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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 method for producing chitin and / or chitosan from insect cuticles, comprising a step of treating insect cuticles with an oxidizing agent, and then, an enzymatic hydrolysis step of cuticles of insects by a proteolytic enzyme.
    公开号:FR3031114A1
    申请号:FR1463511
    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 treating the cuticles of the insects with an oxidant prior to perform the enzymatic hydrolysis. The invention thus relates to a process for producing chitin and / or chitosan from insect cuticles, comprising the following steps: treating insect cuticles with an oxidizing agent, and then (ii) hydrolyzing Enzymatic cuticles of insects by a proteolytic enzyme. "Insects" means insects at any stage of development, such as adult, larval, or pupal stage. Preferably, the insects used in the process according to the invention are edible.
    [0002] More particularly, the insects may be selected from the group consisting of Coleoptera, Diptera, Lepidoptera, Orthoptera, Isoptera, Hymenoptera, Blattoptera, Hemyptères, Heteroptera, Ephemeroptera and Mecoptera, preferably among Coleoptera, Diptera, Orthoptera and Lepidoptera.
    [0003] 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 preferentially again, T molitor.
    [0004] One or more insect species may be used in the process according to the invention, preferably one species of insect. If several species are used, two closely related species, such as, for example, Hermetia illucens and Musca domestica, will be favorably selected. The insects are preferably high and not taken in the wild.
    [0005] For example, insects are raised in an insect farm. Farming insects on a specific farm not only helps to control and eliminate the risks associated with insect-borne diseases, but also to limit the risks associated with the toxicity of food products derived from insects due for example to the presence of insects. insecticides. In addition, livestock can control the quality of the supply of insects and limit supply costs. By "treatment of insect cuticles" is meant not only the treatment of the cuticles once separated from the insects, but also the treatment of the cuticles, including all or part of the other constituents of the insect, including the treatment of 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 only to a portion of the insects comprising the cuticles, for example after extraction, such as crushed insects and then pressed to extract a press cake rich in cuticles, or such as exuvia / moults naturally separated, and collected by a suitable method. The cuticle is the outer layer (or exoskeleton) secreted by the epidermis of insects. It is generally composed 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 comprises a layer of waterproofing wax, as well as proteins and chitin, in a lesser amount; - the exocuticle, which is the middle layer of the cuticle; it is composed mainly 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. 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.
    [0006] 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 total weight of insects, preferably between 2 and 12% by weight on the total weight of insects, preferably of the order of 6% by weight. In the present application, the ranges of values are included limits. Similarly, when "about" or "of the order of" precedes a number, this equates to plus or minus 10% of the value of that number. Preferentially, the treatment of insect cuticles with the oxidizing agent 10 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 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.
    [0007] Note that this ratio also corresponds to the ratio of water weight to insect weight, the density of water being 1.0 g / ml under normal temperature and pressure conditions. Cuticle treatment of insects is followed by enzymatic hydrolysis. According to the invention, 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. Advantageously, the enzymes may be chosen from the following: 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 Netherlands peptidase International BV Pancreative Exo & endo na Lyven Colombelles France peptidase (cocktail proteases + 3031114 6 Enzyme (s) Class Number Provider City Country EC amylases) lzyme BA Protease EC 3.4 .23 Novozyme Bagsvaerd Aspartic Denmark Sumizyme Cocktail na * Takabio - Shin Aichi Enzymatic Japan Nihon * na: not applicable The enzyme or enzyme mixture 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 0.5 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 at the entry into enzymatic hydrolysis step . 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. The process according to the invention makes it possible to obtain a chitin having a degree of purity understood. between 55 and 90%, preferably between 60 and 85%, more preferably of the order of 80% (see Example 2 and Figure 2). In addition, the size of the chitin obtained by the method according to the invention, namely of the order of 80000 g / mol in Example 3 below, is closer to that which exists in the natural state in the insect cuticle.
    [0008] Advantageously, the method according to the invention comprises a step of killing insects. This slaughter step is performed prior to enzymatic hydrolysis. The slaughter stage may be carried out by conventional methods of breeding cold-blooded and / or small-sized animals (crustaceans, fish, snails, etc.), such as cold (freezing), warm (scalding), oxygen deprivation, etc. Preferably, slaughter is by scalding. Scalding reduces insects while lowering the microbial load (reducing the risk of spoilage and sanitation) and by inactivating the internal enzymes of insects that can trigger autolysis, and thus a rapid browning thereof. In addition, scalding is performed so as to cause death as quickly as possible, in the respect of animal welfare, and according to scientific recommendations. 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 to 20 min, preferably 5 to 15 min.
