Fermentation method of yeast for Streptomyces aureus fermentation, fermentation product and application thereof

Abstract

The present invention provides a fermentation method of yeast for Streptomyces aureus fermentation, a fermentation product and an application thereof. The present invention provides a fermentation method of yeast for Streptomyces aureus fermentation, which helps to increase yeast yield and reduce the fermentation cost of Streptomyces aureus. At the same time, the present invention also provides a method for processing yeast fermentation products, by centrifugally concentrating the yeast fermentation products, directly using the yeast milk obtained by centrifugation concentration as a nitrogen source for the fermentation of Streptomyces aureus, and using the supernatant produced by centrifugation concentration as a nitrogen source or a feed culture medium solvent, thereby solving the problems of complicated post-processing steps, high energy consumption and wastewater treatment of yeast fermentation products, reducing the fermentation cost of Streptomyces aureus and alleviating environmental pressure.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a fermentation method of yeast for Streptomyces aureus fermentation, a fermentation product and application thereof. Background Art

[0002] Yeast is a unicellular microorganism that is rich in nutrients, including protein, minerals, B vitamins, etc. Yeast and yeast culture are widely used in long-term fermentation processes such as actinomycetes, Bacillus subtilis and molds due to their rich nutritional composition. Streptomyces aureofaciens ) is a species of Streptomyces ( Streptomyces ) soil microorganisms that produce a variety of antibiotics, the most famous of which is chlortetracycline, also known as aureomycin, a broad-spectrum antibiotic that is active against a variety of Gram-positive and Gram-negative bacteria and some protozoa.

[0003] Yeast powder is used as an organic nitrogen source in the fermentation process of Streptomyces aureus or other antibiotic-producing strains. On the one hand, yeast powder can promote the growth of bacteria and promote the proliferation of bacteria. On the other hand, the addition of yeast powder can appropriately reduce the proportion of nitrogen sources such as soybean cake powder and corn pulp, increase the stability of nitrogen source supply, improve raw material utilization, and greatly reduce the pressure of downstream extraction and environmental protection treatment.

[0004] However, the existing yeast fermentation technology has the problem of low yield. Yeast powder is an indispensable raw material in the fermentation process of Streptomyces aureus, and its use cost is relatively high. How to increase the yeast fermentation yield and reduce the cost of Streptomyces aureus fermentation is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] The invention provides a fermentation method of yeast for Streptomyces aureus fermentation, which is used for improving yeast fermentation yield and reducing Streptomyces aureus fermentation cost.

[0006] In a first aspect, the present invention provides a fermentation method of yeast for Streptomyces aureus fermentation, comprising:

[0007] S1, inoculating yeast cells into a primary seed culture solution for primary culture to obtain a primary seed solution; the primary seed culture solution comprises 50-70 g / L glucose, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L diammonium phosphate, 2-5 g / L triammonium citrate and 3-6 g / L yeast extract;

[0008] S2, inoculating the primary seed solution into a primary seed culture solution for primary culture to obtain a primary seed solution; the primary seed culture solution comprises 50-70 g / L of glucose, 0.7-1.1 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract, 1.5-2.0 g / L of ammonium sulfate and 0.5-1.0 g / L of magnesium sulfate;

[0009] S3, inoculating the primary seed solution into a secondary seed culture solution for secondary culture to obtain a secondary seed solution; the secondary seed culture solution comprises 50-70 g / L of glucose, 0.5-1.0 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract powder, 1.5-1.8 g / L of ammonium sulfate, 0.5-1.0 g / L of magnesium sulfate and 0.2-1.0 g / L of a defoaming agent;

[0010] S4. Inoculating the secondary seed liquid into a fermentation medium for fermentation to obtain a yeast fermentation product; the fermentation medium comprises 80-120 g / L of glucose, 2.6-3.8 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract powder, 4.8-6.5 g / L of ammonium sulfate, 7.3-8.5 g / L of magnesium sulfate and 0.1-0.5 g / L of a defoaming agent.

[0011] The fermentation method as described above, wherein the yeast is Candida magnolii ( Candida magnoliae ), the Candida magnolii ( Candida magnoliae ) is numbered CGMCC2.1919 in the China General Microbiological Culture Collection Center. The use of the magnolia candida helps to increase the yield of yeast fermentation products and reduce the fermentation cost of Streptomyces aureus.

[0012] In the fermentation method as described above, the seed culture medium and fermentation culture medium used in the yeast fermentation process include carbon sources and nitrogen sources necessary for yeast growth, and in order to avoid the Maillard reaction during the sterilization process of the carbon source and the nitrogen source, the carbon source and the nitrogen source are prepared and sterilized separately in the present invention, and then the two are mixed to obtain the final culture medium.

