An integrated method for co-production of soy peptides and soy oligosaccharides using soy molasses

Abstract

The application discloses an integrated method for co-production of soybean peptides and soybean oligosaccharides by using soybean molasses, and belongs to the technical field of deep processing and comprehensive utilization of agricultural and sideline products. The application systematically couples three technologies of enzymatic pretreatment, multi-stage membrane separation and simulated moving bed chromatography purification. Firstly, the high viscosity problem of soybean molasses is solved by specific complex enzyme treatment. Then, the accurate screening of a three-stage membrane system of microfiltration, ultrafiltration and nanofiltration is used to realize efficient coarse separation of proteins and oligosaccharides. Finally, the simulated moving bed technology with suitable chromatographic medium and conditions is used for deep purification according to the different characteristics of the two products, so that high-purity soybean peptides and soybean oligosaccharides are obtained.

Description

Technical Field

[0001] This invention belongs to the technical field of intensive processing and comprehensive utilization of agricultural and sideline products, specifically relating to an integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses. Background Technology

[0002] Soybean molasses is a major byproduct of soybean protein production, with a huge annual output. It contains 25-35 wt% protein, 15-25 wt% functional oligosaccharides, and 20-35 wt% sucrose. Currently, its comprehensive utilization is insufficient, and it is mostly used as animal feed or disposed of as waste, resulting in serious resource waste.

[0003] Existing methods for separating proteins and sugars from soybean molasses, such as isoelectric point precipitation, suffer from problems including low protein recovery rates, demanding separation conditions, easy inactivation of functional components, discontinuous processes, poor product purity, and difficulty in simultaneously obtaining high-purity multi-component solutions. Traditional membrane technologies such as ultrafiltration and nanofiltration face technical bottlenecks when processing high-viscosity, complex soybean molasses systems, including severe membrane fouling, rapid flux decline, and incomplete separation due to molecular weight overlap between sugars and proteins. For example, Chinese patent CN105132335A discloses an ultrafiltration separation method, but it cannot solve the problems of continuous and stable operation and product purity.

[0004] Therefore, developing an efficient and continuous integrated technology that can simultaneously obtain high-purity soybean peptides and high-purity soybean oligosaccharides from soybean molasses is of great significance for enhancing the value of the soybean processing industry chain and realizing the full utilization of resources. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses. Its core lies in the systematic coupling of three technologies: enzymatic pretreatment, multi-stage membrane separation, and simulated moving bed chromatography purification. First, the high viscosity of soybean molasses is overcome through specific complex enzyme treatment. Then, precise sieving using a three-stage membrane system of microfiltration, ultrafiltration, and nanofiltration achieves efficient crude separation of proteins and oligosaccharides. Finally, based on the different characteristics of the two products, deep purification is performed using simulated moving bed technology with appropriate chromatographic media and conditions to obtain high-purity soybean peptides and soybean oligosaccharides.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide an integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses, comprising the following steps: (1) Dilute soybean molasses with water to a dry matter content of 8-15 wt%, adjust the pH to 6.5-8.0, add a compound enzyme, and enzymatically hydrolyze at 40-50℃ for 1-3 hours to obtain an enzymatic hydrolysate; the compound enzyme contains at least pectinase and cellulase. (2) Pass the enzymatic hydrolysate through a microfiltration membrane and collect the microfiltration permeate; pass the microfiltration permeate through an ultrafiltration membrane and collect the ultrafiltration permeate as a protein concentrate; pass the ultrafiltration permeate through a nanofiltration membrane and collect the nanofiltration permeate as an oligosaccharide concentrate. (3) Adjust the pH of the protein concentrate to 6.5-8.5, add protease for enzymatic hydrolysis to obtain soybean peptide hydrolysate, and purify the soybean peptide hydrolysate using a multifunctional simulated moving bed chromatography separation system to obtain the soybean peptide; (4) The oligosaccharide concentrate is decolorized, desalted and concentrated to obtain a pretreated oligosaccharide concentrate. The pretreated oligosaccharide concentrate is separated using an 8-column sequential simulated moving bed chromatography separation system. The residue is dried to obtain the soybean oligosaccharide.

[0007] Preferably, the amount of pectinase added in step (1) is 0.01~0.05% of the substrate weight, and the amount of cellulase added is 0.005~0.02% of the substrate weight.

[0008] Preferably, the complex enzyme in step (1) further includes β-glucanase, and the amount of β-glucanase added is 0.002~0.01% of the substrate mass.

[0009] Preferably, in step (2), the microfiltration membrane has a molecular weight cutoff of 50-100 kDa, an operating pressure of 0.15-0.4 MPa, and an operating temperature of 45-50°C; the ultrafiltration membrane has a molecular weight cutoff of 5-10 kDa, an operating pressure of 0.35-1.0 MPa, and an operating temperature of 50-60°C; and the nanofiltration membrane has a molecular weight cutoff of 0.2-0.5 kDa, an operating pressure of 1.0-1.5 MPa, and an operating temperature of 50-60°C.

