Green and safe mannitol biosynthesis method

Abstract

The invention provides a green and safe mannitol biosynthesis method, and belongs to the technical field of mannitol biosynthesis. The synthesis method comprises the steps of primary seed culture, secondary seed culture and fermentation culture. Secondary seed culture: inoculating the primary seed culture solution into a secondary seed culture medium, and culturing to obtain a secondary seed culture solution; the secondary seed culture medium comprises the following components: glucose, peptone, yeast extract, dipotassium phosphate, glycerol, inositol, trehalose and an additive; two times of seed culture and fermentation culture are adopted, synthesis of saccharomycetes in the mannitol direction is effectively promoted, the fermentation time is shortened, the yield and quality of mannitol are improved, the production efficiency is greatly improved, good biological safety is achieved, culture conditions are mild, dangerous and high-energy-consumption operation such as high temperature and high pressure is avoided, and the method is suitable for industrial production. The safety is high, and the concept of green and safe production is met.

Description

Technical Field

[0001] The invention belongs to the technical field of mannitol biosynthesis, and in particular relates to a green and safe mannitol biosynthesis method. Background Art

[0002] Mannitol is a hexasol, a hexavalent sugar alcohol, and an isomer of sorbitol. Mannitol is a colorless, white needle-shaped or columnar crystalline powder with a refreshing sweet taste, a melting point of 166°C, and a boiling point of 290-295°C. Mannitol is soluble in hot water, pyridine and aniline, but insoluble in ether. It is the only non-hygroscopic crystal.

[0003] Mannitol has a wide range of application values ​​in medicine, food and industry. In the field of medicine, mannitol is a good diuretic. Mannitol injection, as a high-osmotic antihypertensive drug, is a commonly used drug for clinical rescue, especially for brain disease rescue, with the characteristics of rapid blood pressure reduction and accurate efficacy. In the field of food, mannitol has low water absorption and a refreshing sweet taste. It is used to prevent maltose, chewing gum, rice cakes and other foods from sticking, and can also be used as a low-sugar sweetener. In industry, mannitol is an important raw material involved in the production of many products. With the development of various industries, the demand for mannitol continues to grow, and its synthesis method has attracted much attention.

[0004] At present, the synthesis methods of mannitol mainly include extraction from natural seaweed, chemical synthesis and microbial synthesis; The extraction method from natural seaweed mainly uses seaweed as raw material, crushes the seaweed to release the substances in the cells, and then uses hot water extraction or organic solvent extraction to extract mannitol from the seaweed cells into a solution. The extract is filtered, centrifuged and other separation methods to remove insoluble impurities, and then through ion exchange, desorption and crystallization steps, the mannitol product is obtained; however, the supply of seaweed raw materials is unstable and cannot meet large-scale production needs, and the preparation process is complicated and the cost is high. More importantly, more chemical additives are required in the separation process, which poses a great safety hazard and is not conducive to green and safe production.

[0005] The chemical synthesis method usually uses sucrose, glucose or starch as the starting materials and nickel as the catalyst to prepare mannitol through catalytic hydrogenation under high temperature and high pressure conditions. This method has a low yield and contains a large amount of sorbitol by-products. The subsequent separation and purification costs are high, and the high temperature and high pressure reaction conditions have high energy consumption and produce more pollutants, posing a threat to the life and health of personnel and the safety of the production environment, which does not conform to the concept of green and safe production.

[0006] The biosynthesis method generally adopts the microbial fermentation method, specifically, a microbial strain capable of synthesizing mannitol is inoculated into a fermentation medium, and fermented and cultured under suitable temperature and dissolved oxygen conditions. After the fermentation is completed, the mannitol product is obtained through centrifugation, separation and purification.

[0007] Compared with the above two methods, the microbial fermentation method has significant advantages, especially in terms of safe production. It has good biosafety and will not cause harm to operators and the environment. The reaction conditions and temperature avoid the safety risks brought by operating conditions such as high temperature and high pressure, reduce energy consumption, have few by-products during the reaction process, and are highly safe, which is in line with the concept of green and safe production.

[0008] Therefore, developing a green and safe mannitol biosynthesis method through microbial fermentation technology is a key issue that needs to be urgently addressed in this field.

[0009] However, the existing method of synthesizing mannitol using microbial fermentation technology has the defects of low conversion rate and yield, low production efficiency, and cannot meet the needs of large-scale production. In addition, the product quality of mannitol is unstable.

