Bacillus amyloliquefaciens mu2 strain and chlorella protein extraction method using same

Abstract

The present invention relates to: a Bacillus amyloliquefaciens Mu2 strain; and a chlorella protein extraction method using same. A chlorella protein extracted using the strain can be effectively used as an alternative protein material containing an abundance of constituent amino acids and free amino acids.

Description

Bacillus amyloliquefaciens MU2 strain and chlorella protein extraction method using the same

[0001] The present invention relates to a method for extracting chlorella protein using fermentation of Bacillus amyloliquefaciens MU2 strain. * The present invention was supported by the Busan Technopark's Innovative Growth Field-oriented Technology Development Project for Processed Fishery Foods, Development of Future High Value-Added Fishery Foods Based on Blue Food Tech (Project No. 202303370001, Research Period: 2023.04.01~2024.03.31) and the National Research Foundation of Korea's Key Research Institute Support Project, Marine Bionics Convergence Technology Center (Project No. 2021R1A6A1A03039211, Research Period: 2023.03.01~2024.02.29).

[0002] Conventional physical methods for protein extraction, such as sonication, and chemical methods, such as acid-base treatment, can result in low protein extraction yields or quality degradation, such as destruction of physiologically active compounds. Microbial fermentation-based protein extraction utilizes enzymes produced by microorganisms as metabolites to effectively extract proteins. It is currently used in a variety of applications, including the food, pharmaceutical, and environmental industries. Furthermore, it boasts high productivity and efficiency, low production costs, and ease of mass production, making it environmentally friendly with low energy consumption.

[0003] Fermentation technology using Bacillus amyloliquefaciens is utilized in diverse fields, including agriculture, food, and life sciences. Bacillus amyloliquefaciens is known to improve the properties of raw materials through metabolic activity or to extract and produce desired substances, thereby contributing to improved product quality. Furthermore, fermentation technology is recognized as an environmentally friendly production method with a lower environmental impact than other food processing techniques.

[0004] Chlorella contains a balanced mix of protein, various vitamins, minerals, dietary fiber, and chlorophyll, which helps promote healthy metabolism, boost immunity, and prevent acidification of bodily fluids. Chlorella is known to have a protein content of over 50% on a dry weight basis, exceeding that of livestock products like beef. However, chlorella's cell wall makes it difficult to extract the protein itself or break it down into amino acids.

[0005] Accordingly, the inventors of the present invention increased the extraction yield of chlorella protein by fermenting the Bacillus amyloliquefaciens MU2 strain, which has excellent activity of extracellular enzymes such as protease and cellulase, as well as cell wall decomposition enzymes, and confirmed that the content of constituent amino acids, free amino acids, total amino acids, and BCAA of the extracted chlorella protein significantly increased compared to the raw material before fermentation, thereby completing the present invention.

[0006] The purpose of the present invention is to provide a method for producing crude protein derived from microalgae of the genus Chlorella.

[0007] Another object of the present invention is to provide a method for producing branched-chain amino acids from microalgae of the genus Chlorella.

[0008] To achieve the above object, the present invention provides a method for producing crude protein derived from microalgae of the genus Chlorella, comprising (1) a step of inoculating microalgae of the genus Chlorella with Bacillus amyloliquefaciens MU2 strain and fermenting the mixture in a medium; and (2) a step of recovering crude protein from the medium or a culture thereof.

[0009] In addition, the present invention provides a method for producing branched-chain amino acids, comprising the steps of (1) inoculating microalgae of the genus Chlorella with the Bacillus amyloliquefaciens MU2 strain and fermenting the same in a medium; and (2) recovering branched-chain amino acids (BCAA) from the medium or its fermentation material.

[0010] The present invention relates to a Bacillus amyloliquefaciens MU2 strain and a method for extracting chlorella protein using the strain. Chlorella protein extracted using the strain can be usefully used as an alternative protein material rich in the content of constituent amino acids and free amino acids.

[0011] Figure 1 is a graph showing the chlorella protein extraction yield according to the addition of a nitrogen source.

[0012] Figure 2 is a diagram showing the photograph and crude protein content of chlorella raw material and freeze-dried powder after fermentation.

[0013] Hereinafter, the present invention will be described in detail.