    [0009] 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, even more preferably of the order of 1. Preferably, the treatment of insect cuticles with the oxidizing agent can be carried out concomitantly with the scalding and / or after the boiling step, more preferably concomitantly with scalding. When treatment of insect cuticles is performed during scalding, the oxidizing agent is added to the water used to scald insects. Advantageously, the method according to the invention also comprises a step 15 of grinding insects. Preferably, this grinding step takes place after the boiling step. This grinding step aims to reduce the insect particles to facilitate the access of enzymes to the substrate during enzymatic hydrolysis. This step also allows, when followed by a pressing step, to facilitate the removal of the press juice and the isolation of the solid material. Grinding can advantageously be carried out with a mixer mill, such as a knife mill. Preferably, after grinding, the size of the insect particles is less than 1 cm (larger particle size observable by a microscope), preferably less than 0.5 cm, more more preferably, 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, more preferably between 0.7 and 3, even more preferably of the order of 1. Optionally, the treatment of insect cuticles with the oxidizing agent can be carried out concomitantly with grinding. In this case, the oxidizing agent is added to the water used for grinding. Alternatively, cuticle treatment may be performed during scalding, grinding and / or at a specific step of insect cuticle treatment. The process according to the invention may further comprise, preferably before the enzymatic hydrolysis, a pressing step. Although this pressing step can be carried out before the grinding step, this is advantageously carried out just after the grinding step. The aim of this pressing step is to eliminate a fat-rich press juice and to enrich the press cake with a substrate for hydrolysis. Advantageously, the enzymatic hydrolysis may be followed by 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. At this stage, a co-product of interest of chitin is also recovered, namely a hydrolyzate.
    [0010] 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 co-product of the 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.
    [0011] Alternatively, the hydrolyzate may be in liquid form. These hydrolysates may be used as a food or food ingredient especially 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 as a powder, a second grinding can also be carried out. This can also be done in order 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 below. A particularly advantageous process for producing chitin from insect cuticles comprises the following steps: a) the slaughter of insects, b) the grinding of insects, the grinding possibly being preceded or followed by a pressing step c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) recovery of chitin, the insect cuticles being treated with an oxidizing agent, before step c). The preferred embodiments of the various steps a) to d), as well as the treatment with the oxidizing agent, are as indicated above or in the corresponding step in the preferred embodiment hereinafter. The invention also relates to a chitin obtainable by a process according to the invention. This chitin has a structure similar to chitin as naturally occurring in the cuticle of the insect. A particularly advantageous method of producing chitosan from insect cuticles, comprising the following steps: a) the slaughtering of insects, b) the grinding of insects, the grinding optionally being preceded or followed by a pressing step c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) recovery of chitin, e) deacetylation of recovered chitin, f) recovery of chitosan, insect cuticles being treated with an oxidizing agent, before step c).
    [0012] The preferred embodiments of the various steps a) to f), as well as the treatment with the oxidizing agent, are as indicated above or in the corresponding step in the preferred embodiment hereinafter. The invention finally relates to a chitosan that can be obtained by a process according to the invention.
    [0013] Chitin and / or chitosan, which may be obtained by a process according to the invention, may be advantageously used in various applications: in cosmetic and pharmaceutical compositions , as nutraceuticals or dietetics, as biomaterials for treating burns as second skin, for producing corneal dressings or surgical threads, as a filtering, texturing, flocculating and / or adsorbent agent, especially for the filtration and depollution of the skin. 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. - Stage 1: scalding insects This scalding stage 1 makes it possible to kill 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 so rapid browning thereof. 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 scalding step 1 and milling step 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.
    [0014] According to a first embodiment, at the end of the boiling step 1, the oxidizing agent is introduced directly into the boiling tank, after possibly cooling the boiling water to a temperature of 30.degree. of the order of 40 to 60 ° C, preferably of the order of 50 ° C. 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 between 1 to 33% by weight relative to the total weight of insects, preferably 2 to 12% by weight relative to the total weight 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 The insects are removed from the scalding or treatment tank, 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 quantity 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, more preferably between 0.5 and 5, more preferably between 0.7 and 3, still 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, cuticle treatment can take place during scalding step 1 and milling step 2 or during the intermediate cuticle treatment step and during the grinding step.
    [0015] Preferably, after grinding, the size of the insect particles is less than 1 cm (larger particle size observable by 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. This grinding step 2 promotes the access of the enzymes to their substrate.