[0010] From the above, it can be seen that it is very necessary to provide a green and safe mannitol biosynthesis method to improve production efficiency, increase conversion rate and yield, and ensure the product quality of mannitol. Summary of the invention

[0011] In order to solve the technical problems existing in the prior art, the present invention provides a green and safe mannitol biosynthesis method, which improves production efficiency, conversion rate and yield, and ensures the product quality of mannitol.

[0012] In view of the above technical problems, the present invention adopts the following technical solutions: A green and safe mannitol biosynthesis method includes primary seed culture, secondary seed culture and fermentation culture steps, and the specific operations are as follows: 1. Primary seed culture The yeast is placed in a primary seed culture medium for cultivation at a temperature of 25.0-27.0° C. for a time of 31.6-32.5 hours. After the cultivation is completed, a primary seed culture solution is obtained; The yeast is purchased from the market; The components of the primary seed culture medium are glucose 18-22 g / L, peptone 16-19 g / L, yeast extract 10-13 g / L, ammonium sulfate 0.12-0.17 g / L, and magnesium sulfate 0.08-0.12 g / L.

[0013] 2. Secondary seed culture The primary seed culture solution is inoculated into the secondary seed culture medium at an inoculation rate of 5.5-6.5% for culturing at a temperature of 27.6-28.4°C for 19.5-20.5 hours. After the culturing is completed, the secondary seed culture solution is obtained; The components of the secondary seed culture medium are glucose 21.5-22.5 g / L, peptone 18-22 g / L, yeast extract 11-13 g / L, dipotassium hydrogen phosphate 1.4-1.7 g / L, glycerol 1.8-2.1 g / L, inositol 0.10-0.14 mg / L, trehalose 0.9-1.2 g / L, and admixture 0.23-0.27 mg / L; The additive comprises a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.2-1.5.

[0014] 3. Fermentation culture The secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 5.0-7.0% for fermentation culture, the fermentation temperature is controlled to be 29-32°C, the fermentation time is 31.0-33.0h, the ventilation volume is 0.8-1.2vvm, and then the fermentation temperature is lowered to 25-27°C to continue fermentation. When the fermentation reaches 36.0-37.0h, the ventilation volume is lowered to 0.2-0.4vvm, and the fermentation promoter is slowly added at a controllable addition rate of 0.16-0.22g / min / L and an addition time of 1.8-2.2h. After the addition is completed, the fermentation is continued to 46.1-46.5h to obtain a fermentation liquid; The fermentation medium comprises 28.0-29.0 g / L of glucose, 16.4-17.5 g / L of peptone, 4.8-5.2 g / L of yeast extract, 0.4-0.6 g / L of ammonium sulfate, 0.10-0.14 g / L of dipotassium hydrogen phosphate, 1.4-1.7 mg / L of thiamine, 0.10-0.13 g / L of L-glutamic acid, and 0.08-0.12 g / L of DL-methionine; The fermentation promoter comprises a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols, wherein the mass ratio of the deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.4-1.6:0.5-0.7:0.8-1.2.

