A recombinant Bacillus subtilis, a method for preparing a fermentation broth, immobilized cells and a method for preparing the same, and a method and application for synthesizing D-tagatose

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a recombinant Bacillus subtilis, a method for preparing a fermentation broth, immobilized cells and a preparation method, and a method and application for synthesizing D-tagatose. The present invention provides a recombinant Bacillus subtilis ( Bacillus subtle )BLCY-012, with a deposit number of CGMCC No.31950. The recombinant Bacillus subtilis BLCY-012 of the present invention can ferment and produce D-tagatose-4-epimerase, and the D-tagatose-4-epimerase is an intracellular enzyme. The D-tagatose-4-epimerase can prepare D-tagatose using D-fructose as a raw material. The recombinant Bacillus subtilis BLCY-012 is used to prepare immobilized cells, and the immobilized cells are used as enzyme carriers to convert D-fructose into D-tagatose, and the conversion rate can reach 28% to 32%, which is a high conversion rate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant Bacillus subtilis, a method for preparing a fermentation broth, immobilized cells and a preparation method thereof, and a method and application of synthesizing D-tagatose. Background Art

[0002] D-Tagatose is a diastereomer of D-fructose, a naturally occurring six-carbon ketose. Industrial production mainly uses galactose as raw material, and is made through isomerization, decolorization, desalination, concentration, crystallization and other steps. The sweetness of D-Tagatose is 92% of the same amount of sucrose, but the calories are only 38% of sucrose. It also has low hygroscopicity and is a good low-energy food sweetener and filler. It has multiple physiological effects such as inhibiting hyperglycemia, improving intestinal flora, and preventing tooth decay. It is widely used in food, medicine, cosmetics and other fields.

[0003] Since D-tagatose is extremely rare in nature and cannot be directly obtained in large quantities from nature, and chemical preparation methods are polluting and have many by-products, bioconversion has become the most likely preparation method to be industrialized. The existing technology (see "Wang Lifei, Tan Ziyu, Xie Xixian, Zhu Leilei. New enzyme mining and enzymatic properties of tagatose-4-isomerase[J]. Acta Microbiologica Sinica, 2023, 63 (11): 4197-4207.") reported that the conversion rate of catalyzing fructose to tagatose is 14%, and the conversion rate of D-tagatose is low.

[0004] Currently, there is a lack of Bacillus subtilis strains with high D-tagatose conversion efficiency. Summary of the invention

[0005] The invention provides a recombinant Bacillus subtilis, a method for preparing a fermentation broth, immobilized cells and a preparation method thereof, and a method and application for synthesizing D-tagatose. The provided recombinant Bacillus subtilis has a high conversion rate of D-tagatose.

[0006] In order to solve the above technical problems, the following technical solutions are proposed:

[0007] The present invention provides a recombinant Bacillus subtilis ( Bacillus subtilis )BLCY-012, the deposit number is CGMCC No.31950.

[0008] The present invention provides a method for preparing a fermentation broth, comprising: inoculating the recombinant Bacillus subtilis BLCY-012 of the above technical solution into a culture medium for culturing to obtain a fermentation broth;

[0009] During the culturing process, when dissolved oxygen rebound occurs in the fermentation broth, a feed medium is added, wherein the components of the feed medium include glycerol; after the feed medium is added, the glycerol concentration in the fermentation broth is 4-7 g / L;

[0010] When the fermentation liquid OD 600 When the OD value of the fermentation liquid is 45-55, isopropyl-β-D-thiogalactoside is added for the first time for induction. 600 When the concentration of isopropyl-β-D-thiogalactoside is 100-120, isopropyl-β-D-thiogalactoside is added for induction for the second time, and the final concentration of isopropyl-β-D-thiogalactoside in the fermentation broth after the first and second additions is 0.2-0.4 mM, respectively.

[0011] The present invention provides an immobilized cell of recombinant Bacillus subtilis BLCY-012, comprising a manganese-containing immobilized carrier, recombinant Bacillus subtilis BLCY-012 cells, a cross-linking agent solution and water;

[0012] The immobilized carrier is mixed with the manganese-containing solution and then treated to obtain the manganese-containing immobilized carrier;

[0013] The fermentation liquid prepared by the preparation method described in the above technical solution is centrifuged to obtain recombinant Bacillus subtilis BLCY-012 cells;

[0014] The mass ratio of the manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and water is 1: (0.8-1.5): (2.0-3.0);

[0015] The volume of the cross-linking agent solution is 0.2 to 0.35 times the volume of the manganese-containing immobilized carrier; the concentration of the cross-linking agent solution is 0.8% to 1.3%.

[0016] Preferably, the manganese-containing solution comprises a manganese sulfate solution; the molar concentration of the manganese sulfate solution is 30-40 mM.

[0017] The present invention provides a method for preparing immobilized cells according to the above technical solution, comprising:

[0018] The manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and the first part of water are mixed to obtain a first mixture; the first mixture is cross-linked with a cross-linking agent to obtain immobilized cells.

[0019] The present invention provides the recombinant Bacillus subtilis ( Bacillus subtilis ) Use of BLCY-012 or the fermentation broth prepared by the preparation method described in the above technical solution or the immobilized cells described in the above technical solution or the immobilized cells prepared by the preparation method described in the above technical solution in the preparation of D-tagatose.

[0020] The present invention provides a method for synthesizing D-tagatose, comprising: treating the recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-012 or the fermentation liquid prepared by the preparation method described in the above technical solution or the immobilized cells described in the above technical solution or the immobilized cells prepared by the preparation method described in the above technical solution are mixed with a D-fructose solution, and then converted, purified and crystallized to obtain D-tagatose; the crystallization method includes a combination of boiling sugar crystallization and cooling crystallization.

[0021] Preferably, the solid content of the solution to be crystallized is controlled to be 70%-72%, the amount of seed crystals added is 1%-5% of the dry basis mass of the solution to be crystallized, the temperature of boiling sugar is 50-57°C, and the boiling time is 12-16h.

[0022] Preferably, during the cooling crystallization, when the temperature of the sugar boiling liquid is 40-50°C, the temperature is lowered by 0.2-0.5°C per hour; when the temperature of the sugar boiling liquid is 30-39°C, the temperature is lowered by 0.5-1.0°C per hour.

[0023] Preferably, the conversion is completed by an immobilized column, the concentration of the D-fructose solution is 500-600 g / L, the flow rate of the D-fructose solution is 1-2 times the immobilized column / h, and the conversion temperature is 55-60°C.

[0024] Preferably, the purification comprises decolorization, ion exchange, vacuum concentration and chromatographic separation in sequence; the mass ratio of the chromatographic separation is 1g: (3.5-4.0)mL; the processing capacity of the chromatographic separation is 0.025-0.028T.DS / h / m 3 Resin.

[0025] Technical effect of the present invention: The present invention provides a recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-012, the deposit number is CGMCC No. 31950. The recombinant Bacillus subtilis BLCY-012 of the present invention can produce D-tagatose-4-epimerase during the fermentation process. D-tagatose-4-epimerase is an intracellular enzyme. D-tagatose-4-epimerase can prepare D-tagatose using D-fructose as a raw material. The recombinant Bacillus subtilis BLCY-012 is used to prepare immobilized cells, and the immobilized cells are used as enzyme carriers to convert D-fructose into D-tagatose. The conversion rate can reach 28% to 32%, and the conversion rate is high and the conversion amount per unit mass of the immobilized recombinant Bacillus subtilis cells is high.

[0026] Biological Deposit Description

[0027] Bacillus subtilis ( Bacillus subtilis)BLCY-012, deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on September 12, 2024, with the deposit number CGMCC No.31950, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0028] The present invention provides a recombinant Bacillus subtilis ( Bacillus subtilis )BLCY-012, the deposit number is CGMCC No.31950.

[0029] The mainstream synthesis method of D-tagatose in the current prior art is: using D-galactose as a substrate, adding L-arabinose isomerase to convert D-galactose into D-tagatose, but D-galactose comes from the hydrolysis of lactose, 1g of lactose can only hydrolyze 0.5g of D-galactose, the price of lactose itself is relatively high, and the resulting D-galactose is even more expensive, and the conversion rate of L-arabinose isomerase to D-galactose can only reach 50%, resulting in a low unit raw material yield of the product, resulting in high production costs of D-tagatose and a market price of 150 to 180 yuan / kg, which seriously limits the large-scale production and promotion of the product. In the prior art, D-tagatose is prepared using D-fructose as a raw material, and the synthesis conversion rate of D-tagatose is low. The recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-012 can ferment and produce D-tagatose-4-epimerase, and can prepare D-tagatose using D-fructose as raw material. Compared with galactose, D-fructose is easy to obtain as a production raw material, with low cost, which reduces the difficulty of production.

