A method for extracting high-purity nisin

Abstract

The present invention relates to the field of bioengineering technology, and specifically discloses a method for extracting high-purity nisin, the extraction method comprising: after activating nisin with an activation medium, transferring the activated strain into a seed medium for cultivation to obtain a seed liquid, inoculating the seed liquid into a fermentation medium, and obtaining a nisin fermentation liquid after the cultivation is completed; filtering the nisin fermentation liquid, adding a macroporous adsorption resin for static adsorption under stirring conditions, and washing with purified water after complete adsorption to obtain a saturated adsorption resin; pouring the saturated adsorption resin into a resin column, adding a desorbent for dynamic desorption to obtain a desorbent; adding sodium chloride to the desorbent for salting out, layering, filtering, rotary evaporation, and low-temperature drying to obtain the obtained product. The fermentation liquid Nisin obtained by the present invention has a high yield, is thoroughly desorbed by a composite desorbent, has a high total extraction yield of Nisin, has high purity, high color whiteness, and low variability.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and more specifically to a method for extracting high-purity nisin. Background Art

[0002] Nisin, also known as nisin or transliterated as Nisin, is a polypeptide antibiotic extracted from the fermentation product of Streptococcus lactis. After consumption, it is decomposed by enzymes in the human body to produce amino acids. It will not change the normal flora in the human intestine and produce resistance problems such as other antibiotics. It is a world-recognized safe natural biological food preservative and antibacterial agent. Nisin was discovered in 1928, earlier than penicillin. It was originally discovered due to the failure of culturing Bulgarian lactobacillus with milk to make (coagulated) cheese. People found that the impurities in milk lactococci are a protein inhibitor, named Nisin to be consistent with its serogroup Group N Inhibitory Substance. Nisin consists of 34 amino acid residues and has a molecular weight of about 3500 Da. Nisin can inhibit most Gram-positive bacteria and has a strong inhibitory effect on Bacillus spores. It cross-links two alanine residues through thioether bonds, and its active molecules are often dimers or tetramers. After decades of research, many types of nisin have been identified, among which nisin A and nisin Z are the most actively studied. The only difference between the two is the type of amino acid at position 27 in the amino acid sequence. Nisin is generally made into salt preparations of different potency, which can effectively inhibit food spoilage caused by Clostridium botulinum, Staphylococcus aureus, hemolytic Streptococcus, Bacillus subtilis, Bacillus stearothermophilus, etc. High-purity nisin has potential uses in the medical field, especially in the treatment of atopic dermatitis, gastric ulcers and immunodeficiency caused by colitis infection, as well as in the treatment of peptic gastric diseases. Nisin can successfully inhibit Helicobacter pylori in vitro.

[0003] Although nisin has broad potential in food preservation and medical applications, its existing extraction methods still face many challenges. The purity of nisin obtained by traditional fermentation broth extraction methods is generally low, and the color is unstable, which makes it difficult to ensure the consistency and quality of products on the market. In addition, traditional methods usually require the use of a large amount of fermentation broth, resulting in a waste of resources and further increasing production costs. Based on the above statements, the present invention proposes a method for extracting high-purity nisin. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for extracting high-purity nisin. The present invention uses nisin as a production bacterium, activates the production bacterium, and performs seed culture, and then inoculates the production bacterium into a fermentation medium for fermentation to obtain a nisin fermentation broth; through the steps of adsorption by a macroporous adsorption resin, desorption by a composite desorbent, and salting out by adding sodium chloride twice, nisin can be efficiently extracted to obtain a high-purity product, and the use amount of the fermentation broth can be reduced, and the waste of resources can be reduced. The Nisin finally obtained has a high total extraction yield, high purity, high color whiteness and low variability.

[0005] The present invention provides a method for extracting high-purity nisin, which adopts the following technical scheme:

[0006] A method for extracting high-purity nisin comprises the following steps:

[0007] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed liquid, the seed liquid is inoculated into a fermentation medium, and after the cultivation is completed, a nisin fermentation liquid is obtained;

[0008] S2, after filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0-7.0, the temperature to 20-40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain a saturated adsorption resin;

[0009] S3, pouring the saturated adsorption resin into the resin column, adding a desorbent for dynamic desorption, controlling the desorption flow rate to 1-2BV / h, and obtaining a desorption liquid;

[0010] S4. The pH value of the desorption liquid is adjusted to 2.0-3.0, and sodium chloride is added for salting out. The salting out liquid is filtered, and then rotary evaporated and low-temperature dried to obtain high-purity nisin.

[0011] Preferably, the formula of the activation culture medium in step S1 is: peptone 8-12 g / L, beef extract 8-12 g / L, lysine 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, ferrous sulfate heptahydrate 0.3-0.5 g / L, sodium chloride 5-7 g / L, lactose 20-25 g / L, agar 15-18 g / L, pH 6.4-6.8.

