CN116622565A - Bacillus coagulans and application thereof - Google Patents
Bacillus coagulans and application thereof Download PDFInfo
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- CN116622565A CN116622565A CN202310561839.1A CN202310561839A CN116622565A CN 116622565 A CN116622565 A CN 116622565A CN 202310561839 A CN202310561839 A CN 202310561839A CN 116622565 A CN116622565 A CN 116622565A
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- lactic acid
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- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 45
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 44
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims abstract description 136
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 claims description 2
- 239000008267 milk Substances 0.000 claims description 2
- 210000004080 milk Anatomy 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 239000002689 soil Substances 0.000 claims description 2
- 238000012262 fermentative production Methods 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 93
- 230000004151 fermentation Effects 0.000 abstract description 93
- 238000000034 method Methods 0.000 abstract description 28
- 238000004519 manufacturing process Methods 0.000 abstract description 25
- 239000002253 acid Substances 0.000 abstract description 15
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- 239000008103 glucose Substances 0.000 description 26
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- KVZLHPXEUGJPAH-UHFFFAOYSA-N 2-oxidanylpropanoic acid Chemical compound CC(O)C(O)=O.CC(O)C(O)=O KVZLHPXEUGJPAH-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010001539 D-lactate dehydrogenase Proteins 0.000 description 1
- 102100023319 Dihydrolipoyl dehydrogenase, mitochondrial Human genes 0.000 description 1
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- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention relates to the technical field of microorganisms, in particular to bacillus coagulans and application thereof, wherein the bacillus coagulans is named as bacillus coagulans (Bacillus coagulans) TFLY-05 and has been deposited in China center for type culture Collection, and the deposit number is: CCTCC No. M20222058, date of preservation: 2022, 12, 23; deposit place: the invention further provides the application of the bacillus coagulans in the fermentation production of L-lactic acid, the repeated fermentation performance is stable, the fermentation concentration of the L-lactic acid can be stabilized above 160g/L, the maximum L-lactic acid produced by fermentation by adopting a fed-batch feeding process can reach 230g/L, the fermentation time is short, the acid production speed is high, the optical purity of the L-lactic acid is high, the stability of different batches can reach above 99%, and the maximum L-lactic acid can reach 99.8%.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus coagulans and application thereof.
Background
Lactic acid (2-hydroxy propionic acid) is a chiral organic acid, and has two optical isomers, namely L-lactic acid (L-chirality) and D-lactic acid (D-chirality), and has extremely wide application in the fields of food industry, chemical industry, cosmetic industry, medicine, pharmaceutical industry and the like as a high-value functional chemical with green and sustainable characteristics. Based on the wide use of lactic acid and the large demand of the market, the market value of lactic acid reaches $ 26 billion in 2018, and is expected to increase at a composite annual growth rate of 18.7%, and by 2025, the global lactic acid demand will reach approximately 200 ten thousand tons, and the global market value will reach about $ 90 billion.
Lactic acid can be produced by using chemical synthesis methods, microbial fermentation methods, and enzyme catalysis methods. Chemical synthesis or enzymatic catalysis is limited by the sustainable development of petrochemical raw materials, the generation of racemic mixtures, or the industrialized scale, and is not easy to be used as a large-scale industrialized production mode. In contrast, the microbial fermentation method can utilize renewable energy materials as raw materials, and can prepare optically pure L-or D-enantiomer by selecting microorganisms with single L-or D-lactate dehydrogenase activity for fermentation, and has the remarkable characteristics of high efficiency, energy conservation and green sustainable development.
Bacillus coagulans belongs to the genus Bacillus, is a gram-positive bacterium, can utilize homolactic fermentation metabolic pathway to generate L-lactic acid, and is one of the common bacterial strains for industrial production of L-lactic acid at present. The bacillus coagulans can carry out fermentation production of lactic acid at a high temperature of 50-60 ℃, so that an open fermentation method or a non-sterilization culture method can be adopted to generate lactic acid. For example, a Chinese patent (publication No. CN 101173242A) uses a strain of Bacillus coagulans to ferment at high temperature (45-60 ℃) to produce the strain, so that the fermentation medium can not be sterilized, the probability of contamination by mixed bacteria is small, and the energy consumption and the nutrient loss are reduced.
