Preparation method of chitin-glucan complex based mushroom mycelium

Abstract

The present invention provides a method for producing a chitin-glucan complex derived from mushroom mycelium, comprising the steps of: (1) heat-treating mushroom mycelium at pH 7.5 to 8.5 at 60 to 100°C for 2 to 6 hours; (2) reacting flavozyme and amyloglucosidase at pH 4 to 5 at 35 to 45°C for 2 to 6 hours to remove protein and glycogen; and freeze-drying the reaction product of step (2).

Description

Technology Field

[0001] The present invention relates to a method for producing a chitin-glucan complex derived from mushroom mycelium, and more specifically, to a method for producing a chitin-glucan complex derived from mushroom mycelium in which, at the step of culturing the mycelium, it is possible to cultivate the mycelium into an isotropic fibrous structure of a certain size and significantly shorten the cultivation period by maintaining the activity of the mycelium, and a large amount of chitin-glucan complex can be obtained cheaply from the cultured mycelium. Background Technology

[0002] Mushroom mycelium is a non-animal fungal-based biopolymer that has the characteristic of growing continuously in a liquid medium when conditions for asexual reproduction are met, making mass production on an industrial scale possible. Although there are variations depending on the species, nitrogen consumption is not high, so low-cost agricultural and by-products can be used as metabolites.

[0003] Compared to terrestrial animals and plants, these have a very short production cycle of 10 to 15 days, requiring very little energy and water resources, and are sustainable future food and pharmaceutical materials that can practice ESG in an eco-friendly way.

[0004] As glucan, chitin-glucan complex, and manno-protein, which are components of the cell walls of mushroom mycelium attracting industrial attention, β Glucan has the characteristic of being able to replace materials traditionally used for anticancer, immune activity, blood sugar control, antiviral, skin moisturizing and regeneration, chitin-glucan complex has the characteristic of being used for biodegradable films, heavy metal adsorption filters, and mass transfer matrix materials (drug delivery systems), and protein has the characteristic of being able to replace materials traditionally used for food and pharmaceuticals, cosmetics, alternative foods, alternative dairy products, and alternative leather as animal / plant protein substitute materials.

[0005] In particular, the chitin-glucan complex is a major cell wall component of fungi and yeast and consists of (1→4)-2-acetamide-2-deoxy-β-D-glucan (acylation unit) / (1→4)-2-amino-2-deoxy-β-D-glucan (deacylation unit) and (1→3)(1→6)-β-D-glucan.

[0006] Until recently, structures in which chitin is bonded to (1→3)(1→6)-β-D-glucan have not received industrial attention because the presence of various intermolecular or intramolecular hydrogen bonds makes them insoluble in water, acidic or alkaline solutions, and most organic solvents, making processing difficult and causing the chemical structure to decompose before reaching the melting point (Tm) during heat treatment. Additionally, due to properties similar to chitin, chitin-glucan complexes are not suitable for industrial application because treatment with toxic or corrosive polar solvents such as N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) increases solubility but becomes highly chemically unstable (Boureghda et al., 2021).

[0007] Studies have been reported on the synthesis of chitosan-glucan from mushroom fruiting bodies using methods such as deacetylation treatment, hot water extraction followed by treatment with an alkaline solution, or simultaneous treatment with an alkaline solution and an acidic solution, or the extraction of insoluble chitin-glucan complexes and biodegradation to produce films, nanofilms, and nanofilters (Boureghda et al., 2021; Janesch et al., 2020; Nawawi et al., 2019).

[0008] However, the above extraction technology is limited to the use of mushroom fruiting bodies, and no research has been reported on methods using mushroom mycelium that can be produced sustainably in an environmentally friendly manner. Furthermore, because glycogen, one of the major components of the fruiting body, has not been effectively removed, there is an urgent need for the development of a process capable of extracting high-purity chitin-glucan complex. Prior art literature

[0009] (Patent Publication 1) 10-2020-0060734 (2020.06.01) The problem to be solved

[0010] The present invention is the result of research on the development of a chitin-glucan complex extraction technology derived from mushroom mycelium. The purpose of the present invention is to provide a method for producing a chitin-glucan complex derived from mushroom mycelium, which enables the cultivation of mycelium into an isotropic fibrous structure of a certain size during the mycelium cultivation stage and allows for a drastic reduction in the cultivation period by maintaining the activity of the mycelium, and enables the low-cost acquisition of a large amount of chitin-glucan complex from the cultured mycelium. means of solving the problem

[0011] The technical problem of the present invention as described above is achieved by the following means.

