EP4181938A1

EP4181938A1 - Combination of lactobacillus strains and use thereof in animal health

Info

Publication number: EP4181938A1
Application number: EP21755531.7A
Authority: EP
Inventors: Nuria VIECO SAIZ; Ruth RASPOET; Eric Auclair; Djamel Drider; Frédérique GANCEL
Original assignee: Lesaffre et Cie SA; Universite Lille 2 Droit et Sante
Current assignee: Lesaffre et Cie SA; Universite Lille 2 Droit et Sante
Priority date: 2020-07-20
Filing date: 2021-07-19
Publication date: 2023-05-24
Also published as: BR112023000712A2; WO2022018373A1; MX2023000840A; CN116075237A; US20230285476A1; FR3112557A1

Abstract

The present invention relates to a mixture of microorganisms comprising a Lactobacillus reuteri strain and a Lactobacillus salivarius strain, and to the use thereof for preparing an animal feed additive or product and for preventing or treating necrotic enteritis.

Description

COMBINATION OF LACTOBACILLI STRAINS AND ITS USE IN ANIMAL HEALTH

FIELD OF THE INVENTION

The present invention describes a combination of lactobacilli (or lactic acid bacteria), namely a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, and its use for the prevention and/or treatment of necrotic enteritis in animals, in especially the chicken.

PRIOR STATE OF THE TECHNIQUE

In 2017, world meat production increased by 1.25% to reach 323 million tonnes (Mt) and it is expected to increase further by nearly 15% by 2027 (Organisation for Economic Co-operation and Development (OECD) / Food and Agriculture Organization (FAO), 2018). The growth in demand for cameo products is mainly due to population growth, urbanization and rising incomes in developing countries.

The production of poultry meat is the main sector in the meat products industry.

By “poultry”, we mean domestic birds belonging to the gallinaceans or even to the palmipeds which are bred for their meat, their eggs and their feathers. The term "poultry" covers a wide range of birds, ranging from native and commercial chicken breeds to Muscovy ducks, mallards, turkeys, guinea fowl, geese, quails, pigeons, ostriches or pheasants. The most consumed poultry meat is chicken meat.

Poultry meat is mainly produced in large-scale intensive farming. The global poultry industry has become a high profit generating sector whose success is intimately linked to the health of animals and more specifically to their healthy gastrointestinal tract, which ensures the efficient absorption of feed given to poultry.

Indeed, avian diseases, that is to say those affecting birds, can generate significant financial losses, due to the high mortality rate and low feed efficiency. These infections can be of bacterial, fungal, viral or parasitic origin. Thus, the most common bacterial infections in birds are infections due to Escherichia coli, Salmonella spp, Clostridium perfringens, Pasteurella multocida, Staphylococcus aureus, Mycobacterium avium, Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, Mycoplasma iowae, Clostridium sordellii or Clostridium septicum. These are the cause of various diseases such as chronic respiratory disease in poultry, avian cholera, gangrenous dermatitis, necrotic enteritis or avian tuberculosis.

Among the most important agents of enteric disease in poultry are Escherichia coli and bacteria of the genus Clostridium.

Bacteria of the genus Clostridium (or clostridia) are Gram-positive anaerobic bacilli that produce endospores. They are ubiquitous, meaning they are found in the environment and in the gastrointestinal tract of animals. It should be noted that these bacteria are, for the most part, non-pathogenic. Many of these bacteria are widely used in industrial fermentations, for example in the synthesis of chemical compounds such as acetate, butyrate, lactate, ethanol, carbon dioxide or solvents.

Nevertheless, certain bacteria of the genus Clostridium (C.) are known for their pathogenic power. These include C. botulinum, C. tetani, C. difficile, C. perfringens, C. novyi, and C. septicum. Clostridia cause several disorders, including ulcerative enteritis caused by C. colinum or necrotic enteritis caused by C. perfringens.

