CN116064315A - Bacillus albus GXU-2 and method for producing gamma-polyglutamic acid by using same - Google Patents

Bacillus albus GXU-2 and method for producing gamma-polyglutamic acid by using same Download PDF

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CN116064315A
CN116064315A CN202211367797.XA CN202211367797A CN116064315A CN 116064315 A CN116064315 A CN 116064315A CN 202211367797 A CN202211367797 A CN 202211367797A CN 116064315 A CN116064315 A CN 116064315A
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polyglutamic acid
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gxu
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周权能
范晓苏
沈方科
郭霜
黄宏飞
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Guangxi Xida Testing Co ltd
Guangxi University
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Guangxi University
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Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to bacillus albus GXU-2 and a method for producing gamma-polyglutamic acid by using the same. The invention provides a strain of Bacillus albus GXU-2 (Bacillus albus) GXU-2, which has been subjected to biological preservation, the preservation number is CGMCC No.24579, and the fermentation of the Bacillus albus GXU-2 provided by the invention can produce gamma-polyglutamic acid with high yield of more than 70g/L, thereby providing a novel method for the biosynthesis of the gamma-polyglutamic acid.

Description

Bacillus albus GXU-2 and method for producing gamma-polyglutamic acid by using same
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to bacillus albus GXU-2 and a method for producing gamma-polyglutamic acid by using the same.
Background
The gamma-polyglutamic acid (gamma-Polyglutamic acid, gamma-PGA) is polyglutamic acid formed by connecting D-glutamic acid and L-glutamic acid through an amide bond, has the polymerization degree of about 1000-15000, has the relative molecular mass of 5-200 ten thousand daltons, and has the characteristics of water solubility, biological affinity, degradability, high water retention and the like. The gamma-polyglutamic acid is an excellent environment-friendly high polymer material, can be used as a water-retaining agent, a heavy metal ion adsorbent, a flocculating agent, a slow release agent, a drug carrier and the like, and has great commercial value and social value in the fields of cosmetics, environmental protection, foods, medicines, agriculture and the like. The gamma-PGA molecular structure has a large number of free hydrophilic carboxyl groups, so that the gamma-PGA molecular structure has extremely strong moisturizing capability, and can effectively increase the moisturizing capability of skin when being added into cosmetics. In agricultural production, the gamma-PGA can be applied as a fertilizer slow release agent to improve the utilization rate of the fertilizer and promote the yield and income of crops. The gamma-PGA can also be used as a drug carrier for the pharmaceutical industry.
At present, the large-scale production of the gamma-PGA mainly uses a microbial fermentation method, and compared with a chemical synthesis method, the microbial fermentation method has the advantages of short production period, mild conditions, low cost and the like. It has now been found that various strains can be fermented to produce gamma-polyglutamic acid, such as Bacillus subtilis (B.subtilis), bacillus licheniformis (Bacillus paralicheniformis), bacillus pumilus (Bacillus pumilus), bacillus belicus (Bacillus velezensis), and the like. Chinese patent CN110904012B discloses bacillus subtilis (Bacillus subtilis) NT-11 for producing gamma-polyglutamic acid, which is prepared by optimizing fermentation conditions, supplementing glutamine in the middle and later stages of fermentation and the like, and has a high molecular weight gamma-polyglutamic acid product and a yield of about 39.4g/L. Chinese patent CN113462608A discloses a strain of bacillus methylotrophicus (Bacillus methylotrophicus) FEP-225, the yield of γ -polyglutamic acid is 31.2g/L by shake flask fermentation; and fermenting in a fermentation tank, using a micro-nano aeration device, and simultaneously obtaining a secondary seed culture through the expansion culture of the primary seed culture and then inoculating the secondary seed culture into the fermentation tank for fermentation, wherein the highest yield of the obtained gamma-polyglutamic acid is only 51.6g/L. The strain resources are excavated, the strain resource library for producing the gamma-polyglutamic acid is enriched, and the fermentation yield of the gamma-polyglutamic acid is improved.
Disclosure of Invention
The invention aims to provide bacillus albus with high gamma-polyglutamic acid yield and short fermentation period, and the strain resource library for enriching the gamma-polyglutamic acid yield.
The invention provides a bacillus albus GXU-2, wherein the bacillus albus GXU-2 is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 24579.
