CN104232504A - Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain - Google Patents
Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain Download PDFInfo
- Publication number
- CN104232504A CN104232504A CN201410327118.5A CN201410327118A CN104232504A CN 104232504 A CN104232504 A CN 104232504A CN 201410327118 A CN201410327118 A CN 201410327118A CN 104232504 A CN104232504 A CN 104232504A
- Authority
- CN
- China
- Prior art keywords
- gamma
- polyglutamic acid
- strain
- carbon source
- seed
- 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
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a strain for synthesizing gamma-polyglutamic acid. The strain is screened from soil around a chemical fertilizer plant and is classified to be named as Bacillus Subtilis NS-9, the collection number of the strain in the China Center for Type Culture Collection (CCTCC NO.) is M2014142, and the collection date of the strain is April 23, 2014. The invention also discloses a method for efficiently preparing gamma-polyglutamic acid by using the strain for synthesizing gamma-polyglutamic acid. Compared with an existing gamma-polyglutamic acid production technique, the adopted bacillus subtilis microbial strain disclosed by the invention can be used for efficiently producing gamma-polyglutamic acid by virtue of fermentation; by optimizing the culture conditions of the bacillus subtilis strain, particularly replenishing a carbon source, inorganic salts and energy substances in a fermentation process, the content of gamma-polyglutamic acid in a fermentation solution can reach 100-200g/L, so that the method for preparing gamma-polyglutamic acid, disclosed by the invention, is high in efficiency and low in cost.
Description
Technical field
The invention belongs to microorganism and fermentation technical field, be specifically related to a kind of synthesis bacterial strain of gamma-polyglutamic acid-.The invention still further relates to a kind of synthesis bacterium plant height of gamma-polyglutamic acid-that utilizes and imitate the method preparing gamma-polyglutamic acid-.
Background technology
Gamma-polyglutamic acid-(γ-polyglutamic acid, hereinafter referred to as gamma-polyglutamic acid-) is the class homogeneous polyamino acid be formed by connecting by γ-amido linkage by D-Glu and Pidolidone monomer, can be synthesized by fermentable.Because gamma-polyglutamic acid-has splendid film-forming properties, the physics and chemistry becoming the excellences such as fibering, plasticity-, binding property, humidity-preserving type, degradability and biological characteristics, has application potential in industrial or agricultural, makeup and medicine and other fields.
The preparation method of polyglutamic acid has chemical synthesis, extraction method and microbe fermentation method.Microbe fermentation method is lower than the production cost of front two kinds of methods, and the pollution of production process to environment is little, adopts Production by Microorganism Fermentation gamma-polyglutamic acid-so now main.The zymotechnique of gamma-polyglutamic acid-is conducted extensive research, mainly based on the culture medium prescription of liquid submerged fermentation and culture process both at home and abroad.Nanjing University of Technology's screening obtains a strain gamma-polyglutamic acid-superior strain Bacillus Subtilis NX-2, and has done than more comprehensively research liquid fermenting production gamma-polyglutamic acid-, and has applied for patent, and patent publication No. is CN1346891; Hua Zhong Agriculture University has also carried out the screening of gamma-polyglutamic acid generating bacterium and the work of technique, and its bacterial classification and liquids production process have also applied for patent, and patent publication No. is CN1536071; Kubota is separated the strain Bacillus Subtilis F201 obtained in Osaka City University soil, this bacterial strain can reach the production peak of 50g/L under optimal conditions of fermentation, this is the production peak (Kubota H.Biosci Biotec Biochem, 1993:1212 ~ 1213) of bibliographical information.Although the fermentative production of above gamma-polyglutamic acid-achieves larger progress, still there is the production cycle longer, production efficiency is low, the problem that production cost is higher.Therefore, high yield, low cost preparing gamma-polyglutamic acid still need to be studied further.