Necrotic enteritis or necrotizing enteritis is a disease affecting farms in all poultry regions of the world. This pathology is more common in broilers, but laying hens and turkeys can also be affected.

The ability of C. perfringens to cause this pathology depends on the production of certain extracellular toxins and enzymes that cause intestinal cell degradation, such as lecithinases or necrotizing toxins.

For example, C. perfringens produces and secretes alpha-toxin (a-toxin), a lecithinase causing hemolysis and tissue necrosis, or even b-toxin (or Necrotic enteritis toxin B-like or NetB) causing the formation of endospores in the membrane of intestinal cells.

The bacterium C. perfringens is naturally present in the intestine. As an opportunistic bacterium, it needs the intestinal balance to be compromised to colonize, proliferate and express its toxins, and thus cause necrotic enteritis. Studies have shown in particular that simple infection by C. perfringens alone is not sufficient to induce pathology. the Triggering of necrotic enteritis requires the presence of conditions called predisposing factors such as coccidiosis, a disease caused by the parasite Eimeria spp, diet or even immunosuppression or animal stress.

Necrotic enteritis negatively affects the feed conversion rate in farm animals, i.e. the animals must consume more feed to have the same weight gain. This type of necrotic enteritis is problematic as it negatively impacts productivity and profitability resulting in reduced animal size and weight.

The most common signs to identify necrotic enteritis are the appearance of drowsiness in animals, lack of energy and appetite, diarrhea, dehydration or lack of appetite. Chickens usually die within 1-2 hours after symptoms appear. Mortality rates associated with necrotic enteritis are usually between 2 and 10% but can sometimes be as high as 50%. The condition is characterized by the sudden increase in flock mortality, with birds usually dying without warning signs.

Necrotic enteritis is therefore responsible each year for colossal economic losses worldwide, estimated at more than 6 billion dollars in 2015. These losses are due to the costs of disease control measures and the decrease in production in terms of weight and mortality of animals on farms.

Therefore, strategies for the prevention and/or treatment of necrotic enteritis in farm animals are needed.

One of the ways to fight against necrotic enteritis is prevention because, as mentioned before, after infection, the development of the disease is very rapid and mortality is high.

The administration of low concentration antibiotics, that is to say the administration of growth promoters (or AGP for Antibiotics Growth Promoters) such as avoparcin, bacitracin and virginiamycin, is very effective in the prevention and control of necrotic enteritis. These AGPs promote animal growth and improve feed conversion efficiency. It should be noted that these compounds are also used in human medicine, at higher concentrations.

However, the misuse of these antibiotics, particularly in human medicine and veterinarian, caused selective pressure that accelerated the evolution and spread of resistant bacteria. Consequently, their use has been banned as AGP and kept solely for human medicine, particularly in Europe.

After the restrictions on the use of AGPs, alternatives have been developed to prevent and/or treat necrotic enteritis such as the use of NetB toxoids, or vaccination with Eimeria, a unicellular parasite, in order to reduce the prevalence of coccidiosis known to be an important predisposing factor for necrotic enteritis.

There remains, however, a clear need to develop alternatives to overcome the increase in the mortality and morbidity rate linked to the banning of antibiotics in animal feed, presenting performances similar to those obtained with antibiotics in the prevention and/or the treatment of necrotic enteritis.

Due to their properties, lactic acid bacteria are widely used in the food industry as a biological food preservative or in factory farming for the prevention of infectious and zoonotic diseases. These bacteria are also used to improve the performance of animal husbandry through the production of digestive enzymes, volatile fatty acids and/or vitamins, which contribute to increasing the digestibility of nutrients and improving feed conversion rate. These lactic acid bacteria are notably found under the name of probiotics.

Thus, an alternative to the use of antibiotics or AGP is to give animals additives or food products, in particular probiotics.

Broadly, probiotics are defined as live microorganisms that confer a health benefit to the host, when consumed in sufficient concentrations. They can interact with the host to improve immunity, intestinal homeostasis, stimulate metabolism or even reduce the risk of infection by opportunistic pathogens.

