Production method and application of nisin

Abstract

The invention relates to the technical field of antibacterial peptides, and particularly discloses a production method and application of nisin. The production method of the nisin comprises the following steps: mutagenesis, screening and fermentation, and the specific operation of screening comprises the following steps: carrying out primary screening and secondary screening on a mutagenic bacterium suspension obtained after mutagenesis to obtain a single colony with a large inhibition zone, fermenting the single colony in a weak acid environment, detecting the titer after fermenting for a period of time, and screening out a high-yield strain, a method for detecting the titer is a conductivity method or a spectrophotometric method. According to the production method, high-yield strains can be rapidly screened, the nisin production time is shortened, and the product can be used for preparing a food preservative, has a long-term, efficient and stable antibacterial effect and has a lasting preservation effect on foods such as fruits and raw meat.

Description

Technical Field

[0001] The present application relates to the technical field of antimicrobial peptides, and in particular to a production method and application of nisin. Background Art

[0002] Nisin, also known as Nisin, is a natural biological antimicrobial peptide produced by the fermentation of lactococci naturally present in milk and cheese. It has a broad-spectrum antibacterial effect and can effectively inhibit the growth and reproduction of most Gram-positive bacteria and their spores. It is a world-recognized natural biological food preservative and antibacterial agent that is highly efficient, non-toxic, safe, and has no side effects.

[0003] The ability of lactic acid streptococci to produce Nisin is generally low. To achieve industrial production and application, a series of processes such as mutagenesis, screening and fermentation (including high-yield strain screening) are required to obtain a high-yield and stable strain. At present, the most commonly used method for screening high-yield strains is agar diffusion method, with yellow micrococcus as the detection bacteria, and the diameter of the inhibition zone is used to reflect the titer. The advantage of this method is that the titer of the sample is intuitively represented by the application effect. At the same time, the disadvantages of this detection method are also quite obvious. The detection bacteria need to be cultured for more than 16 hours to observe a clear inhibition zone to obtain the result. In industrial production, it is not possible to quickly feedback so as to adjust the process conditions in time. This time-consuming detection method leads to the inconvenience of screening high-yield strains, which leads to the low production efficiency of nisin, becoming a more thorny problem in nisin breeding. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a production method and application of nisin.

[0005] In the first aspect, the present application provides a method for producing nisin, comprising the following steps: mutagenesis, screening and fermentation, wherein the specific operation of the screening is: subjecting the mutagenic bacterial suspension obtained after mutagenesis to primary screening, rescreening to obtain a single colony with a large inhibition zone, placing it in an environment of pH = 5.5-6.5 for fermentation, detecting the titer 16-18 hours after the start of fermentation, and screening out high-yield strains, wherein the method used for detecting the titer is a conductivity method or a spectrophotometric method.

[0006] Specifically, the specific operation of the mutagenesis is: diluting the starting strain 100-1000 times with physiological saline to obtain a bacterial suspension, and then subjecting the bacterial suspension to any one of ultraviolet mutagenesis / microwave mutagenesis / plasma mutagenesis to obtain a mutagenized bacterial suspension.

[0007] The specific operation of ultraviolet mutagenesis is as follows: the bacterial suspension is subjected to ultraviolet irradiation for 2.5 min at an irradiation distance of 30 cm and an ultraviolet irradiation power of 15 W to obtain a mutagenic bacterial suspension.

[0008] The specific operation of microwave mutagenesis is as follows: the diluted bacterial suspension is placed in a culture medium, cultured at a temperature of 30°C to a logarithmic growth phase, and then microwaved for 30 seconds at a power of 750W and a frequency of 2450MHz to obtain a mutagenic bacterial suspension.

[0009] The specific operation of plasma mutagenesis is as follows: in a helium environment with a gas flow rate of 10 L / min, the mutagenic bacterial suspension is subjected to plasma radio frequency treatment with an action distance of 2 mm and a power of 120 W for a treatment time of 60 s to obtain a mutagenic bacterial suspension.

[0010] The specific operations of primary screening and rescreening are as follows: spread the mutagenic bacterial suspension on the primary screening plate, observe the colony morphology after culturing for 36-48 hours, select a single colony and inoculate it on the rescreening plate, and obtain a single colony with a large inhibition zone after culturing for 36-48 hours.

[0011] The specific operation of the conductivity method is as follows: when the fermentation reaches 16-18 hours, the fermented mutagenic bacteria suspension is diluted to 10-20 IU / mL, and cultured together with the yellow micrococcus suspension on a constant temperature shaker for 4-5 hours, wherein the volume ratio of the diluted fermented mutagenic bacteria suspension to the yellow micrococcus suspension is 50:2, and the conductivity is measured, and the titer of the nisin to be tested in the fermented mutagenic bacteria suspension is obtained by comparison with the conductivity standard curve; The conductivity standard solution in the conductivity standard curve is obtained by the following method: weighing a nisin standard and dissolving it with 0.02 mol / mL HCl, then making up the volume to prepare a nisin standard solution with a titer of 2000 IU / mL, and then diluting the nisin standard solution with 0.02 mol / mL HCl to obtain a nisin standard solution with a gradient titer.

[0012] Preferably, the nisin potency in the nisin standard solution in the conductivity method is set in the range of 10-20 IU / mL.

[0013] In a specific embodiment of the present application, the potency gradient of the nisin standard solution in the conductivity method is set to 10 IU / mL, 12 IU / mL, 14 IU / mL, 16 IU / mL, 18 IU / mL and 20 IU / mL, respectively.

[0014] The sequential setting of the potency gradient of the standard working solution in the specific implementation manner of the present application is only one example, but not the entire design scheme. The technical scheme of making appropriate adjustments to the other intermediate concentration points is also within the scope of the method of the present application.

[0015] The yellow micrococcus suspension was obtained by the following method: the yellow micrococcus was inoculated on an LB plate, and cultured at a constant temperature of 30°C for 48 hours, and then a single colony was selected and inoculated into an LB liquid culture medium, and cultured on a shaking table for 20 hours to obtain a yellow micrococcus concentration of 1×10 8 CFU / mL suspension of Micrococcus aureus was aliquoted and refrigerated for later use; The conductivity standard curve is obtained by the following method: conductivity standard solutions of different titers are added to LB culture medium, the addition amount is 500uL, and then 20uL of yellow micrococcus suspension is added respectively. After shaking culture for 4 hours, the conductivity of 6 conductivity standard solutions of different titers is measured, and the known titer and conductivity are plotted, and linear regression is performed to obtain the conductivity standard curve.

