CHITIN AND PROCESS FOR PRODUCING CHITIN AND/OR CHITOSAN BY THE ENZYMATIC AND CHEMICAL PATHWAY

Abstract

The present invention relates to chitin with a differential purity of more than 97.75% and to a process for producing chitin and/or chitosan by the enzymatic and chemical pathway.

Claims

1. Chitin, the purity by difference of which is greater than 97.75%, 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%.

2. Chitin according to claim 1, containing less than 1.2% by weight amino acids relative to the total dry weight of chitin.

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

4. Chitin according to claim 1, the purity by difference of which is greater than or equal to 98.0% and the molar mass of which is greater than or equal to 800 kg.Math.mol.sup.1, wherein the molar mass is determined using the falling ball viscosity measurement.

5. Chitosan, the purity by difference of which is greater than 97.75%, 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%.

6. Chitosan according to claim 5, the purity by difference of which is greater than or equal to 97.9% and the molar mass of which is greater than or equal to 480 kg.Math.mol.sup.1, wherein the molar mass is determined using the falling ball viscosity measurement.

7. Method for obtaining chitin and/or chitosan, from insects, comprising the following steps: separation of the cuticles from the soft part of the insects, enzymatic hydrolysis of the cuticles by a protease, in order to obtain a solid residue, and basic treatment of the solid residue.

8. Method according to claim 7, in which the separation of the cuticles from the soft part of the insects is performed using a belt separator.

9. Method according to claim 7, in which the protease is chosen from the group constituted by aminopeptidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.

10. Method according to claim 9, in which the protease is chosen from serine endopeptidases.

11. Method according to claim 1, in which the basic treatment is carried out with a strong base.

12. Method for obtaining chitin, from insects, comprising the following steps: killing of the insects, separation of the cuticles from the soft part of the insects, enzymatic hydrolysis of the cuticles by a protease, in order to obtain a solid residue, basic treatment of the solid residue, and recovery of the chitin.

13. Chitin that can be obtained using the method according to claim 7.

14. Method for obtaining chitosan, from insects, comprising the following steps: killing of the insects, separation of the cuticles from the soft part of the insects, enzymatic hydrolysis of the cuticles by a protease, in order to obtain a solid residue, basic treatment of the solid residue, recovery of the chitin, deacetylation of the chitin, and recovery of the chitosan.

15. Chitosan that can be obtained using the method according to claim 7.

16. Chitosan that can be obtained using the method according to claim 12.

17. Chitosan that can be obtained using the method according to claim 14.

Description

EXAMPLE 1: METHOD FOR OBTAINING CHITIN ACCORDING TO THE INVENTION

[0251] I. Materials and Methods

[0252] 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:

TABLE-US-00003 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.

[0253] Step 1: Killing of the Insects

[0254] 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.

[0255] The temperature of the larvae after blanching is comprised between 75 C. and 98 C.

[0256] Step 2: Separation of the Soft Part from the Cuticles of the Insects

[0257] 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.

[0258] Advantageously, the separation is performed immediately after killing, such that the larvae do not have time to cool to ambient temperature.

[0259] The belt separator used is a belt separator 601 from the Baader company.

[0260] The diameter of the perforations of the drum is 1.3 mm.

[0261] The soft part of the insects is recovered in a tank.

[0262] The cuticles are recovered using a scraper blade.

[0263] The percentage of dry matter in the cuticles is approximately 35-45%.

[0264] Step 3: Enzymatic Hydrolysis of the Cuticles by a Protease

[0265] 160.0150.007 g wet cuticles obtained in step 2 (Dry Matter=385%), 8 g Prolyve NP (5% of the mass of wet cuticles) and 1.6 L0.02 L hot water are placed in a 2-L three-neck flask equipped with a condenser and a stirrer (Heidolph RZR1). The medium is heated with a bain marie to a temperature of 602 C. for 6 hours. Then, the temperature is brought to 80 C. for 15 min. The reaction medium is then separated using a juicer (Angelia 7500). Throughout the reaction, the reaction medium is subjected to a stirring of 280 rpm. The pH is comprised between 6 and 8, preferably between 6.5 and 7.5. The specific activity of Prolyve NP is 1132.1 IU/g enzyme. 8 g Prolyve NP, i.e. 9056.8 IU, is used per 160.0150.007 g of wet cuticles (Dry Matter=385%). The concentration of the enzyme is therefore approximately 149 IU/g of dry matter of cuticles.