Some probiotic bacteria interfere with or even annihilate the pathogenicity of disease-causing microbial agents, for example by eliminating or inhibiting the growth of pathogenic bacteria in the intestinal lumen. Some produce antibacterial substances capable of competing with pathogens for nutrients, growth factors and attachment sites to the intestinal epithelium. They may also exert immunological functions by modulating the host's immune response to it. thus allowing to better cope with infections.

Most bacterial probiotics belong to lactic acid bacteria. One of the main characteristics of this group of bacteria is their ability to produce lactic acid, in a strain-dependent manner, by homo or hetero-fermentative fermentation of glucose.

Lactic acid bacteria belong to the phylum Firmicutes, class Bacilli and order Lactobacillales. The taxon called lactic acid bacteria includes cocci, Gram-positive bacilli or coccobacilli, non spondees, anaerobes or facultative aerobes with a G+C percentage of less than 50%. These bacteria are acidophilic with an optimum growth pH between 3.5 and 6.5. Most strains have nutritional requirements and require rich media to grow. This group includes 10 genera, the best known of which are Lactobacillus, Pediococcus, Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Carnobacterium.

Within lactic acid bacteria, the most represented genus is that of Lactobacillus (Lb.) with more than 253 species described to date (http://www.bacterio.net/lactobacillus.html).

Lactobacilli are widely used in the development of probiotics, in particular because of their ability to survive the extreme conditions found in the gastrointestinal tract, their good ability to adhere to intestinal cells, making it possible to increase the retention of probiotics in intestine, properties of eliminating or inhibiting the growth of pathogenic bacteria in the intestinal lumen.

Document EP 2287 286 describes in particular that isolates of Lactobacillus {Lb. sakei or Lb. reuteri) possess anti-inflammatory and probiotic properties.

CN 105861399 also describes the use of a specific strain of Lb. plantarum to prevent necrotic enteritis in broiler chickens by inhibiting the growth of C. perfringens.

Document WO2006/133472 describes a complex mixture of 5 microorganisms inhibiting C. perfringens, consisting of E. faecium DSM 16211, L. reuteri DSM 16350, L. salivarius DSM 16351, P. acidilactici DSM 16210 and B. animalis DSM 16284. This document specifies that these specific strains of L. reuteri and L. salivarius are more effective in inhibiting E. coli than C. perfringens. Document WO2016/170280 recommends the use of Bacteroides thetaiotaomicron, optionally combined with L. reuteri and/or L. salivarius, in particular to treat enteritis. KLOSE et al. (Veterinary Microbiology, 2010, 144 (3-4): 515-21) have evaluated the antagonistic activity of various intestinal bacteria, including L. reuteri and L. salivarius, against C. perfringens and report a large heterogeneity between species.

Ren et al. (Microorganisms, 2019, 7(12): 684) reported a synergistic effect between probiotic (L. agilis or L. salivarius) and phytobiotic on the intestinal microbiota of young broiler chickens.

However, there is an obvious need to develop new effective solutions, with a simple formula and containing a minimum of active ingredients, in order to fight against C. perfringens responsible for necrotic enteritis in broiler chickens, for health and economic reasons. .

DISCLOSURE OF THE INVENTION

Definitions

The definitions below correspond to the meaning generally used in the context of the invention and are to be taken into account, unless another definition is explicitly indicated.

Within the meaning of the invention, the articles “un” and “une” are used to refer to one or more (for example at least one) units of the grammatical object of the article. By way of example, “an element” designates at least one element, that is to say one or more elements.

The terms "about" or "approximately", used in reference to a measurable value such as a quantity, a duration, and other analogous values, must be understood as encompassing measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.

