WO2025231132A1

WO2025231132A1 - Probiotic strain for weight management

Info

Publication number: WO2025231132A1
Application number: PCT/US2025/027096
Authority: WO
Inventors: Fumiki Aoki
Original assignee: AB Biotics SA; Kaneka Americas Holding Inc
Current assignee: AB Biotics SA; Kaneka Americas Holding Inc
Priority date: 2024-04-30
Filing date: 2025-04-30
Publication date: 2025-11-06
Anticipated expiration: 2026-10-30

Abstract

Methods to support weight management, promote weight loss and/or to treat overweight, obesity and related conditions or disorders are provided. Methods for regulating stress and anxiety are also provided. Methods comprise administering a probiotic composition comprising the strain Lactiplantibacillus plantarum CECT 7481 (KABP-051) or a derived strain defined herein.

Description

TITLE: PROBIOTIC STRAIN FOR WEIGHT MANAGEMENT

FIELD

[0001] The present disclosure relates to the fields of medicine and microbiology and particularly, to methods for reducing body weight comprising administering a probiotic composition.

BACKGROUND ART

[0002] The prevalence of health problems related to weight and obesity has reached epidemic proportions over the last few decades and recent trend analyses show that the number of subjects who are overweight or obese is continuing to rise worldwide. Because of the multifaceted nature of obesity, there is no single or simple solution to combat this growing epidemic. Novel interventions may thus be necessary to effectively prevent and treat overweight and obesity.

[0003] Obesity, in turn, amplifies the risk of further chronic diseases including type 2 diabetes, cardiovascular disease, coronary artery disease, hypertension, stroke, hypercholesterolemia, cholelithiasis, fatty liver disease, musculoskeletal disorders (including back and joint problems), obstructive sleep apnea, and certain cancers. Many afflicted individuals also experience social consequences, isolation, stress and anxiety.

[0004] Oral administration of probiotic bacteria has been proposed as a way of manipulating the gut ecosystem to favor weight reduction or decrease weight gain. However, the mechanisms by which probiotic supplementation may influence the gut microbiota are largely unknown. A few studies indicate that probiotics may exert an effect on the function of different bacterial species in the gut, but there is currently no clear evidence of compositional alterations of the fecal microbiota in response to probiotic supplementation. Probiotic bacteria such as Lactobacillus and Bifidobacterium species have been proposed fortheir metabolic benefits, such as reducing body fat mass, body weight, and cholesterol levels. Despite some promising effects, the effect sizes of the studies are small and the effects of probiotic supplementation on fat mass or fat percentage are not clear or with non-significance.

[0005] On the other hand, managing stress is a crucial factor in the success of any weight loss strat- egy/therapy. Stress management is often an overlooked aspect of successful weight loss. Stress can profoundly impact physical and mental well-being, making it a significant obstacle to achieving weight loss goals.

[0006] Further, probiotic features are strain-dependent, even among bacteria of the same species.

Therefore, it is important to find those strains able to produce a beneficial effect on the host while having a good performance in all probiotic requirements such as resistance to gastrointestinal conditions, adequate proliferation, and also be suitable for large-scale manufacture.

[0007] US9192634B2 describes a method for the prevention and/or treatment of a disorder caused by oral pathogenesis in an oral cavity of a subject, comprising administering to the subject a composition comprising an effective amount of the isolated strain of Lactobacillus plantarum deposited in the Spanish Type Culture Collection (CECT) under accession number CECT 7481 . The disorder caused by oral pathogenesis is e.g. gingivitis, periodontitis, caries, sensitive teeth, or halitosis.

[0008] Improved compositions and methods of nutritional supplementation to improve weight loss while managing stress in subjects, remain desirable.

BRIEF SUMMARY

[0009] The present disclosure provides methods to support weight management, promote weight loss and/or to treat overweight, obesity and related conditions or disorders, comprising administering a probiotic composition comprising the strain Lactiplantibacillus plantarum CECT 7481 (KABP-051) or a derived strain defined herein.

[0010] Working examples herein provide detailed experimental data of a clinical trial demonstrating that subjects following 12-weeks of supplementation of a probiotic composition comprising CECT 7481 , experienced significant 2-4% improvements in body composition, including loss of body weight (3.3 lbs.); body fat percentage (3%); and waist circumference (3.4 cm), while maintaining muscle mass. Further, EXAMPLE 3 shows the enhanced secretion of GLP-1 (glucagon-like peptide-1) by the strain CECT 7481 , which contributes to regulate body weight. Therefore, this strain shows a potential to support weight management and promote weight loss.

[0011] As discussed, managing stress is a crucial factor in the success of any weight loss strategy. Stress can profoundly impact physical and mental well-being, making it a significant obstacle to achieving weight loss goals. In this sense, it is further advantageous that the probiotic composition provided herein has effects on other relevant parameters and in the well-being of the subject. The probiotic composition has the capacity of significantly improving feelings of fatigue, suggesting that this probiotic strain may enhance energy levels. Of note, the probiotic composition has the capacity of improving stress hormones (9-14% lower cortisol) and microbiome balance (2-7% increase in composite score; 6-20% decrease in Firmicutes/Bacteroidetes ratio; and 14-31 % increase in butyrate kinase).

[0012] These results indicate that a probiotic composition comprising the strain CECT 7481 is an effective way to enhance weight loss, while also helping users “feel better” with reduced fatigue and elevated energy levels while they are embarking on a weight loss regimen. All this leads to the conclusion that the effects of a probiotic composition comprising the strain CECT 7481 is effective in the global management of weight.

[0013] Furthermore, CECT 7481 strain encompasses all main characteristics desirable to exert a beneficial effect in the human gut. These include resistance to gastrointestinal conditions (such resistance to gastric stress and bile salts), long-term stability.

[0014] Accordingly, the probiotic composition described herein can be used for different medical meth- ods/applications in the context of weight management, which are described herein in detail.

[0015] Terms used in the claims and aspects of the present disclosure are understood in its widely and common meaning in this description. Nevertheless, they are defined hereinafter in the Detailed Description.

[0016] Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Furthermore, the present disclosure covers all possible combinations of particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and without intending to be limiting to the present disclosure.

DESCRIPTION OF DRAWINGS

[0017] FIG. 1 shows the results of the clinical trial (CECT 7481 strain vs Placebo) at baseline, week 4, 8 and 12 (W4, W8, W12), regarding body weight (lbs).

[0018] FIG. 2 shows the results in body fat (%).

[0019] FIG. 3 shows the results in waist circumference (cm).

[0020] FIG. 4 shows the results in cortisol levels (salivary, pg/ml).

[0021] FIG. 5 shows the results in microbiome: panel (A) Overall composite score; panel (B) Firmicu- tes/Bacteroidetes (F/B) ratio; panel (C) and (D) Lactobacillus, Bifidobacterium; panel (E) butyrate kinase; panel (F) Akkermansia.

[0022] FIG. 6 shows the PFGE pattern of CECT 7481 strain after digestion with Sfil enzyme (A) and Smal enzyme (B). Order: Marker, CECT 7481 strain.

[0023] FIG. 7 shows the enhancement of GLP-1 secretion by CECT 7481 strain.

DETAILED DESCRIPTION

Definitions [0024] Probiotic: As used herein, the term “probiotic” refers to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. The probiotic can be a variant or a mutant strain of bacterium. Probiotic bacteria can be naturally mutated or genetically engineered modified to retain, enhance or improve desired biological properties, e.g., survivability to provide probiotic properties or to retain, enhance or improve probiotic properties such the ones described herein.

[0025] Derived from: The terms "derived from," "derivative", "variant", "mutant" (e.g., "mutant strain"), or any grammatical variant thereof, as used herein, refer to a component that is isolated from or made using a specified molecule/substance (e.g., a strain of the present disclosure). For example, a bacterial strain that is derived from a first bacterial strain (e.g., a deposited strain) can be a strain that is identical or substantially similar to the first strain. In the case of bacterial strains, the derived strain can be obtained by, e.g., naturally occurring mutagenesis, artificially directed mutagenesis, artificially random mutagenesis or other genetic engineering techniques, and it retains, enhances or improves at least one ability of the deposited strain.

[0026] Excipient/Carrier: The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a e.g., pharmaceutical composition, to further facilitate administration of a compound, e.g., a bacterial strain of the present disclosure. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, e.g., polysorbate. The terms “physiologically acceptable excipient/carrier” and “pharmaceutically acceptable excipient/carrier” which may be used interchangeably, refer to a substance or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these terms.

[0027] Composition: As used herein, the term “composition” refers to a mixture of at least one compound useful within the present disclosure with an excipient/carrier. For example, "pharmaceutical composition" refers to a preparation of the bacteria of the present disclosure with other components such as a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition facilitates the administration of the compound to a patient or subject.

[0028] Identity: As used herein, the term "identity" refers to the overall conservation of the monomeric sequence between polymeric molecules, e.g., between DNA molecules and/or RNA molecules. The term "identical" without any additional qualifiers, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical," is equivalent to describing them as having, e.g., "70% sequence identity.”

[0029] Calculation of the percent identity of two polymeric molecules, e.g., polynucleotide sequences, can be performed, e.g., by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second polynucleotide sequences for optimal alignment). In certain aspects, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the length of the reference sequence. The bases at corresponding base positions, in the case of polynucleotides, are then compared.

[0030] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which can be determined using a mathematical algorithm. Suitable software programs are available for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, which performs a comparison between two sequences using either the BLASTN (used to compare nucleic acid sequences) or BLASTP (used to compare amino acid sequences) algorithm. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs. Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, MAUVE, MUMMER, RAST, etc.

[0031] In certain aspects, the percentage identity (% ID) of a first sequence to a second sequence is calculated as %ID = 100 x where Y is the number of amino acid residues or nucleobases scored as identical matches in the alignment of the first and second sequences (e.g., as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. When comparing complete or near complete genomic nucleobase sequences, % ID is sometimes referred to as ANI (Average Nucleotide Identity). Calculating ANI usually involves the fragmentation of genome sequences, followed by nucleotide sequence search, alignment, and identity calculation.

[0032] Prevent: The terms "prevent," "preventing," "prophylaxis" and variants thereof as used herein, refer, e.g., to

- partially or completely delaying onset of a disease, disorder and/or condition disclosed herein;

- partially or completely delaying onset of one or more symptoms, features, or clinical manifestations, complications, or sequelae of a particular disease, disorder, and/or condition disclosed herein;

- partially or completely delaying onset of one or more symptoms, features, or manifestations, complications, or sequelae of a particular disease, disorder, and/or condition disclosed herein; - partially or completely delaying progression from a particular disease, disorder and/or condition disclosed herein; and/or

- decreasing the risk of developing pathology associated with the disease, disorder, and/or condition disclosed herein.

[0033] Subject: The terms "subject", "patient", "individual", and "host", and variants thereof are used interchangeably herein and refer to any mammalian subject, particularly humans, but also including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications.

