CN114574420B - Method for promoting asexual spore production of Antrodia camphorata through submerged fermentation and culture medium and application thereof - Google Patents
Method for promoting asexual spore production of Antrodia camphorata through submerged fermentation and culture medium and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention discloses a method for promoting asexual spore production of Antrodia camphorata through submerged fermentation, a culture medium and application thereof. The method comprises adding metal ions into a submerged fermentation medium of Antrodia camphorata, and inoculating Antrodia camphorata for fermentation; the metal ion is Ca 2+ 、Fe 2+ Zn (zinc) 2+ One or more of them. Ca alone was added as compared with the control group without metal ion 2+ 、Fe 2+ Zn (zinc) 2+ The method can improve the yield of the asexual spores of the Antrodia camphorata, the 3 metal ions are mixed and added according to the optimized concentration, the yield of the asexual spores of the Antrodia camphorata can be improved by more than 133 percent, the fermentation period is shortened by 24 hours, and the effect of promoting the submerged fermentation spore production speed and the spore production amount of the Antrodia camphorata is extremely remarkable. The method has the advantages of very convenient operation and low cost, and can greatly improve the seed preparation efficiency and reduce the production cost in the industrial production process of the deep fermentation of the Antrodia camphorata, thereby improving the large-scale production efficiency and benefit of the deep fermentation of the Antrodia camphorata.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for promoting asexual spore production of Antrodia camphorate through submerged fermentation, and a culture medium and application thereof.
Background
Antrodia camphorata (Antrodia camphorata or Antrodia cinnamomea), also known as Antrodia camphorata, etc., belongs to Basidiomycetes (Basidiomycetes), polyporaceae (Polyporae), and Polyporus (Antrodia), and is a rare fungus for both medicine and food. Wild Antrodia camphorata fruiting bodies usually grow on the inner wall of a decayed trunk of an Antrodia camphorata (Cinnamomum kanehirai Hay) or fall dead and lodge on a moist surface close to the soil surface, have strong camphor tree fragrance, are extremely bitter in taste, are in a shape of a plate, a bell, a horseshoe or other irregular shapes, and are mainly red in color, so that the wild Antrodia camphorata fruiting bodies are called as forest ruby. Modern researches have shown that Antrodia camphorata has various biological activities of protecting liver, resisting cancer, resisting oxidation, resisting inflammation, resisting virus, reducing blood lipid, regulating immunity, etc., and more than 200 active substances including polysaccharides, diterpenoids, triterpenes, steroids, benzene ring substances, maleic acid and succinic acid derivatives (Antrodin A-E), ubiquinone derivatives (Antroquinone), etc. are separated from fruiting bodies or mycelia of Antrodia camphorata.
The biological activity of the antrodia camphorate is superior, so the market demand is very huge. However, the wild Antrodia camphorata grows very slowly and has very few quantity, and long-term supply and demand cause the fruiting body of the wild Antrodia camphorata to have a high price, and the price per kilogram is up to about 15 ten thousand RMB. Therefore, the large-scale artificial cultivation of Antrodia camphorata is particularly necessary. The common artificial culture techniques of Antrodia camphorate mainly comprise 4 kinds: cultivating the basswood of the camphor tree, culturing in a plate, performing solid-state fermentation and performing submerged fermentation. Wherein, basswood cultivation, plate culture and solid state fermentation are not popular due to the longer period (6 months-5 years) and higher cost. The submerged fermentation method based on asexual spore inoculation only needs 10-14 days in production period, has high production efficiency and is easy for large-scale production, so that the method becomes the most efficient and common artificial culture mode of Antrodia camphorata at present.
The asexual spore production process of Antrodia camphorata is affected by a plurality of factors, such as carbon source, nitrogen source, illumination, dissolved oxygen, pH, exogenous additives and the like, wherein the nutrition limitation of the nitrogen source, the carbon source and the like is generally considered as one of the key factors for inducing the asexual spore production of Antrodia camphorata. Meanwhile, the existing submerged fermentation asexual spore of Antrodia camphorata has low yield and long culture period, so that the preparation time, the efficiency and the cost of the submerged fermentation seeds (asexual spores) of Antrodia camphorata are long, and the submerged fermentation seeds are always one of key factors restricting the large-scale submerged fermentation production of Antrodia camphorata. Therefore, how to remarkably improve the preparation efficiency of the asexual spores of the antrodia camphorata, reduce the cost of the deep fermentation production of the antrodia camphorata, promote the development process of the large-scale industrial production of the deep fermentation of the antrodia camphorata, and is a problem to be solved urgently.
Disclosure of Invention
The invention aims to: aims at solving the problems of long preparation time, low efficiency and high cost of the deep fermentation seeds (asexual spores) of Antrodia camphorata caused by low yield and long culture period of the asexual spores of the prior deep fermentation of Antrodia camphorata. The invention provides a method for promoting asexual spore production of the deep fermentation of Antrodia camphorata, which can greatly improve the yield of asexual spores of Antrodia camphorata, shorten the fermentation period, improve the spore production speed and the spore yield of the deep fermentation of Antrodia camphorata.
