CN109371073A - A kind of method of fermenting and producing nonactin - Google Patents
A kind of method of fermenting and producing nonactin Download PDFInfo
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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Abstract
The present invention relates to a kind of methods of fermenting and producing nonactin, belong to fermentation technical field；Resin is 5-40g/L containing preferred amounts in the fermentation medium；The method of the fermenting and producing nonactin includes that the seed liquor of streptomycete is inoculated in aforesaid fermentation culture medium to carry out liquid fermentation, and after fermented and cultured 96-168 hours, nonactin is extracted from fermentation liquid.The fermentation unit of nonactin is greatly improved in fermentation medium and fermentation process of the present invention, the large-scale production suitable for nonactin.
Description
The application is to applying date 2015.11.05 day, application number 201510744556.6, and " one kind is for mentioning for denomination of invention
The divisional application of the application for a patent for invention of the fermentation medium and fermentation process of high nonactin yield ".
Technical field
The present invention relates to fermentation technical fields, and in particular to a kind of method of fermenting and producing nonactin.
Background technique
Nonactin (Nonactin) is in the macrocyclic polyether ion carrier antibiotic large family referred to as big four lactone of ring
The simplest member of structure.These big four lactones of ring include nonactin, monactin (monactin), dinactin
(dinactin), trinactin (trinactin) and four viable bacterias are plain (tetranactin).Nonactin is a kind of ionophore,
Cation can be transported by biomembrane and artificial membrane, therefore in conjunction with metal ions such as ammonium ion, calcium ion, potassium ions
It can be used for manufacturing ammonium electrode plasma electrodes selective, microelectrode and sensor.In addition, nonactin also have it is antibacterial
Activity；Antitumor activity；And artitumor multi-medicine-resistant；And it is effective Anti-virus agent, may be effectively opened
Send out into AIDS drugs.
Currently, the approach for the acquisition nonactin reported in the world has:
Chemistry is fully synthetic: the fully synthetic of nonactin may be implemented, but because of the complexity of nonactin structure, so that closing
Many at step, total recovery is very low, thus chemistry is fully synthetic that prepare nonactin unrealistic.
Document Identification of antifungal natural products viaSaccharomyces cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and
Mode of action. Med Mycol., 2013,51:280-289 report obtains nonactin using streptomycete fermentation,
7.2L fermentation medium finally obtains 2.7mg nonactin, and yield is only 0.38mg/L.Fermentation medium used are as follows: yeast extract 2
G/L, 5 g/L of brewer's wort, glucose 2 g/L, pH 7.0；Fermentation condition is 250 rpm, and 28 DEG C are cultivated 7 days.Document
Streptokordin, a new cytotoxic compound of the methylpyridine class from a
Marine-derived Streptomyces sp. KORDI-3238. J. Antibiot., 2006,59:234-240 report
Road obtains nonactin using streptomycete fermentation, and 8L fermentation medium finally obtains 17.4mg nonactin, and yield is only
2.18mg/L.Fermentation medium used are as follows: 1 g/L of beef extract, 1 g/L of yeast extract, 2 g/L of acid hydrolyzed casein, glucose 5
G/L, 5 g/L of brewer's wort；Fermentation condition is 200 rpm, and 27 DEG C are cultivated 7 days.
Currently, nonactin production cost is higher, fermentation period is long, yield is low, and fermentation results are undesirable, it is difficult to realize work
Industry.Culture medium and fermentation process must be changed by so that fermentation level is increased.
Summary of the invention
It is an object of the present invention to provide a kind of for improving the fermentation medium and fermentation process of nonactin yield, to solve
The problem of technologies such as certainly prior art fermenting and producing nonactin yield is low.
Realize that technical scheme is as follows:
It is a kind of for improving the fermentation medium of nonactin yield, contain resin in the fermentation medium.
Preferably, resin content is 5-40g/L in the fermentation medium；
It is further preferred that resin content is 10-30g/L in the fermentation medium；
It is further preferred that resin content is 20g/L in the fermentation medium；
In terms of the content is come by round (such as fermentor or shaking flask) practical liquid amount.
Preferably, the resin is in HP-20 resin, XAD-16 resin, XAD-8 resin, D202 resin, XD-5 resin etc.