[0034] Subject in need thereof: As used herein, "subject in need thereof includes subjects, such as mammalian subjects, that would benefit from administration of the probiotic compositions of the present disclosure.

[0035] Therapeutically effective amount: The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to the amount of the probiotic composition of the present disclosure that is sufficient to a produce a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof. Particularly, the terms refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition. A therapeutically effective amount can, e.g., be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disease or condition. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[0036] Treatment: The terms "treat," "treatment," "therapy," as used herein describes the management and care of a subject forthe purpose of combating the disease, condition, or disorder. Treating includes the administration of a probiotic composition as described herein to reduce the severity of a disease or condition disclosed herein; the mitigation/amelioration or elimination of one or more symptoms, complication, or sequelae associated with a disease disclosed herein; to prevent the onset of the symptoms or complications; to eliminate the disease, condition, or disorder; or the provision of beneficial effects to a subject with a condition/disease disclosed herein, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or symptoms, complications, or sequelae thereof.

[0037] The term refers to a clinical or nutritional intervention to prevent the disease or condition; cure the disease or condition; delay onset of the disease or condition; delay onset of a symptom, complication or sequela; reduce the seriousness of the disease or condition; reduce the seriousness of a symptom, complication, or sequela; improve one or more symptoms; improve one or more complications; improve one or more sequelae; prevent one or more symptoms; prevent one or more complications; prevent one or more sequelae; delay one or more symptoms; delay one or more symptoms; delay one or more complications; delay one or more sequelae; mitigate/ameliorate one or more symptoms; mitigate/ameliorate one or more complications; mitigate/ameliorate one or more sequelae; shorten the duration one or more symptoms; shorten the duration one or more complications; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more complications; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more complications; reduce the severity of one or more sequelae; improve the quality of life; increase survival; prevent a recurrence of the disease or condition; delay a recurrence of the disease or condition; or any combination thereof, e.g., with respect to what is expected in the absence of the treatment with the probiotic composition of the present disclosure.

[0038] Symptom: As used herein, the term "symptom" refers to subjective or physical sign, indication, or evidence of disease or physical disturbance observed by the subject. In general, the term refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. In some aspects, a symptom can be a mild symptom, a moderate symptom, orsevere symptom. As used herein, the term "mild symptom" refers to a symptom that is not life threatening and does not require, e.g., intensive care treatment. As used herein, the term "moderate symptom" refers to a symptom that requires monitoring because it may become life threatening and may require, e.g., hospitalization. As used herein, the term "severe symptom" refers to a symptom that is life threatening and requires, e.g., intensive care treatment.

[0039] Complication: As used herein, the term "complication" refers to a pathological process or event occurring during a disease or condition that is not an essential part of the disease or condition; where it may result from the disease/condition or from independent causes. In some aspects, a complication can be chronic or permanent. As used herein, the term "sequela" refers to a long term, chronic, or permanent complication.

[0040] BMI: As used herein the term “BMI” designates body mass index. BMI is a measure of the weight of a person scaled according to height. It is defined as the individual's body weight divided by the square of their height (weight measured in kilograms, height in meters). The formula universally used in medicine produces a unit of measure of kg/m². According to the NIH US Department of Health & Human Services (Overweight and Obesity - Symptoms and Diagnosis, NHLBI, NIH), a BMI below 18.5 indicates underweight, 18.5-24.9 normal -healthy- weight, 25-29.9 overweight, and a BMI of 30 and above indicates obesity. It should be noted that not only obesity but also overweight (BMI 25-29.9) increases the risk of mortality in adults. [0041] Overweight is defined for an adult human as having a BMI between 25 and 29.9.

[0042] Obesity: As used herein, “obesity” means a medical condition in which excess body fat has accumulated to the extent that it may have a negative effect on health. Obesity increases the likelihood of various diseases, particularly heart disease and type 2 diabetes. “Obese” is defined for an adult human as having a BMI greater than 30.

[0043] Weight loss in the context of the present disclosure is a reduction of the total body weight. Weight loss may e.g., refer to the loss of total body mass in an effort to improve fitness, health, and/or appearance.

[0044] Weight management or “weight maintenance” relates to maintaining a total body weight. For example, weight management may relate to maintaining a BMI in the area of 18.5-24.9 which is considered to be normal (healthy).

Methods and uses of the probiotic composition

[0045] It is understood that in this disclosure "probiotic composition" means any probiotic composition as defined herein, e.g., comprising Lactiplantibacillus plantarum CECT 7481 (KABP-051) or a derived strain in the terms defined herein, and any other excipient/carrier, as well as in any product form (e.g., pharmaceutical composition, etc).

[0046] All the methods and uses described herein can be alternatively formulated as a probiotic composition provided herein for use in reducing body weight and the other uses described herein. Also alternatively, this can be formulated as the use of any of the probiotic compositions described herein for the manufacture of a pharmaceutical composition, a nutraceutical composition, a veterinary composition, a food product or a personal care for the treatment, prevention or amelioration of the diseases and conditions, complications and/or seguela thereof disclosed herein. This can be also alternatively formulated as methods of treating, preventing or ameliorating the diseases and conditions, or symptoms, complications and/or seguela described herein of a subject in need thereof comprising administering to the subject the herein described probiotic composition. Finally, the present disclosure also provides the use of the probiotic composition for weight control/weight management.

[0047] The present disclosure provides a method for reducing body weight in a subject, comprising providing (administering) to a subject in need thereof the probiotic composition provided herein.

[0048] Alternatively said, the present disclosure also provides a method to reduce weight gain, reverse weight gain or induce weight loss; a method for weight loss; a method to support weight management; a method to promote weight loss; a method to control or stabilize weight gain; a method of weight maintenance; or a method of regulating body weight of a subject, all comprising administering to a subject in need thereof the probiotic composition provided herein.

[0049] The term “reducing” refers to any indicia of success in the prevention or reduction of weight gain in a patient induced by or associated with the administration of the probiotic composition. The prevention or reduction of weight gain can be measured based on objective parameters, such as the described in the examples provided herein, e.g., the results of a physical examination.

[0050] The term “reversing” refers to any indicia of success in causing the loss of weight gain established prior to the administration of the probiotic composition. The reduction of weight already gained can be measured based on objective parameters, such as the results of a physical examination.

[0051] In an embodiment, the administration of the probiotic composition, particularly for 12 weeks, results in:

(i) a reduction in body weight, particularly a reduction of at least 1.8%;

(ii) a reduction in body fat, particularly a reduction of at least 2.5%;

(iii) a reduction in waist circumference, particularly a reduction of at least 3%;

(iv) an increase (i.e., improvement) in microbiome diversity and resilience in gut, particularly an increase in the Overall composite score of at least 7%;

(v) a reduction (i.e., improvement) of the Firmicutes/Bacteroidetes ratio in gut, particularly a reduction of at least 19%;

(vi) an increase (i.e. improvement) in Lactobacillus species and/or Bifidobacterium species in gut, particularly an increase of at least 15.5% of Lactobacillus and at least 450% of Bifidobacterium;

(vii) an increase (i.e. improvement) in butyrate kinase levels in gut, particularly an increase of at least 14%; and/or

(viii) an increase (i.e. improvement) in Akkermansia levels in gut, particularly an increase of at least 130%.

[0052] In the provided examples, CECT 7481 strain biological impact was assessed through the change of different parameters from baseline to final levels after 12 weeks of treatment, and also through the comparison of final levels at 12 weeks between study groups (placebo vs. CECT 7481).

[0053] In an embodiment, the reduction in body weight after 12 weeks of administration of the probiotic composition is of at least 1.8%, particularly about 1.8%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the reduction in body weight after 12 weeks of treatment is of at least 3 lbs, more particularly of about 3.3 lbs. In an embodiment, the reduction in body weight at 12 weeks of administration compared to control conditions is of at least 1 .5%, particularly of about 1 .5%, i.e., compared to a subject not treated with the probiotic composition (placebo group). [0054] In an embodiment, the reduction in body fat after 12 weeks of administration of the probiotic composition is at least 2.5%, particularly of about 2.9%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition.

[0055] In an embodiment, the reduction in waist circumference after 12 weeks of administration of the probiotic composition is of at least 3%, particularly of about 3.4%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the reduction in body weight after 12 weeks of treatment is of at least 3 cm, more particularly of about 3.4 cm.

[0056] In an embodiment, a reduction in body weight can also be measured by other parameters, such as microbiome diversity in gut. A higher microbiome diversity stands for better health. For example, Overall composite score can be measured (score of markers: Firmicutes/Bacteroidetes (F/B) ratio; Lactobacillus, Bifidobacterium levels, butyrate kinase levels, Akkermansia levels).

[0057] In an embodiment, the administration of the probiotic composition results in a higher microbiome diversity and resilience in gut, as measured e.g. by Overall composite score comprising Firmicutes/Bacteroidetes (F/B) ratio; Lactobacillus, Bifidobacterium, butyrate kinase, and Akkermansia. It is understood that an increase in the Overall composite score means an improvement in the composite score, i.e. an improvement in the microbiome diversity and resilience.

[0058] In an embodiment, the increase (i.e., improvement) in the overall composite score (i.e., overall microbiome diversity and resilience) after 12 weeks of administration of the probiotic composition is of at least 7%, particularly of about 7%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the increase in the composite score after 4 weeks of administration is of at least 1.5%, particularly of about 1.7%. In an embodiment, the increase in the composite score after 8 weeks of administration is of at least 6%, particularly of about 6.3%. In an embodiment, the increase in the composite score over 12 weeks of administration is of between about 1 .5% and about 7%.

[0059] Firmicutes and Bacteroidetes are the two major phyla in the human gut microbiota. In an embodiment, the administration of the probiotic composition results in a reduction (i.e., improvement) of the Firmicutes/Bacteroidetes ratio (F/B ratio) in gut.

[0060] In an embodiment, the reduction (i.e., improvement) in F/B ratio after 12 weeks of administration of the probiotic composition is of least 19%, particularly of about 19.8%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the reduction in F/B ratio after 4 weeks of administration is of at least 5.5%, particularly of about 5.9%. In an embodiment, the reduction in F/B ratio after 8 weeks of administration is of at least 18%, particularly of about 18%. In an embodiment, the reduction in F/B ratio over 12 weeks of administration is of between about 5.5% and about 18%. In an embodiment, the reduction in F/B ratio at 12 weeks of administration compared to control conditions is of at least 7.5%, particularly of about 7.7%, i.e., compared to a subject not treated with the probiotic composition (placebo group).

[0061] Increases in Lactobacillus and Bifidobacterium suggest an increase in beneficial bacteria in the gut. In an embodiment, the administration of the probiotic composition results in an increase (i.e. improvement) in Lactobacillus species and/or Bifidobacterium species in gut.