The invention also provides a culture medium for promoting asexual spore production of the deep fermentation of Antrodia camphorata and application thereof.
The technical scheme is as follows: in order to achieve the aim, the method for promoting asexual spore production of the deep fermentation of the antrodia camphorata adds metal ions into a deep fermentation medium of the antrodia camphorata, and then inoculates the antrodia camphorata for fermentation; the metal ion is Ca 2+ 、Fe 2+ Zn (zinc) 2+ One or more of them.
Wherein, the addition amount of the metal ions in the culture medium is as follows: ca (Ca) 2+ Final concentration of 0.2-10mmol/L, fe 2+ Final concentration of 0.05-0.3mmol/L, zn 2+ The final concentration is 0.05-0.2 mmol/L.
Preferably, the metal ions are added in the following amounts: ca (Ca) 2+ Final concentration 1mmol/L, fe 2+ Final concentration of 0.1mmol/L, zn 2+ The final concentration was 0.1mmol/L.
Wherein the metal ionsCa 2+ 、Fe 2+ And Zn 2+ Simultaneously adding into the Antrodia camphorata submerged fermentation culture medium.
Preferably, the metal ion Ca 2+ 、Fe 2+ And Zn 2+ Simultaneously adding into deep fermentation medium of Antrodia camphorata, wherein the concentration of Ca is 2.012mmol/L respectively 2+ Fe at 0.149mmol/L 2+ And 0.135mmol/L. Wherein, the metal ion adding mode is as follows: before the culture medium is sterilized, the culture medium is added and then sterilized together with the culture medium; or after the culture medium is sterilized, the metal ion solution is sterilized before inoculation and then added; or inoculating Antrodia camphorata after sterilizing the culture medium, and then adding the sterilized metal ion solution.
Wherein the metal ions are mainly added in the form of metal ion compounds.
Preferably, the metal ions are added in the form of chlorides, including CaCl 2 、FeCl 2 、ZnCl 2 One or more of the following.
Preferably, the metal ion compound is added at the time of preparing the medium, and then sterilized together with the medium.
Wherein the Antrodia camphorata is inoculated by mixing Antrodia camphorata spore suspension with a proportion of 1×10 5 personal/mL-5X 10 6 Inoculating the inoculum size of each/mL into a culture medium; after inoculation, the culture is carried out for 8-15d under the conditions of 24-27 ℃ and 50-300 r/min.
Preferably, the suspension of Antrodia camphorate spores is 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; after inoculation, the culture was performed at 26℃and 150r/min for 10d with shaking.
Wherein the formula of the Antrodia camphorate submerged fermentation culture medium is as follows: glucose 1-10%, yeast extract powder 0.1-2%, mgSO4 0.1-1%, KH 2 PO 4 0.1% -1% and an initial pH of 2.0-6.5.
Preferably, the formulation of the Antrodia camphorate submerged fermentation culture medium comprises 2% of glucose, 0.2% of yeast extract powder, 0.15% of MgSO4 and KH 2 PO 4 0.3%, initial pH 4.5. The culture medium for promoting asexual spore production of the deep fermentation of the antrodia camphorata comprises a deep fermentation culture medium of the antrodia camphorata and metal ions, wherein the antrodia camphorata is deepThe formula of the layer fermentation medium comprises 1-10% of glucose, 0.1-2% of yeast extract powder, 0.1-1% of MgSO4 and KH 2 PO 4 0.1% -1%, and the initial pH is 2.0-6.5; the metal ion is Ca 2+ 、Fe 2+ Zn (zinc) 2+ One or more of them has a concentration of Ca in the deep fermentation medium of Antrodia camphorata 2+ Final concentration of 0.2-10mmol/L, fe 2+ Final concentration of 0.05-0.3mmol/L, zn 2+ The final concentration is 0.05-0.2 mmol/L.
The method for promoting asexual spore production of the deep fermentation of the antrodia camphorata is applied to improving the preparation efficiency of asexual spores of the antrodia camphorata and reducing the production cost of the deep fermentation of the antrodia camphorata.
