A kind of preparation method of red rice bran non-starch polysaccharide

Abstract

The invention discloses a method for preparing non-starch polysaccharides from red rice bran, and belongs to the technical field of polysaccharide extraction. The method for preparing non-starch polysaccharides from red rice bran of the invention comprises the following steps: grinding red rice bran with a rice mill, removing fat with anhydrous ethanol; hot water extraction; removing pigments with a macroporous adsorption resin, and removing proteins with a Sevag solution; then precipitating the polysaccharide with ethanol, and removing small molecular impurities such as monosaccharides and oligosaccharides by dialysis after redissolution; further separating and purifying the polysaccharide components with DEAE 52 Cellulose, and obtaining high-purity non-starch polysaccharides from red rice bran after dialysis desalting and freeze-drying of each component. The method can protect the sugar chain integrity of the polysaccharide to the greatest extent in terms of sugar chain structure, obtain high-yield red rice bran polysaccharides with a weight average molecular weight of ten thousand, and enrich the application paths of red rice bran polysaccharides.

Description

Technical Field

[0001] The invention relates to a method for preparing non-starch polysaccharide from red rice bran, and belongs to the technical field of polysaccharide extraction. Background Art

[0002] Red rice is the most produced colored rice in my country. The content of total phenols, protein, flavonoids, γ-aminobutyric acid and other active substances in red rice is significantly higher than that in white rice. It has the effects of improving the body's antioxidant capacity, improving atherosclerosis, lowering cholesterol, anti-cancer, lowering blood sugar, losing weight, and anti-inflammatory.

[0003] Plant polysaccharides have certain antioxidant, hypoglycemic and hypolipidemic effects. They are natural physiologically active substances, and the antioxidant, anti-tumor and hypoglycemic physiological activities of rice bran polysaccharides have been confirmed. Red rice bran resources are abundant, and it is necessary to study the extraction and purification methods of polysaccharides, analyze the structure and function of polysaccharides, and broaden the application channels of polysaccharides.

[0004] When conducting research on the structure and physiological activity of red rice bran polysaccharides and the actual application of red rice polysaccharides, it is necessary to minimize the damage to the glycosidic bonds and groups in the polysaccharide chain structure. First, it is to increase the application channels of red rice bran polysaccharides. The structures of polysaccharides that exert different physiological activities are different. According to the purpose of application, relatively complete red rice bran polysaccharides can be used, and enzymes and other means can be used to specifically degrade polysaccharides to obtain specific polysaccharide fragments to achieve the corresponding purpose of use; second, it is conducive to the analysis of the initial structure and physiological activity of red rice bran polysaccharides. In order to make the structure and function of the extracted polysaccharides closer to their original form in red rice, the original structure and biological activity in red rice are restored as much as possible through experiments, so as to explore the contribution of non-starch water-soluble polysaccharides of red rice bran to the physiological activity of red rice; third, it is to increase the yield of polysaccharides. Polysaccharides exist with a larger molecular weight. Under the same alcohol precipitation and dialysis conditions, more polysaccharides remain in the sample solution, and the polysaccharide yield is increased. However, adding auxiliary means such as ultrasound and enzymes during the extraction and purification process will cause the molecular weight of the polysaccharide to drop sharply. Under the same alcohol precipitation conditions, some polysaccharides cannot be precipitated. Under the same dialysis conditions, some polysaccharide fragments will be released from the dialysis bag, resulting in a decrease in yield.

[0005] The existing polysaccharide extraction methods involve ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction and other methods. Ultrasound is actually a means of polysaccharide degradation, which can easily break the polysaccharide chain. The amylase in the enzyme-assisted extraction method will have a cleavage effect on the "Glc-(1,4)-Glc" structure, causing the polysaccharide chain containing this structure to break; it is difficult to obtain polysaccharide components with a molecular weight of more than 10,000. Summary of the invention

[0006] [Technical issues]

[0007] In the prior art, the glycosidic bond and polysaccharide chain breakages are easily caused during the extraction of non-starch polysaccharides from red rice bran, which makes it impossible for the polysaccharide fragments to be precipitated by alcohol or removed by dialysis, resulting in the loss of polysaccharide information, low yield, and limited application. In addition, there are also problems such as low polysaccharide purity caused by incomplete purification steps, or improper operation methods, which cause secondary degradation of polysaccharide chains, affecting structural characterization, physiological activity research, and practical applications.

