CN114854663A - Method for increasing yield of intracellular flavone of Lonicera Appendula - Google Patents
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Abstract
The invention discloses a method for improving the yield of intracellular flavone of Lonicera Appendula and Phellinus igniarius, and belongs to the field of microbial fermentation. The porous honeysuckle phellinus igniarius (Sanghuangpora ionicola) can produce flavonoid compounds through liquid fermentation, and the yield of intracellular flavone is improved by about 0.47-3.27 times and the yield of flavone is improved by about 1.30-2.83 times compared with the yield of the phellinus igniarius without adding paclobutrazol by adding a certain amount of phytohormone paclobutrazol in a liquid fermentation culture medium of the porous honeysuckle phellinus igniarius (Sanghuangpora ionicola); meanwhile, the invention also provides a culture condition for synthesizing the phellinus igniarius flavone of the lonicera microphylla under the induction of adding paclobutrazol, and provides a certain theoretical and practical basis for improving the research of phellinus fungus flavone.
Description
Technical Field
The invention relates to a method for improving the yield of intracellular flavone of phellinus igniarius of lonicera microphylla, and belongs to the field of microbial fermentation.
Background
Phellinus linteus (Sanghuangporouslongissicola) belongs to the kingdom of Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricales (Agaricaceae), Hymenochaetales (Hymenochaetales), Hymenochaetaceae (Hymenochaetaceae), Phellinus (Sanghuangporous) and is one of the 14 currently known Phellinus Fungi.
Phellinus genus fungi are one of few fungi containing flavonoids in medicinal fungi. Has various biological activities, such as anti-tumor, blood sugar lowering, immunity enhancing, antioxidant, antibacterial, and antiinflammatory etc. However, the wild phellinus igniarius in nature is small in quantity, the artificial cultivation technology of phellinus igniarius is complex, and the period is relatively long, so that the production and the improvement of the yield of flavonoids compounds by a liquid submerged fermentation mode can be researched.
At present, the way of promoting flavone yield is the optimization of fermentation culture medium and fermentation conditions; adding an inducer into a liquid fermentation culture medium, such as precursors for synthesizing flavonoid compounds, such as coumarin, cinnamic acid and the like, but the method has the defects of relatively high production cost; the existing research shows that the addition of the phytohormone with a certain concentration in the fermentation medium can promote the yield of the flavonoid compounds. Therefore, how to greatly improve the fermentation yield of phellinus igniarius flavonoids to further reduce the production cost and realize the wide application of phellinus igniarius flavonoids compounds is a problem which is urgently needed to be solved by fungus liquid fermentation of phellinus igniarius.
Paclobutrazol, chemical name (2RS,3RS) -1- (4-chlorophenyl) -4, 4-dimethyl-2- (1H-1,2, 4-triazol-1-yl) pentan-3-ol, molecular formula C 15 H 20 ClN 3 O, a common plant hormone, has the effects of delaying plant growth, inhibiting stem elongation, shortening internodes, promoting plant tillering, increasing plant stress resistance and the like. Paclobutrazol medicineThe effect of promoting the synthesis of metabolites in bacteria has not been reported. The invention utilizes paclobutrazol as an inducer for producing flavone by liquid fermentation of Lonicera micropore Phellinus, and can obviously improve the yield of intracellular flavone within a certain concentration range.
Disclosure of Invention
The invention aims to provide a method which can stably improve the yield of phellinus igniarius flavone and has a simple production process aiming at the phenomenon that the yield of flavonoid compounds in the existing phellinus igniarius fungi is low.
The invention provides application of paclobutrazol in improving the yield of phellinus fungus flavonoids compounds.
In one embodiment, the Phellinus fungi include, but are not limited to, Phellinus linteus (Sanghuangporus ionicola), Phellinus linteus (Large Lonicera), Phellinus linteus (berk.) Linne, and the like.
