FR3106724A1 - Composition for improving insect production - Google Patents

Abstract

The invention relates to a composition for its use for the prophylaxis of microbial pathologies affecting the growth of farmed insects, said composition consisting essentially of at least one strain of lactic acid bacteria, said strain being chosen from: - a strain NOL11 such as CNCM on March 14, 2012 under the number CNCM I - 4609, and - a strain of lactic acid bacteria belonging to the same operational taxonomic unit (OTU) as said strain NOL11, or a mixture of these strains. Figure for the abstract: no figure

Description

Composition for improving insect production

The invention relates to a composition of microorganisms for breeding insects.

Population growth, urbanization and the rise of the middle classes have increased the global demand for food, animal feed and especially protein. By 2030, more than 9 billion people will need to be fed, as will the billions of animals raised each year for food, recreation and as pets.

To meet this growing demand, the traditional production of feed materials, such as soy and fishmeal, must be intensified, raising the question of resource efficiency and the use alternative protein sources.

New protein solutions for animal and human food must therefore be sought.

As recently mentioned by major international bodies such as the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), one of the many possible responses to the problems of human and animal food safety is to consider the breeding of insects, their consumption and their valorization.

To date, only a small number of insect species have already been domesticated because of their commercial value or that of their derived products, in particular silkworms for their fibers and bees for their honey. Other species are bred to feed pets (e.g. mealworms and crickets) or for recreation (bait for fishing).

Insect farming has many advantages.

Insects are ubiquitous and reproduce rapidly. They also have a high growth rate, low feed conversion rate and low environmental impact throughout their life cycle. In addition, insects can feed on fruits or vegetables that have become unfit for human consumption.

Insects are nutritious, with a high content of high quality proteins, fats and minerals, and are therefore considered a raw material of interest for animal or human nutrition. This nutritional value of insects depends on their life stage (metamorphic stage), their habitat and their diet. However, it is widely accepted that insects provide high quality protein and nutrients compared to beef and fish.

More than 1900 species of insects are currently listed as being edible for humans around the world. However, there are few data on the quantities of insects actually consumed. According to available data, the most commonly consumed insects are beetles ( Coleoptera ) (31%), caterpillars ( Lepidoptera ) (18%), bees, wasps and ants ( Hymenoptera ) (14%), grasshoppers , locusts and crickets ( Orthoptera ) (13%), cicadas, leaf beetles and leafhoppers, scale insects and Hemiptera ( Hemiptera ) (10%), termites ( Isoptera ) (3%), dragonflies ( Odonata ) (3%), flies ( Diptera ) (2%) and other categories (5%).

Insects can be eaten whole, ground into a paste or powdered after drying and grinding. These pastes and powders can be added to other foods low in protein to increase their nutritional value (chocolate bars, biscuits, etc.).

However, European regulations on the human consumption of insects or products derived from insects are still uncertain and their application uneven according to national legislation.

The majority of known edible species of insects are harvested from the wild and eaten whole. However, mini-farms, either domestic or local, are developing in the world (eg mini-farm backed by a pig farm). These mini-farms offer the possibility of increasing the supply of insect protein without compromising wild insect populations.

However, to meet the challenges mentioned above, more and more companies are working on the intensification and industrialization of insect farming.

In particular, the black soldier fly ( Hermetica illucens ) and the mealworm ( Tenebrio molitor ) both combine a large number of qualities sought after for insect production on an industrial scale, namely: high growth rate, short development, high egg-laying rate, ability to live in conditions of high densities, recovery of green waste, sanitizing power of the substrate.

Like any breeding with a commercial purpose, insect breeding must meet performance, quality, animal welfare and health safety requirements for the user, the consumer and the environment.

In insect farms, it is possible to observe occasional mortalities of individuals or more significant mortalities that can affect an entire batch, sometimes very quickly. The causes of individual mortality can be multiple: physiological causes (feeding problem, poor rearing conditions, rearing system poorly adapted to the species), genetic causes (problem of inbreeding, degeneration) or pathological causes.

As far as the pathologies are concerned, these can be linked to a microbial infection of the viral, fungal or bacterial type or even to a parasitic infection.

To meet the new expectations of the world food market and the production requirements of the insect sector, taking into account in particular its specificities, it is necessary to improve the production yields of insects, in particular by securing the growth of insects and limiting their mortality throughout their life cycle.

For example, patent application FR2985664 describes a probiotic composition intended for the prevention of winter mortality in bees, comprising Bacillus , optionally in combination with other species of bacteria.

Other examples of bacterial compositions are known for example from application CN106854627, which describes a mixture of strains of Kocuria marina FE01, Lysinibacillus boronitolerans FE04, and Proteus mirabilis FE008 for its use to optimize the growth of Hermetia illucens during the transformation of chicken manure.

Despite these compositions known from the prior art, the need to propose a composition favorable to the proper development of breeding insects remains.

The invention aims to remedy this lack.

One object of the invention is to provide a microbial composition, as well as a method for improving the performance of insect farms, in particular Black Soldier Flies (BSF) farms.

The objective is to take advantage of the larvae/microbiota relationship in order to improve the conversion efficiency and production yield of BSF larvae, i.e. to improve the growth rate of the larvae and the size of the larvae obtained, and to reduce mortality during rearing.

