KR20210045529A - Method for preparing fermentated ziziphus jujuba seed - Google Patents

Abstract

The present application provides a method for preparing fermented Ziziphus jujuba seeds which comprises the following steps: separating and pulverizing seeds from Ziziphus jujuba; after sterilizing Ziziphus jujuba seed powder, inoculating lactic acid bacteria, yeast or Bacillus subtilis for fermentation to obtain fermented Ziziphus jujuba seeds; and removing impurities after sterilizing the fermented Ziziphus jujuba seeds. The method for preparing fermented Ziziphus jujuba seeds of the present application has an advantage of allowing efficiently utilization of discarded Ziziphus jujuba seed byproducts.

Description

Manufacturing method of fermented jujube seeds {METHOD FOR PREPARING FERMENTATED ZIZIPHUS JUJUBA SEED}

The present application relates to a method of manufacturing a fermented jujube seed.

Jujube seeds are the seed parts in the pulp of jujube and are a by-product of jujube processing produced after processing the jujube pulp. Jujube seed by-products produced during jujube processing account for about 30% by weight of jujube park materials. Since ancient times, jujube has been added to give sweetness like licorice in oriental medicine, and itself has a pharmacological effect that improves the symptoms of stomach cramps, insomnia, indigestion, sub large intestine blood, red blood, and hypersensitivity. Jujube seeds have high nutritional value due to their high content of protein, minerals, and dietary fiber, and have been used as a medicinal ingredient called Sanjoin in oriental medicine. Despite these excellent nutritional and pharmacological values, most of the jujube seed resources are not utilized industrially, and more than two barrels per year are discarded in Korea.

Human skin color is determined by the concentration and distribution of melanin inside the skin. In addition to genetic factors, it is affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, and stress. Melanin is synthesized from melanocytes in the epidermal layer of the skin, and an enzyme called tyrosinase acts on tyrosine, a kind of amino acid, in the melanosome, an organelle within the melanocyte. dopaquinone) and then made through a non-enzymatic oxidation reaction. When such melanin synthesis occurs excessively in the skin, it darkens the skin tone and causes spots, freckles, and the like. Therefore, by inhibiting the synthesis of melanin pigment by inhibiting the tyrosinase activity in the skin, it is possible to realize skin whitening by brightening the skin tone, as well as skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones and genetic causes. It can improve calmness. In addition, melanin itself also exhibits the properties of free radicals.

Skin is an organ where aging occurs primarily, and in order to delay or prevent it, many studies have also been conducted in the cosmetic field. As a representative phenomenon of skin aging, the amount of sebum secretion is reduced, resulting in dry skin, slow cell regeneration, and a lot of aging dead skin cells accumulating, resulting in rough skin. In addition, the amount of collagen synthesis that supports the epidermis is reduced, and elastin (elastic fibers) is denatured, resulting in wrinkles. In addition, the skin color is stained, spots, and pigmentation symptoms such as age spots appear, and the skin protection function is weakened due to the thinning of the epidermis. Other phenomena include an increase in skin troubles due to a decrease in the skin barrier effect associated with a decrease in skin thickness. Research related to skin aging has been largely focused on materials that protect the skin from ultraviolet rays and free radicals, which are the main causes of skin aging, materials for improving skin wrinkles, materials for enhancing skin elasticity, and strengthening the skin epidermis and dermal junction (DEJ). It is classified into related materials and is being studied in depth.

As the skin ages, changes in the dermis appear remarkably, and changes in the dermis are caused by changes in substances having a large molecular weight in the extracellular matrix due to the decrease in the number of fibroblasts and their synthetic ability. The specific changes include separation of collagen bundles, reduction of viscopolysaccharide synthesis, reduction in the number and diameter of collagen and elastic fibers (elastin), decomposition of collagen and elastic fibers, and expansion of blood vessels. In general, among a number of complex factors, such as skin moisture content, collagen content, and immunity against the external environment, the biggest influence on the formation of wrinkles is cola, a collagen-degrading enzyme that reduces collagen production and collagen content. It is the expression level and activity of genease. Collagenase belongs to a group of metalloproteinases (MMPs) that play an important role in the degradation of matrix proteins. Collagenase is known to decompose type 1, 2, 3, and 5 collagen by promoting its expression by ultraviolet rays, growth factors, inflammatory cytokines, and phorbol esters. It is known to play a major role in photoaging caused by.

In addition, elastin fibers form cross-links with collagen and are an important skin component for wrinkle formation, which is involved in skin elasticity. Deficiency and aggregation of elastin fibers, and a marked increase in the activity of elastase, an elastin-degrading enzyme, have been found to be one of the factors of skin wrinkle formation. Elastase is the only enzyme that can break down elastin, and its inhibition is known to fundamentally reduce the improvement of skin wrinkles.

Most of the known anti-wrinkle or whitening substances for skin improvement are limited in usage due to safety problems such as irritation and redness when applied to the skin, or the effect is insignificant, so the effect of substantially improving skin function or wound healing could not be expected. . Therefore, there is an urgent need to develop a new cosmetic composition for skin improvement that is safer to the living body and has a higher effect than the existing cosmetic composition for skin improvement.

