Method for producing nisin through bacterium-enzyme co-immobilization synergistic fermentation

Abstract

The invention relates to a method for producing nisin through bacterium-enzyme co-immobilization and synergistic fermentation. The method comprises the following steps: step 1, preparing immobilized enzyme nanoparticles; step 2, culturing lactococcus lactis; step 3, preparing bacteria and enzyme co-immobilized calcium alginate microspheres; 4, preparing calcium alginate microspheres of which the surfaces are grafted with polylactide layers; step 5, preparing a microcapsule wrapping the immobilized enzyme nanoparticles and lactococcus lactis; and 6, fermenting to produce the nisin. The preparation method comprises the following steps: firstly, immobilizing lactate oxidase by using a metal organic framework to prepare immobilized enzyme nanoparticles, and then preparing the microcapsule containing lactococcus lactis and the immobilized enzyme nanoparticles, thereby effectively relieving the problem of bacterial growth inhibition caused by lactic acid accumulation, enhancing quorum sensing between lactococcus lactis and enhancing the activity of lactococcus lactis. The effect value (the maximum value is 9800IU / mL) of the fermentation liquor is greatly improved, and the recycling and reuse of the lactococcus lactis and the lactate oxidase are realized.

Description

Technical Field

[0001] The technical solution of the present invention can be applied to the field of biotechnology, and specifically relates to the preparation of bacterial cell and enzyme co-immobilized microcapsules, and utilizes the synergistic effect between bacterial enzymes to strengthen the fermentation production of nisin. Background Art

[0002] As people pay more and more attention to food safety issues, the replacement of chemical preservatives by biological preservatives has become an inevitable trend in the development of the food industry. Nisin is a lantibiotic secreted by Lactococcus lactis subsp. lactis, which can strongly inhibit the growth of Gram-positive bacteria and has the advantages of low immunogenicity, good thermal stability, and no cross-resistance when used with antibiotics. So far, nisin is the only bacteriocin approved by the Food and Agriculture Organization of the United Nations / World Health Organization for food preservation. Nisin contains a variety of rare amino acids (e.g., lanthionine, 3-methyllanthionine, and dehydroalanine) and a special thioester ring structure. Its industrial production mainly adopts microbial fermentation. However, low biosynthesis efficiency is the main bottleneck problem restricting the industrial production of nisin. Due to the lack of key enzymes of the Krebs cycle, Lactococcus lactis can only carry out homolactic fermentation, and the large accumulation of lactic acid will inhibit bacterial growth and nisin synthesis. As a quorum sensing signal molecule, nisin can only induce the efficient expression of its own synthetic gene when its concentration reaches a certain threshold. Therefore, developing a suitable method to improve the biosynthesis efficiency of nisin is of great significance for expanding the scale of Lactococcus lactis fermentation, reducing product prices and alleviating food safety issues.

[0003] Gene modification and optimization of culture conditions are common methods to improve the efficiency of nisin biosynthesis. Representative patents include: Chinese invention patent CN202011420240.9 "A method for constructing a high-expression strain of nisin" applied by Roxanna et al.; Chinese invention patent CN202111614279.9 "A method for constructing a genetically engineered bacterium that efficiently secretes Nisin and its engineered bacteria and application" applied by Wu Gang et al.; Chinese invention patent CN202410307798.8 "A Trx-1 protein gene and a method for increasing nisin production" applied by Wang Chao et al.; Chinese invention patent CN201611131569.7 "A method for controlling the pH value of a fermentation culture system to increase nisin production" applied by Qiao Jianjun et al. However, increasing the copy number of genes nisA, nisRK and nisFEG involved in nisin synthesis will aggravate the metabolic burden of the recombinant strain, and insufficient energy supply will lead to slow bacterial proliferation. After constructing a protein single-copy gene recombinant plasmid, the expression stability of the target gene in the host cell is poor and it is easily degraded by the host cell protease. Although maintaining the medium pH above 6.0 is conducive to the growth of Lactococcus lactis, the solubility of nisin is low under this pH condition and cannot continuously induce its own synthesis. In addition, lactate generated by the addition of alkaline substances will reduce the proton permeability of the cell membrane, thereby inhibiting the growth and metabolism of Lactococcus lactis. Previous studies have shown that co-culture of Lactococcus lactis and Saccharomyces cerevisiae can alleviate the acidification of the culture system and thus increase the production of nisin (Doi: 10.1016 / j.lwt.2021.111093). However, due to the different requirements of the two microorganisms for nutrition and environment, it is difficult to maintain synchronous growth in the co-culture system. Bacterial enzyme synergistic fermentation is an emerging biotechnology that uses the catalytic effect of enzymes to improve the stability of the microbial fermentation process, thereby increasing the yield of metabolites. This technology has the advantages of mild reaction conditions, simple carbon source of the culture system, and small bacterial metabolic load. Therefore, the use of bacterial enzymes to synergistically ferment and enhance nisin has good industrial application prospects.

