RU2728458C1 - Method of producing covalent-linked chitosan-melanin complex from black soldier fly hermetia illucens - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents a method for producing a covalent chitosan-melanin complex from dead black soldier fly Hermetia illucens. Essence of the proposed method consists in the fact that the initial dead flies mass is degreased with diethyl ether in the Soxhlet apparatus. Dried and ground mass is demineralised with 1 % HCl at room temperature for 2 hours with stirring, the solid residue is filtered on a porous glass filter, washed with distilled water till neutral pH values and lyophilically dried. Dried mass is treated with 30–50 % solution of NaOH at 90–100 °C for 2 hours with stirring, solid residue is filtered on porous glass filter, washed with distilled water till neutral pH values and lyophilically dried.EFFECT: invention enables to obtain a novel chitosan-melanin complex which combines the properties of these biopolymers.1 cl, 2 ex

Description

The invention relates to biotechnology and can be used to obtain chitosan-melanin complexes linked by a covalent bond.

The black lion fly Hermetia illucens is a unique insect whose larvae are able to process a wide range of organic waste. This technology is already being used on an industrial scale around the world. It is important to note that in addition to the disposal of organic waste and the production of fertilizers, protein and fat, the insect also serves as a source of other important biologically active substances, such as chitin, chitosan and melanin.

Melanin is formed at later stages of insect development and is bound to chitin by strong covalent bonds. Melanin is considered a natural photo- and radioprotector. Depending on the source and method of isolation, melanin has different physicochemical properties that can complement and enhance the biological activity of chitosan, a deacetylated chitin derivative, and be used to solve important scientific and practical problems.

A known method of producing chitosan-melanin complex from the dead bees [MI Selionov and N.V. Pogarskaya "Method of obtaining chitosan-melanin complex from dead bees", RF Patent No. 2382051, 21.07.2008]. The method includes preliminary drying of chitin-melanin raw materials to a moisture content of 4-6%, ensuring its crumbling, then the dried mass is ground to a powdery state, deproteinated with 10% NaOH alkali solution at a temperature of 78-82 ° C for 1.5-2.0 hours ... 50% NaOH solution is added to the reaction mixture, thus bringing its concentration to 30% of the original. The obtained chitin-melanin complex is deacetylated at a temperature of 90-95 ° C for 1.5-2.0 hours, filtered, while the filtered mass is obtained by washing the mass with distilled water until a pH of 7 is reached. Melanin is precipitated from the solution remaining after filtration by the action of concentrated hydrochloric acid until the pH of the treated solution changes to an acidic region and a flocculent precipitate, which is melanin, forms. Melanin is separated from the liquid phase by centrifugation, washed, separately dried chitosan and melanin at a temperature of 50-60 ° C to a moisture content of 8-10% and mixed to obtain the final product of the chitosan-melanin complex.

It is important to note that in this method, chitosan and melanin are obtained separately, and the complexes are obtained by mechanical mixing of two biopolymers.

Also, this method with some modifications was published in two articles [Obtaining a chitosan-melanin complex from the dead bees and its use for the prevention of dysbiosis of young animals (N.V. Pogarskaya, M.I. Selionova) S. 188-190, 2008] and [ N.V. Pogarskaya, M.I. Selionova, V.V. Binatova, Obtaining a chitosan-melanin complex from the dead bees and determining its physicochemical and biological characteristics, Vetkorm. - 2008. - No. 6. - S. 28-29]. Pre-dried to a moisture content of 3-4% bee podmore is ground in a mortar or in a drum mill to a particle size of 3-4 mm. A 10% NaOH solution is added to the resulting powder in a ratio of 1:10 by weight and deacetylation is carried out with constant stirring at 78-82 ° C in a closed vessel without air access for 1.5 hours. Then, after cooling, 50% is added in the same amount. NaOH solution, bringing the concentration of NaOH to 30% of the original, and the deacetylation process is carried out in a closed vessel at 90-97 ° C for 1.5-2.0 hours with periodic stirring. To isolate the chitosan-melanin complex, the resulting hydrolyzate is filtered off and washed until the wash water is neutral (pH 7). The extraction of melanin from the alkaline extract is carried out by precipitation with concentrated hydrochloric acid until the pH of the medium changes to an acidic region, followed by centrifugation of the solution at 5000 rpm for 15 min. The resulting melanin is washed until the wash water is neutral, followed by centrifugation and drying. The resulting melanin is dried simultaneously with the chitosan-melanin complex in an oven at 50 ° C. The output of the chitosan-melanin complex was about 24% by weight of the feedstock. According to the authors, it is impossible to obtain chitosan-melanin complexes from the death of bees, in which the components are connected by a covalent bond, since the destruction of such a complex occurs during the deacetylation reaction.

