CN111304091A - Method for improving ganoderic acid content in ganoderma lucidum cell culture - Google Patents
Method for improving ganoderic acid content in ganoderma lucidum cell culture Download PDFInfo
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Abstract
The invention discloses a method for improving the content of ganoderic acid in ganoderma cell culture; the method comprises the steps of performing protoplast mononucleation on binucleated ganoderma lucidum cells, and performing two-stage culture fermentation by using the mononuclear cells as initial strains to achieve the purpose of improving the contents of monomer ganoderic acid and total ganoderic acid in the cells; compared with a binuclear strain, the method can respectively improve the contents of the monomer ganoderic acid GA-Mk, the monomer ganoderic acid GA-T, GA-Me and the total ganoderic acid in the ganoderma cells by 2.4 to 4 times, 2 to 2.6 times, 1.7 to 2.2 times and 1.5 times, and has the advantages of simple method, easy operation and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for increasing ganoderic acid through mononuclear of ganoderma lucidum cells and two-stage culture.
Background
Ganoderma lucidum, also called Mesona chinensis Benth, is a traditional Chinese medicinal fungus, which is a higher fungus of the genus Ganoderma of the class Hymenomycetes, order Polyporales, family Polyporaceae, the kingdom fungi, Basidiomycotina. Ganoderma has anticancer, blood sugar regulating, blood pressure regulating, liver protecting, and immunity enhancing effects. The tetracyclic triterpene compound ganoderic acid is one of main active ingredients of Ganoderma, and has pharmacological activities of resisting HIV, resisting cancer and resisting oxidation. Studies have shown that different monomeric ganoderic acids have different biological activities, such as: ganoderic acid Mk can inhibit proliferation of HeLa cells and induce apoptosis through the mitochondrial pathway (Liu R M, et al. phytomedine, 2011; 18, 349-211; Tang W, et al. Life Sciences, 2006; 80, 205-211). Ganoderic acid T has the activity of inducing apoptosis of lung cancer cells (Tang W, et al, Life Sciences, 2006; 80, 205-211). While ganoderic acid Me has the activity of inhibiting the metastasis of lung cancer cells (Chen N H, et al. Journal of pharmacological sciences, 2008; 108, 212- & 216 Wang, et al. International Immunopharmacology,2007; 7, 864- & 870). The search shows that the methods for improving the content of the ganoderic acid in the cells are mainly divided into the following categories: firstly, the development of fermentation strategy, for example, the chinese invention application CN1375557A discloses the following: the method for producing the ganoderic acid by two-stage culture of the ganoderic cells in the bioreactor can efficiently produce the ganoderic acid, and the content of the total ganoderic acid reaches 5mg/100mg of dry cell weight; secondly, an inducer is added in the process of culturing, for example, Chinese invention patent CN101692772A discloses a method for increasing the content of ganoderic acid by adding phenobarbital or miconazole in two-stage culturing of ganoderma lucidum cells, the method respectively increases the content of monomer ganoderic acid by 32-55% and 10-28%, and the content of total ganoderic acid is increased by 45% and 15% compared with the reference. Chinese invention patent CN105483197B discloses a method for increasing the content of triterpenes in ganoderma lucidum liquid fermentation by adding calcium ions and salicylic acid, the method can significantly increase the content of triterpenes in ganoderma lucidum mycelia, the content of triterpenes in mycelia reaches 4.89mg/100mg of cell dry weight, which is 48.26% higher than that in a control group. Thirdly, the strains are transformed by genetic engineering technology, for example, Chinese invention patent CN105567578A discloses a method for increasing the content of ganoderic acid in ganoderma lucidum cells by over-expressing SE gene, so that the content of monomer ganoderic acid in SE transformed strains is increased by 20-150% compared with the control. Chinese invention patent CN105296367B discloses a method for improving ganoderic acid content by heterogeneously expressing VGB gene, wherein the content of monomer ganoderic acid of VGB transformed strain is 2.1-3.6 times of that of contrast. Cell mononucleosis is mainly applied to the cross breeding aspect of ganoderma lucidum at present; however, no report has been made on a method for increasing the content of ganoderic acid in a two-stage culture by mononuclear formation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for improving the content of ganoderic acid in two-stage culture through the mononuclear of ganoderma cells.
The method of the invention is that the binuclear ganoderma lucidum cells are subjected to protoplast mononucleation, the mononucleated cells are used as initial strains, and two-stage culture fermentation is carried out at 25-30 ℃, so as to realize the purpose of improving the content of ganoderic acid; through the implementation of the method, the content of the monomer ganoderic acid and the content of the total ganoderic acid in the ganoderma lucidum are both obviously improved, and compared with binuclear ganoderma lucidum cells, the content of the monomer ganoderic acid GA-Mk and the content of the monomer ganoderic acid GA-T, GA-Me and the content of the total ganoderic acid of the mononuclear ganoderma lucidum cells are respectively improved by 2.4 to 4 times, 2 to 2.6 times, 1.7 to 2.2 times and more than 1.5 times.
