EP3104868A2

EP3104868A2 - Use of compositions comprising bifidobacterium animalis ssp. lactis lmg p-28149

Info

Publication number: EP3104868A2
Application number: EP15705283.8A
Authority: EP
Inventors: Johan QUINTENS; Jehan LIENART VAN LIDTH DE JEUDE; Corinne Grangette; Bruno Pot; Kassem MAKKI; Isabelle Wolowczuk; Jeanne ALARD; Véronique LEHRTER-VALENTI
Original assignee: Vesale Pharma SA; Institut Pasteur de Lille
Current assignee: VESALE PHARMA SA
Priority date: 2014-02-14
Filing date: 2015-02-13
Publication date: 2016-12-21
Also published as: JP2017506637A; BR112016018283A2; CA2938790A1; US20160354418A1; CN106535908A; RU2016133852A3; RU2016133852A; RU2673341C2

Abstract

A composition made from at least one probiotic for the use of same in the curative treatment of body weight gain in an overweight human and/or in the preventive treatment of body weight gain in a human who is overweight or has been overweight, said probiotic being Bifidobacterium animalis ssp. Iactis n° LMG P-28149.

Description

Composition comprising Bifidobacterium animalis sso. lactis

The present invention relates to a composition based on at least one probiotic for its use in the curative treatment of body weight gain in overweight humans and / or in the preventive treatment of body weight gain in the body. human being overweight or overweight.

For the purposes of the present invention, the term "overweight" means that the human has an abnormal and / or excessive accumulation of body fat, mainly abdominal, which presents a risk to his health. In the context of the present invention, the term "overweight" also includes the particular and extreme case of the state of obesity in humans.

By "being overweight" is meant in the sense of the present invention that ^humans underwent weight loss and has a weight considered normal (based on BMI or waist / hip) from a period between 3 and 5 years.

It is difficult to develop a simple index that measures overweight and obesity at the panel level of a given population. For example, the scale used to measure the index of overweight and obesity in children and adolescents, because their body experiences a number of physiological changes related to their growth, differs from the one used in 'adult. Therefore, depending on the age, the WHO (World Health Organization) makes available to the public, different methods of measuring body weight according to the age of the individual or the average age of the sample of individuals.

As an illustration, overweight can be evaluated in adults on the basis of Body Mass Index (BMI), which is a simple measure of weight compared to the size commonly used to estimate body weight. overweight and obesity in adults. It corresponds to the weight divided by the square of the size, expressed in kg / m ² . BMI has the advantage of being applicable to both sexes and all adult age groups. However, it should be considered as an approximate indication because it does not necessarily correspond to the same percentage of fat mass according to individuals.

In this context, WHO defines, based on BMI, the following threshold values for determining whether an adult is overweight or obese:

a normal state of weight corresponds to a BMI between 20 and 25; a state of overweight corresponds to a BMI between 25 and 30; and

- a state of obesity corresponds to a BMI equal to or greater than 30.

However, it should be noted that BMI gives no indication as to the distribution of abdominal fat versus subcutaneous fat.

This index, although it has the advantage of standardizing the classification for both sexes and all age groups of adult individuals, can preferably be interpreted in a complementary manner with another indicator which may be, for example, the measurement of the waist of the individual.

Thus, an adult male individual with a waist circumference greater than 100 cm is considered to be overweight. This threshold is 88 cm in the adult woman.

This parameter can be generalized to all age groups by measuring the waist circumference (A) / hip circumference (H).

Overweight is observed for a ratio greater than 1 in the male individual and greater than 0.85 in the female individual. The term "treatment" applies in the context of this invention since overweight in humans and in particular human obesity, has been recognized as a disease by the WHO.

For the purposes of the present invention, the term "curative" is intended to mean the action which aims to reduce the body weight of an overweight human and / or to reduce body weight gain in humans. Overweight. The curative treatment therefore involves the action that aims to achieve, preferably during and / or after diet, a body weight considered normal in humans, that is to say with a lower risk to health, in a predetermined range of values, based for example on the BMI, which is between 20 and 25, preferably equal to 22.

By the term "lesser risk to health", it is to be understood in the context of the present invention that, at a body weight considered normal, the risk of developing a metabolic syndrome is negligible.

In particular, the curative action is primarily aimed at decreasing the abdominal fat in the individual or at least a decrease in the accumulation of abdominal fat.

For the purposes of the present invention, the term "preventive" is intended to mean the action which aims at stabilizing the body weight of an overweight or overweight human being (i.e. has lost weight) and thus to prevent or limit body weight gain in overweight or overweight humans, preferably during and / or after diet. Preventive treatment also involves the action which aims to maintain, preferably during and / or after diet, a stable body weight, preferably considered normal in humans, within a predetermined constant range of values, based, for example , on BMI (as a reminder, between 20-25), but normal body weight can also be interpreted in light of the A / H ratio of less than 0.85 in the female individual, and less than 1, 00 in the male individual. Currently, overweight and obesity worldwide are responsible (because of associated diseases, such as type 2 diabetes or cardiovascular disease) of more deaths than underweight.

In particular, overweight and obesity have reached the proportions of a global epidemic (given that at least 3 million people die each year from overweight or obesity-related diseases) and are no longer limited to rich but now also reach low- and middle-income countries.

It has furthermore been demonstrated that a high BMI, sometimes together with a high A / H ratio, is a factor favoring, on the one hand, the metabolic syndrome which is defined in the context of the present invention as a combination of health risks: high blood pressure, hypertriglyceridemia, low HDL cholesterol, android obesity (eg, abdominal fat accumulation), and elevated blood glucose. These indicators are in fact a basis of definition that is common to the current three main known definitions: that of WHO (published in 1998 and amended in 1999), that of the National Cholesterol Education Program (NCEP-ATPIII) published in 2001, and that of the International Diabetes Federation published in 2005.

In practice, the metabolic syndrome is reflected in the following indicators, in a non-exhaustive manner: an abnormally high insulin level; type 2 diabetes; hypercholesterolemia (associated with a low level of "good" HDL cholesterol) hypertension a significant increase in body weight gain over time, especially for abdominal obesity hypertriglyceridemia; hepatic steatosis; the development of a systemic inflammatory state; and hypertrophy of adipocytes. In addition, the risk of chronic diseases is reflected in the following indicators, in a non-exhaustive way: musculoskeletal disorders, in particular osteoarthritis; or certain types of cancers.

The problem of uncontrolled body weight gain leading to overweight in humans is therefore a recognized societal problem with a significant impact on health worldwide.

It is in this perspective that WHO has established recommendations on diet and exercise in its global strategy to prevent excessive weight gain. Adopted by the World Health Assembly (WHA) in 2004, this strategy sets out the measures necessary to encourage people to follow a healthy diet and exercise regularly.