[0012] Preferably, the formula of the seed culture medium in step S1 is: peptone 8-12 g / L, beef extract 8-12 g / L, yeast extract 5-8 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 5-8 g / L, glucose 20-25 g / L, Tween 80 1-2 mL / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, manganese sulfate tetrahydrate 0.2-0.4 g / L, sodium chloride 10-12 g / L, pH 6.4-6.8.

[0013] Preferably, the formula of the fermentation medium in step S1 is: adding 20-25 g / L of complex nutrients to the seed culture medium.

[0014] Preferably, the activation conditions of the activation medium in step S1 are: static culture at a temperature of 30-37° C. for 20-24 hours.

[0015] Preferably, the culture conditions in the seed culture medium in step S1 are: culture on a shaker at a temperature of 30-37° C. and a rotation speed of 150-180 r / min for 20-24 h.

[0016] Preferably, the culture conditions in the fermentation medium in step S1 are: culture on a shaker at a temperature of 30-37° C. and a rotation speed of 150-180 r / min for 20-24 h.

[0017] Preferably, the composite nutrient is prepared by the following method:

[0018] (1) Pre-drying the microphylla seaweed and the masson pine needles, mixing and grinding them in a mass ratio of 2-4:1, and passing through a 40-60 mesh sieve to obtain a mixed powder;

[0019] (2) Add 50-60°C deionized water to the mixed powder, stir evenly, and soak for 8-10 hours. Heat to boil and extract for 0.5-1 hour, then cool to 30-40°C to obtain the extract. Add the composite bacterial agent, adjust the pH value to 4.5-5.5, seal and ferment for 30-40 hours, then centrifuge and dry to obtain the composite nutrient.

[0020] Preferably, the composite bacterial agent in step (2) comprises Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus oryzae in a live cell count ratio of 5-7:2-3:1.

[0021] Preferably, the viable bacterial count of the composite bacterial agent in the extract of step (2) is 1.8-2.3×10 9 CFU / mL.

[0022] Preferably, the filtration treatment in step S2 refers to: filtering the nisin fermentation broth through a ceramic membrane with a pore size of 0.05-0.1 μm to remove impurities.

[0023] Preferably, the desorbent in step S3 is an acid-organic compound system.

[0024] Preferably, the acid refers to a hydrochloric acid solution with a molar concentration of 1-1.5 mol / L.

[0025] Preferably, the organic compound is ethanol and / or n-butanol.

[0026] Preferably, the desorbent further comprises an ionic liquid.

[0027] Preferably, the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.

[0028] Preferably, the volume ratio of the acid, the organic compound and the ionic liquid is 1:1-2:0.1-0.3.

[0029] Preferably, the salting-out treatment in step S4 specifically refers to: controlling the mass ratio of the desorption liquid after pH adjustment and sodium chloride to be 100:3-6, adding sodium chloride to the desorption liquid after pH adjustment and stirring evenly, standing to separate layers, and extracting the supernatant; controlling the mass ratio of the supernatant and sodium chloride to be 100:8-12, continuing to add sodium chloride to the supernatant and stirring evenly, standing to separate layers, and the upper layer is the salting-out liquid containing nisin.

[0030] In summary, the present invention has the following beneficial effects:

[0031] The invention uses lactic acid streptococcus as a production bacterium, activates the production bacterium, performs seed culture, and then inoculates it into a fermentation medium for fermentation to obtain a nisin fermentation liquid; by adding a composite nutrient rich in nutrients such as protein, polysaccharide, vitamin, inorganic element and trace element into the seed culture medium as the fermentation medium, the lactic acid streptococcus can be provided with all the nutrients required for growth and metabolism, the proliferation and metabolic activities of the lactic acid streptococcus can be promoted, the biosynthesis ability of the bacteria can be enhanced, the synthesis of nisin can be promoted, thereby increasing the yield and activity of Nisin and improving the production efficiency.

[0032] The composite nutrient of the invention uses seaweed and pine needles as base materials, and can effectively release the nutrient components in the base materials through warm water soaking and boiling extraction. The obtained extract is added with a composite bacterial agent obtained by compounding lactobacillus plantarum, brewer's yeast and aspergillus oryzae for fermentation, wherein the lactobacillus plantarum can effectively promote the release of nutrient components, ferment sugars, produce lactic acid, reduce the pH of the fermentation liquid, thereby inhibiting the growth of harmful microorganisms and improving the safety of fermentation; the brewer's yeast is rich in vitamin B group, amino acids and minerals, and as an excellent nutrient source, can promote the growth of other microorganisms; the aspergillus oryzae can effectively decompose complex carbohydrates in the base material, release available sugar sources, produce multiple enzymes such as amylase and cellulase, and further improve the utilization rate of the substrate; the metabolites of the three bacterial agents promote each other, form a good metabolic network, and improve the fermentation effect; the obtained composite nutrient has comprehensive nutrition, can promote the rapid growth and reproduction of lactic acid streptococci, improve the efficiency of cultivation, and then improve the metabolic activity of lactic acid streptococci, and improve the yield and activity of Nisin.

