CN112779302A - Method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid - Google Patents

Method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid Download PDF

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CN112779302A
CN112779302A CN202110291795.6A CN202110291795A CN112779302A CN 112779302 A CN112779302 A CN 112779302A CN 202110291795 A CN202110291795 A CN 202110291795A CN 112779302 A CN112779302 A CN 112779302A
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antrodin
fermentation
antrodia camphorata
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汪雯翰
贾薇
白旭
张赫男
张劲松
杨焱
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Shanghai Academy of Agricultural Sciences
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    • 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
    • C12P17/10—Nitrogen as only ring hetero atom
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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
    • 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
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C

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Abstract

The invention provides a method for preparing Antrodin C and Antrodin A by liquid submerged culture of antrodia camphorata, which comprises the following steps: preparing antrodia camphorata fermentation seed liquid; adding vegetable oil and fermentation precursor into the fermented seed liquid for synchronous fermentation, wherein the fermentation precursor comprises one or more of inositol, phenylalanine and tyrosine. According to the method, when soybean oil is added as the synchronous extraction agent, the fermentation precursor is synchronously added, so that the content of Antrodin C and Antrodin A in the fermentation product can be obviously improved.

Description

Method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid
Technical Field
The invention belongs to the technical field of edible fungus fermentation, and particularly relates to a method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid.
Background
Antrodia camphorata (Taiwanofungus camphoratus), also known as: antrodia camphorata, wild rice in caves camphorata, and Antrodia camphorata, which are rare edible and medicinal fungi native to Taiwan in China. Antrodia camphorata is originally a traditional medicine for Taiwan original residents and is mainly used for eliminating physical discomfort caused by excessive drinking or excessive fatigue.
A large number of researches show that the antrodia camphorata has the physiological activity functions of enhancing the immunity, resisting bacteria, viruses, tumors and allergy, inhibiting platelet aggregation, reducing blood pressure, reducing blood sugar, reducing the gallbladder function, curing intoxication, protecting the liver (detoxifying and promoting the regeneration of liver cells) and the like. Antrodia camphorata is rich in active substances such as triterpenes, quinones and phenols, wherein the components such as Androquinonol and Androdin have definite antitumor activity.
However, the prior art has lower yield of Antrodin C and Antrodin A prepared by fermenting Antrodia camphorata, and how to provide a method for preparing Antrodin C and Antrodin A by submerged culture of Antrodia camphorata liquid is a technical problem to be solved.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The invention provides a method for preparing Antrodin C and Antrodin A by fermenting antrodia camphorata mycelia, which is characterized by comprising the following steps of: the method comprises the following steps:
preparing antrodia camphorata fermentation seed liquid;
adding vegetable oil and fermentation precursor into the fermented seed liquid for synchronous fermentation, wherein the fermentation precursor comprises one or more of inositol, phenylalanine and tyrosine.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by fermenting antrodia camphorata mycelia, the method comprises the following steps: the preparation of the antrodia camphorate fermentation seed liquid comprises the steps of inoculating antrodia camphorate mycelia on a solid culture medium for culture, taking out bacterial lawn, adding the bacterial lawn into a liquid culture medium, and crushing the bacterial lawn into suspension by a homogenizer; and (5) performing shake flask fermentation to obtain the antrodia camphorata seed liquid.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: the Antrodia camphorata fermentation seed liquid is prepared by mixing Antrodia camphorata fermentation seed liquid with an area of 0.5cm2Inoculating the antrodia camphorata mycelia on a potato agar culture medium plate, wherein the diameter of the plate is 9cm, the adding amount of a potato agar culture medium PDA is 20 mL/plate, culturing for 3 weeks at 25 ℃, taking out antrodia camphorata lawn when the diameter of antrodia camphorata colony reaches 7cm, then adding 200mL of potato broth culture medium PDB into the antrodia camphorata lawn, and crushing by using a homogenizer to prepare antrodia camphorata strain suspension; placing 20ml of the suspension into a shake flask with the capacity of 250ml, adding 80ml of potato broth culture medium PDB, and culturing at 25 ℃ for 5 days at 150 rpm to obtain antrodia camphorata seed liquid with the pH value of 6.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: the vegetable oil comprises one or more of soybean oil, peanut oil, rapeseed oil, corn germ oil and olive oil.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: the vegetable oil is pressed vegetable oil.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: the addition concentration of the fermentation precursor is 0.05-0.4 g/l.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: and (3) synchronous fermentation, namely adding the antrodia camphorata seed liquid into a culture medium with the volume 8-12 times that of the antrodia camphorata seed liquid, adding vegetable oil and fermentation precursors accounting for 10-30% of the total volume, adjusting the pH to 6.5-7.5, performing shake flask fermentation for 10-20 days, inactivating at high temperature after the fermentation is finished, and collecting an oil phase.
