CN116200439A

CN116200439A - Method for producing glucosamine by combined fermentation culture

Info

Publication number: CN116200439A
Application number: CN202211678334.5A
Authority: CN
Inventors: 阮澍; 卢正东; 肖其磊; 黄宇; 雷华; 夏炜铠; 黎艳华
Original assignee: Hubei Guangji Pharmaceutical Biotechnology Research Institute Co ltd; Hubei Guangji Pharmaceutical Co ltd
Current assignee: Hubei Guangji Pharmaceutical Biotechnology Research Institute Co ltd; Hubei Guangji Pharmaceutical Co ltd
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-06-02

Abstract

The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a method for producing glucosamine by combined fermentation culture, which comprises the following steps: 1. inoculating the escherichia coli seed liquid into a fermentation medium for fermentation culture; 2. after induction for 5 hours, inoculating bacillus subtilis for fermentation; 3. after 10 hours of combined culture, adding corresponding antibiotics into the fermentation broth to inhibit the growth of bacillus subtilis; 4. after the fermentation is finished, the glucosamine fermentation liquid is obtained. According to the invention, the dialysis bag is used for isolating bacillus subtilis from escherichia coli in the fermentation tank, enzyme systems of the two strains are isolated from direct contact with thalli, only small molecular substances are exchanged, and then the activity of the bacillus subtilis is inhibited, and the escherichia coli can directly utilize the decomposed small molecular substances of the bacillus subtilis to grow and metabolize, so that the escherichia coli is promoted by utilizing the decomposed small molecular substances of the bacillus subtilis, and the phenomenon that the fermentation effect is influenced by competition of the bacillus subtilis in mass propagation can be avoided.

Description

Method for producing glucosamine by combined fermentation culture

Technical Field

The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a method for producing glucosamine by combined fermentation culture.

Background

Glucosamine is involved in the construction of human tissues and cell membranes, an intermediate for the synthesis of proteoglycan macromolecules, which can synthesize mucopolysaccharides, glycoproteins and proteoglycans, in particular those of articular cartilage and synovial fluid molecules; is a natural component of glycoprotein in joint tissue in human and animal bodies. The current production method of glucosamine mainly comprises a chitin hydrolysis method, an enzyme bioconversion method and a microbial fermentation method. The biosynthesis method is industrially utilized, but is limited by strains, recombinant escherichia coli is mainly used as a production strain in China at present, the metabolic pathway is extremely good, and the yield of glucosamine is greatly improved.

The Chinese patent CN112760348A, a fermentation production method of N-acetylglucosamine, utilizes mixed fermentation of escherichia coli and bacillus subtilis to balance and symbiotic. But has several significant drawbacks: 1. the secondary metabolite in the fermentation system is increased by the culture of the two bacteria, which is not beneficial to the purification of the glucosamine in the later period; 2. the two strains are directly mixed to generate competition, the respective metabolites can seriously influence the growth of each other, the fermentation effect can be improved when the bacillus subtilis is just added in the early stage, but the two strains can not reach symbiosis, and the direct competition of the two strains to the substances can influence the growth of each other in the later stage, so that the fermentation effect is rapidly deteriorated; 3. the external environment is not changed, bacillus subtilis cannot easily sleep and form spores, and competition of the two directly mixed strains inevitably occurs in the later stage to cause poor fermentation effect.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to not only utilize small molecular substances decomposed by bacillus subtilis to promote the escherichia coli, but also avoid the competition to the escherichia coli when the bacillus subtilis is propagated in a large quantity to influence the fermentation effect.

The specific principle is that after the induction of escherichia coli, a large amount of glucose invertase is expressed in a period, a large amount of glucose added through feed supplement is quickly converted, but the consumption speed of yeast extract cannot keep up with the consumption speed of glucose, so that the yeast extract is accumulated in a large amount, the carbon-nitrogen ratio in fermentation liquor is unbalanced, the growth of thalli is inhibited, and the thalli amount is slowly increased. At the moment, bacillus subtilis is introduced, and the bacillus subtilis decomposes the composite nitrogen source yeast extract in the fermentation broth to be decomposed into small molecular peptides and amino acids, and the small molecular substances are utilized by escherichia coli to grow and reproduce through a dialysis membrane, so that the purposes of improving the substrate utilization rate and improving the product yield are achieved. After 10 hours of combined culture, antibiotics are added to inhibit the growth and metabolism of the bacillus subtilis, prevent the metabolites of the bacillus subtilis from entering the fermentation tank, and prevent the enzyme activity characteristics of the escherichia coli enzyme system from being influenced. Thus, the bacillus subtilis can be utilized to decompose the composite nitrogen source in the fermentation broth, and the metabolic products of the bacillus subtilis can be prevented from entering the fermentation broth to influence the escherichia coli.

