CN109593808A - Daptomycin fermentation medium and preparation method thereof - Google Patents
Daptomycin fermentation medium and preparation method thereof Download PDFInfo
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Abstract
本发明公开了一种达托霉素发酵培养基及其制备方法。该发酵培养基包括7.2‑11g/100mL的碳源，1.65‑4.0g/100mL的氮源，0.086‑0.5g/100mL的无机盐，0‑0.1g/100mL的微量元素；所述碳源包括麦芽糊精和碳源A，所述麦芽糊精的用量为7.2‑9g/100mL，所述碳源A为糖蜜或红糖，所述碳源A的用量为0‑1.8g/100mL；所述氮源为榴花酵母粉、黄豆粉、豆粕、花生粉和羽毛粉中的一种或多种。本发明的发酵培养基用于培养玫瑰孢链霉菌可以提高达托霉素的产量，同时大大降低生产成本，具有非常好的工业化应用前景。The invention discloses a daptomycin fermentation medium and a preparation method thereof. The fermentation medium includes a carbon source of 7.2-11g/100mL, a nitrogen source of 1.65-4.0g/100mL, an inorganic salt of 0.086-0.5g/100mL, and a trace element of 0-0.1g/100mL; the carbon source includes malt Dextrin and carbon source A, the consumption of the maltodextrin is 7.2-9g/100mL, the carbon source A is molasses or brown sugar, the consumption of the carbon source A is 0-1.8g/100mL; the nitrogen source It is one or more of pomegranate yeast powder, soybean powder, soybean meal, peanut powder and feather powder. The fermentation medium of the present invention can increase the yield of daptomycin when used for culturing Streptomyces roseospora, and at the same time greatly reduce the production cost, and has a very good prospect of industrial application.
Description
Technical field
The present invention relates to a kind of Daptomycin fermentation mediums and preparation method thereof.
Background technique
Daptomycin is a member of lipopeptid class family, belongs to ten lipopeptid structure group of ring, by Streptomyces roseosporus
(Streptomyces roseosporus) ferments and obtains, and is the Cyclic lipopeptide antibiotic that the first success in the current whole world is developed.It reaches
Tobramycin can destroy bacterial cell membrane function in many aspects, and kill gram-positive bacteria, and it is fast that characteristic shows as inhibiting effect
Fast, low with concentration needed for extracorporeal disinfecting effect in vivo, the validity time of antibiotic effect is long.
Space light sea etc. optimizes Daptomycin basal fermentation medium, original basis fermentation medium group subpackage by response phase method
Include glucose 0.75g/100mL, dextrin 1.0g/100mL, soluble starch 1.0g/100mL, peptone 0.85g/100mL, ferment
Mother leaching powder 0.6g/100mL, casein 0.85g/100mL, L-Aspartic acid 0.1g/100mL, glutamic acid 0.05g/100mL,
MgSO4 0.05g/100mL、K2SO40.05g/100mL、K2HPO40.05g/100mL, pH value 7.2-7.4 are pasted after optimization
The optium concentration of essence, soluble starch and L-Aspartic acid is respectively 0.7g/100mL, 0.67g/100mL, 0.04g/100mL.
With this condition, the yield of Daptomycin reaches 185.3mg/L, and 73.7% is improved before relatively optimizing, and (space light sea, Yin Yanli open
Yao response phase method optimizes research [J] chemistry and bioengineering of Daptomycin fermentation medium, 2012.10.011).But it ferments
In culture medium prescription, the material that carbon source should be low using valence as far as possible, to increase economic efficiency, and not only price is higher for soluble starch
And it is comparatively laborious in subsequent processing, starch residual is more.In addition yeast extract and casein have been used in nitrogen source, this is all into
This higher fermentation raw material, then cost can be made to increase suddenly when expanding production, therefore need to be replaced with more cheap raw material.
