Probiotic composition making it possible to promote juvenile livestock growth

Abstract

The probiotic composition comprises at least one lactic acid bacteria strain, preferably Lactobacillus with intestinal tropism, for use in promoting juvenile growth in livestock, with stimulation of linear growth and/or of IGF-1 level. Exemplary lactic acid bacteria include Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus. The strains can be selected in a mouse model. The invention also relates to a probiotic treatment method using this composition.

Claims

1. A method for promoting juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels of animals fed a conventional rearing diet, comprising the administration of a composition or a food comprising at least several grams of a probiotic composition comprising about 10.sup.5 to about 10.sup.12 colony forming units (CFU) Lactobacillus plantarum WJL per gram of probiotic composition to said animal in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

2. The method of claim 1, said method promoting juvenile livestock post-weaning growth.

3. The method of claim 1, wherein the animal is post-weaning livestock.

4. The method of claim 1, said method further comprising measuring serum insulin-like growth factor 1 (IGF-1) levels to determine an increase of IGF-1 levels in treated animals.

5. The method of claim 1, said method stimulating linear growth.

6. The method of claim 1, said method increasing levels of IGF-1.

7. A method of promoting juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels of animals fed a conventional rearing diet, comprising the administration of a composition or a food comprising at least several grams of a probiotic composition comprising about 10.sup.5 to about 10.sup.12 colony forming units (CFU) Lactobacillus plantarum WJL per gram of probiotic composition to said animal, wherein the probiotic composition is administered once a day in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

8. A method of promoting juvenile livestock growth with stimulation of the linear growth and/or of the IGF-1 level of animals fed a conventional rearing diet, comprising the administration of a composition or a food comprising at least several grams of a probiotic composition comprising live bacteria in an amount of about 10.sup.5 to about 10.sup.12 colony forming units (CFU) Lactobacillus plantarum WJL per gram of probiotic composition to said animal in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

9. The method of claim 1, wherein said probiotic composition is administered at a frequency of once per day to once per week.

10. The method of claim 9, wherein said probiotic composition is administered at a frequency of once per day for three to five days each week.

11. The method of claim 1, said method comprising the administration of a composition or a food comprising several grams to several tens of grams of a probiotic composition comprising about 10.sup.5 to about 10.sup.12 CFU Lactobacillus plantarum WJL per gram of probiotic composition to said animal in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

12. The method of claim 7, said method comprising the administration of a composition or a food comprising several grams to several tens of grams of a probiotic composition comprising about 10.sup.5 to about 10.sup.12 CFU Lactobacillus plantarum WJL per gram of probiotic composition to said animal, wherein the probiotic composition is administered once a day in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

13. The method of claim 8, said method comprising the administration of a composition or a food comprising several grams to several tens of grams of a probiotic composition comprising live bacteria in an amount of about 10.sup.5 to about 10.sup.12 CFU Lactobacillus plantarum WJL per gram of probiotic composition to said animal in an amount that promotes juvenile livestock growth with stimulation of the linear growth and/or of IGF-1 levels.

Description

(1) According to an embodiment of the objects of the invention, the bacterial strain is selected from L. plantarum WJL, L. plantarum G821 (CNCM 1-4979), L. plantarum NIZO2877, 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 mode, the strain is selected from L. plantarum WJL, L. plantarum G821, L. casei ATCC 393, L. casei L919, L. fermentum ATCC9338.

(2) In a specific embodiment, it is a matter of bacteria of the species Lactobacillus plantarum, for example strain WJL or strain G821, deposited with the Collection Nationale de Culture de Microorganismes (Pasteur Institute) under registration number CNCM 1-4979 on 11 May 2015. Strain G821 was obtained by experimental evolution (i.e., accumulation and selection of natural variants) of strain L. plantarum NIZO2877.

(3) According to one mode, the compositions according to the invention comprises at least one bacterial strain selected from these groups, which has the required properties and promotes juvenile growth. Of course, the composition of the invention may comprise more than one bacterial strain meeting the needs of the invention. In particular, the composition comprises two or more of these bacterial strains, selected from the same species or from different species.

