CN102559780B - 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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- CN102559780B CN102559780B CN201110311460.2A CN201110311460A CN102559780B CN 102559780 B CN102559780 B CN 102559780B CN 201110311460 A CN201110311460 A CN 201110311460A CN 102559780 B CN102559780 B CN 102559780B
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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 substratum of a kind of sponge symbiotic terreus fermentation preparation Terrein and preparation method thereof.
Background technology
Terrein(terrein) be a kind of cyclopentenone compounds, obtained by terreus fermentation at first, although find apart from modern existing more than 70 years, its many activity are until just found successively this century.The activity of Terrein is very various, not only has the activity of antibacterium, fungi, weeds, the secretion of all right inhibition tumor cell angiogenesis element, check melanin is synthetic, suppress skin keratin hyperplasia, suppress pulp cells inflammatory reaction, promote scleroblast and titanium framework fusion etc.Can predict Terrein and have good application prospect 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.
But existing Terrein preparation method comes with some shortcomings.First; the technology that Terrein is prepared in some chemosynthesis often has overall yield low; need to adopt the extreme reaction conditionss such as High Temperature High Pressure; need expensive reaction reagent; or end product does not possess shortcoming (Lee S, et al.Synthesis and melanin biosynthesis inhibitory activity of (±)-terrein produced by Penicillium sp.20135.Bioorganic and Medicinal Chemistry Letters2005 such as the selectivity of three-dimensional arrangement; 15:471-473).Secondly; adopt the technical production of microorganism fermentation preparation Terrein very limited again; hundreds of μ g/L(Phattanawasin P at least, et al.Weed growth inhibitors from Aspergillus fischeri TISTR3272.Natural Product Research2007; 21:1286-1291), hundreds of mg/L(M.Demasi at most, et al.Studies on Terrein as a New Class of Proteasome Inhibitors.Journal of the Brazilian Chemical Society2010; 21:229-305), fail to reach the g/L magnitude that possesses productive value.
Summary of the invention
The object of the invention is to overcome the above-mentioned shortcoming that prior art exists, substratum of a kind of sponge symbiotic terreus fermentation preparation Terrein and preparation method thereof is provided.The medium component that adopts method of the present invention to produce Terrein to sponge symbiotic terreus is optimized, and the output of Terrein is significantly improved.
The object of the invention is to be achieved through the following technical solutions:
The present invention relates to a kind of substratum for sponge symbiotic terreus fermentation preparation Terrein, described substratum comprises: glucose, maltose, N.F,USP MANNITOL, malt extract, Sodium Glutamate, ammonium chloride and artificial seawater.
Preferably, described sponge symbiotic terreus is terreus (Aspergillus terreus) PF26CGMCC NO.5208.
Preferably, described substratum is specially: in the time that the artificial seawater containing in substratum is 1L, substratum contains glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
Preferably, described artificial seawater is specially, artificial seawater is by the each component of following quality: sodium-chlor 26.518g, magnesium chloride 2.447g, magnesium sulfate 3.305g, calcium chloride 1.141g, Repone K 0.725g, sodium bicarbonate 0.202g, Sodium Bromide 0.083g, be dissolved in 1L distilled water and form; The pH value of described artificial seawater is 7.0～7.2.
The preparation method who the invention still further relates to a kind of above-mentioned substratum for sponge symbiotic terreus fermentation preparation Terrein, comprises the following steps:
The first step, preparation artificial seawater;
Second step adds glucose, maltose, N.F,USP MANNITOL, malt extract, Sodium Glutamate and ammonium chloride stirring and dissolving in the artificial seawater preparing, and then carries out High Temperature Sterilization and obtains substratum.
Preferably, in second step, described substratum is specially, and when the artificial seawater containing when substratum is 1L, substratum contains glucose 28.41g, maltose 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, described preparation artificial seawater is specially, configuration artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g magnesium sulfate, 1.141g calcium chloride, 0.725g Repone K, 0.202g sodium bicarbonate and 0.083g Sodium Bromide to be dissolved in 1L distilled water, obtain, described in the pH value of the artificial seawater for preparing be 7.0～7.2.
