Recombinant bacillus subtilis strain, method for preparing fermentation broth, immobilized cell and preparation method therefor, and method for synthesizing d-tagatose and use thereof

Abstract

Disclosed in the present invention are a recombinant Bacillus subtilis strain, a method for preparing a fermentation broth, an immobilized cell and a preparation method therefor, and a method for synthesizing D-tagatose and the use thereof. Provided in the present invention is recombinant Bacillus subtilis BLCY-012 with the deposit number CGMCC No. 31950. The recombinant Bacillus subtilis BLCY-012 of the present invention can produce D-tagatose 4-epimerase by means of fermentation. The D-tagatose-4-epimerase is an intracellular enzyme. The D-tagatose-4-epimerase enables the preparation of D-tagatose using D-fructose as a raw material. An immobilized cell is prepared by using the recombinant Bacillus subtilis BLCY-012. Using the immobilized cell as an enzyme carrier, D-fructose is converted into D-tagatose, with a conversion rate of 28%-32%.

Description

A recombinant Bacillus subtilis strain, preparation method of fermentation broth, immobilized cells and preparation method, and method and application of D-tagatose synthesis.

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411783519.1, filed on December 6, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention belongs to the field of biotechnology, specifically relating to a recombinant Bacillus subtilis strain, a method for preparing fermentation broth, immobilized cells and their preparation method, and a method and application for synthesizing D-tagatose. Background Technology

[0004] D-Tag sugar is an epimer of D-fructose, a naturally occurring hexose ketose. Industrial production primarily uses galactose as a raw material, involving isomerization, decolorization, desalting, concentration, and crystallization. D-Tag sugar has 92% the sweetness of an equal amount of sucrose, but only 38% of its calories. It also has low hygroscopicity, making it an excellent low-energy food sweetener and filler. Furthermore, it possesses various physiological benefits, including inhibiting hyperglycemia, improving gut microbiota, and preventing tooth decay. It is widely used in the food, pharmaceutical, and cosmetic industries.

[0005] Since D-tagatose is extremely rare in nature and cannot be obtained in large quantities directly from nature, and chemical preparation methods produce pollution and numerous byproducts, bioconversion is the most likely method for industrialization. Existing technologies (see “Wang Lifei, Tan Zijian, Xie Xixian, Zhu Leilei. Discovery of new enzymes and study of enzymatic properties of tagatose-4-isomerase [J]. Acta Microbiologica Sinica, 2023, 63(11):4197-4207.”) report a conversion rate of 14% for catalytic fructose to tagatose, while the conversion rate of D-tagatose is low.

[0006] Currently, there is a lack of Bacillus subtilis strains with high D-tagatose conversion rates. Summary of the Invention

[0007] This invention provides a recombinant Bacillus subtilis strain, a method for preparing fermentation broth, an immobilized cell preparation method, and a method and application for synthesizing D-tagatose. The recombinant Bacillus subtilis strain provided has a high conversion rate of D-tagatose.

[0008] To address the aforementioned technical problems, the following technical solutions are proposed:

[0009] This invention provides a recombinant Bacillus subtilis strain BLCY-012, with accession number CGMCC No.31950.

[0010] The present invention provides a method for preparing fermentation broth, comprising: inoculating the recombinant Bacillus subtilis BLCY-012 of the above-mentioned technical solution into a culture medium for cultivation to obtain fermentation broth;

[0011] During the culture process, when dissolved oxygen rebound occurs in the fermentation broth, fed culture medium is added, the components of which include glycerol; after the addition of the fed culture medium, the glycerol concentration in the fermentation broth is 4-7 g / L.

[0012] When fermentation broth OD 600 When the concentration of β-D-thiogalactoside was 45–55, isopropyl-β-D-thiogalactoside was added for the first time to induce fermentation. When the OD of the fermentation broth reached 45–55, the fermentation broth was... 600 When the concentration was 100-120, isopropyl-β-D-thiogalactoside was added a 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.

[0013] This invention provides an immobilized cell of recombinant Bacillus subtilis BLCY-012, comprising a manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, a cross-linking agent solution, and water;

[0014] The immobilized carrier was mixed with a manganese-containing solution and then processed to obtain a manganese-containing immobilized carrier;

[0015] The fermentation broth prepared by the preparation method described above was centrifuged to obtain recombinant Bacillus subtilis BLCY-012 cells.

[0016] The mass ratio of the manganese-containing immobilized carrier, recombinant Bacillus subtilis BLCY-012 cells, and water is 1:(0.8–1.5):(2.0–3.0).

[0017] The volume of the crosslinking agent solution is 0.2 to 0.35 times the volume of the manganese-containing immobilized carrier; the concentration of the crosslinking agent solution is 0.8% to 1.3%.

[0018] Preferably, the manganese-containing solution includes a manganese sulfate solution; the molar concentration of the manganese sulfate solution is 30-40 mM.

[0019] This invention provides a method for preparing the immobilized cells described in the above technical solution, comprising:

[0020] The manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, and a first portion of water are mixed to obtain a first mixture; the first mixture is then crosslinked with a crosslinking agent to obtain immobilized cells.

[0021] This invention provides the application of the recombinant Bacillus subtilis BLCY-012 described in the above technical solution, 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.

[0022] This invention provides a method for synthesizing D-tagatose, comprising: mixing recombinant Bacillus subtilis BLCY-012 as described in the above technical solution, or fermentation broth prepared by the preparation method described in the above technical solution, or immobilized cells as described in the above technical solution, or immobilized cells prepared by the preparation method described in the above technical solution, with a D-fructose solution, followed by transformation, purification, and crystallization to obtain D-tagatose; wherein the crystallization method includes a combination of boiling crystallization and cooling crystallization.

[0023] Preferably, the solid content of the solution to be crystallized during sugar boiling is controlled at 70% to 72%, the amount of seed crystals added is 1% to 5% of the dry basis mass 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.

[0024] Preferably, during the cooling crystallization, when the temperature of the sugar syrup is 40-50°C, the temperature is reduced by 0.2-0.5°C per hour; when the temperature of the sugar syrup is 30-39°C, the temperature is reduced by 0.5-1.0°C per hour.

[0025] Preferably, the conversion is carried out by an immobilization 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 immobilization column / h, and the conversion temperature is 55-60℃.

[0026] Preferably, the purification includes sequential decolorization, ion exchange, vacuum concentration, and chromatographic separation; the mass ratio of the chromatographic separation is 1 g: (3.5–4.0) mL; and the throughput of the chromatographic separation is 0.025–0.028 T·DS / h / m³. 3 Resin.

[0027] Technical advantages of this invention: This invention provides a recombinant Bacillus subtilis strain BLCY-012, with accession number CGMCC No. 31950. During fermentation, the recombinant Bacillus subtilis strain BLCY-012 of this invention can produce D-tagatose-4-epimerase. This D-tagatose-4-epimerase is an intracellular enzyme that can prepare D-tagatose from D-fructose. Immobilized cells prepared using the recombinant Bacillus subtilis strain BLCY-012 are then used as enzyme carriers to convert D-fructose to D-tagatose, achieving a conversion rate of 28%–32%. This results in a high conversion rate and a high conversion yield per unit mass of immobilized recombinant Bacillus subtilis cells.

[0028] Biological Preservation Instructions

[0029] Bacillus subtilis BLCY-012 was deposited on September 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31950. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0030] This invention provides a recombinant Bacillus subtilis strain BLCY-012, with accession number CGMCC No.31950.

[0031] The current mainstream synthesis method for D-tagatose involves using D-galactose as a substrate and adding L-arabinose isomerase to convert D-galactose into D-tagatose. However, D-galactose is derived from lactose hydrolysis, and only 0.5g of D-galactose can be hydrolyzed from 1g of lactose. Lactose itself is expensive, and the resulting D-galactose is even more so. Furthermore, the conversion rate of D-galactose by L-arabinose is only about 50%, resulting in a low yield per unit of raw material. This leads to high production costs for D-tagatose and a market price of 150-180 yuan / kg, severely limiting the large-scale production and widespread application of this product. Existing technologies using D-fructose as a raw material to prepare D-tagatose also suffer from low conversion rates. The recombinant Bacillus subtilis BLCY-012 of this invention can ferment to produce D-tagatose-4-epimerase, which can prepare D-tagatose using D-fructose as a raw material. Compared with galactose, D-fructose is easier to obtain as a production raw material, has lower cost, and reduces production difficulty.

[0032] The present invention provides a method for preparing fermentation broth, comprising inoculating the recombinant Bacillus subtilis BLCY-012 of the above-mentioned technical solution into a culture medium for cultivation to obtain fermentation broth.

[0033] As an optional implementation method, the present invention does not have any special limitations on the inoculation method, and conventional inoculation methods can be used.

