Method for high-throughput screening of nisin high-yield strain

Abstract

The invention relates to the technical field of microorganisms. The method comprises the following steps: S1, carrying out different mutation breeding treatments on an original strain to obtain a mutant strain library, S2, selecting a single colony by using a selection system, carrying out fermentation culture, carrying out acidification centrifugal filtration, and collecting a filtrate, S3, determining the content of nisin in the filtrate, and carrying out primary screening to obtain the nisin high-yield strain, S4, carrying out fermentation and secondary screening in a test tube, and S5, carrying out high-throughput screening to obtain the nisin high-yield strain. Detecting the content of nisin to obtain a rescreened nisin high-yield strain, and S5, loading into a tank for verification. According to the method, classic mutagenesis, monoclonal high-throughput bacterial selection, high-throughput pore plate screening and ultra-high performance liquid detection are combined, the yield of the screened nisin high-yield strain is increased by 40% or above on a 5L fermentation tank, the method has the advantages of being rapid, accurate and efficient, the screening efficiency is improved, and the breeding period is shortened.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, in particular to a method for high-throughput screening of nisin high-yield strains. Background Art

[0002] As an important natural active antimicrobial peptide, nisin is the only bacteriocin approved for food preservation. Nisin is basically non-toxic to humans and does not produce cross-resistance with medical antibiotics. It has become a promising green food preservative and a potential antibiotic substitute. Its application range is widely distributed in the fields of food industry and biomedicine. The industrial production strain of nisin is Lactococcus lactis. Rapid and efficient breeding of high-yield nisin Lactococcus lactis strains will bring considerable economic benefits to the fermentation production of nisin.

[0003] The methods for industrial selection of nisin high-yield strains mainly include natural separation, physical and chemical mutagenesis, protoplasm fusion, etc. However, no matter which strain screening method is adopted, it is inseparable from the screening steps such as strain culture and product identification and detection. The traditional screening method has a low strain screening amount per unit time and a large detection error, which has great limitations, and a large amount of manual operation makes the screening work extremely time-consuming and laborious. Usually, strain screening is a probabilistic event. Only when the strain screening base is large enough, the probability of obtaining a high-yield strain is large. The key to strain selection lies in a fast and accurate detection method. Therefore, a high-throughput strain culture system and a high-throughput product accurate detection method are the key to obtaining a high-yield nisin strain.

[0004] The detection of nisin generally uses the agar diffusion method, which is based on the antibacterial effect of nisin. Nisin-sensitive bacteria such as Micrococcus luteus are used as indicator bacteria, and the titer of nisin is calculated by detecting the size of the inhibition zone on the solid plate. Even if a large number of mutants are obtained, this method has the disadvantages of being cumbersome, taking a long time to detect, low efficiency, and large errors to screen strains. It is difficult to accurately screen high-yield strains. Therefore, it is necessary to establish a fast and accurate detection method to improve the detection efficiency. In recent years, devices and related technologies for high-throughput screening of microbial strains have continued to develop and mature. High-throughput screening technology uses multi-well plates as carriers, uses an automated operating system to perform strain selection experiments, collects fermentation product data through highly sensitive and high-speed detection instruments, and completes large-scale sample selection, detection, and analysis at the same time. High-throughput screening technology can achieve rapid screening in a large number of mutants, greatly shortening the time required for screening. However, the high-throughput screening method developed specifically for nisin high-yield strains is still less, for example, a method for high-throughput screening strains and its application are disclosed in patent CN109810972A, traditional breeding is combined with high-throughput screening technology, a method for rapidly measuring nisin titer by turbidimetry using Micrococcus luteus as an indicator bacterium is designed, and a high-throughput screening method for nisin high-yield strain breeding is established on this basis. However, the method can not directly quantitatively detect the titer of nisin, but nisin is measured by the correlation between the absorbance of the bacterial concentration of the indicator bacteria and the nisin titer, which is easy to produce errors, and the result is not intuitive enough. Therefore, the simple, fast and efficient high-throughput screening technology for nisin strains developed independently has important practical significance. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention discloses a method for high-throughput screening of nisin high-yielding strains, which comprises: S1: Perform different mutagenesis breeding treatments on the starting strain to obtain a mutant strain library on the plate; S2: Use a high-throughput clone selection system to select single colonies growing on the plate, inoculate them into a multi-well culture plate containing a seed medium for culture, and then transfer them to a multi-well culture plate containing a fermentation medium for culture. After the culture is completed, acidify and centrifuge to collect the filtrate; S3: The nisin content in the filtrate was determined by a high-throughput screening model based on ultra-high performance liquid chromatography detection, and a mutant strain of Lactococcus lactis with high nisin production was initially screened out; S4: inoculating the strain obtained from the preliminary screening into a test tube containing a seed culture medium for culture, and then transferring the strain to a test tube containing a fermentation culture medium for fermentation culture. After the fermentation is completed, the nisin content in the fermentation culture liquid is detected, and the nisin high-yielding strain is re-screened; S5: The above-mentioned re-screened nisin high-yield strains are put into tanks for verification to confirm the fermentation capacity of the re-screened strains.

