Fermentation method of nisin

Abstract

The invention provides a fermentation method for improving titer of nisin, which comprises the following steps of: performing seed culture on a nisin seed solution, transferring the nisin seed solution into a fermentation culture medium, performing fermentation culture, and achieving a manner of improving the titer through a fermentation method of feeding, supplementing materials and introducing air. In the fermentation process, a 5% (g / L) phytic acid solution is added in a fed-batch mode when the period is 10-16 h till fermentation is finished. Before the fermentation period is 14 hours, air is not introduced for culture, the tank pressure is 0 Mpa, 0.01-0.05 vvm of air is introduced from 14 hours, and the tank pressure is 0.03 Mpa until the fermentation is finished. The phytic acid solution is supplemented in the middle and later periods of fermentation, the metabolism speed can be controlled, thallus oxidation death and autolysis are prevented, and the biological value is indirectly improved. A trace amount of air is introduced, so that the generation of lactic acid can be inhibited, tricarboxylic acid circulation is properly enhanced, and the synthesis efficiency of precursor amino acid is improved. Under the combined action of the two, the effects of controlling and prolonging the primary metabolism time, reducing the yield of lactic acid and obviously improving the titer increase in the later period are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method for improving the titer of nisin. Background Art

[0002] Nisin is a polypeptide produced by fermentation of Lactococcus lactis, containing 34 amino acids.

[0003] Nisin has antibacterial effects and has antibacterial effects on most Gram-positive bacteria, especially bacterial spores. It can effectively inhibit the reproduction of Gram-positive bacteria and their spores, and will be quickly digested and absorbed by the human body after ingestion. It is a natural food preservative with high efficiency, non-toxicity, safety and no side effects. The above advantages make nisin widely used in the preservation of dairy products, meat products, canned products and beverages.

[0004] The main production method of nisin is microbial fermentation. The problems existing in the domestic fermentation production of nisin at present are: due to the short fermentation cycle, fast nutrient utilization speed, the sugar metabolism speed is still relatively high in the later stage of fermentation, the titer increase is significantly reduced, the metabolism shifts, and a large amount of lactic acid is produced. Eventually, the fermentation titer remains at a relatively low level for a long time, the production cost is high, and it is not conducive to large-scale production.

[0005] In recent years, a lot of work has been carried out in the domestic research on improving the titer of nisin:

[0006] Chinese Patent CN104789624B (Application No. CN201510190841.8) discloses a method for co-producing calcium lactate by nisin fermentation. The pH of the fermentation broth is controlled by adding a certain concentration of calcium hydroxide suspension, and the titer reaches 12300 - 13030 IU / mL at the end of fermentation.

[0007] Chinese Patent CN201611131569.7, a method for controlling the pH value of the fermentation culture system to improve the yield of nisin, ferments Lactococcus lactis for 6 hours under the condition that the initial pH value of the fermentation medium is 7.2; adjusts the pH value and keeps it constant at pH = 6.2 until the end of fermentation; by adjusting the pH value of the fermentation medium during the fermentation process of Lactococcus lactis, the stability of nisin in the fermentation medium is enhanced, thereby increasing the yield of nisin, and the titer of the final fermentation broth reaches 8980 IU / mL. Summary of the Invention

[0008] In view of the above-mentioned defects or deficiencies in the prior art, the present invention provides a fermentation method of nisin to solve the problems existing in the above-mentioned prior art.

[0009] Its main technical solution is as follows: A fermentation method of nisin, characterized in that: after the nisin streptococcus seed liquid is subjected to seed culture, it is inoculated into a fermenter equipped with a fermentation medium at an inoculation amount of 5-6%, and fermentation culture is carried out, and feeding is added and air is introduced during the fermentation culture process.

[0010] Further, during the fermentation culture process, 5% (g / L) phytic acid solution is added dropwise starting from 10-16 h of the cycle until the fermentation ends.

[0011] Further, during the fermentation culture process, anaerobic culture is carried out before 14 h of the fermentation cycle, the fermenter pressure is 0 Mpa, and after 14 h, 0.01-0.05 vvm of air is introduced, and the fermenter pressure is 0.03 Mpa until the fermentation ends.