However, while this strategy is based on recommendations to prevent weight gain, it does not offer any guidance on the curative and preventive treatment of weight gain in overweight or overweight individuals at risk of recovery. weight. Indeed, currently, the problem of uncontrolled weight gain no longer targets only normoponderal individuals (that is, those who have a normal BMI and / or a normal waist circumference) who may gain weight, but especially individuals already overweight when the WHO indicates that nearly 1, 4 billion people aged 20 years and over are already overweight. Among them, more than 200 million men and nearly 300 million women are obese, and globally, more than one in ten adults in the world is obese.

There is, therefore, a real need for treatment that can reduce or prevent overweight and obesity in overweight humans. Preferably, this treatment must be low-binding when it is known that a main reason related to overweight and obesity is the lack of regular dieting associated with sometimes heavy treatment.

In particular, the present invention is part of a known cause-effect relationship that associates changes in the gut microbiota with the development of obesity (Ley et al., 2006. Nature, 444: 1022-1023; Nadal et al., 2008. Int J. Obes., 33 (7): 758-67).

Numerous studies have been conducted in this context and have fed the current state of the art including a wide range of documents mentioning the use of probiotic-based treatments in the context of overweight and obesity.

In particular, the microbiota has been shown to help keep the host healthy. Indeed, the balanced state of the microbiota contributes to the regulation of intestinal homeostasis and the immune balance, so that any imbalance of the microbiota is interpreted as contributing to the development of the metabolic syndrome, particularly in individuals with genetic predisposition to develop this syndrome (Parks et al., Cell Metab, 2012).

The highlighting of the microbiotic relationship - weight gain has been the driving force for a wide range of studies that have been the subject of various patent applications, the most relevant of which are discussed below.

It is first necessary to mention the international patent application WO2007043933 which proposes the use of the strains of Lactobacillus casei F19 and L acidophilus NCFB 1748, and Bifidobacterium lactis Bb12, in the form of fermented milks, to reduce the Appetite and fat deposits in certain tissues, and thus, control body weight, in humans. However, it seems that it is rather the joint action of calcium and milk proteins which is at the origin of the effect claimed in this application rather than the presence of the strains of bacteria mentioned above. In addition, this effect only targets the expression of genes related to the metabolism of the small intestine and therefore does not relate to the other organs and tissues involved in weight gain.

Next, the document US20100061967A1 also proposes the use of a composition of bacteria for modulating the expression of peptides regulating the satiety mechanism, this modulation taking place exclusively in the gastrointestinal tract.

International Patent Application WO2009153662 discloses the use of a composition based on Bifidobacte esa and Lactobacillus in the treatment of diabetes, a disease listed in the list of indicators associated with the metabolic syndrome induced by overweight or obesity. obesity, based exclusively on the ability of these microorganisms to reduce inflammation of peripheral tissues but without acting on the central nervous system, and thus for example on the mechanism of central regulation of satiety.

In addition, the document US20100150890 informs the use of bacterial probiotics in a composition for stimulating the function of the sympathetic nervous system so that the metabolism, and therefore the energy expenditure, is stimulated. However, it has been shown that sympathetic tone is also active in some obese individuals and thus its activation is not a reliable alternative in the treatment of overweight and obesity.

The patent application US20100111915 presents the generic use of a composition of bacterial probiotics as an alternative in the context of the prevention of obesity in children. According to US20100111915, this use is based on the bifidogenic effects of probiotics, although this document does not provide any tangible basis for establishing that the increase in the number of bifidobacteria in the intestine could be the cause of this preventive action. of the appearance of obesity in children. Document US20050112112 proposes the use of a composition of microorganisms generating sugar polymers, which are non-digestible by humans, from monosaccharides and disaccharides present in the gastrointestinal tract, thus reducing de facto absorption of sugars in the body.

Finally, the document JP 0306028 also provides an inhibitory action of the absorption by the body of cholesterol, and this, by the use of bifidobacteria combined with chitosan.

Unfortunately, the use of the compositions of the state of the art, if it allows a reduction of overweight or obesity and a reduction of the symptoms associated with the metabolic syndrome (we speak for example of remission), it does not allow a long-term maintenance of more than 3 to 5 years (eg healing) of body weight at the desired level of normalcy (ie, corresponding, for example, to a BMI between 20 and 25).

In the context of the present invention, it is necessary to distinguish the remission of healing. An individual is said to be in remission if, during medical examinations (measurement of weight, waist circumference, etc.), no more overweight is detected. But we only talk about healing after a certain amount of time, which varies depending on the type of overweight. In general, when the remission lasts for 3 or 5 years, it is considered that there is healing.

The present invention aims to overcome the lack of the state of the art by providing a composition as described in the beginning characterized in that said probiotic is Bifidobacterium animalis ssp. lactis No. LMG P-28149.

The strain according to the invention was deposited by the BCCM (Belgian Coordinated Collections of Micro-organisms) and the LMG (Laboratorium for Microbiology - Bacteriënverzameling) on January 27, 2014 according to the Budapest Treaty, under the number LMG P-28149. In the context of the present invention, the composition preferably comprises the probiotic is Bifidobacterium animalis ssp. lactis No. LMG P-28149 in the live state. The term "living state" means a concentration of living bacteria in the composition of between 10 ⁸ and 10 ¹³ cfu per gram of composition.

In the context of the present invention, it has been observed that this particular probiotic strain exhibits a significant positive action on satiety and also a restoration of the intestinal microbiota associated with the limitation of body weight gain.

In particular, the role of the presence of the

Bifidobacterium animalis ssp. lactis No. LMG P-28149 in the recolonization of the intestinal tract by Akkermansia muciniphila, a bacterium known for its role in the prevention of obesity in humans whose level is greatly reduced in the overweight or obese individual.

In addition, the anti-inflammatory and immunoregulatory action of the strain according to the invention which is associated with the restoration of the expression of the transcription factor PPARy, which is involved in the function of the proteins, has surprisingly been observed. regulatory T cells of white adipose tissue.

Thus, the composition according to the invention allows in particular the curative or preventive treatment of the disturbance of the intestinal microbiota and the inflammatory diseases associated with this disorder in overweight humans.

The present composition therefore advantageously targets the curative or preventive treatment of the metabolic syndrome related to overweight in humans.

It has thus been demonstrated, according to the present invention, that the composition based on Bifidobacterium animalis ssp. lactis No. LMG P-28149 has the following effects on the body, which are accompanied by a reduction in body weight gain under a high fat diet, preferably during and / or after a diet, or a stabilization of body weight: an improvement in insulin sensitivity; a reduction in the development of abdominal and subcutaneous fat; a reduction in the size of the adipocytes; a reduction in cellular infiltration of white adipose tissue, in particular by pro-inflammatory macrophages; a reduction of inflammatory markers; a limitation of steatosis of the liver; and a limitation or reduction in the rate of "bad" cholesterol.

