Lactobacillus helveticus BGRA43 strain for the treatment of liver steatosis

Abstract

The present invention relates to the use of the Lactobacillus helveticus BGRA43 strain for the prevention and / or treatment of liver diseases and / or liver disorders, preferably metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (N AFLD), and / or for the reduction of liver steatosis. The formulation, preferably for peroral administration, comprising the L. helveticus BGRA43 strain. The formulation comprising the L helveticus BGRA43 strain, which is preferably a probiotic food product intended for human consumption. Lyophilizate of the L helveticus BGRA43 strain, comprising the L. helveticus BGRA43 strain at a concentration from 108 to 1013 CFU / g.

Description

[0001] LACTOBACILLUS HELVETICUS BGRA43 STRAIN FOR THE TREATMENT OF LIVER STEATOSIS

[0002] Technical field of the invention

[0003] The present invention generally relates to the field of formulations exhibiting therapeutic, pharmacological activity. More specifically, present invention relates to the field of probiotic formulations and / or therapeutic formulations comprising probiotic strains, which may be employed in the treatment of liver disorders and diseases, such as metabolic dysfunction- associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), or in the reduction of liver steatosis.

[0004] Technical problem

[0005] The technical problem addressed by the present invention relates to the provision of a Lactobacillus helveticus BGRA43 strain, preferably formulated as a pharmaceutical formulation, dietary supplement, and / or probiotic food product, intended for use in the treatment of liver disorders or diseases. Preferably, the formulation exerts a beneficial effect on metabolic parameters associated with liver function.

[0006] The technical problem addressed by the present invention may also be defined as the need to provide an effective probiotic formulation, safe for use, which exerts a beneficial effect in the prevention and / or treatment of liver disorders and diseases, preferably liver disorders and diseases associated with hepatic fat accumulation. There is a particular need for such a formulation to be free from synthetic components or other pharmacologically active synthetic compounds, thereby offering a healthy and safe alternative to existing market-available products with similar intended use. The present invention is formulation designed in such a way that the Lactobacillus helveticus BGRA43 probiotic strain retains its viability and functional properties, and can be effectively utilized as a constituent in various formulations, including dietary supplements, pharmaceutical products (i.e., medicinal preparations), and / or functional foods.

[0007] The present invention provides a formulation containing Lactobacillus helveticus BGRA43, wherein the formulation is suitable for oral administration and may be used for the treatment of MASLD or for the reduction of liver steatosis.

[0008] Prior Art

[0009] Obesity represents one of the leading health problems of the modern era, as evidenced by the data of the World Health Organization (WHO), over 890 million adults or about 16% of the world's population were classified as obese in 2022 (WHO, Obesity and overweight fact sheet, 2022). Obesity is closely associated with other metabolic disorders, such as insulin resistance and an increased risk of developing MASLD. Over the past decades, MASLD has emerged as the most prevalent liver disease in Western world, with a rapid rise in its complications, including hepatocellular carcinoma. MASLD is now considered the most common chronic liver disease, with a prevalence of about 32% (Teng et al., 2023). The primary pathological feature of MASLD is excessive accumulation of lipids in the liver, while disease progression is marked by the onset of inflammation, steatosis, fibrosis, and hepatocellular carcinoma. Nowadays, as the number of people suffering from obesity and metabolic syndrome increases, so does the incidence of MASLD which continues to rise, which represents a serious challenge to both public health systems and national economies. The treatment of MASLD depends on the disease stage and may involve lifestyle modification (weight loss, dietary changes, and increased physical activity) and / or pharmacotherapy. Current pharmacological interventions include insulin sensitizers, antioxidants and cytoprotective agents, lipid-lowering drugs, among others.

[0010] Given the high prevalence and clinical severity of liver disorders associated with hepatic fat accumulation, numerous clinical studies have recently been conducted to evaluate various therapeutic approaches for the treatment of MASLD and / or liver steatosis. Most of the clinical studies investigating MASLD therapies focus on the effects of different nutritional supplements, such as choline, oligofructose, and inulin, as well as extract of Silybum marianum (milk thistle), and combinations of vitamin B12, vitamin B6, folate, and betaine. Several other clinical studies explore the effects of pharmacological agents on MASLD / NAFLD, including sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., empagliflozin), lisinopril, 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13) inhibitors, semaglutide, and others. Only a three studies (NCT05804422, NCT05821010, and NCT04781933) examine the use of probiotic bacteria, either alone or in combination with prebiotics (i.e., as synbiotics). Modulation of the gut microbiota is increasingly recognized as a relevant therapeutic strategy for managing various systemic diseases, including liver diseases.

[0011] Patent No. US9872844 describes a method for reducing or preventing non-alcoholic steatohepatitis (NASH), wherein the method involves the administration of a composition comprising: a) leucine administered in a dose of 0.25-3.00 g / day, or leucine metabolites in a dose of 0.2-3.0 g / day; b) metformin administered in a dose of 10-1000 mg / day; and c) sildenafil administered in a dose of 0.05-100 mg / day; wherein components a), b), and c) are administered either as a single formulation or separately, wherein components are administered in a sequential manner. However, Patent US9872844 does not suggest or disclose the use of probiotic strains for the reduction and / or prevention of non-alcoholic steatohepatitis.

[0012] The role of the gut microbiota in the development, progression, and treatment of metabolic diseases is becoming increasingly evident. It is well established that certain probiotic bacteria possess functional capacities to either promote or mitigate obesity. For instance, Lactobacillus plantarum has been demonstrated in animal studies to act as an effective probiotic with antiinflammatory properties and beneficial effects on body mass reduction (Mazloom et al., 2019).

[0013] Patent No. US10512661 describes a method for preventing liver cancer in an individual already diagnosed with NAFLD. The method comprises the administration of various genetically modified strains of Lactobacillus spp. using CRISPR-Cas technology, in combination with a minimum daily intake of 6 g of dietary fiber. The dietary fiber is reported to ensure the therapeutic efficacy of the administered Lactobacillus strains, while simultaneously increasing the abundance of Roseburia spp. and decreasing the abundance of Akkermansia spp. within the gut microbiota. Patent US10512661 is based on the use of strains of Lactobacillus spp. for the reduction of LDL cholesterol and triglyceride levels, wherein the strains are specifically engineered using the CRISPR / Cas9 method.

