CN104561143A - Method for improving yield of beta-polymalic acid by fed-batch fermentation - Google Patents

Method for improving yield of beta-polymalic acid by fed-batch fermentation Download PDF

Info

Publication number
CN104561143A
CN104561143A CN201410833031.5A CN201410833031A CN104561143A CN 104561143 A CN104561143 A CN 104561143A CN 201410833031 A CN201410833031 A CN 201410833031A CN 104561143 A CN104561143 A CN 104561143A
Authority
CN
China
Prior art keywords
fermentation
polymalic acid
concentration
solution
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410833031.5A
Other languages
Chinese (zh)
Inventor
乔长晟
范栩嘉
郝华璇
于世云
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Peiyang Biotrans Biotech Co Ltd
Original Assignee
Tianjin Peiyang Biotrans Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Peiyang Biotrans Biotech Co Ltd filed Critical Tianjin Peiyang Biotrans Biotech Co Ltd
Priority to CN201410833031.5A priority Critical patent/CN104561143A/en
Publication of CN104561143A publication Critical patent/CN104561143A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for improving the yield of beta-polymalic acid by fed-batch fermentation, and belongs to the technical field of fermentation engineering. The fed-batch fermentation production method comprises the following steps: causing production strains to enter a fermentation cultivation stage after seed cultivation, performing fermentation cultivation for 36 to 48 hours, starting adding a feeding solution to continue fermentation for 96 to 120 hours in a flowing manner, adding a glucose solution at concentration of 500g/L in the flowing manner to keep residual sugar in fermentation liquor between 4 to 7g/L, and simultaneously feeding 10 milliliters of amino acid solution at concentration of 1 to 5g/L at a time interval of 2 hours. According to the method, the yield of natural polymalic acid can be improved, the production cost of the natural polymalic acid can be reduced, and the yield of beta-polymalic acid is improved by 11 to 20 percent.

