Fermentation culture medium for improving fermentation production of monensin from streptomyces cinnamomi and application of fermentation culture medium

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation culture medium for improving fermentation production of monensin by streptomyces cinnamomi and application of the fermentation culture medium. The fermentation medium comprises a carbon source, a nitrogen source, inorganic salt, growth factors, vegetable oil and water, the carbon source is glucose and maltose, the nitrogen source is soybean meal and casein, and the vegetable oil is sunflower seed oil and methyl oleate; the content of glucose is 2-3%, the content of maltose is 1.5-2.5%, the content of soybean meal is 2.5-3.5%, the content of casein is 0.5-1.5%, and the total content of sunflower seed oil and methyl oleate is 3-4% based on the weight volume percentage concentration of the fermentation culture medium. The fermentation culture medium provided by the invention obviously improves the yield of monensin in a shake flask fermentation stage, and can be applied to industrial production of monensin.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation medium for improving the production of monensin by fermentation of Streptomyces cinnamomi and an application thereof. Background Art

[0002] Monensin is an antibiotic produced by Streptomyces cinnamonensis. It has a broad spectrum of anticoccidial activity and is used for the prevention and treatment of coccidiosis in chickens and the promotion of growth in beef cattle. It has no effect on Gram-negative bacteria, but has a certain inhibitory effect on Staphylococci, Bacillus, Clostridium, Streptococci, and molds (Penicillium, Candida).

[0003] Biological fermentation is the main production method of monensin. At present, relevant research focuses on the selection of biological strains and the optimization of fermentation process at the fermentation tank level, and there are relatively few studies on the shake flask fermentation level. CN117265035A discloses a method for improving the titer of monensin fermentation liquid and the content of monensin A. Monensin is produced by fermentation of Streptomyces cinnamomi, and a combined fatty acid is added to the basic fermentation medium. The combined fatty acid consists of 1 to 2 parts of oleic acid, 2 to 3 parts of linoleic acid, 0.2 to 0.4 parts of stearic acid and 0.4 to 0.6 parts of palmitic acid, so that the titer of monensin reaches 15034μg / mL after 12 days of fermentation in the shake flask system. Breeding of high-yield strains of monensin (Li Ziyong, Li Yunfei, Zeng Xiaoning, etc., China Animal Health, 2021, 23(05): 116-117+119) discloses a fermentation medium containing 3.5% soybean cake powder, 3.5% oral glucose, 2.0% soybean oil, 0.2% ammonium sulfate, 0.03% zinc chloride, 0.007% magnesium sulfate, 0.005% ferrous sulfate, 0.0002 VC, 0.005% dipotassium hydrogen phosphate, and 0.25% calcium carbonate. The average relative titer in the shake flask is 27693 μg / mL. Shake flask fermentation is an important part of monensin fermentation research and can lay the foundation for the scale-up optimization of the fermentation tank level in the later stage. The yield of the shake flask fermentation stage of the above-reported technologies is relatively low, and it is necessary to study more efficient shake flask fermentation media. Summary of the invention

[0004] In view of the technical problem that the potency of existing monensin shake flask fermentation is low, the present invention significantly improves the monensin shake flask fermentation unit by adding growth factors and amino acids of specific composition to the monensin basic fermentation medium, thereby obtaining the present invention.

[0005] In a first aspect, the present invention provides a fermentation medium for improving the fermentation production of monensin by Streptomyces cinnamomi, which comprises a carbon source, a nitrogen source, an inorganic salt, a growth factor, a vegetable oil and water; the carbon source is glucose and maltose, the nitrogen source is soybean meal powder and casein, and the vegetable oil is sunflower oil and methyl oleate; in terms of weight volume percentage concentration of the fermentation medium, the content of glucose is 2-3%, the content of maltose is 1.5-2.5%, the content of soybean meal powder is 2.5-3.5%, the content of casein is 0.5-1.5%, and the total content of sunflower oil and methyl oleate is 3-4%.

