A method for separating stachyose from soybean whey water by fractional filtration

Abstract

This invention discloses a method for separating stachyose from soybean whey through graded filtration, comprising the following steps: Step 1, whey pretreatment; weigh soybeans, soak them in water at a ratio of 1:5 for 14-16 hours, remove the water, add water at 9 times the volume of dry soybeans to grind into a slurry, filter out the soybean residue, heat the soy milk in a water bath to 90°C and maintain for 10 minutes to boil the soy milk, then add a coagulant, with calcium sulfate added at 2% of the dry soybeans, keep warm for 12 minutes, and then press it in a mold, finally collecting the whey; this invention found that 260g of soybeans will produce about 1L of whey, and after graded filtration, about 80mL of crude stachyose extract can be collected, which is then freeze-dried to obtain about 0.8g of solids. The final stachyose purity was determined by HPLC-ELSD to be 42.6%. Although some stachyose is lost during the graded filtration process, the purity of stachyose in the crude stachyose extract obtained by this invention is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of stachyose extraction technology, specifically relating to a method for separating stachyose from soybean yellow pulp water by graded filtration. Background Technology

[0002] During the tofu-making process, a large amount of yellow liquid containing flavonoids and pigments is discharged after pressing and filtration; this is called whey. Whey contains a wealth of nutrients, such as protein, oligosaccharides, minerals, and isoflavones. In recent years, the recovery of functional oligosaccharides from whey has become a research hotspot, and more and more people are beginning to notice the development prospects of whey in extracting stachyose.

[0003] Membrane technology offers advantages such as high separation efficiency, low energy consumption, simple operation, and no secondary pollution, thus showing promising prospects in the separation and purification of oligosaccharides. Membrane separation technology refers to the separation of solution systems on the surface of an organic membrane under pressure. Small molecule solutes, such as solvents, pass through the organic membrane via an asymmetric microporous mechanism, while large molecule solutes or particles are trapped on the membrane surface. The asymmetric structure of the membrane and the high-speed flow of materials and liquids within the device prevent trapped substances from clogging the membrane pores, allowing for long-term membrane use. Ultrafiltration technology offers advantages such as energy efficiency and high performance, making its application in the extraction of soybean oligosaccharides significant economic and social benefits. Nanofiltration membranes typically have a molecular cutoff of 200–1000 D and are widely used in the separation and concentration of food products, especially in the separation and purification of functional oligosaccharides. In summary, due to the advantages of membrane separation technology compared to other separation and purification methods, such as large throughput, high separation efficiency, and no secondary pollution, this study selected a combination of ultrafiltration and nanofiltration for the staged filtration of yellow slurry water. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the existing defects and provide a method for graded filtration and separation of stachyose in soybean slurry water, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for separating stachyose from soybean whey through graded filtration, comprising the following steps:

[0006] Step 1: Pretreatment of yellow slurry;

[0007] Weigh soybeans and soak them in a soybean-to-water ratio of 1:5 for 14-16 hours. After removing the water, add water at a volume of 9 times that of the dry soybeans and grind them into a slurry. Filter out the soybean residue and heat the soy milk in a water bath to 90°C for 10 minutes. Then add a coagulant, with calcium sulfate added at 2% of the dry soybean volume. Keep warm for 12 minutes and then press the mixture into a mold. Finally, collect the yellow slurry. Store the prepared yellow slurry in a refrigerator at 4°C for later use. Pre-treat the soybeans using chitosan flocculation method, with a settling time of 68 minutes, pH=5.5, settling temperature of 30.9°C, and chitosan addition of 0.7 g / L.

