Composition and production method for producing chitin derived from mealworm larvae comprising Bacillus subtilis EG1 strain or culture medium thereof

Abstract

The present invention relates to a composition for producing chitin and a method for producing chitin, comprising a Bacillus subtilis EG1 strain or a culture solution thereof as an active ingredient. Since the Bacillus subtilis EG1 strain or a culture solution thereof according to the present invention has proteolytic and lipolytic enzyme activities, it can be effectively utilized for producing chitin from a mealworm larva substrate.

Description

Technology Field

[0001] The present invention relates to Bacillus subtilis ( Bacillus subtilis The invention relates to an EG1 strain, and more specifically, to a method for producing chitin by biologically removing proteins, lipids, etc. from mealworm larvae using a composition comprising the strain that produces proteolytic enzymes and lipolytic enzymes and the culture medium thereof as active ingredients. Background Technology

[0002] Chitin is a naturally occurring high-molecular-weight polysaccharide, N It is a structure composed of polysaccharides of acetyl-D-glucosamine. Chitin is the second most abundant polymer in nature after cellulose and is found in the exoskeletons of crustaceans, the cuticles of insects, and the cell walls of fungi.

[0003] This chitin is mainly extracted from crab shells and used, and as an important natural resource, its applicability is increasing. Chitinase is used in the production of chitin oligosaccharides, which are the decomposition products of chitin and chitosan. It is used in the process of producing chitin oligosaccharides by decomposing chitin derived from mushroom cell walls, chitin derived from yeast cell walls, chitin derived from fungal cell walls, chitin derived from insect exoskeletons, or chitin derived from crustacean exoskeletons.

[0004] Meanwhile, extracting chitin from mealworm larvae requires a process to remove lipids, proteins, and minerals. Lipids are generally extracted using physical pressing methods or chemical methods involving hexane. For proteins, alkaline solutions or commercial proteolytic enzymes are used, while weakly acidic solutions are employed for mineral removal. However, chemical processes require the removal of the chemicals used, necessitating meticulous management regarding safety.

[0005] Furthermore, in order to use chitin derived from mealworm larvae as a food ingredient requiring safety, it must be produced by biological methods. Therefore, in order to produce chitin from mealworm larvae, there is a need for research on various microorganisms that exhibit good protein-degrading and lipid-degrading activities. The problem to be solved

[0006] Against this background, the inventors, through research efforts on microorganisms capable of producing chitin from mealworm larvae, found Bacillus subtilis ( Bacillus subtilis It was confirmed that the EG1 strain can produce proteolytic enzymes and lipolytic enzymes, and that the strain can be used to produce chitin from which proteins and lipids have been removed from mealworm larvae.

[0007] Accordingly, the object of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipease activity ( Bacillus subtilis It is to provide the EG1 strain.

[0008] Another object of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipease activity ( Bacillus subtilis The present invention provides a composition for producing chitin that includes an EG1 strain or a culture solution thereof as an active ingredient and can produce chitin from a mealworm larva substrate.

[0009] Another object of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipease activity ( Bacillus subtilis ) A culture step of culturing the EG1 strain together with a mealworm larva substrate; and

[0010] The present invention provides a method for producing chitin comprising a separation step of separating mealworm larva-derived chitin from a culture obtained after the above-mentioned culture step.

[0011] Another objective of the present invention is to provide a food composition comprising chitin as an active ingredient, produced according to the chitin production method.

[0012] Another object of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipease activity ( Bacillus subtilis ) It provides a use for the production of mealworm-derived chitin using the EG1 strain. means of solving the problem

[0013] The present invention relates to Bacillus subtilis ( Bacillus subtilis ) This relates to a composition for producing chitin and a method for producing chitin comprising an EG1 strain or a culture solution thereof as an active ingredient, wherein Bacillus subtilis according to the present invention ( Bacillus subtilis The present invention was completed by confirming that the EG1 strain or its culture medium has proteolytic and lipolytic enzyme activities, and thus can produce chitin from a mealworm larva substrate.

