WO2018104336A1

WO2018104336A1 - Lactic acid bacteria of use for treating metabolic diseases, especially type 2 diabetes

Info

Publication number: WO2018104336A1
Application number: PCT/EP2017/081575
Authority: WO
Inventors: Hubert Vidal; Jennifer RIEUSSET; Kassem MAKKI; Maria-Elena MARTINO; Gilles STORELLI; François LEULIER
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique (Cnrs); Université Claude Bernard - Lyon 1; Ens - Ecole Normale Supérieure de Lyon; Institut National Des Sciences Appliquées De Lyon
Priority date: 2016-12-05
Filing date: 2017-12-05
Publication date: 2018-06-14
Also published as: FR3059678A1

Abstract

The invention relates to the use of lactic acid bacteria for treating metabolic diseases, especially type 2 diabetes, and also to a method for screening bacteria capable of improving metabolic health and making it possible to decrease, or slow down, the progression of metabolic diseases. Using drosophila and mouse models, the inventors have demonstrated the beneficial effects of some bacterial strains on the metabolism via the measurement of several parameters, especially the oral glucose test. More particularly, the invention relates to a method for screening bacteria having intestinal tropism in a mammal, in which initially axenic drosophila larvae and/or mice are monocolonized with the bacterial strain to be tested, and these larvae are raised on a hypercaloric nutritional medium, which makes it possible to determine a favourable impact of the bacteria in the context of a hypercaloric diet and to infer a positive effect on metabolic health.

Description

Lactic Bacteria Useful for Treating Metabolic Diseases, Including Type 2 Diabetes

The present invention relates to a method for screening bacteria capable of improving the metabolic health, especially of the adult, and making it possible to reduce or slow the progression of metabolic diseases, especially those related to diet, such as diabetes mellitus. type 2. The present invention also relates to the use of certain lactic acid bacteria to improve the metabolic health, especially of the adult, and to prevent or treat metabolic diseases, especially those related to diet, such as diabetes type 2.

Described as "an additional organ", the intestinal microbial community (or intestinal microbiota) plays a key beneficial role for the host by exerting many biological functions, such as aid in the efficiency of digestion, substrate metabolism , the fight against pathogens, or the establishment and homeostasis of immune responses.

Defined in 2001 by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), probiotics are "living microorganisms that, when ingested in sufficient quantity, have positive effects on health, beyond the traditional nutritional effects.

WO2015173386 describes the combined use of a Drosophila model and a mouse model for screening bacterial strains to promote juvenile human and animal growth in case of undernutrition.

Pasco and Leopold, Plos One 2012, vol 7, 5, e36583, have shown that a hypercaloric diet leads in Drosophila larvae to metabolic alterations resembling the effects of insulin-dependent diabetes and a delay in maturation manifesting themselves. by a metamorphosis entry delay (transition from the larval stage to the pupal stage). The authors conclude that Drosophila could serve as a model for screening genes that predispose to the development of insulin resistance.

The inventors have found that, unexpectedly, bacteria could have a favorable effect making it possible to improve certain metabolic parameters, can be identified on a Drosophila model and confirmed on a mammalian model subjected to a hypercaloric diet (high sugar intake). . The Drosophila model described below is indeed sensitive to these bacteria and this measurably and possibly correlated with a measurable effect on a mammal subjected to a high calorie diet. The inventors furthermore, strains of bacteria selected on these models unexpectedly have comparable activity in Drosophila and mammals, with a favorable effect on the deleterious effects of a hypercaloric diet, both in Drosophila and in the mouse.

The invention thus relates to a method for screening intestinal tropism bacteria in a mammal, including humans, using an axenic Drosophila model. By "axenic" organism is meant an organism (e.g. Drosophila at a stage of its development) raised in an environment devoid of microorganisms and therefore devoid of intestinal flora. In accordance with the invention, this axenic model will then be inoculated with the bacterial strain to be tested, rendering it monoxenic for this strain, and its evolution will be compared to that of a control or reference.

The bacterium has "intestinal tropism" in a mammal, which means that the bacterium has the ability to pass the gastric barrier in this mammal, either naturally or by being administered in a gastro-protected formulation, and is able to persist in the body. 'intestine. This intestinal tropism will allow a bacterium selected according to the invention to produce a favorable effect on metabolic health.

The term "monoxenic" organism means an organism (e.g. Drosophila at a stage of its development) of axenic origin, raised in the presence of a single microorganism and therefore carrying this single microorganism as an intestinal flora.

Advantageously, the strains that responded positively to the Drosophila model test are then tested on a mammalian model, for example a mouse model, preferably having a conventional status, namely a normal (whole) microbiota.

The invention thus proposes a method for screening intestinal tropism bacteria, in which initially initially axenic drosophila embryos are monocolonized, by the bacterial strain to be tested, thanks to which we obtain monoxenic larvae for this bacterium. These larvae are raised with a hypercaloric nutritional medium, that is to say with a high sugar content. At least one larval maturation parameter is measured, and it is determined whether the bacterium has had a significantly favorable effect on the maturation parameter. If appropriate, it is concluded that said strain has a favorable effect on the maturation parameter.

In particular, a favorable effect can be concluded by comparison with pre-established reference data and / or data generated in parallel on axenic or monocolonized larvae by a reference strain. A favorable effect is thus retained when the monocolonized larvae by the strain tested have a maturation parameter significantly improved compared to a control. In particular, control may consist of high axenic larvae under similar nutritional conditions.

A favorable effect is thus retained when the monocolonized larvae by the tested strain have a maturation parameter that is substantially equal to or better than a reference. A reference may especially consist of monocolonized larvae with a reference strain which itself presents a maturation parameter significantly improved over a control corresponding to high axenic larvae under similar nutritional conditions.

