Process for preparing a substrate for rearing insect larvae by enzymatic hydrolysis and substrate obtained

Abstract

Method for preparing a substrate for rearing insect larvae by enzymatic hydrolysis and substrate obtained The invention relates to a method for preparing a substrate for rearing insect larvae from by-products derived from plants, comprising the following steps: a) obtaining by-products derived from plants in the form of particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, if necessary by homogenization, b) mixing the product obtained in a) with an aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase, to obtain a composition whose moisture content is between 55% and 85% by weight relative to the total weight of the mixture, and c) adding at least one enzyme to the mixture b), then homogenization, in which step c) is concomitant with or subsequent to step b). Figure for the abstract: none

Description

Title of the invention: Method for preparing a substrate for breeding insect larvae by enzymatic hydrolysis and substrate obtained

[0001] The present invention relates to a method for preparing a substrate for rearing insect larvae from by-products derived from plants, comprising specific steps a) to c). It also relates to the substrate obtained, and to the method for rearing insects using such a substrate.

[0002] Protein production is one of the key challenges of the coming decades, with a deficit growing worldwide (60Mt by 2030).

[0003] The protein deficit is particularly critical for the aquaculture sector. With almost half of wild fish stocks exploited at biologically unsustainable levels, aquaculture is expanding rapidly to meet growing global demand. According to the FAO's 2016 State of World Fisheries and Aquaculture report, the majority of aquatic products now come from aquaculture, not fisheries. Growing at more than 5% per year, the aquaculture sector is the fastest-growing agri-food sector.

[0004] Insect farming addresses this issue by providing a source of nutrients for animal nutrition and agriculture. These products make it possible to introduce new, more sustainable sources of protein, particularly in aquaculture, for which insect proteins are particularly suitable. Indeed, insect meal is an excellent solution to support the growth of aquaculture, where traditional feed sources (wild fish meal or soybean meal) are insufficient and have a high environmental impact.

[0005] Furthermore, industries for processing plants into derived products include, in particular, starch production (production of starch from cereals, in particular wheat or corn), the sugar industry (production of sugar from sugar cane or beet) or oil production (in particular from palm oil obtained from the fruit of the oil palm, and palm kernel oil obtained from kernels of the fruit of the oil palm). These industries generate plant by-products that can be used as sources of proteins, lipids and potentially carbohydrates, which may be of interest for the breeding of insect larvae, but which have been little used to date. Generally speaking, the processes used include the isolation of the compounds of interest by mechanical methods (grinding, separation of different fractions for starch production or protein preparation), which may include fermentation and / or distillation steps. These processes use water, particularly in the separation steps, and may lead to by-products alongside the main product.

[0006] In particular, palm oil production generates effluents, called "palm oil mill effluent (POME)", which contain organic matter, but also other by-products and wastes that include palm kernel cakes. POME must be subjected to treatment to comply with the standard required by the competent authorities, its degradation by fermentation being responsible for very high emissions of methane, a particularly harmful greenhouse gas. However, due to the concentration of organic matter, the treatment of POME has always been a challenge in the palm oil milling industry. The treatment of POME, when not used for biogas production, is not revenue-generating, requires a substantial cost for palm oil and occupies a large area of ​​land.As for palm kernel cakes, they are generally little, or even very little, valued; they are mainly exported for animal feed, particularly for cattle, used as fertilizer, or burned to produce energy.

[0007] There is therefore a need to improve the recovery of by-products from the processing of plants, in particular from the production of palm oil. In particular, there is a need for upcycling of by-products from the processing of plants, and in particular from the production of palm oil. Preferably, these by-products must be used and / or processed to reduce the environmental impact; used and / or processed on a site close to their place of production; and / or processed to extract their nutrients for optimized animal feed purposes. Finally, there is a need for optimized animal feed, in particular for fish and shrimp.

[0008] The present invention meets these needs.

