CN114181861A - Bacillus belgii for producing gamma-polyglutamic acid by low-ammonia fermentation and application thereof - Google Patents
Bacillus belgii for producing gamma-polyglutamic acid by low-ammonia fermentation and application thereof Download PDFInfo
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Abstract
A Bacillus belgii for realizing low-ammonia fermentation production of gamma-polyglutamic acid and application thereof relate to the technical field of microbiology and bioengineering fermentation, and are classified and named as Bacillus belgii YZHY21.A01(Bacillus velezensis YZHY21.A01) which is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC NO: 61921, the preservation date is 2021, 8 months and 31 days. The invention screens and obtains the Bacillus belgii for producing the gamma-polyglutamic acid by low-ammonia fermentation from the fermented soybeans, and the gamma-polyglutamic acid is fermented and extracted by three steps of strain activation, solid fermentation and purification extraction, thereby realizing high yield of the gamma-polyglutamic acid and reducing the generation of ammonia gas in the fermentation process.
Description
Technical Field
The invention relates to the technical field of fermentation in microbiology and bioengineering, in particular to Bacillus belgii for producing gamma-polyglutamic acid by low-ammonia fermentation and application thereof.
Background
Gamma-polyglutamic acid (gamma-PGA) is a natural high molecular polymer. Because of the physicochemical characteristics of high molecular weight, strong water absorption, high water solubility and the like and the biological properties of good biocompatibility, no environmental toxicity, biodegradability and the like, the gamma-polyglutamic acid has wide application prospect in the fields of agriculture, food processing, medicines and the like, and can be used as a seed water-retaining agent, a beverage thickener, a drug sustained-release agent and the like. In 2019, the global polyglutamic acid market demand reaches 5.8 ten thousand tons.
At present, a microbial fermentation method is the best gamma-PGA preparation method generally adopted in industrial production, the gamma-PGA is prepared by strain screening, culture, separation and purification, the process is simple, the conditions are mild, and large-scale production can be carried out. Polyglutamic acid can be prepared by fermenting various raw materials. Chendaosheng et al use Bacillus amyloliquefaciens, and ferment wheat bran, sweet potato residue, and cassava residue as solid raw materials to obtain polyglutamic acid (patent application number: 201810137031. X). Zhang super et al use Bacillus subtilis, and use sucrose and sodium glutamate as raw materials to ferment to obtain polyglutamic acid with a yield of 37.6g/L (patent application No. 201510287786.4). Wuling Tian et al fermented polyglutamic acid with sugar cane juice and sodium glutamate as raw materials by Bacillus belgii with a yield of 54.11g/L (patent application No. 202010649532.3).
However, the fermentation temperature of the existing microbial method for fermenting the polyglutamic acid is generally 37-40 ℃, and gases with special odor, such as ammonia gas and the like, can be generated in the fermentation process, thereby influencing the production environment. In order to realize the production of gamma-polyglutamic acid under the condition of low ammonia production, a method for constructing rocG, gudB and ureC gene knockout engineering strains (SKRGU1) has been researched, but the large-scale production application is not realized. In addition, the mixed fermentation method can reduce the ammonia production to a certain extent.
Disclosure of Invention
The invention aims to provide Bacillus belgii for producing gamma-polyglutamic acid by low-ammonia fermentation and application thereof, which can reduce the generation of ammonia in the fermentation process while realizing the production of gamma-polyglutamic acid by high-yield fermentation.
The invention obtains a Bacillus belgii for realizing low-ammonia fermentation production of gamma-polyglutamic acid from fermented soybeans by screening, the Bacillus belgii is classified and named as Bacillus velezensis YZHY21.A01, and the Bacillus velezensis YZHY21.A01 is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC NO: 61921, the preservation date is 2021, 8 and 31 days, and the preservation unit addresses are: the Michelson Dai No. 59 building 5, Ministry of sciences, Guangdong province, institute of microbiology, postal code 510070, Ministry of sciences, Michelson, Guangzhou, China.
The invention also provides application of the Bacillus belgii in low-ammonia fermentation production of gamma-polyglutamic acid.
The invention also provides application of the Bacillus belgii in producing nattokinase by low-ammonia fermentation.
The invention also provides a technical scheme for preparing gamma-polyglutamic acid and nattokinase by inoculating the Bacillus beilesiensis YZHY21.A01 into a fermentation medium for fermentation.