[0006] Further, in S1, the starting strain is Lactococcus lactis; The mutagenesis method is one of ultraviolet mutagenesis, chemical mutagenesis, microwave mutagenesis, resistance mutagenesis, plasma mutagenesis at normal temperature and pressure, radiation mutagenesis or magnetic field mutagenesis; Chemical mutagenesis includes ethyl methanesulfonate mutagenesis, 5-bromouracil mutagenesis, and diethyl sulfate mutagenesis; The limiting factors for resistance mutagenesis screening are sugar tolerance, pH tolerance, sodium lactate tolerance, and nisin and nisin derivative tolerance; Furthermore, the multi-well plate culture plate in S2 is selected from: 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.

[0007] Furthermore, the components of the seed culture medium in S2 include: 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, MgSO4 . 7H2O·0.15-0.25g / L, β-glycerophosphate disodium 17-21g / L and 0.3-0.7% (W / V) glucose; The components of the fermentation medium include: sucrose 10-15g / L, soy protein 5-10g / L, yeast extract 10-20g / L, peptone 10-20g / L, KH2PO4·10-20g / L, NaCl·2-5g / L, MgSO4 . 7H2O·0.2-1g / L.

[0008] Furthermore, the multiwell plate culture containing the seed medium in S2 was cultured at 30°C for 8-16 h; The fermentation culture is to transfer the seed liquid to a multi-well plate at an inoculation rate of 5-15% (v / v), and ferment and culture at 30°C for 16-24h; The cradle centrifuge centrifugal filtration uses a multi-well plate filter plate corresponding to the multi-well plate culture plate, with a filter membrane of 0.2-0.45 μm. The conditions for centrifugation to collect the fermentation liquid into the multi-well plate injection plate should be a temperature of 0-10°C, a rotation speed of 2000-4000 r / min, and a centrifugation time of 5-15 min.

[0009] Furthermore, in S3, a chromatographic column of an ultra-high performance liquid chromatography system C18 was used, with acetonitrile-0.1% trifluoroacetic acid water as the mobile phase, a flow rate of 0.1-0.4 mL / min, a detection wavelength of 210-400 nm, and a column temperature of 10-40°C; the injection volume was 1-20 μL, the gradient elution time for a single sample was 2-5 min, and a diode array detector was used as the detector.

[0010] Furthermore, the method for establishing a high-throughput screening model for ultra-high performance liquid chromatography detection of nisin in S3 is as follows: take the nisin standard solution, dilute it step by step to obtain a standard solution with a gradient concentration, perform ultra-high performance liquid chromatography analysis, and the injection volume is 1 μL. A standard curve is prepared with the concentration of nisin as the horizontal axis and the peak area as the vertical axis to obtain a linear equation.

[0011] Furthermore, in S3, the fermentation broth is processed by centrifugation and then collected on a multi-well plate sample plate, and then transferred to an ultra-high performance liquid chromatography system for sample analysis and detection. The obtained peak area of ​​nisin is substituted into a linear equation to calculate the content of nisin in the fermentation broth sample.