[0009] The present invention relates to a method for preparing a breeding substrate. of insect larvae from plant by-products, comprising the following steps:

[0010] a) obtaining by-products from plants in the form of particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, if necessary by homogenization,

[0011] b) mixing the product obtained in a) with an aqueous composition optionally comprising compounds from plants and soluble in the aqueous phase, to obtain a composition whose humidity level is between 55% and 85% by weight relative to the total weight of the mixture, and

[0012] c) adding at least one enzyme to the mixture b), to hydrolyze the mixture b), then homogenization, in which step c) is concomitant or subsequent to step b).

[0013] The method according to the invention makes it possible to valorize by-products derived from plants, preferably from the production of palm oil. Indeed, these by-products are subjected to steps a) to c), which makes it possible to obtain a substrate of major interest for the breeding of insect larvae. Indeed, without wishing to be bound by any theory, high-quality nutrients, compatible with the needs of agriculture, are released from these by-products, and allow the larvae to develop in good conditions. The insects obtained are then easily transformed into animal feed, particularly aquaculture.

[0014] The present invention also relates to a substrate for breeding insect larvae obtained by the method according to the invention. Such a substrate according to the invention can be used for breeding insect larvae.

[0015] The present invention also relates to the use of an aqueous mixture of by-products derived from hydrolyzed plants, said aqueous mixture comprising particles having a D50 of less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, as a substrate for the breeding of insect larvae.

[0016] Finally, the subject of the present invention is a method for rearing insect larvae, comprising the method according to the invention, then, during or after the hydrolysis of step c), a step d) of inoculation of insect larvae into the mixture c) and incubation.

[0017] The method for preparing a substrate for breeding insect larvae from by-products derived from plants according to the invention comprises the following steps:

[0018] a) obtaining by-products from plants in the form of particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, if necessary by homogenization,

[0019] b) mixing the product obtained in a) with an aqueous composition optionally comprising compounds from plants and soluble in the aqueous phase, to obtain a composition whose humidity level is between 55% and 85% by weight relative to the total weight of the mixture, and

[0020] c) adding at least one enzyme to the mixture b), then homogenization,

[0021] in which step c) is concomitant or subsequent to step b).

[0022] The insect larvae are in particular the larvae of the diptera Hermetia Illucens, Musca Domestica, the beetles Tenebrio Molitor, Alphitobius diaperinus as well as the larvae of the crickets Acheta domesticus, Gryllodes sigillatus or Field Cricket Gryllus assimilis. Preferably, the insect larvae are the larvae of Hermetia illucens.

[0023] Plant-derived by-products are co-products obtained during the processing of plants. Preferably, the plants are lignocellulosic, i.e. composed of lignin, cellulose and hemicellulose. Preferably, they are chosen from oilseed plants, cereals, wood and plants from the sugar industry. Preferably, the plant-derived by-products are chosen from oilseed plant cakes, oil mill effluent (POME), decanter cakes, cereal cakes, oilseed seeds, oilseed hulls and skins, cereal co-products such as corn cobs or wheat or rice straw, and sugar manufacturing products, such as sugar cane bagasse or beet pulp.

[0024] Preferably, the by-products derived from plants are chosen from oilseed plant cakes and cereal cakes, preferably from palm kernel cakes (PKC and / or PKE and / or PKM), almond cakes, peanut cakes, safflower cakes, hemp cakes, rapeseed cakes, copra cakes (coconut cakes), cultivated flax cakes, corn cakes (corn germ cakes), rapeseed cakes, walnut cakes, olive cakes (cakes called olive "pomace"), poppy cakes (poppy seed cakes), sesame cakes, soybean cakes and sunflower cakes.