[0004] Existing research on bacterial-enzyme synergistic fermentation has mostly focused on biomass conversion, and there have been no reports on research applied to bacteriocin production. However, free enzymes have problems such as poor stability, difficulty in separation, and inability to be reused during application. Immobilizing enzymes using carrier binding, embedding and cross-linking methods can effectively solve the above problems. Representative patents include: Chinese invention patent CN202410228248.7 "An immobilized acetylcholinesterase, preparation method and application thereof" applied by Yang Dongfeng et al.; Chinese invention patent CN202411484136.4 "An immobilized L-cysteine ​​synthetase, preparation method and application thereof" applied by Zhao Desheng et al. Obviously, in order to enhance the quorum sensing between Lactococcus lactis, the enzyme immobilization technology can be used to immobilize the bacterial cells. Summary of the invention

[0005] The purpose of the present invention is to develop a method for producing nisin by bacterial and enzyme co-immobilization and synergistic fermentation, wherein lactate oxidase is immobilized on a nanoscale, Lactococcus lactis and immobilized enzyme nanoparticles are immobilized on a micrometer scale, and lactate oxidase is used to oxidize lactic acid into pyruvate which promotes the growth of Lactococcus lactis, thereby increasing the yield of nisin.

[0006] The bacterial enzyme co-immobilization synergistic fermentation method proposed in the present invention has a good effect on strengthening the production of nisin, that is, it can greatly improve the efficacy value of the fermentation liquid. At present, this method has not been reported in the literature.

[0007] The technical problem to be solved by the present invention is: a bacterial enzyme co-fixation synergistic fermentation method is proposed, which has a good strengthening effect on the production of nisin, that is, it can greatly improve the efficacy value of the fermentation liquid. The invention alleviates the system acidification problem caused by lactic acid accumulation in the lactococcal fermentation process, and overcomes the defects of low biosynthesis efficiency of lactococcal nisin, difficulty in enzyme recovery, and inability to continuously produce.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation. The bacterial enzyme co-immobilization operation firstly embeds lactococcus lactis and immobilized enzyme nanoparticles with polysaccharide calcification microspheres, then grafts a polymer cross-linking layer on the surface of the microspheres, and prepares bacterial enzyme co-immobilization microcapsules by removing the polysaccharide calcification core; the immobilized enzyme nanoparticles are prepared by immobilizing lactate oxidase in a metal organic framework (MOF).

[0010] Preferably, the present invention uses sodium alginate to prepare the monomer of polysaccharide calcification microspheres.

[0011] Preferably, the present invention adopts visible light-induced graft polymerization to prepare the polymer cross-linking layer on the surface of the polysaccharide calcification microspheres.

[0012] Preferably, the MOF material of the present invention is a manganese-doped zeolite imidazole framework.

[0013] In order to achieve the above object, the present invention provides a method for producing nisin by co-immobilization of bacterial enzymes and synergistic fermentation, comprising the following steps:

[0014] Step 1: Preparation of immobilized enzyme nanoparticles: Dissolve zinc nitrate hexahydrate and manganese nitrate tetrahydrate in deionized water, and then add lactate oxidase solution; Add 2-methylimidazole solution with a pH of 8.0 to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stir to react; After the reaction, collect the precipitate, wash, freeze and dry, and the obtained solid particles are Mn@ZIF-8 nanoparticles encapsulating lactate oxidase;

[0015] Step 2: Cultivation of Lactococcus lactis: Inoculate and culture Lactococcus lactis to obtain a bacterial cell concentration of 3 to 6×10 9 cfu / mL of Lactococcus lactis seed solution;

[0016] Step 3: preparing bacterial enzyme co-immobilized calcium alginate microspheres: mixing the Lactococcus lactis seed solution obtained in step 2 into the sodium alginate solution, and then adding the Mn@ZIF-8 nanoparticles encapsulating lactate oxidase obtained in step 1, and after fully mixing, dripping the mixed solution into the calcium chloride solution to prepare bacterial enzyme co-immobilized calcium alginate microspheres;

[0017] Step 4: Preparation of surface-grafted calcium alginate microspheres: Add the bacterial enzyme co-immobilized calcium alginate microspheres obtained in step 3 to the positively charged polymer solution, stir at 25°C, and centrifuge to obtain bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with the positively charged polymer; Add the bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with the positively charged polymer and a photoinitiator to the grafted monomer solution; Under nitrogen conditions, at a wavelength of 420nm and a light intensity of 8000μW / cm -2 irradiated under visible light irradiation conditions to obtain surface-grafted calcium alginate microspheres;

[0018] Step 5: preparing microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis: adding the surface-grafted calcium alginate microspheres obtained in step 5 into a sodium citrate solution at pH 5.5 for liquefaction to prepare microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis;

[0019] Step 6: Fermentation to produce nisin: The microcapsules obtained in step 5 are inoculated into a fermentation medium for fermentation culture.

[0020] Preferably, in step 1, the titer of the lactate oxidase solution is 200-1000 U / mL; more preferably, it is 200, 400, 600, 800, 1000 U / mL and ranges therebetween.

[0021] Any of the above is preferably that in step 1, the concentration of the 2-methylimidazole solution is 10 to 100 g / L; more preferably 10, 20, 40, 60, 80, 100 g / L and ranges therebetween.

[0022] Preferably, in any of the above items, in step 1, glacial acetic acid is used to adjust the pH of the 2-methylimidazole solution.

[0023] Preferably, in any of the above items, in step 1, 1.88 g of zinc nitrate hexahydrate and 0.07 g of manganese nitrate tetrahydrate are dissolved in every 50 mL of deionized water, and the corresponding amount of lactate oxidase solution added is 5 mL.

[0024] Preferably, in any of the above items, in step 1, the stirring reaction is carried out at a rotation speed of 300 rpm for 60 minutes.

