A method for improving the efficiency of producing sorbose by mixed alcohol fermentation

Abstract

The invention discloses a method for improving the efficiency of producing sorbose by mixed alcohol liquid fermentation, and belongs to the technical field of biomedicine. The method comprises primary seed culture, secondary seed culture and fermentation. In the fermentation, black acetobacter obtained after the secondary seed culture is inoculated into a fermentation medium for fermentation, pH is not controlled during the fermentation process, and the brix of the fermented liquid is detected every 1.5-2.5 hours after 5.5-6.5 hours from the start of the fermentation, and when the brix of the fermented liquid reaches 6-8, a mixed alcohol liquid containing 60-80% of sorbitol is added, and when the brix of the fermented liquid reaches 6-8 again, a mixed alcohol liquid containing 60-80% of sorbitol is added again, and when the brix of the fermented liquid reaches 6-8 for the third time, a pure sorbitol solution containing 48-52% is added until the sorbitol concentration is zero or the sorbose content does not increase any more. The method of the invention can reduce the amount of bacteria in the fermentation process, improve the conversion rate of sorbose, and shorten the fermentation time.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for improving the efficiency of producing sorbose by mixed alcohol solution fermentation. Background Art

[0002] At present, in the production process of vitamin C (VC), the first-step fermentation is to produce sorbose with pure sorbitol as the main raw material, and the second-step fermentation is to produce gulonic acid by consuming sorbose. The formula and process of the first-step fermentation are relatively simple, the fermentation cycle is short, and the fermentation process is relatively mature, which makes sorbitol have a high conversion rate of 97-99% and the output of sorbose is sufficient.

[0003] In the process of chemical synthesis to produce iditol, the ratio of the two enantiomers in the hydrogenation product can be changed by adjusting the catalyst type, hydrogenation temperature and pressure of sorbitol catalytic hydrogenation, and the mixed alcohol solution containing about 64% iditol and about 36% sorbitol can be generated. However, since the two alcohols are difficult to separate, the mixed alcohol solution needs to be subjected to a VC one-step fermentation process to oxidize sorbitol into sorbitol, and then sorbitol and iditol can be easily separated by chromatographic separation technology.

[0004] Although the technology for fermenting and producing sorbitol using pure sorbitol as raw material is relatively mature, the mixed alcohol solution containing 64% iditol and 36% sorbitol affects the fermentation efficiency of Acetobacter nigromitorans, causing a significant increase in the amount of bacteria during the fermentation process, making the ultrafiltration process for removing bacteria and impurities more difficult; at the same time, the conversion rate of sorbitol to sorbitol is reduced, resulting in an increase in the proportion of sorbitol and a decrease in the purity of iditol after chromatographic separation; in addition, the fermentation time is extended, resulting in increased costs. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for improving the efficiency of producing sorbose by mixed alcohol fermentation, which can reduce the amount of bacteria in the fermentation process, improve the conversion rate of sorbose, and shorten the fermentation time.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for improving the efficiency of producing sorbose by mixed alcohol fermentation, comprising the following steps: primary seed culture, secondary seed culture, and fermentation;

[0008] The first-level seed culture is to inoculate black acetobacter in a first-level seed tank culture medium, with an inoculation amount of 5-7%, a culture temperature of 28-32° C., and no pH control during the fermentation process. The fermentation is cultured until the sorbose concentration reaches 160-170 g / L, thereby obtaining black acetobacter obtained after the first-level seed culture;

[0009] In the primary seed culture, the composition of the primary seed tank culture medium is: 24.21-32 mL / L corn steep liquor, 280-370 mL / L mixed alcohol solution containing 60-80% sorbitol, 0.02-0.03 g / L VB5, 0.003-0.004 g / L calcium folinate, VB 12 0.003-0.004g / L, calcium carbonate 0.04-0.06g / L, defoamer 3.4-3.6mL / L, adjust pH=4.6-5;

