Method for simultaneously separating and purifying amylose and amylopectin of tobacco leaves
By using a cellulose DE-52 chromatography column and a gradient elution method with different concentrations of NaCl buffer, the problem of time-consuming separation and purification of amylose and toxic solvents in the prior art is solved, and an efficient, green and safe starch separation and purification effect is achieved.
Patent Information
- Application Number
- CN202510409174.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-06-17
AI Technical Summary
The method of isolating and purifying amylose and amylopectin in the prior art is time-consuming and uses toxic organic solvents, which is not conducive to operation.
Cellulose DE-52 chromatography column was used to perform gradient elution with different concentrations of NaCl buffer, and amylose and amylose were separated by column chromatography. This method does not require the use of toxic organic reagents, and the entire purification process takes only 1 hour.
It realizes efficient separation and purification of amylose and amylopectin, with higher purity, and this method is green and safe, short time-consuming and extremely time-saving.
Smart Images
Figure CN120157776A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of starch, and particularly relates to a method for simultaneously separating and purifying amylose and amylopectin. Background Art
[0002] Starch is widely distributed in various plants. It is a high-molecular carbohydrate polymerized from glucose and can be divided into amylose and amylopectin. In the starch molecule, every 6 glucose residues form a helical cavity, which can complex iodine, so it can react with iodine to produce a color reaction. Amylose has fewer branches and more helical cavities, and produces a blue reaction when encountering iodine; amylopectin has more branches and fewer helical cavities, and produces a purplish-red reaction when encountering iodine.
[0003] Currently, the separation and purification method of amylose and amylopectin is the solvent extraction method, in which n-butanol and isopropanol are commonly used for the separation of the above two starches. For example, the literature [Lu Yali, Wang Mingli, Yan Yan, etc. Separation, purification and identification of amylose and amylopectin in Coix lacryma-jobi starch [J]. Food Industry, 2014, 35(3): 3.] uses a mixed solution of n-butanol - isoamyl alcohol (3:1) to separate amylose and amylopectin. The precipitate is crude amylose, and the centrifugate is crude amylopectin. After subsequent repeated washing with n-butanol and ethanol precipitation, amylose and amylopectin with higher purity can be obtained. However, this method is time-consuming, and one experiment takes several days, and both n-butanol and isoamyl alcohol used have certain toxicity, which is not conducive to operation.
[0004] Therefore, a method for separating and purifying amylose and amylopectin that is fast, green and safe needs to be developed. Summary of the Invention
[0005] In view of the above problems existing in the prior art, the present invention provides a method for separating and purifying amylose and amylopectin. This method uses a cellulose DE-52 chromatography column and different concentrations of NaCl buffer solution for gradient elution of starch, which can effectively separate and purify amylose and amylopectin. Moreover, this method does not need to use toxic organic reagents, is green and safe. Further, this method takes a short time (such as only 1 h), saves a lot of time, and has broad application prospects.
[0006] Specifically, in the first aspect of the present invention, the present invention provides a method for separating amylose and amylopectin in a starch sample, which includes:
[0007] Subjecting the starch sample to be separated to column chromatography to obtain separated amylose and amylopectin;
[0008] Wherein, the stationary phase of the column chromatography is DEAE cellulose, and the eluent of the column chromatography is Tris-HCl buffer solution containing NaCl.
[0009] In some embodiments, the stationary phase of the column chromatography is DEAE cellulose DE-52.
[0010] In some embodiments, the column chromatography comprises:
[0011] (1) Gradient elution is carried out using a Tris-HCl buffer solution containing NaCl with gradually increasing NaCl concentration as the eluent, and the first eluate and the second eluate are collected in sequence;
[0012] (2) The first eluate is desalted and dried to obtain amylose, and the second eluate is desalted and dried to obtain amylopectin.