[0033] The invention can selectively adsorb nisin efficiently by filtering and concentrating the fermentation liquid, remove impurities and other small molecules in the fermentation liquid, achieve purification and decolorization, and improve the concentration and purity of the target product.

[0034] The present invention utilizes a composite desorbent obtained by compounding an acid-organic compound-ionic liquid for desorption, which can promote the release of target molecules under different desorption mechanisms and achieve thorough desorption. The presence of a small amount of ionic liquid in the composite desorbent can adjust the physical properties of the desorbent, improve the solubility of the target molecule, make it easier to desorb, and thus optimize the desorption process.

[0035] The present invention adopts two salting-out treatments to gradually adjust the salt concentration, selectively precipitate the target product, reduce the interference of other non-specific proteins, remove impurities in the first salting-out, and further improve the purity of the target product in the second salting-out, thereby achieving higher concentration of the target product, which is conducive to improving the yield.

[0036] The invention can efficiently extract nisin through the steps of adsorption by macroporous adsorption resin, desorption by composite desorbent, salting out by adding sodium chloride twice and the like, obtain a high-purity product, reduce the usage of fermentation liquid, and reduce resource waste. The Nisin finally obtained has high total extraction yield, high purity, high color whiteness and low variability. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below with reference to the embodiments.

[0038] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0039] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0040] Among them, the formula of the activated culture medium is: peptone 10g / L, beef extract 10g / L, lysine 2g / L, dipotassium hydrogen phosphate 2g / L, ferrous sulfate heptahydrate 0.4g / L, sodium chloride 6g / L, lactose 22g / L, agar 16g / L, pH 6.5.

[0041] The formula of the seed culture medium is: peptone 10g / L, beef extract 10g / L, yeast extract 7g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 6g / L, glucose 22g / L, Tween 80 1.5mL / L, magnesium sulfate heptahydrate 0.5g / L, manganese sulfate tetrahydrate 0.3g / L, sodium chloride 10g / L, pH 6.7.

[0042] The lactic acid streptococcus is lactic acid streptococcus ACCC10653, brand: American ATCC.

[0043] Microphylla seaweed extract and pine needle extract (i.e., Masson pine water-soluble powder) are both provided by Shanyang Lianfeng Biotechnology Co., Ltd.

[0044] Lactobacillus plantarum (Cat. No. BKJ3247), Saccharomyces cerevisiae (Cat. No. BKJ3679), and Aspergillus oryzae (Cat. No. BKJ3728) were all provided by Shanghai Bo Ke Biotechnology Co., Ltd.

[0045] The macroporous adsorption resin is Tulsimer ® ADS-750, provided by Kehaisi (Beijing) Technology Co., Ltd. The pretreatment operation of the macroporous adsorption resin is as follows: the macroporous adsorption resin is fully immersed in ethanol at a volume ratio of 1:3, and stirred at a speed of 100r / min for 10min every 1h. After immersion for 24h to allow the resin to fully swell, ethanol is passed through the resin layer at a flow rate of 4BV / h. After the effluent is diluted with water and becomes no longer turbid, deionized water is passed through the resin layer at a flow rate of 6BV / h to displace the ethanol to obtain the pretreated macroporous adsorption resin.

[0046] The method for determining the bioavailability in the embodiments of the present invention refers to GB 1886.231-2016 "National Food Safety Standard Food Additive Nisin".

[0047] Preparation Examples 1-3 and Comparative Preparation Examples 1-5 provide methods for preparing complex nutrients.

[0048] Preparation Example 1

[0049] The compound nutrients are prepared by the following method:

[0050] (1) Pre-drying the microphylla seaweed and the masson pine needles at 50° C. for 5 h, mixing and grinding them in a mass ratio of 2:1, and passing through a 40-mesh sieve to obtain a mixed powder;

[0051] (2) According to the material-liquid mass ratio of 1:12, the mixed powder was added with 50°C deionized water, stirred evenly, and then soaked for 10 hours. After heating and boiling for 0.5 hours, the temperature was cooled to 30°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus oryzae with a live bacterial count ratio of 5:2:1) was added until the live bacterial count in the extract reached 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0052] Preparation Example 2

[0053] The compound nutrients are prepared by the following method:

[0054] (1) Pre-drying the microphylla seaweed and the masson pine needles at 60° C. for 4 h, mixing and grinding them in a mass ratio of 3:1, and passing through a 50-mesh sieve to obtain a mixed powder;

[0055] (2) According to the material-liquid mass ratio of 1:13.5, the mixed powder was added with 55°C deionized water, stirred evenly, and then soaked for 9 hours. After heating and boiling for 0.8 hours, the temperature was cooled to 35°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus oryzae with a live bacterial count ratio of 6:2.5:1) was added until the live bacterial count in the extract reached 2.0×10 9 CFU / mL, adjust the pH value to 5.0, seal and ferment for 35 hours, centrifuge at 9000r / min for 12 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0056] Preparation Example 3

