CN114574420A - Method for promoting asexual spore production of antrodia camphorata by submerged fermentation, culture medium and application thereof - Google Patents
Method for promoting asexual spore production of antrodia camphorata by submerged fermentation, culture medium and application thereof Download PDFInfo
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Abstract
The invention discloses a method for promoting asexual spore production of antrodia camphorata by submerged fermentation, a culture medium and application thereof. Adding metal ions into an antrodia camphorata submerged fermentation culture medium, and then inoculating antrodia camphorata for fermentation; the metal ion is Ca2+、Fe2+And Zn2+One or more of them. Addition of Ca alone, in comparison with the control group without addition of metal ions2+、Fe2+And Zn2+The 3 kinds of metal ions are mixed and added according to optimized concentration, so that the yield of the antrodia camphorata asexual spores can be improved by more than 133% to the maximum, the fermentation period is shortened by 24 hours, and the deep fermentation sporulation of antrodia camphorata is promotedThe effects of speed and spore yield are extremely remarkable. The method is very convenient to operate and low in cost, can greatly improve the seed preparation efficiency in the deep fermentation industrial production process of the antrodia camphorata and reduce the production cost, and further improves 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 camphorata by submerged fermentation, a culture medium and application thereof.
Background
Antrodia camphorata (Antrodia camphorata or Antrodia cinnamamomea), also known as Antrodia camphorata, Antrodia cinnamomea, Antrodia camphorata and the like, belong to Basidiomycetes (Basidiomycetes), Polyporaceae (Polyporaceae) and Antrodia (Antrodia), and are rare medical and edible dual-purpose mushroom fungi. Wild antrodia camphorata fruiting bodies usually grow on the inner wall of rotten trunks of antrodia camphorata (Cinnamomum kanehirai Hay) or withered and lodged on the wet surface close to the soil surface, have strong camphor tree fragrance, extremely bitter taste, plate-shaped, bell-shaped, horseshoe-shaped or other irregular shapes in appearance, and are mainly red in color, so the wild antrodia camphorata fruiting bodies are called 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 fat, regulating immunity and the like, and more than 200 active substances are separated from antrodia camphorata fruiting bodies or mycelia and comprise polysaccharides, diterpenes, triterpenes, steroids, benzene ring substances, maleic acid, succinic acid derivatives (Antrodin A-E), ubiquinone derivatives (Antroquinonol) and the like.
Because the biological activity of the antrodia camphorata is superior, the market demand is very huge. However, the wild antrodia camphorata grows very slowly, the quantity is very rare, and the supply is not in demand for a long time, so that the price of the wild antrodia camphorata fruiting body is always high, and each kilogram of the wild antrodia camphorata fruiting body is as high as about 15 ten thousand RMB. Therefore, large-scale artificial culture of antrodia camphorata is especially necessary. The common artificial culture techniques of antrodia camphorata mainly include 4 types: culturing basswood of cinnamomum kanehirae, culturing on a plate, performing solid-state fermentation and performing submerged fermentation. Among them, basswood cultivation, plate culture and solid state fermentation cannot be popularized because of long period (6 months-5 years) and high cost. The deep fermentation method based on asexual spore inoculation has the production period of only 10-14 days, has high production efficiency and is easy for large-scale production, so the method becomes the most efficient and common artificial culture method of antrodia camphorata at present.
The asexual spore production process of antrodia camphorata is influenced by various factors, such as carbon source, nitrogen source, illumination, dissolved oxygen, pH, exogenous additives and the like, wherein nutrient limitation of the nitrogen source, the carbon source and the like is generally considered to be one of key factors for inducing asexual spore production of antrodia camphorata. Meanwhile, the low yield and the long culture period of the asexual spores of the deep fermentation of the antrodia camphorata at present lead to long preparation time, low efficiency and high cost of the seeds (the asexual spores) of the deep fermentation of the antrodia camphorata, which are one of the key factors for restricting the large-scale deep fermentation production of the antrodia camphorata. Therefore, how to significantly improve the preparation efficiency of the asexual spores of antrodia camphorata, reduce the cost of the deep fermentation production of antrodia camphorata and promote the development process of the large-scale industrial production of the deep fermentation of antrodia camphorata is an urgent problem to be solved.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems of long preparation time, low efficiency and high cost of antrodia camphorata submerged fermentation seeds (asexual spores) caused by low yield and long culture period of the prior antrodia camphorata submerged fermentation asexual spores. The invention provides a method for promoting asexual spore production of antrodia camphorata by submerged fermentation, which can greatly improve the asexual spore yield of antrodia camphorata, shorten the fermentation period and improve the spore production speed and the spore production quantity of the antrodia camphorata by submerged fermentation.