At present, large-scale industrial fermentation of lactic acid by utilizing bacillus coagulans mainly adopts two modes of batch fermentation or continuous fermentation. Compared with batch fermentation, continuous fermentation can simplify the processes of seed preparation and culture, culture medium configuration, liquid discharge, charging and the like, improve the utilization efficiency and the production efficiency of a fermentation tank and save the production cost.
Disclosure of Invention
In view of the above, the present invention aims to provide bacillus coagulans and an application thereof in fermentation production of L-lactic acid.
In view of the above, the present invention provides a Bacillus coagulans laboratory named Bacillus coagulans (Bacillus coagulans) TFLY-05, which has been deposited with the China center for type culture Collection, accession number: CCTCC No. M20222058, date of preservation: 2022, 12, 23; deposit place: university of martial arts in chinese.
The bacillus coagulans TFLY-05 is obtained by screening and separating from soil in a milk processing plant in Tianjin city.
The bacillus coagulans TFLY-05 is a gram-positive bacterium, facultative anaerobic, and the microspore is in a slender rod shape, the spore end is born, and no flagella exists, and is sensitive to ampicillin, kanamycin, chloramphenicol, tetracycline and erythromycin. The colony is round, the edge is neat, the surface is smooth and glossy, and the colony is transparent. Glucose, sucrose, fructose, dextrin or maltose can be used as a substrate to produce L-lactic acid in a homofermentative manner.
The bacillus coagulans TFLY-05 is a strain of thermophilic bacteria, the optimal growth temperature range is 35-55 ℃, and the optimal growth pH range is 5.5-7.5.
The 16S rDNA sequence of the bacillus coagulans TFLY-05 is shown in SEQ ID NO: 1.
Bacillus coagulans and application thereof, wherein the application refers to application in L-lactic acid fermentation production, and the method comprises the following steps:
step one, using solid bevel activated bacillus coagulans TFLY-05CCTCC NO:M20222058 to prepare bevel seeds;
step two, inoculating and culturing the slant seeds in a seed culture medium to prepare a seed culture solution;
and thirdly, fermenting the seed culture solution in a fermentation medium to produce L-lactic acid.
The specific preparation method of the slant seed comprises the following steps: taking out the ultralow-temperature (-80 ℃) glycerol freezing tube preservation tube of the bacillus coagulans TFLY-05, scribing a slant culture medium (100 mL of 500mL eggplant bottled slant culture medium) after the frozen matter is dissolved, and culturing at 45-55 ℃ for 16-24 hours to ensure that the diameter of a single colony reaches 1-2 mm, thus being capable of being used as slant seeds.
The slant culture medium is LB culture medium, namely Luria-Bertani culture medium, and the formula is: 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride and pH 6.8-7.2.
The seed culture medium comprises a first seed culture medium, a second seed culture medium and a third seed culture medium, wherein the inclined seed is firstly inoculated into the first seed culture medium to carry out liquid shaking culture to obtain a first seed culture solution, then the first seed culture solution is inoculated into the second seed culture medium to carry out seed solution expansion culture in a fermentation tank to obtain a second seed culture solution.
The specific preparation method of the first-stage seed liquid comprises the following steps: washing each cultured eggplant bottle by using 50mL of sterile water, sucking 50mL of bacterial suspension obtained after washing, inoculating the bacterial suspension into 400mL of first-stage seed culture medium, and filling the inoculated culture medium with 450mL of culture medium in a 1L triangular flask. Culturing in a shaking culture table at 45-55 deg.c and rotation speed of 120-180 r/min for 10-18 hr to obtain the first seed liquid.
The formula of the primary seed culture medium is as follows: 80-120 g/L of one or a mixture of sucrose and glucose, 2-5 g/L of one or a mixture of yeast extract and tryptone, 0.2-0.5 g/L of sodium chloride, 0.1-0.3 g/L of monopotassium phosphate, 0.1-0.3 g/L of dipotassium phosphate, 0.5-0.8 g/L of diammonium phosphate, 0.1-0.3 g/L of zinc sulfate, 0.5-2 g/L of calcium carbonate and pH of 5.5-6.5.
The culture temperature is controlled to 45-55 ℃, preferably 48-50 ℃.
The rotating speed of the shaking table is 120-180 r/min, preferably 130-150 r/min.
The pH is 5.5 to 6.5, preferably 6.0 to 6.3.
