CN108676786B

CN108676786B - Culture medium for producing lipase by fermenting torticola terricola and culture method thereof

Info

Publication number: CN108676786B
Application number: CN201810547956.1A
Authority: CN
Inventors: 苏丹; 韦秋婷; 吕国忠
Original assignee: Dalian Minzu University
Current assignee: Dalian Minzu University
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2021-02-23
Anticipated expiration: 2038-05-31
Also published as: CN108676786A

Abstract

The invention relates to a culture medium for producing lipase by fermenting geotrichum and a culture method thereof. The enzyme-producing fermentation medium comprises the following components: 2% (w/v) emulsified canola oil, 2% (w/v) alpha-lactose, 4% (w/v) tryptone, MnSO₄·H₂O 0.1％(w/v)，NH₄NO₃0.1% (w/v), pH 5-10, and the balance water. Compared with the prior art, the fermentation method provided by the invention has mild conditions and is easy to control; provides a culture medium for producing lipase by fermenting the clinopodium terrae C41-3, and has the advantages of short fermentation time and high lipase activity. The fermentation culture conditions explored by the invention enable the enzyme activity to be improved from 102.69U/mL of the basic fermentation culture medium to 6011.69U/mL, have the advantages of high low-temperature lipase activity and the like, are beneficial to large-scale production, and have great significance for improving the industrial application potential of the strain.

Description

Culture medium for producing lipase by fermenting torticola terricola and culture method thereof

Technical Field

The invention relates to a method for producing low-temperature lipase by fungus fermentation, in particular to a culture medium and a method for producing lipase by fermenting torticola virens.

Background

Lipases (ec3.1.1.3), also known as triglyceride hydrolases, catalyze the hydrolysis of triacylglycerides at the interface between insoluble substrate and water. They are ubiquitous in nature and are produced by a variety of plants, animals and microorganisms. Lipases from microbial sources are widely diverse in their catalytic activity and substrate specificity, and microbial lipases are used in a wide range of industrial applications, such as food technology, detergents, biodiesel, chemical industry and biomedical science, among others.

The pH and temperature range of the enzymolysis of the microbial lipase is wider than the range of animal lipase, and the high-purity enzyme preparation is convenient to industrially produce. Although there are many sources of low temperature lipase production available today, only a few microorganisms have been developed for producing low temperature lipase. The use of low temperature lipases has great potential in many ways, such as reducing energy costs, medical applications, detergent additives, and reducing microbial contamination in industrial processes.

The strain of Tolypocladium geodes (Tolypocladium geodes) is a strain of Ascomycota (Ascomycota), Chaetomium (Sordariomy-cetes), Hypocrea (Hypocrea), Paramyces (Ophio-cordipitaceae), Tolypocladium (Tolypocladium), and is a psychrophilic fungus. The campylobacter terraneous is a new Chinese record species, shows the characteristic of high altitude distribution, and is a high-yield lipase strain. Therefore, the campylobacter terricola has great potential in industrial production of lipase. However, the existing fermentation medium or culture method for producing the low-temperature lipase by the torticollis is low in enzyme production activity and harsh in fermentation conditions, and is not beneficial to large-scale production, so that the need for the fermentation medium and the fermentation method for producing the low-temperature lipase by the torticollis is high in the field.

Disclosure of Invention

The invention aims to provide a culture medium and a method for producing low-temperature lipase by fermenting the torticola benthamiana.

The invention adopts the following technical scheme that an enzyme production fermentation culture medium for producing lipase by fermenting the geotrichum C41-3 comprises: 2% (w/v) emulsified canola oil, 2% (w/v) alpha-lactose, 4% (w/v) tryptone, MnSO₄·H₂O 0.1％(w/v)，NH₄NO₃0.1% (w/v), pH 5-10, and the balance water.

Preferably, the pH is 5 and the fermentation temperature is 15-25 ℃, more preferably 15 ℃.

The invention also discloses a method for producing low-temperature lipase by using the culture medium, which comprises the following specific steps:

(1) inoculating 1% of Campylobacter mediterrae in seed culture medium, and shake culturing at 25 deg.C and 150r/min for 3-4d to obtain fermented seed liquid;

(2) inoculating the fermentation seed liquid into an enzyme-producing fermentation culture medium, wherein the liquid loading amount of the enzyme-producing fermentation culture medium is 30%, the inoculation amount is 10%, and performing shake culture at 25 ℃ and a rotation speed of 150r/min for 4d to obtain a fermentation liquid;

(3) centrifuging the fermentation liquor obtained in the step (2), and collecting supernatant to obtain crude enzyme liquid.