Summary of the invention
Technical problem to be solved by this invention is to provide the high yield synthesis bacterial strain of a strain gamma-polyglutamic acid-, and this bacterial strain can synthesize gamma-polyglutamic acid-in a large number.Another technical problem that the present invention will solve is to provide a kind of synthesis bacterium plant height of gamma-polyglutamic acid-that utilizes and imitates the method preparing gamma-polyglutamic acid-.
In order to solve the problems of the technologies described above, the following technical scheme of the present invention:
A kind of synthesis gamma-polyglutamic acid-bacterial strain, screen from fertilizer plant's surrounding soil and obtain, Classification And Nomenclature is subtilis NS-9, i.e. Bacillus Subtilis NS-9, China typical culture collection center deposit number CCTCC NO:M2014142, preservation date on April 23rd, 2014, preservation address is China-Wuhan-Wuhan University, postcode 430072, telephone number (027) 68754052, contact fax (027) 68754833, contact mailbox cctccwhu.edu.cn.
16s rDNA sequential analysis: record 16s rDNA major part sequence 1600bp, see sequence table, by check order arrange compare from the relevant kind GenBank database, build the phylogenetic tree based on 16s rDNA complete sequence.Result shows that this bacterial strain and subtilis (Bacillus Subtilis) homology are 99%.The phylogeny result of study that combining form, cytochemistry composition and 16s rDNA complete series are analyzed, can be subtilis by its classification and orientation, be specially subtilis NS-9, i.e. Bacillus Subtilis NS-9.
Utilize the bacterial strain of above-mentioned synthesis gamma-polyglutamic acid-efficiently to prepare the method for gamma-polyglutamic acid-, step is as follows:
(1) seed culture: choose single colony inoculation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water from flat board, shaking speed 150 ~ 200rpm, cultivates 3 ~ 15h to mid log phase at 30 ~ 37 DEG C;
(2) fermentation culture: seed solution access step (1) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 1 ~ 10%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 10 ~ 100ml/ shaking flask volume 200ml, shaking speed 150 ~ 200rpm, cultivates 24 ~ 48h at 30 ~ 37 DEG C; Carbon source to be added in time when residual sugar content is low to moderate 1-2g/L in fermenting process;
(3) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, centrifugal rotational speed 8000 ~ 10000r/m;
2. micro-filtration removal of impurities: adopt microfiltration membrane filtering thalline and small molecular weight impurity;
3. product precipitation: directly adopt organic solvent to precipitate, collecting precipitation, vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Wherein, in seed culture medium or fermention medium, carbon source is the combination of any one or a few the arbitrary proportion in glucose, sucrose, maltose, citric acid; Preferred carbon source is the combination of any one or a few the arbitrary proportion in glucose, sucrose, maltose; Preferred carbon source is sucrose; Nitrogenous source be organic nitrogen source, inorganic nitrogen-sourced in the combination of one or both arbitrary proportion, organic nitrogen source is the combination of any one or a few the arbitrary proportion in peptone, extractum carnis, yeast powder, corn steep liquor, inorganic nitrogen-sourced is the combination of the arbitrary proportion of any one or two kinds in ammonium chloride, ammonium sulfate, and above-mentioned nitrogenous source can be used alone and also can compositely use; Preferred nitrogenous source is the combination of any one or a few arbitrary proportion in peptone, yeast powder, extractum carnis; Glutaminate is adopt to add the combination containing any one or a few the arbitrary proportion in L-glutamic acid material such as monosodium glutamate, L-glutamic acid, Sodium Glutamate; Inorganic salt are K
2hPO
43H
2o, MgSO
4, NH
4cl, CaCl
22H
2o, MnSO
4h
2the combination of any one or a few the arbitrary proportion in O;
Wherein fermention medium and substratum comprise following density component: carbon source 40 ~ 80g/L, nitrogenous source 5 ~ 10g/L, Sodium Glutamate 70 ~ 90g/L, inorganic salt 0.25 ~ 6g/L, and all the other are water, and pH value is 7.0 ~ 7.5;
In step (3), described microfiltration membrane aperture is 0.22 ~ 0.65um;
In step (3), the organic solvent of employing is one of ethanol, methyl alcohol, propyl alcohol, acetone or multiple combination;
The activation of seed slant culture was also had before step (1) seed culture, the synthesis gamma-polyglutamic acid-bacterial strain NS-9 of cryopreservation is inoculated in slant medium activation, cultivate 20h for 37 DEG C, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0.
