CN118048244A - Phellinus linteus strain Kong Rendong with high polyphenol yield, culture medium and culture method thereof - Google Patents
Phellinus linteus strain Kong Rendong with high polyphenol yield, culture medium and culture method thereof Download PDFInfo
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- 241000001727 Tropicoporus linteus Species 0.000 title claims abstract description 32
- 239000001963 growth medium Substances 0.000 title claims abstract description 25
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 23
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 23
- 238000012136 culture method Methods 0.000 title description 6
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 241000001746 Sanghuangporus lonicericola Species 0.000 claims abstract description 4
- 239000000284 extract Substances 0.000 claims description 34
- 240000000249 Morus alba Species 0.000 claims description 19
- 235000008708 Morus alba Nutrition 0.000 claims description 19
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- 239000008103 glucose Substances 0.000 claims description 15
- 244000061456 Solanum tuberosum Species 0.000 claims description 14
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 14
- 238000009630 liquid culture Methods 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- 239000004475 Arginine Substances 0.000 claims description 12
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 12
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- 235000019198 oils Nutrition 0.000 claims description 12
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- 238000012258 culturing Methods 0.000 claims description 11
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- 238000000034 method Methods 0.000 claims description 10
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- 239000002609 medium Substances 0.000 claims description 9
- 229930003270 Vitamin B Natural products 0.000 claims description 8
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 8
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- 238000001035 drying Methods 0.000 claims description 6
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- 241000205585 Aquilegia canadensis Species 0.000 claims 6
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- 238000012827 research and development Methods 0.000 abstract 1
- 241001570521 Lonicera periclymenum Species 0.000 description 15
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- 239000000243 solution Substances 0.000 description 7
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 4
- 229930003231 vitamin Natural products 0.000 description 4
- 235000013343 vitamin Nutrition 0.000 description 4
- 239000011782 vitamin Substances 0.000 description 4
- 229940088594 vitamin Drugs 0.000 description 4
- 150000003722 vitamin derivatives Chemical class 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
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- 241000245240 Lonicera Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- 240000001980 Cucurbita pepo Species 0.000 description 1
- 235000009852 Cucurbita pepo Nutrition 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
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- 240000002262 Litsea cubeba Species 0.000 description 1
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- 240000005001 Paeonia suffruticosa Species 0.000 description 1
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- 241000001791 Sanghuangporus Species 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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Abstract
The invention discloses a small Kong Rendong Phellinus linteus Sanghuangporus lonicericola strain SiL114 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 41267 in the year of 2024 and 18. The culture medium and culture conditions of the strain are also disclosed. The strain and the culture medium thereof can effectively improve the polyphenol yield in the liquid fermentation process, and lay an important foundation for the related pharmacological research and development.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-yield polyphenol small Kong Rendong Phellinus linteus Sanghuangporus lonicericola strain, a culture medium and a culture method thereof.
Background
A large amount of data and researches prove that the Phellinus linteus has remarkable pharmacological effects, including tumor inhibition, antioxidation, bacteriostasis and anti-inflammation, organism immunity improvement, blood sugar and blood lipid reduction, gastrointestinal tract flora improvement and the like. Phellinus linteus S.lonicoloa (Parmasto) L.W.Zhou & Y.C.Dai, belonging to the kingdom Fungi Fungi, basidiomycota Basidiomycota, agaricus Agaricomycetes, phlebsiella cataria Hymenochaetales, sang Huangkong genus Sanghuangporus Sheng H.Wu, L.W.Zhou & Y.C.Dai, is sold as a species in the group of Morus alba Huang Lei on Lonicera sp. Living and falling woods grown in spring, summer, autumn in areas such as Heilongjiang, jilin, etc., as Phellinus linteus Sanghuangporus sanghuang (root H.Wu, T.Hatt. & Y.C.Dai) in the mountain market.
Disclosure of Invention
The invention provides a small Kong Rendong Phellinus linteus S.ronickellisia strain with high yield of polyphenol, a culture medium and a culture method thereof, so as to obtain the polyphenol efficiently through liquid fermentation, and lay an important foundation for resource protection and development and utilization of corresponding health care products.
