CN102559780A - Culture medium for preparing Terrein by fermenting aspergillus terreus and preparation method thereof - Google Patents
Culture medium for preparing Terrein by fermenting aspergillus terreus and preparation method thereof Download PDFInfo
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- CN102559780A CN102559780A CN2011103114602A CN201110311460A CN102559780A CN 102559780 A CN102559780 A CN 102559780A CN 2011103114602 A CN2011103114602 A CN 2011103114602A CN 201110311460 A CN201110311460 A CN 201110311460A CN 102559780 A CN102559780 A CN 102559780A
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Abstract
The invention discloses a culture medium for preparing Terrein by fermenting aspergillus terreus and a preparation method thereof. The culture medium comprises glucose, maltose, mannitol, malt extract, sodium glutamate, ammonium chloride and artificial seawater. According to a preferred scheme, the culture medium is specifically that: when the culture medium contains 1L of artificial seawater, the culture medium contains 28.41 g of glucose, 23.18 g of maltose, 20.00 g of mannitol, 8.52 g of malt extract, 10.00 g of sodium glutamate and 10.00g of ammonium chloride. The preparation method comprises the following steps of: 1, preparing the artificial seawater; and 2, adding the glucose, the maltose, the mannitol, the malt extract, the sodium glutamate and the ammonium chloride into the prepared artificial seawater and stirring to dissolve, and then performing high-temperature sterilization to obtain the culture medium. Compared with the prior art, the components of the culture medium for producing the Terrein from the aspergillus terreus are successfully optimized, so that the yield of the Terrein is obviously increased.
Description
Technical field
The present invention relates to substratum of a kind of fungus culture technical field and preparation method thereof, specifically is substratum of a kind of sponge symbiotic terreus fermentative prepn Terrein and preparation method thereof.
Background technology
Terrein (terrein) is a kind of cyclopentenone compounds, and being fermented by terreus at first obtains, although year surplus discovery distance the present existing 70, its many activity are up to the discovery successively of ability quilt in this century.Terrein's is active very various, not only has the activity of antibacterium, fungi, weeds, can also suppress the plain secretion of tumour cell angiogenesis; It is synthetic to suppress melanochrome; Suppress the skin keratin hyperplasia, suppress the pulp cells inflammatory reaction, promote scleroblast and titanium framework fusion etc.Can predict Terrein and have good prospects for application in various fields such as medical treatment, beauty treatment, agriculturals in the future, the technology of preparing that certain prerequisite is Terrein makes a breakthrough.
Yet existing Terrein preparation method comes with some shortcomings.At first; The technology that some chemosynthesis prepare Terrein often has overall yield low; Need to adopt extreme reaction conditionss such as HTHP; Need expensive reaction reagent, or end product do not possess shortcomings such as selectivity (Lee S, the et al. Synthesis and melanin biosynthesis inhibitory activity of (±)-terrein produced by of three-dimensional arrangement
PenicilliumSp. 20135. Bioorganic and Medicinal Chemistry Letters 2005; 15:471-473).Secondly, it is very limited again to adopt microbial fermentation to prepare the technical production of Terrein, hundreds of at least μ g/L (Phattanawasin P, et al. Weed growth inhibitors from
Aspergillus fischeriTISTR 3272. Natural Product Research 2007; 21:1286-1291), hundreds of at most mg/L (M. Demasi, et al. Studies on Terrein as a New Class of Proteasome Inhibitors. Journal of the Brazilian Chemical Society 2010; 21:229-305), fail to reach the g/L magnitude that possesses productive value.
Summary of the invention
The objective of the invention is to overcome the above-mentioned shortcoming that prior art exists, substratum of a kind of sponge symbiotic terreus fermentative prepn Terrein and preparation method thereof is provided.Adopt method of the present invention that the medium component that the sponge symbiotic terreus produces Terrein is optimized, make the output of Terrein be significantly improved.
The objective of the invention is to realize through following technical scheme:
The present invention relates to a kind of substratum that is used for sponge symbiotic terreus fermentative prepn Terrein, said substratum comprises: glucose, SANMALT-S, N.F,USP MANNITOL, malt extract, Sodium Glutamate, ammonium chloride and artificial seawater.