As a preferable technical scheme of the invention, the fermentation medium adopts a soybean culture medium. More preferably, the fermentation medium is black bean or soybean medium.
The method for producing the gamma-polyglutamic acid by low-ammonia fermentation by using the Bacillus belgii comprises the following steps:
(1) and (3) activation of thalli: taking out the Bacillus beiLeisi inclined plane from a refrigerated cabinet at 4 ℃, and placing the Bacillus beiLeisi inclined plane in an incubator at 28 ℃ for activation for 2-6 h;
(2) solid fermentation: taking the strain inclined planes in the proper step (1), cleaning the inclined plane lawn with 5mL of sterile water on each inclined plane under the aseptic condition, inoculating 1-4% of the strain amount into a soybean culture medium, and fermenting at a constant temperature of 28-35 ℃ for 24-48 h;
(3) extracting gamma-polyglutamic acid: adding sterile water into a soybean culture medium after fermentation for 24-48 h, adjusting the pH to 7.0-8.0, shaking for 15min, filtering, centrifuging the filtrate (8000r/min, 5min, 25 ℃), removing thalli and a small amount of soybean residues to obtain a supernatant, adding an appropriate amount of ethanol into the supernatant for precipitation, collecting the precipitate, and drying the precipitate to constant weight to obtain gamma-polyglutamic acid;
(4) preparing natto kinase powder: taking the soybean sample collected in the step (3), pre-freezing at-18 ℃ for 12h, freeze-drying at-34 ℃ for 36h, and grinding to obtain nattokinase powder.
More preferably, the volume ratio of the ethanol to the supernatant in the step (3) of the method for producing the gamma-polyglutamic acid is 2-4: 1, and the mass fraction of the ethanol is 95%.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention screens and obtains the Bacillus belgii for producing the gamma-polyglutamic acid by low-ammonia fermentation from the fermented soybeans, and the gamma-polyglutamic acid is fermented and extracted by three steps of strain activation, solid fermentation and purification extraction, thereby realizing high yield of the gamma-polyglutamic acid and low ammonia gas generation amount in the fermentation process.
2. The yield of gamma-polyglutamic acid prepared by fermenting the Bacillus belgii with a soybean culture medium reaches 11.02g/100g, the volume concentration of generated ammonia gas is 0-1333.3 ppm, and even no ammonia gas is generated.
3. Compared with a mixed fermentation method, the ammonia gas production amount in the fermentation process is lower. Compared with the construction of genetic engineering strains, the stability of the fermentation production process is higher, and the method can be applied to large-scale industrial production.
Drawings
FIG. 1 is a photograph of commercially available fermented soybeans.
FIG. 2 is a phylogenetic tree constructed from the 16SrDNA sequence of the Bacillus belgii strain of the present invention.
FIG. 3 is a histogram comparing the contents of polyglutamic acid fermented by Bacillus belgii of the present invention with commercially available histamins produced daily and natto powder produced in a certain country.
FIG. 4 is a bar graph comparing the specific activities of Bacillus belgii, commercially available high-yield bridosin and natto powder produced in some country.
FIG. 5 is a bar graph comparing the volume concentration of ammonia gas produced during fermentation of Bacillus belgii of the present invention with commercially available histatin produced daily and natto powder produced in a country.
Detailed Description
Example 1
Isolation and screening of bacillus beilesiensis yzy21. a01:
the separation and screening comprises the following steps:
(1) and (3) thallus enrichment: adding 1g commercially available fermented soybean (photograph is shown in figure 1) into nutrient broth (liquid content 20mL/250mL triangular flask), heating in water bath at 80 deg.C for 20min, and shake culturing at 30 deg.C for 24h (150 r/min).
(2) Primary screening: adding 1mL of the enrichment solution obtained in the step (1) into 9mL of sterile water to obtain 10-1Sample diluent, and different gradient diluents are obtained in sequence in the method. Each of the dilutions was applied in an amount of 0.1mL to a nutrient agar plate and incubated at a constant temperature of 35 ℃ for 24 hours.
(3) Separation and purification: and selecting the strains with high growth speed, large bacterial colonies and sticky surfaces to corresponding plates, and further separating and purifying.
