Method for obtaining chitin and/or chitosan using two enzymatic hydrolyses

Abstract

The present invention relates to a method for obtaining chitin and/or chitosan from insect cuticles. More particularly, the method according to the present invention comprises a first enzymatic hydrolysis of insect cuticles using at least one endopeptidase, separation from the hydrolysis medium of the hydrolyzed cuticles resulting from the first enzymatic hydrolysis, and a second enzymatic hydrolysis of the hydrolyzed cuticles using at least one endopeptidase, excluding exopeptidase.

Claims

1. Method for obtaining chitin from insect cuticles comprising: performing a first enzymatic hydrolysis of insect cuticles using at least one endopeptidase, separating the hydrolyzed cuticles resulting from the first enzymatic hydrolysis, from the hydrolysis medium, performing a second enzymatic hydrolysis of the hydrolyzed cuticles using at least one endopeptidase, excluding exopeptidase.

2. Method according to claim 1, wherein the at least one endopeptidase used in the second enzymatic hydrolysis is an endopeptidase or a mixture of endopeptidases selected from serine endopeptidases, cysteine endopeptidases, aspartic proteases, metalloendopeptidases and threonine endopeptidases.

3. Method according to claim 1, wherein the at least one endopeptidase used in the second enzymatic hydrolysis is a serine endopeptidase or a mixture of serine endopeptidases, and/or a metalloendoprotease or a mixture of metalloendoproteases, each serine endopeptidase being selected from the group constituted by subtilisin and trypsin, each metalloendoprotease being selected from the group constituted by bacillolysin and thermolysin.

4. Method according to claim 1, wherein the at least one endopeptidase used in the second enzymatic hydrolysis is of bacterial origin.

5. Method according to claim 1, further comprising a step of grinding the insect cuticles prior to the first enzymatic hydrolysis.

6. Method according to claim 1, wherein the insect cuticles are obtained by a step of separating the cuticles from the soft part of the insects.

7. Method according to claim 1, wherein the total time of the hydrolysis steps is less than 6 hours.

8. Method according to claim 1, wherein the total amount of said at least one endopeptidase used is between 10 U and 500 U per gram of insect cuticle, on a dry weight basis.

9. Method according to claim 1, further comprising, after the second hydrolysis, a chemical treatment step.

10. Method of obtaining chitosan comprising a method of obtaining chitin according to claim 1, and further comprising a step of deacetylating the chitin.

Description

[0157] The present invention is illustrated, in a non-limiting way, by the following examples and figures:

[0158] FIG. 1 is a diagram representing the residual protein content of Tenebrio molitor cuticles (in milligrams of protein per gram of solid residue) as a function of three different extraction methods using enzymatic hydrolysis with Alcalase 2.5 L PF.

[0159] FIG. 2 is a diagram representing the residual protein content of Tenebrio molitor cuticles (in milligrams of protein per gram of dry solid residue) as a function of two different extraction methods (simultaneous or sequential+filtration) using enzymatic hydrolysis with different serine endopeptidases.

[0160] FIG. 3 is a diagram representing the residual protein content of Tenebrio molitor cuticles (in milligrams of protein per gram of dry solid residue) according to two different extraction methods (simultaneous or sequential+filtration) using enzymatic hydrolysis with different metalloendopeptidases.

[0161] FIG. 4 is a diagram representing the residual protein content of Tenebrio molitor cuticles (in milligrams of protein per gram of dry solid residue) according to two different extraction methods (simultaneous or sequential+filtration) implementing enzymatic hydrolysis with a protease comprising only an exopeptidase (ProteAX) and/or a protease comprising only a serine endopeptidase (Alcalase 2.5 L PF).

[0162] FIG. 5 is a diagram representing the residual protein content of Tenebrio molitor cuticles (in milligrams of protein per gram of dry solid residue) according to two different extraction methods (simultaneous or sequential+filtration) using enzymatic hydrolysis with a protease comprising a mixture of endopeptidases and an exopeptidase (Sumizyme LP) and/or a protease comprising only a serine endopeptidase (Promod 439L or Alcalase 2.5 L PF).

EXAMPLE 1: OBTAINING GROUND INSECT CUTICLES

[0163] Live larvae of Tenebrio molitor, having received a diet based on cereal coproducts (wheat bran type), are conveyed in a layer of thickness comprised between 2 and 10 cm, on a belt with perforations (1 mm) to a blanching chamber. The larvae are then blanched with water at 92° C. (spray nozzles) for 5 min. The temperature of the larvae after blanching is comprised between 75° C. and 92° C.

