CHITIN, HYDROLYSATE AND METHOD FOR THE PRODUCTION OF ONE OR MORE DESIRED PRODUCTS BY MEANS OF ENZYMATIC HYDROLYSIS, INCLUDING PRE-TREATMENT WITH AN OXIDISING AGENT

Abstract

The invention relates to chitin, a hydrolysate and a method for the production of at least one desired product from insects. More specifically, the invention relates to a method for the production of chitin and/or chitosan from insect cuticles, comprising a step in which insect cuticles are treated with an oxidising agent, followed by a step involving the enzymatic hydrolysis of the insect cuticles using a proteolytic enzyme.

Claims

1. Hydrolysate comprising at least 40% by weight proteins based on the total weight of dry matter, at a maximum 10% by weight ash based on the total weight of dry matter, and a water-soluble protein content larger than 12,400 g/mol less than 50%.

2. Chitin comprising an amino acid content less than or equal to 55% by weight based on the total weight of dry matter, an ash content less than or equal to 3.5% by weight based on the total weight of dry matter, and a purity by difference greater than or equal to 35% or a colorimetric purity greater than or equal to 44%.

3. Method for the production of chitin and/or chitosan from insect cuticles, comprising the following steps: (i) treating the insect cuticles with an oxidizing agent, then (ii) enzymatic hydrolysis of the insect cuticles with a proteolytic enzyme.

4. Method according to claim 3, in which the proteolytic enzyme is a protease.

5. Method according to claim 3 or 4, in which the oxidizing agent is selected from the group constituted by hydrogen peroxide, potassium permanganate, ozone and sodium hypochlorite.

6. Method according to any one of claims 3 to 5, comprising a step of killing the insects.

7. Method according to any one of claims 3 to 6, comprising a step of grinding the insects.

8. Method according to any one of claims 3 to 7, in which the oxidizing agent is hydrogen peroxide.

9. Method according to any one of claims 3 to 8, in which the insect or insects is/are selected from the group constituted by the Coleoptera, the Lepidoptera, the Orthoptera and the Diptera.

10. Method according to any one of claims 4 to 9, in which the protease is selected from the group constituted by aminopeptidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.

11. Method for the production of chitin from insects, comprising the following steps: a) killing the insects, b) grinding the insects, grinding optionally being preceded or followed by a pressing step, c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) recovery of the chitin, the insect cuticles being treated with an oxidizing agent before step c).

12. Chitin obtainable by a method according to any one of claims 1 to 11.

13. Method for the production of chitosan from insects, comprising the following steps: a) killing the insects, b) grinding the insects, grinding optionally being preceded or followed by a pressing step, c) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme, d) recovery of the chitin, e) deacetylation of the recovered chitin, f) recovery of the chitosan, the insect cuticles being treated with an oxidizing agent before step c).

Description

[0192] Other features and advantages of the invention will become clear from the following examples, given by way of illustration, with reference to the figures, which represent respectively:

[0193] FIG. 1 is a flow chart of a preferred embodiment of a method according to the invention,

[0194] FIG. 2 is a diagram comparing the degree of purity for the chitin obtained by an enzymatic method with and without hydrogen peroxide,

[0195] FIG. 3 is a diagram illustrating the influence of the method of extraction (enzymatic or chemical) and treatment with an oxidizing agent on the chitin obtained.

[0196] FIG. 4: Effect of the oxidizing agent on the ash content in the hydrolysate-different enzymes,

[0197] FIG. 5: Effect of the oxidizing agent on the ash content in the hydrolysate-different insects,

[0198] FIG. 6: Effect of the oxidizing agent on the protein content in H. illucens,

[0199] FIG. 7: Effect of the oxidizing agent on the lipid content in the hydrolysate,

[0200] FIG. 8: Effect of the oxidizing agent on the digestibility of the hydrolysate,

[0201] FIG. 9: Amino acid composition of the hydrolysate from a method with an oxidizing agent: TM+Prolyve,

[0202] FIG. 10: Amino acid composition of the hydrolysate from a method with an oxidizing agent: TM+Sumizyme,