(4) Re-screening: the separated and purified strain was inoculated into a soybean solid medium (soybean content: 50g soybean/300 mL Erlenmeyer flask), and left to ferment at a constant temperature of 35 ℃ for 48 hours. Then, the amount of gamma-polyglutamic acid generated by fermentation is measured by adopting an ethanol precipitation method, the enzyme activity of the nattokinase is detected by adopting a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), and the volume concentration of ammonia gas is detected by adopting a gas detector. Screening out bacterial strains which can produce gamma-polyglutamic acid with high yield and can not produce ammonia gas in the fermentation process.
The ethanol precipitation method is specifically implemented as follows: taking 50g of the soybeans fermented for 48 hours in the step (3), adding 50mL of distilled water at 60 ℃, adjusting the pH value to 7.0-8.0, shaking for 15min, and filtering. Adding 95% ethanol (1: 3 ═ V: V), centrifuging (8000r/min, 5min, 25 deg.C), collecting precipitate, oven drying, and weighing. The content of γ -polyglutamic acid (weight of γ -polyglutamic acid/weight of soybean after oven drying) x 100%.
The specific implementation method of the volume concentration of the ammonia gas is as follows: taking the soybeans fermented for 48 hours in the step (3), opening the cotton bottle stopper of the fermentation triangular flask, aligning the gas detection port of the ammonia gas detector with the bottle opening, and recording after the reading is stable.
The specific implementation method for detecting the enzyme activity of the nattokinase by a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) comprises the following steps: preparing standard substances with the concentration of 50U/L, 100U/L, 200U/L, 400U/L and 800U/L, respectively adding 50 μ L of standard solution into 48-well plate, adding 100 μ L of detection antibody labeled by horseradish peroxidase (HRP) into each well, and incubating in 37 ℃ incubator for 60 min. Discarding liquid, filling washing liquid into each hole, washing for 5 times, adding A, B color developing agents 50 mu L into each hole, incubating for 15min in a dark place at 37 ℃, adding stop solution 50 mu L into each hole, measuring the light absorption value at 450nm by an enzyme standard instrument within 15min, and drawing a standard enzyme activity-light absorption value standard curve of a standard product.
Taking a soybean sample fermented for 48h, pre-freezing at the temperature of minus 18 ℃ for 12h, freeze-drying at the temperature of minus 34 ℃ for 36h, grinding, adding a proper amount of normal saline into the powder, mashing, centrifuging (3000r/min, 10min), and taking supernatant. Adding 10 mu L of sample to be detected into the sample hole, adding 40 mu L of sample diluent, measuring the light absorption value of the sample at 450nm according to the method, calculating the enzyme activity (U/L) of the sample to be detected according to the standard curve of the concentration-light absorption value of the standard substance, and further obtaining the specific activity (U/g) of the nattokinase of the sample to be detected.
Example 2
Identification of bacillus beilesiensis yzy21. a01:
extracting and re-screening the DNA of the obtained strain by using a bacterial genome DNA extraction kit, and carrying out PCR amplification on 16S rRNA by using an upstream primer 27F and a downstream primer 1492R. And recovering and purifying the product gel after amplification, and sending the product gel to Guangzhou Egyptian biotechnology limited company for gene sequencing.
Sequencing results show that the length of the 16S rRNA gene of the strain is 1389bp, and the gene sequence is shown as SEQ ID No. 1. The sequencing result is compared with NCBI database, so that the known sequence with the highest homology with the 16S rDNA sequence of the bacterium can be obtained. Through comparison, the similarity with Bacillus velezensis is the highest, and the matching degree reaches 99.93 percent (shown in figure 2). Therefore, the Bacillus subtilis isolated and screened by the invention is considered to be the Bacillus belgii, and is specifically named as Bacillus subtilis YZHY21.A01(Bacillus velezensis YZHY21.A01).
Example 3
Production of gamma-polyglutamic acid and nattokinase powder by low-ammonia fermentation of Bacillus belgii
(1) And (3) activation of thalli: the Bacillus belgii slant was removed from the freezer at 4 ℃ and placed in an incubator at 28 ℃ for activation for 2 h.
(2) Solid fermentation: taking the strain slant in the step (1), cleaning the slant lawn with 5mL of sterile water on each slant under the aseptic condition, inoculating 4% of the slant lawn into a soybean culture medium (containing 50g of wet soybeans per 300mL of triangular flask), and fermenting at constant temperature for 48h, wherein the fermentation temperature is 28 ℃.
