A kind of lactic acid bacteria resistant to low pH and producing D-lactic acid and its application

Abstract

The present invention provides a lactic acid bacterium that is resistant to low pH and produces D-lactic acid and its application, belonging to the field of microbial fermentation technology. The lactic acid bacteria of the present invention are classified and named Lactobacillus sp. YB-303, which has been preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2024835. The present invention uses low-energy nitrogen ion implantation to induce lactic acid bacteria, screens out strains that can produce high D-lactic acid, and under normal culture conditions, no calcium oxide needs to be added for neutralization, and can grow normally under conditions as low as pH 2 to 3. The D-lactic acid yield can reach 160 to 165 g / L, and the optical purity of the produced D-lactic acid reaches more than 99.5%.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a lactic acid bacterium capable of producing D-lactic acid at low pH and application thereof. Background Art

[0002] Lactic acid bacteria refers to a general term for various bacteria that can break down sugars such as glucose into lactic acid, and have a strong ability to metabolize carbohydrates. These strains are relatively common in life, and some still exist in the bodies of humans or animals, and are considered probiotics.

[0003] Lactic acid is an important organic acid, which is widely used in many fields such as food industry, medicine and chemistry. It includes L-lactic acid, D-lactic acid and DL-lactic acid. Among them, D-lactic acid is a basic organic chemical raw material, which can be used in medicine, agriculture, chemical industry, degradable plastics and other fields. In recent years, polylactic acid has been in increasing demand as a green and environmentally friendly source of degradable plastics. D-lactic acid can be obtained by chemical synthesis and microbial fermentation. However, the optical purity of D-lactic acid produced by chemical synthesis is low and the cost is high, while the L-lactic acid produced by microbial fermentation not only has high optical purity, but also mild conditions and low cost. In the fermentation preparation process of D-lactic acid, as the lactic acid production increases, the pH value of the fermentation environment will decrease significantly, and the over-acidic fermentation environment will inhibit the growth and metabolism of the strain. To solve this problem, calcium oxide is usually added to adjust the pH value to about 6.0 to ensure the normal progress of the fermentation process. However, this practice will produce a large amount of calcium sulfate, which not only pollutes the environment, but also increases the economic cost of D-lactic acid extraction.

[0004] In summary, how to solve the tolerance of lactic acid bacteria to the culture environment during the fermentation process, improve the strain's ability to tolerate low pH, produce and accumulate more D-lactic acid, reduce the production of calcium sulfate, reduce costs and environmental pollution, and make it more suitable for industrial production has positive research significance. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a lactic acid bacterium that is resistant to low pH and produces D-lactic acid and its application. The strain screened by the present invention can significantly improve the tolerance of the original strain to the low pH environment, the D-lactic acid produced has high optical purity, and the production process has little pollution to the environment.

[0006] In order to achieve the above objectives, in a first aspect, the present invention provides a lactic acid bacterium that is resistant to low pH and produces D-lactic acid. The lactic acid bacterium is classified and named Lactobacillus sp. YB-303, which has been deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2024835.

[0007] The strain Lactobacillus YB-303 of the present invention is obtained by induction, and the specific steps are as follows:

[0008] The strain YB-1 was inoculated into the plate culture medium by streaking and activated for the second generation. It was cultured at 37°C until OD = 1, diluted with sterile water to OD = 0.6, and then spread on the plate after bacteria were grown. After drying under sterile conditions and observing the absence of cell overlap under a microscope, ion beam implantation was performed. Nitrogen ion beam was selected, the implantation conditions were 10-30 KeV, and the implantation dose was 20x10 13 -35x10 13 ions / cm 3 , the treatment time is 120s. After the injection is completed, take out the plate and wash it with sterile water. Apply it to the plate culture medium and culture it in an anaerobic environment for activation. After a period of culture, the strain is inoculated into the plate culture medium with pH 2.0-3.0 and cultured for 48h. Repeat this process many times to observe and select strains with good growth conditions.

