Mutant strain with high yield of nisin and application of mutant strain

Abstract

The invention belongs to the technical field of microorganism mutation breeding, and particularly relates to a nisin high-yield mutant strain with enhanced production performance. The mutant strain is specifically lactococcus lactis 2686, and the preservation number of the mutant strain is CCTCC (China Center for Type Culture Collection) NO: M 2023219. The strain has relatively high osmotic pressure tolerance, can still normally grow and produce nisin when the osmotic pressure is higher than 0.66 osM / kg H2O, meanwhile, the strain has excellent nisin production capacity, the titer reaches 9834 IU / mL when the strain is fermented in a shake flask for 21 hours, and the titer reaches 21000 IU / mL when the strain is fermented in a fermentation tank for 22 hours. The method is high in yield, good in stability, low in production cost and suitable for further industrial application.

Description

Technical field:

[0001] The invention belongs to the technical field of microbial mutation breeding, and in particular relates to a nisin high-yield mutant strain with enhanced production performance. Background technology:

[0002] Nisin is a natural bioactive antimicrobial peptide that has a strong inhibitory effect on many Gram-positive bacteria, including food spoilage bacteria and pathogenic bacteria, and is therefore used as a food preservative. Nisin is a natural active antibacterial peptide produced by lactic acid bacteria and belongs to the lantibiotic antibiotic. Mature nisin is composed of 34 amino acids and has a strong inhibitory effect on most Gram-positive bacteria and their spores. It also has a killing effect on Gram-negative bacteria when acting together with EDTA and other agents. It is the only bacteriocin approved for food preservation and is widely used in the preservation of dairy products, fermented beverages, meat and meat products.

[0003] At present, nisin is mainly produced by microbial fermentation. However, the strains isolated from nature generally have poor fermentation performance, low production yield and poor stability, which leads to high industrial production costs, which restricts the further application and development of nisin to a certain extent. Therefore, in order to obtain superior strains suitable for large-scale production, with good fermentation performance and stable high yield, we need to further optimize or transform the target strains obtained by screening. Under the current economic and technical conditions, the commonly used microbial breeding methods still mainly include: traditional mutagenesis breeding, genome rearrangement breeding and genetic engineering breeding.

[0004] Mutation breeding is the most widely studied breeding method. It is a method of artificially treating microbial cells with some mutagens or mutagenic factors to accelerate the occurrence of mutations, and combining them with certain specific screening methods to obtain mutant individuals with target traits.

[0005] Mutation breeding can increase the yield of target products, improve product quality, and simplify production processes. It is simple, rapid, and effective, and is currently the main way to obtain high-yield strains. Composite mutagenesis refers to the use of two or more mutagens to act on microorganisms successively or simultaneously, or the repeated use of the same mutagen on microorganisms. It is a commonly used method to improve the production performance of microorganisms. It is generally believed that composite mutagenesis has a synergistic effect. If mutagens are used in a reasonable combination, composite mutagenesis is better than single mutagenesis.

[0006] The present application will adopt traditional mutagenesis methods to carry out mutation breeding of nisin producing bacteria, continue to repeatedly mutagenesis treatment on the obtained high-yield strains, and screen out nisin high-yield strains with improved fermentation levels. Summary of the invention:

[0007] The technical problem to be solved by the present invention is to select and breed a high-yield nisin strain by adopting a series of traditional mutagenesis methods, so as to improve the production level.

[0008] One of the technical solutions provided by the present invention is a strain of Lactococcus lactis selected by mutation, specifically Lactococcus lactis 2686, which was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, Postal Code: 430072) on March 1, 2023, with a deposit number of CCTCCNO: M 2023219.

[0009] The second technical solution provided by the present invention is the application of Lactococcus lactis 2686 described in the first technical solution, especially in the fermentation production of nisin;

[0010] Further, the method for producing nisin by fermentation using Lactococcus lactis 2686 is as follows:

[0011] The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5%-10%, the pH is controlled at 6.3-7.0, the rotation speed is 200-500rpm, the temperature is 28-33°C, and the tank pressure is 0.02-0.06MPa; when the sucrose content drops below 3-7g / L, glucose is added, and the residual sugar concentration is controlled at 0-1.0g / L; 4-8 hours after the start of fermentation, the mixed nitrogen source is added at a rate of 0.2%-0.5% of the fermentation liquid volume per hour, and the fermentation is continued until the nisin titer no longer increases significantly;

[0012] The formula of the fermentation medium is (w / v): sucrose 1%-2.0%, yeast extract powder 0.2%-0.6%, soy protein isolate 0.1%-0.5%, defatted soy powder 0.15%-0.55%, corn steep liquor 1%-3%, disodium hydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.1%-0.5%, and the rest is water.

