CN102140485A - Method for preparing acarbose through microbial fermentation - Google Patents
Method for preparing acarbose through microbial fermentation Download PDFInfo
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- CN102140485A CN102140485A CN201010605821XA CN201010605821A CN102140485A CN 102140485 A CN102140485 A CN 102140485A CN 201010605821X A CN201010605821X A CN 201010605821XA CN 201010605821 A CN201010605821 A CN 201010605821A CN 102140485 A CN102140485 A CN 102140485A
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- acarbose
- validacin
- takeda
- fermention medium
- salt
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Abstract
The invention discloses a method for preparing acarbose through microbial fermentation. The method comprises the following steps: inoculating acarbose producer (CCTCC NO:M 209022) in fermentation medium which is suitable for the strain and contains carbon source, nitrogen source and inorganic salt, fermenting and culturing at 20-32 DEG C for 96-192 hours, obtaining fermentation liquor after fermentation, extracting to obtain acarbose. The method is characterized in that fermenting and culturing are performed for 0-48h and validamycin A solution is added to ensure that the one litre of fermentation medium contains 0.04-0.4g of validamycin A. By supplementing validamycin A in the fermentation process of acarbose, the fermentation level of acarbose can be increased.
Description
(1) technical field:
The invention belongs to fermentation technical field, design the method that a kind of microbial fermentation prepares acarbose, particularly a kind of in the fermenting process of acarbose by adding the method that Validacin (Takeda) improves acarbose output.
(2) background technology:
The acarbose outward appearance is white or pale powder, water soluble, and the pKa value is 5.1, molecular formula is C
25H
43NO
18, molecular weight 645.6, chemical name O-{4, the two deoxidation-4[(1S of 6-, 4R, 5S, 6S)-4,5,6-trishydroxymethyl-2 cyclohexenyl]-α-D-glucopyranosyl }-(1 → 4)-O-α-D-glucopyranosyl-(1 → 4)-D-Glucopyranose.Molecular structural formula is as shown in the formula shown in the I:
By the acarbose chemical structural formula as seen, it is the false tetrose of a kind of compound, comprise acarviose and maltose two portions on the structure, wherein acarviose is by valienamine with unsaturated C7-cyclitol structure and 4-amino-4,6-dideoxy glucose connects by the amino bridging that is similar to the N-glycosidic link and forms, and this acarviose structure plays a major role to the inhibition effect of alpha-glycosidase.
Bayer A.G has obtained acarbose in 1977 from the metabolite of actinoplanes SE50, SE82 and SE18, the biosynthetic process of acarbose in actinoplanes is to finish under the katalysis by a series of enzymes of Acb gene cluster coding, comprise aminocyclitol, 4-amino-4, the synthetic and glycosylation subsequently of 6-dideoxy glucose.
Acarbose nineteen ninety obtained the FDA approval in U.S.'s listing, and was used for the treatment of type ii diabetes at first in Germany's listing in 1996.
Acarbose all has had strong inhibitory effects to the sucrase in the enteron aisle, maltin, dextrinase and glucoamylase, and α-Dian Fenmei there is faint restraining effect, also have good pharmacokinetics character and hypotoxicity, a large amount of clinical study proof acarboses can reduce level of postprandial blood sugar, little to treating diabetes determined curative effect, toxic side effect, also have good effect at aspects such as prevent diabetes cardiovascular complications.Because its efficient, safety, acarbose has been used as a line medicine of treatment of diabetes.
