PRODUCTION METHOD OF CELLULOSE MASK PACK FROM BY-PRODUCT

OF BEER FERMENTATION

Technical field

The present invention relates to a method of producing a base material for a gel- type cosmetic cellulose mask pack, wherein said cellulose is prepared from a yeast waste liquid of beer fermentation.

Background Art

Conventionally, a base material for cellulose mask pack is prepared using cellulose which has been obtained principally from coconuts and appropriately processed to give a base material. However, due to high price of raw coconuts, it has been remained as a problem that production cost for preparing such base material for a mask pack is very high.

Yeast waste liquid of beer fermentation is a waste occurring as a by-product of the beer brewing process by a beer brewing company. Currently, except only few limited cases, a yeast waste liquid of beer fermentation is disposed with extra disposal costs. However, from such yeast waste liquid of beer fermentation, cellulose can be obtained. For microorganisms which can be used for obtaining cellulose from a yeast waste liquid, genus Acetobacter, genus Agrobacterium, genus Rhizobium, genus Pseudomonas and genus Sarcina, etc. have been reported. Among them, many studies has been focused on the microorganisms of genus Acetobacter, because unlike a cell wall polymer that is found in eukaryotes cellulose is secreted as an extracellular fibril by genus Acetobacter so that a mass production of cellulose is possible. Furthermore,

having a characteristic that is distinguished from cellulose derived from plants, microbial cellulose produced by genus Acetobacter draws growing attention not only as food source but also as an advanced material with high industrial value. Microbial cellulose can be mass-produced in relatively short period of time. Further, having high fiber crystallinity, water retentivity, processiblity and tensile strength, etc., it has been developed for various industrial use. Recently, microbial cellulose is used as a cosmetic pad, a medical pad, a carrier for enzyme immobilization and a paper coating agent, etc.

Particularly, US Patent Nos. 5,274,199 and 4,912,049 disclose the use of microbial cellulose for producing an acoustic diaphragm and for producing artificial skin, respectively. Meanwhile, in Korean Patent Application Laid-open Nos. 2003-0015399 and 1997-0014612, a method for producing diet fiber and a method for producing diet drink, both by using microbial cellulose, are disclosed respectively.

As it is discussed in the above, in various areas microbial cellulose can be used as an industrial material or food material, etc. thanks to its excellent physical property. In addition, being an environmentally friendly material, it has a great diversity and an unlimited potential for further development .

On the other hand, cellulose can be obtained from microorganisms with relatively low production yield while cost involved therefor is high. Under such circumstances, the application of microbial cellulose is limited to a very high profit product such as an acoustic diaphragm, etc. until now.

Meanwhile, for producing microbial cellulose from a yeast waste liquid of beer fermentation, culturing cells either by spinner culture or by shaking culture provides a high production yield. However, these methods are disadvantageous in that they

require an expensive apparatus like a fermentation tank, which, therefore makes the cost of equipment very high.

Meanwhile, a method for producing microbial cellulose by spinner culture of Acetobacter xylinum in culture media comprising pepton, a yeast extract, citric acid, glucose, and ethanol, etc. has been known (e.g., Korean Patent Application Laid-open No. 1998-067009). However, because pepton and a yeast extract, etc. that are used for preparing culture media for microbial cellulose are quite expensive, it is economically unfavorable.

Disclosure of Invention

Technical Problem

Inventors of the present invention extensively studied to develop a method of producing a cellulose base material for mask pack, wherein a cellulose-producing microorganism is introduced to a yeast waste liquid of beer fermentation, cellulose is produced using said microorganism by a stationary culture (e.g., static culture), and then thus-obtained cellulose is employed to provide a base material for preparing a mask pack.

As a result, the inventors of the present invention found that a new way of preparing a base material for a cellulose mask pack is possible with significantly reduced cost compared to a conventional mask pack which uses coconut cellulose as a base material. The present invention has been accordingly completed.

Technical Solution

According to the present invention, a stationary culture is adopted to produce microbial cellulose, instead of a spinner culture or a shaking culture.

In order to achieve the above-described purpose, the present invention provides a method for the preparation of a base material which is used for producing a mask pack, comprising steps of inoculating a microorganism which has an ability of producing

cellulose to a yeast waste liquid of beer fermentation and carrying out stationary culture of said microorganisms to produce cellulose; and preparing a base material for a gel-type cellulose mask pack with thus-obtained cellulose.