The method comprises the following specific operation steps:
preparation of primary and secondary ganoderma lucidum cell seed liquid
1. Preparing a potato agar culture medium
2. Activating ganoderma lucidum cells: taking two 1cm Ganoderma lucidum karyon strain inclined planes with an inoculating shovel3The fungus block is placed on a slope of a newly prepared potato agar culture medium and cultured for 5 to 7 days for later use.
3. Preparing liquid seed culture medium
Inoculating cells growing on the inclined plane of a potato agar culture medium into a liquid seed culture medium containing glass beads with the diameter of 5mm by using an inoculation shovel, and performing static culture at the temperature of 25-30 ℃ for 4-5 days to obtain a primary seed culture solution; inoculating the primary seed culture solution into a liquid seed culture medium containing glass beads with the diameter of 5mm, and performing static culture at 25-30 ℃ for 2-3 days to obtain a secondary seed solution.
Secondly, preparation and regeneration of protoplast
1. Preparing citric acid buffer solution
2. Centrifuging at 12000rpm to collect secondary seed liquid cells, washing the collected cells twice with a citric acid buffer solution, and weighing;
3. weighing the muramidase according to the weight of the cells, weighing 0.04g of the muramidase per 200-300mg (wet weight) of the cells, adding a citric acid buffer solution into the weighed muramidase, fixing the volume, filtering by using a sterile 0.22 mu m filter membrane, adding into a centrifuge tube filled with the cells, and mixing uniformly by flicking;
4. placing the uniformly mixed cells in a shaking table with the temperature of 30 ℃ and the rpm of 100, performing enzymolysis for 150min, and reversely shaking once every 30 min;
5. centrifuging the cells subjected to enzymolysis for 5min at 1700g, removing the supernatant, and then washing the residues twice by using a citric acid buffer solution; after the washing is finished, adding a proper amount of citric acid buffer solution, flicking and uniformly mixing, and then filtering by using a 520-mesh nylon net to remove broken hyphae;
6. sucking 100 mu L of the filtrate obtained in the step 5, placing the filtrate on a blood counting plate, observing the filtrate under a microscope, and calculating the number of protoplasts;
7. preparation of protoplast regeneration Medium (CYM)
8. Take 1X 104-1×105The protoplasts were added to 15mL of CYM medium, and the medium was poured into a dish having a diameter of 9cm and cultured at 25-30 ℃ for 3-5 days.
Three, single nucleus identification
Taking out the plate from an incubator at 25-30 ℃, selecting a single colony to be inoculated on a fresh potato agar culture medium inclined plane, taking out a single colony from the inclined plane to be placed on a culture dish poured with the potato agar culture medium after the single colony grows for 4-5 days, obliquely inserting a glass sheet at a position 1cm away from the bacterial block (after sterilization), and placing the culture dish in the incubator at 25-30 ℃ for culturing for 4-6 days.
1. Identification of 4', 6-diamidino-2-phenylindole (DAPI) by nuclear staining
The obtained glass slide with hyphae was stained in 0.1. mu.g/mL DAPI solution for 5min, then the glass slide was removed with forceps, rinsed in 0.01mol/L Phosphate Buffered Saline (PBS) at pH 7 for 5min, and observed under an inverted fluorescence microscope.
2. SNP site identification
Scraping the obtained hyphae with an inoculating spatula, extracting genome DNA by a Cetyl Trimethyl Ammonium Bromide (CTAB) method,
using genomic DNA as a template and using a primer MIP-F: ATGACACGGACCTCATAGCCT;
MIP-R: ATGTCGCTCAGCCTGTCCAA, performing PCR amplification; the PCR product was then recovered and sequenced.
Four, single and double core glossy ganoderma two stage cultivation
1. Preparing fermentation medium
2. Respectively inoculating Ganoderma identified as mononuclear and binuclear Ganoderma to potato culture medium slant, and culturing at 25-30 deg.C for 5-7 days;
3. inoculating cells growing on the inclined plane of a potato culture medium into a liquid seed culture medium by using an inoculation shovel, and performing shake culture at the temperature of 25-30 ℃ for 4-5 days to obtain a fermentation primary seed culture solution;
4. inoculating the primary seed culture solution into a liquid seed culture medium, and performing shake culture at 25-30 deg.C for 2-3 days to obtain a secondary seed solution;
5. respectively inoculating the mononuclear and binuclear secondary seed liquid into a fermentation culture medium, wherein the inoculation amount is that 5mL of secondary seed culture liquid is inoculated into every 50mL of fermentation culture medium, and performing two-stage culture; the two-stage culture comprises inoculating mononuclear Ganoderma cell into liquid seed culture medium, culturing to obtain seed solution, inoculating the seed solution into liquid fermentation culture medium, performing shake culture at 25-30 deg.C for 2-4 days, and continuously standing the fermentation broth at 25-30 deg.C for 8-10 days.
And fifthly, extracting the ganoderic acid and measuring the content of the ganoderic acid.