The specific preparation method of the secondary seed liquid comprises the following steps: inoculating all the cultured primary seed liquid into a full-automatic stirring type ventilation fermentation tank filled with a sterile secondary seed culture medium, wherein the final charging coefficient is 60-75%, the tank pressure is set to be 0.01-0.03 MPa, the culture temperature is controlled to be 45-55 ℃, the pH of the fermentation liquid is controlled to be 5.5-6.5, the stirring rotating speed is controlled to be 200-400 r/min, and the ventilation rate is controlled to be 0.1-0.3 vvm. Culturing for 6-10 h under the above conditions to obtain the secondary seed liquid.
The formula of the secondary culture medium is as follows: 120-160 g/L of one or a mixture of sucrose and glucose, 2-5 g/L of one or a mixture of yeast extract and tryptone, 0.2-0.5 g/L of sodium chloride, 0.1-0.3 g/L of monopotassium phosphate, 0.1-0.3 g/L of dipotassium phosphate, 2-5 g/L of diammonium phosphate and 0.1-0.3 g/L of zinc sulfate.
The culture temperature is controlled to 45-55 ℃, preferably 49-52 ℃.
The pH of the fermentation broth is controlled to be 5.5-6.5, preferably 5.9-6.3.
The specific method for producing L-lactic acid by fermentation in the third step comprises the following steps: all seed liquid cultured in the secondary seed fermentation tank is transferred into a fermentation tank filled with a fermentation medium, the final loading coefficient is 60-75%, the tank pressure is set to be 0.01-0.03 MPa, the culture temperature is controlled to be 45-55 ℃, the pH of the fermentation liquid is controlled to be 5.5-6.5 by adding a neutralizing agent in a process flow, the stirring rotating speed is controlled to be 200-400 r/min, the ventilation rate is controlled to be 0-0.3 vvm, and the fermentation time is 16-24 h. Ending the fermentation when the residual sugar concentration is less than 0.5 g/L.
Samples were taken every 2 hours during fermentation, and the cell density (OD 600), residual sugar concentration, L-lactic acid concentration and D-lactic acid concentration were measured, respectively, to calculate the sugar acid conversion rate, the L-lactic acid optical purity and the L-lactic acid production intensity.
The neutralizing agent is calcium carbonate suspension with the concentration of 300-400 g/L, preferably 320-350 g/L.
The formula of the fermentation medium is as follows: 150-200 g/L glucose, 1-3 g/L yeast extract, tryptone or a mixture thereof, 0.2-0.5 g/L potassium dihydrogen phosphate, 0.2-0.5 g/L dipotassium hydrogen phosphate, 0.5-2 g/L diammonium hydrogen phosphate and 0.1-0.3 g/L zinc sulfate.
The culture temperature is controlled to 45 to 55℃and preferably 50 to 53 ℃.
The pH of the fermentation broth is controlled to be 5.5-6.5, preferably 5.9-6.3.
The glucose is 150-200 g/L, preferably 160-190 g/L.
In the third step, when the Bacillus coagulans TFLY-05 is used for fermentation production of L-lactic acid by taking glucose as a main fermentation carbon source, the sugar acid conversion rate is up to 0.98, the concentration of the obtained L-lactic acid can reach 230g/L at the highest, the production intensity of the L-lactic acid can reach more than 10 g/L.h, and the optical purity of the L-lactic acid can reach 99.8% at the highest.
The residual sugar concentration is the residual concentration of glucose in the fermentation broth at a certain time point in the fermentation process, and the glucose concentration is measured by using a biosensor SBA-40E (institute of biology, academy of sciences of Shandong province).
The method for measuring the concentration of the L-lactic acid and the D-lactic acid comprises the following steps: a Waters e2695 liquid chromatograph and a chiral column (Mitsubishi chemical in Japan, MCI GEL CRS W4.6 ID. Times.50 mm) were used, the absorption wavelength was 254nm, the mobile phase was 2mM CuSO4, the column temperature was 35℃and the flow rate was 0.5mL/min. And drawing standard curves by using L-lactic acid standard substances and D-lactic acid standard substances with different concentration gradients respectively, and then calculating the concentration of the L-lactic acid and the concentration of the D-lactic acid in the fermentation broth respectively.