[0030] The present invention provides a method for preparing a fermentation broth, comprising inoculating the recombinant Bacillus subtilis BLCY-012 of the above technical solution into a culture medium for culturing to obtain a fermentation broth.

[0031] As an optional embodiment, the present invention has no special limitation on the inoculation method, and a conventional inoculation method may be used.

[0032] As an optional embodiment, the present invention inoculates the recombinant Bacillus subtilis BLCY-012 seed liquid in a culture medium for culture. As an optional embodiment, the preparation method of the recombinant Bacillus subtilis BLCY-012 seed liquid of the present invention comprises: streaking the recombinant Bacillus subtilis BLCY-012 on a solid culture medium plate, activating the culture to obtain a single colony; inoculating the single colony in a liquid culture medium for seed culture to obtain a seed liquid. As an optional embodiment, the temperature of the activation culture is 33-37°C, and can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the activation culture is 33, 34, 35, 36 or 37°C. As an optional embodiment, the time of the activation culture is 20-26h, and can also be 22-24h. In a specific embodiment of the present invention, the time of the activation culture is 20, 21, 22, 23, 24, 25 or 26h. As an optional embodiment, the activation culture is an inverted culture. As an optional embodiment, the solid culture medium includes LB solid culture medium. As an optional embodiment, a single colony is obtained, and the present invention inoculates the single colony into an LB liquid culture medium for seed culture to obtain a seed solution. The temperature of the seed culture of the present invention is 33-37°C, and can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the seed culture is 33, 34, 35, 36 or 37°C; the time of the seed culture is 8-12h, and can also be 9-11h. In a specific embodiment of the present invention, the time of the seed culture is 8, 9, 10, 11 or 12h. As an optional embodiment, the liquid culture medium includes an LB liquid culture medium. As an optional embodiment, the liquid volume of the liquid culture medium of the present invention is 1 / 10-1 / 5 of the volume of the culture container. In a specific embodiment of the present invention, the liquid volume of the liquid culture medium is 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6 or 1 / 5 of the volume of the culture container. As an optional embodiment, the rotation speed of the seed culture is 170~220rpm. In a specific embodiment of the present invention, the rotation speed of the seed culture is 170, 180, 190, 200, 210 or 220rpm.

[0033] As an optional embodiment, a seed solution of recombinant Bacillus subtilis BLCY-012 is obtained, and the seed solution is inoculated into a culture medium for cultivation.

[0034] As an optional embodiment, the composition of the culture medium of the present invention includes: 2.5~4g / L corn steep liquor powder, 6~10g / L glycerol, 10.5~13.5g / L potassium dihydrogen phosphate, 1.0~1.5g / L magnesium sulfate heptahydrate, 3.0~5.0g / L diammonium hydrogen phosphate and 1.2~1.8g / L citric acid monohydrate.

[0035] As another implementation, the corn steep powder of the culture medium is 2.5-3 g / L. The function of the corn steep powder is to provide a nitrogen source and synthesize various enzyme systems and various proteins required for life. As another implementation, the glycerol of the culture medium is 6-8 g / L. The function of the glycerol is to provide a carbon source. As another implementation, the potassium dihydrogen phosphate of the culture medium is 10.5-12 g / L. The function of the potassium dihydrogen phosphate is to provide trace elements and provide energy to microorganisms. As another implementation, the magnesium sulfate heptahydrate of the culture medium is 1.0-1.2 g / L. The function of the magnesium sulfate heptahydrate is to provide trace elements. As another implementation, the diammonium hydrogen phosphate of the culture medium is 3.0-4.0 g / L. The function of diammonium hydrogen phosphate is to provide trace elements. As another implementation, the citric acid monohydrate of the culture medium is 1.2-1.5 g / L. The function of citric acid monohydrate is to stabilize pH.

[0036] As an optional embodiment, the inoculation amount of the seed solution of the present invention is 5-15% of the volume of the culture medium. In a specific embodiment of the present invention, the inoculation amount of the seed solution is 5%, 7%, 9%, 11%, 13% or 15% of the volume of the culture medium.

[0037] As an optional embodiment, the culture temperature is 35-37°C. In a specific embodiment of the present invention, the culture temperature is 35, 36 or 37°C. As an optional embodiment, the pH value during the culture process is controlled to be 6.8-7.2, and the dissolved oxygen rate during the culture process is controlled to be 25%-35%.

[0038] During the culture process of the present invention, when dissolved oxygen rebound occurs in the fermentation broth, feed medium is added, and the components of the feed medium include glycerol; the glycerol concentration in the fermentation broth after the feed medium is added is 4-7 g / L. In the culture process of the present invention, the dissolved oxygen rate of the fermentation broth is controlled to be the target dissolved oxygen rate, and when the actual dissolved oxygen rate is higher than the target dissolved oxygen rate and the difference is greater than or equal to 5%, it is considered that dissolved oxygen rebound occurs. The target dissolved oxygen rate of the present invention is 25%-35%. In a specific embodiment of the present invention, the target dissolved oxygen rate is 25%, 26%, 28%, 30%, 33% or 35%. Setting the target dissolved oxygen rate to 25%-35% can promote the growth and reproduction of recombinant Bacillus subtilis BLCY-012 and produce more D-tagatose-4-epimerase. When dissolved oxygen rebound occurs, it means that the nutrients in the culture medium are exhausted. In the present invention, feed medium is added to the fermentation broth when dissolved oxygen rebound occurs; the components of the feed medium of the present invention include glycerol. As an optional embodiment, the composition of the feed medium includes: 400-500 g / L glycerol, 8-10 g / L corn dry pulp powder and 16-20 g / L magnesium sulfate heptahydrate. As another implementation, the feed medium of the present invention uses water as a solvent, and the composition includes: 400-450 g / L glycerol, 8-9 g / L corn dry pulp powder and 17-19 g / L magnesium sulfate heptahydrate. As another implementation, the feed medium uses water as a solvent, and the composition includes: 500 g / L glycerol, 10 g / L corn dry pulp powder and 20 g / L magnesium sulfate heptahydrate. After the feed medium is fed, the glycerol concentration in the fermentation broth of the present invention is 4-7 g / L. The fermentation culture of the feed medium is terminated.

[0039] During the culture process of the present invention, as the culture progresses, the OD 600 When the fermentation liquid OD 600 When the OD value of the fermentation liquid is 45-55, isopropyl-β-D-thiogalactoside (‌IPTG) is added for the first time for induction. 600 When the pH value was 100-120, IPTG was added for the second time for induction. The final concentrations of IPTG in the fermentation broth after the first and second additions were 0.2-0.4 mM, respectively.

[0040] As an optional embodiment, when the fermentation liquid OD 600When the OD of the fermentation liquid is 45-55, first reduce the temperature of the fermentation liquid to 25-28°C, and then add IPTG. The inducer is not resistant to high temperatures, and the inducer can be added after the temperature is reduced to promote the synthesis of D-tagatose-4-isomerase. After reducing to 25-28°C, maintain the temperature until the IPTG induction is completed. The final concentration of IPTG in the fermentation liquid after the first addition of the present invention is 0.2-0.4 mM. As an optional embodiment, the final concentration of IPTG in the fermentation liquid after the first addition of the present invention is 0.25-0.35 mM. When the OD of the fermentation liquid of the present invention is 600 When the pH is 100-120, IPTG is added for induction for the second time, and the final concentration of IPTG in the fermentation broth after the second addition is 0.2-0.4 mM. As an optional embodiment, the final concentration of IPTG in the fermentation broth after the second addition of the present invention is 0.25-0.35 mM. As the fermentation proceeds, the number of microorganisms increases, so it is necessary to supplement the inducer IPTG. Adding IPTG can promote the production of D-tagatose-4-epimerase.

[0041] As an optional embodiment, after adding IPTG for the second time, the OD of the fermentation broth of the present invention is 600 When the highest point is reached, the fermentation culture ends and the fermentation liquid is obtained.

[0042] The D-tagatose-4-epimerase concentration in the fermentation liquid prepared by the invention is high.

[0043] The present invention provides an immobilized cell of recombinant Bacillus subtilis BLCY-012, comprising a manganese-containing immobilized carrier, recombinant Bacillus subtilis BLCY-012 cells, a crosslinking agent solution and water. The mass ratio of the manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and the water is 1: (0.8-1.5): (2.0-3.0).