[0266] 582 g of solid residue is obtained, and a hydrolysate. The solid residue is mixed in order to be homogeneous, weighed, then placed in a refrigerator overnight. The hydrolysate is lyophilized.

[0267] Step 4: Basic Treatment of the Solid Residue

[0268] The solid residue obtained in step 3 (Dry Matter=4416%) and 512.050.02 mL 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 902 C. for 24 hours.

[0269] Approximately 0.02 L aqueous sodium hydroxide solution per gram of dry matter of solid residue is therefore used.

[0270] The ratio of base by dry weight in g:solid residue by dry weight in g:water by weight in g is approximately equal to 0.36:0.45:9.23.

[0271] Step 5: Recovery of the Chitin

[0272] The reaction medium obtained at the end of step 4 is then filtered (SEFAR MEDIFAB 03-60/42 filter) so as to recover the chitin.

[0273] (Optional) Step 6: Washing of the Chitin

[0274] The chitin recovered at the end of step 5 is then rinsed with tepid tap water until neutralization of the pH.

[0275] (Optional) Step 7: Drying of the Chitin

[0276] The chitin is then dried for 24 hours, at 60 C., in a drying oven (Binder, FP53 model).

[0277] 9.60.2 g chitin is thus obtained.

[0278] II. Analysis Methods

[0279] Measurement of the Dry Matter and of the Moisture Content

[0280] The percentage of dry matter and the moisture content are calculated as follows. 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).

[0281] 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.

[0282] The moisture content is obtained by subtracting the percentage of dry matter from the value of 100%.

[0283] This measurement method can also be used to measure the percentage of dry matter and the moisture content of the cuticles.

[0284] Measurement of the Ash Content

[0285] The ash content was determined according to the method from NF V18-101 standard.

[0286] Measurement of the Crude Protein Content

[0287] 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.

[0288] Measurement of the Lipid or Fat Content

[0289] The lipid content is obtained using a method adapted from the EC 152/2009 regulationMethod B-SN.

[0290] Amino Acid Content and Relative Abundance

[0291] 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).

[0292] The relative abundance was calculated by relating each amino acid content to the total amino acid content.

[0293] Total Amino Acid Content

[0294] The total amino acid content was determined by adding up the individual values obtained for each amino acid, including tryptophan.

[0295] Purity by Difference

[0296] 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. For example, a sample which contains 30% amino acids, 10% lipids and 1% ash is consequently attributed a purity by difference of 10030101=59%.

[0297] Molecular Mass of the Chitin

[0298] The molecular mass was determined according to a falling ball viscosity measurement method based on the one described in the following publication: Pacheco et al.; Structural characterization of chitin and chitosan obtained by biological and chemical methods; Biomacromolecules; 12, 3285-3290, 2011.

[0299] In particular, 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:

[]=KM.sup.

[0300] with []: the intrinsic viscosity, M: the molecular mass, K and specific constants of a solvent/polymer system at a given temperature.

[0301] The values for chitin dissolved in the solvent DMAc-LiCl 5% at 25 C. are: K=0.24 mL.Math.g.sup.1 and =0.69 (Pacheco et al.; 2011).

[0302] The intrinsic viscosity corresponds to the specific viscosity when the concentration of the polymer tends towards zero.

[00002]$\left[\right]=\underset{c.fwdarw.0}{\lim }.Math.\left(\frac{.Math.s.Math.p}{c}\right)$

[0303] 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 (t.sub.0) and of the solutions (t) with different concentrations are measured using a falling ball micro viscometer in order to calculate the relative viscosity (.sub.t). From the relative viscosity (.sub.t) it is necessary to calculate the specific (.sub.sp), reduced (.sub.red) and inherent (.sub.inh) viscosities of each concentration of chitin.

[00003]${}_{s.Math.p}={}_r-1;$${}_{r.Math.e.Math.d}=\frac{.Math.s.Math.p}{c};$${}_{\mathrm{inh}}=\frac{\ln \left(.Math.r\right)}{c}$

[0304] 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.