[0015] 4. Post-processing After the fermentation liquid is centrifuged, a primary supernatant is collected, an equal volume of anhydrous ethanol is added to the primary supernatant, and the mixture is centrifuged at 9800-10300 rpm for 15-20 minutes to obtain a secondary supernatant, and the secondary supernatant is crystallized at 3.8-4.2° C. to obtain a mannitol product.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention adopts yeast as the original strain to ferment and synthesize mannitol. First, the yeast is subjected to two seed cultures. In the first seed culture step, under the synergistic culture of ammonium sulfate and magnesium sulfate, sufficient nutrition can be provided for the strain, the metabolic activity of the yeast can be promoted, the number and activity of the strain can be increased, and sufficient strains in good condition can be provided for the second seed culture; in the second seed culture step, the number of yeast is further expanded so that the yeast can adapt to subsequent fermentation conditions, wherein the inositol in the second seed culture medium, as a growth factor for yeast, is conducive to maintaining the integrity of the structure and function of the cell membrane, and combined with trehalose, it can better maintain the stability of the structure and function of the cell and prevent the destruction of the membrane structure, thereby effectively regulating the growth and metabolic activities of the yeast, and the admixture can provide energy for the cells, which has a good promoting effect on the metabolism of the yeast and the synthesis of mannitol; in the fermentation culture step, by reasonably controlling parameters such as temperature and ventilation volume, the yeast is prompted to synthesize mannitol in large quantities, thereby ensuring the mannitol The yield and quality of mannitol can be improved. The ammonium sulfate component can provide ammonium ions for yeast and participate in the nitrogen metabolism process in the cell. Thiamine can act as a coenzyme to participate in the enzymatic reaction in the yeast metabolism process and has an important regulatory effect on the growth and reproduction of yeast. The amino acid component is used as a nitrogen source and participates in amino acid metabolism at the same time, regulating the metabolic balance and protein synthesis in the cell. The present invention adds a fermentation promoter in the middle and late fermentation, which can promote the yeast metabolism to the direction of mannitol synthesis, regulate the permeability of the cell membrane, and promote the metabolic activity, thereby effectively improving the yield and quality of mannitol. The present invention adopts yeast, and after two seed culture and fermentation culture steps, the yeast is effectively promoted to synthesize mannitol, shortens the fermentation time, improves the yield and quality of mannitol, greatly improves the production efficiency, and has good biosafety, does not cause harm to operators and the environment, and the culture conditions are mild, avoiding dangerous and energy-intensive operating conditions such as high temperature and high pressure, and has high safety, which conforms to the concept of green and safe production. 2. The fermentation broth obtained by the synthesis method of the present invention has a mannitol content of 58.2-58.7 g / L and a succinic acid content of 0.84-0.87 g / L. The purity of the mannitol finished product is tested to be 99.47-99.53%. DETAILED DESCRIPTION

[0017] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0018] Example 1 1. Primary seed culture The yeast was placed in a primary seed culture medium for cultivation at a temperature of 26.0°C for a time of 32.0 hours. After the cultivation was completed, a primary seed culture solution was obtained. The yeast is purchased from the market; The components of the primary seed culture medium are 20 g / L glucose, 18 g / L peptone, 11 g / L yeast extract, 0.14 g / L ammonium sulfate, and 0.10 g / L magnesium sulfate.

[0019] 2. Secondary seed culture The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 6.0% for culturing at a temperature of 28.0°C for 20.0 hours. After the culturing was completed, the secondary seed culture solution was obtained; The components of the secondary seed culture medium are glucose 22 g / L, peptone 20 g / L, yeast extract 12 g / L, dipotassium hydrogen phosphate 1.6 g / L, glycerol 2.0 g / L, inositol 0.12 mg / L, trehalose 1.0 g / L, and admixture 0.25 mg / L; The additive comprises a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.3.

[0020] 3. Fermentation culture The secondary seed culture solution was inoculated into the fermentation medium at an inoculation rate of 6.0% for fermentation culture, and the fermentation temperature was controlled to be 30°C, the fermentation time was 32.0 hours, and the ventilation volume was 1.0 vvm. Then the fermentation temperature was lowered to 26°C and the fermentation was continued. When the fermentation reached 36.5 hours, the ventilation volume was lowered to 0.3 vvm, and the fermentation promoter was slowly added at a controllable addition rate of 0.2 g / min / L and an addition time of 2.0 hours. After the addition was completed, the fermentation was continued for 46.3 hours to obtain the fermentation liquid. The fermentation medium comprises 28.5 g / L glucose, 17.0 g / L peptone, 5.0 g / L yeast extract, 0.5 g / L ammonium sulfate, 0.12 g / L dipotassium hydrogen phosphate, 1.5 mg / L thiamine, 0.12 g / L L-glutamic acid, and 0.10 g / L DL-methionine. The fermentation promoter comprises a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols, and the mass ratio of the deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.5:0.6:1.0.

[0021] 4. Post-processing After the fermentation liquid is centrifuged, a primary supernatant is collected, an equal volume of anhydrous ethanol is added to the primary supernatant, and the mixture is centrifuged at 10,000 rpm for 18 minutes to obtain a secondary supernatant, and the secondary supernatant is crystallized at 4.0° C. to obtain a mannitol product.

[0022] The fermentation broth obtained by the method of Example 1 had a mannitol content of 58.7 g / L and a succinic acid content of 0.82 g / L. The purity of the mannitol product was tested to be 99.53%.