Also, the invention relates to a composition for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects, in particular black soldier flies , said composition consisting essentially of at least one strain of bacteria of the Bacillus genus, said strain being chosen in particular from:

- a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and

- a Bacillus strain belonging to the same operational taxonomic unit (OTU) as said NOL01 strain,

or a mixture of these strains.

The invention is based on the surprising observation made by the inventors that specific Bacillus strains, in particular Bacillus strains belonging to the same OTU as the NOL01 strain, and in particular Bacillus subtilis strains, are capable, when placed in contact with breeding insects, to promote the zootechnical performance of breeding, in particular to reduce mortality, to increase yield, etc. In addition, this composition makes it possible to promote a microbiota favorable to insects, thus limiting the development of undesirable microorganisms.

In the invention, the term "improvement of zootechnical performance" means the improvement of technical criteria linked to production such as the daily weight gain of individuals (ADG), the weight of individuals at slaughter, the index of feed consumption or even the viability of farmed insects and their resistance to sanitary episodes (infection rate, mortality rate), the quality of individuals (their protein and lipid content in particular) and their reproductive capacity. The improvement of these technical criteria has an impact on the overall yield of the farm and its economic performance.

The composition according to the invention is “consisting essentially” of said strains of bacteria of the genus Bacillus , which means that the composition contains, as active substance, only strains of bacteria of the genus Bacillus mentioned above. This means that the composition is likely to contain other components, but these additional components do not contribute to the technical effect: that is to say that these components do not, alone, have a significant influence on the improving the zootechnical performance of insect farming, in particular on the treatment or prevention of microbial pathologies affecting the growth of farmed insects.

By “strain of bacteria”, is meant in the invention all the individuals (bacteria) resulting from successive subcultures of a bacterial colony, this set being essentially homogeneous, that is to say that the very great majority of bacteria that make up the strain have the same genotype.

In other words, a strain is a part of a bacterial species different from other bacteria of the same species by a minor but identifiable difference. A strain is also defined as a population of bacteria that descended from a single organism or pure isolate culture. Strains of the same species may differ slightly from each other in many ways.

Advantageously, the invention relates to the aforementioned composition for its use as defined above, where said at least one strain of bacterium of the genus Bacillus , in particular Bacillus subtilis , is chosen from:

• a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and
• a strain of Bacillus , in particular Bacillus subtilis , whose sequence encoding the 16S rRNA has at least 97% identity with the sequence encoding the 16S rRNA of the strain NOL01,

or a mixture of these strains.

The inventors have shown that a NOL01 strain or strains having a genome very similar to this one are capable, by a simple treatment, of preventing and/or treating the microbial pathologies of livestock insects and of improving their performance. zootechnics. More particularly, the inventors were able to demonstrate that the NOL01 strain, and the strains of Bacillus , in particular Bacillus subtilis, having a very similar genome, are capable of preventing or treating microbial pathologies affecting livestock insects or even to improve the zootechnical performance of said breeding insects.

In the invention, when reference is made to a strain deposit at the CNCM, reference is made to a deposit made at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris , France) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

As mentioned above, the composition according to the invention essentially consists of a NOL01 strain or a similar strain, or a mixture thereof. This means that the composition according to the invention consists essentially of said NOL01 strain, or consists essentially of one or more strains belonging to the same operational taxonomic unit (OTU) as the NOL01 strain, or else said NOL01 strain and one or more strains belonging to the same operational taxonomic unit (OTU) as the NOL01 strain. Examples will be given below.

By “a strain of Bacillus belonging to the same operational unit (OTU) as said NOL01 strain” or by “a strain similar” to NOL01, or by “strains having a very similar genome” or “very close” to NOL01, the invention means all the strains of Bacillus , in particular of Bacillus subtilis, having at least 97% of genomic sequences similar to the sequence of genes encoding the 16S rRNA of the strain NOL01.

In biology, the operational taxonomic unit (OTU) is the basic unit of phylogenetic analysis used to group phylogenetically close individuals. The DNA sequences of a given gene, which then serves as a taxonomic marker, are grouped together by data partitioning (clustering) according to their identity. A mainly used gene (reference gene) is 16S rRNA for bacteria. Thus, the OTUs are defined on the basis of an identity threshold chosen by those skilled in the art, which is commonly 97%.

In the invention, "breeding insects" means bees including the Asian bee ( Apis cerana) , the European honey bee ( Apis melifera) , the dwarf bee ( Apis florea) , the spiders including the black tarantula spider ( Brachypelma smithi ), caterpillars including the caterpillar Bunaeopsis spp , ladybugs including the 7-spotted ladybug ( Coccinella septempunctada ), locusts including Locusta migratoria and the desert locust ( Schistocerca gregaria ) , ants including the ant of Colombia ( Atta laevigata ) , crickets including steppe cricket ( Gryllus assimilis ), house cricket ( Acheta domestica ), tropical house cricket ( Gryllodes sigillatus), flies including house fly ( Musca domestica) , black soldier fly ( Hermetia illucens ), mealworms including the lesser mealworm ( Alphitobius diaperinus) , the mealworm (Tenebrio molitor), bedbugs including the giant water bug ( Lethocerus americanus) , beetles including the rhinoceros beetle ( Allomyrina dichotoma ), scorpions including black scorpion ( Heterometrus longimanus ), termites including Mastodermes darwiniensis, worms including silkworm ( Bombyx mori) , bamboo worm ( Omphisa fuscidentalis), palm worm ( Rhynchophorus ferrugineus) , morio worm ( Zophobas morio).