The sugar acid conversion rate refers to the percentage of the amount of L-lactic acid produced and the amount of glucose consumed in the fermentation process, and the calculation method comprises the following steps: sugar acid conversion (%) =l-lactic acid (g/L)/amount of glucose consumed (g/L) ×100%.
Wherein, the amount of glucose consumed (g/L) =total amount of glucose dosed (g/L) -residual sugar (g/L).
The optical purity of the L-lactic acid refers to the percentage of the L-lactic acid in the total amount of the lactic acid, and the calculation method comprises the following steps: l-lactic acid optical purity= [ L-lactic acid content (g/L) -D-lactic acid (g/L) ]/[ L-lactic acid (g/L) +D-lactic acid (g/L) ]. Times.100%
The L-lactic acid production intensity refers to the amount of L-lactic acid produced by a unit volume of fermentation broth in a unit time, and the calculation method comprises the following steps: l-lactic acid production intensity (g/L.h) =L-lactic acid concentration (g/L)/fermentation time (h).
The invention has the beneficial effects that:
1. the strain grows fast and is easy to culture, the sugar acid conversion rate is high, the concentration of residual sugar is low, and the matrix utilization efficiency is high in the fermentation process;
2. the strain fermentation temperature is high (50-53 ℃), so that the cost generated by cooling a sterilization culture medium can be reduced, and the cooling water can be saved in the whole fermentation process;
3. the fermentation production of L-lactic acid can be carried out by adopting a non-sterile culture medium, so that a large amount of energy consumption is saved.
4. The repeated fermentation performance is stable, the fermentation concentration of the L-lactic acid can be stabilized to be more than 160g/L, and the maximum L-lactic acid produced by fermentation by adopting a fed-batch feeding process can reach 230g/L;
5. the fermentation time is short, and the fermentation can be finished within 20 hours by adopting a one-time feeding process;
6. the acid production speed is high, and the highest L-lactic acid production strength can reach 10 g/L.h;
7.L-lactic acid has high optical purity, can be stabilized to be more than 99% in different batches, and can reach 99.8% at most.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of a photomicrograph (1600X) of Bacillus coagulans TFLY-05 of the invention;
FIG. 2 is a schematic diagram showing the fermentation process curve of example 2L-lactic acid according to the present invention.
Detailed Description
The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
EXAMPLE 1 screening of Bacillus coagulans
1 Flat plate preliminary screening
2g of each of the 38 collected natural samples was placed in 50mL of enrichment medium (10 g/L yeast extract, 20g/L glucose) using sterile water, and allowed to stand overnight at 50 ℃. The enriched samples were subjected to gradient dilution to 10-1, 10-2 and 10-3 using sterile water, and 0.2mL of each of the sample dilutions having concentration gradients of 10-1, 10-2 and 10-3 was applied to a sterile solid screening medium (20 g/L yeast extract, 100g/L glucose, 0.4g/L bromocresol green and 1.5% agar), and incubated at 50℃for 48 hours. After colonies developed, representative colonies with yellow circles were picked.
2 shaking bottle re-screening
Representative colonies with yellow circles obtained in the above steps were selected and inoculated into a shake flask fermentation medium (20 g/L yeast extract, 100g/L glucose, 50g/L CaCO 3), and cultured with shaking at 50℃for 48 hours under 200r/min conditions. The fermentation broth was collected by centrifugation and the lactic acid concentration was determined using High Pressure Liquid Chromatography (HPLC).
And placed on an agar plate (yeast extract 20g/L, glucose 100g/L, bromocresol green 0.4g/L, agar powder 15 g/L) at 50 ℃. After 48 hours of incubation, representative colonies with yellow circles were picked and incubated alone in 100mL of medium (yeast extract 20g/L, glucose 100g/L, calcium carbonate 50 g/L) for 48 hours. The supernatant was collected by centrifugation for HPLC analysis to determine the lactic acid concentration in the broth. Simultaneously, the obtained strains are respectively preserved by using an ultralow temperature (-80 ℃) glycerol freezing tube preservation method.
EXAMPLE 2 identification of Bacillus coagulans species
The natural sample is repeatedly screened and compared by the method, and the strain with the number of TFLY-05 is finally obtained, the yield of the L-lactic acid is higher, the strain grows fast in LB culture medium (5 g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride and 15g/L agar powder) plates, the colony is round, the edge is neat, the surface is smooth and glossy, the strain is transparent (figure 1), microscopic examination shows that the bacterial microspore is in a slender bacillus shape, and glucose, sucrose, fructose, dextrin or maltose can be used as a substrate to produce the L-lactic acid in a homofermentation mode.