(4) According to an advantageous embodiment, the bacterium is an L. plantarum. Suitable strains are L. plantarum G821, L. plantarum NIZO2877 and 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 (J H Ryu et al., Science 2008, 319:777-782).

(5) Other examples of suitable strains are as follows: L. casei ATCC 393, L. casei L919 (Koryszewska-Baginska A. et al., 26 Sep. 2013, Genome Announc), L. paracasei ATCC25302, L. paracasei Shirota (Yuki N et al., Int J Food Microbiol. 1 Apr. 1999; 48(1):51-7), L. fermentum ATCC9338, L. rhamnosus L900 (Aleksandrzak-Piekarczyk T. et al., Genome Announc, 15 Aug. 2013), L. rhamnosus L908 (Koryszewska-Baginska A. et al., 20 Feb. 2014, Genome Announc), L. rhamnosus GG (Kankainen M. et al., Proc Natl Acad Sci USA, 6 October 2009).

(6) The present invention thus contributes to the art the teaching that bacterial strains with intestinal tropism promote juvenile growth in animals raised on conventional diets. But the invention is not limited to this teaching; it also gives the skilled person the tools to reliably determine the bacterial strains useful for the invention. Different criteria can be used as a basis for testing, either alone or in combination. These criteria include the serum IGF-1 level in the animal model (e.g., mouse), mouse growth models illustrated, for example, by femur length or linear growth of the animals, or weight gain, notably linked to linear growth and/or to increase in muscle mass and/or to increase in lean mass. On the basis of these or similar criteria, it is possible for the skilled person to develop tests comparing individuals raised in the presence or in the absence of the bacterium to be tested.

(7) An organism (e.g., mouse) raised in an environment free of microorganisms and thus free of intestinal flora is referred to as an “axenic” organism.

(8) An associated axenic organism (e.g., mouse) raised in the presence of a single microorganism and thus carrying this single microorganism as intestinal flora is referred to as a “monoxenic” organism.

(9) According to the invention, it is possible to determine whether a bacterial strain with intestinal tropism can promote growth in the case of a conventional diet, by using an axenic mouse model that allows linear growth monitoring of mice in the presence of the bacterium to be tested in comparison with the absence of microbiota and/or with the presence of a reference bacterial strain. This model can be used as a first-line approach.

(10) According to a feature of the invention, the bacterial strains of the invention are characterised by the fact that they respond positively to the following linear growth test:

(11) from the same mouse line (typically Balb/c mice), a line of axenic parent mice and a line of monoxenic parent mice (associated with the bacterium to be tested) are established, and juveniles are produced which are raised with the parents on a conventional diet comprising about 40% carbohydrates, about 25% proteins and about 9% lipids, until they are weaned (on day 21); to form the group of monoxenic juveniles, parents mono-associated with the bacterial strain to be tested are used,

(12) on day 21: 8 weaned juveniles from each of these two lines are available, forming the monoxenic group and the axenic group, and they are raised on a conventional diet comprising about 40% carbohydrates, about 25% proteins and about 9% lipids,

(13) on day 56: the mean size of the mice is determined for the group in question by measuring from the tip of the nose to the base of the tail of each individual; another possible measurement consists in sacrificing the individuals, removing the femurs and measuring their length,

(14) the lactic acid bacteria strain being considered to respond positively to the test if the mean individual size and/or the mean femur length of the monoxenic group are/is greater, respectively, than the mean individual size and/or femur length of the axenic group, with a p-value of less than 0.05, in Tukey's statistical test.

(15) The present invention thus has as an object a composition comprises at least one bacterial strain with intestinal tropism, in particular a lactic acid bacterium, for use in promoting juvenile growth in a context of a conventional diet, in which the bacterial strain responds positively to the linear growth test in mice. By way of example, the following strain may be cited: L. plantarum WJL. Other strains can be identified among bacteria with intestinal tropism and notably among the species and strains mentioned above, notably among strains L. plantarum G821, 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.