[0013] In the fermentation method as described above, in S1, the concentration of glucose in the primary seed culture solution can be 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L or a range consisting of any two thereof; the concentration of tryptone can be 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, 1.0 g / L, 1.05 g / L, 1.1 g / L, 1.15 g / L, 1.2 g / L or any range between two of them; the concentration of diammonium hydrogen phosphate can be 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, 1.0 g / L, 1.05 g / L, 1.1 g / L, 1.15 g / L, 1.2 g / L or any range between two of them; the concentration of triammonium citrate can be 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L or any range between two of them; the concentration of yeast extract can be 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L or any range between two of them.

[0014] In the fermentation method as described above, in S2, the concentration of glucose in the primary seed culture solution can be 50g / L, 51g / L, 52g / L, 53g / L, 54g / L, 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67g / L, 68g / L, 69g / L, 70g / L or a range consisting of any two thereof; the concentration of potassium dihydrogen phosphate can be 0.7g / L, 0.75g / L, 0.8g / L, 0.85g / L, 0.9g / L, 0.95g / L, 1.0g / L, The concentration of yeast extract powder can be 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, or any range therebetween. The concentration of ammonium sulfate can be 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, or any range therebetween. The concentration of magnesium sulfate can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, or any range therebetween.

[0015] In the fermentation method as described above, in S3, the concentration of glucose in the secondary seed culture solution can be 50g / L, 51g / L, 52g / L, 53g / L, 54g / L, 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67g / L, 68g / L, 69g / L, 70g / L or any combination thereof. The concentration of potassium dihydrogen phosphate can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L or any two thereof; the concentration of yeast extract can be 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L or any two thereof; the concentration of ammonium sulfate can be 1.5 g / L, 1.55 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L or any two thereof; g / L, 1.6 g / L, 1.65 g / L, 1.7 g / L, 1.75 g / L, 1.8 g / L or within the range of any two thereof; the concentration of magnesium sulfate can be specifically 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L or within the range of any two therein; the concentration of the defoaming agent can be specifically 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L or within the range of any two therein.

[0016] In the fermentation method as described above, in S4, the concentration of glucose in the fermentation culture medium can be 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L or a range consisting of any two thereof; the concentration of potassium dihydrogen phosphate can be 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.8 g / L or any range between them; the concentration of yeast extract powder can be 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L or any range between them; the concentration of ammonium sulfate can be 4.8 g / L, 4.9 g / L, 5.0 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6.0 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L or within the range of any two thereof; the concentration of magnesium sulfate can be specifically 7.3g / L, 7.4g / L, 7.5g / L, 7.6g / L, 7.7g / L, 7.8g / L, 7.9g / L, 8.0g / L, 8.1 g / L, 8.2 g / L, 8.3 g / L, 8.4 g / L, 8.5 g / L or within the range of any two thereof; the concentration of the defoaming agent can be specifically 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L or within the range of any two thereof.

[0017] In the fermentation method as described above, in S2, the inoculation amount of the primary seed solution is 0.5-1.0 vol%, the temperature of the primary culture treatment is 29.5-30.5°C, the pH is 5.0-5.5, the ventilation volume is 0.5-0.8 vvm, and the culture time is 20-24 hours.

[0018] In the fermentation method as described above, in S3, the inoculation amount of the primary seed solution is 6-9 vol%, the temperature of the secondary culture treatment is 29.5-30.5°C, the pH is 5.0-5.5, the ventilation volume is 0.5-0.8 vvm, and the culture time is 8-12 hours.

[0019] In the fermentation method as described above, in S4, the inoculation amount of the secondary seed liquid is 10-15 vol%, the fermentation temperature is 29.5-30.5°C, the pH is 5.0-5.5, the sugar content is 5-7%, the ventilation volume is 0.8-1.0 vvm, and the culture time is 36-48 hours.

[0020] In the fermentation method as described above, the sugar content refers to the concentration of soluble sugar in the fermentation broth, which can be obtained by sampling the fermentation broth and testing it with a sugar meter.