In one embodiment, the flavonoids include, but are not limited to, one or more of naringenin, isosakuranetin, hesperetin, methyl phellinus igniarius flavone a, methyl phellinus igniarius flavone B, isomethyl phellinus igniarius flavone B, butin, cherry glycoside, pinobanksin, dihydroquercetin, dihydromyricetin, homoeriodictyol, hispidulin, prunetin, daphnetin, farnesin, myricetin, isoquercitrin, hyperoside, rutin, catechin, kaempferol-3-O-glucuronide, kaempferol-3-O-neohesperidin, rhamnoside, and the like.
In one embodiment, the phellinus linteus of Lonicera miniata includes, but is not limited to, phellinus linteus of Lonicera miniata (Sanghuangporus ionicola), which is a medicinal fungus, species Analyzer.
In one embodiment, the application is the addition of paclobutrazol to a fermentation medium of phellinus fungus.
In one embodiment, the final concentration of paclobutrazol is greater than or equal to 0.03 mg/mL.
In one embodiment, the final concentration of paclobutrazol is 0.10-0.15 mg/mL.
In one embodiment, the paclobutrazol is added to the fermentation medium before inoculation or within 0-48 h of fermentation.
The invention also provides a method for producing flavone by fermenting the phellinus igniarius of the lonicera micropore, which is to ferment the phellinus igniarius of the lonicera micropore in a fermentation culture medium containing paclobutrazol.
In one embodiment, the concentration of paclobutrazol in the fermentation medium is greater than or equal to 0.03 mg/mL.
In one embodiment, the concentration of the paclobutrazol in the fermentation medium is 0.03-0.20 mg/mL.
In one embodiment, the method comprises the steps of activating the strain of phellinus linteus of Lonicera pinicola, using the activated strain for seed liquid preparation, and transferring the seed liquid to a fermentation medium containing paclobutrazol for fermentation to produce intracellular flavonoids.
In one embodiment, the method comprises the steps of activating a strain of phellinus linteus of Lonicera pinicola, using the activated strain for seed liquid preparation, transferring the seed liquid to a fermentation medium, and adding paclobutrazol to the fermentation medium within 0-48 h of fermentation.
In one embodiment, the initial pH of the fermentation medium is 5.5 or more.
In one embodiment, the initial pH of the fermentation medium is 5.5 to 7.5.
In one embodiment, the activation step of phellinus linteus strain is as follows: taking a slant strain block with the diameter of 5mm, inoculating the slant strain block on a PDA plate under the aseptic condition, and performing activated culture for 7-9 days at the culture temperature of 28 ℃.
In one embodiment, the preparation method of the phellinus linteus seed liquid of lonicera microirica comprises the following steps: inoculating 5-6 bacterial slices with the diameter of 2-3mm into a seed culture medium under the aseptic condition, wherein the liquid filling amount is 100mL/250 mL; sterilizing at 115 deg.C for 20 min; the culture conditions are as follows: shaking-culturing at 28 deg.C and 160r/min for 6-7 days to obtain seed liquid.
In one embodiment, the seed medium contains per L: 20g of glucose, 5g of yeast extract, 1.0g of monopotassium phosphate and 0.5g of magnesium sulfate heptahydrate.
In one embodiment, the method for producing intracellular flavonoids by liquid fermentation of phellinus linteus of lonicera minifera comprises the following steps: inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10% (V/V), and carrying out shake culture at the constant temperature of 180r/min at the culture temperature of 28 ℃ for 7-9 days.
In one embodiment, the method further comprises filtering the fermentation broth obtained by fermentation with a 80-mesh sieve, washing, and drying at 60 ℃ to constant weight to obtain the fermented mycelium.
In one embodiment, the fermentation medium contains per L: 20g of glucose, 3g of yeast extract powder, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate and 0.3g of L-phenylalanine.
The invention also claims the flavone-rich phellinus linteus mycelium prepared by the method.
