KR20200067075A - Fermented product of Protaetia brevitarsis extract, its preparation method and use - Google Patents

Abstract

The present invention relates to a fermented product of a solvent extract of Protaetia brevitarsis seulensis, and a manufacturing method thereof. More specifically, the fermented product of a solvent extract of Protaetia brevitarsis seulensishas has antibacterial properties, can be used as a natural preservative as well as an antioxidant composition, a composition for inhibiting lipid oxidation, a cosmetic composition for whitening, an acne prevention and treatment, a dandruff prevention and treatment cosmetic composition and can also be applied to various foods, pharmaceutical compositions, and the like.

Description

Fermented product of white spotted flower radish extract, its manufacturing method and use {Fermented product of Protaetia brevitarsis extract, its preparation method and use}

The present invention relates to a fermentation product of a solvent extract of white spotted flower radish and a method for manufacturing the same. Specifically, the fermentation product of the solvent extract of the white spotted flower radish of the present invention has various functions such as antimicrobial, antioxidant, lipid oxidation suppression, whitening, acne bacteria, dandruff bacteria, food, food additive, cosmetic composition, pharmaceutical It can be used in various fields such as composition.

Edible insects have long been consumed in many countries around the world, including Europe, the Americas, Korea, China, Japan and Southeast Asia. There are more than 1,900 types of edible insects known so far, and they are usually eaten directly in a collected form, or consumed through simple cooking processes such as drying, roasting, and frying.

In 2008, the United Nations Food and Agriculture Organization (FAO) launched a multifaceted study of insect breeding and consumption. In addition to publishing data on the edible insect industry in 2012, since 2013, the world has drawn attention to edible insects as a preparation for eradicating hunger, supplementing nutrition and supplying proteins, and reducing environmental pollution. The insect industry has grown rapidly since the Ministry of Agriculture, Food and Rural Affairs in 2010 recognized the diversified interest in the use and use of insects and the need for industrial growth, and enacted the Act on Fostering and Supporting the Insect Industry. Currently, 7 types of insects are available as food, such as rice locusts, silkworm pupae, baekgangjam, brown larvae, white spotted flower larvae, double-starred crickets, and longevity scarab larvae. The food registration of the American king and pupa is in progress.

Edible insects are easy to breed compared to livestock, and the breeding environment is eco-friendly and has sustainable industrial characteristics. Also, it is rich in protein, unsaturated fatty acid, dietary fiber, minerals such as calcium and iron, and vitamins, and has high nutritional value, so it is enough to replace meat, and is attracting attention as a high-protein, high-nutrition, high-functional food material. .

In Korea, food research using edible insects has been conducted intensively for the past 5-6 years, and the edible insect market has been formed in various ways. The edible cafe develops and sells edible insect confectionery, beverages, and desserts. Papillon's kitchen is also pursuing product development research by large companies such as edible insect pasta, salad, CJ, and Nongshim Co., Ltd. Hanmi Yanghan Co., Ltd. commercialized the powder after degreasing, and Insect Vision Co., Ltd. is launching a variety of products using edible insects such as gosoae guksu, gosoae seasoning, gosoae nurungji, and dog food.

However, consumers still have a feeling of rejection of edible insects, and there are potential risk factors such as odor, taste, microbial contamination, chemical contamination by heavy metals, and allergic symptoms. Accordingly, it is necessary to approach the consumer with familiar foods by reducing the aversion and prejudice of the consumer as a prerequisite for utilizing edible insects. At the same time, it is necessary to secure stability from deterioration by microorganisms, and to solve problems such as removal of heavy metals, odor, and delicacy.

In addition, in order to utilize edible insects in general food, it is necessary to secure safety through toxicological evaluation of edible insects, prepare for contamination and decay by microorganisms such as bacteria and viruses, and research on safe long-term storage distribution and storage methods.

With regard to long-term storage and distribution methods, physical methods such as heating, very high pressure, radioactive rays, PEF (Pulsed Electric Field), and optical pulses in conventional foods Chemical methods such as per-alcohol, chlorine, hydrogen peroxide, etc. are used, and some of them are nitrite, sorbic acid, sodium metabisulfite, and chlorine for convenience and cost. (organochlorine agent) and various synthetic preservatives are used. However, physical methods can weaken the properties of the edible insects themselves, and synthetic preservatives are constantly experiencing problems with regard to safety, such as accumulation in the body, and have a negative effect on the human body depending on the type and amount of substances used.

For this reason, in order to preserve edible insects for a long time, it is necessary to study how to use a natural antimicrobial substance with improved safety and improved shelf life as a preservative, or to manufacture edible insect food itself with such antimicrobial properties.

As one of the traditional natural antibacterial substances, there are nisin, e-polylysine, and natamycin derived from microorganisms, and there are antibacterial peptides produced by lactic acid bacteria, or bacteriocin. . These antimicrobial substances are safe in vivo, and when the antimicrobial substances are ingested into the human body, they are decomposed by proteolytic enzymes present in the digestive organs, so they do not affect beneficial bacteria in the intestine and have no residue, making them suitable for use as natural preservatives such as food. Do.

When the existing material is fermented using microorganisms, new efficacy different from the existing efficacy may be imparted, and a number of harmful ingredients may be changed into safe ingredients. By fermenting the natural product, the components contained in the natural product itself, activation of the natural product component by a converting enzyme, and the creation of new components by the fermentation strain can increase the function of the natural product. In addition, by reducing the molecular weight of nutrients by the action of a degrading enzyme, it is possible to increase absorption or decrease irritation and reduce allergic components.

Recently, research has been actively conducted to search for substances having antibacterial activity from natural materials and apply them to food, but lactic acid bacteria (LAB) separated by GRAS (Generally recognized as safe) have been applied to edible insects. There is no example of fermentation. Produces natural antimicrobial substances using bioconversion technology using lactic acid bacteria and applies them to edible insects to produce fermented products of edible insect extracts with improved antiseptics, antiseptic properties, and preservatives in storage and distribution. Nothing has been known about the fermentation product of the edible insect extract, which has improved the absorption rate and functionality in the body by decomposing amino acids, peptides, fats, and carbohydrates with its own degrading enzyme.

Korean Registered Patent No. 10-2018-0019838 (February 27, 2018) Korean Patent Publication No. 10-2003-0001659 (January 08, 2003)

The present invention relates to a fermented product of lactic acid bacteria of the white spotted flower plain solvent extract.

The present invention comprises the steps of preparing a solvent extract of white spotted flower radish, the step of inoculating and fermenting the lactic acid bacteria in the solvent extract, a method of manufacturing a fermented product of white spotted flower radish solvent extract and white spotted flower plain fabricated therefrom A fermentation product of a solvent extract is provided.

In addition, the present invention provides an antimicrobial composition comprising a lactic acid bacteria fermentation product of the white spotted flower solid solvent extract.

In addition, the present invention provides a composition for decomposing any one of nutrients selected from proteins, fats, carbohydrates, and cellulose, including fermented lactic acid bacteria of white spotted radish solvent extract.

In addition, the present invention provides an antioxidant composition comprising a lactic acid bacteria fermentation product of a white spotted flower radish solvent extract.

In addition, the present invention provides a heavy metal antidote composition comprising a lactic acid bacteria fermentation product of a white spotted flower radish solvent extract.

In addition, the present invention provides a cosmetic composition having one or more uses in the group consisting of whitening, antioxidant, acne prevention and treatment, and dandruff prevention and treatment, including lactic acid bacteria fermentation products of the white spotted radish solvent extract.

The present invention relates to a lactic acid bacteria fermentation product of a white spotted flower radish solvent extract, a method for manufacturing the same, and a use of the lactic acid bacteria fermentation product.

Specifically, the present invention comprises the steps of preparing a solvent extract of the white spotted flower radish, and inoculating and fermenting lactic acid bacteria in the fermentation raw material containing the solvent extract, a method of manufacturing a fermentation product of the white spotted flower solid solvent extract Gives

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to a fermentation product of lactic acid bacteria fermented using a solvent extract of white spotted flower radish as a lactic acid bacteria and a method of manufacturing the fermentation product.

The lactic acid bacteria fermentation product of the solvent extract of the white-spotted flower radish is obtained by inoculating the solvent extract of the white-spotted flower radish and incubating or fermenting it.

The white-spotted flower radish for obtaining the extract of the white-spotted flower radish or the lactic acid bacteria fermentation product of the extract means the larva of the white-spotted flower radish ( Protaetia brevitarsis ), preferably the third- star larvae before forming the pupa It may mean.

The white spotted flower larvae can be fasted for 1 day to 5 days or 3 days to 5 days before powdering. By going through the fasting step, foreign substances in the gut of the white spotted flower larvae can be removed. If the fasting period is less than 1 day, residual foreign matter in the intestine may not be sufficiently removed, and if the fasting is performed for more than 5 days, the larvae may die or malnutrition.

In one embodiment, the white spotted flower larvae are bred for 35 days and then washed in flowing water after 3 days of fasting, for example, 12 to 60 hours at a temperature of 55 to 65°C, for example, 60°C, for example For example, it is dried by hot air for 48 hours and treated with pesticide to be used as an extraction raw material.

The white-spotted flower larvae prepared as the extraction raw material may further undergo a separate sterilization process to remove microorganisms present in the larvae. By passing through the sterilization treatment process, harmful bacteria in the larva can be removed to secure safety as a food or medicine, and a sterilization method can be freely selected and used within the scope for achieving the above object.

For the production of the lactic acid bacteria fermentation product, the solvent extract of the white spotted flower radish is prepared by extracting the white spotted flower larvae, its dried product or pulverized product with an extraction solvent. The white-spotted flower radish used for the preparation of the solvent extract may be a powder powdered with a grinder after drying the white-spotted flower larvae by hot air drying, microwave, freeze drying, or the like. At this time, there is no limitation on the particle size of the powder, and it may be a powder produced by treating a general grinder or a food mixer to prevent strong heat for 1 minute. Alternatively, after freezing the white spotted flower larvae, a powder ground with a buffer may be used. The prepared powder may be used directly for extract preparation, or white spotted flower solid powder stored for 6 months to 1 year at -20°C to -70°C may be used. Alternatively, the white spotted flower larvae may be stored frozen at -70°C without drying, and then watered and crushed living body may be used.

The extraction solvent may be one or more selected from the group consisting of water, alcohols having 1 (C1) to 3 (C3), and water. For example, it may be water, ethanol (EtOH) or an aqueous ethanol solution. The solvent extraction method may be a conventional extraction method, for example, extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, hot water extraction, or the like may be used. Preferably, a hot water extraction method can be used. The solvent extraction time may be 4 to 6 hours, or 5 to 6 hours.

The extraction temperature at the time of the solvent extraction is 10 ℃ to 90 ℃, 20 ℃? To 80°C and 30°C? To 70°C, or 30°C to 60°C. For example, when the solvent is water, the extraction temperature may be 40 to 80°C, 50 to 70°C, 55 to 65°C, for example 60°C, and when the solvent is alcohol (EtOH), the extraction temperature is 10°C to 50℃, 20℃? To 40°C, 25°C? To 35°C, for example, 30°C.

As a specific example, the extraction solvent may be performed at an extraction temperature of 30°C to 80°C using water, or at an extraction temperature of 9°C to 50°C using an aqueous alcohol solution. The alcohol aqueous solution may have an alcohol content of 70 to 100%. For example, the white-spotted radish solvent extract may be a white-spotted radish hot water extract (Hotwater Extract of Protaetia brevitarsis, HePb), or an Ethol Extract of Protaetia brevitarsis (EePb).

