KR101967793B1 - Anti-wrinkle cosmetic composition using Lactobacillus paracasei MK1 and preparation method thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving wrinkles comprising soymilk fermented with soymilk fermented with Lactobacillus paracasei MK1 [Accession Number: KFCC11552P], or a soymilk peptide fraction prepared therefrom. It can be used as a material of the cosmetic composition for improving wrinkles as a natural functional material exhibiting wrinkle improvement activity without exhibiting toxicity to skin cells.

Description

[Anti-wrinkle cosmetic composition using Lactobacillus paracasei MK1 and preparation method thereof]

The present invention relates to a cosmetic composition for improving wrinkles, a manufacturing method thereof, and a lactic acid bacteria used in the manufacturing method.

Collagen, a major component of the extracellular matrix, is a major matrix protein produced by fibroblasts in the skin and is present in the extracellular matrix. In addition, it is an important protein that accounts for about 30% of the total weight of the biological protein and has a solid triple helix structure. Collagen forms most of the organic substances in skin, tendons, bones and teeth, especially in bones and skin (ground skin). In most other sieve structures, it is present as a fibrous inclusion body.

Collagen is a relatively weak immunogen, partly due to the shielding of latent antigenic determinants by the helix structure of collagen, which also makes collagen resistant to protein degradation. The main functions of collagen are known for its mechanical firmness, connective tissue resistance and tissue bonding, support for cell adhesion, and induction of cell division and differentiation (in case of growth of organisms or wound healing) (Van der Rest, etc.). , Ann NY Acad Sci, 1990).

This collagen is reduced by age and photoaging caused by UV irradiation, and it is known that it is closely related to the formation of wrinkles in the skin (Arthur K. Balin et al., Aging and the skin, 1989). In addition, collagen plays an important role in wound healing, and by promoting the synthesis of collagen in the damaged epithelium, wounds can be recovered quickly and without scarring.

Conventionally, in order to take advantage of the skin moisturizing effect and wound healing effect of collagen, products containing collagen in external skin preparations such as cosmetics or ointments have been released. However, these products apply collagen itself to the skin surface, and are a polymer substance called collagen. It could not be said that it shows essential skin function improvement and wound healing function because it cannot be expected to have a moisturizing effect or wound healing effect due to its difficulty in transdermal absorption.

Korean Patent Registration No. 0987518 uses a high molecular weight prepared by inoculating Bacillus subtilis in a medium containing soybean extract and folic acid, fermenting it under aerobic anaerobic conditions, and removing substances with a molecular weight of 500 kDa or less after fermentation. It discloses a cosmetic composition having an effect of promoting collagen biosynthesis including, and Korean Patent Laid-Open No. 2010-0020124 is an active ingredient of soybean fermented product by complex kimchi lactic acid bacteria superior in vitamins and nutrients compared to soybean fermented product by a single strain. A cosmetic composition effective for improving wrinkles is disclosed, and Korean Patent Laid-Open No. 2011-0041178 is effective in improving wrinkles using a fermented product fermented with a mixture of germinated soybeans and sesame seeds, such as Baekguk, Hwangguk, and Bacillus. Disclosed is a cosmetic composition.

However, there has been no report on the anti-wrinkle effect of a low molecular weight soybean peptide or a fraction containing the same.

The present invention is a soybean fermented product having both type I procollagen production activity and tumor necrosis factor-alpha (TNF-α) inhibitory activity that can be used as an active ingredient in a cosmetic composition for improving wrinkles, or a soymilk peptide fraction prepared therefrom, and soy milk An object of the present invention is to provide a method for preparing a peptide fraction, and a novel lactic acid bacteria used in the method for preparing the same.

In order to solve the above problems, the present invention provides a cosmetic composition for improving wrinkles comprising fermented soymilk fermented by inoculating soymilk with Lactobacillus paracasei MK1 [Accession No.: KFCC11552P] as an active ingredient.

The cosmetic composition for wrinkle improvement of the present invention is fermented by inoculating the soymilk hydrolyzate obtained by hydrolyzing the soymilk with a proteolytic enzyme with Lactobacillus paracasei MK1 [Accession Number: KFCC11552P] water; Alternatively, the soymilk fermented product obtained by inoculating Lactobacillus paracasei MK1 [Accession No.: KFCC11552P] to the soymilk is hydrolyzed with a proteolytic enzyme; it is preferable to use as an active ingredient. .

In the cosmetic composition for improving wrinkles of the present invention, the proteolytic enzyme is preferably flabozyme.

In the cosmetic composition for improving wrinkles of the present invention, the fermented soybean milk is preferably a fermented soymilk fermented product obtained by fermenting a dilute soymilk diluted so that the solid content of the soymilk stock solution is 25 to 50% by weight.

In the cosmetic composition for improving wrinkles of the present invention, it is preferable that the soymilk peptide fraction from which the protein or peptide having a molecular weight exceeding 30 kDa is removed from the fermented soybean milk, the fermented product of the soymilk hydrolyzate, or the hydrolyzate of the fermented soybean milk as an active ingredient .

In the cosmetic composition for improving wrinkles of the present invention, the soymilk peptide fraction from which proteins or peptides having a molecular weight exceeding 5 kDa are removed from the fermented soybean milk, the fermented soymilk hydrolyzate, or the hydrolyzate of the soymilk fermented product is preferably used as an active ingredient.

In the cosmetic composition for improving wrinkles of the present invention, the fraction preferably has type I procollagen production activity and tumor necrosis factor-alpha (TNF-α) inhibitory activity.

In the cosmetic composition for improving wrinkles of the present invention, the fraction is preferably not toxic to human dermal fibroblasts and human keratinocytes.

In the cosmetic composition for improving wrinkles of the present invention, it is preferable that the fraction has a free amino acid content of 60 mg% or more in the total fraction, and an essential amino acid content of 50 wt% or more in the free amino acid.

In the cosmetic composition for improving wrinkles of the present invention, it is preferable that the fraction comprises a peptide having an amino acid sequence of SEQ ID NO: 1.

In addition, the present invention is a soybean milk hydrolyzate preparation step of hydrolyzing soymilk with a proteolytic enzyme, flavozyme; Fermentation step of fermenting soy milk hydrolyzate by inoculating and fermenting Lactobacillus paracasei MK1 [Accession No.: KFCC11552P] to the soy milk hydrolyzate; And removing a protein or peptide having a molecular weight of more than 5 kDa from the fermented product of the soymilk hydrolyzate to obtain a soymilk peptide fraction.

In addition, the present invention is a soybean milk fermentation production step of inoculating and fermenting Lactobacillus paracasei MK1 [accession number: KFCC11552P] in soymilk; A step of preparing a hydrolyzate of fermented soybean milk by hydrolyzing the fermented soybean milk with flavozyme, a proteolytic enzyme; And it provides a method for preparing a soymilk peptide fraction for wrinkle improvement comprising a; and removing a protein or peptide having a molecular weight of more than 5 kDa from the hydrolyzate of the fermented soybean milk to obtain a soymilk peptide fraction.

