COMPOSITION OF MICROORGANISMS FOR BREEDING INSECTS

Abstract

A composition for use thereof in the prophylaxis of microbial pathological conditions affecting the growth of insects for breeding, the composition including essentially at least one strain of bacterium of the Bacillus genus, the strain being chosen from: a strain of Bacillus subtilis strain NOL01 as deposited with the CNCM on Mar. 14, 2012 under number CNCM I - 4606, and a strain of Bacillus belonging to the same operational taxonomic unit as the NOL01 strain, or a mixture thereof.

Claims

1-15. (canceled)

16. A method for improving the zootechnical performance of insects for breeding, comprising a step of administering a composition to the insects, said composition consisting essentially of a strain of bacteria of the Bacillus genus, said strain being chosen from: an NOL01 strain as deposited with the Collection Nationale de Cultures de Microorganismes [National Collection of Microorganism Cultures] (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on Mar. 14, 2012 under number CNCM I - 4606, and a strain of Bacillus belonging to the same operational taxonomic unit (OTU) as said NOL01 strain, or a mixture of these strains.

17. The method according to claim 16, wherein the insects for breeding are black soldier flies.

18. The method according to claim 16, wherein strain of Bacillus belonging to the same operational taxonomic unit (OTU) as said NOL01 strain is chosen from: a strain of Bacillus subtilis strain NOL02 as deposited with the CNCM on Jan. 21, 2016 under number CNCM I - 5043, and a strain of Bacillus subtilis strain NOL03 as deposited with the CNCM on Mar. 14, 2012 under number CNCM I - 4607.

19. The method according to claim 16, said composition consisting essentially of one of: the NOL01 strain, the NOL02 strain, the NOL03 strain, the NOL01 strain and the NOL02 strain, the NOL01 strain and the NOL03 strain, the NOL02 strain and the NOL03 strain, and the NOL01 strain, the NOL02 strain and the NOL03 strain.

20. The method according to claim 16, wherein said composition comprises from 10.sup.4 to 10.sup.11 live bacterial colonies of said Bacillus or Bacilli, the bacterial colonies being per mL or g of composition.

21. The method according to claim 16, wherein said bacteria of the genus Bacillus are in vegetative form or in spore form, or both.

22. The method according to claim 16, said composition being in a form capable of being applied to the eggs, the larvae, the prepupa, the pupae or the adult individuals of the insects for breeding, directly or indirectly via the substrate.

23. An insect food comprising a composition said composition consisting essentially of a strain of bacteria of the Bacillus genus, said strain being chosen from: an NOL01 strain as deposited with the Collection Nationale de Cultures de Microorganismes [National Collection of Microorganism Cultures] (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on Mar. 14, 2012 under number CNCM I - 4606, and a strain of Bacillus belonging to the same operational taxonomic unit (OTU) as said NOL01 strain, or a mixture of these strains.

24. A method improve the zootechnical performance of insects for breeding, the method comprising contacting insects for breeding with the insect food as defined in claim 23.

25. A method for the prophylaxis or treatment of microbial pathologies affecting insects for breeding, in particular black soldier flies, comprising a step of contacting the insects for breeding with a composition, said composition consisting essentially of a strain of bacteria of the Bacillus genus, said strain being chosen from: an NOL01 strain as deposited with the Collection Nationale de Cultures de Microorganismes [National Collection of Microorganism Cultures] (CNCM, Institut Pasteur 25-28 rue du Docteur Roux 75724 Paris, France) according to the Budapest Treaty on Mar. 14, 2012 under number CNCM I - 4606, and a strain of Bacillus belonging to the same operational taxonomic unit (OTU) as said NOL01 strain, or a mixture of these strains.

26. The method according to claim 25, wherein said Bacillus strain belonging to the same OTU as said NOL01 strain is chosen from: a strain of Bacillus subtilis strain NOL02 as deposited with the CNCM on Jan. 21, 2016 under number CNCM I - 5043, and a strain of Bacillus subtilis strain NOL03 as deposited with the CNCM on Mar. 14, 2012 under number CNCM I - 4607.

