CN100410364C - Gamma-polyglutamic acid generating bacterium and process of preparing gamma-polyglutamic acid therewith - Google Patents
Gamma-polyglutamic acid generating bacterium and process of preparing gamma-polyglutamic acid therewith Download PDFInfo
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- CN100410364C CN100410364C CNB2006101226405A CN200610122640A CN100410364C CN 100410364 C CN100410364 C CN 100410364C CN B2006101226405 A CNB2006101226405 A CN B2006101226405A CN 200610122640 A CN200610122640 A CN 200610122640A CN 100410364 C CN100410364 C CN 100410364C
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Abstract
The present invention provides one kind of gamma-polyglutamic acid generating bacterium and the process of preparing gamma-polyglutamic acid (gamma-PGA) therewith. The gamma-polyglutamic acid generating bacterium is Bacillus substilis CCTCC M 206102. The preparation process of gamma-PGA includes using Bacillus substilis CCTCC M 206102 as fermenting strain, and shake flask fermentation of sugar material without adding L-glutamic acid. The present invention has relatively low material cost, gamma-PGA output up to 18g/L and excellent industrial application foreground.
Description
Technical field
The present invention relates to a kind of method of producing the microorganism strains of gamma-polyglutamic acid-and utilizing this strain fermentation production gamma-polyglutamic acid-.
Background technology
People such as nineteen thirty-seven Ivanovics at first find to contain gamma-polyglutamic acid-by the folder film of Bacillus anthracis, and (γ-PGA), people such as nineteen forty-two Bovarnick find that some bacillus can accumulate γ-PGA by fermentation culture.Troy in 1973 finds that (hereinafter to be referred as γ-PGA) is a kind of chemical composition of Bacillus anthracis cell folder film to gamma-polyglutamic acid-, and it is a kind of water miscible amide compound, can produce by the mutation of genus bacillus.Its structure is unusual, and it is the amido linkage that is formed at γ-hydroxyl and alpha-amino group by single L-glutamic acid molecule and the polymer that connects into, and its structural formula is as follows:
γ-PGA makes through fermentation from left-handed-L-glutamic acid (bacterial classification that has can add L-L-glutamic acid), is the biopolymer product of a kind of All Pure Nature, multifunctionality, Biodegradable.γ-PGA molecular weight ranges is from 10,000～2, and 000,000, can make different molecular weight and be applied to various different field.Such as γ-PGA tool food safety 1,, can be used as food fibre, protective foods, food thickener, tranquilizer or as the wetting Agent for Printing Inks of used for cosmetic; 2, γ-PGA can make hydrogel, has high water-retaining capacity, and can absorb water reaches 3,500 times, and the utmost point is suitable for the application of agricultural soil and environmentfriendly products; 3, γ-PGA can change into various metallic salt, the different pH values of tool, and special polyanionic surface property is arranged, be applicable to tissue culture and a lot of other food, makeup, biomedicine and industrial application etc.
The biosynthetic research of γ-PGA mainly concentrates on the B.anthracis and B.licheniformisATCC 9945a, B.licheniformis ATCC 9945 bacterial strains such as (being B.subtilis ATCC9945 in the past) of bacillus, need L-L-glutamic acid to make nutrient raw material when this class spawn culture and fermentation and could accumulate γ-PGA, when adding a large amount of L-glutamic acid, the growing amount of γ-PGA is generally at 20gL
-1About.But L-L-glutamic acid is done raw material and is had unfavorable factors such as material price costliness.
Summary of the invention
The objective of the invention is to propose a kind of gamma-polyglutamic acid-that does not need L-glutamic acid to make raw material produces bacterium and utilizes this bacterial strain to prepare the method for gamma-polyglutamic acid-.
Microorganism strains provided by the present invention is that (γ-PGA) produces bacterium--subtilis (Bacillus subtilis) PGA-7 for gamma-polyglutamic acid-through seed selection, this bacterial strain is preserved in Chinese typical culture collection center (CCTCC), is numbered: CCTCCM 206102.This bacterial strain is hereinafter to be referred as Bacillus subtilis PGA-7CCTCC M 206102.