[0006] In some embodiments, the mass ratio of sunflower oil to methyl oleate is 1 to 3:1:3.

[0007] In some embodiments, the inorganic salt is one or a combination of two or more of sodium sulfate, sodium nitrate, manganese chloride, aluminum sulfate, ferrous sulfate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride, magnesium chloride, magnesium sulfate, and potassium dihydrogen phosphate, and the content of the inorganic salt is 0.3-2%; the growth factor comprises L-ascorbic acid, and the content of L-ascorbic acid is 0.001-0.003%.

[0008] In some embodiments, the inorganic salt is sodium sulfate, sodium nitrate, manganese chloride, aluminum sulfate, ferrous sulfate, dipotassium phosphate, and calcium carbonate, and the growth factor is L-ascorbic acid.

[0009] In some embodiments, the fermentation medium comprises the following components in weight volume percentage concentrations: 2-3% glucose, 1.5-2.5% maltose, 2.5-3.5% soybean meal, 0.5-1.5% casein, 0.1-0.5% sodium sulfate, 0.1-0.5% sodium nitrate, 0.01-0.05% manganese chloride, 0.02-0.2% aluminum sulfate, 0.005-0.02% ferrous sulfate, 0.005-0.02% dipotassium hydrogen phosphate, 0.1-0.5% calcium carbonate, 0.001-0.003% L-ascorbic acid, 1-5% vegetable oil, and the remainder is water; the vegetable oil is composed of sunflower oil and methyl oleate in a mass ratio of 1-3:1:3.

[0010] In some embodiments, the pH of the fermentation medium is 6.8 ± 0.2.

[0011] In a second aspect, the present invention provides a method for producing monensin by fermenting Streptomyces cinnamomi, comprising: inoculating fermentation seed liquid of Streptomyces cinnamomi into the aforementioned fermentation medium for shake flask culture to obtain a fermentation liquid containing monensin.

[0012] In some embodiments, during shake flask culture, the vegetable oil in the fermentation medium is added in multiple times. Preferably, 50-60 wt% of the vegetable oil is added for the first time, and then the remaining vegetable oil is added in equal amounts over 2-3 days starting from the third day. The temperature of the shake flask culture is 32±1°C, the time of the shake flask culture is 8-12 days, and the shaker speed of the shake flask culture is 220-250 rpm.

[0013] In some embodiments, the fermentation seed liquid of Streptomyces cinnamomi is prepared as follows: inoculating Streptomyces cinnamomi into a slant culture medium for a first culture to obtain an activated strain; and inoculating the activated strain into a seed culture medium for a second culture to obtain a fermentation seed liquid of Streptomyces cinnamomi; the slant culture medium is composed of the following components in weight volume percentage concentrations: 0.05-0.15% potassium nitrate, 0.025-0.075% sodium chloride, 0.025-0.075% dipotassium hydrogen phosphate, and 0.06% yeast powder. .1~0.3%, magnesium sulfate 0.025~0.075%, ferrous sulfate 0.0005~0.0015%, soluble starch 1~3%, agar powder 1~3%, the balance is water, and the pH is 6.8±0.2; the seed culture medium is composed of the following components in weight volume percentage concentration: glucose 0.25~0.75%, soybean meal powder 1~2%, yeast powder 0.2~0.3%, corn dextrin 1~3%, calcium carbonate 0.05~0.15%, the balance is water, and the pH is 6.8±0.2.

[0014] In some embodiments, the conditions for the first culture are: culture temperature of 32±0.2°C, culture time of 5 to 7 days; the conditions for the second culture are: culture temperature of 32±0.2°C, shaker speed of 220 to 250 rpm, culture time of 20 to 22 h.

