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    • 专利摘要:
    The present invention relates to a process for preparing chitin and / or chitosan from insects. More particularly, the present invention relates to a process for producing chitin and / or chitosan from insect cuticles, comprising a step of pressing cuticles of insects, and then, a step of enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, a cuticle treatment with an oxidizing agent having been carried out prior to the enzymatic hydrolysis.
    公开号:FR3031115A1
    申请号:FR1463513
    申请日:2014-12-31
    公开日:2016-07-01
    发明作者:Nathalie Berezina；Antoine Hubert；Fabrice Berro；Jean-Gabriel Levon；Roux Karine Le
    申请人:Ynsect SAS；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a process for preparing chitin and / or chitosan from insects. More particularly, the invention relates to a process for preparing chitin and / or chitosan by enzymatic hydrolysis of insect cuticles. According to the invention, "chitin" is understood to mean any type of chitinic derivative, that is to say of a polysaccharide derivative comprising N-acetylglucosamine units and D-glucosamine units, in particular the chitin-type copolymers. polypeptides (sometimes referred to as "chitin-polypeptide composite"). Chitin is the second most synthesized polymer in the world after cellulose. Indeed, chitin is synthesized by many species of the living world: it is partly the exoskeleton of crustaceans and insects and the side wall that surrounds and protects mushrooms. More particularly, in insects, chitin thus constitutes 3 to 60% of their exoskeleton. By "chitosan" is meant according to the present invention the deacetylation products of chitin. The usual limit between chitosan and chitin is determined by the degree of acetylation: a compound with a degree of acetylation of less than 50% is called chitosan, beyond that, a compound with a degree of acetylation greater than 50% is named chitin. The applications of chitin and / or chitosan are numerous: cosmetic (cosmetic composition), medical and pharmaceutical (pharmaceutical composition, treatment of burns, biomaterials, corneal dressings, surgical threads), dietary and food, technical (filtering agent, texturizer, flocculant or adsorbent especially for the filtration and depollution of water), etc. Indeed, chitin and / or chitosan are biocompatible, biodegradable and non-toxic materials. The traditional extraction of chitin is carried out chemically from crustaceans, cephalopods, but also, more exceptionally, from fungi. This route employs large amounts of reagents (such as hydrochloric acid, sodium hydroxide and bleaches) which have the effect of denaturing the naturally occurring structure of chitin by example as present in the shell of crustaceans. In addition, most chemical reagents are harmful to humans and the environment and generate large volumes of effluents to be treated. Finally, chitin and / or chitosan derived from crustaceans can generate allergic reactions in sensitive people. Another way of extracting chitin is the enzymatic pathway. This route is considered softer, thus better preserving chitin and / or chitosan. However, the chitin obtained by this route is of a brownish color, requiring purification steps in order to obtain a recoverable powder, that is to say of white color. The existing processes therefore generally include one or more steps (s) to rid the chitin of its impurities, such as an acid demineralization step carried out prior to the enzymatic hydrolysis and / or a bleaching step of the chitin with an oxidizing agent, carried out subsequent to the enzymatic hydrolysis. These two steps of purification of chitin unfortunately have the effect of altering the chemical structure of chitin. The work of the inventors has made it possible to demonstrate that it is possible to obtain a chitin both purer and of structure closer to the original structure of chitin by performing a specific combination of steps prior to the hydrolysis, namely a pressing step and a step of treatment of insect cuticles with an oxidizing agent. The invention thus relates to a process for producing chitin and / or chitosan from insect cuticles, comprising the following steps: (i) pressing cuticles of insects, then, (ii) enzymatic hydrolysis cuticles of insects by a proteolytic enzyme, treatment of cuticles with an oxidizing agent having been carried out prior to enzymatic hydrolysis.
    [0002] By "insects" is meant insects at any stage of development, such as an adult, larval or pupal stage. Preferably, the insects used in the process according to the invention are edible. More particularly, the insects may be selected from the group consisting of Coleoptera, Diptera, Lepidoptera, Isoptera, Orthoptera, Hymenoptera, Blattoptera, Hemiptera, Heteroptera, Ephemeroptera and Mecoptera, preferably among Coleoptera, Diptera, Orthoptera and Lepidoptera. Preferably, the insects are selected from the group consisting of Tenebrio molitor, Hermetia illucens, Galleria mellonella, Alphitobius diaperinus, Zophobas morio, Blattera fusca, Musca domestica, Chrysomya megacephala, Locusta migratoria, Schistocerca gregaria, Acheta domestica and Samia ricini, and more preferably still, T molitor. One or more species of insects may be used in the process according to the invention, preferably a single species of insect. If more than one species is used, two closely related species, for example Hermetia illucens and Musca domestica, will be favorably selected. The insects are preferably high and not taken in the wild. For example, insects are raised in an insect farm. Insect farming in a specific farm not only controls and eliminates the risks associated with insect-borne diseases, but also limits the risks associated with the toxicity of insect-derived food products due to, for example, presence of insecticides. In addition, livestock can control the quality of the supply of insects and limit supply costs.
    [0003] By "insect cuticles" is meant not only the cuticles once separated from the insects, but also the cuticles including all or part of the other constituents of the insect, including the insect in its entirety. Indeed, it is possible to apply the method according to the invention to the complete insect, such as ground insects, or to only a part of the insects comprising the cuticles, for example exuviae and / or moults of the skin. insects, naturally separated and collected by a suitable process. The cuticle is the outer layer (or exoskeleton) secreted by the epidermis of insects. It is generally formed of three layers: the epicuticle, which is the thinnest and outermost layer of the cuticle (less than 4 μm); this layer is impermeable to water and has a layer of waterproofing wax, as well as proteins and chitin, in a smaller quantity; - the exocuticle, which is the middle layer of the cuticle; it consists essentially of hardened proteins, tanned, which are responsible for the rigidity of the cuticle, chitin and possibly melanin; and - the endocuticle, which is a thin, flexible layer made of a mixture of proteins and chitin. The main purpose of pressing insect cuticles is to remove a fat-rich press juice and / or to enrich the substrate press cake for hydrolysis.
    [0004] In the process according to the invention, the pressing of insect cuticles makes it possible to obtain a press cake comprising an oil content of less than or equal to 20%, preferably less than or equal to 15%, more preferably lower. or equal to 12%, even more preferably, less than or equal to 10%.
