CN115820508A - Bacillus licheniformis and application thereof in preparation of low-molecular-weight gamma-polyglutamic acid - Google Patents
Bacillus licheniformis and application thereof in preparation of low-molecular-weight gamma-polyglutamic acid Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to bacillus licheniformis and application thereof in preparation of low-molecular-weight gamma-polyglutamic acid. The invention provides a Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m20221810. The bacillus licheniformis strain can efficiently produce gamma-polyglutamic acid, and the produced gamma-polyglutamic acid is low-molecular-weight gamma-polyglutamic acid. The low molecular weight gamma-polyglutamic acid produced by the strain can be used in the fields of cosmetics, foods and the like, can also be used as a freeze-drying protective agent for microorganisms, can effectively protect bacteria and fungi in the freeze-drying process, and reduces the damage to the microorganisms in the freeze-drying process.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus licheniformis and application thereof in preparation of low-molecular-weight gamma-polyglutamic acid.
Background
Gamma-polyglutamic acid (gamma-PGA) is a biopolymer carrier material, is firstly found in a clamping membrane of bacillus anthracis by Ivanovics and the like, is a secondary metabolite generated by microorganisms (such as bacillus subtilis, bacillus licheniformis and the like) of bacillus in the process of life activity, and can be prepared by a microbial fermentation method.
The molecular weight (Mw) of γ -PGA is mainly 10-10000kDa. γ -PGA of different molecular weights have different functions and application fields, for example: the low molecular weight gamma-PGA (Mw <400 kDa) can improve food mouthfeel, keep skin moist and smooth when being smeared on the skin, can be used in the fields of food and cosmetics, and can be used as a drug carrier for targeting drug delivery to cancer cells and releasing encapsulated active ingredients thereof to inhibit tumor growth; the high molecular weight gamma-PGA (Mw >1 000kDa) has higher viscosity, can be used as a flocculating agent for adsorbing heavy metals in water, and can be used as a thickening agent for preparing biological materials and the like. Currently, the application and the demand of low molecular weight gamma-PGA in the fields of food, cosmetics and medical carriers are more extensive.
The characteristics of different γ -PGA-producing strains are different, and the γ -PGA production and relative molecular mass of the strains are also different. γ -PGA-producing bacteria can be classified into two main groups: bacillus and other species, and the commonly used γ -PGA-producing microorganisms are those of the genus Bacillus, in addition to E.coli which heterologously expresses γ -PGA-synthesizing genes. Among microorganisms of the genus Bacillus, bacillus licheniformis and Bacillus subtilis are commonly used as γ -PGA-producing strains.
At present, the preparation of low molecular weight γ -PGA is mainly achieved through two ways of intracellular regulation and in vitro regulation. Intracellular regulation is mainly through changes in fermentation conditions, such as: adding sorbitol and inorganic salt for stress and modifying the production strain by means of gene modification; however, the current genetic engineering approaches mainly aim at the production of γ -PGA, and the molecular weight is less and immature, for example: pgds and GTT, although capable of reducing molecular weight, may affect the yield of γ -PGA. The in vitro regulation is mainly to add gamma-PGA degrading enzyme to control the molecular weight of the gamma-PGA during fermentation, or to promote the degradation of the gamma-PGA by adopting an ultrasonic method and an acid-thermal degradation method, and has the defect that the hydrolytic molecular weight is difficult to control.
The gamma-PGA producing bacillus licheniformis strains disclosed in the prior art mainly include: the relative molecular mass of the gamma-PGA obtained by fermenting the bacillus licheniformis ATCC 9945A is between 500kDa and 800kDa, and the yield of the gamma-PGA is 5 to 17g/L; another strain of Bacillus lichniformis is fermented for 5 days, the relative molecular mass of gamma-PGA is 300kDa, and the yield is 8.5g/L; the molecular weight of the gamma-PGA synthesized by the Bacillus licheniformis WX-02 is 100-1200kDa. There is still a lack of a strain of bacillus licheniformis that produces gamma-PGA with a high yield and a low molecular weight.
Disclosure of Invention
The invention aims to provide a bacillus licheniformis strain, which can produce gamma-PGA with low molecular weight in high yield.