The growthhabit of Bacillus subtilis PGA-7 CCTCC M 206102 bacterial strains on solid medium has diversity, at LB substratum (peptone 10g, yeast extract paste 5g, NaCl 10g, agar 20g, tap water 1L, on the flat board of adjust pH to 7.0～7.2.), when substratum was moist, bacterium colony was moistening, circular, opaque, the lawn neat in edge, smooth surface; When dry, the coarse gauffer of bacterium colony, concavity, the edge is irregular; When adding a small amount of L-glutamic acid in the substratum, the bacterium colony expansion was obviously cultivated through 3 days, and colony diameter reaches 15-25mm, and the surface is " auricularia auriculajudae shape ".Its physiological and biochemical property sees the following form.
Annotate :+the positive ,-feminine gender
The genetics feature of Bacillus subtilis PGA-7 CCTCC M 206102 bacterial strains: adopt 16S rRNA order-checking to carry out the taxonomy Molecular Identification.The bacterial classification extracting genome DNA adopts acetone to destroy bacteria cell wall membrane lipid structure, and lysate is abolished bacterial cell membrane, discharges nucleic acid in the born of the same parents, removes protein and polysaccharide, dehydrated alcohol deposit D NA, TE damping fluid dissolving DNA through phenol/chloroform/primary isoamyl alcohol.Adopt the 16S rDNA of two kinds of synthetic universal primer PCR amplification bacteriums, primer is as follows: 27F:5 '-AGA GTT TGA TCC TGG CTC AG-3 '; 1541R:5 '-AAG GAA GTG ATG CAG CCG CA-3 '.Directly carry out sequencing behind the pcr amplification product purifying, send GenBank to be Blast relatively (the GenBank accession number of sequencing sequence is DQ415893.2) sequence that obtains, according to the gene series that provides among the GenBank, analyze according to 16S rRNA sequence construct evolutionary tree, wherein homology the highest bacterial strain be B.subtilis MA139 etc., homology be 99% and similarity the highest, the phyletic evolution result of combining form, physiological and biochemical property and 16S rRNA serial analysis, this identification of strains are bacillus bacillus subtilis bacterial classification.
With Bacillus subtilis PGA-7 CCTCC M 206102 bacterial strains provided by the present invention, shake what do not need to add L-glutamic acid fermentation raw material that fermentation can produce gamma-polyglutamic acid-in the bottle, thereby can produce gamma-polyglutamic acid-as fermentation strain with it.
Method of producing gamma-polyglutamic acid-provided by the present invention with Bacillus subtilis PGA-7 CCTCC M 206102 bacterial strains, be characterized in doing fermentation strain, utilize saccharine material to carry out shake flask fermentation with Bacillus subtilis PGA-7 CCTCC M 206102.Said saccharine material can be glucose, fructose, sucrose, maltose etc., does not need to add L-L-glutamic acid in fermentation raw material.Shake flask fermentation 60-96 hour, can produce γ-PGA 10～18g/l.
Utilize the specific embodiment of Bacillus subtilis PGA-7 CCTCC M 206102 bacterial strain shake flask fermentations production γ-PGA as follows:
(1) slant strains activation: (Bacillus subtilis PGA-7 CCTCC M 206102) lines on the LB inclined-plane of new preparation with cultured preservation inclined-plane kind, the LB inclined-plane is composed as follows: peptone 8～12g, yeast extract paste 3～8g, NaCl 9～11g, agar 15～22g, tap water 1L, adjust pH to 6.8～7.4..Cultivated 12～24 hours for 33-38 ℃, refrigerate again in 4 ℃ 1～5 day.
