Fermentation method of sialic acid

Abstract

The invention discloses a fermentation method of sialic acid, and belongs to the technical field of fermentation engineering. According to the sialic acid fermentation method disclosed by the invention, a staged feeding method is adopted, and a fermentation substrate is controlled by detecting the content of N-acetylglucosamine in the fermentation process, so that high-content fermentation of the sialic acid by escherichia coli is realized. The method provided by the invention solves the limitation of acetic acid and other heteroacids on the generation of sialic acid in the escherichia coli fermentation process.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, and more particularly to a method for fermenting sialic acid. Background Art

[0002] Sialic acid, also known as N-acetylneuraminic acid (NeuAc for short), is a type of negatively charged monosaccharide. NeuAc plays a key role in many important physiological and pathological processes. As an important functional monosaccharide, NeuAc has multiple biological activities. First of all, NeuAc can promote neuron sprouting. NeuAc can enter the embryonic and infant nervous system, combine with glycosphingolipids to form gangliosides, and promote the development of the nervous system. NeuAc can improve learning and memory ability, infant bone growth, absorption of vitamins and minerals, and brain and bone development. NeuAc also has anti-inflammatory, anti-cancer, anti-viral, cell adhesion inhibition and as a modified drug carrier. It has important development value in many physiological and pathological processes including cancer metastasis and the field of medicine.

[0003] Sialic acid can be fermented by Escherichia coli engineering bacteria. Glucose in the culture medium is often excessive. Escherichia coli is prone to produce a large amount of acetic acid under oxygen-deficient or aerobic conditions. Accumulated acetic acid not only inhibits the growth of Escherichia coli and reduces the cell yield, but also inhibits the synthesis of exogenous proteins and sugars. Escherichia coli uses glucose in the cell to form a large amount of acetic acid, which reduces the carbon source for synthesizing the required metabolites through the tricarboxylic acid cycle, produces energy dissipation, reduces the substrate conversion rate, and causes a low sialic acid yield. Erythritol mother liquor contains a large amount of nitrogen source, glucose, soluble protein, inorganic salts and glycerol, and subsequent processing is difficult. Using erythritol mother liquor as a culture medium can achieve post-processing of sugar alcohol mother liquor, and by controlling the ratio of glycerol to glucose, the sialic acid yield can be increased to reduce the purpose of acetic acid production.

[0004] Therefore, providing a sialic acid fermentation method is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a sialic acid fermentation method, which adopts a staged feeding method, controls the fermentation substrate by detecting the GlcNAc (N-acetylglucosamine) content during the fermentation process, realizes high-content fermentation of Escherichia coli, and finally achieves a sialic acid content of 50g / L at the end of fermentation; and solves the limitation of sialic acid production by acetic acid and other miscellaneous acids during the fermentation of Escherichia coli.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm is 3-4, and the first-level seed liquid is obtained.

[0007] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm is 3-4, and the secondary seed liquid is obtained.

[0008] (3) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the pressure is less than 15, glycerol is added as feed according to the glucose content, the concentration ratio of glycerol to glucose is controlled to be 1-3:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%.

[0009] (4) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1-3; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1-4; and when OD600nm≥15, the total amount of glucose and glycerol does not exceed 10g / L, and the fermentation is carried out until the sialic acid content does not increase.

[0010] Furthermore, the primary culture medium and the secondary culture medium are both LB culture medium.

[0011] Furthermore, the fermentation medium formula is: erythritol mother liquor adjusted to a soluble solid content of 3-5%, glucose 5g / L, potassium dihydrogen phosphate 3g / L, yeast powder 1.5g / L, trace elements 1mL / L, and polyether defoaming agent 0.15mL / L.

[0012] The parameters of the erythritol mother liquor are as follows: soluble solid content is 40-45%, erythritol content is 180-200 g / L, glycerol content is 30-40 g / L; residual sugar content is 3-5 g / L in terms of glucose.