The characteristics that acarbose has: (1) alleviates blood glucose fluctuation.Blood glucose fluctuation is big, can aggravate response to oxidative stress, the grievous injury great vessels.Acarbose not only can slow down the postprandial blood sugar peak by the absorption that suppresses carbohydrate, and does not cause hypoglycemia, and promptly the what is called peak that disappears goes paddy, alleviates blood glucose fluctuation, further reduces the danger of suffering from cardiovascular disorder.(2) metabolism there is extra benefit.Authority studies show that: acarbose can moderate reduction weight in patients.In addition, what carried out in Europe and Asia studies show that acarbose can also improve insulin sensitivity, improves the disorder of hyperglycemia inductive endothelial function, alleviates body inflammatory reaction etc.(3) can prevent the generation of cardiovascular event.Studies show that the incidence that acarbose significantly reduces any cardiovascular event reaches 35%, wherein the minimizing of myocardial infarction is the most remarkable, and other cardiovascular events also have the trend of minimizing.Acarbose is remarkable to the improvement effect of blood sugar, blood fat and blood pressure, can significantly reduce glycolated hemoglobin, empty stomach and postprandial blood sugar, triglyceride level and systolic pressure level.(4) has outstanding security.Because acarbose is hardly through intestinal absorption, so rarely seen systemic adverse reactions only shows as mild to moderate local gastrointestinal discomfort.The principle of " low dose of initial, dosage " gradually can be avoided most of gastrointestinal reactions if follow.In addition, do not have risk of hypoglycemia and other drug (as angiotensin converting enzyme inhibitor, beta-blockers etc.) during the acarbose single therapy and also do not have drug drug interaction when share, therefore safe, especially be fit to the gerontal patient.
Acarbose has been obtained immense success with its unique mechanism of action, hypoglycemic curative effect, outstanding security and the generation that significantly reduces cardiovascular event stably in the control of hyperglycemia.
Domestic and international at present the fermentation level that mainly improves acarbose by the research of following two aspects to acarbose industrial fermentation process:
(1) concentration of glucose and Fructus Hordei Germinatus oligose in the control fermented liquid.Glucose and Fructus Hordei Germinatus oligose be energy substance be again the precursor substance of acarbose.Glucose just is consumed to the greatest extent in early days in fermentation, and Fructus Hordei Germinatus oligose is utilized in whole fermentation process lentamente, and after glucose exhausts, serves as energy substance.Because maltose can directly mix in the structure of acarbose,, and then influenced the output of acarbose so glucose consumption in early days can cause the minimizing of later stage maltose content.Therefore must strictly control the level of glucose and keep the high density of maltose in fermented liquid to promote the synthetic of acarbose.
(2) suitable osmotic pressure in the control fermented liquid.In the fermenting process of acarbose, osmotic pressure has crucial effect to its output.The osmotic pressure of control nutrient solution helps promoting maltose to intracellular transportation, thereby improves the acarbose yield.
(3) summary of the invention:
The structure of Validacin (Takeda) is suc as formula shown in the II, visible Validacin (Takeda) and acarbose have a similar constructional feature, the contriver finds under study for action, adds Validacin (Takeda) in the fermenting process of acarbose, helps to improve the fermentation unit of acarbose.
The purpose of this invention is to provide a kind of by adding the technology that Validacin (Takeda) improves the acarbose fermentation level.
Technical scheme of the present invention is:
A kind of microbial fermentation prepares the method for acarbose, described method is: acarbose is produced bacterium CCTCC NO:M 209022, be seeded in the fermention medium of the carbonaceous sources that is applicable to described bacterial strain, nitrogenous source, inorganic salt, carried out fermentation culture 96～192 hours (preferred 120～168 hours in 20～32 ℃ of temperature (preferred 28 ℃), most preferably 144 hours), after the fermentation ends, get broth extraction and separate, obtain described acarbose; In the fermenting process, fermentation culture is carried out between 0～48 hour (between preferred 0～12 hour, most preferably between 0～6 hour), add the aqueous solution suc as formula the Validacin (Takeda) shown in the II, making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.04～0.4g.
Described fermentation culture was carried out between 0～48 hour, added the aqueous solution suc as formula the Validacin (Takeda) shown in the II, wherein 0 be meant the aqueous solution that when the fermentation beginning, adds Validacin (Takeda), this is that those skilled in the art understand easily.
It is actinoplanes ZJB-08196 (Actinoplanes sp.ZJB-08196) that acarbose used in the present invention produces bacterium, be preserved in Chinese typical culture collection center, address: China. Wuhan. Wuhan University, 430072, deposit number CCTCC NO:M 209022, preservation date on February 16th, 2009.
The concentration of the aqueous solution of Validacin (Takeda) of the present invention is 1～100g/L, preferred 6.3～12g/L.