It is also possible to associate a reference and a control.

The data obtained with the control group and / or with the reference group may have been obtained previously or, preferably, are generated in parallel, advantageously from the same batch of embryos as that used with the strain to be tested.

The method of screening the Drosophila model of intestinal tropism bacteria to improve maturation parameters may therefore include disposing and raising axenic Drosophila larvae on a nutritional medium, inoculating them with the bacterial strain to test (the larvae are then monoxenic for this strain to be tested), to measure or determine a larval maturation parameter, and to compare the results obtained with the data obtained with high axenic Drosophila larvae on the same nutritional medium (control group and / or with monoxenic Drosophila larvae for a high reference strain on the same nutritional medium (referent or reference group).

The nutritional medium may be any medium capable of allowing the larvae to develop over a period sufficient for comparison, and preferably the nutritional medium makes it possible to drive the larvae at least until metamorphosis. The medium is advantageously a hypercaloric medium, that is to say rich in sugar, for example sucrose, this sugar content can for example go from 20 to 150 g of sugar per liter of medium (before gelling or solidification if solid medium or semi-solid). Typically the nutritional medium is a solid or semi-solid medium containing inactivated yeast and sugar. It may for example comprise inactivated yeast (10 g / l), agar or other gelling agent (7.14 g / l), water (1 L) and sucrose (50 g / l). This medium is contained in suitable containers, for example petri dishes. It may consist, for 1 liter of medium, of 7.14 g of agar, 10 g of inactivated yeast and 50 g of sucrose; the middle is cooked in boiling water for 10 minutes, then allowed to cool for solidification. In contrast, a standard medium will include inactivated yeast, but no added sugar.

For the inoculum, the bacterial strains can be suspended in a saline buffer (eg PBS) and inoculated in a sufficient amount, for example from about 10 ⁷ CFU to about 10 ⁹ CFU, in particular about 10 ⁸ CFU, typically for about 7 ml of nutritional medium, such volume being adapted to a group of standard larvae according to the invention, for example about 40 embryos or larvae.

The maturation parameter may be, for example, the size of the larvae or, preferably, their maturation, namely their entry into metamorphosis. Preferably, groups of several embryos are used, in particular from 20 to 60 embryos per group, typically 40. Each group may advantageously be the subject of several replicas, typically 6 replicas. Means are determined to compare with the control and / or reference values. For example, the determination of J50 is used, ie the time after which 50% of the animals have completed their growth phase and metamorphose. This makes it possible to compare the averages between the test group and the control and / or reference group.

From a practical point of view, Drosophila melanogaster (e.g. Drosophila strain yw, or any other so-called "wild" strain) is preferably used.

The method with the Drosophila model can include the following steps:

(1) a batch of embryos from Drosophila melanogaster (e.g. Drosophila yw, or any other so-called "wild-type" strain) strain is available.

(2) at D0: these embryos are distributed in at least 2 groups (in particular from 20 to 60 embryos, for example 40, preferably made up of several replicas, in particular 6 replicas) on hypercaloric nutritional medium, notably comprising inactivated yeast , agar, water and sucrose, in suitable containers, for example petri dishes. A first group of embryos is inoculated with the bacterial strain to be tested (monoxenic group). For the inoculum, the bacterial strains can be suspended in a saline buffer (eg PBS) and inoculated in a sufficient amount, for example from about 10 ⁷ CFU to about 10 ⁹ CFU, especially about 10 ⁸ CFU per container or petri dish (especially 7 ml volume of nutrient medium).

(2a) An axenic group is inoculated with a sterile saline buffer. (2b) A group involving a reference strain to which the effects of the strain to be tested can be compared, this reference strain monocolonisant a group of embryos.

The culture is conducted at a temperature of between about 24 and about 26 ° C., typically about 25 ° C., preferably with a night-time cycle, for example 12 hours / 12 hours.

This step (2) can advantageously include the control group and the reference group. In this case, step (2) can become:

(2) at D0: these embryos are distributed in at least 2, preferably 3 groups (in particular from 20 to 60 embryos, eg 40, preferably made up of several replicas, in particular 6 replicas) in petri dishes or other container containing nutritional medium comprising inactivated yeast, agar, water and sucrose. A first group of embryos is inoculated with the reference bacterial strain, which has a positive effect in this test; it is thus possible to use the strain L. plantarum WJL.

According to one modality, at least one other group is inoculated with the strain to be tested, one can also test in parallel more than one strain, and thus inoculate as many groups (monoxenic groups). The bacterial strains can be suspended in a saline buffer (eg PBS) and inoculated in a sufficient amount, for example from about 10 ⁷ CFU to about 10 ⁹ CFU, in particular about 10 ⁸ CFU per container or petri dish (especially volume of 7ml of nutrient medium).

In another embodiment, an axenic group is inoculated with a sterile saline buffer.

According to another modality, the two aforementioned groups are included.

The breeding is conducted at a temperature between about 24 and about

26 ° C, typically about 25 ° C, preferably with a night day cycle, for example 12h / 12h.

(3) The number of animals that end their juvenile growth phase under the different inoculation conditions is then determined according to a determined frequency (which defines periods), preferably daily.

For this purpose, it is possible to count the number of pupae formed during the period considered, preferably every day. Entry into pupae signifies the end of the juvenile growth phase and the entry into metamorphosis.

(4) The results of the tested strain are compared with the axenic group and / or with the reference strain. One can reason with the "J50" in each condition, namely the duration after which 50% of the animals have completed their growth phase. The average J50 of each of the groups is then determined, and the averages obtained are compared.

For example, it is possible to graph the cumulative percentage of animals involved in metamorphosis relative to the total number of pupae obtained at the end of the experiment (about 40 pupae, with an initial batch of 40 embryos). These curves make it possible to calculate graphically the "J50" in each condition, namely the duration at which 50% of the animals have completed their growth phase. The average J50 of each of the groups is then determined, and the averages obtained are compared.

