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    • 专利摘要:
    The present invention relates to a method for producing at least one product of interest from insects. More particularly, the present invention relates to a process for producing at least one product of interest from insects, comprising a step of pressing insect cuticles, and then enzymatic hydrolysis of insect cuticles by an enzyme. proteolytic.
    公开号:FR3031113A1
    申请号:FR1463512
    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 method for producing at least one product of interest from insects. More particularly, the invention relates to a method for producing 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 fungi. 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 that is both more pure and structurally closer to the original structure of chitin by performing a step of prior mechanical treatment to hydrolysis. namely, a step of pressing cuticles of insects. The invention thus relates to a process for producing at least one product of interest from insects, comprising the following steps: (i) pressing insect cuticles, then, (ii) enzymatic hydrolysis cuticles of insects by a proteolytic enzyme. By "product of interest" is meant more particularly chitin and / or chitosan and / or a hydrolyzate. By "hydrolyzate" is meant a product which comprises proteins, hydrolysed proteins, peptides, amino acids and / or other protein-derived compounds obtainable by enzymatic hydrolysis of proteins. The product (s) of interest are obtained from insects. "Insects" means insects at any stage of development, such as adult, larval, or pupal stage. Preferably, the insects employed 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, Hemyptères, Heteroptera, Ephemeroptera and Mecoptera, preferably from Coleoptera, Diptera, Orthoptera and Lepidoptera.
    [0002] 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 several species are used, two closely related species, such as, for example, Hermetia illucens and Musca domestica, will be favorably selected.
    [0003] The insects are preferably high and not taken in the wild. For example, insects are raised in an insect farm. The breeding of insects on a specific farm not only makes it possible 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, for example due to presence of insecticides. In addition, livestock can control the quality of the supply of insects and limit supply costs. 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 20 constituents of the insect, including the insect as a whole. 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 cuticles, for example exuviae and / or moults. insects, naturally separated and collected by a suitable process.
    [0004] 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 lm); this layer is impermeable to water and has 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.
    [0005] 3031113 4 The main purpose of pressing insect cuticles is to remove a fat-rich press juice and / or to enrich the press cake with a substrate for hydrolysis. In the process according to the invention, the pressing of insect cuticles makes it possible to obtain a press cake having an oil content of less than or equal to 20%, preferably less than or equal to 15%, more preferably less than or equal to equal to 12%, even more preferably less than or equal to 10%. 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. Moreover, 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 dry matter content of between 30% and 60%, preferentially including between 40% and 55%, and more preferably between 45% and 50%.
    [0006] Any press system can be used to effect 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 the Choquenet type), a plate press, etc. 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. 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 refer to 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 ° C., preferably 45 to 55 ° C. and at a pH of between 6 and 8, preferentially between 6.5 and 7.5.
    [0007] 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.
    [0008] 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 Eccalase 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 peptidases aspartic 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 peptidase Protex 50F P Endo-EC 3.4.21 Genencor Leiden The Netherlands peptidase International BV Pancreaty Exo & endo na Lyven Colombelles France 3031113 6 Enzyme (s) Class Number Provider City Country EC peptidase (cocktail proteases + 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] The process according to the invention makes it possible to obtain a chitin having a degree of purity of between 50 and 90%, preferably between 60 and 85%, more preferably of the order of 80% (see Example 2). and Figure 2). In addition, the process according to the invention makes it possible to obtain a hydrolyzate having a certain number of advantageous properties, in particular in terms of digestibility, lipid and / or protein content, protein size or composition. amino acids. Preferably, the method according to the invention comprises a grinding step prior to the pressing step. 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. Grinding may advantageously be carried out with a blender, such as a knife 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 still, a size between 500 lm and 0.5 cm.
    [0010] In order to facilitate grinding, a quantity of water may 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.
    [0011] Advantageously, the method according to the invention also comprises a step of slaughtering insects prior to the pressing and / or grinding 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.
