Method for obtaining chitosan from insects

Abstract

FIELD: food and agricultural industries.SUBSTANCE: method for obtaining chitosan from cockroaches Nauphoeta cinerea involves grinding the cockroach cake to a fraction of 0.5-1.0 mm, adding 1 M HCl to the ground cake in a ratio of 1:15 weight:volume, followed by processing the resulting mixture in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 30-60°C for 30-60 min, rinsing with distilled water to neutral pH, adding 1 M NaOH to the washed mixture in a ratio of 1:15 mass:volume, followed by processing the resulting mixture using an ultrasonic probe with a frequency of 20 kHz, power of 500 W, pulse mode 5 / 5 s, at a temperature of 50-70 °C for one hour, rinsing with distilled water to neutral pH, followed by drying at 60 °C with the production of chitin. Next, a 50% NaOH solution is added to the resulting chitin in a ratio of 1:20 mass:volume, as well as NaBH4, where the mass of NaBH4 is 0.1% of the volume of the NaOH solution, followed by treatment of the resulting mixture in an ultrasonic bath at 70-100 °C for 3-5 hours, cooling the mixture after the stage to a temperature of 10-20 °C followed by neutralization to pH 7.0 and purification of the resulting product by dialysis against pure water followed by lyophilization of the resulting chitosan. The crushed cake may additionally undergo degreasing with hexane in a 1:3 mass:volume ratio for 2 hours using the circulation extraction method. The obtained chitin is further bleached with 2% hydrogen peroxide in a ratio of 1:10 mass:volume in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 40 °C for 30 minutes.EFFECT: expanding the range of methods for producing natural chitosan, ensuring a high-quality product with a deacetylation degree of up to 93% and improved chelating properties.11 cl, 3 dwg, 2 tbl, 1 ex

Description

[0001] Technical field

[0002] The present invention relates to a method for producing chitosan from Nauphoeta cinerea cockroaches.

[0003] Technology Level

[0004] Chitin and its derivatives, such as deacetylated chitin, known as chitosan, are biocompatible and biodegradable polymers that have the potential to become a major source of bioactive materials, such as in the pharmaceutical, food, waste recycling and reclamation industries [Ezinne C. Achinivu et al. Chitin extracted from various biomass sources: It's not the same / Fluid Phase Equilibria, Volume 552, 15 January 2022, 113286, https: / / doi.org / 10.1016 / j.fluid.2021.113286]. Chitin is synthesized by a vast number of living organisms and is known as the second most abundant natural polysaccharide after cellulose, which consists of a linear chain composed of poly β (1→4) N-acetyl-D-glucosamine. It is commonly found in the shells of crustaceans such as shrimp, crabs, lobsters, etc. Chitin can also be isolated from fungi and insects.Numerous studies have shown that the cell walls of fungi, corals, algae, and nematodes are sources of chitin [F. Hisham et al. Facile extraction of chitin and chitosan from shrimp shell / Volume 42, Part 5, 2021, Pages 2369–2373, https: / / doi.org / 10.1016 / j.matpr.2020.12.329]. Until now, the main commercial sources of chitin have been crab and shrimp shells.

[0005] In industrial processing, chitin production technologies include the use of alkalis, acids, enzyme preparations of animal and plant origin, pressing, high temperatures, freezing, pressure, vacuum, as well as a combination of the listed physical and chemical factors. However, all these processes must be adapted to the source of chitin due to differences in the ultrastructure of the starting material in order to first obtain high-quality chitin and then chitosan (after partial deacetylation) [Islem Younes et al. Chitin and Chitosan Preparation from Marine Sources. Structure, Properties and Applications / Mar Drugs. 2015 Mar 2;13(3):1133-1174. doi: 10.3390 / md13031133].

[0006] A prior art method for obtaining chitin from the larvae of the black soldier fly Hermetia illucens is known, which consists in the fact that the immobilized larvae are processed in an oil press, and 150-200 ml of 85% phosphoric acid are added to 30-50 g of the resulting solid residue, mixed thoroughly and left for 16-20 hours at room temperature, the resulting suspension is filtered under vacuum through a No. 1 glass porous filter and washed with 70-100 ml of 40% phosphoric acid, the combined filtrate is diluted with 2.5-3.0 l of water with vigorous stirring, and the precipitated chitin in the filtrate is washed with water by decantation to a pH of 5.5-6.0 and lyophilized [RU2680691].