[0008] Step 2: Selection of ultrafiltration and nanofiltration membrane elements;

[0009] 2.1 Primary Ultrafiltration Membrane Element: A polyethersulfone ultrafiltration membrane element with a molecular weight cutoff of 10000D was selected to perform primary ultrafiltration on the pretreated yellow slurry water;

[0010] 2.2 Secondary ultrafiltration membrane element: The permeate after ultrafiltration by the primary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 5000D, 6000D, and 8000D respectively. The permeate is collected, and the secondary ultrafiltration membrane element is determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0011] 2.3 Tertiary ultrafiltration membrane elements: The permeate after ultrafiltration by the secondary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 1000D, 2000D, and 3000D respectively. The tertiary ultrafiltration membrane elements are determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0012] 2.4 Nanofiltration membrane element: The permeate after ultrafiltration is subjected to nanofiltration. Polyamide nanofiltration membrane elements with molecular weight cutoffs of 150D, 200D, 300D, 500D, and 600D can be selected. Referring to the molecular weight of stachyose, a nanofiltration membrane element with a molecular weight cutoff of 500D is selected, and the retentate is collected.

[0013] Step 3: Selection of tiered filtration conditions;

[0014] After selecting the ultrafiltration and nanofiltration membrane elements, the staged filtration conditions are selected. The effects of operating pressure, yellow slurry temperature and pH value on the stachyose content after staged filtration are investigated to determine the optimal staged filtration conditions.

[0015] 3.1 Selection of operating pressure: Take an equal volume of pretreated yellow pulp water sample and maintain the sample at 20°C and pH=5.5. Adjust the ultrafiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, and 0.7MPa respectively. Pass the sample through primary, secondary, and tertiary ultrafiltration membrane elements, collect the ultrafiltration permeate, and determine the stachyose retention rate. Under the optimal ultrafiltration pressure, maintain the same temperature and pH conditions for nanofiltration. Adjust the nanofiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, 0.7MPa, 0.9MPa, and 1.1MPa respectively. Pass the ultrafiltration permeate through a nanofiltration membrane element, collect the nanofiltration retentate, and determine the stachyose content in the nanofiltration retentate using high performance liquid chromatography. Calculate the stachyose retention rate to determine the optimal operating pressure for staged filtration of yellow pulp water.

[0016] 3.2 Selection of Yellow Slurry Water Temperature: Take an equal volume of pretreated yellow slurry water sample, adjust the pH of the sample to 5.5, and set the temperatures to 10°C, 20°C, 30°C, and 40°C respectively, controlling the operating pressure to 0.3 MPa. Pass the sample through ultrafiltration and nanofiltration membrane elements in sequence, collect the retentate, and determine its stachyose content. Calculate the stachyose retention rate to determine the optimal temperature for staged filtration of yellow slurry water.

[0017] 3.3 Selection of pH value for yellow pulp water: Take an equal volume of pretreated yellow pulp water sample solution, keep the sample solution at a temperature of 20°C, adjust the pH value to 4.5, 5.5, 6.5, 7.5 and 8.5 respectively, control the operating pressure to 0.3MPa, and pass it through ultrafiltration and nanofiltration membrane elements in sequence. Collect the retentate, determine its stachyose content, calculate the stachyose retention rate, and thus determine the optimal pH value for staged filtration of yellow pulp water;

[0018] Step 4: Determination of stachyose purity;

[0019] The crude stachyose extract after fractionation and filtration was freeze-dried. 5 mg of the dried sample was weighed and added to 10 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.5 mg / mL. 1 mg of stachyose standard was weighed and added to 5 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.2 mg / mL. The solution was filtered through a 0.45 μm filter membrane and analyzed by high performance liquid chromatography with an injection volume of 10 μL.

[0020] Preferably, oligosaccharides in tofu whey can be extracted through ion exchange resin treatment, attapulgite decolorization, vacuum concentration, and protein removal.

[0021] Preferably, oligosaccharides can be extracted from soybean water by high-temperature centrifugation to remove protein, electrodialysis to desalinate, and activated carbon to decolorize, with a final oligosaccharide retention rate of 80.41%.

[0022] Preferably, soybean oligosaccharides in tofu whey can be extracted by ultrafiltration (10000D), activated carbon decolorization, electrodialysis, and ion exchange resin desalination.

[0023] Preferably, soybean oligosaccharide powder can be obtained by extracting oligosaccharides from yellow slurry water through flocculation sedimentation, centrifugation, microfiltration pretreatment, followed by ultrafiltration (10000D) and nanofiltration (500D), with a final stachyose recovery rate of 29.03%.