He Meiru optimizes Daptomycin fermentation medium by orthogonal experiment, it is rear again using design, steepest hill climbing experiment,
The statistical method that CCD experiment combines has carried out the nutrient media components and main external condition that influence Daptomycin fermentation excellent
Change and evaluate, it is determined that Daptomycin shaking flask optimal conditions of fermentation: glucose 1.3g/100mL, dextrin 4.0g/100mL, cheese
Plain 1.2g/100mL, soybean cake powder 0.45g/100mL, L-Aspartic acid 0.26g/100mL, K2SO40.41g/100mL, initially
PH 8., precursor add time 26h, and the predicted value of Daptomycin reaches 184.67mg/L, and fermentation confirmatory experiment measures Daptomycin
Yield be 191.99mg/L, than optimization before improve 2.25 times (He Meiru Streptomyces roseosporus produce Daptomycin zymotechniques
Research [D] Zhejiang University, 2012.).Nevertheless, its final fermentation yield is still lower, it is unable to reach industrialized water
It is flat.
Currently, cost of material used in producing daptomycin by fermentation is higher, it is not able to satisfy the demand of industrialized production.Cause
This, it is necessary to improve fermentation medium, the fermentation medium that exploitation can reduce cost, improve Daptomycin yield.
Summary of the invention
The technical problem to be solved by the present invention is to overcome current fermentation medium culture Streptomyces roseosporus cost of material
The not high status of excessively high and yield, and one kind is provided and can reduce Daptomycin fermentation medium cost and improve Daptomycin production
The fermentation medium of amount.Using fermentation medium culture Streptomyces roseosporus of the invention, production cost is greatly reduced, it is more suitable
In industrialized production, while the fermentation unit of Daptomycin is also improved, improves production efficiency.
The present invention provides a kind of Daptomycin fermentation mediums comprising the carbon source of 7.2-11g/100mL, 1.65-
The nitrogen source of 4.0g/100mL, the inorganic salts of 0.086-0.5g/100mL, the microelement of 0-0.1g/100mL；The carbon source includes
Maltodextrin and carbon source A, the dosage of the maltodextrin are 7.2-9g/100mL；The carbon source A is molasses or brown sugar, dosage
For 0-1.8g/100mL；The nitrogen source is one of pomegranate yeast powder, soybean powder, dregs of beans, peanut powder and feather meal or a variety of.
Wherein, the strain of the Daptomycin fermentation medium culture is generally rose born of the same parents chain as known to those skilled in the art
Mould (Streptomyces roseosporus), preferably rose born of the same parents streptomycete NRRL11379.
Wherein, the carbon source may include glucose, and when the carbon source contains glucose, the proportion of the carbon source is malt
Dextrin 7.2g/100mL, glucose 1.8g/100mL.
Wherein, the carbon source A is preferably molasses, and the dosage of the molasses is preferably 1.0-1.8g/100mL.
Wherein, the nitrogen source is preferably soybean powder；The dosage of the nitrogen source is preferably 1.65-2.2g/100mL, more
It goodly is 1.8-2.2g/100mL.
Wherein, the inorganic salts are that this field is conventional, preferably in potassium chloride, sodium chloride, potassium sulfate and sodium sulphate
It is one or more, it is more preferably sodium sulphate；The dosage of the inorganic salts is preferably 0.086-0.4g/100mL, more preferably for
0.2-0.3g/100mL。
Wherein, the microelement is that this field is conventional, preferably manganese sulfate, zinc sulfate, copper sulphate, cobalt chloride, molybdenum
One of sour sodium, ferrous sulfate, nickel sulfate and potassium bichromate are a variety of, are more preferably cobalt chloride；The use of the microelement
Amount is preferably 0-0.01g/100mL, is more preferably 0.005-0.01g/100mL.
Wherein, the Daptomycin fermentation medium may also include amino acid and/or amino-acid salt, and the amino acid is this
Field is conventional, preferably one of aspartic acid, leucine, glutamic acid, tyrosine and arginine or a variety of, more preferably for
Glutamic acid；The amino-acid salt is that this field is conventional, preferably sodium glutamate；The use of the amino acid and/or amino-acid salt
Amount is preferably 0-0.3g/100mL but is not 0, is more preferably 0.2-0.3g/100mL.