[0014] The present invention will be described in more detail below.

[0016] One aspect of the present invention is a Bacillus subtilis deposited under accession number KACC 92560P having protease and lipase activity ( Bacillus subtilis It is an EG1 strain.

[0017] In one embodiment of the present invention, the strain may be a strain isolated from paddy soil mixed with rice straw, but is not limited thereto.

[0018] In one embodiment of the present invention, Bacillus subtilis according to the present invention ( Bacillus subtilis ) It was confirmed that the EG1 strain has excellent proteolytic and lipolytic enzyme activities when cultured with a mealworm larva substrate.

[0019] In addition, in one embodiment of the present invention, Bacillus subtilis according to the present invention ( Bacillus subtilisThe proteolytic and lipolytic activities of the EG1 strain were compared and analyzed with four strains obtained from the National Institute of Agricultural Sciences' Agricultural Microorganism Bank, and the comparison results confirmed that it has superior proteolytic and lipolytic activities compared to other strains of the same species.

[0020] In the present invention, the strain may include the 16s rRNA base sequence represented by SEQ ID NO. 3, and, for example, may include a base sequence having substantial identity with respect to the 16s rRNA base sequence represented by SEQ ID NO. 3 or a base sequence including the same.

[0021] The term "base sequence" in this specification may be used interchangeably with "nucleotide sequence," "polynucleotide sequence," or "nucleic acid sequence."

[0022] The term "substantial identity" in this specification may mean that by aligning each base sequence with any other base sequence to correspond as much as possible and analyzing the sequences, any other base sequence has at least 70%, at least 90%, or at least 98% sequence homology with each base sequence.

[0024] Another aspect of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipase activity ( Bacillus subtilis ) It is a composition for producing chitin that includes an EG1 strain or a culture solution thereof as an active ingredient and can produce chitin from a mealworm larva substrate.

[0025] The term "culture medium" in this specification refers to a product prepared by filtering out impurities, comprising the cells of microorganisms and substances secreted by growing microorganisms, obtained by inoculating microorganisms into a medium suitable for the growth of microorganisms and inducing their growth in a culture vessel for a certain period of time. Additionally, "culture filtrate" may refer to a product from which microorganisms have been completely removed from the culture medium by performing centrifugation and filtration, but is not limited thereto.

[0026] The term "included as an active ingredient" in this specification refers to a sufficient amount of Bacillus subtilis ( Bacillus subtilis ) This means including the EG1 strain or a culture solution thereof in a composition for producing chitin. In this case, the “desired effect” in the present invention may be the production of chitin from a mealworm larva substrate, and a sufficient amount to achieve such desired effect can be appropriately controlled by a person skilled in the art.

[0027] In the present invention, the strain may include the 16s rRNA base sequence represented by SEQ ID NO. 3, but is not limited thereto.

[0028] In the present invention, the mealworm larva substrate may be one or more selected from the group consisting of mealworm larva pieces and coarsely ground mealworm larvae, but is not limited thereto.

[0029] The term "mealworm (in this specification)" Tenebrio molitor "Larva" refers to a type of insect belonging to the family Tenebrionidae. The larva of the brown mealworm is commonly called a mealworm, and is also referred to by the new name Gosoae. The larval stage refers to the period from hatching from an egg until becoming a pupa. While this may imply growth through more than 10 molts during the larval period, it is not limited to this.

[0030] In one embodiment of the present invention, mealworm larva pieces or coarsely ground mealworm larvae are Bacillus subtilis ( Bacillus subtilis It was confirmed that by culturing with the EG1 strain, proteins and lipids are degraded to produce chitin derived from mealworm larvae.

[0031] In addition, in one embodiment of the present invention, Bacillus subtilis according to the present invention ( Bacillus subtilis The proteolytic and lipolytic activities of the EG1 strain were compared and analyzed with four strains obtained from the National Institute of Agricultural Sciences' Agricultural Microorganism Bank, and the comparison results confirmed that it has superior proteolytic and lipolytic activities compared to other strains of the same species.