    [0005] In the present application, the ranges of values are inclusive. Similarly, when "about" or "of the order of" precedes a number, this equates to plus or minus 10% of the value of that number. Furthermore, in order to enrich the press cake with a substrate for hydrolysis, the pressing of insect cuticles makes it possible to obtain a press cake having a solids content of between 30% and 60%, preferably between 40% and 55%, and more preferably between 45% and 50%. Any press system can be used to carry out the pressing of insect cuticles, such as, for example, a single-screw or twin-screw press (Angel twin-screw press), a filter-press (filter-press of type Choquenet), a plate press, etc.
    [0006] These systems are well known to those skilled in the art who are able to determine the pressing conditions in order to obtain the oil and / or water contents mentioned above. In the process according to the invention, the pressing of insect cuticles is followed by enzymatic hydrolysis.
    [0007] Preferably, the enzymatic hydrolysis is carried out by at least one proteolytic enzyme, preferably a protease. In the present application, the names or suffixes "peptidase" and "protease" are used interchangeably to designate an enzyme lysing a peptide bond of proteins. Advantageously, this is carried out for a period of 4 to 8 hours, preferably for 4 to 5 hours, at a temperature of 40 to 60.degree. C., preferably 45 to 55.degree. C. and at a pH of between 6 and 8. preferably between 6.5 and 7.5. The enzymatic hydrolysis can be carried out with a single protease or alternatively with a mixture of enzymes containing at least one protease, more preferably a mixture of enzymes containing several proteases, such as a mixture containing an endoprotease and an exoprotease, or a protease and a polysaccharase. Preferably, the protease is selected from the group consisting of aminopepidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.
    [0008] Advantageously, the enzymes can be chosen from the following: 3031115 5 Enzyme (s) Class Number Provider City Country EC Flavourzyme Amino-EC Novozyme Bagsvaerd Denmark peptidases 3.4.11.1 Fungal protease EC Bio-Cat Troy United States 500 3.4.11.1 Kojizyme EC Novozyme Bagsvaerd Denmark 3.4.11.1 Protex P Endo-EC 3.4.21 Genencor Leiden Netherlands peptidases International serine BV Chymotrypsin EC Novozyme Bagsvaerd Denmark 3.4.21.1 Protamex EC 3.4.21 Novozyme Bagsvaerd Denmark Elastase EC Novozyme Bagsvaerd Denmark 3.4.21.14 Trypsin EC Novozyme Bagsvaerd Denmark 3.4.21.36 Alcalase EC Novozyme Bagsvaerd Denmark 3.4.21.4 Papain Endo-EC Bio-Cat Troy United States peptidases 3.4.22.2 cysteine Bromelain EC Bio-Cat Troy United States (ananase) 3.4.22.32 Prolyve NP Endo-EC 3.4.23 Lyven Colombelles France aspartic peptidases Pepsin EC Sigma Aldrich Saint-France 3.4.23.1 Quentin-Fallavier Neutral protease Metallo-EC Bio-Cat Troy USA endo- 3.4.24.28 peptida Protex 50FP Endo-EC 3.4.21 Genencor Leiden The Netherlands peptidase International BV Pancreative Exo & endo na Lyven Colombelles France peptidase (cocktail proteases + 3031115 6 Enzyme (s) Class Number Provider City Country EC amylases) Izyme BA Protease EC 3.4 .23 Novozyme Bagsvaerd Denmark aspartic Sumizyme Cocktail na * Takabio - Shin Aichi Japan enzymatic Nihon * na The enzyme or the mixture of enzymes is introduced in an amount ranging from 0.2 to 10% by weight of estimated dry matter, preferably from 0.4 to 8% by weight and more preferably from 0 to 10% by weight. , 5 to 2%. By "estimated dry matter weight", it is more particularly the weight of dry matter of insects or part (s) of insects, as can be estimated when entering the hydrolysis step enzyme. Advantageously, the enzymatic hydrolysis step is carried out in the presence of water, such as fresh water. The amount of water used during the enzymatic hydrolysis is determined as follows: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, still more preferably between 0.7 and 3, even more preferably of the order of 1. It will be noted that this ratio also corresponds to the ratio of the weight of water to the weight of the insect, the density of the water being 1.0 g / mL under normal conditions of temperature and pressure.
    [0009] In the process according to the invention, the enzymatic hydrolysis is preceded by a cuticle treatment step with an oxidizing agent. Preferably, in the process according to the invention, the oxidizing agent used during the treatment of cuticles is chosen from the group consisting of hydrogen peroxide, potassium permanganate, ozone and sodium hypochlorite. More preferably still, hydrogen peroxide. Advantageously, when the oxidizing agent is hydrogen peroxide, the amount of this agent introduced for the treatment of insect cuticles is such that the content of hydrogen peroxide is between 1 and 33% by weight on the weight total of insects, preferably between 2 and 12% by weight on the total weight of insects, preferably of the order of 6% by weight. Preferentially, the treatment of insect cuticles with the oxidizing agent is carried out in the presence of water, such as fresh water. Advantageously, the quantity of water used during the treatment of the cuticles is determined in the following way: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1. The process according to the invention makes it possible to obtain a chitin having a high degree of of purity, such as a purity of between 55 and 95%, preferably between 60 and 95%, more preferably between 70 and 90%, and even more preferably between 80 and 90% (see Example 5 and Figure 7). Preferably, the method according to the invention comprises a grinding step prior to the pressing step.
    [0010] This grinding step is intended to reduce cuticles and / or particulate insects to facilitate enzyme access to the substrate during enzymatic hydrolysis. This step also makes it possible, when followed by a pressing step, to facilitate the elimination of the press juice and the isolation of the solid material.
    [0011] The grinding may advantageously be carried out with a blender, such as a kneader mill. Preferably, at the end of grinding, the size of the insect particles is less than 1 cm (larger size of particle observable using a microscope), preferably less than 0.5 cm, more preferably again, a size of between 500 μm and 0.5 cm. To facilitate grinding, a quantity of water can be added. This quantity of water is determined as follows: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, still more preferably between 0.7 and 3, still more preferably of the order of 1. Advantageously, in the process according to the invention, the treatment of cuticles with an oxidizing agent is carried out before, concomitantly and / or after the step grinding and / or pressing. The process according to the invention may also comprise a step of slaughtering the insects prior to the pressing and / or milling step. This stage of slaughter can be carried out by conventional methods of rearing cold-blooded and / or small-sized animals (crustaceans, fish, snails, etc.), such as cold (freezing), hot ( scalding), oxygen deprivation, etc. Advantageously, the step of killing insects is done by scalding.