The problem to be solved by the present application is to provide a method of preparing a fermented jujube seed contained as an active ingredient in a cosmetic composition or food composition having antioxidant, skin whitening or wrinkle improvement effects.

In order to solve the above-described problem, one embodiment of the present application provides a method of manufacturing a fermented jujube seed.

In one embodiment of the present application, the manufacturing method comprises the steps of separating and pulverizing seeds from jujube; Sterilizing the jujube seed powder and then inoculating the lactic acid bacteria, yeast or Bacillus into fermentation to obtain a fermented jujube seed; And removing impurities after sterilizing the fermented jujube seed.

In one embodiment of the present application, the lactic acid bacteria, yeast or Bacillus bacillus, Lactobacillus plantarum (Lactobacillus plantarum), Bacillus subtilis (Bacillus subtilis), Leuconostoc mecentroid (Leuconostoc mesenteroide ) or Saccharomyces cerevisiae .

In one embodiment of the present application, the powdering may be pulverized by lyophilization, hot air drying, or natural drying, or may be pulverized or pulverized after refrigerated or frozen storage.

In one embodiment of the present application, in the step of obtaining the fermented product, the jujube seed powder and the medium may be mixed in a weight ratio of 1:4 to 1:20, and then sterilized.

In one embodiment of the present application, in the step of obtaining the fermented product, fermentation may be performed by culturing for 1 to 10 days at 100 rpm to 300 rpm, 20 to 45 °C.

In one embodiment of the present application, the step of removing the impurities may be performed by filtering the fermented product to a size of 0.3 µm to 5 µm or centrifuging.

In one embodiment of the present application, the sterilization may be performed at 100° C. to 140° C. for 10 to 30 minutes.

The manufacturing method of fermented jujube seeds of the present application has the advantage of improving manufacturing efficiency and physiological activity, and enabling efficient use of discarded jujube seed by-products, thereby securing competitiveness in terms of manufacturing cost.

In addition, since the fermented jujube seed produced by the method for producing fermented jujube seeds of the present application effectively inhibits melanin production, functional cosmetics for skin whitening, health functional foods that can prevent or improve skin pigmentation disorders, or compositions for external use for skin It can be usefully used as

In addition, the fermented jujube seed produced by the method for producing fermented jujube seeds of the present application effectively promotes collagen synthesis, reduces elastase activity, and thus functional cosmetics for wrinkle improvement, which can prevent or improve skin wrinkles. It can be usefully used as a health functional food or a composition for external skin.

In addition, q fermented jujube produced by the method of preparing fermented jujube seeds according to the present application has high DPPH radical and ABTS radical scavenging activity, and high polyphenol content, which has excellent antioxidant effects.

1 shows a manufacturing process of fermented jujube seeds prepared according to an embodiment of the present application.
1 shows the melanin inhibitory activity of B16F0 cell culture broth of fermented jujube seeds prepared according to an embodiment of the present application.

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

Since the embodiments according to the concept of the present application can be modified in various ways and have various forms, specific embodiments will be described in detail in the present specification. However, this is not intended to limit the embodiments according to the concept of the present application to a specific form of disclosure, it should be understood to include all changes, equivalents, or substitutes included in the spirit and scope of the present application.

The terms used in the present specification are only used to describe specific embodiments and are not intended to limit the present application. Singular expressions include plural expressions unless the context clearly indicates otherwise.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

Unless otherwise defined, all terms including technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present specification. .

In the present application, the term "jujube" is interpreted as including the skin, flesh and seeds of the jujube, and there is no limitation according to the variety. In the present application, it is intended to manufacture a fermented jujube seed that has the efficacy and activity of jujube seeds rather than the various effects and activities of the whole jujube. By separating into, and seeds, a step of preparing a sample of jujube seeds is performed.

The jujube seed sample for each part may be used immediately, but dried (hot air drying, freeze drying, etc.), refrigerated or frozen storage may be used.

The jujube seed sample undergoes a pulverization process, and by this pulverization process, jujube seed powder particles having a uniform size are obtained. In particular, by using powdered jujube seeds, it is possible to maximize a high surface area and thus an extraction effect.

After sterilizing the jujube seed powder, it can be fermented by inoculating the strain.

After mixing the jujube seed powder and the MRS medium in a weight ratio of 1:4 to 1:20, it can be sterilized. Specifically, jujube seed powder and MRS medium may be mixed in a weight ratio of 1:4 to 1:10. More specifically, it can be mixed in a weight ratio of 1:5.

