A modified corn steep liquor for erythromycin fermentation production, its preparation method and application

Abstract

This invention provides a method for preparing improved corn steep liquor, characterized by the following steps: (1) mixing corn steep liquor with an equal volume of water to obtain a corn steep liquor pretreatment solution; (2) taking the corn steep liquor pretreatment solution from step (1), adding HNO3 solution and mixing thoroughly, then adding a certain amount of CaCO3 and reacting thoroughly to obtain a corn steep liquor treatment solution; (3) taking the corn steep liquor treatment solution from step (2), adjusting the pH to 7.0-7.5 to obtain improved corn steep liquor; the amount of HNO3 added in step (2) is 4%-6% (w / v), and the amount of CaCO3 added is 10%-15% (w / v). This invention can extend the shelf life of corn steep liquor without significantly affecting its nutritional components, and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering, specifically to a modified corn steep liquor for erythromycin fermentation production, its preparation method, and its application. Background Technology

[0002] Erythromycin (Er) is an alkaline antibiotic produced by fermentation culture of *Streptomyces erythreus*, isolated from soil on Panay Island in the Philippines in 1952. It is a representative macrolide antibiotic. Erythromycin has broad-spectrum antibacterial activity and exhibits strong inhibitory effects against Gram-positive bacteria, such as Staphylococcus, Streptococcus pyogenes, Streptococcus viridans, Streptococcus pneumoniae, and Bacillus anthracis.

[0003] Current erythromycin fermentation methods mostly employ a multi-stage fermentation model. The main raw materials and auxiliary materials for erythromycin seed, fermentation, and fed-batch culture media include: corn steep liquor, corn starch, dextrin, soybean meal, ammonium sulfate, calcium carbonate, and vegetable oil. Corn steep liquor is a yellowish-brown liquid obtained by concentrating corn kernels after soaking them in sulfurous acid. It contains abundant soluble proteins, amino acids, growth factors, and some precursor substances, and contains approximately 40%–50% solids, which can promote the biometabolism and biosynthesis of erythromycin.

[0004] Due to its complex composition, low pH, and unstable phosphorus solubility, corn steep liquor is one of the most unstable components in erythromycin fermentation feedstocks. It is prone to the growth of putrefactive bacteria and is difficult to store. Therefore, research on pretreatment methods for corn steep liquor is crucial for its successful use as a stable component of the fermentation medium.

[0005] There are currently no reports on methods for pretreating corn steep liquor used in erythromycin fermentation. Summary of the Invention

[0006] To address the above problems, the present invention provides an improved method for preparing corn steep liquor, characterized by comprising the following steps:

[0007] (1) Mix corn syrup and water at a volume ratio of 1:(0.5~2) to obtain corn syrup pretreatment solution;

[0008] (2) Take the corn steep liquor pretreatment liquid from step (1), add HNO3 solution, mix thoroughly, and then add CaCO3. a After complete reaction, corn steep liquor is obtained.

[0009] (3) Take the corn steep liquor from step (2) and adjust the pH to 7.0-7.5 to obtain improved corn steep liquor;

[0010] In step (2), the mass of HNO3 in the HNO3 solution is 4% to 6% (w / v) of the corn steep liquor pretreatment solution;

[0011] The amount of CaCO3 added in step (2) is 10% to 15% (w / v) of the corn steep liquor pretreatment solution.

[0012] As described in the preparation method above, the concentration of the HNO3 solution in step (2) is 10% to 15% (w / v).

[0013] As described above, in step (2), the reaction time after adding CaCO3 is 15 to 20 minutes.

[0014] As described in the preparation method above, the pH value in step (3) is 7.5.

[0015] As described in the preparation method above, the substance used to adjust the pH in step (3) is sodium hydroxide or a solution of sodium hydroxide.

[0016] The present invention also provides an improved corn steep liquor prepared by the aforementioned method.

[0017] The present invention also provides the use of the aforementioned improved corn steep liquor in the culture of erythromycin-producing fungi.

[0018] Furthermore, the erythromycin-producing fungus is *Streptomyces erythromycin*.

[0019] The present invention also provides the use of the aforementioned improved corn steep liquor in the fermentation production of erythromycin.

[0020] The present invention also provides the use of the aforementioned modified corn steep liquor in the preparation of culture media for erythromycin-producing fungi.

[0021] The present invention has the following beneficial effects:

[0022] 1) The nutritional composition of corn steep liquor is slightly altered, and its soluble protein and amino acid content are not significantly different from the original corn steep liquor.

[0023] 2) It can precipitate the phosphorus dissolved in corn steep liquor, reduce the risk of spoilage bacteria growing in corn steep liquor, and extend the shelf life of corn steep liquor.

[0024] 3) The improved corn steep liquor is suitable for erythromycin fermentation production. Experiments have shown that the erythromycin titer obtained by bottle fermentation can reach 8023 u / ml, and the erythromycin titer obtained by tank fermentation can reach 9500 u / ml.

