KR102303173B1 - The method of preparing liquid nuruk with enhaned growth speed of fungus by radio frequency pulse and enhanced enzyme activity by adjusting milling degrees of rice and takju and cosmetic materials containg the liquid nuruk prepared thereby - Google Patents

Abstract

The present invention is a method for producing liquid yeast having high enzymatic activity by using a grain selected from 10 minutes sea bream ~ brown rice, in which the growth rate of the cells is increased by culturing using a high-frequency pulse, and the degree of milling of the rice is adjusted;
By manufacturing using the prepared liquid yeast, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and reduces the non-uniformity of the product, which is a disadvantage of solid yeast, and provides a sensory excellent and differentiated takju, antioxidant action and It is possible to provide cosmetics with anti-wrinkle effect, improving the growth rate of cells using high-frequency pulses and adjusting the milling degree of rice to improve enzyme activity, and for takju or cosmetics containing liquid yeast prepared therefrom it's about

Description

A liquid yeast production method with improved enzyme activity by improving the growth rate of cells using high-frequency pulses and adjusting the milling degree of rice, and Takju or cosmetics containing liquid yeast prepared therefrom of fungus by radio frequency pulse and enhanced enzyme activity by adjusting milling degrees of rice and takju and cosmetic materials containg the liquid nuruk prepared thereby}

The present invention is a method for producing liquid yeast having high enzymatic activity by using a grain selected from 10 minutes sea bream ~ brown rice, in which the growth rate of the cells is increased by culturing using a high-frequency pulse, and the degree of milling of the rice is adjusted;

By manufacturing using the prepared liquid yeast, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and reduces the non-uniformity of the product, which is a disadvantage of solid yeast, and provides a sensory excellent and differentiated takju, antioxidant action and It is possible to provide cosmetics with anti-wrinkle effect, improving the growth rate of cells using high-frequency pulses and adjusting the milling degree of rice to improve enzyme activity, and for takju or cosmetics containing liquid yeast prepared therefrom it's about

Nuruk is used as a fermenting agent for making traditional Korean liquor, such as takju and yakju, and is an important factor in determining the quality of alcohol (So MH. 1995). As for the research on yeast, it is a study on the isolation and identification of useful molds and yeasts inhabiting the yeast (Jo GY et al. 1997, Kim HS et al. 1997) and the quality of brewed wine using improved yeast and traditional yeast in combination with various strains. and physicochemical properties (Jeong ME et al. 2014, Lee TS et al. 2000).

Bacteria present in the yeast include Baekgukgyun (白麴菌), Heukgukkyun (黑麴菌), Hwanggukkyun (黃麴菌), and Honggukkyun (紅麴菌), and these fungi are various types such as α-amylase and glucoamylase. It produces and secretes amylase enzymes to saccharify starch stored in cereals (Jung DH. 2012).

Aspergillus oryzae and Rhizopus oryzae are the most isolated molds from yeast, which can be said to be representative molds of yeast (Ribes JA et al. 2000). Aspergillus oryzae , a yellow yeast mold, has been used for a long time in fermented foods such as soybean paste and soy sauce (Park HK et al. 2008, Oh BH et al. 2006), and is a mold showing high saccharification by producing and secreting α-amylase ( Norihiro T et al. 1989). Aspergillus kawachii, a white yeast mold, is a fungus widely used in the production of shochu, and is one of the most important molds responsible for liquefaction and saccharification in koji.

Traditional Korean yeast include Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus shirousamii, Saccharomyces cerevisiae, Rhizopus sp, Bacillus subtilis and. Numerous microorganisms such as lactic acid bacteria exist, and in particular, it is characterized by a large number of fungi in the genus Aspergilllus , Absidia , and Rhizopus (Kim CJ et al. 1990).

In the production method of yeast, as in the traditional yeast production method, a solid culture method in which grains are treated and then cultured by inoculating yeast mold on the raw material, and a liquid medium by adding other nutrients to water to inoculate and culture the mycelium culturing the yeast mold, etc. There is a liquid culture method.

As for the solid culture method, research on a manufacturing method that can produce a large amount of various enzymes required for brewing (Lee DY. 1969, Lee MK et al. 1994, So MH et al. 2009) has been conducted, but the production of solid yeast is expensive. Because it is solid, it is difficult to uniformly manage temperature and humidity during culture, and since it is cultured in a semi-open type, the risk of contamination with various bacteria is unavoidable.

In general, the liquid culture method has the advantages of easy culture control and quality control of microorganisms, and the efficient production and process of enzymes are simple (Chandran S. 2005, Kang SG et al. 1997). However, it is known that yeast mold grown in liquid culture differs greatly from solid culture in the production of enzymes such as amylase and cellulase, and the overall productivity is lowered. Therefore, there is a need for in-depth research that can produce various types of enzymes at high concentrations by liquid culture.

Various advantages can be expected when yeast is prepared by liquid culture. The first is the simplification of manufacturing facilities. In order to manufacture yeast by solid culture method, large-scale manufacturing equipment is required. In order to secrete and produce enzymes by pulverizing and compressing grain raw materials and then propagating yeast molds, special equipment, facilities, and places in the fermentation room are required, as well as enlargement of the production facility equipment is inevitable.

However, the liquid culture device can use a general ventilation and agitation device that has been used in various fermentation industries so far, and if there is only a small amount of fermenter, it can meet the conditions required for producing liquid yeast. It is also possible to increase the maximum production quantity.

Second, the operability of the manufacturing process is easy. In the production of solid yeast, since the raw material is solid, it is difficult to uniformly manage the temperature and humidity during culture, and since the yeast mold is cultured in a semi-open type, the risk of contamination by various bacteria cannot be avoided.

However, in the production of liquid yeast, the entire process is performed in a liquid phase, so strict culture management is possible, and high-quality yeast can be stably manufactured.

Transfer from the fermentation tank is also easy to manage because a general liquid transfer pump can be used.

Third, it can be expected to develop desired new products. It is difficult to differentiate the quality of traditional liquors sold by small businesses because they use commercially available solid yeast.

However, for the production of liquid yeast, it is possible to easily perform large-scale culture on an actual production scale through continuous seed culture Scale-up from small-scale culture such as Erlenmeyer flasks with only a small amount of excellent yeast seed bacteria without the need for a large amount of yeast.

Therefore, it is possible to quickly make a differentiated traditional wine with unique flavor and outstanding characteristics.

Various brewing methods were established in the Goryeo period after the Three Han and Three Kingdoms period, and it has developed into various types of alcohol such as yakju, takju, and soju (Lee SR. 1986).

Takju is made from grains containing starch such as rice, barley, and sweet potato as raw materials, and is made by parallel fermentation in which saccharification by yeast enzyme and alcohol fermentation by yeast are simultaneously performed (Yu TJ et al. 1999).

In the parallel fermentation process of Takju as shown in Figure 1, α-amylase and glucoamylase, which are starch-decomposing enzymes produced by the yeast mold of yeast, act as enzymes that liquefy and saccharify starch, thereby producing glucose and supplying it to yeast. , yeast consists of a process that simultaneously produces alcohol. α-amylase arbitrarily breaks down the α-1,4 bonds of insoluble starch into small molecules such as dextrin, while glucoamylase acts on starch to α-1,4-linkages and α-1,6-bonds between amylose and amylopectin Glucose is directly produced by hydrolyzing the starch in order from outside the starch molecule.

In addition, it is known that it acts on various high molecular weight dextrins, maltose, etc. to hydrolyze it into glucose (Kim DH. 2012). This saccharification process is the most important in the process of manufacturing alcohol, and parallel fermentation, in which fermentation by yeast proceeds simultaneously with saccharification, has a great effect on takju fermentation.

Alcohol produced in Korea makes fermentation safer, shortens brewing time, and increases alcohol yield compared to brewing with yeast (Lee JS. 1968). However, it is known that the takju prepared in Korea does not have a unique flavor (Han EH et al. 1997) and does not have a harmonious flavor like that produced with yeast (Choi SH et al. 1992). Studies on the quality of takju include quality characteristics of takju (Park CS et al. 2002, Park SS et al. 2011) and quality improvement of takju by using improved yeast (So MH et al. 1999a).