Compare with existing production gamma-polyglutamic acid-technology, the microorganism Bacillus subtilis bacterial strain that the present invention uses can the fermentative production gamma-polyglutamic acid-of rapid, high volume, can fermentative production 50g/L product in 24h fermentation period; By the optimization to bacillus subtilis strain culture condition, particularly add carbon source, inorganic salt and energy matter during the fermentation, make the content of the gamma-polyglutamic acid-in fermented liquid up to 100 ~ 200g/L, thus provide a kind of high efficiency low cost to prepare the method for gamma-polyglutamic acid-.
Embodiment
Technical solution of the present invention is described in detail, content that the present invention may be better understood by following embodiment.For the ease of those skilled in the art will readily understand, concrete material proportion, processing condition and result thereof described in embodiment only for illustration of the present invention, and also should can not limit content required for protection in claims of the present invention.
Embodiment 1
A kind of bacterial strain synthesizing gamma-polyglutamic acid-, screen from fertilizer plant's surrounding soil and obtain, Classification And Nomenclature is subtilis NS-9, i.e. Bacillus Subtilis NS-9, China typical culture collection center deposit number CCTCC NO:M2014142, preservation date on April 23rd, 2014.
16s rDNA sequential analysis: record 16s rDNA major part sequence 1600bp, by check order arrange compare from the relevant kind GenBank database, build the phylogenetic tree based on 16s rDNA complete sequence.Result shows that this bacterial strain and subtilis (Bacillus Subtilis) homology are 99%.The phylogeny result of study that combining form, cytochemistry composition and 16s rDNA complete series are analyzed, can be subtilis by its classification and orientation, be specially subtilis (Bacillus Subtilis) NS-9.
Embodiment 2
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 20ml/ shaking flask volume 250ml, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 30 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 4 times of volume dehydrated alcohols to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 140g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 3
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 20ml/ shaking flask volume 250ml, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is maltose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 30 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 4 times of volumes methanol to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 150g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 4
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 20ml/ shaking flask volume 250ml, shaking speed 220rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, extractum carnis: 5 g/L, Sodium Glutamate: 30 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 4 times of volume propyl alcohol to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 170g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 5
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 20ml/ shaking flask volume 250ml, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 30 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, NaCl:10g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 10000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 4 times of vol acetone to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 100g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 6
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 20ml/ shaking flask volume 250ml, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 80 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 2 times of volume dehydrated alcohols and 2 times of vol acetone to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 200g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 7
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 12h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 200ml/ shaking flask volume 2L, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 80 g/L, K2HPO43H2O:2 g/L, MgSO4:0.25 g/L, NH4Cl:3 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 2 times of volume dehydrated alcohols and 2 times of volumes methanol to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 110g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 8
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 200ml/ shaking flask volume 2L, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is sucrose: 40g/L, peptone: 5 g/L, Sodium Glutamate: 80 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, MnSO
4h
2o:2.5 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 2 times of volume dehydrated alcohols and 2 times of volume propyl alcohol to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 200g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 9