The conclusions are drawn from the comparison (s). The bacterial strain tested may be considered to respond positively to the test if the average of the metamorphosing input J50 of the group inoculated with this strain is less than the average of the metric input J50 of the axenic group, with a lower value of p. or equal to 0.001, preferably less than or equal to 0.0001 in the Mann-Whitney statistical test. The test is performed on the entire data set of the J50 values of the two groups.

The bacterial strain tested can be considered to have a strong effect if the mean of the metamorphosing entry J50 of the group inoculated with this strain is less than the average of the metamorphic entry J50 of the L group plantarum WJL, with a value of p less than or substantially equal to 0.01 in the Mann-Whitney statistical test. The test is performed on the entire data set of the J50 values of the two groups.

The bacterial strain tested can be considered to have a marked effect if the mean of the metamorphosing entry J50 of the group inoculated with this strain is less than the average of the metamorphosis entering J50 of the L. plantarum group NIZ02877, with a value of p less than or substantially equal to 0.01 in the Mann-Whitney statistical test. The test is performed on the entire data set of the J50 values of the two groups.

The bacterial strain to be tested for a favorable effect on certain metabolic parameters can be tested on a mammalian model, in particular mice, having a normal microbiota. This test can be applied immediately or after preselection of the strain on a Drosophila model, including the Drosophila axenic model of the invention. The principle of the method that will be described about mice, can be extrapolated to another mammalian model.

According to one embodiment of the invention, a bacterial strain which has responded positively to the Drosophila model test is then tested on a mammalian model, preferably a mouse model, having a normal microbiota, and receiving a diet rich in sugar and lipids (high-calorie diet), and measuring one or more parameters chosen in particular from blood glucose measurement, glucose tolerance, tolerance to pyruvate , an improvement of one of these parameters signifying a favorable effect of the bacterium on the parameter or parameters of the model, the strain in question can then be selected for its ability to improve one or more of these parameters.

The method can include culturing a group of mice (for example from 5 to 20 mice, typically 10 mice), having a conventional status (possessing an entire microbiota), and receiving a diet rich in sugar and lipids (diet). calorie).

It is also possible to obtain comparative data previously obtained on mice with a conventional status (possessing an entire microbiota), for example from 5 to 20 mice, typically 10 mice, raised with free access to a standard type of food. Preferably, however, this group of mice is raised in parallel with the group under hypercaloric regime.

By standard diet is meant a diet conventionally used to feed laboratory animals without inducing metabolic diseases. A typical diet can consist of 16.9% protein, 4.3% fat and 55.5% carbohydrate (including -1% sucrose and 0% maltodextrin).

As a high calorie diet is meant a diet that differs from the standard by high lipid and sugar content. These diets are typically used to induce obesity, insulin resistance and diabetes in rodents. A typical hypercaloric diet may be composed of 20% protein, 36.1% lipid and 35.1% sucrose and maltodextrin.

Different measures can be performed on the groups of mice. They can be combined two by two or all be made.

A blood glucose measurement can be performed on the groups of mice. One or more measurements can be made, the measurements being of course staggered over time when there are several. Preferably, the blood glucose measurement is preceded by a period of fasting, especially several hours, typically about 6 hours.

A glucose tolerance test may be performed to measure the ability of the animals to store more or less circulating glucose following intraperitoneal injection of the latter. The blood glucose is measured at zero time, then the animals are injected with glucose (for example 2 g / kg), for example intraperitoneally, and different blood glucose measurements are made at different times (eg 15, 30, 60 and 90 minutes post-injection) during the experiment.

A pyruvate tolerance test may be performed to observe liver function that is deregulated by the high calorie diet. As described for the glucose tolerance test, the pyruvate tolerance test consists in injecting pyruvate (for example 1.5 g / kg), for example intraperitoneally. The glucose level is measured at different times, for example 0, 15, 30, 60 and 90 min.

The method with the mouse model can include the following steps:

(1) mice (e.g. from C57BL / 6 and 4 weeks old) with conventional status (possessing an entire microbiota) are reared into two groups of several individuals (eg, 5 to 20 mice, typically mouse), one with free access to a standard type of food for the mice, the other a food rich in sugar and lipids (high calorie diet).

(2) The administration of the bacterial strain to be tested begins at the same time as the placing of the animals under these standard and high calorie diets. Administration of the bacteria orally can be performed several times, for example 5 times (on different days), per week with from about 10 ⁸ CFUs to about ¹⁰ CFUs, typically about 10 ⁹ CFUs per mouse at each administration. , for a total duration of several weeks, typically 10 weeks. The control mice not receiving the bacteria are force-fed with the excipient (saline solution).

(3) The mice are characterized metabolically by measuring the blood glucose, this measurement being preferably carried out after a period of fasting, especially 4 to 8 hours, typically after 6 hours of fasting. The measurement is made by taking a drop of blood, for example at the tail, and using a blood glucose meter or any other appropriate method or tools. These measurements are preferably carried out 2 to 4, typically 4 weeks after the beginning of feeding described above (post-gavage).

(4) A glucose tolerance test can also be performed on these mice in order to measure the ability of the animals to store more or less circulating glucose following an intraperitoneal injection of the latter. The mice are fasted for 4 to 8 hours, typically 6 hours. The blood glucose is measured at zero time, then the animals are injected with glucose (2 g / kg), for example intraperitoneally, and different blood glucose measurements are made at different times (for example 15, 30, 60 and 90 minutes post -injection) during the experiment. (5) Mice can be metabolized again using the same approaches previously described (fasting glucose and / or glucose tolerance test). For example 9 and 10 weeks post-gavage.