[0007] A method for obtaining chitin is known, characterized in that crushed biomass of insect larvae is poured in a ratio of 1:1-1:8, 0.01-1.0% alkali solution, stirred for 5-30 minutes at a temperature of 25-100 ° C, then after inoculation with lactic acid bacteria, fermented under stationary conditions with a fermentation duration of 1.5-7.0 hours at a temperature of 25-45 ° C, chitin is separated, washed with water and additional demineralization is carried out with an acid solution, after which the acid solution is removed, chitin is washed and dried [RU2526936].

[0008] A method for obtaining chitosan from chitin is known from the prior art, which includes the process of deacetylation of chitin pre-crushed to a size of 1-2×2-3 mm in a two-necked reactor equipped with a thermometer, a reflux condenser and connected to a vacuum water-jet pump, in a 40% NaOH solution at a ratio to chitin (1-3):(20-60) at 100°C, a pressure of 300 mm Hg for 1-2 hours [RU2539933].

[0009] A method for extracting chitin from sea cockroaches using acid and alkali is known [CN115232228A].

[0010] The search for new sources of natural chitin, as well as the development of new and effective methods for its isolation, as well as the production of chitosan, is a promising and non-trivial task.

[0011] Disclosure of invention

[0012] The object of the present invention is to develop a new and efficient method for obtaining chitosan from Nauphoeta cinerea cockroaches.

[0013] The said problem is solved by implementing a method for obtaining chitosan from Nauphoeta cinerea cockroaches, which includes the following steps:

[0014] a) grinding cockroach cake to a fraction of 0.5-1.0 mm;

[0015] b) adding 1 M HCl to the cake crushed after step a) in a ratio of 1:15 weight:volume, followed by processing the resulting mixture in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 30-60°C for 30-60 minutes;

[0016] c) rinsing with distilled water to neutral pH;

[0017] d) adding 1 M NaOH to the washed mixture after step c) in a ratio of 1:15 weight:volume, followed by treating the resulting mixture with an ultrasonic probe with a frequency of 20 kHz, a power of 500 W, pulse mode 5 / 5 sec, at a temperature of 50-70 °C for one hour;

[0018] d) washing with distilled water to neutral pH, followed by drying at 60°C and obtaining chitin;

[0019] e) adding to chitin after step d) a 50% NaOH solution in a 1:20 weight:volume ratio, as well as NaBH4, where the mass of NaBH4 is 0.1% of the volume of the NaOH solution, followed by treating the resulting mixture in an ultrasonic bath at 70-100°C for 3-5 hours;

[0020] g) cooling the mixture after step e) to a temperature of 10-20°C, followed by neutralization to pH 7.0;

[0021] z) purification of the obtained product by dialysis against water, followed by lyophilization of the obtained chitosan.

[0022] In particular embodiments of the invention, the grinding of the cake in stage a) is carried out in a knife mill.

[0023] In particular embodiments of the invention, after step a), the crushed cake is additionally defatted with hexane in a weight ratio of 1:3 for 2 hours using the circulation extraction method (i.e. with reverse reflux).

[0024] In particular embodiments of the invention, step b) is carried out with constant stirring.

[0025] In particular embodiments of the invention, the pH in step d) is 13-14.

[0026] In particular embodiments of the invention, after step d), a bleaching step is carried out using 2% hydrogen peroxide in a 1:10 weight:volume ratio in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 40°C for 30 minutes.

[0027] In particular embodiments of the invention, the temperature in step b) is 45°C.

[0028] In particular embodiments of the invention, the treatment in an ultrasonic bath at stage b) is carried out for 45 minutes.

[0029] In particular embodiments of the invention, the temperature in step e) is 90°C.

[0030] In particular embodiments of the invention, the ultrasonic bath treatment of stage e) is carried out for 4 hours.

[0031] In particular embodiments of the invention, the temperature in step d) is 60°C.

[0032] As a result of implementing the invention, the following technical results are achieved:

[0033] - a new and effective method for obtaining chitosan from Nauphoeta cinerea cockroaches has been developed, which is characterized by a low time of the demineralization stage and a high yield of chitosan from chitin;

[0034] - the method according to the invention is a convenient and simple process that ensures reliable results and the production of a high-quality product (high degree of deacetylation up to 93% - a high amount of free amino groups improves the chelating properties of the resulting chitosan, low ash and protein content, i.e. low content of undesirable minerals and allergenicity of the resulting chitosan) with stable characteristics, which is carried out without the use of high temperatures and pressure, in addition, the said method is easily scalable;

[0035] - the method according to the invention expands the arsenal of available means for obtaining chitosan from Nauphoeta cinerea cockroaches.

