CN107446968A - A kind of fermented and cultured accelerator and its application method - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a fermentation culture accelerator and a use method thereof. The accelerator is composed of plant extract, fatty acid, vegetable oil and surfactant, and the accelerator is added to the fermented product. The plant extract is Eucommia leaf extract, tomato juice or orange peel juice; the fatty acid is palmitic acid, hard Fatty acid or oleic acid; Vegetable oil is peanut oil, olive oil or corn oil; The surfactant is Tween80, Span40 or PEG1000. In the process of liquid fermentation, in addition to mycelium proliferating in large quantities, various metabolites such as polysaccharides, polypeptides, alkaloids, polyphenols, inorganic magnesium, nucleic acids, amino acids and vitamins will be produced in the fermentation broth. A large amount of metabolites indicates high mycelium proliferation efficiency. After the accelerator is added in the present invention, the permeability of the cell membrane is changed by the accelerator, thereby increasing extracellular secondary metabolites. The accelerator can effectively promote the activity of glucoamylase, thereby increasing the conversion rate of pachymansin.

Description

A kind of fermentation culture accelerant and using method thereof

technical field

The invention relates to the field of biotechnology, in particular to a fermentation culture accelerator and a use method thereof.

Background technique

Poria cocos is one of the oldest medicinal and edible basidiomycetes in my country, which belongs to the fungal phylum and Basidiomycotina subphylum. In traditional Chinese medicine, Poria cocos has a history of more than 2,000 years of medicinal use. In Shennong’s Compendium of Materia Medica, Poria cocos is listed as the top grade. It has the effect of long-term use to soothe the soul and refresh the mind without hunger and prolong life. According to the Chinese Pharmacopoeia, Poria is sweet, light and flat in nature and flavor, and it is home to the heart, lung, spleen and kidney meridians. It has the effects of diuresis and dampness, invigorating the spleen and calming the heart. It is used for edema, oliguria, insufficiency of the spleen, lack of food, diarrhea, restlessness, palpitation and insomnia. As early as the end of the Spring and Autumn Period in my country, there are records of the use of Poria cocos. Poria cocos is often used as the essence of medicine in traditional Chinese medicine prescriptions. It has a wide range of effects and is known as the holy medicine for dehumidification. Among the prescriptions, the compatibility rate of Poria cocos reaches 70%. Poria cocos is distributed in countries and regions such as China, Japan, Southeast Asia, North America and Australia. Poria resources are abundant in my country and widely distributed, mainly in Hebei, Shanxi, Shaanxi, Shandong, Anhui, Jiangxi, Jiangsu, Zhejiang, Fujian, Henan, Hubei, Hunan, Guangdong, Guangxi, Sichuan, Yunnan, Guizhou and other provinces.

The resources of wild Poria cocos are limited. At present, artificial cultivation methods are mainly used in China to cultivate Poria cocos. The artificial cultivation of Poria cocos has a history of more than 1,500 years in my country, which began in the Southern and Northern Dynasties at the earliest. In recent years, artificial cultivation has been replaced by artificially cultivated pure strains. Liquid fermentation technology is the main content of modern fermentation. The liquid fermentation technology of fungi started with the rise of the antibiotic industry. Researchers believe that fungal liquid fermentation technology has obvious advantages over traditional fungal production methods. Liquid fermentation is to place mycelium in liquid medium, and under the condition of pure culture, force sterile air to shake The way of culturing in bottles or fermenters is the process of mycelium growth and reproduction and metabolite synthesis. Fermentation products include mycelium and fermentation broth, which are obtained through extraction or concentration, spray drying and other processes, and then applied to pharmaceutical raw materials and health food. Liquid fermentation has higher production efficiency than solid fermentation, and can be industrialized continuously and on a large scale, which greatly shortens the production time. In addition, the raw materials have a wide range of sources and low prices, thereby reducing production costs. However, liquid fermentation has disadvantages such as large equipment investment, high technical content, and unstable output. Restricting the industrialized production of the liquid fermentation of medicine and food dual-purpose raw materials such as Poria cocos always.

Contents of the invention

The invention solves the deficiencies of the prior art and provides a fermentation culture accelerant with high yield, stable composition and low cost and a use method thereof.

