CN104975054A - Method for improving yield of 10-deacetylbaccatin III produced by fungi - Google Patents

Abstract

The invention discloses a method for improving yield of 10-deacetylbaccatin III produced by fungi. The method comprises the following steps: culturing in a culture medium to produce 10-deacetylbaccatin III, specifically, culturing at 25 DEG C for three days, then culturing at 10 DEG C for six hours, then culturing at 25 DEG C for 12 hours, and then culturing at 30 DEG C for six hours, and circulating like this at 12 variable temperatures. Therefore, the yield of 10-deacetylbaccatin III produced by fungi is stimulated by circulating excitation of high and low temperatures. Compared with the traditional culturing method, the method provided by the invention has the advantages that the yield of 10-deacetylbaccatin III is obviously improved, the operation is simple, the production cost is low, the purpose of improving the yield is achieved simply by changing the temperature, and the yield of 10-deacetylbaccatin III can be improved by 2.4 times.

Description

A kind ofly improve the method that fungi 10-removes acetyl baccatin III productive rate

Technical field

The invention belongs to fungi Cultivating techniques field, be specifically related to a kind ofly improve the method that fungi 10-removes acetyl baccatin III productive rate.

Background technology

Taxol is anticancer chemotherapeutic agent best at present, especially to women's cancers occurred frequently such as mammary cancer, uterus carcinoma and ovarian cancers, but due to its process for artificial complexity, price is very high, and therefore, this medicine mainly extracts from Ramulus et folium taxi cuspidatae, but the content of taxol of Ramulus et folium taxi cuspidatae is extremely low, and this plant growth is extremely slow, therefore, by the bar card court of a feudal ruler III or 10-go acetyl to cling to main path that III artificial semi-synthetic acquisition taxol in the card court of a feudal ruler is this medicine at present.Although it is high than taxol that the Ramulus et folium taxi cuspidatae mini-bus card court of a feudal ruler III or 10-goes acetyl to cling to card court of a feudal ruler III content, still can not meet the need of market, therefore cause taxol price still expensive, can not fully meet the need of market.Therefore, how producing the bar card court of a feudal ruler III or 10-fast goes acetyl bar card court of a feudal ruler III to become the principal element affecting taxol market value.

In recent years, extraction is found to produce the bar card court of a feudal ruler III or 10-from some endophytes of Ramulus et folium taxi cuspidatae and goes acetyl to cling to card court of a feudal ruler III, this provides basis of reality for producing the semi-synthetic raw material of taxol by fermentation means, but, the Ramulus et folium taxi cuspidatae mini-bus card court of a feudal ruler III or 10-that compare goes acetyl to cling to the content of card court of a feudal ruler III, it is lower that endophyte fermentation produces, and therefore, how to improve its fermentation yield by suitable means and become the key that can these endophytes be applied to production.

Summary of the invention

The object of this invention is to provide and a kind ofly improve the method that fungi 10-removes acetyl baccatin III productive rate, the problem that after solving the former endophyte fermentation existed in prior art, 10-goes acetyl to cling to card court of a feudal ruler III to yield poorly.

The technical solution adopted in the present invention is, a kind ofly improve the method that fungi 10-removes acetyl baccatin III productive rate, comprise the following steps: cultivate wooden mould Trichodermasp. bacterial strain IRB54 in the medium, when mycelia fermentation is after 3 days, carry out Cyclic culture, extraction and isolation is carried out to the nutrient solution obtained, obtains 10-and go acetyl to cling to card court of a feudal ruler III.

Feature of the present invention is also,

Substratum is PDB substratum, and it consists of: water 1000ml, potato 600g, sucrose 280g, pH6.5-7.0.

Cultivating 3 days conditions is in the medium: under 25 DEG C of conditions, cultivate shaking speed is 230r/min-250r/min.

Cyclic culture condition is: low temperature 10 DEG C is cultivated 6 hours, cultivates 12 hours for 25 DEG C, cultivates 6 hours for 30 DEG C, the cycle index of Cyclic culture 12 times, namely 12 days.