    [0012] Advantageously, the step of killing insects is done by scalding. Scalding not only reduces insects, but also lowers the microbial load (reduces the risk of spoilage and sanitation) and inactivates internal enzymes insects that can trigger autolysis, and thus a rapid browning of them. This boiling is carried out so as to cause death as quickly as possible, in 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 soft 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] The method according to the invention may comprise, in addition, a step of treatment of insect cuticles with an oxidizing agent prior to the enzymatic hydrolysis. Preferably, in the process according to the invention, the oxidizing agent used during the treatment of the cuticles is chosen from the group consisting of hydrogen peroxide, potassium permanganate, ozone and hypochlorite. sodium, 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 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. 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 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 treatment of insect cuticles with the oxidizing agent can be carried out during one or more weeks. 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. - 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. 30 - during a specific step of treatment of insect cuticles. 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, chitin can be recovered by pressing or centrifuging the reaction medium of the enzymatic hydrolysis.
    [0014] At this stage, a chitin by-product is also recovered, i.e. a hydrolyzate. 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 below. A particularly advantageous method of producing chitin from insect cuticles comprises the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis insect cuticles by proteolitic enzyme, e) chitin recovery, insect cuticles can optionally be treated with an oxidizing agent prior to step d). The preferred embodiments of the various steps a) to e) as well as the treatment with the oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment hereinafter.
    [0015] The invention also relates to a chitin obtainable by a process according to the invention. Thanks 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 degree of purity of between 50 and 90%, preferably between 60 and 85%, more preferably of the order of 80%. A particularly advantageous method of producing a hydrolyzate from insects comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, 3031113 10 d) enzymatic hydrolysis of insect cuticles by proteolitic enzyme, e) hydrolyzate recovery, insect cuticles optionally being treated with an oxidizing agent prior to step d). The preferred embodiments of the various steps a) to e) 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 hydrolyzate obtainable by a process according to the invention. The hydrolyzate according to the invention has at least one of the following advantageous properties: an excellent digestibility, in particular a pepsic digestibility, greater than 95%, preferably greater than or equal to 98% and more preferably greater than or equal to 99%, pepsic digestibility having been measured according to a method in accordance with Directive 72/188 / EEC; a protein / peptide content greater than or equal to 70%, preferably greater than or equal to 75%, and more preferably greater than or equal to 80%; A lipid content of less than 10%, preferably less than or equal to 5%, more preferably less than or equal to 2%, and even more preferably less than or equal to 1%; and / or - a composition in which the most abundant amino acids are proline, alanine, leucine, glutamine and / or valine. By "more abundant" is meant a relative amount of the amino acid in the composition greater than 5%, preferably greater than 7% by weight on the total weight of amino acids. The high proline content is particularly interesting. In the present application, any reference to a regulation or a directive concerns the said regulation or the said directive as in force on the date of filing of the present application. The hydrolyzate can advantageously be supplemented with additives to balance its nutritional profile in order to be adapted to different types of animals. The hydrolyzate may advantageously 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 especially for animals, or, alternatively, they may be processed, for example, to isolate amino acids. A particularly advantageous method of producing chitosan from insect cuticles comprises the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis cuticles of insects by a protease, e) recovery of chitin, f) deacetylation of recovered chitin, g) recovery of chitosan, insect cuticles which can optionally be treated with an oxidizing agent prior to step d). The preferred embodiments of the various steps a) to g) 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 chitosan obtainable by a process 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 compositions or dietary, as biomaterials for treating burns as a 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 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 autolysis, and thus a rapid browning of these. Insects, preferably larvae, are thus scalded with water for 2 to 20 minutes, preferably 5 to 15 minutes. Preferably, the water is at a temperature of between 95.degree. And 105.degree. C., preferably 100.degree. The amount 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.
    [0016] This intermediate step is preferably carried out with an oxidizing agent selected from the group consisting of hydrogen peroxide (H2O2), potassium permanganate (KMnO4), ozone (O3) and sodium hypochlorite (NaClO4). ), more preferably, hydrogen peroxide. According to a first embodiment, after the scalding step 1, the oxidizing agent is introduced directly into the scalding tank, after possibly cooling the scalding water to a temperature of 1 ° C. 40 to 60 ° C, preferably of the order of 50 ° C. 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.
    [0017] In a second embodiment, the insects are removed from the scalding 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. % by weight on the weight of water, preferably of the order of 6% by weight. Step 3: Grinding Insects are removed from the scalding or treatment tank, sieved, and placed in a grinder, such as a knife mill, to reduce particulate insects.
