CN112143663B - Bacillus belgii strain and application thereof in synthesis of gamma-polyglutamic acid - Google Patents

Bacillus belgii strain and application thereof in synthesis of gamma-polyglutamic acid Download PDF

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CN112143663B
CN112143663B CN202010649532.3A CN202010649532A CN112143663B CN 112143663 B CN112143663 B CN 112143663B CN 202010649532 A CN202010649532 A CN 202010649532A CN 112143663 B CN112143663 B CN 112143663B
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吴凌天
韩靖贤
赵国祥
朱益波
孟佳琪
蔡轶婧
吴金男
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Abstract

The invention discloses a strain of Bacillus belgii, which is classified and named as Bacillus velezensis LT-2 and is preserved in China center for type culture collection with the preservation number of CCTCC No: M2019904 and the preservation date of 2019, 11 months and 7 days. The invention also discloses application of the Bacillus belgii in synthesis of gamma-polyglutamic acid. The strain takes non-grain raw material sugarcane juice as a carbon source, peanut cake powder as an organic nitrogen source and ammonium chloride as an inorganic nitrogen source, the yield of the gamma-polyglutamic acid synthesized by fermentation can reach 47.61g/L, and the production rate is as high as 0.61 g/L/h. The Bacillus bleekensis LT-2 disclosed by the invention can be used for synthesizing gamma-polyglutamic acid without depending on glutamic acid fermentation, the total sugar conversion rate is up to 47.61%, the operation method is simple, the cost is lower, the industrial popularization and application prospect is extremely good, and a new process is provided for the biosynthesis of gamma-polyglutamic acid.

Description

Bacillus belgii strain and application thereof in synthesis of gamma-polyglutamic acid
Technical Field
The invention particularly relates to Bacillus belgii and application thereof in synthesis of gamma-polyglutamic acid, and belongs to the technical fields of microbiology, bioengineering technology and chemical engineering.
Background
Gamma-Polyglutamic acid (gamma-PGA) is composed of 5000 glutamic acid residues with 500-one, and is in a straight-chain fiber shape, and the relative molecular mass is usually 100-1000 kDa. Because the side chain of the water-absorbing agent contains a large amount of free carboxyl with higher activity, the water-absorbing agent has the characteristics of water absorption, moisture retention, chelating elements and the like. Therefore, the gamma-polyglutamic acid has wide application prospect in the fields of daily chemicals, food, environmental protection, agriculture, feed industry and the like. Most of the production strains reported at present are glutamic acid dependent gamma-polyglutamic acid synthesis strains, such as B.subtilis NX-2, B.subtilis RKY3, B.subtilis ZJU-7 and the like, the gamma-polyglutamic acid can be synthesized by adding glutamic acid into a culture medium, and the concentration of the product can reach 20-50 g/L. However, the culture medium needs to be added with a large amount of glutamic acid, so that the cost is high, and the price of the gamma-polyglutamic acid is up to 3000 yuan/kg, so the application of the gamma-polyglutamic acid is mainly limited in the field of daily chemicals, and the application of the gamma-polyglutamic acid in the fields of agriculture and feed is limited. And the other glutamic acid independent type gamma-polyglutamic acid synthesis strain does not need to add glutamic acid additionally, so that the fermentation cost of the gamma-polyglutamic acid is greatly reduced, such as B.subtilis C10, B.methylotrophicus SK19.001, B.amyloliquefaciens LL3 and the like, however, the gamma-polyglutamic acid yield of the strain is extremely low, and no report for production exists so far, and the strain is a research hotspot in the field of the current gamma-polyglutamic acid microbial synthesis.
Figure BDA0002574375010000011
Through retrieval, the existing reported technology only aims at the problems that the beta-bacillus velezensis can ferment and produce the gamma-polyglutamic acid under the condition of adding exogenous glutamic acid, and the raw material utilization rate is low, the production cost is high and the like, so that the large-scale industrial production is difficult, and the large-scale development and application of the gamma-polyglutamic acid are limited. The invention reports a novel method for synthesizing gamma-polyglutamic acid by fermenting the Bacillus bleekensis LT-2 with sugarcane juice as a raw material under the condition of not adding glutamic acid (namely, the glutamic acid is not dependent) for the first time. In addition, the Bacillus belgii used in the invention is a food-safe microorganism and has great industrial application value.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the problem of providing a Bacillus belgii B.velezensis LT-2 which utilizes sugarcane juice to synthesize gamma-polyglutamic acid independently of glutamic acid.
The technical problem to be solved by the invention is to provide the application of the bacillus beilesensis.
The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention obtains a Bacillus velezensis strain B.velezensis from vinasse by screening, the strain is named as Bacillus velezensis LT-2(Bacillus velezensis LT-2) by classification, and the strain is preserved in China center for type culture Collection (CCTCC for short), and the preservation address is as follows: wuhan city, Hubei province, eight mountainous areas, Wuhan university, mountain type culture collection, zip code: 430072, preservation number is CCTCC No: m2019904, and the preservation date is 11/7/2019. The following all refer to this strain as a production strain.
The strain has the following properties:
1. the morphological characteristics and physiological and biochemical characteristics of the colonies are shown in Table 1.
