Optimized method for extracting spirulina protein using bacillus amyloliquefaciens MU2 strain

Abstract

The present invention relates to an optimized method for extracting spirulina protein, using a Bacillus amyloliquefaciens MU2 strain. According to an embodiment of the present invention, a method for producing spirulina protein or a method for producing branched-chain amino acids, each method using a Bacillus amyloliquefaciens MU2 strain, does not cause protein denaturation due to heat treatment compared to conventional techniques, and produces high-content protein and high contents of essential amino acids and branched-chain amino acids, thereby enabling effective use as alternative protein materials.

Description

Optimized extraction method of Spirulina protein using Bacillus amyloliquefaciens MU2 strain

[0001] The present invention relates to an optimized extraction method of spirulina protein using the Bacillus amyloliquefaciance MU2 strain.

[0002] This invention is a study conducted with partial support from the Ministry of Oceans and Fisheries (Project No. 2520000235).

[0003] Spirulina is an organism belonging to the cyanobacteria within the microalgae group, found in alkaline, warm regions of the world, including seawater and freshwater. As a multicellular organism capable of photosynthesis that synthesizes nutrients independently without relying on other organisms, spirulina possesses a distinctive bluish-green color and a spiral shape. Spirulina is cited as an ideal nutritional source and a high-value food due to its high protein content—among the five major nutrients—as well as its abundance of vitamins and minerals and high digestibility.

[0004] Extracting protein from spirulina primarily involves a process of disrupting the cell walls to release the proteins. Common extraction methods include physical disruption via ultrasonic treatment, high-pressure homogenizers, or chemical methods that break down cell walls using enzymes. However, ultrasonic treatment is limited by equipment constraints and carries the risk of protein denaturation if treated excessively, while enzymatic decomposition methods, although capable of extraction under mild conditions, are costly and time-consuming. Therefore, research is continuously being conducted to develop improved methods for the economical and efficient extraction of high-purity protein.

[0005] Protein extraction through microbial fermentation utilizes enzymes produced by microorganisms as metabolic products to effectively extract proteins, and it is currently used in various application fields such as the food, pharmaceutical, and environmental industries. Furthermore, it offers the advantages of high productivity and efficiency, ease of mass production at low costs, and low energy consumption due to its eco-friendliness.

[0006] Fermentation technology utilizing Bacillus amyloliquefaciens plays a significant role in the food, pharmaceutical, agricultural, and industrial sectors due to its high enzyme production capacity, diverse metabolite production characteristics, and eco-friendly production process. It also offers the advantage of stable utilization in fermentation processes as it can survive over a relatively wide temperature range from low to high temperatures. Furthermore, as a microorganism generally recognized as safe for food, it is advantageous for industrial applications due to the lack of restrictions on its use in food products.

[0007] Under the background described above, the inventors extracted spirulina protein using the fermentation of the Bacillus amyloliquefaciens MU2 strain, which has excellent activity of extracellular enzymes such as protease and cellulase, as well as cell wall degrading enzymes, and optimized the extraction conditions. By confirming that the content of constituent amino acids, free amino acids, total amino acids, and branched-chain amino acids of the extracted spirulina protein significantly increased compared to the raw material before fermentation, the present invention was completed.

[0008] Therefore, the objective of the present invention is to provide a method for producing a protein derived from microalgae of the genus Spirulina.

[0009] Another objective of the present invention is to provide a method for producing branched-chain amino acids from microalgae of the genus Spirulina.

[0010] The terms used in this specification are for descriptive purposes only and should not be interpreted as being limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0011] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0012] The present invention will be described in detail below.