The invention also provides a culture medium for promoting asexual spore production of antrodia camphorata by submerged fermentation and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the method for promoting asexual spore production by submerged fermentation of antrodia camphorata, disclosed by the invention, comprises the steps of adding metal ions into an antrodia camphorata submerged fermentation culture medium, and then inoculating antrodia camphorata for fermentation; the metal ion is Ca2+、Fe2+And Zn2+One or more of them.
Wherein the addition amount of the metal ions in the culture medium is as follows: ca2+Final concentration of 0.2-10mmol/L, Fe2+Final concentration of 0.05-0.3mmol/L, Zn2+The final concentration is 0.05-0.2 mmol/L.
Preferably, the addition amount of the metal ions is: ca2+Final concentration 1mmol/L, Fe2+Final concentration 0.1mmol/L, Zn2+The final concentration was 0.1 mmol/L.
Wherein the metal ion Ca2+、Fe2+And Zn2+Simultaneously adding into the Antrodia camphorata submerged fermentation culture medium.
Preferably, the metal ion Ca2+、Fe2+And Zn2+Simultaneously adding into Antrodia camphorata submerged fermentation culture medium, wherein the concentration of Ca is 2.012mmol/L2+0.149mmol/L Fe2+And 0.135 mmol/L. Wherein, the metal ion adding mode is as follows: adding the culture medium before sterilization, and then sterilizing the culture medium and the culture medium together; or after the culture medium is sterilized, adding the metal ion solution after sterilization before inoculation; or inoculating Antrodia camphorata after the culture medium is sterilized, and then adding the metal ion solution after the metal ion solution is sterilized.
Wherein the metal ions are added mainly in the form of metal ion compounds.
Preferably, the metal ions are added in the form of chlorides, including CaCl2、FeCl2、ZnCl2One or more of.
Preferably, the metal ion compound is added at the time of preparing the medium, and then the medium is sterilized.
Wherein the inoculation is performed by inoculating Antrodia camphorata sporeThe sub-suspension is at a ratio of 1 × 105Per mL-5X 106Inoculating the strain/mL into a culture medium; after inoculation, the culture is carried out for 8-15 days under the conditions of 24-27 ℃ and 50-300 r/min.
Preferably, the Antrodia camphorata spore suspension is prepared into a 1 × 106Inoculating the strain/mL into a culture medium; after inoculation, the culture is carried out for 10 days at 26 ℃ and 150r/min under shaking.
Wherein, the formula of the antrodia camphorata submerged fermentation medium is as follows: 1 to 10 percent of glucose, 0.1 to 2 percent of yeast extract powder, MgSO40.1 to 1 percent and KH2PO40.1% -1%, initial pH 2.0-6.5.
Preferably, the formula of the antrodia camphorata submerged fermentation medium comprises 2% of glucose, 0.2% of yeast extract powder, 40.15% of MgSO40 and KH2PO40.3%, initial pH 4.5. The culture medium for promoting asexual spore production by antrodia camphorata submerged fermentation comprises an antrodia camphorata submerged fermentation culture medium and metal ions, wherein the formulation of the antrodia camphorata submerged fermentation culture medium comprises 1-10% of glucose, 0.1-2% of yeast extract powder, 1-1% of MgSO40.1 and KH2PO40.1% -1%, initial pH 2.0-6.5; the metal ion is Ca2+、Fe2+And Zn2+The concentration of one or more of them in the Antrodia camphorata submerged fermentation culture medium is Ca respectively2+The final concentration is 0.2-10mmol/L, Fe2+Final concentration of 0.05-0.3mmol/L, Zn2+The final concentration is 0.05-0.2 mmol/L.
The method for promoting asexual spore production of antrodia camphorata submerged fermentation is applied to improving the preparation efficiency of antrodia camphorata asexual spores and reducing the production cost of antrodia camphorata submerged fermentation.
