CN105483197B - A kind of method of triterpene content in raising Ganoderma lucidum submerged fermentation - Google Patents
A kind of method of triterpene content in raising Ganoderma lucidum submerged fermentation Download PDFInfo
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Abstract
The present invention relates to a kind of methods of triterpene content in raising Ganoderma lucidum submerged fermentation, including prepare CaCl2Aqueous solution and salicylic acid ethanol solution, mycelia Liquid Culture, calcium ion and Induced by Salicylic Acid, mycelia are collected, mycelia triterpene extracts and detection.Calcium ion is added during Ganoderma lucidum submerged fermentation and salicylic acid is induced, the content of triterpene in Ganoderma lucidum mycelium can be significantly improved, the triterpene content of mycelia is higher than control group by 48.26% up to 48.97 mg/g, the quality of ganoderma lucidum is effectively improved, and operating method is simple, at low cost.
Description
Technical field
The present invention relates to a kind of methods of triterpene content in raising edible fungus liquid fermentation, and in particular to a kind of raising ganoderma lucidum
The method of triterpene content in liquid fermentation.
Technical background
Ganoderma lucidum [Ganoderma lucidum(Curtis:Fr.) P.Karst] Gu referred to as seocho, also known as be red sesame, Wan Nian
Gill fungus, red sesame, polyporus lucidus etc., belong on the classification position of fungi mycota (Mycota), Basidiomycotina
(Basidiomycotina), layer bacterium subclass (Hymenomycetes), Aphyllophorales (Aphyllophorales), Polyporaceae
(Polyproraceae), Ganoderma (Ganoderma).Ganodenic acid has good pharmacological action, is mainly manifested in inhibition cancer
Growth, liver protecting, blood pressure lowering, AntiHIV1 RT activity of cell etc..
The quality of ganoderma lucidum quality is closely related with the content of ganodenic acid.Ganoderma lucidum triterpene chemical combination is obtained in production at present
The source of object is mainly the mycelia of mature sporophore and liquid fermentation.The week of the triterpene of ganoderma lucidum is extracted by liquid fermentation technology
Phase is shorter, and easy to operate.With the continuous release of ganoderma lucidum product in the market, it is desirable to more efficiently produce ganodenic acid.
Therefore, the content for how effectively improving triterpene in Ganoderma lucidum submerged fermentation is explored, is with a wide range of applications.
Summary of the invention
The object of the present invention is to provide a kind of methods of triterpene content in raising Ganoderma lucidum submerged fermentation, meet the market requirement,
Also foundation is provided for effective component of glossy ganoderma analysis.
The method of triterpene content, operating procedure are as follows in a kind of raising Ganoderma lucidum submerged fermentation of the invention:
(1) CaCl is prepared2Aqueous solution and salicylic acid ethanol solution: CaCl is used2It is water-soluble that 10mM calcium ion is prepared with sterile water
Liquid；With salicylic acid and ethyl alcohol, 150 μM of salicylic acid ethanol solutions are prepared；
(2) 8 ferfas silk access 100 mycelia Liquid Culture: is got from the plate for cover with Ganoderma lucidum mycelium with 9mm punch
In mL liquid PDA, 28 DEG C, 150rpm shaking table culture 6-7 days, then stationary culture 5-6 days again；The liquid PDA formula are as follows:
200 g potatos boil leachate, and 20 g glucose add water constant volume to 1000mL, 121 DEG C of sterilizing 30min；
(3) the calcium ion aqueous solution 1-5mL and salicylic acid ethanol solution of step (1) calcium ion and Induced by Salicylic Acid: are taken
0.5-1.5mL is mixed, and is added to step (2) in the PDA culture solution of inoculated and cultured using filter membrane degerming method, 28 DEG C of biochemical trainings
Support induction processing 1-2 days in case；
(4) mycelia is collected: collection step (3) induction treated mycelia, and removes inoculation block, is washed with distilled water 3-5
It is secondary, it is placed in 60 DEG C of baking ovens that drying to constant weight；
(5) mycelia triterpene is extracted and is detected: after the mycelia for taking step (4) to dry extracts 2h with alcohol at normal temperature, 150W ultrasound
0.5h, 60 DEG C of rotations are evaporated, and dissolve constant volume with methanol, and detect triterpene content using vanillic aldehyde-ice acetic acid method.
Advantages of the present invention and usefulness: calcium ion is added during Ganoderma lucidum submerged fermentation and salicylic acid is lured
It leads, the content of triterpene in Ganoderma lucidum mycelium can be significantly improved, improve the quality of ganoderma lucidum, and easy to operate at low cost.Ganoderma lucidum three
Terpene is main pharmacological component in ganoderma lucidum, by addition calcium ion and salicylic induction, can effectively improve ganoderma lucidum
The content of triterpene in liquid fermentation, the triterpene content of mycelia are higher than control group by 48.26% up to 48.97 mg/g.
