Lactobacillus plantarum IMC 510 for use in methods of preventing and treating obesity

Abstract

The object of the present invention is the use of the bacterial strain Lactobacillus plantarum IMC 510 in methods for preventing and treating overweight and obesity conditions, in particular, methods for reducing body weight, methods for reducing body mass index (BMI), methods for reducing the ratio of waistline to height, methods for reducing fat mass or methods for reducing blood glucose.

Description

Summary of the invention

[0001] The object of the present invention is the use of the bacterial strain Lactiplantibacillus plantarum IMC 510 in a method for preventing and treating overweight and obesity conditions, in particular, a method for reducing weight, a method for lowering the body mass index (BMI), a method for reducing the ratio of waist circumference to height, a method for reducing fat mass or a method for lowering blood sugar. Background Art

[0002] Obesity is a multifactorial disease caused by socioeconomic, hormonal and neural mechanisms, unhealthy lifestyle, genetic and epigenetic factors. In addition, overweight and obesity are closely related to other chronic diseases, such as type 2 diabetes, cardiovascular disease, certain types of cancer, musculoskeletal diseases and infertility. The World Health Organization (WHO) defines obesity as abnormal or excessive fat accumulation that can impair health and is the result of an imbalance between caloric intake and energy expenditure; it is estimated that by 2035, 39% of adults worldwide will be obese. The criteria used by WHO to diagnose obesity are body mass index (BMI), with individuals being considered overweight when their BMI ≥ 25 and obese when their BMI ≥ 30. Although this index is a useful indicator of the overall obesity of a subject, it should be noted that different fat distributions are associated with different metabolic risks. For this reason, in some cases, assessing abdominal circumference can more accurately classify obesity: if the abdominal circumference is greater than 88 cm for women and greater than 102 cm for men, subcutaneous obesity is present.

[0003] As mentioned above, overweight and obesity are the result of a positive energy balance, i.e., an increase in energy intake and a decrease in energy expenditure. However, many recent studies have highlighted how the human intestinal microbiota (referred to as the gut microbiota or GM) can play a role in the increase in obesity, particularly in terms of energy storage and nutrient absorption. In addition, it has been shown how the intestinal microbiota of obese individuals differs compared to those of lean / normal weight physique. In fact, the GM of adult subjects is mainly characterized by taxa belonging to two phyla, Firmicutes and Bacteroidetes: in subjects of normal weight, there are more Bacteroidetes, while changes in this ratio are associated with the obese state. In fact, it seems that significant changes within the Firmicutes population, where specific changes in the composition of the microbiota are characterized by an increase in Firmicutes and a corresponding decrease in Bacteroidetes, can contribute to a more efficient extraction of energy from food sources, thereby increasing its accumulation in the host's adipose tissue.

[0004] Other clinical studies have shown that individuals with lower bacterial diversity have higher levels of leptin and protein C, are prone to insulin resistance, dyslipidemia, obesity, and display an inflammatory phenotype; all of which lead to a greater susceptibility to weight gain than individuals with higher bacterial diversity.

[0005] Among the functions of intestinal bacteria, the functions of intestinal bacteria include fermentation of indigestible carbohydrate residues, synthesis of short-chain fatty acids (SCFA) and amino acids; these operations can help increase the energy provided to the host. However, on the other hand, it should be noted that the byproducts of bacterial fermentation can also reduce appetite, increase satiety, and lead to weight loss and BMI reduction.

[0006] For all these reasons, the gut microbiota is considered a potentially modifiable factor that could be relevant to the prevention and treatment of excess weight, but its full mechanisms of action and subsequent impact on human health have yet to be conclusively determined.

[0007] In fact, based on the role played by the intestinal microbiota in the human body, three main mechanisms of action of probiotics in the treatment of obesity can be schematically identified: antagonism of the growth of pathogenic microorganisms, competitive adhesion to the intestinal mucosa and epithelium (antimicrobial activity), increase in the production of the intestinal mucus layer and reduction in intestinal permeability (barrier function) and modulation of the gastrointestinal immune system (immunomodulation).

[0008] Experimental evidence has shown that the gut microbiota plays a role in the protection and health of the gastrointestinal tract, regulating mucosal permeability, helping the metabolism of dietary polysaccharides by promoting fermentation and absorption, and is responsible for increased catabolism and increased SCFA production, thereby acting to control fat accumulation and the resulting possible obesity.

[0009] Specifically, the gut microbiota is able to regulate body weight and energy metabolism by acting on SCFA production, controlling bile acid metabolism, and inducing and / or preventing metabolic endotoxemia.

[0010] Therefore, taking these assumptions into account, in addition to the evaluation of the nutritional and dietary patterns to be followed, the effects of probiotic intake have been studied with regard to their ability to positively modulate the composition of the microbiota, stimulate the immune system, and maintain the integrity of the intestinal barrier through a competitive effect on certain pathogenic strains. Studies have shown that probiotics have a positive effect on some obesity-related aspects, such as reducing inflammation within adipose tissue, endotoxemia, adiposity, overall body weight, leptin levels, and energy absorption.

[0011] Based on preliminary studies conducted in experimental animals, most of the currently available preparations for clinical trials contain bacterial strains of the genera Lactobacillus and Bifidobacterium. The clinical studies aim to evaluate the effects of these specific probiotics on the obese state, in particular on the change in body weight, considering that the use of other strains is disadvantageous.

[0012] Therefore, there remains a need for specific bacterial strains that are able to act on specific factors in the gut microbiota that influence the predisposition or development of overweight or obesity conditions.

[0013] To date, there is still a need to obtain alternative and specific probiotic therapies that can act at the level of the intestinal microbiota in obese or overweight subjects, effectively inducing a reduction in body weight and maintaining it at normal levels. Said therapies must have a high degree of acceptance by the subjects involved, be safe to use and, if necessary, also have a positive effect on the levels of biomarkers of obesity-related diseases such as diabetes, hypertension and vascular diseases.