[0012] Furthermore, in S4, when the high-yield strains initially screened were rescreened in test tubes, the seed culture liquid volume was 5-10 mL and cultured at 30°C for 8-16 h; the fermentation culture was carried out in test tubes with a liquid volume of 5-10 mL and an inoculation size of 5-15%. After culture at 30°C for 16-24 h, the fermentation liquid was centrifuged and the nisin content in the fermentation liquid was detected.

[0013] Furthermore, in S5, the high-yield strain screened out was fermented in a 5L fermenter simultaneously with the starting strain, with an inoculation amount of 5-15%. After fermentation at 30°C for 16-24h, the fermentation was terminated and the nisin content in the fermentation broth was detected.

[0014] Beneficial effects of the present invention: The method of the present invention combines classical mutagenesis, monoclonal high-throughput bacterial selection, high-throughput well plate screening, and ultra-high performance liquid phase detection, and provides a method for high-throughput screening of nisin high-yield strains. Compared with traditional breeding methods, the combination of different mutagenesis breeding technologies can establish a large library of selectable Lactococcus lactis mutant strains; monoclonal high-throughput bacterial selection can quickly, stably, and efficiently collect monoclonal strains to be screened, and use an automated operating system to perform multi-well plate high-throughput fermentation; the high-throughput screening model based on ultra-high performance liquid phase detection can collect and accurately analyze fermentation product data on a large scale through highly sensitive, high-speed, and high-precision detection instruments.

[0015] The whole set of methods supports the selection, detection and analysis of large-scale samples at the same time, and establishes corresponding databases to support the optimization and improvement of the screening system and feedback loop. By establishing standardized and automated operating procedures, the selection speed and accuracy of nisin high-yield strains are greatly improved, and the strain biosynthesis efficiency is improved. Compared with traditional selection methods, high-throughput screening has the characteristics of fast, accurate and efficient, improves screening efficiency and shortens the selection cycle. The whole cycle of the present invention lasts for two weeks to complete the screening of 7680 mutants, which is equivalent to the workload of 10 months of traditional screening methods. The nisin yield of the high-yield strains screened out by high-throughput screening is compared with the starting strain on a 5L fermentation tank. On average, it is increased by more than 40%, which significantly improves the screening efficiency and accuracy of industrial nisin high-yield strains, greatly shortens the selection cycle, and has been applied to industrial production, providing an effective method for high-throughput screening of industrial nisin high-yield strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention is a flowchart of a method for high-throughput screening of nisin high-producing strains in an embodiment of the present application.

[0017] Figure 2 This is the standard curve for ultra-high performance liquid phase detection of nisin in the implementation mode of this application.

[0018] Figure 3 This is the result of the 96-well plate high-throughput initial screening in the implementation mode of this application.

[0019] Figure 4 This is the test tube rescreening result in the implementation mode of this application.

[0020] Figure 5 This is the tank verification result in the implementation method of this application. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the specific implementation modes of the present invention will be clearly and completely described below.

[0022] The present invention discloses a method for high-throughput screening of nisin high-yield strains, comprising: S1: Perform different mutagenesis breeding treatments on the starting strain to obtain a mutant strain library on the plate; S2: Use a high-throughput clone selection system to select single colonies growing on the plate, inoculate them into a multi-well culture plate containing a seed medium for culture, and then transfer them to a multi-well culture plate containing a fermentation medium for culture. After the culture is completed, acidify and centrifuge to collect the filtrate; S3: The nisin content in the filtrate was determined by a high-throughput screening model based on ultra-high performance liquid chromatography detection, and a mutant strain of Lactococcus lactis with high nisin production was initially screened out; S4: inoculating the strain obtained from the preliminary screening into a test tube containing a seed culture medium for culture, and then transferring the strain to a test tube containing a fermentation culture medium for fermentation culture. After the fermentation is completed, the nisin content in the fermentation culture liquid is detected, and the nisin high-yielding strain is re-screened; S5: The above-screened nisin high-yield strains were subjected to 5L tank verification to confirm the fermentation capacity of the re-screened strains.

