A Bacillus coagulans for producing L-lactic acid and a method for screening strain mutations

Abstract

The present invention provides a Bacillus coagulans for producing L-lactic acid and a method for screening strain mutations. The Bacillus coagulans was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 21, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26156. Through a series of measures such as mutagenesis, domestication, and screening, the present invention obtains a Bacillus coagulans strain that can tolerate highly toxic straw substrates and high-temperature and low-pH fermentation conditions, avoiding the cost problems caused by pure sugar fermentation, high-temperature sterilization, and strict prevention and control of miscellaneous bacteria during the fermentation process.

Description

Technical Field

[0001] The present invention relates to the field of screening of bioengineering strains, and specifically to a Bacillus coagulans strain producing L-lactic acid and a method for screening strain mutations. Background Art

[0002] L-lactic acid (L(+)-Lactic acid) is an optical isomer of lactic acid and is a chemical raw material widely used in many fields such as environmental protection, food, medicine, leather making, and agriculture. It can be used to synthesize biodegradable polymers such as polylactic acid (PLA) products, such as packaging, films, and plastic products. Compared with traditional plastics, bioplastics have the advantage of being naturally degradable, which can reduce the white pollution problem and reduce human dependence on petroleum.

[0003] Enzymatic method, chemical synthesis method, and microbial fermentation method can be used to prepare L-lactic acid. Among them, the microbial fermentation method has the advantages of low cost, simple process, environmental friendliness, and high product purity. Especially when using agricultural waste straw as the substrate and fermenting with Bacillus coagulans, the cost of fermenting with pure sugar as the substrate can be greatly reduced. At the same time, it can turn waste into treasure, reduce the waste of straw resources and the environmental pollution caused by "straw burning". However, there are several problems that need to be solved urgently in the current microbial fermentation method using straw as the substrate: (1) Through a series of pre-treatments of straw in the early stage, the raw materials contain a large amount of toxic substances, and these inhibitors are highly toxic to cells; (2) Strict control of miscellaneous bacteria contamination is required for low-temperature fermentation, and the operation of high-temperature and high-pressure sterilization greatly increases the fermentation cost; (3) The accumulation of lactic acid during the fermentation process will cause the pH to continuously decrease, affecting cell metabolism and production. The addition of alkaline neutralizing agents such as calcium carbonate and calcium hydroxide not only increases the difficulty of downstream separation and purification processes, raises the production cost, but also causes serious environmental pollution; (4) The traditional Bacillus coagulans has a low utilization rate of xylose, cellobiose, and glucose, and the fermentation time is relatively long; (5) The optical purity of the produced L-lactic acid is low, there are many by-products, and the sugar-acid conversion rate is low. Summary of the Invention

[0004] In order to solve the problems of easy contamination by miscellaneous bacteria, low yield, poor purity, and long fermentation period in the current fermentation of lactic acid by Bacillus coagulans using straw as the substrate, the present invention provides a method for compound mutagenesis and screening of a Bacillus coagulans strain with high temperature resistance, resistance to the toxicity of straw substrate, and resistance to low pH, which can efficiently and quickly obtain the required for industrial production.

[0005] The first aspect of the present invention is to provide a Bacillus coagulans that can efficiently ferment and produce L-lactic acid. The Bacillus coagulans was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 21, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26156.

[0006] The second aspect of the present invention is to provide a method for screening mutants of Bacillus coagulans strains for producing L-lactic acid, and the method includes:

[0007] 1) After resuscitating and culturing the cryopreserved strains, subject them to ultraviolet mutagenesis, and then carry out high-temperature acclimation by culturing at a constant temperature of 38-42 °C;

[0008] 2) Carry out high-temperature acclimation by culturing at a constant temperature of 43-47 °C;

[0009] 3) Carry out high-temperature acclimation by culturing at a constant temperature of 48-52 °C;

[0010] 4) Carry out high-temperature acclimation by culturing at a constant temperature of 53-57 °C;

[0011] 5) Transfer the bacterial suspension and smear it onto a normal culture medium containing CaCO 3 , and place it in an incubator for constant-temperature culture;

[0012] 6) Select 80-100 single colonies according to the morphology and size of the calcium dissolution circle for fermentation experiments,

[0013] 7) Monitor genetic stability and the yield of fermentation products.