TABLE 1 morphological characteristics and physiological and biochemical characteristics of colonies
Figure BDA0002574375010000021
2. 16S rDNA sequence analysis:
the length of the nucleotide sequence of the 16S rDNA gene of the strain is 1396bp, and the gene sequence is shown as SEQID No. 1: as shown. The sequences tested were compared for homology from the Gene Bank database using the BLAST program to construct a phylogenetic tree based on the 16S rDNA full sequence. The results show that: the strain achieves 100% homology with B.velezensis zjt 9. According to the results of morphological observation and analysis of physiological and biochemical experiments of strains, the bacillus subtilis B.velezensis LT-2 is determined to be used in the invention.
The application of the Bacillus belgii B.velezensis LT-2 in synthesizing gamma-polyglutamic acid by utilizing the sugarcane juice independent glutamic acid fermentation is also within the protection scope of the invention.
The specific application method comprises the following steps: inoculating Bacillus belgii LT-2 into a slant solid culture medium, then transferring to a seed culture medium, and finally inoculating into a fermentation culture medium without glutamic acid for aerobic culture, wherein the fermentation liquid is rich in gamma-polyglutamic acid.
Wherein, the fermentation medium comprises the following components: 10-120 g/L of carbon source, 2-20 g/L of nitrogen source, 1.0-8.0 g/L of metal salt, water as solvent and 6.5-7.5 of pH value.
Wherein the carbon source is any one or combination of more of glycerol, glucose, lactose, maltose, sucrose and sugarcane juice;
the nitrogen source is any one or a combination of more of beef extract, corn steep liquor, soybean cake powder, peanut cake powder, peptone, yeast powder, ammonium chloride, ammonium sulfate, urea, diammonium hydrogen phosphate and ammonium nitrate;
the metal salt is any one or combination of magnesium sulfate, monopotassium phosphate, dipotassium phosphate, manganese sulfate and calcium chloride.
Wherein the fermentation medium comprises the following components: 10-80 g/L of sugarcane juice, 1-8 g/L of peanut cake powder, 5-25 g/L of ammonium chloride, 2-8 g/L of monopotassium phosphate, 0.2-1.2 g/L of magnesium sulfate and 0.002-0.008 g/L of manganese sulfate, and adjusting the initial pH of the fermentation liquor to 6.0-7.5 by using ammonia water.
Preferably, the carbon source is one or a combination of sugarcane juice and sucrose.
Preferably, the aerobic culture conditions are: the initial pH is 6.5-7.5, and the culture temperature is 28-32 ℃;
when aerobic culture is shake flask culture, the shake flask inoculation amount is 1-8% (volume fraction), and the culture time is 36-54 h;
when aerobic culture is fermentation tank culture, the inoculation amount of the fermentation tank is 1-15% (volume fraction), the fermentation mode is fed-batch method, the aeration ratio is 1.0-1.4 VVM, and the culture time is 48-96 h.
The bacillus belgii B.velezensis LT-2 and the application of the bacillus belgii in preparing the gamma-polyglutamic acid sequentially comprise the following steps:
1. preparing a culture medium:
(1) the slant culture medium comprises the following components: 5g/L of glucose, 5g/L of peptone, 3g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder and water as a solvent, wherein the pH value is 6.5-7.5, and the preferable pH value is 7;
(2) the liquid seed culture medium is as follows: 2-10 g/L of sucrose, 2g/L of yeast powder, 3g/L of ammonium sulfate, 5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, water as a solvent, 6.5-7.5 of pH value, and preferably 7 of pH value;
(3) the solid seed culture medium is as follows: 2-10 g/L of sucrose, 2g/L of yeast powder, 3g/L of ammonium sulfate, 5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 20g/L of agar powder and water as a solvent, wherein the pH value is 6.5-7.5, and the preferable pH value is 7;
(4) fermentation medium: 10-120 g/L of carbon source, 2-20 g/L of nitrogen source, 1.0-8.0 g/L of metal salt, water as solvent, 6.5-7.5 of pH value, and preferably 7 of pH value;
the carbon source is any one or combination of more of glycerol, sucrose, maltose, sugarcane juice, glucose and lactose, the preferred carbon source is the sugarcane juice, and the most preferred carbon source is that the total sugar concentration in the culture medium after the sugarcane juice is added is 50 g/L;
the nitrogen source comprises an inorganic nitrogen source and an organic nitrogen source, and the organic nitrogen source is any one or a combination of more of beef extract, corn steep liquor, soybean cake powder, peanut cake powder, peptone and yeast powder; the organic nitrogen source is preferably peanut cake powder, and the concentration of the peanut cake powder is 5 g/L; the inorganic nitrogen source is any one or combination of more of ammonium chloride, ammonium sulfate, urea, diammonium hydrogen phosphate and ammonium nitrate, the inorganic nitrogen source is preferably ammonium chloride, and the concentration of the ammonium chloride is preferably 15 g/L;
the metal salt is one or a combination of magnesium sulfate, monopotassium phosphate, dipotassium phosphate, manganese sulfate and calcium chloride, the metal salt is preferably a composition of monopotassium phosphate, magnesium sulfate and manganese sulfate, and the most preferable metal salt concentration is 0.80g/L of magnesium sulfate, 5g/L of monopotassium phosphate and 0.005g/L of manganese sulfate.
The most preferred fermentation medium comprises the following components: 10-80 g/L of sugarcane juice, 1-8 g/L of peanut cake powder, 5-25 g/L of ammonium chloride, 2-8 g/L of monopotassium phosphate, 0.2-1.2 g/L of magnesium sulfate and 0.002-0.008 g/L of manganese sulfate, and adjusting the initial pH of the fermentation liquor to 6.0-7.5 by using ammonia water. Wherein the sugarcane juice is prepared by squeezing fresh sugarcane, and the optimal total sugar concentration of the culture medium is 50 g/L.
