KR20200043013A - Preparation Method of Aplysia kurodai Peptides with High Yield Using Enzyme - Google Patents

Abstract

The present invention provides a method for producing an Aplysia kurodai peptide, the method comprising the steps of: (1) enzymatic degradation by adding protease to the Aplysia kurodai powder; (2) filtering an enzyme decomposition product obtained in step (1) under reduced pressure; and (3) separating the filtrate obtained by step (2) using DEAE-Sephadex chromatography.

Description

Production Method of High Yield Snail Peptide {Preparation Method of Aplysia kurodai Peptides with High Yield Using Enzyme}

The present invention relates to a method for producing a sea snail peptide, and more specifically, a high yield of sea snail peptide, which can be obtained through a simple and inexpensive method with a high yield of a sea snail peptide useful as a component of food additives or cosmetics. It relates to a manufacturing method.

The sea snail is a mollusk from the order of the order of the order of the order of the Aplysia kurodai. The body is about 40 cm long, egg-shaped, and has an off-white speckled pattern on a dark brown background. It has a deer on its head, so it looks like a rabbit and is called a sea hare. It mainly eats green algae and brown algae, and it is a mollusk like shellfish, but it has no shell and instead emits a unique purple-based pigment to protect the body.

Sea snail is a high-protein, low-calorie food that is known to help promote growth and prevent adult diseases. It is also eaten by boiling in the southern regions. As a study on sea snail, we studied the protein and amino acid composition of sea snail meat, and it has been reported that hot water extract of sea snail eggs has anti-cancer activity. In addition, it reported antioxidant and antibacterial effects from sea snail gut extract. There are studies. In addition, sea snails are used in research related to the nervous system because their neural networks are simple and their nerve cells are large.

However, it is true that the sea snail itself has high nutritional and physiologically active ingredients and is of high food science value. However, since it is not provided as an additive to other processed foods, there is a limit in increasing added value, and for this, it is limited. It is required to be processed in the form of, or to extend its use to fields other than food. Conventionally, a method of extracting an extract using sea snail is mainly known as a method for extracting hot water or extracting ethanol, but according to such a method, the extraction yield is not large, and thus, the non-economicity that requires a large amount of sea snail is a problem.

Therefore, the present inventors have been devised to solve the above problems, the purpose of which is to extract high-yielding sea snail extracts, which can be obtained through simple and inexpensive methods with high-yielding peptides of sea snails useful as components of food additives or cosmetics. It is to provide a manufacturing method of.

The technical problem of the present invention as described above is achieved by the following means.

(1) 1) enzymatic decomposition by adding protease to sea snail powder;

2) filtering the enzyme decomposition product obtained in step 1) under reduced pressure; And

3) separating the filtrate obtained by step 2) using DEAE-Sephadex chromatography;

Method for producing a sea snail peptide comprising a.

(2) In the above (1),

Method for producing a sea snail peptide, characterized by eluting and separating the peptide under a concentration of 0.4 ~ 0.5M NaCl using DEAE-Sephadex chromatography.

(3) In the above (1),

Sea snail powder is a powder with odor removed.

10-30% by weight of acidic white clay, 10-30% by weight of silica, 10-30% by weight of diatomaceous earth, 10-30% by weight of pozzolan, 0.1-0.5% by weight of 3-decyloxybenzoic acid decyl ester, methylcellulose 0.1 to 0.5% by weight, 0.1 to 0.2% by weight of kaolinite, 0.1 to 0.2% by weight of hectorite, and 0.1 to 0.2% by weight of beiderite are reacted to remove the odor, characterized in that the snail peptide is removed.

According to the present invention as described above, it is possible to obtain an extract of sea snails useful as a component of food additives or cosmetics through a simple and inexpensive method with high yield, as well as to increase the income of sea snail related businesses and contribute to the promotion of national health. In addition, it is expected to greatly contribute to the growth of the national economy through the export of sea snail peptide.