The culture medium for promoting asexual spore production by antrodia camphorata submerged fermentation is applied to improving the preparation efficiency of antrodia camphorata asexual spores and reducing the production cost of antrodia camphorata submerged fermentation. The slow asexual spore production speed and the low spore production amount of the deep fermentation of the antrodia camphorata are one of the main limiting factors of the deep fermentation industrial production of the antrodia camphorata. Because of the low spore yield, a large volume of fermentation broth is often required for the preparation of seeds (asexual spore suspension of A. camphorata), which greatly increases the raw material cost of the culture medium. In addition, to control the inoculation volume (usually "seedThe volume ratio of the seed solution to the fermentation medium is not higher than 10 percent), and sometimes even the filtered antrodia camphorata spore suspension needs to be concentrated, which greatly increases the probability of bacterial contamination. Therefore, when preparing the antrodia camphorata seeds, the complicated operation and the long spore production period are added, and the raw material, time, labor and material cost of the seed preparation are increased to a great extent. The invention provides a method for promoting asexual spore production of antrodia camphorata submerged fermentation, which is to add Ca with the final concentration of 0.2-10mmol/L into an antrodia camphorata submerged fermentation culture medium2+0.05 to 0.3mmol/L Fe2+Or 0.05 to 0.2mmol/L Zn2+One or more metal ions, and inoculating Antrodia camphorata for fermentation. Addition of Ca alone, in comparison with the control group without addition of metal ions2+The yield of asexual spores of the antrodia camphorata can be improved by more than 87 percent to the maximum extent; addition of Fe alone2+The yield of asexual spores of the antrodia camphorata can be improved by more than 72 percent to the maximum extent; addition of Zn alone2+The yield of asexual spores of the antrodia camphorata can be improved by more than 35 percent to the maximum extent. The 3 metal ions are mixed and added according to the optimized concentration of the response surface and are applied to the antrodia camphorata submerged fermentation, so that the yield of asexual spores of the antrodia camphorata can be improved by more than 133% to the maximum extent, the fermentation period is shortened by 24 hours, the spore yield of the antrodia camphorata submerged fermentation is remarkably improved, and the spore production speed is also remarkably accelerated. The invention discovers that the fermentation period can be obviously shortened while the yield of the asexual spores of the antrodia camphorata is improved, and the subsequent production period can be reduced by shortening the fermentation period, so that the production efficiency is improved and the production cost is reduced. In the prior art, no exogenous additive capable of improving spore yield has the effect of shortening the fermentation period, and the method provided by the invention has the advantages of remarkably improving the yield of asexual spores of antrodia camphorata and shortening the fermentation period, and very having production advantages. In conclusion, the method provided by the invention obviously improves the preparation efficiency of the asexual spores of antrodia camphorata, reduces the industrial production cost of deep fermentation of antrodia camphorata, and greatly improves the industrial production efficiency and benefits of deep fermentation of antrodia camphorata. The main effects of adding the metal ions into the culture medium are 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 antrodia camphorata asexual spores and reduce the production cost.
The inventionThrough multiple optimization findings, the metal ion Ca2+、Fe2+And Zn2+The three substances are added into the deep fermentation medium of the antrodia camphorata simultaneously according to a certain proportion, the effect is the best when the three substances are combined, and the concentration of the combined addition of the three substances is obtained by optimizing the response surface. The addition of a single metal ion can improve a certain yield, but cannot shorten the cycle time. In addition, the invention also tries to add other metal ions, such as Mg2+、Cu2+And Al3+And the results show that: the effect of the metal ions on improving the yield of asexual spores of deep fermentation of antrodia camphorata is not obvious, and the metal ions and Ca are mixed2+、Fe2+And Zn2+The effect is not obviously improved after any two are mixed, wherein the Cu is mixed with the copper2+And Al3+The mixture of (A) and (B) even has the function of inhibiting asexual spore production of deep fermentation of the antrodia camphorata.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the method, metal ions are added into the antrodia camphorata submerged fermentation culture medium for the first time, and then antrodia camphorata is inoculated for fermentation, so that the method provided by the invention can improve the yield of asexual spores of antrodia camphorata submerged fermentation by over 133 percent, and the spore yield is up to 8.6 multiplied by 107One seed/mL, up to 1.0X 10 by inoculation amount6The inoculation volume ratio is only 1.16 percent (calculated by one/mL) and is less than 2 percent, which greatly improves the operability of spore inoculation when the antrodia camphorata is deeply fermented and produced. Due to the high spore yield, the volume of the fermentation liquor of the needed seeds is greatly reduced, concentration is not needed, the preparation procedure of the seeds is simplified, and the contamination risk and the preparation cost of the seeds are reduced. Meanwhile, the method provided by the invention has the very important advantage that the sporulation period can be shortened by 24 hours, and further the production period is shortened. In short, the method obviously improves the preparation efficiency of the antrodia camphorata asexual spores, reduces the preparation cost of antrodia camphorata submerged fermentation production seeds (asexual spores), and further obviously improves the antrodia camphorata submerged fermentation production efficiency and benefit. In addition, the metal ions added in the invention are common metal ions, the price is low and the addition amount is extremely low; meanwhile, the metal ions in the invention can be added when preparing the culture medium, so that the culture medium does not need to be sterilized independently, and the operation is not requiredIs convenient for a long time. Most importantly, the method provided by the invention has extremely obvious effects in the aspects of promoting the spore production speed and the spore production amount. In conclusion, the method provided by the invention has the advantages of convenience in operation, low cost, obvious effect, great development value and good application prospect.
