CN103509746A - Fermentation production method for natural L-(+)-tartaric acid - Google Patents
Fermentation production method for natural L-(+)-tartaric acid Download PDFInfo
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Abstract
The invention discloses pseudomonas putida for producing natural L-(+)-tartaric acid by using 5-keto-D-gluconic acid and salt thereof, and a production method of the pseudomonas putida. The pseudomonas putida mutant strain TUST is preserved in the Common Microorganism Center (CGMCC) of the China Microorganism Culture Preservation and Management Committee on May 28th, 2013, the preservation number is CGMCC No.7648. The natural L-(+)-tartaric acid can be produced from the strain by using 5-keto-D-gluconic acid and salt thereof, the fermentation yield of a shake flask is 1.6g/L, which is 12% of an theoretical value, the fermentation strength is 3% per day, the fermentation yield of a fermentation tank is 7.1g/L, which is 24% of the theoretical value, the fermentation strength is 4% per day, the fermentation product is natural L-(+)-tartaric acid, and moreover the production process is simple and less pollution is caused. Due to adoption of the strain and the production method, a novel process is provided for the production of natural L-(+)-tartaric acid in China, and the economic values of the natural L-(+)-tartaric acid are effectively improved.
Description
Technical field
The invention belongs to microbial fermentation technology field, be specifically related to a kind of natural L-(+)-tartaric fermentation method for producing.
Background technology
L-(+)-tartrate is a kind of natural organic acids, is present in various plants, and as there being high level in the fruit plants such as grape and tamarinds, its of many uses and demand cumulative year after year.In foodstuffs industry, L-(+)-tartrate can be made the first-class of auxiliary and the food dye composition of acidic flavoring agent, swelling agent, antioxidant, and its maximum purposes is drink additive; In pharmaceutical industries, can be used as medical resolving agent; In organic synthesis, can be used for preparing multiple chiral catalyst, or synthesize complicated natural product molecule as chiral source, be very important chiral ligand and chiron; In mirror industry processed, be an important auxiliary agent and reductive agent, can control the formation speed of silver mirror, obtain the coating of homogeneous; In other industry, also can print and dye reserving agent, photographic developer etc.
In early days, L-(+)-tartrate mainly extracts and obtains from by product winestone vinous, but owing to being subject to the restriction of raw material, output cannot satisfy the demands.In recent years, L-(+)-tartaric main production technology was chemical synthesis and half biological synthesis process.Chemical synthesis is owing to producing in process of production a large amount of waste water and the useless non-friendly material of environment that admittedly waits, for later regulation brings many troubles.
Half biological compositional rule mainly utilizes the microorganism with cis-Epoxysuccinic acid hydratase to produce L-(+)-tartrate by enzymatic conversion.At application number, being for example 201010609478.6 discloses a kind of L-(+)-tartaric production method in " a kind of L(+) tartaric production method ", step is: (1) is added to Sodium Hydrogen cis-Epoxysuccinate in the aqueous solution of freezing mother liquor and makes mixing solutions, adjust pH, add red rhodococcus hydrolysis; (2) add calcium sulfate calcification, filter to obtain L (+) calcium tartrate solid and calcification mother liquor, calcification mother liquor is freezing, and centrifugation obtains sal glauberi solid and freezing mother liquor, and freezing mother liquor is back to use step (1); (3) by L-(+)-calcium tartrate solid sulfuric acid solution, filter and obtain L-(+)-aqueous tartaric acid solution, pass through successively positively charged ion, anionite-exchange resin, through concentrated, crystallization, separation and oven dry, obtain L-(+)-tartrate.Calcification mother liquor and calcium sulfate reusable edible that this invention produces in process of production, reduced the problem of environmental pollution, also to a small amount of product L-(+)-calcium tartrate dissolving in calcification mother liquor and not the cis-form epoxy succinic acid sodium of complete reaction reclaim, improved product yield.But in this method, producing the precursor substance cis-form epoxy succinic acid that L-(+)-tartrate uses still needs to adopt chemical method synthetic.
