阅读说明：本技术 一种谷氨酸的发酵方法 (Fermentation method of glutamic acid ) 是由 杨玉岭 满德恩 郭脉海 仇南南 崔光军 王婷婷 杜英慧 王成秋 周明英 李国强 于 2020-12-15 设计创作，主要内容包括：本发明公开了一种谷氨酸的发酵方法,属于谷氨酸发酵技术领域,包括发酵菌株摇瓶培养阶段、一级种子罐扩大培养阶段、二级种子罐扩大培养阶段和发酵阶段,所述发酵阶段采用零底糖浓度的发酵培养基,并以开始就流加的方式替代了高浓度的葡萄糖初始添加的传统发酵方式；本发明的有益效果是：规避了高浓度葡萄糖会在高温灭菌过程中产生的严重的焦糖化反应和局部浓度过高的液氨与高浓度葡萄糖会产生副反应的问题,比现有高浓度的葡萄糖初始添加的传统发酵方式发酵时间上有了进一步地缩短的效果。(The invention discloses a fermentation method of glutamic acid, belonging to the technical field of glutamic acid fermentation, comprising a shake flask culture stage of a fermentation strain, an expanded culture stage of a first-stage seed tank, an expanded culture stage of a second-stage seed tank and a fermentation stage, wherein the fermentation stage adopts a fermentation medium with zero base sugar concentration and replaces the traditional fermentation mode of initial addition of high-concentration glucose in a mode of feeding in a flowing manner at first; the invention has the beneficial effects that: the problems that high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction are solved, and the fermentation time is further shortened compared with the conventional fermentation method for initially adding the high-concentration glucose.)

1. A fermentation method of glutamic acid comprises a shake flask culture stage of a fermentation strain, a first-stage seeding tank amplification culture stage, a second-stage seeding tank amplification culture stage and a fermentation stage, and is characterized by comprising the following specific steps:

(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;

(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;

(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;

(4) the fermentation stage is as follows:

i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;

II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;

III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.

2. The glutamic acid fermentation method according to claim 1, wherein the shake flask culture medium is a bottomless medium comprising 1% soybean meal hydrolysate, 0.1% succinic acid, 0.05% methionine, 0.15% magnesium sulfate, 0.13% betaine, 0.4% potassium dihydrogen phosphate, 0.2% corn steep liquor, 10mg/L ferrous sulfate, 10mg/L manganese sulfate, 300 μ g/L biotin, 200 μ g/L VB1, 0.55% urea, and 0.5% yeast powder.

3. The glutamic acid fermentation method according to claim 1, wherein the first-stage seed tank medium is a non-basal medium comprising, by mass, 0% of glucose, phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.

4. The glutamic acid fermentation method according to claim 2, wherein the secondary seed tank culture medium is a non-substrate medium comprising, by mass, 0% of glucose, a soybean meal hydrolysate: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.

5. The glutamic acid fermentation method according to claim 2, wherein the medium in the fermentation stage is a bottomless medium comprising, by mass, 5-8% of corn steep liquor, soybean meal hydrolysate: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.

The technical field is as follows:

the invention belongs to the technical field of glutamic acid fermentation, and particularly relates to a fermentation method of glutamic acid.

Background art:

the fermentation production of glutamic acid is a biochemical process of decomposing and metabolizing nutrient substances, synthesizing required products and glutamic acid by glutamic acid producing bacteria in the life activity process. Currently, there are Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium scatterum, Brevibacterium flavum, Brevibacterium ammoniagenes, and the like, which are industrially used. In the fermentation production process, a plurality of factors influencing the growth, reproduction, metabolism and synthesis of the glutamic acid producing strain are controlled purposefully through artificial intervention, so that the metabolic synthesis requirement of the glutamic acid strain is finally met, and the aim of increasing the product and reducing the consumption can be achieved.

The glutamic acid producing bacteria are the main body of the reaction process and the biocatalyst of the reaction process, take in the nutrition of the raw materials, and carry out complex biochemical reaction through specific enzyme systems in cells. The reactant in the substrate enters the cell body through the cell wall and the cell membrane, and is subjected to catalytic reaction under the action of enzyme to be converted into a product and released, and the inherent characteristics and the metabolic rule of the cell are key factors influencing biochemical reaction.