[0024] Preferably, soybean wastewater is used as raw material, and oligosaccharides are extracted through ultrafiltration, reverse osmosis and nanofiltration technologies, with a final stachyose recovery rate of 24.70%.

[0025] Compared with the prior art, the present invention provides a method for separating stachyose from soybean whey through graded filtration, which has the following beneficial effects:

[0026] This invention found that 260g of soybeans will produce about 1L of yellow liquid. After graded filtration, about 80mL of crude stachyose extract can be collected. After freeze-drying, about 0.8g of solids is obtained. The final purity of stachyose was determined to be 42.6% by HPLC-ELSD. Although some stachyose was lost during the graded filtration process, the purity of stachyose in the crude stachyose extract obtained by this invention was significantly improved. Attached Figure Description

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0028] Figure 1 This is a schematic diagram of the decay curve of the permeate flux of a secondary ultrafiltration membrane element.

[0029] Figure 2 This is a schematic diagram of the decay curve of the permeate flux of a three-stage ultrafiltration membrane element.

[0030] Figure 3 This is a schematic diagram showing the protein removal rate and stachyose retention rate of a secondary ultrafiltration membrane element.

[0031] Figure 4 A schematic diagram showing the protein removal rate and stachyose retention rate of a three-stage ultrafiltration membrane element;

[0032] Figure 5 A schematic diagram showing the effect of operating pressure on the retention rate of stachyose after ultrafiltration;

[0033] Figure 6 A schematic diagram showing the effect of operating pressure on the retention rate of stachyose after nanofiltration.

[0034] Figure 7This is a schematic diagram showing the effect of yellow pulp water temperature on the stachyose retention rate after graded filtration.

[0035] Figure 8 A schematic diagram showing the effect of pH value of yellow pulp water on the retention rate of stachyose after graded filtration;

[0036] Figure 9 This is the high-performance liquid chromatogram of stachyose standard.

[0037] Figure 10 This is the high-performance liquid chromatogram of crude stachyose. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Please see Figure 1-10 This invention provides a technical solution: a method for separating stachyose from soybean slurry water by graded filtration, comprising the following steps:

[0040] Step 1: Pretreatment of yellow slurry;

[0041] Weigh soybeans and soak them in a soybean-to-water ratio of 1:5 for 14-16 hours. After removing the water, add water at a volume of 9 times that of the dry soybeans and grind them into a slurry. Filter out the soybean residue and heat the soy milk in a water bath to 90°C for 10 minutes. Then add a coagulant, with calcium sulfate added at 2% of the dry soybean volume. Keep warm for 12 minutes and then press the mixture into a mold. Finally, collect the yellow slurry. Store the prepared yellow slurry in a refrigerator at 4°C for later use. Pre-treat the soybeans using chitosan flocculation method, with a settling time of 68 minutes, pH=5.5, settling temperature of 30.9°C, and chitosan addition of 0.7 g / L.

[0042] Step 2: Selection of ultrafiltration and nanofiltration membrane elements;

[0043] 2.1 Primary Ultrafiltration Membrane Element: A polyethersulfone ultrafiltration membrane element with a molecular weight cutoff of 10000D was selected to perform primary ultrafiltration on the pretreated yellow slurry water;

[0044] 2.2 Secondary ultrafiltration membrane element: The permeate after ultrafiltration by the primary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 5000D, 6000D, and 8000D respectively. The permeate is collected, and the secondary ultrafiltration membrane element is determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0045] 2.3 Tertiary ultrafiltration membrane elements: The permeate after ultrafiltration by the secondary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 1000D, 2000D, and 3000D respectively. The tertiary ultrafiltration membrane elements are determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0046] 2.4 Nanofiltration membrane element: The permeate after ultrafiltration is subjected to nanofiltration. Polyamide nanofiltration membrane elements with molecular weight cutoffs of 150D, 200D, 300D, 500D, and 600D can be selected. Referring to the molecular weight of stachyose, a nanofiltration membrane element with a molecular weight cutoff of 500D is selected, and the retentate is collected.