2. Selecting strains:
the deposited strain B.velezensis LT-2 was selected.
3. Activating strains:
inoculating a B.velezensis LT-2 strain to a slant culture medium, performing static culture at 24-36 ℃ for 16-24 h, picking a single colony again, streaking the single colony on the slant culture medium, and performing culture at 24-36 ℃ for 16-24 h to obtain an activated strain for later use;
4. preparing a seed solution:
taking the activated strain in the step (3), inoculating 2-4 rings of the strain into a shake flask containing the seed solution under an aseptic condition, placing the shake flask on a shaking table with the rotation speed of 180rpm, and culturing at the temperature of 24-36 ℃ for 16 hours to obtain a fermented seed solution;
5. and (3) shake flask fermentation culture:
inoculating the seed liquid fermented in the step (4) into a fermentation culture medium in an inoculation amount of 1-8% (v/v) under an aseptic condition, placing the seed liquid on a shaking table with the rotating speed of 200rpm, and culturing for 36-54 h at 24-36 ℃; stopping fermentation when the concentration of the gamma-polyglutamic acid in the fermentation liquor does not rise any more;
6. fermentation culture in a fermentation tank:
inoculating the seed solution fermented in the step (4) into a fermentation tank in an inoculation amount of 1-15% (v/v) under an aseptic condition, wherein the liquid loading amount is 3L/5L, the rotating speed is 200-500 rpm, the aeration ratio is 1.0-1.2 VVM, the culture temperature is 24-36 ℃, the initial pH is 6.5-7.5, and the culture time is 48-96 hours; stopping fermentation when the concentration of the gamma-polyglutamic acid in the fermentation liquor does not rise any more;
7. and (3) determining the content of the gamma-polyglutamic acid:
the concentration of gamma-polyglutamic acid was determined by gel permeation chromatography: and (4) diluting the fermentation liquor obtained in the step (5) or (6) by 5 times, centrifuging at 12,000rpm for 20min to remove thalli, filtering the supernatant by using a filter membrane of 0.22 mu m, and placing the filtrate in a sample injection bottle for later use. Detecting a sample by using a differential detector, and carrying out chromatographic column: superposeTM6; mobile phase: 50mM NaCl: aqueous acetonitrile (4:1, v/v), flow rate: 1.0 mL/min; sample introduction amount: 20 mu L of the solution; calculating gamma-ion in fermentation liquor by contrasting gamma-polyglutamic acid standard substancePolyglutamic acid content.
8. Extracting gamma-polyglutamic acid:
diluting a B.velezensis LT-2 fermentation broth by 5 times, centrifuging at 12,000rpm to remove thalli, concentrating the supernatant into 1/5 with the original volume by rotary evaporation at 65 ℃, adding 3-5 times of anhydrous ethanol, and centrifuging to obtain a precipitate; re-dissolving the precipitate with double distilled water, filtering with 0.22 μm filter membrane to remove insoluble substances such as thallus debris, dialyzing with double distilled water overnight, concentrating the dialysate by rotary evaporation, adjusting pH to 2.5 with HCl, rapidly adding 8 times volume of 1:1(v/v) propanol/ether solution to obtain H-type gamma-polyglutamic acid precipitate, and freeze-drying in vacuum drying oven to obtain pure H-type gamma-polyglutamic acid.
9. Identification of gamma-polyglutamic acid
And identifying the B.velezensis LT-2 fermentation product of the Bacillus belgii by adopting a high performance liquid chromatograph, an infrared spectrometer and a nuclear magnetic resonance spectrometer.
Analysis of hydrolysis Components of the product
Taking 0.1g of a fermentation and purification product of B.velezensis LT-2 in a hydrolysis bottle, adding 3mL of 72% sulfuric acid, mixing, carrying out water bath at 30 ℃ for 60min, taking out, adding 84mL of double distilled water, diluting to 5%, and mixing uniformly. After being treated at 121 ℃ for 1h, the mixture is taken out and cooled, the pH value is adjusted to 6.5, hydrolysate of a fermentation product of the Bacillus belgii B.velezensis LT-2 is obtained, and the hydrolysate is subjected to liquid chromatography analysis.
Measurement of molecular weight
Measuring the molecular weight of the B.velezensis LT-2 fermentation product by using gel permeation chromatography, redissolving the purified product, removing insoluble substances by using a 0.22 mu m filter membrane, and placing the product in a sample bottle for later use. A chromatographic column: shodex Ohpak SB-806M HQ; a chromatographic column: superposeTM6; mobile phase: 0.2M pH 4.0Na2SO4Solution, flow rate: 1.0 mL/min; sample introduction amount: 20 mu L of the solution; and comparing the peak time of the sample with the gamma-polyglutamic acid standard substance to determine the molecular weight of the product.
Analysis of infrared spectrum and nuclear magnetic resonance
Determination of Bacillus belgii B.v by infrared spectrometerInfrared spectrum of the elezensis LT-2 fermentation purified product: grinding and tabletting 0.2mg of a sample and a small amount of KBr to prepare a sample, then measuring an infrared spectrum of the sample by using an infrared spectrometer, and scanning the sample within the range of 4000-400 cm-1. With D2Performing NMR on the purified product of Bacillus belgii B.velezensis LT-2 fermentation with O as solvent1H and NMR13And C, detecting.