[0008] [Technical solution]

[0009] In order to simultaneously reduce the degradation effect on polysaccharide chains, achieve high purity and high yield of polysaccharides, and reduce unnecessary steps to reduce costs and sample preparation time, the present invention provides a method for preparing red rice bran non-starch polysaccharides. Specifically, hot water is used to extract red rice bran water-soluble polysaccharides, aiming to reduce the degradation and destruction effect of the drastic extraction method on the polysaccharide chain structure. At the same time, high-purity red rice bran non-starch water-soluble polysaccharide components are obtained through hot water extraction, decolorization, protein removal, dialysis, anion exchange column chromatography separation and purification, dialysis desalination, and freeze drying. In order to ensure the accuracy of polysaccharide yield and high purity of polysaccharides, the present invention runs through the dialysis operation in the purification step of polysaccharides. After the anion exchange column is eluted with a salt solution, a large amount of salts are also present in the polysaccharide solution, which reduces the effective concentration of the polysaccharide. Dialysis is beneficial to remove the salts in the sample and improve the sample concentration.

[0010] The object of the present invention is to provide a method for preparing non-starch polysaccharides from red rice bran, the method comprising the following steps:

[0011] (1) crushing, defatting, drying and sieving red rice bran;

[0012] (2) mixing the sieved red rice bran in step (1) with water and extracting at a constant temperature of 50° C. to 90° C., centrifuging, and concentrating the supernatant by rotary evaporation;

[0013] (3) adding a macroporous adsorption resin to the polysaccharide solution concentrated in step (2), stirring, filtering, and obtaining a polysaccharide filtrate;

[0014] (4) adding Sevag reagent to the polysaccharide filtrate obtained in step (3), stirring, then transferring to a separatory funnel, standing to separate layers, discarding the lower organic phase, and obtaining a polysaccharide solution; then adding anhydrous ethanol to the polysaccharide solution, standing, centrifuging, discarding the supernatant, and drying the precipitate to obtain purified rice bran non-starch polysaccharide;

[0015] (5) re-dissolving the purified rice bran non-starch polysaccharide obtained in step (4) in water and dialyzing it in a dialysis bag; concentrating the dialyzate by rotary evaporation, and then passing it through a DEAE 52 Cellulose column for elution and separation, and collecting the separated liquid;

[0016] (6) subjecting the separated liquid collected in step (5) to component determination, and combining the same elution peaks into the same component;

[0017] (7) The separated liquids of each group obtained by combining in step (6) are concentrated by rotary evaporation, desalted by dialysis, and freeze-dried to obtain the product.

[0018] In one embodiment, the red rice bran in step (1) is obtained by: using a JGMJ8098 rice grinder to grind 30 g of red rice for 30 seconds to separate the red rice bran; the separated red rice bran powder contains no white matter.

[0019] In one embodiment, the defatting in step (1) is to remove fat and part of the pigment by mixing red rice bran with anhydrous ethanol at a solid-liquid ratio of 1:17 (g / ml) with the assistance of an overhead mechanical stirrer for 4 to 6 hours, and repeating twice.

[0020] In one embodiment, the sieving in step (1) is to grind the dried red rice bran and sieve it through a 80-100 mesh sieve.

[0021] In one embodiment, the leaching time in step (2) is 4 to 8 hours, preferably 5 hours; the leaching temperature is preferably 60°C to 70°C.

[0022] In one embodiment, the solid-liquid ratio of the red rice bran to water in step (2) is 1:20-50.

[0023] In one embodiment, the centrifugation in step (2) is performed at 4000-6000 rpm for 20-35 min.

[0024] In one embodiment, the macroporous adsorption resin in step (3) is AB-8 macroporous adsorption resin.

[0025] In one embodiment, the Sevag reagent in step (4) is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0026] In one embodiment, the volume ratio of the polysaccharide filtrate to the Sevag reagent in step (4) is 2 to 5:1.

[0027] In one embodiment, in step (4), Sevag reagent is added to the polysaccharide filtrate, stirred, and then transferred to a separatory funnel, allowed to stand for stratification, and the lower organic phase is discarded to obtain a polysaccharide solution. The specific steps are as follows: Sevag reagent and polysaccharide solution are mixed in a volume ratio of 1:5, stirred for 25 minutes using an overhead mechanical stirrer at 410 rpm, the mixed solution is placed in a separatory funnel and allowed to stand for 10 minutes, the supernatant is retained, and the supernatant is repeatedly deproteinized 4 times.