The advantageous breeding insects according to the invention are black soldier flies or black soldiers (in English, abbreviated as BSF) or else Hermetia illucens .

In the invention, the term “microbial pathologies affecting said breeding insects” means all the pathologies linked to the infection of breeding by entomopathogens and affecting the growth, development, moulting and/or survival of the insects of 'breeding.

The death of the host insect occurs by the action of toxins emitted by certain germs ( Bacillus thuringiensis ), by bacterial septicemia ( Bacillus popilliae ), following the lysis of the cells of the intestinal epithelium or by destruction of cells fat or blood (viruses, protozoa).

Most entomopathogenic germs are specific, however non-specific bacteria can become pathogenic when rearing conditions become abnormal; thus the enterobacteriaceae Serratia marcescens Bizio, Pseudomonas or even cocci and streptococci bacteria can be deleterious and responsible for mortality in farmed insects. Furthermore, protozoa, such as coccidia or microsporidia, can kill their host at any stage of the development cycle, or considerably reduce the life of the adults, their fecundity as well as the fertility of the eggs.

It is particularly advantageous in the invention for the aforementioned composition to comprise at least two strains of bacteria of the genus Bacillus , and in particular at least two different strains of Bacillus .

It is even more advantageous in the invention for the aforementioned composition to comprise at least three strains of bacteria of the Bacillus genus, and in particular at least three different strains of Bacillus .

Advantageously, the invention relates to the composition for its use as defined above, where said strain of bacterium of the genus Bacillus , in particular Bacillus subtilis , belonging to the same operational taxonomic unit (OTU) as said strain NOL01 is chosen from:

• a NOL02 strain as filed with the CNCM on January 21, 2016 under number CNCM I – 5043, and
• a NOL03 strain as deposited at the CNCM on March 14, 2012 under number CNCM I – 4607.

The NOL02 and NOL03 strains show a very close phylogenetic proximity to the NOL01 strain, insofar as they are all three parts of the same phylogenetic branch, when their DNA, in particular the DNA encoding the 16S rRNA, is compared.

Advantageously, the invention relates to the composition for its use as defined above, said composition consisting essentially of:

- the NOL01 strain, or

- the NOL02 strain, or

- the NOL03 strain, or

- the NOL01 strain and the NOL02 strain, or

- the NOL01 strain and the NOL03 strain, or

- the NOL02 strain and the NOL03 strain, or

- the NOL01 strain, the NOL02 strain and the NOL03 strain.

Advantageously, the invention relates to the composition for its use as defined above, where said composition comprises from 10 ⁴ to 10 ¹¹ live bacterial colonies of said Bacillus , in particular Bacillus subtilis, the bacterial colonies being per mL or g of composition .

In other words, in this advantageous embodiment, if the composition according to the invention is in liquid form, said composition will comprise from 10 ⁴ to 10 ¹¹ bacterial colonies of Bacillus , in particular Bacillus subtilis, per mL of composition, and this for each of the strains when the composition comprises at least two strains.

If, on the other hand, the composition according to the invention is in dehydrated or non-aqueous form (in solid form), said composition will comprise from 10 ⁴ to 10 ¹¹ bacterial colonies of Bacillus per g of composition, and this for each of the strains when the composition comprises at least two strains.

In the invention, "from 10 ⁴ to 10 ¹¹ bacterial colonies" means: approximately 10 ⁴ , approximately 5.10 ⁴ , approximately 10 ⁵ , approximately 5.10 ⁵ , approximately 10 ⁶ , approximately 5.10 ⁶ , approximately 10 ⁷ , approximately 5.10 ⁷ , approximately 10 ⁸ , approximately 5.10 ⁸ , approximately 10 ⁹ , approximately 5.10 ⁹ , approximately 10 ¹⁰ , approximately 5.10 ¹⁰ or approximately 10 ¹¹ bacterial colonies.

Bacterial colonies are defined as per mL (or gram) of bacterial culture. A person skilled in the art easily knows how to determine this number of bacteria, in particular by counting either manually (using a Malassez blade), or by using an automatic cell counter, or by dilution then inoculation on agar and counting, in particular visual, colonies, or by measuring the optical density.

Advantageously, the invention relates to the composition for its use as defined above, where said one or more strains of bacteria of the genus Bacillus , in particular Bacillus subtilis , are in vegetative form or in sporulated form, or both.

In the advantageous embodiments of the invention where the aforementioned composition essentially consists of two strains of bacteria of the Bacillus genus, in particular Bacillus subtilis, belonging to the same operational taxonomic unit (OTU) as said NOL01 strain, said Bacillus are either all under vegetative form, either all in spore form, or one strain is in spore form and the other strain is in vegetative form. If the composition comprises more than two strains of bacteria as defined above, those skilled in the art will understand which combinations are applicable.