Has the advantages that: the invention has the following advantages:
(1) the invention obtains a non-glutamic acid-dependent gamma-polyglutamic acid synthetic strain B.velezensis LT-2 by screening for the first time.
(2) The strain can use sugarcane juice and/or sucrose as a cheap carbon source, and can be used for fermenting and synthesizing gamma-polyglutamic acid without adding glutamic acid, the yield of the gamma-polyglutamic acid can reach 47.61g/L, the molecular weight of the gamma-polyglutamic acid is 1500kDa, the highest substrate conversion rate can reach 47.61%, the production rate is 0.61g/L/h, the production cost is greatly reduced, the operation is simple, and the strain has very important social and economic significance for the production of the gamma-polyglutamic acid and the development and application of sugarcane resources.
Drawings
FIG. 1 shows a 16S rDNA PCR purification agarose gel electrophoresis of B.beijerinckii LT-2 (A) and a phylogenetic tree of B.beijerinckii LT-2 (B).
FIG. 2 is a high performance liquid chromatogram of a glutamic acid standard (A); the high performance liquid chromatogram (B) of the gamma-polyglutamic acid hydrolysate produced by the Bacillus belgii LT-2.
FIG. 3: the left graph is a gamma-polyglutamic acid molecular weight determination standard curve; the right graph shows the distribution of the molecular weight of gamma-polyglutamic acid produced by Bacillus belgii LT-2.
FIG. 4 is an infrared spectrum of gamma-polyglutamic acid produced by Bacillus belgii LT-2.
FIG. 5 is a Nuclear Magnetic Resonance (NMR) chart of gamma-polyglutamic acid production by Bacillus belgii LT-21And (H) map.
FIG. 6 is a Nuclear Magnetic Resonance (NMR) chart of gamma-polyglutamic acid production by Bacillus belgii LT-213And (C) a map.
FIG. 7 Effect of carbon source species on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 8 Effect of carbon source concentration on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 9 Effect of organic nitrogen source species on the synthesis of microbial polysaccharides by Bacillus belgii LT-2.
FIG. 10 Effect of organic nitrogen source concentration on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 11 Effect of inorganic nitrogen source species on the synthesis of microbial polysaccharides by Bacillus belgii LT-2.
FIG. 12 Effect of inorganic nitrogen source concentration on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 13 effect of temperature on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 14 Effect of pH on polysaccharide synthesis by Bacillus belgii LT-2.
FIG. 15 is a graph showing the progress of horizontal synthesis of gamma-polyglutamic acid by Bacillus belgii LT-2 in a shake flask.
FIG. 16 is a graph showing the progress of a batch fed synthesis of gamma-polyglutamic acid in a 50L fermenter.
FIG. 17 is a graph showing the progress of fed batch synthesis of gamma-polyglutamic acid in a 1t fermenter.
FIG. 18 is a process curve of the synthesis of gamma-polyglutamic acid by externally adding sodium glutamate into a 1t fermentation tank.
Detailed Description
The present invention can be better understood from the following examples, however, those skilled in the art will readily appreciate that the descriptions of the examples are only for the purpose of illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1: isolation and screening of B.belgii B.velezensis LT-2.
The composition of the medium used in this example is as follows:
(1) enriching a liquid culture medium: 20g/L of sucrose, 2g/L of yeast powder, 3g/L of ammonium sulfate, 5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.005g/L of manganese sulfate, water as a solvent and a pH value of 6.5-7.5;
(2) solid screening culture medium: 20g/L of sucrose, 2g/L of yeast powder, 3g/L of ammonium sulfate, 5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 20g/L of agar powder and water as a solvent, wherein the pH value is adjusted to 6.5-7.5;
(3) fermentation medium: 50g/L of sucrose, 5g/L of peanut cake powder, 15g/L of ammonium chloride, 5g/L of monopotassium phosphate, 0.8g/L of magnesium sulfate, 0.005g/L of manganese sulfate, water as a solvent and 6.5-7.5 of pH value;
the specific operation process of this embodiment is as follows:
the steps for screening the gamma-polyglutamic acid producing strain are as follows: 2g of each of 50 portions of vinasse is respectively inoculated into a triangular shake flask filled with an enrichment medium, the liquid loading amount is 80mL/500mL, and enrichment culture is carried out for 24h under the conditions of 32 ℃ and 180 rpm. Transferring 3.2mL of culture solution into the same liquid enrichment culture medium, performing the second enrichment culture under the same conditions for 24h, and repeating for 1 time, namely enriching for 3 times. Diluting the third enriched culture fluid to 10 under aseptic conditions-8And 10-9Respectively coating 0.2mL of the strain on a solid screening culture medium, culturing for 24h at 32 ℃, after single colonies grow out, selecting the single colonies which grow quickly and are viscous, inoculating the single colonies into a seed culture medium, culturing to a logarithmic phase, inoculating the single colonies into a fermentation culture medium according to the inoculation amount of 5%, placing the single colonies on a shaking table, culturing for 48h at 32 ℃ at the rotating speed of 200rpm, collecting fermentation liquor, centrifuging, taking supernate, passing the supernate through a 0.22 mu m filter membrane, and measuring the yield of the primary screening strain gamma-polyglutamic acid by using liquid chromatography to obtain the strain with the highest yield.
Example 2: identification of B.belgii B.velezensis LT-2.
