CN111621536A - Fermentation production process of high-yield nisin - Google Patents

Fermentation production process of high-yield nisin Download PDF

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CN111621536A
CN111621536A CN202010462063.4A CN202010462063A CN111621536A CN 111621536 A CN111621536 A CN 111621536A CN 202010462063 A CN202010462063 A CN 202010462063A CN 111621536 A CN111621536 A CN 111621536A
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李珊
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HEBEI SHENGXUE DACHENG PHARMACEUTICAL CO Ltd
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Abstract

The invention provides a fermentation production process of high-yield nisin, which comprises the following steps: after primary seed culture and secondary seed culture, the lactic streptococci liquid is transferred to a fermentation medium for fermentation culture; and then, the sugar concentration is controlled by sugar supplement, after the bacterial concentration is increased to a certain degree and the synthesis of nisin is started, a certain amount of amino acid precursor mixed liquor is added into a fermentation culture medium, and the fermentation is carried out for 22-26 h. The invention determines the culture medium and the culture method for the fermentation production of nisin, and can promote the amino acid precursor substance of cell production by continuous supplement at a certain speed in the feeding process, thereby obviously improving the potency, and the biological potency of nisin is measured to be more than 16000 IU/mL. Effectively removes the influence caused by the action of rich nitrogen, reduces the nutritional ingredients such as impure protein, polypeptide, ammonia nitrogen, phosphorus and the like in the fermentation liquor, effectively reduces the production cost, and is suitable for industrial production.

Description

Fermentation production process of high-yield nisin
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation production process for high-yield nisin.
Background
Nisin (Nisin) is a polypeptide substance produced by fermentation of streptococcus lactis (Lactococcus lactis) and contains 34 amino acids. It can effectively inhibit the propagation of gram-positive bacteria and spores thereof, can be quickly digested and absorbed by human body after being eaten, is a natural food preservative with high efficiency, no toxicity, safety and no side effect, and is widely applied to various foods such as dairy products, meat products, canned products, fruit juice, alcoholic beverages and the like.
The nitrogen source is an important factor affecting the fermentation level of Nisin. Soy peptone, yeast powder, fish meal, cottonseed meal, and the like are generally considered as ideal nitrogen sources for Nisin production. However, the raw materials are expensive, which inevitably increases the production cost.
Chinese patent CN103805660A (application No. 201210443722.5) discloses a method for producing nisin by using cheap raw materials, wherein cheap bean pulp or bean cakes are used as a nitrogen source, pH control and feeding are combined, and the titer reaches 5000IU/mL after fermentation is finished. Chinese patent CN107400688A (application No. 201710425658.0) discloses a method for producing nisin by using food industry waste, wherein corn steep liquor, xylitol residue and soybean protein are subjected to double extraction to prepare basic fermentation liquor, and sucrose and yeast are combined for batch fermentation, and the titer reaches 4823IU/mL after the fermentation is finished.
Although the cost can be reduced by adopting cheap raw materials, the pretreatment cost of the raw materials is increased, and simultaneously, since the streptococcus lactis only utilizes certain components in the nitrogen source and other components are not utilized, the nitrogen-rich phenomenon is caused, the nitrogen-rich phenomenon in the culture medium is more and more serious along with the increase of the content of the nitrogen source, the difficulty of producing bacteria for utilizing effective components in the nitrogen source is more and more high, the titer improvement effect is not obvious, meanwhile, great difficulty is caused to sewage treatment, the production cost is greatly increased, and the industrial production is not facilitated.
Nisin is rich in rare amino acids dehydrated from cysteine, serine and threonine, mainly contributing to the basic structure and modification. If the nitrogen source is rich in the amino acids, the amino acids can be used as precursors of rare amino acids to promote the synthesis of nisin, provide necessary nutrient components for metabolic production of lactic acid bacteria, greatly reduce the dosage of nitrogen sources such as yeast powder and the like in the production process, reduce the content of ammonia nitrogen, phosphorus and the like in fermentation liquor filtrate, relieve the sewage treatment pressure and be suitable for industrial production.
