阅读说明：本技术 一种高产乳酸链球菌素的发酵生产工艺 (Fermentation production process of high-yield nisin ) 是由 李珊 于 2020-05-27 设计创作，主要内容包括：本发明提供了一种高产乳酸链球菌素的发酵生产工艺,包括以下步骤：乳酸链球菌种液经一级种子培养、二级种子培养后转移到发酵培养基中进行发酵培养；然后通过补糖控制糖浓度,在菌浓增长到一定程度,乳酸链球菌素的合成启动后,在发酵培养基中添加一定量的氨基酸前体混合液,发酵22-26h。本发明确定了乳酸链球菌素发酵生产的培养基和培养方法,通过在补料过程中以一定的速率不断的补充能够促进细胞产素的氨基酸前体物质,明显提高了效价,乳酸链球菌素生物效价测定在16000IU/mL以上。有效解除了富氮作用带来的影响,减少了发酵液中杂蛋白和多肽、氨氮、磷等营养成分,有效降低了生产成本,适用于工业化生产。(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.)

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.

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.