The ganoderic acid was extracted from the fermentation product and the content of monomeric ganoderic acid was determined using conventional methods (methods were referred to Xu JW, et al. Applied Microbiology and Biotechnology, 2010: 85, 941-.
Binuclear Ganoderma strain (I) of the present inventionGanoderma lucidum) Is CGMCC5.26, CGMCC5.616 and CGMCC5.542, and the strains are purchased from the common microorganism center (CGMCC) of the China Committee for culture Collection of microorganisms.
The invention has the advantages and technical effects that:
1. the method is utilized to carry out mononucleation on the ganoderma lucidum strain, then the strains subjected to the mononucleation are used for two-stage culture, so that the content of monomer ganoderic acid and the content of total ganoderic acid in ganoderma lucidum cells can be obviously improved, and compared with binuclear ganoderma lucidum, the method can respectively improve the content of monomer ganoderic acid GA-Mk, GA-T, GA-Me and the content of total ganoderic acid in the cells by 2.4 to 4 times, 2 to 2.6 times, 1.7 to 2.2 times and 1.5 times;
2. the method is simple, easy to operate and suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a morphological diagram of a binuclear hyphal cell under a fluorescence microscope;
FIG. 2 is a schematic view showing DAPI staining results of binuclear hyphal cells;
FIG. 3 shows the sequencing results of SNP sites of binuclear hyphal cells;
FIG. 4 is a morphological diagram of a mononuclear hyphal cell under a fluorescence microscope;
FIG. 5 is a DAPI staining pattern of a mononuclear hyphal cell;
FIG. 6 shows the sequencing results of SNP sites of monocytic hyphal cells.
Detailed Description
The present invention is further illustrated in detail by the following examples, but the content of the present invention is not limited thereto, the method in the examples is performed by the conventional method unless otherwise specified, the reagents used are the conventional reagents or the reagents prepared by the conventional method unless otherwise specified, and the strains to which the method of the present invention is applied are not limited to the following three strains of Ganoderma lucidum.
Example 1
Preparation of primary and secondary ganoderma lucidum cell seed liquid
1. Preparing a potato agar culture medium
The formula of the potato agar culture medium comprises the following components: boiling 200g peeled potato in 1L water for 30min, filtering to obtain filtrate, glucose 10g, anhydrous magnesium sulfate 1.5g, potassium dihydrogen phosphate 3g, and vitamin B10.05g of agar and 20g of agar, and the volume is adjusted to 1L by water, and the pH value is 5.5.
2. Activating ganoderma lucidum cells: respectively inoculating with double core strains of Ganoderma (Ganoderma lucidum karst)Ganoderma lucidum) Taking out two 1cm pieces of original culture medium inclined planes of CGMCC5.26, CGMCC5.616 and CGMCC5.5423Placing the fungus blocks on a newly prepared potato agar culture medium inclined plane, and culturing for 5 days at 30 ℃ for later use;
3. preparing liquid seed culture medium
The formula of the seed culture medium is as follows: 35g of glucose, 5g of peptone, 2.5g of yeast powder, 0.5g of anhydrous magnesium sulfate, 1g of monopotassium phosphate and vitamin B10.05g, and the volume is determined by water1L, pH 5.5.
Inoculating cells growing on the inclined plane of a potato agar culture medium into a liquid seed culture medium containing glass beads with the diameter of 5mm by using an inoculation shovel, and standing and culturing for 4 days at the temperature of 30 ℃ to obtain a primary seed culture solution; inoculating the primary seed culture solution into a liquid seed culture medium containing glass beads with the diameter of 5mm, and performing static culture at 30 ℃ for 3 days to obtain a secondary seed solution.
Second, protoplast preparation and regeneration
1. Preparing citric acid buffer solution
The formula of the citric acid buffer solution is as follows: (1) preparing a citric acid solution: weighing 21.91g of mannitol and 4.2g of citric acid, and metering to 200 mL; (2) preparing a sodium citrate solution: weighing 54.78g of mannitol and 14.7g of sodium citrate, and metering to 500 mL; (3) the citric acid solution was slowly poured into the sodium citrate solution and the addition was stopped until the pH was 5.5.
2. Centrifuging at 12000rpm to collect secondary seed liquid cells, washing the collected cells twice with a citric acid buffer solution, and weighing;
3. taking 200mg (wet weight) of each cell, adding a citric acid buffer solution into 0.04g of muramidase, fixing the volume to 1.7mL, filtering by using a sterile 0.22 mu m filter head, adding into a centrifuge tube filled with the cells, and gently and uniformly mixing;
4. placing the uniformly mixed cells in a shaking table with the temperature of 30 ℃ and the rpm of 100, performing enzymolysis for 150min, and reversely shaking once every 30 min;
5. centrifuging the cells subjected to enzymolysis for 5min at 1700g, removing the supernatant, and then washing the residues twice by using a citric acid buffer solution; adding citric acid buffer solution after cleaning, gently flicking and uniformly mixing, and then filtering by using a 520-mesh nylon net to remove broken mycelia;
6. sucking 100 mu L of the filtrate obtained in the step 5, placing the filtrate on a blood counting plate, observing the filtrate under a microscope, and calculating the number of protoplasts;
7. preparation of protoplast regeneration Medium (CYM)
Protoplast regeneration medium (CYM) formulation: 20g of glucose, 2g of peptone, 2g of yeast powder, 0.5g of anhydrous magnesium sulfate, 2.3g of monopotassium phosphate and maltose1g of mannitol, 109.56g of mannitol and 10g of low-melting-point agarose, and adding water to a constant volume of 1L; 8. take 1X 105The protoplasts were added to 15mL of CYM, and the medium was poured into a dish having a diameter of 9cm and cultured in an incubator at 30 ℃ for 4 days.