[0016] The specific operation of the spectrophotometric method is as follows: when the fermentation reaches 16-18 hours, the fermented mutagenic bacterial suspension is diluted to 10-20 IU / mL, and cultured together with the yellow micrococcus suspension in a constant temperature shaker for 4-5 hours, wherein the volume ratio of the diluted fermented mutagenic bacterial suspension to the yellow micrococcus suspension is 50:2, and the OD is measured. 600 , compared with the spectrophotometric standard curve, Obtaining the titer of the nisin to be tested in the fermented mutagenic bacterial suspension; The spectrophotometric standard solution in the spectrophotometric standard curve is obtained by the following method: weighing a nisin standard and dissolving it with 0.02 mol / mL HCl, then making up the volume and preparing a nisin standard solution with a potency of 2000 IU / mL, and then diluting the nisin standard solution with 0.02 mol / mL HCl to obtain a nisin standard solution with a potency gradient; Preferably, the nisin potency in the nisin standard solution in the spectrophotometric method is set in the range of 5-20 IU / mL.

[0017] In a specific embodiment of the present application, the potency gradient of the nisin standard solution in the spectrophotometric method is set to 5 IU / mL, 7.5 IU / mL, 10 IU / mL, 12.5 IU / mL, 15 IU / mL, 17.5 IU / mL and 20 IU / mL, respectively.

[0018] The sequential setting of the potency gradient of the standard working solution in the specific implementation manner of the present application is only one example, but not the entire design scheme. The technical scheme of making appropriate adjustments to the other intermediate concentration points is also within the scope of the method of the present application.

[0019] The spectrophotometric standard curve was obtained by the following method: 500uL of conductivity standard solutions of different titers were added to LB medium, followed by 20uL of yellow micrococcus suspension, and the OD of nisin was measured after shaking culture for 4h. 600 , the known titer and OD 600 Plot and perform linear regression to obtain the spectrophotometric standard curve.

[0020] By adopting the above technical scheme, the present invention firstly utilizes a gradient arrangement of different titer nisin standard solutions and a yellow micrococcus suspension to co-culture on a shaking table, and then determines the OD of nisin after 4 hours of shaking table culture. 600 Or the conductivity of the solution in the system, and then correspond to the known potency, draw a graph, linear regression, get the conductivity standard curve or spectrophotometry standard curve, and then use the conductivity or OD 600 The method uses a standard curve to calculate the titer of the nisin to be tested in the fermented mutagenic bacteria suspension. The conductivity method and spectrophotometry provided in the present application have the advantages of low instrument requirements, good repeatability, and simple steps. Compared with the existing agar diffusion method, the two detection methods provided in the present application can greatly improve the detection efficiency. Compared with the detection results obtained by the agar diffusion method, the detection time can be shortened from the original 16 hours to less than 5 hours. Therefore, the production method of the present application can quickly screen high-yield strains and reduce the time for producing nisin.

[0021] In addition, the present application also adopts the combination of primary screening and secondary screening in fermentation, which expands the screening range to a certain extent, reduces the possibility of missed screening, increases the probability of screening high-yield mutant strains, makes the grown strains more representative, and the results are intuitive, which can effectively reduce the screening workload, improve the screening efficiency, and greatly reduce the time for producing nisin.

[0022] More importantly, the present application strictly controls the potency range of the conductivity standard solution. When the concentration of the standard solution is less than 10 IU / mL or greater than 20 IU / mL, the conductivity result of the solution in the system cannot form a relatively stable linear relationship with the known potency. The potency range of the spectrophotometric standard solution is also strictly controlled in the spectrophotometric method. When the potency is lower than 5 IU / mL, nisin cannot effectively inhibit the growth of Micrococcus aureus. When the potency is higher than 20 IU / mL, nisin has completely inhibited the growth of Micrococcus aureus, and a regression equation cannot be derived.

[0023] Preferably, the specific operation of the fermentation is: continue to ferment the high-yield strain until it enters the logarithmic growth phase, then add 2-2.2wt% of ion exchange resin to the system, and then ferment for 23-24 hours, dynamically desorb the ion exchange resin with an analytical solution, collect the desorbed solution and add sodium chloride until the analytical solution is saturated for salting out, then filter press, elute, and dry to obtain nisin.

[0024] The ion exchange resin is a cation exchange resin D113, and the analytical solution is hydrochloric acid and ethanol in a volume ratio of 1: (1-1.3).

[0025] By adopting the above technical scheme, the cation exchange resin D113 adopted in the present application in fermentation has more functional groups than other ion exchange resins, and can be combined with the amino group on the surface of nisin, so as to achieve a better adsorption effect, and also adsorb more nisin produced in the fermentation process, reduce the concentration of nisin in the fermentation broth, effectively weaken the inhibitory effect of nisin product on cell growth and further synthesis, thereby reducing the time of producing nisin. At the same time, the cation exchange resin D113 with good adsorption effect and the analytical solution with a pH of about 5 can alleviate the problem of excessive acid in the system environment to a certain extent, and protect the equipment from being corroded by acidic substances produced in the fermentation.

[0026] In a second aspect, the present application provides a nisin obtained by the above production method.

[0027] In a third aspect, the present application provides a food preservative, comprising nisin microcapsules and essential oil capsules in a weight ratio of (0.5-0.6):6, wherein the raw materials used for the nisin microcapsules include the above-mentioned nisin, a gelling agent and a calcium chloride aqueous solution, and the essential oil capsules include chitosan, whey protein powder, an emulsifier and essential oil.

[0028] Specifically, the nisin microcapsules are prepared by the following method: dispersing nisin in an acetic acid solution to obtain a 10 g / L nisin dispersion, dispersing a gelling agent in water to obtain a 10 g / L gelling agent dispersion, then dropping the nisin dispersion into an equal amount of the gelling agent dispersion to obtain a mixed solution, then stirring the mixed solution for 30 minutes, adjusting the system pH to 4.4, homogenizing the mixed solution at 10-20 MPa for 10 minutes, then dropping the mixed solution into a calcium chloride solution, stirring for 30 minutes, standing at -4°C for solidification for 1 hour, centrifuging to separate microcapsules, washing away the calcium chloride remaining on the surface of the microcapsules, vacuum freeze-drying for 48 hours, and crushing to obtain nisin microcapsules.