Drawings
FIG. 1 addition of Ca at different concentrations2+Influence on asexual spore production of deep fermentation of Antrodia camphorata; wherein "CK" is a control group without any metal ion added (same below);
FIG. 2 addition of different concentrations of Fe2+Influence on asexual spore production of deep fermentation of Antrodia camphorata;
FIG. 3 addition of Zn in different concentrations2+Influence on asexual spore production of deep fermentation of Antrodia camphorata;
FIG. 4 shows the effect of adding different combinations of two metal ions simultaneously on asexual spore formation in submerged fermentation of Antrodia camphorata;
FIG. 5 is a graph showing interaction between spore production and concentration of each metal ion added in response surface analysis;
FIG. 6 shows the effect of adding 3 optimized combinations of metal ions on asexual spore production by submerged fermentation of Antrodia camphorata;
FIG. 7 is a real comparison graph of spore yields of the combination group optimized by adding 3 kinds of metal ions and the control group.
Detailed Description
The invention is further illustrated by the following figures and examples.
The starting materials and reagents used in the present invention are commercially available unless otherwise specified.
The method for detecting the spore yield of the antrodia camphorata submerged fermentation in the following embodiments is as follows:
filtering the fermentation liquid obtained after deep fermentation of Antrodia camphorata with 4 layers of gauze under aseptic condition to obtain filtrate, i.e. spore suspension (used as seed). Then, 20. mu.L of the spore suspension was aspirated and counted under an optical microscope using a hemocytometer, and the spore yield was calculated from the counted result.
Antrodia camphorata (Antrodia camphorate) strain was purchased from American Type Culture Collection (ATCC) and assigned ATCC 200183.
Example 1
Ca2+The preparation steps of the mother liquor (1mol/L) are as follows:
(1) weighing 11.10g CaCl2Putting the powder into a beaker, and adding 80mL of deionized water;
(2) rolling and stirring with a glass rod until CaCl2Completely dissolving;
(3) dissolving CaCl2Pouring the solution into a 100mL volumetric flask, and determining the volumetric value of 100mL by using deionized water to obtain 1mol/L Ca2+Mother liquor;
(4) adding Ca2+The mother liquor is subpackaged and stored in a refrigerator at the temperature of 20 ℃ below zero for later use.
Example 2
Fe2+The preparation steps of the mother liquor (1mol/L) are as follows:
(1) 12.68g FeCl was weighed2Putting the powder into a beaker, and adding 80mL of deionized water;
(2) rolling and stirring with a glass rod until FeCl2Completely dissolving;
(3) dissolving FeCl2Pouring all the solution into a 100mL volumetric flask, and determining the volumetric value of 100mL by using deionized water to obtain 1mol/L Fe2+Mother liquor;
(4) mixing Fe2+The mother liquor is subpackaged and stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
Example 3
Zn2+The preparation steps of the mother liquor (1mol/L) are as follows:
(1) 13.64g of ZnCl was weighed2Putting the powder into a beaker, and adding 80mL of deionized water;
(2) rolling and stirring with a glass rod until ZnCl is obtained2Completely dissolving;
(3) dissolving ZnCl2Pouring all the solution into a 100mL volumetric flask, and determining the volumetric value of 100mL by using deionized water to obtain 1mol/L Zn2+Mother liquor;
(4) zn is added2++The mother liquor is subpackaged and stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
Example 4
The preparation method of the antrodia camphorata submerged fermentation culture medium comprises the following steps:
(1) preparing a culture medium (mass fraction): 2 percent of glucose, 0.2 percent of yeast extract powder and MgSO4 0.15％、KH2PO40.3 percent of formula is used for preparing an antrodia camphorata submerged fermentation liquid culture medium, and the initial pH value is adjusted to 4.5 by using 1mol/L HCl and 1mol/L NaOH solution.
(2) And (3) sterilizing a culture medium: the culture medium is subpackaged into 250mL conical flasks according to the liquid loading amount of 100mL/250mL (shake flask fermentation) or 5L fermentation tanks according to the liquid loading amount of 3.5L/5L (fermentation tank fermentation), sterilized at 121 ℃ for 20min, and cooled for later use.
Example 5
Separately adding Ca at different concentrations2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, Ca was added to the medium prepared in example 42+The final concentrations of (A) were 0.2mmol/L, 1mmol/L, 5mmol/L and 10mmol/L, respectively. That is, when preparing a medium, 20. mu.L, 100. mu.L, 500. mu.L and 1mL of Ca at a concentration of 1mol/L were added to 100mL of the medium (prepared in example 4), respectively2+The mother liquor (prepared in example 1) is sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared into 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
The method of example 5 was followed while setting the fermentation group without any metal ions added to the medium as a control group, and 3 replicates were set for each group. During the culture process, samples were taken every day for 6-11 days respectively to count spores and determine the spore yield, and the experimental results are shown in FIG. 1.