One or more；Further preferably HP-20 resin, XAD-16 resin.
Also contain carbon source in the fermentation medium.
Preferably, carbon source content is 4.0-15.0g/L in the fermentation medium.
It is further preferred that carbon source content is 6.0-10.0g/L in the fermentation medium.
Preferably, the carbon source is one of oatmeal, glycerol, glucose, cornstarch, maltodextrin etc. or several
Kind.
Also contain nitrogen source in the fermentation medium.
Preferably, nitrogen source content is 0.2-3.5g/L in the fermentation medium.
It is further preferred that nitrogen source content is 0.5-1.5g/L in the fermentation medium.
Preferably, the nitrogen source be bean cake powder, cottonseed meal, corn syrup hydrolyzate, acid hydrolyzed casein, yeast extract/
One or more of powder etc..
Preferably, the carbon source is oatmeal, glycerol and glucose；The nitrogen source is acid hydrolyzed casein, yeast leaching
Cream/powder.
Further, the fermentation medium also includes inorganic salts used in normal fermentation culture medium.
The inorganic salts dosage preferred range is 0.5-2.5g/L；The inorganic salts are preferably calcium carbonate；
Specifically, the fermentation medium contains: resin 15-25g/L, oatmeal 2.5-7.5g/L, glycerol 0.5-5g/L, grape
Sugared 0.1-2.5g/L, yeast extract 0.1-2.5g/L, acid hydrolyzed casein 0.1-1.0g/L, calcium carbonate 0.5-2.5g/L.
In a preferred embodiment of the present invention, the fermentation medium contains: HP-20 resin 10.0 g/L, XAD-
16 resin, 10.0 g/L, 5.0 g/L of oatmeal, 2.5 g/L of glycerol, 0.5 g/L of glucose, 0.5 g/L of yeast extract, sour water solution junket
0.2 g/L of albumen, 1.0 g/L of calcium carbonate.
In another preferred embodiment of the invention, the fermentation medium contains: 25.0 g/L of HP-20 resin, swallow
Oatmeal 7.5g/L, glycerol 5g/L, glucose 2.5g/L, yeast extract 2.5g/L, acid hydrolyzed casein 1g/L, calcium carbonate 2.5g/L.
In another preferred embodiment of the invention, the fermentation medium contains: XAD-16 resin 15.0g/L, swallow
Oatmeal 2.5g/L, glycerol 0.5g/L, glucose 0.1g/L, yeast extract 0.1g/L, acid hydrolyzed casein 0.1g/L, calcium carbonate
0.5g/L。
The fermentation medium pH 6.8-7.5, preferably pH 7.2.
The fermentation medium can be used conventional method and prepare and sterilize；Such as 121 DEG C of sterilizing 30-60 min.
The present invention also provides a kind of methods of fermenting and producing nonactin, aforementioned including the seed liquor of streptomycete to be inoculated in
It is carried out liquid fermentation in fermentation medium, extracts nonactin after fermented and cultured from fermentation liquid.
Preferably, described fermented incubation time 96-168 hours；The fermented and cultured temperature is 28-32 DEG C；Fermented and cultured
PH is 6.8-7.5 in the process.
Conventional method can be used in the preparation of the seed liquor of the streptomycete.
Such as the slant culture of streptomycete is inoculated in seed culture medium, in 28-30 DEG C, revolving speed 180-250rpm is cultivated
2-3 days, obtain seed liquor.
This field conventional medium can be used in the seed culture medium of the streptomycete.
Preferably, the streptomycete seed culture medium contains: 10.0 g/L of brewer's wort, 4.0 g/L of yeast extract, glucose
4.0 g/L；Its pH7.3；30 min of general 121 DEG C of sterilizings.
Conventional method can be used in the method that nonactin is extracted from fermentation liquid.
Preferably, the method that nonactin is extracted from fermentation liquid includes:
By the filtering fermentation liquor, mycelia and filtrate two parts are obtained；(general 5-7 days) are lyophilized in the mycelia, are extracted with methanol
1-3 times, ethyl acetate extracts 1-3 times, and n-hexane extracts 1-2 times, obtains mycelia extracting solution；By the isometric acetic acid second of the filtrate
Ester extracts 1-3 times, obtains filtrate extracting solution；Merge the mycelia extracting solution and filtrate extracting solution, medicinal extract is concentrated under reduced pressure to obtain；It will be described
Medicinal extract carries out column chromatography with silica gel (230-400 mesh), respectively with 0%, 50%, 80%, 100% ethyl acetate-hexane and 100%
Methanol carries out gradient elution, collects 50% and 80% ethyl acetate-hexane eluent, crystallizing at room temperature, and with acetone recrystallization,
Up to (nonactin fine work).
