Probiotics for treating and / or preventing conditions associated with helicobacter pylori colonization

Abstract

The present invention relates to bacterial strains of Lactobacillus acidophilus species and Phytobacterium plantarum species, these bacterial strains are useful in the treatment and / or prevention of conditions associated with Helicobacter pylori colonization of gastric mucosa, inhibition of Helicobacter pylori, treatment and / or prevention of Helicobacter pylori infection, maintenance of gastric microbiota homeostasis, and treatment and prevention of gastric inflammation in a subject. The invention further relates to a composition, a food product, a dietary supplement or a pharmaceutically acceptable formulation comprising said bacterial strain.

Description

Technical Field

[0001] The present invention relates to bacterial strains of the species Lactobacillus acidophilus and Lactiplantibacillus plantarum for use in treating and / or preventing conditions associated with Helicobacter pylori (H. pylori) colonization of the gastric mucosa in a subject. The present invention further relates to compositions, food products, dietary supplements or pharmaceutically acceptable formulations comprising bacterial strains of the species Lactobacillus acidophilus and Lactiplantibacillus plantarum for use in treating and / or preventing conditions associated with Helicobacter pylori colonization of the gastric mucosa in a subject. Background Art

[0002] In recent years, the infection rate of Helicobacter pylori has reached approximately 50%. Helicobacter pylori infection can lead to chronic gastritis, peptic ulcers, gastric lymphoma arising from mucosa-associated lymphoid tissue (MALT lymphoma), and even gastric cancer. Helicobacter pylori is widely considered a key carcinogen in gastric tissue. Currently, the first-line treatment for eradication of Helicobacter pylori is a quadruple therapy consisting of a proton pump inhibitor, two antibiotics, and a bismuth agent.

[0003] However, due to the overuse and misuse of antibiotics, the resistance rate of Helicobacter pylori to antibiotics has increased significantly. In addition, the potential side effects associated with some of the drugs used in the treatment of Helicobacter pylori (such as headache, nausea, vomiting, gastrointestinal dysbiosis and even gastric atrophy) are also well known.

[0004] Significant progress has been made in the development of bacterial strain preparations, which are now widely used in a number of medical applications. Some studies have found that probiotics can not only effectively inhibit Helicobacter pylori infection in vitro, but also significantly reduce Helicobacter pylori colonization in vivo and simultaneously inhibit inflammatory processes.

[0005] Although some probiotic strains appear to help eradicate H. pylori infection, there is a need to identify other more suitable probiotic strains to treat and / or prevent conditions associated with H. pylori colonization of the gastric mucosa and to aid in H. pylori eradication.

[0006] Purpose of the Invention

[0007] In order to overcome the need for other more suitable bacterial strains for the treatment and / or prevention of conditions associated with H. pylori colonization, the inventors of the present invention have investigated different probiotic strains, or combinations of strains, that have excellent stability during passage through the gastrointestinal tract and the ability to adhere to the digestive mucosa.

[0008] Therefore, the object of the present invention is to provide bacterial strains as described herein, methods and compositions, food products, dietary supplements or pharmaceutically acceptable formulations comprising such bacterial strains, which are to be used in the treatment and / or prevention of conditions associated with Helicobacter pylori colonization of the gastric mucosa, inhibition of Helicobacter pylori, treatment and / or prevention of Helicobacter pylori infection, maintenance of gastric microbiota homeostasis and treatment and prevention of gastric inflammation in a subject. Summary of the Invention

[0009] The object of the present invention is to demonstrate the ability of certain bacterial strains to treat and / or prevent disorders associated with H. pylori colonization of the gastric mucosa in a subject.

[0010] Another object of the present invention is to demonstrate the ability of certain bacterial strains to inhibit Helicobacter pylori in a subject.

[0011] Another object of the present invention is to demonstrate the ability of certain bacterial strains to treat and / or prevent Helicobacter pylori infection in a subject.

[0012] Another object of the present invention is to demonstrate the ability of some bacterial strains to maintain gastric microbiota homeostasis in a subject.

[0013] Another object of the present invention is to demonstrate the ability of certain bacterial strains to treat and prevent gastric inflammation in a subject.

[0014] Thus, in one aspect, the present invention provides one or more bacterial strains for use in treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of the strains Lactobacillus acidophilus NCFM (NCFM) strain and the strain Lactobacillus plantarum Lp-115 (Lp-115), or a combination thereof.

[0015] In another aspect, the present invention provides one or more bacterial strains for use in inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0016] In another aspect, the present invention provides one or more bacterial strains for use in treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0017] In another aspect, the present invention provides one or more bacterial strains for use in maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0018] In another aspect, the present invention provides one or more bacterial strains for use in treating and / or preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0019] In a further aspect, the present invention provides a method for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject in need thereof, the method comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0020] In yet another aspect, the present invention provides a method of inhibiting Helicobacter pylori in a subject in need thereof, the method comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0021] In another aspect, the present invention provides a method for treating and / or preventing Helicobacter pylori infection in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0022] In another aspect, the present invention provides a method for maintaining gastric microbiota homeostasis in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0023] In another aspect, the present invention provides a method for treating and / or preventing gastric inflammation in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0024] In a further aspect, the present invention provides the use of one or more bacterial strains for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0025] In another aspect, the present invention provides use of one or more bacterial strains for inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0026] In a further aspect, the present invention provides use of one or more bacterial strains for treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0027] In yet another aspect, the present invention provides use of one or more bacterial strains for maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0028] In another aspect, the present invention provides use of one or more bacterial strains for treating and / or preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Six probiotic strains inhibited the attachment of Helicobacter pylori to AGS cells: (a) Fluorescence microscopy images showing the colonization of Helicobacter pylori P12-GFP on AGS cells after intervention with six probiotic strains (Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La-14, Lactobacillus plantarum Lp-115, Lacticaseibacillus paracasei Lpc-37, Lacticaseibacillus rhamnosus Lr-32, and Lacticaseibacillus rhamnosus GG); (b) Urease activity assay showing that the six tested probiotic strains interfered with the colonization of Helicobacter pylori P12-GFP on AGS cells within 1-7 hours. *(P < 0.05); **(P < 0.01); ns, not significant, compared with H. pylori.

[0030] Figure 2Inhibitory effects of six probiotic strains on Helicobacter pylori-induced inflammation in the AGS cell line. AGS cells were co-infected with six probiotic strains (Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La-14, Lactobacillus plantarum Lp-115, Lactobacillus paracasei Lpc-37, Lactobacillus rhamnosus Lr-32, and Lactobacillus rhamnosus GG) and Helicobacter pylori P12 at a multiplicity of infection (MOI) of 100 for 6 h. mRNA levels (A) Cxcl8 and TNF mRNA levels in cells and (B) IL-8 and TNF-α protein concentrations in the supernatant. Results for each experiment are shown as the mean ± standard deviation of three independent experiments. *(P < 0.05); **(P < 0.01); ***(P < 0.001); ****(P < 0.0001).