[0013] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0014] Furthermore, the glycerol feed is a 30% glycerol aqueous solution.

[0015] The glucose feed is: 300 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L yeast powder, and 2.9 mL / L trace elements.

[0016] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0017] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses a sialic acid fermentation method, glycerol is mainly used to provide a skeleton for cell growth and PEP (phosphoenolpyruvate) supply, glucose is mainly used for the synthesis of GlcNAc (N-acetylglucosamine) and ManNAc (N-acetylmannosamine), and the supply of precursors PEP and ManNAc is balanced by controlling the ratio of glycerol to glucose in stages. In the early stage of fermentation, a high density of bacteria needs to be achieved, so increasing the proportion of glycerol promotes cell growth. During the production of sialic acid in the middle stage of fermentation, increasing the proportion of glucose according to the intermediate GlcNAc can reduce the production of acetic acid. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Recombinant strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE, ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB see patent 202210690425.4.

[0020] Both the primary culture medium and the secondary culture medium were LB medium.

[0021] Example 1 A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc-MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm The value was 3.95, and the first-grade seed solution was obtained.

[0022] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm is 3.80, and the secondary seed solution is obtained.

[0023] (3) Fermentation medium formula: erythritol mother liquor adjusted to 4.3% soluble solid content, glucose 5 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 1 mL / L, polyether defoaming agent 0.15 mL / L; sterilized at 115°C for 20 min.

[0024] The parameters of the erythritol mother liquor are: soluble solid content is 42.3%, erythritol content is 190.65g / L, glycerol content is 35.78g / L; residual sugar content calculated as glucose is 3.35g / L.

[0025] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0026] Feed formula: Glycerol feed is 30% glycerol aqueous solution.

[0027] Glucose feed was: glucose 300 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 2.9 mL / L.

[0028] (4) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the pressure is less than 15, glycerol feed is added according to the glucose content, the concentration ratio of glycerol to glucose is controlled to be 2:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%.

[0029] (5) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:2; and when OD600nm≥15, the total amount of glucose and glycerol does not exceed 10g / L, and the fermentation is carried out until the sialic acid content does not increase.

[0030] GlcNAc detection conditions: Waters 2414 differential detector, BIO RAD Aminex HPX-87H chromatographic column, mobile phase 5 mM sulfuric acid solution, column temperature 60 ° C, flow rate 0.5 mL / min.

[0031] (6) After centrifugation in the lower tank, the sialic acid content and the acetate content were tested. The sialic acid content was 50.25 g / L and the acetate content was 5.33 g / L.

[0032] Example 2 A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm The value was 3.26, and the first-grade seed solution was obtained.

[0033] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm The value was 3.68, and the secondary seed solution was obtained.

[0034] (3) Fermentation medium formula: erythritol mother liquor adjusted to 4.8% soluble solids content, glucose 5 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 1 mL / L, polyether defoaming agent 0.15 mL / L; sterilized at 115°C for 20 min.

[0035] The parameters of the erythritol mother liquor are: soluble solid content is 44.7%, erythritol content is 195.63g / L, glycerol content is 37.23g / L; residual sugar content calculated as glucose is 4.35g / L.

[0036] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0037] Feed formula: Glycerol feed is 30% glycerol aqueous solution.

[0038] Glucose feed was: glucose 300 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 2.9 mL / L.

[0039] (4) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the pressure is less than 15, glycerol feed is added according to the glucose content, the concentration ratio of glycerol to glucose is controlled to be 1:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%.

[0040] (5) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:2; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1; and OD 600nm When the pH value is ≥15, the total amount of glucose and glycerol shall not exceed 10g / L, and the fermentation shall be carried out until the sialic acid content does not increase.

[0041] GlcNAc detection conditions: Waters 2414 differential detector, BIO RAD Aminex HPX-87H chromatographic column, mobile phase 5 mM sulfuric acid solution, column temperature 60 ° C, flow rate 0.5 mL / min.