[0014] Purpose of the Invention

[0015] An object of the present invention is to provide a novel human-derived microorganism that is isolated and characterized for its probiotic properties.

[0016] An object of the present invention is to provide a probiotic strain for use in a method for preventing and treating overweight and / or obesity.

[0017] Another object of the present invention is to provide a probiotic to rebalance the intestinal microbiota, preferably in a subpopulation of obese or overweight subjects.

[0018] These and other objects are achieved by the object of the present invention, which provides bacterial strains as effective probiotics for use in a method for preventing and treating overweight and obesity disorders, in particular a method for reducing weight, a method for reducing the body mass index (BMI), a method for reducing the ratio of waist circumference to height, a method for reducing fat mass or a method for lowering blood sugar. Summary of the invention

[0020] The object of the present invention is a newly isolated, non-genetically modified human microorganism, named Lactobacillus plantarum IMC510, which is deposited in the German Microbiological Collection Center (DSMZ) by the Italian company Synbiotec Srl with the deposit number DSM 32548.

[0021] It is noteworthy that Zheng et al. published a reclassification of the genus Lactobacillus in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4): 2782-2858, 2020. Therefore, Lactobacillus plantarum strain IMC 510 can be identified as Lactobacillus plantarum IMC 510. The above two names are interchangeable because they always refer to the same deposited bacterial strain.

[0022] It is a microaerophilic, Gram-positive, stationary, elongated bacterium. IMC 510 (Lactobacillus plantarum IMC510 DSM 32548) is facultative heterofermentative and produces L-lactic acid by glucose fermentation.

[0023] Colonies (medium HHD, anaerobic conditions, 37°C, 2 days) are round, medium to large, white, shiny, smooth, convex, with gray, transparent edges; under a microscope (MRS broth, 37°C, 12 hours), the microorganisms appear as short to medium-length rods with rounded ends; they exist singly, in pairs or in short chains, arranged in a palisade-like pattern.

[0024] Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) has an optimal growth temperature of 37°C, tolerates pancreatic acid and pancreatic juice well (survival rate of 85% to 95%), and tolerates bile and oxidative stress well (survival rate greater than 95%).

[0025] The assay for viability in response to acid, pancreatic juice, and bile was performed on cells derived from cultures grown in MRS medium for 12 hours at 37°C, washed with sterile saline, and resuspended at approximately 10 9 CFU / ml. In the case of acid, 5 hours of survival at 37°C at pH 3 (citrate buffer) are considered, while for pancreatic juice (pH 8), 3 hours of survival at 37°C are considered. In the case of bile, the method consists of culturing in MRS agar with or without bile salts (at concentrations of 0.1; 0.3; 0.5%), and in MRS agar with or without bile salts No. 3 (at concentrations of 0.05; 0.1; 0.2%). The survival rate is calculated by comparing the logCFU / ml in cultures exposed to adverse conditions with cultures under the same conditions but without the stressor.

[0026] In contrast, oxidative stress was assessed by growing cells in MRS broth at 37°C to 4 μg / cm3 turbidity, placing paper disks moistened with 3% hydrogen peroxide solution on colonies in MRS soft agar, and counting the resulting sterile areas (MRS soft agar, anaerobic conditions, 37°C).

[0027] EFSA (European Food Safety Authority) has assigned QPS (Qualified Permitted Safety) status to this microorganism strain; no antibiotic resistance-related genes or plasmids were detected in this microorganism. Antibiotic susceptibility is also within the parameters recommended by EFSA.

[0028] According to another aspect of the invention, the object of the invention is a probiotic strain based on Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) for use in a method for the prevention and treatment of overweight and obesity.

[0029] Another object of the invention is the bacterial strain Bacillus plantarum IMC 510 for use in the treatment of dysbiosis of the intestinal microbiota in overweight or obese individuals with the aim of reducing body weight and, if desired or necessary, intervening in biomarkers of obesity-related diseases such as diabetes, hypertension and vascular diseases.

[0030] In the present description, dysbiosis of the intestinal microbiota is understood as a condition of alteration in the amount and type of microorganisms present in the gastrointestinal tract compared to normal physiological conditions.

[0031] As previously mentioned, a non-limiting example of the dysbiosis present in obese and overweight individuals involves a high F / B (Firmicutes / Bacteroidetes) ratio, which appears to be associated with more efficient extraction of energy from food sources and subsequent increased accumulation of energy in the host's adipose tissue.

[0032] Overweight and obese subjects were defined as those meeting the aforementioned BMI and abdominal circumference parameters.

[0033] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for weight loss; preferably in overweight and / or obese subjects; more preferably in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota; even more preferably, in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0034] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for body mass index (BMI) reduction; preferably in overweight and / or obese subjects; more preferably in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota; even more preferably, in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0035] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for waist circumference reduction; preferably in overweight and / or obese subjects; more preferably in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota; even more preferably, in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0036] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing the ratio of waist circumference to height; more preferably in overweight and / or obese subjects; more preferably in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota; even more preferably, in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0037] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in the method for reducing fat mass.

[0038] Preferably, the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method of lowering blood glucose; more preferably in overweight and / or obese subjects; more preferably in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota; even more preferably, in overweight and / or obese subjects suffering from dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0039] An object of the present invention is a composition or formulation comprising a mixture comprising or consisting of the bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) and optionally pharmaceutical or food grade additives and / or carriers and / or excipients for use in a method of treatment for the relief of:

[0040] (i) body mass or weight,

[0041] (ii) waist circumference and BMI,

[0042] (iii) waist-to-height ratio,

[0043] (iv) fat mass,

[0044] (v) blood sugar,

[0045] Preferably, the decreases (i) to (v) are observed in a representative subpopulation of overweight or obese subjects.

