Manufacturing method of black rice germinated liquid

Abstract

The present invention relates to a black rice germination liquid having excellent anti-inflammatory effects, which is obtained by subjecting the water used to germinate black rice to low-temperature treatment and room-temperature treatment, and a method for producing the same. The water used to germinate black rice is subjected to low-temperature treatment in an open container at a temperature range of 1 to 5°C for 1 to 10 days, and then to room-temperature treatment in an open container at a temperature range of 10 to 30°C for 30 to 210 days.

Description

[Technical Field]

[0001] The present invention relates to a method for producing black rice germinated liquid having anti-inflammatory effects and the black rice germinated liquid obtained therefrom, and more particularly to a black rice germinated liquid having excellent anti-inflammatory effects obtained by subjecting water used in germinating black rice to low-temperature treatment and room-temperature treatment, and a method for producing the same. [Background technology]

[0002] Recently, it has been reported that native plants also contain various substances that can prevent or treat disease. Due to the cultural need to live a healthy life, research is actively underway to search for substances with diverse physiological functions from the diverse resources on Earth, and among these, much interest is focused on compounds contained in plant resources.

[0003] With the recent aging population and changes in dietary habits, there has been a trend toward an increase in both acute and chronic inflammatory diseases such as rheumatoid arthritis, shingles, and rhinitis (Hyun EA. Anti-inflammatory effect of Salvia officinalis L. extract. Cheju National University. Korea 2003.). Chronic inflammation occurs when the body fails to eliminate substances that induce acute inflammation, such as tuberculosis bacteria, protozoa, fungi, and other parasites that resist host defenses and can persist in tissues for long periods. Onychomycosis, also known as tinea unguium of the fingers and toes, is also an inflammatory nail disease.

[0004] Inflammation is defined as a complex immune response triggered by the innate immune system in response to harmful substances in vascularized tissues, and is typically accompanied by fever and pain. In other words, inflammation is a collective term for the defensive response that occurs in the body when tissues are damaged. It is the body's response to trauma, burns, bacterial invasion, etc., resulting in localized hyperemia, edema, fever, and pain. The inflammatory response is related to the repair process of damaged tissues and includes both the process of removing the causative agents, such as microorganisms and toxins, that caused the tissue damage, and the process of removing necrotic cells and tissues that result from the damage.

[0005] Direct causes of the inflammatory response include: (1) Activation of the complement system (2) Secretion of inflammatory cytokines (3) Fibrinolysis (4) Leukocyte extravasation and phagocytosis (5) Clotting reaction (6) Other inflammation-inducing substances This is caused by the action of immune cells and systems by the immune system. The causes of this immune response include factors that can cause any form of tissue damage, and external factors that can induce inflammation include: (1) Physical causes: burns, frostbite, physical wounds / trauma, foreign bodies, ionizing radiation (2) Biological causes: pathogens, hypersensitivity reactions, stress (3) Chemical causes: toxins, alcohol can be cited as examples.

[0006] While a correlation between chronic inflammation and cancer or other diseases has been reported, efforts are underway to reduce the risk of disease by using dietary interventions to reduce the inflammatory response (Aggarwal BB et al. Inflammation and cancer: How hot is the link 72:1605-1621 (2006)).

[0007] The inflammatory response is a defense mechanism of living tissues against external physical and chemical stimuli and bacterial infections, and is a mechanism for repairing or regenerating damaged tissues (Zamora R, et al. Inducible nitric oxide synthase and inflammatory diseases. Mol. Med. 6:347-373 (2000)). When an inflammatory response occurs in the body, inflammatory cells such as macrophages secrete inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) (Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal. 13:85-94 (2001)). Under normal circumstances, the body neutralizes or eliminates pathogenic factors and regenerates damaged tissues, restoring normal structure and function through the inflammatory response. However, if this does not occur, the body may develop chronic inflammation and other disease states. Inflammatory responses can be triggered by a variety of stimuli, including infectious agents, ischemia, antigen-antibody reactions, fever, or other physical injuries, resulting in clinical symptoms such as erythema, edema, tenderness, and pain. Inflammatory responses occur in three distinct phases, each mediated by a different mechanism. The first is an acute, transient phase characterized by local vasodilation and increased capillary permeability. The second is a delayed, subacute phase characterized by infiltration of leukocytes and phagocytic cells. The third is a chronic, proliferative phase characterized by tissue degeneration and fibrosis. Many other mechanisms are involved in the inflammatory process.