[0305] III. Results

[0306] The properties of the chitin obtained are given in Table 3 below.

TABLE-US-00004 TABLE 3 Properties of the chitin obtained in Example 1 Ash (g/100 g DM*) 1.11 +/ 0.25 Fats (g/100 g DM*) 0 +/ 0.00 Total aa content (g/100 g DM*) 0.72 +/ 0.08 Purity by difference (%) 98.16 +/ 0.33 Molecular mass (kg .Math. mol.sup.1) 642 +/ 125 *DM: dry matter

[0307] The relative abundance of amino acids in the chitin obtained is given in Table 4 below. It is expressed in %.

TABLE-US-00005 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) 0.00 Cysteine (Cys) 6.88 Valine (Val) 16.51 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) 46.78 Lysine (Lys) 29.58 Arginine (Arg) 0.00 Tryptophan (Trp) 0.25 Total 100.00

[0308] Three amino acids appear to be particularly resistant to the method, namely valine, histidine and lysine. In fact they represent more than 93% of the residual amino acids.

EXAMPLE 2: METHOD FOR OBTAINING CHITOSAN ACCORDING TO THE INVENTION

[0309] In order to prepare chitosan, the chitin resulting from step 5, 6 or 7 of Example 1 is used.

[0310] (Optional) Step 8: Grinding of the Chitin

[0311] The chitin was ground in an ultra centrifugal grinding mill with a sieve to a size of 250 m.

[0312] Step 9: Deacetylation of the Chitin

[0313] 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.

[0314] Step 10: Recovery of the Chitosan

[0315] The reaction medium obtained at the end of step 9 is then filtered (SEFAR MEDIFAB 03-60/42 filter) so as to recover the chitosan.

[0316] (Optional) Step 11: Washing

[0317] The chitosan recovered at the end of step 10 is then rinsed with tepid tap water until neutralization of the pH.

[0318] Powdered chitosan is thus obtained.

[0319] The chitosan obtained has a purity by difference greater than 98%.

[0320] The molecular mass of the chitosan obtained is equal to 307+/60 kg.Math.mol.sup.1.

[0321] (Optional) Step 12: Drying

[0322] 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

[0323] I. Materials and Methods

[0324] Step 1: Killing of the Insects

This step is identical to the one in Example 1.

[0325] Step 2: Separation of the Cuticles from the Soft Part of the Insects

This step is identical to the one in Example 1.

[0326] Step 3: Basic Treatment of the Cuticles

[0327] 90.020.02 g wet cuticles obtained in step 2 (Dry Matter=40.10.1%) and 1.800.02 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 902 C. for 48 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 280 rpm.

[0328] Approximately 0.05 L aqueous sodium hydroxide solution per gram of dry matter of cuticles is therefore used.

[0329] 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.

[0330] Step 4: Recovery of the Chitin

[0331] 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.

[0332] (Optional) Step 5: Washing of the Chitin

[0333] The chitin recovered at the end of step 4 is then rinsed with tepid tap water until neutralization of the pH.

[0334] (Optional) Step 6: Drying of the Chitin

[0335] The chitin is then dried for 24 hours, at 60 C., in a drying oven (Binder, FP53 model).

[0336] 6.30.2 g chitin is thus obtained.

[0337] II. Results

[0338] The properties of the chitin obtained at the end of step 6 are given in Table 5 below.

TABLE-US-00006 TABLE 5 Properties of the chitin obtained in Example 3 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 +/ 0.003 Purity by difference (%) 97.61 +/ 0.14 *DM: dry matter **aa: amino acids

[0339] The relative abundance of amino acids in the chitin obtained is given in Table 6 below. It is expressed in %.

TABLE-US-00007 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) 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

[0340] 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 4: OBTAINING CHITIN ACCORDING TO THE INVENTION FROM DIFFERENT INSECTS

[0341] Different insects were used in this example: Tenebrio molitor, Pachnoda marginata, Zophobas mono and Galleria mellonella.

[0342] The cuticles were obtained from the larval stage of the different insects according to steps 1 and 2 described in Example 1.

