Preparation method of dextran standard substance

Abstract

The invention discloses a preparation method of a dextran standard substance, which comprises the following steps: collecting various process sugar material mixed samples of different sugar refineries, carrying out alcohol precipitation, membrane filtration, column purification and other technical means to obtain a special dextran standard substance for the sugar industry, and comparing the standard substance with a commercially available dextran standard substance, the content of the dextran in a sugar refining sample can be more truly reflected, the actual sugar refining condition can be represented, and the detection error is eliminated.

Description

Technical Field

[0001] The invention relates to the technical field of sugar making, and in particular to a method for preparing a dextran standard product dedicated to the sugar making industry. Background Art

[0002] Dextran is a type of polysaccharide produced by the conversion of sucrose by dextran sucrase. Its sugar chain consists of only glucose residues, connected to the main chain by α-1,6-glycosidic bonds, and the side chains are connected to the main chain by one or more of glucose residues or glucose chains (simple or complex) by α-1,2-, α-1,3- or α-1,4-glycosidic bonds, of which α-1,6-glycosidic bonds generally account for more than 65% of the total glycosidic bonds. The presence of dextran has adverse effects on the sugar production process from cane juice clarification to sugar refining, including increasing the optical rotation of the sugar solution, resulting in errors in sucrose content determination, increasing the viscosity of the sugar solution, resulting in difficulty in filtration, hindering the crystallization of sucrose, resulting in a decrease in crystallization rate and crystal deformation. At the same time, sugar products with too high a dextran content are limited in applicability and their selling prices will also be reduced. Therefore, the detection of dextran is of great significance for evaluating the severity of the dextran problem in the sugar production process and judging the effect of preventive and removal measures.

[0003] The current standard methods for dextran detection include 4.7 of GB / T 15108-2017 Raw Sugar (which can only determine the dextran content of raw sugar) and the Immunoturbidimetric Method for Determination of Dextran in Sugarcane and Its Products, DB45 / T 2205-2020. Both methods require the use of dextran standards to prepare a series of standard solutions and draw a working curve for calculating the dextran concentration. The standard specified in the current method is commercially available dextran. Currently available dextran is produced by fermentation of Leuconostoc mesenteroides. About 95% of its glycosidic bonds are α-1,6-glycosidic bonds, and the rest are α-1,3-glycosidic bonds. It has a fixed single fermentation strain, process and purification process, and the dextran structure is single.

[0004] Different microorganisms produce different structures of dextran under different conditions. The raw materials and microbial environment of the sugar factory production line are complex and the conditions are changeable, so the dextran produced has different structures. Therefore, the dextran standards currently available on the market cannot accurately reflect the true composition and structure of dextran in the sugar industry, resulting in errors in the standard curve made after the antibody reacts with dextran, which ultimately leads to errors in the results. Therefore, the standard used to detect dextran should be separated from the actual sugar samples. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a dextran standard product dedicated to the sugar industry. Commercially available dextran is prepared using a single microorganism and conditions, and its structure cannot represent the actual dextran structure in sugar production, resulting in errors in the quantitative detection process. The present invention separates dextran from an actual sugar production sample, and the dextran can represent the actual sugar production situation and eliminate detection errors.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing a dextran standard product comprises the following steps:

[0008] (1) Sampling: Collect sugar material samples from sugar factories with different sugar production processes to prepare mixed samples of sugar materials from multiple processes;

[0009] (2) Alcohol precipitation: Add amylase to the mixed sample for enzymatic hydrolysis, add trichloroacetic acid after enzymatic hydrolysis, shake vigorously, and filter with a filter membrane; the filtrate is subjected to multiple alcohol precipitations to obtain a crude polysaccharide precipitate A;

[0010] (3) Membrane filtration: Dissolve the crude polysaccharide precipitate A in water, use an ultrafiltration membrane to remove small molecular sugars in the solution, then add an equal volume of ethanol, shake vigorously, centrifuge, and collect the precipitate to obtain the crude polysaccharide precipitate B;

[0011] (4) First column separation and purification: dissolving the crude polysaccharide precipitate B in mobile phase A to obtain a crude polysaccharide solution I; separating and purifying the crude polysaccharide solution I using a Sephacryl S-500HR chromatography column, and collecting the eluate; desalting and freeze-drying the eluate to obtain a preliminarily purified dextran powder;

[0012] (5) Second column separation and purification: dissolving the preliminarily purified dextran powder in mobile phase B to obtain a crude polysaccharide solution II; separating and purifying the crude polysaccharide solution II using a Superdex 200PG chromatography column, and collecting the eluate; desalting and freeze-drying the eluate to obtain a high-purity dextran standard powder.

