Preparation process of high-purity fructo-oligosaccharide

Abstract

The invention belongs to the technical field of fructo-oligosaccharide preparation, and relates to a preparation process of high-purity fructo-oligosaccharide, which comprises the following steps: (1) juicing fresh sugarcane to obtain sugarcane juice, and sequentially carrying out activated carbon decoloration, ultrafiltration degumming, high-temperature enzyme deactivation, electrodialysis desalination and enzymolysis reaction on the sugarcane juice to obtain an enzymolysis product; (2) performing membrane separation and purification on the enzymolysis product, and collecting trapped fluid; adsorbing fructo-oligosaccharide in the trapped fluid by using calcium type gel chromatographic resin, then eluting by using deionized water, and collecting eluent; and (3) carrying out double-effect falling film evaporation concentration on the collected eluent to obtain a concentrated solution, carrying out spray drying on the concentrated solution, and screening to obtain a product rich in fructo-oligosaccharide. Through organic combination of physical and biological technologies, various impurities such as pigments, colloids, salts and monosaccharides are removed, meanwhile, target fructo-oligosaccharide is accurately enriched and stably converted, and green, efficient and large-scale preparation of high-purity (more than or equal to 95%) products is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fructooligosaccharide preparation and relates to a preparation process of high-purity fructooligosaccharide. BACKGROUND

[0002] Fructooligosaccharide, as an important functional prebiotic, has a wide application prospect and market value in the fields of food, health care products and medicine due to its multiple health benefits such as selectively promoting the proliferation of intestinal beneficial flora, regulating intestinal microecology, enhancing immunity and promoting mineral absorption. In particular, high-purity (usually referring to the content of fructooligosaccharide being more than 95%) products not only have stronger physiological activity, but also can effectively avoid the adverse effects of high-content impurities such as sucrose, glucose and fructose, such as the applicability limitation for people with diabetes or specific metabolic syndrome, and the processing and storage property problems such as reducing the hygroscopicity and thermal stability of the product, so the demand for high-purity fructooligosaccharide is increasing.

[0003] At present, the production of fructooligosaccharide in industry mainly depends on fructosyltransferase produced by microorganisms (such as Aspergillus niger) to catalyze the transfructosylation reaction of sucrose. However, the core difficulty of the traditional enzymatic process lies in the complexity of the product and the challenge of separation and purification. The reaction product is a complex mixture containing target fructooligosaccharide (mainly trisaccharide GF2 and tetrasaccharide GF3), unreacted substrate sucrose (GF), by-product glucose (G), fructose (F) and a small amount of higher degree of polymerization fructooligosaccharide (such as pentasaccharide GF4). Among them, glucose as an enzyme reaction inhibitor will significantly reduce the conversion efficiency, and its large amount of existence also greatly reduces the functional purity of target fructooligosaccharide in the final product, making it difficult to meet the high standards required for application.

[0004] Therefore, it is urgent to develop a new type, efficient, economical and suitable for large-scale industrialization of high-purity fructooligosaccharide preparation process, which can effectively overcome the problems of insufficient selectivity of enzymatic reaction, high impurity content, high subsequent purification cost, low efficiency and large yield loss in the prior art, to meet the increasing demand of the market for high-quality and healthy ingredients. The present application is a solution to this technical situation. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a preparation process of high-purity fructooligosaccharide, which realizes the green, efficient and large-scale preparation of high-purity (≥95%) product by organically combining physical and biological technologies to remove multiple impurities such as pigments, colloids, salts and monosaccharides, and accurately enrich and stably convert target fructooligosaccharide.

[0006] To achieve this purpose, the technical solutions adopted by the present application are as follows: The present application provides a preparation process of high-purity fructooligosaccharide, which comprises: (I) Fresh sugarcane juice is obtained, and the sugarcane juice is subjected to activated carbon decolorization, ultrafiltration to remove gelatin, high-temperature enzyme inactivation, electrodialysis desalination and enzymatic reaction in sequence to obtain an enzymatic product; (II) The enzymatic product is subjected to membrane separation and purification, and a cut-off liquid is collected; calcium type gel chromatography resin is used to adsorb oligofructose in the cut-off liquid, and then deionized water is used for elution, and an eluate is collected; (III) The collected eluate is subjected to double-effect falling film evaporation concentration to obtain a concentrated liquid, and the concentrated liquid is subjected to spray drying to obtain a product rich in oligofructose after screening.

[0007] The first stage is mainly used for oligofructose. The present application uses fresh sugarcane juice as raw material, removes pigments, phenolic substances and part of small molecule organic matters by activated carbon decolorization, reduces the interference of subsequent steps. Then, ultrafiltration is performed to remove gelatin, and a membrane system with a specific molecular weight cut-off (80-100 kDa) is used to effectively separate colloids, proteins and macromolecular suspensions, significantly reducing the turbidity of the system, creating a clean environment for subsequent membrane separation and chromatographic purification. High-temperature enzyme inactivation completely inactivates the endogenous enzymes in the sugarcane juice by short-time high-temperature treatment, avoiding interference with the subsequent exogenous enzyme catalytic reaction. Electrodialysis desalination deeply removes inorganic salt ions (such as potassium, sodium and chloride ions) by ion selective migration, reducing the conductivity to below 100 μS / cm, which not only eliminates the inhibition of salt on enzyme activity, but also lays a foundation for subsequent precise regulation of the calcium ion concentration of the enzyme hydrolysis system. The final enzymatic reaction is carried out in a low-salt, low-impurity environment, and β-fructofuranosidase efficiently catalyzes the conversion of sucrose to target oligofructose (DP3-DP5), and the enzyme is inactivated to terminate the reaction when the conversion rate reaches 95%, minimizing the generation of by-products glucose and fructose.

[0008] The second stage is used for deep purification of the enzymatic product. First, nanofiltration technology (molecular weight cut-off 250-300 Da) is used to selectively cut off oligofructose (molecular weight > 500 Da) through membrane separation and purification, while monosaccharides (glucose, fructose) and residual small molecule salts are removed, significantly improving the relative content of oligofructose in the cut-off liquid. The obtained cut-off liquid is further purified by calcium type gel chromatography, based on the specific complexation of calcium ions with the hydroxyl groups of oligofructose, achieving fine separation of the target product (DP3-DP5) and residual monosaccharides and disaccharides.

[0009] The third stage is used to complete the efficient conversion of the product. First, double-effect falling film evaporation is used to remove water to a solid content of 40-50 wt% to avoid sugar coking or degradation caused by high temperature. Then, spray drying is used to convert the concentrated liquid into fine powder with good flowability in a very short time, maximizing the retention of the heat-sensitive structure of oligofructose.

[0010] In the whole production process of high-purity oligofructose, the operation units work together to greatly improve the purity of oligofructose. First, the activated carbon decolorization and ultrafiltration remove gel provide a low interference environment for enzymatic hydrolysis. The electrodialysis deep desalination not only eliminates enzyme inhibitors but also provides space for subsequent precise calcium ion regulation, so that the enzymatic reaction can be carried out under high selectivity. The nanofiltration membrane separation as the primary purification efficiently removes small molecule impurities, significantly reduces the load of chromatographic purification, and the calcium type chromatography realizes fine separation based on molecular structure and ion interaction. The two work together to ensure the step-by-step increase of the purity of oligofructose. The double-effect evaporation efficiently concentrates at low temperature and low pressure, directly connects with low-temperature spray drying, avoids repeated heating and cooling of intermediate products, reduces energy consumption, and protects product activity. The whole process uses water as the only solvent, avoids the introduction of organic reagents, and through the organic combination of physical and biological technologies, it removes multiple impurities such as pigments, colloids, salts, and monosaccharides, while accurately enriching and stably converting target oligofructose, realizing the green, efficient, and large-scale preparation of high-purity (≥95%) products.

[0011] As a preferred technical solution of the present application, in step (I), the operation process of activated carbon decolorization includes: The cane juice is subjected to coarse filtration to remove impurities, the fiber residues and large particle suspensions in the cane juice are removed, the filtrate is collected, activated carbon powder is added to the filtrate, mixed, stirred and heated, then the activated carbon powder is filtered out, and a decolorized liquid is obtained.

[0012] The present application realizes multiple effects through the staged physical treatment of cane juice. First, the coarse filtration quickly intercepts the fiber residues and large particle suspensions in the cane juice, effectively reducing the mechanical wear risk and adsorbent loss in the subsequent process. Then, the activated carbon powder is introduced for efficient adsorption under the condition of heating and stirring, which specifically captures soluble pigments (such as flavonoids, polyphenol oxidation products) and phenolic derivatives by virtue of the rich micropore structure of activated carbon powder, while deeply removing pyrogenic substances and small molecule odor components. The heating process not only accelerates the diffusion of pigment molecules to the pores of activated carbon, but also promotes the thermal aggregation and precipitation of colloidal impurities, significantly improving the decolorization efficiency and adsorption saturation, realizing the removal of colored substances, colloidal aggregates and trace metal ions, and finally obtaining a clear decolorized liquid after filtration.