The specific technical scheme adopted is a method for producing glucosamine by combined fermentation culture, which comprises the following steps:

step 1, designing a fermentation tank with an independent dialysis bag, wherein a bag body of the dialysis bag is fixed on the inner wall of the fermentation tank, a bag opening of the dialysis bag is directly communicated with a tank cover of the fermentation tank, and then the bag opening of the dialysis bag is connected with a feeding needle opening through an independent pipeline, and the dialysis molecular weight of the dialysis bag is 5kDa;

step 2, inoculating E.coli ATCC35947 into a fermentation medium, and starting fermentation culture;

step 3, after fermenting until the specific growth rate of the thalli is greater than 0.2, adding IPTG to induce and continue fermenting for 5 hours, and inoculating bacillus subtilis into a dialysis bag by using an inoculating needle after dissolved oxygen begins to rise back;

step 4, continuously feeding after inoculating bacillus subtilis, carrying out combined fermentation culture for 10-15 hours, wherein the pH value of the fermentation liquor starts to be obviously reduced, and inoculating streptomycin and ampicillin into a dialysis bag to inhibit the growth metabolism of the bacillus subtilis;

and 5, after inhibiting the growth metabolism of bacillus subtilis, continuing fermentation culture for 45-50 hours, stopping feeding until the consumption of glucose in the fermentation broth is completed, and stopping fermentation to obtain the glucosamine fermentation broth.

And the fermentation medium in the step 2 comprises 20-30 g/L of carbon source, 5-10 g/L of yeast extract, 0.01-0.1 g/L of calcium salt, 1-5 g/L of citrate, 2-20 g/L of magnesium salt, 5-20 g/L of phosphate and 1-5 g/L of ammonium salt, wherein the pH value is controlled to be 6.8-6.9 by ammonia water, and the fermentation medium is obtained after the total volume is fixed by water and then sterilized.

In addition, E.coli ATCC35947 was inoculated into LB medium in step 2, and after culturing in a shaking table at a constant temperature of 37℃at 220rpm for 14 hours, it was inoculated into the fermentation medium in an inoculum size of 2%.

After fermentation culture for 10 hours in the step 3, the specific growth rate of the thalli exceeds 0.2, and 0.2mM IPTG is added for induction, and after fermentation is continued for 5 hours, the escherichia coli is slowly metabolized and dissolved oxygen starts to rise.

In addition, in the step 3, the bacillus subtilis is firstly inoculated into LB culture medium, cultured for 10 to 15 hours at 220rpm in a shaking table with constant temperature of 37 ℃, and then inoculated into a dialysis bag with an inoculation amount of 0.5 to 0.75 percent.

The LB culture medium comprises 15g/L of glucose, 5g/L of yeast extract, 1g/L of magnesium salt, 4g/L of phosphate and 2g/L of ammonium salt, and is inoculated into a dialysis bag in an inoculation amount of 0.75% after culturing the bacillus subtilis.

And, the components used in the feed supplement in the step 4 at least contain 40% -60% of glucose and 1% -5% of yeast extract, and the total volume is sterilized after the volume is fixed by water.

In addition, the combined fermentation culture condition in the step 4 is that the aeration rate is 1VVM, the pressure of a fermentation tank is 0.04-0.06Mpa, the pH value is 6.8-6.9, the dissolved oxygen amount is maintained at 20-30%, the rotating speed is 300-700 rpm, and the feeding speed is adjusted to maintain the glucose content in the fermentation liquid at 3-12g/L.

In addition, the contents of the access streptomycin and the ampicillin in the step 4 are as follows: 50mg/L streptomycin and 50mg/L ampicillin.

Compared with the prior art, the beneficial effect of this technical scheme lies in:

(1) The utility model provides a fermentation cylinder with independent dialysis bag can avoid two kinds of bacterial strains direct contact through fixed dialysis bag at the fermentation cylinder pipe wall, avoids the high molecular protein to circulate, only the micromolecule metabolite circulate each other, reaches the purpose that the thallus utilized the fermentation substrate fast, thereby avoids the thereby disturbed thallus metabolism chaotic phenomenon of influence of enzyme system simultaneously.