[0014] Patent application CN116064286 relates to a specific strain, Lactobacillus helveticus ZJUIDS11, which has demonstrated various biological effects, including effect relevant to liver diseases. In animal studies described in CN116064286, the strain was administered at concentrations ranging from 109to 1010CFU / mL, over a 12-week period. It was concluded that the strain is suitable for use as an additive in animal feed.

[0015] Patent application CN114145461 describes a composition comprising 60 parts of Lactobacillus plantarum HA-11930, 50 parts of L cose / Rosell-021520, and 30 parts of L. helveticus Rosell- 005210, intended for alleviating conditions associated with fatty liver.

[0016] Patent application W02020041581 discloses a probiotic formulation for the treatment of liver diseases, based on cells derived from one or more genera selected from Lactobacillus,

[0017] Bifidobacterium spp., Bacteroides spp., Enterococcus spp., and Clostridium spp.

[0018] Patent application W02021075663 describes a pharmaceutical composition containing a mixture of L. helveticus and either B. breve or B. longum, in the form of cultures derived from human and / or animal biological material, for the prevention or treatment of non-alcoholic fatty liver disease (NAFLD). The Lactobacillus spp. and Bifidobacterium spp. strains are mixed at a 1:1 ratio and administered at a concentration of 109CFU / day.

[0019] Summary of the Invention

[0020] The present invention provides Lactobacillus helveticus BGRA43 strain intended for the treatment of liver diseases and / or disorders, such as non-alcoholic fatty liver disease and / or metabolic dysfunction-associated steatotic liver disease. The invention provides a strain that is effective at concentrations that are safe for human use. Preferably, the strain is incorporated into a pharmaceutical formulation intended for (per)oral administration or into a probiotic food product. The strain is of natural origin, i.e., it is not genetically modified, and belongs to the species Lactobacillus helveticus, which is considered safe for human consumption. This is the first time in which the L. helveticus BGRA43 strain has been formulated as a pharmaceutical formulation for peroral use, particularly suitable for enhancing physiological liver function and for the treatment of fatty liver and / or liver steatosis.

[0021] Present strain L. helveticus BGRA43 and formulations thereof have demonstrated beneficial pharmacological effects on lipid metabolism in the liver and adipose tissue.

[0022] The invention provides a novel, innovative lyophilizate of the L. helveticus BGRA43 strain. The proposed lyophilizate is suitable for use as a probiotic dietary supplement. Disclosed strain exhibits a pleasant, neutral taste, which makes it particularly suitable for incorporation into oral pharmaceutical formulations and food products. The formulation of the L. helveticus BGRA43 strain ensures that the probiotic strain retains its functional properties and that it can be effectively reconstituted for various applications, including use in dietary supplements, pharmaceutical products, and / or as a component of functional foods.

[0023] Brief Description of the Figures

[0024] Figure 1. Experimental protocol for the peroral administration of a suspension of lyophilized L helveticus BGRA43 (herein abbreviated as LHBGRA43) in mice fed a 60% high-fat diet. Animajs were maintained on either standard chow or a high-fat diet for 14 weeks. During the final 5 weeks, they received either a buffered saline solution (placebo) or a suspension of lyophilized LHBGRA43 by oral gavage.

[0025] Figure 2. Body mass of mice following peroral administration of a suspension of lyophilized L helveticus BGRA43 (LHBGRA43) in animals fed a 60% high-fat diet. C - control; HFD - high-fat diet (60% fat); HFD + B - high-fat diet (60% fat) with LHBGRA43. Data were analyzed using One-way ANOVA followed by Tu key's post hoc test. Statistically significant differences between groups are indicated as ***p < 0.001 (treatments vs. control) and *p < 0.05 (HFD + B vs. HFD).

[0026] Figure 3. Liver mass and relative liver mass (normalized to total body mass, BM) following peroral administration of a suspension of lyophilized L. helvet icus BGRA43 (herein abbreviated as LHBGRA43) in mice fed a 60% high-fat diet. C - control; HFD - high-fat diet (60% fat); HFD + B - high- fat diet (60% fat) with LHBGRA43. Data were analyzed using One-way ANOVA and Tukey's post hoc test. Statistically significant differences between groups are indicated as *p < 0.05, **p < 0.01 (treatments vs. control), and#p < 0.05,##p < 0.01 (HFD + B vs. HFD).

[0027] Figure 4. Morphological characteristics of mice from the control group (C), group fed with 60% fat (HFD), and the treated group fed with 60% fat oraly supplemented with L. helveticus BGRA43 (HFD + B). Notable differences in the amount of visceral white adipose tissue can be observed between the HFD and HFD + B animals.

[0028] Figure 5. Absolute mass of visceral and subcutaneous adipose tissue, as well as relative mass of individual visceral and subcutaneous fat depots normalized to body mass, in mice from the control group (C), group on diet with 60% fat (HFD), and the treated group fed diet with 60% fat oraly supplemented with L. helveticus BGRA43 (herein abbreviated as LHBGRA43) (HFD + B). Data were analyzed using One-way ANOVA followed by Tu key's post hoc test. Statistically significant differences between groups are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 (treatments vs. control), and#p < 0.05 (HFD + B vs. HFD). BM - body mass.

[0029] Figure 6. Positive effect of oral supplementation with L. helveticus BGRA43 (herein abbreviated as LHBGRA43) on liver steatosis in mice fed a 60% high-fat diet. C - control group; HFD - 60% high-fat diet; HFD + B - 60% high-fat diet with LHBGRA43. Liver sections were stained using hematoxylin and eosin (H&E) staining.

[0030] Figure 7. Positive effect of oral supplementation with L. helveticus BGRA43 (herein abbreviated as LHBGRA43) on the protein level of the enzyme SREBPlc in liver of mice fed a 60% high-fat diet. C - control; HFD - diet with 60% of fat; HFD + B - diet with 60% of fat and LHBGRA43. Data were analyzed using One-way ANOVA and Tukey's post hoc test. Statistically significant differences between groups are indicated as **p < 0.01 (HFD vs. C) and **p < 0.01 (HFD + B vs. HFD).

