Method for extracting raffinose from cottonseed processing waste liquid

Abstract

The invention belongs to the technical field of food analysis, and particularly relates to a method for extracting raffinose from cottonseed processing waste liquid. According to the method, the raffinose in the cottonseed processing waste liquid is directly extracted by using an aqueous solution, sephadex LH-20 is used as a column chromatography filler, the molecular sieve effect is utilized, water or ethanol water is used as an eluent, and the raffinose and substances with different molecular weights such as cottonseed protein, salt and cane sugar are eluted in sequence, so that the raffinose is purified. The method is simple in process flow, does not need to add harmful solvents such as acid, alkali and methanol into the system, and has the advantages of simplicity, rapidness and easiness in industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and more specifically, relates to a method for extracting raffinose from cottonseed processing waste liquid. Background Art

[0002] The main methods for extracting raffinose include acid extraction method, alkali extraction method, aqueous organic solvent extraction method, enzyme hydrolysis method, etc. During the extraction process, free gossypol, gossypol-binding protein, and other pigments will be extracted from the cottonseed processing waste liquid together with raffinose, making the color of the extraction liquid dark red. The presence of substances such as gossypol pigment, protein, salt, and sucrose not only makes it difficult for raffinose to crystallize but also affects the final color of the raffinose product. Therefore, after obtaining the raffinose extraction liquid from the raw material, it is necessary to carry out purification by removing phenol and decolorizing, removing protein, desalting, and removing impurities such as sucrose, and finally obtaining the final product through crystallization or precipitation.

[0003] In the prior art, to obtain high-purity raffinose, the raffinose extraction liquid after decolorization, deproteinization, and desalting also needs to be subjected to multiple repeated crystallization treatments. However, multiple crystallizations will cause a large loss of the raffinose product, reduce the total yield of raffinose, and is not conducive to large-scale industrial production. Currently, there have been research reports on the process method of using simulated moving bed to separate raffinose from different raw materials. Using cation exchange resin as the stationary phase, the aqueous solution containing crude raffinose is obtained after elution, crystallization, etc. to obtain the raffinose product. However, this method requires pre-treatment such as decolorization, deproteinization, and desalting of the extraction liquid before high-purity separation of raffinose. Its process route is long, the industrial production cost is high, and the separation efficiency is low. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a method for extracting raffinose from cottonseed processing waste liquid.

[0005] The present invention specifically adopts the following technical solutions:

[0006] The present invention provides a method for extracting raffinose from cottonseed processing waste liquid, comprising the following steps:

[0007] S1. Water is added to the cottonseed processing waste liquid to obtain a premixed solution. The premixed solution is stirred, cooled to room temperature, and then centrifuged to take the supernatant. Activated carbon powder is added to the supernatant, shaken for decolorization, and then filtered by suction to obtain a filtrate. The filtrate is rotary evaporated at 55 - 65 °C to obtain a concentrated solution, and the concentrated solution is dissolved in water to obtain a diluted solution;

[0008] S2. The diluted solution is added to a gel chromatography column filled with Sephadex LH-20 with a height of 600 - 700 mm, and eluted with 1.5 - 2 BV of water at a flow rate of 0.1 - 0.3 BV / h to obtain an eluate. The eluate is rotary evaporated to obtain raffinose.

[0009] The present invention extracts raffinose from cottonseed processing waste liquid by the water extraction method, decolors it with activated carbon powder, and then concentrates it to obtain a raffinose extract. The raffinose extract is directly loaded onto a chromatography column filled with Sephadex LH-20 gel, and after being eluted with water or an ethanol aqueous solution once and then rotary evaporated, crude raffinose can be obtained.

[0010] Further, the mass-volume ratio of cottonseed processing waste liquid to water in the premixed solution is 1 - 2 g : 15 - 25 ml.

[0011] Further, the stirring conditions are 40 - 50 °C and stirring for 20 - 40 min.

[0012] Further, the centrifugation conditions are 10000 - 12000 rpm / min for 20 - 30 min.

