, as shown by in vitro assays that inhibit IL-1β mRNA transcription. It was observed that the inhibitory effect of the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of the Mung bean sprout extract on IL-1β mRNA transcription was at least three times higher than that of the fermentation product obtained in the presence of the bacteria alone. Furthermore, the inhibitory effect of the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of the Mung bean sprout extract on IL-1β mRNA transcription was comparable to the inhibitory effect of mesalazine.

[0071] The fermentation product of Lactobacillus brevis DSM 33682 obtained under anaerobic or aerobic conditions (preferably anaerobic) in the presence of mung bean sprouts is characterized by having an in vivo anti-inflammatory activity. It may be in a solid form obtained by freeze drying or spray drying.

[0072] Surprisingly, it was found that when the lyophilized fermentation product of the present invention was 7 CFU / g is about 1x10 9 CFU / g, in the 2,4,6, trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis animal model, the fermentation product restored colon length to a greater extent than the animal group treated with the fermentation product obtained in the absence of the plant extract, and its effectiveness was found to be comparable to that of mesalazine.

[0073] Will be about 10 7 CFU is about 10 9 The fermentation product in freeze-dried form of CFU was administered to an animal model of ulcerative colitis induced by 2,4,6, trinitrobenzene sulfonic acid (TNBS) and was found to reduce the DAI score (a disease activity index score used to evaluate colitis, including parameters such as weight loss and stool consistency) and inhibit the shortening of the colon (inflammatory index) of the animals in a manner comparable to that of the mesalazine-treated animal group.

[0074] The lyophilized fermentation product of the present invention has a positive effect on TNBS-induced colonic injury in a mouse model. Importantly, the activity of myeloperoxidase (MPO) was reduced in the colon tissue of mice to a level comparable to that of the group treated with mesalazine. Myeloperoxidase (MPO) is a well-known marker of tissue damage and neutrophil infiltration, which can induce mucosal destruction and ulcers closely related to ulcerative colitis.

[0075] The expression levels of pro-inflammatory genes (including TNF-α, IL-1β) and the anti-inflammatory gene IL-10 in TNBS-induced colonic injury tissue in a mouse model were determined. IL-1β and TNF-α are believed to determine the degree of inflammation in IBD patients, and in fact their increase is positively correlated with the severity of IBD. Treatment of TNBS-induced acute colitis mice with mesalazine or the fermentation product of the present invention reduced the expression levels of pro-inflammatory genes in mice.

[0076] It was also surprisingly found that the expression of IL-10, a key factor in the pathogenesis of IBD that reduces TNBS-induced intestinal inflammation, was increased in both mesalazine and the fermentation product of the present invention treated groups in a comparable manner.

[0077] In addition, the fermentation product of the present invention significantly upregulated the expression of tight junction protein ZO-1 in the colon of TNBS-induced colitis mice to a level comparable to that of the mesalazine-treated group, while TNBS induction reduced its expression.

[0078] The differences in the intestinal microbiota of mice induced with TNBS were studied. Surprisingly, it was found that treatment with the fermentation product of the present invention improved the diversity of the intestinal microbiota, and the degree of improvement was comparable to that of the group treated with mesalazine. Specifically, at the phylum level, the administration of the product of the present invention reduced the percentage of Proteobacteria compared to the group treated with TNBS alone, and it is documented that Proteobacteria can produce enterotoxins that often cause gastroenteritis or allergic reactions.

[0079] At the family level, the relative abundance of Lachnospiraceae and Muribaculaceae, which were reduced by TNBS administration, were restored and further enriched in mice treated with the fermentation product of the present invention, and these families were associated with improvements in the ability to produce short-chain fatty acids and the ability to maintain probiotic conditions, respectively. Significant differences were also found in the Lactobacillaceae family, which was significantly reduced by TNBS application, but significantly increased by the application of the fermentation product of the present invention. The ability of Lactobacillaceae to improve inflammatory bowel disease (IBD) and modulate the immune system is particularly significant and well known, which is attributed to multiple factors, such as the production of protective molecules or downregulation of the production and release of proinflammatory cytokines (IL-6, IL-1β, IL-2 and TNF-α).

[0080] The activity of the fermentation product of the present invention was compared in vivo with that of other bacterial products, namely the probiotic mixture VSL#3 (composed of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii subspecies bulgaricus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus salivarius subspecies thermophilus) for maintaining remission in UC patients (Cheng et al., World J Clin Cases. 2020 Apr 26;8(8):1361–1384) and Escherichia coli Nissle for preventing relapse in UC patients (Kruis et al., Gut. 2004 Nov;53(11):1617-23). Surprisingly, the weight loss of mice treated with the fermentation product of the present invention was 50% lower than that of mice treated with VSL#3 or E. coli Nissle. The activity was also demonstrated by measuring the DAI score, colon injury score and colon length, which were improved in a dose-dependent manner in mice treated with the product of the present invention compared to the VSL#3 and E. coli Nissle treatment groups.

[0081] Another object of the present invention is to provide a nutritional or food composition comprising a fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of a plant extract of Phaseolus or Mung bean (preferably Mung bean sprouts) and an excipient suitable for oral administration.

[0082] In particular, the nutritional or food composition comprising a fermentation product of Lactobacillus brevis (DSM 33682) obtained in the presence of a plant extract of Phaseolus or Mung bean (preferably Mung bean sprouts) is in the form of capsules, sachets or granules in solution, or a stick for oral administration.

[0083] The composition may contain other ingredients such as, for example, prebiotics, vitamins (such as vitamin A, vitamin D, vitamin K3, B vitamins), amino acids, and trace elements (such as zinc, selenium, and iron).

[0084] The excipients of the composition of the invention according to the selected form are known to those skilled in the art and can be selected, for example, from the group consisting of diluents, glidants, buffers, stabilizers, lubricants, disintegrants, sweeteners, anticaking agents and preservatives.