[0057] The compound nutrients are prepared by the following method:

[0058] (1) Pre-drying the microphylla seaweed and the masson pine needles at 70° C. for 3 h, mixing and grinding them in a mass ratio of 4:1, and passing through a 60-mesh sieve to obtain a mixed powder;

[0059] (2) According to the material-liquid mass ratio of 1:15, the mixed powder was added with 60°C deionized water, stirred evenly, and then soaked for 8 hours. After heating and boiling for 1 hour, the temperature was cooled to 40°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus oryzae with a live bacterial count ratio of 7:3:1) was added until the live bacterial count in the extract reached 2.3×10 9CFU / mL, adjust the pH value to 5.5, seal and ferment for 40 hours, centrifuge at 10000r / min for 8 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0060] Comparative Preparation Example 1

[0061] The compound nutrients are prepared by the following method:

[0062] (1) Pre-drying the microleaf seaweed at 50°C for 5 hours, crushing the seaweed, and passing through a 40-mesh sieve to obtain seaweed powder;

[0063] (2) According to the material-liquid mass ratio of 1:12, add seaweed powder to 50°C deionized water, stir evenly, and soak for 10 hours. Heat to boil and extract for 0.5 hours, then cool to 30°C to obtain an extract. Add a composite bacterial agent (including Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae with a live bacterial count ratio of 5:2:1) until the live bacterial count in the extract is 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0064] Comparative Preparation Example 2

[0065] The compound nutrients are prepared by the following method:

[0066] (1) Pre-drying the pine needles of Masson pine at 50°C for 5 hours, then crushing them and passing them through a 40-mesh sieve to obtain pine needle powder;

[0067] (2) According to the material-liquid mass ratio of 1:12, pine needle powder was added to 50°C deionized water, stirred evenly, and then soaked for 10 hours. After heating and boiling for 0.5 hours, the temperature was cooled to 30°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae with a live bacterial count ratio of 5:2:1) was added until the live bacterial count in the extract reached 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0068] Comparative Preparation Example 3

[0069] The compound nutrients are prepared by the following method:

[0070] (1) Pre-drying the microphylla seaweed and the masson pine needles at 50° C. for 5 h, mixing and grinding them in a mass ratio of 2:1, and passing through a 40-mesh sieve to obtain a mixed powder;

[0071] (2) According to the material-liquid mass ratio of 1:12, the mixed powder was added with 50°C deionized water, stirred evenly, and then soaked for 10 hours. After heating and boiling for 0.5 hours, the temperature was cooled to 30°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum and Saccharomyces cerevisiae with a live bacterial count ratio of 5:2) was added until the live bacterial count in the extract reached 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0072] Comparative Preparation Example 4

[0073] The compound nutrients are prepared by the following method:

[0074] (1) Pre-drying the microphylla seaweed and the masson pine needles at 50° C. for 5 h, mixing and grinding them in a mass ratio of 2:1, and passing through a 40-mesh sieve to obtain a mixed powder;

[0075] (2) According to the material-liquid mass ratio of 1:12, the mixed powder was added with 50°C deionized water, stirred evenly, and then soaked for 10 hours. After heating and boiling for 0.5 hours, the temperature was cooled to 30°C and kept warm to obtain an extract. A composite bacterial agent (including Lactobacillus plantarum and Aspergillus oryzae with a live bacterial count ratio of 5:1) was added until the live bacterial count in the extract reached 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0076] Comparative Preparation Example 5

[0077] The compound nutrients are prepared by the following method:

[0078] (1) Pre-drying the microphylla seaweed and the masson pine needles at 50° C. for 5 h, mixing and grinding them in a mass ratio of 2:1, and passing through a 40-mesh sieve to obtain a mixed powder;

[0079] (2) According to the material-liquid mass ratio of 1:12, the mixed powder was added with 50°C deionized water, stirred evenly, and then soaked for 10 hours. After heating and boiling for 0.5 hours, the temperature was cooled to 30°C and kept warm to obtain an extract. A composite bacterial agent (including Saccharomyces cerevisiae and Aspergillus oryzae with a live bacterial count ratio of 2:1) was added until the live bacterial count in the extract reached 1.8×10 9 CFU / mL, adjust the pH value to 4.5, seal and ferment for 30 hours, centrifuge at 8000r / min for 15 minutes, take the centrifuge and freeze-dry at -20℃ to obtain the composite nutrient.

[0080] Embodiment 1-4 provides a method for extracting high-purity nisin.