[0010] The secondary seed culture comprises inoculating the black acetobacter obtained after the primary seed culture into the secondary seed tank culture medium, with an inoculation amount of 5-7%, a culture temperature of 28-32° C., and no pH control during the fermentation process. The culture is carried out until the sorbose concentration reaches 160-170 g / L to obtain the black acetobacter obtained after the secondary seed culture;

[0011] In the secondary seed culture, the composition of the secondary seed tank culture medium is: 2.3-3 mL / L corn steep liquor, 280-370 mL / L mixed alcohol solution containing 60-80% sorbitol, 0.01-0.02 g / L VB5, 0.001-0.002 g / L calcium folinate, VB 12 0.0004-0.0006g / L, calcium carbonate 0.02-0.04g / L, defoamer 1.4-1.6mL / L, adjust pH=4.6-5;

[0012] The fermentation comprises inoculating the black acetobacter obtained after culturing the secondary seeds into a fermentation medium in a fermentation tank, with an inoculation amount of 15-18%, a culture temperature of 28-32° C., and performing fermentation. The pH value is not controlled during the fermentation process. After 5.5-6.5 hours from the start of the fermentation, the brix of the fermentation liquid is detected every 1.5-2.5 hours. When the brix of the fermentation liquid reaches 6-8, 100-150 mL / L of a mixed alcohol solution containing 60-80% sorbitol is added. When the brix of the fermentation liquid reaches 6-8 again, 100-150 mL / L of a mixed alcohol solution containing 60-80% sorbitol is added again. When the brix of the fermentation liquid reaches 6-8 for the third time, 100-150 mL / L of a pure sorbitol solution containing 48-52% is added. When the sorbitol concentration is zero or the sorbose content no longer increases, the fermentation is terminated.

[0013] In the fermentation, the fermentation medium is composed of: 4.5-6 mL / L corn steep liquor, 300-400 mL / L mixed alcohol solution containing 60-80% sorbitol, 0.005-0.006 g / L VB5, 0.001-0.002 g / L calcium folinate, VB 12 0.001-0.002g / L, calcium carbonate 0.3-0.5g / L, defoamer 0.7-0.9mL / L, adjust pH=4.6-5;

[0014] The ventilation rate during the fermentation is 1.2-1.5 L / min, the rotation speed is 420-460 rpm, and the tank pressure is 0.035-0.045 MPa;

[0015] The calculation formula of the brix of the fermentation liquid is: solid content of the fermentation liquid % - sorbitol content %;

[0016] The solid content of the corn steep liquor in the primary seed tank culture medium, the corn steep liquor in the secondary seed tank culture medium, and the corn steep liquor in the fermentation culture medium is 35-45%;

[0017] The preparation method of the mixed alcohol solution containing 60-80% sorbitol is as follows: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 60-80%;

[0018] The solid content of the mixed alcohol solution containing 60-80% sorbitol is 50-55%.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The method of improving the efficiency of producing sorbose by mixed alcohol solution fermentation of the present invention, by adding sorbitol to the primary seed tank culture medium and the secondary seed tank culture medium, can allow black Acetobacter to use the mixed alcohol solution of iditol and sorbitol for growth in advance; by adding sorbitol to the fermentation substrate, can adjust the ratio of iditol and sorbitol, increase the proportion of sorbitol, and thus improve the conversion rate of sorbose; by adding the mixed alcohol solution twice and the pure sorbitol solution once during the fermentation process, can further improve the conversion rate of sorbitol; by adjusting the content of corn steep liquor in the primary seed tank culture medium, the secondary seed tank culture medium, and the fermentation medium, can adjust the carbon-nitrogen ratio in the primary seed tank culture medium, the secondary seed tank culture medium, and the fermentation medium to a range suitable for the growth of black Acetobacter, while reducing the amount of bacteria in the fermentation process;

[0021] (2) The method of the present invention for improving the efficiency of producing sorbitol by mixed alcohol liquid fermentation can reduce the amount of bacteria in the fermentation process, improve the conversion rate of sorbitol, shorten the fermentation time, reduce the maximum OD600 value in the fermentation process to 4.2-5.3, reduce the fermentation time to 26-38h, and increase the sorbitol conversion rate to 91.2-97.8%. DETAILED DESCRIPTION

[0022] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0023] The black acetobacter in Examples 1-3 and Comparative Examples 1-4 has a preservation number of GDMCC No: 61006, a Latin name of Gluconobacter oxydans subsp. melanogenus, and is preserved in the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is April 29, 2020.

