KR102681818B1 - Method for preparing spirulina extract with increased branched chain amino acid content - Google Patents

Abstract

The present invention relates to a method for producing a spirulina extract with increased branched chain amino acid content, and specifically to a method for producing a spirulina extract comprising the step of treating spirulina with a proteolytic enzyme and then fermenting it. The spirolina extract prepared by the method of the present invention has an increased content of branched chain amino acids and contains leucine, isoleucine and valine at an optimal mixing ratio.

Description

Method for producing spirulina extract with increased branched chain amino acid content {METHOD FOR PREPARING SPIRULINA EXTRACT WITH INCREASED BRANCHED CHAIN AMINO ACID CONTENT}

The present invention relates to a method for producing a spirulina extract with increased branched chain amino acid content, and specifically to a method for producing a spirulina extract comprising the step of treating spirulina with a proteolytic enzyme and then fermenting it.

Spirulina (Arthrospira platensis) is one of the blue-green algae among microalgae and is found in alkaline warm areas such as seawater and freshwater around the world. Spirulina is a multicellular organism that has the ability to photosynthesize and synthesizes nutrients on its own without relying on other organisms. It has a unique blue-green color and a spiral shape. Among the five major nutrients, spirulina consists of more than 55-65% protein, is rich in vitamins and minerals, and has a high digestion and absorption rate, so it is considered an ideal source of nutrients and a high value-added food.

Meanwhile, branched amino acids refer to Leucine, isoleucine, and Valine, which have hydrocarbon side chains in their structures among Essential Amino Acids. Branched chain is a Chinese word for branched chain. In the market, branched chain amino acids are sometimes called BCAA, taking the first letters of the English name Branched Chain Amino Acid. Branched chain amino acids have the characteristic of being metabolized in skeletal muscles, unlike most amino acids used in the liver. In particular, when leucine, isoleucine, and valine are administered at a ratio of 2:1:1 (weight ratio), protein synthesis in the body has been shown to be higher, so it is considered important when doing muscle growth exercises and is also used as an exercise supplement sought by athletes and the general public who enjoy exercising. It is becoming.

The reasons why BCAAs are considered important when exercising are: first, the amino acids increased by protein breakdown during exercise maintain blood glucose concentration and suppress hypoglycemia; second, they are involved in the production of ATP (Adenosine Phosphate) during exercise; and third, This is because it has the effect of preventing and recovering muscle fatigue. In addition, it is known to have beneficial effects in vivo as an important regulator of insulin signaling and lipid metabolism.

Nevertheless, leucine has no taste or a slightly bitter taste, isoleucine has a bitter taste, and valine has a sweet taste in the D type, but L type has a bitter taste and is difficult to apply to foods, so there is a limit to its application to foods.

Korean Patent Publication No. 2015-0099689 discloses a method of producing seaweed extract by treating seaweed such as spirulina with membrane-degrading enzymes and proteolytic enzymes.

The purpose of the present invention is to provide a method for producing a spirulina extract containing the branched chain amino acids leucine, isoleucine and valine in an optimal mixing ratio by treating protein-rich spirulina using hydrolytic enzymes and microorganisms.

The above task includes preparing a spirulina aqueous solution by adding 8 to 10 times the amount of purified water compared to the weight of spirulina powder to spirulina powder obtained by pulverizing dried spirulina; Adding 5 to 7% by weight of proteolytic enzyme based on the weight of spirulina powder to the spirulina aqueous solution and hydrolyzing it at a temperature of 60 to 90°C for 4 to 6 hours to prepare a hydrolyzate; Fermenting the enzyme hydrolyzate by adding 0.1 to 1.5% by weight of microorganisms based on the weight of spirulina powder; and centrifuging the fermented product and then filtering the supernatant to prepare the spirulina extract.

Preferably, the proteolytic enzyme is alcalase, and the microorganism may be Aspergillus kawachii or Saccharomyces cerevisiae .

Also preferably, the step of deactivating the hydrolyzate may be further included after the step of preparing the hydrolyzate.

Also preferably, the spirulina extract may include leucine, isoleucine, and valine in a weight ratio of 2:1:1.

In the method for producing a spirulina extract according to the present invention, the produced spirulina extract has an increased content of branched chain amino acids and contains leucine, isoleucine and valine at an optimal mixing ratio.

