KR102032033B1 - Fermentation method of lactic acid bacteria using citrus fruit and its use - Google Patents

Abstract

The present invention relates to a method for fermenting lactic acid bacteria using citrus fruit and its use, and more particularly, to Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 ( Citrus fruit fermentation product prepared using Lactobacillus rhamnosus CBG-C14) strain has excellent antioxidant activity and skin regeneration effect, so it is useful for skin regeneration or wound treatment cosmetics, health functional food, medicine, etc. Can be.

Description

Fermentation method of lactic acid bacteria using citrus fruit and its use}

The present invention relates to a method for fermenting lactic acid bacteria using citrus fruit and its use.

The skin is composed of three layers of epidermis, dermis and subcutaneous fat tissue in order from the outside and serves as a primary barrier to protect the human body from the external environment. Of these, the stratum corneum, located in the outermost layer of epidermal tissue, consists of keratin and lipids, inhibits the penetration of harmful substances and evaporates water, and protects the human body from bacteria and external stimuli.

The stratum corneum is stripped every 4 weeks (28 days) to form a new. In addition, fibroblasts mainly distributed in the upper part of the dermis play a role of hair cells to produce collagen and elastin, which are fiber proteins having skin elasticity and protective functions. However, changes in external temperature and humidity, ultraviolet rays, and pollutants, as well as excessive physical and chemical stimuli, stress, and malnutrition from the outside, seriously degrade normal functions such as moisturizing function of the skin, and promote skin aging. To prevent this phenomenon, in order to maintain healthier and more beautiful skin, cosmetics contain bioactive substances from various animals, plants, and microorganisms. come.

Elastase, which is expressed in fibroblasts, is a protease that decomposes elastin, which regulates skin elasticity, and elastin is a protein fiber that gives elasticity to cellular tissues and is an important protein constituting skin connective tissue. to be. Elastase breaks down the collagen and fibrinogen immune system, destroys microorganisms that invade from the outside, breaks down the space and proteins required for cell regeneration, and rapidly increases expression levels and causes excessive wrinkles on the skin as aging progresses.

The skin is increasingly damaged by age or ultraviolet rays, external contaminants and stress, and the ability to protect the skin from these factors is weakened, resulting in poor cellular protection and proliferation. In this case, our body begins the skin regeneration process against the damaged skin. Skin regeneration is a tissue response to injury and is a process of tissue recovery, including chemotaxis, differentiation and replication of cells, synthesis of matrix proteins, angiogenesis and wound reconstruction. It is a complex biological process that includes.

When the skin is damaged, the cell proliferation process is followed by an inflammatory response, which lasts from two days to three weeks after the skin is damaged. At this time, fibroblasts accumulate collagen to fill damaged skin flaws, induce proliferation of fibroblasts, and reconstruct new cells and interstitial components at the wound site. This multiplication process, also called the granulation process, lasts from two days to three weeks. The young skin by the new cells is active in the skin cells, the dermis fibroblast (derived fibroblast) is active in the synthesis and metabolism of collagen (collagen) important for inhibiting the elasticity of the skin and wrinkle formation. In addition, GAGs (Glucosaminoglycans), which are glycoproteins that contain hyaluronic acid, which is essential for skin moisturization, are also active, giving skin elasticity, moisture, and flexibility. In epidermis, basal layer cell proliferation is active, and the production of adhesion proteins such as laminin, integrin and desmosome, which enhance the binding between skin cells, is balanced, thereby strengthening the tissue. It keeps your skin healthy.

Citrus fruits contain various physiologically active substances such as flavonoids, carotenoids, kurmarins and essential oils, and various functional evaluations have been actively conducted in Japan and Korea. In particular, the attention of related industries has been focused on recent research on representative flavonoid components such as hesperidin and naringin among the bioactive substances in citrus peels.