[0013] This operation process ensures that the pigment removal rate is more than 95%, avoids the introduction of organic solvents, and maintains the molecular integrity of sugar components, laying a foundation of low turbidity and low colloidal content for the subsequent ultrafiltration gel removal process, and blocking the pollution and blockage risk of pigments and colloids to the subsequent membrane filtration system from the source.

[0014] In some optional examples, the activated carbon powder is added in an amount of 0.5-1.5 wt% of the filtrate, for example, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt% or 1.5 wt%, but not limited to the listed values, and other values not listed in the range are also applicable.

[0015] In some optional examples, the temperature of the mixing, stirring and heating is 70-80℃, for example, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃ or 80℃, but not limited to the listed values, and other values not listed in the range are also applicable.

[0016] In some optional examples, the mixing, stirring and heating is performed for 20-40 min, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min or 40 min, but not limited to the listed values, and other values not listed in the range are also applicable.

[0017] In some optional examples, the rotation speed of the mixing, stirring and heating is 100-200 rpm, for example, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or 200 rpm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0018] As a preferred technical solution of the present application, in step (I), the operation process of ultrafiltration for removing gelatin includes: The decolorized liquid obtained after decolorization of activated carbon is sent into a polyether sulfone ultrafiltration membrane system for circulation filtration until the turbidity of the filtrate is ≤1.0 NTU, and the filtrate is collected, which is the gelatin-removed liquid.

[0019] The ultrafiltration for removing gelatin provided by the present application is a continuation of the decolorization of activated carbon, and the membrane separation technology is used to realize efficient removal of colloidal impurities. Relying on the circulation filtration of the polyether sulfone ultrafiltration membrane (80-100 kDa molecular weight cut-off), the residual protein polymers, polysaccharide colloids and submicron suspended particles in the decolorized liquid are accurately intercepted, and the sugar small molecules are allowed to freely pass through. The circulation filtration strengthens the shear force on the membrane surface to delay the pollution, and the filtration end point is determined by real-time turbidity monitoring (≤1.0 NTU) to ensure the thoroughness of the removal of colloids.

[0020] This operation process significantly reduces the colloidal load and viscosity in the decolorizing solution, creates a uniform heat transfer environment for the subsequent high-temperature enzyme inactivation process, avoids equipment fouling caused by colloidal thermal denaturation, and further, through the synergistic effect of activated carbon adsorption and ultrafiltration colloidal removal, a low turbidity and low viscosity colloidal removal solution is obtained, which greatly reduces the risk of ion exchange membrane pollution, improves the desalination efficiency, and the extremely low turbidity ensures the accuracy of calcium ion concentration and pH control in the enzymatic reaction, avoiding the shielding effect of colloids on the enzyme active site.

[0021] In some alternative examples, the molecular weight cut-off of the polyether sulfone ultrafiltration membrane system is 80-100 kDa, for example, it can be 80 kDa, 82 kDa, 84 kDa, 86 kDa, 88 kDa, 90 kDa, 92 kDa, 94 kDa, 96 kDa, 98 kDa or 100 kDa, but not limited to the listed values, other values not listed in this range are also applicable.

[0022] In some alternative examples, the pressure of the circulating filtration is 0.4-0.6 MPa, for example, it can be 0.4 MPa, 0.42 MPa, 0.44 MPa, 0.46 MPa, 0.48 MPa, 0.5 MPa, 0.52 MPa, 0.54 MPa, 0.56 MPa, 0.58 MPa or 0.6 MPa, but not limited to the listed values, other values not listed in this range are also applicable.

[0023] In some alternative examples, the temperature of the circulating filtration is 40-50℃, for example, it can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃, but not limited to the listed values, other values not listed in this range are also applicable.

[0024] As a preferred technical solution of the present application, in step (I), the heating temperature of the high-temperature enzyme inactivation is 90-95℃, for example, it can be 90℃, 91℃, 92℃, 93℃, 94℃ or 95℃, but not limited to the listed values, other values not listed in this range are also applicable.

[0025] In some alternative examples, the holding time of the high-temperature enzyme inactivation is 1-3 min, for example, it can be 1.0 min, 1.2 min, 1.4 min, 1.6 min, 1.8 min, 2.0 min, 2.2 min, 2.4 min, 2.6 min, 2.8 min or 3.0 min, but not limited to the listed values, other values not listed in this range are also applicable.

[0026] As a preferred technical solution of the present application, in step (I), the operation process of the electrodialysis desalination comprises: The enzyme inactivation solution obtained after high-temperature enzyme inactivation is diluted with water, and then introduced into an electrodialysis device for cyclic desalination. The conductivity of the effluent is detected by sampling. When the conductivity of the effluent is ≤100 μS / cm, the effluent is collected, which is the desalination solution.

[0027] The electrodialysis desalination step provided by the present application realizes deep removal of ions. After high-temperature enzyme inactivation to eliminate the interference of endogenous enzymes, the enzyme inactivation solution is diluted to a solid content of 8-12 wt%. The electrodialysis cyclic desalination is carried out at an operating voltage of 15-25 V. The inorganic ions such as potassium, sodium and chlorine (especially the cations that inhibit enzyme activity) are efficiently and directionally migrated and removed. At the same time, the integrity of sugar molecules is maintained. The conductivity of the effluent is ≤100 μS / cm as the desalination endpoint, which ensures that the degree of desalination meets the ion sensitivity requirements of subsequent enzymatic reaction.

[0028] This operation process not only removes the soluble salt by-product generated by heat denaturation of the enzyme inactivation solution, but also eliminates the competitive inhibition of the inorganic salt on the activity of β-fructofuranosidase, thereby creating a pure ion environment for the subsequent enzymatic reaction. The trace amount of calcium ions added subsequently can precisely regulate the stability of enzyme molecules, thereby significantly improving the sucrose conversion rate.

[0029] In some optional examples, the enzyme inactivation solution is diluted with water to a solid content of 8-12 wt%, for example, which can be 8.0 wt%, 8.5 wt%, 9.0 wt%, 9.5 wt%, 10.0 wt%, 10.5 wt%, 11.0 wt%, 11.5 wt% or 12.0 wt%, but is not limited to the listed values. Other values not listed in this range are also applicable.

[0030] In some optional examples, the operating voltage of the electrodialysis device is 15-25 V, for example, which can be 15 V, 16 V, 17 V, 18 V, 19 V, 20 V, 21 V, 22 V, 23 V, 24 V or 25 V, but is not limited to the listed values. Other values not listed in this range are also applicable.

[0031] In some optional examples, the flow rate of the enzyme inactivation solution in the electrodialysis device is 30-50 L / h·m 2 membrane area, for example, which can be 30 L / h·m 2 membrane area, 32 L / h·m 2 membrane area, 34 L / h·m 2 membrane area, 36 L / h·m 2 membrane area, 38 L / h·m 2 membrane area, 40 L / h·m 2 membrane area, 42 L / h·m2 Membrane area, 44 L / h·m 2 Membrane area, 46 L / h·m 2 Membrane area, 48 L / h·m 2 Membrane area or 50 L / h·m 2 The membrane area, but not limited to the listed values, applies to other unlisted values ​​within the same range.

[0032] As a preferred technical solution of the present invention, in step (I), the enzymatic hydrolysis reaction operation process includes: Citric acid / acetic acid buffer solution is added dropwise to the desalination solution obtained after electrodialysis to adjust its pH value. Then, calcium chloride solution is added and mixed evenly to obtain the enzymatic hydrolysis substrate. β-fructofuranosidase is added to the enzymatic hydrolysis substrate, and the mixture is stirred and heated to carry out the enzymatic hydrolysis reaction, so that the sucrose in the enzymatic hydrolysis substrate is converted into oligofructose. During the enzymatic hydrolysis reaction, the pH value of the enzymatic hydrolysis system is controlled at 6.3~6.7. The sucrose concentration is sampled and detected during the reaction. When the sucrose conversion rate is ≥95%, the temperature is immediately raised to inactivate the enzyme. After cooling and filtration, the enzymatic hydrolysis product is obtained. For example, it can be 6.3, 6.4, 6.5, 6.6 or 6.7, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0033] The enzymatic hydrolysis reaction is carried out in the pure ionic environment created by electrodialysis desalination, achieving efficient conversion from sucrose to the target fructooligosaccharides. The pH of the desalination solution is stabilized to the optimal enzyme activity range of 6.3-6.7 using a citrate / acetic acid buffer, eliminating the influence of residual acid-base fluctuations on enzyme conformation. The added trace amount of calcium chloride solution can specifically activate the calcium-binding domain of β-fructofuranosidase, enhancing its transglycosylation ability and inhibiting hydrolytic side reactions. Under isothermal heating conditions, the enzyme catalytic system preferentially promotes the conversion of sucrose to DP3-DP5 fructooligosaccharides. The enzymatic hydrolysis reaction is terminated when the sucrose conversion rate is ≥95%, effectively avoiding the risk of enzyme activity inhibition caused by glucose accumulation and excessive hydrolysis of the product.