[0046] The probiotics used according to the present invention are preferably administered orally in the form of capsules, tablets, or sachets, preferably in the form of capsules, which contain not less than 15 billion viable cells (CFU) of Lactobacillus plantarum IMC 510, preferably 15 billion, for example not less than 15 billion to 100 billion viable cells (CFU) of Lactobacillus plantarum IMC 510.

[0047] According to one aspect of the present invention, the administration is preferably performed once a day. According to another aspect of the present invention, the administration is performed at breakfast, regardless of whether or not food is taken. The contents of the capsule can be dispersed in a liquid (e.g., milk or juice) at an intake temperature, or dispersed in a semisolid food (e.g., yogurt).

[0048] The probiotics used according to the present invention are gluten- and lactose-free, free of soy, GMOs, aspartame, acesulfame K, and added flavorings, and are compatible with vegetarian and vegan diets.

[0049] As will be fully demonstrated in the following experimental part, the probiotic bacterial strain Bacillus plantarum IMC 510 used according to the invention is able to:

[0050] (i) reduction in body mass or weight,

[0051] (ii) reduce waist circumference and BMI,

[0052] (iii) reduce waist-to-height ratio,

[0053] (iv) reduce fat mass,

[0054] (v) lower blood sugar,

[0055] Preferably, decreases (i) to (v) are observed in a representative subpopulation of overweight or obese subjects.

[0056] The bacterial strain Bacillus plantarum IMC 510 used according to the present invention has been shown to be effective in influencing the intestinal microbiota (particularly the populations of Lactobacillus spp. and Bifidobacterium spp.) of a subject administered orally and in reducing the F / B ratio.

[0057] Furthermore, the bacterial strain Bacillus plantarum IMC 510 used according to the present invention has been shown to be effective in influencing the intestinal microbiota of subjects administered orally, in particular for the bacterial populations of the genera Hafnia-Obesumbacterium and Rambutsia, a significant decrease was observed, while for the genus Succiniclasticum, an increase was observed.

[0058] According to another aspect of the present invention, the object of the present invention is a method for reducing the body weight of an overweight or obese subject, which comprises administering to said subject in need thereof one or more oral preparations, preferably capsules, containing Lactobacillus plantarum IMC 510 for use according to the present invention. The subject here preferably refers to a human being.

[0059] According to another aspect of the present invention, the object of the present invention is a method for reducing the body mass index (BMI) of an overweight or obese subject, comprising administering to said subject in need thereof one or more oral formulations, preferably capsules, containing Lactobacillus plantarum IMC 510 for use according to the present invention. The subject here preferably refers to a human being.

[0060] According to another aspect of the present invention, the object of the present invention is a method for reducing waist circumference of an overweight or obese subject, comprising administering to said subject in need thereof one or more oral formulations, preferably capsules, containing Lactobacillus plantarum IMC 510 for use according to the present invention. The subject here preferably refers to a human being.

[0061] According to another aspect of the present invention, the object of the present invention is a method for reducing the waist-to-height ratio of an overweight or obese subject, the method comprising administering to the subject in need thereof one or more oral formulations, preferably capsules, containing Lactobacillus plantarum IMC 510 for use according to the present invention. The subject here preferably refers to a human being.

[0062] According to another aspect of the present invention, the object of the present invention is a method for reducing fat mass in overweight or obese subjects, the method comprising administering to said subject in need thereof one or more oral formulations used according to the present invention, preferably capsules containing Lactobacillus plantarum IMC 510. The subject here preferably refers to a human being.

[0063] According to another aspect of the present invention, the object of the present invention is a method for lowering blood sugar in an overweight or obese subject, the method comprising administering to the subject in need thereof one or more oral formulations for use according to the present invention, preferably capsules containing Lactobacillus plantarum IMC 510. The subject herein preferably refers to a human being.

[0064] The probiotics used according to the present invention may be used alone or, if desired or necessary, in combination with other substances, provided that these substances do not counteract or limit the effects of the probiotics.

[0065] The experimental part below describes the results of a clinical study of the probiotics used according to the invention, wherein said use was compared with the effect obtained by administering a placebo.

[0066] Preferred embodiments (FRn) of the present invention are as follows.

[0067] FR1. A bacterial strain called Lactobacillus plantarum IMC 510, deposited by the Italian company Symbio Scientific with the accession number DSM32548 at the German Collection of Microorganisms (DSMZ), is used in a method for preventing and treating overweight and / or obesity.

[0068] FR2. The Lactobacillus plantarum IMC 510 bacterial strain for use according to FR1, characterized in that the subject suffers from dysbiosis of the intestinal microbiota.

[0069] FR3. The Lactobacillus plantarum IMC 510 bacterial strain for use according to FR2, characterized in that the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

[0070] FR4. The bacterial strain Lactobacillus plantarum IMC 510 for use according to the preceding FR, characterized in that it is administered orally.

[0071] FR5. The bacterial strain Lactobacillus plantarum IMC 510 for use according to FR4, characterized in that it is preferably administered in capsules containing an amount of 1 billion CFU to 100 billion CFU, preferably 5 billion CFU to 50 billion CFU, even more preferably 10 billion CFU to 25 billion CFU.

[0072] FR6. The bacterial strain Lactobacillus plantarum IMC 510 for use according to FR5, characterized in that the administration is performed once or more per day, preferably at breakfast.

[0073] FR7. A bacterial strain Lactobacillus plantarum IMC 510 (also known as Lactobacillus plantarum IMC 510), which has been deposited with the German Collection of Microorganisms (DSMZ) under the accession number DSM 32548. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1: Changes in body weight (A), waist circumference (B) and BMI (C) at the scheduled measurement time (blank column at T0, grid-filled column at T90, dashed-filled column at T30 follow-up day) relative to the two groups of volunteers (probiotics and placebo), expressed in %. Asterisks (*) indicate data with statistically significant differences compared with T0 (P<0.05, Student's t test).