The invention takes Bacillus licheniformis (Bacillus licheniformis) which is obtained by screening in soil and produces gamma-polyglutamic acid as an initial strain, and carries out ion beam injection mutagenesis screening to obtain a Bacillus licheniformis strain with high yield of gamma-polyglutamic acid, wherein the strain can efficiently convert precursor glutamic acid into gamma-polyglutamic acid, and gamma-PGA produced by the strain is a low molecular weight product, the molecular weight is less than 265kDa, and the Bacillus licheniformis strain has an excellent effect in freeze-drying protection of microorganisms and can be used as a freeze-drying protective agent of the microorganisms.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides Bacillus licheniformis (Bacillus licheniformis) PGA-BL19, which has been deposited in the chinese typical culture collection center (CCTCC, address: wuhan, wuhan university, zip code 430072) at 11 months and 17 days 2022, with the preservation number being CCTCC NO: m20221810, classified and named Bacillus licheniformis.
The Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 has the characteristic of high yield of gamma-polyglutamic acid, the molecular weight of the gamma-polyglutamic acid produced by fermentation is concentrated below 265kDa, the low molecular weight gamma-polyglutamic acid belongs to the low molecular weight gamma-polyglutamic acid, and the Bacillus licheniformis (Bacillus licheniformis) has wide application in the fields of foods, cosmetics, drug carriers and the like and has excellent effect in the aspect of freeze-drying protection of microorganisms.
In a second aspect, the present invention provides a fermentation product, which is prepared by fermenting and culturing the above-mentioned Bacillus licheniformis (Bacillus licheniformis) PGA-BL19.
The fermentation product contains Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 or Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 and fermentation supernatant thereof.
In a third aspect, the present invention provides a microbial preparation comprising the above-mentioned Bacillus licheniformis (Bacillus licheniformis) PGA-BL19.
The Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 in the above microbial preparation is preferably in the form of viable bacteria.
The formulation of the microbial inoculum is not particularly limited, and can be a formulation commonly used in the field of microbial preparations, such as: solid microbial inoculum or liquid microbial inoculum, wherein the solid microbial inoculum can be lyophilized powder.
The microbial inoculum contains PGA-BL19 of Bacillus licheniformis (Bacillus licheniformis), and adjuvant or carrier acceptable in the field of microbial preparation.
In a fourth aspect, the present invention provides a method for preparing the microbial inoculum, wherein the method comprises a step of culturing Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 to obtain a bacterial solution.
Preferably, the culturing is carried out at 35-37 ℃.
The obtained bacterial liquid can be directly or added with auxiliary materials or carriers allowed in the field of microbial preparations to prepare liquid bacterial agents, and the bacterial bodies in the bacterial liquid can also be collected and mixed with auxiliary materials or carriers allowed in the field of microbial preparations such as freeze-drying protective agents, and the like, and then the mixture is subjected to vacuum freeze-drying to prepare freeze-dried bacterial powder.
In a fifth aspect, the present invention provides the application of the above-mentioned Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 or the microbial inoculum in the preparation of gamma-polyglutamic acid.
Preferably, the molecular weight of the gamma-polyglutamic acid is less than or equal to 300kDa.
Further preferably, the molecular weight of the gamma-polyglutamic acid is less than or equal to 270kDa.
Further preferably, the molecular weight of the gamma-polyglutamic acid is less than or equal to 265kDa.
In a sixth aspect, the invention provides the application of the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 or the microbial inoculum in preparing a microbial freeze-drying protective agent.
The microbial freeze-drying protective agent can also comprise one or more substances with the microbial freeze-drying protection function in addition to the gamma-polyglutamic acid produced by fermenting the PGA-BL19 of the Bacillus licheniformis (Bacillus licheniformis).
In a seventh aspect, the present invention provides a method for preparing gamma-polyglutamic acid, the method comprising: culturing the above-mentioned Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 to obtain a culture solution, and collecting the gamma-polyglutamic acid in the culture solution.
The temperature for the above culture is preferably 35 to 37 ℃. The pH is preferably 6.8 to 7.4. The rotational speed is preferably 100-280rpm.
The above culture is preferably performed under salt stress conditions.