Citrus is one of the flights and (Rutaceae) in citrus fruit eat enjoy worldwide is grown in more than 100 countries on six continents with edible plants (Citrus sp.). Citrus fruits contain a large amount of various biologically active substances such as carotenoids, flavonoids, cellulose, pectin, limonoids, vitamins, etc. Is reported.

Lactic acid bacteria are microorganisms that produce lactic acid and various metabolites by fermenting sugars. Recently, researches as probiotics to promote health and prevent diseases have been widely conducted. It is widely used. In addition, lactic acid bacteria ingested into the human body through fermented foods are introduced into the intestine to support the growth of beneficial strains and inhibit harmful bacteria to maintain the balance of useful microorganisms in the intestine. In addition, antioxidant activity, anti-cancer action, immune activation action, blood cholesterol lowering action is reported to help prevent disease.

On the other hand, Korean Patent Publication No. 2013-0027227 discloses a cosmetic for improving the skin condition using a citrus flower, Korean Patent No. 15183333 discloses a hair loss prevention or hair growth promoting composition comprising a citrus peel extract. However, the lactic acid bacteria fermentation method using the citrus fruit of the present invention and its use has not been disclosed yet.

The present invention has been derived by the above requirements, in the present invention, Lactobacillus plantarum CBG-C21 ( Lactobacillus plantarum CBG-C21, KACC92083P) strain in the extract citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) Citrus fruit fermented products were inoculated with Lactobacillus rhamnosus CBG-C14 and KACC91981P strains, respectively, to increase total polyphenol content, total flavonoid content and antioxidant activity than before fermentation. In addition to better skin regeneration effect than the extract, skin cell regeneration rate is significantly increased compared to citrus fruit fermented product inoculated with the other strains Lactobacillus plantarum (KCTC21004) and Lactobacillus rhamnosus (KCTC3237), respectively. By confirming that, this invention was completed.

In order to solve the above problems, the present invention 1) washing the selected citrus fruit (Citrus fruit); 2) adding water to the citrus fruit washed in step 1), pulverizing and extracting it; And 3) inoculating and fermenting lactic acid bacteria to the citrus fruit extract of step 2). The method provides a method of producing a citrus fruit fermentation product having a skin regeneration effect, comprising the step of fermentation.

In addition, the present invention provides a citrus fruit fermented product having a skin regeneration effect produced by the above production method.

The present invention also provides a cosmetic composition for skin regeneration containing the citrus fruit fermented product as an active ingredient.

The present invention also provides a health functional food composition for skin regeneration containing the citrus fruit fermented product as an active ingredient.

The present invention also provides a pharmaceutical composition for treating wounds containing the citrus fruit fermented product as an active ingredient.

Citrus fruit fermentation products prepared using Lactobacillus plantarum CBG-C21, KACC92083P strains and Lactobacillus rhamnosus CBG-C14 strains of the present invention have antioxidant activity and skin Since the regeneration effect is excellent, it can be usefully used for skin regeneration or wound treatment cosmetics, health functional foods, pharmaceuticals, etc. including the active ingredient.

1 is Lactobacillus plantarum CBG-C21 ( Lactobacillus plantarum CBG-C21, KACC92083P) strain (A) and Lactobacillus rhamnosus CBG-C14 ( Lactobacillus ) in the extract of citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) After inoculating rhamnosus CBG-C14, KACC91981P) strain (B), respectively, it is the result of confirming the pH change of the fermentation product with the fermentation time.
FIG. 2 shows Lactobacillus plantarum CBG-C21 (KACC92083P) strain (A) and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain (B) in citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) extracts After each inoculation, it is a result confirming the viable cell number of the two strains of lactic acid bacteria according to the fermentation time.
Figure 3 shows the Lactobacillus plantarum CBG-C21 (KACC92083P) strain (A) and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain (B) in the extract of citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) Inoculation was carried out to confirm the change in the total polyphenol content before and after fermentation.
FIG. 4 shows Lactobacillus plantarum CBG-C21 (KACC92083P) strain (A) and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain (B) on citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) extracts Each inoculation was the result of confirming the change in the total flavonoid content before and after fermentation.
FIG. 5 shows Lactobacillus plantarum CBG-C21 (KACC92083P) strain (A) and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain (B) in citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) extracts Inoculation was carried out to confirm the change in antioxidant activity before and after fermentation.

