KR20220013345A - Method for Extracting Leghemoglobin from Soybean Root and Using the same into Food - Google Patents

Abstract

The present invention relates to an extracting method of leghemoglobin and a manufacturing method of substitute meat containing leghemoglobin obtained by the extracting method. According to the present invention, leghemoglobin can be extracted from a soybean root by the extracting method, the extracted leghemoglobin substitutes for a food, especially, hemoglobin of meat in substitute meat, and the extracted leghemoglobin can be usefully used to feel texture of meat.

Description

Method for Extracting Leghemoglobin from Soybean Root and Using the same into Food}

The present invention relates to a method for extracting LB (Leghemoglobin) from soybean root and its use in food. It relates to a food composition containing the same.

Nitrogen plays an important role in plant growth, but oxygen in the air inhibits the nitrogen fixation process while the root nodules provide nitrogen to plants. Therefore, in order for plants to utilize nitrogen well, an environment in which oxygen can be avoided is required. However, since a lot of ATP is required for plant growth and high-efficiency energy synthesis using oxygen is also required, the solution to this dilemma is leghemoglobin.

Leghemoglobin is a myoglobin-type unit with strong oxygen affinity found in the roots of plants, and its binding force to oxygen is 10 times higher than that of hemoglobin. Therefore, it is suitable for performing both roles of lowering oxygen and providing oxygen to areas that require respiration so that nitrogen-fixing enzyme works well.

Meanwhile, as meat consumption around the world increases, diseases such as high blood pressure, diabetes, and obesity are induced, and more than 3 billion tons of carbon dioxide is emitted during livestock breeding and feed production, causing serious environmental problems. .

Substitute meat has been proposed as a solution to this problem of meat consumption, but plant-based substitute meat has a disadvantage in that the taste, aroma, and texture are very different from that of actual meat, and thus the preference is lowered. In particular, when the iron contained in the hemoglobin in the blood of the meat is too small, it is difficult to feel the texture of the meat, so an alternative material that can replace the hemoglobin of the meat and feel the texture of the meat is needed.

The present inventors focused on leghemoglobin as a substance applicable to meat substitutes by replacing hemoglobin, and as a result of earnest efforts to develop a method for extracting leghemoglobin from plants, using the extraction method of the present invention from soybean roots By confirming that leghemoglobin can be extracted, the present invention was completed.

Republic of Korea Patent Publication No. 10-2004-0094691

SUMMARY OF THE INVENTION The present invention aims to solve the above problems and other problems related thereto.

An exemplary object of the present invention is to provide a method for extracting leghemoglobin comprising the following steps.

(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;

(b) centrifuging the homogenized mixture;

(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;

(d) dissolving the precipitate in Tris-HCl and centrifuging;

(e) filtering and concentrating the supernatant obtained by the centrifugation; and

(f) extracting leghemoglobin from the concentrate.

Another exemplary object of the present invention is to provide a food composition comprising leghemoglobin extracted by the above method.

Another exemplary object of the present invention is to provide a method for producing an alternative meat containing leghemoglobin extracted by the above method.

Another exemplary object of the present invention is to provide a method for producing leghemoglobin-containing substitute meat comprising the following steps.

(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;

(b) centrifuging the homogenized mixture;

(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;

(d) dissolving the precipitate in Tris-HCl and centrifuging;

(e) filtering and concentrating the supernatant obtained by the centrifugation;

(f) extracting leghemoglobin from the concentrate;

(g) adding the extracted leg hemoglobin after mixing tissue soy protein, coconut oil, mixed concentrated protein, beet powder, garlic powder, onion powder, and purified water; and

(h) fermenting the mixture of step (g) to prepare substitute meat.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem for solving the above-mentioned problems, and another problem not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

As an aspect for achieving the above object, the present invention provides a method for extracting leghemoglobin comprising the following steps.

(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;

(b) centrifuging the homogenized mixture;

(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;

(d) dissolving the precipitate in Tris-HCl and centrifuging;

(e) filtering and concentrating the supernatant obtained by the centrifugation; and

(f) extracting leghemoglobin from the concentrate.