    [0018] In order to facilitate grinding, a quantity of water may 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, still 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 step 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. 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 size of the particles, for example to a size less than 250 This grinding stage 2 promotes the access of the enzymes to their substrate. During this step, it is possible to introduce into the mill the oxidizing agent 20 in order to treat the cuticles according to the modalities indicated in the intermediate step above. When the treatment of cuticles is not carried out concomitantly with grinding, it is possible to add during this step, antioxidant additives commonly used for the preservation and stability of the product. Step 3: Pressing Insect Cuticles The wet paste resulting from grinding step 2 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. Preferably, the pressing step makes it possible to obtain a press cake 30 having an oil content less than or equal to 20%, preferably less than or equal to 15%, more preferably less than or equal to 12%, and even more preferably, less than or equal to 10%. Likewise, 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 30% and 60%. 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 (Angel twin-screw press), a filter press (Choquenet-type filter press). ), a plate press, etc.
    [0019] 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. 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. 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 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 scalding water does not contain any oxidant, the boiling water can be recovered and reintroduced during 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 of from 6.5 to 7.5, at a temperature of 40 to 60 ° C, more particularly of 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 the estimated dry matter entering into hydrolysis, preferably less than 6%, more preferably of the order of 2%.
    [0020] 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 operating mode, 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 out the maximum residue 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: hydrometry, ventilation and air composition 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.
    [0021] 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 lm <117 lm <95 lm <67 pm particles 90% of <310 lm <244 lm <157 lm <159 lm 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 preferentially milled to a particle size of less than 200, more preferably less than 160. Step 10: Deactivation The solid residue, optionally milled in step 9, is then placed in a reactor where is added a solution of concentrated sodium hydroxide 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.
    [0022] 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 3031113 17 The chitosan powder is then dried between 30 and 80 ° C, preferably between 50 and 70 ° C and preferably at about 60 ° C, to obtain a powder having a dry matter content greater than 85%, more particularly greater than 90%. The following steps aim at recovering an oily fraction and a protein fraction from the juice obtained in the pressing step 3 and are therefore only used 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 (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.
    [0023] 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 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, 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).
    [0024] 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 the method according to FIG. FIG. 2 is a chart comparing the degree of purity of chitin obtained by an enzymatic process involving one or more prior grinding and pressing steps. FIG. 3 is a chart comparing the lipid content measured in different fractions. of the intermediate product from which the chitin was extracted, FIG. 4 illustrates the distribution of the lipids in the juice and the press cake obtained by an enzymatic process comprising preliminary steps of grinding and pressing or a preliminary pressing step, FIG. 5 is a diagram illustrating the distribution of proteins / peptides according to their molar masses (in%) in a hydrolyzate according to 6 is a diagram illustrating the relative distribution of the amino acids constituting a hydrolyzate according to the invention, and FIG. 7 is a diagram illustrating the relative abundance of the acids. amines in chitin.
    [0025] EXAMPLE 1 Example of Process According to the Invention 600 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 600 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 600 ml. The liquid thus obtained is pressed with a twin-screw type Angel under the following conditions: Speed = 82 rpm; W (energy) = 3 HP (horsepower) or 2.68 x 106 J; Porosity (approximate) = 0.5 mm in the first part and 0.2 mm in the last part. This gives a press juice and a press cake of 136.49 ± 4.48 g wet weight, of which 100.22 ± 0.22 g are used in the following steps. However, any other type of press could have been used, since this allows the extraction to obtain a press cake substantially similar in terms of water content and / or lipid content. By way of example, other tests were carried out with the Choquenet type filter filter having the following characteristics: Filtration area of the cell = 50 cm 2; Pressure = 2 to 8 bar; Temperature = 20 to 80 ° C, Porosity = 25 to 80 μm; Flow rate at the end of filtration = 100 to 250 mL / h. The press cake is transferred into an Erlen Meyer containing 600 ml of a 1% protease (Prolyve NP conc) solution (based on the wet weight of the press cake), the whole is placed for 4 hours at 45 ° C. ° C with magnetic stirring (at a pH of about 6.5). The 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 lm hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 16.99 ± 1.77 g of chitin are thus obtained by this method. The hydrolyzate (filtrate obtained after hydrolysis) represents, under these conditions, 609.98 ± 10.9 g with a dry matter content of 5.05%, which, after lyophilization, gives 30.8 ± 0.55 g of dry hydrolyzate.