The white-spotted radish hot water extract is mixed with 100 g of white-spotted radish larvae powder ( Protaetia brevitarsis Powder, Pb) in a ratio of 1:9 in water at a ratio of 1:9, followed by ultrasonic and/or vortex chamber, etc. Mixing with, shaking culture at a temperature of 60 ℃ for 4 to 6 hours to obtain an extract, centrifuged and then vacuum filtered (vaccum filter) about 700 ~ 800 ml (70 ~ 80%) of white spotted flower radish hot water extract ( Hotwater Extract of Protaetia brevitarsis, HePb). The white-spotted flower radish hot water extract prepared by the above-described manufacturing method of the present invention may be used as it is in a liquid state, or a powder of the white-spotted flower radish extract obtained by freeze-drying and powdering it may be used.

The white spotted flower solid alcohol extract may be extracted using alcohol (70-100% EtOH). Mix 100 g of white spotted flower larvae larvae powder (Pb) at a ratio of 900 mL (w/v) 1:9, then mix using an ultrasonic or vortex chamber, shake and incubate for 4 to 6 hours at a temperature of 30°C. After obtaining and centrifuging, it may be about 700 to 800 ml (70 to 80%) of white spotted flower extract (Ethanol Extract of Protaetia brevitarsis, EePb) obtained by vacuum filtration. And after volatilizing the alcohol of this white-spotted ignorant alcoholic extract (EePb), washing with water and adding 800 mL of water to the obtained concentrate can be used as a final concentrate of the white-spotted ignorant alcoholic extract.

The lactic acid bacteria fermentation product of the solvent extract of the white spotted flower radish according to the present invention may be used as a raw material for fermentation of the white spotted flower plain solvent solvent, or may be used as a fermentation raw material by processing a post-treatment process such as centrifugation or filtration. . In order to utilize the effective substance of the white spotted flower plain fabric, the whole white spotted flower plain fabric solvent extract for reducing quantitative loss may be used, or only the supernatant of the white spotted flower plain fabric hot water extract may be used.

By preparing the white spotted flower solid solvent extract, it is possible to secure the physiological activity and physical properties of the extract, which were not obtained from the existing white spotted flower solid powder, but also remove the microorganisms contained in the edible insect powder, and then remove it. When used in food or cosmetic compositions, there is an advantage not to affect the physical properties.

Specifically, the white spotted radish solvent extract may be used (i) hot water or alcoholic extract from which some white spotted radish powder is partially removed by centrifugation and filtration, or (ii) the white spotted radish powder is not removed. It may be an extract precipitate.

The fermentation product of the white spotted flower solid solvent extract of the present invention may be prepared by using the white spotted flower solid solvent extract itself or a composition comprising the white spotted flower solid solvent extract as a fermentation raw material.

The additive may be one or more additives selected from the group consisting of a carbon source and a nitrogen source, and preferably may include a carbon source.

The carbon source is not particularly limited, but may be one or more selected from the group consisting of dextrose, glucose, sucluse, lactose, and galactose. It can be dextrose. The amount of the carbon source added is 3 to 20 mass%, 5 to 20 mass%, 7 to 20 mass%, 3 to 15 mass%, 5 to 15 mass%, 7 to 15 mass%, or 7 to the mass of the white spotted radish solvent extract mass. To 10% by mass.

In one embodiment of the present invention, as a result of adding 8% by mass of dextrose to the white spotted radish solvent extract as a fermentation raw material, the growth of lactic acid bacteria was significantly increased compared to the case of using a fermentation raw material without dextrose added. .

Specifically, in the case of Lactobacillus plantarum, when the white spotted flower solid solvent extract alone was cultured without the addition of a carbon source, the LAB Count of 7.0x10 ³ log cfu/ml was shown even after 24 hours of cultivation, showing poor growth, but dextrose When cultivated for 24 hours in a white spotted radish hot water extract with 8% by mass added, it shows LAB count of 2.7x10 ¹¹ log cfu/ml, 6.26x10 ⁹ log when cultured for 24 hours in MRS medium, a general lactic acid bacteria culture medium. It showed about 40 times better than cfu/ml and about 3x10 ⁷ times better proliferation of lactic acid bacteria than dextrose-free white spotted radish solvent extract.

Even in the case of the Weisella paramethenoid KCCM12300P, white spotted flower radish without dextrose showed a proliferation of 3.21x10 ² log cfu/ml level despite 24 hours of incubation, but dextrose-added white spotted flower solid In the hot water extract, the activity of lactic acid bacteria proliferation increased significantly to the level of 5.7x10 ⁹ log cfu/ml, and this value is superior to the activity of 3.0x10 ⁸ log clu/ml of MRS medium.

In the case of the Weisella paramethenoid KCCM12301P strain, LAB Count was 3.21x10 ² log cfu, respectively. /ml and 3.17x10 ⁸ log cfu/ml.

The nitrogen source may be beef extract, peptone, tryptone, skim milk, or the like. In the case of the solvent extract of white spotted flower plain lactic acid bacteria can be cultured without adding a nitrogen source. In addition, sodium acetate, dipotassium sulfate, magnesium sulfate, ammonium citrate, etc. may be added as inorganic salts, but lactic acid bacteria can be cultured without it.

Lactobacillus culture medium is a common medium known in the industry as de Man Rogasa and Sharpe (MRS) medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Mediu (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10 , F-12, α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium. In the present invention, lactic acid bacteria were cultured as a nitrogen source, a carbon source, and a trace element possessed by white spotted flowers. In this case, when carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose are used as a carbon source to help cultivation, lactic acid bacteria can be cultured smoothly.

The lactic acid bacteria fermentation product of the white-spotted flower radish extract according to the present invention can be prepared by inoculating the white-spotted flower-purple solvent extract with lactic acid bacteria. In the process of inoculating and fermenting the lactic acid bacteria, air bubbles may be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. In addition, oxygen or coral-containing gas (air) may be injected to maintain the aerobic state.

Fermentation of lactic acid bacteria in the present invention means that the raw material is transformed or modified by physical and chemical methods, and decomposition process to obtain a desired product in hours or months, which may take years in a natural state. In particular, the fermentation using lactic acid bacteria in the present invention means to selectively decompose and cut the ingredients or medicinal properties of raw materials, and to produce functional and effective substances of lactic acid bacteria using ingredients of raw materials. Through this process, lactic acid bacteria decompose and break the active ingredient into small molecules from the proteins, carbohydrates, fats and minerals contained in white spotted radish to create metabolites. This is called'fermentation of white spotted radish extract'. It is called.

Specifically, in the present invention, the term'fermentation product of white spotted flower radish extract' means (')'fermentation solution' (fermented lactic acid bacteria supernatant) or fermentation of lactic acid bacteria with lactic acid bacteria fermentation of the white spotted flower radish solvent extract or (ii) removal of cells after fermentation of lactic acid bacteria 'Filtrate' (some lactic acid bacteria present only in the supernatant), or (ⅲ)'extracted and precipitated fermentation product' (white spotted flower solid powder, lactic acid bacteria, supernatant) containing the edible insect powder remaining after culture extraction, all of which Contains active ingredients included. These are collectively referred to as fermentation products of white-spotted ignorant solvent extracts.

The lactic acid bacteria fermentation product of the white spotted flower radish provided by the present invention may have one or more of the following characteristics:

i) Superior storage stability compared to unfermented products of white spotted flower radish extract

ii) Antibacterial activity against one or more bacteria selected from the group consisting of Listeria, Staphylococcus aureus, E. coli, Bacillus, Vibrio, Fluorescent bacteria, Acne and Dandruff

iii) decomposition activity against one or more nutrients selected from the group consisting of carbohydrates, proteins, fats and cellulose

iv) Higher fat rancidence inhibitory activity than unfermented white spotted flower radish extract

v) Long-term maintenance of cell safety

vi) 0.5 to 95 parts by weight of heavy metal content based on 100 parts by weight of heavy metal content in white spotted flower solid powder or white spotted flower solid extract and

vii) High anti-melanin activity compared to unfermented white-spotted flower radish extract.

In the lactic acid bacteria fermentation product of the white spotted flower radish solvent extract provided by the present invention and a method for manufacturing the same, the lactic acid bacteria can be used without limitation within the scope for achieving the object of the present invention, and Lactobacillus planter room ( Lactobacillus plantarum , ATCC 10241, NCTC7220), Lactococcus lactics , ATCC 11454, NCIC 8586, Lactobacillus sakei , ATCC 31063, ATCC 15521, Lactobacillus fermentum, ATCC14931, NCDO 1750), Lactobacillus ramno suspension (Lactobacillus rhamnosus, ATCC7469, KCTC3326), Lactobacillus bar pick Russ brevis (Lactobacillus brevis, ATCC15521), Lactobacillus gras mini bus (Lactobacillus graminis, ATACC 51150), Lactobacillus dextrin kusu (Lactobacillus dextrinicus, ATCC 33087), Lactococcus garvieae (ATCC11454, LMG 8162), Enterococcus faecium (ATCC 19634), Enterococcus faecalis (ATCC6057), Leuconostoc citreum , ATCC 49370), Weissella hellenica ( ATCC 51523), Weissella paramesenteroides (ATCC 33313), Pediococcus acidilactici (ATCC33314), Pediococcus pentosace Pediococcus pentosaceus (ATCC 33316), Bifidobacterium from the group consisting of animalis, ATCC 27672) and Bifidobacterium Infante tooth (Bifidobacterium infantis, ATCC15697) may be at least one member selected. The above bacteria can be used by pre-sale from the Korea Research Institute of Bioscience and Biotechnology's Biological Resource Center or the American Type Culture Collection (ATCC). Preferably, one or more lactic acid bacteria strains selected from the group consisting of Lactobacillus planter room (KCCM 12299P), Weisela paramethenoid KCCM 12300P, and Weisella paramethenroid KCCM 12301P may be used.

The Lactobacillus plantarum strain may be a strain comprising a 16S rRNA gene having a sequence identity of 99% or more, 99.5% or more, or 99.9% or more with the base sequence of SEQ ID NO: 3, preferably a sequence It may be a strain comprising a 16S rRNA sequence consisting of the nucleotide sequence of No. 3, more preferably, KCCM12299P strain deposited with the Korea Microbial Conservation Center (KCCM) as of August 10, 2018.

The Weisella paramethenoidal strain may be a strain comprising a 16S rRNA gene base sequence having a sequence homology of 99% or more, 99.5% or 99.9% or more with the base sequence of SEQ ID NO: 4 or SEQ ID NO: 5, and SEQ ID NO: 4 or may be a strain comprising a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 5, preferably the same organization as the accession number KCCM12300P strain or the same as deposited on the Korea Microbiological Conservation Center (KCCM) as of August 10, 2018 It may be a strain deposited with accession number KCCM12301P.

The Lactobacillus plantarum and Weisella paramethenoid strains can be cultured at 30 to 37°C using MRS medium. The Lactobacillus planter room and Weisella paramethenoid strain are 25 to 40°C temperature using a white spotted flower radish extract, a white spotted flower radish hot water extract or a white spotted flower solid alcohol extract as a medium to which a carbon source or a carbon source and a nitrogen source are added. Can be cultured in range.

In the preparation of the extract fermented according to the present invention, the inoculation amount of the lactic acid bacteria may be 1x10 ⁸ to 1x10 ¹⁵ cfu/ml, for example, 1x10 ⁸ to 1x10 ¹² cfu/ml, 1x10 ⁸ to 1x10 ¹⁰ cfu/ml.

When lactic acid bacteria are inoculated to the white spotted flower radish extract and fermented, the fermentation temperature is preferably performed at 25 to 40°C, preferably 30 to 37°C, and the fermentation time is less than 12 hours to 48 hours, and 12 hours to 36 hours. Fermentation for an hour, 12 to 30 hours, or 12 to 24 hours, such as 24 hours. When the temperature and the incubation time are outside the above range, the number of live bacteria of the lactic acid bacteria to the extent that the lactic acid bacteria can maintain the function is not maintained, and there is a fear of the occurrence of dead cells, and it is preferable to ferment culture under the above conditions.

