CN105624229B - A method of improving nimoctin yield - Google Patents
A method of improving nimoctin yield Download PDFInfo
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- CN105624229B CN105624229B CN201410598718.5A CN201410598718A CN105624229B CN 105624229 B CN105624229 B CN 105624229B CN 201410598718 A CN201410598718 A CN 201410598718A CN 105624229 B CN105624229 B CN 105624229B
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Abstract
The present invention provides a kind of method for improving nimoctin yield, its using deposit number be CGMCC No.8731 cyaneogriseus streptomyces (Streptomyces cyaneogriseus) BJX003 as produce bacterial strain, carry out the fermenting and producing of nimoctin, when the concentration of reduced sugar in fermentation liquid is lower than 12-18g/L, the glucose solution containing soybean cake powder is added, and the concentration of reduced sugar is controlled in 12-18g/L；Wherein the concentration of glucose is 300-400g/L in feed supplement liquid, and the concentration of soybean cake powder is 75-125g/L.Nimoctin yield can be made from 3200 μ g/mL or so raising to 3400 μ g/mL or so using the method for the present invention.
Description
Technical field
The present invention relates to field of microbial fermentation, specifically, being related to a kind of method for improving nimoctin yield.
Background technique
In last century the forties, its appearance significantly enhances the mankind and controls disease pest harm for organic chemical pesticides invention
Ability, made major contribution to retrieve crop yield loss, still, the universal toxicity of organic chemical pesticides is higher, safety
Property is low, effect on environment is big, its long-term and a large amount of use has had resulted in well-known environmental pollution, ecological disruption and food
A series of problems, such as product are safe has seriously affected the sustainable development of agricultural economy.In the past more than ten years, various countries are successively not
With degree reducing the dosage of chemical pesticide, in December, 2003, European Union formally forbade 320 kinds of pesticides in the sale of European Union, wherein
Be related to China up to more than 60 kinds, have that omethoate, three victory phosphorus, methoxy pyrethroids, Profenofos, Isoprothiolane, to dislike double spirits etc. chemically synthesized
High toxicity kind, the publication bulletin of the Ministry of Agriculture in 2004: from June 30 this year, must not sell on the market containing acephatemet,
The compound product of parathion, 5 kinds of parathion-methyl, Azodrin and phosphorus ammonium high-toxic organic phosphorus pesticides.With high poison chemical pesticide by
Step is withdrawn from the market, and research and the application of biological pesticide are got the attention.Compared with chemical pesticide, biological pesticide has desinsection
The advantages that mechanism is novel, and Environmental Residues are low, safer to people and animals, is the ideal green pesticide of the following integrated pest control.
Nimoctin (Nemadectin) is ten hexa-atomic macrolide antibiotics, belongs to mibemycin family, with Buddhist nun
Mack spit of fland is that the moxidectin (Moxidectin) of basic half chemical synthesis has excellent effect in terms of driving nematode and antiparasitic
Fruit is current most promising wide spectrum, efficient, novel, without cross-infection biological insecticides, it is highest to be known as current activity
One of insecticide.The compound is by cyaneogriseus streptomyces (Streptomyces cyaneogriseus sp.Noncyanogenus)
Fermentation generates, molecular formula C36H52O8, molecular weight 612.8, sterling is white powder, is had about this drug in foreign countries more
Report, and studies in China is just started to walk, market potential is huge.
Summary of the invention
The object of the present invention is to provide a kind of methods for improving nimoctin yield.
In order to achieve the object of the present invention, the method for a kind of raising nimoctin yield of the invention is with deposit number
Cyaneogriseus streptomyces (Streptomyces cyaneogriseus) BJX003 of CGMCC No.8731 is carried out as production bacterial strain
The fermenting and producing of nimoctin adds the Portugal containing soybean cake powder when the concentration of reduced sugar in fermentation liquid is lower than 12-18g/L
Grape sugar juice, and the concentration of reduced sugar is controlled in 12-18g/L；Wherein the concentration of glucose is 300-400g/L in feed supplement liquid,
The concentration of soybean cake powder is 75-125g/L, is prepared with water.
Cyaneogriseus streptomyces (Streptomyces cyaneogriseus) BJX003 provided by the invention, has been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state, postcode 100101, deposit number CGMCC No.8731, preservation date on January 17th, 2014.
Method above-mentioned, ferment the fermentation medium used are as follows: glucose 40-60g/L, lactose 20-50g/L, yeast powder
20-50g/L, dipotassium hydrogen phosphate 1-3g/L, epsom salt 0.5-2g/L, sodium chloride 8.5-12.5g/L and calcium carbonate 10-30g/
L, pH value 7.2, is prepared with water.
Preferably, fermentation medium are as follows: glucose 50g/L, lactose 35g/L, yeast powder 35g/L, dipotassium hydrogen phosphate 2g/L,
Epsom salt 1.2g/L, sodium chloride 10g/L and calcium carbonate 20g/L.