[0010] Preferably, the microfiltration, ultrafiltration and nanofiltration in step (2) operate in cross-flow mode with a cross-flow velocity of 2~5m / s.

[0011] Preferably, the protease in step (3) is an alkaline protease or papain, and the amount added is 1.8 to 2.5% of the protein mass in the protein concentrate.

[0012] Preferably, the specific steps for purifying the soybean peptide hydrolysate using a multifunctional simulated moving bed chromatography system in step (3) include: adjusting the pH of the soybean peptide hydrolysate to 6.0-7.0 and diluting it to a dry matter content of 10-20 wt% to obtain a feed solution; using a 20-column multifunctional simulated moving bed chromatography system with XAD-7, HP2MG, or SP-850 macroporous adsorption resin as the chromatographic medium; the 20-column multifunctional simulated moving bed chromatography system adopts a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone; the chromatographic columns in each zone are 6, 3, 5, 3, and 3 respectively, and all zones are connected in series; each zone has one inlet and one outlet, and the feed inlets for the adsorption zone, purification zone, desorption zone, and regeneration zone are... The feed inlet for each zone is the first column in a clockwise direction, and the feed inlet for the washing zone is the last column in that zone in a clockwise direction. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves one column position clockwise. This sequential operation achieves continuous operation, continuously collecting soybean peptide fractions from the desorption zone outlet. The materials for the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in order, feed liquid, water, 15-25 vol% ethanol aqueous solution, 85-90 vol% ethanol aqueous solution, and water. The flow rates for each zone are, in order, 1-3 BV / h, 5-8 BV / h, 5-10 BV / h, 5-10 BV / h, and 5-12 BV / h. The operating temperature is 25-40℃, and the switching time is 15-25 min.

[0013] Preferably, step (4) of separating the pretreated oligosaccharide concentrate using an 8-column sequential simulated moving bed chromatography system includes: The pretreated oligosaccharide concentrate is used as the feed liquid. The chromatographic separation medium is a potassium-type, sodium-type, or calcium-type strong acid cation exchange resin. The temperature of the feed liquid and eluent is controlled at 60~70℃. The eluent is water. The mass ratio of the feed liquid to the eluent is 1:2. The circulation volume is 40% of the column volume. The mass ratio of oligosaccharide component / monosaccharide + disaccharide component is controlled at 3. The operation mode of the 8-column sequential simulated moving bed chromatography separation system is as follows: (a) the feed liquid, the eluent, and the product are introduced simultaneously; the eluent is introduced at the upper end of column 1, and component A, mainly containing monosaccharides and disaccharides, is discharged at the lower end of column 2; the feed liquid is introduced at column 5, and the raffinate, mainly containing soybean oligosaccharides, is discharged at the lower end of column 6; (b) no material is introduced or discharged, and a large circulation is performed, with the liquid flow direction from column 1 to column 8; (c) the eluent is introduced at the upper end of column 1, and the raffinate, mainly containing soybean oligosaccharides, is discharged at the lower end of column 6; after the first run, steps (a) to (c) are repeated in sequence, except that the inlet and outlet positions are moved to the next column from the direction of liquid flow, and this procedure is followed thereafter.

[0014] The technical principle of this invention is as follows: Targeted Complex Enzyme Pretreatment System: This invention is the first to design and employ a complex enzyme system containing pectinase, cellulase, and optionally β-glucanase for pretreatment of soybean molasses, which are the main components causing high viscosity (pectin, cellulose, and β-glucan). This treatment specifically degrades these polysaccharides, significantly reducing the viscosity of the feed solution by 40-60%, fundamentally improving the hydrodynamic conditions of subsequent membrane separation, increasing membrane flux by 2-3 times, and greatly reducing membrane fouling.

[0015] Innovative Design and Parameter Optimization of a Three-Stage Membrane Separation System: This invention creatively designs a three-stage membrane separation process and optimizes the molecular weight cutoff and operating parameters of each membrane stage. The first-stage microfiltration (50~100kDa) removes large particulate impurities and protects subsequent membrane modules; the second-stage ultrafiltration (5~10kDa) primarily retains proteins; and the third-stage nanofiltration (0.2~0.5kDa) precisely retains target oligosaccharides (raffinose, stachyose) while allowing most monosaccharides and sucrose to pass through. This design achieves molecular weight gradient separation, resulting in a protein recovery rate ≥90wt%, an oligosaccharide recovery rate ≥85wt%, and minimal cross-contamination.