[0036] Detailed disclosure of the invention

[0037] Brief description of drawings

[0038] Figure 1. FT-IR spectrum of chitin, where CT-ANC is chitin of the adult form of cockroaches of the species Nauphoeta cinerea; CE-AAM is chitin of the adult form of honey bee Ápis melliféra.

[0039] Figure 2. FT-IR spectrum of chitosan, where CS-ANC is chitosan from adult cockroaches of the species Nauphoeta cinerea; CS-AAM is chitosan from adult honeybee Ápis melliféra, CS-SA is chitosan from crustacean shells, China (commercially available standard).

[0040] Figure 3. Scanning electron microscopy data, micrographs of the surface of chitin (CT) and chitosan (CS) obtained by the method according to the invention: a) chitin, 50-fold magnification; b) chitosan, 50-fold magnification; c) chitin, 500-fold magnification; d) chitosan, 500-fold magnification.

[0041] Definitions (terms)

[0042] For a better understanding of the present invention, certain terms used in this description of the invention are provided below. The following definitions apply throughout this document unless otherwise expressly stated.

[0043] In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things." These terms are not intended to be construed as meaning "consists only of."

[0044] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0045] Implementation of the invention

[0046] The invention aims to obtain high-quality, high-molecular-weight deacetylated chitosan from Nauphoeta cinerea cockroaches. A combination of traditional methods and modern physicochemical approaches allows us to propose an improved and efficient method for isolating chitin and chitosan from Nauphoeta cinerea marbled cockroach cake according to the present invention. The cake, after oil extraction, is a good raw material source:

[0047] • partially defatted and deproteinized;

[0048] • retains chitinous exoskeleton;

[0049] • Contains residual protein and minerals.

[0050] The ultrasonic treatment used in the method according to the invention intensifies the processes due to:

[0051] • cavitation, destroying chitin-protein complexes;

[0052] • acceleration of mass transfer of reagents;

[0053] • Reduction of processing time and reagent concentrations.

[0054] Example 1

[0055] Biomass preparation

[0056] • Euthanasia of adult brown marmorated cockroaches (Nauphoeta cinerea) is performed by keeping them at -20°C for 120 minutes.

[0057] • Biomass is homogenized in an aqueous medium in a ratio of 1:2 (weight:volume) in a PM 100 bead mill (Retsch, Germany) at a rotation speed of 400 rpm for 30 minutes.

[0058] • The resulting suspension is pressed on a screw oil press, PITEKA-100, at a pressure of 35-40 MPa and a temperature of 45-50°C.

[0059] 200 g of cockroach cake was ground to a 0.5-1.0 mm particle size using a knife mill. It was then degreased with hexane (hexane volume = 200 g × 3 ml / g = 600 ml) at a 1:3 (weight:volume) ratio for 2 hours using the circulation extraction method in a reverse reflux apparatus. The mixture was heated with continuous return of hexane vapor to the reaction system for 2 hours at a temperature of ~50°C. Next, a demineralization step with ultrasonic (hereinafter referred to as US) treatment was performed. Specifically, 1 M HCl was added to the crushed cake (1:15, weight:volume). 200 g of the crushed cake was treated with 3 liters of 1 M hydrochloric acid solution, gently stirred until the cockroach cake was evenly distributed, after which the resulting mixture was treated in an ultrasonic bath at a frequency of 40 kHz, a power of 300 W at 45°C for 45 minutes with constant stirring. The mixture was then washed with distilled water until a neutral pH was achieved.Then, a deproteinization stage with ultrasound intensification was carried out, namely, 1 M NaOH was added (1 M aqueous NaOH solution was added to the precipitate washed to a neutral pH in a ratio of 1:15 (weight:volume), which for a raw material batch weighing 10 g amounted to 150 ml of alkali solution.) to the washed mixture, gently mixed until homogeneous, after which the resulting mixture was treated with an ultrasonic probe with a frequency of 20 kHz, a power of 500 W, pulse mode 5 / 5 sec, at a temperature of 60 °C for one hour. Next, it was washed with distilled water to a neutral pH (pH 7.0), followed by drying at 60 °C. Afterwards, a bleaching step of the obtained intermediate product (chitin) was carried out using a 2% aqueous solution of hydrogen peroxide (% (w / v) - means 2 grams of H2O2 per 100 ml of the prepared aqueous solution, a standard commercially available solution of 2% hydrogen peroxide) in a ratio of 1:10 weight:volume in an ultrasonic bath at 40°C for 30 minutes.Next came the stage of deacetylation of the obtained chitin to chitosan, namely, 50% (50% is the mass fraction (m / m), that is, 500 grams of NaOH per 1000 grams of the finished solution) NaOH solution (1:20, mass:volume is the mass-volume (m / V) ratio between the mass of chitin (in grams) and the volume of this alkali solution (in milliliters)), NaBH4 (for every 1 g of the obtained chitin, 20 ml of 50% NaOH solution and 20 mg of sodium borohydride are added) were added, followed by treatment of the resulting mixture in an ultrasonic bath at 90°C for 4 hours.