[0085] Diluents suitable for preparing the composition of the present invention are selected from cellulose, microcrystalline cellulose, calcium phosphate, starch, kaolin, anhydrous calcium sulfate or hydrated calcium sulfate or dihydrated calcium sulfate, calcium carbonate, lactose, sucrose, mannitol, polysaccharides, dextran, xyloglucan, starch, natural gums, malt, gelatin and mixtures thereof.

[0086] The buffer or pH corrector is selected from potassium or sodium salts, sodium hydroxide or potassium hydroxide and mixtures thereof.

[0087] The glidant may be selected from the group consisting of talc, microcrystalline cellulose and magnesium carbonate.

[0088] Lubricants suitable for preparing the composition of the present invention are selected from the group consisting of glyceryl dibenzoate, calcium stearate or magnesium stearate, aluminum, sodium stearyl fumarate, hydrogenated vegetable oil, palmitic acid, alcohol, starch, mineral oil, polyethylene glycol, sodium lauryl sulfate, talc, glycerides, sodium benzoate and mixtures thereof.

[0089] Suitable disintegrants for use in preparing the compositions of the present invention are selected from the group consisting of cellulose derivatives such as sodium carboxymethylcellulose (also known as carboxymethylcellulose), cross-linked carboxymethylcellulose (also known as croscarmellose), hydroxypropyl methylcellulose, hydroxypropyl cellulose phthalate, polyvinyl acetate phthalate, povidone, copovidone and sodium starch glycolate.

[0090] Sweeteners suitable for preparing the composition of the present invention are selected from the group consisting of acesulfame potassium salt, maltodextrin, sorbitol, mannitol, isomalt, maltitol, lactitol, xylitol, aspartame, cyclamic acid, cyclamic acid salt, lactose, saccharin and saccharin salt.

[0091] Anticaking agents suitable for use in preparing the compositions of the present invention are selected from the group consisting of silica and talc.

[0092] Preservatives suitable for use in preparing the compositions of the present invention are selected from the group consisting of methylparaben, ethylparaben, sodium edetate, sodium benzoate, potassium sorbate, and mixtures thereof.

[0093] The fermentation product obtained by the fermentation process is in the form of powder or granules, with the content in a sachet ranging from 1 mg to 5 g.

[0094] The composition in the form of a mouth-dissolving stick comprises 1 mg to 1 g of a fermentation product obtained by the fermentation process of the present invention, 0.5 g to 1.5 g of a sweetener selected from isomalt, aspartame, xylitol, lactitol, sodium cyclamate, dextrose, fructose, glucose, lactose and sucrose, 1 mg to 50 mg of a flavoring agent, 1 mg to 50 mg of an anti-caking agent selected from colloidal silicon dioxide and talc, and 0 mg to 50 mg of vitamins.

[0095] The composition obtained by the fermentation process may be in freeze-dried or granulated form and stored in capsules in an amount of 1 mg to 1 g.

[0096] The composition in capsule form comprises 1 mg to 1 g of the fermentation product obtained by the fermentation process of the present invention, and 0 to 20 mg of one or more lubricants selected from talc, magnesium stearate, calcium stearate, stearic acid, hydrogenated vegetable oil, and polyethylene glycol; 0 to 20 mg of an anti-caking agent selected from silicon dioxide and talc; 0 to 300 mg of a sweetener selected from sucrose, sorbitol, mannitol, saccharin, acesulfame potassium, neohesperidin, and maltodextrin; and 0 to 50 mg of vitamins.

[0097] One object of the present invention is to provide a fermentation product itself or a fermentation product contained in an oral composition for use in treating and / or preventing intestinal inflammation.

[0098] The fermentation product or composition of the invention is useful for treating and / or preventing inflammatory bowel diseases or disorders, such as IBD, Crohn's disease, ulcerative colitis or diverticulitis.

[0099] The fermentation product or composition of the present invention is used to treat and / or prevent ulcerative colitis. Preferably, the fermentation product or composition of the present invention is used for subjects affected by ulcerative colitis. More preferably, the fermentation product or composition of the present invention is used for subjects affected by mild to moderate ulcerative colitis.

[0100] The fermentation product or composition can be used in a lyophilized form, or added to a food or pharmaceutically acceptable excipient, and formulated into capsules, soft gelatin capsules, tablets, orally dissolving tablets, stick-shaped granules or sticks, or solutions.

[0101] The fermentation product or composition for treating and / or preventing IBD, Crohn's disease, ulcerative colitis or diverticulitis of the present invention can be used alone or in combination or in conjunction with a concomitant therapy, particularly in combination or in conjunction with an anti-inflammatory drug. Preferably, the fermentation product of the present invention can be used alone or in combination or in conjunction with a non-steroidal anti-inflammatory drug (NSAID), preferably in combination or in conjunction with mesalazine.

[0102] The composition of the present invention can be administered for both cycling therapy and maintenance therapy without any limitation.

[0103] The fermentation product or composition of the present invention is administered 1 to 4 times a day, preferably 1 to 3 times a day, preferably once a day.

[0104] The fermentation product or oral composition of the present invention can be used in an amount of about 1×10 8 CFU is about 1x10 12 The amount of CFU is administered to humans one, two, three or four times per day for a treatment period of at least one week.

[0105] The fermentation product or composition of the present invention is used for subjects with ulcerative colitis or mild to moderate ulcerative colitis who are unresponsive to mesalazine.

[0106] In the context of the present invention, the term treatment refers to alleviating, alleviating, ameliorating or eliminating the symptoms associated with inflammatory bowel disease.

