KR102155716B1 - A carrot fermentation method using lactobacillus plantarum ami-1103 strain - Google Patents

Abstract

The present invention relates to a method for preparing a carrot fermented product using Lactobacillus plantarum and a carrot fermented product prepared by the method. The carrot fermented product may be used in a cosmetic composition.

Description

Carrot fermented product manufacturing method using Lactobacillus plantarum AMI-1103 strain {A CARROT FERMENTATION METHOD USING LACTOBACILLUS PLANTARUM AMI-1103 STRAIN}

The present invention relates to a method for producing a fermented carrot using Lactobacillus plantarum and a fermented carrot prepared by the same method. The carrot fermented product may be used in a cosmetic composition.

The skin is a single membrane covering the outside of the body and plays a role in protecting and regulating internal organs and other internal organs by performing various physiological functions that protect the body from environmental factors such as various external stimuli, obstacles, and dryness. do.

However, as the skin ages, the physiological function of the living body decreases, the secretion of various hormones regulating metabolism decreases, and the function of immune cells and the activity of cells decrease. In addition, the appearance or function of the skin changes due to repeated exposure to light or heat. As a result, free radicals and free radicals and active harmful oxygen increase. Excessive physical and chemical irritation and stress are caused by deterioration of the normal function of the skin and melanin deposition. It damages the skin by promoting the generation of freckles and skin aging.

In order to prevent such skin aging and damage to maintain healthier and more beautiful skin, by adding physiologically active substances obtained from various animals, plants, microorganisms, etc. to cosmetics, it maintains the skin's own function and activates the skin cells to make the skin budding. Efforts to maintain skin health by promoting metabolism are continuing, and cosmetics and external preparations for skin are being developed accordingly.

However, when these substances are applied to the skin, the amount of use of these substances is limited due to safety problems such as irritation, erythema, and redness, or the effect is insignificant, making it difficult to substantially improve skin function. Therefore, there is a need to develop a new composition for external application for skin that is safer to the living body and has higher effect than the existing composition for external application for skin.

Meanwhile, around the world, there is no problem with taste and nutrients, but ugly food campaigns using agricultural products that are thrown away due to their appearance and size are expanding. Recently, carrots are one of the mass-produced non-commodity agricultural products in Jeju, and there is a problem of disposing of farmland. Therefore, research and development and efforts to improve the utilization value of carrots are required.

Korean Patent Publication No. 10-2010-0063459

An object of the present invention is to provide a method for producing a carrot fermented product with enhanced antioxidant activity comprising the step of inoculating and fermenting a carrot extract with Lactobacillus plantarum .

Another object of the present invention is to provide a carrot fermented product prepared by the above manufacturing method.

Another object of the present invention is to provide a cosmetic composition with improved antioxidant activity comprising the fermented carrot.

One aspect of the present invention for achieving the above object relates to a method for producing a fermented carrot with enhanced antioxidant activity, comprising the step of inoculating and fermenting a strain of Lactobacillus plantarum in a carrot extract. .

In the present invention, "carrot ( Daucus carota subsp. sativus )" is a component containing a large amount of ingredients of carotene and vitamin C, which are substances that change into vitamin A in the body. In particular, the carotene has the effect of incapacitating carcinogens and toxic substances, and preventing free radicals from damaging the cells in the body, so the extract extracted from the carrot is often used for keratin relief, skin elasticity maintenance, and skin aging prevention. have.

In particular, carrots are one of the non-commodity wintering vegetables that are recently generated in large quantities in Jeju, and in the present invention, a fermentation process that can enhance the efficacy in the use of carrots has been developed. The value of the resources to be saved was made possible.

In the present invention, “extract” refers to an extract obtained by extraction treatment of carrots, a dilution or concentrate of the extracts, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract It includes extracts of all formulations that can be formed using.

The extract of the present invention may be extracted from natural, hybrid or variant plants of the carrot, and may be extracted from plant tissue culture.

The method of extracting the extract of the present invention is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, a cold needle extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, and these may be performed alone or in combination of two or more methods. Specifically, as an example of a method of extracting the carrot extract, a cold needle extraction method may be used.

The type of the extraction solvent used in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, C ₁ to C ₄ anhydrous or lower alcohol, a mixed solvent of water and lower alcohol, acetone, 1,3-butylene glycol, ethyl acetate, selected from the group consisting of chloroform It can be used alone or in combination of two or more.

