LACTOBACILLUS COMPOSITION ALLOWING THE STIMULATION OF HUMAN AND ANIMAL JUVENILE GROWTH IN CASES OF MALNUTRITION

Abstract

The invention relates to a pharmaceutical or probiotic composition comprising at least one Lactobacillus strain with intestinal tropism, especially selected from the species Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus casei, used to stimulated juvenile growth in cases of malnutrition especially characterised by a protein deficiency. The strains can be selected from a vinegar fly model and/or a mouse model. The invention also relates to a method for probiotic treatment using said composition.

Claims

1. The composition comprising at least one Lactobacillus strain with intestinal tropism, selected from among the species Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, for a use aiming at promoting juvenile growth in the case of malnutrition.

2. The composition according to claim 1, wherein the Lactobacillus strain is selected from among L. plantarum WJL, L. plantarum G821, L. casei ATCC 393, L. casei L919, L. fermentum ATCC9338.

3. The composition according to claim 14, wherein the Lactobacillus strain positively reacts to the following test: a batch of embryos from axenic Drosophila melanogaster parents is available; on D1: these embryos are distributed into at least 2 groups (40 embryos, produced in triplicate i.e. 120 embryos per group) on a nutritional medium comprising maize flour, agar-agar and water, deficient in yeast, one of the embryo groups is further inoculated with a suspension of about 10.sup.8 CFUs of the bacterium to be tested in a saline buffer, which forms the monoxenic group, the other group being the axenic group, the breeding of both groups is conducted at about 25 C. up to D7; on D7: at least 60 Drosophila larvae of each group are recovered, a thermal shock is applied to them; the average of the length of the larvae of each of the group is determined and the obtained averages are compared; the tested bacterial strain is considered as positively reacting to the test if the average of the length of the larvae of the monoxenic group is greater than the average of the length of the larvae of the axenic group with a value p less than or equal to 0.001 in the Mann-Whitney statistical test, conducted on the whole of the set of data of the sizes of the larvae of both groups.

4. The composition according to claim 3, comprising a bacterial strain which, relatively to the strain L. plantarum WJL, in this same test on Drosophila, has a strong effect with an average of the length of the larvae of the monoxenic group with this bacterial strain significantly greater than the average for the monoxenic group with the strain WJL, when the value of p is less than or equal to 0.001 in the Mann-Whitney statistical test, conducted on the whole of the set of data of the sizes of the larvae of both groups.

5. The composition according to claim 1, wherein the Lactobacillus strain positively reacts to the following test: from a same line of mice, a line of axenic parent mice is established and a line of monoxenic parent mice (associated with the bacterium to be tested), and juveniles are produced which are bred with the parents in a conventional nutritional medium up to their weaning (D21); on D21: 8 weaned juveniles issued from each of these two lines are available, forming the monoxenic group and the axenic group, and they are raised in a protein-deficient (8%) and lipides (2.5%) nutritional diet; on D56: the average of the sizes of the mice for each relevant group is determined by measuring the end of the snout to the base of the tail of each individual, and the femurs of the individuals of each group are taken and the latter are measured; the lactic bacterium strain being considered as positively reacting to the test if the average size of the individuals or of the femurs of the monoxenic group is greater than the average size of the individuals or of the femurs, of the axenic group, with a value of p of less than or equal to 0.05 in the Tukey statistical test.

6. The composition according to claim 5, comprising a bacterial strain which, relatively to the strain L. plantarum WJL, in this same test on mice, has a strong effect with an average of the size of the mice or of their sizes of the monoxenic group with this bacterial strain significantly greater than the average for the monoxenic group with the strain WJL, when the value of p is less than or equal to 0.05 in the Tukey statistical test.

7. The composition according to claim 1, wherein the Lactobacillus strain positively reacts to the following test: from a same line of axenic 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 in a conventional nutritional medium up to their weaning (D21), on D21: 8 weaned juveniles are available stemming from each of both of these lines, forming the monoxenic group and the axenic group, and they are raised in a protein-deficient (8%) and lipid-deficient (2.5%) nutritional diet; on D56: blood of the juveniles of each group is sampled and the average IGF-1 serum level is determined for each group; the lactic bacterial strain being considered as positively reacting to the test of the average IGF-1 serum level of the monoxenic group is greater than the average serum level of the axenic group with a value of p of less than or equal to 0.05 in the Tukey's statistical test.