Figure 1 shows the case of treating spirulina aqueous solution with alcalase (Alc), bromelamine (Bro), flavorzyme (Fla), Neutrase (Neu), papain (Pap), and Protamex (Pro). The results of measuring the contents of total polyphenol (a) and crude protein (b) are shown.
Figure 2 shows the aqueous spirulina solution treated with alcalase (Alc), the case of fermentation with Aspergillus kawaii for 24 hours after treatment with alcalase (AK24), and the fermentation of Aspergillus kawaii after treatment with alcalase. When fermented for 36 hours (AK36), when treated with alcalase and fermented for 48 hours with Aspergillus kawaii (AK48), when treated with alcalase and fermented for 24 hours with Saccharomyces cerevisiae (AK48) SC24), total polyphenols when treated with alcalase and fermented with Saccharomys cerevisiae for 36 hours (SC36), and when treated with alcalase and fermented with Saccharomys cerevisiae for 48 hours (SC48) (Figure 2a) shows the results of measuring the contents of crude protein (Figure 2b) and free amino acid (Figure 2c).
Figure 3 shows the case of spirulina aqueous solution treated with alcalase (Alc), the case of treatment with alcalase and then fermented with Aspergillus kawaii for 24 hours (AK24), and the case of fermentation with Aspergillus kawaii after treatment with alcalase. When fermented for 36 hours (AK36), when treated with alcalase and fermented with Aspergillus kawaii for 48 hours (AK48), when treated with alcalase and fermented with Saccharomyces cerevisiae for 24 hours (SC24) ), leucine in the case of treatment with alcalase and fermentation with Saccharomyces cerevisiae for 36 hours (SC36), and with leucine in the case of treatment with alcalase and fermentation with Saccharomyces cerevisiae for 48 hours (SC48) (Figure 3a) ), isoleucine (Figure 3b), and valine (Figure 3c).
Figure 4 shows the case of spirulina aqueous solution treated with alcalase (Alc), the case of treatment with alcalase and fermentation with Aspergillus kawaii for 24 hours (AK24), and the case of fermentation with Aspergillus kawaii after treatment with alcalase. When fermented for 36 hours (AK36), when treated with alcalase and fermented with Aspergillus kawaii for 48 hours (AK48), when treated with alcalase and fermented with Saccharomyces cerevisiae for 24 hours (SC24) ), leucine, isoleucine, when treated with alcalase and fermented with Saccharomyes cerevisiae for 36 hours (SC36), and when treated with alcalase and fermented with Saccharomyes cerevisiae for 48 hours (SC48) It is shown by adding up the valine content.

All technical terms used in the present invention, unless otherwise defined, have the following definitions and correspond to the meaning as generally understood by a person skilled in the art in the relevant field of the present invention. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods are also included in the scope of the present invention.

The term "about" refers to a quantity, level, value, number, frequency, percent, dimension, size, amount, weight or length of 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight or length that varies by 4, 3, 2 or 1%.

Throughout this specification, unless the context otherwise requires, the words "comprise" and "comprising" include a given step or component, or group of steps or components, but any other step or component, or It should be understood that this implies that no step or group of components is excluded.

The present invention relates to a method for producing a spirulina extract with increased branched chain amino acid content.

The method includes the following steps:

Preparing a spirulina aqueous solution by adding 8 to 10 times the amount of purified water compared to the weight of spirulina powder to spirulina powder obtained by pulverizing dried spirulina (S11); Enzymatic hydrolysis step (S12) of adding 5 to 7% by weight of proteolytic enzyme relative to the weight of spirulina powder to the spirulina aqueous solution and hydrolyzing it at a temperature of 60 to 90 ° C. for 4 to 6 hours; Deactivating the hydrolyzate (S13); Fermenting the enzymatic hydrolyzate by adding 0.1 to 1.5% by weight of microorganisms based on the weight of spirulina powder (S14); And preparing a spirulina extract by centrifuging the fermented product and filtering the supernatant (S14).

Each step is described in detail below.

First, spirulina powder is prepared by grinding the dried spirulina, and 8 to 10 times the weight of the powder is added to prepare a spirulina aqueous solution (S11).