[0016] The highly efficient synergy between membrane separation and chromatographic purification: Membrane separation, as a highly efficient primary separation method, removes most impurities, providing a high-quality feed solution for subsequent chromatographic purification. This not only reduces the burden on the chromatographic system and extends the lifespan of the chromatographic media, but also allows the chromatographic process to focus more on the fine separation of the target product, thereby significantly improving the purity and yield of the final product. This synergistic effect of "membrane coarse separation + chromatographic purification" is the key to the high efficiency and high quality of the preparation process of this invention.

[0017] Continuous production design throughout the entire process: The cross-flow membrane filtration and sequential simulated moving bed chromatography used in this invention are both continuous or semi-continuous operation units. By integrating them, near-continuous production from raw materials to products is achieved, increasing production efficiency by more than 50% compared to traditional batch processes, making it more suitable for large-scale industrial production.

[0018] The beneficial technical effects of the present invention are as follows: High resource utilization and high product value: This invention achieves efficient simultaneous extraction and purification of the two main functional components in soybean molasses, with an overall resource utilization rate of 80-85%, producing high-purity and high-activity end products, resulting in significant economic benefits.

[0019] High separation efficiency and high product purity: This invention combines multi-stage membrane separation with dedicated chromatographic purification, resulting in high recovery rates of soybean peptides and oligosaccharides, and product purity exceeding 92%, demonstrating excellent quality.

[0020] Mild process conditions and good product activity: The entire preparation process of this invention is carried out under mild pH and temperature conditions, avoiding the destruction of the functional activity of proteins and oligosaccharides by strong acids, strong alkalis and high temperatures.

[0021] The production process is continuous and highly automated: the process method provided by this invention is easy to automate, has good production stability, and low labor costs.

[0022] Energy saving and environmental protection: The membrane separation process provided by this invention consumes far less energy than traditional evaporation and concentration, and reduces the use of chemical reagents and wastewater discharge, which meets the requirements of green manufacturing. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0024] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0025] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The soybean molasses used in this invention is a byproduct of the production process of soybean protein concentrate, and mainly consists of oligosaccharides, sucrose, and protein; the activity of pectinase is 30,000 u / g, the activity of cellulase is 100,000 u / g, and the activity of β-glucanase is 100,000 u / g.

[0029] Example 1 (1) Pretreatment: Take soybean molasses, dilute it with water to a dry matter content of 10wt%, adjust the pH to 7.0 with NaOH solution, and heat to 45℃. Add 0.03% pectinase and 0.01% cellulase by substrate mass, stir and react for 2h to obtain enzymatic hydrolysate.

[0030] (2) Multistage membrane separation (cross-flow mode, cross-flow velocity of 5 m / s): First-stage microfiltration: The enzymatic hydrolysate is pumped into a 50 kDa ceramic membrane system at an operating pressure of 0.2 MPa and a temperature of 45°C. The permeate is collected. Second-stage ultrafiltration: The microfiltration permeate is pumped into an 8 kDa ultrafiltration membrane system at an operating pressure of 0.4 MPa and a temperature of 50°C. The retentate is collected as a protein concentrate (9.5 wt%), and the permeate is fed to the next stage. Third-stage nanofiltration: The ultrafiltration permeate is pumped into a 0.3 kDa aromatic polyamide nanofiltration membrane system at an operating pressure of 1.2 MPa and a temperature of 50°C. The retentate is collected as an oligosaccharide concentrate (6.2 wt%).

[0031] (3) Enzymatic hydrolysis: Adjust the pH of the protein concentrate to 8.0, add 2.0% alkaline protease by protein mass, stir and hydrolyze in a 55℃ water bath for 3 hours, and control the degree of hydrolysis to about 30% by sampling to obtain soybean peptide hydrolysate.

[0032] (4) Purification of soybean peptides (using a multifunctional simulated moving bed chromatography system): The pH of the soybean peptide hydrolysate was adjusted to 7.0 and diluted to a dry matter content of 10 wt% to obtain the feed solution. A 20-column multifunctional simulated moving bed chromatography system was used, with XAD-7 macroporous adsorption resin as the chromatographic medium. The 20-column multifunctional simulated moving bed chromatography system adopted a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone. The chromatographic columns in each zone were 6, 3, 5, 3, and 3 respectively, and all zones were connected in series. Each zone had one inlet and one outlet. The inlets of the adsorption zone, purification zone, desorption zone, and regeneration zone were the first chromatographic column in the clockwise direction of each zone, and the inlet of the washing zone was the last chromatographic column in the clockwise direction of that zone. The column operates simultaneously in all zones during operation. When the running time reaches the switching time, each column moves clockwise by one column position, and this process is repeated sequentially to achieve continuous operation. Soybean peptide fractions are continuously collected from the desorption zone outlet. The materials in the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in sequence, feed liquid, deionized water, 20 vol% ethanol aqueous solution, 90 vol% ethanol aqueous solution, and deionized water. The flow rates in each zone are 2 BV / h, 6 BV / h, 8 BV / h, 8 BV / h, and 10 BV / h, in sequence. The operating temperature is 30℃, and the switching time is 22 min.