After inoculating lactic acid bacteria in the white spotted flower radish extract of the present invention and ending the fermentation culture step, to separate the culture from the cells or the culture and the precipitate (including the cells), further centrifugation and filtration ( filteration) process. These steps can be appropriately selected and performed according to the needs of the food, cosmetics, feed, and functional fields. Removal of the cells can be carried out through a conventional method for removing cells.

According to an example of the present invention, the step of storing the fermentation product of the white spotted flower radish solvent extract prepared by the above manufacturing method at a low storage temperature for 1 to 400 days may be further included.

According to an example of the present invention, the step of storing the fermentation product of the white spotted flower radish solvent extract prepared by the method of the present invention at a low storage temperature for 1 to 400 days may be further included. The storage temperature of the low temperature may be -70 ℃ to 10 ℃.

Specifically, the fermentation product of the white spotted flower radish solvent extract stored may be in a liquid or powder form, and the storage temperature at the low temperature is 1°C to 10°C, for example, 2°C to 8°C, 3°C? To 7°C and 3°C? To 6°C and 4°C? To 5°C. When stored in the temperature range, the number of lactic acid bacteria contained in the fermentation product of the white-spotted radish solvent extract may be sufficiently maintained to exhibit functionality. When the temperature exceeds the above range, for example, when the temperature is 30°C, the number of lactic acid bacteria rapidly decreases, and the functionality of the white spotted flower radish solvent extract may deteriorate. When storing at the storage temperature of 1°C to 10°C, the storage date of the extract fermentation is preferably 1 day to 90 days, 10 days to 90 days, 20 days to 90 days, or 30 days to 90 days for distribution and storage. When the distribution at the storage temperature and the storage date are exceeded, deterioration of the antibacterial activity, enzyme function, antioxidant function, etc. of the extract fermentation may occur, so it is appropriate to store within the temperature condition and storage date range.

According to an example of the present invention, in the case of a powder of white spotted radish solvent extract, the low temperature storage temperature may be -70°C to -10°C, for example, -70°C to -20°C. In the temperature condition, when the lactic acid bacteria fermentation product of the white spotted flower radish solvent extract of the present invention is stored, it can be stored for 1 day to 400 days, for example, 1 year (365 days), or 360 days. Functional and antioxidant functional properties are maintained.

The fermented lactic acid bacteria fermented by using the solvent extract of the white spotted flower radish according to the present invention as lactic acid bacteria itself, purified by further separation and purification processes, or freeze-dried (frezze dry, lyophilization) to food, cosmetics, feed , Can be used as a functional material.

The fermented product of the white spotted flower radish extract of the present invention has a characteristic that harmful heavy metal components are reduced compared to the white spotted flower solid powder or white spotted flower solid extract without fermentation. Therefore, human body stability may be increased.

The heavy metal may be at least one heavy metal selected from the group consisting of cadmium, mercury, arsenic, selenium, and chromium, preferably cadmium, mercury, and arsenic. It may be one or more selected from the group consisting of.

The fermentation product of the white spotted flower radish extract of the present invention may have a heavy metal content of 0.5 to 95 parts by weight or 1 to 90 parts by weight based on 100 parts by weight of the heavy metal content in the white spotted flower radish powder or white spotted flower radish extract.

Specifically, the content of heavy metals in the fermentation product of the white spotted flower radish extract is 0.5 to 50 parts by weight, 0.5 to 40 parts by weight, 1 to 50 parts by weight, and 1 to 40 parts by weight based on 100 parts by weight of the heavy metal content in the white pointed flower radish powder Parts, or 1.5 to 35 parts by weight.

Specifically, the content of heavy metals in the fermentation product of the white spotted flower radish extract is 50 to 95 parts by weight, 50 to 90 parts by weight, 60 to 95 parts by weight, 60 to 90 parts by weight based on 100 parts by weight of the heavy metal content in the powder of the white spotted flower radish extract Parts by weight, or 65 to 90 parts by weight.

The fermentation product of the white spotted flower radish solvent extract of the present invention can be utilized as a safer edible insect food, cosmetic composition, etc. by effectively reducing the heavy metal contained in the white spotted flower plain powder.

In one embodiment of the present invention, as a result of performing component analysis using a white spotted flower solid powder solution, a white spotted flower solid extract powder solution or a white spotted flower solid extract extract fermentation powder solution, respectively, using an inductively coupled plasma luminescence spectrometer, fermentation The concentrations of cadmium, arsenic, and mercury in water are reduced, and the amounts of selenium and chromium as active ingredients are maintained, and harmful heavy metal components are removed while maintaining the content of active ingredients.

An example of the present invention provides an antimicrobial composition comprising a fermentation product of a white spotted flower radish solvent extract as an active ingredient.

The fermentation product of the white spotted flower radish solvent extract of the present invention is characterized by having antibacterial activity against various bacteria such as Gram-positive bacteria and Gram-negative bacteria. For example, Bicillus subtilis (ATCC 21770), Staphylococcus aureus ( ATCC 29213), Bacillus megaterium (ATCC 14581) as Gram-positive bacteria, Listeria monocytogenes (ATCC 15313) as Gram-negative bacteria, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa , Salmonella enterica (ATCC 9184), Vibrio Parahaemolyticus (ATCC 17802), and the like, may have antibacterial activity against various bacteria.

White spotted flower fermentation of solid solvent extraction Listeria (Listeria), Staphylococcus aureus, according to the present invention (Staphylococcus aureus), Escherichia coli ((Escherichia coli)), Bacillus (Bacillus), Vibrio (Vibrio), Pseudomonas bacterium (Pseudomonas), It is antimicrobial to one or more bacteria selected from the group consisting of yeast, fungi, propionibacterium acnes , and dandruff ( Malassezia furfur ).

In one embodiment of the invention, Listeria ( Listeria monocytogenes, L. monocytogenes , ATCC 15313), Staphylococcus aureus, S. aureus , ATCC 12600, Escherichia coli, E. coli , ATCC 11775, Bacillus megaterium, B. megaterium , ATCC 14579, Vibrio parahaemolyticus, V. parahaemolyticus , ATCC 17802), and fluorescent paper ( Pseudomonas flurorescens, P. flurorescens , ATCC 13525), the antibacterial effect through the size of the inhibition clear zone generated by placing the paper disc with the fermentation of the white spotted flower radish extract As a result of the test, a ring having a diameter similar to that of the control group treated with the lactic acid bacteria-only strain was generated in the fermented product (HePbLp/Wp1) fermented by inoculating both Lactobacillus planterum and Weisella paramesenteroid strains (FIG. 3 ). Through the inhibition ring test results, it was confirmed that the fermentation product of the white spotted flower radish extract provided by the present invention has antibacterial activity against Listeria, Staphylococcus aureus, Escherichia coli, Bacillus, Vibrio, and fluorescent bacteria.

In one embodiment of the present invention, the antibacterial activity against acne bacteria (propionibacterium acnes), and dandruff bacteria (Melassezia puffer) was confirmed through an inhibition ring formation experiment. In the case of the white-spotted flower radish hot water extract that has not been subjected to a fermentation process, an inhibitory ring was not formed, but all fermentations that were fermented using Lactobacillus plantarum or Weissella paramethenoid formed an inhibitory ring (FIG. 4), antibacterial activity against dandruff and acne bacteria was confirmed in the fermentation of white spotted flower radish extract. Therefore, the fermentation product of the white spotted flower radish extract provided by the present ㅂwing can be used for prevention, treatment or improvement of acne and/or prevention, treatment or improvement of dandruff.

Therefore, the fermentation product of the white spotted flower radish solvent extract of the present invention can be utilized as a natural preservative composition or the like due to its excellent antibacterial properties. Therefore, the quality is maintained longer at the same temperature and does not decay compared to the white spotted flower plain powder, so it can be more widely used in various industrial fields such as food, medicine, and cosmetics.

In addition, it was confirmed that the fermentation product of the white-spotted radish solvent extract of the present invention has a significantly lower malodialdehyde (MDA) value compared to the white-spotted radish powder, which can inhibit the acidity of the white-spotted radish. More specifically, the fermentation product of the white spotted radish solvent extract of the present invention may have an MDA production amount of 0.5 to 1.0 times, or 0.7 to 0.95 times compared to the white spotted radish powder. That is, the fermentation product of the white spotted radish solvent extract of the present invention has less lipid rancidity than the white spotted radish powder, and after being stored for a long time, the quality is relatively long and can be utilized in various fields.

An example of the present invention provides a composition for decomposing any one or more nutrients selected from the group consisting of proteins, fats, carbohydrates, and cellulose, which contains a fermentation product of a white spotted radish solvent extract as an active ingredient.

According to one embodiment, the fermentation product of the white spotted flower radish solvent extract of the present invention contains the functionality of the lactic acid bacteria inoculated in the fermentation step, a protease capable of decomposing protein, a lipase capable of decomposing fat, and a carbohydrate It can synthesize and secrete both amylase capable of decomposing and cellulase capable of decomposing cellulose. Therefore, when the fermented product of the white spotted flower radish solvent extract of the present invention is used for food, etc., the nutrients of food can be decomposed into small molecules to improve the absorption efficiency of nutrients into the body.

An example of the present invention provides a composition for antioxidant, which contains the fermentation product of the white spotted flower radish solvent extract as an active ingredient.

According to one embodiment, in the DPPH (1,1-dipheyl-2-picrylhydrazyl) analysis, the fermented product of the white spotted radish solvent extract of the present invention is a conventional preservative BHA (Buylated hydroxyanisole), BHT (bytylated hydroxytoluene), vitamin Compared to C, it was confirmed to have an antioxidant function, and it was confirmed that the ROS of the cell was lost, thereby inhibiting aging. Therefore, it can be used as a pharmaceutical composition for preventing or treating various diseases related to oxidative stress, as a material for health functional food and cosmetic compositions for preventing or improving various diseases related to oxidative stress.

Various diseases related to the oxidative stress include obesity, hypertension, diabetes, arteriosclerosis, liver disease, lung disease, hyperthyroidism, hypothyroidism, chronic fatigue syndrome, heart disease, hyperlipidemia, paralysis, kidney disease, gout, cancer, aging And neurodegenerative diseases.

Another example of the present invention provides a cosmetic composition comprising a fermentation product of a white spotted flower solid solvent extract as an active ingredient.

The cosmetic composition may have one or more uses in the group consisting of whitening, antioxidant, acne prevention and improvement, and dandruff prevention and improvement.

In one embodiment of the present invention, it was confirmed that it exhibits an excellent melanin synthesis inhibitory effect in the fermented product cells of the white spotted radish solvent extract of the present invention compared to the comparative example arbutin. When the melanin content of the untreated cells not treated with any sample is 100%, the melanin content of the white spotted flower radish solvent extract of the present invention is only 60 to 75% when treated with cells, and remarkably It has an excellent whitening effect.

In one embodiment of the present invention, the fermentation product of the white spotted flower radish solvent extract of the present invention has a DPPH inhibition rate of about 30-50% based on 100% of the DPPH inhibition rate of Vit, and the DPPH inhibition rate is maintained even when the storage date is prolonged. As a result, it was confirmed that it was slightly elevated, and the antioxidant efficacy was maintained even after long-term storage. Therefore, the fermentation product of the white spotted flower radish solvent extract of the present invention has excellent storage stability and can be used as a cosmetic composition for antioxidant, anti-aging skin, and promoting skin regeneration.

In another embodiment of the present invention, it was confirmed that the fermentation product of the white spotted flower radish solvent extract of the present invention has excellent antibacterial effects of acne bacterial (Propionibacterium acnes) and dandruff (Malassezia furfur), preventing or treating acne, It may be used as a cleaning composition for preventing or treating dandruff, a cosmetic composition, and a pharmaceutical composition.