1 is a manufacturing process of the sea snail peptide according to the present invention.

In order to achieve the above object, a method for preparing a sea snail peptide according to the present invention,

(1) enzymatic decomposition by adding protease to sea snail powder;

(2) filtering the enzyme decomposition product obtained in step (1) under reduced pressure; And

(3) separating the filtrate obtained by step (2) using DEAE-Sephadex chromatography.

Hereinafter, the contents of the present invention will be described in detail with reference to FIG. 1.

In the present invention, an enzyme extract is obtained to obtain a sea snail peptide rich in physiologically active substances with a high yield in a simple and inexpensive manner, and chromatography is performed on the filtrate obtained through filtration under reduced pressure.

The sea snail powder is preferably removed from the sea snail, and then primary self-steaming; Refrigerating and storing the sea snails that have undergone the primary self-sufficiency; A second self-saturation of the frozen snail; And it is good to be obtained through the step of drying and pulverizing the secondary self-sealing snail.

The first self-sufficiency process includes a process of self-stimulating by adding water of 3 to 5 times the weight of the sea snail using the sea snail with the intestines removed.

The first ripening process is preferably performed at 90 to 100 ° C, and if it is less than 90 ° C, it is difficult to achieve sufficient self-sufficiency, and if it exceeds 100 ° C, there is a fear of elution or destruction of various bioactive substances. high.

At this time, the first self-stay time is preferably performed for 10 to 60 minutes. When the time is exceeded, it is not preferable because various physiologically active substances or functional useful components of sea snails may be destroyed.

The frozen storage of sea snails that have undergone the primary self-saturation is performed at -20 to -18 ° C, which is not only an effect to improve the preservation and to be used when needed, but also to increase the yield of extracts. It is also required for the self-sufficiency process. For this purpose, cryopreservation is preferably performed for 1-2 days.

As a raw material for the primary self-sealing snail that has been frozen and stored, a second self-saturation process is performed by adding water 3 to 5 times the weight of the sea snail.

The second ripening process is preferably performed at 90 to 100 ° C, and if it is less than 90 ° C, self-sufficiency is difficult to achieve, and if it exceeds 100 ° C, there is a fear of elution or destruction of various bioactive substances. high.

At this time, the second self-saturation time is preferably performed for 10-60 minutes as in the first self-saturation process. When the time is exceeded, it is not preferable because various physiologically active substances or functional useful components of sea snails may be destroyed.

The secondary self-sealing snail prepared through the above process is dried in a dry oven and then pulverized using a blender or the like to prepare it as an extraction raw material. At this time, crushing using a blender is preferred to maximize the yield of the extract by crushing to a size having a diameter of about 2-3 mm.

More preferably, after the second self-sealing snail is dried in a dry oven, a swelling sea snail is swelled at 10 to 13 kg / cm 2 and 110 to 130 ° C. for 2 to 10 minutes using a swelling device, and then crushed using a blender. Using the raw material as an extraction material may maximize the extraction yield.

In order to remove the smell of sea snail powder prepared through the above process, in the present invention, 10-30% by weight of acid white clay, 10-30% by weight of silica, 10-30% by weight of diatomaceous earth, 10-30% by weight of pozzolan %, 3-decyloxybenzoic acid decyl ester 0.1-0.5 wt%, methyl cellulose 0.1-0.5 wt%, kaolinite 0.1-0.2 wt%, hectorite 0.1-0.2 wt%, and beiderite 0.1-0.2 wt% It is used as an extraction raw material by removing the odor by reacting by adding.

More specifically, as described above, the odor removal reaction according to the present invention is preferably performed under 100 to 500 rpm for 30 to 60 minutes using a shaking incubator containing water, and then filtered under reduced pressure and dried.

In the present invention, the odor removing agent is added in an amount of 10 to 30% (w / v), preferably 20% (w / v) to the reaction solution containing sea snail powder.

The decompression filtration process may preferably be performed using equipment equipped with 3M paper or any other decompression filtration device commonly used.

