Lactococcus lactis strain with high nisin yield and application

Abstract

The invention discloses a lactococcus lactis strain with high nisin yield and application, and belongs to the technical field of industrial microorganisms. According to the invention, a nisin-tolerant nisin high-producing strain (Lactococcus lactis MN6) is successfully bred on the basis of compound mutation of ethyl methane sulfonate and heavy ion beam and in combination with nisin resistance screening, the strain can tolerate a fermentation environment with the nisin concentration of 6000 IU. ML <-1 >, the nisin yield through shake flask horizontal fermentation can reach 10776 IU. ML <-1 >, which is 9.76 times of the shake flask fermentation yield of original bacteria, the strain has good passage stability, and the strain can be used for producing a high-yield strain of nisin. The method is more suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to a Lactococcus lactis strain with high nisin yield and its application, belonging to the technical field of industrial microorganisms. Background Art

[0002] Nisin is a cationic antibacterial peptide produced by specific Lactococcus lactis fermentation, with the molecular formula C 143 H 228 N 42 O 37 S7. The mature nisin molecule consists of 34 amino acid residues, including four special amino acids: dehydroalanine, methyldehydroalanine, lanthionine, and β-methyllanthionine. Nisin can bind to lipids on the microbial cell membrane to form transmembrane pores, causing cell lysis and death. It can also inhibit the biosynthesis of the microbial cell wall by interacting with the precursors of cell wall synthesis, thereby showing broad-spectrum antibacterial activity against most Gram-positive bacteria and some Gram-negative bacteria, such as Staphylococcus, Streptococcus, and most Clostridium and Bacillus spores. Compared with other antibacterial peptides, nisin has high solubility and strong stability, and still has antibacterial activity after being treated at 121 °C under high temperature and high pressure for 15 min in a solution with a pH of 2. In 1969, nisin was recognized as a safe and non-toxic natural food preservative by the Food and Agriculture Organization of the United Nations and the World Health Organization, and has now been widely used in the field of food preservation. In addition, nisin has also been applied in the fields of daily chemical products and biomedicine because it can inhibit bacteria such as Staphylococcus aureus and Streptococcus sanguinis that cause mastitis, respiratory infections, skin infections, and oral diseases. According to statistics, the market size of nisin increased from 179 million yuan in 2015 to 295 million yuan in 2023, and is expected to reach 440 million yuan in 2024, indicating a stable growth trend in the nisin market and an increasing demand for nisin. Therefore, it is necessary to reduce the production cost of nisin and improve the synthesis efficiency of nisin to meet its growing market demand.

[0003] At present, the nisin production capacity of wild-type strains is poor and cannot meet the requirements of large-scale production. Genetic engineering can be used to improve the nisin synthesis ability of Lactococcus lactis chassis cells by modifying them. For example, overexpressing key genes in the nisin synthesis gene cluster can enhance nisin synthesis. Or by overexpressing some genes related to acid and oxygen tolerance, Lactococcus lactis can better adapt to adverse conditions such as low pH and dissolved oxygen during fermentation, thereby improving its fermentation performance and enhancing nisin synthesis. However, genetic engineering transformation is limited by the complex genetic background of microorganisms, making it difficult to accurately locate key transformation sites. On the other hand, when applying strains to food products, issues such as their safety are often considered. Therefore, it is still necessary to screen food safety microorganisms that can tolerate nisin and produce nisin at high yields. Summary of the Invention

[0004] Aiming at the problem that the yield of current nisin-producing strains is limited by their tolerance, the present invention provides a nisin-tolerant and high-yield Lactococcus lactis MN6, which is classified as Lactococcus lactis. It was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 2, 2024, with the deposit number CGMCC No. 31812 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] In one embodiment, the Lactococcus lactis MN6 can tolerate 6000 IU·mL -1 nisin.

[0006] The present invention also provides a composition containing the Lactococcus lactis MN6.

