CN105001009B - Antrodia camphorata richness terpene Mycelium culture base and application - Google Patents
Antrodia camphorata richness terpene Mycelium culture base and application Download PDFInfo
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Abstract
The present invention provides a kind of Antrodia camphorata richness terpene Mycelium culture base and applications, belong to technical field of biology, are designed by Placket Burman designs, climbing experimental design, response surface excellent ----dissolve the optimal medium formula for preparing antrodia mycelia：Glucose 10.32gL‑1, 42.58 gL of maltose‑1, 15.37 gL of peptone‑1, MgSO4 1.12 g·L‑1, KH2PO4 0.63 g·L‑1, 0.55 gL of ganoderma lucidum water extract‑1, inoculum concentration 3%, after culture 15 days, mycelium triterpene content is up to 36.84 mgg‑1, can be that antrodia mycelia triterpene content in fluid nutrient medium is promoted in actual industrial production.
Description
Technical field
The present invention relates to a kind of Antrodia camphorata richness terpene Mycelium culture base and applications, belong to technical field of biology.
Background technology
Fungi is the second in the world major class biology, is largely divided into microorganism and macro fungi（Food, medicinal fungus）Two classes.Antrodia camphorata
（Antrodia cinnamomea）It is the distinctive rare medicinal fungus kind in China Taiwan, belongs to Basidiomycota
（Basidiomycota）, Aphyllophorales（Polyporales）, plain boiled pork Daedalea section（Fomitopsidaceae）, antrodia karst
（Antrodia）, find to be grown in rotten hollow cinnamomum kanehirai earliest（Cinnamomum kanehirai）Inner wall, early stage Cinnamomum kanahirai hay
Tree is considered as unique host of Antrodia camphorata.Antrodia camphorata bitter, research are confirmed containing a large amount of triterpenes, phenols, polysaccharide etc.
Substance, can be used for treating antiallergy, anti-inflammatory, anti-oxidant, antitumor, liver protection, raising immunity etc. has remarkable efficacy.Cause
This, Antrodia camphorata is high in the market price and supply falls short of demand.
To solve the present situation that Antrodia camphorata fructification is expensive in the market, supply falls short of demand, the present invention is with glucose, malt
Sugar, peptone, KH2PO4、MgSO4、VB1, JUNCAO Ganoderma lucidum water extract, JUNCAO Ganoderma lucidum alcohol extracting thing and grass-cultivated ganoderma lucidum dreg water extraction
Object is screening factor, preferably goes out to significantly improve culture medium prescription and the application of antrodia mycelia triterpene content.
Invention content
In order to further increase the ability that antrodia mycelia generates triterpene compound, present invention offer is a kind of can be into one
Step promotes the culture medium prescription and its application that triterpene compound synthesizes in antrodia mycelia.
Antrodia camphorata richness terpene Mycelium culture base, the culture medium prescription are：Glucose 10-11 gL-1, maltose 42-43
g·L-1, peptone 15-16 gL-1, MgSO4 1-1.5 g·L-1, KH2PO4 0.5-1 g·L-1, ganoderma lucidum water extract 0.5-
1 g·L-1。
Its cultural method is that inoculum concentration is 3%, at 28 DEG C, 120 rpm, dark culturing 15 days or so.
The culture medium prescription is：10 .32gL of glucose-1, 42.58 gL of maltose-1, 15.37 g of peptone
L-1, MgSO4 1.12 g·L-1, KH2PO4 0.63 g·L-1, 0.55 gL of ganoderma lucidum water extract-1。
Antrodia mycelia triterpene extracting method, it is characterised in that：The method is：It takes and crushes uniform Antrodia camphorata mycelia
Appropriate body, using 80% ethyl alcohol as Extraction solvent, solid-liquid ratio 1: 40（w/v）, 1 h of ultrasound, pours into extraction in 70 DEG C of water-baths
Liquid, repetition aforesaid operations are primary, merge extracting solution and simultaneously filter, concentrate, take supernatant constant volume to 50 mL after centrifugation；Take 0.5 mL
It is added in scale test tube, is placed in solvent flashing in 50-60 DEG C of water-bath, 0.3 5% vanillic aldehydes of mL-glacial acetic acid solution and 1 is added
10 mL glacial acetic acid are added after ice water cooling in the perchloric acid of mL, 60 DEG C of 20 min of water-bath, are control with blank after shaking up,
Measurement light absorption value is swept at 540 nm, and the regression equation calculation triterpene content obtained is made using standard items oleanolic acid.
