Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/23—Aspartic endopeptidases (3.4.23)
  • C12Y304/23001—Pepsin A (3.4.23.1)

Definitions

• the present invention relates to the field of valorization of biomass, preferably animal biomass, more preferably the marine biomass and / or entomological.
• the present invention relates to a process for the single-step enzymatic extraction of chitin from elements of animal biomass comprising chitin, preferably from marine and / or entomological co-products, an active enzyme in an acid medium.
• insects are an interesting food resource because of their nutritional qualities.
• the production of insects offers a very interesting ecological alternative compared to the production of other proteins of animal origin.
• carapaces rich in chitin, as in the case of crustaceans.
• Chitin is the second most abundant polysaccharide on the Earth's surface after cellulose. It does not have a single chemical structure but several since it includes polysaccharides composed of N-acetyl-PD-glucosamine units and D-glucosamine units.
• Chitin is partly the exoskeleton of insects and crustaceans, the wall of fungi and bacteria. Chitin thus constitutes 20 to 30% of the shells of crustaceans. In addition to chitin, the crustacean exoskeleton contains 20 to 40% protein, 30 to 60% minerals and 0 to 14% lipids (Waldeck J., Daum G., Bisping B. and Meinhardt F., Appl. Micwbiol Env., 2006, 72 (12), 7879-7885). Chitin thus constitutes 3 to 60% of the carapaces of insects.
• the exoskeleton of insects contains 20 to 80% protein, 1 to 20% minerals and 10 to 50% lipids ("Forest Insects as Food: humans bite back", Proceedings of a workshop on Asia - Pacific resouces and their potential for development, 19-21 February 2008, Chiang Mai, Thailand - FAO).
• the proportions of the different constituents vary according to species, age, genus and can fluctuate according to seasons and environmental conditions.
• the chitin extraction conditions must therefore be adapted according to the raw material used (Tolaimate A., Desbrieres J., Rhazi M. and Alagui A., Polymer, 2003, 44 (26), 7939-7952).
• Chitin is present in co-products of crustaceans and insects in the form of chitin / protein / mineral complexes. It is usually extracted in two stages of "chemical extraction":
• a third optional bleaching step for example using sodium hypochlorite, is often used to remove residual pigments. Washing operations, usually with water, are necessary between these different stages.
• Chitin can then be easily deacetylated, for example using sodium hydroxide, to yield chitosan, also called “chitosan”.
• Chitin is traditionally extracted for a wide range of applications: medical, pharmaceutical, dietary, food, technical (filtration and water depollution), etc.
• chitin, chitosan and their derivatives, especially their oligomers are biocompatible, biodegradable and non-toxic.
• the type of application depends on the physicochemical characteristics of chitin and its derivatives.
• chitosan can be used in particular to make mulching film, protective gels of the stomach, but also for the encapsulation of active ingredients, the filtration of wastewater, the replacement of cartilage, and the regeneration of tissues. ...
• chitin is extracted by lactic fermentation in the presence of bacterial strains for 5 days at 30 ° C.
• the acidification of the medium due to the production of lactic acid by the bacteria, leads to a partial demineralization while the bacteria ensure the deproteinization.
• the pH decreases to 3.5 after 7 days of fermentation.
• the extracted chitin still contains 13% protein and 14% minerals.
• a more pure chitin can then be obtained by performing additional chemical treatments. This type of method does not therefore make it possible to directly obtain a chitin of good quality, limiting its applications.
• Another microbial fermentation study was conducted to extract chitin from shells of red crabs by co-fermenting shells in the presence of two bacteria: on the one hand Lactobacillus paracasei tolerans KCTC-3074, which is an acid-producing bacterium lactic acid, and on the other hand Serratia marcescens FS-3, which is a bacterium producing extracellular proteases (Jung W., Jo G., Kuk J., Kim K. and Park R., Appl Microbiol Biotechnol., 2006 , 71, 234-237).
• Co-fermentation was maintained for 7 days at 30 ° C and resulted in a demineralization percentage of 97.2% and a deproteinization percentage of only 52.6%.