[0044] The present invention mixes an immobilized carrier with a manganese-containing solution and then treats it to obtain a manganese-containing immobilized carrier. As an optional embodiment, the immobilized carrier includes a resin. In a specific embodiment of the present invention, the resin used is D101 resin. As an optional embodiment, the treatment method includes soaking. As an optional embodiment, the soaking time is 10~15h, or 12~14h. As an optional embodiment, the manganese-containing solution includes a manganese sulfate solution, the molar concentration of the manganese sulfate solution is 30~40mM, or 30~35mM, and manganese ions are used as activators of D-tagatose-4-epimerase. Adding manganese sulfate to load manganese ions on the immobilized carrier can maximize the activity of D-tagatose-4-epimerase. As an optional embodiment, before soaking in the manganese sulfate solution, it also includes soaking the immobilized carrier in water, soaking in an alkaline solution, and soaking in an acid solution in sequence. The present invention does not specifically limit the parameters of the water soaking, alkaline solution soaking, and acid solution soaking, and conventional parameters can be used.

[0045] The method for preparing the recombinant Bacillus subtilis BLCY-012 cells of the present invention comprises: centrifuging the fermentation broth to obtain BLCY-012 cells, namely, the recombinant Bacillus subtilis BLCY-012 cells. The method for preparing the fermentation broth of the present invention has been discussed above and will not be repeated here.

[0046] As an optional embodiment, the concentration of the crosslinking agent solution of the present invention is 0.8% to 1.3%, or 1%. The volume of the crosslinking agent solution of the present invention is 0.2 to 0.35 times the volume of the manganese-containing immobilized carrier. In a specific embodiment of the present invention, the volume of the crosslinking agent solution is 0.2, 0.3 or 0.35 of the volume of the immobilized carrier.

[0047] The present invention provides a method for preparing immobilized cells according to the above technical solution, comprising:

[0048] The manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and the first part of water are mixed to obtain a first mixture; the first mixture is cross-linked with a cross-linking agent to obtain immobilized cells.

[0049] As an optional embodiment, the mixing method includes adsorption, the adsorption temperature can be 30-35°C, in a specific embodiment of the present invention, the adsorption temperature can be 30, 31, 32, 33, 34 or 35°C; the adsorption time can be 5-8h, in a specific embodiment of the present invention, the adsorption time can be 5, 6, 7 or 8h. During the adsorption process, the manganese-containing immobilized carrier adsorbs the recombinant Bacillus subtilis BLCY-012 cells.

[0050] As an optional embodiment, the present invention adds a crosslinking agent solution to the first mixture for crosslinking to obtain immobilized cells. In a specific embodiment of the present invention, the crosslinking temperature can be 30, 31, 32, 33, 34 or 35°C; the crosslinking time can be 1 to 2 hours, and in a specific embodiment of the present invention, the crosslinking time can be 1, 1.5 or 2 hours.

[0051] The present invention provides the recombinant Bacillus subtilis ( Bacillus subtilis ) Use of BLCY-012 or the fermentation broth prepared by the preparation method described in the above technical solution or the immobilized cells described in the above technical solution or the immobilized cells prepared by the preparation method described in the above technical solution in the preparation of D-tagatose.

[0052] The present invention provides a method for synthesizing D-tagatose, comprising: treating the recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-012 or the fermentation liquid prepared by the preparation method described in the above technical solution or the immobilized cells described in the above technical solution or the immobilized cells prepared by the preparation method described in the above technical solution are mixed with a D-fructose solution, and then converted, purified and crystallized to obtain D-tagatose; the crystallization method includes a combination of boiling sugar crystallization and cooling crystallization.

[0053] As an optional embodiment, the present invention fills the immobilized cells into an immobilized column, flows a D-fructose solution through the top of the immobilized column, and performs conversion, and the solution flowing out at the bottom of the immobilized column is a D-tagatose conversion solution. The present invention has no particular limitation on the immobilized column, and conventional products can be used.

[0054] As an optional embodiment, the concentration of the D-fructose solution of the present invention is 500-600 g / L; in a specific embodiment of the present invention, the concentration of the D-fructose solution is 500, 550 g / L or 600 g / L. The temperature of the D-fructose solution flowing into the immobilized column is 55-60°C, or 56-58°C; in a specific embodiment of the present invention, the temperature of the D-fructose solution is 55, 56, 58 or 60°C. The volume of the immobilized column of the present invention is referred to as the column volume, and the flow rate of the D-fructose solution of the present invention is 1-2 times the column volume / h, or 1.2-1.8 times the column volume / h. The present invention passes the D-fructose solution through the immobilized column to obtain a D-tagatose conversion liquid with a D-tagatose concentration of 150-195 g / L. The immobilized column contains D-tagatose-4-epimerase, which uses fructose as a raw material to convert fructose into D-tagatose.

[0055] As an optional embodiment, before crystallization, the present invention sequentially purifies the D-tagatose conversion liquid; the purification treatment is refined purification, and the refined purification includes sequentially performing decolorization, ion exchange, vacuum concentration and chromatographic separation to obtain D-tagatose concentrated liquid. As an optional embodiment, the method for decolorization and ion exchange of the present invention includes: first decolorizing the D-tagatose conversion liquid to obtain a decolorized material; and then performing ion exchange on the decolorized material to obtain an ion exchanged material. As an optional embodiment, the decolorization of the present invention includes activated carbon decolorization and filter decolorization, and the activated carbon decolorization can be performed using a granular activated carbon column, and the material temperature when the granular activated carbon column is used for decolorization is 55~65℃, and the filter decolorization can be performed using an Ama filter; the material temperature of the filter decolorization is 50~60℃, and the working pressure of the filter decolorization is 0.2~0.4Mpa. The function of the decolorization is to remove the pigment in the conversion liquid. The ion exchange of the present invention includes sequentially performing cation exchange, anion exchange, cation exchange, anion exchange and cation exchange. The present invention has no special limitation on the resin used for the cation exchange and the resin used for the anion exchange, and conventional commercially available products can be used. The material temperature of the ion exchange of the present invention can be 35-45°C, or 38-43°C; the material flow rate of the ion exchange can be 2-4BV / h, or 2.5-3.5BV / h. The function of the ion exchange is to purify the D-tagatose conversion liquid.

[0056] As an optional embodiment, the present invention performs vacuum concentration and chromatographic separation on the material after ion exchange. As another embodiment, the present invention performs first vacuum concentration, chromatographic separation and vacuum concentration again on the material after ion exchange obtained after ion exchange. The present invention performs chromatographic separation on the material obtained after the first vacuum concentration to obtain a chromatographically separated material, and the chromatographically separated material is vacuum concentrated again to obtain a D-tagatose concentrate. The equipment used for the first vacuum concentration and the second vacuum concentration of the present invention is a six-effect evaporator. The pressures of the first vacuum concentration and the second vacuum concentration of the present invention are -0.06~-0.1Mpa respectively; the temperatures of the first vacuum concentration and the second vacuum concentration of the present invention are 65~75°C respectively. The solid content of the concentrated material obtained after the first vacuum concentration of the present invention is 55%~60%. The solid content of the concentrated material obtained after the second vacuum concentration of the present invention is 71%~73%, that is, the solid content of the D-tagatose concentrate is 71%~73%.

[0057] As an optional embodiment, the chromatographic separation equipment is a simulated moving bed (SMB) chromatogram. The mass ratio of the first vacuum concentrated material for chromatographic separation in the present invention is 1g: (3.5-4.0) mL, or 1g: (3.5-3.8) mL; the resin used in the chromatographic separation is a gel-type polystyrene calcium chromatographic resin, and the chromatographic separation processing capacity is 0.025-0.028 T.DS / h / m 3 Resin, can also be 0.026~0.027T.DS / h / m 3 Resin. T.DS / h / m 3 The resin is the dry basis mass of the material passing through per cubic meter of resin per hour. The yield of D-tagatose after chromatographic separation of the present invention is 80% to 83%.

[0058] As an optional embodiment, the present invention sequentially crystallizes, centrifuges and dries the D-tagatose concentrate to obtain crystalline D-tagatose; the crystalline D-tagatose has a high purity of more than 99.5%.

[0059] As an optional embodiment, the solid content of the D-tagatose concentrate obtained by boiling sugar and crystallizing the present invention is controlled to be 70% to 72%. In a specific embodiment of the present invention, the solid content of the D-tagatose concentrate is controlled to be 70%, 71% or 72%. As an optional embodiment, the present invention adds seed crystals during the process of boiling sugar and crystallizing, and the seed crystals are D-tagatose fine powder, and the D-tagatose fine powder is 180-200 mesh. The amount of the seed crystals added is 1% to 5% of the dry basis weight of the D-tagatose concentrate, and can also be 2% to 3%. In a specific embodiment of the present invention, the amount of the seed crystals added is 1%, 2%, 3% or 5% of the dry basis weight of the solution to be crystallized.

