EP2714918A1

EP2714918A1 - Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium

Info

Publication number: EP2714918A1
Application number: EP12731067.0A
Authority: EP
Inventors: Karine LE ROUX; Jean-Pascal BERGÉ; Régis BARON; Éric LEROY; Abdellah Arhaliass
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut Francais de Recherche pour lExploitation de la Mer (IFREMER); Universite de Nantes
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut Francais de Recherche pour lExploitation de la Mer (IFREMER); Universite de Nantes
Priority date: 2011-05-26
Filing date: 2012-05-25
Publication date: 2014-04-09
Also published as: FR2975706A1; JP2014522240A; ECSP13013114A; WO2012168618A1; FR2975706B1; US20140100361A1

Abstract

The invention relates to a method for the enzymatic extraction of chitin, characterised in that said method is carried out in a single step in which the chitin is obtained by the enzymatic hydrolysis of a raw material consisting of animal biomass including chitin, said enzymatic hydrolysis using an active enzyme in an acid medium. The invention also relates to a method for optimising said method for the enzymatic extraction of chitin. The invention also relates to the chitin that can be obtained by said enzymatic extraction method.

Description

EXTRACTION OF CHITINES IN ONE STEP BY ENZYMATIC HYDROLYSIS IN ACID

FIELD OF THE INVENTION

The present invention relates to the field of valorization of biomass, preferably animal biomass, more preferably the marine biomass and / or entomological. In particular, 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.

STATE OF THE ART

The production and consumption of marine products, and in particular that of crustaceans, especially shrimp, is increasing every year. By-products (heads and shells) generally account for more than 50% of the fresh weight of crustaceans. Their use is therefore an important issue given the volumes concerned but also their slow natural biodegradability. Chitin is the main byproduct of these co-products.

In addition, entomophagy is a common food practice in some countries and is becoming increasingly common worldwide. Indeed, insects are an interesting food resource because of their nutritional qualities. In addition, the production of insects offers a very interesting ecological alternative compared to the production of other proteins of animal origin. Among the byproducts derived from the production of insect proteins are 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. In addition to chitin, 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). Whether for crustaceans or for insects, 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":

demineralization by acid hydrolysis, to remove minerals; then

deproteinization by basic hydrolysis, to eliminate proteins.

The extraction of chitin from marine by-products is currently carried out on an industrial scale by "chemical extraction". The extraction of chitin from insects is not yet very developed but has already been studied, mainly by chemical means (cicada chitin: Sajomsang W. and Gonil P., Mat Science Engineering C, 2010, 30 (3), 357-363, silkworm chrysalis chitin: Paulino A., Simionato J., Garcia J. and Nozaki J., Carbohydrate Polymers, 2006, 64, 98-103; bumblebee: Majtan J., Bilikova K., Markovic O., Grog J., Kogan G. and Simuth J., Int. J. Biol. Macromol., 2007, 40, 237-241)

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. Indeed, 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. In particular, 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. ...

The industrial extraction of chitin from marine co-products is mainly implemented in emerging countries. Traditional chemical extraction uses large amounts of reagents (mainly hydrochloric acid, sodium hydroxide, and bleaches) that are harmful to handlers, equipment, and the environment. In addition, the basic deproteinization step is usually hot and therefore requires a significant energy intake. In addition, the washing steps generate very large volumes of polluted effluents whose recycling is technically difficult and expensive.

One of the problems with current extraction processes is the possibility that chitin is denatured during the process (Crini G., Badot P. and Guibal E., Chitin and Chitosan) Polymer V Application, 2009, Presses University of Franche-Comté). Studies have shown that the extraction of chitin can be done by biological processes, including enzymatic extraction or microbiological fermentation, particularly for the deproteinization step.