There are no particular restrictions on the sterilization process, for example, sterilization using heat sterilization, ultra-high temperature instant sterilization, high temperature long-term sterilization, gamma radiation sterilization, ultra-high pressure sterilization, ultraviolet ray killing, ultrasonic sterilization, electron beam sterilization, or chemical sterilization. The process can be performed, and other methods can be applied as long as it loses/sterilizes the activity of its own microorganisms in the rice bran, and most specifically, a high-temperature/high-pressure sterilization (autoclaving) treatment is performed at 100°C to 140°C. It may be performed for minutes to 30 minutes. The high-temperature/high-pressure sterilization treatment allows the active ingredient to be easily eluted by dissociating the hard jujube seed cell wall tissue, improves fermentation efficiency and speed by the strain, and increases the content of physiologically active substances. It works.

The strain may be Lactobacillus plantarum , Bacillus subtilis , Leuconostoc mesenteroide , or Saccharomyces cerevisiae .

The strain can be inoculated at 0.1% to 10% by weight and 0.5% to 1.0% by weight.

The fermentation may be rotated at 100 rpm to 300 rpm for 1 to 10 days at 20°C to 45°C. Specifically, it can be cultured under the conditions of 120 to 180 rpm at 30 ℃ to 40 ℃.

If the fermentation temperature is less than the above range, it is not economical because equipment for lowering the temperature while the extraction yield of substances useful for skin beauty is lowered, and if the stirring temperature exceeds the above range, substances useful for skin beauty are destroyed or skin whitening , It is not preferable that a material that is not involved in wrinkle improvement, etc. can be extracted together in a high content, so that a fermented product with reduced functionality can be prepared. For example, when the fermentation temperature is in the range of 20 to 45 ℃, since the plant cell tissue is softened, the elution of ingredients such as phenols, which are effective substances having cosmetic activity, is facilitated, so that a fermented product having excellent skin beauty effects can be obtained. There is an effect. In addition, when the culture is performed for less than 1 day, the time is insufficient and it is difficult to completely elute the physiologically active ingredients contained in the jujube seeds. If the fermentation time exceeds 10 days, the physiologically active ingredients contained in the jujube seeds are It does not increase to a significant level, but rather, due to excessive cultivation, a fermented product with reduced antioxidant and skin whitening activity may be prepared.

After fermentation, it can be sterilized so that no further fermentation proceeds. Thereafter, impurities may be removed using a conventional filtration method or apparatus, and for example, a fermented product from which impurities have been removed may be obtained by filtration using a centrifugation method or a micro filter. For example, as the usable micro filter, a filter having pores of 0.3 μm to 5 μm may be used. Specifically, it can be filtered using a filter having a pore size of 3㎛. As another example, it may be centrifuged at 1000 rpm to 2000 rpm for 5 minutes to 20 minutes. Specifically, the precipitate was discarded by centrifugation at 1500 rpm for 10 minutes, and only the supernatant may be collected and used.

Referring to Figure 1, as an embodiment, the step of pulverizing the jujube seeds (S1), the step of mixing the jujube seed powder and the medium (S2), the step of autoclaving (S3), the step of inoculating bacteria (S4), shaking It shows a method for producing a fermented jujube seed, including the step of culturing (S5), the step of autoclaving (S6), and the step of filtering (S7).

The fermented jujube seed may be 0.0025% by weight to 30% by weight, specifically 0.025% by weight or more, and may be 20% by weight or less, 10% by weight or less, or 5% by weight or less, based on the total weight of the composition. At this time, if the content of fermented jujube seeds is less than 0.0025% by weight, the melanin effect of the present application cannot be obtained, and if it exceeds 30% by weight, the effect may not be proportional to the increase in the content, and thus it may be inefficient. There is a problem that this is not secured.

The extract obtained by extracting jujube seeds with an organic solvent such as ethanol has a problem in that a fat-soluble substance that is not suitable for a cosmetic composition is extracted together. In addition, there is a problem in that the solvent must be removed separately. However, the fermented product of jujube seeds of the present application has the advantage that a problem when extracting with an organic solvent does not occur because a fat-soluble substance is not extracted.

The fermented jujube seed prepared according to an exemplary embodiment of the present application may be included as an active ingredient in a cosmetic composition.

In addition to the fermented jujube seed as an active ingredient, to the cosmetic composition, ingredients commonly added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional auxiliary agents such as perfumes, and carriers may be additionally added. .

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, It may be formulated as a powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. In more detail, it may be prepared in the form of a nourishing cream, astringent lotion, a soft lotion, lotion, essence, a nourishing gel or a massage cream.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, gum tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .

When the formulation is a powder or spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane/butane or Propellants such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant gum, etc. may be used.

When the formulation is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate , Alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester, and the like may be used.

The fermented jujube seed prepared according to an embodiment of the present application may be included as an active ingredient in a pharmaceutical composition.

The pharmaceutical composition includes a pharmaceutically acceptable carrier in addition to the fermented jujube seed. Pharmaceutically acceptable carriers included in pharmaceutical compositions are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present application may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The pharmaceutical composition may be administered orally or parenterally, specifically parenteral administration, and more specifically, applied by topical application by application.

