KR20180054055A - Method of fermented mulberry branch extract and beverage and cosmetics containing fermented mulberry branch extract by the same - Google Patents

Abstract

The present invention relates to a fermented Mori Ramulus extract. More specifically, the present invention relates to a method for producing a fermented Mori Ramulus extract, and a beverage and cosmetic composition containing the fermented Mori Ramulus extract produced thereby as an active ingredient. By converting inactive oxyresveratrol glycoside contained in mulberry branches by a fermented mulberry syrup to active aglycone, it is possible to increase absorption of the extract in the body, thereby improving physiological usability. Accordingly, the method enable users to easily obtain antioxidant and anti-aging functions in everyday life.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing a fermented upper limb extract, a fermented upper limb extract, and a beverage and a cosmetic composition containing the fermented upper limb extract as an active ingredient,

The present invention relates to a fermented upper limb extract, and more particularly to a fermented upper limb extract which is obtained by converting an inactive oxyreservatrol glycoside contained in a mulberry branch (upper limb) by fermentation odor into an active oxyreservatrol aglycon, A method of manufacturing a fermented upper limb extract which enables a user to easily provide antioxidant and anti-aging function in everyday life as the fermented upper limb extract is increased, and a beverage and a cosmetic composition .

Despite the fact that the ingredients of the humidifier disinfectant have recently been found to be contained in wet tissues and toothpastes, the stability of the chemical synthetic products has become a hot topic. However, ordinary consumers who do not have professional knowledge recognize dozens of chemical components in their daily life and select harmful components It is still difficult to bet. In particular, the development of products made of naturally occurring phytochemicals harmless to the body has been actively promoted both domestically and externally. Especially, the field of foods containing chemically synthesized substances as main raw materials or chemical synthesis additives consumed by consumers continuously It is attracting attention.

As a raw material for cosmetics or food having an anti-aging effect such as whitening, wrinkles and inflammation, various oriental or natural-derived physiologically active substances are applied. In the past, it has been known that mulberry leaves, , And the development of functional materials and products using mulberry, which is known as a sacred tree that can not be passed down to the root shell (桑 白皮).

Mulberry has been widely used for the treatment of diabetes, differentiations, edema, skin aging, inflammation and whitening in herbal medicine. Especially, the upper branch of the mulberry tree has a tendency to regulate all the winds, promote digestion, , And that the mouth is dry. In addition, recent studies have reported that total cholesterol is reduced by 29% and lipid metabolism in the liver is significantly reduced by administering topical extracts of oxyreservatrix to experimental rats for 4 weeks, Antihypertensive, antiinflammatory, and antiinflammatory effects, the physiological activities of the upper extremities are being actively studied.

The present inventor has also been continuously studying mulberry including the upper part. In the registered patent No. 10-1438543, a method of separating and purifying oxyresveratrol, trans-resveratrol and morasin contained in the mulberry branch was suggested, The present invention provides a method for producing an edible vinegar and an edible vinegar using the extracts of a mulberry tree, .

However, anti-aging and antioxidants derived from natural substances such as olive oil and rhododendron are safe and hypoallergenic substances, but most of them are inactive glycosides. Therefore, when they are not activated, their absorption and utilization rate are low, Accordingly, the present inventors came to the present invention by recognizing that the above problems and in particular, the lack of development of the technology for maximizing the functionality and efficiency of oxyreservatrix among anti-diabetic, anti-inflammatory and anti-aging physiologically active substances contained in the upper extremities.

It is an object of the present invention to overcome the above-mentioned problems, and it is an object of the present invention to maximize the functionality of oxyreservatase contained in the upper limbs and to improve the absorbability and utilization of the body while allowing a small amount of the fermented upper limb extract to be easily applied in daily life. A method for producing a fermented upper limb extract which can be provided, and a beverage and a cosmetic composition containing the fermented upper limb extract thus prepared as an active ingredient.

In order to solve the above-mentioned problem, the present invention is characterized in that the sugar solution and the oligosaccharide are mixed at a weight ratio of 80:20 in a ratio of 1: 1, S1), and 60% ethanol aqueous solution of 10 to 30 times the weight of the dried upper layer was added to the dried upper layer, and the mixture was sonicated by repeating 1 to 5 times at room temperature for 6 hours. After filtration and concentration, (S2) dissolving the upper ethanol extract prepared in S2 in an ionized water of 100 to 250 times the weight of the upper ethanol extract (S3), a step (S3) of mixing the odorant solution prepared in S1 and the dissolution of S3 (Step S4) of obtaining a fermented upper limb extract of 40 to 50 ㅀ Brix by filtration and concentration under reduced pressure after fermentation for 5 weeks in a fermenter including a jar. To provide a method.

The present invention also provides a fermented upper limb extract, which is produced by the above-mentioned method, further characterized by the present invention.

Further, it is another feature of the present invention to provide a beverage and a cosmetic composition comprising the fermented upper body extract in an amount of 0.1 wt% to 5 wt%.

According to the above-described constitution, fermentation conditions capable of maximizing the functionality of oxyreservatrix contained in the upper limb are suggested, so that anti-aging and antioxidative function can be realized even though a small amount of the fermented upper limb extract of the present invention is used, The absorption and availability of the topical extract are improved so that the functionalities are doubled and the functionalities of the fermented upper body extract can be easily provided by the user on a daily basis by being utilized as beverage and cosmetic composition.