From the results of fig. 1, it can be seen that: during fermentation, the spore yield reaches the maximum value at 10d, and 0.2-10mmol/L Ca is added2+Can obviously promote asexual spore production of the antrodia camphorata by submerged fermentation. Wherein, when Ca2+When the final concentration is 1mmol/L, the maximum spore yield (6.76 × 10) of deep fermentation of Antrodia camphorata can be achieved7seed/mL) is improved by 87.21 percent compared with a control group, and the effect is extremely obvious; when Ca is present2+When the final concentration is 0.2mmol/L, the maximum spore yield (5.34 × 10) of deep fermentation of Antrodia camphorata can be achieved7one/mL) was increased by 47.73% compared with the control group, and the effect was very significant. The above results illustrate Ca2+Can obviously promote asexual spore production of deep fermentation of the antrodia camphorata, but has serious concentration dependence, and the higher the adding concentration is, the better the effect is. When the concentration is too high (e.g., 5mmol/L or 10mmol/L), the effect of promoting spore production is rather reduced.
Example 6
Separately adding different concentrations of Fe2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, Fe was added to the medium prepared in example 42+The final concentrations of (A) were 0.05mmol/L, 0.1mmol/L, 0.2mmol/L and 0.3mmol/L, respectively. That is, when preparing a medium, 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), respectively2+The mother liquor (prepared in example 2) is sterilized at 121 ℃ for 20min and cooled, and the Antrodia camphorata spore suspension is prepared according to the proportion of 1 x 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
The method of example 6 was followed while setting the fermentation group without any metal ions added to the medium as a control group, and 3 replicates were set for each group. In the culture process, samples were taken every day for 6-11 days respectively to count spores, and the spore yield was determined, with the experimental results shown in fig. 2.
As can be seen from the results in FIG. 2, the amount of spores produced during the fermentation was maximized at 10d, and 0.05-0.3mmol/L Fe was added2+Can obviously promote asexual spore production of the antrodia camphorata by submerged fermentation. Wherein, when Fe2+When the final concentration is 0.1mmol/L, the maximum spore yield (6.74 × 10) of deep fermentation of Antrodia camphorata can be achieved7seed/mL) is improved by 72.39 percent compared with a control group, the addition amount is extremely little, but the effect is extremely obvious; when Fe2+When the final concentration is 0.2mmol/L, the maximum spore yield (6.01 × 10) of deep fermentation of Antrodia camphorata can be achieved7one/mL) is improved by 53.66 percent compared with a control group, and the effect is very obvious. The above results illustrate Fe2+The method can obviously promote asexual spore production of the deep fermentation of the antrodia camphorata, but has serious concentration dependence, and the higher the adding concentration is, the better the effect is, and the adding amount is strictly controlled. When the concentration is too high (e.g., 0.3mmol/L), the effect of promoting spore production is rather reduced or even eliminated.
Example 7
Separately adding Zn in different concentrations2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, Zn was added to the medium prepared in example 42+The final concentrations of (A) were 0.05mmol/L, 0.1mmol/L and 0.2mmol/L, respectively. That is, when preparing a medium, 5. mu.L, 10. mu.L and 20. mu.L of Zn at a concentration of 1mol/L were added to 100mL of the medium (prepared in example 4), respectively2+The mother liquor (prepared in example 3) is sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared into 1 × 106Inoculating the inoculum size of each/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
The method of example 7 was followed while setting the fermentation groups to which no metal ion was added in the medium as control groups, each group being set to 3 replicates. During the culture process, samples were taken every day for 6-11 days respectively to count spores and determine the spore yield, and the experimental results are shown in FIG. 3.
From the results shown in FIG. 3, it is understood that 0.05 to 0.2mmol/L of Zn was added2+Can obviously promote asexual spore production of the antrodia camphorata by submerged fermentation. Wherein, when Zn2+When the final concentration is 0.1mmol/L, the maximum spore yield (5.08 × 10) of deep fermentation of Antrodia camphorata can be achieved7seed/mL) increased by 35.05% compared with the control group; when Zn2+When the final concentration is 0.05mmol/L, the maximum spore yield (4.62 × 10) of deep fermentation of Antrodia camphorata can be achieved7seed/mL) was increased by 23% over the control, and the addition was very small, but the effect was still significant. The above results illustrate Zn2+The method can obviously promote asexual spore production of deep fermentation of the antrodia camphorata, but the promoting effect has the same serious concentration dependence, and the higher the adding concentration is, the better the effect is, and the adding amount is strictly controlled. When the concentration is too high (e.g., 0.3mmol/L), the effect of promoting spore production is reduced.