[0023] As an embodiment, in S1, the starting strain is Lactococcus lactis; The mutagenesis method is one of ultraviolet mutagenesis, chemical mutagenesis, microwave mutagenesis, resistance mutagenesis, plasma mutagenesis at normal temperature and pressure, radiation mutagenesis or magnetic field mutagenesis; Chemical mutagenesis includes ethyl methanesulfonate mutagenesis, 5-bromouracil mutagenesis, and diethyl sulfate mutagenesis; The limiting factors for resistance mutagenesis screening are sugar tolerance, pH tolerance, sodium lactate tolerance, and nisin and nisin derivative tolerance; As an embodiment, the multi-well plate culture plate in S2 is selected from: 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.

[0024] As an embodiment, the components of the seed culture medium in S2 include: 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, MgSO4 . 7H2O·0.15-0.25g / L, β-glycerophosphate disodium 17-21g / L and 0.3-0.7% (W / V) glucose. The fermentation medium includes: sucrose 10-15g / L, soy protein 5-10g / L, yeast extract 10-20g / L, peptone 10-20g / L, KH2PO4·10-20g / L, NaCl·2-5g / L, MgSO4 . 7H2O·0.2-1g / L.

[0025] As an embodiment, the multi-well plate culture containing the seed culture medium in S2 is cultured at 30° C. for 8-16 hours; The fermentation culture is to transfer the seed liquid to a multi-well plate at an inoculation rate of 5-15% (v / v), and ferment and culture at 30°C for 16-24h; The cradle centrifuge centrifugal filtration uses a multi-well plate filter plate corresponding to the multi-well plate culture plate, with a filter membrane of 0.2-0.45 μm. The conditions for centrifugation to collect the fermentation liquid into the multi-well plate injection plate should be a temperature of 0-10°C, a rotation speed of 2000-4000 r / min, and a centrifugation time of 5-15 min.

[0026] As an embodiment, in S3, a chromatographic column of an ultra-high performance liquid chromatography system C18 is used, with acetonitrile-0.1% trifluoroacetic acid water 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 gradient elution time of 2-5 min, and a diode array detector as the detector. The chromatographic columns of the ultra-high performance liquid chromatography system C18 include: ACQUITY Premier HSS T3 1.8µm 2.1*100mm, ACQUITY UPLCBEH C18 2.1*100mm 1.7µm, InfinityLab Poroshell HPH-C18 2.1*100mm 1.9µm, and Shim-pack Scepter C18 2.1*100mm 1.9µm.

[0027] As an implementation method, the method for establishing a high-throughput screening model for ultra-high performance liquid chromatography detection of nisin in S3 is: take a nisin standard solution, dilute it step by step to obtain a standard solution with a gradient concentration, perform ultra-high performance liquid chromatography analysis, and the injection volume is 1 μL. The concentration of nisin is used as the horizontal axis and the peak area is used as the vertical axis to make a standard curve to obtain a linear equation. Figure 2 As shown, the linear equation Y=0.104X+16.241 (R2=0.9997) was obtained, wherein X is the nisin concentration and Y is the peak area.

[0028] As an embodiment, in S3, the fermentation broth is processed and centrifuged and then collected on a multi-well plate sample plate, which is then transferred to an ultra-high performance liquid chromatography system for sample analysis and detection. The obtained peak area of ​​nisin is substituted into a linear equation to calculate the content of nisin in the fermentation broth sample.

[0029] As an implementation mode, in S4, when the high-yield strains initially screened are rescreened in test tubes, the seed culture liquid volume is 5-10 mL and cultured at 30°C for 8-16 h; the fermentation culture is carried out in test tubes with a liquid volume of 5-10 mL and an inoculation amount of 5-15%. After culture at 30°C for 16-24 h, the fermentation liquid is centrifuged and the nisin content in the fermentation liquid is detected.