(16) In an embodiment of the invention, strain WJL or another strain with a “marked” effect is used as reference strain in order to identify and select bacterial strains having a “marked” effect on juvenile growth, namely an effect close to that of said reference strain, e.g. WJL (effect not significantly different from the reference strain, e.g. WJL), or a “strong” effect on juvenile growth (effect significantly greater than the reference strain, e.g. WJL).

(17) To this end, the mouse test (including 8 mice per condition) is applied to the reference strain, e.g. WJL, and to the strain to be tested (preferably in parallel, or else it is possible to use reference data generated beforehand for strain WJL, for example the data presented in the examples). The mean values obtained for the two strains are then compared. The bacterial strain tested is considered to be a strain with a marked effect if the mean of the size of the individuals and/or of the length of the femurs of the monoxenic group is not significantly different from the corresponding mean for the reference group, e.g. WJL, with a p-value of greater than 0.05 in Tukey's statistical test. The effect is strong if said mean for the strain to be tested is significantly greater than the mean for the reference strain, e.g. WJL, which is the case when the p-value of the statistical test is less than 0.05. The effect is described as intermediate if said mean for the strain to be tested (which was described relative to the axenic mice in the preceding test) is significantly lower than the mean for the reference strain, e.g. WJL, which is the case when the p-value of the statistical test is less than 0.05.

(18) The composition of the invention will preferably comprise at least one bacterial strain having such a marked or strong effect.

(19) The bacterial strains of the invention can be characterised by the fact that they have a positive impact on the serum IGF-1 level. It was therefore possible, on the basis of an axenic mouse model, to show that mice raised on a conventional diet and in the presence of the bacterium (monoxenic mice) had higher growth and, at the same time, a higher serum IGF-1 level, compared to these same mice raised with this conventional diet, but in the absence of the bacterium (axenic mice). This makes it possible to propose a test to determine whether a bacterial strain has the potential to increase the serum IGF-1 level, said test which potentially can be applied in combination with a linear growth test in order to clarify or to refine the effect of the strain on growth.

(20) In this case, the bacterial strains of the invention are characterised by the fact that they respond positively to the following serum IGF-1 level test:

(21) from the same mouse line (typically Balb/c mice) a line of axenic parent mice and a line of monoxenic parent mice (associated with the bacterium to be tested) are established, and juveniles are produced which are raised with the parents on a conventional diet of about 40% carbohydrates, about 25% proteins and about 9% lipids until they are weaned (on day 21); to form the group of monoxenic juveniles, parents mono-associated with the bacterial strain to be tested are used,

(22) on day 21: 8 weaned juveniles from each of these two lines are available, forming the monoxenic group and the axenic group, and they are raised on a conventional diet comprising about 40% carbohydrates, about 25% proteins and about 9% lipids,

(23) on day 56: blood is drawn from the juveniles of each group and the mean serum IGF-1 level is determined for each group; this measurement of the serum IGF-1 level is preferably performed on diluted serum (1:25); commercial ELISA kits for detecting IGF-1 are preferably used, following the manufacturer's instructions,

(24) the lactic acid bacteria strain is considered to respond positively to the test if the mean serum IGF-1 level of the monoxenic group is higher than the mean serum level of the axenic group with a p-value of less than 0.05 in Tukey's statistical test.

(25) The present invention thus has as an object a composition comprising at least one bacterial strain with intestinal tropism, in particular a lactic acid bacterium, for use in promoting juvenile growth under a conventional diet, in which the bacterial strain increases the serum IGF-1 level. It is notably a bacterial strain that responds positively to the IGF-1 test in mice as described above.

(26) The present invention also has as an object a composition comprising at least one bacterial strain with intestinal tropism, in particular a lactic acid bacterium, for use in promoting juvenile growth under a conventional diet, in which the bacterial strain responds positively to the linear growth test in mice and increases the serum IGF-1 level in these same mice. It is notably a bacterial strain that responds positively to the linear growth and IGF-1 tests in mice as described above.

(27) By way of example, the following strain may be cited: L. plantarum WJL. Other strains can be identified among the bacteria with intestinal tropism and notably among the species and strains mentioned above, notably among strains L. plantarum G821, 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.