The glutamic acid fermentation stage is mainly divided into: a glutamic acid producing bacterium proliferation stage and a glutamic acid producing bacterium acid production stage, wherein a biosynthetic pathway of glutamic acid in the acid production stage of the glutamic acid producing bacterium is approximately: glucose is subjected to glycolysis (EMP pathway) and hexose phosphate shunt (HMP pathway) to produce pyruvate, which is then oxidized to acetyl-CoA (acetyl COA), which then enters the tricarboxylic acid cycle to produce alpha-ketoglutarate. Catalysis of alpha-ketoglutarate in glutamate dehydrogenase and with NH₄ ⁺In the presence of a catalyst, glutamic acid is produced.

In the proliferation stage of the glutamic acid producing bacteria, the glutamic acid producing bacteria need to reach a certain OD value in a short time, the fermentation culture medium of the glutamic acid fermentation process of the traditional process contains 10-15% or even higher initial sugar content to meet the requirement of quick growth of the glutamic acid producing bacteria in the initial growth stage, so high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process, a large amount of pigment is generated to burden subsequent processes, a certain amount of glucose is wasted, especially, liquid ammonia needs to be added for regulation in the subsequent process, the local overhigh liquid ammonia and the high-concentration glucose can generate side reaction to generate byproducts to cause product quality reduction, the generated byproducts are similar to the physicochemical property of glutamic acid, and the difficulty of subsequent extraction and purification is increased.

The invention content is as follows:

in order to solve the problems and overcome the defects of the prior art, the invention provides a fermentation method of glutamic acid, which can effectively solve the problems that high-concentration glucose can generate serious caramelization reaction in a high-temperature sterilization process and the high-concentration glucose and liquid ammonia with over-high local concentration can generate side reaction.

The specific technical scheme for solving the technical problems comprises the following steps: the fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a first-stage seed tank, an amplification culture stage of a second-stage seed tank and a fermentation stage;

the method comprises the following specific steps:

(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;

(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;

(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;

the shake flask culture medium is a bottomless sugar culture medium and comprises 1% of soybean meal hydrolysate, 0.1% of succinic acid, 0.05% of methionine, 0.15% of magnesium sulfate, 0.13% of betaine, 0.4% of monopotassium phosphate, 0.2% of corn steep liquor, 10mg/L of ferrous sulfate, 10mg/L of manganese sulfate, 300 mu g/L of biotin, 200 mu g/L of VB1, 0.55% of urea and 0.5% of yeast powder.

The first-stage seeding tank culture medium is a bottomless sugar culture medium and comprises 0% of glucose, 0% of phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.

The secondary seed tank culture medium is a non-glucose culture medium and comprises 0% of glucose and soybean meal hydrolysate in percentage by mass: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.

(4) The fermentation stage is as follows:

i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;

II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;

III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.

The culture medium in the fermentation stage is a bottomless sugar culture medium and comprises 5-8% of corn steep liquor and soybean meal hydrolysate by mass percent: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.

The invention has the beneficial effects that:

the invention creatively sets the fermentation medium with zero-base sugar concentration, and replaces the traditional fermentation mode of high-concentration glucose initial addition by the mode of feeding at the beginning, thereby avoiding the problems that the high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction;

meanwhile, the invention also unexpectedly discovers that the fermentation medium with zero-base sugar concentration is adopted, and the feeding is carried out at a specific flow rate, so that the early growth of the glutamic acid bacteria is not slow due to unilateral reduction of the glucose concentration, and the fermentation time is further shortened compared with the conventional fermentation method for initially adding the high-concentration glucose.

The specific implementation mode is as follows:

in the description of the invention, specific details are given only to enable a full understanding of the embodiments of the invention, but it should be understood by those skilled in the art that the invention is not limited to these details for the implementation. In other instances, well-known structures and functions have not been described or shown in detail to avoid obscuring the points of the embodiments of the invention. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The specific implementation mode of the invention is as follows:

the first embodiment is as follows:

the fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a first-stage seed tank, an amplification culture stage of a second-stage seed tank and a fermentation stage;

the method comprises the following specific steps:

(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;

(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;

(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;

the shake flask culture medium is a bottomless sugar culture medium and comprises 1% of soybean meal hydrolysate, 0.1% of succinic acid, 0.05% of methionine, 0.15% of magnesium sulfate, 0.13% of betaine, 0.4% of monopotassium phosphate, 0.2% of corn steep liquor, 10mg/L of ferrous sulfate, 10mg/L of manganese sulfate, 300 mu g/L of biotin, 200 mu g/L of VB1, 0.55% of urea and 0.5% of yeast powder.