As a preferred scheme of the method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodin, the method comprises the following steps: adding 10 parts by volume of the antrodia camphorata seed liquid into 9 parts by volume of the potato broth culture medium, adding 20 parts by volume of the pressed soybean oil, adding a fermentation precursor, adjusting the pH to 7, rotating at 100-200 rpm, culturing at 20-25 ℃ for 14 days, performing shake flask fermentation, inactivating at 80 ℃ for 2 hours after the fermentation is finished, and collecting an oil phase.
The invention has the beneficial effects that: according to the method, when soybean oil is added as the synchronous extraction agent, the fermentation precursor is synchronously added, so that the content of Antrodin C and Antrodin A in the fermentation product can be obviously improved.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 shows the effect of simultaneous addition of different fermentation precursors on Antrodin C content in fermentation products.
FIG. 2 shows the effect of simultaneous addition of different simultaneous extractants on Antrodin C content in fermentation products.
FIG. 3 shows the effect of simultaneous addition of different simultaneous extractants on Antrodin A content in fermentation products.
FIG. 4 shows the effect of different fermentation precursors on Antrodin C content in fermentation product when soybean oil is squeezed as synchronous extractant.
FIG. 5 shows the effect of different fermentation precursors on Antrodin A content in fermentation product when soybean oil is squeezed as synchronous extractant.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
experimental materials:
solid medium: potato agar medium (PDA);
liquid culture medium: potato broth (PDB);
synchronous extracting agent: one of soybean oil, peanut oil, rapeseed oil, corn germ oil and olive oil; fermentation precursor: one of inositol, phenylalanine, tyrosine;
the experimental materials are all commercial products.
Antrodia camphorata strain: camphoratus strain J1, the strain disclosed in the literature "Antitumor Compounds from the Stout Camphor".
Preparing antrodia camphorata fermentation seed liquid: the area size is 0.5cm2Inoculating Antrodia camphorata lawn on a potato agar culture medium (PDA) plate, wherein the diameter of the plate is 9cm, the adding amount of the potato agar culture medium PDA is 20mL per plate, culturing for 3 weeks at 25 ℃, taking out and adding 200mL of potato broth culture medium PDB when the colony diameter of the Antrodia camphorata reaches 7cm, and crushing by using a homogenizer to prepare suspension; placing 20ml of the suspension into a shake flask with the capacity of 250ml, adding 80ml of potato broth culture medium PDB, and culturing at 25 ℃ for 5 days at 150 rpm to obtain antrodia camphorata seed liquid with the pH value of 6.
Influence of addition of different fermentation precursors on antrodia camphorata Antrodin C yield:
adding 10ml of the antrodia camphorata seed solution into 110ml of potato broth culture medium PDB, adding different fermentation precursors, adjusting the final concentration of the fermentation precursors to be 0.1g/l, adjusting the pH to be 7 by using 0.1mol/l NaOH solution, rotating at 150 rpm, culturing at 25 ℃ for 14d, performing shake flask fermentation, and inactivating at 80 ℃ for 2h after the fermentation is finished. The blank control group was fermented under the same conditions by adding only 120ml of potato broth PDB. The amount of Antrodin C produced in the fermentation broth was determined. See table 1.
Influence of addition of different synchronous extractants on antrodia camphorata Antrodin C and Antrodin a yields:
adding 10ml of the antrodia camphorata seed solution into 90ml of potato broth culture medium PDB, adding 20ml of different synchronous step extractants, adjusting the pH to 7 by using 0.1mol/l NaOH solution, rotating at 150 rpm, culturing at 25 ℃ for 14d, carrying out shake flask fermentation, inactivating at 80 ℃ for 2h after the fermentation is finished, and collecting the synchronous extractants. The yields of Antrodin C and Antrodin A in the simultaneous extraction reagents were determined. The blank control group was fermented under the same conditions using only 120ml of potato broth PDB. See tables 2 and 3.
The influence of different fermentation precursors on the yields of antrodia cinnamomea Antrodin C and Antrodin a when soybean oil is squeezed as a synchronous extractant:
adding 10ml of the antrodia camphorata seed solution into 90ml of potato broth culture medium PDB, adding 20ml of pressed soybean oil, adding different fermentation precursors, adjusting the final concentrations of the fermentation precursors to be 0, 0.05, 0.1, 0.2 and 0.4g/l respectively, adjusting the pH to be 7 by using 0.1mol/l of NaOH solution, rotating at 150 rpm, culturing at 25 ℃ for 14 days, performing shake flask fermentation, inactivating at 80 ℃ for 2 hours after the fermentation is finished, and collecting the pressed soybean oil of the synchronous extractant. The yields of Antrodin C and Antrodin A in the simultaneous extraction reagents were determined. The blank control group was fermented under the same conditions by adding only 120ml of potato broth PDB. See tables 4 and 5.
Detection of fermentation liquor:
and (3) mixing the collected synchronous extracting agent: methanol is mixed according to a volume ratio of 1: 10 mixing, extracting overnight, centrifuging at 4000 rpm, and loading to detect the content of Antrodin C (ADC) and Antrodin A;
and adding 150ml of ethyl acetate into 120ml of fermentation liquor obtained after shaking flask fermentation in the blank control group, putting the fermentation liquor into a shaking table for 150 rpm overnight, dissolving the ethyl acetate by using 10ml of methanol after rotary evaporation and drying, centrifuging at 4000 rpm, and loading to detect the content of Antrodin C (ADC) and Antrodin A.
The peak time of Antrodin C was 8.87min, and the peak time of Antrodin A was 11.85 min.
Liquid phase detection procedure:
time min Acetonitrile% Water%
0-15 50 50
15-15.5 100 0
15.5-25 50 50
The experimental results are as follows:
table 1: effect (mg/l) of different fermentation precursors on Antrodin C content in fermentation products
Different fermentation precursors (0.1g/l) AntrodinC(mg/l) SD P value
Blank control 50.02 3.93
Citric acid 28.10 2.61 0.0013
Maleic acid 24.15 3.02 0.0008
Glutamine 42.15 2.70 0.046
Soy isoflavone 50.57 16.67 0.95
Phenylalanine 62.25 7.03 0.058
Inositol 72.03 28.10 0.25
Tyrosine 71.97 18.44 0.11
As can be seen from Table 1, the addition of 0.1g/l of maleic acid, citric acid or glutamine significantly inhibited the production of Antrodin C, while soybean isoflavone, phenylalanine, inositol or tyrosine did not have significant inhibitory effect on the production of Antrodin C.
The experimental result shows that after the fermentation precursors are respectively added, Antrodin A in the fermentation product cannot be detected, and that the independent addition of the fermentation precursors has no promotion effect on the fermentation production of Antrodin A.
Table 2: influence (mg/l) of different synchronous extractants on Antrodin C content in fermentation product
Different vegetable oils AntrodinC(mg/l) SD P value
Blank control 77.18 14.73
Squeezing soybean oil 29.20 5.20 0.00601
Pressed peanut oil 30.27 12.62 0.013
Pressing rapeseed oil 30.45 4.78 0.006
Squeezing corn germ oil 77.31 13.33 0.99
Squeezing olive oil 33.25 3.09 0.007
As can be seen from table 2, the active ingredients contained in the pressed soybean oil, the pressed peanut oil, the pressed rapeseed oil and the pressed olive oil have a negative regulation effect on the fermentation of Antrodin C, the active substances contained in the pressed corn germ oil do not have a negative regulation effect on the generation of Antrodin C, the active ingredients and fatty acid compositions contained in different pressed vegetable oils are different, and when different pressed vegetable oils are added as an extracting agent, the active substances in the pressed vegetable oils are in contact with antrodia camphorata mycelia in the fermentation process, so that the yield of Antrodin C is affected.
Table 3: influence (mg/l) of different synchronous extractants on Antrodin A content in fermentation product
Different vegetable oils AntrodinA(mg/l) SD P<0.05 significance
Blank control - - -
Squeezing soybean oil 113.22 25.47 a
Pressed peanut oil 87.17 3.67 b
Pressing rapeseed oil 136.73 11.82 a
Squeezing corn germ oil 87.77 1.50 b
Squeezing olive oil 83.84 0.49 b
As can be seen from table 3, there was no Antrodin a produced without adding the pressed vegetable oil, and the addition of the pressed vegetable oil could significantly improve the yield of Antrodin a.
Table 4: influence (mg/l) of adding different fermentation precursors on Antrodin C content in fermentation product when soybean oil is squeezed as synchronous extractant
Figure BDA0002982449880000071
From table 4, it can be seen that the fermentation precursor significantly improves the yield of Antrodin C to different extents under the condition of pressing soybean oil as a synchronous extractant, which indicates that the fermentation precursor can eliminate the inhibiting effect of pressing soybean oil on the yield of Antrodin C, and the addition of the fermentation precursor can significantly promote the production of Antrodin C under the condition of pressing soybean oil as a synchronous extractant.
Table 5: influence (mg/l) of different fermentation precursors on Antrodin A content in fermentation product when soybean oil is squeezed as synchronous extractant
Figure BDA0002982449880000081
From table 5, it can be seen that the addition of different fermentation precursors when soybean oil is used as a synchronous extractant can significantly increase the antrodia camphorata Antrodin a content in the fermentation product.
In conclusion, the method for synchronously adding the fermentation precursor when the soybean oil is added as the synchronous extracting agent can obviously improve the content of Antrodin C and Antrodin A in the fermentation product.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (8)

