WO2020262860A1 - Method for increasing content of tryptophan in bean sprout by using plant elicitor, and extracting tryptophan therefrom with high yield - Google Patents

Abstract

The present invention relates to a method for increasing the content of tryptophan in bean sprouts by using a plant elicitor, and extracting the tryptophan therefrom and, more particularly, to a method for building up tryptophan as a useful component of bean sprouts by using a pine-nut tree extract as a plant elicitor, and extracting the tryptophan therefrom with high yield, wherein the tryptophan has a mental stability function. Therefore, the tryptophan can be produced in high yield wherein the tryptophan is an essential amino acid acting as a natural mood regulator and is a natural control agent that relieves anxiety by inducing a sedative effect and sleep. It is possible to cultivate highly-functional and high-value-added bean sprouts with significantly improved content of physiologically active substances in plants, and is expected to contribute significantly to profit generation of farmers.

Description

Increased tryptophan content and high yield extraction method in bean sprouts using plant eliminator

The present invention relates to a method for increasing the content of amino acids such as tryptophan among functional bean sprouts using an elicitor of plants and a method for extracting high yield.

Agricultural products have played a role as a source of minerals such as vitamins that replenish the energy resources of main grains centered on carbohydrates, but in recent years, the discovery of antioxidants and reinforcement systems of interferon in the dimension of disease prevention through enhancement of the immune system. Although research on the activity of the drug is progressing, it is necessary to develop a formulation that enhances physiologically active substances that have excellent improvement and therapeutic effects in anticancer, antioxidant, and adult diseases in agricultural products that we eat as staple foods, not pharmaceuticals.

As a part of that, the present invention prepares a pine tree extract as an eliminator in a formulation that can be easily transferred to a plant, and is immersed in the germination time of soybeans during cultivation of bean sprouts. Amino acids such as glutamic acid, lysine, and tryptophan contained in the harvested soybean sprouts are enhanced, and amino acids such as tryptophan of high yield and high purity are obtained by extracting the soybean sprouts with proteolytic enzymes after harvesting. Bean sprouts are green sprouts that are not ripe.

Bean sprouts are those grown to some extent by germinating beans without exposure to light. Bean sprouts contain a lot of protein and fat, and have high content of vitamins B1, B2, and ascorbic acid (vitamin C), making them an excellent nutritional food. Changes in components during the germination period generally decreases rapidly in small sugars, but increases in fiber and slightly decreases in protein, but increases in non-protein nitrogen such as amino acids and peptides.

Theoprastus (c. 300 BC) stated that Egyptian beans (chick pea) made the soil more barren than other legumes. He also pointed out that chick pea destroys weeds, especially for male braces.

Pliny (1 year AD) caused chicken pea, barley (Hordeum Vulgare), fenugreek (Trigonella foenum-graecum), and bitter vetch (Vicia ervilia) to wither all corn fields. It was written. It was said that under the walnut tree (Juglans regia) it caused headaches to humans and injured the plants planted by humans, but the soil under the pine trees also prevented other plants from growing. It is also said that walnut, stonepine, spruce, and silver fir (silver fir) contact plants are all detoxified.

Lee and Monsi (1963) found a report by Banzan Kumazawa that, in a Japanese pattern of about 300 years ago, rainwater or dew washed off from pine leaves (Pinus densiflora) harmed the crops growing under the tree. Lee and Monsi demonstrated this through a series of experiments.

Young (1840) argued that shamrocks do not live well in places where shamrocks are continuously cultivated, because the soil makes shamrocks sick. If shamrocks are planted at intervals of 7 to 8 years, the disease can be prevented. Pointed out.

The mechanism of action of Allelopathy is that plants that cannot move with their own forces attack enemies from the outside. It has various defense functions, protects itself, and plays a role in proliferating offspring. A substance that has a physical defense function that makes the epidermis thick, hard, and thorns by creating a wax layer on the surface of the leaf, and it defends against the attack of pathogens according to the metabolism in the body, has antibacterial action, and produces a smell that pests do not like. It has a variety of functions such as repelling action to create a repellent, attracting necessary insects, and insecticidal action with poisonous substances in the body. It maintains life and reproduces offspring, and is more powerful as it grows. By accumulating substances with ingredients, they comply with the providence of Mother Nature and sustain life.