8. The composition according to claim 7, comprising a bacterial strain which, relatively to the strain L. plantarum WJL, in this same test on mice, has a strong effect with an average IGF-1 serum level of the monoxenic group with this bacterial strain significantly greater than the average for the monoxenic group with the strain WJL, when the value of p is less than or equal to 0.05 in the Tukey statistical test.

9. A composition comprising a bacterial strain with intestinal tropism for a use aiming at promoting juvenile growth in the case of malnutrition, wherein the bacterial strain positively reacts to the following test: a batch of embryos of axenic Drosophila melanogaster parents; on D1: these embryos are distributed into at least 2 groups (40 embryos, produced in triplicate i.e. 120 embryos per group) on a nutritional medium comprising maize flour, agar-agar and water, deficient in yeast, one of the groups of embryos is inoculated with a suspension of about 10.sup.8 CFUs of said bacterium in a saline buffer, which forms the monoxenic group test, the other group being inoculated with a suspension of about 10.sup.8 CFUs of a reference bacterium L. plantarum WJL, L. casei ATCC 393, L. fermentum ATCC 9338 or L. paracasei ATCC 25302, in a saline buffer, forming the monoxenic reference group, the raising of both groups is conducted at about 25 C. until D7; on D7: at least 60 Drosophila larvae of each group are recovered, a thermal shock is applied to them; the average of the length of the larvae of each of the groups is determined, and the obtained averages are compared; said bacterial strain having, relatively to the reference bacterium, a strong effect with an average of the length of the larvae of the monoxenic group test with this bacterial strain significantly greater than the average for the monoxenic group with the reference strain, with a value of p of less than or equal to 0.001, in the Mann-Whitney statistical test, conducted on the whole of the set of data of the sets of larvae of both groups.

10. The composition according to claim 1, for use as a drug or probiotic intended for promoting juvenile growth in the case of malnutrition characterized by protein-deficiency.

11. The composition according to claim 1, for use as a drug or probiotic intended to promote juvenile growth in the case of malnutrition characterized by an IGF-1 serum level less than the normal level.

12. The composition according to claim 1, wherein the bacterium is a Lactobacillus plantarum.

13. The composition according to claim 12, wherein the bacterium is a Lactobacillus plantarum G821 deposited at the Collection Nationale de Culture de Microorganismes (Institut Pasteur) under the registration number CNCM I-4979.

14. The composition according to claim 1, containing from 10.sup.5 to 10.sup.12-CFUs of Lactobacillus positively reacting to the test, per gram of composition.

15. A probiotic treatment method for promoting juvenile growth in the case of malnutrition, comprising the administration to a patient in need thereof, of a composition according to claim 1.

16. A method for screening bacteria capable of promoting juvenile growth in the case of malnutrition of the mammals, notably humans, productive animals, pet animals and sport animals, wherein the bacterial strain is subject to the following test: a batch of embryos of axenic Drosophila melanogaster parents is available; these embryos are placed in at least 2 groups on a nutritional medium comprising a nutrient medium for Drosophilas deficient in yeast, one of the groups of embryos is further inoculated with a suspension of the bacterium to be tested, which forms the group of monoxenic individuals; the other group forms the axenic group, the breeding is conducted at a suitable temperature, at the end of a suitable breeding period, Drosophila larvae of each group (axenic larvae and monoxenic larvae) are recovered, the average of the length of the larvae of each of the groups is determined and the obtained averages are compared; the tested bacterial strain is considered as reacting positively to the test if the average of the length of the larvae of the monoxenic group is greater than the average of the length of the larvae of the axenic group with a value of p of less than or equal to 0.001, in the Mann-Whitney test, conducted on the whole of the set of data.