A suitable dosage of the pharmaceutical composition can be prescribed in various ways depending on factors such as the method of formulation, mode of administration, age, weight, sex, pathological condition of the patient, food, time of administration, route of administration, rate of excretion and sensitivity to response. The dosage of the pharmaceutical composition of the present application is in the range of 0.001-100 mg/kg on an adult basis. In addition, in the case of an external preparation, it is recommended to apply once to 5 times a day in an amount of 1.0 to 3.0 ml based on an adult and continue for more than 1 month. However, the dosage does not limit the scope of the present application.

The pharmaceutical composition is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art, or by being incorporated into a multi-dose container. Can be manufactured. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excir, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The fermented jujube seed prepared according to an embodiment of the present application may be included as an active ingredient in a food composition. Food may be provided as, for example, health supplements, functional foods, food additives, and the like.

The term "food" as used in this application refers to a natural product or processed product containing one or more nutrients, and specifically refers to a state that can be eaten directly after a certain degree of processing By meaning, it is intended to include all foods, food additives, functional foods and beverages.

The term "functional food" as used in this application refers to a food group or processed food in which added value is added to the food to act and express the function of the food for a specific purpose by using physical, biochemical, or biotechnological techniques.

The functional food may further include food additives that are acceptable food additives, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of functional foods.

Foods include, for example, various foods, beverages, gum, tea, vitamin complexes, and functional foods. In addition, foods in this application include special nutritional foods (eg, formula, infant food, etc.), processed meat products, fish meat products, tofu, muk, noodles (eg, ramen, noodles, etc.), health supplements, seasoning foods. (E.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), Beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.) are included, but are not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

As a food, it is manufactured as a functional beverage, and a functional beverage is a biological defense of a beverage group or beverage composition that gives added value to act and express the function of the beverage for a specific purpose by using physical, biochemical, and biotechnological techniques. It refers to a beverage designed and processed to sufficiently express the gymnastic function for rhythm control, disease prevention and recovery, etc. to the living body. In particular, it refers to a beverage that improves skin elasticity and promotes collagen synthesis.

Food compositions include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, and organic acids. , A protective colloid thickener, a pH adjuster, a stabilizer, a preservative, a glycerin, an alcohol, a carbonation agent used in carbonated beverages, and the like. These components may be used independently or in combination.

Hereinafter, the present application will be described in more detail through Preparation Examples, Examples, and Experimental Examples, but the following examples are intended to aid understanding of the present application and do not limit the scope of the present application.

< Manufacturing example 1> Preparation of Jujube Seed Sample

Jujube grown in Korea ( Ziziphus jujuba var. spinosa ) was purchased, separated into skin, pulp, and seeds, lyophilized, and pulverized to obtain uniform sized jujube seed powder particles.

< Manufacturing example 2> Jujube seeds Fermented product Produce

Two strains of lactobacillus Lactobacillus plantarum (L. plantarum ) , Leuconostoc mesenteroide (L. mesenteroide) and yeast S accharomyces cerevisiae (S. cerevisiae ) and Bacillus subtilis (B. suntilis) , which are Bacillus subtilis, were sold in the Korean Collection for Type Cultures (KCTC) and used for experiments. L. plantarum In the growth medium it was incubated at 35 ℃ in MRS Broth, L. The growth medium of mesenteroide was cultured at 25℃ with MRS Broth. S. cerevisiae growth medium was cultured at 25℃ using Potato Dextrose Broth, and B. suntilis As a growth medium, Trytic Soy Broth was used and incubated at 35°C.

The jujube seed powder and the medium were mixed in a ratio of 1:4 to 1:20, autoclaved at 121°C for 15 minutes, and then 0.85% of previously cultured bacteria were inoculated. After shaking culture at 35°C and 150rpm for 5 days, it was autoclaved at 121°C for 20 minutes to prevent further fermentation. After completion of the culture, centrifugation was performed at 1,500 rpm for 10 minutes to discard the precipitate and collect only the supernatant to prepare a fermented jujube seed.

< Example 1 to Example 5>

Lactobacillus plantarum (Example 1), Leuconostoc mesenteroide (Example 2), S accharomyces cerevisiae (Example 3) and Bacillus subtilis (Example 4) fermented jujube seeds were prepared according to Preparation Example 2. In addition, Lactobacillus was prepared in a ratio of 1:5 using 200 mg of jujube seed powder and 1,000 mg of medium. A fermented jujube seed broth of plantarum (Example 5) was prepared according to Preparation Example 2.

< Comparative example 1>

Jujube seed powder was extracted with 70% ethanol to prepare a jujube seed extract.

< Experimental example 1> Jujube seeds Fermented DPPH Radical Erasing ability Measure

DPPH assay with the fermented jujube seeds of Examples 1 to 5 was performed by Yoshida et al. It was measured according to the method used by (1989). In a 96 well plate, add 50 µl of deionized water to the control and 50 µl of the sample diluted with deionized water in the test group as a triple. 25 µl of ethanol was added to all wells, and 75µl of DPPH (0.5mM 1,1-diphenyl-2-picrylhydrazyl(DPPH)/ethanol) solution was added and mixed. After reacting at room temperature at 25° C. for 30 minutes, absorbance was measured at 517 nm. As a solvent control, the radical scavenging ability relative to the control was expressed as a percentage.