[0107] Example

[0108] Example 1: Determination of anti-inflammatory activity of plant extracts in vitro and in vivo

[0109] The anti-inflammatory activity of commercial plant extracts of Scutellaria baicalensis, Boswellia serrata, mung bean sprouts, turmeric, tea, or broccoli sprouts (Botanical Cube, Inc.) was analyzed.

[0110] Example 1: Scutellaria baicalensis

[0111] Example 2: Mung Bean (sprouts)

[0112] Example 3 Turmeric

[0113] Example 4 Tea

[0114] Example 5 Broccoli (sprouts)

[0115] Each plant extract was suspended in phosphate buffered saline (PBS) at a concentration of 1.2% (w / v), the pH value was adjusted to 7.4, and the solution was maintained at a temperature of 37°C for 24 hours under anaerobic conditions. After 24 hours, no bacteria grew in the solution. Each solution was centrifuged at a speed of 2500 rpm for 20 minutes, the insoluble residue was discarded, and the supernatant was filtered through a 0.22 μm PES filter.

[0116] Table 1 lists the solutions obtained for each extract.

[0117] Table 1

[0118] Plant Extracts Solution Scutellaria baicalensis Solution Example 1 Mung Bean (sprouts) Solution Example 2 turmeric Solution Example 3 Tea Solution Example 4 Broccoli (sprouts) Solution Example 5

[0119] The anti-inflammatory activity of the plant extract solutions was evaluated by in vitro and in vivo assays.

[0120] (a) The in vitro efficacy of the plant extracts was determined by measuring the amount of IL-10.

[0121] IL-10 values ​​were determined by ELISA assay according to Nemeth et al., J Immunol., 2005 175:8260-8270. 5 Cells / well RAW264.7 macrophages (ATCC) were seeded in 24-well plates (T=37°C, CO2=5% (v / v)). The cells were divided into 5 groups, and each group was treated with 20 μL of plant extract solution for 2 hours (solution examples 1 to 5).

[0122] Cells treated with mesalazine and short-chain fatty acids (propionate, acetate and butyrate) were used as positive controls. 10 μl of lipopolysaccharide (LPS) (Sigma-Aldrich n.2630 (10 μg / mL)) was added to each well and incubated for 24 hours in an incubator at 37°C with CO2 = 5% (v / v) to stimulate immune response. At the end of incubation, the supernatant was collected and the amount of IL-10 was detected by ELISA analysis (Invitrogen kit).

[0123] Table 2 shows the percentage values ​​of IL-10 produced by RAW264.7 macrophages and the percentage increase compared to LPS.

[0124] Table 2

[0125]

[0126] (b) Determination of in vivo efficacy of plant extracts

[0127] Based on the efficacy of the plants in the examples, c1

[0128] Ulcerative colitis was induced in a mouse model by administering 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Yang et al., Scientific Reports, 2016 6:29716).

[0129] Male C57BL / 6 mice aged 6 to 8 weeks were divided into groups of 8. Solutions of the plant extracts (Solutions Example 1 to Example 5) obtained in PBS at a concentration of 1.2% were administered 3 hours before induction with TNBS and then once a day for 3 days.

[0130] - Group A: 100 μL 50% ir + PBS intragastrically

[0131] - Group B: TNBS 2 mg / mouse, intragastrically administered in 100 μL ir+PBS

[0132] - Group C: TNBS 2 mg / mouse, ir in 100 μL + mesalazine 100 mg / kg gavage

[0133] -D group: TNBS 2mg / mouse, in 100μL ir + solution example 1 (Scutellaria baicalensis) / 600μL / mouse, gavage

[0134] -E group: TNBS 2mg / mouse, in 100μL ir + solution example 2 (mung bean) / 600μL / mouse, gavage

[0135] -F group: TNBS 2mg / mouse, in 100μL ir + solution example 3 (turmeric) / 600μL / mouse, gavage

[0136] -G group: TNBS 2mg / mouse, in 100μL ir + solution example 5 (broccoli sprouts) / 600μL / mouse, gavage

[0137] Mice were sacrificed by cervical dislocation 6 hours after the last administration.

[0138] Table 3 shows the parameters of weight loss and colon length of mice at the end of treatment (day 3).

[0139] Table 3

[0140]

[0141] Example 2: Characterization of mung bean sprout matrix

[0142] Based on the efficacy of the plants in Example 1, the green bean extract (Example 2) was selected among the plant extracts used, and its antioxidant activity and redox potential were determined.

[0143] (a) Determination of antioxidant activity.

[0144] Antioxidant activity was determined by the ORAC (oxygen radical absorbance capacity assay) test (Cat. No. STA-345, Cell Biolabs kit) according to the supplier's instructions by measuring the activities of the lipophilic part, the hydrophilic part and the total.

[0145] Table 4 shows the antioxidant activity values ​​obtained for the lipophilic fraction, the hydrophilic fraction and overall.

[0146] Table 4

[0147]

[0148] (b) Determination of redox potential

[0149] 1.0 g of the lyophilized extract of green bean (Example 2) was suspended in 100 mL of 10 mM PBS, pH 7.4, and stirred at 37°C for 30 minutes; the resulting suspension was centrifuged at 9800 x rpm for 20 minutes. TM The redox potential of the supernatant was measured by a digital multimeter (HQ40D, Hach) with MTC101 (Hach MTC101). The redox potential of the mung bean sprout extract was 200.2 mV.

[0150] Example 3: Fermentation of Lactobacillus brevis in the presence of mung bean sprout extract.

[0151] The inoculation concentration was 1.5×10 9 CFU / ml to 4.5x10 9 CFU / ml of Lactobacillus brevis (DSM 33682). The solution was fermented for 12 hours at 37°C + / - 0.5°C, with mild stirring and anaerobic conditions. At the end of the fermentation, the biomass was concentrated to a concentration factor of 7 ± 2 times by centrifugation at 3000 rpm at 4°C for 10 minutes. The obtained biomass was freeze-dried.