[0060] In the process of sugar boiling crystallization of the present invention, the solid content of the sugar boiling liquid will continue to increase due to the continuous evaporation of water from the sugar boiling liquid. In order to control the solid content of the sugar boiling liquid to 70%~72%, it is necessary to add a feeding liquid. As an implementable method, the feeding liquid of the present invention is a D-tagatose solution, and the solid content of the D-tagatose solution is 45%~50%. The temperature of the sugar boiling crystallization of the present invention is 50~57℃, or 52~55℃; the sugar boiling time of the present invention is 12~16h, or 14~15h. The equipment used for sugar boiling crystallization of the present invention is a sugar boiling tank; the sugar boiling time is counted from the time when the D-tagatose concentrated solution enters the sugar boiling tank. After the sugar boiling crystallization of the present invention is completed, a sugar boiling liquid is obtained, and the sugar boiling liquid is cooled and crystallized. When the temperature of the sugar-boiling liquid is 40-50° C., the sugar-boiling liquid of the present invention can be cooled by 0.2-0.5° C. per hour, or by 0.3-0.4° C. per hour; when the temperature of the sugar-boiling liquid is reduced to 30-39° C., the sugar-boiling liquid of the present invention can be cooled by 0.5-1.0° C. per hour, or by 0.6-0.8° C. per hour.

[0061] The present invention has no special restrictions on the centrifugation and drying methods, and conventional methods can be used. In a specific embodiment of the present invention, the centrifugation is performed by a vertical scraper centrifuge, the centrifugation speed is 600-900 rpm, the centrifugation time is 30-40 min, and the washing water volume is 1%-5% of the liquid volume. The drying is performed by a fluidized bed, the inlet air temperature of the fluidized bed is 58-65°C, the cooling temperature is 25-30°C, and the drying time is 20-30 min.

[0062] The method for synthesizing D-tagatose provided by the present invention uses immobilized recombinant Bacillus subtilis cells as enzyme carriers to convert D-fructose into D-tagatose, and the conversion rate can reach 28% to 32%. The unit mass of immobilized recombinant Bacillus subtilis cells can convert 70 to 100 times the mass of D-fructose, and the efficient utilization of recombinant Bacillus subtilis cells is experimentally demonstrated, and the cost of enzyme preparations is greatly reduced.

[0063] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0064] The LB solid medium consisted of 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder and 1 L of distilled water.

[0065] The composition of LB liquid culture medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl and 1 L distilled water.

[0066] In the following examples and comparative examples, the seed crystal is D-tagatose fine powder, and the D-tagatose fine powder has a size of 180-200 meshes.

[0067] Example 1

[0068] (1) Streak the recombinant Bacillus subtilis BLCY-012 on an LB plate and incubate it at 33°C for 20 h to obtain a single colony. The recombinant Bacillus subtilis BLCY-012 was purchased from the National Key Laboratory of Food Science and Resource Mining, Jiangnan University.

[0069] (2) Pick a single colony obtained in step (1) and inoculate it into an LB triangular shake flask, where the amount of liquid culture medium is 1 / 10 of the volume of the triangular shake flask, and culture it at 170 rpm for 8 h to obtain a seed solution.

[0070] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 5% of the volume of the fermentation medium, and the fermentation culture was carried out at 35°C. The pH value during the culture process was 7.0, and the dissolved oxygen rate was 26%. When the dissolved oxygen rate was 31%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to be 5g / L. When the OD of the fermentation liquid reached 50%, the glycerol concentration in the fermentation liquid was controlled to be 50%. 600 When the OD value of the fermentation liquid reached 45, the temperature of the fermentation liquid was lowered to 25°C, and IPTG was added to a final concentration of 0.2 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 0.2 mM for induction. 600 When the highest point is reached, the fermentation culture is stopped to obtain the fermentation liquid.

[0071] The composition of each 1L of fermentation medium is: 2.5g corn steep liquor dry powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate and balance water, and the pH is 7.0.

[0072] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 8 g / L corn pulp powder and 17 g / L magnesium sulfate heptahydrate.

[0073] (4) Using a tubular centrifuge, separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 10° C. and the centrifugal speed is controlled at 8000 rpm.

[0074] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution was 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution was 7.0. Finally, the resin was soaked in a 30 mM manganese sulfate solution for 10 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0075] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0, adsorbed at 30°C for 5 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 30°C, and the cross-linking time was 1 hour, thereby completing the preparation of immobilized cells.

[0076] (6) The immobilized cells obtained in step (5) are filled into the immobilized column, and the D-fructose solution is introduced into the immobilized column through the top of the immobilized column. The liquid flowing out from the bottom of the immobilized column is the D-tagatose conversion liquid, and the D-tagatose solution concentration of the D-tagatose conversion liquid is 154.65 g / L. The solution concentration of the D-fructose solution is 500 g / L, the temperature is 55°C, and the flow rate of the D-fructose solution is 1 column volume / h; the column volume is the volume of the immobilized column, the same below.

[0077] (7) The D-tagatose conversion liquid obtained in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0078] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 55°C, and it is first decolorized using a granular activated carbon column, and then filtered through an Ama filter with a working pressure of 0.2Mpa to obtain a decolorized material.

[0079] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, the material temperature is 35° C., the flow rate is 2BV / h, and the ion exchanged material is obtained.

[0080] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 65°C, the pressure is -0.06Mpa, and after the primary vacuum concentration, the solid content of the material is 55%, and a primary vacuum concentrated material is obtained.

[0081] Chromatographic separation: the vacuum concentrated material obtained once is fed into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g:3.5 mL, with a processing capacity of 0.025 T.DS / h / m 3 The resin had a D-tagatose yield of 80%, and the material after chromatographic separation was obtained.

[0082] The secondary vacuum concentration process is: use a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 65°C, the pressure is -0.06Mpa, and after the secondary vacuum concentration, the solid content of the material is 71%, and D-tagatose concentrated solution is obtained.

[0083] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0084] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled to 70%, and crystal seeds are added. The amount of crystal seeds added is 1% of the dry basis mass of D-tagatose concentrate. The temperature of the crystallization process is controlled to 50°C. During sugar boiling crystallization, the feed liquid is continuously replenished. The feed liquid is D-tagatose solution. The solid content of D-tagatose solution is 45%. The sugar boiling time is 12 hours. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. In the cooling crystallization section, the sugar boiling liquid is cooled by 0.2°C per hour in the range of 40-50°C, and the sugar boiling liquid is cooled by 0.5°C per hour in the range of 30-39°C. It is reduced to 30°C, and the crystallization liquid is obtained after the crystallization is completed.

[0085] Centrifugation was carried out using a vertical scraper centrifuge. The crystallized liquid entered the centrifuge at a speed of 600 rpm and a centrifugal time of 30 min. The crystallized liquid was washed during the centrifugation process, and the amount of water used for washing was 1% of the volume of the liquid.

[0086] Drying: The fluidized bed was used for drying, the air inlet temperature of the fluidized bed was 58°C, the cooling temperature was 30°C, and the drying time was 20 minutes. After drying, D-tagatose was obtained.

[0087] Example 2

[0088] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 37°C for 25 h to obtain a single colony.

[0089] (2) Picking step (1) The single colony obtained by culture was inoculated into an LB triangular shake flask, wherein the amount of liquid culture medium filled was 1 / 5 of the volume of the triangular shake flask, and cultured at 220 rpm for 12 h to obtain a seed solution.

[0090] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 15% of the volume of the fermentation medium, and the fermentation culture was carried out at 37°C. The pH during the culture process was 7.2 and the dissolved oxygen rate was 35%. When the dissolved oxygen rate was 40%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 7 g / L. When the OD of the fermentation liquid reached 0.05, the glycerol concentration in the fermentation liquid was controlled to 0.05. 600 When the OD value of the fermentation liquid reached 55, the temperature of the fermentation liquid was reduced to 28°C, and IPTG was added to induce the fermentation liquid to a final concentration of 0.4 mM. 600 When the OD value of the fermentation liquid reached 120, IPTG was added for the second time to make the final concentration 0.4mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0091] The composition of each 1L of fermentation medium is: 4g corn steep liquor powder, 10g glycerol, 13.5g potassium dihydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 5.0g diammonium hydrogen phosphate, 1.8g citric acid monohydrate and balance water, and the pH is 7.2.

[0092] The composition of the feed medium is: water as solvent, 500 g / L glycerol, 10 g / L corn pulp powder and 20 g / L magnesium sulfate heptahydrate.

[0093] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 4° C. and the centrifugal speed is controlled at 8000 rpm.