[0031] Figure 8. Concentration of ALT enzyme in the serum (A) and triglycerides in the blood (B) of mice following peroral administration of a lyophilized L. helveticus BGRA43 suspension (herein abbreviated as LHBGRA43) in animals on a 60% high-fat diet. C - control; HFD - diet with 60% of fat; HFD + B - diet with 60% of fat and LHBGRA43.. Data were analyzed using One-way ANOVA followed by Tukey's post hoc test. Detailed description of the invention

[0032] The present invention relates to the use of the strain L. helveticus BGRA43 in the prevention and / or treatment of liver diseases and / or disorders. The present strain L. helveticus BGRA43 may have a beneficial effect on biochemical parameters that are elevated in liver diseases and / or disorders, i.e. it may have a beneficial effect on lowering the levels of biochemical parameters such as triglycerides, LDL cholesterol, liver enzymes such as liver transaminases or factors involved in lipogenesis in the liver such as SREBP1c. L. helveticus BGRA43 is a probiotic bacterium that is suitable and safe for suitable for human use for pharmacological and / or nutritional purposes. A detailed characterization of the strain, including the study of antimicrobial and proteolytic activity as well as the study of probiotic and technological characteristics of the strain, has been performed previously (Banina et al., 1998).

[0033] The present invention disclose for the first time the use of the strain L. helveticus BGRA43 for the treatment and / or prevention of liver steatosis. Liver steatosis is a major health problem of Western society and modern lifestyle. The term steatosis refers to the pathological accumulation of fat in the liver cells, which can be caused by various etiologic factors, of which obesity and alcoholism are the most common. In the general population, steatosis most commonly occurs as a concomitant disease of obesity caused by fatty foods and / or foods rich in simple carbohydrates, i.e., sugar. In the context of the present invention, it has also been shown that the use of the strain L. helveticus BGRA43 is particularly effective in the treatment of MASLD. MASLD is characterized by fat accumulation in the liver, which is not related to excessive alcohol consumption, but is usually associated with poor lifestyle habits such as insufficient physical activity and a high-calorie diet. MASLD is common in people with metabolic syndrome, which includes obesity, diabetes, high blood pressure and elevated blood lipid levels. The present strain L. helveticus BGRA43 showed a positive effect in the treatment of MASLD and / or liver steatosis in experiments on an obese mouse model.

[0034] The advantage of using the present strain, L. helveticus BGRA43, in the prevention and / or treatment of diseases and / or disorders of liver function, is that it is a natural strain, i.e. not genetically modified, which complies with national and European standards for use in medicinal and / or food products. The present strain is effective as a monocomponent formulation and [is also suitable for mixing with other probiotic and / or prebiotic components. The present strain showed efficacy in the treatment of MASLD and / or liver steatosis at lower doses and / or in a shorter time interval compared to known probiotic strains.

[0035] Treatment of MASLD is a complex and long-term process which depends on the stage of the disease and involves lifestyle modification, such as changing dietary habits and increasing physical activity, and / or the use of pharmacotherapeutics, such as insulin sensitizers, antioxidants and cytoprotectants, hypolipemic agents, etc. The present strain is the human gastrointestinal isolate L. helveticus BGRA43, which exhibits antimicrobial activity against pathogenic strains such as Clostridium sporogenes, Yersinia enterocolitica, Shigella sonnei, Shigella flekneri and Streptococcus pneumoniae, among others (Strahinic et al., 2013). In addition, bioactive peptides released by L helveticus BGRA43 in fermented milk have been confirmed as modulators of innate immunity, as they modulate the production of the proinflammatory cytokines IL-6 and TNF-α. A novel characteristic of the strain L. helveticus BGRA43 is that it has a ameliorative effect on MASLD and / or liver steatosis, so that after oral administration to obese mice with fatty liver, the liver becomes clean and no longer has accumulated fat droplets.

[0036] The present L. helveticus BGRA43 strain can be used in the treatment of liver diseases and / or disorders, such as metabolic dysfunction-associated steatotic liver disease.

[0037] In the context of the present invention, the L. helveticus BGRA43 strain has been found to have a beneficial effect on conditions associated with an increase in LDL cholesterol and / or triglyceride levels. The strain according to the invention is a safe, natural formulation suitable for use in medicines and / or foods intended for people who wish to reduce their body mass.

[0038] The present L. helveticus BGRA43 strain is particularly suitable for use in diseases associated with liver dysfunction and / or weight gain. The present strain is particularly suitable for use in medicines and foods intended for weight loss. In addition to the surprising effect on weight reduction, the present strain also shows a positive effect on the regulation of biochemical parameters such as triglycerides and / or LDL cholesterol and / or liver enzymes such as ALT (alanine transaminase) in serum and / or blood. Liver function disorders and / or diseases are inextricably linked to changes in the metabolism of fats, carbohydrates and / or proteins, which is reflected in disturbances in the values of biochemical parameters. The use of the present L. helveticus BGRA43 strain achieves a positive effect on the regulation of biochemical parameters, e.g. triglycerides and / or liver enzymes, which may reduce the risk of developing other systemic disorders and / or diseases, such as obesity or type 2 diabetes.

[0039] The present invention describes a formulation comprising the strain L. helveticus BGRA43. The present formulation is intended for the treatment and / or prevention of conditions associated with liver diseases, such as steatosis and / or MASLD.

[0040] Preferably, the formulation containing the strain L. helveticus BGRA43 is intended for oral administration to humans and / or animals. The formulation may be intended for vaginal, rectal, dermal, nasal or enteral administration. Enteral administration refers to the application Of the present formulation where the present formulation passes through the digestive tract. Ini, rectal administration, the present formulation is preferably formulated, for example, as a suppository or as a fecal microbiotic transplant.

[0041] Preferably, the formulation contains the strain L. helveticus BGRA43 in a concentration of about 105-1015CFU / g, preferably in a concentration of about 107-1010, particularly preferably about 108- 109CFU / g. The term CFU / g refers to the number of microorganisms per gram of the formulation (i.e. CFU stands for colony-forming units). A formulation with these characteristics can be formulated as a medical preparation or as a functional food, more precisely as a probiotic preparation with a beneficial effect on the work of the liver and / or the metabolic state pf the organism, in the context of the present invention, it was found that the L. helveticus BGRA43 strain can be used as a monocomponent preparation in lower concentrations than other known strains.