(2) liquid of bacterial classification activation: with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is inoculated in the liquid activation medium, and the liquid activation medium is composed as follows: peptone 8～12g, yeast extract paste 3～8g, NaCl 9～11g, tap water 1L, adjust pH to 6.8～7.4..The liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 32-36 ℃ of reciprocating type shaking table shaking culture 18～36 hours, shaking speed was 80-110r/m, and stroke is 65-75mm.
(3) shake flask fermentation: by 5% inoculum size, getting the cultured liquid activation of 1ml plants, insert in the fermention medium, fermention medium (substratum is formed the explanation of face as follows) is bottled with the 500ml triangle, loading amount 20ml, in 32-48 ℃, stroke 65-80mm, the reciprocating type shaking table shaking culture of rotating speed 90-130r/m 60-96 hour stops fermentation.
(4) the shake flask fermentation mash that finishes of fermentation promptly gets the goods that contain gamma-polyglutamic acid-through extraction process.
The technology that extraction contains the goods of gamma-polyglutamic acid-is common process, generally comprises the centrifugal removal thalline of fermented liquid, gets its supernatant liquor with steps such as ethanol sedimentation extractions.
In step (3), suitable fermention medium composition following (calculating): 60-120g saccharine material, 14-20g nitrogenous source, 1.0-2.0g K by every liter of contained grammes per square metre g/l of substratum
2HPO
4, 0.05-0.15g MgSO
47H
2O, 0.01-0.08g FeCl
36H
2O, 0.1-0.2g CaCl
22H
2O, 0.05-0.104g MnSO
4H
2O, to 6.5-7.0, available NaOH and HCl regulate based on the preceding adjust pH of sterilization in cultivation.Wherein, described saccharine material can be glucose, fructose, sucrose, maltose etc.; Nitrogenous source can be ammonium chloride, ammonium sulfate, ammonium nitrate etc.
The shake flask fermentation mash that finishes of fermentation is the γ-PGA content in the analytical test fermentation liquid by the following method:
To the centrifugal removal thalline of fermented liquid, get supernatant liquor and survey the monomeric content of L-glutamic acid; The hydrochloric acid of supernatant liquor and 6mol/L is added in the glass hydrolysis pipe by 1: 1 (volume ratio), in 110 ℃ of hydrolysis 24h, measure L-glutamic acid total amount in the hydrolyzed solution then behind the vacuum seal; The difference of the L-glutamic acid that records for 2 times is the content of γ-PGA.
Above-mentioned analytical procedure to L-glutamic acid can adopt automatic analyzer for amino acids, or high performance liquid chromatograph (HPLC), or the ply of paper analysis method is measured.The ply of paper analysis method is as follows: get centrifugal fermented supernatant fluid of 10 μ l or its hydrolyzed solution point sample, chromatography spends the night, and with the colour developing of 0.5% triketohydrindene hydrate acetone soln, 65 ℃ of colour developing 10min cut spot 1g/L copper sulfate elutriant (CuSO
45H
2O: 75% ethanol=2: 38) 5mL immersion 20min is above colourless to paper slip, and Λ max=570nm surveys its OD value, relatively calculates corresponding content with the L-glutamic acid typical curve, calculates fermentation liquid again and contains γ-PGA amount.
Characteristics maximum among the present invention are not need L-glutamic acid to make the bacillus subtilis strain fermentative production γ-PGA of raw material with a strain, more in the past than its raw materials cost of microbial fermentation that needs L-glutamic acid to make raw material that adopts usually bigger reduction are arranged; The present invention produces γ-PGA and reaches as high as 18g/l under best shake flask fermentation processing condition.