The said neck fungus is C41-3, Latin literature name is (Tolypocladium geodes), the said neck fungus C41-3 has already submitted the deposit, the concrete information of deposit is as follows:

the name of the depository: china general microbiological culture Collection center (CGMCC);

the address of the depository: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences;

the preservation date is as follows: 24/04/2018;

the preservation number is: CGMCC No. 15667.

The strain of the terrestrial aspergillus is grown in a circular radial shape, the front surface of a colony is flat and snow white, villiform and compact, the back surface of the colony is usually white, or the colony is dark or brown near the origin, and hyphae are white filaments.

Further, the seed culture medium comprises: 200g/L of potato, 20g/L of cane sugar and 1000mL of water, wherein the pH value is 5.

More specifically, the step (1) is as follows: subpackaging the seed culture medium in a triangular flask, sterilizing at 121 ℃ for 30 minutes, and then cooling; inoculating strains with the inoculation amount of 1% into a seed culture medium, and placing the seeds in a shaking table for culture under the culture conditions: culturing at 25 ℃ and 150r/min for 3-4 days to obtain a fermented seed solution;

the basic fermentation culture medium in the step (2): emulsified olive oil 20ml/L, peptone 40g/L, sucrose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, natural pH and the balance of water.

Enzyme-producing fermentation medium: emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, MnSO₄·H₂O1g/L，NH₄NO₃1g/L, pH 5 and the balance of water;

the step (2) is specifically as follows: adding the basic fermentation medium into a culture container according to the liquid loading amount of 30%, sterilizing at 121 ℃ for 30 minutes, and cooling to normal temperature; adding the fermentation seed liquid into a culture container containing an enzyme-producing fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation temperature is 25 ℃, the pH is 5 (namely the pH is natural), the rotation speed is 150r/min, and the fermentation culture is carried out for 4 d.

The step (3) is specifically as follows: centrifuging the fermentation liquor for 10min at 8000-10000r/min and 4 ℃ by using a high-speed refrigerated centrifuge, and collecting supernatant fluid, namely the crude enzyme liquid.

The invention relates to a preparation method of grease emulsion such as emulsified canola oil, emulsified olive oil, emulsified soybean oil, emulsified peanut oil and emulsified sesame oil, which comprises the following steps: dissolving 2% polyvinyl alcohol in deionized water, and heating to dissolve. The oil was added to a 2% solution of polyvinyl alcohol and stirred with a magnetic stirrer for 15min (every 5 min) to a milky white color.

Compared with the prior art, the fermentation method provided by the invention has mild conditions and is easy to control; provides a culture medium for producing lipase by fermenting the clinopodium terrae C41-3, and has the advantages of short fermentation time and high lipase activity. The fermentation culture conditions explored by the invention enable the enzyme activity to be improved from 102.69U/mL of the basic fermentation culture medium to 6011.69U/mL, have the advantages of high low-temperature lipase activity and the like, are beneficial to large-scale production, and have great significance for improving the industrial application potential of the strain.

Drawings

FIG. 1 is a standard curve for p-nitrophenol.

FIG. 2 is a graph showing the effect of different enzyme-producing media on the production of Tolypocladium terrae low-temperature lipase, wherein A is the medium of comparative example 1, B is the medium of comparative example 2, C is the medium of comparative example 3, D is the medium of comparative example 4, E is the medium of comparative example 5, F is the medium of comparative example 6, and G is the medium of comparative example 7.

Detailed Description

The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.

Example 1

(1) Seed culture medium

Seed culture medium: 200g/L of potato, 20g/L of cane sugar and 1000mL of water, and the pH value is natural;

the prepared culture solution is distributed into a plurality of 250ml triangular bottles, each triangular bottle contains 100ml of seed culture medium, the seed culture medium is sterilized for 30 minutes at 121 ℃, and then the seed culture medium is cooled.

Inoculating 3 bacterial blocks (the inoculum size is 1%) with the diameter of 5mm into each triangular flask containing 100ml of seed culture medium, and placing the mixture in a shaking table for culture under the culture conditions: culturing at 25 ℃ and 150r/min for 3-4 days to obtain a fermented seed solution;

(2) enzyme-producing fermentation culture

Fermentation medium: emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, MnSO₄·H₂O 1g/L，NH₄NO₃1g/L, pH 5 and the balance of water;

adding 75ml of fermentation medium into a 250ml triangular flask, sterilizing at 121 ℃ for 30 minutes, and cooling to normal temperature;

adding 7.5ml of fermentation seed liquid into a 250ml triangular flask containing 75ml of fermentation medium according to the inoculation amount of 10%, wherein the fermentation temperature is 25 ℃, the pH value is 5, and the rotation speed is 150r/min, and performing fermentation culture for 4 d;

(3) preparation method of grease emulsion

Dissolving 2% polyvinyl alcohol in deionized water, and heating to dissolve. The oil was added to a 2% solution of polyvinyl alcohol and stirred with a magnetic stirrer for 15min (every 5 min) to a milky white color.