Detailed description of the invention
Fig. 1 is the triterpene content schematic diagram of different incubation time mycelia.In figure, abscissa indicates the number of days of culture, indulges and sits
Mark indicates triterpene content mg/g.
Fig. 2 is the influence schematic diagram for adding calcium ion and salicylic acid to ganodenic acid content.In figure, CK is control, Ca+SA
It indicates to carry out induction processing with method addition calcium ion of the invention and salicylic acid；* it indicates and compares that there were significant differences, P <
0.05；Ordinate indicates triterpene content mg/g.It is not added with calcium ion and salicylic mycelia, triterpene content is 33.03 mg/g；
It adds calcium ion and salicylic mycelia triterpene content is 48.97 mg/g, it is higher than control group by 48.26%.
Specific embodiment
In order to sufficiently disclose the of the invention method for improving the content of triterpene in Ganoderma lucidum submerged fermentation, below in conjunction with example plus
With explanation.
Embodiment 1: a method of improving triterpene content in Ganoderma lucidum submerged fermentation.The following steps are included:
(1) CaCl is prepared2Aqueous solution and salicylic acid ethanol solution: CaCl is used2It is water-soluble that 10 mM calcium ions are prepared with sterile water
Liquid；With salicylic acid and ethyl alcohol, 150 μM of salicylic acid ethanol solutions are prepared；
(2) mycelia Liquid Culture: the ganoderma strain bought using market accesses 90mm's after the inclined-plane PDA is activated
Plate is got in 8 ferfas silk access, 100 mL liquid PDA, 28 DEG C, 150rpm after covering with mycelia on plate with 9mm punch
Shaking table culture 7 days, then stationary culture 5 days again；The liquid PDA formula are as follows: 200 g potatos boil leachate, 20 Portugals g
Grape sugar, adds water constant volume to 1000mL, 121 DEG C of sterilizing 30min；The triterpene content of different incubation time mycelia is as shown in Figure 1；Work as training
It supports by the 13rd day, triterpene content reaches maximum value 33.03mg/g in mycelia.
(3) calcium ion and Induced by Salicylic Acid: taking the calcium ion aqueous solution 1mL and salicylic acid ethanol solution 1mL of step (1),
It mixes, using filter membrane degerming method, that is, uses 1mL asepsis injector via hole diameter for 0.22 μm of sterilized organic filter, be added to
Step (2) is in the PDA culture solution of inoculated and cultured, and induction is handled 1 day in 28 DEG C of biochemical cultivation cases.Add calcium ion and salicylic acid
Influence to ganodenic acid content as shown in Fig. 2, addition calcium ion and salicylic mycelia triterpene content are 48.97 mg/g, than
Control group is high by 48.26%.
(4) mycelia is collected: collection step (3) induction treated mycelia, and removes inoculation block, is washed with distilled water 5 times,
It is placed in 60 DEG C of baking ovens that drying to constant weight；
(5) mycelia triterpene is extracted and is detected: being weighed the mycelia 0.2g of drying, is extracted 2h with 30mL85%(v/v) alcohol at normal temperature
Afterwards, 150W ultrasound 0.5h, 60 DEG C of rotations are evaporated, and are finally settled to 25 mL with methanol dissolution.Using vanillic aldehyde-ice acetic acid method inspection
Survey triterpene content.
The vanillic aldehyde-ice acetic acid method detects triterpene content: taking the sample solution of 0.2mL in tool plug test tube, at 60 DEG C
It is evaporated in water-bath, the perchloric acid of 5% vanillic aldehyde-glacial acetic acid (ready-to-use) and 1mL of 0.4mL is added, 60 DEG C of water-baths after shaking up
15min. adds the glacial acetic acid of 5mL after being cooled to room temperature, 0.5h is stored at room temperature after shaking up, and 546nm carries out the detection of OD value.
Embodiment 2: a method of improving triterpene content in Ganoderma lucidum submerged fermentation.The following steps are included:
(1) CaCl is prepared2Aqueous solution and salicylic acid ethanol solution: CaCl is used2It is water-soluble that 10 mM calcium ions are prepared with sterile water
Liquid；With salicylic acid and ethyl alcohol, 150 μM of salicylic acid ethanol solutions are prepared；
(2) mycelia Liquid Culture: the ganoderma strain bought using market accesses 90mm's after the inclined-plane PDA is activated
Plate is got in 8 ferfas silk access, 100 mL liquid PDA, 28 DEG C, 150rpm after covering with mycelia on plate with 9mm punch
Shaking table culture 6 days, then stationary culture 6 days again；
(3) the calcium ion aqueous solution 5mL and salicylic acid ethanol solution of step (1) calcium ion and Induced by Salicylic Acid: are taken
1.5mL is mixed, and is added to step (2) in the PDA culture solution of inoculated and cultured using filter membrane degerming method, 28 DEG C of biochemical cultures
Induction processing 2 days in case.