The culture medium for promoting asexual sporulation of the deep fermentation of the antrodia camphorata is applied to improving the preparation efficiency of asexual sporulation of the antrodia camphorata and reducing the production cost of the deep fermentation of the antrodia camphorata. The asexual spore production speed of the deep fermentation of the Antrodia camphorata is low, and the low spore production amount is always one of the main limiting factors of the industrial production of the deep fermentation of the Antrodia camphorata. Because of the low spore yield, a large volume of fermentation broth is often required in the preparation of seeds (asexual spore suspension of Antrodia camphorata), which greatly increases the raw material cost of the culture medium. Furthermore, in order to control the inoculation volume (typically the volume ratio of "seed liquid" to "fermentation medium" is not higher than 10%), it is sometimes even necessary to concentrate the filtered suspension of Antrodia camphorata spores, which greatly increases the probability of contamination. Therefore, when preparing the antrodia camphorate seeds, tedious operations and long spore production period greatly increase the cost of raw materials, time, manpower and material resources for preparing the seeds. The invention provides a method for promoting asexual spore production of Antrodia camphorata submerged fermentation, which comprises adding Ca with final concentration of 0.2-10mmol/L into an Antrodia camphorata submerged fermentation culture medium 2+ Fe of 0.05-0.3mmol/L 2+ Or 0.05 to 0.2mmol/L Zn 2+ One or more metal ions of the Antrodia camphorata, and then inoculating the Antrodia camphorata for fermentation. Ca alone was added as compared with the control group without metal ion 2+ The yield of asexual spores of Antrodia camphorata can be improved by more than 87% to the maximum extent; addition of Fe alone 2+ The yield of asexual spores of Antrodia camphorata can be improved by more than 72 percent to the maximum extent; zn is added separately 2+ Can maximize the yield of asexual spores of Antrodia camphorataThe improvement is more than 35%. The 3 metal ions are mixed and added according to the concentration after the response surface optimization and are applied to the deep fermentation of the antrodia camphorata, so that the yield of asexual spores of the antrodia camphorata can be improved by more than 133 percent to the maximum, the fermentation period is shortened by 24 hours, the spore yield of the deep fermentation of the antrodia camphorata is obviously improved, and the spore production speed is also obviously accelerated. The invention can obviously shorten the fermentation period while improving the yield of the asexual spores of the antrodia camphorata, and the shortening of the fermentation period can reduce the subsequent production period, thereby improving the production efficiency and reducing the production cost. The exogenous additive capable of improving the spore yield in the prior art has no effect of shortening the fermentation period, and the invention obviously improves the asexual spore yield of the Antrodia camphorata and shortens the fermentation period at the same time, thereby having production advantages. In conclusion, the preparation method of the asexual spores of the Antrodia camphorata obviously improves the preparation efficiency of the asexual spores of the Antrodia camphorata, reduces the industrial production cost of the deep fermentation of the Antrodia camphorata, and greatly improves the industrial production efficiency and benefit of the deep fermentation of the Antrodia camphorata. The main function of adding metal ions into the culture medium is to improve the spore yield and the spore production speed of the deep fermentation of the Antrodia camphorata, improve the spore yield and the spore production speed, further improve the preparation efficiency of the asexual spores of the Antrodia camphorata and reduce the production cost.
The invention discovers through a plurality of optimizations that the metal ion Ca 2+ 、Fe 2+ And Zn 2+ The three components are added into the Antrodia camphorata submerged fermentation culture medium at the same time according to the proportion, the combined effect is best, and the combined addition concentration of the three components is obtained through response surface optimization. The addition of a single metal ion can improve the yield by a certain amount, but cannot shorten the period. In addition, other metal ions, such as Mg, are also contemplated for use in the present invention 2+ 、Cu 2+ Al and Al 3+ Etc., the results show that: these metal ions have no remarkable effect of improving the yield of submerged fermentation asexual spores of Antrodia camphorata, and are combined with metal ions Ca 2+ 、Fe 2+ And Zn 2+ The effect is not obviously improved after mixing any two by two, wherein the additive is mixed with Cu 2+ Al and Al 3+ Even the mixture of the components can play a role in inhibiting asexual spore production of the deep fermentation of the Antrodia camphorata.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the method provided by the invention can improve the yield of asexual spores of the deep fermentation of Antrodia camphorata by more than 133% and the yield of the asexual spores of the deep fermentation of Antrodia camphorata by up to 8.6X10 7 The inoculation amount of the seed is 1.0X10 per mL 6 The volume ratio of the inoculating liquid is only 1.16 percent and is less than 2 percent calculated by each mL, thus greatly improving the operability of spore inoculation in the deep fermentation production of the antrodia camphorata. Because of the high spore yield, the volume of the fermentation liquid of the needed seeds is greatly reduced, concentration is not needed, the seed preparation procedure is simplified, and the risk of bacterial contamination and the seed preparation cost are reduced. Meanwhile, the method provided by the invention has the very important advantage that the spore production period can be shortened by 24 hours, and the production period is further shortened. In short, the invention obviously improves the preparation efficiency of the asexual spores of the Antrodia camphorata, reduces the preparation cost of seeds (asexual spores) produced by the deep fermentation of the Antrodia camphorata, and further obviously improves the production efficiency and benefit of the deep fermentation of the Antrodia camphorata. In addition, the metal ions added in the invention are common metal ions, and the price is low and the addition amount is extremely low; meanwhile, the metal ions can be added during preparation of the culture medium, and the method is free from separate sterilization and convenient to operate. Most importantly, the method provided by the invention has extremely obvious effect on promoting the spore production speed and the spore production quantity. In conclusion, the method provided by the invention is convenient to operate, low in cost and remarkable in effect, and has great development value and good application prospect.