The invention is realized by the following technical scheme:
The invention discloses a small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 41267 in the year of 2024 and the month of 03.
The invention also discloses a culture method of the small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114, which comprises the following steps:
(1) Inoculating the small Kong Rendong Phellinus linteus S.ronicicola strain on a solid culture medium, activating, and culturing for 7 days in a constant temperature incubator at 24 ℃;
(2) Inoculating the activated strain on a basic liquid culture medium, and carrying out shaking culture at a constant temperature of a shaking table at 24 ℃ for 7 days at 150r/min to prepare seed fermentation suspension;
(3) Inoculating the seed fermentation suspension on a liquid culture medium, and performing shaking culture at a constant temperature of a shaking table at 24 ℃ and 150r/min for 8 days to obtain a solution containing small Kong Rendong Phellinus linteus S.lonicoloa extracellular polyphenol.
Further, the solid medium in step (1) is: peeled potato 300.0g/L, glucose 20.0g/L, agar 20.0g/L, KH 2PO4 1.0.0 g/L, and pH 5.0.
Further, the basic liquid medium in step (2) is: 30.0g/L of peeled potato, 10.0g/L of glucose, 6.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 15.0mL/L of mulberry extract/honeysuckle extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
Further, the liquid medium in the step (3) is: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of mulberry extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
Or the liquid culture medium in the step (3) is as follows: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of honeysuckle extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
Further, the preparation method of the mulberry extract in the steps (2) and (3) comprises the following steps: drying mulberry at 60deg.C for 24 hr, cutting into 1-3cm pieces, mechanically pulverizing with plant pulverizer, sieving with 80 mesh sieve, soaking in ethylene glycol at ratio of 1:50, and treating at 30deg.C for 40min under 300W in ultrasonic crusher to obtain mulberry extract.
Further, the preparation method of the honeysuckle extract in the steps (2) and (3) comprises the following steps: drying the honeysuckle at 60 ℃ for 24 hours, cutting the dried honeysuckle into 1-3cm small pieces, mechanically crushing the small pieces by a plant crusher, sieving the small pieces by a 80-mesh sieve, immersing the small pieces in acetone according to the ratio of 1:50, and treating the small pieces in an ultrasonic crusher at 300W for 40 minutes at 30 ℃ to obtain the honeysuckle extract.
The invention also discloses an application of the small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114 in preparing high-yield fermentation polyphenol.
The invention also discloses an application of the small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114 in preparing medicines for improving human immunity or resisting cancer.
Advantageous effects
The invention discloses a small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114, a culture medium and a culture method, wherein under the same culture condition, a carbon source and a nitrogen source are optimized, and then mulberry extract/honeysuckle extract, arginine and sunflower seed oil are simultaneously added, so that the yield of polyphenol is greatly improved. The culture medium is simple to manufacture, convenient to operate, low in production and processing cost, high in yield of active products such as polyphenol, convenient for commercial culture, and capable of being prepared into medicines and widening the application field.
Strain preservation information
Preservation time: 2024, 03, 18;
preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
preservation number: CGMCC No.41267;
Deposit unit address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, no. 3, chat.Chao, beijing, city;
classification naming: phellinus linteus S.lonicoloa strain SiL114, kong Rendong.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1 screening, identification and purification of strains
Test strain: the small Kong Rendong Phellinus linteus strain SiL114 is collected from the national-grade scenic spot of the Sharpleaf of Paeonia suffruticosa, heilongjiang province, and the host is Lonicera sp. Living Litsea, and is preserved in China general microbiological culture Collection center (CGMCC) No.41267 at 18 months of 2024.