The technical scheme that the present invention solves its technical problem adopts is:

A fermentation culture accelerator, which is composed of plant extract, fatty acid, vegetable oil and surfactant, the accelerator is added to the fermented product, and the plant extract is eucommia leaf extract, tomato juice or orange peel juice; The fatty acid is palmitic acid, stearic acid or oleic acid; the vegetable oil is peanut oil, olive oil or corn oil; the surfactant is Tween80, Span40 or PEG1000.

Add 0.125-0.5% plant extract by mass percentage, 0.125-0.5% fatty acid by mass percentage, 0.125-0.5% vegetable oil by mass percentage and 0.125-0.5% surfactant by mass percentage to the product to be fermented, and the product to be fermented is the strain Poria cocos HD _07-1 .

0.5% by mass of plant extract, 0.5% by mass of fatty acid, 0.25% by mass of vegetable oil and 0.125% by mass of surfactant are added to the fermented product.

0.5% by mass of Eucommia leaf extract, 0.5% by mass of palmitic acid, 0.25% by mass of olive oil and 0.125% by mass of PEG1000 are added to the fermented product.

A method for using a fermentation culture accelerator, comprising the steps of:

A. Addition of culture accelerator

Add accelerator to the original seed of Poria cocos to be fermented to make mixed stock solution;

B. Solid culture of bacteria

Put the mixed stock solution into the strain culture medium, place it in a constant temperature incubator and cultivate it ^at 26°C, and the cultivation time is about 6-8 days;

C. Shake flask seed culture

Under a sterile environment, take seeds from the medium of step B, place them in a shaker and carry out shake flask culture ^at 26°C, and the culture time is 7-14 days to obtain strains;

D. Liquid fermentation culture

Under a sterile environment, the strains were taken from the medium in step C, placed in a shaker for 26 ^° C shake flask culture, and the culture time was 5-6 days to obtain the final product.

The medium of the steps B, C and D is carbon source, nitrogen source, MgSO ₄ ·7H ₂ O and KH ₂ PO ₄ , the carbon source is one or more of glucose, fructose and sucrose, and the nitrogen The source is one or more of peptone and potassium nitrate, and the pH value of the medium is 5-6.

In the steps C and D, a 250mL Erlenmeyer flask is selected for the shaker flask, the inoculation amount in the bottle is 50-100mL, and the rotation speed of the shaker flask is 60-210rpm.

The step C shake flask seed culture is divided into two levels, and the first level shake flask culture takes seeds from the step B medium in a sterile environment, and places them in a shaker to carry out 26 ^° C shake flask culture, with a rotation speed of 150rpm. The culture time is After 7 days, the secondary shake flask culture was carried out, and the shake flask culture was carried out in a shaker at 26° C. ^at a rotation speed of 150 rpm, and the culture time was 4 days to obtain the final product.

The medium in the steps B, C and D is carbon source, nitrogen source, MgSO ₄ ·7H ₂ O and KH ₂ PO ₄ , the carbon source is glucose, and the nitrogen source is a mixture of peptone and potassium nitrate, The pH value of the medium is 5.5.

The beneficial effects of the present invention are:

In the process of liquid fermentation, in addition to mycelium proliferating in large quantities, various metabolites such as polysaccharides, polypeptides, alkaloids, polyphenols, inorganic magnesium, nucleic acids, amino acids and vitamins will be produced in the fermentation broth. A large amount of metabolites indicates high mycelium proliferation efficiency. After the accelerator is added in the present invention, the permeability of the cell membrane is changed by the accelerator, thereby increasing extracellular secondary metabolites. The accelerator can effectively promote the activity of glucoamylase, thereby increasing the conversion rate of pachymansin.

Eucommia ulmoides is a valuable nourishing medicinal material in China. The bark of Eucommia ulmoides has been used as medicine for more than two thousand years. In addition, resources are relatively scarce, so that a large number of eucommia bark has been peeled, resulting in the death of a large number of eucommia trees. Therefore, people study to use Eucommia leaves instead of Eucommia bark as medicine. Modern studies have proved that the medicinal active ingredients of Eucommia leaves are basically the same as those of Eucommia bark, and their medicinal functions are basically the same. Due to the large yield of Eucommia leaves, picking Eucommia leaves will not affect the growth of Eucommia trees. In order to make better use of Eucommia leaves resources, a lot of research has been done on Eucommia leaves in recent years. The results prove that Eucommia leaves and Eucommia bark have similar chemical components and Pharmacological effects have been recorded in the 2010 edition of the "Chinese Pharmacopoeia", but there are currently few marketed Chinese patent medicines using Eucommia leaves as raw materials, and vigorously developing new uses of Eucommia leaves has broad prospects. After years of research, the patent has developed a fermentation accelerator with eucommia leaf extract as the main raw material, combined with fatty acids, vegetable oils, and surfactants. The type and amount of the accelerator were obtained through screening, and the accelerator was determined through experimental optimization. The best combination provides the basis for the high-efficiency fermentation production of Poria cocos polysaccharide. Reduce costs and improve Poria cocos fermentation products at the same time.