[0031] Figure 3 Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 inhibit Helicobacter pylori adhesion in mice: (a) Mice were fed H. pylori SS1 alone or together with L. acidophilus NCFM and / or L. plantarum Lp-115 for 6 weeks; (b) H. pylori colonization was identified by immunohistochemistry (IHC). Uninfected group (200-fold (A) and 400-fold (B); H. pylori group (200-fold (C) and 400-fold (D); H. pylori + NCFM group (200-fold (E) and 400-fold (F); H. pylori + Lp-115 group (200-fold (G) and 400-fold (H); H. pylori + NCFM + Lp-115 group (200-fold (I) and 400-fold (J); (c) H. pylori colonization was identified by qRT-PCR based on the expression of Ure-A and Ure-B. Mice were co-infected with Helicobacter pylori SS1 and Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115. Each experimental result shows the mean ± SD of three independent experiments. *p < 0.05; **(P < 0.01); ***(P < 0.001).

[0032] Figure 4Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 suppress Helicobacter pylori inflammation in mice. (a) Hematoxylin and eosin staining was used to identify Helicobacter pylori inflammation. Uninfected group (100-fold (A) and 200-fold (B); H. pylori group (100-fold (C) and 200-fold (D); H. pylori + NCFM group (100-fold (E) and 200-fold (F); H. pylori + Lp-115 group (100-fold (G) and 200-fold (H); H. pylori + NCFM + Lp-115 group (100-fold (I) and 200-fold (J); (b) mRNA expression levels of IFN-γ, CXCL15, IL-4, and IL-10 in each group. ns(P≥0.05); *(P<0.05); **(P<0.01); ***(P<0.001); ****(P<0.0001).

[0033] Figure 5 Rapid urease test assay in mouse model. Clear samples were negative, and darker samples were positive. Results showed that all samples in the H. pylori group tested positive, and some individual samples in the Lactobacillus and Lactiplantibacillus intervention groups tested negative.

[0034] advantage

[0035] Although the mechanism of action of probiotics in the treatment and prevention of conditions associated with H. pylori colonization of the gastric mucosa is not fully understood, it has been surprisingly found that the ability of probiotics to adhere to the gastric mucosa and their interference with H. pylori adhesion and subsequent downregulation of the expression of immune inflammatory mediators appear to be the mechanism of action.

[0036] Thus, the present inventors have surprisingly discovered that Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 can help treat or prevent conditions associated with H. pylori colonization in subjects by downregulating local Th1 immune responses in the gastric mucosa, inhibiting the pro-inflammatory cytokine IFN-γ, while promoting Th2 responses to produce the anti-inflammatory cytokine IL-4.

[0037] Furthermore, our study also demonstrated that Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 could suppress and maintain gastric microbiota homeostasis in subjects. DETAILED DESCRIPTION

[0038] The following describes detailed aspects of the present invention. Some detailed aspects are discussed in separate sections. This is for ease of reference and is by no means restrictive. Unless the context specifically dictates otherwise, all embodiments described below apply equally to all aspects of the present invention.

[0039] bacteria

[0040] Bacterial strain of the present invention is selected from the bacterial strain of Lactobacillus and Lactobacillus.Preferably, the bacterial strain of Lactobacillus is the bacterial strain of Lactobacillus acidophilus species, Lactobacillus paracasei species and Lactobacillus rhamnosus species.Preferably, the bacterial strain of Lactobacillus is the bacterial strain of Lactobacillus plantarum species.Especially, the bacterial strain is Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La-14, Lactobacillus paracasei Lpc-37, Lactobacillus rhamnosus Lr-32, Lactobacillus rhamnosus GG and Lactobacillus plantarum Lp-115.

[0041] These bacterial strains are commercially available from International Flavors & Fragrances, Inc. (formerly DuPont Nutrition Biosciences Aps, Denmark).

[0042] These bacterial strains have also been deposited by DuPont Nutrition Biosciences of Denmark at the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig, Germany, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, where they are recorded under the following accession numbers:

[0043] 1. strain NCFM (DGCC8698); deposited on March 15, 2021 under accession number DSM33840.

[0044] 2. strain La-14 (DGCC11491); deposited on June 1, 2021 with accession number DSM33880.

[0045] 3. strain Lpc-37 (DGCC4981); deposited on October 5, 2017, under accession number DSM32661.

[0046] 4. strain Lr-32 (DGCC9913); deposited on January 15, 2009 under accession number DSM22193.

[0047] 5. strain Lp-115 (DGCC4715); originally deposited on February 9, 2009, under accession number DSM22266; the deposit has been extended to February 9, 2051, in accordance with Article 9.1 of the Budapest Treaty.

[0048] The bacterial strain Lactobacillus rhamnosus GG (DGCC12645) is the most documented probiotic strain in the world and is commercially available from various sources, including International Flavors & Fragrances (formerly DuPont Nutrition Biosciences, Denmark). It has been described in more than 250 publications.

[0049] Preferably, the bacterial strain used in the present invention is a generally recognized as safe (GRAS) and preferably a GRAS approved bacterial strain. GRAS is a U.S. Food and Drug Administration (FDA) designation that a chemical or substance added to food is considered safe by experts for its intended use and is therefore exempt from the usual U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements.

[0050] In a first aspect, the present invention provides one or more bacterial strains for use in treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0051] In another aspect, the present invention provides one or more bacterial strains for use in inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0052] In another aspect, the present invention provides one or more bacterial strains for use in treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0053] In another aspect, the present invention provides one or more bacterial strains for use in maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0054] In another aspect, the present invention provides one or more bacterial strains for use in treating and preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0055] In another aspect, the present invention also provides a method for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject in need thereof, the method comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0056] In another aspect, the present invention provides a method of inhibiting Helicobacter pylori in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0057] In another aspect, the present invention provides a method for treating and / or preventing Helicobacter pylori infection in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0058] In another aspect, the present invention provides a method for maintaining gastric microbiota homeostasis in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0059] In another aspect, the present invention provides a method for treating and / or preventing gastric inflammation in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0060] In yet another aspect, the present invention provides the use of one or more bacterial strains for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0061] In one aspect, the present invention provides use of one or more bacterial strains for inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0062] In one aspect, the present invention provides use of one or more bacterial strains for treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0063] In another aspect, the present invention provides use of one or more bacterial strains for maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0064] In yet another aspect, the present invention provides use of one or more bacterial strains for treating and / or preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0065] One or more bacterial strains is to be understood as a single strain or more than one strain, such as two strains.

[0066] In a particular aspect, one or more bacterial strains according to the invention are combined with one or more antibiotics. Examples of antibiotics are amoxicillin, clarithromycin, metronidazole, tetracycline and / or bismuth subsalicylate.

[0067] In a particular aspect, the bacterial strain according to the invention is Lactobacillus acidophilus NCFM.

[0068] In another aspect, the bacterial strain is Lactobacillus plantarum Lp-115 strain.

[0069] The bacterial strain Lactobacillus acidophilus NCFM (also referred to as NCFM) and the bacterial strain Lactobacillus plantarum Lp-115 (also referred to as Lp-115) can be used in combination. They can also be used in combination with each other or individually in combination with other strains.

