KR20190023496A - Process for producing fermented algae containing fucose and a cosmetic composition containing the same - Google Patents

Abstract

 And to a cosmetic composition containing the same. More specifically, a) pre-fermentation step by desalting of the hearing and seaweed and addition of whole milk powder; b) mixing with water and fermenting by inoculating lactic acid bacteria and yeast; c) a fermented extract prepared by the above method and a cosmetic composition for improving moisturizing, whitening and skin wrinkles containing the extract as an active ingredient. The composition contains fucose as an active ingredient, which can be expected as antioxidative effect, whitening effect, anti-inflammatory effect, wrinkle improving effect and skin moisturizing effect as cosmetic raw materials.

Description

TECHNICAL FIELD The present invention relates to a fermentation seaweed containing a fucose and a cosmetic composition containing the fucose and a cosmetic composition containing the fucose,

The present invention relates to a fermentation seaweed containing a fucose and a cosmetic composition containing the same, more particularly, to a method for producing fermented seaweed containing fucose, To a cosmetic composition.

Seaweed is a sea bird, which is classified as green algae, brown algae, red algae, and cyanobacteria. In Korea, about 500 species are known, of which only 50 are used for food. Seaweeds vary in depth depending on the ray required for photosynthesis. Generally, the red light is extinguished in a shallow place and the cyan can penetrate to a deep place. The green algae are relatively shallow and live in a bright place, and the brown algae and red algae gradually appear to live in deep and dark places.

Seaweed has low content of protein and fat and low digestibility of carbohydrate, so it has low nutritional value. However, it has high content of various inorganic salts such as potassium iodine and calcium, vitamin A and C, low cholesterol content in blood, And prevention of hardening. Recently, many studies have been conducted as functional food and functional cosmetic raw materials.

The skin is divided into the epidermis, the dermis, and the subcutaneous fat layer. The epidermis consists of the stratum corneum, the transparent layer, the granular layer, the visible layer and the basal layer. The dermis is the connective tissue below the epidermis and consists of the papillary layer and the reticular layer have. The epidermal layer protects against intrusion of bacteria entering from the outside, pulling force, tension, mechanical stimulation, prevents water evaporation, maintains the homeostasis of the human body and prevents evaporation of water to maintain the homeostasis of the human body. The dermis is distributed in many capillaries. The blood vessels regulate the heat loss through contraction and expansion, regulate the body temperature through the eccrine glands, and keep the skin lubricated by sebaceous glands. In addition, collagen, elastin, and hyaluronic acid in the dermis tissue are important fiber tissues that maintain the flexibility and elasticity of the skin.

If the moisture content of the epidermal layer is reduced to less than 10%, dry skin with cracking of the skin occurs. Although the pathogenesis of dry skin has not yet been elucidated, it is known that skin and systemic diseases, hygiene, environment and lifestyle are important, and skin barrier, such as composition change of epidermal lipid component, increase of transdermal water loss, And the change in the stratum corneum that performs the function of the skin.

The inventors of the present invention have studied the possibility of application of various natural products to cosmetic raw materials and found that the seaweed which is a green alga and a brown algae are fermented by using lactobacillus sakei, . As a result of measuring the antioxidative effect, whitening effect, anti-inflammatory effect, wrinkle-reducing effect and skin moisturizing effect from these, it was found that the efficacy as an excellent cosmetic raw material can be expected.

The present invention aims to optimize the production of fermented products as a product by fermentation process using seaweeds such as hearing and seaweed by lactic acid bacteria, and to establish a method of producing seaweed fermented extract and to provide antioxidant, whitening, anti-inflammatory, skin wrinkle, And to provide a cosmetic composition.

In order to achieve the above-mentioned object, the present invention has investigated optimal conditions by examining the content of seaweed, the content of additives, and the optimal culture temperature in fermenting seaweed with lactic acid bacteria, and found that the antioxidant , Whitening, anti-inflammation, wrinkle improvement, and moisturizing effect.

As described above, the present invention is a method for producing an auditory fermentation extract and a wakame fermentation extract. The produced cosmetic composition is a cosmetic composition having antioxidative, whitening, anti-inflammatory, wrinkle-reducing and moisturizing effects.

