CN116694703A - Method for producing curdlan by nitrogen-free gum production fermentation - Google Patents

Abstract

The invention relates to a method for producing curdlan by nitrogen-free gum production fermentation, belonging to the technical field of microbial polysaccharide fermentation. The method comprises the following process steps: inoculating the seed culture solution of the strain producing the curdlan into a fermentation culture medium containing a nitrogen source, and culturing thalli in the first stage; after the thallus culture is finished, separating the fermentation liquor from the thallus, inoculating the separated thallus without nitrogen into a fermentation medium without nitrogen source, performing the second-stage gum-producing fermentation, and after the gum-producing fermentation is finished, performing post-treatment to obtain the curdlan. The method has the advantages of high success rate of gum production during main fermentation, convenient operation and wide selection range of the culture medium for main fermentation.

Description

Method for producing curdlan by nitrogen-free gum production fermentation

Technical Field

The invention relates to the technical field of microbial polysaccharide fermentation, in particular to a method for producing curdlan by nitrogen-free gum production fermentation.

Background

Curdlan (Curdlan), also known as thermogel, a coagulated polysaccharide, is produced by using the bacterium alcaligenes faecalis (a.faecalis var.) of the genus agrobacterium (Agrobacterium biovar) or radiobacter (a.radiobacter) as a producer. The curdlan is water insoluble glucan composed of beta-1, 3-glycosidic bond, and has a molecular formula of (C ₆ H ₁₀ O ₅ ) _n ，n>250 (400-500). The curdlan is a generic name of polysaccharides which can form not only hard and elastic thermoreversible gel but also thermoreversible gel after heating the suspension. 12 months 1996, the U.S. food and drug administration FDA approved and allowed curdlan asIs a direct additive for the food industry. The curdlan is a third food polysaccharide produced by FDA approved fermentation after the xanthan gum and the gellan gum, which provides wider space for further popularization and application of the curdlan. At present, the curdlan has wide application as a food additive, and is widely applied to the preparation of minced fillet products, meat products, rice and flour products, bionic vegetarian products and the like as a gel, a structure modifier, a thickener, a stabilizer and the like. It has freeze thawing resistance, oil packing property, water-repellent property and heat stability.

Several methods for producing curdlan are disclosed, wherein CN108103121a is used for controlling fermentation culture conditions in fermentation culture by adopting a process of controlling pH, supplementing carbon source in several times and adding nonionic surfactant, for shortening fermentation period. CN106701885a is obtained by adding a soluble polysaccharide fermentation accelerator to a fermentation medium and controlling the rotation speed stepwise to increase the yield. The prior art needs to add additional reagent to increase the yield of the curdlan, and increases the cost of enterprises.

The main fermentation of the curdlan is divided into two stages: the first stage is to perform the growth culture of the thalli under the condition of having a nitrogen source and a pH value of about 7.0; and the second stage is to continue fermentation to produce the gum after the nitrogen source is exhausted and the pH is adjusted to about 5.6. However, in the first-stage cell growth phase, there is a problem that nitrogen source cannot be sufficiently consumed, and thus transition to the second stage is impossible, curdlan cannot be produced, and production fails.

Disclosure of Invention

First, the technical problem to be solved

In view of the above-mentioned drawbacks and shortcomings of the prior art, the present invention provides a method for producing curdlan by fermentation without nitrogen production, which solves the technical problem that when curdlan is produced by fermentation, transition to the second stage fails due to insufficient consumption of nitrogen source in the first stage of fermentation, and curdlan cannot be produced.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a method for producing curdlan by nitrogen-free gum production fermentation, which comprises the following steps:

inoculating the seed culture solution of the strain producing the curdlan into a fermentation culture medium containing a nitrogen source, and culturing thalli in the first stage; after the thallus culture is finished, separating the fermentation liquor from the thallus, inoculating the separated thallus without nitrogen into a fermentation medium without nitrogen source, performing the second-stage gum-producing fermentation, and after the gum-producing fermentation is finished, performing post-treatment to obtain the curdlan.

Alternatively, the culture conditions for the cell culture of the first stage are: the inoculation amount is 0.1-2% by volume, the culture temperature is 25-35 ℃, the pH is 6.0-8.0, ventilation is 0.2-2vvm, the stirring speed is 100-1000rpm, and the OD is cultured ₆₆₀ The culture time is 8-24 hours after 4.0-9.0.

Preferably, the OD is cultured ₆₆₀ To 6.0-9.0.