    [0016] In this step, it is possible to introduce into the mill the oxidizing agent 3031114 12 in order to treat the cuticles according to the modalities indicated in the intermediate step above. When the cuticle treatment is not carried out concomitantly with grinding, it is possible to add during this step, antioxidant additives 5 commonly used for the preservation and stability of the product. Step 3 (optional): pressing The wet paste resulting from grinding step 2 is then placed in a press according to a procedure that makes it possible to press and separate a juice comprising both an oily fraction and a protein fraction.
    [0017] If the wet paste resulting from grinding step 2 has been obtained with a water comprising 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.
    [0018] 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.
    [0019] Optionally, and as will be described hereinafter, the protein fraction from the separation step 12 may be reintroduced into this enzymatic hydrolysis step 4 by mixing it with the press cake. Optionally, and in the case where the boiling water contains no oxidant, the boiling 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. The quantity of water introduced in this enzymatic hydrolysis step 4 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, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1. 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 temperature of 40 to 60 ° C, more particularly from 45 to 55 ° C. The quantity of enzymes introduced during the hydrolysis step is less than 10% by weight relative to the total weight of estimated dry matter entering into 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 thermal inactivation step 5 is carried out according to the usual sterilization techniques of the food industry. According to another mode of operation, the enzymatic inactivation is carried out by heating under IR or UV radiation, or microwaves. Step 6: Pressing The solid residue, mainly composed of chitin, is recovered and then pressed by a press 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.
    [0020] 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. 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): Grinding 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 prior crushing of chitin * Measurement of Acetylation Degree DA 10 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 preferably milled 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 according to 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. Chitosan powder is thus obtained.
    [0021] The 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 ...). Step 11 (Optional): Drying The chitosan powder is then dried at 30 to 80 ° C, preferably 50 to 70 ° C and preferably at about 60 ° C, to obtain a powder having a in dry matter 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 step 3 (optional) of pressing and are therefore only implemented when the pressing step 3 has been implemented. and that 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 (hemolymph 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.
    [0022] The centrifugate 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, may be mixed with the press cake from the pressing step 3 just prior to 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 additives commonly used 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, spraying / atomizing ("spray-drying"), which makes it possible to obtain 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).
    [0023] Other characteristics and advantages of the invention will become apparent 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 a method According to the invention, FIG. 2 is a diagram comparing the degree of purity for chitin obtained by an enzymatic process with and without hydrogen peroxide. FIG. 3 is a diagram illustrating the influence of the process of the invention. extraction (enzymatic or chemical) and treatment with an oxidizing agent on the obtained chitin.
    [0024] EXAMPLE 1 Example of a Treatment Process According to the Invention 15 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 30 ml of a solution of hydrogen peroxide at room temperature. 6% previously boiled. After 5 minutes, the beaker is removed from the waterbath, the larvae are dewatered and then ground with a volume of water of 15 ml. The liquid thus obtained is transferred into a 250 ml Erlen Meyer containing 50 ml of a 1% protease solution (Prolve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (pH approximately 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. 0.6 ± 0.05 g of chitin is thus obtained. Example 2: Influence of the presence of the treatment with the oxidant in the process according to the invention 30 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. 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 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 5 to 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. The dry residue thus obtained after use of the hydrogen peroxide is 6.3 ± 0.7% with respect to the initial dry matter, while the dry residue resulting from a process without the hydrogen peroxide is 9 , 75 ± 0.9% relative to the initial dry matter. 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 amounts to 79.9 ± 9% for the product obtained after treatment with peroxide and 51.5 ± 4.9% in the absence of peroxide (see FIG. 2). EXAMPLE 3 Influence of the Sequence of the Different Stages in the Process According to the Invention Obtaining Chitin Enzymatically (Without the Addition of an Oxidant) 50 g of T molitor larvae are introduced into a beaker, placed in a bain-marie 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.
    [0025] Obtaining chitin by means of H 2 O 2 - scalding 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. 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. 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. Thus 1.98 ± 0.22 g of chitin are obtained by this method. Obtaining Chitin Using a Molecular Route with 50% H 2 0 2 T Molitor larvae are placed in a beaker, placed in a 100 ° C water 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 approximately 6 ° C.). , 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.
    [0026] 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 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 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. This residue is then transferred to a 1 L flask supplemented with 500 mL of 1M NaOH and kept under stirring 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. 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 at 90 ° C. for 4 hours. 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 type viscometer and the molecular weight is calculated according to the formula: KM''Q (1) with [q]: intrinsic viscometry in cm3 / 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: reduced viscosity (without units), 20 C: concentration in mg / mL, the reduced viscosity being obtained according to: qr = t / to (3) with t: the measured fall time for the solution in s, 25 to: the time drop measured for 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 70,000 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, close to 80000 g / mol) 3031114 20 and the result obtained is superior to that related to traditional chemical extraction.