[0030] As an implementation mode, in S5, the high-yield strain screened out is fermented in a 5L fermenter simultaneously with the starting strain, with an inoculation amount of 5-15%, and after fermentation at 30°C for 16-24 hours, the fermentation is terminated and the nisin content in the fermentation broth is detected. Example

[0031] The starting strain of Lactococcus lactis of the present invention is hereinafter referred to as LL00. The experimental materials used in the following examples are all commercially available, and the experimental methods not specifically described are conventional experimental methods in the art.

[0032] Culture medium: Seed culture medium (GM17 medium (g / L)): soy peptone 5, beef extract 5, tryptone 5, yeast extract 2.5, ascorbic acid 0.5, MgSO4 . 7H2O·0.25, β-glycerophosphate disodium 19, 0.5% (W / V) glucose.

[0033] Fermentation medium (g / L): sucrose 10, soy protein 5, yeast extract 10, peptone 10, KH2PO4·10, NaCl·2, MgSO4 . 7H2O·0.2.

[0034] Example 1: LL00 strain culture The initial strain of Lactococcus lactis LL00 was streaked on a plate and cultured at 30°C for 12 hours. The activated single colony was picked and inoculated into 10 mL GM17 medium and cultured at 30°C for 12 hours. Then, it was transferred to 50 mL GM17 medium at a 2% inoculation amount and cultured for 6 hours to obtain the initial bacterial solution.

[0035] Example 2: Construction of mutant strain library using different mutagenesis breeding treatments UV mutagenesis: The initial bacterial culture in the logarithmic growth phase was centrifuged at 8000r / min for 3min, the supernatant was removed, resuspended in sterile buffer and diluted to a concentration of 10 4 / mL. Before irradiation, take 1mL of bacterial solution and dilute it 10 times, then take 100μL of the diluted solution and spread it on the GM17 plate as a control. Take 15mL of the diluted bacterial solution in a sterile glass plate, put it into a sterilized rotor, place the plate on a magnetic stirrer, and adjust the speed. The entire device is placed flat under a 15W ultraviolet lamp. Adjust the irradiation distance to 26cm and the irradiation time to 40s. Take 100ul and spread it on the GM17 plate and wrap it with tin foil. The entire operation process is carried out under red light. Incubate the plate at 30℃ in the dark for 2 days, count the number of colonies on the platform, and calculate the lethality to be ~90%.

[0036] Chemical mutagenesis: The initial bacterial culture in the logarithmic growth phase was centrifuged at 8000r / min for 3min, the supernatant was removed, resuspended in sterile buffer and diluted to a concentration of 10 4 / mL. Before mutagenesis, take 1mL of bacterial solution and dilute it 10 times, then take 100μL of the diluted bacterial solution and spread it on the GM17 plate as a control. Add diethyl sulfate solution to the bacterial suspension to make the final concentration reach 1.2%, place it on a shaker, 30℃, and treat it for 30min. After the reaction is completed, add 25% sodium thiosulfate solution to terminate the reaction. Centrifuge immediately after the end, discard the supernatant, and wash the bacteria 3 times with buffer. The treated bacterial solution is diluted to different gradients and spread on the plate, cultured at 30℃ in the dark for 2 days, count the number of colonies on the platform, and calculate the lethality to be ~85%.

[0037] Resistance to mutagenesis: The initial bacterial solution cultured to the logarithmic growth phase was centrifuged at 4000r / min for 10min, the supernatant was removed, resuspended in sterile buffer and diluted to a concentration of 10 4 Before treatment, take 1mL of bacterial solution and dilute it 10 times, then take 100μL of the bacterial solution and spread it on the GM17 plate as a control. Spread the prepared bacterial suspension on the resistance medium containing a final nisin concentration of 20000IU, culture at 30℃ in the dark for 2 days, observe the bacterial growth, and pick the colonies on the platform.