[0033] Another aspect of the present invention is Bacillus subtilis deposited under accession number KACC 92560P having protease and lipase activity ( Bacillus subtilis ) A culture step of culturing the EG1 strain together with a mealworm larva substrate; and

[0034] A method for producing chitin comprising a separation step of separating mealworm larva-derived chitin from a culture obtained after the above-mentioned culture step.

[0035] In the present invention, the strain may include the 16s rRNA base sequence represented by SEQ ID NO. 3, but is not limited thereto.

[0036] In the present invention, the mealworm larva substrate may be one or more selected from the group consisting of mealworm larva pieces and coarsely ground mealworm larvae, but is not limited thereto.

[0037] In the present invention, the culture step may be performed at 30 ℃ to 38 ℃, 30 ℃ to 37 ℃, 30 ℃ to 36 ℃, 30 ℃ to 35 ℃, 31 ℃ to 38 ℃, 31 ℃ to 37 ℃, 31 ℃ to 36 ℃, 31 ℃ to 35 ℃, 32 ℃ to 38 ℃, 32 ℃ to 37 ℃, 32 ℃ to 36 ℃, 32 ℃ to 35 ℃, 33 ℃ to 38 ℃, 33 ℃ to 37 ℃, 33 ℃ to 36 ℃, or 33 ℃ to 35 ℃, and may be performed, for example, at 30 ℃ to 37 ℃, but is not limited thereto.

[0038] In the present invention, the concentration of the mealworm larva substrate may be 1 to 12% (w / v), 1 to 11% (w / v), 1 to 10% (w / v), 2 to 12% (w / v), 2 to 11% (w / v), 2 to 10% (w / v), 3 to 12% (w / v), or 3 to 11% (w / v), and, for example, may be 3 to 10% (w / v) considering the efficiency of protein and lipid degradation, but is not limited thereto.

[0039] In one embodiment of the present invention, mealworm larva pieces or coarsely ground mealworm larvae are Bacillus subtilis ( Bacillus subtilis It was confirmed that by culturing with the EG1 strain, proteins and lipids are degraded to produce chitin derived from mealworm larvae.

[0040] In addition, in one embodiment of the present invention, Bacillus subtilis according to the present invention ( Bacillus subtilis The proteolytic and lipolytic activities of the EG1 strain were compared and analyzed with four strains obtained from the National Institute of Agricultural Sciences' Agricultural Microorganism Bank, and the comparison results confirmed that it has superior proteolytic and lipolytic activities compared to other strains of the same species.

[0042] Another aspect of the present invention is a food composition comprising chitin as an active ingredient, produced according to the chitin production method.

[0043] When the food composition of the present invention is used as a food additive, the food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. Generally, when manufacturing food or beverages, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw materials.

[0044] There are no special restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned substance may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all foods in the conventional sense.

[0045] The above beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, natural sweeteners such as maltose and sucrose, or synthetic sweeteners such as saccharin and aspartame. The proportion of the above natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0046] In addition to the above, the food composition of the present invention may contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art. Effects of the invention

[0047] The present invention relates to Bacillus subtilis ( Bacillus subtilis ) This relates to a composition for producing chitin and a method for producing chitin comprising an EG1 strain or a culture solution thereof as an active ingredient, wherein Bacillus subtilis according to the present invention ( Bacillus subtilis The EG1 strain or its culture medium has proteolytic and lipolytic activity, so it can be effectively utilized for chitin production from mealworm larvae substrates. Brief explanation of the drawing