(2) The combined culture method provided by the invention can fully utilize complex components in the fermentation liquor, such as yeast powder, impurity proteins, organic acids and the like, and insoluble substances in the fermentation liquor can be greatly reduced. The product extraction steps are reduced, and the extraction cost is reduced.

(3) According to the method provided by the invention, the extra antibiotics are added in the later period of the combined culture of the two strains, so that the loss of the thallus of the antibiotics in the middle fermentation period is reduced. Meanwhile, the activity of the bacillus subtilis is restrained in the late logarithmic growth phase of the bacillus subtilis, and the influence of secondary metabolites of the bacillus subtilis on the fermentation production of glucosamine by recombinant escherichia coli is avoided; not only can inhibit the metabolic growth of bacillus subtilis in the dialysis bag, but also can reduce the escherichia coli lost by plasmids in the fermentation liquor, further improves the substrate utilization rate by half, and can continue to ferment for a long time.

Drawings

FIG. 1 is a schematic diagram of a fermenter with a dialysis bag;

reference numerals illustrate: 1. a feeding needle opening, 2, a tank cover of a fermentation tank, 3 and a dialysis bag.

Detailed Description

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1:

a method for producing glucosamine by combined fermentation culture comprises the following steps:

(1) Inoculating recombinant Escherichia coli into LB medium, culturing at 220rpm in shaking table at 37deg.C for 14 hr, inoculating into fermentation medium with 2% inoculum size, and fermenting.

Specifically, the components of the fermentation medium, namely 30g/L glucose, 10g/L yeast extract, 0.05g/L calcium salt, 1g/L citrate, 5g/L magnesium salt, 10g/L phosphate and 5g/L ammonium salt. The volume is fixed to the corresponding volume by water, and the pH is controlled to about 6.8-6.9 by ammonia water in the fermentation process. The culture condition is that the aeration rate is 1VVM, the pressure of a fermentation tank is 0.04-0.06Mpa, the pH value is 6.8-6.9, and the dissolved oxygen amount is maintained at 20% -30%. The rotation speed is 300rpm-700rpm.

(2) After 10 hours of fermentation culture, the specific growth rate of the cells exceeded 0.2, and induction was started by adding 0.2mM IPTG. Inducing for 5h to slow the metabolism of thallus and raising dissolved oxygen.

At the same time of starting the fermentation culture of recombinant escherichia coli, bacillus subtilis is inoculated into an LB culture medium and cultured for 10-15 hours at 220rpm in a shaking table at a constant temperature of 37 ℃. The culture time depends on the amount of the cells.

(3) Fermenting for 15h, inoculating bacillus subtilis with 0.5% of inoculation amount into a fermentation tank through an inoculating needle, carrying out material exchange with the fermentation tank through a dialysis bag, and carrying out combined culture for 10-15h, wherein when the pH of fermentation liquor starts to be obviously reduced and the consumption of ammonia water is increased. Intervention in bacillus subtilis growth metabolism begins.

Specifically, 50mg/L streptomycin and 50mg/L ampicillin antibiotics are prepared and added into a dialysis bag through an inoculating needle, so that the metabolic growth of bacillus subtilis in the dialysis bag can be inhibited, the escherichia coli lost by plasmids in fermentation liquor can be reduced, and the substrate utilization rate is improved by half. The E.coli ATCC35947 is genetically modified, and has genes for producing ammonia sugar and resistance genes for resisting streptomycin and ampicillin, and the escherichia coli with lost plasmids loses the genes for producing ammonia sugar and also loses the genes for resisting antibiotics, so that the escherichia coli can be killed or inhibited by antibiotics, the metabolic growth of bacillus subtilis can be inhibited, the escherichia coli with lost plasmids in fermentation liquor can be reduced, and the normal escherichia coli can not be influenced.

(4) The adding speed of the feed medium is determined according to the glucose content in the fermentation broth, and the glucose content in the fermentation broth is controlled to be 3-12g/L. The formula of the feed medium comprises the following components: glucose 40-60%, yeast extract 1-5%, and water to a certain volume.