[0060] This was followed by chitosan precipitation, namely, cooling the product to room temperature and neutralizing it to pH 7.0. The resulting product was purified by dialysis against pure water, followed by lyophilization of the resulting chitosan. The characteristics of the resulting product are presented in Table 1.

[0061] Table 1. Characteristics of chitin and chitosan obtained by the method of the invention.

[0062] Parameter Meaning Initial mass of cake 200 g Chitin yield 36 grams Chitosan yield 30 g Degree of deacetylation (DD) 93% Mol. mass of chitosan 125 kDa

[0063] Study of the characteristics and properties of chitosan obtained by the method according to the invention.

[0064] Materials and Methods

[0065] List of abbreviations:

[0066] CT - Chitin;

[0067] CS - Chitosan;

[0068] ANC - adult form of cockroaches of the species Nauphoeta cinerea;

[0069] AAM - adult form of the honey bee Ápis melliféra.

[0070] SA - control, commercial chitosan from crustacean shells, China.

[0071] Honey bee dead, sample received from the Moscow State Academy of Veterinary Medicine and Biology - the K.I. Skryabin Moscow State Academy of Veterinary Medicine,

[0072] A study was conducted on the characteristics and properties of chitosan obtained by the method according to the invention, including a study on:

[0073] • Deacetylation degree by FTIR (Fourier-infrared spectroscopy) - the method was used to quantitatively determine the content of free amino groups, which characterizes the efficiency of the deacetylation process of chitin to chitosan.

[0074] Absorption spectra were recorded on a Bruker INVENIO Fourier transform infrared spectrometer in the range of 4000-500 cm -1 Samples were prepared by pressing tablets with potassium bromide. The degree of deacetylation (DD, %) was calculated using the formula: DD% = 100 - [(A1655 / A3450) × 100 / 1.33], where A1655 and A3450 are the optical densities at 1655 cm -1 (amide I) and 3450 cm-1 (hydroxyl group), respectively.

[0075] • Thermal stability by TGA (Thermogravimetric Analysis) - the analysis was used to determine the temperature characteristics and stability of the polymer, which is critical for determining the conditions for its further processing and use. Thermal stability was studied using a ZRT-1 analyzer. Samples of 5–10 mg were heated from 20 to 600°C at a rate of 5°C / min under a nitrogen atmosphere. The onset temperature of decomposition and residual mass at 600°C were determined.

[0077] • (Degrees) of Crystallinity by XRD (X-ray diffraction analysis) - the method was used to assess the degree of ordering of macromolecules, which directly affects the physical, mechanical and sorption properties of the material.

[0078] Diffraction patterns were obtained on a Shimadzu XRD-7000 diffractometer with CuKα radiation (λ = 1.5406 Å) at 30 kV and 40 mA. Scanning was carried out in the range of 2θ = 5-80° at a rate of 10° / min. The crystallinity index (CrI, %) was calculated using the formula: CrI(%)=[(I 110 -Ia m ) / I 110 ]×100, where I 110 - intensity at 2θ ≈ 20° (crystalline region), Ia m - intensity at 2θ ≈ 16° (amorphous region).

[0079] • Surface morphology by SEM (Scanning Electron Microscopy) - the study was conducted to visualize the microstructure and porosity of the surface, which determines the availability of active centers for sorption.

[0080] Surface morphology was examined using a TESCAN MIRA 3 microscope at an accelerating voltage of 20 kV. Samples were sputter-coated with gold in a vacuum. Images were acquired at magnifications of 100× and 1000×.

[0081] • Molecular weight - determination was carried out to establish the length of polymer chains, which affects the viscosity, solubility and functional activity of chitosan.

[0082] The molecular weight was determined by the viscometric method. The intrinsic viscosity [η] was measured in 0.5 M acetate buffer (0.5 M CH3COOH + 0.5 M CH3COONa) at 25°C. The molecular weight (M \ / ) was calculated using the Mark-Houwink equation: [η]=K⋅ M \ / ª, where K = 0.119 and a = 0.59 are constants for the chitosan-acetate buffer system.

[0083] All measurements were performed in triplicate, the results are presented as mean ± standard deviation.

[0084] The results of the studies are presented in Table 2.