[0343] Enzymatic Hydrolysis of the Cuticles by a Protease

[0344] The cuticles are then subjected to an enzymatic hydrolysis step under the following conditions:

[0345] m.sub.1 g wet cuticles obtained in the preceding step (Dry Matter=n.sub.1%), a quantity q.sub.1 (g or mL) of protease and 1.6 L0.02 L hot water are placed in a 2-L three-neck flask equipped with a condenser and a stirrer (Heidolph RZR1), so that the protease concentration is approximately 140 IU/g dry cuticles. The medium is heated with a bain marie to a temperature of 602 C. for 6 hours. Then, the temperature is brought to 80 C. for 15 min. The reaction medium is then separated using a juicer (Angelia 7500). Throughout the reaction, the reaction medium is subjected to a stirring of 300 rpm. The pH is comprised between 6 and 8, preferably between 6.5 and 7.5.

[0346] m.sub.2 g of solid residue is obtained, and a hydrolysate. The different experimental conditions are summarized in Table 7. The solid residue is mixed in order to be homogeneous, weighed, then placed in a refrigerator overnight. The hydrolysate is lyophilized.

TABLE-US-00008 TABLE 7 Experimental conditions of the different enzymatic hydrolyses as a function of the insects used Enzymatic activity Quantity Initial mass Mass m.sub.1 Mass m.sub.2 of (IU/g or mL q.sub.1 of m.sub.1 of n.sub.1 % dry of dry solid residue Insect Protease of protease) protease cuticles (g) matter cuticles (g) obtained (g) T. molitor Prolyve 1132.1 8 g 160.02 0.01 38.08 0.00 60.93 0.00 57.78 2.33 T. molitor Food 486.47 18.6 mL 160.23 0.25 39.50 0.35 63.29 0.59 66.97 4.22 Pro PNL T. molitor Alcalase 952.24 9.5 mL 160.42 0.34 46.98 0.37 75.36 0.60 80.39 13.75 2.5L PF T. molitor Prolyve 1132.1 8 g 160.19 0.15 38.70 0.00 61.99 0.06 76.85 33.56 P. marginata Prolyve 1132.1 8 g 196.23 0.35 33.58 3.38 65.89 6.72 55.53 6.21 Z. morio Prolyve 1132.1 8 g 138.98 0.45 43.60 3.00 60.58 3.98 45.32 2.48 G. mellonella Prolyve 1132.1 8 g 177.05 0.24 38.42 0.99 68.02 1.76 46.02 5.44

[0347] Basic Treatment of the Solid Residue

[0348] M.sub.2 g solid residue obtained in the preceding step (Dry Matter=n.sub.2%) and a volume V.sub.1 (V.sub.1=m.sub.2*n.sub.2*25 mL) of 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 mechanical stirrer (Heidolph RZR1). The medium is heated with an oil bath to a temperature of 902 C. for 48 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 300 rpm.

[0349] Recovery, Washing and Drying of the Chitin

[0350] The chitin is recovered at the end of the step of basic treatment, washed, then dried under the conditions described in steps 5, 6 and 7 of Example 1 respectively.

[0351] The experimental conditions of the basic treatment for different insects are given in Table 8.

TABLE-US-00009 TABLE 8 Experimental conditions of the basic treatment of different insects Ratio of dry base Mass m.sub.2 of Mass m.sub.2 of Volume (g):dry solid Source of the solid residue n.sub.2 % dry dry solid V.sub.1 of residue solid residue Base (g) matter residue (g) base (mL) (g):water (g) T. molitor NaOH 57.78 2.33 43.62 1.67 25.40 10.64 635 266 1:1:25 (Prolyve) T. molitor NaOH 66.97 4.22 30.50 3.84 20.31 1.48 508 37 1:1:25 (Food Pro) T. molitor NaOH 80.39 13.75 28.45 6.16 22.37 2.75 559 69 1:1:25 (Alcalase) T. molitor KOH 76.85 33.56 33.16 16.60 24.51 10.66 613 267 1.4:1:25 (Prolyve) P. marginata NaOH 55.53 6.21 29.61 6.02 16.20 1.68 405 42 1:1:25 (Prolyve) Z. morio NaOH 45.32 2.48 34.24 2.03 15.52 1.30 388 33 1:1:25 (Prolyve) G. mellonella NaOH 46.02 5.44 27.33 4.99 12.46 1.96 311 49 1:1:25 (Prolyve)

[0352] A mass m.sub.3 of chitin is collected. The yield is calculated as follows: m.sub.3/m.sub.1*100 and the results are indicated in Table 9.