[0070] Optionally, the bacterial strains Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115, as well as other bacterial strains when used in aspects of the present invention, are probiotic bacteria.

[0071] The term "probiotic bacteria" is defined as encompassing any non-pathogenic bacteria that, when administered in sufficient quantities to a host with viable bacteria, imparts health benefits to the host. To be classified as "probiotic bacteria," bacteria must survive the upper portion of the host's digestive tract. They are non-pathogenic, non-toxic, and on the one hand, interact with the ecology of the resident microbial communities in the digestive tract and on the other hand, exert their beneficial effects on health via their ability to influence host physiology and the immune system in a positive manner. When administered in sufficient quantities to the host, these probiotic bacteria have the ability to advance through the intestinal tract, thereby maintaining viability and exerting their primary effect in the lumen and / or wall of the host's gastrointestinal tract. Thenceforth, they transiently form a part for the resident microbial communities, and this colonization (or transient colonization) allows probiotic bacteria to play a beneficial role, such as suppressing the potential pathogenic microorganisms present in the microbial communities and interacting with the host in the intestine (including the immune system).

[0072] Thus, in a particular aspect of the invention, the bacterial strain is a probiotic strain.In a particular aspect, the bacterial strains Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 are probiotic strains.

[0073] Fiber / Prebiotics / Plant Extracts

[0074] In one aspect of the invention, the bacterial strain is used in combination with one or more fibers and / or prebiotics.

[0075] Prebiotics are defined as substrates selectively utilized by host microorganisms, thereby imparting health benefits. These prebiotics are normally compositions that beneficially affect the health of the host, thereby improving host health, by selectively stimulating the growth and / or activity of a certain or limited number of bacteria. Prebiotics can be applied to oral routes, but can also be applied to other microbial colonization sites. Typically, prebiotics are carbohydrates (such as oligosaccharides), but the definition does not exclude non-carbohydrates (such as polyphenols) or polyunsaturated fatty acids or can be selectively utilized by a limited number of bacteria to impart health benefits. The most common prebiotic form is nutritionally classified as soluble fiber. To a certain extent, many forms of dietary fiber show a certain level of prebiotic effect.

[0076] Plant extracts refer to products extracted and isolated from plants. Plant extracts are important additives in industry due to their content in bioactive compounds such as polyphenols and carotenoids that have antimicrobial and antioxidant activities.

[0077] In one aspect, prebiotics are selectively fermentable ingredients that allow for specific alterations in both the composition and / or activity of the gastrointestinal microbiota, thereby conferring a well-being and health benefit on the host.

[0078] Suitably, prebiotics, fibers or plant extracts can be used according to the present invention in an amount of 0.01 to 100 g / day, preferably 0.1 to 50 g / day, more preferably 0.5 to 20 g / day. In one embodiment, prebiotics, fibers or plant extracts can be used according to the present invention in an amount of 1 to 10 g / day, preferably 2 to 9 g / day, more preferably 3 to 8 g / day. In another embodiment, prebiotics, fibers or plant extracts can be used according to the present invention in an amount of 5 to 50 g / day, preferably 5 to 25 g / day.

[0079] Examples of dietary sources of prebiotics include soy, inulin sources (such as Jerusalem artichoke, jicama, and chicory root), raw oats, unrefined wheat, unrefined barley, and yacon.

[0080] Examples of suitable prebiotics include alginates, xanthan gum, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharides (GOS), fructooligosaccharides (FOS), polydextrose (e.g. ), Lactitol, L-arabinose, D-xylose, L-rhamnose, D-mannose, L-fucose, inositol, sorbitol, mannitol, xylitol, fructose, carrageenan, alginate, microcrystalline cellulose (MCC), betaine, lactulose, soy oligosaccharides, isomaltulose (Palatinose TM ), isomalto-oligosaccharides, glucooligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, β-glucans, cellobiose, raffinose, gentiobiose, melibiose, xylobiose, cyclodextrins, isomaltose, trehalose, stachyose, panose, pullulan, verbascose, galactomannans, (human) milk oligosaccharides and all forms of resistant starch.

[0081] In one aspect, the combination of one or more bacterial strains according to the invention and one or more prebiotics and / or fibers and / or plant extracts exhibits a synergistic effect (i.e., an effect that is greater than the additive effect of the bacteria used alone) in certain applications.

[0082] In another aspect according to the present invention, one or more bacterial strains are used in combination with one or more fibers and / or prebiotics.

[0083] Suitably, the prebiotic used is polydextrose, lactitol, inositol, L-arabinose, D-xylose, L-rhamnose, D-mannose, L-fucose, sorbitol, mannitol, xylitol, fructose, carrageenan, alginate, microcrystalline cellulose (MCC) or milk oligosaccharides. In a preferred case, the fiber and / or prebiotic is polydextrose. In another preferred case, the fiber and / or prebiotic is a polyphenol.

[0084] In a further aspect, the present invention relates to a composition, a food product, a food ingredient, a dietary supplement or a pharmaceutically acceptable formulation comprising the bacterial strain according to the invention or a mixture thereof and optionally one or more fibers and / or prebiotics.

[0085] Composition

[0086] The term "composition" is used in a broad sense to refer to the manner in which something is composed, i.e., its general composition. In various aspects of the invention, the composition can consist essentially of a single strain of the species Lactobacillus acidophilus. In another aspect of the invention, the composition can consist essentially of a single strain of the species Lactobacillus plantarum. In further aspects of the invention, the composition can comprise strains of both the species Lactobacillus acidophilus and the species Lactobacillus plantarum.

[0087] Alternatively, the composition may comprise other components, such as other bacterial strains, biological and chemical components, active ingredients, metabolites, nutrients, fibers, prebiotics, plant extracts, and the like.

[0088] In one aspect, the bacterial strain according to the invention is in the form of a composition for use in the treatment and / or prevention of a disorder associated with H. pylori colonization of the gastric mucosa in a subject in need thereof.

[0089] In another aspect, the bacterial strain according to the present invention is in the form of a composition for use in inhibiting Helicobacter pylori in a subject in need thereof.

[0090] In a further aspect, the bacterial strain according to the present invention is in the form of a composition for use in the treatment and / or prevention of an Helicobacter pylori infection in a subject in need thereof.

[0091] In another aspect, the bacterial strain according to the invention is in the form of a composition for use in maintaining gastric microbiota homeostasis in a subject in need thereof.

[0092] In another aspect, the bacterial strain according to the present invention is in the form of a composition for use in the treatment and prevention of gastric inflammation in a subject in need thereof.

[0093] According to one aspect of the invention, the composition is a spray-dried, frozen or freeze-dried composition.

[0094] According to another aspect of the present invention, the composition comprises a cryoprotectant.

[0095] In yet another aspect of the present invention, one or more bacterial strains of the species Lactobacillus acidophilus are present in an amount of 10 6 to 10 14 , for example 10 8 to 10 12colony forming units (CFU) / dose, optionally 10 10 The amount of CFU / dose present in the composition.

