Chitin and method for chemically obtaining chitin and/or chitosan

Abstract

The present invention relates to a chitin having a molecular mass of more than 855 kg.Math.mol-1 and to a process for obtaining chitin and/or chitosan by separating cuticles from the soft part of the insect and by a basic treatment of the cuticles.

Claims

1. Chitin isolated or extracted from insects, having (a) a molecular mass greater than or equal to 855 kg.mol.sup.−1 and containing less than 1.5% by weight amino acids relative to the total dry weight of chitin, wherein the molecular mass is determined using the falling ball viscosity measurement; and (b) purity by difference greater than or equal to 95%, wherein the purity by difference is obtained by subtracting the amino acid, lipid and ash impurity contents from the absolute purity value, said absolute purity value being equal to 100%.

2. Chitin according to claim 1, containing less than 3% by weight ash relative to the total dry weight of chitin.

3. Chitosan obtained from chitin isolated or extracted from insects, having a molecular mass greater than or equal to 250 kg.mol.sup.−1 and a purity by difference greater than or equal to 95%, wherein the molecular mass is determined using the falling ball viscosity measurement and wherein the purity by difference is obtained by subtraction of the amino acid, lipid, and ash impurity contents from the absolute purity value, said absolute purity value being equal to 100%.

4. Method for obtaining chitin and/or chitosan, from insects, comprising the following steps: (i.) separation of the cuticles from the soft part of the insects, then (ii.) basic treatment of the cuticles obtained at step (i) for a duration comprised between 5 and 60 hours with an aqueous basic solution, wherein the molar concentration of the base in aqueous solution is comprised between 0.1 and 5 mol.L.sup.−1, (iii.) recovery of the chitin from step (ii), (iv.) optionally, deacetylation of the chitin to produce chitosan, wherein no step of grinding the insects is performed prior to the step of separating the cuticles from the soft part and wherein no step of said method is carried out at a pH lower than 6.

5. Method according to claim 4, in which the separation of the cuticles from the soft part of the insects is performed using a belt separator.

6. Method according to claim 4, in which the separation of the cuticles from the soft part of the insects is performed using a filter press.

7. Method according to claim 4, in which the basic treatment is carried out with a strong base.

8. Method according to claim 4, in which the basic treatment is carried out at a temperature comprised between 60 and 100° C.

9. Method for obtaining chitin, from insects, comprising the following steps: (i.) killing of the insects, (ii.) separation of the cuticles from the soft part of the insects, (iii.) basic treatment of the cuticles obtained at step (ii) for a duration comprised between 5 and 60 hours with an aqueous basic solution, wherein the molar concentration of the base in aqueous solution is comprised between 0.1 and 5 mol.L.sup.−1, (iv.) recovery of the chitin from step (iii), wherein no step of grinding the insects is performed prior to the step of separating the cuticles from the soft part and wherein no step of said method is carried out at a pH lower than 6.

10. Chitin obtained from the method according to claim 4, wherein said chitin has a purity by difference greater than or equal to 95%, wherein the purity by difference is obtained by subtracting the amino acid, lipid and ash impurity contents from the absolute purity value, said absolute purity value being equal to 100%.

11. Method for obtaining chitosan, from insects, comprising the following steps: (i.) killing of the insects, (ii.) separation of the cuticles from the soft part of the insects, (iii.) basic treatment of the cuticles obtained at step (ii) for a duration comprised between 5 and 60 hours with an aqueous basic solution, wherein the molar concentration of the base in aqueous solution is comprised between 0.1 and 5 mol.L.sup.−1, (iv.) recovery of the chitin from step (iii), (v.) deacetylation of the chitin, and (vi.) recovery of the chitosan, wherein no step of grinding the insects is performed prior to the step of separating the cuticles from the soft part and wherein no step of said method is carried out at a pH lower than 6.

12. Chitosan obtained from the method according to claim 4, wherein said chitin recovered in step iii has a purity by difference greater than or equal to 95%, wherein the purity by difference is obtained by subtracting the amino acid, lipid and ash impurity contents from the absolute purity value, said absolute purity value being equal to 100%.