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 200ml/ shaking flask volume 2L, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is maltose: 40g/L, extractum carnis: 5 g/L, Sodium Glutamate: 80 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, MnSO
4h
2o:2.5 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 2 times of volumes methanol and 2 times of vol acetone to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 220g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
Embodiment 10
Utilize the synthesis bacterium plant height of the gamma-polyglutamic acid-of embodiment 1 to imitate the method preparing gamma-polyglutamic acid-, step is as follows:
(1) slant culture: embodiment 1 inoculation of cryopreservation activated in slant medium, cultivates 20h, slant medium component: peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0 for 37 DEG C;
(2) seed culture: by the inoculation after activation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water, shaking speed 200rpm, cultivates 12h to mid log phase at 37 DEG C; Seed culture medium component is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH value 7.0,121 DEG C of sterilizings 20 minutes;
(3) fermentation culture: seed liquor access step (2) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 2%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 200ml/ shaking flask volume 2L, shaking speed 200rpm, cultivates 24h at 37 DEG C; Carbon source is added in time when residual sugar content is very low in fermenting process; Fermention medium component is maltose: 40g/L, extractum carnis: 5 g/L, Sodium Glutamate: 80 g/L, K
2hPO
43H
2o:2 g/L, MgSO
4: 0.25 g/L, NH
4cl:3 g/L, MnSO
4h
2o:2.5 g/L, pH value 7.0,115 DEG C of sterilizings 15 minutes;
(4) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, and centrifugal rotational speed 8000rpm, gets supernatant liquor;
2. micro-filtration removal of impurities: microfiltration membrane filtering thalline and small molecular weight impurity are adopted to supernatant liquor, bacterium liquid must be removed; Again by going the ultra-filtration membrane of bacterium liquid molecular weight cut-off 60KDa to concentrate, obtain concentrated solution;
3. product precipitation: concentrated solution adopts 1 times of volume dehydrated alcohol, 1 vol acetone, 1 times of volumes methanol and 1 times of volume propyl alcohol to precipitate, collecting precipitation, and throw out is gamma-polyglutamic acid-, and weight in wet base content about reaches 280g/1L fermented liquid; Again by throw out vacuum-drying, obtained gamma-polyglutamic acid-finished product.
SEQUENCE LISTING
<110> Zhangshu City Shi Wang bio tech ltd
<120> mono-kind synthesizes the bacterial strain of gamma-polyglutamic acid-and utilizes it efficiently to prepare the method for gamma-polyglutamic acid-
<130> 12
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1610
<212> DNA
<213> bacillus subtilis 16S rDNA
<400> 1
tgcactgggt cgtgattcga gctcggtacc cggggatcct ctagagatta gagtttgatc 60
atggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg gacagatggg 120
agcttgctcc ctgatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa 180
gactgggata actccgggaa accggggcta ataccggatg gttgtttgaa ccgcatggtt 240
caaacataaa aggtggcttc ggctaccact tacagatgga cccgcggcgc attagctagt 300
tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg gtgatcggcc 360
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 420
aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 480
gctctgttgt tagggaagaa caagtaccgt tcgaataggg cggtaccttg acggtaccta 540
accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 600
tgtccggaat tattgggcgt aaagggctcg caggcggttt cttaagtctg atgtgaaagc 660
ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag 720
tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 780
cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 840
ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct 900
tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 960
tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 1020
gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt 1080
cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1140
taagtcccgc aacgagcgca acccttgatc ttagttgcca gcatttagtt gggcactcta 1200
aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1260
tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg 1320
ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg 1380
aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1440
gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt 1500
ttaggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa caaggtaacc 1560
gtaaatcgtc gacctgcagg catgcaagct ggcgtaatca tgtaaattgc 1610
Claims (10)
1. a synthesis gamma-polyglutamic acid-bacterial strain, screen from fertilizer plant's surrounding soil and obtain, Classification And Nomenclature is subtilis NS-9, i.e. Bacillus Subtilis NS-9, China typical culture collection center deposit number CCTCC NO:M2014142, preservation date on April 23rd, 2014.
