阅读说明：本技术 一种阿拉伯木聚糖的制备方法及产品 (Preparation method of arabinoxylan and product ) 是由 牛猛 王朦 唐翠娥 张宾佳 贾才华 赵思明 于 2020-06-18 设计创作，主要内容包括：本发明公开了一种阿拉伯木聚糖的制备方法,具体包括以下步骤：(1)对麦麸去淀粉；(2)采用碱提法得到多糖溶液；(3)对多糖溶液进行脱蛋白处理；(4)对脱蛋白的多糖溶液进行酶处理、透析；(5)对透析后的多糖溶液进行酶解；(6)对未经过酶解的多糖溶液和经过酶解的多糖溶液分别进行醇沉分级,得到不同分子量阿拉伯木聚糖；(7)将不同分子量阿拉伯木聚糖进行二次酶处理、二次透析。本发明制备取方法简单可行,能够提高原材料麦麸的利用率,可得到不同级分的阿拉伯木聚糖,结构各异,具有不同程度的降血糖、抗氧化等功能性作用,医药价值高。(The invention discloses a preparation method of arabinoxylan, which specifically comprises the following steps: (1) removing starch from the wheat bran; (2) obtaining polysaccharide solution by adopting an alkali extraction method; (3) deproteinizing the polysaccharide solution; (4) carrying out enzyme treatment and dialysis on the deproteinized polysaccharide solution; (5) performing enzymolysis on the dialyzed polysaccharide solution; (6) respectively carrying out alcohol precipitation grading on the polysaccharide solution which is not subjected to enzymolysis and the polysaccharide solution which is subjected to enzymolysis to obtain the arabinoxylan with different molecular weights; (7) and (3) carrying out secondary enzyme treatment and secondary dialysis on the arabinoxylan with different molecular weights. The preparation method is simple and feasible, can improve the utilization rate of the raw material wheat bran, can obtain different fractions of arabinoxylan, has different structures, has different degrees of functional effects of reducing blood sugar, resisting oxidation and the like, and has high medical value.)

1. A preparation method of arabinoxylan is characterized by comprising the following steps:

(1) separating the wheat bran from the flour, and removing starch from the wheat bran to obtain de-starch wheat bran;

(2) performing an alkali extraction method on the de-starched wheat bran obtained in the step (1) to obtain a polysaccharide solution;

(3) deproteinizing the polysaccharide solution obtained in the step (2) to obtain a deproteinized polysaccharide solution;

(4) carrying out enzyme treatment and dialysis on the deproteinized polysaccharide solution obtained in the step (3);

(5) performing enzymolysis on the polysaccharide solution dialyzed in the step (4);

(6) respectively carrying out alcohol precipitation grading on the polysaccharide solution which is not subjected to enzymolysis in the step (4) and the polysaccharide solution which is subjected to enzymolysis in the step (5) to obtain arabinoxylan;

(7) and (4) carrying out secondary enzyme treatment and secondary dialysis on the arabinoxylan obtained in the step (6).

2. The method for preparing arabinoxylan according to claim 1, wherein the method for preparing de-starched bran of step (1) comprises the steps of: soaking the wheat bran in 4 deg.C water for 60min, taking out, cleaning, draining, and heating at 50 deg.C for 12 hr to obtain de-starch wheat bran.

3. The method of claim 1, wherein the alkali extraction process of step (2) comprises the steps of ① mixing the de-starched wheat bran with NaOH solution, reacting at 80 deg.C for 90min, cooling to room temperature, centrifuging, wherein the NaOH solution has a molar concentration of 0.15M and a mass/volume ratio of the de-starched wheat bran to the NaOH solution of 1g:15ml, and the NaOH solution contains 0.5 vol% of H₂O₂② adjusting pH of the supernatant obtained in step ① to 4.5 with HCl, centrifuging to obtain supernatant, ③ concentrating the supernatant obtained in step ② under reduced pressure to 1/4 of the original volume, precipitating with ethanol, dissolving the precipitate in water, centrifuging, and repeating the above steps.

4. The method for preparing arabinoxylan according to claim 1, wherein the deproteinization in step (3) is performed by Sevage method, and the method specifically comprises the following steps: adding Sevage reagent into the polysaccharide solution, oscillating vigorously for 1h, centrifuging, collecting water layer, adding Sevage reagent again, repeating for 5-6 times until free protein is completely removed, concentrating the supernatant under reduced pressure, and freeze drying; the ratio of the volume of Sevage reagent added to the volume of polysaccharide solution added was 1: 4.

5. The method for preparing arabinoxylan according to claim 1, wherein the enzymatic treatment of the step (4) comprises the steps of: preparing a polysaccharide solution with the mass fraction of 4%, adding high-temperature-resistant alpha-amylase into the polysaccharide solution, reacting for 2 hours at the temperature of 95 ℃, boiling and cooling to room temperature, wherein the enzyme activity of the high-temperature-resistant alpha-amylase is 100 u/g; adding amyloglucosidase into the solution obtained in the step I, reacting for 4 hours at 55 ℃, boiling for 30min after reaction to inactivate the enzyme, centrifuging, and dialyzing the obtained supernatant with deionized water.

6. The method for preparing arabinoxylan according to claim 1, wherein the enzymatic hydrolysis process of step (5) comprises the steps of: dispersing the polysaccharide solution in NaAc buffer solution to prepare 2% suspension, adding pentosanase, reacting at 50 deg.C and pH5.0 for 2h, and centrifuging to obtain supernatant; the enzyme activity of the pentosanase is 5000 u/g.