Radical scavenging ability of the fermented jujube seed was shown in Table 1 below by confirming the concentration-dependent increase in activity. The DPPH radical scavenging activity of the fermented jujube seed of Example 5 was 69.1% at a concentration of 50%, and the IC ₅₀ was 18.7% DPPH radical scavenging activity.

Sample concentration ( % ) DPPH Erasing ability IC ₅₀ Example 1 100 92.4±1.4 Example 2 100 74.9±0.2 Example 3 100 46.1±3.3 Example 4 100 37.1±5.5 Example 5 One 10.2±1.4 18.7% 5 28.6±0.8 25 61.4±0.7 50 69.1±5.1 100 91.7±3.3

< Experimental example 2> Jujube seeds Fermented ABTS Radical Erasing ability Measure

The ABTS assay was measured by modifying the method of Re et al. (1999) with the fermented jujube seeds of Examples 1 to 5 and Comparative Example 1. That is, after mixing 5 mL of 7 mM ABTS and 88 μL of 140 mM potassium persulfate, the ABTS cation is formed by blocking light for 16 hours at room temperature. Thereafter, this solution was diluted with PBS so that the absorbance value was 1.5 at 414 nm. 190 µl of the prepared diluted solution and 10 µl of the sample were mixed, reacted at room temperature for 6 minutes, and absorbance was measured at 734 nm. As a solvent control, the radical scavenging ability relative to the control was expressed as a percentage.

Radical scavenging activity of the fermented jujube seed was shown in Table 2 below by confirming the concentration-dependent increase in activity. The ABTS radical scavenging activity of jujube seed fermentation broth was 77.6% at the concentration of 1%, and the ABTS radical scavenging activity of _{IC 50 was 0.56%.} When comparing 1% of Comparative Example 1, which is an ethanol extract of jujube seeds, and 1% of Example 1, it can be seen that there is a remarkable difference in the ABTS scavenging activity effect.

As compared with Example 5 in Example 1, Example 5 showed the highest activity, and the following activity was Lactobacillus plantarum (Example 5).

Sample concentration (%) ABTS scavenging ability IC ₅₀ Comparative Example 1 One 14.4±4.8 10 83.9±2.9 100 99.8±0.2 Example 1 100 101.3±2.4 Example 2 100 101.4±0.3 Example 3 100 99.7±0.6 Example 4 100 99.8±0.2 Example 5 0.05 14.6±0.3 0.56% 0.1 20.5±1.2 0.25 30.5±1.6 0.5 48.6±2.2 One 77.6±0.6 5 99.9±0.3

< Experimental example 3> Jujube seeds Mushroom of fermented material Tyrosinase Inhibitory activity measurement

Mushroom Tyrosinase inhibitory activity experiment was performed with the fermented jujube seed of Example 5 with reference to the experimental method of the functional cosmetics evaluation guideline. Tyrosinase is an enzyme that is involved in the most important early rate-determining step in the melanin biosynthesis pathway in the human body, and inhibition of the activity of this enzyme shows the result of inhibiting melanin production. This test is a method of evaluating the effect of a test sample on inhibition of tyrosinase activity by reacting a test sample, purified tyrosinase, and a substrate tyrosine in vitro. First, the sample was dissolved in distilled water, and the jujube seed fermentation broth was diluted to 5, 10, 25, 50% concentration to measure Mushroom Tyrosinase inhibitory activity.

In the test tube, 220 µl of a phosphate buffer (pH 6.5), 20 µl of a sample, and 20 µl of a mushroom tyrosinase solution (1500-2000 U/mL) were sequentially added. To this solution, 40 µl of 1.5 mM tyrosine solution was added and reacted at 37° C. for 10 to 15 minutes, and the absorbance was measured at 490 nm. _{The sample concentration (IC 50} ) when the activity inhibition rate was 50% was calculated. Instead of the sample solution, the solvent in which the sample was dissolved was used as the sample solution, and the results were compared using arbutin as a positive control, and the results are shown in Table 3.

It was found that the inhibitory activity of the jujube seed fermentation broth increased as the concentration increased.

Sample name ( % ) Mushroom Tyrosinase Inhibitory Activity (%) IC ₅₀ IC ₂₀ Example 5 5 10.0 ± 1.0 43.4% 12.0% 10 18.1 ± 0.9 25 38.4 ± 1.6 50 53.6 ± 2.2 Arbutin 5 24.3 ± 4.6

< Experimental example 4> Jujube seeds Cytotoxicity of fermented products Measure

With the fermented jujube seed of Example 5, a melanin inhibitory activity experiment was performed with reference to the experimental method of the functional cosmetics evaluation guideline. This test is a method of evaluating the inhibitory effect of cell melanogenesis on the whitening component. Cells were cultured to quantify the amount of melanin in the cell or the total amount of melanin inside and outside the cell, and compared with the blank sample.

