CN106190854B - A kind of preparation method of desert pseudocystoid and oritavancin intermediate - Google Patents

Abstract

The invention discloses a preparation method of desert pseudocyst and oritavancin intermediate. The high-yielding oritavancin intermediate strain Cystobacter desertessica HS807-AN-2396 (Kibdelosporangium aridum) is preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms, and the preservation number is CGMCC No.10576. The preparation method of the oritavancin intermediate is as follows: fermenting the described oritavancin intermediate CGMCC No.10576 in a fermentation medium to obtain the oritavancin intermediate A82846B from the fermentation broth. The preparation method can realize a high yield of oritavancin intermediate fermentation titer of 2315 mg/L on a 60 m ³ tank, which is beneficial to the industrial production of oritavancin.

Description

A kind of preparation method of desert pseudocystoid and oritavancin intermediate

technical field

The invention belongs to the field of medicine, and in particular relates to a method for preparing an intermediate of Cystoids desertum and oritavancin.

Background technique

Aerobic Gram-positive cocci are important pathogens of bacterial infections. Since the late 1980s, the infections caused by such bacteria have been on the rise. However, the increasing drug resistance of Gram-positive cocci has brought serious challenges to the treatment of infectious diseases. Teicoplanin and vancomycin are currently commonly used glycopeptide drugs for the treatment of drug-resistant Gram-positive bacterial infections. However, with the increasingly widespread clinical application, vancomycin-resistant Staphylococcus aureus (VISA, VRSA) and vancomycin-resistant Enterococcus (VRE) have also appeared in various parts of the world. Therefore, teicoplanin and vancomycin cannot fully meet the clinical needs, and it is necessary to develop and apply new glycopeptide antibiotics.

On August 6, 2014, the U.S. FDA approved Oritavancin (Oritavancin, LY-333328) developed by Medicines, with a trade name of Orbactiv, which is a second-generation sugar developed after Teicoplanin and Vancomycin Peptide antibiotics. Orbactiv is the first and only single-dose antibiotic approved by the U.S. FDA for the treatment of acute bacterial skin and skin structure infections (ABSSSIs). Orbactiv Injection is indicated for the treatment of adult patients with ABSSSIs caused by susceptible strains of Gram-positive bacteria, including: Staphylococcus aureus (methicillin-sensitive and methicillin-resistant strains), Streptococcus pyogenes (Mlicrococcus scarlatinae), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus, and Enterococcus faecalis (vancomycin-susceptible strains).

The chemical synthesis of oritavancin requires the key intermediate A82846B, which is produced by the fermentation of Kibdelosporangium aridum. At present, there are many studies on the drug activity of oritavancin at home and abroad, but there are few reports on the development and production of the intermediate A82846B. U.S. Patent US5843437 (authorization date: December 1, 1998) obtained by Eli Lilly and Company in the 1990s described the strains, fermentation and extraction process for producing A82846B, but its fermentation titer was low. And improving the fermentation level of A82846B has important application value for the industrialization of oritavancin.

ARTP (Atmospheric and Room Temperature Plasma) is a plasma at room temperature at atmospheric pressure. It is a new plasma source developed in recent years. Plasma jet with high concentration of reactive particles (such as helium atoms in excited state, oxygen atoms, nitrogen atoms, OH ^- radicals, etc.). Studies have shown that when an appropriate dose of active particles in the plasma acts on microorganisms, it can change the structure and permeability of microbial cell walls and cell membranes, and cause genetic damage, which in turn will significantly change the microbial gene sequence and its metabolic network, and eventually lead to microbial production. mutation. Compared with traditional mutagenesis methods, the use of atmospheric pressure and room temperature plasma can effectively cause diverse DNA damage, high mutation rate, and easy to obtain mutant strains with good genetic stability; compared with molecular manipulation methods or other chemical mutagenesis methods , Atmospheric pressure and room temperature plasma for microbial mutation breeding has the advantages of simple operation, low cost, and no toxic and harmful substances participating in the mutagenesis process.

NTG (N-methyl-N'-nitro-N-nitrosoguanidine) is a commonly used alkylating agent in microbial breeding and is known as a super mutagen. The alkylating agent has an active alkyl group, which can be transferred to other molecules with high electron density, adding alkyl groups to many positions of the base, thereby changing the hydrogen bond in many ways. Mutations induced by NTG are mainly GC-AT transitions, in addition to small excisions, frameshift mutations, and loss of GC pairs.

Contents of the invention

The technical problem to be solved by the present invention is to provide a kind of desert sporangiocystis and its preparation method for obtaining A82846B by fermentation in view of the defect of low fermentation titer of oritavancin intermediate (A82846B). The cystic fungus can produce the oritavancin intermediate in a high yield, and the fermentation efficiency is high; the preparation method can obtain a large amount of the oritavancin intermediate at a relatively low cost, which is beneficial to industrial production.

The structural formula of A82846B of the present invention is as shown in formula (1):

The present inventors combined two physical and chemical mutagenesis means of ARTP and NTG to carry out compound mutagenesis screening, and obtained the A82846B-producing mutant Cystopsis desertica with high fermentation titer and stable genetic character. And optimize the fermentation medium formula and process parameters of the method of obtaining A82846B by fermenting Cystopsoides desertum.