[0042] Since it is a known strain that is easy to cultivate, the present strain and formulations thereof are particularly suitable for production in large quantities, for example for industrial production.

[0043] Preferably, the formulation contains a lyophilizate of living bacterial cells of the strain L. helveticus BGRA43. The use of the lyophilizate of L. helveticus BGRA43 in the present formulation is particularly suitable because said lyophilizate has favorable technological characteristics for formulation in the form of pharmaceutical and / or food preparations. By using the lyophilizate of the present strain L. helveticus BGRA43, particularly good characteristics are obtained which are particularly suitable for the formulation of pharmaceutical products and / or probiotic food products.

[0044] Preferably, the formulation contains live or inactivated strain L. helveticus BGRA43, metabolic products of strain I. helveticus BGRA43, fragments of cell membranes of strain L. helveticus BGRA43, parts or all of the intracellular matrix, lyophilizate, suspension or otherwise stabilized strain L. helveticus BGRA43. The present strain L. helveticus BGRA43 as well as its metabolites and / or membrane fragments and / or intracellular contents may have a beneficial effect on liver function and / or the metabolic state of the organism, so that they are desirable components; of the present formulation. The present L. helvet icus BGRA43 strain is preferably stabilized by lyophilization, suspension in a suitable solvent or other means to ensure its optimal pharmacological and / or biological effect.

[0045] The present formulation may be formulated in the form of a capsule, a tablet, a powder, a suspension, a gel, a drop, an emulsion, a suspension, an aerosol, a granule, a cream, an ointment and / or a micellar water.

[0046] Preferably, the formulation is formulated for oral administration in the form of a capsule, tablet, powder, gel, suspension, emulsion, droplet or suspension. Preferably, the formulation is formulated in the form of solid pharmaceutical forms such as tablets or capsules, each individual form, e.g., a tablet and / or capsule, containing a single dose of the strain L. helveticus BGRA43. The present formulation may also be formulated in the form of a powder intended for reconstitution immediately prior to administration of the formulation. Preferably, the powder is divided to contain a single dose of L. helveticus BGRA43. In other cases, where it is desirable to adapt the dose to the specific needs of the patient, the present formulation may be provided in the form of a suspension or, for example, in the form of drops, which are particularly suitable for individualized dosing. The present L. helveticus BGRA43 strain is compatible with carriers such as bulking agents, surfactants, stabilizers, antioxidants, sweeteners and / or flavor enchancers and others.

[0047] The present formulation may contain additional probiotic bacteria, fungi, yeasts, prebiotics, postbiotics, vitamins, minerals, plant extracts and / or natural isolates such as polysaccharides from fungi and / or algae. In some formulations, in addition to the strain L. helveticus BGRA43, other probiotic species may also be present, such as species of the genus Lactobacillus, for example L. acidophilus, L. bulgaricus, L. plantarum, Lfermentum, L. casei, Ljohnsonii, L reuteri, L rhamnosus, L paracasei, L salivarius, L crispatus and others, and / or species of the genus Bifidobacterium, for example B. actinocoloniiforme, B. adolescentis, B. angulatum, B. bifidum, B. longum, B. breve, B. pseudoIongum subsp. globosum, B. indicum, B. catenulatim, B. coryneforme, B. animalis, B. kashiwanohense and / or species of the genus Saccharomyces, such as S. cerevisiae or S. boulardii.

[0048] The present formulation may also contain a prebiotic in its composition.

[0049] The present formulation may be formulated as a cosmetic preparation or as a preparation for, topical use. By topical use it is considered the application of the preparation to the skin and / or mucous membranes.

[0050] Preferably, the present oral formulation is formulated as a probiotic food product intended for human and / or animal consumption. The advantage of the present probiotic food product containing the strain L. helveticus BGRA43 is that it is a genetically non-modified, i.e. "non-GMO" natural product that is safe for human consumption. In addition, the bacterial species to which the present strain belongs is listed in the EFSA's QPS (Qualified Presumption of Safety) database, which contains additives that are permitted in human nutrition, but also has GRAS (Generally Recognized as Safe) status from the FDA (Food and Drug Administration). Due to the proven positive effect on the alleviation and / or prevention of liver diseases and / or disorders, the present probiotic product can be considered a functional food. In addition, the present probiotic food product may be recommended as a healthy supplement to a weight loss diet. The advantage of the present probiotic food product is that it not only has a positive effect on the liver and parameters related to liver function, but also has a positive effect on preventing dysbiosis of the gut microbiome, i.e. it has an effect on promoting a healthy gut microbiome.

[0051] Preferably, the formulation for oral administration is formulated as a dairy-based product derivedfrom cow's milk, goat's milk, sheep's milk, donkey's milk, plant milk or other milk or a mixture of at least two types of milk from the group of cow's milk, goat's milk, sheep's milk, donkey's milk and plant milk. Plant-based milk refers to plant-based varieties that do not contain animal milk, e.g. oat milk, soy milk, rice milk, coconut milk, hazelnut milk, almond milk and other plant-based milks. Preferably, the present formulation is formulated for oral administration as yogurt, cream, sour milk, kefir, milk, milkshake, chocolate milk, milk dessert, pudding, mousse, fruit yogurt or ice cream. The investigated strain L. helveticus BGRA43 is subject to fermentation and releases peptides in fermented milk that have an anti-inflammatory effect (Strahinic et al., 2013), which makes it suitable for wider application in nutrition in the form of fermented products, e.g. yogurt, sour milk, etc.

[0052] The present formulation may be formulated as a liquid, semi-solid or solid food. The formulation may contain milk components and may also be vegan, e.g. juice, soft drink, jam, compote, sauce and / or grain-containing foods such as crackers, cereals, flakes, porridge, bread, baked goods and similar products.

[0053] The present formulation may also be formulated as a postbiotic, wherein the formulation contains the cell biomass of the present strain L. helveticus BGRA43 and may contain its components and metabolites.

