A method for preparing high-activity monoside by enzymatic method with sugarcane molasses as raw material

Abstract

This invention discloses a method for preparing highly active monosaccharides using sugarcane molasses as raw material via enzymatic methods. The method includes sequentially diluting and acidifying the sugarcane molasses, flocculating and clarifying it, performing activated carbon-resin combined decolorization, and nanofiltration-ion exchange series desalting to obtain a refined sugar solution with a transmittance ≥85% and a desalting rate ≥90%. Glycerol and sucrose phosphorylase are added to the refined sugar solution, and a directional transglycosylation reaction is carried out under mild conditions, achieving a sucrose conversion rate ≥97% and an α-D-glycerol glucoside selectivity ≥95%. The solution after the transglycosylation reaction is then sequentially subjected to microfiltration to remove the enzyme, nanofiltration to remove small molecule impurities, and refining, decolorizing, and sterilizing to obtain α-D-glycerol glucoside. This invention develops a dedicated pretreatment process adapted to the characteristics of impurities in sugarcane molasses, resulting in a high-purity refined sugar solution that meets the requirements of enzymatic catalysis. A highly efficient directional transglycosylation catalytic system is established to achieve high conversion rate and high selectivity in the synthesis of α-GG from sucrose derived from sugarcane molasses, significantly reducing the raw material cost of production.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a method for preparing highly active monosaccharides. Background Technology

[0002] Sugarcane molasses is a core by-product of the sugarcane sugar industry, with an annual output of over one million tons in my country. Its composition includes 30-40% sucrose, 10-20% reducing sugar, as well as a large amount of ash, pigments, colloidal substances, and high concentrations of metal ions such as potassium, calcium, and magnesium.

[0003] Currently, sugarcane molasses is mostly used in low-value-added alcohol fermentation or feed processing, resulting in extremely low overall resource utilization efficiency. Furthermore, the high-concentration organic wastewater generated during processing is difficult and costly to treat, placing significant environmental pressure on enterprises.

[0004] α-D-glyceroglucoside (α-GG) is a high-performance natural functional monosaccharide with excellent moisturizing, thermal stability, skin barrier repair, and anti-inflammatory effects, and its demand continues to grow in the high-end cosmetics and pharmaceutical excipient sectors. Currently, most α-GG production uses refined sucrose or glucose as substrates, resulting in high raw material costs. If inexpensive sugarcane molasses is used directly as a substrate, its high salt, high pigment, high metal ion, and colloidal impurities strongly inhibit the activity of sucrase phosphorylation, leading to a transglycosylation efficiency typically below 30% and generating a large number of byproducts. Currently, there is no feasible industrial-scale technology solution in the industry. Summary of the Invention

[0005] To address the problem of high salt, high pigment, high metal ion, and colloidal impurities in sugarcane molasses inhibiting sucrose phosphorylation enzyme activity, this invention provides a method for enzymatically preparing highly active monosaccharides from sugarcane molasses. First, a dedicated pretreatment process adapted to the characteristics of sugarcane molasses impurities is developed to obtain a high-purity refined sugar solution that meets the requirements of enzymatic catalysis. Second, an efficient directional transglycosylation catalytic system is established to achieve high conversion rate and high selectivity in the synthesis of α-GG from sucrose derived from sugarcane molasses, significantly reducing the raw material cost of α-GG production. Third, a matching low-energy separation and purification process is developed to obtain high-purity, high-activity α-GG products that meet the requirements of various application fields.

[0006] To achieve the above objectives, the following technical solution is adopted: A method for preparing highly active monosaccharides using sugarcane molasses as raw material via enzymatic process includes the following steps: (1) Pretreatment of sugarcane molasses: Sugarcane molasses is subjected to dilution and acidification, flocculation and clarification, activated carbon-resin combination decolorization, and nanofiltration-ion exchange series desalination treatment in sequence to obtain refined sugar solution with transmittance ≥85% and desalination rate ≥90%. (2) Enzyme-catalyzed transglycosylation: Glycerol and sucrose phosphorylase are added to the refined sugar solution, and a directional transglycosylation reaction is carried out under mild conditions. The sucrose conversion rate is ≥97%, and the α-D-glyceroglucoside selectivity is ≥95%. (3) Separation and purification: The feed solution after the transglycosylation reaction was subjected to microfiltration to remove enzymes, nanofiltration to remove small molecule impurities, and purification, decolorization and sterilization treatment to obtain the target product α-D-glyceroglucoside.

[0007] According to the above scheme, the sugarcane molasses comes from a by-product of sugarcane sugar production, with a total sugar concentration of 65-70°Brix, a sucrose content of 30-40%, a reducing sugar content of 10-20%, an ash content of 7-10%, and an initial light transmittance of 2-5%.

[0008] According to the above scheme, the pretreatment process in step (1) is as follows: first, the sugarcane molasses is diluted with water to a total sugar concentration of 20-30°Brix, and the pH is adjusted to 3.0-4.5 with acid; flocculant is added and stirred for 20-60 minutes, and the supernatant is obtained by centrifugation or filtration; the supernatant is first decolorized by activated carbon adsorption and then further decolorized by anion exchange resin; then, nanofiltration membrane is used to retain sugar components and remove small molecule impurities, and finally, metal ions and salts are removed by cation exchange-anion exchange tandem resin to obtain refined sugar solution.

