CN108823183B - Culture medium and method for producing lipase by fermenting shell fungi - Google Patents
Culture medium and method for producing lipase by fermenting shell fungi Download PDFInfo
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- CN108823183B CN108823183B CN201810803997.2A CN201810803997A CN108823183B CN 108823183 B CN108823183 B CN 108823183B CN 201810803997 A CN201810803997 A CN 201810803997A CN 108823183 B CN108823183 B CN 108823183B
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- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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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 lipase by fermenting shell fungi. The raw materials of the culture medium comprise 1.25-10 g/L of mannitol, 3-24 g/L of ammonium nitrate, 0.1-12.5 g/L of ferric sulfate and 0.00625-0.05 g/L of nicotinic acid; the pH of the culture medium is 6.5-8.0. The method comprises the step of inoculating a fermentation seed solution into the culture medium for culturing, 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 lipase by fermenting the chaetomium has the advantages of high lipase yield, high activity and the like.
Description
Technical Field
The invention relates to a fermentation medium and a process of fungi; in particular to a culture medium and a method for producing lipase by fermenting shell fungi.
Background
Bacteria of the genus Pectinopsis (Torrubiella) Is an important entomogenous fungus that infests mainly scales and arachnids, causing them to be pathogenic to control population numbers, which makes them effective as biocontrol agents. With the continuous progress of research, people find that the species of the chaetomium sp are various and the hosts are different, so that the chaetomium sp has important application value in biological pesticides in recent years.
Lipase is a specific type of lipid-bond hydrolase that catalyzes the hydrolysis of triacylglycerol. The lipase is widely found in plants, animals and microorganisms, and the microbial lipase is an important source of industrial lipase and has important significance in theoretical research.
Disclosure of Invention
The invention aims to provide a culture medium and a method for producing lipase by fermenting shell fungi aiming at the defects of the prior art. The culture medium can be used for producing lipase by fermentation of Cordyceps, and can improve lipase yield.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for producing lipase by fermenting chaetomium, which comprises the following raw materials: 1.25-10 g/L of mannitol, 3-24 g/L of ammonium nitrate, 0.1-12.5 g/L of ferrous sulfate and 0.00625-0.05 g/L of nicotinic acid; the pH of the culture medium is 6.5-8.0.
Preferably, the raw materials of the culture medium comprise: 5 g/L of mannitol, 12 g/L of ammonium nitrate, 0.5 g/L of ferrous sulfate and 0.00625 g/L of nicotinic acid; the pH of the medium was 7.0.
Secondly, the invention also provides a method for producing lipase by fermenting the chaetomium sp, which comprises the following steps:
(a) activating a shell fungus, inoculating the activated shell 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 48 h; 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) into the culture medium for producing lipase by fermenting the shell fungus, wherein the liquid loading amount 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 4 d.
The invention has the beneficial effects that:
1) the culture medium for producing the lipase by fermenting the shell fungi, provided by the invention, can improve the yield of the lipase produced by fermenting the shell fungi;
2) the fermentation method has the advantages of short fermentation period, mild conditions, easy control of production and the like.
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: insect shell fungus
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 the pure insect shell fungi on the flat 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 48h at the rotating speed of 150r/min of a reciprocating shaking table at the temperature of 25 +/-1 ℃ 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: 5 g/L of mannitol, 12 g/L of ammonium nitrate, 0.5 g/L of ferrous sulfate and 0.00625 g/L of nicotinic acid; the pH of the medium was 7.0.
The temperature of the shaking table is 25 +/-1 ℃, and the fermentation period is 4 d.
(3) Lipase activity assay
The main reagents are as follows:
0.5 mol/L trichloroacetic acid: 8.17 g of trichloroacetic acid was weighed to a constant volume of 100mL, stored in a brown bottle and wrapped with tinfoil.
Tris-HCl buffer solution (pH7.5): 50mL of 0.1mol Tris solution was mixed with 40.3 mL of 0.1mol hydrochloric acid, and then diluted to 100mL with water.
