KR0135779B1 - Protoplast hybridoma of lentinula and ganoderma mushroom - Google Patents

Abstract

The present invention relates to a method for producing a protoplast fusion of shiitake mushrooms and ganoderma lucidum mycelium, which are basidiomycetes exhibiting incompatibility between the mycelia, and a protoplast fusion having improved pharmacological activity thus obtained. In other words, the method of producing a protoplast fusion of shiitake mushroom and ganoderma lucidum mushroom by reacting cell hypolytic enzymes with mycelia of shiitake mushroom and ganoderma lucidum to produce respective protoplasts and suspending the protoplast fusion of shiitake mushrooms in an osmotic stabilizer. Protoplast fusion P54 of shiitake mushrooms and ganoderma lucidum prepared according to the present invention is an excellent strain having improved pharmacological activity with faster growth rate than the parent strain.

Description

Protoplast Fusion of Shiitake and Ganoderma Lucidum Mushroom and a Method for Preparing the Same

1 (a), 1 (b) and 1 (c) show the electrophoretic patterns of shiitake mutant strain (LE4), Ganoderma lucidum mutant strain (GL1), and esterase (EST) isoenzymes of several protoplast fusions 2 shows electrophoretic patterns of acid phosphatase (ACP) isoenzymes of shiitake mutant strains (LE4), Ganoderma lucidum mutant strains (GL1), and their protoplast fusions, and 3 (a) and 3 ( b) is a diagram showing the electrophoretic pattern of mitochondrial DNA of shiitake mutant strain (LE4), Ganoderma lucidum mutant strain (GL1), and their protoplast fusions with restriction enzymes EcoR, Hind and Pst.

The present invention relates to a method for producing a protoplast fusion of shiitake mushroom and ganoderma lucidum mycelium, and to a protoplast fusion obtained according to the present invention, and specifically, to enzymatic treatment of mycelia of shiitake mushrooms and ganoderma lucidum which are incompatible with each other. It is a method for inducing protoplast fusion under certain conditions after decomposing the same, and particularly relates to a protoplast fusion of shiitake mushroom and ganoderma lucidum mushroom, which is obtained accordingly and has better anticancer activity than the parent strain.

Shiitake mushroom is a type of fungus belonging to the genus Pleurotus eryngii, Pleurotus eryngii, common mushroom, and has been used as a food because of its excellent taste and aroma. Recently, its efficacy has been proved. The mycelia of shiitake mushrooms and the protein polysaccharide components of fruiting bodies have been reported to have anti-cancer effects, and as an immune enhancer and a T-cell immunoadjuvant, LEM, a culture extract of shiitake mycelium, has been clinically tested for hepatitis B patients. It has been reported to be effective in the treatment of and to inhibit the replication, infection and cytopathological effects of HIV.

Ganoderma lucidum is a fungal fungus belonging to the Democratic arachnoid, Bloxaceae, Bloxaceae, and has been used for many years for inflammatory diseases such as arteriosclerosis, hypertension, stroke, nervous breakdown, gastric ulcer, and indigestion, acute pathogenic hepatitis, and various cancers. . After Kim et al. Found that the Ganoderma lucidum had anti-cancer effects in the fruiting bodies, relegation reported that mycelium polysaccharides had anti-cancer effects against sarcoma 180, and Shin et al. Reported that Ganoderma lucidum heat extract increased the number of immune cells in mice. Sikkim said.

As described above, both shiitake mushrooms and ganoderma lucidum mushrooms have been reported to have excellent effects on the maintenance of health and treatment of diseases. These basidiomycetes have been cultivated by mycelial inoculation in the natural state, but the production capacity has dropped sharply due to the difficulty of stable cultivation due to deterioration of environment and the occurrence of pests. Accordingly, along with the development of the cultivation technology, a new strain of strains capable of stable cultivation under poor environmental conditions is required.

