KR101178860B1 - Nature seasoning using micro algae protein hydrolysate and the manufacturing method thereof - Google Patents

Abstract

PURPOSE: Natural seasonings including microalgae protein hydrolysate, and a producing method thereof are provided to improve the protein-dissolving capacity of microalgae before hydrolyzing the microalgae. CONSTITUTION: A producing method of natural seasonings including microalgae protein hydrolysate comprises the following steps: processing chlorella vulgaris or tetraselmis suecica with ultrasonic waves using an ultrasonic generator in the frequency of 90-100kHz for 4-5 hours for eluting protein; and producing the protein hydrolysate by hydrolyzing the protein for 50-60 minutes using proteinase in the pH of 6-7. The proteinase is alcalase.

Description

Nature seasoning using micro algae protein hydrolysate and the manufacturing method

The present invention relates to a natural seasoning comprising a hydrolyzate of protein extracted from microalgae and a method for producing the same.

Microalgae are aquatic organisms in freshwater and seawater, including blue algae, diatoms, coarse algae, green algae, red algae, yellow brown algae, and silver algae, and there are about 35,000 species reported worldwide. It is estimated that there exists an abnormality. Microalgae, like other plants, are photosynthetic and biosynthesize sugars and proteins in the interior, and are therefore responsible for basic production in freshwater and the ocean, and are important producers of food chains.

Most microalgae contain unique bioactive substances inside the cell wall of each species and can produce large quantities of specific substances depending on the culture environment, so carotenoid pigments, antioxidants, fatty acids, enzymes, functional peptides, biotoxins and Industrially produced for the purpose of using sterols and the like. In addition, it is used for a variety of uses, such as the production of fish and bio-energy of juvenile and sexual organs.

However, microalgae have a thick and hard cell wall like normal plants, and its cell wall consists of cellulose, hemicellulose, pectin, etc., which makes it very difficult to decompose by human carbohydrate degrading enzymes. In addition, since the digestibility of microalgae proteins is lower than that of general plant and animal proteins, the use of internal materials is very limited, and industrial application is very difficult.

With the recent rise of lifestyles that consider the environment and health together, the 'well-being' culture that pursues life in harmony with the mind and body, nature-friendly life, and leisurely life has developed over the years. . In Korea, the concept of well-being began to be introduced at the end of 2002. Now, as the 'Lifestyle Of Health And Sustainability' (LOHAS) era has arrived, the seasoning market has introduced mono sodium glutamate (MSG), chemical compounding seasoning and natural compounding seasoning. After that, it entered the age of natural seasoning. In addition, ingestion of synthetic glutamic acid, which is a predominant chemical condiment, causes headaches, cramps, and asthenia. They avoid chemical seasonings and prefer natural seasonings.

Food containing artificial additives is now becoming an old-fashioned artifact that does not catch the attention of consumers and keeps up with the trend. And the aftermath will affect all foods, and natural seasonings will be valued as a substitute for modern consumers who do not allow any additives that have a reputation as a synthetic seasoning, such as MSG, and the market is increasingly becoming a substitute. Will be expanded.

The present invention, in order to solve the above problems, by increasing the protein dissolution ability by treating the cell wall of the natural material, in particular microalgae, natural seasoning comprising a hydrolyzate produced by hydrolyzing the protein as an active ingredient And to provide a method of manufacturing the same.

The present invention relates to natural seasonings comprising protein hydrolysates of microalgae.

In addition, the present invention comprises the steps of a) eluting the protein by sonicating the microalgae; B) preparing a protein hydrolyzate using protease on the protein;

It relates to a manufacturing method of natural seasoning comprising a.

The natural seasoning method of the present invention has a high food safety because it uses a natural material as compared to the low food safety of the conventional synthetic seasonings, it has a high content of net amino acids and high utilization as a net material. In addition, the amount of protein eluted can be increased by sonication, and the production yield can be improved.

Figure 1 shows a three-dimensional chart of the response surface analysis results for the extraction ability of the internal protein extraction of Chlorella vulgaris by ultrasound.
Figure 2 shows a three-dimensional chart of the response surface analysis results for the extraction ability of the internal protein extraction of Tetraselmis suecica using ultrasonic waves.
Figure 3 Chlorella using Alcalase 2.4L A three-dimensional plot of the response surface analysis results for the degree of hydrolysis during protein hydrolysis of vulgaris is presented.
Figure 4 Tetraselmis using Alcalase 2.4L A three-dimensional plot of the response surface analysis results for the degree of hydrolysis during hydrolysis of suecica is shown.