Next, proteolytic enzyme is added to the spirulina aqueous solution and hydrolyzed for 4 to 6 hours (S12). The proteolytic enzymes include Alcalase, Bromelain, Flavorzyme, Neutrase, Papain, and Protamex from Novozyme. ) may be one type selected from the group consisting of Preferably it may be Alcalase.

Next, the hydrolyzate is heated to deactivate the proteolytic enzyme (S13). The deactivation temperature follows a known method.

Next, microorganisms are added to the deactivated hydrolyzate and fermented (S14). The microorganisms

It may be Aspergillus kawachii or Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae .

In the present invention, spirulina is treated with proteolytic enzymes and then fermented with Saccharomyces cerevisiae, thereby increasing the content of branched-chain amino acids, and in particular, containing leucine, isoleucine, and valine in a mixing ratio that works most optimally in vivo. can do. Preferably, leucine, isoleucine, and valine may be included in a weight ratio of 2:1:1.

The spirulina extract prepared in the present invention can be included in beverages, general foods, health functional foods, etc.

Hereinafter, the present invention will be described in detail through examples and experimental examples, but the scope of the present invention is not limited by these examples.

Experimental Example 1: Component analysis of spirulina proteolytic enzyme-treated hydrolyzate

In order to develop an extraction method to increase the crude protein and amino acid content of Spirulina, a single enzyme was applied to prepare a hydrolyzed extract and experiments were conducted to track down the appropriate enzyme.

In order to maximize the activity of the hydrolytic enzyme, the extract was developed by adjusting the pH according to the activity conditions for each enzyme, proceeding with the reaction at an appropriate temperature, and stopping the reaction. After conducting an experiment using the extract, a hydrolysis process was established based on the experimental results.

1-1. protein hydrolytic enzyme

The experiment using a single enzyme consisted of a control under the same sample conditions without applying the enzyme and the proteolytic enzymes Alcalase, Bromelain, and Flavor from Novozyme. A total of 6 products were applied: Flavourzyme, Neutrase, Papain, and Protamex. The treatment conditions for each enzyme are shown in Table 1 below.

enzyme temperament pH temperature ℃ Alcalase protein 6.5 ~ 8.5 55~70 bromelain 6.0 ~ 8.0 45~50 Flavorzyme 5.0 ~ 7.0 50 Neutraze 5.5 ~ 7.5 45~55 papain 5.0 ~ 7.5 50~70 Protamex 5.5 ~ 7.5 35~60

1-2. Preparation of spirulina hydrolyzate

The enzymes applied in this experiment were used to increase the crude protein and amino acids of spirulina. After preparing an aqueous solution of 10 wt% concentration of spirulina powder, it was homogenized at 18,000 to 25,000 RPM, sterilized and cooled at 100°C/30 minutes, and then used as a control and sample.

The cooled sample was adjusted to the optimal pH for a single enzyme, and then enzymes were added at 1% increments. At this time, the enzyme addition ratio was based on the weight of spirulina powder, which served as a substrate. Samples to which enzymes were added were hydrolyzed in a shaking incubator set at each optimal temperature and 100 to 150 rpm, and after 5 hours, they were deactivated and samples were taken. The supernatant of the sample was recovered through centrifugation at 2,000 rpm for 20 minutes, and the test analysis was performed using the filtered liquid using 5um (Thickness: 0.26mm) filter paper. Total polyphenols were measured using a visible light spectrophotometer (UV/Vis Spectrophotometer), and crude protein was measured using a protein distillation system (Kjeldahl Distillation System).

The experimental conditions are shown in Table 2 below.

Sample name enzyme enzyme concentration Sample concentration processing time 0 Control Enzyme - 10wt% - One Alc Alcalase temperament comparison
1wt% 5 hours 2 Bro AMG 3 Fla Celluclast 4 Neu Flavorzyme 5 Pap Neutrase 6 Pro Protamex

1-3. Determination of total polyphenol content

After mixing 20uL of the sample solution prepared above with 80uL of 7.5% sodium carbonate solution, 100uL of Folin-ciocalteu’s phenol solution was added and reacted for 30 minutes, and the absorbance was measured at 725nm. Tannin was used as a standard solution, and the results were expressed in terms of tannin content in the sample. The results are shown in Figure 1.