[0015] The beneficial effects of the present invention are:

[0016] The fermentation medium provided by the present invention significantly improves the yield in the shake flask fermentation stage by adopting a carbon source, a nitrogen source and a vegetable oil of a defined composition, and uses economical and pollution-free agricultural and sideline product raw materials, which is convenient for transportation and storage, and the fermentation liquid has no harmful impurities remaining, which is beneficial to the later pretreatment and separation and purification of the fermentation liquid, and is beneficial to promoting the industrial production of monensin. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. 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.

[0018] 1. Instruments and Reagents

[0019] 1.1 Strain: Streptomyces cinnamonensis BIOK-Mn-040203 was provided by the Technology Center of Zhejiang Biotech Co., Ltd.

[0020] 1.2 Instruments

[0021] instrument model Manufacturer UV spectrophotometer SP-752 Shanghai Spectrum Instrument Co., Ltd. Electronic balance JA2003 Shanghai Tianping Instrument Factory Constant temperature incubator 2020 Shanghai Zhicheng Analytical Instruments Co., Ltd. High-speed centrifuge <![CDATA[AccuSpinFR M ]]> BACKMAN Clean bench TH-CB-103A Wuxi purification equipment factory Shaker ZHWY-1112B Shanghai Zhicheng Analytical Instruments Co., Ltd. Optical Microscope E100 Nikon Corporation Ultrasonic cleaning machine SB-5200D Ningbo Xinzhi Biotechnology Vertical Pressure Steam Sterilizer YXQ-LS-50S11 Shanghai Boxun Industrial Co., Ltd. Electric constant temperature water bath DK-8D Shanghai Yiheng Technology Co., Ltd.

[0022] 1.3 Reagents

[0023]

[0024]

[0025]

[0026] 2. Detection Method

[0027] 2.1 Main reagent configuration:

[0028] Preparation of 500μg / ml monensin standard solution: accurately weigh 0.05g of monensin standard in a 100ml volumetric flask, add anhydrous ethanol to make up to volume, obtain a final concentration of 500μg / ml monensin standard solution, and store in a refrigerator for later use. Dilute to 20μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 250μg / ml, 500μg / ml, and draw a standard curve y=230.2x-5.3698.

[0029] Preparation of color developer: Weigh 1.5 g of p-dimethylaminobenzaldehyde into a 500 ml volumetric flask, add 300 ml of anhydrous ethanol to dissolve, add 2 ml of concentrated sulfuric acid and shake well, make up to volume with anhydrous ethanol, and store in a brown bottle.

[0030] 2.2 Sample processing:

[0031] Take 2 ml of shake flask fermentation liquid and centrifuge, discard the supernatant, make up to 8 ml with anhydrous ethanol, shake and sonicate for 1 hour, centrifuge, take 40 μl of the supernatant and dilute it 50 times with anhydrous ethanol, pipette 500 μl into a test tube, add 4.5 ml of anhydrous ethanol and 5 ml of color developer, shake well and place in a 70°C water bath for 20 min, cool rapidly, and detect the absorbance at 600 nm with a UV spectrophotometer.

[0032] 2.3 Calculation of fermentation units:

[0033] Monensin concentration = (absorbance × 230.2-5.3698) × 200.

[0034] 3. Definition of terms

[0035] The "%" used in this article refers to the content in weight volume percentage, which can be understood as the weight of the solute in 100 ml of the solution.

[0036] Example 1 Fermentation medium for improving the fermentation of monensin by Streptomyces cinnamomi

[0037] Glucose 2.5%, maltose 2.0%, soybean meal 8488 3.0%, casein 1.0%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 3.5%, the balance is water, pH 6.8.

[0038] Example 2 Method for producing monensin by fermentation with Streptomyces cinnamomi

[0039] 1. Slant culture:

[0040] Slant culture medium (%): potassium nitrate 0.1, sodium chloride 0.05, potassium hydrogen phosphate 0.05, imported yeast powder 0.2, magnesium sulfate 0.05, ferrous sulfate 0.001, soluble starch 2, agar powder 1.8, the balance is water. Adjust pH to 6.8, sterilize at 121℃ for 20min. Take Streptomyces kanamycin as the strain, inoculate into the sterilized slant culture medium, and culture in a 32℃ incubator for 6 days.