A bacterial genome DNA extraction kit is used for extracting genome DNA of B.velezensis LT-2, 16S rDNA sequences are amplified by PCR with an upstream primer 27F and a downstream primer 1492R as shown in figure 1A, products after PCR amplification are subjected to gel recovery and purification, and the gel recovery and purification products are sent to Suzhou Jinzhi Biotech limited for sequencing. The nucleotide sequence length of the 16S rDNA gene of the strain obtained by sequencing is 1396bp, and the gene sequence is shown as SEQID No. 1. BLAST comparison of the sequencing results with known 16S rDNA sequences in GeneBank databases and homology comparison using the BLAST program resulted in the construction of phylogenetic trees based on the 16S rDNA full sequences. The results show that: this strain reached 100% homology with bacillus belgii B. velezensis zjt9 (fig. 1B). According to the results of strain morphology observation and physiological and biochemical experiment analysis, the Bacillus belgii is identified to be used in the invention, and is specifically named as Bacillus belgii B.
Example 3: identification of Bacillus belgii B.velezensis LT-2 fermentation products
Analysis of the hydrolyzate
0.1g of B.velezensis LT-2 fermentation purified product is taken to be put into a hydrolysis bottle, 3mL of 72% sulfuric acid is added to be mixed, water bath is carried out for 60min at the temperature of 30 ℃, double distilled water is added to be diluted to 5% after being taken out, and the mixture is evenly mixed. After being treated at 121 ℃ for 1h, the hydrolysate is taken out and cooled, the pH value is adjusted to 6.5, and the liquid chromatogram result of the hydrolysate which is fermented and purified is shown in figure 2. As can be seen from the amino acid high performance liquid chromatogram, the fermentation product of Bacillus bleekensis LT-2 has 1 amino acid component in total and is consistent with the peak time of a glutamic acid standard product, so that the fermentation product can be preliminarily determined to be gamma-polyglutamic acid.
Measurement of molecular weight
Measuring the molecular weight of the B.velezensis LT-2 fermentation product by using gel permeation chromatography, redissolving the purified product, removing insoluble substances by using a 0.22 mu m filter membrane, and placing the product in a sample bottle for later use. A chromatographic column: shodex Ohpak SB-806M HQ; a chromatographic column: superposeTM6; mobile phase: 0.2M Na pH 4.02SO4Solution, flow rate: 1.0 mL/min; sample introduction amount: 20 mu L of the solution; the peak time of the sample was compared with that of the gamma-polyglutamic acid standard, and it was confirmed from FIG. 3 that the molecular weight of B.velezensis LT-2 fermentation product gamma-polyglutamic acid was 1500 kDa.
Analysis of infrared spectrum and nuclear magnetic resonance
Measuring the infrared spectrum of the B.velezensis LT-2 fermentation and purification product by using an infrared spectrometer, grinding and tabletting a sample of 0.2mg and a small amount of KBr to prepare a sample, measuring the infrared spectrum of the sample by using the infrared spectrometer, and scanning the sample in the range of 4000-400 cm-1. And (3) taking deuterium water as a solvent, and carrying out one-dimensional nuclear magnetic resonance detection on the B.velezensis LT-2 fermentation product. KnotAs shown in FIGS. 4, 5 and 6, the substance has infrared spectrum and NMR compared with gamma-polyglutamic acid standard1H and NMR13The characteristic peak of the B.velezensis LT-2 fermentation product shown by C is consistent with that of standard gamma-polyglutamic acid. B. velezensis LT-2 fermentation product was determined to be gamma-polyglutamic acid.
Example 4: optimization of carbon source variety for fermentation synthesis of gamma-polyglutamic acid by using Bacillus belgii LT-2
This example illustrates the effect of different carbon sources on the preparation of gamma-polyglutamic acid by fermentation of a strain, wherein fermented seed solutions were inoculated with 5% (v/v) of inoculum size into fermentation media containing 40g/L (total sugar concentration in fermentation broth) of glycerol (Gly), glucose (Glc), lactose (Lac), maltose (Mal), sucrose (Suc) and Sugarcane Juice (SJ), respectively, at an initial pH of 6.8, and were subjected to shaking culture at 30 ℃ and 200rpm, wherein the liquid loading volume of the fermentation media was 80mL/500mL in a triangular flask, and the fermentation media were subjected to fermentation culture for 48h, and then fermentation broths with different carbon sources were taken to perform the operation of step 7, thereby calculating the content of gamma-polyglutamic acid. The yield of the gamma-polyglutamic acid is highest by taking the cane sugar and the cane juice as carbon sources, and the cane juice is selected as the optimal carbon source in consideration of lower cost of the cane juice. The yield of the gamma-polyglutamic acid reaches 12.68g/L, the total sugar conversion rate is 31.70 percent, and the production rate is 0.26g/L/h (figure 7).
Example 5: optimization of carbon source concentration for fermentation synthesis of gamma-polyglutamic acid by using Bacillus belgii LT-2
This example illustrates the effect of different total sugar (sugarcane juice) concentrations on the fermentation of strains to produce gamma-polyglutamic acid by inoculating fermented seed solutions at 5% (v/v) into fermentation media with total sugar concentrations of 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, and 80g/L, performing shake culture at 30 deg.C and 200rpm with initial pH of 6.8 and fermentation medium liquid loading of 80mL/500mL triangular shake flask for 48h, respectively taking fermentation liquids with different sugar concentrations, performing step 7, calculating gamma-polyglutamic acid content, and determining when total sugar concentration is 50g/L, the highest total sugar conversion rate is 32.64 percent, the yield of the gamma-polyglutamic acid reaches 16.32g/L, and the production rate is 0.34 g/L/h. Therefore, a sugar concentration of 50g/L was chosen for the subsequent fermentation (FIG. 8).
