CN105368892B - It is a kind of for improving the fermentation medium and fermentation process of nonactin yield - Google Patents
It is a kind of for improving the fermentation medium and fermentation process of nonactin yield Download PDFInfo
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Abstract
The present invention relates to a kind of for improving the fermentation medium and fermentation process of nonactin yield, belongs 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
Technical field
The present invention relates to fermentation technical fields, and in particular to a kind of fermentation medium for improving nonactin yield and
Fermentation process.
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, make
There are many synthesis step, total recovery is very low, thus chemistry is fully synthetic that prepare nonactin unrealistic.
Document Identification of antifungal natural products via Saccharomyces
cerevisiae bioassay:insights into macrotetrolide drug spectrum,potency and
Mode of action.Med Mycol., 2013,51:280-289 report utilizes streptomycete fermentation to obtain nonactin, 7.2L
Fermentation medium finally obtains 2.7mg nonactin, and yield is only 0.38mg/L.Fermentation medium used are as follows: yeast extract 2g/L,
Brewer's wort 5g/L, glucose 2g/L, pH 7.0；Fermentation condition is 250rpm, 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 is obtained using streptomycete fermentation
Nonactin, 8L fermentation medium finally obtain 17.4mg nonactin, and yield is only 2.18mg/L.Fermentation medium used
Are as follows: beef extract 1g/L, yeast extract 1g/L, acid hydrolyzed casein 2g/L, glucose 5g/L, brewer's wort 5g/L；Fermentation condition is
200rpm, 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,
Glucose 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 10.0g/L, XAD-16
Resin 10.0g/L, oatmeal 5.0g/L, glycerol 2.5g/L, glucose 0.5g/L, yeast extract 0.5g/L, acid hydrolyzed casein
0.2g/L, calcium carbonate 1.0g/L.
In another preferred embodiment of the invention, the fermentation medium contains: HP-20 resin 25.0g/L, oat
Piece 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 30-60min.
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: brewer's wort 10.0g/L, yeast extract 4.0g/L, glucose
4.0g/L；Its pH7.3；General 121 DEG C of sterilizings 30min.
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, use methanol
It extracts 1-3 times, ethyl acetate extracts 1-3 times, and n-hexane extracts 1-2 times, obtains mycelia extracting solution；By the isometric second of the filtrate
Acetoacetic 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
The medicinal extract carries out column chromatography with silica gel (230-400 mesh), respectively with 0%, 50%, 80%, 100% ethyl acetate-hexane
And 100% methanol carry out gradient elution, collect 50% and 80% ethyl acetate-hexane eluent, crystallizing at room temperature, Yi Jiyong
Acetone recrystallization is to get (nonactin fine work).
Wherein, the streptomycete is 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, the undesirable deficiency of fermentation level, provides a kind of advantageous
Nonactin is produced in streptomyces griseus grey subspecies (Streptomyces griseus subsp.Griseus) high-efficiency fermenting
(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 greatly
Amplitude improves the fermentation unit of nonactin, the large-scale production suitable for nonactin.Experiment shows to provide in the present invention
Condition of culture under, 20L fermentation medium can get 1.6g nonactin, and yield is up to 80mg/L, and separation purifying technique letter
It is single, it 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 is 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 NX-C18 of Phenomenex Gemini250x 4.6mm；
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, is trained
The condition of supporting: it 30 DEG C of temperature, revolving speed 200rpm, cultivates 2 days；Obtain seed liquor；Seed liquor is connect with the inoculum concentration of 2.5% (V/V)
Kind is fermented in 40 2L shaking flasks equipped with 500mL fermentation medium, 30 DEG C of fermentation temperature, revolving speed 200rpm, fermentation week
Phase 5 days.Sampling (HPLC) measurement after fermentation, nonactin potency 142mg/L.
The seed culture medium composition: brewer's wort 10.0g/L, yeast extract 4.0g/L, glucose 4.0g/L, water surplus；Its
pH7.3；121 DEG C of sterilizing 30min.
Fermentation medium composition: HP-20 resin 10.0g/L, XAD-16 resin 10.0g/L, oatmeal 5.0g/L are sweet
Oily 2.5g/L, glucose 0.5g/L, yeast extract 0.5g/L, acid hydrolyzed casein 0.2g/L, calcium carbonate 1.0g/L, water surplus；Its
pH7.2；121 DEG C of sterilizing 60min.
By above-mentioned filtering fermentation liquor, it is divided into mycelia and filtrate two parts；Mycelia is lyophilized 7 days, is extracted 3 times with 1L methanol,
1L ethyl acetate extracts 3 times, and 1L n-hexane extracts 2 times, obtains mycelia extracting solution；Filtrate is extracted 3 times with isometric ethyl acetate, is obtained
Filtrate extracting solution；Merge the mycelia extracting solution and filtrate extracting solution, medicinal extract about 16g is concentrated under reduced pressure to obtain.By medicinal extract silica gel
(230-400 mesh) carries out column chromatography, each with 0%, 50%, 80%, 100% ethyl acetate-hexane and 100% methanol respectively
1L carries out gradient elution, collects 50% and 80% ethyl acetate-hexane eluent, crystallizing at room temperature, and with acetone recrystallization,
Obtain nonactin fine work 1.6g；Yield 80mg/L.