[0025] Preferably, the by-products derived from plants are those obtained during the production of palm oil:

[0026] Palm oil is an important commodity that has wide applications from the production of cooking oils, foods, confectionery to oleochemicals such as soaps and lubricants as well as biodiesel. Palm oil is derived from the Fresh Fruit Bunches (FFB) of the oil palm tree Elaeis guineensis. The FFB harvested from the oil palm plantation are processed in a palm oil mill to extract crude palm oil (CPO) as the main product. The oil palm fruit nuts (Palm Kernel Crusher, PK) are processed in a Palm Kernel Crushing Plant (KCP) to extract palm kernel oil (PKO) as the main product.When processing FFB to recover crude palm oil, a large volume of effluent is also produced during the various stages of sterilization, FFB pressing, and oil clarification. This effluent typically contains 0.5 to 3% oil, 3 to 6% solids, and 90 to 96% water and is called Palm Oil Mill Effluent (POME). The total volume of effluent is normally 60-70% of the weight ratio of FFB processed in conventional agriculture. Other by-products and waste include palm kernel cakes (PKC; these include palm kernel expeller or PKE, also called, during oil extraction with hexane, palm kernel meal (PKM)), stalks (Empty Fruit bunches or EFB), mesocarp fiber and decanter cake obtained by mechanical or natural decantation of POME (decanter cake).

[0027] Thus, preferably, the by-products derived from plants are chosen from palm kernel cakes (PKC and / or PKE and / or PKM), stalks (Empty Fruit bunches or EFB), mesocarp fiber, palm oil mill effluents, decanter cakes and mixtures thereof.

[0028] In step a), by-products from plants are obtained in the form of particles having a D50 of less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm. If necessary, these by-products from plants can be obtained by homogenization to obtain a product with particles having a D50 of less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm. This homogenization can be carried out by grinding and / or sieving. Indeed, the particle size of the ground material obtained is important for the subsequent development of the larvae. Particles having a D50 strictly greater than 5 mm lead to poorer hydrolysis, poor assimilation of nutrients by the larvae and a lower water retention capacity.On the other hand, particles with a D50 of less than or equal to 5 mm according to the invention can absorb water, creating an ideal mixture which is moist without being liquid (rather semi-liquid or pasty in consistency).

[0029] The homogenization of step a) is advantageously carried out by disc or hammer grinding, preferably by hammer. This grinding and / or sieving step is conventional and known from the prior art.

[0030] The product obtained in a) is then mixed with an aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase: this is step b). The aqueous composition obtained has a humidity level of between 55% and 85% by weight relative to the total weight of the mixture.

[0031] The aqueous composition may consist only of water. According to another embodiment of the invention, the aqueous composition comprises compounds derived from plants and soluble in the aqueous phase; such aqueous compositions are also called “soluble”. Such aqueous compositions comprise an aqueous phase and compounds derived from plants (for example lipids, proteins and / or sugars) soluble in the aqueous phase; these are co-products of the processing of the plants. Preferably, the aqueous composition is a palm oil mill effluent (POME).

[0032] Preferably, the mixing of step b) is carried out with an aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase, to obtain a composition whose humidity level is between 55% and 85% by weight, preferably between 65% and 80% by weight relative to the total weight of the mixture.

[0033] Finally, the mixture obtained in step b) is hydrolyzed, thanks to the addition of at least one enzyme: this is step c). The enzyme that can be used is preferably chosen from hydrolases, more preferably from mannanases, hemicellulases, cellulases, lignases, xylanases, pectinases, carbohydrases, proteases, amylases, lipases, keratinases, phytases and mixtures thereof. Preferably, the enzyme of step c) is chosen from mannanases, cellulases and mixtures thereof. Indeed, preferably at least one mannanase and / or at least one cellulase is used.

[0034] Step c) may be concomitant or subsequent to step b).

[0035] According to a first variant, step c) is carried out at the same time as step b); in this case, the product obtained in a), the aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase, and at least one enzyme, are mixed and then homogenized. According to this embodiment, the aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase can be mixed with at least one enzyme, then the resulting mixture is added to the product obtained in a).

[0036] According to a second variant, step c) is after step b); in this case, the product obtained in a) and the aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase are first mixed, then at least one enzyme is added, and the mixture is homogenized.