13. Chitosan obtained from the method according to claim 11, wherein said chitin recovered in step iv has a purity by difference greater than or equal to 95%, wherein the purity by difference is obtained by subtracting the amino acid, lipid and ash impurity contents from the absolute purity value, said absolute purity value being equal to 100%.

Description

Example 1: Method for Obtaining Chitin According to the Invention

(1) I. Obtention Method

(2) Larvae of Tenebrio molitor were used. On receipt of the larvae, the latter can be stored at 4° C. for 0 to 15 days in their rearing trays before being killed, without major degradation. The weight of the larvae (age) used is variable and as a result their composition can vary, as is illustrated in Table 2 below:

(3) TABLE-US-00002 TABLE 2 Biochemical composition of the larvae of Tenebrio molitor according to their weight. Biomass (insects) mg 23 35 58 80 108 154 Dry matter %* 34 34 34.2 37.9 39.6 39.5 Ash %* 1.59 1.52 1.6 1.75 1.67 1.43 Crude proteins %* 22.6 22.2 22 23.2 23.1 23.2 Lipids %* 6.62 6.88 7.98 10.3 10.9 11.7 *The % are expressed in dry weight relative to the wet weight of larvae. Step 1: Killing of the insects

(4) The living larvae (+4° C. to +25° C.) are conveyed, in a layer with a thickness comprised between 2 and 10 cm, on a perforated conveyor belt (1 mm) to a blanching chamber. The insects are thus blanched in steam (steam nozzles or bed) at 98° C. under forced ventilation or else in water at 92-95° C. (spray nozzles) or in a mixed mode (water+steam). The residence time in the blanching chamber is comprised between 5 seconds and 15 minutes, ideally 5 minutes.

(5) The temperature of the larvae after blanching is comprised between 75° C. and 98° C. Step 2: Separation of the soft part from the cuticles of the insects

(6) The larvae, once blanched, are conveyed to the feed hopper of a belt separator, in order to separate the cuticles from the soft part of the larvae.

(7) Advantageously, the separation is performed immediately after killing, such that the larvae do not have time to cool to ambient temperature.

(8) The belt separator used is a belt separator 601 from the Baader company.

(9) The diameter of the perforations of the drum is 1.3 mm.

(10) The soft part of the insects is recovered in a tank.

(11) The cuticles are recovered using a scraper blade.

(12) The percentage of dry matter in the cuticles is approximately 45%.

(13) The moisture content of the cuticles is therefore approximately 55%. Step 3: Basic treatment of the cuticles

(14) 90.02±0.02 g wet cuticles obtained in step 2 (Dry Matter=40.1±0.1%) and 1.80±0.02 L aqueous sodium hydroxide solution with a concentration equal to 1 mol.Math.L.sup.−1 are placed in a 2-L three-neck flask equipped with a condenser and a mechanical stirrer (Heidolph® RZR1). The medium is heated with an oil bath to a temperature of 90±2° C. for 48 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 280 rpm.

(15) The ratio of base by dry weight in g:cuticles by dry weight in g:water by weight in g is approximately equal to 0.9:0.45:22. Step 4: Recovery of the chitin

(16) The reaction medium obtained at the end of step 3 is then filtered (SEFAR MEDIFAB® 03-60/42 filter) so as to recover the chitin. (Optional) step 5: Washing of the chitin

(17) The chitin recovered at the end of step 4 is then rinsed with tepid tap water until neutralization of the pH. (Optional) step 6: Drying of the chitin

(18) The chitin is then dried for 24 hours, at 60° C., in a drying oven (Binder®, FP53 model).

(19) 6.3±0.2 g chitin is thus obtained.

(20) II. Analysis Methods

(21) Measurement of the Dry Matter and of the Moisture Content

(22) The percentage of dry matter and the moisture content are calculated as follows.

(23) 2 g chitin are weighed into cups, introduced into a drying oven (Binder®, FED 115 model) and dried at 105° C. for 24 h (or until completely dry).

(24) The percentage of dry matter is obtained by making the ratio of the dry mass of chitin after drying to the mass of chitin before drying.

(25) The moisture content is obtained by subtracting the percentage of dry matter from the value of 100%.