In order to achieve the object of the present invention, the present invention

1) washing the selected citrus fruit (Citrus fruit);

2) adding water to the citrus fruit washed in step 1), pulverizing and extracting it; And

3) inoculating fermentation by lactic acid bacteria in the citrus fruit extract of step 2) provides a method for producing a citrus fruit fermentation product having a skin regeneration effect, comprising the step of producing.

In the method of producing a citrus fruit fermentation product having a skin regeneration effect of the present invention, the citrus fruit extract may be extracted using various extraction methods such as solvent extraction, physical extraction, enzymatic extraction, but preferably using a physical extraction method It can be extracted, but is not limited thereto. The extraction method does not destroy the active ingredient, it is possible to obtain a highly active fermented product from citrus fruit.

The citrus fruit extract may include any one of an extract obtained by an extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product.

Further, the citrus fruit may be a tangerine (Citrus unshiu), lemon (Citrus limon), orange (Citrus sinensis), yuzu (Citrus junos), grapefruit (Citrus paradisi) or Hallabong (Citrus hybrid Shiranuhi), but are not limited to, .

In addition, the citrus fruit is preferably aged at least 85%, but is not limited thereto.

In addition, the lactic acid bacteria may be Lactobacillus plantarum strain, Lactobacillus rhamnosus strain or a mixed strain thereof, preferably Lactobacillus plantarum CBG-C21 ( Lactobacillus plantarum CBG-C21) (KACC92083P) strain, Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain (see Korean Patent No. 1655852) or a mixture thereof, but is not limited thereto. The Lactobacillus plan tarum CBG-C21 (Lactobacillus plantarum CBG-C21) (KACC92083P) strain was deposited on July 10, 2015 by the Institute of Agricultural Biotechnology.

In addition, the step 3) may be inoculated with lactic acid bacteria of 1.0 × 10 ⁷ to 1.0 × 10 ⁹ CFU / ㎖ to citrus fruit extract, preferably may be inoculated with lactic acid bacteria of 1.0 × 10 ⁸ CFU / ㎖ This is not restrictive.

In addition, the fermentation in step 3) may be performed for 20 to 28 hours at 32 ~ 42 ℃, preferably may be performed for 24 hours at 37 ℃, but is not limited thereto.

In addition, the number of live bacteria of lactic acid bacteria of the citrus fruit fermentation of step 3) may be 6.0 × 10 ⁸ to 1.0 × 10 ¹⁰ CFU / ㎖, preferably 6.0 × 10 ⁹ CFU / ㎖, but is not limited thereto.

In addition, the step 2) and the step 3) may further include the step of treating the enzyme to the citrus fruit extract, but is not limited thereto.

In addition, the enzyme treatment may be a cellulase, pectinase, or a mixture thereof, but is not limited thereto.

In addition, the present invention provides a citrus fruit fermented product having a skin regeneration effect produced by the above production method.

The present invention also provides a cosmetic composition for skin regeneration containing the citrus fruit fermented product as an active ingredient.

The cosmetic composition for skin regeneration of the present invention includes components conventionally used in cosmetic compositions in addition to the active ingredient, for example, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers , Foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or Lipophilic active agents, conventional adjuvants such as lipid vesicles, and carriers.

The cosmetic composition for skin regeneration of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, oils, powder foundations It may be formulated as emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, Skin, Skin Softener, Skin Toner, Astringent, Lotion, Milk Lotion, Moisture Lotion, Nutrition Lotion, Massage Cream, Nutrition Cream, Eye Cream, Moisture Cream, Hand Cream, Essence, Nutrition Essence, Pack, Cleansing It can be prepared in cosmetic formulations of foams, cleansing water, cleansing lotions, cleansing creams, body lotions, body cleansers, soaps and powders.

