CN112063663A - Fermentation method for high-yield phellinus igniarius extracellular flavonoids - Google Patents
Fermentation method for high-yield phellinus igniarius extracellular flavonoids Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 106
- 230000004151 fermentation Effects 0.000 title claims abstract description 106
- 229930003935 flavonoid Natural products 0.000 title claims abstract description 32
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 32
- 150000002215 flavonoids Chemical class 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000123113 Phellinus igniarius Species 0.000 title abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 41
- 241000001727 Tropicoporus linteus Species 0.000 claims abstract description 38
- 238000012258 culturing Methods 0.000 claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000012528 membrane Substances 0.000 claims abstract description 15
- 238000011081 inoculation Methods 0.000 claims abstract description 13
- 230000001954 sterilising effect Effects 0.000 claims abstract description 11
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 claims abstract description 8
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims abstract description 8
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 claims abstract description 8
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229930016911 cinnamic acid Natural products 0.000 claims abstract description 8
- 235000013985 cinnamic acid Nutrition 0.000 claims abstract description 8
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960001669 kinetin Drugs 0.000 claims abstract description 8
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 29
- 238000011218 seed culture Methods 0.000 claims description 21
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 18
- 239000000725 suspension Substances 0.000 claims description 18
- 239000001888 Peptone Substances 0.000 claims description 17
- 108010080698 Peptones Proteins 0.000 claims description 17
- 235000019319 peptone Nutrition 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 16
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 claims description 15
- 229930003944 flavone Natural products 0.000 claims description 15
- 150000002212 flavone derivatives Chemical class 0.000 claims description 15
- 235000011949 flavones Nutrition 0.000 claims description 15
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 239000007836 KH2PO4 Substances 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 9
- 238000002798 spectrophotometry method Methods 0.000 claims description 7
- 230000003068 static effect Effects 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 abstract description 2
- 229920001282 polysaccharide Polymers 0.000 abstract description 2
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229930000044 secondary metabolite Natural products 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
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- 239000008121 dextrose Substances 0.000 description 2
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
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- 239000002207 metabolite Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
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- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 1
- 229940117954 naringenin Drugs 0.000 description 1
- 235000007625 naringenin Nutrition 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- URFCJEUYXNAHFI-ZDUSSCGKSA-N pinocembrin Chemical compound C1([C@@H]2CC(=O)C3=C(O)C=C(C=C3O2)O)=CC=CC=C1 URFCJEUYXNAHFI-ZDUSSCGKSA-N 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- DJOJDHGQRNZXQQ-AWEZNQCLSA-N sakuranetin Chemical compound C1([C@@H]2CC(=O)C3=C(O)C=C(C=C3O2)OC)=CC=C(O)C=C1 DJOJDHGQRNZXQQ-AWEZNQCLSA-N 0.000 description 1
- RNAPFFYGJWALAQ-UHFFFAOYSA-N sakuranetin Natural products O1C2=CC(C)=CC(O)=C2C(=O)CC1C1=CC=C(O)C=C1 RNAPFFYGJWALAQ-UHFFFAOYSA-N 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a fermentation method for high-yield phellinus linteus extracellular flavonoids, which comprises the following steps: s1, strain preparation; s2, strain inoculation and culture: s3, inoculating and culturing the production fermentation liquor, wherein on the 1 st day of fermentation, 0.01-0.03g/L of prepared cinnamic acid and 0.01-0.02g/L of prepared naphthylacetic acid are added, sterilized by a filter membrane and then aseptically added into a culture medium; fermenting for 3 days, sterilizing the prepared 0.005-0.012g/L kinetin dimethyl sulfoxide solution with filter membrane, and aseptically adding into culture medium; after fermenting for 2 days, performing ultrasonic treatment on the fermentation liquor for 4-8 minutes at 50-100W per day; after 10 days the fermentation was complete, the mycelium was washed with water and freeze dried. The method can obviously improve the content of extracellular polysaccharide of phellinus igniarius and increase the quantity of mycelium.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a fermentation method for high-yield phellinus linteus extracellular flavonoids.