[0013] The mixed nitrogen source comprises (w / v): 0.5%-1.5% corn steep liquor, 0.2%-0.6% yeast extract powder, and the rest is water.

[0014] Beneficial effects:

[0015] The present invention selects a nisin production strain with superior performance, namely Lactococcus lactis 2686, through ultraviolet-light resurrection and ultraviolet-nitrous acid composite mutagenesis. The strain has high osmotic pressure tolerance, can still grow normally and produce nisin when the osmotic pressure is higher than 0.66osM / kg H2O, and has excellent nisin production capacity. After 21h of shake flask fermentation, the titer reaches 9834IU / mL, and after 22h of fermentation in a fermenter, the titer reaches 21000IU / mL. The strain has high yield, good stability, low production cost, and is suitable for further industrial application. Specific implementation method:

[0016] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0017] The composition of some culture media involved in the present invention and embodiments is as follows:

[0018] S1 plate medium includes the following components in mass volume ratio: sucrose 2%, NaCl 0.75%, tryptone 0.8%, yeast powder 0.5%, MgSO4﹒7H2O 0.04%, Na2HPO4﹒12H2O 0.4%, NaH2PO4﹒2H2O 0.2%, agar 1.8%, and the rest is water;

[0019] The SM17 medium includes the following components in a mass volume ratio: polypeptone 0.75%, soy peptone 0.75%, yeast powder 0.3%, beef extract 0.75%, sucrose 2%, vitamin C 0.1%, MgSO4.7H2O 0.04%, sodium β-glycerophosphate 1.5%, and the rest is water.

[0020] The present invention is further explained below by means of specific examples.

[0021] Example 1 First stage mutagenesis screening (UV-light resurrection mutagenesis)

[0022] (1) Mutagenesis

[0023] The nisin-producing strain Lactococcus lactis 1267# stored in the laboratory was used as the starting strain, and S1 plates were coated with 0.75% NaCl solution to make 10 8The bacterial suspension of the order of magnitude is used for subsequent mutagenesis. The specific mutagenesis methods include UV mutagenesis and photoreactivation. The specific time is: UV 30s-photoreactivation 5min-UV 50s-photoreactivation 5min-UV 80s, and the bacterial suspension after UV-photoreactivation mutagenesis is obtained. The mutagenic bacterial suspension is appropriately diluted and applied to the S1 plate, cultured at 32℃ for 60h, and screened after obvious single colonies grow.

[0024] (2) Initial screening

[0025] That is, the agar diffusion method is used for initial screening; the specific agar diffusion method includes the following steps: 1) single bacteria expansion culture, 2) transfer of the expanded single bacteria to the primary SM17 test tube culture medium, 3) transfer of the primary SM17 culture medium to the secondary SM17 culture medium, 4) fermentation in the secondary SM17 culture medium, acidification (adjusting the secondary SM17 test tube culture medium to pH 2.0 with an appropriate amount of 37% hydrochloric acid), inactivation (heating in a water bath to above 85°C for 10 minutes), 5) appropriately diluting the acidified and inactivated secondary SM17 test tube culture medium, and dripping Add to the bioassay plate, 80 μL per well (drop plate: 29 samples per plate, 3 wells for each sample, add standard solution (i.e., use an appropriate amount of nisin standard dissolved in acid water at pH 2.0 to prepare a solution with an efficacy of 2000 IU / mL) drop to 4 wells, 80 μL per well, after the test solution is dried, transfer to a 32°C incubator and culture overnight), 6) Measure the plate, observe the size of the inhibition zone, select the one with the larger inhibition zone to complete the initial screening (use a vernier caliper to measure the size of the inhibition zone, select the one with the larger inhibition zone for re-screening).

[0026] Single bacteria expansion culture medium is S1 plate medium;

[0027] The preparation of the bioassay plate comprises the following steps:

[0028] a. Preparation of plate culture medium, including the following components in mass volume ratio: tryptone 1.0%, yeast powder 0.5%, glucose 0.8%, NaCl 0.5%, Na2HPO4 0.3%, agar 1.5%, divided into 200mL / bottle, sterilized;

[0029] b. Cool to 50°C, add 500 μL of sensitive bacteria suspension (Micrococcus luteus), and shake well;

[0030] c. When laying the board, pay attention to controlling the bubbles and make the board flat and free of bubbles;

[0031] d. Let it cool at low temperature for 2.5 hours. After punching, store it in a refrigerator at 4-8℃.

