High-throughput screening method of high-yield nisin strain and application of high-yield nisin strain

Abstract

The invention relates to the technical field of biology, particularly discloses a high-throughput screening method of a high-yield nisin strain and application of the high-yield nisin strain, and provides a high-yield nisin lactococcus lactis SD-06 with the preservation number being CGMCC No.35340 and the fermentation yield being larger than or equal to 22,400 IU / mL. The invention also provides a high-throughput screening method. The method comprises the following steps: constructing a mutation library through ultraviolet mutagenesis; the method comprises the following steps: rapidly detecting the nisin content in combination with large-scale culture of a deep-well plate and ultra-high performance liquid chromatography, and screening strains with the titer being 1%; after test tube re-screening and fermentation tank verification, high-yield strains are further obtained through mutagenesis; the ultra-high performance liquid chromatography detection adopts a C18 reversed-phase chromatographic column and acetonitrile-0. 1% trifluoroacetic acid for gradient elution, and a linear equation is established to realize accurate quantification; the strain and the method can significantly improve the production efficiency of nisin, and are suitable for the field of food preservative and antibacterial drug preparation.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a high-throughput screening method for a high-yielding nisin strain and its application. Background Art

[0002] Nisin, a natural antimicrobial peptide, is currently the only bacteriocin approved for food preservation. Due to its high safety profile and lack of cross-resistance with medical antibiotics, nisin has become a promising green biopreservative in the food and biopharmaceutical industries. As an industrial nisin production strain, the yield of Lactococcus lactis directly impacts its production efficiency and economic value, making the selection of high-yielding strains of this bacterium crucial.

[0003] Traditional strain selection methods primarily include natural isolation, physical and chemical mutagenesis, and protoplasmic fusion techniques. However, these methods generally suffer from low screening throughput, long detection cycles, and significant error in results. In particular, the commonly used agar diffusion method, while based on the antibacterial properties of nisin, relies on the determination of inhibition zones using indicator bacteria (such as Micrococcus luteus). This method is not only cumbersome and time-consuming, taking up to 18-24 hours, but also lacks accurate quantitative detection, severely limiting the efficiency of screening for high-yielding strains.

[0004] Along with the development of high-throughput screening technology, the automated screening system with multi-well plate as carrier provides new ideas for microbial breeding.Although prior art such as patent CN109810972A attempts to combine high-throughput technology with traditional breeding, the turbidimetry method it adopts still belongs to indirect detection, and is calculated by the correlation of indicator bacteria absorbance and nisin titer, and there are problems such as detection error is large, result is not intuitive.Therefore, develop a kind of high-throughput screening method that can directly, quickly and accurately measure nisin content, become the key technology bottleneck of current nisin high-yield strain breeding.Based on the above statement, the application provides a high-throughput screening method and application thereof of a strain of high-yield nisin bacterial strain. Summary of the Invention

[0005] In order to solve the defects of the prior art such as low nisin production of Lactococcus lactis, low strain screening throughput, long detection cycle, and inaccurate results, the present application provides a high-throughput screening method for a high-nisin-producing strain and its application.

[0006] In a first aspect, the application provides a high-yield nisin strain, which is Lactococcus lactis SD-06, deposited on July 23, 2025 in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Beijing, China, and classified as Lactococcus lactis with a preservation number of CGMCC No. 35340. The nisin yield of the strain is ≥22400 IU / mL.

[0007] Preferably, the 16S rRNA sequence of the Lactococcus lactis SD-06 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the application provides a high-throughput screening method for a high-yield nisin strain, which uses the following technical solution:

[0009] The high-throughput screening method for a high-yield nisin strain includes the following steps:

[0010] S1, performing ultraviolet mutagenesis treatment on a Lactococcus lactis starting strain to obtain a mutant strain library;

[0011] S2, using a high-throughput cloning selection system to select a mutant strain single colony from the mutant strain library and inoculate into a deep well plate containing a seed culture medium for culture, and then transfer to a deep well plate containing a fermentation culture medium for culture to obtain a fermentation broth;

[0012] S3, performing acidification treatment on the fermentation broth, then centrifuging and filtering to collect the supernatant, and using ultra-high performance liquid chromatography to detect the nisin content in the supernatant to select the top 1% of strains to obtain a primary screening strain;

[0013] S4, performing test tube re-screening on the primary screening strain to select the top 10% of strains to obtain a first re-screening strain, and verifying the first re-screening strain under optimized fermentation conditions in a fermentation tank to obtain a second re-screening strain;

[0014] S5, performing chemical mutagenesis and normal temperature and pressure plasma mutagenesis on the second re-screening strain to finally obtain a high-yield nisin strain.