[0025] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0026] The following detailed description of specific embodiments further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0027] Example 1: Preparation method of improved corn steep liquor for erythromycin fermentation production

[0028] The corn syrup was purchased from Yili Heng Hui Starch Co., Ltd., and the production date was October 13, 2017.

[0029] (I) Improved corn steep liquor preparation method

[0030] 1. Corn steep liquor pretreatment: Take corn steep liquor raw material and mix it with an equal volume of process water to obtain corn steep liquor pretreatment solution;

[0031] 2. Corn steep liquor treatment solution: Take corn steep liquor pretreatment solution, add 40% (v / v) of 10-15% (w / v) HNO3 solution of corn steep liquor volume, mix and react thoroughly, then add 10-15% (w / v) CaCO3 and react for 15-20 minutes. After the foam in the reaction process dissipates, the corn steep liquor treatment solution is obtained.

[0032] 3. Improved corn steep liquor: Take the corn steep liquor treatment liquid and adjust the pH to 7.5 using sodium hydroxide to obtain improved corn steep liquor.

[0033] (II) Detection of main components in improved corn steep liquor

[0034] The main components of corn steep liquor before and after the improvement were detected by high performance liquid chromatography (HPLC) and ultraviolet spectrophotometer. The results are shown in Tables 1 and 2. Except for dry matter, the contents of other items in the tables are shown in the supernatant.

[0035] Table 1. Detection of Main Parameters of Corn Stew

[0036]

[0037]

[0038] Table 2. Amino acid content in corn steep liquor

[0039]

[0040] Because the present invention adds an equal volume of production process water and a certain amount of nitric acid during the corn steep liquor processing, there are certain differences in dry matter and reducing sugar. However, the nutritional components and amino acids of the improved corn steep liquor are basically unaffected. The chemical method improves the solubility of phosphorus in the corn steep liquor, which exists stably in the form of precipitation. Microscopic observation shows that there are fewer live bacteria in the corn steep liquor, which can be stably stored for a long time.

[0041] Example 2: Preparation method of modified corn steep liquor for erythromycin fermentation production

[0042] 1. Corn steep liquor pretreatment: Take corn steep liquor raw material and mix it with an equal volume of process water to obtain corn steep liquor pretreatment solution;

[0043] 2. Corn steep liquor treatment solution: Take corn steep liquor pretreatment solution, add 40% (v / v) of 10-15% (w / v) HNO3 solution and mix thoroughly. Then add 10-15% (w / v) CaCO3 and react for 15-20 minutes. After the foam in the reaction process dissipates, the corn steep liquor treatment solution is obtained.

[0044] 3. Improved corn steep liquor: Take the corn steep liquor treatment liquid and adjust the pH to 7.0 using sodium hydroxide to obtain improved corn steep liquor.

[0045] The present invention will be further illustrated by the following experimental examples.

[0046] The erythromycin strains in the following examples are the production strains of erythromycin streptomycin.

[0047] In the following examples, the slant spores were obtained by inoculating erythromycin streptomycin spores that were frozen and stored in sand tubes into sterilized slant culture medium and culturing them at 34°C for 8-9 days using conventional methods.

[0048] Experiment Example 1: Comparative Experiment on the Production of Erythromycin Using Modified Corn Stew Fermentation in Shake Flasks

[0049] (I) Method

[0050] 1. Seed culture:

[0051] Slant spores were inoculated into sterilized seed culture medium and placed on a shaker at 260 rpm / min and 34℃ for 60 hours to obtain erythromycin seed solution.

[0052] The seed culture medium consists of: 3.0% modified corn steep liquor, 3.5% soybean flour, 3.5% starch, 0.6% CaCO3, 0.5% NaCl, 3.5% dextrin, 0.3% ammonium sulfate, an appropriate amount of defoamer, and the remainder is process water. The contents are sterilized and ready for use, by weight percentage.

[0053] 2. Fermentation culture

[0054] The erythromycin seed culture from step 1 was inoculated at a ratio of 20% into sterilized fermentation medium and placed on a shaker at 260 rpm / min. The culture temperature was 32-34℃. After fermentation for 40 hours, 50-60% n-propanol was added, and fermentation was stopped after 155-160 hours to obtain erythromycin fermentation broth.

[0055] The fermentation medium consists of: 4.4% modified corn steep liquor, 3.5% soybean flour, 5.5% starch, 0.6% CaCO3, 0.5% NaCl, 0.3% ammonium sulfate, 0.02% defoamer, and the remainder is process water. All components are sterilized and ready for use.

[0056] 3. Erythromycin potency test

[0057] Erythromycin reacts with sulfuric acid to form a yellow substance with a maximum absorption wavelength of 483 nm. Therefore, quantitative analysis can be achieved by colorimetric determination using a spectrophotometer. The specific method is as follows:

[0058] 3.1 Drawing the Standard Curve

[0059] Prepare working erythromycin standard solutions by measuring 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 30 ml, 3.5 ml, and 4.0 ml of erythromycin standard solution into seven 25 ml volumetric flasks (approximately 64 U / ml - 256 U / ml). Accurately measure 2.0 ml of each working erythromycin standard solution into seven 30 × 200 mm test tubes, accurately add 8 ml of mixed colorimetric solution, shake well, and heat in an 80°C water bath for 6 minutes (starting the timer from the moment of placement). After removal, immediately cool to room temperature with water. Measure the absorbance at 483 nm using a spectrophotometer with purified water as a blank. Plot a standard curve against the added erythromycin potency (U) and absorbance values, and derive the regression equation.