[0062] In an embodiment, the increase (i.e., improvement) in Lactobacillus species levels after 12 weeks of administration is of at least 15.5%, particularly of about 15.9%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the increase in Lactobacillus species levels after 4 weeks of administration is of at least 22%, particularly of about 22%. In an embodiment, the increase in Lactobacillus species levels after 8 weeks of administration is of at least 21 %, particularly of about 21 %. In an embodiment, the increase in Lactobacillus species levels over 12 weeks of administration is of between about 15.5% and about 22%. In an embodiment, the increase in Lactobacillus species levels after 12 weeks of administration of the probiotic composition compared to control conditions is of at least 200%, particularly of about 218%, i.e., compared to a subject not treated with the probiotic composition (placebo group). Alternatively said, the Lactobacillus species levels are increased about 2-fold after 12 weeks of administration compared to control conditions.

[0063] In an embodiment, the increase (i.e., improvement) in Bifidobacterium species levels after 12 weeks of administration of the probiotic composition is of at least 450%, particularly of about 464%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the increase in Bifidobacterium species levels after 4 weeks of administration is of at least 85%, particularly of about 88%. In an embodiment, the increase in Bifidobacterium species levels after 8 weeks of administration is of at least 300%, particularly of about 325%. In an embodiment, the increase in Bifidobacterium species levels over 12 weeks of administration is of between about 85% and about 464%. Alternatively said, the Bifidobacterium species levels are increased about 4-fold after 12 weeks of administration. In an embodiment, the increase in Bifidobacterium species levels after 12 weeks of administration compared to control conditions is of at least 130%, particularly of about 136%, i.e., compared to a subject not treated with the probiotic composition (placebo group).

[0064] Butyrate prevents against body weight gain and fat content. An increase in butyrate kinase, one of the butyrate biosynthetic enzymes, may help increase butyrate in the gut. In an embodiment, the administration of the probiotic composition results in an increase (i.e. improvement) in butyrate kinase levels in gut. [0065] In an embodiment, the increase (i.e., improvement) in butyrate kinase levels after 12 weeks of administration of the probiotic composition is of at least 14%, particularly of about 14.7%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the increase in butyrate kinase levels after 4 weeks of administration is of at least 16%, particularly of about 16%. In an embodiment, the increase in butyrate kinase levels after 8 weeks of administration is of at least 30%, particularly of about 31 %. In an embodiment, the increase in butyrate kinase levels over 12 weeks of administration is of between about 14% and about 31 %.

[0066] In an embodiment, the increase in butyrate kinase levels after 12 weeks of administration compared to control conditions is of at least 22%, particularly of about 22.5%, i.e., compared to a subject not treated with the probiotic composition (placebo group). In an embodiment, the increase in butyrate kinase levels after 8 weeks of administration compared to control conditions is of at least 50%, particularly of about 54%, i.e., compared to a subject not treated with the probiotic composition (placebo group).

[0067] Akkermansia is known to be more abundant in the gut of non-obese people and less abundant in obese people. In an embodiment, the administration of the probiotic composition results in an increase (i.e. improvement) in Akkermansia levels in gut.

[0068] In an embodiment, the increase (i.e., improvement) in Akkermansia levels after 12 weeks of administration of the probiotic composition is of at least 130%, particularly of about 136%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the increase in Akkermansia levels after 4 weeks of administration is of at least 180%, particularly of about 188%. In an embodiment, the increase in Akkermansia levels after 8 weeks of administration is of at least 50%, particularly about 59%. In an embodiment, the increase in Akkermansia levels over 12 weeks of administration is of between about 50% and about 188%. In an embodiment, the increase in Akkermansia levels after 12 weeks of administration compared to control conditions is of at least 65%, particularly of about 67%, i.e., compared to a subject not treated with the probiotic composition (placebo group).

[0069] The present disclosure provides a method for regulating (i.e. treating) stress and anxiety in a subject, comprising providing (administering) to a subject in need thereof the probiotic composition provided herein. In an embodiment, the method for regulating stress and anxiety in a subject is in the context of a weight control/management strategy/therapy. Thus, the probiotic composition has positive effects in reducing body weight and regulating stress and anxiety of the subject submitted to the weight control probiotic treatment. However, the probiotic composition can be used to regulate stress and anxiety in the context of another strategy/treatment for reducing body weight that the subject is following or in any situation. [0070] In another aspect, the present disclosure provides a method for reducing body weight and regulating (i.e., treating) stress and anxiety in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein. In an embodiment, the method is for reducing body weight and regulating stress and anxiety associated to weight control in a subject.

[0071] The obtained effects of the probiotic composition can be viewed as a body composition improvement through stress management, inducing healthier life habits. Thus, in another aspect, the present disclosure provides a method for reducing body weight in a subject, through management of stress in the subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0072] In an embodiment, the administration of the probiotic composition, particularly for 12 weeks, results in:

(i) a reduction in salivary cortisol, particularly a reduction of at least 10.5%;

(ii) an improvement in global well-being (i.e., psychological mood state), particularly an improvement of at least 19%;

(iii) an improvement in anxiety, particularly a reduction of at least 35%;

(iv) an improvement in depression, particularly a reduction of at least 50%;

(v) an improvement in hostility, particularly a reduction of at least 40%;

(vi) an improvement in fatigue, particularly a reduction of at least 50%;

(vii) an improvement in confusion, particularly a reduction of at least 40%; and/or

(viii) an improvement in vigor, particularly an increase of at least 30%.

[0073] Cortisol is the best-known biomarker for stress (lower value correlates to lower stress). In an embodiment, the reduction in salivary cortisol after 12 weeks of administration of the probiotic composition is of at least 10.5%, particularly of about 11 %, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition. In an embodiment, the reduction in salivary cortisol after 4 weeks of administration is at least 9%, particularly of about 9%. In an embodiment, the reduction in salivary cortisol after 8 weeks of administration is at least 14%, particularly of about 14%. In an embodiment, the reduction in salivary cortisol over 12 weeks of administration is of between about 9% and about 14%.

[0074] In an embodiment, the reduction in salivary cortisol after 12 weeks of administration is of at least 70 pg/ml, more particularly about 71 pg/ml. In an embodiment, the reduction in salivary cortisol at 12 weeks of administration compared to control conditions is of at least 14%, particularly of about 14.4%, i.e., compared to a subject not treated with the probiotic composition (placebo group). [0075] In an embodiment, the improvement in global well-being (i.e., psychological mood state) after 12 weeks of administration of the probiotic composition is of at least 19%, particularly of about 19%, i.e., reduction from baseline to 12 weeks after administration of the probiotic composition.

[0076] Psychological Mood State can be measured by questionaries and tests available to the skilled in the art (e.g., Profile of Mood States (POMS), PSS-10, and DASS-21).

[0077] In an embodiment, the improvement in anxiety after 12 weeks of administration is of at least 35%, particularly of about 38%.

[0078] In an embodiment, the improvement in depression after 12 weeks of administration is of at least 50%, particularly of about 54%.

[0079] In an embodiment, the improvement in hostility after 12 weeks of administration is of at least 40%, particularly of about 40%.

[0080] In an embodiment, the improvement in fatigue after 12 weeks of administration is of at least 50%, particularly of about 51 %.

[0081] In an embodiment, the improvement in confusion after 12 weeks of administration is of at least 40%, particularly of about 44%.

[0082] In an embodiment, the improvement (increase) in vigor after 12 weeks of administration is of at least 30%, particularly of about 31 %.

[0083] In an embodiment, the improvement in global well-being (i.e., psychological mood state) after 12 weeks of administration of the probiotic composition compared to control conditions is of at least 170%, particularly of about 171 %, i.e., compared to a subject not treated with the probiotic composition (placebo group). Alternatively said, the improvement in global well-being is increased about 2-fold after 12 weeks of administration compared to control conditions.

[0084] In an embodiment, the improvement in anxiety after 12 weeks of administration compared to control conditions is of at least 80%, particularly of about 80%.

[0085] In an embodiment, the improvement in depression after 12 weeks of administration compared to control conditions is of at least 100%, particularly of about 100%. Alternatively said, the improvement in depression is increased about 1-fold after 12 weeks of administration compared to control conditions. [0086] In an embodiment, the improvement in hostility after 12 weeks of administration compared to control conditions is of at least 90%, particularly of about 90.5%.

[0087] In an embodiment, the improvement in fatigue after 12 weeks of administration compared to control conditions is of at least 1600%, particularly of about 1600%. Alternatively said, the improvement in fatigue is increased about 16-fold after 12 weeks of administration compared to control conditions.

[0088] In an embodiment, the improvement in confusion after 12 weeks of administration compared to control conditions is of at least 380%, particularly of about 389%. Alternatively said, the improvement in confusion is increased about 4-fold after 12 weeks of administration compared to control conditions.

[0089] In an embodiment, the improvement (increase) in vigor after 12 weeks of administration compared to control conditions is of at least 130%, particularly of about 138%.

[0090] GLP-1 is a hormone that plays a crucial role in regulating blood sugar levels and appetite by enhancing insulin secretion in response to food intake, slowing gastric emptying, and promoting satiety. These effects collectively contribute to body weight and fat suppression. In an embodiment, the administration of the probiotic composition results in an enhancement (increase) of GLP-1 secretion.

[0091] In an embodiment, the enhancement (increase) of GLP-1 secretion is of at least 3% when administered at a concentration of at least 0.3 pg/mL of CECT 7481 , particularly of at least 6% and more particularly of at least 9% of increase.

[0092] In one aspect, the present disclosure provides a method for the prevention and/or treatment of overweight, obesity and/or related diseases in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0093] Obesity is a major risk factor for developing a number of diseases and symptoms. According to The Endocrine Society or The Hormone Foundation (http://www.obesityinamerica.org) overweight and obese people are at an increased risk for developing the following conditions: cardiovascular diseases (e.g. atherosclerosis, hypertension, stroke, congestive heart failure, angina pectoris), type 2 diabetes mellitus, obesity-related hypoventilation, back and joint problems, non-alcoholic fatty liver disease, cirrhosis, metabolic syndrome, gastroesophageal reflux disease, reduced fertility, hypothyroidism, dyslipidemia, hyperinsulinemia, cholecystitis, cholelithiasis, osteoarthritis, gout, sleep apnea and other respiratory problems, polycystic ovary syndrome, pregnancy complications, psychological disorders, uric acid nephrolithiasis (kidney stones), stress urinary incontinence and increased incidence of certain cancers (e.g. cancer of the kidney, endometrium, breast, colon and rectum, esophagus, prostate and gall bladder). [0094] Accordingly, in one aspect, the present disclosure provides a method for the prevention and/or treatment of anyone of the above-mentioned diseases or conditions in a subject, which can be considered obesity related-diseases, in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein. In a particular embodiment, the related diseases are metabolic disorders. More particularly, the metabolic disorder is selected from the group consisting of diabetes, hypertension, liver cirrhosis, metabolic syndrome, and cardiovascular diseases.