As can be seen, the composition according to the invention comprising at least the probiotic Bifidobacterium animalis ssp. lactis no. LMG P-28149 affects various factors related to overweight.

In the context of the present invention, it has also been demonstrated the preventive and curative action on weight gain and the action on the metabolic syndrome in overweight humans, not only the specific strain Bifidobacterium animaiis ssp . lactis no. LMG P-28149, whether alive or not, but also compounds derived from this strain.

In this context, the composition according to the invention may further comprise at least one or a combination of at least two probiotic elements, each element being derived from Bifidobacterium animalis ssp. lactis No. LMG P-28149 and being selected from the group consisting of cell wall constituents, cell organelles, nucleic acids, cell membrane constituents, and cell metabolites.

By way of illustration and not limitation, the constituents of the cell wall are selected from the group consisting of peptidoglycans, proteins, polysaccharides, teichoic acid, or a combination thereof.

By way of illustration and without limitation, the metabolites are chosen from the group consisting of organic acids, inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, glycolipids, glycoproteins, vitamins, salts, metals, or a combination thereof.

In a particular embodiment, the composition according to the invention comprises butyrate, or at least one of its derivatives, and / or propionate, or at least one of its derivatives, the induction of said butyrate and / or said propionate being favored by Bifidobacterium animalis ssp. lactis No. LMG P-28149.

Optionally, the composition according to the invention comprises at least one additional probiotic, said additional probiotic being chosen from the group consisting of the following probiotics: Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes, Fibrobacter, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacterium, Lactobacillus, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Catenibacterium, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae (non-pathogenic), Faecalibacterium, Bacteroides , Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, and Clostridium, or a combination thereof.

Preferably, the composition according to the invention may furthermore comprise at least one fungus and / or yeast strain, said strain being chosen from the following group: Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor, and Penicillium.

Preferably, the composition according to the invention comprises a probiotic encapsulation vehicle in which said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic are encapsulated.

Advantageously, said fungus and / or said yeast are also encapsulated in said vehicle. Alternatively, the encapsulating vehicle comprises at least one substance selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel or a combination thereof.

Preferably, said at least one substance is a hydrocolloid.

Advantageously, the composition according to the invention comprises at least one nutritive source selected from the group consisting of a monosaccharide, a polysaccharide, an amino acid, a peptide, a protein, a vitamin , a yeast extract, a halide salt, an alkali metal or alkaline earth metal, an antioxidant, glycerol, zinc acetate, zinc chloride, zinc lactate, ascorbic acid , citric acid, vegetable oil, milk fat, or a combination thereof.

In a particular embodiment, the composition according to the invention is a symbiotic composition comprising at least one prebiotic.

Preferably, said at least one prebiotic is chosen, in a non-limiting manner, from the group consisting of oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, xylooligosaccharides, inulin or its derivatives, lactulose or of its derivatives, mannan-oligosaccharides, or a combination thereof.

Preferably, the composition according to the invention comprises a first enteric coating covering said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic. In particular, the first enteric coating is selected from the group consisting of ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers (such as Eudragit®), or a combination thereof. Alternatively, the composition further comprises a second outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel, cellulose, hemicellulose , ethyl cellulose, carboxymethyl cellulose, or a combination thereof.

In another possible embodiment, the composition according to the invention comprises one or more biocompatible excipients.

The composition according to the invention may be intended for the curative or preventive treatment of excessive body weight gain (that is to say considerably more than the weight gain that accompanies pregnancy) in the pregnant woman or the woman who has given birth or in the man in couvade or having been in couvade.

According to the invention, the composition may be in the form of a food composition, a food based on the composition or a dietary supplement comprising Bifidobacterium animalis ssp. lactis No. LMG P-28149, preferably in the living state.

For the purposes of the present invention, the term "food composition" is intended to mean a composition commonly found on the food market (for example, snacks, prepared meals, beverages, etc.). ).

For the purposes of the present invention, the term "food supplement" is intended to mean a foodstuff whose purpose is to provide an addition of nutrients or substances having a nutritional and / or physiological effect.

According to the invention, the composition may be provided for use and intake orally, or sublingually, but also respiratory, preferably nasal or bronchial, or rectal. The composition may also be a liquid injectable composition based on at least one element derived from the probiotic and intended to be injected subcutaneously.

Preferably, the composition according to the invention may be packaged in the form of tablets, globules, capsules, granules, powders, fluids, liquids, creams or sprays.

According to the invention, the composition can be used for the curative or preventive treatment of excessive body weight gain (that is to say largely exceeding the weight gain that accompanies pregnancy) in the pregnant woman or who has given birth or in the man in couvade or having been in couvade.

Other embodiments and uses of the therapeutic composition according to the invention are indicated in the appended claims.

The present invention also relates to a non-therapeutic cosmetic use of a composition comprising at least probiotic, in the overweight or overweight human, said probiotic being Bifidobactehum animalis ssp. lactis No. LMG P-28149.

Preferably, said composition for a non-therapeutic cosmetic use comprises at least one additional probiotic chosen from the group consisting of the following probiotics: Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes, Fibrobacter, Deferribacteres, Deinococcus , Thermus, Cyanobacteria, Methanobrevibacterium, Lactobacillus, Peptos eptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Catenibacterium, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae (non-pathogenic), Faecalibacterium, Bacteroides , Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, and Clostridium, or a combination thereof. Advantageously, said composition for a non-therapeutic cosmetic use comprises a strain of fungus and / or yeast selected from the group consisting of Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor , and Penicillium.

Preferentially, in said composition for a non-therapeutic cosmetic use, said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic are encapsulated in an encapsulation vehicle.

Advantageously, in said composition for a non-therapeutic cosmetic use, said encapsulation vehicle comprises at least one substance chosen from the group consisting of alginate, chitosan, pectin, pulluian, gelatin, carrageenan, agar gel or a combination thereof.

Advantageously, said composition for a non-therapeutic cosmetic use comprises at least one nutritive source selected from the group consisting of a monosaccharide, a polysaccharide, an amino acid, a peptide, a protein, a vitamin, a yeast extract, an alkali metal or alkaline earth metal halide salt, an antioxidant, glycerol, zinc acetate, zinc chloride, zinc lactate, ascorbic acid, citric acid, vegetable oil, milk fat, or a combination thereof.

Preferably, said composition for non-therapeutic cosmetic use further comprises at least one prebiotic, thereby forming a symbiotic composition.

Preferably, said composition for a non-therapeutic cosmetic use comprises a first enteric coating covering said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic. Advantageously, in said composition for a non-therapeutic cosmetic use, said first enteric coating is selected from the group consisting of ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, Eudragit®, or a combination of them.

Preferably, said composition for a non-therapeutic cosmetic use comprises a second outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel, cellulose, hemicellulose, ethyl cellulose, carboxymethyl cellulose, or a combination thereof.

