Green preparation method of glucosyl-steviol glycoside and waste liquid recovery device

Abstract

This invention relates to the field of green chemical technology, specifically to a green preparation method and wastewater recovery device for glucosyl steviol glycosides. The method includes: sequentially separating the crude wastewater after enzymatic glycoside conversion using ultrafiltration, nanofiltration, and reverse osmosis membranes; recovering enzyme proteins, oligosaccharides, monosaccharides, and purified water in stages; preheating the recovered sugar solution and adding elemental promoters containing nickel, cobalt, and iron before sending it to a two-phase anaerobic reaction system, where it undergoes hydrolysis in the acid-producing phase and conversion in the methanogenic phase to generate biogas; the anaerobic effluent undergoes deep treatment in a downflow aerated biological filter, and finally, sludge-water separation is performed, with the supernatant meeting discharge standards or being reused, and the sludge and backwash wastewater being returned to the acid-producing phase for secondary digestion. This application can achieve efficient recovery and energy conversion of multiple components in wastewater, solving the problem of difficult treatment of complex wastewater.

Description

Technical Field

[0001] This invention relates to the field of green chemical technology, and in particular to a green preparation method for glucosylstevioside and a waste liquid recovery device. Background Technology

[0002] In the steviol glycoside deep processing industry, the enzymatic transglycosylation technology for preparing high-sweetness, low-calorie glucosyl steviol glycosides has become the mainstream process. This process typically uses steviol glycosides and sugars as raw materials, undergoing a transglycosylation reaction catalyzed by specific enzymes, followed by separation and purification using macroporous adsorption resins to obtain the target product. This process generates a large amount of process wastewater, whose main components include unreacted glycosyl donors, a small amount of unconverted steviol glycosides, reaction byproducts, buffer salts, and free enzyme proteins and bacterial fragments. Existing treatment solutions typically employ membrane separation technology for staged treatment of the wastewater. Membrane modules with different molecular weight cutoffs, such as ultrafiltration, nanofiltration, and reverse osmosis, are used to initially separate large-molecule proteins, oligosaccharides, monosaccharides, and water from the wastewater. Some solutions also combine anaerobic fermentation or aerobic biochemical treatment to degrade the concentrated or permeate after separation, achieving compliant wastewater discharge or partial resource recovery.

[0003] However, in existing technologies, it is often difficult to achieve complete harmless treatment and closed-loop utilization of resources for wastewater containing complex sugar components and trace amounts of steviol glycoside derivatives. Because the residual steviol glycosides and their derivatives in the wastewater have stable structures, conventional biodegradation processes are insufficient to completely decompose them, leading to significant fluctuations in effluent quality. Furthermore, the enzymes, proteins, sugars with varying degrees of polymerization, carbon sources, and water resources contained in the wastewater cannot be systematically graded, recovered, and efficiently converted, resulting in raw material waste and excessive load on subsequent treatment processes. Summary of the Invention

[0004] The purpose of this invention is to provide a green preparation method and waste liquid recovery device for glucosyl steviol glycosides, which can solve the technical problems of the difficulty in resource utilization of complex waste liquid generated during the preparation of glucosyl steviol glycosides, the environmental pollution caused by the direct discharge of high-concentration organic wastewater, and the low utilization rate of raw materials.

[0005] The first aspect of this application provides a green preparation method for glucosylstevioside, comprising: using steviol glycoside and a glycosyl donor as raw materials, carrying out a transglycosylation reaction under enzyme catalysis to obtain a transglycosylation solution containing glucosylstevioside; separating the transglycosylation solution through a macroporous adsorption resin, wherein the target product glucosylstevioside is adsorbed by the resin, and collecting the resin permeate rich in unreacted sugars, buffer salts, free enzyme proteins, and bacterial cell debris as crude waste liquid; subsequently eluting the resin with an ethanol-water solution, and collecting the eluent containing the target product; subjecting the crude waste liquid to ultrafiltration, nanofiltration, and reverse osmosis membrane separation in sequence, wherein ultrafiltration retains and recovers free enzyme proteins and bacterial cell debris, nanofiltration retains and recovers disaccharides and higher oligosaccharides, and reverse osmosis retains and recovers monosaccharides; mixing the oligosaccharides retained by nanofiltration with the monosaccharides retained by reverse osmosis and introducing them into an conditioning tank, preheating to 35-40°C, and simultaneously adding an elemental promoter, which includes nickel, cobalt, and iron salts, to supplement the anaerobic microorganisms. The system utilizes coenzyme factors required for the degradation of sugars and steviol glycoside derivatives. Preheated mixed sugar solution is fed into a two-phase anaerobic reaction system. First, it enters the acid-producing phase reactor, where, under a residence time of 12-24 hours, acid-producing bacteria convert sugars into volatile fatty acids and specifically hydrolyze and break the glycosidic bonds of trace steviol glycosides, converting them into small-molecule organic acids. Then, it enters the methanogenic phase reactor, where the small-molecule organic acids are converted into biogas. The effluent from the anaerobic treatment is passed through a downflow aerated biological filter, where the aerobic biofilm attached to the filter media further degrades residual organic matter. The aerobic effluent undergoes precise sludge-water separation; the supernatant is discharged after water quality monitoring and is either discharged as compliant or reused as cleaning water. The liquid permeating through the reverse osmosis membrane is directly reused as purified water in the preparation of resin eluent or in the glycoside conversion reaction section. Backwash wastewater from the aerated biological filter and excess sludge from the anaerobic system are all returned to the acid-producing phase reactor for secondary hydrolysis and digestion.

[0006] The specific steps of the transglycosylation reaction are as follows: steviol glycosides and glycosyl donors are mixed at a mass ratio of 1:2 to 1:4 and added to pure water to prepare a reaction solution with a substrate mass concentration of 10%-20%; cyclodextrin glucosyltransferase is added to the reaction solution at an enzyme amount of 200-500 activity units per gram of steviol glycosides; the reaction is carried out at a constant temperature of 45-55℃ and a pH of 5.5-6.5, with stirring at 100-200 rpm for 12-24 hours to obtain a transglycosylation solution containing glucosyl steviol glycosides; the glycosyl donor is selected from one or more of starch hydrolysate, maltose, or maltotriose.

[0007] Ultrafiltration uses organic or ceramic membranes with a molecular weight cutoff of 5,000-50,000 Daltons, operating at a pressure of 0.2-0.6 MPa. The ultrafiltration concentrate is collected by settling or centrifugation to recover free enzyme proteins and bacterial fragments. Nanofiltration uses nanofiltration membranes with a molecular weight cutoff of 150-300 Daltons, operating at a pressure of 1.0-3.0 MPa. The nanofiltration concentrate is mainly enriched with disaccharides, trisaccharides, and oligosaccharides, and its total sugar concentration reaches 8%-15% before being stored as a carbon source for the methanogenic phase. Reverse osmosis uses high-flux, fouling-resistant reverse osmosis membranes, operating at a pressure of 1.5-4.0 MPa. The reverse osmosis concentrate is enriched with monosaccharides and monovalent salts. The liquid that permeates through the reverse osmosis membrane is purified water, with its chemical oxygen demand reduced to 80-150 mg / L and its conductivity reduced to 500-1500 μS / cm.

