KR102199666B1 - Mycelium Tremella fucifomis TFCUV5 mutant strain having high growth rate and polysaccharide productivity and uses thereof - Google Patents

Abstract

The present invention relates to a mycelium trimella fusiformis TFCUV5 mutant strain having excellent growth rate and polysaccharide productivity, and to a use thereof, and by providing a mycelium trimella fusiformis TFCUV5 mutant strain having excellent growth rate and polysaccharide productivity of the present invention, The derived polymer polysaccharide has excellent feeling of use, thermal stability, and pH stability, but the production of a moisturizer using polysaccharides produced from the fruiting body of white-throated mushrooms has not been easy to use due to instability in supply and demand of the raw material polysaccharide and difficulties in the extraction and purification process. Can be solved. Therefore, it is judged to be more economical to produce high-moisturizing polysaccharides through cultivation using purely isolated and selected mycelium trimella fusformis TFCUV5 mutant strains through the present invention, such as cost reduction and the possibility of producing products of uniform quality. , Very useful for related industries.

Description

Mycelia Trimella fucifomis TFCUV5 mutant strain having high growth rate and polysaccharide productivity and uses thereof {Mycelium Tremella fucifomis TFCUV5 mutant strain having high growth rate and polysaccharide productivity and uses thereof}

The present invention relates to a mycelial trimella fusformis TFCUV5 mutant strain having excellent growth rate and polysaccharide productivity, and uses thereof.

Moisturizing cosmetics using natural polymer polysaccharides are essential materials for cosmetics and have long been popular in the market. Representative moisturizing polysaccharides such as hyaluronic acid, cimaberbot beta-glucan, and leban have been used, but hyaluronic acid is expensive and has low thermal stability, and its use is limited due to difficulties in the fermentation manufacturing process. In the case of the beta-glucan of skirt mushrooms, frequent contamination of the manufacturing process and low production yield are pointed out as problems. In the case of Leban, there is a problem in being widely used due to the decrease in feeling of use due to stickiness and low thermal stability.

High molecular weight polysaccharides derived from white-necked mushrooms have excellent feeling of use, thermal stability, and pH stability, but production of polysaccharide moisturizers using white-necked mushrooms fruiting body has instability in the supply and demand of raw materials and difficulty in extraction and purification processes. Therefore, highly moisturizing polysaccharides through culture using mycelium It is considered to be more economical to produce the product, such as cost reduction and production of uniform quality products.

On the other hand, Korean Patent Publication No. 2008-0079962 discloses'Antidiabetic active extracellular polysaccharide derived from white-necked mushroom mycelium and a method for preparing the same', and Korean Patent Publication No. 2006-0020548 discloses'white color showing hypoglycemic effect. Throat mushroom ( Tremella fuciformis ) Crude polysaccharide extract and a composition for preventing and treating diabetes containing the same, but as in the present invention, as in the present invention, for'mycelium trimella fusformis TFCUV5 mutant strain and its use excellent in growth rate and polysaccharide productivity' Nothing has been disclosed.

The present invention was derived from the above requirements, and in the present invention, in the present invention, the mycelium trimella fusformis TFC6 strain purely isolated from the fruiting body of White-throated mushrooms caused mutations by UV irradiation, and finally, the growth rate and It was possible to isolate the mycelium trimella fusformis TFCUV5 mutant strain (accession number: KCTC18504P) having excellent polysaccharide productivity.

The mycelial trimella fusformis TFCUV5 mutant strain of the present invention was cultivated under the same conditions as the control strain, and the polysaccharide productivity was confirmed. As a result, the maximum of 3.6 g/l at the 6th day of culture in MCM medium, the maximum of 2.6 g/l at 25° C., pH It was confirmed that the maximum 2.7g/l at 7.0, the maximum 3.2g/l at the stirring speed 300rpm, and the maximum 4.3g/l at 2.0vvm air injection volume were confirmed, and based on this, in MCM medium, 25℃, pH 7.0, 300rpm, 2.0 As a result of performing fed-batch culture under the condition of vvm, 8.4 g/L of polysaccharide was produced using about 39.6 g/L of glucose at 108 hours, and the polysaccharide productivity was confirmed to be 1.9 g/L·day. It was confirmed that the polysaccharide production yield was 0.19 g/g compared to the glucose nutrient used, and compared with the previously reported polysaccharide production results of other white mushroom-derived mycelium, the TFCUV5 mutant strain showed very good polysaccharide productivity.

In the meantime, polymer polysaccharides derived from white-throated mushrooms have excellent feeling of use, thermal stability, and pH stability, but the manufacture of moisturizers using polysaccharides produced from white-throated mushrooms has suffered instability in the supply and demand of polysaccharides as a raw material and difficulties in the extraction and purification process. The production of highly moisturizing polysaccharides through culture using the purely isolated and selected mycelium trimela fusformis TFCUV5 mutant strain through the invention confirmed more economical points such as reduction in manufacturing cost and the possibility of manufacturing products of uniform quality. The invention was completed.