The composition includes ingredients commonly added to cosmetic compositions, for example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, Cosmetics or dermatology such as surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives It can be molded into specific formulations, including adjuvants or carriers commonly used in the art.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, packs, soaps, surfactants- It may be formulated as containing cleansing, oil and spray, but is not limited thereto. More specifically, flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, pact, lip gloss It can be prepared in formulations such as rose, lipstick, shadow, shampoo and rinse.

In a preferred embodiment of the present invention, the fermentation product of the white spotted flower radish solvent extract of the present invention can be used as a food composition.

The fermentation product of the white spotted flower radish solvent extract of the present invention may be included in 10 to 100% by weight in the food composition. When the fermentation product of the white spotted flower radish solvent extract of the present invention is included in the food composition, it has stable antimicrobial properties against microorganisms, and antiseptic and antioxidant effects can be expected. Therefore, even if the storage date of food is long, it is possible to maintain equal function and quality for a long time.

Applicable foods are cookies, confectionery, energy bars, bakery, noodles, pudding, chocolate, candy, jelly, beverages, etc., and can be manufactured and commercialized in any of these formulations to achieve the object of the present invention. It is not limited to.

The present invention also provides a preservative composition comprising a fermentation product of a white spotted flower radish solvent extract.

The fermentation product of the white spotted flower radish solvent extract of the present invention can be utilized as a natural preservative. The fermentation product of the white spotted flower radish solvent extract of the present invention has an increased antiseptic effect and antioxidant effect compared to the existing white spotted flower radish powder, and suppresses the growth of microorganisms that occur during general storage, storage, and distribution, thereby preventing product changes. It can be used as a natural preservative in place of sodium nitrite, calcium propionate, sodium propionate, etc., which are used for prevention purposes.

The lactic acid bacteria fermentation product of the white spotted flower radish extract of the present invention has antibacterial properties and can be used as a natural preservative, antioxidant composition, composition for inhibiting lipid oxidation, whitening cosmetic composition, acne prevention and treatment, dandruff prevention and treatment cosmetic composition It can be used as a variety of foods, pharmaceutical compositions, and the like.

1 is a graph showing the results of LAB Counts measured after culturing L. plantarum and two types of W. paramesenteroides lactic acid bacteria in white spotted flower radish hot water extract or MRS medium according to Example 4 of the present invention.
FIG. 2 shows whether each harmful bacteria is detected after storing each fermented product of white-spotted flower radish powder, white-spotted flower radish hot water extract, and white-spotted flower radish hot water extract according to Example 6 of the present invention at 4°C for 6 months. It is a figure showing the result of measurement.
FIG. 3 is a photograph showing the results of antimicrobial analysis for each harmful bacteria of each fermentation product of a white-spotted flower radish hot water extract and a white-spotted flower radish hot water extract according to Example 7-1 of the present invention.
4 is a diagram showing the results of antibacterial analysis of acne and dandruff bacteria for each fermentation product of a white-spotted flower radish hot water extract and a white-spotted flower radish hot water extract according to Example 7-2 of the present invention.
5 is a graph showing the results of analysis of enzyme activity of each fermentation product of a white-spotted flower radish hot water extract and a white-spotted flower radish hot water extract according to Example 8 of the present invention.
6 is, according to Example 9 of the present invention, the white spotted flower radish hot water extract, and white spotted flower radish hot water extract maintains the optimal storage temperature of the number of lactic acid bacteria that can maintain the functionality of the lactic acid bacteria, It is a graph showing the experimental results to confirm.
FIG. 7 shows the antioxidant activity analysis results of each fermentation product of the white-spotted flower radish hot water extract, and the white-spotted flower radish hot water extract according to Example 10 of the present invention.
Figure 8, according to Example 11 of the present invention, the white spotted flower solid powder, white spotted flower plain water hot water extract, and white spotted flower plain water hot water extract, each of the fermentation of the fermentation effect of TBA analysis results, to confirm the results It is the graph shown.
Figure 9, according to Example 12-1 of the present invention, white spotted flower solid powder, white spotted flower solid water hot water extract, and white spotted flower solid water hot water extract of each fermentation of human 3T3-L1 cells to perform a toxicity test It is a graph showing one result.
Figure 10, according to Example 12-2 of the present invention, the white spotted flower solid powder, white spotted flower solid water hot water extract, and white spotted flower solid water hot water extract of each fermentation of the mouse B16F10 cells were subjected to cytotoxicity experiments It is a graph showing the results.
11 is a graph showing the experimental results of evaluating the effect of reducing melanin pigment in each fermentation product of white spotted radish powder, white spotted radish hot water extract, and white spotted radish hot water extract according to Example 13 of the present invention.

The present invention will be described in more detail through the following examples. The examples described below are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Preparation of White Spotted Flower Plain Powder

The larvae of the three-pointed white-flowered ignorant were grown for 35 days and fasted for three days to clean the intestines of the white-spotted larvae. Next, the white spotted flower larvae were washed in running water, and then dried by hot air at 60° C. for 48 hours to treat them.

The dried white spotted flower larvae were prepared as powders using a food grinder. Specifically, in order to prevent heat generated by the use of a food grinder, the pulverization cycle was repeated 5 times, followed by crushing for 1 minute and rest for 3 minutes, to prepare white spotted flower solid powder (Pb).

Example 2. Preparation of white spotted flower plain solvent extract

2-1. White Spotted Flower Plain Hot Water Extract Manufacturing

A hot water extract was prepared using the white spotted radish powder (Pb) prepared in Example 1-1. 100 g of white spotted larvae larvae powder (Pb) and 900 ml of water were mixed at a ratio of 1: 9, and then ultrasonic waves were irradiated at 750 watts and 10 kHz at intervals of 10 seconds for 5 minutes to mix (Sonics Vibra-Cell, USA). . Next, after the mixture was incubated for 4 to 6 hours in 60°C hot water, the cultured extract was centrifuged (3,000 rpm, 10 minutes) and vacuum filtered using 0.45 um corning bottle-top (Sigma, USA). (vacuum filter) to prepare about 800~900ml (80~90%) of White Spotted Flower Plain Hotwater Extract (Hotwater Extract of Protaetia brevitarsis , HePb).

The liquid hot water extract was stored in -70°C and freeze-dried or immediately freeze-dried and powdered to be used in subsequent experiments.

2-2. Preparation of white-spotted flower solid alcohol extract

100 g of white spotted larvae larvae powder (Pb) and alcohol (100% (v/v) EtOH) prepared in Example 1-1 After mixing 900mL (w/v) in a volume ratio of 1: 9, 750 watts and 10 kHz ultrasonic waves were irradiated for 5 minutes at 10 second intervals and mixed (Sonics Vibra-Cell, USA). Next, the mixture was shake cultured at 30°C for 4 hours, and the cultured extract was centrifuged (3,000 rpm, 10 minutes), followed by vacuum filtration using 0.45um corning bottle-top (Sigma, USA). ) To obtain approximately 700-800 ml (70-80%) of alcoholic extract. Subsequently, the alcohol of the alcohol extract was evaporated for 1 hour at 50°C using a rotary evaporator, and then a concentrate of the alcohol extract was prepared. After washing the concentrate of the alcoholic extract 2 or 3 times with water, 800 mL of water was added to finally prepare a squirrel-flowered plain radish alcoholic extract.

Example 3. Separation and identification of lactic acid bacteria for fermentation

3-1. Isolation of strain

Lactobacillus plantarum ( L. plantarum , Lp) and weissella paramesenteroids ( W. paramesenteroides , Wp), which are lactic acid bacteria strains having functions such as antibacterial activity and enzyme activity, for the production of fermented products using effective strains for food and food ) Was used for separation and identification from deep seawater and fermented sawdust, respectively.

Lactobacillus planterum ( L. plantarum ) was isolated from deep ocean water. The deep sea water was taken from November to December 2015 in Taeha, Jeodong, and Hyeonpo, Ulleungdo, and seawater at a depth of 200m was collected from the surface and transported in a refrigerated state. The deep ocean water was spread on MRS agar medium to which 0.1% (v/v) BCP was added and cultured for 1 day at 37°C. Thereafter, the selection of yellow colonies formed by reaction with BCP was repeated to isolate a single species.

The Weisella paramethenoid strain was isolated and identified from fermented sawdust, which is the food of edible insects. The fermented sawdust for feeding of edible insects was purchased from Yeongcheon Insect Industry (Korea). The fermented sawdust stored at 25°C was added to PBS at a concentration of 1% (w/v) and vortexed to mix. The fermented sawdust mixture was smeared on MRS (Difco, USA) agar medium to which 0.1% (v/v) BCP (Bromocresol purple, Sigma, USA) was added, and cultured at 37°C for 1 day. The BCP is acidified by the lactic acid of lactic acid bacteria and functions to form yellow colonies. The process of selecting only the yellow colonies formed by the reaction with BCP and spreading it on MRS agar medium to re-select By repeating, a total of two species were separated.

The separated three strains were cultured in MRS liquid medium, mixed with 50% (v/v) glycerol to make the final glycerol concentration 25%, and then stored at -70°C.

3-2. Identification of strain

The strain isolated in Example 3-2 was identified using nucleotide sequence analysis of 16S rRNA. In order to identify three strains, first, genomic DNA of each strain was isolated using a mini genomic DNA extraction kit (Promega).

Using the isolated genomic DNA as a template, the following 27F and 1492R primer sets and AccPower PCR premix kit (Bioneer, Korea) were used to amplify the 16S rRNA gene regions of the two strains with TaKaRa PCR Thermal Cycler (Japan).

27F primer (SEQ ID NO: 1): 5'- AGAGTTTGATCMTGGCTCAG -3'

1492R primer (SEQ ID NO: 2): 5'- GGTTACCTTGTTACGACTT-3'

The PCR product was purified by PCR clean-up Gel extraction (Macherey Nagel, USA) to analyze the gene sequence by ABI3730 DNA analyzer (Applied Bioxyxtems, USA). The 16S rRNA sequence of the Lactobacillus plantarum strain isolated from the present invention is shown in SEQ ID NO: 3, and the 16S rRNA sequence of two types of Weisella paramethenoid strains are shown in SEQ ID NOs: 4 to 5.

16S rRNA sequence similarity analysis was performed using 16s rRNA sequence information of the isolated Lactobacillus plantarum strain and Weisella paramethenoid strain, and the results are shown in Table 1 below. As shown in Table 1 below, the lactic acid bacteria isolated from the deep sea water showed a sequence similarity of 99% with Lactobacillus planterroom as a result of 16S rRNA analysis, and the lactic acid bacteria isolated from the fermented sawdust had a sequence of 99% with Weisella paramethentheroid. Because of their similarity, they were named Lactobacillus planter room and Weissella paramethentroid, respectively.

number From lactic acid bacteria Isolate strain Proximity 16S rRNA sequence identity One Deep ocean water Lactobacillus plantarum DSW#2-4 (16S rRNA of SEQ ID NO: 3) Lactobacillus plantarum MK311261.1
(SEQ ID NO: 6) 99% 2 Fermented sawdust Weissella paramesenteroides D12
(16S rRNA of SEQ ID NO: 4) Weissella paramesenteroides AB598954.1
(SEQ ID NO: 7) 99% 3 Fermented sawdust Weissella paramesenteroides D20 (16S rRNA of SEQ ID NO: 5) Weissella paramesenteroides LC094432.1
(SEQ ID NO: 8) 99%

The isolated Lactobacillus plantarum strain was deposited with the Korea Microbial Conservation Center (KCCM) on August 10, 2018, and the accession number is KCCM12299P. The two types of Weisella paramethenoid strains were deposited with the Korea Microbial Conservation Center (KCCM) on August 10, 2018, respectively, and Weissella paramesenteroides D12 received accession numbers KCCM12300P and Weissella paramesenteroides D20, respectively, and received KCCM12301P.