The first-stage seeding tank culture medium is a bottomless sugar culture medium and comprises 0% of glucose, 0% of phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.

The secondary seed tank culture medium is a non-glucose culture medium and comprises 0% of glucose and soybean meal hydrolysate in percentage by mass: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.

(4) The fermentation stage is as follows:

i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;

II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;

III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.

The culture medium in the fermentation stage is a bottomless sugar culture medium and comprises 5-8% of corn steep liquor and soybean meal hydrolysate by mass percent: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.

In order to more intuitively show the process advantages of the zero initial sugar concentration culture medium, the zero initial sugar concentration culture medium fermentation method is compared with the method of the comparative example,

comparative example one:

the preparation method is the same as the first embodiment except that: in the preparation process of the comparative example, the fermentation medium adopts a high-concentration base sugar culture medium, and the concentration of the base sugar is 15 percent; wherein the temperature in the early stage of fermentation is maintained at about 33 ℃, the fermentation lasts for about 6-8h, the initial sugar consumption is almost the same, and the sugar feeding is started to ensure that the concentration of residual sugar in the culture medium is always maintained at about 1.0%;

comparative example two:

the preparation method is the same as the first embodiment except that: in the preparation process of the comparative example, the fermentation medium adopts a low-concentration base sugar medium, and the concentration of the base sugar is 8 percent; wherein the temperature in the early stage of fermentation is maintained at about 33 ℃, the fermentation lasts for about 6-8h, the initial sugar consumption is almost the same, and the sugar feeding is started to ensure that the concentration of residual sugar in the culture medium is always maintained at about 1.0%;

the transparency and purity of the glutamic acid were determined according to the quality requirements for the semi-finished product L-glutamic acid (glutamic acid) in the monosodium glutamate industry manual (second edition):

table 1: comparison of influence of zero initial sugar concentration culture medium process on product quality

	Fermentation medium	Sterilization	Fermentation time/h	Light transmittance	Purity/%)
						Example one	Zero concentration base sugar	Lianxiao medicine for curing diabetes	26	8-10	97
Comparative example 1	High concentration base sugar	Lianxiao medicine for curing diabetes	32	1-3	95
						Comparative example No. two	Low-concentration base sugar	Lianxiao medicine for curing diabetes	30	2-5	95

According to the data analysis in the table 1, the following results are obtained:

compared with a high-concentration base sugar culture medium process and a low-concentration base sugar culture medium process, the zero-initial sugar concentration culture medium process has the advantages that the light transmittance of the fermentation liquid is substantially improved, and because the caramelization reaction in the continuous digestion process is avoided due to the reduction of the glucose concentration;

compared with a high-concentration and low-concentration substrate culture medium process and a low-concentration substrate culture medium process, the zero-initial-sugar-concentration culture medium process has the advantages that the content is improved to a certain extent, probably because the zero-initial-sugar-concentration culture medium avoids the reaction of glucose and liquid ammonia to generate ammonium gluconate, and the ammonium gluconate can influence the purity of the product;

it is worth noting that: compared with the high-concentration and low-concentration base sugar culture medium processes, the zero-initial sugar concentration culture medium process has the effect of further shortening the fermentation time, and the difference of the fermentation time is small between the high-concentration base sugar culture medium process and the low-concentration base sugar culture medium process.