The active ingredient contained in the raw material may have a different extraction ratio depending on the polarity of the solvent. In particular, the solvent may preferably be an alcohol having excellent selectivity in the extraction of the physiologically active substance of the raw material, and preferably, an optimal skin improvement effect may be realized by ethanol extraction.

In particular, since water and ethanol have different polarities, the active ingredient extracted according to each polarity may be different, so that the concentration of ethanol may be appropriately controlled so that an optimal skin improvement effect can be realized. However, if the concentration of the ethanol is less than 50%, the active ingredient that exhibits skin improvement effect may not be sufficiently extracted, and if the concentration is more than 80%, an appropriate yield may not be implemented, so that the concentration of ethanol is within the above range. Can be adjusted appropriately in

Specifically, the extraction solvent of the carrot extract may be ethanol, and more specifically, 70% ethanol.

In an embodiment of the present invention, a carrot extract was prepared through cold chim extraction using 70% ethanol.

In the present invention, the cold pickled carrot extract is filtered to remove suspended solid particles, for example, nylon, etc. to filter the particles, or after filtration using a freeze filtration method, etc., or freeze drying, hot air drying, spray drying It can be used after drying using the like.

In the present invention, a method for increasing the utilization of broccoli by improving the antioxidant activity of the carrot extract was studied, and it was confirmed that the antioxidant activity was improved when the carrot extract was fermented using the Lactobacillus plantarum AMI-1103 strain. In particular, it was confirmed that the degree of antioxidant activity was different depending on the extraction solvent and the temperature conditions of the fermentation, and an optimal method for preparing a fermented carrot with excellent antioxidant activity was identified.

Specifically, the Lactobacillus plantarum may be a Lactobacillus plantarum AMI-1103 ( Lactobacillus plantarum AMI-1103) KCCM12384P strain.

In the present invention, the strain showing the highest molecular phylogenetic relationship with Lactobacillus plantarum was identified, named Lactobacillus plantarum AMI-1103, and deposited with the Korea Microbial Conservation Center on November 12, 2018 ( Accession number KCCM12384P).

In the present invention, "fermentation" refers to a process of decomposing organic matter using enzymes possessed by the fermentation strain itself. It is known that raw materials that have undergone a fermentation process through a fermentation strain have an effect of at least two to tens of times greater than before fermentation. In particular, the fermented metabolites contain antioxidants including various amino acids and organic acids that are good for the skin, and have the effect of improving the skin.

In the present invention, “antioxidation” refers to an action of inhibiting oxidation, and in the human body, an oxidation promoting substance and an oxidation inhibitor are in balance, but due to various factors, the balance is lost and the direction of promoting oxidation When inclined toward, oxidative stress is induced in a living body, leading to cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of oxidative stress, are chemically unstable and highly reactive, so they can easily react with various biomaterials such as DNA, proteins, lipids, and carbohydrates. Attacks cause irreversible damage to cells and tissues, or cause mutations, cytotoxicity and cancer, and are also a direct cause of aging. By removing or reducing these reactive oxygen species, antioxidant effects are obtained, preventing aging and maintaining health.

In one embodiment of the present invention, as a result of inoculating carrot extract with Lactobacillus plantarum AMI-1103 strain and measuring free radical scavenging activity to confirm the antioxidant effect, the Lactobacillus plantarum AMI-1103 strain was It was confirmed that the carrot fermented product obtained by fermenting the extract by inoculation significantly increased the antioxidant effect compared to the carrot extract (Table 4 and Table 5).

In one embodiment of the present invention, as a result of confirming the polaronoid content of fermented carrots, it was confirmed that, unlike carrot extract, the content of camperol, a type of polaronoid, was significantly increased (Table 7).

Also specifically, the fermentation may be performed at 15°C or more and 25°C or less.

In one embodiment of the present invention, as a result of low-temperature fermentation of carrot extract at 15 to 25°C, it was confirmed that the free radical scavenging activity was excellent compared to the carrot fermented product fermented at 37 to 42°C, and the content of polaronoids was also high. Confirmed (Tables 4, 5 and 7).

In addition, the carrot fermented product may improve not only the antioxidant activity but also the beneficial bacteria enhancement and/or enhancement promoting efficacy in the ingredient through the process of fermenting the carrot extract into the Lactobacillus plantarum AMI-1103 strain.