Optionally, the fermentation medium containing the nitrogen source comprises the following components in percentage by mass: 2-6% of carbon source, 0.2-1.2% of nitrogen source, K ₂ HPO ₄ 0.05-0.2％，MgSO ₄ ·7H ₂ O 0.05-0.2％，FeCl ₃ 0.001-0.002％，ZnCl ₂ 0.001-0.002%, defoamer 0.05-0.07%, and water for the rest;

the nitrogen source-free fermentation medium comprises the following components in percentage by mass: 3-10% of carbon source, K ₂ HPO ₄ 0.05-0.2％，MgSO ₄ ·7H ₂ O 0.05-0.2％，FeCl ₃ 0.001-0.002％，ZnCl ₂ 0.001-0.002%, defoamer 0.05-0.07% and water for the rest.

Optionally, the nitrogen source is (NH ₄ ) ₂ HPO ₄ Or yeast powder; in the fermentation medium containing the nitrogen source, (NH) ₄ ) ₂ HPO ₄ The addition amount of the yeast powder is 0.2-0.5wt% and the addition amount of the yeast powder is 0.6-1.2wt%.

Optionally, the separated nitrogen-containing culture medium can be recycled for strain fermentation and production of curdlan after component detection, sterilization and nutrient component adjustment.

Optionally, the carbon source comprises glucose and the water comprises deionized water.

Optionally, the separation method comprises aseptic centrifugation or aseptic filtration, wherein the rotation speed of the aseptic centrifugation is 1000g-20000g, and the centrifugation time is 5-60 minutes.

Alternatively, the culture conditions of the gum-producing fermentation of the second stage are: the temperature is 25-35 ℃, the pH is 5.2-5.8, the ventilation is 0.2-2vvm, the stirring speed is 100-1000rpm, and the culture time is 40-120 hours.

Optionally, the post-processing includes:

(1) Alkali solution dissolution: regulating pH of the fermentation liquor to 11.0-13.0 with alkali, stirring for dissolving, centrifuging 1000g-20000g for 1-20 min, and collecting supernatant;

(2) Acid precipitation: adding acid into the supernatant, regulating pH to 6.0-7.0, stirring for precipitation, centrifuging 1000g-20000g for 1-20 min, and collecting precipitate;

(3) Washing and dehydrating: adding alcohol solution, stirring, washing, dehydrating for 30-40 min, centrifuging for 1-20 min with 1000-20000 g, and collecting precipitate;

(4) Drying and pulverizing.

Alternatively, the base comprises sodium hydroxide and the acid comprises hydrochloric acid.

(III) beneficial effects

The beneficial effects of the invention are as follows:

in view of the technical problems that in the fermentation production of the curdlan in the prior art, nitrogen sources in a first stage (thallus culture) of main fermentation cannot be fully used up, natural transition to a second stage (colloid production stage) of main fermentation is difficult, so that the curdlan cannot be produced, great waste and great improvement of production cost are caused, and meanwhile, a large amount of nutrient components such as sugar and the like in a culture medium cannot be consumed and can only be discharged into a sewage treatment system, so that overload load and the like of a sewage pool are caused. The fermentation liquor in the first stage of main fermentation (thallus culture) is subjected to aseptic centrifugation, the thallus and a nitrogen source-free culture medium are separated, and then the nitrogen source-free thallus is added into the nitrogen source-free culture medium in the second stage of main fermentation (colloid production stage) to perform nitrogen-free fermentation, so that the fermentation in the second stage can smoothly produce the curdlan under specific culture conditions and centrifugation conditions.

In the first stage, under the condition of a nitrogen-containing culture medium (total nutrient components), all nutrient components required by normal vital activities of the strain can be provided, so that the strain can be rapidly proliferated to high cell density in a short time to obtain a large number of curdlan-producing zymocyte cells in a logarithmic phase; and then, a large amount of curdlan-producing zymocyte cells are separated from the nitrogen-containing culture medium in a centrifugal way and transferred into a nitrogen-free culture medium of a second stage for further culture, and the curdlan is produced by stress strains under the nitrogen-free condition. On one hand, the efficiency of producing the curdlan can be improved, and the phenomenon that the bacterial strain is hindered to produce the curdlan due to nitrogen in the second stage is avoided; on the other hand, the culture medium after the centrifugation in the first stage can be further recycled to the first stage after the components are adjusted after sterilization, so that the utilization rate of the nutrient components is greatly improved, and the sewage discharge is reduced.