17. A method for screening bacteria capable of promoting juvenile growth in the case of malnutrition, wherein the bacterial strain is subject to the following test: juveniles are available issued from two lines issued from a same mouse strain, i.e. a line of axenic mice and a line of monoxenic mice, they are raised in a protein-deficient and also preferably lipid-deficient nutritional diet; at the end of a suitable breeding period, the average of the values of one or several parameters of each group is determined, these parameters being related to the IGF-1 serum level or to the growth, the lactic bacterial strain is considered as positively reacting to the test if the average IGF-1 level of the monoxenic group is greater than the average level of the axenic group with a value of p of less than or equal to 0.05 in the Tukey statistical test.

18. A composition, a pure culture or an isolate comprising the Lactobacillus plantarum G821 bacterium deposited at the Collection Nationale de Culture de Microorganismes (Institut Pasteur) under the registration number CNCM I-4979.

Description

[0087] The invention will now be described in more details by means of the embodiments of the invention taken as non-limiting examples and referring to the drawing wherein:

[0088] FIG. 1 is a graph showing the growth rate (in cm/day) of mice from D28 to D56; GF: axenic mice; WJL: mice bred in the presence of the strain L. plantarum WJL; NIZO 2877: bred mice in the presence of the strain L. plantarum WJL NIZO 2877; * p<0.05; ***p<0.001;

[0089] FIG. 2 is a graph showing the size of femurs on D56, in GF mice, WJL or NIZO 2877; * p<0.05; ***p<0.001;

[0090] FIG. 3 is a graph showing the serum levels (in ng/ml) of male mice on D56, groups GF, WJL or NIZO 2877* p<0.05; ***p<0.001;

[0091] FIG. 4 is a graph showing the size of larvae of Drosophila at 7 days under conditions of undernourishment: a negative control group (no addition of bacteria on the growth medium after laying), and groups characterized by the addition on the growth medium of the strain L. plantarum NIZO 2877, WJL or G821.

EXAMPLE 1

Functional Screening of Bacteria on Monoxenic Drosophila

[0092] D-1: adult Drosophilae are used (axenic D. melanogaster yw); they are placed in laying cages including a bottom (of the Petri dish type) on which is placed a conventional nutritional medium; laying is ensured by maintaining the adult population in the cage for 1 night at 25 C.

[0093] D0: 6 parts of the nutritional medium are cut out so as to collect 640 embryos, each of the parts are placed on a deficient nutritional medium contained in a Petri dish, three boxes are inoculated with sterile PBS 1 and then the three other ones with a suspension of the bacterium in PBS 1.

[0094] Incubation is performed for 7 days at 25 C.

[0095] D7: Next, from each box, the larvae which have developed therein (the experiment may be utilized from 20 larvae per box, we therefore used hereafter groups of more than 60 individuals) are recovered. The larvae undergo a shock thermal treatment (5 seconds on a hob heated to 100 C.), the size of the larvae is then measured.

[0096] This procedure was applied to different bacterial strains, and the experiments and results are summarized in the following Table 1:

TABLE-US-00001 TABLE 1 Sizes of the larvae at D7 (mm) Statistical analysis Average Comparison/axenic animals Comparison/L. plantarum WJL Interpretation of the (n > 60. Standard Value of p Statistical Value of p Statistical qualitative test mm) deviation (1) significance (1) significance (quantitative level) Axenic 2.693 0.497 <0.0001 **** Lactobacillus plantarum WJL 3.644 0.883 <0.0001 **** GPS (marked) Lactobacillus plantarum NIZO2877 3.398 0.750 <0.0001 **** 0.0001 *** GPS (intermediate) Lactobacillus casei ATCC 393 4.391 0.802 <0.0001 **** 0.0721 ns GPS (marked) Lactobacillus fermentum ATCC 9338 4.133 0.824 <0.0001 **** >0.9999 ns GPS (marked) Lactobacillus fermentum KLD 2.741 0.626 >0.9999 ns <0.0001 **** No effect on growth Lactobacillus fermentum LMG 3.287 0.799 0.1423 ns 0.0015 ** No effect on growth Lactobacillus paracasei ATCC 25302 3.987 0.524 <0.0001 **** 0.4258 ns GPS (marked) Lactobacillus paracasei BL23 2.682 0.482 >0.9999 ns <0.0001 **** No effect on growth Lactobacillus paracasei Shirota 3.407 0.779 <0.0001 **** 0.0984 ns GPS (marked) Lactobacillus delbrueckii 2.540 0.599 >0.9999 ns <0.0001 **** No effect on growth spp. bulgaricus ATCC 11842 Lactobacillus casei L919 4.150 0.712 <0.0001 **** 0.2966 ns GPS (marked) Lactobacillus rhamnosus L900 3.920 0.572 <0.0001 **** >0.9999 ns GPS (marked) Lactobacillus rhamnosus L908 4.112 0.576 <0.0001 **** 0.1227 ns GPS (marked) Lactobacillus rhamnosus GG 4.036 0.488 <0.0001 **** 0.5272 ns GPS (marked) *Significantly greater than the reference value *Significantly less than the reference value GPS = Growth promoter strain

[0097] (1) Statistical Test Used: Mann-Whitney Test.

Indications on the Validation Criteria of the <<Larval Growth>> Phenotype Test

[0098] The strain should at least fulfill criterion 1 in order to be validated and the extent of its activity may be described according to the criterion 1+2.

[0099] Qualitative Criterion 1=Growth Promoting Strain Relatively to the Axenic Condition: A statistical test is applied to the whole of the values of length of the individuals associated relatively to the values of the axenic individuals (at least 60 larvae per condition). A value p of less than 0.001 (Mann-Whitney test) describes the strain as growth promoting. In the present case, the following strains were described as growth-promoting:

Lactobacillus plantarum WJL
Lactobacillus plantarum NIZO2877
Lactobacillus casei ATCC 393
Lactobacillus fermentum ATCC 9338
Lactobacillus paracasei ATCC 25302
Lactobacillus paracasei Shirota
Lactobacillus casei L919
Lactobacillus rhamnosus L900
Lactobacillus rhamnosus L908
Lactobacillus rhamnosus GG

[0100] The following strains do not have any growth-promoting effect:

Lactobacillus fermentum KLD
Lactobacillus fermentum LMG
Lactobacillus paracasei BL23
Lactobacillus delbrueckii spp. bulgaricus ATCC 11842

[0101] Quantitative Criterion 2=Extent of the Growth Promoting Effect (with Criterion 1 Having been Validated Beforehand):

[0102] A statistical test is applied to the whole of the values of length of the individuals associated with the tested strain(s) relatively to the values of the mono-associated individuals with the strain L.plantarum WJL (at least 60 larvae per condition).

[0103] Three scenarii occur: The value p of the statistical test is greater than 0.001 (Mann-Whitney test), the strain will be described as having a <<marked>> effect on the larval growth (similar effect to the strain L.plantarum WJL).

[0104] In the present case, the following strains have a marked effect:

Lactobacillus casei ATCC 393
Lactobacillus fermentum ATCC 9338
Lactobacillus paracasei ATCC 25302
Lactobacillus paracasei Shirota
Lactobacillus casei L919
Lactobacillus rhamnosus L900
Lactobacillus rhamnosus L908
Lactobacillus rhamnosus GG
Lactobacillus plantarum WJL

[0105] The value p of the statistical test is less than 0.001 (Mann-Whitney test), the strain will be described as having an intermediate effect on larval growth if the average size of the individuals is less than those of the individuals associated with L.plantarum WJL (case of the strain L.plantarum NIZO 2877 in the presented example). The strain will be described as having a strong effect if the average size of the individuals is greater than those of the individuals associated with L.plantarum WJL (a non-identified case in the present example).

[0106] Analysis of the <<Larval Growth>> Phenotype Test Example: [0107] 14 Lactobacillus strains were tested (Table 1) in order to determine their growth promoting potential by means of the <<larval growth>> test in Drosophila melanogaster. [0108] These strains are available from ATCC, at the Institut Pasteur of Paris, at the Institut Pasteur of Lille or published in the scientific literature and available from the referee researchers of the mentioned publications (Table 2).