[0014] Among them, after the high-temperature acclimation in steps 1)-4), scrape the colonies with sterile water to form a bacterial suspension

[0015] Preferably, the conditions for ultraviolet mutagenesis are 25-35 W and 25-30 s.

[0016] More preferably, the method of constant-temperature culture in steps 1)-4) is to coat the bacterial solution from the previous step on an enzymolysis culture medium with a concentration range of 40-100%, and the volume of the coated bacterial solution is 100-150 μL; most preferably, the gradient is 20%.

[0017] Most preferably, the steps of the method are as follows:

[0018] 1) Use the well-grown Bacillus coagulans as the original glycerol tube, coat it on a 40% enzymolysis plate, then place it under an ultraviolet lamp for mutagenesis treatment, and then place it in a light-shielded incubator at 40 °C for 24-48 h for high-temperature mutagenesis.

[0019] 2) Scrape the plate strain that is resistant to high temperature at 40 °C and grows well on a 40% enzymolysis plate with 1 mL of sterile water to make bacterial suspension I, suck 125 μL of bacterial suspension I and evenly coat it on a 60% enzymolysis plate, and place it in an incubator at 45 °C in the dark for continued high-temperature mutagenesis culture for 24-48 h.

[0020] 3) The plate strains that can tolerate a high temperature of 45°C and grow well on a 60% enzymatic hydrolysis plate are coated on an 80% enzymatic hydrolysis plate according to the method used in the previous step, and placed in an incubator at 50°C in the dark for high-temperature mutagenesis culture for 24 - 48 h again.

[0021] 4) The plate strains that can tolerate a high temperature of 50°C and grow well on an 80% enzymatic hydrolysis plate are coated on a 100% enzymatic hydrolysis plate according to the method used in the previous step, and placed in an incubator at 55°C in the dark for 24 - 48 h.

[0022] 5) The strains that can tolerate a 100% enzymatic hydrolysis plate and a high temperature of 55°C are continuously sub-coated 5 times under the same culture conditions. Take 125 μL of the final bacterial suspension and coat it on a normal solid medium containing calcium carbonate, and culture it at 55°C for 24 - 48 h. Observe the size of the calcium dissolution circle, pick the single colonies with a larger calcium dissolution circle using a 200 μL pipette tip, and culture them in a normal liquid medium for 24 - 48 h as the seed liquid for fermentation culture.

[0023] 6) The seed liquid with good growth conditions is inoculated into 50 mL of fermentation medium at 20%, and anaerobically fermented at 55°C for 48 - 72 h. Use a high-performance liquid chromatograph to determine the lactic acid production, and select the strains with high lactic acid production during fermentation.

[0024] 7) Re-examine the genetic stability of the selected strains with high fermentation acid production. By continuously sub-coating 5 - 10 times on a 100% enzymatic hydrolysis plate, inoculate the strains after sub-coating 5 - 10 times into a fermenter for seed culture, culture at pH 7.0 and 52 - 55°C for 24 - 48 h. After the seeds grow well, transfer them into the fermentation medium at an inoculation amount of 10%, and ferment at an initial pH 7.0 and 55°C for 48 - 72 h. After fermentation, use a high-performance liquid chromatograph to determine the lactic acid production, and select the strains with higher fermentation acid production.

[0025] The third aspect of the present invention provides the application of the strain described in the first aspect of the present invention in the fermentation preparation of L-lactic acid.

[0026] The fourth aspect of the present invention is to provide a method for the strain described in the first aspect of the present invention to produce L-lactic acid using steam-exploded straw, and the method includes pre-culturing the strain; inoculating and fermenting. In a preferred embodiment, the pre-culturing is to inoculate the strain stored in glycerol into a seed medium, inoculate it into 200 mL of pre-culture medium at 180 - 200 rpm and a temperature of 50 - 58°C for pre-culturing for 12 - 24 h; the fermentation culture refers to directly inoculating the pre-cultured fermentation broth into a medium containing steam-exploded straw, maintaining the temperature at 50 - 58°C, and anaerobically culturing without aeration for 48 - 96 h, and adding supplementary fermentation medium during this period.