The invention also provides the application of the method in producing flavone or flavone-containing products.
Has the advantages that: the invention provides a new application of paclobutrazol in improving the fermentation production of flavonoids compounds by a strain of lonicera microphylla phellinus, and the yield of intracellular flavone in the mycelium of lonicera microphylla phellinus can be improved by about 0.47-3.27 times compared with the yield when no paclobutrazol is added by adding a certain amount of paclobutrazol in a fermentation culture medium of the phellinus microphylla phellinus, the yield of the intracellular flavone can be improved by about 1.30-2.83 times, and the substance can stably promote the synthesis of the phellinus flavone; meanwhile, the invention also provides a basic fermentation condition for producing the flavonoid compound by fermenting the strain of the lonicera microphylla phellinus under the induction condition of paclobutrazol, and provides a certain theoretical and practical basis for improving the research of the phellinus fungus flavone.
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FIG. 1 shows the effect of different exogenous substances on the synthesis and biomass of phellinus linteus flavone of Lonicera japonica; wherein, the biomass has significant differences of #, P <0.05, # #, P <0.01, # #, P < 0.001; significant difference in flavone yield: p <0.05, P <0.01, P < 0.001.
FIG. 2 shows the effect of paclobutrazol concentration on the synthesis and biomass of phellinus linteus flavone in Lonicera japonica; wherein, the biomass has significant differences of #, P <0.05, # #, P <0.01, # #, P < 0.001; significant difference in flavone yield: p <0.05, P <0.01, P < 0.001.
FIG. 3 is a graph showing the effect of paclobutrazol addition time on the synthesis of phellinus linteus flavones and biomass of Lonicera japonica; wherein, the biomass has significant differences of #, P <0.05, # #, P <0.01, # #, P < 0.001; significant difference in flavone yield: p <0.05, P <0.01, P < 0.001.
FIG. 4 is a graph showing the effect of initial pH of the medium on the synthesis of phellinus linteus flavones and biomass of Lonicera japonica; wherein, the biomass has significant differences of #, P <0.05, # #, P <0.01, # #, P < 0.001; significant difference in flavone yield: p <0.05, P <0.01, P < 0.001.
FIG. 5 is a graph showing the variation of the yield of phellinus linteus flavones from Lonicera japonica with fermentation time; compared with the control group, the flavone yield treatment group comprises: p <0.05, P <0.01, P < 0.001.
FIG. 6 is a graph showing the variation of the biomass of phellinus linteus with fermentation time; biomass control group compared to treatment group: p <0.05, P <0.01, P < 0.001.
FIG. 7 is a graph showing the variation of sugar residue in the medium of Lonicera Aperture with fermentation time; residual sugar control group compared to treatment group: p <0.05, P <0.01, P < 0.001.
Detailed Description
Treatment of fermentation products and extraction of intracellular flavonoids:
mycelium → filtration, drying, grinding → dried mycelium powder (50mg) → 90% ethanol soaking for 24h (material-liquid ratio 1:50) → water bath soaking for 1.0h (70 ℃) → ultrasonic extraction for 1.0h (50 ℃) → centrifugation (10000r/min, 10min) → constant volume → crude intracellular flavone extract. Unless otherwise specified, "flavone" as used in the detailed description refers to a flavonoid compound that can be measured according to this measurement method, and includes, but is not limited to, naringenin, isosakuranetin, hesperetin, methyl phellinus flavone a, methyl phellinus flavone B, isomethyl phellinus flavone B, butein, prunin, pinobanksin, dihydroquercetin, dihydromyricetin, homoeriodictyol, hispidulin, prunetin, daphnetin, farnesin, myricetin, isoquercitrin, hyperoside, rutin, catechin, kaempferol-3-O-glucuronide, kaempferol-3-O-neohesperidin, rhamnoside, and the like.