For example, as one of the decompression filtration methods, a vacuum pump can be used to continuously perform while maintaining a vacuum level of 1 atmosphere or less, and a high pressure pump can be used for 10 to 14 liters per minute at a pressure of 45 to 55 kgf / cm ² . The reaction solution may be supplied to the reverse osmosis membrane to remove the salt, and at this time, 1 to 3 liters per minute may be generated in the extract from which the salt is removed.

The proteolytic enzyme is added to the snail powder prepared through the above process to perform an enzymatic reaction. Preferably, protamex is used as the protease. At this time, it is preferable to add protamex so that 0.01 to 10 parts by weight based on 100 parts by weight of sea snail powder.

The enzyme extraction process by protamax generally not only greatly improves the yield of the extract compared to the hot water extraction or organic solvent extraction process, but also can significantly increase the yield of the extract even when other proteolytic enzymes are applied alone. It is preferable in that point.

The enzyme extraction process by protamax is preferably reacted at 35 to 45 ° C, more preferably at 40 ° C for 5 to 10 hours, more preferably at 6 to 7 hours. Under the above conditions, the yield of the extract is maximized.

In the present invention, it is preferable to process for 6 to 7 hours using a low frequency of 1 to 10 kHz in the process of reacting the enzyme to further improve the extraction yield using the enzyme.

More preferably, it is better in terms of yield improvement to treat papain or protex or papain and protex in a predetermined ratio, rather than using protamax alone. Preferably, the composition is mixed in a ratio of protamax: papine (or protex) = 1: 0.5-1: 1 or protamax: papaine: protex = 1: 0.5: 0.5-1: 1: 1 by weight. To do it.

After the enzyme extraction process for the powdered sea snail as described above, the obtained enzyme extract is collected and filtered under reduced pressure. The decompression filtration process may preferably be performed using equipment equipped with 3M paper or any other decompression filtration device commonly used.

For example, as one of the decompression filtration methods, a vacuum pump can be used to continuously perform while maintaining a vacuum level of 1 atmosphere or less, and a high pressure pump can be used for 10 to 14 liters per minute at a pressure of 45 to 55 kgf / cm ² . The salt can be removed by supplying the enzyme extract to the reverse osmosis membrane. At this time, the enzyme extract from which the salt has been removed may generate 1 to 3 liters per minute.

The filtrate obtained through the vacuum filtration process as described above is subjected to a separation process under a concentration gradient of NaCl 0.4-0.5M using DEAE-Sephadex chromatography, and a separation process of sea snail peptide. According to the present invention, it was confirmed that when the NaCl deviates from 0.4 to 0.5M, the separation yield of the sea snail peptide significantly decreases, and further, at the NaCl 1.0M level, polysaccharides are separated.

Through the above process, it is possible to obtain an extract of a sea snail rich in bioactive substances in a simple and inexpensive way with high yield.

Hereinafter, the contents of the present invention will be described in more detail with reference to examples, but these examples are only for understanding the contents of the present invention and should not be interpreted as limiting the scope of the present invention.

[Example 1] Preparation of sea snail powder

As shown in FIG. 1, first, a sea snail was purchased to remove the intestine, and then boiled at 100 ° C. for 30 minutes to perform a primary self-steaming process. The sea snail, which had undergone the primary self-steaming, was frozen and stored at -20 ° C for 1 day, and then boiled at 100 ° C for 30 minutes to perform a second self-steaming process.

After the secondary self-sealing snail prepared through the above process was put in a dry oven and dried, pulverized using a blender and sieved with a mesh to obtain a powder of 2-3 mm in diameter.