[0034] The generated enzymatic hydrolysate is inactivated by immediate heating and then filtered by cooling. This process not only preserves the molecular structure of the target fructooligosaccharide but also removes heat-denatured enzyme proteins and trace precipitates. This provides downstream nanofiltration membrane separation with materials composed of low-protein-load and high-polymerization sugars, significantly reducing the impurity separation pressure in subsequent membrane separation and chromatographic purification processes, thus forming a highly efficient synergy between biotransformation and physical separation.

[0035] In some alternative examples, citric acid / acetic acid buffer is added dropwise to the desalination solution to adjust its pH value to 6-6.5, for example, it can be 6.0, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45 or 6.5, but not limited to the listed values, other values not listed in the range are also applicable.

[0036] In some alternative examples, the ratio of calcium chloride to deionized water in the calcium chloride solution is (1-5) g:100 mL, for example, it can be 1.0 g:100 mL, 1.5 g:100 mL, 2.0 g:100 mL, 2.5 g:100 mL, 3.0 g:100 mL, 3.5 g:100 mL, 4.0 g:100 mL, 4.5 g:100 mL or 5.0 g:100 mL, but not limited to the listed values, other values not listed in the range are also applicable.

[0037] In some alternative examples, the concentration of calcium ions in the enzymatic substrate is 0.5-1 mmol / L, for example, it can be 0.5 mmol / L, 0.55 mmol / L, 0.6 mmol / L, 0.65 mmol / L, 0.7 mmol / L, 0.75 mmol / L, 0.8 mmol / L, 0.85 mmol / L, 0.9 mmol / L, 0.95 mmol / L or 1 mmol / L, but not limited to the listed values, other values not listed in the range are also applicable.

[0038] The present application particularly limits the concentration of calcium ions in the enzymatic substrate to be 0.5-1 mmol / L, which allows calcium ions to fully occupy the metal activation site of β-fructofuranosidase, stabilizes the tertiary structure of the enzyme protein and enhances the transglycosylation activity, and directs the conversion of sucrose to DP3-DP5 oligofructose; at the same time, it avoids excessive calcium ions from competitively occupying the enzyme catalytic center, effectively inhibiting the generation of glucose byproducts, and creating a low monosaccharide content material basis for subsequent chromatographic purification.

[0039] When the concentration of calcium ions is less than 0.5 mmol / L, it will seriously affect the stability of the enzyme structure, the metal activation site is not saturated, leading to loose enzyme molecular conformation, reduced catalytic efficiency, and forced reaction time extension. At the same time, the lack of calcium ions weakens the specific recognition of the enzyme to the fructosyl acceptor, and the hydrolysis pathway is enhanced, making the amount of glucose generated greatly increased, not only intensifying the product inhibition effect, but also causing the target product yield and purity to decrease in subsequent chromatographic purification, as glucose and oligofructose compete for the calcium sites of the resin.

[0040] When the concentration of calcium ions exceeds 1 mmol / L, the excess calcium ions cover the enzyme active center, hinder the substrate from approaching the catalytic site, and reduce the conversion rate of sucrose to fructooligosaccharides; at the same time, the high calcium environment promotes the citric acid buffer to generate a gelatinous calcium citrate precipitate, which wraps the β-fructofuranosidase and reduces the utilization rate of enzyme activity. In addition, the accumulation of residual free calcium in the enzymatic product leads to the early saturation of the calcium ion binding sites of the calcium-type gel chromatography resin in the subsequent chromatographic adsorption stage, which destroys the specific adsorption capacity of the calcium-type gel chromatography resin for fructooligosaccharides.

[0041] In some optional examples, the amount of β-fructofuranosidase added is 40-50 U / g of sucrose in the enzymatic substrate, for example, it can be 40 U / g, 41 U / g, 42 U / g, 43 U / g, 44 U / g, 45 U / g, 46 U / g, 47 U / g, 48 U / g, 49 U / g or 50 U / g, but not limited to the listed values, and other unlisted values within the range are also applicable.

[0042] The present application particularly limits the amount of β-fructofuranosidase added to 40-50 U / g of sucrose in the enzymatic substrate, which can ensure that the sucrose conversion rate is ≥95%, avoiding the accumulation of by-products caused by excessive reaction time, and fully inhibiting the inhibitory effect of glucose on enzyme activity.

[0043] If the enzyme amount is less than 40 U / g of sucrose in the enzymatic substrate, the enzyme active site is not sufficient to bind sucrose molecules, the sucrose conversion rate is low, resulting in a large amount of unconverted sucrose remaining, which directly reduces the product yield, and the unreacted sucrose is difficult to separate in the subsequent nanofiltration purification due to its close molecular weight to fructooligosaccharides, eventually mixing into the finished product, which destroys the product purity in the finished product.

[0044] If the enzyme amount is greater than 50 U / g of sucrose in the enzymatic substrate, the excess enzyme molecules can accelerate the consumption of sucrose in the early stage of catalysis and convert it into fructooligosaccharides, but high enzyme concentration also intensifies the hydrolysis side reaction, leading to an increase in the proportion of glucose and fructose generated. The by-product glucose not only competitively inhibits enzyme activity, but also competes with fructooligosaccharides for resin calcium ion binding sites in the chromatographic purification stage as the accumulation of glucose, which significantly reduces the chromatographic purification efficiency.

[0045] In some optional examples, the temperature of the enzymatic reaction is 50-55°C, for example, it can be 50°C, 50.5°C, 51°C, 51.5°C, 52°C, 52.5°C, 53°C, 53.5°C, 54°C, 54.5°C or 55°C, but not limited to the listed values, and other unlisted values within the range are also applicable.

[0046] In some alternative examples, the temperature is increased to 80-90℃ immediately when the sucrose conversion rate is ≥95%, and the temperature is maintained for 10-20min, wherein the heating temperature can be 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃ or 90℃, and the holding time can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, but not limited to the listed values, and other values not listed in the range are also applicable.

[0047] In some alternative examples, the temperature is decreased to 40-50℃ after the enzyme is inactivated, and then the enzyme solution is filtered by a plate and frame filter, for example, the temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃, but not limited to the listed values, and other values not listed in the range are also applicable.

[0048] As a preferred technical solution of the present application, in step (II), the operation process of membrane separation and purification comprises: The enzyme solution is filtered by a nanofiltration membrane, and the retentate is collected. The retentate is diluted with water to obtain the retentate solution.

[0049] The membrane separation and purification can remove more than 95% of the monosaccharide impurities in the enzyme solution, completely eliminate the competitive occupation of the adsorption sites of the subsequent calcium-type chromatography resin, and ensure the specific binding efficiency of calcium ions and the hydroxyl groups of fructooligosaccharides. In addition, the membrane separation and purification removes residual salts and small pigment molecules, creating a low-conductivity and low-background-interference material basis for chromatography purification.

[0050] In some alternative examples, the molecular weight cut-off of the nanofiltration membrane is 250-300Da, for example, it can be 250Da, 255Da, 260Da, 265Da, 270Da, 275Da, 280Da, 285Da, 290Da, 295Da or 300Da, but not limited to the listed values, and other values not listed in the range are also applicable.

[0051] In some alternative examples, the operation pressure of the filtration is 1-2MPa, for example, it can be 1.0MPa, 1.1MPa, 1.2MPa, 1.3MPa, 1.4MPa, 1.5MPa, 1.6MPa, 1.7MPa, 1.8MPa, 1.9MPa or 2.0MPa, but not limited to the listed values, and other values not listed in the range are also applicable.

[0052] In some optional examples, the operating temperature of the filtration is 30-40℃, for example, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, but not limited to the listed values, and other values not listed in the range are also applicable.

[0053] In some optional examples, the cut-off liquid is diluted to a solid content of 20-30wt%, for example, it can be 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt% or 30wt%, but not limited to the listed values, and other values not listed in the range are also applicable.

[0054] As a preferred technical solution of the present application, in step (II), the calcium-type gel chromatography resin is packed into a chromatography column with a diameter-height ratio of 1:(4-6), for example, it can be 1:4.0, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5.0, 1:5.2, 1:5.4, 1:5.6, 1:5.8 or 1:6.0, but not limited to the listed values, and other values not listed in the range are also applicable.

[0055] In some optional examples, the flow rate of the cut-off liquid is 1-2 BV / h, for example, it can be 1.0 BV / h, 1.1 BV / h, 1.2 BV / h, 1.3 BV / h, 1.4 BV / h, 1.5 BV / h, 1.6 BV / h, 1.7 BV / h, 1.8 BV / h, 1.9 BV / h or 2.0 BV / h, but not limited to the listed values, and other values not listed in the range are also applicable.