[0095] In an embodiment, the administration of the probiotic composition results in:

(i) a reduction in body weight;

(ii) a reduction in body fat;

(iii) a reduction in waist circumference;

(iv) an increase (i.e., improvement) in microbiome diversity and resilience in gut;

(v) a reduction (i.e., improvement) of the Firmicutes/Bacteroidetes ratio in gut;

(vi) an increase (i.e. improvement) in Lactobacillus species and/or Bifidobacterium species in gut;

(vii) an increase (i.e. improvement) in butyrate kinase levels in gut;

(viii) an increase (i.e. improvement) in Akkermansia levels in gut;

(ix) a reduction in salivary cortisol;

(x) an improvement in global well-being (i.e., psychological mood state);

(xi) an improvement in anxiety;

(xii) an improvement in depression;

(xiii) an improvement in hostility;

(xiv) an improvement in fatigue;

(xv) an improvement in confusion;

(xvi) an improvement in vigor; and/or

(xvii) an increase (i.e. enhancement) of GLP-1 secretion.

[0096] In an embodiment, the administration of the probiotic composition for 12 weeks, results in:

(i) a reduction in body weight of at least 1 .8%;

(ii) a reduction in body fat of at least 2.5%;

(iii) a reduction in waist circumference of at least 3%;

(iv) an increase in microbiome diversity and resilience in gut as measured by an increase in the Overall composite score of at least 7%;

(v) a reduction of the Firmicutes/Bacteroidetes ratio in gut of at least 19%;

(vi) an increase in Lactobacillus species and/or Bifidobacterium species in gut of at least 15.5% of Lactobacillus and at least 450% of Bifidobacterium;

(vii) an increase in butyrate kinase levels in gut of at least 14%;

(viii) an increase in Akkermansia levels in gut of at least 130%;

(ix) a reduction in salivary cortisol of at least 10.5%;

(x) an improvement in global well-being or the psychological mood state of at least 19%; (xi) an improvement in anxiety of at least 35%;

(xii) an improvement in depression of at least 50%;

(xiii) an improvement in hostility of at least 40%;

(xiv) an improvement in fatigue of at least 50%;

(xv) an improvement in confusion of at least 40%;

(xvi) an improvement in vigor of at least 30%; and/or

(xvii) an increase of GLP-1 secretion of at least 3%.

[0097] In one aspect, the present disclosure provides a method for reducing body fat in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0098] In one aspect, the present disclosure provides a method for reducing the waist circumference in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0099] In one aspect, the present disclosure provides a method for increasing (i.e., improving) microbiome diversity and resilience in gut in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0100] In one aspect, the present disclosure provides a method for reducing (i.e., improving) the Fir- micutes/Bacteroidetes ratio in gut a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0101] In one aspect, the present disclosure provides a method for increasing (i.e. improving) Lactobacillus species and/or Bifidobacterium species in gut in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0102] In one aspect, the present disclosure provides a method for increasing (i.e. improving) butyrate kinase levels in gut in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0103] In one aspect, the present disclosure provides a method for increasing (i.e. improving) Akker- mansia levels in gut in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0104] In one aspect, the present disclosure provides a method for reducing salivary cortisol in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein. [0105] In one aspect, the present disclosure provides a method for improving the global well-being (i.e., psychological mood state) in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0106] In one aspect, the present disclosure provides a method for improving anxiety in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0107] In one aspect, the present disclosure provides a method for improving depression in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0108] In one aspect, the present disclosure provides a method for improving hostility in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0109] In one aspect, the present disclosure provides a method for improving fatigue in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0110] In one aspect, the present disclosure provides a method for improving confusion in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0111] In one aspect, the present disclosure provides a method for improving vigor in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

[0112] In one aspect, the present disclosure provides a method for increasing (i.e., enhancing) GLP- 1 secretion in a subject, comprising administering to a subject in need thereof the probiotic composition provided herein.

Subject

[0113] In an embodiment, the subject is a mammal. In an embodiment, the mammal is a human.

[0114] One particularly interesting aspect is the use of the probiotic composition provided herein for the treatment of overweight or obesity. It is well known that obesity (BMI 30) but also overweight (BMI 25-29.9) may have serious medical implications and that both obese and overweight individuals may benefit from a weight reduction. Furthermore, even normal or near-normal weight individuals (BM1 18.5- 24.9) who do not suffer under medical implications due to overweight may find it attractive to maintain or strive for an optimal body weight for cosmetic reasons.

[0115] Thus, in an embodiment, the subject is an obese or overweight subject. In an embodiment, the subject is an obese subject. In an embodiment, the subject is an overweight subject. In an embodiment, the subject has a BMI of about 25 to 30 or body fat% of at least 20% or between 20-30% or waist circumference of at least 25 inches or between 25-30 inches.

[0116] In another embodiment, the subject is a non-obese, non-overweight subject having a BMI less than 25.

[0117] In an embodiment, the subject is an obese or overweight human subject under psychological stress.

[0118] In an embodiment, the subject has moderate stress, as determined e.g. by a score in the PSS10 questionnaire between 14 and 26.

[0119] Weight loss can be sustained by continued administration of the disclosed probiotic compositions (e.g., one capsule per day or one capsule with each meal) together with a proper maintenance program, including diet and exercise. For example, a subject may be advised to avoid foods that are high in fat and sugar and focus on consuming a certain quantity of fruits and vegetables. Furthermore, a subject may be advised to undergo a minimum three sessions of 30 minutes of moderate exercise, such as brisk walking, each week. Thus, the probiotic composition can be administered together (concomitantly) with other treatments/strategies to reduce body weight or with other kind oftreatments (e.g., antibiotics).

Probiotic composition

[0120] In one embodiment, the probiotic composition comprises the strain Lactiplantibacillus plantarum CECT 7481 (also referred as KABP-051).

[0121] Lactiplantibacillus plantarum strain CECT 7481 is described in US9192634B2 (previously referred as Lactobacillus plantarum), whose content is incorporated herein by reference in its entirety. The strain was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the Spanish Type Culture Collection (Universitat de Valencia, Campus de Burjassot, Edif. de Investigation, 46100 Burjassot, Valencia, Spain) on 21 .01 .2009 under accession number CECT 7481. The deposited strain is viable and keeps all their features related to their deposit. The strain is commercially available in a product which contains Lactobacillus brevis KABP 052 (CECT 7480) and Pediococcus acidilactici KABP 053 (CECT 8633) besides the strain CECT 7481 .

[0122] Lactiplantibacillus plantarum CECT 7481 was isolated from saliva from 0-5 year-old children. The strain tolerates the challenges of human gastrointestinal tract (gastric conditions and bile salts) and adheres to intestinal epithelium. Genotypic analysis confirmed these features. [0123] As understood by the skilled person in the present context, a bacterial strain has been isolated from its natural environment, i.e., it is not present in its natural environment, so it is free from other organisms and substances present in the natural environment.

[0124] The emergence and spread of resistance to antimicrobials in bacteria pose a threat to human and animal health and present a major financial and societal cost. It has been found by whole genome sequence analysis that strain L. plantarum CECT 7481 does not possess transmissible antibiotic resistance genes to commonly used antibiotics. Overall, these results preclude the risk of a potential transfer of antibiotic resistance to pathogenic species.

[0125] It is clear that by using the deposited strain as starting material, the skilled person in the art can routinely, by conventional mutagenesis or re-isolation techniques, obtain further variants or mutants thereof that retain, enhance or improve the herein described relevant features and advantages of the strain forming the probiotic compositiom of the present disclosure. Thus, the present disclosure also relates to variants/mutants of the strain disclosed herein. In an embodiment, the probiotic composition comprises a bacterial strain derived from the strain L. plantarum CECT 7481 , wherein the derived bacterial strain:

(a) has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain CECT 7481 or has a genome fingerprint of the correspondent deposited strain shown in the pulsed field gel electrophoresis (PFGE) of FIG. 6 (A)(B); and

(b) retains, enhances or improves the ability of the correspondent deposited strain to reduce body weight.

[0126] The ability of the derived bacterial strain to reduce body weight as the deposited strain can be measured with any of the parameters described in the present disclosure (e.g. body fat, waist circumference, etc).

[0127] In a particular embodiment, the bacterial strain derived from the deposited strain has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain; more particularly, % of identity is 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

Particularly the % of ANI is at least 99.5%. More particularly, % of ANI is 99.50%, 99.51 %, 99.52%,

99.53% 99.54% 99.55%, 99.56% 99.57%, 99.58%, 99.59%, 99.60% 99.61 %, 99.62%, 99.63%,

99.64% 99.65% 99.66%, 99.67% 99.68%, 99.69%, 99.70%, 99.71 %, 99.72%, 99.73%, 99.74%,

99.75% 99.76% 99.77%, 99.78% 99.79%, 99.80%, 99.81 %, 99.82%, 99.83%, 99.84%, 99.85%,

99.86% 99.87% 99.88%, 99.89% 99.90%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%,

99.97%, 99.98% or 99.99%. In another embodiment, the % of ANI is at least 99.9%; particularly, % of ANI is 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%. [0128] In some embodiments, the mutant is obtained by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. In an embodiment, the bacterial strain derived from the deposited strain is obtained by using recombinant DNA technology. Thus, another aspect of the present disclosure relates to a method to obtain a strain derived from the deposited strain, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant further retains, enhances or improves at least one ability of the deposited strain disclosed herein.

[0129] In one embodiment, the strain forming part of the probiotic composition is in the form of viable cells. In another embodiment, the strain is in the form of non-viable cells. This can include thermally killed microorganisms or microorganisms killed by exposure to altered pH, sonication, radiation or high pressure. Product preparation is simpler with non-viable cells, as cells can be incorporated easily into dietary, pharmaceuticals cr edible products, and storage requirements are much less limited than viable cells. A probiotic composition comprising the strain of the present disclosure as non-viable cells can comprise products derived from the strain which are in the medium.

[0130] Thus, the probiotic composition can comprise the strain as well as the bioactive compounds released by this strain, the supernatants and the cultures of the strain, the fractions/extracts containing the bioactive compounds, lysates and the formulation of any of these in e.g., food and pharmaceutical formulations.

[0131] For the purposes of the present disclosure, the term bioactive products derived from the strain of the present disclosure, are defined as its cell components, and the compounds and molecules that form part of the strain, such as the metabolites and molecules secreted thereof, such as: intracellular components (e.g., DNA, peptides, fatty acids, etc.) cell-wall components (proteins, peptides, fatty acids, etc.) that confer the desired preventive or therapeutic activity.