(28) In an embodiment of the invention, strain WJL or another strain with a “marked” effect is used as reference strain in order to identify and select bacterial strains having a “marked” effect on the serum IGF-1 level, namely an effect close to that of said reference strain, e.g. WJL (effect not significantly different from the reference strain, e.g. WJL), or a “strong” effect on the serum IGF-1 level (effect significantly greater than the reference strain, e.g. WJL).

(29) To this end, the mouse test (including 8 mice per condition) for measuring the serum IGF-1 level is applied to the reference strain, e.g. WJL, and to the strain to be tested (preferably in parallel, or else it is possible to use reference data generated beforehand for the reference strain, e.g. WJL, for example the data presented in the examples). The mean serum IGF-1 levels obtained for the two strains are then compared. The bacterial strain tested is considered to be a strain with a marked effect if the mean IGF-1 level of the monoxenic group is not significantly different from the mean for the reference group, e.g. WJL, with a p-value of greater than 0.05, in Tukey's statistical test. The effect is strong if said mean for the strain to be tested is significantly greater than the mean for the reference strain, e.g. WJL, when the p-value is less than 0.05. The effect is described as intermediate if said mean for the strain to be tested (described beforehand on the preceding axenic test) is significantly lower than the mean for the reference strain, e.g. WJL, when the p-value is less than 0.05.

(30) The composition of the invention will thus preferably comprise a bacterial strain having such a marked or strong effect, and in particular said bacterial strain has a marked or strong effect both on linear growth and on the serum IGF-1 level.

(31) The composition can be used in domesticated animals in the broad sense, notably including grazing animals (livestock), farmyard animals, aquatic animals, pets. In particular, the composition can be used on mammals, in particular production animals (cattle, sheep, goats, pigs, fowl), pets (dogs, cats) and sporting animals (horses, dromedaries, camels), preferably between weaning and sexual maturity, or between weaning and the adult stage (characterised by reaching adult size, end of skeletal growth), referred to herein as juvenile animals. The composition can be administered to castrated animals, notably before castration and/or in the remaining post-castration period of skeletal growth. It can also be used in fish farming. In an embodiment, the animal is a carnivore. In another embodiment, the animal is a ruminant.

(32) The composition can notably contain an amount of about 10.sup.5 to about 10.sup.12, notably of about 10.sup.6 to about 10.sup.12, preferably of about 10.sup.8 to about 10.sup.12 colony-forming bacterial cells (CFU) according to the invention, per gram of composition. CFU stands for the English technical expression “colony-forming units”. “Per gram of composition” is preferably understood to mean the probiotic composition consisting of bacteria, co-ingredients, and excipients or vectors. “Bacterial cells” is understood to mean a single strain of bacterium in accordance with the invention or a mixture of at least two bacteria, in accordance with the invention.

(33) The composition can notably comprise the one or more lactic acid bacteria in live form. The composition can be in a ready-to-use form or in a form to be mixed or diluted with a food, an excipient or a liquid food such as drinking water.

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

(35) It can be a lyophilised powder, which can be used as such, in powder, granule, tablet, bolus, hard capsule or soft capsule form, or used after incorporation in a suitable vehicle. This composition can comprise a conventional lyophilisation excipient.

(36) The composition can be an oral administration form (for example powder, soft capsule, tablet, bolus) in a gastro-protected form so as to pass through the stomach and release the bacteria in the intestine.

(37) According to an embodiment, the composition is a solid composition, e.g. tablet, bolus, soft capsule, hard capsule, gastro-protected so as to pass through the stomach and release the bacteria in the intestine. According to one mode, the form, notably granules, can be used as feed in aqueous medium, notably for fish.

(38) According to an embodiment, the composition is a liquid or is to be dissolved in a liquid or to be suspended in a liquid, for example drinking water; in this case the composition is initially solid, e.g. powder, granule, dissolvable tablet, for example effervescent tablet, or deliquescent tablet in the liquid.

(39) According to an embodiment, the composition is solid, e.g. powder, granule, tablet, bolus, and intended or suitable for mixing with food.