Along with socioeconomic fast development, people's standard of living has obtained significantly improving, and cooking culture is variation day by day, and people improve food safety attention degree, and the wholesomeness of food is paid attention to increasingly.People are more remarkable to the demand of natural foodstuff additive, so the R and D of natural additive for foodstuff are all devoted in current countries in the world.And at present, domestic not yet discovery utilizes biological direct fermentation synthesis of natural L-(+)-tartaric technique.
Summary of the invention
The object of the present invention is to provide a kind of natural L-of biological process fermentative production (+)-tartaric method, its substrate 5-ketone group-gluconic acid or its salt utilize glucose fermentation to produce and obtain by bacillus of oxidizing glucose, and its product is natural L-(+)-tartrate.L-(+)-tartaric chemosynthesis and half biosynthetic present situation have been changed.
A kind of natural L-of the present invention (+)-tartaric fermentation method for producing, comprises the steps:
The preparation of seed liquor: slant strains is cultivated and obtained seed liquor through switching;
Fermentation culture: seed liquor is accessed to fermentation culture in fermention medium;
Conditions of flask fermentation is: fermention medium loading amount 10～20%, and inoculum size 5～10%, shaking speed 150～200r/min, 28～32 ℃ of temperature, fermentation time is 2～5d.
Fermentor cultivation condition is: 5L fermentor tank liquid amount is 3L, and inoculum size is 5～10%, and rotating speed is 200～400r/min, and temperature is 28～32 ℃, and fermentation time is 6-8d.
Consisting of of described fermention medium: 5-ketone group-Potassium Gluconate 10～100g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 10～20g/L;
Detection method: adopt high performance liquid chromatography (HPLC) method to measure L-(+)-tartaric content, described HPLC testing conditions is:
Chromatographic column shodex DE-613(6.0mm ID * 150mm), moving phase is 10mmol/L HClO
4, flow velocity 0.2mL/min, 30 ℃ of column temperatures, detect wavelength 210nm, sample size 20 μ L;
Instrument model: high performance liquid chromatograph, Agilent 1100, Agilent (China) company limited
Bacterial classification of the present invention is specially pseudomonas putida (Pseudomonas pudita) TUST, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on May 28th, 2013, deposit number is CGMCC No.7648, preservation address: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, postcode: 100101.
This bacterial strain feature is as follows:
This bacterial strain colony colour on LB solid plate is faint yellow, circle, and smooth surface, neat in edge, moistening.It is shaft-like that thalline is, and size is 0.6 – 0.8 * 2.0 – 2.4 μ m, Gram-negative, and for having the aerobic bacteria of mobility, hydrogen peroxide enzyme positive, can utilize Citrate trianion, not gelatin hydrolysate.Optimum growth temperature is 28-30 ℃.
Shown in the list of 16SrDNA sequencing result nucleotides sequence, first adopt DNAMAN software to carry out similarity merger the sequence recording, similarity being greater than to 97% sequence merger is an operating unit, the sequence that represents of choosing in each operating unit utilizes the 16SrDNA gene order in BLAST and GenBank database to carry out homology analysis, choose the above length of 1400bp compare (Clustelx1.83), adopt ortho position to connect (Neighbour Joining) method and carry out Phylogenetic Analysis (MEGA4.1), phylogenetic tree as shown in Figure 1.The homology of the false pseudomonas bacillus of bacterial strain TUST and stench is higher as seen from the figure, can tentatively regard as the false pseudomonas bacillus of stench.
The false pseudomonas bacillus TUST of described stench can utilize 5-keto-D-gluconic acid or its salt to produce L-(+)-tartrate, shake flask fermentation output is 1.6g/L, reach 12% of theoretical value, ferment strength is every day 3%, ferment tank output is 7.1g/L, reach 24% of theoretical value, ferment strength is every day 4%.