[0075] Figure 2 : Weight (kg), waist circumference (cm) and BMI (in kg (m 2 ) -1 The two columns at each time point represent the data of two groups of volunteers: probiotics (grid-filled columns) and placebo (dashed-filled columns).

[0076] Figure 3 : Bacterial concentrations (log CFU / g±SD) of Lactobacillus, Bifidobacterium, Bacteroides-Prevotella-Porphyromonas spp., Staphylococcus spp., Clostridium coccoides-Eubacteriumrectale, and Enterobacteriaceae in fecal samples at the scheduled measurement times (blank column at T0, grid-filled column at T90, and filled column at the follow-up point at T30) relative to the two groups of volunteers (probiotics and placebo). Asterisks (*) indicate data with statistically significant differences (P<0.05) compared with T0; hash marks (#) indicate data with statistically significant differences (P<0.05) compared with the placebo group (according to Tukey's test with one-way analysis of variance).

[0077] Figure 4 : Recovery of Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) relative to total Lactobacillus species in stool samples of subjects belonging to the probiotic group (expressed in %). Asterisks (*) indicate statistically significant differences compared to T0 (P < 0.05, Student's t-test)

[0078] Figure 5 : ORWELL 97 scores of volunteers in two groups (probiotics and placebo) at the scheduled measurement time (T0, T90 day and T30 follow-up day), expressed as mean ± SD. For each time, the three columns represent the total ORWELL score (gray), ORWELL relevance score (dark gray) and ORWELL occurrence score (light gray) in turn.

[0079] Experimental Part I

[0080] Lactobacillus plantarum IMC 510 for use according to the invention: a double-blind randomized clinical trial versus placebo Subjects

[0081] The study involved 19 overweight / obese volunteers, 10 men and 9 women, aged 26 to 73 years, with a BMI of 25 or more. Participants were asked to maintain their usual diet and usual level of physical activity. All volunteer subjects gave informed consent to participate; the procedures followed during the study were in accordance with the ethical standards of the responsible committee for human experimentation and the 1975 Declaration of Helsinki (revised in 1983).

[0082] In addition to being between 26 and 73 years old and having a BMI greater than 25, study participants were required to be willing to cooperate by providing informed consent, having the ability to follow instructions, and in particular, demonstrating willingness to complete study-related questionnaires, records, and diaries. Participants were also required to stop consuming functional foods, dietary supplements containing probiotics, laxatives, and / or weight control substances throughout the study. Other therapies (drugs or nutritional regimens) that affect body weight, food intake, and / or energy expenditure were also prohibited within four weeks prior to treatment with the probiotics of the present invention or placebo. Postmenopausal, pregnant, or breastfeeding women were excluded from selection for this study, as were women who had been enrolled in another obesity treatment regimen.

[0083] Furthermore, exclusion criteria during the study period included noncompliance with the probiotic / placebo intake regimen, the presence of severe illness, and the need for ongoing use of antibiotics and / or laxatives or other probiotics.

[0084] Study Design

[0085] A randomized placebo-controlled study was conducted with an initial phase (run-in) of 4 weeks, followed by a 12-week intervention phase (T90) and a final phase of another 4 weeks (T30 follow-up). Subjects had not received antibiotic treatment within one month before the start of the study.

[0086] In the first phase, the subjects were asked to stop taking any laxatives, dietary fibers and / or probiotics.At the end of the first 4 weeks (T0), the volunteers were divided into an intervention group (treated with the probiotics used according to the invention) and a control group (placebo).

[0087] In other words, the study involves 3 phases: Phase 1 evaluation period (1 month / 4 weeks), during which the subjects recruited must meet the aforementioned inclusion criteria to enter the next phase; Phase 2 intervention period (3 months / 12 weeks), during which the subjects took one capsule containing Lactobacillus plantarum IMC 510 or placebo every day; Phase 3 follow-up period (1 month / 4 weeks).

[0088] At the end of each phase, medical and nutritional examinations were performed, and blood and stool samples were collected at designated times T0 (end of phase 1), T90 (end of phase 2), and T30 follow-up day (end of phase 3). In addition, all subjects received questionnaires at each phase of the study regarding gut health, psychological well-being, and overall quality of life assessment, with a special focus on obesity-related aspects.

[0089] Main Purpose

[0090] The safety of the selected probiotic (Lactobacillus plantarum IMC 510) was evaluated at the first intake (T0), after 3 months of treatment (T90 day) and after the next month of follow-up (T30 follow-up day), as well as the effects on the gut microbiota and weight loss while maintaining the same dietary habits.

[0091] Secondary Purpose

[0092] Evaluation of changes in BMI, waist circumference, blood parameters (e.g., blood glucose), lipid levels, liver function (glutamate oxaloacetate transaminase-GOT and glutamate pyruvate transaminase-GPT, in UIl) -1 ), gut health, quality of life, general well-being, and the level of discomfort caused by overweight and obesity.

[0093] Application

[0094] Lactobacillus plantarum IMC 510 is administered orally in capsules, each capsule containing not less than 1.5 x 10 10 Bacterial cells of the strains under consideration were previously isolated from healthy human subjects and deposited at the German Collection of Microorganisms (DSMZ) by the Italian company Cymbio Scientific (Camerino, Italy).

[0095] Compared to capsules containing probiotics, placebo capsules carried only an inert diluent and were identical in size, shape, and taste to the capsules containing probiotics.

[0096] Physiological measurements

[0097] Volunteers' weight was measured using a calibrated scale after removing their shoes and jackets. Waist circumference was measured at two finger heights below the navel, and height was measured after removing shoes. All measurements were performed at the same time of day, at each control time point: T0 (end of Phase 1), T90 day (end of Phase 2), and T30 follow-up day (end of Phase 3).