The invention also provides application of the bacillus albus GXU-2 in preparing gamma-polyglutamic acid.
The invention also provides a method for producing gamma-polyglutamic acid, which comprises the following steps of:
step S1, seed culture: picking a single colony of the bacillus circulans GXU-2 plate culture, inoculating the single colony in a liquid seed culture medium, and carrying out shaking culture for 12-24 hours at 150-200 rpm at 30-37 ℃ to obtain seed liquid;
step S2, fermentation culture: transferring the seed liquid obtained by culturing in the step S1 into a fermentation medium, wherein the volume ratio of the seed liquid to the fermentation medium is (5-12): 100, culturing at 30-45 deg.c, pH 6.0-7.5, ventilation of 0.8-1.5 vvm, stirring at 150-280 rpm for 24-50 hr, and fermenting to obtain fermentation liquid containing gamma-polyglutamic acid.
Preferably, the seed culture medium comprises 20g/L glucose, 10g/L peptone, 3g/L, naCl g/L beef extract and the balance of distilled water, and is sterilized at 115 ℃ for 20min; the initial pH of the seed medium was 7.0.
Preferably, the fermentation medium comprises 20-150 g/L of carbon source, 3-17 g/L of nitrogen source and 10-100 g/L, K of sodium glutamate 2 HPO 4 2.5g/L and the balance of distilled water, sterilizing for 20min at 115 ℃; the initial pH of the fermentation medium is 6.0-7.5.
Preferably, the carbon source comprises one or more of glucose, sucrose, maltose, fructose and lactose;
the nitrogen source comprises one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor.
The invention provides bacillus albus GXU-2 which is subjected to biological preservation and is preserved in China general microbiological culture Collection center (CGMCC) No.24579, and the preservation date is 2022, 3 and 23. The invention separates 1 strain of bacillus albus GXU-2 from soil, the strain can produce gamma-polyglutamic acid with high yield reaching more than 70g/L, and the strain has wide market application prospect.
Biological preservation information
Bacillus albus (Bacillus albus) GXU-2, deposited at China general microbiological culture Collection center (CGMCC), address: beijing, chaoyang area, north Chenxi way No.1, no. 3, post code: 100101 and the preservation number is CGMCC No.24579.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph showing morphological identification results of Bacillus albus GXU-2;
FIG. 2 is a phylogenetic tree of Bacillus albus GXU-2.
Detailed Description
The invention provides a bacillus albus GXU-2, wherein the bacillus albus GXU-2 is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 24579.
The bacillus albus GXU-2 is obtained by screening from soil, and the 16S rDNA sequence of the bacillus albus GXU-2 is preferably shown as SEQ ID NO.1, and specifically comprises the following steps: 5'-CCCTAATACATGGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCGTAAAGGGG-3'.
The bacillus albus GXU-2 of the invention has round colony on LB solid medium, regular edge, wet lawn, white color and opaqueness (as shown in figure 1). The bacterial cells are in a short rod shape, gram staining is positive, glucose, sucrose, lactose, maltose, fructose and mannitol can be decomposed and utilized, non-hydrolyzable starch can be produced by utilizing glucose, oxidase reaction is negative, nitrate reduction reaction is positive, citrate utilization test is negative, gelatin liquefaction test is positive, high-yield gamma-polyglutamic acid is high, and the bacterial cells have wide market application prospect.
According to the physiological and biochemical characteristics of the bacillus albus GXU-2, the invention also provides the application of the bacillus albus GXU-2 in preparing gamma-polyglutamic acid.
The invention also provides a method for producing gamma-polyglutamic acid, which comprises the following steps of:
step S1, seed culture: picking a single colony of the bacillus circulans GXU-2 plate culture, inoculating the single colony in a liquid seed culture medium, and carrying out shaking culture for 12-24 hours at 150-200 rpm at 30-37 ℃ to obtain seed liquid;
step S2, fermentation culture: transferring the seed liquid obtained by culturing in the step S1 into a fermentation medium, wherein the volume ratio of the seed liquid to the fermentation medium is (5-12): 100, culturing at 30-45 deg.c, pH 6.0-7.5, ventilation of 0.8-1.5 vvm, stirring at 150-280 rpm for 24-50 hr, and fermenting to obtain fermentation liquid containing gamma-polyglutamic acid.