FIG. 1 is a flowchart illustrating a method of manufacturing a fermented upper body extract according to an embodiment of the present invention.
2 is a graph showing the HPLC chromatogram results of the fermented upper body extract according to the fermentation period according to the embodiment of the present invention.
FIG. 3 is a graph showing the results of measurement of cell (HS68) toxicity according to the concentration of the fermented upper body extract according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of measurement of cell (HS68) toxicity according to the concentration of ORT. FIG.
FIG. 5 is a view showing collagen synthesis results of the fermented upper body extract according to an embodiment of the present invention. FIG.
6 is a view showing the result of collagen synthesis of the ORT.
FIG. 7 is a graph showing the collagenase inhibiting activity of the fermented upper body extract according to an embodiment of the present invention. FIG.
Fig. 8 is a graph showing the result of collagenase inhibition activity of ORT. Fig.
FIG. 9 is a graph showing the results of measuring the cell (B16F1) toxicity of the fermented upper body extract according to an embodiment of the present invention.
10 is a diagram showing the result of measurement of the cell (B16F1) toxicity of ORT.
FIG. 11 is a graph showing the results of inhibition of melanin formation by the fermented upper body extract according to an embodiment of the present invention.
Fig. 12 is a graph showing the effect of inhibiting melanin formation of ORT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings.

1. Preparation of fermented upper limb extract

S1: Manufacture of Audi Cyanide

Sugar and oligosaccharide were mixed at a weight ratio of 80:20 in a ratio of 1: 1 by weight to the jar and the mixture was aged at low temperature for 2 weeks to prepare an auditory fluid.

As an example, the above-mentioned Odi is commercially available, and 15 kg of audi and 15 kg of sugar water are put into a jar and then low-temperature aged auditory fluid is prepared. When low temperature aging, microorganisms contained in the audi are added to the sugar The most preferred is the fermented fermented sea tangle extract, which is used for fermenting the upper ethanol extract in the fermentation process.

At this time, when the amount of the sugar is excessively added relative to the weight of the oats, the weight ratio of the oats to the sugar does not satisfy the ratio of 1: 1, so that the activity of the microorganisms is lowered to insufficient fermentation effect, If added in small amounts, molds will form and lead to abnormal fermentation.

S2: Preparation of upper ethanol extract

The dried upper layer was subjected to ultrasonic extraction by repeating 1 to 5 times (preferably 2 times) 6 to 10 hours of 60% aqueous ethanol solution by 10 to 30 times the weight of the dried upper layer, followed by filtration and concentration, Brix (preferably 50 [deg.] Brix) topical ethanol extract.

In the present invention, the ultrasonic extraction is performed at 30 ° C to 35 ° C according to extraction for 6 hours as the extraction time increases. In addition, the reason why the sugar content of the upper ethanol extract is set to be 50 ° Brix is that it can be easily dissolved by ionized water, and the concentration time can be shortened and the manufacturing cost can be reduced.

In the present invention, since the extraction of the active ingredient from the dried upper part is easy compared with the hot water extraction by adopting the ethanol extraction, anti-aging and antioxidative effect can be manifested even though a small amount of the upper part ethanol extract is used and the concentration time is shortened, Can be saved.

S3: dissolution of upper ethanol extract

The upper ethanol extract prepared in S2 is dissolved 50 to 200 times (preferably 100 times) the weight of the upper ethanol extract.

As an example, 300 g of the upper ethanol extract prepared in S2 is dissolved by adding 30 kg of ionized water. The distilled water having almost no inorganic content is used as the ionic water. In the final product of the fermented upper body extract according to the present invention, the effect of color or smell Do not go crazy.

In the present invention, by dissolving the ethanol extract of the upper part of 50 ° Brix with ionized water, the sugar content is lowered to 25 ° Brix because the activation of the microorganisms including lactic acid bacteria is improved during the mixing and fermentation of the aqueous extract of the olive and the upper ethanol, .

S4: Preparation of fermented upper limb extract

The ethanolic solution prepared in S1 and the soluble ethanol extract of the upper part of S3 having a weight of 3 to 10 times the weight of the above-mentioned odor solution are put into a fermenter containing a jar and fermented for 5 weeks, followed by filtration and concentration under reduced pressure to obtain a fermented upper body extract.

As an example, 10 kg of the auditory fluid prepared in S1 and 40 kg of the dissolved ethanol extract of upper part of S3 were mixed and fermented for 5 weeks, and then concentrated under reduced pressure at 60 ° C. for 3 minutes to obtain 5 kg of fermented upper limb extract of 40 to 50 ° Brix Respectively.

In the present invention, fermentation is adopted among the biotransformation techniques to convert inert physiologically active substances contained in the upper extremities into an active form, that is, by converting an inactive polymer into an active low-molecular substance or by using an enzyme produced by a microorganism, To produce a new derivative with higher activity than that of the physiologically active substance. This not only doubles skin aging and antioxidant functions of the above-mentioned physiologically active substances, but also allows the skin to be absorbed more rapidly into the deep part of the skin, thereby enhancing the skin protecting power, And so on.

In addition, the fermentation period is a period set in consideration of the conditions for maximizing the deformation of the inactive oxyreservatable glycosides (ORTG) contained in the upper limb by the active oxyrespetrol aglycon (ORT) through the following experiment.

2. Setting the fermentation period

In the present invention, the content of oxyreservatrol aglycone (ORT), antioxidant activity, antioxidant activity and antioxidant activity, and the fermentation period for maximizing antioxidative function can be measured through HPLC chromatogram experiment, Respectively.