In order to solve the above problems, the present invention is a mycelium trimella fusiformis excellent in growth rate and polysaccharide productivity ( Tremella fucifomis ) TFCUV5 mutant strain (accession number: KCTC18504P) is provided.

In addition, the present invention provides a composition for producing a polysaccharide containing the strain or a culture medium thereof as an active ingredient.

In addition, the present invention provides a skin moisturizing cosmetic composition comprising the strain or a culture solution thereof as an active ingredient.

In addition, the present invention provides a method for producing a polysaccharide comprising the step of culturing the strain.

By providing the mycelium trimella fusformis TFCUV5 mutant strain having excellent growth rate and polysaccharide productivity of the present invention, the high molecular polysaccharide derived from white-throated mushroom has excellent feeling of use, heat stability, and pH stability, but the polysaccharide produced from the fruiting body of white-necked mushroom The preparation of the used moisturizer can solve the problem that it was not easy to use due to instability in supply and demand of polysaccharides as a raw material and difficulties in the extraction and purification process.

Therefore, it is judged to be more economical to produce high-moisturizing polysaccharides through cultivation using purely isolated and selected mycelium trimella fusformis TFCUV5 mutant strains through the present invention, such as cost reduction and the possibility of producing products of uniform quality. , Very useful for related industries.

Figure 1 shows the hyphae (top) and colonies (bottom) of the white-necked mushroom mycelium trimella fusiformis TFC6 strain isolated in the present invention.
Figure 2 shows the taxonomic system diagram of the white-throated mushroom mycelium trimella fusiformis TFC6 strain isolated in the present invention.
Figure 3 shows the growth curve (black line), residual equivalent in the medium (gray line) and the amount of polysaccharide produced (blue bar) after culturing the white tree ear mushroom mycelium trimela fusformis TFC6 strain isolated in the present invention in MCM medium. Is shown.
Figure 4 shows the growth curve (black line), residual equivalent in the medium (gray line), and the amount of polysaccharide produced (blue bar) after culturing the white tree ear mushroom mycelium trimella fusformis TFC6 strain isolated in the present invention in PDA medium. Is shown.
Figure 5 shows the result of irradiating the survival rate over time after UV irradiation on the white-necked mushroom mycelium trimella fusformis TFC6 strain isolated in the present invention.
Figure 6 shows the growth curve (black line) and the amount of polysaccharide produced (blue bar) of the white tree mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention.
Figure 7 shows the amount of polysaccharide produced (blue bar) according to the culture temperature of the white-necked mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention in EPS (g/l).
Figure 8 shows the amount of polysaccharide produced (blue bar) according to the initial pH during cultivation of the white tree mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention in EPS (g/l).
Figure 9 shows the amount of polysaccharide produced according to the stirring speed when cultivating the white tree mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention in EPS (g / l).
FIG. 10 shows the amount of polysaccharide produced according to the amount of air injected in the cultivation of the white-necked mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention in EPS (g/l).
FIG. 11 is a result of examining the glucose consumption and polysaccharide production amount of the culture supernatant after fed-batch culture of the white-necked mushroom mycelium trimela fusformis TFCUV5 mutant strain isolated in the present invention.
Figure 12 is a white-throated mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention This is the result of analyzing the derived polysaccharide amino acid.
Figure 13 is a white-throated mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention This is the result of analyzing the derived polysaccharide structure.
Figure 14 is a white-throated mushroom mycelium trimella fusiformis TFCUV5 mutant strain isolated in the present invention It is the result of analyzing the molecular weight of the derived polysaccharide.

In order to achieve the object of the present invention, the present invention has excellent growth rate and polysaccharide productivity, mycelium trimella fusiformis ( Tremella fucifomis ) TFCUV5 mutant strain (accession number: KCTC18504P) is provided.

In the present invention, the mycelium trimella fusformis TFCUV5 mutant strain, which has excellent growth rate and polysaccharide productivity than the parent strain, is finally induced by inducing mutations by UV irradiation targeting the mycelium trimella fusformis TFC6 strain purely isolated from the fruiting body of White-throated mushrooms. (Accession No.: KCTC18504P) could be separated.

The mycelial trimella fusformis TFCUV5 mutant strain of the present invention was cultivated under the same conditions as the control strain, and the polysaccharide productivity was confirmed. As a result, the maximum of 3.6 g/l at the 6th day of culture in MCM medium, the maximum of 2.6 g/l at 25° C., pH It was confirmed that the maximum 2.7g/l at 7.0, the maximum 3.2g/l at the stirring speed 300rpm, and the maximum 4.3g/l at 2.0vvm air injection volume were confirmed, and based on this, in MCM medium, 25℃, pH 7.0, 300rpm, 2.0 As a result of performing fed-batch culture under the condition of vvm, it was confirmed that 8.4 g/L of polysaccharide was produced using about 39.6 g/L of glucose at 108 hours, and the polysaccharide productivity was 1.9 g/L·day (Fig. 11) Therefore, the strain was deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC) under the accession number KCTC18504P on October 31, 2016.