(4) Preparation of lipase liquid

Taking fermentation liquor, centrifuging the fermentation liquor with a high-speed refrigerated centrifuge at 10000r/min and 4 ℃ for 10min, collecting supernatant as crude enzyme liquid, and obtaining the crude enzyme liquid;

(5) determination of enzyme Activity

The enzyme activity determination method adopts p-nitrophenylpalmitate (p-NPP) as a substrate to measure the lipase activity. One unit of lipase was defined as the amount of enzyme required to release 1. mu. mol p-nitrophenol (p-NP) per minute at 37 ℃ and pH 8.0.

The method comprises the following steps: solution A: 15mg of p-NPP was dissolved in 5ml of isopropanol; solution B: to 45ml of 0.05mol/L Tris-HCl buffer (pH 8.0) were added 0.05g of gum arabic and 0.2ml of Triton X-100.

Taking p-nitrophenol palmitate p-NPP as a substrate, uniformly mixing 5mL of solution A with 45mL of solution B, dividing an enzyme sample into 4 test tubes, respectively adding 1.8mL of mixed solution, and preheating at 37 ℃ for 5 min. mu.L of the enzyme sample solution was added to 3 tubes, and 200. mu.L of the enzyme sample solution denatured by boiling (100 ℃ C., 10min) was added to 1 tube as a control, and mixed well. Reacting in water bath at 37 deg.C for 15min, taking out, and measuring absorbance at wavelength of 410 nm.

The enzyme activity calculation formula is as follows: x is CV/(TV')

In the formula:

X-Lipase Activity, U. m L^-1；

Concentration of C-p-nitrophenol, μmol. mL^-1；

V is the final volume of the reaction solution, mL;

v' -the dosage of enzyme solution, mL;

t-reaction time, min.

(6) And (3) preparing a standard curve:

2.0mmol/LpNP(uL)	0	7.5	15	30	60	90	120	150
									isopropanol (uL)	250	242.5	235	220	190	160	130	100
Substrate buffer (mL)	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25
									pNP concentration (umol/L)	0	6	12	24	48	72	96	120

At a wavelength of 410nmAnd (5) determining an absorbance value. Drawing a standard curve by taking OD410 value as an abscissa and pNP concentration as an ordinate, and processing to obtain a linear regression equation, wherein y is 69.968x-0.993, R²The results are shown in figure 1, 0.9991.

Comparative example

And changing the components of the fermentation culture medium for comparison, and testing the enzyme activity of the components of different fermentation culture media on the low-temperature lipase according to a standard curve. The fermented seed liquids referred to in comparative examples 1 to 7 below were prepared by the method of step (1) of example 1.

Comparative example 1

Seven enzyme-producing fermentation media were prepared for comparison:

(1) emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 20g/L of sucrose, pH 5 and the balance of water.

(2) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, glucose 20g/L, pH 5, and the balance water.

(3) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 20g/L of D-fructose, pH 5 and the balance of water.

(4) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, D (+) -maltose 20g/L, pH 5, the remainder being water.

(5) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 20g/L of soluble starch, 5 of pH and the balance of water.

(6) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 20g/L α -lactose, pH 5, the remainder being water.

(7) Emulsified olive oil 20ml/L, animal peptone 40g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, dextrin 20g/L, pH 5, whichThe balance being water.

The 7 enzyme-producing fermentation medium was sterilized at 121 ℃ for 30 min.

Inoculating 10% fermentation seed solution into the enzyme-producing fermentation culture medium, culturing in a shaker at 25 deg.C and 150r/min for 4d, and measuring enzyme activity. As shown in FIG. 2A, the fermentation enzyme-producing activities of the enzyme-producing fermentation media (2) and (6) were very significant, with the enzyme-producing activity of the enzyme-producing fermentation medium (6) being the highest and the enzyme-producing activity of (2) being the second most; the enzyme production activity of the rest of the enzyme production culture medium is lower, wherein the enzyme production activity of the enzyme production fermentation culture medium (3) is the lowest.

