WO2019227363A1 - Culture medium for producing lipase by fermenting tolypocladium geodes and culture method therefor - Google Patents

Culture medium for producing lipase by fermenting tolypocladium geodes and culture method therefor Download PDF

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WO2019227363A1
WO2019227363A1 PCT/CN2018/089144 CN2018089144W WO2019227363A1 WO 2019227363 A1 WO2019227363 A1 WO 2019227363A1 CN 2018089144 W CN2018089144 W CN 2018089144W WO 2019227363 A1 WO2019227363 A1 WO 2019227363A1
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enzyme
fermentation
medium
producing
water
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French (fr)
Chinese (zh)
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苏丹
韦秋婷
吕国忠
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大连民族大学
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the present invention relates to a method for producing low-temperature lipase by fungal fermentation, and more particularly, the present invention relates to a culture medium and method for producing lipase by fermentation of Geotrichum crassipes.
  • Lipase (EC3.1.1.3), also known as triglyceride hydrolase, catalyzes the hydrolysis of triacylglycerols at the interface between insoluble substrates and water. They are ubiquitous in nature and are produced by a variety of plants, animals, and microorganisms. Lipases derived from microorganisms are widely diversified in terms of their catalytic activity and substrate specificity. Microbial lipases are used in a wide range of industrial applications, such as food technology, detergents, biodiesel, chemical industry and biomedical sciences.
  • the pH and temperature range of microbial lipase hydrolysis is wider than that of animal lipase, which is convenient for industrial production to obtain high-purity enzyme preparations.
  • low-temperature lipase only a few microorganisms have been developed for the production of low-temperature lipase.
  • the use of low-temperature lipase has great potential in many aspects such as reducing energy costs, medical applications, washing additives, and reducing microbial pollution in industrial processes.
  • Tolypocladium geodes are Ascomycota, Sorariomy-cetes, Hypocreales, Ophio-cordycipitaceae, Tolypocladium Fungi are cold-loving fungi.
  • Geotrichum is a new record species in China, showing high altitude distribution characteristics, and is a high-lipase strain. Therefore, Geotrichum crassipes has great potential in the industrial production of lipase.
  • the current fermentation medium or culture method of the benthic bentophyte has low enzyme production activity and harsh fermentation conditions, which is not conducive to large-scale production. Therefore, there is an urgent need in the field for a fermentation culture suitable for low-temperature lipase produced by the benthic bentophyte. And fermentation methods.
  • the purpose of the present invention is to provide a medium and method for producing low-temperature lipase by fermentation of Geotrichum crassipes.
  • the low-temperature lipase produced by fermentation of the medium has high activity, mild fermentation conditions and easy control, which is beneficial to industrial production.
  • the pH is 5 and the fermentation temperature is 15-25 ° C, more preferably 15 ° C.
  • the invention also claims the method for producing low-temperature lipase by using the above-mentioned culture medium.
  • the specific steps are:
  • the fermented seed solution is inoculated into an enzyme-producing fermentation medium, and the amount of the enzyme-producing fermentation medium is 30%, the inoculation amount is 10%, and cultured at 25 ° C and a rotation speed of 150 r / min for 4 days in a shaker. Fermentation broth
  • step (3) The fermentation liquid obtained in step (2) is centrifuged, and the supernatant is collected to obtain a crude enzyme liquid.
  • the terrestrial curved neck mold is C41-3, and the Latin name is Tolypocladium geodes.
  • the terrestrial curved neck mold C41-3 has been submitted for preservation.
  • the specific preservation information is as follows:
  • depository unit General Microbial Center of the China Microbial Collection Management Committee (CGMCC);
  • the strain of Geotrichum crassipes grows in a circular shape.
  • the front of the colony is snow-white, fluffy and dense.
  • the back of the colony is usually white, or it is dark or brown near the origin of the colony, and the mycelium is white and filamentous.
  • the step (1) is: dividing the seed culture medium into a triangle flask, sterilizing at 121 ° C for 30 minutes, and then cooling; inoculating a seed strain with an inoculum amount of 1% into the seed culture medium, Placed in a shaker for cultivation.
  • the culture conditions are: temperature 25 ° C, rotation speed 150r / min, and cultivation for 3 to 4 days to obtain the fermented seed solution.
  • the basic fermentation medium in the step (2) emulsified olive oil 20ml / L, peptone 40g / L, sucrose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, natural pH, The rest is water.
  • Step (2) is specifically: taking the basic fermentation medium into the culture container at 30% of the liquid content, sterilizing at 121 ° C for 30 minutes, and cooling to normal temperature; adding 10% of the inoculation amount to the fermented seed liquid
  • the fermentation temperature was 25 ° C.
  • pH 5 (that is, natural pH)
  • the rotation speed was 150 r / min
  • the step (3) is specifically: centrifuging the fermentation broth with a high-speed refrigerated centrifuge at 8000-10000 r / min and 4 ° C for 10 min, and collecting the supernatant to obtain a crude enzyme solution.
  • the method for preparing emulsified oily emulsified oils such as emulsified canola oil, emulsified olive oil, emulsified soybean oil, emulsified peanut oil, emulsified sesame oil and the like is as follows: 2% of polyvinyl alcohol is dissolved in deionized water and dissolved by heating. Add the fat to the 2% polyvinyl alcohol solution, and use a magnetic stirrer to stir for 15 min (pause every 5 minutes) until it is milky white.
  • the fermentation method provided by the present invention has mild conditions and is easy to control; it provides a culture medium for the production of lipase by the fermentation of C. 1-3, and has the advantages of short fermentation time and high lipase activity.
  • the fermentation culture conditions explored by the present invention increase the enzyme activity from 102.69 U / mL of the basic fermentation medium to 6011.69 U / mL, which has the advantages of high-temperature low-temperature lipase activity, is conducive to large-scale production, and promotes industrial application of the strain The potential is significant.
  • Figure 1 is a standard curve for p-nitrophenol.
  • Figure 2 shows the effects of different enzyme-producing mediums on the production of low-temperature lipase by C. terreus, where A is the medium of Comparative Example 1, B is the medium of Comparative Example 2, C is the medium of Comparative Example 3, and 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.
  • Seed medium 200g / L of potato, 20g / L of sucrose, 1000mL of water, natural pH value;
  • the configured culture solution was dispensed into several 250ml Erlenmeyer flasks, each of the Erlenmeyer flasks contained 100ml of seed medium, sterilized at 121 ° C for 30 minutes, and then cooled.