2. use the bacterial strain of claim 1 efficiently to prepare the method for gamma-polyglutamic acid-, step is as follows:
(1) seed culture: choose single colony inoculation in the aseptic seed substratum containing carbon source, nitrogenous source, glutaminate, inorganic salt and water from flat board, shaking speed 150 ~ 200rpm, cultivates 3 ~ 15h to mid log phase at 30 ~ 37 DEG C;
(2) fermentation culture: seed solution access step (1) obtained is containing in the sterile flasks fermention medium of carbon source, nitrogenous source, glutaminate, inorganic salt and water, inoculum size 1 ~ 10%(seed solution v/ fermentation culture based sols v), shaking flask liquid amount 10 ~ 100ml/ shaking flask volume 200ml, shaking speed 150 ~ 200rpm, cultivates 24 ~ 48h at 30 ~ 37 DEG C; Carbon source to be added in time when residual sugar content is low to moderate 1-2g/L in fermenting process;
(3) extraction and purification of gamma-polyglutamic acid-:
1. fermented liquid is degerming: centrifugal 10min at fermented liquid being down to 4 DEG C, centrifugal rotational speed 8000 ~ 1000r/m;
2. micro-filtration removal of impurities: adopt microfiltration membrane filtering thalline and small molecular weight impurity;
3. product precipitation: directly adopt organic solvent to precipitate, collecting precipitation, vacuum-drying, obtained gamma-polyglutamic acid-finished product.
3. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 2, it is characterized in that, fermention medium and seed culture medium comprise following density component: carbon source 40 ~ 80g/L, nitrogenous source 5 ~ 10g/L, Sodium Glutamate 70 ~ 90g/L, inorganic salt 0.25 ~ 6g/L, all the other are water, and pH value is 7.0 ~ 7.5.
4. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 3, it is characterized in that, in seed culture medium or fermention medium, carbon source is the combination of any one or a few the arbitrary proportion in glucose, sucrose, maltose, citric acid; Nitrogenous source be organic nitrogen source, inorganic nitrogen-sourced in the combination of one or both arbitrary proportion, organic nitrogen source is the combination of any one or a few the arbitrary proportion in peptone, extractum carnis, yeast powder, corn steep liquor, and inorganic nitrogen-sourced is the combination of the arbitrary proportion of any one or two kinds in ammonium chloride, ammonium sulfate; Glutaminate is adopt to add the combination containing any one or a few the arbitrary proportion in L-glutamic acid material such as monosodium glutamate, L-glutamic acid, Sodium Glutamate; Inorganic salt are K
2hPO
43H
2o, MgSO
4, NH
4cl, CaCl
22H
2o, MnSO
4h
2the combination of any one or a few the arbitrary proportion in O.
5. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 4, it is characterized in that, preferred carbon source is the combination of any one or a few the arbitrary proportion in glucose, sucrose, maltose.
6. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 4, it is characterized in that, preferred carbon source is sucrose.
7. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 4, it is characterized in that, preferred nitrogenous source is the combination of any one or a few arbitrary proportion in peptone, yeast powder, extractum carnis.
8. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 1 or 2, it is characterized in that, in step (3), described microfiltration membrane aperture is 0.22 ~ 0.65um.
9. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 1 or 2, it is characterized in that, in step (3), the organic solvent of employing is one of ethanol, methyl alcohol, propyl alcohol, acetone or multiple combination.