[0023] Example 2 1. Primary seed culture The yeast was placed in a primary seed culture medium for cultivation at a temperature of 25.0°C for 32.5 hours. After the cultivation was completed, a primary seed culture solution was obtained. The yeast is purchased from the market; The components of the primary seed culture medium are 18 g / L glucose, 16 g / L peptone, 10 g / L yeast extract, 0.12 g / L ammonium sulfate, and 0.08 g / L magnesium sulfate.

[0024] 2. Secondary seed culture The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 5.5% for culturing at a temperature of 27.6°C for 19.5 hours. After the culturing was completed, the secondary seed culture solution was obtained; The components of the secondary seed culture medium are glucose 21.5 g / L, peptone 18 g / L, yeast extract 11 g / L, dipotassium hydrogen phosphate 1.4 g / L, glycerol 1.8 g / L, inositol 0.10 mg / L, trehalose 0.9 g / L, and admixture 0.23 mg / L; The additive comprises a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.2.

[0025] 3. Fermentation culture The secondary seed culture solution was inoculated into the fermentation medium at an inoculation rate of 5.0% for fermentation culture, and the fermentation temperature was controlled to be 29°C, the fermentation time was 31.0 h, and the ventilation volume was 0.8 vvm. Then the fermentation temperature was lowered to 25°C and the fermentation was continued. When the fermentation reached 36.0 h, the ventilation volume was lowered to 0.2 vvm, and the fermentation promoter was slowly added at a controllable addition rate of 0.16 g / min / L and an addition time of 1.8 h. After the addition was completed, the fermentation was continued for 46.1 h to obtain the fermentation liquid. The fermentation medium comprises 28.0 g / L glucose, 16.4 g / L peptone, 4.8 g / L yeast extract, 0.4 g / L ammonium sulfate, 0.10 g / L dipotassium hydrogen phosphate, 1.4 mg / L thiamine, 0.10 g / L L-glutamic acid, and 0.08 g / L DL-methionine. The fermentation promoter comprises a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols, wherein the mass ratio of the deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.4:0.5:0.8.

[0026] 4. Post-processing After the fermentation liquid is centrifuged, a primary supernatant is collected, an equal volume of anhydrous ethanol is added to the primary supernatant, and the mixture is centrifuged at 9800 rpm for 20 minutes to obtain a secondary supernatant, and the secondary supernatant is crystallized at 3.8° C. to obtain a mannitol product.

[0027] The fermentation broth obtained by the method of Example 2 had a mannitol content of 58.2 g / L and a succinic acid content of 0.87 g / L. The purity of the mannitol product was tested to be 99.47%.

[0028] Example 3 1. Primary seed culture The yeast was placed in a primary seed culture medium for cultivation at a temperature of 27.0°C for a time of 31.6 hours. After the cultivation was completed, a primary seed culture solution was obtained. The yeast is purchased from the market; The components of the primary seed culture medium are 22 g / L glucose, 19 g / L peptone, 13 g / L yeast extract, 0.17 g / L ammonium sulfate, and 0.12 g / L magnesium sulfate.

[0029] 2. Secondary seed culture The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 6.5% for culturing at a temperature of 28.4°C for 20.5 hours. After the culturing was completed, the secondary seed culture solution was obtained; The components of the secondary seed culture medium are glucose 22.5 g / L, peptone 22 g / L, yeast extract 13 g / L, dipotassium hydrogen phosphate 1.7 g / L, glycerol 2.1 g / L, inositol 0.14 mg / L, trehalose 1.2 g / L, and admixture 0.27 mg / L; The additive comprises a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.5.

[0030] 3. Fermentation culture The secondary seed culture solution was inoculated into the fermentation medium at an inoculation rate of 7.0% for fermentation culture, and the fermentation temperature was controlled to be 32°C, the fermentation time was 33.0 h, and the ventilation volume was 1.2 vvm. Then the fermentation temperature was lowered to 27°C and the fermentation was continued. When the fermentation reached 37.0 h, the ventilation volume was lowered to 0.4 vvm, and the fermentation promoter was slowly added at a controllable addition rate of 0.22 g / min / L and an addition time of 2.2 h. After the addition was completed, the fermentation was continued for 46.5 h to obtain the fermentation liquid. The fermentation medium comprises 29.0 g / L glucose, 17.5 g / L peptone, 5.2 g / L yeast extract, 0.6 g / L ammonium sulfate, 0.14 g / L dipotassium hydrogen phosphate, 1.7 mg / L thiamine, 0.13 g / L L-glutamic acid, and 0.12 g / L DL-methionine. The fermentation promoter comprises a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols, and the mass ratio of the deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.6:0.7:1.2.