FIG. 1 shows the growth of Lactobacillus casei according to the time of dry hearing
FIG. 2 shows the growth of Lactobacillus casei according to the time of dry seaweed supplementation
FIG. 3 is a graph showing the fermentation conditions of lactic acid bacteria
Fig. 4 shows changes in the number of lactic acid bacteria depending on the temperature and time of cultivation of lactic acid bacteria
Fig. 5 shows the change in activity of enzyme activity according to the incubation temperature and time of lactic acid bacteria
Fig. 6 is a graph showing the relationship between the number of lactic acid bacteria
Fig. 7 is a graph showing the number of lactic acid bacteria
Fig. 8 shows the comparison of antioxidative activities of fermented seaweed extracts before fermentation
9 shows the comparison of the whitening activity after fermentation of the seaweed fermentation extract
Fig. 10 shows the comparison of the whitening activity after fermentation of the seaweed fermentation extract
11 shows the comparison of the whitening activity of the seaweed fermentation extract before fermentation
Fig. 12 shows the comparison of the whitening activity of the seaweed fermentation extract before fermentation
Fig. 13 shows the comparison of anti-inflammatory activity of fermented seaweed extract before fermentation
Fig. 14 is a graph comparing the inhibitory effect of the seaweed fermentation extract on elastase activity before fermentation
Fig. 15 shows the comparison of the amount of hyaluronic acid after fermentation of the seaweed fermentation extract
FIG. 16 shows the analysis of gene expression patterns before and after fermentation of the seaweed fermentation extract
FIG. 17 shows the results of HPLC

The cosmetic composition of the present invention contains a fucose which is an active ingredient in seaweed. The seaweed used is, but not limited to, the hearing and the seaweed, and can be produced from most seaweeds that maintain their shape in water. The seaweed used in the present invention can be prepared by the following steps.

a) a step in which whole milk powder is added to the dried powder after washing or desalting the hearing or the seaweed

b) Inoculation of lactic acid bacteria by mixing with water in the above step, sterilization by heating, and cooling

c) sterilization and filtration after fermentation with lactic acid bacteria after storage at appropriate temperature

In order to confirm the production conditions of the present invention, the production method was established through Examples 1 to 4 below. Thereafter, various tests were conducted to confirm the efficacy of the seaweed fermentation extract. The antioxidative activity test was carried out in the same manner as in Example 5, whitening activity in Examples 6 to 9, anti-inflammatory activity in Example 10, inhibition of wrinkle formation in Example 11, measurement of hyaluronic acid content in moisturizing in Example 12, The content of fucose as a component was confirmed through Example 14 and the like.

Example 1 Search for Determination of Optimum Fermentation Condition (Seaweed Content)

Lactobacillus sakei , a fermented lactic acid bacterium, was inoculated after sterilization at 80 ℃ for 2 hours with different contents of seaweed (0.1%, 0.5%, 1.0%, 2.0%, and 3.0%) in order to confirm optimal fermentation conditions for lactic acid bacteria. Absorbance was measured every 12 hours and the optimum fermentation conditions were investigated by confirming the amount of lactic acid bacteria.

FIG. 1 shows the results of measurement of dry hearing, while FIG. 2 shows the results in different contents in dry seaweed. When the content of dry hearing was increased, the growth rate was increased in lactic acid bacteria. As the time elapsed, the growth rate decreased. As a result, the growth rate of L. acidophilus was increased as the content of wakame in dried seaweed increased.

≪ Example 2 > Search for confirming optimum fermentation condition (additive content)

(Whole milk, 0.5%, 1.0%, 2.0%) was added to each seaweed (hearing, seaweed) at a concentration of 2.0% in order to confirm the optimum fermentation conditions for the lactic acid bacteria. After sterilization at 80 ° C. for 2 hours, the fermented lactic acid bacteria L after the. sakei inoculum search for an optimal fermentation conditions by measuring the absorbance every 12 hours.