[0021] In the fermentation method described above, in S4, a feed culture solution is added during the fermentation process to adjust the sugar content of the fermentation culture solution and supplement the nutrients required for yeast growth. The concentration of glucose in the feed culture solution can be 400 g / L, 410 g / L, 420 g / L, 430 g / L, 440 g / L, 450 g / L, 460 g / L, 470 g / L, 480 g / L, 490 g / L, 500 g / L, 510 g / L, 520 g / L, 530 g / L, 540 g / L, 550 g / L, 560 g / L, 570 g / L, 580 g / L, 590 g / L, 600 g / L, 610 g / L, 620 g / L, 630 g / L, 640 g / L, g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6.0 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L, or a range between the concentrations of the yeast extract powder. The concentration of ammonium sulfate can be 5.5g / L, 5.6g / L, 5.7g / L, 5.8g / L, 5.9g / L, 6.0g / L, 6.1g / L, 6.2g / L, 6.3g / L, 6.4g / L, 6.5g / L or within the range of any two therein; the concentration of magnesium sulfate can be 6.2g / L, 6.3g / L, 6.4g / L, 6.5g / L, 6.6g / L, 7.0g / L or within the range of any two therein; the concentration of the defoaming agent can be 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L or within the range of any two therein.

[0022] The types of nitrogen sources used in the seed culture solution and the fermentation culture solution provided by the present invention are basically consistent with the types of nitrogen sources used in the fermentation of Streptomyces aureus, which not only helps to improve the fermentation yield of yeast, but also allows the yeast fermentation product to be directly added to the fermentation culture solution of Streptomyces aureus as a nitrogen source, saving the post-processing steps of the yeast fermentation product, and avoiding the influence of yeast fermentation products obtained from different nitrogen sources directly used as nitrogen sources on the fermentation process of Streptomyces aureus.

[0023] In a second aspect, the present invention provides a yeast fermentation product obtained by fermentation according to the above fermentation method.

[0024] In a third aspect, the present invention provides the use of the yeast fermentation product in the following A1)-A2),

[0025] A1) Application in microbial fermentation;

[0026] A2) Application in antibiotic production.

[0027] In the application as described above, the microorganism may specifically be Streptomyces; further, the microorganism is Streptomyces aureus.

[0028] In the application as described above, the antibiotic may specifically be chlortetracycline, oxytetracycline, neomycin sulfate, daptomycin, vancomycin, etc.; further, the antibiotic is chlortetracycline.

[0029] In a fourth aspect, the present invention provides a fermentation method of Streptomyces aureus, comprising: adding the yeast fermentation product during the fermentation process of Streptomyces aureus.

[0030] The fermentation method as described above further comprises: centrifuging and concentrating the yeast fermentation product to obtain yeast milk and supernatant, using the yeast milk as a nitrogen source for the fermentation culture liquid of Streptomyces aureus, and using the supernatant as a nitrogen source for the fermentation culture liquid of Streptomyces aureus or a solvent for the feed culture liquid.

[0031] The yeast milk obtained by centrifugation and concentration of the yeast fermentation product can be directly used as a nitrogen source for the fermentation of Streptomyces aureus, which not only does not affect the fermentation of Streptomyces aureus and the yield of chlortetracycline, but also saves the yeast separation and drying steps, saving energy consumption. The yeast supernatant produced during the centrifugation and concentration of the yeast fermentation product contains a large amount of nutrients such as nitrogen source, carbon source and trace elements. If it is directly discharged, there is an environmental problem. Therefore, the yeast supernatant can be used as a nitrogen source or a solvent for the fermentation culture medium of Streptomyces aureus, avoiding the generation of wastewater and reducing the pressure of environmental protection treatment.

[0032] In a fifth aspect, the present invention provides a method for producing chlortetracycline, comprising: fermenting Streptomyces aureus with the yeast fermentation product, and obtaining chlortetracycline from the fermentation product of Streptomyces aureus.

[0033] The method described above and the extraction process of chlortetracycline can be carried out according to conventional technical means in the art.

[0034] The present invention provides a yeast fermentation method for Streptomyces aureus fermentation, which helps to increase yeast yield and reduce the fermentation cost of Streptomyces aureus. The present invention also provides a method for processing yeast fermentation products, which centrifugally concentrates the yeast fermentation products, directly uses the yeast milk obtained by centrifugation as a nitrogen source for the fermentation of Streptomyces aureus, and uses the concentrated supernatant as a nitrogen source or a feed culture solvent, thereby solving the problems of complicated post-processing steps, high energy consumption and wastewater treatment of yeast fermentation products, reducing the fermentation cost of Streptomyces aureus, and alleviating environmental pressure. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0037] The Candida magnoliae used in the following examples was purchased from the China General Microbiological Culture Collection Center. The number of the Candida magnoliae in the China General Microbiological Culture Collection Center is CGMCC 2.1919.