Intervals: throughout the present description, the various characteristics of the invention can be presented in the form of an interval of values. It should be understood that the description of values in the form of an interval is only intended to make reading easier and should not be interpreted as a rigid limitation of the scope of the invention. Accordingly, the description of a range of values should be considered to specifically disclose all the possible intermediate intervals as well as each of the values within this interval. For example, the description of an interval from 1 to 6 should be considered as specifically describing each of the intervals it comprises, such as the intervals from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this range, for example, 1; 2; 2.7; 3; 4; 5; 5.3 and 6. This definition applies independently of the scope of the interval.

The term "isolated" should be understood in the context of the invention as synonymous with removed or extracted from its environment or natural state. For example, a bacterium strain or an isolated peptide is a bacterium strain or peptide extracted from the natural environment in which it is usually found, be it a living plant or animal for example. . Thus, a strain of bacteria or a peptide naturally present in a living animal is not a strain of bacteria or an isolated peptide within the meaning of the invention, while the same strain of bacteria or peptide, partially or completely separated from the others elements present in its natural context, is for its part “isolated” within the meaning of the invention. A strain of bacteria or an isolated peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

Surprisingly, the Applicant has identified a new specific combination of lactic acid bacteria, useful for combating necrotic enteritis, in particular in farm animals such as chickens.

Thus and according to a first aspect, the present invention relates to a mixture of microorganisms comprising a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius.

Within the meaning of the invention, by "mixture", is meant the association of at least 2 different species of microorganisms, advantageously of at least 2 distinct strains of bacteria, even more advantageously of at least 2 strains of lactic acid bacteria (lactobacilli or Lactobacillus ) distinct.

Thus, a mixture of microorganisms according to the invention may comprise one or more strains of Lb. reuteri and one or more strains of Lb. salivarius, and possibly other microorganisms, including other bacteria.

According to another aspect, the present invention relates to a mixture of lactic acid bacteria consisting of at least one strain of Lb. reuteri and at least one strain of Lb. salivarius. According to a particular embodiment, the mixture of lactic acid bacteria consists of a strain of Lb. reuteri and at least one strain of Lb. salivarius, possibly from two strains of Lb. salivarius. According to another embodiment, the mixture of lactic acid bacteria consists of a strain of Lb. reuteri and a strain of Lb. salivarius.

As demonstrated in the present application, the specific mixture according to the invention is capable of reducing or inhibiting the growth and/or the activity of the bacterium C. perfringens.

Within the meaning of the invention, by “growth of the bacterium C. perfringens”, is meant a set of mechanisms leading to an increase in the dry biomass of bacteria. It is the growth of the bacterial cell in size, mass and/or volume but also the growth of a population by cell division.

Within the meaning of the invention, by “activity of the bacterium C. perfringens”, is meant the pathogenic or even toxigenic activity of this bacterium. These include the production or secretion of toxins, such as NetB and a-toxin, or enzymes. It may also be the ability of C. perfringens to adhere to the gastrointestinal tract or even colonize it.

Advantageously, the mixture according to the invention allows a reduction or even an inhibition of the production and/or of the secretion of the toxins NetB and α-toxin.

According to a particular embodiment, the mixture according to the invention comprises the strain of Lb. reuteri deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020.

According to another particular embodiment, the mixture according to the invention comprises the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020 and/or the strain of Lb. salivarius filed with the CNCM under number 1-5502 dated March 04, 2020.

According to a specific embodiment, the mixture of microorganisms according to the invention comprises or consists of:

- a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius. said strain of Lb. reuteri being the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020; Where

- a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, said strain of Lb. salivarius being the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020 and/or the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020; Where

- the strain of Lb. reuteri deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020 and/or the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020.

According to different embodiments, the mixture according to the invention comprises or consists of:

- the strain of Lb. reuteri deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020; Where

- the strain of Lb. reuteri deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020; Where

- the strain of Lb. reuteri deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020, the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020.

According to a particular embodiment, the mixture contains other microorganisms, advantageously other probiotics. Preferably, the mixture contains other microorganisms chosen from: bacteria of the genus Lactobacillus, bacteria of the genus Bifidobacterium, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Pediococcus, bacteria of the genus Bacillus, yeasts, and combinations thereof.