• the chitin obtained was not characterized in this study but the low rate of deproteinization is limiting as to its use.
• Another microbial fermentation study was conducted by the same team, with a bacterial strain producing proteases to deproteinize and demineralize marine co-products (Jo G., Jung W., Kuk J., Oh K., Kim Y. and Park R., Carbohydrate Polymers, 2008, 74, 504-508).
• a fermentation test was conducted for 7 days at 30 ° C., in the presence of 10% of bacterial strain, and led to a deproteinization and demineralization rate of 84% and 47%, respectively.
• Demineralization is like previously due to the decrease in pH over time (pH 5.6 after 7 days of fermentation), related to acid production by bacteria.
• the low degree of purity of the chitins obtained is limiting as to its applications and this method, like the previous one, has the disadvantage of requiring a very long reaction time.
• the best yields of two-step demineralization and deproteinization were obtained by Waldeck (Waldeck J., Daum G., Bisping B. and Meinhardt F., Appl., Microbiol., 2006, 72 (12)). 7879-7885).
• the reaction time is relatively long.
• the extraction of chitin by fermentation therefore leads to a chitin with a higher level of residual proteins than in the case of chemical extraction and additional treatments are often necessary to improve the demineralization.
• the reaction times are much longer than by the chemical route.
• the extraction of chitin by biological means can also be done by enzymatic extraction.
• a chitin extraction method comprising the removal of proteins by the enzymatic activity of fish viscera has been proposed in International Patent Application WO 86/06082.
• the process described in this patent application comprises the extraction of shrimp shell chitin by demineralization with an acid followed by deproteinization using fish viscera, optionally pre-ensiled at pH 1.2. 2.5.
• the raw material i.e. shrimp shells
• the raw material is first ensiled in a solution of sulfuric acid.
• Silage preserves the raw material before use and allows its demineralization.
• the pre-ensiled carcasses are brought into contact with the fish viscera for deproteinization.
• the characteristics of the chitin obtained by this two-step process are not specified.
• Enzymatic extraction can also be done using a purified enzyme, most often a proteolytic enzyme. This is the case, for example, in the study conducted by N. Winned with the use of chymotrypsin or papain to extract chitin from shrimp shells (Gagné N. "Production of chitin and chitosan from crustacean waste and their use of food processing aids", 1993, McGill University - Montreal, thesis manuscript). After a conventional chemical demineralization step, the proteins present are hydrolysed by the enzymes. The optimal conditions for deproteinization in particular use a pH of 8.0-8.7 for chymotrypsin and papain. Under the conditions used, the residual protein level is very low (1.3% and 2.8% for chymotrypsin and papain respectively).
• a method of producing chitin using an enzymatic hydrolysis step has been patented (CN1715255).
• This process offers a global approach to the exploitation of the raw material since compounds other than chitin are also extracted from shrimp shells.
• this process comprises an enzymatic hydrolysis step followed by extraction with a solvent.
• the solid part obtained is then brought into the presence of hydrochloric acid to ensure the demineralization and finish extracting the chitin.
• All presently described chitin enzymatic extraction methods use an independent conventional chemical demineralization step, before or after the enzymatic hydrolysis step.
• the Applicant has conducted research to achieve a total recovery of the parts of the animal biomass containing chitin, including a total valuation of marine byproducts, especially shells of shellfish and entomological biomass, especially shells insects.
• coextraction methods have been studied in view of their advantages over targeted extractions.
• the subject of the invention is therefore a process for the enzymatic extraction of chitin, characterized in that the said process is carried out in a single step, hereinafter referred to as the "single step", in which the chitin is obtained by enzymatic hydrolysis of biomass.
• animal comprising chitin, said enzymatic hydrolysis using an active enzyme in an acidic medium.
• said single step is an enzymatic hydrolysis whose function is to simultaneously deproteinize and demineralize marine by-products.
• said acid-active enzyme is a protease with a broad spectrum of activity in an acidic medium, preferably pepsin or a stable acidic protease.