[0029] Optionally, the gelling agent may be one of grapefruit peel pectin or lemon peel pectin.

[0030] Preferably, the gelling agent is grapefruit peel pectin.

[0031] Preferably, the calcium chloride concentration in the calcium chloride aqueous solution is 4.5-6wt%.

[0032] The essential oil capsule is prepared by the following method: dissolving chitosan in an acetic acid solution under a water bath condition of 50°C, adjusting the pH value of the system to 5.5 after cooling to room temperature, adding whey protein powder into the system, adding an emulsifier after complete dissolution, stirring for 15 minutes to obtain a mixed solution, dissolving essential oil in anhydrous ethanol, adding dropwise into the mixed solution, stirring for 30 minutes, homogenizing for 10 minutes under the condition of 10-20 MPa, adding sodium tripolyphosphate solution dropwise into the reaction system, homogenizing for 10 minutes, then centrifuging at a speed of 12000 rpm for 5 minutes, taking the precipitate, vacuum freeze-drying for 48 hours, and crushing to obtain the essential oil capsule, wherein the weight ratio of the chitosan, whey protein powder, emulsifier and essential oil is 1:1:2.2:(1.08-1.12).

[0033] Optionally, the emulsifier is any one of polysorbate 80, soy protein isolate, gum arabic, xanthan gum and soy lecithin.

[0034] Preferably, the emulsifier is polysorbate 80.

[0035] Preferably, the weight ratio of the chitosan, whey protein powder, emulsifier and essential oil is 1:1:1.5:1.11.

[0036] By adopting the above technical scheme, the present application will utilize the combination of nisin with high titer obtained by an efficient production method with a gelling agent and a calcium chloride aqueous solution, and obtain a nisin microcapsule with antibacterial and fresh-keeping effect, which can play a good fresh-keeping effect on food (fruit, raw meat, etc.). The capsule shell is a natural biopolymer encapsulation layer, which can separate nisin from enzymes, lipids and other substances in food, and reduce the possibility that the antibacterial activity and long-term antibacterial stability of nisin are negatively affected. And the nisin microcapsule and the essential oil capsule are mixed for use, and by adjusting the type of essential oil in the essential oil capsule, the food preservative can have an antibacterial effect on different strains, and the essential oil used includes but is not limited to cinnamon essential oil, oregano essential oil, rosemary essential oil and thyme essential oil, and in the specific embodiment of the present application, only cinnamon essential oil is used as an example for illustration.

[0037] The present application utilizes chitosan, whey protein powder, an emulsifier and cinnamon essential oil to prepare a cinnamon essential oil capsule with both anti-Gram-positive bacteria ability and anti-Gram-negative bacteria ability. The cinnamon essential oil capsule is mixed with nisin microcapsules for use, which can not only reduce the usage of nisin microcapsules, but also effectively improve the antibacterial ability of the food preservative and expand the antibacterial range. Therefore, the food preservative of the present application has both good economic applicability and high-efficiency antibacterial property.

[0038] Furthermore, the present application uses grapefruit peel pectin as a gelling agent, which has a more excellent gelling ability than lemon peel pectin. According to experimental data, it can increase the encapsulation rate of nisin to 79.1%. The present application also strictly controls the calcium chloride concentration of the calcium chloride aqueous solution used in the preparation of nisin microcapsules, thereby increasing the encapsulation rate of nisin microcapsules. If the calcium chloride concentration is too low, it cannot react with the active groups in the gelling agent and effectively encapsulate; if the calcium chloride concentration is too high, the thickness of the encapsulation layer will increase, greatly reducing the space inside the encapsulation layer that can accommodate nisin, which will also affect the encapsulation effect and reduce the encapsulation rate.

[0039] Finally, the present application emulsifies the essential oil, improves the water solubility of cinnamon essential oil, reduces its volatility, allows it to be more fully in contact with microorganisms during food preservation, and exerts a better antibacterial effect. Compared with other emulsifiers, the emulsion particle size range of the essential oil emulsion obtained after emulsification of polysorbate-80 is the smallest, and the emulsification effect is better. The present application also controls the weight ratio of chitosan, whey protein powder, emulsifier and essential oil, further improving the encapsulation rate of the essential oil capsule. If the amount of essential oil is too large, more free essential oil will adhere to the surface of the obtained essential oil capsule, causing the essential oil capsule to aggregate and reduce the encapsulation rate; if the amount of chitosan and whey protein powder is too large, the thickness of the encapsulation layer will increase and the encapsulation rate will be reduced.

[0040] In summary, this application has the following beneficial technical effects: 1. The conductivity method and spectrophotometry provided in this application have the advantages of low instrument requirements, good repeatability, and simple steps. Compared with the existing agar diffusion method, the two detection methods provided in this application can greatly improve the detection efficiency and reduce the production time of nisin by 11.0-12.9 hours; 2. The present application combines high-valence nisin obtained by an efficient production method, a gelling agent and a calcium chloride aqueous solution, and produces a nisin microcapsule with antibacterial and fresh-keeping effects, which can have a good fresh-keeping effect on food (fruits, raw meat, etc.); 3. The food preservative of the present application mixes nisin microcapsules with essential oil capsules, which reduces the usage of nisin microcapsules and can effectively enhance the antibacterial ability of the food preservative and expand the antibacterial range. Therefore, the food preservative has both good economic applicability and high antibacterial efficiency. DETAILED DESCRIPTION

[0041] Material Source Unless otherwise specified, the raw materials used in this application are all commercially available products, specifically: The starting strain was provided by Beijing Wanjia Reference Material Research Center, with the accession number BWCC27230; Nisin standard was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., with a titer of ≥900 IU / mL; Micrococcus xanthus was provided by Yunguan Biotechnology Co., Ltd., with the deposit number NCIMB 8166; M17 liquid culture medium was purchased from Haibo Biotechnology Co., Ltd.; The macroporous resin NKA-II was purchased from Shandong Shoucheng Chemical Co., Ltd.; Cation exchange resin D113 was purchased from Hebei Nijia Chemical Co., Ltd.; Lemon peel pectin and grapefruit peel pectin were purchased from Solebow Technology Co., Ltd., with a methoxy content of ≥6%; Cinnamon essential oil was purchased from Henan Zhengzhou Xuemailong Food Co., Ltd. with a purity of >90%; Chitosan, whey protein powder, soy protein isolate, gum arabic, and xanthan gum were all purchased from Henan Wanbang Industrial Co., Ltd. The CAS number of chitosan is 9012-76-4, the main components of whey protein powder include β-lactoglobulin, α-lactalbumin, immunoglobulin, and lactoferrin, the CAS number of soy protein isolate is 9010-10-0, the CAS number of gum arabic is 9000-01-5, and the CAS number of xanthan gum is 11138-66-2; Sodium tripolyphosphate was purchased from Suzhou Dongbai Chemical Co., Ltd.; Polysorbate-80 was purchased from Chengdu Kelong Chemical Co., Ltd., CAS No. 9005-65-6; Soybean lecithin was purchased from Hunan Tairen Pharmaceutical Co., Ltd., CAS No. 8002-43-5.