Advantageously, said composition for a non-therapeutic cosmetic use further comprises one or more biocompatible excipients.

Other forms of non-therapeutic cosmetic use of the composition according to the invention are indicated in the appended claims.

The present invention also relates to a culture medium of the probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149 comprising at least one protein source and at least one source of carbohydrates, said culture medium being characterized in that it further comprises glutathione.

Glutathione is a tripeptide involved in maintaining the redox potential of cell cytoplasm and in a number of detoxification and elimination reactions of reactive oxygen species.

Surprisingly, it has been shown, in the context of the present invention, that such a culture medium comprising glutathione optimizes the efficacy of the strain LMG P-28149 of the probiotic Bifidobacterium animalis ssp. lactis, for which it has been observed a curative or preventive action on body weight gain in overweight or overweight humans.

Preferably, the culture medium according to the invention comprises glutathione in a concentration of between 20 and 30 g / l of culture medium.

Advantageously, according to the invention, said protein source of said culture medium is selected from the group consisting of a whey peptone, a casein peptone, a plant or bacterial peptone, and a combination of these. this.

Preferably, according to the invention, said source of carbohydrates of said culture medium comprises at least one sugar or a mixture of sugars, which are chosen from the group consisting of lactose, glucose, galactose, fructose and maltodextrin. , starch, trehalose, maltotriose, and a combination thereof.

Preferably, according to the invention, said culture medium further comprises at least one amino sugar, for example glucosamine or galactosamine.

Preferably, according to the invention, said culture medium further comprises at least one yeast extract.

Preferably, according to the invention, said culture medium further comprises at least one egg extract.

Other forms of the culture medium according to the invention are indicated in the appended claims.

The present invention also relates to a process for the fermentation manufacture of probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149, said method comprising at least one step of culturing said probiotic in a culture medium according to the invention.

Other forms of the process according to the invention are indicated in the appended claims. Other features and advantages of the invention will emerge from the description given below, by way of non-limiting example and with reference to the examples described below.

These examples show results obtained in mice which have been supplemented by results obtained in vitro from immune cells isolated from human blood.

Although the results of the tests given in Examples 1 to 4 below were obtained in the mouse, it is understood that similar results expected in humans.

In particular, it has been demonstrated in the context of the present invention, a strain-specific effect on body weight gain in the mammal, in particular in rodents.

Indeed, taking the LMG P-28149 strain of probiotic Bifldobacterium animalis ssp. Lactis, alone or in combination with at least one other probiotic, induces a decrease in body weight gain together with an improvement in inflammatory and metabolic parameters in the mammal, especially the overweight rodent, including insulin resistance. .

In particular, it has been demonstrated, in the context of the present invention, the metabolic protective effects of the composition based on the probiotic strain according to the invention which are moreover associated with a restoration, within the adipose tissues, of the expression of PPARy and cellular remodeling, and a protective effect against hepatic steatosis.

The composition based on the strain according to the invention also allows a modulation of the expression of the receptors bound to the transport of fatty acids, and in particular a restoration of the G protein-coupled receptors (GPR41 and GPR43) involved in the transport of short-chain fatty acids (SCFA), which are important players in the satiety mechanism induced by nutrient uptake. In addition, the positive action of the strain according to the invention on the size of the adipocytes and on the production of pro-inflammatory cytokines and chemokines (MCP-1, IL-6, TNF-alpha, etc.) is demonstrated. some of them are directly responsible for insulin resistance.

It has been observed that the strain according to the invention also makes it possible to restore the lipid profile and the glucose metabolism in the mammal, preventing the risk of developing diseases directly related to the metabolic syndrome, such as dyslipidemia (and therefore type diabetes). 2) and hyperglycemia.

Finally, the link between the taking of the probiotic strain according to the invention and the recolonization of the intestinal tract by YAkkermansia, a bacterium known for its role in the prevention of obesity in humans and greatly diminished, has been demonstrated. in the overweight or obese individual.

Figure 1 illustrates, in the mouse developing overweight and then obesity, the action of Ls33 on (A) the evolution of body weight gain (in%), (B) glucose tolerance (GT) ), (C) the weight (mass) of the epididymal adipose tissue (EWAT), and (D) the weight (mass) of the subcutaneous adipose tissue (SCWAT). The data are expressed on average (from 10 to 15 mice per group) + standard deviation to the mean (SEM). ** p <0.01; *** p <0.001. * corresponds to the comparison between the lipid-enriched diet (HFD) and the control diet (LFD) with identical interventional treatment (measurement of the effect of the diet).

Figure 2 illustrates in the developing mouse overweight and then obesity, the action of the Mix on (A) the change in body weight gain (in%), (B) the cumulative intake of food (in gram / day / mouse), (C) insulin tolerance (IT), and (D) GT. The data are expressed on average (from 5 to 14 mice per group) + standard deviation to the mean (SEM). ** p <0.01; *** p <0.001;^# p <0.05; ^m p>0.01;^## p <0.001. * corresponds to the comparison between the HFD diet and the LFD regimen with identical interventional treatment (measurement of the effect of the scheme). * corresponds to the comparison between the Mix vs the phosphate buffered saline (PBS) identical regime (measuring the effect of the probiotic or mixture of probiotics according to the invention).

Figure 3 illustrates in the developing mouse overweight and then obesity, the action of the Mix on (A) the weight (mass) of EWAT, (B) the weight (mass) of SCWAT, (C) the amount of leptin in the blood (ng / ml), (D) the amount of adiponectin in the blood (μ / τη \), (E) the histology of EWAT (sections representative of each of the experimental groups), and ( F) the size distribution of adipocytes in EWATs. The data are expressed on average (10 to 15 mice per group) + standard deviation to the mean (SEM). ** p <0.01; *** p <0.001;^# p <0.05; ^ O.01; ** p <0.001. Refers to the comparison between the HFD and LFD regimens with identical interventional treatment (measure of the effect of the plan). Corresponds to the comparison between the Mix vs the PBS with identical regime (measurement of the effect of the probiotic or mixture of probiotics according to the invention).

FIG. 4 illustrates in the mouse during the development of overweight then obesity, the action of the Mix on (A) the cellular composition of the EWATs (expression analysis of monocyte / macrophage specific genes (F4 / 80, CD68 , CD11b, CD11c), regulatory T cells (FoxP3) and chemokine MCP-1), (B) the presence of macrophages in EWATs (F4 / 80 specific immunofluorescent staining and quantification in terms of integrated density [IntDen] ), (C) inflammation of the EWATs (gene expression analysis of pro-inflammatory cytokines Tnfa, IL-1a, IL-6 and IL-17), and (D) expression of PPAR gamma (PPARy) at the level of messenger RNA (left, gene expression) and protein (center and right, westem-blot and quantification [arbitrary unit: AU]). The data are expressed on average (from 5 to 14 mice per group) + standard deviation to the mean (SEM). ** p <0.01; ^* ** p <0.001; ^m p <0.01;^# p <0.001. Corresponds to the comparison between the HFD diet and the identical interventional LFD diet (measurement of the effect of the diet), "corresponds to the comparison between the Mix vs the PBS with identical diet (measurement of the effect of the probiotic or mixture of probiotics.