As shown in FIG. 3, when 2.0% of seaweed dried powder was contained in the same amount and the content was different in the whole powdered milk, the sensory and the seaweed contents were both high when the powdered milk containing 2.0% And the number of lactobacillus was increased in a short time when 1.0% of total milk powder was contained than 0.5% of whole milk powder, and it was suitable for fermentation when 1.0% of whole milk powder was contained.

≪ Example 3 > Searching for optimum fermentation condition (culture temperature)

Optimal incubation temperature was determined by physical conditions to optimize fermentation. The optimum fermentation conditions were confirmed by measuring total bacterial counts of lactic acid bacteria at different cultivation temperature and containing 2.0% of seaweed powder and 1.0% of whole milk powder.

As a result, as shown in FIG. 4 and FIG. 6, the highest number of lactic acid bacteria was found at 30 ° C in the hearing, and the seaweed was slightly higher at 28 ° C, and the growth conditions of lactic acid bacteria at 25 ° C and 35 ° C were good there was.

≪ Example 4 > Search for confirmation of optimal fermentation conditions (enzyme measurement)

Optimal incubation temperature was determined by physical conditions to optimize fermentation. The optimum fermentation condition was confirmed by measuring the activity of α-amylase enzyme with the same culture temperature of 2.0% for seaweed powder and 1.0% for whole milk powder. α-amylase activity was measured using a SALIVARY α-AMYLASE kit. This method uses 2-chloro-p-nitrophenol coupled with the maltotriose, which is a pigmented substrate, and 2-chloro-p-nitrophenol is produced by enzymatic reaction of α-amylase with this substrate. Can be measured.

As a result of measuring the activity of α-amylase as another method for confirming the optimal culture temperature, the results were as shown in FIG. 5 and FIG. 7. As in the measurement results of total viable cell count, The enzyme activity of α-amylase was measured at 28 ℃ and it was confirmed that it was the optimal temperature for fermentation of lactic acid bacteria.

Example 5 Comparison of post-fermentation antioxidant activity of seaweed fermentation extract (DPPH assay)

Antioxidative activity of fermented seaweed extracts was investigated by DPPH assay. The DPPH radical scavenging ability of fermented hearing and seaweed before fermentation was measured. For the DPPH assay, 60 μl of the sample was mixed with 240 μl of 0.2 mM DPPH (2,2-diphenyl-1-picrylhydrazyl), allowed to stand at 37 ° C for 30 minutes, and the absorbance was measured at 517 nm using a microplate reader . Blank was used as a sample solvent, DPPH reagent was used as a control dilution solvent instead of a sample, and L-ascorbic acid was used as a positive control. DPPH radicals are purple, and when reacted with antioxidants, they are reduced by antioxidants and become pale yellow. The antioxidant activity is measured by measuring the color change.

As shown in FIG. 8, the hearing loss was reduced to 43% before fermentation at 400 ㎍ / ㎖, but increased to 69% after fermentation. The seaweed decreased to 33% before fermentation at 400 ㎍ / After that, it showed a scavenging ability at 65%, and it was confirmed that DPPH radical scavenging ability increased after fermentation before fermentation.

Example 6: Comparison of post-fermentation activity of a seaweed fermentation extract (Tyrosinase inhibition assay)

After the fermentation, tyrosinase inhibition assay was performed to confirm the whitening activity. In this assay, B16F1 mouse melanoma cells were cultured and seeded in a 12-well plate at 1 × 10 ⁵ cells / well. The cells were cultured for 24 hours. The medium was removed and the samples were seeded at 100 μg / , 200 [mu] g / ml, 400 [mu] g / ml) and cultured for 48 hours. Arbutin is used as a positive control and incubated for 48 hours. Each well is washed with 10 mM PBS and suspended in 100 μl of 10 mM PBS containing 1% Triton X-100. This suspension is vortexed and centrifuged at 1,000 rpm for 5 minutes. The supernatant is used as the active enzyme solution. 40 μl of this enzyme solution was added to a 96-well plate, and 100 μl of L-dopa (2 mg / ml) was added. After incubation at 37 ° C for 1 hour, the absorbance was measured at 405 nm to determine the activity of tyrosinase Calculate with the following formula.