(26) This measurement method can also be used to measure the percentage of dry matter and the moisture content of the cuticles.

(27) Measurement of the Ash Content

(28) The ash content was determined according to the method from NF V18-101 standard.

(29) Measurement of the Crude Protein Content

(30) The protein content is obtained using the Dumas method, with the conversion coefficient of 6.25, adapted from the NF EN ISO 16634-1 standard.

(31) Measurement of the Lipid or Fat Content

(32) The lipid content is obtained using a method adapted from the EC 152/2009 regulation-B Method-SN.

(33) Amino Acid Content and Relative Abundance

(34) The amino acid content of the chitin according to the invention is preferably determined according to the NF EN ISO 13904 method or a method adapted from the EC 152/2009 regulation of 27-01-2009-SN (these two methods being equivalent) for tryptophan, and according to the NF EN ISO 13903 method or a method from the EC 152/2009 regulation of 27-01-2009-SN for the other amino acids (these two methods being equivalent).

(35) The relative abundance was calculated by relating each amino acid content to the total amino acid content.

(36) Total Amino Acid Content

(37) The total amino acid content was determined by adding up the individual values obtained for each amino acid, including tryptophan.

(38) Purity by Difference

(39) For this measurement, the known impurity contents (amino acids, lipids and ash) were subtracted from the absolute purity value (100%) in order to obtain the value of the estimated purity by difference. Thus, for example, a sample which contains 30% amino acids, 10% lipids and 1% ash is consequently attributed a purity by difference of 100−30−10−1=59%.

(40) Molecular Mass of the Chitin

(41) The method for determining the molecular mass of the chitin is based on viscosity measurements of diluted solutions of chitin. The dilutions are carried out in dimethylacetamide containing 5% lithium chloride (DMAc-LiCl 5%). In fact, the polymers in solution increase the viscosity of a solvent. The viscosity of the polymer in solution depends on its concentration and its molecular mass. The relationship is defined by the Mark-Houwink-Sakurada equation:
[η]=KM.sup.α

(42) with [η]: the intrinsic viscosity, M: the molecular mass, K and a specific constants of a solvent/polymer system at a given temperature.

(43) The values for chitin dissolved in the solvent DMAc-LiCl 5% at 25° C. are: K=0.24 mL.Math.g.sup.−1 and α=0.69 (Pacheco et al.; 2011).

(44) The intrinsic viscosity corresponds to the specific viscosity when the concentration of the polymer tends towards zero.

(45) $\left[\eta \right]=\underset{c.fwdarw.0}{\lim }\left(\frac{\eta \mathrm{sp}}{c}\right)$

(46) In order to measure the specific viscosity of the chitin, five solutions with weak concentrations are prepared in DMAc-LiCl 5%. The flow times of the solvent alone (to) and of the solutions (t) with different concentrations are measured using a falling ball micro viscometer in order to calculate the relative viscosity (η.sub.r). From the relative viscosity OM, it is necessary to calculate the specific (η.sub.sp), reduced (η.sub.red) and inherent (η.sub.inh) viscosities of each concentration of chitin.

(47) ${\eta }_{\mathrm{sp}}={\eta }_r-1;{\eta }_{\mathrm{red}}=\frac{\eta \mathrm{sp}}{c};{\eta }_{\mathrm{inh}}=\frac{\ln \left(\eta r\right)}{c}$

(48) The intrinsic viscosity is then calculated by plotting the curves “concentration vs. reduced viscosity” (positive slope) and “concentration vs. inherent viscosity” (negative slope). Each of these curves is extrapolated from zero concentration. The y-intercept corresponds to the intrinsic viscosity. Similar results are expected from the two curves. Finally, in order to determine the molecular mass of the chitin, the Mark-Houwink-Sakurada equation is applied.

(49) III. Results

(50) The properties of the chitin obtained at the end of step 6 are given in Table 3 below.