[0033] (5) Oligosaccharide purification (using a sequential simulated moving bed chromatographic separation system): The oligosaccharide concentrate is decolorized with activated carbon and desalted by ion exchange, and then concentrated to a dry matter content of 60% to obtain the feed solution. The chromatographic system uses an 8-column configuration, operates in a sequential simulated moving bed mode, uses potassium-type strong acid cation exchange resin as the chromatographic separation medium, controls the temperature of the feed solution and eluent at 65℃, and uses deionized water as the eluent. The specific operation consists of the following three steps: a: Simultaneous feeding of feed liquid, eluent, and discharge; this step is also called all-in, all-out. Eluent enters at the top of column 1, and component A (monosaccharide and disaccharide components) exits at the bottom of column 2. Feed liquid enters at column 5, and a portion of component B (oligosaccharide component) exits at the bottom of column 6, i.e., oligosaccharide fraction. b: The material does not enter or exit, but undergoes a large circulation, with the liquid flow direction from column 1 to column 8; c: This step is a small loop, only the eluent deionized water is introduced. The eluent enters at the upper end of column 1, and another part of component B (oligosaccharide component) exits at the lower end of column 6, that is, the oligosaccharide fraction; After the first run, repeat steps a to c in sequence, except that the inlet and outlet positions are moved to the next column in the direction of liquid flow. Follow this procedure for all subsequent runs.

[0034] The ratio of the mass of the system feed liquid to the total mass of the eluent in the two injections is 1:2 (the amount of eluent in the two injections is allocated based on a mass ratio of oligosaccharide component / monosaccharide + disaccharide component of 3). The circulation volume is 40% of the column volume. By controlling the concentration and purity of the outlet feed liquid, the mass ratio of oligosaccharide component / monosaccharide + disaccharide component is kept at 3.

[0035] (6) Post-processing: Soybean peptide fractionation: After desalting and vacuum concentration to remove ethanol, the product is spray-dried to obtain a white powdery soybean peptide product.

[0036] Oligosaccharide fraction: After decolorization and concentration, vacuum drying is performed to obtain a white crystalline soybean oligosaccharide product.

[0037] Product testing results: Soybean peptide products: The peptide content is 93.5 wt%, and the proportion of peptides with a molecular weight of 1000~3000 Da is 68.1 wt%.

[0038] Soybean oligosaccharide product: The oligosaccharide fraction concentration is 22.5wt%, the total content of raffinose and stachyose is 91.8wt%, the total content of monosaccharides and disaccharides is 2.5wt%, and the purity of soybean oligosaccharides is 93.2wt%.

[0039] Example 2 (1) Pretreatment: Take soybean molasses, dilute it with water to a dry matter content of 12wt%, adjust the pH to 7.5 with NaOH solution, and heat to 48℃. Add 0.04% pectinase, 0.015% cellulase and 0.005% β-glucanase by substrate mass, stir and react for 2.5h to obtain enzymatic hydrolysate.

[0040] (2) Multistage membrane separation (cross-flow mode, cross-flow velocity of 3 m / s): First-stage microfiltration: The enzymatic hydrolysate is pumped into an 80 kDa ceramic membrane system at an operating pressure of 0.4 MPa and a temperature of 48°C. The permeate is collected. Second-stage ultrafiltration: The microfiltration permeate is pumped into a 5 kDa ultrafiltration membrane system at an operating pressure of 0.8 MPa and a temperature of 55°C. The retentate is collected as a protein concentrate (8.8 wt%), and the permeate is fed to the next stage. Third-stage nanofiltration: The ultrafiltration permeate is pumped into a 0.2 kDa aromatic polyamide nanofiltration membrane system at an operating pressure of 1.5 MPa and a temperature of 55°C. The retentate is collected as an oligosaccharide concentrate (7.1 wt%).

[0041] (3) Enzymatic hydrolysis: Adjust the pH of the protein concentrate to 7.8, add 1.8% of the protein mass of papain, stir and hydrolyze in a 52℃ water bath for 4 hours, and control the degree of hydrolysis to about 25% by sampling to obtain soybean peptide hydrolysate.