In the invention, the temperature of shake culture in the step S1 is 30-37 ℃, preferably 32-36 ℃, and more preferably 35 ℃; the shake culture time in the step S1 is 12-24 hours, preferably 15-20 hours, and more preferably 16-18 hours; the rotational speed of the shaking culture in the step S1 is 150 to 200rpm, preferably 160 to 180rpm. When the inoculation is carried out in the step S1, the inoculation amount is not strictly required, and the conventional operation is carried out.
In the invention, the liquid seed culture medium preferably comprises 20g/L of glucose, 10g/L of peptone, 3g/L, naCl g/L of beef extract and the balance of distilled water, and the culture medium is sterilized at 115 ℃ for 20min; the initial pH of the seed medium was 7.0. The invention has no strict requirement on the sources of all components in the liquid seed culture medium, and can be obtained conventionally. According to the invention, in the step S1, the bacillus albus GXU-2 is cultivated to the mid-logarithmic growth phase by seed cultivation, so that the activity of the bacillus albus GXU-2 is improved.
After the seed liquid of the bacillus albus GXU-2 is obtained, the seed liquid of the bacillus albus GXU-2 is inoculated into a fermentation medium for fermentation culture, and fermentation liquid containing gamma-polyglutamic acid is obtained. The volume ratio of the seed liquid of the bacillus albus GXU-2 to the fermentation medium is (5-12): 100, preferably (6 to 10): 100, more preferably 8:100. the fermentation culture according to the invention preferably comprises aerobic fermentation.
In the present invention, the temperature of the fermentation culture is 30 to 45 ℃, preferably 32 to 42 ℃, more preferably 35 to 40 ℃, and even more preferably 36 to 38 ℃; the fermentation culture time is 24-50 hours, preferably 30-50 hours, more preferably 36-50 hours, more preferably 42-50 hours, and most preferably 48-50 hours; the aeration rate of the fermentation culture is 0.8-1.5 vvm. In the specific implementation process of the invention, the ventilation is preferably specifically determined according to the fermentation culture time, and when the fermentation culture time is 24 hours, the ventilation is preferably 0.8-1.0 vvm, and more preferably 1.0vvm; when the fermentation time is 48 hours, the ventilation is 1.0 to 1.2vvm, more preferably 1.2vvm. The gas introduced in the invention is preferably air. The stirring speed of the fermentation culture of the present invention is 150 to 280rpm, preferably 160 to 250rpm, more preferably 170 to 220rpm, and even more preferably 180 to 200rpm.
In the invention, the fermentation medium preferably comprises 20-150 g/L of carbon source, 3-17 g/L of nitrogen source and 10-100 g/L, K of sodium glutamate 2 HPO 4 2.5g/L and the balance of distilled water, sterilizing for 20min at 115 ℃; the content of the carbon source is preferably 25 to 75g/L or 80 to 140g/L, more preferably 30 to 70g/L or 85 to 120g/L, still more preferably 40 to 60g/L or 90 to 110g/L, and most preferably 50g/L or 100g/L; the nitrogen source is preferably 5 to 17g/L, more preferably 6 to 15g/L, and still more preferably 8 to 10g/L; the sodium glutamate is preferably 20 to 100g/L, more preferably 30 to 100g/L, still more preferably 40 to 100g/L, and most preferably 90 to 100g/L.
In the present invention, the carbon source preferably includes one or more of glucose, sucrose, maltose, fructose, lactose, more preferably one of glucose, sucrose, and maltose, or a mixture of fructose and lactose; the nitrogen source preferably includes one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor, and more preferably ammonium sulfate, or ammonium sulfate and corn steep liquor.
In the present invention, the fermentation medium has an initial pH of 6.0 to 7.5, preferably 6.5 to 7.0, and more preferably 7.0. The invention has no strict requirement on the sources of the components in the fermentation medium, and can be obtained conventionally. The invention does not need to adjust pH in the fermentation culture process.
The fermentation culture of bacillus albus obtained by fermenting the seed liquid of bacillus albus GXU-2 is rich in gamma-polyglutamic acid, the yield of the gamma-polyglutamic acid detected by the fermentation culture reaches more than 70g/L, and the fermentation process is simple, convenient to operate, simple in culture medium components and low in cost, and can realize industrialized mass production.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Unless otherwise specified, the culture medium and the identification method used in the implementation process are conventional methods.