• the enzyme concentration used for the hydrolysis is from 0.1 to 75%, preferably from 5 to 30%, more preferably from approximately 23 to approximately 27% by weight relative to the mass of estimated protein in the raw material.
• the acidic medium is obtained by the presence of an acid, preferably a food acid, more preferably phosphoric acid or formic acid.
• said animal biomass comprising chitin comprises marine by-products, preferably marine by-products derived from crustaceans, preferably shrimps, crabs or krill, or cephalopods, preferably squid. or cuttlefish.
• said animal biomass comprising chitin comprises insect by-products, preferably insect by-products derived from coleopterans or hymenoptera.
• said method further comprises operations for washing, drying and / or grinding the raw material, preferably washing with water, cold drying and / or grinding operations leading to fragments smaller than 1 mm in size.
• said method also further comprises operations for treating the reaction medium at the end of the enzymatic hydrolysis, said operations comprising solid and liquid phase separation, rinsing and / or drying operations. the insoluble part, preferably filtration, rinsing with water and / or drying in an oven.
• the subject of the invention is also a process for optimizing said enzymatic extraction process of chitin, characterized in that said optimization method comprises at least one of the following steps: a) choice of the pH of the acidic medium in the range enz ⁇ pH 0-2, preferably pH enz ⁇ 0-l, 5, preferably pH enz ⁇ 0-l, wherein enz pH is the pH at which the enzyme exhibits maximum activity, b) selecting the temperature of the acid medium in the range T in ⁇ 0-20 ° C, preferably T enz ⁇ 0-15 ° C, preferably T enz ⁇ 0-10 ° C, where T enz is the temperature at which the enzyme exhibits maximum activity, c) determination of the mineral and protein content of the raw material, d) calculation of the acid concentration to be used in the reaction medium, as a function of the mineral content of the raw material, being understood that according to a preferred embodiment, the pH is chosen so that the reaction medium is maintained throughout the enzymatic hydrolysis at
• the invention further relates to the chitin obtainable by the method of the invention.
• the subject of the invention is also chitosan capable of being obtained by deacetylation of the chitin according to the invention.
• the subject of the invention is also a composition comprising chitin according to the invention and / or chitosan according to the invention.
• the subject of the invention is also a pharmaceutical composition comprising chitin according to the invention and / or chitosan according to the invention, a cosmetic composition comprising chitin according to the invention and / or chitosan according to the invention, a medical device comprising chitin according to the invention and / or chitosan according to the invention
• the invention also relates to a food product, a nutraceutical composition, a dietary composition, a food supplement or a functional food comprising chitin according to the invention and / or chitosan according to the invention.
• the subject of the invention is also a composition comprising chitin according to the invention and / or chitosan according to the invention for its use in the treatment, filtration and / or depollution of water.
• the subject of the invention is also a texturing agent comprising the chitin according to the invention and / or the chitosan according to the invention.
• Chrosin refers to polysaccharides of N-acetylglucosamines and glucosamines
• Chitosan refers to products of deacetylation of chitin.
• the border between chitosan and chitin corresponds to a degree of acetylation of 50%: below, the compound is called chitosan, beyond, chitin.
• chitosan refers to products of deacetylation of chitin.
• the border between chitosan and chitin corresponds to a degree of acetylation of 50%: below, the compound is called chitosan, beyond, chitin.
• chitosan refers to all organic matter of animal origin.
• Marine co-products refers to parts not used by the agri-food industry in marine products, including shells and heads of crustaceans.
• Entomological co-products or “insect co-products” refers to the parts not used by the agri-food industry in entomological products, including the shells and heads of insects.
• Degree of polymerization refers to the length of a polymer chain, especially chitin.
• the degree of polymerization corresponds to the number of constituent monomer units of the polymer chain.
• Crystalstallinity index refers to the proportion of matter in the crystalline state.
• Demineralization refers to a process of removing minerals.
• Deproteinization refers to a process of protein removal.