[0038] The liquid sugar (containing 60% glucose) used in the following examples was sourced from Inner Mongolia Jinhe Starch Co., Ltd.; diammonium hydrogen phosphate was sourced from Tianjin Shengao Chemical Reagent Co., Ltd.; the defoamer (polydimethylsiloxane emulsion) was sourced from Nanjing Golden Key Biotechnology Co., Ltd.; the yeast extract powder (FM916) was sourced from Angel Yeast Co., Ltd.; magnesium sulfate was sourced from Jiangsu Kolondo Food Ingredients Co., Ltd.; ammonium sulfate was sourced from Shandong Dongju Chemical Co., Ltd.; potassium dihydrogen phosphate was sourced from Tangshan Fengnan District Chengfeng Technology Co., Ltd.; ammonium citrate was sourced from Jiangsu Shayingxi Industrial Co., Ltd.; and glucose was sourced from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.

[0039] Example 1

[0040] This embodiment provides a fermentation method of Candida magnoliae, comprising the following steps:

[0041] 1. Preparation of culture medium

[0042] 1.1. Preparation of primary seed culture medium

[0043] Glucose is used as a carbon source, glucose is mixed with tap water to obtain 0.5L of a primary carbon source culture solution with a natural pH value, and the solution is sterilized with high-pressure steam at 115°C for 15-20min before use; trypsin, diammonium phosphate, triammonium citrate, yeast extract powder and tap water are mixed to obtain 1.5L of a primary nitrogen source culture solution with a natural pH value, and the solution is sterilized with high-pressure steam at 115°C for 15-20min before use; the primary carbon source culture solution and the primary nitrogen source culture solution are combined when they are cooled to below 40°C, and the mixture is evenly mixed to obtain a primary seed culture solution, including 60g / L glucose, 1g / L trypsin, 1g / L diammonium phosphate, 3g / L triammonium citrate and 5g / L yeast extract powder.

[0044] 1.2. Preparation of primary seed culture medium

[0045] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 200 L of a primary carbon source culture solution with a natural pH, and sterilized by high-pressure steam at 115°C for 15-20 min before use; ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder and tap water are mixed to obtain 100 L of a primary nitrogen source culture solution with a natural pH, and sterilized by high-pressure steam at 115°C for 15-20 min before use; the primary carbon source culture solution and the primary nitrogen source culture solution are combined when they are cooled to below 40°C, and mixed evenly to obtain a primary seed culture solution, including 65 g / L glucose, 1.8 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate and 5 g / L yeast extract powder.

[0046] 1.3. Preparation of secondary seed culture medium

[0047] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 2000L of a secondary carbon source culture solution with a natural pH, and sterilized by high-pressure steam at 115°C for 15-20min for use; ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder, and a defoamer are mixed with tap water to obtain 1000L of a secondary nitrogen source culture solution with a natural pH, and sterilized by high-pressure steam at 115°C for 15-20min for use; the secondary carbon source culture solution and the secondary nitrogen source culture solution are combined when they are cooled to below 40°C, and mixed evenly to obtain a secondary seed culture solution, including 65g / L glucose, 1.8g / L ammonium sulfate, 1g / L potassium dihydrogen phosphate, 0.7g / L magnesium sulfate, 5g / L yeast extract powder, and 0.2g / L defoamer.

[0048] 1.4. Preparation of fermentation medium

[0049] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 15000L of fermentation carbon source culture solution with natural pH, and sterilized by high-pressure steam at 115°C for 15-20min before use; ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder, and defoaming agent are mixed with tap water to obtain 5000L of fermentation nitrogen source culture solution with natural pH, and sterilized by high-pressure steam at 115°C for 15-20min before use; when the fermentation carbon source culture solution and the fermentation nitrogen source culture solution are cooled to below 40°C, they are combined and mixed evenly to obtain a fermentation culture solution, including 110g / L glucose, 6.3g / L ammonium sulfate, 3.2g / L potassium dihydrogen phosphate, 8g / L magnesium sulfate, 5g / L yeast extract powder, and 0.2g / L defoaming agent.

[0050] 1.5. Preparation of fed-batch culture medium

[0051] Liquid sugar (containing 60% glucose) was used as a carbon source, and the liquid sugar was mixed with tap water to obtain 4500 L of a feed carbon source culture solution containing natural pH, and sterilized by high-pressure steam at 115°C for 15-20 min for use; ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder, and a defoaming agent were mixed with tap water to obtain 1500 L of a feed nitrogen source culture solution containing natural pH, and sterilized by high-pressure steam at 115°C for 15-20 min for use; when the feed carbon source culture solution and the feed nitrogen source culture solution were cooled to below 40°C, they were combined and mixed evenly to obtain a feed culture solution including 480 g / L glucose, 6.0 g / L ammonium sulfate, 4.0 g / L potassium dihydrogen phosphate, 7.0 g / L magnesium sulfate, 6.0 g / L yeast extract powder, and 0.4 g / L defoaming agent.