Three, single nucleus identification
The plate was removed from the 30 ℃ incubator, a single colony was picked and inoculated onto a fresh potato agar medium slant, and after 4 days of growth, a piece was removed from the slant and placed on a petri dish with the potato agar medium poured over, and a glass slide (sterilized) was inserted obliquely at 1cm from the block, and the dish was placed at 30 ℃ for 5 days of culture.
1. DAPI nuclear staining identification
Staining the glass sheet with hyphae in 0.1 μ g/mL DAPI solution for 5min, then clamping the glass sheet with forceps, rinsing in 0.01mol/L PBS buffer solution with pH of 7 for 5min, and immediately observing under an inverted fluorescence microscope (FIGS. 1, 2, 4, 5); it can be seen from the figure that when DAPI stained cells are excited by ultraviolet light with wavelength of 358 nm, the cell nucleus emits blue light with wavelength of 461 nm; after the binuclear strain is dyed, two blue-light-emitting cell nucleuses appear on one hypha, and only one hypha of the mononuclear cell exists.
2. SNP site identification
Scraping the hyphae obtained in the last step by using an inoculating shovel, extracting genome DNA by using a CTAB method, and then using a primer MIP-F: ATGACACGGACCTCATAGCCT; MIP-R: ATGTCGCTCAGCCTGTCCAA PCR amplification was carried out using 25. mu.L of 2 XTaq PCR Mastermix II, 2. mu.L of MIP-F, 2. mu.L of MIP-R, 1. mu.L of DNA, 20. mu.L of ddH2O；
The PCR amplification conditions were: (1) 94 ℃ for 5 min; (2) 30S at 94 ℃; (3) at 55 ℃, 30S; (4) 72 ℃, 30S; (5) 72 ℃ for 5 min; (6) at 4 ℃ for 30 min; (2) circulating for 30 times until (4), then recovering PCR products and sequencing; the results are shown in FIGS. 3 and 6; several overlapping peaks and double peaks appear at several sites on the sequence peak diagram of the binuclear strain, and the arrow points to the position in FIG. 3, while the corresponding position of the sequence peak diagram of the mononuclear strain has only a single peak (FIG. 6).
After three kinds of binuclear strains of the lucid ganoderma, namely CGMCC5.26, CGMCC5.616 and CGMCC5.542, are subjected to mononucleation, the mononuclear strains, namely CGMCC5.26-1, CGMCC5.616-1 and CGMCC5.542-1, corresponding to the three strains are obtained respectively.
Example 2: two-stage culture of monokaryon and binuclear Ganoderma
1. Preparing fermentation medium
The formula of the fermentation medium is as follows: 35g of lactose, 5g of peptone, 5g of yeast powder, 0.5g of anhydrous magnesium sulfate, 1g of monopotassium phosphate and vitamin B10.05g, and the volume is adjusted to 1L by water, and the pH value is 6.0;
2. respectively inoculating the binuclear strain identified as CGMCC5.26 and the mononuclear strain identified as CGMCC5.26-1 on the slant of a potato agar culture medium, and culturing at 30 ℃ for 5 days;
3. inoculating cells growing on the inclined plane of the potato culture medium into a liquid seed culture medium by using an inoculation shovel, and performing shake culture at the temperature of 30 ℃ for 5 days to obtain a fermentation primary seed culture solution;
4. inoculating the primary seed culture solution into a liquid seed culture medium, and performing shake culture at 30 ℃ for 2 days to obtain a secondary seed solution;
5. respectively inoculating the secondary seed liquid of the mononuclear bacterial strain and the secondary seed liquid of the binuclear bacterial strain into a fermentation culture medium, wherein the inoculation amount is 5mL of secondary seed culture liquid inoculated into every 50mL of fermentation culture medium, and performing shake culture at 30 ℃ for 2 days;
6. pouring the fermentation culture solution into culture dishes with the diameter of 9cm respectively, and standing and culturing for 9 days at the temperature of 30 ℃;
7. extraction and analysis of Total Ganoderic acid
Centrifuging the fermentation liquor obtained in the step 6 to collect cells, drying for 5 days at 37 ℃, weighing and grinding, weighing 25mg of ground dry cell powder, adding 0.5mL of ethanol solution with volume concentration of 70% to soak overnight, carrying out ultrasonic treatment at 4 ℃ for 1.5h, centrifuging for 5min at 10000rpm, sucking supernatant, carrying out vacuum drying at 40 ℃ for two days, adding 250 muL of water-dispersed vortex, adding 250 muL of chloroform to extract the dispersed water phase twice, collecting and combining chloroform phases, totaling 0.5mL, mixing 0.5mL of sodium bicarbonate solution with mass concentration of 5% and chloroform phase, carrying out vortex three times, collecting and combining the upper water phase, totaling 1.5mL, adjusting the pH of the water phase to be less than 3.0 by using 2mol/L of hydrochloric acid, then extracting twice by using chloroform with the same volume as the water phase, collecting and combining the chloroform phases, carrying out drying at room temperature, dissolving by using 500 muL of absolute ethyl alcohol, and measuring the.