[0013]

[0014] To achieve the above objective, one aspect of the present invention comprises the step of (a) preparing a spirulina aqueous solution by mixing spirulina powder, obtained by grinding microalgae of the genus Spirulina, with 15 to 25 times the weight of the powder in purified water;

[0015] (b) a step of preparing a fermented product by inoculating the above spirulina aqueous solution with the microalgae strain Bacillus amyloliquefaciens MU2 (KCTC 15499BP) and fermenting it; and

[0016] (c) A method for producing a protein derived from the genus Spirulina microalgae, comprising the step of recovering the protein from the above fermented product.

[0017] The term "fermentation" used in the present invention refers to the decomposition of organic matter using enzymes possessed by microorganisms. In the present invention, a spirulina extract can be fermented using microorganisms, and the microorganism may specifically be a Bacillus sp. microorganism, specifically Bacillus amyloliquefaciens, and more specifically, Bacillus amyloliquefaciens MU2 strain deposited under accession number KCTC 15499BP.

[0018] In one embodiment of the present invention, the microalgae of the genus Spirulina in step (a) may be one or more selected from the group consisting of Spirulina platensis, Spirulina maxima, and Spirulina fusiformis, but are not limited thereto.

[0019] In one embodiment of the present invention, the method for producing a protein derived from the genus Spirulina microalgae may increase the protein extraction yield by exploring optimized protein extraction conditions using a Box-Behnken design (BBD) of the response surface method (RSM).

[0020] The Response Surface Method (RSM) is a statistical technique used in experimental design and optimization, utilized to find optimal conditions by analyzing the interactions between various variables. RSM design methods primarily include Central Composite Design, Box-Behnken Design, and 3-level Full Factorial Design. RSM is mainly used in process optimization, product development, and chemical process control, and can save time and costs by predicting and optimizing responses to changes in variables.

[0021] The Box-Behnken Design described above is one of the RSM design methods used to evaluate interactions between variables and perform optimization. In the Box-Behnken design, each variable is designed to be tested at three levels: low, medium, and high, and the experimental points are placed at the center points rather than at the boundaries of the variable combinations. For example, if there are three variables, the medium level is fixed for one variable, while the low and high levels are used for the remaining two variables. In addition to these combinations, experiments at the center point of all variables are repeated multiple times to explore optimal conditions. In one embodiment of the present invention, the protein extraction yield of Spirulina microalgae was increased by exploring optimal conditions for the inoculation ratio, fermentation time, and fermentation temperature using the Box-Behnken design.

[0022] Based on this optimized design, in one embodiment of the present invention, the fermentation in step (b) may be carried out at 20 to 50°C, preferably 25 to 45°C, and more preferably 30 to 40°C.

[0023] In addition, in one embodiment of the present invention, the fermentation in step (b) may be carried out for 1 to 100 hours, preferably 6 to 30 hours, more preferably 18 to 24 hours.

[0024] In addition, in one embodiment of the present invention, the inoculation in step (b) may be performed at 1 to 10 (weight / volume)%, preferably 2 to 7 (weight / volume)%, more preferably 3 to 5 (weight / volume)%.

[0025]

[0026] Another aspect of the present invention comprises the step of (a) preparing a spirulina aqueous solution by mixing spirulina powder, obtained by grinding microalgae of the genus Spirulina, with 15 to 25 times the weight of the powder in purified water;

[0027] (b) a step of preparing a fermented product by inoculating the above spirulina aqueous solution with the microalgae strain Bacillus amyloliquefaciens MU2 (KCTC 15499BP) and fermenting it; and

[0028] (c) A method for producing branched-chain amino acids, comprising the step of recovering branched-chain amino acids from the fermented product.

[0029] The term "branched chain amino acid" used in this invention refers to an amino acid among essential amino acids that has a hydrocarbon side chain within its structure, and includes valine, isoleucine, and leucine. "Branched chain" is a Chinese character term for "branched chain," and in the market, branched chain amino acids are also referred to as BCAA, derived from the first letters of their English name, Branched Chain Amino Acid. Unlike most amino acids, which are utilized in the liver, branched chain amino acids have the characteristic of being metabolized in skeletal muscle; therefore, they are considered important for muscle growth exercises and are utilized as exercise supplements sought after by athletes and the general public who enjoy exercise. When branched chain amino acids are produced using the Bacillus amyloliquefaciens MU2 strain according to one embodiment of this invention, there is an advantage that the content of branched chain amino acids is increased compared to conventional spirulina extracts.