(3) Extracting gamma-polyglutamic acid: adding sterile water into a soybean culture medium fermented for 48 hours, adjusting the pH to 7.0-8.0, shaking for 15min, and filtering. The filtrate was centrifuged (8000r/min, 5min, 25 ℃), and the cells and a small amount of soybean residue were removed to obtain a supernatant. Adding 95% ethanol (1: 3 ═ V: V) into the supernatant for precipitation, finally collecting the precipitate, and drying the precipitate to constant weight to obtain the gamma-polyglutamic acid.
(4) Natto kinase powder: taking a soybean sample fermented for 48 hours, pre-freezing for 12 hours at the temperature of minus 18 ℃, freeze-drying for 36 hours at the temperature of minus 34 ℃, and grinding.
Comparative examples
The same fermentation steps as in example 3 were carried out using hispidulin produced daily and natto powder produced in some country, respectively. The fermentation product is detected (example 1 detection method), analyzed and compared, and the result shows that:
in example 3, the content of gamma-polyglutamic acid produced by fermentation of Bacillus beleisi YZHY21.A01 was 11.02%, which was 104% of commercially available homofunosin produced on a daily basis and 131% of natto powder produced in a certain country (shown in FIG. 3).
② the other product of the fermented soybean, namely the specific activity of the nattokinase reaches 61 percent of the commercially available homodesmosine produced in daily life and the natto powder produced in a certain country, and is about 0.6U/g (shown in figure 4).
③, the volume concentration of ammonia gas is 1333.3ppm after the fermentation of 48h in the example 3, 6666.7ppm after the fermentation of 48h by adopting Gaoqiao natto essence, and 12000.0ppm after the fermentation of 48h by adopting natto powder produced in a certain country. By comparison, it was found that the ammonia production by fermentation using Bacillus belgii YZHY21.A01 of the present invention was significantly reduced compared to the other two commercially available microbial agents (shown in FIG. 5).
Example 4
(1) And (3) activation of thalli: the Bacillus belgii slant was removed from the freezer at 4 ℃ and placed in an incubator at 28 ℃ for activation for 2 h.
(2) Solid fermentation: taking the strain slant in the step (1), cleaning the slant lawn with 5mL of sterile water on each slant under the aseptic condition, inoculating 4% of inoculum size into black bean culture medium (bean amount: 50g wet black bean/300 mL triangular flask), fermenting at constant temperature for 48h, and fermenting at 28 ℃.
(3) Extracting gamma-polyglutamic acid: adding sterile water into the black bean culture medium fermented for 48 hours, adjusting the pH to 7.0-8.0, shaking for 15min, and filtering. The filtrate was centrifuged (8000r/min, 5min, 25 ℃), and the cells and a small amount of black bean residue were removed to obtain a supernatant. Adding 95% ethanol (1: 3 ═ V: V) into the supernatant for precipitation, finally collecting the precipitate, and drying the precipitate to constant weight to obtain the gamma-polyglutamic acid.
(4) Natto kinase powder: taking a black bean sample fermented for 48 hours, pre-freezing at the temperature of minus 18 ℃ for 12 hours, freeze-drying at the temperature of minus 34 ℃ for 36 hours, and grinding.