Wherein, the streptomycete be preferably streptomyces griseus grey subspecies (Streptomyces griseus subsp.Griseus), deposit number CGMCC 4.181 can be from China General Microbiological culture presevation administrative center (China
General Microbiological Culture Collection Center, CGMCC) it buys.
The beneficial effects of the present invention are:
The present invention solves that nonactin production cost is higher, and the undesirable deficiency of fermentation level provides one kind and is conducive to ash
Color streptomycete grey subspecies (Streptomyces griseus subsp. Griseus) high-efficiency fermenting production nonactin
(Nonactin) fermentation medium and corresponding fermentation process.By dexterously adding resin in the fermentation medium, promote
Into the output increased of nonactin, and by the fermentation parameters such as preferred fermentation medium and optimization culture temperature, pH, thus
The fermentation unit of nonactin is greatly improved, the large-scale production suitable for nonactin.Experiment shows to mention in the present invention
Under the condition of culture of confession, 20 L fermentation mediums can get 1.6g nonactin, and yield is up to 80 mg/L, and isolates and purifies work
Skill is simple, can be used for the industrialized production of nonactin.
Detailed description of the invention
Fig. 1 is 1 nonactin of experimental example1H-NMR map；
Fig. 2 is 1 nonactin of experimental example13C-NMR map.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Each component in following embodiment in culture medium prescription is commercial goods.
Experimental strain be streptomyces griseus grey subspecies (Streptomyces griseus subsp. Griseus)
CGMCC4.181, can be from China General Microbiological culture presevation administrative center (China General Microbiological
Culture Collection Center, CGMCC) it buys.
The HPLC testing conditions of fermentation liquid nonactin content are as follows in following embodiment and comparative example:
Instrument: 2690 high performance liquid chromatograph of Waters；
Chromatographic column: 5 μm of 110,250 x of NX-C18,4.6 mm of Phenomenex Gemini；
Mobile phase: A methanol, B water.Flow velocity 1mL/min；Gradient setting is as follows:
| Time (min) | A% | B% |
| 0 | 40 | 60 |
| 20 | 90 | 10 |
| 25 | 40 | 60 |
| 30 | 40 | 60 |
Detector: 2100 evaporative light scattering detector of PL-ELS.
Embodiment 1
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, cultivates item
Part: it 30 DEG C of temperature, 200 rpm of revolving speed, cultivates 2 days；Obtain seed liquor；By seed liquor with 2.5%(V/V) inoculum concentration be inoculated in
It ferments in 40 2L shaking flasks equipped with 500 mL fermentation mediums, 30 DEG C of fermentation temperature, 200 rpm of revolving speed, fermentation period 5
It.Sampling (HPLC) measurement after fermentation, nonactin potency 142mg/L.
The seed culture medium composition: 10.0 g/L of brewer's wort, 4.0 g/L of yeast extract, 4.0 g/L of glucose, water surplus；
Its pH7.3；121 DEG C of 30 min of sterilizing.
The fermentation medium composition: 10.0 g/L of HP-20 resin, 10.0 g/L of XAD-16 resin, 5.0 g/ of oatmeal
L, 2.5 g/L of glycerol, 0.5 g/L of glucose, 0.5 g/L of yeast extract, 0.2 g/L of acid hydrolyzed casein, 1.0 g/L of calcium carbonate,
Water surplus；Its pH7.2；121 DEG C of 60 min of sterilizing.
