CN108841798B - Culture medium and method for producing superoxide dismutase through paecilomyces fermentation - Google Patents
Culture medium and method for producing superoxide dismutase through paecilomyces fermentation Download PDFInfo
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Abstract
The invention relates to a fermentation culture medium and a process of fungi, in particular to a culture medium and a method for producing superoxide dismutase by fermenting paecilomyces varioti. The raw materials of the culture medium contain 5-40 g/L of fructose, 3.2-25.6 g/L of ammonium sulfate, 2.6-20.8 g/L of aluminum chloride and 0.02-0.16 g/L of vitamin C; the pH of the medium is 6.8-7.7. The method comprises the steps of inoculating fermentation seed liquid into the culture medium for producing the superoxide dismutase to culture, wherein the liquid loading capacity of a 250mL triangular flask is 100mL, the inoculation amount is 10% (v/v), and the culture is carried out at 25 ℃ and the rotating speed of 150 r/min. The fermentation method has the advantages of short fermentation period, mild conditions and easy control; the culture medium for producing superoxide dismutase by paecilomyces fermentation has the advantages of high yield, high activity and the like of the superoxide dismutase.
Description
Technical Field
The invention relates to a fermentation medium and a process of fungi; more particularly, the invention relates to a culture medium and a method for producing superoxide dismutase by fermenting paecilomyces.
Background
Paecilomyces (A)Paecilomyces) Are important members of the entomopathogenic fungi that infest predominantly lepidopteran insects and plant nematodes, sensitizing them to control population numbers, making it an effective biocontrol agent. With the continuous and deep research, people find that the metabolite of paecilomyces has various types and special action mechanism and has important application value in biopesticides or medicines in recent years.
Superoxide dismutase (Superoxide dismutase) is an antioxidant enzyme that can eliminate harmful substances produced by organisms during metabolism. Superoxide dismutase is widely distributed in animals, plants and microorganisms, and has an important function of clearing free radicals in organisms. The research on the superoxide dismutase is more beneficial to the disclosure of the biological antioxidant mechanism and the application of the superoxide dismutase.
Disclosure of Invention
The invention aims to provide a culture medium and a method for producing superoxide dismutase by fermenting paecilomyces in order to overcome the defects in the prior art. When the culture medium is used for producing superoxide dismutase by fermenting paecilomyces, the yield of the superoxide dismutase can be improved; the fermentation method has short period; the condition is mild and easy to control.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for producing superoxide dismutase by fermenting paecilomyces comprises the following raw materials: 5-40 g/L of fructose, 3.2-25.6 g/L of ammonium sulfate, 2.6-20.8 g/L of aluminum chloride and 0.02-0.16 g/L of vitamin C; the pH of the medium is 6.8-7.7.
Preferably, the raw materials of the culture medium comprise: 20g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminum chloride and 0.16 g/L of vitamin C; the pH of the medium was 7.4.
Secondly, the invention also provides a method for producing superoxide dismutase by fermenting paecilomyces, which comprises the following steps:
(a) activating a Paecilomyces fungus, inoculating the fungus into a seed culture medium, and culturing to obtain a fermentation seed solution; temperature of the shaking table: the temperature is 25 +/-1 ℃, and the seed culture period is 72 hours; the seed culture medium contains the following raw materials: 200g/L of potato and 20g/L of glucose; the preparation method comprises the following steps: accurately weighing 200g peeled and cut potato, heating and decocting, filtering with eight layers of gauze, adding 20g glucose into potato juice, and diluting to 1000 ml.
(b) Inoculating the fermentation seed liquid prepared in the step (a) on a culture medium for producing superoxide dismutase by fermenting the paecilomyces fungus, wherein the liquid loading of a 250mL triangular flask is 100mL, the inoculation amount is 10% (v/v), and the culture is carried out at 25 ℃ and the rotating speed of 150r/min for 25 h.
The invention has the beneficial effects that:
1) the culture medium for producing the superoxide dismutase by the paecilomyces fungus fermentation can improve the yield of the superoxide dismutase produced by the paecilomyces fungus fermentation;
2) the fermentation method has the advantages of short fermentation period, mild conditions, easy control of production and the like.