[0164] After blanching, the larvae are conveyed to the feed hopper of a belt separator (belt separator 601 from Baader), in order to separate the cuticles from the soft part of the larvae. The diameter of the drum perforations is 1.3 mm. The separation is carried out immediately after the killing so that the larvae do not have time to cool to room temperature. The soft part of the insects is collected in a tank and the cuticles are collected using a scraper blade.

[0165] The cuticles are then ground in the presence of water (in a weight ratio of cuticles to water of 1:6, for example 1200 mL water to 200 g cuticles) using a Thermomix Vorwerk for 1 minute at speed 10, leading to ground cuticles with a size of about 1-2 mm. They are then filtered on a Büchner with a metal filter with a mesh size of 500 μm, and washed with water (same volume of water as used for grinding, i.e. 1200 mL water in this example).

[0166] Lastly, the washed cuticles are pressed (Angelia 7500, Angel) to remove excess water so as to obtain a dry matter content of 52% ±5 by weight.

Example 2: Effect of Filtration between Two Enzymatic Extractions with a Protease of Bacterial Origin—Alcalase 2.5 L PF

[0167] Alcalase 2.5 L PF is a serine endopeptidase derived from Bacillus licheniformis and marketed by Novozymes.

[0168] A total of 260 U of Alcalase 2.5 L PF is added to a mixture of 8.6 g of ground cuticles (wet weight) obtained according to the method described in Example 1, and 86 g of tap water, using three different methods. The pH of the reaction medium before the addition of protease in these three methods is 7.3. The three methods are carried out at a temperature of 55° C. and the results are shown in FIG. 1:

“Alcalase 1X”: the 260 U Alcalase are added in a single time and the hydrolysis reaction is left for 5 h.
“Alcalase/Alcalase No filtration”: 130 U Alcalase are added, the first hydrolysis is left for 2 h, then 130 U Alcalase are added again and the second hydrolysis is left for 3 h.
“Alcalase/Alcalase”: 130 U Alcalase are added, the first hydrolysis is left for 2 h and the reaction medium is then filtered (Büchner filter with a 160 μm metal mesh). The recovered cuticles are then mixed with 86 g tap water and 130 U Alcalase are added again, then the second hydrolysis is left for 3 h.

[0169] After inactivation of the enzyme for 15 min at 85° C., the reaction media are filtered (Büchner filter with a 160 μm metal mesh).

[0170] The residual protein content is determined by the BCA (BiCinchoninic acid Assay) method, using 2 mg of dry solid residue. The reaction medium obtained from the BCA method is diluted with water with a dilution factor of 4 prior to spectrophotometer measurement.

[0171] Thus, for a same total amount of added protease, deproteinization is slightly less efficient when two hydrolyses are carried out successively and without filtration compared to a single hydrolysis (respectively 41 mg and 38 mg of residual protein per gram of solid residue).

[0172] However, when two hydrolyses are carried out successively and with filtration between the two hydrolyses, the degree of deproteinization is significantly increased compared to the hydrolysis performed in a single time (31 mg and 38 mg of residual protein per gram of solid residue, respectively).

[0173] Therefore, a filtration step between two successive hydrolyses has the effect of increasing the degree of deproteinization of insect cuticles, and thus the purity of the resulting chitin.

[0174] Example 3: Method According to the Invention and Comparative Method—serine endopeptidase

[0175] The serine endopeptidases used in this Example 3 are subtilisin and pancreatin. The subtilisin is from two different suppliers: Alcalase 2.5 L PF (Novozymes) and Promod 439L (Biocatalysts), and is derived from Bacillus licheniformis. The pancreatin is from Sigma-Aldrich and is derived from Sus scrofa domesticus.

[0176] Two different methods were used: simultaneous (comparative method) and sequential+filtration (method according to the invention), the results of which are presented in FIG. 2.

[0177] Simultaneous Method

[0178] Protease is added to a mixture of 2 g of ground cuticles (dry weight), obtained according to the method described in Example 1, with 39.8 g of tap water at pH 7.3.

[0179] The simultaneous method was carried out by adding a total of 40 U of protease(s) per gram of cuticle (dry weight) to the mixture of cuticle and water, for a reaction time of 4 h and a temperature depending on the protease used.

[0180] Alcalase 2.5 L PF/Alcalase 2.5 L PF: a single hydrolysis step with 40 U of Alcalase 2.5 L PF per gram of cuticle (dry weight) and at a temperature of 55° C.

[0181] Promod 439 L/Promod 439 L: a single hydrolysis step with 40 U Promod 439 L per gram of cuticles (dry weight) and at a temperature of 57° C.