[0009] In the optimized scheme, during dilution and acidification, deionized water is added to dilute to a total sugar concentration of 150-250 g / L. The pH of the system is adjusted to 3.0-4.0 using 10%-20% dilute sulfuric acid. After stirring evenly, the mixture is allowed to stand for 20-30 minutes to complete the acidification. Sugarcane molasses contains high concentrations of colloids, polysaccharide complexes, and metal ions such as potassium, calcium, and magnesium. Conventional dilution at too low a concentration can easily cause excessive hydration of the colloids, making subsequent flocculation and sedimentation difficult; while at too high a concentration, the system has high viscosity and ionic strength, which directly inhibits subsequent enzyme activity. This invention uses a narrow dilution range of 150-250 g / L, combined with rapid acidification using 10%-20% high-concentration dilute sulfuric acid, rather than simple pH adjustment: the high-concentration acid can directionally break down the colloids in the molasses. The cross-linked structure of metal ions promotes the early instability of colloids; at the same time, it avoids the introduction of a large amount of water, which would lead to uneven dilution of salt ions and local acid-base imbalance; the critical window for micro-aggregation of colloids is 20-30 min. Too short a time will result in insufficient aggregation, while too long a time will easily lead to re-dissolution. This is an unconventional residence time design for the characteristics of molasses colloids, which provides a key structural basis for subsequent efficient flocculation.

[0010] In the optimized scheme, during flocculation and clarification, 0.1-0.3 g / L of polyaluminum chloride is added as a flocculant to the acidified sugar solution. First, the solution is rapidly stirred at 200-300 rpm for 5-10 minutes, then the speed is adjusted to 50-100 rpm and stirred slowly for 10-15 minutes. After stirring, the solution is allowed to settle for 30-60 minutes, and the supernatant is filtered to remove colloidal and large particle impurities. Molasses colloids exhibit high negative charge, high viscosity, and multi-scale distribution characteristics. Conventional flocculation easily leads to problems such as small flocs, slow settling, turbid supernatant, and low light transmittance. Furthermore, metal ions easily form insoluble complex salt precipitates with the flocculant, encapsulating the sugar and causing sugar loss. This invention employs low-dose polyaluminum chloride (0.1-0.3 g / L), two-stage variable-speed stirring, and a critical settling window of 30-60 min, which are not conventional flocculation parameters: high-speed rapid stirring instantly breaks the colloidal stability layer and promotes rapid adsorption of flocculants; low-speed slow stirring controls the slow growth of flocs, forming dense large flocs and avoiding floc breakage and turbidity; the 30-60 min settling period is for molasses-specific colloids. The complete settling range of salt-based composite flocs varies; a short range results in fine floc residue, while a long range leads to floc decomposition and turbidity. This combination can increase the colloid removal rate from the conventional 60%-70% to ≥90%, while controlling the sugar loss rate to ≤5%, making it suitable for molasses colloids. The unconventional parameter synergistic design of metal-ion composite systems is not a standard choice in this field.

[0011] In the optimized solution, activated carbon During resin combination decolorization, 1%-3% (by weight / volume) of powdered activated carbon is added to the supernatant. The system temperature is controlled at 50-60℃, and the mixture is stirred and adsorbed for 20-30 minutes. The activated carbon is then removed by filtration. The resulting filtrate is then passed through a macroporous adsorption resin column, with the flow rate controlled at 1-2 BV / h. The decolorized sugar solution is collected, at which point the transmittance of the sugar solution is ≥85%. Molasses pigments are mainly composed of large-molecule Maillard products, phenolic polymers, and caramel color complexes. Conventional single activated carbon decolorization can only remove small-molecule pigments, leaving high levels of large-molecule pigment residues and transmittance often below 60%. Conventional resin decolorization is easily clogged by colloidal residues, resulting in low decolorization efficiency and short resin life. This invention employs a combination of 50-60℃ medium-temperature activated carbon pre-decolorization, macroporous resin fine decolorization, and a low flow rate of 1-2 BV / h, which is not a conventional decolorization combination. 50-60℃ is the conformational activation temperature for molasses macromolecular pigments, promoting pigment molecule expansion and exposing adsorption sites, increasing the activated carbon adsorption capacity by over 40%. 1%-3% activated carbon represents a narrow range balancing decolorization efficiency and sugar adsorption loss; below 1% decolorization is insufficient, and above 3% sugar loss is significant. The 1-2 BV / h low flow rate represents the diffusion equilibrium time of macromolecular pigments within the resin channels; above 2 BV / h adsorption is insufficient, and below 1 BV / h efficiency is low. This combination can increase light transmittance from below 60% to ≥85% and pigment removal rate to ≥92%, representing an unconventional temperature range specifically for molasses macromolecular complex pigments. dose The flow rate coordination design is not simply a matter of adding up conventional processes.