[0012] Further, the culture conditions of the fermenter are as follows: 20% (g / L) sodium hydroxide solution is automatically added dropwise during the fermentation process to maintain the pH at 6.0-6.2; glucose solution is added to control the glucose concentration at 0.5-0.8 g / 100 mL; the culture temperature is 29-30 °C until the fermentation ends.

[0013] Further, the mass concentration (g / L) of each component of the fermentation medium is: glucose 1-2%, bovine bone peptone 0.4-0.6%, soybean cake powder 1.0-2.0%, yeast extract powder 1-1.5%, corn steep liquor 1-3%, magnesium sulfate heptahydrate 0.01-0.02%, zinc sulfate heptahydrate 0.005-0.01%, sodium chloride 0.1-0.3%, liquid antifoaming agent 0.03-0.05%, and water, with the pH being natural.

[0014] Further, the fermentation medium is prepared in proportion, sterilized at 115 °C for 20 min with sterile steam and then cooled to room temperature, and kept under pressure with sterile air.

[0015] The beneficial effects of the present invention are as follows:

[0016] This method determines the culture conditions during fermentation, including: pH control, glucose control, feeding by continuous addition, air injection, medium ratio, tank pressure, etc. Phytic acid, also known as inositol hexaphosphate, is an organic phosphorus compound and can be used as an antioxidant. Supplementing phytic acid solution can control the metabolic rate in the later stage of fermentation, prevent the oxidation and autolysis of bacteria, and indirectly improve the biological potency. Reasons for lactic acid production: Because the consumption rate of glucose is fast in the later stage, pyruvate accumulates, the metabolic flux biases towards lactic acid, and excessive lactic acid is produced, resulting in a significant reduction in the content of precursor amino acids of the target product. Therefore, the synthesis rate of the target product decreases rapidly, inhibiting the production of potency. Micro-air injection can inhibit the production of lactic acid, appropriately enhance the tricarboxylic acid cycle, and improve the synthesis efficiency of precursor amino acids. In the middle and later stages of nisin fermentation, the present invention injects air microscopically and supplements phytic acid solution. Under the combined action of the two, it plays a role in controlling and prolonging the primary metabolism time, reducing the lactic acid production, and significantly increasing the potency increase in the later stage. Description of the Drawings

[0017] Other features, objects, and advantages of the present application will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0018] Figure 1 It is a schematic diagram of the change in the increase of nisin during the fermentation process of the embodiment and the comparative example of the present invention. Detailed Embodiments

[0019] The following further describes the present application in detail with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the relevant invention and do not limit the invention. In addition, it should be noted that only the parts related to the invention are shown in the drawings for the convenience of description.

[0020] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0021] The fermentation method for improving the potency of nisin adopts the following technological steps:

[0022] The nisin seed liquid is transferred to the fermentation medium for fermentation culture after seed culture, and the method of feeding by continuous addition and air injection is used to improve the potency.

[0023] During the fermentation culture process, 5% (g / L) phytic acid solution is continuously added starting from 10 - 16 h of the cycle until the fermentation ends.

[0024] During the fermentation culture process, anaerobic culture is carried out before 14 h of the fermentation cycle, the tank pressure is 0 Mpa, and after 14 h, air is introduced at a rate of 0.01 - 0.05 vvm, and the tank pressure is 0.03 Mpa until the fermentation ends.

[0025] During the fermentation culture process, the culture conditions in the fermenter are as follows: During the fermentation process, 20% (g / L) sodium hydroxide solution is automatically added dropwise to maintain the pH at 6.0 - 6.2. Glucose solution is added to control the glucose concentration at 0.5 - 0.8 g / 100 mL. The culture temperature is 29 - 30 °C until the fermentation ends.

[0026] Among them:

[0027] (1) Fermentation medium: The mass concentration (g / L) of each component of the fermentation medium is as follows: glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with the pH being natural.

[0028] The fermentation medium is prepared in proportion, sterilized with sterile steam at 115 °C for 20 min, cooled to room temperature, and kept under pressure with sterile air.