B16F0, which is a melanocyte, was used to examine the melanin production inhibitory ability of the sample. The B16F0 cell line was cultured in a cell culture flask in a CO ₂ incubator at _{37° C., 95% humidity, and 5% CO 2 in a medium in which 10% FBS was added to DMEM.}

The B16F0 cell line was dispensed into a 96-well plate at a concentration of 1×10⁴/well, and cultured in a CO _{2 incubator controlled at 37° C., 95% humidity, and 5% CO 2 for 24 hours.} Samples were prepared by concentration in a new medium, treated with cell lines, and cultured for 24 hours. To measure the viability of the cell line, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using MTS reagent. The viability of the cells was expressed as the absorbance of the sample-treated group compared to the control group not treated with the sample. Alpha-MSH was used to induce melanin production. Control (-) was treated as an untreated group, and only alpha-MSH was treated at a concentration of 2 μM in the Control (+) group. The jujube seed fermentation broth was treated by diluting the jujube seed fermentation broth with the same concentration of alpha-MSH. After incubation for 72 hours, toxicity was measured.

Toxicity was observed at the highest concentration of 0.5%, and the extracellular/intracellular melanin content was measured in a concentration range of 0.0025% to 0.25%, and is shown in Table 4 below.

Sample name (%) Cell Viability (%) Control (-) 100 ± 5.9 Control (+) 100 ± 4.5 Example 5 0.0025 101.0 ± 7.7 0.005 101.2 ± 4.8 0.025 99.4 ± 2.2 0.05 99.5 ± 8.9 0.1 97.2 ± 8.2 0.25 102.1 ± 6.6 0.5 77.7 ± 1.9* Arbutin 0.01 97.7 ± 9.1 0.1 102.7 ± 8.7 *: p <0.05, compared with Control group

< Experimental example 4> Jujube seeds Fermented Extracellular Melanin content measurement

The B16F0 cell line was dispensed into a 6-well plate at 1±10 ⁵ cells/well and cultured in a CO ₂ incubator controlled at 37° C., 95% humidity, and _{5% CO 2 for 24 hours.} Samples were prepared by concentration in DMEM medium containing no phenol red, treated with cell lines, and cultured for 24 hours. The medium was treated with a-MSH to promote the melanin production process. Cell medium is transferred to another container, and the remaining cells are washed with PBS and recovered, and the amount of protein is determined using cell lysis buffer. As for the medium, absorbance was measured at 490 nm, and the amount of melanin was calculated from a calibration curve using synthetic melanin. The amount of melanin was converted into the amount of melanin per certain number of cells or the amount of melanin per certain protein, and compared with the results of a control treated with a solvent in which the sample was dissolved. Control (-) was a non-treatment group, and only alpha-MSH was treated at a concentration of 2 μM in the Control (+) group, and the results of measuring the B16F0 extracellular melanin content of the fermentation broth of jujube seeds of Example 5 are shown in Table 5 below and FIG. Indicated.

When the concentration of fermented jujube was 0.1%, the inhibitory activity was 37.0%, and at the concentration of 0.25%, the inhibitory activity was 53.8%. Arbutin, a positive control, showed high activity of 35.4% at a concentration of 0.01% and 62.7% at a concentration of 0.1%. Jujube seed fermentation broth 0.1% and arbutin 0.01% showed similar melanin production inhibitory ability, confirming the activity of about 1/5.

In terms of extracellular melanin content. The unit price per 1 kg of arbutin is around 160,000 won, and the price of fermented jujube seeds is about 1/10 of this, and it was confirmed that the cost reduction effect was about 2 times in terms of economy.

Sample name (%) Extracellular melanin content (mg/g protein) Extracellular melanin
Inhibitory activity (%) IC ₅₀
(%) IC ₁₅
(%) Control (+) 246.4 ± 1.4 0 ± 0.6 Example 5 0.0025 219.8 ± 1.2 10.8 ± 0.5 0.22 0.004 0.005 200 ± 1.8 18.8 ± 0.7 0.025 193.8 ± 1.3 21.4 ± 0.5 0.05 187.1 ± 1.0 24.1 ± 0.4 0.1 155.2 ± 0.7 37 ± 0.3 0.25 113.9 ± 1.7 53.8 ± 0.7 Arbutin 0.01 159.1 ± 1.5 35.4 ± 0.6 0.1 91.8 ± 0.3 62.7 ± 0.1 *: p <0.05, compared with Control group

< Experimental example 5> Jujube seeds Fermented Tyrosinase Inhibitory activity measurement

B16F10 cells were dispensed into a 6 well plate at ^{a concentration of 1±10 5} cells/1.5ml/well, incubated for 24 hours for adhesion and stabilization, and then 4μM Tyrosine and jujube seed fermentation broth at a concentration of 0.0025 ~ 0.25%. After treatment, the cells were further cultured for 48 hours in a 5% CO2 incubator at 37°C. After incubation, the cells were suspended in 100 µl of 10 mM PBS containing 1% Triton X-100, vortexed, and centrifuged at 1,000 rpm for 5 minutes, and the supernatant was used as an enzyme solution for measuring activity. To measure the activity of tyrosinase, 100 µl of L-DOPA (2 mg/ml) as a substrate was added to a 96 well plate, 40 µl of enzyme solution was added, and then reacted in a 5% CO2 incubator at 37°C for 1 hour. Measurements were made at 405 nm using a Microplate Reader. The inhibition rate of tyrosinase activity for Example 5 and Comparative Example 1 was calculated as a percentage of the absorbance of the control group and is shown in Table 6 below.