FIG. 5 illustrates in the mouse during the development of overweight then obesity, the action of the Mix on (A) factors involved in lipid metabolism (expression analysis of the FABP1, APO Cil, and CD36 genes) (B) short-chain fatty acid (SCCC or SCFA) receptors, by expression analysis of the GPR41 and GPR43 genes). The data are expressed on average (from 5 to 14 mice per group) + standard deviation to the mean (SEM). ** p <0.01; *** p <0.001; ** p <0.01;^# p <0.001. Corresponds to the comparison between the HFD diet and the LFD regimen with identical interventional treatment (measurement of the effect of the diet), "corresponds to the comparison between the Mix versus the PBS at identical diet (measurement of the effect of the probiotic or mixture probiotics).

Figure 5 (C) illustrates the level of production of total SCFA (SCFA) (left) and proportional levels of acetate, butyrate and propionate (right) after 24 and 48h incubation with the probiotic mix mix in the Model simulating the intestinal ecosystem (SHIME), in vitro. *** compared to time 0.

6 illustrates in mice overweight being developed and obesity, the effect of Mix on (A) the composition of the intestinal microbiota and in particular the population of Bifidobacteria and ¹ Akkermansia muciniphila. (B) The Bifidobacterium species detected in HFD mice treated or not with the mix was determined by TGGE analysis for 5 mice representative of the HFD-PBS and HFD-Mix groups. The markers M1 or M2 correspond to the mixtures of the indicated strains. * P <0.05; ** p <0.01;^# p <0 _s 05; "" PO.01. Corresponds to the comparison between HFD vs. LFD with identical interventional treatment (measure of plan effect), "corresponds to the comparison between the Mix vs Se PBS at identical regime (measurement of the effect of the probiotic or mixture of probiotics according to the invention),

Figure 7 illustrates in the developing mouse overweight and then obesity, the action of the Mix on (A) the weight (mass) of the pancreas, liver and spleen, (B) the accumulation of droplets lipid (steatosis, see histological sections representative of each of the experimental groups), and (C) different markers of inflammation or involved in lipid metabolism or insulin response (mcp-1 expression analysis, IL-6, TNFα, IL-10, IL-17, srebp-1c, APOCII Fabp1 and IRS2). The data are expressed on average (from 5 to 14 mice per group) + standard deviation to the mean (SEM). ** p <0.01; *** p <0.001;^## p <0.01;^# p <0.001;^#p> 0.0001. Refers to the comparison between the HFD and LFD regimens with identical interventional treatment (measure of the effect of the plan). * corresponds to the comparison between the Mix vs the PBS at identical speed (measurement of the effect of the probiotic or mixture of probiotics according to the invention).

Figures 8a and 8b illustrate the impact of probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149 according to the invention on the weight gain over time depending on whether the probiotic was cultured (FIG. 8a) or uncultivated (FIG. 8b) in a culture medium according to the invention. LFD = low fat diet; HFD = high fat diet; HFD B. lactis = fat-rich diet associated with taking the composition according to the invention when the probiotic is cultured in the presence of glutathione (FIG. 8a) or in the absence of glutathione (FIG. 8b).

The following protocol was observed to harvest the in vivo results discussed in Examples 1 to 4 which follow.

In vivo protocols

Mice, strains of bacteria, and réaimes

The tests were performed on male C57BL / 6J mice, 5 weeks old at the beginning of the experiment. The Ls33 strain of Lactobacillus L salivarius was provided by Danisco (Madison, WI, USA).

The mixture of probiotics (referenced as Mix in the examples which follow) comprises two different strains: a strain of L rhamnosus LMG S-28148 and a strain of Bifidobacterium animalis ssp. lactis No. LMG P-28149 in a cfu (colony-forming units) ratio of 1: 1 (10 ⁹ total cfu). The diets imposed on the mice come in a first low-fat diet (LFD: D12450B, 10% kcal from fat) and a second high-fat diet (HFD: D12492, 60% kcal from fat) . The diets were provided by the Society Research Diet.

Animal Treatment Protocol

At 5 consecutive days per week, each mouse was orally administered with 30 μl of Ls33 - 10 ⁹ cfu (colony-forming units or, the number of colony-forming units present in the oral composition) in sterilized water ( ½0) (LS33-H ₂ 0), sterilized water (group H ₂ 0) or mixture of probiotics (mix group, and 5 August ¹⁰ cfu for each strain) in phosphate buffered saline (PBS) (mix group -PBS or PBS group).

After one week of treatment, mice treated with Ls33, water, Mix, or PBS were randomly assigned to LFD (n = 5 per group) or HFD (n = 15 per group). Body weight and food intake were recorded weekly.

At sacrifice, blood, epididymal white (EWAT) and subcutaneous adipose tissue (SCWAT), liver, spleen, small intestine, and pancreas were collected. Insulin and glucose tolerance tests Glucose tolerance (GTT) and insulin (ITT) tests were performed after 12 and 14 weeks of diet, respectively. The animals were fasted for 6 hours before administration of intraperitoneal (IP) glucose (D-glucose, g / kg body weight) (GTT) or insulin (0.75 lU / kg). body weight) (ITT). Glucose levels in the blood were measured by a commercially available automatic glucose meter (eg, a ACCU-CHEK® performa) on a blood sample taken from the tail before glucose injection (GTT) or insulin (ITT) and at different times after glucose injection (GTT) or insulin (ITT).

Blood tests

Plasma levels of leptin, adiponectin, MCP-, and insulin were measured using commercially available ELISA kits. Levels of non-esterified fatty acids (NEFA), triglycerides, glycerol, HDL cholesterol, and LDL cholesterol were determined using commercially available assay kits (for example, the Abcam kit developed by Cambridge , GB). Histologic and immunohistochemical analyzes

The liver and white adipose tissue samples (EWAT) were fixed in a solution of 4% paraformaldehyde, then paraffin-embedded and finally cut, deposited on a slide and then stained with hematoxylin eosin (H & E). Morphometric analysis of white adipose tissue (EWAT), at least 10 fields (representing about 100 adipocytes) per section was performed using the Image J imaging program (NIH image, National Center for Biotechnology Information). Analyzes of gene expression Total RNA fragments of white adipose tissue, liver and intestine were extracted (according to the method known to those skilled in the art) and then retro-transcribed (a quantity of 1 μg was retranscribed for each of the above-mentioned tissue categories). The quantitative real-time PCR (Polymerase Chain Reaction) technique (RT-qPCR) was carried out according to a protocol known to those skilled in the art.