Background
Phellinus linteus is a precious large-scale medicinal wood-rotting fungus growing for many years and is called forest gold by modern people. The application history of phellinus igniarius in China has been 2000 years, the name of phellinus igniarius appears on the Shen nong Ben Cao Jing at the earliest time, and the name of phellinus igniarius appears in the medicine property treatise at the beginning of Tang and is used up to now. The outline of materia Medica of sang Huang Ming Dynasty and other medical books are also recorded. Except for the records of ancient Chinese books, the book of 500 years ago of Korea (the integrated prescription of the village medicines) and the book of 400 years ago (the eastern medicine and treasure inspection) are both named as sanghuang such as lingdan miao medicine. Phellinus linteus grows mainly on the trunk of a living Phellinus linteus, and the Phellinus linteus is rather dependent on the growth of the living Phellinus linteus, which is also the reason why the Phellinus linteus fruiting body is difficult to be artificially cultivated at present. Phellinus linteus is called as "forest gold" by modern people. Phellinus igniarius is a medicinal fungus with the highest effective rate in the current internationally recognized biological cancer treatment medicaments.
Compared with other edible and medicinal fungi, the flavonoid compound is a unique component of phellinus igniarius. At present, there are more than 20 kinds of flavonoids extracted and identified from phellinus linteus, such as naringenin, sakuranetin, dihydrochrysin, 7-methoxy dihydrochrysin, benzyl dihydroflavonoids, etc. With the increase of researchers and the improvement of research equipment, people know phellinus igniarius deeply, and researches show that different types of phellinus igniarius have obvious differences in flavonoid component content. Researches find that the flavonoid compounds have the functions of improving cardiovascular and cerebrovascular circulation, removing in-vivo free radicals, resisting aging, inhibiting lipid peroxidation, protecting skin, resisting cancer, protecting liver, resisting allergy and improving autoimmunity.
China is a large export country of wild medicinal fungi, but the research on phellinus igniarius is still in the initial stage at present, and the technology is not mature. Because the limited phellinus igniarius wild resources in China have long growth period and poor stability of effective components, are excessively collected and are less and less, the phellinus igniarius wild resources are difficult to become stable industrial product sources, and the market requirements can not be met far away. The biological product prepared by the biological fermentation technology is the basis for overcoming the best way of utilizing wild resources with limited biomass and realizing the development and utilization of useful metabolites. Compared with the traditional sporocarp cultivation, the method for preparing the secondary metabolite of phellinus igniarius through liquid fermentation has the advantages of short production period, more metabolites, easy quality control, large-scale production and the like. The fermentation culture medium for preparing the secondary metabolite of the phellinus igniarius by liquid fermentation has important significance, and can directly influence the activity of the secondary metabolite of the phellinus igniarius.
Chinese patent document CN108611381A discloses a liquid fermentation culture method beneficial to the enrichment of flavonoids in phellinus igniarius, which comprises mother strain preparation and phellinus igniarius liquid culture, wherein the phellinus igniarius liquid culture comprises the following specific steps: taking 180-210g of potato, 17-22g of glucose and peptone, and fixing the volume to 1L by using distilled water, wherein the final concentration of the peptone is 2-8g/L and the natural pH value, preparing culture media, respectively subpackaging the culture media in 250mL conical flasks, sterilizing at high temperature, standing at normal temperature and sterilizing by ultraviolet; inoculating 500 mu L of phellinus igniarius mother liquor into a liquid culture medium in a clean bench for inoculation, and culturing in a shaking culture box at 25-28 ℃ and at the rotation speed of 150-180 revolutions per minute, wherein 8-12 bacterial balls are arranged in each culture medium in 72 hours, the diameter of each bacterial ball is 1.7-1.9cm, the color of the bacterial bodies is dark yellow, 35-45 bacterial balls are arranged in each culture medium after culturing for one week, the diameter of each bacterial ball is 0.7-0.9cm, and the color of the bacterial bodies is yellow brown; step three, culturing for 15 days until the mycelia are mature, filtering the culture solution, filtering the mycelia, filling the mycelia into a small conical bottle, sealing a filter membrane, putting the bottle into a refrigerator with the temperature of minus 85-minus 75 ℃, and freeze-drying the bottle; step four, weighing 0.5030-0.5320g of mycelium, adding the mycelium into 10mL of 95% ethanol by mass fraction, placing the mixture into a centrifuge tube, performing ultrasonic treatment for 50-75min at 66-78 ℃, centrifuging for 8-12min, taking filtrate, performing rotary evaporation, and adding 2mL of chromatographic methanol for dissolution to obtain a finished product. The culture medium in the method is simple and convenient to configure, and the yield of the flavonoid compounds in the phellinus igniarius is improved by preparing the culture medium from the basic PDA culture medium, the potatoes, the glucose and the water and adding a proper amount of peptone.