Molecular biological identification of bacillus coagulans strains:
(1) Bacillus coagulans chromosome extraction
The SDS cracking method is used, and the specific steps are as follows:
(1) inoculating the separated strain into LB liquid culture medium (5 g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride) for culturing for 12h, centrifuging to collect the strain, and washing the strain once with sterile water; (2) suspending the microspore with 0.4mL sterile water, adding lysozyme to a final concentration of 10mg/mL, mixing, and then carrying out warm bath at 37 ℃ for 10min; (3) respectively adding 50 mu L of 10% SDS (sodium dodecyl sulfate) solution, 10 mu L of proteinase K (10 mg/mL) and 10 mu L of RNaseA (10 mg/mL), uniformly mixing, and then carrying out warm bath at 65 ℃ for 20-30 min until the solution is in a transparent state, and reversing and uniformly mixing every 3-5 min; (4) equal volumes of phenol were added: gently mixing chloroform with 1min, centrifuging at 4deg.C for 5min, and collecting supernatant; (5) adding equal volume chloroform into the supernatant, extracting, gently mixing, centrifuging at 4deg.C for 5min at 10,000r/min, and collecting supernatant; (6) adding 2.5 times volume of absolute ethyl alcohol into the supernatant, precipitating at room temperature for 20min, centrifuging at 10000r/min at 4 ℃ for 10min, and collecting nucleic acid precipitate; (7) adding 1mL of 75% ethanol to wash the precipitate, centrifuging at 4 ℃ for 5min at 10000 r/min; (8) the supernatant was aspirated off, the Ep tube was back-buckled on a clean paper towel and the DNA pellet was dried at room temperature for 15min. 100. Mu.L TE buffer was added and the mixture was kept at-20 ℃.
(2) PCR amplification and sequencing of Bacillus coagulans 16S rDNA Gene
The strain 16S rDNA gene fragment was obtained by using a PCR method with the chromosomal DNA obtained by SDS-cleavage as a template and the 16S rDNA region universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 142R: 5 '-TACCTTGTTACGACTT-3') as an amplification primer.
The PCR amplification system (50. Mu.L) is shown in Table 1 below.
TABLE 1PCR amplification System (50. Mu.L)
The PCR product is connected with a vector pUCm-T, transformed into Escherichia coli JM109 competent microspores, recombinant plasmids are extracted, and the sequence determination of a target gene is completed by general biology (Anhui) Co., ltd., and the determined 16S rDNA gene sequence is SEQ ID NO.1.
(3) Phylogenetic tree construction
The sequencing result of the 16S rDNA gene fragment of the strain is compared with the determined sequence through an online database Blast/blastp (http:// www.ncbi.nlm.nih.gov/Blast), similar sequences are selected and multiple comparison is carried out by using BioEdit software, the comparison result is constructed into a phylogenetic tree through a software MEGA5.1 by utilizing a Neighbor-Joing (NJ) method (figure 2), the isolated strain is identified as bacillus coagulans according to the clustering result, and the bacillus coagulans is named as bacillus coagulans (Bacillus coagulans) TFLY-05 which is preserved in China center for type culture collection, and the preservation number is: CCTCC No. M20222058, date of preservation: 2022, 12, 23; deposit place: university of martial arts in chinese.
EXAMPLE 2 fermentation of L-lactic acid by Bacillus coagulans TFLY-05
Taking out the ultralow temperature (-80 ℃) glycerol freezing tube preservation tube of the bacillus coagulans TFLY-05, and streaking two bottles of slant culture medium (100 mL of 500mL eggplant bottle slant culture medium) after the frozen matter is dissolved, wherein the culture temperature is 50 ℃, and the culture time is 18h. Washing each cultured eggplant bottle by using 50mL of sterile water, respectively inoculating 50mL of bacterial suspension obtained by washing into 400mL of sterile primary seed culture medium, and filling the culture medium with 450mL of culture medium in a 1L triangular flask after inoculation. Placing the inoculated strain into a constant-temperature shake culture shaking table for culture, wherein the temperature of the shaking table is set to be 50 ℃, the rotating speed of the shaking table is 120r/min, and the culture is carried out for 12 hours. Two bottles of seed liquid were collected and 500mL of the primary seed liquid was inoculated into a 10-L fully automatic stirred aerated fermenter containing 5.5L of sterile secondary seed medium. The tank pressure is set to be 0.01MPa, the culture temperature is controlled to be 49-52 ℃, the pH of fermentation liquor is controlled to be 6.0-6.3, the stirring rotation speed is set to be 300r/min, the ventilation is controlled to be 0.1vvm, and the secondary seed liquor is obtained after 8 hours of culture.