[0012] In the optimized scheme, nanofiltration In ion exchange desalination, the decolorized sugar solution is first passed through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, with the operating pressure controlled at 1.0-2.0 MPa and the operating temperature at 30-40℃. This concentrates and removes some small-molecule salt impurities. The concentrated solution obtained from nanofiltration is then passed sequentially through a cation exchange resin column and an anion exchange resin column, with the column flow rate controlled at 1-1.5 BV / h. The refined sugar solution is collected, and the desalination rate is tested to be ≥90%. Molasses contains mainly potassium, sodium, calcium, and magnesium ions, as well as sulfates and phosphates. It has high ionic strength and easily forms hydrated ion complexes with sugar molecules. Conventional direct ion exchange is prone to rapid resin saturation, frequent regeneration, desalination rates below 70%, and residual enzyme activity inhibition. Conventional nanofiltration can only remove some salts and cannot deeply remove metal ions. This invention employs a narrow molecular weight cutoff nanofiltration (200-300 Da), a medium temperature of 30-40℃, and a medium pressure of 1.0-2.0 MPa, synergistically combined with ion exchange, unlike conventional desalting combinations. The 200-300 Da range represents a precise sieving range for small molecule salts and sucrose / oligosaccharides in molasses, selectively retaining sugars while allowing small molecule salts to pass through, reducing the subsequent ion exchange load by more than 50%. The 30-40℃ range is for molasses salts... The dissociation temperature of the glycoconjugate promotes salt ion release and improves nanofiltration desalination efficiency; 1.0-2.0 MPa is a pressure window that balances desalination rate and membrane life; a flow rate of 1-1.5 BV / h is the resin contact time for deep removal of metal ions. This combination can increase the desalination rate from the conventional below 70% to ≥90%, with metal ion residue <50 ppm and enzyme activity inhibition rate reduced to <5%, making it suitable for high-salt molasses. Unconventional membranes in high metal ion systems The resin-assisted desalination design is not a conventional technology choice in this field.

[0013] According to the above scheme, the enzyme-catalyzed glycosylation process in step (2) is as follows: glycerol is added in the refined sugar solution at a molar ratio of sucrose to glycerol of 1:3-1:8, the amount of sucrose phosphorylase added is 10-100U / g sucrose, the reaction temperature is 20-45℃, the reaction pH is 5.5-7.0, and the reaction time is 12-24h.

[0014] Molasses-refined sugar solutions still contain trace amounts of residual pigments, metal ions, and organic acid impurities, which readily induce sucrose phosphorylase to undergo secondary hydrolysis, preferentially hydrolyzing sucrose into glucose and fructose, significantly reducing the selectivity of monosaccharide products. Conventional feeding methods often involve excessive addition of glycerol or enzymes, resulting in high costs for raw materials and enzymes, and a large accumulation of hydrolysis byproducts. This invention specifies a sucrose to glycerol molar ratio of 1:3-1:8, an enzyme dosage of 10-100 U / g sucrose, a controlled temperature of 20-45℃, a pH of 5.5-7.0, and a reaction time of 12-24 h. This is a targeted parameter combination aimed at addressing the problem of side hydrolysis induced by trace impurities in molasses: when the molar ratio of glycerol is below 1:3, there are insufficient acceptors and the transglycosylation reaction is limited; when the molar ratio is above 1:8, excessive glycerol inhibits enzyme conformational activity. The enzyme dosage, temperature, and pH form a narrow-range synergistic barrier, which can significantly inhibit ineffective sucrose hydrolysis and directionally promote the main transglycosylation reaction, ultimately achieving a sucrose conversion rate of ≥97% and an α-D-glyceroglucoside selectivity of ≥95%. This ratio is not a conventional random parameter selection in the field and overcomes the technical challenge of side reactions caused by trace impurities in molasses.

[0015] According to the above scheme, the separation and purification process in step (3) is as follows: after the enzyme-catalyzed transglycosylation reaction is completed, the enzyme preparation is first removed by microfiltration membrane to obtain clear liquid, and then the residual glycerol and monosaccharide small molecule impurities are removed by nanofiltration membrane. Finally, after decolorization and sterilization filtration by food-grade activated carbon, α-D-glyceroglucoside product with a purity ≥95% is obtained.

[0016] The reaction system contains target monoglycosides, free glycerol, glucose, fructose, trace pigments, and soluble protein impurities. The molecular weights of these components are relatively similar, and conventional stepwise purification methods often result in product loss, difficulty in retaining small-molecule impurities, and insufficient product transmittance. This invention employs an integrated route combining fractional membrane separation with deep activated carbon purification. Utilizing the characteristics of fractional sieving, it achieves stepwise separation of the product from multiple impurities, avoiding high-cost purification methods such as conventional extraction and chromatography, and achieving both high recovery rate and high product purity.

[0017] In the optimized scheme, during microfiltration enzyme removal, the reaction system temperature is adjusted to 40-50℃, the pH is adjusted to 6.0-7.0, and a 0.22μm pore size organic microfiltration membrane is used for circulating cross-flow filtration. The operating pressure is controlled at 0.1-0.3MPa, and the circulation filtration is carried out for 1-2 hours to completely remove the sucrose phosphorylase preparation and a small amount of residual insoluble impurities in the reaction system. The clear microfiltration permeate is collected, and the retained enzyme preparation is recovered and reused after elution with phosphate buffer.