The skin is formed from the surface into three layers of epidermis, dermis and subcutaneous tissue, and skin cells are composed of fibroblasts, macrophages, mast cells and plasma cells.

Fibroblasts are cells that produce collagen, elastin, hyaluronic acid, etc., and function to form the skin structure. In addition, when damage is added to the skin tissue, fibroblasts induce the production of cells that start the production of extracellular matrix such as collagen in the damaged area and cells that update the extracellular matrix, and interact with the matrix during the wound healing process. plays an important role.

These fibroblasts are characterized by slowing cell proliferation and irregular division due to UV rays, wind, stress, aging, etc. Therefore, when an abnormality occurs in fibroblasts, problems such as the occurrence of wrinkles or blemishes, deterioration of skin elasticity, and other skin aging phenomena, such as slow wound healing, may occur.

Skin aging can be divided into time-dependent intrinsic aging (intrinsic aging), which occurs naturally as people age, and photoaging (photoaging) caused by ultraviolet (UV) radiation in the environment (Seo JY. 2001, Gilchrest B.A. 1990).

The biggest cause of skin aging is UV rays exposed to the skin on the face, back of hands, and back of the neck that have been exposed to sunlight for a long time. UV-induced reactive oxygen species (ROS) accelerate skin aging by causing photo-oxidative damage to the skin.

Reactive oxygen species (ROS) are oxygen species with great oxidizing power, _{and not only oxygen-centered radicals such as superoxide anioin radical (O 2 ·} -) and hydroxyl radical ( _· OH), but also hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ ), and peroxyl radicals (ROO _· ) and alkoxyl radicals (RO _· ) generated by their reaction with biological components are included (Fantone JC et al. 1982, Davies KJA. 1987).

In particular, ¹ O ₂ and ·OH are known to play an important role in the photodamage of the skin. Although ROS is a natural by-product of oxygen metabolism, the level of ROS in vivo increases due to environmental factors such as ultraviolet rays or stress (Foote CS. 1976). The role of ROS is also important in preventing photoaging and developing a protective agent from UV rays.

Since _{ROS including O 2} are associated with photoaging, reduction of ROS after UV exposure by antioxidants (Oikarinen A et al. 1985, Kligman LH. 1992) can prevent and minimize photoaging (Kim EH et al. 2009, Kim). JE et al. 2011, Scharffetter-Kochanek K. 1997).

As antioxidants that can control free radicals, commonly used phenol-based synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have been widely used for their excellent antioxidant power and economic feasibility. A lot of research is underway to develop and apply new natural antioxidants. At the same time, the development of functional cosmetics with whitening, anti-aging (wrinkle) and UV protection functions using various natural materials is being actively carried out according to the nature-oriented and environmentally friendly consumption trend (Jeong HR et al. 2011, Nguyen DH et al. 2007).

In the present invention, as shown in FIG. 2, yeast was prepared by a liquid culture method to compensate for the disadvantages of solid yeast, and the activities of α-amylase and glucoamylase, which are enzymes required for liquid culture of yeast mold, were measured and the liquid culture method of yeast mold. was studied, and yeast culture was prepared based on what was recorded in the old literature, and its properties were tested by using the stone method. In addition, takju was prepared using this liquid yeast and lime, and the quality characteristics of takju were tested, and anti-wrinkle and antioxidant properties were tested by freeze-drying the liquid yeast to examine its applicability as a cosmetic material.

Registered Patent Publication No. 10-1261800 (Registration Date April 30, 2013) Laid-Open Patent Publication No. 10-2015-0128381 (published on November 18, 2015) Registered Patent Publication No. 10-1429161 (Registration date August 05, 2014)

The present invention is a method for producing liquid yeast having high enzymatic activity by using a grain selected from 10 minutes sea bream ~ brown rice, in which the growth rate of the cells is increased by culturing using a high-frequency pulse, and the degree of milling of the rice is adjusted;

By preparing using the prepared liquid yeast, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and provides a sensually excellent and differentiated takju while reducing the non-uniformity of the product, which is a disadvantage of solid yeast, and provides antioxidant action And it is an object of the invention to provide a cosmetic having an anti-wrinkle effect.

In order to achieve the above object,

the present invention

Put any one or two or more types of yeast raw materials selected from brown rice, 3 minute sea bream, 7 minute sea bream, or 10 minute sea bream in a high-frequency incubator,

Inoculate the yeast raw material with a spore suspension of yeast mold so that the number of spores becomes 1 × 10 ⁵ pieces/mL,

After high-frequency pulse treatment during incubation,

Enzyme activity is improved by improving the growth rate of cells using high-frequency pulses to produce liquid yeast by continuously culturing for 40 to 60 hours at a culture temperature of 28 ~ 32 ℃ and a stirring speed of 180 ~ 300 rpm and adjusting the degree of milling of rice. It provides a method for producing liquid yeast.

And, by using the liquid yeast prepared through the liquid yeast manufacturing method, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and reduces the non-uniformity of the product, which is a disadvantage of solid yeast, while sensually excellent and differentiated takju , to provide a cosmetic having antioxidant and anti-wrinkle effects.

Liquid yeast prepared according to the manufacturing method of the present invention has high enzyme activity by culturing using high-frequency pulses to speed up the growth rate of cells, and adjusting the grain so that the degree of milling of rice is selected from sea bream ~ brown rice for 10 minutes.

In addition, by preparing takju using liquid yeast according to the present invention, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and reduces the non-uniformity of the product, which is a disadvantage of solid yeast, while providing sensually excellent and differentiated takju can provide

And, by manufacturing a cosmetic using the liquid yeast according to the present invention, it is possible to provide a cosmetic having an antioxidant action and anti-wrinkle effect.

1 is a view showing the parallel fermentation process of Takju.
Figure 2 is a flow chart of cosmetic materials and takju using liquid yeast according to the present invention.
3 is a schematic diagram of a high-frequency incubator according to the present invention.
Figure 4 is a cytotoxicity graph for each concentration and degree of liquid yeast.
Figure 5 is a graph of the PIP content by liquid yeast degree and different concentrations.
6 is a graph of the relative concentration of PCOLCE according to the degree of liquid yeast and different concentrations.
7 is a graph of DPPH radical scavenging activity according to the degree and concentration of liquid yeast.
8 is a graph of SOD activity for each concentration and degree of liquid yeast.

About the liquid yeast production method with improved enzyme activity by improving the growth rate of cells using high-frequency pulses and the degree of milling of rice according to the present invention, and specific technical contents of takju or cosmetics containing liquid yeast prepared therefrom Let's take a look.

The liquid yeast manufacturing method according to the present invention

Put any one or two or more types of yeast raw materials selected from brown rice, 3 minute sea bream, 7 minute sea bream, or 10 minute sea bream in a high-frequency incubator,

Inoculate the yeast raw material with a spore suspension of yeast mold so that the number of spores becomes 1 × 10 ⁵ pieces/mL,

After high-frequency pulse treatment during incubation,

Liquid yeast is prepared by continuously culturing for 40 to 60 hours at a culture temperature of 28 ~ 32 ℃ and a stirring speed of 180 ~ 300 rpm.

The high-frequency incubator 1 has a structure in which the incubator 20 is mounted on the high-frequency generator 10 as shown in FIG. 3 .

The high frequency generator 10 includes a high frequency display device 101 , a high frequency operation device 102 , and a high frequency operation lever 103 .

The incubator 20 includes a culture vessel 201 forming a hollow vessel shape, an impeller 202 installed in the center of the culture vessel 201, and spaced apart from the bottom by a predetermined width to perform agitation, and the A stirring rod 203 installed in the upper vertical direction of the impeller 202, a stirrer 204 installed at the end of the stirring rod 203 to provide rotational power, and the inner side wall surface of the culture vessel 201 It is installed in the electrode terminal 205 for outputting a high-frequency pulse, and includes a culture solution 206 filled in the culture vessel (201).

The liquid yeast production is made together with the high-frequency pulse processing of the high-frequency incubator (1).

The high-frequency pulse treatment is performed during the process of culturing yeast raw materials by inoculating a spore suspension of yeast mold, and is treated three times at a high frequency of 240 to 270 kHz at 10-minute intervals, incubated for 5-7 hours, and then at 10-minute intervals It is characterized in that it is processed three times with a high frequency of 240 ~ 270 kHz.