Example 8
Simultaneously adding Ca2+And Fe2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, Ca was added to the medium prepared in example 4 to a final concentration of 1mmol/L2+And 0.1mmol/L Fe2+. That is, when preparing a culture medium, 1mL of Ca at a concentration of 1mol/L was added to 100mL of the culture medium (prepared in example 4)2+Mother liquor (prepared in example 1) and 10. mu.L of Fe at a concentration of 1mol/L2+The mother liquor (prepared in example 2) is sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared according to the proportion of 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
Example 9
While adding Ca2+And Zn2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, Ca was added to the medium prepared in example 4 to a final concentration of 1mmol/L2+And 0.1mmol/L Zn2+. That is, when preparing a culture medium, 1mL of Ca at a concentration of 1mol/L was added to 100mL of the culture medium (prepared in example 4)2+Mother liquor (prepared in example 1) and 10. mu.L of Zn at a concentration of 1mol/L2+The mother liquor (prepared in example 3) is sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared into 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
Example 10
With addition of Fe2+And Zn2+Effect on asexual spore formation in submerged fermentation of Antrodia camphorata, the medium prepared in example 4 was simultaneously supplemented with+Fe at a final concentration of 0.1mmol/L2+And 0.1mmol/L Zn2+. That is, when preparing a medium, 10. mu.L of Fe at 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 1mol/L Zn2+The mother liquor (prepared in example 3) is sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared into 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
The method of example 8, example 9 and example 10 was followed, and 3 replicates of each group were set to fermentation groups in which no metal ion was added to the medium as a control group. At 10d of fermentation, samples were taken for spore counting and spore production was determined, and the results are shown in FIG. 4.
From the results in FIG. 4, it is clear that Ca is contained in combination2+、Fe2+And Zn2+Any two metal ions in the antrodia camphorata can obviously promote asexual spore production through submerged fermentation. Wherein Ca with a final concentration of 1mmol/L is added simultaneously2+And 0.1mmol/L Fe2+In practice, the submerged fermentation spore yield of Antrodia camphorata (7.59X 10)7one/mL) is improved by 106.05% compared with a control group when the fermentation is carried out for 10 days, and the effect is extremely obvious; while adding Ca to a final concentration of 1mmol/L2+And 0.1mmol/L Zn2+In practice, the submerged fermentation spore yield of Antrodia camphorata (7.15 × 10)7one/mL) is improved by 94.01 percent compared with a control group when the fermentation is carried out for 10 days, and the effect is extremely obvious; when Fe with a final concentration of 0.1mmol/L is added simultaneously2+And 0.1mmol/L Zn2+In practice, the submerged fermentation spore yield of Antrodia camphorata (6.48 × 10)7one/mL) was improved by 78.60% compared to the control group at 10d of fermentation, but the effect was very significant. The above results show that Ca is added at an appropriate concentration simultaneously2+、Fe2+And Zn2+Any two metal ions in the strain can obviously promote asexual spore production of antrodia camphorata by submerged fermentation, so that the spore production is improved by about 78-106% compared with that of a control group, and the effect is extremely obvious.
Example 11
Optimization of simultaneous addition of Ca by response surface method2+、Fe2+And Zn2+The optimum concentration of each metal ion is used. First, Ca was added at a concentration of 3 for each metal ion2+、Fe2+And Zn2+The levels of the response surface factors (c) are shown in the following table (table 1).
TABLE 1 level table of each metal ion response surface factor
Subsequently, we designed 3-factor 3 level combination test groups using the addition concentration of each metal ion as an independent variable and the maximum spore yield as a dependent variable by using the software "Design expert 11", and the group Design results are shown in the following table (table 2).
TABLE 2 design table for each group of metal ion response surfaces
Finally, Ca prepared in example 1 was added to the culture medium prepared in example 42+Mother liquor, Fe prepared in example 22+Mother liquor and Zn prepared in example 32+The mother liquor was added with Ca at the concentrations corresponding to the combined concentrations of the respective groups in Table 22+、Fe2+And Zn2+Sterilizing at 121 deg.C for 20min, cooling, and mixing with Antrodia Camphorata spore suspension at 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
Meanwhile, a fermentation group without adding metal ions in the culture medium is set as a control group, and each group is set with 3 replicates. During the culture process, sampling every day in 6-11 days respectively to count spores, measuring spore yield, and analyzing by using Design expert 11 software according to the spore yield data of each group to obtain an interaction diagram (figure 5) among all factors and a ternary quadratic regression equation between the spore yield and each factor.