[0025] Any of the above is preferably that the step in step 1 is: prepare immobilized enzyme nanoparticles: dissolve 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate in 50mL of deionized water, and then slowly add 5mL of lactate oxidase solution. Prepare 2-methylimidazole solution and adjust the pH to 8.0. Slowly add 2-methylimidazole solution to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stir for 60 minutes at a speed of 300rpm. The reaction solution is centrifuged, and the collected precipitate is washed and freeze-dried to obtain the solid particles, which are Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0026] Preferably, in any of the above items, in step 2, the Lactococcus lactis is at least one of L. lactis ATCC11454, L. lactis SM526, L. lactis F44, L. lactis N8, L. lactis WNC20, L. lactis CHCC5826 or L. lactis ATCC6242. However, the present invention is not limited to the above strains.

[0027] Any of the above is preferably that in step 2, the culturing step of Lactococcus lactis is: inoculating the Lactococcus lactis onto a slant culture medium, culturing at a temperature of 30°C for 18 hours to obtain slant bacteria; inoculating the slant bacteria into a seed culture medium, culturing at a speed of 150 rpm and a temperature of 30°C for 18 hours, centrifuging the culture solution, collecting bacterial cells, and diluting the bacterial cells to 3 to 6×10 9cfu / mL.

[0028] Preferably, any of the above items is that the slant culture medium consists of: 20 g / L sodium dihydrogen phosphate, 15 g / L peptone, 15 g / L yeast extract powder, 15 g / L sucrose, 2 g / L sodium chloride, 2 g / L magnesium sulfate and 18 g / L agar, the pH is 6.9, and the solvent is deionized water.

[0029] Preferably, any of the above items is that the seed culture medium consists of 20 g / L sodium dihydrogen phosphate, 15 g / L peptone, 15 g / L yeast extract, 15 g / L sucrose, 2 g / L sodium chloride and 2 g / L magnesium sulfate, the pH is 6.9, and the solvent is deionized water.

[0030] Preferably, any of the above items is that the PBS phosphate buffer is composed of: dissolving 0.24 g potassium dihydrogen phosphate and 1.44 g sodium dihydrogen phosphate in 800 mL distilled water, adjusting the pH to 7.4 with 0.1 mol / L hydrochloric acid solution, and making up to 1 L with distilled water.

[0031] Any of the above is preferably that in step 2, the bacterial cell concentration is diluted to 3-6×10 9 cfu / mL.

[0032] Any of the above is preferably that in step 3, the volume ratio of the Lactococcus lactis seed solution to the sodium alginate solution is 0.01:1 to 0.2:1; further preferably, 0.01:1, 0.05:1, 0.1:1, 0.15:1, 0.2:1 and ranges therebetween.

[0033] Preferably, in any of the above items, in step 3, the concentration of the Mn@ZIF-8 nanoparticles encapsulating lactate oxidase is 0.02-0.10 g / L; more preferably, 0.02, 0.04, 0.06, 0.08, 0.10 g / L and ranges therebetween.

[0034] Preferably, in any of the above items, in step 3, the concentration of the sodium alginate solution is 5 to 20 g / L.

[0035] Any of the above is preferably that in step 3, a mixed solution containing Lactococcus lactis, Mn@ZIF-8 nanoparticles encapsulating lactate oxidase, and sodium alginate is added dropwise to a 2% calcium chloride solution at a temperature of 34°C at a rate of 3 to 10 mL / min to obtain bacterial enzyme co-fixed calcium alginate microspheres. If the speed is too fast, the particle size of the microspheres will be too small, and the subsequent preparation of microcapsules and the fermentation cell density will be too small, and the product secretion rate will be slow. If the speed is too slow, the particle size of the microspheres will be too large, and the microcapsule volume is too large and easy to rupture. The speed at which the mixed solution is added to the 2% calcium chloride solution is further preferably 3, 4, 5, 6, 7, 8, 9, 10 mL / min and the range therebetween, and more preferably 5 mL / min.

[0036] Any of the above is preferably that in step 3, the bacterial enzyme co-immobilized calcium alginate microspheres are prepared according to the following steps: using a disposable 5 mL syringe, a solution containing Lactococcus lactis, Mn@ZIF-8 nanoparticles encapsulating lactate oxidase and sodium alginate is added dropwise to a 2% calcium chloride solution at a rate of 5 mL / min, and the preferred operating temperature is 34°C.

[0037] Preferably, in any of the above items, in step 4, the positively charged polymer is one of polyethyleneimine, polyacrylamine hydrochloride or polylysine.

[0038] Preferably, in any one of the above items, in step 4, the mass fraction of the positively charged polymer is 1%.

[0039] Preferably, in any of the above items, in step 4, the photoinitiator is a thioxanthone derivative.

[0040] Preferably, in any of the above items, in step 4, the grafting monomer is one of polyurethane acrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate or polylactide.

[0041] Preferably, in any of the above items, in step 4, the photoinitiator is thioxanthone catechol-O,O'-diacetic acid.

[0042] Preferably, in any of the above items, in step 4, the grafting monomer is polylactide; and what is obtained in step 4 is calcium alginate microspheres with a polylactide layer grafted on the surface.

[0043] Preferably, in any of the above items, in step 4, the concentration of the photoinitiator added to the polylactide solution is 1 g / L.