[0009] The strains screened in the previous step were made into cell suspensions and the concentration was adjusted to 10 -6 / ml. Take 0.1ml of cell suspension and spread it on the sterilized plate. After drying, perform nitrogen ion beam implantation at 20-30KeV and an implantation dose of 20x10 14 -35x10 14 ions / cm 3 After the injection, wash the plate with sterile water, spread the washed bacterial solution on the solid culture medium, and invert and culture at 37℃ for 36-48h.

[0010] The low pH-tolerant lactic acid bacteria of the present invention are facultative anaerobic bacteria, and the suitable growth temperature is 37° C. They can grow normally under low pH conditions, and the pH tolerance can reach 2-3, which effectively improves the ability of lactic acid bacteria to resist adverse external environments.

[0011] In a second aspect, the present invention provides a bacterial agent, comprising the above-mentioned lactic acid bacteria that are resistant to low pH and produce D-lactic acid; the bacterial agent may also include nutrients, carriers, fillers, stabilizers or active agents required by the strain.

[0012] In a third aspect, the present invention provides the use of the above-mentioned low-pH-tolerant lactic acid bacteria or bacterial agent for producing D-lactic acid in fermentation production of D-lactic acid under low pH conditions.

[0013] Preferably, the pH value of the low pH condition is 2-3. During the process of lactic acid bacteria producing lactic acid, the pH of the system will gradually decrease. The strains of the present invention have high activity in the whole process, that is, when the pH of the system gradually decreases from 8-2, and can produce D-lactic acid within the above pH range.

[0014] Preferably, the yield of D-lactic acid is 160-165 g / L.

[0015] Preferably, the method for producing D-lactic acid by fermentation comprises the following steps:

[0016] 1) Plate culture: The strain is inoculated onto a plate culture medium and cultured at 37°C for 24-48 hours to obtain plate culture colonies.

[0017] 2) Slant culture: Inoculate the colony cultured on the plate in step 1) into the slant culture medium for culture at 37°C for 22-24 hours to obtain a slant culture;

[0018] 3) Seed culture: inoculate the slant culture in step 2) into the seed culture medium for cultivation at 37°C for 12-20 hours to obtain a seed culture solution;

[0019] 4) Fermentation culture: The seed culture solution in step 3) is inoculated into the fermentation medium at an inoculation amount of 5-15% v / v, the fermentation temperature is 37° C., and the static fermentation culture is performed for 30-50 hours.

[0020] Preferably, the plate culture medium in step 1) contains the following components by mass: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.5-2% agar; the pH of the culture medium is 6.0-6.5;

[0021] In step 2), the slant culture medium contains the following components by weight: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.8-2% agar; the pH of the culture medium is 6.0-6.5;

[0022] In step 3), the seed culture medium contains the following components by weight: 4-5% glucose, 1-1.5% beef extract, 0.5-1% yeast extract, 1-1.5% peptone, 0.2-0.5% dipotassium hydrogen phosphate, and 0.05-0.08% magnesium sulfate; the pH of the culture medium is 6.0-6.5;

[0023] In step 4), the fermentation medium contains the following components by mass: 15-20% glucose, 1-1.5% yeast extract, 0.5-0.8% sodium acetate, 0.2-0.4% dipotassium hydrogen phosphate, and 0.05-0.1% magnesium sulfate; the pH of the medium is 6.0-6.5.