Among the researches on the fermentation route, those carried out by Beaney use as a study matrix the exoskeleton of the Nephrops norvegius shrimp (Beaney P., Lizardi-Mendoza J. and Healy M., J. Chem., Tech Biotech. 2005, 80, 145-150). In this study, 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. In this study, the pH decreases to 3.5 after 7 days of fermentation. However, under these conditions, 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). After a 6-day fermentation of 42 to 55 ° C, followed by a lactic acid treatment of 3h, the residual protein content is less than 10% and the demineralization rate is equal to 98.8%. As in the study by Jo et al., 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. In addition, 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. In particular, 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, is first ensiled in a solution of sulfuric acid. Silage preserves the raw material before use and allows its demineralization. In a second step, 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).

The same type of process was used by Synowiecki and Al Khateeb to carry out the enzymatic digestion of shrimp shells, previously demineralized with hydrochloric acid, using alkalase, at 55 ° C. and pH 8.5 ( Synowiecki J. and Al Khateeb, Food Chemistry, 2000, 68, 147-152)

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. In particular, 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. Thus, even if the deproteinization step is carried out by a biological process, there is always a chemical step requiring washing and producing polluted effluents and may affect the properties of the extracted chitin. Current processes are therefore unsatisfactory and there is a need for chitin extraction methods that are simple, fast, efficient, inexpensive and more environmentally friendly. These methods must make it possible to produce chitines whose purity is compatible with a use in agri-food, dietary or cosmetic. In addition, chitin produced must meet specifications necessary to be transformed into chitosan, oligo-chitosan or glucosamines, etc. In particular, the degree of polymerization of chitin must be sufficiently high and it must not be denatured during the process.

In addition, other compounds are potentially valuable from co-products of crustaceans and insects, including nutraceutical, dietary or cosmetic. Indeed, these marine by-products and insects contain soluble compounds such as lipids, pigments, sugars, mineral salts, amino acids or peptides. Targeted extractions of these soluble compounds have been developed, such as, for example, the extraction of pigments and in particular astaxanthin which is used in food processing (US Pat. No. 7,241,463). However, these methods targeted to the extraction of soluble compounds require additional steps to lead to the extraction of chitin.

In the present methods of extracting chitin, the latter is obtained in solid form and the liquid extraction phases, containing the potentially interesting soluble compounds, are not valued. This lack of recovery can be explained in particular by the low quality of the soluble compounds present in the liquid phases. In fact, in these chitin extraction processes, the soluble compounds are often degraded because of the relatively drastic conditions used.

There is therefore a need for purification of chitin by a method both more respectful of its initial structure and likely to be associated with the co-extraction of soluble products.

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. In particular, coextraction methods have been studied in view of their advantages over targeted extractions. ABSTRACT

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.

According to one embodiment, said single step is an enzymatic hydrolysis whose function is to simultaneously deproteinize and demineralize marine by-products.

According to one embodiment, said acid-active enzyme is a protease with a broad spectrum of activity in an acidic medium, preferably pepsin or a stable acidic protease.

According to one embodiment, 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.

According to one embodiment, the acidic medium is obtained by the presence of an acid, preferably a food acid, more preferably phosphoric acid or formic acid.

According to one embodiment, 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.

According to one embodiment, said animal biomass comprising chitin comprises insect by-products, preferably insect by-products derived from coleopterans or hymenoptera.

According to one embodiment, 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. According to one embodiment, 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 pH chosen in step a), e) calculation of the proportion of enzyme to be used in relation to the protein content of the raw material, f) determination of the duration of the reaction to obtain the desired chitin or chitin derivatives.

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.

DEFINITIONS

In the present invention, the following terms are defined as follows:

"Chitin" 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. "Animal biomass" 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.

"Crystallinity 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.

"Moisture 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.

"About" in front of a number means plus or minus 10% of the face value of that number. Unless otherwise stated, percentages are percentages by mass.

DETAILED DESCRIPTION

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. In the present invention, 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.

Since 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.

According to one embodiment, the method of the present invention may comprise, in addition to the enzymatic acid hydrolysis step, preparation and treatment operations:

- preparation of the raw material,

preparation of a reaction medium according to the optimal conditions of enzymatic activity and comprising at least one acid,

mixture of the raw material prepared in the reaction medium, homogenization and addition of the enzyme,

enzymatic hydrolysis step at controlled temperature, pH and stirring: simultaneous deproteinization and demineralization reactions for an optimized duration,

separation of the soluble and insoluble parts of the "reaction juice",

washing of the "insoluble" part, drying and conditioning,

- optionally, characterization of the extracted products.