[0008] The specific steps for separation using macroporous adsorption resin are as follows: The transglycosylation solution is first passed through an adsorption column packed with macroporous adsorption resin at a flow rate of 1-2 BV / h. The target product, glucosylstevioside, is adsorbed by the resin. The liquid rich in unreacted sugars, salts, and free enzyme proteins is discharged as resin permeate and collected as crude waste liquid. After adsorption saturation, the resin column is forward-washed or back-washed with pure water to remove residual sugars and impurities in the resin interstices. The washing water is added to the crude waste liquid. Subsequently, the resin is eluted with a 50%-70% (v / v) ethanol aqueous solution at a flow rate of 0.5-1 BV / h. The ethanol eluent containing glucosylstevioside is collected and subsequently concentrated and de-ethanolified to obtain the target product.

[0009] The specific steps of membrane separation are as follows: First, the crude waste liquid is pumped into an ultrafiltration membrane separation system, where it undergoes cross-flow filtration using a polyvinylidene fluoride (PVDF) or polyethersulfone (PES) ultrafiltration membrane. The ultrafiltration retentate is concentrated and recovered to obtain a mixture of free enzyme protein and bacterial fragments. The ultrafiltration permeate then enters the nanofiltration stage. Next, the ultrafiltration permeate is pumped into a nanofiltration membrane separation system, where it is separated using a nanofiltration membrane. The nanofiltration retentate is concentrated and recovered to obtain a solution of disaccharides and higher oligosaccharides rich in maltose and maltotriose. The nanofiltration permeate then enters the reverse osmosis stage. Finally, the nanofiltration permeate is pumped into a reverse osmosis membrane separation system, where it is separated using a reverse osmosis composite membrane. The reverse osmosis retentate is concentrated and recovered to obtain a monosaccharide solution whose main component is glucose. The liquid that permeates through the reverse osmosis membrane is purified water.

[0010] The specific steps for preheating and element addition are as follows: A mixture of nanofiltration and reverse osmosis retentate is introduced into an equalization tank. The wastewater temperature is precisely increased and maintained at 35-40℃ through internal and external tubular heat exchangers. A mechanical agitator is installed in the equalization tank at a speed of 60-80 rpm for homogenization. Simultaneously, an element promoter is continuously or intermittently added to the equalization tank via a metering pump. The element promoter is an aqueous solution prepared from nickel chloride, cobalt chloride, and ferrous sulfate in a mass ratio of nickel chloride:cobalt chloride:ferrous sulfate equal to 1:1.5:2.5. The dosage is controlled according to the ratio of 0.05-0.15 micrograms of nickel ions, 0.075-0.225 micrograms of cobalt ions, and 0.125-0.375 micrograms of ferrous ions per milligram of chemical oxygen demand in the wastewater.

[0011] The second aspect of this application provides a waste liquid recovery device for the green preparation of glucosylstevioside, applied to the green preparation method of glucosylstevioside described in any one of the above claims, comprising: a transglycosylation reaction unit, a resin adsorption separation unit, a membrane separation and recovery unit, a preheating and conditioning unit, a two-phase anaerobic treatment unit, a downflow aerated biological filter unit, and a sludge-water separation and reuse unit connected in sequence by pipelines; the transglycosylation reaction unit includes a transglycosylation reaction vessel with a temperature control jacket and a stirrer; the resin adsorption separation unit includes a macroporous adsorption resin column, and an eluent collection tank and a crude waste liquid collection tank connected to the bottom outlet of the resin column; the membrane separation and recovery unit includes an ultrafiltration membrane module, a nanofiltration membrane module, and a reverse osmosis membrane module connected in sequence by pipelines, each membrane module having an inlet, a retentate outlet, and a permeate outlet, and the retentate outlet of the ultrafiltration membrane module containing ligase protein recovery. The system includes a collection tank, a nanofiltration membrane module whose retentate outlet is connected to an oligosaccharide recovery tank, a reverse osmosis membrane module whose retentate outlet is connected to a monosaccharide recovery tank, and a reverse osmosis membrane module whose permeate outlet is connected to a pure water reuse network. The outlets of the oligosaccharide and monosaccharide recovery tanks merge and connect to the inlet of the regulating and preheating unit. The regulating and preheating unit includes a regulating tank equipped with a heating device, a stirring device, and an element dosing device. The outlet of the regulating tank connects to a two-phase anaerobic treatment unit. The two-phase anaerobic treatment unit includes a sequentially connected acid-producing reactor and a methanogenic reactor. A biogas collection pipe is located at the top of the methanogenic reactor. The downflow aerated biological filter unit includes a downflow aerated biological filter with an aeration pipe at the bottom. The sludge-water separation and reuse unit includes a sludge-water separator, an online water quality monitor, and a sludge return pipeline. The end of the sludge return pipeline connects to the inlet of the acid-producing reactor.

[0012] In the membrane separation and recovery unit, booster pumps are installed between the ultrafiltration membrane module and the nanofiltration membrane module, and between the nanofiltration membrane module and the reverse osmosis membrane module; flow regulating valves are installed on the outlet pipes of the oligosaccharide recovery tank and the monosaccharide recovery tank, and the two high-concentration sugar solutions merge on the pipes before entering the regulating tank.

[0013] The acid-producing phase reactor is a completely mixed anaerobic reactor, equipped with a mechanical agitator, an online pH detection probe, and a temperature sensor, and has an exhaust pipe at the top; the methanogenic phase reactor is an internal circulation anaerobic reactor, with a water distributor, an anaerobic sludge bed zone, a suspended sludge zone, and a three-phase separator arranged from bottom to top inside.

[0014] The sludge-water separator is a high-efficiency sedimentation tank. The supernatant outlet of the sludge-water separator is connected to an online water quality monitoring instrument via a pipeline. The online water quality monitoring instrument has a conductivity detection probe and a TOC detection probe. The effluent that meets the standards is discharged or connected to the cleaning water reuse pipeline. The permeate outlet of the reverse osmosis membrane module is connected to the inlet of the eluent preparation tank of the macroporous adsorption resin column in the resin adsorption separation unit and the pure water inlet of the glycoside conversion reactor via a pure water reuse pipeline. The backwash drain outlet of the downflow aerated biological filter unit and the bottom sludge discharge outlet of the methanogenic phase reactor are respectively connected to a total sludge return pipeline via pipelines with sludge discharge valves. A sludge return pump is installed on the total sludge return pipeline. The end of the total sludge return pipeline is directly cut into the upper feed inlet of the acidogenic phase reactor, forming a completely closed sludge secondary digestion loop.