    [0012] Scalding not only reduces insects, but also lowers the microbial load (reduces the risk of spoilage and sanitation) and inactivates the internal enzymes of the insects that can trigger autolysis, and so rapid browning of these. This boiling is carried out so as to cause death as quickly as possible, in the respect of animal welfare, and according to scientific recommendations. Advantageously, the insects are slaughtered, for example by scalding, then crushed before being pressed. Preferably, the boiling step is carried out in water, such as fresh water, at a temperature of 95 to 105 ° C., preferably of the order of 100 ° C. and for a period of 2 hours. at 20 min, preferably 5 to 15 min. The quantity of water introduced at this boiling step is determined as follows: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, still more preferably between 0.7 and 3, still more preferably of the order of 1.
    [0013] Advantageously, the treatment of cuticles with an oxidizing agent is carried out concomitantly and / or after the slaughtering step and in particular concomitantly and / or after scalding. More particularly, the treatment of insect cuticles with the oxidizing agent can be carried out during one or more of the following steps: - concomitantly with scalding and / or after the boiling step, more preferably concomitantly with scalding. More particularly, when insect cuticle treatment is performed during scalding, the oxidizing agent may be advantageously added to the water used to scald insects. 25 - before, concomitantly and / or after grinding. More particularly, when insect cuticle treatment is performed during grinding, the oxidizing agent may be advantageously added to the water used for grinding. - before and / or concomitantly with pressing. - during a specific step of treatment of cuticles of insects.
    [0014] After the enzymatic hydrolysis, the process according to the invention may further comprise a thermal inactivation step aimed at inactivating the enzyme or the enzyme mixture used during the enzymatic hydrolysis. At the end of a process according to the invention, the chitin can be recovered by pressing or centrifugation of the reaction medium of the enzymatic hydrolysis.
    [0015] At this stage, a co-product of interest of chitin is also recovered, i.e. a hydrolyzate. By "hydrolyzate" is meant a product which comprises proteins, hydrolysed proteins, peptides, amino acids and / or other compounds derived from a protein, obtainable by enzymatic hydrolysis of proteins. The invention also relates to a hydrolyzate obtainable as a by-product of enzymatic hydrolysis by any of the processes according to the invention. This hydrolyzate may advantageously be supplemented with additives to balance its nutritional profile in order to be adapted to different types of animals. The hydrolyzate can be concentrated and then dried to obtain a dried hydrolyzate. Alternatively, the hydrolyzate may be in liquid form. These hydrolysates may be used as a food or a food ingredient particularly for animals, or, alternatively, they may be processed, for example, to isolate amino acids. A preferred embodiment of a method according to the invention is more detailed below. In particular, this preferred embodiment describes various advantageous steps for a process according to the invention, such as steps of gentle purification of chitin: a second pressing, washing, filtration and drying possible. Finally, since chitin is generally marketed in the form of a powder, a second grinding can also be carried out. This can also be done to promote the deacetylation reaction, which makes it possible to prepare chitosan from chitin. The conditions of the deacetylation reaction are more fully described in step 10 of the preferred embodiment detailed hereinafter. A particularly advantageous method of producing chitin from insects comprises the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis of cuticles of insects by a proteolytic enzyme, e) the recovery of chitin, the insect cuticles being treated with an oxidizing agent, before step d).
    [0016] The preferred embodiments of the various steps a) to e) and the treatment of insect cuticles with an oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment of the present invention. after. The invention also relates to a chitin obtainable by a process according to the invention. Due to the mild conditions employed in the process according to the invention, this chitin has a structure similar to chitin as naturally occurring in the cuticle of the insect while having a high degree of purity, such as a degree of purity of between 55 and 95%, preferably between 60 and 95%, more preferably between 70 and 90%, and even more preferably between 80 and 90%.
    [0017] A particularly advantageous method of producing chitosan from insects comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis of insects, cuticles of insects by a proteolytic enzyme, e) recovery of chitin, f) deacetylation of recovered chitin, g) recovery of chitosan, insect cuticles being treated with an oxidizing agent, prior to step d). Preferred embodiments of the various steps a) to g) and treatment of insect cuticles with an oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment hereinafter. The invention also relates to a chitosan obtainable by a method according to the invention. The chitin and / or chitosan likely to be obtained by a process according to the invention can be advantageously used in various applications: in cosmetic, pharmaceutical, nutraceutical or cosmetic compositions; dietary compounds, as biomaterials for treating burns as second skin, for producing corneal dressings or surgical threads, as a filtering, texturing, flocculating and / or adsorbent agent, in particular for filtering and depolluting water. According to a preferred embodiment of the invention, the method comprises the following steps, schematically described in FIG. 1. It will be noted that certain steps are indicated as optional in this preferred embodiment. Step 1: scalding insects This scalding step 1 makes it possible to kill the insects while lowering the microbial load (reducing the risk of spoilage and sanitation) and by inactivating the internal enzymes of the insects that can trigger an autolysis, and thus a rapid browning of these. Insects, preferably larvae, are thus scalded with water for 2 to 20 min, preferably 5 to 15 min. Preferably, the water is at a temperature of between 95.degree. And 105.degree. C., preferably 100.degree. The quantity of water introduced at this boiling step 1 is determined as follows: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, still more preferably between 0.7 and 3, even more preferably of the order of 1. In this step, it is also possible to treat insect cuticles using a water of scalding comprising the oxidizing agent according to the modalities indicated in the intermediate step below. Intermediate step (optional): treatment of the cuticles with the oxidizing agent It is possible to introduce into the process a specific step of treatment of the cuticles with the oxidizing agent. Advantageously, this intermediate cuticle treatment step is carried out between the boiling step 1 and the grinding stage 2. This intermediate step is preferably carried out with an oxidizing agent chosen from the group consisting of hydrogen peroxide (H2O2), potassium permanganate (KMnO4), ozone (O3) and sodium hypochlorite (NaClO). more preferably hydrogen peroxide. According to a first embodiment, at the end of the boiling step 1, the oxidizing agent is introduced directly into the boiling tank, after cooling of the boiling water to a temperature of 10 ° C. 40 to 60 ° C, preferably of the order of 50 ° C.
