CN105368892A - Fermentation medium for increasing yield of nonactin and fermentation method - Google Patents
Fermentation medium for increasing yield of nonactin and fermentation method Download PDFInfo
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Abstract
The invention relates to a fermentation medium for increasing the yield of nonactin and a fermentation method and belongs to the field of the fermentation technique. Preferably, the content of resin in the fermentation medium is 5-40 g/L. The method for fermentation production of nonactin comprises the steps of inoculating the fermentation medium with a streptomycete seed solution for liquid fermentation, and extracting nonactin from fermentation liquor after fermentation culture is conducted for 96-168 h. The fermentation medium and the fermentation method greatly increase the number of fermentation units of nonactin and are suitable for large-scale production of nonactin.
Description
Technical field
The present invention relates to fermentation technical field, being specifically related to a kind of fermention medium for improving nonactin output and fermentation process.
Background technology
Nonactin (Nonactin) is the simplest member of structure in the macrocyclic polyether ion carrier antibiotic extended familys being referred to as large ring four lactone.These large ring four lactones comprise nonactin, monactin (monactin), dinactin (dinactin), trinactin (trinactin) and four viable bacteria elements (tetranactin).Nonactin is a kind of ionophore, in conjunction with metal ions such as ammonium ion, calcium ion, potassium ions, can transport positively charged ion by microbial film and artificial rust, therefore may be used for manufacturing ammonium electrode plasma electrodes selective, microelectrode and sensor.In addition, nonactin also has antibacterial activity; Antineoplastic activity; And artitumor multi-medicine-resistant; And be effective Anti-virus agent, effectively may be developed to anti-acquired immunodeficiency syndrome drug.
At present, the approach of the acquisition nonactin reported in the world has:
Chemistry is complete synthesis: the complete synthesis of nonactin can realize, but because the complicacy of nonactin structure, make synthesis step a lot, total recovery is very low, and thus chemical complete synthesis to prepare nonactin unrealistic.
Document IdentificationofantifungalnaturalproductsviaSaccharomyce scerevisiaebioassay:insightsintomacrotetrolidedrugspectr um, potencyandmodeofaction.MedMycol., 2013,51:280 – 289 reports, streptomycete fermentation is utilized to obtain nonactin, 7.2L fermention medium finally obtains 2.7mg nonactin, and yield is only 0.38mg/L.Fermention medium used is: yeast extract paste 2g/L, wort 5g/L, glucose 2g/L, pH7.0; Fermentation condition is 250rpm, cultivates 7 days for 28 DEG C.Document Streptokordin, anewcytotoxiccompoundofthemethylpyridineclassfromamarine-derivedStreptomycessp.KORDI-3238.J.Antibiot., 2006,59:234 – 240 reports, streptomycete fermentation is utilized to obtain nonactin, 8L fermention medium finally obtains 17.4mg nonactin, and yield is only 2.18mg/L.Fermention medium used is: extractum carnis 1g/L, yeast extract paste 1g/L, acid hydrolyzed casein 2g/L, glucose 5g/L, wort 5g/L; Fermentation condition is 200rpm, cultivates 7 days for 27 DEG C.
At present, nonactin production cost is higher, and fermentation period is long, yield is low, and fermentation results is undesirable, is difficult to realize industrialization.Fermentation level is increased and must change substratum and fermentation process.
Summary of the invention
The object of the invention is to provide a kind of fermention medium for improving nonactin output and fermentation process, in order to solve the problem of the technology such as prior art fermentative production nonactin yield is low.
Realize technical scheme of the present invention as follows:
For improving a fermention medium for nonactin output, containing resin in described fermention medium.
Preferably, in described fermention medium, resin content is 5-40g/L;
Further preferably, in described fermention medium, resin content is 10-30g/L;
Still more preferably, in described fermention medium, resin content is 20g/L;
Described content comes in fermenting container (such as fermentor tank or shaking flask) actual liquid amount.
Preferably, described resin is one or more in HP-20 resin, XAD-16 resin, XAD-8 resin, D202 resin, XD-5 resin etc.; More preferably HP-20 resin, XAD-16 resin.
Also containing carbon source in described fermention medium.
Preferably, in described fermention medium, carbon source content is 4.0-15.0g/L.