8. Extraction analysis of monomeric ganoderic acid
Centrifuging the fermentation liquid obtained in the step 6 to collect cells, drying for 5 days at 37 ℃, weighing and grinding, weighing 100mg of ground dry cell powder, adding 2mL of ethanol solution with volume concentration of 70% to soak overnight, performing ultrasound at 4 ℃ for 2.5 h, centrifuging at 12000rpm for 5min, sucking supernatant, performing vacuum drying at 40 ℃, adding 200 mu L of methanol, performing vortex, filtering with a 0.22 mu m filter membrane, and analyzing the content of three monomers, namely ganoderic acid by high performance liquid chromatography.
As shown in Table 1, the cell dry weight of the binuclear strain was 5.882. + -. 0.02g/L, and the cell dry weights of the monomeric ganoderic acids GA-Mk, GA-T, GA-Me and total ganoderic acids were 19.63. + -. 0.194. mu.g/100 mg, 62.881. + -. 1.963. mu.g/100 mg, 5.682. + -. 0.252. mu.g/100 mg and 1.299. + -. 0.043mg/100mg, respectively.
The cell dry weight of the monocyte strain is 12.155 + -0.072 g/L, and the cell dry weights of the monomeric ganoderic acids GA-Mk, GA-T, GA-Me and total ganoderic acids are 84.214 + -6.404 μ g/100mg, 163.936 + -7.638 μ g/100mg, 12.235 + -1.751 μ g/100mg and 1.991 + -0.029 mg/100mg, respectively.
Compared with a binuclear strain, the cell dry weight, the monomer ganoderic acid and the total ganoderic acid of the mononuclear strain are obviously improved, the cell dry weight of the mononuclear strain is 2.3 times that of the binuclear strain, the contents of the monomer ganoderic acid GA-Mk and GA-T, GA-Me of the mononuclear strain are 4.3, 2.6 and 2.2 times that of the binuclear strain respectively, and the total ganoderic acid content of the mononuclear strain is 1.5 times that of the binuclear strain.
TABLE 1
Example 3: two-stage culture of monokaryon and binuclear Ganoderma
1. Preparing fermentation medium
The formula of the fermentation medium is as follows: 35g of lactose, 5g of peptone, 5g of yeast powder and 0.5g of anhydrous magnesium sulfateg. 1g of monopotassium phosphate and vitamin B10.05g, and the volume is adjusted to 1L by water, and the pH value is 5.5;
2. respectively inoculating the binuclear strain identified as CGMCC5.616 and the mononuclear strain identified as CGMCC5.616-1 on the slant of a potato agar culture medium, and culturing for 5 days at 28 ℃;
3. inoculating cells growing on the inclined plane of the potato culture medium into a liquid seed culture medium by using an inoculation shovel, and performing shake culture at 28 ℃ for 5 days to obtain a fermentation primary seed culture solution;
4. inoculating the primary seed culture solution into a liquid seed culture medium, and performing shake culture at 28 ℃ for 2 days to obtain a secondary seed solution;
5. respectively inoculating the secondary seed liquid of the mononuclear bacterial strain and the secondary seed liquid of the binuclear bacterial strain into a fermentation culture medium, wherein the inoculation amount is 5mL of secondary seed culture liquid inoculated into every 50mL of fermentation culture medium, and performing shake culture at 28 ℃ for 3 days;
6. pouring the fermentation culture solution into culture dishes with the diameter of 9cm respectively, and standing and culturing for 8 days at 28 ℃;
7. extraction and analysis of Total Ganoderic acid
Centrifuging the fermentation liquor obtained in the step 6 to collect cells, drying for 5 days at 37 ℃, weighing and grinding, weighing 25mg of ground dry cell powder, adding 0.5mL of ethanol solution with volume concentration of 70% to soak overnight, carrying out ultrasonic treatment at 4 ℃ for 1.5h, centrifuging at 10000rpm for 5min, sucking supernatant, carrying out vacuum drying at 40 ℃ for two days, adding 250 muL of water dispersion vortex, adding 250 muL of chloroform to extract the dispersed water phase twice, collecting and combining chloroform phases, totaling 0.5mL, mixing 0.5mL of sodium bicarbonate solution with mass concentration of 5% and chloroform phase, carrying out vortex three times, collecting and combining the upper water phase, totaling 1.5mL, adjusting the pH of the water phase to be below 3.0 by using 2mol/L of hydrochloric acid, then extracting twice by using chloroform with the same volume as the water phase, collecting and combining the chloroform phases, drying at room temperature, dissolving by using 500 muL of absolute ethyl alcohol, and measuring the light absorption value at 245.