Beneficial effect:
First, by rhizosphere soil sampling, sieve to such an extent that a strain utilizes 5-ketone group-D glyconic acid or its salt to produce L-(+)-tartaric pseudomonas putida, the method is biological fermentation process, product is natural L-(+)-tartrate, and this bacterial strain production L-(+)-tartrate technique is simple, pollutes few.The second, utilize bacterial classification of the present invention, shake-flask culture 2～5d, L-(+)-tartaric output is theoretical value 12%, ferment strength is every day 3%, fermentor cultivation 6～8d, L-(+)-tartrate output is theoretical value 24%, ferment strength is every day 4%.。Three, the present invention has filtered out and can utilize 5-ketone group-D glyconic acid or its salt to produce L-(+)-tartaric new bacterial strain, tunning is natural L-(+)-tartrate, met the demand of people to natural additive for foodstuff, and provide new technique for domestic L-(+)-tartaric production, thereby create more economic benefit.
Accompanying drawing explanation:
Fig. 1 is the phylogenetic tree of pseudomonas putida CGMCC No.7648
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used 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 are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1: utilize 5-ketone group gluconate to produce the screening of L-(+)-tartrate bacterial classification
The collection of 1 Rhizosphere sampling
During sampling, get each point normal plant 4-5 cave, band soil digs out root, cuts over-ground part, and Liu Gen and the soil sample that is attached to root pack sample sack into, seal sack, put into 4 ℃ of refrigerators, separated in order to microorganism.
2 separated and cultivations
(1) substratum
Enrichment medium: peptone 20g/L, K
2hPO
41.5g/L, glycerine 10g/L, MgSO
41.5g/L, agar 15g/L, pH7.2.
Isolation medium: same enrichment medium.
(2) separation and Culture
Prepare soil diluent: take the Rhizosphere Soil of 1.0g, put into 99ml sterilized water and the 250ml triangular flask of granulated glass sphere is housed, shaking table vibration 5min makes soil dispersed, becomes soil supension (10
-2).With the aseptic suction nozzle of 1ml, therefrom draw 0.5ml soil supension, inject and divide in advance the test tube that 4.5ml sterilized water is housed, pressure-vaccum, vibration even (10
-3).Use the same method, prepared and diluted degree is 10 respectively
-4, 10
-5, 10
-6soil supension.
Coating: draw 100 μ l respectively from each concentration soil diluent, put on KB culture medium flat plate, with aseptic coated with glass device coating evenly, each concentration is made 3 flat boards.
Cultivate: KB culture medium flat plate is placed in to 30 ℃ of thermostat containers and cultivates 48h, observe the bacterium colony mean number on three flat boards of same extent of dilution, determine optimum dilution degree
3 separation and purification
Get and at 30 ℃, cultivate the KB culture medium flat plate of 48 hours, the bacterium colony that has fluorescence to produce at the lower picking of UV-light (256nm), three rides on KB culture medium flat plate, flat-plate inverted is placed in to 30 ℃ of thermostat containers and cultivates 24h, after purifying 2-3 time of then colonies typical being rule on KB culture medium flat plate, obtain single bacterium colony.
Embodiment 2L-(+)-tartaric fermentation culture
From activated inclined plane, picking one ring TUST thalline is seeded in the 250mL triangular flask that 50mL seed culture medium is housed and cultivates, and 30 ℃, 170r/min shaking table cultivation 24h, obtain seed liquor.Seed culture medium consists of: peptone 20g/L, K
2hPO
41.5g/L, glycerine 10g/L, MgSO
41.5g/L, agar 15g/L, pH7.2,121 ℃ of high pressure steam sterilization 20min.
Seed liquor is with in 10% inoculum size access fermention medium, and 30 ℃, 170r/min shake flask fermentation, cultivate 5d.Consisting of of described fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, Semen Maydis powder 10g/L.
After fermentation 5d, L-(+)-tartrate output is 0.3g/L, reaches 2% of theoretical value, and ferment strength is every day 0.4%.