Molecular fungi, which are difficult to fuse with hyphae due to complex genetic patterns and incompatibility between the same strains, have not developed much genetic research at the molecular level and the development of the resulting strains compared to higher plants. In recent years, protoplast fusion of hyphae has been attempted to create new hybrids within and between species. As a result, protoplast fusion and nuclear transfer have been used as useful methods in mushrooms.

Protoplasts are free of cell walls, and somatic hybridization can be achieved even in the first place by using a fusion aid to facilitate fusion. For protoplast fusion, many protoplasts must be able to be ejected first, and the protoplasts must be able to regenerate the cell wall and return to the original mycelial state. With the commercialization of cell wall degrading enzymes, studies on protoplasts have progressed, and further studies on protoplast outflow and regeneration of mushrooms currently being edible have been carried out, and shiitake mushrooms have been studied by Ushiyama et al. (1977).

Early protoplast fusion was very low in efficiency due to homogenous centrifugal fusion, and did not occur in the original period. Polyethylene glycol was then discovered as a fusion aid for higher plants, and protoplast fusion using PEG has been widely used across bacterial, fungal, plant and animal cells. The mechanism of membrane fusion by PEG is not yet known, but it is estimated that the protoplasts are fused by Ca 2+ acting as the protein particles of the two membranes are moved to form a lipid-rich region. Protoplast fusion has been successful in developing new recombinants and industrially useful microbial strains in fungi whose genetic background is not well known. In particular, the advantages of the protoplast fusion method in basidiomycetes are that the preparation process is easy, the protoplasts can be reduced to normal, genetic analysis of species whose life history is not revealed, and between species that are not hybridized. Recombinant can be obtained and can be used as a breeding method of industrially useful strains.

US Patent No. 4,940,839 discloses a method for producing hybrids by protoplast fusion and regeneration under certain conditions of cultivated tomato Lycopersicon esculentum and wild tomato, and US Pat. The present invention discloses a method of making yeast strains with improved fermentation, and in US Pat. No. 5,231,007, yeast strains having improved riboflavin production capacity are obtained through mutagenesis and protoplast fusion of Candida famata strains.

As described above, many attempts have been made for the purpose of breeding, but no protoplasts have yet been attempted for the purpose of developing pharmacological activity of basidiomycetes. Therefore, in the present invention, the physiological activity of the two mushrooms is increased by using shiitake mushrooms and ganoderma lucidum, which have been commonly used for food and medicinal use since ancient times, and a protoplast fusion having new genetic traits having bacteriological significance. The present invention has been made.

In other words, the object of the present invention, using shiitake mushrooms, which have excellent taste and aroma in basidiomycetes and have strong anticancer activity, and mycelium of Ganoderma lucidum mushrooms known to have physiological effects such as anticancer action and lowering blood pressure, It is to provide a method for forming a protoplast fusion between these two basidiomycetes.

Another object of the present invention is to provide a protoplast fusion of shiitake mushrooms and ganoderma lucidum which is formed by the above method and has increased pharmacological activity compared to the parent strain.

Method for producing a protoplast of shiitake mushroom and ganoderma lucidum mushroom of the present invention to achieve the above object is to produce the respective protoplasts by reacting the cell wall degrading enzyme to the mycelia of shiitake mushroom and ganoderma lucidum mushroom, the protoplast of shiitake mushroom and ganoderma lucidum The mixture is mixed with PEG solution to produce a protoplast fusion, and the protoplast fusion is characterized in that the suspension is cultivated in an osmotic stabilizer.

Another object of the present invention is achieved by a protoplast fusion of shiitake mushrooms and ganoderma lucidum prepared according to the above method.

In particular, in the present invention, the protoplast fusion P54 (Accession No. KFCC-10825) of shiitake mushroom and ganoderma lucidum having a greater anticancer effect than the parent strain is preferred.