[0018] Under conventional room-temperature microfiltration conditions, enzyme proteins are prone to spatial conformational denaturation and adhesion to membrane pores, causing membrane blockage. This results in insufficient enzyme retention and the inability to recover and reuse enzymes, leading to significant enzyme loss with waste liquid and increased production costs. This invention limits the pretreatment conditions to 40-50℃ and pH 6.0-7.0, which is the steady-state range for sucrose phosphorylase, reducing protein viscosity and membrane adsorption. Combined with low-pressure cross-flow circulation filtration at 0.1-0.3 MPa for 1-2 hours, the cross-flow continuously removes deposited proteins from the membrane surface, achieving near-complete enzyme retention. Enzymes can also be recovered and recycled via buffer elution. This multi-parameter synergy of temperature, pH, pressure, and circulation time is an unconventional design that solves the problem of enzyme membrane adsorption loss, distinguishing it from conventional single-use dead-end microfiltration processes.

[0019] In the optimized scheme, when removing small molecule impurities by nanofiltration, the permeate from microfiltration to remove enzymes is passed into a nanofiltration membrane system with a molecular weight cutoff of 150-300 Da. The operating pressure is controlled at 1.0-2.5 MPa and the operating temperature at 30-45℃ for continuous nanofiltration separation. During the nanofiltration process, 1-2 times the volume of purified water from the microfiltration permeate is added for dialysis to thoroughly remove residual unreacted glycerol, glucose, fructose, and other small molecule impurities from the system. The nanofiltration retentate is then collected, at which point the crude purity of α-D-glyceroglucoside in the nanofiltration retentate reaches over 95%.

[0020] The molecular weight of α-D-glyceroglucoside is relatively close to that of glycerol and other small monosaccharide molecules. Conventional fixed-volume single-pass nanofiltration is insufficient to remove these small molecules, and excessive concentration can lead to product loss through co-osmosis with water molecules. This invention uses a 150-300 Da molecular weight cutoff membrane to match the critical difference in molecular weight between the product and impurities. Temperature control at 30-45℃ is used to reduce the viscosity of the feed solution and improve sieving efficiency. Continuous dialysis nanofiltration with 1-2 times the volume of purified water is employed, gradually washing out free small molecules through gradient dialysis, achieving deep impurity removal while ensuring a high retention rate of the target glycoside. This membrane selection combined with dynamic dialysis is not a commonly used nanofiltration method in the industry, overcoming the technical shortcomings of conventional concentrated nanofiltration's incomplete impurity removal.

[0021] In the optimized scheme, during the purification, decolorization, and sterilization process: the above nanofiltration retentate is heated to 50-60℃, and 0.5%-1.5% of food-grade powdered activated carbon is added to the total mass of the retentate. The stirring speed is controlled at 100-200 rpm, and the mixture is kept warm and stirred for decolorization for 30-60 minutes. After decolorization, the activated carbon and any microorganisms that may be present are removed by filtration through a 0.22μm sterile-grade microporous membrane. A clear filtrate with a purity of ≥95% and a transmittance of ≥98% is collected.

[0022] After nanofiltration, trace amounts of bound phenolic pigments remain. At room temperature, these pigments form weak complexes with glycoside molecules, which are difficult to remove using conventional low-temperature activated carbon adsorption. This invention uses a 50-60℃ heating method to break the weak pigment-glycoside complexes, exposing adsorption sites for the free pigments. A narrow parameter matching is achieved with 0.5%-1.5% activated carbon addition, a stirring rate of 100-200 rpm, and an adsorption time of 30-60 minutes, maximizing decolorization while avoiding product loss due to activated carbon adsorption. A final 0.22μm sterilization filtration simultaneously removes carbon and provides sterilization. This entire temperature control-carbon addition-stirring coupled solution is a targeted, unconventional refining design, resulting in a final product with significantly higher transmittance and purity than conventional room-temperature activated carbon refining processes.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: Firstly, in terms of raw material utilization, using sugarcane molasses, a cheap byproduct of the sugar industry, as a raw material to replace refined sugar used in traditional processes can reduce the raw material cost of α-GG production by more than 60%, realize the high-value transformation of sugarcane molasses, and increase its added value by more than 30 times compared with traditional application scenarios. At the same time, it can avoid the generation of a large amount of high-concentration organic wastewater during the traditional sugarcane molasses fermentation and processing, significantly reduce environmental treatment costs, and achieve outstanding environmental benefits.

[0024] Secondly, at the pretreatment process level, the aforementioned dilution acidification, flocculation clarification, and activated carbon... Resin combination decolorization and nanofiltration Ion exchange tandem desalination is not a simple superposition of conventional separation processes in this field, but rather a method specifically designed for the unique colloids of sugarcane molasses. Macromolecular pigments High salt A high-metal-ion quadruple inhibition system precisely identifies technical pain points that are difficult to address with conventional purification methods, through narrow-range parameter control and temperature... time Flow rate coordination, targeted destruction of colloidal structure, salt A series of unconventional techniques, including precise sugar sieving, have led to a customized purification process chain adapted to the complex molasses system. This process chain overcomes the inherent defects of conventional purification methods, such as low transmittance, incomplete desalting, strong enzyme activity inhibition, and significant sugar loss. It achieves technical results of "transmittance ≥85%, desalting rate ≥90%, and enzyme activity inhibition rate <5%", which cannot be easily conceived or simply modified by those skilled in the art based on existing technologies. It possesses outstanding substantive characteristics and significant progress.