[0027] In addition, the present invention naturally provides the application of the aforementioned Bacillus coagulans in the process of straw fermentation to produce lactic acid.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Through a series of measures such as mutagenesis, domestication, and screening, the present invention obtains a strain of Bacillus coagulans that can tolerate highly toxic straw substrates and high-temperature and low-pH fermentation conditions, avoiding the cost problems caused by pure sugar fermentation, high-temperature sterilization, and strict prevention and control of miscellaneous bacteria during the fermentation process. In addition, low-pH fermentation can avoid the addition of a large amount of alkaline neutralizing agents, reduce the difficulty of downstream lactic acid separation and purification, and the environmental pollution caused by by-products, and can also significantly reduce the production cost of L-lactic acid. At the same time, this strain also has the characteristics of high total sugar utilization rate and high lactic acid product purity, and is extremely suitable for industrial production. Specific Embodiments

[0030] The objects and functions of the present invention and the methods for achieving these objects and functions will be clarified below by referring to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The essence of the specification is only to help those skilled in the relevant art comprehensively understand the specific details of the present invention.

[0031] Example 1: Strain Mutation and Screening

[0032] 1. Culture Medium and Its Preparation

[0033] 1) Ratio of normal solid plate medium: Glucose 30 g / L, yeast powder 5 g / L, sodium chloride 0.5 g / L, potassium dihydrogen phosphate 1 g / L, ammonium sulfate 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.1 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 15 g / L (normal liquid medium does not need to add agar), and the rest is purified water. Use 2 mol / L sodium hydroxide to adjust the pH to 7.0.

[0034] 2) Ratio of enzymolysis plate medium: Based on the standard of containing 30 g / L glucose in 100% enzymolysis solution, add the required amount of glucose to be supplemented according to the corresponding ratio (for example, for an enzymolysis medium with a volume fraction of 50%, only an additional 15 g / L of glucose needs to be added), yeast powder 5 g / L, sodium chloride 0.5 g / L, potassium dihydrogen phosphate 1 g / L, ammonium sulfate 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.1 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 15 g / L, and the rest is purified water. Use 2 mol / L sodium hydroxide to adjust the pH to 7.0.

[0035] Prepare enzymatically hydrolyzed plate media of 40%, 60%, and 80% in the same way.

[0036] 3) Pre-culture medium: Glucose 30 - 50 g / L, yeast extract 3 - 7 g / L, sodium chloride 1 - 3 g / L, ammonium sulfate 1 - 3 g / L, calcium carbonate 5 - 10 g / L.

[0037] 4) Composition of fermentation medium: Add 5 g / L of corn steep liquor to the enzymatically hydrolyzed solution of straw substrate material, and adjust the pH to 7.0 with 20% sodium hydroxide.

[0038] Among them, the straw substrate material is steam-exploded straw material, and the preparation method is: Coarsely powder the dry straw material to 3 - 8 mm, fully mix it with an equal mass of water, put it into the steam explosion tank, input high-pressure steam, when the temperature reaches 210 °C, maintain for 15 - 30 min, then open the valve at the bottom of the steam explosion tank, instantaneously release the material, crush, cool, and store for later use.

[0039] 2. Strain mutation and domestication

[0040] (1). Take a glycerol tube of Bacillus coagulans containing a mixed strain, take 125 μL of the bacterial solution and evenly coat it on a 40% enzymatically hydrolyzed plate, then place it 35 cm away from a 30 W ultraviolet lamp for mutagenesis treatment for 25 - 30 s, and place it in an incubator at 40 °C for constant temperature culture for 24 - 48 h for high-temperature mutagenesis. Then add sterile water to scrape the bacteria to form a bacterial suspension.