The present invention carries out between 0～48 hour in fermentation culture, adds the aqueous solution suc as formula the Validacin (Takeda) shown in the II, and the adding mode of the aqueous solution of described Validacin (Takeda) is one of following: (1) disposable adding; (2) intermittently add in batches; (3) flow continuous the adding.
The aqueous solution of described adding Validacin (Takeda), making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.04～0.4g, preferably making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.04～0.25g, and most preferably making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.08～0.15g.
Comparatively concrete, the method of the invention is: aseptic transfer low temperature glycerine pipe CCTCCNO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ activate 2～3 days, picking colony is seeded to seed culture medium, 28 ℃ of temperature, ventilate to stirring or shake and cultivate 24～96 hours (preferred 72 hours) down, obtain seed liquor; Seed liquor and fermention medium are seeded to fermention medium with the inoculum size of volume ratio 1～10% (preferred 3～5%), 28 ℃ of temperature, carried out fermentation culture 96～192 hours (preferred 120～168 hours) under ventilation stirring or the concussion, be cultured to 0～48 hour (during preferred 0～12h), the aqueous solution that adds 6.3～12g/L Validacin (Takeda), making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.04～0.25g; After the fermentation ends, the fermented liquid of acquisition gets described acarbose through extraction separation.
Fermention medium of the present invention contains and can be utilized carbon source, nitrogenous source, inorganic salt by described bacterial strain, and described carbon source is one or several following arbitrary combination: glucose, lactose, maltose, dextrin, starch, glycerine, N.F,USP MANNITOL, sorbyl alcohol; Described nitrogenous source is one or several following arbitrary combination: gravy, yeast extract paste, dry yeast, soyflour, corn steep liquor, peptone, urea, ammonia salt (as ammonium sulfate, ammonium chloride, ammonium nitrate, Ammoniom-Acetate etc.), peptide (as dipeptides, tripeptides etc.); Described inorganic salt are one or several following arbitrary combination: Na salt, K salt, Ca salt, Mg salt, Fe salt, Mn salt, Zn salt, Co salt or Ni salt.Described fermention medium can also contain amino acids (as L-glutamic acid, aspartic acid, Methionin, glycine, methionine(Met) etc.), VITAMIN (as V
B1, V
B2, V
B12, V
C, V
E, nicotinic acid etc.) and/or nucleic acid class (as purine, pyrimidine and derivative thereof etc.) material.
It is pH6.0～8.0 that described fermention medium is regulated initial pH value with mineral acid or organic acid, bases, is preferably 6.8～7.0.
Comparatively concrete, the fermention medium that the embodiment of the invention adopts is composed as follows: malt syrup 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L solvent is a water, initial pH 7.0.
The seed culture medium that the embodiment of the invention adopts is composed as follows: W-Gum 15.0g/L, soybean cake powder 40.0g/L, glycerine 20.0g/L, K
2HPO
40.1g/L, CaCO
32.0g/L solvent is a water, initial pH 7.0.
Fermention medium of the present invention, seed culture medium and the Validacin (Takeda) aqueous solution all need sterilising treatment after preparation is finished, and adopt 121 ℃ of sterilization 30min usually.
More specifically, recommend the method for the invention to carry out as follows:
(1) aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ of activation are after 2～3 days, picking colony is seeded to seed culture medium, 28 ℃ of temperature, ventilate to stirring or shake down and cultivated 24～96 hours, obtain seed liquor, described seed culture medium is composed as follows: W-Gum 15.0g/L, soybean cake powder 40.0g/L, glycerine 20.0g/L, K
2HPO
40.1g/L, CaCO
32.0g/L solvent is a water, initial pH 7.0.
(2) seed liquor and fermention medium are seeded to fermention medium with the inoculum size of volume ratio 1～10% (preferred 3～5%), 28 ℃ of temperature, carried out fermentation culture 96～192 hours (preferred 120～168 hours) under ventilation stirring or the concussion, be cultured to 0～48 hour (during preferred 0～12h), (aqueous solution of preferred 6.3～12g/L) Validacin (Takeda)s, the add-on of the aqueous solution of described Validacin (Takeda) are that to make in every liter of fermention medium the amount that adds Validacin (Takeda) be 0.04～0.25g to disposable adding 1～100g/L; Described fermention medium is composed as follows: malt syrup 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L solvent is a water, initial pH 7.0.