According to the invention, other components for promoting fermentation and gum production are not required to be added, and enterprises do not need to pay for purchasing additional reagents. The invention can realize high yield of the curdlan and reduce the sewage treatment cost. The method has the advantages of high success rate of gum production during main fermentation, convenient operation, wide selection range of the culture medium of main fermentation and wide application prospect.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments for better explaining the present invention.

In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The curdlan-producing strains used in examples 1 to 8 and comparative examples 1 to 5 below were rhizobium ATCC31750.

Example 1

a. Shake flask seed liquid culture

1) The curdlan glycerol bacterial liquid is inoculated into 100g/500mL of culture medium, and the inoculation amount is 0.5% (by volume).

The culture medium comprises the following components in percentage by mass: white granulated sugar 2.0%, yeast powder 1.0%, mgSO ₄ ·7H ₂ O0.04%, calcium carbonate 0.3%, deionized water to a total of 100.0%.

2) Placing on a shaking table for culturing. The culture conditions are as follows; the culture was carried out at 30℃and 180rpm for 20 hours.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 3.5%, yeast powder 1%, K ₂ HPO ₄ 0.4％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 16 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.91, 149mg/kg amino nitrogen and 1.05wt% residual sugar.

c. Sterile centrifugation of nitrogen-free cells

1) The broth obtained after the completion of the first stage of main fermentation was centrifuged at 4000g for 20 minutes under aseptic conditions.

2) Collecting the precipitate, wherein the precipitate is nitrogen-free thallus.

d. In the second stage of main fermentation, gum-producing fermentation

All the nitrogen-free bacteria obtained by centrifugal separation are inoculated into the culture medium of the second stage of main fermentation.

The formula of the culture medium in the second stage is as follows by mass percent: glucose 5%, K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05% deionized water to a total of 100.0%.

The culture conditions are as follows: the incubation time was 72 hours at 30℃with pH5.6, aeration 0.6vvm, stirring speed 600rpm.

e. Extraction of curdlan

1) Alkali solution dissolution: and adding 0.3mol/L NaOH solution with the volume being 2.5 times that of the fermentation liquor into the fermentation liquor obtained in the second stage, regulating the pH of the fermentation liquor to 11.0-13.0, and stirring for dissolution. The supernatant was collected by centrifugation at 11000g for 10 minutes.

2) Acid precipitation: adding 3mol/L HCl solution into the supernatant, adjusting pH to 6.0-7.0, and stirring for precipitation. The pellet was collected by centrifugation at 11000g for 10 minutes.

3) Washing and dehydrating: the mixture was washed with 75% by volume of an alcoholic solution and dehydrated for 30 minutes with stirring. Centrifuge at 5000g for 10 min and collect the precipitate.

f. Drying, pulverizing, and packaging

1) Oven drying at 60deg.C overnight.

2) Pulverizing, sieving, and packaging to obtain final product.

Example 2

The steps a, c, d, e, f of this example are the same as those of example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 3.5%, yeast powder 0.6%, K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 20 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 7.98, 31.5mg/kg amino nitrogen and 1.1wt% residual sugar.

Example 3

The steps a, c, d, e, f of this example are the same as those of example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 3.5%, yeast powder 0.6%, K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.05％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 24 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.06, 30.5mg/kg amino nitrogen and 1.0wt% residual sugar.

Example 4

The steps a, c, d, e, f of this example are the same as those of example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 3.5% (NH) ₄ ) ₂ HPO ₄ 0.23％，K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 16 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.26, amino nitrogen 37.3mg/Kg, residual sugar 1.4wt%.

Example 5

The steps a, c, d, e, f of this example are the same as those of example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 3.5% (NH) ₄ ) ₂ HPO ₄ 0.23％，K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 24 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.4, 35.1mg/kg amino nitrogen and 1.3wt% residual sugar.

Example 6

The steps a, b, d, e, f of this example are the same as those of example 1.

c. Sterile centrifugation of nitrogen-free cells

1) The broth obtained after the completion of the first stage of main fermentation was centrifuged at 4000g for 10 minutes under aseptic conditions.

2) Collecting the precipitate, wherein the precipitate is nitrogen-free thallus.

Example 7

The steps a, b, d, e, f of this example are the same as those of example 1.

c. Sterile centrifugation of nitrogen-free cells

1) The fermentation broth obtained after the completion of the first stage of main fermentation was centrifuged at 6000g for 10 minutes under aseptic conditions.

2) Collecting the precipitate, wherein the precipitate is nitrogen-free thallus.