[0028] In one embodiment, anhydrous ethanol is added to the polysaccharide solution in step (4), wherein the volume ratio of the polysaccharide solution to anhydrous ethanol is 1:4.

[0029] In one embodiment, the drying temperature in step (4) is 45-60°C.

[0030] In one embodiment, the dialysis in the dialysis bag in step (5) refers to dialysis in a dialysis bag with a molecular weight cut-off of 3500Da for 60 to 72 hours.

[0031] In one embodiment, in step (5), the DEAE 52 Cellulose column is eluted and separated using ultrapure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution, 0.8 mol / L NaCl solution, and 1.0 mol / L NaCl solution as eluents, and the volume of the eluents of each concentration gradient is 100 ml, and 4 ml / tube is used to receive the eluent.

[0032] In one embodiment, the dialysis desalination in step (7) is to concentrate each component by rotary evaporation and place it in a 1000Da dialysis bag for dialysis for 60 to 72 hours.

[0033] Another object of the present invention is to provide a red rice bran non-starch polysaccharide prepared by the above method.

[0034] The third object of the present invention is to provide an application of the above method or non-starch polysaccharide from red rice bran in preparing functional food.

[0035] The fourth object of the present invention is to provide a method for increasing the content of red rice bran polysaccharides with a weight average molecular weight of ten thousand, the method comprising the following steps:

[0036] (1) crushing, defatting, drying and sieving red rice bran;

[0037] (2) mixing the sieved red rice bran in step (1) with water and extracting at a constant temperature of 50° C. to 90° C., centrifuging, and concentrating the supernatant by rotary evaporation;

[0038] (3) adding a macroporous adsorption resin to the polysaccharide solution concentrated in step (2), stirring, filtering, and obtaining a polysaccharide filtrate;

[0039] (4) adding Sevag reagent to the polysaccharide filtrate obtained in step (3), stirring, then transferring to a separatory funnel, standing to separate layers, discarding the lower organic phase, and obtaining a polysaccharide solution; then adding anhydrous ethanol to the polysaccharide solution, standing, centrifuging, discarding the supernatant, and drying the precipitate to obtain purified rice bran non-starch polysaccharide;

[0040] (5) re-dissolving the purified rice bran non-starch polysaccharide obtained in step (4) in water and dialyzing it in a dialysis bag; concentrating the dialyzate by rotary evaporation, and then passing it through a DEAE 52 Cellulose column for elution and separation, and collecting the separated liquid;

[0041] (6) subjecting the separated liquid collected in step (5) to component determination, and combining the same elution peaks into the same component;

[0042] (7) The separated liquids of each group obtained by combining in step (6) are concentrated by rotary evaporation, desalted by dialysis, and freeze-dried to obtain the product.

[0043] Beneficial effects of the present invention:

[0044] The invention uses the mildest hot water extraction method to extract red rice bran polysaccharide, avoiding the degradation of polysaccharide chains by auxiliary means such as ultra-high temperature and ultrasound; at the same time, starch is prevented from mixing in the rice bran grinding stage, and no amylase is needed to remove starch, which not only saves the cost of removing starch, but also prevents amylase from breaking glycosidic bonds and causing polysaccharide chain breakage; the mildest Sevag reagent is used for protein removal, avoiding secondary degradation of polysaccharide in the impurity removal process, and the polysaccharide obtained by the method of the invention has a peak time concentrated in 12.923-20.132 min, and a relative molecular mass of 1039-4.08×10 6 Da.

[0045] (1) With respect to the prior art, the preparation method of red rice bran polysaccharide of the present invention can protect the chain structure of rice bran polysaccharide from being destroyed; and obtain rice bran polysaccharide with a weight average molecular weight of more than 10,000, and a weight average molecular weight of 6.70×10 5 Da.

[0046] (2) The present invention avoids using α-amylase and saccharifying enzyme to remove starch, protects the polysaccharide structure and reduces the purification cost and time at the same time, and judges the degree of grinding by the color of the grinding powder and red rice, so as to avoid the starch in the endosperm from mixing into the rice bran powder, thereby avoiding the mixing of starch impurities. The polysaccharide purified by α-amylase and saccharifying enzyme has a peak time of 19.361 to 23.056 min, a relative molecular weight of 496 to 3950 Da, and a weight average molecular weight of 1377 Da, which proves that the use of α-amylase and saccharifying enzyme can significantly destroy the chain structure of the water-soluble non-starch polysaccharide in red rice bran.