(40) According to an embodiment, the composition is solid, in a ready-to-use form without the need for mixing with food, but can nevertheless be mixed with food, for example a palatable tablet or bolus.

(41) The invention also has as an object a food for animal-breeding containing at least one bacterium or composition of the invention. By way of example, the food comprises the composition of the invention mixed with at least carbohydrates, proteins and lipids. The food can be in a liquid, emulsion or solid form, and among the solid forms mention may be made, by way of example, of granules, boluses, flakes, silage added to the composition, fodder added to the composition. In particular, the food can be a complete food, a functional food, a milk replacer, a formulated food, a concentrated supplement.

(42) The invention also has as an object a probiotic treatment method for promoting juvenile growth in livestock, comprising the administration to an animal according to the invention of a composition according to the invention. Preferably, the composition is administered via the oral route. Preferably, the composition is administered several times during a period ranging from weaning to sexual maturity.

(43) The invention also has as an object the animal-breeding method integrating said probiotic treatment.

(44) Said probiotic treatment or breeding method comprises the administration of a sufficient amount of a composition as described above to the young animal, preferably post-weaning. The method will comprise one or more administrations, which can be spread over the growth period of the subject (until sexual maturity), of doses of the composition of the invention. The doses can be divided to facilitate administration. The frequency of administration is notably between one dose (single or divided) every day and one dose every month. Typically, the frequency of administration will be between one dose (single or divided) every day and one dose every week, or even every 2, 3, 4, 5 or 6 days. Each dose (single or divided) notably represents several grams to several tens of grams of composition.

(45) The method notably comprises the administration of a composition comprising an amount of about 10.sup.5 to about 10.sup.12, in particular of about 10.sup.6 to about 10.sup.12, preferably of about 10.sup.8 to about 10.sup.12 colony-forming bacterial cells (CFU) per gram of composition (“bacterial cells” means a single strain of bacterium in accordance with the invention or a mixture of at least two bacteria in accordance with the invention).

(46) The method preferably comprises the administration of a composition comprising the one or more lactic acid bacteria in live form.

(47) The form of the composition can be solid or liquid and take any of the forms presented above.

(48) In an embodiment, the composition of the invention, notably the composition used in the treatment method, comprises at least one bacterial strain that is not naturally occurring in the treated species. In this configuration, when there are several different bacteria, it is sufficient that one of them is not naturally occurring. On the other hand, this bacterium will have intestinal tropism in the animal species and meet the definition of active strains according to the invention.

(49) The invention also relates to a method for screening bacteria capable of promoting juvenile growth under a conventional diet, using an axenic mouse model.

(50) The method includes the following steps: juveniles are provided from two lines derived from the same mouse strain (typically Balb/c mice), namely a line of axenic parent mice and a line of monoxenic parent mice (associated with the bacterium to be tested), they are raised on a conventional diet comprising about 40% carbohydrates, about 25% proteins and about 9% lipids, at the end of a suitable rearing period, the mean value of one or more parameters is determined for each group, said parameters being related to growth (for example weight gain, linear growth for example by measuring the size of the individuals or the length of their femurs), and/or to the serum IGF-1 level, the lactic acid bacteria strain is considered to respond positively to the test if the mean value of the parameter measured in the monoxenic group is higher than the mean value of the parameter measured in the axenic group with a p-value of less than 0.05 in Tukey's statistical test.

(51) Preferably, the screening method adopts the features of the mouse test of linear growth or of serum IGF-1 level described above.

(52) The screening method can also be a comparative test with a reference strain, for example strain WJL, and this test thus adopts the features described above for the mouse tests.

(53) The various tests described are quite capable of being performed with a different diet, as long as it is a diet suitable for mice.

(54) The invention will now be described in greater detail using embodiments of the invention taken as non-limiting examples.