[0015] Preferably, the seed culture medium in step S2 comprises soybean peptone 3-8 g / L, beef extract 3-8 g / L, tryptone 3-8 g / L, yeast extract 1.5-3.5 g / L, ascorbic acid 0.2-0.7 g / L, MgSO4·7H2O 0.15-0.25 g / L, β-glycerophosphate disodium 17-21 g / L, and glucose 0.3-0.7% (w / v).

[0016] The fermentation medium contains: sucrose 10-15 g / L, soy peptone 5-10 g / L, yeast extract 10-20 g / L, peptone 10-20 g / L, KH2PO4 10-20 g / L, NaCl 2-5 g / L, and MgSO4·7H2O 0.2-1 g / L.

[0017] Preferably, in step S2, the seed culture liquid volume is 0.8-1.2 mL, and the culture is carried out at 28-32°C for 8-16 h; the fermentation culture liquid volume is 0.8-1.2 mL, and the seed liquid is transferred to a deep-well plate containing fermentation medium at an inoculum size of 5-15% (v / v). After culture at 28-32°C for 16-24 h, the nisin content in the fermentation liquid is detected.

[0018] Preferably, the deep-well plate in step S2 is selected from one of a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 192-well plate or a 384-well plate.

[0019] Preferably, the acidification treatment in step S3 is to add 5-7M hydrochloric acid to the fermentation broth to adjust the pH to 2.0-3.0; the centrifugation parameters are: temperature 0-10°C, speed 2000-4000 rpm, time 5-15 min; and filtration uses a 0.22-0.45 μm microporous filter membrane.

[0020] Preferably, the ultra-high performance liquid chromatography detection in step S3 comprises the following steps:

[0021] A high-throughput screening model for ultra-high performance liquid chromatography (UPLC) detection was established to obtain a linear equation for calculating nisin content. The supernatant was added to a multi-well plate injection plate compatible with the deep-well plate. The multi-well plate injection plate was transferred to an ultra-high performance liquid chromatography system for gradient elution and detection. The nisin peak area obtained from the test sample was substituted into the linear equation to calculate the nisin content in the fermentation broth.

[0022] Preferably, the high-throughput screening model for ultra-high performance liquid chromatography detection is established by the following method:

[0023] The nisin standard solution was diluted stepwise to obtain a standard solution with a gradient concentration. A 1 μL injection volume was used for ultra-performance liquid chromatography analysis. A standard curve was prepared with nisin concentration as the abscissa and peak area as the ordinate. The linear equation was Y=0.12X+209.42, R²=1, where X is the nisin concentration and Y is the peak area.

[0024] Preferably, the ultra-high performance liquid chromatography system uses a C18 reverse phase chromatography column with a column size of 2.1×100 mm and a particle size of 1.7-1.9 μm, with acetonitrile-0.1% trifluoroacetic acid aqueous solution as the mobile phase for gradient elution, a flow rate of 0.1-0.4 mL / min, a detection wavelength of 210-400 nm, a column temperature of 10-40°C, an injection volume of 1-20 μL, a single sample analysis time of 2-5 min, and a diode array detector.

[0025] Preferably, during the test tube rescreening in step S4, the seed culture liquid volume is 5-10 mL, and the culture is carried out at 28-32°C for 8-16 hours; the fermentation culture liquid volume is 5-10 mL, and the seed liquid is transferred to a test tube containing fermentation medium at an inoculum size of 5-15% (v / v), and after culture at 28-32°C for 16-24 hours, the nisin content in the fermentation liquid is detected.

[0026] Preferably, the fermentation tank optimization fermentation conditions in step S4 includes:

[0027] Fed-batch fermentation was carried out in a 5-L fermentor. Fermentation medium was initially selected and fed during fermentation. The feed medium contained 500-600 g / L glucose, 20-40 g / L yeast extract, and 0.5-2 g / L MgSO4·7H2O. Fermentation conditions were as follows: inoculum size 5-15% (v / v), pH 6.5-7.0, temperature 28-32°C, fermentation time 16-24 h, feeding rate 1.5-3 mL / (L / h), and residual sugar concentration maintained at 0.5-1 g / L. After fermentation, the nisin titer in the fermentation broth was determined by high-performance liquid chromatography.