(3) after the fermentation ends, the fermented liquid of acquisition carries out extraction separation, gets described acarbose.
Most preferred, recommend the method for the invention to carry out as follows:
(1) aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ of activation are after 2 days, picking colony is seeded to seed culture medium, 28 ℃ of temperature, ventilate to stirring or shake down and cultivated 72 hours, obtain seed liquor, described seed culture medium is composed as follows: W-Gum 15.0g/L, soybean cake powder 40.0g/L, glycerine 20.0g/L, K
2HPO
40.1g/L, CaCO
32.0g/L solvent is a water, initial pH 7.0.
(2) seed liquor and fermention medium are seeded to fermention medium with the inoculum size of volume ratio 5%, 28 ℃ of temperature, carried out fermentation culture 144 hours under ventilation stirring or the concussion, during being cultured to 0～6h, the aqueous solution of disposable adding 6.3～12g/L Validacin (Takeda), the add-on of the aqueous solution of described Validacin (Takeda) are that to make in every liter of fermention medium the amount that adds Validacin (Takeda) be 0.15g; Described fermention medium is composed as follows: malt syrup 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L solvent is a water, initial pH 7.0.
(3) after the fermentation ends, the fermented liquid of acquisition carries out extraction separation, gets described acarbose.
The method that the middle fermented liquid of step of the present invention (3) carries out extraction separation is: fermented liquid is regulated pH value to 3, carries out centrifugal then or filtration, obtains clarifying acarbose filtrate; Adopt strongly acidic cation-exchange absorption, wash-out; It is refining through desalination, high resolving power Zeo-karb to collect liquid, neutralization, and lyophilize makes acarbose.
Acarbose concentration detects the HPLC method that adopts in the fermented liquid.Sample pretreatment: the fermented liquid vibration evenly pipettes 5.0ml and places the 10ml centrifuge tube, and centrifugal 5 minutes of 5000rpm abandons precipitation, collects supernatant; Supernatant mixed with dehydrated alcohol in 1: 4 by volume, and centrifugal 10 minutes of 10000rpm collects clarifying supernatant liquor, 0.45 μ m micro-filtrate membrane filtration, and filtrate adopts island Feng HPLC to analyze; HPLC moving phase preparation: accurately take by weighing 0.300g KH
2PO
3, 0.350g Na
2HPO
3, dissolve, be settled to 500 milliliters, 0.45 μ m micro-filtrate membrane filtration with ultrapure water; Filtrate was mixed with trifluoroacetic acid aqueous solution in 30: 70 by volume, ultrasonic degas; HPLC analysis condition: island Feng LC-20AT pump, island Feng SPD-20A ultraviolet-visible(light)detector, 250mm * 4.6mm Hyersil nh 2 column (Yi Lite, Dalian), 40 ℃ of column oven temperature; Flow rate of mobile phase 1.0mL/min, sample size are 20.0 μ L, and UV detects wavelength 210nm.
Beneficial effect of the present invention is: utilize the inventive method, by add Validacin (Takeda) in the acarbose fermenting process, improved the fermentation level of acarbose.As can be seen, compare with the situation of not adding Validacin (Takeda) from the embodiment of the invention, the fermentation titer of acarbose is the highest to improve 51%.
(4) embodiment:
With specific embodiment the present invention program is described further below, but protection scope of the present invention is not limited thereto.