And (3) measuring the flavone content: the content of flavone is measured by adopting sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method, the measuring system is 100uL sample bath liquid, absolute ethyl alcohol is added to 200uL, and then 40uL 5% NaNO is added in sequence 2 Shaking the solution, and covering tin foil paper for light-proof reaction for 6 min; then adding 10% Al (NO) 3 ) 3 Shaking the solution 40uL evenly, and reacting for 6min at room temperature; then adding 400uL of 4% NaOH solution, and reacting for 10 min; finally, 320uL of absolute ethyl alcohol is added to the solution to be constant volume of 1.0 mL. Absolute ethanol was added as a blank control, and absorbance was measured at 510nm wavelength.
Flavone yield (mg/g): the yield of flavone is total flavone content (mg)/quality of phellinus linteus mycelium of Lonicera japonica.
Biomass (mg/L): expressed as dry weight of the mycelium of phellinus linteus in unit volume of fermentation broth.
The addition mode of the exogenous substances is as follows: firstly, preparing a mother solution with a certain concentration from a foreign substance by using a dimethyl sulfoxide (DMSO) solution, sterilizing by adopting a filtration mode, adding a certain volume of the mother solution of the foreign substance into a sterile liquid fermentation culture medium, and adjusting to a required final concentration.
Seed medium (per 1L): 20g of glucose, 5g of yeast extract, 1.0g of monopotassium phosphate and 0.5g of magnesium sulfate heptahydrate, and the pH value is natural.
Fermentation medium (per L): 20g of glucose, 3g of yeast extract powder, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.3g of L-phenylalanine and pH of 6.9 +/-0.2.
Example 1 Effect of different exogenous Agents on the Synthesis and Biomass of Phellinus linteus flavones of Lonicera japonica
a. Activating strains: inoculating the slant strain of the phellinus igniarius of the Lonicera microphylla on a PDA (personal digital assistant) plate under the aseptic condition for activation culture for 7-9 days, wherein the culture temperature is 28 ℃;
b. seed culture: inoculating 5-6 fungus pieces with diameter of 2-3mm into sterilized seed culture medium in triangular flask with liquid loading capacity of 100mL/250mL, and performing shake culture at 28 deg.C and constant temperature of 160r/min for 6-7 days to obtain seed solution;
c. fermentation culture: adding 100mL of fermentation medium into a 250mL triangular flask, and sterilizing at 115 ℃ for 20 min; dissolving with DMSO, preparing into exogenous substance solution with mother solution concentration of 30mg/mL, and sterilizing by filtration, wherein the exogenous substance comprises paclobutrazol (PP333), naphthylacetic acid (NAA), indoleacetic acid (IAA), abscisic acid (ABA), and gibberellin (GA 3); adding 100uL of the exogenous mother liquor to adjust the final concentration to 0.03mg/mL under aseptic conditions before inoculation, and adding the same volume of sterile water subjected to filter sterilization as a CK group and DMSO solution as a solvent control group (DMSO group); inoculating the seed solution obtained by the culture in the step b into a liquid fermentation culture medium according to the inoculation amount of 10% (V/V), carrying out shake culture at the constant temperature of 28 ℃ and 180r/min for 7 days, filtering by using a 80-mesh sieve, washing the culture medium with distilled water, and drying at 60 ℃ to constant weight to obtain a fermentation mycelium;
and after the fermentation is finished, measuring the yield of intracellular flavone of each group of phellinus linteus mycelium. The results show (figure 1), the flavone yield and biomass have no significant difference when sterile water and DMSO are added, which indicates that the induction of the exogenous substance actually increases the flavone yield, and in addition, the flavone yields of the paclobutrazol and the solvent control group are 10.744mg/g and 5.679mg/g respectively, so that the flavone yield is improved by 89.19%; combining biomass, the yield of flavone and the yield of flavone are 64.455mg/L and 28.050mg/L respectively, and the yield is improved by 129.79%.
