Method for extracting chlorella protein

Abstract

The present invention relates to a method for extracting chlorella protein. In order to derive optimal conditions for obtaining chlorella protein, optimal protease treatment conditions according to the present invention were established by using response surface methodology (RSM), and thus it is possible to provide a chlorella protein extract in which the functional components and physiological activity effects of chlorella are enhanced.

Description

Chlorella protein extraction method

[0001] The present invention relates to a method for extracting chlorella protein. This invention was made possible with the support of 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: April 1, 2023 - March 31, 2024) and the National Research Foundation of Korea's Key Research Institute Support Project, Marine Bionics Convergence Technology Center (Project No. 2021R1A6A1A03039211, Research Period: March 1, 2023 - February 29, 2024).

[0002]

[0003] Chlorella is a type of freshwater algae plankton belonging to the Chlorellaceae family, a green algae plant. It is a spherical or oval single-celled organism with a diameter of less than 10㎛ that is commonly found in rivers, lakes, and wetlands. In addition, Chlorella does not have flagella and thus has no motility, and its cells consist of one nucleus and cup-shaped chloroplasts. The grown cells rapidly proliferate through a unique cell division, and usually four daughter cells form endospores. Currently, about 10 species are known, including Pyrenoidosa, Bulgaris, and Ellipsoidea.

[0004] Chlorella is rich in carbohydrates, dietary fiber, and minerals, and is known to contain over 50% protein (based on dry weight). Chlorella protein possesses physiological activities such as immune-boosting, antioxidant, and anti-inflammatory properties. It also contains both essential and non-essential amino acids, providing the body with essential nutrients.

[0005] However, chlorella has a rigid cell wall, making it difficult to extract the proteins within it. Methods for extracting chlorella proteins include frezze-thawing, sonication, acid-base treatment, and heat treatment. However, these conventional physicochemical methods are known to consume excessive energy, damage physiologically active substances, and cause protein denaturation.

[0006] In addition, a method for extracting a liquid extract from chlorella is generally known, which involves adding hot water of 90℃ to chlorella powder to extract only the useful substances rich in nutrients contained within the cell membrane of chlorella and then liquefying them. However, in the case of the above method, the culture solution in which chlorella has been grown in advance is powdered, and then the powder is dissolved in water to manufacture the chlorella-dissolved solution again. Therefore, unnecessary processes are repeated, and in addition, a process such as adjusting the pH of the aqueous solution to acidify it so that the chlorella powder dissolved in water does not precipitate, is added, so there is a problem in that the extraction process is extremely inefficient.

[0007] Therefore, effective solutions are needed to overcome these problems.

[0008] Under these circumstances, the inventors of the present invention sought to develop an environmentally friendly chlorella protein extraction method that not only increases protein extraction yield but also prevents protein denaturation. As a result, they identified optimal conditions for chlorella protein extraction using microbial proteases through RSM analysis. Their statistical significance demonstrated that chlorella protein could be extracted in high yield and concentration, thereby completing the present invention.

[0009] Accordingly, one object of the present invention is to provide a method for extracting chlorella protein.

[0010] In addition, another object of the present invention is to provide a method for producing a chlorella extract.

[0011] In addition, another object of the present invention is to provide a chlorella extract prepared by the above method.

[0012] In addition, another object of the present invention is to provide a processed food containing a chlorella extract.

[0013]

[0014] Other objects and advantages of the present invention will become more apparent from the detailed description of the invention and the claims below.

[0015]

[0016] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0017] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0018]

[0019] Hereinafter, the present invention will be described in detail.

[0020]

[0021] According to one aspect of the present invention, there is provided a method for extracting chlorella protein, comprising the step of adding protease to a chlorella suspension and reacting the protease at a pH of 8.1 to 10 and a temperature of 40°C to 50°C for 40 to 60 minutes.

[0022] The above chlorella suspension method can be performed by any method known in the art.

[0023] The protease may be any protease known in the art as long as it can achieve the purpose of the present invention, and may be, for example, at least one selected from the group consisting of microbial protease, cellulase, viscozyme L, papain, kozyme, alkalise, esperase, neutrase, and phlegmozyme, but is not limited thereto, and microbial protease is preferred.

[0024] The above microbial protease may be any microbial protease known in the art as long as it can achieve the purpose of the present invention, but one derived from Bacillus is preferred.

[0025] The above Bacillus-derived protease can be treated at a concentration of 0.1 to 99%, but is preferably treated at a concentration of 1 to 5%, most preferably 3%.

[0026] In one embodiment of the present invention, 3% protease is treated in a chlorella suspension relative to the chlorella solid content of the chlorella suspension, and the optimal conditions designed and modeled according to BBD in RSM for optimization are derived as follows: a protease treatment temperature of 45.45°C; a pH of 9.01; and a time of 49.85 minutes. Accordingly, by reacting and then extracting (reacting) under the optimal conditions derived through the RSM analysis, the yield of chlorella protein can be improved.