8. Extraction analysis of monomeric ganoderic acid
Centrifuging the fermentation liquid obtained in the step 6 to collect cells, drying at 37 ℃ for 5 days, weighing and grinding, weighing 100mg of ground dry cell powder, adding 2mL of ethanol solution with volume concentration of 70% for soaking overnight, performing ultrasonic treatment at 4 ℃ for 2.5 h, centrifuging at 12000rpm for 5min, sucking supernatant, performing vacuum drying at 40 ℃, adding 200 mu L of methanol, performing vortex, filtering with a 0.22 mu m filter membrane, and analyzing the content of three monomers, namely ganoderic acid by high performance liquid chromatography.
As shown in Table 2, the cell dry weight of the binuclear strain was 10.451. + -. 0.112 g/L, and the monomeric ganoderic acids GA-Mk, GA-T, GA-Me and total ganoderic acids were 25.451. + -. 0.889. mu.g/100 mg cell dry weight, 131.343. + -. 0.773. mu.g/100 mg cell dry weight, 66.617. + -. 7.355. mu.g/100 mg cell dry weight and 0.562. + -. 0.011mg/100mg cell dry weight, respectively.
The cell dry weight of the monocyte strain is 12.512 + -0.639 g/L, and the cell dry weights of the monomeric ganoderic acid GA-Mk, GA-T, GA-Me and total ganoderic acid are 73.617 + -0.577 μ g/100mg, 292.911 + -2.721 μ g/100mg, 115.151 + -4.327 μ g/100mg and 0.863 + -0.014 mg/100mg, respectively.
Compared with a binuclear strain, the cell dry weight, the monomer ganoderic acid and the total ganoderic acid of the mononuclear strain are obviously improved, the cell dry weight of the mononuclear strain is 1.2 times that of the binuclear strain, the contents of the monomer ganoderic acid GA-Mk and GA-T, GA-Me of the mononuclear strain are 2.9, 2.2 and 1.7 times that of the binuclear strain respectively, and the total ganoderic acid content of the mononuclear strain is 1.5 times that of the binuclear strain.
TABLE 2
Example 4: two-stage culture of monokaryon and binuclear Ganoderma
1. Preparing fermentation medium
The formula of the fermentation medium is as follows: 35g of lactose, 5g of peptone, 5g of yeast powder, 0.5g of anhydrous magnesium sulfate, 1g of monopotassium phosphate and vitamin B10.05g, and the volume is adjusted to 1L by water, and the pH value is 5.8;
2. respectively inoculating the binuclear strain identified as CGMCC5.616 and the mononuclear strain identified as CGMCC5.616-1 on the slant of a potato agar culture medium, and culturing for 5 days at 25 ℃;
3. inoculating cells growing on the inclined plane of the potato culture medium into a liquid seed culture medium by using an inoculation shovel, and performing shake culture at 25 ℃ for 5 days to obtain a fermentation primary seed culture solution;
4. inoculating the primary seed culture solution into a liquid seed culture medium, and performing shake culture at 25 ℃ for 2 days to obtain a secondary seed solution;
5. respectively inoculating the secondary seed liquid of the mononuclear bacterial strain and the secondary seed liquid of the binuclear bacterial strain into a fermentation culture medium, wherein the inoculation amount is 5mL of secondary seed culture liquid inoculated into every 50mL of fermentation culture medium, and performing shake culture at 25 ℃ for 4 days;
6. pouring the fermentation culture solution into culture dishes with the diameter of 9cm respectively, and standing and culturing for 10 days at 25 ℃;
7. extraction and analysis of Total Ganoderic acid
Centrifuging the fermentation liquor obtained in the step 6 to collect cells, drying for 5 days at 37 ℃, weighing and grinding, weighing 25mg of ground dry cell powder, adding 0.5mL of ethanol solution with volume concentration of 70% to soak overnight, carrying out ultrasonic treatment at 4 ℃ for 1.5h, centrifuging at 10000rpm for 5min, sucking supernatant, carrying out vacuum drying at 40 ℃ for two days, adding 250 muL of water dispersion vortex, adding 250 muL of chloroform to extract the dispersed water phase twice, collecting and combining chloroform phases, totaling 0.5mL, mixing 0.5mL of sodium bicarbonate solution with mass concentration of 5% and chloroform phase, carrying out vortex three times, collecting and combining the upper water phase, totaling 1.5mL, adjusting the pH of the water phase to be below 3.0 by using 2mol/L of hydrochloric acid, then extracting twice by using chloroform with the same volume as the water phase, collecting and combining the chloroform phases, drying at room temperature, dissolving by using 500 muL of absolute ethyl alcohol, and measuring the light absorption value at 245.