A known method of obtaining chitin from the larvae of the black lioness Hermetia illucens [S.A. Lopatin, A. Sh. Khairov, V.P. Varlamov, I.V. Sokolov "Method for obtaining chitin from the larvae of the black lioness Hermetia illucens", RF Patent No. 2680691, 25.02.2019]. The method of obtaining chitin from the larvae of the black lioness Hermetia illucens involves the processing of immobilized larvae on an oil press. To 30-50 g of the obtained solid residue, add 150-200 ml of 85% phosphoric acid, mix thoroughly and leave for 16-20 hours at room temperature. The resulting suspension is filtered under vacuum through a porous glass filter No. 1 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. The precipitated chitin precipitate in the filtrate is washed with water by decantation to pH 5.5-6.0 and lyophilized. The invention makes it possible to simplify the process of obtaining chitin from the larvae of the black lioness Hermetia illucens, as well as to obtain chitin in an amorphous form.

Also in [A. Khayrova, S. Lopatin, V. Varlamov, Black soldier fly Hermetia illucens as a novel source of chitin and chitosan // International Journal of Sciences, Vol. 8, pp. 81-86, 2019] shows that treatment of chitin from black lion cubs with 50% alkali makes it possible to obtain chitosan with a degree of deacetylation of 90%, while treatment with a weaker 30% alkali does not lead to deacetylation of chitin, and an increase in the concentration of NaOH leads to an increase in the content protein in the complex. This can be explained by the fact that more concentrated alkali is more viscous, which does not allow it to effectively wet matter and penetrate into it.

A known method of obtaining melanin from the larvae of a black lioness [RF Patent No. 2692260]. The method of obtaining melanin from the larvae of Hermetia illucens black lion flies aged 25 to 35 days consists in the fact that first the larvae are thoroughly washed and dried, then placed in an extruder and extrusion is carried out at a temperature of 37-38 ° C by cold pressing for one minute. , upon completion of extrusion, the resulting extrudate is allowed to settle before separation into fractions at a temperature of 0 to -15 ° C and the melanin layer obtained on the surface is separated mechanically, to re-obtain the melanin layer, the remaining mixture is kept at room temperature and the melanin layer obtained on the surface is separated mechanically method, after which, at the final stage, the remaining mixture is frozen, then it is heated to room temperature and the newly obtained melanin layer on the surface is separated mechanically, under certain conditions. The method allows you to obtain melanin by natural processing of larvae, without resorting to aggressive effects of chemical reagents.

This paper does not describe a method for determining impurities in the final product. According to the author, the melanin content was 88-90%, which indicates the presence of impurities. Fat, which is lighter than melanin, forms on the surface and forms part of the final product.

The above methods make it possible to obtain each of the biopolymers — chitin, chitosan and melanin — separately. Accordingly, to obtain a chitin- or chitosan-melanin complex, it is additionally required to combine biopolymers by mechanical means.

Closest to the claimed is a method of obtaining a chitosan-melanin complex from a black lion fly N. illucens [A.I. Bastrakov et al., Black lion fly Hermetia illucens in artificial breeding - a renewable source of the melanin-chitosan complex, News of the Ufa Scientific Center of the Russian Academy of Sciences, 2016, No. 4, pp. 77-79]. Nevertheless, the authors of the work do not describe the method itself, but refer to the work of N.V. Pogarskaya [Pogarskaya N.V., Selionova M.I., Binatova V.V. Production of chitosan-melanin complex from dead bees and identification of its physicochemical and biological characteristics, Vetkorm, 2008, no. 6, pp. 28-29.], Which indicates that the complexes are a mechanical mixture of biopolymers. Pigments were identified based on the assessment of their solubility, spectral and paramagnetic properties, which also indicates that melanin was obtained separately and then added to chitosan. It should be noted that according to our data, 30% of alkali is not enough to remove impurities (protein, fat) from the dead, so the final product is not pure.

From the above, it follows that previously in the literature, no methods have been described for obtaining covalently bound chitosan-melanin complexes from the black lion fly Hermetia illucens, and the content of impurities in these complexes has not been determined. We have developed a method for obtaining covalent chitosan-melanin complexes from the podmore Hermetia illucens.

Podmore Hermetia illucens served as a starting material for the production of chitosan-melanin complexes. The feedstock contained more than 30% protein, about 5% ash and 27% fat. The ash content was determined gravimetrically, the fat mass fraction in the sample was determined by extraction with an organic solvent in a Soxhlet apparatus, and the protein content by analysis of amino acids (free and bound) was determined by ion-exchange chromatography with post-column derivatization of samples with ninhydrin.

In order to overcome the above disadvantages, it is proposed to obtain covalently-bound chitosan-melanin complexes from the black lion fly Hermetia illucens (hereinafter referred to as H. Illucens) the use of H. Illucens (with a content of more than 30% protein, about 5% ash and 27 % fat).