[Implementation 2]

The powder obtained in Example 1 was placed in a shake incubator containing 5 times the volume of water, to which 20% by weight of acid white clay, 20% by weight of silica, 30% by weight of diatomaceous earth, 29.2% by weight of pozzolan, 3-decyloxybenzoic 20% (w / v) of 0.1% by weight of decyl ester, 0.1% by weight of methylcellulose, 0.2% by weight of kaolinite, 0.2% by weight of hectorite, and 0.2% by weight of beiderite was added and reacted at 200 rpm for 30 minutes. After carrying out, it was filtered under reduced pressure and dried to obtain a sea snail powder with odor removed.

[Example 3] Preparation of sea snail peptide

After adding 30 g of the snail powder sample obtained in Example 1 to a 1000 ml flask, 300 mg of Protamex and 450 ml of distilled water were added, and then reacted in a 40 ° C. water bath for 6 hours. After the enzymatic reaction was completed, vacuum filtration was performed while washing distilled water using 3M paper, and sea snail peptides were separated at a concentration of 0.4 to 0.5M NaCl using DEAE-Sephadex chromatography.

[Example 4] Preparation of sea snail peptide

A sea snail peptide was prepared in the same manner as in Example 3, except that 200 mg of Protamex and 100 mg of papain were used as an enzyme.

[Example 5] Preparation of sea snail peptide

A sea snail peptide was prepared in the same manner as in Example 3, except that 200 mg of Protamex and 100 mg of Protex were used as an enzyme.

[Example 6] Preparation of sea snail peptide

In Example 3, a sea snail peptide was prepared by the same procedure, except that 200 mg of Protamex, 100 mg of papain, and 100 mg of Protex were used as an enzyme.

[Example 7] Preparation of sea snail peptide

After adding 30 g of the snail powder sample obtained in Example 2 to a 1000 ml flask, 300 mg of Protamex and 450 ml of distilled water were added, and then reacted in a 40 ° C. water bath for 6 hours. After the enzymatic reaction was completed, vacuum filtration was performed while washing distilled water using 3M paper, and sea snail peptides were separated at a concentration of 0.4 to 0.5M NaCl using DEAE-Sephadex chromatography.

sample Product weight yield Example 3 15.2 g 42.3% Example 4 14.8 g 41.4% Example 5 15.1 g 42.2% Example 6 15.6 g 42.5% Example 7 15.2 g 42.3% Comparative Example 1 8.29 g 28.5% Comparative Example 2 4.50 g 14.2% Comparative Example 3 4.20 g 13.8%

From the results of the above experiment, it was confirmed that the yield of the extraction method using the protamax enzyme was much better than the extraction method using ethanol [Table 2].

In the case of Example 7, as a result of sensory evaluation (five-point scale) of 20 men and women in their 20s and 30s, it was confirmed that the smell was significantly reduced compared to other examples [Table 3].

division test results Example 3 4.0 Example 4 4.1 Example 5 4.1 Example 6 4.1 Example 7 4.7 Comparative example 3.2

As described above, although described with reference to preferred embodiments of the present invention, those skilled in the art will variously modify and change the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. You can understand that you can.

Claims (3)

(1) enzymatic decomposition by adding protease to sea snail powder;
(2) filtering the enzyme decomposition product obtained in step (1) under reduced pressure; And
(3) separating the filtrate obtained by step (2) using DEAE-Sephadex chromatography;
Method for producing a sea snail peptide comprising a. According to claim 1,
A method for producing a sea snail peptide, characterized by eluting and separating a peptide under a concentration of 0.4 to 0.5 M NaCl using DEAE-Sephadex chromatography. According to claim 1,
Sea snail powder is a powder with odor removed.
10-30% by weight of acidic white clay, 10-30% by weight of silica, 10-30% by weight of diatomaceous earth, 10-30% by weight of pozzolan, 0.1-0.5% by weight of 3-decyloxybenzoic acid decyl ester, methylcellulose 0.1 to 0.5% by weight, 0.1 to 0.2% by weight of kaolinite, 0.1 to 0.2% by weight of hectorite, and 0.1 to 0.2% by weight of beiderite are added to react to remove the odor, characterized in that the snail peptide production method.

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