More preferably, the treatment is performed three times at a high frequency of 240 kHz or 270 kHz at intervals of 10 minutes, and the treatment is repeated three times at intervals of 10 minutes after incubation for 6 hours.

The yeast raw material is milled brown rice, 3 minute sea bream, 7 minute sea bream, or 10 minute sea bream, respectively, washed, immersed in water for 50 ~ 70 minutes, drained for 20 ~ 40 minutes, and then at 115 ~ 130 ℃ After sterilization for 10 to 30 minutes, use the one prepared by cooling.

The yeast raw material is used according to the degree of rice milling, and 100% unpolished is brown rice, 97.4% is 3 minutes sea bream, 94.4% is 7 minutes sea bream, and 92.0% is selected from 10 minutes sea bream.

As such, the degree of milling of rice affects enzyme activity, and the higher the degree of milling, the lower the activity, and the lower the degree of milling, the higher the activity.

The yeast mold uses any one or two or more selected from Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoling.

Molds present in traditional yeast include Baekgukgyun, Heukgukgyun, Hwanggukgyun, and Honggukgyun. The most isolated molds from yeast are Rhizopus oryzae and Aspergillus oryzae , which represent yeast. Aspergillus kawachii is widely used in the production of soju. These fungi secrete and produce various amylase enzymes such as α-amylase and glucoamylase to saccharify starch. plays a role

Hereinafter, examples and test examples of a liquid yeast manufacturing method with improved growth rate by using a high frequency pulse of the present invention and takju or cosmetics containing liquid yeast prepared therefrom will be described.

[ Liquid yeast production ]

1. Strains used

The yeast mold used in this experiment were white yeast mold Aspergillus kawachii (KCCM 11460), black yeast mold Aspergillus niger (KCCM 60315), yellow yeast mold Aspergillus oryzae (KCCM 11372), and red yeast mold Monascus kaoling mold purchased from the Korea Microbiology Center. (KCCM 60154) was used, and as the yeast solution, yeast provided by the Yesan Traditional Yeast Research Institute was suspended in an aqueous solution, and yeast fungus separated from the suspension was used. The received yeast mold was used after culturing for 5 days in a thermostat at 30°C using PDA (potato dextrose agar) medium.

2. Yeast, yeast mold culture and production of liquid yeast

Preparation of liquid yeast is shown in Table 1 below.

Mixing ratio of different types of microorganisms and liquid koji according to the degree of milling of rice

The high-frequency incubator 1 shown in FIG. 3 was used. After milling the rice into brown rice (100%), 3 minutes sea bream (97.4%), 7 minutes sea bream (94.4%), and 10 minutes sea bream (92.0%), respectively, washed and soaked for 1 hour, drained water for 30 minutes, and then a 1,000 mL flask 100g each, put 300g each of distilled water in a 500mL Erlenmeyer flask, sterilize and cool at 121℃ for 15 minutes, and incubate the yeast solution for one day at a high frequency of 240 or 270kHz 3 times at 10-minute intervals and incubate for 6 hours After that, the high frequency was set to 240 or 270 kHz and repeated 3 times at 10-minute intervals.

As a yeast solution, a suspension of Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoling was inoculated into the culture medium so that the number of ^{spores was 1 × 10 5 /mL.} And the high frequency-treated yeast solution was cultured for 48 hours at a culture temperature of 30 °C and a stirring speed of 200 rpm.

3. Measurement of α-amylase activity

The α-amylase activity of liquid yeast was measured as follows.

1) Sample preparation

Mcllvaine buffer was used by titrating a citric acid solution _{in 0.1N Na 2} HPO _{4 to pH 6.0-7.0.} The iodine solution was used by dissolving 0.2 g of iodine and 2 g of potassium iodide in 100 mL of 1N hydrochloric acid solution.

2) Experimental method

0.05 mL of the sample was diluted 200-fold in 10 mL of distilled water. 5 mL of 1% soluble starch solution was added to 1 mL of the diluted sample, 13 mL of Mcllvaine buffer was added, 1 mL of 0.1% calcium chloride solution was added, and then reacted at 37°C for 30 minutes. Take 0.2 mL, add 10 mL of iodine solution, and measure the absorbance at 600 nm.

For blank measurement, 5 mL of 1% soluble starch solution was added to 1 mL of 200-fold diluted sample, 13 mL of Mcllvaine buffer was added, and then 1 mL of 0.1% calcium chloride solution was added. After reacting at 100° C. for 30 minutes, absorbance was measured at 600 nm. The α-amylase content in the sample was calculated by creating a standard curve using α-amylase Ultrapure as a standard material.

3) Calculation method

The α-amylase activity value was calculated using the following formula.

units/mg = { (DO-D)/DO }×10

here,

DO = absorbance of control solution

D = absorbance of solution after enzymatic reaction

4. Measurement of glucoamylase activity

The glucoamylase activity of liquid yeast was measured as follows. A sample cultured at 30°C was collected and used as a sample for measuring enzyme activity.

The glucoamylase activity was measured using the glucose C Ⅱ-kit (Wako Pure Chemical Industries, Osaka Japan) saccharification power fractionation quantification kit.

1) Sample preparation

A reagent for measuring glucoamylase activity was prepared as follows.

A. 0.1 mol/L acetate buffer (pH 4.5) solution

B. As a substrate solution, add 40 mL of distilled water to 1 g of water-soluble starch, heat to dissolve, cool, add 50 mL of solution A, and then add distilled water to make a total of 100 mL

C. 0.05 mol/L acetate buffer (pH 5.5)

D. 0.6 mol/L NaOH solution

E. Enzyme solution

Take exactly 100 mg of glucoamylase, add 1 mL of ion-exchanged water, and then take 50 µL of this solution to make a total of 50 mL with solution C.

F. Coloring solution

Chromogen solution (glucose C Ⅱ-Kit, Wako)

G. 2 g/L glucose standard solution

2) Experimental method

Take 2 mL of solution B in a beaker, preheat at 37°C for 5 minutes, add 0.5 mL of solution E, and warm at 37°C for 15 minutes. Here, a solution in which 0.5 mL of solution D was added was used as a' solution, and a solution obtained by mixing 2 mL of solution B, 0.5 mL of solution D and 0.5 mL of solution E in a beaker was used as solution b'.

For the measurement sample solution, 3 mL of solution F was taken and pre-warmed to 37°C for 5 minutes, then 20 µL of a' solution was added and treated at 37°C for 5 minutes. In addition, Blank 1 took 3 mL of F solution, preheated at 37°C for 5 minutes, added 20 µL to b' solution, and reacted at 37°C for 5 minutes. For Blank 2, 3 mL of F solution was pre-heated at 37°C for 5 minutes, then 20 µL of distilled water was added and reacted at 37°C for 5 minutes. And as for the standard solution, after taking 3 mL of solution F, pre-heating at 37°C for 5 minutes, adding 20 µL of solution G, and reacting at 37°C for 5 minutes.

3) Calculation method

The glucoamylase activity was calculated using the following formula.

The sample solution, Blank 1, and the standard solution prepared above were used for measurement using Blank 2 solution as a control solution. The absorbance was measured at 505 nm using a UV/Vis spectrophotometer (JASCO, V-550), and calculated by the following equation. Glucoamylase 1 units was defined as the amount of enzyme that produced 1 μmol of glucose for 1 minute at pH 4.5 and 37°C.

units/mg = 2 × {(E1-E0)/E2} × (10 ³ /180.16) × 3.0 × 1/15 × 1/0.5 × 1/S × 1000

here,

E1: Absorbance of sample solution

E0: Absorbance of Blank 1

E2: Absorbance of standard solution

3.0: total solution amount

15: reaction time (min)

0.5: E solution amount

180.16: molecular weight of glucose

S: glucoamylase ingested amount

5. Total sugar measurement

Total sugar content was measured by the phenol-sulfuric acid method (Kang KH et al. 1998). 2 mL of the sample was placed in a test tube, and 1 mL of a 5% (v/v) phenol reagent (Shinyo Pure Chemical Co. Ltd., Osaka, Japan) solution was added thereto. After adding 5 mL of 95% sulfuric acid (Deajung Chemical & Metals Co. Korea), it was left at room temperature for 30 minutes at room temperature, and then absorbance was measured at 470 nm using a UV/Vis spectrophotometer (JASCO. V-550). did. For quantification of sugar, the total sugar content (mg/mL) was obtained by comparing it with the standard curve prepared by the above method using glucose as a standard material.