[0037] Preferably, the enzyme is present in the mixture in an effective amount. By "effective amount" is meant an amount capable of significantly hydrolyzing the mixture. Preferably, the enzyme is present in an amount expressed in units of enzymatic activity present per gram of dry matter of substrate ("units / g of substrate"). The enzymatic activity used is that used by the supplier to characterize the effectiveness of its enzyme. Preferably, the enzyme is present in an amount of at least 0.5 units / g of substrate, preferably at least 1 unit / g of substrate, preferably at least 1.5 units / g of substrate. Preferably, the enzyme is present in an amount of between 0.5 and 2000 units / g of substrate, preferably between 1 and 1800 units / g of substrate, preferably between 1.5 and 1700 units / g of substrate.

[0038] Finally, the mixture obtained is homogenized.

[0039] Preferably, the hydrolysis is carried out at a temperature between 25°C and 90°C, preferably between 25°C and 70°C. Preferably, the hydrolysis is carried out for a period of between 5 minutes and 10 days, preferably between 5 minutes and 3 days.

[0040] At the end of step c) of the method according to the invention, a substrate for breeding insect larvae is thus obtained. This substrate has several advantages: it allows larvae to live, move and feed in a single environment, without the need to change this environment or provide other sources of nutrients to the larvae. In addition, it has a semi-liquid or pasty and homogeneous consistency that allows the larvae to have access to nutrients and to drink throughout the breeding period without the risk of drowning. At the end of the breeding, the substrate has partially dried, which allows the larvae to be separated from the substrate and recovered without difficulty.

[0041] The invention therefore also relates to a substrate for breeding insect larvae obtained by the method according to the invention. This “substrate”, also called “breeding medium”, is a composition intended for breeding insect larvae; it is nutritious and serves as a physical support for the larvae due to its particular properties. The invention therefore also relates to the use of the substrate according to the invention for breeding insect larvae.

[0042] The invention also relates to a method for rearing insect larvae, comprising the method according to the invention, then, during or after the hydrolysis of step c), a step d) of inoculation of insect larvae into the mixture c) and incubation. The mixture obtained at the end of step c) corresponds to the substrate obtained according to the invention.

[0043] Preferably, according to a first variant, the inoculation is carried out by depositing the larvae, preferably at the young larvae stage, on the mixture obtained during the hydrolysis of step c). Subsequently, the incubation of the insect larvae rearing process is carried out for 1 to 60 days at a temperature between 25°C and 45°C. According to such a variant, the hydrolysis reaction takes place during the inoculation and the incubation. Typically, according to this first variant, the insect larvae are inoculated at the same time, or within a time interval of a few minutes (typically 5 to 30 minutes) as the addition of the enzyme(s) (step c).

[0044] Preferably, according to a second variant, the inoculation is carried out by depositing the larvae, preferably at the young larvae stage, on the mixture obtained at the end of step c). Subsequently, the incubation of the insect larvae rearing process is carried out for 1 to 60 days at a temperature between 25°C and 45°C. According to such a variant, the hydrolysis reaction takes place before the inoculation and the incubation. Preferably, according to this variant, the hydrolysis of step c) takes place for 1 to 7 days, preferably at a temperature between 25°C and 90°C, preferably between 25°C and 70°C. Then the inoculation and the incubation take place.

[0045] The incubation of insect larvae is carried out under suitable temperature and humidity conditions. Optionally, the larvae are recovered after 1 to 60 days of incubation. An advantage of the substrate and its use is that it does not need to be changed or renewed. A complete breeding cycle can therefore be carried out on a single batch of substrate.

[0046] Generally, the larvae are deposited on the breeding substrate at the young larvae stage, then grow at a temperature between 25 and 45°C, at a humidity level between 30 and 90%, for a period between 1 and 60 days, depending on the insect in question. The insects can then be collected at the larval or pupal development stage. The larvae can be used to produce high-protein feed (particularly for aquaculture), oils; the digested substrate is called frass, and can be used as organic fertilizer; the pupae can be used to produce reproductive adults, in order to generate new generations of insects.