  • the inoculation amount of 10% 7.5 ml of fermented seed solution was added to a 250 ml triangular flask containing 75 ml of fermentation medium, the fermentation temperature was 25 ° C, the pH was 5, the rotation speed was 150 r / min, and the fermentation was cultured for 4 days;
  • the enzyme activity measurement method uses p-nitrophenyl palmitate (p-NPP) as a substrate to measure lipase activity.
  • p-NPP p-nitrophenyl palmitate
  • One unit of lipase is defined as the amount of enzyme required to release 1 ⁇ mol of p-nitrophenol (p-NP) per minute at 37 ° C, pH 8.0.
  • Solution A 15 mg of p-NPP was dissolved in 5 ml of isopropanol; Solution B: 45 ml of 0.05 mol / L Tris-HCl buffer (pH 8.0) was added with 0.05 g of acacia gum and 0.2 ml of Triton X-100.
  • V′ the amount of enzyme solution, mL
  • the absorbance was measured at a wavelength of 410 nm.
  • the standard curve is drawn with the OD410 value as the abscissa and the pNP concentration as the ordinate.
  • the composition of the fermentation medium was changed as a comparison, and according to the standard curve, the composition of different fermentation mediums was tested for the enzyme-producing activity of low-temperature lipase.
  • the fermented seed liquid involved in the following Comparative Examples 1-7 was prepared by the method of step (1) of Example 1.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, sucrose 20g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, glucose 20g / L, pH 5, and the rest is water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, D-fructose 20g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, D (+)-maltose 20g / L, pH 5, the rest are water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, soluble starch 20g / L, pH 5, and the rest is water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, ⁇ -lactose 20g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, dextrin 20g / L, pH 5, the rest is water.
  • the above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
  • the enzyme-producing fermentation mediums (2) and (6) had extremely significant enzyme-producing activities. Among them, the enzyme-producing fermentation medium (6) had the highest enzyme-producing activity and (2) had only the enzyme-producing activity. Secondly, the remaining enzyme-producing medium fermenting enzyme production activity is relatively low, of which the enzyme-producing fermentation medium (3) has the lowest activity.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, tryptone 40g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, soy peptone 40g / L, pH 5, and the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, animal peptone 40g / L, pH 5, and the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, beef paste 40g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, yeast extract 40g / L, pH 5, and the rest is water.
  • the five enzyme-producing fermentation mediums were sterilized at 121 ° C for 30 minutes.
  • the enzyme-producing fermentation medium (1) increased the activity of the fermentation enzyme of Geotrichum crassipes.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, NH 4 NO 3 1g / L, pH 5, the rest is water .
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, urea 1g / L, pH 5, and the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, MgSO 4 ⁇ 7H 2 O 1g / L, KH 2 PO 4 1g / L, NH 4 Cl 1g / L, pH 5, and the rest is water.
  • the above four kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, CaSO 4 ⁇ 2H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, CuSO 4 ⁇ 5H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, MgSO 4 ⁇ 7H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, MnSO 4 ⁇ 4H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, ZnSO 4 ⁇ 7H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, FeSO 4 ⁇ 7H 2 O 1g / L, pH 5, the rest is water.
  • Emulsified olive oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, pH 5, the rest is water.
  • the above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
  • the five enzyme-producing fermentation mediums were sterilized at 121 ° C for 30 minutes.
  • the low-temperature lipase was produced by the above-mentioned enzyme-producing fermentation medium.
  • the emulsified canola oil was added to the fermentation medium, and the highest enzyme-producing activity was 4839.33 U / mL; peanut oil and soybean oil are next, and the commonly used olive oil fermentation enzyme production activity is 3688.54U / mL, which is inferior to soybean oil; sesame oil fermentation production enzyme activity is the lowest.
  • Emulsified canola oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 ⁇ H 2 O 1g / L, pH 4, the rest is water.
  • Emulsified canola oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 ⁇ H 2 O 1g / L, pH 6, the rest is water.
  • Emulsified canola oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 ⁇ H 2 O 1g / L, pH 9, the rest is water.
  • Emulsified canola oil 20ml / L, ⁇ -lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 ⁇ H 2 O 1g / L, pH 10, the rest is water.
  • the above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
  • the fermentation temperature was 25 ° C.
  • the above 6 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 min.

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Abstract

Provided are a culture medium for producing lipase by fermenting Tolypocladium geodes and a culture method therefor. The culture medium consists of: 2％ (w/v) of emulsified canola oil, 2％ (w/v) of alpha-lactose, 4％ (w/v) of tryptone, 0.1％ (w/v) of MnSO 4·H 2O, and 0.1％ (w/v) of NH 4NO 3, pH = 5-10, and the balance of water; the enzyme activity of the lipase is improved to 6011.69 U/mL from 102.69 U/mL of a basic fermentation culture medium, and large-scale production thereof can be achieved.

Description

一种地生弯颈霉发酵生产脂肪酶的培养基及其培养方法Culture medium for producing lipase by terrestrial curved neck mold fermentation and culture method thereof 技术领域Technical field
本发明涉及一种真菌发酵产低温脂肪酶的方法，更具体地，本发明涉及一种地生弯颈霉发酵生产脂肪酶的培养基及方法。The present invention relates to a method for producing low-temperature lipase by fungal fermentation, and more particularly, the present invention relates to a culture medium and method for producing lipase by fermentation of Geotrichum crassipes.
背景技术Background technique
脂肪酶(Lipase，EC3.1.1.3)又称甘油三酯水解酶，催化三酰基甘油酯在不溶性底物与水之间界面的水解。它们本质上是普遍存在的，并且由各种植物，动物和微生物产生。由微生物来源的脂肪酶在其催化活性和底物特异性方面广泛多样化，微生物脂肪酶用于广泛的工业应用，如食品技术，洗涤剂，生物柴油，化学工业和生物医学科学等。Lipase (EC3.1.1.3), also known as triglyceride hydrolase, catalyzes the hydrolysis of triacylglycerols at the interface between insoluble substrates and water. They are ubiquitous in nature and are produced by a variety of plants, animals, and microorganisms. Lipases derived from microorganisms are widely diversified in terms of their catalytic activity and substrate specificity. Microbial lipases are used in a wide range of industrial applications, such as food technology, detergents, biodiesel, chemical industry and biomedical sciences.