Raw material

For the purposes of the present invention, the term "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.

According to one embodiment, 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.

According to one embodiment, 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.

The operation of preparation of the raw material must allow to preserve its qualities while meeting the requirements of the process.

According to one embodiment of the present invention, the preparation of the raw material comprises cleaning, drying and / or grinding operations.

According to one embodiment, the raw material is cleaned with water.

According to one embodiment, 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. According to one embodiment, the raw material is milled to obtain fragments with a maximum diameter of about 10 mm, preferably less than about 1 mm in diameter.

According to one embodiment, 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

For the purpose of the present invention, the term "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. According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, 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. The chosen temperature must not cause the degradation of the enzyme or inhibit its action. Advantageously, the temperature of the reaction medium is lower than T _enz so as to limit the energy consumption.

According to one embodiment, 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. In the case where the enzyme is pepsin, the pH of the reaction medium is preferably from about 1.9 to about 2.1.

According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, the reaction medium is ready to be used when the temperature and pH conditions chosen for the enzymatic hydrolysis reaction are stabilized. According to a first embodiment, the reaction medium comprises at least one acid. According to a second embodiment, the reaction medium further comprises a solvent such as water or an aqueous solution. According to one embodiment of the present invention, the acid used is preferably a food acid, preferably phosphoric acid or formic acid.

When the acid used in the enzymatic hydrolysis step is a food 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.

According to one embodiment, 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 .

According to one embodiment, 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.

Enzyme

According to one embodiment, 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.

According to one embodiment, 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.

Reaction conditions

According to one embodiment, 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. According to one embodiment, 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.

According to one embodiment, 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. In particular, it is taken into account that as the size of the raw material fragments decreases, the solvent absorption increases and therefore it is necessary to increase the volume of reaction medium.

The addition of the raw material to the acid reaction medium can lead to foaming due to the production of carbon dioxide due to the presence of calcium carbonate in the exoskeleton of crustaceans and insects. According to one embodiment, 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. According to one embodiment, 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. In this embodiment, 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.

According to a first embodiment, the enzyme is added directly into the homogenized reaction medium optionally containing the raw material.

According to a second embodiment, 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.

According to one embodiment, the enzymatic hydrolysis reaction is carried out with stirring so as to optimize the contact between the raw material and the enzyme. According to one embodiment, the initial pH and temperature conditions of the reaction medium are kept constant throughout the duration of the enzymatic hydrolysis reaction. According to another embodiment, the initial pH and / or temperature conditions of the reaction medium are not kept constant during the duration of the enzymatic hydrolysis reaction.

According to one embodiment, the enzymatic hydrolysis reaction is carried out in a reactor equipped with a device for regulating the temperature. According to a first embodiment, said reactor is a jacketed reactor in which circulates a heat transfer fluid, the temperature of said fluid can be controlled. According to a second embodiment, said reactor is provided with a heating resistor, the temperature of said resistance being controllable.

According to a first embodiment, the pH is stable throughout the duration of the enzymatic hydrolysis. According to a second embodiment, 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.

According to one embodiment, 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.

According to one embodiment, 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.

According to one embodiment, the enzymatic reaction produces a reaction juice comprising soluble and insoluble parts. Separation of products

According to one embodiment, the soluble and insoluble portions of the reaction juice are separated by any suitable means known to those skilled in the art.

According to a first embodiment, the soluble and insoluble parts are separated by filtration. According to one embodiment, the filtration is carried out by a filtration system preserving the integrity of the structures of the extracted compounds. According to another embodiment, the filtration is carried out by a filtration system on a membrane press. According to another embodiment, the filtration is carried out on filter cloth, preferably on cloth to be blotted. In a second embodiment, the soluble and insoluble portions are separated by centrifugation.