Phytochemical is a compound word of ‘phyto’ meaning vegetable and ‘chemical’ meaning chemistry, and refers to a phytochemical that has physiological activity that helps health. Although it is not an essential nutrient that must be ingested to sustain life, it can adversely affect health if consumption of phytochemicals is continuously insufficient. Recently, as research on this has been actively conducted, it is receiving more attention as an ingredient necessary for maintaining health. In addition, phytochemicals do not only play an important role in health, but also give the unique taste, aroma, and color of plants to express the unique personality of each food.

Research to find physiologically active substances from natural products has been conducted by many researchers from a long time ago, and plant resources are widely used, especially as therapeutics, preventive measures, or health supplements for diseases. Currently, in the case of Korea, with the increase of economic growth and national income, various adult diseases are on the rise, and interest in health functional foods with preventive medicine is rapidly increasing. The market size of health functional foods in 2007 is estimated at KRW 3 trillion, which is a leap of 2.7 times compared to KRW 1.1 trillion in 2001. On the other hand, due to the recent saturation of the food market, it is showing a contrast of 1% annual growth.

Free radicals cause physiological disorders in plants by peroxidation of lipids, degradation of cell membranes and proteins, destruction of chloroplasts, and aging. In other words, it makes them dry. So, to protect itself from strong sunlight, it creates a protective film on the skin, leaves, and stems of fruits that are directly exposed to UV rays. This protective film is made of powerful antioxidants and acts to prevent destruction by free radicals.

In addition, they must overcome various stresses that cause viruses or fungi to infiltrate and eat themselves and make them sick, and endure extreme natural conditions such as floods, drought, heat waves, and cold.

The types of phytochemicals that have been identified so far reach over 10,000, and their efficacy varies by type. The most famous ingredient among countless phytochemicals and their representative foods are as follows.

Among the secondary metabolites, essential oils collectively refer to antimicrobial substances that plants release into the air to protect themselves from bacteria, microorganisms, and pests. It is composed of 50 kinds of terpene-based substances, which are highly volatile antibiotics with a low boiling point, and mainly all parts of conifers such as pine, pine, fir, cedar, cypress, globule, etc.(wood, bark, needles) ). In general, phytoncide released from plants of the same species or genus is almost constant and regularly produced in the type, composition, and content of constituents, but even if it is the same species according to the environment and type in which the plant grows or the difference in harvest time Separately, there is a difference in the composition and content of the ingredients. The main components of phytoncide are α-pinene, β-pinene, myrcene, β-thujene and bornylacetate, but it contains dozens to hundreds of trace components such as terpenoid, poly terpenoid, and phenol. As a mixture, these ingredients have antibacterial, insecticidal, allelopathy and inhibit the growth of bacteria. Phytoncide is a substance that does not directly affect the growth and differentiation of plants, but exhibits various functions, and is a substance that can be widely applied to medicine, flavoring, and household products. In particular, in recent years, it has unique pharmacological effects such as anti-inflammatory, sterilization, deodorization, insecticide, nervous stability, hormone balance, and cardiovascular disease prevention, and promotes blood circulation by dissolving cholesterol, thus preventing aging, hyperlipidemia and cholesterol reduction It has been reported to exhibit excellent effects.

Tryptophan is a kind of essential amino acid and has been widely used as a raw material for medicines such as infusions and health foods because of its feed additive, sleep effect, and mental stability effect. Tryptophan (also known as L-tryptophan) is one of the essential amino acids that act as a natural mood regulator because it has the ability to help the body naturally produce and balance certain hormones. Consuming tryptophan-rich foods or supplements can relieve anxiety by inducing sedation and sleep. 5-hrdoxytryptophan (5HTP) is achieved by increasing the body's production of serotonin, the main pleasing hormone.

Serotonin works by transmitting signals between nerve cells and changing brain functions that affect mood and sleep. Amino acid therapy is very useful when certain amino acids are used to treat diseases such as sleep disorders, depression, fatigue, anxiety and sexual dysfunction.

An important part of the use of amino acids is completely natural, requires no prescription and most of the time does not cause side effects. Because of its ability to increase serotonin, ingesting 5HTP synthesized from tryptophan and more L-tryptophan itself is used to treat a number of diseases.