Example 1
Isolation, identification and preservation of bacillus albus:
1. isolation of bacillus albus:
1.1 sample treatment: placing 5g soil sample into a sterilizing triangular flask, adding 50mL sterilized water, shaking at 30deg.C with 200rpm for 30min, standing for precipitation, and gradient diluting the supernatant with sterilized water with dilution multiple of 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 And 10 8 。
1.2 preliminary screening of the Gamma-polyglutamic acid producing Strain: select step 1.1 dilution 10 3 ～10 8 The multiplied samples are respectively coated on a preliminary screening flat-plate culture medium in an amount of 100 mu L, and are subjected to inversion culture at 37 ℃; the primary screening flat-plate culture medium is LB solid culture medium, and comprises the following components: 10g/L peptone, 5g/L, naCl g/L yeast powder, 20g/L agar powder and the balance distilled water, and sterilizing the primary screening flat-plate culture medium at pH 7.0 and 121 ℃ for 30min.
1.3 rescreening of the Gamma-polyglutamic acid producing Strain: selecting single colony which is in emulsion form and can be drawn from the primary screening flat plate culture medium, transferring to a fresh liquid re-screening culture medium, and carrying out shaking culture at 37 ℃ and 200rpm for 48 hours to obtain fermentation liquor; the liquid re-screening culture medium comprises the following components: glucose 20g/L, peptone 10g/L, beef extract 3g/L, naCl g/L and distilled water for the rest, wherein the pH value of the liquid re-screening culture medium is 7.0, the temperature is 121 ℃, and the sterilization is carried out for 30min.
1.4 extraction and detection of gamma-polyglutamic acid: centrifuging to remove thalli in the fermentation liquid in the step 1.3, taking supernatant, adding 4 times of pre-cooled absolute ethyl alcohol, placing into a refrigerator at the temperature of 4 ℃ for precipitation for 12 hours, taking out, centrifuging at the temperature of 4 ℃ for 10 minutes, removing supernatant, washing the precipitate with 75% ethanol for 3 times, drying at the temperature of 105 ℃ to constant weight, taking out, cooling to room temperature to obtain a gamma-polyglutamic acid crude product, and weighing to measure the gamma-polyglutamic acid yield.
1.5 identification of gamma-polyglutamic acid: weighing 2g of the gamma-polyglutamic acid product in the step 1.4, adding ultrapure water for re-dissolution, then fixing the volume to 100mL, filtering with a 0.22 mu m filter membrane to remove impurities, taking 5mL of filtered sample, adding an equal amount of 6mol/L hydrochloric acid solution, carrying out oil bath hydrolysis for 12 hours at 110 ℃, adding 6mol/L NaOH solution for neutralization until the pH value is 7.0 after hydrolysis, analyzing the hydrolysate by high performance liquid chromatography after dilution, simultaneously analyzing a glutamic acid standard substance by high performance liquid chromatography, comparing the liquid chromatography peak time of the sample hydrolysate and the glutamic acid standard substance, and identifying the prepared gamma-polyglutamic acid.
Screening results of gamma-polyglutamic acid-producing strains:
diluting and coating soil samples into a primary screening flat plate culture medium, and then, carrying out primary screening on the obtained strain, selecting single bacterial colonies which are in emulsion and can be drawn into wires, respectively transferring the single bacterial colonies into a fresh liquid secondary screening culture medium, culturing for 12 hours, and then, carrying out gamma-polyglutamic acid extraction and detection according to the steps 1.4 and 1.5 to obtain the strain with the highest gamma-polyglutamic acid yield, wherein the number is GXU-2.
2. Identification of Bacillus albus
2.1 morphological identification
Single colonies of GXU-2 strain were inoculated on LB solid medium (composed of peptone 10g/L, yeast powder 5g/L, naCl g/L, agar powder 20g/L and the balance distilled water) with an inoculating needle, the initial pH of the medium was 7.0, sterilized at 121℃for 30min, and the colony morphology was observed by inversion culture at 37℃and the results were shown in FIG. 1. As can be seen from FIG. 1, the colony of GXU-2 strain on LB solid medium is round, the edge is clean, the lawn is moist, the surface is smooth, white and opaque.