Utilize the research of L-glutamic acid as fermentation substrate, correlative study both at home and abroad is abundanter, the compare non-glutamic acid dependent form fermentation of its productive rate is higher, but consider from the raw materials for production angle, do not need L-glutamic acid to have advantage on the cost as the bacterial strain of fermentation substrate, have more the industrial applications prospect, so the present invention is expected to provide another kind of feasible method for industrial fermentation production γ-PGA.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
The inclined-plane kind of using in the following examples adopts following method to cultivate: Bacillus subtilis PGA-7 CCTCC M206102 lines on the LB inclined-plane of new preparation, and the LB inclined-plane is composed as follows: peptone 10g, yeast extract paste 5g, NaCl 10g, agar 20g, tap water 1L, adjust pH is to 7.2..Cultivated 17 hours, and refrigerated in 4 ℃ of refrigerators standby again for 337 ℃.
Embodiment one:
[composition of preservation slant medium (g/L): peptone 10, NaCl 10, yeast extract paste 5, agar 20, pH7.0 to get interior 1 day the inclined-plane kind of preservation of 4 ℃ of refrigerators.], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is transferred on the fermention medium of new configuration, and (g/L) is as follows for the composition of fermention medium: 80g fructose, 18g NH
4Cl, 1.5g K
2HPO
4, 0.05gMgSO
47H
2O, 0.01g FeCl
36H
2O, 0.15g CaCl
22H
2O, 0.05g MnSO
4H
2O, and be 6.5,120 ℃ with the pH value (before the sterilization) that NaOH and HCl regulate substratum and sterilized 20 minutes.Fermention medium is loaded in the 500ml triangular flask, and loading amount 20ml is in 76mm stroke, the reciprocating type shaking table shaking culture of rotating speed 130r/m, after 96 hours, after shake flask fermentation finished, fermented liquid adopted 20 through 37 ℃ of constant temperature culture, 000 rev/min centrifugal 10 minutes, measure γ-PGA content with Paper Chromatography.Adopt this method shake flask fermentation to produce γ-PGA and can reach 18g/L.
Embodiment two:
[composition of preservation slant medium (g/L): peptone 8, NaCl 11, yeast extract paste 6, agar 20, pH7.0 to get interior 2 days the inclined-plane kind of preservation of 4 ℃ of refrigerators.], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is transferred on the fermention medium of new configuration, and (g/L) is as follows for the composition of fermention medium: 90g sucrose, 20g (NH
4)
2SO
4, 1.0g K
2HPO
4, 0.06g MgSO
47H
2O, 0.01g FeCl
36H
2O, 0.15g CaCl
22H
2O, 0.05g MnSO
4H
2O, and be 6.5,120 ℃ with the pH value (before the sterilization) that NaOH and HCl regulate substratum and sterilized 20 minutes.Fermention medium is loaded in the 500ml triangular flask, and loading amount 20ml is in 76mm stroke, the reciprocating type shaking table shaking culture of rotating speed 120r/m, after 90 hours, after shake flask fermentation finished, fermented liquid adopted 20 through 37 ℃ of constant temperature culture, 000 rev/min centrifugal 10 minutes, measure γ-PGA content with Paper Chromatography.Adopt this method shake flask fermentation to produce γ-PGA and can reach 16.2g/L.
Embodiment three:
[composition of preservation slant medium (g/L): peptone 12, NaCl 9, yeast extract paste 4, agar 20, pH7.0 to get interior 3 days the inclined-plane kind of preservation of 4 ℃ of refrigerators.], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is transferred on the fermention medium of new configuration, and (g/L) is as follows for the composition of fermention medium: 60g glucose, 20g NH
4NO
3, 1.0g K
2HPO
4, 0.01gMgSO
47H
2O, 0.05g FeCl
36H
2O, 0.2g CaCl
22H
2O, 0.104g MnSO
4H
2O, and be 7.0,120 ℃ with the pH value (before the sterilization) that NaOH and HCl regulate substratum and sterilized 20 minutes.Fermention medium is loaded in the 500ml triangular flask, and loading amount 20ml is in 65mm stroke, the reciprocating type shaking table shaking culture of rotating speed 90r/m, after 60 hours, after shake flask fermentation finished, fermented liquid adopted 20 through 37 ℃ of constant temperature culture, 000 rev/min centrifugal 10 minutes, measure γ-PGA content with Paper Chromatography.Adopt this method shake flask fermentation to produce γ-PGA and can reach 12.6g/L.