6. Results

In order to investigate the enzyme activity of liquid yeast according to the degree of milling of rice and the relationship with sugar, nuruk solution and Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoliang were added to liquid cultured yeast and yeast and their α-amylase , glucoamylase activity and total sugar were measured.

1) Changes in α-amylase activity

Table 2 shows the results of α-amylase activity according to the milling degree of rice and the types of yeast and yeast mold. The activity of each enzyme showed different aspects. The enzymatic activity of yeast is very important in its liquefaction and saccharification power. The activity of α-amylase according to the type of koji fungus showed the highest 8.82 and 8.72 units/mL in the Aspergillus niger and Aspergillus kawachii treated group in liquid brown rice koji at 24 hours of culture, followed by 8.00 units/mL in the Aspergillus oryzae treatment group. indicated. The activity of α-amylase according to the degree of polishing was 8.82-8.73 units/mL in the Aspergillus niger-treated group and 8.72-8.58 units/mL in the Aspergillus kawachii-treated group, showing a high trend regardless of the degree of polishing. Aspergillus oryzae-treated group showed a lower activity as the degree of milling increased, indicating 8.00-4.23 units/mL. In the Nuruk-treated group, there was no significant change at 4.43-3.79 units/mL depending on the degree of milling, while in the Monascus kaoliang-treated group, it was 5.11-1.91 units/mL, indicating that the higher the degree of milling, the lower the activity of α-amylase.

At 48 hours of incubation, similar results to those at 24 hours were shown, showing the highest activity at 8.83 units/mL in the Aspergillus niger-treated group of liquid brown rice yeast, and 8.73 units/mL in the Aspergillus kawachii-treated group. Aspergillus oryzae treated group showed 8.16 units/mL, which was slightly increased compared to the 24 hours of culture. Enzyme activity according to the degree of polishing was 8.73-8.59 units/mL in the Aspergillus kawachii-treated group and 8.83-8.73 units/mL in the Aspergillus niger-treated group, indicating a high trend regardless of the degree of milling. Aspergillus oryzae treated group was 8.16-5.07 units/mL, which showed a tendency to decrease the activity of α-amylase as the degree of polishing increased. showed a decreasing trend. This is thought to be because the content of total sugar liberated from rice during liquid culture increases as the degree of milling increases (Ruiter GJ. 1997).

Changes in α-amylase activity according to various types of liquid yeast, the degree of milling and incubation time of rice

2) Changes in glucoamylase activity

Table 3 shows the results of each glucoamylase activity according to the milling degree of rice and the types of yeast and yeast mold. The glucoamylase activity according to the type of mold and the degree of milling showed the highest activity in liquid brown rice koji at 24 hours of culture in the order of Aspergillus oryzae treatment group > Nuruk treatment group > Monascus kaoliang treatment group > Aspergillus niger treatment group > Aspergillus kawachii treatment group, and Aspergillus oryzae treatment group showed 3,013 units /mL showed the highest enzyme activity. In the production of liquid yeast using Aspergillus oryzae mold, it was found that the enzyme activity was different depending on each sample and the degree of milling. . In the Nuruk treatment group, liquid brown rice koji showed 2,665 units/mL, but liquid white rice koji decreased to 1,197 units/mL. In the Monascus kaoliang treatment group, liquid brown rice yeast showed 1,781 units/mL, and liquid white rice yeast showed a very low activity than liquid brown rice yeast at 216 units/mL. .

At 48 hours of culture, Aspergillus oryzae-treated group showed the highest enzyme activity in liquid yeast with brown rice added at 3,048 units/mL. Most of the experimental groups showed a tendency to decrease enzyme activity according to the degree of milling, and liquid white rice yeast was 1,423 units/mL in the Aspergillus oryzae-treated group, which was significantly reduced after 24 hours. In the Monascus kaoliang treatment group, liquid brown rice yeast showed 1,477 units/mL, but liquid white rice yeast showed 206 units/mL. In the Aspergillus niger-treated group, the enzyme activity decreased according to the degree of milling.

Changes in glucoamylase activity according to different types of liquid koji and rice with different degrees of milling and incubation time

3) Total sugar and enzyme activity

Table 4 shows the results of each total sugar according to the milling degree of rice and the types of yeast and yeast mold. In the case of liquid culture of yeast mold, α-amylase and glucoamylase activity according to the degree of milling of rice showed a close relationship with sugar. Both α-amylase and glucoamylase showed higher enzymatic activity as the degree of milling was lower, indicating the best enzymatic activity in liquid brown rice koji. Also, the total sugar content shown in Table 4 showed an inverse correlation (Ruiter GJ et al. 1997, Sugimoto T et al. 2011). It is desirable to control the total sugar (glucose) concentration to a low concentration in glucoamylase production by culturing Aspergillus niger (Guido M et al. 2007, VanKuyk P et al. 2008). When cultured with brown rice, the total sugar (glucose) concentration in the medium is It is judged that the concentration is controlled to a high level capable of producing α-amylase and glucoamylase. In liquid culture, enzyme production starts when the glycogen content in the cells decreases in the presence of an α-amylase-inducing substance, and the glycogen content is affected by the glucose concentration and the growth rate of the cells in the medium (Sudo S et al. 1993). . In addition, even in solid culture, α-amylase production starts from the time when the glycogen content in the cells is reduced. The reason why the productivity of α-amylase is higher in solid culture than in liquid culture is because of the difference in glycogen degradation in cells between solid culture and liquid culture. That is, in solid culture, the glycogen content in the cells decreases immediately after the start of the culture, so the start time of enzyme production is quick. Because the cells proliferate logarithmically, a high α-amylase production rate is reached, whereas in liquid culture, the production rate increases due to a decrease in the glycogen content in the cells only after reaching a relatively late growth phase (Sudo S et al. 1994, Sudo S et al. . 1995). As described above, the reason that the decrease in the initial cell glycogen content in the conventional liquid culture is delayed is because the carbon source supplied to the yeast is abundant. It is judged that the content shows a tendency to increase.

Therefore, the difference in productivity of α-amylase and glucoamylase depending on the degree of milling of rice is because the content of total sugar varies depending on the physical state of the carbon source used for culture. As shown in Tables 3 and 4, brown rice used for the medium as a carbon source showing high enzyme productivity in liquid culture of yeast mold is covered with an outer shell, so it is judged that the culture is progressing while excessive elution of starch into the medium is suppressed. . In addition, the difference in enzyme activity according to the degree of milling can apply the new methodology established in liquid culture to general grain raw materials. The high industrial value of solid koji lies in its ability to produce numerous enzymes in large quantities and at high concentrations at the same time. Liquid yeast using brown rice was confirmed to enable high production of starch degrading enzymes.

Total sugar change and milling time for different types of liquid yeast, rice

7. Conclusion

α-amylase and glucoamylase activity of liquid yeast prepared using Nuruk liquid and Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoliang to investigate the relationship between the enzyme activity and total sugar content of liquid yeast according to the degree of milling of rice. The measurement results are as follows.

1) The activity of α-amylase according to the type of yeast mold was high at 8.82 and 8.72 units/mL in the Aspergillus niger-treated and Aspergillus kawachii-treated groups in liquid brown rice koji at 24 hours of culture. was shown to be low. Even at 48 hours of incubation, when brown rice was cultured with Aspergillus niger, the highest activity was shown at 8.83 units/mL, and the higher the degree of milling, the lower the activity of α-amylase.

2) The activity of glucoamylase according to the degree of milling was highest in liquid brown rice yeast at 24 hours of culture in the order of Aspergillus oryzae treatment group > Nuruk treatment group > Monascus kaoliang treatment group > Aspergillus niger treatment group > Aspergillus kawachii treatment group, and Aspergillus oryzae treatment group showed 3,013 units/ mL showed high enzymatic activity. As the degree of milling increased, glucoamylase activity decreased, so that liquid brown rice yeast showed the highest enzyme activity, and liquid white rice yeast showed the lowest enzyme activity. Even at 48 hours of culture, the highest enzyme activity was shown in liquid koji containing brown rice.