[0052] 2. Fermentation culture of Magnolia candida

[0053] The Candida magnoliae yeast was inoculated into 2 L of primary seed culture solution and cultured in a shake flask for 18-24 hours to obtain a primary seed solution;

[0054] The primary seed liquid was inoculated into the primary seed culture liquid at an inoculum rate of 0.66 vol% for primary culture, stirring was turned on during the whole process, the temperature was controlled at 30.0±0.5°C, the pH value was controlled to 5.3-5.5 with ammonia water, the ventilation rate was 0.6 vvm, and the culture was carried out for 24 hours to obtain the primary seed liquid, and the wet weight of yeast in the primary seed liquid was 64.2 g / L;

[0055] The first-level seed liquid was inoculated into the second-level seed culture liquid at an inoculum rate of 9.1 vol% for second-level culture under the same culture conditions as the first-level culture conditions for 11 hours to obtain the second-level seed liquid, in which the wet weight of yeast in the second-level seed liquid was 137.2 g / L;

[0056] The secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 14.2 vol% for fermentation. Stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5°C, the pH value was controlled to be between 5.0-5.5 with ammonia water, the sugar content of the fermentation medium was controlled to be 4.5%-5.5% by adding feed culture medium, the ventilation volume was 1.0 vvm, and the culture was carried out for 44 hours. After the culture was completed, a yeast fermentation liquid was obtained.

[0057] The yeast fermentation liquid was filtered, and the bacteria were collected and weighed. The wet weight of the yeast was 442.7 g / L, and the dry matter content after drying was 145.3 g / L. The fermentation liquid was a milky white liquid.

[0058] Example 2

[0059] This embodiment provides a fermentation method of Candida magnoliae, comprising the following steps:

[0060] 1. Preparation of culture medium

[0061] The preparation methods of the primary seed solution, the first-level seed culture solution, the second-level seed culture solution and the feed culture solution in this embodiment are the same as those in Example 1. The preparation of the fermentation medium includes: using liquid sugar (containing 60% glucose) as a carbon source, mixing the liquid sugar with tap water to obtain 15,000 L of a fermentation carbon source culture solution with a natural pH, and sterilizing it with high pressure steam at 115° C. for 15-20 min for use; mixing ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder, and a defoamer with tap water to obtain 5,000 L of a fermentation nitrogen source culture solution with a natural pH, and sterilizing it with high pressure steam at 115° C. for 15-20 min for use; combining the fermentation carbon source culture solution and the fermentation nitrogen source culture solution when they are cooled to below 40° C., and mixing them uniformly to obtain a fermentation culture solution comprising 100 g / L glucose, 6.1 g / L ammonium sulfate, 3.2 g / L potassium dihydrogen phosphate, 8 g / L magnesium sulfate, 5 g / L yeast extract powder, and 0.2 g / L defoamer.

[0062] 2. Fermentation culture of Magnolia candida

[0063] The primary seed solution provided in Example 1 was inoculated into the primary seed culture solution at an inoculum rate of 0.66% for primary culture, stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5°C, the pH value was controlled to 5.3-5.5 with ammonia water, the ventilation rate was 0.6vvm, and the culture was carried out for 24 hours to obtain the primary seed solution, and the wet weight of yeast in the primary seed solution was 88.3g / L;

[0064] The first-level seed liquid was inoculated into the second-level seed culture liquid at an inoculum amount of 9.1 vol% for second-level culture under the same culture conditions as the first-level culture conditions for 9 hours to obtain the second-level seed liquid, in which the wet weight of yeast in the second-level seed liquid was 141.6 g / L;

[0065] The secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 14.2 vol% for fermentation, stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5℃, the pH value was controlled between 5.0-5.5 with ammonia water, the sugar content of the fermentation medium was controlled to 4.5%-5.5% by adding feed culture medium, the ventilation volume was 0.8vvm, and the culture was carried out for 46 hours. After the culture was completed, the yeast fermentation liquid was obtained. The yeast fermentation liquid was filtered, the bacteria were collected and weighed, the wet weight of the yeast was 410.0g / L, the dry matter content after drying was 148.6g / L, and the fermentation liquid was milky white liquid.