[0152] Total gain is 3.5x 10 11 CFU / g to 8.0x 10 11 CFU / g of 6.7 kg of lyophilized product (Lio-Prob1).

[0153] Example 4: Fermentation of Lactobacillus brevis in the absence of a plant substrate (comparative)

[0154] The inoculum concentration in 600L MRS medium was 1.5 x 10 9 CFU / ml to 4.5x10 9 CFU / ml of Lactobacillus brevis (DSM 33682). The solution was fermented for 12 hours at 37°C + / - 0.5°C, with mild stirring and anaerobic conditions. At the end of the fermentation, the biomass was concentrated to a concentration factor of 7 ± 2 times by centrifugation at 3000 rpm at 4°C for 10 minutes. The obtained biomass was freeze-dried.

[0155] Gain 6.7kg with 3.5x10 11 CFU / g to 8.0x 10 11 CFU / g of lyophilized product (Lio-Prob2).

[0156] The lyophilisate of Lio-Prob2 was analyzed in comparison with the product obtained by fermentation in the presence of mung beans (Example 3).

[0157] Example 5: In vitro activity determination of Lactobacillus brevis fermentation products in the presence of mung bean sprout extract

[0158] The fermentation product of Lactobacillus brevis DSM 33682 in freeze-dried form obtained in the presence of mung bean sprout extract according to Example 5 (Lio-Prob1) and the product obtained without mung bean sprout extract according to Example 6 (Lio-Prob2) were analyzed for anti-inflammatory activity by determining the amount of IL-1β mRNA.

[0159] 3x10 5 Cells / well RAW264.7 macrophages (ATCC) were seeded in 24-well plates.

[0160] The cells were divided into three groups and co-cultured with 20 μl of the above lyophilized fermentation product (diluted to a final concentration of 6×10 7 CFU / ml to 7.2x10 8 CFU / ml) or mesalazine (Sigma-Aldrich Y0000297) (0.1 mM) were incubated together. 10 μl of Sigma-Aldrich n.2630 lipopolysaccharide (LPS) (10 μg / mL) was added to each well and incubated in a 37°C 5% CO2 incubator for 24 hours.

[0161] At the end of incubation, macrophage RNA was extracted and analyzed by real-time PCR to detect the gene transcription level of the proinflammatory cytokine IL-1β, which was calculated as the mean value of the treatment group compared with macrophages treated only with LPS. Specifically, total RNA was isolated from each sample using the miniRNeasy kit (QiaGen GmbH, Hilden, Germany), and 1 μg of total RNA was reverse transcribed using the IScriptTM cDNA Synthesis kit (BioRad, Hercules, CA, USA). Real-time PCR was performed using SsoAdvanced UniversalSYBR Greensuper Mix (BioRad) by the BioRad CFX96 TouchTM real-time PCR detection system. The expression level of each mRNA was evaluated using the ΔΔCT method, and Gapdh was used as a housekeeping gene for normalization. The following primers were used.

[0162] IL-1β forward primer: 5′-CGAGGCAGTATCACTCATTG-3′;

[0163] IL-1β reverse primer: 5′-CGTTGCTTGGTTCTCCTTGT-3′;

[0164] GAPDH forward primer 5′-AACTTTGGCATTGTGGAAGG-3′;

[0165] GAPDH reverse primer 5′-CACATTGGGGGTAGGAACAC-3′.

[0166] Table 5 shows the inhibition levels of IL-1β mRNA transcription by the tested products after real-time PCR analysis.

[0167] Table 5

[0168]

[0169] Example 6: Determination of in vivo efficacy of Lactobacillus brevis fermentation products in the presence of mung bean sprout extract

[0170] The efficacy of the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence (Lio-Prob1) and absence (Lio-Prob2) of mung bean sprout extract was determined by measuring the DAI score and colon length.

[0171] Ulcerative colitis was induced with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Yang et al., Scientific Reports, 2016 6:29716) in 6- to 8-week-old male C57BL / 6 mice (Envigo) (8 mice / group).

[0172] The mice were divided into several groups of 8 each, and the freeze-dried fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of mung bean extract as obtained in Example 3 (Lio-Prob1) or the freeze-dried fermentation product of Lactobacillus brevis DSM 33682 obtained in Example 6 (Lio-Prob2) was orally administered to the mice starting 3 hours before the administration of TNBS and then every 24 hours for the next three days.

[0173] - Group A: 100 μL 50% rectal administration + PBS gavage

[0174] - Group B: TNBS 2 mg / mouse, intragastrically administered in 100 μL ir+PBS

[0175] - Group C: TNBS 2 mg / mouse, ir in 100 μL + mesalazine 100 mg / kg gavage;

[0176] -D group: TNBS 2mg / mouse, ir+Lio-Prob1 in 100μL; 2mg / 500μL / mouse, gavage

[0177] -E group: TNBS 2mg / mouse, ir+Lio-Prob1 in 100μL; 1mg / 500μL / mouse, gavage

[0178] -F group: TNBS 2mg / mouse, ir+Lio-Prob1 in 100μL; 0.5mg / 500μL / mouse, gavage

[0179] -G group: TNBS 2mg / mouse, ir+Lio-Prob1 in 100μL; 0.25mg / 500μL / mouse, gavage

[0180] -H group: TNBS 2mg / mouse, ir+Lio-Prob1 in 100μL; 0.125mg / 500μL / mouse, gavage

[0181] -L group: TNBS 2 mg / mouse, ir+Lio-Prob2 in 100 μL; 0.8 mg / 500 μL / mouse, 6 hours after the last oral administration, the mice were killed by cervical dislocation.