In summary, the present invention utilizes liquid fermentation technology to produce Poria cocos polysaccharide, and optimizes the fermentation and process of the polysaccharide; liquid fermentation has the advantages of short fermentation cycle, high yield of active ingredients, and easy industrial production. Adding promoters is one of the important ways to increase the biosynthesis of fungal metabolites, through which the promoters can change the permeability of the cell membrane, thereby increasing extracellular secondary metabolites. Studies have shown that adding accelerators to fungal liquid fermentation media such as Ganoderma lucidum and Cordyceps militaris can increase the biosynthesis of polysaccharides, but there is no report on the selection and addition of accelerators for Poria cocos liquid fermentation. In this application, starting from the selection and addition amount of the accelerator, the accelerator composed of eucommia extract, fatty acid, vegetable oil, and surfactant was added to the Poria cocos fermentation medium, and the accelerator was optimized through experiments through investigation and screening of its type and concentration. Determine the best combination of accelerators, and successfully develop a high-efficiency polysaccharide fermentation accelerator, which provides the basis for the efficient fermentation and production of Poria cocos polysaccharides.

detailed description

A fermentation culture accelerator, which is composed of plant extract, fatty acid, vegetable oil and surfactant, the accelerator is added to the fermented product, and the plant extract is eucommia leaf extract, tomato juice or orange peel juice; The fatty acid is palmitic acid, stearic acid or oleic acid; the vegetable oil is peanut oil, olive oil or corn oil; the surfactant is Tween80, Span40 or PEG1000. 0.125-0.5% by mass of plant extract, 0.125-0.5% by mass of fatty acid, 0.125-0.5% by mass of vegetable oil and 0.125-0.5% by mass of surfactant are added to the fermented product.

Preferably, 0.5% by mass of plant extract, 0.5% by mass of fatty acid, 0.25% by mass of vegetable oil and 0.125% by mass of surfactant are added to the product to be fermented.

Preferably, 0.5% by mass of eucommia leaf extract, 0.5% by mass of palmitic acid, 0.25% by mass of olive oil and 0.125% by mass of PEG1000 are added to the fermented product.

A method for using a fermentation culture accelerator, comprising the steps of:

A. Addition of culture accelerator

Add accelerator to the original seed of Poria cocos to be fermented to make mixed stock solution;

B. Solid culture of bacteria

Put the mixed stock solution into the strain culture medium, place it in a constant temperature incubator and cultivate it at 26 ⁰ C, and the cultivation time is about 6-8 days;

C. Shake flask seed culture

Under a sterile environment, take seeds from the medium of step B, place them in a shaker for 26 ^° C shake flask culture, and the culture time is 7-14 days to obtain strains;

D. Liquid fermentation culture

Under a sterile environment, the strains were taken from the medium in step C, placed in a shaker for 26 ^° C shake flask culture, and the culture time was 5-6 days to obtain the final product.

The medium of the steps B, C and D is carbon source, nitrogen source, MgSO ₄ ·7H ₂ O and KH ₂ PO ₄ , the carbon source is one or more of glucose, fructose and sucrose, and the nitrogen The source is one or more of peptone and potassium nitrate, and the pH value of the medium is 5-6. In the steps C and D, a 250mL Erlenmeyer flask is selected for the shaker flask, the inoculum amount in the bottle is 50-100mL, and the rotation speed of the shaker flask is 60-210 rpm.

Preferably, the step C shake flask seed culture is divided into two levels, and the first level shake flask culture takes seeds from the medium of step B under a sterile environment, and places them in a shaker for 26°C shake flask culture ^at a speed of 150rpm The culture time is 7 days, and then the secondary shake flask culture is carried out, and the shake flask culture is carried out at 26 ⁰ C in a shaker, and the rotation speed is 150 rpm, and the culture time is 4 days to obtain the final product.