微生物脂肪酶酶解作用的pH和温度范围比动物脂肪酶的范围更宽，便于工业化生产获得高纯度酶制剂。虽然，现在有许多可用的低温脂肪酶的产生源，但只有少数微生物已被开发用于生产低温脂肪酶。低温脂肪酶的使用在降低能源成本、医疗应用、洗涤添加剂和降低工业过程中的微生物污染等多方面具有很大的潜力。The pH and temperature range of microbial lipase hydrolysis is wider than that of animal lipase, which is convenient for industrial production to obtain high-purity enzyme preparations. Although there are many available sources of low-temperature lipase, only a few microorganisms have been developed for the production of low-temperature lipase. The use of low-temperature lipase has great potential in many aspects such as reducing energy costs, medical applications, washing additives, and reducing microbial pollution in industrial processes.
地生弯颈霉(Tolypocladium geodes)为子囊菌门(Ascomycota)、粪壳菌纲(Sordariomy-cetes)、肉座菌目(Hypocreales)、线虫草科(Ophio-cordycipitaceae)、弯颈霉属(Tolypocladium)真菌，是喜冷真菌。地生弯颈霉为中国新纪录种，表现出高海拔分布的特点，同时是脂肪酶高产菌株。所以地生弯颈霉在脂肪酶工业化生产中具有很大的潜力。但是目前地生弯颈霉发酵培养基或培养方法产酶活性低、发酵条件苛刻，不利于大规模生产，因此，本领域亟需一种适用于地生弯颈霉产低温脂肪酶的发酵培养基及发酵方法。Tolypocladium geodes are Ascomycota, Sorariomy-cetes, Hypocreales, Ophio-cordycipitaceae, Tolypocladium Fungi are cold-loving fungi. Geotrichum is a new record species in China, showing high altitude distribution characteristics, and is a high-lipase strain. Therefore, Geotrichum crassipes has great potential in the industrial production of lipase. However, the current fermentation medium or culture method of the benthic bentophyte has low enzyme production activity and harsh fermentation conditions, which is not conducive to large-scale production. Therefore, there is an urgent need in the field for a fermentation culture suitable for low-temperature lipase produced by the benthic bentophyte. And fermentation methods.
发明内容Summary of the Invention
本发明的目的在于提供一种地生弯颈霉发酵生产低温脂肪酶的培养基及方法，该培养基发酵生产的低温脂肪酶活力高，发酵条件温和且易于控制，有利于工业化生产。The purpose of the present invention is to provide a medium and method for producing low-temperature lipase by fermentation of Geotrichum crassipes. The low-temperature lipase produced by fermentation of the medium has high activity, mild fermentation conditions and easy control, which is beneficial to industrial production.
本发明采用如下技术方案，一种地生弯颈霉C41-3发酵生产脂肪酶的产酶发酵培养基包含：乳化芥花油2％(w/v)，α-乳糖2％(w/v)，胰蛋白胨4％(w/v)，MnSO 4·H 2O 0.1％(w/v)，NH 4NO 3 0.1％(w/v)，pH＝5-10，其余为水。 The present invention adopts the following technical scheme. An enzyme-producing fermentation medium for the production of lipase by the fermentation of C.1-33 C. 1-3 includes: emulsified canola oil 2% (w / v), α-lactose 2% (w / v ), Tryptone 4% (w / v), MnSO 4 · H 2 O 0.1% (w / v), NH 4 NO 3 0.1% (w / v), pH = 5-10, the rest is water.
优选的，pH＝5，发酵温度为15-25℃，更优选15℃。Preferably, the pH is 5 and the fermentation temperature is 15-25 ° C, more preferably 15 ° C.
本发明同时请求保护采用上述培养基产低温脂肪酶的方法，具体步骤为：The invention also claims the method for producing low-temperature lipase by using the above-mentioned culture medium. The specific steps are:
(1)将接种量为1％的地生弯颈霉接种于种子培养基中于25℃，转速150r/min摇床培养3-4d得到发酵种子液；(1) Inoculating the terrigenous bentophyte with a 1% inoculation amount in a seed medium at 25 ° C and shaking at 150r / min for 3-4 days to obtain a fermented seed solution;
(2)将发酵种子液接种于产酶发酵培养基中，产酶发酵培养基的装液量为30％，接种量为10％，于25℃，转速150r/min下摇床培养4d，得发酵液；(2) The fermented seed solution is inoculated into an enzyme-producing fermentation medium, and the amount of the enzyme-producing fermentation medium is 30%, the inoculation amount is 10%, and cultured at 25 ° C and a rotation speed of 150 r / min for 4 days in a shaker. Fermentation broth
(3)将步骤(2)所得发酵液离心、收集上清即为粗酶液。(3) The fermentation liquid obtained in step (2) is centrifuged, and the supernatant is collected to obtain a crude enzyme liquid.
所述地生弯颈霉为C41-3，拉丁文学名为(Tolypocladium geodes)，所述的地生弯颈霉C41-3已提交保藏，具体保藏信息如下：The terrestrial curved neck mold is C41-3, and the Latin name is Tolypocladium geodes. The terrestrial curved neck mold C41-3 has been submitted for preservation. The specific preservation information is as follows:
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；Name of the depository unit: General Microbial Center of the China Microbial Collection Management Committee (CGMCC);
保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；Preservation unit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences;
保藏日期：2018年04月24日；Deposit date: April 24, 2018;
保藏编号：CGMCC No.15667。Deposit number: CGMCC No. 15667.
地生弯颈霉菌株呈圆形辐射状生长，菌落正面平展呈现雪白色，绒毛状，致密，菌落背面通常白色，或在菌落起源附近呈深色或褐色，菌丝为白色丝状。The strain of Geotrichum crassipes grows in a circular shape. The front of the colony is snow-white, fluffy and dense. The back of the colony is usually white, or it is dark or brown near the origin of the colony, and the mycelium is white and filamentous.
进一步的，所述的种子培养基组成为：马铃薯200g/L、蔗糖20g/L、水1000mL，pH＝5。Further, the composition of the seed medium is: 200 g / L of potato, 20 g / L of sucrose, 1000 mL of water, and pH = 5.