The typical application of tryptophan is a mental illness that affects daily function by causing various cognitive disorders and physical symptoms with depression as the main symptoms of depression and depressive mood. It is caused by a combination of biological, psychological, and social factors. Among neurotransmitters, dopamine, serotonin, norepinephrine, etc. are substances necessary for positive and positive thinking. As health functional foods, Seongyohancho and curcumin are most commonly known.

In particular, in women's premenstrual syndrome and menopausal syndrome, due to the hormonal imbalance in which mental and physical symptoms occur repeatedly, mental symptoms are easily irritable, aggressive tendency, depression, anxiety, and memory loss. Physically, breast pain, swelling, headache, Since symptoms such as hot flashes appear, calcium, magnesium, vitamin B6, vitamin D, red shamrock, and chastity tree extract are most commonly used as health functional foods. Especially, red shamrock is called female hormone along with soybeans. It contains a lot of genistein.

Soybeans (soybeans) are becoming popular as a health food among modern people because they contain various useful physiologically active substances. Among the useful physiologically active substances, there are many isoflovonoids such as genistein and daidzein. It attracts attention and is in the spotlight as a research subject.

Various legume foods are not only rich in fiber, but also contain a lot of tryptophan.You can get 60-90 mg/100 g of tryptophan in beans, and in addition to tryptophan, you can get various B vitamins and iron through soy consumption. And, it is a crucial help in converting tryptophan to niacin (vitamin B3).

Genistein has excellent anticancer effects in in vitro, animal and clinical trials, has a high preventive effect on breast cancer and prostate, and is effective in preventing and inhibiting the progression of osteoporosis caused by estrogen deficiency in postmenopausal women. It is known that there is.

A study has been published that Daidzein is effective in preventing osteoporosis in the elderly and women by inhibiting the reabsorption of calcium from bone to blood. Isoflavonoid is a major component of soybeans in lowering cardiovascular disease.

Tryptophan is effective in improving sleep, reducing stress, and reducing depression, and research support for a method of increasing tryptophan that is technically and economically feasible is needed. Bean sprouts are loved by many people as a food with a market size of about 800 billion won every year in Korea. However, since cultivation is carried out in a high humidity environment, there are many diseases, and a problem occurred every summer, especially in summer, due to the use of pesticides in various cultivation plants.

Since soybean sprouts are highly contaminated by microorganisms, there is a loss in which a large portion of the harvested bean sprouts is discarded in a short shelf life of 1 to 2 days after harvest. Therefore, the purpose of developing a cultivation method that reduces the use of pesticides when cultivating bean sprouts, and at the same time confirming whether the content of amino acids such as tryptophan is increased, and establishing optimal conditions for increasing the content of tryptophan, is intended to develop a health functional food.

Thus, the present invention further relates to a functional bean sprouts formulation composition using a walnut green skin extract.

Agricultural products have played a role as a source of minerals such as vitamins that supplement the energy resources of main grains centered on carbohydrates, but recently, the discovery of antioxidants and reinforcement systems of interferon in the dimension of disease prevention through enhancement of the immune system. Although research on the activity of walnut is progressing, it is related to the development of a formulation that enhances physiologically active substances with excellent improvement and treatment effects for anticancer, antioxidant, and adult diseases in agricultural products that we eat as staple foods, not pharmaceuticals. When continued fertilization during subsequent growth, it was confirmed that allelopathic action occurred and the content of tryptophan was increased, and the present invention was completed.

[Prior technical literature]

[Patent Literature]

(Patent Document 1) [Patent Document 0001] Published Patent Publication No. 10-2013-0097494

(Patent Document 2) [Patent Document 0002] Published Patent Publication No. 10-2017-0095478

In the present invention, it was found that the extract of the by-product of the pine tree is effective in increasing the secondary metabolism product of the plant, that is, phytochemical.

Therefore, in the present invention, it is intended to enhance tryptophan in bean sprouts by providing a composition for a functional enhancer of the extract of pine pine by-product.

In addition, the purpose of extracting tryptophan in a high yield of bean sprouts with enhanced functionality by using pine pine extract is aimed at.