In addition, the present invention fermented soymilk to produce a soymilk peptide having type I procollagen production activity and tumor necrosis factor-alpha (TNF-α) inhibitory activity Lactobacillus paracasei (Lactobacillus paracasei) MK1 [Accession No: KFCC11552P] Provides.

The cosmetic composition for improving wrinkles of the present invention comprises 0.00001 to 50% by weight of the fermented soybean milk in an amount of 0.00001 to 50% by weight, preferably, 0.0001 to 20% by weight, more preferably 0.1 to 10% by weight, based on the total weight of the composition. . However, the composition as described above is not necessarily limited thereto, and may vary depending on the patient's condition and progression.

Ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the fermented soybean milk as an active ingredient, for example, stabilizers, solubilizers, vitamins, pigments, and flavors. Phosphorus adjuvants and carriers.

The formulation of the cosmetic composition of the present invention is not particularly limited, and may be appropriately selected depending on the intended purpose. For example, the cosmetic composition of the present invention may be prepared in a formulation such as an ointment for external use of the skin, a lotion, a soft lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, a pack, an emulsion, an oil gel, and the like.

The novel lactic acid bacteria of the present invention ferment soy milk to produce a soymilk peptide having both type I procollagen production activity and tumor necrosis factor-alpha (TNF-α) inhibitory activity, and a soymilk fermented product or soymilk peptide prepared using the lactic acid bacteria The fraction is a natural functional material that exhibits anti-wrinkle activity without showing toxicity to skin cells, and can be used as a material for a cosmetic composition for improving wrinkles.

1A to 1D show a protein band pattern according to SDS-PAGE in Experimental Example 3. In FIGS. 1A to 1D, M represents a molecular weight marker, C represents soy milk, and in Figure 1A 1. GK1; 2. GK2; 3. GK3; 4. GK4; 5. GK5; 6. OJ1; 7. OJ2 is shown, and in Fig. 1B, 1. CRJ; 2. JJ; 3. CMK2; 4. K1; 5. CCK; 6. MK1 is shown, and in Fig. 1c, 1. SULK 2. radish 3. K2; 4. kimchis; 5. GOM is shown, and in Fig. 1D, 1. NJ3; 2. NJ1; 3. SJ; 4. It shows CMK1.
Figure 2 is a photograph of the morphology of the MK1 strain magnified 30,000 times with an electron scanning microscope.
3 shows the phylogenic tree of the MK1 strain.
4A to 4C are graphs showing titratable acidity, pH, and number of bacteria according to culture temperature and time in 1) of Experimental Example 5, respectively.
Figures 5a and 5b show the SDS-PAGE protein band pattern of the culture supernatant according to the culture temperature in 1) of Experimental Example 5, in common M is a molecular weight marker, in Figure 5a 1. 25 ℃, 0 hr; 2. 25° C., 16 hr; 3. 25° C., 24 hr; 4. 30° C., 0 time; 5. 30° C., 16 hr; 6. 30° C., 24 hr, and 1. 37° C., 0 hr in FIG. 5B; 2. 37° C., 16 hr; 3. 37° C., 24 hr; 4. 40° C., 0 hr; 5. 40° C., 16 hr; 6. It shows 40 degreeC and 24 hr.
6A to 6C are graphs showing titratable acidity, pH, and number of bacteria according to dilution concentration and time in 2) of Experimental Example 5, respectively.
7A to 7D show the SDS-PAGE protein band pattern of the culture supernatant of 100%, 75%, 50% and 25% soymilk, respectively, depending on the dilution concentration in 2) of Experimental Example 5, in common M is a molecular weight marker To, 1. 0 hr; 2. 6 hr; 3. 12 hr; 4. 18 hr; 5. Indicate the incubation time of 24 hr.
8A to 8J are graphs of MTT analysis that can confirm cytotoxicity to HaCaT cells according to the type and concentration of a sample.
9A to 9E are graphs measuring the amount of I-type procollagen production, FIG. 9A is a 50 μM treatment of ascorbic acid as a positive control, and FIG. 9B is 50S, 50% soymilk not fermented with an MK1 strain, and FIGS. 9C to Figure 9e is a comparison of 50SFMKUF5, 50SMKP, 50SFMKUF30, 50SPMK and 50SFMK by concentration with ascorbic acid as a positive control.
Figures 10a to 10e are graphs measuring the production amount of TNF-α, Figure 10a is dexamethasone (DXMS) 1 μM treatment as a positive control, Figure 10b is 50S, 50% soymilk not fermented with MK1 strain, Figures 10c to Figure 10e is a comparison of 50SFMKUF5, 50SMKP, 50SFMKUF30, 50SPMK and 50SFMK with the positive control dexamethasone by concentration.
11A and 11B are results of peptide mapping of the 50SFMKUF5 fraction using LC/MS-MS in Experimental Example 9, and FIG. 11A is an LC chromatogram, and FIG. 11B is an MS chromatogram.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

Experimental material

Soymilk was obtained from Yonsei University's milk, BCP plate count agar was purchased from Kyokuto Pharmaceutical Co., Ltd. (Japan), and skim milk, MRS broth and agar were purchased from Difco Co. (USA). Azo-casein and bovine serum albumin were purchased and used from Sigma-Aldrich (USA), and a protein assay reagent was used by Bio-rad (USA). The API kit was manufactured by bioMerieux Ltd. (France). Protamex and flavorzyme were manufactured by Novozymes (Denmark). The Procollagen type-I C-Peptide (PIP) EIA kit was manufactured by Takara Bio (MK101, Japan) and TNF-α was measured by Invitrogen (#KHC3012, USA).

Experimental Example 1: Primary screening of lactic acid bacteria having high protease activity

In order to obtain a low molecular weight peptide for improving wrinkles or an active substance other than a peptide from soy milk, microorganisms with high protease activity were searched among lactic acid bacteria that are safe and excellent in physiological activity.

After purchasing various fermented foods such as kimchi and salted fish, dilute it in sterile physiological saline (0.88%(w/v) NaCl), spread the diluted solution on a BCP plate count agar added with skim milk, and incubate at 37°C for 2 days. , Lactobacillus forming a transparent clear zone around the colony was isolated. It was confirmed that out of a total of 180 lactic acid bacteria, 22 strains formed a clear zone above a certain level. These strains were subcultured in BCP plate count agar and purely isolated.