In the advantageous embodiments of the invention where the aforementioned composition essentially consists of three strains of Bacillus , in particular Bacillus subtilis, belonging to the same operational taxonomic unit (OTU) as said strain NOL01, or these three strains are in sporulated form, either these three strains are in vegetative form, or one strain is in vegetative form and the other two in sporulated form, or one strain is in sporulated form and the other two in vegetative form.

In addition, the same strain of bacteria according to the invention can be in the vegetative form, or in the sporulated form, or form a mixture of bacteria in the vegetative form and in the sporulated form.

Those skilled in the art will know which form is the most suitable for each of the strains used in the invention.

Advantageously, the invention relates to the composition for its use as defined above, in combination with at least one strain of lactic acid bacteria, in particular at least one strain of Lactococcus lactis .

The inventors have found that the composition according to the invention can be supplemented with at least one strain of lactic acid bacteria, in particular at least one strain of Lactococcus lactis , in order to improve the properties of preventing mortality, preserving performance in the event of microbial pathology or improving the technical performance of insect farms.

It is particularly advantageous in the invention for the aforementioned composition to comprise at least two strains of bacteria of the genus Bacillus , and in particular at least two different strains of Bacillus , and at least one strain of lactic acid bacteria, in particular of Lactococcus lactis.

It is even more advantageous in the invention for the aforementioned composition to comprise at least three strains of bacteria of the Bacillus genus, and in particular at least three different strains of Bacillus , and at least one strain of lactic acid bacteria, in particular of Lactococcus lactis .

Advantageously, the invention relates to the aforementioned composition for its use as defined above, where said strain of lactic acid bacteria is a lactic acid bacteria strain NOL11 as deposited at the CNCM on March 14, 2012 under number CNCM I – 4609.

More advantageously, the invention relates to the following compositions for their use as defined above:

- the NOL01 strain, in association with the NOL11 strain, or

- the NOL02 strain, in association with the NOL11 strain, or

- the NOL03 strain, in association with the NOL11 strain, or

- the NOL01 strain and the NOL02 strain, in association with the NOL11 strain, or

- the NOL01 strain and the NOL03 strain, in association with the NOL11 strain, or

- the NOL02 strain and the NOL03 strain, in association with the NOL11 strain, or

- the NOL01 strain, the NOL02 strain and the NOL03 strain, in association with the NOL11 strain.

Even more advantageously, said at least one strain of lactic acid bacteria is present in a proportion of 10 ⁴ to 10 ¹¹ live bacterial colonies, the bacterial colonies being per mL or g of composition.

The definition of the amount of bacterium of the genus Bacillus , in particular Bacillus subtilis , mentioned above applies mutatis mutandis to the strain of lactic acid bacteria.

In another advantageous embodiment, the invention relates to the composition supplemented as abovementioned for its use as defined above, where said abovementioned composition and said strain of lactic acid bacteria are used simultaneously, separately or spread over time.

Advantageously, the invention relates to the abovementioned composition for its use as defined above, said composition being applied, or in a form to be or capable of being applied, to eggs, larvae, prepupa, pupae or pupae. adult individuals of breeding insects, directly or indirectly via the breeding substrate.

Advantageously, the invention relates to the aforementioned composition for its use as defined above, said composition being applied to the eggs, larvae, prepupae, pupae or adult individuals of farmed insects.

The objective is that the insects, at one or more stages of their development, be in direct contact with the composition according to the invention so that the pathogens cannot affect their growth, and that the maximum of them can reach the pupa or even adult stage depending on the production.

Advantageously, preference will be given to early direct contact of the insects with the composition according to the invention.

Also, is it possible to apply the composition according to the invention directly to the eggs.

The invention also relates to insect feed comprising a composition as defined above, in particular for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said livestock insects, that is to say microbial pathologies affecting the growth, development, moulting and/or survival of livestock insects.

It is particularly advantageous for the composition according to the invention to be integrated into food allowing the growth of insects at their different stages of development, in particular for the nutrition of the larvae after hatching and before empupation.

The food according to the invention therefore consists of nutritional supports conventionally used for breeding insects, for example food by-products, such as peelings of fruits or vegetables such as bananas, carrots, broccoli, courgettes, cereals (wheat, corn, etc.).

The aforementioned food allows:

- bringing the composition according to the invention into indirect contact with insects at their different stages of development, insofar as the insects develop in their growth substrate,

- an absorption of the bacteria constituting the composition according to the invention, during the consumption of the nutritional substrate, or else,

- a modification of the food by the bacteria of the composition, so that the nutrients resulting from the action of the said bacteria are more easily bioavailable for the breeding insects.

Even more advantageously, the composition according to the invention is present in the aforementioned food at the rate of 100 g to 1 kg per ton of food.

In another aspect, the invention relates to the use of a composition consisting essentially of at least one strain of bacteria of the Bacillus genus, said strain being chosen in particular from:

- a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and

- a Bacillus strain belonging to the same operational taxonomic unit (OTU) as said NOL01 strain,

or a mixture of these strains,

for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects, in particular black soldier flies.