<Formula 1>

Inhibition rate (%) = {1 - (Absorbance of no added group of absorbance of sample added group)} X 100

As a result, as shown in Fig. 9, the activity of tyrosinase was inhibited in a concentration-dependent manner in the hearing and seaweed fermentation extracts. In addition, in the case of 400 μg / ㎖ of hearing, enzyme activity inhibition ability was 64% after fermentation, 76% after fermentation before fermentation, and enzyme activity inhibition ability was 64% after fermentation at 400 ㎍ / And the activity inhibition was increased.

<Example 7> Comparison of post-fermentation activity of fermented seaweed extract (L-DOPA Oxidation inhibition assay)

L-DOPA Oxidation inhibition assay was performed to confirm whitening activity after fermentation. For this assay, add 0.1 M phosphate buffer (pH 6.5) and sample to a 96-well plate and add 1.5 mM tyrosine or 10 mM L-DOPA in that order. Arbutin was used as a positive control and 0.1 M phosphate buffer was used as a negative control. Add 500 U / ml of Mushroom Tyrosinase and incubate at 37 ° C for 15 minutes. After the reaction, the absorbance is measured at 490 nm using an ELISA reader.

As a result, as shown in FIG. 10, the inhibitory effect on L-DOPA Oxidation was increased from 65% in fermentation to 73% in fermentation at 400 μg / , And the enzyme activity inhibition was increased from 41% to 55% after fermentation at 400 ㎍ / ㎖ in the seaweed before fermentation.

<Example 8> Cellulol melanin synthesis inhibition assay of fermented seaweed extract before fermentation.

The amount of melanin in the melanocytes was measured by measuring the content of melanin in the cells. 1 × 10 ⁵ B16F1 cells were plated on a cell culture dish and incubated for 24 hours. After 3 days of treatment, the cells were washed with 1 × phosphate buffered saline (PBS), and then incubated for 1 hour with 10% dimethyl sulfoxide (DMSO) N sodium hydroxide (NaOH) solution at 80 ° C for one hour. Absorbance was measured at 475 nm using a multi-plate reader. The total amount of protein was measured and then the amount of melanin per a certain amount of protein was calculated.

The fermentation efficiency of the fermented extract (CF-F) and the seaweed fermented extract (US-F) was compared before fermentation. As a result, as shown in FIG. 11, arbutin, a positive control, inhibited melanin synthesis by 50% at a concentration of 50 μg / ml and 83% at a concentration of 100 μg / ml. The sensory evaluation showed that melanin synthesis was suppressed from 47% before fermentation to 50% after fermentation of seaweed fermented extract, while that of audible fermented extract increased from 49% to 57% after fermentation.

Example 9 Comparison of post-fermentation activity of seaweed fermentation extract (Extracellular melanin synthesis inhibition assay).

Extracellular melanin synthesis inhibition assay was performed by dividing 1 × 10 ⁵ B16F1 cells in a cell culture dish for 24 hours and then culturing the media for 3 days. The absorbance was measured at 475 nm using a multi-plate reader, Respectively.

As a result, as shown in FIG. 12, arbutin, a positive control, inhibited melanin synthesis by 48% when treated with 50 μg / ml and 71% when treated with 100 μg / ml. In the seaweed fermented extract, the inhibitory effect of the fermented extract was increased 42% before fermentation to 51% after fermentation, and that of seaweed fermented extract also increased from 42% before fermentation to 44% after fermentation.

Example 10: Comparison of anti-inflammatory activity of fermented seaweed extract before fermentation.

The most representative indicator of inflammatory response in cells is NO (nitro oxide). Nitric oxide (NO) is a fast speed to nitrate (NO ^3-), and nitrite (NO ^2-) biological conversion, so a measure of the total NO ^3- / ^2- to NO in nitric oxide generation to be produced by NO synthase (NOS) Can be used as a marker. Nitrate Reductase can be used to reduce NO ³ to NO ^{2 -,} and then measure the amount of NO ^{2 - with} the Griess reagent. It is known that the color reaction using Griess reagent is inhibited by NADPH, which is essential for NO synthase and nitrate reductase reaction.