    [0026] EXAMPLE 2 Influence of the Mechanical Steps Preceded by Enzymatic Hydrolysis on the Degree of Purity of Chitin Obtained Various types of mechanical pretreatment were tested, grinding ("grinding 1") alone, grinding followed by pressing, grinding followed by pressing and a second grinding ("grinding 2"), as well as pressing alone.
    [0027] For pressing, the Angel press described in Example 1 was used. 1. Material and methods 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.
    [0028] 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). 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 lm 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 grinding followed by pressing 10 200 g of T molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of boiling water. 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 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 temperature of pH about 6.5). The 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 lm hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 4.71 ± 0.11 g of chitin are thus obtained by this method. Production of chitin with a first grinding ("grinding 1") followed by pressing and a second grinding ("crushing 2") 200 g of T molitor larvae are introduced into a beaker, placed in a water bath to 100 ° C and containing 200 mL of water previously boiled.
    [0029] 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., then milled 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). The 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 lm hot. 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.
    [0030] Production of chitin with pressing only 200 200 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 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 water bath at 90 ° C to deactivate the enzymes, the solution is then filtered at 0.450.5 lm hot. The chitin thus obtained is dried for 24 hours at 70 ° C.
    [0031] 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, about 5% of the initial dry matter. Measurement of lipid content 2 g of sample are placed in a beaker, 0.2 g of Na 2 SO 4 and 15 ml of CHCl 3 / MeOH (2/1 v / v) are added thereto. 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 Figure 2, 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 degree of 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. 3). 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, 7 at 15%, whereas the level of lipids in the hydrolyzate varies from 11 to 47% (Figure 3). 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 10% lipids, and - the lipid content of the hydrolyzate will be around 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 lipids, 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 similar lipid content in chitin is obtained, namely 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% of impurities on the one hand and 16% of 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 4). It thus clearly appears 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 carried out. Example 3 Analysis of the Hydrolyzate A detailed analysis of the hydrolyzate obtained in Example 1 was carried out. 1. Content of glucosamines The content of the hydrolyzate with glucosamines and certain other sugars was analyzed by gas chromatography after methanolysis and sillilation. Procedure: 10 mg of the sample and 50 μg of internal standard are placed in 500 μl of a 3N methanol / hydrochloric acid mixture for 4 hours at 110 ° C (or 24 hours at 130 ° C). The mixture is then neutralized with Ag2CO3. 50 μl of acetic anhydride are added to re-acetylate any osamines present. After dark overnight and at room temperature, the samples are centrifuged (15 min, 3000 rpm) and the supernatant is evaporated. The compounds are then dissolved in 100 μl of pyridine and incubated overnight at room temperature with 100 μl of BSTFA (Supelco). The reagents are then evaporated and the residue taken up in 700 μl of CH 2 Cl 2 and injected into CPG. Temperature program: 1 minute at 120 ° C, ramp 1.5 ° C / minute up to 180 ° C, then 2 ° C / minute up to 200 ° C. Detection: FID 25 Column: HP-5MS (30 m, 0.25 mm internal diameter) Internal standard: myo-inositol The different contents were measured and calculated in two different ways (Table 2). It appears that the glucosamine is contained in the hydrolyzate at 0.1-0.15% by weight and with a ratio of 0.04-0.05 relative to glucose.