[0060] 3.2 Determination of chemical potency

[0061] Take an appropriate amount of the supernatant from the fermentation broth after filtration or centrifugation, and dilute it with a diluent to prepare a solution of approximately 100-200 U / ml erythromycin. Use this solution as the test sample. Accurately measure 2.0 ml of the test solution into a 30×200 mm test tube, then accurately add 8 ml of the mixed colorimetric solution. Shake well and heat in an 80℃ water bath for 6 minutes (starting the timer from the moment of placement). After removal, immediately cool to room temperature with water. Use a spectrophotometer with purified water as a blank to measure the absorbance at a wavelength of 483 nm. Simultaneously, use the regression equation of the standard curve.

[0062] Calculation formula: Fermentation broth potency (U / ml) = Potency calculated from regression line equation × Dilution factor

[0063] (II) Results

[0064] The potency of erythromycin in the bottle is 8023 u / ml.

[0065] Experiment Example 2: Production of Erythromycin from Modified Corn Stew in a 50L Mechanically Stirred Fermentation Tank

[0066] (I) Method

[0067] 1. Seed culture:

[0068] Slant spores were inoculated into a sterilized 15L seed tank culture medium, sterile air was introduced (aeration ratio 0.8-1.2 V / V / min), the mixture was stirred at 180-500 rpm / min, and the culture temperature was 34℃ for 50-60 hours to obtain erythromycin seed solution.

[0069] The seed culture medium consists of: 3.0% modified corn steep liquor, 3.5% soybean flour, 3.5% starch, 0.6% CaCO3, 0.5% NaCl, 3.5% dextrin, 0.3% ammonium sulfate, 0.02% defoamer, and the remainder is process water. The total weight percentage is 9 kg, which is sterilized and ready for use.

[0070] 2. Fermentation culture

[0071] The erythromycin seed culture from step 1 was inoculated at a ratio of 20% into a sterilized 50L fermenter fermentation medium. Sterile air was introduced (aeration ratio of 0.8-1.4V / V / min), and the mixture was stirred at 180-700 rpm / min. The culture temperature was 32-34℃. After fermentation for 15 hours, 40-50% glucose, 50-60% n-propanol, and soybean oil were added. After 155-160 hours of fermentation, the fermentation was stopped to obtain the erythromycin fermentation broth.

[0072] Fermentation medium composition: 4.4% modified corn steep liquor, 3.5% soybean flour, 5.5% starch, 0.6% CaCO3, 0.5% NaCl, 0.3% ammonium sulfate, 0.02% defoamer, and the remainder is process water. The total weight percentage is 28 kg, which is sterilized and ready for use.

[0073] The optimal amount of glucose to be added is 45%, with the total sugar content in the fermentation broth controlled at 0.3-0.6%. The optimal amount of n-propanol to be added is 50%, with the propanol content in the fermentation broth controlled at 0.02-0.05%.

[0074] 3. Erythromycin potency test

[0075] The detection method is the same as in Section 3 of Experimental Example 1.

[0076] (II) Results

[0077] The potency of erythromycin in the container is 9500 u / ml.

[0078] In summary, the corn steep liquor of the present invention exhibits minimal changes in nutritional composition, has a long shelf life, and is suitable for erythromycin fermentation production, yielding erythromycin with high potency.

Claims

1. The use of improved corn steep liquor in the culture of erythromycin-producing fungi, characterized in that, The erythromycin-producing fungus is *Streptomyces erythromycin*; the method for preparing the improved corn steep liquor includes the following steps: (1) Mix corn syrup and water at a volume ratio of 1:(0.5~2) to obtain corn syrup pretreatment solution; (2) Take the corn steep liquor pretreatment liquid from step (1), add HNO3 solution and mix thoroughly, then add CaCO3 and react thoroughly to obtain corn steep liquor treatment liquid; the concentration of the HNO3 solution is 10%~15% (w / v); the reaction time after CaCO3 is 15~20 minutes; the mass of HNO3 in the HNO3 solution is 4%~6% (w / v) of the corn steep liquor pretreatment liquid; the amount of CaCO3 added is 10%~15% (w / v) of the corn steep liquor pretreatment liquid; (3) Take the corn steep liquor from step (2) and adjust the pH to 7.0-7.5 to obtain improved corn steep liquor.

2. The use as described in claim 1, characterized in that, The pH value in step (3) is 7.

5.

3. The use as described in claim 1, characterized in that, The substance used to adjust the pH in step (3) is sodium hydroxide or a sodium hydroxide solution.

4. The use as described in claim 1, characterized in that, The improved corn steep liquor is used in the fermentation production of erythromycin.

5. The use as described in claim 4, characterized in that, The modified corn steep liquor is used in the culture medium for erythromycin-producing fungi.

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