Experimental Example 2: Secondary screening according to protease activity, acidity and pH

Table 1 shows the protease activity was measured by applying the azo-casein method for the second screening of the first selected lactic acid bacteria. The first selected strains were inoculated into MRS broth and cultured at 37° C. for 48 hours, and then the culture solution was centrifuged at 16,100×g for 5 minutes to remove cells, and the supernatant was taken and used as a coenzyme. Azo-casein was used as a substrate used to measure protease activity. After dissolving 2 g of azo-casein in 100 mL of 100 mM phosphate buffer solution (pH 7.0), 500 μL of azo-casein substrate solution was added at 40°C, 5 Preheated for minutes. 50 μL of the coenzyme was mixed with the preheated azo-casein substrate solution and reacted at 40° C. for 10 minutes to release the azo group. To this, 1 mL of 12% (w/v) trichloroacetic acid was added to inactivate the residual activity, and unreacted azo-casein was precipitated at 4° C. for 30 minutes and then centrifuged at 16,100×g for 10 minutes. After centrifugation, 500 μL of the supernatant was taken, and the same amount of 0.5 M NaOH solution was added to measure the absorbance at 440 nm. At this time, 1 unit of enzyme activity was set as an amount to increase the absorbance at 440 nm per minute by 0.001 under the given conditions.

division Protease activity ( mU / mL · min ) GK1
GK2
GK3
GK4
GK5
GOM
K1
K2
NJ1
NJ2
NJ3
MK1
SJ
BK
SULK
OJ1
OJ2
JJ
CRJ
CCK
CMK1
CMK2 140
71
164
98
77
114
2
73
134
96
118
126
128
138
112
120
168
130
116
116
102
152

Protease isolated from kimchi in a study by Min et al. (Min SG, Kim JH, Kim TW, Kim KN. Isolation and identification of protease producing bacteria in kimchi. Korean J. Food Sci. Technol. 35: 660-670 (2003)) Was isolated and identified. The strain identified as Lactobacillus delbruekii was 58 mU/mg, and the protease activity of Leuconostoc citreum , another lactic acid strain, was 5 mU/mg.

However, as shown in Table 1, the strains of 22 strains selected in Experimental Example 1 exhibited high protease activity. In the strain showing the highest protease activity, OJ1 was measured to have high protease activity at 168 mU/mL·min, followed by GK3, CMK2, and GK1 (each 164, 152, 140 mU/mL·min) in that order. . In addition, K1, which showed the lowest activity, was 2 mU/mL·min, and it was confirmed that even the strains isolated from the same source (English abbreviations are indicated identically) have different protease activities.

In addition, the 22 strains selected in Experimental Example 1 were cultured for 48 hours at 37°C using soymilk sterilized at 121°C for 15 minutes as a medium, and then the appropriate acidity and pH were measured and shown in Table 2, respectively. The growth and fermentation characteristics of lactic acid bacteria were confirmed. For titratable acidity, take 9 ml of the culture solution in 10 ml of distilled water, dilute it, add 500 μL of 0.1% phenolphthalein solution, and titrate with 0.1 N NaOH for 30 seconds until the pale red color does not disappear, and the consumption is expressed as% titration acidity. I got it. Acidity was expressed as lactic acid (0.1N NaOH 1 mL = lactic acid 0.009 g), and the pH of the remaining culture was measured using a pH meter (FE-20K, METTLER TOREDO, USA).

division pH Titratable acidity (%) GK1
GK2
GK3
GK4
GK5
GOM
K1
K2
NJ1
NJ2
NJ3
MK1
SJ
BK
SULK
OJ1
OJ2
JJ
CRJ
CCK
CMK1
CMK2 5.10
4.84
4.95
4.78
4.71
6.44
4.76
5.24
4.59
ND
4.60
4.10
4.58
6.55
4.90
4.61
4.65
5.05
4.64
4.38
4.82
4.67 0.11
0.17
0.14
0.17
0.17
0.00
0.16
ND ¹⁾
0.01
ND
0.19
0.28
0.18
0.03
0.13
0.18
0.18
0.11
0.17
0.19
0.16
0.18

¹⁾ ND stands for Not detemided

As shown in Table 2, the pH of MK1 showed the lowest pH of 4.10, and showed a higher acidity than other strains with an appropriate acidity of 0.28%. On the other hand, the soymilk fermented by inoculating the BK strain had a pH of 6.55, so that acid production of lactic acid bacteria did not occur at all, and titratable acidity was also measured at only 0.03%. This could be estimated that the BK strain was almost impossible to grow in soy milk.

Experimental Example 3: Protein quantification and third screening using SDS-PAGE pattern

For the third screening of the first selected lactic acid bacteria, cells were removed by centrifugation at 16,100×g for 5 minutes using soymilk sterilized at 121°C for 15 minutes as a medium in Experimental Example 2 and cultured for 48 hours at 37°C. After taking the supernatant, the protein was quantified using Bio-rad's protein assay reagent, and it is shown in Table 3 (Bradford, 1976).

division Protein concentration (μg/μL) GK1
GK2
GK3
GK4
GK5
GOM
K1
K2
NJ1
NJ2
NJ3
MK1
SJ
BK
SULK
OJ1
OJ2
JJ
CRJ
CCK
CMK1
CMK2 1.10
1.08
1.04
0.99
1.08
16.90
1.10
1.24
1.10
ND ¹⁾
1.20
0.74
1.10
ND
1.10
1.11
1.17
1.18
0.90
1.15
1.40
1.12

¹⁾ ND stands for Not detemided

MK1 showed the best results in the results of pH and titratable acidity, and as a result of protein measurement using the bradford assay, it was possible to measure the amount of protein as low as 0.74 μg/μL. In contrast to MK1, in the case of the GOM strain, the protein amount was measured as 16.90 μg/μL, as can be seen from the results of pH and titration acidity, and the highest protein amount could be observed. This can be judged as a result showing that the protein present in the soymilk has been degraded or not degraded by the strains.

In addition, after removing the cells by centrifugation, the supernatant was loaded on SDS-PAGE so that the concentration of the sample was 10 μg/μL, subjected to electrophoresis, and then the protein band pattern on the SDS-PAGE was confirmed, and the results are shown in FIGS. 1A to 1D. Shown in.

When soymilk was fermented with MK1, it could be confirmed that numerous major bands exist around 14.3 kDa. This is a pattern in which proteins present in soy milk are hydrolyzed and major bands disappear, indicating the degree of proteolytic activity.

By synthesizing the results of the protease activity, pH, titratable acidity and SDS-PAGE pattern of Experimental Examples 1 to 3 above, fermentation of soy milk was performed to hydrolyze the protein in soy milk with protease activity to be a candidate for a strain for producing a low-molecular peptide having anti-wrinkle activity. The MK1 strain was selected.

Experimental Example 4: Identification of MK1 strain

1) Morphological observation

As a result of observing the morphology of the MK1 strain using a scanning electron microscope (SEM), the length of the bacterial body as a bacillus was measured to be about 0.5×1-1.5 μm.

2) Confirmation of sugar metabolism of lactic acid bacteria

The glucose metabolism of the MK1 strain was investigated using the API 50 CHL system (BioMerieux, Marcy, IEtoile, France) and shown in Table 4.