[0044] Preferably, in any one of the above items, in step 4, the mass fraction of the grafting monomer is 10%.

[0045] Any of the above items is preferably that step 4 is performed as follows: add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction is 1%), stir the reaction at a temperature of 25°C for 20min, centrifuge and wash, and sterile store for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction is 10%). Move into a visible light lamp irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0046] Preferably, in any of the above items, the microcapsules encapsulating the immobilized enzyme nanoparticles and Lactococcus lactis in step 5 are prepared according to the following steps: calcium alginate microspheres with a polylactide layer grafted on the surface are added to a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes.

[0047] Preferably, in any of the above items, in step 6, the culture condition is a rotation speed of 50 to 250 rpm, more preferably 50, 100, 150, 200, 250 rpm and ranges therebetween.

[0048] Preferably, in any of the above items, in step 6, the culture temperature is 20-35°C, more preferably 20, 25, 30, 35°C and ranges therebetween.

[0049] Any of the above items is preferably that in the sixth step, the microcapsules are inoculated into the fermentation medium at a volume ratio of 1-5% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and the culture is carried out for 24 hours under the conditions of an initial pH of 5.0-7.6, a rotation speed of 50-250 rpm and a temperature of 20-35°C, and the potency value of the fermentation liquid is determined.

[0050] Preferably, in any of the above items, the volume ratio of Lactococcus lactis seed solution to fermentation medium is 1, 2, 3, 4 or 5%.

[0051] The fermentation medium consists of 15 g / L potassium dihydrogen phosphate, 15 g / L peptone, 15 g / L yeast extract powder, 42 g / L sucrose, 2 g / L sodium chloride and 2 g / L magnesium sulfate, and the solvent is deionized water.

[0052] In any of the above, preferably, the initial pH of the fermentation medium is 5.0 to 7.6, more preferably 5.0, 5.5, 6.0, 6.5, 7.0, or 7.6.

[0053] Preferably, any of the above items is that the potency value of the fermentation broth is determined by a double-dose agar diffusion method, and the specific steps are as follows: in a sterile environment, a nisin standard solution and a fermentation broth solution are injected into the small holes on the solidified detection medium, and cultured at a temperature of 35°C for 24 hours, and the diameter of the inhibition zone is measured with a vernier caliper, and the potency of the fermentation broth is calculated according to Formula 1.

[0054]

[0055] In the above formula, l sh and l sl The diameters of the inhibition zones when the fermentation liquid to be tested was diluted 300 times and 600 times, respectively. bh and l bl The diameters of the inhibition zones when the standard solution was diluted 300 times and 600 times, respectively. sh and C bh are the titers of the fermentation broth to be tested and the standard solution respectively. k is the ratio of the high dose multiple to the low dose multiple.

[0056] In a preferred embodiment of the present invention, a method for producing nisin by co-immobilization of bacterial enzymes and synergistic fermentation is provided, comprising the following steps:

[0057] In the first step, 2-methylimidazole was used as a ligand to immobilize lactate oxidase to prepare immobilized enzyme nanoparticles;

[0058] In the second step, Lactococcus lactis ATCC11454 was used as the starting bacteria, inoculated onto the slant culture medium, and cultured at 30°C for 18 hours to obtain slant bacteria; the slant bacteria were inoculated into the seed culture medium, cultured at a speed of 150 rpm and a temperature of 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to a certain concentration with PBS phosphate buffer.

[0059] The third step is to add the solution containing Lactococcus lactis, immobilized enzyme nanoparticles and sodium alginate into 2% calcium chloride solution at a certain rate to prepare bacteria-enzyme co-immobilized calcium alginate microspheres.

[0060] The fourth step is to adsorb positively charged polymers on the surface of calcium alginate microspheres. Based on the layer-by-layer self-assembly method, photoinitiators are used to initiate polymerization of grafted monomers under the irradiation of visible light to graft a cross-linked polymer layer on the surface of the calcium alginate microspheres.

[0061] The fifth step is to reduce calcium alginate to sodium alginate to prepare microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0062] Step 6: Inoculate the microcapsules into the fermentation medium, culture for 24 hours under the conditions of an initial pH of 5.0 to 7.6, a rotation speed of 50 to 250 rpm and a temperature of 20 to 35° C., and determine the potency value of the fermentation liquid.

[0063] Preferably, the first step of immobilizing enzyme nanoparticles is prepared as follows: 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate are dissolved in 50mL of deionized water, and then 5mL of lactate oxidase solution is slowly added. A 2-methylimidazole solution with a concentration of 10-50g / L is prepared, and the pH is adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution is slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred for 60 minutes at a speed of 300rpm. The reaction solution is centrifuged, and the collected precipitate is washed and freeze-dried to obtain solid particles, which are manganese-doped zeolite imidazole framework (Mn@ZIF-8) nanoparticles encapsulating lactate oxidase.