Specifically, the carrot fermented product may further include the use of maintaining the balance of microbiome in the skin.

In the present invention, “microbiome” is a compound word of microbiota and genome and refers to a community of microorganisms and the entire genome that coexist in humans, animals and plants, soil, sea, lake, rock wall, atmosphere, etc. . Among them, the microbiome in the skin is a microbial group that forms a complex interrelationship between microbes and microbes, and hosts and microbes existing in the skin, and refers to a part of the human microbiome.

The “maintaining the balance of microbiome in the skin” refers to maintaining the balance of microbiome in the skin by inhibiting the proliferation and/or activity of harmful bacteria in the skin while promoting the activity and survival of beneficial bacteria in the skin.

More specifically, the maintenance of the microbiome balance may be achieved by promoting skin beneficial bacteria.

In the present invention, "promoting skin beneficial bacteria" means that there are more than 2 million microorganisms of about 1,000 types per 2 cm ² in the skin, of which, by promoting the promotion of beneficial bacteria in the skin and activating the growth of beneficial bacteria, the balance of microorganisms in the skin is maintained. It means letting go.

In one embodiment of the present invention, it was confirmed that the carrot fermented product prepared by inoculating the carrot extract with Lactobacillus plantarum AMI-1103 strain promotes the growth of Leuconostoc mesenteorides bacteria, a kind of beneficial bacteria. (Table 8), it was confirmed that the fermented carrot can be used for maintaining the balance of microbiome in the skin.

Another aspect of the present invention may relate to a carrot fermented product prepared by the above manufacturing method.

Another aspect of the present invention may relate to a cosmetic composition with improved antioxidant activity, including a fermented carrot prepared by the above manufacturing method.

The'carrot fermented product' and'antioxidant activity' are the same as described above.

In one embodiment of the present invention, it was confirmed that the carrot fermentation product is safe for skin because it is not cytotoxic, and thus it can be used in a cosmetic composition (Table 3).

Specifically, the cosmetic composition may further include a use for maintaining the balance of microbiome in the skin.

In an embodiment of the present invention, it was confirmed that the carrot fermentation promotes the proliferation of Leuconostoc mesenteorides bacteria, which is a kind of beneficial bacteria (Table 8), and the skin condition by maintaining the microbiome balance in the skin It was confirmed that it can be used as a cosmetic composition for use that can effectively improve.

The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nutrient lotion, flexible lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil, suspension, emulsion It can be prepared in a formulation selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray, but is limited thereto. no.

The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , A chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately blended, but are not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. May be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohard It may include a propellant such as locarbon, propane/butane or dimethyl ether, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention 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, and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan May be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but are not limited thereto. These may be used alone or in combination of two or more.

In addition, the composition of the present invention may be used as a transdermal administration method such as direct application or spraying on the skin, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue.

The amount of use of the composition of the present invention may be appropriately adjusted according to individual differences or formulations such as age and degree of lesions, and may be used from one week to several months by applying one or several times a day to the skin.

The carrot fermented product prepared by inoculating the Lactobacillus plantarum strain of the present invention has excellent antioxidant activity. In addition, since it has an excellent effect of promoting beneficial bacteria in the skin, it can be used in various ways for improving skin conditions.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Lactobacillus planta room AMI-1103 ( Lactobacillus plantarum AMI-1103) strain identification

1-1. Lactobacillus Planta Room AMI-1103( Lactobacillus plantarum AMI-1103) Isolation of strain

After crushing and homogenizing Jeju-do kimchi, 1.0 g of this was taken, suspended in 9.0 ml of sterilized physiological saline (0.85% NaCl), diluted stepwise, and plated on the MRS growth medium of Table 1 and each of the corresponding selective medium. After culturing at 37° C. for 24 to 48 hours, strains having different colonies were selected and purified through several pure culture processes. The purely isolated strain was spread on MRS solid medium and cultured at 37°C for 24 hours, and the purely isolated colony was cultured on MRS liquid medium and mixed with 20% (v/v) glycerol in a ratio of 1:1. Stored in a -80 ℃ ultra-low temperature freezer.