Further preferably, in described fermention medium, carbon source content is 6.0-10.0g/L.
Preferably, described carbon source is one or more in rolled oats, glycerine, glucose, W-Gum, maltodextrin etc.
Also containing nitrogenous source in described fermention medium.
Preferably, in described fermention medium, nitrogenous source content is 0.2-3.5g/L.
Further preferably, in described fermention medium, nitrogenous source content is 0.5-1.5g/L.
Preferably, described nitrogenous source is one or more in bean cake powder, cottonseed meal, corn syrup hydrolyzate, acid hydrolyzed casein, yeast extract/powder etc.
As preferably, described carbon source is rolled oats, glycerine and glucose; Described nitrogenous source is acid hydrolyzed casein, yeast extract/powder.
Further, described fermention medium also comprises the inorganic salt used in normal fermentation substratum.
Described inorganic salt consumption preferred range is 0.5-2.5g/L; Described inorganic salt are preferably calcium carbonate;
Particularly, described fermention medium contains: resin 15-25g/L, rolled oats 2.5-7.5g/L, glycerine 0.5-5g/L, glucose 0.1-2.5g/L, yeast extract paste 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, described fermention medium contains: HP-20 resin 10.0g/L, XAD-16 resin 10.0g/L, rolled oats 5.0g/L, glycerine 2.5g/L, glucose 0.5g/L, yeast extract paste 0.5g/L, acid hydrolyzed casein 0.2g/L, calcium carbonate 1.0g/L.
In another preferred embodiment of the present invention, described fermention medium contains: HP-20 resin 25.0g/L, rolled oats 7.5g/L, glycerine 5g/L, glucose 2.5g/L, yeast extract paste 2.5g/L, acid hydrolyzed casein 1g/L, calcium carbonate 2.5g/L.
In another preferred embodiment of the present invention, described fermention medium contains: XAD-16 resin 15.0g/L, rolled oats 2.5g/L, glycerine 0.5g/L, glucose 0.1g/L, yeast extract paste 0.1g/L, acid hydrolyzed casein 0.1g/L, calcium carbonate 0.5g/L.
Described fermention medium pH6.8-7.5, preferred pH7.2.
Described fermention medium can adopt ordinary method to prepare and sterilizing; Such as 121 DEG C of sterilizing 30-60min.
The present invention also provides a kind of method of fermentative production nonactin, comprises the seed liquor of streptomycete to be inoculated in aforesaid fermentation substratum to carry out liquid fermenting, extracts nonactin after fermentation culture from fermented liquid.
Preferably, described fermented incubation time 96-168 hour; Described fermentation culture temperature is 28-32 DEG C; In fermentation culture process, pH is 6.8-7.5.
The preparation of the seed liquor of described streptomycete can adopt ordinary method.
Such as the slant culture of streptomycete is inoculated in seed culture medium, in 28-30 DEG C, rotating speed 180-250rpm cultivates 2-3 days, obtains seed liquor.
The seed culture medium of described streptomycete can adopt this area conventional medium.
Preferably, described streptomycete seed culture medium contains: wort 10.0g/L, yeast extract paste 4.0g/L, glucose 4.0g/L; Its pH7.3; General 121 DEG C of sterilizing 30min.
The described method extracting nonactin from fermented liquid can adopt ordinary method.
Preferably, the described method extracting nonactin from fermented liquid comprises:
By described filtering fermentation liquor, obtain mycelia and filtrate two portions; By described mycelia freeze-drying (general 5-7 days), with methanol extraction 1-3 time, ethyl acetate is extracted 1-3 time, and normal hexane extraction 1-2 time, obtains mycelia extracting solution; By described filtrate with equal-volume extraction into ethyl acetate 1-3 time, obtain filtrate extracting solution; Merge described mycelia extracting solution and filtrate extracting solution, concentrating under reduced pressure obtains medicinal extract; Described medicinal extract silica gel (230-400 order) is carried out column chromatography, gradient elution is carried out respectively with 0%, 50%, 80%, 100% ethyl acetate-hexane and 100% methyl alcohol, collect 50% and 80% ethyl acetate-hexane elutriant, crystallizing at room temperature, and with acetone recrystallization, obtain (nonactin fine work).