[0054] The present invention also provides a lyophilizate of the strain L. helveticus BGRA43. It is preferred that the present lyophilizate contains the strain L. helveticus BGRA43 in a concentration of about 105-1015CFU / g, preferably about 108-1013, more preferably about 1011-1012CFU / g. The present lyophilizate is suitable as an intermediate product, which can be easily formulated into a medicinal preparation or a food product, depending on the application. The freeze-drying process ensures the preservation of the pharmacological characteristics of the strain as well as the elimination of water, which is advantageous due to the preservation of microbiological quality and / or the reduction of contamination possibilities.

[0055] In contrast to known formulations, the lyophilizate of L. helveticus strain BGRA43 is effective as a monocomponent formulation and other probiotic strains or prebiotics can be added to it, e.g., dietary fibers such as cellulose, xylose, pectin, inulin and others.

[0056] Preferably, the lyophilizate of the strain L. helveticus BGRA43 contains additional substances, preferably cryoprotectants such as glycerol, sucrose or trehalose. The term cryoprotectants refers to chemical compounds that prevent damage to the biological functions of the strain L. helveticus BGRA43, i.e. they prevent cell damage caused by the expansion of water during the freezing process, i.e. freezing.

[0057] Examples

[0058] Example 1. Effects of L. helveticus strain BGRA43 in animal experiments

[0059] To investigate the effects of oral administration of the probiotic bacterial strain L. helveticus

[0060] BGRA43 on metabolic parameters (lipid status, obesity and liver steatosis), a mouse model of obesity induced by a 60% fat diet was selected.

[0061] The protocol began with the random selection of 2.5-month-old male animals, their housing in cages and their adaptation to the new living conditions for one week. At the end of this period, the experiment began for 14 weeks, during which the animals were fed a standard or high-fat diet, and for the last 5 weeks they received phosphate-buffered saline (PBS) or a suspension of L. helveticus BGRA43 in PBS by oral gavage. The mice were divided into the following experimental groups (n = 8-10 animals per group) (Figure 1): 1) Control group (C) - mice reared under standard laboratory conditions with unlimited access to drinking water and commercial chow with a fat content of 10%, and administered PBS orally daily during the experiment; 2) Placebo group (HFD) - mice reared under standard laboratory conditions with unrestricted access to drinking water and commercially available food with a fat content of 60% and administered PBS by oral gavage daily during the experiment; 3) Test group (HFD + B) - mice reared under standard laboratory conditions with unrestricted access to drinking water and commercially available food with a fat content of 60% and administered a suspension of lyophilized L. helveticus BGRA43 in PBS was administered daily by oral gavage.

[0062] The food intake and body mass of the animals were measured once a week and 3-5 fecal samples were collected during the last three weeks of the trial. 14 weeks after the start of the experiment, the animals were killed by rapid decapitation. Blood was taken from the body for biochemical analyzes, and tissue (liver, adipose tissue) was removed for further histological and molecular biological analyzes. Effect of L. helveticus BGRA43 on the lipid status, metabolic parameters and liver function parameters of the experimental animals.

[0063] The results obtained showed that the body mass of the animals from the HFD group was significantly higher compared to the control group (Figure 2), but also that the treatment with L. helveticus BGRA43 had a positive effect on the reduction of body mass and the reduction of absolute and relative liver mass (Figure 3). When the morphological characteristics of the experimental animals were examined, it was found that treatment with L. helveticus BGRA43 had a favorable effect on the amount of white adipose tissue (Figure 4).

[0064] After determining the mass of all individual white adipose tissue depots, it was found that L helveticus BGRA43 treatment had a favorable effect on reducing the mass of the subcutaneous white adipose tissue depot (Figure 5), while it had no significant effect on visceral adipose tissue depots compared to the HFD group receiving a diet containing 60% fat.

[0065] After histologic analysis of the liver, the effect of the diet with 60% fat was clearly visible in the HFD group of animals in terms of the accumulation of fat droplets and the appearance of steatosis on the sections (Figure 6). However, in the group of HFD + B animals that received a high-fat diet but also received a suspension of L. helveticus BGRA43 during the experiment, a positive effect of the bacteria on clearing the liver of fat droplets is clearly visible.

[0066] Further analysis has shown that treatment with L. helveticus BGRA43 affects the key factor in the process of de novo lipogenesis in the liver, SREBPlc (Figure 7), by lowering its protein level and thereby preventing the process of fat droplet formation in the hepatocytes.

[0067] The present strain resulted in favorable effects on lipid status, metabolic parameters and liver function parameters of experimental animals when applied for 5 weeks, which is a shorter period of time compared to other strains of L. helveticus (Kim et al., 2023).

[0068] Example 2. Determination of alanine transaminase levels in serum and triglycerides in blood

[0069] The one-way ANOVA and post-hoc Tukey's test showed that treatment with L. helveticus BGRA43 in animals fed a 60% fat diet resulted in a reduction in serum ALT levels in these animals compared to animals fed a 60% fat diet alone (Figure 8A, p=0.08, HFD + B vs. HFD), suggesting that treatment with L. helveticus BGRA43 has a positive effect on the levels of liver enzymes such as alanine transaminase (ALT), i.e. a positive effect on liver function and metabolic parameters related to liver function.

[0070] Treatment with L. helveticus BGRA43 in animals fed a 60% fat diet causes a reduction in blood triglyceride levels in these animals compared to animals fed a control diet and a 60% fat diet (Figure 8B, HFD + B vs. C, HFD + B vs. HFD), suggesting that treatment with L. helveticus BGRA43 has a positive effect on blood triglyceride levels as a metabolic parameter of lipid status related to liver function.

[0071] Material and methods

[0072] The origin of the strain Lactobacillus helveticus BGRA43. L. helveticus BGRA43 isolated from the human gastrointestinal tract (Strahinic et al., 2013a) was used for the experiment. The bacteria were grown in DeMan, Rogosa and Sharpe medium (MRS, Merck, Darmstadt, Germany) at 37°C under anaerobic conditions (Anaerocult A, Merck). As previously reported, BGRA43 achieves growth of approximately 3 x 108 CFU / ml in overnight culture (Strahinic et al., 2013b). The present bacterial strain, L. helveticus BGRA43, was deposited in the Belgian Coordinated Collection of Microorganisms (BCCM), Ghent, Belgium (Lozo et al., 2011). The present strain, L. helveticus BGRA43, was deposited in the BCCM collection on 8 May 2007 under the deposit number LMG P-24226 and is the property of the Institute of Molecular Genetics and Genetic Engineering of the University of Belgrade (IMGGI).