[0007] In one embodiment, the composition includes but is not limited to microbial preparations.

[0008] In one embodiment, the microbial preparation contains live cells of the Lactococcus lactis MN6.

[0009] The present invention also provides a method for preparing the bacteriocin nisin, which is to ferment the Lactococcus lactis MN6 in a medium at 28 - 30 °C for at least 24 h.

[0010] In one embodiment, the method is to culture Lactococcus lactis MN6 at 30 °C and 150 rpm for 12 - 16 h to prepare a seed solution, and then transfer the cultured seed solution to a fermentation medium at an inoculation amount of 3% and ferment at 30 °C and 150 rpm for 24 - 26 h.

[0011] In one embodiment, the seed medium contains: 5 g / L of glucose, 5 g / L of bovine peptone, 5 g / L of yeast extract powder, 1 g / L of KH2PO4, 2 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and the pH is 7.2.

[0012] In one embodiment, the fermentation medium contains: 30 g / L of sucrose, 16 g / L of soy peptone, 8 g / L of yeast extract powder, 5 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O, 0.2% of Tween-80, and the pH is 7.2.

[0013] The present invention also provides the application of the Lactococcus lactis MN6 in the fields of food, medicine, agriculture or industry.

[0014] In one embodiment, the application includes fermenting to produce nisin or a product containing nisin.

[0015] In one embodiment, the application includes preparing a feed additive containing nisin.

[0016] In one embodiment, the application includes preparing a preservative or antibacterial agent containing nisin.

[0017] Beneficial effects:

[0018] (1) Through iterative composite mutagenesis of wild-type Lactococcus lactis strains with ethyl methanesulfonate (EMS) and heavy ion beams, and using nisin resistance as the screening condition, after primary screening with microplates and secondary screening with shake flasks, a high nisin-producing strain Lactococcus lactis MN6 was obtained, which can tolerate 6000 IU·mL -1 of nisin, and its yield in shake flask fermentation can reach 10776 IU·mL -1 , which is 9.76 times the yield of the original strain in shake flask fermentation. After 6 subcultures of this strain, the yield can be stably maintained at 10712 IU·mL -1 or above, showing good subculture stability.

[0019] (2) Fermenting the strain obtained by screening in a 5 L fermentor can further increase the nisin yield to 12676 IU·mL -1 .

[0020] Biological material preservation

[0021] Lactococcus lactis MN6, classified and named as Lactococcus lactis, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 2, 2024, with the deposit number CGMCC No. 31812 and the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0022] Figure 1 Growth of the original strain CGMCC1.2829 and the high-yield strain MN6 under nisin stress conditions; among them, (a) 2000 IU·mL -1 Comparison of the growth of the original strain and the high-yield strain under nisin stress conditions; (b) 6000 IU·mL -1 Comparison of the growth of the original strain and the high-yield strain under nisin stress conditions.

[0023] Figure 2 Cell morphological differences between the original strain and the high-yield strain under 2000 IU·mL -1 nisin stress conditions; among them, (a) FESEM×10.0k cell morphology of the original strain; (b) FESEM×10.0k cell morphology of the high-yield strain; (c) FESEM×30.0k cell morphology of the original strain; (d) FESEM×30.0k cell morphology of the high-yield strain.

[0024] Figure 3 Differences in the titer of flask fermentation and the batch fermentation process in a 5L fermenter between the original strain and the high-yield strain; among them, (a) Nisin titers of the original strain and the high-yield strain in flask fermentation; (b) Parameters of the fermentation process of MN6 in a 5L fermenter; (c) Parameters of the fermentation process of the original strain CGMCC1.2829 in a 5L fermenter. Detailed Embodiments

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0026] The experimental materials used in the following examples are all conventional biochemical reagents unless otherwise specified.