[0024] The method for expanding the culture of the black acetobacter in Example 1-3 and Comparative Example 1-4 before use is as follows: take a glycerol tube from an ultra-low temperature refrigerator, take 100uL of the bacterial solution with a pipette after melting, evenly apply it on the plate culture medium, put it upside down in an incubator at 30°C for 48h, observe that there are no foreign bacteria, inoculate all of the 1 flat plate of bacterial lawn into a 750mL thorn bottle, the thorn bottle contains 200mL of seed liquid culture medium, and then culture, the culture temperature is 30°C, the rotation speed is 220rpm, culture to OD600≥3, take samples for microscopic examination, after no foreign bacteria, according to 5% inoculation, transfer to the shake flask seed liquid for culture, the culture temperature is 30°C, the rotation speed is 220rpm, and culture to OD600≥3;

[0025] The formula of the plate culture medium is: glucose 10%, yeast extract 0.6%, calcium carbonate 1.1%, agar powder 2%;

[0026] The formula of the seed liquid culture medium is: 20% sorbitol, 0.6% yeast extract, 0.15% calcium carbonate, pH=5.

[0027] Example 1 Adding a mixed alcohol solution containing 60% sorbitol

[0028] A method for improving the efficiency of producing sorbose by mixed alcohol fermentation, specifically comprising:

[0029] 1. Primary seed culture: The composition of the primary seed tank culture medium is: corn steep liquor 32mL / L, mixed alcohol solution containing 60% sorbitol 370mL / L, VB50.02g / L, calcium folinate 0.003g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol concentration reaches 165g / L, then transplant;

[0030] 2. Secondary seed culture: The composition of the secondary seed tank culture medium is: corn syrup 3mL / L, mixed alcohol solution containing 60% sorbitol 370mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 120.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol concentration reaches 165g / L, then transplant;

[0031] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 6mL / L, mixed alcohol solution containing 60% sorbitol 400mL / L, VB50.005g / L, calcium folinate 0.001g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH=4.8, do not control pH during the process, black acetic acid bacteria inoculation amount 16%, culture temperature 30 ° C, fermentation, fermentation ventilation volume 1.4 L / min, rotation speed 450 rpm, tank pressure 0.04 MPa; 6 hours after the start of fermentation, every 2 hours to detect the brix of the fermentation liquid (the calculation formula of the brix of the fermentation liquid is: solid content % of the fermentation liquid - sorbitol content %), when the brix of the fermentation liquid reaches 7, add 100 mL / L of a mixed alcohol solution containing 60% sorbitol, when the brix of the fermentation liquid reaches 7 again, add 100 mL / L of a mixed alcohol solution containing 60% sorbitol, when the brix of the fermentation liquid reaches 7 for the third time, add 100 mL / L of a pure sorbitol solution containing 50%, until the sorbitol concentration is zero or the sorbitol content no longer increases, the fermentation is terminated.

[0032] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0033] The preparation method of the mixed alcohol solution containing 60% sorbitol used in this embodiment is as follows: sorbitol is added to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and the proportion of sorbitol in the solute is adjusted to 60%;

[0034] The solid content of the mixed alcohol solution containing 60% sorbitol used in this example is 50% (the total solid content is measured by Abbe refractometer).