Preferably, said sponge symbiotic terreus be terreus (
Aspergillus terreus) PF26 CGMCC NO.5208.
Preferably, said substratum is specially: when the artificial seawater that contains in the substratum was 1L, substratum contained glucose 28.41g, SANMALT-S 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
Preferably, said artificial seawater is specially, and artificial seawater is by the each component of following quality: sodium-chlor 26.518g, magnesium chloride 2.447g, sal epsom 3.305g, calcium chloride
1.141g, Repone K 0.725g, sodium hydrogencarbonate
0.202g, Sodium Bromide 0.083g, be dissolved in the 1L zero(ppm) water and form; The pH value of said artificial seawater is 7.0～7.2.
The invention still further relates to a kind of preparation method of the above-mentioned substratum that is used for sponge symbiotic terreus fermentative prepn Terrein, may further comprise the steps:
The first step, the preparation artificial seawater;
Second step added glucose, SANMALT-S, N.F,USP MANNITOL, malt extract, Sodium Glutamate and ammonium chloride and stirring and dissolving in the artificial seawater for preparing, carry out High Temperature Sterilization then and obtain substratum.
Preferably; In second step; Said substratum is specially, and when the artificial seawater that contains when substratum was 1L, substratum contained glucose 28.41g, SANMALT-S 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
Preferably, in the first step, said preparation artificial seawater is specially, and the configuration artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g sal epsom, 1.141g calcium chloride
, 0.725g Repone K, 0.202g sodium hydrogencarbonate and 0.083g Sodium Bromide be dissolved in the 1L zero(ppm) water, promptly gets, the pH value of the said artificial seawater for preparing is 7.0～7.2.
Preferably, in second step, said High Temperature Sterilization is under 115 ℃ of environment, to sterilize 30 minutes.
Compared with prior art, the beneficial effect that has of the present invention is: adopt the medium component that the sponge symbiotic terreus is produced Terrein of the inventive method success to optimize.Shaking on bottle level, bacterial classification is being inoculated into respectively in minimum medium and the best medium, comparing the growing state and the situation of producing Terrein of bacterial classification in two kinds of substratum; The output of Terrein of fermentation 24d reaches 3.71g/L in best medium, is 2.1 times of output (1.73g/L) in the minimum medium of not optimizing; This shows, is shaking on bottle level, and the output of Terrein is significantly improved.
Description of drawings
Fig. 1 is the comparison diagram of sponge symbiotic terreus fermentation Terrein output in different minimum mediums;
Fig. 2 is the comparison diagram of sponge symbiotic terreus fermentation Terrein output in minimum medium and best medium.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated, present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
What relate to Terrein in following examples is specially terreus Aspergillus terreus bacterial strain PF26; Separation is Phakellia fusca sponge from island, South China Sea Yongxing; Its preservation information is: this bacterial strain is submitted the common micro-organisms center preservation of CGMCC China Committee for Culture Collection of Microorganisms on August 31st, 2011, and deposit number is CGMCC NO.5208; The depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The preparation of four kinds of minimum mediums is following:
1) minimum medium one: 30g sucrose, 2g SODIUMNITRATE, 10mL phosphoric acid buffer, 1mL iron salt solutions are dissolved in the 1L artificial seawater;
The component of described phosphoric acid buffer and concentration content are: the K of 0.4mol/L
2HPO
4, the KH of 0.2mol/L
2PO
4, solvent is a zero(ppm) water, the pH of this phosphoric acid buffer is 7.1.
The component of described iron salt solutions and concentration content are: the FeSO of 0.06mol/L
4, 0.04mol/L trisodium citrate, solvent are zero(ppm) water.
2) minimum medium two: 10g glucose, 20g SANMALT-S, 20g N.F,USP MANNITOL, 3g malt extract, 10g Sodium Glutamate and 10g chloride leach are in the 1L artificial seawater;
3) minimum medium three: the 40g maltodextrin, and 30g SANMALT-S, 40g sucrose, the 2g Sodium Glutamate, the 1g yeast extract, 1g soy peptone and 2g Tryptones are dissolved in the 1L artificial seawater;
4) minimum medium four: the 50g lactose, and 20g glucose, 10g glycerine, 6g soy peptone and 4g yeast extract are dissolved in the 1L artificial seawater.