One of the technical solutions of the present invention is: a desert cyst bacterium (Kibdelosporangium aridum), which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is: CGMCC No.10576.

The bacterial strain A.orientalis A82846 was isolated from soil samples in Haiti, the bacterial strain HS807-O-13 obtained by natural isolation, and the bacterial strain HS807-O-13 was screened by mutagenesis to obtain the bacterial strain HS807-A-639, and the bacterial strain HS807- A-639 was screened by compound mutagenesis of ARTP and NTG, and the strain CGMCC No.10576 with high yield of A82846B was obtained. The strain was identified, and the result was Kibdelosporangium aridum, named HS807-AN-2396. The strain was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 11, 2015, and received the registration number CGMCC No.10576 of the preservation center, which has the following microbiological characteristics:

1. Morphological characteristics

When the bacterial strain cultured in the solid medium is observed under a microscope, the bacterial strain is filamentous in different lengths, the diameter of mycelium is 0.1-1.0 μm, white spores are produced, and Gram staining is positive.

2. Characteristics of Cultivation

When the temperature is 14-37°C and the pH is 5.0-7.5, the colony grows well, and if it exceeds this range, the growth will be poor or no growth will occur.

3. Physiological characteristics

D-glucose, maltose, D-lactose are well utilized as carbon sources, but D-xylose, L-arabinose and glycine are not. Ammonium nitrogen sources can be effectively utilized; however, nitrate nitrogen sources are difficult to utilize. Can grow well on degradants other than adenine; in terms of degradation, only casein and tyrosine can be degraded. And can carry out gelatin liquefaction and can produce hydrogen sulfide.

The second technical solution of the present invention is: a method for preparing oritavancin intermediate A82846B, fermenting the described Cystopsis desertica CGMCC No.10576 in the fermentation medium, and obtaining oritavancin from the fermentation liquid Intermediate A82846B.

The fermentation time of the present invention is a conventional time in the field, preferably 168-240 hours, more preferably 192-212 hours, most preferably 202-210 hours. The fermentation temperature in the present invention is a conventional temperature in the field, preferably 25-34°C, more preferably 28-32°C.

The fermentation is carried out in a shake flask, and the volume of the shake flask is a conventional volume in the art, preferably 250 mL. The rotation speed is a conventional rotation speed in the art, preferably 250 rpm. The amplitude is conventional in the art, preferably 5 cm. The humidity of the fermentation is the conventional humidity in the field, preferably 40-60%.

The fermentation is carried out in a small-scale fermenter, and the volume of the small-scale fermenter is a conventional volume in the art, preferably 50L. The stirring speed is a conventional speed in the field, preferably 200-600 rpm. The dissolved oxygen amount is the conventional dissolved oxygen amount in this field, preferably 30-50%, and the percentage is conventional in the field, preferably the oxygen content of the fermented liquid in the fermenter accounts for the saturated oxygen content at this temperature. percentage of volume. The air flow rate is a conventional air flow rate in the field, preferably 0.5-1.0 vvm.

The fermentation is carried out in a pilot-scale fermenter, and the volume of the pilot-scale fermenter is a conventional volume in the field, preferably 5000L. The stirring speed is a conventional speed in the field, preferably 30-150 rpm. The dissolved oxygen amount is the conventional dissolved oxygen amount in this field, preferably 30-40%, and the percentage is conventional in the field, preferably the oxygen content of the fermented liquid in the fermenter accounts for the saturated oxygen content at this temperature. percentage of volume. The air flow rate is a conventional air flow rate in the field, preferably 0.2-1.0vvm.

The fermentation is carried out in an industrialized fermenter, and the volume of the industrialized fermenter is a conventional volume in the field, preferably 60m ³ . The stirring speed is a conventional speed in the field, preferably 30-120 rpm. The dissolved oxygen amount is the conventional dissolved oxygen amount in this field, preferably 30-35%, and the percentage is conventional in the field, preferably the oxygen content of the fermented liquid in the fermenter accounts for the saturated oxygen content at this temperature. percentage of volume. The air flow rate is a conventional air flow rate in the field, preferably 0.1-0.8vvm.

The inoculum amount of the fermented seed solution is a conventional inoculum amount in the art, preferably 10-12%, and the percentage is volume percentage. The seed liquid is obtained by a method comprising the following steps: cultivating the described Cystopsis desertica CGMCC No.10576 in a seed medium.

Wherein, the culture time is a conventional time in the field, preferably 37-44 hours, more preferably 41-43 hours. The culture temperature is a conventional temperature in the field, preferably 28-32°C.

The culture is carried out in shake flasks, the volume of which is conventional in the art, preferably 250 mL. The rotation speed is a conventional rotation speed in the art, preferably 250 rpm. The amplitude is conventional in the art, preferably 5 cm. The humidity of the fermentation is the conventional humidity in the field, preferably 40-60%.

The cultivation is carried out in a small-scale seed tank, and the volume of the small-scale seed tank is a conventional volume in the art, preferably 15L. The stirring speed is a conventional speed in the field, preferably 200-600 rpm. The dissolved oxygen amount is the conventional dissolved oxygen amount in this field, preferably 30-50%, and the percentage is conventional in the field, preferably the oxygen content of the seed medium in the seed tank accounts for the saturated content at this temperature. percentage of oxygen. The air flow rate is a conventional air flow rate in the field, preferably 0.5-1.5vvm.