    [0018] The hydrogen peroxide as commercially available is usually in the form of an aqueous solution, for example a 30% by weight solution based on the total weight of water. The amount of hydrogen peroxide introduced for the treatment is such that the content of hydrogen peroxide is from 1 to 33% by weight relative to the total weight of insects, preferably from 2 to 12% by weight on the weight. total of insects, preferably of the order of 6% by weight. In a second embodiment, the insects are removed from the boiling tank, sieved and reintroduced into a tank. The hydrogen peroxide is then introduced into the tank in the form of a dilute aqueous solution, the content of hydrogen peroxide then being between 1 and 33% by weight relative to the weight of water, preferably 2 to 12% by weight on the weight of water, preferably of the order of 6% by weight. Step 2: Grinding Insects are removed from the scalding or treatment tank, then sieved, and placed in a grinder, such as a knife mill, to reduce particulate insects. To facilitate grinding, a quantity of water can be added. This amount of water is similar to that introduced during the boiling step 1: the ratio of the volume of water in ml to the weight in g of insect is preferably between 0.3 and 10, plus 15 preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1. It is also possible to keep the scalding water and / or the water of the stage intermediate to perform this step. This water is likely to contain the oxidant. In this case, treatment of the cuticles can take place during scalding step 1 and milling step 2 or during the intermediate cuticle treatment step and during the grinding step. Preferably, at the end of grinding, the size of the insect particles is less than 1 cm (larger particle size observable by means of a microscope), preferably less than 0.5 cm. It is not necessary to excessively reduce the particle size, for example to a size less than 250 μm.
    [0019] This milling step 2 promotes the access of the enzymes to their substrate. During this step, it is possible to introduce into the mill the oxidizing agent in order to treat the cuticles according to the conditions indicated in the intermediate step above. When the cuticle treatment is not carried out concomitantly with grinding, it is possible to add in this step, antioxidant additives commonly used for the preservation and stability of the product. Step 3: Pressing Insect Cuticles The wet paste resulting from step 2 of grinding is then placed in a press according to a procedure which makes it possible to press and separate a juice comprising both an oily fraction and a fraction. protein.
    [0020] Preferably, the pressing step makes it possible to obtain a press cake comprising an oil content of less than or equal to 20%, preferably less than or equal to 15%, more preferably less than or equal to 12%, and still more preferably more preferably, less than or equal to 10%.
    [0021] Similarly, the pressing step makes it possible to obtain a press cake having a solids content of between 30% and 60%, preferably between 40% and 55%, and more preferably between 45% and 50%. %. Any press system can be used to carry out the pressing step, such as, for example, a single-screw or twin-screw press (twin-screw press of the Angel type), a filter press (press-type press). Choquenet), a plate press, etc. These systems are well known to those skilled in the art who is able to determine the pressing conditions in order to obtain the oil and / or water contents mentioned above.
    [0022] If the wet paste resulting from grinding step 2 has been obtained with water containing the oxidant, it may be advantageous to remove at least a portion of this oxidant before the pressing step 3. This pressing step 3 may possibly be carried out before step 2 grinding from scalded insects. However, it is advantageous to carry out the pressing step 3 after the grinding step 2. This pressing step 3 thus makes it possible to obtain a press juice and a press cake. Step 4: Enzymatic hydrolysis The wet paste resulting from the milling step 2 or the press cake resulting from the pressing step 3 is placed in a hydrolysis tank with water. Optionally, and as will be described below, the protein fraction from the separation step 12, can be reintroduced in this step 4 of enzymatic hydrolysis, by mixing it with the press cake. Optionally, and in the case where the scalding water contains no oxidant, the scalding water may be recovered and reintroduced in the hydrolysis step. Indeed, this water contains insect fractions solubilized by the action of this scalding and the use thereof during the hydrolysis reduces losses. Optionally, this water from boiling can be degreased, some waxes may have dissolved in water.
    [0023] The amount of water introduced in this enzymatic hydrolysis step 4 is similar to that introduced during scalding step 1: the ratio of water volume in ml to weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, still more preferably between 0.7 and 3, even more preferably of the order of 1.
    [0024] The enzymatic hydrolysis is carried out with a protease, such as a commercial protease for 4 to 8 hours, more particularly for 4 to 5 hours, at a pH of 6 to 8, more preferably from 6.5 to 7.5, at a pH of 40 to 60 ° C, more preferably 45 to 55 ° C. The amount of enzymes introduced during the hydrolysis step is less than 10% by weight relative to the total weight of dry matter estimated entering hydrolysis, preferably less than 6%, more preferably of the order of 2%. Proteolytic hydrolysis results in the production of a soluble phase containing the peptides, glucosamines and oligochitins and a solid residue formed of chitin, mainly chitin-polypeptide copolymer. Step 5: Thermal Inactivation In order to stop the activity of the enzymes of the reaction and to stabilize the soluble phase of the hydrolysis, a thermal inactivation is carried out by heating this juice between 80 and 105 ° for 10 to 25 minutes, preferentially 15 to 20 minutes. According to one procedure, this heat inactivation step 5 is carried out according to the usual sterilization techniques of the food industry. According to another operating mode, the enzymatic inactivation is carried out by heating under IR or UV radiation, or microwaves. Step 6: Pressing The solid residue, predominantly composed of chitin, is recovered and then pressed by a press in order to wring this residue to the maximum to reinject in the soluble phase this press. The pressed residue thus formed is composed essentially of chitin, mainly in the form of chitin-polypeptide copolymer. Steps (optional) 7 and 8: washing and drying The solid residue is then washed, filtered, washed again and then dried by conventional technologies known to those skilled in the art. Advantageously, the drying system is designed to protect the structure of the chitin-polypeptide copolymer: the hydrometry, the ventilation and the composition of the air are controlled. Advantageously, the drying can be carried out in a ventilated oven at a temperature of 60 to 80.degree. C., preferably of the order of 70.degree.