Drawings
FIG. 1 effect of different carbon sources on the activity of superoxide dismutase produced by fermentation of Paecilomyces fungi;
FIG. 2 effect of different nitrogen sources on the activity of superoxide dismutase produced by fermentation of Paecilomyces fungi;
FIG. 3 effect of different metal ions on the activity of superoxide dismutase produced by fermentation of Paecilomyces fungi;
FIG. 4 effect of different vitamins on the activity of superoxide dismutase produced by fermentation of Paecilomyces fungi;
FIG. 5 Effect of different pH on the activity of superoxide dismutase produced by fermentation of Paecilomyces fungi.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
(1) Preparation of fermented seeds
(A) Strains and culture media
Strain: paecilomyces fungi
The seed culture medium comprises the following raw materials: 200g/L of potato and 20g/L of glucose; the preparation method comprises the following steps: accurately weighing 200g peeled and cut potato, heating and decocting, filtering with eight layers of gauze, adding 20g glucose into potato juice, and diluting to 1000 ml.
(B) Seed liquid preparation
Transferring pure Paecilomyces fungi on the plate into a plurality of 250mL triangular flasks, wherein 100mL seed culture media are filled in the triangular flasks, and then culturing and culturing for 72h at 25 +/-1 ℃ in a reciprocating shaking table rotating speed of 150r/min to obtain a fermentation seed solution.
(2) Fermentation culture
Inoculating the fermentation seed liquid prepared in the step (B) into a culture medium, wherein the liquid loading of a 250mL triangular flask is 100mL, the inoculation amount is 10%, and culturing is carried out at 25 ℃ and the rotating speed of 150 r/min;
the raw materials of the fermentation medium comprise: 20g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminum chloride, 0.16 g/L of vitamin C and pH 7.4.
The temperature of the shaking table is 25 +/-1 ℃, and the fermentation period is 25 h.
(3) Determination of superoxide dismutase Activity
Determination of autoxidation: at 25 ℃, adding 10 mu L of 50 mmol/L pyrogallol into 4.5 mL of 50 mmol/L, pH 8.2.2 Tris-HCl buffer solution, quickly shaking up, pouring into a cuvette, measuring the A value once every 30s at 325 nm by taking the Tris-HCl buffer solution as a blank control for 6 times, and controlling the autoxidation rate to be about 0.07 OD/min.
And (3) measuring enzyme activity: absorbing the fermentation liquor, centrifuging for 10 min under 10000 r/m, obtaining a supernatant as a crude enzyme solution, adding a certain amount of enzyme solution to be detected into 4.5 mL of 50 mmol/L Tris-HCl buffer solution with the pH value of 8.2 at 25 ℃, preheating for 20 min, adding 10 mu L of 50 mmol/L pyrogallol, quickly shaking up, pouring into a cuvette, taking Tris-HCl buffer solution as a blank control, detecting the A value once every 30s at the wavelength of 325 nm, and detecting for 6 times in total. Under these conditions, the amount of enzyme inhibiting the pyrogallol autoxidation rate by 50% per minute was defined as 1 activity unit.
Inhibition rate/% = (a0-AS)/AS × 100
SOD unit activity (U/ml) = (A0-AS)/(AS x 50%) × 4.5/V × N
Wherein A0 is the autoxidation rate;
AS is the oxidation rate after adding the sample liquid;
v is the sample addition volume;
and N is the dilution factor of the sample.
The determination by this method was that the fungus Paecilomyces was in basal fermentation medium: 20g of glucose, 0.4g of yeast powder, 0.7g of ammonium sulfate, 0.5g of corn flour, 0.5g of calcium carbide and 0.05g of magnesium sulfide, wherein the content of superoxide dismutase produced on the corn flour is 7.85 +/-0.31U/mL.
In this way, the paecilomyces fungus was determined to be in the fermentation medium of example 1: 20g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminum chloride, 0.16 g/L of vitamin C and 303.56 +/-4.62U/mL of superoxide dismutase produced at the pH of 7.4.
Optimization experiment:
determination of optimal composition of culture medium by single factor experiment
(1) The raw materials of the seed culture medium comprise: 200g/L of potato and 20g/L of glucose; the preparation method comprises the following steps: accurately weighing 200g peeled and cut potato, heating and decocting, filtering with eight layers of gauze, adding 20g glucose into potato juice, and diluting to 1000 ml.
(2) Preparing fermented seeds: inoculating the activated paecilomyces fungus strain into a seed culture medium, and culturing at 25 +/-1 ℃ at a rotating speed of 150r/min for 72h to obtain a seed solution.
On the basis of a basic fermentation culture medium, only one condition is changed, different culture media are prepared, the seed liquid is inoculated into 250mL of strains according to the same inoculum size (10 mL), 100mL of culture liquid is filled, and the strains are cultured and cultured for 25h at 25 +/-1 ℃ and the rotating speed of 150r/min to obtain the fermentation liquid.
Preparation of enzyme solution: absorbing the fermentation liquor, and centrifuging for 10 min under the condition of 10000 r/m, wherein the supernatant is the crude enzyme liquid. The enzyme activity was measured by the method described in example 1.