From the results of fig. 5, it can be seen that the response surface graph of any two-factor interaction between the three factors (three metal ions) is a perfect arc-shaped curved surface, which indicates that the center point and step length of each factor level are designed reasonably, and the optimal additive concentration is near the center point. Then, the 'Design expert 11' software is used for simulating a ternary quadratic regression equation as follows: the sporulation yield was 8.485+ 276.85A + 13.89B + 401.35C + 15.416A B-137.5A-8.08B C-972.5A2-3.75*B2-1350*C2. The extremum is obtained according to the regression equation, and the sporulation yield is maximum when A is 0.14874, B is 2.0121998, and C is 0.135. Namely, the optimal addition concentrations of the three metal ions are respectively as follows: ca2+Final concentration 2.012mmol/L, Fe2+Final concentration 0.149mmol/L and Zn2+The final concentration was 0.135 mmol/L.
Example 12
Method for promoting asexual spore production by submerged fermentation of Antrodia camphorata by adding metal ions, and application thereof2+、Fe2+And Zn2+Three kinds of metal ionsThe mixture was mixed at the optimized concentration of example 11 and added to the medium prepared in example 4 in the amount of Ca2+Final concentration 2.012mmol/L, Fe2+Final concentration 0.149mmol/L and Zn2+The final concentration was 0.135 mmol/L. That is, when preparing a medium, 2.012mL of Ca at a concentration of 1mol/L was added to 1000mL of the medium (prepared in example 4)2+Stock solution (prepared in example 1) 149. mu.L of 1mol/L Fe2+Mother liquor (prepared in example 2) and 135. mu.L of Zn at a concentration of 1mol/L2+Mother liquor (prepared in example 3), stirred evenly and then subpackaged into 100mL of shake bottles, sterilized at 121 ℃ for 20min and cooled, and then the Antrodia camphorata spore suspension is prepared according to the proportion of 1 × 106Inoculating the strain/mL into a culture medium; placing the inoculated shake flask in a shaking table at 26 ℃, and carrying out shaking culture at 150r/min for 11 days.
The method of example 12 was followed, and 3 replicates were taken for each sampling and spore counting, while the control group was a fermentation group to which no metal ions were added. In the fermentation process, samples were taken every day during 6-11 days (wherein samples were taken twice at 9 d) to count spores, and the amount of spores produced was determined, with the experimental results shown in fig. 6. Meanwhile, 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 in FIG. 6, 2.012mmol/L Ca was added simultaneously2+0.149mmol/L Fe2+0.135mmol/L Zn of2+The optimized combination of the three metal ions can obviously promote asexual spore production of the deep fermentation of the antrodia camphorata, and the promotion effect is simultaneously shown to obviously improve the maximum spore yield and the spore production speed. Wherein, the time for the submerged fermentation spore yield of the antrodia camphorata to reach the maximum value is shortened by 24 hours from 10 days to 9 days; simultaneously, the maximum spore yield of deep fermentation of the antrodia camphorata is from 3.71 multiplied by 107Increase to 8.67X 10/mL7The seed/mL is improved by 133.62%, and the promotion effect is extremely obvious. When Ca is added alone2+、Fe2+And Zn2+When one of the metal ions is contained in the solution, 1mmol/L Ca is added2+The effect of the method is best, the asexual spore yield of the deep fermentation of the antrodia camphorata can be improved by 87.21 percent to the maximum extent, and the effect is far less than the promotion effect of 133.62 percent in the embodiment; when Ca is added simultaneously2+、Fe2+And Zn2+When two kinds of metal ions are contained in (1 mmol/L) Ca is added2+And 0.1mmol/L Fe2+The effect of the method is best, the asexual spore yield of the antrodia camphorata submerged fermentation can be improved by 106.05% to the maximum extent, but the promotion effect is also obviously lower than 133.62% in the embodiment. In addition, in the embodiment, the three optimal proportion combined addition modes significantly promote the yield of the asexual spores of deep fermentation of antrodia camphorata, and simultaneously shorten the fermentation period by 24 hours, so that the preparation efficiency of the asexual spores of antrodia camphorata is further greatly improved, the production cost is significantly reduced, and the effect is particularly valuable.
In addition, as can be seen from the 2-fold dilution of spore on the hemocytometer under the microscope in FIG. 7, Ca was added to the medium in comparison with the control group in which no metal ion was added to the medium2+、Fe2+And Zn2+The tendency of the spore concentration to increase is also very pronounced after the mixture of (1). The above results show that Ca2+、Fe2+And Zn2+The combination is carried out according to the concentration optimized according to the response surface and is applied to the preparation of antrodia camphorata submerged fermentation seeds (asexual spores), the seed preparation efficiency can be obviously improved, the raw material, time, labor and material cost for preparing the seeds are reduced, the production efficiency and the benefit of antrodia camphorata submerged fermentation are further improved, and the antrodia camphorata submerged fermentation method has great application value and wide application prospect.