[0012] 1. (1) A step of heat-treating mushroom mycelium at pH 7.5~8.5 at 60~100℃ for 2~6 hours;

[0013] (2) a step of removing protein and glycogen by reacting flavozyme and amyloglucosidase at pH 4~5 and 35~45℃ for 2~6 hours; and

[0014] (3) A method for producing a chitin-glucan complex derived from mushroom mycelium, comprising the step of freeze-drying the reaction product of step (2).

[0015] 2. In the above 1,

[0016] A method for producing a chitin-glucan complex derived from mushroom mycelium, characterized in that the pH adjustment in step (2) includes adjusting NaHCO3, Na2H3CO6, NaOH, and CH3COOH to 1-5% (w / v) each.

[0017] 3. In the above 1, at step (2),

[0018] A method for producing a chitin-glucan complex derived from mushroom mycelium, characterized by using a complex enzyme mixed with flavozyme, kozyzyme, and nutase as the proteolytic enzyme. Effects of the invention

[0019] As described above, according to the present invention, in the step of culturing mycelia, it is possible to cultivate them into an isotropic fibrous structure of a certain size, and by maintaining the activity of the mycelia, the culture period can be drastically shortened. By culturing mycelia with an isotropic fibrous structure, a large amount of chitin-glucan complex can be obtained at a low cost from the cultured mycelia. Brief explanation of the drawing

[0020] FIG. 1 shows the microstructure of a mycelium according to an embodiment of the present invention and a scanning electron microscope (SEM). (A) Mycelium grown in a spherical shape, (B) ×50, (C) ×500. Figure 2 is a diagram showing the production process of a mushroom mycelium-derived chitin-glucan complex according to the present invention. Specific details for implementing the invention

[0021] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but can be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0022] In describing the embodiments of the present invention, specific descriptions of known functions or configurations will be omitted if it is determined that such detailed descriptions could unnecessarily obscure the essence of the invention. Furthermore, the terms described below are defined in consideration of their functions in the embodiments of the present invention, and these definitions may vary depending on the intentions or practices of the user or operator. Therefore, such definitions should be based on the content throughout this specification.

[0023] The method for producing a mushroom mycelium-derived chitin-glucan complex according to the present invention is,

[0024] (1) A step of heat-treating mushroom mycelium at pH 7.5~8.5 and 60~100℃ for 2~6 hours;

[0025] (2) a step of removing protein and glycogen by reacting flavozyme and amyloglucosidase at pH 4~5 and 35~45℃ for 2~6 hours; and

[0026] (3) Includes a step of freeze-drying the reaction product of step (2).

[0027] The contents of the present invention will be explained in more detail below.

[0028] Step 1: Strain culture

[0029] The mushrooms usable in this invention do not require any special limitations, and any various edible mushrooms such as shiitake mushrooms, king oyster mushrooms, and oyster mushrooms are sufficient, preferably shiitake mushrooms ( Lentinus edodes )am.

[0030] In the present invention, the mycelium of the mushroom is cultured by separating tissue from the mushroom fruiting body. The mushroom tissue is obtained by splitting the mushroom under sterile conditions, preferably using the thick part where the cap and the stem are connected.

[0031] Preferably, the internal tissue is cut into squares with a thickness of 1 to 2 mm and a length of 2 to 4 mm using a sterile surgical scalpel. The separated tissue is preferably inoculated onto PDA (potato dextrose agar) medium and cultured at 20 to 30°C, preferably 25°C.

[0032] When the mycelium grows to about 2-3 cm, subculture it 2-3 times on a new medium. When the mycelium has grown to about 70-80% of the Petri dish surface area, use it as a spawn for mycelial culture.

[0033] Step 2: Culture of organized strains

[0034] To cultivate a cultured strain, mushroom mycelial spawn grown in a Petri dish is cut into square pieces, preferably 2-3 mm in size, with a surgical scalpel.

[0035] The size of the spawn particles is preferably adjusted to <100 µm, 200-1,000 µm, and 2-3 mm, and the spawn particles of 10-100 µm are homogenized at 10,000-13,000 rpm, preferably 12,000 rpm for 10-30 seconds, preferably 20 seconds, and the spawn particles of 200-1,000 µm are homogenized at 6,000-8,000 rpm for 10-30 seconds, preferably 20 seconds.