After the pineapple extract is prepared in a formulation that is easy to transfer to plants, the formulation is immersed in the germination period of beans during cultivation of bean sprouts. In the soybean sprouts harvested in this way, amino acids such as glutamic acid, lysine, and tryptophan are enhanced. Essential amino acids include valine, leucine, isoleucine, methionine, threonine, lysine, phenylalanine, tryptophan, histidine, arginine, cystine, aspartic acid, glutamic acid, proline, serine, glycine, and alanine. Among them, tryptophan-enhanced bean sprouts are harvested and then extracted in high yield using proteolytic enzymes to produce tryptophan in high yield. Green sprouts that are not ripened are also applicable.

The object of the present invention is to prepare a formulation for enhancing the functionality of agricultural products such as beans by using pine pine among conifers, among which is an immature, mature conifer extract, and injecting the washed beans into a cultivation container, water Preparing a functional enhancer by mixing 10 to 50% by weight of the prepared pine tree extract with respect to 100 parts by weight, and adding the prepared functional enhancer to the beans put into the cultivation container 5 to 7 times a day at intervals of 3 to 4 hours It characterized in that it consists of a step of cultivating bean sprouts by sufficiently fertilizing, and extracting tryptophan from the grown bean sprouts.

The present invention not only has the effect of providing bean sprouts with increased functionality using the pine tree extract, but also improves the value of agricultural products in terms of nutritional, functional, and quality, and can maximize the use of functional agricultural products, and glutamic acid of bean sprouts , Lysine, tryptophan, etc., especially tryptophan content can be increased. Therefore, it is possible to obtain bean sprouts that have a sedative effect to improve sleep or control depression and mood disorders, and by extracting them, tryptophan, a natural antidepressant, can be mass produced.

1 is a flow chart of a method for manufacturing pine pine extract microcapsules according to the present invention.

Hereinafter, the present invention will be described in more detail based on Examples and Experimental Examples. However, the following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

The present invention corresponds to all kinds of coniferous trees such as pine, pine, fir, cedar, cypress, globule, etc., but in the present invention, pine, among them, conifers surrounding the seeds, coniferous or immature, mature conifer extract after shelling the pine nuts It is characterized in that it is applied by microencapsulation using a white pine tree extract and walnut green skin extract.

The present invention uses a capsule in which a pine tree extract is collected inside a cell crosslinked using chitosan, the capsule is harmless to the human body, has phase stability, and has an effect of controlling the release characteristics of the pine tree extract.

A method of preparing the pine pine extract microcapsules used in the present invention is as follows.

The first step of preparing an oil-in-water emulsion:

1) The first step is to prepare an aqueous chitosan solution. Since chitosan is not dissolved in an aqueous solution in a neutral but basic atmosphere, it must be dissolved in an aqueous solution in an acidic atmosphere. The type of acid used to make the acidic atmosphere aqueous solution is not particularly limited, and the type may be selected according to the intended use, and acetic acid is preferably used. The acidic aqueous solution is preferably pH 2 to pH 4. The concentration of the chitosan solution is a factor that affects the thickness of the capsule in the encapsulation process. Since acetic acid is used to produce chitosan microcapsules for sustained release of chitosan, a thick capsule wall is required, and 2 to 4 wt% acetic acid is used. desirable. The concentration of chitosan is most preferably 2 to 5%.

Chitosan is added to an aqueous solution in an acidic atmosphere and stirred to prepare an aqueous chitosan solution. The chitosan is preferably used in an amount of 10 to 30% by weight, more preferably 20% by weight.

2) The second step is to obtain a chitosan solution containing a surfactant by dissolving a surfactant in the chitosan aqueous solution obtained in 1) in order to increase the dispersibility of the pine tree extract. The type of surfactant used in this process is not particularly limited, but tween 80 or tween 20, etc. can be used. When the tween 80 is used alone, it is preferable to maximize the dispersing power by mixing span 80 since the liquid phase may be gelled as a whole due to insufficient dispersing power. If a mixture of Tween 80 and span 80 is used, a capsule solution that maintains a homogeneous phase for more than 1 month can be obtained, and the optimal amount is 1 to 3% by weight of the two surfactants, tween 80 and span 80, and preferably 2 % By weight.