The effects of different carbon sources, nitrogen sources, vitamins, pH, and metal ions on superoxide dismutase production are shown in FIGS. 1-5.
Determination of optimal fermentation conditions by orthogonal experiments
Based on the culture medium determined by the single-factor experiment, the concentration and the pH value of each component are changed, different culture media (shown in table 1) are prepared, the strain with the same inoculation amount (10 mL) is inoculated into 250mL, 100mL of culture solution is filled, and the culture is carried out for 25 hours at the temperature of 25 +/-1 ℃ and the rotating speed of 150 r/min. The enzyme activity was then determined as in example 1, with the results shown in Table 2. And selecting the optimal result of the orthogonal experiment and the experiment group number with the highest enzyme yield to carry out verification experiment. The medium preparation method, preparation of crude enzyme solution, and enzyme activity measurement method in examples were the same as those in example 1 to avoid redundancy.
TABLE 1 orthogonal experimental design
TABLE 2 results of orthogonal experiments
Note: i1 is the sum of all enzyme activities of each factor level 1, II 2 is the sum of all enzyme activities of each factor level 2, III 3 is the sum of all enzyme activities of each factor level 3, IV 4 is the sum of all enzyme activities of each factor level 4, R is the range, and the number of the enzyme activities represents the average value (mean) +/-standard deviation (S.D) of 3 repetitions.
TABLE 3 media optimization validation test
The analysis of the orthogonal result shows that: the best factor combination is A3B3C3D4E3I.e. 20g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminum chloride and vitamins0.16 g/L of the element C, 7.4 of pH, and the result is consistent with the verification test. Table 2 the very different values R of the change factors are carbon source (1421.68), nitrogen source (1062.85), metal ion (1288.77), vitamin (1824.53) and pH (1588.04), respectively, indicating that the changes of carbon source, metal ion, vitamin and pH are closely related to the culture medium of the fungus of paecilomyces for producing superoxide dismutase, especially the change of vitamin has a greater influence on the fungus for producing enzyme. In contrast, the change in the nitrogen source has less influence on the enzyme production.
The fermentation production result shows that the yield of the paecilomyces fungus superoxide dismutase is 303.56 +/-4.62U/mL, the embodiment shows that the shaking flask experiment process is good, and compared with the shaking flask experiment process under the condition of a basic culture medium, the enzyme activity of the paecilomyces fungus fermentation superoxide dismutase (the enzyme activity of the basic culture medium for producing the superoxide dismutase is 7.85 +/-0.31U/mL) is improved by 38.67 times.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A culture medium for producing superoxide dismutase by fermenting paecilomyces is characterized in that: the raw materials of the culture medium comprise: 5-40 g/L of fructose, 3.2-25.6 g/L of ammonium sulfate, 2.6-20.8 g/L of aluminum chloride and 0.02-0.16 g/L of vitamin C; the pH of the medium is 6.8-7.7.
2. The culture medium for producing superoxide dismutase by fermentation with paecilomyces as claimed in claim 1, wherein: the raw materials of the culture medium comprise: 20g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminum chloride and 0.16 g/L of vitamin C; the pH of the medium was 7.4.
3. A method for producing superoxide dismutase using the medium of claim 1 or 2, characterized in that: comprises the following steps:
(a) activating paecilomyces fungi, and culturing in a seed culture medium to obtain a fermented seed solution;
(b) inoculating the fermentation seed solution obtained in step (a) into the medium of claim 1 or 2, wherein the amount of the culture in a 250mL Erlenmeyer flask is 100mL and the inoculation amount is 10% (v/v), and culturing is carried out at 25 ℃ and at a rotation speed of 150 r/min.
4. The method for producing superoxide dismutase as claimed in claim 3, wherein: the seed culture medium of the step (a) contains the following raw materials: 200g/L of potato and 20g/L of glucose.
5. The method for producing superoxide dismutase as claimed in claim 3, wherein: the specific conditions of the culture in the step (a) are as follows: temperature of the shaking table: the temperature is 25 +/-1 ℃, and the seed culture period is 72 h.
6. The method for producing superoxide dismutase as claimed in claim 3, wherein: the incubation time in step (b) was 25 h.
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| CN102084780A (en) * | 2010-05-31 | 2011-06-08 | 陈卫东 | Method for culturing cordyceps militaris sporocarp with high-content bioactive substances |
| CN103211212A (en) * | 2013-04-11 | 2013-07-24 | 天津天狮生物发展有限公司 | Cordyceps mycelia and preparation method thereof |
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