[0094] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 40 mM manganese sulfate solution for 15 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0095] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:1.5:3.0, and adsorbed at 35°C for 8 hours. Then, chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.35 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 35°C, and the cross-linking time was 2 hours. The preparation of immobilized cells was completed.

[0096] (6) The immobilized cells obtained in step (5) are filled into an immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion solution, wherein the D-tagatose solution concentration of the D-tagatose conversion solution is 191 g / L. The D-fructose solution concentration is 600 g / L, the temperature is 60° C., and the D-fructose solution flow rate is 2 times the column volume / h.

[0097] (7) The D-tagatose conversion liquid obtained in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0098] Decolorization is as follows: the material temperature of the D-tagatose conversion liquid is 65°C, and a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 60°C, and then filtered through an Ama filter with a working pressure of 0.4Mpa to obtain a decolorized material.

[0099] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, the material temperature of the D-tagatose conversion liquid is 45° C., the flow rate is 4BV / h, and the ion exchanged material is obtained.

[0100] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 75°C, the pressure is -0.1Mpa, and after the primary vacuum concentration, the solid content of the material is 60%, and the primary vacuum concentrated material is obtained.

[0101] Chromatographic separation: the vacuum concentrated material obtained once was put into the simulated moving bed (SMB) chromatography at a mass ratio of 1g:4.0mL, with a processing capacity of 0.028T.DS / h / m 3 The resin had a D-tagatose yield of 83%, and the material after chromatographic separation was obtained.

[0102] The secondary vacuum concentration process is: use a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 75°C, the pressure is -0.1Mpa, and after the secondary vacuum concentration, the solid content of the material is 73%, and D-tagatose concentrated solution is obtained.

[0103] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0104] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific sugar boiling crystallization stage is as follows: the solid content of the D-tagatose concentrate is controlled at 72%, and crystal seeds are added. The amount of crystal seeds added is 5% of the dry basis weight of the D-tagatose concentrate. The temperature of the crystallization process is controlled at 57°C. The feed liquid is continuously replenished during the sugar boiling crystallization. The feed liquid is a D-tagatose solution. The solid content of the D-tagatose solution is 50%, and the sugar boiling time is 16 hours. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization stage. The cooling crystallization stage is in the range of 40~50°C, and the temperature drops by 0.5°C per hour. In the range of 30~39°C, the temperature drops by 1.0°C per hour. When it drops to 30°C, the crystallization ends.

[0105] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 900 rpm for 40 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 5% of the volume of the liquid.

[0106] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 65° C., the cooling temperature was set at 25° C., and the drying time was set at 30 min. After drying, D-tagatose was obtained.

[0107] Comparative Example 1

[0108] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 35°C for 21 h to obtain a single colony.

[0109] (2) Pick a single colony obtained in step (1) and inoculate it into an LB triangular shake flask, where the amount of liquid culture medium is 1 / 7 of the volume of the triangular shake flask, and culture it at 33°C and 200 rpm for 9 hours to obtain a seed solution.

[0110] (3) Fermentation culture: The seed liquid described in step (2) was inoculated into the fermentation medium, the inoculation amount was 7% of the volume of the fermentation medium, and the fermentation culture was carried out at 35°C. The pH value during the culture process was 7.0 and the dissolved oxygen rate was 25%. When the dissolved oxygen rate was 30%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to be 2 g / L. When the OD value of the fermentation liquid was 0.04, the glycerol concentration in the fermentation liquid was controlled to be 0.03. 600 When the temperature of the fermentation liquid reached 45, the temperature was lowered to 26°C, and IPTG was added to a final concentration of 0.3 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 0.2 mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0111] The composition of each 1L of fermentation medium is: 2.5g corn steep liquor dry powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate and balance water, pH 7.0.

[0112] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 8 g / L corn pulp powder and 18 g / L magnesium sulfate heptahydrate.

[0113] (4) Using a tubular centrifuge, separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 15° C. and the centrifugal speed is controlled at 8000 rpm.

[0114] (5) Preparation of immobilized cells: D101 resin was used for immobilized cells. The pretreatment process of the resin was as follows: soak the resin in distilled water 4 times the volume of D101 resin for 4 h, filter out the distilled water, soak the resin in a 5% NaOH solution for 5 h, filter out the NaOH solution, wash the D101 resin with distilled water until the pH of the washing solution reached 7.0, soak the resin in a 5% HCl solution for 5 h, filter out the HCl solution, wash the resin with distilled water until the pH of the washing solution reached 7.0, soak the resin in a 30 mM manganese sulfate solution for 10 h, filter out the excess liquid, and complete the pretreatment of the resin.

[0115] The pretreated resin, the cells in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.5, adsorbed at 30°C for 6 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.3 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 31°C, and the cross-linking time was 1 hour, thereby completing the preparation of immobilized cells.

[0116] (6) The immobilized cells obtained in step (5) are filled into an immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion liquid, wherein the D-tagatose concentration of the D-tagatose conversion liquid is 142.39 g / L. The D-fructose liquid concentration is 550 g / L, the temperature is 57° C., and the flow rate of the D-fructose liquid is 1.5 times the column volume / h.

[0117] (7) The D-tagatose conversion liquid obtained in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0118] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, and the granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 55°C, and then filtered through an Ama filter with a working pressure of 0.3Mpa to obtain the decolorized material.

[0119] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with a material temperature of 40° C. and a flow rate of 3BV / h to obtain an ion exchanged material.

[0120] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the primary vacuum concentration, the solid content of the material is 58%, and the primary vacuum concentrated material is obtained.

[0121] Simulated moving bed (SMB) chromatography separation is as follows: the obtained primary vacuum concentrated material is fed into the simulated moving bed (SMB) chromatography according to the mass ratio of 1g:3.5mL, and the processing capacity is 0.026T.DS / h / m 3 The resin had a D-tagatose yield of 82% and the material after chromatographic separation was obtained.

[0122] The secondary vacuum concentration process is: use a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrated solution is obtained.

[0123] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried.

[0124] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, with sugar boiling crystallization being performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of the D-tagatose concentrate is controlled at 71%, and crystal seeds are added. The amount of crystal seeds added is 1% of the dry basis mass of the D-tagatose concentrate. The temperature of the crystallization process is controlled at 55°C. The feed liquid is continuously replenished during sugar boiling crystallization. The feed liquid is a D-tagatose solution. The solid content of the D-tagatose solution is 45%, and the sugar boiling time is 15h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then the cooling crystallization section is entered. The cooling crystallization section is to cool down by 0.2°C per hour in the range of 40~50°C, and by 0.5°C per hour in the range of 30~39°C. When the temperature drops to 30°C, the crystallization ends.

[0125] Centrifugation was carried out using a vertical scraper centrifuge. The crystallized liquid entered the centrifuge at a speed of 600 rpm and a centrifugal time of 30 min. The crystallized liquid was washed during the centrifugation process, and the amount of water used for washing was 1% of the volume of the liquid.

[0126] The drying was carried out by fluidized bed drying. The centrifuged product entered the fluidized bed, and the air inlet temperature was set at 60°C, the cooling temperature was set at 30°C, and the drying time was set at 20 minutes. After the drying was completed, D-tagatose was obtained.

[0127] Comparative Example 2

[0128] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 35°C for 25 h to obtain a single colony.

[0129] (2) Pick a single colony obtained in the culture in step (1) and inoculate it into an LB triangular shake flask, wherein the amount of liquid culture medium filled is 1 / 7 of the volume of the triangular shake flask, and culture it at 33°C and 200 rpm for 10 h to obtain a seed solution.

[0130] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 10% of the volume of the fermentation medium, and the fermentation culture was carried out at 36°C. The pH during the culture process was 7.0 and the dissolved oxygen rate was 30%. When the dissolved oxygen rate was 35%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 5g / L. When the OD of the fermentation liquid reached 50%, the glycerol concentration in the fermentation liquid was controlled to 50 ... 600 When the OD value of the fermentation liquid reached 50, the temperature of the fermentation liquid was reduced to 26°C, and IPTG was added to a final concentration of 0.1 mM for induction. 600 When the OD value of the fermentation liquid reached 150, IPTG was added for the second time to make the final concentration 0.1mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0131] The composition of each 1L of fermentation medium is: 3.0g corn steep liquor dry powder, 8g glycerol, 12.0g potassium dihydrogen phosphate, 1.2g magnesium sulfate heptahydrate, 4.0g diammonium hydrogen phosphate, 1.5g citric acid monohydrate and balance water, and the pH is 7.0.

[0132] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 10 g / L corn pulp powder and 18 g / L magnesium sulfate heptahydrate.

