A green and safe method for mannitol biosynthesis

Abstract

The present invention provides a green and safe method for synthesizing mannitol, belonging to the technical field of mannitol biosynthesis; the synthesis method includes steps of primary seed culture, secondary seed culture and fermentation culture; in the secondary seed culture, the primary seed culture solution is inoculated into a secondary seed culture medium to obtain a secondary seed culture solution; the components of the secondary seed culture medium are glucose, peptone, yeast extract, dipotassium hydrogen phosphate, glycerol, inositol, trehalose, and an additive; the present invention adopts two-stage seed culture and fermentation culture, effectively promotes the synthesis of yeast in the direction of mannitol, shortens the fermentation time, improves the yield and quality of mannitol, greatly improves the production efficiency, and has good biological safety. The culture conditions are mild, avoiding dangerous and energy-consuming operations such as high temperature and high pressure, with high safety, and conforming to the concept of green and safe production.

Description

Technical Field

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

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

[0003] Mannitol has wide application values in the fields of medicine, food, and industry; in the medical field, mannitol is a good diuretic. Mannitol injection, as a hypertonic antihypertensive drug, is a commonly used drug in clinical rescue, especially in the rescue of brain diseases, with the characteristics of rapid antihypertensive effect and accurate curative effect; in the food field, mannitol has low water absorption and a refreshing sweet taste, and is used for anti - sticking of foods such as maltose, chewing gum, and rice cakes, 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;

[0004] With the development of various industries, the demand for mannitol continues to grow, and its synthesis methods have attracted much attention.

[0005] Currently, the synthesis methods of mannitol mainly include extraction from natural seaweeds, chemical synthesis method, and microbial synthesis method;

[0006] The extraction method from natural seaweeds mainly uses seaweeds as raw materials, crushes the seaweeds to release the substances inside the cells, and then uses hot - water extraction or organic - solvent extraction to extract mannitol from the seaweed cells into the solution. The extract is separated by filtration, centrifugation and other separation means to remove insoluble impurities, and then through ion exchange, concentration, and crystallization and other steps to obtain mannitol products; however, the supply of seaweed raw materials is unstable, cannot meet the large - scale production demand, and the preparation process is complex and the cost is high. More importantly, a large amount of chemical aids are needed in the separation process, which poses a great safety hazard and is not conducive to green and safe production.

[0007] The chemical synthesis method usually uses sucrose, glucose or starch as the starting raw material, uses nickel as the catalyst, and under high - temperature and high - pressure conditions, prepares mannitol by catalytic hydrogenation; this method has a low yield, contains a large amount of sorbitol by - products, and the subsequent separation and purification cost is high. Moreover, the high - temperature and high - pressure reaction conditions consume a lot of energy, produce more pollutants, pose a threat to the life and health of personnel and the safety of the production environment, and do not conform to the concept of green and safe production.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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

[0013] 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.

[0014] In view of the above technical problems, the present invention adopts the following technical solutions:

[0015] 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:

[0016] 1. Primary seed culture

[0017] 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;

[0018] The yeast is purchased from the market;

[0019] The composition of the primary seed culture medium is as follows: glucose 18 - 22 g / L, peptone 16 - 19 g / L, yeast extract 10 - 13 g / L, ammonium sulfate 0.12 - 0.17 g / L, magnesium sulfate 0.08 - 0.12 g / L.

[0020] 2. Secondary seed culture

[0021] The primary seed culture solution is inoculated into the secondary seed culture medium at an inoculation amount of 5.5 - 6.5% for culture. The culture temperature is 27.6 - 28.4 °C, and the culture time is 19.5 - 20.5 h. After the culture is completed, the secondary seed culture solution is obtained.

[0022] The composition of the secondary seed culture medium is as follows: 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, additive 0.23 - 0.27 mg / L.

[0023] The additive is a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.2 - 1.5.

[0024] 3. Fermentation culture

[0025] The secondary seed culture solution is inoculated into the fermentation medium at an inoculation amount of 5.0 - 7.0% for fermentation culture. The fermentation temperature is controlled at 29 - 32 °C, the fermentation time is 31.0 - 33.0 h, and the aeration rate is 0.8 - 1.2 vvm. Then, the fermentation temperature is reduced to 25 - 27 °C for continuous fermentation. When the fermentation reaches 36.0 - 37.0 h, the aeration rate is reduced to 0.2 - 0.4 vvm, and the fermentation promoter is slowly added at a rate of 0.16 - 0.22 g / min / L for 1.8 - 2.2 h. After the addition is completed, the fermentation continues until 46.1 - 46.5 h to obtain the fermentation broth.