According to one embodiment, 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. According to one embodiment, the insoluble portion is rinsed with a solvent. According to a first embodiment, the solvent is water or an aqueous solution. This embodiment is preferred in the case where chitins are then used for food applications. In a second embodiment, 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. This second embodiment is preferred in the case where a bleaching of chitin is desired. In this embodiment, the bleaching agents used are in accordance with the legislation.

Chitins are very hygroscopic products whose biological activity can be degraded by an increase in temperature.

According to one embodiment, 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.

According to one embodiment, the filtered insoluble part is neutralized with sodium hydroxide. According to one embodiment, the insoluble portion is lyophilized. According to one embodiment, 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. According to a particular embodiment, 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.

According to a first embodiment, the soluble part is centrifuged. According to a second embodiment, the soluble part is dialyzed and ultrafiltered. According to third, 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.

Extraction yields

Control of the reaction medium (enzyme concentration, pH and temperature) as a function of the raw material used 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 mass yield of extraction of insoluble parts (Rdt) depends on the nature of the raw material, the acid and the enzyme used and is calculated from the following formula:

Yield = 100 * (weight in dried insolubles) / (weight of dry raw material) According to one embodiment, the insoluble portion contains mainly chitin as well as proteins and residual minerals that have not been removed during the enzymatic hydrolysis reaction.

The application of the conventional chemical extraction treatment to the insoluble parts obtained by the process of the present invention makes it possible to estimate the level of residual impurities in the insoluble part. Indeed, this treatment eliminates the majority of proteins and residual minerals.

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:

Purity = 100 * [(insoluble part mass after treatment) /

According to one embodiment, 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%.

According to one embodiment, 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%.

According to one embodiment, 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%.

According to one embodiment, 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%.

According to one embodiment, 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.

According to one embodiment, 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. Characteristics of extracted chitin

According to one embodiment, 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 (pattern distribution, α, β and γ form) depend on the nature of the raw material and not on the characteristics of the process according to the invention.

According to one embodiment, the chitins extracted by the process of the present invention have a shape close to the natural form of chitin. In other words, the chitins extracted by the method of the present invention are not or slightly denatured with respect to the natural chitin.

According to one embodiment, 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. According to one embodiment, the degree of polymerization of chitin is estimated by calculation from the average molecular weight of said chitin. According to one embodiment, 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).

According to one embodiment, 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 .

According to one embodiment, 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%.

According to one embodiment, 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%.

Soluble compounds

According to one embodiment, 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.

According to one embodiment, 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. EXAMPLES The invention will be better understood on reading the following example, which non-limitatively illustrates the present invention.

Example 1 Enzymatic hydrolysis by pepsin in the presence of phosphoric acid Equipment

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

5 g of raw material, prepared as described above, are weighed. The composition of the dry extract is determined according to the methods of analysis described below. A solution of phosphoric acid at 0.94 mol.L- ¹ (25 ml) is preheated to 30 ° C. and then added to the raw material.The mixture is stirred for 5 minutes to homogenize. pH meter should be stable and be between 1.9 and 2.1.

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.

After 6 hours of incubation, the mixture is filtered on fabric to be blotted and rinsed thoroughly with distilled water. The retentate is resuspended in distilled water, the mixture is stirred for 10 min before being filtered and rinsed again with water. The solid fraction obtained is transferred to a cup and dried overnight at 90 ° C. in an oven. The mass of dry extract obtained (m = 1.29g) makes it possible to calculate the mass yield of extraction, ie 30.26%.