The cultivation is carried out in a pilot-scale seed tank, and the volume of the pilot-scale seed tank is a conventional volume in the field, preferably 500L. The stirring speed is a conventional speed in the field, preferably 30-150 rpm. The dissolved oxygen is the conventional dissolved oxygen in this field, preferably 30-40%, and the percentage is conventional in the field, preferably the oxygen content of the seed medium in the seed tank accounts for the saturated content at this temperature. percentage of oxygen. The air flow rate is a conventional air flow rate in the field, preferably 0.2-1.0vvm.

The cultivation is carried out in an industrialized seed tank, and the volume of the industrialized seed tank is conventional in the field, preferably 15 m ³ . The stirring speed is a conventional speed in the art, preferably 50-200 rpm. The dissolved oxygen amount is the conventional dissolved oxygen amount in this field, preferably 30-50%, and the percentage is conventional in the field, preferably the oxygen content of the seed medium in the seed tank accounts for the saturated content at this temperature. percentage of oxygen. The air flow rate is a conventional air flow rate in the field, preferably 0.2-1.0vvm.

The seed culture medium is a conventional seed culture medium in the field, preferably, it includes the following components (g/L): glucose 5.0-15.0, soluble starch 10.0-30.0, soybean meal powder 10.0-30.0, yeast Extract powder 2.0-8.0, hydrolyzed casein 2.0-8.0, sodium chloride 0.1-0.5, calcium carbonate 0.9-1.5, pH 6.8-7.5; more preferably include the following components (g/L): glucose 10.0, Soluble starch 20.0, soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.

Preferably, the fermentation is carried out in a small-scale fermenter, a pilot-scale fermenter or an industrial fermenter, which also includes the following steps: adding L-tyrosine; and/or, adding L-valline acid.

Wherein, the content of the L-tyrosine in the fermentation medium is 0.0-30.0 g/L, preferably 4.0-6.0 g/L. The content of the L-valine in the fermentation medium is 0.0-20.0 g/L, preferably 2.0-4.0 g/L.

The third technical solution of the present invention is: a fermentation medium for obtaining the oritavancin intermediate A82846B by fermenting the described Cystopsis desertica CGMCC No.10576, which includes the following components (g/L) : organic carbon source 75.0～155.0, organic nitrogen source 24.0～58.0 and inorganic salt 1.9～6.3; described organic carbon source is one or more in glucose, maltodextrin, sucrose, molasses and cornstarch; described The organic nitrogen source is one or more of soybean cake powder, cottonseed cake powder, hydrolyzed casein, yeast extract powder and peptone; the inorganic salt is one or two of sodium chloride and calcium carbonate .

Preferably, the fermentation medium described in the fermentation medium further includes one or more of the following components (g/L): 0.04-0.22 trace elements, 0.0-50.0 amino acids and/or defoaming Dose 0.2 to 0.8. The trace elements are one or two of manganese sulfate tetrahydrate and cobalt chloride hexahydrate. The amino acid is one or more of L-tyrosine, L-valine, L-glutamic acid and L-leucine, preferably, the amino acid is L-tyrosine and one or both of L-valine. The defoamer is a fermentation defoamer, preferably, the defoamer is a fermentation defoamer THIX-298, purchased from Yantai Hengxin Chemical Technology Co., Ltd.

The pH of the fermentation medium is a conventional pH in the field, preferably 6.0-7.5, more preferably 6.5-7.8.

More preferably, the fermentation medium includes the following components (g/L): glucose 10.0-30.0, maltodextrin 60.0-100.0, molasses 5.0-25.0, hydrolyzed casein 2.0-10.0, L-tyrosine 0～30.0, L-valine 0～20.0, yeast extract powder 2.0～8.0, cottonseed cake powder 20.0～40.0, sodium chloride 0.4～0.8, calcium carbonate 1.5～5.5, manganese sulfate tetrahydrate 0.02～0.14, six Cobalt chloride hydrochloride 0.02～0.08 and defoamer 0.2～0.8, pH 6.5～7.8; Optimally, the fermentation medium includes the following components (g/L): glucose 20.0, maltodextrin 80.0, Molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, hexahydrate Cobalt Chloride 0.05 and THIX-2980.2, pH 7.5.

The fourth technical solution of the present invention is: a method for obtaining the described Cystopsis desertica CGMCC No.10576, which includes the following steps, cultivating the described Cystopsis desertica CGMCC No.10576 in the culture medium That's it.