[0036] Preferably, to obtain strains with a uniform particle size, filtration is performed sequentially using nylon fiber filter paper with pore sizes of 100, 200, and 1,000 μm.

[0037] Mycelial culture is inoculated into PDB (potato dextrose broth) medium and cultured at 20–30°C, preferably 25°C, for 4–6 days, preferably 5 days, and then subcultured in YGM medium (yeast=0.5%, glucose=2.0%, KH2PO4=0.05%, MgSO4·7H2O=0.05%).

[0038] In the YGM medium, culture is performed at 20 to 30°C, preferably 25°C, for 4 to 6 days, preferably 5 days. Here, in the PDB medium, mycelia are inoculated into the YGM medium after obtaining strains of 100 µm or larger using a 100 µm sterile nylon fiber filter paper. The filter air supply volume in the PDB medium and the YGM medium is controlled to a constant level, preferably 0.10 to 0.20 vvm, more preferably 0.15 vvm.

[0039] In the culture medium obtained through the above process, mycelia forming a ball shape are obtained as shown in Fig. 1.

[0040] Step 3: CGC extraction from mushroom mycelium

[0041] The decomposition of the cell walls of the mushroom mycelium is carried out by treating with heat, enzymes, an alkaline solution, and an acidic solution, respectively. The heat treatment temperature is preferably adjusted to 60–100°C, and the reaction is carried out for 2–6 hours while maintaining a pH of 7.5–8.5.

[0042] Subsequently, enzymatic treatment is carried out, and in the present invention, a flavorzyme and amyloglucosidase are used. The former degrades proteins, and the latter sequentially removes glycogen. Preferably, during enzymatic treatment, the pH is 4 to 5, the temperature is 35 to 45°C, and the reaction time is 2 to 6 hours. The flavorzyme and amyloglucosidase are each added in an amount of 0.1 to 1.0 weight%.

[0043] Preferably, a mixed enzyme of kozyzyme and nutase is used together with flavozyme, and it is preferable to use a mixed enzyme composed of a weight ratio of 1:1:1 to 2:1:1. More preferably, the first part of the reaction is preferably treated with flavozyme alone, and the second part is preferably performed by treating with the mixed enzyme.

[0044] In the embodiment of the present invention, NaHCO3, Na2H3CO6, NaOH, and CH3COOH are used to control pH during the enzyme treatment process, and the concentration of each component is adjusted to 1-5% (w / v).

[0045] Finally, the insoluble chitin-glucan complex is centrifuged, and then a high-purity chitin-glucan complex (CGC) is obtained using a dialysis membrane (3 kDa, 5 kDa, 10 kDa).

[0046] The contents of the present invention will be explained in more detail below with reference to the embodiments; however, these embodiments are presented only to aid in understanding the present invention and should not be interpreted as limiting the scope of the present invention.

[0047] [Example 1] Acquisition of mushroom mycelium

[0048] (1) Strain culture

[0049] shiitake mushrooms used in the experiment ( Lentinus edodes Mycelium was cultured by isolating tissue from shiitake mushroom fruiting bodies using the following method. Under sterile conditions, the shiitake mushrooms were split, and the internal tissue of the thick section connecting the cap and stem was cut into squares approximately 1 mm thick and 3 mm long using a sterile surgical scalpel. The isolated tissues were inoculated onto PDA (potato dextrose agar) medium and cultured at 25°C. When the mycelium grew to about 2-3 cm, it was subcultured 2-3 times on a fresh medium. When the mycelium grew to about 70-80% of the Petri dish area, it was used as spawn for mycelial culture.

[0050] (2) Culture of organized strains

[0051] To culture the organized strain, shiitake mushroom mycelial spawn grown in Petri dishes was cut into square pieces of 2-3 mm using a surgical scalpel, and the process was carried out as follows. The spawn particle sizes were adjusted to <100 µm, 200-1,000 µm, and 2-3 mm. Spawn particles of 10-100 µm were homogenized at 12,000 rpm for 20 seconds, and particles of 200-1,000 µm were homogenized at 6,000-8,000 rpm for 20 seconds. To secure strains with uniform particle sizes, the samples were filtered sequentially using nylon fiber filter paper with pore sizes of 100, 200, and 1,000 µm.