Wherein, one preferred version of Daptomycin fermentation medium includes the maltodextrin of 7.5g/100mL for it,
The molasses of 1.5g/100mL, the soybean powder of 2.0g/100mL, the iron ammonium sulfate of 0.086g/100mL, the sulfuric acid of 0.3g/100mL
Sodium, the cobalt chloride of 0.005g/100mL, the glutamic acid of 0.3g/100mL.
The ingredients such as the carbon source, nitrogen source and inorganic salts that can be utilized can also be added in fermentation medium of the present invention,
As long as being there is no mutual antagonism between each component in those ingredients and the aforementioned Daptomycin fermentation medium of the present invention
Can, these ingredients can disposably be added in culture medium in advance, realize the high yield quantization of Daptomycin.
Wherein, the preparation method of the fermentation medium is that this field is conventional, and each component is dissolved with water, is preferably steamed
Distilled water dissolution, then with high-temperature sterilization, preferable sterilising conditions are 121 DEG C, 20min.
The present invention also provides a kind of methods of producing daptomycin by fermentation comprising following steps: by rose born of the same parents streptomycete
(Streptomyces roseosporus) is inoculated in aforementioned Daptomycin fermentation medium fermented and cultured production Daptomycin.
Wherein, it when producing Daptomycin with aforementioned Daptomycin fermentation medium fermented and cultured rose born of the same parents streptomycete, uses
Conventional method, i.e., be inoculated in prior culture media for rose born of the same parents streptomycete, then carries out normal fermentation.
Preferably, the method includes the steps: the rose born of the same parents streptomycete seed liquor inoculum concentration of 5-15% in mass ratio is connect
Kind in foregoing fermentation medium, fermentation flask is then cultivated into 5-6 on 28-32 DEG C, the shaking table of revolving speed 200-280rpm
It.
Wherein, further preferably include the steps that harvesting Daptomycin after producing daptomycin by fermentation, the harvest is mould up to holding in the palm
The method that element uses takes supernatant i.e. after being centrifuged again for the methanol of 4 times of volumes is added into supernatant by fermentation liquid centrifuging and taking supernatant
?.
As known to those skilled in the art, with the rose born of the same parents streptomycete seed liquor fermented and cultured production Daptomycin it
Before, rose born of the same parents' streptomyces species are subjected to actication of culture according to this field is conventional, are generally comprised the steps:
(1) rose born of the same parents streptomycete is inoculated on slant medium and is activated；
(2) colony inoculation that activation obtains is obtained into seed liquor in seed culture medium culture.
Preferably, the actication of culture includes the following steps:
(1) rose born of the same parents streptomycete is inoculated on slant medium and is activated；
(2) by the obtained colony inoculation of activation in seed culture medium, on 25-30 DEG C, the shaking table of revolving speed 200-280rpm
Culture obtains seed liquor in 40-46 hours, and the seed culture medium includes 2.0-4.0g/100mL TSB pancreas peptone soybean broth,
3.0-4.0g/100mL maltodextrin, the pH of the seed culture medium is 6.8-7.0.
It is highly preferred that the actication of culture includes the following steps:
(1) rose born of the same parents streptomycete is inoculated on slant medium to cultivate in 30 DEG C of incubators and is activated, it is described oblique
Face culture medium includes 0.4g/100mL yeast extract, 1.0g/100mL fructus hordei germinatus leaching powder, 0.4g/100mL glucose and 1.5g/
100mL agar, the pH of the slant medium are 7.2-7.4；
(2) by the obtained colony inoculation of activation in seed culture medium, in cultivating 40- on 28 DEG C, the shaking table of revolving speed 240rpm
Seed liquor is obtained within 46 hours, the seed culture medium includes 3.0g/100mL TSB pancreas peptone soybean broth, 3.5g/100mL
Maltodextrin, the pH of the seed culture medium are 6.8-7.0.