Wherein, the medium is a conventional medium in the art, and it is enough to be able to grow the described Cystopsis desertica CGMCCNo.10576, preferably ISP2, Gaoshi No. 1, YMS or ISP9 medium, more preferably Preferably, it is the fermentation medium described in the description of the present invention, and most preferably it is the seed culture medium described in the description of the present invention. The temperature of the cultivation is a conventional temperature in this field, and it is sufficient to be able to grow the CGMCC No.10576, preferably 14-37°C, more preferably 25-34°C, and most preferably The ground temperature is 28-32°C. The pH of the culture is a conventional pH in the field, and it is enough to grow the CGMCC No.10576, preferably 5.0-7.5, more preferably 6.0-7.5, and most preferably 6.5 ~7.8.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effects of the present invention are: the ARTP and NTG compound mutagenesis A82846B high-yield strain CGMCC No.10576 provided by the present invention has stable genetic properties, good reproducibility, easy amplification, and excellent industrial application value. The method for producing A82846B by fermentation according to the present invention comprehensively adjusts the fermentation formula and fermentation control process, and performs supplementation and residual control for L-tyrosine and L-valine to realize the fermentation titer on a ^60m3 tank The high yield of 2315mg/L is higher than the existing literature reports in terms of scale and technical level, which is conducive to the industrial production of oritavancin.

Biological Material Deposit Information

The HS807-AN-2396 of the present invention has been preserved in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC) on March 11, 2015, and the preservation address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postcode : 100101, the deposit number is: CGMCC No.10576, the biological material (strain) is HS807-AN-2396, and the classification name is Kibdelosporangium aridum.

Description of drawings

Figure 1 is the breeding lineage of Cystopsis deserticus HS807-AN-2396, and the numbers in Figure 1 indicate the corresponding A82846B fermentation titer of the strain.

Fig. 2 is the HPLC profile of the fermentation broth of Example 1 (the Rt of A82846B is 5.588).

Fig. 3 is the fermented liquid ESI/MS pattern of embodiment 1.

Fig. 4 is the fermentation metabolism curve of the 60m ³ tank of embodiment 15.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The preparation of embodiment 1 high-yield bacterial strain Cystobasidium deserticus HS807-AN-2396 (Kibdelosporangium aridum)

The strain A.orientalis A82846 was isolated from soil samples in Haiti (for strain A.orientalisA82846 see: Oliver Puk, Petra Huber, Daniel Bischoff, etc., Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908: Function of a Halogenase and a Haloperoxidase/Perhydrolase, Chemistry & Vol.9, 225–235, February, 2002), and then the HS807-O-13 strain was naturally isolated, and the HS807-O-13 strain was screened by mutagenesis to obtain the starting strain HS807-A-639. The bacterial suspension of HS807-A-639 was prepared and filtered with filter paper, and the cell dispersion degree of microscopic examination was over 95%, to ensure that most of them were single cells, and then used for mutagenesis treatment.

ARTP mutagenesis treatment: adjust the plasma generating gas-helium flow rate to 12.5L/min; the distance between the plasma launch port and the iron sheet containing the sample is 2mm; the plasma irradiation power is 100w, and the irradiation time is 30s, and the obtained ARTP irradiated bacterial suspension.

Accurately weigh 10.0 mg of NTG and dissolve it in 1 mL of acetone, add the above-mentioned ARTP-irradiated bacterial suspension to make the final concentration of NTG reach 1.0 mg/L. Slowly shake at 4°C for 20 minutes, centrifuge to discard the supernatant, disperse with sterile saline, and repeat washing twice to terminate the reaction.

Pipette the liquid medium containing the mutagen into a petri dish containing 750mg/L A82846B (obtained from Zhejiang Hisun Pharmaceutical Co., Ltd.) and a petri dish containing 2.0g/L L-tyrosine for coating culture . The culture temperature is 28-32°C, and the culture period is 8 days. Calculation results showed that the lethal rate of compound mutagenesis reached 89%.

Pick a single colony from the resistant plate after compound mutagenesis, and put it into a seed bottle for culture, wherein the seed medium is (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed Casein 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2, cultured at 30°C for 3 days, and then cultured in a 250mL fermentation shaker flask, wherein the fermentation medium was (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, chlorine hexahydrate Cobalt 0.05 and THIX-2980.2, pH 7.5. Fermented at 30°C for 8 days, and the content of A82846B was detected by HPLC. A high-yield mutant strain HS807-AN-2396 was obtained, and the shake flask fermentation titer was 1325mg/L, and the shake flask fermentation titer was 1263mg/L after double screening (that is, shaking the flask again according to the same conditions to carry out fermentation culture). The HPLC spectrum is shown in FIG. 2 . The fermentation broth obtained by fermentation is separated and purified (see Tsuji N. for the steps of separation and purification; T. Kamigauchi, M. Kobayashi & Y. Terui: New glycopeptide antibiotics: II. The isolation and structures of chloroorienticins. J. Antibiotics 41: 1506-1510, 1988 ), the ESI/MS spectrum of the purified A82846B is shown in Figure 3.

Pick a single colony from the starting strain HS807-A-639, and culture it in a seed bottle. The seed medium is (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed Casein 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2, cultured at 30°C for 3 days, and then cultured in a 250mL fermentation shaker flask, wherein the fermentation medium was (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, chlorine hexahydrate Cobalt 0.05 and THIX-2980.2, pH 7.5. Fermented at 30°C for 8 days, and the content of A82846B was detected by HPLC. The shake flask fermentation titer was 683mg/L, and the shake flask fermentation titer was 656mg/L after re-screening (that is, shake the flask again according to the same conditions to carry out fermentation culture). From the fermentation results, the fermentation titer of the high-yield mutant strain HS807-AN-2396 was 93% higher than that of the starting strain HS807-A-639.