[0032] (3) Rescreening (fermentation broth corresponding to samples with large inhibition zones)

[0033] The rescreening, i.e., rescreening and determining the potency of nisin by high performance liquid chromatography, specifically comprises the following steps: 1) preparing a nisin mobile phase, 2) preparing a nisin standard solution, 3) detecting the peak area of ​​the nisin standard product and calculating the potency per unit area, and 4) detecting the peak area of ​​the primary screening sample and calculating the potency.

[0034] The high titer strains obtained by HPLC rescreening were repeated through the above steps, from mutagenesis to screening, until strain 2443# with stable genetic traits and a test tube fermentation titer increased by more than 28% was screened. High-yield strain 2443# was frozen and stored in glycerol tubes.

[0035] Example 2 Second stage mutagenesis screening (ultraviolet-nitrous acid combined mutagenesis)

[0036] (1) Mutagenesis

[0037] The high-yield strain 2443# obtained by mutation was used as the starting strain, and S1 plates were coated with 0.75% NaCl solution to make 10 8 Order of magnitude bacterial suspension. Used for subsequent composite mutagenesis. Specific mutagenesis methods include UV mutagenesis and nitrite mutagenesis, with specific concentrations and times of UV 30s-photoreactivation 5min-UV 50s-photoreactivation 5min-0.3M nitrite action 6min. Obtain the bacterial suspension after UV-photoreactivation-nitrite mutagenesis. Appropriately dilute the mutagenic bacterial suspension and apply it to the S1 plate, culture at 32℃ for 60h, and screen after obvious single colonies grow.

[0038] (2) Primary screening - secondary screening

[0039] Repeat the initial screening and rescreening steps of Example 1 to obtain those with high titers.

[0040] Repeat the above steps, from mutagenesis to screening, until the high-yield strain 2686# with stable genetic traits is screened, and the high-yield strain is frozen and stored in glycerol tubes. The specific screening results are as follows:

[0041] Table 1 Comparison of liquid phase titer between mutagenic bacteria and starting strains

[0042]

[0043]

[0044] As shown in Table 1, after six rounds of recursive mutagenesis, the high-yield strain 2686# obtained had a titer increased by more than 36% compared with the starting strain 1267#.

[0045] Table 2 The stability of high-yield strain 2686#

[0046] No. HPLC titer IU / mL First Generation 3530 Second Generation 3545 Third Generation 3519 Fourth Generation 3540 Fifth Generation 3528

[0047] As shown in Table 2, the high-yield strain 2686# has good stability in passage, relatively stable genetic traits, and is promptly frozen and stored in glycerol tubes. On March 1, 2023, Lactococcus lactis 2686 (i.e., 2686# obtained by the above screening) was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China, Postal Code: 430072), with the deposit number: CCTCC NO: M 2023219.

[0048] Example 3 Acid resistance comparison

[0049] The growth and metabolic performance of the starting strain 1267# and the mutant strain 2686# were compared and analyzed. During the fermentation process of Lactococcus lactis to produce nisin, the pH in the culture environment gradually decreased. In order to extend the production time of nisin without adjusting the pH, the production strain needs to have a certain acid resistance. The starting strain 1267# and the mutant strain 2686# were inoculated into a test tube culture medium containing SM17, and cultured at 32°C in an incubator for 24 hours, and samples were taken at regular intervals (specifically subject to the following table) to detect indicators such as pH, OD600, and titer. The results are shown in Table 3.

[0050] From the results, we can see that when the pH of the culture medium is lower than 4.5, the OD600 of the mutant strain 2686# is still increasing, indicating that the strain still has the ability to grow under conditions where the pH is lower than 4.5, while the OD600 of the control starting strain 1267# basically stops increasing when the pH reaches about 4.5, indicating that the starting strain cannot grow normally under conditions where the pH is lower than 4.5.

[0051] It can be seen that the mutant strain 2686# is more tolerant to low pH environment than the starting strain 1267#, and the growth and metabolic performance of the thalline is significantly enhanced. In synchronization with the growth situation, the process of nisin production by the mutant strain is also extended, and the final nisin output is also significantly improved. It can be seen that the acid resistance of the mutant strain 2686# obtained by the present invention is significantly enhanced, and the corresponding nisin production performance is also enhanced, which is more suitable for industrial production process.