[0048] FIG. 1 is a schematic diagram of a process for producing chitin and fermented protein from mealworm larvae according to one embodiment of the present invention. FIG. 2 shows a culture medium cultured on day 0, day 3, and day 7 by inoculating a Bacillus subtilis EG1 strain onto a mealworm larva piece or coarsely ground mealworm larva substrate according to one embodiment of the present invention. Figure 3 shows a polarized light microscope image of a mealworm larva chitin product produced by culturing mealworm larva pieces on a Bacillus subtilis EG1 strain according to one embodiment of the present invention. Figure 4 shows a polarized light microscope image of a mealworm larva chitin product produced by culturing coarsely ground mealworm larvae on a Bacillus subtilis EG1 strain according to one embodiment of the present invention. Figure 5 shows the activity graph of a proteolytic enzyme produced by a Bacillus subtilis EG1 strain for extracting chitin from mealworm larvae according to one embodiment of the present invention. Figure 6 shows the activity graph of a lipase produced by a Bacillus subtilis EG1 strain for extracting chitin from mealworm larvae according to one embodiment of the present invention. Figure 7 shows the expression pattern of a proteolytic enzyme produced by a Bacillus subtilis EG1 strain for extracting chitin from mealworm larvae according to one embodiment of the present invention. Figure 8 shows the expression pattern of a lipase produced by a Bacillus subtilis EG1 strain for extracting chitin from mealworm larvae according to one embodiment of the present invention. FIG. 9 shows graphs of the activity of proteolytic enzymes and lipolytic enzymes produced by Bacillus subtilis EG1 strain and four comparative strains for extracting chitin from mealworm larvae according to one embodiment of the present invention. Specific details for implementing the invention

[0049] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings so that those skilled in the art can easily implement the present invention. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art.

[0050] The drawings and embodiments of this specification are intended to enable a person skilled in the art to easily understand and practice the invention. Content that may obscure the essence of the invention may be omitted from the drawings and embodiments, and the invention is not limited to the drawings and embodiments.

[0051] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0052] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0054] Example 1: Isolation and Identification of Bacillus subtilis EG1 Strain

[0055] 1-1. Isolation of Strains

[0056] Bacillus subtilis, which has excellent proteolytic enzyme production or proteolytic activity ( Bacillus subtilis The isolation process of the EG1 strain is as follows.

[0057] First, paddy soil mixed with rice straw was collected, and 1 g of the collected soil sample was suspended in sterile distilled water and activated at 30 ℃ for 30 minutes. The supernatant was then diluted 10-fold with sterile water four times. 1x10 4 After paddy soil samples mixed with rice straw diluted by a factor of 10 were plated onto skim milk agar plates (10 g skim milk per liter; 0.5 g yeast extract; 1.0 g K2HPO4; 0.5 g MgSO4·7H2O; 0.1 mg FeSO4·7H2O; 0.1 mg MnSO4·H2O; 0.1 mg ZnSO4·7H2O; 15 g agar), the plated plates were incubated in a constant temperature and humidity incubator for 48 hours.

[0058] Then, among the cultured colonies, those that were highly active in degrading proteins contained in the skim milk medium and morphologically different were primarily selected and purely isolated by streaking on TSB agar.

[0059] In addition, the selected primary isolates were inoculated onto Tween 80 agar plates (10 g of skim milk per liter; 0.5 g of yeast extract; 1.0 g of K2HPO4; 0.5 g of MgSO4·7H2O; 0.1 mg of FeSO4·7H2O; 0.1 mg of MnSO4·H2O; 0.1 mg of ZnSO4·7H2O; 15 g of agar) to finally select strains with relatively high lipid-degrading activity.

[0061] 1-2. Identification of Strains

[0062] For sequence analysis, the strain isolated in 1-1 above was cultured in TSB broth, and 1.5 mL of the culture medium was centrifuged and washed with 0.8% sterile physiological saline. Then, chromosomal DNA was extracted using a genomic DNA kit and used as template DNA for PCR. 9 F primers and 1412 R primers were used to amplify the bacterial 16S rRNA, and the primer sequences used are shown in Table 1 below.

[0063] Sequence number division Sequence (5'→3') 1 9 F primer GAG TTT GAT CCT GGC TCA G 2 1412 R primer ACG GCT ACC TTG TTA CGA CTT

[0064] After performing PCR, the amplified products were verified via electrophoresis and purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). Nucleotide sequencing was performed at Geno Tech Co. (Daejeon, Korea) using ABI PRISM Big Dye™ Terminator Cycle Sequencing Kits (Applied Biosystems, USA) and an ABI PRISM 3730xl Analyzer (Applied Biosystems). A homology search was conducted on NCBI using the provided sequences, and the above strain Bacillus subtilis It was sympathized with.