    [0025] Optionally, these steps may include a terminal delipidation step: the solid residue is treated with HCl to remove the last lipid residues, including cuticular waxes. The following steps 9 to 11 aim at converting chitin into chitosan and are therefore only used when the desired product is chitosan. Step 9 (Optional): Bravado The dried solid residue, predominantly chitin, is then milled, for example in a knife mill. The production of chitosan from chitin, by the deacetylation reaction, depends largely on the size of the chitin particles. Thus a very fine grinding of the dried solid residue before deacetylation makes it possible to significantly increase the yields and the speed of the deacetylation reaction, as illustrated in Table 1 below: Grinding 30 s Grinding 45 s Grinding 60 s Grinding 120 s 50% of <174 pm <117 pm <95 pm <67 pm particles 90% of <310 pm <244 pm <157 pm <159 pm particles DA * 99% 90% 85% 80% Table 1: Effectiveness of the deacetylation according to the pre-grinding of chitin 15 * Measurement of the degree of acetylation DA The conditions of the deacetylation carried out in the test reported in Table 1 are as follows: 4 h reaction, 100 ° C., NaOH in aqueous solution at 30 ° C. % by volume, in an estimated chitin ratio: NaOH solution equal to 1:20. Therefore, the solid residue is preferentially ground to a particle size of less than 200 μm, more preferably less than 160 μm. Step 10: Deactivation The solid residue, optionally ground in step 9, is then placed in a reactor where a solution of concentrated sodium hydroxide is added for 4 to 24 hours, and preferably 6 to 18 hours. Sodium hydroxide in aqueous solution at a content ranging from 30% to 40% is added in a weight ratio in g of milled chitin / volume in mL of sodium hydroxide in aqueous solution of between 1: 50 to 1: 10, preferably of the order of 1:20. The tank is then heated, the deacetylation temperature being between 80 and 150 ° C, preferably between 90 and 120 ° C and more preferably at 100 ° C.
    [0026] Thus, powdered chitosan is obtained. Chitosan can then undergo any operation known to those skilled in the art to functionalize it, in particular by the addition of radicals (carboxylation, hydroxylation ...) 5 - Step 11 (optional): drying The chitosan powder is then dried between 30 and 80.degree. C., preferably between 50 and 70.degree. C. and preferably at about 60.degree. C., in order to obtain a powder having a solids content of greater than 85%, more particularly greater than 90%. The following steps are aimed at recovering an oily fraction and a protein fraction from the juice obtained in the pressing step 3 and are therefore used only when such recovery is desired. Step 12: Separation The press juice undergoes one or more separation steps, in order to separate the oily fraction (insect oils) from the protein fraction (haemolymph proteins from insects). These steps can be performed by any oil separation technology well known to those skilled in the art, such as centrifugation, decantation, reverse osmosis separation, ultrafiltration, supercritical CO2, etc. or a combination of several of these technologies. The separation of the oily fraction can be complex, given the presence of oils of very different compositions in insects, the fatty acids possibly having short chains (C2-05) as well as very long chains (for example, for waxes:> C25). The rise in temperature above the melting point of these oils (about 38 ° C.) during the centrifugation makes it possible to solubilize this cream and to facilitate the separation of the oily fraction from the rest of the juice.
    [0027] The centrifugate is then decanted according to a procedure (decanter or tricantor type), in order to better separate the oils and proteins. These steps thus make it possible to obtain an oily fraction. The protein fraction, once separated from the oily fraction, can be mixed with the press cake from the pressing step 3 just before the hydrolysis step 4. Indeed, often more than 20% of the proteins are lost in the juice during the pressing step 3, hence the interest of recovering this fraction and subjecting it to the hydrolysis step. Step 13 (Optional): Concentration According to one procedure, the concentration is carried out by evaporation in vacuo of the aqueous portion. The concentrate has a solids content greater than 10%, preferably greater than 20%. This operation facilitates drying and commonly used additives for product shelf life and stability can be added at this stage. Step 14 (Optional): Drying The concentrate is finally dried by technologies known to those skilled in the art, such as, for example, by spray-drying, which makes it possible to obtain an extract. that is to say a dry concentrate powder rich in peptides and glucosamines, the glucosamines being in particular derived from the partial hydrolysis of chitin by H 2 O 2 (essentially).
    [0028] Other features and advantages of the invention will appear in the examples which follow, given by way of illustration, with reference to the figures, which represent respectively: FIG. 1 is a diagram of a preferred embodiment of the method according to FIG. FIG. 2 is a diagram comparing the degree of purity of chitin obtained by an enzymatic process with and without hydrogen peroxide; FIG. 3 is a diagram illustrating the influence of the extraction process (FIG. enzymatic or chemical) and treatment with an oxidizing agent on the chitin obtained, - Figure 4 is a diagram comparing the degree of purity of chitin obtained by an enzymatic process comprising one or more preliminary steps of grinding (s) and pressing Fig. 5 is a diagram comparing the lipid level measured in different fractions of the intermediate product from which chitin has been extracted. distribution of lipids in the juice and the press cake obtained by an enzymatic process comprising preliminary grinding and pressing steps or a prior pressing step; FIG. 7 is a diagram comparing the degree of purity of the chitin obtained by a enzymatic process comprising one or more preliminary stages of grinding (s), pressing and treatment with an oxidizing agent. In the following examples, any reference to a regulation or a directive concerns the regulation or the said directive as in force on the filing date of the present application.
    [0029] EXAMPLE 1 Example of Process According to the Invention 50 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100.degree. C. and containing 50 ml of a solution of hydrogen peroxide at 50.degree. 6% 5 previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 100 mL. The liquid thus obtained is passed through a bi-screw type press, which results in 17.89 ± 0.87 g of press cake with a solids content of 20.9 ± 1.3% and in 389, 49 ± 6.53 g of press juice with a dry matter content of 4.04 ± 0.05%. 10 g (wet weight) of press cake thus obtained are transferred into an Erlen Meyer containing 50 ml of water and 0.1 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with stirring. magnetic (at a pH of about 6.5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. 0.64 ± 0.05 g of chitin are thus obtained by this method. EXAMPLE 2 Influence of the Presence of Cuticle Treatment with the Oxidant on the Degree of Purity of the Chitin Obtained 25 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 50 mL of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 25 ml. In the case of the reaction with hydrogen peroxide, the liquid thus obtained is placed for 1 hour in the presence of a solution of hydrogen peroxide, then transferred into a 250 ml Erlen Meyer containing a protease solution ( Sumizyme LP) at 4%, otherwise, it is transferred directly into the Erlen Meyer containing the protease solution. The whole is placed for 4 hours at 45 ° C. with magnetic stirring (pH approximately 6.5). Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. The dry residue thus obtained after use of the hydrogen peroxide is 6.3 ± 0.7% relative to the initial dry matter, while the dry residue resulting from a process without the hydrogen peroxide is 9, 75 ± 0.9% based on initial dry matter.