Preferably, the Lactobacillus bacterium is chosen from the group consisting of: Lb acidophilus, Lb. lactis, Lb. helveticus, Lb. brevis, Lb. casei, Lb. plantarum, Lb. salivarius, advantageously another strain of Lb salivarius, Lb. reuteri, advantageously another strain of Lb reuteri, Lb. bifidus, lb. bulgaricus, Lb. caucasicus, Lb. rhamnosus, Lb. gasseri, Lb. sakei, Lb. fermentum, and combinations thereof.

Preferably, the Bifidobacterium bacterium is chosen from the group consisting of: B. bifidum, B. longum, B. infantis, B.breve, B. adolescentis, B. animalis, B. lactis, and combinations thereof.

Preferably, the Streptococcus bacterium is chosen from the group consisting of: S. thermophilus, S. lactis, S. cremoris, S. diacetylcatis, and combinations thereof.

Preferably, the Enterococcus bacterium is chosen from the group consisting of: E.faecium, E.faecalis, and their combinations

Preferably, the Pediococcus bacterium is P. acidilactici.

Preferably, the Bacillus bacterium is chosen from the group consisting of: B. subtilis, B. velezensis, B. licheniformis, B. coagulans, B. pumilus, and their combinations.

Preferably, the yeast is chosen from the group consisting of: Candida Kefyr, Saccharomyces florentinus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. boulardii, and combinations thereof.

According to another aspect, the present invention relates to a composition comprising a mixture of microorganisms as defined above.

In addition, the composition according to the invention may comprise the usual adjuvants or excipients used in the field considered, such as hydrophilic or lipophilic thickeners or gelling agents, palatables, hydrophilic or lipophilic additives, preservatives, antioxidants, diluents, vitamins, minerals, suspending agents, cellulosic derivatives, absorbents, cryoprotective agents or dyes.

Of course, those skilled in the art will take care to choose this or these possible adjuvants or excipients, and to adjust their quantity, in such a way that the advantageous properties of the composition according to the invention are not, or substantially not, altered by the proposed addition.

According to one embodiment, the composition according to the invention comprises nutritive substances which can be used as a support and/or prebiotic substances advantageously chosen from fructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides, xylo-oligosaccharides, vitamins, in particular vitamin E.

According to another embodiment, the composition according to the invention comprises at least one compound chosen from the following group: zeolites, calcium carbonate, calcium sulphate, magnesium carbonate, talc, trehalose, chitosan , shellac, albumin, starch, skimmed milk powder, whey, whey powder, maltodextrins, lactose, inulin, dextroses, celluloses, clays including sepiolite, derivatives of yeasts and cereals, vegetable oils or a solvent chosen from water or a physiological solution.

According to a particular embodiment, the composition according to the invention comprises a coating material advantageously chosen from maltodextrins, guar seed flour, gum arabic, alginates, modified starch and starch derivatives, dextrins, cellulose derivatives such as cellulose ester and cellulose ether, proteins such as gelatin, albumin, casein, gluten, gum arabic, gum tragacanth, lipids such as waxes, paraffin, stearic acid, mono- and diglycerides.

The mixture or composition according to the invention may be in the form of a powder, a capsule, a spray, a solution, an emulsion, a suspension or a dispersion.

Advantageously, the mixture or the composition according to the invention is in dry or liquid form, in particular in freeze-dried, dried, pressed, liquid or frozen form, advantageously in freeze-dried form.

According to a preferred embodiment, the mixture or composition according to the invention is intended to be administered orally. For this, the mixture or the composition may be in various suitable galenic forms, for example in the form of a lyophilisate to be poured and dissolved in drinking water, of a liquid to be poured over food or in water. drink, tablets, powder packaged in capsules or any other suitable form. Advantageously, it is a powder or lyophilisate to be poured and dissolved in the drinking water of the animals.