10. efficiently prepare the method for gamma-polyglutamic acid-as claimed in claim 1 or 2, it is characterized in that, the activation of seed slant culture was also had before step (1) seed culture, the synthesis gamma-polyglutamic acid-bacterial strain NS-9 of cryopreservation is inoculated in slant medium activation, cultivates 20h, slant medium component: peptone 10g/L for 37 DEG C, yeast powder 5g/L, sodium-chlor 10g/L, agar powder 15g/L, pH value 7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410327118.5A CN104232504A (en) | 2014-07-10 | 2014-07-10 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410327118.5A CN104232504A (en) | 2014-07-10 | 2014-07-10 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104232504A true CN104232504A (en) | 2014-12-24 |
Family
ID=52221414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410327118.5A Pending CN104232504A (en) | 2014-07-10 | 2014-07-10 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104232504A (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282252A (en) * | 2015-06-01 | 2017-01-04 | 山东建筑大学 | A kind of high density fermentation prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid- |
| CN106591190A (en) * | 2016-12-16 | 2017-04-26 | 大连理工大学 | Bacillus and application in preparing Gama-polyglutamic acid |
| CN106589350A (en) * | 2016-11-10 | 2017-04-26 | 北京赛诺膜技术有限公司 | Method for refining small molecule polyglutamic acid by using ultra-filtration technology |
| CN106834190A (en) * | 2017-03-11 | 2017-06-13 | 鲁东大学 | Culture medium, bacterial strain and the method for polyglutamic acid are produced with pea protein wastewater fermentation |
| CN106947724A (en) * | 2017-05-22 | 2017-07-14 | 中国科学院成都生物研究所 | A kind of method of increase γ polyglutamic acid zymotic fluid dissolved oxygens |
| CN107287254A (en) * | 2017-07-14 | 2017-10-24 | 武汉乐阳生物科技有限公司 | The zymotechnique of γ polyglutamic acids |
| CN108752625A (en) * | 2018-06-19 | 2018-11-06 | 广州无添加主义化妆品有限公司 | The method for preparing different molecular weight polyglutamic acid |
| CN109182404A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
| CN109234328A (en) * | 2017-07-10 | 2019-01-18 | 北京化工大学 | A method of producing gamma-polyglutamic acid |
| CN109943495A (en) * | 2018-09-05 | 2019-06-28 | 浙江惠嘉生物科技股份有限公司 | One plant height produces bacillus subtilis and its fermentation process of gamma-polyglutamic acid |
| CN110373346A (en) * | 2019-06-10 | 2019-10-25 | 上海市农业科学院 | Bacillus subtilis Bacillus sp.A-5 and application thereof |
| CN110628688A (en) * | 2016-12-23 | 2019-12-31 | 南京轩凯生物科技有限公司 | Bacillus subtilis for producing gamma-polyglutamic acid and application thereof |
| CN113073121A (en) * | 2021-04-06 | 2021-07-06 | 江苏南创化学与生命健康研究院有限公司 | Nano carbon material containing high-polymerization-degree polyphosphate and preparation method of high-polymerization-degree polyphosphate |
| CN115058352A (en) * | 2022-03-28 | 2022-09-16 | 四川师范大学 | Bacillus subtilis and method for producing agricultural gamma-polyglutamic acid by using same |
| CN115261419A (en) * | 2022-07-23 | 2022-11-01 | 广州市博之越精细化工有限公司 | Preparation method of gamma-polyglutamic acid sodium solution |
| CN115820758A (en) * | 2023-02-07 | 2023-03-21 | 山东嘉禾海洋生物科技有限公司 | Polyglutamic acid fermentation liquor and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644677A (en) * | 2004-12-29 | 2005-07-27 | 浙江大学 | Bacillus and its use of preparation of gama-polycysteine |
| CN1908178A (en) * | 2005-08-06 | 2007-02-07 | 加拿大时代科技投资管理有限公司 | Method of low cost preparing gamma-polyglutamic acid utilizing farm and side line products solid fermentation |
| 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 |