The above-mentioned high-yielding mutant strain HS807-AN-2396 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC) on March 11, 2015, and the preservation number was: CGMCC No.10576, biological material (strain) It is HS807-AN-2396, and the classification name is Kibdelosporangium aridum.

Morphological and culture characteristics of embodiment 2 bacterial strain HS807-AN-2396 (CGMCC No.10576)

The experiments were carried out with reference to the relevant contents in the "Streptomyces Identification Manual", "Classification and Identification of Actinomycetes", "Common Bacterial System Identification Manual" and "Molecular Cloning Experiment Guide".

Relevant symbols indicate explanation: 0: no growth; 1: weak growth; 2: able to grow, with a few spores; 3: good growth, with a large number of spores; 4: best growth, with abundant spores; +: positive; -: feminine.

Culture characteristics: Nine mediums of ISP1, ISP2, ISP3, ISP4, ISP5, Gaoshi No. 1, calcium malate, YMS and Cha's (obtained from Zhejiang Hisun Pharmaceutical Co., Ltd.) were used, and cultured at 28°C for 6～ After 8 days, observe the color and pigment situation of mycelia.

Table 1 Culture characteristics of bacterial strain HS807-AN-2396 on 9 kinds of media

The results in Table 1 show that the improved strain grows well on ISP2, Gaoshi No. 1, and YMS, and shows different appearances, pigments, and sporulation on different media.

Physiological and biochemical characteristics of Example 3 bacterial strain HS807-AN-2396 (CGMCC No.10576)

The culture conditions were all cultured at 28°C for 6-8 days.

a) Carbon source: ISP9 was used as the basal medium, and the final concentrations of various carbon sources were all 1.0%, and the stated percentages were mass percentages.

b) Inorganic nitrogen source: ISP9 was used as the basic medium, and the concentrations of potassium nitrate and ammonium sulfate were both 0.1%.

The utilization of carbon source and inorganic nitrogen source is shown in Table 2. The results of Table 2 show that strain HS807-AN-2396 has different degrees of utilization of different carbon sources, and can well utilize D-glucose, maltose, and D-lactose, but D-xylose, L-arabinose and glycine are not utilized. Ammonium nitrogen sources can be effectively utilized; however, nitrate nitrogen sources are difficult to utilize.

The utilization of carbon source and nitrogen source of table 2 bacterial strain HS807-AN-2396

carbon source growth situation carbon source growth situation Inorganic nitrogen source growth situation D-glucose 3 Salicin 2 ammonium sulfate + D-Raffinose 2 D-lactose 3 potassium nitrate - D-xylose 0 Galactose 2 D-sorbitol 2 Inositol 2 L-arabinose 0 Mannitol 2 glycerin 2 Glycine 0 maltose 3 Xylan 2 D-fructose 1 inulin 2 D-sucrose 2 D 2

c) Degradation test: GYEA (pH 6.8) was used as the basal medium, and the concentrations of various degradation products are shown in Table 3. Table 3 shows that strain HS807-AN-2396 can grow well on several degradants except adenine; in terms of degradation, strain HS807-AN-2396 can only degrade casein and tyrosine. The percentages stated in Table 3 are mass percentages.

The degradation test result of table 3 bacterial strain HS807-AN-2396

Degradants Degradant concentration result Degradants Degradant concentration result adenine 0.5% 2,- casein 1.0% 4, + Guanine 0.5% 4,- Tyrosine 1.0% 4, + Xylan 0.4% 4,- Tween-40 1.0% 4,- Hypoxanthine 0.4% 4,- Tween-60 1.0% 4,- Tween-80 1.0% 4,-

d) Catalase test uses YMS medium.

e) M.R and V-P experiments adopt the method of "Common Bacterial System Identification Manual". Table 4 demonstrates that strain HS807-AN-2396 is capable of gelatin liquefaction and capable of producing hydrogen sulfide.

Table 4 The main physiological and biochemical characteristics of strain HS807-AN-2396

Pilot projects result Pilot projects result Pilot projects result

gelatin liquefaction + peptonization of milk - Cellulose Utilization - starch hydrolysis - Nitrate reduction - Catalase - curdled milk - hydrogen sulfide generation + V.P experiment - M.R. experiment -

Illustrate by the data of embodiment 2～3, described desert pseudocystis CGMCC No.10576 has the following microbiological characteristics:

1. Morphological characteristics

When the strains cultured in solid medium were observed under a microscope, the cells were filamentous in different lengths, with a size of 0.4-1.0 μm, producing white spores, and Gram staining was positive.

2. Characteristics of Cultivation

When the temperature is 14-37°C and the pH is 5.0-7.5, the colony grows well, and if it exceeds this range, the growth will be poor or no growth will occur.

3. Physiological characteristics

D-glucose, maltose, D-lactose are well utilized as carbon sources, but D-xylose, L-arabinose and glycine are not. Ammonium nitrogen sources can be effectively utilized; however, nitrate nitrogen sources are difficult to utilize. Can grow well on degradants other than adenine; in terms of degradation, only casein and tyrosine can be degraded. And can carry out gelatin liquefaction and can produce hydrogen sulfide.

16S rDNA sequence analysis of Example 4 bacterial strain HS807-AN-2396 (CGMCC No.10576)

16S rDNA sequence analysis: The 16S rDNA sequence of the strain HS807-AN-2396 was compared with related sequences in GenBank by BLAST, and the results are shown in Table 5.