[0042] (6) After centrifugation in the lower tank, the sialic acid content and the acetate content were tested. The sialic acid content was 35.25 g / L, and the acetate content was 12.53 g / L.

[0043] Example 3 A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm The value is 3.55, and the first-grade seed solution is obtained.

[0044] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm The value was 3.92, and the secondary seed solution was obtained.

[0045] (3) Fermentation medium formula: erythritol mother liquor adjusted to 4.5% soluble solid content, glucose 5 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 1 mL / L, polyether defoaming agent 0.15 mL / L; sterilized at 115°C for 20 min.

[0046] The parameters of the erythritol mother liquor are: soluble solid content is 43.2%, erythritol content is 190.73 g / L, glycerol content is 39.62 g / L; residual sugar content calculated as glucose is 4.43 g / L.

[0047] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0048] Feed formula: Glycerol feed is 30% glycerol aqueous solution.

[0049] Glucose feed was: glucose 300 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 2.9 mL / L.

[0050] (4) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the pressure is less than 15, glycerol feed is added according to the glucose content, the concentration ratio of glycerol to glucose is controlled to be 3:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%.

[0051] (5) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:3; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:4; and OD 600nm When the pH value is ≥15, the total amount of glucose and glycerol shall not exceed 10g / L, and the fermentation shall be carried out until the sialic acid content does not increase.

[0052] GlcNAc detection conditions: Waters 2414 differential detector, BIO RAD Aminex HPX-87H chromatographic column, mobile phase 5 mM sulfuric acid solution, column temperature 60 ° C, flow rate 0.5 mL / min.

[0053] (6) After centrifugation in the lower tank, the sialic acid content and the acetate content were tested. The sialic acid content was 30.25 g / L, and the acetate content was 22.53 g / L.

[0054] Comparative Example 1 (using only glucose as feed) A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm The value was 3.77, and the first-grade seed solution was obtained.

[0055] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm The value was 4.03, and the secondary seed solution was obtained.

[0056] (3) Preparation and sterilization of fermentation medium: glucose 12 g / L, yeast powder 12 g / L, peptone 6 g / L, dipotassium hydrogen phosphate trihydrate 5 g / L, potassium dihydrogen phosphate 3 g / L, trace elements 1 mL / L, defoamer (polyether defoamer) 0.15 mL / L, make up to 10 L, and sterilize at 115 °C for 20 min.

[0057] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0058] (4) Feed preparation and sterilization: Glucose feed formula: glucose 300 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 2.9 mL / L, sterilized at 115 °C for 20 min.

[0059] (5) The fermentation medium and secondary seed liquid were connected to the fermentation tank, and the inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. During the fermentation process, the dissolved oxygen was controlled at 10%-25%. After the initial sugar was exhausted, glucose was continued to be fed to control the glucose content to 3-5 g / L. The tank was removed after the sialic acid content stopped increasing.

[0060] (6) After centrifugation in the lower tank, the sialic acid content and the acetate content were tested. The sialic acid content was 20.15 g / L and the acetate content was 19.23 g / L.

[0061] Comparative Example 2 A sialic acid fermentation method, the specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::PgprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm The value was 4.32, and the first-grade seed solution was obtained.

[0062] (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm The value was 3.95, and the secondary seed solution was obtained.

[0063] (3) Fermentation medium formula: erythritol mother liquor adjusted to 4.7% soluble solids content, glucose 5 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 1 mL / L, polyether defoaming agent 0.15 mL / L; sterilized at 115°C for 20 min.

[0064] The parameters of the erythritol mother liquor are: soluble solid content is 42.6%, erythritol content is 185.73 g / L, glycerol content is 35.26 g / L; residual sugar content calculated as glucose is 3.77 g / L.

[0065] Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

[0066] Feed formula: Glycerol feed is 30% glycerol aqueous solution.