In addition, the step of reacting the cell wall degrading enzyme to the mycelia of shiitake mushroom and Ganoderma lucidum mushroom, the mycelia of shiitake mushroom and Ganoderma lucidum in 0.6M sucrose solution containing Novozyme 234 and cellulase Onozukaal-10 pH 4 It is preferable to make the reaction within 6 to about 30 to 5 hours.

In addition, the step of mixing the shiitake mushroom and Ganoderma lucidum protoplasts and reacting by adding a PEG solution to produce a protoplast fusion, 30% PEG 4,000 solution containing 10mM calcium ion to a mixture of shiitake mushroom and Ganoderma lucidum protoplasts It is preferred to react for 30 to 10 15 minutes while shaking.

Hereinafter, the method for forming the protoplast fusion of shiitake mushroom and ganoderma lucidum and the characteristics of the protoplast fusion of shiitake mushroom and ganoderma lucidum will be described in detail.

[Selection of Nutritional Components by Mutagenesis]

Prior to forming the protoplast fusion of the present invention, a trophogenic mutant strain to be used as a label for screening the fusion is formed. In other words, each group of shiitake mushrooms and ganoderma lucidum are cultured in the minimum and medium plates after UV treatment to select the mycelia that grow on the complete medium but do not grow on the minimum medium. In the present invention, a paraamino benzoic acid mutant strain of shiitake mushroom and a hypoxanthine mutant strain of ganoderma lucidum are selected and used for protoplast fusion.

[Production of protoplasts]

Nutrient mutant strains of shiitake mushrooms and ganoderma lucidum mushrooms were shaken with a solution of cell wall degrading enzyme added with an osmotic stabilizer for a certain period of time, filtered and separated from the mycelia residues and suspended in an osmotic stabilizer.

Factors affecting the production of protoplasts include the bell tower and concentration of cell wall degrading enzyme, reaction time and temperature, concentration of osmotic stabilizer, and culture time and pH of hyphae. In the present invention, commercially available Novozyme 234 and cellulase Onozuka al-10 are used as cell wall degrading enzymes of shiitake mushroom and Ganoderma lucidum mycelium, respectively, and mixed at a concentration of 5 15 mg / ml to react within about 30 to 5 hours. desirable. As the osmotic stabilizer, the stability is high at the quantum of 0.6M sudang, and the pH is about 3 7 is suitable, especially 4 5 is optimal. On the other hand, shiitake mushrooms were cultured for 7 to 8 days, and for Ganoderma lucidum for 3.5 days, mycelia were the highest in protoplast formation.

[Protoplast Fusion]

Protoplasts of trophic mutant strains of shiitake mushrooms and ganoderma lucidum were adjusted to 1 106-8 protoplast / ml, respectively, mixed at the same ratio and centrifuged to remove supernatant. A PEG solution is added to the precipitated mixed protoplasts and reacted for 30 to 10 15 minutes with shaking, followed by the addition of an osmotic stabilizer and centrifugation to remove the PEG solution.

When PEG is used to induce protoplast fusion, the molecular weight and concentration of PEG, calcium ion concentration, etc. are important factors. In basidiomycete, PEG of 25 30% molecular weight 4,000 or 6,000 is preferably used. It is also preferred to add calcium ions.

[Regeneration of protoplasts]

Osmotic stabilizer is added to the fused protoplasts, diluted to an appropriate ratio, and then inoculated in a regeneration medium and incubated for 30 to 20 30 days.

Cell wall regeneration of protoplasts is a uniform phenomenon that occurs in all protoplasts, and the success or failure of protoplast fusion depends on the degree of protoplast regeneration. Factors influencing the regeneration of mycelia include the type of osmotic stabilizer, the method of application on the medium and the dry state of the medium. Osmotic stabilizers used for protoplast regeneration are often the same as those used for protoplast production. In the present invention, it is preferable that the soft agar added with 0.6 M sucrose as an osmotic stabilizer is mixed with the protoplasts and cultured.

The following examples illustrate the preparation of the protoplast fusions of the invention in more detail.