In the method according to an embodiment of the present invention, the polysaccharide may be composed of mannose, xylose, glucuronic acid, fucose and glucose, and preferably mannose, xylose, glucuronic acid, fucose and glucose are each It may be a polysaccharide having a composition of 40.3, 22.0, 19.3, 17.1, 1.3%, but is not limited thereto.

In addition, the present invention provides a composition for producing a polysaccharide containing the strain or a culture medium thereof as an active ingredient.

In the composition according to an embodiment of the present invention, the strain is trimella fusiformis ( Tremella fucifomis ) may be a strain, preferably mycelium trimella fuciformis ( Tremella fucifomis ) TFCUV5 mutant strain (accession number: KCTC18504P) may be, but is not limited thereto.

In addition, the present invention provides a skin moisturizing cosmetic composition comprising the strain or a culture solution thereof as an active ingredient.

The strain or a culture solution thereof of the present invention is preferably a lyophilized powder form, but is not limited thereto.

The cosmetic composition of the present invention is a solution, gel, anhydrous product of a solid or paste, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranular sphere or an ionic liposome, a nonionic vesicle dispersant in the form, a cream , Skin, lotion, powder, ointment, essence, spray, or can be provided in the form of a conceal stick. In addition, it may be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition is a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, an interface Actives, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or cosmetics It may contain adjuvants commonly used in the cosmetic field, such as any other ingredients commonly used.

In the composition according to an embodiment of the present invention, the strain is trimella fusiformis ( Tremella fucifomis ) may be a strain, preferably mycelium trimella fuciformis ( Tremella fucifomis ) TFCUV5 mutant strain (accession number: KCTC18504P) may be, but is not limited thereto.

The cosmetic composition for moisturizing skin of the present invention may further include a known skin moisturizing component in addition to the strain or a culture solution thereof. If additional ingredients that help skin health are included, the effect of the cosmetic composition for moisturizing skin of the present invention can be further enhanced, and when ingredients that help skin health are added, skin safety and formulation according to combination use Ease of fire and stability of active ingredients can be considered.

In addition, the present invention provides a method for producing a polysaccharide comprising the step of culturing the strain.

The method of producing the polysaccharide of the present invention may further include the step of recovering the polysaccharide from the culture medium of the strain. The step of recovering the polysaccharide from the culture medium of the strain may use conventional separation techniques, such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, and the like, and usually separate substances of high purity. In order to do this, they can be used in combination.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a specific method.

In the method according to an embodiment of the present invention, the culture is in MCM medium prepared by adding glucose as a carbon source and yeast extract as a nitrogen source, 24 to 26°C, pH 6.5 to 9.5, 270 to 330 rpm, 1.8 to In the MCM medium prepared by adding glucose as a carbon source and yeast extract as a nitrogen source, preferably performing fed-batch culture under conditions of 2.2 vvm, under conditions of 25°C, pH 7, 300 rpm, and 2 vvm It may be to perform fed-batch culture, but is not limited thereto.

In the method according to an embodiment of the present invention, the MCM (Mushroom complete medium) medium is an MCM medium generally used in the art for culturing strains, glucose 20g/L, yeast extract 2g/L are added, and peptone 2g/L, MgSO ₄ ·7H ₂ O 0.5g/L, K ₂ HPO ₄ 1g/L, KH ₂ PO ₄ 0.46 g/L may be added to prepare a medium, but is not limited thereto.

In the method according to an embodiment of the present invention, the glucose may be added at 18 to 22 g/l, the yeast extract at 1.8 to 2.2 g/l, preferably glucose is 20 g/l, and the yeast extract is 2 g/l. It may be added as l, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. In the present invention White-throated mushroom mycelium Trimella Push Formis TFC6 Isolation of strains

In order to isolate the white-necked mushroom mycelium, the white-necked mushroom fruiting bodies were tapped on potato agar medium (PDA, pH 5.5) to induce spores to fall, and this was cultured in an incubator at 25°C. As shown in FIG. 1, when a colony that grows viscous on the 7th day after culture was secured and observed under a microscope, a yeast-like cell morphology was confirmed (FIG. 1), and this was purely isolated.

The 18S rRNA gene sequence was analyzed for molecular biological identification of the newly isolated white-necked mushroom mycelium trimella fusformis TFC6 strain. After separating chromosomal DNA from a single colony using a genomic DNA extraction kit (Invitrogen, Germany), the nucleotide sequence of the ITS region and large subunit rRNA gene was PCR method using the genomic DNA as a template using AccuPower® Pfu PCR PreMix. Was amplified. Primers used to perform PCR are shown in Table 1 below.