Example 4. Confirmation of optimum conditions for fermentation of white spotted flower radish extract

In the preparation of the fermentation product of the white spotted flower radish extract, an experiment was conducted to confirm whether it is necessary to add the carbon source under the optimum conditions and the optimal incubation time to effectively effect the fermentation of lactic acid bacteria.

First, three types of media were prepared. The three medium is a white spotted flower radish hot water extract (HePb) 1L medium prepared in Example 2, HePb 1L and 2% (w/v) dextrose (D-glucose, Sigma, USA) 80mL mixed medium , And MRS medium which is a commonly used lactic acid bacteria medium. Three types of lactic acid bacteria L.plantarum ( KCCM12299P ), W.paramesenteroides (KCCM12300P), and W.paramesenteroides (KCCM12301P) were inoculated in 1 mL at a concentration of 1x10 ⁸ cfu/ml, respectively for 24 hours at 30°C or 48 hours at 30°C. Incubated for an hour. The optimal fermentation conditions were confirmed by measuring LAB Counts (log cfu/ml) after 24 hours and 48 hours, and the measurement results are shown in FIG. 1 and Table 2 below.

Culture conditions LAB Counts by culture time
(log cfu/ml) Culture medium Strain 24h culture 48h culture Hot water extract (HePb) L. plantarum KCCM12299P 8.0x10 ³ 6.10x10 ² W. paramesenteroides KCCM12300P 3.21x10 ² 1.01x10 W. paramesenteroides KCCM12301P 3.21x10 ² 2.01x10 HePb + dextrose L. plantarum KCCM12299P 2.7x10 ¹¹ 1.01x10 ⁹ W. paramesenteroides KCCM12300P 5.7x10 ⁹ 7.0x10 ⁸ W. paramesenteroides KCCM12301P 3.17x10 ⁸ 2.7x10 ⁷ MRS L. plantarum KCCM12299P 6.26x10 ⁹ 1.27x10 ⁷ W. paramesenteroides KCCM12300P 3.0x10 ⁸ 6.0x10 ⁷ W. paramesenteroides KCCM12301P 1.11x10 ⁹ 1.91x10 ²

The LAB Counts was diluted with 1% (v/v) of the culture in a solid medium prepared by adding agarose (BD Difco, USA) to each liquid medium, and 1 ml was applied to the LAB plate, and then 24 hours at 30°C. It was performed by measuring the number of colonies formed by culturing.

As can be seen in FIG. 1, when L.plantarum was inoculated into MRS medium, LAB Counts were measured as 6.26x10 ⁹ /ml(6.26E+09) after incubation for 24 hours, but 4.53x10 for W paramesenteroides KCCM12301P ^It was measured as ⁸ /ml (4.53E+08), and when cultured for 48 hours, it was found that the number of lactic acid bacteria amplification was greatly reduced. L. plantarum was inoculated into HePb medium not containing dextrose and the number of lactic acid bacteria in the culture cultured for 24 hours was only 8.0x10 ³ /ml (8.0E+03), and the deposit number was also obtained when W. paramesenteroides were inoculated. Both the KCCM12300P and KCCM12301P strains only proliferated to about 3.21x10 ² /ml (3.21E+02).

On the other hand, L. plantarum was inoculated into a medium containing dextrose and HePb, and the number of lactic acid bacteria in the culture cultured for 24 hours was 2.7x10 ^{11 /} ml (2.7E+ ¹¹ ), and proliferation occurred actively, and cultured for 48 hours. Later it appeared as 1.3x10 ¹⁰ (1.30E+10).

Therefore, it was confirmed that the medium containing dextrose and white spotted flower radish extract significantly increased the growth of lactic acid bacteria than the medium without dextrose. In addition, the number of lactic acid bacteria decreased after 48 hours of inoculation with L.plantarum or W. paramesenteroides in all medium conditions, and it was confirmed that fermentation culture at 30° C. for 24 hours after inoculation with lactic acid bacteria was appropriate.

Therefore, in the subsequent experiments, dextrose was added to the white spotted flower plain hot water extract to inoculate lactic acid bacteria and fermented and cultured at 30° C. for 24 hours to prepare a fermented product.

Example 5. Preparation of fermentation product of white spotted flower solid solvent extract

The white spotted flower radish prepared in Example 2 was mixed with dextrose in the hot water extract or the alcoholic extract to prepare a medium, and then inoculated and cultured with lactic acid bacteria to prepare a fermentation product of the white spotted flower radish extract.

80 ml of 2% (w/v) dextrose (D-glucose, Sigma, USA) in 1 L (pH6.5~7.5) of white spotted flower radish hot water extract prepared in Example 2 (about 8% of the weight of the hot water extract) To prepare a culture medium for lactic acid bacteria culture. The prepared medium was inoculated with 1 mL of lactic acid bacteria isolated and identified in 1x10 ⁸ cfu/ml Example 3-2, and incubated at 30° C. for 24 hours to prepare a fermented product of white spotted flower radish extract.

The prepared fermentation product was powdered and stored in the same manner as the solvent extract of white spotted flower radish, and then dissolved in water as needed in the experiment.

Table 3 below shows the types and deposit numbers of the inoculated lactic acid bacteria, and the fermentation product of the white spotted flower radish extract prepared by inoculating the lactic acid bacteria. Specifically, the control sample 1 is a white spotted flower solid powder (Pb), the control sample 2 is a white spotted flower solid heat water extract (HePb) powder, and the sample 1 is a fermentation powder of a white spotted flower solid water hot water extract inoculated with L. plantarum (HePbLp), Sample 2 is fermentation powder of white spotted radish extract inoculated with W. paramesenteroides KCCM12300P (HePbWp0), Sample 3 is fermentation powder of white spotted radish extract inoculated with W. paramesenteroides KCCM 12301P (HePbWp1) , And Sample 4 showed the fermentation powder (HePbLp/Wp1) of the white spotted flower radish extract inoculated with the mixed strains of L. plantarum and W. paramesenteroides .

division denomination Type of lactic acid bacteria Control Sample 1 Pb Non-fermentation Control Sample 2 HePb Non-fermentation Sample 1 HePbLp Lactobacillus planter room KCCM12299P ( Lactobacillus plantarum ) Sample 2 HePbWp0 Weissella paramesenteroides KCCM12300P Sample 3 HePbWp1 Weissella paramesenteroides KCCM12301P Sample 4 HePbLp/Wp1 Lactobacillus planter room and wayella paramethenoid KCCM12301P

Example 6. White Spotted Flower Plain Of extract Fermentation Microbial storage stability analysis

6-1. Confirmation of microbial storage stability of fermented white spotted flower radish extract

To confirm whether the fermentation product of the white spotted flower radish extract or the white spotted flower radish extract of the present invention inhibits the growth of harmful microorganisms and has storage stability against microorganisms, after storing each fermented product for a certain period of time, An experiment was conducted to check whether a specific food poisoning bacteria was present.

The control samples 1 and 2 and the samples 1 and 3 of Table 3 were stored at 4° C. for 6 months, respectively, and then diluted in water so as to be 0.01 g/ml to obtain a 1% (w/v) homogeneous solution sample. It was prepared. 1 mL of each sample prepared was Listeri , Staphylococcus aureus , S. aureus , yeast and fungus (Fungi), Escherichia coli ( E.coli ), petrifilmTM (3M, for measurement) USA) and incubated at 37° C. for 24 hours, it was confirmed whether Listeria, Staphylococcus aureus, yeast and fungus, and E. coli were detected, and the results are shown in FIG. 2.

As shown in Figure 2, HePbLp of Sample 1 and HePbWp1 of Sample 3 did not detect Listeria, Staphylococcus aureus, yeast and fungi, and E. coli even after storage at 4°C for 6 months, HePbLp of Sample 1, Sample 3 It was confirmed that HePbWp1 of inhibits the growth of these bacteria and has storage stability. On the other hand, Staphylococcus aureus was detected in Control Sample 2 (HePb), and Listeria, Staphylococcus aureus, yeast and fungus, and E. coli were detected in Control Sample 1 (Pb). Thus, compared to the white spotted flower solid powder or white spotted flower solid extract, it was confirmed that the white spotted flower solid extract of the present invention has excellent growth inhibitory ability against microorganisms and storage stability against microorganisms.

6-2. Measurement of the number of microorganisms according to the storage temperature of white spotted flower solid powder

An experiment was conducted to confirm the storage stability of white spotted flower solid powder (Pb) against microorganisms. The white spotted flower solid powder was stored at -20°C or 28°C for 6 months, and then diluted in water so as to be 0.01 g/ml to prepare a 1% (w/v) homogeneous solution sample. 1 mL of the sample is petrifilm ^TM (3M, USA) for measuring Listeria, Staphylococcus aureus, S. aureus, yeast and fungi, Escherichia coli, E.coli And incubated for 24 hours at 37° C., to confirm the number of colonies formed. Strains showing typical colonies were calculated by strain X dilution factor, and the results are shown in Table 4 below.

Storage temperature Listeria Staphylococcus aureus Coliform Yeast and mold Other -20℃ 0 0 0 0 2 28℃ 3 X 10 ⁶ 1.05 X 10 ⁸ ∞ 0 ∞

As shown in Table 4, in the case of white-spotted radish powder (Pb), microorganisms hardly proliferated at -20°C, but markedly at 28°C, such as Listeria, Staphylococcus aureus, E. coli, yeast and fungi, and other microorganisms Many were detected. Therefore, it was confirmed that the white spotted flower solid powder had poor storage stability against microorganisms at 28°C.

Example 7. Confirmation of antibacterial effect of fermentation of white-spotted flower radish extract

7-1. Antibacterial activity check

An experiment was conducted to confirm the antibacterial activity of the fermentation of the white-spotted flower radish hot water extract or the white-spotted flower radish extract of the present invention.

Listeria monocytogenes (L. monocytogenes , ATCC 15313), a test strain for confirming the antibacterial effect, Staphylococcus aureus, S. aureus , ATCC 12600, Escherichia coli, E. coli , ATCC 11775, Bacillus megaterium, B. megaterium , ATCC 14579, Vibrio parahaemolyticus, V. parahaemolyticus , ATCC 17802), and fluorescent bacteria ( Pseudomonas flurorescens, P. flurorescens , ATCC 13525) were prepared. Each test strain was inoculated into LB medium and pre-cultured at 37° C. for 24 hours. Subsequently, 20 ml of 1% (w/v) LB agar was dispensed into a Petri dish and solidified to prepare a plate medium, and then 200 μL of each of the previously cultured test strains was added and evenly sterilized on a medium with a sterile glass rod. After application, it was dried for 30 minutes. A paper disc (8 mm, Toyo Roshi Kaicha Ltd, Japan) was placed on the dried medium.

After diluting the powder of the control sample 2 (HePb) and sample 4 (HePbLp/Wp1) of Table 3 in water so as to be 0.01 g/ml, respectively, to prepare a 1% (w/v) homogeneous solution sample, each 1 30 μL of the% (w/v) homogeneous solution sample was injected into the previously prepared paper disc. After injection, the cells were cultured at 37° C. for 24 hours, and the diameter of the inhibition clear zone around the paper disc was measured to analyze the antibacterial properties. As a control, antibacterial activity was analyzed using a medium in which 30 μL of L. plantarum, or W. paramesenteroides KCCM12301P strains were injected into a medium containing test strains. The antibacterial analysis results are shown in FIG. 3.