[0042] (4) Purification of soybean peptides (using a multifunctional simulated moving bed chromatography system): The pH of the soybean peptide hydrolysate was adjusted to 6.5 and diluted to a dry matter content of 15 wt% to obtain the feed solution. A 20-column multifunctional simulated moving bed chromatography system was used, with HP2MG macroporous adsorption resin as the chromatographic medium. The 20-column multifunctional simulated moving bed chromatography system adopted a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone. The chromatographic columns in each zone were 6, 3, 5, 3, and 3 respectively, and all zones were connected in series. Each zone had one inlet and one outlet. The inlets of the adsorption zone, purification zone, desorption zone, and regeneration zone were the first chromatographic column in the clockwise direction of each zone, and the inlet of the washing zone was the last chromatographic column in the clockwise direction of that zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position. This process is repeated sequentially to achieve continuous operation, continuously collecting soybean peptide fractions from the desorption zone outlet. The materials for the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in sequence, feed liquid, deionized water, 25 vol% ethanol aqueous solution, 90 vol% ethanol aqueous solution, and deionized water. The flow rates for each zone are 1.5 BV / h, 5 BV / h, 7 BV / h, 7 BV / h, and 9 BV / h, in sequence. The operating temperature is 30℃, and the switching time is 25 min.

[0043] (5) Oligosaccharide purification (using a sequential simulated moving bed chromatographic separation system): The oligosaccharide concentrate is decolorized with activated carbon and desalted by ion exchange, then concentrated to a dry matter content of 55% to obtain the feed solution. The chromatographic system uses an 8-column configuration, operates in a sequential simulated moving bed mode, uses sodium-type strong acid cation exchange resin as the chromatographic separation medium, controls the temperature of the feed solution and eluent at 65℃, and uses deionized water as the eluent. The specific operation consists of the following three steps: a: Simultaneous feeding of feed liquid, eluent, and discharge; this step is also called all-in, all-out. Eluent enters at the top of column 1, and component A (monosaccharide and disaccharide components) exits at the bottom of column 2. Feed liquid enters at column 5, and a portion of component B (oligosaccharide component) exits at the bottom of column 6, i.e., oligosaccharide fraction. b: The material does not enter or exit, but undergoes a large circulation, with the liquid flow direction from column 1 to column 8; c: This step is a small loop, only the eluent deionized water is introduced. The eluent enters at the upper end of column 1, and another part of component B (oligosaccharide component) exits at the lower end of column 6, that is, the oligosaccharide fraction; After the first run, repeat steps a to c in sequence, except that the inlet and outlet positions are moved to the next column in the direction of liquid flow. Follow this procedure for all subsequent runs.

[0044] The ratio of the mass of the system feed liquid to the total mass of the eluent in the two injections is 1:1.8 (the amount of eluent in the two injections is allocated based on a mass ratio of oligosaccharide component / monosaccharide + disaccharide component of 2.8). The circulation volume is 38% of the column volume. By controlling the concentration and purity of the outlet feed liquid, the mass ratio of oligosaccharide component / monosaccharide + disaccharide component is kept at 2.8.

[0045] (6) Post-processing: Same as in Example 1.

[0046] Product testing results: Soybean peptide products: The peptide content is 91.2 wt%, and the proportion of peptides with a molecular weight of 1000~3000 Da is 76.3 wt%.

[0047] Soybean oligosaccharide product: Oligosaccharide fraction concentration is 21.2wt%, total content of raffinose and stachyose is 93.7wt%, total content of monosaccharides and disaccharides is 1.9wt%, and purity of soybean oligosaccharides is 95.8wt%.

[0048] Example 3 (1) Pretreatment: Take soybean molasses, dilute it with water to a dry matter content of 8wt%, adjust the pH to 6.5 with NaOH solution, and heat to 50℃. Add 0.02% pectinase, 0.01% cellulase and 0.003% β-glucanase by substrate mass, stir and react for 1.5h to obtain enzymatic hydrolysate.

[0049] (2) Multistage membrane separation (cross-flow mode, cross-flow velocity is 4m / s): First-stage microfiltration: The enzymatic hydrolysate is pumped into a 100 kDa organic microfiltration membrane at a cross-flow velocity of 4 m / s, operating pressure of 0.3 MPa, and temperature of 50°C. The permeate is collected. Second-stage ultrafiltration: The microfiltration permeate is pumped into a 10 kDa ultrafiltration membrane system at an operating pressure of 1.0 MPa and temperature of 60°C. The retentate is collected as a protein concentrate (7.5 wt%), and the permeate is fed to the next stage. Third-stage nanofiltration: The ultrafiltration permeate is pumped into a 0.5 kDa aromatic polyamide nanofiltration membrane system at an operating pressure of 1.2 MPa and temperature of 60°C. The retentate is collected as an oligosaccharide concentrate (8.5 wt%).

[0050] (3) Enzymatic hydrolysis: Adjust the pH of the protein concentrate to 8.5, add 2.5% alkaline protease by protein mass, stir and hydrolyze in a 58℃ water bath for 2.5h, and control the degree of hydrolysis to about 32% by sampling to obtain soybean peptide hydrolysate.