[0026] The composition of the fermentation medium is as follows: glucose 28.0 - 29.0 g / L, peptone 16.4 - 17.5 g / L, yeast extract 4.8 - 5.2 g / L, ammonium sulfate 0.4 - 0.6 g / L, dipotassium hydrogen phosphate 0.10 - 0.14 g / L, thiamine 1.4 - 1.7 mg / L, L - glutamic acid 0.10 - 0.13 g / L, DL - methionine 0.08 - 0.12 g / L.

[0027] The fermentation promoter is a mixture of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols, and the mass ratio of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols is 100:1.4 - 1.6:0.5 - 0.7:0.8 - 1.2.

[0028] 4. Post-treatment

[0029] After centrifuging the fermentation broth, the primary supernatant is collected. Equal volume of absolute ethanol is added to the primary supernatant, and then centrifuged at 9800 - 10300 rpm for 15 - 20 min to obtain the secondary supernatant. The secondary supernatant is crystallized at 3.8 - 4.2 °C to obtain the finished product of mannitol.

[0030] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0031] 1. The present invention uses yeast as the original strain for fermentative synthesis of mannitol. First, the yeast is subjected to two-stage seed culture. In the first-stage seed culture step, the synergistic culture of ammonium sulfate and magnesium sulfate can provide sufficient nutrients for the strain, promote the metabolic activities of the yeast, increase the quantity and activity of the strain, and provide sufficient and well-conditioned strains for the second-stage seed culture. In the second-stage seed culture step, the quantity of yeast is further expanded to enable the yeast to adapt to the subsequent fermentation conditions. Among them, inositol in the second-stage seed culture medium acts as a growth factor for the yeast, which is beneficial to maintaining the integrity of the structure and function of the cell membrane. Combined with trehalose, it can preferably maintain the stability of the cell structure and function, prevent the destruction of the membrane structure, and thus can effectively regulate the growth and metabolic activities of the yeast. The additive 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 aeration volume, the yeast is promoted to synthesize a large amount of mannitol, ensuring the yield and quality of mannitol. The ammonium sulfate component can provide ammonium ions for the yeast and participate in the nitrogen metabolism process in the cell. Thiamine can participate in the enzymatic reactions in the metabolic process of the yeast as a coenzyme and has an important regulatory effect on the growth and reproduction of the yeast. The amino acid component acts as a nitrogen source and simultaneously participates in amino acid metabolism to regulate the intracellular metabolic balance and protein synthesis. The present invention adds a fermentation promoter in the middle and later stages of fermentation, which can promote the metabolism of the yeast to proceed in the direction of mannitol synthesis, regulate the permeability of the cell membrane, and promote the progress of metabolic activity, thereby effectively increasing the yield and quality of mannitol. The present invention uses yeast, and through two-stage seed culture and fermentation culture steps, effectively promotes the synthesis of yeast in the direction of mannitol, shortens the fermentation time, increases the yield and quality of mannitol, greatly improves the production efficiency, and has good biological safety, which will not cause harm to operators and the environment. Moreover, the culture conditions are mild, avoiding dangerous and energy-consuming operating conditions such as high temperature and high pressure, with high safety, and conforming to the concept of green and safe production;

[0032] 2. The fermentation broth obtained by using the synthesis method of the present invention has a mannitol content of 58.2 - 58.7 g / L, a succinic acid content of 0.84 - 0.87 g / L, and the purity of the tested mannitol finished product is 99.47 - 99.53%. Detailed Embodiments

[0033] For a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed embodiments of the present invention are now described.

[0034] Example 1

[0035] 1. Primary seed culture

[0036] The yeast was cultured in the primary seed culture medium at a culture temperature of 26.0 °C for 32.0 h. After the culture ended, the primary seed culture broth was obtained;

[0037] The yeast was purchased from the market;

[0038] The composition of the primary seed culture medium was glucose 20 g / L, peptone 18 g / L, yeast extract 11 g / L, ammonium sulfate 0.14 g / L, and magnesium sulfate 0.10 g / L.