The present invention provides a natural seasoning containing protein hydrolyzate of microalgae.

In the present invention, the protein hydrolyzate of the microalgae refers to a hydrolyzate produced by ultrasonically treating the microalgae to elute the protein and using the protease to the eluted protein.

The microalgae may be, but are not limited to, green algae (Chlorophyte), brown algae (Phaeophyceae), red algae (Rhodophyte), cyanophyta, diatoms (Bacillariophycea), dynaphytes or Haptophyta. .

In addition, the green alga is Chlorella vulgaris ( Chlorella vulgaris ), coccyx bird may include Tetrselmis suecica .

The microalgae, in particular, can be eluted protein by treating the cell wall of the microalgae, it is possible to prepare a hydrolyzate using a protease to the eluted protein. The present invention is to provide a natural seasoning containing the hydrolyzate.

The microalgal protein hydrolyzate may include 5-30 parts by weight based on 100 parts by weight of the natural seasoning. The present invention also provides a method for producing natural seasonings comprising microalgal protein hydrolysates. More specifically,

a) eluting the protein by sonicating the microalgae; B) preparing a protein hydrolyzate using protease on the protein;

It provides a method of producing a natural seasoning comprising a.

The microalgae cell wall can be sonicated to elute the protein.

The microalgae include, but are not limited to, green algae (Chlorophyte), brown algae (Phaeophyceae), red algae (Rhodophyte), cyanophyta, diatoms (Bacillariophycea), warpweed algae (Dinophyta), or flax algae (Haptophyta). Can be.

In addition, the green alga is Chlorella vulgaris ( Chlorella vulgaris ), coccyx bird may include Tetrselmis suecica .

In the present invention, it is possible to calculate the optimum conditions of extraction frequency, ultrasonic extraction time and extraction temperature of the protein eluting (or extraction) in step a), in an embodiment of the present invention (Response Surface Methodology, RSM) Can be calculated using That is, as a result of calculating the extraction frequency, the ultrasonic extraction time and the extraction temperature as independent variables, the ultrasonic treatment can be performed at 26 ~ 28 ℃ for 4-5 hours at a frequency of 90-100 kHz in the ultrasonic generator.

The response surface analysis method is a regression analysis method that finds an optimal condition of factors that give an optimal response value from a change pattern of a response variable according to a change of factor level on the assumption of a secondary model. It is used.

In addition, in the present invention, the optimal condition for hydrolysis in step b) may be calculated using Response Surface Methodology (RSM). That is, as a result of calculating the concentration, hydrolysis time and pH of the protease as independent variables, the concentration of the protease may be 2 to 3%, the hydrolysis time is 50 to 60 minutes, and the pH may be 6 to 7. .

The protease may include an alcalase, and may use 2 to 3 L of the alkalase, and may be hydrolyzed at 45 to 55 ° C.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Microalgal Proteins Hydrolyzate  Produce

Example  1-1. Preparation of Microalgal Solids

Chlorella in the microalgae used in the experiment vulgaris was purchased directly from Daesang Co., Ltd. in 2010, Tetraselmis suecica was cultured directly at the Marine Biology Research and Education Center. The microalgae purchased and cultured were centrifuged to remove the culture solution. More specifically, the purchased and cultured microalgae were centrifuged at 7,000 rpm for 15 minutes to separate the supernatant and solids, and after removing the supernatant, only the solids were used. Stored.

Example  1-2. Extraction of Microalgae-Derived Proteins

Ultrasonic treatment was performed on the solids of the obtained microalgae to calculate the optimal extraction conditions of the internal protein, and the microalgae proteins were extracted by applying the extraction optimum conditions.

More specifically, 900 mL of water was added to 100 g, and ultrasonic extraction was performed at a frequency of 35-170 kHz, 1-6 hours, and a temperature range of 25-45 ° C. The optimum extraction conditions were calculated through Response Surface Methodology, and the results are shown in Table 1 and FIGS. 1 and 2.