(1) Changes in ORT content during fermentation period

HPLC (Waters e2695 HPLC System, Milford, Mass., USA) was used for the HPLC analysis of the surface component of oxytetracycline aglycon (ORT) of the fermented upper limb extract according to the production method of the present invention. 100 ml of the fermented upper limb extract was collected, concentrated under reduced pressure, dissolved in distilled water, and passed through a 0.25 μm membrane filter (Waters, Milford, Mass., USA). The HPLC analysis conditions were as follows: column; (0.05% H ₃ PO ₄ in H ₂ O), Solvent B (CH ₃ CN: H ₂ O: ₁ : ₁ ), mobile phase: YMC-Pack Pro C ₁₈ column (5 μm, 46 * 250 mm, YMC Inc., USA) MeOH = 1: 1: 1.15, v / v / v) (linear gradient elution: A-> B, 65 min), flow rate; 0.8 mL / min, injection amount: 10 mu L, and detector: ultraviolet ray (UV _{310 nm} ).

On the other hand, the peaks of the oxyreveratrol glycoside (ORTG) and the oxyresveratrol aglycone (ORT) separated by HPLC were compared with the retention time of the previously isolated reference material, The content of functional indicator substances was calculated from the calibration curves prepared using standard substances and shown in Table 1. The content of functional index components such as ORTG and ORT of the fermented upper body extract according to the fermentation period is as shown in Fig.

The oxyserberatrol glycoside (ORTG) refers to the above-mentioned inactive-type physiologically active substance or an inactive-type polymer substance, and is an active low-molecular substance converted into an active form by the fermentation of oxyresvetrol aglycon (ORT) It is a substance that ultimately maximizes the content in the invention.

Surface component Content (g / 100 g, extract) Control 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks Oxyresveratrol glycoside (Mulberroside A) 3.46 ± 0.07 2.46 ± 0.05 2.14 + 0.06 1.54 + - 0.05 0.46 + 0.03 0.12 + 0.03 0.11 + 0.03 Oxyresveratrol aglycone (Oxyresvaratrol) 5.76 + 0.04 6.65 + 0.14 6.78 + - 0.10 7.91 + 0.07 8.49 + 0.07 9.03 + - 0.12 8.90 + - 0.13

According to Table 1, ORTG, which is an inactive glycoside, was present as a main functional substance of non-fermented upper limb extract (Control), but its content tended to decrease as fermentation progressed. On the contrary, as the ORTG was fermented by the action of the microorganisms contained in the AUDI solution, the content of the low molecular weight ORT increased until the fifth week of fermentation, and then decreased from the sixth week of the fermentation. This is because, when the fermentation reaches the fifth week, it is used as a raw material for other microorganisms such as acetic acid bacteria present in the fermented upper body extract or further decomposed. In particular, the ORT content of the fermented upper limb extract was 9.03 ± 0.12, which was about twice that of non-fermented upper limb extract (Control, 5.76 ± 0.04), indicating that the antioxidant and antioxidant functionalities were also doubled.

As can be seen from FIG. 2, the peak of ORTG (Rt = 12.8) in the non-fermented upper limb extract (Control) HPLC chromatogram showed a tendency to decrease with the fermentation period, and the ORT (Rt = 32.3) Respectively. At Rt = 22.7, the fermented product increased sharply in the first week of fermentation, decreased in the third week, and then increased again after the fourth week. In the case of Rt = 35 or more, resveratrol and morasin components were detected more Respectively.

That is, by converting an inactive ORTG into an active ORT through fermentation, it is possible not only to improve the absorption rate and utilization rate in a later product, but also to obtain resveratrol and morasin in addition to ORT, thereby doubling the anti-aging and antioxidant function . Considering the purpose of extracting maximal amount of oxirebesterol aglycon (ORT) from the upper part, the fermentation period is preferably 5 weeks in which the ORT content increased by about twice as much as that in non-fermentation.

(2) Changes in physiological activity by fermentation period

Table 2 shows the results of measuring the change in physiological activity according to the fermentation period with the measurement sample concentration of the fermented upper limb extract of the present invention at 5 mg / ml.

Physiological activity Inhibition rate (%) Control 1 week 3 weeks 5 weeks 6 weeks Antioxidant ¹ 14.81 24.87 47.61 55.30 40.92 Skin aging ² 41.39 35.48 27.51 78.11 46.89 Skin aging ³ 13.0 35.30 62.52 67.74 65.98

As shown in Table 2, the antioxidant ¹ showed the highest inhibition rate of 55.30% at the 5th week after the fermentation, and the non-fermented topical extract (Control) was 14.81% as a result of DPPH radical scavenging activity measurement. (Control), and decreased to 40.92% from 6 weeks.

In addition, it showed a skin anti-aging ² Tyrosinase inhibitory non-fermented extract of the upper limbs (Control) is a high inhibition rate than the fermented tea 1 weeks (35.48%) and the fermentation Week 2 (27.51%) 41.39% as active as the measurement result. However, in the fermentation period of 5 weeks (78.11%), the fermentation period was about twice that of the fermentation period. In the 6 weeks fermentation period (46.89%), the fermentation period was slightly lower than the 5 week fermentation period. appear.