• Depolymerization refers to the reduction of the length of the polymer chain of chitin.
• Deacetylation refers to the elimination of acetyl groups and corresponds to the passage of chitin to chitosan.
• Measurement content refers to the mass percentage of water contained in a sample.
• Protein content refers to the mass percentage of protein contained in a sample.
• Mineral content refers to the mass percentage of minerals in a sample.
• Chitin content refers to the mass percentage of chitin contained in a sample.
• the present invention relates to a process for the single-step enzymatic extraction of chitin from a raw material derived from animal biomass and comprising chitin, preferably a raw material consisting of marine by-products and / or entomological co-products, using an active enzyme in an acid medium, preferably a protease, the acid used being preferably a food acid, this process also making it possible to extract soluble compounds such as lipids, pigments, sugars, salts minerals, amino acids or peptides.
• the two key steps of the traditional chitin extraction process namely, acid demineralization and deproteinization in an alkaline medium, are fused in a single step.
• This one-step fusion is permitted through the use of an enzyme whose optimal pH of activity is acidic: the enzyme ensures the deproteinization, while the acidic pH allows for simultaneous demineralization.
• the method of the invention comprises only a single key step, it has the advantage of reducing the losses of matter related to rinsing between the two steps of the traditional process. This process also makes it possible to reduce the consumption of reagents and solvents and to limit the production of polluted effluents. This process is both inexpensive and environmentally friendly.
• the conditions used in the process of the present invention are such that the biological activity of the chitins, as well as their native structure, are better preserved than in the extraction methods existing to date.
• the method of the present invention has the advantage that it allows the destructuration of the crustacean and / or insect co-product matrix by separation of chitin, proteins and minerals, these three major compounds being initially strongly bound .
• the method according to the invention comprises an enzymatic hydrolysis step in an acidic medium simultaneously ensuring demineralization and deproteinization.
• the minerals and proteins are separated from the solid phase and entrained in the liquid phase.
• the method of the present invention may comprise, in addition to the enzymatic acid hydrolysis step, preparation and treatment operations:
• raw material is intended to mean animal biomass comprising chitin used for extracting chitin, preferably marine by-products used to extract chitin and / or entomological co-products. used to extract chitin.
• the raw material used in the process of the present invention comprises marine by-products, preferably crustaceans, shrimps, crabs, krill, more preferentially shells and crustacean heads; according to a mode of Particular embodiment of the invention, the raw material is derived from cephalopods, preferably squid or cuttlefish.
• the raw material used in the process of the present invention comprises entomological co-products, preferably beetles such as the beetle Tenebrio molitor, hymenopterans such as the fly Hermetia illucens, more preferentially carapaces and caries. insect heads.
• beetles such as the beetle Tenebrio molitor
• hymenopterans such as the fly Hermetia illucens, more preferentially carapaces and caries. insect heads.
• the preparation of the raw material comprises cleaning, drying and / or grinding operations.
• the raw material is cleaned with water.
• the raw material is dried for 1h to 36h preferably for about 18h, preferably in ventilated air, preferably at a temperature of 5 to 35 ° C, more preferably about 12 ° C.
• the raw material is milled to obtain fragments with a maximum diameter of about 10 mm, preferably less than about 1 mm in diameter.
• the raw material preferably prepared by cleaning, drying and grinding, is stored before extraction at a temperature between -30 and -10 ° C, preferably at -20 ° C, preferably by limiting the presence of oxygen.
• Reaction medium preferably a temperature between -30 and -10 ° C, preferably at -20 ° C, preferably by limiting the presence of oxygen.
• reaction medium is intended to mean the medium in which the enzymatic hydrolysis reaction in acidic medium takes place.
• the preparation of the reaction medium must take into account the activity conditions of the enzyme used, such as temperature, solvent and pH. The choice of these conditions makes it possible to optimize the reaction time and the yields.
• the reaction medium is maintained during the enzymatic hydrolysis at a temperature of between 2 and 80 ° C., preferably between 35 and 45 ° C., more preferably between approximately 37 and approximately 40 ° C.