[0098] Use questionnaires to assess health status

[0099] Gut Health

[0100] Gut health and stool consistency (key parameters describing normal or impaired bowel habits) were self-assessed by individual subjects at the end of Phase 2 through questions about "change in the number of daily bowel movements" and "change in the number of eggs (stool portions) corresponding to equal volumes by visual estimation". Specifically, subjects were then asked to record whether their gut health (in terms of bowel regularity and stool volume) remained the same, improved, or worsened during the three months of probiotic (or placebo) administration and the subsequent one-month follow-up period compared to the previous period. The degree of change was recorded on a combined scale of a 10-point Likert scale (-5, 0, +5).

[0101] Secondary parameters such as ease of defecation, bloating, constipation, abdominal pain, intestinal cramping, feeling of incomplete defecation, incontinence, and halitosis were also measured using the same Likert numerical scale. Stool consistency was determined by the Bristol Stool Shape Scale.

[0102] Health status related quality of life

[0103] The subjects' health-related quality of life (HRQoL) was assessed using the self-completed Mental Health General Index (PGWBI), which is a questionnaire that generally measures mental health and discomfort, and consists of 22 items covering six dimensions (anxiety, depression, self-control, positive attitude, general health, and vitality). These questions allow for multiple answers, with a score range of 0 to 5 (5 being the highest score). The overall PGWBI score is composed of the sum of all items in the multiple dimensions; the higher the score (up to 100), the higher the mental health.

[0104] ORWELL Questionnaire

[0105] The Obesity-Related Wellness (ORWELL) questionnaire is a psychometric test that assesses obesity-specific quality of life. The domains investigated include physical health, emotional state, and interpersonal life. For each item considered, participants were asked to rate the occurrence / severity of symptoms (prevalence) and the subjective relevance of symptom-related impairment in life (relevance) according to a 4-point Likert scale (0, not at all; 1, a little; 2, not too much; 3, very much). The score for each item was then calculated as the product of prevalence and relevance. The total score obtained (ORWELL 97) was the sum of the scores of the individual items; the sum of the scores for symptom prevalence (ORWELL 97-O) and relevance (ORWELL 97-R) in the individual items was also calculated. The higher the ORWELL score, the lower the perception of quality of life.

[0106] Characterization of the gut microbiota

[0107] Stool samples

[0108] Fecal samples were collected from each subject in the placebo and probiotic treatment groups at time 0 (T0), corresponding to the date of starting probiotic / placebo supplementation (end of period 1 to start of period 2), after 90 days of probiotic / placebo supplementation (T90, end of period 2), and after a 30-day follow-up period (T30 follow-up, end of period 3). Feces were frozen at -80°C until microbiota analysis (enumeration of selected bacterial groups by real-time quantitative PCR and colonization of probiotic strains).

[0109] DNA extraction

[0110] DNA was extracted from all stool samples using a stool DNA isolation kit (NorgenBiotek Corp., Thorold, Canada) according to the manufacturer's instructions for stool samples. The quantity and purity of the extracted DNA were checked with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and then stored at -20°C until used for molecular analysis.

[0111] Real-time PCR

[0112] Selected bacterial strains were quantified in the feces of all subjects using a real-time quantitative PCR (qPCR) procedure. Specifically, the target bacterial groups included: Lactobacillus, Bifidobacterium, Bacteroides-Prevotella-Porphyromonas, Staphylococcus, Clostridium cocci-Eubacterium rectum group, and Enterobacteriaceae. Real-time quantitative PCR amplification was performed using specific primers and SYBR Green, using the iCycleriQ Real-Time Detection System (Stratagene) associated with MXP software, following the conditions and standard curves for each bacterial group.

[0113] Lactobacillus plantarum IMC 510 in feces

[0114] To confirm the presence of the probiotic strains used in the present invention in the intestines of the treated subjects, stool samples were analyzed by counting lactobacilli on MRS agar (de Man, Rogosa and Sharpe agar, Oxoid, Basingstoke, UK) by 10-fold dilution method. Ten to twenty percent of the total colonies of each sample were randomly selected from the counted agar plates after 48 to 72 hours of aerobic incubation at 37°C, then isolated and tested for purity. DNA extracted from the selected colonies using a modified benzyl chloride method was analyzed by RAPD (random amplification of polymorphic DNA) technique.

[0115] Blood samples

[0116] Blood samples were collected from each subject at the same time points as stool samples: at time 0 (T0), corresponding to the date of start of probiotic / placebo supplementation (end of period 1 to start of period 2), after 90 days of probiotic / placebo supplementation (T90 day, end of period 2), and after a 30-day follow-up period (T30 follow-up day, end of period 3).

[0117] Statistical analysis

[0118] The results are expressed as mean ± standard deviation (SD) or standard error (SE). The statistical significance of the differences between the probiotic group and the placebo group was analyzed using the Student's t-test. Significant differences between means were determined by Tukey's test after 1-way ANOVA. P values ​​less than 0.05 were considered statistically significant. For bowel habits, P < 0.05 reflected a significant difference, and four multiple comparison tests were used.

[0119] result

[0120] Nineteen volunteers were recruited for the clinical study. There were no withdrawals, exclusions, or adverse events in either group (probiotics and placebo), and the compliance with supplementation was 100%. The baseline demographic data of the participants who completed the study are shown in Table 1.

[0121] Table 1: Demographics and baseline characteristics of the study population. Values ​​are expressed as mean ± DS

[0122]

[0123] *Student's t-test, P < 0.05 is a significant level

[0124] It is obvious from the data in the table that the two groups of subjects (probiotic group and placebo group) have balanced characteristics in terms of baseline characteristics (such as gender, height, waist circumference and weight). Only the age parameter showed a significant difference between the two groups (P<0.05, Student's t test).

