阅读说明：本技术 鼠李糖脂发酵培养基及其制备方法、应用 (Rhamnolipid fermentation medium and preparation method and application thereof ) 是由 史正军 张祥胜 袁峰均 周一波 于 2019-09-23 设计创作，主要内容包括：本发明涉及一种鼠李糖脂发酵培养基及其制备方法、应用。其中,鼠李糖脂发酵培养基包括如下质量份的原料组分：尿素2-3份、磷酸二氢钾6-8份、氯化钾1-2份、酵母1-2份、镁盐0.1-0.3份、钙盐0.01-0.03份、碳源20-100份、以及水900-1100份,其中,所述镁盐选自硫酸镁、氯化镁和硝酸镁中的至少一种,所述钙盐选自氯化钙和碳酸钙中的至少一种,所述碳源选自植物油和甘油中的至少一种。上述鼠李糖脂发酵培养基中各原料组分配比合理,经实验验证,铜绿假单胞菌能够充分利用此培养基为基础原料制得产率高的鼠李糖脂。(The invention relates to a rhamnolipid fermentation medium, a preparation method and application thereof. The rhamnolipid fermentation medium comprises the following raw material components in parts by mass: 2-3 parts of urea, 6-8 parts of monopotassium phosphate, 1-2 parts of potassium chloride, 1-2 parts of yeast, 0.1-0.3 part of magnesium salt, 0.01-0.03 part of calcium salt, 20-100 parts of carbon source and 900 parts of water, wherein the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, the calcium salt is selected from at least one of calcium chloride and calcium carbonate, and the carbon source is selected from at least one of vegetable oil and glycerol. The rhamnolipid fermentation medium has reasonable raw material component proportion, and the pseudomonas aeruginosa can fully utilize the medium as a basic raw material to prepare the rhamnolipid with high yield through experimental verification.)

1. The rhamnolipid fermentation medium is characterized by comprising the following raw material components in parts by mass:

wherein the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, the calcium salt is selected from at least one of calcium chloride and calcium carbonate, and the carbon source is selected from at least one of vegetable oil and glycerol.

2. The rhamnolipid fermentation medium of claim 1, characterized by comprising the following raw material components in parts by mass:

3. the rhamnolipid fermentation medium of claim 1, wherein the vegetable oil is selected from at least one of peanut oil, soybean oil, linseed oil, castor oil and rapeseed oil, fried cargo oil.

4. A preparation method of a rhamnolipid fermentation medium is characterized by comprising the following steps:

mixing the raw material components of the rhamnolipid fermentation medium of any one of claims 1-3, and discharging after sterilization treatment.

5. The method for preparing a rhamnolipid fermentation medium according to claim 4, wherein in the step of mixing the raw material components of the rhamnolipid fermentation medium according to any one of claims 1 to 3, urea, monopotassium phosphate, potassium chloride, magnesium salts, calcium salts and water are mixed to be uniform to obtain a mixed solution; adding yeast and a carbon source into the mixed solution.

6. A fertilizer auxiliary agent, characterized in that the raw material of the fertilizer auxiliary agent comprises the rhamnolipid fermentation medium of any one of claims 1-3 or the medium prepared by the preparation method of the rhamnolipid fermentation medium of any one of claims 4-5.

7. The preparation method of the fertilizer additive is characterized by comprising the following steps:

enlarging and culturing pseudomonas aeruginosa to prepare a seed solution;

inoculating the rhamnolipid fermentation medium according to the inoculation amount of 5-10% of the mass percent of the seed liquid into any one of claims 1-3 for fermentation to obtain fermentation liquid; or inoculating the seed solution into the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to any one of claims 4 to 5 according to the inoculation amount of 5 to 10 percent by mass of the seed solution, and fermenting to obtain fermentation liquor;

adjusting the pH value of the fermentation liquor to 8.0-10.0, heating to 80-100 ℃, preserving heat, and filtering to obtain the fertilizer auxiliary agent.