[0024] The beneficial effects of the present invention include at least:

[0025] Compared with wild strains, the lactobacillus YB-303 obtained by the present invention through ion beam engineering technology screening effectively improves the ability of the strain to withstand low pH environment, has good genetic stability and improved D-lactic acid production ability, and the optical purity of the product has also been significantly improved, with a production cycle of 2-3d, a yield of 160-165g / L, and an optical purity of more than 99.5%. After the lactic acid bacteria are induced to change, under normal culture conditions and acidic culture conditions, calcium oxide does not need to be added for neutralization, and can grow normally under conditions as low as pH 2-3, reducing costs, reducing the pollution of waste calcium sulfate to the environment, and can be put into large-scale industrial production, which has great research significance and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the acid production curve of YB-303 lactobacillus. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below by specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0028] Biodeposit Information:

[0029] The lactic acid bacteria is classified and named Lactobacillus sp. YB-303, and has been deposited in the China Center for Type Culture Collection on April 30, 2024. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: M2024835.

[0030] Example 1 Mutagenesis and screening of low pH tolerant strains

[0031] Starting strain screening:

[0032] The original strain YB-1 was inoculated into the plate culture medium by streaking and activated for the second generation. It was cultured at 37°C until OD = 1, diluted with sterile water to OD = 0.6, and then spread on a blank plate. After drying under sterile conditions and observing the absence of cell overlap under a microscope, ion beam implantation was performed. Nitrogen ion beam was selected, the implantation conditions were 10-30KeV, and the implantation dose was 20x10 13 -35x10 13 ions / cm 3, the treatment time is 120s. After the injection is completed, take out the plate and wash it with sterile water. Apply it to the plate culture medium and culture it in an anaerobic environment for activation. After a period of culture, the strain is inoculated into the plate culture medium with pH 2.0-3.0 and cultured for 36-48h. Repeat this process many times to observe and select strains with good growth conditions.

[0033] Rescreening:

[0034] The strains screened in the previous step were made into cell suspensions and the concentration was adjusted to 10 -6 / ml. Take 0.1ml of cell suspension and spread it on the sterilized plate. After drying, perform nitrogen ion beam implantation at 20-30KeV and an implantation dose of 20x10 14 -35x10 14 ions / cm 3 After the injection, wash the plate with sterile water, spread the washed bacterial solution on the plate culture medium, and invert and culture at 37℃ for 36-48h.

[0035] The induced strain was inoculated into the seed culture medium and cultured at 37°C for 12-20 hours. The volume of the shake flask was 10-30 ml. The bacterial solution in the seed culture medium was inoculated into the fermentation medium and cultured anaerobically for 30-50 hours. The yield of D-lactic acid in the fermentation liquid was detected. Repeat the rescreening steps until the target strain YB-303 was screened out.

[0036] The plate culture medium comprises the following components by weight: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.5-2% agar, and the pH of the culture medium is 6.0-6.5.

[0037] The slant culture medium comprises the following components by weight: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.8-2% agar, and the pH of the culture medium is 6.0-6.5.

[0038] The seed culture medium comprises the following components by weight: 4-5% glucose, 1-1.5% beef extract, 0.5-1% yeast extract, 1-1.5% peptone, 0.2-0.5% dipotassium hydrogen phosphate, and 0.05-0.08% magnesium sulfate, and the pH of the culture medium is 6.0-6.5.

[0039] The fermentation medium contains the following components by mass: 15-20% glucose, 1-1.5% yeast extract, 0.5-0.8% sodium acetate, 0.2-0.4% dipotassium hydrogen phosphate, and 0.05-0.1% magnesium sulfate, and the pH of the medium is 6.0-6.5.

[0040] The strains obtained by screening were Gram-stain positive and catalase test negative. The colony morphology was white, with a smooth surface, slightly raised, and obvious rough edges. The strains were mostly round in morphology, and a few were irregular in shape.

[0041] Example 2 Verification of low pH tolerance of screened strains

[0042] At 37°C, the mutant strain YB-303 and the original strain YB-1 were diluted and spread on plate culture media with pH 1, 2, 3, 4, 5, 6, and 7 for culture. The growth conditions were observed after 48 hours. Table 1 shows the number of surviving strains in different pH plates.