As a result, as shown in FIG. 13, LPS was treated and each sample was treated to induce an inflammatory reaction. When the extract was treated with 100 ㎍ / ㎖ of the extract before the fermentation, the release of nitric oxide was decreased by 50% before fermentation and 34% after fermentation. Seaweed fermentation extract also inhibited the production of nitrogen compounds after fermentation from 46% to 43%.

Example 11: Comparison of inhibition of elastase formation after fermentation of fermented seaweed extracts.

Elastase inhibition assay was performed to measure the amount of p-nitroanilide produced from substrates at 37 ° C for 30 min using n-succinyl- (L-Ala) ₃ -p-nitroanilide as a substrate. Prepare each sample at a constant concentration, and take 40 μl of the solution in a 96-well plate. Add 40 μl of 2.5 U / ml porcine pancreas elastase solution in 50 mM Tris-HCl buffer (pH 8.6). (L-Ala) 3-pnitroanilide (0.5 mg / ml) dissolved in 50 mM Tris-HCl buffer (pH 8.6) was added to the reaction mixture for 30 minutes and the reaction was stopped for 5 minutes. And the absorbance at 410 nm is measured. Ursolic acid (UA) is used as a positive control for wrinkle improvement activity evaluation.

In order to examine the wrinkle-reducing effect of the seaweed fermentation extract, ursolic acid was used as a positive control group as shown in Fig. At the concentration of 200 ㎍ / ㎖, auditory extracts showed 59% before fermentation and 60% after fermentation, but elastase was not increased but changed. At the concentration of 200 ㎍ / ㎖, the seaweed extract also changed to 49% before fermentation and 56% after fermentation.

<Example 12> Comparison of hyaluronic acid amount after fermentation of fermented seaweed extract

HaCat cells were seeded in 6 well plates at a concentration of 2 x 10 ⁵ cells / ml. Twenty-four hours later, the cells were washed 2 times with serum-free DMEM, then serum-free DMEM was added and the seaweed fermentation extract was treated. After 24 hours, centrifugation was carried out at 15,000 xg for 5 minutes, and the supernatant was removed and stored at -20 ° C until ELISA. The supernatant was added to the plate coated with the hyaluronic acid anti-body and reacted, and then the calibration curve was drawn and measured.

As a result, as shown in FIG. 15, HaCat cells were cultured in order to determine the amount of hyaluronoc acid, and the seaweed fermentation extract was treated and measured using culture filtrate. In the untreated group, 144 ng / ml hyaluronic acid was measured. In the case of 100 μg / ㎖ of the extract of the sea mustard extract (US), it was 186 ng / ml after the fermentation (CF-F) and 197 ng / ml and increased to 188 ng / ml after fermentation (CF-F).

<Example 13> Gene expression pattern before fermentation of seaweed fermentation extract

B16F1 cells producing melanin were treated with 100 nM of α-MSH to induce melanogenesis and treated with seaweed extract. TYR (Tyrosinase), MITF (Microphthalmia-associated transcription factor) and TRP-1 (Tyrosinase related protein-1) were used as primers using the nucleotide sequences shown in Table 1 below.

As a result, as shown in FIG. 16A, it was confirmed that the expression level of TYR associated with whitening was increased when α-MSH was treated. There was no significant change in hearing and seaweed extracts after treatment with α-MSH. However, when the fermented extract was treated, the appearance of the fermented extract was significantly reduced, and the fermented extract of the seaweed fermented was also reduced. MITF also increased the expression level when α-MSH was treated, but there was no significant change in the extract before fermentation, but the polyadenylation pattern was decreased in the auditory fermentation extract and the seaweed fermentation extract. In the case of TRP-1, it was also confirmed that the expression patterns of TRP-1 were decreased in the auditory and seaweed fermentation extracts.