[0052] Table 3 Comparison of growth and metabolic performance between mutant strain 2686# and starting strain 1267#

[0053]

[0054] Example 4 Osmotic pressure resistance comparison

[0055] The growth and metabolic performance of the starting strain 1267# and the mutant strain 2686# were further compared and analyzed. During the production of nisin by lactic acid streptococci, lactic acid is continuously produced in the fermentation broth, and the pH value of the fermentation system is continuously reduced, which inhibits the growth and metabolic performance of the bacteria to a certain extent. Therefore, when the pH of the culture broth is lower than 6.0, liquid alkali needs to be added in time to reach a pH of about 6.5. However, while adjusting the pH, the osmotic pressure of the culture broth gradually increases. In order to extend the production time of nisin, the production strain needs to have a certain resistance to osmotic pressure.

[0056] The starting strain 1267# and the mutant strain 2686# were inoculated into the shake flask fermentation medium, and cultured at 32°C in the incubator for 24 hours. The pH was adjusted every 2 hours, that is, when the pH dropped below 6.0, liquid alkali was added to pH 6.5 in time. Samples were taken at regular intervals (specific details are subject to the table below) to test the osmotic pressure, OD600, titer and other indicators.

[0057] The formula of shake flask fermentation medium is as follows: sucrose 1.5%, yeast extract powder 0.6%, soy protein isolate 0.3%, defatted soy flour 0.15%, corn steep liquor 1.5%, disodium hydrogen phosphate 0.3%, magnesium sulfate 0.3%, and the rest is water.

[0058] As shown in the results (Table 4), when the osmotic pressure of the culture solution is higher than 0.66osM / kg H2O, the mutant strain 2686# can still grow, while the control starting strain 1267# can not grow again when the osmotic pressure reaches 0.66osM / kg H2O. It is shown that the mutant strain 2686# can tolerate a higher osmotic pressure environment compared with the starting strain 1267#, and the growth and metabolism performance of the thalline is significantly enhanced. Synchronous with the growth situation, the process of the mutant strain producing nisin is also extended, and the output of final nisin is also significantly improved. It can be seen from this that the osmotic pressure tolerance of the mutant strain 2686# obtained by the present invention is significantly enhanced, and the corresponding nisin production performance is also enhanced, which is more suitable for industrial production process.

[0059] Table 4 Further comparison of growth and metabolic performance of mutant strain 2686# and starting strain 1267#

[0060]

[0061] Example 5 Comparison of fermentation performance of 10L fermenter

[0062] The starting strain 1267# and the mutant strain 2686# were used as experimental strains, and the nisin production performance was compared under the fermentation conditions of a 10L fermenter, as follows:

[0063] The secondary seed shake flask culture mode was adopted, and the culture medium formula was the same: sucrose 1.5%, yeast powder 0.5%, Na2HPO4 0.3%, MgSO4 0.15%, soy peptone 0.2%, and the rest was water. The temperature was 32 degrees, the speed was 150rpm, and when the pH of the primary seed dropped to 5.5-6.0, the secondary seed was transferred at a ratio of 3%, and the culture method was the same as the first level. When the OD of the secondary seed reached 3.5 or above, the fermentation medium was transferred.

[0064] Fermentation tank culture: inoculation amount 5%, pH controlled at 6.8, rotation speed 300 rpm, temperature 32°C, tank pressure 0.04 MPa; fermentation until nisin titer no longer increases significantly.

[0065] The fermentation medium formula of a 10L fermenter is as follows: sucrose 1%, yeast extract powder 0.3%, soy protein isolate 0.3%, defatted soy flour 0.15%, corn steep liquor 1%, disodium hydrogen phosphate 0.3%, magnesium sulfate 0.3%, and the rest is water.

[0066] The following table is a comparative analysis of the fermentation results of the starting strain 1267# and the mutant strain 2686# in a 10L tank:

[0067] Table 5 Comparison of 10L tank fermentation results of starting strain 1267# and mutant strain 2686#

[0068]

[0069]

[0070] Example 6 Comparison of sugar supplementation fermentation performance

[0071] The starting strain 1267# and the mutant strain 2686# were used as experimental strains, and the nisin production performance was compared under the fermentation conditions of a 10L fermenter (sugar supplementation), as follows:

[0072] The secondary seed shake flask culture mode was adopted, and the culture medium formula was the same: sucrose 1.5%, yeast powder 0.5%, Na2HPO4 0.3%, MgSO4 0.15%, soy peptone 0.2%, and the rest was water. The temperature was 32 degrees, the speed was 150rpm, and when the pH of the primary seed dropped to 5.5-6.0, the secondary seed was transferred at a ratio of 3%, and the culture method was the same as the first level. When the OD of the secondary seed reached 3.5 or above, the fermentation medium was transferred.