[0034] As an optional embodiment, the present invention inoculates the recombinant Bacillus subtilis BLCY-012 seed culture into a culture medium for cultivation. As an optional embodiment, the preparation method of the recombinant Bacillus subtilis BLCY-012 seed culture of the present invention includes: streaking the recombinant Bacillus subtilis BLCY-012 onto a solid culture medium plate to activate and obtain single colonies; inoculating the single colonies into a liquid culture medium for seed culture to obtain a seed culture. As an optional embodiment, the activation culture temperature is 33–37°C, and can also be 35–36°C; in specific embodiments of the present invention, the activation culture temperature is 33, 34, 35, 36, or 37°C. As an optional embodiment, the activation culture time is 20–26 h, and can also be 22–24 h; in specific embodiments of the present invention, the activation culture time is 20, 21, 22, 23, 24, 25, or 26 h. 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 implementation, to obtain a single colony, the present invention inoculates the single colony into LB liquid medium for seed culture to obtain a seed solution. The seed culture temperature of the present invention is 33–37°C, and can also be 35–36°C; in specific embodiments of the present invention, the seed culture temperature is 33, 34, 35, 36, or 37°C; the seed culture time is 8–12 hours, and can also be 9–11 hours; in specific embodiments of the present invention, the seed culture time is 8, 9, 10, 11, or 12 hours. As an optional implementation, the liquid medium includes LB liquid medium. As an optional implementation, the liquid volume of the present invention is 1 / 10 to 1 / 5 of the culture container volume; in specific embodiments of the present invention, the liquid volume of the liquid medium is 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, or 1 / 5 of the culture container volume. As an optional implementation, the seed culture rotation speed is 170-220 rpm. In specific embodiments of the present invention, the seed culture rotation speed is 170, 180, 190, 200, 210 or 220 rpm.

[0035] As an optional implementation method, a recombinant Bacillus subtilis BLCY-012 seed culture is obtained, and the seed culture is inoculated into a culture medium for cultivation.

[0036] As an optional implementation, the culture medium of the present invention comprises: 2.5-4 g / L corn steep liquor powder, 6-10 g / L glycerol, 10.5-13.5 g / L potassium dihydrogen phosphate, 1.0-1.5 g / L magnesium sulfate heptahydrate, 3.0-5.0 g / L diammonium hydrogen phosphate and 1.2-1.8 g / L citric acid monohydrate.

[0037] In another embodiment, the corn steep liquor powder in the culture medium is 2.5–3 g / L. The corn steep liquor powder serves to provide a nitrogen source for the synthesis of various enzyme systems and proteins essential for life. In another embodiment, the glycerol in the culture medium is 6–8 g / L. The glycerol serves to provide a carbon source. In another embodiment, the potassium dihydrogen phosphate in the culture medium is 10.5–12 g / L. The potassium dihydrogen phosphate serves to provide trace elements and energy for microorganisms. In another embodiment, the magnesium sulfate heptahydrate in the culture medium is 1.0–1.2 g / L. The magnesium sulfate heptahydrate serves to provide trace elements. In another embodiment, the diammonium hydrogen phosphate in the culture medium is 3.0–4.0 g / L. The diammonium hydrogen phosphate serves to provide trace elements. In another embodiment, the citric acid monohydrate in the culture medium is 1.2–1.5 g / L. The citric acid monohydrate serves to stabilize the pH.

[0038] As an optional implementation, the inoculation amount of the seed solution in this invention is 5-15% of the culture medium volume. In specific embodiments of this invention, the inoculation amount of the seed solution is 5%, 7%, 9%, 11%, 13%, or 15% of the culture medium volume.

[0039] As an optional implementation, the culture temperature is 35-37°C. In specific embodiments of the present invention, the culture temperature is 35, 36, or 37°C. As an optional implementation, the pH value is controlled at 6.8-7.2 during the culture process, and the dissolved oxygen rate is controlled at 25%-35% during the culture process.

[0040] During the cultivation process described in this invention, when dissolved oxygen rebound occurs in the fermentation broth, fed culture medium is added. The components of the fed culture medium include glycerol; after adding the fed culture medium, the glycerol concentration in the fermentation broth is 4–7 g / L. This invention controls the dissolved oxygen rate of the fermentation broth to a target dissolved oxygen rate during cultivation. 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 has occurred. The target dissolved oxygen rate described in this invention is 25%–35%. In specific embodiments of this 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, producing more D-tagatose-4-epimerase. When dissolved oxygen rebound occurs, it indicates that the nutrients in the culture medium are exhausted. In this invention, when dissolved oxygen rebound occurs, fed culture medium is added to the fermentation broth; the components of the fed culture medium described in this invention include glycerol. In one optional embodiment, the fed-batch culture medium comprises: 400-500 g / L glycerol, 8-10 g / L corn syrup powder, and 16-20 g / L magnesium sulfate heptahydrate. In another embodiment, the fed-batch culture medium uses water as a solvent and comprises: 400-450 g / L glycerol, 8-9 g / L corn syrup powder, and 17-19 g / L magnesium sulfate heptahydrate. In yet another embodiment, the fed-batch culture medium uses water as a solvent and comprises: 500 g / L glycerol, 10 g / L corn syrup powder, and 20 g / L magnesium sulfate heptahydrate. After the fed-batch culture medium is added, the glycerol concentration in the fermentation broth is 4-7 g / L. The fermentation culture is then terminated upon the addition of the fed-batch culture medium.

[0041] During the cultivation process described in this invention, as the cultivation proceeds, the OD of the fermentation broth... 600 It continues to increase, when the OD of the fermentation broth... 600 When the temperature reaches 45–55°C, isopropyl-β-D-thiogalactoside (IPTG) is added for the first time to induce fermentation. The fermentation broth OD is then adjusted accordingly. 600 When the concentration was 100-120, IPTG was added a 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.

[0042] As an optional implementation method, when the fermentation broth OD 600When the temperature reaches 45-55°C, the fermentation broth temperature is first lowered to 25-28°C before IPTG is added. The inducer is not heat-resistant; it must be added after cooling to promote the synthesis of D-tagatose-4-epimerase. After lowering to 25-28°C, this temperature is maintained until IPTG induction is complete. In this invention, the final IPTG concentration in the fermentation broth after the first addition is 0.2-0.4 mM. As an optional embodiment, the final IPTG concentration in the fermentation broth after the first addition is 0.25-0.35 mM. In this invention, when the fermentation broth OD... 600 When the concentration reaches 100-120, IPTG is added a second time for induction, and the final IPTG concentration in the fermentation broth after the second addition is 0.2-0.4 mM. As an optional embodiment, the final IPTG concentration in the fermentation broth after the second addition is 0.25-0.35 mM. As fermentation progresses, the number of microorganisms increases, thus requiring the addition of the inducer IPTG. Adding IPTG can promote the production of D-tagatose-4-epimerase.

[0043] As an optional implementation, after the second addition of IPTG, the OD of the fermentation broth of this invention... 600 When the highest point is reached, the fermentation process ends, and the fermentation broth is obtained.

[0044] The fermentation broth prepared by this invention has a high concentration of D-tagatose-4-epimerase.

[0045] This invention provides an immobilized cell of recombinant Bacillus subtilis BLCY-012, comprising a manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, a cross-linking agent solution, and water. The mass ratio of the manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, and water in this invention is 1:(0.8–1.5):(2.0–3.0).

[0046] This invention involves mixing an immobilized carrier with a manganese-containing solution and then processing the mixture to obtain a manganese-containing immobilized carrier. As an optional embodiment, the immobilized carrier includes a resin. In a specific embodiment of this invention, D101 resin is used. As an optional embodiment, the processing method includes soaking. As an optional embodiment, the soaking time is 10–15 hours, or 12–14 hours. As an optional embodiment, the manganese-containing solution includes a manganese sulfate solution with a molar concentration of 30–40 mM, or 30–35 mM. Manganese ions act as an activator of D-tagatose-4-epomerase; adding manganese sulfate to load manganese ions onto the immobilized carrier maximizes the activity of D-tagatose-4-epomerase. As an optional embodiment, before soaking in the manganese sulfate solution, the immobilized carrier is further subjected to sequential soaking in water, alkaline solution, and acid solution. This invention does not specifically limit the parameters for the water soaking, alkaline solution soaking, and acid solution soaking; conventional parameters are acceptable.

[0047] The method for preparing recombinant Bacillus subtilis BLCY-012 cells according to the present invention includes: centrifuging the fermentation broth to obtain BLCY-012 cells, i.e., recombinant Bacillus subtilis BLCY-012 cells. The preparation method of the fermentation broth of the present invention has been described above and will not be repeated here.

[0048] As an optional implementation, the concentration of the crosslinking agent solution of the present invention is 0.8% to 1.3%, or it can be 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 specific embodiments of the present invention, the volume of the crosslinking agent solution is 0.2, 0.3, or 0.35 times the volume of the immobilized carrier.