[0042] The present application is further described in detail below with reference to embodiments and comparative examples.

[0043] Example 1 A method for producing nisin comprises the following steps: S1, ultraviolet mutagenesis: the starting strain was diluted 1000 times with physiological saline to obtain a bacterial suspension, and the bacterial suspension was subjected to ultraviolet irradiation for 2.5 min at an irradiation distance of 30 cm and an ultraviolet irradiation power of 15 W to obtain a mutagenic bacterial suspension; S21, preparation of a conductivity standard curve: 0.1 g of nisin standard was weighed and dissolved with 0.02 mol / mL HCl, and then the volume was fixed to 50 mL to prepare a 2000 IU / mL nisin standard solution, and then the potency of the nisin standard solution was diluted to 10 IU / mL, 12 IU / mL and 10 IU / mL with 0.02 mol / mL HCl, respectively. L, 14IU / mL, 16IU / mL, 18IU / mL and 20IU / mL, inoculate yellow micrococcus on LB plate, culture at 30℃ for 48h, then select single colony and inoculate into LB liquid culture medium, culture on shaking table for 20h, refrigerate after packaging for later use, add 6 different titer nisin standard solutions to LB culture medium, the addition amount is 500uL, then add 20uL of yellow micrococcus suspension respectively, after shaking table culture for 4h, measure the conductivity of 6 different titer conductivity standard solutions, plot known titer and conductivity, linear regression, and obtain conductivity standard curve; S22, screening: After the M17 liquid culture medium is sterilized, add the nisin standard to make the nisin titer 5000IU / mL, and use it as the primary screening plate. Then add yellow micrococcus to the sterilized M17 culture medium to make the colony count reach 1.0×10 6 cfu / mL, as a rescreening plate, firstly spread the mutagenic bacterial suspension on the primary screening plate, observe the colony morphology after culturing for 36 hours, select a single colony and inoculate it on the rescreening plate, and obtain a single colony with a large inhibition zone after culturing for 36 hours, then inoculate the single colony on M17 liquid culture medium, and ferment it under the environment of pH=6.5. When the fermentation reaches 18 hours, take the fermented mutagenic bacterial suspension and dilute it to 20IU / mL, mix it with the yellow micrococcus suspension at a volume ratio of 50:2, and then culture it on a constant temperature shaker for 4 hours, measure the conductivity, and compare it with the conductivity standard curve prepared in step S21, and obtain the titer of the tested nisin in the fermented mutagenic bacterial suspension, and screen out the high-yield strain; S3, fermentation: until entering the logarithmic growth phase, add 2wt% of macroporous resin NKA-II to the system, and ferment for 24 hours, then use analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1) to dynamically desorb the macroporous resin NKA-II, collect the desorbed liquid and add sodium chloride until the analytical solution is saturated to perform salting out, followed by filter pressing, elution, and drying to obtain high-titer nisin.

[0044] Example 2 A method for producing nisin comprises the following steps: S1. Microwave mutagenesis: The diluted bacterial suspension was placed in a culture medium and cultured at 30°C until the logarithmic growth phase, and then microwaved for 30 seconds at a power of 750 W and a frequency of 2450 MHz to obtain a mutagenic bacterial suspension; S21. Prepare a conductivity standard curve: weigh 0.1 g of nisin standard and dissolve it with 0.02 mol / mL HCl, then dilute to 50 mL to prepare a 2000 IU / mL nisin standard solution, and then dilute the nisin standard solution with 0.02 mol / mL HCl to 10 IU / mL, 12 IU / mL, 14 IU / mL, 16 IU / mL, 18 IU / mL and 20 IU / mL, respectively. Inoculate Micrococcus aureus to On the LB plate, the culture was cultured at a constant temperature of 30°C for 48 hours, and then a single colony was selected and inoculated into the LB liquid culture medium, and cultured on a shaking table for 20 hours. After being packaged and refrigerated for later use, 6 different titer nisin standard solutions were added to the LB culture medium, and the addition amount was 500uL, and then 20uL of yellow micrococcus suspension was added respectively. After culturing on a shaking table for 4 hours, the conductivity of the 6 different titer conductivity standard solutions was measured, and the known titer was plotted against the conductivity, and linear regression was performed to obtain the conductivity standard curve; S22, screening: After the M17 liquid culture medium is sterilized, add the nisin standard to make the nisin titer 5000IU / mL, and use it as the primary screening plate. Then add yellow micrococcus to the sterilized M17 culture medium to make the colony count reach 1.0×10 6 cfu / mL, as a rescreening plate, first spread the mutagenic bacterial suspension on the primary screening plate, observe the colony morphology after culturing for 42 hours, select a single colony and inoculate it on the rescreening plate, and obtain a single colony with a large inhibition zone after culturing for 32 hours, then inoculate the single colony on M17 liquid culture medium, ferment under a pH=5.5 environment, and when the fermentation reaches 17 hours, take the fermented mutagenic bacterial suspension and dilute it to 20IU / mL, mix it with the yellow micrococcus suspension at a volume ratio of 50:2, and then culture it on a constant temperature shaker for 4 hours, measure the conductivity, and compare it with the conductivity standard curve prepared in step S21, and obtain the titer of the tested nisin in the fermented mutagenic bacterial suspension, and screen out the high-yield strain; S3. Fermentation: The high-yield strain obtained in step S22 is continued to be fermented until it enters the logarithmic growth phase, and then 2.2 wt% of the macroporous resin NKA-II is added to the system. After fermentation for another 24 hours, the macroporous resin NKA-II is dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1.3), the desorbed solution is collected and sodium chloride is added until the analytical solution is saturated for salting out, followed by filter pressing, elution, and drying to obtain high-titer nisin.