[0028] Preferably, the chemical mutagenesis in step S5 includes ethyl methanesulfonate mutagenesis, 5-bromouracil mutagenesis and diethyl sulfate mutagenesis.

[0029] In a third aspect, the present application provides the use of a high-yield nisin strain in food preservation and the preparation of antibacterial drugs.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. Breakthrough progress in the selection and breeding of high-yield strains: Through a multi-round precision screening strategy combining UV mutagenesis, chemical mutagenesis, and normal temperature and pressure plasma mutagenesis, a high-yielding nisin strain, Lactococcus lactis SD-06, with potential for industrial application was successfully identified. This strain achieved a breakthrough fermentation titer of 22,400 IU / mL, a 21.4% increase compared to the original starting strain. This significant titer increase has reached the leading level among similar studies internationally. This strain provides an efficient and stable production strain for large-scale industrial production of nisin, significantly improving the efficiency and accuracy of screening for high-yielding industrial nisin strains and significantly shortening the selection cycle. It has already been applied to industrial production in 230-ton fermentation tanks, providing an effective method for high-throughput screening of high-yielding nisin strains for industrial use.

[0032] 2. High-throughput screening platform achieves technological upgrades: A high-throughput screening system based on deep-well plate culture combined with ultra-performance liquid chromatography (UPLC) detection has been established, increasing the single-batch screening throughput to over 50 times that of traditional shake flask screening methods. Through standardized operating procedures, the reliability and reproducibility of screening results are ensured, significantly shortening the selection cycle for superior strains and providing an efficient technical platform for the rapid screening of high-yield strains.

[0033] 3. Detection systems have been upgraded to be more intelligent and efficient: By optimizing ultra-high performance liquid chromatography (UPLC) detection methods, the detection time for a single sample has been significantly reduced from over 20 minutes with traditional HPLC to 2-3 minutes. A single device can now continuously test 720 samples per day, increasing detection efficiency by more than 10 times. This system offers advantages such as rapid detection, high data accuracy, and excellent repeatability, effectively supporting the efficient advancement of large-scale strain selection and breeding, and providing strong technical support for industrial strain development.

[0034] 4. Significant reduction in screening costs: This application uses a deep-well plate culture system combined with automated detection technology to reduce the cost of single strain screening to less than 1 / 10 of the traditional shake flask method. The miniaturized culture system not only saves more than 80% of the culture medium usage, but also reduces labor costs by approximately 60% through standardized operating procedures. This technical solution significantly reduces the R&D investment in high-yield strains, making large-scale strain selection more economically feasible and providing an affordable technical path for companies to carry out strain improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The flowchart of the high-throughput screening method for a high-yielding nisin strain in the embodiment of the present application is shown.

[0036] Figure 2 This is the standard curve for nisin ultra-high performance liquid chromatography detection in the embodiment of this application.

[0037] Figure 3 This is the result of high-throughput initial screening in a deep-well plate after UV mutagenesis in the embodiment of this application.

[0038] Figure 4 This is the result of test tube rescreening after UV mutagenesis in the embodiment of this application.

[0039] Figure 5 This is the canning verification result after ultraviolet mutagenesis in the implementation mode of this application.

[0040] Figure 6 This is the result of high-throughput initial screening in a deep-well plate after chemical mutagenesis in the embodiment of this application.

[0041] Figure 7 This is the result of test tube rescreening after chemical mutagenesis in the embodiment of this application.

[0042] Figure 8 This is the result of tank verification after chemical mutagenesis in the implementation mode of this application.

[0043] Figure 9 This is the initial high-throughput screening result of deep-well plate after plasma mutagenesis at normal temperature and pressure in the embodiment of this application.

[0044] Figure 10 This is the test tube rescreening result after normal temperature and pressure plasma mutagenesis in the embodiment of this application.

[0045] Figure 11 This is the tank verification result after normal temperature and pressure plasma mutagenesis in the implementation mode of this application. DETAILED DESCRIPTION

[0046] The present application will be further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are only used to explain the present application and are not intended to limit the scope of the present application.

[0047] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0049] The starting strain of Lactococcus lactis described in this application is an industrial production strain provided by Zhejiang New Silver Elephant Bioengineering Co., Ltd., hereinafter referred to as LL00.