Comparative example 2

Five enzyme-producing fermentation media were prepared for comparison:

(1) emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, tryptone 40g/L, pH 5, and the balance water.

(2) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, soy peptone 40g/L, pH 5, and water in balance.

(3) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, animal peptone 40g/L, pH 5, and water for the rest.

(4) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 40g/L beef extract, 5 of pH and the balance of water.

(5) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, yeast extract powder 40g/L, pH 5, and the balance water.

The 5 enzyme-producing fermentation medium was sterilized at 121 ℃ for 30 min.

10% of fermentation seed liquid is respectively added into the enzyme-producing fermentation culture medium, and the enzyme activity is measured after the fermentation seed liquid is cultured for 4 days in a shaking table at 25 ℃ and 150 r/min. As a result, as shown in FIG. 2B, the activity of the fermentation medium (1) for producing an enzyme was higher in the case of a fermentation of Tolypocladium flexneri.

Comparative example 3

Four enzyme-producing fermentation media were prepared for comparison:

(1) emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄ 1g/L，NH₄NO₃1g/L, pH 5, and the balance water.

(2) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄1g/L, 1g/L urea, pH 5, and the balance water.

(3) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄ 1g/L，NH₄Cl1g/L, pH 5, remainder water.

(4) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄ 1g/L，(NH₄)₂SO₄1g/L, pH 5, and the balance water.

The 4 enzyme-producing fermentation medium was sterilized at 121 ℃ for 30 min.

10% of fermentation seed liquid is respectively added into the enzyme-producing fermentation culture medium, and the enzyme activity is measured after the fermentation seed liquid is cultured for 4 days in a shaking table at 25 ℃ and 150 r/min. As a result, as shown in FIG. 2C, the pair of inorganic nitrogen sources (NH) by Tolypocladium meyeri C41-3 was observed₄)₂SO₄、NH₄Cl、NH₄NO₃The utilization ratio of (1) is higher, wherein NH₄NO₃The enzyme production activity of the fermentation is up to 5052.35U/mL.

Comparative example 4

Seven enzyme-producing fermentation media were prepared for comparison:

(1) emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, CaSO₄·2H₂O1 g/L, pH 5, the remainder being water.

(2) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, CuSO₄·5H₂O1 g/L, pH 5, the remainder being water.

(3) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, MgSO₄·7H₂O1 g/L, pH 5, the remainder being water.

(4) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, MnSO₄·4H₂O1 g/L, pH 5, the remainder being water.

(5) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, ZnSO₄·7H₂O1 g/L, pH 5, the remainder being water.

(6) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，FeSO₄·7H₂O1 g/L, pH 5, the remainder being water.

(7) Emulsified olive oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, pH 5, the rest is water.

10% of fermentation seed liquid is respectively added into the enzyme-producing fermentation culture medium, and the enzyme activity is measured after the fermentation seed liquid is cultured for 4 days in a shaking table at 25 ℃ and 150 r/min. As can be seen from FIG. 2D, Fe is compared with the activity of the enzyme produced by fermentation without adding metal ions²⁺、Cu²⁺、Ca²⁺、Mg²⁺、Mn²⁺The fermentation enzyme production of the bent neck mold C41-3 is promoted, the enzyme production fermentation culture mediums (1), (2), (4) and (6) have high enzyme production activity, wherein the enzyme production fermentation culture medium (4) Mn²⁺The promotion effect is most obvious, and is as high as 4988.59U/mL; zn in the enzyme-producing fermentation medium (5)²⁺The enzyme production activity of the fermentation is inhibited.

Comparative example 5

Four enzyme-producing fermentation media were prepared for comparison:

(1) alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, emulsified sesame oil 20ml/L, pH 5, and the balance of water.

(2) Alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, emulsified soybean oil 20ml/L, pH 5, and the balance of water.

(3) Alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, emulsified canola oil 20ml/L, pH 5, and the balance water.

(4) Alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, emulsified olive oil 20ml/L, pH 5, and the balance water.

(5) Alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, emulsified peanut oil 20ml/L, natural pH and the balance of water.

10% of fermentation seed liquid is respectively added into the enzyme-producing fermentation culture medium, and the enzyme activity is measured after the fermentation seed liquid is cultured for 4 days in a shaking table at 25 ℃ and 150 r/min. As shown in FIG. 2E, the looper fungus produced low temperature lipase in the presence of the enzyme-producing fermentation medium, wherein the enzyme-producing activity of the fermentation was highest at 4839.33U/mL when the emulsified canola oil was added to the fermentation medium; the activity of the common olive oil fermentation production enzyme is 3668.54U/mL, which is inferior to the peanut oil and the soybean oil; the enzyme production activity of sesame oil fermentation is lowest.