Description

A kind of fed-batch fermentation improves the method for Beta-polymalic acid output
Technical field:
The invention belongs to microbial fermentation engineering technical field, be specifically related to a kind of method adopting supplying technics to improve Aureobasidium pullulans fermentative production Beta-polymalic acid output.
Background technology:
The macromolecular material research history of existing 40 years so far, last century, the seventies started flourish, and the research of macromolecular material all has very important meaning in science or in practical value.Along with the develop rapidly of macromolecular material, its importance is day by day simultaneously significant, and its weak point also appears in face of people, and the artificial synthesized polymer material of major part is difficult to degraded at occurring in nature.In today that environment goes from bad to worse, people start to pay close attention to the problem of environmental pollution that macromolecular material causes because not decomposing.Along with the enhancing of environmental consciousness, people increase the requirement of the degradability of material gradually, are using now or be in the product of development for us using whether possessing biodegradable as important judgement criteria.In order to solve an above-mentioned difficult problem, people begin one's study and have manufactured various degradation material.
Polymalic acid (PMLA) is a kind of water-soluble aliphatic poly ester compound, with ester group on main chain, and can be spontaneous or enzymatic reaction and degrading occurs under water surrounding.Because have the three-dimensional arrangement that this is special, polymalic acid has possessed some excellent character, comprising: biocompatibility, height water-soluble, Bioabsorbable, biodegradability, can chemically derived property and non-immunogenicity etc., polymalic acid and derivative thereof are subject to the great attention of people as the novel bioabsorbable polymer material of a class.
Biological process synthesis polymalic acid can utilize reproducible Biological resources fermentative production, with low cost, synthesis condition is gentle, and the molecular weight of product is relatively high, from fermented liquid, extraction purification step is simple, just can obtain polymalic acid calcium salt by methyl alcohol or ethanol repeated precipitation.Therefore biological process synthesis tool has great advantage, but its development and application receives high cost, inefficient restriction, and that goes back that neither one optimizes at present can the route of suitability for industrialized production, only has a small amount of natural polymalic acid and synthetic polymalic acid to provide.If can improve the output of natural polymalic acid, reduce its production cost, its Application Areas will be developed further.
Summary of the invention:
Main purpose of the present invention is to provide the novel method that a kind of supplying technics produces Beta-polymalic acid, it is that the glucose control fermented liquid residual sugar of 500g/L is at 4g/L-7g/L that the method adds concentration by stream, stream adds the amino acid solution that concentration is 1g/L-5g/L simultaneously, described amino acid solution comprises at least one in aspartic acid, Histidine, leucine, Threonine, solve the problem that in batch fermentation, Beta-polymalic acid productive rate is lower, significantly improve the productive rate of Beta-polymalic acid, reduce production cost.
Technical scheme provided by the invention is specific as follows:
1. the preparation of substratum
(1) activation medium is potato glucose agar medium (PDA): potato 200g/L, glucose 20g/L, agar 20g/L, pH nature, 121 DEG C of high pressure steam sterilization 20min;
Seed culture medium consists of: sucrose 140g/L, yeast extract paste 3g/L, ammonium sulfate 1g/L succinic acid 2g/L, corn steep liquor 1g/L, K 2cO 30.4g/L, MgSO 47H 2o 0.1g/L, KH 2pO 41g/L, ZnSO 47H 2o 0.05g/L, all the other are water pH7.0.
Fermention medium consists of: sucrose 100-200g/L, SODIUMNITRATE 6g/L, potassium primary phosphate 0.1-0.3g/L, Repone K 0.5, manganous sulfate 0.05-0.1g/L, peptone 35g/L, bitter salt 0.3g/L, calcium carbonate 20g/L.
2.CGMCC3336 actication of culture: strain transfer on PDA slant medium, 25 DEG C of activation culture 3 ~ 5d;
3. seed liquor preparation: choose well-grown inclined-plane seed stroke-physiological saline solution and wash lower spore, prepare spore concentration about 10 6the spore suspension of individual/ml, is seeded to by 10% (v/v) inoculum size and is equipped with in the 500ml triangular flask of 100ml liquid seed culture medium, 25 DEG C, cultivates 2 ~ 3d under 200rpm condition, obtained seed liquor.
4. ferment tank: gained seed liquor is connected in 10L fermentor tank with the amount of 10% (v/v) and carries out fermentation culture, under control pH6.0-7.0 and dissolved oxygen concentration 60%-70% condition, in the substratum of carbonaceous sources, nitrogenous source, inorganic salt and trace element, cultivate Aureobasidium pullulans CGMCC NO.3336; First sugar adopts sucrose, fermentation 36-48h starts flow feeding solution, the glucose solution being added 500g/L by stream makes fermented liquid residual sugar maintain 4-7g/L, added every two hours the amino acid solution 10mL that concentration is 1-5g/L simultaneously, described amino acid solution comprises at least one in aspartic acid, leucine, Histidine, Threonine, continues fermentation 96-120h.
Beneficial effect:
Improve the fermentation production rate of Beta-polymalic acid by flow feeding solution, efficiency of pcr product in fermentation period, reduces cost, by Beta-polymalic acid output increased 11-20%.
Embodiment:
The production method of polymalic acid in the present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1: continuously ferment production Beta-polymalic acid
Fermentative production Beta-polymalic acid as follows:
1. the preparation of substratum
(1) activation medium is: potato glucose agar medium (PDA): potato 200g/L, glucose 20g/L, agar 20g/L, pH nature, 121 DEG C of high pressure steam sterilization 20min;
Seed culture medium consists of: sucrose 140g/L, yeast extract paste 3g/L, ammonium sulfate 1g/L succinic acid 2g/L, corn steep liquor 1g/L, K 2cO 30.4g/L, MgSO 47H 2o 0.1g/L, KH 2pO 41g/L, ZnSO 47H 2o 0.05g/L, all the other are water, pH7.0.
Fermention medium consists of: sucrose 150g/L, SODIUMNITRATE 6g/L, potassium primary phosphate 0.2g/L, Repone K 0.5, manganous sulfate 0.75g/L, peptone 35g/L, bitter salt 0.3g/L, calcium carbonate 20g/L.
2.CGMCC3336 actication of culture: strain transfer on PDA slant medium, 25 DEG C of activation culture 4d;
3. seed liquor preparation: choose well-grown inclined-plane seed stroke-physiological saline solution and wash lower spore, prepare spore concentration about 10 6the spore suspension of individual/ml, is equipped with in the 500ml triangular flask of 100ml liquid seed culture medium by the inoculation of 10 (%v/v) inoculum size, 25 DEG C, cultivates 2d under 200rpm condition, obtained seed liquor.
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 65%.Initial sucrose concentration 150g/L, fermentation period is 120h.In fermented liquid, Beta-polymalic acid content is 40.81g/L.
Embodiment 2: producing beta-polymalic acid by feed-batch fermentation
1. the preparation of substratum: with embodiment 1
2. actication of culture: with embodiment 1
3. seed liquor preparation: with embodiment 1
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 65%.
Initial sucrose concentration 150g/L, it is 500g/L that fermentation 36h starts glucose concn in the feed solutions that stream adds, and maintains fermented liquid residual sugar at 4g/L-7g/L, adds every 2 hours stream the aspartic acid solution that concentration is 1.7g/L, each stream adds 10ml, continues fermentation 112h.In fermentation ends fermented liquid, polymalic acid content is 45.33g/L.
Embodiment 3: producing beta-polymalic acid by feed-batch fermentation
1. the preparation of substratum: with embodiment 1
2. actication of culture: with embodiment 1
3. seed liquor preparation: with embodiment 1
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 62%.
Initial sucrose concentration 150g/L, fermentation 40h starts the feed solutions that stream adds, and the glucose concentration that wherein stream adds is 500g/L, maintain fermented liquid residual sugar at 4g/L-7g/L, adding a concentration every 2 hours stream is the histidine solution of 1g/L, and each stream adds 10ml, continues fermentation 112h.In fermentation ends fermented liquid, polymalic acid content is 45.26g/L.
Embodiment 4: producing beta-polymalic acid by feed-batch fermentation
1. the preparation of substratum: with embodiment 1
2. actication of culture: with embodiment 1
3. seed liquor preparation: with embodiment 1
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 62%.
Initial sucrose concentration 150g/L, fermentation 48h starts the feed solutions that stream adds, and the glucose concentration that wherein stream adds is 500g/L, maintain fermented liquid residual sugar at 4g/L-7g/L, adding a Threonine solution concentration every 2 hours stream is 2.5g/L, and each stream adds 10ml, continues fermentation 96h.In fermentation ends fermented liquid, polymalic acid content is 46.03g/L.
Embodiment 5: producing beta-polymalic acid by feed-batch fermentation
1. the preparation of substratum: with embodiment 1
2. actication of culture: with embodiment 1
3. seed liquor preparation: with embodiment 1
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 62%.
Initial sucrose concentration 150g/L, fermentation 48h starts the feed solutions that stream adds, and the glucose concentration that wherein stream adds is 500g/L, maintain fermented liquid residual sugar at 4g/L-7g/L, adding a concentration every 2 hours stream is the leucine solution of 4.35g/L, and each stream adds 10ml, continues fermentation 112h.In fermentation ends fermented liquid, polymalic acid content is 46.03g/L.
Embodiment 6: producing beta-polymalic acid by feed-batch fermentation
1. the preparation of substratum: with embodiment 1
2. actication of culture: with embodiment 1
3. seed liquor preparation: with embodiment 1
4. ferment tank: gained seed liquor is connected to the amount of 10% (v/v) in the 10L fermentor tank that 5L fermented liquid is housed and carries out fermentation culture initial speed 400r/min, air flow 1.0vvm, tank pressure, at 0.01-0.1Mpa, holds oxygen series connection mixing control DO 62%.
Initial sucrose concentration 150g/L, fermentation 36h starts the feed solutions that stream adds, the glucose concentration that wherein stream adds is 500g/L, maintains fermented liquid residual sugar at 4g/L-7g/L, adds an amino acid solution every two hours stream, each 10ml, described amino acid solution contains the leucine that concentration is 4.35g/L, the Threonine of 2.5g/L, the Histidine of 1g/L, the aspartic acid of 1.7g/L, continues fermentation 104h.In fermentation ends fermented liquid, polymalic acid content is 49.78g/L.