[0064] Further preferred steps are:

[0065] The first step is to prepare immobilized enzyme nanoparticles

[0066] Dissolve 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate in 50mL of deionized water, then slowly add 5mL of lactate oxidase solution (titer 200-1000U / mL). Prepare a 2-methylimidazole solution with a concentration of 20-100g / L, and adjust the pH to 8.0 with glacial acetic acid. Slowly add the 2-methylimidazole solution to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stir at 300rpm for 60 minutes. Centrifuge the reaction solution, wash the collected precipitate, and freeze-dry the solid particles to obtain the Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0067] Step 2: Cultivation of Lactococcus lactis

[0068] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0069] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0070] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.01:1 to 0.2:1, and then the immobilized enzyme nanoparticles (concentration of 0.02 to 0.10 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing the lactococcus lactis, the immobilized enzyme nanoparticles and the sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0071] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0072] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction 10%). Move into a visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0073] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0074] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0075] Step 6: Fermentation to produce nisin

[0076] The microcapsules were inoculated into the fermentation medium at a volume ratio of 1-5% of the lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 5.0-7.6, a rotation speed of 50-250 rpm, and a temperature of 20-35° C. After the fermentation was completed, the fermentation liquid was collected and the potency value was determined.

[0077] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0078] (1) The present invention uses Mn@ZIF-8 to immobilize lactate oxidase. Lactate oxidase can oxidize lactic acid secreted by Lactococcus lactis into pyruvic acid, and manganese ions can decompose hydrogen peroxide generated in the reaction process into oxygen and water. Pyruvic acid promotes the growth of Lactococcus lactis, and the generated oxygen can increase the dissolved oxygen level of the system, which alleviates the acidification problem of the fermentation system and is beneficial to improving the biosynthesis efficiency of nisin.

[0079] (2) Under visible light conditions, the enzyme and microorganisms are co-fixed in the microcapsule by layer-by-layer self-assembly method. The operating conditions of photopolymerization are mild and will not affect the enzyme activity and bacterial activity. The polylactide layer can greatly improve the mechanical properties of the microcapsule, and the grafted polymer capsule layer of polylysine and polylactide has semi-permeable membrane properties, allowing nutrients to penetrate inward, thereby achieving the continuous growth of Lactococcus lactis in the microcapsule.

[0080] (3) Immobilizing Lactococcus lactis using microcapsules is beneficial to increasing the concentration of nisin inside the microcapsules, thereby strengthening the quorum sensing between Lactococcus lactis, shortening the start-up cycle of nisin biosynthesis, and improving the biosynthesis efficiency of nisin. In addition, the capsule layer of the microcapsule allows nisin to diffuse outward, thereby avoiding the problem of nisin accumulation inhibiting the growth of Lactococcus lactis.

[0081] (4) The present invention uses bacterial enzyme co-immobilized microcapsules for fermentation production of nisin, which does not require additional carbon source and only requires regular replacement of the culture medium. The microcapsules can be recycled, have low operating costs, do not generate secondary pollution, and are beneficial to the separation and purification of nisin.

[0082] The invention provides a method for producing nisin by bacteria-enzyme co-immobilization synergistic fermentation. After the fermentation is completed, the potency value of the fermentation liquid can reach up to 9800 IU / mL, which is significantly higher than five operation schemes of free enzyme-free bacteria synergistic fermentation, free enzyme-immobilized bacteria synergistic fermentation, immobilized enzyme-free enzyme synergistic fermentation, immobilized enzyme-immobilized bacteria synergistic fermentation and free enzyme-bacteria co-immobilization synergistic fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is the preferred preparation process of bacterial enzyme co-immobilized microcapsules of the present invention.

[0084] Figure 2 This is the titer change curve of the fermentation broth under the conditions of bacterial enzyme co-immobilization fermentation and Lactococcus lactis fermentation under the same inoculation amount in the preferred embodiment 4 of the present invention.

[0085] Figure 3 This is the inhibition zone diagram of the preferred embodiment 1 of the present invention.

[0086] Figure 4 This is the inhibition zone diagram of the preferred embodiment 4 of the present invention.

[0087] Figure 5 This is the inhibition zone diagram of the preferred embodiment 6 of the present invention.

[0088] Figure 6 This is the inhibition zone diagram of the preferred embodiment 8 of the present invention. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0090] like Figure 1 The figure shows the preferred preparation process of bacterial enzyme co-immobilized microcapsules of the present invention. Among them, solution 1 is 2-methylimidazole solution; solution 2 is a mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase; solution 3 is a mixed solution of Lactococcus lactis seed solution, sodium alginate and immobilized enzyme nanoparticles; solution 4 is a 2% calcium chloride solution.

[0091] In a preferred embodiment of the present invention, the titer change curves of the bacterial enzyme co-immobilized fermentation broth are consistent, therefore, Figure 2 In the present invention, the titer change curve of the fermentation broth of the bacterial enzyme co-immobilization fermentation in the preferred embodiment 4 and the fermentation of Lactococcus lactis under the same inoculation amount conditions are randomly selected as examples.

[0092] In the preferred embodiment of the present invention, the antibacterial circles have the same technical effect, so the antibacterial circle diagrams of Examples 1, 4, 6, and 8 are randomly selected and displayed in Figures 3 to 6 Table 1 shows the diameters of the inhibition zones of the fermentation broth samples in various embodiments.

[0093] In the following examples, the potency value of the fermentation broth was determined by a double-dose agar diffusion method, and the specific steps were as follows: in a sterile environment, a nisin standard solution and a fermentation broth solution were injected into the small holes on the solidified detection medium, and cultured at a temperature of 35° C. for 24 hours. The diameter of the inhibition zone was measured with a vernier caliper, and the potency of the fermentation broth was calculated according to Formula 1.