[0035] Example 2 Adding a mixed alcohol solution containing 70% sorbitol

[0036] A method for improving the efficiency of producing sorbose by mixed alcohol fermentation, specifically comprising:

[0037] 1. Primary seed culture: The composition of the primary seed tank culture medium is: corn steep liquor 27.43 mL / L, mixed alcohol solution containing 70% sorbitol 320 mL / L, VB50.02 g / L, calcium folinate 0.003 g / L, VB 120.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0038] 2. Secondary seed culture: The composition of the secondary seed tank culture medium is: corn steep liquor 2.6mL / L, mixed alcohol solution with sorbitol accounting for 70% 320mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 12 0.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0039] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 5.2 mL / L, mixed alcohol solution containing 70% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH=4.8, do not control pH during the process, black acetic acid bacteria inoculation amount 16%, culture temperature 30 ° C, fermentation, fermentation ventilation volume 1.4 L / min, rotation speed 450 rpm, tank pressure 0.04 MPa; fermentation control process is the same as in Example 1, to be detected when the fermentation liquid brax (the calculation formula of the fermentation liquid brax is: the solid content of the fermentation liquid % - sorbitol content %) reaches 7, add 70% sorbitol containing 100 mL / L of mixed alcohol solution, when the fermentation liquid brax reaches 7 again, add 70% sorbitol containing 100 mL / L of mixed alcohol solution, when the fermentation liquid brax reaches 7 for the third time, add 50% pure sorbitol solution 100 mL / L, until the sorbitol concentration is zero or the sorbitol content no longer increases, end the fermentation;

[0040] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0041] The preparation method of the mixed alcohol solution containing 70% sorbitol used in this embodiment is as follows: sorbitol is added to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and the proportion of sorbitol in the solute is adjusted to 70%;

[0042] The solid content of the mixed alcohol solution containing 70% sorbitol used in this example is 50% (the total solid content is measured by Abbe refractometer).

[0043] Example 3 Adding a mixed alcohol solution containing 80% sorbitol

[0044] A method for improving the efficiency of producing sorbose by mixed alcohol fermentation, specifically comprising:

[0045] 1. Primary seed culture: The composition of the primary seed tank culture medium is: corn steep liquor 24.21mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.02g / L, calcium folinate 0.003g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0046] 2. Secondary seed culture: The composition of the secondary seed tank culture medium is: corn steep liquor 2.3mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 12 0.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0047] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 4.5 mL / L, mixed alcohol solution containing 80% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH = 4.8, do not control pH during the process, the fermentation tank inoculation amount is 16%, the culture temperature is 30 ° C, fermentation, the ventilation volume during fermentation is 1.4 L / min, the rotation speed is 450 rpm, and the tank pressure is 0.04 MPa; the fermentation control process is the same as in Example 1, when the brix of the fermented liquid to be detected (the calculation formula of the brix of the fermented liquid is: the solid content% of the fermented liquid-the sorbose content%) reaches 7, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added, and when the brix of the fermented liquid reaches 7 again, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added again, and when the brix of the fermented liquid reaches 7 for the third time, 100 mL / L of a pure sorbitol solution containing 50% is added, and the fermentation is terminated when the sorbitol concentration is zero or the sorbose content no longer increases.

[0048] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0049] The preparation method of the mixed alcohol solution containing 80% sorbitol used in this embodiment is as follows: sorbitol is added to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and the proportion of sorbitol in the solute is adjusted to 80%;

[0050] The solid content of the mixed alcohol solution containing 80% sorbitol used in this example is 50% (the total solid content is measured by Abbe refractometer).

[0051] Comparative Example 1

[0052] The method of Example 3 is modified in that in the first step of primary seed culture, the addition of the mixed alcohol solution containing 80% sorbitol is omitted in the primary seed tank culture medium. Specifically:

[0053] 1. Primary culture: The composition of the primary seed tank culture medium is: corn steep liquor 24.21mL / L, 50% pure sorbitol solution 220mL / L, VB50.02g / L, calcium folinate 0.003g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0054] 2. Secondary culture: The composition of the secondary seed tank culture medium is: corn steep liquor 2.3mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 12 0.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0055] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 4.5 mL / L, mixed alcohol solution containing 80% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH = 4.8, do not control pH during the process, the fermentation tank inoculation amount is 16%, the culture temperature is 30 ° C, fermentation, the ventilation volume during fermentation is 1.4 L / min, the rotation speed is 450 rpm, and the tank pressure is 0.04 MPa; the fermentation control process is the same as in Example 1, when the brix of the fermentation liquid to be detected (the calculation formula of the brix of the fermentation liquid is: the solid content% of the fermentation liquid-the sorbose content%) reaches 7, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added, and when the brix of the fermentation liquid reaches 7 again, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added again, and when the brix reaches 7 for the third time, 100 mL / L of a pure sorbitol solution containing 50% is added, and the fermentation is terminated when the sorbitol concentration is zero or the sorbose content no longer increases.

[0056] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0057] The preparation method of the mixed alcohol solution containing 80% sorbitol used in this comparative example is: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 80%;

[0058] The solid content of the mixed alcohol solution containing 80% sorbitol used in this comparative example is 50%.

[0059] Comparative Example 2

[0060] The method of Example 3 is modified in that in the second step of the secondary seed culture, the addition of the mixed alcohol solution containing 80% sorbitol is omitted in the secondary seed tank culture medium. Specifically:

[0061] 1. Primary culture: The composition of the primary seed tank culture medium is: corn steep liquor 24.21mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.02g / L, calcium folinate 0.003g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0062] 2. Secondary culture: The composition of the secondary seed tank culture medium is: corn steep liquor 2.3mL / L, 50% pure sorbitol solution 220mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 12 0.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% in the secondary seed tank, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0063] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 4.5 mL / L, mixed alcohol solution containing 80% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 120.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH = 4.8, do not control pH during the process, the fermentation tank inoculation amount is 16%, the culture temperature is 30 ° C, fermentation, the ventilation volume during fermentation is 1.4 L / min, the rotation speed is 450 rpm, and the tank pressure is 0.04 MPa; the fermentation control process is the same as in Example 3, when the brix of the fermentation liquid to be detected (the calculation formula of the brix of the fermentation liquid is: the solid content% of the fermentation liquid-the sorbitol content%) reaches 7, add 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80%, and when the brix of the fermentation liquid reaches 7 again, add 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80%, and when the brix reaches 7 for the third time, add 100 mL / L of a pure sorbitol solution containing 50%, and when the sorbitol concentration is zero or the sorbitol content no longer increases, the fermentation is terminated.

[0064] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0065] The preparation method of the mixed alcohol solution containing 80% sorbitol used in this comparative example is: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 80%;

[0066] The solid content of the mixed alcohol solution containing 80% sorbitol used in this comparative example was 50% (the total solid content was measured by Abbe refractometer).

[0067] Comparative Example 3

[0068] The method of Example 3 is modified in that the addition of 50% pure sorbitol solution is omitted in the primary fermentation step 3. Specifically:

[0069] 1. Primary seed culture: The composition of the primary seed tank culture medium is: corn steep liquor 24.21mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.02g / L, calcium folinate 0.003g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0070] 2. Secondary seed culture: Secondary seed tank culture medium: corn steep liquor 2.3mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 120.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% of black acetobacter, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0071] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 4.5 mL / L, mixed alcohol solution containing 80% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH = 4.8, do not control pH during the process, the fermentation tank inoculation amount is 16%, the culture temperature is 30 ° C, fermentation, the ventilation volume during fermentation is 1.4 L / min, the rotation speed is 450 rpm, and the tank pressure is 0.04 MPa; the fermentation control process is the same as in Example 1, and when the brix of the fermentation liquid to be detected (the calculation formula of the brix of the fermentation liquid is: the solid content% of the fermentation liquid-the sorbitol content%) reaches 7, 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80% is added, and when the brix of the fermentation liquid reaches 7 again, 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80% is added again, until the sorbitol concentration is zero or the sorbitol content no longer increases, the fermentation is terminated.