8. The method for preparing a fertilizer auxiliary according to claim 7, wherein in the step of preparing the seed solution by expanding and culturing pseudomonas aeruginosa, slant strain is inoculated into a nutrient broth culture medium by using an inoculating loop, the slant strain is the pseudomonas aeruginosa and is subjected to expanding and culturing to prepare a primary seed solution, and the primary seed solution is inoculated into the culture medium and is subjected to expanding and culturing to prepare a secondary seed solution.

9. The method for preparing a fertilizer auxiliary agent according to claim 8, wherein in the step of preparing the first-stage seed solution by inoculating the slant strain into the culture medium for scale-up culture, the slant strain is inoculated into the broth culture medium for shake-flask culture for 8h to 12h, and the culture temperature is 28 ℃ to 30 ℃.

10. The method for preparing fertilizer auxiliary agent according to claim 8, wherein in the step of preparing the secondary seed solution by inoculating the primary seed solution into the culture medium in an expanding way, the primary seed solution is inoculated into the rhamnolipid fermentation culture medium according to any one of claims 1-3 according to the inoculation amount of 1-5% by mass; or inoculating the primary seed liquid into the culture medium prepared by the method for preparing the rhamnolipid fermentation culture medium according to any one of claims 4 to 5 according to the inoculation amount of 1 to 5 percent by mass;

performing shake flask culture or aeration culture for 24-48 h at 28-30 deg.C.

Technical Field

The invention relates to the field of fermentation engineering, and in particular relates to a rhamnolipid fermentation medium, and a preparation method and application thereof.

Background

The rhamnolipid is a biosurfactant produced by fermenting pseudomonas aeruginosa, has strong surface and interface activity, low toxicity and biodegradability, and is widely applied to the fields of petroleum, environmental protection, chemical industry, food, medicine, agriculture and the like. Rhamnolipid is an anionic surfactant, is mainly formed by combining hydrophilic carbohydrate and hydrophobic long-chain fatty acid or hydroxy fatty acid in a covalent bond mode, and is mainly applied to fertilizers or pesticide auxiliaries to play roles in emulsification, dispersion, wetting and the like. The rhamnolipid is usually prepared by fermenting Pseudomonas aeruginosa (Pseudomonas aeruginosa) with raw material culture media such as glucose, vegetable oil, dipotassium hydrogen phosphate and the like.

The pesticide adjuvant is an important novel functional material, has indispensable functions in the aspects of improving the quality of a fertilizer product, increasing the function of the fertilizer product, improving the application effect of the fertilizer, improving the utilization rate of the fertilizer and the like, can assist traditional fertilizer production enterprises to reduce the energy consumption of fertilizer production, improve the production efficiency of the fertilizer, increase the economic benefits of the enterprises, accelerate the structure adjustment and the industrial upgrading of the fertilizer product, and has very important significance in promoting the development of modern agriculture, improving the utilization rate of resources and improving the influence of fertilizer application on the environment.

The traditional rhamnolipid fermentation medium has high raw material component cost, and the yield of rhamnolipid in the prepared fermentation liquid is low.

Disclosure of Invention

Therefore, the rhamnolipid fermentation medium which can improve the yield of rhamnolipid and has low raw material cost is provided.

A rhamnolipid fermentation medium comprises the following raw material components in parts by mass:

wherein the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, the calcium salt is selected from at least one of calcium chloride, calcium nitrate and calcium carbonate, and the carbon source is selected from at least one of vegetable oil and glycerol.

In one embodiment, the material comprises the following raw material components in parts by mass:

in one embodiment, the vegetable oil is selected from at least one of peanut oil, soybean oil, linseed oil, castor oil, rapeseed oil, and fried cargo oil.

The invention provides a preparation method of a rhamnolipid fermentation medium, which comprises the following steps:

the raw material components of the rhamnolipid fermentation medium are mixed, sterilized and discharged.

In one embodiment, in the step of mixing the raw material components of the rhamnolipid fermentation medium according to any one of the present invention, urea, potassium dihydrogen phosphate, potassium chloride, magnesium salt, calcium salt and water are mixed to be uniform to obtain a mixed solution; adding yeast and a carbon source into the mixed solution.

The invention also provides a fertilizer auxiliary agent, and the raw material of the fertilizer auxiliary agent comprises the rhamnolipid fermentation culture medium or the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium.