[0041] 2. Seed cultivation:

[0042] Seed culture medium: glucose 0.5, soybean meal 8488 1.5, imported yeast powder 0.25, corn dextrin 2.0, calcium carbonate 0.1, the balance is water. Adjust pH to 6.8, sterilize at 121℃ for 20min. Take the strain cultured on the slant, inoculate it into the sterilized seed culture medium, and culture it on a shaking table at 32℃ for 21h to obtain the fermentation seed liquid.

[0043] 3. Shake bottle fermentation:

[0044] The fermentation seed liquid is inoculated into the following culture medium: glucose 2.5%, maltose 2.0%, soybean meal 84883.0%, casein 1.0%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 2%, and the balance is water. The culture is carried out in a shaking incubator at 32°C for 10 days, and 0.5% sunflower oil: methyl oleate (2:1) is added daily from the third day to the fifth day (accounting for the volume of the fermentation liquid), to obtain a fermentation liquid containing monensin.

[0045] Experimental Example: Screening test of the fermentation medium formula of the present invention

[0046] 1. Screening of carbon sources

[0047] 1.1 Effect of glucose addition on monensin fermentation units

[0048] The basal culture medium was composed of 4.5% glucose, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil. With 4.5% glucose as the control, shake flask culture media with glucose concentrations of 3.5%, 4.0%, 4.5%, 5.0% and 5.5% were prepared and cultured in a shaking incubator at 32°C for 10 days.

[0049]

[0050] The results showed that the fermentation medium with 4.5% glucose had the highest monensin fermentation unit.

[0051] 1.2 Partial replacement of glucose

[0052] The basic culture medium is based on 4.5% glucose, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate, and 2.0% soybean oil. 1% corn starch, corn flour, maltodextrin, corn dextrin, maltose, lactose, sucrose, fructose, mannose, and glycerol are used to replace 1% glucose, and the basic formula is used as the control.

[0053]

[0054]

[0055] The results showed that corn dextrin, maltose and sucrose instead of glucose were beneficial to increase the fermentation unit of monensin.

[0056] 1.3 Partial replacement of corn dextrin

[0057] The basic culture medium is based on 3.5% glucose, 1% corn dextrin, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate, and 2.0% soybean oil. The amount of corn dextrin replaced is increased to 1.5%, 2%, and 2.5%.

[0058]

[0059] The results showed that the fermentation unit of monensin was higher when the addition amount of corn dextrin was 2.5%.

[0060] 1.4 Partial replacement of sucrose

[0061] The basic culture medium is based on 3.5% glucose, 1% sucrose, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate, and 2.0% soybean oil. The amount of sucrose replacement is increased to 1.5%, 2%, and 2.5%.

[0062] Sucrose partial replacement (%) 1.0 1.5 2.0 2.5 Fermentation unit (μg / mL) 20961 20782 19145 18707

[0063] The results showed that the fermentation unit of monensin was higher when the sucrose addition was 1%.

[0064] 1.5 Partial replacement of maltose

[0065] The basic culture medium is based on 3.5% glucose, 1% maltose, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil, and the amount of maltose replaced is increased to 1.5%, 2% and 2.5%.

[0066]

[0067] The results showed that the fermentation unit of monensin was the highest when the maltose addition was 2%.

[0068] 2. Screening of nitrogen sources

[0069] 2.1 Effects of different soy products on monensin fermentation units

[0070] The basic culture medium is composed of 2.5% glucose, 2.0% maltose, 4% soybean meal, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil. The soybean meal is completely replaced by cold soybean powder, hot soybean powder and soybean meal 8488, and culture is carried out at 32°C in a shaking incubator for 10 days.

[0071]

[0072] The results showed that the fermentation medium supplemented with soybean meal 8488 had the highest monensin fermentation unit.