[0047] (3) The research object of non-starch polysaccharides in red rice bran is limited to the range of polysaccharides. In the operation, polysaccharides are screened out by "dialysis". If the polysaccharide structure is destroyed and the molecular weight is sharply reduced, part of the broken polysaccharides will seep out of the dialysis bag during the dialysis process, resulting in loss of information, incomplete characterization of the polysaccharide structure, and reduced available polysaccharide fragments. The present invention can protect the original structure of polysaccharides to the greatest extent and ensure the integrity of polysaccharide information.

[0048] (4) After separating and purifying the polysaccharide components, the present invention further performs a desalting treatment to ensure the improvement of the effective quality of the polysaccharide, while avoiding the interference of salt ions on chemical signals in nuclear magnetic resonance scanning and methylation experiments, thereby improving the purity of the polysaccharide final product and improving the effective polysaccharide quality during the use of the polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The present invention is a flow chart for preparing the non-starch polysaccharide from red rice bran;

[0050] Figure 2 The ultraviolet spectrum scanning diagram of the red rice bran non-starch polysaccharide prepared in Example 1 of the present invention at 190-400nm;

[0051] Figure 3 This is a molecular weight distribution diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Example 1 of the present invention;

[0052] Figure 4 This is the molecular weight distribution diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Comparative Example 1;

[0053] Figure 5 This is the molecular weight distribution diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Comparative Example 2;

[0054] Figure 6 This is the molecular weight distribution diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Comparative Example 3;

[0055] Figure 7 This is the molecular weight distribution diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Comparative Example 4;

[0056] Figure 8 This is a monosaccharide composition diagram of the purified rice bran non-starch polysaccharide obtained in step (4) of Example 1 of the present invention. DETAILED DESCRIPTION

[0057] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0058] 1. Determination of polysaccharide content in samples

[0059] Place 1g of D-anhydrous glucose in a 105℃ oven and dry for 2-3h to constant weight, take 0.175g of D-anhydrous glucose and dilute to 1000ml with deionized water to prepare a 0.175mg / ml glucose standard solution. Use 1.25g of phenol to dilute to 25ml to prepare a 5% phenol solution.

[0060] Prepare 2 ml each of 0, 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1.0 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml, and 2.0 mg / ml glucose standard solutions, add 1 ml of 5% phenol, add 7 ml of concentrated sulfuric acid, shake well, react at 40°C for 30 min, measure the absorbance at 490 nm, and calculate the glucose standard curve.

[0061] Take 2 ml of sample solution, add 1 ml of 5% phenol, add 7 ml of concentrated sulfuric acid, shake well and react at 40°C for 30 min, measure the absorbance at 490 nm, insert it into the standard curve, and calculate the polysaccharide content.

[0062] 2. Determination of monosaccharide composition of rice bran polysaccharides

[0063] 20 mg of rice bran polysaccharide sample was added to 4 mol / L trifluoroacetic acid solution, hydrolyzed at 105°C for 5 h, and then TFA was blown dry using a nitrogen blower. The hydrolyzate was dissolved with 500 μl of ultrapure water, and 300 μl of 0.3 mol / L NaOH and 0.5 mol / L PMP methanol solution were added. The reaction was carried out at 70°C for 1 h, and 300 μl of 0.3 mol / L HCl was added to make the solution pH 7. 2.5 ml of chloroform was added and vortexed to extract the polysaccharide derivatization product. The lower organic phase was aspirated and repeated 3 times. The results were determined using a Waters high performance liquid chromatography-ultraviolet detector (HPLC-UV).

[0064] The specific conditions are: chromatographic column: C18; mobile phase: acetonitrile: 0.05 mol / L phosphate buffer (PH = 6.8) = 18:82; UV detector wavelength is 245 nm; column temperature: 30°C; flow rate: 1 ml / min; sample elution time t = 45 min.

[0065] Macroporous adsorption resin AB-8 was purchased from Donghong Chemical Co., Ltd.

[0066] DEAE 52 Cellulose was purchased from Solarbio.