3) α-amylase and glucoamylase activity showed higher enzyme productivity as the degree of milling was lower, and showed an inverse correlation with total sugar content. Liquid brown rice yeast showed the best enzyme productivity, and it was preferable to control the total sugar (glucose) concentration to a low concentration for the production of α-amylase and glucoamylase in liquid culture.

Next, let's look at examples and test examples for takju or cosmetics containing liquid yeast according to the present invention.

[ Takju ]

1. Material

The glutinous rice used in this experiment was purchased from the market and the bottled water was Jeju Samdasoo.

2. Preparation and fermentation of Takju

Table 5 shows the preparation ratio of takju for examining the quality characteristics of takju using liquid yeast according to the present invention. After immersing 1,600 g of glutinous rice in water for 6 hours, the water was removed for 1 hour, and then Godubap was made in a steaming pot and allowed to cool. Add 160 g of each culture medium of Nuruk, Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoliang to Godubap, mix with 1,920 g of water and 480 g of lime, and ferment in a fermenter at 25°C for 7 days, stirring twice daily for the first 2 days of fermentation. did it Changes in pH, acidity, sugar content, color, and alcohol content according to the elapsed days from 5 hours after fermentation were investigated at 1-day intervals, and sensory evaluation was performed after fermentation was completed.

Takju mixing ratio

3. pH and acidity; sugar content and alcohol content; chromaticity measurement

The chromaticity was measured repeatedly 3 times using a colorimeter (Colorimeter, CR-300, Minolta Co. Japan) of Takju fermented for 7 days at 25°C, and the average value was calculated as the Hunter value (L: lightness, a: redness, b: yellowness).

4. Sensory Test

The sensory characteristics evaluation of Takju prepared with liquid yeast was conducted with 15 trained panelists with excellent discernment, fully explaining the purpose of this experiment, educating the method and evaluation characteristics of the sensory test, Five characteristics of sour taste, aroma, and overall preference were evaluated using a 7-point scoring method. The evaluation score was evaluated as 7 points for very good and 1 point for very dislike, indicating the average value of each.

5. Results

1) Changes in pH and acidity

Acidic components related to the pH of Takju are not only factors that can easily identify changes in the components of Takju, but are also an important indicator component that can identify the fermentation progress of Takju because they are complexly generated during the alcohol production process.

Table 6 shows the pH change of Takju during the fermentation process. Looking at the pH between each sample group, on the fermentation start day, Takju in the Aspergillus oryzae treatment group had the highest pH of 5.52 among the sample groups, and the Nuruk treatment group had the lowest pH at 5.21. On the second day of fermentation, the Nuruk treatment group had the lowest level, and the Monascus kaoliang treatment group> Aspergillus oryzae treatment group> Aspergillus kawachii treatment group> Aspergillus niger treatment group showed the lowest. Looking at the change in pH according to the fermentation period, it was 5.21-5.52 on the first day of fermentation, but markedly decreased to 4.28-4.46 on the first day of fermentation, and there was no significant change after gently lowering to 3.65-3.81 until the third day of fermentation. The difference in pH between each sample group was not significant (p<0.05), but when looking at the change by fermentation period, the average from day 0 to day 3 was significantly lowered from 5.40 to 3.72 on average (p<0.05), and from day 5 to day 7 There was no significant change (p<0.05). These results are consistent with the research results showing that pH is rapidly lowered due to a rapid increase in acid in the early stage of fermentation of takju, usually during the fermentation of malsul.

Acidity is directly related to the taste and smell of Takju along with volatile aroma components and affects the preservation. Table 7 shows the acidity content of Takju during the fermentation process. The acidity immediately after fermentation is mainly derived from yeast or raw materials, but as fermentation proceeds, various organic acids produced by the action of microorganisms such as yeast and lactic acid bacteria in Takju are produced, so the acidity content increases. The proliferation of lactic acid bacteria lowers the pH, prevents contamination by various bacteria, and activates the growth of yeast bacteria. The acidity content of the sample sections did not show a significant difference, and the change in acidity according to the fermentation period was similar to 0.07-0.11% on the first day of fermentation, but sharply increased to 0.42-0.48% on the first day of fermentation, and then gradually increased or After 2 days of fermentation, Nuruk treatment group > Aspergillus kawachii treatment group > Aspergillus oryzae treatment group > Monascus kaoliang treatment group > Aspergillus niger treatment group showed high in the order. The acidity content on the 4th day of fermentation was 0.69-0.73%, which increased significantly from the start of fermentation to the 4th day (p<0.05). The acidity content on the 7th day after fermentation was completed was 0.77-0.82, and there was no significant difference (p<0.05). When comparing the changes in the pH and acidity content of Takju in this experiment, the reason that the pH does not decrease despite the increase in the acidity content is that secondary metabolites of amino acids generated due to protein decomposition increase during fermentation and increase the pH. It is thought that this is because the change is buffered, and it is consistent with the results of So MH (1999b) et al.

Changes in pH of Takju by various types of liquid yeast during fermentation

Changes in acidity of Takju due to various types of liquid yeast during fermentation

2) Changes in sugar content and alcohol content

Table 8 shows the sugar content of Takju during the fermentation process. The sugar content on the first day of fermentation was 2.0-2.2 °Brix, but on the first day of fermentation, it rapidly increased to 18.2-19.1 °Brix, showing the maximum content. Thereafter, it gradually decreased and significantly decreased until the 3rd day of fermentation (p<0.05). There was no significant change from the 4th day to the 6th day of fermentation (p<0.05), and it was expressed as 13.4-14.4 °Brix on the 7th day of fermentation. This is considered to be related to the result that pH and acidity content did not change significantly (p<0.05) from 3-4 days of fermentation. The sugar content of each sample was low in the Nuruk, Aspergillus kawachii, and Aspergillus niger treated groups on the first day of fermentation, and the sugar content in the Aspergillus oryzae and Monascus kaoliang treated groups was high. However, on the 7th day of fermentation, the sugar content in the Nuruk-treated group and the Monascus kaoliang-treated group was high, and the Aspergillus kawachii-treated group, Aspergillus niger-treated group, and Aspergillus oryzae-treated group showed low sugar content. There was no significant difference in sugar content between each sample group (p<0.05).

After fermentation, the enzyme action in the yeast decomposes the raw material starch into sugar and is used as a fermentation substrate for yeast, increasing the alcohol content for a certain period of time. Table 9 shows the alcohol content during the fermentation process of takju fermented by different types of yeast and yeast mold. During Takju fermentation, the alcohol content immediately after fermentation was 0.3-0.4%. On the first day of fermentation, the Nuruk, Aspergillus kawachii, and Aspergillus niger treated groups showed a moderate increase to 1.6-1.9%, whereas the Aspergillus oryzae treated group showed a sharp increase to 2.4%. Aspergillus oryzae treated group increased rapidly to 7.8% even on the 3rd day of fermentation, and then increased gently in all sample groups, reaching a maximum of 8.7-9.6% on the 7th day of fermentation. The results of this experiment are consistent with the report of Han EH (1997) that the alcohol component of takju is actually generated from the beginning of fermentation, and the alcohol content in the last stage of fermentation in takju fermentation with different types of yeast was found to be 8.2-12.6%. was similar. The alcohol content of Takju using Aspergillus oryzae was 9.6%, which was higher than that of Monascus kaoliang treated group, 8.7%. Aspergillus oryzae treatment group > Aspergillus niger treatment group > Aspergillus kawachii treatment group > Nuruk treatment group > Monascus kaoliang treatment group showed a high trend in alcohol content by sample. It has been reported that during the fermentation process of Takju using rice milling by-products, the sugar content on the second day of fermentation was higher than that of the start of soaking, because the reducing sugar caused by amylase secreted from the yeast microorganisms activated yeast cell proliferation rather than alcohol production by yeast. there is a bar The tendency of the sugar content to decrease in all sample groups after 1 day of fermentation is considered to be the result of a large amount of sugar being converted to alcohol by alcohol fermentation rather than conversion to sugar by saccharification enzyme. Although the amount of lime added to each sample group and the activity of yeast in limestone are the same during the production of takju, the enzyme activity of the yeast mold growing in the liquorice is different, indicating that the alcohol content of the takju is different.