TABLE-US-00002 TABLE 2 Public collection and/or publication with sequence of the genome Lactobacillus plantarum Kim et al., Genome Announc. Nov. 21, 2013, WJL 1(6). Pil: e00937-13 Lactobacillus plantarum NIZO (2877) NIZO2877 Lactobacillus casei ATCC (393) ATCC 393 Lactobacillus fermentum ATCC (9338) ATCC 9338 Lactobacillus fermentum Institut Pasteur of Lille (A5.20) KLD Lactobacillus fermentum Institut Pasteur of Lille (A5.16) LMG Lactobacillus paracasei ATCC (25302) ATCC 25302 Lactobacillus paracasei Institut Pasteur of Lille (A3.6) et Maz et al., BL23 J. Bacteriol., May 2010; 192(10): 2647-8 Lactobacillus paracasei Institut Pasteur of Lille (A3.5) Shirota Lactobacillus delbrueckii ATCC (11842) et van de Guchte M, et al., spp. bulgaricus Proc. Natl Acad Sci USA Jun. 13, 2006 Lactobacillus casei Koryszewska-Baginska A et al., Genome L919 Announc, Sep. 26, 2013 Lactobacillus rhamnosus Aleksandrzak-Piekarczyk T et al. Genome L900 Announc, Aug. 15, 2013 Lactobacillus rhamnosus Koryszewska-Baginska A et al. Genome L908 Announc, Feb. 20, 2014 Lactobacillus rhamnosus ATCC (53103) and Kankainen M et al., GG Proc Natl Acad Sci USA, Oct. 6, 2009 [0109] The size of a minimum of 60 individuals per condition is studied on day 7 after association with the bacterial strain to be tested, and then cultivation of the juvenile individuals in a yeast-deficient nutritional medium. Table 1 shows the average and the standard deviation of these sets of data. [0110] The statistical analysis of these results gives the possibility of qualifying according to criterion 1 of the <<larval growth test>>, 10 strains of different species of Lactobacilli. This selection brings to light a strictly strain-specific functional effect and is not necessarily related to a given species of Lactobacillus. [0111] Statistical analysis of the results gives the possibility according to criterion 2, of describing as <<intermediate>> the effect of the strain L.plantarum NIZO 2877 while the 9 other strains promoting growth have a <<marked>> effect.

Example 2

Description of a Strain with a Strong Effect on the Drosophila Model

[0112] The Drosophila test of Example 1 is reproduced with 3 bacterial strains: [0113] Lactobacillus plantarum WJL [0114] Lactobacillus plantarum NIZO2877 [0115] Lactobacillus plantarum G821

[0116] The analysis of the results (see FIG. 4) shows the following effects:

Axenic versus NIZO 2877 p<0.001 (***)
NIZO 2877 versus WJL p<0.0001 (****)
G821 versus WJL p<0.0001 (****)
Axenic versus WJL ou G821 p<0.0001 (****)

Conclusion:

[0117] Lactobacillus plantarum WJL: marked effect [0118] Lactobacillus plantarum NIZO2877: intermediate effect [0119] Lactobacillus plantarum G821: strong effect.

Example 3

Functional Screening of Bacteria on MicePhenotype Analysis

[0120] Study in mice of the juvenile growth promoting effect of two strains with <<intermediate>> and <<marked>> effect (L. plantarum NIZO2877 and L.plantarum WJL, respectively) identified by means of the <<larval growth>> test of Example 1.

[0121] The male progeny (a minimum of 15 individuals) of three groups of individuals stemming from a same axenic mouse colony was studied, the first group consists in axenic juveniles (group GF), the second one in juveniles issued from mono-associated parents with the strain L.plantarum WJL (group WJL), and the third one in juveniles stemming from mono-associated parents with the strain L.plantarum NIZO2877 (group NIZO 2877). The parents and juveniles are cultivated in a conventional nutritional medium until weaning of the juveniles (J21), and then the weaned juveniles are cultivated in a protein-deficient and lipid-deficient (8% and 2.5% respectively) nutritional medium up to D56. [0122] Conventional nutritional medium: 23% proteins and 5% lipids. [0123] Deficient nutritional medium: 8% proteins and 2.5% of lipids.