1-4. Crude protein content determination

2 g of potassium sulfate and 12 mL of concentrated sulfuric acid as decomposition accelerators were added to 1 g of the sample solution and carbonized in a Kjeldahl decomposition device until the sample solution changed from light off-white to colorless. The crude protein content was measured by distilling, neutralizing, and adding the carbide dropwise using a protein distillation quantitative device. The results are shown in Figure 2.

1-5. result

Looking at Figure 1, total polyphenols all showed an increase compared to the control dog after enzymatic hydrolysis, and among them, proteolytic enzymes showed the highest effect in that order: alcalase, bromelain, and neutrase. The crude protein content of alcalase and bromelain increased by nearly two times compared to the control group. In order to increase active ingredients such as total polyphenols and crude protein of Spirulina through this single enzyme application experiment, proteolytic enzymes, especially alcalase, are suitable. Accordingly, Experiment Example 2 below was conducted on spirulina hydrolyzate using alcalase.

Experimental Example 2: Optimization of conditions for Alcalase treatment using response surface analysis

A response surface analysis experiment was conducted to track the optimal conditions for Alcalase. The experimental conditions were designed based on central synthesis planning, enzyme concentration and treatment time were set as independent variables, and total polyphenol and crude protein results were set as dependent variables. The experiment was conducted with a total of 15 treatments, and the treatments were conducted in a random order to reduce statistical errors. After preparing an aqueous solution of 10% concentration of spirulina, it was homogenized at 18,000 to 25,000 RPM, sterilized and cooled at 100°C/30 minutes, and then used as a control and sample.

The cooled sample was adjusted to the optimal pH of Alcalase and then added based on the concentration of each enzyme set as an independent variable based on the weight of spirulina, which served as the substrate. Samples to which enzymes were added were hydrolyzed in a shaking incubator set at each optimal temperature and 100 to 150 rpm, and samples were taken after deactivation after each treatment time set as an independent variable. The supernatant of the sample was recovered through centrifugation at 2,000 rpm for 20 minutes, and the test analysis was performed using the filtered liquid using 5um (Thickness: 0.26mm) filter paper. Response surface analysis conditions are shown in Table 3 below.

Total polyphenols were measured using a visible light spectrophotometer (UV/Vis Spectrophotometer), crude protein was measured using a protein distillation system (Kjeldahl Distillation System), and each result was used as a dependent variable to ultimately perform response surface analysis. The results were shown through The results are shown in Table 4.

treatment area independent variable
(Independent variable) dependent variable
(Dependent variable) enzyme concentration
(%) processing time
(hr) Total polyphenol content
(mg/L) Crude protein content
(mg/g) free amino acid
(mg/g) One 4 5 494.2 1808.5 323.8 2 4 One 526.9 1468.8 110.7 3 4 3 512.7 624.6 180.0 4 6 3 351.4 1887.5 206.2 5 4 3 561.0 1070.7 178.3 6 4 3 625.6 1358.9 184.7 7 2 3 383.3 1153.0 132.4 8 6 One 489.6 1458.5 107.8 9 6 5 871.6 2766.0 513.1 10 4 3 661.1 1811.9 174.0 11 2 One 386.1 1153.0 76.7 12 4 3 590.0 1448.2 179.5 13 2 5 557.5 1129.0 257.6 14 4 3 571.4 1479.1 183.9

Based on the above analysis results, when enzymatically hydrolyzing spirulina using alcalase, the optimal condition was to add 6 wt% of alcalase and react for 5 hours to increase total polyphenol and crude protein.

Experimental Example 3: Analysis of spirulina extract components using hydrolytic enzyme and fermentation treatment

After preparing an aqueous solution of 10 wt% concentration of spirulina powder, it was homogenized at 18,000 to 25,000 RPM, sterilized and cooled at 100°C/30 minutes, and then used as a control and sample. After adjusting the cooled sample to the optimal pH of Alcalase, 6 wt% of Alcalase was added based on the weight of spirulina, which was the substrate, and this was determined in the response surface analysis of Experimental Example 2. This is a value derived from optimization conditions.