Acarbose concentration detects the HPLC method that adopts in the fermented liquid.Sample pretreatment: the fermented liquid vibration evenly pipettes 5.0ml and places the 10ml centrifuge tube, and centrifugal 5 minutes of 5000rpm abandons precipitation, collects supernatant; Supernatant mixed with dehydrated alcohol in 1: 4 by volume, and centrifugal 10 minutes of 10000rpm collects clarifying supernatant liquor, 0.45 μ m micro-filtrate membrane filtration, and filtrate adopts island Feng HPLC to analyze; HPLC moving phase preparation: accurately take by weighing 0.300g KH
2PO
3, 0.350g Na
2HPO
3, dissolve, be settled to 500 milliliters, 0.45 μ m micro-filtrate membrane filtration with ultrapure water; Filtrate was mixed with trifluoroacetic acid aqueous solution in 30: 70 by volume, ultrasonic degas; HPLC analysis condition: island Feng LC-20AT pump, island Feng SPD-20A ultraviolet-visible(light)detector, 250mm * 4.6mm Hyersil nh 2 column (Yi Lite, Dalian), 40 ℃ of column oven temperature; Flow rate of mobile phase 1.0mL/min, sample size are 20.0 μ L, and UV detects wavelength 210nm.
Embodiment 1: the production of acarbose (triangular flask concussion fermentation)
The preparation seed culture medium, its substratum is composed as follows: W-Gum 15.0g/L, soybean cake powder 40.0g/L, glycerine 20.0g/L, K
2HPO
40.1g/L, CaCO
32.0g/L, with the tap water preparation, initial pH 7.0.Sterilized 30 minutes for 121 ℃.
The preparation fermention medium, its substratum is composed as follows: malt syrup 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L with the tap water preparation, pH 7.0.121 ℃ of sterilization 30min.
The preparation Validacin (Takeda) aqueous solution: with tap water preparation Validacin (Takeda) concentration is the Validacin (Takeda) solution of 6.3g/L, sterilizes 30 minutes for 121 ℃.
The preparation of seed: aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ of activation are after 2 days, picking colony is inoculated in the 500ml triangular flask and does seed culture, the volume of seed culture medium is 50ml in the bottle, shaking speed 200rpm, cultivated 72 hours down at 28 ℃, obtain seed liquor.
The fermentation of acarbose: get the above fermention medium 300mL for preparing, pour in 6 500mL triangular flasks, pour the 50mL fermention medium in every bottle, 121 ℃ of sterilization 30min.Every bottle of 2.5ml seed liquor that makes more than inserting is respectively fermented, and leavening temperature is 28 ℃, shaking speed 200rpm.At fermentation 0h, the 6.3g/L Validacin (Takeda) aqueous solution 0.3ml, 0.6ml, 1.2ml, 2.0ml, the 3.2ml that add above-mentioned preparation therein in 5 fermentation shake flask respectively, other one is shaken bottle and is not added the validamycin A aqueous solution in contrast, total fermentation time after 144 hours with adopting the HPLC method to measure the concentration of acarbose in the fermented liquid.
The results are shown in table 1.
Table 1:
As can be seen from the above table, in the shake flask fermentation process, add the fermentation level that Validacin (Takeda) can improve acarbose.
Embodiment 2: the production of acarbose (stirred reactor fermentation)
Seed culture medium, fermention medium are prepared with embodiment 1.
The preparation Validacin (Takeda) aqueous solution: with tap water preparation Validacin (Takeda) concentration is the Validacin (Takeda) solution of 12g/L, sterilizes 30 minutes for 121 ℃.
The preparation of seed: aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ of activation are after 2 days, picking colony is inoculated in the 500ml triangular flask and does seed culture, the volume of seed culture medium is 50ml in the bottle, shaking speed 200rpm, cultivate 72h down at 28 ℃, obtain seed liquor.
The fermentation of acarbose:
Get the above fermention medium 24L for preparing, be respectively charged in the stirred fermentor of 3 15L, each fermentor tank 8L fermention medium of packing into, 121 ℃ of sterilization 30min, inserting the seed liquor that makes more than the 0.4L in each fermentor tank ferments, 28 ℃ of leavening temperatures, stirring velocity 150rpm, air flow 1.0vvm.No. 1 fermentor tank does not add validamycin A solution in contrast, ferments 144 hours; The Validacin (Takeda) aqueous solution of the 12.0g/L that No. 2 ferment tanks prepared more than the disposable adding 100.0ml after 6 hours continues fermentation 138 hours; , divide to add 12.0g/L Validacin (Takeda) solution for 4 times after 6 hours at No. 3 ferment tanks, add 25.0ml at every turn, added once total fermentation time 144 hours in per 4 hours.After the fermentation ends, the fermentation level of acarbose obtains in the mensuration fermented liquid result such as following table 2.