[0096] Although it is not required that the composition comprises any support, diluent or excipient, such support, diluent or excipient can be added and used in a manner familiar to those skilled in the art. Examples of suitable excipients include, but are not limited to, microcrystalline cellulose, rice maltodextrin, silicon dioxide and magnesium stearate. The composition of the present invention may also comprise a cryoprotectant component (e.g., glucose, sucrose, lactose, trehalose, sodium ascorbate and / or other suitable cryoprotectants).

[0097] The terms "composition" and "formulation" are used interchangeably.

[0098] The compositions used in various aspects of the present invention can take the form of solid, liquid, solution or suspension formulations. Examples of solid formulations include, but are not limited to, tablets, pills, capsules, granules and powders that can be wettable, spray-dried or freeze-dried / lyophilized. The compositions can contain flavorings or coloring agents. The compositions can be formulated for immediate release, delayed release, modified release, sustained release, pulsed release or controlled release applications.

[0099] For example, if the composition of the present invention is used in tablet form, the tablet may also contain one or more of the following: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate and glycine; disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and gum arabic; and lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0100] Examples of other acceptable carriers for preparing the compositions include, for example, water, saline solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides and fatty acid diglycerides, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0101] For aqueous suspensions and / or elixirs, the compositions of the invention may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and / or suspending agents, and with diluents such as water, propylene glycol and glycerin, and combinations thereof.

[0102] For illustrative purposes, specific non-limiting examples of compositions that can be used in various aspects of the present invention are described below. These examples include, but are not limited to, food products, food ingredients, functional foods, dietary supplements, pharmaceutical compositions or formulations, and medicaments.

[0103] Food products

[0104] Bacterial strains according to the present invention can take the form of food products or be a part of food products. Herein, the term "food" is used in a broad sense and covers food and beverages for humans and food and beverages for animals (i.e., feed). Preferably, the food product is suitable for and designed for human consumption.

[0105] In one aspect, the bacterial strain according to the invention is in the form of a food product for use in the treatment and / or prevention of disorders associated with H. pylori colonization of the gastric mucosa in a subject in need thereof.

[0106] In another aspect, the bacterial strain according to the present invention is in the form of a food product for use in inhibiting Helicobacter pylori in a subject in need thereof.

[0107] In a further aspect, the bacterial strain according to the present invention is in the form of a food product for use in the treatment and / or prevention of an Helicobacter pylori infection in a subject in need thereof.

[0108] In another aspect, the bacterial strain according to the invention is in the form of a food product for use in maintaining gastric microbiota homeostasis in a subject in need thereof.

[0109] In another aspect, the bacterial strain according to the present invention is in the form of a food product for use in the treatment and prevention of gastric inflammation in a subject in need thereof.

[0110] Depending on the use and / or the mode of application and / or the mode of administration, the food may be in the form of a liquid, a solid or a suspension.

[0111] When in the form of a food product, the composition may comprise or be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.

[0112] For example, the food product of the present invention may take the form of one of the following:

[0113] Juice; beverages containing whey protein: health tea or herbal tea, cocoa beverage, milk beverage, lactic acid bacteria beverage, yogurt and / or yogurt beverage, cheese, ice cream, water ice, dessert, sweet food, biscuit, cake, cake mix or cake filling, snack food, fruit filling, cake or donut frosting, ready-to-eat bakery filling cream, cookie filling, ready-to-eat bakery filling, reduced-calorie filling, adult nutritional beverage, acidified soy / juice beverage, nutrition or health bar, beverage powder, calcium-fortified soy milk, calcium-fortified coffee beverage or fermented vegetable products (such as but not limited to Korean kimchi and sauerkraut).

[0114] Optionally, when the product is a food product, bacterium of the present invention should be kept effective in the normal "sell by" date or the "cut-off" date of the food product sold by the retailer. Preferably, the effective time should exceed such date, until the normal fresh phase when the food deteriorates and becomes obvious ends. Required time span and normal shelf life will change with foodstuff, and those of ordinary skill in the art will recognize that the shelf life time will change according to the kind of foodstuff, the size of foodstuff, storage temperature, processing conditions, packaging material and packaging equipment of foodstuff.

[0115] Food ingredients

[0116] The bacterial strain according to the present invention may take the form of or be part of a food ingredient and / or feed ingredient.

[0117] As used herein, the term "food ingredient" or "feed ingredient" includes compositions that are functional foods or foodstuffs or that can be added to functional foods or foodstuffs as nutritional and / or health supplements for humans and animals.

[0118] Depending on the use and / or the mode of application and / or the mode of administration, the food ingredient may be in the form of a liquid, a suspension or a solid.

[0119] Functional foods

[0120] The bacterial strain according to the invention may be in the form of or be part of a functional food.

[0121] As used herein, the term "functional food" means a food that is not only capable of providing a nutritional effect, but is also capable of delivering additional beneficial effects to the consumer.

[0122] Thus, functional foods are ordinary foods that have incorporated therein components or ingredients (such as those described herein) that impart a specific function (eg, a medical or physiological benefit) to the food rather than a purely nutritional effect.

[0123] While there is no legal definition of functional foods, most parties involved in this area agree that they are foods marketed as having specific health benefits beyond basic nutritional effects.

[0124] Some functional foods are nutraceuticals. Here, the term "nutraceutical" means a food that not only provides nutritional benefits and / or flavor satisfaction but also delivers therapeutic (or other beneficial) effects to the consumer. Nutraceuticals straddle the traditional dividing line between food and medicine.

[0125] dietary supplements

[0126] The bacterial strain according to the invention may take the form of a dietary supplement or may itself be used in combination with a dietary supplement, also referred to herein as a food supplement.

[0127] As used herein, the term "dietary supplement" refers to a product for ingestion that contains a "dietary ingredient" intended to increase the nutritional value or health benefits of (to supplement) the diet. "Dietary ingredients" may include, but are not limited to, one or any combination of the following: a microorganism, a probiotic (e.g., a probiotic bacteria), a vitamin, a mineral, a herb or other plant product, an amino acid, a dietary substance, concentrate, metabolite, component, or extract that is used by a person to supplement the diet by increasing total dietary intake.

[0128] In one aspect, the bacterial strain according to the invention is in the form of a dietary supplement for use in the treatment and / or prevention of disorders associated with H. pylori colonization of the gastric mucosa in a subject in need thereof.

[0129] In another aspect, the bacterial strain according to the present invention is in the form of a dietary supplement for use in inhibiting Helicobacter pylori in a subject in need thereof.

[0130] In a further aspect, the bacterial strain according to the present invention is in the form of a dietary supplement for use in the treatment and / or prevention of Helicobacter pylori infection in a subject in need thereof.

[0131] In another aspect, the bacterial strain according to the invention is in the form of a dietary supplement for use in maintaining gastric microbiota homeostasis in a subject in need thereof.

[0132] In another aspect, the bacterial strain according to the present invention is in the form of a dietary supplement for use in the treatment and prevention of gastric inflammation in a subject in need thereof.

[0133] Dietary supplements can come in many forms, such as tablets, capsules, soft gels, gel caps, liquids, or powders. Some dietary supplements can help ensure you get enough essential nutrients from your diet; others may help reduce your risk of disease.