[0125] Changes in weight, BMI, and waist circumference (mean values) are summarized in Figure 1 The probiotic group showed statistically significant decreases in all parameters relative to T0 (P<0.05). In contrast, the placebo group never showed statistically significant decreases.

[0126] like Figure 2 As shown, at the end of 90 days of treatment, subjects in the probiotic group had a significant reduction in weight (-3.39 kg), waist circumference (-4.29 cm), and BMI (-1.15 kg / m2 Even after the follow-up for the next 30 days, the trend of maintaining weight loss and waist circumference reduction was maintained.

[0127] A real-time quantitative PCR (qPCR) procedure was used to quantify selected bacterial groups from the feces of the volunteers. Specifically, the target bacterial groups were Bacteroides-Prevotella-Porphyromonas, Staphylococcus, Clostridium cocci-Eubacterium rectum group, Lactobacillus, Bifidobacterium and Enterobacteriaceae. Figure 3 Shown are changes in microbiota composition in the probiotic and placebo groups at the end of the treatment period (T90) and after the next 30-day follow-up (T30 follow-up).

[0128] exist Figure 3 In the data, it can be seen that the supplementation of the probiotics used in the present invention significantly increased the concentration of Lactobacillus on day T90 compared to T0 (P<0.05). In addition, it can be seen that the level of Bifidobacterium was also higher after 90 days of treatment; although there was no statistical significance compared to T0, it was statistically significant compared to the values ​​recorded in the placebo group. In contrast, no significant changes in other bacterial species were found between the two groups during the treatment period. However, after the treatment period, the proportion of subjects with increased or decreased Bifidobacterium concentrations tended to be different between the two groups (Chi-square, P=0.01). Specifically, it was found that 10 of the 12 subjects (83.3%) in the probiotic group had increased levels of Bifidobacterium, while 5 of the 7 subjects (71.4%) in the placebo group had an opposite trend of decreased Bifidobacterium levels.

[0129] As previously reported, Firmicutes and Bacteroidetes are two key bacterial phyla of the human gut microbiota that have been shown to interact with the homeostasis of obesity; in particular, a higher F / B ratio has been found in obese people. The present study showed an increase in the relative abundance of Firmicutes (which includes Staphylococcus, Clostridium cocci-rectal Eubacterium group and Lactobacillus) in overweight and obese subjects in the placebo group, and therefore a higher F / B ratio, while in the probiotic group, the administration of the probiotics used according to the present invention seemed to be able to maintain a lower F / B ratio (also due to an increase in Bacteroides-Prevotella-Porphyromonas).

[0130] To confirm the survival of the administered probiotics during intestinal transit in the treated subjects, recovery of Bacillus plantarum IMC 510 was performed from all stool samples (collected at T0, T90 days and T30 follow-up day). Figure 4As can be seen from the histogram, this strain was found in the stool of all participants in the probiotic group, not only at the end of the 90-day treatment period (T90) but also at the end of the follow-up period (T30). This particular treatment strain was not present in any stool samples collected at T0 before the start of administration.

[0131] In more detail: The mean value of the recovery percentage of the probiotic Lactobacillus plantarum IMC 510 reached 31.12% of the total Lactobacilli at the end of the administration period (T90 day) and remained at a high level of 18.50% even after a 30-day follow-up period (T30 follow-up day).

[0132] In the bowel health questionnaire, significant changes in bowel habits considered as the primary outcome are listed in Table 2 .

[0133] Table 2: Mean absolute changes in bowel habit frequency scores, stool consistency, and PGWBI questionnaire scores after the 3-month probiotic / placebo supplementation period (T90 day) and after the next month follow-up (T30 follow-up day)

[0134]

[0135] * Changes in bowel habits were assessed using a ten-point Likert scale (-5, 0, 5);

[0136] a Assessment was done according to the Bristol stool classification;

[0137] b The total score ranges from 0 to 100 (100 is the highest score)

[0138] # There was a statistical difference from T0; There was a statistically significant difference compared with placebo at T90 day; There was a statistically significant difference compared with placebo on the T30 follow-up day (Student's t test, P<0.05).

[0139] In general, subjects administered with the probiotics used according to the invention showed significantly higher values ​​in the scoring scale related to bowel habits (P<0.05). The health status-related quality of life was assessed with the overall PGWBI score; the probiotic group showed statistically higher values ​​(P<0.05) than the PGWBI index values ​​recorded at the beginning of treatment (T0) after 90 days of treatment (T90 days) and after a subsequent 30-day follow-up (T30 follow-up day).

[0140] To assess obesity-related quality of life, patients were asked to self-assess using the ORWELL 97 questionnaire to assess the subjective relevance and intensity of physical and psychological discomfort. Then, the questionnaires of all 19 overweight / obese volunteers were evaluated to determine the changes in symptom onset and / or severity over time (T0, T90 days, T30 follow-up days) (incidence) and the subjective relevance of symptom-related impairment in personal life (relevance). The data obtained were as follows Figure 5 shown.

[0141] ORWELL 97 total score is the sum of the scores of individual items. In addition, in order to understand Figure 5 The results shown show that it is important to note that higher ORWELL 97 scores are associated with lower quality of life, analyzed using Student's t-test with a significance level of P < 0.05.

[0142] The total score values ​​of the subjects in the probiotic group showed a downward trend over time, although it was not significant (P>0.05), indicating a slight improvement in quality of life. This trend was not obvious in the placebo group.

[0143] In conclusion, the experimental data presented show that, in contrast to the results recorded in the case of the placebo group, the administration of the probiotic Bacillus plantarum IMC 510 used according to the invention resulted in a statistically significant reduction in body weight, BMI and waist circumference of the subjects in the probiotic group. In particular, the reduction in waist circumference is an important factor, as it relates to a direct measure of fat distribution and is closely related to the atherosclerotic lipid profile.