[0066] Example 3

[0067] This embodiment provides a fermentation method of Candida magnoliae, comprising the following steps:

[0068] 1. Preparation of culture medium

[0069] The preparation methods of the primary seed culture solution, the first-level seed culture solution, the second-level seed culture solution and the feed culture solution in this embodiment are the same as those in Example 1. The preparation of the fermentation medium includes: using liquid sugar (containing 60% glucose) as a carbon source, mixing the liquid sugar with tap water to obtain 15,000 L of a fermentation carbon source culture solution with a natural pH, and sterilizing it with high pressure steam at 115° C. for 15-20 min for use; mixing ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, yeast extract powder, and a defoaming agent with tap water to obtain 5,000 L of a fermentation nitrogen source culture solution with a natural pH, and sterilizing it with high pressure steam at 115° C. for 15-20 min for use; combining the fermentation carbon source culture solution and the fermentation nitrogen source culture solution when they are cooled to below 40° C., and mixing them uniformly to obtain a fermentation culture solution comprising 100 g / L glucose, 6.3 g / L ammonium sulfate, 3.2 g / L potassium dihydrogen phosphate, 7.6 g / L magnesium sulfate, 5 g / L yeast extract powder, and 0.2 g / L defoaming agent.

[0070] 2. Fermentation culture of Magnolia candida

[0071] The primary seed solution provided in Example 1 was inoculated into the primary seed culture solution at an inoculum rate of 0.66 vol% for primary culture, stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5° C., the pH value was controlled to 5.3-5.5 with ammonia water, the ventilation rate was 0.6 vvm, and the culture was carried out for 24 hours to obtain the primary seed solution, and the wet weight of yeast in the primary seed solution was 94.0 g / L;

[0072] The first-level seed liquid was inoculated into the second-level seed culture liquid at an inoculum rate of 9.1% for second-level culture under the same culture conditions as the first-level culture conditions for 9 hours to obtain the second-level seed liquid, in which the wet weight of yeast in the second-level seed liquid was 152.8 g / L;

[0073] The secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 14.2 vol% for fermentation, stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5℃, the pH value was controlled between 5.0-5.5 with ammonia water, the sugar content of the fermentation medium was controlled to 4.5%-5.5% by adding feed culture medium, the ventilation volume was 1.0 vvm, and the culture was carried out for 46 hours. After the culture was completed, the yeast fermentation liquid was obtained. The yeast fermentation liquid was filtered, the bacteria were collected and weighed, the wet weight of the yeast was 422.0 g / L, the dry matter content after drying was 157.0 g / L, and the fermentation liquid was milky white liquid.

[0074] Comparative Example 1

[0075] The fermentation method of Candida magnoliae provided in this comparative example comprises the following steps:

[0076] 1. Preparation of culture medium

[0077] 1.1. Preparation of primary seed culture medium

[0078] The same as the primary seed culture solution in Example 1.

[0079] 1.2. Preparation of primary seed culture medium

[0080] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 200 L of a primary carbon source culture solution with a natural pH, and sterilized with high-pressure steam at 115°C for 15-20 min for use; triammonium citrate, diammonium hydrogen phosphate, peptone, corn steep liquor, yeast extract powder and tap water are mixed to obtain 100 L of a primary nitrogen source culture solution with a natural pH, and sterilized with high-pressure steam at 115°C for 15-20 min for use; when the primary carbon source culture solution and the primary nitrogen source culture solution are cooled to below 40°C, they are combined and mixed evenly to obtain a primary seed culture solution, including 60 g / L glucose, 2 g / L triammonium citrate, 2 g / L diammonium hydrogen phosphate, 1 g / L tryptone, 4.4 g / L corn steep liquor, and 5 g / L yeast extract powder.

[0081] 1.3. Preparation of secondary seed culture medium

[0082] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 2000L of a secondary carbon source culture solution with a natural pH, and the solution is sterilized by high-pressure steam at 115°C for 15-20min for use; triammonium citrate, diammonium hydrogen phosphate, tryptone, corn steep liquor, yeast extract powder and tap water are mixed to obtain 1000L of a secondary nitrogen source culture solution with a natural pH, and the solution is sterilized by high-pressure steam at 115°C for 15-20min for use; when the secondary carbon source culture solution and the secondary nitrogen source culture solution are cooled to below 40°C, they are combined and mixed evenly to obtain a secondary seed culture solution, including 60g / L glucose, 2g / L triammonium citrate, 2g / L diammonium hydrogen phosphate, 1g / L tryptone, 4.4g / L corn steep liquor, 5g / L yeast extract powder, and 0.2g / L defoaming agent.