[0045] Example 3 A method for producing nisin comprises the following steps: S1. Plasma mutagenesis: In a helium environment with a gas flow rate of 10 L / min, the mutagenic bacterial suspension was subjected to plasma radio frequency treatment at an action distance of 2 mm and a power of 120 W for 60 s to obtain a mutagenic bacterial suspension; S21. Prepare a spectrophotometric standard curve: weigh 0.1 g of nisin standard and dissolve it with 0.02 mol / mL HCl, then dilute to 50 mL to prepare a 2000 IU / mL nisin standard solution, and then dilute the nisin standard solution with 0.02 mol / mL HCl to 5 IU / mL, 7.5 IU / mL, 10 IU / mL, 12.5 IU / mL, 15 IU / mL, 17 .5IU / mL and 20IU / mL, inoculate yellow micrococcus on LB plate, culture at 30℃ for 48h, then select single colony and inoculate into LB liquid culture medium, culture on shaking table for 20h, divide and refrigerate for later use, add 7 kinds of different titers of nisin standard solution to LB culture medium, add 500uL, then add 20uL of yellow micrococcus suspension, culture on shaking table for 4h, measure OD of nisin. 600 , the known titer and OD 600 Plot and linear regression to obtain the spectrophotometric standard curve; S22, screening: After the M17 liquid culture medium is sterilized, add the nisin standard to make the nisin titer 5000IU / mL, and use it as the primary screening plate. Then add yellow micrococcus to the sterilized M17 culture medium to make the colony count reach 1.0×10 6 cfu / mL, as the rescreening plate, first spread the mutagenic bacterial suspension on the primary screening plate, observe the colony morphology after culturing for 36 hours, select a single colony and inoculate it on the rescreening plate, and obtain a single colony with a large inhibition zone after culturing for 48 hours. Then inoculate the single colony on M17 liquid culture medium and ferment it under pH=6. When the fermentation reaches 16 hours, take the fermented mutagenic bacterial suspension and dilute it to 20IU / mL, mix it with the yellow micrococcus suspension at a volume ratio of 50:2, and culture it on a constant temperature shaker for 4 hours, and measure the OD 600 , comparing with the spectrophotometric standard curve prepared in step S21, obtaining the titer of the nisin to be tested in the fermented mutagenic bacterial suspension, and screening out high-yield strains; S3, fermentation: the high-yield strain obtained in step S22 is continued to be fermented until it enters the logarithmic growth phase, and then 2.1wt% of the macroporous resin NKA-II is added to the system. After fermentation for another 24 hours, the macroporous resin NKA-II is dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1.15), the desorbed solution is collected and sodium chloride is added until the analytical solution is saturated for salting out, followed by filter pressing, elution, and drying to obtain high-titer nisin.

[0046] Example 4 A method for producing nisin, which is different from Example 1 in that the macroporous resin NKA-II in step S3 is replaced by a cation exchange resin D113, and the rest is the same as Example 1.

[0047] Example 5 A method for producing nisin, which is different from that of Example 1 in that: in step S21, the potency of the nisin standard solution is diluted to 5 IU / mL, 10 IU / mL, 15 IU / mL, 20 IU / mL, 25 IU / mL and 30 IU / mL respectively with 0.02 mol / mL HCl, and the rest is the same as that of Example 1.

[0048] Example 6 A method for producing nisin, which is different from that of Example 3 in that: in step S21, the potency of the nisin standard solution is diluted to 2.5 IU / mL, 6 IU / mL, 9.5 IU / mL, 13 IU / mL, 16.5 IU / mL, 20 IU / mL and 23.5 IU / mL with 0.02 mol / mL HCl, and the rest is the same as that of Example 3.

[0049] Comparative Example 1 The difference from Example 1 is that: Step S21 is removed. Step S22 is specifically as follows: after the M17 liquid culture medium is sterilized, a nisin standard is added to make the nisin titer 5000 IU / mL, which is used as the primary screening plate, and then a yellow micrococcus is added to the sterilized M17 culture medium after sterilization to make the colony count reach 1.0×10 6cfu / mL, as the rescreening plate, first spread the mutagenic bacterial suspension on the primary screening plate, observe the colony morphology after culturing for 36 hours, select a single colony and inoculate it on the rescreening plate, and obtain a single colony with a large inhibition zone after culturing for 36 hours. Then inoculate the single colony on M17 liquid culture medium and ferment it under pH=6.5. When the fermentation reaches 18 hours, take the fermented mutagenic bacterial suspension and dilute it to 20IU / mL. Refer to GB According to the records in 1886.231-2023, the titer of the tested nisin in the fermented mutagenic bacterial suspension was obtained after determination, and the high-yielding strain was screened out. The high-yielding strain was then fermented until it entered the logarithmic growth phase, and 2 wt% of the macroporous resin NKA-II was added to the system. After fermentation for 24 hours, the macroporous resin NKA-II was dynamically desorbed using an analytical solution (hydrochloric acid and ethanol in a volume ratio of 1:1), the desorbed solution was collected and sodium chloride was added until the analytical solution was saturated for salting out, followed by filter pressing, elution, and drying to obtain high-titer nisin.

[0050] Comparative Example 2 The difference from Example 1 is that: Step S1 is specifically, after the M17 liquid culture medium is sterilized, a nisin standard is added to make the nisin titer 10000IU / mL, as a screening plate, step S22 is specifically, the mutagenic bacterial suspension is coated on the screening plate, the colony morphology is observed after culturing for 36 hours, and then the single colony with a large inhibition zone is inoculated into the M17 liquid culture medium, and fermented under a pH = 6.5 environment, and the rest is the same as Example 1.

[0051] Performance Testing According to the records in GB 1886.231-2023, the potency of the high-titer nisin obtained in the example was detected by agar diffusion method, and the time difference (with "-" indicating a decrease) for obtaining equivalent nisin after mutagenesis, screening and fermentation of equal amounts of nisin strains was recorded.