[0047] Step 3: Selection of tiered filtration conditions;

[0048] After selecting the ultrafiltration and nanofiltration membrane elements, the staged filtration conditions are selected. The effects of operating pressure, yellow slurry temperature and pH value on the stachyose content after staged filtration are investigated to determine the optimal staged filtration conditions.

[0049] 3.1 Selection of operating pressure: Take an equal volume of pretreated yellow pulp water sample and maintain the sample at 20°C and pH=5.5. Adjust the ultrafiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, and 0.7MPa respectively. Pass the sample through primary, secondary, and tertiary ultrafiltration membrane elements, collect the ultrafiltration permeate, and determine the stachyose retention rate. Under the optimal ultrafiltration pressure, maintain the same temperature and pH conditions for nanofiltration. Adjust the nanofiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, 0.7MPa, 0.9MPa, and 1.1MPa respectively. Pass the ultrafiltration permeate through a nanofiltration membrane element, collect the nanofiltration retentate, and determine the stachyose content in the nanofiltration retentate using high performance liquid chromatography. Calculate the stachyose retention rate to determine the optimal operating pressure for staged filtration of yellow pulp water.

[0050] 3.2 Selection of Yellow Slurry Water Temperature: Take an equal volume of pretreated yellow slurry water sample, adjust the pH of the sample to 5.5, and set the temperatures to 10°C, 20°C, 30°C, and 40°C respectively, controlling the operating pressure to 0.3 MPa. Pass the sample through ultrafiltration and nanofiltration membrane elements in sequence, collect the retentate, and determine its stachyose content. Calculate the stachyose retention rate to determine the optimal temperature for staged filtration of yellow slurry water.

[0051] 3.3 Selection of pH value for yellow pulp water: Take an equal volume of pretreated yellow pulp water sample solution, keep the sample solution at a temperature of 20°C, adjust the pH value to 4.5, 5.5, 6.5, 7.5 and 8.5 respectively, control the operating pressure to 0.3MPa, and pass it through ultrafiltration and nanofiltration membrane elements in sequence. Collect the retentate, determine its stachyose content, calculate the stachyose retention rate, and thus determine the optimal pH value for staged filtration of yellow pulp water;

[0052] Step 4: Determination of stachyose purity;

[0053] The crude stachyose extract after fractionation and filtration was freeze-dried. 5 mg of the dried sample was weighed and added to 10 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.5 mg / mL. 1 mg of stachyose standard was weighed and added to 5 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.2 mg / mL. The solution was filtered through a 0.45 μm filter membrane and analyzed by high performance liquid chromatography with an injection volume of 10 μL.

[0054] In this invention, oligosaccharides can preferably be extracted from tofu whey through ion exchange resin treatment, attapulgite decolorization, vacuum concentration, and protein removal.

[0055] In this invention, preferably, oligosaccharides can be extracted from soybean water by high-temperature centrifugation to remove protein, electrodialysis to desalinate, and activated carbon to decolorize, with a final oligosaccharide retention rate of 80.41%.

[0056] In this invention, preferably, soybean oligosaccharides can be extracted from tofu whey by ultrafiltration (10000D), activated carbon decolorization, electrodialysis, and ion exchange resin desalination.

[0057] In this invention, preferably, soybean oligosaccharide powder can be obtained by extracting oligosaccharides from yellow slurry water through flocculation sedimentation, centrifugation, microfiltration pretreatment, followed by ultrafiltration (10000D) and nanofiltration (500D), with a final stachyose recovery rate of 29.03%.

[0058] In this invention, preferably, soybean wastewater is used as raw material, and oligosaccharides are extracted through ultrafiltration, reverse osmosis and nanofiltration technologies, with a final stachyose recovery rate of 24.70%.