In addition, the anti-aging activity of skin antioxidant ³ was found to be 13.0% for non-fermented upper body extract (control), about 3 times for fermentation 1 week (35.30%) and 5 weeks for fermentation (67.74% (5-fold increase) and decreased from 6 weeks (65.98%) of fermentation.

That is, the result of measuring the physiological activity of the fermented upper limb extract was similar to that of the ORT content, and the fermentation period of 5 weeks in which the ORT content and other antioxidant and antioxidant functional substance contents were confirmed to the maximum showed the anti- It is best suited for implementation.

(3) Changes in physicochemical quality characteristics according to fermentation period

The results of measuring the physicochemical quality characteristics of the fermented upper limb extract according to the fermentation period of the present invention are shown in Table 3.

characteristic Fermentation period (week) Control 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks Sugar content
(° Brix) 8 7 9 10 10 9 9 Acidity
(pH) 0.1
(4.1) 1.2
(3.2) 1.5
(3.4) 1.2
(3.5) 1.6
(3.7) 1.5
(3.9) 0.6
(4.1)
Chromaticity L 88.90 55.43 36.59 43.36 41.18 50.65 29.25 a 11.43 51.75 49.11 49.70 43.02 38.28 22.64 b 6.71 24.08 29.66 29.29 22.97 20.80 11.91

The sugar content of the nonfermenting upper limb extract (control) was 8 ° Brix, which was slightly decreased to 7 ° rix in the first week of fermentation, but increased from the 2nd week of fermentation to 10 ° Brix in the third and fourth weeks of fermentation. The car maintained a 9 ° Brix.

The acidity of the non - fermented upper limb extract (Control) was pH4.1, which decreased to pH 3.2 after 1 week of fermentation, then gradually increased with fermentation period and became pH4.1 at 6 weeks of fermentation.

In the control, the lightness (L) was the highest at 88.90 and decreased to 36.59 at the 2nd week of fermentation and increased to 50.65 at the 5th week of fermentation. The lowest lightness Respectively. In addition, the redness (a) showed the lowest value of 11.43 in the upper limb extract (Control) without fermentation, and decreased to 51.75 in the 1st week of fermentation, but decreased until the 6th week of fermentation (22.64). In the case of yellowness (b), the top extract (Control) without fermentation tended to be lowest at 6.71 and tended to increase until the second week of fermentation (29.66), but tended to decrease from the third week of fermentation (29.29).

Therefore, considering the application of the fermented upper limb extract of the present invention as a beverage and cosmetic composition, the fermented upper body extract of fermented 5th week of fermentation is most preferable in terms of sugar content, acidity and chromaticity as processability.

In the mean time, all the experimental results were expressed as mean standard deviation. Statistical analysis was performed using the SPSSPackage Program (IBM, USA). All experimental results were independent experiments at least three times, and statistical significance was tested by one-way ANOVA and statistically significant at p <0.05.

3. Measurement of physiological activity of fermented upper limb extract

The physiological activity of the fermented upper limb extract according to the present invention is compared with non-fermented upper limb extract (Control) and other surface components from the following experiments and verified.

(1) DPPH radical scavenging activity

The reducing power of each sample was measured by electron donating effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH). That is, 0.5 mL of 0.4 mM DPPH solution was added to 1 mL of the extract prepared by concentration, vortexed for 10 seconds, and reacted at 37 for 30 minutes. Then, the absorbance of the reaction solution was measured at 517 nm using a spectrophotometer, Are shown in Table 4.

sample DPPH radical scavenging activity (IC ₅₀ , 占 퐂 / mL) Non-fermented upper limb extract (Control) 925.34 ± 31.82 Fermented upper limb extract 230.76 + - 21.70 Oxyresveratrol (indicator component) 7.02 + - 0.43 L-Ascorbic acid (vitamin C, positive control) 5.08 ± 0.11

The DPPH radical scavenging activity was 230.76 ± 21.70, which was about 4 times higher than that of non-fermented upper limb extract (Control, 925.34 ± 31.82) in the fermented upper body extract according to the present invention. As a result, ORT and various active ingredients.

(2) Xanthine oxidase inhibitory activity (Superoxide anion radical scavenging activity)

Add 0.1 mL of the sample solution and 0.6 mL of 0.1 M potassium phosphate buffer, pH 7.5, and add 0.2 mL of 2 mM xanthine solution, add 0.1 mL of 0.2 unit / mL xanthine oxidase, incubate at 37 ° C for 5 minutes, add 1 mL of 1N HCl After the reaction was completed, the uricacid produced in the reaction solution was measured at 292 nm in absorbance. Xanthin oxidase inhibitory activity was expressed as the absorbance reduction rate of the sample solution added group and no addition group.

sample Superoxide anion Radical scavenging activity (IC ₅₀ , ㎍ / mL) Non-fermented upper limb extract (Control) 74.49 + - 2.85 Fermented upper limb extract 25.5 ± 1.23 Oxyresveratrol (indicator component) 829.39 ± 27.94 Epigallocatechingallate (EGCG, positive control) 71.04 + 2.03

The superoxide anion radical scavenging activity of the fermented upper body extract according to the present invention was 25.5 ± 1.23, which was about three times higher than that of the non-fermented upper body extract (Control, 74.49 ± 2.85).