[0050] Seed culture medium (GM17 medium): 5 g / L soy peptone, 5 g / L beef extract, 5 g / L tryptone, 2.5 g / L yeast extract, 0.5 g / L ascorbic acid, 0.25 g / L MgSO4·7H2O, 19 g / L disodium β-glycerophosphate, and 0.5% (w / v) glucose.

[0051] GM17 solid plate: Add 15g / L agar powder to the seed culture medium (GM17 medium) and pour the plate.

[0052] Fermentation medium: sucrose 10 g / L, soy peptone 5 g / L, yeast extract 10 g / L, peptone 10 g / L, KH2PO4 10 g / L, NaCl 2 g / L, MgSO4·7H2O 0.2 g / L.

[0053] Feed medium: glucose 600 g / L, yeast extract 30 g / L, MgSO4·7H2O 1.5 g / L.

[0054] Example 1: LL00 starting strain culture

[0055] The Lactococcus lactis starting strain LL00 was streaked onto a GM17 solid plate and incubated in a 30°C constant temperature incubator for 12 hours. After colonies grew, a single representative colony was selected and inoculated into 10 mL of GM17 medium. The activated culture was then incubated in a 30°C constant temperature incubator for 12 hours for activation. Subsequently, the activated culture was transferred to 50 mL of GM17 medium at a 2% (v / v) inoculum and incubated at 30°C with shaking for 6 hours to obtain the LL00 initial culture (OD 600 =0.6).

[0056] Example 2: Construction of mutant strain library by UV mutagenesis

[0057] 1. Pretreatment of bacterial solution: 600 =0.6) was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in sterile phosphate buffer (PBS, 0.1 M, pH 7.0) and the cell concentration was adjusted to 10 6 CFU / mL to obtain the resuspended LL00 bacterial solution.

[0058] 2. Non-mutagenized control setting: Take 1 mL of resuspended LL00 bacterial solution and perform gradient dilution. Pipette 100 μL of the dilution solution and spread it on a GM17 solid plate. Incubate at 30°C for 24 h as a non-mutagenized control for subsequent calculation of the lethality.

[0059] 3. UV mutagenesis: Transfer 15 mL of the resuspended LL00 bacterial solution to a sterile glass dish with a diameter of 9 cm, add a sterile magnetic rotor, place the dish on a magnetic stirrer, adjust the speed to 200 rpm, adjust the vertical distance between the 15 W UV lamp and the liquid surface in the sterile glass dish to 26 cm, and set the irradiation time gradient to 30 s, 60 s, 90 s, 120 s and 150 s respectively to obtain UV mutagenesis bacterial solution.

[0060] 4. Mutation library screening: After treatment at each time point, the UV mutagenized bacterial solution was immediately protected from light and graded diluted. 100 μL was spread on a GM17 solid plate, with three replicates for each time gradient. The plate was incubated at 30°C in the dark for 48 hours, the number of colonies was counted, and the lethality rate was calculated:

[0061] Lethality rate (%) = 100 × [1 - (CFU / mL of treatment group) / (CFU / mL of control group)];

[0062] Where: CFU / mL of treatment group = number of colonies on plate × dilution factor of treatment group × 10;

[0063] CFU / mL of the control group = number of colonies on the plate × dilution factor of the control group × 10.

[0064] The irradiation time with a lethality of 90% was selected as the optimized parameter for subsequent large-scale mutagenesis. After large-scale UV mutagenesis, a mutant strain library growing on multiple plates was obtained.

[0065] Example 3: High-throughput selection and cultivation of mutagenic strains

[0066] 1. Preparation of primary culture plates: Use a BioTek continuous dispenser to dispense sterile GM17 medium into 96-well deep-well plates at 1 mL / well. Set up a positive control (well A1 inoculated with the original strain LL00) and a negative control (well H12 filled with sterile culture medium) on each plate. Label them as primary culture plates (An, n = 1-96).

[0067] 2. Single clone isolation: Using the QpixXT 400 series fully automated clone selection system, we selected colonies from the mutant strain library for primary screening based on colony diameters greater than 0.5 mm, as there is a positive correlation between nisin production and Lactococcus lactis biomass. Single colonies were inoculated into the corresponding wells of primary culture plates (An, n = 1-96).

[0068] 3. Activation culture of the strain: Place the An plate in a 30°C constant temperature incubator and culture for 11 hours.