Statistical analyzes

The data are expressed as mean ± standard deviation (SEM). Statistical analyzes were performed using the Kruskal Wallis test followed by the Mann-Whitney U test. Differences between the experimental groups are considered to be statistically significant when the p value is less than 0.05.

The following protocol was observed to harvest the in vitro results discussed in Examples 5 and 6.

In vitro protocol

Human microbial ecosystem simulator - SHIME model

The SHIME reactor which simulates the human gastrointestinal tract was set up according to a procedure well known to those skilled in the art.

The Mix was added to the reactor and short chain fatty acid (SCCC or SCFA) productions were measured at time t = 0, t = 24 h, and t = 48h incubation at 37 ° C. C under anaerobic atmosphere.

Evaluation of the anti-inflammatory effect of the strain Bifidobacterium animalis ssp. lactis no. LMG P-28149 on human blood cells

Fresh human blood, obtained from four healthy donors, was diluted 1: 1 with PBS-Ca (GIBCO), deposited on a layer of Ficoil (GIBCO) and centrifuged at 400 xg for 30 minutes at 20 ° C.

Peripheral blood mononuclear cells (PBMC) were isolated from the blood after centrifugation and suspended in a final volume of 50 ml using PBS-Ca, and then washed three times at 750 xg for 10 minutes at 20 ° C. The PBMCs were then resuspended in RPMI medium (GIBCO), supplemented with 10% (v / v) decomplemented calf serum (serum heated at 56 ° C for 30 minutes), 1% (w / v) L- glutamine (GIBCO), and gentamycin (30pg / ml) (GIBCO). PBMCs were counted and their number adjusted to 2 x 10 ⁶ cells per ml. The preparation is dispensed (1 ml) into 24-well culture plates.

Example 1: Ls33 vs Mix

This example illustrates the impact of Ls33 on the development of overweight and obesity in mice.

Figure 1 illustrates, for a course of 15 weeks in Ls33 or water and a low fat diet (LFD) diet or high fat diet (HFD), the following indicators:

(A) the change in body weight gain, expressed as a percentage of the weight measured at day d = 0;

(B) glucose tolerance test (GTT) performed after 12 weeks of dieting. Glucose levels (in mg / dl) were measured in the mice, directly after a fasting period of 6 hours, at the times t (in min) indicated on the graph, after intraperitoneal injection (IP) of glucose (which corresponds to the time t = 0).

(C) the weight (mass) of epididymal adipose tissue (EWAT) (in g) after 15 weeks of diet (weighed during sacrifice);

- (D) the weight (mass) of the subcutaneous adipose tissue

(SCWAT) (in g) after 15 weeks of dieting (weighed at sacrifice). As the results presented in this figure show, the Ls33 strain, despite its anti-inflammatory activity demonstrated in vitro and in other pathological models (ie intestinal inflammations), shows no effect (positive or negative) on the various indicators A at D.

Conversely, administration of the Mix mixture shows significant protective effects. These results are illustrated in Figure 2.

This figure illustrates, after 17 weeks of treatment with the mixture of probiotics (Mix) or PBS and for a low-fat diet (LFD) or high-fat diet (HFD), the corresponding results:

(A) the change in body weight gain (expressed as a percentage of the weight measured at d = 0);

(B) cumulative food intake per mouse per day (g / day / mouse);

(C) the insulin tolerance test (ITT) performed 14 weeks after starting the diet: blood glucose levels were measured after intraperitoneal injection (IP) of insulin. The results show normalized levels (as a% of glucose levels measured before injection) + SEM and averages + SEM of AUC values for each normalized glucose curve after insulin injection ;

(D) Glucose Tolerance Test (GTT) performed after 12 weeks of regimen. Glucose levels (in mg / dl) were measured in mice after IP glucose injection and AUC values were calculated.

For the group of HFD-Mix mice, a lower body weight gain (gain of 80.97% + 4.96%) was observed than that obtained in the HFD-PBS group (gain of 113.51% + 4.89%) (see Figure 2A). As shown in FIG. 2B, the mice in the HFD group treated with the mixture of Mix probiotics showed a decrease in food intake (Food intake, FI). Interestingly, the beneficial effects observed on body weight gain and the IF index appeared rapidly after the start of treatment (from the fourth week).

Mix administration improves glucose homeostasis for the HFD group as shown by the lower glucose and insulin levels in Table 1 below:

Table 1

Groups / LFD-PBS LFD-Mix HFD-PBS HFD-Mix

indicators

Glucose (mg dt) 180.4 ± 30.88 160.3 ± 28.96 231, 67 ± 21, 7 * 198.43 ± 15 **

Insulin (ng ml) 0.72 ± 0.37 0.98 ± 0.24 2.96 ± 0.57 * 1, 57 ± 0.23 *

NEFA (mmol / l) 0.85 ± 0.06 1, 07 ± 0.07 1, 06 ± 0.03 0.9 ± 0.08

Glycerol (mmol / l) 0.13 ± 0.06 0.24 ± 0.05 0.38 ± 0.1 0.34 ± 0.05

Triglyceride (mmol / l) 0.4 ± 0.009 0.46 ± 0.02 0.41 ± 0.03 0.41 ± 0.03

Cholesterol (mmol / l) 1, 35 ± 0.05 1, 33 ± 0.05 1, 94 ± 0.04 *** 1, 72 ± 0.3 ***

HDL (mmol / 1) 1, 09 ± 0.04 1, 07 ± 0.04 1, 5 ± 0.01 *** 1, 35 ± 0.02 ***

LDL (mmol / l) 0.25 ± 0.01 0.26 ± 0.01 0.45 ± 0.04 *** 0.36 ± 0.02 ***

The data in this table are expressed as mean ± SEM (corresponding to 5 and 14 mice per group). * P <0.05; *** p <0.001; * p <0.05; ** p <0.01; *** P <0.001. * corresponds to the comparison between the HFD system and the LFD system with identical interventional treatment (measure of the effect of the plan). * corresponds to the comparison between the Mix vs the PBS at identical speed (measurement of the effect of the probiotic or the mixture of probiotics).

Moreover, as shown in Table 1, if the effects of Mix remain limited to NEFA, glycerol and triglyceride levels, it demonstrates a cholesterol lowering action since it is associated with a decrease in total cholesterol and HDL cholesterol levels. .

In addition, the HOMA-IR index (fasting insulin level / fasting glucose level) / 22.5) was significantly reduced in mice of the HDF-Mix group, compared to the control group (32.04 + 5.86 vs 81, 57 + 19.63; p <0.01), indicating improved insulin sensitivity, confirmed by the results of the insulin tolerance test (see Figure 2C).