The invention also provides a preparation method of the fertilizer additive, which comprises the following steps:

enlarging and culturing pseudomonas aeruginosa to prepare a seed solution;

inoculating the rhamnolipid into a rhamnolipid fermentation culture medium according to the inoculation amount of 5-10% of the seed liquid by mass percent, and fermenting to obtain fermentation liquor; or inoculating the seed solution into the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to the inoculation amount of 5-10% of the seed solution by mass percent for fermentation to obtain fermentation liquor;

adjusting the pH value of the fermentation liquor to 8.0-10.0, heating to 80-100 ℃, preserving heat, and filtering to obtain the fertilizer auxiliary agent.

In one embodiment, in the step of preparing the seed solution by expanding and culturing the pseudomonas aeruginosa, a slant strain is inoculated into a nutrient broth culture medium by using an inoculating loop, the slant strain is the pseudomonas aeruginosa and is subjected to expanding and culturing to prepare a primary seed solution, and the primary seed solution is inoculated into the culture medium and is subjected to expanding and culturing to prepare a secondary seed solution.

In one embodiment, in the step of preparing the first-stage seed liquid by inoculating the slant strain into the culture medium for scale-up culture, the slant strain is inoculated into a meat soup culture medium and cultured for 8-12 h in a shaking way, and the culture temperature is 28-30 ℃.

In one embodiment, in the step of preparing the secondary seed liquid by the amplification culture of the primary seed liquid into the culture medium, the primary seed liquid is inoculated into the rhamnolipid fermentation culture medium according to any one of the invention according to the inoculation amount of 1-5% by mass; or inoculating the primary seed liquid into the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to the inoculation amount of 1-5% by mass;

performing shake flask culture or aeration culture for 24-48 h at 28-30 deg.C.

The inventor of the application discovers that the traditional rhamnolipid fermentation medium mostly contains diphosphates consisting of monopotassium phosphate and dipotassium phosphate, so that the raw material cost is high, pseudomonas aeruginosa can not fully utilize nutrient substances in the medium, and the rhamnolipid prepared by fermentation has low yield.

Based on the method, the formula of the traditional rhamnolipid fermentation medium is improved by the inventor, the raw material components in the rhamnolipid fermentation medium are reasonable in proportion, and the pseudomonas aeruginosa can make full use of the medium as a basic raw material to prepare the rhamnolipid with high yield through experimental verification. In addition, the inventor selects cheap monopotassium phosphate, urea, potassium chloride and the like as the raw materials of the rhamnolipid fermentation medium, so that the raw material cost is greatly reduced.

Drawings

FIG. 1 is a graph comparing rhamnolipid content in fertilizer adjuvants of example 1, example 3, comparative example 3 and comparative example 4 of the present invention;

fig. 2 is a graph comparing rhamnolipid content in fertilizer adjuvants of example 3, comparative example 1 and comparative example 2 of the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a rhamnolipid fermentation medium which comprises the following raw material components in parts by mass:

wherein the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, the calcium salt is selected from at least one of calcium chloride and calcium carbonate, and the carbon source is selected from at least one of vegetable oil and glycerol.

In one embodiment, the invention provides a rhamnolipid fermentation medium, which comprises the following raw material components in parts by mass:

in one embodiment, the yeast is at least one of a low cost food grade yeast powder, yeast extract, and yeast extract.

In one embodiment, the vegetable oil is selected from at least one of peanut oil, soybean oil, linseed oil, castor oil, rapeseed oil, and fried cargo oil.

In one example, it was experimentally verified that when the calcium salt is calcium chloride, pseudomonas aeruginosa produces higher amounts of rhamnolipids when fermented in the culture medium of the present application than when the calcium salt is calcium carbonate.

In one embodiment, urea, monopotassium phosphate, magnesium sulfate and calcium chloride are all fertilizer-grade reagent materials, the yeast powder is food-grade yeast powder, the vegetable oil is fried cargo oil, waste oil and the like, the raw materials are low in cost, the cost can be reduced, and the yield of rhamnolipid is not obviously influenced under the same condition.