Advantageously, the invention relates to the aforementioned use where said Bacillus strain belonging to the same OTU as said NOL01 strain is chosen from:

- a strain of Bacillus subtilis strain NOL02 as filed with the CNCM on January 21, 2016 under number CNCM I – 5043, and

- a strain of Bacillus subtilis strain NOL03 as filed with the CNCM on March 14, 2012 under number CNCM I – 4607.

Even more advantageously, the invention relates to the aforementioned use, said composition consisting essentially of:

- the NOL01 strain, or

- the NOL02 strain, or

- the NOL03 strain, or

- the NOL01 strain and the NOL02 strain, or

- the NOL01 strain and the NOL03 strain, or

- the NOL02 strain and the NOL03 strain, or

- the NOL01 strain, the NOL02 strain and the NOL03 strain.

According to the aforementioned use, it is advantageous for the composition to comprise from 10 ⁴ to 10 ¹¹ live bacterial colonies of said Bacillus( s) , the bacterial colonies being per mL or g of composition.

It is also even more advantageous for said one or more strains of bacteria of the genus Bacillus to be in vegetative form or in spore form, or both.

Even more advantageously, the invention relates to the aforementioned use, said composition being used in combination with at least one strain of lactic acid bacteria, in particular a strain of Lactococcus lactis , in particular a lactic acid bacterium strain NOL11 as deposited at the CNCM on March 14, 2012 under number CNCM I – 4609, in particular at a rate of 10 ⁴ to 10 ¹¹ live bacterial colonies, the bacterial colonies being per mL or g of composition.

Advantageously, the invention relates to the use as defined above, said composition being applied or in a form capable of being applied, directly or indirectly via the breeding substrate, to the eggs, the larvae, the prepupae, the pupae or adult individuals of breeding insects.

Provision is also made for the use of the aforementioned food, for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects.

The invention also relates to a composition for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects, in particular black soldier flies, said composition consisting essentially into at least one strain of lactic acid bacteria, in particular Lactococcus lactis , said strain being chosen in particular from:

- a NOL11 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4609, and

- a strain of lactic acid bacteria belonging to the same operational taxonomic unit (OTU) as said NOL11 strain,

or a mixture of these strains.

The composition according to the invention is "consisting essentially" of said strains of lactic acid bacteria, which means that the composition contains, as active substance, only strains of lactic acid bacteria. This means that the composition is likely to contain other components, but these additional components do not contribute to the technical effect: that is to say that these components do not, alone, have a significant influence on the improving the zootechnical performance of insect farming, in particular on the treatment or prevention of microbial pathologies affecting the growth of farmed insects.

It may be particularly advantageous if the genetic proximity of lactic acid bacteria is more than 97% genome identity, compared to the 16S rRNA of the NOL11 strain.

Advantageously, the invention relates to an above-mentioned composition for its above-mentioned use, wherein said composition comprises from 10 ⁴ to 10 ¹¹ living bacterial colonies of the said strain or strains of lactic acid bacteria, the bacterial colonies being per mL or g of composition.

Advantageously, the invention relates to an aforementioned composition for its aforementioned use, in combination with at least one strain of bacteria of the genus Bacillus , in particular at least one strain of Bacillus subtilis.

Advantageously, the invention relates to an aforementioned composition for its aforementioned use, where said strain of Bacillus is chosen in particular from:

- a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and

- a Bacillus strain belonging to the same operational taxonomic unit (OTU) as said NOL01 strain,

or a mixture of these strains.

Advantageously, the invention relates to an aforementioned composition for its aforementioned use, wherein said Bacillus strain belonging to the same OTU as said NOL01 strain is chosen from:

- a strain of Bacillus subtilis strain NOL02 as filed with the CNCM on January 21, 2016 under number CNCM I – 5043, and

- a strain of Bacillus subtilis strain NOL03 as filed with the CNCM on March 14, 2012 under number CNCM I – 4607.

Advantageously, the invention relates to an aforementioned composition for its aforementioned use, wherein said at least one strain of Bacillus is chosen from:

- the NOL01 strain, or

- the NOL02 strain, or

- the NOL03 strain, or

- the NOL01 strain and the NOL02 strain, or

- the NOL01 strain and the NOL03 strain, or

- the NOL02 strain and the NOL03 strain, or

- the NOL01 strain, the NOL02 strain and the NOL03 strain.

Advantageously, the invention relates to a composition mentioned above for its use as defined above, where said composition comprises from 10 ⁴ to 10 ¹¹ living bacterial colonies of said Bacillus( s), the bacterial colonies being per mL or g of composition.

More advantageously, the invention relates to a composition mentioned above for its use as defined above, said composition being applied, or in a form to be or capable of being applied, to eggs, larvae, prepupae, pupae or adult individuals of breeding insects, directly or indirectly via the breeding substrate.

The invention further relates to an insect feed comprising a composition as defined above,

in particular for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects.

It is also envisaged the use of a composition consisting essentially of at least one strain of lactic acid bacteria, said strain being chosen in particular from:

- a NOL11 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4609, and

- a strain of lactic acid bacteria belonging to the same operational taxonomic unit (OTU) as said NOL11 strain,

or a mixture of these strains,

for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects, in particular black soldier flies.