The medium used for the above culture preferably comprises the following components: 130-150g/L of glucose or sucrose or glycerol, 30-40g/L of peptone or yeast powder or potassium nitrate, 1.5-2.5g/L of dipotassium hydrogen phosphate or potassium dihydrogen phosphate, 50-80g/L of sodium glutamate, 3-5g/L of citric acid monohydrate, 0.4-0.6g/L of magnesium sulfate or calcium chloride or manganese sulfate, 8-12g/L of sodium chloride and 6.5-7.2 of pH.
The gamma-polyglutamic acid in the culture solution can be collected by removing thalli from the culture solution, adding ethanol into the supernatant for precipitation, centrifugally collecting the precipitate, dialyzing and desalting to obtain the gamma-PGA. Wherein, the addition amount of ethanol is preferably 1-5 times of the supernatant.
In an eighth aspect, the invention provides a gamma-polyglutamic acid, wherein the molecular weight of the gamma-polyglutamic acid is less than or equal to 270kDa, the mass percentage of the gamma-polyglutamic acid with the molecular weight of 265kDa-35kDa is 86-97%, the mass percentage of the gamma-polyglutamic acid with the molecular weight of 18kDa or more and less than 35kDa is 1.5-2.5%, and the balance is the gamma-polyglutamic acid with the molecular weight of less than or equal to 10 kDa.
Preferably, the molecular weight of the gamma-polyglutamic acid is less than or equal to 265kDa, wherein the mass percentage of the gamma-polyglutamic acid with the molecular weight of 265kDa-35kDa is 94-97%, the mass percentage of the gamma-polyglutamic acid with the molecular weight of more than or equal to 18kDa and less than 35kDa is 1.5-2.0%, and the balance is the gamma-polyglutamic acid with the molecular weight of less than or equal to 10 kDa.
Preferably, the above-mentioned gamma-polyglutamic acid is produced by fermenting Bacillus licheniformis (Bacillus licheniformis) PGA-BL19.
Further preferably, the above-mentioned gamma-polyglutamic acid is prepared by the above-mentioned method for preparing gamma-polyglutamic acid.
In a ninth aspect, the present invention provides a microbial lyoprotectant, wherein the lyoprotectant comprises the above-mentioned gamma-polyglutamic acid.
The microbial lyoprotectant may further comprise one or more substances having a microbial lyoprotectant function, in addition to the gamma-polyglutamic acid.
The invention has the beneficial effects that: the bacillus licheniformis provided by the invention can efficiently produce gamma-polyglutamic acid, and the produced gamma-polyglutamic acid is low-molecular-weight gamma-polyglutamic acid.
The low molecular weight gamma-polyglutamic acid produced by the bacillus licheniformis can be used in the fields of cosmetics, foods and the like and can also be used as a freeze-drying protective agent for microorganisms. Experiments prove that the low molecular weight gamma-polyglutamic acid has a good protective effect on bacteria such as lactobacillus plantarum and the like in a freeze-drying process, can be applied to a probiotic freeze-dried powder preparation process, has an excellent protective effect on freeze-drying of fungi such as mortierella alpina and the like, and can be applied to strain preservation and a microbial inoculum preparation process.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 obtainment of high-yield Low-molecular-weight Gamma-PGA Bacillus licheniformis
The bacillus licheniformis bacterial suspension is coated on a sterile plate, is dried in sterile air for standby in a super clean bench, and is subjected to plasma mutagenesis on a nitrogen ion injection device in an important laboratory of ion beam bioengineering of Chinese academy of sciences. N is a radical of + Ion source implantation energy 15keV, implantation dose 10X 10 14 ion/cm 2 The implantation was performed in 5s pulses at an interval of 20s for a total of 15s, and the degree of vacuum in the target chamber was about 10 -3 Pa. The coated plate is placed on an implanter target table for ion beam implantation. After mutagenesis, the cells were eluted with 2mL of sterile water, diluted in gradient, plated on a plate containing a prescreening medium, and cultured at 37 ℃ for 16 hours. And selecting sticky colonies, activating by secondary seeds, and inoculating to a re-screening fermentation medium for preservation by using the inoculation amount of 1%. The strains obtained by primary screening were inoculated into a 24-well plate (liquid content 3mL/10 mL) containing a seed medium, 3 strains were arranged in parallel, and cultured at 37 ℃ at 250r/min for 12 hours. The seed solution was transferred to a 24-well plate (liquid loading 3mL/10 mL) containing a fermentation medium at 4% and cultured at 37 ℃ at 150r/min for 48 hours.