In one embodiment, the water is deionized water, and experiments prove that when the deionized water is selected, the rhamnolipid produced by the pseudomonas aeruginosa through the fermentation of the culture medium disclosed by the application is higher in yield. It will of course be appreciated that the water may also be tap water.

The inventor improves the formula of the traditional rhamnolipid fermentation medium, the raw material components in the rhamnolipid fermentation medium are reasonable in proportion, and the pseudomonas aeruginosa can fully utilize the medium as a basic raw material to prepare the rhamnolipid with high yield through experimental verification. In addition, the inventor selects cheap monopotassium phosphate, urea, potassium chloride and the like as the raw materials of the rhamnolipid fermentation medium, so that the raw material cost is greatly reduced.

The invention provides a preparation method of a rhamnolipid fermentation medium, which comprises the following steps:

the raw material components of the rhamnolipid fermentation medium are mixed, sterilized and discharged.

In one embodiment, in the step of mixing the raw material components of the rhamnolipid fermentation medium of claim, urea, monopotassium phosphate, potassium chloride, magnesium salt, calcium salt and water are mixed to be uniform to obtain a mixed solution; adding yeast and a carbon source into the mixed solution.

In one embodiment, the sterilization process is steam sterilization at 120-122 ℃ for 15-20 min, although steam autoclaving may also be used.

The invention also provides a fertilizer auxiliary agent, and the raw material of the fertilizer auxiliary agent comprises the rhamnolipid fermentation culture medium or the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium.

The fertilizer auxiliary agent contains the rhamnolipid fermentation medium, has low raw material cost, can be used as a root irrigation agent and a disinfectant, prevents and treats soil-borne diseases, and promotes the growth of crops.

The invention also provides a preparation method of the fertilizer additive, which comprises the following steps:

enlarging and culturing pseudomonas aeruginosa to prepare a seed solution;

inoculating the seed solution into the rhamnolipid fermentation medium according to the inoculum size of 5-10% of the mass percent of the seed solution to ferment to obtain fermentation liquor, then adjusting the pH of the fermentation liquor to 8.0-10.0, heating to 80-100 ℃, preserving heat, and filtering to obtain a fertilizer auxiliary agent; or

Inoculating the seed solution into the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to the inoculation amount of 5-10% of the mass percent of the seed solution, fermenting to obtain fermentation liquor, then adjusting the pH of the fermentation liquor to 8.0-10.0, heating to 80-100 ℃, preserving heat, and filtering to obtain the fertilizer auxiliary agent.

The rhamnolipid fermentation medium is inoculated into the rhamnolipid fermentation medium according to the inoculum size of 5-10% of the mass percent of the seed liquid for fermentation to obtain fermentation liquid, wherein the mass percent of the seed liquid accounts for 5-10% of the mass of the seed liquid and the total mass of the rhamnolipid fermentation medium.

Wherein, the Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a hydrocarbon degrading bacterium capable of synthesizing a surfactant rhamnolipid, and the rhamnolipid can be synthesized by fermentation by utilizing the rhamnolipid fermentation culture medium. The pseudomonas aeruginosa of the invention can be selected from pseudomonas aeruginosa known to those skilled in the art.

In one embodiment, the time of the heat preservation treatment is 15min-25 min.

In one embodiment, in the step of preparing the seed solution by expanding and culturing the pseudomonas aeruginosa, a slant strain is inoculated into a nutrient broth culture medium by using an inoculating loop, the slant strain is the pseudomonas aeruginosa and is subjected to expanding and culturing to prepare a primary seed solution, and the primary seed solution is inoculated into the culture medium and is subjected to expanding and culturing to prepare a secondary seed solution. At this time, the secondary seed solution is also the final seed solution. The slant strains are subjected to secondary amplification culture to prepare a secondary seed solution, which is more beneficial to subsequent fermentation of pseudomonas aeruginosa. It can be understood that the first-stage seed solution may not be inoculated into the culture medium for amplification culture to prepare a second-stage seed solution, but the first-stage seed solution is directly inoculated into the rhamnolipid fermentation culture medium or the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to the invention according to the inoculum size of 5-10% by mass for fermentation to obtain the fermentation liquid.