8. Extraction analysis of monomeric ganoderic acid
Centrifuging the fermentation liquid obtained in the step 6 to collect cells, drying at 37 ℃ for 5 days, weighing and grinding, weighing 100mg of ground dry cell powder, adding 2mL of ethanol solution with volume concentration of 70% for soaking overnight, performing ultrasonic treatment at 4 ℃ for 2.5 h, centrifuging at 12000rpm for 5min, sucking supernatant, performing vacuum drying at 40 ℃, adding 200 mu L of methanol, performing vortex, filtering with a 0.22 mu m filter membrane, and analyzing the content of three monomers, namely ganoderic acid by high performance liquid chromatography.
As shown in Table 3, the cell dry weight of the binuclear strain was 9.311. + -. 0.672g/L, and the cell dry weights of monomeric ganoderic acid GA-Mk, GA-T, GA-Me and total ganoderic acid were 27.068. + -. 1.079. mu.g/100 mg, 95.072. + -. 0.773. mu.g/100 mg, 41.541. + -. 2.167. mu.g/100 mg and 0.557. + -. 0.03mg/100mg, respectively. The cell dry weight of the monocyte strain is 11.778 + -0.268 g/L, and the cell dry weights of the monomeric ganoderic acid GA-Mk, GA-T, GA-Me and total ganoderic acid are 64.496 + -1.215 μ g/100mg, 188.263 + -0.447 μ g/100mg, 73.718 + -1.083 μ g/100mg and 0.813 + -0.015 mg/100mg, respectively.
Compared with a binuclear strain, the cell dry weight, the monomer ganoderic acid and the total ganoderic acid of the mononuclear strain are obviously improved, the cell dry weight of the mononuclear strain is 1.3 times that of the binuclear strain, the contents of the monomer ganoderic acid GA-Mk and GA-T, GA-Me of the mononuclear strain are 2.4, 2.0 and 1.8 times that of the binuclear strain respectively, and the total ganoderic acid content of the mononuclear strain is 1.5 times that of the binuclear strain;
TABLE 3
Sequence listing
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Claims (2)
1. A method for improving the content of ganoderic acid in ganoderma cell culture is characterized in that: the binuclear ganoderma lucidum cells are subjected to protoplast mononucleation, and the mononuclear cells are used as initial strains to be subjected to two-stage culture fermentation, so that the purpose of improving the content of ganoderic acid is achieved.
2. The method according to claim 1, wherein the content of ganoderic acid in the cultured ganoderma lucidum cells is increased by: the two-stage culture and fermentation comprises inoculating mononuclear Ganoderma cell into liquid seed culture medium, culturing to obtain seed solution, inoculating the seed solution into liquid fermentation culture medium, performing shake culture at 25-30 deg.C for 2-4 days, and continuously standing and culturing the fermentation broth at 25-30 deg.C for 8-10 days.
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Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375557A (en) * | 2002-03-29 | 2002-10-23 | 华东理工大学 | Two-stage glossy ganoderma cell culturing process of producing ganoderic acid in bioreactor |
| EP1366078A2 (en) * | 2000-09-26 | 2003-12-03 | Genentech, Inc. | Ige receptor antagonists |
| CN101084934A (en) * | 2007-06-22 | 2007-12-12 | 广州汉方现代中药研究开发有限公司 | Liquid capsule of lucidum spore oil and preparation method |
| CN101352144A (en) * | 2008-09-16 | 2009-01-28 | 福建农林大学 | Cross breeding method of Ganoderma lucidum |
| CN101361764A (en) * | 2008-09-01 | 2009-02-11 | 北京世纪博康医药科技有限公司 | Composition containing brown alga polysaccharide sulfuric ester and glossy ganoderma polysaccharide and use thereof |
| WO2009091842A2 (en) * | 2008-01-14 | 2009-07-23 | Active Organics, Lp | Anti-glycation hydrolysate of g. lucidum |
| CN101559073A (en) * | 2009-04-29 | 2009-10-21 | 吉林大学 | Cordyceps and ganoderma lucidum polysaccharides compound preparation with immune synergism |
| KR101034567B1 (en) * | 2009-11-13 | 2011-05-12 | 이소영 | Drug delivery system and compositions of preventing loss of hair and promoting growth of hair |
| CN103026901A (en) * | 2012-12-28 | 2013-04-10 | 福建农林大学 | Method for germinating ganoderma lucidum (leyss.fr) karst basidiospores |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
| CN103184282A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings |
| CN103828600A (en) * | 2014-02-25 | 2014-06-04 | 福建农林大学 | Breeding method for excellent white agrocybe cylindracea |