[0067] Example 1

[0068] A method for extracting non-starch polysaccharides from red rice bran specifically comprises the following steps:

[0069] (1) The red rice bran is separated, and each 30g of red rice is ground for 30s until there is no red rice bran on the surface of the red rice; the red rice bran after stripping is added with anhydrous ethanol at a solid-liquid ratio of 1:17, and stirred for 3h by an overhead stirrer for degreasing, and the mixture is repeated twice; then, the mixture is dried in an oven at 45°C, and then crushed by a food grinder and passed through a 100-mesh sieve;

[0070] (2) The rice bran sieved in step (1) was mixed with water at 60° C. in a mass volume ratio of 1:20 (g: ml), stirred at 60° C. for 5 h, and then centrifuged at 4000 rpm for 35 min to obtain an extract; the extract was concentrated to 200 mL by rotary evaporation at 55° C.; macroporous adsorption resin AB-8 was added to the concentrate and stirred for 2 h to adsorb red rice pigment, and then the macroporous adsorption resin was removed by suction filtering; and this was repeated 4 times;

[0071] (3) adding 40 ml of Sevag reagent prepared by trichloromethane and n-butanol in a volume ratio of 4:1 to the filtrate after decolorization in step (2), stirring at a speed of 410 rpm for 25 min using an overhead mechanical stirrer; then transferring the mixture to a separating funnel, standing and stratifying, removing the lower organic phase, and repeating the above operation for a total of 5 times with the upper aqueous phase to finally obtain a polysaccharide aqueous solution;

[0072] (4) adding anhydrous ethanol to the polysaccharide aqueous solution obtained in step (3) at a volume ratio of 1:4, standing at 4° C. for 12 h, centrifuging at 4000 rpm for 15 min, discarding the supernatant, washing the precipitate with anhydrous ethanol, and drying in an oven at 45° C. to obtain purified rice bran non-starch polysaccharide;

[0073] (5) grinding the purified rice bran non-starch polysaccharide prepared in step (4) into powder using a mortar, then dissolving it in 100 ml of water, and dialyzing it in a dialysis bag with a molecular weight cut-off of 3500 Da for 72 h;

[0074] (6) The dialysate obtained in step (5) was concentrated to 20 ml by rotary evaporation at 55° C. (vacuum degree: 0.1 MPa), and the concentrate was loaded onto a DEAE 52 Cellulose column. Polysaccharides were eluted using 100 ml each of ultrapure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution, 0.8 mol / L NaCl solution, and 1.0 mol / L NaCl in the above order, and the eluate was collected in 5 ml centrifuge tubes (4 ml / tube).

[0075] (7) Determine the polysaccharide content in each tube of step (6), draw an elution curve, and combine the eluates of the same elution peak to obtain 5 groups of refined polysaccharide components; then concentrate the combined polysaccharide eluate of each group at 55°C by rotary evaporation, and dialyze in a 1000Da dialysis bag for 48 hours; the dialyzate is further concentrated by rotary evaporation at 55°C, and freeze-dried at -30°C and 10Pa to obtain 5 groups of rice bran non-starch polysaccharide components.

[0076] Example 2

[0077] The only difference from Example 1 is that step (2) is adjusted to: the sieved rice bran in step (1) is mixed with 60° C. water in a mass volume ratio of 1:35 (g:ml); the rest is the same as Example 1.

[0078] Example 3

[0079] The only difference from Example 1 is that step (2) is adjusted to: the sieved rice bran in step (1) is mixed with 60° C. water in a mass volume ratio of 1:50 (g:ml); the rest is the same as Example 1.

[0080] Comparative Example 1

[0081] The only difference from Example 1 is that step (2) is adjusted to: the sieved rice bran in step (1) is mixed with 95° C. water in a mass volume ratio of 1:20 (g:ml); the rest is the same as Example 1.

[0082] Comparative Example 2

[0083] The only difference from Example 1 is that step (2) is adjusted to: the sieved rice bran in step (1) is mixed with 40° C. water in a mass volume ratio of 1:20 (g:ml); the other steps are the same as in Example 1.

[0084] Comparative Example 3

[0085] The only difference from Example 1 is that step (2) is adjusted from "step (1) the sieved rice bran is mixed with 60° C. water in a mass volume ratio of 1:20 (g: ml), and stirred at 60° C. for 5 h" to "step (1) the sieved rice bran is mixed with 60° C. water in a mass volume ratio of 1:20 (g: ml), and then ultrasonicated at 150 W for 1 h"; the other steps are the same as in Example 1.