[0052] Table 1 Group Time difference in production / h Example 1 -11.7 Example 2 -11.8 Example 3 -11.8 Example 4 -12.9 Example 5 -11.0 Example 6 -11.1 Comparative Example 1 -0.2 Comparative Example 2 -8.5 Data Analysis: As can be seen from Table 1, the production time of the production method of Examples 1-6 is reduced by 11.0-12.9 hours compared with the agar diffusion method, which proves that the present invention utilizes a gradient arrangement of different titer nisin standard solutions and a yellow micrococcus suspension to co-culture on a shaking table, and by measuring the OD of nisin 600 Or the conductivity of the solution in the system, obtain the conductivity standard curve or the spectrophotometric standard curve, and then use the conductivity or OD 600The method uses a standard curve to calculate the titer of the nisin to be tested in the fermented mutagenic bacterial suspension, which greatly improves the detection efficiency. The production method of the present application can quickly screen high-yield strains and reduce the time for producing nisin; The time difference used for production in Example 4 is significantly greater than that in Example 1. In the present application, cation exchange resin D113 is used in fermentation. Compared with the macroporous resin NKA-II, it has more functional groups and can combine with the amino groups on the surface of nisin, thereby achieving a better adsorption effect, and adsorbing more nisin produced during the fermentation process, reducing the concentration of nisin in the fermentation broth, and effectively weakening the inhibitory effect of nisin products on cell growth and further synthesis, thereby reducing the time for producing nisin; The time difference used for production in Example 5 is shorter than that in Example 1, which proves that the present application improves the detection efficiency by strictly controlling the potency range of the conductivity standard solution. When the concentration of the standard solution is less than 10 IU / mL or greater than 20 IU / mL, the conductivity result of the solution in the system cannot form a relatively stable linear relationship with the known potency. The time difference used for production in Example 6 is less than that in Example 3, which proves that the present application improves the detection efficiency by strictly controlling the titer range of the spectrophotometric standard solution. When the titer is lower than 5 IU / mL, nisin cannot effectively inhibit the growth of yellow micrococcus. When the titer is higher than 20 IU / mL, nisin has completely inhibited the growth of yellow micrococcus, and the regression equation cannot be obtained; the time difference used for production in Comparative Examples 1-2 is less than that in Example 1, which proves that the present application expands the screening range to a certain extent by adopting the combination of primary screening and rescreening in fermentation, reduces the possibility of missed screening, increases the probability of screening high-yield mutant strains, makes the grown strains more representative, and the results are intuitive, which can effectively reduce the screening workload and improve the screening efficiency. The conductivity method is used instead of the agar diffusion method, which can greatly improve the detection efficiency, quickly screen high-yield strains, and reduce the production time of nisin.

[0053] In the following application examples, the present application is explained by taking the nisin in Example 4, which has the largest time difference in production, as an example.

[0054] Application Example 1 A food preservative is prepared by the following method: 1. Preparation of Nisin Microcapsules: Preparation of core material: Dispersing the nisin obtained in Example 4 in an acetic acid solution to obtain a 10 g / L nisin dispersion; Preparation of wall material: Disperse the gelling agent (lemon peel pectin) in water to obtain a 10 g / L gelling agent dispersion; Preparation of microcapsules: Add the nisin dispersion dropwise into an equal amount of the gelling agent dispersion to obtain a mixed solution, stir for 30 minutes, adjust the system pH to 4.4, homogenize for 10 minutes under 10 MPa, add the mixed solution dropwise into the calcium chloride solution, stir for 30 minutes, place at -4°C to solidify for 1 hour, centrifuge to separate microcapsules, wash off the residual calcium chloride on the surface of the microcapsules, vacuum freeze-dry for 48 hours, and crush to obtain nisin microcapsules; 2. Preparation of essential oil capsules: Preparation of wall material: 1 kg of chitosan was dissolved in acetic acid solution under 50°C water bath condition, and the pH of the system was adjusted to 5.5 after cooling to room temperature, 1 kg of whey protein powder was added to the system, and after complete dissolution, 2.2 kg of emulsifier (soy protein isolate) was added, and a mixture was obtained after stirring for 15 minutes; Preparation of microcapsules: 1.08 kg of cinnamon essential oil was dissolved in 54 kg of anhydrous ethanol, added dropwise to the above mixture, stirred for 30 min, homogenized for 10 min under 10 MPa, 2 g / L of sodium tripolyphosphate solution was added dropwise to the reaction system, homogenized for 10 min, then centrifuged at 12000 rpm for 5 min, the precipitate was vacuum freeze-dried for 48 h, and crushed to obtain essential oil capsules; III. Evenly mix the nisin microcapsules and the essential oil capsules at a weight ratio of 0.5:6 to obtain a food preservative.

[0055] Application Example 2 A food preservative is prepared by the following method: 1. Preparation of Nisin Microcapsules: Preparation of core material: Dispersing the nisin obtained in Example 4 in an acetic acid solution to obtain a 10 g / L nisin dispersion; Preparation of wall material: Disperse the gelling agent (lemon peel pectin) in water to obtain a 10 g / L gelling agent dispersion; Preparation of microcapsules: Add the nisin dispersion dropwise into an equal amount of the gelling agent dispersion to obtain a mixed solution, stir for 30 minutes, adjust the system pH to 4.4, homogenize at 20 MPa for 10 minutes, add the mixed solution dropwise into the calcium chloride solution, stir for 30 minutes, place at -4°C to solidify for 1 hour, separate the microcapsules by centrifugation, wash off the residual calcium chloride on the surface of the microcapsules, vacuum freeze-dry for 48 hours, and crush to obtain nisin microcapsules; 2. Preparation of essential oil capsules: Preparation of wall material: 1 kg of chitosan was dissolved in acetic acid solution under 50°C water bath condition, and the pH of the system was adjusted to 5.5 after cooling to room temperature, 1 kg of whey protein powder was added to the system, and after complete dissolution, 2.2 kg of emulsifier (soy protein isolate) was added, and a mixture was obtained after stirring for 15 minutes; Preparation of microcapsules: 1.12 kg of cinnamon essential oil was dissolved in 22.4 kg of anhydrous ethanol, and added dropwise to the above mixed solution, stirred for 30 min, homogenized for 10 min under 20 MPa, 5 g / L sodium tripolyphosphate solution was added dropwise to the reaction system, homogenized for 10 min, and then centrifuged at 12000 rpm for 5 min. The precipitate was vacuum freeze-dried for 48 h, and crushed to obtain essential oil capsules; III. Evenly mix the nisin microcapsules and the essential oil capsules at a weight ratio of 0.6:6 to obtain a food preservative.

[0056] Application Example 3 A food preservative, which is different from Application Example 1 in that the lemon peel pectin in step I is replaced with grapefruit peel pectin, and the rest is the same as Application Example 1.

[0057] Application Example 4 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 4wt%, and the rest is the same as Application Example 1.