Run Time (hr) Temperature (℃℃) Frequency
(kHz) Chlorella vulgaris Tetraselmis
suecica Protein contents
(mg / mL) Protein contents
(mg / mL) One 2 20 170 0.62 1.77 2 6 45 35 1.60 2.54 3 4 32.5 35 1.89 2.82 4 6 32.5 170 1.71 2.40 5 2 45 170 1.52 1.64 6 4 38.75 72 2.39 3.55 7 2 45 170 1.52 1.63 8 2 32.5 100 1.58 2.49 9 2 20 35 0.95 1.94 10 6 45 100 1.78 2.58 11 2 45 35 1.23 1.85 12 6 20 170 1.60 2.33 13 2 20 170 0.61 1.76 14 2 45 35 1.22 1.88 15 4 20 35 1.53 2.52 16 6 45 170 1.65 2.02 17 4 20 100 2.54 3.79 18 6 20 35 2.09 2.67 19 6 20 35 2.10 2.65 20 2 20 35 0.93 1.93

Chlorella Quadratic models according to the extraction frequency, ultrasonic extraction time and extraction temperature of vulgaris are as follows.

Y (protein content, mg / mL ) = 2.40 + 0.31 A + 0.068 B - 0.030 C - 0.42 (A) 2 - 0.072 (B) 2 - 0.53 (C) 2 - 0.24 AB - 0.042 AC + 0.12 BC

Where, A = extraction time (hr), B = temperature (℃), C = frequency (kHz)

Thus Chlorella Optimum condition for protein extraction of vulgaris was 95.8 kHz, 27.16 ℃, extraction time was 4.99 hr.

Meanwhile , Tetraselmis The quadratic model according to the extraction frequency, ultrasonic extraction time and extraction temperature of suecica is as follows.

Y (protein content, mg / mL ) = 3.53 + 0.28 A - 0.11 B - 0.12 C - 0.64 (A) 2 - 0.12 (B) 2 - 0.71 (C) 2 - 0.067 AB - 0.038 AC 0.053 BC

Where, A = extraction time (hr), B = temperature (℃), C = frequency (kHz)

therefore Tetraselmis The optimum protein extraction condition of suecica was 95.8 kHz, 27.16 ℃ temperature and extraction time was 4.99 hr.

Example  1-3. Microalgal Proteins Hydrolyzate  Produce

Alcalase 2.4L was added to the obtained microalgae extract to calculate optimal protein hydrolysis conditions. Protein hydrolysates were prepared by applying the optimum conditions, and the protein hydrolysates were lyophilized to prepare microalgal protein hydrolysates.

More specifically, the microalgae from which the protein was extracted were preheated in a constant temperature water bath at 52.5 ° C., and hydrolyzed at a pH range of 6-7 with alcalase 2.4L at a concentration of 1-3%, a reaction time of 10-60 min, and a pH of 6-7. Optimal hydrolysis conditions were calculated through Response Surface Methodology, and the results are shown in Table 2 and FIGS. 3 and 4.

Run Time (hr) Temperature (℃℃) Frequency
(kHz) Chlorella vulgaris Tetraselmis
suecica Degree of hydrolysis (%) Degree of hydrolysis (%) One One 10 7 20.88 19.87 2 2.5 47.5 6.5 38.11 36.01 3 One 35 6.5 24.71 24.31 4 2 35 7 34.38 31.18 5 3 10 7 25.35 23.9 6 2 35 7 34.34 31.14 7 3 10 7 25.36 24 8 One 60 7 31.9 31.2 9 3 60 7 38.96 36.86 10 3 35 6 34.37 33.37 11 2 60 6 37.47 36.17 12 One 10 7 20.81 19.85 13 3 60 7 38.98 36.8 14 2 10 6.5 22.79 21.49 15 3 60 6 38.94 36.04 16 3 10 6 24.65 23.25 17 One 60 7 32.18 31.28 18 One 60 6 30.58 30.08 19 One 10 6 20.15 19.16 20 2 10 6 22.29 21.09

Chlorella The quadratic model according to enzyme concentration, hydrolysis time and pH of vulgaris is as follows.

Y (DH%) = 33.26 + 3.94 A + 5.73 B + 0.33 C - 2.00 (A) 2 - 1.22 (B) 2 + 1.52 (C) 2 - 1.50 AB - 0.36 AC - 0.33 BC

Where, A = enzyme concentration, (%, v / w), B = hydrolysis time (min),

C = pH

Thus Chlorella The optimum hydrolysis condition of vulgaris was 2.49% alcalase concentration, 6.01 pH, 58.70 min hydrolysis time.

Meanwhile , Tetraselmis The quadratic model according to the enzyme concentration, hydrolysis time and pH of suecica is as follows.