[0029] (2) Fermentation culture: The cultured seed liquid is inoculated into the fermenter filled with the fermentation medium at an inoculation amount of 5 - 6%. The culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, 20% (g / L) sodium hydroxide solution is automatically added dropwise to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, 50 - 60% (g / L) glucose is added to control the glucose concentration within this range. At 10 - 16 h of the cycle, 5% (g / L) phytic acid solution is added dropwise, and the addition rate is 0.9 ml / L / h. Anaerobic culture is carried out before 14 h, the tank pressure is 0 Mpa, air is introduced at 0.01 - 0.05 vvm starting from 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29 - 30 °C until the fermentation ends.

[0030] (3) Using the above fermentation process, the culture cycle is about 20 h. After the fermentation ends, a nisin fermentation broth is obtained; using this method, the nisin titer range is 18700 U / mL to 20000 U / mL at 20 h.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and in combination with the specific embodiments of the examples.

[0032] Example 1:

[0033] The fermentation method for improving the titer of nisin adopts the following specific process.

[0034] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with natural pH.

[0035] Prepare the fermentation medium in proportion, sterilize it at 115 °C for 20 min, then cool it to room temperature, and maintain the pressure with sterile air.

[0036] (2) Inoculate the well - cultured seed liquid into the fermenter filled with the fermentation medium at an inoculation amount of 5 - 6%. The culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, start to supplement 50 - 60% (g / L) glucose to control the glucose concentration within this range. Start to add and supplement 5% (g / L) phytic acid solution at 10 h, and the supplementation rate is 0.9 ml / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start to introduce 0.01 vvm air at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29 - 30 °C until the fermentation ends.

[0037] (3) Using the above - mentioned fermentation process, the culture period is 20 h. After the fermentation ends, obtain the nisin fermentation broth. After detection, the titer of nisin in the fermentation broth is 18753 U / mL.

[0038] Example 2:

[0039] The fermentation method for improving the titer of nisin adopts the following specific process.

[0040] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with natural pH.

[0041] Prepare the fermentation medium in proportion, sterilize it at 115 °C for 20 min, then cool it to room temperature, and maintain the pressure with sterile air.

[0042] (2) Inoculate the cultured seed liquid into a fermenter filled with fermentation medium at an inoculation amount of 5-6%, and the culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0-6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5-0.8 g / 100 mL, start to supplement 50-60% (g / L) glucose to control the glucose concentration within this range. At the 13th hour of the cycle, start to flow in and supplement 5% (g / L) phytic acid solution at a feeding rate of 0.9 mL / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start to introduce 0.03 vvm air at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29-30 °C until the fermentation ends.

[0043] (3) Using the above fermentation process, the culture period is 20 h. After the fermentation ends, a nisin fermentation broth is obtained. After detection, the titer of nisin in the fermentation broth is 19921 U / mL.

[0044] Example 3:

[0045] The fermentation method for improving the titer of nisin adopts the following specific process.

[0046] (1) Fermentation medium: The volume of the medium is 60 L. The components of the fermentation medium (g / L): glucose 1-2%, bovine bone peptone 0.4-0.6%, soybean cake powder 1.0-2.0%, yeast extract powder 1-1.5%, corn steep liquor 1-3%, magnesium sulfate heptahydrate 0.01-0.02%, zinc sulfate heptahydrate 0.005-0.01%, sodium chloride 0.1-0.3%, liquid antifoaming agent 0.03-0.05%, and water, with the pH being natural.

[0047] Prepare the fermentation medium according to the ratio, sterilize it at 115 °C for 20 min, then cool it to room temperature, and keep it under pressure with sterile air.

[0048] (2) Inoculate the cultured seed liquid into a fermenter filled with fermentation medium at an inoculation amount of 5-6%, and the culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0-6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5-0.8 g / 100 mL, start to supplement 50-60% (g / L) glucose to control the glucose concentration within this range. At the 16th hour of the cycle, start to flow in and supplement 5% (g / L) phytic acid solution at a feeding rate of 0.9 mL / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start to introduce 0.05 vvm air at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29-30 °C until the fermentation ends.

[0049] (3) Using the above fermentation process, the cultivation period is 20 h. After fermentation ends, a nisin fermentation broth is obtained. After detection, the nisin titer in the fermentation broth is 19019 U / mL.