[0013] Further, the mass fraction of the activated carbon powder in the supernatant is 4% - 6%.

[0014] Further, the oscillation decolorization conditions are 40 - 60 °C for 1 - 2 h.

[0015] Further, the bed volume of the gel chromatography column is 100 - 150 mL.

[0016] Further, the sample loading amount of the dilution liquid is 0.01 - 0.02 BV.

[0017] The present invention has the following beneficial effects:

[0018] The present invention directly extracts raffinose from cottonseed processing waste liquid using an aqueous solution, uses dextran gel Sephadex LH-20 as the column chromatography packing material, utilizes the molecular sieve effect, and uses water or ethanol water as the eluent, so that raffinose and substances with different molecular weights such as cottonseed protein, salts, and sucrose are eluted successively to achieve the purification of raffinose. This method has a simple process flow, does not require the addition of harmful solvents such as acids, alkalis, and methanol to the system, and has the advantages of being simple, fast, green, and easy for industrial production. Description of the Drawings

[0019] Figure 1 It is the standard curve graph of raffinose.

[0020] Figure 2 It is the separation curve graph of raffinose when the column height is 400 mm.

[0021] Figure 3 It is the separation curve graph of raffinose when the column height is 700 mm.

[0022] Figure 4 It is the separation curve graph of raffinose when the elution flow rate is 0.42 BV / h.

[0023] Figure 5 It is the separation curve diagram of raffinose when the elution flow rate is 0.32 BV / h.

[0024] Figure 6 It is the separation curve diagram of raffinose when the elution flow rate is 0.22 BV / h.

[0025] Figure 7 It is the separation curve diagram of raffinose when the elution solvent is 20% ethanol water.

[0026] Figure 8 It is the separation curve diagram of raffinose when the elution solvent is 30% ethanol water.

[0027] Figure 9 It is the separation curve diagram of raffinose when the elution solvent is pure distilled water.

[0028] Figure 10 It is the separation curve diagram of raffinose when the sample loading is 0.02 BV.

[0029] Figure 11 It is the separation curve diagram of raffinose when the sample loading is 0.05 BV.

[0030] Figure 12 It is the separation curve diagram of raffinose when the sample loading is 0.1 BV.

[0031] Figure 13 It is the chromatogram of the crude raffinose separated and purified under the optimal process conditions determined by HPLC-ELSD. Detailed implementation manners

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Example 1

[0034] I. Materials and reagents.

[0035] The cottonseed processing waste liquid comes from the plant protein processing production line of TaiKun Bio-Protein Technology Co., Ltd. in Changji Prefecture, Xinjiang. Sephadex LH-20 gel and ordinary chromatography columns are both purchased from Shanghai Titan Scientific Co., Ltd. The 0.22μm water-based needle filter head is purchased from Urumqi Baiheng Dazheng Instrument and Equipment Co., Ltd. Chromatographic grade raffinose (purity HPLC≥98%) is purchased from Beijing Solarbio Science & Technology Co., Ltd. Chromatographic grade sucrose (purity HPLC≥99.5%) and stachyose (purity≥70%) are both purchased from Shanghai Aladdin Co., Ltd. HPLC grade water is Wahaha drinking pure water, purchased from Aksu Wahaha Beverage Co., Ltd. Acetonitrile (chromatographic pure) is purchased from Sigma-Aldrich Co., USA. The raffinose standard is purchased from Shanghai Adamas Beta Chemical Reagent Co., Ltd. Absolute ethanol is purchased from Tianjin Xinbot Chemical Co., Ltd.

[0036] II. Experimental methods.

[0037] 1. Preparation of extraction solution.

[0038] Accurately weigh 5.0 g of cottonseed processing waste liquid, add 100 mL of water and stir at 45°C for 30 min. After cooling to room temperature, centrifuge at 11000 r / min for 25 min. Take the supernatant, add activated carbon powder accounting for 5% of the supernatant mass fraction to the supernatant, and oscillate and decolorize at 50°C for 1 h, then filter to obtain the filtrate. Concentrate the filtrate by rotary evaporation at 60°C until the volume remains unchanged, and then dilute it with ultrapure water to a concentration of 200 mg / mL to obtain the extraction solution.