(55) The male offspring (minimum 8 individuals) of three groups of individuals from the same colony of axenic mice were studied, the first group consisting of axenic juveniles (germ-free (GF) group), the second of juveniles from parents mono-associated with strain L. plantarum WJL (WJL group), and the third of juveniles from parents mono-associated with strain L. plantarum NIZO2877 (NIZO2877 group). The parents and juveniles are raised on a conventional diet (40% carbohydrates, 25.1% proteins, 9.1% lipids and 3646 kcal/kg) until the juveniles are weaned (day 21 post-birth), then the weaned juveniles are raised on a conventional diet until day 56.

(56) Four parameters illustrating the juvenile growth of these individuals were studied: the primary parameter being linear growth or size increase (measured from the nose to the base of the tail) for a period of 35 days following weaning (days 21 to 56), then three secondary parameters, namely (1) weight gain for a period of 35 days following weaning (days 21 to 56), (2) femur length of a set of individuals (at least 8 individuals) representative of the population tested on day 56, and finally (3) the serum level on day 56 of growth factor IGF-1 in at least 8 individuals.

(57) Statistical analyses were performed using the t-test with the GraphPad software (GraphPad PRISM 5.04, San Diego, USA); values of p<0.05 are considered significant.

(58) (1) Weight and Size Increase:

(59) The mice were anaesthetised by brief exposure to isoflurane in order to measure their weight and size (from the nose to the base of the tail) on day 21 and day 56.

(60) TABLE-US-00001 TABLE 1 Weight gain, days 21-56, in g/day Lp GF Lp WJL NIZO2877 0.272286 0.357619 0.319337 0.231714 0.369333 0.353623 0.242857 0.353333 0.31648 0.34 0.341905 0.291051 0.263429 0.343333 0.283623 0.311429 0.359048 0.250765 0.331429 0.293333 0.276765 0.322857 0.301905 0.385051 0.308571 0.345051 0.345051 0.373623 0.327908 Mean 0.291619111 0.339976125 0.322360667 Standard error of 0.01328949 0.009779005 0.011782513 the mean Standard 0.03986847 0.027659203 0.040815822 deviation t-test for unpaired series Lp WJL vs. GF p = 0.0118 Lp WJL vs. Lp NIZO2877 p = 0.3014 Lp NIZO2877 vs. GF p = 0.1008 Lp = L. plantarum

(61) TABLE-US-00002 TABLE 2 Size increase, days 21-56, in cm/day Lp GF Lp WJL NIZO2877 0.054286 0.072508 0.056122 0.062857 0.063937 0.061837 0.057143 0.075365 0.061837 0.06 0.078222 0.061837 0.054286 0.069651 0.056122 0.057143 0.081079 0.05898 0.057143 0.063937 0.05898 0.06 0.066794 0.05898 0.048571 0.061837 0.067551 0.073265 0.061837 Mean 0.056825444 0.071436625 0.06159875 Standard error 0.001383791 0.002286716 0.001382733 of the mean Standard 0.004151373 0.006467809 0.004789929 deviation t-test for unpaired series Lp WJL vs. GF p < 0.0001 Lp WJL vs. Lp NIZO2877 p = 0.0010 Lp NIZO2877 vs. GF p = 0.0275

(62) (2) Femur Length:

(63) The mice are sacrificed on day 56; a femur is removed and stripped of muscle and its length is measured with a Vernier caliper.

(64) TABLE-US-00003 TABLE 3 Femur length in mm on day 56 GF Lp WJL Lp NIZO2877 13 13.7 13.2 13.1 13.8 13.3 13.1 13.6 13.4 13.3 13.8 13.2 13.7 13.5 13.2 13.5 13.8 13.6 13.5 13.5 13.3 13.7 13.6 13.6 13.8 13.6 14 13.5 Mean 13.3625 13.6625 13.475 Standard 0.09808433 0.046049275 0.073983004 error of the mean Standard 0.277424378 0.130247018 0.256284643 deviation t-test for unpaired series Lp WJL vs. GF p = 0.0151 Lp WJL vs. Lp NIZO2877 p = 0.0735 Lp NIZO2877 vs. GF p = 0.3641

(65) (3) Serum IGF-1 Levels:

(66) IGF-1 levels are measured on serum obtained from the blood of mice sacrificed on day 56. The measurement is performed on diluted serum (1:25) using the ELISA Ready-SET-Go Kit (eBioscience, USA), following the manufacturer's instructions.