Drawings
FIG. 1 addition of Ca at various concentrations 2+ Influence on asexual spore production of the deep fermentation of Antrodia camphorate; wherein "CK" is a control group to which no metal ion was added (hereinafter);
FIG. 2 addition of Fe at different concentrations 2+ Influence on asexual spore production of the deep fermentation of Antrodia camphorate;
FIG. 3 addition of Zn of different concentrations 2+ Influence on asexual spore production of the deep fermentation of Antrodia camphorate;
FIG. 4 is the effect of simultaneous addition of different combinations of two metal ions on asexual sporulation of Antrodia camphorate;
FIG. 5 is a graph showing interactions between the response surface analysis of spore yield and the addition concentration of each metal ion;
FIG. 6 influence of addition of 3 metal ion optimized combinations on asexual spore production of Antrodia camphorate submerged fermentation;
fig. 7 shows a physical comparison of spore yields of the 3 metal ion-optimized combination groups and the control group.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The raw materials and the reagents in the invention are commercially available unless otherwise specified.
The detection method of the spore yield of the Antrodia camphorate submerged fermentation in the following embodiment comprises the following steps:
filtering fermentation broth after deep fermentation of Antrodia camphorata with 4 layers of gauze under aseptic condition to obtain filtrate as spore suspension (which can be used as seed). Then 20. Mu.L of spore suspension was aspirated and counted under an optical microscope using a hemocytometer, and the spore yield was calculated from the counting result.
The Antrodia camphorate (Antrodia camphorate) strain was purchased from the American type culture Collection (American Type Culture Collection, ATCC) under the number ATCC 200183.
Example 1
Ca 2+ The preparation steps of the mother solution (1 mol/L) are as follows:
(1) Weigh 11.10g CaCl 2 The powder was placed in a beaker and 80mL deionized water was added;
(2) Grinding and stirring with glass rod until CaCl 2 Completely dissolving;
(3) Dissolving CaCl 2 Pouring all the solution into a 100mL volumetric flask, and fixing the volume value to 100mL by deionized water to obtain 1mol/L Ca 2+ A mother liquor;
(4) Ca is added with 2+ And (5) split charging the mother solution, and placing the mother solution in a refrigerator at the temperature of minus 20 ℃ for standby.
Example 2
Fe 2+ The preparation steps of the mother solution (1 mol/L) are as follows:
(1) Weigh 12.68g FeCl 2 The powder was placed in a beaker and 80mL deionized water was added;
(2) Grinding and stirring with a glass rod until FeCl 2 Completely dissolving;
(3) Dissolving FeCl 2 Pouring all the solution into a 100mL volumetric flask, and fixing the volume value to 100mL by deionized water to obtain 1mol/L Fe 2+ A mother liquor;
(4) Fe is added to 2+ And (5) split charging the mother solution, and placing the mother solution in a refrigerator at the temperature of minus 20 ℃ for standby.
Example 3
Zn 2+ The preparation steps of the mother solution (1 mol/L) are as follows:
(1) 13.64g ZnCl was weighed out 2 The powder was placed in a beaker and 80mL deionized water was added;
(2) Grinding and stirring with glass rod until ZnCl 2 Completely dissolving;
(3) Dissolving ZnCl 2 Pouring all the solution into a 100mL volumetric flask, and fixing the volume value to 100mL by deionized water to obtain 1mol/L Zn 2+ A mother liquor;
(4) Zn is added 2++ And (5) split charging the mother solution, and placing the mother solution in a refrigerator at the temperature of minus 20 ℃ for standby.
Example 4
The preparation method of the Antrodia camphorate submerged fermentation medium comprises the following steps:
(1) Culture medium preparation (mass fraction): according to glucose 2%, yeast extract powder 0.2%, mgSO 4 0.15％、KH 2 PO 4 A submerged fermentation liquid medium of Antrodia camphorata is prepared according to a formula of 0.3%, and the initial pH is adjusted to 4.5 by using 1mol/L HCl and 1mol/L NaOH solution.
(2) Sterilizing a culture medium: the culture medium is subpackaged into 250mL conical flasks (shake flask fermentation) according to the liquid loading amount of 100mL/250mL or into a 5L fermentation tank (fermentation tank fermentation) according to the liquid loading amount of 3.5L/5L, sterilized at 121 ℃ for 20min, and cooled for later use.
Example 5
Adding Ca with different concentrations separately 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorate, ca is added into the culture medium prepared in example 4 2+ The final concentration of (C) was 0.2mmol/L, 1mmol/L, 5mmol/L and 10mmol/L, respectively. That is, the culture medium was prepared in 100mL of the culture medium (example 4)20. Mu.L, 100. Mu.L, 500. Mu.L and 1mL of Ca at a concentration of 1mol/L were added 2+ Mother liquor (prepared in example 1) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
The procedure of example 5 was followed, while the fermentation groups without any metal ions added to the medium were set as control groups, each group being set with 3 replicates. During the cultivation, samples were taken daily for each period of 6-11 days for spore count, and the spore yield was measured, and the experimental results are shown in FIG. 1.