[0085] Table 2. Results of the study of the characteristics of chitin and chitosan obtained by the method of the invention.

[0086] Parameter Nauphoeta cinerea (claimed method) Crustaceans (Shrimp) (Luo et al., 2019, Table 1) Chitin yield, % 18 ~25* Chitosan yield, % of chitin 82 ~75* Degree of deacetylation (DD), % 93 91.2 Mol. mass of chitosan, kDa 125 162.0 Ash content, % 0.8 0.95 Fat sorption (FBC), % 500 438 Viscosity (2% solution), cPz 300 >2000 Chitin crystallinity, % 55 85.21 ** - Values ​​generally accepted for shrimp shells are provided for reference, as yields are not reported in Luo et al. (2019).

[0087] Fourier transform infrared spectroscopy (FT-IR) was used to analyze the chitin obtained after step d) according to the claimed method and the chitin from the adult form of the honeybee Ápis melliféra. The FT-IR spectra are shown in Figures 1 and 2. The obtained IR spectra matched the test substance and the standard, indicating that the obtained substance is chitosan.

[0088] The surface of the chitin and chitosan obtained by the method of the invention was also examined. Surface micrographs of the chitin (CT) and chitosan (CS) obtained by the method of the invention (SEM data) are shown in Figure 3. The SEM data clearly demonstrate that the obtained product has a highly developed nanoporous structure. This unique morphology directly underlies the improved sorption properties.

[0089] Thus, as a result of implementing the method according to the invention, chitosan was obtained from marbled cockroaches Nauphoeta cinerea, which is characterized by the following advantages:

[0090] 1. High degree of deacetylation (93%)

[0091] - More free amino groups (-NH2);

[0092] - Improved chelating properties.

[0093] 2. Optimum average molecular weight (125 kDa)

[0094] - High solubility;

[0095] - High availability of active centers.

[0096] 3. Low ash content (0.5-1.0%)

[0097] - Minimum mineral impurities;

[0098] - Large active surface.

[0099] 4. Improved sorption characteristics:

[0100] - Cu 2+ : 120-150 mg / g (20-30% higher than analogues);

[0101] - Fat: 450-500% (15-20% higher than standard samples).

[0102] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

1. A method for obtaining chitosan from cockroaches Nauphoeta cinerea, comprising the following steps: a) grinding of cockroach cake to a fraction of 0.5-1.0 mm; b) adding 1 M HCl to the cake crushed after step a) in a ratio of 1:15 weight:volume, followed by processing the resulting mixture in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 30-60°C for 30-60 min; c) rinsing with distilled water until neutral pH; d) adding 1 M NaOH to the washed mixture after step c) in a ratio of 1:15 mass:volume, followed by treatment of the resulting mixture using an ultrasonic probe with a frequency of 20 kHz, a power of 500 W, pulse mode 5 / 5 s, at a temperature of 50-70°C for one hour; d) washing with distilled water to neutral pH, followed by drying at 60°C and obtaining chitin; e) adding to chitin after step d) a 50% NaOH solution in a 1:20 weight:volume ratio, as well as NaBH4, where the mass of NaBH4 is 0.1% of the volume of the NaOH solution, followed by treatment of the resulting mixture in an ultrasonic bath at 70-100°C for 3-5 hours; g) cooling the mixture after step e) to a temperature of 10-20°C, followed by neutralization to pH 7.0; c) purification of the obtained product by dialysis against pure water, followed by lyophilization of the obtained chitosan.

2. The method according to claim 1, in which the grinding of the cake in stage a) is carried out in a knife mill.

3. The method according to claim 1, wherein after step a) the crushed cake is additionally defatted with hexane in a ratio of 1:3 mass:volume for 2 hours by the circulation extraction method.

4. The method according to claim 1, wherein step b) is carried out with constant stirring.

5. The method according to claim 1, wherein the pH in step d) is 13-14.

6. The method according to claim 1, wherein after step d) a bleaching step is carried out using 2% hydrogen peroxide in a ratio of 1:10 weight:volume in an ultrasonic bath with a frequency of 40 kHz, a power of 300 W at 40°C for 30 minutes.

7. The method according to claim 1, wherein the temperature in step b) is 45°C.

8. The method according to claim 1, wherein the treatment in an ultrasonic bath in step b) is carried out for 45 minutes.

9. The method according to claim 1, wherein the temperature in step e) is 90°C.

10. The method according to claim 1, wherein the ultrasonic bath treatment of step e) is carried out for 4 hours.

11. The method according to claim 1, wherein the temperature in step d) is 60°C.

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