3) The third process is a step of dispersing 10 to 50% by weight of the pine tree extract in 10 to 20% by weight of the surfactant-containing chitosan aqueous solution obtained in 2) above in which the surfactant is sufficiently dissolved, and a solution of the pine tree extract solution is added to the surfactant-containing chitosan aqueous solution. After addition, an oil-in-water emulsion was prepared by adding 10 to 20% by weight of walnut green peel extract to the pine nut extract, surfactant and chitosan aqueous solution obtained by stirring at rpm 7,000 to 10,000, preferably rpm 9,000. The suggested stirring speed is an example and does not specifically limit the stirring speed. When the walnut extract is less than 10% by weight, the component to be strengthened in the plant is not strengthened, and when it is more than 20% by weight, the plant does not grow or wither.

A second step of preparing a multiphase emulsion;

1) The first step is to dissolve a surfactant (span or tween) in an oily liquid (pine conifer extract oil), and the oily liquid and surfactant are preferably mixed in a weight ratio of 2:5. The oily liquid and surfactant are not particularly limited, and types suitable for use are used. In the mixing ratio, there is no effect below the allowable range, that is, tryptophan and amino acids are not increased, and the crop does not grow above the allowable range.

2) In the second step, the oily liquid in which the surfactant obtained in step 1) is sufficiently dissolved is stirred at rpm 7,000 to 10,000, preferably rpm 9,000, and the oil-in-water emulsion prepared in the first step is added to obtain a multiphase emulsion. . The suggested stirring speed is an example and does not specifically limit the stirring speed.

The type of the surfactant is not particularly limited, but span 80 or span 20 and mineral oil may be used, and it is preferable to use span 80 and mineral oil. The tween-based surfactant used in the manufacture of oil-in-water cannot be used. Since the viscosity of oil-in-water to be dispersed in the oil phase is high due to the viscosity of the chitosan solution and the tween-based surfactant, the finished capsule solution is separated into layers when using the surfactant used in the aqueous phase. In addition to poor phase stability, the crosslinking agent is not properly mixed. Therefore, it is preferable to use a span-based surfactant used in the aqueous phase. In consideration of the viscosity of the oil-in-water, the span 80 is preferably 10 to 30 g, more preferably 10 g. It is preferable to use 50 to 200 g of mineral oil, more preferably 100 g.

There is no effect below the allowable range of the surfactant, and tryptophan and amino acids do not increase, but crops do not grow above the allowable range.

Step 3 manufacturing step of pine pine extract microcapsules;

The multiphase emulsion prepared in the second step is stirred at rpm 7,000 to 10,000, preferably rpm 9,000 to prepare nano-sized microcapsules of pine needles extract. The suggested stirring speed is an example and does not specifically limit the stirring speed.

The size of the pine pine extract nano-sized microcapsules is preferably 4.5 to 12 ㎛. If it exceeds 12 ㎛, the capsule wall becomes thick, making it difficult to release the core material, resulting in insufficient deodorization and antibacterial effects due to a small amount of initial release. In addition, the amount of the core material is reduced, resulting in less release, and no clear effect can be seen. If it is less than ㎛, the wall of the microcapsule becomes thin and the capsule is easily broken or the initial release amount is increased, and after a long period of time, the remaining core material disappears, and a clear effect cannot be seen. The most preferred microcapsule size is 4.7 μm.

There is no effect below the allowable range, and tryptophan and amino acids do not increase, but the crop does not grow above the allowable range.

[Example 1] Extraction of pine nuts by-product

For extraction of the pine pine by-product, the dried pine pine by-product is pulverized into an appropriate size, and 200 g of the pine pine by-product and a total of 3.0 L of 95% ethanol aqueous solution are added and immersed in a 5 L beaker. Then, it was filtered through filter paper to obtain an extract. The extraction process was repeated 3 times.

● Pine tree extract obtained by using a supercritical system (see Korean Patent Publication No. 10-2017-0095478).

● Pine tree extract obtained by fermentation (refer to Publication No. 10-2013-0097494).

[Example 2] Pine tree extract microcapsule preparation-1

To 2 g of 2% acetic acid, 20 g of 4% chitosan was added and stirred to prepare an aqueous chitosan solution. The chitosan aqueous solution was dissolved by mixing 2 g of tween 80 and span 80. The solution was stirred at 9,000 rpm for 10 minutes, and 2 g of pine nut extract was added to prepare an oil-in-water emulsion.

After mixing 10 g of Span 80 and 100 g of mineral oil, mixing with 11 g of oily liquid, stirring at 9,000 rpm, and adding 24 g of the obtained oil-in-water emulsion to prepare a multiphase emulsion.