Y (DH%) = 32.17 + 3.30 A + 6.52 B + 0.55 C - 2.38 (A) 2 - 2.55 (B) 2 + 0.54 (C) 2 + 0.77 AB - 0.27 AC - 0.24 BC

Where, A = enzyme concentration, (%, v / w), B = hydrolysis time (min),

C = pH

Thus Chlorella The optimal hydrolysis conditions for vulgaris were pH of 6.01 and hydrolysis time of 58.53 min at 2.91% alcalase concentration.

By applying the respective optimum conditions producing microalgae hydrolyzate, lyophilized at -80 ℃ After removing the solids for 15 minutes was centrifuged at 7,000 rpm Chlorella 14.68 g of vulgaris protein hydrolyzate and 14.2 g of Tetraselmis suecica protein hydrolyzate were prepared.

< Example  2> Microalgal Proteins Hydrolyzate  Net ingredient analysis

Example  2-1. Microalgal Proteins Hydrolyzate  Amino acid analysis

The composition and free amino acids of the microalgal hydrolyzate prepared in Example 1-3 were analyzed.

In general, since the free amino acid is used as an important quality index as a degradation product of protein having the most important effect on flavor, the composition of microalgae protein hydrolysates and the composition of free amino acids were analyzed.

More specifically, 20 mL of 6 N HCl was added to 100 mg of the microalgae protein hydrolysate and decomposed in a 105 ° C constant temperature dryer for 24 hours. The decomposed sample was concentrated with a reduced pressure concentrator (R-114, BUCHI, Flawil, Swiss), and then analyzed with an amino acid analyzer (L-8800, Hitachi, Tokyo, Japan). The free amino acid was added to 40 g of 75% ethanol in 1 g of microalgal hydrolyzate and stirred for 24 hours, followed by centrifugation (10,000 × g, 15 min) to separate the supernatant and solids, followed by stirring for 24 hours, followed by centrifugation ( 10,000 × g, 15 min) to separate the supernatant and the solids, and from the third time, stirring and centrifugation was repeated for 1 hour until the supernatant became colorless. The supernatant was concentrated with a decompression concentrator (BUCHI) and then purified by 25 mL of distilled water and analyzed with an amino acid analyzer (L-8800, Hitachi, Tokyo, Japan).

Amino acid Tetraselmis suecica Chlorella vulgaris Free Compositional Free Compositional Asp 8.8 4.1 6.1 8.1 Thr 5.6 6.8 4.4 5.1 Ser 5.9 7.1 1.6 4.1 Glu 12.6 5.2 14.9 7.0 Gly 4.0 4.3 3.2 6.6 Ala 8.9 5.8 15.3 10.3 Val 3.8 8.5 6.0 6.4 Cys 5.1 1.4 0.3 9.1 Met 2.6 1.6 3.8 1.6 Ile 6.2 6.1 3.4 4.4 Leu 7.4 8.9 13.8 9.4 Tyr 2.4 7.1 4.2 3.5 Phe 3.9 7.8 7.4 4.2 Trp 5.8 9.4 3.4 - Hylys 0.1 - 0.2 - Lys 3.0 6.5 2.0 6.2 His 0.5 0.8 1.3 1.8 Arg 6.2 5.0 3.8 6.3 Pro 7.0 3.7 4.9 5.9 Hypro 0.2 - - - Total (%) 100 100 100 100

The constituent amino acid is Chlorella vulgaris Hydrolyzate was found in the order of Ala (10.3%), Leu (9.4%), Cys (9.1%), and Asp (8.1%), followed by Tetraselmis. suecica Hydrolyzate was the highest in Phe (9.4%), Ile (8.9%), Val (8.5%) and Tyr (7.8%).

On the other hand, the contents of sweet amino acid threonine, proline, serine, glycine, alanine and lysine are Chlorella. vulgaris hydrolyzate 28.2%, Tetraselmis The suecica hydrolyzate was 30.4%, and the content of aspartic acid, glutamic acid and cysteine, which had a rich and salty taste, was Chlrella. vulgaris 21.3% hydrolyzate, Tetraselmis The suecica hydrolyzate was 26.6%, and 49.5% and 57% of the total amino acids were good in terms of taste, and the bitterness and the contents of amino acids such as methionine, isoleucine and leucine were Chlorella. vulgaris 20.9%, Tetraselmis suecica was 16.1%. In general, the bitter taste produced during the enzymatic hydrolysis of proteins is closely related to the content of hydrophobic amino acids. The hydrophobic amino acid content of the prepared microalgal protein hydrolyzate has a great influence on the bitter taste of about 20% of all amino acids. It was not crazy.

Therefore, according to the results of this study, microalgal protein hydrolyzate is considered to be suitable for use as a flavoring ingredient of seasonings.