[0069] 4. Preparation of secondary culture plates: Use a Multidrop dispenser to transfer the bacterial solution from the An plate to a 96-well deep-well plate containing 1 mL of fermentation medium at a 20% inoculum volume. Mark it as a secondary culture plate (Bn, n = 1-96).

[0070] 5. Expand the culture: Place the Bn plate in a 30°C constant temperature incubator for 20 hours.

[0071] Example 4: Acid treatment of fermentation broth and sample preparation

[0072] 1. Preparation of bacterial solution treatment plates: Accurately pipette 400 μL of fermentation bacterial solution from each well of plate Bn and add it to the corresponding treatment plates Cn.

[0073] 2. Acid treatment: Use a dispenser to dispense 20 μL of 6 M hydrochloric acid into treatment plate Cn. Use an electric dispenser to repeatedly pipette and aspirate to mix 10 times. Be careful to avoid bubbles during the process. Mark it as acid treatment plate (Cn, n = 1-96).

[0074] 3. pH adjustment and heat treatment: Adjust the pH of the mixed solution in the Cn plate to 2.0, then place the Cn plate in a 95°C dry bath thermostat for heat treatment for 30 minutes, and cover the plate opening with a sealing film to prevent evaporation.

[0075] 4. Sample collection: Immediately cool in an ice bath for 5 minutes after heat treatment, centrifuge at 3000 rpm for 5 minutes using a 96-well plate centrifuge, and filter using a 0.22 μm microporous filter membrane. Collect the filtrate into a 96-well sample plate, marked as Dn plate (n=1-96).

[0076] Example 5: Construction of a high-throughput screening model

[0077] 1. Chromatographic analysis conditions: This application uses an Agilent 1290 ultra-high performance liquid chromatography system with the following configuration parameters:

[0078] Column: InfinityLab Poroshell HPH-C18 (2.1 × 100 mm, 1.9 µm);

[0079] Detector: PDA diode array detector (detection wavelength 220 nm);

[0080] Mobile phase: Phase A: 0.1% trifluoroacetic acid aqueous solution (v / v), Phase B: acetonitrile;

[0081] Elution procedure: gradient elution, the volume ratio of phase B to phase A is 10-90%, and the elution time of a single sample is 3 minutes;

[0082] Flow rate: 0.4 mL / min;

[0083] Column temperature: 30°C;

[0084] Injection volume: 1 μL

[0085] 2. Preparation of standard: Dissolve the nisin standard in ultrapure water to prepare a 1 mg / mL stock solution. After filtering the stock solution through a 0.22 μm filter membrane, dilute it to prepare 5 standard solutions with different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL).

[0086] 3. Verification of analytical method: According to the above chromatographic analysis conditions, the prepared standard solution at each concentration point was injected three times in parallel, and the mean peak area was used as the vertical axis (Y) and the concentration of nisin was used as the horizontal axis (X). Linear regression was performed to obtain the following results: Figure 2 The standard curve equation shown is Y=0.12X+209.42 (R 2 =1).

[0087] 4. Application of the method: This method can achieve single-sample analysis time of less than 3 minutes and continuous detection of 96-well sample plates (including system cleaning) in less than 6 hours. It is suitable for rapid screening of nisin production in a mutant library of more than 1000 strains.

[0088] Example 6: High-throughput screening and analysis of mutant strains

[0089] 1. Sample testing process: Transfer the 96-well sample plate to the ultra-high performance liquid chromatography detection system for testing. The injection volume is 5 μL. Set nisin standard and blank culture medium as quality control samples on each plate. The system automatically records the chromatographic peak area for quantitative calculation.

[0090] 2. Quantitative analysis: Substitute the peak area into the standard curve equation Y = 0.12X + 209.42 to calculate the nisin concentration and convert it into the potency (IU / mL). A total of 7990 mutant strain fermentation products were tested and a yield distribution heat map was drawn ( Figure 3 ) Screen out strains with a yield 150% higher than that of the wild type and a peak shape that meets the standard.

[0091] 3. Rescreening of high-yield strains: Sort the strains obtained from the heat map screening by titer, and select the 80 candidate strains in the top 1% of titer. Add an equal volume of 50% glycerol (final concentration 25%) to the corresponding Bn plate wells and mix them evenly. Pipette out the strains and place them in storage tubes. Store them at -80°C (labeled as LLB-001 to LLB-080) for subsequent screening.