• the temperature of the reaction medium is adapted to the enzyme used so that said enzyme has a quasi-optimal activity for the duration of the enzymatic hydrolysis.
• the reaction medium is maintained during the enzymatic hydrolysis at a temperature in the range T in ⁇ 0 to 20 ° C, preferably T in ⁇ 0 to 15 ° C, preferably T enz ⁇ 0 to 10 ° C, where T enz is the temperature at which the enzyme exhibits maximum activity.
• T enz is the temperature at which the enzyme exhibits maximum activity.
• the chosen temperature must not cause the degradation of the enzyme or inhibit its action.
• the temperature of the reaction medium is lower than T enz so as to limit the energy consumption.
• the pH of the reaction medium is from 0.5 to 6.5, preferably from 1.8 to 3.8, more preferably from about 1.9 to about 2.1.
• the pH of the reaction medium is preferably from about 1.9 to about 2.1.
• the pH of the reaction medium is acidic and its value is adapted to the enzyme used so that said enzyme has an optimal activity.
• the pH of the reaction medium is within the pH range enz ⁇ 2, preferably pH enz ⁇ l, 5, preferably pH enz ⁇ l, enz where pH is the pH at which the enzyme has a maximum activity.
• the chosen pH must be acidic so that the extraction yield of chitin is sufficient.
• the reaction medium is ready to be used when the temperature and pH conditions chosen for the enzymatic hydrolysis reaction are stabilized.
• the reaction medium comprises at least one acid.
• the reaction medium further comprises a solvent such as water or an aqueous solution.
• the acid used is preferably a food acid, preferably phosphoric acid or formic acid.
• the products extracted by the method of the present invention have the advantage of being used more easily in the agri-food or cosmetics fields.
• the concentration of acid in the reaction medium is from 0.1 to 6 mol.L 1 , preferably from 0.8 to 2.8 mol.L 1 , more preferably from 0.9 to 1 mol. .L "1 .
• the acid concentration in the reaction medium is adapted to the mineral content of the raw material used so that the pH of the reaction medium is acidic and remains constant throughout the enzymatic hydrolysis.
• the enzyme used in the present invention is an acid-active enzyme, preferably a protease with a broad spectrum of activity in an acid medium, preferably pepsin or a stable acid protease.
• the enzyme concentration in the reaction medium is adapted to the protein content of the raw material used. According to one embodiment, the enzyme concentration is from 0.1 to 75%, preferably from 5 to 30%, more preferably from about 23 to about 27% by weight relative to the weight of proteins estimated in the material. first.
• the raw material is mixed with the reaction medium and the resulting mixture is optionally homogenized by stirring for 0 to 30 minutes, preferably for 3 to 10 minutes, more preferably for about 5 minutes.
• the ratio between the weight of raw material prepared and the volume of reaction medium is from 1:60 to 2: 1, preferably from 1: 7 to 1: 3, more preferably equal to 1: 5.
• the ratio between the weight of raw material prepared and the volume of reaction medium is adapted to the size of the prepared raw material fragments.
• the container used to carry out the enzymatic hydrolysis has a volume adapted to prevent overflow of the foam that can form.
• the risk of foam production increases as the temperature of the acid before mixing increases.
• the temperature of the reaction medium before the addition of the raw material is 5 to 65 ° C, preferably 20 to 30 ° C, more preferably about 25 ° C.
• the temperature of the reaction medium is chosen to be less than the temperature at which the enzymatic hydrolysis reaction must be conducted and this in order to limit the formation of foam during the addition of the raw material.
• the enzyme is added directly into the homogenized reaction medium optionally containing the raw material.
• the enzyme is solubilized in water, or in a solution, preferably an aqueous solution, and is then added to the homogenized reaction medium containing the raw material.
• the enzymatic hydrolysis reaction is carried out with stirring so as to optimize the contact between the raw material and the enzyme.
• the initial pH and temperature conditions of the reaction medium are kept constant throughout the duration of the enzymatic hydrolysis reaction.