Primer sequences used in PCR Gene name Base sequence TYR
(Tyrosinase) F: GAG AAC TAA CTG GGG ATG AG
R: ATC TGC TAT CCC TGT GAG TG, MITF
(Microphthalmia-associated transcription factor) F: AGG GGA AAA CTT GGT GTG AG
R: TAG AAA CGC TGG AAG GAA GG, TRP-1
(Tyrosinase related protein-1) F: GCA CTG ATG ACT TGA TGG GA
R: CAG TGA AAG TGT GCA GGA GG IL-1? F: AGC AGC ACA TCA ACA AGA GC
R: AGC AGC ACA TCA ACA AGA GC, TNF-a F: GGG ACA GTG ACC TGG ACT GT
R: GCA GAG GTT CAG TGA TGT AG, COX-2 F: GCC TTC TCC AAC CTC TCC TA
R: ACC TCT CCA CCA ATG ACC TG,  iNOS F: GCT GTG CTC CAT AGT TTCA
R: CTT GTC ACC ACC AGC AGT AG.

As shown in FIG. 16B, in order to examine the expression pattern of inflammation-related genes involved in inflammation, Raw 264.7 cells were treated with LPS for the induction of inflammatory reaction. IL-1β and TNF -α, COX-2, and iNOS, respectively.

The nucleotide sequence of the primer was used as shown in Table 1 above. In the case of IL-1β, the expression pattern of LPS-treated seaweed extract did not change significantly, but the expression pattern of IL-1β was remarkably decreased when the fermented extract was treated. When TNF-α was also treated with fermented extract, Respectively. In the case of COX-2 and iNOS, the expression pattern was significantly reduced in the extracts and fermented extracts.

<Example 14> Confirmation of fucose content using HPLC

Fucoidan, which is an effective substance of seaweed, is a viscous polysaccharide form and it is difficult to analyze by HPLC. Fucoidan was confirmed to be a monosaccharide form fucose after fermentation. To confirm the contents of fucose as an active ingredient, analysis was conducted under the conditions shown in Table 2 below. As a result, as shown in FIG. 17, it was confirmed that the fucose monosaccharide was contained in the pericarp fermentation extract and the seaweed fermentation extract, considering that there was a peak in the same time zone as the standard product fucose (FIG. 17A).

Post Course Analysis Condition Column Agilent Zorbax SB-C18 4.6 x 250 mm, 5 m Solventa H ₂ O (20%) Solvent B CH ₃ CN (80%) Run time 30min Flow rate 1 ml / min Injection vol. 310 쨉 l Wave length 254 nm Oven temp. 30 ℃

Claims (7)

A process for preparing fermented seaweed containing fucose and a cosmetic composition containing the same. The method according to claim 1,
The fermentation seaweed is fermented with Lactobacillus sakei as green algae or brown algae such as Ecklonia cava or Undaria pinnatifida. The method according to claim 1,
(1) pre-fermentation step by desalting of the hearing or the seaweed and addition of whole milk powder; And
(2) mixing and sterilizing with water, inoculating the lactic acid bacteria to ferment the seaweed;
The method of manufacturing a seaweed fermented product according to claim 1, The method of claim 3,
Characterized in that it contains 0.01% to 5.0% of hearing or sea mustard in the pre-fermentation step; In the fermentation step, sterilization is carried out at 80 ° C for 2 hours, followed by cooling to 40 ° C. The lactic acid bacteria inoculation is inoculated at 1 × 10 ³ cells / ml to 1 × ^{10 9} cells / ml and fermented at a temperature of 25 ° C to 37 ° C &Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; 5. The method of claim 4,
Wherein the fermented product or the dry powder is added in an amount of 0.01 to 100% based on the total weight of the cosmetic product. 6. The method of claim 5,
The emulsion, facust or solid anhydrous product of the oil-in-water (O / W) type or water-in-oil type (W / O) type, Such as an oil dispersion in an aqueous phase using a sphere, a lipid vesicle form in an ionic or non-ionic form, an ointment, a cleansing foam, a cleansing water, a pack or a body oil, and an aqueous or milky makeup base, foundation, Wherein the cosmetic composition is formulated into one of a color cosmetics product line comprising skin cover, lipstick, lip gloss, face powder, two way cake, eye shadow, mascara, cheek color and eyebrow pencil. 6. The method of claim 5,
The cosmetic composition is characterized by an antioxidant effect, a whitening effect, an anti-inflammatory effect, a wrinkle-reducing effect, and a skin moisturizing effect.

Images