[0072] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0073] The preparation method of the mixed alcohol solution containing 80% sorbitol used in this comparative example is: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 80%;

[0074] The solid content of the mixed alcohol solution containing 80% sorbitol used in this comparative example was 50% (the total solid content was measured by Abbe refractometer).

[0075] Comparative Example 4

[0076] The method of Example 3 was modified in that the amount of corn syrup was changed. Specifically:

[0077] 1. Primary seed culture: The composition of the primary seed tank culture medium is: corn steep liquor 19.4 mL / L, mixed alcohol solution containing 80% sorbitol 280 mL / L, VB50.02 g / L, calcium folinate 0.003 g / L, VB 12 0.003g / L, calcium carbonate 0.05g / L, defoamer 3.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% in the first-level seed tank, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0078] 2. Secondary seed culture: The composition of the secondary seed tank culture medium is: corn steep liquor 1.84mL / L, mixed alcohol solution containing 80% sorbitol 280mL / L, VB50.01g / L, calcium folinate 0.001g / L, VB 12 0.0005g / L, calcium carbonate 0.03g / L, defoamer 1.5mL / L, adjust pH=4.8, do not control pH during the process, inoculate 6% in the secondary seed tank, culture temperature 30℃, culture until sorbitol reaches 165g / L, then transplant;

[0079] 3. Fermentation: The composition of the fermentation medium is: corn steep liquor 3.3 mL / L, mixed alcohol solution containing 80% sorbitol 300 mL / L, VB5 0.005 g / L, calcium folinate 0.001 g / L, VB 12 0.001 g / L, calcium carbonate 0.4 g / L, defoaming agent 0.8 mL / L, adjust pH = 4.8, do not control pH during the process, the fermentation tank inoculation amount is 16%, the culture temperature is 30 ° C, fermentation, the ventilation volume during fermentation is 1.4 L / min, the rotation speed is 450 rpm, and the tank pressure is 0.04 MPa; the fermentation control process is the same as in Example 3, when the brix of the fermentation liquid to be detected (the calculation formula of the brix of the fermentation liquid is: the solid content% of the fermentation liquid-the sorbitol content%) reaches 7, add 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80%, and when the brix of the fermentation liquid reaches 7 again, add 100 mL / L of a mixed alcohol solution in which sorbitol accounts for 80%, and when the brix reaches 7 for the third time, add 100 mL / L of a pure sorbitol solution containing 50%, and when the sorbitol concentration is zero or the sorbitol content no longer increases, the fermentation is terminated.

[0080] The solid content of corn steep liquor used in the first-stage seed tank culture medium, the second-stage seed tank culture medium, and the third-stage fermentation culture medium is 40%;

[0081] The preparation method of the mixed alcohol solution containing 80% sorbitol used in this comparative example is: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 80%;

[0082] The solid content of the mixed alcohol solution containing 80% sorbitol used in this comparative example was 50% (the total solid content was measured by Abbe refractometer).

[0083] The fermentation time required for step 3 in Example 1-3 and Comparative Example 1-4 (i.e., the time for complete conversion of sorbitol), the sorbitol conversion rate, and the maximum OD600 value of the bacteria during the fermentation process were tested, and the test results are as follows:

[0084]

[0085] Comparing the results of Example 3 with those of Comparative Example 1, the fermentation time of Comparative Example 1 was prolonged, the maximum OD600 value increased, and although the sorbitol conversion rate decreased, the decrease was not large; comparing the results of Example 3 with those of Comparative Example 2, the fermentation time of Comparative Example 2 was prolonged, the sorbitol conversion rate decreased, and the maximum OD600 value increased; comparing the results of Example 3 with those of Comparative Example 3, although the fermentation time of Comparative Example 3 was shortened, the sorbitol conversion rate decreased, and the maximum OD600 value increased; comparing the results of Example 3 with those of Comparative Example 4, although the maximum OD600 value of Comparative Example 4 decreased, the fermentation time was greatly prolonged, and the sorbitol conversion rate was greatly reduced. This shows that the fermentation conditions of Example 3 are better than those of Comparative Examples 1-4.