Wherein, described streptomycete is preferably streptomyces griseus grey subspecies (Streptomycesgriseussubsp.Griseus), deposit number CGMCC4.181, can buy from Chinese General Microbiological Culture preservation administrative center (ChinaGeneralMicrobiologicalCultureCollectionCenter, CGMCC).
Beneficial effect of the present invention is:
The invention solves nonactin production cost higher, the deficiency that fermentation level is undesirable, provides one and is conducive to the fermention medium of streptomyces griseus grey subspecies (Streptomycesgriseussubsp.Griseus) high-efficiency fermenting production nonactin (Nonactin) and corresponding fermentation process.By adding resin in the fermentation medium dexterously; facilitate the output increased of nonactin; and by fermentation parameters such as preferred fermention medium and optimization culture temperature, pH, thus increased substantially the fermentation unit of nonactin, be applicable to the large-scale production of nonactin.Experiment shows, under culture condition provided by the invention, 20L fermention medium can obtain 1.6g nonactin, and yield is up to 80mg/L, and separation purifying technique is simple, may be used for the suitability for industrialized production of nonactin.
Accompanying drawing explanation
Fig. 1 is experimental example 1 nonactin
1h-NMR collection of illustrative plates;
Fig. 2 is experimental example 1 nonactin
13c-NMR collection of illustrative plates.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Each component in following examples in culture medium prescription is commercial goods.
Experimental strain is streptomyces griseus grey subspecies (Streptomycesgriseussubsp.Griseus) CGMCC4.181, can buy from Chinese General Microbiological Culture preservation administrative center (ChinaGeneralMicrobiologicalCultureCollectionCenter, CGMCC).
In following examples and comparative example, the HPLC testing conditions of fermented liquid nonactin content is as follows:
Instrument: Waters2690 high performance liquid chromatograph;
Chromatographic column: PhenomenexGemini5 μm NX-C18
250x4.6mm;
Moving phase: A methyl alcohol, B water.Flow velocity 1mL/min; Gradient is arranged as following table:
| Time (min) | A％ | B％ |
| 0 | 40 | 60 |
| 20 | 90 | 10 |
| 25 | 40 | 60 |
| 30 | 40 | 60 |
Detector: PL-ELS2100 light scattering detector.
Embodiment 1
The slant culture of experimental strain streptomyces griseus grey subspecies CGMCC4.181 is inoculated in seed culture medium, culture condition: temperature 30 DEG C, rotating speed 200rpm, cultivates 2 days; Obtain seed liquor; Seed liquor is inoculated in 40 with the inoculum size of 2.5% (V/V) be equipped with in the 2L shaking flask of 500mL fermention medium and ferment, leavening temperature 30 DEG C, rotating speed 200rpm, fermentation period 5 days.Sample (HPLC) after fermentation ends to measure, nonactin is tired 142mg/L.
Described seed culture medium composition: wort 10.0g/L, yeast extract paste 4.0g/L, glucose 4.0g/L, water surplus; Its pH7.3; 121 DEG C of sterilizing 30min.
Described fermention medium composition: HP-20 resin 10.0g/L, XAD-16 resin 10.0g/L, rolled oats 5.0g/L, glycerine 2.5g/L, glucose 0.5g/L, yeast extract paste 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, be divided into mycelia and filtrate two portions; By mycelia freeze-drying 7 days, with 1L methanol extraction 3 times, 1L ethyl acetate extracted 3 times, and 1L normal hexane extraction 2 times, obtains mycelia extracting solution; Filtrate, with equal-volume extraction into ethyl acetate 3 times, obtains filtrate extracting solution; Merge described mycelia extracting solution and filtrate extracting solution, concentrating under reduced pressure obtains medicinal extract and is about 16g.Medicinal extract silica gel (230-400 order) is carried out column chromatography, gradient elution is carried out respectively with 0%, 50%, 80%, 100% ethyl acetate-hexane and each 1L of 100% methyl alcohol, collect 50% and 80% ethyl acetate-hexane elutriant, 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, culture condition: temperature 30 DEG C, rotating speed 200rpm, cultivates 2 days; Obtain seed liquor; Seed liquor is inoculated in 40 with the inoculum size of 3% (V/V) be equipped with in the 2L shaking flask of 500mL fermention medium and ferment, leavening temperature 28 DEG C, rotating speed 250rpm, fermentation period 6 days.Sample (HPLC) after fermentation ends to measure, nonactin is tired 125mg/L.