[0025] Thirdly, at the level of enzyme catalysis, sucrose phosphorylase is used to catalyze the directional transglycosylation reaction. The reaction conditions are at room temperature and pressure, which is green and safe. The sucrose conversion rate is ≥97%, and the α-GG selectivity is ≥95%, which is far higher than the conversion rate of less than 30% when using sugarcane molasses as a substrate. Moreover, the amount of by-products generated is extremely small, which greatly reduces the cost and difficulty of subsequent separation and purification. At the same time, the enzyme's action conditions are stable and it can be repeatedly recycled, further reducing the cost of the catalysis process.

[0026] Fourth, in terms of product performance, the final α-GG product has a purity of ≥95%, fully retains the high bioactivity of natural α-GG, and has excellent moisturizing, skin barrier repair, and anti-inflammatory effects. Its moisturizing performance is not affected by changes in environmental humidity. It is non-sticky in high humidity environments and can lock in moisture for a long time in low humidity environments. Its thermal stability is far superior to conventional moisturizers such as hyaluronic acid and glycerin. It is suitable for the processing requirements of multiple fields and can be directly added to various products such as sensitive skin repair cosmetics, moisturizing essences, sugar-free baked goods, functional beverages, drug freeze-dried powder protectants, and pharmaceutical excipients.

[0027] Fifth, in terms of industrial impact, this invention uses no high-risk reagents throughout the entire process, and its emissions of waste are far lower than those of traditional production processes. It aligns with green manufacturing and dual-carbon development goals, which not only extends the sugar industry's industrial chain and enhances the profitability and risk resistance of sugar companies, but also significantly reduces the market price of α-GG, promoting its widespread application in more civilian scenarios and driving the development of multiple related industries such as biomanufacturing, daily chemicals, food, and biopharmaceuticals. It has significant economic, social, and ecological benefits. Attached Figure Description

[0028] Figure 1 : Schematic diagram of the separation and purification process of sugarcane molasses in this invention.

[0029] Figure 2 Flowchart of the enzymatic preparation of highly active monosaccharides in this invention.

[0030] Figure 3 : An example diagram illustrating the application of the highly active monosaccharides of this invention. Detailed Implementation

[0031] The following embodiments further illustrate the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0032] A specific implementation method for separating and purifying sugarcane molasses has been provided, see Appendix. Figure 1As shown. In the specific embodiment, the sugarcane molasses used was taken from the final molasses by-product of a sugarcane sugar processing enterprise in Guangxi, China. According to the SN / T 5735-2024 standard, the measured values ​​were: total sugar concentration 67°Brix, sucrose content 35wt%, reducing sugar content 12wt%, ash content 8.0wt%, and initial light transmittance at room temperature 3.2%.

[0033] Example 1 First, weigh 100 kg of sugarcane molasses, add purified water and stir to dilute to a total sugar concentration of 25°Brix. Adjust the pH of the system to 3.8 using food-grade sulfuric acid. After stirring evenly, add polyaluminum chloride flocculant at a concentration of 0.15% of the total mass of the diluted molasses solution. Control the reaction temperature at 25°C and stir at 80 r / min for 35 min. Then, use a plate and frame filter press to filter and remove flocculation and sediment, and collect the clear supernatant. At this time, the transmittance of the supernatant is 42%.

[0034] The clarified supernatant was first passed through an adsorption column packed with food-grade columnar activated carbon for primary decolorization. The flow rate was controlled at 1.5 BV / h and the adsorption temperature at 30℃. After primary decolorization, the transmittance of the sugar solution increased to 72%. Then, the sugar solution was passed through a D315 macroporous anion exchange resin column for secondary decolorization. The flow rate was controlled at 2 BV / h and the operating temperature at 25℃. After secondary decolorization, the transmittance of the sugar solution reached 86%.

[0035] The decolorized sugar solution was passed through a nanofiltration membrane with a molecular weight cutoff of 200 Da for processing. The operating pressure was 1.2 MPa and the operating temperature was 30 °C. During the nanofiltration process, purified water with a volume twice that of the sugar solution was added for dialysis. The retained sugar component solution was collected, and the permeate was discharged to remove impurities such as small molecule salts and free monosaccharides. The initial desalination rate of the sugar solution after nanofiltration reached 55%.

[0036] Finally, the retentate obtained from nanofiltration was first passed through a 001×7 strong acid cation exchange resin column, with the influent flow rate controlled at 1 BV / h and the operating temperature at 25℃, to remove metal cations such as potassium, calcium, and magnesium ions. Then, it was passed through a D201 strong basic anion exchange resin column, with the influent flow rate controlled at 1 BV / h and the operating temperature at 25℃, to remove anionic salts. The final refined sugar solution was tested and found to have a transmittance of 91%, a total desalination rate of 94%, and a sucrose retention rate of 92%, which fully met the substrate requirements for the subsequent sucrose phosphorylase catalytic reaction.

[0037] Example 2 Weigh 100 kg of sugarcane molasses, add purified water and stir to dilute to a total sugar concentration of 20°Brix. Adjust the pH of the system to 3.0 using food-grade hydrochloric acid. After stirring evenly, add polyacrylamide flocculant at a concentration of 0.1% of the total mass of the diluted molasses solution. Control the reaction temperature at 30°C and stir at 100 r / min for 20 min. Then, use a disc centrifuge to remove the flocculated precipitate and collect the clear supernatant. At this time, the transmittance of the supernatant is 38%.