[0043] Table 1 Comparison of acid resistance between high-yield strains and starting strains

[0044] PH 1 2 3 4 5 6 7 YB-1 0 2 30 234 About 600 About 8000 More than 1000 YB-303 36 456 About 100 About 600 Around 900 More than 1000 More than 1000

[0045] As shown in Table 1, the survival rate of the mutant strains in a low pH environment is higher than that of the original strains, and the growth state of the surviving strains is better than that of the original strains.

[0046] Example 3 Screening of acid production curves of strains

[0047] Draw the acid production curve of YB-303 lactobacillus, such as Figure 1 shown.

[0048] from Figure 1 It can be seen that as the fermentation time increases, the change in OD value shows that the bacterial growth rate is relatively fast. After 34 hours, the OD value begins to enter a stable period, and the strain also enters a high-speed acid production stage. Lactic acid fermentation belongs to the primary metabolic process. With the accumulation of biomass, the production of D-lactic acid increases rapidly. When the fermentation end point is reached at 48 hours, the acid production reaches about 155g / L and the conversion rate reaches about 96%.

[0049] Example 4 Verification of genetic stability of screening strains

[0050] Subculture experiment: In order to ensure the genetic stability of the selected strains, the genetic stability was investigated. The experimental process of each generation: natural separation of mutant strains → single colony → slant → seed bottle → shake bottle fermentation, and measurement of D-lactic acid production. A total of 6 generations were passed, and 3 parallel samples were made for each generation. The verification results are shown in Table 2.

[0051] Table 2 Genetic stability experiment of high-yield mutants

[0052] Serial number Acid production of high-yield mutants (g / L) F1 154.82 F2 152.23 F3 151.47 F4 153.06 F5 154.13 F6 150.92 Control YB-1 109.68

[0053] As shown in Table 2, the acid production is basically maintained at around 150-155 g / L, indicating that the mutant strain has good genetic stability and its acid production capacity is increased by 37%-43% compared with the original strain, so it can be used as a strain for further research and development.

[0054] Example 5 YB-303 lactobacillus fermentation to produce D-lactic acid

[0055] The culture medium formula described in this embodiment (by mass percentage):

[0056] Slant culture medium: 0.5% glucose, 1.0% peptone, 0.3% beef extract, 0.3% yeast extract, 0.05% magnesium sulfate heptahydrate, 1.5% agar, and the rest is water. The pH of the culture medium is 6.0.

[0057] Seed culture medium: 6% glucose, 1.0% peptone, 1.0% yeast extract, 0.05% magnesium sulfate heptahydrate, and the rest water, the pH of the culture medium is 6.0. Glucose is decomposed at 0.05 MPa for 30 min.

[0058] Fermentation medium: 15% glucose, 1.5% yeast extract, 0.05% magnesium sulfate heptahydrate, and the rest water. The pH of the medium is 6.0.

[0059] The screened mutant strains were inoculated into slant culture medium for puncture culture at a temperature of 37°C for 24 hours.

[0060] The slant culture was inoculated into the seed medium at 37°C, the volume of the 250mL shake flask was 30mL, and the culture was carried out at a shaker speed of 80 rpm for 18 hours;

[0061] The seed liquid was inoculated into a 5L fermentation tank filled with 3L fermentation medium, with an inoculation volume of 10% (v / v), a fermentation temperature of 37°C, a stirring speed of 100rpm for the first 0-24h, and static fermentation culture for 24-48h. After 48h, the D-lactic acid content in the fermentation liquid was detected, reaching 163g / L, which was 49.5% higher than the original starting bacteria under the same culture conditions, and the optical purity reached 99.5%.

[0062] High performance liquid chromatography was used to detect lactic acid in the fermentation broth. The relevant equipment and parameters are as follows:

[0063] Chromatograph: Agilent Technologies 1260 Infinity II;

[0064] Detector: RID;

[0065] Separation column: Aminex HPX-87H Column 300×7.8mm;

[0066] Mobile phase: 0.005 M sulfuric acid;

[0067] Flow rate: 0.5mL / min;

[0068] Injection volume: 20 μL. Lactic acid retention time is about 14 min.