[0058] Application Example 5 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 4.5wt%, and the rest is the same as Application Example 1.

[0059] Application Example 6 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 5wt%, and the rest is the same as Application Example 1.

[0060] Application Example 7 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 5.5wt%, and the rest is the same as Application Example 1.

[0061] Application Example 8 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 6wt%, and the rest is the same as Application Example 1.

[0062] Application Example 9 A food preservative, which is different from Application Example 1 in that the calcium chloride concentration of the calcium chloride aqueous solution in step I is 6.5wt%, and the rest is the same as Application Example 1.

[0063] Application Example 10 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 1.09 kg, and the rest is the same as Application Example 1.

[0064] Application Example 11 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 1.10 kg, and the rest is the same as Application Example 1.

[0065] Application Example 12 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 1.11 kg, and the rest is the same as Application Example 1.

[0066] Application Example 13 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 1.12 kg, and the rest is the same as Application Example 1.

[0067] Application Example 14 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 0.8 kg, and the rest is the same as Application Example 1.

[0068] Application Example 15 A food preservative, which is different from Application Example 1 in that: the amount of cinnamon essential oil added in step II is 1.2 kg, and the rest is the same as Application Example 1.

[0069] Application Examples 16-19 A food preservative, which is different from Application Example 1 in that the soy protein isolate in step II is replaced by polysorbate 80, gum arabic, xanthan gum and soy lecithin respectively, and the rest is the same as Application Example 1.

[0070] Comparative application example 1 The difference from Application Example 1 is that step II is removed, and the rest is the same as Application Example 1.

[0071] Comparative Application Example 2 The difference from Application Example 1 is that step I is removed, and in step III, a 50wt% nisin solution and essential oil capsules are uniformly mixed in a weight ratio of 0.5:6, and the rest are the same as Application Example 1.

[0072] Comparative Application Example 3 The difference from Application Example 1 is that in step II, the emulsifier is removed, the amount of cinnamon essential oil is 3.28 kg, and the rest is the same as Application Example 1.

[0073] Performance Testing 1. Determination of capsule encapsulation efficiency: refer to the record in "Bai Shuangyan, Yang Yufan, Zou Hao, et al. Study on the preparation process of tea polyphenol chitosan microcapsules [J]. Grain and Oils, 2023, 36(8): 101-104." to determine the encapsulation efficiency of nisin microcapsules and essential oil capsules in the application example, and record the results in Table 2; 2. Application performance determination: The food preservatives obtained in Application Examples 1-19 and Comparative Application Example 1 are dissolved in water to obtain a preservative dispersion, and then raw meat preservation tests and fruit preservation tests are carried out. Beef is selected as raw meat, and mango is selected as fruit. Specifically: 1. Raw meat preservation: In a sterile environment, the beef is trimmed and cut into small pieces of 50 g, and the cut beef is immersed in different preservative dispersions for 5 minutes, taken out and allowed to stand for 10 minutes, and then air-dried and placed in a sterile device and stored at 4°C. Each sample is made in parallel three times, and the uncoated sample is used as a blank control. The weight loss rate and total colony count of the sample are measured every 3 days, and the results are recorded in Table 3, wherein the weight loss rate = (W0-W1) / W0×100%, W0 is the initial weight of the sample, W1 is the weight of the same sample after refrigeration for 15 days, and the total colony count is measured according to the method of GB / T 4789.2-2016; 2. Fruit preservation: Select mangoes with no mechanical damage, no pests and diseases, moderate size and similar maturity after immediate picking, wash with tap water and then soak in sodium hypochlorite solution with volume fraction of 2% for 2 min, take out and dry naturally, randomly distribute the dried mangoes into several groups, each group has 100 fruits, and each group of mangoes is placed in different containers for soaking treatment, and the soaking time is 2 min. The control group mangoes are soaked in distilled water for 2 min. After air drying, each mango is individually packaged in a polyethylene bag and stored at room temperature for eight days. The decay rate and respiration intensity of mangoes are measured subsequently, which are all recorded in Table 3, wherein decay rate=N0 / N×100%, N0 is the number of mangoes with black spots on the epidermis, and N is the total number of mangoes (100). The respiration intensity is measured as follows: about 0.5 kg of mangoes are taken from each group and placed in a 2L airtight container, sealed for 0.5 h, and then the carbon dioxide content in the container is measured with a portable carbon dioxide meter (measuring frequency 4h / time), and the time when its respiration intensity reaches the peak value is recorded.