Preferably, the medium in the steps B, C and D is carbon source, nitrogen source, MgSO ₄ ·7H ₂ O and KH ₂ PO ₄ , the carbon source is glucose, and the nitrogen source is peptone and potassium nitrate, etc. The quality is mixed, and the pH value of the medium is 5.5.

The comparison experiment is as follows:

1. Strains to be fermented

Strains: Poria cocos HD _07-1 , Institute of Biology, Gansu Academy of Sciences.

2. Instruments and equipment

HYG-1 rotary constant temperature and speed regulating shaker cabinet (Shanghai Xinrui Automation Equipment Co., Ltd.); LDZS-2 low-speed automatic balancing centrifuge (Beijing Medical Centrifuge Factory); JYD-150 intelligent ultrasonic cell pulverizer (Shanghai Zhixin Instrument Co., Ltd.); 752 UV-Vis Spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd.): Electronic Balance (Shanghai Tianping Instrument Factory); PHS-3B Precision pH Meter (Shanghai Leici Instrument Factory); DHP-9272 Electric constant temperature incubator (Shanghai-J Heng Technology Co., Ltd.)

3. Comparison of fermentation effects

(l) Effect of fermentation temperature on Poria cocos liquid fermentation

Put 80mL liquid shake flask fermentation medium in a 250mL Erlenmeyer flask, and place it at 8 different temperatures of 20 ⁰ C, 22 ⁰ C, 24 ⁰ C, 26 ⁰ C, 28 ⁰ C, 30 ⁰ C, 34 ⁰ C Shake flask shaking culture was carried out under the following conditions, the amount of bacteria in each Erlenmeyer flask was measured, and the optimum temperature of Poria cocos liquid fermentation culture was determined to be ²⁶²⁶ °C.

(2) The influence of the rotating speed of shake flask on the liquid fermentation of Poria cocos

Put 80mL liquid shake flask fermentation medium in a 250mL Erlenmeyer flask at a culture temperature of 26°C, place it ^at 60rpm, 90rpm, 120rpm, 150rpm, 180rpm, and 210rpm for shaking culture in a shaker flask, and measure each The amount of bacteria in the triangular flask was determined to be 150 rpm for the optimal rotation speed of the liquid fermentation culture.

(3) Effect of nitrogen source type on Poria cocos liquid fermentation

In the basic fermentation medium, keeping other components unchanged, under the optimal fermentation conditions, only the type of nitrogen source is changed, and the following nitrogen sources are added separately in the fermentation culture bottle: peptone, potassium nitrate, peptone and potassium nitrate in equal proportions Mixed, cultured in a liquid shake flask, compared the amount of bacteria produced and the content of fermented polysaccharides (including exopolysaccharides and intracellular polysaccharides), and selected the best nitrogen source as a mixture of peptone and potassium nitrate in equal proportions.

(5) Effect of carbon source type on Poria cocos liquid fermentation

In the basic fermentation medium, keep other components unchanged, and only change the type of carbon source under the optimal fermentation conditions. The following three carbon sources are separately added to the fermentation culture bottle: glucose, sucrose and fructose, and shaken by liquid. Bottle culture, compare the amount of bacteria produced and the content of fermented polysaccharides (the determination of polysaccharides is the same as above), and choose the best carbon source as glucose.

4. Analysis and detection method

(l) Determination of bacterial mass

Take 100 mL of fermentation broth, pour it into a centrifuge tube, centrifuge at 4000 rpm for 15 min, dry at 100 ⁰ C to constant weight, weigh, and express the biomass by the amount of dry bacteria.

(2) Determination of residual glucose content in fermentation broth

The sugar content in the sample and the reaction between DNS and reducing sugar can be determined by colorimetry.

(3) Determination of polysaccharides (phenol-sulfuric acid method)

Determination of phenol-sulfuric acid standard curve method: take 1mL of diluted sample solution and add it to a 20mL test tube, then add 2mL of 5% phenol, 6mL of concentrated sulfuric acid, let it stand for 30min, measure its absorbance value at a wavelength of 490nm, compare it with the standard curve, and calculate the polysaccharide content.