[0008] Inflammatory responses can be observed in almost all clinical diseases, and it is known that enzymes related to inflammatory responses play an important role in carcinogenesis. Therefore, the treatment of inflammatory diseases is one of the challenges facing modern medicine. Among these inflammatory diseases, some are bacterial diseases that can be treated causally with antibiotics, but most are known to be intractable diseases for which no specific treatment exists, as they are caused by tissue damage due to an autoimmune response.

[0009] The anti-inflammatory effects of most non-steroidal anti-inflammatory drugs (NSAIDs) are mediated by inhibiting COX enzyme activity (Vane et al., Annual Review of Pharmacology and Toxicology, 38:97-120, 1998).

[0010] COX-1 is an enzyme that is constantly present in tissues such as the stomach and kidney and is involved in maintaining normal homeostasis, while COX-2 is transiently and rapidly expressed intracellularly in response to mitogens and cytokines during inflammation and other immune responses. NSAIDs, used to treat acute or chronic inflammatory diseases such as rheumatoid arthritis, are known to cause side effects such as gastrointestinal disorders by inhibiting not only COX-2 enzyme but also COX-1 enzyme (Masferrer et al., Proceedings of the National Academy of Sciences of the United States of America, 91:3228-3232, 1994; Seibert et al., Proceedings of the National Academy of Sciences of the United States of America, 91:12013-12017, 1994).

[0011] In particular, stimulation of macrophages with cytokines, tumor necrosis factor (TNF-α), and lipopolysaccharide (LPS) activates the inflammatory transcription factor nuclear factor-kB (NF-kB), which in turn induces the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in the production of excessive amounts of nitric oxide (NO) and prostaglandin E2 (PGE2), leading to inflammation (Nishida T et al. Geranylgeranylacetone induces cyclooxygenase-2 expression in cultured rat gastric epithelial cells through NF-kappa B. Digestive Diseases and Sciences. 52:1890-1896 (2007)).

[0012] The synthetic anti-inflammatory drugs developed to date can be broadly divided into steroids (hydrocortisone, prednisolone, betamethasone) and non-steroids (aspirin, indomethacin, ibuprofen). Most of these drugs have side effects, such as gastrointestinal, renal, and cardiac disorders (Dagne JM, et al. Cardiovascular side effects: from light to shadow. Currpharm Des. 12: 917-975 (2006) and Makins R, Ballinger A. Gastrointestinal side effects of drugs. Expert Opin Drug Saf. 2: 421-429 (2003)). Therefore, there is a need to develop safer and more effective anti-inflammatory therapeutic agents derived from natural products. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention aims to provide a black rice germination liquid having excellent anti-inflammatory effects, which is obtained by subjecting water used for germinating black rice to low-temperature treatment and room-temperature treatment, and a method for producing the same. [Means for solving the problem]

[0014] The black rice germination liquid according to the present invention is obtained by subjecting the water used for germinating black rice to low-temperature treatment in an open container within a temperature range of 1 to 5°C for 1 to 10 days, and then subjecting it to room temperature treatment in an open container within a temperature range of 10 to 30°C for 30 to 210 days.

[0015] In addition, the method for producing black rice germination liquid according to the present invention is characterized by comprising: (1) a black rice germination step in which black rice is soaked in water to germinate; (2) a germination liquid separation step in which the germinated black rice is separated from the germination liquid used for germinating the black rice; (3) a low-temperature treatment step in which the germination liquid is treated at low temperature in an open container within a temperature range of 1°C to 5°C for 1 to 10 days; and (4) a room-temperature treatment step in which the low-temperature treated germination liquid is treated at room temperature in an open container within a temperature range of 10°C to 30°C for 30 to 210 days. [Effects of the Invention]

[0016] According to the present invention, there is an effect of providing a black rice germination liquid having excellent anti-inflammatory effects and a method for producing the same, which is obtained by subjecting water used for germinating black rice to low-temperature treatment and room-temperature treatment. [Brief explanation of the drawings]