Claims (4)

1. the method for a producing beta-polymalic acid by feed-batch fermentation, it is characterized in that, fermentation culture stage is entered after production bacterial classification is carried out seed culture, start flow feeding solution after fermentation culture 36-48 hour and continue fermentation 96-120h, adding concentration by stream is that the glucose solution of 500g/L makes fermented liquid residual sugar maintain 4-7g/L, adds the amino acid solution 10mL that concentration is 1-5g/L every two hours simultaneously.
2. the method for a kind of producing beta-polymalic acid by feed-batch fermentation as claimed in claim, it is characterized in that, described amino acid solution comprises at least one in aspartic acid, leucine, Histidine, Threonine.
3. the method for a kind of producing beta-polymalic acid by feed-batch fermentation as claimed in claim, it is characterized in that, described production bacterial strain is Aureobasidium pullulans, numbering CGMCC NO.3376.
4. the method for a kind of producing beta-polymalic acid by feed-batch fermentation as claimed in claim, it is characterized in that, fermentation stage is specific as follows:
Aureobasidium pullulans CGMCC NO.3336 seed liquor is connected in 10L fermentor tank with the amount of volume ratio 10% and carries out fermentation culture, under control pH6.0-7.0 and dissolved oxygen concentration 60%-70% condition, fermentation 36-48h starts flow feeding solution, the glucose solution being added 500g/L by stream makes fermented liquid residual sugar maintain 4-7g/L, added every two hours the amino acid solution 10mL that concentration is 1-5g/L simultaneously, described amino acid solution comprises at least one in aspartic acid, leucine, Histidine, Threonine, continues fermentation 96-120h.
CN201410833031.5A 2014-12-29 2014-12-29 Method for improving yield of beta-polymalic acid by fed-batch fermentation Pending CN104561143A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410833031.5A CN104561143A (en) 2014-12-29 2014-12-29 Method for improving yield of beta-polymalic acid by fed-batch fermentation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410833031.5A CN104561143A (en) 2014-12-29 2014-12-29 Method for improving yield of beta-polymalic acid by fed-batch fermentation