[0182] The disease activity index of C57BL / 6 mice was measured according to the method reported by Yang et al. Scientific Reports, 2016 6: 29716, expressed as DAI score. The disease index is based on average weight loss, stool consistency and fecal bleeding (value 0 = no effect on body weight, normal stool consistency, no fecal bleeding; value 1 = weight loss of 1% to 5%; value 2 = weight loss between 5% and 10%, soft feces, slight bleeding; value 3 = weight loss between 10% and 15%; value 4 = weight loss greater than 15%, diarrhea and bleeding).

[0183] Table 6 shows the DAI scores and colon lengths of each treatment animal group.

[0184] Table 6

[0185]

[0186] *P<0.05, ****P<0.0001 vs group B (ANOVA); ns=not significant.

[0187] Example 7: Dose response assay for treatment of mice

[0188] C57BL / 6 male mice (ENVIGO, Italy) aged 7 to 8 weeks were housed under standard conditions: light / dark cycle (12 / 12 h), ambient temperature 20±2° C., relative air humidity 55%, with ad libitum access to food (Mucedola RF18) and water.

[0189] After 7 days of adaptive feeding, the mice were randomly divided into the following different groups: normal control group (control group, n = 8); untreated TNBS-induced colitis mice (TNBS group, n = 8); TNBS-induced colitis mice were subdivided according to the following treatment groups, with 8 mice in each treatment group:

[0190] Group A: ethanol solution + PBS

[0191] Group B: TNBS 2mg / mouse + PBS

[0192] Group C: TNBS 2mg / mouse + mesalazine

[0193] Group D: TNBS 2mg / mouse + Lio-Prob1 (80mg / 20ml)

[0194] Group E: TNBS 2mg / mouse + Lio-Prob1 (40mg / 20ml)

[0195] Group F: TNBS 2mg / mouse + Lio-Prob1 (20mg / 20ml)

[0196] Group G: TNBS 2mg / mouse + Lio-Prob1 (10mg / 20ml)

[0197] Group H: TNBS 2mg / mouse + Lio-Prob1 (5mg / 20ml)

[0198] Colitis was induced in mice using TNBS as previously described (Yang et al., Scientific Reports, 2016 6:29716).

[0199] All animals were orally administered different treatments 3 hours after colitis induction for 4 days. During the 4-day experimental period, the mice were regularly checked for body weight and diarrhea. At the end of the 4th day, all mice were sacrificed by cervical dislocation to collect samples.

[0200] The extent of macroscopic damage to the colon was determined using a previously established scoring system (Yang et al., Scientific Reports, 2016 6:29716; Morris et al., Gastroenterology 1989, 96, 795–803) (no damage: 0 points; congestion without ulcers: 1 point; congestion and wall thickening without ulcers: 2 points; one ulcer site without wall thickening: 3 points; more than two ulcer sites: 4 points; inflammation extending 0.5 cm or major damage: 5 points; inflammation extending 1 cm or severe damage: 6 to 10 points (for every additional 0.5 cm of damage, increase by 1 point, with a maximum score of 10).

[0201] Then, colon tissue was collected, fixed in 4% buffered paraformaldehyde solution, then placed in paraffin, and stained with H&E staining solution according to known practices. Finally, it was observed and photographed under an optical microscope. The colon mucosal injury index (CMDI) score was evaluated in a double-blind manner (Siegmund et al. Am J Physiol Regul Integr Comp Physiol. 2001, 281, R1264-73; Obermeier et al., Clin Exp Immunol. 1999, 116, 238–245). CMDI was calculated by combining the histological scores of inflammatory cell infiltration and tissue damage, ranging from 0 (no change) to 6 (extensive cell infiltration and tissue damage), as shown below:

[0202] Inflammatory cell infiltration Fraction Occasional inflammatory cells in the lamina propria 0 Increased number of inflammatory cells in the lamina propria 1 Inflammatory cells confluent, extending into the submucosa 2 Infiltrate extends across the wall 3 Tissue damage Fraction No mucosal damage 0 Discrete lymphoepithelial lesion 1 Surface mucosal erosion or focal ulcer 2 Extensive mucosal damage extending into the deeper bones of the intestinal wall. 3

[0203] FIG1 shows the parameters of weight loss, DAI score, colon length and colon injury score of mice in the treatment groups.

[0204] After overall evaluation of the test of administering increasing doses of Lio-Prob 1 of Example 3 to the colitis mouse model, a dose of 12 mg / kg was selected, which had a 50% improvement effect on ulcerative colitis mice.

[0205] A dose of 12 mg / kg in mice is equivalent to a dose of 60 mg in humans.

[0206] Example 8: Determination of myeloperoxidase (MPO) activity in vivo in mice with TNBS-induced colitis

[0207] C57BL / 6 male mice (ENVIGO, Italy) aged 7 to 8 weeks were housed under standard conditions: light / dark cycle (12 / 12 h), ambient temperature 20±2° C., relative air humidity 55%, with ad libitum access to food (Mucedola RF18) and water.

[0208] After 7 days of adaptive feeding, the mice were randomly divided into the following different groups: normal control group (control group, n = 8); untreated TNBS-induced colitis mice (TNBS group, n = 8); TNBS-induced colitis mice treated with 100 mg / kg mesalazine (n = 8); TNBS-induced colitis mice treated with 12 mg / kg Lio-Prob1 of Example 3 (n = 8).

[0209] Colitis was induced in mice using TNBS as previously described (Yang et al., Scientific Reports, 2016 6:29716).

[0210] Three hours after colitis induction, mice in each group were orally administered the corresponding treatment for 4 days. During the 4-day experimental period, the body weight and diarrhea of ​​mice were checked regularly. At the end of the 4th day, all mice were killed by cervical dislocation to collect samples.