Wherein, the degree of activation described in step (1) preferably cultivates base matrix and fades to rosiness.
Wherein, in the step of amount of inoculation described in step (2) is preferably 0.8cm × 1.5cm size (1) after activation
Slant medium.
Wherein, preferably microscopy mycelia dissipates seed liquor described in step (2) around at bulk, and mycelia is longer, without miscellaneous
Bacterium, pH7.2-7.6.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) it is compared with existing Daptomycin fermentation medium, Daptomycin fermentation medium used in the present invention
Carbon nitrogen source mainly uses agricultural and sideline product.According to the quotation in Alibaba: 4000 yuan/ton of soybean cake powder, 50,000 yuan of casein/
Ton, 40,000 yuan/ton of yeast extract, 30,000 yuan/ton of peptone, it is known that low in raw material price used herein, and source
Abundant, chemical property stabilization, transport and storage are convenient, are suitable for industrialized production, while the use of agricultural and sideline product makes fermentation liquid
Middle nonhazardous substance residual, can reduce pollution, reduces cost, convenient for the processing of later stage fermentation liquid.
(2) fermentation yield can be improved for cultivating rose born of the same parents streptomycete in fermentation medium of the invention, in original fermentation
In liquid, the yield of Daptomycin at least up to 220mg/L or so, up to 396mg/L, has reached industrialized level.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
In following embodiments, used rose born of the same parents streptomycete is purchased from Agricultural Research Service
Culture Collection (american agriculture studies Culture Collection Center, ARS), rose born of the same parents' strepto- that number is NRRL11379
Bacteria strain.
In following embodiments, the model of used instrument and producer are as shown in table 1 below.
Table 1
In following embodiments, the specification of used reagent and producer are as shown in table 2 below.
Table 2
In following embodiments, using following methods culture rose born of the same parents streptomycete:
(1) aseptically, it will be inoculated into the freeze-drying pipe for preserving Streptomyces roseosporus with sterilized transfer needle
It is cultivated in eggplant bottle equipped with slant medium；The group of the slant medium becomes 0.4g/100mL yeast extract, 1.0g/
100mL fructus hordei germinatus leaching powder, 0.4g/100mL glucose and 1.5g/100mL agar, pH 7.2-7.4；Inclined-plane culture third day is visible
Surface rosiness thallus, the 4th day or the 5th day, color burn, and see that a small amount of canescence spore generates, it is straight to continue culture
A large amount of canescence spores, production of the culture base matrix due to Streptomyces roseosporus pigment itself are generated to can see media surface
Rosiness is changed into, at this point, aseptically, gently scratching the slant medium with spore with disinfection inoculation shovel, taking
The slant medium for covering with spore of 0.8cm × 1.5cm thin as far as possible or so, is inoculated in seed culture medium；
(2) after being inoculated in seed culture medium, seed is obtained in cultivating 40-46 hours on 28 DEG C, the shaking table of revolving speed 240rpm
Liquid, the seed culture medium are made of 3g/100mL TSB pancreas peptone soybean broth, 3.5g/100mL maltodextrin, and described kind
The pH of sub- culture medium is 6.8-7.0；
(3) (2) resulting seed liquor in mass ratio 10% is inoculated in the fermented and cultured prepared with the formula of each embodiment
In base, fermentation flask is cultivated 5-6 days on 30 DEG C, the shaking table of revolving speed 240rpm then, obtains fermentation liquid.