2.2 physiological and biochemical identification:
the GXU-2 strain bacterial cells are in a short rod shape, gram staining is positive, glucose, sucrose, lactose, maltose, fructose and mannitol can be decomposed and utilized, non-hydrolyzable starch can be produced by utilizing glucose, oxidase reaction is negative, nitrate reduction reaction is positive, citrate utilization test is negative, and gelatin liquefaction test is positive.
2.3 molecular biological identification
Extracting genomic DNA of GXU-2 strain, adopting bacterial universal primer (27F, 14992R), PCR amplifying 16S rDNA sequence, and sequencing to obtain 16S rDNA sequence as shown in SEQ ID NO.1, length is 1440bp. The results of the alignment analysis of the sequences in the GeneBank database at NCBI using BLAST software showed up to 99% homology of the 16S rDNA sequence of the GXU-2 strain to Bacillus albus. By combining morphological and physiological biochemical identification results, GXU-2 is identified as Bacillus albus, and the phylogenetic tree is shown in figure 2.
3. Preservation of bacillus albus:
and (3) performing strain preservation on the strain GXU-2 identified as bacillus albus in the step (2), wherein the preservation information is as follows:
preservation unit: china general microbiological culture Collection center (CGMCC);
preservation address: beijing, chaoyang area, north Chenxi way No.1, no. 3, post code: 100101;
classification naming: bacillus albus GXU-2;
preservation number: CGMCC No.24579
Preservation time: 2022, 03 and 23.
Example 2
A method for producing gamma-polyglutamic acid by fermentation of bacillus albus comprises the following steps:
1. seed culture
Bacillus albus GXU-2 (Bacillus albus GXU-2) obtained in example 1 was spread on LB solid medium (composed of peptone 10g/L, yeast powder 5g/L, naCl g/L, agar powder 20g/L and the balance of distilled water, pH 7.0 of the medium, sterilized at 121 ℃ C. For 30 min), cultured upside down at 37 ℃ C.), and single colony was picked up and inoculated on liquid seed medium (composed of glucose 20g/L, peptone 10g/L, beef extract 3g/L, naCl g/L and the balance of distilled water, pH 7.0 of the medium at the initial pH 115 ℃ C. And sterilized at 20 min), and cultured with shaking at 37 ℃ C. For 16h to mid-log growth at 220rpm to obtain seed liquid.
2. Fermentation culture
Inoculating the seed solution obtained in step 1 to fermentation medium (prepared from glucose 50g/L, (NH) 4 ) 2 SO 4 7g/L, 8g/L corn steep liquor, 50g/L, K sodium glutamate 2 HPO 4 2.5g/L and the balance of distilled water, the initial pH of the culture medium is 7.0, the sterilization is carried out for 30min at 121 ℃, and the shaking culture is carried out for 24h by a 220rpm shaking table under the conditions of the temperature of 37 ℃ and the ventilation rate of 1.0 vvm.
3. The gamma-polyglutamic acid content was measured in the same manner as in step 1.4 of example 1, and as a result, 42.71g/L was obtained.
Examples 3 to 6
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the temperature during the fermentation culture in step 2 of examples 3 to 6 was different from that in example 2 and is specifically shown in Table 1.
TABLE 1 fermentation temperature and post-fermentation gamma-polyglutamic acid content in step 2 of examples 3 to 6
Example 2 Example 3 Example 4 Example 5 Example 6
Fermentation temperature (. Degree. C.) 37 30 42 25 45
Gamma-polyglutamic acid content (g/L) 42.71 36.71 38.63 20.26 36.75
Examples 7 to 10
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the ventilation amount during the fermentation culture in step 2 of examples 7 to 10 was different from that in example 2 and is specifically shown in Table 2.
TABLE 2 ventilation and post-fermentation gamma-polyglutamic acid content in step 2 of examples 7 to 10
Figure BDA0003923946870000081
Figure BDA0003923946870000091
Examples 11 to 14
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the stirring speed during the fermentation culture in step 2 of examples 11 to 14 was different from that of example 2 and is specifically shown in Table 3.
TABLE 3 stirring speed and post-fermentation gamma-polyglutamic acid content at step 2 in examples 11 to 14
Figure BDA0003923946870000092
Examples 15 to 19
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the fermentation time during the fermentation culture in step 2 of examples 15 to 19 was different from that of example 2 and is specifically shown in Table 4.