Fruiting body shape characteristics: the basidiomycete fruits are perennial, have no handle cover shape, are single, are fresh in wood, have no smell and smell, and are woody after being dried; the fungus cover is semicircular, the surface is cracked by fine fluff at the initial stage and coarse and irregular at the later stage, a thin shell is formed, the length is 8cm, the width is 8cm, the thickness of the basal part is 3cm, the fungus cover is black brown or gray brown, concentric ring grooves are formed, the edge is blunt, the fungus cover is pale yellow in young, the fungus cover is yellow brown or dark brown in mature, the sterile edge of the yellow brown is obvious, and the width can reach 5mm; the surface of the orifice is yellow brown to rusty brown, has refraction, has thin and full edges and is nearly circular, and the number of the orifice is 8/mm; the fungus meat is yellow brown when fresh, and wood is in a shape of a bolt after being dried, and the thickness of the wood is 1cm; the fungus tube is multi-layered, obvious in layering, golden brown in young, light brown in mature and 20mm long.
Microstructure characteristics: a mycelium two-system is simply separated, has no pseudo-dextrin reaction, has bluphilic reaction and turns black in potassium hydroxide; the germ hyphae of the fungus meat account for a majority of 3.7-4.5 mu m, and are colorless to pale yellow or golden yellow, and thin wall to thick wall; the skeleton hypha occupies a small number, 4.3-5 mu m, is golden yellow, has a thick wall and a cavity, is not separated and branched, and is regular in arrangement; the bacterial tube reproduction hypha accounts for a few, 1.8-2.6 mu m, is colorless, thin-walled and occasionally branched; the skeleton hypha is mostly 2.4-3.2 mu m, is golden yellow, is thick-walled and has a medium-degree cavity, is occasionally separated, is not branched and is arranged approximately parallel to the fungus tube; the fruiting layer has a large number of bristles, a gourd shape, sharp top end, brown color, thick wall, length 15-21×width 5-7 μm; the sub-layers have crystals and diamond shapes; the basidiomycete has a narrow cylindrical shape, 4 basidiomycete stems are arranged at the top end, the basidiomycete stems are simply separated, and the length is 7.6-9.7 times the width is 4.4-5.6 mu m; the basidiospores are widely elliptical, yellow brown, free of dextrin-like reaction, free of blue-philic reaction, thick-walled, smooth, 3.3-4.2 (-4.6) long (3.1-) x wide (2.3-) 2.4-3.3 (-3.6) mu m, average length of 3.97 mu m, average width of 2.94 mu m, and aspect ratio of 1.35 (30 spores total).
Identification of strain molecules: the isolated strain SiL114 was subjected to genomic DNA extraction using a rapid, non-toxic plant DNA extraction kit (FH PLANT DNA KIT) from german company. PCR amplification is carried out on the transcription spacer (internal transcribed spacer, ITS) by taking the transcription spacer as a template, and the ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3')/ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') genes are taken as primers, wherein the total system is 30.0 mu L: 2X EASYTAQ PCR Supermix 15.0. Mu.L, primer ITS 5. Mu. Mol/L1.0. Mu.L, primer ITS4 10. Mu. Mol/L1.0. Mu.L, genomic DNA 1.0. Mu.L, deionized water 12.0. Mu.L. The procedure is: pre-denaturation at 95℃for 3min; denaturation at 94℃for 40s, annealing at 54℃for 45s, elongation at 72℃for 1min, and repetition of 34 cycles; finally, the temperature is prolonged for 10min at 72 ℃; preserving heat at 4 ℃. The PCR product was sent to Beijing Liuhua macrogene technologies Co., ltd for sequencing.
And (3) comparison: the sequencing results were Blast aligned in NCBI (National Center for Biotechnology Information) database (https:// www.ncbi.nlm.nih.gov /), and the similarity to Phellinus linteus of Kong Rendong was highest, so strain SiL114 was identified as Sanghuangporus lonicericola (Parmasto) L.W.Zhou & Y.C.Dai, genBank accession number PP447545.
EXAMPLE 2 cultivation of strains
Preparing a solid culture medium: peeled potato 300.0g/L, glucose 20.0g/L, agar 20.0g/L, potassium dihydrogen phosphate 1.0g/L, pH 5.0,1X 10 5 Pa, and autoclaving for 30min.
Solid culture: activating on solid culture medium, and culturing in a constant temperature incubator at 24 ℃ for 7 days.