In case the formulation of the cosmetic composition of the present invention is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluoro hydro Propellant such as carbon, propane / butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester as carrier components , Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

The present invention also provides a health functional food composition for skin regeneration containing the citrus fruit fermented product as an active ingredient.

In the health functional food composition for skin regeneration of the present invention, there is no particular limitation on the kind of the food. Examples of foods to which the above-mentioned substances may be added include dairy products, various soups, drinks, meat, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and the like includes all the health functional foods in the conventional sense.

The health functional food may be prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrups and beverages, but is not limited thereto and may be manufactured and processed in any form according to the law.

Citrus fruit fermentation products having a skin regeneration effect of the present invention can be added to food as it is, or used with other foods or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use thereof. In general, the amount of the citrus fruit fermentation product in the health food may be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The functional beverage composition of the present invention is not particularly limited to other ingredients contained in the indicated proportions other than the citrus fruit fermented product as essential ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above citrus fruit fermented product of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, such as colorants and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the citrus fruit fermented product of the present invention may contain a pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination.

The present invention also provides a pharmaceutical composition for treating wounds containing the citrus fruit fermented product as an active ingredient.

The pharmaceutical composition for wound treatment of the present invention may further include a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition.

Pharmaceutical dosage forms of the compositions according to the invention can be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the citrus fruit fermentation product include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Various compounds or mixtures including calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be mentioned. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the citrus fruit fermentation. The mixture is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.

The pharmaceutical composition for wound treatment containing the citrus fruit fermentation product of the present invention as an active ingredient may be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention more specifically, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Production Example  1. Citrus Fruit Extract and Its Fermented products  Produce

After selecting and washing more than 85% of ripe citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong), 1,000 parts by weight of water is added to 1 part by weight of the washed citrus fruits and crushed by a crusher. The extract was obtained and then filtered using cotton cloth. The filtered citrus fruit extracts were sterilized at 121 ° C. for 15 minutes, followed by Lactobacillus plantarum CBG-C21 ( Lactobacillus plantarum CBG-C21, KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 ( Lactobacillus rhamnosus CBG-C14, KACC91981P) The strains were inoculated at 1.0 × 10 ⁸ CFU / mL concentrations and fermented at 37 ° C. for 24 hours to obtain respective citrus fruit fermentation products (hereinafter referred to as “samples”).

Citrus fruit water extract and citrus fruit ethanol extract used as a comparative control were screened and washed citrus fruit by the same method as described above, and added 500 parts by weight of water and ethanol to 1 part by weight of citrus fruit, respectively, at 80 ° C. for 6 hours. During extraction.

Production Example  2. Citrus Fruits Treated with Cellulase Fermented products  Produce

After selecting and washing more than 85% of ripe citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong), 1,000 parts by weight of water is added to 1 part by weight of the washed citrus fruits and crushed by a crusher. The extract was obtained and then filtered using cotton cloth. The filtered citrus fruit extract was treated with cellulase and then sterilized at 121 ° C. for 15 minutes, followed by Lactobacillus plantarum CBG-C21 and KACC92083P strains and Lactobacillus rhamnosus CBG-C14 ( Lactobacillus rhamnosus CBG-C14, KACC91981P) strains were inoculated at a concentration of 1.0 × 10 ⁸ CFU / mL and fermented at 37 ° C. for 24 hours to obtain respective citrus fruit fermentation products (hereinafter referred to as “samples”).