Claims (4)
1. a gamma-polyglutamic acid-produces bacterium, it is characterized in that this bacterial strain is subtilis (Bacillussubtilis) the PGA-7 CCTCC M206102 through seed selection.
2. the production method of a gamma-polyglutamic acid-, it is characterized in that doing fermentation strain with subtilis (Bacillus subtilis) PGA-7CCTCC M206102, utilize saccharine material to carry out shake flask fermentation, described saccharine material is glucose, fructose, sucrose or maltose.
3. the production method of gamma-polyglutamic acid-according to claim 2 is characterized in that said shake flask fermentation produces the technological process of gamma-polyglutamic acid-and be:
(1) slant strains activation: PGA-7CCTCC M206102 lines on the LB inclined-plane of new preparation with cultured preservation inclined-plane kind subtilis (Bacillus subtilis), the LB inclined-plane is composed as follows: peptone 8～12g, yeast extract paste 3～8g, NaCl 9～11g, agar 15～22 g, tap water 1L, adjust pH to 6.8～7.4, cultivated 12～24 hours for 33-38 ℃, refrigerate again in 4 ℃ 1～5 day;
(2) liquid of bacterial classification activation: with 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be inoculated in the liquid activation medium, the liquid activation medium is composed as follows: peptone 8～12g, yeast extract paste 3～8g, NaCl 9～11g, tap water 1L, adjust pH to 6.8～7.4, the liquid activation medium is loaded in the 250ml triangular flask, the triangular flask loading amount is 20ml, in 32-36 ℃ of reciprocating type shaking table shaking culture 18～36 hours, shaking speed was 80-110r/m, and stroke is 65-75mm;
(3) shake flask fermentation: by 5% inoculum size, get the cultured liquid activation of 1ml and plant, insert in the fermention medium, fermention medium is bottled with the 500ml triangle, and loading amount 20ml is in 32-48 ℃, stroke 65-80mm, the reciprocating type shaking table shaking culture of rotating speed 90-130r/m 60-96 hour stops fermentation;
(4) the shake flask fermentation mash that finishes of fermentation promptly gets the goods that contain gamma-polyglutamic acid-through extraction process.
4. the production method of gamma-polyglutamic acid-according to claim 3 is characterized in that in step (3), and described fermention medium composition is calculated as by every liter of contained grammes per square metre of substratum: 60-120g saccharine material, 14-20g nitrogenous source, 1.0-2.0gK
2HPO
4, 0.05-0.15g MgSO
47H
2O, 0.01-0.08g FeCl
36H
2O, 0.1-0.2g CaCl
22H
2O, 0.05-0.104gMnSO
4H
2O, adjust pH is to 6.5-7.0 before the sterilization, and described saccharine material is glucose, fructose, sucrose or maltose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN106750387A (en) * | 2016-12-30 | 2017-05-31 | 广东迪美新材料科技有限公司 | A kind of method that high temperature spray-drying prepares γ polyglutamic acid powder |
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| CN101417894B (en) * | 2008-04-09 | 2010-10-27 | 广东省微生物研究所 | Method for producing fertilizer additive agent containing gamma-polyglutamic acid |
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| CN101864073B (en) * | 2010-05-25 | 2012-01-18 | 上海应用技术学院 | Method for extracting Gamma-polyglutamic acid from fermentation broth |
| CN102911896B (en) * | 2012-09-28 | 2014-07-30 | 广西南宁智天生物科技有限公司 | Bacillus subtilis for producing gamma-polyglutamic acid by high-temperature fermentation and application of bacillus subtilis for producing gamma-polyglutamic acid by high-temperature fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
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| CN106750387A (en) * | 2016-12-30 | 2017-05-31 | 广东迪美新材料科技有限公司 | A kind of method that high temperature spray-drying prepares γ polyglutamic acid powder |
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