Example 2 Effect of paclobutrazol addition concentration on the Synthesis and Biomass of phellinus linteus flavones of Lonicera japonica
The culture medium and the culture method are the same as the example 1, except that the paclobutrazol is used as an exogenous substance in the step c, the paclobutrazol is prepared into a mother solution with the concentration of 0-200mg/mL by a DMSO solution, after bacteria are filtered out, a certain volume of the mother solution is added into the fermentation culture medium, and the final concentrations are respectively 0.03, 0.05, 0.10, 0.15 and 0.20 mg/mL.
And after the fermentation is finished, measuring the yield of the intracellular flavone of the phellinus linteus mycelium of the Lonicera miniata. The results show (FIG. 2) that at biomass concentrations of 0.10 and 0.15mg/mL, flavone yields of 23.821mg/g and 24.269mg/g were respectively achieved, and flavone yields were 3.19-fold and 3.27-fold, respectively, relative to the solvent control (5.679mg/g) in example 1; the yield of flavone is 107.542mg/L and 106.995mg/L respectively when the biomass concentration is 0.10 and 0.15mg/mL, and is 2.83 times and 2.81 times of that of DMSO group respectively; when the final concentration of biomass is 0.20mg/mL, the flavone yield reaches 18.788mg/g, and the flavone yield is 83.166 mg/L.
Example 3 Effect of paclobutrazol addition time on the Synthesis and Biomass of Phellinus linteus flavones of Lonicera Aphylla
The culture medium and the culture method are the same as in example 1, except that in the step c, paclobutrazol is used as an exogenous substance, a mother solution with the concentration of 200mg/mL is prepared by using a DMSO solution, and after bacteria are filtered out, a certain volume of paclobutrazol mother solution is added in the fermentation stages of 0, 24, 48, 72, 96, 120 and 144h respectively to adjust the final concentration of paclobutrazol in the fermentation medium to be 0.10 mg/mL.
And after the fermentation is finished, measuring the yield of the intracellular flavone of the phellinus linteus mycelium of the Lonicera miniata. The result shows (figure 3), adding exogenous material at day 0, the growth of thallus is inhibited obviously, but the flavone yield can reach 26.223mg/g, which is improved by 1.86 times compared with DMSO group, the flavone yield at 24h and 48h of fermentation is 24.361mg/g and 23.363mg/g, which is improved by 1.66 times and 1.55 times respectively compared with DMSO group; the biomass at 0, 24 and 48h is 91.174mg/L, 120.841mg/L and 120.228mg/L respectively, which are respectively improved by 0.78 times, 1.36 times and 1.35 times compared with DMSO group; after more than 96 hours, the flavone yield is lower than 15.474mg/g, and the flavone yield is lower than 85.503 mg/L.
Example 4 Effect of initial pH of the Medium on the Synthesis and Biomass of Phellinus linteus flavones of Lonicera Aphylla
The culture medium and the culture method are the same as in example 1, except that in the step c, paclobutrazol is taken as a foreign substance, a mother solution with a certain concentration is prepared by using a DMSO solution, the mother solution is added into a fermentation culture medium after filtration sterilization, the final concentration of the paclobutrazol in the fermentation culture medium is 0.10mg/mL, and the pH is adjusted to 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5 or 11.5 by using 1mol/L NaOH and 1mol/L HCl respectively.
And after the fermentation is finished, measuring the yield of the intracellular flavone of the phellinus linteus mycelium of the Lonicera miniata. The result shows (figure 4) that when the pH is 5.5-11.5, the flavone yield reaches 26.003-29.200 mg/g; when the pH value is 5.5-7.5, the flavone yield reaches 26.003-29.200 mg/g, which is improved by 1.84-2.19 times compared with DMSO group, and the flavone yield reaches 62.931-101.116 mg/L; at pH 7.5, flavone yield was increased 0.98 times compared to DMSO group.