[0013] Preferably, step (1) specifically comprises: collecting sugar cane, mixed juice, molasses, and white sugar samples from sulfite sugar factories, sugar cane, mixed juice, molasses, and white sugar samples from carbonate sugar factories, raw sugar, raw sugar dilute juice, molasses, and refined sugar samples from refined sugar factories, and sugar beets, exudate juice, molasses, and soft sugar samples from sugar beet factories; juicing sugar cane and sugar beets, and then mixing and dissolving all liquid and solid materials to obtain mixed samples of various process sugar materials.

[0014] Preferably, in step (2), the volume ratio of the amylase to the mixed sample is 3:1000, and the enzymatic hydrolysis conditions are 55° C. for 15 min.

[0015] Preferably, in step (2), the conditions for subjecting the filtrate to multiple alcohol precipitation are: adding an equal volume of 95% ethanol to the filtrate, shaking vigorously at 4°C, and centrifuging to obtain a precipitate; adding deionized water to the precipitate, and then adding an equal volume of 95% ethanol to the deionized water, shaking vigorously at 4°C, and centrifuging to obtain a precipitate, repeating the operation, and centrifuging 3 times in total to obtain a crude polysaccharide precipitate A.

[0016] Preferably, in step (3), the molecular weight cut-off of the ultrafiltration membrane is 1 kDa.

[0017] Preferably, in step (4), the composition of the mobile phase A is: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, and the solvent is water.

[0018] Preferably, in step (4), the conditions for separation and purification using a Sephacryl S-500HR chromatography column are: using mobile phase A as the eluent, the amount of mobile phase A is 7 to 8 times the volume of the Sephacryl S-500HR chromatography column, the flow rate is 1 mL / min, the loading amount of the crude polysaccharide solution I is 5 mL, and the eluate is collected for 100 to 200 minutes.

[0019] Preferably, in step (5), the composition of the mobile phase B is: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, 5.844 g / L sodium chloride, and the solvent is water.

[0020] Preferably, in step (5), the conditions for separation and purification using a Superdex 200PG chromatography column are: using mobile phase B as the eluent, the amount of mobile phase B is 5 to 6 times the volume of the Superdex 200PG chromatography column, the flow rate is 0.5 mL / min, the loading amount of the crude polysaccharide solution II is 5 mL, and the eluate is collected for 112 to 136 minutes.

[0021] The present invention also provides a dextran standard prepared according to the above preparation method.

[0022] The present invention has the following beneficial effects:

[0023] The present invention collects mixed samples of various process sugar materials from different sugar factories, and obtains a dextran standard product dedicated to the sugar industry through technical means such as alcohol precipitation, membrane filtration, and column purification. Compared with the commercially available dextran standard product, the standard product can more truly reflect the dextran content in the sugar production sample, can represent the actual situation of sugar production, and eliminate detection errors.

[0024] The standard product of the present invention and the standard product required by the original standard were used to determine the dextran content of commercially available white sugar / raw sugar according to the methods of "GB / T15108-2017 Raw Sugar" and "DB45 / T 2205-2020 Determination of Dextran in Sugarcane and Its Products - Immunoturbidimetric Method". The results showed that the results of the dextran content determination using the standard product of the present invention were closest to the results of the separation weighing method, and could more truly reflect the dextran content in commercially available white sugar / raw sugar. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Example 1 Preparation of Dextran Standard

[0027] 1. Solution formulation

[0028] Mobile phase A: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, solvent is water;

[0029] Mobile phase B: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, 5.844 g / L sodium chloride, the solvent is water.

[0030] 2. Preparation of Standards

[0031] (1) Sampling and alcohol precipitation

[0032] Sampling was carried out at 10 sulfite sugar factories, 1 carbonate sugar factory, 3 refined sugar factories and 2 beet sugar factories according to the table below.