[0038] The clarified supernatant was first passed into an adsorption tank filled with powdered activated carbon for primary decolorization. The amount of activated carbon added was 0.5% of the total mass of the supernatant. After stirring and adsorption for 30 minutes, the activated carbon was removed by filtration. The transmittance of the resulting primary decolorized sugar solution increased to 70%. Then, the primary decolorized sugar solution was passed into a D301 macroporous anion exchange resin column for secondary decolorization. The influent flow rate was controlled at 3 BV / h and the operating temperature was 30℃. After secondary decolorization, the transmittance of the sugar solution reached 85%.

[0039] The decolorized sugar solution was passed through a nanofiltration membrane device with a molecular weight cutoff of 150 Da for processing. The operating pressure was 1.5 MPa and the operating temperature was 35 °C. During the nanofiltration process, 1.5 times the volume of purified water was added for dialysis. The retained sugar component solution was collected. The initial desalination rate of the sugar solution after nanofiltration reached 58%.

[0040] Finally, the retentate obtained from nanofiltration was first passed through a 732 strong acid cation exchange resin column, with the influent flow rate controlled at 1.5 BV / h and the operating temperature at 30℃, to remove metal cations. Then, it was passed through a 717 strong base anion exchange resin column, with the influent flow rate controlled at 1.5 BV / h and the operating temperature at 30℃, to remove anionic salts. The final refined sugar solution was tested and found to have a transmittance of 88%, a total desalination rate of 92%, and a sucrose retention rate of 90%.

[0041] Example 3 Weigh 100 kg of sugarcane molasses, add purified water and stir to dilute to a total sugar concentration of 30°Brix. Adjust the pH of the system to 4.5 using food-grade citric acid. After stirring evenly, add a compound flocculant of polyaluminum chloride and polyacrylamide, with a total addition amount of 0.2% of the total mass of the molasses dilution. Control the reaction temperature at 20℃ and stir at 60 r / min for 60 min. Then filter the mixture using a plate and frame filter press to remove flocculation and precipitate, and collect the clear supernatant. At this time, the transmittance of the supernatant is 45%.

[0042] The clarified supernatant was first passed through an adsorption column packed with food-grade columnar activated carbon for primary decolorization. The influent flow rate was controlled at 1 BV / h and the adsorption temperature at 25℃. After primary decolorization, the transmittance of the sugar solution increased to 74%. Then, the primary decolorized sugar solution was passed through a D315 macroporous anion exchange resin column for secondary decolorization. The influent flow rate was controlled at 1.5 BV / h and the operating temperature at 20℃. After secondary decolorization, the transmittance of the sugar solution reached 87%.

[0043] The decolorized sugar solution was passed through a nanofiltration membrane device with a molecular weight cutoff of 250 Da for processing. The operating pressure was 1.0 MPa and the operating temperature was 25°C. During the nanofiltration process, 2.5 times the volume of purified water was added for dialysis. The retained sugar component solution was collected. The initial desalination rate of the sugar solution after nanofiltration reached 52%.

[0044] Finally, the retentate obtained from nanofiltration was first passed through a 001×7 strong acid cation exchange resin column, with the influent flow rate controlled at 0.8 BV / h and the operating temperature at 20℃, to remove metal cations. Then, it was passed through a D201 strong base anion exchange resin column, with the influent flow rate controlled at 0.8 BV / h and the operating temperature at 20℃, to remove anionic salts. The final refined sugar solution was tested and found to have a transmittance of 92%, a total desalination rate of 95%, and a sucrose retention rate of 93%.

[0045] A specific embodiment also provides a method for preparing highly active monosaccharides using sugarcane molasses as raw material via enzymatic method, wherein the process for preparing highly active monosaccharides via enzymatic method is attached. Figure 2 As shown. Specifically: Example 4 Sugarcane molasses pretreatment: Sugarcane molasses, a byproduct of sugar production, was taken and tested. Its sucrose content was found to be 35 wt%, reducing sugar content 12 wt%, and ash content 8 wt%. First, deionized water was added to dilute the molasses to a sugar concentration of 22 Brix. The pH of the system was adjusted to 3.5 using 0.8 mol / L hydrochloric acid. After stirring evenly, the mixture was allowed to stand for 25 minutes. Then, polyaluminum chloride flocculant was added to the acidified sugar solution at a rate of 0.2% of the total sugar solution mass. The mixture was first stirred rapidly at 250 rpm for 12 minutes, then the stirring speed was adjusted to 80 rpm for 5 minutes. Afterward, the mixture was allowed to settle for 40 minutes. The lower layer of precipitated colloidal and particulate impurities was removed by filtration, and the clear upper layer was collected. Next, activated carbon-resin combination decolorization was performed. Powdered activated carbon was first added to the clarified liquid at a rate of 1% of the clarified liquid mass. The system temperature was controlled at 55°C. After stirring and adsorption for 25 minutes, the activated carbon was removed by filtration to obtain the first decolorized liquid. The first decolorized liquid was then passed into a D101 macroporous adsorption resin column at a flow rate of 1.5 BV / h, and the column temperature was controlled at 45°C. The outflowing second decolorized liquid was collected. At this time, the transmittance of the second decolorized liquid was measured to be 89%. Finally, nanofiltration-ion exchange tandem desalination was performed. The secondary decolorized solution was passed through a nanofiltration membrane with a molecular weight cutoff of 180 Da, and the operating pressure was controlled at 2.0 MPa and the operating temperature at 35 °C. The solution was concentrated to a sugar content of 32 Brix to obtain the nanofiltration concentrate. The nanofiltration concentrate was then passed sequentially through a 001×7 type cation exchange resin column and a D301 type anion exchange resin column, with the column flow rate controlled at 1.2 BV / h and the column temperature at 28 °C. The purified sugar solution was collected, and the transmittance of the purified sugar solution was found to be 92%, and the desalination rate was 94%, which met the requirements for subsequent reactions.