Characteristics MK1 Characteristics MK1 Glycerol
Erythritol
D-Arabinose
L-Arabinose
D-Ribose
D-Xylose
L-Xylose
D-Adonitol
Methyl-β-D-xylopyranoside
D-Galactose
D-Glucose
D-Fructose
D-Mannose
L-Sorbose
L-Rhamnose
Dulcitol
Inositol
D-Mannitol
D-Sorbitol
Methyl-α-D-mannopyranoside
Methyl-α-D-glucopyranoside
N-Acetyl glucosamine
Amygdalin
Arbutin
Esculin ferric citrate -
-
-
-
+
-
-
-
-
+
+
+
+
-
-
-
-
+
-
-
-
+
+
+
+ Salicin
D-Cellobiose
D-Maltose
D-Lactose (bovine origin)
D-Melibiose
D-Saccharose (sucrose)
D-Trehalose
Inulin
D-Melezitose
D-Raffinose
Amidon (starch)
Glycogen
Xylitol
Gentiobiose
D-Turanose
D-Lyxose
D-Tagatose
D-Fucose
L-Fucose
D-Arabitol
L-Arabitol
Potassium Gluconate
Potassium 2-ketogluconate
Potassium 5-ketogluconate
-
-
-
-
-
+
+
+
+
-
-
-
-
-
+
-
+
-
-
-
-
-
-
-

As shown in Table 4, monosaccharides glucose, galactose, fructose, ribose, maanose, tagatose and disaccharides saccharose, trehalose, and turanose were used, and polysaccharides inulin and melezitose, sugar alcohols mannitol, cyan glycosides amygdalin, arbutin and It was confirmed that raffinose glycosides, N-acetyl glucosamin, and esculin ferric citrate, were used.

3) Molecular biological identification

For final identification, the 16S rRNA nucleotide sequence of the MK1 strain was determined, analyzed using the blastn program of the National Center for Biotechnology Information (NCBI), and then homology with the type strain registered in GenBank was compared.

The 16S rRNA nucleotide sequence of the MK1 strain is shown in SEQ ID NO: 1, and as a result of comparing this nucleotide sequence with the 16S rRNA nucleotide sequence of GenBank using the BLASTN program in NCBI, it showed high homology with the genus Lactobacillus, and Clustal X and Mega Using the 5 program, a phylogenetic tree was created by comparing and analyzing the homology of base sequences with various species belonging to each Lacobacillus gene, and is shown in FIG. 3.

MK1 is isolated from kimchi and has a high similarity to Latobacillus paracasei and L. casei. Among them, it shows the highest homology with Latobacillus paracasei JCM813, so the final selected strain was named Lactobacillus paracasei MK1 and named Korea. It was deposited with the Microbial Conservation Center on April 4, 2013, and was given the deposit number KFCC11552P.

In addition, as a result of examining the general characteristics of the Lactobacillus paracasei MK1, it is a Gram-positive aspore-forming bacteria, has no motility, and does not produce catalase activity and gas.

Experimental Example 5: Growth conditions of MK1 strain

1) Growth rate according to culture temperature

MK1 cells were inoculated into soymilk and cultured at 37°C, and the cells were inoculated with 100% soymilk at 2% (v/v) and cultured at 25, 30, 37, and 40°C for 24 hours. The degree of growth according to the culture temperature was measured using pH and SDS-PAGE.

As a result of observing the optimum culture temperature of L. paracasei MK1 with appropriate acidity and pH, when fermented at 30℃ for 24 hours, the highest acidity was 0.98%, and in the case of pH, it was measured at a pH of 5.64, which is higher than the remaining temperature at 25℃. Became. After fermentation at 30, 37, and 40° C. for 24 hours, the pH was 4.56, 4.56, and 4.54, respectively, and it was confirmed that the pH was similar for each experimental group (FIGS. 4A and 4B ).

The number of viable cells measured after inoculation of L. paracasei MK1 in soymilk was found that the experimental group fermented at 30℃ entered the logarithmic growth phase first and entered the death phase when fermented for 24 hours. Despite entering the death phase, the number of viable cells was higher than that of the experimental plots at other temperatures. When fermented at 40°C, the number of viable cells was 8.37 log CFU/mL, at 37°C 8.74 log CFU/mL, and at 25°C, 8.98 similar to 30°C. It was measured as log CFU / mL, the number of viable cells at 30 °C was measured as 9.03 log CFU / mL (Fig. 4c).

The protein resolution of soy milk was confirmed by SDS-PAGE. Compared to 0 hours, the SDS-PAGE pattern of the 24 hour culture solution at 30, 37 and 40°C except for the sample at 25°C is shown in Fig. As can be seen in 13 and 14, it was observed that the degree of decomposition was similar (FIGS. 5A and 5B).

In addition , due to the lactic acid formation of L. paracasei MK1, the physical properties of soy milk formed curd from 12 hours after inoculation, and the hardness of the curd increased as the fermentation time passed. As a result of the final analysis, the most optimal fermentation temperature for soy milk fermentation was determined to be 30°C.

2) Growth rate according to the dilution concentration of soy milk

MK1 cells were inoculated into soymilk and cultured at 37°C, and the cells were inoculated at 25, 50, 75, and 100% soymilk at 2% (v/v) and cultured at 30°C for 24 hours. The degree of growth according to the dilution of soy milk was measured using pH and SDS-PAGE.

In 100% soymilk (solid content 10.8%), the titratable acidity was 0.90% at 18 hours of fermentation, and the remaining 75% or less group was observed to continuously increase the titratable acidity after 24 hours (Fig. 6a). .

Unlike the appropriate acidity, the pH and the number of viable cells, which did not differ significantly between the experimental sections, were measured at a final pH of about 4.6 after 24 hours fermentation, and the number of viable cells was 8.86 (100% soy milk) to 9.12 (75% soy milk) log CFU/mL. As a result, the dilution of soymilk did not appear to have a significant effect (FIGS. 6b and 6c), but as a result of confirming the protein resolution by SDS-PAGE, in the case of 100% and 75% soymilk, a band of 44.3 kDa or more remained after fermentation for 24 hours. And, when 50% soymilk was fermented for 24 hours, a faint band did not appear in a portion of 44.3 kDa or more (FIGS. 7a to 7c). 25% soymilk was also found to have good resolution, but was judged to be disadvantageous compared to 50% soymilk in terms of productivity (FIG. 7d).

Manufacturing example:

1) Manufacture of fermented soy milk

Soymilk fermentation broth is prepared by inoculating colony of L. paracasei MK1 in sterilized soymilk or soymilk hydrolyzate, and then 2%(v/v) of soymilk fermentation broth is inoculated with soymilk or 50% soymilk, and fermented at 30℃ for 30 hours. I did. The fermentation product was centrifuged and the supernatant was used as a sample.