After 54L of fermentation medium is filled into a full-automatic ventilation type fermentation tank with the total volume of 100L and high-temperature sterilization is carried out, 6L of secondary seed liquid is completely transferred into the fermentation tank when the sterilization medium is cooled to 50 ℃, so that the total filling volume after inoculation is 60L, namely the filling volume is 60 percent. In the fermentation process, the tank pressure is set to be 0.01MPa, the culture temperature is controlled to be 51 ℃, the pH of fermentation liquor is controlled to be 6.1, and the stirring rotating speed is controlled to be 300r/min. During the fermentation period, the ventilation rate is controlled to be 0.2vvm within 0 to 5 hours; 6 to 10 hours, and controlling the ventilation rate to be 0.1vvm; 11 th to 18 th, the ventilation is 0.
Samples were taken every 2 hours during fermentation, and the cell density (OD 600), residual sugar concentration, L-lactic acid concentration and D-lactic acid concentration were measured, respectively, to calculate the sugar acid conversion rate, the L-lactic acid optical purity and the L-lactic acid production intensity.
Under the fermentation conditions, the concentration of the L-lactic acid obtained by fermenting for 18 hours is 170g/L, the production intensity of the L-lactic acid is 9.4 g/L.h, the sugar acid conversion rate is 0.98, and the purity of the L-lactic acid is 99.6%. The L-lactic acid fermentation process curve is shown in FIG. 2.
The high temperature sterilization refers to a method of treating the culture medium with high temperature (118 ℃) for 25 minutes to kill all microbial microspores in the culture medium.
The slant culture medium is LB culture medium, namely Luria-Bertani culture medium, and the formula is as follows: yeast extract 5g/L, peptone 10g/L, sodium chloride 10g/L, pH 7.
The formula of the primary seed culture medium is as follows: sucrose sugar 50g/L, glucose 50g/L, yeast extract 3g/L, tryptone 1g/L, sodium chloride 0.3g/L, potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, diammonium hydrogen phosphate 0.5g/L, zinc sulfate 0.15g/L, calcium carbonate 0.5g/L, and pH6.
The formula of the secondary seed culture medium is as follows: 70g/L of sucrose, 70g/L of glucose, 3g/L of yeast extract, 0.5g/L of tryptone, 0.1g/L of sodium chloride, 0.25g/L of monopotassium phosphate, 0.25g/L of dipotassium phosphate, 3g/L of diammonium phosphate and 0.15g/L of zinc sulfate.
The formula of the fermentation medium is as follows: 180g/L glucose, 3g/L yeast extract, 0.2-0.5 g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 2.5g/L diammonium hydrogen phosphate and 0.2g/L zinc sulfate.
Example 2
This example differs from example 1 in that the fermentation medium is not autoclaved in this example. Other process conditions are unchanged. After 18 hours of fermentation culture, the concentration of the L-lactic acid is 167g/L, the production intensity of the L-lactic acid is 9.3 g/L.h, the sugar acid conversion rate is 0.95, and the purity of the L-lactic acid is 99.2%.
The step of not sterilizing the fermentation medium at high temperature means that the fermentation medium is directly and rapidly heated to 50 ℃ without high-temperature treatment, cooling, and the like after preparation, and then secondary seed liquid is added for fermentation.
Example 3
This example differs from example 1 in that the fermentation medium is not autoclaved but boiled in this example. Other process conditions are unchanged. After 18h of fermentation culture, the concentration of the L-lactic acid obtained by fermenting for 18h is 170g/L, the production intensity of the L-lactic acid is 9.4 g/L.h, the sugar acid conversion rate is 0.98, and the purity of the L-lactic acid is 99.6 percent under the fermentation condition.
The boiling treatment refers to heating the fermentation medium to 100 ℃ and maintaining for 20min after the preparation of the fermentation medium is completed.