(51) TABLE-US-00003 TABLE 3 Properties of the chitin obtained in Example 1 Ash (g/100 g DM*) 1.71 +/− 0.11 Fats (g/100 g DM*)   0 +/− 0.00 Total aa** content (g/100 g DM*) 0.68 +/− 0.03 Purity by difference (%) 97.61 +/− 0.14  Molecular mass (kg .Math. mol.sup.−1) 1015.5 +/− 98.29  *DM: dry matter **aa: amino acids

(52) The relative abundance of amino acids in the chitin obtained is given in Table 4 below. It is expressed in %.

(53) TABLE-US-00004 TABLE 4 Relative abundance of amino acids in the chitin obtained in Example 1 Threonine (Thr) 0.00 Serine (Ser) 0.00 Proline (Pro) 0.00 Glycine (Gly) 0.00 Glutamic acid (Glu) 0.00 Alanine (Ala) 12.48 Cysteine (Cys) 5.87 Valine (Val) 8.07 Methionine (Met) 0.00 Isoleucine (Ile) 0.00 Aspartic acid (Asp) 0.00 Leucine (Leu) 0.00 Tyrosine (Tyr) 0.00 Phenylalanine (Phe) 0.00 Histidine (His) 44.77 Lysine (Lys) 28.62 Arginine (Arg) 0.00 Tryptophan (Trp) 0.19 Total 100.00

(54) 5 amino acids appear to be particularly resistant to the method, namely alanine, cysteine, valine, histidine and lysine. Histidine, lysine and alanine are the 3 most resistant amino acids. In fact they represent more than 85% of the residual amino acids.

Example 2: Method for Obtaining Chitosan According to the Invention

(55) In order to prepare chitosan, the chitin resulting from step 4, 5 or 6 of Example 1 is used. (Optional) step 7: Grinding of the chitin The chitin was ground in an ultra centrifugal grinder with a sieve to a size of 250 μm. Step 8: Deacetylation of the chitin

(56) The chitin is then placed in a reactor, where a concentrated caustic soda solution is added. The sodium hydroxide in aqueous solution at a level of 50% (i.e. a concentration of sodium hydroxide in aqueous solution of 12.5 mol/L) is added in a ratio of weight in g ground chitin/volume in mL sodium hydroxide in aqueous solution equal to 1:50. The tank is then heated to a temperature of 100° C. The deacetylation reaction is carried out for 2 hours twice, with an intermediate step of neutralization of the pH. Step 9: Recovery of the chitosan

(57) The reaction medium obtained at the end of step 8 is then filtered (SEFAR MEDIFAB® 03-60/42 filter) so as to recover the chitosan. (Optional) step 10: Washing

(58) The chitosan recovered at the end of step 9 is then rinsed with tepid tap water until neutralization of the pH.

(59) Powdered chitosan is thus obtained.

(60) The molecular mass of the chitosan obtained is equal to 350+/−26 kg.Math.mol.sup.−1.

(61) The purity by difference of the chitosan obtained is greater than 97%. (Optional) step 11: Drying

(62) The chitosan powder is then dried at 60° C. in order to obtain a powder having a dry matter content greater than 85%.

Example 3: Comparative Method for Obtaining Chitin

(63) This comparative method does not comprise a step of separating the soft part from the cuticles of the insects (step 2 of the method according to the invention), but does comprise a step of grinding then pressing the insects (step 2 of the comparative method).

(64) I. Materials and Methods Step 1: Killing of the insects
This step is identical to the one in Example 1. Step 2: Grinding then pressing of the insects

(65) Following step 1, 600 g larvae are introduced into a beaker containing 450 mL water, and mixed using a Thermomix® mixer, at maximum speed for 30 seconds. The thus-obtained liquid is pressed with a twin screw press of the Angel type under the following conditions:

(66) Speed=82 rpm;

(67) W (energy)=3 HP (horsepower) i.e. 2.68×10.sup.6 J;

(68) Porosity (approximate)=0.5 mm in the first part and 0.2 mm in the last part.