[0038] Microwave mutagenesis: The initial bacterial solution cultured to the logarithmic growth phase was centrifuged at 4000r / min for 10min, the supernatant was removed, resuspended in sterile buffer and diluted to a concentration of 10 4 / mL. Before microwave, take 1mL of bacterial solution and dilute it 10 times, then take 100μL of bacterial solution and spread it on GM17 plate as a control. Put the prepared bacterial suspension directly into the microwave oven and treat it with high-intensity microwave (850W) for 30s. Take it out and cool it in ice water every 10s to eliminate the thermal effect. Dilute the bacterial solution after microwave and spread it on the plate with different gradients, culture it at 30℃ in the dark for 2 days, count the number of colonies on the platform, and calculate the lethality rate to be ~80%.

[0039] Plasma mutagenesis at room temperature and pressure: The initial bacterial solution cultured to the logarithmic growth phase was centrifuged at 4000r / min for 10min, the supernatant was removed, resuspended in sterile buffer and diluted to a concentration of 10 4 / mL. Before plasma mutagenesis, take 1mL of bacterial solution and dilute it 10 times, then take 100μL of bacterial solution and spread it on the GM17 plate as a control. Take 10uL of bacterial solution and spread it evenly on the sterilized and cooled slide, then place it in a plasma mutagenizer at room temperature and pressure, with high-purity He as the working gas, RF power of 100W, gas flow of 10SLM, the distance between the emission source and the slide is 3mm, and the treatment time is 40s. Immediately after the end, take out the slide and put it into an EP tube filled with sterile buffer, use a vortex shaker to shake for 1min, so that the bacteria on the slide are eluted to obtain mutant bacterial solution, and the bacterial solution after plasma mutagenesis is diluted with different gradients to spread on the plate, and cultured at 30℃ in the dark for 2 days. The number of colonies on the platform is counted, and the lethality is calculated to be ~80%.

[0040] Example 3: Fully automated high-throughput monoclonal bacterial picking The QpixXT fully automatic high-throughput clone selection system was used to select single colonies of mutant strains grown on the plates after the above-mentioned different mutagenesis breeding treatments. The GM17 culture medium was dispensed into 96-well plates at 1 mL per well using a continuous dispenser. Since there is a certain positive correlation between the yield of nisin and the biomass of Lactococcus lactis, based on the principle that the colony diameter is greater than 0.5 mm, about 8,000 colonies were selected from the above-mentioned mutant plates using QpixXT for initial screening, and single colonies were inoculated into the corresponding 96-well plate wells. Each 96-well plate is provided with an initial strain control and a blank control, marked as plate An (n=1…80). Using the fully automatic high-throughput clone selection system, the selection of 80 plates with a total of about 80x96=7,680 mutant strains can be completed within 4 days, marked as plates A1-A80 respectively.

[0041] Example 4: Multiwell plate high-throughput fermentation and bacterial liquid collection and processing After the An plate was cultured in a 30°C incubator for 11 hours, it was transferred to a 96-well culture plate containing 1 mL of fermentation medium at a 20% inoculation volume and cultured, and labeled as plate Bn (n=1…80). Plate B was cultured in a 30°C incubator for 20 hours.

[0042] Use a continuous dispenser to dispense 0.02M HCl into a new 96-well plate, labeled Cn (n=1…8). Draw a certain volume of bacterial solution from plate B and inject it into plate C (the mass ratio of fermented bacterial solution to 0.02M HCl is 1:24). Use a pipette to blow and mix the diluted bacterial solution, adjust the pH to 2, and heat at 95℃ for 30 minutes. Then filter and centrifuge plate C at 3000r / min for 5 minutes to collect the filtrate into a 96-well sample plate.

[0043] Example 5: Construction of a high-throughput screening model based on ultra-high performance liquid chromatography detection The present invention uses an Agilent ultra-high performance liquid chromatograph 1290, the chromatographic column used is InfinityLabPoroshell HPH-C18 2.1*100mm 1.9µm, and a PDA diode array detector is used as a detector. The mobile phase is acetonitrile-0.1% trifluoroacetic acid water in a volume ratio of 10-90%, gradient elution, a flow rate of 0.4mL / min, a detection wavelength of 220nm, a column temperature of 30°C, an injection volume of 1μL, and a single sample elution time of 3min.