[0134] Pharmaceutical preparations

[0135] The bacterial strains according to the present invention can be used as pharmaceuticals or for the preparation of pharmaceuticals.Herein, the term "pharmaceutical" is used in a broad sense and covers pharmaceuticals for humans as well as pharmaceuticals for animals (ie veterinary applications).

[0136] In a preferred aspect, the pharmaceutical product is for human use.

[0137] Pharmaceuticals may be used for therapeutic purposes, which may be curative, palliative, palliative or preventive in nature.

[0138] The drug may be in the form of a compressed tablet, tablet, powder, capsule, ointment, suppository or drinkable solution.

[0139] When used as a medicine or for preparing a medicine, the bacterial strain of the present invention can be used in combination with one or more of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, or a pharmaceutically active ingredient.

[0140] Depending on the use and / or mode of application and / or mode of administration, the medicinal product may be in the form of a liquid or as a solid.

[0141] The bacterial strain of the lactobacillus acidophilus species and the plant lactobacillus species used in the present invention itself can constitute a pharmaceutically active ingredient. In one embodiment, lactobacillus acidophilus constitutes a unique active ingredient. In another embodiment, plant lactobacillus constitutes a unique active ingredient. In another embodiment, there is a bacterial strain of lactobacillus acidophilus species and plant lactobacillus species. Alternatively, bacterial strain of the present invention can be at least one in multiple (that is, two or more) pharmaceutically active ingredients (for example, other bacterial strains).

[0142] medicine

[0143] The bacterial strain according to the invention may take the form of a medicament.

[0144] As used herein, the term "drug" encompasses drugs for both human and animal use in human and veterinary medicine. Furthermore, as used herein, the term "drug" means any substance that provides a therapeutic, preventive, and / or beneficial effect. As used herein, the term "drug" is not necessarily limited to substances that require marketing approval, but can include substances that can be used in cosmetics, health supplements, foods (including, for example, feed and beverages), probiotic cultures, and natural remedies. Furthermore, as used herein, the term "drug" encompasses products designed for incorporation into animal feed (e.g., livestock feed and / or pet food).

[0145] Medical food

[0146] The bacterial strain according to the present invention may take the form of a medical food.

[0147] “Medical Food” means a food that is formulated for consumption or administration, with or without a physician’s supervision, and is intended for use in the specific dietary management or condition for which unique nutritional requirements have been established through medical evaluation based on generally accepted scientific principles.

[0148] dose

[0149] The bacterial strain according to the present invention may be present in different forms (ie in a composition, a food product, a dietary supplement or a pharmaceutically acceptable formulation) in an amount of 10 6 to 10 14 Colony forming units (CFU) of one or more bacterial strains per dose or per gram of composition, food product, dietary supplement or pharmaceutically acceptable formulation, and more particularly, in an amount of 10 8 to 10 12 CFU of one or more bacterial strains per dose or per gram of composition, food product, dietary supplement, or pharmaceutically acceptable formulation.

[0150] Optionally, the composition, food product, dietary supplement or pharmaceutically acceptable formulation comprises about 10 10 CFU of one or more bacterial strains per dose or per gram of composition, food product, dietary supplement, or pharmaceutically acceptable formulation.

[0151] One or more bacterial strains (eg, Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115) may be present in an amount of about 10 6 to about 10 14 CFU of bacterial strain / dose, preferably about 10 8 to about 10 12The term "per dose" means that this amount of bacteria is provided to the subject per day or per intake (preferably per day). For example, if the bacteria are administered in a food product, such as in yogurt, the yogurt may contain about 10 CFU of the bacterial strain per dose. 6 to 10 14 Alternatively, however, this amount of bacteria may be divided into multiple administrations, each administration consisting of a smaller microbial load, as long as the total amount of bacterial strain received by the subject at any particular time (e.g., per 24-hour period) is about 10 6 to about 10 14 CFU of bacteria, optionally 10 8 to about 10 12 CFU of bacteria.

[0152] In one aspect, the bacterial strain of the invention is present in a composition, food product, dietary supplement, or pharmaceutically acceptable formulation in a therapeutically effective amount.

[0153] According to the present invention, the effective amount of at least one bacterial strain may be at least 10 6 CFU of bacteria / dose, optionally about 10 8 to about 10 12 CFU of bacteria / dose, e.g. about 10 10 CFU of bacteria / dose.

[0154] In one embodiment, the Lactobacillus acidophilus or Lactobacillus plantarum strain can be used at about 10 6 to about 10 14 CFU of bacteria / day, optionally about 10 8 to about 10 12 CFU of bacteria / day. Thus, an effective amount may be about 10 6 to about 10 14 CFU of bacteria / day, optionally about 10 8 to about 10 12 CFU of bacteria / day.

[0155] In a specific embodiment, 1×10 9 CFU of a single bacterial strain or 1.5 × 10 9 The amount of CFU of multiple bacterial strains.

[0156] Effects / Subjects / Medical Indications

[0157] In one embodiment, as used herein, the term "subject" refers to a mammal, including, for example, livestock (e.g., cattle, horses, pigs, and sheep) and humans. In one embodiment, the subject is a human. In one embodiment, the subject is a female. In one embodiment, the subject is a male. In another embodiment, the subject is a dog (e.g., a member of the genus Canis) or a cat (e.g., a member of the genus Felis or Panthera). In preferred embodiments, one or more bacterial strains and compositions, food products, dietary supplements, or pharmaceutically acceptable formulations are for use in humans.

[0158] Other embodiments of the present invention

[0159] Additional embodiments of the present invention include, but are not limited to, those set forth below:

[0160] Example 1. One or more bacterial strains for use in treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0161] Example 2. One or more bacterial strains for use in inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0162] Example 3. One or more bacterial strains for use in treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0163] Example 4. One or more bacterial strains for use in maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0164] Example 5. One or more bacterial strains for use in treating and preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0165] Embodiment 6. One or more bacterial strains for use according to any one of embodiments 1-5, wherein the one or more bacterial strains are combined with one or more antibiotics.

[0166] Example 7. One or more bacterial strains for use according to Example 6, wherein the antibiotic is but not limited to amoxicillin, clarithromycin, metronidazole, tetracycline and / or bismuth subsalicylate.

[0167] Embodiment 8. One or more bacterial strains for use according to any one of embodiments 1-7, wherein the one or more bacterial strains are Lactobacillus acidophilus NCFM.

[0168] Embodiment 9. The one or more bacterial strains for use according to any one of embodiments 1-7, wherein the one or more bacterial strains are Lactobacillus plantarum Lp-115.

[0169] Embodiment 10. One or more bacterial strains for use according to any one of embodiments 1 to 7, wherein the Lactobacillus acidophilus NCFM strain and the Lactobacillus plantarum Lp-115 strain are used in combination.

[0170] Embodiment 11. One or more bacterial strains for use according to any one of embodiments 1-10, wherein the one or more bacterial strains are used in combination with one or more fibers and / or prebiotics.

[0171] Embodiment 12. One or more bacterial strains for use according to embodiment 11, wherein the fiber and / or the prebiotic is polydextrose and / or polyphenol.