As used herein, the term 'leghemoglobin' is a protein found in root nodules where nitrogen is fixed in legumes, and has the structure of Formula 1 below.

[Formula 1]

Substitute meat with plant ingredients has a disadvantage in that the taste, aroma, and texture are very different from actual meat, so the preference is lowered. In particular, if the iron contained in the hemoglobin in the blood of the actual meat is too small, it is difficult to feel the texture of the meat. Plant-derived leghemoglobin acts as a substitute for hemoglobin in meat while providing a sense of texture in meat.

In the present invention, the phosphate buffer may have a concentration of 0.01 M to 0.2 M, a concentration of 0.05 M to 0.15 M, and specifically 0.1 M.

In the present invention, the polyvinylpyrrolidone may be added in an amount of 0.1 to 0.5 parts by weight, 0.2 to 0.4 parts by weight, and specifically 0.3 parts by weight per 1 part by weight of the root knot.

In the present invention, the centrifugation in step (b) may be centrifugation at 3,000 rpm to 5,000 rpm, 10 minutes to 90 minutes, specifically, centrifugation at 4,000 rpm for 50 minutes.

In the present invention, the ammonium sulfate may be 55 to 80% by weight, specifically 60 to 75% by weight, more specifically 70% by weight.

In the present invention, the step (d) may be to dissolve the precipitate in Tris-HCl having a concentration of 0.01 M to 0.2 M for 1 hour to 10 hours, specifically, the precipitate is Tris having a concentration of 0.05 M to 0.15 M It may be dissolved in -HCl for 3 hours to 8 hours, and more specifically, the precipitate may be dissolved in Tris-HCl having a concentration of 0.1 M for 5 hours.

In the present invention, the centrifugation in step (d) may be centrifugation at 3,000 rpm to 5,000 rpm, 10 minutes to 90 minutes, specifically, centrifugation at 4,000 rpm for 50 minutes.

Specifically, in the embodiment of the present invention, it was confirmed that leghemoglobin could be extracted from soybean root nodules through the above extraction method.

As another aspect of the present invention, the present invention provides a food composition comprising leghemoglobin extracted by the above method.

The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material. The food composition of the present invention may be prepared in any form, and specifically, health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, pieces, pastes, syrups, gels, jellies, bars, etc.; It may be one or more formulations selected from the group consisting of beverages, gums and candies, but is not particularly limited thereto.

In addition, the food composition of the present invention may contain food additives in addition to the active ingredient. Food additives can be generally understood as substances that are added to and mixed with or infiltrated into food in manufacturing, processing, or preserving food, and their safety must be ensured since they are consumed daily and for a long period of time with food. The food additives are divided into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as the food composition of the present invention meets the intended purpose. In addition, the food composition of the present invention may contain physiologically active substances or minerals known in the art for the purpose of functional and nutritional supplementation and guaranteed stability as food additives in addition to the food additives. The physiologically active substances or minerals are not particularly limited as long as the food composition of the present invention meets the intended purpose.

In the food composition of the present invention, the food additives as described above may be included in an effective amount to achieve the purpose of the addition according to the type of product. or the Food Additives Ordinance.

In the present invention, the food may be substitute meat.

As used herein, the term "substitute meat" refers to artificial meat made using raw materials other than meat, and is divided into substitute meat made by culturing animal cells and substitute meat using non-animal ingredients. In the present invention, the meat substitute may be meat substitute using non-animal ingredients.

In the present invention, the shape of the substitute meat is not particularly limited, and examples of the substitute meat include, but are not limited to, burger patties, sausages, bacon, nuggets, jerky, meatballs, ham, and the like.

As another aspect of the present invention, the present invention provides a method for producing substitute meat containing leg hemoglobin extracted according to the extraction method.

As another aspect of the present invention, the present invention provides a method for producing leghemoglobin-containing substitute meat comprising the following steps.