• the initial pH and / or temperature conditions of the reaction medium are not kept constant during the duration of the enzymatic hydrolysis reaction.
• the enzymatic hydrolysis reaction is carried out in a reactor equipped with a device for regulating the temperature.
• said reactor is a jacketed reactor in which circulates a heat transfer fluid, the temperature of said fluid can be controlled.
• said reactor is provided with a heating resistor, the temperature of said resistance being controllable.
• the pH is stable throughout the duration of the enzymatic hydrolysis.
• the pH is adjusted, during the enzymatic hydrolysis reaction, to the pKa value between the acid used and the calcium carbonate by addition of a concentrated solution of acid, acid being the same as that used in the reaction medium.
• the duration of the enzymatic hydrolysis is 30 minutes to 24 hours, preferably 1 to 12 hours, preferably 3 hours to 8 hours, more preferably about 6 hours. According to one embodiment, the duration of the enzymatic hydrolysis is adapted to the activity of the enzyme used to carry out the enzymatic hydrolysis reaction, the acid employed and the raw material.
• the duration of the enzymatic hydrolysis is adapted according to the characteristics desired for the final products, such as the degree of purity, the degree of polymerization and the degree of acetylation.
• the enzymatic reaction produces a reaction juice comprising soluble and insoluble parts. Separation of products
• the soluble and insoluble portions of the reaction juice are separated by any suitable means known to those skilled in the art.
• the soluble and insoluble parts are separated by filtration.
• the filtration is carried out by a filtration system preserving the integrity of the structures of the extracted compounds.
• the filtration is carried out by a filtration system on a membrane press.
• the filtration is carried out on filter cloth, preferably on cloth to be blotted.
• the soluble and insoluble portions are separated by centrifugation.
• the insoluble portion of the reaction juice contains very predominantly chitin and the soluble part contains various compounds such as lipids, pigments, sugars, mineral salts, amino acids or peptides.
• the insoluble portion is rinsed with a solvent.
• the solvent is water or an aqueous solution. This embodiment is preferred in the case where chitins are then used for food applications.
• the insoluble portion is first rinsed with water or an aqueous solution and then with a bleaching agent such as hydrogen peroxide, sodium hypochlorite or potassium persulfate and is rinsed again with water or an aqueous solution.
• a bleaching agent such as hydrogen peroxide, sodium hypochlorite or potassium persulfate
• Chitins are very hygroscopic products whose biological activity can be degraded by an increase in temperature.
• the insoluble portion filtered and rinsed is then dried for 8 to 16 hours, preferably for about 12 hours, in an oven whose temperature is preferably less than 100 ° C, preferably between 50 and 95 ° C, more preferably about 90 ° C.
• the filtered insoluble part is neutralized with sodium hydroxide.
• the insoluble portion is lyophilized.
• the dried and / or lyophilized insoluble part is packaged in containers such as glass or plastic bottles or vacuum bags and preferably stored at room temperature in a dry place.
• the insoluble part (chitin) is stored at a temperature below room temperature, preferably at a temperature of -30 to 0 ° C, more preferably from -20 to -10 ° C, more preferably at about -20 ° C.
• the soluble part is centrifuged.
• the soluble part is dialyzed and ultrafiltered.
• one embodiment of the compounds of the neutralized soluble portion are extracted with organic solvents. The organic or aqueous solvents are then evaporated to obtain the compounds of interest.
• the technique for treating the soluble phase depends on the nature of the compounds to be valorized.
• reaction medium enzyme concentration, pH and temperature
• control of the reaction medium makes it possible to control the yield and the biochemical and physicochemical characteristics of the chitins obtained. Theoretically, extending the hydrolysis time tends to reduce the degree of polymerization.
• the insoluble portion contains mainly chitin as well as proteins and residual minerals that have not been removed during the enzymatic hydrolysis reaction.