[0013] In some embodiments, step (1) includes:
[0014] Gradient elution is carried out in sequence using buffer B, buffer C, buffer D, and buffer E as the eluent, and the first eluate and the second eluate are collected in sequence;
[0015] Wherein:
[0016] The buffer B is a Tris-HCl buffer solution containing 0.4 M NaCl, preferably a 0.05 M, pH 7.5 Tris-HCl buffer solution containing 0.4 M NaCl;
[0017] The buffer C is a Tris-HCl buffer solution containing 0.6 M NaCl, preferably a 0.05 M, pH 7.5 Tris-HCl buffer solution containing 0.6 M NaCl;
[0018] The buffer D is a Tris-HCl buffer solution containing 0.8 M NaCl, preferably a 0.05 M, pH 7.5 Tris-HCl buffer solution containing 0.8 M NaCl;
[0019] The buffer E is a Tris-HCl buffer solution containing 1.0 M NaCl, preferably a 0.05 M, pH 7.5 Tris-HCl buffer solution containing 1.0 M NaCl.
[0020] In some embodiments, the volume ratio of the buffer B, the buffer C, the buffer D, and the buffer E is 1:1:1:1.
[0021] In some embodiments, when the buffer B, the buffer C, the buffer D, and the buffer E are used as the eluent, the volume ratio of the eluates collected in sequence is 1:1:1:1.
[0022] In some embodiments, the ratio of the volume of buffer B to the volume of the eluate collected when buffer B is used as the eluent is 1:1.
[0023] In some embodiments, when buffer B is used as the eluent, buffer B is divided into 5 equal volumes and added dropwise separately, and eluates B-1, B-2, B-3, B-4, and B-5 are collected in sequence; when buffer C is used as the eluent, buffer C is divided into 5 equal volumes and added dropwise separately, and eluates C-1, C-2, C-3, C-4, and C-5 are collected in sequence; when buffer D is used as the eluent, buffer D is divided into 5 equal volumes and added dropwise separately, and eluates D-1, D-2, D-3, D-4, and D-5 are collected in sequence; when buffer E is used as the eluent, buffer E is divided into 5 equal volumes and added dropwise separately, and eluates E-1, E-2, E-3, E-4, and E-5 are collected in sequence.
[0024] In some embodiments, the volume ratio of eluates B-1, B-2, B-3, B-4, and B-5 is 1:1:1:1:1.
[0025] In some embodiments, the volume ratio of eluates C-1, C-2, C-3, C-4, and C-5 is 1:1:1:1:1.
[0026] In some embodiments, the volume ratio of eluates D-1, D-2, D-3, D-4, and D-5 is 1:1:1:1:1.
[0027] In some embodiments, the volume ratio of eluates E-1, E-2, E-3, E-4, and E-5 is 1:1:1:1:1.
[0028] In some embodiments, the addition dropwise is achieved by using a pipette.
[0029] In some embodiments, before the gradient elution, pre-rinsing is performed with buffer A. In some embodiments, buffer A is a Tris-HCl buffer. In some embodiments, buffer A is a 0.05 M Tris-HCl buffer with a pH of 7.5.
[0030] In some embodiments, the volume ratio of buffer A before the gradient elution to buffer B during the gradient elution is 7:7.5.
[0031] In some embodiments, the ratio of the volume of buffer A before the gradient elution to the volume of the pre-rinse solution collected using buffer A is 7:8.
[0032] In some embodiments, before the gradient elution, buffer A is pre-divided into 5 portions according to a volume ratio of 1:1.5:1.5:1.5:1.5 and added dropwise respectively, and eluent A is collected successively. 前 -1, A 前 -2, A 前 -3, A 前 -4 and A 前 -5.
[0033] In some embodiments, the eluent A 前 -1, A 前 -2, A 前 -3, A 前 -4, A 前 -5 has a volume ratio of 2:1.5:1.5:1.5:1.5.
[0034] In some embodiments, after the gradient elution, it further includes: rinsing with buffer A. In some embodiments, buffer A is Tris-HCl buffer. In some embodiments, buffer A is 0.05 M Tris-HCl buffer with a pH of 7.5.
[0035] In some embodiments, the volume ratio of buffer A after the gradient elution to buffer B during the gradient elution is 1:1.
[0036] In some embodiments, the volume ratio of buffer A after the gradient elution to the volume of the eluent collected by rinsing with buffer A is 1:1.
[0037] In some embodiments, after the gradient elution, it further includes: dividing buffer A into 5 equal-volume portions and adding them dropwise respectively, and eluent A is collected successively. 后 -1, A 后 -2, A 后 -3, A 后 -4 and A 后 -5.