Strain Optional badge Growth medium Lactobacillus strain
Lactobacillus spp. LBS
(Lactobacillus selection) MRS Lactococcus strain
Lactococcus spp. M17 MRS Pediococcus strain
Pediococcus spp. PSM MRS Leukonose strain
Leuconostoc spp. PES-30 MRS

1-2. Identification of Lactobacillus plantarum AMI-1103 strain

The isolated strain was identified through 16S rDNA sequence analysis. Genomic DNA of the isolated strain was used as a template after DNA extraction using ZR Bacterial DNA MiniPrepTMkit (ZYMORESEARCH). Universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYT ACCTTGTT ACGACTT-3') were used as primers for PCR reactions to amplify 16S rDNA, and PCR conditions were initial denaturation 5 Minutes, and denaturation at 94°C for 45 seconds, annealing at 55°C for 60 seconds, and extension cycles at 72°C for 60 seconds were performed 35 times. The 16S rDNA sequence was confirmed by requesting Microgen Co., Ltd., and the strain corresponding to the food raw material microorganism/grass (GRAS, Generally Recognized as Safe Substance) was first selected.

Sequence number primer order One 27F 5'-AGAGTTTGATCMTGGCTCAG-3' 2 1492R 5'-TACGGYT ACCTTGTT ACGACTT-3'

Comparison of the nucleotide sequence of the strain obtained above and the nucleotide sequence listed in the GenBank database, Lactobacillus spp and 90 - the nucleotide sequence represented by the degree of similarity of the 99.86% level, showed that Lactobacillus sp. In addition, when compared with the base sequence of Lactobacillus plantarum JCM 1149 strain, it was analyzed that the similarity was 99%, and it was generally accepted as a standard for dividing species based on the 16S rDNA gene sequence (Stackebrandt & Goebel, 1994). ; Stackebrandt & Ebers, 2006), was identified as belonging to the species Lactobacillus plantarum. The identified strain was named Lactobacillus plantarum AMI1103, and on November 12, 2018, a patent was deposited with the Korea Microbial Conservation Center (address: 45 Yurim Building, 2ga-gil, Hongje-nae, Seodaemun-gu, Seoul, Korea) and entrusted KCCM12384P. A single colony of Lactobacillus plantarum AMI-1103, the strain isolated in the above example, was added to each flask and cultured in MRS medium at 37° C. for 48 hours. The suspension cultured bacterial solution was used after centrifugation to obtain a supernatant, filtered through a 0.22 μm filter to remove the cells.

Example 2. Preparation of low-temperature fermented carrots using Lactobacillus plantarum strain of carrot extract extracted with distilled water

2-1. Preparation of carrot extract using distilled water as a solvent

After washing the carrots with water, it was completely dried at room temperature and pulverized to obtain 100 g of a pulverized product. 100 g of the pulverized product was placed in a flask and extracted by cold soaking at 25° C. for 3 days with 1000 g of distilled water (extraction solvent). The chilled extract was filtered through a filter having a pore size of 0.2 μm to prepare a carrot extract.

2-2. Preparation of fermented carrots using low temperature (15-25℃) fermentation process

The carrot extract prepared in Example 2-1 was inoculated with Lactobacillus plantarum AMI-1103. Thereafter, a carrot fermentation product was prepared by fermenting at 250 rpm for 72 to 96 hours at a temperature of 15-25°C.

Example 3. Preparation of high-temperature fermented carrots using Lactobacillus plantarum strain of carrot extract extracted with distilled water

3-1. Preparation of carrot extract using distilled water as a solvent

A carrot extract using distilled water as a solvent was prepared in the same manner as in 2-1.

3-2. Preparation of fermented carrots using high temperature (37-42℃) fermentation process

The carrot extract prepared in Example 3-1 was inoculated with Lactobacillus plantarum AMI-1103. Then, a carrot fermentation product was prepared by fermenting at 250 rpm for 72 to 96 hours at a temperature of 37-42°C.

Example 4. Preparation of Carrot Low-Temperature (15-25°C) Fermentation Using Ethanol-Extracted Carrot Extract Lactobacillus Plantarum Strains

4-1. Preparation of Carrot Extract Using Ethanol as a Solvent

After washing the carrots with water, it was completely dried at room temperature and pulverized to obtain 100 g of a pulverized product. For each pulverized product 100 g, 20 times volume of ethanol 70% was used as a solvent, followed by immersion extraction at room temperature for 7 days. After filtering the extract through a 250 mesh filter and a 3 μm filter, ethanol was removed using a concentrator to prepare a carrot extract.