[0027] (1) Detection of Nisin Titer:

[0028] Preparation of the titer determination plate: The sterilized titer detection medium was cooled to about 50 °C and the suspension of Micrococcus luteus, the indicator bacterium, was added so that the final concentration of the indicator bacterium suspension in the medium was 10 6 cells·mL -1, dispense the titer detection medium into sterile 25 mL beakers, with 20 mL of the titer detection medium in each plate. The plates can be stacked no more than three at a time. Ensure the plates are level and place them in the laminar flow hood to wait for the plates to solidify. Then punch holes evenly.

[0029] Standard sample treatment: Weigh the known titer silver elephant standard sample with an analytical balance and dissolve it in 0.02 M HCl to make a nisin solution of 2000 IU·mL -1 After filtering with a sterile filter membrane, store it in a -20 °C refrigerator.

[0030] Sample treatment: Adjust the pH of the fermentation broth to about 2 with 6 M HCl, boil it in a water bath for 3 - 5 min, centrifuge at 12000 rpm for 3 - 5 min, and place the supernatant in a 4 °C refrigerator for standby. Dilute the supernatant with 0.02 M HCl according to the standard curve range.

[0031] Detect the nisin titer in the fermentation broth: Add 80 μL of the standard sample and the sample to each well on the titer determination plate. After adding the samples, place the plate in an incubator at 30 °C for 20 - 24 h, then measure the diameter of the inhibition zone, and detect the nisin titer through the standard curve.

[0032] (2) Biomass determination method:

[0033] Take 1 mL of the fermentation broth, centrifuge at 12000 rpm for 3 min to collect the bacteria. Resuspend and dilute the bacteria with 0.85% NaCl solution of the same volume as the fermentation broth, and measure its absorbance value at 600 nm.

[0034] (3) Culture medium:

[0035] (1) Seed culture medium: Glucose 5 g / L, bovine bone peptone 5 g / L, yeast extract powder 5 g / L, KH2PO4 1 g / L, NaCl 2 g / L, MgSO4·7H2O 0.2 g / L, pH 7.2; Add 2% agar to the solid culture medium.

[0036] (2) Fermentation culture medium: Sucrose 30 g / L, soy peptone 16 g / L, yeast extract powder 8 g / L, KH2PO4 5 g / L, MgSO4·7H2O 0.2 g / L, Tween - 80 0.2%, pH 7.2.

[0037] (3) Indicator bacteria growth culture medium: Tryptone 8 g / L, yeast extract 5 g / L, glucose 5 g / L, Na2HPO4·12H2O 2 g / L, NaCl 5 g / L, pH 6.8.

[0038] (4) Potency detection medium: Tryptone 8 g / L, Yeast extract 5 g / L, Glucose 5 g / L, Na2HPO4·12H2O 2 g / L, NaCl 5 g / L, Tween-20 1%, Agar 1.5%, pH 6.8.

[0039] Example 1 Screening of Nisin-Tolerant Lactococcus lactis Mutants with High Nisin Yield

[0040] (1) Screening of nisin-tolerant high-yield mutants by EMS mutagenesis

[0041] Lactococcus lactis CGMCC 1.2829 obtained from the China Center for Type Culture Collection was used as the starting strain. After activation by streak plate, it was inoculated into the seed medium and cultured until the mid-log phase. An appropriate amount of cells was collected by centrifugation at 12,000 rpm for 3 min, and the collected cells were resuspended in phosphate buffer with pH = 7.2 to prepare a cell suspension for standby. A certain amount of ethyl methyl sulfonate (EMS) solution was added to the cell suspension to make its final concentration 1%, and it was mutagenized in a 30°C water bath for 50 min and then diluted and spread on a solid plate of the seed medium with 2000 IU·mL -1 nisin resistance, and cultured at 30°C for 24 - 48 h. Subsequently, mutants on the 2000 IU·mL -1 nisin resistance plate were picked by a colony automatic picker and fermented in a 48-well plate. The mutants were initially screened by the microbial turbidimetry method.