Preferably, in second step, described High Temperature Sterilization is sterilizing 30 minutes under 115 ℃ of environment.
Compared with prior art, the beneficial effect that the present invention has is: adopt the inventive method successfully the medium component of sponge symbiotic terreus product Terrein to be optimized.In shaking flask level, bacterial classification is inoculated into respectively in minimum medium and best medium, relatively the situation of the growing state of bacterial classification and product Terrein in two kinds of substratum; The output of Terrein of 24d of fermenting in best medium reaches 3.71g/L, is 2.1 times of output (1.73g/L) in the minimum medium of not optimizing; This shows, in shaking flask level, the output of Terrein is significantly improved.
Accompanying drawing explanation
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, the present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
What in following examples, relate to Terrein is specially terreus Aspergillus terreus bacterial strain PF26, separate from South China Sea Yongxing Island Phakellia fusca sponge, its preservation information is: this bacterial strain is submitted the center preservation of CGMCC China Committee for Culture Collection of Microorganisms common micro-organisms on August 31st, 2011, and deposit number is CGMCC NO.5208; Depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Being formulated as follows of four kinds of minimum mediums:
1) minimum medium one: 30g sucrose, 2g SODIUMNITRATE, 10mL phosphoric acid buffer, 1mL iron salt solutions are dissolved in 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 distilled 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 is distilled water.
2) minimum medium two: 10g glucose, 20g maltose, 20g N.F,USP MANNITOL, 3g malt extract, 10g Sodium Glutamate and 10g chloride leach are in 1L artificial seawater;
3) minimum medium three: 40g maltodextrin, 30g maltose, 40g sucrose, 2g Sodium Glutamate, 1g yeast extract, 1g soy peptone and 2g Tryptones are dissolved in 1L artificial seawater;
4) minimum medium four: 50g lactose, 20g glucose, 10g glycerine, 6g soy peptone and 4g yeast extract are dissolved in 1L artificial seawater.
Above four kinds of substratum are all in 115 ℃ of sterilizing 30min, wherein, and the each component that described artificial seawater comprises following quality: sodium-chlor (NaCl) 26.518g, magnesium chloride (MgCl
2) 2.447g, magnesium sulfate (MgSO
4) 3.305g, calcium chloride (CaCl
2) 1.141g, Repone K (KCl) 0.725g, sodium bicarbonate (NaHCO
3) 0.202g, Sodium Bromide (NaBr) 0.083g be dissolved in 1L distilled water, the pH value of described artificial seawater is 7.0～7.2.
Ferment with above-mentioned four kinds of substratum respectively, bacterial classification inoculum size is 4 × 10
6individual spore/mL, liquid amount 100mL in 250mL triangular flask cultivates pH nature in 28 ℃, the constant-temperature table of 180rpm.