Solution A: 150 mg of p-NPP was dissolved in 50mL of isopropanol and sonicated to allow complete dissolution of the p-NPP in the isopropanol and stored at 4 ℃ until use.
Solution B: 2.22 g of Triton-100 and 0.56 g of Arabic gum were added to Tris-HCl buffer (pH7.5), mixed by a magnetic stirrer, and the mixture was made to 500 mL and stored at 4 ℃ for further use.
300 mL of enzyme solution was added to an EP tube and centrifuged at 10000 r/min for 15 min. 2.8 mL of solution B was added to a 25 mL tube with a ground glass stopper, mixed with 0.1 mL of solution A, and then preheated at 37 ℃ for 5 min. Preheating the centrifuged enzyme solution at 37 deg.C for 10 min, adding 100 μ L of centrifuged enzyme supernatant into preheated glass test tube containing solution A and solution B, and preheating at 37 deg.C for 10 min. Adding 1.5 mL of 0.5 moL/L trichloroacetic acid, preheating at 37 ℃ for 5 min, adding 1.5 mL of NaOH, and mixing uniformly. The absorbance values were determined at 410 nm. The enzyme solution inactivated by boiling in water for 10 min was added to the control group, and each measurement was repeated 3 times, and the average value was taken. 1 enzyme activity unit (U) is defined as: the amount of enzyme required for the conversion of the substrate to 1. mu. mol fat per minute at 40 ℃ pH7.
Enzyme activity (U/ml) = AV ÷ (2.868V' T139.11).
In the formula:
a is the absorbance of the sample;
v' enzyme volume;
v is the total volume of the reaction;
t is reaction time;
according to this method, the chaetomium fungus is cultured in a basal fermentation medium: 50 g/L emulsified olive oil, 1.0 g/L K2HPO4，0.1 g/L CaCl2，0.5 g/L NaCl，0.1 g/L MgSO4，1.0 g/L (NH4)2SO4And the yield of lipase on 10.0 g/L glucose is 2.43 +/-0.18U/mL.
In this way, the chaetomium fungus was tested in the fermentation medium of example 1: 5 g/L of mannitol, 12 g/L of ammonium nitrate, 0.5 g/L of ferrous sulfate and 0.00625 g/L of nicotinic acid; the lipase produced at pH 7.0 of this medium was 18.49. + -. 0.37U/mL.
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 strain of the fungus of the genus chaetomium to a seed culture medium, and culturing at 25 +/-1 ℃ and the rotating speed of 150r/min for 48 hours to obtain seed liquid.
On the basis of a basic fermentation culture medium, only one condition is changed, different culture media are prepared, strains with the same inoculum size (10 mL) are inoculated into 250mL, 100mL of culture solution is filled, and the culture solution is cultured and cultured for 4 days at 25 +/-1 ℃ and the rotating speed of 150r/min to prepare fermentation liquor.
Preparation of enzyme solution: and (4) absorbing the fermentation liquor, and centrifuging for 15 min under the condition of 10000 r/min to obtain supernatant fluid which is the crude enzyme solution. The enzyme activity was measured by the method described in example 1.
The effects of different carbon sources, nitrogen sources, vitamins, pH, metal ions on lipase production are shown in tables 4-8.
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 4d 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 A3B3C2D1E2Namely 5 g/L of mannitol, 12 g/L of ammonium nitrate, 0.5 g/L of ferrous sulfate, 0.00625 g/L of nicotinic acid and pH 7.0, and the result is in accordance with the verification test. Table 2 the very different values R of the variation factors are carbon source (19.03), nitrogen source (13.07), metal ion (11.09), vitamin (18.8) and pH (15.79), respectively, indicating that the variation of carbon source, nitrogen source, vitamin and pH is closely related to the culture medium for producing lipase by the chaetomium fungus, especially the variation of carbon source has a greater influence on the enzyme production by the fungus. In contrast, changes in metal ions have less effect on enzyme production.
The lipase produced by the fruiting shell fungi through fermentation is 18.49 +/-0.37U/mL, and 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 shell fungi for producing the lipase (the enzyme activity of the basic culture medium for producing the lipase is 2.43 +/-0.18U/mL) is improved by 7.6 times.