Example 8

The steps a, b, d, e, f of this example are the same as those of example 1.

c. Sterile centrifugation of nitrogen-free cells

1) The fermentation broth obtained after the completion of the first stage of main fermentation was centrifuged at 8000g for 10 minutes under aseptic conditions.

2) Collecting the precipitate, wherein the precipitate is nitrogen-free thallus.

Comparative example 1

a. Shake flask seed liquid culture

1) The curdlan glycerol bacterial liquid is inoculated into 100g/500mL of culture medium, and the inoculation amount is 0.5% (by volume).

The culture medium comprises the following components in percentage by mass: white granulated sugar 2.0%, yeast powder 1.0%, mgSO ₄ ·7H ₂ O0.04%, calcium carbonate 0.3%, deionized water to a total of 100.0%.

2) Placing on a shaking table for culturing. The culture conditions are as follows: the culture was carried out at 30℃and 180rpm for 20 hours.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0%, yeast powder 0.6%, K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 16 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 7.82, amino nitrogen 44mg/kg, residual sugar 4.4wt%.

c. In the second stage of main fermentation, gum-producing fermentation

The pH of the fermentation broth was adjusted to 5.6 with 10% by volume hydrochloric acid solution and the second stage fermentation was continued in the main fermenter.

Culture conditions: the temperature was 30℃and the pH was 5.6, and the stirring speed was 600rpm with aeration of 0.6 vvm. The culture was continued for 72 hours after the pH was adjusted to 5.6.

d. Extraction of curdlan

1) Alkali solution dissolution: and adding 0.3mol/L NaOH solution with the volume being 2.5 times that of the fermentation liquor into the fermentation liquor obtained in the second stage, regulating the pH of the fermentation liquor to 11.0-13.0, and stirring for dissolution. The supernatant was collected by centrifugation at 11000g for 10 minutes.

2) Acid precipitation: adding 3mol/L HCl solution into the supernatant, adjusting pH to 6.0-7.0, and stirring for precipitation. The pellet was collected by centrifugation at 11000g for 10 minutes.

3) Washing and dehydrating: the mixture was washed with 75% by volume of an alcoholic solution and dehydrated for 30 minutes with stirring. Centrifuge at 5000g for 10 min and collect the precipitate.

e. Drying, pulverizing, and packaging

1) Oven drying at 60deg.C overnight.

2) Pulverizing, sieving, and packaging to obtain final product.

Comparative example 2

The steps a, c, d, e of this comparative example are the same as comparative example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0%, yeast powder 0.6%, K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 24 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.01, amino nitrogen 30.2mg/kg, residual sugar 4.1wt%.

Comparative example 3

The steps a, c, d, e of this comparative example are the same as comparative example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0% (NH) ₄ ) ₂ HPO ₄ 0.23％，K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. CulturingThe time was 16 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.21, 36.5mg/kg amino nitrogen and 4.3wt% residual sugar.

Comparative example 4

The steps a, c, d, e of this comparative example are the same as comparative example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0% (NH) ₄ ) ₂ HPO ₄ 0.23％，K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 24 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 8.45, 34.7mg/kg amino nitrogen and 4.2wt% residual sugar.

Comparative example 5

The steps a, c, d, e of this comparative example are the same as comparative example 1,

b. in the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0%, yeast powder 0.5%, K ₂ HPO ₄ 0.4％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 16 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 6.50, 28.1mg/kg amino nitrogen and 5.1wt% residual sugar.

Comparative example 6

The steps a, c, d, e of this comparative example are the same as comparative example 1.

b. In the first stage of main fermentation, thallus culture

The shake flask seed liquid was transferred to the primary fermenter for cell culture at an inoculum size of 5% (by volume).

The culture medium formula in the first stage comprises the following components in percentage by mass: glucose 7.0% (NH) ₄ ) ₂ HPO ₄ 0.115％，K ₂ HPO ₄ 0.1％，MgSO ₄ ·7H ₂ O 0.04％，FeCl ₃ 0.001％，ZnCl ₂ 0.001%, defoamer 0.05%, deionized water to a total of 100.0%.

The culture conditions are as follows: temperature 30 ℃, pH7.0, aeration 0.6vvm, stirring speed 600rpm. The culture time was 24 hours, at which time the OD of the cells in the fermentation broth was measured ₆₆₀ 4.22, 19.5mg/kg amino nitrogen and 5.4wt% residual sugar.