Chinese patent document CN101702984A discloses a phellinus igniarius mycelium liquid fermentation method for increasing the yield of phellinus igniarius flavone by using fungal (Fungus) elicitor, which comprises (1) preparation of fungal elicitor; (2) liquid fermentation culture of phellinus igniarius; (3) and (3) carrying out induced culture on the phellinus igniarius cells by using fungal elicitors. Wherein the liquid fermentation culture of the phellinus igniarius: inoculating the phellinus igniarius strain preserved on the inclined plane to a PDA (PDA dextrose agar) plate culture medium for activation culture at the culture temperature of 25-30 ℃ for 5-10 days, then inoculating the strain blocks into a triangular flask filled with a PD (dextrose agar) liquid fermentation culture medium for fermentation culture at the shaking table rotating speed of 100-180 r/min at the temperature of 25-30 ℃ for 5-8 days. The invention uses the fungal elicitor for the liquid fermentation production of the phellinus igniarius flavone to improve the yield of the flavone in the phellinus igniarius.
Chinese patent document CN103695316A discloses a liquid fermentation method for increasing the yield of flavone in phellinus igniarius mycelia, which comprises the following steps: (1) culturing a phellinus igniarius liquid fermentation strain, inoculating a phellinus igniarius solid strain preserved on an inclined plane to a PDA (potato dextrose agar) plate medium), performing activated culture at the culture temperature of 25-30 ℃ for 5-7 days, then inoculating phellinus igniarius blocks into a triangular flask filled with a PD liquid culture medium for fermentation culture at the table rotating speed of 150-; (2) liquid fermentation culture of phellinus igniarius, namely homogenizing cultured phellinus igniarius liquid fermentation strains, inoculating a bacterial liquid into a PD liquid fermentation culture medium added with magnesium ions for fermentation culture, wherein the rotating speed of a shaking table is 100 plus one year per minute at the culture temperature of 25-30 ℃, the culture time is 5-10 days, and low-intensity ultrasonic irradiation is adopted every day to obtain phellinus igniarius mycelia with high flavone content. The invention applies a low-intensity ultrasonic field to the liquid fermentation production of the flavone in the phellinus igniarius mycelium to improve the yield of the flavone in the phellinus igniarius mycelium.
Disclosure of Invention
In view of the above, the present invention is directed to a fermentation method for producing phellinus linteus extracellular flavonoids in high yield, which is different from the prior art method for producing phellinus linteus extracellular flavonoids in high yield.
The adopted technical scheme is as follows:
a fermentation method for high-yield phellinus linteus extracellular flavonoids comprises the following steps:
s1, strain preparation:
inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at constant temperature of 25-30 deg.C for 9-12 days, and placing in 2-5 deg.C refrigerator;
s2, strain inoculation and culture:
inoculating the activated mycelium of the strain of the step S1 into a seed culture solution, and performing static culture at 25-30 ℃ for 6-8 days to obtain a strain seed solution;
s3, inoculating and culturing production fermentation liquor:
washing the mycelium in the strain seed liquid obtained in the step S2 with water under an aseptic condition, smashing the mycelium, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 8-12 mg/mL; inoculating the hypha suspension into a culture medium for producing fermentation liquor according to the inoculation amount of 180 and 220 mg/L; adding 0.01-0.03g/L prepared cinnamic acid and 0.01-0.02g/L prepared naphthylacetic acid, sterilizing with filter membrane, and adding into culture medium;
fermenting for 3 days, sterilizing the prepared 0.005-0.012g/L kinetin dimethyl sulfoxide solution with filter membrane, and aseptically adding into culture medium;
after fermenting for 2 days, performing ultrasonic treatment on the fermentation liquor for 4-8 minutes at 50-100W per day;
after 10 days the fermentation was complete, the mycelium was washed with water and freeze dried.
Further, in S2, the seed culture solution contained 30g of glucose, 5g of peptone and KH per liter2PO41.0g、MgSO40.5g、VB10.5g。
Further, in S3, the fermentation broth was prepared to contain 30g of glucose, 5g of peptone and KH per liter2PO41.0g、MgSO40.5g、VB10.5g。
Further, in S1, the phellinus linteus strain is selected from i.sanghuanghuang of qiandao lake, hangzhou, zhejiang.
Further, in S1, Phellinus Linteus strain was inoculated on PDA slant culture medium, cultured at constant temperature of 25 ℃ for 10 days, and then placed in a refrigerator at 4 ℃ for further use.