[0033]

[0034]

[0035] Juice the sugar cane and beet, mix all the liquid and solid materials, dissolve them, and obtain mixed samples of various process sugar materials. Take 100 mL of the mixed sample, add 0.3 mL of amylase, react at 55°C for 15 min, add 6 mL of trichloroacetic acid solution (100 g / L), shake violently, filter with a 0.45 μm membrane, add 106 mL of 95% ethanol (4°C), shake violently, centrifuge at 10,000 r / min at 4°C for 15 min, take the precipitate, add 50 mL of deionized water to dissolve the precipitate, add 50 mL of 95% ethanol (4°C), shake violently, centrifuge at 10,000 r / min at 4°C for 15 min, take the precipitate, repeat the operation, centrifuge 3 times in total, and obtain crude polysaccharide precipitate A.

[0036] (2) Membrane filtration

[0037] Dissolve the crude polysaccharide precipitate A in water to prepare a 20 mg / mL solution. Use a 1 kDa ultrafiltration membrane to treat the solution to ensure the removal of small molecular sugars. Add an equal volume of 95% ethanol (4°C), shake vigorously, centrifuge at 10,000 r / min at 4°C for 15 minutes, and take the precipitate to obtain the crude polysaccharide precipitate B.

[0038] (3) First column separation and purification

[0039] Use mobile phase A to dissolve crude polysaccharide precipitate B and prepare a 20 mg / mL solution. Use AKTA Explorer 100 protein purifier for purification, the column is Sephacryl S-500HR with an inner diameter of 16 mm, and the column volume is 120 mL. Operation parameters: flow rate 1 mL / min, pass 600 mL of mobile phase A, load 5 mL of crude polysaccharide solution, and pass 300 mL of mobile phase A. Collect one tube for every 5 mL (60 tubes in total). Determine the total sugar content of each tube by sulfuric acid-phenol method (for specific operations, refer to 7.2-7.5 of "Determination of total sugar content in edible fungi" in GB / T 15672-2009) to analyze the polysaccharide separation.

[0040] The 5 peak tubes were subjected to total acid hydrolysis (all polysaccharides can be hydrolyzed into monosaccharides) and monosaccharide component analysis. The process is as follows: 0.287 mL of sulfuric acid was added to each tube, and the tube was placed in a boiling water bath for 2 hours. Sodium hydroxide was added to neutralize the tube until it was neutral. Subsequent operations and chromatographic conditions were carried out in accordance with "NY / T 3902-2021 Ion Chromatography for the Determination of Arabinose, Galactose, Glucose, Fructose, Maltose and Sucrose in Fruits, Vegetables and Their Products", and the four monosaccharides of arabinose, galactose, glucose and fructose were determined. The results showed that the third peak had only one monosaccharide, glucose, and the remaining peaks had two or more monosaccharides, indicating that only the third peak was dextran, and the rest were heteropolysaccharides. Therefore, the collection tube number was confirmed to be tubes 21-40, i.e. 100-200 min.

[0041] peak Start and End Peak tube Peak area ratio Peak monosaccharide composition 1 Tubes 5-15 No. 10 21％ 84% glucose, 11% arabinose, 5% galactose 2 Tubes 15-18 No. 16 8％ 75% glucose, 13% arabinose, 12% galactose 3 Tubes 21-40 Tube No. 31 62％ 100% Glucose 4 Tube No. 47-52 Tube No. 49 7％ 71% glucose, 29% arabinose 5 Tubes 52-58 Tube No. 55 2％ 94% glucose, 6% galactose

[0042] The collected 100 mL effluent (100-200 min) was dialyzed and desalted using a dialysis bag, and then freeze-dried to obtain preliminarily purified dextran powder.

[0043] (4) Second column separation and purification

[0044] Dissolve the preliminarily purified dextran powder with mobile phase B and prepare a 20 mg / mL solution. Use AKTAExplorer100 protein purifier for purification, the column is Superdex 200PG with an inner diameter of 16 mm, and the column volume is 120 mL. Operation parameters: flow rate 0.5 mL / min, pass 600 mL of mobile phase B, load 5 mL of crude polysaccharide solution, and pass 80 mL of mobile phase B. Collect one tube for every 4 mL (20 tubes in total). Determine the total sugar content of each tube by sulfuric acid-phenol method (refer to 7.2-7.5 of "GB / T 15672-2009 Determination of total sugar content in edible fungi" for specific operations), and the polysaccharide separation can be analyzed.