Table 1: effect comparison of examples and comparative examples

The results in Table 1 show that the gel yield was higher (higher than 3.7%) for 8 examples in which nitrogen-free cells were inoculated into the medium of the gel-producing fermentation after centrifugation of the cell culture broth. The longer the cell culture time is, the higher the gel yield is. Under the same bacterial culture time, the higher the centrifugal rotation speed is, the higher the gel yield is; the longer the centrifugation time, the higher the gum yield.

Comparative examples 1 to 4 demonstrated that the cell culture broth was not centrifuged, and the pH was directly adjusted to perform gum-producing fermentation, and after 72 hours of fermentation, no curdlan was obtained, and the gum yield was 0%.

Comparative example 5 demonstrates that even if the amount of nitrogen source in the first stage medium is reduced, the unconsumed nitrogen source remains in the system after the first stage culture is completed, and the natural transition to the second stage is not possible. If the nitrogen source amount in the first stage is excessively reduced, the growth of the bacteria in the first stage is affected, and the subsequent production of the curdlan is also affected.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. A method for producing curdlan by nitrogen-free gum production fermentation is characterized by comprising the following steps:

inoculating the seed culture solution of the strain producing the curdlan into a fermentation culture medium containing a nitrogen source, and culturing thalli in the first stage; after the thallus culture is finished, separating the fermentation liquor from the thallus, inoculating the separated thallus without nitrogen into a fermentation medium without nitrogen source, performing the second-stage gum-producing fermentation, and after the gum-producing fermentation is finished, performing post-treatment to obtain the curdlan.

2. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the culture conditions of the cell culture in the first stage are: the inoculation amount is 0.1-2% by volume, the culture temperature is 25-35 ℃, the pH is 6.0-8.0, ventilation is 0.2-2vvm, the stirring speed is 100-1000rpm, and the OD is cultured ₆₆₀ The culture time is 8-24 hours after 4.0-9.0.

3. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the fermentation medium containing nitrogen source comprises the following components in percentage by mass: 2-6% of carbon source, 0.2-1.2% of nitrogen source, K ₂ HPO ₄ 0.05-0.2％，MgSO ₄ ·7H ₂ O0.05-0.2％，FeCl ₃ 0.001-0.002％，ZnCl ₂ 0.001-0.002%, defoamer 0.05-0.07%, and water for the rest;

the fermentation medium without nitrogen source comprises the following components in percentage by massThe components in proportion: 3-10% of carbon source, K ₂ HPO ₄ 0.05-0.2％，MgSO ₄ ·7H ₂ O0.05-0.2％，FeCl ₃ 0.001-0.002％，ZnCl ₂ 0.001-0.002%, defoamer 0.05-0.07% and water for the rest.

4. A method for producing curdlan by fermentation without nitrogen production according to claim 3, wherein the nitrogen source is (NH ₄ ) ₂ HPO ₄ Or yeast powder; in the fermentation medium containing the nitrogen source, (NH) ₄ ) ₂ HPO ₄ The addition amount of the yeast powder is 0.2-0.5wt% and the addition amount of the yeast powder is 0.6-1.2wt%.

5. A method of producing curdlan by nitrogen-free gum production fermentation as claimed in claim 3, wherein the carbon source comprises glucose and the water comprises deionized water.

6. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the separation method comprises aseptic centrifugation or aseptic filtration, the rotational speed of the aseptic centrifugation is 1000g-20000g, and the centrifugation time is 5-60 minutes.

7. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the separated nitrogen-containing culture medium can be recycled for bacterial strain fermentation and curdlan production after component detection, sterilization and nutrient component adjustment.

8. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the culture conditions of the gum production fermentation of the second stage are: the temperature is 25-35 ℃, the pH is 5.2-5.8, the ventilation is 0.2-2vvm, the stirring speed is 100-1000rpm, and the culture time is 40-120 hours.

9. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 1, wherein the post-treatment comprises:

(1) Alkali solution dissolution: regulating pH of the fermentation liquor to 11.0-13.0 with alkali, stirring for dissolving, centrifuging 1000g-20000g for 1-20 min, and collecting supernatant;

(2) Acid precipitation: adding acid into the supernatant, regulating pH to 6.0-7.0, stirring for precipitation, centrifuging 1000g-20000g for 1-20 min, and collecting precipitate;

(3) Washing and dehydrating: adding alcohol solution, stirring, washing, dehydrating for 30-40 min, centrifuging for 1-20 min with 1000-20000 g, and collecting precipitate;

(4) Drying and pulverizing.

10. The method for producing curdlan by nitrogen-free gum production fermentation according to claim 9, wherein the base comprises sodium hydroxide and the acid comprises hydrochloric acid.