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Bacillus subtilis and application thereof in production of gamma-polyglutamic acid

A technology of Bacillus subtilis and polyglutamic acid, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of increasing the ionic strength of the medium, high viscosity of the fermentation liquid, complex process, etc., and improve the efficiency of the enzymatic reaction , increase the concentration of dissolved oxygen, improve the effect of accumulation

Active Publication Date: 2020-03-24
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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AI Technical Summary

Problems solved by technology

The disadvantage is that the molecular weight of the product is only 750kDa~1500kDa, and the multi-strain mixed fermentation process is complex, the fermentation conditions are not easy to control when the two strains grow in harmony, the dominant strains are difficult to control during the fermentation process, and the yield is unstable. Large-scale production requires further research
[0006] Chinese patent CN103710404B discloses a production method of high molecular weight γ-polyglutamic acid, using Bacillus licheniformis CGMCC NO. Add glucose, calcium chloride, manganese sulfate, magnesium sulfate and other feed solutions to control the fermentation pH and increase the ionic strength of the medium to obtain γ-PGA with a molecular weight of 1000kDa~4000kDa, but the molecular weight range of the product obtained by this method is still scattered and small , to a certain extent, cannot satisfy the application of γ-PGA in the field of polymer material technology
[0007] Chinese patent CN101597627B discloses a production method of polymer γ-polyglutamic acid, which is characterized in that the production rate of γ-PGA is increased to 31.8g / L by using Bacillus natto through nutrient depletion and external environmental stress. It can reach more than 3000kDa, the extraction process is complicated, and it is impossible to achieve large-scale production
The content of PGA in this patent is relatively low, and a dialysis step is required, and the process is complicated, which is not conducive to large-scale production
And the molecular weight detection of this patent uses the GPC method, the detection range of molecular weight of this method is small, the accuracy and stability are not as good as the multi-angle laser light scattering method, and compared with the multi-angle laser light scattering method, the measured molecular weight is higher
[0009] At present, the fermentation production process and yield of low molecular weight γ-PGA (below 1000kDa) have been extensively studied at home and abroad. Shortcomings lead to less research on high molecular weight γ-PGA, especially ultra-high molecular weight (3000kDa~6000kDa) γ-PGA, which seriously hinders the development and application of γ-PGA in the field of polymer material technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Screening, separation and purification and identification of embodiment 1 bacterial classification

[0061] The Bacillus subtilis NT-11 CCTCC NO: M 2019383 of the present invention is obtained by screening from orchard soil rich in organic matter, and its specific acquisition process is as follows:

[0062] (1) Collection of samples for sieving: from forests, farmland, orchards, vegetable gardens and other places rich in organic soil, scrape appropriate amount of soil samples with a sterile shovel, and place them in sterile glass bottles; from commercially available natto, bean paste , fermented soy sauce, soy sauce and other products, use a sterile spoon or dropper to collect an appropriate amount of samples and place them in a sterile glass bottle.

[0063] (2) Primary screening and separation and purification of strains:

[0064] Properly dissolve or dilute the collected samples with sterile water, filter with sterile filter paper, remove large particles of impuriti...

Embodiment 2

[0069] (1) Strain activation: Under sterile conditions, streak and inoculate Bacillus subtilis NT-11 CCTCC NO: M 2019383 on the plate medium, activate and culture at 35°C for 48 hours, pick a single colony and inoculate it on the slant medium of the test tube On the 35°C, the activation culture was continued for 48 hours to obtain fully activated slant seeds in test tubes.

[0070] The composition of plate medium and test tube slant medium is: glucose 5g / L, sodium glutamate 5 / L, peptone 20g / L, magnesium sulfate 0.1g / L, dipotassium hydrogen phosphate 1g / L, agar 18g / L, pH6.0, water balance.

[0071] (2) Seed liquid preparation: Under sterile conditions, pick fully activated test tube slant seeds and inoculate them in a 500 mL Erlenmeyer flask containing 150 mL of liquid seed medium, and incubate with shaking at 150 r / min and 35 °C for 40 h to obtain seed liquid.

[0072] The composition of the liquid seed medium is: glucose 5g / L, sodium glutamate 5g / L, peptone 20g / L, magnesium ...

Embodiment 3

[0077] (1) Strain activation: Under sterile conditions, streak and inoculate Bacillus subtilis NT-11 CCTCC NO: M 2019383 on a specific plate medium, activate and culture at 36°C for 20 hours, pick a single colony and inoculate it on the slant of a specific test tube On the culture medium, the activation culture was continued at 36° C. for 20 h to obtain fully activated test tube slant seeds.

[0078] Plate medium and test tube slant medium are composed of: glucose 15g / L, sodium glutamate 10g / L, peptone 5g / L, magnesium sulfate 0.3g / L, dipotassium hydrogen phosphate 0.8g / L, agar 20g / L, pH6.5, water balance.

[0079] (2) Preparation of seed solution: under sterile conditions, pick fully activated slanted seeds of test tubes and inoculate them into a 500 mL Erlenmeyer flask containing 180 mL of liquid seed medium, and incubate with shaking at 200 r / min at 36°C for 20 hours to obtain a seed solution.

[0080] The composition of liquid seed medium is: glucose 15g / L, sodium glutamat...

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Abstract

The invention discloses bacillus subtilis and an application of bacillus subtilis in production of gamma-polyglutamic acid. Bacillus subtilis NT-11 (Bacillus subtilis) CCTCC NO: M 2019383 is taken asa fermentation strain, and glutamine is taken as a precursor. pH, stirring speed, tank pressure and ventilation quantity are regulated and controlled in the middle stage of fermentation, so that morebeneficial conditions are provided for the fermentation process. Thallus growth is promoted, and ultrahigh molecular weight gamma-polyglutamic acid is synthesized. The fermentation broth is subjectedto alcohol precipitation, redissolution, centrifugation, filtration, alcohol precipitation again, drying and other extraction processes to obtain an ultrahigh molecular weight gamma-polyglutamic acidproduct, and the molecular weight of the ultrahigh molecular weight gamma-polyglutamic acid product is detected to be 3000 to 6000 kDa. According to the invention, the method is simple in process, easy to operate, low in cost and high in yield; the prepared polyglutamic acid is high in purity, clear in molecular weight, ultrahigh, relatively stable and controllable; and industrial large-scale production can be achieved.

Description

technical field [0001] The present invention relates to a kind of Bacillus subtilis ( Bacillus subtilis ), and also relates to a fermentation production method of gamma-polyglutamic acid, in particular to a fermentation production method of ultra-high molecular weight gamma-polyglutamic acid, belonging to the technical field of gamma-polyglutamic acid preparation. Background technique [0002] γ-polyglutamic acid (γ-PGA for short) is composed of L-glutamic acid (L-Glutamic acid), D-glutamic acid (D-glutamic acid) through glutamic acid monomer α-amino and Polyamino acid compound formed by γ-carboxyl cross-linking. It is a non-toxic, harmless to the environment and human amino acid polymer, its molecular weight is generally between 100kDa ~ 1000kDa, equivalent to about 500 ~ 5000 glutamic acid monomers. The main chain of γ-PGA contains a large number of free carboxyl groups, which can undergo various reactions such as cross-linking, chelation, and derivatization. It also has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/02C12R1/125
CPCC12N1/20C12P13/02C12N1/205C12R2001/125
Inventor 孙元军陆震魏玉洁陈雯雯石艳丽栾贻宏郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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