[0030] In the present invention, the method for recovering protein or branched-chain amino acids from the fermentation product may be carried out using suitable methods known in the art. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof may be used, but are not limited to these examples. Furthermore, the step of recovering the crude protein or branched-chain amino acids may include an additional purification step and may be carried out using suitable methods known in the art.

[0031] A method for producing spirulina protein or branched-chain amino acids using the Bacillus amyloliquefaciens MU2 strain according to one embodiment of the present invention can be usefully used as an alternative protein material because, compared to existing technology, there is no denaturation of the protein due to heat treatment, the protein content is high, and the content of essential amino acids and branched-chain amino acids is high.

[0032] Figure 1 shows the extraction yield according to independent variable conditions for Spirulina protein extraction. (A): Fermentation temperature (°C), (B): Fermentation time (h), (C): Inoculation ratio (%).

[0033] Figure 2 shows the extraction yield according to the correlation between independent variable conditions for spirulina protein extraction. (A): Fermentation temperature–Fermentation time, (B): Fermentation temperature–Inoculation ratio, (C): Fermentation time–Inoculation ratio.

[0034] The present invention will be explained in more detail below through specific embodiments. The details described in the following embodiments describe a preferred embodiment of the present invention, and the scope of the present invention is not limited by the details described in the following embodiments.

[0035]

[0036] Example 1. Comparison of Spirulina Protein Extraction Yields for Various Bacillus Strains

[0037] Protein extraction yields were examined by applying Bacillus sp. strains commonly used for fermentation to the fermentation of spirulina. Spirulina powder and distilled water were mixed in a 1:20 (w / v) ratio to prepare a 5% suspension, and then 1% of a bacterial culture of 8 log CFU / mL or higher was inoculated and fermented at 37°C for 24 hours. The results are shown in Table 1.

[0038] NO.SpeciesProtein extraction yield(%)1Bacillus amyloliquefaciensMU260.35±2.102Bacillus altitudinisAC11.250.44±2.673Bacillus altitudinisLNPC851.47±1.714Bacillus subtilisMMS53.53±2.015Bacillus subtilisHY-8852.80±1.186Bacillus subtilisZIM353.24±2.847Bacillus velezenisisZY153.79±1.868Bacillus rugosusSPB754.61±1.459Bacillus subtilisPEBSB30704030149.02±2.2610Bacillus vallismortisSAB1253.56±2.0611Bacillus safensis52.09±2.26

[0039]

[0040] As shown in Table 1, it was confirmed that among various Bacillus strains, the protein extraction yield was highest at 60.35±2.10% when Spirulina protein was extracted using Bacillus amyloliquefaciens MU2.

[0041]

[0042] Example 2. Comparison of Spirulina Protein Extraction Yields Using Enzymes

[0043] The extraction yield of Bacillus amyloliquefaciance MU2, which has the highest spirulina protein extraction yield, was compared with the protein extraction yield of industrially used enzymes. The treatment conditions of the enzymes used and the resulting protein extraction yields are shown in Table 2.

[0044] Enzyme Type Treatment Temperature (°C) Treatment Time (h) Initial pH Protein Extraction Yield (%) Alcalase 5038 37.96±0.39 Microbial protease 50310 34.88±0.81 Viscozyme 5035 9.19±1.02 Cellulase 5035 8.59±0.69 Papain 5037 28.38±2.96 Bromelain 5038 20.79±1.57

[0045]

[0046] As shown in Table 2, the highest extraction yield was observed with Alcalase treatment at 37.96±0.39, but it was confirmed to be more than 20% lower than the extraction yield of Bacillus amyloliquefaciens MU2, confirming that fermentation using this strain is superior for extracting Spirulina protein.