Table 2:
As can be seen from the above table, in the ferment tank process, add the fermentation level that Validacin (Takeda) can improve acarbose.
Embodiment 3: the extraction of acarbose
Get 100 milliliters of fermented liquids that fermentation is finished in No. 12 bottles of embodiment, regulate pH value to 3, centrifugal 10 minutes of 3000 * g abandons precipitation, collects supernatant liquor; Clarified supernatant is just separated through the macroporous cation exchange.Chromatography column adopts 2.5 * 30 centimetres of glass columns (U.S. BIO-Rad).Just separate chromatography and select 80 microns UNOsphere S of particle diameter filler, wet method dress post, about 22.3 centimetres of bed height, medium is with 5.0 * 10
-2M hydrochloric acid diluted acid makes the transition into H
+Type.Flow rate of mobile phase is set to 440 milliliters/hour (linear velocity 1.5 cm per minute), fully after the balance, gets 2.0 ml concns and be sample on the concentrated solution of 5 gram acarboses/rise concentrated solution, 7 column volumes of ultrapure water drip washing, and wash-out adopts 5.0 * 10
-3M hydrochloric acid/8 column volumes of column volume gradient elution, fraction collection, the identical elution peak of merging are collected liquid and are analyzed through HPLC, repeat 5 batches of above-mentioned purification operations; The acarbose component of collecting is adjusted to pH3 after the ion exchange chromatography desalination, make with extra care then, selects 25 microns Macro-Prep 25S of particle diameter filler to carry out cation-exchange chromatography, wet method dress post, and about 21.7 centimetres of bed height, medium is with 5.0 * 10
-2M hydrochloric acid diluted acid makes the transition into H
+Type.Flow rate of mobile phase is set to 290 milliliters/hour (linear velocity 1.0 cm per minute), fully after the balance, gets 1.0 ml concns and be sample on the concentrated solution of 7.3 gram acarboses/rise concentrated solution, 5 column volumes of ultrapure water drip washing, and wash-out adopts 5.0 * 10
-3M hydrochloric acid/9 column volumes of column volume gradient elution, fraction collection, collection liquid are analyzed through HPLC; Repeat 7 batches of above-mentioned purification operations.Merge acarbose and collect liquid, material is through resin cation (R.C.) and resin anion(R.A) absorbed portion pigment, inorganic ion, neutralization, and lyophilize must 4.63 * 10
-2The gram acarbose, sample purity 98.3%, extraction process is calculated comprehensive yield 71.4%.
Claims (10)
1. a microbial fermentation prepares the method for acarbose, described method is: acarbose is produced bacterium CCTCC NO:M 209022, be seeded to the carbonaceous sources that is applicable to described bacterial strain, nitrogenous source, in the fermention medium of inorganic salt, carried out fermentation culture 96～192 hours 20～32 ℃ of temperature, after the fermentation ends, getting broth extraction separates, obtain described acarbose, it is characterized in that, fermentation culture is carried out between 0～48h, adding is suc as formula the aqueous solution of the Validacin (Takeda) shown in the II, and making the quality that adds Validacin (Takeda) in every liter of fermention medium is 0.04～0.4g;
2. the method for claim 1, the concentration that it is characterized in that the aqueous solution of described Validacin (Takeda) is 1～100g/L.
3. the method for claim 1 is characterized in that carrying out between 0～12h in fermentation culture, adds the aqueous solution suc as formula the Validacin (Takeda) shown in the II, and the adding mode of the aqueous solution of described Validacin (Takeda) is one of following: (1) disposable adding; (2) intermittently add in batches; (3) flow continuous the adding.
4. the method for claim 1 is characterized in that in the described fermention medium, and described carbon source is one or several following arbitrary combination: glucose, lactose, maltose, dextrin, starch, glycerine, N.F,USP MANNITOL, sorbyl alcohol; Described nitrogenous source is one or several following arbitrary combination: gravy, yeast extract paste, dry yeast, soyflour, corn steep liquor, peptone, urea, ammonia salt, peptide; Described inorganic salt are one or several following arbitrary combination: Na salt, K salt, Ca salt, Mg salt, Fe salt, Mn salt, Zn salt, Co salt or Ni salt.