[0081] Example 1

[0082] A method for extracting high-purity nisin comprises the following steps:

[0083] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0084] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0085] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0086] S13, adding 20 g / L of the composite nutrient in Preparation Example 1 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30° C. and a speed of 180 r / min for 24 h. After the culturing, a nisin fermentation liquid with a biological titer of 18970 IU / ml was obtained;

[0087] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0088] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0089] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0090] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0091] S31, controlling the volume ratio of hydrochloric acid solution, ethanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to be 1:1:0.1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L, ethanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to prepare a desorbent;

[0092] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0093] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0094] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. The salting-out liquid was filtered and placed in a rotary evaporator for rotary evaporation. The rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 316g of nisin milky white powder, and the biological potency test result was 39986IU / mg.

[0095] Table 1 Comprehensive performance indexes of nisin obtained in Example 1

[0096]

[0097] Example 2

[0098] A method for extracting high-purity nisin comprises the following steps:

[0099] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0100] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 34° C. for 22 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0101] S12, transfer the activated strain into the seed culture medium at an inoculum size of 2%, and culture on a shaker at 34°C and 160 rpm for 22 h to obtain a seed solution;

[0102] S13, adding 22 g / L of the composite nutrient in Preparation Example 2 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 6%, and culturing the culture medium on a shaker at a temperature of 34° C. and a speed of 160 r / min for 22 h. After the culturing, a nisin fermentation liquid with a biological titer of 20835 IU / ml was obtained;

[0103] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 5.0, the temperature to 30°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0104] S21, taking 40 L of nisin fermentation liquid, heating it to 80° C., filtering it through a ceramic membrane with a pore size of 0.1 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0105] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 25% of the total volume of the nisin concentrated solution, adjusting the pH to 5.0 with a 1.1 mol / L hydrochloric acid solution, controlling the stirring speed to 110 r / min at a temperature of 30° C., stirring and adsorbing for 2.5 hours, adding 8% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0106] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0107] S31, controlling the volume ratio of hydrochloric acid solution, n-butanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to be 1:1.5:0.2, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.2 mol / L, n-butanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to prepare a desorbent;

[0108] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3.5:1, the desorption flow rate to be 1BV / h, adding the desorbent for dynamic desorption, taking an instantaneous sample for detection during the desorption process, and obtaining a desorption liquid after the desorption is completed;

[0109] S4. The pH of the desorption liquid is adjusted to 2.5, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0110] The pH of the desorption liquid was adjusted to 2.5, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:5. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 4 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:10. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 4 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 35°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 35°C, dried to constant weight, and then crushed to obtain 329g of nisin milky white powder, and the biological potency test result was 39245IU / mg.

[0111] Table 2 Comprehensive performance indexes of nisin obtained in Example 2

[0112]

[0113] Example 3

[0114] A method for extracting high-purity nisin comprises the following steps:

[0115] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0116] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 37° C. for 20 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0117] S12, transfer the activated strain into the seed culture medium at an inoculum rate of 3%, and culture it on a shaker at 37°C and 150 r / min for 20 h to obtain a seed solution;

[0118] S13, adding 25 g / L of the composite nutrient in Preparation Example 3 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 8%, and culturing the culture medium on a shaker at a temperature of 37° C. and a speed of 150 r / min for 20 h. After the culturing, a nisin fermentation liquid with a biological titer of 22438 IU / ml was obtained;

[0119] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 7.0, the temperature to 20°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain a saturated adsorption resin. The specific operation is as follows:

[0120] S21, taking 40 L of nisin fermentation liquid, heating it to 80° C., filtering it through a ceramic membrane with a pore size of 0.1 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0121] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 30% of the total volume of the nisin concentrated solution, adjusting the pH to 7.0 with 1 mol / L hydrochloric acid solution, controlling the stirring speed to 120 r / min at a temperature of 20° C., stirring and adsorbing for 2 hours, adding 5% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0122] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 2BV / h, and obtain the desorption liquid, which is as follows:

[0123] S31, controlling the volume ratio of hydrochloric acid solution, ethanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to be 1:2:0.3, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L, ethanol, and 1-butyl-3-methylimidazolium tetrafluoroborate to prepare a desorbent;

[0124] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 4:1, the desorption flow rate to be 2BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0125] S4, the pH of the desorption liquid is adjusted to 3.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0126] The pH of the desorption liquid was adjusted to 3.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:6. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 3 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:12. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 3 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 40°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 40°C, dried to constant weight, and then crushed to obtain 345g of nisin milky white powder, and the biological potency test result was 38458IU / mg.

[0127] Table 3 Comprehensive performance indexes of nisin obtained in Example 3

[0128]

[0129] Example 4

[0130] A method for extracting high-purity nisin comprises the following steps:

[0131] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0132] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0133] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0134] S13, adding 20 g / L of the composite nutrient in Preparation Example 1 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30° C. and a speed of 180 r / min for 24 h. After the culturing, a nisin fermentation liquid with a biological titer of 18970 IU / ml was obtained;

[0135] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0136] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0137] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0138] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0139] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0140] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0141] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0142] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 303g of nisin milky white powder, and the biological potency test result was 36897IU / mg.