[0144] The safety of the treatment was confirmed in this study, which recorded no significant changes or physiological abnormalities in lipid metabolism-related parameters (such as triglycerides, total cholesterol, low-density lipoprotein or high-density lipoprotein) in the volunteers.

[0145] Clinical studies also show that at the end of the administration period, the ratio of Firmicutes to Bacteroidetes was lower in the probiotic group, while the F / B ratio was higher in the placebo group, indicating a tendency to overweight / obesity. This fact further confirms the potential of the probiotic strain Bacillus plantarum IMC 510 used according to the present invention for the prevention / treatment of overweight and obesity, in particular for the treatment of dysbiosis of the intestinal microbiota of overweight or obese subjects by acting on the F / B ratio.

[0146] Finally, it is well known how obesity and overweight conditions affect the quality of life of subjects with these conditions by implying a reduced ability to carry out activities, early fatigue, as well as increased anxiety, depression and generally low self-esteem. The self-assessment questionnaire used in this study makes it possible to demonstrate how these aspects related to the psychological and relational areas of the subjects also have significant improvements; in fact, the set of scores recorded for the probiotic group records a positive evolution in aspects related to general health, health / energy / emotional state and general quality of life.

[0147] Experimental Part II

[0148] The applicant conducted a second study in which the aim was to evaluate the effects of supplementation with Lactobacillus plantarum IMC 510 on anthropometric parameters, biochemical parameters, gut microbiota (GM) composition and function, gastrointestinal symptoms and general symptoms in overweight / obese subjects.

[0149] A group of 40 overweight / obese subjects were randomly assigned to take the probiotic Lactobacillus plantarum IMC 510 or placebo daily for 3 months. Anthropometric and biochemical parameters, self-administered questionnaires, plasma and stool samples were obtained from each participant before and after the administration period. GM was characterized by 16S rRNA sequencing, while short-chain fatty acids (SCFA) and medium-chain fatty acids (MCFA) in stool were analyzed by gas chromatography-mass spectrometry.

[0150] Compared with placebo, probiotic supplementation resulted in significant reductions in body weight, body mass index (BMI), waist circumference, waist-to-height ratio, and blood glucose. In addition, probiotic administration resulted in significant reductions in Hafnia and Rambutsia genera and increases in Succinicobacteria, whereas placebo administration resulted in reductions in Actinomycetaceae, increases in Alloprevotella spp., and increases in proinflammatory caproic acid and heptanoic acid levels.

[0151] Due to the effects of Bacillus plantarum IMC 510 in increasing certain beneficial intestinal bacteria and reducing waist circumference, fasting blood glucose levels, and gastrointestinal symptoms in obese subjects, supplementation with Bacillus plantarum IMC 510 may be an effective strategy for preventing or treating obesity in the future.

[0152] This clinical study aimed to evaluate the effects of a probiotic formulation based on Lactobacillus plantarum IMC 510 on anthropometric and biochemical parameters, GM composition and function, and gastrointestinal and systemic clinical manifestations in overweight / obese subjects.

[0153] Materials and methods

[0154] Study population

[0155] Forty subjects (20 males and 20 females) with a mean age of 51.6 ± 13.2 years were enrolled in this study. If the participants were between 18 and 65 years old and had a BMI ≥ 25 kg / m 2 Exclusion criteria were as follows: continuous use of probiotics within 2 months before enrollment; use of other therapies (drugs or nutritional regimens) that affect body weight, food intake, and / or energy expenditure; pregnancy or breastfeeding; participation in another obesity treatment program.

[0156] Study Design

[0157] This study was a parallel, randomized, double-blind controlled trial. Before randomization, there was a 2-week run-in period to assess the commitment, motivation, and willingness of the participants. After the run-in period, eligible participants were randomized to two groups, each designated to take the probiotic Lactobacillus plantarum IMC 510 or placebo for 3 months. During the intervention period, all participants were encouraged not to change their lifestyle, exercise, or dietary habits. The study procedures were approved by the Regional Ethics Committee of Tuscany, Azienda Ospedaliera Universitaria Careggi (14592_spe), which are registered on clinicaltrials.gov (identifier: NCT05358301), and adhered to the principles of the Declaration of Helsinki and the Data Protection Act.

[0158] Weight and body composition were measured at each clinical assessment. Weight and height were measured using a stadiometer. BMI was calculated as weight (kg) / height (m 2 ), WHtR (waist circumference / height ratio) was calculated as waist circumference (cm) / height (cm). If the participant’s BMI was ≥25 kg / m 2 And <30kg / m 2 Participants were classified as overweight if their BMI was ≥ 30 kg / m 2 Body composition was measured using a bioelectrical impedance analyzer (AKERN, Model 101 Sport Edition).

[0159] Intervention

[0160] The experimental probiotic dietary supplement used in this clinical study is manufactured by the Italian company Symbio Scientific (Camerino, Macerata, Italy). Each capsule contains the probiotic strain Lactobacillus plantarum IMC 510 at a concentration of 15 billion viable cells (CFU / capsule). The intake is N = 1 capsule / day, preferably at breakfast, for 3 months. The capsules can be opened and their contents dispersed in cold liquids or semi-solid foods. The capsules should be stored in the original container or in a clean, covered container out of the reach of children. When stored in a clean, dry environment without direct sunlight, the shelf life of probiotic products is at least 24 months. At the beginning of the study, participants were given a sample of the probiotic supplement in a sealed box, which was sufficient for the entire duration of the study (90 capsules, 3 months).

[0161] Placebo dietary supplements (i.e., capsules without added probiotics) were administered at N=1 capsule / day, with the same intake pattern as the experimental probiotics. Probiotics and placebo looked identical to ensure double blinding of the physician and participants.