Preparing a basic liquid culture medium: peeled potato 30.0g/L, glucose 10.0g/L, wheat bran 6.0g/L, potassium dihydrogen phosphate 1.0g/L, zinc sulfate heptahydrate 0.3g/L, magnesium sulfate heptahydrate 0.6g/L, mulberry extract/honeysuckle extract 15.0mL/L, arginine 11.0mg/L, sunflower seed oil 1.2mL/L, vitamin B1.0 mg/L, pH 5.0,1 × 5 Pa, and autoclaved for 30min.
The liquid culture medium components are respectively prepared (specific components are shown in table 1): peeled potato 30.0g/L, glucose 10.0g/L, wheat bran 4.0g/L, potassium dihydrogen phosphate 1.0g/L, zinc sulfate heptahydrate 0.3g/L, magnesium sulfate heptahydrate 0.6g/L, mulberry extract 20.0mL/L, arginine 11.0mg/L, sunflower seed oil 1.2mL/L, vitamin B1.0 mg/L, pH 5.0,1 X10 5 Pa, and autoclaving for 30min.
Peeled potato 30.0g/L, glucose 10.0g/L, wheat bran 4.0g/L, potassium dihydrogen phosphate 1.0g/L, zinc sulfate heptahydrate 0.3g/L, magnesium sulfate heptahydrate 0.6g/L, honeysuckle extract 20.0mL/L, arginine 11.0mg/L, sunflower seed oil 1.2mL/L, vitamin B1.0 mg/L, pH 5.0,1 ×10 5 Pa, and autoclaved for 30min.
The preparation method of the mulberry extract comprises the following steps: drying mulberry at 60deg.C for 24 hr, cutting into 1-3cm pieces, mechanically pulverizing with plant pulverizer, sieving with 80 mesh sieve, soaking in ethylene glycol at ratio of 1:50, and treating at 30deg.C for 40min under 300W in ultrasonic crusher to obtain mulberry extract.
The preparation method of the honeysuckle extract comprises the following steps: drying the honeysuckle at 60 ℃ for 24 hours, cutting the dried honeysuckle into 1-3cm small pieces, mechanically crushing the small pieces by a plant crusher, sieving the small pieces by a 80-mesh sieve, immersing the small pieces in acetone according to the ratio of 1:50, and treating the small pieces in an ultrasonic crusher at 300W for 40 minutes at 30 ℃ to obtain the honeysuckle extract.
Liquid culture: taking 250mL triangular flask to split 100mL of basic liquid culture medium, inoculating 5 bacterial cakes with the diameter of 1.0cm, and culturing for 7 days at the constant temperature shaking table 24 ℃ under 150r/min in an oscillating way. Preparing the culture into seed fermentation suspension by using an internal cutting type refiner at 5000r/min and 1min, and fully oscillating for standby. The seed fermentation suspension was added to a 250mL triangular flask containing 100mL of liquid medium at an inoculum size of 10.0% (V/V), and the mixture was subjected to shaking culture at a constant temperature of shaking table 24℃for 8 days at 150r/min to obtain a solution containing small Kong Rendong Phellinus linteus S.lonicoloa extracellular polyphenol.
In the liquid culture process, the basic liquid culture medium and the extract added in the liquid culture medium are the same in kind.
Extraction of polyphenols: centrifuging the fermentation liquor for 20min by 12000r/min to obtain supernatant, and concentrating in vacuum to 1/3 of the original volume; adding 3 times of pre-cooled 95% ethanol, standing overnight at 4deg.C, and removing precipitate (removing polysaccharide) to obtain supernatant; adding chloroform with 3 times of volume for extraction, removing upper layer (removing impurities) to obtain lower layer water solution; then adding 3 times of ethyl acetate for extraction, concentrating and freeze-drying to prepare the small Kong Rendong Phellinus linteus S.lonicoloa extracellular polyphenol water solution.