EXAMPLE

Strains used in the examples of the present invention are shiitake mushrooms and ganoderma lucidum mushroom, the medium (a) composition for shiitake mushrooms is MgSO 4, 7H 2 O 0.5g, KH 2 PO 4 0.46g, K 2 HPO 1.0g, mineral solution 10ml, peptone 2.0g, yeast Extract 2.0g, glucose 50g, agar 20g, the composition of the Ganoderma lucidum mushroom medium (a) is 5.0g of yeast extract and glucose 20g, the complete medium is a medium (a) to remove the mineral solution and peptone and Each glucose is 5.0g.

[Selection of Nutritional Components by Mutation]

Take the mycelium of shiitake mushrooms and ganoderma lucidum from the medium, suspend them in sterile distilled water, and dispense 10ml into sterilized Petri dishes and irradiate ultraviolet rays for a certain time while gently shaking with a magnetic stirrer (lamp: 39erg / sec, cm2, 10cm )do. After UV treatment, in order to prevent reactivation by light, it is left in the cow for more than 10 minutes, diluted to the concentration of 10-1, 10-2, 10-3, smeared in a complete solid medium and incubated for 30 to 3 10 days. The mycelia growing on the medium are 12 strains each to complete and medium plates for 5 to 7 days, and then the mycelia growing on the complete medium but not on the minimum medium are selected as nutrient-forming mutants. Paraaminobenzoic acid-uremic mutants, and Ganoderma lucidum, select hypoxanthine-urea mutant strains.

[Production of protoplasts]

7 Addition of Novozyme 234 and Cellulase Onozuka al-10 at a concentration of 15 and 10 mg / ml to 0.6M Sudang solution added to the mycelia incubated for 8 days was performed at 30 to 4 hours with shaking at about 100 rpm. React for a while. After the reaction, the protoplasts were separated using a glass filter, 0.6M sucrose solution was added, centrifuged at 1000 rpm for 5 minutes to remove the enzyme solution, washed twice more, and then suspended in 0.6M sucrose solution. In the case of GL, protoplasts were generated by treating Novozyme 234 and cellulase Onozuka al-10 at concentrations of 10 mg / ml for about 3 hours in GL1 mycelia cultured for 3.5 days.

[Protoplast Fusion]

The separated protoplasts of LE4 and GL1 were adjusted to 1 106-8 protoplast / ml, respectively, and mixed at the same ratio, followed by centrifugation at 1000 rpm for 15 minutes to remove the supernatant. 1 ml of pre-warmed 30% PEG 4000 solution (10 mM CaCl 2, 2H 2 O, 50 mM glycine, pH 8.0) was added to the precipitated mixed protoplasts and reacted for 30 to 10 15 minutes with shaking. After the reaction, 5 ml of 0.6 M sucrose solution was added and centrifuged to remove the PEG solution.

0.6M Sudang solution was added to the fused protoplasts, diluted at a rate of 10-1, 10-2, and 10-3, and then inoculated in a regeneration medium and incubated for 25 to 30 60 days. Convergence rate is measured by comparing the number of cells grown on the complete medium with the number of cells grown by complementary phenomena on the minimum medium.

According to the method as described above, in the present invention, between the nutritive mutant strain LE4 of shiitake mushroom (wrinkle fungus) and the nutritive mutant strain GL1 of Ganoderma lucidum (Pleurotus eryngii), which have different necks and show incompatibility between hyphae Several complementary autotrophic fusions were prepared. These fusions are named L4G1P2S1, L4G1P22, L4G1P92, etc., respectively (-S1, -S2, etc. mean different sectors naturally separated in one fusion), and are related to the other object of the present invention. In particular, as described below, the fusion L4G1P22, which has a higher anticancer effect than the parent strain, has been deposited with the Korean spawn association as accession number KFCC-10825 dated June 16, 1994, P-54, abbreviated as P-54 below. do.