[0094]

[0095] In the above formula, l sh and l sl The diameters of the inhibition zones when the fermentation liquid to be tested was diluted 300 times and 600 times, respectively. bh and l bl The diameters of the inhibition zones when the standard solution was diluted 300 times and 600 times, respectively. sh and C bh are the titers of the fermentation broth to be tested and the standard solution respectively. k is the ratio of the high dose multiple to the low dose multiple.

[0096] Example 1

[0097] The first step is to prepare immobilized enzyme nanoparticles

[0098] 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate were dissolved in 50mL of deionized water, followed by the slow addition of 5mL of lactate oxidase solution (titer 200U / mL). A 2-methylimidazole solution with a concentration of 20g / L was prepared, and the pH was adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution was slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred at a speed of 300rpm for 60 minutes. The reaction solution was centrifuged, and the collected precipitate was washed and freeze-dried to obtain the solid particles, which were Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0099] Step 2: Cultivation of Lactococcus lactis

[0100] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0101] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0102] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.01:1, and then the immobilized enzyme nanoparticles (concentration of 0.02 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing the lactococcus lactis, the immobilized enzyme nanoparticles and the sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0103] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0104] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction is 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,O′-diacetic acid to 50mL of polylactide solution (mass fraction is 10%). Move into the visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0105] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0106] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0107] Step 6: Fermentation to produce nisin

[0108] The microcapsules were inoculated into the fermentation medium at a volume ratio of 1% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm, and a temperature of 35°C. After the fermentation was completed, the fermentation broth was collected, and the potency value was 2600-3200 IU / mL. The inhibition zone in Example 1 is as follows Figure 3 shown.

[0109] Example 2

[0110] The first step is to prepare immobilized enzyme nanoparticles

[0111] 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate were dissolved in 50mL of deionized water, followed by the slow addition of 5mL of lactate oxidase solution (titer 400U / mL). A 2-methylimidazole solution with a concentration of 40g / L was prepared, and the pH was adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution was slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred at a speed of 300rpm for 60 minutes. The reaction solution was centrifuged, and the collected precipitate was washed and freeze-dried to obtain the solid particles, which were Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0112] Step 2: Cultivation of Lactococcus lactis

[0113] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0114] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0115] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.05:1, and then the immobilized enzyme nanoparticles (concentration of 0.04 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing lactococcus lactis, immobilized enzyme nanoparticles and sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0116] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0117] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction 10%). Move into a visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0118] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0119] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0120] Step 6: Fermentation to produce nisin

[0121] The microcapsules were inoculated into the fermentation medium at a volume ratio of 2% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm, and a temperature of 35° C. After the fermentation was completed, the fermentation broth was collected, and the potency value was 3300-5700 IU / mL.

[0122] Example 3

[0123] The first step is to prepare immobilized enzyme nanoparticles

[0124] 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate were dissolved in 50mL of deionized water, followed by the slow addition of 5mL of lactate oxidase solution (titer 600U / mL). A 2-methylimidazole solution with a concentration of 60g / L was prepared, and the pH was adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution was slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred at a speed of 300rpm for 60 minutes. The reaction solution was centrifuged, and the collected precipitate was washed and freeze-dried to obtain the solid particles, which were Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0125] Step 2: Cultivation of Lactococcus lactis

[0126] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0127] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0128] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.1:1, and then the immobilized enzyme nanoparticles (concentration of 0.06 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing lactococcus lactis, immobilized enzyme nanoparticles and sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0129] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0130] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction 10%). Move into a visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0131] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0132] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0133] Step 6: Fermentation to produce nisin

[0134] The microcapsules were inoculated into the fermentation medium at a volume ratio of 3% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm, and a temperature of 35° C. After the fermentation was completed, the fermentation broth was collected, and the potency value was 5800-7800 IU / mL.

[0135] Example 4

[0136] The first step is to prepare immobilized enzyme nanoparticles

[0137] 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate were dissolved in 50mL of deionized water, and then 5mL of lactate oxidase solution (titer 800U / mL) was slowly added. A 2-methylimidazole solution with a concentration of 80g / L was prepared, and the pH was adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution was slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred for 60 minutes at a speed of 300rpm. The reaction solution was centrifuged, and the collected precipitate was washed and freeze-dried to obtain the solid particles, which were Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0138] Step 2: Cultivation of Lactococcus lactis

[0139] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0140] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0141] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.15:1, and then the immobilized enzyme nanoparticles (concentration of 0.08 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing the lactococcus lactis, the immobilized enzyme nanoparticles and the sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0142] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0143] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction 10%). Move into a visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0144] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0145] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0146] Step 6: Fermentation to produce nisin

[0147] The microcapsules were inoculated into the fermentation medium at a volume ratio of 4% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm, and a temperature of 35°C. The titer change curve of the fermentation broth under the conditions of bacterial enzyme co-immobilization fermentation and Lactococcus lactis fermentation with the same inoculation amount is shown in Figure 2. Figure 2 After the fermentation was completed, the fermentation broth was collected and the potency was 7900-8800 IU / mL. Figure 4 shown.