[0073] Lyophilised isolate L. helveticus BGRA43.

[0074] For the experiments, the isolate was used in freeze-dried form, i.e. as a lyophilisate, which was obtained from the company Phytonet doo in Belgrade. The investigated strain of the bacterium L. helveticus was produced by Lactosan GmbH & Co. KG under the serial number M-20-021C (production date 19 / 02 / 2020).

[0075] Preparation of L. helveticus BGRA43 lyophilisate. 1. Fermentation of the probiotic culture. Before lyophilisation, the probiotic culture is fermented in a first step. The probiotic strain is first cultivated in a nutrient-rich medium under optimal growth conditions to promote the proliferation of viable cells. Once the culture has reached the desired cell density and a certain biomass, the probiotic biomass is separated from the cell medium by centrifugation.

[0076] 2. Formulation of the mixture for lyophilisation. The harvested probiotic biomass is then resuspended in a cryoprotective solvent, often referred to as a cryoprotectant. This solvent may contain substances such as glycerol, sucrose or trehalose, which protect the cells from damage during the freezing and drying phase. The concentration of the cryoprotectant was adjusted to ensure maximum viability of the probiotic strain after lyophilisation.

[0077] 3. Freezing. The probiotic mixture undergoes a freezing process in which the temperature is rapidly lowered to freeze the water content of the cells. This step is crucial as it prevents the formation of large ice crystals that can damage the cell structure. The freezing process is usually carried out at a specific temperature and for a specific duration.

[0078] 4. Primary drying (sublimation phase). After freezing, the frozen mixture is placed in a lyophiliser, where the primary drying phase begins. Under reduced pressure, the frozen water (ice) sublimates directly into vapour without passing through the liquid phase. During this phase, the temperature in the chamber is gradually increased while the vacuum pressure is maintained. This removes most of the water content from the probiotic cells over time.

[0079] 5. Secondary drying (desorption phase). After primary drying, the lyophilised product undergoes secondary drying to remove residual water molecules bound to cellular structures. This is achieved by further increasing the temperature to values above zero under low pressure conditions. The aim is to reduce the total moisture content and water activity to a level that ensures the long-term stability of the probiotics. This phase is carried out until the desired dryness and water activity is achieved.

[0080] 6. Packaging and storage. At the end of the lyophilisation process, the dried probiotic powder is sealed in hermetically sealed containers to prevent the absorption of moisture. The containers are then stored under recommended conditions, preferably at sub-zero temperatures, to ensure the viability and effectiveness of the probiotic strain.

[0081] Animal treatment.

[0082] Male C57BL / 6J mice, 2.5 months old at the beginning of the treatment, were used for the experiment. The animals were randomly housed in pair in a cage with a perforated plexiglass barrier that provided physical separation while allowing communication and minimising social isolation stress. After a week of adaptation to the new living conditions, a 14-week experiment began in which the animals were fed a standard or high-fat diet. In the last 5 weeks, the animals received phosphate-buffered saline (PBS) or a suspension of lyophilised bacteria Lactobacillus helveticus BGRA43 (LHBGRA43) in PBS via oral gavage. The mice were divided into three experimental groups (n = 8-10 animals per group):

[0083] 1. Control group (C) - mice fed a commercial diet with a 10 % fat content (D12450B, Research Diets, Inc., NJ, USA) and received 100 μl PBS via oral gavage daily during the experiment.

[0084] 2. Placebo group (HFD) - mice fed a commercial diet with a 60% fat content of 60% (D12492, Research Diets, Inc., NJ, USA) and received 100 μl PBS via oral gavage daily during the experiment.

[0085] 3. Test group (HFD + B) - mice fed a commercial diet containing 60 % fat (D12492, Research Diets, Inc., NJ, USA) and received 2x108 cells of Lactobacillus helveticus BGRA43 in 100 μl PBS by oral gavage daily.

[0086] All experimental animals had ad libitum access to food and water and were housed at a constant temperature of 22°C and a defined day-night cycle with a 12-hour shift. The animals were under constant veterinary care and monitoring.

[0087] Food intake was measured daily and body mass of the animals was measured weekly. At the end of 14-week treatment, the animals were sacrificed by rapid decapitation. Liver, visceral fat depots (epididymal, perirenal, retroperitoneal and mesenteric) and subcutaneous fat depots (anterior and posterior) were excised and measured. Parts of the liver tissue were prepared and stored for further histological and molecular biological analyses.

[0088] Histological analysis of the liver. In order to preserve cell morphology, a small liver lobe was fixed in 3.5% formaldehyde for 24 hours at 4°C immediately after sacrifice. Before embedding in paraffin, the tissue was dehydrated in a series of ethanol with increasing concentration: 30 % ethanol (2x30 min), 50 % ethanol (2x30 min), 70 % ethanol (5 days), 96 % ethanol (18 h) and 100 % ethanol (2x1 h), at room temperature. To enable impregnation of the tissue with paraffin, the ethanol was replaced by xylene (2x5 min), an anhydrous agent in which the paraffin dissolves. The tissue was then transferred to containers with melted paraffin (52 - 57’C) containing 7 % wax and left in the thermostat for 210 min (with a paraffin change after 90 min). The prepared paraffin blocks with tissue were moulded and glued to wooden supports, which were attached to the appropriate part of a rotary microtome (Lameris, USA). The sections with a thickness of 5 μm were placed on glass slides and deparaffinised with xylene (2x5 min). The specimens were then treated with a series of alcohols of decreasing ' concentration: 100 % ethanol (5 min), 96 % ethanol (5 min), 70 % ethanol (5 min), 50 % ethanol (5 min) and water (5 min).

[0089] Routine histological analysis of paraffin sections of the liver was performed after staining the preparation with haematoxylin and eosin. The staining consisted of a series of immersions followed by water washes: 1% haematoxylin (2 min), water (2x1 min), 0.3% NH4OH (2x2 s), water (2x1 min), 1% eosin (1 min), water (3x1 min). Sections were then dehydrated with a series of alcohols of increasing concentration: 50% ethanol (1 min), 70% ethanol (1 min), 96% ethanol (3x1 min) and 100% ethanol (3 min) and illuminated with xylene (1x2 min).