10-go acetyl cling to card court of a feudal ruler III extraction and isolation step comprise:

1), nutrient solution through four layers of filtered through gauze, filtered liquid is placed in 70 DEG C of low pressure rotatory evaporators and distills, and as liquid residual 10ml, is placed in the chloroform/methanol mixed solution that volume ratio is 3:2, and fully mixes, for subsequent use;

2), filter after mycelium residue added quartz sand and fully ground, then will grind residue abundant drying in 50 DEG C of baking ovens, dried residue being placed in volume ratio is that the chloroform/methanol mixed solution of 3:2 fully mixes, 40 DEG C of ultrasonic wave 30 minutes, for subsequent use;

3), by 1) in chloroform/methanol mixed solution under the condition of 45 DEG C, carry out vacuum rotating distillation removing chloroform and methyl alcohol, obtain paste extract;

4) extract is dissolved in the chloroform of 30ml again, and then carry out extracting with the water of 30ml, organic phase chloroform is evaporated by rotary evaporation under 40 DEG C of conditions, produces dry extract, is 10-and goes acetyl to cling to card court of a feudal ruler III crude extract;

5) this dry extract is dissolved in the methyl alcohol of 5ml, and by the membrane filtration of 0.45lm, filtered extract solution for standby detects in high performance liquid phase;

6) to the chloroform/methanol mixed solution of mycelium residue extract, repeating step 1)-step 5) 10-that extracts goes acetyl to cling to card court of a feudal ruler III.

The invention has the beneficial effects as follows: present method improves endophyte by the physical method of temperature cycle and produces 10-and go acetyl to cling to the productive rate of card court of a feudal ruler III, method simply and do not increase any investment.Present method improves endophyte tunning by temperature cycle method, and comparing with general chemical process to reduce has containing the pollution of chemicals to tunning, reduces extraction purification cost.

The present invention carries out a strain by temperature cycle method and produces the cultivation that 10-removes acetyl bar card court of a feudal ruler III, and the output making its 10-go acetyl to cling to card court of a feudal ruler III significantly improves, and reaches 2.4 times of original contrast output.

Accompanying drawing explanation

Fig. 1 is the bacterium colony figure of wooden mould Trichodermasp. bacterial strain IRB54 and spore and sporophore figure thereof.

Wherein, A) be bacterium colony figure; Figure B is bacterium colony conidium and conidiophore thereof.

Embodiment

Below in conjunction with embodiment, the present invention is described in detail.

The invention provides and a kind ofly improve the method that fungi 10-removes acetyl baccatin III productive rate, comprise the following steps:

Cultivate wooden mould Trichodermasp. bacterial strain IRB54 in the medium, when mycelia fermentation is after 3 days, carry out 10 DEG C 6 hours, 25 DEG C 12 hours, 30 DEG C of 6 h cycle is cultivated, and makes the growth of bacterial strain IRB54 be subject to the stimulation of high/low temperature, thus increases the output of secondary fungus material.

Wherein, substratum is PDB substratum, and it consists of: water 1000ml, potato 600g, sucrose 280g, pH6.5-7.0;

Culturing process is: first cultivate 3 days under 25 DEG C of conditions, cultivation shaking speed is 230r/min-250r/min.The initial temperature that temperature cycle is cultivated is low temperature 10 DEG C 6 hours, then 25 DEG C 12 hours, be then 30 DEG C and cultivate 6 hours.The number of times of temperature cycle is 12 times, is equivalent to 12 days.

It is that the IRB54 bacterial strain of the mould Trichodermasp. of wooden mould wood is (open that the selected product 10-cultivated goes acetyl to cling to card court of a feudal ruler III fungi, reference: Lietal. (2015) IsolationandIdentificationofa10-DeacetylBaccatin-III-Pro ducingEndophytefromTaxuswallichiana.Appl.Biochem.Biotech nol.175:2224-2231), the bacterium colony figure of wooden mould Trichodermasp. bacterial strain IRB54 and spore and sporophore figure thereof are as shown in Figure 1.

10-go acetyl cling to card court of a feudal ruler III extraction and isolation step comprise:

1), nutrient solution through four layers of filtered through gauze, filtered liquid is placed in 70 DEG C of low pressure rotatory evaporators and distills, and as liquid residual about 10ml, is placed in chloroform/methanol (3:2, v/v) mixed solution, and fully mixes, for subsequent use.

2) after, filtering, mycelium residue is added quartz sand and is fully ground, then will grind residue fully dry in 50 DEG C of baking ovens, and dried residue will be placed in chloroform/methanol (3:2, v/v) mixed solution and fully mix, 40 DEG C of ultrasonic wave 30 minutes, for subsequent use.

3) by 1) in chloroform/methanol mixed solution under the condition of 45 DEG C, carry out vacuum rotating distillation removing chloroform and methyl alcohol, obtain paste extract.

4) extract is dissolved in the chloroform of 30ml again, and then carry out extracting with the water of 30ml, organic phase chloroform is evaporated by rotary evaporation under 40 DEG C of conditions, produces dry extract (10-goes acetyl to cling to card court of a feudal ruler III crude extract).

5) this dry extract is dissolved in the methyl alcohol of 5ml, and by the membrane filtration of 0.45lm, filtered extract solution for standby detects in high performance liquid phase.