Further, in S2, the activated mycelium of the strain obtained in step S1 is inoculated into a seed culture solution, and the strain is cultured at a static state at 25 ℃ for 7 days to form a strain seed solution.
Further, in S3, washing mycelium in the strain seed solution of S2 under aseptic condition, crushing, adding sterile water to prepare mycelium suspension with mycelium concentration of 10 mg/mL; inoculating the hypha suspension into a culture medium for producing fermentation liquor according to the inoculation amount of 200 mg/L.
Further, in S3, after fermentation is finished after 10 days, the mycelium is washed, frozen, dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
Further, in S3, after the fermentation day 2, the fermentation broth was sonicated at 25 ℃ for 5 minutes at 50-100W per day.
The invention has the beneficial effects that:
the invention adopts a method different from the prior art to highly yield the content of phellinus igniarius extracellular flavonoids:
in the first aspect, cinnamic acid is a phenylpropanoid compound, and the parent nucleus structure can be used as a precursor substance of a flavonoid compound.
In the second aspect, a certain amount of naphthylacetic acid is added, so that the synthesis of flavonoids can be improved, and key genes for synthesizing flavonoids can be activated.
In the third aspect, kinetin (or cytokinin) promotes cell division and accelerates fermentation speed; in particular, dimethyl sulfoxide also changes the permeability of cell membranes and promotes the transfer of active substances.
In the fourth aspect, ultrasound can increase the permeability of the cell membrane of the mycelium, promote the transfer of active substances from inside to outside of the cell, and promote cell division.
In conclusion, the technical effect of high yield of the content of the phellinus igniarius extracellular flavonoids is realized by the application of the four aspects.
Detailed Description
The present invention is described in detail below by way of specific comparative examples and examples, but the use and purpose of these exemplary embodiments are merely to exemplify the present invention, and do not constitute any limitation to the actual scope of the present invention in any form, and do not limit the scope of the present invention thereto.
COMPARATIVE EXAMPLE 1 (as a control group)
A liquid fermentation process comprising the steps of:
(1) preparing a seed culture solution and a production fermentation liquid: the culture solution and the fermentation liquor comprise the following components in percentage by weight:
1) seed culture (L): 30g of glucose, 5g of peptone and KH2PO41.0g，MgSO40.5g，VB10.5g，
2) Production of fermentation broth (L): glucose 30g, peptone 5g, KH2PO41.0g，MgSO40.5g，VB10.5g，
(2) Preparing strains: inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at 25 deg.C for 10 days, and storing in 4 deg.C refrigerator;
(3) inoculating and culturing strains: inoculating the activated and cultured mycelium of the strain in the step (2) into the seed culture solution in the step (1), and statically culturing at 28 ℃ for 7 days to obtain strain seed solution;
(4) inoculating and culturing production fermentation liquor: washing the mycelium in the strain seed liquid in the step (3) under an aseptic condition, smashing, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 10 mg/mL; inoculating the hypha suspension into the fermentation liquor produced in the step (1)2) according to the inoculation amount of 200 mg/L.
(5) Fermenting at 25 deg.C for 10 days, washing mycelium with water, freeze drying, and weighing; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
Example 1
A fermentation method for high-yield phellinus linteus extracellular flavonoids comprises the following steps:
(1) preparing a seed culture solution and a production fermentation liquid:
1) seed culture (L): 30g of glucose, 5g of peptone and KH2PO41.0g，MgSO40.5g，VB10.5g，
2) Production of fermentation broth (L): glucose 30g, peptone 5g, KH2PO41.0g，MgSO40.5g，VB10.5g，
(2) Preparing strains: inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at 25 deg.C for 10 days, and storing in 4 deg.C refrigerator;
(3) inoculating and culturing strains: inoculating the activated and cultured mycelium of the strain in the step (2) into the seed culture solution in the step (1), and statically culturing at 28 ℃ for 7 days to obtain strain seed solution;
(4) inoculating and culturing production fermentation liquor: washing the mycelium in the strain seed liquid in the step (3) under an aseptic condition, smashing, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 10 mg/mL; inoculating the hypha suspension into the culture medium of the fermentation liquid produced in the step (1)2) according to the inoculation amount of 200mg/L, adding prepared cinnamic acid (0.01g/L) and naphthylacetic acid (0.01g/L), sterilizing by a filter membrane, and then aseptically adding into the culture medium.