(4) mycelia is collected: with gauze collection step (3) induction treated mycelia, and being removed inoculation block, is washed with distillation
It washs 3 times, is placed in 60 DEG C of baking ovens that drying to constant weight.
(5) mycelia triterpene is extracted and is detected: being weighed the mycelia 0.2g of drying, is extracted 2h with 30mL85%(v/v) alcohol at normal temperature
Afterwards, 150W ultrasound 0.5h, 60 DEG C of rotations are evaporated, and finally dissolve 25 mL of constant volume with methanol, and using vanillic aldehyde-ice acetic acid method inspection
Survey triterpene content.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (3)
1. a kind of method for improving triterpene content in Ganoderma lucidum submerged fermentation, it is characterised in that operating procedure is as follows:
(1) CaCl is prepared2Aqueous solution and salicylic acid ethanol solution: CaCl is used210mM calcium ion aqueous solution is prepared with sterile water；With
Salicylic acid and ethyl alcohol prepare 150 μM of salicylic acid ethanol solutions；
(2) 8 ferfas silk 100 mL liquid of access mycelia Liquid Culture: are got from the plate for cover with Ganoderma lucidum mycelium with 9mm punch
In body PDA, 28 DEG C, 150rpm shaking table culture 6-7 days, then stationary culture 5-6 days again；The liquid PDA formula are as follows: 200 g
Potato boils leachate, 20 g glucose plus water constant volume to 1000mL, 121 DEG C of sterilizing 30min；
(3) the calcium ion aqueous solution 1-5mL and salicylic acid ethanol solution 0.5- of step (1) calcium ion and Induced by Salicylic Acid: are taken
1.5mL is mixed, and is added to step (2) in the PDA culture solution of inoculated and cultured using filter membrane degerming method, 28 DEG C of biochemical cultivation cases
Middle induction is handled 1-2 days；
(4) mycelia is collected: collection step (3) induction treated mycelia, and removes inoculation block, is washed with distilled water 3-5 times, is set
Drying to constant weight in 60 DEG C of baking ovens；
(5) mycelia triterpene is extracted and is detected: after the mycelia for taking step (4) to dry extracts 2h with alcohol at normal temperature, 150W ultrasound 0.5h,
60 DEG C of rotations are evaporated, and dissolve constant volume with methanol, and detect triterpene content using vanillic aldehyde-ice acetic acid method.
2. a kind of method for improving triterpene content in Ganoderma lucidum submerged fermentation according to claim 1, it is characterised in that described
Mycelia Liquid Culture: being accessed in 100 mL liquid PDA with 9mm punch from 8 ferfas silk is got on the plate for cover with Ganoderma lucidum mycelium,
28 DEG C, 150rpm shaking table culture 7 days, then stationary culture 5 days again.
3. a kind of method for improving triterpene content in Ganoderma lucidum submerged fermentation according to claim 1, it is characterised in that described
Calcium ion and Induced by Salicylic Acid: taking calcium ion aqueous solution 1mL and salicylic acid ethanol solution 1mL, mixes, is added using filter membrane degerming method
Enter into the PDA culture solution of inoculated and cultured, induction processing 1 day in 28 DEG C of biochemical cultivation cases.
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| CN105506049B (en) * | 2016-02-02 | 2018-11-27 | 华中农业大学 | A method of improving the ganodenic acid content intracellular of ganoderma lucidum mycelium liquid fermentation |
| CN106538237A (en) * | 2016-10-18 | 2017-03-29 | 福建农林大学 | A kind of raising Ganoderma sporophore yield and polysaccharide, the processing method of triterpene content |
| CN109168964B (en) * | 2018-11-28 | 2020-09-01 | 山东农业大学 | Method for increasing content of ganoderma triterpene in ganoderma lucidum fruiting body, compound inducer and application thereof |
| CN109938040B (en) * | 2019-04-22 | 2021-10-01 | 山东省农业科学院生物技术研究中心 | Method for improving peanut root rot resistance by using salicylic acid and calcium |
| CN110982868B (en) * | 2019-11-29 | 2023-09-19 | 贵州中医药大学 | Co-culture method for improving triterpene content of ganoderma lucidum and application thereof |
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