CN108841798A - A kind of Paecilomyces varioti fermentation generates the culture medium and method of superoxide dismutase - Google Patents
A kind of Paecilomyces varioti fermentation generates the culture medium and method of superoxide dismutase Download PDFInfo
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- CN108841798A CN108841798A CN201810802571.5A CN201810802571A CN108841798A CN 108841798 A CN108841798 A CN 108841798A CN 201810802571 A CN201810802571 A CN 201810802571A CN 108841798 A CN108841798 A CN 108841798A
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- culture medium
- fermentation
- superoxide dismutase
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- paecilomyces varioti
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
Abstract
The present invention relates to the fermentation medium of fungi and techniques, and in particular to a kind of Paecilomyces varioti fermentation generates the culture medium and method of superoxide dismutase.The raw material of the culture medium contains fructose 5-40 g/L, ammonium sulfate 3.2-25.6 g/L, aluminium chloride 2.6-20.8 g/L, vitamin C 0.02-0.16 g/L；The pH of the culture medium is 6.8-7.7.This method is cultivated by the way that fermentation seed liquid to be inoculated in the culture medium of above-mentioned production superoxide dismutase, and the triangular flask liquid amount of 250mL is 100mL, inoculum concentration 10%（v/v）, cultivated under 25 DEG C, 150 r/min of revolving speed.Fermentation process of the invention has short fermentation period, mild condition, easily controllable；What is provided generates the culture medium of superoxide dismutase for Paecilomyces varioti fermentation, has many advantages, such as that superoxide dismutase yield is high, vigor is high.
Description
Technical field
The present invention relates to the fermentation medium of fungi and techniques；More particularly it relates to which a kind of Paecilomyces varioti fermentation produces
The culture medium and method of raw superoxide dismutase.
Background technique
Paecilomyces varioti (Paecilomyces) be insect pathogenic fungus important member, it mainly infects lepidopterous insects and plant
Object nematode keeps its susceptible to control population quantity, this enables it to become effective biocontrol agent.With the continuous depth of research
Enter, in recent years it is found that the metabolite wide variety of Paecilomyces varioti, the mechanism of action are special, in biological pesticide or pharmaceutically has
Significant application value.
Superoxide dismutase (Superoxide dismutase) system's one kind can eliminate organism in metabolic mistake
The antioxidase of the harmful substance generated in journey.Superoxide dismutase is distributed widely in animal, plant and microorganism, can be clear
Play a significant role except biological interior free yl.Research superoxide dismutase will be relatively beneficial to taking off for biological antioxidant mechanism
Show and the application of the enzyme.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of Paecilomyces varioti fermentation generation superoxide dismutase
The culture medium and method of enzyme.When culture medium of the invention generates superoxide dismutase for Paecilomyces varioti fermentation, super oxygen can be improved
The yield of compound mutase；The fermentation process period is short；Mild condition, it is easily controllable.
For achieving the above object, the present invention adopts the following technical scheme that：
A kind of Paecilomyces varioti fermentation generates the culture medium of superoxide dismutase, and raw material includes：Fructose 5-40 g/L, ammonium sulfate
3.2-25.6 g/L, aluminium chloride 2.6-20.8 g/L, vitamin C 0.02-0.16 g/L；The pH of the culture medium is 6.8-7.7.
Preferably, the raw material of the culture medium includes：20 g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminium chloride,
0.16 g/L of vitamin C；The pH of the culture medium is 7.4.
Secondly, the present invention also provides the method that a kind of fermentation of Paecilomyces varioti generates superoxide dismutase, the method packet
Containing following steps：
（a）It is cultivated being inoculated into seed culture medium after a kind of activation of paecilomyces fungi, fermentation seed liquid is made；Shaking table temperature
Degree：25 DEG C ± 1 DEG C, the seed culture period is 72h；The seed culture medium contains following raw material：Potato 200g/L, glucose
20g/L；Preparation method is：The potato 200g for stripping and slicing of having peeled is accurately weighed, extractive, eight layers of filtered through gauze, to murphy juice are heated
Middle addition 20g glucose, and it is diluted to 1000ml.
（b）By step（a）In fermentation seed liquid obtained be inoculated in a kind of paecilomyces fungi fermentation and generate super oxygen
On the culture medium of compound mutase, the triangular flask liquid amount of 250mL is 100mL, inoculum concentration 10%（v/v）, in 25 DEG C, revolving speed
It is cultivated under 150r/min, incubation time 25h.
The beneficial effects of the present invention are：
1）The culture medium of superoxide dismutase is generated provided by the present invention for paecilomyces fungi fermentation, which can mention
High paecilomyces fungi fermentation generates the yield of superoxide dismutase；
2）Fermentation process of the invention has the advantages that short fermentation period, mild condition, easily controllable generation.