[0073] 2.2 Effect of soybean meal 8488 addition on monensin fermentation unit

[0074] With glucose 2.5%, maltose 2.0%, soybean meal 8488 4%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25% and soybean oil 2.0% as the basal culture medium, shake flask culture medium with soybean meal concentration of 3.0%, 3.5%, 4.5% and 5.0% was prepared, and cultured in a shaking incubator at 32°C for 10 days.

[0075]

[0076] The results showed that the fermentation unit of monensin was the highest when the addition amount of soybean meal 8488 was 4.0%.

[0077] 2.3 Effects of different nitrogen source components replacing soybean meal 8488 on monensin fermentation units

[0078] The basic medium is composed of 2.5% glucose, 2.0% maltose, 4% soybean meal 8488, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate, and 2.0% soybean oil. 1% cottonseed meal, peanut meal, Ruzhou yeast powder, Angel yeast powder, fish meal, feather meal, Dolphin brand peptone, soybean peptone, fish meal peptone, fish paste, Ruzhou peptone, casein, yeast paste LM801, and corn steep liquor are used to partially replace soybean meal 8488.

[0079]

[0080] The results showed that the fermentation medium supplemented with Dolphin peptone, Ruzhou peptone and casein had higher monensin fermentation units.

[0081] 2.4 Effect of partial replacement of soybean meal 8488 with Dolphin peptone on monensin fermentation units

[0082] The basic culture medium is based on 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% Dolphin brand peptone, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil. The replacement amount of Dolphin brand peptone is increased to 1.5%, 2.0% and 2.5% respectively.

[0083]

[0084] The results showed that the fermentation medium supplemented with 1% Dolphin brand peptone had higher monensin fermentation units.

[0085] 2.5 Effect of partial replacement of soybean meal 8488 with Ruzhou peptone on monensin fermentation units

[0086] The basic culture medium is based on 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% Ruzhou peptone, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil. Ruzhou peptone is used to replace the two and the content is increased to 1.5%, 2.0% and 2.5%.

[0087] Partial replacement of Ruzhou peptone (%) 1.0 1.5 2 2.5 Fermentation unit (μg / mL) 21755 21086 22130 6921

[0088] The results showed that when the addition amount of Ruzhou peptone was 2%, the fermentation unit of monensin in the fermentation medium was higher.

[0089] 2.6 Effect of partial replacement of soybean meal 8488 with casein on monensin fermentation units

[0090] The basic culture medium is based on 2.5% glucose, 2.0% maltose, 3% soybean meal, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil. Casein is used to replace the two and the content is increased to 1.5%, 2.0% and 2.5%.

[0091]

[0092] The results showed that the monensin fermentation unit of the fermentation medium was higher when the casein addition was 1%.

[0093] 3. Screening of vegetable oil

[0094] 3.1 Effects of replacing soybean oil with different vegetable oils on monensin fermentation units

[0095] The basal culture medium was composed of 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% soybean oil, with sunflower oil, cottonseed oil, corn oil and peanut oil used instead of soybean oil respectively.

[0096] Soybean oil replacement (%) Soybean Oil Sunflower Oil Cottonseed oil Corn Oil Peanut Oil Fermentation unit (μg / mL) 22721 22953 4343 22562 21092

[0097] The results showed that the fermentation medium supplemented with sunflower oil had higher monensin fermentation units than that supplemented with soybean oil.

[0098] 3.2 Effect of sunflower oil addition on monensin fermentation units

[0099] The basal culture medium is composed of 2.5% glucose, 2.0% maltose, 3% soybean meal, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 2.0% sunflower oil, and the amount of sunflower oil is adjusted to 2.0%, 2.5%, 3.0%, 3.5% and 4.0% respectively.

[0100]

[0101] The results showed that the fermentation medium with 3.5% sunflower oil added had a high monensin fermentation unit.