(5) On the 3 rd day of fermentation, the prepared kinetin dimethyl sulfoxide solution (0.005g/L) is added into the culture medium aseptically after being sterilized by a filter membrane.
(6) After 2 days of fermentation, the fermentation broth is subjected to ultrasonic treatment for 5 minutes at room temperature of 25 ℃ every day and with ultrasonic power of 50W.
(7) After 10 days, the fermentation is finished, and the mycelium is washed, freeze-dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
Example 2
A fermentation method for high-yield phellinus linteus extracellular flavonoids comprises the following steps:
(1) preparing a seed culture solution and a production fermentation liquid:
1) seed culture (L): 30g of glucose, 5g of peptone and KH2PO41.0g，MgSO40.5g，VB10.5g，
2) Production of fermentation broth (L): glucose 30g, peptone 5g, KH2PO41.0g，MgSO40.5g，VB10.5g，
(2) Preparing strains: inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at 25 deg.C for 10 days, and storing in 4 deg.C refrigerator;
(3) inoculating and culturing strains: inoculating the activated and cultured mycelium of the strain in the step (2) into the seed culture solution in the step (1), and statically culturing at 28 ℃ for 7 days to obtain strain seed solution;
(4) inoculating and culturing production fermentation liquor: washing the mycelium in the strain seed liquid in the step (3) under an aseptic condition, smashing, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 10 mg/mL; inoculating the hypha suspension into the fermentation liquor produced in the step (1)2) according to the inoculation amount of 200mg/L, adding prepared cinnamic acid (0.02g/L) and naphthylacetic acid (0.015g/L), sterilizing by a filter membrane, and aseptically adding into a culture medium.
(5) On the 3 rd day of fermentation, the prepared kinetin dimethyl sulfoxide solution (0.008g/L) is added into the culture medium aseptically after being sterilized by a filter membrane.
(6) After the fermentation day 2, the fermentation broth was sonicated at 25 ℃ for 5 minutes at room temperature of 70W per day.
(7) After 10 days, the fermentation is finished, and the mycelium is washed, freeze-dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
Example 3
A fermentation method for high-yield phellinus linteus extracellular flavonoids comprises the following steps:
(1) preparing a seed culture solution and a production fermentation liquid:
1) seed culture (L): 30g of glucose, 5g of peptone and KH2PO41.0g，MgSO40.5g，VB10.5g，
2) Production of fermentation broth (L): glucose 30g, peptone 5g, KH2PO41.0g，MgSO40.5g，VB10.5g，
(2) Preparing strains: inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at 25 deg.C for 10 days, and storing in 4 deg.C refrigerator;
(3) inoculating and culturing strains: inoculating the activated and cultured mycelium of the strain in the step (2) into the seed culture solution in the step (1), and statically culturing at 28 ℃ for 7 days to obtain strain seed solution;
(4) inoculating and culturing production fermentation liquor: washing the mycelium in the strain seed liquid in the step (3) under an aseptic condition, smashing, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 10 mg/mL; inoculating hypha suspension into the fermentation liquor produced in the step (1)2) according to the inoculation amount of 200mg/L, adding prepared cinnamic acid (0.03g/L) and naphthylacetic acid (0.012g/L), sterilizing with a filter membrane, and aseptically adding into a culture medium.
(5) On the 3 rd day of fermentation, the prepared kinetin dimethyl sulfoxide solution (0.010g/L) is added into the culture medium aseptically after being sterilized by a filter membrane.
(6) After the fermentation day 2, the fermentation broth was sonicated at 25 ℃ for 5 minutes at room temperature 90W per day.
(7) After 10 days, the fermentation is finished, and the mycelium is washed, freeze-dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
Example 4
A fermentation method for high-yield phellinus linteus extracellular flavonoids comprises the following steps:
(1) preparing a seed culture solution and a production fermentation liquid:
1) seed culture (L): 30g of glucose, 5g of peptone and KH2PO41.0g，MgSO40.5g，VB10.5g，
2) Production of fermentation broth (L): glucose 30g, peptone 5g, KH2PO41.0g，MgSO40.5g，VB10.5g，
(2) Preparing strains: inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at 25 deg.C for 10 days, and storing in 4 deg.C refrigerator;
(3) inoculating and culturing strains: inoculating the activated and cultured mycelium of the strain in the step (2) into the seed culture solution in the step (1), and statically culturing at 28 ℃ for 7 days to obtain strain seed solution;
(4) inoculating and culturing production fermentation liquor: washing the mycelium in the strain seed liquid in the step (3) under an aseptic condition, smashing, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 10 mg/mL; inoculating hypha suspension into fermentation liquor produced in the step (1)2) according to the inoculation amount of 200mg/L, adding prepared cinnamic acid (0.02g/L) and naphthylacetic acid (0.02g/L), sterilizing with a filter membrane, and aseptically adding into a culture medium.