(69) A press juice and a press cake of 133.1±0.1 g by wet weight are thus obtained. Step 3: Basic treatment of the press cake

(70) 90.06±0.05 g press cake obtained in step 2 (Dry Matter=48±1%) and 1.80±0.02 L aqueous sodium hydroxide solution with a concentration equal to 1 mol.Math.L.sup.−1 are placed in a 2-L three-neck flask equipped with a condenser and a mechanical stirrer (Heidolph® RZR1). The medium is heated with an oil bath to a temperature of 90±2° C. for 48 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 280 rpm. Step 4: Recovery of the chitin

(71) The reaction medium obtained at the end of step 3 is then filtered (SEFAR MEDIFAB® 03-60/42 filter) so as to recover the chitin. Step 5: Washing of the chitin

(72) The chitin recovered at the end of step 4 is then rinsed with tepid tap water until neutralization of the pH. Step 6: Drying of the chitin

(73) The chitin is then dried for 24 hours, at 60° C., in a drying oven (Binder®, FP53 model).

(74) 8.1±0.7 g chitin is thus obtained.

(75) II. Results

(76) The analysis methods are identical to those described in Example 1.

(77) The properties of the thus-obtained chitin are given in Table 5 below.

(78) TABLE-US-00005 TABLE 5 Properties of the chitin obtained in Example 3 Ash (g/100 g DM*) 1.825 +/− 0.40 Fats (g/100 g DM*)    0 +/− 0.00 Total aa** content (g/100 g DM*) 1.105 +/− 0.18 Molecular mass (kg .Math. mol.sup.−1)  799.5 +/− 53.03 *DM: dry matter **aa: amino acids

(79) The relative abundance of amino acids in the chitin obtained is given in Table 6 below. It is expressed in %.

(80) TABLE-US-00006 TABLE 6 Relative abundance of amino acids in the chitin obtained in Example 3 Threonine (Thr) 0.00 Serine (Ser) 0.00 Proline (Pro) 0.00 Glycine (Gly) 0.00 Glutamic acid (Glu) 0.00 Alanine (Ala) 26.56 Cysteine (Cys) 4.96 Valine (Val) 13.95 Methionine (Met) 0.00 Isoleucine (Ile) 0.00 Aspartic acid (Asp) 0.00 Leucine (Leu) 0.00 Tyrosine (Tyr) 0.00 Phenylalanine (Phe) 0.00 Histidine (His) 38.71 Lysine (Lys) 15.30 Arginine (Arg) 0.00 Tryptophan (Trp) 0.52 Total 100.00

(81) 5 amino acids appear to be particularly resistant to the method, namely alanine, cysteine, valine, histidine and lysine. Histidine, lysine and alanine are the 3 most resistant amino acids. The respective relative abundances of alanine and of valine are clearly greater than those calculated in Example 1.

Example 4: Obtaining Chitin According to the Invention from Different Insects

(82) Different insects were used in this example: Tenebrio molitor, Pachnoda marginata, Zophobas mono and Galleria mellonella.

(83) The cuticles were obtained from the larval stage of the different insects according to steps 1 and 2 described in Example 1.

(84) The cuticles are then subjected to the following basic treatment: m.sub.1 g wet cuticles obtained in the preceding step (Dry Matter=n.sub.1%) and 1.80±0.02 L aqueous sodium hydroxide (or potassium hydroxide) solution with a concentration equal to 1.0 mol.Math.L.sup.−1 are placed in a 2-L three-neck flask equipped with a condenser and a stirrer (Heidolph® RZR1). The medium is heated with an oil bath to a temperature of 90±2° C. for 48 hours (24 h for G. mellonella). Throughout the reaction, the reaction medium is subjected to a stirring of 300 rpm. Approximately 0.05 L aqueous sodium hydroxide (or potassium hydroxide) solution per gram of dry matter of solid residue is therefore used.

(85) After this basic treatment, steps 4 to 6 of the method from Example 1 are then performed (for P. marginata step 5 is performed with distilled water).

(86) The experimental conditions are summarized in Table 7, in particular the ratio of base:cuticles:water is a ratio by dry weight in g for the base and the cuticles, and by weight in g for the water.