[0162] Table 3

[0163]

[0164]

[0165] Effects of intervention on body composition

[0166] Table 4 shows the changes in anthropometric parameters after the intervention and control periods. There were no significant differences between the two groups. The group that underwent probiotic supplementation had significant (p<0.05) reductions in body weight (-0.7 kg, p=0.026), BMI (-0.2, p=0.032), waist circumference (-0.9 cm, p=0.043), and WHt ratio (-0.01, p=0.041). In addition, although not statistically significant, but close to significance, the probiotic group also had a reduction in fat mass (-0.9 kg, p=0.075).

[0167]

[0168] Table 4

[0169] Effects of intervention on biochemical parameters

[0170] Regarding biochemical parameters, the general linear model for repeated measures showed that probiotics led to a significant decrease in blood glucose (-3.9 g / L; p = 0.033) and a significant increase in uric acid (0.2 mg / dL; P = 0.040) after 3 months of intervention (Table 5).

[0171]

[0172] Table 5

[0173] Therefore, in the present study, we evaluated the effects of a novel probiotic formulation based on Lactobacillus plantarum IMC 510 on anthropometric and biochemical parameters, GM composition and function, and gastrointestinal and general symptoms in overweight / obese subjects.

[0174] First, regarding anthropometric parameters, we found that probiotic supplementation resulted in a slight decrease in body weight, BMI, WHt ratio, and fat mass, confirming our previous results showing a significant net decrease in body weight gain, BMI, and white adipose tissue weight in obese rats after 84 days of supplementation with Lactobacillus plantarum IMC 510 and in human subjects after 3 months of administration of Lactobacillus plantarum IMC 510.

[0175] Furthermore, in agreement with Coman and colleagues, probiotic supplementation also resulted in a significant reduction in waist circumference in volunteers compared with placebo. A reduction in waist circumference provides important cardiovascular benefits in obese subjects, and several strains of Lactobacillus and Bifidobacterium have been reported to have anti-obesity effects in obese adults. On the other hand, waist circumference and WHtR are used in clinical practice as markers of visceral fat, which is closely associated with obesity and related metabolic diseases. Waist circumference and WHt ratio, rather than BMI, are key indicators of obesity severity and are more closely associated with obesity-related metabolic diseases. Probiotics appear to be able to modify fat distribution in obese / overweight subjects without significantly changing body weight, as reflected in a reduction in waist circumference and WHt ratio, meaning that the use of probiotics appears to be beneficial for this type of patients.

[0176] A 3-month supplementation seems to be the starting point for observing a trend towards a positive effect of probiotic use on body weight and / or fat mass (waist circumference and WHt ratio). It should be noted that an initial weight loss of ≥±1% over a 3-month period is clinically relevant and is associated with a significant reduction in some cardiovascular risk factors, such as blood glucose, as shown in our data. Thus, the probiotic intervention resulted in a significant reduction in blood glucose compared with placebo, whereas other blood parameters did not change significantly. In agreement with our results, a meta-analysis conducted by Kocsis et al. reported that probiotic supplementation resulted in a significant reduction in fasting blood glucose levels in patients with type 2 diabetes.

[0177] However, our data clearly indicate that the potential probiotic strain Bacillus plantarum IMC 510, in addition to increasing some beneficial gut bacteria, has the effect of reducing body weight, waist circumference, fasting glucose levels, and gastrointestinal symptoms in obese individuals.

Claims

1. A bacterial strain named Lactobacillus plantarum IMC 510, deposited by the Italian company Symbio Technol. under the accession number DSM 32548 at the German Collection of Microorganisms (DSMZ), for use in a method for preventing and treating overweight and / or obesity in a subject; wherein: The subject suffers from dysbiosis of the intestinal microbiota.

2. The bacterial strain Lactobacillus plantarum IMC 510 according to claim 1, wherein The dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

3. The bacterial strain Lactobacillus plantarum IMC 510 according to claim 1 or 2, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing body weight.

4. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing body mass index (BMI).

5. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing waist circumference.

6. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing the waist-to-height ratio.

7. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for reducing fat mass.

8. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The bacterial strain Lactobacillus plantarum IMC 510 (Lactobacillus plantarum IMC 510 DSM 32548) is used in a method for lowering blood sugar.

9. The bacterial strain Lactobacillus plantarum IMC 510 for use according to any one of the preceding claims, wherein The strains were administered orally.

10. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The strain is preferably administered in the form of capsules containing an amount of 1 billion CFU to 100 billion CFU, preferably 5 billion CFU to 50 billion CFU, even more preferably 10 billion CFU to 25 billion CFU.

11. The bacterial strain Lactobacillus plantarum IMC 510 according to claim 9 or 10, wherein The administration is performed once or more per day, preferably at breakfast.

12. The bacterial strain Lactobacillus plantarum IMC 510 according to any one of the preceding claims, wherein The administration preferably occurs in overweight and / or obese subjects; more preferably in overweight and / or obese subjects exhibiting dysbiosis of the intestinal microbiota; even more preferably in overweight and / or obese subjects exhibiting dysbiosis of the intestinal microbiota, wherein the dysbiosis is characterized by a high F / B (Firmicutes / Bacteroidetes) ratio.

13. A composition comprising a mixture comprising or consisting of the bacterial strain Lactobacillus plantarum IMC 510 (also known as Lactobacillus plantarum IMC 510) as claimed in any one of the preceding claims and optionally pharmaceutical or food grade additives and / or carriers and / or excipients.

14. A bacterial strain Lactobacillus plantarum IMC 510 (also known as Lactobacillus plantarum IMC 510), which has been deposited with the German Collection of Microorganisms (DSMZ) under the accession number DSM 32548.

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