Testing conditions: adopt Agilent 1100 high performance liquid chromatographs, shodex DE-613(6.0mm ID * 150mm) chromatographic column, with 10mmol/L HClO
4for moving phase, flow velocity 0.2mL/min, 30 ℃ of column temperatures, detect wavelength 210nm, and high performance liquid chromatography (HPLC) method of sample size 20 μ L detects.
Embodiment 3L-(+)-tartaric fermentation culture
Fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 10g/L, its fermentation and testing conditions are all with embodiment 2, and fermentation time is 4d, and L-(+)-tartrate output is 1.2g/L, reaches 10% of theoretical value, and ferment strength is every day 2.5%.
Embodiment 4L-(+)-tartaric fermentation culture
Fermention medium: 5-ketone group-Potassium Gluconate 10g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 20g/L.Its fermentation and testing conditions are all with embodiment 2, and fermentation time is 4d, and L-(+)-tartrate output is 0.2g/L, reaches 5% of theoretical value, and ferment strength is every day 1.25%.
Embodiment 5L-(+)-tartaric fermentation culture
Fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 20g/L, its fermentation and testing conditions are all with embodiment 2, and fermentation time is 4d, and L-(+)-tartrate output is 1.6g/L, reaches 12% of theoretical value, and ferment strength is every day 3%
Embodiment 6L-(+)-tartaric acid fermentation tank is cultivated
Seed liquor accesses in fermention medium with 10% inoculum size, 30 ℃, 300r/min fermentor cultivation 6d.Consisting of of described fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 20g/L.Testing conditions is with embodiment 2, and fermentation time is 6d, and L-(+)-tartrate output is 7.1g/L, reaches theoretical value and obtains 24%, and ferment strength is every day 4%.
Claims (8)
1. a strain utilizes 5-keto-D-gluconic acid or its salt to produce natural L-(+)-tartaric pseudomonas putida TUST, it is characterized in that, and described pseudomonas putida (Pseudomonas pudita) TUST, deposit number is CGMCC No.7648.
2. a natural L-(+)-tartaric fermentation method for producing, it is characterized in that, take 5-keto-D-gluconic acid or its salt is substrate, and by pseudomonas putida (Pseudomonas pudita), TUST obtains natural L-(+)-tartrate through biological fermentation.
3. a kind of natural L-as claimed in claim 2 (+)-tartaric fermentation method for producing, is characterized in that, comprises the steps:
The preparation of seed liquor: pseudomonas putida TUST slant strains is cultivated and obtained seed liquor through switching;
Fermentation culture: seed liquor is accessed to fermentation culture in fermention medium; Fermentation culture is carried out in shaking flask or fermentor tank; Described conditions of flask fermentation is: fermention medium loading amount 10～20%, and inoculum size 5～10%, shaking speed 150～200r/min, 28～32 ℃ of temperature, fermentation time is 2-5d; Fermentor tank condition is: 5L fermentor tank liquid amount is 3L, and inoculum size is 5～10%, and rotating speed is 200～400r/min, and temperature is 28～32 ℃, and fermentation time is 6-8d.
4. a kind of natural L-(+)-tartaric fermentation method for producing as claimed in claim 2 or claim 3, is characterized in that: described fermention medium consists of: 5-ketone group-Potassium Gluconate 10～100g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 10～20g/L.
5. a kind of natural L-(+)-tartaric fermentation method for producing as claimed in claim 2 or claim 3, is characterized in that: comprise the steps:
The preparation of seed liquor: slant strains is cultivated and obtained seed liquor through switching;
Fermentation culture: seed liquor is accessed to fermentation culture in fermention medium; Fermentation culture is carried out in shaking flask; Described conditions of flask fermentation is: fermention medium loading amount 20%, and inoculum size 10%, shaking speed 170r/min, 30 ℃ of temperature, fermentation time is 4d;
Fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 20g/L.