As can be seen from the results of fig. 1: in the fermentation process, the spore yield reaches the maximum value at the 10d, and 0.2-10mmol/L Ca is added 2+ Can obviously promote asexual spore production of the deep fermentation of the Antrodia camphorata. Wherein, when Ca 2+ When the final concentration is 1mmol/L, the maximum spore yield (6.76X10) of the deep fermentation of Antrodia camphorata can be achieved 7 The volume per mL) is increased by 87.21 percent compared with a control group, and the effect is extremely remarkable; when Ca is 2+ When the final concentration is 0.2mmol/L, the maximum spore yield (5.34×10) of the deep fermentation of Antrodia camphorata can also be achieved 7 The volume per mL) is 47.73 percent higher than that of a control group, and the effect is very obvious. The above results illustrate Ca 2+ Can obviously promote asexual spore production of the deep fermentation of the antrodia camphorata, but has serious concentration dependence, and the effect is better when the concentration is not higher. When the concentration is too high (e.g., 5mmol/L or 10 mmol/L), the effect of promoting sporulation is rather lowered.
Example 6
Independently add Fe with different concentrations 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorata, fe is added into the culture medium prepared in example 4 2+ The final concentration of (C) was 0.05mmol/L, 0.1mmol/L, 0.2mmol/L and 0.3mmol/L, respectively. That is, 5. Mu.L, 10. Mu.L, 20. Mu.L and 30. Mu.L of Fe at a concentration of 1mol/L were added to 100mL of the medium (prepared in example 4) when the medium was prepared 2+ Mother liquor (prepared in example 2) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
The procedure of example 6 was followed, while the fermentation groups without any metal ions added to the medium were set as control groups, each group being set with 3 replicates. During the culture, samples were taken daily for each period of 6 to 11 days for spore count, and the spore yield was measured, and the experimental results are shown in FIG. 2.
As can be seen from the results of FIG. 2, during the fermentation, the spore yield reached the maximum at 10d, and 0.05-0.3mmol/L Fe was added 2+ Can obviously promote asexual spore production of the deep fermentation of the Antrodia camphorata. Wherein when Fe 2+ When the final concentration is 0.1mmol/L, the maximum spore yield (6.74×10) of the deep fermentation of Antrodia camphorata can be achieved 7 The number of the additive per mL) is improved by 72.39% compared with a control group, the additive amount is extremely small, but the effect is extremely remarkable; when Fe is 2+ When the final concentration is 0.2mmol/L, the maximum spore yield (6.01X10) of the deep fermentation of Antrodia camphorata can also be achieved 7 The volume per mL) is improved by 53.66 percent compared with the control group, and the effect is very obvious. The above results illustrate Fe 2+ Can obviously promote asexual spore production of the deep fermentation of the antrodia camphorata, but has serious concentration dependence, and has better effect when the concentration is higher than that of the antrodia camphorata, and the addition amount is strictly controlled. When the concentration is too high (e.g., 0.3 mmol/L), the effect of promoting sporulation is rather lowered or even eliminated.
Example 7
Independently adding Zn with different concentrations 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorate, zn is added into the culture medium prepared in example 4 2+ The final concentration of (C) was 0.05mmol/L, 0.1mmol/L and 0.2mmol/L, respectively. That is, 5. Mu.L, 10. Mu.L and 20. Mu.L of Zn having a concentration of 1mol/L were added to 100mL of the medium (prepared in example 4) when the medium was prepared 2+ Mother liquor (prepared in example 3) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
The procedure of example 7 was followed, except that the fermentation group to which no metal ion was added in the medium was used as a control group, and 3 replicates were set for each group. During the cultivation, samples were taken daily for each period of 6-11 days for spore count, and the spore yield was measured, and the experimental results are shown in FIG. 3.
As is clear from the results of FIG. 3, zn was added in an amount of 0.05 to 0.2mmol/L 2+ Can obviously promote asexual spore production of the deep fermentation of the Antrodia camphorata. Wherein, when Zn 2+ When the final concentration is 0.1mmol/L, the maximum spore yield (5.08X10) of the deep fermentation of Antrodia camphorata can be achieved 7 The number of the cells per mL) is increased by 35.05% compared with a control group; when Zn is 2+ When the final concentration is 0.05mmol/L, the maximum spore yield (4.62X10) of the deep fermentation of Antrodia camphorata can be achieved 7 The number of the additive per mL) is increased by 23% compared with the control group, the additive amount is extremely small, but the effect is still obvious. The above results illustrate Zn 2+ Can obviously promote asexual spore production of the deep fermentation of the antrodia camphorata, but the promoting effect has serious concentration dependency, and the effect is better when the concentration is not higher, and the adding amount is strictly controlled. When the concentration is too high (e.g., 0.3 mmol/L), the effect of promoting sporulation is decreased.