[0047]

[0048] Example 3. Optimization of Fermentation Conditions for Spirulina Protein Extraction

[0049] 3-1. Assessment of Single and Dual Variable Impact

[0050] To optimize the conditions for extracting spirulina protein using Bacillus amyloliquefaciens, independent variable experiments were conducted with fermentation temperature (°C), fermentation time (h), and inoculation ratio (%) as variables, and the results are shown in Figure 1.

[0051] As shown in Figure 1, it was confirmed that the highest protein extraction yield was observed at a fermentation temperature of 30 to 40°C, a fermentation time of 12 to 24 hours, and a bacterial inoculation ratio of 3 to 5 (weight / volume)%.

[0052] Based on the experimental results, the effects of two of the three variables were evaluated using the response surface method (RSM), and the results are shown in Figure 2.

[0053]

[0054] 3-2. Exploring Optimized Fermentation Conditions

[0055] Based on the results shown in Example 3-1, an experiment was conducted by designing a model as shown in Table 3 using the Box-Behnken design (BBD).

[0056] Fermentation Temperature (°C) Fermentation Time (h) Inoculation Ratio (%) Protein Extraction Yield (%) 1 30 245 68.43±0.85 235 244 71.37±0.87 335 244 71.41±0.78 435 244 71.23±0.77 535 18 365.53±0.46 640 245 70.28±0.96 735 244 71.66±0.69 840 30 468.81±0.70 930 18468. 50±0.86103030465.16±0.73114018468.58±0.93124024367.82±0.60133024364.69±0.60143530364.67±0.44153518568.69±1.09163524471.12±1.10173530568.46±0.69

[0057]

[0058] As shown in Table 3, the experimental results confirmed that the validity and reproducibility of the model were high, and based on this, the fermentation conditions of Bacillus amyloliquefaciens for Spirulina protein extraction were optimized. The results of the ANOVA test performed on the optimized model are shown in Table 4.

[0059] Sum of Squares Mean SquareF value PvalueModel 9 2.8 10.3 17 6.91 < 0.0001 Significant A (Fermentation Temperature) 9.5 2 9.5 27 1.03 < 0.0001 B (Fermentation Time) 2.2 2.2 16.4 10.004 9 C (Inoculation Ratio) 21.5 8 21.5 81 60.96 < 0.0001 AB 3.2 3.2 23.8 50.001 8 AC 0.4 0.9 60.4 0.9 63.0 60.1 24 BC 0.1 0.1 0.1 0.7 5 190.4 14 6A 2 7.297.2954.360.0002B2 21.9221.92163.53<0.0001C 2 21.0921.09157.28<0.0001Residual0.93850.1341Lack of fit0.77220.25746.190.0552not significantPure error0.16620.0416Cor Total93.74R 2 0.99Adj R-Squared0.9771Coefficient of Variation (%)0.5337PRESS12.62Standard Deviation0.3662Adequate precision24.3754

[0060]

[0061] As shown in Table 4, R of the protein extraction condition optimization model according to the present invention 2 The value was 0.99, the Adj R-Squared was 0.9771, and the adequate precision was 24.3754, confirming that this model represents the entire data well and has high reliability. In addition, the optimal conditions for Sparulina protein extraction and the results of the reproducibility evaluation derived from the above results are shown in Table 5.

[0062] Derived Optimal Conditions Predicted Values ​​Desirability Measured Values ​​Inoculation Ratio (%) Fermentation Time (h) Fermentation Temperature (°C) 4.28 24.05 35.39 71.8 21.00 71.80 ± 0.66

[0063]

[0064] Example 4. Amino acid analysis of protein extracted under optimized conditions

[0065] Spirulina protein extracted using optimized fermentation conditions as described in Example 3 was freeze-dried, and the free amino acids were analyzed, and the results are shown in Table 6.