Similarly, the mice treated with the Mix are less intolerant to glucose injections (see FIG. 2D).

Example 2: Action of the Mix on the inflammation of white adipose tissues

Figure 3 illustrates the effects of the mix on:

(A) the weight (mass) of the EWAT (in g);

- (B) the weight (mass) of the SCWAT (in g);

(C) the amounts of leptin in the blood (in ng / ml) measured by ELISA (enzyme immunoassay method) in the serum of mice previously fasted for 6 hours;

(D) the amounts of adiponectin in the blood (in μg / ml) measured by the ELISA method in the serum of mice previously fasted for 6 hours;

(E) Epididymal adipose tissue histology (presentation of hematoxylin and eosin stained sections representative of each of the experimental groups). The scale is 100 μιτι. Black arrows indicate the presence of cellular infiltration; and

(F) the size distribution of the EWAT adipocytes. The results are presented in% of adipocytes by size class (0-20 μιη, 20-40, etc.), while Figure 7 illustrates the action of the Mix on:

- (A) the weight (mass) of the pancreas, liver, and spleen (in g);

(B) the accumulation of lipid droplets ^(/ .e. Steatosis) in the liver (presentation of sections stained with H & E, representative of each of the experimental groups). The scale is 00 μιτι; and (C) the expression of genes encoding pro and anti-inflammatory cytokines and receptors involved in the transport and metabolism of lipids, in the liver.

In comparison with the HFD control groups, the HFD-Mix group shows a marked decrease in EWAT and SCWAT fat masses (see Figures 3A and 3B).

A marked decrease in the weights (masses) of the pancreas, liver, and spleen is also observed in individuals in the HFD-Mix group (see Figure 7A).

In addition, while the mice of the HFD control group developed hepatic steatosis, the administration of the mixture of probiotics Mix makes it possible to limit the presence of lipid droplets in the tissues (see Figure 7B).

Consistent with decreasing white adipose tissue mass, leptin blood levels appear to be significantly lower in the HFD-Mix group (see Figure 3C), while adiponectin levels tend to be higher (see figure 3D).

The histology of epididymal adipose tissue showed a higher density in small adipocytes in mice of the HFD-Mix group (see Figure 3E and 3F).

It should be noted that, in the mice in the control group, the adipose tissues are markedly infiltrated by cells surrounding the adipocytes (marked by an arrow in FIG. 3E), the tissue samples taken from the mice of the group having received the probiotic mix are less marked by this infiltration.

In the development of obesity, macrophages are recruited into white adipose tissue while FoxP3 ⁺ CD4 ⁺ regulatory T cells leave these tissues.

Figure 4 illustrates in overweight mice the action of the Mix on inflammation of adipose tissue. As shown in Figure 4A, in comparison with the control LFD tissues, the expression levels of several specific markers of monocytes / macrophages (F4 / 80, Cd68, Cd11b, and Cd11c) are increased in the mice of the HFD group then that the rates relative to the Foxp3 marker are decreased.

Administration of the mix probiotic mixture significantly decreases HFD-induced expression of monocyte / macrophage markers and increases expression levels of the Foxp3 marker.

The impact of the Mix mixture, and therefore of the probiotic strain according to the invention, on the recruitment of macrophages in adipose tissue is supported by the results illustrated in FIG. 4B.

In accordance with the decrease in the recruitment of macrophages together with an increase in the accumulation of anti-inflammatory Treg cells, it is shown in this example that the epididymal adipose tissues in the mice treated with the Mix mixture have a lower level of inflammation than the control tissues, as demonstrated by the reduced levels of expression for the messenger RNAs specific for Γ II6, Tnfa, ΓΙΜα, and ΓΙΙ-17 (see Figure 4C).

In addition, it is shown that the Mix-based treatment limits the decrease in PPARy (both at the level of the messenger RNA and the protein) induced by the HFD diet (see FIG. 4D). Example 3: Action of the Mix on Lipid Metabolism of the Small Intestine and the Production of Short Chain Fatty Acids (AGCC / SCFA)

It is demonstrated in this example that gene expression levels (GPR41 and GPR43) that are responsible for the short-chain fatty acid (SCFAs) transport are decreased in the small intestine of the submitted mice. to the HFD scheme, which indicates that in the HFD group, the AGCCs are They are able to act positively since the latter are less detected, while they are significantly increased in animals treated with the Mix mixture under the HFD regime (see FIG. 5B).

Conversely, the administration of the Mix has the effect of limiting the increase of genes involved in lipid metabolism induced by a fatty diet (see Figure 5A).

Example 4: Mix action on the microbiota and level of colonization by Akkermansia muciniphila

An analysis of certain bacteria of the microbiota was carried out by quantitative PCR (qPCR) from the caecal content of the mice that were subjected to the HFD diet, with or without a Mix intake, and secondly, mice subjected to the diet. LFD jointly or not to take a Mix.

The results illustrated in FIG. 6 show a change in the composition of the microbiota of the HFD-Mix mice, with in particular a restoration of the level of colonization by Akkermansia muciniphila in the mice subjected to the HFD diet and treated with the probiotic strain or the mixture probiotics according to the invention.

Example 5: action of the Mix on the production of AGCC: use of the SHIME model

In this example, a dynamic in vitro model of gut simulation, which is intended to replicate the microbial ecosystem of the human gut, has been employed.

As shown by the results obtained from this simulation (see FIG. 5C), the use of the strain LMG P-28149 of the probiotic Bifidobacterium animalis ssp. Lactis allows an increase, within 48 hours after inoculation, of the production of total SCCCs (total SCFAs), favoring the production in the intestine of butyrate and propionate, two metabolites that promote satiety induced by taking of nutrients in humans. The levels of butyrate and propionate produced in the ascending colon associated with the SHIME reactor before (T0) and after (T24h and T48h) incubation of the Mix mixture according to the invention are shown in Table 2. These data are expressed in mmol / L + SED .

Table 2

Period / T0 T24h T48h

products

Acetate 3.53 ± 0.38 10.96 ± 0.91 3.10 ± 0.16

Propionate 1, 10 ± 0.03 3.87 ± 0.51 7.07 ± 0.16

Isobutyrate 0.00 ± 0.00 0.24 ± 0.05 1.30 ± 0.05

Butyrate 0.69 ± 0.07 3.25 ± 0.27 12.27 ± 0.22

Isovalerate 0.11 ± 0.00 0.50 ± 0.05 2.44 ± 0.04

Isocaproate 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Caproate 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Total 5, 44 ± 0.33 18.84 + 1.46 26.19 ± 0.36

Demonstration of the importance of glutathione in the culture medium of the strain Bifidobacterium animalis ssp. lactis No. LMG P-28149

Figures 8a and 8b illustrate the impact of probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149 according to the invention on the weight gain over time depending on whether the probiotic was cultured (FIG. 8a) or uncultivated (FIG. 8b) in a culture medium according to the invention.