Example 8
Simultaneously adding Ca 2+ And Fe (Fe) 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorata, ca with a final concentration of 1mmol/L is added into the culture medium prepared in example 4 2+ And 0.1mmol/L Fe 2+ . That is, when preparing the medium, 1mL of Ca was added to each 100mL of the medium (prepared in example 4) at a concentration of 1mol/L 2+ Mother liquor (prepared in example 1) and 10. Mu.L of Fe at a concentration of 1mol/L 2+ Mother liquor (prepared in example 2) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
Example 9
Simultaneously adding Ca 2+ And Zn 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorata, ca with a final concentration of 1mmol/L is added into the culture medium prepared in example 4 2+ And 0.1mmol/L Zn 2+ . That is, when preparing the medium, 1mL of Ca was added to each 100mL of the medium (prepared in example 4) at a concentration of 1mol/L 2+ Mother liquor (prepared in example 1) and 10. Mu.L of Zn with a concentration of 1mol/L 2+ Mother liquor (prepared in example 3) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculating amount of each/mL into the culture mediumThe method comprises the steps of carrying out a first treatment on the surface of the The inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
Example 10
At the same time add Fe 2+ And Zn 2+ Influence on asexual spore production of deep fermentation of Antrodia camphorate, simultaneous addition of the culture medium prepared in example 4 + Fe with final concentration of 0.1mmol/L 2+ And 0.1mmol/L Zn 2+ . That is, when preparing the medium, 10. Mu.L of Fe having a concentration of 1mol/L was added to 100mL of the medium (prepared in example 4) 2+ Mother liquor (prepared in example 2) and 10. Mu.L of Zn with a concentration of 1mol/L 2+ Mother liquor (prepared in example 3) was sterilized at 121deg.C for 20min and cooled, and then the suspension of Antrodia camphorate spores was stirred at a rate of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
The methods of example 8, example 9 and example 10 were carried out in the same manner, and the fermentation groups without metal ions added to the medium were used as control groups, and 3 replicates were set for each group. At 10d of fermentation, samples were taken for spore count, and the spore yield was measured, and the experimental results are shown in FIG. 4.
As can be seen from the results of FIG. 4, ca at the same time 2+ 、Fe 2+ Zn (zinc) 2+ Any two metal ions in the preparation can obviously promote asexual spore production of the deep fermentation of Antrodia camphorate. Wherein, when Ca with a final concentration of 1mmol/L is added simultaneously 2+ And 0.1mmol/L Fe 2+ When the Antrodia camphorate is subjected to submerged fermentation, the spore yield (7.59 multiplied by 10) 7 The volume per mL) is increased by 106.05 percent compared with a control group when fermentation is carried out for 10 days, and the effect is extremely remarkable; when Ca with a final concentration of 1mmol/L is added simultaneously 2+ And 0.1mmol/L Zn 2+ When the Antrodia camphorate is subjected to submerged fermentation, the spore yield (7.15 multiplied by 10) 7 The fermentation rate is 94.01% higher than that of a control group in 10d, and the effect is extremely remarkable; when Fe with the final concentration of 0.1mmol/L is added simultaneously 2+ And 0.1mmol/L Zn 2+ When the Antrodia camphorate is subjected to submerged fermentation, the spore yield (6.48 multiplied by 10) 7 The number of the organic matters per mL) is increased by 78.60 percent compared with the control group at the fermentation time of 10d, but the effect is very remarkable. The above results demonstrate that Ca was added at the same time at the appropriate concentration 2+ 、Fe 2+ Zn (zinc) 2+ Any two metal ions in the composition can obviously promote the deep hair of Antrodia camphorataThe asexual spore production is improved by about 78 to 106 percent compared with the control group, and the effect is extremely remarkable.
Example 11
Optimization of simultaneous Ca addition by response surface method 2+ 、Fe 2+ Zn (zinc) 2+ In this case, the optimum concentration of each metal ion is added. First, the added concentration of each metal ion was set to 3 levels, ca 2+ 、Fe 2+ Zn (zinc) 2+ The response surface factor levels of (a) are shown in the following table (table 1).
TABLE 1 response surface factor level table for each metal ion
Subsequently, we designed 3 factor 3 horizontal combination test groups with the addition concentration of each metal ion as independent variable and the maximum spore yield as dependent variable using "Design expert 11" software, and the group Design results are shown in the following table (table 2).
TABLE 2 design table of each metal ion response surface group
Finally, ca prepared in example 1 was added to the medium prepared in example 4 2+ Mother liquor, fe prepared in example 2 2+ Mother liquor and Zn prepared in example 3 2+ The mother liquor was added with Ca at the corresponding concentration according to the combined concentration of each group in Table 2 2+ 、Fe 2+ Zn (zinc) 2+ Sterilizing at 121deg.C for 20min, cooling, and mixing with Antrodia camphorata spore suspension at a ratio of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
Meanwhile, a fermentation group without metal ions added to the culture medium was set as a control group, and 3 replicates were set for each group. In the culture process, spores are sampled every day during 6-11d respectively, spore yield is measured, analysis is carried out by using Design expert 11 software according to each group of spore yield data, and an interaction diagram (figure 5) among all factors and a ternary quadratic regression equation between the spore yield and all the factors are obtained.