Table 5 Homology of strain HS807-AN-2396 and related strains

Through the apparent characteristic test of the strain HS807-AN-2396 (CGMCC No.10576), it was found that the classification-related parameters of the strain and Kibdelosporangium aridum were very close, and the 16S rDNA of the strain HS807-AN-2396 was tested Sequencing, the sequencing results are shown in the sequence table SEQ ID No.1. The 16S rDNA sequence of the bacterial strain HS807-AN-2396 as shown in the sequence table SEQ ID No.1 was compared with the 16S rDNA sequence of Kibdelosporangium aridum, and the comparison results showed that the bacterial strain HS807-AN- The homology between 2396 and Kibdelosporangium aridum is as high as 98.9%: the evolutionary relationship between the strain Kibdelosporangium aridumstrain DSM 43828 and the strain HS807-AN-2396 is the closest, and the homology between the two is 98.9% . Therefore, the strain HS807-AN-2396 was identified as Kibdelosporangium aridum.

Example 5 Fermentation of bacterial strain HS807-AN-2396 (CGMCC No.10576) in 250 mL shake flask without amino acid medium

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.

The amount of seed medium in the seed bottle is 20mL/250mL, the culture temperature is 32°C, the culture humidity is 50-60%, the shaker amplitude is 5cm, the shaker speed is 250rpm, and the culture period is 39h.

Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolyzed casein 6.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08 And cobalt chloride hexahydrate 0.05, pH7.3.

The amount of transplantation from the seed bottle to the fermentation bottle is 10%, and the stated percentage is a volume percentage.

The loading capacity of the fermentation medium in the fermentation bottle is 20mL/250mL, the culture temperature is 28°C, the culture humidity is 40-50%, the shaker amplitude is 5cm, the shaker speed is 250rpm, and the culture period is 212h.

After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 923mg/L.

Example 6 Fermentation of strain HS807-AN-2396 (CGMCC No. 10576) in a medium supplemented with L-tyrosine and L-valine in a 250 mL shake flask.

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.

The filling capacity of the seed bottle is 20ml/250ml, the culture temperature is 28°C, the culture humidity is 40-50%, the shaker amplitude is 5cm, the shaker speed is 250rpm, and the culture period is 37h.

Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, Sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08 and cobalt chloride hexahydrate 0.05, pH7.3.

The amount of transplantation from the seed bottle to the fermentation bottle is 10%, and the stated percentage is a volume percentage.

The loading capacity of the fermentation medium in the fermentation bottle is 20mL/250mL, the culture temperature is 32°C, the culture humidity is 50-60%, the shaker amplitude is 5cm, the shaker speed is 250rpm, and the culture cycle is 210h.

After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 1210mg/L.

Experimental Technology Research on the Fermentation of the 7th Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on the 50L Tank

Based on the process parameters obtained from the 250ml shake flask, and according to the characteristics of the strain HS807-AN-2396 described in Example 1, the fermentation process in a 50L tank was designed and optimized, and the growth and metabolism of the mutant strain and the best production capacity of A82846B were investigated.

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (purchased from Yantai Hengxin Chemical Technology Co., Ltd.) 0.2, pH 7.2. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 43h.

Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, Sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.6, pH7.3.

The amount of inoculation from the seed tank to the fermenter is 12%, and the stated percentage is a volume percentage.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 28-32°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 206h.

Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 4.0-6.0g/L.

During the fermentation process, the detection of total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer is carried out at the same time. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 2156mg/L.

Experimental Technology Research on the Fermentation of Example 8 Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on a 50L Tank

Seed medium (g/L): glucose 5.0, soluble starch 10.0, soybean cake powder 10.0, yeast extract powder 2.0, hydrolyzed casein 2.0, sodium chloride 0.1, calcium carbonate 0.9, THIX-2980.2, pH7.5.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 10.0, corn starch 100.0, sucrose 5.0, hydrolyzed casein 2.0, L-glutamic acid 30.0, L-leucine 20.0, yeast extract powder 2.0, soybean cake powder 20.0, carbonic acid Calcium 5.5, manganese sulfate tetrahydrate 0.02, THIX-2980.8, pH 6.5.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 28-32°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 168h.

Fermentation feeding control: feed L-valine, and control the residual amount of L-valine at 0.0-20.0g/L.

During the fermentation process, the total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer were detected at the same time, and the above fermentation conditions can be fermented to obtain A82846B. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 2110mg/L.

Experimental Technology Research on Fermentation of Example 9 Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on a 50L Tank

Seed medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, yeast extract powder 8.0, hydrolyzed casein 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH 6.8.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, hydrolyzed casein 10.0, yeast extract powder 8.0, cottonseed cake powder 40.0, sodium chloride 0.8, cobalt chloride hexahydrate 0.02, THIX- 2980.8, pH7.8.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 28-32°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 240h.

Fermentation feeding control: feed L-tyrosine, and control the residual amount of L-tyrosine at 0.0-30.0g/L.

During the fermentation process, the total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer were detected at the same time, and the above fermentation conditions can be fermented to obtain A82846B. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 1967mg/L.