[0017] [Figure 1] This is a copy of the test report analyzing the bacteria present in the black rice germination liquid before low-temperature treatment and room-temperature treatment. [Figure 2] 1 is a copy of a test report for analyzing bacteria present in germinated black rice liquid obtained after low-temperature treatment and room-temperature treatment according to the present invention. [Figure 3] 1 is a copy of a test report for analyzing bacteria present in germinated black rice liquid obtained after low-temperature treatment and room-temperature treatment according to the present invention. [Figure 4a]1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 4b] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 4c] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 5] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 6] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 7] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 8] 1 is a photograph showing the experimental results of the antifungal activity of each example of the present invention. [Figure 9] 1 shows photographs of experimental results of antiviral activity in each example of the present invention. [Figure 10] 1 shows photographs of experimental results of antiviral activity in each example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] In its best form, the present invention provides a black rice germination liquid having anti-inflammatory effects, which is obtained by subjecting water used for germinating black rice to low-temperature treatment in an open container within a temperature range of 1°C to 5°C for 1 to 10 days, and then subjecting it to room temperature treatment in an open container within a temperature range of 10°C to 30°C for 30 to 210 days.

[0019] In addition, the present invention provides, as its best mode, a method for producing black rice germinated liquid having anti-inflammatory effects, which comprises: (1) a black rice germination step in which black rice is soaked in water to germinate; (2) a germinated liquid separation step in which the germinated black rice is separated from the germinated liquid used for germinating the black rice; (3) a low-temperature treatment step in which the germinated liquid is treated at low temperature in an open container within a temperature range of 1°C to 5°C for 1 to 10 days; and (4) a room-temperature treatment step in which the low-temperature treated germinated liquid is treated at room temperature in an open container within a temperature range of 10°C to 30°C for 30 to 210 days.

[0020] The present invention will now be described in detail with reference to specific examples.

[0021] The black rice germination liquid according to the present invention is obtained by subjecting the water used for germinating black rice to low-temperature treatment in an open container within a temperature range of 1 to 5°C for 1 to 10 days, and then subjecting it to room temperature treatment in an open container within a temperature range of 10 to 30°C for 30 to 210 days.

[0022] Rice, a grass family crop, is one of the world's three major grains along with wheat and corn, and is one of the most important food resources in the world. Rice is also an important source of energy as it is easy to store, has low moisture and protein content, and is high in carbohydrates, so it does not spoil easily.

[0023] Anthocyanins in black rice, also known as black rice, are known to have the aforementioned strong antioxidant effects as well as cancer prevention and immune-boosting effects. Black rice, a type of colored rice, is processed into various foods due to its unique color and aroma, and its consumption is gradually increasing. The aroma of black rice is attributed to alcohols such as ethanediol and guaiacol, ketones such as hexadecanoic acid, hexanal, and acetic acid, aldehydes, and organic acids. Its main pigments are reported to be glycosides such as cyanidin-3-glucoside (C3G) and peonidin-3-glucoside (P3G).

[0024] In particular, the pigment components of black rice contain polyphenolic compounds with diverse structures and molecular weights, and these polyphenolic compounds have been confirmed to have physiological activities such as antioxidant, antibacterial, and anticancer properties. Black rice, also popular as brown rice, has a higher dietary fiber content than regular brown rice, a unique flavor, and high protein, vitamin B, and mineral contents. According to Li Shizhen's Compendium of Materia Medica, black rice has the effects of clearing the stomach and promoting circulation, nourishing yin and strengthening the kidneys, strengthening the liver, and brightening the eyes and activating blood, and is also significantly effective in preventing and treating dizziness, anemia, and gray hair, as well as eye diseases, polyuria, constipation, cardiovascular diseases, and other diseases.

[0025] Daily consumption of black rice is known to improve the body's overall regulatory functions, strengthen the immune system, and promote anti-aging, disease prevention, and beauty for women. Black rice is particularly effective against anemia, which is common among pregnant women, and is extremely beneficial for skeletal development in children. Feeding black rice to infants as baby food also contributes significantly to their healthy development. Black rice also contains a large amount of selenium, which is effective in preventing cancer. A Chinese study (November 1992) reported that the long-term administration of black rice to 40 rectal cancer patients resulted in an improvement in their condition. In particular, caravans traveling China's Silk Road and residents of Xinjiang Province have long enjoyed the nutritious and healthy porridge made with black rice, adlay, and jujube as a way to beat the heat of the Taklamakan Desert. In addition to its beautiful color, black rice also has a fragrant aroma and is effective against diabetes, constipation, and gastrointestinal disorders. It is also richer in vitamins B, C, and E than regular rice, making it a healthy black grain food that is good for the human body.