Production Example  3. Pectinase  Processed citrus fruit Fermented products  Produce

After selecting and washing more than 85% of ripe citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong), 1,000 parts by weight of water is added to 1 part by weight of the washed citrus fruits and crushed by a crusher. The extract was obtained and then filtered using cotton cloth. After treating the filtered citrus fruit extract with pectinase and sterilizing for 15 minutes at 121 ℃, Lactobacillus plantarum CBG-C21 ( Lactobacillus plantarum CBG-C21, KACC92083P) strain and Lactobacillus rhamnosus CBG- C14 ( Lactobacillus rhamnosus CBG-C14, KACC91981P) strains were inoculated at a concentration of 1.0 × 10 ⁸ CFU / mL and fermented at 37 ° C. for 24 hours to obtain respective citrus fruit fermentation products (hereinafter referred to as 'samples').

Example  Citrus fruit Fermented product  pH and Lactic Acid Bacteria Change

In Example 1, Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) in the citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) extract prepared in Preparation Example 1 After inoculating each strain, fermented products were collected every 2 hours while fermenting for 24 hours to measure pH, and every 3 hours, the plate count medium (Plate Count Agar with Brom Cresol Purple) was added to the viable count of lactic acid bacteria. It measured using. As a result, Lactobacillus plantarum CBG-C21 strain and Lactobacillus rhamnosus CBG-C14 strain of the present invention for citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) extracts as disclosed in Figures 1 and 2 When inoculated, all of the pH value was reduced with the fermentation time, it was confirmed that the number of lactic acid bacteria viable bacteria. Through this, all of the lactic acid bacteria strains used in the present invention were able to survive and develop at pH 2 to 4, and were confirmed to have excellent acid resistance.

Example  2. citrus fruit Fermented product  Changes in Total Polyphenols, Total Flavonoid Contents and Antioxidant Activity

In Example 2, the Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) for the citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) extract prepared in Preparation Example 1 ), The total polyphenol content, total flavonoid content and antioxidant activity before and after fermentation were measured and compared.

(1) total polyphenol content

The total polyphenol compound content was measured by slightly modifying the Folin-Denis method using a phenomenon in which the phenolic substance reacted with phosphomolybdate to appear blue.

Citrus fruit fermentation prepared by inoculating citrus fruit (citrus fruits, lemon, orange, citron, grapefruit and Hallabong) extract with Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain Water was prepared at 50 mg / mL, 20 μL was taken from this, diluted with 1.4 mL of distilled water in a 2 mL tube, and shaken by addition of 0.1 mL of Folin-Denis reagent. After 3 minutes, 0.2 ml of saturated sodium carbonate solution was added, the mixture was mixed well with 2 ml of the total solution, and left at room temperature for 1 hour, followed by spectrophotometer (DU 650, Beckman Coulter Inc., Miami, FL, USA) was used to measure absorbance at 725 nm. At this time, the total polyphenol compound was determined from the standard curve prepared using tannic acid.

As a result, when the fermentation using the Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain for the citrus fruit as shown in Figure 3, respectively, than the total polyphenols It was confirmed that the content was increased.

(2) total flavonoid content

Total flavonoid content was determined in 0.5 ml of citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) extracts according to Moreno et al., 2000, J. Enthrophamacol., 71, 109-114. Add 0.1 ml of aluminum nitrate and 1M potassium acetate, add 4.3 ml of ethanol, mix and leave at room temperature for 40 minutes, and use ELISA reader (VERSA, USA) at 415 nm. Absorbance was measured. The total flavonoid content was calculated from the standard curve prepared using quercetin.

As a result, when the fermentation using the Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain for the citrus fruit extract as shown in Figure 4, respectively, than the total flavonoids It was confirmed that the content was increased.

(3) antioxidant activity

Citrus fruit fermented by inoculating citrus fruit (citrus, lemon, orange, citron, grapefruit and Hallabong) extracts with Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain, respectively. Was dissolved by using 80% methanol. 0.2 ml of the citrus fruit extract and the fermented product were added to the test tube, and 0.8 ml of 0.2 mM DPPH solution (dissolved in 80% methanol) was added thereto, mixed for 10 seconds by a vortex mixer, and left at room temperature for 30 minutes. Absorbance was measured at 525 nm. Electron Donating Ability (EDA) was calculated using the following equation, and the required sample concentration was calculated as EC ₅₀ (Efficient Concentration) until the initial DPPH concentration was reduced by 50%.