Example  2-2. Microalgal Proteins Hydrolyzate  Nucleic Acid Related Substance Analysis

Nucleic acid-related material of the microalgal protein hydrolyzate prepared in Example 1-3 was analyzed.

Among the nucleic acid-related substances, IMP is relatively stable to heat and is known as a taste component in nucleotides, and especially when coexisted with glutamic acid among amino acids, it has been shown to exhibit a strong umami due to synergy. AMP also shows little synergy in itself, but synergistic effect when coexisted with glutamic acid.

More specifically, 10 ml of 10% perchloric acid was added to 10 g of the microalgal hydrolyzate of the microalgae, followed by crushing. The supernatant was separated by centrifugation at 5000 rpm for 10 minutes, and the precipitate was treated three times in the same manner. It was. The combined supernatant was adjusted to pH 6.5 with 5 N KOH solution, centrifuged at 10,000 rpm for 10 minutes, then purified by 100 mL with perchloric acid and filtered with a 0.45 μm syringe filter (ADVATEC, Tokyo, Japan). The filtrate was analyzed by high-performance liquid chromatography (HPLC, Agilent 1100 HPLC Chemstation, Agilent, Santa Clara, CA. USA). The column used bondapak C18 (ø 39 × 300 mm), mobile phase used 1% triethylamine / phosphate (pH, 6.5), flow rate 0.8 mL / min, OD 254 nm, 0.001-1.0 M concentration. Standard calibration curves were prepared by using nucleic acid-related substances (ATP, ADP, AMP, IMP, inosine, hypoxanthinee), and the peak area of each sample solution was converted to calculate the amount of nucleic acid-related substances in the sample.

The nucleic acid-related substances from microalgae protein hydrolyzate of IMP was the highest net ingredient in the range of 0.34-0.57, Chlorella vulgaris has AMP, Tetraselmis Suecica was next highest in hypoxanthine. Therefore, the microalgae hydrolyzate prepared in this study has higher IMP content than hypoxanthine, which has a bitter taste.

< Example  3> Preparation of Microalgae Natural Seasoning

The microalgae natural seasoning was prepared by adding various subsidiary materials to the microalgal protein hydrolyzate prepared in the above 1-3.

More specifically, 15 g of microalgae protein hydrolyzate is a natural salt of 10 g, nucleic acid 2.5 g, glucose 5 g, red pepper powder 2.5 g, onion powder 0.5 g, ginger powder 0.25 g, garlic powder 1 g, miso powder 1.5 g To prepare a natural seasoning by mixing.

< Example  4> Sensory Evaluation of Natural Seasonings of Microalgae

The sensory evaluation of the natural microalgae prepared in Example 3 was carried out.

More specifically, the sensory test was carried out by adding 30 g of microalgae seasoning to 70 mL of water and cooking it in boiling water for 5 minutes. A total of 10 males and four females in their 20s among graduate students and undergraduate students majoring in food science A five-step scoring method (five points: very good, four points: good, three points: moderate, two points: bad, one point) for four items: taste, smell, color, and overall preference : Very bad). As a control, commercial beef seasonings, anchovy seasonings, and seafood complexing seasonings were cooked under the same conditions and evaluated. All experiments were repeated three times.

The statistical significance test for the sensory test result was Duncan's multiple range test using Statistical Packages for Social Science (SPSS, Chicago, IL, USA) within 5% of the significance level (p <0.05). Was investigated. The data are shown as the mean value and the standard deviation of each experimental value, and the sensory test results for the microalgae natural seasonings are shown in Table 5.

Compared to the microalgae seasonings and commercial seasonings on the scale of taste, color, aroma, and overall preference, the results were slightly lower in color, but the taste and aroma were similar or higher when compared to other natural seasonings. As there is no significant difference (p <0.05), microalgal protein hydrolyzate can be used as food seasoning material such as natural seasoning.

Claims (9)

delete delete delete a) eluting the protein by sonication at 26-28 ° C. for 4-5 hours at a frequency of 90-100 kHz in a Chlorella vulgaris or Tetraselmis suecica sonicator; And
b) preparing a protein hydrolyzate using protease on the protein;
Natural seasoning manufacturing method comprising a. delete delete delete The method of claim 4, wherein the proteolytic enzyme of step b) is an alkalase. The method of claim 4, wherein the hydrolyzate of step b) is produced by hydrolysis of 2-3% protease, pH 6-7, 50-60 minutes.

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