Universal primer used for strain identification primer Sequence (SEQ ID NO) Product size ITS region ITS1 5'-TCCGTAGGTGAACCTGCGG-3' (3) 295bp ITS4 5'-TCCTCCGCTTATTGATATGC-3'(4) Large subunit
rRNA gene NL1 5'-GCATATCAATAAGCGGAGGAAAAG-3'(5) 570bp NL4 5'-GGTCCGTGTTTCAAGACGG-3' (6)

The nucleotide sequence of the PCR product was confirmed by Solgent Co., Ltd., and 295 bp (SEQ ID NO: 1) and 570 bp (SEQ ID NO: 2) were obtained as effective nucleotide sequences for the ITS region and the large subunit rRNA gene, respectively. This nucleotide sequence was confirmed by NCBI blast and EZbiocloud EzFungi. It was 99.8% consistent with CCJ1531. Based on the result of nucleotide sequence homology analysis, the isolated white-necked mushroom mycelium was treated with Trimella fusformis ( Tremella fuciformis ) was named TFC6 strain (Fig. 2).

Example 2. Separated from the present invention White-throated mushroom mycelium Trimella Push Formis TFC6 Polysaccharide production and polysaccharide composition analysis of strain

Polysaccharide production was confirmed from the isolated trimella fusformis TFC6 strain. The pre-culture was carried out in PDA (potato starch 4g/L, glucose 20g/L) medium at 25°C, 200 rpm for 3 days, and then the cells were recovered through centrifugation. This was again performed with PDA and MCM (Mushroom complete medium, glucose 20g/L). L, yeast extract 2g/L, peptone 2g/L, MgSO ₄ 7H ₂ O 0.5g/L, K ₂ HPO ₄ 1g/L, KH ₂ PO ₄ 0.46g/L) Initial inoculation concentration in the medium at OD ₆₀₀ It was inoculated to be 0.3, and cultured with shaking at 200 rpm at 25° C. and sampled every 24 hours to check the OD value, the amount of residual equivalent in the medium, and the amount of polysaccharide produced (FIGS. 3 and 4 ).

The OD value for the growth curve of the Trimela fusformis TFC6 strain was measured by absorbance at 600 nm, and the residual glucose in the medium was centrifuged at 12000 rpm for 15 minutes to separate cells and supernatant, and the separated supernatant was 0.2 After filtering with a μm filter, it was used with a high-performance liquid chromatography flow rate of 0.6 mL/min, a mobile phase 0.25 mM H ₂ SO ₄ , an ion exchange column (300×78mm, Aminex HPX-87H, Bio-Rad), and a differential refractive detector (RID). The amount of glucose was analyzed under the conditions used. The polysaccharide produced in the medium was measured by the ethanol precipitation method. Each culture was centrifuged at 12000 rpm for 15 minutes to separate the cells and the supernatant, treated with ethanol 3 times (37.5 ml) in 12.5 ml of the separated supernatant from the cells, mixed well, and reacted at 4°C for 16 hours to precipitate polysaccharides. Induced. This was centrifuged at 12000 rpm for 15 minutes to recover the precipitated polysaccharide, and freeze-dried to measure its weight.

As a result, as shown in Figs. 3 and 4, it was confirmed that both media used in the growth curve experiment using PDA and MCM media passed through the logarithmic growth phase on the first 1-3 days of culture and reached a plateau on the fourth day of culture. However, the growth in MCM medium showed up to 35 with OD ₆₀₀ value, which was more than 2 times higher than the maximum 14.7 in PDA medium, and the resulting polysaccharide was also about 2 g/l (4-6 days) in MCM medium. The maximum polysaccharide produced in about 0.9 g / l (6th day) was higher than. Afterwards, Trimella Pushformis The polysaccharide production amount experiment using the TFC6 strain was performed using MCM medium.

Sugar composition of polysaccharide produced by trimella fusformis TFC6 strain Party content (%) Fucos 15.0 Glucose 2.3 Xylose 21.0 Mannose 41.5 Glucuronic acid 20.2 gun 100

Example 3. Mycelium of the present invention Trimella Push Formis TFC6 Isolation of high polysaccharide mutant strains from strain mutants

For the generation of UV mutations, the kill rate by UV of the TFC6 strain of white tree mushroom was confirmed. After plating 10 ⁶ cells/plate of cells in MCM solid medium, they were exposed for 0, 1, 2, 3, 4, 5, 6, 10, 15 minutes at a distance of about 30 cm from the UV lamp. After that, the cells were cultured at 25° C. for about 7 days for growth, and the number of colonies grown after the culture was counted to check the death rate. As shown in FIG. 5, when the UV exposure time was 5 minutes or more, growing colonies could not be confirmed, and when exposed to UV for about 3 minutes and 30 seconds, it was confirmed that the survival rate was 0.01% (mortality rate 99.99%). Thereafter, in order to cause a mutation, the white-necked mushroom strain spread on the medium was exposed to UV under the same conditions for 3 minutes and 30 seconds. After smearing the white-necked mushroom mycelium 10 ⁶ cells on each plate, exposure to UV for 3 minutes and 30 seconds to induce mutations, and 880 single colonies were obtained. Afterwards, for the stabilization of the mutated gene, subcultures were performed up to the third stage while dropping out the small colonies. As a result, 200 colonies were obtained after the third passage.