Disclosure of Invention
The invention provides a fermentation production process of high-yield nisin, which can improve the titer, reduce the contents of ammonia nitrogen, phosphorus and the like in a fermentation liquid filtrate, relieve the sewage treatment pressure and reduce the production cost, and aims to solve the problems in the prior art.
The technical scheme adopted by the invention is as follows: a fermentation production process of high-yield nisin comprises the following steps: after primary seed culture and secondary seed culture, the lactic streptococci liquid is transferred to a fermentation medium for fermentation culture; and then, the sugar concentration is controlled by sugar supplement, after the bacterial concentration is increased to a certain degree and the synthesis of nisin is started, a certain amount of amino acid precursor mixed liquor is added into a fermentation culture medium, and the fermentation is carried out for 22-26 h.
Further, the primary seed culture comprises the following specific steps: inoculating the lactic acid streptococcus strain liquid into a first-stage seed tank for culture, wherein the inoculation amount is 0.15-0.25%, the liquid is diluted by 5 times and detected under OD600nm, and when the light absorption value reaches more than 0.6, the pH value is 6.0-6.5, and the culture time is 8-12h, the first-stage seed liquid is obtained; the secondary seed culture comprises the following specific steps: inoculating the first-stage seed solution into a second-stage seed tank for culturing, wherein the inoculation amount is 3-5%, the feed liquid is diluted by 5 times of OD600nm for detection, and the seeds are transferred when the light absorption value reaches above 0.6, the pH value is 6.0-6.5, and the culture time is 4-8 h.
Further, the fermentation culture comprises the following specific steps: inoculating the cultured secondary seed liquid into a fermentation tank filled with a fermentation culture medium for culturing, wherein the inoculation amount is 5-8%, the culture period is 22-26h, and the nisin fermentation liquid is obtained after the fermentation is finished.
Further, the culture medium components in the first-stage seeding tank and the second-stage seeding tank are as follows by weight percentage: 0.5-1.0% of glucose, 0.5-1.0% of bovine bone peptone, 0.5-1.0% of yeast extract powder and KH2PO40.3-0.7％，NaCl 0.1-0.3％，MgSO4·7H2O 0.01-0.03％。
Further, the fermentation medium comprises the following components in percentage by weight: 1.0-1.5% of glucose, 0.5-1.0% of yeast extract powder and KH2PO40.1-0.3％，NaCl 0.1-0.3％，MgSO4·7H20.01 to 0.03 percent of O, 0.2 to 0.8 percent of corn steep liquor dry powder and 800.1 to 0.2 percent of tween-E.
Further, the amino acid precursor mixed liquor comprises the following components in percentage by weight: serine 0.01-0.03%, threonine 0.01-0.03%, and cysteine 0.01-0.03%.
Further, the conditions of the primary seed culture and the secondary seed culture are as follows: keeping the temperature at 30 + -1 deg.C, and culturing under 0.04 + -0.05 Mpa.
Further, the conditions of the fermentation culture are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
Further, the sugar supplementing and sugar concentration controlling method comprises the following specific steps: sugar supplement is carried out by adopting a fed-batch method, wherein: glucose is reduced to 0.5% to supplement sugar, and the intermediate sugar concentration is controlled to 0.3-0.5%.
Further, the addition mode of the amino acid precursor mixed liquor in the fermentation process is as follows: feeding at the rate of 0.2-0.5% fermentation liquid volume after fermenting for 6-10 h.