TABLE 4 fermentation time and post-fermentation gamma-polyglutamic acid content in step 2 of examples 15 to 19
Figure BDA0003923946870000093
Examples 20 to 24
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the concentration of glucose during the fermentation culture in step 2 of examples 20 to 24 was different from that in example 2 and is specifically shown in Table 5.
TABLE 5 glucose concentration and post-fermentation gamma-polyglutamic acid content at step 2 in examples 20 to 24
Figure BDA0003923946870000094
Examples 25 to 29
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that (NH) was obtained in the fermentation culture process of step 2 in examples 20 to 24 4 ) 2 SO 4 And the corn steep liquor at a concentration different from that of example 2, are specifically listed in Table 6.
Table 6 step 2 (NH) in examples 25 to 29 4 ) 2 SO 4 And corn steep liquor concentration and post-fermentation gamma-polyglutamic acid content
Figure BDA0003923946870000101
Examples 30 to 39
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the concentrations of sodium glutamate and glucose during the fermentation culture in step 2 of examples 30 to 39 were different from those in example 2 and are specifically shown in Table 7.
TABLE 7 concentration of sodium glutamate at step 2 and content of gamma-polyglutamic acid after fermentation in examples 30 to 39
Figure BDA0003923946870000102
Examples 40 to 42
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the kind of carbon source was different from example 2 and the quality was the same in the fermentation culture process of step 2 in examples 40 to 42, as shown in Table 8.
TABLE 8 carbon source type and post-fermentation gamma-polyglutamic acid content in step 2 of examples 40 to 42
Figure BDA0003923946870000103
Examples 43 to 47
Gamma-polyglutamic acid was produced in the same manner as in example 2 except that the kind and amount of nitrogen source used in the fermentation culture in step 2 of examples 43 to 47 were different from those of example 2 and are specifically shown in Table 9.
TABLE 9 Nitrogen source species and post-fermentation gamma-polyglutamic acid content in step 2 of examples 43 to 47
Figure BDA0003923946870000111
According to the embodiment, the bacillus albus GXU-2 is cultivated, the obtained fermentation culture is rich in gamma-polyglutamic acid, the yield is more than 70g/L, the fermentation process is simple, the operation is convenient, the components of the culture medium are simple, the cost is low, and the industrial mass production can be realized.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (6)

1. A strain of Bacillus albus GXU-2 is characterized in that the Bacillus albus GXU-2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 24579.
2. The use of bacillus albus gu-2 according to claim 1 for the preparation of gamma-polyglutamic acid.
3. A method for producing gamma-polyglutamic acid, characterized by fermenting the bacillus albus gu-2 according to claim 1 to obtain gamma-polyglutamic acid, comprising the following steps:
step S1, seed culture: picking a single colony of the bacillus circulans GXU-2 plate culture, inoculating the single colony in a liquid seed culture medium, and carrying out shaking culture for 12-24 hours at 150-200 rpm at 30-37 ℃ to obtain seed liquid;
step S2, fermentation culture: transferring the seed liquid obtained by culturing in the step S1 into a fermentation medium, wherein the volume ratio of the seed liquid to the fermentation medium is (5-12): 100, culturing at 30-45 deg.c, pH 6.0-7.5, ventilation of 0.8-1.5 vvm, stirring at 150-280 rpm for 24-50 hr, and fermenting to obtain fermentation liquid containing gamma-polyglutamic acid.
4. The method for producing gamma-polyglutamic acid according to claim 3, wherein the seed medium comprises 20g/L of glucose, 10g/L of peptone, 3g/L, naCl g/L of beef extract and the balance of distilled water, and is sterilized at 115℃for 20min; the initial pH of the seed medium was 7.0.
5. The method for producing gamma-polyglutamic acid according to claim 3, wherein the fermentation medium comprises a carbon source of 20 to 150g/L, a nitrogen source of 3 to 17g/L, and sodium glutamate of 10 to 100g/L, K 2 HPO 4 2.5g/L and the balance of distilled water, sterilizing for 20min at 115 ℃; the initial pH of the fermentation medium is 6.0-7.5.
6. The method of producing gamma-polyglutamic acid according to claim 5, wherein the carbon source comprises one or more of glucose, sucrose, maltose, fructose, lactose;
the nitrogen source comprises one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor.
CN202211367797.XA 2022-11-03 2022-11-03 Bacillus albus GXU-2 and method for producing gamma-polyglutamic acid by using same Pending CN116064315A (en)

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