After subculturing the UV mutant strain of the TFC6 strain of white oyster mushroom to the third stage for stabilization, 84 large-growing colonies were selected, divided into three sets, and cultured in MCM medium, 25°C, 200 rpm for 4 days, and then OD value The amount of hyperpolysaccharide produced was measured. Basically, because the amount of polysaccharide production was different for each set, two strains with the highest polysaccharide production amount were selected for each set, and each strain was selected according to the colony number, TFCUV-5, TFCUV-7, TFCUV-36, TFCUV-38, and It was named TFCUV-59, and TFCUV-82.

In the TFCUV5 mutant strain, which showed the highest polysaccharide production amount among the mutant strains of the TFC6 strain, the amount of polysaccharide production was confirmed under the same culture conditions according to the culture time. As a result, it was confirmed that the TFCUV5 mutant strain produced up to 3.6 g/l of polysaccharide on the 6th day of culture (FIG. 6).

Example 4. Polysaccharide high-producing mycelium isolated in the present invention Trimella Push Formis Fermentation process study of TFCUV5 mutant strain

1) Influence of carbon and nitrogen sources

The effects of different carbon sources and nitrogen sources on the polysaccharide production of the obtained trimella fusformis TFCUV5 mutant strain were investigated. 20 g/L of carbon sources (glucsoe, maltose, frutose, sucrose, lactose, mannitol, sorbitol, xylose) and 2 g/L of nitrogen sources (yeast extract, tryptone, soy peptone, ammonium sulfate, ammonium nitrate) were each prepared in the polysaccharide-producing MCM medium. Alternatively, it was cultured in a 250 ml flask at 25° C. and 200 rpm. As a result, as shown in Table 4, when glucose and sucrose were used as carbon sources, a high level of polysaccharide was produced. Meanwhile, when yeast extract was used as a nitrogen source, the maximum polysaccharide production was shown. Compared with the organic nitrogen source, the inorganic nitrogen source showed relatively higher cell production and polysaccharide production.

Effect of Polysaccharide Production by Carbon and Nitrogen Sources of Trimela fusformis TFCUV5 Mutant Strains Cell dry weight
(g/L) Polysaccharide
(g/L) Carbon source Glucose 37 2.5 Maltose 28 1.8 Fructose 25 1.7 Sucrose 33 2.3 Lactose 21 1.5 Mannitol 10 0.5 Sorbitol 8 0.3 Xylose 19 1.0 Nitrogen source Yeast extract 38 2.6 Tryptophan 29 2.1 Soy peptone 30 2.0 Ammonium sulfate 15 1.2 Ammonium nitrate 16 1.0

2) Influence of temperature

The effect of temperature on the productivity of polysaccharides was investigated using the Trimela fusformis TFCUV5 mutant strain. TFCUV5 strain in a 250 ml flask (MCM medium, glucose 20 g/L, yeast extract 2 g/L, peptone 2 g/L, MgSO ₄ 7H ₂ O 0.5 g/L, K ₂ HPO ₄ 1 g/L, KH ₂ PO ₄ 0.46 g /L) was used to batch culture at 200 rpm at different temperatures (25, 28, 31, 34°C), and at the same time, the amount of polysaccharide produced in the culture supernatant was investigated. As a result, as shown in FIG. 7, the highest 2.6 g/L polysaccharide (EPS) was produced at a culture temperature of 25°C.

3) Effect of initial pH

The effect of initial pH on polysaccharide productivity was investigated using the Trimela fusformis TFCUV5 mutant strain. TFCUV5 strain in a 250 ml flask (MCM medium, glucose 20 g/L, yeast extract 2 g/L, peptone 2 g/L, MgSO ₄ 7H ₂ O 0.5 g/L, K ₂ HPO ₄ 1 g/L, KH ₂ PO ₄ 0.46 g /L) was used to change the initial pH (5.0, 6.0, 7.0, 8.0, 9.0) to batch culture at 25°C and 200 rpm, and at the same time, the amount of polysaccharide produced in the culture supernatant was investigated. As a result, as shown in FIG. 8, there was no significant difference at the initial pH of 7.0, 8.0, and 9.0. The highest 2.7 g/L polysaccharide was produced at an initial pH of 7.0.

4) Influence of stirring speed

The effect of agitation speed on polysaccharide productivity was investigated using the Trimela fusformis TFCUV5 mutant strain. TFCUV5 strain in a 5L fermenter (MCM medium, glucose 20g/L, yeast extract 2g/L, peptone 2g/L, MgSO ₄ 7H ₂ O 0.5g/L, K ₂ HPO ₄ 1g/L, KH ₂ PO ₄ 0.46 g /L) was used to vary the stirring speed (100, 200, 300, 400, 500 rpm) to perform batch culture at an initial pH of 7.0, 25° C., 1.0 vvm, and at the same time, the amount of polysaccharide produced in the culture supernatant was investigated. As a result, polysaccharides were not produced at all when the stirring speed was 100 rpm as shown in FIG. 9. The highest 3.2 g/L polysaccharide was produced at 300 rpm.