[0090] Morphological tissue analysis was performed in the appropriate field using a Leitz DMRB microscope (Leica Microsystems Germany) equipped with a Leica MC190 HD camera. The system was controlled with the Leica Application Suite (LAS) 4.11.0 software (Leica Microsystems, Germany).

[0091] Preparation of nuclear fraction of the liver.

[0092] A portion of the frozen liver from each animal was weighed and homogenised with an electric homogeniser at 8000 rpm (30 s / 30 s pause / 30 s, Janke Kunkel T25) in four volumes of cold homogenisation buffer (20 mM Tris HCI pH 7.2, 1 mM EDTA Na2, 1 mM EGTA Na2, 10% glycerol, 50 mM NaCI, 2 mM DTT, 20 mM Na2MoO4, 0,15 mM spermine, 0,15 mM spermidine, 0,1 mM PMSF,

[0093] 5 μg / ml antipain, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 5 mM Na4O2P7, 20 mM β-glycerophosphate and 25 mM NaF). The homogenates were filtered through gauze and centrifuged (2000xg, 15 min, 4°C, Eppendorf 5804 / R). Cytoplasmic and microsomal fractions were separated in the supernatant, while the precipitates were used to prepare the nuclear fractions. The sediments were washed twice by resuspension in four volumes of HEPES buffer (25 mM HEPES pH 7.6, 1 mM EDTA Na2, 1 mM EGTA Na2, 10% glycerol, 50 mM NaCI, 2 mM DTT, 20mM Na2MoO4, 0,15 mM spermine, 0,15 mM spermidine, 0,1 mM PMSF, 5 μg / ml antipain, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 5 mM Na4O2P7, 20 mM β-glycerophosphate i 25 mM NaF) and centrifuged (4000xg, 10 min, 4°C, Eppendorf 5804 / R). During the 90-minute incubation on ice in the same volume of NUN buffer (25 mM HEPES pH 7.6, 1 M urea, 300 mM NaCI, 1% Nonidet P 40, 2 mM DTT, 20 mM Na2MoO4, 0.15 mM spermine, 0.15 mM spermidine, 0.1 mM PMSF, 5 μg / ml antipain, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 5 mM Na4O2P7, 20 mM β-glycerophosphate and 25 mM NaF), the samples were often mixed vigorously on a vibrating mixer. After centrifugation (8000xg, 10 min, 4°C, Eppendorf 5804 / R), the obtained supernatants were frozen at 80°C and used as the nuclear fraction.

[0094] The determination of protein concentration in cell extracts was done using the Spektor colorimetric method. A standard curve was generated by measuring the absorbance of a bovine serum albumin solution of known concentrations (BSA, Sigma Aldrich). On 96-well microtitre plates, 180 μl of Spektor reagent (0.01 % Coomassie Brilliant Blue G, 4.8 % ethanol and 8.5 % phosphoric acid) was added to 20 μl of appropriately diluted sample, and after 5 min of incubation, the absorbance was measured at 595 nm using a Multiskan Spectrum spectrophotometer (Thermo

[0095] Electron Corporation, Finland).

[0096] Electrophoresis on polyacrylamide gel (SDS-PAGE).

[0097] Separation of proteins by molecular mass was performed by electrophoresis on polyacrylamide gels in a Mini-Protean II Electrophoresis Cell system (Bio-Rad Laboratories, Hercules, CA). Each gel consisted oftwo functionally distinct parts, an upper gel for concentration (4% acrylamide / 0.14% bisacrylamide, 0.1% SDS, 125 mM Tris-HCI, pH 6.8) and a lower gel for separation of proteins with molecular masses between 30 and 170 kDa (8% acrylamide / 0.27% bisacrylamide, 0.1% SDS, 375 mM Tris-HCI, pH 8.8). 0.05 % ammonium persulphate and 0.033 % TEMED were used as catalysts for gel polymerisation.

[0098] Samples were prepared by boiling for 5 minutes at 100 °C in an equal volume of a reducing, 2-fold concentrated sample buffer (125 mM Tris, pH 6.8 with 20 wt% glycerol, 4 wt% SDS and 10% β- mercaptoethanol) and then loaded onto a gel and separated at a constant voltage of 120 V at 4 °C for approximately 90 min (buffer for reservoirs: 192 mM glycine, 0.1% SDS and 25 mM Tris-HCI, pH 8.3).

[0099] Western blot method.

[0100] Protein transfer from the gels to PVDF membranes was performed in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) overnight at a constant electricity of 135 mA per gel at 4°C in 25 mM Tris buffer, pH 8.3, containing 192 mM glycine and 20% (v / z) methanol. After transfer, the PVDF membranes were stained with a 1% solution of

[0101] Ponceau S in 5% acetic acid to check the efficiency of the transfer. The sites on the membranes to which the proteins did not bind were blocked by incubation for one hour at room temperature in PBS buffer (1.5 mM KH2PO4, 6.5 mM Na2HPO4, 2.7 mM KCI, 0.14 M NaCI, pH 7.2) with 1% fat-free milk powder. For the detection of sterol regulatory element binding protein 1c (SREBPlc) and lamin B1, primary antibodies from Santa Cruz Biotechnology (sc-366 for SREBPlc, sc-374015 for lamin B1) were used at a dilution of 1:500. The membranes were incubated with the primary antibodies overnight at 4 °C with agitation, then washed four times for 5 min with PBS buffer containing 0.1% Tween 20 and incubated with secondary anti-rabbit (ab6721, Abeam, 1:20000) and anti-mouse (ab97046, Abeam, 1:30000) antibodies conjugated to peroscidase for one hour at room temperature. Lamin B1 was used for each sample as a control for equal loading of the samples on the gel. Before performing the enzymatic reaction, the membranes were washed with PBS buffercontaining 0.1 % Tween 20 and then with pure PBS buffer. Immunopositive bands on the membranes were visualised by incubation with a solution of luminol and 1 % hydrogen peroxide and scanned using the iBright FL1500 fluorescence detection scanner (Thermo Fisher Scientific, USA). Quantitative analysis of the relative integrated optical densities of the immunopositive bands was performed using the iBright Analysis Software (Thermo Fisher Scientific, USA).