As can be seen by comparing FIGS. 8a and 8b, and more particularly the HFD B. lactis curves, when a high-fat diet is associated with a setting of the composition according to the invention in which the probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149 is obtained by culturing in a medium of culture comprising glutathione, a significant decrease in weight gain over time is observed (FIG. 8a), which is not the case when taking a composition according to the invention which comprises the probiotic obtained by placing in culture in a culture medium not comprising glutathione (FIG. 8b).

It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made without departing from the scope of the appended claims.

Abreviations list

AGCC (SCFA): Short Chain Fatty Acid

EWAT: epididymal white adipose tissue

GT (T): (Test) glucose tolerance

HDL: High density lipids

H & E: Hematoxylin and Eosin

HFD: High fat diet

BMI: Body Mass Index

(HOMA) -IR: (homeostatic model) to appreciate insulin resistance

IT (T): (Test) insulin tolerance

(V) LDL: Lipid at (very) low density

LFD: Low fat diet

NEFA: Non-esterified fatty acid

PBS: Phosphate phosphate buffer

PBMC: Mononuclear peripheral blood cells

SCWAT: White subcutaneous adipose tissue

SEM: Standard deviation

SHIME: Simulator of the Human Intestinal Microbial Ecosystem

WAT: White adipose tissue

Claims

1. A composition based on at least one probiotic for use in the curative treatment of body weight gain in overweight humans and / or in the preventive treatment of body weight gain in overweight humans or having been overweight, characterized in that said probiotic is Bifidobacterium animalis ssp. lactis No. LMG P-28149.

2. Composition according to claim 1, for use according to claim 1, comprising at least one additional probiotic chosen from the group consisting of the following probiotics: Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes, Fibrobacters , Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacterium, Lactobacillus, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Catenibacterium, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae (non-pathogenic), Faecalibacterium, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, and Clostridium, or a combination thereof.

The composition of claim 1 or 2 for use according to claim 1, comprising a fungus and / or yeast strain selected from the group consisting of Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergilius, Rhizopus, Mucor, and Penicillium.

The composition of any preceding claim for use according to claim 1, wherein said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic are encapsulated in an encapsulation vehicle.

The composition according to claim 4, for use according to claim 1, wherein said encapsulating vehicle comprises at least one substance selected from the group consisting of aiginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel or a combination thereof.

A composition according to any one of the preceding claims for use according to claim 1, comprising at least one nutrient source selected from the group consisting of monosaccharide, polysaccharide, amino acid, peptide. , a protein, a vitamin, a yeast extract, an alkali or alkaline earth metal halide salt, an antioxidant, glycerol, zinc acetate, zinc chloride, lactate zinc, ascorbic acid, citric acid, vegetable oil, milk fat, or a combination thereof.

The composition of any of the preceding claims for use according to claim 1, further comprising at least one prebiotic, thereby forming a symbiotic composition.

The composition of any one of claims 1 to 8 for use according to claim 1, comprising a first enteric coating covering said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic.

The composition of claim 8 for use according to claim 1, wherein said first enteric coating is selected from the group consisting of ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, Eudragit® , or a combination thereof.

The composition of any of the preceding claims for use according to claim 1 comprising a second outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel, cellulose, hemicellulose, ethyl cellulose, carboxymethyl cellulose, or a combination of those -this.

A composition according to any one of the preceding claims for use according to claim 1, further comprising one or more biocompatible excipients.

12. Non-therapeutic cosmetic use of a composition comprising at least probiotic, in the overweight or overweight human, said probiotic being Bifidobacterium animalis ssp. lactis No. LMG P-28149.

13. Non-therapeutic cosmetic use of a composition according to claim 12, said composition comprising at least one additional probiotic chosen from the group consisting of the following probiotics: Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes , Fibrobacteria, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacterium, Lactobacillus, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Catenibacterium, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae (nonpathogenic ), Faecalibacterium, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, and Clostridium, or a combination thereof.

14. Non-therapeutic cosmetic use of a composition according to claim 12 or 13, said composition comprising a fungus and / or yeast strain selected from the group consisting of Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor, and Penicillium.

15. Non-therapeutic cosmetic use of a composition according to any one of claims 12 to 14, wherein said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic are encapsulated in an encapsulation vehicle.

16. The non-therapeutic cosmetic use of a composition according to claim 15, wherein said encapsulating vehicle comprises at least one substance selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel or a combination thereof.

17. Non-therapeutic cosmetic use of a composition according to any one of claims 12 to 16, said composition comprising at least one nutritive source selected from the group consisting of a monosaccharide, a polysaccharide, an amino acid, a peptide, a protein, a vitamin, a yeast extract, an alkali or alkaline earth metal halide salt, an antioxidant, glycerol, zinc acetate, zinc, zinc lactate, ascorbic acid, citric acid, vegetable oil, milk fat, or a combination thereof.

18. Non-therapeutic cosmetic use of a composition according to any one of claims 12 to 17, said composition further comprising at least one prebiotic, thus forming a symbiotic composition.

19. Non-therapeutic cosmetic use of a composition according to any one of claims 12 to 18, said composition comprising a first enteric coating covering said Bifidobacterium animalis ssp. lactis No. LMG P-28149 and optionally said at least one additional probiotic.

20. The non-therapeutic cosmetic use of a composition according to claim 19, wherein said first Enteric coating is selected from the group consisting of ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, udragit®, or a combination thereof.

21. A non-therapeutic cosmetic use of a composition according to any one of claims 12 to 20, said composition comprising a second outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar gel, cellulose, hemicellulose, ethyl cellulose, carboxymethyl cellulose, or a combination thereof.

22. Non-therapeutic cosmetic use of a composition according to any one of claims 12 to 21, said composition further comprising one or more biocompatible excipients.

23. Culture medium of probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149 comprising at least one protein source and at least one carbohydrate source, said culture medium being characterized in that it further comprises glutathione.

24. Culture medium according to claim 23, characterized in that the glutathione is present in a concentration of between 20 and 30 g / l of culture medium.

25. Culture medium according to claim 23 or 24, characterized in that said source of carbohydrates comprises at least one sugar or a mixture of sugars, which are chosen from the group consisting of lactose, glucose, galactose, fructose, maltodextrin, starch, trehalose, maltotriose, and a combination thereof.

26. Culture medium according to any one of claims 23 to 25, characterized in that it further comprises at least one amino sugar, for example glucosamine or gaiactosamine.

27. Culture medium according to any one of claims 23 to 26, characterized in that it further comprises at least one yeast extract.

28. Fermentation process of probiotic Bifidobacterium animalis ssp. lactis No. LMG P-28149, said method comprising at least one step of culturing said probiotic in a culture medium according to any one of claims 23 to 27.