更为具体的，所述步骤(1)为：将种子培养基分装于三角瓶中，121℃灭菌30分钟，然后冷却；将接种量为1％的菌种接种于种子培养基中，置于摇床培养，培养条件：温度25℃，转速150r/min，培养3～4d即可得到发酵种子液；More specifically, the step (1) is: dividing the seed culture medium into a triangle flask, sterilizing at 121 ° C for 30 minutes, and then cooling; inoculating a seed strain with an inoculum amount of 1% into the seed culture medium, Placed in a shaker for cultivation. The culture conditions are: temperature 25 ° C, rotation speed 150r / min, and cultivation for 3 to 4 days to obtain the fermented seed solution.
所述步骤(2)中基础发酵培养基：乳化橄榄油20ml/L，蛋白胨40g/L，蔗糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，pH自然，其余为水。 The basic fermentation medium in the step (2): emulsified olive oil 20ml / L, peptone 40g / L, sucrose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, natural pH, The rest is water.
产酶发酵培养基：乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，MnSO 4·H 2O 1g/L，NH 4NO 3 1g/L，pH＝5，其余为水； Enzyme-producing fermentation medium: emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, MnSO 4 · H 2 O 1g / L, NH 4 NO 3 1g / L, pH = 5, the rest For water
步骤(2)具体为：取基础发酵培养基按30％的装液量加入到培养容器中，121℃灭菌30分钟，冷却到常温；按10％的接种量，将发酵种子液加入到含产酶发酵培养基的培养容器中，发酵温度为25℃，pH＝5(即pH自然)，转速150r/min，发酵培养4d。Step (2) is specifically: taking the basic fermentation medium into the culture container at 30% of the liquid content, sterilizing at 121 ° C for 30 minutes, and cooling to normal temperature; adding 10% of the inoculation amount to the fermented seed liquid In the culture vessel of the enzyme-producing fermentation medium, the fermentation temperature was 25 ° C., pH = 5 (that is, natural pH), the rotation speed was 150 r / min, and the fermentation was cultured for 4 days.
所述步骤(3)具体为：将发酵液用高速冷冻离心机于8000-10000r/min，4℃ 的条件下离心10min，收集上清液即为粗酶液。The step (3) is specifically: centrifuging the fermentation broth with a high-speed refrigerated centrifuge at 8000-10000 r / min and 4 ° C for 10 min, and collecting the supernatant to obtain a crude enzyme solution.
本发明涉及的乳化芥花油、乳化橄榄油、乳化大豆油、乳化花生油、乳化芝麻油等油脂乳化液的制备方法具体为：将2％的聚乙烯醇溶于去离子水中，加热溶解。再将油脂加入到2％的聚乙烯醇溶液中，使用磁力搅拌器搅拌15min(每5分钟暂停一次)，至乳白色。The method for preparing emulsified oily emulsified oils such as emulsified canola oil, emulsified olive oil, emulsified soybean oil, emulsified peanut oil, emulsified sesame oil and the like is as follows: 2% of polyvinyl alcohol is dissolved in deionized water and dissolved by heating. Add the fat to the 2% polyvinyl alcohol solution, and use a magnetic stirrer to stir for 15 min (pause every 5 minutes) until it is milky white.
与现有技术相比，本发明提供的发酵方法有条件温和，易于控制；提供用于地生弯颈霉C41-3发酵生产脂肪酶的培养基，具有发酵时间短、脂肪酶活力高的优点。本发明摸索到的发酵培养条件，使酶活力从基础发酵培养基的102.69U/mL提高到6011.69U/mL，具有低温脂肪酶活力高等优点，有利于规模化生产，对提升该菌株的工业应用潜力有重大的意义。Compared with the prior art, the fermentation method provided by the present invention has mild conditions and is easy to control; it provides a culture medium for the production of lipase by the fermentation of C. 1-3, and has the advantages of short fermentation time and high lipase activity. . The fermentation culture conditions explored by the present invention increase the enzyme activity from 102.69 U / mL of the basic fermentation medium to 6011.69 U / mL, which has the advantages of high-temperature low-temperature lipase activity, is conducive to large-scale production, and promotes industrial application of the strain The potential is significant.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为对硝基苯酚标准曲线。Figure 1 is a standard curve for p-nitrophenol.
图2为不同产酶培养基对地生弯颈霉低温脂肪酶产量的影响，其中A是对比例1的培养基，B是对比例2的培养基，C是对比例3的培养基，D是对比例4的培养基，E是对比例5的培养基，F是对比例6的培养基，G是对比例7的培养基。Figure 2 shows the effects of different enzyme-producing mediums on the production of low-temperature lipase by C. terreus, where A is the medium of Comparative Example 1, B is the medium of Comparative Example 2, C is the medium of Comparative Example 3, and 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.
具体实施方式Detailed ways
下面通过具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。The following describes the present invention in detail through specific embodiments, but does not limit the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, and reagents used can be obtained from commercial sources.
实施例1Example 1
(1)种子培养基(1) Seed medium
种子培养基：马铃薯200g/L、蔗糖20g/L、水1000mL，pH值自然；Seed medium: 200g / L of potato, 20g / L of sucrose, 1000mL of water, natural pH value;
将配置好的培养液分装于若干个250ml的三角瓶中，每个三角瓶装种子培养基100ml，121℃灭菌30分钟，然后冷却。The configured culture solution was dispensed into several 250ml Erlenmeyer flasks, each of the Erlenmeyer flasks contained 100ml of seed medium, sterilized at 121 ° C for 30 minutes, and then cooled.