[0026] Black rice, which is made by polishing brown rice, retains its germ and contains more nutrients than regular rice. Its blackness is about five times greater than that of regular rice. Black rice is rich in natural pigments such as anthocyanins, as well as vitamin E and other inorganic salts such as iron, zinc, and selenium, making it known for its excellent ability to scavenge active oxygen. Consuming black rice is expected to help prevent skin aging due to its high content of antioxidants, such as anthocyanins and oryzafuran, which scavenge active oxygen. Black rice, with its physiological and pharmacological benefits, is attracting attention. Black rice is primarily produced in southern Asia and China, and is now also grown in parts of Korea. It is known for its excellent medical properties. The purple pigment in black rice is particularly stable against high temperatures and sunlight, making it a popular food ingredient in bread, porridge, noodles, and other processed foods.

[0027] Below we will introduce some examples of how black rice can be used as a food ingredient.

[0028] Korean Patent Registration No. 451126 discloses noodles made from black rice, characterized by their main ingredients being 55% to 70% by weight of rice flour made by grinding rice or black rice to 140 mesh to 180 mesh, 20% to 28% by weight of wheat flour, 5% to 8% by weight of edible salt, and 5% to 10% by weight of water, as well as a method for producing the noodles.

[0029] Korean Patent Publication No. 2000-75359 discloses a rice snack made from black rice, which is made by adding water to a mixture of black rice and brown rice so that the moisture content is 18%, and then swelling the resulting dough, and a method for making the same.

[0030] Korean Patent Publication No. 2002-92334 discloses black rice cake for rice cake soup, which is made by mixing and grinding 1.5L-1.8L of raw water, 1.5kg-2.5kg of black rice, 1.5kg-2.5kg of barley, 6kg-7kg of polished rice, 400g-500g of potato starch, and 0.4g-0.5g of refined salt, and then uniformly mixing the mixture and steaming it with steam at 100℃-120℃.

[0031] Korean Patent Publication No. 2001-105020 discloses noodles containing plant extracts, which are prepared by mixing a plant food in a liquid or powder state with wheat flour, kneading, rolling, and shaping the noodles, and a method for preparing the noodles.

[0032] Korean Patent Publication No. 2002-42384 discloses a method for producing bread containing black rice flour, which is made by mixing black rice and wheat flour in a certain ratio, adding an appropriate amount of water, and kneading and fermenting the mixture to produce bread.

[0033] In the present invention, black rice, also referred to as black rice, is known to be rich in anthocyanins and inorganic salts and to be capable of effectively neutralizing active oxygen in the body. As an example, Heugjinju (Oryza sativa cv. Heugjinjubyeo), cultivated in Jindo, can be used as the black rice. Heugjinju has a deep purple pericarp and is reported to contain the anthocyanin compounds cyanidin and mervidin; their glycosides (cyanidin glucoside, mervidin glucoside, delpinidin glucoside), alkaloids, and phenolic compounds; and 2-hydroxy-4-methoxypyridine. Furthermore, black rice is rich in minerals and has alkaline properties. Alkaline foods neutralize the body's oxidation caused by various pollutants and food oxides and have the effect of preventing various inflammatory diseases. In particular, black rice has the highest selenium content among minerals, and selenium in black rice is known to not only activate liver cells but also inhibit their destruction.

[0034] The milling precision of black rice can be anywhere from 9-minute to 10-minute. Milling precision refers to the degree to which the rice is harvested. Based on brown rice and polished rice, the entire rice, including the outer bran layer and rice bran layer (see Rice Structure and Nutrient Distribution), is divided into 10 equal parts. 10-minute milling refers to rice that has been completely removed. Brown rice that has not had any of the bran layer removed is called 0-minute milling. Therefore, 5-minute milling indicates that approximately 50% of the outer bran layer and rice bran layer have been removed. Milling precision is sometimes expressed by weight, with the outer bran layer and rice bran layer generally accounting for approximately 8-9% of the total weight of brown rice. This varies depending on the variety and does not apply uniformly to all rice. However, 10-minute milling refers to rice that has been milled to 8% of its total weight for ease of milling. In other words, 1-minute polishing can be considered as polishing approximately 0.8% of the total weight of brown rice. 5-minute polishing rice available on the market is polishing approximately 4% of the total weight of brown rice, and 7-minute polishing rice is polishing approximately 5.6% of the total weight of brown rice. As mentioned above, when polishing rice for 9 to 10 minutes, the rice germ necessary for germination remains, so germination is possible.