Application of the antrodia mycelia culture medium in antrodia mycelia culture.
Present invention solves the technical problem that used technical solution is：
1 antrodia mycelia culture and triterpene assay
Antrodia mycelia condition of culture is 28 DEG C, and it is dry under the conditions of 55 DEG C to collect mycelium for dark culturing 15-28 days
To constant weight, the mycelium after drying measures its triterpene content using vanillic aldehyde-ice acetic acid method.
2 Placket-Burman are designed
Plackett-Burman（PB）Design is a kind of effective ways screened for key factor in multivariable.Experiment
It is designed using 11 Plackett-Burman, by lot of documents information, 9 factors of assessment is chosen on the basis of previous experiments
To the difference of triterpene content in antrodia Mycelia body.Each factor sets 2 levels：- 1 represents low-level, and+1 represents
High level（Table 1）.The level value of different factors is set according to forefathers' experimental result, utilizes Design Expert 8.0.6 softwares
Result is analyzed, the factor for significantly affecting Antrodia camphorata Mycelia body triterpene compound content is filtered out.
1 Plackett-Burman design levels of table and influence factor (gL-1)
2 PB contrived experiments of table and result
PB designs the result shows that antrodia mycelia triterpene content from 15.36-47.3 mgg-1（Table 2）, wherein Portugals A-
Grape sugar, B- maltose, D- peptones, E-KH2PO4, H- MgSO4Antrodia camphorata mycelium triterpene is contained with K- JUNCAO Ganoderma lucidums water extract
Measurer has significant facilitation；And A- glucose has the function of negative correlation to the growth of mycelium triterpene content.Analysis result
Show A- glucose, B- maltose, D- peptones, E-KH2PO4, H-MgSO4In a model with K- JUNCAO Ganoderma lucidums water extracts
Importance is up to 95% or more, is considered the principal element that can significantly affect antrodia mycelia triterpene content（Table 3）,
It can carry out next step experiment.
3 PB design variables influence factor of table and interpretation of result
The climbing experiment of 3 steepests
Steepest hill climbing test is climbing direction with the change direction of Plackett-Burman test values, is tested and is tied according to PB
Fruit determines change step to the effect value of each factor（Table 4）, most fast approaches optimum value range, is with mycelial biomass content
Investigation object screens highest processing and is set as the central point of next step response surface analysis.
The climbing experimental design of 4 steepest of table and result (gL-1)
Table 4 the result shows that, when glucose be 20 gL-1, 40 gL of maltose-1, 20 gL of peptone-1, KH2PO4For
0.8 g·L-1, MgSO4For 0.8 gL-1, JUNCAO Ganoderma lucidum water extract is 0.75 gL-1When, mycelium triterpene content
Therefore this central point that face is tested in response is carried out experimental design by highest.
4 response surface experimental designs
According to above experimental result, response surface design is carried out by Box-Behnken（Table 5）, and result is subjected to ANOVA
Analysis（Table 6）.The result shows that the model has conspicuousness（p<0.05）, F values are 2.16, are shown with this experimental data good
Correlation.Check the related coefficient for the model for meeting efficiency（R2=0.5866）Coefficient is determined with adjustment（Adj R2=0.3153),
Show that equation model is preferable.C.V. value indicates the accuracy of experiment, and C.V. values are higher, and the reliability of experiment is lower, this research
Middle C.V.=6.84% illustrates that experimental implementation is credible.Therefore, which can be as the influence of the trend prediction biomass of assessment.