[0027]

[0028] In addition, according to another aspect of the present invention, the present invention provides a method for producing a chlorella extract, including a step of adding protease to a chlorella suspension and reacting the same at a pH of 8.1 to 10 and a temperature of 40°C to 50°C for 40 to 60 minutes, and a chlorella extract produced by the method.

[0029] In the present invention, when manufacturing chlorella extract, it is recommended that the pH of the chlorella suspension be adjusted to 8.1 to 10.

[0030] In the above, protease is added to the chlorella suspension, and the reaction is carried out at 1 to 5%, preferably 3%, of the chlorella solid content of the chlorella suspension, and the reaction is carried out for an optimal time at the optimal temperature and optimal pH for protease treatment derived according to RSM.

[0031] In addition, the chlorella extract of the present invention can be obtained by reacting a chlorella suspension with protease under optimal conditions derived through RSM analysis, obtaining a chlorella extract, and filtering the filtrate using a filtration means. At this time, any filtration means commonly used in the technical field of the present invention can be used as the filtration means. For example, the chlorella extract can be filtered using a filtration means such as filter paper, filter cloth, or ultrafiltration.

[0032] The chlorella extract of the present invention can be obtained by freeze-drying a chlorella extract filtrate filtered by a filtration means to obtain a dried chlorella product. At this time, the freeze-drying can be performed under drying conditions commonly used in the technical field of the present invention.

[0033]

[0034] In addition, the present invention can provide processed foods containing the chlorella extract. There is no particular limitation on the type of the processed foods. Examples of foods to which the chlorella extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all processed foods in the conventional sense.

[0035] In addition, the present invention may include a health supplement containing the chlorella extract prepared above.

[0036] The health supplement containing the chlorella extract of the present invention can use the chlorella extract prepared above in liquid form, or the chlorella extract can be powdered and used in powder form.

[0037] The health supplement containing the chlorella extract of the present invention can be easily obtained by adding a process of adding the chlorella extract prepared by the above-mentioned method in liquid or powder form to the health supplement manufacturing process, or by adding a process of adding the chlorella extract of the present invention described above after the manufacturing of the food as a base material. At this time, the health supplement containing the chlorella extract may add additives usable in food science to improve taste, smell, etc., if necessary.

[0038] The content of the chlorella extract in the health supplement containing the chlorella extract of the present invention varies depending on the type or preference of the food, but when used in the form of a tablet, capsule, pill, powder, etc., the chlorella extract may be contained in an amount of 0.1 to 10 wt% in liquid or powder form.

[0039]

[0040] The present invention solves the problems of existing organic solvent extraction methods in extracting chlorella proteins, such as human harm, environmental toxicity, high cost, and low selectivity, and establishes optimal protease treatment conditions using response surface methodology (RSM) to derive optimal conditions, thereby providing a chlorella protein extract with enhanced functional components and physiological activity of chlorella.

[0041] Figure 1 shows a 3D response surface model of protein extraction yield according to temperature and pH of the present invention.

[0042] Figure 2 shows a 3D response surface model of protein extraction yield as a function of temperature and time.

[0043] Figure 3 shows a 3D response surface model of protein extraction yield according to pH and time of the present invention.

[0044]

[0045] The following examples are provided solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[0046]

[0047] Example 1. Enzyme screening for chlorella protein extraction

[0048] The present inventors, in order to select an enzyme suitable for effectively extracting protein from chlorella, mixed Chlorella pyrenoidosa with distilled water to prepare a 5% suspension (w / v), and treated it with various types of enzymes such as cellulase (cellulase from Trichoderma reesei), viscozyme L (viscozyme L from Aspergillus sp.), microbial protease (protease from Bacillus spp.), papain (papain from Carica papaya), and bromelain (bromelain from pineapple stem) (added at 3% based on the chlorella solid content), and confirmed the chlorella protein extraction yield according to the treatment time.

[0049] As a result, as shown in Table 1 below, it was confirmed that microbial-derived protease had the best extraction efficiency.

[0050] Accordingly, a Bacillus-derived protease was selected as an effective enzyme for extracting protein from chlorella and used in the following examples.

[0051]

[0052] [Table 1]

[0053]

[0054]

[0055] Example 2. Optimization of Bacillus-derived protease treatment conditions for chlorella protein extraction using RSM.

[0056] The present inventors applied Response surface methodology (RSM) as a statistical experimental method to derive optimization of microbial protease conditions for effective protein extraction from chlorella.

[0057] At this time, a single factor experiment was performed on the conditions of protease treatment time (minutes), pH, and temperature (℃) to derive an optimized model of protease treatment conditions for chlorella protein extraction using RSM, and Box-Behnken Design (BBD) was used.