| CN103881954A (en) * | 2012-12-20 | 2014-06-25 | 山东福瑞达生物科技有限公司 | Gamma-polyglutamic acid production gene engineering bacterial and method for producing high-yield gamma-polyglutamic acid through gamma-polyglutamic acid production gene engineering bacterial |
-
2014
- 2014-07-10 CN CN201410327118.5A patent/CN104232504A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644677A (en) * | 2004-12-29 | 2005-07-27 | 浙江大学 | Bacillus and its use of preparation of gama-polycysteine |
| CN1908178A (en) * | 2005-08-06 | 2007-02-07 | 加拿大时代科技投资管理有限公司 | Method of low cost preparing gamma-polyglutamic acid utilizing farm and side line products solid fermentation |
| 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 |
| CN103881954A (en) * | 2012-12-20 | 2014-06-25 | 山东福瑞达生物科技有限公司 | Gamma-polyglutamic acid production gene engineering bacterial and method for producing high-yield gamma-polyglutamic acid through gamma-polyglutamic acid production gene engineering bacterial |
Non-Patent Citations (3)
| Title |
|---|
| JIN HUANG等: "High yield and cost-effective production of poly(c-glutamic acid) with Bacillus subtilis", 《ENGINEERING IN LIFE SCIENCES》 * |
| 刘晓鸥等: "枯草芽孢杆菌TKPG011聚谷氨酸发酵培养基的优化", 《现代食品科技》 * |
| 吕萌等: "补料发酵枯草芽孢杆菌合成γ-聚谷氨酸的研究", 《生物工程》 * |
Cited By (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282252A (en) * | 2015-06-01 | 2017-01-04 | 山东建筑大学 | A kind of high density fermentation prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid- |
| CN106589350A (en) * | 2016-11-10 | 2017-04-26 | 北京赛诺膜技术有限公司 | Method for refining small molecule polyglutamic acid by using ultra-filtration technology |
| CN106591190A (en) * | 2016-12-16 | 2017-04-26 | 大连理工大学 | Bacillus and application in preparing Gama-polyglutamic acid |
| CN106591190B (en) * | 2016-12-16 | 2019-07-16 | 大连理工大学 | A kind of bacillus and its preparing the application in gamma-polyglutamic acid |
| CN110628688A (en) * | 2016-12-23 | 2019-12-31 | 南京轩凯生物科技有限公司 | Bacillus subtilis for producing gamma-polyglutamic acid and application thereof |
| CN106834190A (en) * | 2017-03-11 | 2017-06-13 | 鲁东大学 | Culture medium, bacterial strain and the method for polyglutamic acid are produced with pea protein wastewater fermentation |
| CN106947724A (en) * | 2017-05-22 | 2017-07-14 | 中国科学院成都生物研究所 | A kind of method of increase γ polyglutamic acid zymotic fluid dissolved oxygens |
| CN106947724B (en) * | 2017-05-22 | 2020-05-19 | 中国科学院成都生物研究所 | Method for increasing dissolved oxygen of gamma-polyglutamic acid fermentation liquor |
| CN109234328B (en) * | 2017-07-10 | 2022-10-21 | 北京化工大学 | Method for producing gamma-polyglutamic acid |
| CN109234328A (en) * | 2017-07-10 | 2019-01-18 | 北京化工大学 | A method of producing gamma-polyglutamic acid |
| CN107287254A (en) * | 2017-07-14 | 2017-10-24 | 武汉乐阳生物科技有限公司 | The zymotechnique of γ polyglutamic acids |
| CN107287254B (en) * | 2017-07-14 | 2021-06-29 | 武汉光华时代生物科技有限公司 | Fermentation process of gamma-polyglutamic acid |
| CN108752625A (en) * | 2018-06-19 | 2018-11-06 | 广州无添加主义化妆品有限公司 | The method for preparing different molecular weight polyglutamic acid |
| CN109943495A (en) * | 2018-09-05 | 2019-06-28 | 浙江惠嘉生物科技股份有限公司 | One plant height produces bacillus subtilis and its fermentation process of gamma-polyglutamic acid |
| CN109182404A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
| CN110373346A (en) * | 2019-06-10 | 2019-10-25 | 上海市农业科学院 | Bacillus subtilis Bacillus sp.A-5 and application thereof |
| CN110373346B (en) * | 2019-06-10 | 2023-05-23 | 上海市农业科学院 | Bacillus subtilis sp.A-5 and application thereof |
| CN113073121A (en) * | 2021-04-06 | 2021-07-06 | 江苏南创化学与生命健康研究院有限公司 | Nano carbon material containing high-polymerization-degree polyphosphate and preparation method of high-polymerization-degree polyphosphate |