[0102] Determination of alanine transaminase levels in serum and triglycerides in blood.

[0103] In mouse serum samples, the concentration of alanine transaminase (ALT) was measured with a Mindray Animal Technology BS-240 VET (Netherlands) using a commercially available ALT / GPT kit (#11533, BioSystems S.A., Costa Brava, Spain) according to the manufacturer's instructions. Blood triglycerides were measured using Accutrend®strips (F. Hoffmann-La Roche AG, Basel, Switzerland).

[0104] References

[0105] Banina, A., Vukasinovic, M., Brankovic, S., Fira, D., Kojic, M., Topisirovic, L, 1998. Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production. J. Appl. Microbiol. 84, 593-599. https: / / doi.Org / 10.1046 / j.1365-2672.1998.00386

[0106] Kim, H., Lee, K.; Kim, L-Y., Shim, J-J., Lim, J., Kim J-Y., Lee, J-L., 2023. Lactobacillus helveticus isolated from raw milk improves liver function, hepatic steatosis, and lipid metabolism in non-alcoholic fatty liver disease mouse model. Microorganisms. 11, 2466. https: / / www.mdpi.com / 2076-

[0107] 2607 / 11 / 10 / 2466

[0108] Lozo, J., Strahinic, I., Dalgalarrondo, M., Chobert, J.M., Haertle, T., Topisirovic, L., 2011. Comparative analysis of β-casein proteolysis by PrtP proteinase from Lactobacillus paracasei subsp. paracasei BGHN14, PrtR proteinase from Lactobacillus rhamnosus BGT10 and PrtH proteinase from Lactobacillus helveticus BGRA43. Int. Dairy J. 21, 863-868. https: / / doi.Org / 10.1016 / j.idairyj.2011.05.002

[0109] Mazloom, K., Siddiqi, I., Covasa, M., 2019. Probiotics: How effective are they in the fight against obesity? Nutrients, https: / / doi.org / 10.3390 / null020258 Strahinic, I., Lozo, J., Terzic-Vidojevic, A., Fira, D., Kojic, M., Golic, N., Begovic, J., Topisiroyic, L, 2013a. Technological and probiotic potential of BGRA43 a natural isolate of Lactobacillus helveticus. Front. Microbiol, https: / / doi.org / 10.3389 / fmicb.2013.00002

[0110] Strahinic, I., Lukic, J., Terzic-Vidojevic, A., Lozo, J., Kojic, M., Topisirovic, L., 2013b. Use of Lactobacillus helveticus BGRA43 for Manufacturing Fermented Milk Products. Food Technol. Biotechnol. 51, 257-265.

[0111] Teng, M.L.P., Ng, C.H., Huang, D.Q., Chan, K.E., Tan, D.J.H., Lim, W.H., Yang, J.D., Tan, E., Muthiah, M.D., 2023. Global incidence and prevalence of nonalcoholic fatty liver disease. Clin. Mol. Hepatol. https: / / doi.org / 10.3350 / CMH.2022.0365

Claims

Claims1. Use of the Lactobacillus helveticus BGRA43 strain for the prevention and / or treatment of liver diseases and / or liver disorders.

2. Use according to claim 1, wherein the liver disease and / or disorder is liver steatosis.

3. Use according to claim 1, wherein the liver disease and / or disorder is non-alcoholic fatty liver disease.

4. Use according to claim 1, wherein the liver disease and / or disorder is metabolic dysfunction- associated steatotic liver disease.

5. Use according to claim 1, wherein the liver disease and / or disorder is associated with increased levels of LDL cholesterol and / or triglycerides.

6. Use according to claim 1, wherein the liver disease and / or disorder is associated with and / or accompanied by an increase in body mass.

7. A formulation containing the L. helveticus BGRA43 strain.

8. The formulation according to claim 7, wherein the formulation is intended for peroral, vaginal, rectal, dermal, nasal, or enteral administration.

9. The formulation according to claim 7, wherein the L. helveticus BGRA43 strain is present in a concentration of about 10sto 1015CFU / g, preferably about 108to 109CFU / g.

10. The formulation according to claim 7, wherein the strain is a live or inactivated L. helveticus BGRA43 strain.

11. The formulation according to claim 7, wherein it comprises metabolic products of the L. helveticus BGRA43 strain, cell membrane fragments of the L. helveticus BGRA43 strain, parts or the entirety of the intracellular matrix of the L. helveticus BGRA43 strain, lyophilizate of the L. helveticus BGRA43 strain, suspension of the L. helveticus BGRA43 strain, or stabilized form of the L. helveticus BGRA43 strain.

12. The formulation according to claim 7, wherein it is formulated in the form of a capsule, tablet, powder, suspension, gel, drops, emulsion, aerosol, granules, cream, ointment, and / or micellar water.

13. The formulation according to claim 7, wherein it further contains additional probiotic bacteria, prebiotics, postbiotics, vitamins, minerals, plant extracts, or natural isolates.

14. The formulation according to claim 7, wherein formulation is a cosmetic preparation or preparation for topical application.

15. The formulation according to claim 7, wherein formulation is a probiotic food product intended for human and / or animal consumption.

16. The formulation according to claim- 15, wherein the probiotic food product is a dairy-based product derived from cow's milk, goat's milk, sheep's milk, donkey's milk, plant-based milk, or a mixture of at least two types selected from the group consisting of cow's, goat's, sheep's, donkey's, and plant-based milk.

17. The formulation according to claim 15, wherein the probiotic food product is yogurt, sour cream, fermented milk, kefir, milk, milkshake, chocolate milk, dairy dessert, pudding, mousse, fruit yogurt, or ice cream.

18. A lyophilizate of the L. helveticus BGRA43 strain, wherein it contains the L. helveticus BGRA43 strain at a concentration of 108to 1013CFU / g.

19. The lyophilizate of the L. helveticus BGRA43 strain according to claim 18, wherein it contains additional substances, preferably cryoprotectants such as glycerol, sucrose, or trehalose.