Adopt HPLC method to measure Terrein content, Figure 1 shows that the comparison diagram of sponge symbiotic terreus fermentation Terrein output in above four kinds of different minimum mediums, as shown in Figure 1: in above four kinds of substratum, Terrein content is respectively: 0.49g/L, 1.73g/L, 0.52g/L and 0.38g/L.Obtaining thus reasonable minimum medium formula (formula 1) is: 10g glucose, 20g maltose, 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 formula 1 of determining at above-described embodiment 1, first apply Plackett-Burman design and pick out the factor larger to Terrein yield effect: glucose, maltose and malt extract.Their addition level is as shown in table 1:
The high low value of each factor in table 1Plackett-Burman experiment
Filter out after important factor, application responds Surface Method, further optimizes the optimum of glucose, maltose and malt extract and cultivates concentration.First find central point concentration by experiment of single factor, be respectively 20g glucose, 40g maltose and 6g malt extract, secondly determine that with central composite design their addition level is as shown in table 2:
The encoded radio of the central composite design middle important factor of table 2 and actual value concentration
The component of optimizing the best medium (formula 2) obtaining is: 28.41g glucose, 23.18g maltose, 20.00g N.F,USP MANNITOL, 8.52g malt extract, 10.00g Sodium Glutamate and 10.00g ammonium chloride, prepare with 1L artificial seawater, described 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, magnesium sulfate (MgSO
4) 3.305g, calcium chloride (CaCl
2) 1.141g, Repone K (KCl) 0.725g, sodium bicarbonate (NaHCO
3) 0.202g, Sodium Bromide (NaBr) 0.083g, be dissolved in 1L distilled water; The pH value of described artificial seawater is 7.0～7.2.Figure 2 shows that the comparison diagram of sponge symbiotic terreus fermentation Terrein output in minimum medium and best medium, in figure, solid line represents Terrein output, dotted line represents dry cell weight, light color represents minimum medium, the dark best medium that represents, as shown in Figure 2: carry out shake flask fermentation with 2 substratum of filling a prescription, Terrein output and dry cell weight are all high compared with minimum medium (formula 1), and in best medium, the output of Terrein reaches 3.71g/L; 2.1 times of output (1.73g/L) in the minimum medium of not optimizing.This shows, in shaking flask level, the output of Terrein is significantly improved.
Claims (6)
1. for a substratum of sponge symbiotic terreus fermentation preparation Terrein, it is characterized in that, described substratum comprises: glucose, maltose, N.F,USP MANNITOL, malt extract, Sodium Glutamate, ammonium chloride and artificial seawater;
Described substratum is specially: in the time that the artificial seawater containing in substratum is 1L, substratum contains glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g;
Described artificial seawater is specially, artificial seawater is by each component of following quality: sodium-chlor 26.518g, magnesium chloride 2.447g, magnesium sulfate 3.305g, calcium chloride 1.141g, Repone K 0.725g, sodium bicarbonate 0.202g, Sodium Bromide 0.083g, be dissolved in 1L distilled water and form; The pH value of described artificial seawater is 7.0～7.2.
2. the substratum for sponge symbiotic terreus fermentation preparation Terrein according to claim 1, is characterized in that, described sponge symbiotic terreus is terreus (Aspergillus terreus) PF26 CGMCC NO.5208.
3. a preparation method for the substratum for sponge symbiotic terreus fermentation preparation Terrein according to claim 1, is characterized in that, comprises the following steps:
The first step, preparation artificial seawater;
Second step adds glucose, maltose, N.F,USP MANNITOL, malt extract, Sodium Glutamate and ammonium chloride stirring and dissolving in the artificial seawater preparing, and then carries out High Temperature Sterilization and obtains substratum.
4. the preparation method of the substratum for sponge symbiotic terreus fermentation preparation Terrein according to claim 3, it is characterized in that, in second step, described substratum is specially, when the artificial seawater containing when substratum is 1L, substratum contains glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g.
5. the preparation method of the substratum for sponge symbiotic terreus fermentation preparation Terrein according to claim 4, it is characterized in that, in the first step, described preparation artificial seawater is specially, preparation artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g magnesium sulfate, 1.141g calcium chloride, 0.725g Repone K, 0.202g sodium bicarbonate and 0.083g Sodium Bromide to be dissolved in 1L distilled water, obtain, described in the pH value of the artificial seawater for preparing be 7.0～7.2.
6. according to the preparation method of the substratum for sponge symbiotic terreus fermentation preparation Terrein described in claim 3,4 or 5, it is characterized in that, in second step, described High Temperature Sterilization is sterilizing 30 minutes under 115 ℃ of environment.
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| 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 |
| CN103497975B (en) * | 2013-07-02 | 2015-04-22 | 上海交通大学 | Application of vorinostat and method for promoting synthesis of (+)-Terrein by sponge-associated aspergillus terreus |
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| 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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| 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 (2)
| 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》.2001,摘要. |
| 碳源和氮源对中生菌素发酵影响的研究;田云龙;《中国优秀硕士学位论文全文数据库》;20011129;摘要 * |
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