The condition of fermentation are as follows: 26-30 DEG C, revolving speed 150-220rpm, ventilation ratio 1:0.3-0.7, tank presses 0.03-0.04MPa,
Dissolved oxygen control is 40% or more, fermentation period 192-264hr.
The invention has the following advantages that
It (one) can be fermentation using the glucose during cyaneogriseus streptomyces BJX003 fermenting and producing nimoctin, added
Process provides sufficient carbon source；Soybean cake powder belongs to effect nitrogen source late, in fermentation early stage due to endobacillary various albumen enzyme system shapes
At and it is not perfect, the ability utilized to it is lower, when enter ferment the middle and later periods, to its Utilization ability enhance, add soya bean at this time
Cake powder can provide sufficient nitrogen source for fermentation process.
(2) nimoctin yield can be made from 3200 μ g/mL or so raising to 3400 μ g/mL or so using the method for the present invention.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Culture medium involved in following embodiment and condition of culture are as follows:
Slant medium composition: soluble starch 50g/L, glucose 40g/L, dipotassium hydrogen phosphate 0.5g/L, seven water sulfuric acid
Magnesium 5g/L, sodium chloride 5g/L, calcium carbonate 1g/L and agar 20g/L composition, pH value 7.2 are prepared with water；It 115 DEG C, sterilizes 20 points
Clock.PH to 7.2 is adjusted with 100g/L sodium hydroxide solution before sterilizing.Inclined-plane culture condition: cyaneogriseus streptomyces BJX003 is inoculated with
Onto slant medium, at 28 DEG C, envionmental humidity is cultivated 10-12 days under the conditions of being 50-60%.
Seed culture medium composition: glucose 10g/L, maltodextrin 20g/L, beancake powder 15g/L, yeast powder 10g/L, sulphur
Sour magnesium 1g/L, dipotassium hydrogen phosphate 1g/L, are prepared with water.The pH value of seed culture medium is 7.2,121 DEG C, and sterilize 30min.Seed training
The condition of supporting: take the good lawn 1cm of inclined-plane culture2, transferred fill 30mL sterilizing seed culture medium seed bottle in,
Under conditions of 28 DEG C, envionmental humidity 50-60%, revolving speed 180-200rpm, radius of turn 50mm, shaken cultivation 44-
48 hours (to logarithmic growth phase), obtain shake-flask seed culture solution.Shake-flask seed culture solution is connected to by 1% (v/v) inoculum concentration
In 50L seeding tank equipped with 25L seed culture medium, at 28 DEG C, revolving speed 200rpm, ventilation ratio 1:0.5, tank presses 0.05MPa, dissolved oxygen
Control cultivates 20 hours under conditions of 40% or more, obtains seed culture fluid.
The method of the raising nimoctin yield of embodiment 1
Inclined-plane culture, seed culture and fermented and cultured are carried out to cyaneogriseus streptomyces BJX003.
Fermentation culture method: seed culture fluid is taken to be inoculated in by the inoculum concentration of 5% (v/v) equipped with 25L fermentation medium
In 50L fermentor, at 28 DEG C, revolving speed 200rpm, ventilation ratio 1:0.5, tank presses 0.03MPa, item of the dissolved oxygen control 40% or more
Under part, fermented and cultured 240 hours, fermentation liquid is obtained.In fermentation process, when the concentration of reduced sugar in fermentation liquid is lower than 15g/L,
The glucose solution containing soybean cake powder is added, and the concentration of reduced sugar is controlled in 15g/L；Wherein glucose in feed supplement liquid
Concentration is 350g/L, and the concentration of soybean cake powder is 100g/L, is prepared with water.
Ferment the fermentation medium used are as follows: glucose 50g/L, lactose 35g/L, yeast powder 35g/L, dipotassium hydrogen phosphate
2g/L, epsom salt 1.2g/L, sodium chloride 10g/L and calcium carbonate 20g/L, pH value 7.2 are prepared with water.
The method of the raising nimoctin yield of embodiment 2
Inclined-plane culture, seed culture and fermented and cultured are carried out to cyaneogriseus streptomyces BJX003.
Fermentation culture method: seed culture fluid is taken to be inoculated in by the inoculum concentration of 5% (v/v) equipped with 25L fermentation medium
In 50L fermentor, at 26 DEG C, revolving speed 180rpm, ventilation ratio 1:0.3, tank presses 0.04MPa, item of the dissolved oxygen control 40% or more
Under part, fermented and cultured 192 hours, fermentation liquid is obtained.In fermentation process, when the concentration of reduced sugar in fermentation liquid is lower than 12g/L,
The glucose solution containing soybean cake powder is added, and the concentration of reduced sugar is controlled in 12g/L；Wherein glucose in feed supplement liquid
Concentration is 300g/L, and the concentration of soybean cake powder is 75g/L.