[0038] In some embodiments, the eluent A 后 -1, A 后 -2, A 后 -3, A 后 -4, A 后 -5 has a volume ratio of 1:1:1:1:1.
[0039] In some embodiments, before loading the starch sample to be separated, the chromatography column is pre-treated by rinsing with deionized water successively (for example, rinsing with 3 column volumes of deionized water) and equilibrating with buffer A (for example, equilibrating the column with 3 column volumes of buffer A). In some embodiments, buffer A is a Tris-HCl buffer. In some embodiments, buffer A is a 0.05 M Tris-HCl buffer with a pH of 7.5.
[0040] For the above loading, as is well known to those skilled in the art, dry loading or wet loading can be used, and both loading methods are technical means well known to those skilled in the art. In this application, the starch sample can be loaded by wet loading. For example, the starch sample can be pre-prepared as a starch solution and loaded by wet loading (for example, taking 1 mL of the starch solution for loading). An exemplary method for preparing the starch solution is as follows: Take 0.5 g of starch (such as tobacco leaf starch), add 90 mL of 1 mM NaOH aqueous solution, heat to boiling and then simmer for 5 min until completely dissolved and the solution is clear and transparent. After cooling to room temperature, add 1 mL of buffer A, adjust the pH to 7.5 slightly, and make up deionized water to 100 mL.
[0041] In some embodiments, after gradient elution and rinsing with buffer A, it further includes: removing impurities and re-equilibrating the column with buffer A (for example, removing impurities and re-equilibrating the column with 5 column volumes of buffer A). In some embodiments, buffer A is a Tris-HCl buffer. In some embodiments, buffer A is a 0.05 M Tris-HCl buffer with a pH of 7.5.
[0042] In some embodiments, the eluate collected when buffer B and buffer C are used as eluents contains the first eluate, and the eluate collected when buffer C and buffer D are used as eluents contains the second eluate.
[0043] In some embodiments, the mixed solution of eluates B-2, B-3, B-4, B-5, C-1 and C-2 is the first eluate, and the mixed solution of eluates C-4, C-5, D-1, D-2, D-3, D-4 and D-5 is the second eluate.
[0044] In some embodiments, the mixed solution of eluates that show a blue color when reacting with iodine is the first eluate, and the mixed solution of eluates that show a red color when reacting with iodine is the second eluate.
[0045] In some embodiments, the color reaction with iodine is achieved using an iodine reagent.
[0046] In some embodiments, the iodine reagent is dilute iodine solution.
[0047] In some embodiments, the method for preparing the dilute iodine solution includes: sucking 0.16 mL of the original iodine solution, adding 1.6 g of potassium iodide, dissolving with deionized water, and then making up the volume to 40 mL.
[0048] In some embodiments, the method for preparing the original iodine solution includes: weighing 1.1 g of iodine and 2.2 g of potassium iodide, completely dissolving them with a small amount of deionized water, and then making up the volume to 50 mL.
[0049] In some embodiments, the desalting is carried out using a dialysis bag with a molecular weight cut-off of 1 kD.
[0050] In some embodiments, the drying is freeze-drying.
[0051] In some embodiments, before packing the column, the stationary phase is pre-swollen and activated.
[0052] In some embodiments, in the column chromatography, the ratio of the packing volume of the stationary phase to the volume of the chromatography column is 0.7:1 to 0.8:1, preferably 0.73:1 to 0.75:1.
[0053] In some embodiments, in the column chromatography, the ratio of the packing height of the stationary phase to the height of the chromatography column is 0.7:1 to 0.8:1, preferably 0.72:1 to 0.74:1.
[0054] In some embodiments, the starch is tobacco leaf starch.