These woody fusions are able to distinguish military growth visually when incubated for 30 to 60 days. The regeneration time of the intracellular fusion is 10 20 days and that of the intercellular fusion is 20 30 days. Able to know. In addition, the frequency of fusion was 0.0067%, which was much lower than that of species and genus fusion.

Protoplast fusions of the present invention are novel strains that differ in form and growth, biochemical and pharmacological activity from the parent strain.

Morphological Aspects of Fusion

LE4 is pure white with many aerobic hyphae and does not form pigments, and there is clamp connection at the time of microscopic observation, while GL1 also has pure white mycelium and no pigments at the beginning of cultivation, but forms reddish brown pigments on a completely flat medium when cultured for 7 days or more. And a clamp connection is observed. The difference in the shape of the parent strain was used for fusion selection by visual observation and microscopic observation.

Protoplast fusions were classified as having one or more different forms, and most of them had one or more forms. The results of morphology, degree of growth of each fusion, section formation, ring formation and clamp connection are shown in (Table 1), and the mycelial growth rate is shown in (Table 2). The growth rate of P54 was faster than that of any parent strain.

[Biochemical Properties of Fusions]

The biochemical properties of the fusions in the present invention are evaluated by examining the isotope electrophoresis patterns of esterase and acid phosphatase. That is, after culturing the parent strain and the fusions used in the fusion in a liquid medium for 14 days and 30 days, only the mycelia were taken and non-modified polyamide gel (T = 10%, C = 5%) was used for intracellular esterase and acid phosphatase. Perform electrophoresis on).

a) L: L. edodes, G: G. lucidum, not N: L and G

b) ++++: maximum yield display

c) F: fast growth, M: moderate growth, S: slow growth

d) +: Brown cow production

c) +: Clamp present,-: No clamp. *: Not detected

Figures 1 (a), 1 (b) and 1 (c) show the electrophoretic patterns of shitake mushroom mutants (LE4), ganoderma lucidum mutants (GL1), and esterase (EST) isoenzymes of their protoplast fusions Drawing. Here, the fusions can be divided into those similar to those of one parent and exhibiting a new aspect, and the characteristics of both mothers could not be observed. In the case of fusions between strains that are not flexible, it is possible that the cells may become unstable due to coexistence of different substances and cytoplasm, increase of nucleus, and chromosomal abnormalities. It can be seen that they are separated from the parent or rearranged in a wide range of genes, and thus have new properties different from those of the parent.

Figure 2 shows the electrophoretic pattern of acid phosphatase (ACP) isozymes of shiitake mutant strain (LE4), ganoderma lucidum mutant strain (GL1), and their protoplast fusions. Here, it can be seen that L4G1P2S1 of the three isolates of the fusion L4G1P2 had almost the same composition as GL1, but P2S2 and P2S3 were completely different from the two parent strains. In other words, the fusion was found to change the function of enzymes and organelles involved in cell wall synthesis during the fusion and recombination process, it can be estimated that there was a change in cell wall skeleton and robustness.

[Characteristics of Fusion Protein Polysaccharide]

Distilled water was added to the silica sand obtained by filtering the cultures of the parent strains and fusions, hot water extracted, concentrated and precipitated with ethanol. The precipitate was dialyzed in distilled water, and the supernatant obtained by centrifugation was concentrated and lyophilized to obtain a sample.

Monosaccharide Analysis

The sample is hydrolyzed with 3% hydrochloric acid-methanol and analyzed for monosaccharides by gas chromatography.

3 (a) and 3 (b) are gas chromatographic patterns showing monosaccharides constituting shiitake mushroom strain (LE4), ganoderma lucidum strain (GL1), and hydrothermal extracting protein polysaccharides of their protoplast fusions. The content% of each monosaccharide was calculated from the peak area of the gas chromatography on the basis of 8%, and is shown in the following Table 3. Here, LE4 and GL1 show differences in the content of xylose and arabinose. The fusions L4G1P2S1 and S3 are similar to GL1, but S2 is different from the two parent strains, such as the absence of arabinose. Particularly, L4G1P6, 22, and 38 had a high amount of mannose, but 49 were very small, and 92 had no xylose and mannose.