Detailed description of the invention
The influence for the superoxide dismutase activity that Fig. 1 different carbon source generates paecilomyces fungi fermentation；
The influence for the superoxide dismutase activity that Fig. 2 different nitrogen sources generate paecilomyces fungi fermentation；
The influence for the superoxide dismutase activity that Fig. 3 different metal ions generate paecilomyces fungi fermentation；
The influence for the superoxide dismutase activity that Fig. 4 difference vitamin generates paecilomyces fungi fermentation；
The influence for the superoxide dismutase activity that Fig. 5 difference pH generates paecilomyces fungi fermentation.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
（1）The preparation of fermentation seed
（A）Strain and culture medium
Strain：Paecilomyces fungi
Seed culture based raw material includes：Potato 200g/L, glucose 20g/L；Preparation method is：Accurately weigh stripping and slicing of having peeled
Potato 200g, heats extractive, and 20g glucose is added into murphy juice, and is diluted to 1000ml for eight layers of filtered through gauze.
（B）Seed liquor preparation
Purebred paecilomyces fungi on plate is transferred in multiple 250mL triangular flasks, wherein being equipped with 100mL seed culture
Base, then at 25 DEG C ± 1 DEG C reciprocating 150 r/min of concussion shaking speed culture culture 72h after to get fermentation seed liquid.
（2）Fermented and cultured
By step（B）In fermentation seed liquid obtained be inoculated in culture medium, the triangular flask liquid amount of 250mL is 100mL, inoculation
Amount is 10%, is cultivated under 25 DEG C, 150 r/min of revolving speed；
The raw material of fermentation medium includes：20 g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminium chloride, vitamin C
0.16 g/L, pH 7.4.
Shaking table temperature is 25 DEG C ± 1 DEG C, fermentation period 25h.
（3）Superoxide dismutase enzyme activity determination
Autoxidizable measurement：At 25 DEG C, 10 μ will be added in the Tris-HCl buffer solution of 4.5 mL, 50 mmol/L, pH 8.2
The pyrogallol of 50 mmol/L of L, shakes up rapidly, pours into cuvette, using Tris-HCl buffer solution as blank control, 325
An A value is surveyed every 30s under nm wavelength, is surveyed 6 times altogether, it is desirable that autoxidation rate control is in 0.07 OD/min or so.
The measurement of enzyme activity：Fermentation liquid to be drawn, 10 min are centrifuged under the conditions of 10000 r/m, supernatant is crude enzyme liquid,
25 DEG C, by 4.5 mL, 50 mmol/L, a certain amount of enzyme solution to be measured, preheating 20 are added in the Tris-HCl buffer solution of pH8.2
Min is added the pyrogallol of 10 μ L, 50 mmol/L, shakes up rapidly, pour into cuvette, is sky with Tris-HCl buffer solution
White control surveys an A value every 30 s under 325 nm wavelength, surveys 6 times altogether.With this condition, inhibit pyrogallol per minute
The enzyme amount of autoxidation rate 50% is set to 1 unit of activity.
Inhibiting rate/%=(A0-AS)/AS × 100
SOD unit vigor (U/ml)=(A0-AS)/(AS × 50%) × 4.5/V × N
In formula:A0 is autoxidation rate;
AS is that oxidation rate after sample liquid is added;
V is injection volume;
N is sample extension rate.
It according to said method measures, paecilomyces fungi is in basal fermentation medium：Glucose 20 g, yeast powder 0.4g, sulfuric acid
It is 7.85 ± 0.31 U/ that superoxide dismutase is produced on ammonium 0.7g, corn flour 0.5g, calcium carbide 0.5g, magnesium sulfide 0.05g
mL。
It according to said method measures, fermentation medium of the paecilomyces fungi in embodiment 1：20 g/L of fructose, ammonium sulfate 12.8
G/L, 10.4 g/L of aluminium chloride, it is 303.56 ± 4.62 U/ that superoxide dismutase is produced on vitamin C 0.16 g/L, pH 7.4
mL。
Optimization experiment：
Experiment of single factor determines the optimal ingredient of culture medium
（1）The raw material of seed culture medium includes：Potato 200g/L, glucose 20g/L；Preparation method is：It accurately weighs and has peeled
The potato 200g of stripping and slicing, heats extractive, and 20g glucose is added into murphy juice, and is diluted to 1000ml for eight layers of filtered through gauze.
（2）The production of fermentation seed：Paecilomyces fungal bacterial strain after activation is seeded in seed culture medium, 25 DEG C ±
1 DEG C, 72h is cultivated under 150 r/min of revolving speed to get seed liquor.
On the basis of basal fermentation medium, only change one of condition, configure different culture mediums, by seed liquor
By identical inoculum concentration（10ml）Strain is inoculated in 250mL, and 100mL culture solution, at 25 DEG C ± 1 DEG C, 150 r/ of revolving speed are housed
Culture culture 25h, obtains fermentation liquid under min.