[0044] The method for establishing a high-throughput screening model for nisin ultra-high performance liquid chromatography detection is as follows: take a nisin standard solution, dilute it step by step to obtain 5 standard solutions with different concentration gradients; analyze according to the detection method of the present invention, the injection volume is 1 μL, and a standard curve is prepared with the concentration of nisin as the horizontal axis and the peak area as the vertical axis, and the following is obtained: Figure 2 The linear equation shown is Y = 0.104X + 16.241 (R2 = 0.9997), where X is the nisin concentration, Y is the peak area, and the correlation coefficient is 0.9997, indicating a good linear relationship.

[0045] The 96-well sample plate was transferred to the ultra-high performance liquid chromatography detection system and 1-20 μL was injected for analysis and detection. The peak area of ​​nisin obtained by ultra-high performance liquid chromatography detection of the sample was substituted into the above linear equation to calculate the content of nisin in the sample to be tested. A total of 7680 mutant strain library nisin fermentation product data were collected, and the mutant strains of Lactococcus lactis with high nisin production were analyzed and preliminarily screened. The preliminary screening results are as follows Figure 3 As shown. The strains obtained from the initial screening were sorted by titer, and about 80 strains in the top 1% of the titer were selected for rescreening. The remaining bacterial liquid in the corresponding wells of plate B to be rescreened was added with 50% glycerol for seed preservation. The entire initial screening and testing process took two weeks, and a total of 7,680 mutant strains were screened, and 80 high-yield mutant strains were initially screened for rescreening. The workload is equivalent to 10 months of workload for traditional screening methods.

[0046] Example 6: Test tube rescreening The 80 high-yield nisin strains to be rescreened were streaked on GM17 plates and cultured at 30°C for 24 hours. Single colonies were picked and inoculated into test tubes containing 10 mL of seed culture medium. Each strain had 3 parallel samples, and an initial strain control and a blank control were set up respectively. After culturing at 30°C for 12 hours, they were transferred to test tubes containing 10 mL of fermentation medium at a rate of 5-10%. After culturing at 30°C for 20 hours, the fermentation broth was collected, acidified, centrifuged and filtered to detect the nisin content, and the high-yield strains were rescreened. The rescreening results are as follows: Figure 4 The strains obtained by rescreening were sorted by titer, and about 8 strains in the top 10% of the titer were selected and labeled LL01-08 for canning verification.

[0047] Example 7: Tank loading verification The 8 strains of high-yield nisin to be tested in the tank were inoculated into a 500mL shake flask containing 350mL seed culture medium, cultured at 150r / min and 30℃ for 20h, and then transferred to a 5L fermenter with a liquid volume of 3.5L and an inoculation volume of 10%. Liquid alkali was used to control the pH at 6.5-7, and the feed was started after culturing at 30℃ for 5h to control the residual sugar concentration at 0.5-1g / L. After fermentation continued for 18h, sampling was started to determine the titer, and sampling was performed every 2h until the titer stopped rising, and the fermentation was terminated. The fermentation time was usually 22h. The experimental results are as follows: Figure 5 As shown, the results showed that the nisin production of LL01-08 was significantly improved compared with the starting strain LL00, among which LL03, LL04 and LL06 were increased by 41.1%, 44.5% and 46.6% respectively compared with the starting strain LL00, the highest titer reached 19856 IU / mL, and the average nisin fermentation unit was increased by more than 40%, indicating that the present invention provides an effective method for high-throughput screening of nisin high-yield strains.