[0056] The present application particularly limits the flow rate of the cut-off liquid to 1-2 BV / h, which balances the complexation reaction of oligofructose (DP3-DP5) with calcium ions of the calcium-type resin, and fully adsorbs the target component; at the same time, it maintains a laminar flow state to avoid spectral band broadening caused by turbulent flow. Online HPLC can accurately capture the elution window of DP3-DP5 peak area of up to 90% under stable flow rate, realizing high-purity collection of the target component. In addition, the flow rate of 1-2 BV / h cooperates with the diameter-height ratio of 1:(4-6) to control the pressure drop in the column, which not only ensures uniform permeability of the resin bed, but also prevents elution peak distortion caused by flow rate fluctuations.

[0057] If the flow rate of the retentate is lower than 1 BV / h, the residence time of the fructooligosaccharides in the column is too long, resulting in serious peak tailing, the elution range of DP3~DP5 and the high polymerization degree components (DP6+) overlapping, the 90% peak area threshold of the online HPLC monitoring containing more DP6+ impurities, and the product polymerization degree specificity being destroyed. In addition, under the ultra-low flow rate, the coordination bond stability of calcium ions and the resin decreases, part of the active sites desorb, the selective binding capacity of the hydroxyl group of the fructooligosaccharides is weakened, and the separation efficiency is reduced.

[0058] If the flow rate of the retentate is higher than 2 BV / h, the fluid shear force hinders the formation of stable complexes of fructooligosaccharides and calcium ions, part of DP3~DP5 penetrates the chromatographic column without being fully adsorbed, directly reducing the product yield. At the same time, too high flow rate of the retentate causes vortex diffusion, making the elution peak front steep and the tailing, and the online HPLC is forced to expand the collection window to compensate for the peak shape distortion, resulting in the mixing of monosaccharide impurities. In addition, too high linear velocity will also increase the pressure difference at the column head, destroy the structure of the resin particles or cause bed cracking, causing permanent loss of column efficiency.

[0059] In some optional examples, the elution speed of the deionized water is 5~10 mL / min, for example, it can be 5.0 mL / min, 5.5 mL / min, 6.0 mL / min, 6.5 mL / min, 7.0 mL / min, 7.5 mL / min, 8.0 mL / min, 8.5 mL / min, 9.0 mL / min, 9.5 mL / min or 10 mL / min, but not limited to the listed values, and other unlisted values within the range are also applicable.

[0060] The present application particularly limits the elution speed of the deionized water to 5~10 mL / min, and in the elution process, the moderate flow rate of the deionized water promotes the dissociation of the coordination bond between the calcium ions and the hydroxyl group of the fructooligosaccharides, avoids incomplete desorption of the target product, and prevents the instantaneous concentration fluctuation caused by too high flow rate, so that the online HPLC can accurately capture the elution peak shape change of DP3~DP5, when the peak area of the target component reaches the 90% threshold of the total sugar, the concentration distribution is in a narrow and symmetrical elution range, thereby ensuring the accuracy of the collection time.

[0061] When the elution speed of deionized water is lower than 5 mL / min, the oligofructose is difficult to desorb at low flow rate due to its large molecular weight, large number of hydroxyl groups and high binding energy with calcium ions, and part of the target product will be retained in the deep pore of the resin and cannot be eluted, directly reducing the yield of the target product. At the same time, the elution peak is seriously tailing at ultra-low flow rate, and the elution time of DP3~DP5 is significantly prolonged, which causes the elution interval of DP3~DP5 to overlap with that of higher degree of polymerization impurities (such as DP6+). At this time, the 90% peak area threshold interval monitored by online HPLC contains more high degree of polymerization impurities, resulting in a decrease in the purity of the target product in the product.

[0062] When the elution speed of deionized water is higher than 10 mL / min, the high flow rate causes a turbulent effect, which can destroy the stability of the calcium ions and the functional groups of the resin, cause local calcium ions to dissolve out, and weaken the subsequent adsorption capacity. At the same time, the high flow rate causes the front of the elution peak to be steep and the tail to be tailing, the elution time window of each component of DP3~DP5 is compressed and overlaps with each other, and the detection response speed of the online HPLC is difficult to match the transient process. When the system recognizes the 90% peak area signal, part of the DP5 component has actually penetrated, and part of the DP3 has not been eluted, causing incomplete collection of the target product. In addition, the high-speed mobile phase intensifies the pressure difference in the column, and long-term operation will cause the resin particles to break or the bed layer to crack, resulting in irreversible loss of column efficiency.

[0063] In some optional examples, during the deionized water elution process, the components of the eluent discharged are monitored by online HPLC, when the peak area of oligofructose DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluent is collected, and when the peak area of oligofructose DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the collection is stopped, and the collected eluent is the eluent rich in oligofructose.

[0064] As a preferred technical solution of the present application, in step (III), the double-effect falling film evaporation concentration is carried out in a double-effect falling film evaporator, and the double-effect falling film evaporator comprises a one-effect evaporator and a two-effect evaporator connected in series.

[0065] In some optional examples, the operating temperature of the one-effect evaporator is 70~80℃, for example, it can be 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃ or 80℃, but is not limited to the listed values, and other values not listed in the range are also applicable.

[0066] In some optional examples, the operating pressure of the primary evaporator is -0.08 ~ -0.09 MPa, for example, can be -0.08 MPa, -0.081 MPa, -0.082 MPa, -0.083 MPa, -0.084 MPa, -0.085 MPa, -0.086 MPa, -0.087 MPa, -0.088 MPa, -0.089 MPa or -0.09 MPa, but not only limited to the listed values, other values not listed in the range of values are also applicable.

[0067] In some optional examples, the operating temperature of the secondary evaporator is 60 ~ 70℃, for example, can be 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃ or 70℃, but not only limited to the listed values, other values not listed in the range of values are also applicable.

[0068] In some optional examples, the operating pressure of the secondary evaporator is -0.085 ~ -0.098 MPa, for example, can be -0.085 MPa, -0.086 MPa, -0.087 MPa, -0.088 MPa, -0.089 MPa, -0.09 MPa, -0.091 MPa, -0.092 MPa, -0.093 MPa, -0.094 MPa, -0.095 MPa, -0.096 MPa, -0.097 MPa or -0.098 MPa, but not only limited to the listed values, other values not listed in the range of values are also applicable.

[0069] In some optional examples, the solid content of the concentrated solution is 40 ~ 50wt%, for example, can be 40wt%, 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, 49wt% or 50wt%, but not only limited to the listed values, other values not listed in the range of values are also applicable.

[0070] The present application realizes efficient concentration of eluent by double-effect falling film evaporation concentration, wherein the first-effect evaporator removes more than 60% of water in the eluent under high temperature (70-80 DEG C) and low pressure (-0.05--0.06 MPa) conditions, and the steam condensation latent heat directly drives the low-temperature evaporation of the second-effect evaporator, forming a thermal energy cascade reuse, which saves more than 40% of energy compared with single-effect evaporation. The stepwise temperature control (high-temperature first-effect to low-temperature second-effect) avoids the Maillard reaction and coking risk of leucrose under continuous high temperature, and completely retains the molecular structure activity of DP3-DP5. At the same time, the low-temperature concentration environment of the second-effect evaporator inhibits the browning reaction of residual trace monosaccharides, ensuring the whiteness and solubility of the final powder product. After double-effect falling film evaporation concentration to 40-50% solid content, the atomization efficiency of subsequent spray drying is improved, and the adhesion and crystallization of high-concentration concentrated liquid on the inner wall of the spray drying tower are avoided.

[0071] As a preferred technical solution of the present application, in step (III), the inlet air temperature of the spray drying is 170-180 DEG C, for example, it can be 170 DEG C, 171 DEG C, 172 DEG C, 173 DEG C, 174 DEG C, 175 DEG C, 176 DEG C, 177 DEG C, 178 DEG C, 179 DEG C or 180 DEG C, but not limited to the listed values, other values not listed in this range are also applicable.

[0072] In some optional examples, the outlet air temperature of the spray drying is 75-85 DEG C, for example, it can be 75 DEG C, 76 DEG C, 77 DEG C, 78 DEG C, 79 DEG C, 80 DEG C, 81 DEG C, 82 DEG C, 83 DEG C, 84 DEG C or 85 DEG C, but not limited to the listed values, other values not listed in this range are also applicable.

[0073] In some optional examples, the feeding rate of the concentrated liquid is 20-40 L / h, for example, it can be 20 L / h, 22 L / h, 24 L / h, 26 L / h, 28 L / h, 30 L / h, 32 L / h, 34 L / h, 36 L / h, 38 L / h or 40 L / h, but not limited to the listed values, other values not listed in this range are also applicable.

[0074] In some optional examples, the mesh number of the screen is 80-100 mesh, for example, it can be 80 mesh, 82 mesh, 84 mesh, 86 mesh, 88 mesh, 90 mesh, 92 mesh, 94 mesh, 96 mesh, 98 mesh or 100 mesh, but not limited to the listed values, other values not listed in this range are also applicable.