[0050] Example 4:

[0051] The fermentation method for increasing the nisin titer adopts the following specific process.

[0052] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with the pH being natural.

[0053] Prepare the fermentation medium according to the ratio, sterilize it at 115 °C for 20 min and then cool it to room temperature, and maintain the pressure with sterile air.

[0054] (2) Inoculate the cultured seed liquid into a fermenter filled with the fermentation medium at an inoculation amount of 5 - 6%, and the cultivation conditions and process are as follows: After fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, start to supplement 50 - 60% (g / L) glucose to control the glucose concentration within this range. Start to add and supplement 5% (g / L) phytic acid solution at the 10th h of the cycle, and the supplementation rate is 0.9 ml / L / h. Do not aerate for cultivation before 14 h, the tank pressure is 0 Mpa, start to introduce air at 0.03 vvm at 14 h, the tank pressure is 0.03 Mpa, and the cultivation temperature is 29 - 30 °C until fermentation ends.

[0055] (3) Using the above fermentation process, the cultivation period is 20 h. After fermentation ends, a nisin fermentation broth is obtained. After detection, the nisin titer in the fermentation broth is 19876 U / mL.

[0056] Example 5:

[0057] The fermentation method for increasing the nisin titer adopts the following specific process.

[0058] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with natural pH.

[0059] Prepare the fermentation medium in proportion, sterilize it at 115°C for 20 min and then cool it to room temperature, and keep it under pressure with sterile air.

[0060] (2) Inoculate the cultured seed liquid into a fermenter filled with the fermentation medium at an inoculation amount of 5 - 6%, and the culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, start to supplement 50 - 60% (g / L) glucose to control the glucose concentration within this range. Start to add 5% (g / L) phytic acid solution at 16 h of the cycle, and the feeding rate is 0.9 ml / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start to introduce air at 0.03 vvm at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29 - 30°C until the fermentation ends.

[0061] (3) Using the above fermentation process, the culture period is 20 h. After the fermentation ends, a nisin fermentation broth is obtained. After detection, the nisin titer in the fermentation broth is 18979 U / mL.

[0062] Example 6:

[0063] The fermentation method for improving the nisin titer adopts the following specific process.

[0064] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with natural pH.

[0065] Prepare the fermentation medium in proportion, sterilize it at 115°C for 20 min and then cool it to room temperature, and keep it under pressure with sterile air.

[0066] (2) Inoculate the well-cultured seed liquid into a fermenter filled with fermentation medium at an inoculation amount of 5 - 6%, and the culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, start adding 50 - 60% (g / L) glucose to control the glucose concentration within this range. Start adding 5% (g / L) phytic acid solution at 13 h, and the feeding rate is 0.9 mL / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start aerating with 0.01 vvm air at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29 - 30 °C until the fermentation ends.

[0067] (3) Using the above fermentation process, the culture period is 20 h. After the fermentation ends, a nisin fermentation broth is obtained. After detection, the titer of nisin in the fermentation broth is 18,876 U / mL.

[0068] Example 7:

[0069] The fermentation method for improving the titer of nisin adopts the following specific process.

[0070] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with natural pH.

[0071] Prepare the fermentation medium according to the proportion, sterilize it at 115 °C for 20 min, then cool it to room temperature, and maintain the pressure with sterile air.

[0072] (2) Inoculate the well-cultured seed liquid into a fermenter filled with fermentation medium at an inoculation amount of 5 - 6%, and the culture conditions and process are as follows: After the fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, start adding 50 - 60% (g / L) glucose to control the glucose concentration within this range. Start adding 5% (g / L) phytic acid solution at 13 h, and the feeding rate is 0.9 mL / L / h. Do not aerate the culture before 14 h, the tank pressure is 0 Mpa, start aerating with 0.05 vvm air at 14 h, the tank pressure is 0.03 Mpa, and the culture temperature is 29 - 30 °C until the fermentation ends.

[0073] (3) Using the above fermentation process, the culture period is 20 h. After fermentation ends, a nisin fermentation broth is obtained. After detection, the nisin titer in the fermentation broth is 19,629 U / mL.