Experimental Technology Research on Fermentation of Example 10 Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on a 50L Tank

Seed medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, yeast extract powder 8.0, hydrolyzed casein 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH 6.8.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 30.0, maltodextrin 100.0, molasses 5.0, hydrolyzed casein 10.0, yeast extract powder 8.0, cottonseed cake powder 20.0, sodium chloride 0.4, calcium carbonate 1.5, manganese sulfate tetrahydrate 0.14 , Cobalt chloride hexahydrate 0.08, pH7.5.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 28-32°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 240h.

Fermentation feeding control: feed L-tyrosine, and control the residual amount of L-tyrosine to 0.0-30.0g/L.

During the fermentation process, the total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer were detected at the same time, and the above fermentation conditions can be fermented to obtain A82846B. After the fermentation was completed, the fermentation titer was detected by HPLC, and A82846B reached 2016mg/L.

Experimental Technology Research on the Fermentation of Example 11 Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on a 50L Tank

Seed medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, yeast extract powder 8.0, hydrolyzed casein 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH 6.8.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, hydrolyzed casein 10.0, yeast extract powder 8.0, cottonseed cake powder 40.0, sodium chloride 0.4, calcium carbonate 1.5, pH 5.0.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 28-32°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 240h.

Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 2.0-6.0g/L.

During the fermentation process, the total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer were detected at the same time, and the above fermentation conditions can be fermented to obtain A82846B. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 1954mg/L.

Example 12 Fermentation small test process research on bacterial strain HS807-AN-2396 (CGMCC No.10576) on 50L tank

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (purchased from Yantai Hengxin Chemical Technology Co., Ltd.) 0.2, pH 7.2. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 43h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 3.0, L-valine 3.0, L-glutamic acid 3.0, L-leucine Acid 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.2, pH7.5.

The amount of inoculation from the seed tank to the fermenter is 12%, and the stated percentage is a volume percentage.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 25°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 206h.

Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 4.0-6.0g/L.

During the fermentation process, the detection of total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer is carried out at the same time. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 1791mg/L.

Experimental Technology Research on the Fermentation of Example 13 Bacterial Strain HS807-AN-2396 (CGMCC No.10576) on a 50L Tank

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (purchased from Yantai Hengxin Chemical Technology Co., Ltd.) 0.2, pH 7.2. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Seed tank control process: culture temperature 28-32°C, stirring speed 200-600rpm, dissolved oxygen 30%-50%, air flow 0.5-1.5vvm, culture period 43h. Wherein, the filling capacity of the seed culture medium in the seed tank is 10L/15L.

Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 30.0, L-valine 20.0, peptone 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.2, pH 6.0.

The amount of inoculation from the seed tank to the fermenter is 12%, and the stated percentage is a volume percentage.

The loading capacity of the fermentation medium in the fermenter is 35L, the culture temperature is 34°C, the stirring speed is 200-600rpm, the dissolved oxygen is 30-50%, the air flow rate is 0.5-1.0vvm, and the culture period is 206h. Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 4.0-6.0g/L.

During the fermentation process, the detection of total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer is carried out at the same time. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 1563mg/L.

Example 14 Fermentation pilot-scale process research on bacterial strain HS807-AN-2396 (CGMCC No.10576) on 5000L tank

Based on the process parameters obtained in the 50L fermenter, and according to the characteristics of the strain HS807-AN-2396 described in Example 1, the fermentation process in a 5000L tank was designed and optimized, and the growth and metabolism of the mutant strain and the optimal production capacity of A82846B were investigated.

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-2980.2, pH 7.2.

Seed tank control process: culture temperature 28-32°C, stirring speed 30-150rpm, dissolved oxygen 30-40%, air flow 0.2-0.1vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 300L/500L.

Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, sodium chloride 0.6, calcium carbonate 3.5. Manganese sulfate tetrahydrate 0.08, cobalt chloride hexahydrate 0.05 and defoamer THIX-2980.4, pH7.5.

The amount of transplanting from the seed tank to the fermenter is 10%, and the percentage is volume percentage. The loading capacity of the fermentation medium in the fermenter is 3500L: the culture temperature is 28-32°C, the stirring speed is 30-150rpm, the dissolved oxygen is 30-40%, the air flow rate is 0.2-1.0vvm, and the culture period is 202h.

Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 4.0-6.0g/L.

During the fermentation process, the detection of total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer is carried out at the same time. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 2199mg/L.

Example 15 Fermentation Industrialization Scale-Up of Bacterial Strain HS807-AN-2396 (CGMCC ^No.10576 ) on 60m Tank

Based on the process parameters obtained from the 5000L fermenter, and according to the characteristics of the strain HS807-AN-2396 described in Example 1, a 60m ³ tank fermentation process was designed and optimized to investigate the growth and metabolism of the mutant strain and the best production capacity of A82846B .

Seed medium (g/L): glucose 10.0, soluble starch 20.0, hot-pressed soybean cake powder 20.0, yeast extract powder 5.0, hydrolyzed casein 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-2980.2, pH 7.2.

Seed tank control process: culture temperature 28-32°C, stirring speed 50-200rpm, dissolved oxygen 30-50%, air flow 0.2-1.0vvm, culture period 41h. Wherein, the filling capacity of the seed culture medium in the seed tank is 5m ³ /15m ³ .

Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolyzed casein 6.0, L-tyrosine 12.0, L-valine 3.0, yeast extract powder 5.0, cottonseed cake powder 30.0, Sodium chloride 0.6, calcium carbonate 3.5, manganese sulfate tetrahydrate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.2, pH7.5.

The amount of inoculation from the seed tank to the fermenter is 12%, and the stated percentage is a volume percentage. The capacity of the fermentation medium in the fermenter is 42m ³ : the culture temperature is 28-32°C, the stirring speed is 30-120rpm, the dissolved oxygen is 30-35%, the air flow rate is 0.1-0.8vvm, and the culture period is 192h.

Fermentation feeding control: add L-tyrosine continuously, and control the residual amount of L-tyrosine at 4.0-6.0g/L.

During the fermentation process, the detection of total sugar, reducing sugar, amino nitrogen, mycelium concentration and fermentation titer is carried out at the same time. Refer to Figure 4 for the fermentation metabolic curve of the HS807-AN-2396 fermented strain HS807-AN-2396 in a 60m ³ tank according to the above-mentioned culture and fermentation conditions. After the fermentation was finished, the fermentation titer was detected by HPLC, and A82846B reached 2315mg/L.

It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (17)

1. a kind of desert pseudocyst bacterium (Kibdelosporangium aridum), which is characterized in that it is deposited in the micro- life of China Object culture presevation administration committee common micro-organisms center, deposit number are as follows: CGMCC No.10576.

2. a kind of method for preparing oritavancin intermediate A 82846B, which is characterized in that by desert as described in claim 1 Pseudocyst bacterium CGMCC No.10576 ferments in the fermentation medium, and oritavancin intermediate A 82846B is obtained from fermentation liquid.

3. method according to claim 2, which is characterized in that the time of the fermentation is 168~240 hours；The fermentation Temperature be 25~34 DEG C.

4. method as claimed in claim 3, which is characterized in that the fermentation carries out in shaking flask, revolving speed 250rpm；Amplitude For 5cm；The humidity of the fermentation is 40~60%.

5. method as claimed in claim 3, which is characterized in that the fermentation carries out in lab scale fermentor；Speed of agitator is 200~600rpm；Dissolved oxygen amount is 30~50%；Air mass flow is 0.5~1.0vvm.

6. method as claimed in claim 3, which is characterized in that the fermentation carries out in pilot scale fermentation tank, and speed of agitator is 30~150rpm；Dissolved oxygen amount is 30~40%；Air mass flow is 0.2~1.0vvm.

7. method as claimed in claim 3, which is characterized in that the fermentation carries out in industrialization fermentor, speed of agitator For 30~120rpm；Dissolved oxygen amount is 30~35%；Air mass flow is 0.1~0.8vvm.

8. method according to claim 2, which is characterized in that the inoculum concentration of the seed liquor of the fermentation is 10~12%, institute The percentage stated is percent by volume；The seed liquor of the fermentation is obtained by the method included the following steps, namely: will be wanted such as right Desert pseudocyst bacterium CGMCC No.10576 described in asking 1 is cultivated in seed culture medium.

9. method according to claim 8, which is characterized in that the time of the culture is 37~44 hours, the culture Temperature is 28~32 DEG C.

10. method as claimed in claim 9, which is characterized in that the culture carries out in shaking flask, revolving speed 250rpm；Vibration Width is 5cm；The humidity of the fermentation is 40~60%.

11. method as claimed in claim 9, which is characterized in that the culture carries out in lab scale seeding tank, and speed of agitator is 200~600rpm；The dissolved oxygen amount of the lab scale fermentor is 30~50%；Air mass flow is 0.5~1.5vvm.

12. method as claimed in claim 9, which is characterized in that the culture carries out in pilot scale seeding tank, and speed of agitator is 30~150rpm；Dissolved oxygen amount is 30~40%；Air mass flow is 0.2~1.0vvm.

13. method as claimed in claim 9, which is characterized in that the culture carries out in industrialization seeding tank, speed of agitator For 50~200rpm；Dissolved oxygen amount is 30~50%；Air mass flow is 0.2~1.0vvm.

14. method according to claim 8, which is characterized in that the seed culture medium includes following constituent (g/ L): glucose 5.0~15.0, soluble starch 10.0~30.0, soybean cake powder 10.0~30.0, extraction from yeast powder 2.0~ 8.0, caseinhydrolysate 2.0~8.0, sodium chloride 0.1~0.5 and calcium carbonate 0.9~1.5, pH6.8~7.5.

15. method according to claim 2, which is characterized in that the fermentation is in lab scale fermentor, pilot scale fermentation tank or industry Change and carried out in fermentor, is include thed steps that as follows:

Add l-tyrosine；Or, adding Valine.

16. a kind of method for obtaining desert pseudocyst bacterium CGMCC No.10576 as described in claim 1, which is characterized in that Desert pseudocyst bacterium CGMCC No.10576 as described in claim 1 is cultivated in the medium.

17. method as claimed in claim 14, which is characterized in that the seed culture medium includes following constituent (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, extraction from yeast powder 5.0, caseinhydrolysate 5.0, chlorination Sodium 0.3, calcium carbonate 1.2 and fermentation defoaming agent THIX-2980.2, pH7.2.