TABLE 4 Effect of different carbon sources on the Lipase Activity of the Coccidium fungus
TABLE 5 Effect of different Nitrogen sources on Lipase Activity of Pestichopus fungi
TABLE 6 Effect of different Metal ions on Lipase Activity of Pestichopus fungi
TABLE 7 Effect of different pH's on Lipase Activity of Pestichopus fungi
TABLE 8 Effect of different vitamins on Lipase Activity of Cochloa fungi
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 lipase by fermenting shell fungi is characterized in that: the raw materials of the culture medium comprise: 1.25-10 g/L of mannitol, 3-24 g/L of ammonium nitrate, 0.1-12.5 g/L of ferrous sulfate and 0.00625-0.05 g/L of nicotinic acid; the pH of the culture medium is 6.5-8.0.
2. The culture medium for producing lipase by fermentation of chaetomium according to claim 1, characterized in that: the raw materials of the culture medium comprise: 5 g/L of mannitol, 12 g/L of ammonium nitrate, 0.5 g/L of ferrous sulfate and 0.00625 g/L of nicotinic acid; the pH of the medium was 7.0.
3. A method for producing lipase using the medium of claim 1 or 2, characterized in that: comprises the following steps:
(a) activating the eumycete fungi, and culturing in seed culture medium to obtain fermented seed liquid;
(b) inoculating the fermentation seed liquid prepared in the step (a) into a culture medium, wherein the liquid loading of a 250mL triangular flask is 100mL, the inoculation amount is 10% (v/v), and culturing is carried out at 25 ℃ and the rotating speed of 150 r/min.
4. The production method according to claim 3, characterized in that: the seed culture medium of the step (a) contains the following raw materials: 200g/L of potato and 20g/L of glucose.
5. The production method according to claim 3, characterized in that: 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 48 h.
6. The production method according to claim 3, characterized in that: the incubation time in step (b) was 4 days.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2928860A1 (en) * | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Biopesticide composition comprising a fungal pesticide and a surfactant system |
| CN106857256A (en) * | 2017-02-21 | 2017-06-20 | 淮北师范大学 | The method that beautiful dew breeding potential is improved based on callus induction Regeneration Ways |
| CN107828669A (en) * | 2017-12-12 | 2018-03-23 | 福建农林大学 | A kind of culture medium and method of worm shell category fungi fermentation production zonal contribution ratio |
| CN107937412A (en) * | 2017-11-06 | 2018-04-20 | 吉林农业大学 | A kind of method that drought resistance of maize is improved by transgenosis |
| EP3330349A1 (en) * | 2016-12-02 | 2018-06-06 | The Procter & Gamble Company | Cleaning compositions including enzymes |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2928860A1 (en) * | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Biopesticide composition comprising a fungal pesticide and a surfactant system |
| EP3330349A1 (en) * | 2016-12-02 | 2018-06-06 | The Procter & Gamble Company | Cleaning compositions including enzymes |
| CN106857256A (en) * | 2017-02-21 | 2017-06-20 | 淮北师范大学 | The method that beautiful dew breeding potential is improved based on callus induction Regeneration Ways |
| CN107937412A (en) * | 2017-11-06 | 2018-04-20 | 吉林农业大学 | A kind of method that drought resistance of maize is improved by transgenosis |
| CN107828669A (en) * | 2017-12-12 | 2018-03-23 | 福建农林大学 | A kind of culture medium and method of worm shell category fungi fermentation production zonal contribution ratio |
Non-Patent Citations (2)
| Title |
|---|
| Monoseptic growth of fungal lipase producersunder minimized sterile conditions:Cultivation of Phialemonium curvatum in350 L scale;Susann Barig et al.;《Eng. Life Sci.》;20111231;第11卷(第4期);第387-394页 * |
| 华根霉产脂肪酶发酵培养基的研究;徐岩等;《工业微生物》;20000930;第30卷(第3期);第8-11页 * |
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