[0086] Comparative Example 4

[0087] The only difference from Example 1 is that after decolorization in step (2), the pH of the decolorized filtrate is adjusted to 5.9 using 0.1 mol / L HCl, 1 mg of α-amylase is added at T = 58°C, and the reaction is carried out for 2 h; then the pH is adjusted to 4.3 using 0.1 mol / L HCl, 80 μl of saccharifying enzyme is added at T = 60°C, and the reaction is carried out for 1 h; the other steps are the same as in Example 1.

[0088] Comparative Example 5

[0089] The only difference from Example 1 is that the dialysis process in step (7) is omitted, and the combined polysaccharide components are directly freeze-dried.

[0090] Take 30 mg of one of the above-mentioned freeze-dried rice bran polysaccharide components, dissolve it in a 50 ml centrifuge tube with 5 ml ultrapure water, shake well, then take 0.84935 g AgNO3 and dissolve it in 5 ml ultrapure water, shake and evenly, that is, prepare 5 ml 0.1 mol / L AgNO3, add silver nitrate solution to the polysaccharide solution, shake well to allow the reaction to proceed fully, centrifuge at 3500 rpm for 5 min to obtain a silver chloride precipitate, and after centrifugation, place the precipitate in an oven at 105°C to dry, weigh the mass of the silver chloride precipitate to be 73 mg, which is converted to a mass of original sodium chloride to be 29.79 mg, indicating that 29.79 mg of the 30 mg sample is salt, which will cause a strong shielding effect on the nuclear magnetic resonance signal and reduce the effective mass of the polysaccharide.

[0091] Results

[0092] 1. Analysis of the purity of rice bran non-starch polysaccharides

[0093] Take 3 ml of 1 mol / L rice bran non-starch polysaccharide numbered as component 5 prepared in Example 1 and scan it at 190-400 nm ultraviolet spectrum; Figure 2 As shown, the results show that the rice bran non-starch polysaccharide has no absorption peaks at 245nm, 260nm and 280nm, which proves that this group of rice bran polysaccharides does not contain red rice pigment, nucleic acid and protein, and the rice bran non-starch polysaccharide extract has a high purity.

[0094] 2. Determination of non-starch polysaccharide components in rice bran

[0095] The composition of the purified red rice bran non-starch polysaccharide obtained after dialysis in step (5) of Example 1 was detected by HPLC-UV; the results are as follows: Figure 8 As shown, the monosaccharide composition of red rice bran polysaccharide is: glucose (Glc), galactose (Gal), arabinose (Ara), mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), and the ratio is glucose (Glc): galactose (Gal): arabinose (Ara): mannose (Man): glucuronic acid (GlcA): galacturonic acid (GalA) = 1.74: 1.27: 0.7: 0.39: 0.29: 0.14.

[0096] 3. Determination of molecular weight distribution of non-starch polysaccharides in rice bran

[0097] The purified red rice bran non-starch polysaccharide obtained after dialysis in step (5) of Example 1 and Comparative Examples 1 to 4 was dissolved in water to obtain a solution with a mass fraction of 1%, and the solution was measured by a Waters high performance liquid chromatography system with a differential detector. The specific conditions were as follows: chromatographic column: Ultrahydroge TMLiniear 300mm×7.8mm: mobile phase: 0.1mol / L NaNO3: flow rate: 0.5mL / min, column temperature: 40℃, detector: RI; the results are shown in Table 1 and Table 2:

[0098] Table 1 Quality and yield of polysaccharides obtained by different extraction methods

[0099]

[0100] Table 2 Molecular weight distribution of polysaccharides obtained by different extraction methods

[0101]

[0102]

[0103] From the data in the table, it can be seen that the molecular weights of the polysaccharide products obtained by 60°C hot water extraction, 95°C hot water extraction, 40°C hot water extraction, 150W ultrasonic extraction, and amylase impurity removal extraction are quite different, among which the products with weight average molecular weights above 10,000 account for 70.23%, 13.65%, 18.29%, 5.58%, and 1.21%, respectively; the non-starch polysaccharide of rice bran prepared in Example 1, of which 70.23% of the polysaccharides have molecular weights concentrated in the range of 1039 to 4.08×10 6 Da, weight average molecular weight is 6.70×10 5 Da, the remaining small molecular weight polysaccharides can be removed by subsequent dialysis.