(6) A pyruvate tolerance test may be performed to observe liver function that is deregulated by the high calorie diet. As described for the glucose tolerance test, the pyruvate tolerance test consists of injecting pyruvate (for example 1.5 g / kg) intraperitoneally. The glucose level is measured at different times, for example 0, 15, 30, 60 and 90 min.

The animals are sacrificed (for example at the end of the week postgavage) and the different tissues can be collected in order to carry out further experiments such as serum assays in the blood (insulin), in particular by ELISA. The insulin assay can be performed on pure plasma (standard mouse) or ¼ diluted (high-calorie mice) using specific commercial ELISA kits for insulin detection according to the manufacturer's instructions.

The lactic bacterium strain is considered to respond positively to the test if the fasting glucose and insulin levels as well as the possible metabolic tests are significantly lower than the corresponding control groups (mice receiving the excipient), with a probability of error value. p less than 0.05 in the Mann-Whitney test.

The screening may also include a group of mice that will be inoculated with a reference bacterial strain, which has a positive effect in this test; it is thus possible to use the strain L. plantarum WJL. The bacterial strain tested is considered to respond very positively to the test if the fasting glucose and insulin levels as well as any metabolic tests are significantly lower or substantially equal to this reference group, with a probability value of error p less than 0.05 in the Mann-Whitney test.

In particular, the invention provides strains of intestinal tropism bacteria belonging to the following families: Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae, Bifidobacteriaceae. According to one embodiment, the invention uses one or more strains of the genus Lactobacillus, in particular one of the following species, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus.

More particularly, they are bacteria belonging to the species Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus. According to a modality, the strain is selected from the species Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei.

According to one embodiment, the bacterial strain is selected from L. plantarum WJL, L. plantarum G821 (CNCM I-4979), L. plantarum NIZ02877, L. casei

ATCC 393, L. casei L919, L. paracasei ATCC25302, L. paracasei Shirota, L. fermentum ATCC9338, L. rhamnosus L900, L. rhamnosus L908, L. rhamnosus GG. According to one modality, the strain is selected from L. plantarum WJL, L. plantarum G821, L. casei ATCC 393, L. casei L919, L. fermentum ATCC9338.

The strains that were selected on the Drosophila model, on the mouse model or on both models are considered to have a favorable effect on the metabolic health of the target mammal, including man. The mouse model or the like also has the advantage of giving precise indications of the measured parameter (s) such as blood glucose, the glucose tolerance test and the pyruvate tolerance test. This makes it possible to predict a favorable effect of the strain in the target mammal on blood sugar, glucose tolerance and / or tolerance to pyruvate (ability to lower or contain glucose levels in a patient who is healthy or has glucose intolerance or in type 2 diabetes, to improve tolerance to glucose and / or pyruvate, to improve the status of an individual with glucose intolerance or type 2 diabetes)).

In particular, a strain of bacterium that has led to a favorable effect on the Drosophila model and on the mammalian model is retained as having a favorable effect on metabolic health.

The bacteria responding positively to the method of the invention are useful for reducing metabolic diseases, in particular glucose intolerance and / or type 2 diabetes, or slowing their progression.

In order to select bacteria intended to reduce or slow down progression, metabolic diseases, in particular glucose intolerance and / or type 2 diabetes, it is preferred to follow at least two of the aforementioned parameters in the mammalian model. preferably the three parameters: fasting glucose, glucose tolerance and pyruvate tolerance.

The subject of the present invention is also a composition comprising at least one strain of bacterium having an intestinal tropism, in particular a lactic bacterium of the species L. plantarum, for use in the prevention and / or control of treatment of metabolic diseases, in particular type 2 diabetes. By way of example, mention may be made of the following strain: L. plantarum WJL. L. plantarum WJL (Eun-Kyoung Kim et al., Genome Announcements, November / December 2013, Vol.1, No. 6 e00937-13, GenBank AUTE00000000, Lactobacillus plantarum WJL, whole genome shotgun sequencing project). This strain WJL was initially isolated and can be isolated from Drosophila (JH Ryu et al., Science 2008, 319: 777-782). Other strains can be identified among intestinal tropism bacteria and in particular among the species and strains mentioned above, in particular among the strains L. plantarum G821 and L. plantarum NIZ02877. The strain G821 is disclosed in WO2015173386 and deposited in the National Collection of Culture of Microorganisms (Institut Pasteur) under the registration number CNCM I-4979 on May 1, 2015. The strain G821 was obtained by experimental evolution, it is that is, by accumulation and selection of natural variants of L. plantarum strain NIZ02877 (NIZO collection (2877)).

The composition may especially contain from about 10 ⁵ to about 10 ¹² , especially from about 10 ⁶ to about 10 ¹² , preferably from about 10 ⁸ to about 10 ¹² bacterial colony forming (CFU) cells according to the present invention. invention, per gram of composition. The term CFU means "colony forming units" according to the English technical expression. By gram of composition is preferably meant the probiotic formed bacteria, co-ingredients, and excipients or vectors. By bacterial cells is meant a single strain of bacteria according to the invention or a mixture of at least two bacteria, according to the invention.

The composition may especially comprise the lactic acid bacteria or bacteria in living form. The composition may be in ready-to-use form or mixed or diluted with a food, excipient or food liquid such as drinking water.

It can be a bacterial suspension, which can be frozen and thawed before use.

It can be a freeze-dried powder, which can be used as such, in the form of powder, granules, tablet, bolus, capsule, capsule, or used after being taken back into a suitable vehicle. This composition may comprise a conventional freeze-drying excipient.

The composition may be a form of oral administration (eg, powder, capsule, tablet, bolus) in a gastro-protected form to pass the stomach and release the bacteria in the intestine.