[0039] 2. Chromatography preparation.

[0040] (1) Chromatographic conditions: The chromatographic column is Prevail carbohydrate ES (250 mm×4.6 mm, 5 μm particle size), the mobile phase is acetonitrile: water, with a volume ratio of 70:30, isocratic elution, the column oven temperature is 35°C, the mobile phase flow rate is 1.0 mL / min, the injection volume is 20 μL, the ELSD drift tube temperature is 40°C, and the nitrogen gas flow rate is 1.5 L / min.

[0041] (2) Preparation of standard solutions: Accurately weigh 20.0 mg of chromatographic grade raffinose, sucrose, and stachyose into 10 mL volumetric flasks respectively, and dilute to a constant volume with ultrapure water to obtain single-standard standard stock solutions with the concentrations of raffinose, sucrose, and stachyose all being 2.0 mg / mL, and store them in the refrigerator at 4°C in the dark. Then dilute the stock solutions with ultrapure water to obtain raffinose, sucrose, and stachyose standard solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, and filter them through a 0.22μm water-based needle filter head before injecting into the HPLC system.

[0042] (3) Standard curve and linear range: The linear relationships of raffinose, sucrose, and stachyose were determined by a calibration curve of five points. With the logarithm of the peak area (A) as the ordinate value and the logarithm of the mass concentration (C, mg / mL) of the standard solution as the abscissa value, the standard curves of raffinose, sucrose, and stachyose for HPLC-ELSD were plotted. As Figure 1 shown in Table 1, the regression equation of raffinose was Y = 1.56143X + 6.56288, and the linear relationships of raffinose, sucrose, and stachyose were good in the concentration range of 0.1 mg / mL to 0.5 mg / mL (R 2 = 0.9986 - 0.9999). The regression equation was used for the subsequent determination of the contents of raffinose, sucrose, and stachyose.

[0043] Table 1: Regression equations, correlation coefficients, and linear ranges of raffinose, sucrose, and stachyose.

[0044] Sugar compound Regression equation <![CDATA[R 2 > Linear range (mg / mL) Raffinose Y = 1.56143X + 6.56288 0.9999 0.1～0.5 Sucrose Y = 1.57128X + 6.82736 0.9997 0.1～0.5 Stachyose Y = 1.51566X + 6.25992 0.9986 0.1～0.5

[0045] 3. Optimization of the conditions for the separation and purification of raffinose by gel column chromatography.

[0046] (1) Packing material and column packing of the gel chromatography column: Using Sephadex LH-20 gel as the packing material, the gel chromatography column was packed by the wet packing method.

[0047] (2) Influence of the column height of the chromatography column on the separation and purification of raffinose: At room temperature, 0.02 BV (BV, i.e., the volume of the gel column) of the extract was respectively loaded onto chromatography columns with column heights of 400 mm and 700 mm. Using ethanol with a mass fraction of 20% as the elution solvent, the eluate was collected at a flow rate of 0.32 BV / h with an automatic collector at 1.5 mL / tube, and the concentrations of raffinose, sucrose, and stachyose in each tube of the eluate were determined by HPLC-ELSD to investigate the influence of chromatography columns with different column heights on the separation and purification of raffinose.

[0048] (3) Influence of the elution flow rate on the separation and purification of raffinose: Using ethanol with a mass fraction of 20% as the elution solvent, 0.02 BV of the extract was loaded onto the chromatography column, and the elution flow rates were set at 0.22 BV / h, 0.32 BV / h, and 0.42 BV / h respectively. The eluate was collected at 1.5 mL / tube with an automatic collector, and the concentrations of raffinose, sucrose, and stachyose in each tube of the eluate were determined by HPLC-ELSD to investigate the influence of different elution flow rates on the separation and purification of raffinose.