(67) TABLE-US-00004 TABLE 4 IGF-1 level in ng/mL on day 56 GF Lp WJL Lp NIZO2877 67.2375 45.51875 69.89375 44.83125 79.70625 61.3 47.6125 71.6125 42.39375 42.4875 72.425 48.55 39.3 38.55 39.55 44.01875 40.425 31.8 42.1125 59.175 53.45625 39.3 47.76875 51.4875 41.32248 41.14375 50.11075 40.3 34.41869 26.7375 31.5581 34.39375 41.00821 Mean 43.48601769 56.89765625 45.08385417 Standard 2.402432601 5.675518549 3.594197638 error of the mean Standard 8.662093929 16.05279061 12.45066584 deviation t-test for unpaired series Lp WJL vs. GF p = 0.0217 Lp WJL vs. Lp NIZO2877 p = 0.0802 Lp NIZO2877 vs. GF p = 0.7111

(68) The results illustrate a “marked” effect of strain L. plantarum WJL on linear growth (higher mean value and value of p<0.05 compared to the axenic condition) and an “intermediate” effect of strain L. plantarum NIZO2877 (higher mean value and value of p<0.05 compared to the axenic condition and lower mean value and value of p<0.05 compared to the L. plantarum WJL condition). The effect of strain L. plantarum WJL is confirmed with the secondary parameters, namely weight gain, IGF-1 level and femur length compared to the axenic condition (higher mean value and value of p<0.05). However the intermediate effect of strain L. plantarum NIZO2877 is not confirmed on the secondary parameters of the study. The secondary parameters cannot therefore be used to identify the quantitative effect of the strain tested; only the primary parameter (linear growth) makes this possible.

(69) All these results demonstrate, by scientific evidence, the juvenile growth-promoting effect of certain Lactobacillus strains and exemplify a “marked” effect or a “moderate” effect of certain strains on linear growth. A “strong” effect will be obtained with certain strains, said strong effect corresponding to a higher mean linear growth value and a value of p<0.05 compared to the L. plantarum WJL condition.

(70) Strains are available at the ATCC, the Pasteur Institute of Paris, the Pasteur Institute of Lille, or published in the scientific literature and available from the principal investigators of the publications mentioned:

(71) TABLE-US-00005 Public collection and/or publication with genome sequence Lactobacillus plantarum WJL Kim et al., Genome Announc. 21 Nov. 2013, 1(6).Pil: e00937-13 Lactobacillus plantarum NIZO2877 NIZO (2877) Lactobacillus casei ATCC 393 ATCC (393) Lactobacillus fermentum ATCC 9338 ATCC (9338) Lactobacillus fermentum KLD Pasteur Institute of Lille (A5.20) Lactobacillus fermentum LMG Pasteur Institute of Lille (A5.16) Lactobacillus paracasei ATCC 25302 ATCC (25302) Lactobacillus paracasei BL23 Pasteur Institute of Lille (A3.6) and Mazé et al., J. Bacteriol., May 2010; Lactobacillus paracasei Shirota 192(10): 2647-8 Lactobacillus delbrueckii spp. bulgaricus Pasteur Institute of Lille (A3.5) Lactobacillus casei L919 ATCC (11842) and van de Guchte M, et al., Proc. Natl Acad Sci USA 13 Jun. Lactobacillus rhamnosus L900 2006 Lactobacillus rhamnosus L908 Koryszewska-Baginska A et al., Genome Announc, 26 Sep. 2013 Lactobacillus rhamnosus GG Aleksandrzak-Piekarczyk T et al. Genome Announc, 15 Aug. 2013 Lactobacillus plantarum G821 Koryszewska-Baginska A et al. Genome Announc, 20 Feb. 2014 ATCC (53103) and Kankainen M et al., Proc Natl Acad Sci USA, 6 Oct. 2009 CNCM I-4979

(72) All public documents cited herein are incorporated by reference. Similarly, the skilled person may refer to these various documents and to the deposited commercial strains to which reference is made herein.