As can be seen from the results of fig. 5, the response surface patterns of the interaction between any two of the three factors (three metal ions) are all perfect arch surfaces, which means that the center point and the step length of each factor level are designed very reasonably, and the optimal addition concentration is near the center point. Then, a ternary quadratic regression equation is simulated by using Design expert 11 software as follows: sporulation = 8.485+276.85 a +13.89 b +401.35 c +15.416 a b-137.5 a c-8.08 b c-972.5 a 2 -3.75*B 2 -1350*C 2 . Extremum was found from this regression equation, solving that when a=0.14874, b=2.012688, c=0.135, the sporulation amount was maximum. Namely, the optimal addition concentrations of the three metal ions are respectively as follows: ca (Ca) 2+ Final concentration 2.012mmol/L, fe 2+ Final concentration 0.149mmol/L and Zn 2+ The final concentration was 0.135mmol/L.
Example 12
Method for promoting asexual spore production of Antrodia camphorata by deep fermentation by adding metal ions and application thereof, and Ca is prepared by 2+ 、Fe 2+ And Zn 2+ The three metal ions were mixed at the optimized concentration of example 11 and added to the culture medium prepared in example 4 in the amounts of Ca, respectively 2+ Final concentration 2.012mmol/L, fe 2+ Final concentration of 0.149mmol/L and Zn 2+ The final concentration was 0.135mmol/L. That is, when preparing the medium, 2.012mL of Ca having a concentration of 1mol/L was added to 1000mL of the medium (prepared in example 4) 2+ Mother liquor (prepared in example 1), 149. Mu.L of Fe with concentration of 1mol/L 2+ Mother liquor (prepared in example 2) and 135. Mu.L of Zn with a concentration of 1mol/L 2+ Mother liquor (prepared in example 3), stirring, packaging into shaking bottles of 100mL each, sterilizing at 121deg.C for 20min, cooling, and mixing with Antrodia camphorata spore suspension of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; the inoculated shake flask was placed in a shaking table at 26℃and cultured with shaking at 150r/min for 11 days.
A fermentation group without metal ions added to the medium was used as a control group in the same manner as in example 12, and 3 replicates were taken each time the spores were counted. During the fermentation, samples were taken daily (two samples were taken at 9 d) for each 6-11d period, and spore production was measured, and the experimental results are shown in FIG. 6. At the same time, the spore counting lenses of the two groups of samples were photographed in real objects, and the results are shown in fig. 7.
As is clear from the results of FIG. 6, 2.012mmol/L of Ca was added simultaneously 2+ Fe at 0.149mmol/L 2+ And 0.135mmol/L Zn 2+ The optimized combination of the three metal ions can obviously promote asexual spore production of the deep fermentation of the Antrodia camphorata, and the promoting effect simultaneously shows that the maximum spore production amount and the spore production speed are obviously improved. Wherein, the time for reaching the maximum value of the spore yield of the deep fermentation of the Antrodia camphorata is advanced from 10d to 9d, and the time is shortened by 24h; simultaneously, the maximum spore yield of the deep fermentation of the Antrodia camphorata is 3.71 multiplied by 10 7 The number of cells per mL was increased to 8.67×10 7 The per mL is increased by 133.62 percent, and the promoting effect is extremely remarkable. When Ca is added alone 2+ 、Fe 2+ And Zn 2+ When one metal ion is added, 1mmol/L Ca is added 2+ The effect of (2) is best, the asexual sporulation yield of the deep fermentation of the antrodia camphorata can be improved by 87.21 percent to the maximum, and the effect is far less than 133.62 percent of the promotion effect in the embodiment; when Ca is added at the same time 2+ 、Fe 2+ And Zn 2+ When two metal ions in (1 mmol/L) Ca are added 2+ And 0.1mmol/L Fe 2+ The effect of (2) is best, the asexual sporulation yield of the deep fermentation of the antrodia camphorata can be improved by 106.05 percent to the maximum, but the promoting effect is obviously lower than 133.62 percent in the embodiment. In addition, the combined adding mode of the three optimal proportions in the embodiment obviously promotes the yield of the asexual spores of the deep fermentation of the antrodia camphorata, shortens the fermentation period by 24 hours, greatly improves the preparation efficiency of the asexual spores of the antrodia camphorata, obviously reduces the production cost, and has extremely high effect.
In addition, as can be seen from the spore physical pattern of FIG. 7 after dilution by 2 times on the microscope on the blood cell counting plate, ca was added compared with the control group without metal ions in the medium 2+ 、Fe 2+ And Zn 2+ The tendency of the spore concentration to increase is also very pronounced after the mixture of (a). The above results indicate that Ca 2+ 、Fe 2+ Zn (zinc) 2+ The method combines the concentrations according to the optimized response surface and is applied to the preparation of the Antrodia camphorata submerged fermentation seeds (asexual spores), so that the seed preparation efficiency can be remarkably improved, the cost of raw materials, time, manpower and material resources for preparing the seeds can be reduced, the production efficiency and benefit of the Antrodia camphorata submerged fermentation can be further improved, and the method has great application value and wide application prospect.
Example 13
Example 13 is the same as example 5 except that: inoculating Antrodia camphorata to obtain Antrodia camphorata spore suspension 1×10 5 Inoculating the inoculum size of each/mL into a culture medium; after inoculation, the culture was performed at 24℃and 50r/min with shaking for 15d.