Change in equivalent weight of Takju by various types of liquid yeast during fermentation

Changes in Takju alcohol content according to the type of liquid yeast during fermentation

3) Chromaticity change

Table 10 shows the changes in the brightness (L-value) of Takju during the fermentation process. The L value indicating the brightness of Takju was not significantly changed by sample group (p<0.05), and it was found to be 60.66-66.25 at the start of fermentation. Aspergillus oryzae treatment group had the highest L value, and Nuruk treatment group> Monascus kaoliang treatment group> Aspergillus kawachii treatment group> Aspergillus niger treatment group Brightness was higher. On the first day of fermentation, the brightness increased to 67.08 in the Aspergillus niger-treated group, but as the fermentation progressed, the overall decreased slightly. indicated.

Changes in the redness (a-value) of Takju are shown in Table 11. The a value of the sample section was the lowest in the Aspergillus niger treatment group at -0.48 and the highest in the Nuruk treatment group at -0.14 at the start of fermentation. The a value of the Monascus kaoliang-treated group increased significantly from the second day of fermentation, and on the seventh day of fermentation, it was 0.64, which was higher than that of other samples, indicating a significant difference from other samples (p<0.05). did not show

The change in yellowness (b-value) of Takju is shown in Table 12. The b-value indicating the yellowness of Takju was 4.58-7.02 at the start of fermentation. The Nuruk treatment group had the highest yellowness, and the Aspergillus oryzae treatment group > Monascus kaoliang treatment group > Aspergillus kawachii treatment group > Aspergillus niger treatment group showed the highest yellowness. From the 2nd day of fermentation, the b value of the Monascus kaoliang-treated group increased, and on the 7th day of fermentation, it was 7.27, which was higher than that of other samples, indicating a significant difference (p<0.05). There was no significant difference in yellowness according to the fermentation period.

Monascus kaoliang, a red yeast infection, had an effect as the fermentation progressed, so that the redness a value was higher than that of other samples, and the yellowness b value also showed a tendency to increase.

Changes in L-value of Takju according to the type of liquid yeast during fermentation

Change of Takju a-value by various kinds of liquid yeast during fermentation

Change of b-value of Takju by various kinds of liquid yeast during fermentation

4) Sensory test

The sensory evaluation of Takju was performed after fermentation was completed by different types of yeast and yeast mold. Table 13 shows the results of five items for color, flavor, sweetness, acidity, and overall-acceptability. As for the color, the Aspergillus kawachii treatment group, Aspergillus niger treatment group, and Monascus kaoliang treatment group showed 5.07, and the Nuruk treatment group and Aspergillus oryzae treatment group showed 5.13, and there was no significant change in the entire sample section (p<0.05). As for the flavor, the Aspergillus oryzae treatment group was significantly higher with 5.53 (p<0.05), followed by the Aspergillus kawachii treatment group with 5.20, and the Monascus kaoliang treatment group with 4.47 the lowest. In terms of sweetness, Aspergillus oryzae treatment showed the highest value at 5.93, and Aspergillus kawachii and Nuruk treatment showed no significant change at 5.60 (p<0.05). Aspergillus niger treated group showed 5.20, Nuruk treatment showed 4.60, and Monascus kaoliang treated group showed the lowest at 4.20. The acidity of Aspergillus oryzae and Aspergillus kawachii treated groups was high at 5.13 and 5.07, respectively, and Nuruk treatment showed the lowest at 3.93.

The overall acceptability was the highest in Aspergillus oryzae treatment with 5.80, followed by 5.07 for Aspergillus kawachii treatment, 4.13 for Aspergillus niger treatment, 4.33 for Nuruk treatment, and 4.07 for Monascus kaoliang treatment. It was. The overall order of preference was Aspergillus oryzae treatment group > Aspergillus kawachii treatment group > Nuruk treatment group > Aspergillus niger treatment group > Monascus kaoliang treatment group, indicating a significant change in the sample interval (p<0.05). When looking at pH, acidity, sugar content, alcohol content, and sensory characteristics comprehensively, it is judged that the Aspergillus oryzae treatment was the most preferred. This shows that Takju made with Aspergillus oryzae had the highest preference, and it is considered to have useful value in improving the quality of Takju made from liquid yeast and to suggest the possibility of commercialization.

Sensory evaluation of Takju by various types of liquid yeast on the 7th day of fermentation

4) Conclusion

After preparing Takju using liquid yeast prepared with Nuruk, Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoliang, the quality characteristics during fermentation were studied.

1) The pH of Takju was 5.21-5.52 on the first day of fermentation, but it decreased significantly to 4.28-4.46 on the first day of fermentation, and there was no significant change after gently lowering to 3.65-3.81 by the third day of fermentation. There was no significant difference in pH between each sample group (p<0.05), and when looking at the change according to the fermentation period, the average of 5.40 to 3.72 was significantly lowered from the 0th day to the 3rd day (p<0.05), but from the 5th day Until the 7th day, there was no significant change (p<0.05) with about 3.68.

2) The sugar content was 2.0-2.2 °Brix on the first day of fermentation, but it increased rapidly to 18.2-19.1 °Brix on the first day of fermentation, showing the maximum value. Thereafter, it gradually decreased and significantly decreased until the third day of fermentation (p<0.05). There was no significant change from the 4th day to the 6th day of fermentation (p<0.05), and it gradually decreased to 13.4-14.4 °Brix on the 7th day of fermentation.

3) Alcohol content was found to be 8.7-9.6% on the 7th day of fermentation, with Aspergillus oryzae treatment group having the highest Takju (9.6%), Aspergillus niger treatment group>Aspergillus kawachii treatment group>Nuruk treatment group>Monascus kaoliang treatment group.

4) The L value was 60.66-66.25 at the start of fermentation, the highest in the Aspergillus oryzae treated group, and gradually decreased as the fermentation period progressed to 55.60-57.76 on the 7th day of fermentation. Monascus kaoliang, a red yeast infection, had an effect as the fermentation progressed, so that the redness a value was higher than that of other samples, and the yellowness b value also showed a tendency to increase.

5) There was no significant difference in color between samples in the sensory test (p<0.05). The Aspergillus oryzae treatment group had a significantly higher aroma (5.53) (p<0.05), and the sweetness level of the Aspergillus oryzae treatment group was 5.93 (p<0.05), and the overall preference was also higher in the Aspergillus oryzae treatment group at 5.80, higher than the other samples. got it

[ cosmetics ]

1. Cell Lines and Reagents

Human fibroblasts (fibroblast, CCD 986sk, ATCC, CRL-1947) used in this experiment, human keratinocytes (kerinocyte, HaCaT, Konkuk University), Procollagen Type I C-Peptide (PIP) EIA kit (Takara, MK101. Japan) ), RNA isolation (FastLane Cell one-step buffer set) Kit (Qiagen, 216213. USA), QuantiTect primer assays (PCOLCE, Qiagen, QT01005725), and DPPH (Sigma, D9132-1G. USA) were used. For cell culture, Dulbecco's modified eagle's medium (DMEM, Gibco, 11995-073. USA), fetal bovine serum (FBS, Gibco, 16000-044. USA), Phosphate buffered saline (PBS, Welgene. Korea), antibiotic-antimycotic ( Gibco, 15240-062. USA) and 0.5% Trypsin-EDTA (10X, Gibco, 15400-054. USA) were used. Housekeeping genes GAPDH required for gene analysis were used. Vitamin C-AA-2G (ascorbic acid 2-glucoside, DAMY chemical) was used as a reagent. For other products, special grade reagents were used.

2. Skin cell safety test (MTT assay)

Nuruk (Y), Aspergillus oryzae (O), Aspergillus niger (N), Aspergillus kawachii (K) and Monascus kaoling (M) were cultured in brown rice and white rice. Liquid brown rice koji (1) and liquid white rice koji (2) were frozen. After drying, it was used as a test material for skin cell safety, anti-wrinkle efficacy, and antioxidant activity.