[0086] Unless otherwise specified, all percentages used in the present invention are by mass.

[0087] 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 method for improving the efficiency of producing sorbose by mixed alcohol fermentation, characterized in that: The method comprises the following steps: primary seed culture, secondary seed culture, and fermentation; The first-level seed culture is to inoculate black acetobacter in a first-level seed tank culture medium, with an inoculation amount of 6%, a culture temperature of 30° C., and no pH control during the fermentation process. The fermentation is cultured until the sorbose concentration reaches 165 g / L, thereby obtaining black acetobacter obtained after the first-level seed culture; The deposit number of the black acetobacter is GDMCC No: 61006; In the primary seed culture, the composition of the primary seed tank culture medium is: 24.21 mL / L corn steep liquor, 280 mL / L mixed alcohol solution containing 80% sorbitol, 0.02-0.03 g / L VB5, 0.003-0.004 g / L folinic acid calcium, VB 12 0.003-0.004g / L, calcium carbonate 0.04-0.06g / L, defoamer 3.4-3.6mL / L, adjust pH=4.6-5; The secondary seed culture is to inoculate the black acetobacter obtained after the primary seed culture into the secondary seed tank culture medium, with an inoculation amount of 6%, a culture temperature of 30° C., and no pH control during the fermentation process, and culture until the sorbose concentration reaches 165 g / L to obtain the black acetobacter obtained after the secondary seed culture; In the secondary seed culture, the composition of the secondary seed tank culture medium is: 2.3 mL / L corn steep liquor, 280 mL / L mixed alcohol solution containing 80% sorbitol, 0.01-0.02 g / L VB5, 0.001-0.002 g / L calcium folinate, VB 12 0.0004-0.0006g / L, calcium carbonate 0.02-0.04g / L, defoamer 1.4-1.6mL / L, adjust pH=4.6-5; The fermentation comprises inoculating the raw black acetobacter obtained after the secondary seed culture into the fermentation medium in the fermentation tank, the inoculation amount is 16%, the culture temperature is 30°C, and the pH is not controlled during the fermentation process. After 6 hours from the start of the fermentation, the brix of the fermentation liquid is detected every 2 hours. When the brix of the fermentation liquid reaches 7, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added. When the brix of the fermentation liquid reaches 7 again, 100 mL / L of a mixed alcohol solution containing 80% sorbitol is added. When the brix of the fermentation liquid reaches 7 for the third time, 100 mL / L of a pure sorbitol solution containing 50% is added. When the sorbitol concentration is zero or the sorbose content no longer increases, the fermentation is terminated. In the fermentation, the fermentation medium is composed of: 4.5 mL / L corn steep liquor, 300 mL / L mixed alcohol solution containing 80% sorbitol, 0.005-0.006 g / L VB5, 0.001-0.002 g / L calcium folinate, VB 12 0.001-0.002g / L, calcium carbonate 0.3-0.5g / L, defoamer 0.7-0.9mL / L, adjust pH=4.6-5; The aeration rate during the fermentation was 1.4 L / min, the rotation speed was 450 rpm, and the tank pressure was 0.04 MPa; The calculation formula of the brix of the fermentation liquid is: solid content of the fermentation liquid % - sorbitol content %; The preparation method of the mixed alcohol solution containing 80% sorbitol is as follows: adding sorbitol to a mixed alcohol solution containing 64% iditol and 36% sorbitol, and adjusting the proportion of sorbitol in the solute to 80%; The solid content of the mixed alcohol solution containing 80% sorbitol is 50%.

2. The method for improving the efficiency of producing sorbose by mixed alcohol fermentation according to claim 1, characterized in that: The solid contents of the corn steep liquor in the primary seed tank culture medium, the corn steep liquor in the secondary seed tank culture medium, and the corn steep liquor in the fermentation culture medium are 35-45%.

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