[0065] The above Bacillus subtilis ( Bacillus subtilis The 16S rRNA base sequence of the EG1 strain is shown in Table 2 below, and the above Bacillus subtilis ( Bacillus subtilis The EG1 strain was deposited with the National Institute of Agricultural Sciences Agricultural Microorganism Bank on October 12, 2023, and was assigned the accession number KACC 92560P.

[0066] Sequence number division Sequence (5'→3') 3 Bacillus subtilisEG1 16S rRNA tggagtgcgggtgctatacatgcaagtcgagcggacagatgggagcttgc tccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctg taagactgggataactccgggaaaccggggctaataccggatggttgttt gaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagat ggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaa cgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggac gaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgt aaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtacc ttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgc ggtaatacgcaggtggcaagcgttgtccggaattattgggcgtaaagggc tcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaatt ccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcga aggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggag cgaacaggattagataccctggtagtccacgccgtaaacgatgagtgcta agtgttagggggtttccgccccttagtgctgcagctaacgcattaagcac tccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccc cttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcg tgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttg ccagcattcagttgggcactctaaggtgactgccggtgacaaaccggagg aaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagcc aatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgc gtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatac gttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaaca cccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgacagaagttg

[0068] Example 2: Isolation of Chitin and Fermented Proteins After Culture of Bacillus subtilis EG1 Strain in Mealworm Larvae Substrate

[0069] Bacillus subtilis EG1 strain was inoculated into mealworm larval pieces (2 or 3 pieces of each individual mealworm larva) and coarsely ground mealworm larvae (mealworm larval substrate passed through a 10-mesh sieve), respectively, at a mealworm larval substrate concentration of 5% (w / v), and cultured at 30°C for 7 days. The mealworm larval culture medium contained nutrient medium GM1, and the composition of GM1 was 10 g / L glucose, 0.5 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, and 5.0 g / L NaCl. The culture results are shown in Figure 2.

[0070] As a result of the culture, as shown in Figure 2, the protein from the mealworm larvae was decomposed and extracted through the culture of the Bacillus subtilis EG1 strain, mealworm larvae pieces, and coarsely ground mealworm larvae substrate, showing that the suspension became stronger as the culture period increased, and it was confirmed that the protein was decomposed in the mealworm larvae substrate used for culture.

[0072] And, the process of isolating (extracting) chitin or fermented protein from the culture medium after inoculating and culturing the Bacillus subtilis EG1 strain on a mealworm larva substrate is as follows (Fig. 1).

[0073] First, the culture medium after cultivation is passed through a filter mesh of 200 mesh or larger to separate the fermented protein and the mealworm larva solids. The mealworm larva solids separated from the culture medium are washed to remove protein residues, lipid residues, and microorganisms, and are washed by applying physical impact through high-pressure water spraying. Finally, the mixture is finished with primary distilled water and dried to separate (extract) the chitin derived from mealworm larvae.

[0074] In addition, the culture medium passed through a filter mesh of 200 mesh or larger is centrifuged to separate it into a supernatant and a precipitate. The centrifuged supernatant contains peptides and fermented proteins produced by the reaction between the Bacillus subtilis EG1 strain and mealworm larvae, while the precipitate contains the EG1 strain and insoluble proteins. Depending on the intended use, the fermented proteins can be separated (extracted) by selecting whether or not to remove the Bacillus subtilis EG1 strain.

[0075] Mealworm larvae substrates collected daily from the culture medium were prepared using the process presented in Fig. 1, and the results observed with a polarizing microscope are shown in Figs. 3 and 4.

[0076] As a result of observation, as shown in Figures 3 and 4, it was confirmed that the light transmittance increases as the protein of the mealworm larva decomposes.