Example 6: optimization of organic nitrogen source variety for fermentation synthesis of gamma-polyglutamic acid by using Bacillus belgii LT-2
This example illustrates the effect of different organic nitrogen sources on the preparation of gamma-polyglutamic acid by fermentation of bacterial strains, wherein the fermented seed liquid is inoculated into 5g/L fermentation medium of Beef Extract (BE), Corn Steep Liquor (CSL), soybean cake powder (SC), peanut cake Powder (PM), Peptone (PT) and yeast powder (YEP) at an inoculum size of 5% (v/v), performing shaking culture at 30 deg.C and 200rpm with initial pH of 6.8 and fermentation medium liquid loading of 80mL/500mL triangular shake flask, performing fermentation culture for 48h, respectively taking different organic nitrogen source fermentation liquids, performing the operation of step 7, calculating gamma-polyglutamic acid content, when the beef extract and the peanut cake powder are used as organic nitrogen sources, the yield of the gamma-polyglutamic acid is the highest, the yield is equivalent and is about 16.89g/L, the total sugar conversion rate is 33.78% at the highest, and the production rate is 0.35 g/L/h. Considering that the peanut cake powder is the leftovers of peanut oil production, the price is lower, which is beneficial to reducing the production cost. Therefore, peanut cake flour was chosen as the best organic nitrogen source (fig. 9).
Example 7: optimization of concentration of gamma-polyglutamic acid peanut cake powder synthesized by fermentation of Bacillus belgii LT-2
This example illustrates the effect of different concentrations of peanut cake powder on the preparation of gamma-polyglutamic acid by fermentation of strains, and is to inoculate fermented seed liquid with an inoculum size of 5% (v/v) into a fermentation medium containing 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L and 8g/L of peanut cake powder, respectively, with an initial pH of 6.8, shake-culture at 30 ℃, 200rpm, a fermentation medium loading volume of 80mL/500mL of triangular shake flask, fermentation-culture for 48h, take the peanut cake powder fermentation liquid with each concentration, respectively, perform the operation of step 7, and calculate the content of gamma-polyglutamic acid. When the concentration of the peanut cake powder is 5g/L, the yield of the gamma-polyglutamic acid reaches 17.31g/L at most, the total sugar conversion rate is 34.62 percent, and the production rate is 0.36 g/L/h. Therefore, 5g/L peanut cake flour was selected for subsequent fermentation (FIG. 10).
Example 8: optimization of gamma-polyglutamic acid inorganic nitrogen source species synthesized by fermentation of Bacillus belgii LT-2
This example illustrates the effect of different inorganic nitrogen sources on the preparation of gamma-polyglutamic acid by bacterial strain fermentation, the fermented seed solution is inoculated into 5g/L fermentation medium of ammonium chloride (A), ammonium sulfate (B), urea (C), diammonium phosphate (D) and ammonium nitrate (E) at an inoculum size of 5% (v/v), the initial pH value is 6.8, shaking culture is performed at 30 ℃ and 200rpm, the liquid loading amount of the fermentation medium is 80mL/500mL triangular shake flask, fermentation culture is performed for 48h, different inorganic nitrogen source fermentation liquids are taken respectively to perform the operation of step 7, the content of gamma-polyglutamic acid is calculated, the yield of gamma-polyglutamic acid reaches 17.88g/L when ammonium chloride is used as an inorganic nitrogen source, the total sugar conversion rate is 35.76%, and the production rate is 0.37 g/L/h. Therefore, ammonium chloride was chosen as the optimum inorganic nitrogen source (FIG. 11).
Example 9: optimization of concentration of gamma-polyglutamic acid ammonium chloride synthesized by fermentation of Bacillus belgii LT-2
This example illustrates the effect of different ammonium chloride concentrations on the preparation of gamma-polyglutamic acid by bacterial strain fermentation, the fermented seed liquid is inoculated into fermentation media containing 3g/L, 6g/L, 9g/L, 12g/L and 15g/L of ammonium chloride with an inoculum size of 5% (v/v), the initial pH value is 6.8, shaking culture is carried out at 30 ℃ and 200rpm, the liquid loading amount of the fermentation media is 80mL/500mL triangular shake flasks, fermentation culture is carried out for 48h, ammonium chloride fermentation liquids with various concentrations are respectively taken to carry out the operation of step 7, and the content of gamma-polyglutamic acid is calculated. When the concentration of ammonium chloride is 15g/L, the yield of the gamma-polyglutamic acid is 18.91g/L at most, the total sugar conversion rate is 37.82 percent, and the production rate is 0.39 g/L/h. Therefore, 15g/L ammonium chloride was selected for subsequent fermentation (FIG. 12).
Example 10: optimization of temperature for fermentation synthesis of gamma-polyglutamic acid by using Bacillus belgii LT-2
This example illustrates the effect of different temperatures on the yield of gamma-polyglutamic acid prepared by fermentation of Bacillus belgii LT-2, wherein 5% (v/v) of the seed solution after fermentation is inoculated into a fermentation medium, the initial pH value is 6.8, the seed solution is subjected to shaking culture at 30 ℃ and 200rpm, the culture temperatures are 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃ and 34 ℃ respectively, the liquid loading amount of the fermentation medium is 80mL/500mL of triangular shake flask, the fermentation culture is performed for 48h, the fermentation liquid at each temperature is taken to perform the operation of step 7, the content of gamma-polyglutamic acid is calculated, and the highest position of the content of gamma-polyglutamic acid at 30 ℃ is 19.20g/L, the total sugar conversion rate is 38.40%, and the production rate is 0.40 g/L/h. Therefore, 30 ℃ was selected as the optimum fermentation temperature (FIG. 13).