    [0030] The degree of purity of chitin is determined in comparison with the mass of dry residue obtained relative to that resulting from a chemical extraction, 5% of the initial dry matter. It thus stands at 79.9 ± 9% for the product obtained after treatment with peroxide and at 51.5 ± 4.9% in the absence of peroxide (see Figure 2).
    [0031] EXAMPLE 3 Influence of the Sequence of the Execution Sequence of Cuticle Treatment with Oxidant and Enzymatic Hydrolysis Obtaining Enzymatic Chitin (Without the Addition of Oxidant) 50 g T-Molitor Larvae introduced into a beaker, placed in a water bath at 100 ° C and containing 50 ml of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 100 mL. The liquid thus obtained is transferred to a 500 mL Erlen Meyer containing 150 mL of a 1% protease solution (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (pH approx. , 5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. Thus 1.656 ± 0.021 g of chitin are obtained by this method.
    [0032] Obtaining Chitin Using Enzyme with H202 - Scalding 50 g of T molitor larvae are placed in a beaker, placed in a water bath at 100 ° C. and containing 50 ml of water. a solution of 6% H 2 O 2 in water, previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then mixed with a volume of water of 100 ml. The liquid thus obtained is transferred into a 500 ml Erlen Meyer containing 150 ml of a 1% protease solution (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring. The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. Thus 1.98 ± 0.22 g of chitin are obtained by this method. Obtaining Chitin Using a Molecular Pathway with 50% H 2 O 2 50 g T Molitor larvae are introduced into a beaker, placed in a 100 ° C bath and containing 50 ml of water. water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered, and then ground with a volume of water of 100 ml. The liquid thus obtained is transferred into a 500 ml Erlen Meyer containing 150 ml of a 1% protease solution (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (pH approx. , 5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The residue is then placed for 1 hour at 65 ° C in a solution of 6% H 2 O 2. The chitin thus obtained is filtered (0.45-0.5 μm) and then dried for 24 hours at 70 ° C. Thus 1.304 ± 0.091 g of chitin are obtained by this method. Obtaining chitin by chemical means 50 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C and containing 50 ml of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 60 ml. The liquid thus obtained is transferred with 50 ml of water into a 1 L flask. 500 ml of 1M HCl are added thereto and the whole is stirred for 1 hour at 90 ° C. The reaction medium is then filtered and washed with water until a clear residue is obtained. This residue is then transferred to a 1 L flask supplemented with 500 mL of 1M NaOH and stirred at 90 ° C for 24 hours. The reaction medium is then filtered and washed until a clear filtrate is obtained, the residue is finally dried for 24 hours at 70 ° C. Thus 0.944 ± 0.005 g of chitin is obtained by this method.
    [0033] Determination (by viscometry) of the molecular weight of the obtained chitin A flask containing 1 g of chitin and 10 ml of 1 M NaOH is placed for 4 hours at 90 ° C. The mixture is then filtered (0.45-0.5 μm) and the thus washed residue is placed at 70 ° C. for 24 hours. Preparation of the solvent: 5 g of LiCl are placed in 100 ml of N, N-dimethylacetamide, with stirring and for 4-5 hours (until complete dissolution). The stock solution is obtained by dissolving 0.2 mg of chitin in 1 mL of solvent. From this stock solution, daughter solutions with concentrations of 0.1 mg / mL, 0.08 mg / mL and 0.04 mg / mL are prepared. The viscosity of these different solutions is then measured in triplicate with an Ostwald viscometer and the molecular weight is calculated according to the formula: [111 = KM''Q (1) with intrinsic viscosimetry in cm 3 / g, M,: molar mass of chitin in g / mol (or Da), and the Mark-Houvink coefficients a = 0.71 and K = 0.000893, the intrinsic viscosity being obtained according to: [rerir / C (2) with 10: reduced viscosity (without units), C: concentration in mg / ml, the reduced viscosity being obtained according to: qr = t / to (3) with 15 t: the measured fall time for the solution in s, to: the falling time measured for the solvent in s. It can be seen in Figure 3 that the size of the molecule of chitin obtained is a function of the extraction method used. Thus, the chemical method damages the integrity of the molecule (M, obtained is less than 70000 g / mol), but the most drastic treatment is that which consists in bleaching chitin with hydrogen peroxide after hydrolysis, even enzymatic (M, less than 9000 g / mol). The method according to the invention (enzymatic hydrolysis with the addition of hydrogen peroxide during or just after scalding, that is to say at the beginning of the process), certainly, decreases the size of the molecule with respect to that which can be found initially in the insect (M, chitin by simple enzymatic hydrolysis is 130000 g / mol), but to a much lesser extent (M, of nearly 80000 g / mol) and the This result is superior to that of traditional chemical extraction. EXAMPLE 4 Influence of the pre-enzymatic hydrolysis pressing step on the degree of purity of the chitin obtained Various types of mechanical pretreatment were tested, grinding ("grinding 1") alone, grinding followed by pressing milling followed by pressing and second milling ("milling 2"), as well as pressing alone.
    [0034] For pressing, an Angel press has been used under the following conditions: Speed = 82 rpm; - VV (energy) = 3 HP (horsepower) or 2.68 x 106 J; 5 - Porosity (approximate) = 0.5 mm in the first part and 0.2 mm in the last part. 1. Material and methods 10 Production of chitin with grinding 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C and containing 200 ml of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are drained and then mixed with a volume of water of 200 mL. The liquid thus obtained is transferred into an Erlen Meyer containing 2 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of about 6.5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. Thus 8.13 ± 0.27 g of chitin is obtained by this method. Production of chitin with milling followed by pressing 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water boiled beforehand.
    [0035] After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered, and then mixed with a volume of water of 200 mL. The liquid thus obtained is passed through a bi-screw type press. 30 g of the press cake thus obtained are transferred into an Erlen Meyer containing 150 ml of water and 0.3 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of about 6.5).
    [0036] The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under hot conditions. The chitin thus obtained is dried for 24 hours at 70 ° C. This gives 4.71 ± 0.11 g of chitin by this method.