EDA (%) = (1-absorbance of sample / absorbance of control) × 100

As a result, as shown in Figure 5, when the fermentation using the Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain for the citrus fruit extract as shown in Figure 5, the antioxidant activity than before It was confirmed that this increased.

Example  3. citrus fruit Fermented product  Skin regeneration efficacy evaluation

Fermentation of citrus fruits (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) fermented with Lactobacillus plantarum CBG-C21 (KACC92083P) strain prepared in Preparation Example 1 and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain In order to confirm the skin regeneration effect of water (hereinafter referred to as 'sample'), a wound healing assay was performed.

First, keratinocytes (HaCaT) were adjusted to 5 × 10 ⁵ cells / well using DMEM medium to which 10% FBS was added, and then inoculated into 6-well plates and incubated for 18 hours. Scratch the cells grown in the 6-well plate to remove a portion of the cells from the bottom of the 6-well plate, and then sample the 1%, 10%, 20%, 30%, 40%, 50%, 60% and 70 The cells were incubated at 18% concentration for 18 hours. The degree of regeneration of the cells was confirmed, and the results are shown in Tables 1 to 5 below. Cell regeneration rate was calculated by the following formula, the hyaluronic acid (Hyaluronic Acid) was used as a positive control, the negative control means that nothing was treated, the experiment was carried out in three iterations.

% Cell regeneration rate = (sample cell regeneration rate / negative control cell regeneration rate) × 100

As a result, citrus fruit (citrus fruits, lemons, oranges, citron, grapefruit and Hallabong) fermented using Lactobacillus plantarum CBG-C21 (KACC92083P) strain and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strain of the present invention. It was confirmed that the rate of cell regeneration increased in all concentrations of water up to 40% treatment concentration (Table 1 and Table 3).

In addition, the cell regeneration rate of each citrus fruit fermentation fermented with the Lactobacillus plantarum CBG-C21 (KACC92083P) strain of the present invention and the Lactobacillus plantarum KCTC21004 strain, a control group thereof, showed the highest cell regeneration rate. At% treatment concentration, the citrus fruit fermentation product produced using the Lactobacillus plantarum CBG-C21 (KACC92083P) strain of the present invention was compared with the citrus fruit fermentation product produced using the Lactobacillus plantarum KCTC21004 strain as a control. Higher cell regeneration effect (Table 2), and Lactobacillus rhamnosus CBG-C14 (KACC91981P) strains also had a higher cell regeneration rate than citrus fruit fermented with the control control Lactobacillus rhamnosus KCTC3237 strain. It was confirmed that the skin regeneration effect is higher (Table 4).

In addition, in order to compare the efficacy between the citrus fruit extract and the fermented product on the cell regeneration efficacy, the control group used the citrus water extract and ethanol extract prepared in Preparation Example 1, the test group was Lactobacillus plantarum CBG-C21 Citrus fruit fermented with the strain was compared at 40% treatment concentration showing the highest activity in cell activity. As a result, as shown in Table 5 below, the cell regeneration effect of the citrus fruit fermented by the Lactobacillus plantarum CBG-C21 (KACC92083P) strain is more than 10% superior to the citrus fruit water extract and ethanol extract It was confirmed.