[0132] As used herein “lysate” refers to a sample of the probiotic strain which has been subject to lysis. A lysate can contain one or more soluble metabolites of the probiotic strain useful in the methods described herein. Lysis can occur by chemical or physical disruption, e.g., by addition of an osmotic agent or enzyme to the bacteria, or by the application of physical pressure, e.g., through sonic disruption. The lysate can be a mixture comprising the cellular contents of the probiotic strain. For example, one or more of fragments of cell wall or cell membrane, proteins, nucleic acids, carbohydrates, and organelles (disrupted or intact). The lysate can be suspended, e.g., in aqueous medium.

[0133] The present disclosure thus provides a method of producing a culture supernatant from the strain, the method comprising a step of culturing the isolated strain and separating the supernatant from the culture, thereby producing the culture supernatant. [0134] The present disclosure also provides a method of producing a fraction/extract or a metabolite from strain cells (comprising bioactive compounds of the strain), the method comprising a step of lysing the cells of the isolated strain and separating the extract of the lysed cells, thereby producing the fraction/extract or the metabolite.

[0135] The strain disclosed herein is produced by cultivating (or fermenting) the bacteria in a suitable artificial medium and under suitable conditions. By the expression, “artificial medium” is understood to be a medium containing natural substances, and optionally synthetic chemicals such as the polymer polyvinyl alcohol which can reproduce some of the functions of serums. Common suitable artificial media are nutrient broths that contain the elements including a carbon source (e.g., glucose), a nitrogen source (e.g., amino acids and proteins), water and salts needed for bacterial growth. Growth media can be liquid form or often mixed with agar or another gelling agent to obtain a solid medium. The strain can be cultivated alone to form a pure culture, or as a mixed culture together with other microorganisms, or by cultivating bacteria of different types separately and then combining them in the desired proportions. After cultivation, and depending on the final formulation, the strain can be used as purified bacteria, or alternatively, the bacterial culture or the cell suspension can be used, either as such or after an appropriate post-treatment. In this description, the term “biomass” is understood to be the bacterial strain culture obtained after cultivation (or fermentation as a term synonymous to cultivation).

[0136] In an embodiment, the strain is fermented in an artificial medium and submitted to a post-treatment after fermentation, to obtain bacterial cells, and the resulting bacterial cells are in a liquid medium or in a solid form. Particularly, the post-treatment is selected from the group consisting of drying, freezing, freeze-drying, fluid bed-drying, spray-drying and refrigerating in liquid medium, and more particularly, is freeze-drying.

[0137] By the term “post-treatment” is to be understood in the present context, any processing carried out on the biomass with the aim of obtaining storable bacterial cells. The objective of the post-treatment is decreasing the metabolic activity of the cells in the biomass, and thus, slowing the rate of cellular deleterious reactions. As a result of the post-treatment, the bacterial cells can be in solid or liquid form. In solid form, the stored bacterial cells can be a powder or granules. In any case, both the solid and liquid forms containing the bacterial cells are not present in nature, hence, are not naturally-occurring, since they are the result of artificial post-treatment process(es). The post-treatment processes can in particular embodiments require the use of one or more of so-called post-treatment agents. In the context of the present disclosure, the expression “post-treatment agent” refers to a compound used to perform the herein described post-treatment processes. Among the post-treatment agents are to be included, without limitation, dehydrating agents, bacteriostatic agents, cryoprotective agents (cryoprotectants), inert fillers (also known as lyoprotectants), carrier material (also known as core material), etc., used either alone or in combination. [0138] There are two basic approaches to decrease the metabolic activity of the bacterial cells, and thus, two approaches to carry out the post-treatment. The first one is decreasing the rate of all chemical reactions, which can be done by lowering the temperature by refrigerating or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers. Alternatively, decreasing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of the bacterial cells, namely a bacteriostatic agent, abbreviated Bstatic.

[0139] The second approach to carry out the post-treatment is to remove water from the biomass, a process which can involve sublimation of water using a lyophilizer. Suitable techniques to remove water from the biomass are drying, freeze-drying, spray-drying or fluid bed-drying. Post-treatments that result in solid form can be drying, freezing, freeze-drying, fluid bed-drying, or spray-drying.

[0140] In an embodiment, the post-treatment is freeze-drying, which involves the removal of water from frozen bacterial suspensions by sublimation under reduced pressure. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most water, and secondary drying to remove bound water. Due to objective and expected variability of industrial processes for manufacturing and isolation of lyophilized bacterial cultures, the latter commonly contain a certain amount of inert filler also known as lyoprotectant. Its role is to standardize the content of live probiotic bacteria in the product. The following inert fillers in commercially available lyophilized cultures are used: sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic fillers. Optionally, other stabilizing or freezeprotecting agents like ascorbic acid, are also used to form a viscous paste, which is submitted to freeze- drying. In any case, the so-obtained material can be grinded to appropriate size, including to a powder.

[0141] Alternatively to having biomass preserved in solid form, biomass can be also preserved in liquid form. This can be done by adding a bacteriostatic agent as described above to stop bacteria growth to the culture medium or with an intermediate step of harvesting cells, re-suspending the pellet in saline solution with a bacteriostatic agent, and optionally refrigerating it.

[0142] Sometimes, as described for instance above in the fluid bed-drying process, the probiotic composition is subjected to an immobilization and/or coating, or encapsulation process in order to improve the shelf life and/or functionalities. Several techniques for immobilization, coating or encapsulation of bacteria are known in the art.

[0143] In other embodiments, the probiotic composition is formulated for sustained-release administration e.g., by means of the encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. The suitable sustained-release forms as well as materials and methods fortheir preparation are well known in the state of the art. Thus, the orally administrable form of any of the probiotic compositions of the present disclosure is in a sustained-release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semisynthetic or synthetic polymers, water-insoluble or modified, waxes, fats, fatty alcohols, fatty acids, natural, semisynthetic or synthetic plasticizers or a combination of two or more of the same. Enteric coatings can be applied using conventional processes known to those skilled in the art.

[0144] The effective amount of colony forming units (cfu) for the strain in the probiotic composition can be determined by the skilled in the art and will depend upon the final formulation. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells as revealed by microbiological counts on agar plates.

[0145] As known by the skilled person, the effective amount of colony units can also be measured by the effective amount of active fluorescent units. The term “active fluorescent unit" ("afu") is defined as the number of bacterial cells as revealed by flow cytometry counts in a gate specific for fluorescence characteristics of presumed live cells. Therefore, the skilled person would consider the above-mentioned specific quantities of cfu to be about the same quantity of afu.

[0146] In one embodiment, the probiotic composition is a solid composition. In another embodiment, the probiotic composition is a liquid composition.

[0147] In an embodiment, the probiotic composition comprises a cryoprotectant. Particularly, the probiotic composition comprises at least one cryoprotectant that is an allergen-free cryoprotectant. In some embodiments, the probiotic composition comprises at least one cryoprotectant such as maltose, trehalose, mannitol (particularly, d-mannitol), saccharose, lactose, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, anhydrous dextrose, starch, cellulose and inulin. In a particular embodiment, the cryoprotectant and/or the pharmaceutically acceptable carrier is selected from the group consisting of trehalose, D-mannitol, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, starch, and cellulose. Particularly, the starch is corn, maize starch and/or potato starch.

[0148] More particularly, the probiotic composition further comprises a pharmaceutically acceptable carrier chosen from an emulsion, a suspension, a gel, a paste, granules, a powder, and a gum. Particularly, the carrier is an allergen-free carrier.

[0149] In some embodiments, the probiotic composition comprises one or more carriers selected from the group consisting of: maltodextrin, cellulose, starches, inulin, lactose, trehalose, dextrose, sodium citrate, sodium ascorbate, D-mannitol, L-cysteine, magnesium sulphate, sodium glutamate, histidine and marie acid.

Product forms of the probiotic composition Pharmaceutical/nutraceutical forms

[0150] In some embodiments, the probiotic composition described herein is in a pharmaceutical/nutraceutical form, such as a capsule, a powder, a suspension, or a tablet.

[0151] The term “pharmaceutical form" is understood in its widest meaning, including any composition that comprises an active ingredient, in this case, the probiotic composition described herein together with at least a pharmaceutically (also referred as nutraceutically or veterinary) acceptable excipient. The term "pharmaceutical form" is not limited to medicaments but includes e.g., pharmaceutical compositions, nutraceutical compositions or veterinary compositions. A pharmaceutical form can adopt different names depending on the product regulatory approval route and also depending on the country.

[0152] A nutraceutical composition can also be named e.g., as food supplement ordietary supplement. A nutraceutical composition is understood as a preparation or product intended to supplement the diet, made from compounds usually used in foodstuffs, which provide nutrients or beneficial ingredients that are not usually ingested in the normal diet or can not be consumed in sufficient quantities. Nutraceutical compositions are usually sold “over the counter”, i.e., without prescription.

[0153] In some embodiments, the probiotic composition is formulated as pharmaceutical form in which the strain is the only active agent or is mixed with one or more other active agents and/or are mixed with pharmaceutically/nutraceutically/veterinary acceptable excipients. Particularly, the additional active agent or agents are other probiotic bacteria which are not antagonistic to the strain forming the probiotic composition of the present disclosure. Depending on the formulation, the strain can be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture which has been post-treated, and alone or together with suitable carriers or other ingredients.

[0154] The term "pharmaceutically/nutraceutical/veterinary acceptable" is art-recognized, and includes excipients, compounds, materials, compositions, carriers, vehicles and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., human or animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical/nutraceutical/veterinary texts.

[0155] Thus, some embodiments of the present disclosure relate to a pharmaceutical composition, a nutraceutical composition, and a veterinary composition comprising the probiotic composition described herein together with at least a pharmaceutically/nutraceutically/veterinary acceptable excipient as described above. [0156] Some non-limiting examples of materials which can serve as pharmaceutically/nutraceuti- cally/veterinary acceptable excipients or carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; or phosphate buffer solutions.

[0157] Excipients are selected, without limitation, from the group comprising: fillers/diluents/bulking agents, binders, antiadherents, disintegrants, coatings, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colors, or tensides.

[0158] Fillers are selected, without limitation, from the group comprising: inulin, oligofructose, pectin, modified pectins, microcrystalline cellulose, lactose, starch, maltodextrin, saccharose, glucose, fructose, mannitol, xylitol, non-crystallizing sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharmacologically acceptable fillers, and mixtures of these substances. At dosage form of oral suspension, fillers or diluents are selected from the group comprising: vegetable oil, oleic acid, oleyl alcohol, liquid polyethylene glycol, other pharmacologically acceptable inert liquids, or mixtures of these substances.

[0159] Binders are used in solid dosage forms, e.g., to hold the ingredients in a tablet together, to ensure that tablets and granules can be formed with required mechanical strength, and to give volume to low active dose tablets. Binders in solid dosage forms like tablets are: lactose, sucrose, corn (maize) starch, modified starches, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethylcellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP) also known as povidone, poly-ethylene glycol, sorbitol, maltitol, xylitol and dibasic calcium phosphate; other suitable pharmacologically acceptable binders, or mixtures of these substances.