Example 11: optimization of pH value of gamma-polyglutamic acid synthesized by fermentation of Bacillus belgii LT-2
This example illustrates the effect of different pH values on the preparation of gamma-polyglutamic acid by strain fermentation, inoculating fermented seed liquid in a fermentation medium at 5% (v/v) inoculum size, performing shaking culture at 30 ℃ and 200rpm, wherein the liquid loading volume of the medium is 80mL/500mL triangular shake flask, the pH values of the culture medium are 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, the fermentation broth at each pH value is taken to perform the operation of step 7, and the content of gamma-polyglutamic acid is calculated by a standard curve, so that the maximum content of gamma-polyglutamic acid is 19.52g/L when the pH value is 7.0, the total sugar conversion rate is 39.04%, and the production rate is 0.41 g/L/h. Therefore, pH 7.0 was chosen as the optimum fermentation pH (FIG. 14).
Example 12: optimization of metal salt species and concentration thereof required for fermentation synthesis of gamma-polyglutamic acid by using Bacillus belgii LT-2
This example illustrates the effect of different metal salts and their concentrations on the preparation of gamma-polyglutamic acid by fermentation of bacterial strains, wherein the fermented seed liquid is inoculated with 5% (v/v) of metal salts at the following concentrations, respectively, by single-factor variable experiments as follows:
magnesium sulfate: 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2g/L and a control group, wherein the optimal concentration of the magnesium sulfate is 0.80 g/L;
potassium dihydrogen phosphate: 1.2, 3, 4, 5, 6, 7, and 8g/L and a control group, the optimum concentration of monopotassium phosphate is 5 g/L;
dipotassium hydrogen phosphate: 1.2, 3, 4, 5, 6, 7, and 8g/L and a control group, the optimum concentration of dipotassium hydrogen phosphate was 0g/L (no addition);
manganese sulfate 0.001, 0.002, 0.003, 0.004, 0.005 and 0.006g/L and a control group, wherein the optimal concentration of manganese sulfate is 0.005 g/L;
calcium chloride: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60g/L and the control group, the optimum concentration of calcium chloride was 0g/L (not added).
For fermentation culture media of different experimental groups, setting 3 groups of parallel controls for metal salts with different concentrations, setting the initial pH value to be 7.0, carrying out shaking culture at 30 ℃ and 200rpm, setting the liquid loading amount of the culture media to be 80mL/500mL triangular shake flasks, carrying out fermentation culture for 48h, and carrying out the operation of the step 7 on fermentation liquid with each metal salt concentration to calculate the content of the gamma-polyglutamic acid. When the strain produced gamma-polyglutamic acid under the above-mentioned optimum metal salt conditions, the yield of gamma-polyglutamic acid was 20.61g/L, the total sugar conversion rate was 41.22%, and the production rate was 0.43g/L/h (FIG. 15).
Example 13: fed-batch synthesis of gamma-polyglutamic acid by 50L fermentation tank
Inoculating Bacillus belgii B.velezensis LT-2 strain to a slant culture medium, standing and culturing at 32 ℃ for 16h, picking out a single colony again, streaking onto the slant culture medium, and culturing at 32 ℃ for 16h to obtain an activated strain for later use; inoculating 2 rings of activated strains into a shake flask containing a seed culture medium under aseptic conditions, placing the shake flask on a shaking table with the rotation speed of 180rpm, and culturing at 32 ℃ for 16h to obtain a fermented seed solution; inoculating the seed solution into a sterile fermentation medium according to an inoculation amount of 5% by volume, wherein the inoculation amount is 50g/L of sugarcane juice (the total sugar concentration of the sugarcane juice after being added into the fermentation broth is shown, and the sugarcane juice is supplemented when the total sugar concentration in the fermentation broth is lower than 5 g/L), 5g/L of peanut cake powder, 15g/L of ammonium chloride, 0.80g/L of magnesium sulfate, 5g/L of potassium dihydrogen phosphate and 0.005g/L of manganese sulfate. The total liquid loading of the fermentation tank is 30L, the fermentation temperature is 30 ℃, the stirring speed is 200rpm, and the ventilation volume is 1.2VVM for fermentation; the initial pH value of the fermentation is 7.0, a pH automatic control device is started in the fermentation process, and the pH value of the fermentation liquor is controlled to be about 7.0 by using ammonia water or hydrochloric acid; the fermentation time was 96 hours. Sampling every 6 hours to determine the concentration of the gamma-polyglutamic acid in the fermentation liquor. Determination and analysis: the fermentation broth is taken and centrifuged at 12,000rpm for 2 minutes, the supernatant is taken and diluted by proper times to detect the content of the gamma-polyglutamic acid in the fermentation broth, and the yield of the gamma-polyglutamic acid after one feeding is found to reach 43.87g/L, the total sugar conversion rate is 43.87%, and the production rate is 0.46g/L/h (figure 16).