[0074] Comparative Example 1

[0075] (1) Fermentation medium: The volume of the medium is 60 L. Components of the fermentation medium (g / L): glucose 1 - 2%, bovine bone peptone 0.4 - 0.6%, soybean cake powder 1.0 - 2.0%, yeast extract powder 1 - 1.5%, corn steep liquor 1 - 3%, magnesium sulfate heptahydrate 0.01 - 0.02%, zinc sulfate heptahydrate 0.005 - 0.01%, sodium chloride 0.1 - 0.3%, liquid antifoaming agent 0.03 - 0.05%, and water, with the pH being natural.

[0076] Prepare the fermentation medium in proportion, sterilize it at 115 °C for 20 min and then cool it to room temperature, and maintain the pressure with sterile air.

[0077] (2) Inoculate the well - cultured seed liquid into a fermenter filled with the fermentation medium at an inoculation amount of 5 - 6%. The culture conditions and process are as follows: After fermentation starts, when the pH of the fermentation broth naturally drops to 6.0 - 6.2, automatically add 20% (g / L) sodium hydroxide solution to maintain the pH within this range. When the glucose concentration in the fermentation broth drops to 0.5 - 0.8 g / 100 mL, add 50 - 60% (g / L) glucose to control the glucose concentration within this range. Do not aerate during cultivation, the tank pressure is 0 Mpa, and the cultivation temperature is 29 - 30 °C until fermentation ends.

[0078] (3) Using the above fermentation process, the culture period is 20 h. After fermentation ends, a nisin fermentation broth is obtained. After detection, the nisin titer in the fermentation broth is 17,213 U / mL.

[0079] Figure 1 is a schematic diagram showing the change in the increase rate of nisin titer during the fermentation process of the above - mentioned examples and comparative examples. From the above - mentioned examples and Figure 1 it can be seen that this method has an obvious effect on increasing the increase rate of nisin titer in the middle and late stages.

[0080] Table 1 Nisin titers among each example and comparative example

[0081]

[0082] The above description is only a preferred embodiment of the present application and an explanation of the technical principles applied. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the technical features (but not limited to) disclosed in the present application that have similar functions.

Claims

1. A fermentation method of nisin, characterized in that: After the Lactococcus lactis seed culture solution is subjected to seed culture, it is inoculated into a fermenter filled with a fermentation medium at an inoculation amount of 5-6% for fermentation culture, and feeding is carried out and air is introduced during the fermentation culture process.

2. The fermentation method of nisin according to claim 1, characterized in that: During the fermentation culture process, a 5% (g / L) phytic acid solution is fed and supplemented starting at 10-16 h of the cycle until the fermentation ends.

3. The fermentation method of nisin according to claim 1, characterized in that: During the fermentation culture process, anaerobic culture is carried out before 14 h of the fermentation cycle, the fermenter pressure is 0 Mpa, and after 14 h, 0.01-0.05 vvm of air is introduced, and the fermenter pressure is 0.03 Mpa until the fermentation ends.

4. A fermentation method of nisin according to claim 1, characterized in that: The culture conditions of the fermenter are as follows: during the fermentation process, a 20% (g / L) sodium hydroxide solution is automatically fed to maintain the pH at 6.0-6.2; a glucose solution is supplemented to control the glucose concentration at 0.5-0.8 g / 100 mL; the culture temperature is 29-30 °C until the fermentation ends.

5. A fermentation method of nisin according to any one of claims 1-4, characterized in that: The mass concentration (g / L) of each component of the fermentation medium is as follows: glucose 1-2%, bovine bone peptone 0.4-0.6%, soybean cake powder 1.0-2.0%, yeast extract powder 1-1.5%, corn steep liquor 1-3%, magnesium sulfate heptahydrate 0.01-0.02%, zinc sulfate heptahydrate 0.005-0.01%, sodium chloride 0.1-0.3%, liquid antifoaming agent 0.03-0.05%, and water, with the pH being natural.

6. The fermentation method of nisin according to claim 5, characterized in that: The fermentation medium is prepared proportionally, sterilized with sterile steam at 115 °C for 20 min, cooled to room temperature, and kept under pressure with sterile air.

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