By above-mentioned filtering fermentation liquor, it is divided into mycelia and filtrate two parts；Mycelia is lyophilized 7 days, extracts 3 with 1 L methanol
Secondary, 1 L ethyl acetate extracts 3 times, and 1 L n-hexane extracts 2 times, obtains mycelia extracting solution；Filtrate extracts 3 with isometric ethyl acetate
It is secondary, obtain filtrate extracting solution；Merge the mycelia extracting solution and filtrate extracting solution, about 16 g of medicinal extract is concentrated under reduced pressure to obtain.Medicinal extract is used
Silica gel (230-400 mesh) carries out column chromatography, each with 0%, 50%, 80%, 100% ethyl acetate-hexane and 100% methanol respectively
1 L carries out gradient elution, collects 50% and 80% ethyl acetate-hexane eluent, crystallizing at room temperature, and with acetone recrystallization,
Obtain 1.6 g of nonactin fine work；80 mg/L of yield.
Embodiment 2
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, cultivates item
Part: it 30 DEG C of temperature, 200 rpm of revolving speed, cultivates 2 days；Obtain seed liquor；By seed liquor with 3%(V/V) inoculum concentration be inoculated in 40
It ferments in a 2L shaking flask that 500 mL fermentation mediums are housed, 28 DEG C of fermentation temperature, 250 rpm of revolving speed, fermentation period 6
It.Sampling (HPLC) measurement after fermentation, nonactin potency 125mg/L.
The seed culture medium is the same as embodiment 1.
The fermentation medium composition: HP-20 resin 25.0 g/L, oatmeal 7.5g/L, glycerol 5g/L, glucose
2.5g/L, yeast extract 2.5g/L, acid hydrolyzed casein 1g/L, calcium carbonate 2.5g/L, water surplus；Its pH7.2；121 DEG C of sterilizings 60
min。
Nonactin is extracted from fermentation liquid according to the same manner as in Example 1, obtains 1.5 g of nonactin fine work；Yield
75 mg/L。
Embodiment 3
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, cultivates item
Part: it 30 DEG C of temperature, 200 rpm of revolving speed, cultivates 2 days；Obtain seed liquor；By seed liquor with 2%(V/V) inoculum concentration be inoculated in 40
It ferments in a 2L shaking flask that 500 mL fermentation mediums are housed, 28 DEG C of fermentation temperature, 250 rpm of revolving speed, fermentation period 6
It.Sampling (HPLC) measurement after fermentation, nonactin potency 98mg/L.
The seed culture medium is the same as embodiment 1.
The fermentation medium composition: XAD-16 resin 15.0g/L, oatmeal 2.5g/L, glycerol 0.5g/L, glucose
0.1g/L, yeast extract 0.1g/L, acid hydrolyzed casein 0.1g/L, calcium carbonate 0.5g/L, water surplus；Its pH7.2；121 DEG C of sterilizings
60 min。
Nonactin is extracted from fermentation liquid according to the same manner as in Example 1, obtains 1.2 g of nonactin fine work；Yield
60 mg/L。
Comparative example 1:
Nonactin fermentation liquid is made according to the same manner as in Example 1, difference is only that in fermentation medium without HP-20 tree
Rouge and XAD-16 resin.Sampling (HPLC) measurement after fermentation, nonactin potency 4mg/L.
Comparative example 2:
Nonactin fermentation liquid is made according to the same manner as in Example 2, difference is only that in fermentation medium without HP-20 tree
Rouge.Sampling (HPLC) measurement after fermentation, nonactin potency 3mg/L.
Nonactin fermentation liquid is made according to the same manner as in Example 3, difference, which is only that in fermentation medium, to be free of
XAD-16 resin.Sampling (HPLC) measurement after fermentation, nonactin potency 1mg/L.
Experimental example 1
Nonactin made from embodiment 1 is colorless needle crystals；The structure of nonactin passes through1H-NMR and13C-NMR is able to really
Recognize；And be further confirmed by HRMS, HRMS detects [M+Na]+ 759.4293 quasi-molecular ion peak, with no viable bacteria
The calculating molecular weight [M+Na] of element+759.4295 coincideing；Its1H-NMR and13C-NMR spectrogram is shown in attached drawing 1 and attached drawing 2 respectively.