    [0037] Production of Chitin with First Grinding ("Grinding 1") followed by Pressing and Second Grinding ("Grinding 2") 200 g of T molitor larvae are introduced into a beaker, placed in a bath -marie at 100 ° C and containing 200 mL of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then mixed with a volume of water of 200 ml. The liquid thus obtained is passed through a bi-screw type press. The press cake thus obtained is dried for 24 hours in an oven at 70 ° C. and then ground at 250 μm. 10 g of the powder thus obtained are transferred into an Erlen Meyer containing 50 ml of water and 0.1 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of about 6.5).
    [0038] The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. 4.93 ± 0.12 g of chitin are thus obtained by this method.
    [0039] Production of chitin with pressing only 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C and containing 200 ml of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then passed through a bi-screw type press. 90 g of press cake thus obtained are transferred into an Erlen Meyer containing 450 ml of water and 0.9 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a temperature of pH about 6.5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.450.5 μm under hot conditions. The chitin thus obtained is dried for 24 hours at 70 ° C.
    [0040] This gives 6.48 ± 0.28 g of chitin by this method. Production of chitin by chemical means 50 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C and containing 50 ml of water, previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered, and then mixed with a volume of water of 60 ml. The liquid thus obtained is transferred to a 1 L container and 500 mL of a 1M HCl solution is added. The whole is placed at 90 ° C with stirring for 1 hour. The contents are then filtered and the solid residue is transferred to a 1 L flask containing 500 mL of a 1M NaOH solution, the whole is placed at 90 ° C with stirring for 24 hours. The residue is then filtered and placed in a ventilated oven at 70 ° C. for 24 hours. Thus 0.944 ± 0.005 g of chemically purified chitin is obtained. Calculation of degree of purity The degree of purity of chitin is determined in comparison with the mass of dry residue obtained relative to that resulting from a chemical extraction, approximately 5% of the initial dry matter. Lipid Level Measurement 2 g of sample were placed in a beaker, 0.2 g of Na 2 SO 4 and 15 ml of CHCl 3 / MeOH (2/1 v / v) were added. The whole is placed under magnetic stirring for 20 minutes, then the solution is filtered, the residue is placed again in the beaker with 10 ml of CHCl 3 / MeOH (2/1 v / v). The whole is placed under magnetic stirring for 15 minutes, then the solution is filtered, the solvent phases are combined and evaporated to constant weight. The lipid content is determined as the mass percentage after extraction-evaporation relative to the initial mass of the sample (2 g). 2. Results As can be seen in FIG. 4, the process has an influence on the purity of the obtained chitin, the best results being obtained with minimal pressing. The best result is obtained with milling followed by pressing, namely chitin having a degree of purity of 78% and the worst with grinding alone, namely a chitin having a purity of 48%. A more refined analysis of the intermediate product from which chitin has been extracted shows that a low level of lipids is favorable to a higher purity of the chitin obtained (FIG. 5). The term "intermediate product" is intended to mean the product entering into enzymatic hydrolysis, that is to say from the last step before hydrolysis, namely, according to the production processes mentioned above, the grinding stage 1 or 2 or the pressing step. The analysis of the lipid content in chitin and the hydrolysis juice makes it possible to note that, depending on the initial lipid content present in the intermediate product, the lipid content in chitin is relatively stable, from 7 to 15%, while the level of lipids in the hydrolyzate varies from 11 to 47% (Figure 5). More particularly, if the intermediate product has a lipid content of 35%, then: - the chitin that will result from the hydrolysis will be only 50% pure and will contain 5 10% lipids, and - the lipid content of the hydrolyzate will be close to 40%. On the other hand, if the lipid content of the intermediate product is 7%, then: the chitin obtained after hydrolysis will have a purity of 80% and have a lipid content of 8% and the hydrolyzate will also have a low lipid content , of the order of 10%. This indicates that when the initial lipid level is high, higher than 12%, the enzyme responsible for hydrolysis is made to hydrolyze not only the proteins, but also the lipids by catalytic promiscuity. Thus a lipid level in the similar chitin is obtained ie 8.6 and 7.9% in cases where the initial lipids were 35 and 7% respectively. On the other hand, the purity of chitin passes in this case from 48 to 84% respectively. Thus, of the 52% impurities on the one hand and 16% on the other, 8% on average are due to lipids, the amount of proteins that remain attached to chitin is therefore 38 points higher in the case where more lipids were present in the intermediate product subjected to hydrolysis. Finally, the importance of grinding upstream of pressing can also be studied (Figure 6). It is thus clear that the distribution of lipids between the cake and the press juice is much more efficient, 12.9 versus 87.1 versus 42.7 versus 57.3, when a preliminary grinding has been done.
    [0041] Example 5: Influence of the combination of steps prior to enzymatic hydrolysis on the degree of purity of chitin obtained Various types of pretreatment were tested, grinding alone, grinding followed by pressing, treatment of cuticles with oxidizing agent (H 2 O 2) followed by grinding and treatment of the cuticles with an oxidizing agent (H 2 O 2) followed by milling and pressing. For pressing, the Angel type press described in Example 4 was used under the following conditions: - Speed = 82 rpm; 35 - VV (energy) = 3 HP (horsepower) or 2.68 x 106 J; 3031115 26 - Porosity (approximate) = 0.5 mm in the first part and 0.2 mm in the last part. 1. Material and methods 5 Production of chitin with grinding 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C and containing 200 ml of water previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered, and then mixed with a volume of water of 200 mL. The liquid thus obtained is transferred into an Erlen Meyer containing 2 g of protease (Prolyve), the whole is placed for 4 hours at 45 ° C with magnetic stirring (at a pH of about 6.5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. 8.13 ± 0.27 g of chitin are thus obtained by this method. Production of chitin with milling followed by pressing 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water boiled beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 200 mL. The liquid thus obtained is passed through a bi-screw type press. 30 g of the press cake thus obtained are transferred into an Erlen Meyer containing 150 ml of water and 0.3 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at one pH about 6.5). The Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. This gives 4.71 ± 0.11 g of chitin by this method.