[0035] Germination of black rice can be carried out by soaking it in water at a temperature of 16°C to 23°C for 24 to 48 hours, which can be understood as the normal temperature conditions that mainly correspond to winter in Korea.

[0036] Germination of black rice can be carried out by soaking it in water at a temperature of 23°C to 30°C for 15 to 24 hours, which can be understood as the room temperature condition that mainly corresponds to the summer in the Republic of Korea.

[0037] The above-mentioned germination temperature and germination time can be understood to be suitable conditions for germinating black rice seeds to a size of about 0.5 mm to 1.5 mm.

[0038] Water is stirred during the germination of black rice, and it is preferable to lift the water from the bottom to the top and then drop it from the top to the bottom again. The stirring of water serves to prevent the water from stagnating and spoiling. This is understood to be because the water is purified by its own power and contact with oxygen, etc., which are naturally generated as it flows without stagnating, preventing spoilage. Furthermore, this can be understood to be the same and / or similar to the fact that naturally flowing water has a higher self-purifying ability than stagnant water, and water stirring can be understood to be a method that is also applied in water purification processes.

[0039] The water used during germination is preferably drinking water or table water, and may be tap water.

[0040] Before low-temperature treatment and room-temperature treatment, the germination liquid itself contained 92,000,000 general bacteria (CFU / g) and 1,100,000 Escherichia coli (CFU / g) as shown in Figure 1. However, after low-temperature treatment and room-temperature treatment according to the present invention, it contained three general bacteria and three fungi in particular, as shown in the test report in Figure 2. As shown in Figure 3, it was confirmed that the general bacteria present were Citrobacter murliniae, Pseudacidovorax intermedius, and Paenirhodobacter enshiensis, and the fungi present were Candida intermedia, Candida tropicalis, and Meyerzyma sp.

[0041] In addition, the method for producing black rice germination liquid according to the present invention is characterized by comprising: (1) a black rice germination step in which black rice is soaked in water to germinate; (2) a germination liquid separation step in which the germinated black rice is separated from the germination liquid used for germinating the black rice; (3) a low-temperature treatment step in which the germination liquid is treated at low temperature in an open container within a temperature range of 1°C to 5°C for 1 to 10 days; and (4) a room-temperature treatment step in which the low-temperature treated germination liquid is treated at room temperature in an open container within a temperature range of 10°C to 30°C for 30 to 210 days.

[0042] The black rice germination step (1) involves soaking the black rice in water to germinate. The germination of black rice and black rice can be understood to be the same and / or similar to those described above. Seed germination requires a specific temperature, sufficient oxygen, and adequate moisture. While some species require light, others require darkness, most plants do not require light. Once germination begins, various enzymes become active, increasing respiration, breaking down stored nutrients and converting them into more readily available forms. These nutrients are then utilized for the rapid development of meristematic tissues such as the shoot and root. In seeds, stored nutrients are contained in the endosperm (e.g., in grasses), episperm (e.g., in lotus flowers), and cotyledons (e.g., in legumes) in the form of green powder, fat, protein, etc. As germination progresses, stored nutrients decrease, but chlorophyll is formed in the cotyledons and primary leaves, allowing for gradual photosynthesis, and the roots begin to absorb nutrients and water, causing the seed to deviate from heterotrophic growth based on stored nutrients and begin to grow autotrophically on its own.As a result, new components that were not present before germination may be produced.

[0043] The germinant liquid separation step (2) involves separating the germinated black rice produced in the black rice germination step (1) from the germinant liquid used for germinating the black rice. The germinated black rice obtained may be used alone or in cooking with other rice. Cooking germinated black rice increases the digestibility and absorption rate of the resulting black rice. The present invention is characterized by germinating black rice, using the germinated black rice for its intended purpose, and utilizing the germinant liquid obtained as a by-product. That is, the water supplied for germination of the black rice is recovered as a germinant liquid after germination of the black rice, and this by-product is utilized. The germinant liquid obtained is black in color. This suggests that black rice contains a particularly large amount of anthocyanins, and that many effective and unidentified physiologically active substances have been extracted.

[0044] The low-temperature treatment step (3) comprises subjecting the germination solution obtained in the germination solution separation step (2) to a low-temperature treatment in an open container at a temperature within the range of 1°C to 5°C for 1 to 10 days. The pH of the germination solution before and after the low-temperature treatment is within the range of 4.5 to 5.5.