Determination of polyphenols: respectively sucking 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7mL of 0.08g/L of pyrogallol standard solution and 0.3mL of 1.0mol/L of Fu Lin Fen reagent, mixing, placing for 8min in a dark place, adding 0.6mL of 10% (m/V) sodium carbonate solution, uniformly mixing, reacting for 30min at 25 ℃ in a dark place, fixing the volume to 5.0mL by deionized water, and measuring the light absorption value at 750 nm; setting 3 parallels, and averaging; taking the concentration of the pyrogallol in the reaction system as an abscissa and the light absorption value as an ordinate as a standard curve, and calculating a regression equation as follows: y= 0.2350x-0.1830, and the correlation coefficient R 2 = 0.9968. And (3) measuring the polyphenol content of the sample according to a standard curve manufacturing method, wherein a reaction system of replacing supernatant and a Fu Lin Fen reagent with deionized water is used as a blank and a reference respectively, and the polyphenol content is calculated according to the o-phloroglucinol equivalent in the culture solution.
Results:
The levels of the factors shown in Table 1 and the results of the test (other ingredients: glucose 10.0g/L, potassium dihydrogen phosphate 1.0g/L, zinc sulfate heptahydrate 0.3g/L, magnesium sulfate heptahydrate 0.6g/L, vitamin B1.0 mg/L)
As can be seen from Table 1 above, the extracts added to the medium are different and the polyphenol yields in the fermentation broth are different; the same extract was added, the amount of extract added was different, and the yield of polyphenols in the fermentation broth was also different.
Analysis shows that the liquid culture medium for promoting the improvement of extracellular polyphenol in the fermentation broth aiming at the mulberry extract is as follows: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of mulberry extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
Aiming at the honeysuckle extract, the liquid culture medium for promoting the improvement of extracellular polyphenol in the fermentation broth is as follows: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of honeysuckle extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
According to the invention, the culture medium is optimized, so that the yield of extracellular polyphenol is obviously improved, the large-scale culture of the strain and the extraction of the product are facilitated, the development of medicinal products by utilizing fermentation products can be promoted, and the application field of the strain is widened.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. A Phellinus linteus Sanghuangporus lonicericola strain SiL114 of Kong Rendong is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 41267 at 18/2024.
2. A method of culturing the small Kong Rendong Phellinus linteus s.lonicoloa strain SiL114 as defined in claim 1, comprising the steps of:
(1) Inoculating the small Kong Rendong Phellinus linteus S.ronicicola strain on a solid culture medium, activating, and culturing for 7 days in a constant temperature incubator at 24 ℃;
(2) Inoculating the activated strain on a basic liquid culture medium, and carrying out shaking culture at a constant temperature of a shaking table at 24 ℃ for 7 days at 150r/min to prepare seed fermentation suspension;
(3) Inoculating the seed fermentation suspension on a liquid culture medium, and performing shaking culture at a constant temperature of a shaking table at 24 ℃ and 150r/min for 8 days to obtain a solution containing small Kong Rendong Phellinus linteus S.lonicoloa extracellular polyphenol.
3. The method for culturing the small Kong Rendong Phellinus linteus strain Sil114 according to claim 2, wherein the solid medium in the step (1) is: peeled potato 300.0g/L, glucose 20.0g/L, agar 20.0g/L, KH 2PO4 1.0.0 g/L, and pH 5.0.
4. The method for culturing the small Kong Rendong Phellinus linteus strain Sil114 according to claim 2, wherein the basic liquid medium in the step (2) is: 30.0g/L of peeled potato, 10.0g/L of glucose, 6.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 15.0mL/L of mulberry extract/honeysuckle extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
5. The method for culturing the small Kong Rendong Phellinus linteus strain Sil114 according to claim 2, wherein the liquid medium in the step (3) is: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of mulberry extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
6. The method for culturing the small Kong Rendong Phellinus linteus strain Sil114 according to claim 2, wherein the liquid medium in the step (3) is: 30.0g/L of peeled potato, 10.0g/L of glucose, 4.0g/L of wheat bran, 1.0g/L of monopotassium phosphate, 0.3g/L of zinc sulfate heptahydrate, 0.6g/L of magnesium sulfate heptahydrate, 20.0mL/L of honeysuckle extract, 11.0mg/L of arginine, 1.2mL/L of sunflower seed oil, 1.0mg/L of vitamin B and pH 5.0.