[0104] The rice bran polysaccharide prepared in Comparative Example 1 has 86.35% of the polysaccharide molecular weight concentrated in the range of 522-3930Da, and the weight-average molecular weight is 1197Da. The weight-average molecular weight of the polysaccharide extracted under this high temperature condition is significantly reduced compared with the polysaccharide extracted at 60°C, and the maximum molecular weight is also significantly smaller than the polysaccharide extracted at 60°C.

[0105] The rice bran polysaccharide prepared in Comparative Example 2 has 81.71% of the polysaccharide molecular weight concentrated in the range of 2951 to 1.74×10 4 The weight average molecular weight is 3957. Under low temperature conditions, many large molecular weight polysaccharides cannot be dissolved, resulting in reduced extraction efficiency.

[0106] The rice bran polysaccharide prepared in Comparative Example 3 has a molecular weight of 91.15% polysaccharide concentrated in the range of 936 to 1.91×10 4 Da, and the weight average molecular weight is 3802Da; when the polysaccharide is extracted with the help of ultrasound, the molecular weight distribution and weight average molecular weight of the polysaccharide are significantly lower than those of the polysaccharide not extracted with ultrasound in Example 1.

[0107] In comparative example 4, the molecular weight distribution after starch removal by α-glycosidase and saccharifying enzyme is shown in which 87.02% of the polysaccharides are concentrated in the range of 496-3950Da, and the weight average molecular weight is 1377Da. This indicates that the participation of amylase and saccharifying enzyme will lead to a sharp decrease in the molecular weight of polysaccharides, destroying glycosidic bonds and breaking polysaccharide chains; it is impossible to obtain rice bran polysaccharides with a molecular weight of more than 10,000.

[0108] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for increasing the content of red rice bran polysaccharides with a weight average molecular weight of 10,000, characterized in that: The method comprises the following steps: (1) crushing, defatting, drying and sieving red rice bran; the red rice bran is obtained by: using a JGMJ8098 rice grinding machine, grinding 30 g of red rice for 30 seconds to separate the red rice bran; the separated red rice bran powder contains no white matter; (2) mixing the sieved red rice bran in step (1) with water and extracting at a constant temperature of 60° C. for 5 h, centrifuging, and taking the supernatant for rotary evaporation concentration; the solid-liquid ratio of the red rice bran to water is 1:20-50; (3) adding AB-8 macroporous adsorption resin to the polysaccharide solution concentrated in step (2), stirring, filtering, and obtaining a polysaccharide filtrate; (4) adding Sevag reagent to the polysaccharide filtrate obtained in step (3), stirring, then transferring to a separatory funnel, standing to separate layers, discarding the lower organic phase, and obtaining a polysaccharide solution; then adding anhydrous ethanol to the polysaccharide solution, standing, centrifuging, discarding the supernatant, and drying the precipitate to obtain purified rice bran non-starch polysaccharide; (5) re-dissolving the purified rice bran non-starch polysaccharide obtained in step (4) in water and dialyzing it in a dialysis bag; concentrating the dialyzate by rotary evaporation, and then passing it through a DEAE 52 Cellulose column for elution and separation, and collecting the separated liquid; (6) subjecting the separated liquid collected in step (5) to component determination, and combining the same elution peaks into the same component; (7) The separated liquids of each group obtained by combining in step (6) are concentrated by rotary evaporation, desalted by dialysis, and freeze-dried to obtain the product.

2. The method according to claim 1, characterized in that The defatting in step (1) is to mix red rice bran and anhydrous ethanol at a solid-liquid ratio of 1:17 g / ml, stir for 4 to 6 hours with the assistance of an overhead mechanical stirrer to remove fat and part of the pigment, and repeat twice.

3. The method according to claim 1, characterized in that The Sevag reagent in step (4) is a mixture of chloroform and n-butanol in a volume ratio of 4:

1.

4. The method according to claim 1, characterized in that: Step (4) Add Sevag reagent to the polysaccharide filtrate, stir, then transfer to a separatory funnel, let stand to separate layers, discard the lower organic phase, and obtain the polysaccharide solution. The specific steps are as follows: Sevag reagent and polysaccharide solution are mixed in a volume ratio of 1:5, stirred at 410 rpm with an overhead mechanical stirrer for 25 minutes, the mixed solution is placed in a separatory funnel and allowed to stand for 10 minutes, the supernatant is retained, and the supernatant is repeatedly deproteinized 4 times.

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