As shown in Figure 3, when inoculated with the control group L. plantarum, or W. paramesenteroides KCCM12301P strain, a circular ring was generated because the test strain did not grow around the paper disk due to the antibacterial effect of the strain itself, For sample 4 (HePbLp/Wp1), a ring with a diameter similar to that of the control group was produced. Therefore, it was confirmed that the antibacterial effect of the fermented product provided by the present invention on the test strain was excellent. On the other hand, when the HePb sample as the control sample 2 was treated, almost no circular ring was generated around the paper disc.

Therefore, the hot water extract of the white spotted flower radish has almost no antibacterial activity, but in the case of the fermentation product of the extract of the white spotted flower radish, the antibacterial activity of the lactic acid bacteria itself is maintained and the listeria and yellow grapes are passed through the fermentation step after inoculation of lactic acid bacteria. It was confirmed that antimicrobial activity against cocci, Escherichia coli, Bacillus, Vibrio, and Pseudomonas was present.

7-2. Antibacterial activity against acne and dandruff bacteria

In the same experimental method as in Example 7-1, Sample 1 in Table 3 above (HePbLp) and sample 3 (HePbWp1), and the control sample 2 of the hot water extract (HePb) of acne bacteria ( Propionibacterium acnes , ATCC 6919), and dandruff ( Malassezia furfur, ATCC 14521) was confirmed antibacterial activity, the results 4 is shown.

As shown in FIG. 4, the control sample 2 (HePb) hardly killed acne and dandruff, and thus a round inhibitory ring was hardly generated around the paper disc, but HePbLp in sample 1 and HePbWp1 in sample 3 were controlled around the paper disc. A round ring of a diameter similar to that was produced.

Thus, in the case of the fermentation product of the extract of the white spotted flower radish of the present invention, it was confirmed that it has an excellent antibacterial effect against acne and dandruff by going through a fermentation step after inoculation of lactic acid bacteria. Thus, it was confirmed that the fermentation product of the white spotted flower radish extract of the present invention can be used as a material for improving or treating acne and dandruff.

Example 8. Enzyme activity analysis of fermentation product of white spotted flower radish extract

An experiment was conducted to confirm the decomposition ability of proteins, fats, carbohydrates, and cellulose of the fermented product of the white spotted flower radish extract or the white spotted flower radish extract of the present invention.

First, as a substrate for testing enzyme activity, 1% (w/v) skim milk (MB cell), tributyrin (MB cell), 0.2% (w/v) starch (starch, MB cell) in MRS medium ), 0.2% (w / v) methylcellulose (methylcellulose, Sigma) was added to each plate was prepared.

Next, after diluting the powder of the control sample 2 (HePb) or sample 4 ( HePbLp/Wp1) of Table 3 in water to be 0.01 g/ml, respectively, to prepare a 1% (w/v) homogeneous solution sample, 100 μl of each 1% (w/v) homogeneous solution sample was placed in a paper disc of the plate and incubated at 30° C. for 24 hours. As a control, L. plantarum , or W. paramesenteroides KCCM12301P strain was inoculated into the prepared substrate plate and cultured under the same conditions.

In each of the experimental plates, it was observed whether the skim milk, tribtiline, starch, and methyl cellulose decompose, and the results are shown in FIG. 5. The production of amylase and cellulase was observed by staining with 0.2% (w/v) Congo Red after culture. When the skim milk is decomposed, protease is formed, when tribtiline is decomposed, lipase is formed, when starch is decomposed, amylase is formed, and when cellulose is decomposed, cellulase is formed. Fig. 5 shows the results of measurement of enzyme production.

As can be seen in Figure 5, when inoculating the control groups, Lactobacillus (KCCM12299P), Weisella (KCCM12301P) strain itself, protease, lipase, amylase, cellulase enzymes were all generated, sample 4 of the present invention (HePbLp/ Wp1) also confirmed that all four enzymes were produced. However, in the control sample 2 (HePb), the substrate decomposition activity of the enzyme was not observed.

Therefore, it was confirmed that the fermentation product of the white spotted flower radish extract of the present invention includes protease, lipase, amylase, and cellulase, and can decompose proteins, carbohydrates, and fat components to lower the molecular weight. Therefore, the fermentation product of the white spotted flower radish extract of the present invention can improve the absorption efficiency of each component in the body by decomposing a large molecule into a small molecule.

Example 9. Confirmation of the number of live bacteria according to the storage temperature of the fermentation product of the white spotted flower radish extract

In the fermentation product of the white spotted flower radish extract prepared in Example 5 of the present invention, an experiment was conducted to confirm the optimal storage temperature, in which the number of lactic acid bacteria was maintained, to the extent that the functionality of lactic acid bacteria could be maintained.

The control samples 2 (HePb), sample 1 ( HePbLp), sample 3 (HePbWp1) , and sample 4 (HePbLp/Wp1) of Table 3 were stored at 4°C (refrigeration temperature) or 30°C for 30 days in total. As a control, the number of lactic acid bacteria in the fermentation product powder of the white spotted flower radish extract immediately after being prepared by the method of Example 5 was measured.

Next, the powders of the 1st, 7th, 15th, and 30th days were collected from the day stored at each temperature and diluted in water to 10 mg/ml to prepare a 0.1% (w/v) sample solution. The prepared 0.1% (w/v) sample was spread on MRS medium (including BCP), incubated at 37° C. for 24 hours, and the number of live cells (LAB counts) was measured, and the results are shown in Tables 5 and 6 below. Showed on.

As shown in FIG. 6, the lowest number of viable cells at 4° C. storage temperature was Sample 4 (HePb/Wp1) powder 1 day after storage, and the viable cell count was 1.93×10 ¹¹ (log cfu/ml). It was. The most viable cell count was Sample 4 (HePbLp/Wp1) powder 30 days after storage, and the viable cell count of 4.93 x 10 ¹¹ was measured. At 4°C, it was confirmed that the number of viable cells did not significantly decrease over the course of 30 days after storage, and the number of viable cells was maintained relatively similarly. When each sample powder was stored at 30°C, the number of viable cells decreased in all powders after 7 days of storage, and after 30 days, sample 1 (HePbLp) was 240, sample 3 (HePbWp1) was 0, and sample 4 In the case of (HePbLp/Wp1), the number of viable cells was significantly reduced to 260.

Therefore, in order to maintain the functionality of the lactic acid bacteria of the fermentation product of the white flower spotted flower radish extract of the present invention, a certain number of live bacteria should be maintained, and the fermentation product or powder thereof of the white spotted flower radish extract should be stored at a temperature of 4°C or lower. It was confirmed appropriate.

Example 10. Confirmation of antioxidant activity according to storage conditions

Free radical scavenging activity analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) to confirm the antioxidant effect according to the storage temperature and storage period of the fermented white spotted flower radish extract of the present invention.

Control sample 1 (Pb), control sample 2 (HePb), sample 1 in Table 3 (HePbLp), Sample 3 (HePbWp1), and sample 4 (HePbLp/Wp1) Each was stored at a storage temperature of 3°C (refrigeration temperature) or 30°C for 30 days.

Powders after 1 and 30 days from the day of storage at each temperature were collected and diluted in 1 mL of sterile water at a concentration of 10 mg/ml to prepare a 0.1% (w/v) sample solution. Stock solution (100 mM Tri-HCl, Ph 7.4) was prepared by dissolving DPPH in 1 mM methanol, and diluted to 100 μM concentration in analytical experiments.

Subsequently, 10 μL of each sample solution of 0.1% (w/v) concentration prepared prior to the 96-well plate was dispensed, and the change in antioxidant effect over time was measured by DPPH inhibition (%). As control, ascorbic acid (vit C) and BHT (butyl hidroxytoluene) were used.

Antioxidant effect was confirmed by adding 100 μM DPPH to the well plate by adding 100 μL and blocking it for 10-30 minutes at room temperature, then measuring the absorbance by irradiating light at a wavelength of 517 nm. As a negative control, absorbance was measured by treating 100 uL DPPH in methanol or water to perform zero treatment. After measuring the absorbance, the DPPH inhibition rate was calculated using Equation 1 below, and the experimental results are shown in FIG. 7. The measured DPPH inhibition (%) is shown in Figure 7 and Table 6 below.

[Equation 1]

DPPH inhibition rate (scavenging activity, %) = (1-OD sample/OD blind) x 100

In Equation 1, OD smaple means absorbance of each sample, and OD blind means absorbance of a DPPH solution without adding any sample.

sample Storage conditions DPPH inhibition (%) Control Sample 1 (Pb) 4℃, 1 day 42 4℃, 30 days 36 30℃, 1 day 36 30℃, 30 days 27 Control Sample 2 (HePb) 4℃, 1 day 50.6 4℃, 30 days 48.04 30℃, 1 day 50.12 30℃, 30 days 55.4 Sample 1 (HePbLp) 4℃, 1 day 34.7 4℃, 30 days 37.2 30℃, 1 day 33.2 30℃, 30 days 42.5 Sample 3 (HePbWp1) 4℃, 1 day 44.6 4℃, 30 days 43.9 30℃, 1 day 43.3 30℃, 30 days 42.3 Sample 4 (HePbLp/Wp1) 4℃, 1 day 41.4 4℃, 30 days 41.7 30℃, 1 day 41.7 30℃, 30 days 43.1 Control (VitC) none 95.1 Control (BHT) none 91.8

As shown in FIG. 7, in the case of white spotted radish powder, the DPPH inhibition rate measured after 1 day from the storage date at 42C was 42%, but the DPPH inhibition rate decreased to 36% after 30 days. The DPPH inhibition rate measured after 1 day at 36C was 36%, but decreased to 27% after 30 days. In the case of the control sample (HePb), the DPPH inhibition rate measured after 1 day at 4°C was 50.6%, but decreased slightly to 48.04% after 30 days, and the DPPH inhibition rate after 30 days at 30°C was 50.12% but 30 days Later it increased slightly to 55.4%. Sample 1 (HePbLp), sample 3 (HePbWp1), and sample 4 (HePbLp/Wp1) had DPPH inhibition rates of 34.7%, 44.6%, and 41.4%, respectively, measured 1 day after storage at 4°C, and after 30 days, It was confirmed that it was slightly increased to 37.2%, 43.9%, and 41.7%, respectively. Under the conditions of 30°C, the DPPH inhibition rate after 1 day from storage was 33.2%, 43.3%, and 41.7%, respectively. After 30 days, it was confirmed that the DPPH inhibition rate increased to 42.5%, 42.3%, and 43.1%, respectively.

Therefore, it was confirmed that the fermentation product of the white spotted radish extract of the present invention, compared to the white spotted radish powder, irrespective of the storage temperature, the antioxidant effect was not decreased and increased even after 30 days after storage.

Example 11. Confirmation of the effect of inhibiting fat rancidity of the fermentation product of the white spotted flower radish extract

To confirm the efficacy of inhibiting fatty acid plaques from the fermentation of the white spotted flower radish extract, the malodialdehyde (MDA) value first generated during the oxidation process of fats and oils was measured using TBA. TBA reacts with malodialdehyde (MDA), which is first produced during the oxidation process of fats and oils to form a red pigment. MDA is produced when oxidative decomposition of polyunsaturated fatty acids, which are intermediate products of lipid oxidation, by free radicals.

Control sample 1 (Pb), control sample 2 (HePb), sample 1 of Table 3 above (HePbLp), Sample 3 (HePbWp1), and sample 4 The powder of (HePbLp/Wp1) was stored for 30 days at a storage temperature of 3°C (refrigeration temperature) or 30°C, respectively. The powder after 1 day and 30 days from the day of storage at each temperature was collected and diluted in 1 mL of sterile water at a concentration of 10 mg/ml to prepare a 0.1% (w/v) sample solution.