[0055] Beneficial effects
[0056] The method of the present invention uses a cellulose DE-52 chromatography column and gradient elution with different concentrations of NaCl buffer solution to separate and purify amylose and amylopectin (such as tobacco leaf amylose and tobacco leaf amylopectin). Compared with the commonly used n-butanol-isopropanol extraction method, the amylose and amylopectin obtained have higher purity. Moreover, the method of the present invention does not need to use toxic organic reagents, and the whole purification process only takes 1 h, which saves a lot of time compared with the commonly used n-butanol-isopropanol extraction method (at least 7 days). Description of the drawings
[0057] Figure 1 It is the separation effect diagram of amylose and amylopectin eluted with a concentration gradient of NaCl buffer solution using cellulose DE-52 packing;
[0058] Figure 2 It is the comparison diagram of the original starch (tobacco leaf starch), amylose, and amylopectin;
[0059] Figure 3The separation effect diagram of amylose and amylopectin using cellulose DE-52 packing material and eluting with NaCl-free buffer solution;
[0060] Figure 4 The separation effect diagram of amylose and amylopectin using Sephadex G-100 packing material and eluting with a concentration gradient of NaCl buffer solution;
[0061] Figure 5 The separation effect diagram of amylose and amylopectin using macroporous adsorption resin D301 packing material and eluting with a concentration gradient of NaCl buffer solution;
[0062] Figure 6 The separation effect diagram of amylose and amylopectin using anion exchange resin 717 packing material and eluting with a concentration gradient of NaCl buffer solution. Detailed implementation mode
[0063] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limiting the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art.
[0064] To achieve the object of the present invention, the present invention adopts the following technical solutions:
[0065] Use a cellulose DE-52 chromatography column to cooperate with different concentrations of NaCl buffer solution gradient elution of tobacco leaf starch to separate and purify tobacco leaf amylose and tobacco leaf amylopectin. After tobacco leaf amylose and tobacco leaf amylopectin pass through DE-52 column chromatography, they are desalted through a 1kD dialysis bag and then freeze-dried to form tobacco leaf amylose powder and tobacco leaf amylopectin powder respectively.
[0066] In some embodiments, the method for making the cellulose DE-52 chromatography column is as follows: Swell and activate cellulose DE-52 with reference to the instructions, and then install it in an empty column with an inner diameter of 21.4 mm and a total volume of 27 mL. The height of the packing in the column is 55 mm, the packing volume is 20 mL, and the packing is rinsed with 3 times the column volume of deionized water and then equilibrated with 3 times the column volume of buffer A.
[0067] In some embodiments, the gradient elution method with different concentrations of NaCl buffer is as follows: Take 1 mL of tobacco leaf starch solution for loading. After the tobacco leaf starch solution is completely immersed in the packing material, use a pipette to add 1 mL of buffer A and collect the first tube of 2 mL of liquid. Thereafter, the 2nd to 5th tubes are sequentially rinsed with 1.5 mL of buffer A and collected, 1.5 mL per tube; the 6th to 10th tubes are sequentially rinsed with 1.5 mL of buffer B and collected, 1.5 mL per tube; the 11th to 15th tubes are sequentially rinsed with 1.5 mL of buffer C and collected, 1.5 mL per tube; the 16th to 20th tubes are sequentially rinsed with 1.5 mL of buffer D and collected, 1.5 mL per tube; the 21st to 25th tubes are sequentially rinsed with 1.5 mL of buffer E and collected, 1.5 mL per tube; the 26th to 30th tubes are sequentially rinsed with 1.5 mL of buffer A and collected, and the column is purified and re-equilibrated with 5 times the column volume of buffer A to facilitate entry into the next purification cycle.
[0068] Among them, the preparation method of buffer A: Dilute 0.4 M, pH 7.5 Tris-HCl with deionized water to 0.05 M.
[0069] The preparation method of buffer B: Take 125 mL of 0.4 M, pH 7.5 Tris-HCl, add 500 mL of deionized water and 23.4 g of NaCl, dissolve and then make up the volume to 1 L.
[0070] The preparation method of buffer C: Take 125 mL of 0.4 M, pH 7.5 Tris-HCl, add 500 mL of deionized water and 35.1 g of NaCl, dissolve and then make up the volume to 1 L.
[0071] The preparation method of buffer D: Take 125 mL of 0.4 M, pH 7.5 Tris-HCl, add 500 mL of deionized water and 46.8 g of NaCl, dissolve and then make up the volume to 1 L.
[0072] The preparation method of buffer E: Take 125 mL of 0.4 M, pH 7.5 Tris-HCl, add 500 mL of deionized water and 58.5 g of NaCl, dissolve and then make up the volume to 1 L.
[0073] The present invention will be further explained below with specific embodiments.