More than four kinds of substratum all in 115 ℃ of sterilization 30min, wherein, said artificial seawater comprises the each component of following quality: sodium-chlor (NaCl) 26.518g, magnesium chloride (MgCl
2) 2.447g, sal epsom (MgSO
4) 3.305g, calcium chloride (CaCl
2) 1.141g, Repone K (KCl) 0.725g, sodium hydrogencarbonate (NaHCO
3) 0.202g, Sodium Bromide (NaBr) 0.083g be dissolved in the 1L zero(ppm) water, the pH value of said artificial seawater is 7.0～7.2.
Ferment with above-mentioned four kinds of substratum respectively, the bacterial classification inoculation amount is 4 * 10
6Individual spore/mL, liquid amount 100mL in the 250mL triangular flask cultivates the pH nature in 28 ℃, the constant temperature shaking table of 180rpm.
Adopt the HPLC method to measure Terrein content; Shown in Figure 1ly be the comparison diagram of sponge symbiotic terreus fermentation Terrein output in above four kinds of different minimum mediums; Can be known by Fig. 1: Terrein content is respectively in above four kinds of substratum: 0.49g/L; 1.73g/L, 0.52g/L and 0.38g/L.Obtaining reasonable minimum medium prescription (prescription 1) thus is: 10g glucose, 20g SANMALT-S, 20g N.F,USP MANNITOL, 3g malt extract, 10g Sodium Glutamate, 10g ammonium chloride, the preparation of 1L artificial seawater, pH nature.1 ferment to fill a prescription, Terrein output is 1.73g/L.
On the basis of the reasonable minimum medium prescription 1 that the foregoing description 1 is confirmed, at first the factor bigger to the Terrein yield effect picked out in Using P lackett-Burman design: glucose, SANMALT-S and malt extract.Their addition level is as shown in table 1:
The high low value of each factor in the table 1 Plackett-Burman experiment
After filtering out important factor, the application responds Surface Method is further optimized the optimum of glucose, SANMALT-S and malt extract and is cultivated concentration.At first find central point concentration, be respectively 20g glucose, 40g SANMALT-S and 6g malt extract, secondly confirm that with central composite design their addition level is as shown in table 2 through experiment of single factor:
The encoded radio and the actual value concentration of important factor during table 2 is central composite design
The component of optimizing the best medium (prescription 2) that obtains is: 28.41g glucose, 23.18g SANMALT-S, 20.00g N.F,USP MANNITOL, 8.52g malt extract, 10.00g Sodium Glutamate and 10.00g ammonium chloride; Prepare with the 1L artificial seawater; Said artificial seawater is specially, and artificial seawater is made up of the each component of following quality: sodium-chlor (NaCl) 26.518g, magnesium chloride (MgCl
2) 2.447g, sal epsom (MgSO
4) 3.305g, calcium chloride (CaCl
2) 1.141g, Repone K (KCl) 0.725g, sodium hydrogencarbonate (NaHCO
3) 0.202g, Sodium Bromide (NaBr) 0.083g, be dissolved in the 1L zero(ppm) water; The pH value of said artificial seawater is 7.0～7.2.Shown in Figure 2ly be the comparison diagram of sponge symbiotic terreus fermentation Terrein output in minimum medium and best medium; Solid line is represented Terrein output among the figure, and dotted line is represented dry cell weight, light color expression minimum medium; Dark expression best medium; Can be known by Fig. 2: carry out shake flask fermentation with 2 substratum of filling a prescription, Terrein output and dry cell weight are high than minimum medium (prescription 1) all, and the output of Terrein reaches 3.71g/L in the best medium; Be 2.1 times of output (1.73g/L) in the minimum medium of not optimizing.This shows, is shaking on bottle level, and the output of Terrein is significantly improved.
Claims (8)
1. a substratum that is used for sponge symbiotic terreus fermentative prepn Terrein is characterized in that said substratum comprises: glucose, SANMALT-S, N.F,USP MANNITOL, malt extract, Sodium Glutamate, ammonium chloride and artificial seawater.