[0083] 1.4. Preparation of fermentation medium

[0084] Liquid sugar (containing 60% glucose) is used as a carbon source, and the liquid sugar is mixed with tap water to obtain 15000L of fermentation carbon source culture solution with natural pH, and the solution is sterilized by high-pressure steam at 115°C for 15-20min for use; triammonium citrate, diammonium hydrogen phosphate, tryptone, corn steep liquor, yeast extract powder and tap water are mixed to obtain 5000L of fermentation nitrogen source culture solution with natural pH, and the solution is sterilized by high-pressure steam at 115°C for 15-20min for use; when the fermentation carbon source culture solution and the fermentation nitrogen source culture solution are cooled to below 40°C, they are combined and mixed evenly to obtain a fermentation culture solution, including 110g / L glucose, 3g / L triammonium citrate, 3g / L diammonium hydrogen phosphate, 1g / L tryptone, 4.4g / L corn steep liquor, 5g / L yeast extract powder, and 0.2g / L defoaming agent.

[0085] 1.5. Preparation of fed-batch culture medium

[0086] Liquid sugar (containing 60% glucose) was used as a carbon source, and the liquid sugar was mixed with tap water to obtain 4500 L of a feed carbon source culture solution with a natural pH value, and the solution was sterilized with high pressure steam at 115°C for 15-20 min for later use; triammonium citrate, diammonium hydrogen phosphate, corn steep liquor, yeast extract powder and tap water were mixed to obtain 1500 L of a feed nitrogen source culture solution with a natural pH value, and the solution was sterilized with high pressure steam at 115°C for 15-20 min for later use; when the feed carbon source culture solution and the feed nitrogen source culture solution were cooled to below 40°C, they were combined and mixed evenly to obtain a feed culture solution comprising 480 g / L glucose, 3 g / L ammonium citrate, 3 g / L diammonium hydrogen phosphate, 5 g / L corn steep liquor, 6 g / L yeast extract powder, and 0.8 g / L defoaming agent.

[0087] 2. Fermentation culture of Magnolia candida

[0088] The Candida magnoliae yeast was inoculated into 2 L of primary seed culture solution and cultured in a shake flask for 18-24 hours to obtain a primary seed solution;

[0089] The primary seed liquid was inoculated into the primary seed culture liquid for primary culture, stirring was started during the whole process, the temperature was controlled at 30.0±0.5°C, the pH value was controlled to 5.3-5.5 with ammonia water, the ventilation volume was 0.6vvm, and cultured for 24 hours to obtain the primary seed liquid, and the wet weight of yeast in the primary seed liquid was 80.0g / L;

[0090] The first-level seed liquid was inoculated into the second-level seed culture liquid at an inoculum amount of 9.1 vol% for second-level culture under the same culture conditions as the first-level culture conditions for 18 hours to obtain the second-level seed liquid, in which the wet weight of yeast in the second-level seed liquid was 184.0 g / L;

[0091] The secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 14.2 vol% for fermentation, stirring was turned on throughout the process, the temperature was controlled at 30.0±0.5℃, the pH value was controlled between 5.0-5.5 with ammonia water, the sugar content of the fermentation medium was controlled to 4.5%-5.5% by adding feed culture medium, the ventilation volume was 1.0 vvm, and the culture was carried out for 40 hours. After the culture was completed, the yeast fermentation liquid was obtained. The yeast fermentation liquid was filtered, the bacteria were collected and weighed, the wet weight of the yeast was 222.9 g / L, the dry matter content after drying was 114.2 g / L, and the fermentation liquid was milky white liquid.

[0092] According to the data provided in Examples 1-3 and Comparative Example 1, the production capacity of the optimized culture medium formula of the present invention is increased by 30.8% compared with that before optimization, and the wet weight of yeast when placed in the tank is above 400 g / L. This formula improves product quality, effectively reduces production costs, and has excellent yeast production capacity.

[0093] Example 4

[0094] The yeast fermentation product obtained in Example 1 was used as a raw material, and yeast milk was obtained after concentration. The yeast milk was used as a nitrogen source in the fermentation of Streptomyces aureus. Four groups of experiments were set up, and the fermentation cycle and volume are shown in Table 1. At the same time, yeast powder (purchased from Angel Yeast Co., Ltd.) was used as a nitrogen source control. The weight of dry matter in the yeast milk was the same as that of the yeast powder. The data shown in Table 1 were obtained by statistics.

[0095]

[0096] According to Table 1, when the yeast milk provided in Example 1 of the present invention is used as a nitrogen source, the fermentation index of Streptomyces aureus is basically consistent with that of the control group, indicating that the yeast milk provided in Example 1 of the present invention has no obvious effect on the fermentation of Streptomyces aureus, but since the yeast milk yield is increased, the yeast fermentation method provided by the present invention is helpful to reduce the fermentation cost of Streptomyces aureus.

[0097] Example 5

[0098] In this example, the yeast fermentation product obtained by fermentation in Example 1 was centrifuged and concentrated to obtain the yeast centrifugal supernatant, which was used as a nitrogen source for fermentation with Streptomyces aureus to prepare chlortetracycline.