[0172] Embodiment 13. One or more bacterial strains for use according to any one of embodiments 1-12, wherein the one or more bacterial strains are in the form of a composition, a food product, a dietary supplement, or a pharmaceutically acceptable formulation.

[0173] Embodiment 14. One or more bacterial strains for use according to embodiment 13, wherein the pharmaceutically acceptable formulation is a medicament.

[0174] Embodiment 15. One or more bacterial strains for use according to embodiment 13, wherein the food product is a medical food product.

[0175] Example 16. A method for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject in need thereof, the method comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0176] Example 17. A method of inhibiting Helicobacter pylori in a subject in need thereof, the method comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0177] Example 18. A method for treating and / or preventing Helicobacter pylori infection in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0178] Example 19. A method of maintaining gastric microbiota homeostasis in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0179] Example 20. A method for treating and / or preventing gastric inflammation in a subject in need thereof, comprising administering to the subject one or more bacterial strains, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0180] Embodiment 21. The method of any one of embodiments 16-20, wherein the one or more bacterial strains are combined with one or more antibiotics.

[0181] Embodiment 22. The method of embodiment 21, wherein the antibiotic is amoxicillin, clarithromycin, metronidazole, tetracycline, and / or bismuth subsalicylate.

[0182] Embodiment 23. The method of any one of embodiments 16-22, wherein the strain is Lactobacillus acidophilus NCFM.

[0183] Embodiment 24. The method according to any one of embodiments 16-22, wherein the strain is Lactobacillus plantarum Lp-115.

[0184] Embodiment 25. The method according to any one of embodiments 16-22, wherein Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 are used in combination.

[0185] Embodiment 26. The method of any one of embodiments 16-25, wherein one or more bacterial strains are used in combination with one or more fibers and / or prebiotics.

[0186] Embodiment 27. The method of embodiment 26, wherein the fiber and / or the prebiotic is polydextrose and / or polyphenol.

[0187] Embodiment 28. The method of any one of embodiments 16-28, wherein the one or more bacterial strains are in the form of a composition, a food product, a dietary supplement, or a pharmaceutically acceptable formulation.

[0188] Embodiment 29. The method of embodiment 28, wherein the pharmaceutically acceptable formulation is a drug.

[0189] Embodiment 30. The method of embodiment 28, wherein the food product is a medical food product.

[0190] Example 31. Use of one or more bacterial strains for treating and / or preventing a condition associated with Helicobacter pylori colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0191] Example 32. Use of one or more bacterial strains for inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0192] Example 33. Use of one or more bacterial strains for treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0193] Example 34. Use of one or more bacterial strains for maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0194] Example 35. Use of one or more bacterial strains for treating and / or preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

[0195] Embodiment 36. The use of one or more bacterial strains according to any one of embodiments 31-35, wherein the one or more bacterial strains are combined with one or more antibiotics.

[0196] Embodiment 37. The use of one or more bacterial strains according to embodiment 36, wherein the antibiotic is but not limited to amoxicillin, clarithromycin, metronidazole, tetracycline and / or bismuth subsalicylate.

[0197] Embodiment 38. Use of one or more bacterial strains according to any one of embodiments 31-37, wherein the strain is Lactobacillus acidophilus NCFM.

[0198] Embodiment 39. The use of one or more bacterial strains according to any one of embodiments 31-37, wherein the strain is Lactobacillus plantarum Lp-115.

[0199] Embodiment 40. The use of one or more bacterial strains according to any one of embodiments 31-37, wherein Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 are used in combination.

[0200] Embodiment 41. The use of one or more bacterial strains according to any one of embodiments 31-40, wherein the one or more bacterial strains are used in combination with one or more fibers and / or prebiotics.

[0201] Embodiment 42. Use of one or more bacterial strains according to embodiment 41, wherein the fiber and / or the prebiotic is polydextrose and / or polyphenol.

[0202] Embodiment 43. The use of one or more bacterial strains according to any one of embodiments 31-42, wherein the one or more bacterial strains are in the form of a composition, a food product, a dietary supplement, or a pharmaceutically acceptable formulation.

[0203] Embodiment 44. The use of one or more bacterial strains according to embodiment 43, wherein the pharmaceutically acceptable formulation is a drug.

[0204] Embodiment 45. Use of one or more bacterial strains according to embodiment 43, wherein the food product is a medical food product.

[0205] Examples

[0206] The following examples are provided to demonstrate and further illustrate specific embodiments and aspects of the present invention and should not be construed as limiting the scope of the invention.

[0207] Materials and Methods

[0208] Bacterial strains, cell lines, and animals

[0209] H. pylori P12 and H. pylori P12-GFP strains were obtained from the Max Planck Institute for Infection Biology (MPIIB) in Berlin, Germany, and H. pylori SS1 was obtained from the University of Western Australia (UWA), Australia.

[0210] Helicobacter pylori strains were cultured in Columbia medium plates containing 7% sterile defibrinated sheep blood (Bianzhen, Nanjing), 20 μg / ml vancomycin (Meilunbio), 10 μg / ml polymyxin (Meilunbio), 10 μg / ml amphotericin B (Meilunbio), and 10 μg / ml trimethoprim (Sigma), and then placed in an incubator containing 5% O2 and 10% CO2, cultured at 37°C, subcultured every 3 days, and used for experimental use after subculture.

[0211] Lactobacillus, Lactobacillus, and Lactobacillus strains were provided by Danisco (China) Holding Co., Ltd., namely, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La-14 (La-14), Lactobacillus plantarum Lp-115 (Lp-115), Lactobacillus paracasei Lpc-37 (Lpc-37), Lactobacillus rhamnosus Lr-32 (Lr-32), and Lactobacillus rhamnosus GG. After identification of bacterial morphology using a Gram staining kit (Solarbio), the strains were anaerobically cultured in MRS broth (Solarbio) at 37° C. for 48 h and then subcultured.

[0212] Human gastric adenocarcinoma cell line (AGS) was purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). AGS cells were cultured in RPMI 1640 medium (Thermo) supplemented with 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO2.

[0213] Fifty male C57BL / 6 mice (specific pathogen-free, 4 weeks old) were purchased from Zhejiang Vital River Laboratory Animal Technology Co. Ltd. All animals were housed under standard conditions.

[0214] In vitro Helicobacter pylori and Lactobacillus coinfection model

[0215] Helicobacter pylori P12 and Lactobacillus were cultured overnight in BHI (Thermo Fisher) and MRS broth. After centrifugation at 5000 x rpm for 8 min and at 4000 x rpm for 5 min, the supernatant was discarded and the bacteria were harvested and resuspended in 1 ml of serum-free RPMI 1640 medium.

[0216] Overnight cultured AGS cells were co-infected with Lactobacillus (MOI=100) and H. pylori P12 (MOI=100) for 6 h. After the incubation period, supernatants were harvested for ELISA and cells were harvested for RNA isolation.