| CN104017738A (en) * | 2014-05-13 | 2014-09-03 | 昆明理工大学 | High-yield engineering strain kmust-SQS for ganoderic acid |
| CN105012356A (en) * | 2014-04-30 | 2015-11-04 | 北京冠瑞金生物科技有限公司 | Application of ganoderic acid A to anti-depression |
| CN105296367A (en) * | 2015-10-09 | 2016-02-03 | 昆明理工大学 | Ganoderic acid high-producing engineering strain kmust-VGB-1 |
| CN105567578A (en) * | 2016-01-06 | 2016-05-11 | 昆明理工大学 | Ganoderic acid high yield engineering strain kmust-SE |
| CN105886413A (en) * | 2016-05-23 | 2016-08-24 | 昆明理工大学 | High-yield engineering strain kmust-Lae of ganoderic acid |
| CN106010989A (en) * | 2016-05-23 | 2016-10-12 | 昆明理工大学 | High-ganodenic-acid-yield engineering strain kmust-Crz |
| CN107083337A (en) * | 2017-06-13 | 2017-08-22 | 上海市农业科学院 | The preparation method and protoplast of a kind of edible mushroom protoplast |
| CN109402211A (en) * | 2018-10-16 | 2019-03-01 | 昆明理工大学 | A kind of method of monomer ganoderic acid content in raising glossy ganoderma cell |
| CN110904262A (en) * | 2019-12-16 | 2020-03-24 | 上海市农业科学院 | Method for creating nucleoplasm hybrid of shiitake mushroom cultivation strain by using cytoplasm and nucleus molecular markers |
-
2019
- 2019-11-28 CN CN201911186696.0A patent/CN111304091A/en active Pending
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1366078A2 (en) * | 2000-09-26 | 2003-12-03 | Genentech, Inc. | Ige receptor antagonists |
| CN1375557A (en) * | 2002-03-29 | 2002-10-23 | 华东理工大学 | Two-stage glossy ganoderma cell culturing process of producing ganoderic acid in bioreactor |
| CN101084934A (en) * | 2007-06-22 | 2007-12-12 | 广州汉方现代中药研究开发有限公司 | Liquid capsule of lucidum spore oil and preparation method |
| WO2009091842A2 (en) * | 2008-01-14 | 2009-07-23 | Active Organics, Lp | Anti-glycation hydrolysate of g. lucidum |
| CN101361764A (en) * | 2008-09-01 | 2009-02-11 | 北京世纪博康医药科技有限公司 | Composition containing brown alga polysaccharide sulfuric ester and glossy ganoderma polysaccharide and use thereof |
| CN101352144A (en) * | 2008-09-16 | 2009-01-28 | 福建农林大学 | Cross breeding method of Ganoderma lucidum |
| CN101559073A (en) * | 2009-04-29 | 2009-10-21 | 吉林大学 | Cordyceps and ganoderma lucidum polysaccharides compound preparation with immune synergism |
| KR101034567B1 (en) * | 2009-11-13 | 2011-05-12 | 이소영 | Drug delivery system and compositions of preventing loss of hair and promoting growth of hair |
| CN103026901A (en) * | 2012-12-28 | 2013-04-10 | 福建农林大学 | Method for germinating ganoderma lucidum (leyss.fr) karst basidiospores |
| CN103184282A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
| CN103828600A (en) * | 2014-02-25 | 2014-06-04 | 福建农林大学 | Breeding method for excellent white agrocybe cylindracea |
| CN105012356A (en) * | 2014-04-30 | 2015-11-04 | 北京冠瑞金生物科技有限公司 | Application of ganoderic acid A to anti-depression |
| CN104017738A (en) * | 2014-05-13 | 2014-09-03 | 昆明理工大学 | High-yield engineering strain kmust-SQS for ganoderic acid |
| CN105296367A (en) * | 2015-10-09 | 2016-02-03 | 昆明理工大学 | Ganoderic acid high-producing engineering strain kmust-VGB-1 |
| CN105567578A (en) * | 2016-01-06 | 2016-05-11 | 昆明理工大学 | Ganoderic acid high yield engineering strain kmust-SE |
| CN105886413A (en) * | 2016-05-23 | 2016-08-24 | 昆明理工大学 | High-yield engineering strain kmust-Lae of ganoderic acid |
| CN106010989A (en) * | 2016-05-23 | 2016-10-12 | 昆明理工大学 | High-ganodenic-acid-yield engineering strain kmust-Crz |
| CN107083337A (en) * | 2017-06-13 | 2017-08-22 | 上海市农业科学院 | The preparation method and protoplast of a kind of edible mushroom protoplast |
| CN109402211A (en) * | 2018-10-16 | 2019-03-01 | 昆明理工大学 | A kind of method of monomer ganoderic acid content in raising glossy ganoderma cell |
| CN110904262A (en) * | 2019-12-16 | 2020-03-24 | 上海市农业科学院 | Method for creating nucleoplasm hybrid of shiitake mushroom cultivation strain by using cytoplasm and nucleus molecular markers |
Non-Patent Citations (3)
| Title |
|---|
| ELINE POLAK等: ""Asexual Sporulation in Coprinus cinereus: Structure and Development of Oidiophores and Oidia in an Amut Bmut Homokaryon"", 《FUNGAL GENETICS AND BIOLOGY》 * |
| WEN-XIAN ZHANG等: ""Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum"", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| 丛倩倩等: ""灵芝原生质体单核菌株的制备及形态特性比较"", 《食用菌》 * |
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