[0074] Preparation of tobacco leaf starch: Refer to the method in the literature [Wang Tao, He Fan, Tian Binqiang, etc. Optimization of the extraction process of flue-cured tobacco leaf starch [J]. Tobacco Science & Technology, 2012(6):4.] to extract and prepare tobacco leaf starch.
[0075] Preparation of the original iodine solution: Weigh 1.1 g of iodine and 2.2 g of potassium iodide, dissolve them completely with a small amount of deionized water, make up the volume to 50 mL, store it in a brown bottle and keep it away from light. The solution can be stored at room temperature for 1 month.
[0076] Preparation of the dilute iodine solution: Pipette 0.16 mL of the original iodine solution, add 1.6 g of potassium iodide, dissolve it with deionized water and make up the volume to 40 mL, store it in a brown bottle. Use it immediately after preparation.
[0077] Preparation of buffer A (0.05 M Tris-HCl buffer with pH 7.5): Dilute 0.4 M Tris-HCl (purchased from Shanghai Yuanye, product number R22648-500 ml, the same below) with deionized water to 0.05 M.
[0078] Preparation of buffer B (0.05 M Tris-HCl buffer containing 0.4 M NaCl with pH 7.5): Take 125 mL of 0.4 M Tris-HCl with pH 7.5, add 500 mL of deionized water and 23.4 g of NaCl, dissolve and then make up the volume to 1 L.
[0079] Preparation of buffer C (0.05 M Tris-HCl buffer containing 0.6 M NaCl with pH 7.5): Take 125 mL of 0.4 M Tris-HCl with pH 7.5, add 500 mL of deionized water and 35.1 g of NaCl, dissolve and then make up the volume to 1 L.
[0080] Preparation of buffer D (0.05 M Tris-HCl buffer containing 0.8 M NaCl with pH 7.5): Take 125 mL of 0.4 M Tris-HCl with pH 7.5, add 500 mL of deionized water and 46.8 g of NaCl, dissolve and then make up the volume to 1 L.
[0081] Preparation of buffer E (0.05 M Tris-HCl buffer containing 1.0 M NaCl with pH 7.5): Take 125 mL of 0.4 M Tris-HCl with pH 7.5, add 500 mL of deionized water and 58.5 g of NaCl, dissolve and then make up the volume to 1 L.
[0082] Tobacco leaf starch solution: Take 0.5 g of tobacco leaf starch, add 90 mL of 1 mM NaOH aqueous solution, heat to boiling and then simmer for 5 min until it is completely dissolved and the solution is clear and transparent. After cooling to room temperature, add 1 mL of buffer A, adjust the pH to 7.5 slightly, and make up the volume to 100 mL with deionized water.
[0083] Example 1: The method for separating and purifying amylose and amylopectin of the present invention
[0084] Taking tobacco leaf starch as an example, the separation and purification of amylose and amylopectin were carried out.
[0085] (1) Column packing
[0086] The anion exchange packing material (DEAE cellulose DE-52, purchased from Shanghai Yuanye, product number S14024-100g) was swollen and activated with reference to the instruction manual, and then packed into a column (the empty column used was purchased from Tianyan Biotechnology, which was a general B-type empty column, numbered DB02, with an inner diameter of 21.4 mm and a total volume of 27 mL). After packing, the height of the packing material was 55 mm and the volume of the packing material was 20 mL. The packing material was rinsed with 3 column volumes of deionized water and then the column was equilibrated with 3 column volumes of buffer A.
[0087] (2) Sample loading, sample collection and detection
[0088] 1 mL of the tobacco leaf starch solution was loaded onto the column. After the tobacco leaf starch solution was completely immersed in the packing material, 1 mL of buffer A was added dropwise with a pipette to collect the first tube (2 mL). Thereafter, the 2nd to 5th tubes were rinsed and collected with 1.5 mL of buffer A in sequence (1.5 mL per tube);
[0089] The 6th to 10th tubes were rinsed and collected with 1.5 mL of buffer B in sequence (1.5 mL per tube);
[0090] The 11th to 15th tubes were rinsed and collected with 1.5 mL of buffer C in sequence (1.5 mL per tube);
[0091] The 16th to 20th tubes were rinsed and collected with 1.5 mL of buffer D in sequence (1.5 mL per tube);
[0092] The 21st to 25th tubes were rinsed and collected with 1.5 mL of buffer E in sequence (1.5 mL per tube);
[0093] The 26th to 30th tubes were rinsed and collected with 1.5 mL of buffer A in sequence (1.5 mL per tube), and the column was decontaminated and re-equilibrated with 5 column volumes of buffer A to facilitate entry into the next purification cycle. The obtained target sample solution was desalted through a 1 kD dialysis bag and then freeze-dried to form amylose and amylopectin respectively.