Described seed culture medium is with embodiment 1.
Described fermention medium composition: HP-20 resin 25.0g/L, rolled oats 7.5g/L, glycerine 5g/L, glucose 2.5g/L, yeast extract paste 2.5g/L, acid hydrolyzed casein 1g/L, calcium carbonate 2.5g/L, water surplus; Its pH7.2; 121 DEG C of sterilizing 60min.
From fermented liquid, extract nonactin by the method identical with embodiment 1, obtain 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, culture condition: temperature 30 DEG C, rotating speed 200rpm, cultivates 2 days; Obtain seed liquor; Seed liquor is inoculated in 40 with the inoculum size of 2% (V/V) be equipped with in the 2L shaking flask of 500mL fermention medium and ferment, leavening temperature 28 DEG C, rotating speed 250rpm, fermentation period 6 days.Sample (HPLC) after fermentation ends to measure, nonactin is tired 98mg/L.
Described seed culture medium is with embodiment 1.
Described fermention medium composition: XAD-16 resin 15.0g/L, rolled oats 2.5g/L, glycerine 0.5g/L, glucose 0.1g/L, yeast extract paste 0.1g/L, acid hydrolyzed casein 0.1g/L, calcium carbonate 0.5g/L, water surplus; Its pH7.2; 121 DEG C of sterilizing 60min.
From fermented liquid, extract nonactin by the method identical with embodiment 1, obtain nonactin fine work 1.2g; Yield 60mg/L.
Comparative example 1:
Obtain nonactin fermented liquid by the method identical with embodiment 1, difference is only in fermention medium not containing HP-20 resin and XAD-16 resin.Sample (HPLC) after fermentation ends to measure, nonactin is tired 4mg/L.
Comparative example 2:
Obtain nonactin fermented liquid by the method identical with embodiment 2, difference is only in fermention medium not containing HP-20 resin.Sample (HPLC) after fermentation ends to measure, nonactin is tired 3mg/L.
Obtain nonactin fermented liquid by the method identical with embodiment 3, difference is only in fermention medium not containing XAD-16 resin.Sample (HPLC) after fermentation ends to measure, nonactin is tired 1mg/L.
Experimental example 1
The nonactin that embodiment 1 is obtained is colorless needle crystals; The structure warp of nonactin
1h-NMR and
13c-NMR is confirmed; And be confirmed further by HRMS, HRMS detects [M+Na]
+759.4293 quasi-molecular ion peaks, with the calculating molecular weight [M+Na] of nonactin
+759.4295 coincide; Its
1h-NMR and
13c-NMR spectrogram is shown in accompanying drawing 1 and accompanying drawing 2 respectively.
The nucleus magnetic resonance modal data of product nonactin:
1H-NMR(CDCl
3，500MHz)δ4.97(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-CH
3)，1.08(3H，d，J＝7.0Hz，2-CH
3)。
13C-NMR(CDCl
3，125MHz)δ174.4(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-CH
3)，13.0(q，2-CH
3)。
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. for improving a fermention medium for nonactin output, it is characterized in that, containing resin in described fermention medium.
2. fermention medium according to claim 1, is characterized in that, in described fermention medium, resin content is 5-40g/L; Be preferably 10-30g/L; More preferably 20g/L.
3. fermention medium according to claim 1 and 2, is characterized in that, described resin is one or more in HP-20 resin, XAD-16 resin, XAD-8 resin, D202 resin, XD-5 resin etc.
4. the fermention medium according to any one of claim 1-3, is characterized in that, in described fermention medium, carbon source content is 4.0-15.0g/L; Be preferably 6.0-10.0g/L;
Preferably, described carbon source is one or more in rolled oats, glycerine, glucose, W-Gum, maltodextrin;
Preferably, in described fermention medium, nitrogenous source content is 0.2-3.5g/L; More preferably 0.5-1.5g/L;
Preferably, described nitrogenous source is one or more in bean cake powder, cottonseed meal, corn syrup hydrolyzate, acid hydrolyzed casein, yeast extract/powder etc.;
Preferably, described fermention medium also comprises the inorganic salt used in normal fermentation substratum; Further preferably, described inorganic salt amount ranges is 0.5-2.5g/L; Preferably, described inorganic salt are preferably calcium carbonate.