According to a particular embodiment, the mixture or the composition according to the invention contains the lactobacilli according to the invention at a final concentration of between 10 ⁵ and 10 ⁹ CFU ('Colony Forming Unit or unit forming a colony)/mL of the mixture or of the composition according to the invention, advantageously between 10 ⁶ and 10 ⁸ CFU/mL, for example at a final concentration of 10 ⁷ CFU/mL.

This concentration can be understood as the concentration of each microorganism, advantageously bacteria, even more advantageously lactobacillus, present in the mixture or the composition, advantageously in the drinking water of the animals. Preferably, it is the concentration of all the microorganisms, advantageously bacteria, even more advantageously lactobacilli, present in the mixture or the composition. Thus and by way of example, for a final concentration of lactobacilli of 10 ⁷ CFU/mL, a mixture according to the invention may comprise or consist of a strain of Lb. reuteri and a strain of Lb. salivarius at a concentration of 0.5x10 ⁷ CFU/mL each.

According to another aspect, the invention relates to the use of a mixture or of a composition according to the invention as an additive or food product for animal purposes.

Within the meaning of the invention, by “additive or food product”, is meant a composition whose purpose is to supplement the traditional diet and comprising nutrients or other substances having a nutritional or physiological effect. As already said, it may be an additive to drinking water or to foods such as, for example, cereals and/or legumes such as soya. Conventionally, the term "drink" is used for ingested products in liquid form and the term "food" for those in solid form.

The mixture or the composition according to the invention can be added to the drink or to the food of extemporaneously, or can be introduced in particular into foods at the time of their manufacture, for example by mixing or coating.

According to a particular embodiment, the mixture or the composition according to the invention is added to the beverage or to the food so that the lactobacilli represent from 10 ⁸ to 10 ¹⁴ CFU/kg of the beverage or of the food, advantageously 10 ¹⁰ to 10 ¹² CFU/kg.

According to another aspect, the invention relates to the use of a mixture or a composition as described above, as well as beverages and foods containing them, for combating necrotic enteritis in animals, at know to prevent and/or treat this pathology.

In a known manner and as described in the examples, the efficacy on necrotic enteritis can be evaluated by determining a lesion score in the intestines of the animals:

- score 0 corresponds to a healthy intestine;

- score 1 corresponds to a thin and friable intestine;

- score 2 corresponds to thin and friable intestine with the appearance of small necrotic spots; and

- score 3 corresponds to an intestine showing large lesions visible through the outer wall of the intestinal tract.

Prevention and/or treatment of necrotic enteritis can also be assessed by monitoring animal weight. As mentioned previously, intestinal damage decreases feed conversion which induces weight loss in animals.

According to a preferred embodiment, the animal targeted by the present invention is a poultry, advantageously a chicken, preferably a broiler (or farmed chicken).

As already said and preferably, the mixture or composition according to the invention, optionally integrated into the drink or the food, is administered or ingested orally.

The intake can be systematic for all animals from birth or can be decided upon the appearance of symptoms or even death in the farm. Advantageously, the treatment is carried out as a preventive measure, that is to say before the appearance of any symptoms, as soon as the animals are born or after a few days. Even more advantageously, the treatment continues until the death of the animals, which generally occurs at 40 days for poultry.

In addition, the intake can take place once a day, or even at every drink intake or of food, or perhaps separated by several days. Advantageously, it is taken daily.

A preferred dosage corresponds to daily administration, in the form of a food supplement at a concentration of 10 ⁷ CFU/mL (equivalent to 10 ⁷ CFU/g), throughout the life of the animal.

According to another aspect, the invention relates to strains of lactobacilli of interest due to their inhibitory activity on the growth and/or activity of Clostridium perfringens listed below: strain Lb. reuteri filed with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020; the Lb strain. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020; the Lb strain. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020.

EXAMPLES OF REALIZATION OF THE INVENTION

The way in which the invention can be implemented and the resulting advantages will emerge better from the examples of embodiment which follow, given by way of indication and not limitation, in support of the appended figure.