[0051] (4) Purification of soybean peptides (using a multifunctional simulated moving bed chromatography system): The pH of the soybean peptide hydrolysate was adjusted to 7.0 and diluted to a dry matter content of 12 wt% to obtain the feed solution. A 20-column multifunctional simulated moving bed chromatography system was used, with SP-850 macroporous adsorption resin as the chromatographic medium. The 20-column multifunctional simulated moving bed chromatography system adopted a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone. The chromatographic columns in each zone were 6, 3, 5, 3, and 3 respectively, and all zones were connected in series. Each zone had one inlet and one outlet. The inlets of the adsorption zone, purification zone, desorption zone, and regeneration zone were the first chromatographic column in the clockwise direction of each zone, and the inlet of the washing zone was the last chromatographic column in the clockwise direction of that zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves clockwise by one column position. This process is repeated sequentially to achieve continuous operation, continuously collecting soybean peptide fractions from the desorption zone outlet. The materials in the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in sequence, feed liquid, deionized water, 20 vol% ethanol aqueous solution, 85 vol% ethanol aqueous solution, and deionized water. The flow rates in each zone are 3 BV / h, 8 BV / h, 10 BV / h, 10 BV / h, and 12 BV / h, in sequence. The operating temperature is 35℃, and the switching time is 18 min.

[0052] (5) Oligosaccharide purification (using a sequential simulated moving bed chromatographic separation system): The oligosaccharide concentrate is decolorized with activated carbon and desalted by ion exchange, and then concentrated to a dry matter content of 60% to obtain the feed solution. The chromatographic system uses an 8-column configuration, operates in a sequential simulated moving bed mode, uses sodium-type strong acid cation exchange resin as the chromatographic separation medium, controls the temperature of the feed solution and eluent at 70℃, and uses deionized water as the eluent. The specific operation is divided into the following three steps: a: Simultaneous feeding of feed liquid, eluent, and discharge; this step is also called all-in, all-out. Eluent enters at the top of column 1, and component A (monosaccharide and disaccharide components) exits at the bottom of column 2. Feed liquid enters at column 5, and a portion of component B (oligosaccharide component) exits at the bottom of column 6, i.e., oligosaccharide fraction. b: The material does not enter or exit, but undergoes a large circulation, with the liquid flow direction from column 1 to column 8; c: This step is a small loop, only the eluent deionized water is introduced. The eluent enters at the upper end of column 1, and another part of component B (oligosaccharide component) exits at the lower end of column 6, that is, the oligosaccharide fraction; After the first run, repeat steps a to c in sequence, except that the inlet and outlet positions are moved to the next column in the direction of liquid flow. Follow this procedure for all subsequent runs.

[0053] The ratio of the mass of the system feed liquid to the total mass of the eluent in the two injections is 1:2.2 (the amount of eluent in the two injections is allocated based on a mass ratio of oligosaccharide component / monosaccharide + disaccharide component of 3.2). The circulation volume is 42% of the column volume. By controlling the concentration and purity of the outlet feed liquid, the mass ratio of oligosaccharide component / monosaccharide + disaccharide component is kept at 3.2.

[0054] (6) Post-processing: Same as in Example 1.

[0055] Product testing results: Soybean peptide products: The peptide content is 90.1 wt%, and the proportion of peptides with a molecular weight of 1000~3000 Da is 71.5 wt%.

[0056] Soybean oligosaccharide product: Oligosaccharide fraction concentration is 24.5wt%, total content of raffinose and stachyose is 92.5wt%, total content of monosaccharides and disaccharides is 2.8wt%, and purity of soybean oligosaccharides is 94.3wt%.

[0057] Example 4 (1) Pretreatment: Take soybean molasses, dilute it with water to a dry matter content of 12wt%, adjust the pH to 7.5 with NaOH solution, and heat to 45℃. Add 0.04% pectinase, 0.015% cellulase and 0.005% β-glucanase by substrate mass, stir and react for 2.5h to obtain enzymatic hydrolysate (viscosity reduced by about 55% compared to soybean molasses).

[0058] (2) Multistage membrane separation (cross-flow mode, cross-flow velocity of 3 m / s): First-stage microfiltration: The enzymatic hydrolysate is pumped into a 100 kDa ceramic membrane system at an operating pressure of 0.15 MPa and a temperature of 45°C. The permeate is collected. Second-stage ultrafiltration: The microfiltration permeate is pumped into a 10 kDa ultrafiltration membrane system at an operating pressure of 0.35 MPa and a temperature of 50°C. The retentate is collected as a protein concentrate (8.8 wt%), and the permeate proceeds to the next stage. Third-stage nanofiltration: The ultrafiltration permeate is pumped into a 0.5 kDa aromatic polyamide nanofiltration membrane system at an operating pressure of 1.0 MPa and a temperature of 50°C. The retentate is collected as an oligosaccharide concentrate (5.8 wt%).