Through detection (the detection method in example 1), the content of the gamma-polyglutamic acid produced by fermentation in the embodiment is 4.20%; the volume concentration of ammonia gas in the fermentation process is 0ppm, and no ammonia gas is generated in 48 hours of fermentation.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Sequence listing
<110> Anhui Yuanzhihui Biotechnology Limited
<120> Bacillus belgii for producing gamma-polyglutamic acid by low-ammonia fermentation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1389
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaggttacc tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg gcggtgtgta 60
caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc gattccagct 120
tcacgcagtc gagttgcaga ctgcgatccg aactgagaac agatttgtgg gattggctta 180
acctcgcggt ttcgctgccc tttgttctgt ccattgtagc acgtgtgtag cccaggtcat 240
aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc ggcagtcacc 300
ttagagtgcc caactgaatg ctggcaacta agatcaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca ctctgccccc 420
gaaggggacg tcctatctct aggattgtca gaggatgtca agacctggta aggttcttcg 480
cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg 540
agtttcagtc ttgcgaccgt actccccagg cggagtgctt aatgcgttag ctgcagcact 600
aaggggcgga aaccccctaa cacttagcac tcatcgttta cggcgtggac taccagggta 660
tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt cagttacaga ccagagagtc 720
gccttcgcca ctggtgttcc tccacatctc tacgcatttc accgctacac gtggaattcc 780
actctcctct tctgcactca agttccccag tttccaatga ccctccccgg ttgagccggg 840
ggctttcaca tcagacttaa gaaaccgcct gcgagccctt tacgcccaat aattccggac 900
aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt ggctttctgg 960
ttaggtaccg tcaaggtgcc gccctatttg aacggcactt gttcttccct aacaacagag 1020
ctttacgatc cgaaaacctt catcactcac gcggcgttgc tccgtcagac tttcgtccat 1080
tgcggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg 1140
tggccgatca ccctctcagg tcggctacgc atcgtcgcct tggtgagccg ttacctcacc 1200
aactagctaa tgcgccgcgg gtccatctgt aagtggtagc cgaagccacc ttttatgtct 1260
gaaccatgcg gttcaaacaa ccatccggta ttagccccgg tttcccggag ttatcccagt 1320
cttacaggca ggttacccac gtgttactca cccgtccgcc gctaacatca gggagcaagc 1380
tcccatctg 1389
Claims (7)
1. The Bacillus belgii classified name of Bacillus velezensis YZHY21.A01(Bacillus velezensis YZHY21.A01) for producing gamma-polyglutamic acid by low-ammonia fermentation is preserved in Guangdong province microorganism strain collection center, and the collection number is GDMCC NO: 61921, the preservation date is 2021, 8 and 31 days, and the preservation unit addresses are: the Michelson Dai No. 59 building 5, Ministry of sciences, Guangdong province, institute of microbiology, postal code 510070, Ministry of sciences, Michelson, Guangzhou, China.
2. Use of Bacillus belgii according to claim 1 for the low ammonia fermentation production of gamma-polyglutamic acid.
3. Use of Bacillus belgii according to claim 1 for the production of nattokinase by low ammonia fermentation.
4. The use according to claim 2 or 3, wherein the gamma-polyglutamic acid and nattokinase are prepared by inoculating Bacillus belgii YZHY21.A01 into a fermentation medium for fermentation.
5. The use of claim 4, wherein the fermentation medium is a soy medium.
6. The use of claim 5, wherein the method for producing gamma-polyglutamic acid by low ammonia fermentation using Bacillus belgii comprises:
(1) and (3) activation of thalli: taking out the Bacillus beiLeisi inclined plane from a refrigerated cabinet at 4 ℃, and placing the Bacillus beiLeisi inclined plane in an incubator at 28 ℃ for activation for 2-6 h;
(2) solid fermentation: taking the strain inclined planes in the proper step (1), cleaning the inclined plane lawn with 5mL of sterile water on each inclined plane under the aseptic condition, inoculating 1-4% of the strain amount into a soybean culture medium, and fermenting at a constant temperature of 28-35 ℃ for 24-48 h;
(3) extracting gamma-polyglutamic acid: adding sterile water into a soybean culture medium after fermentation for 24-48 h, adjusting the pH to 7.0-8.0, shaking for 15min, filtering, centrifuging the filtrate, removing thalli and a small amount of soybean residues to obtain a supernatant, adding an appropriate amount of ethanol into the supernatant for precipitation, collecting the precipitate, and drying the precipitate to constant weight to obtain gamma-polyglutamic acid;
(4) preparing natto kinase powder: taking the soybean sample collected in the step (3), pre-freezing at-18 ℃ for 12h, freeze-drying at-34 ℃ for 36h, and grinding to obtain nattokinase powder.
7. The use according to claim 6, wherein the volume ratio of the ethanol to the supernatant in the step (3) is 2-4: 1, and the mass fraction of the ethanol is 95%.
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| CN112143663A (en) * | 2020-07-08 | 2020-12-29 | 常熟理工学院 | Bacillus belgii strain and application thereof in synthesis of gamma-polyglutamic acid |
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| CN112143663A (en) * | 2020-07-08 | 2020-12-29 | 常熟理工学院 | Bacillus belgii strain and application thereof in synthesis of gamma-polyglutamic acid |
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