[0015] This application provides a green preparation method and waste liquid recovery device for glucosyl steviol glycosides. The method involves sequentially separating the crude waste liquid generated after separating the transglycosylation solution through macroporous adsorption resin using ultrafiltration, nanofiltration, and reverse osmosis membrane separation. This allows for the fractional retention and recovery of free enzyme proteins, bacterial fragments, disaccharides and higher oligosaccharides, monosaccharides, and purified water, thereby achieving the targeted enrichment of biocatalysts and carbon sources in the waste liquid. The recovered oligosaccharide and monosaccharide mixture is preheated to 35-40℃ and an elemental promoter containing nickel, cobalt, and iron salts is added to supplement the coenzyme factors required for anaerobic microbial metabolism, significantly enhancing the activity of the microbial community. Utilizing a two-phase anaerobic reaction system, the process first... The acid phase converts sugars into volatile fatty acids and specifically hydrolyzes and breaks the glycosidic bonds of trace amounts of steviol glycosides. Subsequently, in the methanogenic phase, small-molecule organic acids are converted into biogas, thus avoiding the inhibition of subsequent treatment by recalcitrant derivatives and achieving energy recovery. Building upon this, a downflow aerated biofilter further degrades recalcitrant organic matter and residual pollutants, achieving deep purification of the effluent. Finally, through precise sludge-water separation and a full-process reflux mechanism, backwash wastewater and residual sludge are returned to the acidogenic phase for secondary hydrolysis and digestion, and purified water is reused in the process stages. This effectively solves the problems of complex wastewater that is difficult to treat for resource recovery and the direct discharge of high-concentration organic wastewater. This scheme constructs a complete closed loop from product separation to wastewater classification, resource recovery, energy conversion, and deep purification, significantly improving raw material utilization, ensuring the stability and reliability of effluent quality, and achieving the goals of green production and zero emissions.

[0016] This application, through integrated process design and equipment configuration, forms a logically rigorous and stable technical system. It not only achieves high-value recycling of by-products and complete decomposition of waste, but also achieves the unity of material recycling and energy cascade utilization at the system level, which has significant industrial application value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 This is a flowchart of a green preparation method for glucosylstevioside according to the present invention.

[0019] Figure 2 This is a structural diagram of the waste liquid recovery device for the green preparation of glucosylsteviosides according to the present invention.

[0020] Figure 3 This is a cross-sectional structural diagram of the methane-producing reactor of the present invention.

[0021] In the diagram: Reactor 101, Resin Adsorption Separation Unit 102, Eluent Collection Tank 103, Coarse Waste Liquid Collection Tank 104, Ultrafiltration Membrane Module 105, Nanofiltration Membrane Module 106, Reverse Osmosis Membrane Module 107, Enzyme Protein Recovery Tank 108, Oligosaccharide Recovery Tank 109, Monosaccharide Recovery Tank 110, Equalization Tank 111, Heating Device 112, Stirring Device 113, Element Dosing Device 114, Acid-Generating Phase Reactor 115, Methanogenic Phase Reactor 116, Biogas Collection Pipe 117, Downflow Aerated Biological Filter 118, Aeration Pipeline 119, Sludge-Water Separator 120, Online Water Quality Monitor 121, Booster Pump 122, Flow Control Valve 123, Water Distributor 124, Anaerobic Sludge Bed Zone 125, Suspended Sludge Zone 126. Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0023] First embodiment: This invention provides a green preparation method for glucosylsteviosides. The overall technical solution is as described in the embodiments of this application, mainly including the following core technical elements: a target product extraction process coupled with enzymatic glycoside conversion reaction and macroporous adsorption resin separation; a waste liquid component fractionation and recovery strategy based on a three-stage membrane system of ultrafiltration-nanofiltration-reverse osmosis; a two-phase anaerobic bioconversion system with added nickel, cobalt, and iron element promoters; and a deep purification and closed-loop material reuse mechanism in a downflow aerated biofilter. These technical elements work together to constitute the overall technical solution of this invention.

[0024] Please see Figure 1 The first aspect of this invention provides a green preparation method for glucosylstevioside, comprising: S101 uses steviol glycosides and glycosyl donors as raw materials to carry out a transglycosylation reaction under enzyme catalysis to obtain a transglycosylation solution containing glucosyl steviol glycosides; The specific steps of the transglycosylation reaction are as follows: steviol glycosides and glycosyl donors are mixed at a mass ratio of 1:2 to 1:4 and added to pure water to prepare a reaction solution with a substrate mass concentration of 10%-20%; cyclodextrin glucosyltransferase is added to the reaction solution at an enzyme activity of 200-500 activity units per gram of steviol glycosides; the reaction is carried out at a constant temperature of 45-55℃ and a pH of 5.5-6.5, with stirring at 100-200 rpm for 12-24 hours to obtain a transglycosylation solution containing glucosyl steviol glycosides; the glycosyl donor is selected from one or more of starch hydrolysate, maltose, or maltotriose.

[0025] By strictly controlling the mass ratio of steviol glycosides to glycosyl donors and the substrate concentration, sufficient reaction driving force was ensured while avoiding excessive system viscosity that could affect mass transfer efficiency. Cyclodextrin glucosyltransferase was selected and reacted under mild conditions of 45-55℃ and pH 5.5-6.5, which maintained the enzyme's high activity and stability while reducing the formation of byproducts. This specific combination of parameters significantly improved the transglycosylation reaction rate and the yield of glucosyl steviol glycosides, reducing the amount of unreacted raw materials at the source, thereby reducing the organic load on the subsequent waste liquid treatment unit and laying the foundation for the economy and cleanliness of the entire green preparation system.

[0026] S102 separates the transglycosylation solution through a macroporous adsorption resin. The target product, glucosylstevioside, is adsorbed by the resin. The resin permeate, rich in unreacted sugars, buffer salts, free enzyme proteins, and bacterial cell fragments, is collected as crude waste liquid. Subsequently, the resin is eluted with an aqueous ethanol solution, and the eluent containing the target product is collected. The transglycosylation solution is first passed through an adsorption column packed with macroporous adsorption resin at a flow rate of 1-2 BV / h. The target product, glucosylstevioside, is adsorbed by the resin. The liquid rich in unreacted sugars, salts, and free enzyme proteins is discharged as resin permeate and collected as crude waste liquid. After adsorption saturation, the resin column is forward-washed or back-washed with pure water to remove residual sugars and impurities in the resin interstices. The washing water is added to the crude waste liquid. Subsequently, the resin is eluted with an aqueous ethanol solution with a volume concentration of 50%-70% at a flow rate of 0.5-1 BV / h. The ethanol eluent containing glucosylstevioside is collected and then concentrated and de-ethanolified to obtain the target product. In this invention, by controlling the adsorption flow rate at 1-2 BV / h, sufficient contact time between the transglycosylation solution and resin particles is ensured, maximizing the utilization of the resin's adsorption capacity and reducing the breakthrough loss of the target product. The pure water washing step after adsorption saturation effectively removes sugars and salts trapped in the resin gaps, preventing impurities from entering the eluent and affecting product purity. At the same time, the washing water is incorporated into the crude waste liquid to maintain material balance. Using a 50%-70% ethanol aqueous solution and elution at a low flow rate of 0.5-1 BV / h, the target product is gently desorbed by changing the polarity. This yields a high-purity glucosylstevioside eluent while avoiding severe swelling damage to the resin skeleton, providing a stable input source for the subsequent homogenization treatment of the waste liquid.