2) Preparation of soy milk hydrolyzate

The sterilized soy milk or fermented soy milk was preheated to 50°C before enzyme treatment, and after adding 2 g of enzyme per kg of protein to protamex and flavourzyme, reacted with vigorous shaking at 50°C for 4 hours, and then reacted at 80°C for 10 minutes. It was deactivated by heating.

3) Preparation of ultrafiltration fraction

The sterilized soymilk, fermented soymilk, or hydrolyzate of soybean milk, pore size 0.22 μm, filtrate filtered by membrane filteration using a nitrocellulose filter membrane (Sigma, USA) was used for ultrafiltration. The ultrafiltration device used the Lascale ^™ TFF system of Millipore (USA), and the cartridge was carried out using BIOMAX, 30 K and 5 K polyethersul-fone (50 cm ² ) among Millipore's Pellicon XL filters.

A sample of Table 5 was prepared by combining one or two or more of the methods 1) to 3) above.

Sample name Treatment method 50S
50SMK
50SMKUF30
50SMKUF5
SF
SP
50SMKF
50SMKFUF30
50SMKFUF5

50SMKP
50SMKPUF30
50SMKPUF5

50SFMK
50SFMKUF30
50SFMKUF5

50SPMK
50SPMKUF30
50SPMKUF5

100S
100SMK
100SMKUF30
100SMKUF5
100SMKF
100SMKFUF30

100SMKFUF5

100SMKP
100SMKPUF30
100SMKPUF5

100SFMK
100SFMKUF30
100SFMKUF5

100SPMK
100SPMKUF30
100SPMKUF5 50% Soymilk
50% Soymilk → Fermented with L. paracasei MK1
50% Soymilk → Fermented with L. paracasei MK1 → UF 30 kDa
50% Soymilk → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa
100% Soymilk → Flavourzyme
100% Soymilk → Protamex
50% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme
50% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme → UF 30 kDa
50% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme → UF 30 kDa → UF 5 kDa
50% Soymilk → Fermented with L. paracasei MK1 → Protamex
50% Soymilk → Fermented with L. paracasei MK1 → Protamex → UF 30 kDa
50% Soymilk → Fermented with L. paracasei MK1 → Protamex → UF 30 kDa → UF 5 kDa
50% Soymilk → Flavorzyme → Fermented with L. paracasei MK1
50% Soymilk → Flavorzyme → Fermented with L. paracasei MK1 → UF 30 kDa
50% Soymilk → Flavorzyme → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa
50% Soymilk → Protamex → Fermented with L. paracasei MK1
50% Soymilk → Protamex → Fermented with L. paracasei MK1 → UF 30 kDa
50% Soymilk → Protamex → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa
100% Soymilk
100% Soymilk → Fermented with L. paracasei MK1
100% Soymilk → Fermented with L. paracasei MK1 → UF 30 kDa
100% Soymilk → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa
100% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme
100% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme → UF 30 kDa
100% Soymilk → Fermented with L. paracasei MK1 → Flavourzyme → UF 30 kDa → UF 5 kDa
100% Soymilk → Fermented with L. paracasei MK1 → Protamex
100% Soymilk → Fermented with L. paracasei MK1 → Protamex → UF 30 kDa
100% Soymilk → Fermented with L. paracasei MK1 → Protamex → UF 30 kDa → UF 5 kDa
100% Soymilk → Flavorzyme → Fermented with L. paracasei MK1
100% Soymilk → Flavorzyme → Fermented with L. paracasei MK1 → UF 30 kDa
100% Soymilk → Flavorzyme → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa
100% Soymilk → Protamex → Fermented with L. paracasei MK1
100% Soymilk → Protamex → Fermented with L. paracasei MK1 → UF 30 kDa
100% Soymilk → Protamex → Fermented with L. paracasei MK1 → UF 30 kDa → UF 5 kDa

Experimental Example 6: Solid content

Before measuring the antioxidant, cell growth and toxicity to skin cells, procollagen production amount, and TNF-α production amount of the samples of Preparation Example, the solid content of each sample was measured and shown in Table 6.

Sample name Solid content (%) sample Solid content (%) sample Solid content (%) 50S
50SMK
50SMKUF30
50SMKUF5
SF
SP
50SMKF
50SMKFUF30
50SMKFUF5
50SMKP
50SMKPUF30
3.51
1.42
1.30
1.13
ND ¹⁾
ND
1.65
1.38
1.30
1.45
1.34
50SMKPUF5
50SFMK
50SFMKUF30
50SFMKUF5
50SPMK
50SPMKUF30
50SPMKUF5
100S
100SMK
100SMKUF30
100SMKUF5
1.18
1.97
1.65
1.37
2.70
2.34
1.85
ND
2.58
2.05
1.70
100SMKF
100SMKFUF30
100SMKFUF5
100SMKP
100SMKPUF30
100SMKPUF5
100SFMK
100SFMKUF30
100SFMKUF5
100SPMK
100SPMKUF30
100SPMKUF5 3.30
2.96
2.45
3.10
1.66
1.36
3.31
2.88
2.58
4.10
3.22
2.45

¹⁾ ND stands for Not detemided

As shown in Table 6, 100SF, 100SP, and 100S (100% soymilk) were excluded because filtration was impossible.

100SPMK, which has the highest solids content, is a sample fermented with L. paracasei MK1 after protamex treatment of 100% soymilk. It showed a high solids content of 4.10%, and 50SMKUF5, which showed the lowest solids content of 1.13%, contained 50% soymilk in L. By fermentation with paracasei MK1, a fraction of less than 5 kDa was obtained by ultrafiltration, and proteins of large molecular weight were removed.

The overall trend was that the samples prepared using 100% soymilk showed a difference of 1.5-1.7 times in solid content compared to 50% soymilk, and the solid content tended to decrease as fermentation and ultrafiltration proceeded. This is thought that the initial dilution factor also affects the solid content, and it is thought that the ultrafiltration has a slight effect on the solid content.

Experimental Example 7: Cytotoxicity

1) cell culture

In order to evaluate whether the dermal cell line involved in collagen production, Normal Human dermal fibroblast (ATCC, VA) and the epidermal cell line Immotalized human keratinocyte (HaCaT), were 1% antibiotic- Dulbecco's modified essential medium (DMEM, Welgene, Korea) containing antimycotic (AA, Welgene, Korea) and 10% fetal bovine serum (FBS, Welgene, Korea) was incubated at _{37°C under 5% CO 2 conditions.}

2) MTT analysis

MTT assay is a method that reflects the concentration of metabolically active cells by measuring the absorbance of formazan (purple) produced by reducing MTT (yellow) by mitochondrial succinate dehydrogenase in the mitochondria of cells. After diluting by 1/2 at 0.02%, treating the cells at various concentrations and incubating for 24 hours, Fibroblast and HaCaT cells were used, and cells without sample treatment were used as a control.