[0049] (4) Influence of elution solvent on the separation and purification of raffinose: 0.02 BV of the extract was loaded onto the chromatography column, and ethanol with a mass fraction of 20%, ethanol with a mass fraction of 30%, and pure distilled water were used as elution solvents respectively. An automatic collector was used to collect the eluate at 1.5 mL / tube, and the concentrations of raffinose, sucrose, and stachyose in each tube of eluate were determined by HPLC-ELSD to investigate the influence of different elution solvents on the separation and purification of raffinose.

[0050] (5) Influence of sample loading volume on the separation and purification of raffinose: 0.02 BV, 0.05 BV, and 0.1 BV of the sample extract were respectively loaded onto the chromatography column. An automatic collector was used to collect the eluate at 1.5 mL / tube, and the concentrations of raffinose, sucrose, and stachyose in each tube of eluate were determined by HPLC-ELSD to investigate the influence of different sample loading volumes on the separation and purification of raffinose.

[0051] (6) Data statistics and analysis: All experimental data were in triplicate. Data analysis was performed using Microsoft Excel 2010 (Microsoft Corporation, USA), and graphs were plotted using Origin 8.6 software.

[0052] III. Experimental results.

[0053] 1. Influence of column height of the chromatography column on the separation and purification of raffinose.

[0054] The separation mechanism of gel column chromatography is mainly the volume exclusion effect. The larger molecular weight stachyose moves through the gel bed in the shortest time and elutes first, followed by raffinose and sucrose. As Figure 2 、 Figure 3 and Table 2 show, when the column height is 400 mm, the three peaks overlap severely and the separation effect is poor; when the column height is 700 mm, the peaks of the stachyose and sucrose components are relatively separated from the raffinose component, the separation effect is enhanced, and the proportion of raffinose at the peak is significantly increased. This indicates that a longer chromatography column is more conducive to the separation of raffinose, and finally 700 mm was determined as the separation column height.

[0055] Table 2: Influence of column height of the chromatography column on the separation effect of raffinose.

[0056] Column height (mm) Raffinose / total recovered oligosaccharides (%) Raffinose / total oligosaccharides at peak (%) 400 67.70 73.52 700 69.85 84.12

[0057] 2. Influence of elution flow rate on the separation and purification of raffinose.

[0058] As Figure 4 、 Figure 5 、 Figure 6As shown in Table 3, when the elution flow rate is 0.42 BV / h, the three peaks coincide; when the elution flow rate is 0.22 BV / h, the raffinose peak and the sucrose peak are relatively separated; when the elution flow rate is 0.32 BV / h, the three peaks overlap less, and at this time, raffinose accounts for 83.72% of the total oligosaccharide mass at the peak. Too high an elution flow rate will increase the column pressure, resulting in overlapping elution peaks; too low a flow rate will increase the lateral diffusion of the raffinose component in the gel bed, resulting in peak broadening and also reducing the working efficiency. Therefore, 0.32 BV / h is selected as the optimal elution flow rate for separation.

[0059] Table 3: Influence of elution flow rate on the separation effect of raffinose.

[0060] Elution flow rate (BV / h) Raffinose / total recovered oligosaccharides (%) Raffinose / total oligosaccharides at peak (%) 0.42 70.79 76.75 0.32 71.46 83.72 0.22 69.95 82.05

[0061] 3. Influence of elution solvent on the separation and purification of raffinose.

[0062] Sephadex LH-20 is a dextran gel with both hydrophilic and lipophilic properties. The polarity of the substance to be separated and the elution solvent plays an important role in the separation process. As Figure 7 、 Figure 8 、 Figure 9 and Table 4 show, when ethanol with a mass fraction of 20% and ethanol with a mass fraction of 30% are used as elution solvents, the three peaks basically overlap and the separation effect is poor; when pure distilled water is used as the elution solvent, the raffinose peak and the sucrose peak can be basically separated, and at this time, raffinose accounts for 85.90% of the total oligosaccharide mass at the peak. Increasing the proportion of ethanol in the elution solvent will weaken its polarity, and under the reverse phase distribution of the gel, it will lead to an increase in the retention time of substances with high polarity, which is not conducive to the separation of raffinose and sucrose. Therefore, pure distilled water is selected as the optimal elution solvent.