[0066] Amino acids Concentration of total amino acid (mg / 100 g) Fermented Spirulina Powder Aspartic acid 74.43±1.35 1.50 2.01±5.23 Glutamic acid acid102.47±0.782,038.55±2.87Serine5.20±0.9847.03±2.14Histidine3.87±0.17157.85±1.43Arginine12.58±0.871,204.27±3.49Glycine4. 33±0.2751.07±1.04threonine6.08±0.3266.90±1.00Alanine12.61±0.86656.76±5.17Tyrosine6.35±0.11159.74±0.38Cystine25.95±1.042,15 0.30±5.68Methionine5.26±0.01318.92±4.00Valine2.73±0.13274.85±1.11Phenylalanine5.82±0.10417.63±7.69Isoleucine6.07±0.25469.4 0±2.86Leucine4.00±0.14755.32±2.81Lysine3.75±0.08613.59±9.50∑E AA37.58903074.4625∑NEAA238.73017762.7064TAA281.514410884.1945

[0067]

[0068] As shown in Table 6, the analysis results confirmed that the content of both essential and non-essential amino acids in fermented spirulina powder increased significantly by more than 40 times compared to raw spirulina (P < 0.05). In particular, the content of branched-chain amino acids (BCAAs) such as valine, isoleucine, and leucine was found to have increased by nearly 120 times compared to raw spirulina. [Accession No.]

[0069] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0070] Trustee Number: KCTC15499BP

[0071] Date of Trust: 20230711

[0072]

[0073] A method for producing spirulina protein or branched-chain amino acids using the Bacillus amyloliquefaciens MU2 strain according to one embodiment of the present invention has industrial applicability as it can be usefully used as an alternative protein material because, compared to existing technology, there is no denaturation of the protein due to heat treatment, the protein content is high, and the content of essential amino acids and branched-chain amino acids is high.

Claims

1. (a) A step of preparing a spirulina aqueous solution by mixing spirulina powder, obtained by grinding microalgae of the genus Spirulina, with 15 to 25 times the weight of the powder in purified water; (b) a step of preparing a fermented product by inoculating the above spirulina aqueous solution with the microalgae strain Bacillus amyloliquefaciens MU2 (KCTC 15499BP) and fermenting it; and (c) A method for producing a protein derived from the genus Spirulina microalgae, comprising the step of recovering the protein from the above fermented product.

2. In Paragraph 1, A method for producing a protein derived from a genus of microalgae, characterized in that the genus of microalgae in step (a) above is selected from the group consisting of genus of microalgae of genus of spirulina, genus of microalgae of spirulina, genus of microalgae of spirulina, and genus of microalgae of spirulina fusiformis.

3. In Paragraph 1, A method for producing a protein derived from the genus Spirulina microalgae, characterized in that the fermentation in step (b) above is carried out at 30 to 40°C.

4. In Paragraph 1, A method for producing a protein derived from the genus Spirulina microalgae, characterized in that the fermentation in step (b) above is carried out for 18 to 24 hours.

5. In Paragraph 1, A method for producing a protein derived from the genus Spirulina microalgae, characterized in that the inoculation in step (b) above is performed at 3 to 5 (weight / volume)%.

6. (a) A step of preparing a spirulina aqueous solution by mixing spirulina powder, obtained by grinding microalgae of the genus Spirulina, with 15 to 25 times the weight of the powder in purified water; (b) a step of preparing a fermented product by inoculating the above spirulina aqueous solution with the microalgae strain Bacillus amyloliquefaciens MU2 (KCTC 15499BP) and fermenting it; and (c) A method for producing branched-chain amino acids, comprising the step of recovering branched-chain amino acids from the fermented product.

7. In Paragraph 6, A method for producing branched-chain amino acids, wherein the branched-chain amino acid is one or more selected from the group consisting of valine, isoleucine, and leucine.

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