(87) TABLE-US-00007 TABLE 7 Experimental conditions of the basic treatment of different insects Mass m′.sub.1 Initial of dry Ratio of mass m.sub.1 of n.sub.1 % dry cuticles base:cuti- Insect Base cuticles (g) matter (g) cles:water T. molitor NaOH 90.03 ± 0.02 40.06 ± 0.00 36.06 ± 0.01   2:1:50 T. molitor KOH 90.33 ± 0.18 41.23 ± 1.63 37.24 ± 1.44 2.7:1:48 P. marginata NaOH 110.62 ± 0.58  31.58 ± 1.24 34.93 ± 1.55 2.1:1:51 Z. morio NaOH 75.11 ± 0.07 45.29 ± 1.63 34.02 ± 1.24 2.1:1:53 G. mellonella NaOH 100.63 ± 0.56  34.44 ± 1.40 34.65 ± 1.40 2.1:1:52

(88) A mass m.sub.2 of chitin is finally collected. The yield is calculated as follows: m.sub.2/m′.sub.1*100 and the results are indicated in Table 8.

(89) TABLE-US-00008 TABLE 8 Chitin extraction yields for different insects Mass m.sub.2 of Insect chitin obtained (g) Yield (%) T. molitor 6.28 ± 0.19 17.4 ± 0.5 T. molitor 8.12 ± 0.32 21.8 ± 1.3 P. marginata 7.95 ± 0.24 22.8 ± 1.2 Z. morio 5.93 ± 0.78 17.4 ± 2.2 G. mellonella 7.05 ± 0.54 20.4 ± 1.5

(90) The chitins are then analyzed according to the analysis methods indicated in point II of Example 1. The analysis results are given in Table 9, in which the ash, lipid and amino acid contents correspond to contents in grams per 100 g dry matter.

(91) TABLE-US-00009 TABLE 9 Properties of the chitins obtained from different insects Molar Purity by Source of mass Amino difference chitin (kDa) Ash Lipids acids (%) T. molitor 1015 ± 98  1.71 ± 0.11   0 ± 0.00 0.68 ± 0.03 97.61 ± 0.12 (NaOH) T. molitor 886 ± 49 2.58 ± 0.04 0.71 ± 0.40 0.32 ± 0.01 96.39 ± 0.40 (KOH) P. 1932 ± 66  2.56 ± 0.13 0.33 ± 0.25 0.25 ± 0.07 96.86 ± 0.29 marginata Z. morio 865 ± 51 1.57 ± 0.00 0.32 ± 0.01 0.535 ± 0.19  97.575 ± 0.19  G. 1626 ± 107 1.635 ± 0.06    0 ± 0.00 0.18 ± 0.06 98.185 ± 0.19  mellonella

(92) The method according to the invention makes it possible to obtain a chitin with a high molecular mass, also having a high purity, whatever the insect or basic treatment used.

(93) The relative abundance of amino acids in the chitins obtained is given in Table 10 below and is expressed in percent.

(94) TABLE-US-00010 TABLE 10 Relative abundance of amino acids in the chitins obtained from different insects T. molitor T. molitor (NaOH) (KOH) P. marginata Z. morio G. mellonella threonine 0.000 0.000 31.955 38.261 0.000 serine 0.000 0.000 0.000 0.000 0.000 proline 0.000 0.000 0.000 0.000 0.000 glycine 0.000 0.000 0.000 0.000 0.000 glutamic acid 0.000 0.000 0.000 3.733 0.000 alanine 12.476 24.845 0.000 23.330 0.000 cysteine 5.871 0.000 0.000 0.000 0.000 valine 8.073 0.000 0.000 23.330 0.000 methionine 0.000 0.000 0.000 0.000 0.000 isoleucine 0.000 0.000 0.000 0.000 0.000 aspartic acid 0.000 0.000 0.000 0.000 0.000 leucine 0.000 0.000 0.000 0.000 0.000 tyrosine 0.000 0.000 0.000 11.198 0.000 phenylalanine 0.000 0.000 0.000 0.000 0.000 histidine 44.767 0.000 0.000 0.000 0.000 lysine 28.622 74.534 67.905 0.000 99.174 arginine 0.000 0.000 0.000 0.000 0.000 tryptophan 0.191 0.621 0.140 0.149 0.826 total 100.000 100.000 100.000 100.000 100.000

(95) The conditions of the basic treatment (NaOH or KOH) as well as the insects used influence the amino acid composition of the resulting chitin.