Measuring the yield and molecular weight of the gamma-PGA after fermentation, and screening strains of the gamma-PGA with high yield and low molecular weight.
The yield of γ -PGA and the molecular weight of γ -PGA were determined as follows:
determination of γ -PGA production: measured according to QB/T5189-2017;
gamma-PGA molecular weight determination: the molecular weight of γ -PGA was measured by aqueous Gel Permeation Chromatography (GPC) using three columns connected in series, and the column numbers used were: waters Ultrahydrogel TM 2000 (7.8 mm. Times.300 mm), waters Ultrahydrogel TM 250 (7.8 mm. Times.300 mm) and Waters Ultrahydrogel TM120 (7.8 mm. Times.300 mm). The detection mode is as follows: deionized water was used as the mobile phase, the flow rate was 0.6mL/min, the column temperature was 65 ℃ and the detection was performed by a differential refractive index detector (RI). And (3) taking the glucans with different molecular masses as standard samples, establishing a standard equation between the molecular mass and the retention time, and calculating the molecular weight of the gamma-PGA of the sample.
Continuously screening to obtain a bacillus licheniformis strain with high yield of gamma-PGA and lower molecular weight of the produced gamma-PGA, and naming the strain as PGA-BL19.
Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 has been preserved in China center for type culture Collection (CCTCC, address: wuhan, wuhan university, zip code 430072) in 2022 at 11 months and 17 days, with the preservation number of CCTCC NO: m20221810, classified and named Bacillus licheniformis.
EXAMPLE 2 fermentative culture of the Strain and preparation of Low molecular weight Gamma-PGA (1)
The method for producing the low molecular weight gamma-PGA by fermenting and culturing the Bacillus licheniformis PGA-BL19 comprises the following steps:
1. seed culture
Activating strains: inoculating Bacillus licheniformis PGA-BL19 on slant culture medium, culturing at 37 deg.C and humidity of 60% for 16-18h, and preparing mature slant seed.
The slant culture medium comprises the following components: 60g/L of sucrose, 60g/L of peptone, 80g/L of sodium glutamate, 10g/L of sodium chloride, 1g/L of magnesium sulfate, 1g/L of calcium chloride and 15g/L of agar, wherein the pH value is 6.5, and the sterilization is carried out for 20min at 115 ℃.
Preparing a seed culture medium and activating and culturing seeds:
preparing a first-level seed solution: the colony of the inclined plane is eluted by 1-2mL of sterilized 0.85% physiological saline, inoculated into a 250mL triangular flask containing 25mL of liquid culture medium according to the inoculation amount of 0.1% -0.2%, and cultured for 14h at 37 ℃ and 200rmp until the logarithmic phase.
Preparing a secondary seed liquid: the prepared primary seed solution is inoculated into a 250mL triangular flask containing 50mL of liquid culture medium according to the inoculation amount of 0.3 percent, and is cultured for 12h at 37 ℃ and 200rmp until the logarithmic phase.
The primary and secondary seed culture media consist of: 10g/L glucose, 5g/L yeast powder, 0.25g/L magnesium sulfate, 2g/L dipotassium hydrogen phosphate and 10g/L sodium glutamate, wherein the pH value is 7.5, and the sterilization is carried out for 20min at 115 ℃.
2. Fermentation culture
Preparing a fermentation medium: 140g/L of glucose, 35g/L of yeast powder, 2g/L of dipotassium phosphate, 60g/L of sodium glutamate, 4g/L of citric acid monohydrate, 0.5g/L of calcium chloride, 10g/L of sodium chloride and pH6.8.