The beneficial effects obtained by the invention are as follows: the invention determines the culture medium and the culture method for the fermentation production of nisin, and can promote the amino acid precursor substance of cell production by continuous supplement at a certain speed in the feeding process, thereby obviously improving the potency, and the biological potency of nisin is measured to be more than 16000 IU/mL. The method effectively removes the influence caused by the nitrogen-rich effect, reduces the nutritional ingredients such as impure protein, polypeptide, ammonia nitrogen, phosphorus and the like in the fermentation liquor, accelerates the extraction and filtration speed, reduces the wastewater treatment pressure, effectively reduces the production cost, and is suitable for industrial production.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1: first-order seed culture: the volume of the culture medium is 80L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.5 percent of glucose, 0.5 percent of bovine bone peptone and yeast extract powder0.5，KH2PO40.3，NaCl 0.1，MgSO4·7H2O0.01, and pH7.0 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
120mL of cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 8.5h, the feed liquid is diluted by 5 times and the absorbance value under OD600nm is 0.618, the pH value is 6.26, and the seeds are transplanted.
Secondary seed culture: the volume of the medium was 2T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.5 percent of glucose, 0.5 percent of bovine bone peptone, 0.5 percent of yeast extract powder and KH2PO40.3，NaCl 0.1，MgSO4·7H2O0.01, and pH7.0 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
80L of the cultured primary seed liquid is inoculated into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 6.5h, the feed liquid is diluted by 5 times of OD600nm, the light absorption value reaches 0.61, the pH value is 6.21, and the seeds are transferred.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 0.5 percent of yeast extract powder and KH2PO40.1，NaCl 0.2，MgSO4·7H2O0.02, corn steep liquor dry powder 0.2, Tween-800.1, and pH6.87 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2T cultured secondary seed liquid into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control: sugar supplement: when the fermentation culture is carried out for 4 hours, the glucose is reduced to 0.52 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3 to 0.5 percent.
Amino acid precursor mixture: the fermentation was incubated for 6h and fed-through at a rate of 0.2% volume of broth. After the fermentation culture is finished, the fermentation period is 22h, and the biological value of nisin is 16388U/mL.
Example 2
First-order seed culture: the volume of the medium was 120L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, pH6.99 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
240mL of cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 9.5h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.619, the pH value is 6.28, and the seeds are transferred.
Secondary seed culture: the volume of the culture medium is 2.5T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, pH6.98 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
And inoculating 120L of cultured primary seed liquid into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 5.5h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.613, the pH value is 6.21, and the seeds are transplanted.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is calculated by weight percentage,the composition is prepared according to the following proportion (%): 1.25% glucose, 0.75% yeast extract powder, KH2PO40.3，NaCl 0.3，MgSO4·7H2O0.03, corn steep liquor dry powder 0.5, Tween-800.15, and pH6.86 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2.5T cultured secondary seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control: sugar supplement: when the fermentation culture is carried out for 4.5h, the glucose is reduced to 0.51 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3-0.5 percent.
Amino acid precursor mixture: the fermentation was incubated for 8h and fed-through at a rate of 0.3% volume of broth. And (5) finishing the fermentation culture, wherein the fermentation period is 22h, and the biological value of the nisin is detected to be 16852U/mL.
Example 3
First-order seed culture: the volume of the medium was 160L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder and KH2PO40.7，NaCl 0.3，MgSO4·7H2O0.03, pH6.96 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
400mL of the cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 8.5h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.608, the pH value is 6.25, and the seeds are transplanted.
Secondary seed culture: the volume of the medium was 3T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder,KH2PO40.7，NaCl 0.3，MgSO4·7H2o0.03, pH7.0 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
160L of cultured primary seed liquid is inoculated into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 4.5h, the feed liquid is diluted by 5 times of OD600nm, the absorbance reaches 0.613, the pH value is 6.20, and the seeds are transferred.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: glucose 1.5, yeast extract powder 1.0, KH2PO40.3，NaCl 0.3，MgSO4·7H2O0.03, corn steep liquor dry powder 0.8, Tween-800.2, and pH6.92 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2.5T cultured secondary seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 3.5h, the glucose is reduced to 0.53 percent, the sugar is supplemented, and the concentration of the intermediate control sugar is 0.3 to 0.5 percent.