3) Analysis result

The viability of fibroblast cells was not dependent on the concentration of the sample. In the case of fibroblast, the concentration increased and the survival rate increased. In particular, 100% soymilk was fermented with L. paracasei MK1 after protamex enzyme treatment and 5 kDa through ultrafiltration through ultrafiltration. 100SPMKUF5, the filtrate filtered below, showed the highest growth rate of 129.67% among other samples at a concentration ^{of 2.50×10 -2.} In addition, 50SMKF showed a cell survival rate of 122.85% or more from a concentration ^{of 3.13×10 -3.} The samples treated with 100% soymilk and samples treated with 50% soymilk could not find any difference, but at ^{concentrations higher than 1.25×10 -2} , almost all samples had a higher viability than cells that were not treated with the sample. Showed (data not attached).

In the case of HaCaT cells with an anti-inflammatory response, the sample and the survival rate of which the survival rate was high at the final concentration of 0.1% were 50SFMKUF5 103.71%, 50SMKP 102.02%, 50SFMKUF30 101.83%, 50SPMK 101.45%, 100SMK 101.41%, 50SFMK 100.7%, and 100SPMK. 100.23%, except for these samples, showed the lowest survival rate of 59.63% with 100SMKFUF5, and most of them showed cytotoxicity (FIGS. 8A to 8J).

In addition, in the overall HaCaT cells, it was found that the cell viability of the sample proceeded with 100% soymilk was lower than that of the sample proceeded with 50% soymilk.

Experimental Example 8: Analysis of I-type procollagen production

1) Cell culture

Dulbecco's modified essential medium containing 1% antibiotic-antimycotic (AA, Welgene, Korea) and 10% fetal bovine serum (FBS, Welgene, Korea), which is a dermal cell line involved in collagen production (ATCC, VA). DMEM, Welgene, Korea) was incubated at 37°C under 5% CO _{2 conditions.}

2) Analysis of I-type procollagen production

^{Fibroblast was inoculated with 2×10 5} cells in a 6 well plate and incubated for 24 hours, and then the medium was removed and then starvation was performed for 24 hours by treatment with a serum-free medium. After starvation, the medium was removed and washed with PBS (phosphate buffered saline, Amresco, USA). In the presence of PBS, irradiation was performed at 312 nm at 25 mJ, the sample was added to DMEM medium containing no FBS, and the sample was treated for each concentration, followed by further incubation for 48 hours. After cultivation, the collected medium was measured for collagen produced using a procollagen type-I C-Peptide (PIP) EIA kit (Takara Bio, Inc., MK101, Japan). As a positive control, 50 μM ascorbic acid was used, as a negative control, a test group that was not UV-treated, and a sample treated with only the medium was used as a control.

3) Analysis result

Samples treated with 50% soymilk, which did not show cytotoxicity to HaCat cells of Experimental Example 7, 50SFMKUF5, 50SMKP, 50SFMKUF30, 50SPMK and 50SFMK were compared with ascorbic acid as a positive control, and at a concentration of 0.1, 0.01, 0.001% relative to the solid content. Samples were treated, and 50S not fermented with MK1 were compared together as a control, and are shown in FIGS. 9A to 9E.

The positive control when treated with 50 μM ascorbic acid was measured as 197.76% at 50 μM, and the sample higher than the control was 50 SPMK measured as 246.94% when the solid content was 0.1%. Except for 50SPMK, a sample within a range similar to that of the control was identified as 50SFMKUF5, and 50SFMKUF5 was a sample of a fraction of less than 5 kDa through ultrafiltration after fermenting with L. paracasei MK1 after treating 50% soy milk with flavourzyme. 50SFMKUF5 showed a slightly lower amount of procollagen of 164.23% than that of the control group, but not less than 160% of procollagen could be confirmed at all concentrations.

Experimental example 9: TNF -α production analysis

1) Analysis of the amount of TNF-α produced

^{HaCaT cells were inoculated with 1×10 5} cells in a 12 well plate and cultured for 24 hours, and then, in order to induce TNF-α, samples were treated by concentration with UV irradiation and cultured for an additional 24 hours. The group without UV irradiation and the group treated with 1 μM/mL of dexamethasone after UV irradiation were used as controls and compared with the results of the sample. After irradiation at 312 nm with 12.5 mJ, the sample was cultured for 24 hours, and then TNF-α secreted into the medium was measured for absorbance at 450 nm using a kit (Invitrogen, #KHC3012, USA).

2) Analysis result

The control group was 100% UV-irradiated HaCaT, and the positive control group used dexamethasone, which is a strong anti-inflammatory agent. Samples 50SFMKUF5, 50SMKP, 50SFMKUF30, 50SPMK and 50SFMK samples treated with 50% soymilk that did not show cytotoxicity to HaCat cells of Experimental Example 7 are shown in FIGS. 10A to 10E in comparison with the positive control dexamethasone.

The amount of TNF-α produced in the positive control treated with dexamethasone was 65.58%, and the samples showing lower values than the positive control at all concentrations (0.1, 0.02, 0.004, 0.0008%) were 50SFMKUF30 and 50SFMKUF5, of which 50SFMKUF5 also had a concentration of 0.02%. In 23.44% of TNF-α was detected, 50SFMKUF5 was confirmed to inhibit the production of TNF-α.

Experimental example 10: 50 SFMKUF5 And 5 kDa Component analysis of the following fractions

1) Free amino acid analysis

The selected 50% soymilk dilution was subjected to ultrafiltration to analyze free amino acids for five samples of 50SMKUF5, 50SMKFUF5, 50SMKPUF5, 50SFMKUF5, and 50SPMKUF5 fractions of less than 5 kDa through ultrafiltration, and are shown in Table 7.