[0069] The optical purity of lactic acid was detected by high performance liquid chromatography. The relevant equipment and parameters are as follows:

[0070] Chromatograph: Agilent Technologies 1260 Infinity;

[0071] Detector: wavelength 254nm, sensitivity 0.32AUFS;

[0072] Separation column: MCI GEL-CRS10 W (3u) 4.6ID × 50nm;

[0073] Mobile phase: 0.002M copper sulfate

[0074] Flow rate: 0.5mL / min;

[0075] Injection volume: 20 μL.

[0076] The sample was diluted to a total lactic acid concentration of 0.5-1 g / L before testing. The retention time of D-lactic acid was about 11 min, and the retention time of L-lactic acid was about 13 min. The optical purity of D-lactic acid was calculated based on its peak area.

[0077] The above studies show that the strain of the present invention is significantly superior to the original strain without mutation in terms of D-lactic acid production speed and product optical purity. The strain of the present invention has increased acid production to about 163g / L and optical purity to 99.5%. The present invention provides a novel strain with low fermentation cost, fast D-lactic acid production speed and high product optical purity. This strain can produce D-lactic acid with high optical purity, can reduce production costs, and achieve the purpose of reducing environmental pollution, providing a better potential choice for the industrial microbial fermentation production of D-lactic acid.

[0078] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A lactic acid bacterium tolerant to low pH and producing D-lactic acid, characterized in that: The lactic acid bacteria is classified and named as Lactobacillus sp. YB-303, and has been deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M2024835.

2. A bacterial agent, characterized in that The invention comprises the lactic acid bacteria capable of producing D-lactic acid at low pH as claimed in claim 1.

3. Use of the low pH tolerant D-lactic acid producing lactic acid bacteria according to claim 1 or the bacterial agent according to claim 2 in fermentation production of D-lactic acid under low pH conditions, characterized in that: The low pH condition has a pH value of 2-3.

4. The use according to claim 3, characterized in that: The yield of D-lactic acid is 160-165 g / L.

5. The use according to claim 3, characterized in that: The method for producing D-lactic acid by fermentation comprises the following steps: 1) Plate culture: Inoculate the strain onto a plate culture medium and culture at 37°C for 24-48 hours to obtain plate culture colonies; 2) Slant culture: Inoculate the colony cultured on the plate in step 1) into the slant culture medium for culture at 37°C for 22-24 hours to obtain a slant culture; 3) Seed culture: inoculate the slant culture in step 2) into the seed culture medium for cultivation at 37°C for 12-20 hours to obtain a seed culture solution; 4) Fermentation culture: The seed culture solution in step 3) is inoculated into the fermentation medium at an inoculation amount of 5-15% v / v, the fermentation temperature is 37° C., and the static fermentation culture is performed for 30-50 hours.

6. The use according to claim 5, characterized in that: In step 1), the plate culture medium contains the following components by weight: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.5-2% agar; the pH of the culture medium is 6.0-6.5; In step 2), the slant culture medium contains the following components by weight: 2-3% glucose, 0.5-1% yeast extract, 1-2% peptone, and 1.8-2% agar; the pH of the culture medium is 6.0-6.5; In step 3), the seed culture medium contains the following components by weight: 4-5% glucose, 1-1.5% beef extract, 0.5-1% yeast extract, 1-1.5% peptone, 0.2-0.5% dipotassium hydrogen phosphate, and 0.05-0.08% magnesium sulfate; the pH of the culture medium is 6.0-6.5; In step 4), the fermentation medium contains the following components by mass: 15-20% glucose, 1-1.5% yeast extract, 0.5-0.8% sodium acetate, 0.2-0.4% dipotassium hydrogen phosphate, and 0.05-0.1% magnesium sulfate; the pH of the medium is 6.0-6.5.

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