A microorganism which has an ability of producing cellulose is preferably selected from a group consisting of genus Acetobacter, genus Gluconacetobacter, genus Agrobacterium, genus Rhizobium and genus Pseudomonas. It is preferred to use Gluconacetobacter hansenii (Deposit No; KCTC10505BP).

Preferably, the above-described stationary culture consists of the following steps:

(a) pre-culturing the microorganism which has an ability of producing cellulose and then inoculating the resulting culture supernatant to a yeast waste liquid of beer fermentation,

(b) culturing said microorganisms in the yeast waste liquid of beer fermentation to produce microbial cellulose in a lump state, (c) inoculating a part of the resulting culture supernatant to a new yeast waste liquid of beer fermentation, and (d) producing microbial cellulose from said inoculated culture liquid. The reason why it is important to use a stationary culture is that, for a mass production of cellulose to be used as a commercial product, equipments such as a fermentation tank, etc. are required for a spinner culture and a shaking culture. A fermentation tank is a very expensive equipment. Thus, if microbial cellulose is produced using such an expensive fermentation tank, not much of production costs can be saved compared to a conventional method in which cellulose is produced from coconuts.

On the other hand, when microbial cellulose is produced via stationary culture, expensive equipments like a fermentation tank are not required at all. As a result,

commercial application of the method of the present invention becomes possible.

Meanwhile, it is preferred to add a protease during a culture process of a yeast waste liquid of beer fermentation. By doing so, disintegrated yeast waste of beer fermentation can be first digested by a protease, thus contributing to the improvement in productivity of microorganisms that produce microbial cellulose.

According to the present invention, by culturing said microorganisms in a medium comprising ethanol, a mutant (CeI ^" mutant) which does not produce cellulose during culture process can be inhibited while the proper microorganisms are induced to produce microbial cellulose in a lump state, so that the yield for producing microbial cellulose and the efficiency for an separating process can be maximized, hi addition, because microbial cellulose is produced by the microorganisms which have been cultured in culture media that corresponds to the supernatant of a yeast waste liquid as a byproduct of beer fermentation, both the production costs and the waste processing costs can be reduced. According to the present invention, when ethanol is present in the culture media at a concentration of less than 0.5% (v/v), it is difficult to control the occurrence of a mutant that cannot produce cellulose (e.g., CeI ^" mutant). On the other hand, when the ethanol concentration is above 15% (v/v), growth of the microorganisms is inhibited, and therefore reducing the productivity of the process of the present invention. According to the present invention, it is further preferred to use

Glucoπacetobacter hansenii PJK (Deposit No.; KCTCl 0505BP), which has an ability of producing cellulose, as a microorganism for fermentation.

Advantageous Effects

According to the present invention, microbial cellulose is first produced using a yeast waste liquid of beer fermentation as a culture medium, and then the resulting cellulose is used for preparing a mask pack. Thus, production costs to prepare a base material for a mask pack can be significantly reduced. Furthermore, waste processing costs for the yeast by-products of beer fermentation can be also reduced. Still furthermore, the method of the present invention involves a stationary culture for culturing cellulose, so that an extra equipment such as a fermentation apparatus that is required in a spinner culture or a shaking culture is not necessary at all. Thus, the costs of equipment for commercial production can be also dramatically reduced according to the present invention.

Brief Description of the Drawings

Figure 1 is a photographic image of a yeast waste liquid of beer fermentation. Figure 2 is a photographic image showing the production of cellulose from a yeast waste liquid of beer fermentation.

Figure 3 is a graph showing the difference in XRD between the cellulose obtained from coconuts and the cellulose obtained from a yeast waste liquid of beer fermentation. Figure 4 is a graph showing the production of microbial cellulose from a yeast waste liquid of beer fermentation over time.

Figure 5 is a photographic image showing the base material made of cellulose that has been produced from a yeast waste liquid of beer fermentation.

Figure 6 is a photographic image showing the face mask pack made with the cellulose base material that has been produced from a yeast waste liquid of beer fermentation.