[0042] Fermentation culture conditions in 48-well plate: The liquid loading volume is 1 mL, at 30°C, 200 rpm, and the fermentation time is 28 h.

[0043] Detecting the nisin potency of mutants by microbial turbidimetry method: After the fermentation of Lactococcus lactis in 48-well plate was completed, 6M HCl was added to each well to make the pH of the fermentation broth in the well about 2.0, boiled in a boiling water bath for 3 - 5 min, and then centrifuged at 3500 rpm for 15 min. The supernatant was diluted by a certain multiple in a new sterile 48-well plate. Then, 950 μL of the indicator bacteria cell suspension and 50 μL of the fermentation supernatant were added to the new sterile 48-well plate, cultured at 30°C, 200 rpm for 6 h, and then the OD of the indicator bacteria was detected by an enzyme-labeled instrument. 600 . According to the OD 600 value of the indicator bacteria corresponding to the mutant bacteria and the OD 600 value of the indicator bacteria in the control group, calculate ΔOD 600 , and its calculation formula is as follows, ΔOD 600 = OD 600(control) - OD 600(mutant) . According to this calculation formula, the larger the ΔOD 600 , the higher the nisin potency of the corresponding mutant strain.

[0044] Preparation of the indicator bacteria suspension: The indicator bacteria is Micrococcus flavus ATCC 10240 (obtained from ATCC). The inoculation loop is dipped into the bacterial liquid in the glycerol tube and streaked on the solid plate of the indicator bacteria growth medium, and cultured in an incubator at 30 °C for 24 h. One loop of the indicator bacteria is inoculated from the indicator bacteria plate with colonies into the indicator bacteria liquid medium, and cultured on a shaker at 30 °C and 200 rpm for 12 h. The cultured indicator bacteria liquid is inoculated into the fresh indicator bacteria growth medium at an inoculation amount of 10%.

[0045] Through the microbial turbidimetry method, select 10 mutant strains with the largest ΔOD 600 value for preservation and perform the next round of EMS mutagenesis and coat the nisin resistance plate at 4000 IU·mL -1 . And so on, through iterative mutagenesis with EMS, 462, 185, 464, 459, and 464 mutant strains were screened on the solid plates of the seed media with nisin resistance of 2000, 4000, 6000, 8000, and 10000 IU·mL -1 respectively. Subsequently, for the 10 mutant strains with the largest ΔOD 600 value screened on each concentration of the resistance plate, a total of 50 mutant strains at five resistance concentrations were subjected to shake flask re-screening. The nisin titer of the high-yield strain L. lactis E429 obtained by re-screening was 2527 IU·mL -1 after shake flask fermentation at 30 °C and 150 rpm for 24 - 26 h, which was 2.29 times that of the original strain in shake flask fermentation.

[0046] (2) Screening of nisin-tolerant high-yield mutant strains by heavy ion beam mutagenesis

[0047] The iterative use of the chemical mutagen EMS makes the strain develop a certain tolerance to it and reduces the mutation rate. Therefore, another mutagenesis method is selected for combined use to further improve its nisin titer. Heavy ion beam mutagenesis is widely used in the field of microbial breeding due to its advantages such as high positive mutation rate, wide mutation spectrum, non-toxic and harmless. The high-yield strain L. lactis E429 obtained by EMS mutagenesis is used as the starting strain for heavy ion beam mutagenesis treatment. The starting strain is streaked on the slant medium and cultured at 30 °C for a certain time. The bacterial lawn on the slant is washed off with 0.85% NaCl solution to prepare a bacterial suspension, and the OD 600 value of the bacterial suspension is controlled at about 1.0. 1 mL of the bacterial suspension is dispensed into an irradiation dish with a diameter of 35 mm, and the irradiation dish is sealed with a sealing film. The carbon ion beam 12C provided by HIRFL is used 6+Radiation treatment was carried out with radiation doses of 30 Gy, 60 Gy, 90 Gy, 120 Gy, 150 Gy, and 180 Gy, and a radiation dose rate of 80 Gy·min -1 .