[0045] The two peak tubes were subjected to total acid hydrolysis (all polysaccharides can be hydrolyzed into monosaccharides) and monosaccharide component analysis. The process is as follows: 0.287 mL of sulfuric acid was added to each tube, and the tube was placed in a boiling water bath for 2 hours. Sodium hydroxide was added to neutralize the tube until it was neutral. Subsequent operations and chromatographic conditions were carried out in accordance with "NY / T 3902-2021 Ion Chromatography for the Determination of Arabinose, Galactose, Glucose, Fructose, Maltose and Sucrose in Fruits, Vegetables and Their Products". The four monosaccharides of arabinose, galactose, glucose and fructose were determined. The results showed that the second peak had only one monosaccharide, glucose, and the first peak had glucose and galactose, indicating that only the second peak was dextran and the first was a heteropolysaccharide. Therefore, the collection tube number was confirmed to be tubes 15-17, i.e. 112-136 min.

[0046] peak Start and End Peak tube Peak area ratio Peak monosaccharide composition 1 Tubes 11-13 No. 12 8％ 88% glucose, 12% galactose 2 Tubes 15-17 No. 16 92％ 100% Glucose

[0047] The collected 15 mL effluent (112-136 min) was dialyzed and desalted using a dialysis bag, and then freeze-dried to obtain dextran standard powder.

[0048] Example 2 Dextran Standard Purity Verification

[0049] Dissolve the high-purity dextran standard powder in commercially available PBS buffer (0.01M pH = 5.5) to prepare a 20 mg / mL solution. Take 10 mL of the solution, add 1 mL of dextranase solution (dextranase can specifically hydrolyze dextran), and react at 55°C for 30 minutes. The solution before and after the reaction was analyzed by high-performance liquid chromatography, and the chromatographic conditions refer to the analysis method of dextran in the Chinese Pharmacopoeia. The results show that the solution before the reaction has only one peak at 12-18 minutes, and the peak of the solution after the reaction appears after 20 minutes, and the peak of 12-18 minutes completely disappears, indicating that the original substances are all dextran.

[0050] Example 3 Application of Dextran Standards

[0051] Commercially available white sugar / raw sugar was purchased and the dextran content was determined using two methods (three methods for raw sugar).

[0052] Method 1 (separation and weighing method): The total polysaccharides in the sugar product are separated by alcohol precipitation, and the proportion of dextran is calculated by the HPLC data before and after dextranase treatment, so as to calculate the content of dextran in the sugar product. The specific steps are as follows:

[0053] (1) Dissolve the sample in water to a concentration of 500 g / L, add anhydrous ethanol to a final concentration of 80%, let stand at 4°C for 24 h, centrifuge at 12,000 r / min for 20 min, take the precipitate, re-dissolve the precipitate with a small amount of water, add anhydrous ethanol again for alcohol precipitation, let stand, centrifuge, repeat twice. Freeze-dry to obtain the total polysaccharide in the sample, weigh it, and measure the water content using a rapid moisture analyzer.

[0054] (2) Add a small amount of dextranase (dextranase can specifically hydrolyze dextran) and react at 55°C for 30 minutes. The solution before and after the reaction is analyzed by high performance liquid chromatography. The chromatographic conditions refer to the analytical method for dextran in the Chinese Pharmacopoeia. Before the reaction, there is only one peak in the solution. After the reaction, the peak area decreases and a new peak appears in the small molecule range, indicating that dextran is degraded to form small molecule sugars. The ratio of the peak area reduction can be used to calculate the ratio of dextran in the total polysaccharide, and the dextran content in the sample can be calculated using this ratio.

[0055] Method 2: 4.7 of GB / T 15108-2017 Raw Sugar (only the dextran content of raw sugar can be determined), the standard substances used include the standard substances required by the original standard and the standard substances prepared by the present invention.

[0056] Method 3: "DB45 / T 2205-2020 Determination of glucan in sugarcane and its products - Immunoturbidimetric method", the standards used include the standards required by the original standard and the standards prepared by the present invention.

[0057] The following table shows the content data of dextran measured by different methods, in mg / kg.

[0058]

[0059] Note: *y is absorbance, x is dextran concentration; #y is turbidity value, x is dextran concentration.