[0182] Alcalase 2.5 L PF/Promod 439 L: a single hydrolysis step with 20 U of Alcalase 2.5 L PF and with 20 U of Promod 439 L per gram of cuticles (dry weight), and at a temperature of 56° C.

[0183] Pancreatin/Pancreatin: a single hydrolysis step with 40 U of Pancreatin per gram of cuticle (dry weight) and at a temperature of 45° C.

[0184] Sequential Method+Filtration

[0185] Protease is added to a mixture of 2 g of ground and washed cuticles (dry weight), obtained according to the method described in Example 1, with 39.8 g of tap water at pH 7.3.

[0186] The sequential +filtration method was also performed by adding a total of 40 U of protease per gram of cuticle (dry weight) to the mixture of cuticle and water, for a total reaction time of 4 h and a temperature depending on the protease used. However, in this method, a first hydrolysis is performed with 20 U of protease per gram of cuticles (dry weight) for 2 h, then the hydrolysis medium is filtered (160 μm mesh); the recovered hydrolyzed cuticles are then mixed again with 39.8 g of tap water at a pH of 7.3 and a second hydrolysis is again performed with 20 U of protease per gram of cuticles (dry weight) for 2 h.

[0187] Alcalase 2.5 L PF/Alcalase 2.5 L PF: first hydrolysis with 20 U of Alcalase 2.5 L PF per gram of cuticle (dry weight) at a temperature of 55° C., then filtration of the hydrolysis medium, and second hydrolysis with 20 U of Alcalase 2.5 L PF per gram of cuticle (dry weight) at a temperature of 55° C. Promod 439L/Promod 439L: first hydrolysis with 20 U of Promod 439 L per gram of cuticles (dry weight) at a temperature of 57° C., then filtration of the hydrolysis medium, and second hydrolysis with 20 U of Promod 439 L per gram of cuticles (dry weight) at a temperature of 57° C.

[0188] Alcalase 2.5 L PF/Promod 439 L: first hydrolysis with 20 U Alcalase 2.5 L PF per gram of cuticles (dry weight) at a temperature of 55° C., then filtration of the hydrolysis medium, and second hydrolysis with 20 U of Promod 439 L per gram of cuticles (dry weight) at a temperature of 57° C.

[0189] Pancreatin/Pancreatin: first hydrolysis with 20 U Pancreatin per gram of cuticle (dry weight) at a temperature of 45° C., then filtration of the hydrolysis medium, and second hydrolysis with 20 U Pancreatin per gram of cuticle (dry weight) at a temperature of 45° C.

[0190] In both methods (simultaneous or sequential+filtration), the protease is then inactivated for 15 min at 85° C., and the reaction medium is filtered (160 μm mesh size).

[0191] The residual protein content is determined by the BCA (BiCinchoninic acid Assay) method, using 2 mg of dry solid residue. The reaction medium obtained from the BCA method is diluted with water with a dilution factor of 4 prior to spectrophotometer measurement.

[0192] FIG. 2 shows that when two successive hydrolyses are performed with the protease and with filtration between the two hydrolyses (sequential+filtration), the degree of deproteinization is significantly increased compared to hydrolysis performed in a single time (simultaneous), this being for the three proteases tested (see Alcalase 2.5 L PF/Alcalase 2.5 L PF, Promod 439 L/Promod 439 L and Pancreatin/Pancreatin), or for alternating proteases (see Alcalase 2.5 L PF/Promod 439 L).

[0193] Therefore, a filtration step between two hydrolyses with a serine endopeptidase has the effect of significantly increasing the degree of deproteinization of the insect cuticles, and thus the purity of the resulting chitin.

Example 4: Method According to the Invention and Comparative Method—metalloendopeptidase

[0194] The metalloendopeptidases used in this Example 4 are bacillolysin and thermolysin. The bacillolysin is FoodPro PNL which is from Dupont Danisco and is derived from Bacillus amyloliquefaciens. The thermolysin is Corolase 2TS from AB Enzymes and is derived from Bacillus stearothermophilus.

[0195] The simultaneous and sequential+filtration methods of Example 3 were replicated with these metalloendopeptidases and at a temperature of 50° C. for FoodPro PNL and 60° C. for Corolase 2TS. The results are presented in FIG. 3.

[0196] FIG. 3 shows that when two successive hydrolyses are performed with FoodPro PNL or Corolase 2TS and with filtration between the two hydrolyses (sequential+filtration), the degree of deproteinization is significantly increased compared to hydrolysis performed in a single time (simultaneous).