6. a kind of natural L-(+)-tartaric fermentation method for producing as claimed in claim 2 or claim 3, is characterized in that: comprise the steps:
The preparation of seed liquor: slant strains is cultivated and obtained seed liquor through switching;
Fermentation culture: seed liquor is accessed to fermentation culture in fermention medium; Fermentation culture is carried out in fermentor tank; Described ferment tank condition is: 5L fermentor tank liquid amount is 3L, and inoculum size is 10%, and rotating speed is 300r/min, and temperature is 30 ℃, and fermentation time is 6d;
Fermention medium: 5-ketone group-Potassium Gluconate 60g/L, MgSO
40.25g/L, KH
2pO
40.6g/L, urea 2g/L, corn steep liquor 10～20g/L.
7. a kind of natural L-(+)-tartaric fermentation method for producing as claimed in claim 2 or claim 3, it is characterized in that: the shake flask fermentation output that described pseudomonas putida TUST utilizes 5-keto-D-gluconic acid or its salt to produce L-(+)-tartrate or its salt is 1.6g/L, reach 12% of theoretical value, ferment strength reaches every day 3%, ferment tank output is 7.1g/L, reach 24% of theoretical value, ferment strength reaches every day 4%.
8. the application of a kind of natural L-as claimed in claim 2 (+)-tartaric fermentation method for producing in producing L-(+)-tartrate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923866A (en) * | 2014-04-25 | 2014-07-16 | 杭州宝晶生物股份有限公司 | Pseudomonas koreensis and method for preparing mesotartaric acid or salt thereof by using same |
| CN110591954A (en) * | 2019-09-25 | 2019-12-20 | 杭州宝晶生物股份有限公司 | Sphingobacterium and application and method thereof in catalytic synthesis of L (+) -tartaric acid or salt thereof |
| CN111088198A (en) * | 2020-01-21 | 2020-05-01 | 杭州宝晶生物股份有限公司 | Pseudomonas fuscogongensis, application thereof and method for catalytically synthesizing L (+) -tartaric acid or salt thereof |
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| EP0311835B1 (en) * | 1987-10-16 | 1993-01-20 | Toray Industries, Inc. | Process for producing d-(-)-tartaric acid |
| CN101407773A (en) * | 2008-10-07 | 2009-04-15 | 南京工业大学 | Pseudomonas putida GNA5 strain and method for preparing D-glucosamine acid by using same |
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2013
- 2013-10-15 CN CN201310481586.3A patent/CN103509746B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0311835B1 (en) * | 1987-10-16 | 1993-01-20 | Toray Industries, Inc. | Process for producing d-(-)-tartaric acid |
| CN101407773A (en) * | 2008-10-07 | 2009-04-15 | 南京工业大学 | Pseudomonas putida GNA5 strain and method for preparing D-glucosamine acid by using same |
Non-Patent Citations (1)
| Title |
|---|
| 袁建锋: "基于5-酮基-D-葡萄糖酸生物制造L-(+)-酒石酸的研究进展", 《现代化工》, no. 9, 30 September 2013 (2013-09-30), pages 13 - 17 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923866A (en) * | 2014-04-25 | 2014-07-16 | 杭州宝晶生物股份有限公司 | Pseudomonas koreensis and method for preparing mesotartaric acid or salt thereof by using same |
| CN110591954A (en) * | 2019-09-25 | 2019-12-20 | 杭州宝晶生物股份有限公司 | Sphingobacterium and application and method thereof in catalytic synthesis of L (+) -tartaric acid or salt thereof |
| CN111088198A (en) * | 2020-01-21 | 2020-05-01 | 杭州宝晶生物股份有限公司 | Pseudomonas fuscogongensis, application thereof and method for catalytically synthesizing L (+) -tartaric acid or salt thereof |
| CN111088198B (en) * | 2020-01-21 | 2021-03-30 | 杭州宝晶生物股份有限公司 | Pseudomonas fuscogongensis, application thereof and method for catalytically synthesizing L (+) -tartaric acid or salt thereof |
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