[0203] FIG. 11: Amino acid composition of the hydrolysate from a method with an oxidizing agent: TM+Novozyme,

[0204] FIG. 12: Amino acid composition of the hydrolysate from a method with an oxidizing agent: TM+Neutrase,

[0205] FIG. 13: Amino acid composition of the hydrolysate from a method with an oxidizing agent: HI+Prolyve,

[0206] FIG. 14: Amino acid composition of the hydrolysate from a method with an oxidizing agent: GM+Prolyve,

[0207] FIG. 15: Amino acid composition of the hydrolysate from a method with an oxidizing agent: AD+Prolyve

[0208] FIG. 16: Effect of the oxidizing agent on the ash content in chitin,

[0209] FIG. 17: Effect of the oxidizing agent on the lipid content in chitin,

[0210] FIG. 18: Lipid content in chitin,

[0211] FIG. 19: Effect of the oxidizing agent on the amino acid content in chitin,

[0212] FIG. 20: TM+Prolyve: relative abundance of amino acids of chitin,

[0213] FIG. 21: TM+Sumizyme: relative abundance of amino acids of chitin,

[0214] FIG. 22: TM+Novozyme: relative abundance of amino acids of chitin,

[0215] FIG. 23: TM+Neutrase: relative abundance of amino acids of chitin,

[0216] FIG. 24: HI+Prolyve: relative abundance of amino acids of chitin,

[0217] FIG. 25: GM+Prolyve: relative abundance of amino acids of chitin,

[0218] FIG. 26: AD+Prolyve: relative abundance of amino acids of chitin,

[0219] FIG. 27: Effect of the oxidizing agent on the colorimetric purity of chitin obtained from T. molitor-different enzymes,

[0220] FIG. 28: Effect of the oxidizing agent on the colorimetric purity of chitin obtained with hydrolysis in the presence of Prolyve—different insects,

[0221] FIG. 29: Purity by difference of chitin obtained by different methods,

[0222] FIG. 30: Degree of crystallinity of chitins obtained.

EXAMPLE 1: EXAMPLE OF A TREATMENT METHOD ACCORDING TO THE INVENTION

[0223] 15 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 30 mL of a 6% solution of hydrogen peroxide brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then ground with a volume of water of 15 mL. The liquid thus obtained is transferred to a 250-mL Erlenmeyer flask containing 50 mL of a 1% solution of protease (Prolyve), the whole is placed under magnetic stirring for 4 hours at 45° C. (pH approximately 6.5). The Erlenmeyer flask is then placed in a water bath at 90° C. for 15 minutes in order to deactivate the enzymes, and then the solution is filtered hot at 0.45-0.5 μm. The chitin thus obtained is dried for 24 hours at 70° C. This gives 0.6±0.05 g of chitin.

EXAMPLE 2: INFLUENCE OF THE PRESENCE OF THE TREATMENT WITH OXIDANT IN THE METHOD ACCORDING TO THE INVENTION

[0224] 25 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 50 mL of water brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then ground with a volume of water of 25 mL. In the case of the reaction with hydrogen peroxide, the liquid thus obtained is placed in the presence of a solution of hydrogen peroxide for 1 hour, and then transferred to a 250-mL Erlenmeyer flask containing a 4% solution of protease (Sumizyme LP), or otherwise it is transferred directly to the Erlenmeyer flask containing the protease solution. The whole is placed under magnetic stirring for 4 hours at 45° C. (pH approximately 6.5). The Erlenmeyer flask is then placed for 15 minutes in a water bath at 90° C. in order to deactivate the enzymes, and the solution is then filtered hot at 0.45-0.5 μm. The chitin thus obtained is dried for 24 hours at 70° C.

[0225] The dry residue thus obtained after using hydrogen peroxide is 6.3±0.7% relative to the initial dry matter, whereas the dry residue resulting from a method without hydrogen peroxide is 9.75±0.9% relative to the initial dry matter.

[0226] The degree of purity of the chitin is determined compared to the weight of dry residue obtained relative to that resulting from chemical extraction, 5% of the initial dry matter. It is thus established at 79.9±9% for the product obtained after treatment with peroxide and at 51.5±4.9% in the absence of peroxide (see FIG. 2).