A baffle shake flask with the liquid loading capacity of the fermentation medium of 200mL/1L is adopted, the second-stage seed liquid in the step 1 is inoculated into the fermentation medium according to the inoculation amount of 10 percent, the second-stage seed liquid is subjected to shaking culture at the constant temperature of 37 ℃ for 48h, and the rotating speed of 0-12h is 120rmp; the rotating speed is 200rmp within 12-24 h; the rotating speed is 250rmp after 24-48 h. Culturing for 48h, and detecting that the yield of the fermentation liquid gamma-polyglutamic acid reaches 45.8g/L after fermentation is finished, wherein the content of the gamma-polyglutamic acid with the molecular weight of more than or equal to 265kDa and more than or equal to 35kDa accounts for 96.5%, the content of the gamma-polyglutamic acid with the molecular weight of more than 35kDa and more than or equal to 18kDa accounts for 1.7%, and the balance is the gamma-polyglutamic acid with the molecular weight of less than or equal to 10 kDa.
3. Extraction of gamma-PGA from fermentation liquor
And centrifuging the fermentation liquor to remove thalli, adding ethanol with the volume 2 times that of the fermentation liquor into the supernatant, centrifuging and collecting precipitates after precipitation, dialyzing and desalting to obtain the gamma-PGA.
EXAMPLE 3 fermentative culture of the Strain and preparation of Low molecular weight Gamma-PGA (2)
The low molecular weight gamma-PGA is produced by performing fermentation culture on the Bacillus licheniformis PGA-BL19, and the method of the fermentation culture is different from the example 2 only in that: fermenting and culturing in a 7L fermentation tank with liquid loading amount of 1/2, shaking and culturing at 37 deg.C for 48h, and rotating at 150rmp for 0-12 h; the rotating speed is 220rmp within 12-24 h; the rotating speed is 250rmp after 24-48 h. Culturing for 48h, and detecting that the yield of the fermentation liquid gamma-polyglutamic acid reaches 48.8g/L after fermentation is finished, wherein the content of the gamma-polyglutamic acid with the molecular weight of more than or equal to 265kDa and more than or equal to 35kDa accounts for 94.6%, the content of the gamma-polyglutamic acid with the molecular weight of more than 35kDa and more than or equal to 18kDa accounts for 1.9%, and the balance is the gamma-polyglutamic acid with the molecular weight of less than or equal to 10 kDa.
Example 4 use of low molecular weight γ -PGA for lyophilization protection of microorganisms
The γ -PGA prepared in example 2 was used for the freeze-drying protection of Lactobacillus plantarum and Mortierella alpina, and the specific methods and results were as follows:
1. freeze-drying protection of lactobacillus plantarum
The lactobacillus plantarum seed culture medium and the activated culture medium adopt MRS culture medium, the lactobacillus plantarum grows for 48h in MRS solid culture medium, grows for 16h to logarithmic phase in liquid culture medium, the thalli are collected by centrifugation, washed for 2 times by sterile water, resuspended by 2.5 percent of gamma-polyglutamic acid prepared in example 2, frozen at-80 ℃ overnight, and freeze-dried into powder. Sealing and placing at-20 ℃ for two days, adopting gradient dilution, counting plates, and calculating the survival rate N% = N1/N0 x 100 of the freeze-dried strain; wherein, N1: counting the viable count of the freeze-dried powder; n0: viable count was counted before lyophilization.
2. Freeze-drying protection of mortierella alpina
The Mortierella alpina seed culture medium and the activation culture medium adopt PDA culture medium, mortierella alpina is cultured on PDA slant culture medium for 4 days, cultured in liquid culture medium for 3 days to mature period, centrifuged to collect thallus, washed with sterile water for 2 times, resuspended with 2.5% of the gamma-polyglutamic acid prepared in example 2, frozen at-80 deg.C overnight, and lyophilized into powder. Sealing and placing at-20 ℃ for two days, adopting gradient dilution, counting plates, and calculating the survival rate of the freeze-dried strain N% = lgN1/lgN0 x 100%, wherein N1: counting the viable count of the freeze-dried powder; n0: viable count was counted before lyophilization.
The results of the experiment are shown in table 1.