In order to solve the problem of how to further shorten the fermentation time, the method also comprises a method for shortening the fermentation time of glutamic acid, wherein in the early stage of the fermentation stage, on the basis of a zero-initial-sugar-concentration culture medium process, sugar, liquid ammonia and an antifoaming agent are added in a flowing manner, and meanwhile, disodium adenosine triphosphate is added in a flowing manner, and the flowing amount is 5-8 g/L; the method comprises the following specific steps:

example two

The fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a primary seed tank, an amplification culture stage of a secondary seed tank and a fermentation stage, and is characterized in that the fermentation stage comprises:

(1) performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;

(2) when the temperature of the culture medium is reduced to 38 ℃, transplanting common glutamic acid bacteria in a secondary seed tank, fermenting when inoculation is finished, wherein sugar, liquid ammonia, adenosine disodium triphosphate and a defoaming agent are added in the fermentation process, and the adding amount of the adenosine disodium triphosphate is 5-8 g/L; and the concentration of residual sugar in the culture medium is maintained at about 0.6-1.0% all the time, the fermentation lasts for about 8-10h, the OD is increased to 0.8-1.0, the fermentation temperature is raised to 37 ℃, and the fermentation is shifted to the acid production stage;

(3) fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.

Comparative example three:

the preparation method is the same as the second embodiment, except that: in the preparation process of the comparative example, no disodium adenosine triphosphate is fed;

comparative example four:

the preparation method is the same as the third embodiment except that: in the preparation process of the comparative example, no disodium adenosine triphosphate is fed;

comparing the fermentation time and the comprehensive acid yield when the OD of the fermentation liquor reaches 0.8-1.0;

table 2: comparison of the influence of medium with zero initial sugar concentration and adenosine disodium triphosphate on the product quality

	Fermentation medium	Adenosine disodium triphosphate	Fermentation time/h	Acid yield/%	Sugar acid conversion/%
						Example one	Zero concentration base sugar	Is free of	26	18-20.5	70-72
Example two	Zero concentration base sugar	Is provided with	23	19-21	70-72.5
						Comparative example 1	High concentration base sugar	Is free of	32	16-19	67-70
Comparative example No. two	Low-concentration base sugar	Is free of	30	17-20	69-72
						Comparative example No. three	High concentration base sugar	Is provided with	31	16.5-18.5	68-70
Comparative example No. four	Low-concentration base sugar	Is provided with	29	18-20	68-71

According to the data analysis of the table 2, the following results are obtained:

the disodium adenosine triphosphate is fed in the early stage of fermentation, the fermentation time can be improved to a certain extent for the above examples and comparative examples, but the effect is small for a high-concentration primary sugar culture medium, wherein the effect is more remarkable along with the reduction of the glucose concentration, and particularly, the effect can be shortened by nearly 3 hours for a zero-primary sugar culture medium;

by adding disodium adenosine triphosphate, the difference between the acid yield and the sugar acid conversion rate is not large, but the high-concentration primary sugar culture medium has poor effect on both the acid yield and the sugar acid conversion rate compared with a zero-primary sugar culture medium.

Therefore, the invention creatively adds the adenosine disodium triphosphate in the initial fermentation stage, can quickly improve the absorption of the strains on the glucose and achieves the aim of quick growth of the strains;

this is probably because disodium adenosine triphosphate is capable of powering many of the vital activities of the fermenting energy-producing microorganisms, which require the motive force of transmembrane proton gradient production, and therefore these microorganisms also have ATPases to consume ATP to produce a transmembrane proton gradient.

Particularly, the magical discovery shows that the acid yield and the fermentation time are further improved by adding the adenosine disodium triphosphate in the zero-substrate-sugar-concentration fermentation medium process of the invention compared with the traditional high-concentration substrate fermentation method in the comparative example I.

In summary, the following steps:

(1) the invention creatively sets the fermentation culture medium with zero base sugar concentration, and replaces the traditional fermentation mode of the initial addition of high-concentration glucose by the mode of feeding at the beginning, thereby avoiding the problems that the high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction;

(2) the fermentation medium with zero-base sugar concentration is adopted, and the feeding mode is started at a specific flow rate, so that the effect that the early growth of the glutamic acid bacteria is slow because the glucose concentration is reduced unilaterally is surprisingly found, and the fermentation time is further shortened compared with the conventional fermentation mode in which the high-concentration glucose is initially added;

(3) the invention creatively adds the adenosine disodium triphosphate in the initial fermentation stage, can quickly improve the absorption of the strains on glucose, and achieves the aim of further and quickly growing the strains.