The nuclear magnetic resonance modal data of product nonactin:
1H-NMR (CDCl3, 500 MHz) δ 4.97 (1H, m, H-8), 4.01 (1H, m, H-3), 3.85 (1H,
M, H-6), 2.50 (1H, m, H-2), 1.98 (1H, m, H-5), 1.92 (1H, m, H-4), 1.76 (2H,
M, H-7), 1.61 (1H, m, H-4), 1.49 (1H, m, H-5), 1.23 (3H, d,J=6.3 Hz, 8-CH3),
1.08 (3H, d,J=7.0 Hz, 2-CH3)。
13C-NMR (CDCl3, 125 MHz) and δ 174.4 (s, C-1), 80.2 (d, C-3), 76.6 (d, C-
6), 69.3 (d, C-8), 45.4 (d, C-2), 42.5 (t, C-7), 31.6 (t, C-5), 28.3 (t, C-
4), 20.7 (q, 8-CH3), 13.0 (q, 2-CH3)。
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 (7)
1. a kind of for improving the fermentation medium of nonactin yield, which is characterized in that the fermentation medium contains: HP-
20 resin 25.0 g/L, oatmeal 7.5g/L, glycerol 5g/L, glucose 2.5g/L, yeast extract 2.5g/L, acid hydrolyzed casein
1g/L, calcium carbonate 2.5g/L.
2. fermentation medium according to claim 1, which is characterized in that the fermentation medium composition: HP-20 resin
25.0 g/L, oatmeal 7.5g/L, glycerol 5g/L, glucose 2.5g/L, yeast extract 2.5g/L, acid hydrolyzed casein 1g/L, carbon
Sour calcium 2.5g/L, water surplus；Its pH7.2.
3. fermentation medium according to claim 1 or 2, which is characterized in that the strepto- for fermenting and producing nonactin
Bacterium be streptomyces griseus grey subspecies (Streptomyces griseus subsp. Griseus), deposit number CGMCC
4.181。
4. a kind of method of fermenting and producing nonactin, which is characterized in that wanted including the seed liquor of streptomycete is inoculated in right
It asks and is carried out liquid fermentation in the described in any item fermentation mediums of 1-3, extract nonactin after fermented and cultured from fermentation liquid.
5. according to the method described in claim 4, it is characterized in that, described fermented incubation time 96-168 hours；And/or
The fermented and cultured temperature is 28-32 DEG C；And/or
PH is 6.8-7.5 during fermented and cultured.
6. the method for fermenting and producing nonactin according to claim 4 or 5, which is characterized in that the streptomycete seed
Culture medium contains: 10.0 g/L of brewer's wort, 4.0 g/L of yeast extract, 4.0 g/L of glucose；Its pH7.3.
7. according to the method for the described in any item fermenting and producing nonactins of claim 4-6, which is characterized in that the streptomycete
For streptomyces griseus grey subspecies (Streptomyces griseus subsp. Griseus), deposit number CGMCC
4.181。
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| CN201811594585.9A CN109371073B (en) | 2015-11-05 | 2015-11-05 | Method for producing non-viable bacteria element by fermentation |
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| US7416870B2 (en) * | 2002-08-22 | 2008-08-26 | Wisconsin Alumni Research Foundation | Methods of directing C-O bond formation utilizing a type II polyketide synthase system |
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| WOO A J等: "Nonactin Biosynthesis: the Product of nonS Catalyzes the Formation of the Furan Ring of Nonactic Acid", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
Also Published As
| Publication number | Publication date |
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| CN109628520A (en) | 2019-04-16 |
| CN109628520B (en) | 2022-02-01 |
| CN109576319A (en) | 2019-04-05 |
| CN109371073B (en) | 2021-11-05 |
| CN109576318A (en) | 2019-04-05 |
| CN109576318B (en) | 2022-02-01 |
| CN109576319B (en) | 2022-02-01 |
| CN105368892A (en) | 2016-03-02 |
| CN105368892B (en) | 2018-12-18 |
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Application publication date: 20190222 Assignee: Guilin Aijie Electronic Technology Co.,Ltd. Assignor: GUILIN University OF ELECTRONIC TECHNOLOGY Contract record no.: X2022450000224 Denomination of invention: A method of producing avidin by fermentation Granted publication date: 20211105 License type: Common License Record date: 20221206 Application publication date: 20190222 Assignee: Guilin Xinjiatianxia Food Co.,Ltd. Assignor: GUILIN University OF ELECTRONIC TECHNOLOGY Contract record no.: X2022450000222 Denomination of invention: A method of producing avidin by fermentation Granted publication date: 20211105 License type: Common License Record date: 20221206 |