[0031] 4. Post-processing After the fermentation liquid is centrifuged, a primary supernatant is collected, an equal volume of anhydrous ethanol is added to the primary supernatant, and the mixture is centrifuged at 10,300 rpm for 15 minutes to obtain a secondary supernatant, and the secondary supernatant is crystallized at 4.2° C. to obtain a mannitol product.

[0032] The fermentation broth obtained by the method of Example 3 had a mannitol content of 58.5 g / L and a succinic acid content of 0.84 g / L. The purity of the mannitol product was tested to be 99.50%.

[0033] Comparative Example 1 Based on Example 1, the changes are as follows: In the secondary seed culture step, the "glycerol, inositol, trehalose" components were omitted; In the fermentation culture step, the operation step of "slowly adding 23.0 g of fermentation promoter and controlling the addition rate to 0.2 g / min" was omitted; The rest of the operations are the same.

[0034] The fermentation liquid obtained by the method of Comparative Example 1 had a mannitol content of 49.2 g / L and a succinic acid content of 1.66 g / L. The purity of the mannitol finished product was tested to be 94.52%.

[0035] Comparative Example 2 Based on Example 1, the changes are as follows: In the secondary seed culture step, the "admixture" component in the secondary seed culture medium is omitted; Omit the ingredients "thiamine, L-glutamic acid, DL-methionine" in the fermentation medium; The rest of the operations are the same.

[0036] The fermentation broth obtained by the method of Comparative Example 2 had a mannitol content of 51.4 g / L and a succinic acid content of 1.51 g / L. The purity of the mannitol finished product was tested to be 95.36%.

[0037] The invention adopts yeast as the original strain to ferment and synthesize mannitol. First, the yeast is subjected to two seed cultures. In the first seed culture step, under the coordinated culture of ammonium sulfate and magnesium sulfate, sufficient nutrition can be provided for the strain, the metabolic activity of the yeast can be promoted, the number and activity of the strain can be increased, and sufficient strains in good condition can be provided for the second seed culture. In the second seed culture step, the number of yeast is further expanded so that the yeast can adapt to subsequent fermentation conditions. In the second seed culture step, inositol in the second seed culture medium is used as a growth factor for yeast, which is beneficial to maintaining the integrity of the structure and function of the cell membrane. In combination with trehalose, the stability of the structure and function of the cell can be better maintained, and the destruction of the membrane structure can be prevented, thereby effectively regulating the growth and metabolic activities of the yeast. The admixture can provide energy for the cells, and has a good promoting effect on the metabolism of the yeast and the synthesis of mannitol. In the fermentation culture step, by reasonably controlling parameters such as temperature and ventilation volume, the yeast is prompted to synthesize a large amount of mannitol, thereby ensuring the mannitol. The invention has the advantages of high yield and high quality, wherein the ammonium sulfate component can provide ammonium ions for yeast and participate in the nitrogen metabolism process in the cell, thiamine can act as a coenzyme to participate in the enzymatic reaction in the yeast metabolism process, and has an important regulatory effect on the growth and reproduction of yeast, and the amino acid component, as a nitrogen source, participates in amino acid metabolism at the same time, and regulates the metabolic balance and protein synthesis in the cell; the invention adds a fermentation promoter in the middle and late fermentation period, which can promote the yeast metabolism to proceed in the direction of mannitol synthesis, regulate the permeability of the cell membrane, and promote the metabolic activity, thereby effectively improving the yield and quality of mannitol; the invention adopts yeast, and effectively promotes the synthesis of mannitol by yeast through two seed culture and fermentation culture steps, shortens the fermentation time, improves the yield and quality of mannitol, greatly improves the production efficiency, and has good biosafety, does not cause harm to operators and the environment, and has mild culture conditions, avoids dangerous and energy-intensive operating conditions such as high temperature and high pressure, has high safety, and conforms to the concept of green and safe production.