(4) Optimizing Poria cocos liquid fermentation medium

On the basis of obtaining the most suitable Poria cocos liquid culture conditions, the nitrogen source type, carbon source type, pH, and liquid volume (mL/250mL) were selected as the main influencing conditions, and the optimum Poria cocos liquid fermentation medium was determined through multiple experiments. The best cultivation formula.

Table 1 Experimental design and results

Through the screening test in Table 1, the prescription obtained in Test 7 is the best prescription, that is, the nitrogen source is a mixture of peptone and potassium nitrate in equal proportions, the carbon source is glucose, pH5.5, and measured under the conditions of 80 mL of shake bottle liquid The highest total sugar content is 28.98g/L.

5. Effect comparison of accelerators

Plant extracts: Eucommia leaf extract, tomato juice or orange peel juice.

Fatty acids: Palmitic, Stearic or Oleic.

Vegetable oils: peanut, olive, or corn oil.

Surfactant: Tween80, Span40 or PEG100.

In the comparison test, the Eucommia ulmoides leaf extract was selected from the product of Xi Anao Biotechnology Co., Ltd., and the tomato juice and orange peel juice were freshly squeezed. Palmitic acid is selected from Nanjing Jiaguan Chemical Co., Ltd., stearic acid is selected from Zhengzhou Chuangmei Chemical Products Co., Ltd., and oleic acid is selected from Zhengzhou Dongda Chemical Products Co., Ltd. products. The peanut oil is selected from the product of Shenzhen Chuanhong Food Co., Ltd., the olive oil is selected from the product of Gansu Longnan Xiangyu Oil Olive Development Co., Ltd., and the corn oil is selected from the product of Hebei Xingbo Pharmaceutical Co., Ltd. Sorbitan monooleate polyoxyethylene ether Tween80 is selected from Jiangsu Haian Petrochemical Factory, sorbitan palmitic acid monoester Span40 is selected from Guangzhou Runhua Fine Chemical Factory, polyethylene glycol PEG1000 is selected from Jiangsu Haian Petrochemical plant products.

Accelerator formulation screening

The added mass fraction is all: 0.25%, and the formulation screening of plant extracts, fatty acids, vegetable oils, and surfactants is carried out. The basal medium without any promoter was used as the control group.

Table 2 Screening tests and results of accelerator types

Through the screening test in Table 2, the prescription obtained in Test 6 is the best prescription, that is, the plant extract is Eucommia leaf extract, the fatty acid is palmitic acid, the vegetable oil is olive oil, and the surfactant is PEG1000. The highest total sugar content measured under the conditions was 37.88g/L.

Screening of Accelerator Concentration

Based on the sixth group, prepare by equal weight, set the added concentration: 0.125%, 0.25%, 0.5%, and use the basal medium without adding accelerator as the control group.

Table 3 The screening and results of accelerator addition concentration

Through the screening test in Table 3, the prescription obtained in Test 4 is the best prescription, that is, the concentration of eucommia leaf extract is 0.50%, the concentration of palmitic acid is 0.50%, the concentration of olive oil is 0.25%, and the concentration of PEG1000 is 0.125%. The highest total sugar content measured under the above conditions was 41.84g/L.

6. Results and discussion

The medium not only provides the necessary nutrients for the growth, reproduction, metabolism and product synthesis of microorganisms, but also provides the necessary environmental conditions for their growth. For different microorganisms with different growth stages, different metabolites, etc., the medium used is different. Because the ratio and composition of the medium are suitable or not, it will have a great impact on the growth of microorganisms, the refinement of products, the choice of solvents and the quality of finished products. The components of the medium generally include carbon source, nitrogen source, inorganic salts, growth factors and water. Carbon source and nitrogen source, as the basis of fermentation medium, are important components of mycelium cell structure. The main raw material for various metabolites and intracellular storage substances; it is also an important substance that constitutes mycelium amino acids, proteins, and nucleic acids. This patent finds through research that the results show that glucose should be selected as the carbon source, and other types of carbon sources are not ideal for the amount of cells of Poria cocos and the yield of polysaccharides fermented by shake flasks. At the same time, the culture temperature, pH, liquid volume, shaker speed, and inoculum volume have a great influence on the growth of mycelia. If the inoculation amount is too large, the mycelium will grow vigorously and proliferate too fast in the early stage of fermentation, which will increase the consumption of nutrients in the medium, thus affecting the growth of the bacteria in the later stage of fermentation, which is not conducive to the acquisition of metabolites. Too few mycelium clusters will prolong the delay period, thereby prolonging the fermentation cycle and increasing production costs to a certain extent. The results of this experiment can be seen in Table 1.