[0133] (4) Using a tubular centrifuge, separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 10° C. and the centrifugal speed is controlled at 8000 rpm.

[0134] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 35 mM manganese sulfate solution for 12 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0135] The pretreated resin, the cells in step (4), and purified water were mixed in a mass ratio of 1:1.0:2.0, adsorbed at 32°C for 6 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 32°C, and the cross-linking time was 1.5 hours, thereby completing the preparation of immobilized cells.

[0136] (6) The immobilized cells obtained in step (5) are filled into an immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion solution, wherein the D-tagatose concentration of the D-tagatose conversion solution is 113.6 g / L. The solution concentration of the D-fructose solution is 550 g / L, the temperature is 58° C., and the flow rate of the D-fructose solution is 2 times the column volume / h.

[0137] (7) The D-tagatose conversion liquid obtained in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0138] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 55°C, and then filtered through an Ama filter with a working pressure of 0.3Mpa to obtain a decolorized material.

[0139] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with a material temperature of 40° C. and a flow rate of 3BV / h to obtain an ion exchanged material.

[0140] The vacuum concentration process is as follows: a six-effect evaporator is used to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after one vacuum concentration, the solid content of the material is 58%, and a vacuum concentrated material is obtained.

[0141] Chromatographic separation: the vacuum concentrated material obtained once was put into a simulated moving bed (SMB) chromatographic separation at a mass ratio of 1g:3.8mL, with a processing capacity of 0.026T.DS / h / m 3 The resin had a D-tagatose yield of 82% and the material after chromatographic separation was obtained.

[0142] The secondary vacuum concentration process is: using a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrated solution is obtained.

[0143] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0144] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled at 72%, seed crystals are added, and the amount of seed crystals added is 3% of the dry basis mass of D-tagatose concentrate. The temperature of the crystallization process is controlled at 52°C, and the feed liquid is continuously replenished during sugar boiling crystallization. The feed liquid is D-tagatose solution, and the solid content of D-tagatose solution is 48%. The sugar boiling time is 14h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. The cooling crystallization section is in the range of 40~50°C, and the temperature is reduced by 0.3°C per hour. In the range of 30~39°C, the temperature is reduced by 0.8°C per hour. When it is reduced to 30°C, the crystallization is completed and the crystallization liquid is obtained.

[0145] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 800 rpm for 35 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 2% of the volume of the liquid.

[0146] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 60° C., the cooling temperature was set at 28° C., and the drying time was set at 25 min. After drying, D-tagatose was obtained.

[0147] Comparative Example 3

[0148] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 35°C for 24 h to obtain a single colony.

[0149] (2) Pick a single colony obtained in the culture in step (1) and inoculate it into an LB triangular shake flask, wherein the amount of liquid culture medium filled is 1 / 5 of the volume of the triangular shake flask, and culture it at 200 rpm for 10 hours to obtain a seed solution.

[0150] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 10% of the volume of the fermentation medium, and the fermentation culture was carried out at 36°C. The pH value during the culture process was 7.0, and the dissolved oxygen rate was 30%. When the dissolved oxygen rate was 35%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 5 g / L. When the OD of the fermentation liquid reached 50%, the glycerol concentration in the fermentation liquid was controlled to 50 ... 600 When the OD value of the fermentation liquid reached 50, the temperature of the fermentation liquid was lowered to 25°C, and IPTG was added to a final concentration of 1.0 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 1.0 mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0151] The composition of each 1L of fermentation medium is: 2.5g corn steep liquor dry powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate and balance water, pH 7.0.

[0152] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 10 g / L corn pulp powder and 20 g / L magnesium sulfate heptahydrate.

[0153] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 4° C. and the centrifugal speed is controlled at 8000 rpm.

[0154] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 35 mM manganese sulfate solution for 15 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0155] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0, adsorbed at 30°C for 6 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 30°C, and the cross-linking time was 1 hour, thereby completing the preparation of immobilized cells.

[0156] (6) The immobilized cells obtained in step (5) are filled into an immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion liquid, wherein the D-tagatose concentration of the D-tagatose conversion liquid is 130.25 g / L. The solution concentration of the D-fructose solution is 500 g / L, the temperature is 55° C., and the flow rate of the D-fructose solution is 1 column volume / h. (7) The D-tagatose conversion liquid obtained in step (6) is sequentially subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration to obtain a D-tagatose concentrated solution.

[0157] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 55°C, and then filtered through an Ama filter with a working pressure of 0.3Mpa to obtain a decolorized material.

[0158] The ion exchange process is to pass the decolorized material through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin, and cation exchange resin in sequence, with a material temperature of 40° C. and a flow rate of 3BV / h to obtain an ion-exchanged material.

[0159] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the primary vacuum concentration, the solid content of the material is 58%, and the primary vacuum concentrated material is obtained.

[0160] Simulated moving bed (SMB) chromatography separation is as follows: the obtained primary vacuum concentrated material is fed into the simulated moving bed (SMB) chromatography according to the mass ratio of 1g:3.5mL, and the processing capacity is 0.025T.DS / h / m 3 Resin, D-tagatose yield was 80%, and the material after chromatographic separation was obtained.

[0161] The secondary vacuum concentration process is: using a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrated solution is obtained.

[0162] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0163] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled at 70%, seed crystals are added, and the amount of seed crystals added is 1% of the dry basis mass of D-tagatose concentrate. The temperature of the crystallization process is controlled at 50°C, and the feed liquid is continuously replenished during sugar boiling crystallization. The feed liquid is D-tagatose solution, and the solid content of D-tagatose solution is 45%. The sugar boiling time is 12h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. The cooling crystallization section is in the range of 40~50°C, and the temperature is reduced by 0.2°C per hour. In the range of 30~39°C, the temperature is reduced by 0.5°C per hour. When it is reduced to 30°C, the crystallization is completed and the crystallization liquid is obtained.

[0164] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 600 rpm for 30 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 1% of the volume of the liquid.

[0165] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 58° C., the cooling temperature was set at 25° C., and the drying time was set at 20 min. After drying, D-tagatose was obtained.

[0166] Comparative Example 4

[0167] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 35°C for 23 h to obtain a single colony.

[0168] (2) Picking step (1) The single colony obtained in the culture was inoculated into an LB triangular shake flask, wherein the amount of liquid culture medium filled was 1 / 8 of the volume of the triangular shake flask, and cultured at 200 rpm for 10 h to obtain a seed solution.

[0169] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 10% of the volume of the fermentation medium, and the fermentation culture was carried out at 37°C. The pH value during the culture process was 7.0 and the dissolved oxygen rate was 30%. When the dissolved oxygen rate was 35%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 6 g / L. When the OD value of the fermentation liquid was 0.04, the glycerol concentration in the fermentation liquid was controlled to 0.06. 600 When the temperature reached 50, the temperature was lowered to 27°C, and IPTG was added to make the final concentration 0.3 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 0.3 mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0170] The composition of each 1L of fermentation medium is: 4g corn steep liquor dry powder, 10g glycerol, 13.5g potassium dihydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 5.0g diammonium hydrogen phosphate, 1.8g citric acid monohydrate and balance water, pH 7.0.

[0171] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 8 g / L corn pulp powder and 18 g / L magnesium sulfate heptahydrate.

[0172] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 4° C. and the centrifugal speed is controlled at 8000 rpm.

[0173] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0.

[0174] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0, adsorbed at 32°C for 6 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.35 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 1%, the cross-linking temperature was 32°C, and the cross-linking time was 1.5 hours, thereby completing the preparation of immobilized cells.

[0175] (6) The immobilized cells described in step (5) are filled into the immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion solution, wherein the D-tagatose solution concentration of the D-tagatose conversion solution is 163.24 g / L. The solution concentration of the D-fructose solution is 550 g / L, the temperature is 60° C., and the flow rate of the D-fructose solution is 1 column volume / h.

[0176] (7) The D-tagatose conversion liquid obtained in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0177] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 60°C, and then filtered through an Ama filter, the material temperature is 60°C, and the working pressure of the Ama filter is 0.3Mpa to obtain a decolorized material.

[0178] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with a material temperature of 40° C. and a flow rate of 3BV / h to obtain an ion exchanged material.

[0179] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the primary vacuum concentration, the solid content of the material is 60%, and the primary vacuum concentrated material is obtained.

[0180] Chromatographic separation: the vacuum concentrated material obtained once was put into a simulated moving bed (SMB) chromatography for separation at a mass ratio of 1g:4.0mL, with a processing capacity of 0.025T.DS / h / m 3 The resin had a D-tagatose yield of 82% and the material after chromatographic separation was obtained.