A total of 10 types of liquid brown rice yeast 5 types and liquid white rice yeast 5 types were freeze-dried and used. After pulverizing each of the freeze-dried liquid yeast, filtered through a 200 mesh sieve, dissolved in distilled water at a concentration of 5 mg/mL, and diluted to the required concentration (0.01, 0.05, 0.1, 0.5 mg/mL) for the experiment.

1) Cell culture

The human skin cell line (keratinocyte line HaCaT) was cultured at 37°C and 5% CO2 in a 100 mm culture dish using Dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotic-antimycotic. did. When these cells reached 80% of the culture dish, they were subcultured and used for the experiment.

2) Cell culture test method

An assay using MTT (3-(4,5-dimethyl thiazol-2yl)-2,5 diphenyl-2H-tetrazolium bromide), which reacts specifically to cellular mitochondrial dehydratase, was tested.

Cells were seeded in a 24-well plate at 1×105 cells/well and then cultured under cell culture conditions until the number of cells reached 80%. After removing the medium, it was washed with 10% phosphate buffered saline (PBS), and then a new medium not containing 10% FBS was added, treated with the prepared yeast lyophilized material, and further cultured for 24 hours. After incubation, 50 uL of MTT solution (6.6 mg/mL, 3-(4,5-dimethyl thiazol-2yl)-2,5 diphenyl-2H-tetrazolium bromide solution) was added to each well and incubated for 3 hours. The culture medium was removed. Dimethylsulfoxide was added 200 uL at a time, shaken for 10 minutes, and then 100 uL each was taken in a 96-well plate and absorbance was measured at 540 nm with a microplate reader. Cytotoxicity was expressed as a percentage based on the absorbance of the control group using pure water.

Cell safety (%) = (absorbance of the experimental group / absorbance of the control group) × 100

3. Anti-wrinkle efficacy evaluation

1) Procollagen synthesis assay

Procollagen synthesis analysis was performed using the Procollagen Type I C-Peptide (PIP) EIA Kit. After dispensing human fibroblasts (CCD 986sk, fibroblast) in a 48-well plate at 5×10⁴cells/well with Dulbecco's modified eagle's medium (DMEM) containing 10% Fatal bovine serum (FBS) and 1% antibiotic-antimycotic, After culturing at 37°C and 5% CO2 for 24 hours, it was replaced with a starvation medium and treated with test substances. After 48 hours of treatment with the test substance, 100 uL of antibody-POD conjugate solution was added to each well while the antibody-coated microplate was placed on ice. After transferring the culture solution from the cell culture well to a microtube, centrifugation was performed to precipitate the cells, and 20 uL of the supernatant was placed in each well of an antibody-coated microplate, covered with wrap, and incubated at 37°C for 3 hours. After removing the solution contained in each well, it was washed 4 times with 300 uL of ice-cold PBS. After the last wash, PBS was completely removed, and 100 uL substrate solution was added to each well, and then reacted at room temperature while covering with foil for 15 minutes. After confirming the color change, 100 uL stop solution (1N-H2SO4) was added to each well and absorbance was measured at 450 nm.

Intracellular collagen synthesis rate (%) = (absorbance of each sample solution / absorbance of negative control group) × 100

2) Procollagen expression promotion action

① Cell culture

Human fibroblasts (CCD986sk, fibroblast) were treated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic in a 100 mm culture dish at 37°C and 5% CO2 condition. cultured. When human fibroblasts were cultured more than 100% of the culture dish, they were aliquoted into a 96-well plate at a concentration of 1×10⁴cells/well, and then cultured under cell culture conditions until the cells reached 80% or more of the culture dish. After removing the medium, the samples were processed by concentration after exchange with FBS-free-DMEM medium (medium not containing FBS). It was further cultured for 24 hours in cell culture conditions to determine what effect each material had on the expression of the wrinkle-related gene, Procollagen C-endopeptidase enhancer (PCOLCE). Experimental method was performed by real-time PCR method.

② RNA isolation

RNA isolation was performed using FastLane Cell one-step buffer set. After removing the medium, wash the cells with FCW (cell wash buffer), add 50 uL cell processing mixture (a mixture of Buffer FCPW with Buffer FCPL and gDAN Wipeout buffer added), incubate at room temperature for 5 minutes, and then transfer to a new microtube. After transfer, the reaction was carried out at 75 °C for 5 minutes.

③ Real-time PCR analysis

To analyze the genes (MMPs) involved in wrinkle formation, PCR was performed with the Rotor Gene Q real-time PCR Machine (Qiagen. Co) according to the method of 2X QuantiTect SYBR Green RT-PCR Master mix using RNA extracted from cells as a template. did. The primers used in the experiment were QuantiTect® primer assays. The relative expression rates of genes were normalized with housekeeping genes. Real-time PCR conditions are shown in Table 14.

Operating conditions for real-time PCR of cell culture

4. Antioxidant activity evaluation

1) Measurement of DPPH free radical scavenging activity

The compound DPPH generates free radicals in ethanol. A method of investigating the antioxidant activity of natural substances by measuring the electron donating ability of DPPH, which is the ability to inhibit oxidation by donating electrons to free radicals, has been widely used (Leteliera ME et al. 2008).

Samples of a certain concentration (0.01, 0.05, 0.1 mg/mL) were mixed with DPPH, respectively, to determine how much the amount of free radicals decreased. After adding 0.5 mL of 0.1 mM DPPH solution to 0.4 mL of ethanol, and 0.1 mL of each sample, vigorously vortexing for 10 seconds, and reacting in a cool and dark place for 30 minutes, absorbance was measured at 517 nm using a microplate reader. Antioxidant activity was expressed as a percentage based on the absorbance of the control group using ethanol, and vitamin C 0.1% (AA-2G, Ascorbic acid 2-glucoside) was used as a positive control group.

Free radical activity inhibition rate (%) = {1-(reaction absorbance of each sample solution/reaction absorbance of blank sample solution)} × 100

2) SOD-like activity measurement

SOD-like activity was measured according to the method of Marklund S et al. (1974) by measuring the amount of pyrrogallol, which catalyzes the conversion to hydrogen peroxide (H2O2), and expressed as SOD-like activity. To 0.2 mL of the sample solution, 3.0 mL of Tris-HCl buffer (50 mM Tris + 10 mM EDTA, pH 8.5) and 0.2 mL of 7.2 mM pyrogallol were added, reacted at 25°C for 20 minutes, and then 0.1 mL of 1 N HCl was added. The reaction was stopped and the amount of oxidized pyrrogallol in the reaction solution was measured at 420 nm. The SOD-like activity was expressed as a percentage of the absorbance decrease in the group with and without the sample solution. As a positive control, Vitamin C 0.1% (AA-2G, Ascorbic acid 2-glucoside) was used.

SOD-like activity (%) = {(reaction absorbance of blank sample solution - reaction absorbance of each sample solution)/reaction absorbance of blank sample solution} × 100

5. Results

1) Cell safety against human keratinocyte cell line (HaCaT)

The first step in the functional search for various functional cosmetic raw materials is to select a concentration range that does not show cytotoxicity in the cell types that make up the skin (keratinocyte, melanocyte, fibroblast, etc.). For this purpose, when dissolving or diluting the test material, DMEM medium containing no FBS was used. In the search for these concentrations, the MTT test method was used as a method of examining the effects on cell growth and proliferation or toxicity after treatment of various concentrations of test substances in cell culture while culturing the cells.

The results of evaluating the cellular safety of the samples in the human keratinocyte cell line, HaCaT, are shown in FIG. 4 (the cytotoxicity graph for each concentration and degree of liquid yeast). Each sample was tested at concentrations of 0.01, 0.05, 0.1, and 0.5 mg/mL. As a result, it was confirmed that there was no cytotoxicity in most samples when treated up to a concentration range of 0.1 mg/mL. It was confirmed that the O-1, O-2, N-1, and M-2 samples were reduced to less than 80% cell viability when treated at a concentration of 0.5 mg/mL. In general, when the cell viability is 80% or more, it is judged that there is no toxicity, so 0.01, 0.05, and 0.1 mg/mL that do not show toxicity were determined and used in the experiment.