After years of research, this patent has developed a fermentation accelerator that uses Eucommia extract as the main raw material and is equipped with fatty acids, vegetable oils, and surfactants. The type and amount of the accelerator were obtained through screening, and the optimum value of the accelerator was determined through experimental optimization. The best combination, the results are shown in Table 2 and Table 3, which provides the basis for the efficient fermentation and production of Poria cocos polysaccharide. To sum up, when we study the fermentation of pachyrhin, we explore the effect of some compound additives on the growth of fungi, so as to screen and obtain suitable fermentation accelerators, which is very effective for increasing yield and reducing production costs.

Claims (9)

1. A fermenting culture accelerant, it is characterized in that by plant extract, fatty acid, vegetable oil and tensio-active agent form accelerant, add accelerant in the fermented product, described plant extract is eucommia leaf extract, tomato juice or orange peel juice; the fatty acid is palmitic acid, stearic acid or oleic acid; the vegetable oil is peanut oil, olive oil or corn oil; the surfactant is Tween80, Span40 or PEG1000. 2. A kind of fermentation culture accelerant according to claim 1, it is characterized in that adding mass percent 0.125-0.5% plant extract, mass percent 0.125-0.5% fatty acid, mass percent 0.125-0.5% vegetable oil in the said to-be-fermented product and a mass percentage of 0.125-0.5% surfactant, the fermented product is strain Poria HD _07-1 . 3. A kind of fermentation culture accelerant according to claim 2, it is characterized in that adding mass percentage 0.5% plant extract, mass percentage 0.5% fatty acid, mass percentage 0.25% vegetable oil and mass percentage 0.125% surface active agent. 4. according to claim 3, a kind of fermentation culture accelerant is characterized in that adding mass percent 0.5% Eucommia leaf extract, mass percent 0.5% palmitic acid, mass percent 0.25% olive oil and mass percent in the described to-be-fermented product 0.125% PEG1000. 5. according to the using method of a kind of fermentation culture accelerant described in any one of claim 1 to 4, it is characterized in that comprising the steps: A. Addition of culture accelerator Add accelerator to the original seed of Poria cocos to be fermented to make mixed stock solution; B. Solid culture of bacteria Put the mixed stock solution into the strain culture medium, place it in a constant temperature incubator and cultivate it ^at 26°C, and the cultivation time is about 6-8 days; C. Shake flask seed culture Under a sterile environment, take seeds from the medium of step B, place them in a shaker for 26 ^° C shake flask culture, and the culture time is 7-14 days to obtain strains; D. Liquid fermentation culture Under a sterile environment, the strains were taken from the medium in step C, placed in a shaker for 26 ^° C shake flask culture, and the culture time was 5-6 days to obtain the final product. 6. The method for using a fermentation culture accelerator according to claim 5, characterized in that the medium in steps B, C and D is carbon source, nitrogen source, MgSO ₄ 7H ₂ O and KH ₂ PO _4. The carbon source is one or more of glucose, fructose and sucrose, the nitrogen source is one or more of peptone and potassium nitrate, and the pH value of the culture medium is 5-6. 7. the using method of a kind of fermentation culture accelerant according to claim 6, it is characterized in that shake flask selects 250mL triangular flask for use in the described step C and D, inoculum size 50-100mL in the bottle, shake flask rotating speed 60-210 rpm. 8. the using method of a kind of fermentation culture accelerant according to claim 5, it is characterized in that described step C shaking flask seed culture is divided into two grades, and the first grade shake flask is cultivated from step B culture medium under aseptic environment The seeds were taken from the medium and placed in a shaker for 26 ⁰ C shake flask culture with a rotation speed of 150 rpm for a period of ⁷ days. 4d to get the final product. 9. The method for using a fermentation culture accelerator according to claim 5, characterized in that the medium in steps B, C and D is carbon source, nitrogen source, MgSO ₄ 7H ₂ O and KH ₂ PO _4. The carbon source is glucose, the nitrogen source is a mixture of equal masses of peptone and potassium nitrate, and the pH value of the culture medium is 5.5.