In one embodiment, in the step of preparing the first-stage seed liquid by inoculating the slant strain into the culture medium for scale-up culture, the slant strain is inoculated into a meat soup culture medium and cultured for 8-12 h in a shaking way, and the culture temperature is 28-30 ℃.

In one embodiment, in the step of preparing the secondary seed solution by inoculating the primary seed solution into the culture medium for amplification culture, the primary seed solution is inoculated into the rhamnolipid fermentation culture medium according to the inoculation amount of 1-5% by mass, and the rhamnolipid fermentation culture medium is subjected to shake flask culture for 24-48 h at the culture temperature of 28-30 ℃.

In one embodiment, in the step of preparing the secondary seed solution by inoculating the primary seed solution into the culture medium for expansion culture, the primary seed solution is inoculated into the culture medium prepared by the preparation method of the rhamnolipid fermentation culture medium according to the mass percent of 1-5%, and the culture temperature is 28-30 ℃ for 24-48 h by shake flask culture or aeration culture.

In one embodiment, in the step of inoculating the rhamnolipid fermentation medium according to any one of the present invention with an inoculum size of 5-10% by mass of a seed solution, the fermentation conditions are fermentation at 28-32 ℃ for 3-5 days, and further, the rhamnolipid fermentation medium according to any one of the present invention with an inoculum size of 5% by mass of the seed solution.

In this embodiment, the fermentation device is not limited to a shake flask or a fermentation tank, and further, if the shake flask is used for fermentation, the volume of the fermentation liquid is one fifth of the volume of the shake flask, and if the fermentation tank is used for fermentation, the volume of the fermentation liquid is 70% of the volume of the fermentation tank. Is more beneficial to full fermentation.

In one embodiment, in the step of inoculating the rhamnolipid fermentation medium according to any one of the present invention again according to the inoculation amount of 5-10% by mass of the seed solution, the fermentation condition is fermentation at 28-32 ℃ for 3-5 days, and further, the rhamnolipid fermentation medium according to any one of the present invention is inoculated according to the inoculation amount of 5% by mass of the seed solution.

In this embodiment, the fermentation device is not limited to a shake flask or a fermentation tank, and further, if the shake flask is used for fermentation, the volume of the fermentation liquid is one fifth of the volume of the shake flask, and if the fermentation tank is used for fermentation, the volume of the fermentation liquid is 70% of the volume of the fermentation tank. Is more beneficial to full fermentation.

In one embodiment, in the step of inoculating the culture medium prepared by the method for preparing the rhamnolipid fermentation culture medium according to any one of the invention with the inoculum size of 5-10% by mass of the seed solution, the fermentation condition is that the rhamnolipid fermentation culture medium is fermented for 3-5 days at 28-32 ℃, and further, the rhamnolipid fermentation culture medium according to any one of the invention with the inoculum size of 5% by mass of the seed solution is inoculated.

The culture medium prepared by the method for preparing the rhamnolipid fermentation culture medium is inoculated to 5-10% of the seed liquid by mass percent, and the seed liquid by mass percent accounts for 5-10% of the total mass of the seed liquid and the rhamnolipid fermentation culture medium.

The preparation method of the fertilizer additive is simple to operate, and the fertilizer additive prepared by using the rhamnolipid fermentation medium can be used as a root irrigation agent and a disinfectant, can prevent and treat soil-borne diseases and can promote the growth of crops.

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Design of experiments

Preparing materials:

the strain for experiment is Pseudomonas aeruginosa strain Z-2, which is disclosed in CN104087525A (a novel marine fermentation strain producing biosurfactant).

Broth culture medium purchased from Hangzhou microbial agents, Inc.

Except for other provisions, except for comparative example 5, the raw materials of the reagents used in the following examples are all purchased from the market, urea, potassium dihydrogen phosphate, magnesium sulfate and calcium chloride are all fertilizer-grade reagent materials, the yeast powder is food-grade yeast powder, waste frying oil is selected from vegetable oil, and the raw materials of the rhamnolipid fermentation medium are low in cost.