TABLE 1 Freeze-drying protection of different molecular weights of gamma-PGA against Lactobacillus plantarum and Mortierella alpina
Freeze drying is a drying method in which a suspension of cells is frozen below the freezing point to convert it into a solid crystalline state, and then the solid is sublimated at a low temperature and a low pressure (vacuum) to dehydrate it. Generally, the cell membrane of the microorganism is damaged in the drying process, so that a large amount of cells die, and therefore, a protective agent needs to be added, the uniformity of the bacterial liquid density is improved, and the death of viable bacteria caused by a mechanical effect and a solute effect in the freeze-drying process is reduced. The cryoprotectant added before lyophilization is a crucial determinant factor influencing the activity of the lyophilized bacterial powder, and can influence the activity and stability of cells in a storage period and the survival rate of the cells when being rehydrated.
In addition to the wide application of gamma-polyglutamic acid in the fields of cosmetics, foods and medicines, the literature discloses that gamma-polyglutamic acid can be applied to the freeze-drying protection of probiotics, for example, CN112899204B discloses a probiotic freeze-dried shell composite protective agent containing gamma-polyglutamic acid; the document 'freeze-drying protection effect of high molecular polymer gamma-PGA on bifidobacterium longum' discloses that the gamma-PGA and whey powder can play a better freeze-drying protection effect after being compounded. These studies all involve the formulation of gamma-polyglutamic acid with other substances as cryoprotectants for microorganisms. After physical and Chemical experiments in the literature "Relationship between the anti-freeze Activities and the Chemical Structures of Oligo-and Poly (glutamic acid) s", it was found that the freezing resistance of 20kDa gamma-PGA is the best, and indeed for bacteria, the freeze-drying protection effect of the prepared gamma-PGA and the ultra-low molecular weight gamma-PGA on microorganisms is not obvious, but compared with bacteria, fungi are more easily damaged by ice crystals during the freeze-drying and pre-freezing process due to the large cell volume and the lack of cell wall protection morphology. Therefore, the fungi are more easily inactivated in the freeze-drying process, the cell death rate is higher, and the performance requirement on the freeze-drying protective agent is higher. The bacillus licheniformis PGA-BL19 disclosed by the invention can be used for generating a gamma-polyglutamic acid product with a proper molecular weight range and distribution, and the product can improve the protection effect of the gamma-polyglutamic acid on thalli in a freeze-drying process.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. Bacillus licheniformis (Bacillus licheniformis) PGA-BL19, characterized by that, it is preserved in China center for type culture Collection, the preserving number is CCTCC NO: m20221810.
2. A bacterial preparation comprising the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 according to claim 1.
3. The method for preparing the microbial inoculum according to claim 2, which comprises the step of culturing Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 to obtain a bacterial solution.
4. Use of the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 according to claim 1 or the bacterial agent according to claim 2 for the preparation of gamma-polyglutamic acid.
5. The use according to claim 4, wherein the gamma-polyglutamic acid has a molecular weight of 300kDa or less.
6. Use of the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 as claimed in claim 1 or the bacterial agent as claimed in claim 2 for preparing a freeze-drying protective agent for microorganisms.
7. A method for preparing gamma-polyglutamic acid, comprising: culturing the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 of claim 1 to obtain a culture solution, and collecting the gamma-polyglutamic acid in the culture solution.
8. The gamma-polyglutamic acid is characterized in that the molecular weight of the gamma-polyglutamic acid is less than or equal to 270kDa, wherein the mass percentage of the gamma-polyglutamic acid with the molecular weight of 265kDa-35kDa is 86-97%, the mass percentage of the gamma-polyglutamic acid with the molecular weight of more than or equal to 18kDa and less than 35kDa is 1.5-2.5%, and the balance is the gamma-polyglutamic acid with the molecular weight of less than or equal to 10 kDa.
9. The gamma-polyglutamic acid of claim 8, wherein the gamma-polyglutamic acid is produced by fermenting the Bacillus licheniformis (Bacillus licheniformis) PGA-BL19 of claim 1.
10. A microbial lyoprotectant, said lyoprotectant comprising the gamma-polyglutamic acid of claim 8 or 9.
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| CN117089496A (en) * | 2023-08-22 | 2023-11-21 | 轩凯生物科技(山东)有限公司 | Bacillus licheniformis with phage resistance and application thereof in preparation of polyglutamic acid |
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