    [0032] 3031113 24 Methanolysis at 110 ° C Methanolysis at 130 ° C (3/0 mass Ratio molar% mass by weight Molar ratio Glucose 1.6 ± 0.3 1 2 ± 0.4 1 Mannose 0.3 ± 0.05 0.15 ± 0.005 0.4 ± 0.08 0.15 ± 0.007 Glucosamine 0.1 ± 0.02 0.04 ± 0.007 0.15 ± 0.04 0.05 ± 0.003 Table 2: Distribution of glucosamine, mannose and glucose in the blood Hydrolyzate 2. Protein size The size of the proteins / peptides of the hydrolyzate was evaluated by HPLC, Shimadzu 20A apparatus, at room temperature, on a Superdex Peptide 10/300 GL column, in acetonitrile buffer (ACN) 30% 0.1% trifluoroacetic acid (TFA) at a flow rate of 0.4 ml / min Detection was carried out at 205 nm and the sample volume injected was 20 μl. The peptides of the press juice prepared in Example 1 were also evaluated under identical conditions for comparison purposes.The comparison of the protein size between the press juice and the hydro lysate (FIG. 5) clearly shows that the essential, 76%, of the peptides present in the hydrolyzate have a molar mass of between 130 and 900 Da. On the other hand, the portion of the proteins that are not easily digested (with a molar mass greater than 1300 Da) is reduced from 31% to 2%. Similarly, the proportion of peptides having a bitterness character (of molecular weight less than 130 Da) increases from 38% to 15%. The digestibility and palatability criteria are therefore improved by the proposed enzymatic treatment. 3. Digestibility The pepsic digestibility rate of the hydrolyzate is estimated at 99.6% of total protein. It has been measured by an external laboratory, the method used complies with the directive 72/199 / EEC and has been practiced without degreasing. 4. Protein content The protein content of the hydrolyzate is estimated at 84.8%. It was measured by an external laboratory, according to the Kjeldahl method with the correction coefficient of 6.25. The method used is in accordance with EC regulation 152/2009. 5. Lipid content The lipid content of the hydrolyzate is estimated to be 0.7 ± 0.5%. It was measured by an external laboratory according to the so-called "hydrolysis" method adapted from Regulation EC 152/2009 (process B). 6. Amino Acid Composition The hydrolyzate obtained after treatment with the various enzymes was analyzed for its amino acid composition (Figure 6). The preponderance of proline is observed, together with the presence of the hydroxy proline (HYP) - proline metabolite and amino acid absent in some organisms, eg crustaceans. However, proline and its metabolite, hydroxyproline, play an essential role in metabolism, in particular by allowing the synthesis of other amino acids such as arginine and glutamate. If most mammals are able to synthesize proline, the production of this amino acid by newborns, birds and fish is insufficient, therefore supplementation with proline and hydroxyproline is sometimes put in place to increase the growth of certain animals. Moreover, proline is the first amino acid contained in mammalian milk, it is present at 12%, but its content in plant proteins is much lower, only 2.9% in soy and 0.8% in corn. Thus, in this hydrolyzate, there is a proline content as high as in milk and much higher than that found in vegetable proteins. The other amino acids present in significant amounts are alanine, leucine and glutamate (with glutamine). Their relative amounts are greater than 9%. On the other hand, the sulfur-containing amino acids such as cysteine and methionine are in a small amount, of the order of 0.5%. The method used to determine these results (acid hydrolysis) did not allow the detection of amino acids such as tryptophan and arginine.
    [0033] On the other hand, asparagine has been completely converted to aspartic acid and glutamine to glutamic acid and what is observed under the ASP and GLU peaks is actually the sum of aspartic acid and asparagine, respectively. and glutamic acid and glutamine on the other hand, initially present in the hydrolyzate.
    [0034] Example 4: Chitin Assay A detailed amino acid analysis of the chitin obtained in Example 1 was performed. The total amino acid content is 32.3 g per 100 g copolymer (determined as the sum of amino acids). The amino acids predominantly present are valine, glycine, alanine and tyrosine (FIG. 7). The analyzes were subcontracted and the results obtained according to the method NF EN ISO 13904 for tryptophan and NF EN ISO 13903 for the other amino acids.
    权利要求:
    Claims (13)
    [0001]
    REVENDICATIONS1. A method of producing at least one product of interest from insects, comprising the following steps: (i) pressing cuticles of insects, then, (ii) enzymatic hydrolysis of insect cuticles by proteolytic enzyme.
    [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, comprising a step of killing the insects prior to the pressing step and / or grinding.
    [0004]
    4. Method according to any one of claims 1 to 3, further comprising a step of treatment of insect cuticles with an oxidizing agent prior to enzymatic hydrolysis.
    [0005]
    5. Method according to any one of claims 1 to 4, wherein the insect (s) is (are) selected from the group consisting of Coleoptera, Lepidoptera, Orthoptera and Diptera.
    [0006]
    The method according to any one of claims 1 to 5, wherein the protease is selected from the group consisting of aminopepidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.
    [0007]
    The method according to any one of claims 1 to 6, wherein the product of interest is chitin and / or chitosan.