In following embodiments, Daptomycin fermentation unit is surveyed using efficient liquid phase, method particularly includes:
(1) pre-treatment: fermentation liquid 12000rpm centrifugation takes supernatant, the methanol of 4 times of volumes is added, again centrifuging and taking supernatant；
(2) Agilent 1200G4290AA integration liquid chromatograph, chromatographic condition: chromatography HPLC detection method: are used
Column: Agilent Eclipse Plus C18 (4.6mm × 250mm, 5 μm)；Mobile phase is the acetonitrile-containing 0.1% trifluoroacetic acid
Water (volume ratio 45:55)；Flow velocity: 1.0mlmin-1；Column temperature: 30 DEG C；Detection wavelength: 223nm；Sample volume: 20 μ L.This
The retention time of Daptomycin main peak is 9.5min or so under part.
Influence of the 1 maltodextrin dosage of embodiment to Daptomycin yield
Carbon source kind is maltodextrin, glucose in fermentation medium, and dosage is respectively 7.2g/100mL, 1.8g/
100mL, remaining group is divided into pomegranate yeast powder 1.65g/100mL, iron ammonium sulfate 0.086g/100mL, pH are adjusted to 7.5.Culture medium
Component is as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when the dosage of maltodextrin, glucose is respectively 7.2g/100mL, 1.8g/100mL, Daptomycin fermentation is single
Position can maintain and existing fermentation unit is comparable horizontal and can reduce production cost.
Influence of the ratio of 1 maltodextrin of comparative example and glucose to Daptomycin yield
Carbon source kind is maltodextrin, glucose in fermentation medium, and the dosage of glucose is 1.8g/100mL, remaining group
It is divided into pomegranate yeast powder 1.65g/100mL, iron ammonium sulfate 0.086g/100mL, pH are adjusted to 7.5.Change the use of maltodextrin
Amount, each nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when glucose dosage is 1.8g/100mL in carbon source, and the dosage of maltodextrin is not 7.2g/100mL, though
Cost of material is so reduced, but Daptomycin fermentation unit is substantially less than the existing attainable fermentation unit of institute.
Influence of 2 carbon source kind of embodiment to Daptomycin yield
Carbon source is the maltodextrin of 7.2g/100mL in fermentation medium, remaining group is divided into pomegranate yeast powder 1.65g/
100mL, iron ammonium sulfate 0.086g/100mL, pH are adjusted to 7.5.1.8g/100mL is added in addition on the basis of maltodextrin
A kind of carbon source, each nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result can also be added molasses or brown sugar as carbon source on the basis of maltodextrin, reduce cost and further mention
High yield, the fermentation unit highest of Daptomycin when molasses are added.
Influence of 2 sucrose of comparative example to Daptomycin yield
Nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result is added sucrose on the basis of maltodextrin and reduces costs as carbon source, but do not ensure that Daptomycin
Yield.
3 carbon source of embodiment is with the influence for comparing Daptomycin yield
Carbon source dosage is 9g/100mL in fermentation medium, and carbon source kind is maltodextrin and molasses, remaining group is divided into pomegranate
Flower yeast powder 1.65g/100mL, iron ammonium sulfate 0.086g/100mL, pH are adjusted to 7.5.Change the ratio of maltodextrin, molasses,
Each nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, on the basis of controlling cost, the amount of maltodextrin is 7.5g/100mL, the amount of molasses is 1.5g/100mL
When, Daptomycin fermentation unit highest.
Influence of the 4 nitrogen source type of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, iron ammonium sulfate 0.086g/100mL,
PH is adjusted to 7.5.
On the basis of above-mentioned fermentation medium, the nitrogen source of 1.65g/100mL is added, changes the type of nitrogen source, it is each to cultivate
Base component is as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, on the basis of controlling cost, when nitrogen source is the soybean powder of 1.65g/100mL, Daptomycin fermentation unit is most
It is high.
3 yeast extract LM801 of comparative example, influence of the fish meal protein peptone to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, iron ammonium sulfate 0.086g/100mL,
PH is adjusted to 7.5.
On the basis of above-mentioned fermentation medium, yeast extract LM801 or the fish meal protein peptone of 1.65g/100mL are added, respectively
Nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, Daptomycin fermentation unit is higher when nitrogen source is yeast extract or fish meal protein peptone, but cost is excessively high, no
Utilize industrialized production.