EXAMPLE 3: INFLUENCE OF THE SEQUENCE OF THE DIFFERENT STEPS IN THE METHOD ACCORDING TO THE INVENTION

[0227] Obtaining Chitin Enzymatically (without Adding Oxidant)

[0228] 50 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 50 mL of water brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then ground with a volume of water of 100 mL. The liquid thus obtained is transferred to a 500-mL Erlenmeyer flask containing 150 mL of 1% protease solution (Prolyve), and the whole is placed under magnetic stirring for 4 hours at 45° C. (pH approximately 6.5). The Erlenmeyer flask is then placed for 15 minutes in a water bath at 90° C. in order to deactivate the enzymes, and the solution is then filtered hot at 0.45-0.5 μm. The chitin thus obtained is dried for 24 hours at 70° C. 1.656±0.021 g of chitin is obtained by this method.

[0229] Obtaining Chitin Enzymatically with Addition of H.sub.2O.sub.2 During Scalding

[0230] 50 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 50 mL of a 6% solution of H.sub.2O.sub.2 in water brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then mixed with a volume of water of 100 mL. The liquid thus obtained is transferred to a 500-mL Erlenmeyer flask containing 150 mL of 1% protease solution (Prolyve), and the whole is placed under magnetic stirring for 4 hours at 45° C. The Erlenmeyer flask is then placed in a water bath at 90° C. for 15 minutes in order to deactivate the enzymes, and then the solution is filtered hot at 0.45-0.5 μm. The chitin thus obtained is dried for 24 hours at 70° C. 1.98±0.22 g of chitin is obtained by this method.

[0231] Obtaining Chitin Enzymatically with Addition of H.sub.2O.sub.2 after Hydrolysis

[0232] 50 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 50 mL of water brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then ground with a volume of water of 100 mL. The liquid thus obtained is transferred to a 500-mL Erlenmeyer flask containing 150 mL of 1% protease solution (Prolyve), and the whole is placed under magnetic stirring for 4 hours at 45° C. (pH approximately 6.5). The Erlenmeyer flask is then placed in a water bath at 90° C. for 15 minutes in order to deactivate the enzymes, and then the solution is filtered hot at 0.45-0.5 μm. The residue is then placed in a 6% solution of H.sub.2O.sub.2 for 1 hour at 65° C. The chitin thus obtained is filtered (0.45-0.5 μm), and then dried for 24 hours at 70° C. 1.304±0.091 g of chitin is obtained by this method.

[0233] Obtaining Chitin Chemically

[0234] 50 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100° C. containing 50 mL of water brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then ground with a volume of water of 60 mL. The liquid thus obtained is transferred with 50 mL of water to a 1 L bottle. 500 mL of 1M HCl is added and the whole is stirred for 1 hour at 90° C. The reaction mixture is then filtered and washed with water until a clear residue is obtained. This residue is then transferred to a 1 L bottle, to which 500 mL of 1M NaOH is added, and the whole is stirred at 90° C. for 24 hours. The reaction mixture is then filtered and washed until a clear filtrate is obtained, and the residue is finally dried for 24 hours at 70° C. 0.944±0.005 g of chitin is obtained by this method.

[0235] Determination (by Viscometry) of the Molecular Weight of the Chitin Obtained

[0236] A bottle containing 1 g of chitin and 10 mL of 1M NaOH is held for 4 hours at 90° C. The mixture is then filtered (0.45-0.5 μm) and the residue thus washed is held for 24 hours at 70° C.

[0237] Preparation of the solvent: 5 g of LiCl is placed in 100 mL of N,N-dimethylacetamide, under stirring for 4-5 hours (until completely dissolved).