[0075] Compared with the prior art, the present application has the following advantages: In the whole production process of high-purity oligofructose, the operation units work together to greatly improve the purity of oligofructose. First, activated carbon decolorization and ultrafiltration remove gel to provide a low-interference environment for enzymatic hydrolysis. Electrodialysis deep desalination not only eliminates enzyme inhibitors but also provides space for precise calcium ion regulation, allowing the enzymatic reaction to proceed under high selectivity. Nanofiltration membrane separation as primary purification efficiently removes small molecular impurities, significantly reducing the load of chromatographic purification. Calcium-type chromatography realizes fine separation based on molecular structure and ionic interaction, and the two work together to ensure the step-by-step increase of oligofructose purity. Double-effect evaporation efficiently concentrates at low temperature and low pressure, directly linking to low-temperature spray drying to avoid repeated heating and cooling of intermediates, reducing energy consumption and protecting product activity. The whole process uses water as the only solvent, avoiding the introduction of organic reagents. Through the organic combination of physical and biological technologies, the target oligofructose is precisely enriched and stably converted while removing multiple impurities such as pigments, colloids, salts, and monosaccharides, achieving green, efficient, and large-scale preparation of high-purity (≥95%) products. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 The preparation process flow chart of high-purity oligofructose is provided for embodiments 1-13 of the present application. DETAILED DESCRIPTION

[0077] The technical solutions of the present application will be described in detail below in combination with specific embodiments and drawings. The embodiments described herein are specific specific embodiments of the present application, used to illustrate the concept of the present application; these descriptions are all explanatory and exemplary, and should not be understood as limiting the embodiments of the present application and the protection scope of the present application. In addition to the embodiments described herein, those skilled in the art can also employ other technical solutions that are obvious based on the disclosure of the claims and the specification of the present application, which include technical solutions that make any obvious substitutions and modifications to the embodiments described herein.

[0078] Embodiment 1 The present embodiment provides a preparation process of high-purity oligofructose, as shown in Figure 1 The preparation process comprises: (1) Fresh sugarcane juice is taken to obtain sugarcane juice, and the sugarcane juice is coarsely filtered to remove impurities, remove fiber residues and large particle suspensions in the sugarcane juice, collect the filtrate, add activated carbon powder to the filtrate, the amount of activated carbon powder added is 0.5wt% of the mass of the filtrate, mix and stir at a heating temperature of 70℃ and a stirring speed of 100rpm for 40min, then filter out the activated carbon powder to obtain a decolorized liquid; The decolorized liquid obtained after decolorization of activated carbon is sent into a polyether sulfone ultrafiltration membrane system with a molecular weight cut-off of 80 kDa for circulation filtration, the pressure for circulation filtration is 0.4 MPa, the temperature for circulation filtration is 40℃, and the filtrate is collected when the turbidity of the filtrate reaches 1.0 NTU, which is the glue removal liquid; The glue removal liquid is heated to 90℃ and kept for 3 min to complete high-temperature enzyme inactivation, and the enzyme inactivated liquid is obtained; The enzyme inactivated liquid obtained after high-temperature enzyme inactivation is diluted with water to a solid content of 8wt%, and then is sent into an electrodialysis device for circulation desalination, the operating voltage of the electrodialysis device is 15V, and the flow rate of the enzyme inactivated liquid in the electrodialysis device is 30L / h·m 2 The membrane area, and the conductivity of the effluent is detected by sampling, and the effluent is collected when the conductivity of the effluent reaches 100μS / cm, which is the desalted liquid; The pH value of the desalted liquid obtained after electrodialysis desalination is adjusted to 6 by adding citric acid / acetic acid buffer solution, and then calcium chloride solution is added, the ratio of calcium chloride to deionized water in the calcium chloride solution is 1g:100mL, and the enzyme substrate is obtained after mixing uniformly, the concentration of calcium ions in the enzyme substrate is 0.5mmol / L; β-fructofuranosidase is added to the enzyme substrate, the amount of β-fructofuranosidase added is 40U / g sucrose in the enzyme substrate, and the enzyme substrate is mixed and stirred at 50℃ to occur enzyme reaction, so that the sucrose in the enzyme substrate is converted into fructooligosaccharides, the pH value of the enzyme system is controlled at 6.3 during the enzyme reaction process, the sucrose concentration is detected by sampling during the reaction process, and the temperature is immediately raised to 80℃ and kept for 20min when the sucrose conversion rate reaches 95%, and then the temperature is lowered to 40℃, and the filtrate is obtained by sending into a plate and frame filter, which is the enzyme product; (2) The enzyme product is filtered through a nanofiltration membrane with a molecular weight cut-off of 250Da, the operating pressure for filtration is 1MPa, and the operating temperature is 30℃, and the retentate is collected, and the retentate is diluted with water to a solid content of 20wt% to obtain the retentate liquid; The calcium type gel chromatography resin (calcium type carboxymethyl cellulose gel, separation range 400-3000 Da, model ZXC-800Ca, purchased from Zhejiang Zhengguang Industry Co., Ltd.) is loaded into a chromatography column with a ratio of 1:4, the cut-off liquid is passed into the chromatography column at a flow rate of 1 BV / h, the low oligofructose in the cut-off liquid is adsorbed by the calcium type gel chromatography resin, then the calcium type gel chromatography resin adsorbed with oligofructose is eluted with deionized water, the elution speed of deionized water is 5 mL / min, during the deionized water elution process, the components of the eluent are monitored online by HPLC, when the peak area of oligofructose DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluent is collected, when the peak area of oligofructose DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the collection is stopped, and the collected eluent is the eluent rich in oligofructose; (3) The collected eluent is sent into a double-effect falling film evaporator for concentration, and a concentrated liquid with a solid content of 40wt% is obtained, wherein the operating temperature of the first-effect evaporator is 70°C, and the operating pressure is -0.08MPa (gauge pressure), and the operating temperature of the second-effect evaporator is 60°C, and the operating pressure is -0.085MPa (gauge pressure); The concentrated liquid is subjected to spray drying, the inlet air temperature of the spray drying is 170°C, the outlet air temperature is 75°C, the feeding rate of the concentrated liquid is 20L / h, and the powder obtained after the spray drying is sieved through an 80-mesh screen to obtain the product rich in oligofructose.

[0079] Example 2 The present embodiment provides a preparation process of high-purity oligofructose, as shown in Figure 1 The preparation process comprises the following steps: (1) Fresh sugarcane juice is obtained by juicing fresh sugarcane, the sugarcane juice is subjected to coarse filtration to remove impurities, fiber residues and large particle suspensions in the sugarcane juice, and the filtrate is collected, activated carbon powder is added to the filtrate, the amount of the activated carbon powder added is 0.8wt% of the mass of the filtrate, and the mixture is mixed and stirred at a heating temperature of 72°C and a stirring speed of 120rpm for 35min, then the activated carbon powder is filtered out, and a decolorized liquid is obtained; The decolorized liquid obtained after the activated carbon decolorization is sent into a polyether sulfone ultrafiltration membrane system with a molecular weight cut-off of 85kDa for circulation filtration, the circulation filtration pressure is 0.45MPa, and the circulation filtration temperature is 42°C, until the turbidity of the filtrate reaches 0.8NTU, the filtrate is collected, and the filtrate is the glue-removed liquid; The glue-removed liquid is heated to 91°C and kept for 2.5min to complete the high-temperature enzyme inactivation, and the enzyme-inactivated liquid is obtained; The enzyme inactivation liquid obtained after high temperature enzyme inactivation is diluted with water to a solid content of 9wt%, and then is circulated and desalted in an electrodialysis device, the operating voltage of the electrodialysis device is 18V, and the flow rate of the enzyme inactivation liquid in the electrodialysis device is 35L / h·m 2 The membrane area, the conductivity of the effluent is sampled and detected, and when the conductivity of the effluent reaches 80μS / cm, the effluent is collected, which is the desalted liquid; The desalted liquid obtained after the electrodialysis desalination is added with citric acid / acetic acid buffer solution to adjust the pH value to 6.1, and then a calcium chloride solution is added, the ratio of calcium chloride to deionized water in the calcium chloride solution is 2g:100mL, and after uniform mixing, an enzymatic substrate is obtained, the concentration of calcium ions in the enzymatic substrate is 0.6mmol / L; β-fructofuranosidase is added to the enzymatic substrate, the amount of β-fructofuranosidase added is 42U / g sucrose in the enzymatic substrate, and the enzymatic substrate is mixed and stirred at 51℃ to occur an enzymatic reaction, so that the sucrose in the enzymatic substrate is converted into fructooligosaccharides, the pH value of the enzymatic system is controlled at 6.4 during the enzymatic reaction, the sucrose concentration is detected during the reaction, and when the sucrose conversion rate reaches 96%, the temperature is immediately increased to 82℃ and kept for 18min, and then the temperature is decreased to 42℃, and the enzymatic product is obtained by filtering in a plate and frame filter; (2) The enzymatic product is filtered through a nanofiltration membrane with a molecular weight cut-off of 260Da, the operating pressure of the filtration is 1.2MPa, and the operating temperature is 32℃, the retentate is collected, and the retentate is diluted with water to a solid content of 22wt% to obtain a retentate liquid; The calcium type gel chromatography resin is packed into a chromatography column with a diameter-height ratio of 1:4.5, the retentate liquid is passed into the chromatography column at a flow rate of 1.2BV / h, the calcium type gel chromatography resin adsorbs the fructooligosaccharides in the retentate liquid, and then deionized water is used to elute the calcium type gel chromatography resin adsorbed with the fructooligosaccharides, the elution speed of the deionized water is 6mL / min, during the deionized water elution, the composition of the eluent discharged is monitored online by HPLC, when the peak area of the fructooligosaccharides DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluent is collected, and when the peak area of the fructooligosaccharides DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the collection is stopped, and the collected eluent is the eluent rich in fructooligosaccharides; (3) The collected eluent is sent into a double-effect falling film evaporator for concentration to obtain a concentrated liquid with a solid content of 42wt%, wherein the operating temperature of the first-effect evaporator is 72℃, and the operating pressure is-0.082MPa (gauge pressure), the operating temperature of the second-effect evaporator is 62℃, and the operating pressure is-0.088MPa (gauge pressure); The concentrate was spray-dried at an inlet air temperature of 172°C and an outlet air temperature of 78°C. The feed rate of the concentrate was 25 L / h. The powder obtained after spray drying was sieved through an 85-mesh sieve to obtain a product rich in fructooligosaccharides.