7. The method for culturing small Kong Rendong Phellinus linteus strain Sil114 according to claim 4 or 5, wherein the preparation method of the mulberry extract is as follows: drying mulberry at 60deg.C for 24 hr, cutting into 1-3cm pieces, mechanically pulverizing with plant pulverizer, sieving with 80 mesh sieve, soaking in ethylene glycol at ratio of 1:50, and treating at 30deg.C for 40min under 300W in ultrasonic crusher to obtain mulberry extract.
8. The method for culturing the small Kong Rendong Phellinus linteus strain Sil114 as set forth in claim 4 or 6, wherein the preparation method of the honeysuckle extract is as follows: drying the honeysuckle at 60 ℃ for 24 hours, cutting the dried honeysuckle into 1-3cm small pieces, mechanically crushing the small pieces by a plant crusher, sieving the small pieces by a 80-mesh sieve, immersing the small pieces in acetone according to the ratio of 1:50, and treating the small pieces in an ultrasonic crusher at 300W for 40 minutes at 30 ℃ to obtain the honeysuckle extract.
9. Use of the small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114 as defined in claim 1 for the preparation of high-yield polyphenols.
10. An application of a small Kong Rendong Phellinus linteus S.lonicoloa strain SiL114 as claimed in claim 1 in preparing medicines for improving human immunity or resisting cancer.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000066822A (en) * | 1999-04-21 | 2000-11-15 | 이영호 | Method for culturing Phellinus linteus on the solid medium |
| JP2003259857A (en) * | 2002-03-08 | 2003-09-16 | Oubiken:Kk | Liquid culture for mycelium of phellinus linteus |
| JP2006271339A (en) * | 2005-03-30 | 2006-10-12 | Naris Cosmetics Co Ltd | Liquid culture medium for phellinus linteus |
| CN113249226A (en) * | 2021-01-29 | 2021-08-13 | 上海理工大学 | Fermentation method for improving biomass and total triterpene content of phellinus igniarius |
| CN114854663A (en) * | 2022-05-27 | 2022-08-05 | 中南林业科技大学 | Method for increasing yield of intracellular flavone of Lonicera Appendula |
| CN116948836A (en) * | 2023-06-05 | 2023-10-27 | 北京林业大学 | Ganoderma lucidum strain with high flavone yield, culture medium, culture method and application thereof |
| CN117126745A (en) * | 2023-05-30 | 2023-11-28 | 北京林业大学 | Medicinal fungus ganoderma lucidum, culture medium suitable for high-yield extracellular polyphenol of medicinal fungus ganoderma lucidum, and culture method and application thereof |
-
2024
- 2024-03-26 CN CN202410346509.5A patent/CN118048244A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000066822A (en) * | 1999-04-21 | 2000-11-15 | 이영호 | Method for culturing Phellinus linteus on the solid medium |
| JP2003259857A (en) * | 2002-03-08 | 2003-09-16 | Oubiken:Kk | Liquid culture for mycelium of phellinus linteus |
| JP2006271339A (en) * | 2005-03-30 | 2006-10-12 | Naris Cosmetics Co Ltd | Liquid culture medium for phellinus linteus |
| CN113249226A (en) * | 2021-01-29 | 2021-08-13 | 上海理工大学 | Fermentation method for improving biomass and total triterpene content of phellinus igniarius |
| CN114854663A (en) * | 2022-05-27 | 2022-08-05 | 中南林业科技大学 | Method for increasing yield of intracellular flavone of Lonicera Appendula |
| CN117126745A (en) * | 2023-05-30 | 2023-11-28 | 北京林业大学 | Medicinal fungus ganoderma lucidum, culture medium suitable for high-yield extracellular polyphenol of medicinal fungus ganoderma lucidum, and culture method and application thereof |
| CN116948836A (en) * | 2023-06-05 | 2023-10-27 | 北京林业大学 | Ganoderma lucidum strain with high flavone yield, culture medium, culture method and application thereof |
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