[0238] The stock solution is obtained by dissolving 0.2 mg of chitin in 1 mL of solvent. Dilutions with concentrations of 0.1 mg/mL, 0.08 mg/mL and 0.04 mg/mL are prepared from this stock solution. The viscosity of these various solutions is then measured in triplicate with a viscometer of the Ostwald type and the molecular weight is calculated from the formula:

[η]=KM.sub.w.sup.α  (1)

[0239] with

[0240] [η]: intrinsic viscosity in cm.sup.3/g,

[0241] M.sub.w: molar mass of the chitin in g/mol (or Da), and

[0242] the Mark-Houwink coefficients α=0.71 and K=0.000893,

the intrinsic viscosity being obtained from:

[η]=η.sub.r/C  (2)

[0243] with

[0244] η.sub.r: reduced viscosity (without units),

[0245] C: concentration in mg/mL,

the reduced viscosity being obtained from:

η.sub.r=t/t.sub.0  (3)

[0246] with

[0247] t: the falling time measured for the solution in s,

[0248] t.sub.0: the falling time measured for the solvent in s.

[0249] It can be seen from FIG. 3 that the size of the chitin molecule obtained is a function of the method of extraction used. Thus, the chemical method damages the integrity of the molecule (M.sub.w obtained is below 70000 g/mol), but the harshest treatment is that which consists of bleaching the chitin with hydrogen peroxide after hydrolysis, even enzymatic hydrolysis (M.sub.w below 9000 g/mol).

[0250] The method according to the invention (enzymatic hydrolysis with addition of hydrogen peroxide during or just after scalding, i.e. at the beginning of the method) does decrease the size of the molecule relative to what can be found initially in the insect (M.sub.w of chitin by simple enzymatic hydrolysis is 130000 g/mol), but to a much smaller extent (M.sub.w close to 80000 g/mol) and the result obtained is greater than that associated with conventional chemical extraction.

EXAMPLE 4: CHARACTERIZATION OF THE HYDROLYSATE AND THE CHITIN ACCORDING TO THE METHOD OF PRODUCTION USED

[0251] I. Material and Methods

[0252] a) Material

[0253] Insects

[0254] Various insects were studied: [0255] a coleopteron: Tenebrio molitor (T. molitor or TM), [0256] a lepidopteron: Galleria mellonella (G. melonella or GM), [0257] a dipteron: Hermetia illucens (H. illucens or HI), and [0258] an orthopteron: Acheta domesticus (A. domesticus or AD).

[0259] Enzymes

[0260] Various enzymes were used in the hydrolysis step.

[0261] This measurement of the enzymatic activity is based on the principle of measurement of tyrosine release at 275 nm during hydrolysis of casein by a proteolytic enzyme (Valley Research SAPU Assay method, by Karen PRATT).

[00001]$\frac{\mathrm{SAPU}}{g}=\frac{\left(\Delta .Math..Math.A-i\right)\times 11}{m\times 30\times C\times 1}$

[0262] SAPU/g=one spectrophotometric unit of protease

[0263] ΔA=correlated absorbance

[0264] i=y-axis at origin

[0265] 11=final reaction volume

[0266] M=slope of the calibration curve

[0267] 30=reaction time (in minutes)

[0268] C=concentration of the enzyme (g/mL) in the enzyme solution added

[0269] 1=1 mL volume of the enzyme solution added

[0270] The calibration curve is obtained by measuring the absorbance of tyrosine solutions of different concentrations in a phosphate buffer (0.2 M, pH 7).

[0271] 5 mL of a solution of casein (0.7% w/v, phosphate buffer 0.2 M, pH 7, heated for 30 minutes at 90° C. and with 3.75 g/L.sub.solution added) is incubated with 1 mL of the enzyme solution (0.15 g in 100 mL of glycine buffer, 0.05M) to be tested at 37° C. for 30 minutes. Then 5 mL of TCA solution is added (18 g TCA, 11.35 g of anhydrous sodium acetate, 21 mL of glacial acetic acid, made up with demineralized water to 1 litre of solution), mix on a vortex, filter and measure the absorbance at 275 nm.

[0272] The control is prepared identically but without adding enzyme, 1 mL of demineralized water is added instead in order to have the same reaction volume.

[0273] The activities thus measured for the various enzymes used (Prolyve NP, Novozyme 37071, Neutrase and Sumizyme) are listed in Table 3.