[0039] 2. Secondary seed culture

[0040] The primary seed culture broth was inoculated into the secondary seed culture medium at an inoculation amount of 6.0% for culture. The culture temperature was 28.0 °C and the culture time was 20.0 h. After the culture ended, the secondary seed culture broth was obtained;

[0041] The composition of the secondary seed culture medium was 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 an additive 0.25 mg / L;

[0042] The additive was a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin was 1:1.3.

[0043] 3. Fermentation culture

[0044] The secondary seed culture broth was inoculated into the fermentation medium at an inoculation amount of 6.0% for fermentation culture. The fermentation temperature was controlled at 30 °C, the fermentation time was 32.0 h, and the ventilation rate was 1.0 vvm. Then the fermentation temperature was reduced to 26 °C for continued fermentation. When the fermentation reached 36.5 h, the ventilation rate was reduced to 0.3 vvm, and the fermentation promoter was slowly added at a rate of 0.2 g / min / L for 2.0 h. After the addition was completed, the fermentation continued until 46.3 h to obtain the fermentation broth;

[0045] The components of the fermentation medium are as follows: glucose 28.5 g / L, peptone 17.0 g / L, yeast extract 5.0 g / L, ammonium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.12 g / L, thiamine 1.5 mg / L, L-glutamic acid 0.12 g / L, DL-methionine 0.10 g / L;

[0046] The components of the fermentation promoter are a mixture of deionized water, inulin, chitosan oligosaccharide and tea polyphenols, and the mass ratio of deionized water, inulin, chitosan oligosaccharide and tea polyphenols is 100:1.5:0.6:1.0.

[0047] 4. Post-treatment

[0048] After centrifuging the fermentation broth, collect the first supernatant. Add an equal volume of absolute ethanol to the first supernatant and centrifuge at 10000 rpm for 18 min to obtain the second supernatant. Crystallize the second supernatant at 4.0 °C to obtain the finished product of mannitol.

[0049] For the fermentation broth obtained by the method of Example 1, the mannitol content is 58.7 g / L, the succinic acid content is 0.82 g / L, and the purity of the finished product of mannitol is tested to be 99.53%.

[0050] Example 2

[0051] 1. Primary seed culture

[0052] Place the yeast in the primary seed medium for cultivation. The cultivation temperature is 25.0 °C and the cultivation time is 32.5 h. After the cultivation is completed, obtain the primary seed culture solution;

[0053] The yeast is obtained by purchasing from the market;

[0054] The components of the primary seed medium are as follows: glucose 18 g / L, peptone 16 g / L, yeast extract 10 g / L, ammonium sulfate 0.12 g / L, magnesium sulfate 0.08 g / L.

[0055] 2. Secondary seed culture

[0056] Inoculate the primary seed culture solution into the secondary seed medium at an inoculation amount of 5.5% for cultivation. The cultivation temperature is 27.6 °C and the cultivation time is 19.5 h. After the cultivation is completed, obtain the secondary seed culture solution;

[0057] The components of the secondary seed medium are as follows: 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, additive 0.23 mg / L;

[0058] The component of the admixture is a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.2.

[0059] 3. Fermentation culture

[0060] The secondary seed culture solution is inoculated into the fermentation medium at an inoculation amount of 5.0% for fermentation culture. The fermentation temperature is controlled at 29 °C, the fermentation time is 31.0 h, and the ventilation rate is 0.8 vvm. Then the fermentation temperature is reduced to 25 °C for continuous fermentation. When the fermentation reaches 36.0 h, the ventilation rate is reduced to 0.2 vvm, and the fermentation promoter is slowly added. The addition rate is controlled at 0.16 g / min / L, and the addition time is 1.8 h. After the addition is completed, fermentation is carried out until 46.1 h to obtain the fermentation broth;

[0061] The component of the fermentation medium is 28.0 g / L of glucose, 16.4 g / L of peptone, 4.8 g / L of yeast extract, 0.4 g / L of ammonium sulfate, 0.10 g / L of dipotassium hydrogen phosphate, 1.4 mg / L of thiamine, 0.10 g / L of L-glutamic acid, and 0.08 g / L of DL-methionine;

[0062] The component of the fermentation promoter is a mixture of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols, and the mass ratio of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols is 100:1.4:0.5:0.8.