[0060] The above results show that the results of the dextran content determination using the standard product of the present invention are closest to the results determined by the separation weighing method, and can more truly reflect the dextran content in commercially available white sugar / raw sugar.

[0061] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a dextran standard product, characterized in that: The following steps are involved: (1) Sampling: Collect sugar material samples from sugar factories with different sugar production processes to prepare mixed samples of sugar materials from multiple processes; (2) Alcohol precipitation: Add amylase to the mixed sample for enzymatic hydrolysis, add trichloroacetic acid after enzymatic hydrolysis, shake vigorously, and filter with a filter membrane; the filtrate is subjected to multiple alcohol precipitations to obtain a crude polysaccharide precipitate A; (3) Membrane filtration: Dissolve the crude polysaccharide precipitate A in water, use an ultrafiltration membrane to remove small molecular sugars in the solution, then add an equal volume of ethanol, shake vigorously, centrifuge, and collect the precipitate to obtain the crude polysaccharide precipitate B; (4) First column separation and purification: dissolving the crude polysaccharide precipitate B in the mobile phase A to obtain a crude polysaccharide solution I; The crude polysaccharide solution I is separated and purified by Sephacryl S-500HR chromatography column, and the eluate is collected; the eluate is desalted and freeze-dried to obtain a preliminarily purified dextran powder; (5) Second column separation and purification: dissolving the preliminarily purified dextran powder in mobile phase B to obtain a crude polysaccharide solution II; separating and purifying the crude polysaccharide solution II using a Superdex 200PG chromatography column, and collecting the eluate; desalting and freeze-drying the eluate to obtain a high-purity dextran standard powder.

2. The preparation method according to claim 1, characterized in that: Step (1) specifically comprises: collecting sugar cane, mixed juice, molasses, and white sugar samples from sulfite process sugar factories, sugar cane, mixed juice, molasses, and white sugar samples from carbonate process sugar factories, raw sugar, raw sugar dilute juice, molasses, and refined sugar samples from refined sugar factories, and sugar beets, exudate juice, molasses, and soft sugar samples from sugar beet factories; juicing sugar cane and sugar beets, and then mixing and dissolving all liquid and solid materials to obtain mixed samples of various process sugar materials.

3. The preparation method according to claim 1, characterized in that: In step (2), the volume ratio of the amylase to the mixed sample is 3:1000, and the enzymatic hydrolysis conditions are 55° C. for 15 min.

4. The preparation method according to claim 1, characterized in that: In step (2), the conditions for subjecting the filtrate to multiple alcohol precipitation are: adding an equal volume of 95% ethanol to the filtrate, shaking violently at 4°C, and centrifuging to obtain a precipitate; adding deionized water to the precipitate, and then adding an equal volume of 95% ethanol to the deionized water, shaking violently at 4°C, and centrifuging to obtain a precipitate, repeating the operation, and centrifuging 3 times in total to obtain a crude polysaccharide precipitate A.

5. The preparation method according to claim 1, characterized in that: In step (3), the molecular weight cut-off of the ultrafiltration membrane is 1 kDa.

6. The preparation method according to claim 1, characterized in that: In step (4), the composition of the mobile phase A is: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, and the solvent is water.

7. The preparation method according to claim 1, characterized in that: In step (4), the conditions for separation and purification using a Sephacryl S-500HR chromatography column are: using mobile phase A as the eluent, the amount of mobile phase A is 7 to 8 times the volume of the Sephacryl S-500HR chromatography column, the flow rate is 1 mL / min, the loading amount of the crude polysaccharide solution I is 5 mL, and the eluate is collected for 100 to 200 minutes.

8. The preparation method according to claim 1, characterized in that: In step (5), the composition of the mobile phase B is: 2.236 g / L disodium hydrogen phosphate, 4.109 g / L sodium dihydrogen phosphate, 5.844 g / L sodium chloride, and the solvent is water.

9. The preparation method according to claim 1, characterized in that: In step (5), the conditions for separation and purification using a Superdex 200PG chromatography column are: using mobile phase B as the eluent, the amount of mobile phase B is 5 to 6 times the volume of the Superdex 200PG chromatography column, the flow rate is 0.5 mL / min, the loading amount of the crude polysaccharide solution II is 5 mL, and the eluent is collected for 112 to 136 minutes.

10. The dextran standard prepared according to the preparation method according to any one of claims 1 to 9.

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