(3) Measurement of collagenase inhibitory activity

The collagenase inhibitory activity was determined by the addition of 4 mM CaCl ₂ to 0.1 M Tris-HCl buffer (pH 7.5) to determine the inhibitory effect on skin aging. 0.3 mg / mL), 0.15 mL of collagenase (0.2 mg / mL) was added to the mixture, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 0.5 mL of 6% citric acid was added to stop the reaction After adding 1.5 mL of ethylacetate, the supernatant was measured for absorbance at 320 nm. The absorbance of the supernatant was determined by the absorbance of the sample solution and the no-added sample.

sample Collagenase inhibitory activity (IC ₅₀ , ㎍ / mL) Non-fermented upper limb extract (Control) 175.38 ± 7.56 Fermented upper limb extract 85.73 ± 0.73 Oxyresveratrol (indicator component) 23.84 + - 2.93 EGCG (positive control) 476.93 + - 15.92

The collagenase inhibitory activity of the fermented upper body extract according to the present invention was 85.73 ± 10.73 μg / mL, which was about twice that of non-fermented upper body extract (175.38 ± 17.56), and the ORT (23.84 ± 2.93 μg / mL) ) Showed about 20 times more inhibitory activity than EGCG (476.93 ± 5.92 ㎍ / mL), which is positive control group, and collagenase inhibitory activity of the fermented upper limb extract seems to be due to inclusion of the above index components.

(4) Elastase inhibitory activity

The amount of p- nitoanilide was measured at 37 for 30 min using N-succinyl- (L-Ala) -3-p-nitoanilide as a substrate for the inhibition of porcine pancreas elastase. Prepare each test solution to a constant concentration, add 0.1 ml of each solution to a test tube, add 0.05 ml of elastase (0.6 units / ml, pancreatic solution, Type I: From Porcine Pancreas) dissolved in 50 mM Tris-HCl buffer (pH 8.6) 0.1 ml of N-succinyl- (L-Ala) 3-p-nitoanilide (1 mg / ml) dissolved in 50 mM Tris-HCl buffer (pH 8.6) was added and reacted for 30 minutes to measure the absorbance at 410 nm Respectively. Elastase inhibitory activity was expressed as the absorbance reduction ratio of the sample solution and the non - added sample.

sample Elastase inhibitory activity (IC ₅₀ , / / mL) Non-fermented upper limb extract (Control) 103.63 + - 2.45 Fermented upper limb extract 157.12 ± 3.78 Oxyresveratrol (indicator component) 523.82 + - 12.82 Oleanolic acid (positive control) 84.29 + - 5.34

The Elastase inhibitory activity of the fermented upper body extract according to the present invention was 157.12 ± 3.78 μg / mL, lower than that of the nonfermentative upper body extract (103.63 ± 2.45 μg / mL), and the functional index component, ORT (523.82 ± 12.82 μg / mL) Was lower than the positive control oleanolic acid (84.29 ± 5.34 μg / mL). Therefore, the fermented upper limb extract and surface components showed almost no inhibitory effect of Elastin on the skin elasticity.

(5) Measurement of tyrosinase inhibitory activity

To confirm skin whitening effect, tyrosinase inhibitory activity was measured as follows. In a test tube, add 0.2 mL of a solution containing 10 mM L-DOPA in 0.5 mL of 1 / 15M sodium phosphate buffer at pH 6.8, and 0.1 mL of a sample solution, and add 0.2 mL of 110 Unit / mL mushroom tyrosinase. And the DOPA chrome produced in the reaction solution was measured at 475 nm. Tyrosinase inhibitory activity was expressed as the absorbance reduction rate of the sample solution added group and the no added group.

sample Tyrosinase inhibitory activity (IC _50, / / mL) Non-fermented upper limb extract (Control) 375.39 ± 6.58 Fermented upper limb extract 184.67 + - 7.83 Oxyresveratrol (indicator component) 0.26 + 0.03 Arbutin (positive control) 555.03 + - 57.50

The tyrosinase inhibitory activity of the fermented upper limb extract according to the present invention was 184.67 ㅁ 7.83 ㎍ / mL, which was about twice that of the nonfermenting upper limb extract (375.39 ± 16.58 ㎍ / mL). The ORT (0.26 ± 0.03 ㎍ / mL) showed about 2,000 times higher inhibitory effect than arbutin (555.03 ± 57.50 μg / mL), which is currently used as a whitening cosmetic raw material. Therefore, it was confirmed that the fermented upper limb extract and ORT can be used as a cosmetic whitening material.

(6) Measurement of? -Glucosidase inhibitory activity

Α-glucosidase inhibitory activity on tyrosinase activation associated with skin melanin synthesis was measured as follows. In other words, 10 L of sample solution or 0.05 M sodium phosphate buffer (pH 6.5) was added to 50 L of α-glucosidase (2 U / mL), and the mixture was reacted at room temperature for 5 minutes. Then, 5 mM p- nitrophenylglucoside a p -NPG) p -NP product, after addition of 50 L to the reaction for 5 minutes at room temperature by measuring the absorbance at 405 nm is coming out from the glass substrate, p -NPG was measured. At this time, the? -Glucosidase inhibitory activity was calculated according to the following formula. That is, the activity of α-glucosidase inhibition (%) = 1- (A / B) 100, A: the absorbance of the sample at 405 nm, and B: the absorbance of the control at 405 nm. Antioxidant activity was measured by IC ₅₀ values.

sample α-glucosidase inhibitory activity (IC ₅₀ , ㎍ / mL) Non-fermented upper limb extract (Control) 2,045.92 + - 72.04 Fermented upper limb extract 438.36 ± 21.83 Oxyresveratrol (indicator component) 112.24 + - 3.75 Acarbose (positive control) 447.73 ± 18.90

The α-glucosidase inhibitory activity of the fermented upper limb extract according to the present invention was 438.36 ± 21.83 μg / mL, which was 2.3 times higher than that of the non-fermented upper limb extract (2,045.92 ± 72.04 μg / mL), and the functional index component ORT (112.24 ± 3.75 ㎍ / mL) showed about four times more inhibitory activity than acarbose (447.73 ± 18.90 ㎍ / mL) positive control. Therefore, it was confirmed that the fermented upper limb extract and ORT can be used as a whitening cosmetic raw material as an α-glucosidase inhibitor.