Figure 1 shows the effectiveness of the bacterial mixture according to the invention in terms of chicken weight. The letters (a, b, c) indicate statistically significant differences.

FIG. 2 represents the effectiveness of the bacterial mixture according to the invention in terms of the lesion score of the chickens. The letters (a, b, c) indicate statistically significant differences.

Demonstration of the effect of bacterial melanea according to the invention on necrotial enteritis

The experiments were carried out using the strain of Lb. reuteri deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated 04 March 2020.

The strain of C. perfringens used in the example is a strain of C. perfringens isolated from a chicken suffering from necrotic enteritis.

1/ Materials and methods:

The capacity of the combination of Lb. reuteri and Lb. salivarius according to the invention to prevent necrotic enteritis was evaluated in vivo on broiler chickens from a cross of the Cobb 500 (female) and Hubbard M99 (male) lines.

The in vivo experiments took place for 17 days on 150 chickens (30 per experimental condition). The trial started on hatch day. During the tests, the lactobacilli were administered alone {Lb. reuteri, Lb. salivarius) or in combination {Lb. reuteri + Lb. salivarius), by gavage once a day, on days 1 and 2, then on day 10 to day 13.

1-1/ Chicken environment and diet

The chickens were reared in cages giving an average surface area of 432 cm ² per chicken. The cages were placed on several levels in an air-conditioned room maintained at an ambient temperature throughout the study, and qualified for tests with a biological risk level 2. Lighting was provided 24/24 hours during all the duration of the study. The birds were given water and food ad-libitum, that is, they were fed to satiety.

The diet for the first 9 days is based on corn and soy. After day 10, the growth diet includes wheat (Tables 1 and 2). Both diets are given as a mash.

Table 1 represents the composition of diets by ingredients (g/100g)

Table 2 represents the approximate composition of diets (g/100g) 1-2/ Vaccination

With the exception of the non-challenged group (not having received any administration of C. perfringens), the chickens were vaccinated on day 1 with “Advent@9X”. This vaccine contains live oocysts of Eiineria acervulina, E. maxima and E. tenell, gentamicin and amphotericin B as preservatives. The aim is to help prevent avian coccidiosis caused by these pathogens. At the ^9th day, the chickens were vaccinated intraocular way against infectious bursal disease by Intervet bursal Vac-G603 is a vaccine containing live attenuated viruses.

1-3/ Treatment of the different batches Five different treatments were tested. Each treatment was repeated six times and each replicate contains 5 chickens.

These treatments are summarized below:

- T-: Control group not supplemented with lactobacilli and not challenged with C. perfringens;

- T+: Positive control group, not supplemented with lactobacilli and challenged with C. perfringens;

- lbs. reuteri: supplemented with Lb. reuteri and challenged with C. perfringens,

- lbs. salivarius: supplemented with Lb. salivarius and challenged with C. perfringens,

- lbs. reuteri + Lb. salivarius: supplemented with a mixture of Lb. reuteri and Lb. salivarius and challenged with C. perfringens.

1-4/ Administration of C. perfringens

The wild-type C. perfringens strain was cultured in thioglycollate broth overnight at 37°C perfringens was then administered at a concentration of 10 ⁷ CFU/mL in a 3 mL oral gavage dose administered in sterile thioglycollate, at using a 20 mL syringe and a 20 gauge dosing needle, from 14 ^th to 16 ^th days of age. After administration, birds were hand-restrained for 5-10s to confirm delivery of the correct dose and freedom from stress.

1-5/ Administration of lactic acid bacteria

The strains of lactic acid bacteria were cultured in MRS broth (from Man-Rogosa-Sharpe) at 37° C. overnight. The ^1st and ^2nd days of age, 250 to 500 pL suspension of Lactobacillus than 10 ⁷ CFU / mL (final concentration alone or the mixture of two strains Lb and Lb salivarius reuteri, that is to say 0 5x10 ⁷ CFU/mL each concerning the mixture) were administered orally to the birds. For the 10 ^th to 13 ^th days of age, the dosage is increased to 1 mL, while maintaining the concentration at 10 ⁷ CFU/mL. The control groups (T- and T+) received the same volume of sterile PBS solution. Negative control birds were treated first to reduce the risk of cross contamination.