(5) On the 3 rd day of fermentation, the prepared kinetin dimethyl sulfoxide solution (0.012g/L) is added into the culture medium aseptically after being sterilized by a filter membrane.
(6) After the fermentation day 2, the fermentation broth was sonicated at 25 ℃ for 5 minutes at room temperature 100W per day.
(7) After 10 days, the fermentation is finished, and the mycelium is washed, freeze-dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
The amount of mycelia and the content of extracellular flavonoids measured in the control group (i.e., comparative example 1) and examples 1 to 4 are shown in Table 1 below.
TABLE 1 comparison of mycelial mass with extracellular flavone content
As can be seen from the above table, the method of the present invention significantly increased the content of extracellular polysaccharides of Phellinus linteus and increased the amount of mycelia, relative to the control group (i.e., comparative example 1).
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (9)
1. A fermentation method for high-yield phellinus linteus extracellular flavonoids is characterized by comprising the following steps:
s1, strain preparation:
inoculating Phellinus Linteus strain on PDA slant culture medium, culturing at constant temperature of 25-30 deg.C for 9-12 days, and placing in 2-5 deg.C refrigerator;
s2, strain inoculation and culture:
inoculating the activated mycelium of the strain of the step S1 into a seed culture solution, and performing static culture at 25-30 ℃ for 6-8 days to obtain a strain seed solution;
s3, inoculating and culturing production fermentation liquor:
washing the mycelium in the strain seed liquid obtained in the step S2 with water under an aseptic condition, smashing the mycelium, and adding aseptic water to prepare a mycelium suspension with the mycelium concentration of 8-12 mg/mL; inoculating the hypha suspension into a culture medium for producing fermentation liquor according to the inoculation amount of 180 and 220 mg/L; adding 0.01-0.03g/L prepared cinnamic acid and 0.01-0.02g/L prepared naphthylacetic acid, sterilizing with filter membrane, and adding into culture medium;
fermenting for 3 days, sterilizing the prepared 0.005-0.012g/L kinetin dimethyl sulfoxide solution with filter membrane, and aseptically adding into culture medium;
after fermenting for 2 days, performing ultrasonic treatment on the fermentation liquor for 4-8 minutes at 50-100W per day;
after 10 days the fermentation was complete, the mycelium was washed with water and freeze dried.
2. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein the seed culture broth in S2 contains 30 g/L glucose, 5g/L peptone and KH2PO41.0g，MgSO40.5g，VB10.5g。
3. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein the fermentation broth in S3 is prepared to contain 30g of glucose, 5g of peptone and KH per liter2PO41.0g，MgSO40.5g，VB10.5g。
4. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein in S1, the phellinus linteus strain is selected from I.sanghuanghuang of Qiandao lake, Hangzhou, Zhejiang.
5. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein in S1, phellinus linteus strain is inoculated on PDA slant culture medium, cultured at constant temperature of 25 ℃ for 10 days, and then placed in a refrigerator of 4 ℃ for later use.
6. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein in S2, the activated mycelium of the strain of step S1 is inoculated into a seed culture solution, and the strain seed solution is formed by static culture at 25 ℃ for 7 days.
7. The fermentation method of high-yield phellinus linteus extracellular flavonoids according to claim 1, wherein in S3, the mycelium in the strain seed solution of step S2 is washed with water under aseptic conditions, broken and added with sterile water to prepare a mycelium suspension with a mycelium concentration of 10 mg/mL; inoculating the hypha suspension into a culture medium for producing fermentation liquor according to the inoculation amount of 200 mg/L.
8. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein in S3, after fermentation is completed after 10 days, the mycelia are washed with water, freeze-dried and weighed; the fermentation liquor adopts spectrophotometry to determine the content of flavone.
9. The fermentation method of high-yielding phellinus linteus extracellular flavonoids according to claim 1, wherein in S3, after the fermentation day 2, the fermentation broth is sonicated at 25 ℃ for 5 minutes at 50-100W per day at room temperature.
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