As can be seen from the table, linear coefficient（X3, X6）, interaction coefficent（X1X4, X1X6）With significant difference（p<0.05）, and other are then
Without significant difference（p>0.05）.In our current research, the sequence for influencing hypha biomass factor is：Ganoderma lucidum water extract>Peptone>
Maltose>Glucose> MgSO4> KH2PO4.Data result (Y) and the matrix of RSM experiments determine the influence of 6 variables, wrap
Include glucose (X1), maltose (X2), peptone (X3)、MgSO4（X4）、KH2PO4（X5）、JCGLWE（X6）It is suitble to following second order more
Item formula equation（Eq. 1）：
（Eq.1）
5 Box-Behnken of table designs and result
6 ANOVA of table analyses and mycelium triterpene are assessed containing coefficient of discharge
By showing that liquid fermentation antrodia mycelia triterpene culture medium condition is to the optimization of condition of culture：Glucose
10.32 g·L-1, 42.58 gL of maltose-1, 15.37 gL of peptone-1, MgSO4 1.12 g·L-1, KH2PO4 0.63
g·L-1, 0.5 gL of ganoderma lucidum water extract-1When, antrodia mycelia triterpene content may be up to 36.84 mg/g.Pass through verification
Experiment, it is 36.84 ± 0.103 mgg that liquid fermentation, which produces antrodia mycelia triterpene yield actual value,-1(n=3), actual value
It is consistent with predicted value result, far it is higher by control group（25 gL of glucose-1, 5 gL of peptone-1, 3 gL of maltose-1）Institute
Production antrodia mycelia triterpene content is 28.47 mgg-1, therefore, which has preferable predictability, can be used as and promote ox
The theoretical foundation of Antrodia camphorata mycelium triterpene content liquid fermentation medium optimization.
The benefit of the present invention：By the screening and combination to a number of factors, select to antrodia mycelia triterpene content shadow
It is glucose, peptone, maltose, MgSO to ring larger factor4、KH2PO4With ganoderma lucidum water extract, culture medium prescription is Portugal
Grape sugar 10-11 gL-1, maltose 42-43 gL-1, peptone 15-16 gL-1, MgSO4 1-1.5 g·L-1, KH2PO4
0.5-1 g·L-1, ganoderma lucidum water extract 0.5-1 gL-1When, antrodia mycelia only can be made by the adjustment of culture medium prescription
A large amount of triterpene substances are produced, product quality is promoted, the maximum amount of Antrodia camphorata mycelia can be obtained in industrialized production and application
The triterpene substance of body.
Specific implementation mode
Embodiment 1
The antrodia mycelia culture medium of the present embodiment is prepared in the following proportions：10 g of glucose, 4.2 g of maltose, egg
White peptone 15 g, MgSO4 1g, KH2PO40.6 g, in 1000 mL water, adjust pH value is 0.5 g constant volumes of ganoderma lucidum water extract
5.5, every 100 mL is dispensed into 250 mL triangular flasks, and sterilize 20 min at 121 DEG C, spare after cooling.
Cultural method：At 28 DEG C, 120 rpm, dark culturing 15 days.
Through measuring, mycelia triterpene content is 35.67 mgg-1。
Embodiment 2
The antrodia mycelia culture medium of the present embodiment is prepared in the following proportions：100 g of glucose, 42 g of maltose, egg
White peptone 15 g, MgSO4 10g, KH2PO46 g, ganoderma lucidum water extract 5g constant volumes are in 10 L water, and it is 5.5 to adjust pH value, every 1 L
In packing to triangular flask, sterilize 20 min at 121 DEG C, spare after cooling.
Cultural method：At 28 DEG C, 120 rpm, dark culturing 15 days.