1. A method for preparing Antrodin C and Antrodin A by liquid submerged culture of Antrodia camphorata is characterized by comprising the following steps: the method comprises the following steps:
preparing antrodia camphorata fermentation seed liquid;
adding vegetable oil and fermentation precursor into the fermented seed liquid for synchronous fermentation, wherein the fermentation precursor comprises one or more of inositol, phenylalanine and tyrosine.
2. The method of preparing Antrodin C and Antrodin a by liquid submerged culture of antrodia camphorata according to claim 1, wherein: the preparation of the antrodia camphorate fermentation seed liquid comprises the steps of inoculating antrodia camphorate mycelia on a solid culture medium for culture, taking out bacterial lawn, adding the bacterial lawn into a liquid culture medium, and crushing the bacterial lawn into suspension by a homogenizer; and (5) performing shake flask fermentation to obtain the antrodia camphorata seed liquid.
3. The method of preparing Antrodin C and Antrodin a by submerged culture of antrodia camphorata liquid according to claim 1 or 2, wherein: the Antrodia camphorata fermentation seed liquid is prepared by mixing Antrodia camphorata fermentation seed liquid with an area of 0.5cm2Inoculating Antrodia camphorata lawn on a potato agar culture medium plate, wherein the diameter of the plate is 9cm, the adding amount of the potato agar culture medium PDA is 20 mL/plate, culturing for 3 weeks at 25 ℃, taking out the Antrodia camphorata lawn when the diameter of the Antrodia camphorata colony reaches 7cm, and then taking out the Antrodia camphorata lawn and adding 200mL of potato brothCrushing the culture medium PDB by using a homogenizer to prepare antrodia camphorata mycelium suspension; placing 20ml of the antrodia camphorata mycelium suspension into a shake flask with the capacity of 250ml, adding 80ml of potato broth culture medium PDB, culturing at the speed of 150 rpm and the temperature of 25 ℃ for 5 days to obtain antrodia camphorata seed liquid, wherein the pH value of the antrodia camphorata seed liquid is 6.
4. The method of preparing Antrodin C and Antrodin a by submerged culture of antrodia camphorata liquid according to claim 1 or 2, wherein: the vegetable oil comprises one or more of soybean oil, peanut oil, rapeseed oil, corn germ oil and olive oil.
5. The method of preparing Antrodin C and Antrodin a by liquid submerged culture of antrodia camphorata according to claim 4, wherein: the vegetable oil is pressed vegetable oil.
6. The method of preparing Antrodin C and Antrodin a by submerged culture of antrodia camphorata liquid according to claim 1 or 2, wherein: the addition concentration of the fermentation precursor is 0.05-0.4 g/l.
7. The method of preparing Antrodin C and Antrodin a by submerged culture of antrodia camphorata liquid according to claim 1 or 2, wherein: and (3) synchronous fermentation, namely adding the antrodia camphorata seed liquid into a culture medium with the volume of 8-12 times, adding vegetable oil accounting for 10-30% of the total volume and a fermentation precursor, adjusting the pH to 6.5-7.5, performing shake flask fermentation for 10-20 days, performing high-temperature inactivation after the fermentation is finished, and collecting an oil phase.
8. The method of preparing Antrodin C and Antrodin a by liquid submerged culture of antrodia camphorata according to claim 7, wherein: adding 10 parts by volume of the antrodia camphorata seed liquid into 9 parts by volume of the potato broth culture medium, adding 20 parts by volume of the pressed soybean oil, adding a fermentation precursor, adjusting the pH to 7, rotating at 100-200 rpm, culturing at 20-25 ℃ for 14 days, performing shake flask fermentation, inactivating at 80 ℃ for 2 hours after the fermentation is finished, and collecting an oil phase.
CN202110291795.6A 2021-03-18 2021-03-18 Method for preparing Antrodin C and Antrodin A by submerged culture of antrodia camphorata liquid Withdrawn CN112779302A (en)

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Application publication date: 20210511