[0080] Example 3 This embodiment provides a process for preparing high-purity fructooligosaccharides, such as... Figure 1 As shown, the preparation process includes: (1) Take fresh sugarcane juice and extract it. Then, filter the sugarcane juice to remove impurities, remove the fiber residue and large particulate matter in the sugarcane juice, collect the filtrate, add activated carbon powder to the filtrate, the amount of activated carbon powder added is 1 wt% of the mass of the filtrate, mix and stir at a heating temperature of 75℃ and a stirring speed of 150 rpm for 30 min, then filter out the activated carbon powder to obtain the decolorized liquid. The decolorized solution obtained after decolorization with activated carbon is sent to a polyethersulfone ultrafiltration membrane system with a molecular weight cutoff of 90 kDa for circulation filtration. The circulation filtration pressure is 0.5 MPa and the circulation filtration temperature is 45°C. The filtrate is collected when the turbidity of the filtrate reaches 0.8 NTU, which is the degummed solution. The degumming solution was heated to 92°C and kept at that temperature for 2 minutes to complete the high-temperature enzyme inactivation, resulting in an enzyme-inactivated solution. The enzyme-inactivating solution obtained after high-temperature enzyme inactivation was diluted with water to a solid content of 10 wt%, and then circulated into an electrodialysis unit for desalination. The operating voltage of the electrodialysis unit was 20 V, and the flow rate of the enzyme-inactivating solution in the electrodialysis unit was 40 L / h·m. 2 The membrane area is used to sample and test the conductivity of the effluent. When the conductivity of the effluent reaches 80 μS / cm, the effluent is collected, which is the desalination solution. Citric acid / acetic acid buffer was added dropwise to the desalination solution obtained after electrodialysis to adjust its pH to 6.2. Then, calcium chloride solution was added, with a calcium chloride to deionized water ratio of 3g:100mL. After mixing evenly, the enzymatic hydrolysis substrate was obtained, with a calcium ion concentration of 0.7mmol / L. β-fructofuranosidase was added to the enzymatic hydrolysis substrate at a concentration of 45 U / g of sucrose in the substrate. The mixture was stirred and heated at 52°C to induce an enzymatic hydrolysis reaction, converting the sucrose in the substrate into fructooligosaccharides. The pH of the enzymatic hydrolysis system was controlled at 6.5 during the reaction. The sucrose concentration was measured during the reaction. When the sucrose conversion rate reached 97%, the temperature was immediately raised to 85°C and held for 15 min. The temperature was then lowered to 45°C and filtered through a plate and frame filter press to obtain the enzymatic hydrolysis product. (2) The enzymatic product is filtered through a nanofiltration membrane with a molecular weight cut-off of 270 Da, the operating pressure is 1.5 MPa, the operating temperature is 35°C, the cut-off phase is collected, and the cut-off phase is diluted with water to a solid content of 25wt%, thereby obtaining a cut-off liquid; The calcium-type gel chromatography resin is loaded into a chromatography column with a diameter-height ratio of 1:5, the cut-off liquid is passed into the chromatography column at a flow rate of 1.5 BV / h, the calcium-type gel chromatography resin adsorbs the oligofructose in the cut-off liquid, then deionized water is used to elute the calcium-type gel chromatography resin adsorbed with oligofructose, the elution rate of deionized water is 7 mL / min, and during the deionized water elution process, online HPLC is used to monitor the composition of the eluent discharged, when the peak area of oligofructose DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluent collection starts, and when the peak area of oligofructose DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the eluent collection stops, and the collected eluent is the oligofructose-rich eluent; (3) The collected eluent is sent into a double-effect falling film evaporator for concentration, thereby obtaining a concentrated liquid with a solid content of 45wt%, wherein the operating temperature of the first-effect evaporator is 75°C, the operating pressure is -0.085 MPa (gauge pressure), the operating temperature of the second-effect evaporator is 65°C, and the operating pressure is -0.09 MPa (gauge pressure); The concentrated liquid is subjected to spray drying, the inlet air temperature of the spray drying is 175°C, the outlet air temperature is 80°C, the feeding rate of the concentrated liquid is 30 L / h, and after the spray drying, the powder obtained is sieved through a 90-mesh screen to obtain the oligofructose-rich product.

[0081] Example 4 The present embodiment provides a preparation process of high-purity oligofructose, as shown in Figure 1 The preparation process comprises: (1) Fresh sugarcane juice is obtained by juicing, the sugarcane juice is subjected to coarse filtration to remove impurities, thereby removing fiber residues and large-particle suspended matters in the sugarcane juice, the filtrate is collected, activated carbon powder is added to the filtrate, the amount of the activated carbon powder added is 1.2wt% of the mass of the filtrate, the mixture is stirred at a heating temperature of 78°C and a stirring speed of 180 rpm for 25 min, then the activated carbon powder is filtered out, thereby obtaining a decolorized liquid; The decolorized liquid obtained after the activated carbon decolorization is sent into a polyether sulfone ultrafiltration membrane system with a molecular weight cut-off of 95 kDa for circulation filtration, the pressure for the circulation filtration is 0.55 MPa, the temperature for the circulation filtration is 48°C, and the filtrate is collected when the turbidity of the filtrate reaches 0.6 NTU, thereby obtaining a gum-removed liquid; The gum-removed liquid is heated to 93°C and kept for 1.5 min to complete the high-temperature enzyme inactivation, thereby obtaining an enzyme-inactivated liquid; The enzyme inactivation liquid obtained after high temperature enzyme inactivation is diluted with water to a solid content of 11wt%, and then is circulated and desalted in an electrodialysis device, the operating voltage of the electrodialysis device is 22V, and the flow rate of the enzyme inactivation liquid in the electrodialysis device is 45L / h·m 2 The membrane area, the conductivity of the effluent is sampled and detected, and when the conductivity of the effluent reaches 60μS / cm, the effluent is collected, which is the desalted liquid; The desalted liquid obtained after the electrodialysis desalination is added with citric acid / acetic acid buffer solution to adjust the pH value to 6.3, and then a calcium chloride solution is added, the ratio of calcium chloride to deionized water in the calcium chloride solution is 4g:100mL, and after uniform mixing, an enzymatic substrate is obtained, the concentration of calcium ions in the enzymatic substrate is 0.8mmol / L; β-fructofuranosidase is added to the enzymatic substrate, the amount of β-fructofuranosidase added is 48U / g sucrose in the enzymatic substrate, and the mixture is heated under stirring at 53℃ to occur enzymatic reaction, so that the sucrose in the enzymatic substrate is converted into fructooligosaccharides, and the pH value of the enzymatic system is controlled at 6.6 during the enzymatic reaction, the sucrose concentration is detected during the reaction, and when the sucrose conversion rate reaches 98%, the temperature is immediately increased to 88℃ and kept for 12min, and then the temperature is decreased to 48℃, and the mixture is filtered in a plate and frame filter to obtain an enzymatic product; (2) The enzymatic product is filtered through a nanofiltration membrane with a molecular weight cut-off of 280Da, the operation pressure of the filtration is 1.8MPa, and the operation temperature is 38℃, the retentate is collected, and the retentate is diluted with water to a solid content of 28wt% to obtain a retentate liquid; The calcium type gel chromatography resin is packed into a chromatography column with a diameter-height ratio of 1:5.5, the retentate liquid is passed into the chromatography column at a flow rate of 1.8BV / h, the calcium type gel chromatography resin adsorbs the fructooligosaccharides in the retentate liquid, and then deionized water is used to elute the calcium type gel chromatography resin adsorbed with the fructooligosaccharides, the elution speed of the deionized water is 8mL / min, during the elution of the deionized water, the components of the eluent discharged are monitored online by HPLC, when the peak areas of fructooligosaccharides DP3, DP4 and DP5 reach 90% of the total sugar peak area, the eluent is collected, and when the peak areas of fructooligosaccharides DP3, DP4 and DP5 are less than 80% of the total sugar peak area, the collection is stopped, and the collected eluent is the eluent rich in fructooligosaccharides; (3) The collected eluent is sent into a double-effect falling film evaporator for concentration to obtain a concentrated liquid with a solid content of 48wt%, wherein the operation temperature of the first-effect evaporator is 78℃, and the operation pressure is-0.088MPa (gauge pressure), and the operation temperature of the second-effect evaporator is 68℃, and the operation pressure is-0.095MPa (gauge pressure); The concentrate was spray-dried at an inlet air temperature of 178°C and an outlet air temperature of 82°C. The feed rate of the concentrate was 35 L / h. The powder obtained after spray drying was sieved through a 95-mesh sieve to obtain a product rich in fructooligosaccharides.