Subsequently, benzene (benzene), 0.7% (v/v) TBA solution, and acetic acid (acetic acid) were mixed with each sample solution by 1 ml to prepare a TBA solution mixture. After mixing and stirring the mixture, the resulting lower layer was recovered, and the recovered lower layer was left at 95°C for 10 minutes, and absorbance was measured at a wavelength of 530 nm in a spectrophotometer. At this time, instead of the sample, distilled water was added in the same amount as the sample and used as a control. Subsequently, the same experiment was performed by changing the storage temperature to 30°C, and the results of measurement of malodialdehyde (MDA) (nmol/μg) are shown in FIGS. 8 and 7.

sample Storage conditions MDA (nmol/ug) Control Sample 1 (Pb) 4℃, 1 day 238.8 4℃, 30 days 222.4 30℃, 1 day 212.6 30℃, 30 days 239.6 Control Sample 2 (HePb) 4℃, 1 day 233.5 4℃, 30 days 236.9 30℃, 1 day 194.8 30℃, 30 days 208.3 Sample 1 (HePbLp) 4℃, 1 day 196.8 4℃, 30 days 195.6 30℃, 1 day 201.4 30℃, 30 days 204,1 Sample 3 (HePbWp1) 4℃, 1 day 184.4 4℃, 30 days 179.3 30℃, 1 day 198.7 30℃, 30 days 190.8 Sample 4 (HePbLp/Wp1) 4℃, 1 day 196.0 4℃, 30 days 191.9 30℃, 1 day 194.4 30℃, 30 days 196.4 Control group (distilled water) none 0

According to FIG. 8, in the case of the control sample 2 (HePb), the MDA was measured higher 30 days after 1 day from the storage date in both the 4°C and 30°C temperature conditions, whereas the sample 1 (HePbLp), sample 3, (HePbWp1 ), and in the case of Sample 4 (HePbLp/Wp1), it was confirmed that the levels of malodialdehyde (MDA) after 1 day and 30 days at both 4° C. and 30° C. temperature conditions were maintained at a similar level. Therefore, it was found that the fermentation product of the white spotted flower radish extract of the present invention can suppress the acidity of the fat of the white spotted flower radish through the fermentation process of lactic acid bacteria.

Example 12. Cell line toxicity test of fermentation product of white spotted flower radish extract

12-1. Cell line toxicity test in mast cells according to storage temperature

In human mast cells 3T3-L1 (ATCC CRL-3242), a cell line toxicity test was performed according to the temperature and storage date of the fermentation product of the white spotted flower radish extract of the present invention.

Cell line toxicity of 3T3-L1 cell was measured using Cell counting kit-8 (CCK-8, Dojindo). To prepare a 3T3-L1 cell cell line for use in the experiment, the 3T3-L1 cell cell line was prepared in Dulbecco's modified Eagle's Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% (w/v) Penicillin-streptomycin. Cultured at 37 ℃, 5% CO ₂ culture conditions were used in subsequent experiments.

Control sample 1 (Pb), control sample 2 (HePb), sample 1 (HePbLp), sample 3 (HePbWp1), and sample 4 (HePbLp/Wp1) were each placed in 3T3-L1 cell cells at 4°C or 30°C, respectively. After daily storage, samples collected after 1 day from the storage date and samples collected after 30 days were added to 3T3-L1 cell cells at 1 mg/ml, and cell viability (% of control) was measured. 9 and Table 8. The cell viability was expressed by setting the cell viability of the control at 100%, and the relative value was displayed. As a control, a new DMEM medium was added to 3T3-L1 cells.

sample Storage conditions Cell viability (%) Control Sample 1 (Pb) 4℃, 1 day 100.2 4℃, 30 days 101.2 30℃, 1 day 103.9 30℃, 30 days 128.2 Control Sample 2 (HePb) 4℃, 1 day 96.4 4℃, 30 days 99.8 30℃, 1 day 98.9 30℃, 30 days 122.4 Sample 1 (HePbLp) 4℃, 1 day 104.7 4℃, 30 days 97.9 30℃, 1 day 100.2 30℃, 30 days 117.1 Sample 3 (HePbWp1) 4℃, 1 day 123.7 4℃, 30 days 117.7 30℃, 1 day 130.8 30℃, 30 days 132.4 Sample 4 (HePbLp/Wp1) 4℃, 1 day 140.8 4℃, 30 days 124.0 30℃, 1 day 128.9 30℃, 30 days 135.6 Control (DMEM medium) none 100.0

As shown in FIG. 9, it was confirmed that 3T3L1 cells treated with each sample maintained 100% of the number of cells compared to the control group or slightly proliferated cells even after 30 days from the storage date at both 4°C and 30°C temperature conditions. Therefore, it was confirmed that the fermentation product of the white spotted flower radish extract of the present invention has low toxicity and safety in the human mast cell line 3T3-L1 cell line regardless of the storage temperature.

12-2. Cell line toxicity test in Raw264.7 cell

Toxicity experiments of the fermentation of the white spotted flower radish extract of the present invention were performed on the mouse cell line Raw264.7 cell (ATCC TIB-71).

Cell line toxicity of Raw264.7 cells was measured using Cell counting kit-8 (CCK-8, Dojindo). To prepare the Raw264.7 cell cell line for use in the experiment, the Raw264.7 cell cell line was used in Dulbecco's modified Eagle's Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% (w/v) Penicillin-streptomycin. After incubation at 37°C and 5% CO ₂ conditions, it was used in subsequent experiments.

The control samples 1 (Pb), control samples 2 (HePb), samples 1 and 3 (HePbLp and HePbWp1), and sample 4 (HePbLp/Wp1) were respectively 4°C and 30°C in Raw264.7 cells? The cells were stored for 30 days under conditions, and after each day, samples collected after 1 day and samples collected after 30 days were added to Raw264.7 cells, a macrophage cell line of cultured rats at 1 mg/ml, and then cell viability (% of control) and the measurement results are shown in Fig. 10 and Table 9. Cell viability (%) in the control was set to 100% using cells to which new DMEM medium was added as a control, and cell viability of each sample was calculated.

sample Storage conditions Cell viability (%) Control Sample 1 (Pb) 4℃, 1 day 105.7 4℃, 30 days 100.2 30℃, 1 day 103.1 30℃, 30 days 111.2 Control Sample 2 (HePb) 4℃, 1 day 104.5 4℃, 30 days 103.1 30℃, 1 day 105.3 30℃, 30 days 110.3 Sample 1 (HePbLp) 4℃, 1 day 101.8 4℃, 30 days 105.9 30℃, 1 day 105.3 30℃, 30 days 114.8 Sample 3 (HePbWp1) 4℃, 1 day 103.8 4℃, 30 days 100.9 30℃, 1 day 101.4 30℃, 30 days 110.4 Sample 4 (HePbLp/Wp1) 4℃, 1 day 105.1 4℃, 30 days 102.2 30℃, 1 day 100.4 30℃, 30 days 112.2 Control (DMEM medium) none 100.0

As shown in Figure 10, Raw264.7 cells treated with each sample can be confirmed that the cell number is maintained at about 100% compared to the control group or the cells proliferate slightly, even after 30 days from the storage date at both 4℃ or 30℃ temperature conditions. there was. Therefore, it was confirmed that the fermentation product of the white spotted flower radish extract of the present invention has low toxicity and safety to the Raw264.7 cell line regardless of the storage temperature.

Example 12. Confirmation of Heavy Metal Reduction Effect of Fermentation of White Spotted Flower Plain Extract

White Spotted Flower Plain Powder (Control Sample 1, Pb), White Spotted Flower Plain Hot Water Extract (Control Sample 2, HePb) and Fermented White Spotted Flower Plain Extract (Sample 1 (HePbLp), and Sample 3 (HePbWp1) )) was performed to confirm the effect of reducing heavy metals.

After the control sample 1 (Pb), control sample 2 (HePb), sample 1 and sample 2 (HePbLp, HePbWp0) of Table 3 were dissolved in water to prepare a 1% by weight solution, each was induced by inductively coupled plasma. Injected into the spectrometer, the luminescence intensity of two elements of arsenic and cadmium was measured, and the concentration of the element of interest was quantified by substituting it into the regression equation of the calibration curve. When the concentration of the element of interest in the sample solution in the inductively coupled plasma was quantified, the concentration of the element of interest in the sample was calculated according to Equation 2 below.

[Equation 2]

Elemental concentration in the sample (ug/g) ={(g/L x final amount ml x dilution factor) / sample weight (g) x 1000} x specification (g)

In the case of arsenic (As) and cadmium (Cd) in accordance with the general test method of the food industry, it was dissolved in water and prepared as a 1% (w/w) solution, treated with nitric acid, and inductiviely coupled plasma atomic emission spectrometry. , ICP-MS, KHSI-A-158) was performed (Korea Polymer Testing Institute, KOPTRI). When the concentration of the element of interest in the sample solution in the inductively coupled plasma was quantified, the concentration of the element of interest in the sample was calculated according to Equation 3 below.

[Equation 3]

Elemental concentration of interest in the sample (mg/kg) = test solution concentration (ug/kg) x final volume (ml)/sample collection (g) x dilution factor x 1/1000

In the case of mercury, the sample prepared by the above method was analyzed with a Mercyry analyzer Hydra-C (KHSI-A-060). When the concentration of the element of interest in the sample solution was quantified in the analyzer, the concentration of the element of interest in the sample was calculated according to the following equation (4).

[Equation 4]

Concentration of the element of interest in the sample (ng/g) = detection amount (ng) / sample collection amount (g)

In the case of chromium and selenium, a certain amount of sample is put into microwaves (Microwave MARS CEM, KHSI-A-063), decomposed by treatment with nitric acid, etc. Atomic Emission Spectrometry, ICP-MS agilent 7700, KHSI-A-059) was used to analyze the components (Korea Polymer Testing Institute, KOPTRI).

Table 5 shows the results of the analysis of the five heavy metals obtained according to the above method.

Item Control Sample 1
(Pb) Control Sample 2
(HePb) Sample 1
(HePbLp) Sample 3
(HePbWp0) One Cadmium (Cadimium, mg/kg) 0.1326 0.0583 0.0443 0.0475 2 Mercury (Mercury, mg/kg) 0.0236 0.0027 0.0021 0.0024 3 Arsenic (mg/kg) 10.0192 0.2387 0.2102 0.1662 4 Selenium (ug/100g) 21.44 20.89 17.23 20.78 5 Chromium (ug/100g) 27.28 26.03 23.89 23.01

In Table 10, the control sample 1 (Pb) was cadmium, mercury, arsenic, selenium, and chromium, respectively, measured at 0.1326 mg/kg, 0.0236 mg/kg, 10.0192 mg/kg, 21.44 ug/100g, 27.28 ug/100g, and food The standard value of heavy metals for edible insects presented in the Food and Drug Administration's Food Code was 0.05 mg/kg cadmium, 0.5 mg/kg mercury, and 3 times and 10 times higher than 0.1 mg/kg arsenic. In Control Sample 2 (HePb), cadmium, mercury, arsenic, selenium, and chromium were reduced by about 2 to 10 times to 0.0583 mg/kg, 0.0027 mg/kg, 0.2387 mg/kg, 20.89 ug/100g, and 26.03 ug/100, respectively. However, selenium and chromium did not decrease significantly.

In the case of Sample 1 and Sample 2 (HePbLp, HePbWp0), cadmium (0.0443, 0.0475 mg/kg, respectively), mercury (0.0021,0.0024 mg/kg, respectively), arsenic (0.2102, 0.1662) is more than Control Sample 2 (HePb). Small quantities were measured, and selenium and chromium were not significantly reduced. Selenium is used as a nutritional supplement and is known for its antioxidant, immune-enhancing, hair and eye health enhancements, thyroid function improvement, and heart health-enhancing functions, and is recommended as a daily recommended dose of 55mg for adults. Chromium enhances insulin action as a glucose tolerance factor, and is an essential nutrient involved in protein, carbohydrate, and lipid metabolism, and is known for its effects on energy generation, blood sugar control, and prevention of coronary artery disease.