When the jujube seed fermentation broth was at 0.1% concentration, it showed 95.2% inhibitory activity, and at 0.25% concentration, it showed a high inhibitory activity of 98.7%. Arbutin, a positive control, showed high activity of 99.5% at the concentration of 0.01% and 99.8% at the concentration of 0.1%. Similar Tyrosinase inhibitory activity was confirmed in 0.1% of jujube seed fermentation broth and 0.01% of arbutin, and IC ₅₀ was confirmed to be 0.05%. It can be seen that the whitening effect is very high even when only about 0.1% of the fermented jujube seed is used. It can be seen that Comparative Example 1, which is an ethanol extract of jujube seeds, has no tyrosinase inhibitory activity.

Sample name (%) Tyrosinase inhibitory activity (%) IC ₅₀
(%) IC ₂₀
(%) Control (+) 0.0 ± 0.4 0.05 0.02 Comparative Example 1 100 0 ± 0 Example 5 0.0025 -14.6 ± 0.7 0.005 -2.7 ± 0.6 0.025 15.5 ± 1.8 0.05 67.8 ± 0.4 0.1 95.2 ± 2.3 0.25 98.7 ± 1.3 Arbutin 0.01 99.5 ± 0.0 0.1 99.8 ± 0.1 *: p <0.05, compared with Control group

< Experimental example 6> Jujube seeds Fermented Collagenase Inhibitory activity measurement

For collagenase inhibitory activity, Invitrogen's EnzChek® Gelatinase/Collagenase Assay Kit (250-2000 Assays) reagent was purchased and used. First, a DQ collagen stock solution (1 mg/ml) was prepared by adding 1.0 ml of DDW to a 1 mg DQ collagen vial. The reaction buffer was diluted by adding 18 ml of DDW to 2 ml of 10 x reaction buffer. Next, a collagenase enzyme reagent was prepared. It was diluted with a reaction buffer to a final concentration of 0.2 U/ml with a working solution. Samples were prepared in a 96 well plate in a triple manner by 50 µl. Add 20 µl of DQ collagen and 30 µl of reaction buffer to the sample blank, 30 µl of working solution to the sample, and leave it for 1 to 2 hours at room temperature in a state of blocking light. The fluorescence intensity was measured with an ELISA plate reader (VICTOR X3™, PerkinElmer, US) at nm, calculated according to Equation 3 below, and shown in Table 7.

As a result of measuring the collagenase inhibitory activity of the fermented jujube seeds of Example 5 and Comparative Example 1, the inhibitory activity of 46.2% was confirmed at 0.5% concentration, and it was found that the collagenase inhibitory activity increased as the concentration increased. When compared with adenosine, which is a functional ingredient for improving wrinkles, it can be seen that the concentration of 50 ug/ml and the concentration of 0.1% jujube seed fermentation broth have similar inhibitory activity. The IC ₅₀ was confirmed to be 0.5%, and it can be confirmed that the wrinkle improvement effect is very good even when only about 0.5% of the fermented jujube seed is used. In Comparative Example 1, which is an ethanol extract of jujube seeds, it can be seen that the activity of 100% is 60.09±0.51, which is significantly lower than that of 1% of Example 1.

Sample name (%) Collagenase inhibitory activity (%) IC ₅₀ Comparative Example 1 100 60.09 ± 0.51 Example 1 0.01 8.2 ± 0.4 0.5% 0.05 12.9 ± 0.2 0.1 17.5 ± 1.6 0.5 46.2 ± 0.7 One 99.6 ± 0.1 Adenosine
(ug/ml) 0.1 1.9 ± 3.9 118.4 ug/ml 10 2.7 ± 2.3 50 21.7 ± 5.7

< Experimental example 7> Jujube seeds Fermented Elastase Inhibitory activity measurement

For elastase inhibitory activity, Invitrogen's EnzChek® Elastase Assay Kit (600 Assays) reagent was purchased and used. First, DQ elastin stock solution (1 mg/ml) was prepared by adding 1.0 ml of DDW to a 1 mg DQ Elastin vial. The reaction buffer was diluted by adding 18 ml of DDW to 2 ml of 10 x reaction buffer. Next, an elastase enzyme reagent was prepared. It was diluted with a reaction buffer to a final concentration of 0.2 U/ml with a working solution. Prepare a sample in a 96 well plate in a triple manner by 50 µl each. Add 50 µl of DQ elastin and 50 µl of reaction buffer to the sample blank, 50 µl of working solution to the sample, and leave it for 1 hour at room temperature in a state of blocking light. The fluorescence intensity was measured with an ELISA plate reader (VICTOR X3™, PerkinElmer, US) and calculated according to Equation 4 below, and the results are shown in Table 8 below.