[0077] In summary, it was confirmed that by culturing the mealworm larva substrate together with the Bacillus subtilis EG1 strain, substances such as proteins from the mealworm larva substrate are degraded to produce mealworm larva-derived chitin.

[0079] Example 3: Confirmation of protease activity of Bacillus subtilis EG1 strain according to mealworm larva substrate

[0080] The activity characteristics of the proteolytic enzyme produced by the Bacillus subtilis EG1 strain were investigated as follows.

[0081] First, to investigate the proteolytic activity characteristics of Bacillus subtilis EG1 strain according to the size of the mealworm larva substrate (mealworm larva pieces and coarsely ground mealworm larva), 50 μL of culture medium collected daily after 7 days of culture was used, and 500 μL of substrate buffer containing 0.65% (w / v) casein was mixed with 50 mM phosphate buffer (pH 7.5) and incubated in a water bath at 37 ℃ for 1 hour, and the reaction was stopped by adding 500 μL of 110 mM trichloroacetic acid (TCA).

[0082] Then, the solution from which the reaction had stopped was centrifuged at 10,000 rpm for 10 minutes, 200 μL of the supernatant was transferred to a new container, and 500 μL of 500 mM Na2CO3 solution and 100 μL of 500 mM Folin's phenol reagent were added and reacted at 37 °C for 30 minutes. After the reaction was finished, the absorbance was measured at 660 nm using a spectrophotometer.

[0083] At this time, the proteolytic enzyme activity unit (unit, U) according to the present method was defined as the amount of enzyme that produces 1 nmole of tyrosine per minute, and the results are shown in Fig. 5.

[0084] As a result of measurement, as shown in Figure 5, it was confirmed that the proteolytic enzyme activity of the mealworm larva pieces was measured at 549.2 unit / mg P on day 7, and the maximum activity of the coarsely ground mealworm larvae was measured at 689.6 unit / mg P on day 4.

[0085] Specifically, it was confirmed that the protein degradation activity in coarsely ground mealworm larvae increased until day 4 and then decreased, while in mealworm larvae pieces it continued to increase until day 7. Therefore, it was confirmed that the protein degradation efficiency of the Bacillus subtilis EG1 strain differs depending on the size of the mealworm larvae substrate or the shape of the mealworm larvae.

[0087] Example 4: Confirmation of Lipase Activity of Bacillus subtilis EG1 Strain According to Mealworm Larva Substrate

[0088] The activity characteristics of the lipase produced by the Bacillus subtilis EG1 strain were investigated as follows.

[0089] First, to investigate the lipase activity characteristics of Bacillus subtilis EG1 strain according to the size of the mealworm larva substrate (mealworm larva fragments and coarsely ground mealworm larvae), 100 μL of culture medium collected daily after 7 days of culture was used. 800 μL of 50 mM Tris-HCl buffer (pH 8.0) and 100 μL of 10 mM p-nitrophenyl-laurate (pNPL) were mixed and incubated in a water bath at 37 ℃ for 1 hour. Subsequently, 100 μL of 0.5 M CaCl2 and 400 μL of 100 mM Tris-HCl buffer (pH 12.0) were added to terminate the reaction. After centrifuging the solution at 10,000 xg for 15 minutes, the supernatant was taken and the absorbance was measured using a spectrophotometer at 410 nm.

[0090] At this time, the lipase activity unit (unit, U) according to the present method was defined as the amount of enzyme producing 1 μmole of para-nitrophenol (p-nitrophenol) per minute, and this is shown in Fig. 6.

[0091] As a result of measurement, as shown in Figure 6, it was confirmed that lipase activity reached a maximum of 24.6 unit / mg P on day 1 for the p-nitrophenyl-laurate substrate in the mealworm larva pieces, and a maximum of 8.9 unit / mg P on day 1 for the coarsely ground mealworm larvae. Furthermore, it was confirmed that after day 1 of culture, lipase activity showed a decreasing trend until day 6 of culture in both the mealworm larva pieces and the coarsely ground mealworm larvae, and then increased again on day 7.