[0052] Mycelial culture was performed by inoculating into PDB medium and culturing at 25°C for 5 days, followed by subculturing into YGM medium (yeast=0.5%, glucose=2.0%, KH2PO4=0.05%, MgSO4·7H2O=0.05%). In the YGM medium, culture was carried out at 25°C for 5 days. Here, mycelia from the PDB medium were inoculated into the YGM medium after securing strains larger than 100 µm using a 100 µm sterile nylon fiber filter paper. The filter air supply rate for both the PDB and YGM media was constantly controlled to 0.15 vvm, and the culture volume was 800 ml.

[0053] As shown in Figure 1, the experimental results confirmed that a ball-shaped mycelium was formed (A), and through scanning electron microscope (SEM) images taken at 50x (B) and 500x (C) magnifications, it was confirmed that an isotropic fibrous mycelium structure was obtained.

[0054] (3) Decomposition of mushroom mycelial cell walls and separation of substances

[0055] The degradation of the mushroom mycelial cell walls was carried out by heat treatment, enzyme treatment, alkaline solution, and acidic solution treatment, respectively. The heat treatment temperatures were adjusted to 60, 80, and 100°C, respectively, and reacted for 4 hours. The pH at this time was 8.0. Subsequently, enzyme treatment was performed, and 1 wt% each of flavourzyme and amyloglucosidase were added to sequentially remove proteins and glycogen. The pH at this stage was 4–5, the temperature was 40°C, and the reaction time was 4 hours; to control the pH, the concentrations of NaHCO3, Na2H3CO6, NaOH, and CH3COOH were adjusted to 1–5% (w / v). The insoluble chitin-glucan complex was centrifuged, and high-purity chitin-glucan complex was obtained using dialysis membranes (3 kDa, 5 kDa, 10 kDa).

[0056] [Example 2] Acquisition of mushroom mycelium

[0057] Mushroom mycelium was obtained by carrying out the same procedure as in Example 1, except that a complex enzyme composed of Flavozyme, Kozyzyme, and Nutrase in a weight ratio of 1:1:1 was added.

[0058] [Example 3] Acquisition of mushroom mycelium

[0059] Mushroom mycelium was obtained by carrying out the same procedure as in Example 1, except that 1% by weight of Flavozyme was added during the first half of the reaction period, and 1% by weight of a complex enzyme composed of Flavozyme, Kozyzyme, and Nutrase in a weight ratio of 1:1:1 was added during the second half of the reaction period.

[0060] [Experimental Example]

[0061] To analyze the CGC content, two types of acid hydrolysis processes were performed on the dried samples according to the embodiment of the present invention as per the method presented by Farinha et al. Glucose monomers from the beta-glucan fraction were obtained by hydrolyzing the samples with 99% tetrafluoroacetic acid (TFA) at 120°C for 2 hours, and glucosamine monomers present in the chitin fraction were obtained by hydrolyzing the samples with HCl at 120°C for 5 hours. Then, the two hydrolysis products were used to quantify the constituent monosaccharides by liquid chromatography (HPLC) using a CarboPac PA10 column (Dionex) equipped with an amperometric detector. Analysis was performed at 30°C with a 4 mM NaOH eluent and a flow rate of 0.9 mL / min. Glucose (Sigma), mannose (Sigma), and glucosamine (Sigma), which underwent the same hydrolysis process, were used as standards for polymer samples. The measurement results are shown in Table 1 below.

[0062] Sample CGC(wt%) Comparative Example 1 10.12±0.10 Comparative Example 2 11.14±0.09 Example 1 18.60±0.13 Example 2 20.25±0.11 Example 3 22.58±0.10

[0063] The foregoing description is merely an illustrative explanation of the technical concept of the present invention, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these embodiments. The scope of protection of the present invention shall be interpreted by the claims below, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

Claims

Claim 1 (1) a step of heat-treating mushroom mycelium at pH 7.5~8.5 at 60~100℃ for 2~6 hours; (2) a step of removing protein and glycogen by reacting flavozyme and amyloglucosidase at pH 4~5 and 35~45℃ for 2~6 hours; and (3) a step of freeze-drying the reaction product of step (2). A method for producing a chitin-glucan complex derived from mushroom mycelium. Claim 2 A method for producing a chitin-glucan complex derived from mushroom mycelium, characterized in that, in step (2) of claim 1, the pH adjustment includes adjusting NaHCO3, Na2H3CO6, NaOH, and CH3COOH to 1-5% (w / v) each. Claim 3 A method for producing a mushroom mycelium-derived chitin-glucan complex according to claim 1, characterized in that, in step (2), kozyzyme and nutase are reacted together with flavozyme and amyloglucosidase to remove protein and glycogen.

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