[0099] Specifically, the fermentation product obtained by fermentation in Example 1 was separated by a centrifuge, and the supernatant was collected and used as a nitrogen source in the fermentation of Streptomyces aureus. Two groups of experiments were set up, and the fermentation cycle and volume are shown in Table 2; at the same time, chlortetracycline fermentation medium without yeast fermentation supernatant was used as a control, and the data shown in Table 2 were obtained by statistics.

[0100]

[0101] Example 6

[0102] In this example, the yeast fermentation product obtained by fermentation in Example 1 was centrifuged and concentrated to obtain yeast centrifugal supernatant as a feed culture solvent. After adding other feed culture components, the contents of C, N, and P in the culture solution after defoaming and the pH value were counted. At the same time, a feed culture solvent without using yeast fermentation supernatant was used as a control. The experimental data are summarized in Table 3.

[0103]

[0104] As shown in Table 3, the yeast supernatant obtained by the yeast fermentation method provided by the present invention is used to replace a small amount of tap water in the intermediate feed, and the parameters after detection and elimination are required to be within the normal control range, thereby avoiding the generation of wastewater and reducing the pressure of environmental protection treatment.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation method of Streptomyces aureus, characterized in that: include: The yeast fermentation product is centrifuged and concentrated to obtain yeast milk and supernatant, which are added into the fermentation process of Streptomyces aureus, wherein the yeast is Candida magnoliensis, and the number of the Candida magnoliensis in the China General Microbiological Culture Collection Center is CGMCC 2.1919; The yeast fermentation product is prepared by the following method: S1, inoculating yeast cells into a primary seed culture solution for primary culture to obtain a primary seed solution; the primary seed culture solution comprises 50-70 g / L glucose, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L diammonium phosphate, 2-5 g / L triammonium citrate and 3-6 g / L yeast extract; S2, inoculating the primary seed solution into a primary seed culture solution for primary culture to obtain a primary seed solution; the primary seed culture solution comprises 50-70 g / L of glucose, 0.7-1.1 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract, 1.5-2.0 g / L of ammonium sulfate and 0.5-1.0 g / L of magnesium sulfate; S3, inoculating the primary seed solution into a secondary seed culture solution for secondary culture to obtain a secondary seed solution; the secondary seed culture solution comprises 50-70 g / L of glucose, 0.5-1.0 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract powder, 1.5-1.8 g / L of ammonium sulfate, 0.5-1.0 g / L of magnesium sulfate and 0.2-1.0 g / L of a defoaming agent; S4. Inoculating the secondary seed liquid into a fermentation medium for fermentation to obtain a yeast fermentation product; the fermentation medium comprises 80-120 g / L of glucose, 2.6-3.8 g / L of potassium dihydrogen phosphate, 3.0-8.0 g / L of yeast extract powder, 4.8-6.5 g / L of ammonium sulfate, 7.3-8.5 g / L of magnesium sulfate and 0.1-0.5 g / L of a defoaming agent.

2. The fermentation method according to claim 1, characterized in that In S2, the inoculation amount of the primary seed solution is 0.5-1.0 vol%, the temperature of the primary culture treatment is 29.5-30.5°C, the pH is 5.0-5.5, the ventilation volume is 0.5-0.8 vvm, and the culture time is 20-24 hours.

3. The fermentation method according to claim 1, characterized in that In S3, the inoculation amount of the primary seed solution is 6-9 vol%, the temperature of the secondary culture treatment is 29.5-30.5°C, the pH is 5.0-5.5, the ventilation volume is 0.5-0.8 vvm, and the culture time is 8-12 hours.

4. The fermentation method according to claim 1, characterized in that In S4, the inoculation amount of the secondary seed liquid is 10-15 vol%, the fermentation temperature is 29.5-30.5°C, the pH is 5.0-5.5, the sugar content is 5-7%, the ventilation volume is 0.8-1.0 vvm, and the culture time is 36-48 hours.

5. The fermentation method according to claim 1, characterized in that In S4, a feed culture medium is added during the fermentation process, wherein the feed culture medium includes 400-500 g / L of glucose, 3.3-4.1 g / L of potassium dihydrogen phosphate, 5.0-6.5 g / L of yeast extract, 5.5-6.5 g / L of ammonium sulfate, 6.2-7.0 g / L of magnesium sulfate and 0.1-0.5 g / L of a defoaming agent.

6. A method for producing chlortetracycline, characterized in that: include: The fermentation method according to any one of claims 1 to 5 is used to ferment Streptomyces aureus, and chlortetracycline is obtained from the fermentation product of the Streptomyces aureus.

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