27. The method according to claim 24, said composition consisting essentially of one of: the NOL01 strain, the NOL02 strain, the NOL03 strain, the NOL01 strain and the NOL02 strain, the NOL01 strain and the NOL03 strain, the NOL02 strain and the NOL03 strain, and the NOL01 strain, the NOL02 strain and the NOL03 strain.

28. The method according to claim 25, wherein said composition comprises from 10.sup.4 to 10.sup.11 live bacterial colonies of said Bacillus or Bacilli, the bacterial colonies being per mL or g of composition.

29. The method according to claim 25, wherein said one or more strains of bacteria of the genus Bacillus are in vegetative form or in spore form, or both.

30. The method according to claim 25, said composition being in a form capable of being applied to the eggs, the larvae, the prepupa, the pupae or the adult individuals of the insects for breeding, directly or indirectly via the substrate.

31. A for the prophylaxis or treatment of microbial pathologies affecting said insects for breeding, the method comprising contacting insects for breeding with the insect food as defined in claim 23.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0157] FIG. 1 is a box plot representing the individual weights of a representative sample of 30 larvae per fattening tank (in g) for the control batches (Control, D1+H.sub.2O and D3+H.sub.2O - B) compared with those of the test batches (D1+CI and D3+CI - A), on D14. t-test: p-value = 0.0002434.

[0158] FIG. 2 is a box plot representing the masses of pupae per fattening tank (in kg) for the control groups (Control, D1+H.sub.2O and D3+H.sub.2O - B) compared to those of the test groups (D1+CI and D3+CI - A), on sorting day D28. t-test: p-value = 0.001243.

[0159] FIG. 3 is a box plot representing the survival rates per fattening tank (in %) for the control groups (Control, D1 +H.sub.2O and D3+H.sub.2O - B) compared to those of the test groups (D1+CI and D3+CI - A), on sorting day D28. t-test: p-value = 0.03293.

[0160] FIG. 4 is a box plot representing the estimated number of pupae per fattening tank for the control groups (Control, D1+H.sub.2O and D3+H.sub.2O - B) compared to that of the test groups (D1+CI and D3+CI - A), on sorting day D28.

EXAMPLES

Example 1

[0161] The objective of this test is to demonstrate the effect of a composition according to the invention (Cl) on the increase in production yields of BSF larvae for breeding.

[0162] This test shows the results obtained under experimental conditions on 30 batches divided into 5 modalities (i.e. 6 repetitions per modality).

1/ Material and Methods

[0163] The 5 modalities vary according to 2 criteria: [0164] Spraying of a composition according to the invention or not: the control groups undergo either no spraying (Control) or a spraying of water (Dx+H.sub.2O); the test groups are sprayed with a composition according to the invention (Dx+CI), [0165] Spraying frequency: when spraying occurs, it is carried out once a day (D1+CI and D1+ H.sub.2O) or once every 3 days (D3+CI and D3+ H.sub.2O).

[0166] The substrate, common to the 5 modalities, is composed of plant raw materials that are ground (using a grinder) in order to mix together the raw materials (fruit or vegetable peelings such as bananas, carrots, broccoli, zucchini, cereals (wheat, corn, etc.)) in such a way that the substrate is nutritionally suitable to meet the average nutritional needs of an insect larva.

[0167] Upstream, the eggs come from nest boxes and are collected daily. On D0, they are placed in incubation tanks with a capacity of 300 g of substrate (average pH 5.8) for incubation (30° C. - 70% humidity) for 6 days. Approximately 0.100 g of BSF eggs are thus cultured, corresponding approximately to 8 clutches, in each of the 30 incubation tanks. The average number of inoculated eggs per incubation tank is 5200 eggs.

[0168] 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 (mean pH 5.9) is added.

[0169] The fattening tanks are then placed in the production area.

[0170] On D28, the test ends with individuals at the pupal stage.

[0171] The composition according to the invention sprayed for the D1+CI and D3+CI groups consists of the following three strains of Bacillus subtilis: NOL01 , NOL02 and NOL03.