According to one embodiment, the composition is a solid composition, eg tablet, bolus, capsule, capsule, gastro-protected to pass the stomach and release the bacteria in the intestine. According to one modality, the form, in particular granules, can be used for feeding in an aqueous medium, for fish in particular.

According to one embodiment, the composition is a liquid or is to dissolve in a liquid or to suspend it in a liquid, for example drinking water, in this case the composition is initially solid, eg powder, granulate, compressed dissolvable, eg effervescent tablet, or deliquescent tablet in the liquid.

According to one embodiment, the composition is solid, e.g. powder, granule, tablet, bolus, and intended or capable of being mixed with a food.

According to one embodiment, the composition is solid, in a ready-to-use form without the need for mixing with a foodstuff, but which can nevertheless be mixed with a foodstuff, for example a tablet or an appetizing bolus.

The invention also relates to a probiotic treatment method for preventing and / or treating metabolic diseases, in particular type 2 diabetes, comprising administering to a mammal including humans, a composition according to the invention. 'invention. By mammal, in addition to humans, we mean livestock, including large animals, cattle and pork for example, sports animals, including horse, and pets, including cat and dog. According to one characteristic of the invention, the mouse and / or, more generally, rodents are excluded. Preferably, the composition is administered orally.

This probiotic treatment comprises administering a sufficient amount of a composition as described above to the human or mammalian patient. The method will include the administration in one or more times, which may be staggered over the treatment period, doses of composition according to the invention. Doses can be fractionated to facilitate administration. The frequency of administration is in particular between a dose (single or fractional) every day and a dose every month. Typically, the frequency of administration will be between a dose (single or fractional) every day and a dose every week or every 2, 3, 4, 5 or 6 days. Each dose (single or fractional) represents in particular several grams to several tens of grams of composition.

The method includes administering a composition comprising from about 10 ⁵ to about 10 ¹² , including from about 10 ⁶ to about 10 ¹² , preferably from about 10 ⁸ to about 10 ¹² bacterial cells. forming CFU colonies per gram of composition (bacterial cells means a single strain of bacteria according to the invention or a mixture of at least two bacteria, according to the invention).

The method preferably comprises administering a composition comprising the lactic acid bacteria or bacteria in living form.

The form of the composition may be solid or liquid and take any of the forms presented supra.

The invention will now be described in more detail with the aid of embodiments taken by way of non-limiting examples and with reference to the drawings in which:

FIG. 1 is a graph derived from the data of Table 1 representing the effect of different strains of L. plantarum (strains WJL and NIZ02877) on the correction of the metamorphic entry time of drosophila larvae fed on a yeast base and sucrose (10 g of inactivated yeast per 50 g of sucrose). These results demonstrate that both strains have the ability to buffer the deleterious effects of high calorie nutrition on the date of entry into metamorphosis and that the strain WJL is more efficient than the strain NIZ02877. The strain WJL has a marked effect and the strain NIZ02877 a moderate effect ( ^** p <0.01).

Figure 2 combines three graphs from the data in Tables 2A, B and C showing the impact of L. plantarum strains on the metabolic parameters of mice under normal (Standard Calory Diet, SCD) diet. Panel A: As early as 4 weeks of treatment, L. plantarum® improves fasting glucose (6 hours of pre-measurement) and (BC panels) after 9 weeks of glucose tolerance treatment (IP-GTT = glucose tolerance test) intraperitoneally AUC: area under the curve). Strain NIZ02877 showed no significant effect on these parameters. ^* P <0.05; ^** p <0.01.

Figure 3 groups together six graphs from the data in Tables 3 A, B, C, D, E and F showing the impact of the daily administration of 10 ⁹ CFU of L. plantarum, on the metabolism of mouse glucose under high calory diet (HCD) .. 9 weeks of administration markedly improve (A) fasting glucose, (BC) glucose tolerance (GTT; AUC: area under the curve), ( DE) tolerance to pyruvate (PTT AUC: area under the curve) and (F) fasting insulinemia demonstrating marked antidiabetic properties of L. plantarurrf strain ^, on glucose metabolism ( ^* p <0) , 05; ^** p <0.01; ^*** p <0.001). Figure 4 shows that Lp ^WJL increases pancreatic islet size in standard diet mice.

Figure 5 shows that Lp ^WJL stimulates the L cell density of ileum in mice with standard diet (quantification of GLP1 positive cells, n = 6 mice per group).

EXAMPLES

Example 1: Drosophila Model Referring to Figure 1

The Drosophila test is typically conducted in the following manner: a batch of embryos from Drosophila melanogaster (Drosophila yw strain) relatives is available;

at D0: we divide these embryos into at least 3 groups (40 embryos, made into 6 replicates for a total of 240 embryos per group) in petri dishes containing nutritional medium including inactivated yeast, agar, water and sucrose. We study at least 3 groups: a first group of embryos is inoculated with the reference bacterial strain L. plantarurr ^^, and one or more group (s) are inoculated with the strain (s) to be tested (groups monoxenic, in the embodiment of strain NIZ02877). For the inoculum, the bacterial strains are suspended in a saline buffer (eg PBS) and are inoculated at a level of about 10 ⁸ CFU per petri dish. Finally, the group forming the axenic group is inoculated with a sterile saline buffer. The breeding is conducted at about 25 ° C, with a night day cycle of 12h / 12h. Typically, the nutritional medium consists, for 1 liter of medium, of 7.14 g of agar, 10 g of inactivated yeast and 50 g of sucrose. The medium is cooked in boiling water for 10 minutes, then allowed to cool for solidification.

The number of animals that complete their juvenile growth phase under the different inoculation conditions is then determined daily. To do this we count the number of pupae formed each day. The entrance into metamorphosis marks the end of the juvenile growth phase.

We then graphically represent (Figure 1) the date at which 50% of the animals entered metamorphosis.