[0094] Effect test: 20 μL of dilute iodine solution was pre-added to the microplate, and 150 μL of the eluate was added in sequence according to the collection order, blown and mixed evenly, and the color was developed. The results were as Figure 1 shown. As Figure 1 can be seen, under the method of the present invention, the solutions in the 7th to 12th tubes showed a blue color after being developed with iodine solution and were judged to be amylose; the solutions in the 14th to 20th tubes showed a red color after being developed with iodine solution and were judged to be amylopectin. As Figure 2It can be seen that the iodine solution color reactions of the isolated amylose and amylopectin are significantly different from that of the original starch. Amylose is bluer than the original starch, and amylopectin is redder than the original starch, indicating that the method of the present invention can effectively separate amylose and amylopectin in tobacco leaf starch. The solutions in tubes 7 - 12 were combined as the amylose solution, and the solutions in tubes 14 - 20 were combined as the amylopectin solution. After desalting the above two solutions through a 1kD dialysis bag and then freeze-drying, tobacco leaf amylose and tobacco leaf amylopectin were respectively formed.
[0095] Comparative Example 1: Influence of different elution solvents on the separation and purification of amylose and amylopectin
[0096] All the eluent in Example 1 was replaced with buffer A, that is, no NaCl gradient elution was used, and the column packing and other operation steps remained unchanged. The results are as Figure 3 shown.
[0097] It can be Figure 3 seen that using the buffer without NaCl for elution cannot separate amylose from amylopectin, indicating that NaCl gradient elution is very necessary.
[0098] Comparative Example 2: Influence of different column packings on the separation and purification of amylose and amylopectin
[0099] The anion exchange packing (DEAE cellulose DE-52) in Example 1 was respectively replaced with Sephadex G-100, macroporous adsorption resin D301, and anion exchange resin 717, and other steps remained unchanged. The test results are as Figure 4 (Sephadex G-100, purchased from Shanghai Yuanye, product number S14034-100g), Figure 5 (Macroporous adsorption resin D301, purchased from Shanghai Yuanye, product number S26726-500g), Figure 6 (Anion exchange resin 717, purchased from Shanghai Yuanye, product number S14158-1kg) shown.
[0100] It can be Figures 4 to 6 seen that none of these three packings can separate amylose and amylopectin, indicating that the packing DEAE cellulose DE-52 used in the present invention cannot be replaced by other packings.
[0101] Comparative Example 3: Effect comparison between the method of the present invention and existing methods
[0102] Taking tobacco starch as an example, the purification methods of amylose and amylopectin in the present invention are the same as those in Example 1; the existing purification methods (n-butanol-isopropanol extraction method) of amylose and amylopectin and their steps refer to the literature [Lu Yali, Wang Mingli, Yan Yan, etc. Separation, purification and identification of amylose and amylopectin in coix starch [J]. Food Industry, 2014, 35(3): 3.].
[0103] Blue value and iodine affinity are often used to characterize the purity of starch. The blue value of amylose is generally 0.8 - 1.2, while that of amylopectin is only 0.08 - 0.22; the iodine affinity of amylose is generally between 19% - 20%, while that of amylopectin is below 1%. The blue value and iodine affinity of the amylose and amylopectin purified by the two methods were measured respectively, and the measurement steps refer to the literature [Li Li. Research on the analytical determination method of amylose content in corn starch [D]. Henan University of Technology, 2018: 14 - 15.]. The results are shown in Table 1.