[0172] The composition according to the invention is supplied frozen for breeding and packaged in a 20 mL bottle. After thawing, the 20 mL vial is diluted in 1 liter of water in the reservoir of a sprayer and the preparation is stored at 4° C. until use. The reservoir is shaken before each daily use or every 3 days.

[0173] 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 (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).

[0174] The data is collected: [0175] every day: recording of the temperature of the incubation or fattening tanks (in °C) [0176] at D14 and D17, mass of larvae (in g) [0177] at sorting = D28, mass of pupae (in g).

2/ Results and Conclusions

[0178] The results are presented in FIGS. 1 to 4.

[0179] FIG. 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.sub.2O and D3+H.sub.2O) on D14 after spraying.

[0180] FIG. 2 shows an average mass of the pupae of the test groups (D1+Cl and D3+CI) significantly higher than that of the control groups (Control, D1+H.sub.2O and D3+H.sub.2O) on D28.

[0181] FIG. 3 shows an average survival rate for the test groups (D1+Cl and D3+CI) significantly higher (+0.08 points) than that of the control groups (Control, D1+H.sub.2O and D3+H.sub.2O) at D28.

[0182] FIG. 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).

[0183] 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 (reduced mortality) of the groups of BSF treated, whether daily or every 3 days. This results in a significant gain in final biomass.

Example 2

[0184] The objective of this test is to demonstrate the effect of a composition according to the invention on the production of BSF larvae for breeding in the event of a bacterial pathology harmful to production.

[0185] 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/ Material and Methods

[0186] As in example 1, these 5 modalities 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.sub.2O) and the frequency of this spraying (daily or every 3 days).

[0187] The incubation and fattening of the 40 batches is identical to example 1.

[0188] The composition of the substrate common to the 5 modalities is identical to that of example 1.

[0189] The composition according to the invention sprayed for groups D1+CI and D3+CI and its implementation are identical to those of example 1 apart from the number of sprays per tank.

[0190] 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

[0191] On D11, significant mortality was observed in certain batches of the test due to contamination of the farm by a pathogenic bacterium, Bacillus thuringiensis, commonly used in organic farming as a natural insecticide. This contamination affected 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.sub.2O and D3+H.sub.2O).

3/ Results

[0192] From D0 to D14, no difference in temperature or pH change was observed in the fattening tanks of the test batches (D1+CI and D3+CI) versus the control batches (Control, D1+H.sub.2O and D3+H.sub.2O). [0193] On D14, no significant difference was observed between the mass of the larvae of the different lots according to the “Frequency of spraying” modality, daily or every 3 days. [0194] On D14, a significantly positive difference (+15.4%) was observed in the average mass of the larvae of the test lots (D1 +Cl and D3+CI) compared to that of the control lots (Control, D1+H.sub.2O and D3+ H.sub.2O). [0195] It was 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 exhibit the pathology than the tanks without spraying (Control) or with water spraying (D1+H.sub.2O and D3+H.sub.2O). The appearance of the first symptoms of the pathology in the Test tanks led to the test being stopped on D14. [0196] The larvae of the Control lots, 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 lots.

4/ Conclusions

[0197] Despite bacterial contamination causing high mortality of the larvae, the application of the composition according to the invention makes it possible to continue fattening of the BSF larvae for 3 days longer compared to the control lots: these 3 additional days have a real economic interest for an insect protein production sector 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 meal before the prepupal phase, around D14.

Example 3

[0198] The objective of this test is to compare the effect of different compositions on the production yields of BSF larvae for breeding, and therefore on zootechnical performance.

[0199] This test shows the results obtained under experimental conditions on 72 lots divided into 3 modalities and 2 batches.

1/ Material and Methods

[0200] The 3 modalities vary according to the composition of the substrate used: [0201] Control T-: common substrate (without addition); [0202] L: common substrate + composition consisting of the strain of lactic acid bacteria Lactococcus lactis spp lactis 1 strain NOL11, said strain being deposited with the CNCM under number CNCM I - 4609; [0203] B+L: common substrate + composition consisting of the combination of strains NOL01, NOL02, NOL03 and NOL11.