[0082] Example 5 This embodiment provides a process for preparing high-purity fructooligosaccharides, such as... Figure 1 As shown, the preparation process includes: (1) Take fresh sugarcane to extract juice, and filter the sugarcane juice to remove impurities, remove the fiber residue and large particulate suspended matter in the sugarcane juice, collect the filtrate, add activated carbon powder to the filtrate, the amount of activated carbon powder added is 1.5wt% of the mass of the filtrate, mix and stir at a heating temperature of 80℃ and a stirring speed of 200rpm for 20min, then filter out the activated carbon powder to obtain the decolorized liquid; The decolorized solution obtained after decolorization with activated carbon is sent to a polyethersulfone ultrafiltration membrane system with a molecular weight cutoff of 100kDa for circulation filtration. The circulation filtration pressure is 0.6MPa and the circulation filtration temperature is 50℃. The filtrate is collected when the turbidity of the filtrate reaches 0.6NTU, which is the degummed solution. Heat the degumming solution to 95°C and keep it at that temperature for 1 minute to complete the high-temperature enzyme inactivation and obtain the enzyme-inactivated solution. The enzyme-inactivating solution obtained after high-temperature enzyme inactivation was diluted with water to a solid content of 12 wt%, and then circulated into an electrodialysis unit for desalination. The operating voltage of the electrodialysis unit was 25 V, and the flow rate of the enzyme-inactivating solution in the electrodialysis unit was 50 L / h·m. 2 The membrane area is used to sample and test the conductivity of the effluent. When the conductivity of the effluent reaches 60 μS / cm, the effluent is collected, which is the desalination solution. Citric acid / acetic acid buffer was added dropwise to the desalination solution obtained after electrodialysis to adjust its pH to 6.5. Then, calcium chloride solution was added, with a calcium chloride to deionized water ratio of 5g:100mL. After mixing evenly, the enzymatic hydrolysis substrate was obtained, with a calcium ion concentration of 1mmol / L. β-fructofuranosidase was added to the enzymatic hydrolysis substrate at a concentration of 50 U / g of sucrose in the substrate. The mixture was stirred and heated at 55°C to induce an enzymatic hydrolysis reaction, converting the sucrose in the substrate into fructooligosaccharides. The pH of the enzymatic hydrolysis system was controlled at 6.7 during the reaction. The sucrose concentration was measured during the reaction. When the sucrose conversion rate reached 97%, the temperature was immediately raised to 90°C and held for 10 min. The temperature was then lowered to 50°C and filtered through a plate and frame filter press to obtain the enzymatic hydrolysis product. (2) The enzymatic product is filtered through a nanofiltration membrane with a molecular weight cut-off of 300 Da, the operating pressure is 2 MPa, the operating temperature is 40℃, the retentate is collected, and the retentate is diluted with water to a solid content of 30wt%, to obtain a retentate solution; The retentate solution is passed into the chromatographic column at a flow rate of 2 BV / h, and the calcium type gel chromatographic resin adsorbs the fructooligosaccharides in the retentate solution. Subsequently, deionized water is used to elute the calcium type gel chromatographic resin adsorbed with fructooligosaccharides, and the elution rate of deionized water is 10 mL / min. During the deionized water elution process, online HPLC is used to monitor the composition of the eluate, and when the peak area of oligofructose DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluate is collected. When the peak area of oligofructose DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the collection is stopped. The collected eluate is the eluate rich in fructooligosaccharides; (3) The collected eluate is sent into a double-effect falling film evaporator for concentration, to obtain a concentrated solution with a solid content of 50wt%, wherein the operating temperature of the first-effect evaporator is 80℃, and the operating pressure is -0.09 MPa (gauge pressure). The operating temperature of the second-effect evaporator is 70℃, and the operating pressure is -0.098 MPa (gauge pressure). The concentrated solution is subjected to spray drying, the inlet air temperature is 180℃, the outlet air temperature is 85℃, and the feeding rate of the concentrated solution is 40 L / h. After spray drying, the powder is sieved through a 100-mesh screen to obtain a product rich in fructooligosaccharides.

[0083] Example 6 This example provides a preparation process of high-purity fructooligosaccharides, which is different from Example 1 in that in step (1), the concentration of calcium ions in the enzymatic substrate is adjusted to 0.1 mmol / L, and other process parameters and operation steps are completely the same as those of Example 1.

[0084] Example 7 This example provides a preparation process of high-purity fructooligosaccharides, which is different from Example 1 in that in step (1), the concentration of calcium ions in the enzymatic substrate is adjusted to 1.5 mmol / L, and other process parameters and operation steps are completely the same as those of Example 1.

[0085] Example 8 This example provides a preparation process of high-purity fructooligosaccharides, which is different from Example 1 in that in step (1), the amount of β-fructofuranosidase added is adjusted to 30 U / g of sucrose in the enzymatic substrate, and other process parameters and operation steps are completely the same as those of Example 1.

[0086] Example 9 The embodiment provides a preparation process of high-purity fructooligosaccharide, which is different from the embodiment 1 in that the addition amount of the beta-fructofuranosidase is adjusted to 60 U / g of sucrose in the enzymatic substrate in step (1), and other process parameters and operation steps are completely same as those of the embodiment 1.

[0087] Embodiment 10 The embodiment provides a preparation process of high-purity fructooligosaccharide, which is different from the embodiment 1 in that the flow rate of the retentate is adjusted to 0.5 BV / h in step (2), and other process parameters and operation steps are completely same as those of the embodiment 1.

[0088] Embodiment 11 The embodiment provides a preparation process of high-purity fructooligosaccharide, which is different from the embodiment 1 in that the flow rate of the retentate is adjusted to 2.5 BV / h in step (2), and other process parameters and operation steps are completely same as those of the embodiment 1.

[0089] Embodiment 12 The embodiment provides a preparation process of high-purity fructooligosaccharide, which is different from the embodiment 1 in that the elution speed of the deionized water is adjusted to 1 mL / min in step (2), and other process parameters and operation steps are completely same as those of the embodiment 1.

[0090] Embodiment 13 The embodiment provides a preparation process of high-purity fructooligosaccharide, which is different from the embodiment 1 in that the elution speed of the deionized water is adjusted to 15 mL / min in step (2), and other process parameters and operation steps are completely same as those of the embodiment 1.

[0091] The purity and yield of fructooligosaccharide in the products prepared in the embodiments 1-13 are detected, and the test steps are as follows: (1) Product purity Different polymeric sugars are separated by using an amino column, and the content ratio of DP3-DP5 to total sugar is quantitatively analyzed by a refractive index detector (RID).

[0092] (1.1) Instruments and reagents Chromatograph: Agilent 1260 HPLC (with an automatic sampler); Chromatographic column: Waters XBridge Amide (4.6x250mm, 3.5um); Mobile phase: acetonitrile: water = 75:25 (v / v), isocratic elution; Flow rate: 1.0 mL / min; Column temperature: 30 DEG C; Detector: refractive index detector (RID), temperature 40 DEG C; Standards: glucose, fructose (monosaccharide standard), sucrose (disaccharide standard), fructooligosaccharide DP3 (1-kestose), DP4 (nystose), DP5 (standard purchased from Sigma).

[0093] (1.2) Operation steps Sample pretreatment: accurately weigh 0.1 g of sample, dilute with ultrapure water to 10 mL, and pass through a 0.22 μm water filter membrane; Preparation of standard curve: prepare a mixed standard solution of glucose, fructose, sucrose, DP3, DP4, and DP5 (concentration gradient: 0.5-10 mg / mL); HPLC analysis: sample injection amount is 10 μL, and the running time is 25 min (monosaccharide retention time is about 8-10 min, DP3 is about 12 min, DP4 is about 14 min, and DP5 is about 16 min).

[0094] The purity of fructooligosaccharide in the product is calculated using the following formula: Purity = (sum of DP3, DP4, and DP5 peak areas / total sugar peak area) x 100%; (2) Product yield The sucrose content in the cane juice is determined, and the fructooligosaccharide content in the product is calculated according to the total mass of the product obtained after spray drying and the purity of fructooligosaccharide in the product.