[0143] Table 4 Comprehensive performance indexes of nisin obtained in Example 4

[0144]

[0145] In order to verify the comprehensive performance of nisin obtained in Examples 1-4 of the present invention, the applicant set up Comparative Examples 1-7, which are as follows:

[0146] Comparative Example 1

[0147] A method for extracting high-purity nisin comprises the following steps:

[0148] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0149] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0150] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0151] S13, adding 20g / L of the composite nutrient in Comparative Preparation Example 1 to the seed culture medium to prepare a fermentation medium, inoculating the seed liquid into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30°C and a speed of 180r / min for 24h, and after the culturing, obtaining a nisin fermentation liquid with a biological titer of 15328IU / ml;

[0152] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0153] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0154] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0155] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0156] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0157] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0158] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0159] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 253g of nisin milky white powder, and the biological potency test result was 34195IU / mg.

[0160] Table 5 Comprehensive performance indexes of nisin obtained in comparative example 1

[0161]

[0162] Comparative Example 2

[0163] A method for extracting high-purity nisin comprises the following steps:

[0164] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0165] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0166] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0167] S13, adding 20g / L of the composite nutrient in Comparative Preparation Example 2 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30°C and a speed of 180r / min for 24h. After the culturing, a nisin fermentation liquid with a biological titer of 14492IU / ml was obtained;

[0168] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0169] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0170] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0171] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0172] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0173] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0174] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0175] The pH of the desorption solution was adjusted to 2.0, and the mass ratio of the desorption solution after pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption solution after pH adjustment and stirring to dissolve, the solution was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted; the mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the solution was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out solution containing nisin. After filtering the salting-out solution, it was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 238g of nisin milky white powder, and the biological potency test result was 33277IU / mg.

[0176] Table 6 Comprehensive performance indexes of nisin obtained in comparative example 2

[0177]

[0178] Comparative Example 3

[0179] A method for extracting high-purity nisin comprises the following steps:

[0180] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0181] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0182] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0183] S13, adding 20g / L of the composite nutrient in comparative preparation example 3 to the seed culture medium to prepare a fermentation medium, inoculating the seed liquid into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30°C and a speed of 180r / min for 24h. After the culturing, a nisin fermentation liquid with a biological titer of 16785IU / ml was obtained;

[0184] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0185] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0186] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0187] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0188] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0189] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0190] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0191] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 271g of nisin milky white powder, and the biological potency test result was 34668IU / mg.

[0192] Table 7 Comprehensive performance indexes of nisin obtained in comparative example 3

[0193]

[0194] Comparative Example 4

[0195] A method for extracting high-purity nisin comprises the following steps:

[0196] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0197] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0198] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0199] S13, adding 20g / L of the composite nutrient in Comparative Preparation Example 4 to the seed culture medium to prepare a fermentation medium, inoculating the seed liquid into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30°C and a speed of 180r / min for 24h. After the culturing, a nisin fermentation liquid with a biological titer of 16129IU / ml was obtained;

[0200] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0201] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0202] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0203] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0204] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0205] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0206] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0207] The pH of the desorption solution was adjusted to 2.0, and the mass ratio of the desorption solution after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption solution after the pH adjustment and stirring to dissolve, the solution was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted; the mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the solution was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out solution containing nisin. After filtering the salting-out solution, it was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 262g of nisin milky white powder, and the biological potency test result was 34397IU / mg.

[0208] Table 8 Comprehensive performance indexes of nisin obtained in Comparative Example 4

[0209]

[0210] Comparative Example 5

[0211] A method for extracting high-purity nisin comprises the following steps:

[0212] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0213] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0214] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0215] S13, adding 20g / L of the composite nutrient in comparative preparation example 5 to the seed culture medium to prepare a fermentation medium, inoculating the seed liquid into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30°C and a speed of 180r / min for 24h. After the culturing, a nisin fermentation liquid with a biological titer of 15864IU / ml was obtained;

[0216] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0217] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0218] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0219] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0220] S31, controlling the volume ratio of hydrochloric acid solution to ethanol to be 1:1, and uniformly mixing hydrochloric acid solution with a molar concentration of 1.5 mol / L and ethanol to prepare a desorbent;

[0221] S32, pouring the saturated adsorption resin into the resin column, controlling the volume ratio of the desorbent and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1BV / h, adding the desorbent to perform dynamic desorption, taking an instantaneous sample to detect after the desorption is completed during the desorption process, and obtaining a desorption liquid;

[0222] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0223] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 259g of nisin milky white powder, and the biological potency test result was 34275IU / mg.