TABLE-US-00010 TABLE 9 Chitin extraction yields for different insects Source of the Mass m.sub.3 of chitin solid residue obtained (g) Yield (%) T. molitor 9.62 0.66 15.8 1.1 (Prolyve + NaOH) T. molitor 12.35 0.14 19.5 0.4 (Food Pro + NaOH) T. molitor 14.36 0.09 19.1 0.1 (Alcalase + NaOH) T. molitor 13.12 0.30 21.2 0.5 (Prolyve + KOH) P. marginata 12.57 0.48 19.2 1.5 (Prolyve + NaOH) Z. morio 10.65 1.00 17.6 1.6 (Prolyve + NaOH) G. mellonella 10.89 0.60 16.0 1.0 (Prolyve + NaOH)

[0353] The chitins are then analyzed according to the analysis methods indicated in point II of Example 1. The analysis results are given in Table 10, in which the ash, lipid and amino acid contents correspond to contents in grams per 100 g dry matter.

TABLE-US-00011 TABLE 10 Properties of the chitins obtained from different insects and different proteases Source of Molar Purity by chitin mass (kDa) Ash Lipids Amino acids difference (%) T. molitor 666 167 1.115 0.25 0 0.00 0.725 0.08 98.16 0.26 (Prolyve + NaOH) T. molitor 986 110 1.36 0.06 0.27 0.01 0.605 0.33 97.765 0.34 (Food Pro + NaOH) T. molitor 929 78 0.85 0.07 0.285 0.02 0.31 0.01 98.555 0.08 (Alcalase + NaOH) T. molitor 895 76 1.37 0.05 0 0.00 0.74 0.02 97.89 0.05 (Prolyve + KOH) P. marginata 782 105 1.105 0.39 0.225 0.08 0.32 0.01 98.35 0.40 (Prolyve + NaOH) Z. morio 693 76 1.03 0.31 0.28 0.08 0.33 0.20 98.36 0.38 (Prolyve + NaOH) G. mellonella 498 51 1.255 0.13 0 0.00 0.305 0.01 98.44 0.13 (Prolyve + NaOH)

[0354] The method according to the invention makes it possible to obtain a chitin having a high purity, whatever the insect or the hydrolysis conditions (protease and base) used.

[0355] The relative abundance of amino acids in the chitins obtained is given in Table 11 below and is expressed in percent.

TABLE-US-00012 TABLE 11 Relative abundance of amino acids in the chitins obtained from different insects T. molitor T. molitor (Food T. molitor T. molitor P. marginata Z. morio G. mellonella (Prolyve + Pro + (Alcalase + (Prolyve + (Prolyve + (Prolyve + (Prolyve + NaOH) NaOH) NaOH) KOH) NaOH) NaOH) NaOH) threonine 0.000 18.122 14.400 18.970 60.681 46.892 29.402 serine 0.000 3.295 0.000 0.000 0.000 0.000 0.000 proline 0.000 0.000 0.000 0.000 0.000 0.000 0.000 glycine 0.000 4.942 0.000 0.000 0.000 0.000 0.000 glutamic acid 0.000 9.061 22.400 10.840 10.892 15.126 0.000 alanine 0.000 4.119 0.000 0.000 0.000 0.000 0.000 cysteine 6.879 0.000 0.000 0.000 0.000 0.000 0.000 valine 16.510 0.000 0.000 0.000 0.000 13.614 0.000 methionine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 isoleucine 0.000 0.000 0.000 29.133 0.000 0.000 0.000 aspartic acid 0.000 4.119 9.600 0.000 0.000 0.000 0.000 leucine 0.000 0.000 0.000 6.775 0.000 0.000 0.000 tyrosine 0.000 8.237 0.000 0.000 28.007 19.664 0.000 phenylalanine 0.000 4.119 0.000 4.743 0.000 0.000 6.534 histidine 46.777 4.942 0.000 2.710 0.000 4.538 0.000 lysine 29.580 38.715 52.800 26.423 0.000 0.000 63.705 arginine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 tryptophan 0.255 0.329 0.800 0.407 0.420 0.166 0.359 total 100.000 100.000 100.000 100.000 100.000 100.000 100.000

[0356] The conditions of the enzymatic hydrolysis (protease used), 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

[0357] Grinding of the Chitin

[0358] Each chitin obtained in Example 4 is finely ground in a centrifugal grinder (Retsch ZM200) with a strainer with pores of 250 m.