The averages obtained are compared: the bacterial strain tested is considered to respond positively to the test if the mean of the metamorphosing entry J50 of the group inoculated with this strain is less than the average of the meteorological input J50 of the axenic group, with a value of p less than or equal to 0.01 in the Mann-Whitney statistical test. The test is performed on the entire data set of the J50 values of the two groups.

Figure 1 shows the data obtained in terms of J50 of animals entered in metamorphosis relative to the total number of pupae obtained at the end of the experiment, in the example Axenic, L. plantarurr ^^ and L. plantarum ^NÎZ ° ²⁸⁷⁷

Calculated J50's are shown in Table 1 (raw data in Figure 1)

0

Example 2: mouse model

Mice from line C57BL / 6 and 4 weeks old were used in this study. Animals have a conventional status

(possessing an entire microbiota) and are reared under controlled sanitary conditions. They have free access to two types of food: the first is a standard type of food and the second is rich in sugar and fat (high calorie diet). at 4 weeks of age: 10 mice per experimental group are studied. The bacterial strains L. plantarum WJL and L. plantarum NIZ02877 were started at the same time as the animals were put on diets, standard and high calorie. The administration of the bacteria orally is carried out 5 times a week with 10 ⁹ CFU / mouse / day for a total duration of 10 weeks. The control mice not receiving the bacteria are force-fed with the excipient (saline solution).

- At 3 weeks post-gavage: the mice are characterized metabolically by a measurement of blood glucose after 6 hours of fasting. The measurement is made by taking a drop of blood at the tail and using a blood glucose meter. A glucose tolerance test is also performed on these mice in order to measure the capacity of the animals to store more or less circulating glucose following an intraperitoneal injection of the latter. The mice are fasted for 6 hours. The blood glucose is measured at zero time, then the animals are injected with glucose (2 g / kg) intraperitoneally and different blood glucose measurements are made at different times (15, 30, 60 and 90 minutes postinjection) during the experiment.

at 9 and 10 weeks post-gavage: the mice are characterized again metabolically using the same approaches described previously (fasting glucose and glucose tolerance test). However, a pyruvate tolerance test is performed in order to observe the liver function which is deregulated by the hypercaloric diet. As described for the glucose tolerance test, the pyruvate tolerance test consists of injecting pyruvate (1.5 g / kg) intraperitoneally. The glucose level is measured at time 0, 15, 30, 60 and 90 min.

at the end of the post-gavage week: the animals are sacrificed and the different tissues are collected in order to carry out more in-depth experiments such as serum blood tests (insulin). The insulin assay is performed on pure plasma (standard mouse) or diluted to ¼ (hypercaloric mice) using commercial ELISA kits specific for insulin detection following the manufacturer's instructions.

The lactic acid bacterium strain being considered to respond positively to the test if the fasting glucose and insulin levels as well as the metabolic tests are significantly lower than the corresponding control groups (vehicle receiving mice), with a probability value of error p less than 0.05 in the Mann-Whitney test.

Results obtained with L. plantarum WJL and L. plantarum strains NIZ02877, and an untreated control: Table 2A: Raw Data Figure 2A

Table 2B: Raw Data Figure 2B

Time (min) TO T15 T30 T45 T60 T90

100 223.7 164.0 139.5 132.5 108.8

100,278.8 176.3 161.9 163.6 137.3

100 338.1 178.8 188.1 179.7 129.7

100 256.8 145.5 135.6 122.0 113.6

100 224.1 179.3 178.4 163.8 135.3

100 201.8 151.3 147.8 136.3 131.9

100 201.9 147.2 148.1 139.8 125.9

100 182.8 143.3 117.9 121.6 111.9

100 237.9 173.3 158.6 136.2 122.4 100 178.2 127.2 123.1 119.0 112.2

100 255.8 167.3 153.1 144.2 115.9

100 231.1 169.7 139.3 145.1 119.7

100 181.7 171.4 134.1 162.7 146.8

100,295.5 158.0 170.5 161.6 126.8

100 226.9 162.0 153.7 136.1 125.9

Table 2C: Raw Data Figure 2C

SCD-PBS SCD-WJL SCD- # 2877

5017.5 4800 6120

8100 4507.5 6322.5

9652.5 4080 6825

5595 5962.5 7267.5

7065 3735 5152.5

SCD-PBS SCD-WJL SCD- # 2877 Statistical Analysis

PBS vs.

Average 7086 4617 6337 WJL s. PBS # 2877

Value

Standard Deviation 1432.2 611.4 567 Value of p Significance of p Significance

Nb 5 5 5 <0.01 ** n / a n / a

Table 3A: Raw Data Figure 3A

SCD-PBS HCD-PBS HCD-WJL

155 182 156

128,177 155

122 155 148

120 165 164 107 167 141

148,168 157

143,174 157

146,165 187

132 183 150

139 131

SCD-PBS HCD-PBS HCD-WJL Statistical Analysis

Average 134 170.7 154.6 HCD-PBS vs. SCD-PBS HCD-WJL vs. HCD-PBS

Standard Deviation 12.2 7.4 9.7 Value of p Significance Value of p Significance

Number 10 9 10 <0.0001 * * * * <0.01 **

Table 3B: Raw Data Figure 3B

Time (min) TO T15 T30 T60 T90

100 210.2 244.1 257.6 254.2

100 157.8 161.7 176.6 145.3

100 270.7 250.9 237.1 140.5

100 299.3 197.1 183.2 139.4

(/)