[0014] The present invention provides a method for producing crude protein derived from microalgae of the genus Chlorella, comprising the steps of (1) inoculating microalgae of the genus Chlorella with Bacillus amyloliquefaciens MU2 strain and fermenting the mixture in a medium; and (2) recovering crude protein from the medium or a culture thereof.

[0015] In addition, the present invention provides a method for producing branched chain amino acids, comprising the steps of (1) inoculating microalgae of the genus Chlorella with the Bacillus amyloliquefaciens MU2 strain and fermenting the same in a medium; and (2) recovering branched chain amino acids (BCAA) from the medium or a culture thereof.

[0016] In the present invention, the microalgae of the genus Chlorella may be any one selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana, and Chlorella protothecoides, but is not limited thereto.

[0017] In the present invention, the Amyloliquefaciens MU2 strain may be deposited under the accession number KCTC 15499BP and may have a 16S rRNA gene sequence of sequence number 1.

[0018] In the present invention, the medium may be used without limitation as long as it is a medium used for conventional chlorella cultivation. For example, sugar sources that may be used may include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or as a mixture, and are not limited thereto. Nitrogen sources that may be used may include peptone, yeast extract, beef extract, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture, and are not limited thereto. The nitrogen source may be added to the medium at 1 to 5 (weight / volume)%, but is not limited thereto. Examples of nitrogen sources that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. Additionally, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate, which are necessary for growth. Additionally, essential growth substances such as amino acids and vitamins may be used. Furthermore, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch or continuous manner during the culture process, but are not limited thereto.

[0019] The pH of the culture can be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid, in an appropriate manner. Furthermore, foaming can be suppressed using antifoaming agents such as fatty acid polyglycol esters. Oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain aerobic conditions. The temperature of the culture can be between 20 and 45°C, specifically between 30 and 40°C. Cultivation can be continued until the desired crude protein or branched-chain amino acid is produced to a maximum extent. For this purpose, the culture time can be between 10 and 120 hours. The crude protein or branched-chain amino acid can be released into the culture medium or contained within the cells, but is not limited thereto.

[0020] In the present invention, the method for recovering the crude protein or branched-chain amino acid from the medium or culture can be performed using a suitable method known in the art. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof can be used, but the present invention is not limited to these examples. In addition, the step of recovering the crude protein or branched-chain amino acid can include an additional purification step, and can be performed using a suitable method known in the art.

[0021] In the present invention, branched-chain amino acids refer to amino acids having a branched alkyl group in the side chain, and include valine, leucine, and isoleucine.

[0022] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0023] <Example 1> Confirmation of chlorella protein extraction yield by Bacillus spp. strain

[0024] The protein extraction yield was examined by fermenting chlorella with each Bacillus spp. strain.

[0025] Specifically, chlorella was mixed with distilled water to prepare a 5% (w / v) suspension, and then 1% of the suspension was inoculated with a bacterial culture solution of 8 log CFU / mL or more, fermented at 37°C for 24 hours, and the fermented solution was centrifuged (10,000 g, 20 min) and the supernatant was used as a sample.

[0026] Crude protein content was calculated using the bicinchoninic acid assay (BCA; Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL, USA), and the calculation formula is as follows:

[0027] Protein extraction yield (%) = P f / P c × 100%

[0028] [P c = Protein content of chlorella powder, P f = Protein content of fermented chlorella powder]

[0029] As a result, as shown in Table 1, the extraction yield of Bacillus amyloliquefaciens MU2 was the highest at 31.45±1.21%.

[0030]

[0031] <Example 2> Exploration of a Chlorella Fermentation Medium Using Bacillus amyloliquefaciens MU2

[0032] To increase the extraction yield of Bacillus amyloliquefaciens MU2, which has the highest chlorella protein extraction yield, a chlorella fermentation medium was explored.

[0033] Specifically, the chlorella protein extraction yield was compared by adding 1, 2, 3, 4, and 5% of nitrogen sources (yeast extract, beef extract, peptone). The protein extraction yield was calculated using the same method as in Example 1.

[0034] As a result, as shown in Fig. 1, the chlorella protein extraction yield was found to be the highest when 1% yeast extract was added.