Amino acid precursor mixture: the fermentation was incubated for 10h and fed-through at a rate of 0.4% volume of broth. After the fermentation culture is finished, the fermentation period is 22h, and the biological value of nisin is detected to be 16924U/mL.
Comparative example 1
First-order seed culture: the volume of the culture medium is 80L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.5 percent of glucose, 0.5 percent of bovine bone peptone, 0.5 percent of yeast extract powder and KH2PO40.3，NaCl 0.2，MgSO4·7H2O0.02, pH7.0 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
120mL of cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 9h, the feed liquid is diluted by 5 times and the absorbance value under OD600nm is 0.607, the pH value is 6.19, and the seeds are transferred.
Secondary seed culture: the volume of the medium was 2T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.5 percent of glucose, 0.5 percent of bovine bone peptone, 0.5 percent of yeast extract powder and KH2PO40.3，NaCl 0.2，MgSO4·7H2O0.02, pH6.98 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
80L of the cultured primary seed liquid is inoculated into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 6h, the feed liquid is diluted by 5 times of OD600nm, the light absorption value reaches 0.615, the pH value is 6.17, and the seeds are transferred.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 0.75 percent of yeast extract powder and KH2PO40.2，NaCl 0.2，MgSO4·7H2O0.02, corn steep liquor dry powder 0.5, Tween-800.1, and pH6.89 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2T cultured secondary seed liquid into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4 hours, the glucose is reduced to 0.55 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3 to 0.5 percent.
After the fermentation culture is finished, the fermentation period is 22h, and the biological value of the nisin is 12536U/mL.
Comparative example 2
First-order seed culture: the volume of the medium was 120L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, pH6.95 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
240mL of cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 8h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.611, the pH value is 6.31, and the seeds are transplanted.
Secondary seed culture: the volume of the culture medium is 2.5T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, pH6.96 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
And inoculating 120L of cultured primary seed liquid into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 5h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.613, the pH value is 6.22, and the seeds are transplanted.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.25% glucose, 0.75% yeast extract powder, KH2PO40.2，NaCl 0.2，MgSO4·7H2O0.02, corn steep liquor dry powder 0.5, Tween-800.15, and pH6.9 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2.5T cultured secondary seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4.5h, the glucose is reduced to 0.51 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3-0.5 percent.
1.5% corn steep liquor: the fermentation was incubated for 8h and fed-through at a rate of 0.3% volume of broth.
After the fermentation culture is finished, the fermentation period is 22h, and the biological value of the nisin is 13274U/mL.
Comparative example 3
First-order seed culture: the volume of the medium was 160L.
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder and KH2PO40.7，NaCl 0.3，MgSO4·7H2O0.03, and pH7.01 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
400mL of the cultured Streptococcus lactis seed solution was inoculated into a primary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 8.5h, the feed liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.624, the pH value is 6.17, and the seeds are transplanted.
Secondary seed culture: the volume of the medium was 3T.
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder and KH2PO40.7，NaCl 0.3，MgSO4·7H2O0.03, pH7.0 after digestion.
And (3) sterilization: sterilizing at the temperature of 121-.
160L of cultured primary seed liquid is inoculated into a secondary seed tank. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. When the culture time is 4.5h, the feed liquid is diluted by 5 times of OD600nm, the light absorption value reaches 0.627, the pH value is 6.23, and the seeds are transplanted.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: glucose 1.5, yeast extract powder 1.0, KH2PO40.3，NaCl 0.3，MgSO4·7H2O0.03, corn steep liquor dry powder 0.8, Tween-800.2, and pH6.92 after digestion.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the 2.5T cultured secondary seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4 hours, the glucose is reduced to 0.5 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3 to 0.5 percent.
1.0% yeast extract powder and corn steep liquor mixture: the fermentation was incubated for 8h and fed-through at a rate of 0.4% volume of broth.
And (5) finishing the fermentation culture, wherein the fermentation period is 22h, and the biological value of the nisin is detected to be 13411U/mL.