[0217] Helicobacter pylori attachment to AGS cells

[0218] AGS cells were cultured in two 12-well plates and incubated with Lactobacillus (MOI = 100) and Helicobacter pylori P12-GFP (MOI = 100) for 6 h. At the end of the incubation, one plate of cells was washed three times with PBS and photographed using fluorescence and white light. Urease activity assay reagent was prepared before the end of infection: Concentrated sodium hydroxide was added to PBS (pH = 7.4) to adjust the solution (pH = 6.8), and urea (Solybold) and phenol red (Solybold) were weighed and added to a concentration of 110 mmol / L urea and 10 mg / L phenol red, respectively, and then dissolved by vigorous shaking. 1 ml of urease detection reagent was added to another 12-well plate. After 1-7 h of reaction, 100 μl of culture medium was aspirated into a 96-well plate to record the absorbance value at 540 nm.

[0219] Quantitative real-time PCR (qRT-PCR)

[0220] AGS cells and mouse gastric mucosal tissue were lysed using RNAisoplus (TaKaRa, Japan). cDNA was converted using the ReverTra Ace qPCR RT Kit (TOYOBO, Shanghai) according to the manufacturer's instructions. qRT-PCR was performed using 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a Roche Lightcycler 480 II system based on the manufacturer's recommendations. Primer sequences were designed using NCBI Primer-BLAST (Table S1). 2 -ΔΔCt Methods Relative gene expression was determined. All experiments were repeated three times.

[0221] Table S1. Primers used for quantification of inflammatory factors in AGS cells and mouse gastric tissue

[0222]

[0223] Quantification of cytokine profiles by ELISA

[0224] After infection, the final culture medium was collected, centrifuged at 12000rpm for 5min, cell debris and bacteria were removed, and the supernatant was collected. Blood of mice was collected from the orbital venous plexus of each group of mice and incubated overnight at 4°C, and then the sample was centrifuged at 3500rpm for 20min to collect the supernatant. The concentrations of IL-8 and TNF-α were detected by human IL-8 (interleukin-8) ELISA kit (Elabscience, Wuhan, China) and human TNF-α (tumor necrosis factor α) ELISA kit (Elabscience, Wuhan, China). The serum levels of mouse IL-4, IL-10 and IFN-γ were measured by V-PLEX proinflammatory cytokine detection combination 1 (mouse) kit (V-PLEX Proinflammatory Panel 1 (mouse) Kit) (Meso Scale Discovery, Rockville, Maryland, USA). Operated according to the specific steps in the manual. All experiments were repeated 3 times.

[0225] A model of Helicobacter pylori infection and intervention with bacterial strains Lactobacillus and Lactobacillus was established in mice.

[0226] Specific pathogen-free C57BL / 6 mice (male; 4 weeks old) were obtained from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. and fed with Laboratory Rodent Diet 5001. After 1 week of adaptive feeding, 50 mice were randomly divided into 5 groups:

[0227] ·Not infected;

[0228] Helicobacter pylori SS1;

[0229] Helicobacter pylori SS1 + Lactobacillus acidophilus NCFM;

[0230] Helicobacter pylori SS1 + Lactobacillus plantarum Lp-115;

[0231] Helicobacter pylori SS1 + Lactobacillus acidophilus NCFM + Lactobacillus plantarum Lp-115.

[0232] The control group was gavaged with PBS, and the experimental group was only treated with Helicobacter pylori SS1 (1*10 9 CFU / ml or compared with the corresponding bacterial strains (1*10 9 CFU / m1) combined oral administration, once every 2 days, for 6 weeks ( Figure 3 a).

[0233] Rapid Urease Test (RUT)

[0234] The gastric mucosa of the mice was removed and a rapid urease test was performed using a rapid urease kit (Sanqiang Biochemical Co., Ltd., Fujian, China). The color change was determined. When the solution turned pink or red, the urease test was positive; when it remained yellow, the urease test was negative.

[0235] Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC)

[0236] Gastric tissue was fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4 mm sections, and then stained by HE method and immunohistochemistry method, respectively. Chronic gastritis histological grading scale: There are five histological changes to be graded: Helicobacter pylori, chronic inflammation, activity, atrophy and intestinal metaplasia, each of which is divided into four grades: none, mild, moderate and severe. According to the new Sydney system, the degree of inflammation and lymphocyte infiltration of gastric tissue after Helicobacter pylori infection was evaluated. Immunohistochemistry of Helicobacter pylori was performed using rabbit anti-Helicobacter pylori polyclonal antibody (Cell Marque) (Zhongshan Jinqiao Company (ZSGB), Beijing, China) to identify Helicobacter pylori colonization of mouse gastric mucosa.

[0237] statistics

[0238] All statistical analyses were performed using GraphPad Prism 9 software. During the processing of experimental data, values ​​that deviated from the mean of the same group of data by more than three times the standard deviation were considered outliers and were removed. We performed one-way analysis of variance on the raw data and 1g-transformed data to compare multiple groups. Bonferroni and Tukey tests were performed to calculate statistical significance between multiple groups. For all statistical analyses, p values ​​< 0.05 were considered significant.

[0239] result

[0240] Six strains of Lactobacillus and Lactobacillus inhibited the attachment of H. pylori to AGS cells.

[0241] To compare the effects of six probiotic strains in intervening in H. pylori attachment, AGS cells were co-infected with H. pylori P12-GFP and six probiotic bacterial strains (MOI = 100) for 6 hours. After infection, cells and GFP-positive H. pylori were measured under a fluorescence microscope. The number of GFP-positive H. pylori in the six probiotic strain intervention group was less than that in the H. pylori only infection group. In addition, compared with the other Lactobacillus intervention groups, the Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 groups had less cellular stress on AGS cells ( Figure 1 a).

[0242] To further quantify the inhibitory effects between different probiotic strains, we used the urease activity assay as described above to treat the co-infection plates and recorded the absorbance values ​​at 540 nm. From 1 to 7 h, the absorbance values ​​of the six probiotic strain intervention groups decreased to varying degrees compared with the H. pylori only infection group ( Figure 1 b). Therefore, it was demonstrated that these six probiotic strains can effectively reduce H. pylori colonization, and in addition, L. acidophilus NCFM and L. plantarum Lp-115 have less cytotoxicity to AGS cells.

[0243] Six probiotic strains inhibited Helicobacter pylori-induced inflammation in AGS cells.

[0244] Helicobacter pylori infection induces inflammation of the gastric mucosa and activates cytokines (such as IL-8 and TNF-a). To compare the inhibitory effects of the six probiotic strains, we performed a model of co-infection of H. pylori with six Lactobacillus strains (MOI = 100) in AGS cells for 6 hours and examined the mRNA and protein levels of IL-8 and TNF-a. It showed that the mRNA expression of IL-8 and TNF-α in the six probiotic strain intervention group was significantly reduced compared with the mRNA expression of IL-8 and TNF-α in the H. pylori only infection group ( Figure 2 a).

[0245] Consistently, the levels of IL-8 and TNF-α in the cell supernatant were also significantly decreased in the probiotic strain intervention group compared with the H. pylori only infection group ( Figure 2 b) The combination of mRNA and protein level data suggests that L. acidophilus NCFM and L. plantarum Lp-115 have superior intervention effects compared to other strains that require further validation in mouse models of H. pylori infection.