[0046] Enzymatic catalytic glycosylation: Take the above refined sugar solution, adjust the sucrose concentration in the sugar solution to 200 g / L, add glycerol according to the molar ratio of sucrose to glycerol 1:4, stir evenly, adjust the pH of the system to 6.5 with 0.1 mol / L phosphate buffer, add sucrose phosphorylase, the amount of enzyme added is 8 U enzyme activity per gram of sucrose, control the reaction temperature at 40℃, the stirring speed at 120 rpm, take samples for testing after 15 hours of reaction, at this time the sucrose conversion rate is 97.8% and the selectivity of α-D-glyceroglucoside is 96.2%, stop the reaction.

[0047] Separation and purification: First, the feed solution after the transglycosylation reaction was passed through a microfiltration membrane with a pore size of 0.22 μm, and the operating pressure was controlled at 0.4 MPa to remove sucrose phosphorylase and residual macromolecular impurities, resulting in a microfiltration permeate. Then, the microfiltration permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 100 Da, and the operating pressure was controlled at 2.5 MPa and the operating temperature at 32 °C to remove unreacted small molecule impurities such as glycerol, glucose, and fructose, resulting in a crude α-D-glyceroglucoside concentrate. Subsequently, the crude concentrate was passed through a granular activated carbon purification column at a flow rate of 0.8 BV / h for decolorization. The effluent was collected and spray-dried, with the inlet air temperature controlled at 165 °C and the outlet air temperature controlled at 85 °C, finally yielding a white powdery α-D-glyceroglucoside product, whose purity was determined to be 96.5% by high performance liquid chromatography.

[0048] Example 5 The preparation steps in this embodiment are basically the same as those in Example 1, except that in the sugarcane molasses dilution and acidification step, the sugar content after dilution is 20 Brix, the pH value is adjusted to 3.0, the flocculant addition is 0.1%, the activated carbon addition is 0.5%, the nanofiltration molecular weight cutoff is 150 Da, the sucrose to glycerol molar ratio in the enzyme catalysis step is 1:3, the enzyme addition is 5 U per gram of sucrose, the reaction temperature is 35℃, the reaction time is 18 hours, the final sucrose conversion rate is 97.2%, the α-D-glyceroglucoside selectivity is 95.3%, and the purity of the obtained product is 95.8%.

[0049] Example 6 The preparation steps in this embodiment are basically the same as those in Example 1, except that in the sugarcane molasses dilution and acidification step, the sugar content after dilution is 25 Brix, the pH value is adjusted to 4.0, the flocculant addition is 0.3%, the activated carbon addition is 1.5%, the nanofiltration molecular weight cutoff is 200 Da, the sucrose to glycerol molar ratio in the enzyme catalysis step is 1:6, the enzyme addition is 10 U per gram of sucrose, the reaction temperature is 45℃, the reaction time is 12 hours, the final sucrose conversion rate is 98.1%, the α-D-glyceroglucoside selectivity is 95.7%, and the purity of the obtained product is 96.1%.

[0050] The detailed implementation also provides application examples of the highly active monosaccharides prepared in the embodiments, see appendix. Figure 3 As shown: Application Example 1 The α-D-glyceroglucoside obtained in Example 4 was added to the basic moisturizing essence formula at a mass fraction of 2%, as shown in the appendix. Figure 3As shown. Without adjusting other formula components, the prepared moisturizing essence was tested on 30 subjects with sensitive skin for 28 consecutive days. The subjects' skin stratum corneum moisture content increased by an average of 42%, transepidermal water loss decreased by an average of 35%, and the improvement rate of facial redness and inflammation reached 29%. No subjects experienced allergic or irritation reactions, which proves that the product has excellent moisturizing, skin barrier repair and anti-inflammatory effects and can be safely applied in the field of high-end cosmetics.

[0051] Application Example 2 The α-D-glyceroglucoside obtained in Example 4 was added to the sugar-free chewing gum base at a mass fraction of 5%. The resulting sugar-free chewing gum had a moderate sweetness and a refreshing taste. Tests showed that it could inhibit the reproduction rate of Streptococcus mutans in the oral cavity by 41%. After 12 months of sealed storage at room temperature, there were no quality problems such as clumping or flavor changes. It can be widely used in the field of sugar-free food.

[0052] Application Example 3 The α-D-glyceroglucoside prepared in Example 4 was added as a freeze-dried powder protectant to the probiotic freeze-dried powder formulation at a dosage of 8% of the total mass of the formulation. Compared with the control group with an equal amount of sucrose, the survival rate of the probiotics after freeze-drying increased by 27%, and the survival rate after accelerated storage at 37°C for 3 months increased by 43%, confirming that it can be used as a high-quality stabilizer and protectant in the field of pharmaceutical excipients.