Best Mode

Herein below, the present invention is explained in greater detail in view of the examples. However, the following examples are only to illustrate the present invention, and should not be construed as a limitation of the invention. Especially, although in the following examples a publicly known microorganism of Gluconacetobacter hansenii PJK (deposited with Korea Research Institute of Bioscience and Biotechnology; Deposit No. KCTC 10505BP) is used, it should be obvious to a skilled person in the art that the same results can be obtained for other microorganisms which can also produce cellulose. Example 1: Culture of Gluconacetobacter hansenii PJK in ethanol-comprising culture media

Culture media having the composition as described in the following Table 1 was prepared (pH 5). The media solution was sterilized at 121 ^° C for 15 min and then 5OmL of sterilized solution was introduced to a 250 mL Erlenmeyer flask. Gluconacetobacter hansenii PJK, which can produce microbial cellulose, was inoculated to said flask. The flask was pre-cultured in a shaker incubator at 30 ^° C with revolution speed of 200rpm for 1 day. Then, the supernatant of the pre-culture solution was collected and inoculated to a new media (5OmL) comprising 1% (v/v) ethanol in 250 mL flask to have a final concentration of 5% (v/v). The resulting mixture was subjected to a shake culture at 30 ^° C with revolution speed of 200rpm for five days.

Table 1

Components Amount (g/L)

Glucose (SIGMA, U.S.A.) 1O g

Yeast extract (DIFCO, U.S.A.) 1O g

Pepton (DIFCO, U.S.A.) 7 g

Succinic acid (WACO PURE CHEMICAL, U. S.A.) 0.2 g

Acetic acid (Oriental Chemical Company, South Korea) 1.5 ml

After culturing, the culture solution was recovered and then centrifuged at 4000rpm for 20 min. Thereafter, the supernatant was removed. After the washing with distilled water and the centrifuge twice under the same condition as described above, the remains were frozen at -50 ^° C until the constant weight is obtained. By doing so, dry weight of microbial cellulose comprising cell bodies was obtained first. 0.3N Sodium hydroxide solution (2OmL) was then added to said cellulose comprising cell bodies and the mixture was boiled for 5 min. As a result, all the cells were lysised. Thus-obtained pure microbial cellulose which contains no intact cells was sufficiently washed until it becomes neutral. Then the microbial cellulose was subjected to a freeze-dry again to obtain its dry weight. Dry weight of the cell bodies was calculated by measuring the difference between the dry weight of the microbial cellulose in which the cell bodies are comprised and the dry weight of the pure microbial cellulose. As a result of such measurement, it was found that the dry weight for the cell bodies and the microbial cellulose obtained from the culture media comprising ethanol was 3.34g/L and 2.31 g/L, respectively.

Example 2: Production of microbial cellulose in a lump state using ethanol- comprising culture media

Appearance of the microbial cellulose which has been produced in the above Example 1 is shown in Figure 2. From Day 2 of the culture, the microbial cellulose which has been produced in ethanol-comprising culture media was starting to form a lump in which small globular cellulose pellets were slowly aggregated to each other. On Day 3 of the culture, a new cellulose film in a form of pellicle was formed and covered the entire surface of the microbial cellulose lump. The film was then getting thicker and thicker, which eventually yielded one big lump as shown in Figure 2.

Example 3: Occurrence of a variant (CeI ^" mutant) which does not produce microbial cellulose in ethanol-comprising culture media According to the same procedure as described in the above Example 1, supernatant of the pre-culture solution was inoculated to 5OmL culture media comprising l%(v/v) ethanol to have a final concentration of 5%(v/v) and then incubated at 30 ^° C , 200rpm. The cells were then sampled every day. Thus-obtained culture supernatant was diluted in a saline solution (e.g., xlO ⁵ dilution) and then plated to a solid culture media which has a composition as described in the above Table 1. Culture was carried out at 30 ^° C for two days.

By determining the shape of colony that has been produced on said solid culture media, frequency of occurrence of variant which did not produce any microbial cellulose was calculated (e.g., Number of CeI ^" mutant colony/Number of whole colony). The results are summarized in the following Table 2. Because in solid culture media cellulose-producing cells form a rough-type colony while the cells which do not produce any cellulose form a smooth-type colony, the variant (e.g., CeI ^" mutant) can be easily discerned with the naked eye (Valla, S. and Kjosbakken, J., J. General Microb. 128:1401-

8, 1981).

Table 2

As it is indicated in the above Table 2, a variant which does not produce microbial cellulose was hardly found for the microbes cultured in l%(v/v) ethanol- comprising media for three days.