4. Cell experiment of fermented upper limb extract

(1) Cell culture

HS68 cells, normal human fibroblasts, and B16 melanoma cells, a melanoma cell line, were purchased from ATCC. (10% fetal bovine serum, FBS, Life Tech Inc., USA), 1% antibiotic antimycotic (100 U / mL penicillin and 50 g / mL streptomycin, Life Tech Inc., USA) in a 37, 5% CO ₂ incubator.

(2) Cytotoxicity measurement

In this experiment, the MTT [3- (4,5-dimethythiazyl-2-yl) -2,5-diphenytetrazolium bromide, Sigma] assay was used to determine the concentration of samples treated with HS68 and B16F1 cells Respectively. HS68 cells and B16F1 cells were plated in 96-well plates at 1 × 10 ⁴ cells / well and 5 × 10 ³ cells / well, respectively, and cultured for 24 h at 37 ° C under 5% CO ₂ . After the culture, the culture medium was removed, and the samples were diluted in the medium by the concentration, changed, and cultured for 24 h and 72 h, respectively. Cell viability was determined by MTT assay after incubation.

① Non-fermented upper limb extract (Control) and fermented upper limb extract

Concentration (/ / mL) Cell viability (%, N = 3, mean ± SD) Non-fermented upper limb extract (Control) Fermented upper limb extract 0 100 100 One 92.323 + 2.359 97.504 + - 4.372 10 95.704 + 4.701 101.764 ± 7.344 50 89.353 +/- 2.410 97.055 ± 4.882 100 94.676 ± 1.916 102.601 + - 6.941 500 89.511 + - 8.825 86.157 ± 9.779 1000 89.689 ± 7.628 112.133 ± 9.278

As shown in Table 10 and FIG. 3, it was confirmed that cytotoxicity of fibroblasts (HS68 cells) did not show up to 1000 μg / mL in both non-fermented upper body extract and fermented upper body extract.

②Oxyresveratrol

Concentration (/ / mL) Cell viability (%, N = 3, mean ㅁ standard deviation) 0 100 One 99.662 + - 8.576 10 93.928 + 7.681 20 90.040 ± 6.147 50 77.737 ± 3.469 100 45.735 + 0.389

According to Table 11 and FIG. 4, Oxyresveratrol showed cell viability of 90% or more up to a concentration of 20 μg / mL, but showed a tendency to decrease from a concentration of 50 μg / mL or more. % Cell viability, and cytotoxicity of fibroblast (HS68 cells) was observed.

(3) Synthesis effect of collagen (Type 1 procollagen)

HS 68 cells were seeded in 24-well plates at 1 × 10 ⁵ cells / well and cultured for 24 h under 5% CO ₂ and 37 conditions. For each concentration, the samples were replaced with diluted serum-free medium and incubated for 24 h. The amount of collagen precursor C-terminal was measured by using Procollagen type I C-peptide EIA kit (Takara, Japan).

① non-fermented upper limb extract and fermented upper limb extract

The synthesis effect of collagen (Type 1 procollagen) of the non-fermented upper limb extract and the fermented upper limb extract is as shown in FIG. The non-fermented upper limb extract (Control) showed collagen synthesis effect in a concentration dependent manner at 1 μg / mL to 10 μg / mL. On the other hand, although the collagen synthesis effect was lower than that of the non-fermented extract, the fermented upper limb extract showed a collagen synthesis effect in a concentration dependent manner at 1 μg / mL to 50 μg / mL. Thus, it is considered that appropriate concentration control is required to exhibit the desirable effect of improving the wrinkles of the fermented upper body extract.

② Oxyresvestrol

The synthesis effect of oxyreservatrol collagen (Type 1 procollagen) is as shown in Fig. According to this, Oxyresveratrol showed a collagen synthesis effect in a concentration dependent manner at 0 / / mL to 5 / / mL, but the effect tended to decrease at a higher concentration.

(4) Inhibitory effect on collagenase (MMP-1) production

HS68 cells were seeded in 24-well plates at 1 × 10 ⁵ cells / well and cultured for 24 h under 5% CO ₂ and 37 conditions. After washing with phosphate-buffered saline (PBS), serum was removed from the medium, and 15 J / cm ² UVA (ELC-500, Lightning Enterprises, USA) was added. After irradiation with UVA, the sample was replaced with serum-free medium diluted by concentration and cultured for 48 hr. The amount of MMP-1 liberated in the medium was measured using a MMP-1, Human, Biotrak ELISA System (GE Healthcare, UK) and corrected for the total protein content.