in

at. 100 257.4 232.6 220.6 241.1

at

u 100 468.6 295.2 219.0 182.9

100 298.5 189.6 180.6 153.7

100 256.2 158.7 162.8 91.7

100 261.5 190.4 160.9 112.2

100 237.9 124.8 118.0 99.4

100 275.6 257.6 243.3 227.6

100 331.5 332.2 388.6 356.4

100 296.1 310.5 331.5 330.4

100 296.1 323.8 331.5 298.9

CQ

Q. 100 284.4 284.4 284.4 257.8

Q

U 100 315.0 346.8 493.6 279.2

100,290.3 340.9 340.9 315.3

100 230.8 230.8 230.8 230.8

100 339.5 348.8 321.5 305.2

100,274.0 274.0 274.0 274.0

100,285.9 268.4 239.0 220.9

_l 100 209.7 259.7 233.1 216.2

§ 100 297.1 263.4 191.4 182.9

Q

U 100 300.0 317.4 266.3 243.8

X

100,277.6 214.4 207.5 204.0

100 374.2 297.5 289.3 278.0 100 262.9 247.1 242.4 241.2

_SC D-100 342.9 264.6 258.9 248.0

S _P B 100 304.2 249.7 243.6 242.4

100,245.3 198.8 186.3 178.9

Table 3C: Raw Data Figures 3C

SCD-PBS HCD-PBS HCD-WJL

16614.4 16624.4 16457.6

7060.5 29375.8 14717.5

13338.4 25006.9 13234.3

10100.4 24360.5 19689.6

15005.3 19944.3 13194.0

17764.3 31868.5 22415.1

10231.3 25576.7 16394.1

6241.7 14711.5 18934.3

7562.5 25316.9 17168.2

3419.3 19571.9 10653.7

SCD-PBS HCD-PBS HCD-WJL Statistical Analysis

Average 10733.8 23235.7 16285.8 HCD-PBS vs. SCD-PBS HCD-WJL vs. HCD-PBS

Value

Standard deviation 3957.4 4418.2 2668.8 Value of p Significance of p Significance

Nb 10 10 10 <0.001 * * * <0.05 *

3D table: raw data 3D figures

Time (min) TO T15 T30 T60 T90

83 87.0 83.0 103.0 90.0 77 150.0 154.0 144.0 130.0 88 144.0 165.0 188.0 186.0

83 137.0 153.0 146.0 131.0

99 157.0 175.0 175.0 115.0

78 130.0 160.0 140.0 129.0

109 149.0 151.0 171.0 137.0

88 152.0 172.0 176.0 132.0

96 145.0 166.0 174.0 159.0

92 141.0 171.0 149.0 121.0

145,268.0 248.0 305.0 264.0

107 199.0 220.0 248.0 200.0

115 187.0 217.0 239.0 203.0

CQ 98 170.0 184.0 241.0 209.0

Q.

Q 130 178.0 223.0 273.0 244.0

(_)

X 126 224.0 265.0 169.0 195.0

152,208.0223.0 310.0 255.0

177,231.0 286.0 272.0,245.0

211,330.0 321.0 313.0 281.0

103,127.0 159.0 236.0 233.0

100 155.0 170.0 207.0 200.0

98,114.0 136.0 154.0 148.0

121 207.0 188.0 192.0 183.0

½ 88 148.0 172.0 195.0 134.0

Q 116 133.0 168.0 171.0 159.0

X

107 161.0 204.0 214.0 224.0

135,197.0 166.0 202.0 185.0

142 198.0 222.0 285.0 227.0

97 117.0 134.0 171.0 202.0

Average 110.7 155.7 171.9 202.7 189.5

HCD-WJL

Standard deviation 14.2 28.0 19.7 26.2 27.7 Number 10 10 10 10 10

Table 3E: Raw Data Figures 3E

Table 3F: Raw Data Figures 3F

SCD-PBS HCD-PBS HCD-WJL Statistical Analysis

Mean 0.5 4.1 2.2 HCD-PBS vs. SCD-PBS HCD-WJL vs. HCD-PBS

Value

Standard deviation 0.2 1.9 1.1 Value of p Significance P Significance

Nb 10 8 9 <0.001 * * * <0.05 * In conventional mice under normal diet 4 weeks of treatment with L. plantarum strain improves glucose fasting and from 9 weeks of glucose tolerance treatment.

In the conventional high-calorie diet, 9 weeks of administration of the L. plantarum strain significantly improves fasting glucose, glucose tolerance, pyruvate tolerance, and fasting insulinemia. marked functional properties of this strain on glucose metabolism.

Example 3: Strains are available at the ATCC, at the Institut Pasteur in Paris, at the Institut Pasteur in Lille or published in the scientific literature and available from the referent researchers of the publications mentioned:

Example 4 (Figures 4 and 5)

The metabolic adaptations of the body induced by Lp ^wjl imply an increase in insulin production and pancreatic islet size in conventional mice.

In order to investigate the metabolic adaptations induced by Lp ^wjl , conventional C57BL / 6 mice were fed a standard diet supplemented daily by the oral administration of 109 CFUs of Lp ^wjl .

The treatment does not modify the evolution of the body mass and does not modify the food intake under these optimal nutritional conditions (data not presented). However, after 10 weeks, while blood glucose is maintained at a normal level (data not shown), fasting insulinaemia is higher (data not shown), and the intraperitoneal glucose tolerance test indicates better tolerability at baseline. glucose in Lp ^wjl- treated mice (data not shown). These effects are not observed when the strain ^NIZ02877 is administered (data not shown). These observations thus demonstrate the strain-specific action of Lp ^wjl on the host metabolism.