2. the substratum that is used for sponge symbiotic terreus fermentative prepn Terrein according to claim 1 is characterized in that, said sponge symbiotic terreus be terreus (
Aspergillus terreus) PF26 CGMCC NO.5208.
3. the substratum that is used for sponge symbiotic terreus fermentative prepn Terrein according to claim 1 and 2; It is characterized in that; Said substratum is specially: when the artificial seawater that contains in the substratum was 1L, substratum contained glucose 28.41g, SANMALT-S 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
4. the substratum that is used for sponge symbiotic terreus fermentative prepn Terrein according to claim 1 and 2; It is characterized in that; Said artificial seawater is specially, and artificial seawater is by the each component of following quality: sodium-chlor 26.518g, magnesium chloride 2.447g, sal epsom 3.305g, calcium chloride
1.141g, Repone K 0.725g, sodium hydrogencarbonate
0.202g, Sodium Bromide 0.083g, be dissolved in the 1L zero(ppm) water and form; The pH value of said artificial seawater is 7.0～7.2.
5. a preparation method who is used for the substratum of sponge symbiotic terreus fermentative prepn Terrein according to claim 1 is characterized in that, may further comprise the steps:
The first step, the preparation artificial seawater;
Second step added glucose, SANMALT-S, N.F,USP MANNITOL, malt extract, Sodium Glutamate and ammonium chloride and stirring and dissolving in the artificial seawater for preparing, carry out High Temperature Sterilization then and obtain substratum.
6. the preparation method who is used for the substratum of sponge symbiotic terreus fermentative prepn Terrein according to claim 5; It is characterized in that; In second step; Said substratum is specially, and when the artificial seawater that contains when substratum was 1L, substratum contained glucose 28.41g, SANMALT-S 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
7. the preparation method who is used for the substratum of sponge symbiotic terreus fermentative prepn Terrein according to claim 6; It is characterized in that; In the first step; Said preparation artificial seawater is specially, and the configuration artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g sal epsom, 1.141g calcium chloride
, 0.725g Repone K, 0.202g sodium hydrogencarbonate and 0.083g Sodium Bromide be dissolved in the 1L zero(ppm) water, promptly gets, the pH value of the said artificial seawater for preparing is 7.0～7.2.
8. according to claim 5, the 6 or 7 described preparing methods that are used for the substratum of sponge symbiotic terreus fermentative prepn Terrein, it is characterized in that in second step, said High Temperature Sterilization is under 115 ℃ of environment, to sterilize 30 minutes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497975A (en) * | 2013-07-02 | 2014-01-08 | 上海交通大学 | Application of vorinostat and method for promoting synthesis of (+)-Terrein by sponge-associated aspergillus terreus |
| CN103497976A (en) * | 2013-07-02 | 2014-01-08 | 上海交通大学 | Artificial seawater and culture medium for producing (+)-Terrein by fermentation of sponge-associated aspergillus terreus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766081A (en) * | 2005-09-01 | 2006-05-03 | 上海交通大学 | Blending culture for sponge and intergrowth microbe, culturing colony monitoring method |
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2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766081A (en) * | 2005-09-01 | 2006-05-03 | 上海交通大学 | Blending culture for sponge and intergrowth microbe, culturing colony monitoring method |
Non-Patent Citations (1)
| Title |
|---|
| 田云龙: "碳源和氮源对中生菌素发酵影响的研究", 《中国优秀硕士学位论文全文数据库》, 29 November 2001 (2001-11-29) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497975A (en) * | 2013-07-02 | 2014-01-08 | 上海交通大学 | Application of vorinostat and method for promoting synthesis of (+)-Terrein by sponge-associated aspergillus terreus |
| CN103497976A (en) * | 2013-07-02 | 2014-01-08 | 上海交通大学 | Artificial seawater and culture medium for producing (+)-Terrein by fermentation of sponge-associated aspergillus terreus |
| CN103497976B (en) * | 2013-07-02 | 2015-08-19 | 上海交通大学 | To grow nonparasitically upon another plant altogether the artificial seawater of terreus fermentative production (+)-Terrein and substratum for sponge |
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