[0224] Table 9 Comprehensive performance indexes of nisin obtained in comparative example 5

[0225]

[0226] Comparative Example 6

[0227] A method for extracting high-purity nisin comprises the following steps:

[0228] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0229] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0230] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0231] S13, adding 20 g / L of the composite nutrient in Preparation Example 1 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30° C. and a speed of 180 r / min for 24 h. After the culturing, a nisin fermentation liquid with a biological titer of 18970 IU / ml was obtained;

[0232] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0233] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0234] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0235] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0236] Pour the saturated adsorption resin into the resin column, control the volume ratio of the hydrochloric acid solution with a mass concentration of 1.5 mol / L and the saturated adsorption resin to be 3:1, the desorption flow rate to be 1 BV / h, add the hydrochloric acid solution for desorption, take an instantaneous sample for detection during the desorption process, and obtain the desorption liquid after the desorption is completed;

[0237] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0238] The pH of the desorption liquid was adjusted to 2.0, and the mass ratio of the desorption liquid after the pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption liquid after the pH adjustment and stirring to dissolve, the mixture was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted. The mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the mixture was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out liquid containing nisin. After filtering the salting-out liquid, the mixture was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 244g of nisin milky white powder, and the biological potency test result was 31240IU / mg.

[0239] Table 10 Comprehensive performance indexes of nisin obtained in Comparative Example 6

[0240]

[0241] Comparative Example 7

[0242] A method for extracting high-purity nisin comprises the following steps:

[0243] S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed solution, and the seed solution is inoculated into a fermentation medium. After the cultivation is completed, a nisin fermentation solution is obtained. The specific operation is as follows:

[0244] S11, transferring the preserved lactic acid streptococci to an activation culture medium, and culturing the culture medium at 30° C. for 24 hours to restore the physiological activity of the lactic acid streptococci and obtain an activated strain;

[0245] S12, transfer the activated strain into the seed culture medium at an inoculation rate of 1%, and culture it on a shaker at a temperature of 30°C and a speed of 180r / min for 24h to obtain a seed solution;

[0246] S13, adding 20 g / L of the composite nutrient in Preparation Example 1 to the seed culture medium to prepare a fermentation medium, inoculating the seed solution into the fermentation medium at an inoculum amount of 4%, and culturing the culture medium on a shaker at a temperature of 30° C. and a speed of 180 r / min for 24 h. After the culturing, a nisin fermentation liquid with a biological titer of 18970 IU / ml was obtained;

[0247] S2. After filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0, the temperature to 40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain saturated adsorption resin. The specific operation is as follows:

[0248] S21, taking 40L of nisin fermentation liquid, heating it to 85°C, filtering it through a ceramic membrane with a pore size of 0.05 μm, and obtaining a nisin concentrate after removing macromolecular impurities;

[0249] S22, pretreating the macroporous adsorption resin to obtain a pretreated macroporous adsorption resin, adding the pretreated macroporous adsorption resin to the nisin concentrated solution of step S21, controlling the added volume of the pretreated macroporous adsorption resin to account for 20% of the total volume of the nisin concentrated solution, adjusting the pH to 3.0 with a 1.2 mol / L hydrochloric acid solution, controlling the stirring speed to 100 r / min at a temperature of 40° C., stirring and adsorbing for 3 hours, adding 10% of the total volume of the nisin concentrated solution of the pretreated macroporous adsorption resin, repeating the above stirring and adsorption operation, sampling once every 1 hour during the adsorption process, until the biological potency content in the residual liquid to be adsorbed is determined to be 0, stopping the adsorption, washing with purified water, and obtaining a saturated adsorption resin;

[0250] S3, pour the saturated adsorption resin into the resin column, add the desorbent for dynamic desorption, control the desorption flow rate to 1BV / h, and obtain the desorption liquid, which is as follows:

[0251] The saturated adsorption resin is poured into the resin column, the volume ratio of ethanol to the saturated adsorption resin is controlled to be 3:1, the desorption flow rate is 1BV / h, ethanol is added for desorption, and an instantaneous sample is taken during the desorption process for detection, and a desorption liquid is obtained after the desorption is completed;

[0252] S4, the pH of the desorption liquid is adjusted to 2.0, sodium chloride is added for salting out, the salting out liquid is filtered, rotary evaporated, and low-temperature dried to obtain high-purity nisin, as follows:

[0253] The pH of the desorption solution was adjusted to 2.0, and the mass ratio of the desorption solution after pH adjustment to sodium chloride was controlled to be 100:3. After adding sodium chloride to the desorption solution after pH adjustment and stirring to dissolve, the solution was allowed to stand for 5 hours to separate the layers, and the upper clear liquid was extracted; the mass ratio of the upper clear liquid to sodium chloride was controlled to be 100:8. After adding sodium chloride to the upper clear liquid and stirring to dissolve, the solution was allowed to stand for 5 hours again to separate the layers, and the upper layer was a salting-out solution containing nisin. After filtering the salting-out solution, it was placed in a rotary evaporator for rotary evaporation, and the rotary evaporation temperature was controlled to be 30°C. The nisin wet powder obtained by rotary evaporation was placed in a vacuum oven at 30°C, dried to constant weight, and then crushed to obtain 251g of nisin milky white powder, and the biological potency test result was 33178IU / mg.