[0211] 1 cm of mouse colon mucosal scrapings were suspended in potassium phosphate buffer (pH 6.0) containing hexadecyltrimethylammonium bromide buffer (SigmaAldrich H5882) and supplemented with a protease inhibitor cocktail (Sigma Aldrich 539136). The samples were then homogenized on ice and sonicated. The suspension was then centrifuged at 10000 g for 10 minutes at 4°C, and the supernatant was diluted in potassium phosphate buffer (pH 6.0) containing 0.167 mg O-dianisidine (Sigma Aldrich D3252) and 0.0005% (vol / vol) H2O2. The absorbance change at 450 nm was recorded using a spectrophotometer (Tecan NanoQuant model InfiniteM200) (Alex et al., Inflammatory bowel diseases 2009, 15, 341–352; Chinand Barrett, Dig Dis Sci. 1994, 39, 513–525).

[0212] FIG2 shows the MPO values ​​of each treatment group.

[0213] Example 9: Relative quantitative real-time PCR of colon cells

[0214] RNA from colon cells of C57BL / 6 male mice treated in Example 8 was isolated using Trizol (Life-technologies). First-strand cDNA was synthesized from 1 μg of total RNA using reverse transcriptase (Bi-oLine n.BIO 65053) in a 20 μl reaction. Real-time PCR was performed using SYBR green Master Mix (#1725150, Biorad). GAPDH was used as an internal reference. The primers used are listed in Table 7. Relative transcript mRNA levels were calculated.

[0215] Table 7-qPCR experimental primer list

[0216]

[0217] FIG. 3 shows mRNA of pro-inflammatory cytokines measured in the colon of mice treated with TNBS-induced colitis.

[0218] FIG. 4 shows immune cell mRNA measured in the colon of mice treated with TNBS-induced colitis.

[0219] Example 10: Western blot analysis of colon cells

[0220] Mucosa was scraped from the colon of C57BL / 6 male mice treated according to Example 8. Then it was immersed in urea extraction buffer (6M urea, 0.1% Triton X-100, 10mM Tris, pH 8.0, 1mM DTT, 5mM MgCl2, 5mM EGTA, 150mM NaCl), supplemented with PMSF (Sigma Aldrich n.93482) and protease inhibitor cocktail (CellSignaling n.58715) to prevent protein degradation, and then sonicated for 25 seconds. According to the instructions of the manufacturer (Bioradn.5000006), protein concentration was determined by Bradford assay. Then, under denaturing and reducing conditions, acrylamide gel electrophoresis was performed on the protein extraction samples. Different acrylamide concentrations were used according to the weight of the protein to be detected, as shown below:

[0221] -8% acrylamide gel for ZO-1 (Abclonaln.A0659) and NK1.1 (Abclonaln.A8189)

[0222] - 10% acrylamide gel for Gata-3 (Abclonal n. A5711) and T-bet (Abclonal n. A23414).

[0223] - 15% acrylamide gel for MCP1 (Abclonal n. A7277).

[0224] The proteins were then transferred to nitrocellulose filters and unsaturated binding sites were blocked with 5% skim milk at room temperature for 1 hour. The filters were then incubated overnight at 4°C with antibodies specific for the detection protein and species-specific HRP-conjugated secondary antibodies (Invitrogen n.G21234). Actin and / or GAPDH were used as internal controls. Immunoreactive bands were detected using a chemiluminescence kit according to the manufacturer's instructions (Life technologies) and images were acquired using a C280 Azure Biosystemy documentation system. Bands were analyzed for density using ImageJ software.

[0225] FIG. 5 shows ZO-1 protein levels measured in the colon of mice treated with TNBS-induced colitis.

[0226] FIG. 6 shows the levels of immune cells and chemokines measured in the colon of mice treated with TNBS-induced colitis.

[0227] Example 11: Fecal DNA extraction and luminal microbiome analysis

[0228] Feces of C57BL / 6 male mice treated according to Example 8 were collected, snap frozen in liquid nitrogen and stored at -80°C. Bacterial DNA was extracted from each sample using the FastDNASPIN kit for soil and the FastPrep instrument (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions.

[0229] The V4-V5 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced to obtain the microbial composition of the analyzed samples. Amplicon libraries were generated using primers based on 515FB (5'-GTGYCAGCMGCCGCGGTAA-3') / 926R (5'-CCGYCAATTYMTTTRAGTTT-3') (Walters, mSystems 2015, 1, e00009-15). Sequencing equipment, methods and chemical reagents were based on Illumina MiSeq instruments, using 2x300bp double-end v3 chemical assays, see Comeau (Comeau et al., mSystems. 2017, 2: e00127-16).

[0230] All results are based on sequence reads and operational taxonomic units.

[0231] FIG. 7 shows beta diversity analysis of gut microbiota measured in feces of mice treated with TNBS-induced colitis.

[0232] Example 12: Fermentation of Lactobacillus brevis in the presence of mung bean sprout extract with VSL#3 and Escherichia coli In vivo efficacy assay of Nissle

[0233] C57BL / 6 male mice (ENVIGO, Italy) aged 7 to 8 weeks were housed under standard conditions: light / dark cycle (12 / 12 h), ambient temperature 20±2° C., relative air humidity 55%, with ad libitum access to food (Mucedola RF18) and water.