Therefore, it was confirmed that the fermentation product of the white spotted flower radish extract of the present invention has an effect of removing harmful heavy metal components while maintaining the content of active ingredients such as selenium and chromium due to lactic acid fermentation.

Example 13. Confirmation of melanin pigment reduction effect of fermentation of white-spotted flower radish extract

In order to confirm the effect of reducing the melanin pigment of the fermentation of the white spotted radish extract of the present invention, the fermentation of the white spotted radish extract was treated on the B16F10 cells of the mouse, and the content of melanin was quantitatively analyzed.

To prepare cells for use in the experiment, rat B16F10 cells were cultured in DMEM medium containing 10% FBS. The cultured B16F10 cells 3 x 10 ⁵ cells were dispensed into 6-well plates and cultured for 12 hours.

Subsequently, each of the control sample 1 (Pb), control sample 2 (HePb), and sample 4 (HePbLp/Wp1) was stored at 4°C for 30 days, and 1 day, 30 day powder was collected from the storage date, and 1 mg Dissolved in /ml water to prepare a 0.1% sample solution.

The previously prepared, B16F10 cells were treated with 200 nmol a-MSH (a-melanin sitmulating hormone, sigma) and each 0.1% (w/v) sample solution prepared above in a cultured 6-well plate, and 24 in an incubator at 37°C. Incubated for an hour. As a control (Control), a cell group treated with only 200 nmol a-MSH (control), as a comparative example, 200 nmol a-MSH (a-melanin sitmulating hormone, sigma) and arbutin (albutin 0.1% (w/v)) It was used together. After completing the culture, the cells were collected, lysed in 1N NaOH with 10% DMSO added, incubated at 80° C. for 1 hour, and absorbance was measured at 475 nm. 11 and Table 11 show the results showing the relative absorbance in each sample with 100% absorbance when B16F10 cells were treated with only a-MSH.

sample Condition Melanin content (%) Control No treatment 100 Control Sample 1 Pb, 4℃, 1 day storage 87.9 Pb, 4℃, 30 days storage 75.7 Control Sample 2 HePb, 4℃, 1 day storage 77.3 HePb, 4℃, 30 days storage 77.8 Sample 4 HePbLp/Wp1, 4℃, 1 day storage 64.5 HePbLp/Wp1, 4℃, 30 days storage 71.5 Comparative example Albutin 68.7

As shown in FIG. 11, in the case of Sample 4 (HePbLp/Wp1), the melanin content of B16F10 cells was reduced to about 70% after storage at 4° C. for 1 day. The results are superior to the inhibitory effect of melanin pigment than the comparative example albutin (albutin), it was confirmed the excellent whitening effect of the fermentation of the white-spotted flower radish extract of the present invention.

Korea Microbial Conservation Center KCCM12299P 20180810 Korea Microbial Conservation Center KCCM12300P 20190810 Korea Microbial Conservation Center KCCM12301P 20190810

<110> Marine Industry Research institute for East sea <120> Fermented product of Protaetia brevitarsis extract, its preparation method and use <130> DPP20191806KR <150> KR 10-2018-0153921 <151> 2018-12-03 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cmtggctca 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1472 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum KCCM12299P 16SrRNA <400> 3 gaagcagtgg cgggtgctat acatgcagtc gaacgaactc tggtattgat tggtgcttgc 60 atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca 120 gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg 180 gtccgagctt gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct 240 agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg 300 gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360 cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaaaggt ttcggctcgt 420 aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480 taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta cgtggcaagc 540 gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600 gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac 660 agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720 ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt 780 agataccctg gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc 840 ttcagtgctg cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa 900 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960 acgcgaagaa ccttaccagg tcttgacata ctatgcaaat ctaagagatt agacgttccc 1020 ttcggggaca tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattaag ttgggcactc 1140 tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc gaactcgcga 1260 gagtaagcta atctcttaaa gccattctca gttcggattg taggctgcaa ctcgcctaca 1320 tgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagtcgg tggggtaacc 1440 ttttaggaac cagccgctaa gtgacagaat gg 1472 <210> 4 <211> 1345 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides KCCM12300P 16SrRNA <400> 4 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaactg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcatttagtt 1140 gggcactcta gtgagactgc cggtgacaaa ccggaggaag gtgggggatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcag ttcggattgt aggctgcaac 1320 tcgcctacat gaagtcggaa tcgct 1345 <210> 5 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides KCCM12301P 16SrRNA <400> 5 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaattg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcattcagtt 1140 gggcactcta gtgagactgc cggtgacaac cggaggaggg ggggggaatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcaa tttcggaatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgct 1347 <210> 6 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum reference 16SrRNA <400> 6 cgcagggcgg gtgctataca tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc 60 atgatttaca tttgagtgag tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa 120 gcgggggata acacctggaa acagatgcta ataccgcata acaacttgga ccgcatggtc 180 cgagtttgaa agatggcttc ggctatcact tttggatggt cccgcggcgt attagctaga 240 tggtggggta acggctcacc atggcaatga tacgtagccg acctgagagg gtaatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360 aatggacgaa agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa 420 actctgttgt taaagaagaa catatctgag agtaactgtt caggtattga cggtatttaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc 600 ttcggctcaa ccgaagaagt gcatcggaaa ctgggaaact tgagtgcaga agaggacagt 660 ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggctgtctgg tctgtaactg acgctgaggc tcgaaagtat gggtagcaaa caggattaga 780 taccctggta gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttc 840 agtgctgcag ctaacgcatt aagcattccg cctggggagt acggccgcaa ggctgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg 960 cgaagaacct taccaggtct tgacatacta tgcaaatcta agagattaga cgttcccttc 1020 ggggacatgg atacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttattat cagttgccag cattaagttg ggcactctgg 1140 tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg atggtacaac gagttgcgaa ctcgcgagag 1260 taagctaatc tcttaaagcc attctcagtt cggattgtag gctgcaactc gcctacatga 1320 agtcggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagtcggtgg ggtaaccttt 1440 taggaaccag ccgcctaagt gacagaatgg 1470 <210> 7 <211> 1345 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides 16S rRNA reference AB598954.1 <400> 7 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaactg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcatttagtt 1140 gggcactcta gtgagactgc cggtgacaaa ccggaggaag gtgggggatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcag ttcggattgt aggctgcaac 1320 tcgcctacat gaagtcggaa tcgct 1345 <210> 8 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides 16S rRNA reference LC094432.1 <400> 8 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaattg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcattcagtt 1140 gggcactcta gtgagactgc cggtgacaac cggaggaggg ggggggaatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcaa tttcggaatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgct 1347

Claims (21)

Lactic acid bacteria fermentation product of white spotted flower plain solvent extract. The fermentation product of claim 1, wherein the extraction solvent for the preparation of the white-spotted flower solid solvent extract is at least one selected from the group consisting of alcohol and water having 1 to 3 carbon atoms. The method of claim 1, wherein the lactic acid bacteria fermentation product is produced by culturing lactic acid bacteria in a fermentation raw material prepared by adding one or more selected from the group consisting of carbon sources and nitrogen sources to the white spotted flower solid solvent extract. water. According to claim 3, The carbon source is one or more selected from the group consisting of dextrose (dextrose), glucose (glucose), sucrose (sucluse), lactose (lactose), and galactose (galactose), fermentation. The method according to claim 1, wherein the heavy metal content of the lactic acid bacteria fermentation product of the white-spotted radish solvent extract is 0.5 to 50 parts by weight based on 100 parts by weight of the heavy metal content in the extract raw material of the white-spotted radish,
The heavy metal is one or more heavy metals selected from the group consisting of arsenic, cadmium and mercury. The method of claim 1, wherein the lactic acid bacteria are Lactobacillus plantarum, Lactococcus lactics , Lactobacillus sakei , Lactobacillus fermentum , Lactobacillus fermentum , Lactobacillus fermentum ( Lactobacillus rhamnosus ), Lactobacillus brevis , Lactobacillus graminis , Lactobacillus dextrinicus , Lactobacillus dextrinicus , Lactococcus garvieae , Lactococcus garvieae Enterococcus faecium , Enterococcus faecalis , Leuconostoc citreum , Weissella hellenica , Weissella paramesenteroides , Pediococcus escidactactic One or more lactic acid bacteria selected from the group consisting of Pediococcus acidilactici , Pediococcus pentosaceus , Bifidobacterium animalis , and Bifidobacterium infantis Phosphorus, fermentation products. Preparing a solvent extract of white spotted flower plain fabric; And
Including the step of inoculating and fermenting the lactic acid bacteria in the fermentation raw material containing the solvent extract,
Method for producing lactic acid bacteria fermentation product of white spotted flower solid solvent extract. The method according to claim 7, wherein the solvent is at least one selected from the group consisting of alcohol and water having 1 to 3 carbon atoms. The method of claim 7, wherein in the preparation of the solvent extract, the extraction temperature is 30°C to 90°C, and the extraction time is 4 to 6 hours. The method according to claim 7, wherein the fermentation raw material is one or more selected from the group consisting of a white spotted flower solid solvent extract and a carbon source and a nitrogen source. The method of claim 10, wherein the carbon source is at least one selected from the group consisting of dextrose, glucose, sucluse, lactose, and galactose. The method of claim 10, wherein the nitrogen source is at least one selected from the group consisting of beef extract, peptone, tryptone, and skim milk. The method of claim 7, wherein the lactic acid bacteria are Lactobacillus plantarum, Lactococcus lactics , Lactobacillus sakei , Lactobacillus fermentum , Lactobacillus fermentum , Lactobacillus fermentum ( Lactobacillus rhamnosus ), Lactobacillus brevis , Lactobacillus graminis , Lactobacillus dextrinicus , Lactobacillus dextrinicus , Lactococcus garvieae , Lactococcus garvieae Enterococcus faecium , Enterococcus faecalis , Leuconostoc citreum , Weissella hellenica , Weissella paramesenteroides , Pediococcus escidactactic One or more lactic acid bacteria selected from the group consisting of Pediococcus acidilactici , Pediococcus pentosaceus , Bifidobacterium animalis , and Bifidobacterium infantis Phosphorus, manufacturing method. The method of claim 7, wherein the fermenting step is fermentation at a temperature of 25 to 45° C. for 12 to 48 hours. The method according to claim 7, further comprising the step of storing the lactic acid bacteria fermentation product of the white spotted radish solvent extract at a storage temperature of 1°C to 10°C for 1 day to 90 days. An antimicrobial composition comprising a lactic acid bacteria fermentation product of a white spotted flower solid solvent extract according to any one of claims 1 to 6. 17. The method of claim 16 wherein the antimicrobial agent is, Listeria, Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Bacillus (Bacillus), Vibrio (Vibrio), Pseudomonas bacterium (Pseudomonas), yeast (Yeast), fungus (Fungi ), acne ( Propionibacterium acnes ), and dandruff ( Malassezia furfur ) having an antimicrobial effect against at least one microorganism selected from the group consisting of, antimicrobial composition. Claim 1 to claim 6, wherein the white spotted flower radish solvent extract of at least one nutrient selected from the group consisting of proteins, fats, carbohydrates, and cellulose, including the fermentation product of the solvent extract, nutrition The composition of the disintegrant of the component. An antioxidant composition comprising a lactic acid bacteria fermentation product of a white-spotted flower solid solvent extract according to any one of claims 1 to 6. A cosmetic composition for skin whitening comprising a lactic acid bacteria fermentation product of a white spotted flower solid solvent extract according to any one of claims 1 to 6. A cosmetic composition for preventing and improving acne, comprising a lactic acid bacteria fermentation product of a white spotted flower solid solvent extract according to any one of claims 1 to 6.

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