[0038] Mannitol was synthesized by the method of Comparative Example 1. The glycerol, inositol and trehalose components were omitted in the secondary seed culture step, resulting in poor structural and functional stability of the cells, reducing the growth metabolic activity of the yeast, and omitting the step of adding a fermentation promoter in the fermentation culture step, which failed to provide sufficient nutrients for the fermentation activity, resulting in unstable metabolic activity, weakening the synthesis of mannitol by the yeast, reducing the mannitol content in the fermentation broth, and reducing the purity of the product; Comparative Example 2 omits the admixture component in the secondary culture step, reduces the supply of cellular energy, and cannot effectively promote metabolic activity. Comparative Example 2 also omits the thiamine and amino acid components in the fermentation medium, resulting in incomplete enzymatic fermentation, insufficient nutrients, and incomplete cell metabolism, which ultimately increases the content of by-products, reduces the mannitol content, and has low product purity.

[0039] Unless otherwise specified, all percentages used in the present invention are by mass.

[0040] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A green and safe mannitol biosynthesis method, characterized in that: The method comprises the steps of primary seed culture, secondary seed culture and fermentation culture; The secondary seed culture step comprises: inoculating the primary seed culture solution into the secondary seed culture medium to obtain the secondary seed culture solution; The components of the secondary seed culture medium are glucose, peptone, yeast extract, dipotassium hydrogen phosphate, glycerol, inositol, trehalose, and additives; The fermentation and culturing step comprises: inoculating the secondary seed culture liquid into the fermentation medium, fermenting at 29-32° C. for 31.0-33.0 hours, with an aeration volume of 0.8-1.2 vvm, lowering the fermentation temperature to 25-27° C. and continuing the fermentation, and when the fermentation reaches 36.0-37.0 hours, lowering the aeration volume to 0.2-0.4 vvm, adding a fermentation promoter, controlling the addition rate to 0.16-0.22 g / min / L, and adding for 1.8-2.2 hours, and after the addition is completed, fermenting to 46.1-46.5 hours to obtain a fermentation liquid; The fermentation medium comprises glucose, peptone, yeast extract, ammonium sulfate, dipotassium hydrogen phosphate, thiamine, L-glutamic acid and DL-methionine; The fermentation promoting agent comprises a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols.

2. A green and safe mannitol biosynthesis method according to claim 1, characterized in that: The primary seed culture step comprises placing the yeast in a primary seed culture medium for culture at a temperature of 25.0-27.0° C. for a time of 31.6-32.5 hours, and obtaining a primary seed culture solution after the culture is completed; The components of the primary seed culture medium are glucose 18-22 g / L, peptone 16-19 g / L, yeast extract 10-13 g / L, ammonium sulfate 0.12-0.17 g / L, and magnesium sulfate 0.08-0.12 g / L.

3. A green and safe mannitol biosynthesis method according to claim 1, characterized in that: The secondary seed culture step is to inoculate the primary seed culture solution into the secondary seed culture medium at an inoculation rate of 5.5-6.5% for culture at a temperature of 27.6-28.4° C. for a culture time of 19.5-20.5 h. After the culture is completed, the secondary seed culture solution is obtained.

4. A green and safe mannitol biosynthesis method according to claim 3, characterized in that: The components of the secondary seed culture medium are glucose 21.5-22.5 g / L, peptone 18-22 g / L, yeast extract 11-13 g / L, dipotassium hydrogen phosphate 1.4-1.7 g / L, glycerol 1.8-2.1 g / L, inositol 0.10-0.14 mg / L, trehalose 0.9-1.2 g / L, and admixture 0.23-0.27 mg / L; The additive comprises a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.2-1.

5.

5. A green and safe mannitol biosynthesis method according to claim 1, characterized in that: The fermentation medium comprises 28.0-29.0 g / L of glucose, 16.4-17.5 g / L of peptone, 4.8-5.2 g / L of yeast extract, 0.4-0.6 g / L of ammonium sulfate, 0.10-0.14 g / L of dipotassium hydrogen phosphate, 1.4-1.7 mg / L of thiamine, 0.10-0.13 g / L of L-glutamic acid, and 0.08-0.12 g / L of DL-methionine.

6. A green and safe mannitol biosynthesis method according to claim 1, characterized in that: In the fermentation promoter, the mass ratio of the deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.4-1.6:0.5-0.7:0.8-1.

2.

7. A green and safe mannitol biosynthesis method according to claim 1, characterized in that: The post-treatment step is to collect a primary supernatant after centrifuging the fermentation liquid, add an equal volume of anhydrous ethanol to the primary supernatant, centrifuge at 9800-10300 rpm for 15-20 minutes to obtain a secondary supernatant, and crystallize the secondary supernatant at 3.8-4.2° C. to obtain a mannitol product.