5. the method for claim 1 is characterized in that described fermention medium also contains amino acids, VITAMIN and/or nucleic acid material.
6. as claim 1,4 or 5 described methods, it is characterized in that described fermention medium initial pH value is pH 6.0～8.0.
7. the method for claim 1 is characterized in that described fermention medium is composed as follows: maltose 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L solvent is a water, initial pH 7.0.
8. the method for claim 1, it is characterized in that described method is: aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ activate 2～3 days, picking colony is seeded to seed culture medium, 28 ℃ of temperature, ventilate to stirring or shake down and cultivated 24～96 hours, obtain seed liquor; Seed liquor and fermention medium are seeded to fermention medium with the inoculum size of volume ratio 1～10%, 28 ℃ of temperature, carried out fermentation culture 96～192 hours under ventilation stirring or the concussion, during being cultured to 0～12 hour, the aqueous solution that adds 6.3～12g/L Validacin (Takeda), the add-on of the aqueous solution of described Validacin (Takeda) are that to make the amount that adds Validacin (Takeda) in every liter of fermention medium be 0.04～0.4g; After the fermentation ends, get broth extraction and separate, get described acarbose.
9. the method for claim 1 is characterized in that described method carries out as follows:
(1) aseptic transfer low temperature glycerine pipe CCTCC NO:M 209022 bacterial strains are on fresh, sterile solid is dull and stereotyped, 28 ℃ activate 2～3 days, picking colony is seeded to seed culture medium, 28 ℃ of temperature, ventilate to stirring or shake down and cultivated 24～96 hours, obtain seed liquor, described seed culture medium is composed as follows: W-Gum 15.0g/L, soybean cake powder 40.0g/L, glycerine 20.0g/L, K
2HPO
40.1g/L, CaCO
32.0g/L solvent is a water, initial pH7.0;
(2) seed liquor and fermention medium are seeded to fermention medium with the inoculum size of volume ratio 1～10%, 28 ℃ of temperature, carried out fermentation culture 96～192 hours under ventilation stirring or the concussion, during being cultured to 0～12 hour, disposable adding 6.3～12g/L Validacin (Takeda) aqueous solution, the add-on of the aqueous solution of described Validacin (Takeda) are that to make in every liter of fermention medium the amount that adds Validacin (Takeda) be 0.04～0.15g; Described fermention medium is composed as follows: maltose 80.0g/L, glucose 20.0g/L, soybean cake powder 10.0, g/L, corn steep liquor 5.0g/L, FeCl
30.1g/L, CaCl
22.0g/L, CaCO
36.0g/L solvent is a water, initial pH 7.0;
(3) after the fermentation ends, get broth extraction and separate, get described acarbose.
10. method as claimed in claim 9, it is characterized in that fermented liquid separates as follows in the described step (3): fermented liquid is regulated pH value to 3, carries out centrifugal then or filtration, obtains clarifying acarbose filtrate; Adopt strongly acidic cation-exchange absorption, wash-out; It is refining through desalination, high resolving power Zeo-karb to collect liquid, neutralization, and lyophilize makes acarbose.