[0092] 4. Data quality control: The SIMS 3.0 system was used to automatically collect and analyze data, and duplicate samples with peak area RSD greater than 5% and abnormal data with retention time offset greater than 0.1 min were eliminated to ensure the reliability of the screening results.

[0093] Example 7: Rescreening and verification of high-yield strains

[0094] 1. Rescreening and culture process: The 80 high-yield strains (LLB-001 to LLB-080) obtained in the initial screening were streaked and separated on GM17 solid plates. After static culture in a constant temperature incubator at 30°C for 24 hours, typical single colonies were picked and inoculated into test tubes containing 10 mL of seed culture medium. Three replicates were set up for each strain. At the same time, the original strain LL00 control and blank culture medium control were set up. After static culture in a constant temperature incubator at 30°C for 12 hours to reach the logarithmic growth phase, the inoculation amount was transferred to 10 mL of fermentation medium at 8%, and fermentation was completed by shaking culture at 30°C for 20 hours.

[0095] 2. Potency analysis: The fermentation broth was collected, acidified with 6M hydrochloric acid, and the pH was adjusted to 2.0. The broth was then heat-treated at 95°C for 30 minutes and immediately cooled in an ice bath for 5 minutes. The supernatant was collected after centrifugation at 3000 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm microporous filter membrane and the filtrate was collected for ultra-high performance liquid chromatography. The nisin potency was calculated based on the standard curve. The rescreening results were as follows: Figure 4 shown.

[0096] 3. Verification of preferred strains: The top 10% high-yield strains were selected by potency for the next step of 5L fermentation tank amplification verification. A total of 8 high-yield strains were numbered LL01-08.

[0097] Example 8: Verification of high-yield strains in 5L fermentation tanks

[0098] 1. Fermentation process parameters: The above eight high-yielding strains (LL01-08) and the starting strain LL00 were inoculated from glycerol storage tubes at a 2% (v / v) inoculum size into a test tube containing 10 mL of seed medium. After static incubation at 30°C for 12 h, the inoculum size was inoculated into a 500 mL shake flask containing 350 mL of seed medium at a 2% (v / v) inoculum size. After shaking incubation at 30°C and 150 rpm for 20 h, the inoculum size was transferred to a 5 L fermentor (working volume 3.5 L) at a 10% inoculum size. The pH was controlled at 7.0 by automatic flow-feeding of liquid caustic soda. After incubation at 30°C for 5 h, feed medium feeding was started at a feed rate of 2 mL / (Lh). The residual sugar concentration was controlled at 0.5 g / L. After 18 h of fermentation, samples were taken every 2 h for titer detection until the plateau phase.

[0099] 2. Analysis of fermentation results: Figure 5 As shown, the nisin production of all verified strains was significantly higher than that of the starting strain LL00, among which LL02 performed best with a fermentation titer of 19856 IU / mL. This strain was selected as the starting strain for the next round of chemical mutagenesis studies.

[0100] Example 9: Chemical mutagenesis screening

[0101] 1. Pretreatment of bacterial solution: 600 = 0.6) was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in sterile phosphate buffered saline (PBS, 0.1 M, pH 7.0) and the cell concentration was adjusted to 10 6 CFU / mL to obtain the resuspended LL02 bacterial solution.

[0102] 2. Non-mutagenized control setting: Take 1 mL of resuspended LL02 bacterial solution and perform gradient dilution. Pipette 100 μL of the dilution solution and spread it on a GM17 solid plate. Incubate in a 30°C constant temperature incubator for 24 h as a non-mutagenized control for subsequent calculation of the lethality.

[0103] 3. DES mutagenesis: Add 1.2% (v / v) diethyl sulfate (DES) to 15 mL of resuspended LL02 bacterial solution. After shaking at 30°C and 150 rpm for 30 min, immediately add an equal volume of 25% sodium thiosulfate to terminate the reaction. Collect the cells by centrifugation at 8000 rpm for 3 min, wash the cells three times with sterile phosphate buffer (PBS, 0.1 M, pH 7.0) to remove residual mutagen, and resuspend the cells in 15 mL of sterile phosphate buffer to obtain DES-induced bacterial solution.