[0102] 3.3 Effect of the ratio of soybean oil to methyl oleate on the fermentation unit of monensin

[0103] The control culture medium was composed of 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 3.5% soybean oil. The ratios of soybean oil and methyl oleate were adjusted to 3:1, 2:1, 1:1, 1:2 and 1:3, respectively, and the total addition amount of soybean oil and methyl oleate was 3.5%.

[0104]

[0105] The results showed that the fermentation medium with soybean oil:methyl oleate = 2:1 had the highest monensin fermentation unit.

[0106] 3.4 Effect of the ratio of sunflower oil to methyl oleate on the fermentation unit of monensin

[0107] The control culture medium was composed of 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 3.5% sunflower oil. The ratios of sunflower oil and methyl oleate were adjusted to 3:1, 2:1, 1:1, 1:2 and 1:3, respectively, and the total addition amount of sunflower oil and methyl oleate was 3.5%.

[0108]

[0109] The results showed that the fermentation unit of monensin in the fermentation medium with sunflower oil:methyl oleate=2:1 was the highest.

[0110] 3.5 Effect of the ratio of corn oil to methyl oleate on the fermentation unit of monensin

[0111] The control culture medium was composed of 2.5% glucose, 2.0% maltose, 3% soybean meal 8488, 1% casein, 0.22% sodium sulfate, 0.22% sodium nitrate, 0.033% manganese chloride, 0.07% aluminum sulfate, 0.01% ferrous sulfate, 0.0019% L-ascorbic acid, 0.0075% dipotassium hydrogen phosphate, 0.25% calcium carbonate and 3.5% corn oil. The ratios of corn oil and methyl oleate were adjusted to 3:1, 2:1, 1:1, 1:2 and 1:3, respectively, and the total addition amount of corn oil and methyl oleate was 3.5%.

[0112]

[0113] The results showed that the fermentation medium with corn oil:methyl oleate=2:1 had the highest monensin fermentation unit.

[0114] 4. Screening of vegetable oil addition methods

[0115] Control group: glucose 2.5%, maltose 2.0%, soybean meal 8488 3%, casein 1%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 3.5%.

[0116] Experimental group 1: glucose 2.5%, maltose 2.0%, soybean meal 8488 3%, casein 1%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 3.5%. From the third day, 0.5% (accounting for the volume of the fermentation liquid) was added every day until the fifth day.

[0117] Experimental group 2: glucose 2.5%, maltose 2.0%, soybean meal 8488 3%, casein 1%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 2.0%. From the third day, 0.5% (based on the volume of the fermentation liquid) was added every day until the fifth day.

[0118] Group Control group Experimental Group 1 Experimental Group 2 Fermentation unit (μg / ml) 27648 25203 29855

[0119] The results showed that the oil supplementation method of experimental group 2 was beneficial to improve the fermentation unit of monensin.

[0120] Therefore, the optimal fermentation medium formula is: glucose 2.5%, maltose 2.0%, soybean meal 84883.0%, casein 1.0%, sodium sulfate 0.22%, sodium nitrate 0.22%, manganese chloride 0.033%, aluminum sulfate 0.07%, ferrous sulfate 0.01%, L-ascorbic acid 0.0019%, dipotassium hydrogen phosphate 0.0075%, calcium carbonate 0.25%, sunflower oil: methyl oleate (2:1) 3.5%, the balance is water. After testing, the shake flask fermentation unit is 27648μg / mL, which is 49.82% higher than the shake flask fermentation unit of 18454μg / mL of the basic medium. By reducing the initial oil amount and replenishing oil in batches from the third to the fifth day, the shake flask fermentation unit is increased to 29855μg / mL, which is 61.78% higher than the basic medium.

[0121] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A fermentation medium for improving the fermentation of monensin by Streptomyces cinnamomi, characterized in that: The fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt, a growth factor, a vegetable oil and water; the carbon source is glucose and maltose, the nitrogen source is soybean meal and casein, and the vegetable oil is sunflower oil and methyl oleate; Calculated in terms of weight volume percentage concentration of the fermentation medium, the content of glucose is 2-3%, the content of maltose is 1.5-2.5%, the content of soybean meal powder is 2.5-3.5%, the content of casein is 0.5-1.5%, and the total content of sunflower oil and methyl oleate is 3-4%.