5) Influence of air injection volume

The effect of air injection on the productivity of polysaccharides was investigated using the Trimela fusformis TFCUV5 mutant strain. TFCUV5 strain in a 5L fermenter (MCM medium, glucose 20g/L, yeast extract 2g/L, peptone 2g/L, MgSO ₄ 7H ₂ O 0.5g/L, K ₂ HPO ₄ 1g/L, KH ₂ PO ₄ 0.46 g /L) was used to vary the amount of air injection (0.5, 1.0, 2.0vvm) to perform batch culture at an initial pH of 7.0, 25°C, and 300 rpm, and at the same time, the amount of polysaccharide produced in the culture supernatant was investigated. As a result, as shown in FIG. 10, when the air injection amount was 0.5vvm, no polysaccharide was produced. The highest 4.3 g/L polysaccharide was produced at 2.0 vvm.

6) fed-batch culture

Fed-batch cultivation was performed using trimella fusformis TFCUV5 mutant strain. 5L fermentation tank (MCM medium, glucose 20g/L, yeast extract 2g/L, peptone 2g/L, MgSO ₄ 7H ₂ O 0.5g/L, K ₂ HPO ₄ 1g/L, KH ₂ PO ₄ 0.46g/L) By using the initial pH 7.0, 25 ℃, 300rpm, was cultured at 2.0vvm, and at the same time, the amount of glucose remaining in the culture supernatant and the amount of polysaccharide produced were examined. As a result, as shown in FIG. 11, 8.4 g/L of polysaccharide was produced using about 39.6 g/L of glucose at 108 hours of culture (FIG. 11), and the polysaccharide productivity was 1.9 g/L·day. The polysaccharide production yield compared to the glucose nutrient used was found to be 0.19g/g glucose. Compared with the results of polysaccharide production of other white mushroom-derived mycelium reported in the literature, the TFCUV5 mutant showed excellent polysaccharide production (Jant et al., 2008, Journal of Mushroom Science and Production March;6(1);27) -31).

Example 5. White-throated mushroom mycelium Trimella Push Formis TFCUV5 Analysis of the composition of polysaccharides derived from mutant strains

The sugar composition of the polysaccharide obtained through cultivation of the Trimela fusformis TFCUV5 mutant strain was analyzed. The analysis method is as follows. 100 µg of a sample and 400 µl of 2M TFA were placed in a microcentrifuge tube, respectively, placed in a 100°C heat block, hydrolyzed for 4 hours, cooled to room temperature, and dried in a vacuum centrifuge. Thereafter, the sample was dissolved in 2 ml of third water, filtered through a 0.2 μm filter, and then hydrolyzed with glucuronic acid, and 100 μg of the sample and 400 μl of 2M TFA were respectively added to a microcentrifuge tube, and placed in a heat block at 100°C. After hydrolysis for 4 hours, it was cooled to room temperature, dried in a vacuum centrifugal concentrator, dissolved in 1 ml of third order water, and filtered through a 0.2 μm filter.

HPAEC conditions Neutral Cathedral Glucuronic acid Mode Ion exchange chromatography detection Integrated amperometry menstruum 2 mM NaOH 100 mM NaOH/150 mM
Sodium acetate Flow rate (ml/min) 0.5 1.0 Column CarboPac PA20 column (3 x 150 mm, Dionex, 060142) and
AminTrap (3x30 ㎜, Dionex
060146) CarboPac PA1 column (4×250
Mm, Dionex, 035391) and guide column (4×50 mm, Dionex,
043096) Temperature, ℃ Column oven, 30; Autosampler, 8

The value measured by HPAEC was converted into weight, and the value was calculated by dividing it by the amount of protein (0.5 or 1 μg) used for analysis. At this time, the molecular weight of each monosaccharide used for conversion was assumed to be composed of a polymer, and a value obtained by subtracting 18D of the molecular weight of water that escaped from each glycosidic bonding was used. As a result, as shown in Table 6 below, mannose, xylose, glucuronic acid, fucose, and glucose exhibited a composition of 40.3, 22.0, 19.3, 17.1, and 1.3%, respectively.

Sugar composition of polysaccharide produced by trimella fusformis TFCUV5 mutant strain Party content(%) Fucos 17.1 Glucose 1.3 Xylose 22.0 Mannose 40.3 Glucuronic acid 19.3 gun 100.0

As a result of measuring the general microbial content of the polysaccharide obtained through cultivation of the Trimela fusformis TFCUV5 mutant strain according to the Food Code (2015) microbial test method, it was found to be negative.