TABLE-US-00003 TABLE 2 Correspondence of the activities and enzyme masses of the enzymes used enzyme Prolyve Novozyme Neutrase Sumizyme Desired enzymatic 3789.52 3789.52 3789.52 3789.52 activity Enzymatic 3789.52 1662.35 2213.24 3237.19 activity/g m (g) 1.00 2.28 1.71 1.17

[0274] b) Methods of Production

[0275] Method of Production with Grinding Only (Denoted “Grinding” in the Figures)

[0276] 600 g of fresh insects (either larvae in the case of T. molitor, G. melonella or H. illucens; crickets in the case of A. domesticus) are introduced into a chamber, where they are killed with steam (115° C., 5 minutes). The insects are then introduced into a mixer and 75 mL of water is added per 100 g of insects, and the whole is then mixed. 100 g (wet weight) of product thus obtained is then introduced into a three-necked flask equipped with a condenser and a mechanical stirrer, and a proteolytic enzyme with an activity equivalent to 3789 SAPU is then added. The reaction is then heated to 45° C. for 4 hours. The temperature is then raised to 90° C. for 15 minutes, and the reaction mixture is finally filtered (0.40-0.45 μm). The residue is dried for 24 hours at 70° C.: this is therefore chitin obtained by the enzymatic route of purification; the filtrate is frozen and lyophilized: this is therefore the hydrolysate.

[0277] The method is identical whatever insect or enzyme is studied.

[0278] Method of Production with Grinding and Oxidizing Treatment of the Insect Cuticles (Designated “Grinding+H.sub.2O.sub.2” in the Figures)

[0279] 600 g of fresh insects (whether larvae in the case of T. molitor, G. melonella or H. illucens; crickets in the case of A. domesticus) are introduced into a chamber, where they are killed with the vapour of a water/H.sub.2O.sub.2 mixture (6%) (115° C., 5 minutes). The insects are then introduced into a mixer and 75 mL of water is added per 100 g of insects, and the whole is then mixed. 100 g (wet weight) of the product thus obtained is then introduced into a three-necked flask equipped with a condenser and a mechanical stirrer, and a proteolytic enzyme with activity equivalent to 3789 SAPU is then added. The reaction is then heated at 45° C. for 4 hours. The temperature is then raised to 90° C. for 15 minutes, and the reaction mixture is finally filtered (0.40-0.45 μm). The residue is dried for 24 hours at 70° C.: this is therefore chitin obtained by purification by the enzymatic route; the filtrate is frozen and lyophilized: this is therefore the hydrolysate.

[0280] The method is identical whatever insect or enzyme is studied.

[0281] c) Analyses

[0282] Measurement of the Ash Content

[0283] The ash content was determined by the method based on EC Regulation 152/2009 dated 27 Jan. 2009.

[0284] Measurement of the Protein Content

[0285] The protein content is obtained by the Dumas method, with a conversion factor of 6.25, adapted from standard NF EN ISO 16634-1.

[0286] Measurement of the Lipid Content

[0287] The lipid content is obtained by a method adapted from EC Regulation 152/2009-method B-SN.

[0288] Pepsin Digestibility

[0289] Pepsin digestibility is measured by the method described in Directive 72/199/EC.

[0290] Relative Abundance of Amino Acids

[0291] The abundance of the amino acids was determined by a method derived from EC Regulation 152/2009 dated 27 Jan. 2009-SN. The tryptophan content was determined separately by a method adapted from EC Regulation 152/2009 dated 27 Jan. 2009-SN. The relative abundance was calculated by relating the content of each amino acid to the amino acid content.

[0292] Amino Acid Content

[0293] The amino acid content was determined by adding together the individual values obtained for each amino acid, including tryptophan.

[0294] Colorimetric Purity

[0295] The colour of the sample was estimated by analysing photographs of samples using the ImageJ software according to the three colours red, green and blue (RGB), their average representing an assessment of the true colour. A sample of prawn chitin marketed by Chitine France was taken as the standard (100% purity) and the colorimetric purity of the samples produced was calculated as a percentage of this colour (ratio of the colour of the sample to the colour of the standard).

[0296] Purity by Difference

[0297] For this measurement, the quantities of known impurities (amino acids, lipids and ash) were subtracted from the value of absolute purity (100%) to obtain the value of the purity estimated by difference; i.e. a sample that contains 30% proteins, 10% lipids and 1% ash is therefore assigned a purity of 100−30−10−1=59%.