Influence of the 5 nitrogen source dosage of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 1.65g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, change the dosage of nitrogen source soybean powder, each nutrient media components are as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when soybean powder dosage is 2g/100mL, Daptomycin fermentation unit highest.
Influence of 6 Inorganic Salts of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, different types of inorganic salts of 0.1g/100mL, each culture medium group are added
Divide as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the sodium sulphate of 0.1g/100mL, Daptomycin fermentation unit highest.
Influence of 4 calcium carbonate of comparative example to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, calcium carbonate 0.1g/100mL, pH are adjusted to 7.5.
According to the formula of above-mentioned fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to above
Experimental result, when adding the calcium carbonate of 0.1g/100mL, Daptomycin fermentation unit is significantly reduced.
Influence of the 7 inorganic salts dosage of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, inorganic salts sodium sulphate is added, changes its dosage, each nutrient media components are as follows
Shown in table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the sodium sulphate of 0.3g/100mL, Daptomycin fermentation unit highest.
Influence of the 8 microelement type of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, sodium sulphate 0.3g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, different types of microelement of 0.005g/100mL is added, it is each to cultivate
Base component is as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the cobalt chloride of 0.005g/100mL, Daptomycin fermentation unit highest.
Influence of the 9 microelement dosage of embodiment to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, sodium sulphate 0.3g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, microelement cobalt chloride is added, changes its dosage, each nutrient media components are such as
Shown in following table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the cobalt chloride of 0.008g/100mL, Daptomycin fermentation unit highest.
Influence of embodiment ten amino acid (salt) type to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, sodium sulphate 0.3g/100mL, 0.008g/100mL cobalt chloride, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, different types of amino acid (salt) of 0.1g/100mL is added, it is each to cultivate
Base component is as shown in the table:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the glutamic acid of 0.1g/100mL, Daptomycin fermentation unit highest.
Influence of embodiment 11 amino acid (salt) dosage to Daptomycin yield
Fermentation medium: maltodextrin 7.5g/100mL, molasses 1.5g/100mL, soybean powder 2.0g/100mL, sulfuric acid are sub-
Iron ammonium 0.086g/100mL, sodium sulphate 0.3g/100mL, cobalt chloride 0.008g/100mL, pH are adjusted to 7.5.
On the basis of above-mentioned fermentation medium, glutamic acid is added, its dosage, each nutrient media components such as following table institute are changed
Show:
According to the formula of above-mentioned each fermentation medium, shake flask fermentation 5 days, fermentation unit is surveyed using efficient liquid phase.According to
Upper experimental result, when adding the glutamic acid of 0.3g/100mL, Daptomycin fermentation unit highest.
Claims (10)
1. a kind of Daptomycin fermentation medium, which is characterized in that it includes the carbon source of 7.2-11g/100mL, 1.65-4.0g/
The nitrogen source of 100mL, the inorganic salts of 0.086-0.5g/100mL, the microelement of 0-0.1g/100mL；
The carbon source includes maltodextrin and carbon source A, and the dosage of the maltodextrin is 7.2-9g/100mL, and the carbon source A is
Molasses or brown sugar, the dosage of the carbon source A are 0-1.8g/100mL；
The nitrogen source is one of pomegranate yeast powder, soybean powder, dregs of beans, peanut powder and feather meal or a variety of.
2. Daptomycin fermentation medium as described in claim 1, which is characterized in that the Daptomycin fermentation medium training
Feeding strain is rose born of the same parents streptomycete (Streptomyces roseosporus), preferably rose born of the same parents streptomycete
NRRL11379；
And/or the carbon source A is molasses.
3. Daptomycin fermentation medium as claimed in claim 2, which is characterized in that the dosage of the molasses is 1.0-
1.8g/100mL。
4. Daptomycin fermentation medium as described in claim 1, which is characterized in that the carbon source includes glucose, institute
The proportion for stating carbon source is maltodextrin 7.2g/100mL, glucose 1.8g/100mL.