[0045] The room temperature treatment step (4) comprises treating the germination solution that has undergone the low temperature treatment step (3) in an open container at room temperature within a temperature range of 10°C to 30°C for 30 to 210 days. After the room temperature treatment, the pH of the germination solution rises to 7.5 to 8.

[0046] The pounding precision of black rice can be 9 minutes to 10 minutes. The pounding precision can be understood to be the same and / or similar to that described above.

[0047] Germination of black rice can be carried out by soaking it in running water for 24 to 48 hours at 16 to 23 degrees Celsius, which can be understood as the normal temperature conditions that mainly correspond to winter in Korea.

[0048] Germination of black rice can be carried out by soaking it in running water for 15 to 24 hours at 23 to 30 degrees Celsius, which can be understood as the room temperature condition that mainly corresponds to the summer in Korea.

[0049] The germination temperature and germination time are understood to be suitable conditions for germinating black rice to a size of about 0.5 mm to 1.5 mm.

[0050] It is preferable to stir the water during germination of the black rice, as stirring the water prevents the water from stagnating and spoiling.

[0051] The water used during germination is preferably drinking water or table water, and may be tap water.

[0052] Preferred examples of the present invention and comparative examples will be described below.

[0053] The following examples are intended to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0054] Example 1 and Example 2 500 g of black rice (Oriza sativa L., variety: Cheongpung Heugchalbyeo) milled to approximately 9 minutes was poured into 5 L of tap water and allowed to germinate for 15 to 48 hours at 20°C ± 3°C with slow stirring. The germinated black rice and germination liquor were harvested. The black germination liquor was separated from the germinated black rice and poured into an open container. The pH was measured (approximately 5.5; this is referred to as "Example 1") and then subjected to low-temperature treatment in a refrigerator maintained at 2°C ± 0.1°C for 48 hours. After low-temperature treatment, the germination liquor was stored in an open container and subjected to room temperature treatment (approximately 20°C ± 3°C) for 50 days. The pH of the germination liquor was measured and found to be approximately 7.8 (this is referred to as "Example 2").

[0055] The bacteria present in the obtained black rice germinated liquid according to the present invention were measured, and as a result, it was found to contain three types of general bacteria and three types of fungi, as shown in Figures 2 and 3. As shown in Figure 3, it was confirmed that the general bacteria included Citrobacter murliniae, Pseudacidovorax intermedius, and Paenirhodobacter enshiensis, and the fungi included Candida intermedia, Candida tropicalis, and Meyerzyma sp.

[0056] For the convenience of storage and use, the black rice germinated liquids of Examples 1 and 2 were paper filtered, freeze-dried, powdered, and stored, and diluted with distilled water immediately before use.

[0057] Comparative Example 1 Brown rice germination liquid was produced in the same manner as in the previous example, except that brown rice (Oriza sativa L., variety: Akibare) milled to a milling precision of about 9 minutes was used instead of black rice milled to a milling precision of about 9 minutes. However, it was frequently confirmed that the brown rice spoiled during the low-temperature treatment and room-temperature treatment, resulting in the generation of a foul odor.

[0058] Comparative Example 2 Black rice germinated liquid was produced in the same manner as in Example 1, except that black rice (same as in Example 1) that had been milled to a milling precision of approximately 9 minutes was used and low-temperature treatment was not performed. However, since low-temperature treatment was not performed, it was confirmed that the black rice germination liquid spoiled during the room temperature treatment and produced a foul odor.

[0059] Comparative Example 3 Black rice (same as in Example 1) milled for approximately 9 minutes was used and germination was carried out as in Example 1, but the germination liquid was obtained without low-temperature or room-temperature treatment after germination. The initial pH of the obtained black rice germination liquid was approximately 4.5, and it was confirmed that there were 92,000,000 general bacteria (CFU / g) and 1,100,000 E. coli (CFU / g).

[0060] Experimental Example 1: Antifungal Activity Experiment (1) 1. Experimental Strains Trichophyton rubrum (KCTC 6375), a type of fungus known to cause ringworm, was provided by Daegu University of Korean Medicine and was subcultured at least three times before use in the antifungal activity experiments.

[0061] 2. Media Used SDA (Sabouraud dextrose agar) was used to confirm growth patterns, subculture and antifungal activity.