[0041] (2). Pipette 125 μL of the bacterial suspension in (1) and evenly coat it on a 60% enzymatically hydrolyzed plate, place it in an incubator at 45 °C for constant temperature culture for 24 - 48 h and continue high-temperature mutagenesis. Then add sterile water to scrape the bacteria to form a bacterial suspension.

[0042] (3). Pipette 125 μL of the bacterial suspension in (2) and evenly coat it on an 80% enzymatically hydrolyzed plate, place it in an incubator at 50 °C for constant temperature culture for 24 - 48 h and continue high-temperature mutagenesis. Then add sterile water to scrape the bacteria to form a bacterial suspension.

[0043] (4). Take 125 μL of the bacterial suspension in (3) and evenly coat it on a 100% enzymatically hydrolyzed plate, place it in an incubator at 55 °C for constant temperature culture for 24 - 48 h for plate domestication.

[0044] (5). For the strain that can tolerate the 100% enzymatically hydrolyzed plate and 55 °C, continuously streak it 5 times under the same culture conditions. Take 125 μL of the bacterial suspension and streak it onto a plate containing 3 - 5 g of CaCO 3In a normal culture medium, place it in an incubator and incubate at a constant temperature of 55°C for 24 - 48 h. Observe the size of the calcium dissolution circle. Use a 200 μL pipette tip to pick a single colony with a larger calcium dissolution circle and culture it in a normal liquid culture medium for 24 - 48 h as the seed liquid for fermentation culture.

[0045] (6) Inoculate 100 picked single colonies into shake flasks containing the culture medium for seed culture, incubate at 55°C for 24 - 48 h, and perform anaerobic fermentation at 55°C for 48 - 72 h. Use a high-performance liquid chromatograph to determine the lactic acid production, and select the strains with high lactic acid production.

[0046] (7) Select 30 bottles of well-grown seed liquid and inoculate them into fermentation shake flasks with 100% enzymolysis solution. Set the temperature to 55°C and the initial pH to 7.0. Ferment for 48 h, and select 6 strains with strong acid production ability according to the pH drop during fermentation. The results are shown in the following table:

[0047] Table 1: pH drop during shake flask fermentation of strains screened by high-temperature mutagenesis and ultraviolet mutagenesis

[0048]

[0049] (8) Use glycerol tubes to preserve several strains with relatively high lactic acid production screened in the previous step. Inoculate the strains into the enzymolysis solution culture medium of steam-exploded straw substrate material with a 100% content. After culturing at a temperature of 55°C for 24 - 48 h, inoculate them into a seed tank at the same temperature for seed expansion culture. After culturing for 24 - 48 h, take an inoculum of 10% of the fermentation broth volume and inoculate it into a fermentation tank containing 15% straw substrate material. Use 20% sodium hydroxide to adjust the pH value of the fermentation broth to 7.0, and perform anaerobic fermentation for 48 - 72 h to measure the lactic acid concentration. The lactic acid concentration is shown in Table 2 below. The above experiment uses Bacillus coagulans starting strain as a control.

[0050] Table 2: Lactic acid content in the fermentation tank of steam-exploded straw substrate

[0051] Screening bacteria number Lactic acid production concentration (g / L) 0425-1 36.21 0425-7 35.45 0425-15 38.73 0516-4 43.14 0525-7 35.57 0525-8 39.16 Original bacteria 25.43

[0052] Example 2: Strain stability

[0053] The genetic stability of the strain with the highest lactic acid content in the fermenter was investigated. The strain 0516-4 with the highest lactic acid content in the fermenter and the original strain were subcultured on a solid medium of straw material enzymatic hydrolysate with 100% content. They were cultured at a temperature of 52 - 55 °C for 24 - 48 h and transferred and coated for 7 - 8 generations. After each generation of seeds was inoculated into the seed tank for seed expansion culture for 24 - 48 h, an inoculum of 10% of the fermentation broth volume was inoculated into the fermenter with straw substrate material, and fermented anaerobically for 48 - 72 h. Then, the lactic acid production concentration was measured, and the lactic acid production abilities of each generation of the strain 0516-4 and the original strain were compared. The results are shown in Table 3 below.