Analyzes

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:

for the raw material: treatment for 60 minutes with 1N HCl at room temperature, then with 1.25N NaOH at 90 ° C for 120 minutes and finally bleaching with 33% hydrogen peroxide and acetone;

for hydrolysis products, treatment is limited to NaOH treatment

1.25 N at 90 ° C for one hour. 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). Thus, 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 radiation is produced in a copper tube (Cu K i = 1.5405 A) and the beams produced are recorded every 10 min. From the spectra obtained, 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)

Results and discussion

After 6 hours of enzymatic hydrolysis by pepsin in the presence of phosphoric acid, the mass yield of extraction is 30.26 ± 0.32%. 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

totally dry:

% by mass 0% 25% 40% 30% ND ND raw material

prepared: 14.55% 21.25% 34% 25.5% 3.5% 1.2%

% mass

dry extract :

% by mass 0% 0.99 10.98 88.42 ND ND

(+ 0.03%) (+ 1.01%) (+ 1.22%)

for 100g of

material

first dry 0g 0,30g 3,32g 26,76g ND ND

ND: not determined

Table 1

Taking into account the composition of the dry 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 (Table 1) 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. Thus, 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).

Claims

1. Process for the enzymatic extraction of chitin, characterized in that said process is carried out in a single step in which the chitin is obtained by enzymatic hydrolysis of a raw material consisting of animal biomass comprising chitin, said enzymatic hydrolysis an active enzyme in an acid medium.

The method of claim 1, wherein said single step is enzymatic hydrolysis having the function of simultaneously deproteinizing and demineralizing said raw material.

3. A process according to claims 1 to 2 wherein said acid-active enzyme is a broad-spectrum protease of acidic activity, preferably pepsin or a stable acidic protease.

4. Process according to any one of claims 1 to 3, wherein the enzyme concentration used for the hydrolysis is from 0.1 to 75%, preferably from 5 to 30%, more preferably from about 23 to about 27% by mass relative to the mass of proteins estimated in the raw material.

5. Method according to claims 1 to 4, wherein the acidic medium is obtained by the presence of an acid, preferably a food acid, more preferably phosphoric acid or formic acid.

6. Process according to claims 1 to 5, wherein said animal biomass comprising chitin comprises marine by-products, preferably marine coproducts derived from crustaceans, preferably shrimps, crabs or krill, or cephalopods, preferably squid or cuttlefish.

The method according to claims 1 to 6, wherein said animal biomass comprising chitin comprises insect by-products, preferably insect by-products derived from beetles or hymenoptera.

The process according to claims 1 to 7, further comprising washing, drying and / or grinding the raw material, preferably water washing, cold drying and / or grinding operations. .

9. Process according to claims 1 to 8, further comprising processing operations of the reaction medium at the end of the enzymatic hydrolysis, said operations comprising solid and liquid phase separation operations, then rinsing and / or drying operations. the insoluble part.

10. Process for optimizing the enzymatic extraction process of chitin described in any one of claims 1 to 9, characterized in that said optimization process comprises at least one of the following steps: a) choice of the pH of the acidic medium in the pH range _enz ± 2, preferably pH _enz ± l, 5, preferably pH _enz ± l, _enz where pH is the pH at which the enzyme exhibits maximum activity, b) selecting the temperature of the acid medium in the range T _in z ± 20 ^o C, preferably T _in z ± 15 ⁰ C, preferably T _in z ± 10 ^o C, where T _enz is the temperature at which the enzyme has a maximum of 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, depending on the mineral content of the raw material, that the pH is maintained while during the enzymatic hydrolysis at the pH chosen in step a), e) calculation of the proportion of enzyme to be used in relation to the protein content of the raw material, f) determining the reaction time to obtain the desired chitin or chitin derivatives.

11. Chitin obtainable by the process described in any one of claims 1 to 10.

12. Chitosan obtainable by deacetylation of chitin according to claim 11.

13. Composition comprising chitin according to claim 11 and / or chitosan according to claim 12.

A pharmaceutical composition comprising chitin according to claim 11 and / or chitosan according to claim 12.

Cosmetic composition comprising chitin according to claim 11 and / or chitosan according to claim 12.

Medical device comprising chitin according to claim 11 and / or chitosan according to claim 12.

17. Food product, nutraceutical composition, dietary composition, food supplement or functional food comprising chitin according to claim 11 and / or chitosan according to claim 12.

18. Composition comprising chitin according to claim 11 and / or chitosan according to claim 12 for its use in the treatment, filtration and / or depollution of water.

Texturing agent comprising chitin according to claim 11 and / or chitosan according to claim 12.