| CN113073121B (en) * | 2021-04-06 | 2023-05-05 | 江苏南创化学与生命健康研究院有限公司 | Nanocarbon material containing high-polymerization-degree polyphosphate and preparation method of high-polymerization-degree polyphosphate |
| CN115058352A (en) * | 2022-03-28 | 2022-09-16 | 四川师范大学 | Bacillus subtilis and method for producing agricultural gamma-polyglutamic acid by using same |
| CN115058352B (en) * | 2022-03-28 | 2023-08-11 | 四川师范大学 | Bacillus subtilis and method for producing agricultural gamma-polyglutamic acid by using same |
| CN115261419A (en) * | 2022-07-23 | 2022-11-01 | 广州市博之越精细化工有限公司 | Preparation method of gamma-polyglutamic acid sodium solution |
| CN115261419B (en) * | 2022-07-23 | 2023-07-21 | 广州市博之越精细化工有限公司 | Preparation method of gamma-sodium polyglutamate solution |
| CN115820758A (en) * | 2023-02-07 | 2023-03-21 | 山东嘉禾海洋生物科技有限公司 | Polyglutamic acid fermentation liquor and preparation method and application thereof |
| CN115820758B (en) * | 2023-02-07 | 2023-04-14 | 山东嘉禾海洋生物科技有限公司 | Polyglutamic acid fermentation liquor and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104232504A (en) | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain | |
| CN101948785B (en) | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium | |
| CN106635869B (en) | A method of producing surfactin using bacillus amyloliquefaciens | |
| CN100410364C (en) | Gamma-polyglutamic acid generating bacterium and process of preparing gamma-polyglutamic acid therewith | |
| CN102965311B (en) | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid | |
| CN109161492A (en) | A kind of gamma-polyglutamic acid generating bacterium and the method for efficiently synthesizing gamma-polyglutamic acid | |
| CN102911974B (en) | Method for producing gamma-polyglutamic acid though hot fermentation of bacillus subtilis | |
| CN101875910A (en) | Bacillus amyloliquefaciens for producing gamma-polyglutamic acid | |
| CN110016446A (en) | One plant of Bei Laisi bacillus and its method for producing polyglutamic acid | |
| CN101838619A (en) | Mutagenized strain Bacillus licheniformis TKPG091 for generating great amount of gamma-poly glutamic acid | |
| CN102533885B (en) | Method for producing gamma-polyglutamic acid through adding sodium chloride (NaCl) in fermentation process | |
| CN107118973B (en) | A method of utilizing micro-organisms parahydroxyben-zaldehyde | |
| CN104694590A (en) | Method for producing gamma-polyglutamic acid by fermenting and bacterial strain for producing gamma-polyglutamic acid | |
| CN106434475B (en) | One streptomycete category polysaccharide degradation bacteria and its cultural method and application | |
| CN1346891A (en) | Process for prepering gamma-polyglutamic acid and polyglutamates | |
| CN103255067B (en) | Aureobasidium Pullulan producing pullulan with high yield by utilizing xylose and application of Aureobasidium Pullulan | |
| CN108220352A (en) | A kind of method of raw material fermentation production gamma-polyglutamic acid | |
| CN102911896B (en) | 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 | |
| CN110129225A (en) | γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method | |
| CN103820508A (en) | Application of rare earth element in production of gamma-polyglutamic acid by bacillus licheniformis fermentation | |
| CN107760732A (en) | A kind of production method of agriculture level γ polyglutamic acids | |
| CN101701237B (en) | Method for producing alpha-glucosyl eugenol by fermentation | |
| CN106631440A (en) | Compound microbial organic fertilizer for taxus chinensis as well as preparation method and application thereof | |
| CN102206597B (en) | Gamma-polyglutamic acid synthetic bacteria and fermentation method thereof | |
| CN101643712A (en) | Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141224 |
|
| RJ01 | Rejection of invention patent application after publication |