[0254] Table 11 Comprehensive performance indexes of nisin obtained in comparative example 7

[0255]

[0256] It can be seen from the data shown in Tables 1-11 above that the comprehensive performance of the nisin obtained in Examples 1-4 is significantly better than that in Comparative Examples 1-7. The nisin obtained in Examples 1-4 has obvious advantages in terms of Nisin production, total extraction yield, purity, activity and safety, and has broad market prospects.

[0257] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for extracting high-purity nisin, characterized in that: The following steps are involved: S1. After activating lactic acid streptococci using an activation medium, the activated strain is transferred into a seed medium for cultivation to obtain a seed liquid, the seed liquid is inoculated into a fermentation medium, and after the cultivation is completed, a nisin fermentation liquid is obtained; S2, after filtering the nisin fermentation broth, add macroporous adsorption resin, adjust the pH to 3.0-7.0, the temperature to 20-40°C, and perform static adsorption under stirring conditions. After the adsorption is complete, wash with purified water to obtain a saturated adsorption resin; S3, pouring the saturated adsorption resin into the resin column, adding a desorbent for dynamic desorption, controlling the desorption flow rate to 1-2BV / h, and obtaining a desorption liquid; S4, adjusting the pH of the desorption liquid to 2.0-3.0, adding sodium chloride for salting out, filtering the salting out liquid, rotary evaporating, and drying at low temperature to obtain high-purity nisin; The formula of the seed culture medium in step S1 is: peptone 8-12 g / L, beef extract 8-12 g / L, yeast extract 5-8 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 5-8 g / L, glucose 20-25 g / L, Tween 80 1-2 mL / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, manganese sulfate tetrahydrate 0.2-0.4 g / L, sodium chloride 10-12 g / L, pH 6.4-6.8; The formula of the fermentation medium in step S1 is: adding 20-25 g / L of complex nutrients to the seed culture medium; The composite nutrient is prepared by the following method: (1) pre-drying the microphylla seaweed and the masson pine needles, mixing and grinding them in a mass ratio of 2-4:1, and passing through a 40-60 mesh sieve to obtain a mixed powder; (2) adding deionized water at 50-60° C. to the mixed powder, stirring evenly, and then soaking for 8-10 hours, heating and boiling for extraction for 0.5-1 hour, cooling to 30-40° C. to obtain an extract, adding a composite bacterial agent, adjusting the pH value to 4.5-5.5, sealing and fermenting for 30-40 hours, and then centrifuging and drying to obtain a composite nutrient; The composite bacterial agent in step (2) is composed of Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus oryzae in a ratio of viable bacteria of 5-7:2-3:1; the viable bacteria content of the composite bacterial agent in the extract of step (2) is 1.8-2.3×10 9 CFU / mL; In step S1, the lactic acid streptococcus is lactic acid streptococcus ACCC10653; The macroporous adsorption resin in step S3 is ADS-750; In step S3, the desorbent is composed of an acid-organic compound system and an ionic liquid; the acid is a hydrochloric acid solution with a molar concentration of 1-1.5 mol / L; the organic compound is ethanol and / or n-butanol; the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate, and the volume ratio of the acid, the organic compound and the ionic liquid is 1:1-2:0.1-0.3; The salting-out treatment in step S4 specifically refers to: controlling the mass ratio of the pH-adjusted desorption liquid and sodium chloride to be 100:3-6, adding sodium chloride to the pH-adjusted desorption liquid and stirring evenly, standing to separate layers, and extracting the upper clear liquid; controlling the mass ratio of the upper clear liquid and sodium chloride to be 100:8-12, continuing to add sodium chloride to the upper clear liquid and stirring evenly, standing to separate layers, and the upper layer is the salting-out liquid containing nisin.

2. The method for extracting high-purity nisin according to claim 1, characterized in that: The formula of the activation culture medium in step S1 is: 8-12 g / L peptone, 8-12 g / L beef extract, 1-3 g / L lysine, 1-3 g / L dipotassium hydrogen phosphate, 0.3-0.5 g / L ferrous sulfate heptahydrate, 5-7 g / L sodium chloride, 20-25 g / L lactose, 15-18 g / L agar, and pH 6.4-6.

8.

3. The method for extracting high-purity nisin according to claim 2, characterized in that: The activation conditions of the activation medium in step S1 are: static culture at a temperature of 30-37° C. for 20-24 hours; The culture conditions in the seed culture medium are: at a temperature of 30-37° C., at a speed of 150-180 r / min, and incubated on a shaking table for 20-24 hours; The culture conditions in the fermentation medium are: at a temperature of 30-37° C. and a rotation speed of 150-180 r / min, the culture is shaken on a shaker for 20-24 hours.

4. The method for extracting high-purity nisin according to claim 1, characterized in that: The filtration treatment in step S2 refers to filtering the nisin fermentation broth through a ceramic membrane with a pore size of 0.05-0.1 μm to remove impurities.

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