The preparation of enzyme solution：Fermentation liquid is drawn, 10 min are centrifuged under the conditions of 10000 r/m, supernatant is crude enzyme liquid.Enzyme
Measuring method living is measured by method in embodiment 1.
The influence of different carbon source, nitrogen source, vitamin, pH, metal ion to superoxide dismutase is produced, the result is shown in Figure 1-5.
Orthogonal experiment determines optimal fermentation condition
On the basis of the culture medium that experiment of single factor determines, changes the concentration and pH value of each ingredient, configure different culture mediums
（Such as table 1）, by same amount of inoculum concentration（10ml）Strain is inoculated in 250mL, and 100mL culture solution is housed, at 25 DEG C ± 1 DEG C,
Culture culture 25h under 150 r/min of revolving speed.Then enzyme activity is measured by 1 step of embodiment, experimental result is shown in Table 2.It chooses orthogonal
Optimal result and the highest experiment group number of its yield of enzyme are tested, confirmatory experiment is carried out.In order to avoid the culture in burdensome embodiment
Basigamy method, the production of crude enzyme liquid, enzyme activity determination method are identical as the method for embodiment 1.
1 Orthogonal Experiment and Design of table
2 orthogonal experiments of table
Note：I 1 be the sum of all enzyme activities of each factor level 1, and II 2 be all enzyme activities of each factor level 2
The sum of, III 3 be each factor level 3 the sum of all enzyme activities, IV 4 for each factor level 4 all enzyme activities it
Be with, R it is very poor, the number of enzyme activity represents 3 duplicate average value (mean) ± standard deviations (S.D).
3 medium optimization verification test of table
Orthogonal interpretation of result shows：Best factor combination is A3B3C3D4E3, i.e. 20 g/L of fructose, 12.8 g/L of ammonium sulfate, chlorination
10.4 g/L of aluminium, vitamin C 0.16 g/L, pH 7.4, and result is consistent with verification test.2 changed factor of table it is very poor
Value R is respectively carbon source (1421.68), nitrogen source（1062.85), metal ion (1288.77), vitamin (1824.53) and
PH (1588.04) shows variation and the paecilomyces fungi superoxide dismutase of carbon source, metal ion, vitamin and pH
The culture medium of enzyme is closely bound up, and especially influence of the variation of vitamin to the fungi producing enzyme is bigger.In contrast, nitrogen source changes
The influence become to producing enzyme is smaller.
It is 303.56 ± 4.62U/mL, the embodiment table that fermentation, which generates result paecilomyces fungi superoxide dismutase,
Bright shake flat experiment technique of the invention is good, and compared under the conditions of basal medium, paecilomyces fungi fermentation generates superoxides
The enzyme activity (it is 7.85 ± 0.31 U/mL that basal medium, which produces the enzyme activity of superoxide dismutase) of mutase improves 38.67
Times.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (6)
1. the culture medium that a kind of Paecilomyces varioti fermentation generates superoxide dismutase, it is characterised in that：The raw material of culture medium includes：Fruit
Sugared 5-40 g/L, ammonium sulfate 3.2-25.6 g/L, aluminium chloride 2.6-20.8 g/L, vitamin C 0.02-0.16 g/L；The culture
The pH of base is 6.8-7.7.
2. the culture medium that Paecilomyces varioti fermentation according to claim 1 generates superoxide dismutase, it is characterised in that：Culture
The raw material of base includes：20 g/L of fructose, 12.8 g/L of ammonium sulfate, 10.4 g/L of aluminium chloride, 0.16 g/L of vitamin C；The culture
The pH of base is 7.4.
3. a kind of method for generating superoxide dismutase using culture medium described in as claimed in claim 1 or 22, it is characterised in that：Packet
Containing following steps：
（a）The activation of Paecilomyces varioti fungi is followed by cultivating into seed culture medium, obtains fermentation seed liquid；
（b）By step（a）In fermentation seed liquid obtained be inoculated in culture medium, the triangular flask liquid amount of 250mL is 100mL, is connect
Kind amount is 10%（v/v）, cultivated under 25 DEG C, revolving speed 150r/min.
4. production method according to claim 3, it is characterised in that：Step（a）The seed culture medium contains following
Raw material：Potato 200g/L, glucose 20g/L.
5. production method according to claim 3, it is characterised in that：Step（a）The actual conditions of the culture are：Shaking table
Temperature：25 DEG C ± 1 DEG C, the seed culture period is 72h.
6. production method according to claim 3, it is characterised in that：Step（b）In incubation time be 25h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111826383A (en) * | 2020-07-16 | 2020-10-27 | 昆明理工大学 | Application of Danbo black soybean superoxide dismutase gene in improving plant aluminum tolerance |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111826383A (en) * | 2020-07-16 | 2020-10-27 | 昆明理工大学 | Application of Danbo black soybean superoxide dismutase gene in improving plant aluminum tolerance |
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