Embodiment 2
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, is trained
The condition of supporting: it 30 DEG C of temperature, revolving speed 200rpm, cultivates 2 days；Obtain seed liquor；Seed liquor is inoculated with the inoculum concentration of 3% (V/V)
It ferments in 40 2L shaking flasks equipped with 500mL fermentation medium, 28 DEG C of fermentation temperature, revolving speed 250rpm, 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 25.0g/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 sterilizing 60min.
Nonactin is extracted from fermentation liquid according to the same manner as in Example 1, obtains nonactin fine work 1.5g；Yield
75mg/L。
Embodiment 3
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, is trained
The condition of supporting: it 30 DEG C of temperature, revolving speed 200rpm, cultivates 2 days；Obtain seed liquor；Seed liquor is inoculated with the inoculum concentration of 2% (V/V)
It ferments in 40 2L shaking flasks equipped with 500mL fermentation medium, 28 DEG C of fermentation temperature, revolving speed 250rpm, 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
60min。
Nonactin is extracted from fermentation liquid according to the same manner as in Example 1, obtains nonactin fine work 1.2g；Yield
60mg/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 resins 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 resins.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 obtained
With confirmation；And be further confirmed by HRMS, HRMS detects [M+Na]+759.4293 quasi-molecular ion peak, with no work
The calculating molecular weight [M+Na] of rhzomorph+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, 500MHz) and 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=6.3Hz, 8-CH3), 1.08 (3H, d, J=7.0Hz, 2-CH3)。
13C-NMR(CDCl3, 125MHz) 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, 10.0 g/L, 10.0 g/L of XAD-16 resin, 5.0 g/L of oatmeal, 2.5 g/L of glycerol, 0.5 g/L of glucose, ferment
Female 0.5 g/L of cream, 0.2 g/L of acid hydrolyzed casein, 1.0 g/L of calcium carbonate.
2. fermentation medium according to claim 1, which is characterized in that the fermentation medium composition: 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, yeast extract 0.5
G/L, 0.2 g/L of acid hydrolyzed casein, 1.0 g/L of calcium carbonate, water surplus；Its pH 7.2.
3. fermentation medium according to claim 1 or 2, which is characterized in that the strepto- for fermenting and producing nonactin
Bacterium is 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 is carried out liquid fermentation in fermentation medium described in asking 1 or 2, extracts nonactin after fermented and cultured from fermentation liquid.
5. the method for fermenting and producing nonactin according to claim 4, which is characterized in that the 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 training
Feeding base contains: 10.0 g/L of brewer's wort, 4.0 g/L of yeast extract, 4.0 g/L of glucose；Its pH 7.3.
7. the method for fermenting and producing nonactin according to claim 4 or 5, which is characterized in that the streptomycete is grey
Streptomycete grey subspecies (Streptomyces griseus subsp. Griseus), deposit number CGMCC 4.181.
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| CN201510744556.6A CN105368892B (en) | 2015-11-05 | 2015-11-05 | It is a kind of for improving the fermentation medium and fermentation process of nonactin yield |
| CN201811594642.3A CN109628520B (en) | 2015-11-05 | 2015-11-05 | Fermentation medium for increasing yield of non-viable bacteria and application thereof |
| CN201811595822.3A CN109576318B (en) | 2015-11-05 | 2015-11-05 | Method for extracting non-viable bacteria from fermentation liquor |
| CN201811594585.9A CN109371073B (en) | 2015-11-05 | 2015-11-05 | Method for producing non-viable bacteria element by fermentation |
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| CN201811595822.3A Division CN109576318B (en) | 2015-11-05 | 2015-11-05 | Method for extracting non-viable bacteria from fermentation liquor |
| CN201811595830.8A Division CN109576319B (en) | 2015-11-05 | 2015-11-05 | Fermentation medium without viable bacteria |
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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 |
| CN104480174A (en) * | 2014-12-26 | 2015-04-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing virginiamycin through streptomyces virginiae fermentation and feeding method of culture medium |
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| GB606569A (en) * | 1945-07-30 | 1948-08-17 | Louis Sandy Sanders | Media for use in making camera copy and methods of preparing same |
| SU417032A1 (en) * | 1972-06-14 | 1974-05-05 | Н. К. Соловьева, Н. П. Фадеева, С. М. Руда В. И. Фролова , А. Д. Кузовков | PRODUCER NONACTINE |
| CN1876827A (en) * | 2006-04-14 | 2006-12-13 | 浙江大学宁波理工学院 | Method for improving pristinamycine fermentation output |
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| CN103243134B (en) * | 2013-04-15 | 2015-04-22 | 陕西科技大学 | Fermentation production method based on epothilone B metabolic pathways |
| CN104131054B (en) * | 2014-06-17 | 2017-02-01 | 安徽丰原发酵技术工程研究有限公司 | Fermentation culture medium and fermentation method for improving enramycin yield |
| CN104561180B (en) * | 2014-12-26 | 2017-12-15 | 宁夏泰瑞制药股份有限公司 | A kind of culture medium and feed process for being mutated Avid kyowamycin fermenting and producing doractin |
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| 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 |
| CN109371073A (en) | 2019-02-22 |
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