在每个装种子培养基100ml的三角瓶中接入接入3个直径为5mm菌块(接种量1％)，置于摇床培养，培养条件：温度25℃，转速150r/min，培养3～4d即可得到发酵种子液；Into each 100ml triangular flask containing seed culture medium, insert 3 pieces of bacteria with a diameter of 5mm (inoculation amount 1%), and place them in a shaker for cultivation. The culture conditions are: temperature 25 ° C, rotation speed 150r / min, culture 3 ～ 4d can get the fermented seed liquid;
(2)产酶发酵培养(2) Enzymatic fermentation culture
发酵培养基：乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L， MnSO 4·H 2O 1g/L，NH 4NO 3 1g/L，pH＝5，其余为水； Fermentation medium: emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, MnSO 4 · H 2 O 1g / L, NH 4 NO 3 1g / L, pH = 5, the rest is water ;
取发酵培养基75ml加入到250ml的三角瓶中，121℃灭菌30分钟，冷却到常温；75ml of fermentation medium was added to a 250ml Erlenmeyer flask, sterilized at 121 ° C for 30 minutes, and cooled to normal temperature;
按10％的接种量，将7.5ml发酵种子液加入到含75ml发酵培养基的250ml三角瓶中，发酵温度为25℃，pH＝5，转速150r/min，发酵培养4d；According to the inoculation amount of 10%, 7.5 ml of fermented seed solution was added to a 250 ml triangular flask containing 75 ml of fermentation medium, the fermentation temperature was 25 ° C, the pH was 5, the rotation speed was 150 r / min, and the fermentation was cultured for 4 days;
(3)油脂乳化液的制备方法(3) Preparation method of fat emulsion
将2％的聚乙烯醇溶于去离子水中，加热溶解。再将油脂加入到2％的聚乙烯醇溶液中，使用磁力搅拌器搅拌15min(每5分钟暂停一次)，至乳白色。2% of polyvinyl alcohol was dissolved in deionized water and dissolved by heating. Add the fat to the 2% polyvinyl alcohol solution, and use a magnetic stirrer to stir for 15 min (pause every 5 minutes) until it is milky white.
(4)脂肪酶液制备(4) Preparation of lipase solution
取发酵液，将发酵液用高速冷冻离心机于10000r/min，4℃的条件下离心10min，收集上清液即为粗酶液，取得粗酶液；Take the fermentation broth, centrifuge the fermentation broth with a high-speed refrigerated centrifuge at 10,000 r / min and 4 ° C for 10 min, and collect the supernatant as the crude enzyme solution to obtain the crude enzyme solution;
(5)酶活力的测定(5) Determination of enzyme activity
酶活测定方法采用对硝基苯基棕榈酸酯(p-NPP)作为底物测量脂肪酶活性。将一个单位的脂肪酶定义为在37℃，pH 8.0条件下，每分钟释放出1μmol对硝基苯酚(p-NP)所需要的酶的量。The enzyme activity measurement method uses p-nitrophenyl palmitate (p-NPP) as a substrate to measure lipase activity. One unit of lipase is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (p-NP) per minute at 37 ° C, pH 8.0.
方法：溶液A：15mg p-NPP溶于5ml异丙醇中；溶液B：在45ml的0.05mol/L Tris-HCl缓冲液(pH 8.0)中加入0.05g***胶和0.2ml Triton X-100。Methods: Solution A: 15 mg of p-NPP was dissolved in 5 ml of isopropanol; Solution B: 45 ml of 0.05 mol / L Tris-HCl buffer (pH 8.0) was added with 0.05 g of acacia gum and 0.2 ml of Triton X-100.
以对硝基苯酚棕榈酸酯p-NPP为底物，将5ml的溶液A与45ml溶液B均匀混合，一个酶样品分为4只试管，分别加入混合液1.8mL，37℃预热5min。3只试管加入200μL酶样品溶液，另外1只加入200μL经煮沸变性(100℃，10min)的酶样品溶液作为对照，混合均匀。37℃水浴反应15min，取出后于波长410nm下测定吸光度值。Using p-nitrophenol palmitate p-NPP as a substrate, 5 ml of solution A and 45 ml of solution B were mixed uniformly. An enzyme sample was divided into 4 test tubes, and 1.8 mL of the mixed solution was added to each, and preheated at 37 ° C for 5 minutes. Add 200 μL of enzyme sample solution to 3 test tubes, and add 200 μL of enzyme sample solution denatured by boiling (100 ° C, 10min) as a control, and mix well. The reaction was carried out in a 37 ° C water bath for 15 minutes, and the absorbance was measured at a wavelength of 410 nm after taking out.
酶活计算公式：X＝CV/(TV′)Enzyme calculation formula: X = CV / (TV ′)
式中：In the formula:
X—脂肪酶活力，U·mL -1； X-lipase activity, U · mL -1 ;
C—对硝基苯酚浓度，μmol·mL -1； C—p-nitrophenol concentration, μmol · mL -1 ;
V—反应液终体积，mL；V—final volume of reaction solution, mL;
V′—酶液的用量，mL；V′—the amount of enzyme solution, mL;
T—反应时间，min。T—Reaction time, min.
(6)标准曲线的制作：(6) Preparation of standard curve:
2.0mmol/LpNP(uL)2.0mmol / LpNP (uL) 00 7.57.5 1515 3030 6060 9090 120120 150150
异丙醇(uL)Isopropanol (uL) 250250 242.5242.5 235235 220220 190190 160160 130130 100100
底物缓冲液(mL)Substrate buffer (mL) 2.252.25 2.252.25 2.252.25 2.252.25 2.252.25 2.252.25 2.252.25 2.252.25
pNP浓度(umol/L)pNP concentration (umol / L) 00 66 1212 24twenty four 4848 7272 9696 120120
在波长410nm处测定吸光度值。以OD410值为横坐标，pNP浓度为纵坐标绘制标准曲线，经处理得到的线性回归方程，y＝69.968x-0.993，R 2＝0.9991，结果见图1。 The absorbance was measured at a wavelength of 410 nm. The standard curve is drawn with the OD410 value as the abscissa and the pNP concentration as the ordinate. The linear regression equation obtained after processing is y = 69.968x-0.993, and R 2 = 0.9991. The results are shown in Figure 1.
对比例Comparative example
改变发酵培养基的组成成分作为对比，根据标准曲线测试不同发酵培养基其组成对低温脂肪酶的产酶酶活。下述对比例1-7中涉及到的发酵种子液采用实施例1步骤(1)的方法制备。The composition of the fermentation medium was changed as a comparison, and according to the standard curve, the composition of different fermentation mediums was tested for the enzyme-producing activity of low-temperature lipase. The fermented seed liquid involved in the following Comparative Examples 1-7 was prepared by the method of step (1) of Example 1.
对比例1Comparative Example 1
制作七种产酶发酵培养基作为对比：For comparison, seven enzyme-producing fermentation media were prepared:
(1)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，蔗糖20g/L，pH＝5，其余为水。 (1) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, sucrose 20g / L, pH = 5, the rest is water.
(2)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，葡萄糖20g/L，pH＝5，其余为水。 (2) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, glucose 20g / L, pH = 5, and the rest is water.