Example 4: Production of microbial cellulose in ethanol-comprising culture media using a continuous batch culture method According to the same procedure as described in the above Example 1, supernatant of the pre-culture solution was inoculated to 5OmL culture media comprising 1% (v/v) ethanol to obtain the cell concentration of 5% (v/v). Subsequently, a continuous batch culture was carried out at 30 ^° C, 200rpm. The production amount of microbial cellulose, which was obtained after five days of culturing, is summarized in the following Table 3.

Table 3

As it is indicated in the above Table 3, when the microbes that have been cultured in the culture media comprising 1% (v/v) ethanol for three days were used for a continuous batch culture, the microbes' ability of producing microbial cellulose is maintained up to the fifth batch culture.

Example 5: Composition analysis for a yeast waste liquid of beer fermentation

A yeast waste liquid of beer fermentation was centrifuged at 4000rpm. The resulting supernatant was collected and then sterilized at 121 ^° C for 15min. Subsequently, GC (gas chromatography) analysis of the supernatant indicates that the yeast waste liquid of beer fermentation comprised ethanol and acetic acid at the concentration of 8.3% (v/v) and 6.9% (v/v), respectively. Further, it comprised glucose at the concentration of 0.28g/L. Dry weight of the solid mass was 122g/L.

Example 6: Culture of Gluconacetobacter hansenii PJK using a yeast waste liquid of beer fermentation as culture media

A yeast waste liquid of beer fermentation was centrifuged at 4000rpm. The resulting supernatant (5OmL) was taken into a 25OmL Erlenmeyer flask and then sterilized at 121 ^° C for 15min. After carrying out the pre-culture the same as described in the above Example 1, the culture supernatant was inoculated to a supernatant of a

yeast waste liquid of beer fermentation. Shake culture was carried out at 30 ^° C, 200rpm. After the culturing, samples were taken every day to measure the production amount of microbial cellulose using the same method as described in Example 2 (see, Figure 4). In addition, frequency of occurrence of variant which does not produce cellulose (e.g., CeI ^" mutant) was determined using the same method as described in the above Example 4 (see, Table 4).

Table 4

As it can be seen from Figure 4, on Day 16 of the culture, the production amount of microbial cellulose (i.e., 1.37g/L) was very similar to the production amount of cellulose that has been produced from the ethanol-free culture media (e.g., Comparative example 1). In Figure 4, filled circle (•) represents the dry weight of cellulose while

filled triangle (A) and filled square (■) represent pH and glucose concentration, respectively. Further, the above Table 4 indicates that there are hardly any of CeI ^" mutant present, which does not produce cellulose.

Example 7: Stationary culture A yeast waste liquid of beer fermentation was (4L) was homogenized with a high-pressure homogenizer. After the sterilization at 121 ^° C for 15 min, it was introduced to a cultivator having a diameter of approximately 40cm. Gluconacetobacter hansenii PJK and a protease (Aldrich) were also added to the cultivator and the mixture was subjected to a stationary culture. Twenty days later, a layer of microbial cellulose having a thickness of about 18mm was evenly formed over the surface of the cultivator (see, Figure 2).

Example 8: Method of preparing a mask pack using microbial cellulose which has been produced from stationary culture of a yeast waste liquid of beer fermentation

The cellulose obtained from the above Example 7 was isolated and washed with water. Water can be any of distilled water, super-distilled water, or purified tap water. The cellulose was cut to yield pieces having thickness of 3 mm. Thus-obtained cellulose pieces were pressed until they would have thickness of about 0.3mm. They were then immersed in water to have 3mm thickness again. Subsequently, thus-obtained cellulose was again pressed to have 0.3mm thickness. Such procedure was repeated three to five times. The final 0.3mm-thick cellulose base material which has been obtained therefrom is shown in Figure 5. Figure 6 shows said cellulose base material cut and prepared into a desired shape.

Industrial applicability

According to the present invention, by using a yeast waste liquid of beer fermentation, costs involved with a production of a base material for a gel-type cellulose mask pack can be dramatically reduced compared to prior methods which use food materials such as coconuts to produce cellulose. Furthermore, by producing cellulose with a stationary culture using a yeast waste liquid of beer fermentation, only a small production cost is required therefor.