[0104] As can be seen from Table 1, the blue value and iodine affinity of the amylose purified by the method of the present invention are slightly higher than those of the method in the comparative literature, indicating that there are more helical regions in the amylose purified by the method of the present invention that can complex iodine. Since the better the linearity of amylose, the higher the purity, therefore, compared with the prior art, the purity of the amylose purified by the method of the present invention is higher; at the same time, the blue value and iodine affinity of the amylopectin purified by the present invention are slightly lower than those of the comparative literature, indicating that the linearity of the amylopectin purified by the method of the present invention is worse. Since the worse the linearity of amylopectin, the more branches and the higher the purity, therefore, compared with the prior art, the purity of the amylopectin purified by the method of the present invention is also higher.
[0105] Table 1 Comparison of the effects of purifying amylose and amylopectin by different methods
[0106]
[0107] Therefore, the method of the present invention can well replace the n-butanol-isopropanol extraction method in the literature. At the same time, it is worth mentioning that after the crude separation of amylose and amylopectin by the extraction method (the cycle is 24h), it is necessary to carry out 6 - 10 times of repeated extractions, each time taking 24h, and the entire purification cycle requires at least 7 days, with too high time cost. However, the method of the present invention only requires 1h in the column passing stage, and its purification efficiency is increased by 168 times. Moreover, it does not require the large use of strongly volatile organic solvents n-butanol and isopropanol, saving material costs and being green and healthy, which is exactly the advanced point of the present invention.
[0108] It should be understood that the inventions described herein are not limited to specific methodologies, experimental protocols or reagents, as these can vary. The discussions and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.
Claims
1. A method for separating amylose and amylopectin in a starch sample, comprising: The starch sample to be separated is separated by column chromatography to obtain separated amylose and amylopectin; Wherein, the stationary phase of the column chromatography is DEAE cellulose, and the eluent of the column chromatography is Tris-HCl buffer containing NaCl.
2. The method according to claim 1, wherein: The stationary phase of the column chromatography is DEAE cellulose DE-52.
3. The method according to claim 1 or 2, wherein: The column chromatography comprises: (1) using a Tris-HCl buffer containing NaCl with a gradually increasing NaCl concentration as an eluent for gradient elution, and collecting a first eluate and a second eluate in sequence; (2) Desalting and drying the first eluate to obtain amylose, and desalting and drying the second eluate to obtain amylopectin.
4. The method according to claim 3, wherein: Step (1) comprises: Using buffer B, buffer C, buffer D, and buffer E as eluents in sequence for gradient elution, and collecting the first eluate and the second eluate in sequence; in: The buffer B is a Tris-HCl buffer containing 0.4M NaCl, preferably a 0.05M Tris-HCl buffer containing 0.4M NaCl and pH 7.5; The buffer C is a Tris-HCl buffer containing 0.6M NaCl, preferably a 0.05M Tris-HCl buffer containing 0.6M NaCl and pH 7.5; The buffer D is a Tris-HCl buffer containing 0.8M NaCl, preferably a 0.05M Tris-HCl buffer containing 0.8M NaCl and pH 7.5; The buffer E is a Tris-HCl buffer containing 1.0 M NaCl, preferably a 0.05 M Tris-HCl buffer containing 1.0 M NaCl and pH 7.
5.
5. The method according to claim 4, wherein: The volume ratio of the buffer B, the buffer C, the buffer D, and the buffer E is 1:1:1:1; Preferably, when the buffer B, the buffer C, the buffer D, and the buffer E are used as eluents, the volume ratio of the eluates collected sequentially is 1:1:1:1; Preferably, the ratio of the volume of the buffer solution B to the volume of the eluate collected when the buffer solution B is used as the eluent is 1:
1.
6. The method according to claim 5, wherein: When the buffer B is used as the eluent, the buffer B is divided into 5 equal volumes and added dropwise respectively, and the eluents B-1, B-2, B-3, B-4 and B-5 are collected in sequence; preferably, the volume ratio of the eluents B-1, B-2, B-3, B-4 and B-5 is 1:1:1:1:1; When the buffer C is used as the eluent, the buffer C is divided into 5 equal volumes and added dropwise respectively, and the eluents C-1, C-2, C-3, C-4 and C-5 are collected in sequence; preferably, the volume ratio of the eluents C-1, C-2, C-3, C-4 and C-5 is 1:1:1:1:1; When the buffer D is used as the eluent, the buffer D is divided into 5 equal volumes and added dropwise respectively, and the eluents D-1, D-2, D-3, D-4 and D-5 are collected in sequence; preferably, the volume ratio of the eluents D-1, D-2, D-3, D-4 and D-5 is 1:1:1:1:1; When the buffer E is used as the eluent, the buffer E is divided into 5 equal volumes and added dropwise respectively, and the eluents E-1, E-2, E-3, E-4 and E-5 are collected in sequence; preferably, the volume ratio of the eluents E-1, E-2, E-3, E-4 and E-5 is 1:1:1:1:
1.