[0059] Example 1: Pretreatment of Yellow Slurry

[0060] Weigh soybeans and soak them in a soybean-to-water ratio of 1:5 for 14-16 hours. Remove the water, add water at a ratio of 9 times the volume of dry soybeans, grind into a slurry, filter out the soybean residue, and heat the soy milk in a water bath to 90°C for 10 minutes. Then add a coagulant (calcium sulfate at 2% of the dry soybean volume), keep warm for 12 minutes, and press into a mold. Finally, collect the yellow slurry. Store the prepared yellow slurry at 4°C for later use. Pretreatment is performed using chitosan flocculation method, with a settling time of 68 minutes, pH=5.5, settling temperature of 30.9°C, and chitosan addition of 0.7 g / L.

[0061] Example 2: Selection of Ultrafiltration and Nanofiltration Membrane Elements

[0062] 1. Primary ultrafiltration membrane element: A polyethersulfone ultrafiltration membrane element with a molecular weight cutoff of 10000D is selected to perform primary ultrafiltration on the pretreated yellow slurry water;

[0063] 2. Secondary ultrafiltration membrane element: The permeate after ultrafiltration by the primary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 5000D, 6000D, and 8000D respectively. The permeate is collected, and the secondary ultrafiltration membrane element is determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0064] 3. Tertiary ultrafiltration membrane elements: The permeate after ultrafiltration by the secondary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 1000D, 2000D, and 3000D respectively. The tertiary ultrafiltration membrane elements are determined by comparing the permeate flux, protein removal rate, and stachyose retention rate.

[0065] 4. Nanofiltration membrane element: The permeate after ultrafiltration is subjected to nanofiltration. Polyamide nanofiltration membrane elements with molecular weight cutoffs of 150D, 200D, 300D, 500D, and 600D can be selected. Based on the molecular weight of stachyose, a nanofiltration membrane element with a molecular weight cutoff of 500D is selected, and the retentate is collected.

[0066] The formulas for calculating protein removal rate, stachyose retention rate, and osmotic flux are as follows:

[0067]

[0068] Where: W1—protein content in the treated sample solution; W0—protein content in the original solution.

[0069]

[0070] Where: M1—stachyose content in the treated sample solution; M0—stachyose content in the original solution.

[0071]

[0072] Where: L—volume of liquid permeated; m 2 —Effective area of ​​the membrane; h—Running time

[0073] Results and Analysis

[0074] 1. Decrease curves of permeate flux of secondary and tertiary ultrafiltration membrane elements

[0075] like Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 The figures show the decay curves of permeate flux for secondary and tertiary ultrafiltration membrane elements, respectively. As can be seen from the figures... Figure 1 and Figure 2The permeation flux of membrane elements with different molecular weight cutoffs decreases continuously with the increase of permeate volume. As the permeate volume increases, the permeation flux decay rate of high molecular weight membrane elements is faster than that of low molecular weight membrane elements. Therefore, selecting membrane elements with a small molecular weight cutoff is beneficial for the long-term use of the membrane.

[0076] 2. Protein removal rate and stachyose retention rate of secondary and tertiary ultrafiltration membrane elements

[0077] like Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 The figures show the protein removal rate and stachyose retention rate of secondary and tertiary ultrafiltration membrane elements, respectively. As can be seen from the figures, with the increase of the molecular weight cutoff of the membrane element, the protein removal rate decreases while the stachyose retention rate increases. Figure 3 It can be seen that compared with the ultrafiltration membrane element with a molecular weight cutoff of 5000D, the protein removal rate in the permeate of 6000D and 8000D membrane elements was significantly decreased (p<0.05). Regarding stachyose retention, compared with the ultrafiltration membrane element with a molecular weight cutoff of 5000D, the stachyose retention rate in the permeate of 6000D membrane elements was not significantly different, while the stachyose retention rate in the permeate of 8000D membrane elements was significantly different. Figure 4 It can be seen that compared with the 1000D molecular weight cutoff, the protein removal rate in the permeate of 2000D and 3000D membranes decreased significantly (p<0.05). As for the stachyose retention rate, compared with the 1000D molecular weight cutoff ultrafiltration membrane element, the stachyose retention rate in the permeate of 2000D membranes was not significantly different, while the stachyose retention rate in the permeate of 3000D membranes was significantly different. In summary, ultrafiltration membranes with molecular weight cutoffs of 5000D and 1000D were selected as secondary and tertiary ultrafiltration membrane elements, respectively.