(3)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，D-果糖20g/L，pH＝5，其余为水。 (3) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, D-fructose 20g / L, pH = 5, the rest is water.
(4)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，D(+)-麦芽糖20g/L，pH＝5，其余为水。 (4) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, D (+)-maltose 20g / L, pH = 5, the rest are water.
(5)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，可溶性淀粉20g/L，pH＝5，其余为水。 (5) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, soluble starch 20g / L, pH = 5, and the rest is water.
(6)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，α-乳糖20g/L，pH＝5，其余为水。 (6) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, α-lactose 20g / L, pH = 5, the rest is water.
(7)乳化橄榄油20ml/L，动物蛋白胨40g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，糊精20g/L，pH＝5，其余为水。 (7) Emulsified olive oil 20ml / L, animal peptone 40g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, dextrin 20g / L, pH = 5, the rest is water.
将上述7种产酶发酵培养基于121℃灭菌30min。The above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇床中培养4d，然后测酶活。结果如图2A所示，产酶发酵培养基(2)、(6)的 发酵产酶活力极显著，其中产酶发酵培养基(6)的产酶活力最高，(2)的产酶活力仅次之；其余的产酶培养基发酵产酶活力比较低，其中产酶发酵培养基(3)的发酵产酶活力最低。10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, cultured in a shaker at 25 ° C. and 150 r / min for 4 days, and then the enzyme activity was measured. The results are shown in FIG. 2A. The enzyme-producing fermentation mediums (2) and (6) had extremely significant enzyme-producing activities. Among them, the enzyme-producing fermentation medium (6) had the highest enzyme-producing activity and (2) had only the enzyme-producing activity. Secondly, the remaining enzyme-producing medium fermenting enzyme production activity is relatively low, of which the enzyme-producing fermentation medium (3) has the lowest activity.
对比例2Comparative Example 2
制作五种产酶发酵培养基作为对比：For comparison, five enzyme-producing fermentation media were prepared:
(1)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，胰蛋白胨40g/L，pH＝5，其余为水。 (1) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, tryptone 40g / L, pH = 5, the rest is water.
(2)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，大豆蛋白胨40g/L，pH＝5，其余为水。 (2) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, soy peptone 40g / L, pH = 5, and the rest is water.
(3)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，动物蛋白胨40g/L，pH＝5，其余为水。 (3) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, animal peptone 40g / L, pH = 5, and the rest is water.
(4)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，牛肉膏40g/L，pH＝5，其余为水。 (4) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, beef paste 40g / L, pH = 5, the rest is water.
(5)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，酵母浸粉40g/L，pH＝5，其余为水。 (5) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, yeast extract 40g / L, pH = 5, and the rest is water.
将上述5种产酶发酵培养基于121℃灭菌30min。The five enzyme-producing fermentation mediums were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇床中培养4d后测酶活。结果如图2B所示，产酶发酵培养基(1)使地生弯颈霉发酵酶活力更高。10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, and the enzyme activity was measured after culturing in a shaker at 25 ° C and 150 r / min for 4 days. As a result, as shown in FIG. 2B, the enzyme-producing fermentation medium (1) increased the activity of the fermentation enzyme of Geotrichum crassipes.
对比例3Comparative Example 3
制作四种产酶发酵培养基作为对比：For comparison, four enzyme-producing fermentation media were prepared:
(1)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，NH 4NO 3 1g/L，pH＝5，其余为水。 (1) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, NH 4 NO 3 1g / L, pH = 5, the rest is water .
(2)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，尿素1g/L，pH＝5，其余为水。 (2) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, urea 1g / L, pH = 5, and the rest is water.
(3)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，NH 4Cl 1g/L，pH＝5，其余为水。 (3) Emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, NH 4 Cl 1g / L, pH = 5, and the rest is water.
(4)乳化橄榄油20ml/L，α-乳糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 1g/L，(NH 4) 2SO 4 1g/L，pH＝5，其余为水。 (4) emulsified olive oil 20ml / L, α-lactose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, (NH 4 ) 2 SO 4 1g / L, pH = 5, The rest is water.
将上述4种产酶发酵培养基于121℃灭菌30min。The above four kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇 床中培养4d后测酶活。结果如图2C所示，地生弯颈霉C41-3对无机氮源(NH 4) 2SO 4、NH 4Cl、NH 4NO 3的利用率都是比较高，其中NH 4NO 3使其发酵产酶活力高达5052.35U/mL。 10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, and the enzyme activity was measured after culturing in a shaker at 25 ° C and 150 r / min for 4 days. The results are shown in FIG. 2C. The utilization rate of the inorganic nitrogen source (NH 4 ) 2 SO 4 , NH 4 Cl, and NH 4 NO 3 was high by the benthic fungus C41-3. Among them, NH 4 NO 3 caused Enzyme production activity was as high as 5052.35 U / mL.
对比例4Comparative Example 4
制作七种产酶发酵培养基作为对比：For comparison, seven enzyme-producing fermentation media were prepared:
(1)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，CaSO 4·2H 2O 1g/L，pH＝5，其余为水。 (1) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, CaSO 4 · 2H 2 O 1g / L, pH = 5, the rest is water.
(2)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，CuSO 4·5H 2O 1g/L，pH＝5，其余为水。 (2) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, CuSO 4 · 5H 2 O 1g / L, pH = 5, the rest is water.
(3)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，MgSO 4·7H 2O 1g/L，pH＝5，其余为水。 (3) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, MgSO 4 · 7H 2 O 1g / L, pH = 5, the rest is water.
(4)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水。 (4) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water.
(5)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，ZnSO 4·7H 2O 1g/L，pH＝5，其余为水。 (5) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, ZnSO 4 · 7H 2 O 1g / L, pH = 5, the rest is water.
(6)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，FeSO 4·7H 2O 1g/L，pH＝5，其余为水。 (6) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, FeSO 4 · 7H 2 O 1g / L, pH = 5, the rest is water.
(7)乳化橄榄油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，pH＝5，其余为水。(7) Emulsified olive oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, pH = 5, the rest is water.