[0093] Example 5: Confirmation of the electrophoretic pattern of proteases produced in the culture medium of mealworm larvae of Bacillus subtilis EG1 strain

[0094] The electrophoretic pattern of proteolytic enzyme activity was determined using a 0.1% (w / v) gelatin substrate on a 10% (w / v) polyacrylamide separation gel according to the method of Heussen and Dowdle (1980).

[0095] Specifically, after electrophoresis, the gel was immersed in a 1% (v / v) Triton X-100 solution and incubated at 25°C for 30 minutes to remove SDS, and then the gel was immersed in a 50 mM Tris-HCl (pH 7.5) solution containing 10 mM CaCl2 and incubated at 37°C for 16 hours. After incubation, the gel was stained with 0.25% Coomassie brilliant blue R-250, and after decolorizing several times with a decolorizing reagent, the active band was identified, which is shown in Figure 7.

[0096] As confirmed, as shown in Figure 7, a total of nine isoenzymes (P1, P2, P3, P4, P5, P6, P7, P8, and P9) of the proteases of the Bacillus subtilis EG1 strain were identified on the SDS-PAGE gel, and the protease expression patterns showed different aspects depending on the culture date.

[0097] In addition, the P1, P2, P3, P4, P5, P6, and P9 isoenzymes were expressed during the early stages of culture, while the P7 isoenzyme was expressed starting from day 3 of culture and the P8 isoenzyme was expressed starting from day 5 of culture.

[0098] In addition, the P6 isoenzyme showed strong activity on the second day of culture and exhibited a characteristic of decreasing expression as the culture period continued. The results showed that P1, P2, and P9 isoenzymes were expressed as major activity bands, while P3, P4, P5, P6, P7, and P8 isoenzymes were expressed as minor activity bands. The results showing that the expression patterns of P1, P2, P5, P7, and P8 isoenzymes increased or newly expressed with increasing culture days appear to be isoenzymes associated with the trend of increasing proteolytic activity by day in Figure 5.

[0100] Example 6: Confirmation of the electrophoretic pattern of lipase produced in the culture medium of mealworm larvae of Bacillus subtilis EG1 strain

[0101] The electrophoretic pattern of lipase activity was confirmed according to the method of Diaz et al. (1999). After electrophoresis on a 10% (w / v) polyacrylamide separation gel, the gel was immersed in a 2.5% (v / v) Triton X-100 solution and incubated at 25°C for 30 minutes to remove SDS. The gel was then washed with 50 mM phosphate buffer (pH 7.0) and incubated again for 1 minute in 50 mM phosphate buffer (pH 7.0) where the 4-methylumbelliferyl (MUF)-butyrate reaction substrate was at a concentration of 100 μM. After incubation, the active band was confirmed under a UV lamp with a wavelength of 365 nm, which is shown in Figure 8.

[0102] As confirmed, as shown in Figure 8, a total of two isoenzymes (L1 and L2) of the lipase of the Bacillus subtilis EG1 strain were identified on the SDS-PAGE gel. The expression of the L1 isoenzyme showed an increasing pattern as the culture period increased, while the L2 isoenzyme showed a tendency to be expressed relatively strongly on the first and second days of culture and then decrease.

[0103] In addition, Aspergillus niger Lipase (AL) was used as a positive control. The molecular weight of AL is 45 kDa or less, and the other active band can be seen as esterase.

[0104] In the case of the Bacillus subtilis EG1 strain, the L1 isoenzyme appears to be an esterase, and the L2 isoenzyme appears to be a lipase, as it showed an expression pattern similar to the lipolytic activity in Figure 6.

[0105] From the results of the above examples, it is suggested that the Bacillus subtilis EG1 strain according to the present invention or the culture medium thereof has activity for degrading proteins and lipids from mealworm larva substrates, and thus can be effectively used for the production of chitin derived from mealworm larvae.