    权利要求:
    Claims (11)
    [0001]
    REVENDICATIONS1. A process for producing chitin and / or chitosan from insect cuticles comprising the following steps: (i) treating insect cuticles with an oxidizing agent, and then (ii) enzymatically hydrolyzing the cuticles of insects by a proteolytic enzyme.
    [0002]
    The method of claim 1, wherein the proteolytic enzyme is a protease.
    [0003]
    3. The method of claim 1 or 2, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide, potassium permanganate, ozone and sodium hypochlorite.
    [0004]
    4. Method according to any one of claims 1 to 3, comprising a step of killing insects.
    [0005]
    5. Method according to any one of claims 1 to 4, comprising a step of grinding insects.
    [0006]
    The process according to any one of claims 1 to 5, wherein the oxidizing agent is hydrogen peroxide.
    [0007]
    The method of any one of claims 1 to 6, wherein the insect (s) is / are selected from the group consisting of Coleoptera, Lepidoptera, Orthoptera and Diptera.
    [0008]
    The method according to any one of claims 2 to 7, wherein the protease is selected from the group consisting of aminopepidases, metal locarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.
    [0009]
    9. Process for the production of chitin from insects, comprising the following steps: a) the slaughter of insects, b) the grinding of insects, the grinding optionally being preceded or followed by a pressing step, c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) the recovery of chitin, the insect cuticles being treated with an oxidizing agent, before step c).
    [0010]
    10. Chitin obtainable by a process according to any one of claims 1 to 9. 3031114 22
    [0011]
    11. A method of producing chitosan from insects, comprising the following steps: a) slaughter of the insects, b) grinding of the insects, the grinding optionally being preceded or followed by a pressing step, c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) recovery of chitin, e) deacetylation of recovered chitin, f) recovery of chitosan, insect cuticles being treated with an oxidizing agent before step c).
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    同族专利:
    公开号 | 公开日
    CN107257860A|2017-10-17|
    WO2016108034A1|2016-07-07|
    US20180002452A1|2018-01-04|
    EP3240903A1|2017-11-08|
    CA2970718A1|2016-07-07|
    FR3031114B1|2018-01-26|
    CN107257860B|2019-04-02|
    EP3240903B1|2020-05-06|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2012168618A1|2011-05-26|2012-12-13|Ifremer |Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium|
    CN102898544A|2012-10-25|2013-01-30|南京大地冷冻食品有限公司|Method for extracting chitosan from environment-friendly insects|FR3089759A1|2018-12-12|2020-06-19|Ynsect|Water-soluble extract and process for obtaining it from insect cuticles|
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    CN101144097B|2007-09-18|2010-12-01|重庆百奥帝克微生态科技有限公司|Method for preparing chitin and its chitosan and chitosan oligosaccharide|
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    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|EP3370543A4|2015-11-03|2019-07-31|Aspire Food Group USA, Inc.|Insect products and methods of manufacture thereof|
    WO2021090982A1|2019-11-07|2021-05-14|농업회사법인푸디웜주식회사|Method for preparing black soldier fly-derived chitosan|
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    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 |
    2016-07-01| PLSC| Publication of the preliminary search report|Effective date: 20160701 |
    2016-12-26| PLFP| Fee payment|Year of fee payment: 3 |
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1463511A|FR3031114B1|2014-12-31|2014-12-31|PROCESS FOR PRODUCTION OF CHITINE BY ENZYMATIC HYDROLYSIS WITH PRIOR TREATMENT WITH OXIDIZING AGENT|
    FR1463511|2014-12-31|FR1463511A| FR3031114B1|2014-12-31|2014-12-31|PROCESS FOR PRODUCTION OF CHITINE BY ENZYMATIC HYDROLYSIS WITH PRIOR TREATMENT WITH OXIDIZING AGENT|
    EP15830825.4A| EP3240903B1|2014-12-31|2015-12-30|Method for the production of chitin and/or chitosan by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent|
    PCT/FR2015/053782| WO2016108034A1|2014-12-31|2015-12-30|Chitin, hydrolysate and method for the production of one or more desired products by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent|
    US15/541,174| US20180002452A1|2014-12-31|2015-12-30|Chitin, hydrolysate and method for the production of one or more desired products by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent|
    CA2970718A| CA2970718A1|2014-12-31|2015-12-30|Chitin, hydrolysate and method for the production of one or more desired products by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent|
    CN201580077200.XA| CN107257860B|2014-12-31|2015-12-30|Chitin, hydrolysate and by means of including the method for being used to produce one or more desired products with the mode of the pretreated enzymatic hydrolysis of oxidant|
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