① Non-fermented upper limb extract (Control) and fermented upper limb extract

density
(쨉 g / mL)
MMP-1 (%, N = 3, mean ± standard deviation) P value Non-fermented upper limb extract Fermented upper limb extract Non-fermented upper limb extract Fermented upper limb extract 0 100 100 One 95.376 ± 17.188 102.235 ± 2.749 10 99.214 ± 5.800 90.561 7.827 0.105 50 101.002 ± 10.774 53.164 ± 2.792 <0.001 100 81.526 + 2.590 44.802 + 4.023 <0.001 <0.001

The results of inhibiting the collagenase (MMP-1) production inhibition of the non-fermented upper limb extract (control) and the fermented upper limb extract are shown in Table 12 and FIG. According to the results, except for 1 μg / mL of the non-fermented upper body extract and fermented upper body extract, the inhibitory effect of the fermented upper body extract on the MMP-1 production was higher than that of the non-fermented upper body extract, From ㎍ / mL, the inhibitory effect was about twice as high as that of the non-fermented upper limb extract, and the excellent wrinkle-reducing effect of the fermented upper limb extract can be confirmed.

② Oxyresveratrol

Concentration (/ / mL) MMP-1 (%, N = 3, mean ± standard deviation) P value 0 100 One 80.144 + - 5.507 0.003 5 58.883 + - 2.546 <0.001 10 55.490 ± 3.099 <0.001 20 40.083 + - 4.172 <0.001

The results of inhibitory effect of Oxyresveratrol on collagenase (MMP-1) production, which is an index component of the upper limb, are shown in Table 13 and FIG. According to the results, oxyresveratrol significantly inhibited collagenase (MMP-1) in a concentration-dependent manner at a concentration of 1 μg / mL to 20 μg / mL.

(5) Melanin production inhibitory effect

Melanin production was measured by Oka et al. B16F1 cells were seeded in 6-well plates at 5 × 10 ⁴ cells / well and cultured for 24 h at 37 ° C in 5% CO ² . After the medium was removed, the sample was diluted in the medium by concentration, and α-MSH was added to the final concentration of 200 nM for 3 days. After incubation, the culture medium was removed, washed with PBS, and 1N NaOH containing 10% DMSO was added to dissolve the intracellular melanin in a 50 ° C incubator. Absorbance was measured at 490 nm using a microplate reader and corrected for total protein content.

(A) Cytotoxicity test (48 hours sample treatment)

① Non-fermented upper limb extract (Control) and fermented upper limb extract

Concentration (/ / mL) Cell viability (%, N = 3, mean ± SD) Non-fermented upper limb extract Fermented upper limb extract 0 100 100 One 99.826 ± 5.103 104.546 ± 2.098 10 102.824 ± 3.243 105.700 ± 2.652 50 98.248 ± 4.324 113.391 + 4.739 100 102.621 + 1.834 110.625 + - 6.357 200 108.454 + - 6.244 108.217 ± 3.321 500 106.484 + - 6.508 111.217 ± 5.815

The results of the cytotoxicity tests of the non-fermented upper limb extract (control) and the fermented upper limb extract are shown in Table 14 and FIG. Thus, it was confirmed that neither the fermented upper limb extract nor the non-fermented upper limb extract of the present invention exhibited cytotoxicity up to a concentration of 500 μg / mL.

② Oxyresveratrol

Concentration (/ / mL) Cell viability (%, N = 3, mean ± SD) Oxyresveratrol 0 100 One 97.741 + - 4.421 5 98.013 + 13.221 10 102.387 ± 12.200 20 98.149 ± 11.394 50 17.741 ± 0.944

The results of the cytotoxicity test of oxirsveratrol are shown in Table 15 and FIG. 10, and the cell survival rate was 90% or more to 20 μg / mL, but the cell survival rate rapidly decreased from 50 μg / mL to 20% And the experimental concentration was determined to be 20 占 퐂 / mL.

(B) Melanogenesis inhibitory effect (N = 1)

① Non-fermented upper limb extract (Control) and fermented upper limb extract

FIG. 11 shows the results of inhibition of melanin formation by the non-fermented upper limb extract (control) and fermented upper limb extract. As shown in FIG. 11, the non-fermented upper limb extract of melanin, which is one of the determinants of skin whitening, It was confirmed that the amount of melanin was kept almost unchanged. In contrast, the fermented upper limb extract tended to show a slight increase in melanin at 10 μg / mL in cells, but decreased to 92.771% up to 100 μg / mL. In addition, the effect of inhibiting the formation of melanin was somewhat greater in a concentration-dependent manner up to 100 μg / mL in the extracellular medium. At this time, the fermented upper limb extract showed an effect similar to arbutin, which is well known as an extracellular whitening substance, and decreased to 92.771% in cells up to 100 μg / mL, which is similar to 92.285% of arbutin The fermented upper limb extract according to the invention can be confirmed to have excellent whitening action.

② Oxyresveratrol

FIG. 12 shows the results of inhibition of melanin production by oxyresveratrol. As a result, it inhibited melanogenesis in a concentration dependent manner at 1 to 10 μg / mL in cells and extracellularly. In particular, it inhibited melanin production more than arbutin, Respectively.