4-2. Preparation of fermented carrots using low temperature (15-25℃) fermentation process

The carrot extract prepared in Example 4-1 was inoculated with Lactobacillus plantarum AMI-1103. Thereafter, a carrot fermentation product was prepared by fermenting at 250 rpm for 72 to 96 hours at a temperature of 15-25°C.

Example 5. Preparation of high-temperature (37-42°C) fermented product of carrots using Lactobacillus plantarum strain of carrot extract extracted with ethanol

5-1. Preparation of Carrot Extract Using Ethanol as a Solvent

A carrot extract using ethanol as a solvent was prepared in the same manner as in 4-1.

5-2. Preparation of fermented carrots using high temperature (37-42℃) fermentation process

The carrot extract prepared in Example 5-1 was inoculated with Lactobacillus plantarum AMI-1103. Then, a carrot fermentation product was prepared by fermenting at 250 rpm for 72 to 96 hours at a temperature of 37-42°C.

Comparative Example 1. Carrot extract extracted with distilled water

A carrot extract using distilled water as a solvent was prepared in the same manner as in 2-1, and a carrot extract extracted with distilled water was used as Comparative Example 1.

Comparative Example 2. Carrot extract extracted with ethanol

A carrot extract using ethanol as a solvent was prepared in the same manner as in 4-1, and a carrot extract extracted with ethanol was used as Comparative Example 2.

Experimental Example 1. Skin keratinocyte culture

HaCaT cells, which are skin keratinocytes, are Dr. Pre-sale from CG Hyun (Jeju National University, Korea) and use Dulbecco's Modified Eagle's Medium (DMEM) medium containing 100 units/ml penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C, It was cultured in a 5% CO ₂ incubator, and subculture was performed every 3 to 4 days.

Experimental Example 2. Cytotoxicity evaluation (EZ-cytox assay)

In order to confirm the skin stability of Examples 1 to 4, cytotoxicity evaluation was performed using the EZ-cytox assay method. EZ-cytox assay is a representative method of measuring cell viability using the principle of producing orange water-soluble formazan by reacting with dehydrogenase of living cells.

Specifically, in order to confirm the toxicity in skin keratinocytes, HaCaT cells were dispensed into a 96-well plate at 5 X 10 ⁴ cells/mL using DMEM medium supplemented with 10% FBS, under conditions of 37°C and 5% CO _2. After reacting for 18 hours at, the absorbance was measured at 450 nm using a microplate reader. The average absorbance value for each sample group was obtained, and the cell viability was evaluated by comparing it with the absorbance value of the control group.

Cell viability (% control) Concentration (ug/ml) 0 50 100 200 Example 1 100 102 103 102 Example 2 100 101 103 103 Example 3 100 105 105 106 Example 4 100 102 102 104

As shown in Table 3, it was confirmed that the fermented carrots of Examples 1 to 4 exhibited excellent cell viability irrespective of the concentration, and had little toxicity to skin keratinocytes and thus had high stability.

Experimental Example 3. Antioxidant effect confirmation

3-1. DPPH scavenging activity confirmation

The antioxidant activity of Examples 1 to 4 and Comparative Examples 1 and 2 was confirmed. For the evaluation of antioxidant activity, DPPH (1,1-diphenyl1-2-picry1hydrazy1) was used to measure free radical scavenging activity. Specifically, after adding 20 μL of the extracts of Examples 1 to 4 and Comparative Examples 1 and 2 to 180 μL of the DPPH solution, each of the extracts of Examples 1 to 4 and Comparative Examples 1 and 2 was added, reacting at room temperature for 30 minutes by blocking light from the outside, and then measuring the absorbance at 517 nm for the control group. Antioxidant activity was shown as a rate of decrease in absorbance. At this time, as a control for determining the rate of decrease in absorbance, ascorbic acid, an antioxidant, was used, and the same amount as the sample was added and measured in the same manner. Then, DPPH scavenging activity was calculated according to Equation 1 below.