Example 4
The difference between this example and example 1 is that the stirring speed was set to 200r/min after the second seed liquid was inoculated into the fermentation medium in this example. During the fermentation, the ventilation rate is controlled to be 0.2vvm within 0 to 12 hours; 13 to 18h, the ventilation is 0. Other process conditions are unchanged. After 18 hours of fermentation culture, the concentration of the obtained L-lactic acid is 165g/L, the production intensity of the L-lactic acid is 9.2 g/L.h, the sugar acid conversion rate is 0.97, and the purity of the L-lactic acid is 99.6%.
Example 5
This example differs from example 1 in that the secondary medium and the fermentation medium differ in composition, and the other process conditions are unchanged. After 44 hours of fermentation culture, the concentration of the obtained L-lactic acid is 180g/L, the production intensity of the L-lactic acid is 10 g/L.h, the sugar acid conversion rate is 0.98, and the purity of the L-lactic acid is 99.8%.
The formula of the secondary seed culture medium is as follows: glucose 150g/L, yeast extract 3g/L, tryptone 0.5g/L, sodium chloride 0.1g/L, potassium dihydrogen phosphate 0.25g/L, dipotassium hydrogen phosphate 0.25g/L, diammonium hydrogen phosphate 3g/L, and zinc sulfate 0.15g/L.
The formula of the fermentation medium is as follows: 190g/L glucose, 4g/L yeast extract, 0.2-0.5 g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 2.5g/L diammonium hydrogen phosphate and 0.2g/L zinc sulfate.
Example 6
This example differs from example 5 in the composition of the fermentation medium, the feed process and the fermentation time, the other process conditions being unchanged. After the fermentation, the concentration of the obtained L-lactic acid is 230g/L, the production intensity of the L-lactic acid is 9.6 g/L.h, the sugar acid conversion rate is 0.96, and the purity of the L-lactic acid is 99.7%.
The formula of the fermentation medium is as follows: 170g/L glucose, 4g/L yeast extract, 0.2-0.5 g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 2.5g/L diammonium hydrogen phosphate and 0.2g/L zinc sulfate.
The feeding process refers to the process of fermenting according to the fermentation control process described in the embodiment 5 until 12h, and 15L of glucose solution with the concentration of 500g/L is fed into the fermentation tank at one time.
The fermentation time is 24 hours.
Example 7
This example differs from example 6 in that the secondary seed liquid volume, the fermentation medium charge volume, the glucose solution feed volume are different, and the other process conditions are unchanged. After fermentation culture for 24 hours, fermentation is finished, the concentration of the L-lactic acid is 210g/L, the production intensity of the L-lactic acid is 8.75 g/L.h, the sugar acid conversion rate is 0.96, and the purity of the L-lactic acid is 99.7%.
In this example, two bottles of seed liquid were collected and 500mL of the primary seed liquid was inoculated into a 10-L full-automatic stirred-tank aeration fermenter containing 7.5L of sterile secondary seed medium, so that the total charge volume after inoculation was 7L, i.e., the charge volume was 70%.
In this example, 63L of fermentation medium was placed in a fully automatic aeration fermenter having a total volume of 100L, and after high-temperature sterilization, 7L of secondary seed liquid was completely transferred into the fermenter when the sterilization medium was cooled to 50℃to a total volume of 70L, i.e., a liquid loading amount of 70%.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The present invention is intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
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Claims (4)
1. Bacillus coagulans and use thereof, characterized in that the Bacillus coagulans laboratory is designated Bacillus coagulans (Bacillus coagulans) TFLY-05, which has been deposited with the China center for type culture Collection, accession number: CCTCC No. M20222058, date of preservation: 2022, 12, 23; deposit place: university of martial arts in chinese.
2. The bacillus coagulans and application thereof according to claim 1, wherein the bacillus coagulans TFLY-05 is obtained by screening and separating from soil in a milk processing plant in Tianjin.
3. The bacillus coagulans and the application thereof according to claim 1, wherein the bacillus coagulans TFLY-05 is a strain of thermophilic bacteria, the optimal growth temperature range is 35-55 ℃, and the optimal growth pH range is 5.5-7.5.
4. Use of bacillus coagulans according to any of claims 1-3, characterized in that it is applied in the fermentative production of L-lactic acid.
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