Through measuring, mycelia triterpene content is 38.98 mgg-1。
Embodiment 3
The antrodia mycelia culture medium of the present embodiment is prepared in the following proportions：500 g of glucose, 210 g of maltose, egg
White peptone 75 g, MgSO4 50g, KH2PO430 g, for ganoderma lucidum water extract 25g constant volumes in 50 L water, it is 5.5, every 5 to adjust pH value
L is dispensed into small-sized fermentation tank, and sterilize 20 min at 121 DEG C, and internal stirring ventilation is spare after inoculation.
Cultural method：At 28 DEG C, 120 rpm, cultivate 15 days.
Through measuring, mycelia triterpene content is 43.98 mgg-1。
Embodiment 4
The antrodia mycelia culture medium of the present embodiment is prepared in the following proportions：Glucose 10.32g, maltose 42.58
G, peptone 15.37 g, MgSO4 1.12 g, KH2PO40.63 g, 0.55 g of ganoderma lucidum water extract, constant volume is in 1000mL water
In, it is 5.5 to adjust pH value, is dispensed into 250mL triangular flasks per 100mL, and sterilize 20min at 121 DEG C, spare after cooling.
Cultural method：At 28 DEG C, 120 rpm, cultivate 15 days.
Through measuring, mycelia triterpene content is 36.84 mgg-1。
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 should all belong to the covering scope of the present invention.
Claims (4)
1. Antrodia camphorata richness terpene Mycelium culture base, it is characterised in that：The culture medium prescription is：500 g of glucose, maltose
210 g, peptone 75 g, MgSO4 50g, KH2PO430 g, ganoderma lucidum water extract 25g constant volumes are in 50 L water.
2. Antrodia camphorata richness terpene Mycelium culture base according to claim 1, it is characterised in that：Its cultural method is inoculum concentration
It is 3%, at 28 DEG C, 120 rpm, dark culturing 15 days.
3. Antrodia camphorata richness terpene Mycelium culture base according to claim 1, it is characterised in that：The culture medium prescription is：
Glucose 10.32gL-1, 42.58 gL of maltose-1, 15.37 gL of peptone-1, MgSO4 1.12 g·L-1, KH2PO4
0.63 g·L-1, 0.55 gL of ganoderma lucidum water extract-1。
4. a kind of a kind of Antrodia camphorata richness terpene Mycelium culture base answering in antrodia mycelia culture as described in claim 1
With.
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| CN108048336A (en) * | 2017-12-30 | 2018-05-18 | 佛山科学技术学院 | A kind of nitrogen source fluid nutrient medium of Antrodia camphorata and its application |
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| TW201720917A (en) * | 2015-12-02 | 2017-06-16 | Ultra-Microrigin Biomedical Technology Co Ltd | Cultivation method for raising triterpenoid component of Antrodia cinnamomea to cultivate for one month to be able to harvest Antrodia cinnamomea with large amount of triterpenoid without needing to use Antrodia basswood for cultivation |
| CN107242026B (en) * | 2017-04-27 | 2020-06-02 | 永腾生技有限公司 | Culture method and culture medium for white antrodia cinnamomea fruiting bodies |
| CN107573122B (en) * | 2017-09-27 | 2018-12-14 | 江苏农林职业技术学院 | The liquid fermentation culturing method and culture medium of Antrodia camphorata |
| CN108157065A (en) * | 2018-01-05 | 2018-06-15 | 江苏农林职业技术学院 | A kind of mycelial cultural method of Antrodia camphorata and its application |
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| CN111937678B (en) * | 2020-08-28 | 2022-04-05 | 青岛国海生物制药有限公司 | Antrodia camphorata culture medium and preparation method thereof |
| CN113913359B (en) * | 2021-11-18 | 2024-03-01 | 福建农林大学 | Solid culture medium for promoting growth of antrodia mycelium and synthesis of triterpene |
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