Publications (1)

Publication Number Publication Date
CN104561143A true CN104561143A (en) 2015-04-29

Family

ID=53078221

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410833031.5A Pending CN104561143A (en) 2014-12-29 2014-12-29 Method for improving yield of beta-polymalic acid by fed-batch fermentation

Country Status (1)

Country Link
CN (1) CN104561143A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154389A (en) * 2011-01-06 2011-08-17 中国科学院过程工程研究所 Process for producing beta-polymalic acid by feed-batch fermentation
CN103045662A (en) * 2012-12-31 2013-04-17 天津北洋百川生物技术有限公司 Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics
CN103757063A (en) * 2013-12-25 2014-04-30 天津北洋百川生物技术有限公司 Fermentation medium for greatly increasing yield of beta-polymalic acid by adding vitamins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154389A (en) * 2011-01-06 2011-08-17 中国科学院过程工程研究所 Process for producing beta-polymalic acid by feed-batch fermentation
CN103045662A (en) * 2012-12-31 2013-04-17 天津北洋百川生物技术有限公司 Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics
CN103757063A (en) * 2013-12-25 2014-04-30 天津北洋百川生物技术有限公司 Fermentation medium for greatly increasing yield of beta-polymalic acid by adding vitamins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王丽燕等: "聚苹果酸的发酵培养条件优化", 《生物加工过程》 *

Similar Documents

Publication Publication Date Title
CN101948785B (en) Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium
CN103665371B (en) A kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid
CN102154395A (en) Method for extracting gamma-polyglutamic acid by inorganic salt/organic solvent coprecipitation effect
CN102220248B (en) Bacterial strain for producing PMLA [Poly (Beta-L-malic acid)] and method for producing PMLA by fermentation of bacterial strain
CN103695485B (en) High-efficiency production method of gamma-polyglutamic acid
CN101560537B (en) Fermentation method for producing high viscocity xanthan gum by xanthomonas campestris
CN109609408B (en) Gamma-polyglutamic acid high-yield strain and method for preparing gamma-polyglutamic acid by using strain for liquid fermentation
CN103993042A (en) Method for combined production of bioethanol and pullulan from lignocellulose substances
CN102286564A (en) Method for culturing halophilic microorganism to produce ectoin and hydroxyl ectoin
CN102112617A (en) Process for co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by fermentation of yeast pichia pastoris
CN103789363A (en) Method for improving poly-malic acid yield
CN102268465B (en) Method for massively producing gamma-poly-glutamic acid
CN105624218A (en) Method for increasing yield of aureobasidium pullulans synthesized polymalic acid
CN104805143B (en) A kind of method for preparing low molecule amount γ polyglutamic acids
CN103045662B (en) Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics
CN107557407B (en) Method for regulating and controlling molecular weight of schizophyllan of schizophyllum commune fermentation product
CN102154389A (en) Process for producing beta-polymalic acid by feed-batch fermentation
CN101696434A (en) New process for fermenting Beta-polymalic acid
CN113881734A (en) Culture medium for producing pullulan through microbial fermentation and application of culture medium
CN110129225A (en) γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method
CN103757063A (en) Fermentation medium for greatly increasing yield of beta-polymalic acid by adding vitamins
CN110042129B (en) Preparation method of agricultural gamma-polyglutamic acid
CN102181509A (en) Method for producing vernine
CN104561143A (en) Method for improving yield of beta-polymalic acid by fed-batch fermentation
CN102605009B (en) Method for improving strength and concentration of butane diacid generated from anaerobic fermentation

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20150429