Comparative example 6

Seven enzyme-producing fermentation media were prepared for comparison:

(1) emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 4, the remainder being water.

(2) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 5, the remainder being water.

(3) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 6, the remainder being water.

(4) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 7, remainder water.

(5) Milk20ml/L of canola oil, 20g/L of alpha-lactose, 40g/L of tryptone and NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 8, the remainder being water.

(6) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 9, the remainder being water.

(7) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·H₂O1 g/L, pH 10, remainder water.

10% of fermentation seed liquid is respectively added into the enzyme-producing fermentation culture medium, and the enzyme activity is measured after the fermentation seed liquid is cultured for 4 days in a shaking table at 25 ℃ and 150 r/min. The results are shown in FIG. 2F, and the activity of the lipase produced by fermentation is maintained at a high level within the pH range of 5-10; the enzyme activity is greatly reduced when the pH value is less than 5. Therefore, the pH range of the produced lipase by fermentation of the Tolypocladium flexuosum C41-3 is 5-10, and when the pH value is 5, the activity of the produced enzyme by fermentation is the highest and reaches 6011.69U/mL.

Comparative example 7

Six enzyme-producing fermentation media were prepared for comparison:

(1) emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, pH 5, balance water, fermentation temperature 5 ℃.

(2) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, pH 5, balance water, fermentation temperature 10 ℃.

(3) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, pH 5, balance water, fermentation temperature 15 ℃.

(4) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O 1g/L, pH 5, water for the rest, fermentation temperature 20 ℃.

(5) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, pH 5, balance water, fermentation temperature 25 ℃.

(6) Emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, NH₄NO₃ 1g/L，MnSO₄·4H₂O1 g/L, pH 5, balance water, fermentation temperature 30 ℃.

The 6 enzyme-producing fermentation medium was sterilized at 121 ℃ for 30 min.

And (3) inoculating 10% of fermentation seed liquid into each enzyme-producing fermentation culture medium, culturing for 3d in a shaking table at 150r/min, and measuring enzyme activity. As shown in FIG. 2G, the fermentation enzyme productivity of the Tolypocladium virens was higher at the above fermentation temperatures at 15 deg.C, 20 deg.C and 25 deg.C, wherein the enzyme productivity was the highest at 15 deg.C and the enzyme productivity was the second only at 25 deg.C; no enzyme was produced at 5 ℃.

The above description is only for the purpose of creating a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. An enzyme-producing fermentation culture medium for producing lipase by fermenting the clinopodium geodes (Tolypocladium geodes) C41-3, which is characterized in that the preservation number of the clinopodium geodes C41-3 is CGMCC No.15667, and the fermentation culture medium is: emulsified canola oil 2% (w/v), alpha-lactose 2% (w/v), tryptone 4% (w/v), MnSO4 · H2O 0.1% (w/v), NH4NO30.1% (w/v), pH = 5-10, and the balance water.

2. A method for producing low-temperature lipase by fermentation, which is characterized by utilizing the torticola virginiana C41-3 of claim 1 for fermentation production, and comprises the following steps:

(1) subpackaging the seed culture medium in a triangular flask, sterilizing at 121 ℃ for 30 minutes, and then cooling; inoculating strains with the inoculation amount of 1% into a seed culture medium, and placing the seeds in a shaking table for culture under the culture conditions: culturing at 25 ℃ and 150r/min for 3-4 days to obtain a fermented seed solution; the preservation number of the campylobacter terrae C41-3 is CGMCC No. 15667; the seed culture medium comprises the following components: potato 200g/L, sucrose 20g/L, water 1000mL, pH = 5;

(2) adding the fermentation seed liquid into a culture container containing an enzyme-producing fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation temperature is 25 ℃, the pH =5, and the rotation speed is 150r/min, and performing shake culture for 4d to obtain a fermentation liquid; wherein, the enzyme-producing fermentation culture medium: emulsified canola oil 20ml/L, alpha-lactose 20g/L, tryptone 40g/L, MnSO 4. H2O 1g/L, NH4NO 31 g/L, pH =5, and the balance water;

3. The method according to claim 2, characterized in that the step (3) is in particular: centrifuging the fermentation liquor for 10min at 8000-10000r/min and 4 ℃ by using a high-speed refrigerated centrifuge, and collecting supernatant fluid, namely the crude enzyme liquid.