[0063] 4. Post-treatment

[0064] The fermentation broth is centrifuged, and the primary supernatant is collected. An equal volume of absolute ethanol is added to the primary supernatant and centrifuged at 9800 rpm for 20 min to obtain the secondary supernatant. The secondary supernatant is crystallized at 3.8 °C to obtain the mannitol finished product.

[0065] For the fermentation broth obtained by the method of Example 2, the mannitol content is 58.2 g / L, the succinic acid content is 0.87 g / L, and the purity of the mannitol finished product is tested to be 99.47%.

[0066] Example 3

[0067] 1. Primary seed culture

[0068] The yeast is cultured in the primary seed medium. The culture temperature is 27.0 °C, and the culture time is 31.6 h. After the culture is completed, the primary seed culture solution is obtained;

[0069] The yeast is obtained by purchasing from the market;

[0070] The component of the primary seed medium is 22 g / L of glucose, 19 g / L of peptone, 13 g / L of yeast extract, 0.17 g / L of ammonium sulfate, and 0.12 g / L of magnesium sulfate.

[0071] 2. Secondary seed culture

[0072] The primary seed culture solution is inoculated into the secondary seed culture medium at an inoculation amount of 6.5%, and cultured at a temperature of 28.4 °C for 20.5 h. After the culture is completed, the secondary seed culture solution is obtained;

[0073] The components of the secondary seed culture medium are as follows: 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 additive 0.27 mg / L;

[0074] The component of the additive is a mixture of pyridoxine and riboflavin, and the mass ratio of pyridoxine to riboflavin is 1:1.5.

[0075] 3. Fermentation culture

[0076] The secondary seed culture solution is inoculated into the fermentation medium at an inoculation amount of 7.0% for fermentation culture. The fermentation temperature is controlled at 32 °C, the fermentation time is 33.0 h, and the ventilation rate is 1.2 vvm. Then, the fermentation temperature is reduced to 27 °C for continued fermentation. When the fermentation reaches 37.0 h, the ventilation rate is reduced to 0.4 vvm, and the fermentation promoter is slowly added at a rate of 0.22 g / min / L for 2.2 h. After the addition is completed, the fermentation continues until 46.5 h to obtain the fermentation broth;

[0077] The components of the fermentation medium are as follows: glucose 29.0 g / L, peptone 17.5 g / L, yeast extract 5.2 g / L, ammonium sulfate 0.6 g / L, dipotassium hydrogen phosphate 0.14 g / L, thiamine 1.7 mg / L, L-glutamic acid 0.13 g / L, and DL-methionine 0.12 g / L;

[0078] The component of the fermentation promoter is a mixture of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols, and the mass ratio of deionized water, inulin, chitosan oligosaccharide, and tea polyphenols is 100:1.6:0.7:1.2.

[0079] 4. Post-treatment

[0080] The fermentation broth is centrifuged, and the primary supernatant is collected. An equal volume of absolute ethanol is added to the primary supernatant, and centrifuged at 10300 rpm for 15 min to obtain the secondary supernatant. The secondary supernatant is crystallized at 4.2 °C to obtain the mannitol finished product.

[0081] For the fermentation broth obtained by the method of Example 3, the mannitol content is 58.5 g / L, the succinic acid content is 0.84 g / L, and the purity of the mannitol finished product is tested to be 99.50%.

[0082] Comparative Example 1

[0083] Based on Example 1, the changes are as follows:

[0084] In the secondary seed culture step, the components of "glycerol, inositol, trehalose" are omitted;

[0085] In the fermentation culture step, the operation steps of "slowly adding 23.0 g of fermentation promoter and controlling the addition rate at 0.2 g / min" are omitted;

[0086] The remaining operations are the same.

[0087] For the fermentation broth obtained by the method of Comparative Example 1, the mannitol content is 49.2 g / L, the succinic acid content is 1.66 g / L, and the purity of the tested mannitol product is 94.52%.