Advantageously, the invention relates to the aforementioned use, where said composition comprises from 10 ⁴ to 10 ¹¹ bacterial colonies of said lactic acid bacteria(s), the bacterial colonies being per mL or g of composition.

More advantageously, the invention relates to the aforementioned use, where said composition is in combination with at least one strain of bacteria of the genus Bacillus , in particular at least one strain of Bacillus subtilis , in particular

- a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and

- a Bacillus strain belonging to the same operational taxonomic unit (OTU) as said NOL01 strain,

or a mixture of these strains.

More advantageously, the invention relates to the aforementioned use, where said Bacillus strain belonging to the same OTU as said NOL01 strain is chosen from:

- a strain of Bacillus subtilis strain NOL02 as filed with the CNCM on January 21, 2016 under number CNCM I – 5043, and

- a strain of Bacillus subtilis strain NOL03 as filed with the CNCM on March 14, 2012 under number CNCM I – 4607.

Advantageously, the invention relates to the aforementioned use, where said composition is used in combination with:

- the NOL01 strain, or

- the NOL02 strain, or

- the NOL03 strain, or

- the NOL01 strain and the NOL02 strain, or

- the NOL01 strain and the NOL03 strain, or

- the NOL02 strain and the NOL03 strain, or

- the NOL01 strain, the NOL02 strain and the NOL03 strain.

Advantageously, the invention relates to the use as defined above, where said composition comprises from 10 ⁴ to 10 ¹¹ living bacterial colonies of said Bacillus(s) , the bacterial colonies being per mL or g of composition.

Advantageously, the invention relates to the use as defined above, said composition being applied or in a form capable of being applied to the eggs, larvae, prepupae, pupae or adult individuals of farmed insects, directly or indirectly via the substrate.

The invention also relates to the use of an insect feed mentioned above for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects.

In another aspect, the invention relates to a method for improving the zootechnical performance of farmed insects, in particular a method for the prophylaxis or treatment of microbial pathologies affecting said farmed insects, in particular black soldier flies, comprising a step of bringing into contact directly or indirectly via the rearing substrate, in particular, eggs, larvae, prepupae, pupae or adult individuals of the rearing insects, with any one of the aforementioned compositions, in particular

a composition consisting essentially of at least one strain of bacteria of the genus Bacillus, said strain being chosen from: a strain NOL01 and a strain of Bacillus belonging to the same OTU as said strain NOL01, or a mixture of these strains,

Or

a composition consisting essentially of at least one strain of lactic acid bacteria, chosen from: an NOL11 strain and a strain of lactic acid bacteria belonging to the same OTU as said NOL11 strain, or a mixture of these strains,

or a mixture of these compositions.

The invention will be better understood in the light of the following figures and drawings.

Brief description of figures

Figure 1 is a box plot diagram representing the individual weights of a representative sample of 30 larvae per fattening tank (in g) for the control batches (Control, D1+H2O and D3+ H2O - B) compared to those of the test batches (D1+CI and D3+CI - A), on D14. t-test: p-value = 0.0002434.

Figure 2 is a box plot diagram representing the masses of pupae per fattening tank (in kg) for the control groups (Control, D1+H2O and D3+H2O - B) compared to those of the test groups (D1+ CI and D3+CI - A), on sorting day D28. t-test: p-value = 0.001243.

Figure 3 is a box plot diagram representing the survival rates per fattening tank (in %) for the control groups (Control, D1+H20 and D3+H2O - B) compared to those of the test groups (D1+ CI and D3+CI - A), on sorting day D28. t-test: p-value = 0.03293.

Figure 4 is a box plot diagram representing the estimated number of pupae per fattening tank for the control groups (Control, D1+H2O and D3+H2O - B) compared to that of the test groups (D1+CI and D3 +CI - A), on sorting day D28.

Examples

Example 1

The objective of this test is to demonstrate the effect of a composition according to the invention (CI) on the increase in production yields of BSF larvae in breeding.

This test shows the results obtained under experimental conditions on 30 batches divided into 5 modalities (i.e. 6 repetitions per modality).

1/ Materials and methods:

The 5 modalities vary according to 2 criteria:

- Spraying of a composition according to the invention or not: the control groups undergo either no spraying (Control) or a spraying of water (Jx+H ₂ O); the test groups are sprayed with a composition according to the invention (Jx+CI),

- Frequency of spraying: when there is spraying, it is carried out once a day (D1+CI and D1+ H ₂ O) or once every 3 days (D3+CI and D3+ H ₂ O).

The substrate, common to the 5 modalities, is composed of ground plant raw materials (using a grinder) in order to mix the raw materials together (fruit or vegetable peelings such as bananas, carrots, broccoli, courgettes, cereals (wheat, corn, etc.) in such a way that the substrate is nutritionally suitable to meet the average nutritional needs of an insect larva.

Upstream, the eggs come from nests and are collected daily. On D0, they are placed for incubation (30°C – 70% humidity) for 6 days, in incubation tanks with a capacity of 300g of substrate (average pH 5.8). Approximately 0.100g of BSF eggs are thus cultured, corresponding to approximately 8 clutches, in each of the 30 incubation tanks. The average number of inoculated eggs per tank at incubation is 5200 eggs.