[0148] Example 5

[0149] The first step is to prepare immobilized enzyme nanoparticles

[0150] 1.88g of zinc nitrate hexahydrate and 0.07g of manganese nitrate tetrahydrate were dissolved in 50mL of deionized water, followed by the slow addition of 5mL of lactate oxidase solution (titer 1000U / mL). A 2-methylimidazole solution with a concentration of 100g / L was prepared, and the pH was adjusted to 8.0 with glacial acetic acid. The 2-methylimidazole solution was slowly added to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stirred at a speed of 300rpm for 60 minutes. The reaction solution was centrifuged, and the collected precipitate was washed and freeze-dried to obtain the solid particles, which were Mn@ZIF-8 nanoparticles encapsulating lactate oxidase.

[0151] Step 2: Cultivation of Lactococcus lactis

[0152] Lactococcus lactis ATCC11454 was inoculated on a slant culture medium and cultured at 30°C for 18 hours to obtain slant cells; the slant cells were inoculated into a seed culture medium and cultured at 150 rpm and 30°C for 18 hours, the culture solution was centrifuged, the bacterial cells were collected, and the bacterial cells were diluted to 3-6×10 with PBS phosphate buffer. 9 cfu / mL.

[0153] Step 3: Preparation of bacterial enzyme co-immobilized calcium alginate microspheres

[0154] The lactococcus lactis seed solution and the sodium alginate solution were mixed at a volume ratio of 0.2:1, and then the immobilized enzyme nanoparticles (concentration of 0.10 g / L) were added. After thorough mixing, a disposable 5 mL syringe was used to drop the solution containing lactococcus lactis, immobilized enzyme nanoparticles and sodium alginate into a 2% calcium chloride solution at a speed of 5 mL / min at a temperature of 34°C to obtain bacterial enzyme co-immobilized calcium alginate microspheres.

[0155] Step 4: Preparation of calcium alginate microspheres with a polylactide layer grafted on the surface

[0156] Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres to 50mL of polylysine solution (mass fraction 1%), stir and react at 25℃ for 20min, centrifuge and wash, and store aseptically for later use. Add 0.5g of bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with polylysine and 0.05g of thioxanthone catechol-O,Oˊ-diacetic acid to 50mL of polylactide solution (mass fraction 10%). Move into a visible light irradiation device (wavelength 420nm, light intensity 8000μW / cm -2 ) for 2 hours to obtain calcium alginate microspheres with a polylactide layer grafted on the surface.

[0157] Step 5: Preparation of microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis

[0158] Calcium alginate microspheres with a polylactide layer grafted on the surface were added into a 5% sodium citrate solution (pH 5.5) and liquefied for 15 minutes to obtain microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis.

[0159] Step 6: Fermentation to produce nisin

[0160] The microcapsules were inoculated into the fermentation medium at a volume ratio of 5% of the Lactococcus lactis seed solution to the fermentation medium in the fourth step, and cultured for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm, and a temperature of 35° C. After the fermentation was completed, the fermentation broth was collected, and the potency value was 8900-9800 IU / mL.

[0161] Example 6

[0162] Example 6 is similar to Example 4, except that the fermentation to produce nisin was carried out under the conditions of an initial pH of 5.0, a rotation speed of 150 rpm and a temperature of 35°C for 24 hours. After the fermentation was completed, the fermentation broth was collected and the titer was 3200 to 4900 IU / mL. Figure 5 shown.

[0163] Example 7

[0164] Example 7 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 5.5, a rotation speed of 150 rpm and a temperature of 35° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 5000-5900 IU / mL.

[0165] Example 8

[0166] Example 8 is similar to Example 4, except that the fermentation to produce nisin was carried out under the conditions of an initial pH of 6.0, a rotation speed of 150 rpm and a temperature of 35°C for 24 hours. After the fermentation was completed, the fermentation broth was collected and the titer was 6000 to 6900 IU / mL. Figure 6 shown.

[0167] Example 9

[0168] Example 9 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 6.6, a rotation speed of 150 rpm and a temperature of 35° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 7000-7900 IU / mL.

[0169] Example 10

[0170] Example 10 is similar to Example 4, except that the fermentation to produce nisin is carried out for 24 hours under the conditions of an initial pH of 7.0, a rotation speed of 150 rpm and a temperature of 35° C. After the fermentation is completed, the fermentation broth is collected, and the technical effect is equivalent to the initial pH of 7.6.

[0171] Embodiment 11

[0172] Example 11 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 7.6, a rotation speed of 50 rpm and a temperature of 35° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 4000-6400 IU / mL.

[0173] Example 12

[0174] Example 12 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 7.6, a rotation speed of 100 rpm and a temperature of 35° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 6500-7800 IU / mL.

[0175] Embodiment 13

[0176] Example 13 is similar to Example 4, except that the fermentation to produce nisin is carried out for 24 hours under the conditions of an initial pH of 7.6, a rotation speed of 200 rpm and a temperature of 35° C. After the fermentation is completed, the fermentation broth is collected, and the technical effect is equivalent to the value of the rotation speed of 100 rpm.

[0177] Embodiment 14

[0178] Example 14 is similar to Example 4, except that the fermentation to produce nisin is carried out for 24 hours at an initial pH of 7.6, a rotation speed of 250 rpm and a temperature of 35° C. After the fermentation is completed, the fermentation broth is collected, and the technical effect is equivalent to the value at a rotation speed of 50 rpm.

[0179] Embodiment 15

[0180] Example 15 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm and a temperature of 20° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 2000-3900 IU / mL.

[0181] Example 16

[0182] Example 16 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm and a temperature of 25° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 4000-5900 IU / mL.