[0359] Deacetylation of the Chitin

[0360] A mass of ma g of the chitin obtained after the grinding step and a volume V2 (V2=m.sub.4*50 mL) of aqueous sodium hydroxide (or potassium hydroxide) solution with a concentration equal to 19.0 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 1002 C. for 2 hours. Throughout the reaction, the reaction medium is subjected to a stirring of 300 rpm.

[0361] Recovery of the Chitosan

[0362] 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.

[0363] Washing of the Chitosan

[0364] The chitosan recovered is then rinsed with tepid tap water until neutralization of the pH.

[0365] 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.

[0366] Drying of the Chitosan

[0367] 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 m.sub.5 of chitosan is collected. The yield is calculated as follows: m.sub.5/m.sub.4*00 and the results are summarized in Table 12. In particular, Table 12 refers to a 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.

TABLE-US-00013 TABLE 12 Masses of chitin, volume of base and yield for obtaining chitosan depending on the insects Mass m.sub.5 Initial mass Volume V.sub.2 of chitosan Source of m.sub.4 of chitin of base Ratio of obtained chitin Base (g) used (mL) base:chitin:water (g) Yield (%) T. molitor NaOH 9.98 0.04 499 2 38:1:50 5.59 1.24 56.0 12.6 (Prolyve + NaOH) T. molitor NaOH 15.04 0.01 752 1 38:1:50 10.35 0.06 68.8 0.5 (Food Pro + NaOH) T. molitor NaOH 18.02 0.02 901 1 38:1:50 12.58 0.07 69.8 0.5 (Alcalase + NaOH) T. molitor KOH 18.58 0.18 929 9 53:1:50 12.70 0.02 68.4 0.8 (Prolyve + KOH) P. marginata NaOH 27.71 3.32 1385 166 38:1:50 19.26 2.55 69.5 0.9 (Prolyve + NaOH) Z. morio NaOH 23.51 0.04 1176 2 38:1:50 16.60 0.53 70.6 2.4 (Prolyve + NaOH) G. mellonella NaOH 18.58 1.39 929 70 38:1:50 12.85 1.00 69.2 0.2 (Prolyve + NaOH)

[0368] The chitosans are then analyzed according to the analysis methods indicated in point II of Example 1. The analysis results are given in Table 13, 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.

TABLE-US-00014 TABLE 13 Properties of the chitosans obtained from different insects Source of Molar Purity by chitosan mass (kDa) Ash Lipids Amino acids difference (%) T. molitor 302 15 0.97 0.04 0.485 0.01 0.24 0.06 98.305 0.07 (Prolyve + NaOH) T. molitor 453 14 0.35 0.02 0.205 0.04 0.37 0.03 99.08 0.05 (Food Pro + NaOH) T. molitor 566 23 0.525 0.08 0.165 0.01 0.32 0.10 98.99 0.13 (Alcalase + NaOH) T. molitor 496 7 1.48 0.18 0.255 0.15 0.285 0.05 97.98 0.24 (Prolyve + KOH) P. marginata 395 19 1.485 0.50 0.48 0.24 0.285 0.09 97.75 0.56 (Prolyve + NaOH) Z. morio 359 19 0.725 0.06 0.495 0.02 0.28 0.07 98.50 0.10 (Prolyve + NaOH) G. mellonella 349 10 0.705 0.02 0.67 0.00 0.30 0.04 98.325 0.05 (Prolyve + NaOH)

[0369] As the method according to the invention makes it possible to obtain a chitin with a high purity, the resulting chitosan also has this feature, even under different deacetylation conditions.