[0048] After mutagenesis, it was spread on a nisin-resistant plate of 12000 IU·mL -1 . Based on the preliminary screening results of the microbial turbidimetry method, 10 mutant strains with the largest ΔOD 600 value were preserved and subjected to the next round of heavy ion beam mutagenesis. They were successively spread on solid plates of seed medium with nisin resistance of 14000, 16000, 18000, and 20000 IU·mL -1 . 465, 464, 464, 464, and 464 mutant strains were screened on these five concentration-resistant plates respectively. Subsequently, 50 mutant strains with the largest ΔOD 600 value at each nisin resistance concentration were selected for flask rescreening. Finally, a high-yield strain was obtained on the nisin-resistant plate of 20000 IU·mL -1 . The nisin titer of its flask fermentation for 24 - 26 h reached 10776 IU·mL -1 , which was 9.76 times the flask fermentation yield of the original strain. This mutant strain was named Lactococcus lactis MN6 and was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No. 31812.

[0049] Example 2 Comparison of the tolerance of the high-yield strain L. lactis MN6 and the original strain CGMCC1.2829 to nisin

[0050] The difference in nisin tolerance between the original strain CGMCC1.2829 and the high-yield strain MN6 was evaluated by spot plate experiment. As Figure 1 shown, after gradient dilution of the cultures of the original strain and the high-yield strain cultured to the mid-logarithmic phase, 10 μL of bacterial liquid at different dilution gradients was spotted on solid plates of seed medium with nisin resistance concentrations of 2000 and 6000 IU·mL -1 . The results showed that the growth of the high-yield strain MN6 on the nisin-resistant plate was better than that of the original strain CGMCC1.2829. Under the nisin stress condition of 6000 IU·mL -1 , the growth of the original strain CGMCC1.2829 was significantly inhibited, and the colonies were sparse, while the growth of the high-yield strain MN6 was hardly affected. Therefore, the nisin resistance of the high-yield strain MN6 obtained by nisin resistance screening was significantly enhanced.

[0051] Nisin inhibits Gram-positive bacteria mainly by inhibiting cell wall synthesis and binding to lipids in the cell membrane to form pores. Therefore, the tolerance of Lactococcus lactis to nisin can be judged by observing the cell morphology of Lactococcus lactis under nisin stress conditions. As Figure 2 shown, under the condition of 2000 IU·mL -1 nisin stress, there are more ruptured cells of the original strain than those of the high-yield strain under the same magnification (×10.0k) field of view. Therefore, it can be inferred that the tolerance of the high-yield strain to 2000 IU·mL -1 nisin is significantly stronger than that of the original strain.

[0052] Verification of the passage stability of strain MN6 in Example 3

[0053] The obtained strain was streaked on a solid seed medium and cultured at 30 °C for 1-2 d. A single colony was picked and inoculated into a liquid seed medium and cultured at 30 °C and 150 rpm for 12-16 h. The obtained seed liquid was streaked on a new solid seed medium. This process is the subculture.

[0054] The seed liquid of each subculture was inoculated into a shake flask fermentation medium at an inoculation amount of 3%, and fermented at 30 °C and 150 rpm for 24-26 h. The nisin titer in the fermentation broth was detected. The results are shown in Table 1. The obtained nisin-tolerant high-yield mutant Lactococcus lactis MN6 could stably maintain a yield of 10712 IU·mL -1 or more after 6 subcultures.