Example 5: Obtaining Chitosan According to the Invention from Different Insects

(96) Grinding of the chitin

(97) Each chitin obtained in Example 4 is finely ground in a centrifugal grinder (Retsch® ZM200) with a strainer with pores of 250 μm. Deacetylation of the chitin

(98) A mass of m.sub.3 g of the chitin obtained after the grinding step and a volume V.sub.1 (V.sub.1=m.sub.3*50 mL) of aqueous sodium hydroxide (or potassium hydroxide) solution with a concentration equal to 19 mol.Math.L.sup.−1 are placed in a 2-L three-neck flask equipped with a condenser and a stirrer (Heidolph® RZR1). The medium is heated with an oil bath to a temperature of 100±2° C. for 2 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 300 rpm. Recovery of the chitosan

(99) The reaction medium obtained at the end of the deacetylation step is then filtered (SEFAR MEDIFAB® 03-60/42 filter) so as to recover the chitosan. Washing of the chitosan

(100) The chitosan recovered is then rinsed with tepid tap water until neutralization of the pH.

(101) It is then made to react again in a three-neck flask containing the same quantity of aqueous sodium hydroxide (or potassium hydroxide) solution and the 3 preceding steps (deacetylation, recovery and washing) are repeated identically. Drying of the chitosan

(102) The steps of deacetylation, recovery and washing are therefore carried out twice in total. At the end of these steps, the chitosan is dried for 24 hours, at 60° C., in a drying oven (Binder®, FP53 model). A mass ma of chitosan is collected. The yield is calculated as follows: m.sub.4/m.sub.3*100 and the results are summarized in Table 11. In particular, Table 11 refers to the ratio of base:chitin:water, which is a ratio by dry weight in g for the base and the chitin, and by weight in g for the water.

(103) TABLE-US-00011 TABLE 11 Masses of chitin, volume of base and yield for obtaining chitosan depending on the insects Volume Mass m.sub.4 of Initial V.sub.1 of Ratio of chitosan Source of mass m.sub.3 of base used base:chi- obtained chitin Base chitin (g) (mL) tin:water (g) Yield (%) T. molitor NaOH 12.02 ± 0.24 601 ± 12 38:1:50 8.83 ± 0.25 73.4 ± 0.6 (NaOH) T. molitor KOH 13.73 ± 0.49 686 ± 24 53:1:50 9.17 ± 0.27 66.9 ± 4.3 (KOH) P. NaOH 16.78 ± 3.51  839 ± 175 38:1:50 11.11 ± 2.52  66.1 ± 1.2 marginata Z. morio NaOH 13.91 ± 0.28 696 ± 14 38:1:50 9.61 ± 0.09 69.1 ± 2.1 G. NaOH 16.80 ± 0.01 840 ± 0  38:1:50 11.19 ± 0.60  66.6 ± 3.6 mellonella

(104) The chitosans are then analyzed according to the analysis methods indicated in point II of Example 1. The analysis results are given in Table 12, in which the ash, lipid and amino acid contents correspond to contents in grams per 100 g dry matter. In this table, the lipid content measured is at the detection limit.

(105) TABLE-US-00012 TABLE 12 Properties of the chitosans obtained from different insects Molar Purity by Source of mass Amino difference chitosan (kDa) Ash Lipids acids (%) T. molitor 379 ± 31 2.305 ± 0.29  0.455 ± 0.01 0.245 ± 0.08 96.995 ± 0.30  (NaOH) T. molitor 593 ± 9  1.81 ± 0.11 0.245 ± 0.01 0.375 ± 0.01 97.57 ± 0.11 (KOH) P. 561 ± 74 2.71 ± 0.03 0.385 ± 0.05 0.215 ± 0.01 96.69 ± 0.06 marginata Z. morio 430 ± 11 1.24 ± 0.16  0.53 ± 0.01  0.26 ± 0.06 97.97 ± 0.17 G. 483 ± 29 0.99 ± 0.04 0.665 ± 0.02 0.265 ± 0.01 98.08 ± 0.05 mellonella

(106) As the method according to the invention makes it possible to obtain a chitin with a high molecular mass and purity, the resulting chitosan also has these features, even under different deacetylation conditions.