[0234] After 7 days of adaptive feeding, mice were randomly assigned to different groups as follows:

[0235] Group 1: Vehicle (50% ethanol solution) ir + PBS 1 mL / mouse oral administration

[0236] Group 2: TNBS 2 mg / mouse ir + PBS 1 mL / mouse oral administration

[0237] Group 3: TNBS 2 mg / mouse ir + mesalazine 1 mL / mouse (100 mg / Kg) oral administration

[0238] Group 4: TNBS 2mg / mouse ir+VSL#3 (Alfasigma) 5x10 10 CFU / Kg / mouse Oral administration

[0239] Group 5: TNBS 2 mg / mouse ir+VSL#3 5x 10 9 CFU / Kg / mouse oral

[0240] Group 6: TNBS 2 mg / mouse ir+VSL#3 5x 10 8 CFU / Kg / mouse oral

[0241] Group 7: TNBS 2mg / mouse ir+E. coli Nissle (Cadigroup) 5x 10 10 CFU / Kg / mouse Oral administration

[0242] Group 8: TNBS 2mg / mouse ir+E. coli Nissle 5x 10 9 CFU / Kg / mouse Oral administration

[0243] Group 9: TNBS 2mg / mouse ir+E. coli Nissle 5x 10 8 CFU / Kg / mouse Oral administration

[0244] Group 10: TNBS 2mg / mouse ir+Lio-Prob1 5x 10 10 CFU / Kg / mouse Oral administration

[0245] Group 11: TNBS 2mg / mouse ir+Lio-Prob1 5x 10 9 CFU / Kg / mouse Oral administration

[0246] Group 12: TNBS 2mg / mouse ir+Lio-Prob1 5x 10 8 CFU / Kg / mouse Oral administration

[0247] Colitis was induced in mice using TNBS as previously described (Yang et al., Scientific Reports, 2016 6:29716).

[0248] Three hours after colitis induction, mice in each group were orally administered the corresponding treatment for 4 days. During the 4-day experimental period, the body weight and diarrhea of ​​mice were checked regularly. At the end of the 4th day, all mice were killed by cervical dislocation to collect samples.

[0249] Table 8 lists the weight loss of mice in different treatment groups.

[0250] Table 8

[0251]

[0252] Table 9 lists the DAI scores measured for each treatment group, and the measurement method is as described in Example 6.

[0253] Table 9

[0254]

[0255] *P<0.05 vs Group 2 (ANOVA); ns=not significant

[0256] Table 10 lists the colon injury scores measured for each treatment group, and the measurement method is as described in Example 7.

[0257] Table 10

[0258]

[0259] *P<0.05, **P<0.01, ***P<0.0001 vs. Group 2 (ANOVA); ns=not significant.

[0260] Example 13: Analysis of the supernatant of fermentation of Lactobacillus brevis DSM33682 in the presence of mung bean sprouts by mass spectrometry (LC-MS) Fluid characterization

[0261] Samples Prob1 (Lactobacillus brevis DSM 33682 fermented in the presence of mung bean sprouts), Prob2 (Lactobacillus brevis DSM 33682 fermented without substrate) and Example 2 (mung bean extract) were centrifuged (14000 rpm, 5 min, 4°C) and diluted 1:10 with water.

[0262] 10 μl of the sample was injected into an Acquity UPLC-BEH C18 column (1.7 μm; 100×2.1 mm, Waters).

[0263] The tests were performed on a liquid chromatography system (Xevo G2-XS coupled to an Acquity H-Class UPLC system) coupled to a mass spectrometer (LC-MS).

[0264] Chromatographic separation was achieved using a linear gradient of water + 0.1% (v / v) formic acid (solvent A) and acetonitrile + 0.1% (v / v) formic acid (solvent B) at a flow rate of 0.3 ml / min, as shown in Table 11.

[0265] Table 11

[0266] Time (minutes) Solvent A% Solvent B% 0 95 5 5 95 5 45 5 95 50 5 95 53 95 5 60 95 5

[0267] Ionization was performed using an electrospray ionization (ESI) source. The settings were as follows

[0268] parameter value Capillary 0.80kV cone 40V Source temperature 120℃ Desolvation temperature 400℃ Airflow in the cone 25L / h Desolvation gas flow 500L / h

[0269] Data acquisition was performed in positive ion mode with MS scans from 50 m / z to 2000 m / z.

[0270] The obtained chromatogram was qualitatively analyzed using Unifi 1.9.3.0 software (Waters) and a spectrum was created by subtracting Prob2 and Example 2 from Prob1, as shown in Table 12.

[0271] Table 12

[0272] m / z(monoisotope) RT(time) Charge MW (monoisotope), Da 575.384 39.06 +1 574.376 605.395 44.27 +2 1208.774 611.854 15.76 +2 1221.692 633.344 15.96 +2 1264.672

[0273] Example 14: Analysis of isoflavones present in fermentation products.

[0274] A sample of the fermentation product of Lactobacillus brevis DSM 33682 (Lio-Prob1) obtained in the presence of mung bean sprouts was dissolved in DMSO, and the resulting solution was subjected to HPLC analysis according to the following conditions:

[0275] -Column: C18 (ZorbaxEclipse XDB-C18 4.6x250mm (5μm))

[0276] - Temperature: 35℃

[0277] -Flow rate: 1.0ml / min

[0278] -Injection volume: 5μl

[0279] -λ:250nm

[0280] -Mobile phase A: water 0.1% HCOOH

[0281] -Mobile phase B: acetonitrile

[0282] minute Phase A Phase B 0 90 10 20 70 30 25 50 50 30 25 75 31 90 10 35 90 10

[0283] Table 13 describes the percentage of isoflavones present in the samples.

[0284] Table 13

[0285] Isoflavones ％ Soybean Flavonoids 0.0278 Genistein 0.00864 Isoflavones (expressed as soy flavonoids) 0.00436

[0286] Example 15: Bag-packed preparation

[0287] Take the preparation of the orally disintegrating stick containing 60 mg or 240 mg of the Lio-Prob1 fermentation product of Example 3 as an example. Table 14 shows the qualitative and quantitative compositions.

[0288] Table 14

[0289] Element Rod 1 Stick 2 Lio-Prob1 fermentation product (Example 3) 0.060g 0.240g Isomalt-E953 1.145g 0.965g Vanilla essence 0.017g 0.017g Silicon dioxide (E551) 0.015g 0.015g Cholecalciferol (Vitamin D) (0.25%) 0.013g 0.013g

[0290] Example 16: Preparation of capsule composition

[0291] Take the preparation of capsules containing 240 mg of the Lio-Prob1 fermentation product of Example 3 as an example.