[0062] 3. Antifungal activity verification (Paper disc agar diffusion method) The antifungal activity was verified using the paper disc agar diffusion method. The test strain was harvested using a 5mm cork borer and placed in the center of the medium. A paper disc was attached to the medium so that it would not come off. 50μl of samples containing 5mg / ml and 10mg / ml of black rice germination liquid (solid content) from Examples 1 and 2, respectively, was then poured into the culture vessel. The vessel was then cultured at 25°C for at least 7 days, after which the formation of growth inhibition rings was confirmed. Sterile water was used as a control to examine the effect of the black rice germination liquid powder (solid content).

[0063] The antifungal activity of the samples in Examples 1 and 2, each at a concentration of 5 mg / ml and 10 mg / ml, was tested using the paper disc agar diffusion method. The results are shown in Figure 4 and Table 1. After five days, the antifungal activity of the black rice germ liquid inhibited the growth of Trichophyton rubrum (KCTC 6375), with the growth rates reaching 80 mm and 150 mm at 5 mg / ml and 10 mg / ml, respectively, for the Example and 70 mm and 140 mm at 5 mg / ml and 10 mg / ml, respectively, for Example 2 (Figure 4a). After seven days, the growth rates reached 70 mm and 130 mm at 5 mg / ml and 10 mg / ml, respectively, for Example 1, and 50 mm and 110 mm at 5 mg / ml and 10 mg / ml, respectively, for Example 2, demonstrating similar antifungal activity in Examples 1 and 2 (Figure 4b).

[0064] [Table 1]

[0065] In Figure 4, P is the strain (Trichophyton rubrum KCTC 6375); C is distilled water as a control; 1 is 5 mg / ml (Example 1); 2 is 10 mg / ml (Example 1); 3 is 5 mg / ml (Example 2); and 4 is 10 mg / ml (Example 2).

[0066] Experimental Example 2: Antifungal Activity Experiment (2) The culture in Experimental Example 1 was continued for a long period of 10 days or more. As a result of the experiment, it was confirmed that an unknown white mold grew in Example 2 after 10 days of culture, and that the antifungal activity of this unknown mold significantly inhibited the growth of Trichophyton rubrum, as shown in Table 2 and Figure 4c below.

[0067] [Table 2]

[0068] The confirmation of the unknown white mold observed in Example 2 and the results of experiments conducted at concentrations of 5 mg / ml and 10 mg / ml for Examples 1 and 2 are shown in Table 3 and Figure 5. While the unknown white mold was not observed in Example 1, the growth of the unknown white mold was confirmed in Example 2, and the concentration of the unknown mold was also significantly higher between the 5 mg / ml and 10 mg / ml concentrations.

[0069] [Table 3]

[0070] In Figure 5, P is the strain (Trichophyton rubrum); C is distilled water as a control; 1 is 5 mg / ml (Example 1); 2 is 10 mg / ml (Example 1); 3 is 5 mg / ml (Example 2); and 4 is 10 mg / ml (Example 2).

[0071] In addition, to investigate the significant correlation between the white unknown mold found in Example 2 and the concentration, the experimental results for Example 2 at concentrations of 1 mg / ml, 5 mg / ml, and 10 mg / ml are shown in Table 4 and FIG. 6.

[0072] As a result, it was confirmed that as the concentration of Example 2 increased, the concentration of the unknown white mold and the antifungal activity against Trichophyton rubrum tended to increase relatively.

[0073] [Table 4]

[0074] In FIG. 6, P is the strain (Trichophyton rubrum); C is distilled water as a control; 1 is 1 mg / ml (Example 2); 2 is 5 mg / ml (Example 1); and 3 is 10 mg / ml (Example 2).

[0075] As a result of the experiment, it was confirmed that after 5 days, the growth of Trichophyton rubrum was inhibited by the antifungal activity of Examples 1 and 2, and after 7 days, similar activity was observed in Examples 1 and 2.

[0076] Experimental Example 3: Antifungal Activity Experiment (3) The antibacterial effect of black rice germ liquid against the tinea fungus Trichophyton rubrum (KCTC6375) was measured using the method of Chandrasekaran and Venkatesalu (Journal of Ethnopharmacology, 91, 105-108 (2004)). The fungus was cultured in Sabouraud dextrose broth at 28°C for 3 days and then diluted to an absorbance of 0.6 at 450 nm. 50 μl of the diluted fungus was inoculated into 1 ml of medium containing the extract. The extract was diluted two-fold to concentrations of 10%, 20%, and 40% in the medium. After culturing at 28°C for 3 days, the fungus growth was observed, and the results are shown in Figures 7 and 8. Figure 8 is a magnified photograph of Figure 7.