Claims (10)

1. A fermentation production process of high-yield nisin is characterized by comprising the following steps: the method comprises the following steps: after primary seed culture and secondary seed culture, the lactic streptococci liquid is transferred to a fermentation medium for fermentation culture; and then, the sugar concentration is controlled by sugar supplement, after the bacterial concentration is increased to a certain degree and the synthesis of nisin is started, a certain amount of amino acid precursor mixed liquor is added into a fermentation culture medium, and the fermentation is carried out for 22-26 h.
2. The fermentation production process of high yield nisin according to claim 1, wherein: the first-stage seed culture comprises the following specific steps: inoculating the lactic acid streptococcus strain liquid into a first-stage seed tank for culture, wherein the inoculation amount is 0.15-0.25%, the liquid is diluted by 5 times and detected under OD600nm, and when the light absorption value reaches more than 0.6, the pH value is 6.0-6.5, and the culture time is 8-12h, the first-stage seed liquid is obtained; the secondary seed culture comprises the following specific steps: inoculating the first-stage seed solution into a second-stage seed tank for culturing, wherein the inoculation amount is 3-5%, the feed liquid is diluted by 5 times of OD600nm for detection, and the seeds are transferred when the light absorption value reaches above 0.6, the pH value is 6.0-6.5, and the culture time is 4-8 h.
3. The fermentation production process of high yield nisin according to claim 1, wherein: the fermentation culture comprises the following specific steps: inoculating the cultured secondary seed liquid into a fermentation tank filled with a fermentation culture medium for culturing, wherein the inoculation amount is 5-8%, the culture period is 22-26h, and the nisin fermentation liquid is obtained after the fermentation is finished.
4. The fermentation production process of high yield nisin according to claim 2, wherein: the culture medium components in the first-stage seeding tank and the second-stage seeding tank are as follows according to weight percentage: 0.5-1.0% of glucose, 0.5-1.0% of bovine bone peptone, 0.5-1.0% of yeast extract powder and KH2PO40.3-0.7％，NaCl 0.1-0.3％，MgSO4·7H2O 0.01-0.03％。
5. The fermentation production process of high yield nisin according to claim 3, wherein: the fermentation medium comprises the following components in percentage by weight: 1.0-1.5% of glucose, 0.5-1.0% of yeast extract powder and KH2PO40.1-0.3％，NaCl 0.1-0.3％，MgSO4·7H20.01 to 0.03 percent of O, 0.2 to 0.8 percent of corn steep liquor dry powder and 800.1 to 0.2 percent of tween-E.
6. The fermentation production process of high yield nisin according to claim 1, wherein: the amino acid precursor mixed solution comprises the following components in percentage by weight: serine 0.01-0.03%, threonine 0.01-0.03%, and cysteine 0.01-0.03%.
7. The fermentation production process of high yield nisin according to claim 2, wherein: the conditions of the first-stage seed culture and the second-stage seed culture are as follows: keeping the temperature at 30 + -1 deg.C, and culturing under 0.04 + -0.05 Mpa.
8. The fermentation production process of high yield nisin according to claim 3, wherein: the conditions of the fermentation culture are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.0-6.3 by adjusting liquid alkali.
9. The fermentation production process of high yield nisin according to claim 1, wherein: the sugar-supplementing sugar concentration control method comprises the following specific steps: sugar supplement is carried out by adopting a fed-batch method, wherein: glucose is reduced to 0.5% to supplement sugar, and the intermediate sugar concentration is controlled to 0.3-0.5%.
10. The fermentation production process of high yield nisin according to claim 1, wherein: the addition mode of the amino acid precursor mixed liquor in the fermentation process is as follows: feeding at the rate of 0.2-0.5% fermentation liquid volume after fermenting for 6-10 h.
CN202010462063.4A 2020-05-27 2020-05-27 Fermentation production process of high-yield nisin Pending CN111621536A (en)

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