[0035] <Example 3> Analysis of color and crude protein content of fermented chlorella protein

[0036] Green color has been reported to suppress appetite in studies, so the pigment of chlorella was removed through fermentation and the crude protein content after fermentation was compared.

[0037] The freeze-dried fermented chlorella supernatant was used as a sample, and the crude protein content was calculated using the same method as in Example 1.

[0038] As a result, as shown in Fig. 2, the protein content of chlorella before fermentation was 60.0±1.8 g / 100 g per dry weight, but the protein content of chlorella after fermentation was confirmed to be 78.2±1.2 g / 100 g per dry weight.

[0039] The above results show that the crude protein content in chlorella after fermentation increases compared to before fermentation.

[0040] <Example 4> Amino acid analysis of extracted protein

[0041] The constituent amino acids and free amino acids of chlorella protein extracted using fermentation were analyzed.

[0042] Specifically, the contents of constituent amino acids and free amino acids were analyzed using chlorella raw material, enzyme treatment (3% protease; pH 9.1, 45.6°C, 49.8 min), and fermented chlorella powder.

[0043] The composition and free amino acid content were determined using an amino acid auto-analyzer (L-8900; Hitachi High-Techonologies Corp., Tokyo, Japan).

[0044] As a result of analyzing the composition of amino acids, as shown in Table 2, both essential and non-essential amino acid contents in fermented chlorella powder were significantly increased compared to raw chlorella (P<0.05).

[0045]

[0046] [EAA: Essential Amino Acid, NEAA: Non-Essential Amino Acids, TAA: Total Amino Acid, Different letters above the numbers indicate a significant difference in the results (P<0.05)]

[0047] In addition, as a result of analyzing the free amino acids, as shown in Table 3, the contents of essential and non-essential amino acids in the fermented chlorella powder were significantly increased compared to the original chlorella, similar to the constituent amino acids, and in particular, the contents of valine, leucine, and isoleucine, which are branched chain amino acids (BCAA), were increased by approximately 30, 59, and 83 times, respectively, compared to the original chlorella.

[0048]

[0049] [EAA: Essential Amino Acid, NEAA: Non-Essential Amino Acids, TAA: Total Amino Acid, Different letters above the numbers indicate a significant difference in the results (P<0.05)]

[0050] Therefore, it is expected that Bacillus amyloliquefaciens MU2, which has an excellent chlorella protein extraction yield and significantly higher composition amino acid content, free amino acid content, total amino acid content, and BCAA production ability, can be usefully utilized in the development of alternative proteins.

[0051] [Accession number]

[0052] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0053] Accession number: KCTC15499BP

[0054] Date of acceptance: 20230711

[0055] [Correction pursuant to Rule 91, May 3, 2024]

Claims

1. (1) A step of inoculating microalgae of the genus Chlorella with Bacillus amyloliquefaciens MU2 strain and fermenting it in a medium; and (2) A method for producing crude protein derived from microalgae of the genus Chlorella, comprising a step of recovering crude protein from the medium or a culture thereof.

2. In paragraph 1, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the above Bacillus amyloliquefaciens MU2 strain is deposited under the accession number KCTC 15499BP.

3. In paragraph 1, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the microalgae of the genus Chlorella are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides.

4. In paragraph 1, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the medium contains a nitrogen source.

5. In paragraph 4, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the nitrogen source is at least one selected from the group consisting of peptone, yeast extract, beef extract, malt extract, corn steep liquor, soybean meal, and urea and inorganic compounds.

6. In paragraph 4, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the nitrogen source is added at 1 to 5 (weight / volume)%.

7. In paragraph 1, A method for producing crude protein derived from microalgae of the genus Chlorella, characterized in that the above fermentation is performed at 30 to 40°C for 10 to 120 hours. 8.(1) A step of inoculating microalgae of the genus Chlorella with Bacillus amyloliquefaciens MU2 strain and fermenting it in a medium; and (2) A method for producing branched chain amino acids, comprising a step of recovering branched chain amino acids (BCAA) from the medium or a culture thereof.

9. In paragraph 8, A method for producing a branched-chain amino acid, wherein the branched-chain amino acid is at least one selected from the group consisting of valine, isoleucine, and leucine.

Images