The essence of the method for obtaining a covalently bound chitosan-melanin complex from a black lion fly H. illucens is as follows: 500 g of a black lion fly is defatted with diethyl ether in a Soxhlet apparatus, dried at room temperature in a fume hood and 325 g of defatted dead are obtained. Then the obtained fat-free dead water is ground in a mortar and fractionated through a sieve. The particle size is 0.2-0.5 mm (yield = 275 g). Next, 275 g of crushed and defatted mass is demineralized with 2.5 l of 1% HCl at room temperature for 2 hours with stirring. The solid residue is filtered on a porous glass filter No. 2, washed with distilled water to neutral pH values and lyophilized. Get 200 g of demineralized matter, containing less than 0.3% ash. From the obtained 200 g of demineralized matter take only 60 g, treated with 400-600 ml of 30-50% NaOH solution at 100 ° C for 2 hours with stirring, the solid residue is filtered on a porous glass filter. The obtained chitosan-melanin complex is washed with distilled water to pH 5.5-6.0 and freeze-dried. Get 11-23 g of covalently bound chitosan-melanin complex containing 2.2-4.3% protein.

The technical result of this method is to obtain a new product - chitosan-melanin complex, which combines the properties of these biopolymers.

The main difference of this method is that, in contrast to the previous methods of obtaining chitosan-melanin complexes, the resulting product is not a mechanical mixture of two compounds - chitosan and melanin, but a chemical compound, where both biopolymers are connected to each other by covalent bonds. In addition, in this method, the completeness of the removal of impurities is controlled, which has not previously been encountered in the literature. An important difference of the proposed method is the introduction of the initial stage of degreasing with an organic solvent. It was found that after treatment with 10% NaOH (90-100 ° C, 2 hours), the podmore contained 26% fat. When treated with 30% NaOH (90-100 ° C, 2 hours), 8% fat remained, and even when treated with 50% NaOH with alkali under the same conditions, fat is not completely removed (5.4% remains). It is possible that in addition to triglycerides, wax, which is not hydrolyzed by alkali, can also be contained in the black lioness. The presence of fat in biomass leads to a deterioration in its wettability and incomplete removal of ash and protein. As a result of treatment with diethyl ether, fat was removed from the feedstock.

Melanin content was shown from EPR data. The melanin content in terms of DOPA-melanin in the original submore was 10%, and in the complexes it exceeded 14%.

Determination of free amino groups in the resulting complexes was carried out using the reaction with ninhydrin according to the method [Taylor I., Hovard A.G. Measurement of primary amine groups on surface-modified silica and their role in metal binding // Analytica Chimica Acta. 1993. V. 271. No. 1. R.77-82]. The results of titration of amino groups show that after treatment with 50% NaOH, the concentration of amino groups significantly increases as compared to the matter treated with 30% NaOH, which confirms the occurrence of the deacetylation reaction of chitin.

Example 1.

500 g of the black lion fly dead fly is defatted with diethyl ether in a Soxhlet apparatus, dried at room temperature in a fume hood and 325 g of fat free dead fly is obtained. Then the drug is crushed in a mortar and fractionated through a sieve. The particle size is 0.2-0.5 mm (yield = 275 g).

275 g of crushed and defatted mass is demineralized with 2.5 l of 1% HCl at room temperature for 2 hours with stirring. The solid residue is filtered on a porous glass filter No. 2, washed with distilled water to neutral pH values and lyophilized. Get 200 g of demineralized matter, containing less than 0.3% ash.

60 g of demineralized matter is deproteinated by treatment with 600 ml of 50% NaOH solution at 100 ° C for 2 hours with stirring, the solid residue is filtered on a porous glass filter No. 2. The obtained chitosan-melanin complex is washed with distilled water to pH 5.5-6.0 and freeze-dried. 11 g of covalently bound chitosan-melanin complex containing 3.5% protein are obtained.

Example 2.

Analogously to example 1, but after the demineralization step 60 g of demineralized matter is deproteinated by treatment with 600 ml of 30% NaOH solution at 100 ° C for 2 hours with stirring. The solid residue is filtered on a porous glass filter No. 2. The obtained chitosan-melanin complex is washed with distilled water to pH 5.5-6.0 and freeze-dried. Get 13.5 g of a covalently bound chitosan-melanin complex with a protein content of 2.2%.

Example 3.

Analogously to example 1, but after the demineralization step, 60 g of demineralized matter are treated with 400 ml of 50% NaOH solution at 90 ° C for 2 hours with stirring. The solid residue is filtered on a glass porous filter No. 2, washed with distilled water to pH 5.5-6.0 and lyophilized. Get 23 g of covalent chitosan-melanin complex containing 4.3% protein.

Claims (1)

A method of obtaining a covalent chitosan-melanin complex from a black lion fly Hermetia illucens, which consists in the fact that 200-500 g of submarine is defatted with diethyl ether in a Soxhlet apparatus, dried at room temperature, crushed and fractionated through a sieve to 0.2-0.5 mm , demineralized with 1000-2500 ml of 2.5 L of 1% HCl at room temperature for 2 hours with stirring, the solid residue is filtered on a porous glass filter, washed with distilled water to neutral pH values and lyophilized, then the dried mass is treated with 30-50% NaOH solution at 90-100 ° C for 2 hours with stirring, the solid residue is filtered on a porous glass filter, washed with distilled water to pH 5.5-6.0 and lyophilized.