[0160] Antiadherents are used to reduce the adhesion between the powder (granules) and the punch faces and thus prevent sticking to tablet punches. They are also used to help protect tablets from sticking. The most commonly used is magnesium stearate.

[0161] As disintegrants and superdisintegrants in solid dosage forms like tablets and capsules, the following substances, without limitation, are used: cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, and formaldehyde-casein, other suitable pharmacologically acceptable disintegrant and superdisintegrant, or their mixtures. [0162] Coatings in the case of solid dosage forms, such as tablets and granules for capsule filling, protect the ingredients from deterioration by moisture in the air, make large, unpleasant-tasting tablets easier to swallow and/or in the case of enteric coatings ensure intact passage through a strong acidic medium of gastric juice (pH around 1), and which allow release in duodenum or ileum (small intestine). For most coated tablets, a cellulose ether hydroxypropyl methylcellulose (HPMC) film coating is used. Occasionally, other coating materials are used, e.g., synthetic polymers and co-polymers like polyvinylacetate phthalate (PVAP); co-polymers of methyl acrylate-metacrylic acid; co-polymers of methyl metacrylate-metacrylic acid; shellac, corn protein zein or other polysaccharides; waxes or wax-like substances such as beeswax, stearic acid; higher fatty alcohols like cetyl or stearyl alcohol; solid paraffin; glycerol monostearate; glycerol distearate, or their combinations. Capsules are coated with gelatin or hydroxypropyl methylcellulose.

[0163] Enteric coatings control the rate of drug release and determine where the drug will be released in the digestive tract. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers and their mixtures, also in combination with other above-mentioned coatings.

[0164] An anticaking agent is an additive placed in powdered or granulated materials to prevent the formation of lumps (caking) and for easing packaging, transport, and consumption. As anti-caking agents in solid dosage forms like tablets, capsules, or powders, the following are used: magnesium stearate, colloidal silicon dioxide, talc, other pharmacologically acceptable anticaking agents, or their mixtures.

[0165] Lubricants are used in solid dosage forms, in particular in tablets and capsules, to prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine, and also in hard capsules. As lubricants talc or silica, and fats, e.g., vegetable stearin, magnesium stearate or stearic acid, and mixtures thereof, are the most frequently used lubricants in tablets or hard gelatin capsules.

[0166] Sweeteners are added to make the ingredients more palatable, especially in solid dosage forms, e.g., chewable tablets, as well as in liquids dosage forms, like cough syrup. Sweeteners can be selected from artificial, natural or synthetic or semi-synthetic sweeteners; non-limiting examples of sweeteners are aspartame, acesulfame potassium, cyclamate, sucralose, saccharine, sugars or any mixture thereof.

[0167] Flavors can be used to mask unpleasant tasting active ingredients in any dosage form. Flavorings can be natural (e.g., fruit extract) or artificial. For example, to improve: (1) a bitter product, mint, cherry or anise can be used; (2) a salty product, peach or apricot or liquorice can be used; (3) a sour product, raspberry; and (4) an excessively sweet product, vanilla. [0168] Except auxiliary substances from the class of excipients, the formulation from the present disclosure can contain other pharmacologically active or nutritive substances including, but not limited, to prebiotics, vitamins, such as vitamin D (calciferol) in the pharmaceutically acceptable chemical form, salt or derivatives; minerals in the form of pharmaceutically and nutritive acceptable chemical form; and L-amino acids.

[0169] In certain embodiments, the probiotic composition further comprises one or more prebiotics. Prebiotics resist breakdown in the gastrointestinal tract and increase probiotic functionality by acting as a food source for naturally occurring and added beneficial bacteria. Prebiotics promote the growth and proliferation of beneficial bacteria in the digestive system. In such embodiments where one or more prebiotics is present, such prebiotics are present in a quantity between about 10 to 99.9 percent weight. Particularly, the concentration of prebiotics ranges from about 15 to 25 weight percent. Examples of prebiotics are fructo-oligosaccharides (e.g., inulin), galacto-oligosaccharides, xylo-oligosaccharides, arabinoxylan-oligosaccharides, pectins, beta-glucans, human milk oligosaccharides (e.g., Lacto-N- tetraose) gum acacia, resistant starch, chicory root extract or partially hydrolyzed guar gum.

[0170] In each case the presentation of the probiotic composition will be adapted to the type of administration used by means known by the person skilled in the art. Thus, the probiotic composition can be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount. The probiotic composition can be thus formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders (such as those derived from lyophilization (freeze-drying) or air-drying), granules, solutions, suppositories, gels or microspheres. In a particular embodiment, the probiotic composition is formulated for administration in liquid form or in solid form.

[0171] Pharmaceutical unit dosage forms of the compounds of this disclosure are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., bolus injection), topical, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or nonaqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. The pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). [0172] In an embodiment, the probiotic composition is in solid form such as tablets, lozenges, sweets, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, tablet, pills, troches, gastro-resistant tablets and capsules, dispersible strips and films. More particularly, the probiotic composition is in form of a capsule, a powder, a tablet, a pill, lozenges, sachets, or granules.

[0173] In an embodiment, the probiotic composition is in form of a powder which is put in contact with an aqueous phase to form a solution. The aqueous phase can comprise fibers such as inulin. The two components (the powder and the aqueous phase) can be in separate compartments/containers and the two components are mixed for /n situ reconstitution. In an embodiment where the probiotic formulation is delivered as e.g., a capsule or tablet, the probiotic composition can further comprise one or more excipients to facilitate the manufacturing process by preventing the ingredients from adhering to machines. Moreover, such excipients render the capsule or tablet form easier to swallow and digest through the intestinal tract. The excipients can be a vegetable stearate, magnesium stearate, steric acid, ascorbyl palmitate, retinyl palmitate, or hyproxypropyl methylcellulose. In a particular embodiment, the excipients are present in an amount between about 0 to 30 weight percent.

[0174] In an embodiment, the probiotic composition is in form of gelatin capsules. In a particular embodiment, the probiotic composition is in the form of a vegetable capsule and comprises hydroxypropyl methylcellulose (HPMC).

[0175] In another embodiment, the probiotic composition is in liquid form such as oral solutions, drops, suspensions (e.g., oil), emulsions and syrups. Particularly, the probiotic composition is in form of drops. More particularly, the probiotic composition is in form of oily drops.

[0176] In some embodiments, the probiotic composition is in the form of an oily suspension to be administered alone or mixed with a liquid. The oily suspension comprises at least one edible oil such as olive oil, maize oil, soybean oil, linseed oil, sunflower oil or rice oil. The oil is present in a quantity of at least 70% weight/weight. In a particular embodiment, the oily suspension also comprises at least one excipient which is an emulsifier, stabilizer or anti-caking agent, in an amount of 0.1-15% w/w. Suitable agents are silicon dioxide, silica gel, colloidal silica, precipitated silica, talc, magnesium silicate, lecithin, pectin, starch, modified starches, konjac gum, xanthan gum, gellan gum, carrageenan, sodium alginate, mono- or diglycerides of fatty acids such as glycerol monostearate or glycerol monooleate and citric acid esters of mono- or diglycerides.

[0177] In an embodiment, the e.g., capsule, sachet or stick, tablet or pill have a weight of about 150 mg to about 8000 mg. More particularly, the capsule has a weight of about 200 mg to about 600 mg. More particularly, the sachet or stick has a weight of about 1 .5 g to about 6 g. More particularly, the tablet or pill have a weight of about 400 mg to about 1200 mg. [0178] In an embodiment, the e.g., spray, oily drops have a volume of about 3 ml to about 50 ml. More particularly, the spray has a volume of about 5 ml to about 50 ml. More particularly, the oil drops have a volume of about 3 ml to about 30 ml.

[0179] Regarding the preparation of the formulations of the present disclosure, it is within the scope of ordinary person skilled in the art and will depend upon the final dosage formulation. For instance, and without limitation, when the final dosage form is an oral solid one, such as tablets, capsules, powder, granules, oral suspension, etc. the process for preparation of solid dosage forms of the formulation includes homogenization of: (1) the active ingredient(s), comprising post-treated probiotic bacteria in an effective amount; (2) with one or more excipients to form homogeneous mixture which is, e.g., according to requirements, subjected to lubrication with magnesium stearate or other lubricants yielding final dosage form of powder. Such homogeneous powder is filled into ordinary gelatin capsules or, alternatively, into gastro-resistant capsules. In the case of tablets, they are manufactured by direct compression or granulation. In the first case, a homogeneous mixture of active ingredients and suitable excipients such as anhydrous lactose, non-crystallizing sorbitol, and others is prepared. In the second case, tablets are processed of the mixture in granulated form. Granules are prepared by granulation process of active ingredients of the formulation with suitable fillers, binders, disintegrants, and small amount of purified water. Such prepared granules are sieved and dried until the water content of <1 % w/w.

[0180] Regarding the process for preparation of liquid dosage forms (e.g., oral suspension), it involves homogenization of the active ingredient(s) of the formulation comprising post-treated probiotic bacteria in an effective amount in an inert liquid diluent (filler) such as various vegetable oils like sunflower, soybean or olive oil; oleic acid; oleyl alcohol; liquid polyethylene glycols like PEG 200, PEG 400 or PEG 600; or other inert pharmacologically acceptable liquids. The process further involves treatment of homogeneous mixture with one or more processes selected from the group comprising: (1) stabilization of the formulation, by addition and homogenization of suspension stabilizers like beeswax, colloidal silicon dioxide, etc.; (2) sweetening of the formulation, by addition and homogenization of sweetener; (3) flavoring of the formulation, by addition and homogenization of flavoring.

Food products

[0181] In some embodiments, the probiotic composition is in the form of a food product or an edible composition, such as infant formulas or food, milk-based fermented products (e.g., yogurt, cheese, curd), vegetable-based fermented products, breads, bars (e.g., energetic bars), spreads, biscuits, syrups, beverages, dressings, sauces, fillings, soups, ice creams, oils, dressings or confectionaries. [0182] The term “food product or edible composition” are used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal, particularly a human, but excluding pharmaceutical, nutraceutical and veterinary products. Such foods products can be in solid or liquid/drinkable form. Further, the food product can contain all customary additives, including but not limited to, proteins, vitamins, minerals, trace elements, and other nutritional ingredients.

[0183] In an embodiment, the probiotic composition is included in an infant formula or food. Particularly, the probiotic composition is included in a beverage.

[0184] Examples of other food products are meat products, chocolate spreads, fillings and frostings, chocolate, confectionery, baked goods, sauces and soups, fruit juices and coffee Whiteners. The food product particularly comprises a carrier material such as oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. In a particular embodiment the strain is encapsulated or coated. Particularly, milks can be either of animal or vegetable origin.