2. The fermentation medium according to claim 1, characterized in that The mass ratio of the sunflower seed oil to methyl oleate is 1-3:1:

3.

3. The fermentation medium according to claim 1, characterized in that The inorganic salt is one or a combination of two or more of sodium sulfate, sodium nitrate, manganese chloride, aluminum sulfate, ferrous sulfate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride, magnesium chloride, magnesium sulfate, and potassium dihydrogen phosphate, and the content of the inorganic salt is 0.3-2%; the growth factor contains L-ascorbic acid, and the content of the L-ascorbic acid is 0.001-0.003%.

4. The fermentation medium according to claim 3, characterized in that The inorganic salts are sodium sulfate, sodium nitrate, manganese chloride, aluminum sulfate, ferrous sulfate, dipotassium hydrogen phosphate and calcium carbonate, and the growth factor is L-ascorbic acid.

5. The fermentation medium according to claim 4, characterized in that The fermentation medium comprises the following components in weight volume percentage concentrations: 2-3% glucose, 1.5-2.5% maltose, 2.5-3.5% soybean meal, 0.5-1.5% casein, 0.1-0.5% sodium sulfate, 0.1-0.5% sodium nitrate, 0.01-0.05% manganese chloride, 0.02-0.2% aluminum sulfate, 0.005-0.02% ferrous sulfate, 0.005-0.02% dipotassium hydrogen phosphate, 0.1-0.5% calcium carbonate, 0.001-0.003% L-ascorbic acid, 1-5% vegetable oil, and the balance is water; the vegetable oil is composed of sunflower oil and methyl oleate in a mass ratio of 1-3:1:

3.

6. The fermentation medium according to claim 1, characterized in that The pH of the fermentation medium is 6.8±0.

2.

7. A method for producing monensin by fermentation with Streptomyces cinnamomi, characterized in that: The method comprises: inoculating fermentation seed liquid of Streptomyces cinnamomi into the fermentation medium according to any one of claims 1 to 6 for shake flask culture to obtain fermentation liquid containing monensin.

8. The method according to claim 7, characterized in that In the shake flask culture, the vegetable oil in the fermentation medium is added in multiple times; the temperature of the shake flask culture is 32±1° C., the time of the shake flask culture is 8 to 12 days, and the shaking speed of the shake flask culture is 220 to 250 rpm.

9. The method according to claim 7, characterized in that: The fermentation seed liquid of Streptomyces cinnamomi is prepared by the following method: inoculating Streptomyces cinnamomi into a slant culture medium for a first culture to obtain an activated strain; and inoculating the activated strain into a seed culture medium for a second culture to obtain a fermentation seed liquid of Streptomyces cinnamomi; The slant culture medium is composed of the following components in weight volume percentage concentrations: 0.05-0.15% potassium nitrate, 0.025-0.075% sodium chloride, 0.025-0.075% dipotassium hydrogen phosphate, 0.1-0.3% yeast powder, 0.025-0.075% magnesium sulfate, 0.0005-0.0015% ferrous sulfate, 1-3% soluble starch, 1-3% agar powder, and the balance is water, with a pH of 6.8±0.2; The seed culture medium is composed of the following components in weight volume percentage concentrations: 0.25-0.75% glucose, 1-2% soybean meal powder, 0.2-0.3% yeast powder, 1-3% corn dextrin, 0.05-0.15% calcium carbonate, and the balance is water, with a pH of 6.8±0.

2.

10. The method according to claim 9, characterized in that The conditions for the first culture are: culture temperature of 32±0.2°C, culture time of 5 to 7 days; the conditions for the second culture are: culture temperature of 32±0.2°C, shaker speed of 220 to 250 rpm, culture time of 20 to 22 hours.

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