[0058] As a result, 5% chlorella suspension mixed with Chlorella pyrenoidosa and distilled water was treated with 3% microbial protease relative to the chlorella solids, and the experiment was designed according to BBD in RSM for optimization, and the independent variable (X i ) dependent variable (Y) according to pH (X1), temperature (X2) and time (X3) i ) The protein extraction yield (Y1) results were obtained, and the optimal conditions modeled by setting the protein extraction yield with the R value were derived.

[0059] More specifically, the experimental levels and ranges of independent variables designed for model optimization are shown in Table 2, the second-order polynomial model equation is shown in Table 3, and the ANOVA for variance and adequacy analysis of the second-order model is shown in Table 4.

[0060] In Table 2, the extraction conditions of chlorella protein were coded into three levels of -1, 0, and +1 to optimize them, and the ranges of independent variables were set as temperature 40, 50, and 60℃; pH 9.0, 10.0, and 11.0; and time 30, 40, and 50 minutes.

[0061]

[0062] [Table 2]

[0063]

[0064]

[0065] The results of the experiment according to the conditions presented in RSM and the quadratic equation derived for each dependent variable are as shown in Table 3.

[0066]

[0067] [Table 3]

[0068]

[0069]

[0070] To verify the significance of the model presented in this invention, ANOVA analysis was conducted, and the results are shown in Table 4. It was found that the models for all dependent variables developed in this study were significant.

[0071]

[0072] [Table 4]

[0073]

[0074]

[0075] The optimal extraction conditions for protein decomposition enzyme treatment using RSM are shown in Table 5 below.

[0076]

[0077] [Table 5]

[0078]

[0079]

[0080] Example 3. Verification of optimal protease treatment conditions using RSM.

[0081] In order to verify the optimal conditions predicted through modeling using RSM in the above example, the inventors measured the yield of chlorella protein extracted by treating with protease according to the suggested optimal conditions, and the results are shown in Table 6 below.

[0082] This indicates that the model optimized using RSM is appropriate, as the deviation between the actual and predicted values ​​in all variables is less than 5% (p< 0.05).

[0083]

[0084] [Table 6]

[0085]

[0086]

[0087] As a result, the optimal conditions modeled according to the present invention were derived as a protease treatment temperature of 45.45°C; a pH of 9.01; and a time of 49.85 minutes, and it was confirmed that this was a statistically significant model.

[0088] Therefore, this proves that the optimal protease treatment conditions of the present invention, derived based on the RSM analysis of the present invention, can excellently extract chlorella proteins.

[0089]

[0090] In summary, enzyme treatment can break down the cell walls of chlorella, converting the proteins into easily digestible forms, and increasing the content of nutrients such as amino acids, vitamins, and minerals. However, effective protein extraction requires adjusting the type of enzyme, amount of enzyme, and treatment time. Currently, costly and time-consuming research is being conducted to effectively extract chlorella proteins using enzymes, such as research on enzymes for chlorella protein extraction and research on long-term treatment for protein extraction after the research. However, problems such as reduced efficiency compared to time and protein denaturation are occurring. Therefore, the present invention effectively extracts chlorella proteins using relatively inexpensive microbial-derived protein-degrading enzymes, and provides an industrial method that can reduce costs and time compared to existing studies through optimization of enzyme treatment conditions and statistical verification. In addition, when treated under the optimal conditions of the present invention, the hard cell walls of chlorella composed of cellulose are softened and made soft, making them easily dissolved in the human body, thereby significantly increasing the digestibility and absorption rate.

[0091] In addition, in the present invention, in extracting chlorella proteins, a RSM analysis-based extraction method was established to solve the problems of existing extraction methods, such as organic solvent extraction methods, such as human harm, environmental toxicity, high cost, and low selectivity, and to produce an extract with enhanced functional components and physiological activity of chlorella using a safe and inexpensive Bacillus-derived protease.

[0092]

[0093] Although the present invention has been described with reference to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims encompass such modifications and variations as fall within the spirit of the invention.

Claims

1. A method for extracting chlorella protein, comprising the step of adding protease to a chlorella suspension and reacting the mixture at a pH of 8.1 to 10 and a temperature of 40°C to 50°C for 40 to 60 minutes.

2. In paragraph 1, A method for extracting chlorella protein, characterized in that the protease is at least one selected from the group consisting of microbial protease, cellulase, viscozyme L, papain, kozyme, alkalise, esperase, nutraze, and flavonoid.

3. In paragraph 2, A method for extracting chlorella protein, characterized in that the above microbial protease is derived from Bacillus.

4. In paragraph 3, The above Bacillus-derived protease is characterized in that the concentration is 0.1 to 99%. Method for extracting chlorella protein.

5. A method for producing a chlorella extract, comprising the step of adding protease to a chlorella suspension and reacting the mixture at a pH of 8.1 to 10 and a temperature of 40°C to 50°C for 40 to 60 minutes.

6. Chlorella extract manufactured by the method of Article 5.

7. Processed food containing the chlorella extract of Article 6.

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