[0088] Comparative Example 2

[0089] Based on Example 1, the changes are as follows:

[0090] In the secondary seed culture step, the "additive" component in the secondary seed culture medium is omitted;

[0091] The components of "thiamine, L-glutamic acid, DL-methionine" in the fermentation medium are omitted;

[0092] The remaining operations are the same.

[0093] For the fermentation broth obtained by the method of Comparative Example 2, the mannitol content is 51.4 g / L, the succinic acid content is 1.51 g / L, and the purity of the tested mannitol product is 95.36%.

[0094] The present invention uses yeast as the original strain for fermentative synthesis of mannitol. First, the yeast is subjected to two-stage seed cultivation. In the first-stage seed cultivation step, the co-cultivation of ammonium sulfate and magnesium sulfate can provide sufficient nutrients for the strain, promote the metabolic activities of the yeast, increase the quantity and activity of the strain, and provide sufficient and in good condition strains for the second-stage seed cultivation. In the second-stage seed cultivation step, the quantity of the yeast is further increased to enable the yeast to adapt to the subsequent fermentation conditions. Among them, inositol in the second-stage seed culture medium serves as a growth factor for the yeast, which is beneficial to maintaining the integrity of the structure and function of the cell membrane. Combined with trehalose, it can preferably maintain the stability of the cell structure and function, prevent the damage of the membrane structure, and thus can effectively regulate the growth and metabolic activities of the yeast. The additive 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 cultivation step, by reasonably controlling parameters such as temperature and ventilation volume, the yeast is prompted to synthesize a large amount of mannitol, ensuring the yield and quality of mannitol. The ammonium sulfate component therein can provide ammonium ions for the yeast and participate in the nitrogen metabolism process in the cells. Thiamine can participate in the enzymatic reactions in the metabolic process of the yeast as a coenzyme and has an important regulatory effect on the growth and reproduction of the yeast. The amino acid component serves as a nitrogen source and simultaneously participates in amino acid metabolism to regulate the intracellular metabolic balance and protein synthesis. In the middle and late stages of fermentation in the present invention, a fermentation promoter is added, which can promote the metabolism of the yeast to proceed in the direction of mannitol synthesis, regulate the permeability of the cell membrane, and promote the progress of metabolic activity, thereby effectively increasing the yield and quality of mannitol. The present invention uses yeast, and through two-stage seed cultivation and fermentation cultivation steps, it effectively promotes the synthesis of yeast in the direction of mannitol, shortens the fermentation time, increases the yield and quality of mannitol, greatly improves the production efficiency, and has good biosafety, causing no harm to operators and the environment. Moreover, the cultivation conditions are mild, avoiding dangerous and energy-consuming operating conditions such as high temperature and high pressure, with high safety, and conforming to the concept of green and safe production.

[0095] Mannitol was synthesized by the method of Comparative Example 1. In the second-stage seed cultivation step, the components of glycerol, inositol and trehalose were omitted, resulting in poor stability of the cell structure and function, reducing the growth and metabolic activity of the yeast. And in the fermentation cultivation step, the addition step of the fermentation promoter was omitted, which could not provide sufficient nutrients for the fermentation activity, resulting in unstable metabolic activity, weakening the synthesis of the yeast in the direction of mannitol, reducing the mannitol content in the fermentation broth, and lowering the product purity.

[0096] Comparative Example 2 omitted the admixture components in the secondary culture step, reducing the provision of cell energy and unable to effectively promote metabolic activity. Moreover, Comparative Example 2 also omitted the thiamine and amino acid components in the fermentation medium, resulting in incomplete enzymatic reactions, insufficient nutrient components, and incomplete cell metabolism during the fermentation process. Ultimately, the content of by-products increased, the mannitol content decreased, and the product purity was relatively low.

[0097] Unless otherwise specified, the percentages used in the present invention are all mass percentages.

[0098] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A green and safe mannitol biosynthesis method, characterized in that: It includes primary seed culture, secondary seed culture, fermentation culture and post-processing steps; The secondary seed culture step comprises: inoculating the primary seed culture solution 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 a culturing time of 19.5-20.5 h, and obtaining the secondary seed culture solution after the culturing is completed; 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 admixture comprises a mixture of pyridoxine and riboflavin, wherein the mass ratio of pyridoxine to riboflavin is 1:1.2-1.5; 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 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.

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 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.