• the degree of purity of chitin (purity D) is estimated gravimetrically by measuring the mass of the insoluble sample before and after treatment of the insoluble parts with sodium hydroxide at 1.25 mol.L -1. 90 ° C. for 1 h As mentioned above, this treatment makes it possible to eliminate the residual proteins and minerals
• the estimated degree of purity is calculated from the following formula:
• the estimated degree of purity of chitin (D ° purity) is greater than 75%, preferably greater than 80%, more preferably greater than 85%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 80%. 90%.
• the mass content of residual proteins in the dried insoluble part is less than 20%, preferably less than 15%, preferably less than 10%, more preferably less than 5%.
• the mass proportion of proteins eliminated by the process of the present invention is greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%.
• the mass content of residual minerals in the dried insoluble part is less than 5%, preferably less than 3%, more preferably less than 1%. According to one embodiment, the mass proportion of minerals removed by the process of the present invention is greater than 95%, preferably greater than 97%, more preferably greater than 99%.
• an additional bleaching operation is carried out on the insoluble part, making it possible to remove pigments as well as a part of the residual proteins and minerals.
• an additional deacetylation operation is carried out on the insoluble part, making it possible to produce chitosan and to eliminate a part of the residual proteins.
• the chitins extracted by the method of the present invention can be used as is or converted into chitosan, chitin oligomers, chitosan oligomers or N-acetylated glucosamines or not.
• the process of the present invention provides a wide range of chitin quality with respect to the degree of purity and polymerization.
• the other characteristics depend on the nature of the raw material and not on the characteristics of the process according to the invention.
• the chitins extracted by the process of the present invention have a shape close to the natural form of chitin.
• the chitins extracted by the method of the present invention are not or slightly denatured with respect to the natural chitin.
• the estimated degree of purity of the chitins extracted by the process of the present invention is greater than 85%, preferably greater than 90%, more preferably greater than 95%.
• the degree of purity of the chitins obtained by the method of the present invention is sufficient to be able to convert them in the form of chitosan, chitin oligomers, chitosan oligomers or glucosamines.
• the degree of polymerization of chitin is estimated by calculation from the average molecular weight of said chitin.
• the average molecular weight of the chitins is estimated by calculation from the intrinsic viscosity. The intrinsic viscosity can be determined by the method described by Poirier et al. (Pear, M. and Chrlet, G., Carbohydrate Polymers, 2002, 50, 363-370).
• the degree of polymerization of the chitins extracted by the method of the present invention is from 1.10 3 to 1 ⁇ 10 9 , preferably from 1 ⁇ 10 4 to 1 ⁇ 10 7 , more preferably from 1 ⁇ 10 5 to 1 ⁇ 10 6 .
• the degree of acetylation of the chitins extracted by the process of the present invention is from 80% to 100%, preferably from 90% to 98%, more preferably from 95% to 97%.
• the crystallinity index of the chitins extracted by the process of the present invention is from 10% to 70%, preferably from 20% to 50%, more preferably from 30% to 40%.
• the soluble products extracted by the method of the present invention may be peptides, pigments, sugars and mineral salts.
• the use of food acid in this process allows these compounds to be exploited in the agri-food, dietary and nutraceutical fields.
• the present invention therefore has the advantage of limiting the amount of waste since all products other than chitin extracted by the process of the invention can also be used and recovered.
• the pigments extracted by the process of the present invention are astaxanthin. DESCRIPTIONS OF FIGURES
• Figure 1 shows a schematic of the chitin extraction process according to the invention.
• the raw material used is the raw Penaeus vannamei shrimp exoskeleton.
• the raw material is dried at 12 ° C in ventilated air and ground to give fragments smaller than 1 mm.
• the prepared raw material is stored at -20 ° C under vacuum.
• the reagent used to maintain the acidic pH is phosphoric acid.
• the acid concentration is calculated based on the initial mineral content in the prepared raw material. For an initial mineral content of 25% by weight relative to the dry weight of raw material, a solution of phosphoric acid at 0.94 mol.L -1 is used in order to maintain the pH of the reaction medium around 2.