[0054] Table 3: Investigation on the genetic stability of strain 0516-4 and the original strain

[0055]

[0056]

[0057] Bacillus coagulans was subjected to ultraviolet mutagenesis, high-temperature induction, subculture domestication, fermentation screening, etc. In lactic acid fermentation with steam-exploded straw material as the substrate, the acid production ability of the screened strain was more than 50% higher than that of the original strain. After subculture on a solid medium of steam-exploded straw enzymatic hydrolysate, the difference in the production capacity of the fermenter between the fifth generation and the original strain, the second generation, and the third generation was within 5%, indicating good genetic stability. This proved that the lactic acid-producing Bacillus coagulans screened by the present invention that is resistant to straw substrate has a large increase in lactic acid production efficiency in the fermenter and good genetic stability.

[0058] Example 3: Fermentation application of the strain

[0059] 1) Preparation of steam-exploded straw: The dry straw material was coarsely ground to 3 - 8 mm, fully mixed with an equal mass of water, and then put into the steam explosion tank. High-pressure steam was input. When the temperature reached 210 °C, it was maintained for 15 - 30 min. Then, the bottom valve of the steam explosion tank was opened, and the material was instantaneously released, pulverized, cooled, and stored for use.

[0060] 2) Pre-culture of Bacillus coagulans 0516-4: The glycerol-preserved bacterial solution was inoculated into the pre-culture medium, and inoculated into 200 mL of the pre-culture medium at 180 - 200 rpm and a temperature of 50 - 58 °C for pre-culture for 12 - 24 h.

[0061] 3) Inoculation and fermentation: Directly inoculate the pre-cultured bacterial liquid in step 2) into the medium containing the steam-exploded straw prepared in step 1), maintain the temperature at 50 - 58 °C, and anaerobically culture for 48 - 96 h without aeration. Adjust the pH of the initial medium to 7.0, and keep the pH dropping naturally during the fermentation process without adding alkali solution for adjustment. During this process, add and supplement the fermentation medium; wherein the fermentation medium contains 100 - 200 g / L of steam-exploded straw; after the fermentation ends, centrifuge to obtain the fermentation supernatant.

[0062] 4) Take the fermentation supernatant, detect the concentrations of L-lactic acid and D-lactic acid, and calculate the yield and optical purity.

[0063]

[0064]

[0065] Among them, the contents of L-lactic acid and D-lactic acid are detected by an HPLC chromatograph, where the chromatograph is an Agilent 1200 and the chromatographic column is Sumichiral OA5000. The chromatographic conditions are that the mobile phase is 1 mM CuSO 4 , the flow rate is 1 - 1.5 Ml / min, the detection wavelength is 254 nm UV, the column temperature is 30 - 35 °C, and the injection volume is 10 - 30 μL.

[0066] After calculation, when the strain obtained by the mutation method of the present invention ferments steam-exploded straw, the yield of L-lactic acid can reach 67.3 g / L, the optical purity is 98.4%, and the comprehensive yield is 94.3%.

Claims

1. A Bacillus coagulans for producing L-lactic acid ( Bacillus coagulans ), which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 21, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26156.

2. Use of the Bacillus coagulans according to claim 1 in the fermentation preparation of L-lactic acid.

3. Method for producing L-lactic acid by the Bacillus coagulans according to claim 1 using steam-exploded straw, said method comprising pre-culturing of the strain; inoculating and fermenting culture.

4. According to the method of claim 3, said pre-culturing is inoculating the glycerol cryopreserved bacterial solution into the pre-culture medium, and pre-culturing for 12-24 h in the pre-culture medium inoculated at 180-200 rpm and at a temperature of 50-58 °C.

5. According to the method of claim 3 or 4, wherein said fermenting culture means directly inoculating the pre-cultured fermentation broth into the medium containing steam-exploded straw, maintaining the temperature at 50-58 °C, anaerobically culturing for 48-96 h without aeration, and adding supplementary fermentation medium during this period.

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