[0095] The product yield is calculated using the following formula: Product yield (%) = [fructooligosaccharide content in the product / (sucrose content in the cane residue x theoretical conversion rate)] x 100%; The theoretical conversion rate is 0.95.

[0096] The test results are shown in Table 1.

[0097] Table 1 Product purity and yield

[0098] As can be seen from the test data of Example 1, Example 6, and Example 7, the calcium ion concentration in the enzyme substrate is adjusted to 0.1 mmol / L and 1.5 mmol / L, respectively. When the calcium ion concentration is too low (0.1 mmol / L), the enzyme structure stability is insufficient, resulting in a decrease in catalytic efficiency, incomplete sucrose conversion, an increase in glucose byproduct, and a significant decrease in purity and yield. While a too high calcium ion concentration (1.5 mmol / L) covers the enzyme active center, inhibits the conversion reaction, and may form calcium citrate precipitate, which interferes with subsequent chromatography adsorption, also causing a decrease in purity and yield.

[0099] As can be seen from the test data of Example 1, Example 8 and Example 9, the addition amount of β-fructofuranosidase is adjusted to 30 U / g and 60 U / g, and when the enzyme amount is insufficient (30 U / g), the sucrose conversion rate is reduced, and the residual sucrose is difficult to separate in subsequent purification, resulting in a decrease in product purity and yield loss; while the enzyme amount is too high (60 U / g), it exacerbates the hydrolysis side reaction, and the generation of glucose and fructose increases, which not only inhibits the enzyme activity, but also competes for the adsorption sites in chromatographic purification, further reducing the purity and yield.

[0100] As can be seen from the test data of Example 1, Example 10 and Example 11, the flow rate of the retentate is adjusted to 0.5 BV / h and 2.5 BV / h, and low flow rate (0.5 BV / h) causes chromatographic peak tailing, and the elution interval of target oligofructose (DP3~DP5) overlaps with higher degree of polymerization impurities (such as DP6+), resulting in a decrease in purity; at the same time, adsorption instability causes a decrease in yield. High flow rate (2.5 BV / h) causes the target components to penetrate the chromatographic column without being fully adsorbed, resulting in a significant decrease in yield, and impurities are mixed due to elution peak distortion, which also affects the purity.

[0101] As can be seen from the test data of Example 1, Example 12 and Example 13, the elution speed of deionized water is adjusted to 1 mL / min and 15 mL / min, and when the elution speed is too low (1 mL / min), desorption is slow, and part of the target product is retained in the chromatographic column, resulting in a decrease in yield, and impurities are mixed due to elution peak tailing, resulting in a decrease in purity; when the elution speed is too high (15 mL / min), it causes turbulent flow effect, which destroys the stability of calcium ions and resin, and the elution peak shape is poor, the target components are not fully collected, the yield is reduced, and the pressure difference in the column caused by high flow rate may also damage the column efficiency, and both the purity and the yield are negatively affected.

[0102] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A process for the preparation of high purity oligofructose, characterized in that, The preparation process comprises: (I) taking fresh sugarcane juice, obtaining the sugarcane juice, and sequentially performing activated carbon decolorization, ultrafiltration gel removal, high-temperature enzyme inactivation, electrodialysis desalination and enzymatic reaction on the sugarcane juice to obtain an enzymatic reaction product; (II) performing membrane separation and purification on the enzymatic reaction product to collect a retentate; adsorbing oligofructose in the retentate by using a calcium type gel chromatography resin, and then eluting and collecting the eluate by using deionized water; (III) performing double-effect falling film evaporation concentration on the collected eluate to obtain a concentrated solution, performing spray drying on the concentrated solution, and screening to obtain a product rich in oligofructose.

2. The process for the preparation of high purity oligofructose according to claim 1, characterized in that, In step (I), the operation process of the activated carbon decolorization comprises: performing coarse filtration on the sugarcane juice to remove fiber residues and large particle suspensions in the sugarcane juice, collecting a filtrate, adding activated carbon powder to the filtrate, mixing, stirring and heating, and then filtering out the activated carbon powder to obtain a decolorized solution; the amount of the activated carbon powder added is 0.5-1.5 wt% of the mass of the filtrate; the temperature of the mixing, stirring and heating is 70-80℃; the time of the mixing, stirring and heating is 20-40 min; the rotating speed of the mixing, stirring and heating is 100-200 rpm.

3. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (I), the operation process of the ultrafiltration gel removal comprises: feeding the decolorized solution obtained after the activated carbon decolorization into a polyether sulfone ultrafiltration membrane system for cyclic filtration until the turbidity of the filtered solution is ≤1.0 NTU, and then collecting the filtered solution, which is a gel-removed solution; the molecular weight cut-off of the polyether sulfone ultrafiltration membrane system is 80-100 kDa; the pressure of the cyclic filtration is 0.4-0.6 MPa; the temperature of the cyclic filtration is 40-50℃.

4. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (I), the heating temperature of the high-temperature enzyme inactivation is 90-95℃; the holding time of the high-temperature enzyme inactivation is 1-3 min.

5. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (I), the operation process of the electrodialysis desalination comprises: diluting the enzyme inactivation solution obtained after the high-temperature enzyme inactivation with water, and then feeding it into an electrodialysis device for cyclic desalination, sampling and detecting the conductivity of the effluent, and collecting the effluent when the conductivity of the effluent is ≤100 μS / cm, which is a desalted solution; the enzyme inactivation solution is diluted with water to a solid content of 8-12 wt%; the operating voltage of the electrodialysis device is 15-25 V; The flow rate of the enzyme-killing solution in the electrodialysis device is 30-50 L / h·m 2 Membrane area.

6. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (I), the operation process of the enzymatic reaction comprises: adding a citric acid / acetic acid buffer solution to the desalted solution obtained after the electrodialysis desalination to adjust the pH value thereof, and then adding a calcium chloride solution, mixing uniformly to obtain an enzymatic substrate; adding β-fructofuranosidase to the enzymatic substrate, mixing, stirring and heating to cause an enzymatic reaction, so that the sucrose in the enzymatic substrate is converted into oligofructose, and the pH value of the enzymatic system is controlled at 6.3-6.7 during the enzymatic reaction; sampling and detecting the sucrose concentration during the reaction, and immediately inactivating the enzyme when the sucrose conversion rate is ≥95%; and then cooling and filtering to obtain the enzymatic reaction product; the pH value of the desalted solution is adjusted to 6-6.5 by adding a citric acid / acetic acid buffer solution thereto; the ratio of calcium chloride to deionized water in the calcium chloride solution is (1-5) g:100 mL; The concentration of calcium ions in the enzymatic substrate is 0.5-1 mmol / L; The amount of the added β-furanofructosidase is 40-50 U / g of sucrose in the enzymatic substrate; The temperature of the enzymatic reaction is 50-55 ℃; When the sucrose conversion rate is ≥95%, the temperature is immediately increased to 80-90 ℃ and kept for 10-20 min; After the enzyme inactivation is completed, the temperature is decreased to 40-50 ℃, and then the filtration is performed in a plate and frame filter.

7. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (II), the operation process of the membrane separation and purification comprises: The enzymatic product is filtered through a nanofiltration membrane, and the retentate is collected, and the retentate is diluted with water to obtain the retentate liquid; The molecular weight cut-off of the nanofiltration membrane is 250-300 Da; The operation pressure of the filtration is 1-2 MPa; The operation temperature of the filtration is 30-40 ℃; The retentate is diluted with water to a solid content of 20-30 wt%.

8. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (II), the calcium-type gel chromatography resin is loaded into a chromatography column with a diameter-height ratio of 1:(4-6); The flow rate of the retentate liquid is 1-2 BV / h; The elution rate of the deionized water is 5-10 mL / min; During the deionized water elution process, online HPLC is used to monitor the composition of the eluent, when the peak area of oligofructose DP3, DP4 and DP5 reaches 90% of the total sugar peak area, the eluent is collected, and when the peak area of oligofructose DP3, DP4 and DP5 is less than 80% of the total sugar peak area, the collection is stopped, and the collected eluent is the eluent rich in oligofructose.

9. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (III), the double-effect falling film evaporation concentration is performed in a double-effect falling film evaporator, and the double-effect falling film evaporator comprises a one-effect evaporator and a two-effect evaporator connected in series; The operation temperature of the one-effect evaporator is 70-80 ℃; The operation pressure of the one-effect evaporator is -0.08--0.09 MPa; The operation temperature of the two-effect evaporator is 60-70 ℃; The operation pressure of the two-effect evaporator is -0.085--0.098 MPa; The solid content of the concentrated liquid is 40-50 wt%.

10. The process for preparing high purity oligofructose according to claim 1, characterized in that, In step (III), the inlet air temperature of the spray drying is 170-180 ℃; The outlet air temperature of the spray drying is 75-85 ℃; The feeding rate of the concentrated liquid is 20-40 L / h; The mesh number of the screen is 80-100 meshes.

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