(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;

(b) centrifuging the homogenized mixture;

(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;

(d) dissolving the precipitate in Tris-HCl and centrifuging;

(e) filtering and concentrating the supernatant obtained by the centrifugation;

(f) extracting leghemoglobin from the concentrate;

(g) adding the extracted leg hemoglobin after mixing tissue soy protein, coconut oil, mixed concentrated protein, beet powder, garlic powder, onion powder, and purified water; and

(h) fermenting the mixture of step (g) to prepare substitute meat.

In the present invention, the tissue soy protein may be a protein extracted from soybeans.

In the present invention, the mixed protein may include proteins extracted from two or more selected from soybeans, mung beans, red beans and peas.

In the present invention, the step (g) is 10 to 30 parts by weight of coconut oil, 40 to 60 parts by weight of mixed concentrated protein, 10 to 30 parts by weight of beet powder, 10 to 30 parts by weight of garlic powder per 100 parts by weight of tissue soy protein. , 10 to 30 parts by weight of onion powder, 100 to 200 parts by weight of purified water, specifically, 15 to 25 parts by weight of coconut oil per 100 parts by weight of tissue soy protein, 45 to 55 parts by weight of mixed concentrated protein, beets 15 to 25 parts by weight of powder, 15 to 25 parts by weight of garlic powder, 15 to 25 parts by weight of onion powder, and 120 to 180 parts by weight of purified water may include mixing, more specifically, 20 parts by weight of coconut oil per 100 parts by weight of tissue soy protein It may include mixing in parts by weight, 50 parts by weight of the mixed concentrated protein, 20 parts by weight of beet powder, 20 parts by weight of garlic powder, 20 parts by weight of onion powder, and 150 parts by weight of purified water.

In the present invention, the leg hemoglobin in step (g) may be added in an amount of 0.1 to 10 parts by weight per 100 parts by weight of tissue soy protein, specifically 1 to 5 parts by weight, and more specifically 2 to 4 parts by weight. may be added as parts.

In the present invention, the fermentation of step (h) may be performed at 1 to 10 ℃ 1 to 10 hours, specifically 3 to 8 ℃ 3 to 8 hours may be performed, more specifically 4 to 5 ℃ It can be carried out for 4 to 6 hours.

Leg hemoglobin can be extracted from soybean root by the extraction method according to the present invention, and the extracted leg hemoglobin can be usefully used to replace the hemoglobin of meat in food, particularly substitute meat, and to feel the texture of meat.

1 is a soybean root nodule obtained by hydroponically cultivating soybeans.
2 shows the process of mixing the phosphate buffer into the soybean root nodules.
3 shows a process of homogenizing the soybean root nodules by adding polyvinylpyrrolidone.
4 shows the process of treating the separated supernatant with ammonium sulfate.
Figure 5 shows the process of dissolving the precipitate by stirring with Tris-HCl.
Figure 6 shows the process of pressure filtration of the supernatant.
7 is a result of confirming the leg hemoglobin extracted by SDS-PAGE.
8 is a result of confirming the leghemoglobin extracted by Western-blot.
9 is a result of confirming leghemoglobin (leghemoglobin A, leghemoglobin C1, leghemoglobin C2, and leghemoglobin C3) extracted by LC-MS.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Extraction of leghemoglobin from soybean root nodules

As shown in FIG. 1, soybeans were grown hydroponically to grow soybean root nodules. Thereafter, as shown in FIG. 2 , 0.1 M phosphate buffer was mixed with soybean root nodules in an amount of 80 to 100 mL per 1 g of soybean root nodules. Thereafter, as shown in FIG. 3 , 0.3 g of polyvinylpyrrolidone per 1 g of root nodules was added and homogenized, and the mixture was centrifuged at 4,000 rpm for 50 minutes.

Thereafter, as shown in FIG. 4 , the supernatant was separated and treated with 55 to 80 wt % ammonium sulfate overnight, and then the precipitate was separated.

Then, as shown in FIG. 5, the precipitate was dissolved by stirring with 0.1M Tris-HCl containing 9 times 0.1 mM EDTA for 5 hours, followed by centrifugation at 4,000 rpm for 50 minutes. Thereafter, the supernatant was pressure filtered and concentrated as shown in FIG. 6 using a syringe equipped with a filter.