6) to the chloroform/methanol mixed solution of mycelium residue extract, the 10-using identical method to extract goes acetyl to cling to card court of a feudal ruler III.

7) by 5) and 6) in respective the obtained 5ml methanol solution being dissolved with extract be mixed into 10ml, for subsequent use.

10-using 99.8% goes acetyl to cling to card court of a feudal ruler III as standard substance, carries out 10-go acetyl to cling to the mensuration of card court of a feudal ruler III content by high performance liquid phase method to said extracted from fermented liquid and mycelial extract.

Compared with the cultural method (control group) adopting the method common with prior art, its productive rate is as shown in table 1, adopts the productive rate of present method greatly to improve.

Table 1 circulating temperature is cultivated and contrast 10-goes acetyl to cling to the productivity ratio of card court of a feudal ruler III comparatively (unit: mg/l)

The 10-that this invention improves wooden mould Trichoderma sp. bacterial strain IRB54 by temperature cycle method goes acetyl to cling to the output of card court of a feudal ruler III, successful, simple to operate.10-goes acetyl bar card court of a feudal ruler III to belong to alkyl compound, it is typical secondary metabolite, the experimental results shows, under microorganism is in higher temperatures or lower temperature environments bar, its secondary metabolite amount can increase thereupon, and the present invention stimulates the increase of inducing 10-to go acetyl bar card court of a feudal ruler III output according to the circulation high/low temperature by going acetyl to cling to the bacterial strain IRB54 of card court of a feudal ruler III to product 10-, result shows, the method successful, simple possible.

Another advantage of this invention is different from the method that tradition adds precursor, the method reaches by regulating temperature the increase increasing Secondary Metabolites of Microorganisms, not only save investment, and environmental protection, because extract increases before some, may bring extraction cost increase and medicine investment increase, and if some add precursors can not thoroughly remove may to human body produce murder by poisoning.

Claims (5)

1. one kind is improved the method that fungi 10-removes acetyl baccatin III productive rate, it is characterized in that, comprise the following steps: cultivate wooden mould Trichoderma sp. bacterial strain IRB54 in the medium, when mycelia fermentation is after 3 days, carry out Cyclic culture, extraction and isolation is carried out to the nutrient solution obtained, obtains 10-and go acetyl to cling to card court of a feudal ruler III.

2. raising fungi 10-according to claim 1 goes the method for acetyl baccatin III productive rate, and it is characterized in that, described substratum is PDB substratum, and it consists of: water 1000ml, potato 600g, sucrose 280g, pH 6.5-7.0.

3. raising fungi 10-according to claim 1 goes the method for acetyl baccatin III productive rate, it is characterized in that, described cultivation in the medium 3 days conditions are: under 25 DEG C of conditions, cultivate shaking speed is 230r/min-250r/min.

4. raising fungi 10-according to claim 1 goes the method for acetyl baccatin III productive rate, it is characterized in that, described Cyclic culture condition is: low temperature 10 DEG C is cultivated 6 hours, cultivate 12 hours for 25 DEG C, cultivate 6 hours for 30 DEG C, the cycle index of described Cyclic culture 12 times, namely 12 days.

5. raising fungi 10-according to claim 1 goes the method for acetyl baccatin III productive rate, it is characterized in that, described 10-go acetyl cling to card court of a feudal ruler III extraction and isolation step comprise:

1), nutrient solution through four layers of filtered through gauze, filtered liquid is placed in 70 DEG C of low pressure rotatory evaporators and distills, and as liquid residual 10ml, is placed in the chloroform/methanol mixed solution that volume ratio is 3:2, and fully mixes, for subsequent use;

2), filter after mycelium residue added quartz sand and fully ground, then will grind residue abundant drying in 50 DEG C of baking ovens, dried residue being placed in volume ratio is that the chloroform/methanol mixed solution of 3:2 fully mixes, 40 DEG C of ultrasonic wave 30 minutes, for subsequent use;

3), by 1) in chloroform/methanol mixed solution under the condition of 45 DEG C, carry out vacuum rotating distillation removing chloroform and methyl alcohol, obtain paste extract;

4) extract is dissolved in the chloroform of 30ml again, and then carry out extracting with the water of 30ml, organic phase chloroform is evaporated by rotary evaporation under 40 DEG C of conditions, produces dry extract, is 10-and goes acetyl to cling to card court of a feudal ruler III crude extract;

5) this dry extract is dissolved in the methyl alcohol of 5ml, and by the membrane filtration of 0.45lm, filtered extract solution for standby detects in high performance liquid phase;

6) to the chloroform/methanol mixed solution of mycelium residue extract, repeating step 1)-step 5) 10-that extracts goes acetyl to cling to card court of a feudal ruler III.