[Equation 1]

DPPH scavenging activity (%) = [1-(absorbance of the sample treatment group in the Example or Comparative Example / absorbance of the sample untreated group)] X 100

DPPH elimination (%) Concentration (ug/ml) 125 250 500 Example 1 6.1 6.5 6.9 Example 2 5.9 6.2 6.5 Example 3 9.8 15.5 19.8 Example 4 8.9 11.4 15.4 Comparative Example 1 3.5 3.8 5.9 Comparative Example 2 3.7 4.9 6.6

As shown in Table 4, when the carrot extract was fermented using the Lactobacillus plantarum strain (Examples 1 to 4), it was confirmed that the DPPH scavenging ability was excellent.

3-2. ABTS ⁺ Confirmation of radical scavenging activity

ABTS ⁺ [2,2'-Azino-Beads (3-ethyl benzothiazoline-6-sulfonic acid) (ABTS ⁺ [2,2'-Azino-bis (3-ethyl benzothiazoline-6-sulfonic acid)]) radical scavenging activity Is capable of measuring both hydrogen-donating antioxidants and chainbreaking antioxidants, and is a method applicable to both the aqueous and organic phases.

The 7 mM ABTS solution was mixed so that potassium persulfate became 2.45 mM, reacted in the dark for about 24 hours, and the ABTS ⁺ solution was mixed with the sample and reacted in the dark for 6 minutes to measure the absorbance at 734 nm. The resulting value was expressed as a percentage (%) of radical scavenging activity through calculation using Equation 1 above by comparing the extract sample-treated group and the untreated group, and BHT was used as a positive control group.

ABTS ⁺ Elimination (%) Concentration (μg/ml) 125 250 500 Example 1 9.8 10.5 14.5 Example 2 9.8 12.5 18.5 Example 3 12.2 20.2 31.5 Example 4 10.4 16.8 27.4 Comparative Example 1 7.1 10.5 14.8 Comparative Example 2 7.3 12.0 18.6

As shown in Table 5, when the carrot extract was fermented using the Lactobacillus plantarum strain (Examples 1 to 4), it was confirmed that ABTS ⁺ scavenging ability was excellent.

Through the experiment to confirm the DPPH and ABTS ⁺ scavenging ability, it was confirmed that the antioxidant effect of the carrot fermented carrot extract fermented using the Lactobacillus plantarum strain was excellent, especially when the carrot extract extracted with ethanol was fermented at low temperature. It was confirmed that the antioxidant effect was significantly increased.

Experimental Example 4. Quantitative analysis of camphorol (kaempferol)

Quantitative analysis of the flavonoids of Examples 1 to 4 and Comparative Examples 1 and 2 was performed as follows using a liquid chromatography-mass spectrometer (LC-MS/MS).

As the analysis column, a C ₁₈ (3.0 mm × 150 mm, 3.0 μm) column was selected in consideration of the characteristics of the carrot fermentation product. The mobile phase was analyzed by gradient elution using acetonitrile containing 0.1% formic acid and 0.1% aqueous formic acid solution. The carrot fermented product, which is the target component, was appropriately dissolved in an aqueous methanol solution to prepare 10 mg/ml. Using the prepared standard solution, a calibration curve was prepared by using the ratio of the peak area to the concentration of the standard product, and the linearity of the prepared calibration curve was determined by obtaining the correlation coefficient (r ² ).

division Regression analysis Linearity range (μg/mL) Correlation coefficient (r ² ) Kaempferol y = 1,506,502.7605x-137,079.6369 0.5-10 0.9975

The correlation coefficient (r ² ) was 0.9975, showing good linearity. Using LC-ESI-MS/MS MRM mode analysis, peak identification and content analysis of major components in carrot extract were performed. As a result, camphorol components at m/z 286.9 in the form of [M+H] ⁺ which is a cationic mode. The molecular ion peak of was confirmed. The content analysis results for the identified camphorrol components are shown in Table 7 below.

sample Content per 1 mg of sample (μg) Kaempferol Example 1 0.009 Example 2 0.005 Example 3 0.016 Example 4 0.009 Comparative Example 1 - Comparative Example 2 -

As shown in Table 7, in the carrot extract (Comparative Examples 1 and 2), camphorol, a kind of flavonoid, was hardly present, but when the carrot extract was fermented using a Lactobacillus plantarum strain (Examples 1 to 4) It was confirmed that camphor roll was detected. In particular, it can be seen that when the carrot extract using ethanol is fermented at low temperature, the content of camphorol significantly increases.

The above results suggest that when the carrot extract is fermented under specific conditions, the antioxidant effect can be improved by increasing the flavonoid content.