[0049] This invention provides a method for preparing the immobilized cells described in the above technical solution, comprising:

[0050] The manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, and a first portion of water are mixed to obtain a first mixture; the first mixture is then crosslinked with a crosslinking agent to obtain immobilized cells.

[0051] As an optional implementation, the mixing method includes adsorption, and the adsorption temperature can be 30-35°C. In specific embodiments of the present invention, the adsorption temperature can be 30, 31, 32, 33, 34, or 35°C; the adsorption time can be 5-8 hours. In specific embodiments of the present invention, the adsorption time can be 5, 6, 7, or 8 hours. During the adsorption process, the manganese-containing immobilized carrier adsorbs recombinant Bacillus subtilis BLCY-012 cells.

[0052] As an optional implementation, the present invention adds a crosslinking agent solution to the first mixture to perform crosslinking, thereby obtaining immobilized cells. In specific embodiments 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 specific embodiments of the present invention, the crosslinking time can be 1, 1.5, or 2 hours.

[0053] This invention provides the application of the recombinant Bacillus subtilis BLCY-012 described in the above technical solution, or the fermentation broth prepared by the preparation method 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.

[0054] This invention provides a method for synthesizing D-tagatose, comprising: mixing recombinant Bacillus subtilis BLCY-012 as described in the above technical solution, or fermentation broth prepared by the preparation method described in the above technical solution, or immobilized cells as described in the above technical solution, or immobilized cells prepared by the preparation method described in the above technical solution, with a D-fructose solution, followed by transformation, purification, and crystallization to obtain D-tagatose; wherein the crystallization method includes a combination of boiling crystallization and cooling crystallization.

[0055] As an optional implementation, this invention involves filling the immobilized cells into an immobilization column, through which a D-fructose solution flows in for conversion, and the solution flowing out from the bottom of the immobilization column is the D-tagatose conversion solution. This invention does not have any particular limitations on the immobilization column; conventional products can be used.

[0056] As an optional implementation, the concentration of the D-fructose solution in this invention is 500–600 g / L; in specific embodiments of this 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 specific embodiments of this invention, the temperature of the D-fructose solution is 55, 56, 58, or 60°C. The volume of the immobilized column in this invention is simply referred to as the column volume, and the flow rate of the D-fructose solution in this invention is 1–2 times the column volume / h, or 1.2–1.8 times the column volume / h. This invention obtains a D-tagatose conversion solution with a D-tagatose concentration of 150–195 g / L by passing the D-fructose solution through the immobilized column. The immobilized column contains D-tagatose-4-epimerase, which uses fructose as a raw material to convert fructose into D-tagatose.

[0057] As an optional implementation, before crystallization, the D-tagatose conversion solution is sequentially purified. This purification process is a fine purification, which includes sequential decolorization, ion exchange, vacuum concentration, and chromatographic separation to obtain a concentrated D-tagatose solution. As an optional implementation, the decolorization and ion exchange methods of the present invention include: first decolorizing the D-tagatose conversion solution to obtain a decolorized material; then subjecting the decolorized material to ion exchange to obtain an ion-exchanged material. As an optional implementation, the decolorization includes activated carbon decolorization and filter decolorization. The activated carbon decolorization can be performed using a granular activated carbon column, with a material temperature of 55–65°C. The filter decolorization can be performed using an Ama filter, with a material temperature of 50–60°C and a working pressure of 0.2–0.4 MPa. The purpose of decolorization is to remove pigments from the conversion solution. The ion exchange described in this invention includes sequential cation exchange, anion exchange, cation exchange, anion exchange, and cation exchange. This invention does not have specific limitations on the resins used for cation exchange and anion exchange; any conventional commercially available product can be used. The material temperature for ion exchange can be 35–45°C or 38–43°C; the material flow rate for ion exchange can be 2–4 BV / h or 2.5–3.5 BV / h. The purpose of the ion exchange is for the purification of D-tagatose conversion solution.

[0058] As an optional implementation, the present invention performs vacuum concentration and chromatographic separation on the ion-exchange material. As another implementation, the present invention performs initial vacuum concentration, chromatographic separation, and secondary vacuum concentration on the ion-exchange material. The material obtained after the initial vacuum concentration is subjected to chromatographic separation to obtain a chromatographically separated material, which is then subjected to secondary vacuum concentration to obtain a D-tagatose concentrate. The equipment used for the initial and secondary vacuum concentrations in this invention is a six-effect evaporator. The pressures for the initial and secondary vacuum concentrations are -0.06 to -0.1 MPa, and the temperatures are 65 to 75°C, respectively. The solid content of the concentrated material obtained after the initial vacuum concentration is 55% to 60%. The solid content of the concentrated material obtained after the secondary vacuum concentration is 71% to 73%, i.e., the solid content of the D-tagatose concentrate is 71% to 73%.

[0059] As an optional implementation, the chromatographic separation equipment is a simulated moving bed (SMB) chromatograph. The mass ratio of the initial vacuum-concentrated material for chromatographic separation in this invention is 1 g:(3.5–4.0) mL, or alternatively 1 g:(3.5–3.8) mL; the resin used in the chromatographic separation is a gel-type polystyrene-calcium chromatographic resin, and the chromatographic separation throughput is 0.025–0.028 T·DS / h / m³. 3 The resin can also be 0.026~0.027T.DS / h / m 3 Resin. The T.DS / h / m 3 The resin refers to the dry basis mass of the material passing through per cubic meter of resin per hour. The yield of D-tagagose after chromatographic separation in this invention is 80% to 83%.

[0060] As an optional implementation, the present invention sequentially crystallizes, centrifuges and dries the D-tagatose concentrate to obtain crystalline D-tagatose; the crystalline D-tagatose has high purity, with a purity of over 99.5%.

[0061] As an optional implementation, the solid content of the D-tagatose concentrate obtained by sugar boiling and crystallization in this invention is controlled to be 70%–72%. In specific embodiments of this invention, the solid content of the D-tagatose concentrate is controlled to be 70%, 71%, or 72%. As an optional implementation, seed crystals are added during the sugar boiling and crystallization process. The seed crystals are D-tagatose fine powder, with a mesh size of 180–200 mesh. The amount of seed crystals added is 1%–5% of the dry basis mass of the D-tagatose concentrate, or it can be 2%–3%. In specific embodiments of this invention, the amount of seed crystals added is 1%, 2%, 3%, or 5% of the dry basis mass of the solution to be crystallized.

[0062] During the sugar boiling and crystallization process described in this invention, the solid content of the sugar boiling solution continuously increases due to the continuous evaporation of water. To control the solid content of the sugar boiling solution to 70%–72%, a replenishing solution needs to be added. As one feasible method, the replenishing solution described in this invention is a D-tagatose solution with a solid content of 45%–50%. The sugar boiling and crystallization temperature described in this invention is 50–57°C, or 52–55°C; the sugar boiling time is 12–16 hours, or 14–15 hours. The equipment used for sugar boiling and crystallization described in this invention is a sugar boiling tank; the sugar boiling time is counted from the time the D-tagatose concentrate enters the sugar boiling tank. After the sugar boiling and crystallization is completed, a sugar boiling solution is obtained, which is then cooled and crystallized. When the temperature of the sugar syrup is 40-50°C, the sugar syrup of the present invention cools down by 0.2-0.5°C per hour, or by 0.3-0.4°C per hour; when the temperature of the sugar syrup is reduced to 30-39°C, the sugar syrup of the present invention cools down by 0.5-1.0°C per hour, or by 0.6-0.8°C per hour.

[0063] The present invention does not specifically limit the centrifugation and drying methods; conventional methods can be used. In a specific embodiment of the present invention, the centrifugation is performed using a vertical scraper centrifuge at a speed of 600–900 rpm for 30–40 minutes, and the washing water volume is 1%–5% of the liquid volume. The drying is performed using a fluidized bed at an inlet air temperature of 58–65°C, a cooling temperature of 25–30°C, and a drying time of 20–30 minutes.

[0064] The method for synthesizing D-tagatose provided by this invention uses immobilized recombinant Bacillus subtilis cells as enzyme carriers to convert D-fructose into D-tagatose, with a conversion rate of 28% to 32%. Each unit mass of immobilized recombinant Bacillus subtilis cells can convert 70 to 100 times the mass of D-fructose. This experiment demonstrates the efficient utilization of recombinant Bacillus subtilis cells and significantly reduces the cost of enzyme preparations.

[0065] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] The LB solid medium consists of: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar powder, and 1L distilled water.

[0067] The LB liquid culture medium consists of: 10g tryptone, 5g yeast extract, 10g NaCl, and 1L distilled water.

[0068] In the following examples and comparative examples, the seed crystals are D-tagatose fine powder, which is 180-200 mesh.