[0055] Table 1 Passage stability of high-yield mutant MN6

[0056]

[0057] Flask fermentation of the original strain and high-yield strain in Example 4

[0058] After the Lactococcus lactis plate was streaked and activated for 24 h, it was inoculated into a seed medium and cultured overnight. The seed culture was transferred to a fermentation medium at an inoculation amount of 3% and fermented at 30 °C and 150 rpm for 24-26 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3-5 min to collect the cells. The cells were resuspended and diluted with 0.85% NaCl solution of the same volume as the fermentation broth, and the absorbance was measured at 600 nm. Another 1 mL of the fermentation broth was adjusted to pH 2.0±0.2 with 6M HCl, boiled in boiling water for 3-5 min, centrifuged at 12000 rpm for 3-5 min, and the supernatant was transferred to a clean centrifuge tube and stored in a 4 °C refrigerator for detecting the nisin titer.

[0059] As Figure 3As shown in a, the nisin titer in the MN6 shake flask fermentation was 10776 IU·mL -1 , which was 9.76 times the nisin titer (1104 IU·mL -1 ) obtained by fermenting CGMCC1.2829.

[0060] Batch fermentation of the original strain and the high-yield strain in a 5L fermenter in Example 5

[0061] Compare the differences in OD 600 , nisin titer, pH, lactic acid production and residual sugar between CGMCC1.2829 and MN6 during batch fermentation on a 5L scale.

[0062] The specific process of fermenter fermentation is as follows: Prepare the seed liquid according to the method of Example 4, transfer the activated seed liquid to a 5L tank at an inoculation amount of 3%, and after the fermentation starts, rely on the dissolved oxygen linkage to control the dissolved oxygen DO during the fermentation process at 40%. When the pH drops naturally from 7.0 to 6.0, control the pH at 6.0 with 50% ammonia water until the fermentation ends.

[0063] The results are as shown in Figure 3 b and 3c. Compared with MN6, during the entire batch fermentation process, the sucrose consumption rate of the original strain CGMCC1.2829 was significantly faster, and it was basically consumed up in 8h. Also, the pH drop rate was significantly faster, and more lactic acid was produced. At the end of fermentation, the OD 600 and nisin titer of MN6 reached 14.24 and 12676 IU·mL -1 respectively, which were 1.61 times and 3.57 times that of the strain CGMCC1.2829. Comparing the nisin titer per unit OD 600 of MN6 and the original strain, that is, the nisin production intensity of the two strains, the nisin titer per unit OD 600 of MN6 could reach 890 IU·mL -1 , which was 2.22 times that of the original strain and was more suitable for industrial production.

[0064] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Lactococcus lactis MN6 was deposited at the China General Microbiological Culture Collection Center on September 2, 2024, with the deposit number CGMCC No. 31812.

2. A composition comprising the Lactococcus lactis MN6 as claimed in claim 1.

3. A microbial preparation comprising the Lactococcus lactis MN6 as claimed in claim 1.

4. The microbial agent according to claim 3, wherein, The microbial preparation contains viable cells of the Lactococcus lactis MN6.

5. A method for preparing bacteriocin nisin, characterized in that, Ferment the Lactococcus lactis MN6 as claimed in claim 1 or the microbial preparation as claimed in any one of claims 3 to 4 in a culture medium at 28 - 30 °C for at least 24 h.

6. The method according to claim 5, characterized in that, Culture the Lactococcus lactis MN6 at 28 - 30 °C for 12 - 16 h to prepare a seed solution, and then transfer the seed solution to a fermentation medium and culture at 28 - 30 °C for 24 - 26 h.

7. The method according to claim 5 or 6, characterized in that, The culture medium for fermentation contains: sucrose, soy peptone, yeast extract powder, KH2PO4, MgSO4·7H2O, Tween - 80.

8. Use of the Lactococcus lactis MN6 as claimed in claim 1, or the composition as claimed in claim 2, or the microbial preparation as claimed in any one of claims 3 to 4, or the method as claimed in any one of claims 5 to 7 in the fields of food, medicine, agriculture or industry.

9. The application according to claim 8, characterized in that, The use includes fermentative production of nisin or products containing nisin.

10. The application according to claim 8, characterized in that, The use includes preparation of a feed additive, preservative or antibacterial agent containing nisin.

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