[0073] Fermentation tank culture: inoculation amount 5%, pH controlled at 6.8, rotation speed 300 rpm, temperature 32°C, tank pressure 0.04 MPa; when the sucrose content drops below 5 g / L, start adding 60% glucose solution and control the residual sugar concentration at about 0-0.5 g / L; ferment until the nisin titer no longer increases significantly.

[0074] The formula of the fermentation tank culture medium is: sucrose 1%, yeast extract powder 0.3%, soy protein isolate 0.3%, defatted soy powder 0.15%, corn steep liquor 1%, disodium hydrogen phosphate 0.3%, magnesium sulfate 0.3%, and the rest is water.

[0075] Table 6 Comparison of sugar-supplemented fermentation results between starting strain 1267# and mutant strain 2686#

[0076] No. Sugar supplementation fermentation high performance liquid phase potency IU / mL Fermentation time Starting strain 1267# 12500 22 Mutant strain 2686# 16500 22

[0077] Example 7 Comparison of nitrogen source supplementation fermentation performance

[0078] The starting strain 1267# and the mutant strain 2686# were used as experimental strains, and the nisin production performance was compared under the fermentation conditions of a 10L fermenter (nitrogen source supplementation), as follows:

[0079] The secondary seed shake flask culture mode was adopted, and the culture medium formula was the same: sucrose 1.5%, yeast powder 0.5%, Na2HPO4 0.3%, MgSO4 0.15%, soy peptone 0.2%, and the rest was water. The temperature was 32 degrees, the speed was 150rpm, and when the pH of the primary seed dropped to 5.5-6.0, the secondary seed was transferred at a ratio of 3%, and the culture method was the same as the first level. When the OD of the secondary seed reached 3.5 or above, the fermentation medium was transferred.

[0080] Fermentation tank culture: inoculation amount 5%, pH controlled at 6.8, rotation speed 300 rpm, temperature 32°C, tank pressure 0.04 MPa; when the sucrose content drops below 5 g / L, 60% glucose is added, and the residual sugar concentration is controlled at about 0-0.5 g / L; 4 hours after the start of fermentation, mixed nitrogen source is added at a rate of 0.3% of the fermentation liquid volume per hour; fermentation is continued until the nisin titer no longer increases significantly.

[0081] The formula of the fermentation tank culture medium is: sucrose 1%, yeast extract powder 0.3%, soy protein isolate 0.3%, defatted soy powder 0.15%, corn steep liquor 1%, disodium hydrogen phosphate 0.3%, magnesium sulfate 0.3%, and the rest is water.

[0082] The composition of the mixed nitrogen source is: 1% corn steep liquor, 0.3% yeast extract powder, and the rest is water.

[0083] Table 7 Comparison of nitrogen source fermentation results between starting strain 1267# and mutant strain 2686#

[0084] No. Nitrogen source supplementation fermentation high-performance liquid phase potency IU / mL Fermentation time Starting strain 1267# 15500 22 Mutant strain 2686# 21000 22

[0085] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A strain of Lactococcus lactis, characterized in that Specifically, it is Lactococcus lactis 2686, with a preservation number of CCTCC NO: M 2023219.

2. Use of Lactococcus lactis according to claim 1.

3. The use according to claim 2, characterized in that It is used in the production of nisin.

4. The use according to claim 3, characterized in that The method for producing nisin by fermentation of Lactococcus lactis 2686 is as follows: The inoculation amount is 5%-10%, the pH is controlled at 6.3-7.0, the rotation speed is 200-500rpm, the temperature is 28-33°C, and the tank pressure is 0.02-0.06MPa; when the sucrose content drops below 3-7g / L, glucose is added, and the residual sugar concentration is controlled at 0-1.0g / L; 4-8h after the start of fermentation, the mixed nitrogen source is added at a rate of 0.2%-0.5% of the fermentation liquid volume per hour, and the fermentation is continued until the nisin titer no longer increases significantly.

5. The use according to claim 4, characterized in that The formula of the fermentation medium is: sucrose 1%-2.0%, yeast extract powder 0.2%-0.6%, soy protein isolate 0.1%-0.5%, defatted soy powder 0.15%-0.55%, corn steep liquor 1%-3%, disodium hydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.1%-0.5%, and the rest is water.

6. The use according to claim 4, characterized in that The mixed nitrogen source comprises: 0.5%-1.5% corn steep liquor, 0.2%-0.6% yeast extract powder and the rest water.

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