[0059] (3) Enzymatic hydrolysis: Adjust the pH of the protein concentrate to 7.5, add 2.5% of the protein mass of papain, stir and hydrolyze in a 58℃ water bath for 2.5h, and control the degree of hydrolysis to about 28% by sampling to obtain soybean peptide hydrolysate.

[0060] (4) Purification of soybean peptides (using a multifunctional simulated moving bed chromatography system): The pH of the soybean peptide hydrolysate was adjusted to 6.5 and diluted to a dry matter content of 10 wt% to obtain the feed solution. A 20-column multifunctional simulated moving bed chromatography system was used, with XAD-7 macroporous adsorption resin as the chromatographic medium. The 20-column multifunctional simulated moving bed chromatography system adopted a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone. The chromatographic columns in each zone were 6, 3, 5, 3, and 3 respectively, and all zones were connected in series. Each zone had one inlet and one outlet. The inlets of the adsorption zone, purification zone, desorption zone, and regeneration zone were the first chromatographic column in the clockwise direction of each zone, and the inlet of the washing zone was the last chromatographic column in the clockwise direction of that zone. The column operates simultaneously in all zones during operation. When the running time reaches the switching time, each column moves clockwise by one column position, and this process is repeated sequentially to achieve continuous operation. Soybean peptide fractions are continuously collected from the desorption zone outlet. The materials in the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in sequence, feed liquid, deionized water, 18 vol% ethanol aqueous solution, 85 vol% ethanol aqueous solution, and deionized water. The flow rates in each zone are 2 BV / h, 6 BV / h, 8 BV / h, 8 BV / h, and 10 BV / h, in sequence. The operating temperature is 40℃, and the switching time is 22 min.

[0061] (5) Oligosaccharide purification (using a sequential simulated moving bed chromatographic separation system): The oligosaccharide concentrate is decolorized with activated carbon and desalted by ion exchange, then concentrated to a dry matter content of 60% to obtain the feed solution. The chromatographic system uses an 8-column configuration, operates in a sequential simulated moving bed mode, uses calcium-type strong acid cation exchange resin as the chromatographic separation medium, controls the temperature of the feed solution and eluent at 68℃, and uses deionized water as the eluent. The specific operation consists of the following three steps: a: Simultaneous feeding of feed liquid, eluent, and discharge; this step is also called all-in, all-out. Eluent enters at the top of column 1, and component A (monosaccharide and disaccharide components) exits at the bottom of column 2. Feed liquid enters at column 5, and a portion of component B (oligosaccharide component) exits at the bottom of column 6, i.e., oligosaccharide fraction. b: The material does not enter or exit, but undergoes a large circulation, with the liquid flow direction from column 1 to column 8; c: This step is a small loop, only the eluent deionized water is introduced. The eluent enters at the upper end of column 1, and another part of component B (oligosaccharide component) exits at the lower end of column 6, that is, the oligosaccharide fraction; After the first run, repeat steps a to c in sequence, except that the inlet and outlet positions are moved to the next column in the direction of liquid flow. Follow this procedure for all subsequent runs.

[0062] The ratio of the mass of the system feed liquid to the total mass of the eluent in the two injections is 1:2 (the amount of eluent in the two injections is allocated based on a mass ratio of oligosaccharide component / monosaccharide + disaccharide component of 3). The circulation volume is 40% of the column volume. By controlling the concentration and purity of the outlet feed liquid, the mass ratio of oligosaccharide component / monosaccharide + disaccharide component is kept at 3.

[0063] (6) Post-processing: Same as in Example 1.

[0064] Product testing results: Soybean peptide products: The peptide content is 92.5 wt%, and the proportion of peptides with a molecular weight of 1000~3000 Da is 73.8 wt%.

[0065] Soybean oligosaccharide product: Oligosaccharide fraction concentration is 23.8wt%, total content of raffinose and stachyose is 93.3wt%, total content of monosaccharides and disaccharides is 2.1wt%, and purity of soybean oligosaccharides is 95.2wt%.

[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses, characterized in that, Includes the following steps: (1) Dilute soybean molasses with water to a dry matter content of 8-15 wt%, adjust the pH to 6.5-8.0, add a compound enzyme, and enzymatically hydrolyze at 40-50℃ for 1-3 hours to obtain an enzymatic hydrolysate; the compound enzyme contains at least pectinase and cellulase. (2) Pass the enzymatic hydrolysate through a microfiltration membrane and collect the microfiltration permeate; The microfiltration permeate is passed through an ultrafiltration membrane and collected as a protein concentrate. The ultrafiltration permeate is passed through a nanofiltration membrane and collected as a concentrate of oligosaccharides. (3) Adjust the pH of the protein concentrate to 6.5-8.5, add protease for enzymatic hydrolysis to obtain soybean peptide hydrolysate, and purify the soybean peptide hydrolysate using a multifunctional simulated moving bed chromatography separation system to obtain the soybean peptide; (4) The oligosaccharide concentrate is decolorized, desalted and concentrated to obtain a pretreated oligosaccharide concentrate. The pretreated oligosaccharide concentrate is separated using an 8-column sequential simulated moving bed chromatography separation system. The residue is dried to obtain the soybean oligosaccharide.

2. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, In step (1), the amount of pectinase added is 0.01~0.05% of the substrate mass, and the amount of cellulase added is 0.005~0.02% of the substrate mass.

3. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, The complex enzyme in step (1) also includes β-glucanase, and the amount of β-glucanase added is 0.002~0.01% of the substrate mass.

4. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, The microfiltration membrane in step (2) has a molecular weight cutoff of 50~100kDa, an operating pressure of 0.15~0.4MPa, and an operating temperature of 45~50℃; the ultrafiltration membrane has a molecular weight cutoff of 5~10kDa, an operating pressure of 0.35~1.0MPa, and an operating temperature of 50~60℃; and the nanofiltration membrane has a molecular weight cutoff of 0.2~0.5kDa, an operating pressure of 1.0~1.5MPa, and an operating temperature of 50~60℃.

5. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, In step (2), the microfiltration, ultrafiltration and nanofiltration operate in cross-flow mode with a cross-flow velocity of 2~5m / s.

6. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, The protease mentioned in step (3) is an alkaline protease or papain, and the amount added is 1.8 to 2.5% of the protein mass in the protein concentrate.

7. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, The specific steps for purifying the soybean peptide hydrolysate using a multifunctional simulated moving bed chromatography system in step (3) include: adjusting the pH of the soybean peptide hydrolysate to 6.0-7.0 and diluting it to a dry matter content of 10-20 wt% to obtain the feed solution; using a 20-column multifunctional simulated moving bed chromatography system with XAD-7, HP2MG, or SP-850 macroporous adsorption resin as the chromatographic medium; the 20-column multifunctional simulated moving bed chromatography system adopts a 5-zone design, divided into an adsorption zone, a purification zone, a desorption zone, a regeneration zone, and a washing zone; the chromatographic columns in each zone are 6, 3, 5, 3, and 3 respectively, and all zones are connected in series; each zone has one inlet and one outlet, and the inlets of the adsorption zone, purification zone, desorption zone, and regeneration zone are all... The feed inlet of the washing zone is the first column in the clockwise direction in each zone, and the feed inlet of the washing zone is the last column in the clockwise direction in that zone. During operation, all zones work simultaneously. When the running time reaches the switching time, each column moves one column position clockwise. This process is repeated sequentially to achieve continuous operation. Soybean peptide fractions are continuously collected from the desorption zone outlet. The materials for the adsorption zone, purification zone, desorption zone, regeneration zone, and washing zone are, in order, feed liquid, water, 15-25 vol% ethanol aqueous solution, 85-90 vol% ethanol aqueous solution, and water. The flow rates for each zone are, in order, 1-3 BV / h, 5-8 BV / h, 5-10 BV / h, 5-10 BV / h, and 5-12 BV / h. The operating temperature is 25-40℃, and the switching time is 15-25 min.

8. The integrated method for co-producing soybean peptides and soybean oligosaccharides using soybean molasses according to claim 1, characterized in that, Step (4) involves separating the pretreated oligosaccharide concentrate using an 8-column sequential simulated moving bed chromatography system, including: The pretreated oligosaccharide concentrate is used as the feed liquid. The chromatographic separation medium is a potassium-type, sodium-type, or calcium-type strong acid cation exchange resin. The temperature of the feed liquid and eluent is controlled at 60~70℃. The eluent is water. The mass ratio of the feed liquid to the eluent is 1:

2. The circulation volume is 40% of the column volume. The mass ratio of oligosaccharide component / monosaccharide + disaccharide component is controlled at 3. The operation mode of the 8-column sequential simulated moving bed chromatography separation system is as follows: (a) the feed liquid, the eluent, and the product are introduced simultaneously; the eluent is introduced at the upper end of column 1, and component A, mainly containing monosaccharides and disaccharides, is discharged at the lower end of column 2; the feed liquid is introduced at column 5, and the raffinate, mainly containing soybean oligosaccharides, is discharged at the lower end of column 6; (b) no material is introduced or discharged, and a large circulation is performed, with the liquid flow direction from column 1 to column 8; (c) the eluent is introduced at the upper end of column 1, and the raffinate, mainly containing soybean oligosaccharides, is discharged at the lower end of column 6; after the first run, steps (a) to (c) are repeated in sequence, except that the inlet and outlet positions are moved to the next column from the direction of liquid flow, and this procedure is followed thereafter.