将上述7种产酶发酵培养基于121℃灭菌30min。The above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇床中培养4d后测酶活。由图2D可知，相对于未添加金属离子的发酵产酶活力来说，Fe 2+、Cu 2+、Ca 2+、Mg 2+、Mn 2+对地生弯颈霉C41-3的发酵产酶起到了促进的作用，产酶发酵培养基(1)、(2)、(4)、(6)发酵产酶活力高，其中产酶发酵培养基(4)Mn 2+的促进作用最明显，高达4988.59U/mL；产酶发酵培养基(5)中Zn 2+则是抑制了发酵产酶活力。 10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, and the enzyme activity was measured after culturing in a shaker at 25 ° C and 150 r / min for 4 days. It can be seen from FIG. 2D that compared to the fermentative enzyme production activity without added metal ions, the fermentative production of Fe2 + by Cu 2+ , Ca 2+ , Mg 2+ , and Mn 2+ on terrestrial curved neck mold C41-3 Enzymes play a promoting role, and the enzyme-producing fermentation medium (1), (2), (4), (6) has high enzyme-producing activity, and the enzyme-producing fermentation medium (4) Mn 2+ has the most obvious promotion effect. , Up to 4988.59 U / mL; Zn 2+ in the enzyme-producing fermentation medium (5) inhibited the enzyme-producing activity of fermentation.
对比例5Comparative Example 5
制作四种产酶发酵培养基作为对比：For comparison, four enzyme-producing fermentation media were prepared:
(1)α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，乳化芝麻油20ml/L，pH＝5，其余为水。 (1) α-lactose 20 g / L, tryptone 40 g / L, NH 4 NO 3 1 g / L, MnSO 4 · H 2 O 1 g / L, emulsified sesame oil 20 ml / L, pH = 5, the rest is water.
(2)α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，乳化大豆油20ml/L，pH＝5，其余为水。 (2) α-lactose 20 g / L, tryptone 40 g / L, NH 4 NO 3 1 g / L, MnSO 4 · H 2 O 1 g / L, emulsified soybean oil 20 ml / L, pH = 5, and the rest is water.
(3)α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，乳化芥花油20ml/L，pH＝5，其余为水。 (3) α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, emulsified canola oil 20ml / L, pH = 5, the rest is water.
(4)α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，乳化橄榄油20ml/L，pH＝5，其余为水。 (4) α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, emulsified olive oil 20ml / L, pH = 5, the rest is water.
(5)α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，乳化花生油20ml/L，pH自然，其余为水。 (5) α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, emulsified peanut oil 20ml / L, natural pH, the rest is water.
将上述5种产酶发酵培养基于121℃灭菌30min。The five enzyme-producing fermentation mediums were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇床中培养4d后测酶活。结果如图2E所示，地生弯颈霉在上述产酶发酵培养基作用下都产生低温脂肪酶，其中向发酵培养基中加入乳化芥花油时，发酵产酶活力最高，达到4839.33U/mL；花生油和大豆油次之，常用的橄榄油发酵产酶活力为3668.54U/mL，次于大豆油；芝麻油发酵产酶活力最低。10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, and the enzyme activity was measured after culturing in a shaker at 25 ° C and 150 r / min for 4 days. The results are shown in FIG. 2E. The low-temperature lipase was produced by the above-mentioned enzyme-producing fermentation medium. The emulsified canola oil was added to the fermentation medium, and the highest enzyme-producing activity was 4839.33 U / mL; peanut oil and soybean oil are next, and the commonly used olive oil fermentation enzyme production activity is 3688.54U / mL, which is inferior to soybean oil; sesame oil fermentation production enzyme activity is the lowest.
对比例6Comparative Example 6
制作七种产酶发酵培养基作为对比：For comparison, seven enzyme-producing fermentation media were prepared:
(1)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝4，其余为水。 (1) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 4, the rest is water.
(2)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝5，其余为水。 (2) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 5, the rest is water.
(3)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝6，其余为水。 (3) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 6, the rest is water.
(4)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝7，其余为水。 (4) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 7, the rest is water.
(5)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝8，其余为水。 (5) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 8, the rest is water.
(6)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝9，其余为水。 (6) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 9, the rest is water.
(7)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·H 2O 1g/L，pH＝10，其余为水。 (7) Emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · H 2 O 1g / L, pH = 10, the rest is water.
将上述7种产酶发酵培养基于121℃灭菌30min。The above 7 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 minutes.
向上述产酶发酵培养基各接入10％的发酵种子液，在25℃、150r/min的摇床中培养4d后测酶活。结果如图2F表明，在pH 5～10范围内，发酵产脂肪酶活 力维持在较高水平；pH值小于5时的酶活力大幅度降低。所以地生弯颈霉C41-3发酵产脂肪酶pH范围是5～10，当pH值为5时，发酵产酶活力最高，达到6011.69U/mL。10% of the fermented seed solution was added to each of the above-mentioned enzyme-producing fermentation medium, and the enzyme activity was measured after culturing in a shaker at 25 ° C and 150 r / min for 4 days. The results, as shown in Figure 2F, show that in the pH range of 5 to 10, the activity of fermenting lipase is maintained at a high level; when the pH value is less than 5, the enzyme activity is greatly reduced. Therefore, the pH range of lipase produced by C. 1-3 C. 1-3 was 5-10. When the pH value was 5, the enzyme production activity was the highest, reaching 6011.69 U / mL.
对比例7Comparative Example 7
制作六种产酶发酵培养基作为对比：For comparison, six enzyme-producing fermentation media were prepared:
(1)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为5℃。 (1) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 5 ° C.
(2)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为10℃。 (2) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 10 ° C.
(3)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为15℃。 (3) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 15 ° C.
(4)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为20℃。 (4) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 20 ° C.
(5)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为25℃。 (5) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 25 ° C.
(6)乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，NH 4NO 3 1g/L，MnSO 4·4H 2O 1g/L，pH＝5，其余为水，发酵温度为30℃。 (6) emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, NH 4 NO 3 1g / L, MnSO 4 · 4H 2 O 1g / L, pH = 5, the rest is water, The fermentation temperature was 30 ° C.
将上述6种产酶发酵培养基于121℃灭菌30min。The above 6 kinds of enzyme-producing fermentation medium were sterilized at 121 ° C for 30 min.