[0078] Example 3: Selection of tiered filtration conditions

[0079] After selecting the ultrafiltration and nanofiltration membrane elements, the staged filtration conditions are selected. The effects of operating pressure, yellow slurry temperature and pH value on the stachyose content after staged filtration are investigated to determine the optimal staged filtration conditions.

[0080] 1. Selection of operating pressure: Take an equal volume of pretreated yellow pulp water sample and maintain the sample at 20°C and pH=5.5. Adjust the ultrafiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, and 0.7MPa respectively. Pass the sample through primary, secondary, and tertiary ultrafiltration membrane elements, collect the ultrafiltration permeate, and determine the stachyose retention rate. Under the optimal ultrafiltration pressure, maintain the same temperature and pH conditions for nanofiltration. Adjust the nanofiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, 0.7MPa, 0.9MPa, and 1.1MPa respectively. Pass the ultrafiltration permeate through a nanofiltration membrane element, collect the nanofiltration retentate, and determine the stachyose content in the nanofiltration retentate using high performance liquid chromatography (HPLC). Calculate the stachyose retention rate to determine the optimal operating pressure for staged filtration of yellow pulp water.

[0081] 2. Selection of temperature for yellow pulp water: Take an equal volume of pretreated yellow pulp water sample, adjust the pH of the sample to 5.5, and set the temperature to 10°C, 20°C, 30°C, and 40°C respectively, controlling the operating pressure to 0.3 MPa. Pass the sample through ultrafiltration and nanofiltration membrane elements in sequence, collect the retentate, and determine its stachyose content. Calculate the stachyose retention rate to determine the optimal temperature for staged filtration of yellow pulp water.

[0082] 3. Selection of pH value for yellow pulp water: Take an equal volume of pretreated yellow pulp water sample solution, keep the sample solution at a temperature of 20°C, adjust the pH value to 4.5, 5.5, 6.5, 7.5 and 8.5 respectively, control the operating pressure to 0.3MPa, and pass it through ultrafiltration and nanofiltration membrane elements in sequence. Collect the retentate, determine its stachyose content, calculate the stachyose retention rate, and thus determine the optimal pH value for staged filtration of yellow pulp water.

[0083] Results and Analysis

[0084] 1. Effect of operating pressure on stachyose retention rate in staged filtration

[0085] like Figure 5 and Figure 6 As shown, the effect of operating pressure on the retention rate of stachyose in staged filtration is... Figure 5 It can be seen that as the operating pressure of ultrafiltration increases, there is no significant difference in stachyose retention rate (p>0.05). Figure 6 It can be seen that the stachyose retention rate did not differ significantly with the continuous increase of the nanofiltration operating pressure (p>0.05). Since there was no significant difference in the stachyose retention rate under different operating pressures, the effect of operating pressure on the stachyose retention rate of staged filtration was not considered. It is only necessary to ensure that it is carried out under normal operating pressure.

[0086] 2. Effect of yellow pulp water temperature on stachyose retention rate after fractional filtration

[0087] from Figure 7It can be seen that the retention rate of stachyose changed significantly with the continuous increase of the temperature of the yellow slurry. As the temperature of the yellow slurry increased, the retention rate of stachyose first increased and then decreased. When the temperature reached 30°C, the retention rate of stachyose was the highest and then decreased significantly. This may be because as the temperature increases, the viscosity of the yellow slurry decreases and the porosity of the membrane surface increases. However, when it reaches a certain temperature, it will increase the membrane pores, causing some solute to pass through the filter membrane, resulting in a decrease in the stachyose content. Therefore, 30°C was selected as the optimal temperature condition for the staged filtration of yellow slurry.