Administration

[0185] A therapeutically effective dose level will depend on many factors. In addition, it is well-known within the skill of the art to start doses of the active composition at relatively low levels, and increase the dosage until the desired effect is achieved. Efficacy of the methods provided herein can be determined using any appropriate method. For example, weight gain can be determined and evaluated with any one of several objective, standard instruments known in the art, which include scales and instruments that measure body fat percentage. The simple instrument of a scale is routinely used by all professional health care practitioners. More sophisticated instruments to measure body fat percentage operate based on skin-fold methodology and measurement of body density or electrical resistance.

[0186] Clinicians, physicians, and other health care professionals can administer the probiotic composition to a subject in need thereof according to a method provided herein. In some cases, a single administration of the probiotic composition can be sufficient. In other cases, more than one administration of the probiotic composition is performed at various intervals (e.g., once per week, twice per week, daily, monthly) or according to any other appropriate treatment regimen. The duration of treatment can be a single dose or periodic multiple doses for as long as administration of the probiotic composition provided herein is tolerated by the subject.

[0187] In some embodiments, the probiotic composition is administered in a single dose or repeated dose at specific time intervals, e.g., can be administered daily for a specific number of days or according to a specific dosing schedule. In an embodiment, the probiotic composition is administered from 10 days to 90 days. More particularly, it is administered from 10 days to 60 days or from 15 to 45 days, more particularly for 30 days. In another embodiment, the probiotic composition is administered for at least 12 weeks or for at least 90 days.

[0188] In some embodiments, the probiotic composition is administered from once every three days to thrice a day, particularly, once or twice a day.

[0189] In an embodiment, the probiotic composition contains a single (unit) dose of the strain. Suitable doses of the strain (intact or lysed) can be in the range 10⁴ to 10¹² colony forming units (cfu), e.g. one of 10⁴ to 10¹⁰, 10⁴ to 10⁸, 10⁶ to 10¹², 10⁶ to 10¹⁰, or 10⁶ to 10⁸ cfu. Particularly, the probiotic composition comprises the strain in an amount of about 1x10⁹ cfu.

[0190] In an embodiment, the probiotic composition comprises the strain in an amount between 10⁴ cfu and 10¹² cfu per gram or milliliter of composition, particularly between 10⁶ and 10¹² cfu/g or cfu/ml or between 10⁷ and 10¹² cfu/g or cfu/ml.

[0191] In an embodiment, the probiotic composition comprises the strain in an amount between 10⁴ cfu and 10¹² cfu per daily dose, and particularly in an amount of about 10⁹ cfu.

[0192] In an embodiment, the probiotic composition comprises: a freeze-dried bacterial biomass comprising from about 10⁴ cfu to about 10¹² cfu of the strain; particularly from about 10⁵ cfu to about 10¹² cfu of the strain; more particularly from about 10⁹ cfu to about 10¹² cfu of the strain; more particularly about 10¹¹ cfu.

[0193] In some embodiments, the probiotic composition can be administered orally, rectally, parenterally, topically, ocularly, aurally, nasally, intravaginally or to the buccal cavity, to give a local and/or a systemic effect. Particularly, the probiotic composition is orally administered.

EXAMPLES

EXAMPLE 1. Randomized placebo-controlled clinical study of Lactiplantibacillus plantarum CECT 7481 (KABP-051)

[0194] The objective of the study was the evaluation of the effects of dietary supplementation with the probiotic strain (Lactiplantibacillus plantarum CECT 7481) on measures of psychological mood state, gut microbiome status, stress hormones, and body composition (body weight, body fat and muscle mass) in a population of healthy subject with moderate overweight and stress.

1.1. Clinical study design Table 1 :

1.2. Results. Subject characteristics and summary of the results.

[0195] Female = 25 (CECT 7481) 124 (Placebo)

Male = 4 (CECT 7481) 14 (Placebo) Height = 65.2 inches (CECT 7481) 165.5 inches (Placebo)

Weight = 183.4 lbs (CECT 7481) / 182.5 lbs (Placebo)

[0196] Following 12-weeks of supplementation, the CECT 7481 group experienced significant 2-4% improvements in body composition, including loss of body weight (3.3 lbs); body fat percentage (3%); and waist circumference (3.4 cm), while maintaining muscle mass. In addition, subjects in the

CECT 7481 group showed significant improvements in feelings of fatigue, suggesting that this probiotic strain may enhance energy levels. [0197] The CECT 7481 group also experienced improvements in stress hormones (9-14% lower cortisol) and microbiome balance (2-7% increase in composite score; 6-20% decrease in F/B ratio; and 14-31 % increase in butyrate kinase).

[0198] These results indicate that the CECT 7481 strain is an effective way to enhance weight loss, while also helping users “feel better” with reduced fatigue and elevated energy levels while they are embarking on a weight loss regimen.

1.3. Results. Body Weight (lbs)

[0199] A multilevel model analysis of covariance was performed to assess the interaction between treatment group (supplement vs. control) across time. Height was used as a covariate to account for any differential effect that my occur as a function of subject height. Random subject-level effects were used to account for the repeated measures on an individual person. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0200] There was a significant omnibus effect of supplementation across time. The post-hoc contrasts indicated that supplementation resulted in a decreased body weight compared to controls across time, specifically Week 0 to Week 12 and Week 4 to Week 12. The net effect was a loss of 3.8 lbs. and 2.5 lbs., respectively.

[0201] Results are shown in FIG. 1 and summarized below:

Placebo = 182.5 (baseline), 182.4 (w4), 182.8 (w8), 182.8 (w12) - No change in Body Weight

CECT 7481 = 183.4 (baseline), 182.1 (w4), 181.9 (w8), 180.1 (w12) - 1.8% (~2%) loss in body weight

1.4. Results. Body fat (%)

[0202] A multilevel model analysis of covariance was performed to assess the interaction between treatment group (supplement vs. control) across time. Height was used as a covariate to account for any differential effect that my occur as a function of subject height. Random subject-level effects were used to account for the repeated measures on an individual person. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0203] There was a significant omnibus effect of supplementation across time. The post-hoc contrasts indicated that supplementation resulted in a decreased body fat percentage compare to controls across time, specifically Week 0 to Week 12. The net effect was a decrease in body fat percentage by 3% across the study period. [0204] Results are shown in FIG. 2 and summarized below:

Placebo = 35.9 (baseline), 36.3 (w4), 36.1 (w8), 36.3 (w12) - No change in Body Fat %

CECT 7481 = 37.4 (baseline), 37.0 (w4), 36.9 (w8), 36.3 (w12) - 2.94% (~3%) loss in Body Fat %

1.5. Results. Waist circumference (cm)

[0205] A multilevel model analysis of covariance was performed to assess the interaction between treatment group (supplement vs. control) across time. Height was used as a covariate to account for any differential effect that my occur as a function of subject height. Random subject-level effects were used to account for the repeated measures on an individual person. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0206] There was a significant omnibus effect of supplementation across time. The post-hoc contrasts indicated that supplementation resulted in a decreased waist circumference compare to controls across time, specifically Week 0 to Week 8 and Week 0 to Week 12. The net effect was a decrease in waist circumference by of 1 .7 and 3.4 units, respectively.

[0207] Results are shown in FIG. 3 and summarized below

Placebo = 98.4 (baseline), 98.2 (w4), 97.8 (w8), 97.4 (w12) - ~1 % loss in Waist Circumference

CECT 7481 = 99.3 (baseline), 97.9 (w4), 96.9 (w8), 95.9 (w12) - 3.4% (~4%) loss in Waist Circumference

1.6. Results. Cortisol (salivary, pg/ml)

[0208] Cortisol is the best-known biomarker for stress (lower value corelates to lower stress). A multilevel model analysis was performed to assess the interaction between treatment group (supplement vs. control) across time. Random subject-level effects were used to account for the repeated measures on an individual person. Initial models indicated significant error heterogeneity which was remediated by a natural log transform of the outcome variable (cortisol). The estimates and standard errors have been exponentiated back to their original units to aide in their lay interpretation. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0209] Results are shown in FIG. 4 and summarized below:

Placebo = ~4-16% increase in salivary cortisol over 12-weeks Baseline = 583

Week 4 = 607 (+4%) Week 8 = 604 (+4%)

Week 12 = 674 (+16%)

[0210] CECT 7481 : 9-14% decrease in salivary cortisol over 12-weeks

Baseline = 648

Week 4 = 590 (-9%)

Week 8 = 556 (-14%)

Week 12 = 577 (-11 %)

1.7. Results. Microbiome

[0211] A multilevel model analysis was performed to assess the interaction between treatment group (supplement vs. control) across time for Overall composite score, F/B ratio, Lactobacillus, Bifidobacterium, butyrate kinase, and Akkermansia. Random subject-level effects were used to account for the repeated measures on an individual person. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0212] Higher microbiome diversity stands for better health. Overall composite score (of markers = Firmicutes/Bacteroidetes (F/B) ratio; Lactobacillus, Bifidobacterium, butyrate kinase, Akkermansia, S. thermophilus) was analyzed from stool samples of the subjects. Results are shown in FIG. 5 panel (A) and summarized below:

[0213] Placebo:

Baseline = 6.03

Week 4 = 6.00 (-0.5%)

Week 8 = 6.04 (+0.2%)

Week 12 = 6.03 (+0.1 %)

[0214] CECT 7481 : ~2-7% improvement (increase) in overall microbiome diversity and resilience Baseline = 5.87

Week 4 = 5.97 (+1.7%)

Week 8 = 6.24 (+6.3%)

Week 12 = 6.29 (+7.1 %)

[0215] F/B Ratio (Firmicutes/Bacteroidetes). Firmicutes and Bacteroidetes are the two major phyla in the human gut microbiota. The ratio (F/B) has been used as a biomarker of human health status. F/B has been shown to correlate with obesity. Results are shown in FIG. 5 panel (B) and Table 2, and summarized below: Table 2:

[0216] ~6-20% improvement (decrease) in F/B ratio (Firmicutes/Bacteroidetes)

No differences in Bacteroidetes

~20% improvement (decrease) in Firmicutes (suggesting positive effects for weight loss)

[0217] Lactobacillus, Bifidobacterium. Increases in Lactobacillus and Bifidobacterium suggest an increase in beneficial bacteria in the gut. Results are shown in FIG. 5 panel (C), (D) and Table 3, and summarized below:

Table 3:

[0218] ~20% improvement (increase) in Lactobacillus species

~up to 4-fold improvement (increase) in Bifidobacterium levels

[0219] Butyrate kinase. Butyrate prevents against body weight gain and fat content. An increase in butyrate kinase, one of the butyrate biosynthetic enzymes, may help increase butyrate in the gut. Results are shown in FIG. 5 panel (E) and summarized below:

[0220] Placebo:

Baseline = 0.104

Week 4 = 0.099 (-5%)

Week 8 = 0.093 (-10%)

Week 12 = 0.102 (-3%) [0221] CECT 7481 : ~14-31 % improvement (increase) in Butyrate Kinase (potentially associated with weight loss)

Baseline = 0.109

Week 4 = 0.127 (+16%)

Week 8 = 0.144 (+31 %)

Week 12 = 0.125 (+14%)

[0222] Akkermansia. Akkermansia is known to be more abundant in the gut of non-obese people and less abundant in obese people. Results are shown in FIG. 5 panel (F) and summarized below:

[0223] Placebo: Baseline = 20.84 Week 4 = 19.79 (-5%) Week 8 = 31 .33 (+50%) Week 12 = 16.95 (-18%)

[0224] CECT 7481 :

Baseline = 12.05

Week 4 = 34.76 (+188%)

Week 8 = 19.13 (+59%)

Week 12 = 28.46 (+136%)

1.8. Results. Psychological mood state

[0225] Changes in mood/focus/energy related to microbiome changes was assessed using the re- search-validated Profile of Mood States (POMS) questionnaire to measure 6 primary psychological factors (tension, mood, irritability, fatigue, confusion, and vigor) plus the combined global mood state as an indication of subjective well-being. The POMS methodology has been used in ~3,000 studies, and its validity is well established. The POMS profile uses 65 adjective-based intensity scales scored on a 0-4 hedonic scale (e.g. “not at all” to “extremely”). The output of the POMS questionnaire is an assessment of the positive and negative moods of each healthy subject at baseline and post-supplementation - it is not used as a diagnostic tool for assessing psychological disorders such as depression or anxiety.