[0292] Table 15 shows the qualitative-quantitative composition.

[0293] Table 15

[0294] Element Weight(mg) Lio-Prob1 fermentation product (Example 3) 240 Maltodextrin 91 Cholecalciferol (Vitamin D 0.25%) 13 Magnesium salt of fatty acid (magnesium stearate) E470b 3 Silicon dioxide (E551) 3 Vegetable Capsule (Hydroxypropyl Methylcellulose-E464 and Calcium Carbonate-E170) 96

Claims

1. A fermentation product obtained by a fermentation process of the probiotic strain Lactobacillus brevis deposited with the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the accession number DSM 33682 in the presence of at least one plant extract selected from the group consisting of Scutellaria baicalensis Georgi, Boswellia serrata, Phaseolus, Phaseolus radiatus, Mung bean sprouts, Curcuma longa, Camellia sinensis, broccoli sprouts and mixtures thereof.

2. The fermentation product according to claim 1, wherein The probiotic Lactobacillus brevis DSM 33682 was added at 1×10 8 CFU / ml to 1x10 10 The amount of CFU / ml was inoculated into a culture medium supplemented with a plant extract selected from the group consisting of Scutellaria baicalensis, Boswellia serrata, Phaseolus, Mung bean, Mung bean sprouts, Turmeric, tea, broccoli sprouts and a mixture thereof.

3. The fermentation product according to claim 2, wherein The plant extract is a Phaseolus extract, a Mung bean extract or a Mung bean sprout extract at a concentration of 5 g / L to 15 g / L relative to the fermentation liquid.

4. The fermentation product according to claim 3, wherein The Phaseolus extract or the Mung bean extract or the Mung bean sprout extract is characterized in that the oxidation potential is reduced by 100 mV to 400 mV when in a phosphate buffer at a concentration of 0.5% (w / v) to 20% (w / v).

5. The fermentation product according to claim 2, characterized in that Compounds with molecular weights between 200 Daltons and 2000 Daltons are included.

6. The fermentation product according to any one of claims 1 to 5, characterized in that It is obtained by a method comprising the following steps: (a) Lactobacillus brevis DSM 33682 was cultured at 1×10 8 CFU / ml to 1x10 10 The amount of CFU / ml was placed in the culture medium; (b) adding Phaseolus extract, Mung bean extract or Mung bean sprout extract to the culture medium at a concentration of 5 g / L to 15 g / L; (c) under anaerobic or aerobic conditions at a temperature of 30°C to 40°C with mild agitation for 8 hours to 24 hours; (d) centrifuging at a speed of less than 4000 rpm for 5 minutes to 40 minutes; (e) Freeze-drying or freezing.

7. The fermentation product according to claims 1 to 6, characterized in that The invention relates to a fermentation product comprising said Lactobacillus brevis DSM 33682 and a fermentation supernatant containing bacterial metabolites of a fermentation carried out in the presence of a plant.

8. A nutritional composition or food composition comprising the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of Phaseolus extract, Mung bean extract or Mung bean sprout extract as claimed in claim 1, and an excipient suitable for oral administration.

9. The composition according to claim 8, further comprising prebiotics, vitamins, amino acids and mineral salts selected from the group consisting of salts of selenium, zinc and iron.

10. The composition according to claim 8, which is in the form of an orally dissolving stick, wherein the amount of the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of mung bean sprout extract is 1 mg to 1 g, and there is also 0.5 g to 1.5 g of a sweetener selected from isomalt, aspartame, xylitol, lactitol, sodium cyclamate, dextrose, fructose, glucose, lactose and sucrose, 1 mg to 50 mg of a flavoring agent, 1 mg to 50 mg of an anti-caking agent selected from colloidal silicon dioxide and talc, and 0 mg to 50 mg of a vitamin.

11. The composition according to claim 8, which is in the form of capsules, wherein the amount of the fermentation product of Lactobacillus brevis DSM 33682 obtained in the presence of mung bean sprout extract is 1 mg to 1 g, and there is also 0 to 20 mg of one or more lubricants selected from talc, magnesium stearate, calcium stearate, stearic acid, hydrogenated vegetable oil and polyethylene glycol; 0 to 20 mg of an anti-caking agent selected from silicon dioxide and talc; 0 to 300 mg of a sweetener selected from sucrose, sorbitol, mannitol, saccharin, acesulfame potassium, neohesperidin and maltodextrin; 0 mg to 50 mg of vitamins.

12. The composition according to claim 8, for use in treating and / or preventing intestinal inflammation.

13. The composition according to claim 12, for use in the treatment and / or prevention of IBD, Crohn's disease, ulcerative colitis or diverticulitis.

14. The composition according to claim 12, used alone or in combination or conjunction with a concomitant therapy.

15. A method for obtaining a fermentation product of Lactobacillus brevis DSM33682 in the presence of a Phaseolus extract, a Mung bean extract or a Mung bean sprout extract, comprising the following steps: (a) Lactobacillus brevis DSM 33682 was cultured at 1×10 8 CFU / ml to 1x10 10 The amount of CFU / ml was placed in the culture medium; (b) adding mung bean sprout extract at a concentration of 5 g / L to 15 g / L to the culture medium; (c) under anaerobic or aerobic conditions at a temperature of 30°C to 40°C with mild agitation for 8 hours to 24 hours; (d) centrifuging at a speed of less than 4000 rpm for 20 minutes to 40 minutes; (e) Freeze-drying or freezing.

16. A method for obtaining a pharmaceutical or nutritional composition comprising a fermentation product according to claims 1 to 6 in the presence of at least one plant extract and a pharmaceutical or food excipient.

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