(5) After the antibiotics are added to inhibit the growth of bacillus subtilis, continuing fermentation culture, stopping feeding when the culture is completed for 45-50 hours, and stopping fermentation until the consumption of glucose in the fermentation broth is completed, thus obtaining the glucosamine fermentation broth.

The whole fermentation culture period is 50-55h, and fermentation can be continuously started according to the same method after the period is finished.

The fermentation liquor obtained in the example is detected by liquid chromatography, wherein the glucosamine content is 120g/L, and is improved by 15g/L compared with the glucosamine content when the fermentation liquor is singly cultured.

Example 2:

the specific process of the method for producing glucosamine by combined fermentation culture is basically the same as that of the example 1, except that:

the bacillus subtilis seed culture medium is different, and the specific components are as follows: 15g/L glucose, 5g/L yeast extract, 1g/L magnesium salt, 4g/L phosphate and 2g/L ammonium salt. The components of the culture medium are the same as the components of the fermentation broth as far as possible, so that the adaptation period time of the bacillus subtilis is reduced.

The inoculation amount of the bacillus subtilis is correspondingly increased to 0.75%, the bacillus subtilis thallus amount is increased, the components of the culture medium are not enriched before the culture medium is changed, and the inoculation amount needs to be increased.

The fermentation liquor obtained in the example is detected by liquid chromatography, wherein the glucosamine content is 125g/L, and is improved by 20g/L compared with the glucosamine content when the fermentation liquor is singly cultured.

A fermenter with independent dialysis bags for the method is shown in figure 1, wherein the bag body of the dialysis bag 3 is fixed on the inner wall of the fermenter, the bag opening of the dialysis bag is directly communicated with the tank cover 2 of the fermenter, the bag opening of the dialysis bag is connected with the feeding needle opening 1 of the fermenter by an independent pipeline, and the dialysis molecular weight of the dialysis bag is 5kDa.

Claims

1. A method for producing glucosamine by combined fermentation culture, which is characterized by comprising the following steps:

2. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: the fermentation medium in the step 2 comprises the components of 20-30 g/L of carbon source, 5-10 g/L of yeast extract, 0.01-0.1 g/L of calcium salt, 1-5 g/L of citrate, 2-20 g/L of magnesium salt, 5-20 g/L of phosphate and 1-5 g/L of ammonium salt, the pH value is controlled to be 6.8-6.9 by ammonia water, and the fermentation medium is obtained after the total volume is fixed by water and then sterilized.

3. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: e.coli ATCC35947 in step 2 was inoculated into LB medium, cultured in a shaking table at a constant temperature of 37℃at 220rpm for 14 hours, and then inoculated into the fermentation medium in an inoculum size of 2%.

4. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: after fermentation culture for 10 hours in the step 3, the specific growth rate of the thalli exceeds 0.2, 0.2mM IPTG is added for induction, and after fermentation is continued for 5 hours, the escherichia coli is slowly metabolized and dissolved oxygen begins to rise.

5. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: in the step 3, bacillus subtilis is firstly inoculated into LB culture medium, cultured for 10-15h at 220rpm in a shaking table at a constant temperature of 37 ℃, and then inoculated into a dialysis bag with an inoculum size of 0.5-0.75%.

6. The method for producing glucosamine by combined fermentation and culture according to claim 5, wherein the steps of: the components of the LB culture medium are 15g/L glucose, 5g/L yeast extract, 1g/L magnesium salt, 4g/L phosphate and 2g/L ammonium salt, and the bacillus subtilis is inoculated into a dialysis bag in an inoculation amount of 0.75% after being cultured.

7. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: the components used in the material supplementing in the step 4 at least contain 40% -60% of glucose and 1% -5% of yeast extract, and the total volume is sterilized after the volume is fixed by water.

8. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: in the step 4, the combined fermentation culture condition is that the aeration rate is 1VVM, the pressure of a fermentation tank is 0.04-0.06Mpa, the pH value is 6.8-6.9, the dissolved oxygen amount is maintained at 20-30%, the rotating speed is 300-700 rpm, and the feeding speed is adjusted to maintain the glucose content in the fermentation liquid at 3-12g/L.

9. The method for producing glucosamine by combined fermentation culture according to claim 1, wherein: the contents of the access streptomycin and the ampicillin in the step 4 are as follows: 50mg/L streptomycin and 50mg/L ampicillin.