[0226] A multilevel model analysis was performed to assess the interaction between treatment group (supplement vs. control) across time for POMS Global Mood. Random subject-level effects were used to account for the repeated measures on an individual person. Initial models indicated significant error heterogeneity which was remediated by a natural log transform of the outcome variable (Global Mood). The estimates and standard errors have been exponentiated back to their original units to aide in their lay interpretation. When a significant interaction effect was observed, a “contrast of contrasts” approach was performed whereby the differences between groups were compared at each time point. Adjustments for multiple comparisons were made via the Kenward-Roger method.

[0227] Equivalent model and analysis were applied for the analysis of POMS tension/anxiety, depression, anger and hostility, vigor, fatigue confusion, and vigor. Results are shown in Table 4.

Table 4:

* For anxiety, depression, hostility, fatigue and confusion, lower number indicates lower symptom (or status), representing an improvement. For vigor, a higher number represents an improvement.

[0228] Global well-being score is calculated as [sum of all negative factors] - [sum of all positive factors, such as vigor]. Therefore the global score reflects a general improvement.

EXAMPLE 2

[0229] Macrorestriction analysis of genomic DNA of L. plantarum CECT 7481 digested with Sfil and Smal and resolved by pulsed-field gel electrophoresis (PFGE).

[0230] The strain of L. plantarum CECT 7481 was as a pure culture plate and it was subcultured in MRS agar plate, and incubated at 37 °C for 24 h under 5% CO2. The study of PFGE was made following the methodology described by Rodas A. M., Ferrer S., Pardo I. 2005. "Polyphasic study of wine Lactobacillus strains: taxonomic implications", International Journal of Systematic and Evolutionary Microbiology, 55: 197-207, changing the times of incubation of cellular lysis accordingly. Two restriction enzymes Sfil y Smal were used for characterization. The band pattern is shown in FIG. 6(A)(B).

EXAMPLE 3. Evaluation of GLP-1 Secretion enhancement of Lactiplantibacillus plantarum CECT 7481 (KABP-051) [0231] The objective of the study was the evaluation of the enhancement of GLP-1 (glucagon-like peptide-1) secretion by Lactiplantibacillus plantarum CECT 7481 (KABP-051) using cultured enteroendocrine cells (STC-1 cells).

[0232] GLP-1 is a hormone that plays a crucial role in regulating blood sugar levels and appetite by enhancing insulin secretion in response to food intake, slowing gastric emptying, and promoting satiety. These effects collectively contribute to body weight and fat suppression.

[0233] STC-1 cells were seeded in a 24-well plate at a density of 2 x 10⁵ cells/well and cultured for 48 hours using DMEM (Dulbecco's Modified Eagle Medium) with 10% fetal bovine serum (FBS), 10 U/ml penicillin, 100 pg/ml streptomycin and 0.25 pg/ml amphotericin B in a CO2 incubator at 37 °C. After the initial culture period and cell wash, DMEM (no glucose, no glutamine) supplemented with 1 mM glucose was added to each well, then test samples were added with concentrations ranging from 0 to 3 pg/mL. The cells were then incubated for 6 hours in a CO2 incubator at 37 °C, and the supernatants were collected. The concentration of GLP-1 in the collected supernatants were measured using an ELISA method (GLP-1 ELISA Kit Wako (High Sensitive)).

[0234] As a result, the group treated with CECT 7481 showed a significantly higher GLP-1 secretion compared to the vehicle (0 pg/mL) control (p<0.01 , t-test), indicating that CECT 7481 promotes GLP-1 secretion. Results are shown in FIG. 7 and summarized below:

0 pg/mL of CECT 7481 = 1

0.3 pg/mL of CECT 7481 = 1 .03 (+3%)

1 pg/mL of CECT 7481 = 1.07 (+7%)

3 pg/mL of CECT 7481 = 1 .09 (+9%)

[0235] Additionally, the same evaluations were conducted for other strains. In this analysis, a sample concentration of 30 pg/mL was used to compare GLP-1 secretion against the vehicle control (0 pg/mL). None of the evaluated strains, including different Lactiplantibacillus plantarum strains, showed statistically significant GLP-1 secretion compared to the vehicle control (P>0.05, t-test), indicating that the effect on GLP-1 secretion is specific to the CECT 7481 strain. Results are shown in Table 5.

Table 5:

[0236] It is to be appreciated that the Detailed Description section is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0237] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0238] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0239] The breadth and scope of the present disclosure should not be limited by any of the abovedescribed exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1 . A method for reducing body weight in a subject, comprising administering to a subject in need thereof a probiotic composition comprising:

Lactiplantibacillus plantarum strain deposited under the Budapest Treaty in the Spanish Type Culture Collection, under accession number CECT 7481 , or a bacterial strain derived thereof, wherein the derived bacterial strain:

(a) has a genome fingerprint of the correspondent deposited strain CECT 7481 shown in the pulsed field gel electrophoresis of FIG. 6(A)(B) ; and

(b) retains the ability of the correspondent deposited strain CECT 7481 to reduce body weight.

2. The method according to claim 1 , for further regulating stress and anxiety in the subject.

3. The method according to claim 2, wherein the administration of the probiotic composition results in:

(i) a reduction in body weight;

(ii) a reduction in body fat;

(iii) a reduction in waist circumference;

(iv) an increase in microbiome diversity and resilience in gut;

(v) a reduction of the Firmicutes/Bacteroidetes ratio in gut;

(vi) an increase in Lactobacillus species and/or Bifidobacterium species in gut;

(vii) an increase in butyrate kinase levels in gut;

(viii) an increase in Akkermansia levels in gut;

(ix) a reduction in salivary cortisol;

(x) an improvement in the global well-being of the subject or the psychological mood state;

(xi) an improvement in anxiety;

(xii) an improvement in depression;

(xiii) an improvement in hostility;

(xiv) an improvement in fatigue;

(xv) an improvement in confusion;

(xvi) an improvement in vigor; and/or

(xvii) an increase of GLP-1 secretion.

4. The method according to claim 3, wherein the administration of the probiotic composition for 12 weeks, results in:

(i) a reduction in body weight of at least 1 .8%;

(ii) a reduction in body fat of at least 2.5%;

(iii) a reduction in waist circumference of at least 3%;

(iv) an increase in microbiome diversity and resilience in gut as measured by an increase in the Overall composite score of at least 7%; (v) a reduction of the Firmicutes/Bacteroidetes ratio in gut of at least 19%;

(vii) an increase in butyrate kinase levels in gut of at least 14%;

(viii) an increase in Akkermansia levels in gut of at least 130%;

(ix) a reduction in salivary cortisol of at least 10.5%;

(x) an improvement in global well-being or the psychological mood state of at least 19%;

(xi) an improvement in anxiety of at least 35%;

(xii) an improvement in depression of at least 50%;

(xiii) an improvement in hostility of at least 40%;

(xiv) an improvement in fatigue of at least 50%;

(xv) an improvement in confusion of at least 40%;

(xvi) an improvement in vigor of at least 30%; and/or

(xvii) an increase of GLP-1 secretion of at least 3%.

5. The method according to claim 1 , comprising Lactiplantibacillus plantarum strain deposited under the accession number CECT 7481 .

6. The method according to claim 1 , wherein the probiotic composition comprises the strain in an amount from 10⁴ to 10¹² colony forming units.

7. The method according to claim 1 , wherein the probiotic composition comprises one or more carriers selected from the group consisting of maltodextrin, cellulose, starch, inulin, lactose, trehalose, dextrose, sodium citrate, sodium ascorbate, D-mannitol, L-cysteine, magnesium sulphate, sodium glutamate, histidine and marie acid.

8. The method according to claim 1 , wherein the probiotic composition is in the form of capsule, a powder, a suspension, or a tablet.

9. The method according to claim 1 , wherein the probiotic composition comprises the strain in an amount about 1x10⁹ colony forming units and maltodextrin.

10. The method according to claim 1 , wherein the probiotic composition is administered to the subject once daily for at least 12 weeks.

11 . The method according to claim 1 , wherein the subject is an obese or overweight human subject.

12. The method according to claim 1 , wherein the subject is an obese or overweight human subject under psychological stress.

13. The method according to claim 1 , wherein the subject has a Body Mass Index of about 25 to 30 or body fat% of at least 20% or waist circumference of at least 25 inches.

14. A method for the prevention and/or treatment of overweight, obesity and/or related diseases, comprising administering to a subject in need thereof the probiotic composition comprising:

(a) has a genome fingerprint of the correspondent deposited strain CECT 7481 shown in the pulsed field gel electrophoresis of FIG. 6(A)(B); and

15. The method according to claim 14, wherein the obesity related disease is selected from the group consisting of cardiovascular disease, atherosclerosis, hypertension, stroke, congestive heart failure, angina pectoris, type 2 diabetes mellitus, obesity-related hypoventilation, back and joint problems, nonalcoholic fatty liver disease, cirrhosis, metabolic syndrome, gastroesophageal reflux disease, reduced fertility, hypothyroidism, dyslipidemia, hyperinsulinemia, cholecystitis, cholelithiasis, osteoarthritis, gout, sleep apnea, polycystic ovary syndrome, pregnancy complications, psychological disorders, uric acid nephrolithiasis, stress urinary incontinence and cancer.

16. The method according to claim 15, wherein the obesity related disease is selected from the group consisting of type 2 diabetes mellitus, hypertension, liver cirrhosis, metabolic syndrome, and cardiovascular disease.