Example 6. White-throated mushroom mycelium Trimella Push Formis TFCUV5 Analysis of polysaccharide amino acids derived from mutant strains

The amino acids of the polysaccharide obtained through the culture of the Trimela fusformis TFCUV5 mutant strain were analyzed, and the analysis method is as follows.

[Sample preparation for general amino acid analysis]

1. Put 100 ㎍ of a sample in a sample tube and dried.

2. A sample tube, 200 µl of 6 M HCl, and 2 µl of phenol were added to the reaction vial.

3. Hydrolyzed at 110° C. for 24 hours.

4. After the reaction was completed, 25 nmol of norleucine was added and dried again.

5. According to Pico·Tag™ Manual, it was labeled with PITC and dried.

6. The sample was dissolved in 400 µl of HPLC solvent A.

[Sample preparation for cysteine analysis]

1. Put 100 ㎍ of a sample in a sample tube and dried.

2. 10 µl of a solution of formic acid: hydrogen peroxide = 10:1 was added to the sample tube and reacted at room temperature for 30 minutes, followed by drying.

3. A sample tube, 200 µl of 6 M HCl, and 2 µl of phenol were added to the reaction vial.

4. Hydrolyzed at 110° C. for 24 hours.

5. After the reaction was completed, 25 nmol of norleucine was added and dried again.

6. According to Pico·Tag™ Manual, it was labeled with PITC and dried.

7. The sample was dissolved in 400 µl of HPLC solvent A.

[Sample preparation for tryptophan analysis]

1. Put 100 ㎍ of a sample in a sample tube and dried.

2. Put 20 µl of 4M methanesulfonic acid into the sample tube and 100 µl of the third order into the reaction vial.

3. Hydrolyzed at 110° C. for 24 hours.

4. After the reaction was completed, 25 nmol of norleucine was added and dried again.

5. According to Pico·Tag™ Manual, it was labeled with PITC and dried.

6. The sample was dissolved in 400 µl of HPLC solvent A.

HPLC solvent Time (minutes) A: 140 mM sodium acetate
trihydrate/0.1% TEA, pH 5.9
in 6% acetonitrile (%) B: 60% acetonitrile in water
(%) Early 100 0 9.0 86 14 9.2 80 20 17.5 54 46 17.7 0 100 21.0 100 0

HPLC conditions Neutral Cathedral Mode Reverse phase chromatography detection UV254nm injection volume (µl) 2 mM NaOH Flow rate (ml/min) 1.0 Column Pico Tag column, 4 ㎛, 3.9 x 300 ㎜ (Waters, WAT01950) Temperature, ℃ Column oven, 46; Autosampler, 4

The analyzed amino acid results are shown in Table 9 and FIG. 12 below. Histidine and tryptophan were not detected. BCA analysis showed that the 1.5 µg BSA eq/100 µg TFC value and the amino acid sum of 1.21 g/100 g were almost identical, indicating that the protein content of the sample was at the level of 1.2%.

Amino acid composition Amino
acid TFC
(pmol) IS corrected
(pmol) mol(%) g/100g Cys 4.8 5.7 1.8 0.02 Asp 24.7 27.3 8.6 0.13 Glu 30.1 33.3 10.4 0.17 Ser 29.0 32.1 10.0 0.11 Gly 62.2 68.7 21.5 0.16 His - - - - Arg 2.3 2.5 0.8 0.02 Thr 23.2 25.6 8.0 0.10 Ala 29.9 33.1 10.4 0.09 Pro 28.5 31.5 9.9 0.12 Tyr 3.9 4.3 1.4 0.03 Val 11.6 12.9 4.0 0.05 Met 2.5 2.7 0.8 0.01 Ile 13.3 14.7 4.6 0.07 Leu 8.2 9.1 2.9 0.04 Phe 4.4 4.8 1.5 0.03 Trp - - - - Lys 9.9 10.9 3.4 0.06 Total 288.6 319.2 100 1.21

Example 7. Trimella Push Formis TFCUV5 Analysis of polysaccharide structure derived from mutant strain

In order to confirm the functional groups of polysaccharides isolated from the culture of the Trimela fusformis TFCUV5 mutant strain, a thin film was prepared using the KBr disk method, and then FT-IR (JASCO FT-IR 4100) analysis was performed. As a result of performing FT-IR analysis, it was confirmed that the polysaccharide isolated from the culture medium of the Trimela fusformis TFCUV5 mutant strain contained a hydroxy group, a carboxyl group, an O-acetyl group, and a glucuronic acid (FIG. 13). Strong and broad peaks around 3325.04 cm ^-1 represents a hydroxy group, two peaks appear symmetrically in the peak and near 1411.92 ~ 1374.39 cm ^-1 appear to 1720.99 cm ^-1 exhibited a carboxy group. In addition, 1249.38 cm ^-1 and 1022.28 cm ^-1 each represented an O-acetyl group and glucuronic acid (FIG. 13). These results were similar to the previously reported IR spectrum of polysaccharides derived from the genus Trimela.