Experimental Example 5. Confirmation of the effect of promoting beneficial skin bacteria

In order to confirm the effect of the carrot fermentation on the proliferation of beneficial skin bacteria, an experiment was performed to confirm the effect of promoting the growth of Leuconostoc mesenteorides bacteria.

First, Leukonostock mecenteroides was isolated from Kefir grain using a sorting medium. Using 0.002% BPB-MRS medium, anaerobic culture was performed at 30° C. for 72 hours. Thereafter, lactic acid bacteria colonies were selected and then identified by performing 16S rRNA sequencing.

Two days ago, the turbidity of the bacteria that had been awakened beforehand was adjusted to 2.0, and then injected into a tube containing 3 mL of MRS broth, diluted with 10 ¹ CFU/mL, and the initial number of bacteria was measured. Thereafter, 5 mg of the compositions of Examples 1 to 4 were each added to 3 mL MRS broth and sonicated for 5 minutes (operation of 50 seconds, intervals of 10 seconds, repeated 5 times), and 0.1% of DMSO was injected. In the same manner, the compositions of Comparative Examples 1 and 2 were added to 3 mL MRS broth, and the number of bacteria (CFU/mL) was measured after incubation.

division Number of bacteria (CFU/mL) Example 1 3.16 x 10 ⁷ Example 2 3.14 x 10 ⁷ Example 3 3.25 x 10 ⁷ Example 4 3.18 x 10 ⁷ Comparative Example 1 4.31 x 10 ⁶ Comparative Example 2 4.46 x 10 ⁶

As shown in Table 8, when the Leukonostock mecenteroides strains were cultured in Examples 1 to 4, the growth was significantly increased compared to those cultured in Comparative Examples 1 and 2. In particular, fermented carrots using ethanol as an extraction solvent (Examples 3 and 4) significantly increased the beneficial bacteria promoting effect compared to fermented carrots (Examples 1 and 2) using purified water as an extraction solvent. This suggests that it affects the promotion of beneficial bacteria.

Preparation Example 1. Essence Preparation

A cosmetic composition of an essence formulation was prepared as shown in Table 9 below (unit: wt%).

ingredient Content (% by weight) Carrot fermented product of Examples 1 to 4 5 Cetearyl alcohol 2 Cetearyl glucoside One Jojoba seed oil 5 Silica 0.5 glycerin 10 Purified water To 100

Preparation Example 2. Cream Preparation

As shown in Table 10 below, a cream-formed cosmetic composition was prepared (unit: wt%).

ingredient Content (% by weight) Carrot fermented product of Examples 1 to 4 5 Stearic acid 15 ethanol One Potassium hydroxide 0.7 glycerin 5 Propylene glycol 3 antiseptic Appropriate amount incense Appropriate amount Purified water to 100

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the technical field to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM12384P 20181112

<110> AMI COSMETIC CO., LTD. <120> A CARROT FERMENTATION METHOD USING LACTOBACILLUS PLANTARUM AMI-1103 STRAIN <130> 19PP31269 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_27F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer_1492R <400> 2 tacggytacc ttgttacgac tt 22

Claims (9)

As a method for producing a carrot fermented product with enhanced antioxidant activity, comprising the step of inoculating and fermenting a carrot extract with Lactobacillus plantarum strain,
The extraction solvent of the carrot extract is ethanol,
The fermentation method for producing a carrot fermented product is made at 15 ℃ or more and 25 ℃ or less. The method of claim 1,
The extraction solvent of the carrot extract is 70% ethanol, a method for producing a fermented carrot. The method of claim 1,
The Lactobacillus plantarum is Lactobacillus plantarum AMI-1103 ( Lactobacillus plantarum AMI-1103) KCCM12384P strain, Carrot fermented production method. delete The method of claim 1,
The carrot fermented product further comprises a use of maintaining the microbiome balance in the skin, the method for producing a carrot fermented product. The method of claim 5,
The maintenance of the microbiome balance is made through the promotion of skin beneficial bacteria, a method for producing a fermented carrot. Carrot fermented product with improved antioxidant activity prepared by the method of claim 1. A cosmetic composition comprising the fermented carrot of claim 7 with improved antioxidant activity. The method of claim 8,
The cosmetic composition is a cosmetic composition that further comprises the use of maintaining the microbiome balance in the skin.