[0088] 3. Effect of pH value of yellow pulp water on stachyose retention rate after staged filtration

[0089] from Figure 8 It can be seen that the stachyose retention rate changed significantly with the continuous increase of the pH value of the yellow slurry (p<0.05). The stachyose retention rate was the highest when the pH value of the yellow slurry was 6.5, and then decreased significantly. This may be because when the pH value of the yellow slurry is greater than the isoelectric point of the protein, both the protein and the membrane element are negatively charged. The two charges are mutually repulsive, which reduces their concentration and polarization on the membrane surface. As the pH value of the yellow slurry continues to increase, the turbidity of the sample solution increases, the protein is more likely to precipitate, and the resistance on the membrane surface increases. Therefore, pH=6.5 was selected as the optimal pH value for the staged filtration of yellow slurry.

[0090] By comparing the permeate flux, protein removal rate, and stachyose retention rate of membrane elements with different molecular weight cutoffs, membrane elements with molecular weight cutoffs of 5000 and 1000D were selected as the ultrafiltration membrane elements for the second and third stages of graded filtration. Referring to relevant studies and the molecular weight of stachyose, polyethersulfone ultrafiltration membranes and polyamide nanofiltration membranes with molecular weight cutoffs of 10000 and 500D were selected as the first-stage ultrafiltration and nanofiltration membrane elements for graded filtration. Finally, membrane elements with molecular weight cutoffs of 10000, 5000, 1000, and 500D were selected for graded filtration.

[0091] By comparing the effects of operating pressure, yellow slurry temperature, and pH on the stachyose retention rate of staged filtration, the optimal staged filtration conditions were determined. Since operating pressure had no significant effect on the stachyose retention rate, a temperature of 30°C and a pH of 6.5 were selected as the optimal operating conditions for staged filtration.

[0092] Example 4: Determination of Stachyose Purity

[0093] The crude stachyose extract after fractionation and filtration was freeze-dried. 5 mg of the dried sample was weighed and added to 10 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.5 mg / mL. 1 mg of stachyose standard was weighed and added to 5 mL of acetonitrile (40%) to prepare a solution with a concentration of 0.2 mg / mL. The solution was filtered through a 0.45 μm filter membrane and analyzed by high performance liquid chromatography with an injection volume of 10 μL.

[0094] Results and Analysis

[0095] like Figure 9 and Figure 10 The figure shows the high-performance liquid chromatogram of stachyose, in which... Figure 9 The chromatogram of stachyose standard. Figure 10 This is the chromatogram of freeze-dried crude stachyose. Figure 9 and Figure 10 The retention times of the chromatographic peaks of stachyose in the chromatograms were basically consistent, approximately 29.60 min. Calculations showed that the purity of crude stachyose after freeze-drying was 42.6%.

[0096] This invention found that 260g of soybeans will produce about 1L of yellow liquid. After grading and filtration, about 80mL of crude stachyose extract can be collected. After freeze-drying, about 0.8g of solids are obtained. The final purity of stachyose is 42.6% after HPLC-ELSD determination. Although some stachyose is lost during the grading and filtration process, the purity of stachyose in the final crude stachyose extract is significantly improved.