7. The method according to any one of claims 3 to 6, wherein: Before the gradient elution, the washing was performed in advance with buffer A; Preferably, the buffer A is Tris-HCl buffer; Preferably, the buffer A is 0.05M Tris-HCl buffer at pH 7.5; Preferably, the volume ratio of the buffer A before the gradient elution to the buffer B during the gradient elution is 7:7.5; Preferably, the ratio of the volume of the buffer A before the gradient elution to the volume of the eluent collected by eluting with the buffer A is 7:8; Preferably, before the gradient elution, the buffer A is pre-divided into 5 portions according to a volume ratio of 1:1.5:1.5:1.5:1.5 and added dropwise, and the eluent A is collected in sequence. 前 -1, A 前 -2, A 前 -3.A 前 -4 and A 前 -5; Preferably, the eluent A 前 -1, A 前 -2, A 前 -3.A 前 -4, A 前 The volume ratio of -5 is 2:1.5:1.5:1.5:1.
5.
8. The method according to any one of claims 3 to 7, wherein: After the gradient elution, the method further comprises: eluting with buffer A; Preferably, the buffer A is Tris-HCl buffer; Preferably, the buffer A is 0.05M Tris-HCl buffer at pH 7.5; Preferably, the volume ratio of the buffer A after the gradient elution to the buffer B during the gradient elution is 1:1; Preferably, the ratio of the volume of the buffer A after the gradient elution to the volume of the eluent collected by eluting with the buffer A is 1:1; Preferably, after the gradient elution, the step further comprises: dividing the buffer A into 5 equal volumes and adding them dropwise respectively, and collecting the eluent A in sequence. 后 -1, A 后 -2, A 后 -3.A 后 -4 and A 后 -5; Preferably, the eluent A 后 -1, A 后 -2, A 后 -3.A 后 -4, A 后 -5 has a volume ratio of 1:1:1:1:
1.
9. The method according to any one of claims 1 to 8, wherein: The method further has one or more technical features selected from the following (i)-(v): (i) before loading the starch sample to be separated, the chromatography column is preliminarily subjected to a deionized water elution treatment and a buffer A equilibration treatment in sequence; preferably, the buffer A is a Tris-HCl buffer; more preferably, the buffer A is a 0.05 M, pH 7.5 Tris-HCl buffer; (ii) after the gradient elution is performed with buffer A, the column is further treated with buffer A for impurity removal and re-equilibration; preferably, the buffer A is Tris-HCl buffer; more preferably, the buffer A is 0.05 M Tris-HCl buffer at pH 7.5; (iii) the eluate collected when the buffer B and the buffer C are used as eluents comprises the first eluate, and the eluate collected when the buffer C and the buffer D are used as eluents comprises the second eluate; (iv) a mixture of the eluents B-2, B-3, B-4, B-5, C-1 and C-2 is the first eluent, and a mixture of the eluents C-4, C-5, D-1, D-2, D-3, D-4 and D-5 is the second eluent; (v) A mixture of eluents that develop blue color when exposed to iodine is the first eluent, and a mixture of eluents that develop red color when exposed to iodine is the second eluent.
10. The method according to any one of claims 1 to 9, wherein: The method further has one or more technical features selected from the following (i)-(v): (i) the desalting is carried out using a 1 kD dialysis bag; (ii) the drying is freeze drying; (iii) the stationary phase is swollen and activated before being loaded into the column; (iv) in the column chromatography, the ratio of the volume of the stationary phase filler to the volume of the chromatography column is 0.7:1 to 0.8:1, preferably 0.73:1 to 0.75:1; (v) In the column chromatography, the ratio of the height of the stationary phase filler to the height of the chromatography column is 0.7:1 to 0.8:1, preferably 0.72:1 to 0.74:1.