On the 7th day (D7), the young larvae and their substrate are transferred to 30 fattening tanks to continue their development, to which 8 kg of fresh substrate (average pH 5.9) are added.

The fattening tanks are then placed in the production area.

On D28, the test ends with individuals at the pupal stage.

The composition according to the invention sprayed for the J1+CI and J3+CI groups consists of the following three strains of Bacillus subtilis : NOL01, NOL02 and NOL03.

The composition according to the invention is brought frozen to the farm and packaged in a 20 ml bottle. After thawing, the 20 ml vial is diluted in 1 liter of water in the tank of a sprayer and the preparation is stored at 4°C until use. The tank is shaken before each daily use or every 3 days.

For the groups with spraying, spraying is carried out per incubation or fattening tank (i.e. approximately 2 ml), either of the composition according to the invention, or of water without composition according to the invention from D1 to D17 (this which corresponds to 17 sprays of approximately 2 ml per container for batches D1+CI and D1+ H20 and 7 sprays of approximately 2 ml per container for batches D3+CI and D3+H20).

The data is collected:

- every day: reading of the temperature of the incubation or fattening tanks (in °C)

- at D14 and D17, mass of larvae (in g)

- at sorting = D28, mass of pupae (in g).

2/ Results and conclusions

The results are presented in figures 1 to 4.

Figure 1 shows an individual average weight of the larvae of the test batches (D1+CI and D3+CI) significantly higher than that of the control groups (Control, D1+H ₂ O and D3+H ₂ O) on D14 after spraying.

Figure 2 shows an average mass of the pupae of the test groups (D1+CI and D3+CI) significantly higher than that of the control groups (Control, D1+H ₂ O and D3+H ₂ O) at D28.

Figure 3 shows an average survival rate for the test groups (D1+CI and D3+CI) significantly higher (+0.08 points) than that of the control groups (Control, D1+H ₂ O and D3+H ₂ O) at D28.

Figure 4 shows a greater average number of pupae at D28 in the test tanks than the control tanks, which reflects a lower mortality rate for the test groups (reduction in mortality).

These data show that the supply of the composition according to the invention via the substrate has a beneficial effect on the weight gain and the final number of individuals (reduction of mortality) of the groups of BSF treated, whether daily or every the 3 days. This results in a significant gain in final biomass.

Example 2

The objective of this test is to demonstrate the effect of a composition according to the invention on the production of farmed BSF larvae in the event of a bacterial pathology harmful to production.

This test shows the results obtained under experimental conditions on 40 batches divided into 5 modalities (i.e. 8 repetitions per modality) during an unexpected episode of bacterial contamination of the farm.

1/ Materials and methods:

As in example 1, these 5 methods vary according to 2 criteria, namely the spraying on the rearing substrate of a composition according to the invention or not (absence of spraying, spraying of a composition according to the invention or spraying of H ₂ O) and the frequency of this spraying (daily or every 3 days).

The incubation and fattening of the 40 batches is identical to example 1.

The composition of the substrate common to the 5 modalities is identical to that of example 1.

The composition according to the invention sprayed for groups J1+CI and J3+CI and its implementation are identical to those of Example 1 except for the number of sprays per tank.

Thus, for the groups with spraying, 10 sprayings are carried out per incubation or fattening tank (i.e. approximately 10 ml), either of the composition according to the invention, or of water without composition according to the invention, on a daily basis. or every 3 days depending on the batch.

2/ Incident:

On D11, significant mortality is observed in certain batches of the trial due to contamination of the farm by a pathogenic bacterium, Bacillus thuringiensis , commonly used in organic farming as a natural insecticide. This contamination affects all of the 40 tanks in the test, both the test batch tanks (D1+CI and D3+CI) and the control batch tanks (Control, D1+H ₂ O and D3+H ₂ O ).

3/ Results:

- From D0 to D14, no difference in temperature or pH change is observed in the fattening tanks of the test batches (D1+CI and D3+CI) versus the control batches (Control, D1+H ₂ O and J3+H ₂ O).

- On D14, no significant difference was observed between the mass of the larvae of the different batches according to the "Frequency of spraying" modality daily or every 3 days.

- On D14, a significantly positive difference (+15.4%) is observed in the average mass of the larvae of the test batches (D1+CI and D3+CI) compared to that of the control batches (Control, D1+H ₂ O and D3 +H ₂ O).

- It is observed that the mortality did not occur at the same speed in all the tanks, the fattening tanks with spraying of the composition according to the invention having taken approximately 3 days longer to declare the pathology than the tanks without spray (Control) or with water spray (J1+H ₂ O and J3+H ₂ O). The appearance of the first symptoms of the pathology in the Test tanks led to the trial being stopped on D14.

- The larvae of the Control batches, that is to say without spraying the composition according to the invention or with spraying water, showed a higher and earlier mortality rate (from D11) than the test batches.

4/ Conclusion:

Despite bacterial contamination causing high mortality of the larvae, the application of the composition according to the invention makes it possible to continue the fattening of the BSF larvae for 3 days longer compared to the control batches: these 3 additional days have a real economic interest for an insect protein production chain due to the continued growth of the treated larvae. Indeed, except in the context of rearing for reproduction, the larvae are slaughtered for recovery in oil or cake before the prepupal phase, around D14.