[0204] Table 1 presents the test device, in number of lots per modality, according to the production batch considered:

TABLE-US-00001 Modality T- B. lactic (L) B+L Batch 1 18 18 Batch 2 18 18 Total 36 18 18

[0205] The composition of the substrate common to the 3 modalities is identical to that of example 1.

[0206] Bacterial compositions L and L+B are added to this common substrate, for lots L and L+B, up to 50 g per 100 kg of substrate. The bacterial compositions are provided in powder form and incorporated into the substrate at the time of its preparation according to conventional techniques known in the art.

[0207] The incubation of the 72 lots differs from that of example 1 on the following points: [0208] 250 g of substrate per incubation tank [0209] 15 laying units per incubation tank

[0210] Measurements to take at start-up and during incubation: [0211] The pH, density and dry matter are recorded during the manufacture of each of the different substrates (D0). [0212] The temperature and relative humidity of the incubator are recorded daily. [0213] The weight of the incubation trays is also recorded daily. [0214] On leaving incubation (D7), the trays are weighed one last time, the mass of 30 larvae is recorded before transfer to the fattening tanks.

[0215] 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 harvest day on D17.

[0216] Measurements to take during fattening:

[0217] The temperature and the relative humidity of the fattening area are 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 is measured on D10, D13, D15 and on harvest day (D17).

[0218] Measurements to be taken on harvest day at D17:

[0219] On harvest day (D17), each tank is subject to the following measurements: [0220] The mass of the tank before sorting (in Kg) [0221] The mass of larvae collected (in Kg) [0222] The mass of amendment harvested (in Kg) [0223] The mass of 30 larvae.

[0224] On harvest day D17, the results concerning the individual weight, the survival rate of the larvae and the mortality are recorded and are expressed as a percentage relative to the control lot T-.

[0225] Table 2 shows all the results of this example:

TABLE-US-00002 Strains Individual weight Survival Yield of larvae Lactococcus + 25%*** - 34%*** - 18%*** Lactococcus and Bacillus - 3% + 11% + 9.2% The data are expressed as a percentage relative to the control T- (common substrate). ***: significant variation compared to the control (p value <0.05)

[0226] The results obtained during this test evaluating the improvement of zootechnical performance show quite surprising results: [0227] i) it is noted that the individual weight is increased in the Lactococcus lots, but that this significant difference is no longer observed when the two bacterial types are used simultaneously, [0228] ii) survival significantly collapses by more than a third in the Lactococcus lots, and it does not vary significantly from the control in the Bacillus + Lactococcus lots, and [0229] iii) the yield of the larvae is significantly reduced in the Lactococcus lots and is unchanged in the Bacillus + Lactococcus lots compared with the control.

[0230] Table 3 shows all the results of the three previous examples:

TABLE-US-00003 Strains Tests Individual weight Survival Yield of larvae Bacillus Example 1: Example 2: + 3.45% + 13.4%*** + 17%*** 3 more days*** + 20.7%*** n/d Lactococcus Example 3: + 25%*** - 34%*** - 18%*** Lactococcus and Bacillus Example 3: - 3% + 11% + 9.2% The data are expressed as a percentage relative to the control T- (common substrate) n/d: not determined ***: significant variation compared to the control (p value <0.05)

[0231] These three examples show that, very surprisingly, the sole use of a substrate comprising Bacillus strains according to the invention allows: [0232] increased/improved zootechnical performances, evaluated on the basis of the survival of the breeding, its yield and the weight of the individuals, whereas [0233] the use of strains of Lactococcus has a predominantly detrimental effect on said performance.

[0234] Indeed, although the individual weight of the larvae is significantly better in this last lot, this is linked to the fact that a high mortality occurred and therefore that the remaining larvae had more substrate to gain weight (less density per substrate unit).

[0235] Furthermore, the combination of the two species of bacteria ( Bacillus subtilis and Lactococcus lactis) does not make it possible to improve the performance obtained with the Bacillus strains alone, these latter results being comparable to those of the control (no bacteria).

[0236] Therefore, a composition according to the invention indeed shows particularly interesting and surprising properties on zootechnical performance as demonstrated by these tests.