[0027] S103 sequentially separates crude waste liquid through ultrafiltration, nanofiltration, and reverse osmosis membranes. Ultrafiltration retains and recovers free enzyme proteins and bacterial fragments, nanofiltration retains and recovers disaccharides and oligosaccharides, and reverse osmosis retains and recovers monosaccharides. Ultrafiltration uses organic or ceramic membranes with a molecular weight cutoff of 5,000-50,000 Daltons, operating at a pressure of 0.2-0.6 MPa. The ultrafiltration concentrate is recovered by settling or centrifugation to remove free enzyme proteins and bacterial fragments. Nanofiltration uses nanofiltration membranes with a molecular weight cutoff of 150-300 Daltons, operating at a pressure of 1.0-3.0 MPa. The nanofiltration concentrate is mainly enriched with disaccharides, trisaccharides, and oligosaccharides, and is stored as a carbon source for the methanogenic phase after the total sugar concentration reaches 8%-15%. Reverse osmosis uses high-flux, fouling-resistant reverse osmosis membranes, operating at a pressure of 1.5-4.0 MPa. The reverse osmosis concentrate is enriched with monosaccharides and monovalent salts. The liquid that permeates through the reverse osmosis membrane is purified water, with its chemical oxygen demand reduced to 80-150 mg / L and its conductivity reduced to 500-1500 μS / cm. In this invention, by setting the ultrafiltration molecular weight cutoff to 5000-50000 Daltons, large molecules such as free enzyme proteins and bacterial fragments are effectively intercepted, preventing them from clogging the downstream nanofiltration and reverse osmosis membrane channels, while simultaneously enabling the recovery and reuse of biocatalysts. A nanofiltration membrane with a molecular weight cutoff of 150-300 Daltons is used to precisely retain oligosaccharides such as maltose, enriching them to a high concentration of 8%-15% as a high-quality carbon source for the anaerobic system, thus avoiding carbon source loss. Combined with a high-flux, anti-fouling reverse osmosis membrane operating at 1.5-4.0 MPa pressure, not only is low-COD and low-conductivity process water obtained, but also the effective retention of monosaccharides is ensured. The optimized matching of operating pressures at each membrane stage balances flux and energy consumption, guaranteeing the long-term stable operation of the membrane system in complex wastewater environments.

[0028] The crude waste liquid is first pumped into an ultrafiltration membrane separation system, where it undergoes cross-flow filtration using a polyvinylidene fluoride or polyethersulfone ultrafiltration membrane. The ultrafiltration retentate is concentrated and recovered to obtain a mixture of free enzyme protein and bacterial cell fragments. The ultrafiltration permeate then enters the nanofiltration stage. The ultrafiltration permeate is then pumped into a nanofiltration membrane separation system, where it is separated using a nanofiltration membrane. The nanofiltration retentate is concentrated and recovered to obtain a solution of disaccharides and higher oligosaccharides rich in maltose and maltotriose. The nanofiltration permeate then enters the reverse osmosis stage. The nanofiltration permeate is then pumped into a reverse osmosis membrane separation system, where it is separated using a reverse osmosis composite membrane. The reverse osmosis retentate is concentrated and recovered to obtain a monosaccharide solution whose main component is glucose. The liquid that permeates through the reverse osmosis membrane is purified water. In this invention, by using ultrafiltration membranes made of polyvinylidene fluoride or polyethersulfone and performing cross-flow filtration, the shear force is used to remove deposits from the membrane surface, significantly alleviating the membrane fouling problem of waste liquid containing bacterial fragments and extending the operating cycle. The ultrafiltration, nanofiltration and reverse osmosis three-stage membrane modules are connected in series in descending order of molecular weight to construct a clear material flow path, which enables the stepwise separation and enrichment of large molecular weight enzymes, medium molecular weight oligosaccharides and small molecular weight monosaccharides. This continuous membrane separation process avoids the risk of material deterioration caused by intermediate storage tanks, and the purified water produced by reverse osmosis can be directly reused in the front end of the process, greatly reducing the consumption of fresh water and improving the resource utilization rate of the entire preparation process.

[0029] S104 mixes the oligosaccharides retained by nanofiltration with the monosaccharides retained by reverse osmosis and introduces them into the conditioning tank, preheating them to 35-40°C. At the same time, elemental promoters are added, including nickel, cobalt and iron salts, to supplement the coenzyme factors required for anaerobic microorganisms to degrade sugars and steviol glycoside derivatives. The mixture of nanofiltration and reverse osmosis retentate is introduced into the equalization tank. The wastewater temperature is precisely raised and maintained at 35-40℃ through internal and external tubular heat exchangers. A mechanical agitator is installed in the equalization tank at a speed of 60-80 rpm for homogenization. At the same time, an elemental promoter is continuously or intermittently added to the equalization tank through a metering pump. The elemental promoter is an aqueous solution prepared by nickel chloride, cobalt chloride, and ferrous sulfate in a mass ratio of nickel chloride:cobalt chloride:ferrous sulfate of 1:1.5:2.5. The dosage is controlled according to the ratio of 0.05-0.15 micrograms of nickel ions, 0.075-0.225 micrograms of cobalt ions, and 0.125-0.375 micrograms of ferrous ions per milligram of chemical oxygen demand in the wastewater. In this invention, by preheating the mixed sugar solution to the optimal mesophilic anaerobic range of 35-40℃, the metabolic enzyme activity of acid-producing and methanogenic bacteria is significantly activated. Mechanical stirring at 60-80 rpm ensures uniform mixing of the sugar solution and trace elements, preventing excessively high local concentrations that could cause toxicity, and also avoids damage to the anaerobic sludge floc structure. In particular, the addition of nickel, cobalt, and iron element promoters in a specific ratio (1:1.5:2.5) specifically supplements the key metal cofactors required for the synthesis of hydrogenase, coenzyme F430, and vitamin B12, greatly enhancing the hydrolytic capacity and tolerance of the anaerobic bacteria to recalcitrant steviol glycoside derivatives. The dynamic addition strategy based on COD enables precise control of nutrient supply, significantly improving the start-up speed and operational stability of the anaerobic system.

[0030] S105 feeds the preheated mixed sugar solution into a two-phase anaerobic reaction system. First, it enters the acid-producing phase reactor, where, under a residence time of 12-24 hours, acid-producing bacteria convert the sugars into volatile fatty acids and specifically hydrolyze and break the glycosidic bonds of trace steviol glycosides, converting them into small-molecule organic acids. Then, it enters the methanogenic phase reactor, where the small-molecule organic acids are converted into biogas. The effluent from the S106 anaerobic treatment process passes through a downflow aerated biological filter, and then the residual organic matter is further degraded by the aerobic biofilm attached to the surface of the filter media. S107 performs precise mud-water separation on the effluent after aerobic treatment. The separated supernatant is discharged after water quality monitoring and meets the standards or is reused as cleaning water. The liquid that passes through the reverse osmosis membrane is directly reused as purified water in the resin eluent preparation or glycoside conversion reaction section. The backwash wastewater generated by the aerated biological filter and the excess sludge generated by the anaerobic system are all returned to the acid-producing phase reactor for secondary hydrolysis and digestion.