    [0008]
    The process of any one of claims 1 to 6, wherein the product of interest is a hydrolyzate.
    [0009]
    9. Process for producing chitin from insect cuticles, comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis of cuticles insects by a proteolitic enzyme, e) recovery of chitin, the insect cuticles can optionally be treated with an oxidizing agent before step d). 3031113 28
    [0010]
    10. Chitin obtainable by a process according to any one of claims 1 to 7, 9.
    [0011]
    11. Process for producing a hydrolyzate from insects, comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis of insects, insect cuticles by a proteolitic enzyme; e) recovery of the hydrolyzate, the insect cuticles being optionally treated with an oxidizing agent prior to step d).
    [0012]
    12. A hydrolyzate obtainable by a process according to any one of claims 1 to 6, 8, 11.
    [0013]
    13. A method of producing chitosan from insect cuticles, comprising the following steps: a) killing of insects, b) grinding of insects, c) pressing of insects, d) enzymatic hydrolysis of insects, insect cuticle by protease, e) recovery of chitin, f) deacetylation of recovered chitin, g) recovery of chitosan, insect cuticles optionally being treated with an oxidizing agent prior to step d).
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    同族专利:
    公开号 | 公开日
    CN107428849A|2017-12-01|
    US20180016357A1|2018-01-18|
    EP3240902A1|2017-11-08|
    AU2015373263A1|2017-08-03|
    HUE050766T2|2021-01-28|
    ES2819858T3|2021-04-19|
    BR112017014340A2|2018-01-02|
    JP2021020911A|2021-02-18|
    MX2017008814A|2018-02-21|
    AU2015373263B2|2020-07-09|
    CA2970707A1|2016-07-07|
    JP2018502203A|2018-01-25|
    SG10201911348UA|2020-01-30|
    FR3031113B1|2018-03-16|
    EP3240902B1|2020-07-29|
    KR20170106355A|2017-09-20|
    AU2020200381A1|2020-02-13|
    SG11201705391UA|2017-08-30|
    WO2016108033A1|2016-07-07|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1463512|2014-12-31|
    FR1463512A|FR3031113B1|2014-12-31|2014-12-31|PROCESS FOR PRODUCTION OF PRODUCTOF INTEREST FROM INSECT BY ENZYMATIC HYDROLYSIS|FR1463512A| FR3031113B1|2014-12-31|2014-12-31|PROCESS FOR PRODUCTION OF PRODUCTOF INTEREST FROM INSECT BY ENZYMATIC HYDROLYSIS|
    KR1020177020950A| KR20170105023A|2014-12-31|2015-12-30|Beetle powder|
    PE2017001181A| PE20171076A1|2014-12-31|2015-12-30|COMPOSITION CONTAINING CHITIN AND DIGERIBLE PROTEINS|
    US15/541,186| US20180016357A1|2014-12-31|2015-12-30|Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis|
    US15/541,140| US20180000121A1|2014-12-31|2015-12-30|Beetle powder|
    HUE15828828A| HUE050766T2|2014-12-31|2015-12-30|Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis|
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    CN201580077226.4A| CN107428849A|2014-12-31|2015-12-30|Chitin, hydrolysate and the mode by means of enzymatic hydrolysis are used for the method that one or more desired products are produced from insect|
    CA2970724A| CA2970724A1|2014-12-31|2015-12-30|Beetle powder|
    PT15830828T| PT3240905T|2014-12-31|2015-12-30|Beetle powder|
    DK15830828.8T| DK3240905T3|2014-12-31|2015-12-30|Bill Pulver|
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    CL2017001750A| CL2017001750A1|2014-12-31|2017-06-30|Coleoptera dust.|
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    ECIEPI201743318A| ECSP17043318A|2014-12-31|2017-07-06|COMPOSITION CONTAINING CHITIN AND DIGERIBLE PROTEINS|
    ECIEPI201743316A| ECSP17043316A|2014-12-31|2017-07-06|COLEOPTERUS POWDER|
    HK18105880.2A| HK1246097A1|2014-12-31|2018-05-07|Composition containing chitin and digestible proteins|
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    AU2020200381A| AU2020200381B2|2014-12-31|2020-01-20|Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis|
    JP2020168003A| JP2021020911A|2014-12-31|2020-10-02|Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis|
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