[0197] Therefore, a filtration step between two hydrolyses with a metalloendopeptidase has the effect of significantly increasing the degree of deproteinization of insect cuticles, and thus the purity of the resulting chitin.

Example 5: Influence of the Nature of the Protease: Requirement for the Presence of an endopeptidase in each Hydrolysis Step

[0198] In order to study the influence of the endo- or exopeptidase nature on the degree of deproteinization, a protease comprising mainly aminopeptidase (exopeptidase) was used (ProteAX derived from Aspergillus oryzae and sold by Amano Enzyme; IUB number stated by the supplier: 3.4.11.1), as well as subtilisin (Alcalase 2.5 L PF).

[0199] The simultaneous and sequential+filtration methods of Example 3 were replicated, with the results shown in FIG. 4: [0200] ProteAX/ProteAX: the temperature is 50° C. [0201] Alcalase 2.5 L PF/ProteAX: for the simultaneous method, the temperature is 52.5° C.; for the sequential method, Alcalase 2.5 L PF is added first at a temperature of 55° C., and ProteAX is added second at a temperature of 50° C. [0202] ProteAX/Alcalase 2.5 L PF: for the simultaneous method, the temperature is 52.5° C.; for the sequential method, ProteAX is added first at a temperature of 50° C., and Alcalase 2.5 L PF is added second at a temperature of 55° C.

[0203] FIG. 4 shows that when two successive hydrolyses are performed with ProteAX and with filtration between the two hydrolyses, the rate of deproteinization is significantly decreased compared to hydrolysis performed in a single time. Moreover, the fact that two successive hydrolyses are performed with ProteAX before or after Alcalase 2.5 L PF does not make it possible to increase the degree of deproteinization compared to a hydrolysis performed in a single time with both proteases (simultaneous addition): the use of ProteAX in a sequential method+filtration therefore significantly decreases the degree of deproteinization.

[0204] Therefore, in the method according to the invention, the presence of an endopeptidase is necessary in each hydrolysis step in order to increase the deproteinization rate.

Example 6: Influence of the Nature of the Protease in the Second Enzymatic Hydrolysis: Requirement for a Protease not including an exopeptidase in the Second Hydrolysis Step

[0205] In order to study the influence of the use of a protease comprising both endopeptidases and an exopeptidase on the degree of deproteinization, a protease comprising a mixture of oryzin, aspergillopepsin I (endopeptidases) and leucyl aminopeptidase (exopeptidase) was used (Sumizyme LP derived from Aspergillus oryzae and sold by Shin Nihon Chemical), optionally alternating with subtilisin (Alcalase 2.5 L PF and Promod 439 L).

[0206] The simultaneous and sequential+filtration methods of Example 3 were replicated, with the results shown in FIG. 5: [0207] Sumizyme LP/Sumizyme LP: the temperature is 45° C. [0208] Promod 439L/Sumizyme LP: for the simultaneous method, the temperature is 50° C.; for the sequential method, Promod 439L is added first at a temperature of 57° C., and Sumizyme LP is added second at a temperature of 45° C. [0209] Sumizyme LP/Promod 439 L: for the simultaneous method, the temperature is 50° C.; for the sequential method, Sumizyme LP is added first at a temperature of 45° C., and Promod 439 L is added second at a temperature of 57° C. [0210] Sumizyme LP/Alcalase 2.5 L PF: for the simultaneous method, the temperature is 50° C.; for the sequential method, Sumizyme LP is added first at a temperature of 45° C., and Alcalase 2.5 L PF is added second at a temperature of 55° C.

[0211] When two successive hydrolyses are performed with Sumizyme LP and with filtration between the two hydrolyses, the degree of deproteinization is similar to the hydrolysis performed in a single time. Furthermore, a second hydrolysis with Sumizyme LP after a first hydrolysis with Promod 439 L followed by filtration significantly decreases the degree of deproteinization compared to a hydrolysis carried out in a single time.

[0212] However, if the first hydrolysis is performed with Sumizyme LP and the second with Promod 439 L in the sequential+filtration method (Sumizyme LP/Promod 439 L), then the degree of deproteinization is significantly increased compared to the hydrolysis performed at a single time. The case is similar when Promod 439L is replaced by Alcalase 2.5 L PF (Sumizyme LP/Alcalase 2.5 L PF).

[0213] Therefore, when filtration is carried out between two enzymatic hydrolyses, it is not necessary for the protease used in the first hydrolysis not to comprise exopeptidase. However, this condition is imperative for the protease used in the second hydrolysis, and allows the degree of deproteinization to be significantly increased.