Amino acid
Concentration of free amino acid(mg/100 g (%)) 50SMKUF5 50SMKFUF5 50SMKPUF5 50SFMKUF5 50SPMKUF5 Aspartic acid 1.8 (4.5) 1.9 (4.1) 1.9 (4.5) 2.6 (3.9) 1.2 (2.8) Glutamic acid 12.5 (31.0) 13.3 (28.4) 11.2 (27.0) 7.2 (10.7) 4.6 (10.5) Asparagine 0.4 (1.0) 0.6 (1.2) 0.4 (0.9) 1.0 (1.5) 0.2 (0.4) Serine 0.2 (0.6) 0.5 (1.1) 0.3 (0.6) 0.2 (0.3) 0.1 (0.2) Glutamine 0.0 (0.0) 0.5 (1.1) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) Glycine 0.8 (2.0) 0.7 (1.5) 0.7 (1.7) 0.6 (0.8) 1.2 (2.7) Histidine 1.6 (3.9) 1.6 (3.4) 1.7 (4.2) 1.3 (2.0) 1.2 (2.8) Arginine 10.9 (27.0) 10.2 (21.8) 10.7 (25.8) 11.9 (17.7) 9.6 (22.1) Threonine 0.3 (0.6) 0.7 (1.6) 0.4 (1.0) 0.5 (0.8) 0.5 (1.1) Alanine 2.4 (6.0) 2.5 (5.3) 2.5 (6.1) 2.9 (4.3) 3.1 (7.1) Proline 2.9 (7.2) 2.7 (5.9) 3.8 (9.1) 1.5 (2.2) 1.0 (2.3) Tyrosine 1.5 (3.7) 0.7 (1.6) 1.5 (3.6) 3.1 (4.5) 1.4 (3.2) Valine 0.3 (0.7) 0.7 (1.4) 0.3 (0.7) 2.2 (3.3) 1.7 (4.0) Methionine 0.0 (0.0) 0.7 (1.6) 0.0 (0.0) 0.3 (0.5) 0.5 (1.2) Cysteine 0.0 (0.0) 0.1 (0.3) 0.1 (0.2) 0.1 (0.2) 0.0 (0.0) Isoleucine 0.0 (0.0) 0.6 (1.2) 0.2 (0.4) 2.4 (3.6) 2.1 (4.8) Leucine 0.4 (0.9) 2.1 (4.5) 0.6 (1.4) 11.2 (16.6) 4.7 (10.8) Phenylalanine 0.6 (1.5) 2.4 (5.1) 1.0 (2.4) 15.0 (22.3) 6.4 (14.6) Tryptophan 2.7 (6.7) 2.6 (5.6) 3.0 (7.3) 2.7 (4.0) 3.9 (8.9) Lysine 1.0 (2.5) 1.7 (3.6) 1.2 (3.0) 0.6 (0.8) 0.2 (0.4) TA ^{1 )} 40.2 (100) 46.8 (100) 41.4 (100) 67.3 (100) 43.6 (100) EA ^{2 )} 6.9 (16.8) 13.1 (28.0) 8.4 (20.4) 36.2 (53.9) 21.2 (43.6)

¹⁾ TA: Total free amino acid

²⁾ EA: Essential amino acid (Thr+Val+Met+Ile+Leu+Phe+Lys+Trp+His)

50SMKUF5 without enzyme treatment showed the lowest value of 40.2 mg/100 g, and the free amino acid content of 50SFMKUF5 and 50SPMKUF5 fermented after enzyme treatment was higher than 50SMKFUF5 and 50SMKPUF5 enzyme treatment after fermentation. The free amino acid content of 50SFMKUF5 was the highest at 67.3 mg/100 g, and 50SMKFUF5 was measured at 46.8 mg/100 g. This is because L. paracasei MK1 could more effectively cleave the protein of soy milk by enzymatic treatment first, so it is thought that the samples subjected to the enzyme treatment had higher free amino acid content than the samples without the enzyme treatment.

As for the content of essential amino acids among free amino acids, 50SFMKUF5 showed the highest content of 36.2 mg/100 g, accounting for 53.9% of the total amino acids. On the other hand, 50SMKUF5 was found to have a difference of about 6 times that of 50SFMKUF5, the highest at 6.9 mg/100 g. In addition, 50SMKUF5 without enzyme treatment was found to have a low content of essential amino acids in samples treated with flavourzyme or protamex. Flavourzyme-treated samples 50SMKFUF5 and 50SFMKUF5 had an essential amino acid content of 13.1 and 36.2 mg/100 g, respectively, and protamex-treated samples 50SMKPUF5 and 50SPMKUF5 had an essential amino acid content of 8.4 and 21.2 mg/100 g. Samples showed higher essential amino acid content. It is judged that there is a difference in the range in which the enzyme acts on the substrate, and L. paracasei MK1 is used in soymilk fermentation. , This suggests that flavouzyme is more effective than protamex.

2) Total nitrogen and TCA dissolved nitrogen content analysis

Changes in nitrogen content by fermentation and enzyme treatment were measured through the total nitrogen content of the sample and the dissolved nitrogen content of TCA. The total nitrogen content can estimate the amount of all peptides or proteins present in the sample, and the content of nitrogen dissolved in TCA is meaningful to estimate the content of nitrogen compounds generated by hydrolysis of proteins by protease.

division Total nitrogen
(mg N/100 mL) TCA soluble nitrogen
(mg N/100 mL) TN / SN(%) 50S
50SMKUF5
50SMKFUF5
50SMKPUF5
50SFMKUF5
50SPMKUF5 2.82±0.17
0.34±0.07
0.60±0.14
0.40±0.09
1.22±0.23
1.60±0.22 0.12±0.03
0.12±0.05
0.36±0.03
0.23±0.01
0.45±0.02
0.82±0.06 4.39
36.17
59.59
58.02
36.93
51.16

As shown in Table 8, 50S without any treatment showed a total nitrogen content of 2.82 mg N/100 mL, and 50SPMKUF5 was measured as 1.60 mg N/100 mL. 50SMKUF5, which showed the lowest value in terms of total nitrogen content, was found to be 0.34 mg N/100 mL. This showed that the maximum total nitrogen content was reduced by up to less than 50%. In addition, 50SMKUF5 fermented with L. paracasei MK1, the final selection strain, and 50SMKFUF5 and 50SMKPUF5 fermented after fermentation, had no significant difference in total nitrogen content. The total nitrogen content of 50SPMKUF5 was measured to be more than twice that of 50SMKUF5, 50SMKFUF5, and 50SMKPUF5.

Through the nitrogen content of TCA dissolved, the content of non-protein nitrogen by enzyme treatment was observed, and as a result, it was observed that 50SFMKUF5 and 50SPMKUF5 were 0.45 and 0.82 mg N/100 mL, respectively, similar to the total nitrogen content. The first enzyme treatment was determined to have a high content of non-proteinaceous nitrogen. Since the soymilk was first hydrolyzed due to an enzyme agent, it is believed that the nitrogen content of TCA dissolved is high, and the enzyme treatment after fermentation is considered to have little effect from the enzyme agent because hydrolysis has already occurred due to the final selected strain. It was found that the content of samples 50S and 50SMKUF5 did not differ significantly.

3) General component analysis

In order to analyze the difference between the 50SFMKUF5 fraction selected through Experimental Examples 7 and 8 and the general ingredients of 100% soymilk, the general ingredients of 100% soymilk are shown in Table 9.

division
content 100% soy milk 50SFMKUF5 Saccharide (g/100 g)
Carbohydrate (g/100 g)
Crude protein (g/100 g)
Crude lipid (g/100 g)
Solid (%) 0.71
3.40
4.30
2.40
10.8 ND ¹⁾
ND
0.30
0.00
1.37

¹⁾ ND stands for Not detemided

Sugars per 100 g were 0.71 g, carbohydrates 3.40 g, crude protein 4.30 g, crude fat 2.40 g, and the solid content was measured to be 10.8%. The crude protein of 50SFMKUF5 was found to be 0.30 g, the content of crude fat was 0.00 g, and the solid content was 1.37%. All general ingredients showed lower values than 100% soymilk, which could be judged by the decrease due to fermentation and ultrafiltration.