It was confirmed whether leghemoglobin was extracted from the concentrate using SDS-PAGE.

Specifically, the SDS-PAGE gel was composed of 10% Separating Gel and Stacking Gel. The same amount of 2×SDS sample buffer (0.5 M Tris-HCl buffer, pH 6.8, containing 10% SDS, 4% 2-mercaptoethanol, 20% glycerol, 0.05% bromo-phenol blue, and 10 mM EDTA) was added to the concentrate. Then, it was heated at 95 °C for 15 minutes. After electrophoresis using an electrophoresis device, the gel was stained with a staining solution (0.025 Coomassie brilliant blue R-250, 50% methanol and 5% acetic acid) for more than 4 hours, followed by a decolorization solution (7% Glacial acetic acid and 5% acetic acid). % methanol).

As a result, it was confirmed as shown in FIG. 7 that leghemoglobin was extracted.

In addition, it was confirmed by Western-blotting that leghemoglobin was detected as shown in FIG. 8, and leghemoglobin of leghemoglobin A, leghemoglobin C1, leghemoglobin C2 and leghemoglobin C3 was extracted using LC-MS as shown in FIG. 9 using LC-MS. Confirmed.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (13)

Extraction method of leghemoglobin comprising the steps of:
(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;
(b) centrifuging the homogenized mixture;
(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;
(d) dissolving the precipitate in Tris-HCl and centrifuging;
(e) filtering and concentrating the supernatant obtained by the centrifugation; and
(f) extracting leghemoglobin from the concentrate.
According to claim 1,
The phosphate buffer is at a concentration of 0.01 M to 0.2 M, the extraction method of leghemoglobin.
According to claim 1,
The polyvinylpyrrolidone is added in an amount of 0.1 to 0.5 parts by weight per 1 part by weight of the root nodules, the method of extracting leghemoglobin.
According to claim 1,
The centrifugation of step (b) is centrifugation at 3,000 rpm to 5,000 rpm, 10 minutes to 90 minutes, the extraction method of leg hemoglobin.
According to claim 1,
The ammonium sulfate is 55 to 80% by weight, the extraction method of leghemoglobin.
The method of claim 1,
The step (d) is to dissolve the precipitate in Tris-HCl having a concentration of 0.01M to 0.2M for 1 hour to 10 hours, the method of extracting leghemoglobin.
According to claim 1,
The centrifugation in step (d) is centrifugation at 3,000 rpm to 5,000 rpm, 10 minutes to 90 minutes, the extraction method of leg hemoglobin.
A food composition comprising leghemoglobin extracted by the method of claim 1.
9. The method of claim 8,
The food is an alternative meat, a food composition.
10. The method of claim 9,
The substitute meat is at least one selected from the group consisting of burger patties, sausages, bacon, nuggets, jerky, meatballs, and ham, a food composition.
A method for producing substitute meat containing leghemoglobin extracted according to the extraction method of any one of claims 1 to 7.
A method for producing leghemoglobin-containing substitute meat comprising the steps of:
(a) mixing a phosphate buffer with soybean root nodules and homogenizing by adding polyvinylpyrrolidone;
(b) centrifuging the homogenized mixture;
(c) treating the supernatant obtained by the centrifugation with ammonium sulfate to separate the precipitate;
(d) dissolving the precipitate in Tris-HCl and centrifuging;
(e) filtering and concentrating the supernatant obtained by the centrifugation;
(f) extracting leghemoglobin from the concentrate;
(g) adding the extracted leghemoglobin after mixing tissue soy protein, coconut oil, mixed concentrated protein, beet powder, garlic powder, onion powder, and purified water; and
(h) fermenting the mixture of step (g) to prepare substitute meat.
13. The method of claim 12,
The substitute meat is at least one selected from the group consisting of burger patties, sausages, bacon, nuggets, jerky, meatballs, and ham, a method for producing substitute meat.

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