[0097] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for separating stachyose from soybean slurry water by graded filtration, characterized in that, Includes the following steps: Step 1: Pretreatment of yellow slurry; Weigh soybeans and soak them in a soybean-to-water ratio of 1:5 for 14-16 hours. After removing the water, add water at a volume of 9 times that of the dry soybeans and grind them into a slurry. Filter out the soybean residue and heat the soy milk in a water bath to 90°C for 10 minutes. Then add a coagulant, with calcium sulfate added at 2% of the dry soybean volume. Keep warm for 12 minutes and then press the mixture into a mold. Finally, collect the yellow slurry. Store the prepared yellow slurry in a refrigerator at 4°C for later use. Pre-treat the soybeans using chitosan flocculation method, with a settling time of 68 minutes, pH=5.5, settling temperature of 30.9°C, and chitosan addition of 0.7 g / L. Step 2: Selection of ultrafiltration and nanofiltration membrane elements; 2.1 Primary Ultrafiltration Membrane Element: A polyethersulfone ultrafiltration membrane element with a molecular weight cutoff of 10000D was selected to perform primary ultrafiltration on the pretreated yellow slurry water; 2.2 Secondary ultrafiltration membrane element: The permeate after ultrafiltration by the primary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 5000D, 6000D, and 8000D respectively. The permeate is collected, and the secondary ultrafiltration membrane element is determined by comparing the permeate flux, protein removal rate, and stachyose retention rate. The secondary ultrafiltration membrane element is an ultrafiltration membrane element with a molecular weight cutoff of 5000D. 2.3 Tertiary ultrafiltration membrane element: The permeate after ultrafiltration by the secondary membrane element is passed through ultrafiltration membrane elements with molecular weight cutoffs of 1000D, 2000D, and 3000D respectively. The tertiary ultrafiltration membrane element is determined by comparing the permeate flux, protein removal rate, and stachyose retention rate. The tertiary ultrafiltration membrane element is an ultrafiltration membrane element with a molecular weight cutoff of 1000D. 2.4 Nanofiltration membrane element: The permeate after ultrafiltration is subjected to nanofiltration. Polyamide nanofiltration membrane elements with molecular weight cutoffs of 150D, 200D, 300D, 500D, and 600D can be selected. Referring to the molecular weight of stachyose, a nanofiltration membrane element with a molecular weight cutoff of 500D is selected, and the retentate is collected. Step 3: Selection of tiered filtration conditions; After selecting the ultrafiltration and nanofiltration membrane elements, the staged filtration conditions are selected. The effects of operating pressure, yellow slurry temperature and pH value on the stachyose content after staged filtration are investigated to determine the optimal staged filtration conditions. 3.1 Selection of operating pressure: Take an equal volume of pretreated yellow pulp water sample and maintain the sample at 20°C and pH=5.

5. Adjust the ultrafiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, and 0.7MPa respectively. Pass the sample through primary, secondary, and tertiary ultrafiltration membrane elements, collect the ultrafiltration permeate, and determine the stachyose retention rate. Under the optimal ultrafiltration pressure, maintain the same temperature and pH conditions for nanofiltration. Adjust the nanofiltration operating pressure to 0.1MPa, 0.3MPa, 0.5MPa, 0.7MPa, 0.9MPa, and 1.1MPa respectively. Pass the ultrafiltration permeate through a nanofiltration membrane element, collect the nanofiltration retentate, and determine the stachyose content in the nanofiltration retentate using high performance liquid chromatography. Calculate the stachyose retention rate to determine the optimal operating pressure for staged filtration of yellow pulp water. 3.2 Selection of Yellow Slurry Water Temperature: Take an equal volume of pretreated yellow slurry water sample, adjust the pH of the sample to 5.5, and set the temperatures to 10°C, 20°C, 30°C, and 40°C respectively, controlling the operating pressure to 0.3 MPa. Pass the sample through ultrafiltration and nanofiltration membrane elements in sequence, collect the retentate, and determine its stachyose content. Calculate the stachyose retention rate to determine that the optimal temperature for staged filtration of yellow slurry water is 30°C. 3.3 Selection of pH value for yellow pulp water: Take an equal volume of pretreated yellow pulp water sample solution, keep the sample solution at a temperature of 20°C, adjust the pH value to 4.5, 5.5, 6.5, 7.5 and 8.5 respectively, control the operating pressure to 0.3MPa, and pass it through ultrafiltration and nanofiltration membrane elements in sequence. Collect the retentate, determine its stachyose content, calculate the stachyose retention rate, and thus determine that the optimal pH value for staged filtration of yellow pulp water is 6.

5. Step 4: Determination of stachyose purity; The crude stachyose extract after fractionation and filtration was freeze-dried. 5 mg of the dried sample was weighed and added to 10 mL of acetonitrile to prepare a solution with a concentration of 0.5 mg / mL. 1 mg of stachyose standard was weighed and added to 5 mL of acetonitrile to prepare a solution with a concentration of 0.2 mg / mL. The solution was filtered through a 0.45 μm filter membrane and analyzed by high performance liquid chromatography with an injection volume of 10 μL.

Images