Increased insulin concentrations in standard diet mice suggest a treatment effect on insulin secretion by pancreatic beta cells. The analysis of pancreatic islets in animals revealed a strong impact of Lp ^wjl administration on mean islet size (Figure 4) with the increase in the number of large or medium islets and a reduction in the number of small islets ( Figure 4). In agreement with the lack of effect on glucose tolerance and insulinemia, the administration of ^NIZ02877 does not change the size of the islets (Figure 4). The immunodetection of pancreatic hormones demonstrates that the increase in islet size is not associated with observable changes in islet organization or insulin / glucagon ratio (data not shown). Since Lp ^wjl was previously shown to maintain the activity of the GH / IGF1 axis through its growth promoting activity under chronic undernutrition conditions, the inventors assessed whether the increase in pancreatic islet size was due to stimulation by somatotropic hormones. Neither serum IGFI level nor the expression of IGFI and GH receptors or their classical mediators (SOCS2 and 3) in the islets was affected in Lp ^wjl- treated mice (data not shown), which suggests that another mechanism contributes to the enlargement of the islet size. The function of the pancreas to secrete insulin was then evaluated in vivo by the oral glucose test and ex vivo using isolated islets (data not shown). For both experiments, treatment with Lp ^wjl significantly improved insulin secretion after the glucose test, suggesting increased islet sensitivity to the secretory effect of the islets. In conclusion, the increase in islet size and the improved response to oral glucose testing suggest stimulation of the incretin gut-pancreas axis in Lp ^wjl- treated mice.

Lp ^wjl increases the secretion of GLP1 and the number of cells producing intestine

In order to evaluate the incretin pancreas-intestine axis, the inventors measured the production of GLP1 in the blood of the portal vein 15 minutes after oral glucose loading, a test that significantly doubles the production of GLP1 in control mice ( data not shown). Importantly, when the mice are treated with Lp ^wjl , an increase of four to five times the level of GLP1 in the portal vein is already observed under basal conditions and remains high, although it is not further increased, glucose response (data not shown). This result suggests a marked increased ability to produce GLP1 in Lp ^wjl- treated mice. This characteristic is radically less pronounced, especially in basal conditions, under administration of ^NIZ02877

(data not shown). In line with these observations, the number of L cells produce GLP-1 was significantly higher in mice treated with Lp ^WJL ileum, but not treated mice with | _p ^NIZ02877 (Figure 5). The treatments do not affect the organization of the intestinal barrier, as assessed by histological examination of the crypt and villi of the ileum (data not shown). Similar results are found in the animal's colon, with an increase in the number of positive GLP1 cells and a normal appearance of colon histology, albeit with a visible increase in the length of the crypt of the colon under treatment Lp ^wjl (data not shown).

In order to evaluate whether the increased production of GLP1 observed and the concentration in the portal vein in the presence of Lp ^wjl contributes directly to the improved glucose tolerance of the animals, the inventors used Exendin-9-39, a well-characterized inhibitor of the receptor. of GLP1. As expected, infusion of Exendi-9-39 prior to oral glucose tolerance test significantly altered glucose tolerance as evidenced by an increase in area under the test curves (data not shown). The effect is more pronounced in mice treated with ^NIZ02877 as assessed by AUC delta values (data not shown). This observation clearly indicates that the improved glucose tolerance of Lp ^wjl- treated mice is largely dependent on the increased level of GLP1 and the improved pancreatic incretin-pancreatic axis.

Claims

claims

1. A composition comprising at least one strain of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus rhamnosus, for use in decreasing or slowing the progression of metabolic diseases.

A composition for use in decreasing, or slowing the progression of, metabolic diseases according to claim 1, wherein the metabolic disease is type 2 diabetes.

3. The composition of claim 1, wherein L. plantarum is L. plantarum WJL or L. plantarum NIZ02877.

4. A method for screening intestinal tropism in a mammal, which may decrease, or slow the progression, of metabolic diseases, in particular type 2 diabetes, in which the initially axenic larvae of Drosophila are monocolonized by the strain of bacterium to be tested, these larvae are raised with a high-calorie nutritional medium, at least one larva maturation parameter is measured, it is determined whether the bacterium has had a significantly favorable effect on the maturation parameter, and one concludes if necessary that said strain has a favorable effect on ripening.

5. The method according to claim 4, wherein it is determined whether the bacterium leads to a significantly improved maturation parameter compared to a control consisting of high axenic larvae under similar nutritional conditions.

6. Method according to claim 4, wherein it is determined whether the bacterium leads to a maturation parameter substantially equal or improved compared to a reference consisting of monocolonized larvae by a reference strain which itself has a growth parameter improvement. significant compared to a control corresponding to high axenic larvae under similar nutritional conditions.

7. Method according to any one of claims 4 to 6, wherein is raised axenic Drosophila embryos on a nutritional medium, they are inoculated with the bacterial strain to be tested (the larvae from these embryos are then monoxenic for this strain). to be tested), a larval maturation parameter is measured, and the results obtained are compared with the data obtained with high axenic Drosophila larvae on the same nutritional medium (control group) and / or with monoxenic Drosophila larvae for one year. high reference strain on the same nutrient medium (reference group).

8. Method according to any one of claims 4 to 7, wherein J0: one distributes embryos in at least 2 groups on nutritional medium hypercaloric, including inactivated yeast, agar, water and sucrose, in suitable containers, a first group of embryos is inoculated with the bacterial strain to be tested (monoxenic group), the group forming the Axenic group is inoculated with sterile saline buffer.

The method according to any one of claims 4 to 8, wherein a bacterial strain which has responded positively to the Drosophila model test is then tested on a mammalian model, preferably a mouse model, having a normal microbiota, and receiving a food rich in sugar and lipids (high calorie diet), and measuring one or more parameters selected from blood glucose measurement, glucose tolerance test, pyruvate tolerance test, and selecting a strain that has improved one of these parameters.

10. The method of claim 9, wherein there is control with mice with a whole and high microbiota with free access to a standard type of food.

1 1. A method according to any one of claims 4 to 10, wherein a bacterial strain which has a favorable effect on the Drosophila model and / or the mammalian model is retained as having a favorable effect on metabolic health.