5. fermention medium according to claim 1, it is characterized in that, described fermention medium contains: resin 15-25g/L, rolled oats 2.5-7.5g/L, glycerine 0.5-5g/L, glucose 0.1-2.5g/L, yeast extract paste 0.1-2.5g/L, acid hydrolyzed casein 0.1-1.0g/L, calcium carbonate 0.5-2.5g/L; Preferably, described fermention medium pH6.8-7.5.
6. fermention medium according to claim 5, it is characterized in that, described fermention medium contains: HP-20 resin 10.0g/L, XAD-16 resin 10.0g/L, rolled oats 5.0g/L, glycerine 2.5g/L, glucose 0.5g/L, yeast extract paste 0.5g/L, acid hydrolyzed casein 0.2g/L, calcium carbonate 1.0g/L;
Or described fermention medium contains: HP-20 resin 25.0g/L, rolled oats 7.5g/L, glycerine 5g/L, glucose 2.5g/L, yeast extract paste 2.5g/L, acid hydrolyzed casein 1g/L, calcium carbonate 2.5g/L;
Or described fermention medium contains: XAD-16 resin 15.0g/L, rolled oats 2.5g/L, glycerine 0.5g/L, glucose 0.1g/L, yeast extract paste 0.1g/L, acid hydrolyzed casein 0.1g/L, calcium carbonate 0.5g/L.
7. a method for fermentative production nonactin, is characterized in that, comprises in the fermention medium seed liquor of streptomycete be inoculated in described in any one of claim 1-6 and carries out liquid fermenting, extract nonactin after fermentation culture from fermented liquid;
Preferably, described fermented incubation time 96-168 hour;
Preferably, described fermentation culture temperature is 28-32 DEG C;
Preferably, in fermentation culture process, pH is 6.8-7.5.
8. the method for fermentative production nonactin according to claim 7, is characterized in that, described streptomycete seed culture medium contains: wort 10.0g/L, yeast extract paste 4.0g/L, glucose 4.0g/L; Its pH7.3.
9. the method for the fermentative production nonactin according to claim 7 or 8, is characterized in that, the described method extracting nonactin from fermented liquid comprises:
By described filtering fermentation liquor, obtain mycelia and filtrate two portions; By described mycelia freeze-drying, with methanol extraction 1-3 time, ethyl acetate is extracted 1-3 time, and normal hexane extraction 1-2 time, obtains mycelia extracting solution; By described filtrate with equal-volume extraction into ethyl acetate 1-3 time, obtain filtrate extracting solution; Merge described mycelia extracting solution and filtrate extracting solution, concentrating under reduced pressure obtains medicinal extract; Described medicinal extract silica gel is carried out column chromatography, carry out gradient elution with 0%, 50%, 80%, 100% ethyl acetate-hexane and 100% methyl alcohol respectively, collect 50% and 80% ethyl acetate-hexane elutriant, crystallizing at room temperature, and with acetone recrystallization, to obtain final product.
10. the method for the fermentative production nonactin according to any one of claim 7-9, is characterized in that, described streptomycete is streptomyces griseus grey subspecies (Streptomycesgriseussubsp.Griseus), deposit number CGMCC4.181.
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| 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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| CN201811595830.8A Division CN109576319B (en) | 2015-11-05 | 2015-11-05 | Fermentation medium without viable bacteria |
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| CN108753861A (en) * | 2018-06-08 | 2018-11-06 | 福建省微生物研究所 | A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin |
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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 |
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- 2015-11-05 CN CN201811594642.3A patent/CN109628520B/en active Active
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| CN108753861A (en) * | 2018-06-08 | 2018-11-06 | 福建省微生物研究所 | A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin |
Also Published As
| Publication number | Publication date |
|---|---|
| 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 |
| CN105368892B (en) | 2018-12-18 |
| CN109371073A (en) | 2019-02-22 |
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