    [0042] Production of chitin with treatment of cuticles with an oxidizing agent IL, _12L2)) followed by grinding 50 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 50 ml. a 6% hydrogen peroxide solution previously boiled. After 5 minutes, the beaker is removed from the water bath, the larvae are dewatered and then ground with a volume of water of 100 ml. The liquid thus obtained is transferred into an Erlen Meyer containing 150 ml of water and 0.5 g of protease (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of about 6 ° C.). , 5). Erlen Meyer is then placed for 15 minutes in a 90 ° C water bath to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm under heat. The chitin thus obtained is dried for 24 hours at 70 ° C. Thus 1.98 ± 0.22 g of chitin are obtained by this method. Production of chitin with treatment of cuticles with an oxidizing agent followed by grinding and pressing This test is carried out according to Example 1. Calculation of purity The purity of chitin is calculated gravimetrically as a function of the rate of chitin, initially contained in the larvae, ie about 5% of the dry weight of the larvae. 2. Results The results are shown in FIG. 7. The purity of the chitin obtained by enzymatic hydrolysis strongly depends on the pretreatment method used. Thus, when only grinding is carried out before the enzymatic hydrolysis, this process gives rise to only a purity of 48%, whereas the addition of a pressing step or treatment of the cuticles with an oxidizing agent allows to obtain a purity of 50-78%. Finally, the combination of a pressing step with the cuticle treatment with an oxidizing agent makes it possible to reach a chitin having a degree of purity of 88%. Synergistic effect resulting from the combination of a pressing step with a cuticle treatment step with an oxidizing agent Table 2 below shows the results presented in Figure 7.
    [0043] 3031115 28 Grinding only Grinding + H202 + H202 + pressing Grinding grinding + pressing Purity of 48% 78% 50% 88% chitin obtained Improvement of - + 63% + 4% + 83% purity of chitin compared to grinding alone Table 2: Improved purity of chitin according to the pretreatment method used Table 2 shows the synergistic effect of the combination of a pressing step and a treatment step with an oxidizing agent.
    权利要求:
    Claims (12)
    [0001]
    REVENDICATIONS1. A process for producing chitin and / or chitosan from insect cuticles, comprising the steps of: (i) pressing cuticles of insects, and then (i) enzymatically hydrolyzing insect cuticles by proteolytic enzyme, a cuticle treatment with an oxidizing agent having been carried out prior to the enzymatic hydrolysis.
    [0002]
    2. Method according to claim 1, comprising a grinding step prior to the pressing step.
    [0003]
    3. Method according to claim 1 or 2, characterized in that the treatment of cuticles with an oxidizing agent is performed before, concomitantly and / or after the grinding and / or pressing step.
    [0004]
    The method of any one of claims 1 to 3, wherein the proteolytic enzyme is a protease.
    [0005]
    The process according to any one of claims 1 to 4, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide, potassium permanganate, ozone and sodium hypochlorite.
    [0006]
    6. Method according to any one of claims 1 to 5, comprising a step of killing the insects prior to the pressing step and / or grinding.
    [0007]
    7. Method according to claim 6, characterized in that the step of killing insects is a scalding step.
    [0008]
    8. Method according to claim 6 or 7, characterized in that the treatment of cuticles with an oxidizing agent is carried out concomitantly and / or after the slaughtering step.
    [0009]
    The method according to any of claims 1 to 8, wherein the insect (s) is / are selected from the group consisting of Coleoptera, Lepidoptera, Orthoptera and Diptera.
    [0010]
    10. A method of producing chitin from insects, comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, 3031115 30 d) enzymatic hydrolysis of cuticles of insects by a proteolytic enzyme, e) the recovery of chitin, the insect cuticles being treated with an oxidizing agent, before step d). 5
    [0011]
    11. Chitin obtainable by a process according to any one of claims 1 to 10.
    [0012]
    12. Process for the production of chitosan from insects, comprising the following steps: a) killing of insects, b) breaking of insects, c) pressing of insects, d) enzymatic hydrolysis of cuticles, insects by a proteolytic enzyme, e) recovery of chitin, f) deacetylation of recovered chitin, g) recovery of chitosan, insect cuticles being treated with an oxidizing agent, before step d) .
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    CN104059953A|2013-09-29|2014-09-24|天津天狮生物发展有限公司|Chitosan enzymatic hydrolysis method|
    CN103694372A|2013-12-06|2014-04-02|中山奈德生物科技有限公司|Method for preparing chitin by use of insects|CN108208345A|2016-12-15|2018-06-29|丰益生物技术研发中心有限公司|A kind of technique that fermented bean dregs production is carried out the environment suitable for edible fat production factory|
    FR3075204B1|2017-12-15|2020-11-20|Ynsect|CHITINE AND PROCESS FOR OBTAINING CHITINE AND / OR CHITOSAN BY CHEMICAL MEANS|
    法律状态:
    2015-11-20| PLFP| Fee payment|Year of fee payment: 2 |
    2016-07-01| EXTE| Extension to a french territory|Extension state: PF |
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1463513A|FR3031115B1|2014-12-31|2014-12-31|PRODUCTION OF CHITINES FROM INSECTS BY ENZYMATIC HYDROLYSIS COMPRISING A COMBINATION OF PRELIMINARY STEPS|
    FR1463513|2014-12-31|FR1463513A| FR3031115B1|2014-12-31|2014-12-31|PRODUCTION OF CHITINES FROM INSECTS BY ENZYMATIC HYDROLYSIS COMPRISING A COMBINATION OF PRELIMINARY STEPS|
    PCT/FR2015/053783| WO2016108035A1|2014-12-31|2015-12-30|Chitin, hydrolysate and production of at least one desired product from insects by means of enzymatic hydrolysis, comprising a combination of steps performed prior to the enzymatic hydrolysis|
    CN201580077221.1A| CN107278208B|2014-12-31|2015-12-30|Chitin, hydrolysate and production of at least one target product from insects by means of enzymatic hydrolysis|
    CA2970640A| CA2970640A1|2014-12-31|2015-12-30|Chitin, hydrolysate and production of at least one desired product from insects by means of enzymatic hydrolysis, comprising a combination of steps performed prior to the enzymatic hydrolysis|
    EP15830826.2A| EP3240904B1|2014-12-31|2015-12-30|Production of chitin and/or chitosan from insects by means of enzymatic hydrolysis, comprising a combination of steps performed prior to the enzymatic hydrolysis|
    US15/541,162| US10995124B2|2014-12-31|2015-12-30|Chitin, hydrolysate and production of at least one desired product from insects by means of enzymatic hydrolysis, comprising a combination of steps performed prior to the enzymatic hydrolysis|
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