[0181] The secondary vacuum concentration process is as follows: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrate is obtained.

[0182] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0183] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled at 70%, seed crystals are added, and the amount of seed crystals added is 3% of the dry basis weight of D-tagatose concentrate. The temperature of the crystallization process is controlled at 55°C, and the feed liquid is continuously replenished during sugar boiling crystallization. The feed liquid is D-tagatose solution, and the solid content of D-tagatose solution is 48%. The sugar boiling time is 15h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. The cooling crystallization section is in the range of 40~50°C, and the temperature is reduced by 0.2°C per hour. In the range of 30~39°C, the temperature is reduced by 0.5°C per hour. When it is reduced to 30°C, the crystallization is completed and the crystallization liquid is obtained.

[0184] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 800 rpm for 35 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 2% of the volume of the liquid.

[0185] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 60° C., the cooling temperature was set at 26° C., and the drying time was set at 25 min. After drying, D-tagatose was obtained.

[0186] Comparative Example 5

[0187] (1) Streak the recombinant Bacillus subtilis on an LB plate and culture it upside down at 35°C for 23 h to obtain a single colony.

[0188] (2) Pick a single colony obtained in step (1) and inoculate it into an LB triangular shake flask, where the amount of liquid culture medium is 1 / 7 of the volume of the triangular shake flask, and shake culture at 200 rpm for 10 h to obtain a seed solution.

[0189] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 10% of the volume of the fermentation medium, and the fermentation culture was carried out at 36°C. The pH value during the culture process was 7.0, and the dissolved oxygen rate was 30%. When the dissolved oxygen rate was 35%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 5 g / L. When the OD of the fermentation liquid reached 50%, the glycerol concentration in the fermentation liquid was controlled to 50 ... 600 When the temperature reached 50, the temperature was lowered to 26°C, and IPTG was added to a final concentration of 0.3 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 0.3 mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0190] The composition of the fermentation medium is: 3.0 g / L corn steep liquor powder, 8 g / L glycerol, 12.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 4.0 g / L diammonium hydrogen phosphate, 1.5 g / L citric acid monohydrate and balance water, pH 7.0.

[0191] The composition of the feed medium is: water as solvent, 400 g / L glycerol, 8 g / L corn pulp powder and 16 g / L magnesium sulfate heptahydrate.

[0192] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 4° C. and the centrifugal speed is controlled at 8000 rpm.

[0193] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 35 mM manganese sulfate solution for 12 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0194] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.5, adsorbed at 32°C for 7 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.3 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 8%, the cross-linking temperature was 32°C, and the cross-linking time was 1.5 hours, thereby completing the preparation of immobilized cells.

[0195] (6) The immobilized cells obtained in step (5) are filled into an immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion solution, wherein the D-tagatose concentration of the D-tagatose conversion solution is 124.13 g / L. The solution concentration of the D-fructose solution is 550 g / L, the temperature is 58° C., and the flow rate of the D-fructose solution is 1 column volume / h.

[0196] (7) The D-tagatose conversion liquid described in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain a D-tagatose concentrated liquid.

[0197] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 55°C, and then filtered through an Ama filter with a working pressure of 0.3Mpa to obtain a decolorized material.

[0198] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin, and cation exchange resin, with a material temperature of 40° C. and a flow rate of 3BV / h to obtain an ion exchanged material.

[0199] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the primary vacuum concentration, the solid content of the material is 58%, and the primary vacuum concentrated material is obtained.

[0200] Simulated moving bed (SMB) chromatography separation is as follows: the obtained primary vacuum concentrated material is fed into the simulated moving bed (SMB) chromatography at a mass ratio of 1g:4.0mL, with a processing capacity of 0.026T.DS / h / m 3 The resin had a D-tagatose yield of 81%, and the material after chromatographic separation was obtained.

[0201] The secondary vacuum concentration process is: using a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 70°C, the pressure is -0.08Mpa, and after the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrated solution is obtained.

[0202] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0203] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled at 71%, the amount of seed crystals added is 2% of the dry basis weight of D-tagatose concentrate, the temperature of the crystallization process is controlled at 55°C, the feed liquid is continuously replenished during sugar boiling crystallization, the feed liquid is D-tagatose solution, the solid content of D-tagatose solution is 48%, and the sugar boiling time is 15h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. The cooling crystallization section is in the range of 40~50°C, the temperature is reduced by 0.2°C per hour, in the range of 30~39°C, the temperature is reduced by 0.5°C per hour, and it is reduced to 30°C. The crystallization is completed and the crystallization liquid is obtained.

[0204] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 600 rpm for 30 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 2% of the volume of the liquid.

[0205] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 60° C., the cooling temperature was set at 28° C., and the drying time was set at 25 min. After drying, D-tagatose was obtained.

[0206] Comparative Example 6

[0207] (1) Streak the recombinant Bacillus subtilis on an LB plate and incubate it upside down at 35°C for 24 h to obtain a single colony;

[0208] (2) Pick a single colony obtained in step (1) and inoculate it into an LB triangular shake flask, wherein the amount of liquid culture medium is 1 / 6 of the volume of the triangular shake flask, and culture at 180 rpm for 9 hours to obtain a seed solution;

[0209] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, the inoculation amount was 8% of the volume of the fermentation medium, and the culture was carried out at 36°C, the pH during the culture process was 7.0, and the dissolved oxygen rate was 30%. When the dissolved oxygen rate was 35%, it was considered that the dissolved oxygen rebound occurred. When the dissolved oxygen rebound occurred, the feed medium was added. After the feed medium was added, the glycerol concentration in the fermentation liquid was controlled to 5 g / L. When the OD of the fermentation liquid was 600 When the OD value of the fermentation liquid reached 50, the temperature of the fermentation liquid was reduced to 27°C, and IPTG was added to a final concentration of 0.3 mM for induction. 600 When the OD value of the fermentation liquid reached 100, IPTG was added for the second time to make the final concentration 0.3 mM for induction. 600 When the maximum point is reached, the fermentation culture is stopped.

[0210] The fermentation medium consists of 4 g / L corn steep liquor powder, 10 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 5.0 g / L diammonium hydrogen phosphate, 1.8 g / L citric acid monohydrate and the remainder water, pH 7.0.

[0211] The feed medium composition is: water as solvent, 450 g / L glycerol, 8 g / L corn pulp powder and 16 g / L magnesium sulfate heptahydrate.

[0212] (4) Using a tubular centrifuge, separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, the centrifugal temperature is controlled at 10° C. and the centrifugal speed is controlled at 8000 rpm.

[0213] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in distilled water 4 times the volume of the resin for 4 h. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 h. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 5% HCl solution for 5 h. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution reached 7.0. The resin was then soaked in a 40 mM manganese sulfate solution for 15 h. The excess liquid was filtered out to complete the pretreatment of the resin.

[0214] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0, adsorbed at 30°C for 5 hours, and then chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times the volume of the pretreated resin, the mass concentration of the chitosan solution was 0.5%, the cross-linking temperature was 30°C, and the cross-linking time was 2 hours, thereby completing the preparation of immobilized cells.

[0215] (6) The immobilized cells described in step (5) are filled into the immobilized column, and the D-fructose solution is passed through the immobilized column to obtain a D-tagatose conversion liquid, wherein the D-tagatose solution concentration of the D-tagatose conversion liquid is 170.28 g / L. The concentration of the D-fructose solution is 550 g / L, the temperature is 58°C, and the flow rate of the D-fructose solution is 1 column volume / h. (7) The D-tagatose conversion liquid obtained in step (6) is sequentially subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration to obtain a D-tagatose concentrated solution.

[0216] The decolorization process is as follows: the material temperature of the D-tagatose conversion liquid is 60°C, a granular activated carbon column is first used for decolorization, and then the material temperature of the D-tagatose conversion liquid is adjusted to 55°C, and then filtered through an Ama filter with a working pressure of 0.3Mpa to obtain a decolorized material.

[0217] The ion exchange process is to pass the decolorized material through a cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin in sequence, with a material temperature of 40° C. and a flow rate of 3 BV / h to obtain an ion-exchanged material.

[0218] The primary vacuum concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, the concentration temperature is 70°C, the pressure is -0.1Mpa, and after the primary vacuum concentration, the solid content of the material is 58%, and the primary vacuum concentrated material is obtained.

[0219] Chromatographic separation: the obtained vacuum concentrated material is put into the simulated moving bed (SMB) chromatography according to the mass ratio of 1g:3.8mL to separate D-tagatose, and the processing capacity is 0.027T.DS / h / m 3 The resin had a D-tagatose yield of 81%, and the material after chromatographic separation was obtained.