[0067] Glucose feed was: glucose 300 g / L, potassium dihydrogen phosphate 3 g / L, yeast powder 1.5 g / L, trace elements 2.9 mL / L.

[0068] (4) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the pressure is less than 15, glycerol feed is added according to the glucose content, the concentration ratio of glycerol to glucose is controlled to be 4:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%.

[0069] (5) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:4; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:5; and OD 600nmWhen it is ≥15, the total amount of glucose and glycerol does not exceed 10 g / L, and the fermentation is terminated when the sialic acid content no longer increases.

[0070] GlcNAc detection conditions: Waters 2414 differential refractometer, BIO RAD Aminex HPX-87H chromatographic column, mobile phase is 5 mM sulfuric acid solution, column temperature is 60 °C, and flow rate is 0.5 mL / min.

[0071] (6) After centrifugation treatment upon termination of fermentation, the sialic acid content and acetate content are detected. The sialic acid content is 25.26 g / L, and the acetate content is 25.33 g / L.

[0072] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sialic acid fermentation method, characterized in that: The specific steps are as follows: (1) Primary seed culture: The recombinant Escherichia coli strain Ecoli. BL21(DE3)ΔnanATEK,ΔnagABE, ΔmanXYZ::P gprE -NeuC, ΔpoxB::P trc -MovNeuB was inoculated into the primary culture medium at an inoculum size of 1% and cultured at 37°C and 220 rpm until OD 600nm 3-4, to obtain the first-grade seed solution; (2) Secondary seed culture: Transfer the primary seed solution to the secondary culture medium with an inoculation rate of 1% and culture at 37°C and 220 r / min until the OD 600nm is 3-4, and the secondary seed solution is obtained; (3) The fermentation medium and secondary seed liquid were added to the fermentation tank. The inoculation amount of the secondary seed liquid was 1%. The fermentation temperature was controlled at 37°C and the pH was 7. The glycerol and glucose contents, OD 600nm When the temperature is less than 15, glycerol is added as feed according to the glucose content, and the concentration ratio of glycerol to glucose is controlled to be 1-3:1, and the total amount of glucose and glycerol does not exceed 30g / L, and the dissolved oxygen is controlled at 10-15%; (4) OD 600nm When the concentration is ≥15, the dissolved oxygen is controlled at 15-20%, the pH is controlled at 7, and glucose feeding is started. During the fermentation process, the ratio of glycerol to glucose is controlled according to the content of intermediate GlcNAc: When the GlcNAc content is greater than or equal to 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1-3; when the GlcNAc content is less than 5g / L, the glycerol: glucose concentration ratio is controlled to be 1:1-4; and when OD600nm≥15, the total amount of glucose and glycerol does not exceed 10g / L, and the fermentation is carried out until the sialic acid content does not increase.

2. A sialic acid fermentation method according to claim 1, characterized in that, The primary culture medium and the secondary culture medium are both LB culture medium.

3. A sialic acid fermentation method according to claim 1, characterized in that: The fermentation medium formula is: erythritol mother liquor adjusted to 3-5% soluble solid content, glucose 5g / L, potassium dihydrogen phosphate 3g / L, yeast powder 1.5g / L, trace elements 1mL / L, polyether defoamer 0.15mL / L; The parameters of the erythritol mother liquor are: soluble solid content of 40-45%, erythritol content of 180-200 g / L, glycerol content of 30-40 g / L; residual sugar content in terms of glucose of 3-5 g / L; Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.

4. A sialic acid fermentation method according to claim 1, characterized in that, The glycerol feed is a 30% glycerol aqueous solution; The glucose feed is: glucose 300g / L, potassium dihydrogen phosphate 3g / L, yeast powder 1.5g / L, trace elements 2.9mL / L; Trace element formula: ferrous sulfate heptahydrate 5g / L, manganese sulfate pentahydrate 3g / L, zinc sulfate heptahydrate 0.3g / L.