[Example 3] Pine tree extract microcapsule preparation-2

Beta-cyclodextrin is added to an aqueous ethanol solution of 1:2 mixture of ethanol and water, and dissolved by stirring while raising the temperature. At the same time, span 80 and tween 80 are added to dissolve it together. At this time, 20 ml of an aqueous ethanol solution per 2 g of beta-cyclodextrin is used, and 0.5 w/v% of span 80 and tween 80 is added. The amount of the emulsifiers span 80 and tween 80 added is 1 w/v% of the total solvent, but the addition ratio of the two emulsifiers can vary depending on the characteristics of the supported material.

[Example 4] Method for producing tryptophan-enhanced bean sprouts

To 100 to 500 g of soybeans, preferably to 200 g of pineapple extract nano-sized microcapsules and 1 to 10% of distilled water, a liquid, preferably 2% of a solution was added thereto for 12 to 36 hours, preferably 24 After soaking time, put the beans in the bean sprout grower. Fertilize 1 to 2 L, preferably 1 L of the mixed solution every day until the bean sprouts grow at least once, preferably 5 to 7 times, at intervals of 3 to 4 hours to grow bean sprouts to produce functional bean sprouts do.

[Example 5] Tryptophan Extraction Method from Bean Sprouts Enriched with Tryptophan

In order to extract tryptophan from bean sprouts grown in the same manner as in Example 4, an ultrasonic extraction method was used. Soybean sprouts are immersed in an alkali solution (choose of sodium hydroxide, potassium hydroxide, or ammonia) in 1 to 10%, preferably 2% for 24 to 36 hours, preferably 24 hours, and then extracted using an ultrasonic extractor, and the pH is acidified. Using a substance (hydrochloric acid, acetic acid, etc.), the pH is adjusted to 5-7 neutral, preferably pH 6.

[Experimental Example 1] Bean sprouts experiment

An experiment was conducted to confirm the increase in physiologically active substances of bean sprouts using pine pine extract.

Soybean seeds were fertilized through an automatic cultivation machine before harvesting after immersion in pine pine extract and general agricultural water, and then tryptophan content was analyzed by HPLC, and the analysis results are shown in Table 1 below.

	Pine tree extract	General agricultural water
Bean seeds	0.224%	0.085%

Claims (6)

1. Method for producing pine pine extract microcapsules consisting of the following steps.

   1. Step 1: Preparation of oil-in-water emulsion

   1) To prepare an aqueous chitosan solution containing 10 to 30 wt% chitosan and 2 to 4 wt% acetic acid,

   2) After adding 1 to 3% by weight of surfactant to the aqueous chitosan solution to obtain an aqueous chitosan solution containing a surfactant,

   3) dispersing 10 to 50% by weight of the pine tree extract by stirring at 7,000 to 10,000 rpm in the aqueous chitosan solution containing the surfactant of 2), adding and homogenizing 10 to 20% by weight of walnut extract to obtain an oil-in-water emulsion;

   2. Step 2: Preparation of multi-phase emulsion

   1) An oily liquid was prepared by mixing the pine cone conifer extract in an oil with a surfactant in a weight ratio of 2:5,

   2) preparing a multiphase emulsion by adding the oil-in-water emulsion obtained in step 1 to the oily liquid of 1) and stirring at 7,000 to 10,000 rpm; And

   3. 3rd step: Preparation of microcapsules of pine pine extract

   1) This is a step of preparing microcapsules of pine needles extract by stirring the multiphase emulsion obtained in the second step at 7,000 ~ 10,000 rpm.
2. The method of claim 1,

   The surfactant is Span 20, Span 80, or a mixture of span and mineral oil.
3. The method of claim 1,

   In the first step, the second step and the third step, the stirring is performed at 7,000 ~ 10,000 rpm.
4. The method of claim 1,

   The size of the microcapsules is 4.5 ~ 12 ㎛ manufacturing method.
5. A method of cultivating bean sprouts with enhanced tryptophan by fertilizing the pine pine microcapsules obtained according to claim 1 on beans 5 to 7 times a day at intervals of 3 to 4 hours.
6. A method of obtaining tryptophan by extracting the bean sprouts obtained according to claim 5 with a proteolytic enzyme.

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