[0077] 7 and 8, Trichophyton floating matter was observed in Comparative Example 4 (an ungerminated liquid obtained by pouring 5 L of ordinary tap water into 500 g of black rice milled for about 7 minutes and soaking the rice for about 24 hours at a temperature of 20°C ± 3°C while slowly stirring) and the medium liquid, but no Trichophyton floating matter was observed in Example 1. As a result, 40% was confirmed as the minimum inhibitory concentration (MIC).

[0078] Experimental Example 4: Antiviral activity experiment Example 1 was used for the antiviral activity experiment of black rice germinated liquid.

[0079] The antiviral activity of Comparative Example 4 and Example 1 against herpes simplex virus (HSV) was tested using Vero cells as follows.

[0080] Vero cells (1 × 10) were cultured in a 24-well TC plate. 5 Eighteen to 20 hours after seeding with 100 μg / ml and 200 μg / ml HSV-1 (MOI: 2) from Comparative Example 4 and Example 1, respectively, were incubated at 37°C for 1 hour and then inoculated onto Vero cells. As with Comparative Example 4 and Example 1, 2 hours after inoculation with HSV-1, the inoculum was removed from the Vero cells, washed three times with PBS, and replaced with DMEM supplemented with 10% FBS. The negative control group contained only DMEM supplemented with 10% FBS. After 48 hours, the level of virus infection was measured under a microscope. To measure cell viability, 20 μl of MTT reagent was added, incubated for 1 hour, and then the absorbance at 450 nm was measured.

[0081] The results of the antiviral activity analysis against herpes simplex virus using images taken 48 hours after infection are as follows: As can be seen from Figures 9 and 10, the virus infection rate was significantly reduced in cells infected with herpes simplex virus and then treated with black rice germ solution (Figure 9), and the cell viability was also confirmed to be more than five times higher than in Comparative Example 4 and the virus-infected group (Figure 10).

[0082] In Figures 9 and 10, Cell-1 (C-1) refers to Vero cells (uninfected group), Cell-2 (C-2) refers to Vero cells (HSV-infected group), C-100 refers to Comparative Example 4 (concentration of 100 μg / ml), C-200 refers to Comparative Example 4 (concentration of 200 μg / ml), E-100 refers to Example 1 (concentration of 100 μg / ml), and E-200 refers to Example 1 (concentration of 200 μg / ml).

[0083] In Comparative Examples 1 and 2, usable germinant solution could not be obtained due to spoilage, and therefore could not be used for the experiment. In Comparative Example 3, the initial bacterial state and pH change were confirmed using the germinant solution obtained as described above.

[0084] Although the present invention has been described in detail above with reference to only the specific examples, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention, and it is to be understood that such modifications and variations are within the scope of the appended claims.

Claims

1. (1) the black rice germination stage, in which black rice is soaked in water to germinate; (2) A germination liquid separation step for separating the germinated black rice from the germination liquid used for germinating the black rice; (3) A low-temperature treatment step in which the germination solution is subjected to a low-temperature treatment in an open container at a temperature range of 1°C to 5°C for 1 to 10 days; and (4) A method for producing black rice germinated liquid, comprising: a room temperature treatment step of treating the low-temperature treated germinated liquid in an open container at a temperature within the range of 10°C to 30°C for 30 to 210 days.

2. The method for producing black rice germinated liquid according to claim 1, wherein the pH is within the range of 4.5 to 5.5 before, during or after the low-temperature treatment, and increases to a pH within the range of 7.5 to 8 after the room temperature treatment.

3. 2. The method for producing black rice germinated liquid according to claim 1, wherein the black rice is milled for 9 to 10 minutes.

4. 2. The method for producing black rice germinated liquid according to claim 1, wherein the germination of black rice is carried out by soaking the black rice in water at a temperature of 23°C to 30°C for 15 to 24 hours.

5. 2. The method for producing black rice germinated liquid according to claim 1, wherein the germination of black rice is carried out by soaking the black rice in water at a temperature of 16°C to 23°C for 24 to 48 hours.

6. The method for producing black rice germinated liquid according to claim 1, wherein the water is stirred during the germination of the black rice.

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