5. Preparation of Beverage and Cosmetic Composition Using Fermented Upper Extract

The beverage and cosmetic composition is prepared according to the following description so that the anti-aging and antioxidant functionalities of the fermented topical extract of the present invention, which have been verified above, can be easily provided by the user in daily life.

ingredient Content (% by weight) Purified water 40 to 80 Liquid fructose 10 to 25 Blueberry concentrate 1 to 10 Raspberry concentrate 1 to 10 Polydextrose 1 to 5 Isomaltooligosaccharide 1 to 5 Fermented upper limb extract (or ORT) 1 to 5 Vitamin C 0.1 to 1 Garcinia cambogia bark extract 0.1 to 1 Enzyme treatment Stevia 0.1 to 1 Collagen 0.1 to 1 Herbal extracts 0.01 to 0.1 Chrysanthemum 0.01 to 0.05 TOTAL 100

The raw materials suitable for the food and food additives standard including the ingredients of Table 16 were used and used. The ingredients were added and mixed within the range of the contents shown in the table to prepare a beverage mixture containing the fermented upper body extract, It is dissolved by stirring for 30 minutes. The completely dissolved beverage mixture is sterilized (HTST) at 90 ° C to 110 ° C (preferably 98 ° C) for 10 seconds to 20 seconds (preferably 15 seconds) and then filtered using a 0.5 micro filter. In the above, the sterilized and filtered liquid is filled and sealed in the automatic filling machine in a predetermined amount, and the sealed product is cooled to have a product temperature of 50 ° C, and then the packaging and shipping proceed.

As an example, in the present invention, a mixture of 68.25 wt% of purified water, 15 wt% of liquid fructose, 5 wt% of blueberry concentrate, 5 wt% of raspberry concentrate, 2 wt% of polydextrose, 2 wt% of isomaltooligosaccharide, , An extract of fermented upper body including 0.5% by weight of vitamin C, 0.2% by weight of Garcinia cambogia bark extract, 0.2% by weight of enzyme treated stevia, 0.1% by weight of collagen, 0.05% by weight of a complex herb extract and 0.05% &Lt; / RTI &gt;

Beverage (fermented milk extract) pH 3.12 ± 0.00 Brix 25.85 ± 0.01 Acidity (acetic acid%) 1.81 + 0.02

Table 17 shows the physicochemical quality characteristics of the beverage liquid using the fermented upper body extract prepared according to the present invention. The crude liquid itself has a pH of 3.11 and an acidity of 1.81 ± 0.02. Sourness and strong sweetness due to 25.85 ± 0.01 ° Brix are felt. Therefore, users who require continuous intake in daily life can dilute them with water containing ionized water and water, and can ingest them according to their preference.

Thus, in the present invention, a sensory evaluation was conducted on 30 beak sensory evaluation drinks by 7 points scale (7: very good, 4 is excellent) to beverages obtained by diluting 2 to 5 times of water with respect to the weight of beverage containing fermented upper limb extract : Normal, 1: very bad), and the results are shown in Table 18.

division Beverage (fermented milk extract) Fermented upper limb extract
Used beverage (1: 4 dilution) color 5.50 + - 0.75 5.57 + - 0.95 smell 4.50 ± 1.10 5.50 ± 1.21 flavor 5.21 ± 1.08 6.24 ± 1.28 Overall likelihood 5.02 ± 0.18 6.08 ± 1.18

According to Table 18 above, the diluted beverage was subjected to an improved sensory evaluation in terms of color, smell, taste, and overall acceptability compared to the undiluted solution.

In the case of color, the difference between the undiluted solution and the diluted beverage was insufficient.

In the case of odor, it is assumed that the raw juice is 4.50 ± 1.10 and the diluted beverage is 5.50 ± 1.21, and that a slight sour odor derived from the raw juice is somewhat irritating to the olfactory sense of the evaluation stage, and by diluting the raw juice, .

In particular, the taste of the diluted beverage was 6.24 ± 1.28, which was rated as good as 7 and the overall taste of the diluted beverage due to the excellent sensory evaluation of the color, smell and taste was also 6.08 ± 1.18, .

In addition, the beverage blend liquid or the fermented upper limb extract according to the present invention can be manufactured in the form of an ampoule, so that it can be continuously consumed while being easily carried by the user.

The fermented topical extract may be formulated into a skin, lotion and cream formulation containing 0.1 wt% to 5 wt% based on the total weight of the cosmetic composition, so that the extract may be directly absorbed by the skin of the user to provide antioxidant and antioxidant functionalities.

Claims (4)

A step (S1) of adding a mixture of sugar and an oligosaccharide at a weight ratio of 80:20 in a ratio of 1: 1 by weight to the jar and aging the mixture at low temperature for 2 weeks (S1);
To the dried upper layer was added 10 ~ 30 times of 60% aqueous ethanol solution to the dried upper layer weight, and the mixture was sonicated for 1 to 5 times for 6 hours, followed by filtration and concentration to prepare 40 ~ 60 ㅀ Brix upper ethanol extract Step S2;
(S3) dissolving the upper-layer ethanol extract prepared in S2 in ionized water at 100 to 250 times the weight of the upper-layer ethanol extract;
[0051] The step (S4) comprises fermenting the oocyte solution prepared in S1 and the dissolved upper ethanol extract of S3 into a fermenter containing a jar and fermenting the mixture for 5 weeks, followed by filtration and concentration under reduced pressure to obtain a fermented upper body extract of 40 to 50 ㅀ Brix Wherein the fermented topical extract is prepared by a method comprising the steps of: A fermented topical extract prepared by the method of claim 1. A beverage comprising the fermented upper limb extract of claim 2 as an active ingredient, wherein the fermented upper limb extract comprises 0.1 wt% to 5 wt%. A cosmetic composition comprising the fermented upper limb extract of claim 2 as an active ingredient, wherein the fermented upper limb extract comprises 0.1 wt% to 5 wt%.

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