Example 3

The objective of this test is to compare the effect of different compositions according to the invention on the production yields of farmed BSF larvae.

This test shows the results obtained under experimental conditions on 108 batches divided into 4 modalities and 2 batches.

1/ Materials and methods:

The 4 methods vary according to the composition of the substrate used:

- Control T-: common substrate (without addition);

- L: common substrate+composition according to the invention consisting of the strain of lactic acid bacteria Lactococcus lactis spp lactis 1 strain NOL11, said strain being deposited at the CNCM under the number CNCM I-4609;

- B: common substrate + composition according to the invention consisting of the following three strains of Bacillus subtilis : NOL01, NOL02, NOL03, said strains being deposited at the CNCM under the respective numbers: CNCM I - 4606, CNCM I-5043 and CNCM I – 4607;

- B+L: common substrate + composition according to the invention consisting of the combination of strains NOL01, NOL02, NOL03 and NOL11.

Table 1 presents the test device, in number of batches per modality, according to the production batch considered:

Modality T- B. lactic (L) Bacillus (B) B+L Batch 1 18 18 18 Batch 2 18 18 18 Total 36 36 18 18

The composition of the substrate common to the 4 modalities is identical to that of example 1.

To this common substrate is added, for batches L, B and L+B, the composition according to the invention, up to 50 g per 100 kg of substrate. The composition according to the invention is provided in the form of a powder and incorporated into the substrate at the time of its preparation according to conventional techniques known in the art.

The incubation of the 108 batches differs from that of example 1 on the following points:

- 250g of substrate per tank at incubation

- 15 spawning units per tank at incubation

Steps to take at start-up and during incubation:

- The pH, density and dry matter are recorded during the manufacture of each of the different substrates (D0).

- The temperature and relative humidity of the incubator will be recorded daily.

- The weight of the incubation trays will also be recorded daily.

- At the end of incubation (D7), the trays will be weighed one last time, the mass of 30 larvae will be recorded before transfer to the fattening tanks.

On D7, the incubation tanks are transferred to fattening tanks containing 15 kg of fresh substrate. The fattening tanks are then placed in the production area and kept in optimal breeding conditions until the day of harvest on D17.

Steps to take when fattening:

The temperature and relative humidity of the fattening area will be recorded daily as well as the temperature inside the tanks, the mass of 30 larvae and an observation of good health. The pH of each tank will be measured on D10, D13, D15 and on the day of harvest (D17).

Measures to be taken on the day of harvest at D17:

On harvest day (D17), each bin will be subject to the following measures:

- The mass of the bin before sorting (in Kg)

- The mass of larvae collected (in Kg)

- The mass of amendment harvested (in Kg)

- The mass of 30 larvae

Claims (9)

Composition for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting the said farmed insects, in particular black soldier flies, the said composition consisting essentially of at least one strain of lactic acid bacteria, said strain being chosen from:
- a NOL11 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4609, and
- a strain of lactic acid bacteria belonging to the same operational taxonomic unit (OTU) as said NOL11 strain,
or a mixture of these strains. A composition for use according to claim 1, wherein said composition comprises from 10 ⁴ to 10 ¹¹ live bacterial colonies of said strain(s) of lactic acid bacteria, the bacterial colonies being per mL or g of composition. Composition for its use according to claim 1 or claim 2, in combination with at least one strain of bacteria of the Bacillus genus, in particular at least one strain of Bacillus subtilis . Composition for its use according to claim 3, wherein said strain of Bacillus is chosen from:
- a NOL01 strain as deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on March 14, 2012 under number CNCM I – 4606, and
- a Bacillus strain belonging to the same operational taxonomic unit (OTU) as said NOL01 strain,
or a mixture of these strains. Composition for its use according to claim 3 or claim 4, wherein said strain of Bacillus belonging to the same OTU as said strain NOL01 is chosen from:
- a strain of Bacillus subtilis strain NOL02 as filed with the CNCM on January 21, 2016 under number CNCM I – 5043, and
- a strain of Bacillus subtilis strain NOL03 as filed with the CNCM on March 14, 2012 under number CNCM I – 4607. Composition for its use according to any one of Claims 3 to 5, in which the said at least one strain of Bacillus is chosen from
- the NOL01 strain, or
- the NOL02 strain, or
- the NOL03 strain, or
- the NOL01 strain and the NOL02 strain, or
- the NOL01 strain and the NOL03 strain, or
- the NOL02 strain and the NOL03 strain, or
- the NOL01 strain, the NOL02 strain and the NOL03 strain. A composition for use according to any one of claims 3 to 6, wherein said composition comprises from 10 ⁴ to 10 ¹¹ live bacterial colonies of said Bacillus(s) , the bacterial colonies being per mL or g of composition. Composition for its use according to any one of Claims 1 to 7, the said composition being in a form suitable for being applied to the eggs, the larvae, the prepupa, the pupae or the adult individuals of the breeding insects, directly or indirectly through the rearing substrate. Insect food comprising a composition as defined in any one of claims 1 to 2, in particular according to any one of claims 1 to 7,
in particular for its use for improving the zootechnical performance of farmed insects, in particular for the prophylaxis or treatment of microbial pathologies affecting said farmed insects.