4) Peptide analysis

The 50SFMKUF5 fraction selected through Experimental Examples 7 and 8 was subjected to peptide mapping using LC/MS-MS. The LC chromatogram is shown in FIG. 11A, and most of the peaks were present at the retention time of 19.03-46.54 minutes. The MS chromatogram selecting the interval between 19.25-48.28 minutes of retention time is shown in FIG. 11B. The major peak between the peptide peaks having a molecular weight of 953.48-4770.20 kDa has an m/z value of 726.87, and this peak was identified as a peptide with a molecular weight of 1453.74 kDa. This peptide was found as Glycinin G2 as a result of searching on the data base derived from soybean (NCBI, USA), and its amino acid sequence was confirmed as APEFLKEAFGVN.

Hereinafter, according to the following composition, each of the soap-type, lotion-type, cream-type, pack-type, and cosmetic liquid-type formulations were prepared.

Manufacturing Example 1. Soap type

50SFMKUF5 fraction 5

Maintenance 75

Sodium hydroxide 5

Spices 10

Remaining amount of purified water

(Unit: wt%)

Manufacturing Example 2. Lotion type

50SFMKUF5 fraction 3.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Water-soluble collagen (1% aqueous solution) 1.00

Sodium citrate 0.10

Citric acid 0.05

Licorice extract 0.20

1,3-butylene glycol 3.00

Remaining amount of purified water

(Unit: wt%)

Manufacturing Example 3. Cream type

50SFMKUF5 fraction 1.00

Polyethylene glycol monostearate 2.00

Self-emulsifying glycerin monostearate 5.00

Cetyl alcohol 4.00

Squalene 6.00

Tri2-ethylhexane glyceryl 6.00

Sphingolipid 1.00

1.3-butylene glycol 7.00

Remaining amount of purified water

(Unit: wt%)

Manufacturing Example 4. Packed

50SFMKUF5 fraction 5.00

Polyvinyl alcohol 13.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Lauroylhydroxyproline 1.00

Water-soluble collagen (1% aqueous solution) 2.00

1,3-butylene glycol 3.00

Ethanol 5.00

Remaining amount of purified water

(Unit: wt%)

Manufacturing Example 5. Essence liquid type

50SFMKUF5 fraction 2.00

Hydroxyethylene cellulose (2% aqueous solution) 12.00

Xanthan gum (2% aqueous solution) 2.00

1,3-butylene glycol 6.00

Dark glycerin 4.00

Sodium hyaluronate (1% aqueous solution) 5.00

Remaining amount of purified water

The above formulations represent a form using the soymilk fermentation broth or soymilk peptide fraction according to the present invention, and the soymilk fermentation broth or soymilk peptide fraction according to the present invention may be used in various formulations used in the art, and this is also the scope of the present invention. Belongs to.

Korea Microorganism Conservation Center (domestic) KFCC11552P 20130404

<110> YONSEI UNIVERSITY BKBIO.CO.,Ltd. Gachon University of Industry-Academic cooperation Foundation LCS Biotech Co., Ltd. <120> Anti-wrinkle cosmetic composition using Lactobacillus paracasei MK1 and preparation method thereof <130> HPC3939 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 1284 <212> DNA <213> Lactobacillus paracasei <400> 1 tttggaaaca gatgctaata ccgcatagat ccaacaaccg catggttctt ggctgaaaga 60 tggcgtaagc tatcgctttt ggatggaccc gcggcgtatt agctagttgg tgaggtaatg 120 gctcaccaag gcgatgatac gtagccgaac tgagaggttg atcggccaca ttgggactga 180 gacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat ggacgcaagt 240 ctgatggagc aacgccgcgt gagtgaagaa ggctttcggg tcgtaaaact ctgttgttgg 300 agaagaatgg tcggcagagt aactgttgcc ggcgtgacgg tatccaacca gaaagccacg 360 gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc cggatttatt 420 gggcgtaaag cgagcgcagg cggtttttta agtctgatgt gaaagccctc ggcttaaccg 480 aggaagcgca tcggaaactg ggaaacttga gtgcagaaga ggacagtgga actccatgtg 540 tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc tgtctggtct 600 gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac cctggtagtc 660 catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt gccgcagcta 720 acgcattaag cattccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 780 gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 840 caggtcttga catcttttga tcacctgaga gatcaggttt ccccttcggg ggcaaaatga 900 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 960 agcgcaaccc ttatgactag ttgccagcat ttagttgggc actctagtaa gactgccggt 1020 gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1080 cacacgtgct acaatggatg gtacaacgag ttgcgagacc gcgaggtcaa gctaatctct 1140 taaagccatt ctcagttcgg actgtaggct gcaactcgcc tacacgaagt cggaatcgct 1200 agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1260 tcacaccatg agagttgtaa cacc 1284 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 50SFMKUF5 <400> 2 Ala Pro Glu Phe Leu Lys Glu Ala Phe Gly Val Asn 1 5 10

Claims (10)

Soymilk hydrolysed with Flavorzyme (flavourzyme) inoculated with Lactobacillus paracasei MK1 [Accession No .: KFCC11552P] to fermentation product of soymilk hydrolysates exceeding molecular weight of 5 kDa. Anti-wrinkle cosmetic composition comprising a fermented product of soy milk hydrolyzate from which proteins or peptides have been removed.
delete delete delete The method of claim 1, wherein the fermented product of soybean milk hydrolyzate or the hydrolyzate of soymilk fermented product has type I procollagen production activity and tumor necrosis factor-alpha (TNF-α) inhibitory activity, and human dermal fibroblasts and human keratinocytes. Wrinkle improvement cosmetic composition characterized in that it does not exhibit toxicity.
6. The wrinkle according to claim 5, wherein the fermented product of the soymilk hydrolyzate or the hydrolyzate of the soymilk fermentate has a free amino acid content of 60 mg or more in 100 g total and an essential amino acid content of 50% by weight or more in the free amino acid. Improvement cosmetic composition.
The cosmetic composition for improving wrinkles according to claim 6, wherein the fermented product of the soymilk hydrolyzate or the hydrolyzate of the soymilk fermentate contains a peptide having the amino acid sequence of SEQ ID NO: 2.
A soymilk hydrolyzate manufacturing step of hydrolyzing soymilk with a proteinase, flavorzyme;
A fermentation step of soymilk hydrolyzate inoculating the soymilk hydrolysate by inoculating Lactobacillus paracasei MK1 [Accession No .: KFCC11552P] and fermenting the soymilk hydrolysate; And
Removing the protein or peptide having a molecular weight of more than 5 kDa from the fermented product of the soy milk hydrolyzate. delete delete

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