[0031] In this invention, an integrated pathway of "product separation - waste liquid classification - resource recovery - energy conversion - deep purification - closed-loop reuse" is constructed. Multi-level membrane gradient retention is used to achieve the targeted separation and recovery of enzyme proteins, oligosaccharides, monosaccharides and purified water. This solves the problem of high-concentration organic waste liquid being difficult to recover as a resource and directly polluting the environment when discharged directly in traditional processes. In particular, by adding elemental promoters containing nickel, cobalt and iron and adopting a two-phase anaerobic system with division of labor and cooperation, the acid-producing phase specifically hydrolyzes the recalcitrant steviol glycoside derivatives into small molecule organic acids, avoiding their inhibition of methanogenic bacteria. This significantly improves biogas production and system stability, and achieves near-zero wastewater discharge and high-value utilization of by-products.

[0032] Second Embodiment Please see Figures 2-3 This invention provides a green wastewater recovery device for the preparation of glucosylstevioside, comprising: a transglycosylation reaction unit, a resin adsorption separation unit 102, a membrane separation and recovery unit, a preheating and conditioning unit, a two-phase anaerobic treatment unit, a downflow aerated biological filter unit, and a sludge-water separation and reuse unit, all connected sequentially by pipes; the transglycosylation reaction unit includes a transglycosylation reactor 101 with a temperature control jacket and a stirrer; the resin adsorption separation unit 102 includes a macroporous adsorption resin column, and an eluent collection tank 103 and a crude wastewater collection tank 104 connected to the bottom outlet of the resin column; the membrane separation and recovery unit includes an ultrafiltration membrane module 105, a nanofiltration membrane module 106, and a reverse osmosis membrane module 107 connected sequentially by pipes, each membrane module having an inlet, a retentate outlet, and a permeate outlet; the retentate outlet of the ultrafiltration membrane module 105 is connected to an enzyme protein recovery tank 108, the retentate outlet of the nanofiltration membrane module 106 is connected to an oligosaccharide recovery tank 109, and the reverse osmosis membrane module... The retentate outlet of component 107 is connected to the monosaccharide recovery tank 110, and the permeate outlet of reverse osmosis membrane module 107 is connected to the pure water reuse pipeline network; the outlets of oligosaccharide recovery tank 109 and monosaccharide recovery tank 110 merge and are connected to the inlet of the regulating and preheating unit; the regulating and preheating unit includes a regulating tank 111, which is equipped with a heating device 112, a stirring device 113, and an element dosing device 114; the outlet of regulating tank 111 is connected to the two-phase anaerobic treatment unit; the two-phase anaerobic... The treatment unit includes an acid-producing phase reactor 115 and a methanogenic phase reactor 116 connected in sequence. The top of the methanogenic phase reactor 116 is equipped with a biogas collection pipe 117. The downflow aerated biological filter unit includes a downflow aerated biological filter 118, with an aeration pipe 119 at the bottom. The sludge-water separation and reuse unit includes a sludge-water separator 120, an online water quality monitor 121, and a sludge return pipeline. The end of the sludge return pipeline is connected to the inlet of the acid-producing phase reactor 115.

[0033] By connecting units such as glycoside transoxidation, resin separation, membrane fractionation and recovery, anaerobic fermentation and deep oxidation sequentially through pipelines to form an integrated production line, the entire life cycle management from raw material input to waste disposal is realized. Each unit has a clear function and is closely connected. For example, enzyme proteins, oligosaccharides and monosaccharides are collected through dedicated recovery tanks, and purified water is directly reused through the pipeline network, thus constructing a dual circulation system of matter and energy. In particular, the connection design between the sludge return pipeline and the acid-producing phase reactor 115 ensures that backwash wastewater and residual sludge can be returned to the front end of the system for secondary hydrolysis, completely eliminating the discharge of solid waste. The device has a compact structure and a high degree of automation, effectively solving the problem of low treatment efficiency caused by the dispersion and low integration of existing equipment.

[0034] Furthermore, booster pumps 122 are installed between the ultrafiltration membrane module 105 and the nanofiltration membrane module 106, and between the nanofiltration membrane module 106 and the reverse osmosis membrane module 107 in the membrane separation and recovery unit; flow regulating valves 123 are respectively installed on the outlet pipes of the oligosaccharide recovery tank 109 and the monosaccharide recovery tank 110, and the two high-concentration sugar solutions merge on the pipes before entering the equalization tank 111. In this invention, by adding booster pumps 122 between each stage of membrane modules, the attenuation of the effluent pressure of the upstream membrane is compensated, ensuring that the nanofiltration and reverse osmosis membrane modules can operate stably within the set high pressure range, maintaining a high desalination rate and retention efficiency, and overcoming the problem of flux reduction caused by insufficient pressure; at the same time, flow regulating valves 123 are installed at the outlets of the oligosaccharide and monosaccharide recovery tanks, which can flexibly adjust the mixing ratio and feed rate of different concentration sugar solutions according to the real-time load of the anaerobic system, avoiding the inhibition of anaerobic bacteria by shock loads, and realizing precise control of carbon source supply and flexible adjustment of system operation.

[0035] Furthermore, the acid-producing phase reactor 115 is a completely mixed anaerobic reactor, equipped with a mechanical agitator, an online pH detection probe, and a temperature sensor, and has an exhaust pipe at the top; the methanogenic phase reactor 116 is an internal circulation anaerobic reactor, with a water distributor 124, an anaerobic sludge bed zone 125, a suspended sludge zone 126, and a three-phase separator arranged from bottom to top inside. In this invention, the acid-producing phase adopts a completely mixed design combined with mechanical stirring, achieving uniform mass transfer of materials within the reactor, eliminating dead zones, and facilitating the rapid conversion of complex sugars into volatile fatty acids by acid-producing bacteria; the methanogenic phase adopts an internal circulation structure, utilizing the rising water flow to drive sludge recirculation, enhancing the contact efficiency between microorganisms and substrates, and achieving efficient separation of gas, liquid, and solid phases with the three-phase separator, preventing sludge loss; the built-in online pH and temperature detection probes support real-time feedback control, maintaining the optimal metabolic environment. This differentiated reactor structure design significantly improves the adaptability of the two-phase anaerobic system to fluctuating influent and its overall treatment efficiency.

[0036] Furthermore, the sludge-water separator 120 is a high-efficiency sedimentation tank. The supernatant outlet of the sludge-water separator 120 is connected to the online water quality monitoring instrument 121 through a pipeline. The online water quality monitoring instrument 121 has a conductivity detection probe and a TOC detection probe. The qualified effluent is discharged or connected to the cleaning water reuse pipeline. The permeate outlet of the reverse osmosis membrane module 107 is connected to the inlet of the eluent preparation tank of the macroporous adsorption resin column in the resin adsorption separation unit 102 and the pure water inlet of the glycoside conversion reactor 101 through a pure water reuse pipeline. The backwash drain of the downflow aerated biological filter unit 118 and the bottom sludge discharge port of the methanogenic phase reactor 116 are respectively connected to a total sludge return pipeline through pipelines with sludge discharge valves. A sludge return pump is installed on the total sludge return pipeline. The end of the total sludge return pipeline is directly cut into the upper feed port of the acidogenic phase reactor 115, forming a completely closed sludge secondary digestion loop.