As a result of measuring the Elastase inhibitory activity of the fermented jujube seeds of Example 5 and Comparative Example 1, the concentration of 0.00025% of the Elastase inhibitor, which is a comparative control, and 30% of the fermented jujube seed, appear to have similar Elastase inhibitory activity. In addition, it was found that the fermentation broth of jujube seeds increased the Elastase inhibitory activity as the concentration increased, and the IC ₅₀ was confirmed to be 16.9%. It can be seen that even if only about 16.9% of fermented jujube seed is used, it is very effective in improving wrinkles. It can be seen that Comparative Example 1, which is an ethanol extract of jujube seeds, has no elastase inhibitory activity.

Sample name (%) Elastase inhibitory activity (%) IC ₅₀ Comparative Example 1 100 0 ± 3.26 Example 5 One 9.8 ± 0.4 16.9% 10 37.5 ± 1.1 20 58.1 ± 1.8 30 79.3 ± 0.4 Elastase inhibitor 0.00025 74.9 ± 2.8 - 0.0025 96.2 ± 3.5 0.025 108.2 ± 2.0 0.25 103.3 ± 2.8

< Experimental example 8> Jujube seeds Fermented Determination of polyphenol and flavonoid content

The total polyphenol content was requested to be analyzed by an authorized institution and proceeded with the "Health Functional Food Code" Article 4. 3-64 Total Polyphenol Test Method, and Example 5 and Comparative Example 1 are shown in Table 9 below.

Sample name (%) Polyphenol content (㎛/ml GAE) Example 5 (100%) 5.19±0.51 Comparative Example 1 1.7

The total flavonoid content was requested to be analyzed by an accredited institution and proceeded with the "Health Functional Food Code" III.3.6.3 Total Flavonoid Test Method, and is shown in Table 10 below. Flavonoids are _{a generic term for phenolic compounds that have a pale yellow or yellow color with C 6} -C ₃ -C ₆ as a basic skeleton, and are widely distributed in nature, have high antioxidant activity by effectively removing reactive oxygen species, and have antiviral and anti-inflammatory effects. It is known that there are. It is known that only higher plants than vegetables and some green algae are synthesized, and animals have no synthetic ability, so they exist only in plants, and are known to exist in almost all parts such as leaves, flowers, fruits, stems, and roots of vegetables and related plants. Flavonoids include flavonol-based quercetin, kaempferol, and myricetin, and flavone-based apigenin, luteolin, limonin, and momilin are known. And, among flavonoids, quercetin is known to inhibit the oxidation and cytotoxicity of low-density lipoprotein (LDL) in vitro.

Sample name (%) Flavonoid content (mg/ml QE) Example 5 (100%) 0.699±0.04

In the above, the present application has been described in detail with reference to preferred embodiments and experimental examples, but the present application is not limited to the above embodiments and experimental examples, and those having ordinary knowledge in the art within the spirit and scope of the present application Various modifications and changes are possible by the user.

Those of ordinary skill in the field to which the present application belongs will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

As described above, a specific part of the present application has been described in detail, and it is clear that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present application is not limited thereto. Accordingly, it will be said that the substantial scope of the present application is defined by the appended claims and their equivalents.

Claims (6)

Separating and pulverizing the seeds from the jujube;
Sterilizing the jujube seed powder and then inoculating the lactic acid bacteria, yeast or Bacillus into fermentation to obtain a fermented jujube seed; And
A method for producing a fermented jujube seed comprising a; step of removing impurities after sterilizing the fermented jujube seed. The method according to claim 1,
The powdering step,
Freeze drying, hot air drying or natural drying to crush, or
A method for producing fermented jujube seeds, characterized in that pulverized or crushed to powder after refrigerated or frozen storage. The method according to claim 1,
The lactic acid bacteria, yeast or Bacillus bacillus,
Lactobacillus plantarum , Bacillus subtilis , Leuconostoc mecentroid mesenteroide ) or Saccharomyces cerevisiae (Saccharomyces cerevisiae). The method according to claim 1,
In the step of obtaining the fermented product,
After mixing the jujube seed powder and the medium in a weight ratio of 1:4 to 1:20, sterilized,
Method for producing a fermented jujube seed, characterized in that the fermentation is carried out by culturing for 1 to 10 days at 100 rpm to 300 rpm, 20 to 45 ℃. The method according to claim 1,
The step of removing the impurities,
A method for producing a fermented jujube seed, characterized in that the fermented product is filtered or centrifuged to a size of 0.3 µm to 5 µm. The method according to claim 1,
The sterilization method for producing fermented jujube seeds, characterized in that performed for 10 to 30 minutes at 100 ℃ to 140 ℃.

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