[0069] Example 1

[0070] (1) Recombinant Bacillus subtilis BLCY-012 was streaked on LB agar plates and incubated upside down at 33°C for 20 h to obtain single colonies. Recombinant Bacillus subtilis BLCY-012 was purchased from the National Key Laboratory of Food Science and Resource Exploration, Jiangnan University.

[0071] (2) Pick the single colony obtained in step (1) and inoculate it into an LB Erlenmeyer flask, wherein the liquid culture medium is 1 / 10 of the volume of the Erlenmeyer flask, and incubate it with shaking at 170 rpm for 8 hours to obtain the seed culture.

[0072] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at a volume of 5% of the fermentation medium. Fermentation was carried out at 35°C with a pH of 7.0 and a dissolved oxygen rate of 26%. When the dissolved oxygen rate reached 31%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 5 g / L. When the OD of the fermentation broth reached 5 g / L, the glycerol concentration in the fermentation broth was controlled at 5 g / L. 600 When the temperature reached 45°C, the fermentation broth temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.2 mM for induction. When the fermentation broth OD... 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 0.2 mM for induction. When the OD of the fermentation broth reaches 100... 600 Fermentation is stopped when the peak is reached, and the fermentation broth is obtained.

[0073] Each 1L of fermentation medium consists of: 2.5g corn steep liquor powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate, and the remainder water, with a pH of 7.0.

[0074] The feed culture medium consisted of water as the solvent, 400 g / L glycerol, 8 g / L corn syrup powder, and 17 g / L magnesium sulfate heptahydrate.

[0075] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 10°C and the centrifugation speed at 8000 rpm.

[0076] (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 with a volume of 4 times the resin for 4 hours. After filtering out the distilled water, the resin was first soaked in a 5% NaOH solution for 5 hours. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution was 7.0. Then, the resin was soaked in a 5% HCl solution for 5 hours. 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 30mM manganese sulfate solution for 10 hours. The excess liquid was filtered out to complete the pretreatment of the resin.

[0077] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0 and adsorbed at 30°C for 5 hours. Then, chitosan solution was added for cross-linking. The volume of chitosan solution was 0.2 times that of the pretreated resin, the mass concentration of chitosan solution was 1%, the cross-linking temperature was 30°C, and the cross-linking time was 1 hour. The preparation of immobilized cells was completed.

[0078] (6) Fill the immobilized cells obtained in step (5) into the immobilization column. Enter the immobilization column through the top of the column with D-fructose solution. The liquid flowing out from the bottom of the column is the D-tagatose conversion solution. The concentration of D-tagatose solution in the D-tagatose conversion solution is 154.65 g / L. The concentration of D-fructose solution is 500 g / L, the temperature is 55℃, and the flow rate of D-fructose solution is 1 column volume / h. The column volume refers to the immobilization column volume, and the same applies below.

[0079] (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, first vacuum concentration, chromatographic separation and second vacuum concentration in sequence to obtain D-tagatose concentrate.

[0080] The decolorization process involved using a granular activated carbon column to decolorize the D-tagatose conversion solution at a temperature of 55°C, followed by filtration through an Ama filter at a working pressure of 0.2 MPa to obtain the decolorized material.

[0081] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, the material temperature is 35℃ and the flow rate is 2 BV / h, to obtain the ion-exchanged material.

[0082] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 65℃ and a pressure of -0.06 MPa. After one vacuum concentration, the solid content of the material is 55%, and the material is obtained after one vacuum concentration.

[0083] Chromatographic separation was performed as follows: the obtained vacuum concentrated material was introduced into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 3.5 mL, with a throughput of 0.025 T.DS / h / m. 3 The resin, with a D-tagagose yield of 80%, yielded the chromatographically separated material.

[0084] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 65℃ and a pressure of -0.06 MPa. After the secondary vacuum concentration, the solid content of the material is 71%, and D-tagatose concentrate is obtained.

[0085] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0086] The crystallization process employs a combination of boiling crystallization and cooling crystallization, first involving boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 70%, adding seed crystals at 1% of the dry weight of the D-tagatose concentrate, maintaining a crystallization temperature of 50℃, and continuously replenishing the solution with D-tagatose solution containing 45% solids. The boiling process lasts for 12 hours. After boiling crystallization, a boiling solution is obtained, which then proceeds to the cooling crystallization stage. During the cooling crystallization stage, the boiling solution is cooled by 0.2℃ per hour within the 40–50℃ range, and by 0.5℃ per hour within the 30–39℃ range, until it reaches 30℃, at which point crystallization is complete, yielding the crystallized solution.

[0087] The centrifugation was performed using a vertical scraper centrifuge. The crystallized liquid was fed into the centrifuge at a speed of 600 rpm for 30 minutes. The crystallized liquid was rinsed during the centrifugation process, and the amount of water used for rinsing was 1% of the volume of the liquid.

[0088] Drying: A fluidized bed drying process was used, with an inlet air temperature of 58℃, a cooling temperature of 30℃, and a drying time of 20 min. D-tagatose was obtained upon completion of drying.

[0089] Example 2

[0090] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 37°C for 25 h to obtain single colonies.

[0091] (2) Pick the single colonies obtained from step (1) and inoculate them into LB Erlenmeyer flasks, where the liquid culture medium is 1 / 5 of the volume of the Erlenmeyer flask. Shake and culture at 220 rpm for 12 h to obtain seed culture.

[0092] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 15% of the fermentation medium volume. Fermentation culture was carried out at 37°C with a pH of 7.2 and a dissolved oxygen rate of 35%. When the dissolved oxygen rate reached 40%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 7 g / L. When the OD of the fermentation broth reached 7 g / L, the glycerol concentration in the fermentation broth was controlled at 7 g / L. 600 When the concentration reached 55, the temperature of the fermentation broth was lowered to 28°C, and IPTG was added to a final concentration of 0.4 mM for induction. When the OD of the fermentation broth... 600 When the concentration reached 120, IPTG was added a second time to bring the final concentration to 0.4 mM for induction. The fermentation broth OD was then adjusted. 600 Fermentation should be stopped when the peak value is reached.

[0093] Each 1L of fermentation medium consists of: 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 the remainder water, with a pH of 7.2.

[0094] The feed culture medium consisted of water as the solvent, 500 g / L of glycerol, 10 g / L of corn syrup powder, and 20 g / L of magnesium sulfate heptahydrate.

[0095] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 4°C and the centrifugation speed at 8000 rpm.

[0096] (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 with a volume of 4 times the resin for 4 hours. After filtering out the distilled water, the resin was first soaked in a 5% NaOH solution for 5 hours. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution was 7.0. Then the resin was soaked in a 5% HCl solution for 5 hours. 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 40mM manganese sulfate solution for 15 hours. The excess liquid was filtered out to complete the pretreatment of the resin.

[0097] 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 chitosan solution was 0.35 times that of the pretreated resin, the mass concentration of chitosan solution was 1%, the cross-linking temperature was 35°C, and the cross-linking time was 2 hours to complete the preparation of immobilized cells.

[0098] (6) The immobilized cells obtained in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The concentration of the D-tagatose solution in the D-tagatose conversion solution is 191 g / L. The concentration of the D-fructose solution is 600 g / L, the temperature is 60℃, and the flow rate of the D-fructose solution is 2 column volumes / h.

[0099] (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, first vacuum concentration, chromatographic separation and second vacuum concentration in sequence to obtain D-tagatose concentrate.

[0100] The decolorization process is as follows: The material temperature of the D-tagatose conversion solution is 65℃. First, granular activated carbon column is used for decolorization. Then, the material temperature of the D-tagatose conversion solution is adjusted to 60℃, and then filtered through an Ama filter with an operating pressure of 0.4 MPa to obtain the decolorized material.

[0101] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin. The material temperature of the D-tagatose conversion solution is 45℃ and the flow rate is 4 BV / h to obtain the ion-exchanged material.

[0102] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 75℃ and a pressure of -0.1 MPa. After one vacuum concentration, the solid content of the material is 60%, and the material is obtained after one vacuum concentration.

[0103] Chromatographic separation was performed as follows: the obtained vacuum concentrated material was introduced into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 4.0 mL, with a throughput of 0.028 T.DS / h / m. 3 The resin yielded 83% D-tagagose, and the chromatographically separated material was obtained.

[0104] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 75℃ and a pressure of -0.1 MPa. After the secondary vacuum concentration, the solid content of the material is 73%, and D-tagatose concentrate is obtained.

[0105] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0106] The crystallization process employs a combination of boiling crystallization and cooling crystallization, with boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 72%, adding seed crystals at a rate of 5% of the dry weight of the D-tagatose concentrate, and maintaining a controlled crystallization temperature of 57°C. During boiling crystallization, a D-tagatose solution with a solid content of 50% is continuously replenished. The boiling time is 16 hours. After boiling crystallization, a boiling solution is obtained, which then enters the cooling crystallization stage. The cooling crystallization stage involves decreasing the temperature by 0.5°C per hour within the 40–50°C range, and by 1.0°C per hour within the 30–39°C range, until the temperature reaches 30°C, at which point crystallization ends.