Measurement of Cell Regeneration by Treatment of Citrus Fruit Fermented with Lactobacillus Plantarum CBG-C21 (KACC92083P) Strains sample Cell renewal rate (%) positivity
Control Test group One% 10% 20% 30% 40% 50% 60% 70% Citrus 82.3 86.1 85.7 88.3 90.4 92.1 92.3 92.1 92.3 lemon 82.3 83.4 84.3 87.2 89.6 90.2 90.3 90.5 89.4 Orange 82.3 84.2 84.6 86.3 88.6 91.7 91.3 91.4 90.5 Citron 82.3 83.1 84.7 87.6 90.4 90.4 90.3 90.2 89.9 grapefruit 82.3 82.6 85.3 87.5 90.8 91.1 91.2 90.9 90.8 Hallabong 82.3 83.3 85.1 86.3 89.7 92.7 92.3 92.4 92.6

Comparison of Cell Regeneration Rates of Citrus Fruit Ferment Prepared by Lactobacillus Plantarum Strains division Citrus lemon Orange Citron grapefruit Hallabong Comparative control
(KCTC21004) 84.2 83.7 85.1 82.4 83.6 84.2 Test group
(CBG-C21) 92.1 90.2 91.7 90.4 91.1 92.7

Measurement of Cell Regeneration by Treatment of Citrus Fruit Fermentation Fermented with Lactobacillus rhamnosus CBG-C14 (KACC91981P) Strain sample Cell renewal rate (%) positivity
Control Test group One% 10% 20% 30% 40% 50% 60% 70% Citrus 82.3 85.4 85.0 87.6 89.7 91.4 91.6 91.4 91.6 lemon 82.3 82.7 83.6 86.5 88.9 89.5 89.6 89.8 88.7 Orange 82.3 83.5 83.9 85.6 87.9 91.0 90.5 90.7 89.8 Citron 82.3 82.4 84.0 86.9 89.7 89.7 89.6 89.5 89.2 grapefruit 82.3 81.9 84.6 86.8 90.1 90.4 90.5 90.2 90.1 Hallabong 82.3 82.6 84.4 85.6 89.0 92.0 91.6 91.7 91.9

Comparison of Cell Regeneration Rates of Citrus Fruit Ferment Prepared by Lactobacillus rhamnosus Strains division Citrus lemon Orange Citron grapefruit Hallabong Comparative control
(KCTC3237) 84.5 84.0 85.4 82.7 83.9 84.5 Test group
(CBG-C14) 91.4 89.5 91.0 89.7 90.4 92.0

Institute of Agricultural Biotechnology KACC92083P 20150710

Claims (11)

1) washing the selected citrus fruit (Citrus fruit);
2) adding water to the citrus fruit washed in step 1), pulverizing and extracting it; And
3) Lactobacillus plantarum CBG-C21 (KACC92083P) strain, Lactobacillus rhamnosus CBG-C14 ( Lactobacillus rhamnosus CBG-C14) (KACC91981P) strain or these in the citrus fruit extract of step 2) Inoculating the mixed strain of the step of fermentation for 20 to 28 hours at 32 ~ 42 ℃; Method of producing a citrus fruit fermented product having a skin regeneration effect comprising the. The method of claim 1, wherein the citrus fruit is characterized in that the citrus fruits (Citrus unshiu), lemon (Citrus limon), orange (Citrus sinensis), yuzu (Citrus junos), grapefruit (Citrus paradisi) or Hallabong (Citrus hybrid Shiranuhi) Citrus fruit fermented product having a skin regeneration effect. delete delete delete The method of claim 1, further comprising treating the citrus fruit extract with an enzyme between step 2) and step 3). The method of claim 6, wherein the enzyme treatment is a cellulase, pectinase or a method of producing a citrus fruit fermentation product having a skin regeneration effect, characterized in that the mixed treatment. Citrus fruit fermented product having a skin regeneration effect produced by the method of any one of claims 1, 2, 6 and 7. A cosmetic composition for skin regeneration comprising the citrus fruit fermentation product of claim 8 as an active ingredient. A health functional food composition for skin regeneration comprising the citrus fruit fermentation product of claim 8 as an active ingredient. A pharmaceutical composition for treating wounds, comprising the citrus fruit fermentation product of claim 8 as an active ingredient.