[0124] Three parameters illustrating the juvenile growth of these individuals were studied: (1) the increase in size for a period of 28 days following weaning (from D28 to D56), (2) the size of the femurs of a batch of individuals (a minimum of 9 individuals) representative of the tested population at D56 and finally (3) the serum level at D56 of the IGF-1 growth factor in at least 8 individuals.

[0125] The statistical analyses were carried out by using Tukey's test by using the piece of software GraphPad (GraphPad Prism 5.04, San Diego, USA); values of p<0.05 are considered as significant.

[0126] (1) Increase in Size:

[0127] The mice were anesthetized by brief exposure to isoflurane in order to allow measurement of the size of the mice (from the snout to the base of the tail) on D28 and D56.

TABLE-US-00003 TABLE 3 growth rate (size increase per day in cm from D28 to D56) - Nb = number of mice GF WJL NIZO 2877 Standard Standard Standard Average deviation Nb Average deviation Nb Average deviation Nb 0.007321 0.009878 15 0.031114 0.009376 17 0.016429 0.011071 25 [0128] These data are collected in FIG. 1.

[0129] (2) Size of the Femurs:

[0130] The mice were sacrificed on D56, a femur was sampled, released from the muscle and its length was measured with the Vernier caliper.

TABLE-US-00004 TABLE 4 Length of the femurs in mm on D56 in males GF WJL NIZO 2877 10.2 11.2 10.9 10.2 11.6 11 10.5 11.9 10.5 9.9 11.6 11.2 10.4 12 11 10 11.4 11 10.2 12 11 10.2 11.7 11.2 9.8 12 11 11.9 11.6 11 [0131] These data are collected in FIG. 2.

[0132] (3) Serum Titers of IGF-1:

[0133] The IGF-1 titers are measured on the serum obtained from the blood of the sacrificed mice at D56. The measurement is conducted on diluted serum (1:25) by using the ELISA Ready-Set-Go kit (eBioscience, USA), by following the instructions of the manufacturer.

TABLE-US-00005 TABLE 5 Serum IGF-1 titer in pg/ml (dual measurement from ELISA plates) GF WJL NIZO 2877 4526.75 4343.75 8823.25 8606.5 7291.25 6998.75 2825.5 2614.75 9258.5 9122.25 5128 5102 2667.5 2773 8471.25 8660.5 4343.75 4396 3455.25 3219.5 7025.25 7078.25 5232.75 5180.5 7131.5 6839.5 11063 9557.3 8471.25 8282.5 2164.2 2057.55 15125 14409 4866.5 4761.75 3140.75 3035.75 10023 9372 6363.25 6152.25 2509 2456.15 11735 12026 7211.25 6680.5 2588.25 2614.75 4213.25 4161 4432.5 4601.3 4770.5 4517 4013.3 3930 4940.3 3846.5 3597.3 3846.5

[0134] These data are collected in FIG. 3.

[0135] The results illustrate a <<marked>> effect of the strain L.plantarum WJL on the growth rate (value p<0.001 relative to the axenic condition) and an <<intermediate>> effect of the strain L.plantarum NIZO2877 (value p<0.05 relatively to the axenic condition and value p<0.001 relatively to the condition L.plantarum WJL). These effects are confirmed with the <<IGF-1 level>> parameter (same ranges of p values of the different tests), but reinforced with the <<femur size>> parameter (value p<0.001 between the axenic conditions and L.plantarum NIZO 2877).

[0136] The whole of these results demonstrates by scientific proof, the juvenile growth promoting effect of certain strains of Lactobacilles also positively reacting to the <<larval growth>> test.

[0137] All the public documents quoted here are incorporated by reference. Also, one skilled in the art may refer to these diverse documents and to the deposits of commercial strains to which reference is made here.