1-6/ Measurements taken on birds

The study was completed on 17 ^th day. The performance of the birds were measured at 0, 10 ^th, 14 ^th and 17 ^th days of the experimental period by recording the weight of the birds (in g) (Figure 1) and the feed consumption for each cage. At the end of the trial, all birds were euthanized by CO2 asphyxiation. Necrotic enteritis lesions were sought in the intestines and analyzed as described by Prescott et al. (1978, Can. Vet. J. 19, 181-183) (Figure 2). The experimental protocol is summarized in Table 3 below. X corresponds to the day of administration of a product.

1-7/ Statistical analysis

Statistical comparisons between the different results obtained were made by ANOVA analysis of variance using Statgraphics® Centurion XVI software.

2/ Results:

The results are shown in Table 4 below.

Index letters (a, b, c) indicate statistically significant differences. Groups with the same letter do not differ significantly.

The results in Figure 1 show that the chickens of the group treated with Lb. reuteri or Lb. salivarius show a non-significant increase in their weight (709.79g and 707.50g, respectively) compared to the positive control group (T+). This increase does not make it possible to find a weight of the chickens similar to that of the negative control group (T-).

On the contrary, the chickens of the group treated with the mixture of Lb. reuteri and Lb. salivarius have similar weights (753.13g) to those of the group of non-challenged chickens (765g), which are significantly different from the infected group (699.58g).

Concerning the intestinal lesions caused by the administration of C. perfringens (Figure 2), it appears from the data that the administration of Lb. reuteri or Lb. salivarius alone to groups of challenged chickens does not significantly reduce the lesion score compared to the T+ group.

A significant decrease in intestinal lesions is obtained on the chickens of the group treated with the Lb mixture. reuteri and Lb. salivarius which present a score of lesions (1.00) similar to that of the group of non-challenged T- chickens (0.88) and significantly different from that of the infected T+ group (1.67).

In conclusion, these in vivo data show that supplementation with Lb. reuteri and Lb. salivarius in combination produced a protective effect against necrotic enteritis in chickens.

Claims

1/ Mixture of microorganisms comprising a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius.

2 / mixture according to claim 1 characterized in that it consists of a strain of Lb. reuteri and at least one strain of Lb. salivarius, advantageously from a strain of Lb. reuteri and a strain of Lb. salivarius.

3 / mixture according to one of claims 1 to 2, characterized in that the strains have an inhibitory activity on the growth and / or the activity of Clostridium perfringens.

4 / mixture according to one of claims 1 to 3, characterized in that the strain of Lb. reuteri is the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 on March 04, 2020.

5 / mixture according to one of claims 1 to 4, characterized in that the strain of Lb. salivarius is the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020 or the strain deposited at the CNCM ( National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020.

6 / Composition comprising a mixture according to one of claims 1 to 5.

7 / mixture according to one of claims 1 to 5 or composition according to claim 6 which is in freeze-dried form.

8 / Use of a mixture according to one of claims 1 to 5 or 7, or of a composition according to one of claims 6 to 7, as an additive or food product for animal purposes.

9 / mixture according to one of claims 1 to 5 or 7, or composition according to one of claims 6 to 7, for use in the prevention and / or treatment of necrotic enteritis in animals. 10 / mixture or composition for its use according to claim 9, characterized in that the mixture or composition is administered orally.

11 / mixture or composition for its use according to claim 9 or 10, characterized in that the animal is a poultry, advantageously a chicken, even more advantageously a broiler.

12/ Strain corresponding to: the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 04, 2020; or the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 04, 2020; or - the strain deposited at the CNCM (National Collection of Cultures of Microorganisms,

Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 04, 2020.