Ferment the fermentation medium used are as follows: glucose 40g/L, lactose 20g/L, yeast powder 20g/L, dipotassium hydrogen phosphate
1g/L, epsom salt 0.5g/L, sodium chloride 8.5g/L and calcium carbonate 10g/L, pH value 7.2 are prepared with water.
The method of the raising nimoctin yield of embodiment 3
Inclined-plane culture, seed culture and fermented and cultured are carried out to cyaneogriseus streptomyces BJX003.
Fermentation culture method: seed culture fluid is taken to be inoculated in by the inoculum concentration of 5% (v/v) equipped with 25L fermentation medium
In 50L fermentor, at 30 DEG C, revolving speed 220rpm, ventilation ratio 1:0.7, tank presses 0.03MPa, item of the dissolved oxygen control 40% or more
Under part, fermented and cultured 264 hours, fermentation liquid is obtained.In fermentation process, when the concentration of reduced sugar in fermentation liquid is lower than 18g/L,
The glucose solution containing soybean cake powder is added, and the concentration of reduced sugar is controlled in 18g/L；Wherein glucose in feed supplement liquid
Concentration is 400g/L, and the concentration of soybean cake powder is 125g/L.
Ferment the fermentation medium used are as follows: glucose 60g/L, lactose 50g/L, yeast powder 50g/L, dipotassium hydrogen phosphate
3g/L, epsom salt 2g/L, sodium chloride 12.5g/L and calcium carbonate 30g/L, pH value 7.2 are prepared with water.
The extraction of 4 nimoctin of embodiment and yield detection
1, extracting method is as follows:
Fermentation liquor pretreatment: taking 1ml fermentation liquid in centrifuge tube, and 9ml methanol is added, after mixing ultrasound 15 minutes
(ultrasonic power 200w) is centrifuged 15min in 4000rpm, spare then by 0.22 μm of membrane filtration of supernatant.
2, HPLC is analyzed:
Using 1100 type efficient liquid phase of Agilent
Chromatograph, 150mm × 4.6mm (id) stainless steel column, inside fills out the C18 of Reliasil Cl8 model Wondasil
Reversed-phase column, takes above-mentioned filtrate, 20 μ L of sample volume by 25 DEG C of column temperature；With methanol: water (volume ratio 17:3) is that mobile phase is separated,
Flow velocity 1mL/min is detected at wavelength 240nm using UV detector and is automatically formed separating spectrum.
The result shows that the fermentation yield of nimoctin is respectively 3427 μ g/mL, 3385 μ g/mL and 3410 in embodiment 1-3
μg/mL。
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of method for improving nimoctin yield, which is characterized in that the bluish grey chain for being CGMCC No.8731 with deposit number
Mould (Streptomyces cyaneogriseus) BJX003 carries out the fermenting and producing of nimoctin as production bacterial strain, when
When the concentration of reduced sugar is lower than 18g/L in fermentation liquid, the glucose solution containing soybean cake powder is added, and by the concentration of reduced sugar
Control is in 12-18g/L；Wherein the concentration of glucose is 300-400g/L in feed supplement liquid, and the concentration of soybean cake powder is 75-125g/
L。
2. the method according to claim 1, wherein the fermentation medium that fermentation uses are as follows: glucose 40-60g/
L, lactose 20-50g/L, yeast powder 20-50g/L, dipotassium hydrogen phosphate 1-3g/L, epsom salt 0.5-2g/L, sodium chloride 8.5-
12.5g/L and calcium carbonate 10-30g/L, pH value 7.2 are prepared with water.
3. the method according to claim 1, wherein the fermentation medium that fermentation uses are as follows: glucose 50g/L,
Lactose 35g/L, yeast powder 35g/L, dipotassium hydrogen phosphate 2g/L, epsom salt 1.2g/L, sodium chloride 10g/L and calcium carbonate 20g/
L。
4. method according to claim 1-3, which is characterized in that the condition of fermentation are as follows: 26-30 DEG C, revolving speed
150-220rpm, ventilation ratio 1:0.3-0.7, tank press 0.03-0.04MPa, and dissolved oxygen control is 40% or more, fermentation period 192-
264hr。
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| CN107815477A (en) * | 2016-09-12 | 2018-03-20 | 牡丹江佰佳信生物科技有限公司 | A kind of method of fermenting and producing moxidectin |
| CN110387390B (en) * | 2019-06-27 | 2021-06-01 | 华东理工大学青岛创新研究院 | Fermentation medium and fermentation process |
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| WO1993018779A1 (en) * | 1992-03-23 | 1993-09-30 | Merck & Co., Inc. | A strain of streptomyces avermitilis glycosylates avermectin compounds |
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Denomination of invention: A Method to Increase the Yield of Nimoxetine Effective date of registration: 20230522 Granted publication date: 20190108 Pledgee: Mudanjiang New Area Branch of Longjiang Bank Co.,Ltd. Pledgor: MUDANJIANG BIOSEEN BIOLOGICAL CO.,LTD. Registration number: Y2023230000041 |
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