[0220] The secondary vacuum concentration process is: use a six-effect evaporator to perform secondary vacuum concentration on the material after chromatographic separation, the concentration temperature is 70°C, the pressure is -0.1Mpa, and the secondary concentration is performed until the solid content of the material is increased to 72%, thereby obtaining a D-tagatose concentrate.

[0221] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0222] The crystallization is a combination of sugar boiling crystallization and cooling crystallization, where sugar boiling crystallization is performed first and then cooling crystallization. The specific section of sugar boiling crystallization is as follows: the solid content of D-tagatose concentrate is controlled at 70%, seed crystals are added, and the amount of seed crystals added is 3% of the dry basis weight of D-tagatose concentrate. The temperature of the crystallization process is controlled at 55°C, and the feed liquid is continuously replenished during sugar boiling crystallization. The feed liquid is D-tagatose solution, and the solid content of D-tagatose solution is 48%. The sugar boiling time is 15h. After the sugar boiling crystallization is completed, the sugar boiling liquid is obtained, and then it enters the cooling crystallization section. The cooling crystallization section is in the range of 40~50°C, and the temperature is reduced by 0.3°C per hour. In the range of 30~39°C, the temperature is reduced by 0.8°C per hour. When it is reduced to 30°C, the crystallization is completed and the crystallization liquid is obtained.

[0223] A vertical scraper centrifuge was used for centrifugation. The crystallization liquid was centrifuged at a speed of 700 rpm for 35 min. The crystallization liquid was washed during the centrifugation process. The amount of water used for washing was 3% of the volume of the liquid.

[0224] Fluidized bed drying was used for drying. The centrifuged product entered the fluidized bed. The air inlet temperature was set at 60° C., the cooling temperature was set at 27° C., and the drying time was set at 25 min. After drying, D-tagatose was obtained.

[0225] The conversion rate of D-tagatose and the purity data of D-tagatose in Examples 1-2 and Comparative Examples 1-6 are shown in Table 1.

[0226] Table 1 Conversion rate of D-tagatose and purity of D-tagatose in Examples 1 to 2 and Comparative Examples 1 to 6

[0227]

[0228] It can be seen that in Comparative Example 1, the glycerol concentration in the fermentation broth after feeding was adjusted, and the other variables were all within the protection range. The factor of glycerol concentration would affect the conversion rate of D-fructose to D-tagatose by using the immobilized recombinant Bacillus subtilis cells as carriers to a certain extent.

[0229] Comparative Example 2 reduced the IPTG concentration in the fermentation broth, and the other variables were all within the protection range. By comparing with Examples 1 and 2, it was proved that the factor of reduced IPTG concentration affected the conversion rate of D-fructose to D-tagatose using immobilized recombinant Bacillus subtilis cells as carriers.

[0230] Comparative Example 3 increased the IPTG induction content in the fermentation broth, and the other variables were all within the protection range. By comparing with Examples 1 and 2, it was proved that the increase in IPTG concentration affected the conversion rate of D-fructose to D-tagatose using immobilized recombinant Bacillus subtilis cells as carriers.

[0231] In Comparative Example 4, manganese sulfate was not used to soak the resin during the preparation of the immobilized cells, and the other variables were all within the protection range. By comparing with Examples 1 and 2, it was proved that the conversion amount of the immobilized recombinant Bacillus subtilis cells per unit mass was significantly reduced without soaking the resin in manganese sulfate.

[0232] In the preparation step of immobilized cells in Comparative Example 5, the mass concentration of the chitosan cross-linking agent was increased, and the other variables were all within the protection range. By comparing with Examples 1 to 2, it was proved that increasing the mass concentration of the chitosan cross-linking agent significantly reduced the conversion amount of immobilized recombinant Bacillus subtilis cells per unit mass, and reduced the conversion rate.

[0233] In the preparation step of immobilized cells in Comparative Example 6, the mass concentration of chitosan crosslinking agent was reduced, and the other variables were all within the protection range. By comparing with Examples 1 to 2, it was proved that increasing the mass concentration of chitosan crosslinking agent significantly reduced the transformation amount of immobilized recombinant Bacillus subtilis cells per unit mass.

[0234] In summary, the present invention uses immobilized recombinant Bacillus subtilis cells as enzyme carriers to convert D-fructose into D-tagatose, and the conversion rate can reach 28% to 32%. The unit mass of immobilized recombinant Bacillus subtilis cells can convert 70 to 100 times the mass of D-fructose, and the purity of the finished D-tagatose reaches more than 99.5%. The efficient utilization of recombinant Bacillus subtilis cells is experimentally demonstrated, and the cost of enzyme preparations is greatly reduced.

[0235] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant Bacillus subtilis ( Bacillus subtilis )BLCY-012, the deposit number is CGMCC No.31950.

2. A method for preparing a fermentation broth, characterized in that: include: Inoculating the recombinant Bacillus subtilis BLCY-012 of claim 1 into a culture medium for culturing to obtain a fermentation broth; During the culturing process, when dissolved oxygen rebound occurs in the fermentation broth, a feed medium is added, wherein the components of the feed medium include glycerol; after the feed medium is added, the glycerol concentration in the fermentation broth is 4-7 g / L; When the fermentation liquid OD 600 When the OD value of the fermentation liquid is 45-55, isopropyl-β-D-thiogalactoside is added for the first time for induction. 600 When the pH value was 100-120, isopropyl-β-D-thiogalactoside was added for the second time for induction. The final concentrations of isopropyl-β-D-thiogalactoside in the fermentation broth after the first and second additions were 0.2-0.4 mM, respectively.

3. An immobilized cell of a recombinant Bacillus subtilis BLCY-012, characterized in that: The method comprises a manganese-containing immobilized carrier, recombinant Bacillus subtilis BLCY-012 cells, a cross-linking agent solution and water; The immobilized carrier is mixed with the manganese-containing solution and then treated to obtain the manganese-containing immobilized carrier; Centrifuging the fermentation broth prepared by the preparation method of claim 2 to obtain recombinant Bacillus subtilis BLCY-012 cells; The mass ratio of the manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and water is 1: (0.8-1.5): (2.0-3.0); The volume of the cross-linking agent solution is 0.2 to 0.35 times the volume of the manganese-containing immobilized carrier; the concentration of the cross-linking agent solution is 0.8% to 1.3%.

4. The immobilized cells according to claim 3, characterized in that The concentration of the cross-linking agent solution is 1%.

5. The immobilized cells according to claim 3, characterized in that The manganese-containing solution includes a manganese sulfate solution; the molar concentration of the manganese sulfate solution is 30-40 mM.

6. The method for preparing immobilized cells according to any one of claims 3 to 5, characterized in that: include: The manganese-containing immobilized carrier, the recombinant Bacillus subtilis BLCY-012 cells and the first part of water are mixed to obtain a first mixture; the first mixture is cross-linked with a cross-linking agent to obtain immobilized cells.

7. The recombinant Bacillus subtilis according to claim 1 ( Bacillus subtilis ) Use of BLCY-012 or the fermentation broth prepared by the preparation method of claim 2 or the immobilized cells according to any one of claims 3 to 5 or the immobilized cells prepared by the preparation method of claim 6 in the preparation of D-tagatose.

8. A method for synthesizing D-tagatose, characterized in that: include: The immobilized cells according to any one of claims 3 to 5 or the immobilized cells prepared by the preparation method according to claim 6 are mixed with a D-fructose solution, and then converted, purified and crystallized to obtain D-tagatose; the crystallization method includes a method of combining sugar boiling crystallization with cooling crystallization; the solid content of the solution to be crystallized in the sugar boiling crystallization is controlled to be 70% to 72%, the amount of seed crystals added is 1% to 5% of the dry basis weight of the solution to be crystallized, the sugar boiling temperature is 50 to 57°C, and the sugar boiling time is 12 to 16 hours; during the cooling crystallization, when the temperature of the sugar boiling solution is 40 to 50°C, the temperature is reduced by 0.2 to 0.5°C per hour; when the temperature of the sugar boiling solution is 30 to 39°C, the temperature is reduced by 0.5 to 1.0°C per hour.

9. The method according to claim 8, characterized in that The conversion is completed by an immobilized column, the concentration of the D-fructose solution is 500-600 g / L, the flow rate of the D-fructose solution is 1-2 times the immobilized column / h, and the conversion temperature is 55-60°C.

10. The method according to claim 8, characterized in that The purification includes decolorization, ion exchange, vacuum concentration and chromatographic separation in sequence; the mass ratio of the chromatographic separation is 1g: (3.5-4.0)mL, and the processing capacity of the chromatographic separation is 0.025-0.028T.DS / h / m 3 Resin.

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