[0107] The centrifugation was carried out using a vertical scraper centrifuge at a speed of 900 rpm for 40 minutes. The crystallized liquid was rinsed during the centrifugation process, and the amount of water used for rinsing was 5% of the liquid volume.

[0108] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 65°C, a cooling temperature of 25°C, and a drying time of 30 min. After drying, D-tagatose was obtained.

[0109] Comparative Example 1

[0110] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 35°C for 21 h to obtain single colonies.

[0111] (2) Pick the single colonies obtained in step (1) and inoculate them into LB Erlenmeyer flasks, where the liquid culture medium is 1 / 7 of the volume of the Erlenmeyer flask. Incubate at 33℃ and 200rpm for 9 hours to obtain seed culture.

[0112] (3) Fermentation culture: The seed culture described in step (2) is inoculated into the fermentation medium at a volume of 7% of the fermentation medium volume. Fermentation is carried out at 35°C with a pH of 7.0 and a dissolved oxygen rate of 25%. When the dissolved oxygen rate reaches 30%, it is considered that dissolved oxygen rebound has occurred. When dissolved oxygen rebound occurs, fed culture medium is started. After feeding culture medium is added, the glycerol concentration in the fermentation broth is controlled at 2 g / L. When the OD of the fermentation broth reaches 2 g / L... 600 When the temperature reached 45°C, the fermentation broth temperature was lowered to 26°C, and IPTG was added to a final concentration of 0.3 mM for induction. When the fermentation broth OD... 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 0.2 mM for induction. When the OD of the fermentation broth reaches 100... 600 Fermentation should be stopped when the peak value is reached.

[0113] Each 1L of fermentation medium consists of: 2.5g corn steep liquor powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate, and the remainder water, pH 7.0.

[0114] The feed culture medium consisted of water as the solvent, 400 g / L glycerol, 8 g / L corn syrup powder, and 18 g / L magnesium sulfate heptahydrate.

[0115] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 15°C and the centrifugation speed at 8000 rpm.

[0116] (5) Preparation of immobilized cells: D101 resin was used for immobilization. The pretreatment process of the resin was as follows: The resin was soaked in distilled water at a volume of 4 times that of D101 resin for 4 hours. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 hours. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the resulting washing solution was 7.0. The resin was then soaked in a 5% HCl solution for 5 hours. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the resulting washing solution was 7.0. Finally, the resin was soaked in a 30mM manganese sulfate solution for 10 hours. Excess liquid was filtered out, and the pretreatment of the resin was completed.

[0117] The pretreated resin, the cells described in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.5 and adsorbed at 30°C for 6 hours. Then, chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.3 times that 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 to complete the preparation of immobilized cells.

[0118] (6) The immobilized cells obtained in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The D-tagatose concentration of the D-tagatose conversion solution is 142.39 g / L. The D-fructose concentration is 550 g / L, the temperature is 57℃, and the D-fructose flow rate is 1.5 times the column volume / h.

[0119] (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, first vacuum concentration, chromatographic separation and second vacuum concentration in sequence to obtain D-tagatose concentrate.

[0120] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, a granular activated carbon column was used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 55℃, and then filtered through an Ama filter at a working pressure of 0.3 MPa to obtain the decolorized material.

[0121] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with the material temperature at 40℃ and the flow rate at 3 BV / h, to obtain the ion-exchanged material.

[0122] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After one vacuum concentration, the solid content of the material is 58%, and the material is obtained after one vacuum concentration.

[0123] Simulated moving bed (SMB) chromatography separation was performed as follows: the obtained primary vacuum concentrated material was introduced into the simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 3.5 mL, with a throughput of 0.026 T.DS / h / m 3 The resin yielded 82% D-tagagose, and the chromatographically separated material was obtained.

[0124] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrate is obtained.

[0125] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged and dried.

[0126] The crystallization process employs a combination of boiling crystallization and cooling crystallization, with boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 71%, adding seed crystals at 1% of the dry weight of the D-tagatose concentrate, and maintaining a crystallization temperature of 55°C. During boiling crystallization, a D-tagatose solution with a solid content of 45% is continuously replenished, and the boiling time is 15 hours. After boiling crystallization, a boiling solution is obtained, which then enters the cooling crystallization stage. The cooling crystallization stage involves decreasing the temperature by 0.2°C per hour within the 40–50°C range, and by 0.5°C per hour within the 30–39°C range, until the temperature reaches 30°C, at which point crystallization ends.

[0127] The centrifugation was performed using a vertical scraper centrifuge. The crystallized liquid was fed into the centrifuge at a speed of 600 rpm for 30 minutes. The crystallized liquid was rinsed during the centrifugation process, and the amount of water used for rinsing was 1% of the volume of the liquid.

[0128] Fluidized bed drying was used. The centrifuged product was fed into a fluidized bed with an inlet air temperature of 60°C, a cooling temperature of 30°C, and a drying time of 20 min. After drying, D-tagatose was obtained.

[0129] Comparative Example 2

[0130] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 35°C for 25 h to obtain single colonies.

[0131] (2) Pick the single colonies obtained in step (1) and inoculate them into LB Erlenmeyer flasks, where the liquid culture medium is 1 / 7 of the volume of the Erlenmeyer flask. Incubate at 33℃ and 200rpm for 10h to obtain seed culture.

[0132] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 10% of the fermentation medium volume. Fermentation culture was carried out at 36℃, with a pH of 7.0 and a dissolved oxygen rate of 30% during the culture process. When the dissolved oxygen rate reached 35%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 5 g / L. When the OD of the fermentation broth reached 5 g / L, the glycerol concentration in the fermentation broth was controlled at 5 g / L. 600 When the concentration reached 50, the temperature of the fermentation broth was lowered to 26°C, and IPTG was added to a final concentration of 0.1 mM for induction. When the OD of the fermentation broth reached 50, the temperature was lowered to 26°C. 600 When the concentration reached 150, a second addition of IPTG was made to bring the final concentration to 0.1 mM for induction. The fermentation broth OD was then adjusted. 600 Fermentation should be stopped when the peak value is reached.

[0133] Each 1L of fermentation medium consists of: 3.0g corn steep liquor powder, 8g glycerol, 12.0g potassium dihydrogen phosphate, 1.2g magnesium sulfate heptahydrate, 4.0g diammonium hydrogen phosphate, 1.5g citric acid monohydrate, and the remainder water, with a pH of 7.0.

[0134] The feed culture medium consisted of water as the solvent, 400 g / L glycerol, 10 g / L corn syrup powder, and 18 g / L magnesium sulfate heptahydrate.

[0135] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 10°C and the centrifugation speed at 8000 rpm.

[0136] (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 with a volume of 4 times the resin for 4 hours. After filtering out the distilled water, the resin was soaked in NaOH solution with a mass concentration of 5% for 5 hours. 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 HCl solution with a mass concentration of 5% for 5 hours. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution was 7.0. The resin was then soaked in 35mM manganese sulfate solution for 12 hours. Excess liquid was filtered out to complete the pretreatment of the resin.

[0137] The pretreated resin, the cells described in step (4), and purified water were mixed in a mass ratio of 1:1.0:2.0 and adsorbed at 32°C for 6 hours. Then, chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times that 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, thus completing the preparation of immobilized cells.

[0138] (6) The immobilized cells obtained in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The D-tagatose concentration of the D-tagatose conversion solution is 113.6 g / L. The concentration of the D-fructose solution is 550 g / L, the temperature is 58℃, and the flow rate of the D-fructose solution is 2 column volumes / h.

[0139] (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, first vacuum concentration, chromatographic separation and second vacuum concentration in sequence to obtain D-tagatose concentrate.

[0140] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, granular activated carbon columns were used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 55℃, and then filtered through an Ama filter with an operating pressure of 0.3 MPa to obtain the decolorized material.

[0141] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with the material temperature at 40℃ and the flow rate at 3 BV / h, to obtain the ion-exchanged material.

[0142] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After one vacuum concentration, the solid content of the material is 58%, and the material is obtained after one vacuum concentration.

[0143] Chromatographic separation was performed as follows: the obtained vacuum concentrated material was introduced into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 3.8 mL for separation, with a throughput of 0.026 T.DS / h / m 3 The resin yielded 82% D-tagagose, and the chromatographically separated material was obtained.

[0144] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrate is obtained.

[0145] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0146] The crystallization process employs a combination of boiling crystallization and cooling crystallization, with boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 72%, adding seed crystals at 3% of the dry weight of the D-tagatose concentrate, and maintaining a crystallization temperature of 52℃. During boiling crystallization, a D-tagatose solution with a solid content of 48% is continuously replenished. The boiling time is 14 hours. After boiling crystallization, a boiling solution is obtained, which then enters the cooling crystallization stage. The cooling crystallization stage involves a temperature reduction of 0.3℃ per hour within the 40–50℃ range, and a temperature reduction of 0.8℃ per hour within the 30–39℃ range, decreasing the temperature to 30℃ to complete the crystallization and obtain the crystallized solution.