[0107] Example 7: Confirmation of protease activity of Bacillus subtilis EG1 strain and control strain

[0108] To compare the proteolytic enzyme activities produced by the Bacillus subtilis EG1 strain with four strains obtained from the Agricultural Microorganism Bank of the National Institute of Agricultural Sciences, the proteolytic enzyme activities of the Bacillus subtilis EG1, KACC 10579, KACC 10806, KACC 10854, and KACC 13751 strains were investigated, respectively Tryptic Soy Broth( 50 μL of a culture sample cultured in TSB medium for 30 hours was used, and measured using the method of Example 3. The strains used are shown in Table 3 below, and the measurement results are shown in Figure 9.

[0109] scientific name division Bacillus subtilis EG1 Bacillus subtilis KACC 10579 Bacillus subtilis KACC 10806 Bacillus subtilis KACC 10854 Bacillus subtilis KACC 13751

[0110] As a result of measurement, as shown in Figure 9, when comparing proteolytic enzyme activity with other strains, the EG1 strain of the present invention showed the highest activity, with an activity value of 50.2 unit / mg P. In addition, the KACC 13751 strain showed the second best activity, with a value of 25.1 unit / mg P, confirming that the EG1 strain was twice as high.

[0112] Example 8: Confirmation of lipase activity of Bacillus subtilis EG1 strain and control strain

[0113] To compare the lipolytic enzyme activities produced by the Bacillus subtilis EG1 strain with four strains obtained from the Agricultural Microorganism Bank of the National Institute of Agricultural Sciences, the proteolytic enzyme activities of the Bacillus subtilis EG1, KACC 10579, KACC 10806, KACC 10854, and KACC 13751 strains were investigated, respectively Tryptic Soy Broth ( 100 μL of a culture sample cultured for 30 hours in TSB medium was used, and measured using the method of Example 4 above. The strains used are shown in Table 3 above, and the measurement results are shown in Figure 9.

[0114] As a result of measurement, as shown in Figure 9, the lipase activity of the Bacillus subtilis EG1 strain of the present invention was measured to be 35.3 unit / mg P for the p-nitrophenyl-laurate substrate, and the KACC 10806 strain showed an activity value of 51.2 unit / mg P.

[0115] When combining the results of the above examples, it can be confirmed that the Bacillus subtilis EG1 strain of the present invention not only has balanced proteolytic enzyme activity and lipolytic enzyme activity compared to other strains, but also that the Bacillus subtilis EG1 strain, which has relatively superior proteolytic enzyme activity, is the most suitable for the production of chitin from mealworm larvae substrates.

[0116] Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC92560P Date of Deposit: 2023-10-12

Claims

Claim 1 delete Claim 2 delete Claim 3 Bacillus subtilis deposited under accession number KACC 92560P, having protease and lipease activity ( Bacillus subtilis A composition for producing chitin that includes an EG1 strain or a culture solution thereof as an active ingredient and can produce chitin from a mealworm larva substrate. Claim 4 A composition for producing chitin according to claim 3, wherein the strain comprises a 16S rRNA base sequence represented by SEQ ID NO.

3. Claim 5 A composition for producing chitin according to paragraph 3, wherein the mealworm larva substrate is one or more selected from the group consisting of mealworm larva pieces and coarsely ground mealworm larvae. Claim 6 Bacillus subtilis deposited under accession number KACC 92560P, having protease and lipease activity ( Bacillus subtilis A method for producing chitin comprising: a culture step of culturing an EG1 strain together with a mealworm larva substrate; and a separation step of separating mealworm larva-derived chitin from the culture obtained after the culture step. Claim 7 A method for producing chitin according to claim 6, wherein the strain comprises a 16S rRNA base sequence represented by SEQ ID NO.

3. Claim 8 A method for producing chitin according to claim 6, wherein the mealworm larva substrate is one or more selected from the group consisting of mealworm larva pieces and coarsely ground mealworm larvae. Claim 9 A method for producing chitin according to claim 6, wherein the concentration of the mealworm larva substrate in the culture step is 3 to 10% (w / v). Claim 10 A method for producing chitin according to claim 6, wherein the culture step is performed at 30 ℃ to 37 ℃. Claim 11 delete

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