[0048] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for high-throughput screening of nisin high-producing strains, characterized in that: include S1: Perform different mutagenesis breeding treatments on the starting strain to obtain a mutant strain library on the plate; S2: Use a high-throughput clone selection system to select single colonies growing on the plate, inoculate them into a multi-well culture plate containing a seed medium for culture, and then transfer them to a multi-well culture plate containing a fermentation medium for culture. After the culture is completed, acidify and centrifuge to collect the filtrate; S3: The nisin content in the filtrate was determined by a high-throughput screening model based on ultra-high performance liquid chromatography detection, and a mutant strain of Lactococcus lactis with high nisin production was initially screened out; S4: inoculating the strain obtained from the preliminary screening into a test tube containing a seed culture medium for culture, and then transferring the strain to a test tube containing a fermentation culture medium for fermentation culture. After the fermentation is completed, the nisin content in the fermentation culture liquid is detected, and the nisin high-yielding strain is re-screened; S5: The above-mentioned re-screened nisin high-yield strains are put into tanks for verification to confirm the fermentation capacity of the re-screened strains.

2. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: In S1, the starting strain is Lactococcus lactis; The mutagenesis method is one of ultraviolet mutagenesis, chemical mutagenesis, microwave mutagenesis, resistance mutagenesis, plasma mutagenesis at normal temperature and pressure, radiation mutagenesis or magnetic field mutagenesis; Chemical mutagenesis includes ethyl methanesulfonate mutagenesis, 5-bromouracil mutagenesis, and diethyl sulfate mutagenesis; The limiting factors for resistance mutagenesis screening are sugar tolerance, pH tolerance, sodium lactate tolerance, nisin and nisin derivative tolerance.

3. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: The multi-well plate culture plate in S2 is selected from: 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.

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

5. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: The multiwell plate culture containing seed medium in S2 was cultured at 30°C for 8-16 h; The fermentation culture is to transfer the seed liquid to a multi-well plate at an inoculation rate of 5-15% (v / v), and ferment and culture at 30°C for 16-24h; The cradle centrifuge centrifugal filtration uses a multi-well plate filter plate corresponding to the multi-well plate culture plate, with a filter membrane of 0.2-0.45 μm. The conditions for centrifugation to collect the fermentation liquid into the multi-well plate injection plate should be a temperature of 0-10°C, a rotation speed of 2000-4000 r / min, and a centrifugation time of 5-15 min.

6. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: In S3, a C18 chromatographic column of an ultra-high performance liquid chromatography system was used, with acetonitrile-0.1% trifluoroacetic acid water as the mobile phase, a flow rate of 0.1-0.4 mL / min, a detection wavelength of 210-400 nm, and a column temperature of 10-40°C; the injection volume was 1-20 μL, the gradient elution time for a single sample was 2-5 min, and a diode array detector was used as the detector.

7. A method for high-throughput screening of nisin high-producing strains according to claim 6, characterized in that: The method for establishing a high-throughput screening model for ultra-high performance liquid chromatography detection of nisin in S3 is as follows: take the nisin standard solution, dilute it step by step to obtain a standard solution with a gradient concentration, and perform ultra-high performance liquid chromatography analysis with an injection volume of 1 μL. Use the concentration of nisin as the horizontal axis and the peak area as the vertical axis to draw a standard curve and obtain the linear equation.

8. A method for high-throughput screening of nisin high-producing strains according to claim 7, characterized in that: In S3, the fermentation broth is processed and centrifuged and then collected on a multi-well plate sample plate, which is then transferred to an ultra-high performance liquid chromatography system for sample analysis and detection. The obtained peak area of ​​nisin is substituted into a linear equation to calculate the content of nisin in the fermentation broth sample.

9. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: In S4, when the high-yield strains initially screened were rescreened in test tubes, the seed culture was filled with 5-10 mL of liquid and cultured at 30°C for 8-16 h. The fermentation culture was carried out in test tubes with a liquid volume of 5-10 mL and an inoculation size of 5-15%. After culture at 30°C for 16-24 h, the fermentation liquid was centrifuged and the nisin content was detected.

10. A method for high-throughput screening of nisin high-producing strains according to claim 1, characterized in that: In S5, the high-yield strain screened out was fermented in a 5L fermenter simultaneously with the starting strain, with an inoculation amount of 5-15%. After fermentation at 30°C for 16-24 hours, the fermentation was terminated and the nisin content in the fermentation broth was detected.

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