[0147] Centrifugation was performed using a vertical scraper centrifuge at 800 rpm for 35 minutes. The crystallized liquid was rinsed during centrifugation, and the amount of water used for rinsing was 2% of the liquid volume.

[0148] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 60°C, a cooling temperature of 28°C, and a drying time of 25 min. After drying, D-tagatose was obtained.

[0149] Comparative Example 3

[0150] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 35°C for 24 hours to obtain single colonies.

[0151] (2) Pick the single colonies obtained in step (1) and inoculate them into an LB Erlenmeyer flask, wherein the liquid culture medium is 1 / 5 of the volume of the Erlenmeyer flask, and incubate at 200 rpm for 10 h to obtain the seed culture.

[0152] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 10% of the fermentation medium volume. Fermentation culture was carried out at 36℃, with a pH of 7.0 and a dissolved oxygen rate of 30% during the culture process. When the dissolved oxygen rate reached 35%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 5 g / L. When the OD of the fermentation broth reached 5 g / L, the glycerol concentration in the fermentation broth was controlled at 5 g / L. 600 When the concentration reached 50, the temperature of the fermentation broth was lowered to 25°C, and IPTG was added to a final concentration of 1.0 mM for induction. When the OD of the fermentation broth reached 50, the temperature was lowered to 25°C. 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 1.0 mM for induction. When the OD of the fermentation broth reaches 100... 600 Fermentation should be stopped when the peak value is reached.

[0153] Each 1L of fermentation medium consists of: 2.5g corn steep liquor powder, 6g glycerol, 10.5g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 3.0g diammonium hydrogen phosphate, 1.2g citric acid monohydrate, and the remainder water, pH 7.0.

[0154] The feed culture medium consists of water as the solvent, 400 g / L glycerol, 10 g / L corn syrup powder, and 20 g / L magnesium sulfate heptahydrate.

[0155] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 4°C and the centrifugation speed at 8000 rpm.

[0156] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in 4 times the volume of distilled water for 4 hours. After filtering out the distilled water, the resin was soaked in 5% NaOH solution for 5 hours. 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 5% HCl solution for 5 hours. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution was 7.0. The resin was then soaked in 35mM manganese sulfate solution for 15 hours. Excess liquid was filtered out to complete the pretreatment of the resin.

[0157] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0 and adsorbed at 30°C for 6 hours. Then, chitosan solution was added for cross-linking. The volume of the chitosan solution was 0.2 times that 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 to complete the preparation of immobilized cells.

[0158] (6) The immobilized cells obtained in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The D-tagatose concentration of the D-tagatose conversion solution is 130.25 g / L. The concentration of the D-fructose solution is 500 g / L, the temperature is 55℃, and the flow rate of the D-fructose solution is 1 column volume / h. (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, one vacuum concentration, chromatographic separation, and two vacuum concentrations in sequence to obtain the D-tagatose concentrate.

[0159] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, granular activated carbon columns were used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 55℃, and then filtered through an Ama filter with an operating pressure of 0.3 MPa to obtain the decolorized material.

[0160] The ion exchange process involves sequentially passing the decolorized material through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin, and cation exchange resin. The material temperature is 40℃, and the flow rate is 3 BV / h, resulting in the ion-exchanged material.

[0161] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After one vacuum concentration, the solid content of the material is 58%, and the material is obtained after one vacuum concentration.

[0162] The simulated moving bed (SMB) chromatography separation was performed as follows: the obtained primary vacuum concentrated material was introduced into the simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 3.5 mL, with a throughput of 0.025 T.DS / h / m. 3 The resin, with a D-tagagose yield of 80%, yielded the chromatographically separated material.

[0163] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrate is obtained.

[0164] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0165] The crystallization process employs a combination of boiling crystallization and cooling crystallization, first involving boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 70%, adding seed crystals at a rate of 1% of the dry weight of the D-tagatose concentrate, maintaining a crystallization temperature of 50°C, and continuously replenishing the solution with D-tagatose solution containing 45% solids. The boiling time is 12 hours. After boiling crystallization, the resulting sugar solution is cooled and then proceeds to the cooling crystallization stage. The cooling crystallization stage involves decreasing the temperature by 0.2°C per hour within the 40–50°C range, and by 0.5°C per hour within the 30–39°C range, until the temperature reaches 30°C, at which point crystallization ends, yielding the crystallized solution.

[0166] Centrifugation was performed using a vertical scraper centrifuge at 600 rpm for 30 minutes. The crystallized liquid was rinsed during centrifugation, and the amount of water used for rinsing was 1% of the liquid volume.

[0167] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 58°C, a cooling temperature of 25°C, and a drying time of 20 min. After drying, D-tagatose was obtained.

[0168] Comparative Example 4

[0169] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 35°C for 23 hours to obtain single colonies.

[0170] (2) Pick the single colonies obtained in step (1) and inoculate them into LB Erlenmeyer flasks, where the liquid culture medium is 1 / 8 of the volume of the Erlenmeyer flask. Shake and culture at 200 rpm for 10 h to obtain seed culture.

[0171] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 10% of the fermentation medium volume. Fermentation culture was carried out at 37°C with a pH of 7.0 and a dissolved oxygen rate of 30% during the culture process. When the dissolved oxygen rate reached 35%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 6 g / L. When the OD of the fermentation broth reached 6 g / L, the glycerol concentration in the fermentation broth was controlled at 6 g / L. 600 When the concentration reached 50, the temperature was lowered to 27°C, and IPTG was added to a final concentration of 0.3 mM for induction. When the OD of the fermentation broth reached 50, the temperature was lowered to 27°C. 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 0.3 mM for induction. When the OD of the fermentation broth reaches 100... 600 Fermentation should be stopped when the peak value is reached.

[0172] The composition of each 1L fermentation medium is as follows: 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 the balance water, pH 7.0.

[0173] The feed culture medium consisted of water as the solvent, 400 g / L glycerol, 8 g / L corn syrup powder, and 18 g / L magnesium sulfate heptahydrate.

[0174] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 4°C and the centrifugation speed at 8000 rpm.

[0175] (5) Preparation of immobilized cells: D101 resin was used. The pretreatment process of the resin was as follows: the resin was soaked in 4 times the volume of distilled water for 4 hours. After filtering out the distilled water, the resin was soaked in 5% NaOH solution for 5 hours. After filtering out the NaOH solution, the D101 resin was washed with distilled water until the pH of the washing solution was 7.0. Then the resin was soaked in 5% HCl solution for 5 hours. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution was 7.0.

[0176] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0 and adsorbed at 32°C for 6 hours. Then, chitosan solution was added for cross-linking. The volume of chitosan solution was 0.35 times that of the pretreated resin, the mass concentration of chitosan solution was 1%, the cross-linking temperature was 32°C, and the cross-linking time was 1.5 hours to complete the preparation of immobilized cells.

[0177] (6) The immobilized cells described in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The concentration of the D-tagatose solution in the D-tagatose conversion solution is 163.24 g / L. The concentration of the D-fructose solution is 550 g / L, the temperature is 60℃, and the flow rate of the D-fructose solution is 1 column volume / h.

[0178] (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, first vacuum concentration, chromatographic separation and second vacuum concentration in sequence to obtain D-tagatose concentrate.

[0179] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, granular activated carbon columns were used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 60℃, and then filtered through an Ama filter at a material temperature of 60℃ and a working pressure of 0.3 MPa to obtain the decolorized material.

[0180] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin and cation exchange resin, with the material temperature at 40℃ and the flow rate at 3 BV / h, to obtain the ion-exchanged material.

[0181] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After one vacuum concentration, the solid content of the material is 60%, and the material is obtained after one vacuum concentration.

[0182] Chromatographic separation was performed as follows: the obtained vacuum concentrated material was introduced into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 4.0 mL for separation, with a throughput of 0.025 T.DS / h / m. 3 The resin yielded 82% D-tagagose, and the chromatographically separated material was obtained.

[0183] The secondary vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After secondary vacuum concentration, the solid content of the material is 72%, resulting in D-tagatose concentrate.

[0184] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0185] The crystallization process employs a combination of boiling crystallization and cooling crystallization, with boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 70%, adding seed crystals at 3% of the dry weight of the D-tagatose concentrate, and maintaining the crystallization temperature at 55°C. During boiling crystallization, a D-tagatose solution with a solid content of 48% is continuously replenished, and the boiling time is 15 hours. After boiling crystallization, a boiling solution is obtained, which then enters the cooling crystallization stage. The cooling crystallization stage involves decreasing the temperature by 0.2°C per hour within the 40–50°C range, and by 0.5°C per hour within the 30–39°C range, until the temperature reaches 30°C, at which point crystallization ends, yielding the crystallized solution.