2) Evaluation of anti-wrinkle efficacy of liquid yeast

① Procollagen synthesis assay efficacy evaluation

The results of the anti-wrinkle procollagen synthesis assay efficacy evaluation are the same as in FIG. 5 (a graph of the PIP content by liquid yeast degree and different concentrations). In this experiment, the anti-wrinkle effect was measured by comparing the effects of samples on procollagen synthesis using the Procollagen Type I C-Peptide EIA Kit. Each sample was at a concentration of 0.01, 0.05, and 0.1 mg/mL. As a result, when K-1 was treated at a concentration of 0.1 mg/mL, the amount of PIP increased by about 25%, and when M-1 was treated at a concentration of 0.05 mg/mL, it increased by about 37%. Also, when Y-1 and Y-2 were treated with 0.1 mg/mL, they increased by about 49% and 39%, respectively. Except for M-1, Y-1, and Y-2, the remaining experimental groups showed similar amounts of PIP to the control group. Through this, it was found that Y-1, Y-2, K-1, and M-1 increased procollagen synthesis.

2) Procollagen expression promotion action

The result of comparing the expression level of Procollagen C-endopeptidase enhancer (PCOLCE), a wrinkle-related gene, is as shown in FIG. 6 (a graph of the degree of liquid yeast and the relative concentration of PCOLCE at different concentrations). As a result, it was confirmed that when O-2 was treated at a concentration of 0.1 mg/mL, the expression level of PCOLCE was increased by about 2 times compared to the control. This is an increase of about 25% compared to when the positive control, TGFβ-1, was treated with 10 ng/mL. And, when Y-2 and M-2 were treated with 0.1 mg/mL concentration, PCOLCE expression levels were increased by 45% and 53%, respectively. Most of the other samples showed similar expression levels to the control group. These results indicate that Y-2, O-2, and M-2 increase PCOLCE expression to indicate anti-wrinkle efficacy.

3) Evaluation of antioxidant activity of liquid yeast

① DPPH free radical scavenging action

Free radicals are known as the causative substances of skin aging. In this experiment, 1,1-Diphenyl-2-picryhydrazyl (DPPH, Sigma D9132-1G) in ethanol was tested for the scavenging action on free radicals to determine the degree of direct action of the antioxidant effect.

In order to proceed with the antioxidant experiment, the free radical activity inhibition rate was confirmed with samples of various concentrations (0.01, 0.05, 0.1 mg/mL), and the results are shown in FIG. same. It was confirmed that DPPH free radical scavenging activity was present in most samples. When the DPPH free radical scavenging activity was conducted, the reaction color of all 10 samples changed from purple to yellow, and most of the samples started to show weak DPPH free radical scavenging activity even at low concentrations. Among a total of 10 samples, Y-1 had the highest DPPH free radical scavenging activity (53%). Next, K-1 and O-1 samples showed free radical scavenging activity of about 28 and 23%, respectively.

② SOD-like activity

In the SOD-like activity reaction, pyrrogallol is automatically oxidized by superoxide radicals present in water to form a brown substance, which is analyzed with a spectrophotometer, and uses the principle that the oxidation rate of pyrrogallol is lowered in the presence of a substance with superoxide trapping activity. Thus, the superoxide trapping activity can be measured indirectly.

The results of confirming the SOD-like activity for samples of various concentrations (0.01, 0.05, 0.1 mg/mL) are shown in FIG. As a result, although most samples did not show SOD-like activity, about 10% of SOD-like activity was confirmed in Y-1 and M-2 samples. The positive control, vitamin C, showed about 38% SOD-like activity. In comparison, M-2 and Y-1 samples showed about 1/3 SOD-like activity.

6. Conclusion

Nuruk (Y). Aspergillus kawachii (K), Aspergillus niger (N), Aspergillus oryzae (O), and Monascus kaoling (M) were cultured using brown rice and white rice as a medium, and liquid brown rice koji (1), liquid white rice koji (2) were freeze-dried. Efficacy as a cosmetic material was evaluated for a total of 10 liquid yeast samples.

1) As a result of the cell safety test, O-1, O-2, N-1, and M-2 showed cytotoxicity at concentrations of 0.5 mg/mL or more, and all samples were tested at concentrations of 0.01, 0.05, and 0.1 mg/mL. It showed no cytotoxicity.

2) As a result of measuring the amount of PIP for anti-wrinkle-related efficacy evaluation, when K-1 was treated at a concentration of 0.1 mg/mL, the amount of PIP increased by about 25%, and M-1 at a concentration of 0.05 mg/mL It increased by about 37% when treated with When Y-1 and Y-2 were treated with 0.1 mg/mL, they increased by about 49% and 39%, respectively. As a result of comparing the mRNA expression level of PCOLCE related to wrinkles, when Y-2, O-2, and M-2 were treated at a concentration of 0.1 mg/mL, the expression level of PCOLCE increased by 2 times, 1.4 times, and 1.5 times, respectively. . It is determined that Y-2, O-2, and M-2 increase PCOLCE expression to exhibit anti-wrinkle efficacy.

3) As a result of testing on DPPH free radical scavenging activity to evaluate antioxidant activity, it was confirmed that most samples had DPPH free radical scavenging activity. Among them, DPPH free radical scavenging activity of Y-1 was the highest at 53% . As a result of measuring SOD-like activity, most samples did not show SOD-like activity, but SOD-like activity of about 10% was confirmed in Y-1 and M-2 samples.

Overall, most of the samples showed antioxidant effects by scavenging DPPH free radicals rather than SOD-like activity, and among them, Y-1, Y-2, K-1, O-2, M-1, and M-2 synthesize procollagen. It was confirmed that the anti-wrinkle effect and the antioxidant effect were simultaneously exhibited by promoting DPPH free radical scavenging activity.

In the production of liquid yeast according to the present invention, by culturing using a high-frequency pulse to speed up the growth rate of the cells, and by adjusting the grain so that the degree of milling of the rice is selected from sea bream ~ brown rice for 10 minutes, it has high enzyme activity,

By using the liquid yeast prepared in this way, it has excellent physicochemical properties including pH, acidity, sugar content, and alcohol, and provides sensually excellent and differentiated takju while reducing the non-uniformity of the product, which is a disadvantage of solid yeast,

It is possible to provide cosmetics having antioxidant and anti-wrinkle effects, and thus has great industrial applicability.

Claims (6)

Put any one or two or more types of yeast raw materials selected from brown rice, 3 minute sea bream, 7 minute sea bream, or 10 minute sea bream in a high-frequency incubator,
Inoculate the yeast raw material with a spore suspension of yeast mold so that the number of spores becomes 1 × 10 ⁵ pieces/mL,
After high-frequency pulse treatment during incubation,
In the production of liquid yeast by culturing continuously for 40 to 60 hours at a culture temperature of 28 ~ 32 ℃, a stirring speed of 180 ~ 300 rpm,

The high-frequency pulse treatment is performed during the process of culturing yeast raw materials by inoculating a spore suspension of yeast mold, and treating high-frequency waves of 240 to 270 kHz three times at 10-minute intervals, culturing for 5-7 hours, and then culturing at 10-minute intervals A liquid yeast manufacturing method with improved enzyme activity by improving the growth rate of cells using high-frequency pulses and adjusting the degree of milling of rice, characterized in that the high-frequency wave of 240 ~ 270 kHz is processed three times with a high-frequency pulse.
delete The method according to claim 1,
Yeast raw material is milled rice into brown rice, 3 minute sea bream, 7 minute sea bream or 10 minute sea bream, washed, immersed in water for 50 ~ 70 minutes, drained for 20 ~ 40 minutes, and then 115 ~ 130 ℃ A liquid yeast production method with improved enzyme activity by improving the growth rate of cells using high-frequency pulses and adjusting the degree of milling of rice, characterized in that it was prepared by cooling after sterilization for 10 to 30 minutes.
The method according to claim 1,
Aspergillus kawachii, Aspergillus niger, Aspergillus oryzae, and Monascus kaoling are any one or two or more selected from Aspergillus kawachii, Aspergillus niger, Monascus kaoling. Liquid yeast manufacturing method.
Takju, characterized in that it is prepared using the liquid yeast prepared by the manufacturing method of claim 1.
Cosmetics, characterized in that it is manufactured including the liquid yeast prepared by the manufacturing method of claim 1.

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