Example 14: 1t fermentation tank fed-batch synthesis of gamma-polyglutamic acid
Inoculating Bacillus belgii B.velezensis LT-2 seed liquid into an aseptic fermentation medium according to the inoculation amount of 8% by volume: 50g/L of sugarcane juice (the total sugar concentration after the sugarcane juice is added into the fermentation liquid, and the sugarcane juice is supplemented when the total sugar concentration in the fermentation liquid is lower than 5 g/L), 5g/L of peanut cake powder, 15g/L of ammonium chloride, 0.80g/L of magnesium sulfate, 5g/L of potassium dihydrogen phosphate and 0.005g/L of manganese sulfate. The total liquid loading of the fermentation tank is 700L, the fermentation temperature is 30 ℃, the stirring speed is 260rpm, and the ventilation volume is 1.2VVM for fermentation; the initial pH value of the fermentation is 7.0, a pH automatic control device is started in the fermentation process, and the pH value of the fermentation liquor is controlled to be about 7.0 by using ammonia water or hydrochloric acid; the fermentation time was 96 hours. Sampling every 6 hours to determine the concentration of the gamma-polyglutamic acid in the fermentation liquor. Determination and analysis: the fermentation liquor is taken and centrifuged for 2 minutes at 12,000rpm, the supernatant is taken to be diluted by proper times, the yield of the gamma-polyglutamic acid in the fermentation liquor is detected to reach 47.61g/L, the total sugar conversion rate is 47.61 percent, and the production rate is 0.61g/L/h (figure 17).
Example 14: synthesis of gamma-polyglutamic acid by exogenously adding sodium glutamate
Inoculating the fermented B.velezensis LT-2 seed liquid into a sterile fermentation medium according to the inoculation amount of 8% by volume: 50g/L of sugarcane juice (the total sugar concentration after the sugarcane juice is added into the fermentation liquor), 40g/L of sodium glutamate, 5g/L of peanut cake powder, 15g/L of ammonium chloride, 0.80g/L of magnesium sulfate, 5g/L of monopotassium phosphate and 0.005g/L of manganese sulfate. In the middle, the total liquid loading of the fermentation tank is 700L, the fermentation temperature is 30 ℃, the stirring speed is 260rpm, and the ventilation volume is 1.2VVM for fermentation; the initial pH value of the fermentation is 7.0, a pH automatic control device is started in the fermentation process, and the pH value of the fermentation liquor is controlled to be about 7.0 by using ammonia water or hydrochloric acid; the fermentation time was 80 hours. Sampling every 6 hours to determine the concentration of the gamma-polyglutamic acid in the fermentation liquor. Determination and analysis: and (3) centrifuging the fermentation liquor at 12,000rpm for 2 minutes, taking the supernatant to dilute by proper times, and detecting that the yield of the gamma-polyglutamic acid in the fermentation liquor reaches 54.11g/L and the production rate is 0.68g/L/h (figure 18).
Finally, it should also be noted that the above-mentioned list is only a specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the teachings of the present invention are to be considered within the scope of the present invention.
Sequence listing
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ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt agccgacctg 240
agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300
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ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 480
gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa 540
gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg ggaacttgag 600
tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa 660
caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg 720
agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag 780
ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg 840
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Claims (8)

1. The Bacillus belgii is classified and named as Bacillus velezensis LT-2, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019904 and the preservation date of 2019, 11 months and 7 days.
2. Use of the Bacillus belgii of claim 1 for the preparation of gamma-polyglutamic acid.
3. The use according to claim 2, wherein gamma-polyglutamic acid is prepared by inoculating Bacillus velezensis LT-2 into a fermentation medium and performing aerobic culture.
4. The use according to claim 3, wherein the fermentation medium comprises the following components: 10-120 g/L of carbon source, 2-20 g/L of nitrogen source, 1.0-8.0 g/L of metal salt, water as solvent and 6.5-7.5 of pH value.
5. The use of claim 4, wherein the carbon source is any one or a combination of glycerol, glucose, lactose, maltose, sucrose and sugarcane juice;
the nitrogen source is any one or a combination of more of beef extract, corn steep liquor, soybean cake powder, peanut cake powder, peptone, yeast powder, ammonium chloride, ammonium sulfate, urea, diammonium hydrogen phosphate and ammonium nitrate;
the metal salt is any one or combination of magnesium sulfate, monopotassium phosphate, dipotassium phosphate, manganese sulfate and calcium chloride.
6. Use according to claim 3, wherein the fermentation medium comprises the following components: 10-80 g/L of sugarcane juice, 1-8 g/L of peanut cake powder, 5-25 g/L of ammonium chloride, 2-8 g/L of monopotassium phosphate, 0.2-1.2 g/L of magnesium sulfate and 0.002-0.008 g/L of manganese sulfate, and adjusting the initial pH of the fermentation liquor to 6.0-7.5 by using ammonia water.
7. The use of claim 4, wherein the carbon source is one or a combination of sugarcane juice and sucrose.
8. The use according to claim 3, wherein the aerobic culture conditions are: the initial pH is 6.5-7.5, and the culture temperature is 28-32 ℃;
when aerobic culture is shake flask culture, inoculating 1-8 mL of seed solution into every 100mL of fermentation liquor by using a shake flask for 36-54 h;
when aerobic culture is fermentation tank culture, the inoculation amount of the fermentation tank is 1-15 mL of seed liquid inoculated in every 100mL of fermentation liquid, the fermentation mode is a fed-batch method, the aeration ratio is 1.0-1.4 VVM, and the culture time is 48-96 h.
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