向上述产酶发酵培养基各接入10％的发酵种子液，在150r/min的摇床中培养3d后测酶活。结果如图2G所示，地生弯颈霉在以上发酵温度中，在发酵温度为15℃、20℃、25℃中的发酵产酶能力都比较高，其中在15℃下的产酶活力最高，25℃下的产酶活力仅次之；在5℃中不产酶。10% of the fermented seed solution was added to each of the above enzyme-producing fermentation mediums, and the enzyme activity was measured after being cultured in a shaker at 150 r / min for 3 days. The results are shown in FIG. 2G. Among the above fermentation temperatures, the fermentation ability of S. crassipes at 15 ° C, 20 ° C, and 25 ° C was relatively high, and the enzyme production activity was highest at 15 ° C. The enzyme-producing activity at 25 ° C was second; no enzyme was produced at 5 ° C.
以上所述，仅为本发明创造较佳的具体实施方式，但本发明创造的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明创造披露的技术范围内，根据本发明创造的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明创造的保护范围之内。The above description is only the preferred embodiment of the present invention, but the scope of protection of the present invention is not limited to this. Any person skilled in the technical field is within the technical scope disclosed by the present invention. The created technical solution and its inventive concept are equivalently replaced or changed, and should all be covered by the protection scope of the invention.

Claims (6)

  1. 一种地生弯颈霉发酵生产脂肪酶的产酶发酵培养基，其特征在于，发酵培养基包含：乳化芥花油2％(w/v)，α-乳糖2％(w/v)，胰蛋白胨4％(w/v)，MnSO 4·H 2O 0.1％(w/v)，NH 4NO 30.1％(w/v)，pH＝5-10，其余为水。 An enzyme-producing fermentation medium for producing lipase by fermentation of Geotrichum crassipes, characterized in that the fermentation medium comprises: emulsified canola oil 2% (w / v), α-lactose 2% (w / v), Tryptone 4% (w / v), MnSO 4 · H 2 O 0.1% (w / v), NH 4 NO 3 0.1% (w / v), pH = 5-10, the rest was water.
  2. 采用权利要求1所述培养基产低温脂肪酶的方法，其特征在于，具体包括以下步骤：The method for producing low-temperature lipase by using the medium according to claim 1, characterized in that it specifically comprises the following steps:
    (1)将接种量为1％的地生弯颈霉接种于种子培养基中于25℃，转速150r/min摇床培养3-4d得到发酵种子液；(1) Inoculating the terrigenous bentophyte with a 1% inoculation amount in a seed medium at 25 ° C and shaking at 150r / min for 3-4 days to obtain a fermented seed solution;
    (2)将发酵种子液接种于产酶发酵培养基中，产酶发酵培养基的装液量为30％，接种量为10％，于25℃，转速150r/min下摇床培养4d，得发酵液；(2) The fermented seed solution is inoculated into an enzyme-producing fermentation medium, and the amount of the enzyme-producing fermentation medium is 30%, the inoculation amount is 10%, and cultured at 25 ° C and a rotation speed of 150 r / min for 4 days in a shaker. Fermentation broth
    (3)将步骤(2)所得发酵液离心、收集上清即为粗酶液。(3) The fermentation liquid obtained in step (2) is centrifuged, and the supernatant is collected to obtain a crude enzyme liquid.
  3. 根据权利要求2所述的方法，其特征在于，所述的种子培养基组成为：马铃薯200g/L、蔗糖20g/L、水1000mL，pH＝5。The method according to claim 2, wherein the composition of the seed medium is: 200 g / L of potato, 20 g / L of sucrose, 1000 mL of water, and pH = 5.
  4. 根据权利要求2所述的方法，其特征在于，所述步骤(1)为：将种子培养基分装于三角瓶中，121℃灭菌30分钟，然后冷却；将接种量为1％的菌种接种于种子培养基中，置于摇床培养，培养条件：温度25℃，转速150r/min，培养3～4d即可得到发酵种子液。The method according to claim 2, characterized in that the step (1) is: dividing the seed culture medium into a triangle flask, sterilizing at 121 ° C for 30 minutes, and then cooling; and inoculating a 1% bacteria strain The seed is inoculated in the seed medium and cultured in a shaker. The culture conditions are: temperature 25 ° C, rotation speed 150 r / min, and culture for 3 to 4 days to obtain a fermented seed solution.
  5. 根据权利要求2所述的方法，其特征在于，所述步骤(2)具体为：取基础发酵培养基按30％的装液量加入到培养容器中，121℃灭菌30分钟，冷却到常温；按10％的接种量，将发酵种子液加入到含产酶发酵培养基的培养容器中，发酵温度为25℃，pH＝5，转速150r/min，发酵培养4d；The method according to claim 2, characterized in that said step (2) is specifically: taking the basic fermentation medium at 30% of the liquid content and adding it to the culture container, sterilizing at 121 ° C for 30 minutes, and cooling to normal temperature ; According to the 10% inoculation amount, add the fermented seed liquid to the culture container containing the enzyme-producing fermentation medium, the fermentation temperature is 25 ° C., pH = 5, the rotation speed is 150 r / min, and the fermentation is cultured for 4 days;
    其中，基础发酵培养基：乳化橄榄油20ml/L，蛋白胨40g/L，蔗糖20g/L，MgSO 4·7H 2O 1g/L，KH 2PO 41g/L，pH自然，其余为水； Among them, the basic fermentation medium: emulsified olive oil 20ml / L, peptone 40g / L, sucrose 20g / L, MgSO 4 · 7H 2 O 1g / L, KH 2 PO 4 1g / L, natural pH, the rest is water;
    产酶发酵培养基：乳化芥花油20ml/L，α-乳糖20g/L，胰蛋白胨40g/L，MnSO 4·H 2O 1g/L，NH 4NO 31g/L，pH＝5，其余为水。 Enzyme-producing fermentation medium: emulsified canola oil 20ml / L, α-lactose 20g / L, tryptone 40g / L, MnSO 4 · H 2 O 1g / L, NH 4 NO 3 1g / L, pH = 5, the rest For water.
  6. 根据权利要求2所述的方法，其特征在于，所述步骤(3)具体为：将发酵液用高速冷冻离心机于8000-10000r/min，4℃的条件下离心10min，收集上清液即为粗酶液。The method according to claim 2, characterized in that the step (3) is specifically: centrifuging the fermentation broth with a high-speed refrigerated centrifuge at 8000-10000 r / min and 4 ° C for 10 min, and collecting the supernatant, that is, For crude enzyme solution.
PCT/CN2018/089144 2018-05-31 2018-05-31 Culture medium for producing lipase by fermenting tolypocladium geodes and culture method therefor WO2019227363A1 (en)

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Citations (4)

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