[0037] By linking the high-efficiency sedimentation tank with the online water quality monitoring instrument 121, which has conductivity and TOC detection functions, real-time judgment and diversion control of effluent quality are realized, ensuring the safety of recycled water. The reverse osmosis permeate is directly recycled for eluent preparation and glycoside conversion reaction, forming an internal closed loop of water resources and greatly saving fresh water consumption. In particular, the backwash drainage and residual sludge are combined through the main pipeline and pumped back to the acid-producing reactor 115, constructing a closed sludge secondary digestion loop, ensuring that all solid waste containing organic matter is hydrolyzed and digested within the system.

[0038] Unless otherwise specified, all materials, reagents and instruments used in the embodiments of this invention can be obtained through commercial channels.

[0039] Key raw materials include: steviol glycosides (purity ≥95%), maltose (food grade), starch hydrolysate (DE value 10-15), nickel chloride (analytical grade), cobalt chloride (analytical grade), ferrous sulfate (analytical grade), and anhydrous ethanol (chromatographic grade).

[0040] The biological materials include: cyclodextrin glucosyltransferase (CGTase, activity ≥5000 U / g), acid-producing anaerobic sludge (taken from the anaerobic digester of an urban wastewater treatment plant), and methanogenic anaerobic granular sludge (taken from a UASB reactor for brewing wastewater).

[0041] Characterization and testing methods included: the content of glucosylsteviosides was determined by high performance liquid chromatography (HPLC); chemical oxygen demand (COD) was determined by the potassium dichromate method; conductivity was determined by a conductivity meter; total organic carbon (TOC) was determined by a TOC analyzer; biogas production was determined by a wet gas flow meter; and volatile fatty acids (VFA) were determined by gas chromatography. Example 1:

[0042] This embodiment provides an optimal process for the green preparation of glucosylstevioside and the resource utilization treatment of waste liquid.

[0043] 1. Glycoside transfection: Stevioside and maltose were mixed at a mass ratio of 1:3, and purified water was added to prepare a reaction solution with a substrate mass concentration of 15%. Cyclodextrin glucosyltransferase was added to the reaction solution at a dosage of 350 activity units per gram of steviol glycoside. The reaction was carried out at a constant temperature of 50℃ and a pH of 6.0 with stirring at 150 rpm for 18 hours to obtain a glycoside transfection solution containing glucosyl steviol glycoside.

[0044] 2. Resin Separation: The transglycosylation solution was passed through an adsorption column packed with D101 macroporous adsorption resin at a flow rate of 1.5 BV / h. The target product was adsorbed, and the resin permeate was collected as crude waste liquid. After adsorption saturation, the resin column was backwashed with pure water, and the wash water was added to the crude waste liquid. Subsequently, the resin was eluted with a 60% (v / v) ethanol aqueous solution at a flow rate of 0.8 BV / h. The ethanol eluent containing glucosylstevioside was collected, concentrated, and then dealcoholized to obtain the target product. The product yield was measured to be 88.5%.

[0045] 3. Membrane Separation and Recovery: The crude waste liquid is pumped into an ultrafiltration membrane separation system, where cross-flow filtration is performed using a PVDF ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons at an operating pressure of 0.4 MPa. The ultrafiltration retentate is recovered to obtain a mixture of free enzyme proteins and bacterial cell fragments. The ultrafiltration permeate enters the nanofiltration stage, where separation is performed using a nanofiltration membrane with a molecular weight cutoff of 200 Daltons at an operating pressure of 2.0 MPa. The nanofiltration retentate is concentrated to a total sugar concentration of 12% before storage. The nanofiltration permeate enters the reverse osmosis stage, where separation is performed using an antifouling reverse osmosis membrane at an operating pressure of 2.5 MPa. The reverse osmosis retentate is enriched with monosaccharides, and the permeate is purified water (COD 95 mg / L, conductivity 800 μS / cm).

[0046] 4. Anaerobic Treatment Preparation: The nanofiltration retentate and reverse osmosis retentate are mixed at a volume ratio of 1:1 and introduced into the equalization tank, then heated to 38°C using a shell-and-tube heat exchanger. A mechanical stirrer is turned on and homogenized at 70 rpm. An elemental promoter (nickel chloride:cobalt chloride:ferrous sulfate = 1:1.5:2.5) is added via a metering pump, with the dosage controlled at 0.1 μg nickel ions, 0.15 μg cobalt ions, and 0.25 μg ferrous ions per milligram of COD.

[0047] 5. Two-phase anaerobic fermentation: The pretreated mixed sugar solution is fed into a two-phase anaerobic reaction system. First, it enters the acid-producing phase reactor (completely mixed type) for 18 hours, where it is converted into volatile fatty acids and trace amounts of steviol glycoside derivatives are hydrolyzed. Then, it enters the methanogenic phase reactor (internal circulation type) to convert small-molecule organic acids into biogas.

[0048] 6. Deep Purification and Reuse: Anaerobic effluent passes through a downflow aerated biological filter. The effluent then enters a high-efficiency sedimentation tank for sludge-water separation. The supernatant, after water quality monitoring (COD < 50 mg / L, TOC < 20 mg / L), is reused as cleaning water. Reverse osmosis permeate is reused for resin eluent preparation. Backwash wastewater and excess sludge are returned to the acidogenic reactor.

[0049] The purity of the glucosylstevioside obtained in this embodiment reached 96.2%. After waste liquid treatment, the biogas yield reached 0.45m³. 3 / kgCOD, the effluent quality is stable and meets the standards, and the enzyme and protein recovery rate is 92% and the sugar resource recovery rate is over 85%.

[0050] This embodiment successfully achieved efficient preparation of the target product and comprehensive resource utilization of waste liquid through optimized combination of process parameters, verifying the feasibility and superiority of the technical solution. Example 2:

[0051] This embodiment aims to verify the effect of the lower limit of the mass ratio of steviol glycoside to glycosyl donor on the technical effect during the transglycosylation reaction.

[0052] With all other preparation conditions the same as in Example 1, only the mass ratio of the defined steviol glycoside to the glycosyl donor was adjusted from 1:3 to 1:2 to obtain a transglycosylation solution containing glucosyl steviol glycoside, and then the separation and waste liquid treatment were carried out according to the steps of Example 1.

[0053] The results showed that the product yield under these conditions was 82.1%, slightly lower than in Example 1, but the concentration of unreacted sugars in the waste liquid was reduced. The subsequent anaerobic system operated stably, and the biogas production rate remained at 0.42 m³ / s. 3 Even at / kgCOD, the technical effects of this invention can still be achieved.

[0054] This embodiment demonstrates that the technical solution of the present invention has good feasibility under the condition of a mass ratio lower limit of 1:2. Example 3:

[0055] This embodiment aims to verify the effect of the upper limit of the mass ratio of steviol glycoside to glycosyl donor on the technical effect during the transglycosylation reaction.