[0063] Table 4: Influence of elution solvent on the separation effect of raffinose.

[0064] Elution solvent Raffinose / total recovered oligosaccharides (%) Raffinose / total oligosaccharides at peak (%) 20% ethanol in water 71.65 77.33 30% ethanol in water 71.03 77.25 Pure distilled water 71.99 85.90

[0065] 4. Influence of sample loading amount on the separation and purification of raffinose.

[0066] As Figure 10 、 Figure 11 、 Figure 12 and Table 5 show, the sample loading amount has a great influence on the separation effect of raffinose. When the injection volume is 0.05 BV and 0.1 BV, the peak separation effect of the three sugars is poor, the three peaks basically overlap, and there is an obvious tailing phenomenon; when the injection volume is 0.02 BV, the peaks of the three sugars are basically separated, and raffinose accounts for 85.19% of the total oligosaccharide at the peak. The results show that when the sample loading amount is large, it will lead to peak overlap and affect the separation effect of raffinose. Therefore, the optimal sample loading amount is selected as 0.02 BV.

[0067] Table 5: Influence of sample loading amount on the separation effect of raffinose.

[0068] Sample loading volume (BV) Raffinose / total recovered oligosaccharides (%) Raffinose / total oligosaccharides at peak (%) 0.02 71.87 85.19 0.05 72.53 83.64 0.1 72.25 84.31

[0069] 5. Optimal process conditions for separating and purifying raffinose by gel column chromatography.

[0070] In summary, the optimal process conditions for separating and purifying raffinose by gel column chromatography are as follows: elution flow rate of 0.32 BV / h, pure distilled water as the elution solvent, and sample loading amount of 0.02 BV. Under these conditions, the recovery rate of raffinose is 80.96%, raffinose accounts for 71.87% of the total recovered oligosaccharides, and raffinose accounts for 85.19% of the total oligosaccharide mass fraction at the peak of the separation curve. Repeatedly load the extraction solution three times under the above optimal process conditions, collect the eluate, and obtain the crude raffinose after freeze-drying. Determine the crude raffinose by HPLC-ELSD method. As Figure 13 shown, the purity of raffinose can reach 88.84%.

[0071] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent redundancy, the present invention describes preferred embodiments.

[0072] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A method for extracting raffinose from cottonseed processing wastewater, characterized in that: The following steps are involved: S1. Add water to the cottonseed processing waste liquid to obtain a premixed liquid, stir the premixed liquid, cool it to room temperature, centrifuge to obtain a supernatant, add activated carbon powder to the supernatant, shake and decolorize it, and then filter it to obtain a filtrate, evaporate the filtrate at 55-65° C. to obtain a concentrated liquid, and dissolve the concentrated liquid in water to obtain a diluted liquid; S2. Add the diluted solution to a 600-700 mm high gel chromatography column filled with Sephadex LH-20, and elute with 1.5-2 BV of water at a flow rate of 0.1-0.3 BV / h to obtain an eluate, and rotary evaporate the eluate to obtain raffinose.

2. A method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The mass volume ratio of cottonseed processing waste liquid and water in the premixed liquid is 1-2g:15-25ml.

3. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The stirring condition is 40-50° C. and stirring for 20-40 min.

4. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The centrifugal conditions are 10000-12000 rpm / min, 20-30 min.

5. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The activated carbon powder accounts for 4% to 6% of the mass of the supernatant.

6. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The shaking decolorization condition is 40-60° C., 1-2 h.

7. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The bed volume of the gel chromatography column is 100-150 mL.

8. The method for extracting raffinose from cottonseed processing waste liquid according to claim 1, characterized in that: The loading volume of the diluent is 0.01-0.02 BV.

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