5. Daptomycin fermentation medium as described in claim 1, which is characterized in that the nitrogen source is soybean powder；
And/or the inorganic salts are one of potassium chloride, sodium chloride, potassium sulfate and sodium sulphate or a variety of, preferably sulfuric acid
Sodium；
And/or the microelement be manganese sulfate, zinc sulfate, copper sulphate, cobalt chloride, sodium molybdate, ferrous sulfate, nickel sulfate and
One of potassium bichromate is a variety of, preferably cobalt chloride.
6. Daptomycin fermentation medium as described in claim 1, which is characterized in that the Daptomycin fermentation medium is also
Including amino acid and/or amino-acid salt, the amino acid is in aspartic acid, leucine, glutamic acid, tyrosine and arginine
It is one or more, preferably glutamic acid；The amino-acid salt is sodium glutamate；
The dosage of the amino acid and/or amino-acid salt is 0-0.3g/100mL but is not 0, preferably 0.2-0.3g/
100mL。
7. the Daptomycin fermentation medium as described in any one of claim 1~6 claim, which is characterized in that the nitrogen
The dosage in source is 1.65-2.2g/100mL, preferably 1.8-2.2g/100mL；
And/or the dosage of the inorganic salts is 0.086-0.4g/100mL, preferably 0.2-0.3g/100mL；
And/or the dosage of the microelement is 0-0.01g/100mL, preferably 0.005-0.01g/100mL.
8. Daptomycin fermentation medium as described in claim 1, which is characterized in that the Daptomycin fermentation medium packet
Include the maltodextrin of 7.5g/100mL, the molasses of 1.5g/100mL, the soybean powder of 2.0g/100mL, the sulfuric acid of 0.086g/100mL
Ferrous ammonium, the sodium sulphate of 0.3g/100mL, the cobalt chloride of 0.005g/100mL, the glutamic acid of 0.3g/100mL.
9. a kind of method of producing daptomycin by fermentation, which is characterized in that it includes the following steps: any with claim 1~8
Daptomycin fermentation medium fermented and cultured rose born of the same parents streptomycete described in produces Daptomycin.
10. method as claimed in claim 9, which is characterized in that it is comprising steps of press quality for rose born of the same parents' streptomycete seed liquor
It is inoculated in culture Daptomycin fermentation medium as described in any one of claims 1 to 8 than the inoculum concentration of 5-15%, is then existed
28-32 DEG C, cultivate 5-6 days on the shaking table of revolving speed 200-280rpm；
Preferably, the method further includes the steps that actication of culture, the step of actication of culture, includes:
(1) rose born of the same parents streptomycete is inoculated on slant medium and is activated；
(2) it by the obtained colony inoculation of activation in seed culture medium, is cultivated on 25-30 DEG C, the shaking table of revolving speed 200-280rpm
Seed liquor is obtained within 40-46 hours, the seed culture medium includes 2.0-4.0g/100mLTSB pancreas peptone soybean broth, 3.0-
4.0g/100mL maltodextrin, the pH of the seed culture medium are 6.8-7.0；
Preferably, the step of actication of culture, includes:
(1) rose born of the same parents streptomycete is inoculated on slant medium to cultivate in 30 DEG C of incubators and is activated, the inclined-plane training
Feeding base includes 0.4g/100mL yeast extract, 1.0g/100mL fructus hordei germinatus leaching powder, 0.4g/100mL glucose and 1.5g/100mL fine jade
Rouge, the pH of the slant medium are 7.2-7.4；
(2) small in culture 40-46 on 28 DEG C, the shaking table of revolving speed 240rpm by the obtained colony inoculation of activation in seed culture medium
When obtain seed liquor, the seed culture medium includes 3.0g/100mL TSB pancreas peptone soybean broth, 3.5g/100mL malt
Dextrin, the pH of the seed culture medium are 6.8-7.0.
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