Example 13
Example 13 is the same as example 5 except that: inoculating Antrodia camphorata, and mixing with the spore suspension of Antrodia camphorata at 1 × 105Inoculating the strain/mL into a culture medium; after inoculation, the culture is carried out for 15 days under the conditions of 24 ℃ and 50 r/min.
Example 14
Example 14 is the same as example 5 except that: inoculating Antrodia camphorata, and mixing with Antrodia camphorata spore suspension at 5 × 106Inoculating the strain/mL into a culture medium; after inoculation, the culture is carried out for 8 days under the conditions of 27 ℃ and 300 r/min.
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+、Fe2+And Zn2+Sterilizing at 121 deg.C for 20min, cooling, and mixing with Antrodia Camphorata spore suspension at 1 × 106Inoculating the strain/mL into a culture medium; fermenting with single control cabinet 5L duplex fermentation tank at 26 deg.C, 150r/min and 1.8m3Fermenting for 11 days under the condition of ventilation volume.
Claims (10)
1. A method for promoting asexual spore production by submerged fermentation of Antrodia camphorata is characterized in that metal ions are added into a submerged fermentation culture medium of Antrodia camphorata, and then the Antrodia camphorata is inoculated for fermentation; the metal ion is Ca2+、Fe2+And Zn2+One or more of them.
2. The method for promoting asexual spore production by submerged fermentation of antrodia camphorata according to claim 1, wherein the metal ions are added in the culture medium in an amount of: ca2+The final concentration is 0.2-10mmol/L, Fe2+Final concentration of 0.05-0.3mmol/L, Zn2+The final concentration is 0.05-0.2 mmol/L.
3. The method for promoting asexual spore production by submerged fermentation of Antrodia camphorata according to claim 1, wherein the metal ion Ca2+、Fe2+And Zn2+Preferably, the culture medium is added into the Antrodia camphorata submerged fermentation medium at the same time.
4. The method for promoting asexual spore formation in submerged fermentation of antrodia camphorata according to claim 1, wherein the metal ions are added in a manner of: adding the culture medium before sterilization, and then sterilizing the culture medium and the culture medium together; or after the culture medium is sterilized, adding the metal ion solution after sterilization before inoculation; or inoculating Antrodia camphorata after the culture medium is sterilized, and then adding the metal ion solution after the metal ion solution is sterilized.
5. The method for promoting asexual spore production by submerged fermentation of antrodia camphorata according to claim 1, wherein the metal ions are mainly added in the form of metal ion compounds comprising CaCl2、FeCl2、ZnCl2One or more of.
6. The method for promoting asexual spore production by submerged fermentation of Antrodia camphorata according to claim 1, wherein the Antrodia camphorata is inoculated by suspending Antrodia camphorata spores in a 1 x 10 manner5Per mL-5X 106Inoculating the strain/mL into a culture medium; after inoculation, the culture is carried out for 8-15 days under the conditions of 24-27 ℃ and 50-300 r/min.
7. The method for promoting asexual spore production by submerged fermentation of antrodia camphorata according to claim 1, wherein the submerged fermentation medium comprises: 1 to 10 percent of glucose, 0.1 to 2 percent of yeast extract powder, MgSO40.1 to 1 percent and KH2PO40.1% -1%, initial pH 2.0-6.5.
8. The culture medium for promoting asexual spore production of antrodia camphorata submerged fermentation is characterized by comprising an antrodia camphorata submerged fermentation culture medium and metal ions, wherein the antrodia camphorata submerged fermentation culture medium comprises the following formula: 1 to 10 percent of glucose, 0.1 to 2 percent of yeast extract powder, MgSO40.1 to 1 percent and KH2PO40.1% -1%, initial pH 2.0-6.5; the metal ion is Ca2+、Fe2+And Zn2+One or more of them, their concentration in Antrodia camphorata submerged fermentation culture medium is Ca respectively2+The final concentration is 0.2-10mmol/L, Fe2+Final concentration of 0.05-0.3mmol/L, Zn2+The final concentration is 0.05-0.3 mmol/L.
9. The method for promoting asexual spore production by submerged fermentation of antrodia camphorata according to claim 1, which is applied to improving the efficiency of preparing asexual spores of antrodia camphorata and reducing the production cost of submerged fermentation of antrodia camphorata.
10. The use of the culture medium for promoting asexual spore production by submerged fermentation of Antrodia camphorata according to claim 8 for improving the efficiency of preparing asexual spore 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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| 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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| Title |
|---|
| 李华祥 等: "钙离子调控樟芝深层发酵无性产孢及其分子机制", 生物工程学报, vol. 33, no. 7, pages 1 * |
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