[Protein analysis]

The protein content of the sample was measured according to the Bradford method, and the analysis results of the proteins constituting the hydrothermal extract protein polysaccharide of shiitake mutant strain (LE4), ganoderma lucidum mutant strain (GL1), and their protoplast fusions are shown in Table 4 below. Shown in Here, the amount of protein is 64.0 g / ml for LE4 and 54.36 g / ml for GL1, where the fusion L4G1P18 is 2.2 2.6-fold for both parent strains, and 49, 55, 82 and 92 are 1.5 for the parent strain. It was about 1.9 times and 61 cases were not contained at all.

[Anticancer effect experiment]

Solid carcinoma was induced by subcutaneous transplantation of 180 cells of mouse carcoma into the left groin of 5 weeks-old ICR mice (male, 20-25 g). From day 3 after cancer cell transplantation, the feed dissolved in physiological saline was administered intraperitoneally once daily for 10 days for 20 mg / kg, and the saline in the control group and 20 mg / kg of crestin in the positive control group were administered daily. On the 28th day after the cancer cell transplantation, the experimental animals were killed and solid tumors were extracted to obtain the average tumor weight. The percentage of tumor inhibition (I.R.%) was calculated according to the following formula and is shown in (Table 5).

Here, Cw and Tw mean the average tumor weight of the control group and the sample administration group, respectively.

In Table 5, the tumor inhibition percentages of the parent strains LE4 and GL1 were 62.4% and 51.3%, respectively, higher than those of the positive control group, 41.4%. Among the fusion strains, P54 was 71.7%, which showed a significantly increased anticancer activity in the confidence range of 99.9% compared to the parent strain.

[Method of Treating Restriction Enzyme of Mitochondrial DNA]

Total DNA was isolated from the cultured mycelia of the parent strain and fusion, purified by ultracentrifugation in CsCl-EtBr, followed by ultracentrifugation in CsCl-bisbenzimid to separate the mitochondrial DNA. EcoR, Hind, Pst ( KOSKO) was treated with -Hind (KOSKO) as a label and electrophoresed to compare the patterns.

Figures 4 (a), 4 (b) and 4 (c) show the mitochondrial DNA of shiitake mutant strain (LE4), ganoderma lucidum mutant strain (GL1), and their protoplast fusions with restriction enzymes EcoR, Hind and Pst. An electrophoretic pattern is shown. Here, in shiitake mushroom strains, there were about 6 mitochondrial DNA fragments cut by EcoR and about 4 pieces cut by Pst, but about 14 were cut by Hind. Ganoderma lucidum strains had about 12 mitochondrial DNA fragments cut by EcoR, 22 by Hind, and 4 by Pst. Among the fusion strains, L4G1P2S2 formed only one band between the first and second bands of -Hind, and was not cleaved by the restriction enzymes EcoR, Hind and Pst. Thus, mitochondrial DNA of shiitake mushrooms and ganoderma lucidum was recombined to produce new mitochondria. It was found. In addition, the fusion L4G1P2S3 showed the same size and location of Ganoderma lucidum mushrooms when electrophoresed with restriction enzymes. Therefore, mitochondria of shiitake mushrooms were presumed to be eliminated because mitochondria of Ganoderma lucidum were predominant in the isolation process after cytoplasmic fusion. .

As described above, the fusion obtained by protoplast fusion between shiitake mushrooms and Ganoderma lucidum mushrooms with different necks and incompatibility between hyphae was able to confirm that the exchange of genetic material occurred. Excellent strains with pharmacological activity can be obtained.

Claims (1)

Protoplast fusion of shiitake mushrooms and ganoderma lucidum mushroom, protoplast fusion P54 having an anticancer effect (Accession No. KFCC-10825).