[0298] Measurement of the Degree of Crystallinity

[0299] The measurements were carried out according to the WAXS (wide angle X-ray scattering) technique on Bruker D8 Advance apparatus (A25 DaVinci design) equipped with a Lynxeye XE detector. The results were interpreted following the method described in Loelovich, M. Res. Rev.: J. Chem. 2014, 3, 7-14.

[0300] II. Hydrolysate

[0301] a) Ash

[0302] Adding the oxidizing agent contributes to a decrease in the ash content in the hydrolysate, whatever the insects (FIG. 5) and the enzyme used (FIG. 4). This decrease can reach 20% when Novozyme is used and 23.4% when the experiment is carried out with Acheta domesticus (A. domesticus).

[0303] b) Protein Content

[0304] Adding the oxidizing agent makes it possible to increase the content of proteins in the hydrolysate (FIG. 6), in particular for insects rich in lipids, such as flying insects.

[0305] c) Lipid Content

[0306] Adding the oxidizing agent can have a significant effect on the lipid content in the hydrolysate (FIG. 7), and this decrease can reach close to 21% in certain cases (TM+Sumizyme).

[0307] d) Pepsin Digestibility

[0308] Digestibility is little affected by adding the oxidizing agent, for all the tests carried out, and the pepsin digestibility of the hydrolysates that have undergone treatment by adding the oxidizing agent in the method is above 93% whatever the insect or enzyme used (FIG. 8).

[0309] e) Abundance of Amino Acids

[0310] The hydrolysates resulting from the method with treatment with an oxidizing agent predominantly consist of aspartic acid, glutamate and proline, and to a smaller extent valine, lysine, leucine, serine and alanine, whatever the insect or enzyme studied (FIGS. 9-15).

[0311] III. Chitin

[0312] a) Ash

[0313] Adding the oxidizing agent also has a beneficial effect on the decrease in the ash content in the chitin obtained, whatever the insect or enzyme studied (FIG. 16). The decrease observed can thus reach 35.7% relative to the ash content present in the method without a pressing step.

[0314] b) Lipid Content

[0315] In contrast to the hydrolysate, adding the oxidizing agent has a significant effect on the lipid content of the chitin. In fact, as the oxidizing agent is added before grinding the insects, it essentially affects the tannins and the waxes present on the surface of the cuticle. The decrease can thus reach as much as 30% in certain cases (FIG. 17).

[0316] The lipid content of the chitin thus obtained is below 29% whatever the insect, and even below 8.5% in the case of T. molitor (FIG. 18).

[0317] c) Content and Relative Abundance of Amino Acids

[0318] Adding the oxidizing agent contributes slightly to a decrease in protein load of the chitin (FIG. 19).

[0319] Regarding the relative abundance of amino acids bound to chitin obtained from the method with an oxidative step, it can be stated that for all of the insects, alanine, tyrosine and proline, and to a smaller extent valine, glycine, leucine and serine are the main amino acids attached to the chitin, and their content is on average between 20 and 40% of the total amino acids (FIGS. 20 to 26).

[0320] d) Colorimetric Purity

[0321] Owing to its action on the tannins essentially present on the surface of the cuticles, the oxidizing agent plays an important role in improving the colorimetric purity of the chitin obtained, regardless of which insect or enzyme is studied (FIGS. 27 and 28). There is quite particularly marked improvement in the case of G. melonella, the purity finally obtained even exceeding 100%, i.e. greater than that of the commercial chitin used as the standard (FIG. 28).

[0322] e) Purity by Difference

[0323] Owing to its effect on the content of ash, lipids and amino acids in the chitin, the oxidizing agent plays an important role in improving the purity by difference of the chitin (FIG. 29).

[0324] f) Degree of Crystallinity

[0325] Treatment with an oxidizing agent tends to make the chitin more amorphous (FIG. 30), whatever insect is studied. The degree of crystallinity, i.e. the ratio of the crystalline and amorphous areas, of the chitin obtained is between 0.29 and 0.32.