[0186] Centrifugation was performed using a vertical scraper centrifuge at 800 rpm for 35 minutes. The crystallized liquid was rinsed during centrifugation, and the amount of water used for rinsing was 2% of the liquid volume.

[0187] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 60°C, a cooling temperature of 26°C, and a drying time of 25 min. After drying, D-tagatose was obtained.

[0188] Comparative Example 5

[0189] (1) Recombinant Bacillus subtilis was streaked on LB plates and incubated upside down at 35°C for 23 hours to obtain single colonies.

[0190] (2) Pick the single colony obtained in step (1) and inoculate it into an LB Erlenmeyer flask, wherein the liquid culture medium is 1 / 7 of the volume of the Erlenmeyer flask, and shake it at 200 rpm for 10 h to obtain the seed culture.

[0191] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 10% of the fermentation medium volume. Fermentation culture was carried out at 36℃, with a pH of 7.0 and a dissolved oxygen rate of 30% during the culture process. When the dissolved oxygen rate reached 35%, it was considered that dissolved oxygen rebound had occurred. When dissolved oxygen rebound occurred, fed culture medium was started. After feeding culture medium was added, the glycerol concentration in the fermentation broth was controlled at 5 g / L. When the OD of the fermentation broth reached 5 g / L, the glycerol concentration in the fermentation broth was controlled at 5 g / L. 600 When the concentration reached 50, the temperature was lowered to 26°C, and IPTG was added to a final concentration of 0.3 mM for induction. When the OD of the fermentation broth reached 50, the temperature was lowered to 26°C. 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 0.3 mM for induction. When the OD of the fermentation broth reaches 100... 600 Fermentation should be stopped when the peak value is reached.

[0192] The fermentation medium consisted of: 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 the remainder water, with a pH of 7.0.

[0193] The feed culture medium consists of: water as solvent, 400 g / L glycerol, 8 g / L corn syrup powder, and 16 g / L magnesium sulfate heptahydrate.

[0194] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 4°C and the centrifugation speed at 8000 rpm.

[0195] (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 at a volume of 4 times the resin volume for 4 hours. After filtering out the distilled water, the resin was soaked in a 5% NaOH solution for 5 hours. 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 hours. 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 35mM manganese sulfate solution for 12 hours. Excess liquid was filtered out to complete the pretreatment of the resin.

[0196] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.5 and adsorbed at 32°C for 7 hours. Then, chitosan solution was added for cross-linking. The volume of chitosan solution was 0.3 times that of the pretreated resin, the mass concentration of chitosan solution was 8%, the cross-linking temperature was 32°C, and the cross-linking time was 1.5 hours to complete the preparation of immobilized cells.

[0197] (6) The immobilized cells obtained in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The D-tagatose concentration of the D-tagatose conversion solution is 124.13 g / L. The concentration of the D-fructose solution is 550 g / L, the temperature is 58℃, and the flow rate of the D-fructose solution is 1 column volume / h.

[0198] (7) The D-tagatose conversion solution described in step (6) is subjected to decolorization, ion exchange, primary vacuum concentration, chromatographic separation, and secondary vacuum concentration in sequence to obtain D-tagatose concentrate.

[0199] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, granular activated carbon columns were used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 55℃, and then filtered through an Ama filter with an operating pressure of 0.3 MPa to obtain the decolorized material.

[0200] The ion exchange process is as follows: the decolorized material is passed sequentially through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin, and cation exchange resin. The material temperature is 40℃ and the flow rate is 3 BV / h to obtain the ion-exchanged material.

[0201] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After one vacuum concentration, the solid content of the material is 58%, and the material is obtained after one vacuum concentration.

[0202] The simulated moving bed (SMB) chromatography separation was performed as follows: the obtained primary vacuum concentrated material was introduced into the simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 4.0 mL, with a throughput of 0.026 T.DS / h / m. 3 The resin yielded 81% D-tagagose, and the chromatographically separated material was obtained.

[0203] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.08 MPa. After the secondary vacuum concentration, the solid content of the material is 72%, and D-tagatose concentrate is obtained.

[0204] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0205] The crystallization process employs a combination of boiling crystallization and cooling crystallization, first involving boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 71%, adding seed crystals at 2% of the dry weight of the D-tagatose concentrate, maintaining a crystallization temperature of 55℃, and continuously replenishing the solution with D-tagatose solution containing 48% solids. The boiling time is 15 hours. After boiling crystallization, the resulting liquid is boiled sugar. This liquid then enters the cooling crystallization stage, where the temperature decreases by 0.2℃ per hour within the 40–50℃ range, and by 0.5℃ per hour within the 30–39℃ range, until reaching 30℃. Crystallization then ends at this temperature, yielding the crystallized liquid.

[0206] Centrifugation was performed using a vertical scraper centrifuge at 600 rpm for 30 minutes. The crystallized liquid was rinsed during centrifugation, and the amount of water used for rinsing was 2% of the liquid volume.

[0207] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 60°C, a cooling temperature of 28°C, and a drying time of 25 min. After drying, D-tagatose was obtained.

[0208] Comparative Example 6

[0209] (1) Streaking of recombinant Bacillus subtilis on LB plates and incubating at 35°C upside down for 24 h to obtain single colonies;

[0210] (2) Pick the single colony obtained in step (1) and inoculate it into an LB Erlenmeyer flask, wherein the liquid culture medium is 1 / 6 of the volume of the Erlenmeyer flask, and shake it at 180 rpm for 9 h to obtain the seed culture.

[0211] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation volume of 8% of the fermentation medium volume. The culture was carried out at 36°C with a pH of 7.0 and a dissolved oxygen rate of 30%. A dissolved oxygen rebound was considered to have occurred when the dissolved oxygen rate reached 35%. When a dissolved oxygen rebound occurred, fed culture medium was started. After feeding the culture medium, the glycerol concentration in the fermentation broth was controlled at 5 g / L. When the OD of the fermentation broth... 600 When the concentration reached 50, the temperature of the fermentation broth was lowered to 27°C, and IPTG was added to a final concentration of 0.3 mM for induction. When the OD of the fermentation broth reached 50, the temperature was lowered to 27°C. 600 When the concentration reaches 100, IPTG is added a second time to bring the final concentration to 0.3 mM for induction. The fermentation broth OD... 600 Fermentation should be stopped when the peak value is reached.

[0212] The fermentation medium consisted 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 balance being water, with a pH of 7.0.

[0213] The feed culture medium consists of: water as solvent, 450 g / L glycerol, 8 g / L corn syrup powder, and 16 g / L magnesium sulfate heptahydrate.

[0214] (4) Use a tubular centrifuge to separate the fermentation broth obtained in step (3) and collect the cells. During the centrifugation process, control the centrifugation temperature at 10°C and the centrifugation speed at 8000 rpm.

[0215] (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 with a volume of 4 times the resin for 4 hours. After filtering out the distilled water, the resin was soaked in NaOH solution with a mass concentration of 5% for 5 hours. 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 HCl solution with a mass concentration of 5% for 5 hours. After filtering out the HCl solution, the resin was washed with distilled water until the pH of the washing solution was 7.0. The resin was then soaked in 40mM manganese sulfate solution for 15 hours. Excess liquid was filtered out to complete the pretreatment of the resin.

[0216] The pretreated resin, the cells obtained in step (4), and purified water were mixed in a mass ratio of 1:0.8:2.0 and adsorbed at 30°C for 5 hours. Then, chitosan solution was added for cross-linking. The volume of chitosan solution was 0.2 times that of the pretreated resin, the mass concentration of chitosan solution was 0.5%, the cross-linking temperature was 30°C, and the cross-linking time was 2 hours to complete the preparation of immobilized cells.

[0217] (6) The immobilized cells described in step (5) are packed into an immobilization column, and the D-fructose solution is passed through the immobilization column to obtain the D-tagatose conversion solution. The concentration of the D-tagatose solution in the D-tagatose conversion solution is 170.28 g / L. The concentration of the D-fructose solution is 550 g / L, the temperature is 58℃, and the flow rate of the D-fructose solution is 1 column volume / h. (7) The D-tagatose conversion solution obtained in step (6) is subjected to decolorization, ion exchange, one vacuum concentration, chromatographic separation, and two vacuum concentrations in sequence to obtain the D-tagatose concentrate.

[0218] The decolorization process involved: the material temperature of the D-tagatose conversion solution was 60℃. First, granular activated carbon columns were used for decolorization. Then, the material temperature of the D-tagatose conversion solution was adjusted to 55℃, and then filtered through an Ama filter with an operating pressure of 0.3 MPa to obtain the decolorized material.