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Cited By (12)
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| CN102399837A (en) * | 2011-09-29 | 2012-04-04 | 浙江工业大学 | Method for synthesizing acarbose through microbial fermentation |
| CN102603822A (en) * | 2012-02-21 | 2012-07-25 | 河北华荣制药有限公司 | Method for improving purity of acarbose |
| CN102978261A (en) * | 2012-08-24 | 2013-03-20 | 河北华荣制药有限公司 | High malt syrup and method for improving acarbose fermentation unit by using high malt syrup |
| CN103088089A (en) * | 2013-01-10 | 2013-05-08 | 伊犁川宁生物技术有限公司 | Method for fermenting acarbose |
| WO2014057407A2 (en) * | 2012-10-12 | 2014-04-17 | Mahesh Kandula | Compositions and methods for the treatment of diabetes and prediabetes |
| CN104745661A (en) * | 2015-04-10 | 2015-07-01 | 江南大学 | Method for establishing and analyzing scale metabolism network model of actinoplanetes genomes |
| CN106167814A (en) * | 2016-08-31 | 2016-11-30 | 河北华荣制药有限公司 | A kind of method improving acarbose fermentation unit |
| CN109355333A (en) * | 2018-12-05 | 2019-02-19 | 山东鲁抗医药股份有限公司 | A kind of preparation method of acarbose |
| CN110541017A (en) * | 2019-10-12 | 2019-12-06 | 山东鲁抗医药股份有限公司 | Method for improving production of acarbose |
| CN110564794A (en) * | 2019-10-12 | 2019-12-13 | 山东鲁抗医药股份有限公司 | Fermentation method of acarbose |
| CN112300229A (en) * | 2020-11-06 | 2021-02-02 | 苏州第四制药厂有限公司 | Method for purifying acarbose from acarbose fermentation liquor |
| CN114686546A (en) * | 2020-12-30 | 2022-07-01 | 杭州中美华东制药有限公司 | Method for improving acarbose fermentation unit |
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Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399837A (en) * | 2011-09-29 | 2012-04-04 | 浙江工业大学 | Method for synthesizing acarbose through microbial fermentation |
| CN102399837B (en) * | 2011-09-29 | 2013-07-31 | 浙江工业大学 | Method for synthesizing acarbose through microbial fermentation |
| CN102603822B (en) * | 2012-02-21 | 2013-07-03 | 河北华荣制药有限公司 | Method for improving purity of acarbose |
| CN102603822A (en) * | 2012-02-21 | 2012-07-25 | 河北华荣制药有限公司 | Method for improving purity of acarbose |
| CN102978261B (en) * | 2012-08-24 | 2019-04-05 | 河北华荣制药有限公司 | A kind of high maltose syrup and the method using its raising acarbose fermentation unit |
| CN102978261A (en) * | 2012-08-24 | 2013-03-20 | 河北华荣制药有限公司 | High malt syrup and method for improving acarbose fermentation unit by using high malt syrup |
| WO2014057407A2 (en) * | 2012-10-12 | 2014-04-17 | Mahesh Kandula | Compositions and methods for the treatment of diabetes and prediabetes |
| WO2014057407A3 (en) * | 2012-10-12 | 2014-06-26 | Mahesh Kandula | Compositions and methods for treatment of diabetes and prediabetes |
| CN103088089B (en) * | 2013-01-10 | 2014-07-16 | 伊犁川宁生物技术有限公司 | Method for fermenting acarbose |
| CN103088089A (en) * | 2013-01-10 | 2013-05-08 | 伊犁川宁生物技术有限公司 | Method for fermenting acarbose |
| CN104745661A (en) * | 2015-04-10 | 2015-07-01 | 江南大学 | Method for establishing and analyzing scale metabolism network model of actinoplanetes genomes |
| CN106167814B (en) * | 2016-08-31 | 2019-08-09 | 河北华荣制药有限公司 | A method of improving acarbose fermentation unit |
| CN106167814A (en) * | 2016-08-31 | 2016-11-30 | 河北华荣制药有限公司 | A kind of method improving acarbose fermentation unit |
| CN109355333A (en) * | 2018-12-05 | 2019-02-19 | 山东鲁抗医药股份有限公司 | A kind of preparation method of acarbose |
| CN109355333B (en) * | 2018-12-05 | 2020-10-27 | 山东鲁抗医药股份有限公司 | Preparation method of acarbose |
| CN110541017A (en) * | 2019-10-12 | 2019-12-06 | 山东鲁抗医药股份有限公司 | Method for improving production of acarbose |
| CN110564794A (en) * | 2019-10-12 | 2019-12-13 | 山东鲁抗医药股份有限公司 | Fermentation method of acarbose |
| CN112300229A (en) * | 2020-11-06 | 2021-02-02 | 苏州第四制药厂有限公司 | Method for purifying acarbose from acarbose fermentation liquor |
| CN114686546A (en) * | 2020-12-30 | 2022-07-01 | 杭州中美华东制药有限公司 | Method for improving acarbose fermentation unit |
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