[0246] Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 suppress Helicobacter pylori colonization in mice.

[0247] To further verify whether Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 can attenuate H. pylori colonization and gastric inflammation in vivo, C57BL / 6 mice were co-infected with Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 and H. pylori SS1 for 6 weeks ( Figure 3 a). After infection, mice were euthanized and gastric tissues were assessed for H. pylori infection by rapid urease test ( Figure 5 ). H. pylori attachment on gastric tissue was then analyzed by immunohistochemistry (IHC) assay ( Figure 3 b), which showed that the Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 intervention groups had considerably less H. pylori attachment compared to the H. pylori only infection group. To further verify the H. pylori colonization in the different groups, the mRNA expression of Ure-A and Ure-B in the gastric tissues of all groups was tested ( Figure 3 c) The results showed that the Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 intervention groups had lower Ure-A and Ure-B expression compared to the H. pylori-only infection group. This further demonstrates that Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 can inhibit H. pylori colonization in the gastric mucosa in vivo.

[0248] Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 suppress Helicobacter pylori-induced inflammation in mice.

[0249] To further investigate whether Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 can attenuate H. pylori colonization and gastric inflammation in vivo, gastric tissues from the five groups were then analyzed by hematoxylin-eosin staining (HE staining). Compared with the uninfected group, the gastric lamina propria of most mice in the H. pylori-infected group showed more lymphocyte and plasma cell infiltration and neutrophil infiltration during the active phase, and some mice also had local thinning, reduced glandular glands, and thickening of the muscularis mucosa; compared with the H. pylori group, the inflammatory cells in the infiltrative lamina propria of the gastric mucosa of the H. pylori + NCFM group, the H. pylori + Lp-115 group, and the H. pylori + NCFM + Lp-115 group were reduced to varying degrees, indicating that Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 can improve H. pylori-induced gastric inflammation ( Figure 4 a).

[0250] To further verify whether strains NCFM and / or Lp-115 have the ability to inhibit Helicobacter pylori-induced Th1 inflammation, the expression of IL-4, CXCL15, IL-10, and IFN-γ was detected by qRT-PCR. It showed that Helicobacter pylori infection significantly increased the expression of IL-4, CXCL15, IL-10, and IFN-γ, while after the intervention of strains NCFM and Lp-115, IFN-γ in each intervention group decreased to varying degrees, and CXCL15 was slightly decreased in the Lp-115 intervention group; in addition, the expression of IL-4 increased to varying degrees in the Lp-115 and NCFM+Lp-115 intervention groups. Compared with the mRNA expression of IL-10 in the Helicobacter pylori group, the mRNA expression of IL-10 in the NCFM and Lp-115 intervention groups did not change ( Figure 4 b). In conclusion, it was shown that NCFM and / or Lp-115 can inhibit H. pylori-induced Th1 inflammation and activate Th2 inflammation in C57BL / 6 mice.

[0251] discuss

[0252] In our study, we screened six probiotic strains and found that Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 could inhibit Helicobacter pylori attachment in vitro in the AGS cell line and in vivo in the C57 / BL6 mouse model, representing the first validation study of these two strains. This study demonstrated for the first time that Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 could inhibit H. pylori P12-induced IL-8 and TNF-α expression at both the mRNA and protein levels in the AGS cell line. Furthermore, in the C57BL / 6 mouse model of H. pylori SS1 infection, this study demonstrated that Lactobacillus acidophilus NCFM and / or Lactobacillus plantarum Lp-115 could suppress IFN-γ but increase IL-4.

[0253] This study demonstrated that intervention with Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 can alleviate the host inflammatory response induced by Helicobacter pylori infection by downregulating the local Th1 immune response in the gastric mucosa, inhibiting the pro-inflammatory cytokine IFN-γ, and promoting the Th2 response to produce the anti-inflammatory cytokine IL-4. Therefore, Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 may play an important role in promoting T cell differentiation into Th2 cells to balance HP-induced Th1 inflammation.

[0254] In this study, it was also demonstrated from the AGS cell model that some Lactobacillus strains were harmful, including Lactobacillus rhamnosus Lr-32, Lactobacillus paracasei Lpc-37, Lactobacillus rhamnosus GG and Lactobacillus acidophilus La-14, while Lactobacillus acidophilus NCFM and Lactobacillus plantarum Lp-115 were safe.

[0255] In summary, this study demonstrated that strain NCFM and strain Lp-115 have the ability to inhibit H. pylori attachment and host inflammatory responses in cell lines and mouse models, and therefore show unique value in the treatment of H. pylori, such as treating and / or preventing conditions associated with H. pylori colonization of the gastric mucosa, treating and / or preventing H. pylori infection, maintaining gastric microbiota homeostasis, and treating and / or preventing gastric inflammation in subjects.

[0256] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for practicing the present invention that would be apparent to those skilled in the biochemistry and biotechnology or related fields are intended to fall within the scope of the following claims.

Claims

1. One or more bacterial strains for use in treating and / or preventing a condition associated with Helicobacter pylori (H. pylori) colonization of the gastric mucosa in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactiplantibacillus plantarum Lp-115 strain, or a combination thereof.

2. One or more bacterial strains for use in inhibiting Helicobacter pylori in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

3. One or more bacterial strains for use in treating and / or preventing Helicobacter pylori infection in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

4. One or more bacterial strains for use in maintaining gastric microbiota homeostasis in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

5. One or more bacterial strains for use in treating and preventing gastric inflammation in a subject, wherein the one or more bacterial strains are selected from the group consisting of Lactobacillus acidophilus NCFM strain and Lactobacillus plantarum Lp-115 strain, or a combination thereof.

6. One or more bacterial strains for use according to any one of claims 1 to 5, wherein the one or more bacterial strains are combined with one or more antibiotics.

7. One or more bacterial strains for use according to claim 6, wherein the antibiotic is but not limited to amoxicillin, clarithromycin, metronidazole, tetracycline and / or bismuth subsalicylate.

8. One or more bacterial strains for use according to any one of claims 1 to 7, wherein the one or more bacterial strains are Lactobacillus acidophilus NCFM.

9. One or more bacterial strains for use according to any one of claims 1 to 7, wherein the one or more bacterial strains are Lactobacillus plantarum Lp-115.

10. One or more bacterial strains for use according to any one of claims 1 to 7, wherein the Lactobacillus acidophilus NCFM strain and the Lactobacillus plantarum Lp-115 strain are used in combination.

11. One or more bacterial strains for use according to any one of claims 1 to 10, wherein the one or more bacterial strains are used in combination with one or more fibers and / or prebiotics.

12. One or more bacterial strains for use according to claim 11, wherein the fiber and / or the prebiotic is polydextrose and / or polyphenol.

13. The one or more bacterial strains for use according to any one of claims 1 to 12, wherein the one or more bacterial strains are in the form of a composition, a food product, a dietary supplement or a pharmaceutically acceptable formulation.

14. One or more bacterial strains for use according to claim 13, wherein the pharmaceutically acceptable formulation is a medicament.

15. One or more bacterial strains for use according to claim 13, wherein the food product is a medical food product.

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