Samples to which enzymes were added were deactivated after 5 hours, which was determined as the optimal time in response surface analysis, in a shaking incubator set at each optimal temperature and 100 to 150 rpm. For the deactivated sample , Aspergillus kawachii (AK) and Saccharomyces cerevisiae (SC) dry yeast were added at a content of 1% of the substrate for 24 hours, 36 hours, and 48 hours, respectively. After culturing for a while, sterilized samples were taken.

The supernatant of the sample was recovered through centrifugation at 2,000 rpm for 20 minutes, and the test analysis was performed using the filtered liquid using 5um (Thickness: 0.26mm) filter paper.

Total polyphenols were measured using a visible light spectrophotometer (UV/Vis Spectrophotometer), crude protein was measured using a protein distillation system (Kjeldahl Distillation System), and an automatic amino acid analyzer (L-8800, Hitachi, Japan) was used. Free amino acids were measured.

3-1. Total polyphenol content test

After mixing 20uL of the sample solution prepared above with 80uL of 7.5% sodium carbonate solution, 100uL of Folin-ciocalteu’s phenol solution was added and reacted for 30 minutes, and the absorbance was measured at 725nm. Tannin was used as a standard solution, and the results were expressed in terms of tannin content in the sample.

3-2. Crude protein experiment

2 g of potassium sulfate and 12 mL of concentrated sulfuric acid as decomposition accelerators were added to 1 g of the sample solution and carbonized in a Kjeldahl decomposition device until the sample solution changed from light off-white to colorless. The crude protein content was measured by distilling, neutralizing, and adding the carbide dropwise using a protein distillation quantitative device. The results are shown in Figure 2.

3-3. Free amino acid measurement experiment

45 mL of 50% ethanol solution was added to 2 g of the sample solution, shaken for 3 hours, and then ethanol was removed using a reduced pressure concentrator. Distilled water was added to the ethanol-free filtrate to make 100 mL, and the free amino acids were measured using an automatic amino acid analyzer and 43 types of free amino acid standards. The measurement conditions are as shown in Table 5 below, and the results are shown in Figures 3 and 4.

Free Amino acid Instrument L-8800, Hitachi, Japan Column Ion exchange column (4.6 mm×60 mm) Detector UV Oven Temperature 30 - 70℃ Coil Temperature 135℃ Flow rate 0.35mL/min

3-4. result

Looking at Figure 2, it was confirmed that total polyphenol and crude protein increased overall when microbial fermentation was performed after alcalase treatment. In the case of free amino acids, Aspergillus kawaii (AK) increased as fermentation time increased, and Saccharomyces cerevisiae (SC) tended to decrease, but when only branched chain amino acids were checked among 43 types of free amino acids, Saccharomyces cerevisiae (SC) showed a tendency to increase. The increase in Myces cerevisiae (SC) was higher.

Looking at Figure 3a, the increase in leucine showed no significant difference between Aspergillus kawaii (AK) and Saccharomyces cerevisiae (SC). 3B and 3C, the increase in isoleucine and valine was higher in the Saccharomyces cerevisiae (SC) treatment group, and the Saccharomyces cerevisiae (SC) treatment group for 48 hours had higher levels of leucine, isoleucine, and valine. It was confirmed that the content ratio was close to 2:1:1, increasing at the best ratio for in vivo action.

Claims (4)

Preparing a spirulina aqueous solution by adding 8 to 10 times the amount of purified water compared to the weight of spirulina powder to spirulina powder obtained by pulverizing dried spirulina;
Adding 5 to 7% by weight of proteolytic enzyme based on the weight of spirulina powder to the spirulina aqueous solution and hydrolyzing it at a temperature of 60 to 90°C for 4 to 6 hours to prepare a hydrolyzate;
Preparing a fermented product by adding 0.1 to 1.5% by weight of microorganisms relative to the weight of spirulina powder to the hydrolyzate and fermenting it; and
Producing a spirulina extract by centrifuging the fermented product and filtering the supernatant,
The proteolytic enzyme is alcalase, and the microorganism is Saccharomyces cerevisiae ,
A method for producing a spirulina extract, characterized in that the spirulina extract contains leucine, isoleucine and valine in a weight ratio of 2:1:1. The method of producing a spirulina extract according to claim 1, further comprising the step of deactivating the hydrolyzate after preparing the hydrolyzate. delete delete