[0104] 4. Screening of mutant library: Perform gradient dilution of the DES mutagenic bacterial solution, take 100 μL and spread on GM17 solid plates, set up three parallel plates, incubate in a 30°C constant temperature incubator in the dark for 48 hours, count the number of colonies and calculate the lethality rate:

[0105] Lethality rate (%) = 100 × [1 - (CFU / mL of treatment group) / (CFU / mL of control group)];

[0106] Where: CFU / mL of treatment group = number of colonies on plate × dilution factor of treatment group × 10;

[0107] CFU / mL of the control group = number of colonies on the plate × dilution factor of the control group × 10.

[0108] The plate with a lethality rate of 85% was selected to construct a mutant strain library, and a monoclonal picking system and deep-well plate were used for initial screening (the results were as follows Figure 6 ), ultra-high performance liquid chromatography, test tube rescreening (results such as Figure 7 )、Tank verification screening (results such as Figure 8 ) obtained an excellent mutant strain LL12 with a titer of 21227 IU / mL, which will be used as the starting strain for normal temperature and pressure plasma (ARTP) mutagenesis.

[0109] Example 10: Normal Temperature and Normal Pressure Plasma Mutation Screening

[0110] 1. Pretreatment of bacterial solution: 600 = 0.6) was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in sterile phosphate buffered saline (PBS, 0.1 M, pH 7.0) and the cell concentration was adjusted to 10 6 CFU / mL to obtain the resuspended LL12 bacterial solution.

[0111] 2. Non-mutagenized control setting: Take 1 mL of resuspended LL12 bacterial solution and perform gradient dilution. Pipette 100 μL of the dilution solution and spread it on a GM17 solid plate. Incubate in a 30°C constant temperature incubator for 48 h as a non-mutagenized control for subsequent calculation of the lethality.

[0112] 3. ARTP mutagenesis: Using the ARTP mutagenesis system (He working gas, flow rate 10 SLM, RF power 100 W), 10 μL of resuspended LL12 bacterial solution was evenly spread on a sterile slide, and gradient treatment was performed at a treatment distance of 3 mm for 0 s, 30 s, 60 s, 90 s, and 120 s. After the treatment, the slide was transferred to a 2 mL EP tube containing 1 mL of pre-cooled GM17 medium using sterile tweezers. The tube was vortexed for 30 s to fully elute the bacteria, and the tube was allowed to stand in a 30°C constant temperature incubator away from light for 2 h to recover the ARTP mutagenic bacterial solution.

[0113] 4. Screening of mutant library: Stratify the dilution of ARTP mutagenic bacterial solution, take 100 μL and spread it on GM17 solid plate, set up 3 parallel plates, incubate in a 30°C constant temperature incubator in the dark for 48 hours, count the number of colonies and calculate the lethality rate:

[0114] Lethality rate (%) = 100 × [1 - (CFU / mL of treatment group) / (CFU / mL of control group)];

[0115] Where: CFU / mL of treatment group = number of colonies on plate × dilution factor of treatment group × 10;

[0116] CFU / mL of the control group = number of colonies on the plate × dilution factor of the control group × 10.

[0117] The plate with a lethality rate of 80% was selected to construct a mutant strain library, and a monoclonal picking system and deep-well plate were used for initial screening (the results were as follows Figure 9 ), ultra-high performance liquid chromatography, test tube rescreening (results such as Figure 10 )、Tank verification screening (results such as Figure 11) obtained a high-yield mutant strain LL21 with a titer of 22400 IU / mL, named SD-06, and deposited it on July 23, 2025 at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 35340 and the classification name Lactococcus lactis.

[0118] The 16S rRNA sequence of Lactococcus lactis SD-06 is shown in SEQ ID NO: 1:

[0119]

[0120] The above specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A high-yielding nisin strain, characterized in that: The strain is Lactococcus lactis SD-06, which was deposited on July 23, 2025 at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 35340, and the classification name is Lactococcus lactis. The nisin production of this strain is ≥22400 IU / mL.

2. A high-throughput screening method for the high-yielding nisin strain according to claim 1, characterized in that: The following steps are involved: S1. Performing UV mutagenesis on the starting strain of Lactococcus lactis to obtain a mutant strain library; S2. Using a high-throughput cloning system, single colonies of mutant strains were selected from the mutant strain library and inoculated into deep-well plates containing seed culture medium for cultivation. The colonies were then transferred to deep-well plates containing fermentation culture medium to obtain fermentation broth. S3, acidifying the fermentation broth, collecting the supernatant by centrifugation, detecting the nisin content in the supernatant by ultra-high performance liquid chromatography, and screening the strains with the top 1% titer to obtain the primary screening strains; S4. The primary screened strains were rescreened in test tubes to obtain the top 10% strains in terms of titer to obtain the primary rescreened strains. The primary rescreened strains were then verified in a fermentation tank under optimized fermentation conditions to obtain the secondary rescreened strains. S5. The secondary screened strains were subjected to chemical mutagenesis and normal temperature and normal pressure plasma mutagenesis to finally obtain a high-yield nisin strain.