Claims

1. A method for preparing highly active monosaccharides using sugarcane molasses as raw material via enzymatic process, characterized in that... Includes the following steps: (1) Pretreatment of sugarcane molasses: Sugarcane molasses is subjected to dilution and acidification, flocculation and clarification, activated carbon-resin combination decolorization, and nanofiltration-ion exchange series desalination treatment in sequence to obtain refined sugar solution with transmittance ≥85% and desalination rate ≥90%. (2) Enzyme-catalyzed transglycosylation: Glycerol and sucrose phosphorylase are added to the refined sugar solution, and a directional transglycosylation reaction is carried out under mild conditions. The sucrose conversion rate is ≥97%, and the α-D-glyceroglucoside selectivity is ≥95%. (3) Separation and purification: The feed solution after the transglycosylation reaction was subjected to microfiltration to remove enzymes, nanofiltration to remove small molecule impurities, and purification, decolorization and sterilization treatment to obtain the target product α-D-glyceroglucoside.

2. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material according to claim 1, characterized in that... The sugarcane molasses is derived from a byproduct of sugarcane sugar production, with a total sugar concentration of 65-70°Brix, a sucrose content of 30-40%, a reducing sugar content of 10-20%, an ash content of 7-10%, and an initial light transmittance of 2-5%.

3. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... In step (1), during the dilution and acidification process, deionized water is added to dilute the total sugar concentration to 150-250 g / L. The pH of the system is adjusted to 3.0-4.0 using 10%-20% dilute sulfuric acid. After stirring evenly, the system is allowed to stand for 20-30 minutes to complete the acidification.

4. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... In step (1), during flocculation and clarification, add 0.1-0.3 g / L of polyaluminum chloride as a flocculant to the acidified sugar solution. First, stir rapidly at 200-300 rpm for 5-10 min, then adjust the speed to 50-100 rpm and stir slowly for 10-15 min. After stirring, let it stand and settle for 30-60 min, filter and take the supernatant to remove colloidal and large particle impurities.

5. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... Activated carbon in step (1) During resin decolorization, 1%-3% (by mass / volume) of powdered activated carbon is added to the supernatant. The system temperature is controlled at 50-60℃. After stirring and adsorption for 20-30 minutes, the activated carbon is removed by filtration. The resulting filtrate is then passed through a macroporous adsorption resin column, and the flow rate through the column is controlled at 1-2 BV / h. The decolorized sugar solution is collected, at which point the transmittance of the sugar solution is ≥85%.

6. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material according to claim 1, characterized in that... Nanofiltration in step (1) During ion exchange desalination, the decolorized sugar solution is first passed through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, with the operating pressure controlled at 1.0-2.0 MPa and the operating temperature at 30-40℃. This concentrates and removes some small molecule salt impurities. The concentrated solution obtained from nanofiltration is then passed sequentially through a cation exchange resin column and an anion exchange resin column, with the column flow rate controlled at 1-1.5 BV / h. The purified sugar solution is then collected, and the desalination rate is tested to be ≥90%.

7. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... The enzyme-catalyzed transglycosylation process in step (2) is as follows: glycerol is added in the refined sugar solution at a molar ratio of sucrose to glycerol of 1:3-1:8, the amount of sucrose phosphorylase added is 10-100U / g sucrose, the reaction temperature is 20-45℃, the reaction pH is 5.5-7.0, and the reaction time is 12-24h.

8. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... In step (3), when removing enzymes by microfiltration, the temperature of the reaction system is adjusted to 40-50℃ and the pH is adjusted to 6.0-7.

0. An organic microfiltration membrane with a pore size of 0.22μm is used for circulating cross-flow filtration. The operating pressure is controlled at 0.1-0.3MPa. The filtration is carried out for 1-2 hours to completely remove the sucrose phosphorylase preparation and a small amount of residual insoluble impurities in the reaction system. The clear microfiltration permeate is collected. The retained enzyme preparation is washed with phosphate buffer and then recycled for reuse.

9. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... In step (3), when removing small molecule impurities by nanofiltration, the permeate from microfiltration to remove enzymes is passed into a nanofiltration membrane system with a molecular weight cutoff of 150-300 Da. The operating pressure is controlled at 1.0-2.5 MPa and the operating temperature at 30-45℃ for continuous nanofiltration separation. During the nanofiltration process, 1-2 times the volume of purified water from the microfiltration permeate is added for dialysis to thoroughly remove unreacted small molecule impurities such as glycerol, glucose, and fructose remaining in the system. The nanofiltration retentate is collected, and at this time, the crude purity of α-D-glyceroglucoside in the nanofiltration retentate is above 95%.

10. The method for preparing highly active monosaccharides using sugarcane molasses as a raw material by enzymatic method as described in claim 1, characterized in that... In step (3), during the purification, decolorization, and sterilization process: the above nanofiltration retentate is heated to 50-60℃, and 0.5%-1.5% of food-grade powdered activated carbon is added to the total mass of the retentate. The stirring speed is controlled at 100-200 rpm, and the retentate is kept warm and stirred for 30-60 min for decolorization. After decolorization, the activated carbon and any microorganisms that may be present are removed by filtration through a 0.22μm sterilization-grade microporous membrane. A clear filtrate with a purity of ≥95% and a transmittance of ≥98% is collected.

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