[0047] Finally, the invention also relates to the use of an aqueous mixture of by-products derived from hydrolyzed plants, said aqueous mixture comprising particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, as a substrate for the breeding of insect larvae. The aqueous mixture of by-products derived from hydrolyzed plants comprises particles having a size less than or equal to 5 mm: this size makes it possible to obtain a good quality substrate for the breeding of insect larvae. Preferably, this aqueous mixture is obtained by steps a) to c) above, and therefore includes enzymatic hydrolysis.

[0048] The substrates, methods and uses according to the invention are preferably implemented in installations which are geographically close to the plant processing plants. Indeed, this makes it possible to avoid the transport of large volumes of by-products. Preferably, these methods and uses are implemented using by-products from pipes and / or transport racks directly connected to the plant processing plants, and bringing the by-products to the installations in which they are transformed to give the substrates for breeding insect larvae. Preferably, the plant transformations (for example for the production of oil, starch, bioethanol or proteins) are carried out "continuously", that is to say that the by-products are not stored for more than 24 hours between their obtaining as by-products and their use in the methods according to the invention.This also helps reduce storage costs for these by-products.

[0049] The invention is now illustrated by the following non-limiting examples.

[0050] Example 1: examples of implementations of methods according to the invention and substrates obtained Materials & Methods

[0051] Experiments were carried out on the enzymatic hydrolysis of the PKE by-product: Substrate preparation protocol:

[0052] The PKE by-product is ground to obtain a particle size of less than 2 mm (step a). The product resulting from step a) is mixed with water previously mixed with the enzyme (concomitant steps b and c) in the following mass proportions: 1 / 3 PKE to 2 / 3 water. For the majority of the hydrolysis (step c), the temperature is 30°C. Inoculation:

[0053] Young larvae are inoculated into the substrate and are raised for 7 days (step d) under a temperature and humidity controlled atmosphere. Inoculation is carried out by depositing the larvae at the young larvae stage, on the substrate obtained during hydrolysis of step c) (i.e. no waiting between steps c) and d). The same quantity of larvae is used for each experiment.

[0054] After 7 days, the average weight of the larvae is measured. Enzymes:

[0055] The enzymes used are as follows:

[0056] [Tables 1] Provide your commercial name Type of enzyme Abbreviation Enzymatic activity (in units / g of enzyme, ie u / g) Enzymes L imited Cellulase 15,000L Cellulase Cellulase ES 15000 u / g Amano Enzymes Mannanase B GM “Ama no”10 Mannanase of Aspergillus niger Mannanase AE 10000 u / g Sigma Aid rich Hémicellu lase of Asper gillus niger Hemicellulase Hemicellulase 300 u / g Amano Enzymes Cellulase A ”Amano”3 Cellulase from A. niger Cellulase A AE 30000 u / g Amano Enzymes Cellulase T ”Amano”4 Cellulase from T. viride Cellulase T AE 280 u / g Amano Enzymes Pectinase PL pectinase from A. niger (cellulase and Endo-polygalac turonase) Pectinase 2000 u / g Biocatalyst Cellulase 13 L - C013L Cellulase from Trichoderma sp. Results

[0057] The results are as follows:

[0058] [Tables2] Tested Enzymes Grams of enzyme added per 100 grams of PKE dry matter Minimum enzyme units u / g of substrate dry matter Average initial larval weight (mg) Average final larval weight (mg) % difference in final weight compared to untreated PKE control (control) - 26 122.06 0% Cellulase A AE 3.13 939 26 144.59 15.6% Cellulase T AE 3.13 9 26 162.33 24.8% Pectinase 3.13 63 26 144.09 15.3% Cellulase 13 L 3.13 47 26 154.61 21.1% Carbohydrase 3.13 63 26 134.11 9%