[0074] Table 2 Table 3 Group Weight loss rate % Total colony count (log CFU / g) Decay rate % Time to peak respiratory intensity / h Application Example 1 3.65 5.15 28 172 Application Example 2 3.67 5.16 28 168 Application Example 3 3.32 5.05 23 180 Application Example 4 3.66 5.15 28 172 Application Example 5 3.36 5.10 24 180 Application Example 6 3.37 5.11 24 184 Application Example 7 3.35 5.11 25 180 Application Example 8 3.36 5.09 24 180 Application Example 9 3.68 5.18 28 168 Application Example 10 3.66 5.15 28 172 Application Example 11 3.15 4.85 23 180 Application Example 12 3.14 4.82 23 176 Application Example 13 3.15 4.87 22 180 Application Example 14 4.23 7.05 31 156 Application Example 15 4.31 7.08 30 156 Application Example 16 3.14 4.85 23 180 Application Example 17 3.65 5.15 28 172 Application Example 18 3.64 5.18 28 176 Application Example 19 3.66 5.16 27 172 Comparative application example 1 12.96 9.05 61 48 Comparative Application Example 2 11.54 7.85 54 124 Comparative Application Example 3 8.65 6.98 48 144 Data Analysis: As can be seen from Table 3, the encapsulation rate of nisin microcapsules in Application Examples 1-2 of the present application is 78.4-78.5%, and the encapsulation rate of essential oil capsules is 92.5-92.6%, which proves that the present application prepares a nisin microcapsule with antibacterial and fresh-keeping effects by combining nisin with a gelling agent and a calcium chloride aqueous solution, and prepares a cinnamon essential oil capsule with both anti-Gram-positive bacteria and anti-Gram-negative bacteria ability by using chitosan, whey protein powder, emulsifier and cinnamon essential oil. The weight loss rate of beef in Example 1-2 was as low as 3.65-3.67%, the total colony count in beef was as low as 5.15-5.16 logCFU / g, the mango rot rate was as low as 28%, and the time for the respiration intensity to reach the peak was as long as 168-172 hours, which proved that the food preservative obtained by mixing nisin microcapsules with nisin microcapsules in the present application had good antibacterial effect and a large antibacterial range, and that the fruit and raw meat could be soaked and treated with antibacterial agents to have a longer fresh-keeping time. The nisin microcapsule encapsulation rate of Application Example 3 is significantly higher than that of Application Example 1, while the mango rot rate, beef weight loss rate and total colony count are significantly lower than those of Application Example 1, and the time for the respiration intensity to reach the peak value is also longer than that of Application Example 1, which proves that the application uses grapefruit peel pectin as a gelling agent, which has a better gelling ability than lemon peel pectin, and can significantly improve the nisin microcapsule encapsulation rate, thereby optimizing the preservation effect of the food preservative; The difference between Application Examples 4-9 is that the present application adjusts the calcium chloride concentration of the calcium chloride aqueous solution in step I, wherein the nisin microcapsule encapsulation rate of Application Examples 5-8 is significantly higher, while the mango rot rate, beef weight loss rate and total bacterial count are lower, and the respiration intensity reaches the peak time longer, proving that the present application improves the encapsulation rate of nisin microcapsules by strictly controlling the calcium chloride concentration of the calcium chloride aqueous solution, thereby optimizing the preservation effect of the food preservative. If the calcium chloride concentration is too low, it cannot react with the active groups in the gelling agent and effectively encapsulate. If the calcium chloride concentration is too high, the thickness of the encapsulation layer will increase, greatly reducing the space inside the encapsulation layer that can accommodate nisin, which will also affect the encapsulation effect and reduce the encapsulation rate. The difference between Application Examples 10-15 is that the present application adjusts the amount of cinnamon essential oil added in step II, wherein the essential oil capsule encapsulation rate of Application Examples 11-13 is significantly higher, while the mango rot rate, beef weight loss rate and total colony count are lower, and the respiration intensity reaches the peak time longer, proving that the present application further improves the encapsulation rate of the essential oil capsule by strictly controlling the weight ratio of chitosan, whey protein powder, emulsifier and essential oil, thereby optimizing the preservation effect of the food preservative. If the amount of essential oil is too large, more free essential oil will adhere to the surface of the prepared essential oil capsule, causing the essential oil capsule to aggregate and reduce the encapsulation rate. If the amount of chitosan and whey protein powder is too large, the thickness of the encapsulation layer will increase and the encapsulation rate will be reduced. The difference between Application Examples 16-19 is that the present application adjusts the type of emulsifier, wherein the mango rot rate, beef weight loss rate and total bacterial count of Application Example 16 are all lower, and the time for the respiration intensity to reach the peak value is longer, proving that compared with other emulsifiers, the polysorbate-80 in the present application has the smallest emulsion particle size range in the essential oil emulsion obtained after emulsification, and has a better emulsification effect, further improving the water solubility of cinnamon essential oil and reducing its volatility, so that it can be more fully in contact with microorganisms during food preservation, and exert a better antibacterial effect; The mango rot rate, beef weight loss rate and total bacterial count of Application Example 1 are much higher than those of Application Example 1, and the time for the respiration intensity to reach the peak value is much shorter than that of Application Example 1, which proves that the food preservative obtained by mixing nisin microcapsules with nisin microcapsules in this application has good antibacterial effect and a large antibacterial range, and can make fruits and raw meat have a longer preservation time after soaking and antibacterial treatment. The mango rot rate, beef weight loss rate and total bacterial count of Application Example 2 are much higher than those of Application Example 1, and the time for the respiration intensity to reach the peak is much shorter than that of Application Example 1, which proves that the present application can separate nisin from enzymes, lipids and other substances in food by encapsulating nisin, thereby reducing the possibility of negative impact on the antibacterial activity and long-term antibacterial stability of nisin, thereby greatly improving the preservation effect of the food preservative; The mango rot rate, beef weight loss rate and total colony count of Application Example 3 are much higher than those of Application Example 1, and the time for the respiration intensity to reach the peak is much shorter than that of Application Example 1, which proves that the present application improves the water solubility of cinnamon essential oil and reduces its volatility by emulsifying the essential oil, so that it can be more fully in contact with microorganisms during food preservation and exert a better antibacterial effect.

[0075] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for producing nisin, comprising the following steps: Mutagenesis, screening and fermentation, characterized in that: the specific operation of the screening is: subjecting the mutagenic bacterial suspension obtained after mutagenesis to primary screening, rescreening to obtain a single colony with a large inhibition zone, placing it in an environment of pH = 5.5-6.5 for fermentation, detecting the titer 16-18 hours after the start of fermentation, and screening out high-yield strains, wherein the method used to detect the titer is a conductivity method or a spectrophotometer.

2. The method for producing nisin according to claim 1, characterized in that: The nisin potency in the nisin standard solution in the conductivity method is set in the range of 10-20 IU / mL.

3. The method for producing nisin according to claim 1, characterized in that: The nisin titer in the nisin standard solution in the spectrophotometric method is set in the range of 5-20 IU / mL.

4. The method for producing nisin according to claim 1, characterized in that: The specific operation of the fermentation is: continue to ferment the high-yield strain until it enters the logarithmic growth phase, then add 2-2.2wt% of ion exchange resin to the system, and then ferment for 23-24 hours and dynamically desorb the ion exchange resin with an analytical solution, collect the desorbed solution and add sodium chloride until the analytical solution is saturated to perform salt precipitation, then filter press, elute, and dry to obtain nisin.

5. Nisin obtained by the production method according to any one of claims 1 to 4.

6. A food preservative, characterized in that: The invention comprises nisin microcapsules and essential oil capsules in a weight ratio of (0.5-0.6):6, wherein the raw materials used for the nisin microcapsules include the nisin according to claim 5, a gelling agent and a calcium chloride solution, and the essential oil capsules include chitosan, whey protein powder, an emulsifier and essential oil.

7. A food preservative according to claim 6, characterized in that: The gelling agent is grapefruit peel pectin.

8. A food preservative according to claim 6, characterized in that: The calcium chloride concentration in the calcium chloride aqueous solution is 4.5-6wt%.

9. A food preservative according to claim 6, characterized in that: The emulsifier is polysorbate 80.

10. A food preservative according to claim 6, characterized in that: The weight ratio of the chitosan, whey protein powder, emulsifier and essential oil is 1:1:1.5:1.11.

Images