• the acid protease employed is pepsin (CAS 9001-75-6, supplier: Sigma, activity: 8112 U / mg). It is stored as a powder at + 4 ° C. It is solubilized in distilled water 15 min before introduction into the reactive medium.
• the amount of enzyme added in this example corresponds to 25% of the protein mass estimated in the initial raw material. Thus, for a sample of 5 g of raw material having a moisture content of about 15% and a protein content close to 40%, this corresponds to about 8.5% of enzyme relative to the raw material, ie a quantity of pepsin of 0.43 g. Protocol
• Pepsin (0.43 g), previously solubilized in 1 ml of water, is added to the reaction medium. The mixture is heated at 40 ° C. on a heating plate and then incubated in an oven maintained at 40 ° C. ⁇ 1 ° C.
• the moisture content of the sample is measured gravimetrically by measuring the mass of the sample before and after overnight at 105 ° C.
• the mineral content is determined gravimetrically by measuring the mass of the sample before and after incineration at 600 ° C for 6 hours.
• the protein content is estimated by gas chromatography by assaying the total amino acids. It can also be measured by a colorimetric assay (Lowry, BSA, Bradford or Coomassie blue) or by Kjeldahl assay.
• the chitin content can be measured gravimetrically by measuring the mass of the sample before and after the following treatments:
• treatment is limited to NaOH treatment
• the molecular weight of chitin is estimated by calculation from intrinsic viscosity.
• the intrinsic viscosity can be determined by the method described by Poirier et al., which is based on Mark-Houvink's law (Poirier, M. and Charlet, G., Carbohydrate Polymers, 2002, 50, 363-370).
• the intrinsic viscosity was determined by measuring the reduced viscosity from solutions of different chitin concentrations in N, N-dimethylacetamide containing 5% LiCl.
• the apparatus used is a capillary viscometer called Ubbelhode.
• the constant K of the viscometer is 0.3 cSt / s.
• the measurement volume is 15mL.
• the degree of polymerization is calculated from the molecular weight of chitin.
• the degree of acetylation is estimated by NMR of the proton liquid, according to the method described by Einbu A., Varum K., 2008.
• the chitin (20 mg) is solubilized in ImL of DCl (7.6N in D 2 0, Euriso -top) with magnetic stirring at room temperature for 5 hours.
• the 1H NMR analysis is carried out at 300 ° K using a Brucker ALS300 spectrometer (300MHz, TMSP reference O.OOppm).
• the degree of acetylation is then calculated from the intensity of the characteristic proton NMR signals, according to the formula given by Einbu et al.
• the crystallinity index is determined by X-ray diffraction.
• the diffractometer used is a D8 Discover from Brucker-axs (Karlsruhe, Germany).
• the method of calculating the crystallinity index is based on the ratio between the areas of the crystalline zones on the total area (Osario-Madrazo A., David L., Trombotto S., Lucas JM, Peniche-Covas C. and Domard A., Carbohydrate Polymers, 2011, 83, 1730-1739)
• the composition of the dry extract obtained can be compared with that of the totally dried raw material or with the prepared raw material used in this example (Table 1): moisture minerals protein chitin lipids sugars raw material
• the quantities of minerals and proteins eliminated by the process are respectively 98.5% and 91.7%.
• the residual contents of minerals and proteins are those present in the final product, without bleaching step.
• the application of a bleaching agent or a washing with sodium hydroxide improves the degree of purity.
• the degree of acetylation measured by NMR is in this example of the order of 95%.
• the molecular weight of this sample is of the order of 10 5 to 10 6 g / mol and the crystallinity index of 35%. These characteristics are close to those of native chitin.
• the performance of this example can be improved by increasing the amount of pepsin used.
• the experiment was carried out with a pepsin concentration of 41% relative to the amount of protein present in the raw material, instead of 25% previously.
• the degree of purity in chitin increases (96.78% instead of 88.42%) because the deproteinization is improved (92.00% of proteins eliminated) as well as the demineralization (99.23% of eliminated minerals).

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