[0059] [Tables3] Tested Enzymes Grams of enzyme added per 100 grams of PKE dry matter Minimum enzyme units u / g of substrate dry matter Mean initial larval weight (mg) Mean final larval weight (mg) % difference in final weight compared to the untreated PKE control (control) - 23 129.3 0% Hemicellulase 0.7 2 23 138.5 6.6% Mannanase AE 0.7 70 23 142.5 9.3% Cellulase T AE 0.7 2 23 153.2 15.6% Cellulase ES 0.7 105 23 148.7 13.%

[0060] The enzymes tested allow the hydrolysis of PKE substrate and the release of nutrients allowing the larvae to reach a higher average weight after 7 days than when they are fed on non-hydrolyzed PKE.

[0061] Conclusion: These experiments showed that enzymatic hydrolysis improves the efficiency of the substrate for larval growth. These enzymes allow efficient hydrolysis of the substrate, while maintaining a food texture suitable for the rearing of insect larvae.

Claims

Claims

1. A method for rearing insect larvae, comprising: - a method for preparing a substrate for rearing insect larvae from by-products derived from plants, comprising the following steps: a) obtaining by-products derived from plants in the form of particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, if necessary by homogenization, b) mixing the product obtained in a) with an aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase, to obtain a composition whose moisture content is between 55% and 85% by weight relative to the total weight of the mixture, and c) adding at least one enzyme to the mixture b), to hydrolyze the mixture b), then homogenization, in which the hydrolysis c) is carried out at a temperature between 25°C and 70°C,and is carried out for a period of between 5 minutes and 3 days, in which step c) is concomitant with or subsequent to step b), and - a step d) of inoculation of insect larvae into the mixture c) and incubation, in which the inoculation is carried out by depositing the larvae on the mixture obtained during the hydrolysis of step c), and subsequently, the incubation of the insect larvae rearing process is carried out for 1 to 60 days at a temperature of between 25°C and 45°C.,

2. A method according to claim 1, wherein the by-products from plants are co-products obtained during the processing of plants, preferably the plants are lignocellulosic; preferably the plants are chosen from oilseed plants, cereals, wood and plants from the sugar industry; preferably the by-products from plants are chosen from oilseed plant cakes, oil mill effluents, decanter cakes, cereal cakes, oilseed seeds, oilseed hulls and skins, cereal co-products such as corn cobs or wheat or rice straw, and sugar manufacturing products, such as sugarcane bagasse or beet pulp.

3. A method according to claim 2, wherein the cakes are chosen from palm kernel, almond, peanut, safflower, hemp, rapeseed, copra, cultivated flax, corn, rape, walnut, olive, poppy, sesame, soybean and sunflower cakes.

4. Method according to one of claims 1 to 3, in which: the mixture b) is carried out with an aqueous composition optionally comprising compounds derived from plants and soluble in the aqueous phase, to obtain a composition whose humidity level is between 65% and 80% by weight relative to the total weight of the mixture.

5. Method according to one of claims 1 to 4, in which the enzyme of step c) is chosen from mannanases, hemicellulases, cellulases, lignases, xylanases, pectinases, carbohydrases, proteases, amylases, lipases, keratinases, phytases and mixtures thereof, preferably the enzyme of step c) is chosen from mannanases, cellulases and mixtures thereof.

6. Substrate for rearing insect larvae obtained by the method according to one of claims 1 to 5.

7. Method for rearing insect larvae according to one of claims 1 to 5, wherein: the inoculation is carried out by depositing the larvae at the young larvae stage on the mixture obtained during the hydrolysis of step c), and subsequently, the incubation of the method for rearing insect larvae is carried out for 1 to 60 days at a temperature between 25°C and 45°C.

8. Use of an aqueous mixture of by-products from hydrolyzed plants, said aqueous mixture comprising particles having a D50 less than or equal to 5 mm, preferably less than or equal to 2 mm, even more preferably less than or equal to 1.5 mm, as a substrate for the breeding of Hermetia Illucens larvae.

9. Use of the substrate according to claim 6, for the breeding of Hermetia Illucens larvae.