[0183] Embodiment 17

[0184] Example 17 is similar to Example 4, except that the fermentation to produce nisin is carried out under the conditions of an initial pH of 7.6, a rotation speed of 150 rpm and a temperature of 30° C. for 24 hours. After the fermentation is completed, the fermentation broth is collected, and the titer is 6000-7800 IU / mL.

[0185] Table 1 Diameters of inhibition zones of fermentation broth samples in various embodiments

[0186]

[0187] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for producing nisin by co-immobilization of bacterial enzymes and synergistic fermentation, comprising the following steps: Step 1: Preparation of immobilized enzyme nanoparticles: Dissolve zinc nitrate hexahydrate and manganese nitrate tetrahydrate in deionized water, and then add lactate oxidase solution; Add 2-methylimidazole solution with a pH of 8.0 to the mixed solution of zinc nitrate hexahydrate, manganese nitrate tetrahydrate and lactate oxidase, and stir to react; After the reaction, the collected precipitate is washed, frozen, and dried to obtain solid particles, which are Mn@ZIF-8 nanoparticles encapsulating lactate oxidase; Step 2: Cultivation of Lactococcus lactis: Inoculate and culture Lactococcus lactis to obtain a bacterial cell concentration of 3 to 6×10 9 cfu / mL of Lactococcus lactis seed solution; Step 3: preparing bacterial enzyme co-immobilized calcium alginate microspheres: mixing the Lactococcus lactis seed solution obtained in step 2 into the sodium alginate solution, and then adding the Mn@ZIF-8 nanoparticles encapsulating lactate oxidase obtained in step 1, and after fully mixing, dripping the mixed solution into the calcium chloride solution to prepare bacterial enzyme co-immobilized calcium alginate microspheres; Step 4: Preparation of surface-grafted calcium alginate microspheres: Add the bacterial enzyme co-immobilized calcium alginate microspheres obtained in step 3 to the positively charged polymer solution, stir at 25°C, and centrifuge to obtain bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with the positively charged polymer; Add the bacterial enzyme co-immobilized calcium alginate microspheres adsorbed with the positively charged polymer and a photoinitiator to the grafted monomer solution; Under nitrogen conditions, at a wavelength of 420nm and a light intensity of 8000μW / cm -2 irradiated under visible light irradiation conditions to obtain surface-grafted calcium alginate microspheres; Step 5: preparing microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis: adding the surface-grafted calcium alginate microspheres obtained in step 5 into a sodium citrate solution at pH 5.5 for liquefaction to prepare microcapsules encapsulating immobilized enzyme nanoparticles and Lactococcus lactis; Step 6: Fermentation to produce nisin: The microcapsules obtained in step 5 are inoculated into a fermentation medium for fermentation culture.

2. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 1, characterized in that: In step 1, the titer of the lactate oxidase solution is 200 to 1000 U / mL.

3. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 2, characterized in that: In step 1, the concentration of the 2-methylimidazole solution is 10-100 g / L.

4. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 3, characterized in that: In step 1, glacial acetic acid is used to adjust the pH of the 2-methylimidazole solution; 1.88 g of zinc nitrate hexahydrate and 0.07 g of manganese nitrate tetrahydrate are dissolved in every 50 mL of deionized water, and the corresponding amount of lactate oxidase solution added is 5 mL; the stirring reaction condition is stirring at a speed of 300 rpm for 60 minutes.

5. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 4, characterized in that: In step 2, the starting strain of Lactococcus lactis is at least one of L. lactis ATCC11454, L. lactis SM526, L. lactis F44, L. lactis N8, L. lactis WNC20, L. lactis CHCC5826 or L. lactis ATCC6242.

6. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 1, characterized in that: In step 3, the volume ratio of the Lactococcus lactis seed solution to the sodium alginate solution is 0.01:1-0.2:1; the concentration of the Mn@ZIF-8 nanoparticles encapsulating lactate oxidase is 0.02-0.10 g / L; and the concentration of the sodium alginate solution is 5-20 g / L.

7. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 6, characterized in that: In step 3, a mixed solution containing Lactococcus lactis, Mn@ZIF-8 nanoparticles encapsulating lactate oxidase and sodium alginate is added dropwise to a 2% calcium chloride solution at a rate of 3 to 10 mL / min at a temperature of 34° C. to prepare bacterial enzyme co-immobilized calcium alginate microspheres.

8. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 1, characterized in that: In step 4, the positively charged polymer is one of polyethyleneimine, polyacrylamine hydrochloride or polylysine; the mass fraction of the positively charged polymer is 1%; the photoinitiator is a thioxanthone derivative; and the grafting monomer is one of polyurethane acrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate or polylactide.

9. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation as claimed in claim 8, characterized in that: The photoinitiator is thioxanthone catechol-O,Oˊ-diacetic acid; the grafted monomer is polylactide; the concentration of the photoinitiator added to the polylactide solution is 1g / L; the mass fraction of the grafted monomer is 10%; step 4 obtains calcium alginate microspheres with a polylactide layer grafted on the surface.

10. The method for producing nisin by bacterial enzyme co-immobilization and synergistic fermentation according to claim 1, characterized in that: In step 6, the initial pH of the fermentation medium is 5.0-7.6; the culture conditions are a rotation speed of 50-250 rpm and a temperature of 20-35°C.

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