Example 5 Effect of fermentation time on the Synthesis and Biomass of Phellinus linteus flavones of Lonicera Aphylla
The culture medium and the culture method are the same as example 1, except that in the step c, paclobutrazol is used as an exogenous substance, a mother solution with a certain concentration is prepared by using a DMSO solution, the mother solution is added into a fermentation culture medium after filtration sterilization, the final concentration of the paclobutrazol in the fermentation culture medium is 0.10mg/mL, the change conditions of the PP333 group and the CK group along with time in the aspects of thallus growth, product generation, substrate consumption and the like are compared by using a non-added (CK group) as a control, and fermentation samples cultured for 1,2, 3, 4, 5, 6, 7, 8, 9 and 10 days are respectively detected. After sampling every day, the yield, biomass and residual sugar content of the total flavonoids in the cells of the phellinus linteus mycelium of Lonicera Aperture are measured.
The result shows that the flavone yield of the PP333 group is obviously higher than that of a control group (figure 5) from day 2, the yield of 19.023mg/g can be achieved after the culture is carried out for 5 days, the yield is 58.084mg/L, the yield is 21.946mg/g on the 8 th day of the culture, and the yield is 88.238 mg/L. The biomass is in a gradual increasing trend along with the increase of the culture time, but the increase speed is lower than that of a CK group (figure 6), the glucose consumption curve can be in one-to-one correspondence (figure 7), namely the glucose consumption of the CK group is faster than that of a treatment group, the glucose of the CK group is basically completely consumed after 6 days, and in experiments, the culture medium is dark in color after the control group is cultured for 6-7 days, and the cells are autolyzed. Therefore, the end time of fermentation of the phellinus linteus of Lonicera japonica is preferably controlled to be 6-7 days when no exogenous substances are added; the culture time is preferably controlled to 6-8 days when exogenous materials are added.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Application of paclobutrazol in improving the yield of flavonoids compounds in phellinus fungus is provided.
2. The use of claim 1, wherein the Phellinus fungus includes, but is not limited to, Phellinus linteus (Sanghuangporus lonicera), Phellinus macrocarpus, Phellinus linteus, or Phellinus linteus.
3. The use according to claim 1 or 2, wherein the use is the addition of paclobutrazol to a fermentation medium of phellinus fungus such that the final concentration of paclobutrazol in the fermentation medium is 0.03mg/mL or more.
4. A method for producing flavone by fermenting Lonicera microphylla Phellinus Linteus is characterized in that the Lonicera microphylla Phellinus Linteus is fermented in a fermentation culture medium containing paclobutrazol; the concentration of the paclobutrazol in the fermentation medium is more than or equal to 0.03 mg/mL.
5. The method as claimed in claim 4, wherein the strain of the phellinus linteus is activated, and the activated seed solution is transferred to a fermentation medium containing paclobutrazol for fermentation to produce intracellular flavonoids.
6. The method according to claim 4, wherein the strain of the phellinus igniarius of Lonicera pinicola is activated, the activated seed solution is transferred to a fermentation medium, and the paclobutrazol is added after the fermentation for a period of time to continue the fermentation.
7. The method of claim 6, wherein paclobutrazol is added to the fermentation medium within 0-48 hours of fermentation.
8. The method according to any one of claims 4 to 7, wherein the initial pH of the fermentation medium is 5.5 or more.
9. The method according to any one of claims 4 to 8, wherein the fermentation medium comprises: glucose, yeast extract powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and L-phenylalanine.
10. A flavone-rich mycelium of phellinus linteus of Lonicera japonica prepared by the method of any one of claims 4 to 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115418321A (en) * | 2022-10-09 | 2022-12-02 | 安徽利民生物科技股份有限公司 | Phellinus igniarius mycelium liquid fermentation method for high yield of hyperoside |
| CN115418321B (en) * | 2022-10-09 | 2024-02-06 | 安徽利民生物科技股份有限公司 | Phellinus linteus mycelium liquid fermentation method for high production of hyperin |
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