[0056] With all other preparation conditions the same as in Example 1, only the mass ratio of the defined steviol glycoside to the glycosyl donor was adjusted from 1:3 to 1:4 to obtain a transglycosylation solution containing glucosyl steviol glycoside, and then the separation and waste liquid treatment were carried out according to the steps of Example 1.

[0057] The results showed that under these conditions, the product yield was 86.8%, and the concentration of unreacted sugars in the waste liquid was high. After membrane concentration, these sugars were used as a carbon source in the anaerobic system, increasing the biogas yield to 0.48 m³.3 / kgCOD, the system is operating normally.

[0058] This embodiment demonstrates that, under the condition of a mass ratio of up to 1:4, the technical solution of the present invention can effectively treat high-concentration organic waste liquid and achieve energy recovery.

[0059] Scale settings: Comparative Example 1: Blank Control Group. Glucosylsteviosides were prepared using conventional enzymatic transglycosylation, but without subsequent membrane separation, fractionation, and two-phase anaerobic treatment. The crude waste liquid from the transglycosylation solution, after resin separation, was directly treated and discharged using the aerobic activated sludge process.

[0060] Comparative Example 2: Group with missing element accelerators. The same process flow as in Example 1 was used, but nickel, cobalt, and iron element accelerators were not added during the conditioning tank stage; only heating and stirring were performed.

[0061] Comparative Example 3: Single-phase anaerobic treatment group. The same process flow as in Example 1 was used, but the two-phase anaerobic reaction system was replaced with a single upflow anaerobic sludge blanket (UASB) reactor, without separating the acid-producing phase and the methanogenic phase.

[0062] Comparative Example 4: Control group of commercially available products. The production process of glucosyl steviol glycosides (acid-base method or ordinary enzymatic method) commonly used in the market was adopted, and the waste liquid was discharged after conventional coagulation sedimentation + aerobic treatment.

[0063] Test items: 1. Yield of glucosylstevioside (%); 2. COD (mg / L) of the effluent after wastewater treatment; 3. Biogas production rate (m³) 3 / kgCOD); 4. Enzyme protein recovery rate (%); 5. System operating energy consumption (kWh / m³) 3 Waste liquid).

[0064] The test results are shown in Table 1 below: Table 1 Results of the effect test Effect Analysis: As shown in Table 1, the yields of glucosylsteviosides obtained in Examples 1 to 3 were all above 82%, significantly higher than that of the commercially available process in Comparative Example 4. This indicates that the optimized transglycosylation reaction conditions of this invention effectively improved the product yield. Regarding wastewater treatment, the effluent COD of Example 1 was only 42 mg / L, far superior to the conventional aerobic treatment effects (COD > 180 mg / L) of Comparative Examples 1 and 4. This confirms the excellent degradation capability of the "membrane separation + two-phase anaerobic + deep oxidation" combined process of this invention for complex organic wastewater.

[0065] Through the synergistic combination of various technical features, this invention not only achieves high yield preparation of the target product, but also achieves near-zero discharge of waste liquid and resource recovery of energy.

[0066] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method of green production of glucosyl-steviol glycosides, Its features are, This includes: using steviol glycosides and glycosyl donors as raw materials, carrying out a transglycosylation reaction under enzyme catalysis to obtain a transglycosylation solution containing glucosyl steviol glycosides; The transglycosylation solution was separated by a macroporous adsorption resin. The target product, glucosylstevioside, was adsorbed by the resin. The resin permeate, rich in unreacted sugars, buffer salts, free enzyme proteins, and bacterial fragments, was collected as crude waste liquid. Subsequently, the resin was eluted with an aqueous ethanol solution, and the eluent containing the target product was collected. The crude waste liquid is subjected to ultrafiltration, nanofiltration and reverse osmosis membrane separation in sequence, wherein ultrafiltration retains and recovers free enzyme proteins and bacterial fragments, nanofiltration retains and recovers disaccharides and above oligosaccharides, and reverse osmosis retains and recovers monosaccharides. The oligosaccharides retained by nanofiltration and the monosaccharides retained by reverse osmosis are mixed and introduced into the conditioning tank and preheated to 35-40°C. At the same time, elemental promoters are added, which include nickel, cobalt and iron salts, to supplement the coenzyme factors required by anaerobic microorganisms to degrade sugars and steviol glycoside derivatives. The preheated mixed sugar solution is fed into a two-phase anaerobic reaction system. First, it enters the acid-producing phase reactor, where, under a residence time of 12-24 hours, acid-producing bacteria convert the sugars into volatile fatty acids and specifically hydrolyze and break the glycosidic bonds of trace amounts of steviol glycosides, converting them into small-molecule organic acids. Then, it enters the methanogenic phase reactor, where the small-molecule organic acids are converted into biogas. The effluent after anaerobic treatment is passed through a downflow aerated biological filter, and then the residual organic matter is further degraded by the aerobic biofilm attached to the surface of the filter media. The effluent from aerobic treatment undergoes precise mud-water separation. The separated supernatant is discharged after water quality monitoring to meet standards or reused as cleaning water. The liquid that permeates through the reverse osmosis membrane is directly reused as purified water in the preparation of resin eluent or in the glycoside conversion reaction section. The backwash wastewater generated by the aerated biological filter and the excess sludge generated by the anaerobic system are all returned to the acid-producing phase reactor for secondary hydrolysis and digestion.

2. The green preparation method of glucosylstevioside as described in claim 1, characterized in that, The specific steps for the transglycosylation reaction are as follows: Stevioside and glycosyl donor are mixed at a mass ratio of 1:2 to 1:4 and added to pure water to prepare a reaction solution with a substrate mass concentration of 10%-20%. Cyclodextrin glucosyltransferase is added to the reaction solution at an enzyme activity of 200-500 activity units per gram of steviol glycoside. The reaction is carried out at a constant temperature of 45-55℃ and a pH of 5.5-6.5, with stirring at 100-200 rpm for 12-24 hours to obtain the glycoside-containing solution containing glucosyl steviol glycoside. The glycosyl donor is selected from one or more of starch hydrolysate, maltose, or maltotriose.

3. The green preparation method of glucosylstevioside as described in claim 2, characterized in that, The ultrafiltration process uses an organic or ceramic membrane with a molecular weight cutoff of 5,000-50,000 Daltons and an operating pressure of 0.2-0.6 MPa. The ultrafiltration concentrate is then allowed to stand or centrifuged to recover free enzyme proteins and bacterial cell fragments. Nanofiltration uses nanofiltration membranes with a molecular weight cutoff of 150-300 Daltons and an operating pressure of 1.0-3.0 MPa. The nanofiltration concentrate is mainly enriched with disaccharides, trisaccharides and oligosaccharides. After the total sugar concentration reaches 8%-15%, it is stored as a carbon source for the methanogenic phase. The reverse osmosis uses a high-flux, fouling-resistant reverse osmosis membrane with an operating pressure of 1.5-4.0 MPa. The reverse osmosis concentrate is enriched with monosaccharides and monovalent salts, and the liquid that passes through the reverse osmosis membrane is purified water with a chemical oxygen demand reduced to 80-150 mg / L and conductivity reduced to 500-1500 μS / cm.