Example 8. Trimella Push Formis TFCUV5 Molecular weight analysis of polysaccharides derived from mutant strains

The average molecular weight of the polysaccharide isolated from the culture of the Trimela fusformis TFCUV5 mutant strain was determined by a high-performance liquid chromatography-MALS (Multi-angle light scattering) system (Wyatt Technology, Santa Babara, CA) in a SEC column (TSK-gel-GMPWXL, CA). 30 cm × 7.8 mm, 13 μm, Tosoh). The polysaccharide isolated from the culture of the Trimela fusformis TFCUV5 mutant strain was dissolved in phosphate buffered saline (pH 7.3 to 7.5) to prepare and analyze a sample at a concentration of 3 mg/mL. The prepared samples were analyzed under the conditions of high performance liquid chromatography flow rate 0.5 mL/min, injection volume 100 μl, mobile phase phosphate buffered saline solution (pH 7.3-7.5), detector DAWN HELEOS II, Optilab rEX. When calculating the molecular weight, the dn/dc value (specific refractive index increment) was determined to be 0.15 mL/g with reference to the literature. The calculation of the average molecular weight of polysaccharides isolated from the culture of the Trimela fusformis TFCUV5 mutant strain was analyzed using ASTRA 6 software (Wyatt Technology). The average molecular weight of the polysaccharide analyzed by the above method was confirmed to be 1.79 × 10 ⁶ (±0.721%) (FIG. 14 ).

Korea Research Institute of Bioscience and Biotechnology KCTC18504P 20161031

<110> Korea Research Institute of Bioscience and Biotechnology Microbiome Co., Ltd. <120> Mycelium Tremella fucifomis TFCUV5 mutant strain having high growth rate and polysaccharide productivity and uses thereof <130> PN16449 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 295 <212> DNA <213> Tremella fucifomis <400> 1 ggccgtgagg ccttccaaca cctgtgcaca tcggaccgcg cccccgggcc gggctgcctt 60 cacacaaaca tatgtcaaga acgtaatgca tcataacatg aaacaacttt caacaacgga 120 tctcttggct ctcgcatcga tgaagaacgc agcgaattgc gaaaagtaat gtgaattgca 180 gaattcagtg aatcatcgaa tctttgaacg caccttgcgc cttttggtat tccgaaaggc 240 atgcctgttt gagtgtcatg tagactcaac cccccgggtt tctgacccgg cggtg 295 <210> 2 <211> 570 <212> DNA <213> Tremella fucifomis <400> 2 ccctagtacg gcgagcgaac cgggaagagc tcaaatttga aatctggcgt cctccgggcg 60 tccgagttgt aatctacaga ggcgttttcc gcgccggtcc gcgtccaagt cccttggaac 120 agggtatcaa agagggtgac aatcccgtac ttgacacgac cgccggtgct ttgtgatacg 180 tcttctaaga gtcgagttgt ttgggaatgc agctcaaaac gggtggtaaa ttccatctaa 240 ggctaaatat tggcgagaga ccgatagcga acaagtaccg tgagggaaag atgaaaagca 300 ctttggaaag agagttaaac agcacgtgaa attgttgaaa gggaaacgat tgaagtcagt 360 cgtgtccggg gggttcagcc ggttctgccg gtgcattcct ctcggacggg tcaacatcgg 420 ttttgcccgg cggaaaaggg cgtgaggaat gtggcacctc cgggtgtgtt atagcctcgc 480 gtcgcatacg tcgggcggga ccgaggaacg cagctcgcct tcacggccgg ggttcgccca 540 cgtacgagct taggatgttg acataatggc 570 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tccgtaggtg aacctgcgg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcctccgctt attgatatgc 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcatatcaat aagcggagga aaag 24 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggtccgtgtt tcaagacgg 19

Claims (8)

As a mycelium trimella fucifomis TFCUV5 mutant strain (accession number: KCTC18504P) having excellent growth rate and polysaccharide productivity,
The polysaccharide strain, characterized in that consisting of mannose, xylose, glucuronic acid, fucose and glucose. delete A composition for producing polysaccharides containing the strain of claim 1 or a culture solution thereof as an active ingredient. A cosmetic composition for moisturizing skin comprising the strain of claim 1 or a culture solution thereof as an active ingredient. The method of claim 4, wherein the cosmetic composition is a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic agent. Or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles. Cosmetic composition for moisturizing skin, characterized in that. A method for producing a polysaccharide comprising the step of culturing the strain of claim 1. The method of claim 6, wherein the cultivation is performed in MCM medium prepared by adding glucose as a carbon source and yeast extract as a nitrogen source, under conditions of 24 to 26°C, pH 6.5 to 9.5, 270 to 330 rpm, and 1.8 to 2.2 vvm. A method, characterized in that performing fed-batch culture. The method of claim 7, wherein the glucose is 18 to 22 g/l and the yeast extract is added at 1.8 to 2.2 g/l.

Images