3. The high-throughput screening method for high-yield nisin strains according to claim 2, characterized in that: The seed culture medium in step S2 comprises: 3-8 g / L soy peptone, 3-8 g / L beef extract, 3-8 g / L tryptone, 1.5-3.5 g / L yeast extract, 0.2-0.7 g / L ascorbic acid, 0.15-0.25 g / L MgSO4·7H2O, 17-21 g / L β-glycerophosphate disodium, and 0.3-0.7% (w / v) glucose; The fermentation medium contains: sucrose 10-15 g / L, soy peptone 5-10 g / L, yeast extract 10-20 g / L, peptone 10-20 g / L, KH2PO4 10-20 g / L, NaCl 2-5 g / L, and MgSO4·7H2O 0.2-1 g / L.

4. The high-throughput screening method for high-yield nisin strains according to claim 2, characterized in that: In step S3, the acidification treatment is to add 5-7M hydrochloric acid to the fermentation broth to adjust the pH to 2.0-3.0; the centrifugation parameters are: temperature 0-10°C, rotation speed 2000-4000 rpm, time 5-15 min; and filtration is performed using a 0.22-0.45 μm microporous filter membrane.

5. The high-throughput screening method for high-yield nisin strains according to claim 2, characterized in that: The ultra-high performance liquid chromatography detection in step S3 comprises the following steps: A high-throughput screening model for ultra-high performance liquid chromatography (UPLC) detection was established to obtain a linear equation for calculating nisin content. The supernatant was added to a multi-well plate injection plate compatible with the deep-well plate. The multi-well plate injection plate was transferred to an ultra-high performance liquid chromatography system for gradient elution and detection. The nisin peak area obtained from the test sample was substituted into the linear equation to calculate the nisin content in the fermentation broth.

6. The high-throughput screening method for high-yield nisin strains according to claim 5, characterized in that: The high-throughput screening model for ultra-high performance liquid chromatography detection was established by the following method: The nisin standard solution was diluted stepwise to obtain a standard solution with a gradient concentration. A 1 μL injection volume was used for ultra-performance liquid chromatography analysis. A standard curve was prepared with nisin concentration as the abscissa and peak area as the ordinate. The linear equation was Y=0.12X+209.42, R²=1, where X is the nisin concentration and Y is the peak area.

7. The high-throughput screening method for high-yield nisin strains according to claim 5, characterized in that: The ultra-high performance liquid chromatography system uses a C18 reverse-phase column with a column size of 2.1×100 mm and a particle size of 1.7-1.9 μm. Gradient elution is performed with acetonitrile-0.1% trifluoroacetic acid aqueous solution as the mobile phase, a flow rate of 0.1-0.4 mL / min, a detection wavelength of 210-400 nm, a column temperature of 10-40°C, an injection volume of 1-20 μL, a single sample analysis time of 2-5 minutes, and a diode array detector.

8. The high-throughput screening method for high-yield nisin strains according to claim 2, characterized in that: The fermentation tank optimization fermentation conditions in step S4 include: Fed-batch fermentation was carried out in a 5-L fermentor. Fermentation medium was initially selected and fed during fermentation. The feed medium contained 500-600 g / L glucose, 20-40 g / L yeast extract, and 0.5-2 g / L MgSO4·7H2O. Fermentation conditions were as follows: inoculum size 5-15% (v / v), pH 6.5-7.0, temperature 28-32°C, fermentation time 16-24 h, feeding rate 1.5-3 mL / (L / h), and residual sugar concentration maintained at 0.5-1 g / L. After fermentation, the nisin titer in the fermentation broth was determined by high-performance liquid chromatography.

9. The high-throughput screening method for high-yield nisin strains according to claim 2, characterized in that: The chemical mutagenesis in step S5 includes ethyl methanesulfonate mutagenesis, 5-bromouracil mutagenesis and diethyl sulfate mutagenesis.

10. Use of the high-nisin-producing strain according to claim 1 in food preservation and preparation of antibacterial drugs.

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