[0219] The ion exchange process involves sequentially passing the decolorized material through cation exchange resin, anion exchange resin, cation exchange resin, anion exchange resin, and cation exchange resin. The material temperature is 40℃, and the flow rate is 3 BV / h, resulting in the ion-exchanged material.

[0220] The vacuum concentration process is as follows: the ion-exchange material is concentrated using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.1 MPa. After one vacuum concentration, the solid content of the material is 58%, and the material is obtained after one vacuum concentration.

[0221] Chromatographic separation was performed as follows: the obtained vacuum concentrated material was introduced into a simulated moving bed (SMB) chromatograph at a mass ratio of 1 g: 3.8 mL to separate D-tagatose, with a throughput of 0.027 T.DS / h / m. 3 The resin yielded 81% D-tagagose, and the chromatographically separated material was obtained.

[0222] The secondary vacuum concentration process is as follows: the material after chromatographic separation is concentrated twice using a six-effect evaporator at a concentration temperature of 70℃ and a pressure of -0.1 MPa. The secondary concentration is carried out until the solid content of the material increases to 72%, thus obtaining D-tagatose concentrate.

[0223] (8) The D-tagatose concentrate obtained in step (7) is crystallized, centrifuged, and dried. The specific process is as follows:

[0224] The crystallization process employs a combination of boiling crystallization and cooling crystallization, with boiling crystallization followed by cooling crystallization. Specifically, the boiling crystallization stage involves controlling the solid content of the D-tagatose concentrate to 70%, adding seed crystals at 3% of the dry weight of the D-tagatose concentrate, and maintaining the crystallization temperature at 55°C. During boiling crystallization, a D-tagatose solution with a solid content of 48% is continuously replenished, and the boiling time is 15 hours. After boiling crystallization, a boiling solution is obtained, which then enters the cooling crystallization stage. The cooling crystallization stage involves a temperature reduction of 0.3°C per hour within the 40–50°C range, and a temperature reduction of 0.8°C per hour within the 30–39°C range, decreasing the temperature to 30°C, at which point crystallization ends, yielding the crystallized solution.

[0225] Centrifugation was performed using a vertical scraper centrifuge at 700 rpm for 35 minutes. The crystallized liquid was rinsed during centrifugation, and the amount of water used for rinsing was 3% of the liquid volume.

[0226] The drying process was carried out using fluidized bed drying. The centrifuged material was fed into the fluidized bed with an inlet air temperature of 60°C, a cooling temperature of 27°C, and a drying time of 25 min. After drying, D-tagatose was obtained.

[0227] The conversion rate and purity data of D-tagatose in Examples 1-2 and Comparative Examples 1-6 are shown in Table 1.

[0228] Table 1. Conversion rate and purity of D-tagatose in Examples 1-2 and Comparative Examples 1-6

[0229] It can be seen that after adjusting the glycerol concentration in the fermentation broth of Comparative Example 1, all other variables were within the protection range. The glycerol concentration factor will affect the conversion rate of D-fructose to D-tagatose by immobilized recombinant Bacillus subtilis cells as a carrier to a certain extent.

[0230] Comparative Example 2 reduced the IPTG concentration in the fermentation broth, while all other variables remained within the protection range. By comparing with Examples 1 and 2, it was demonstrated that the reduction in IPTG concentration affected the conversion rate of D-fructose to D-tagatose using immobilized recombinant Bacillus subtilis cells as a carrier.

[0231] Comparative Example 3 increased the IPTG-induced content in the fermentation broth, while all other variables were within the protection range. By comparing with Examples 1 and 2, it was demonstrated that the increased IPTG concentration affected the conversion rate of D-fructose to D-tagatose using immobilized recombinant Bacillus subtilis cells as a carrier.

[0232] In Comparative Example 4, the resin was not soaked with manganese sulfate during the preparation of the immobilized cells, and all other variables were within the protection range. By comparing with Examples 1 and 2, it was demonstrated that the absence of manganese sulfate soaking in the resin significantly reduced the conversion yield of immobilized recombinant Bacillus subtilis cells per unit mass.

[0233] In Comparative Example 5, the concentration of chitosan crosslinking agent was increased in the preparation steps of immobilized cells, while all other variables were within the protection range. By comparing with Examples 1 and 2, it was demonstrated that increasing the concentration of chitosan crosslinking agent significantly reduced the conversion yield and conversion rate of immobilized recombinant Bacillus subtilis cells per unit mass.

[0234] In Comparative Example 6, the mass concentration of chitosan crosslinking agent was reduced in the preparation steps of immobilized cells, while all other variables were within the protection range. By comparing with Examples 1 and 2, it was demonstrated that increasing the mass concentration of chitosan crosslinking agent significantly reduced the transformation yield of immobilized recombinant Bacillus subtilis cells per unit mass.

[0235] In summary, this invention uses immobilized recombinant Bacillus subtilis cells as enzyme carriers to convert D-fructose into D-tagatose, achieving a conversion rate of 28%–32%. Each unit mass of immobilized recombinant Bacillus subtilis cells can convert 70–100 times its mass of D-fructose, and the purity of the D-tagatose product reaches over 99.5%. This experiment demonstrates the efficient utilization of recombinant Bacillus subtilis cells and significantly reduces the cost of enzyme preparations.

[0236] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant Bacillus subtilis strain BLCY-012, with accession number CGMCC No.31950.

2. A method for preparing fermentation broth, characterized in that, include: The recombinant Bacillus subtilis BLCY-012 of claim 1 was inoculated into a culture medium and cultured to obtain a fermentation broth; During the culture process, when dissolved oxygen rebound occurs in the fermentation broth, fed culture medium is added, the components of which include glycerol; after the addition of the fed culture medium, the glycerol concentration in the fermentation broth is 4-7 g / L. When fermentation broth OD 600 When the concentration of β-D-thiogalactoside was 45–55, isopropyl-β-D-thiogalactoside was added for the first time to induce fermentation. When the OD of the fermentation broth reached 45–55, the fermentation broth was... 600 When the concentration was 100-120, isopropyl-β-D-thiogalactoside was added a 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 line of recombinant Bacillus subtilis BLCY-012, characterized in that, Includes a manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, a cross-linking agent solution, and water; The immobilized carrier was mixed with a manganese-containing solution and then processed to obtain a manganese-containing immobilized carrier; Centrifuge the fermentation broth prepared by the method described in claim 2 to obtain recombinant Bacillus subtilis BLCY-012 cells; The mass ratio of the manganese-containing immobilized carrier, recombinant Bacillus subtilis BLCY-012 cells, and water is 1:(0.8–1.5):(2.0–3.0). The volume of the crosslinking agent solution is 0.2 to 0.35 times the volume of the manganese-containing immobilized carrier; the concentration of the crosslinking agent solution is 0.8% to 1.3%.

4. 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.

5. The method for preparing immobilized cells according to claim 3 or 4, characterized in that, include: The manganese-containing immobilization carrier, recombinant Bacillus subtilis BLCY-012 cells, and a first portion of water are mixed to obtain a first mixture; the first mixture is then crosslinked with a crosslinking agent to obtain immobilized cells.

6. The application of the recombinant Bacillus subtilis BLCY-012 of claim 1, or the fermentation broth prepared by the preparation method of claim 2, or the immobilized cells of claim 3 or 4, or the immobilized cells prepared by the preparation method of claim 5, in the preparation of D-tagatose.

7. A method for synthesizing D-tagatose, characterized in that, include: The fermentation broth prepared by the recombinant Bacillus subtilis BLCY-012 of claim 1 or the preparation method of claim 2, or the immobilized cells prepared by the preparation method of claim 3 or 4 or the preparation method of claim 5, is mixed with D-fructose solution, and then transformed, purified and crystallized to obtain D-tagatose; the crystallization method includes a combination of boiling crystallization and cooling crystallization.

8. The method of claim 7, wherein, The solid content of the solution to be crystallized during sugar boiling is controlled at 70%–72%, the amount of seed crystals added is 1%–5% of the dry basis mass of the solution to be crystallized, the sugar boiling temperature is 50–57°C, and the sugar boiling time is 12–16 hours; during the cooling crystallization, when the temperature of the sugar boiling solution is 40–50°C, the temperature is reduced by 0.2–0.5°C per hour; when the temperature of the sugar boiling solution is 30–39°C, the temperature is reduced by 0.5–1.0°C per hour.

9. The method of claim 7, wherein, The conversion was carried out using an immobilized column, with the concentration of the D-fructose solution being 500–600 g / L, the flow rate of the D-fructose solution being 1–2 times the immobilized column / h, and the conversion temperature being 55–60 °C.

10. The method of claim 7, wherein, The purification process includes sequential decolorization, ion exchange, vacuum concentration, and chromatographic separation; the mass ratio of the chromatographic separation is 1 g: (3.5–4.0) mL, and the throughput of the chromatographic separation is 0.025–0.028 T·DS / h / m³. 3 Resin.

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