Example 14
Example 14 is the same as example 5 except that: inoculating Antrodia camphorata to obtain Antrodia camphorata spore suspension at a ratio of 5×10 6 Inoculating the inoculum size of each/mL into a culture medium; after inoculation, the culture was performed at 27℃and 300r/min for 8d with shaking.
Example 15
Example 15 is the same as example 12 except that: ca was added simultaneously to 3.5L of the medium (prepared in example 4) 2+ 、Fe 2+ And Zn 2+ Sterilizing at 121deg.C for 20min, cooling, and mixing with Antrodia camphorata spore suspension at a ratio of 1×10 6 Inoculating the inoculum size of each/mL into a culture medium; fermenting with single control cabinet 5L duplex fermenter, inoculating at 26deg.C, 150r/min and 1.8m 3 Fermentation is carried out for 11 days under the aeration condition/h.
Claims (8)
1. A method for promoting asexual spore production of Antrodia camphorata by submerged fermentation is characterized in that metal ions are added into an Antrodia camphorata submerged fermentation culture medium, and then Antrodia camphorata is inoculated for fermentation; the metal ion is Ca 2+ 、Fe 2+ Zn (zinc) 2+ Any two or three of these; the addition amount of the metal ions in the culture medium is as follows: ca (Ca) 2+ Final concentration of 0.2-10mmol/L, fe 2+ Final concentration of 0.05-0.3mmol/L, zn 2+ The final concentration is 0.05-0.2 mmol/L.
2. The method for promoting asexual spore production by deep fermentation of Antrodia camphorata according to claim 1, wherein the metal ions are added in the following manner: before the culture medium is sterilized, the culture medium is added and then sterilized together with the culture medium; or after the culture medium is sterilized, the metal ion solution is sterilized before inoculation and then added; or inoculating Antrodia camphorata after sterilizing the culture medium, and then adding the sterilized metal ion solution.
3. The method for promoting asexual sporulation by deep fermentation of Antrodia camphorata according to claim 1, wherein the metal ions are added in the form of metal ion compounds including CaCl 2 、FeCl 2 、ZnCl 2 One or more of the following.
4. The method for promoting asexual sporulation by deep fermentation of Antrodia camphorata according to claim 1, wherein the Antrodia camphorata is inoculated by 1×10 of Antrodia camphorata spore suspension 5 personal/mL-5X 10 6 Inoculating the inoculum size of each/mL into a culture medium; after inoculation, 8-15d is cultivated by shaking at 24-27 ℃ and 50-300 r/min.
5. The method for promoting asexual spore production by deep fermentation of Antrodia camphorata according to claim 1, wherein the formula of the culture medium for deep fermentation of Antrodia camphorata is as follows: glucose 1-10%, yeast extract powder 0.1-2%, mgSO4 0.1-1%, KH 2 PO 4 0.1% -1% and an initial pH of 2.0-6.5.
6. The culture medium for promoting asexual spore production of the deep fermentation of the antrodia camphorata is characterized by comprising a deep fermentation culture medium of the antrodia camphorata and metal ions, wherein the formula of the deep fermentation culture medium of the antrodia camphorata is as follows: glucose 1-10%, yeast extract powder 0.1-2%, mgSO4 0.1-1%, KH 2 PO 4 0.1% -1%, and the initial pH is 2.0-6.5; the metal ion is Ca 2+ 、Fe 2+ Zn (zinc) 2+ Any two or three of them are Ca in deep fermentation medium of Antrodia camphorata 2+ Final concentration of 0.2-10mmol/L, fe 2+ Final concentration of 0.05-0.3mmol/L, zn 2+ The final concentration is 0.05-0.3 mmol/L.
7. An application of the method for promoting asexual production of Antrodia camphorata by submerged fermentation in improving the preparation efficiency of asexual spores of Antrodia camphorata and reducing the production cost of submerged fermentation of Antrodia camphorata according to claim 1.
8. The use of the medium for promoting asexual production of Antrodia camphorata by submerged fermentation according to claim 6 for improving the preparation efficiency of asexual spores of Antrodia camphorata and reducing the production cost of submerged fermentation of Antrodia camphorata.
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| CN102174461A (en) * | 2011-03-07 | 2011-09-07 | 江南大学 | Method for preparing asexual spores of Antrodia camphorata based on submerged fermentation and application thereof |
| CN104087631A (en) * | 2014-07-15 | 2014-10-08 | 江苏阜丰生物科技有限公司 | Method for producing Antrodia camphorata extracellular polysaccharides by deep liquid fermentation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174461A (en) * | 2011-03-07 | 2011-09-07 | 江南大学 | Method for preparing asexual spores of Antrodia camphorata based on submerged fermentation and application thereof |
| CN104087631A (en) * | 2014-07-15 | 2014-10-08 | 江苏阜丰生物科技有限公司 | Method for producing Antrodia camphorata extracellular polysaccharides by deep liquid fermentation |
Non-Patent Citations (1)
| Title |
|---|
| 钙离子调控樟芝深层发酵无性产孢及其分子机制;李华祥 等;生物工程学报;第33卷(第7期);第1.2.1节；第1.1.3节；第2.1节 * |
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