4. The green preparation method of glucosylstevioside as described in claim 3, characterized in that, The specific steps for separation using the macroporous adsorption resin are as follows: The transglycosylation solution is first passed through an adsorption column packed with macroporous adsorption resin at a flow rate of 1-2 BV / h. The target product, glucosylstevioside, is adsorbed by the resin. The liquid rich in unreacted sugars, salts, and free enzyme proteins is discharged as resin permeate and collected as crude waste liquid. After adsorption saturation, the resin column is washed with pure water in either forward or backwash to remove residual sugars and impurities in the resin gaps. The washing water is then added to the crude waste liquid. The resin was then eluted with an aqueous ethanol solution of 50%-70% (v / v) at a flow rate of 0.5-1 BV / h. The ethanol eluent containing glucosylstevioside was collected and subsequently concentrated and de-alcoholized to obtain the target product.

5. The green preparation method of glucosylstevioside as described in claim 4, characterized in that, The specific steps for implementing membrane separation are as follows: The crude waste liquid is first pumped into an ultrafiltration membrane separation system, and cross-flow filtration is performed using a polyvinylidene fluoride or polyethersulfone ultrafiltration membrane. The ultrafiltration retentate is concentrated and then recovered to obtain a mixture of free enzyme protein and bacterial cell fragments. The ultrafiltration permeate enters the nanofiltration stage. The ultrafiltration permeate is pumped into a nanofiltration membrane separation system, where nanofiltration membranes are used for separation. The nanofiltration retentate is concentrated and then recovered to obtain a solution of disaccharides and oligosaccharides rich in maltose and maltotriose. The nanofiltration permeate then enters the reverse osmosis stage. The nanofiltration permeate is pumped into a reverse osmosis membrane separation system, where a reverse osmosis composite membrane is used for separation. The reverse osmosis retentate is concentrated and recovered to obtain a monosaccharide solution whose main component is glucose. The liquid that permeates through the reverse osmosis membrane is the purified water.

6. The green preparation method of glucosylstevioside as described in claim 5, characterized in that, The specific steps for preheating and element addition are as follows: The mixture of nanofiltration retentate and reverse osmosis retentate is introduced into the equalization tank. The wastewater temperature is precisely increased and maintained at 35-40℃ through the internal and external tubular heat exchangers of the tank. A mechanical stirring paddle is installed in the equalization tank to homogenize the mixture at a speed of 60-80 rpm. At the same time, an element promoter is continuously or intermittently added to the equalization tank through a metering pump. The element promoter is an aqueous solution prepared by nickel chloride, cobalt chloride, and ferrous sulfate in a mass ratio of nickel chloride:cobalt chloride:ferrous sulfate equal to 1:1.5:2.

5. The dosage is controlled according to the ratio of 0.05-0.15 micrograms of nickel ions, 0.075-0.225 micrograms of cobalt ions, and 0.125-0.375 micrograms of ferrous ions per milligram of chemical oxygen demand in the wastewater.

7. A green waste liquid recovery device for the preparation of glucosylsteviosides, characterized in that, The method for green preparation of glucosylstevioside according to any one of claims 1-6 comprises: The system consists of a transglycosylation reaction unit, a resin adsorption and separation unit, a membrane separation and recovery unit, a conditioning and preheating unit, a two-phase anaerobic treatment unit, a downflow aerated biological filter unit, and a sludge-water separation and reuse unit, all connected sequentially by pipelines. The transglycosylation reaction unit includes a transglycosylation reaction vessel equipped with a temperature control jacket and a stirrer; The resin adsorption separation unit includes a macroporous adsorption resin column, and an eluent collection tank and a crude waste liquid collection tank connected to the water outlet at the bottom of the resin column. The membrane separation and recovery unit includes an ultrafiltration membrane module, a nanofiltration membrane module, and a reverse osmosis membrane module connected sequentially by pipelines. Each membrane module is equipped with an inlet, a retentate outlet, and a permeate outlet. The retentate outlet of the ultrafiltration membrane module is connected to an enzyme protein recovery tank, the retentate outlet of the nanofiltration membrane module is connected to an oligosaccharide recovery tank, the retentate outlet of the reverse osmosis membrane module is connected to a monosaccharide recovery tank, and the permeate outlet of the reverse osmosis membrane module is connected to a pure water reuse pipeline network. The outlets of the oligosaccharide recovery tank and the monosaccharide recovery tank merge and are then connected to the inlet of the regulating preheating unit; The regulating and preheating unit includes an regulating tank, which is equipped with a heating device, a stirring device and an element dosing device. The outlet of the regulating tank is connected to the two-phase anaerobic treatment unit. The two-phase anaerobic treatment unit includes an acid-producing phase reactor and a methanogenic phase reactor connected in sequence, with a biogas collection pipe at the top of the methanogenic phase reactor. The downflow aerated biological filter unit includes a downflow aerated biological filter with an aeration pipe at the bottom; The sludge-water separation and reuse unit includes a sludge-water separator, an online water quality monitoring instrument, and a sludge return pipeline. The end of the sludge return pipeline is connected to the inlet of the acid-producing reactor.

8. The waste liquid recovery device for the green preparation of glucosylstevioside as described in claim 7, characterized in that, In the membrane separation and recovery unit, booster pumps are provided between the ultrafiltration membrane module and the nanofiltration membrane module, and between the nanofiltration membrane module and the reverse osmosis membrane module; The outlet pipes of the oligosaccharide recovery tank and the monosaccharide recovery tank are respectively equipped with flow regulating valves, and the two high-concentration sugar solutions merge on the pipes before entering the regulating tank.

9. The waste liquid recovery device for the green preparation of glucosylstevioside as described in claim 8, characterized in that, The acid-producing reactor is a completely mixed anaerobic reactor, equipped with a mechanical stirring paddle, an online pH detection probe, and a temperature sensor, and an exhaust pipe at the top. The methanogenic phase reactor is an internal circulation anaerobic reactor, which consists of a water distributor, an anaerobic sludge bed zone, a suspended sludge zone, and a three-phase separator, arranged from bottom to top.

10. The waste liquid recovery device for the green preparation of glucosylstevioside as described in claim 9, characterized in that, The mud-water separator is a high-efficiency sedimentation tank. The supernatant outlet of the mud-water separator is connected to an online water quality monitoring instrument through a pipeline. The online water quality monitoring instrument has a conductivity detection probe and a TOC detection probe. The qualified effluent is discharged or connected to the cleaning water reuse pipeline. The permeate outlet of the reverse osmosis membrane module is connected to the inlet of the eluent preparation tank of the macroporous adsorption resin column in the resin adsorption separation unit and the pure water inlet of the transglycosylation reactor via a pure water recycling pipeline. The backwash outlet of the downflow aerated biological filter unit and the bottom sludge outlet of the methanogenic phase reactor are respectively connected to a total sludge return pipeline through pipes equipped with sludge discharge valves. A sludge return pump is installed on the total sludge return pipeline, and the end of the total sludge return pipeline is directly cut into the upper feed inlet of the acidogenic phase reactor, forming a completely closed sludge secondary digestion loop.

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