7. The method for preparing arabinoxylan according to claim 1, wherein the alcohol precipitation fractionation of the step (6) comprises the steps of:

and A, carrying out alcohol precipitation grading on the polysaccharide solution subjected to enzymolysis in the step (5) to obtain a polysaccharide solution:

and B, carrying out alcohol precipitation grading on the polysaccharide solution which is not subjected to enzymolysis in the step (4) to obtain a polysaccharide solution:

and C, respectively standing the polysaccharide solutions obtained by the alcohol precipitation and classification in the step A, B at 4 ℃ overnight, centrifuging to obtain precipitates, dissolving the precipitates in water, and freeze-drying and storing the obtained crude polysaccharide.

8. The method for preparing arabinoxylan according to claim 7, wherein the secondary enzymatic treatment of the step (7) comprises the steps of: respectively preparing the crude polysaccharide freeze-dried and stored in the step (6) into polysaccharide solutions with the mass fraction of 0.5%, adding lichenase, reacting for 1h at the temperature of 40 ℃, cooling, centrifuging, dialyzing the obtained supernatant with deionized water, and freeze-drying and storing; the enzyme activity of the lichenase is 20 u/g.

9. The method for preparing arabinoxylan according to claim 1, wherein the dialysis duration in the steps (4) and (7) is about 3500Da and 36 h.

10. Arabinoxylan produced by the method of producing arabinoxylan according to any of claims 1 to 9.

Technical Field

The invention belongs to the technical field of food processing, and particularly relates to a preparation method of arabinoxylan and a product.

Background

Arabinoxylan (AX) is a non-starch polysaccharide of great research value, is mainly present in seed coats of cereals such as wheat, rye, barley, rice, corn, etc., and is one of the main components constituting plant cell walls. AX has a complex structure and wide molecular weight distribution, and the basic structure comprises a xylan main chain and an alpha-L-arabinofuranosyl side chain, wherein the xylan main chain is formed by connecting beta-D-xylopyranose residues through beta- (1 → 4) glycosidic bonds, and the relative quantity and the substitution condition of the arabinose residues are different mainly due to the difference of properties (solubility, solution viscosity, gelatination, enzyme action degree and the like) of arabinoxylan.

AX is generally divided into two main classes according to its solubility in water: water-soluble arabinoxylans (Water-extractable arabinoxylans, WEAX) and Water-insoluble arabinoxylans (Water-insoluble arabinoxylans, WUAX). WUAX, although not extractable by water, is under alkaline conditions, especially in hot lye (KOH, NaOH, Ba (OH)₂) WUAX is sometimes also referred to as a base to extract arabinoxylan. The methods of extraction of AX are also hereby mainly divided into three main categories: water/hot water extraction, alkaline extraction, and enzymatic extraction. In contrast, the extraction rate of water extraction is generally low; although the extraction rate of the alkaline extraction is high, certain functional groups in the AX structure, such as feruloyl groups, protein groups and the like, are usually damaged, and the functional properties of the AX structure can be weakened; the conditions of the enzymatic extraction are mild, partial degradation can be caused to the sugar chain structure of AX, but functional groups in the AX can not be affected generally.

The research on the arabinoxylan at home and abroad shows that the AX is widely used in the field of food, and can be used as a stabilizer and a thickener of the food due to the characteristics of high viscosity and high water binding capacity. In addition, AX has biological activities of reducing serum oxide, maintaining blood glucose level, resisting oxidation, reducing postprandial blood glucose response, enhancing immunity, etc. The functional properties of AX depend on the properties of AX itself. For example, ferulic acid enables AX to have unique oxidative gelation properties, can be used as a free radical scavenger, and has the functional characteristics of oxidation resistance, antibiosis, antiphlogosis and the like; a low relative molecular mass AX affects dough water holding power, and a high relative molecular mass AX significantly affects dough extensibility. How to separate AX to obtain AX of different molecular weight is a matter of concern in the art.

Disclosure of Invention

The invention aims to provide a preparation method of arabinoxylan and a product thereof, wherein the method integrates the advantages of an alkali extraction and alcohol precipitation method, an enzymolysis method and a membrane separation technology, overcomes the defects of high quality and low cost of a single method and difficulty in fusion, can keep the polysaccharide structure less damaged and high purity, achieves the effect of multi-stage separation, can reduce energy consumption and processing cost, and can obtain the arabinoxylan with different molecular weights.

In order to achieve the above object, the present invention provides a method for preparing arabinoxylan, comprising the steps of:

(1) separating the wheat bran from the flour, and removing starch from the wheat bran to obtain de-starch wheat bran;

(2) performing an alkali extraction method on the de-starched wheat bran obtained in the step (1) to obtain a polysaccharide solution;

(3) deproteinizing the polysaccharide solution obtained in the step (2) to obtain a deproteinized polysaccharide solution;

(4) carrying out enzyme treatment and dialysis on the deproteinized polysaccharide solution obtained in the step (3);

(5) performing enzymolysis on the polysaccharide solution dialyzed in the step (4);

(6) respectively carrying out alcohol precipitation grading on the polysaccharide solution which is not subjected to enzymolysis in the step (4) and the polysaccharide solution which is subjected to enzymolysis in the step (5) to obtain arabinoxylan;

(7) and (4) carrying out secondary enzyme treatment and secondary dialysis on the arabinoxylan obtained in the step (6).

Preferably, the method for preparing the de-starched wheat bran in the step (1) comprises the following steps: soaking the wheat bran in 4 deg.C water for 60min, taking out, cleaning, draining, and heating at 50 deg.C for 12 hr to obtain de-starch wheat bran.

Preferably, the alkali extraction method of step (2) comprises ① mixing the de-starched wheat bran with NaOH solution, reacting at 80 deg.C for 90min, and coolingCentrifuging the mixture after the temperature is reduced to room temperature, wherein the molar concentration of the NaOH solution is 0.15M, the mass-volume ratio of the de-starched wheat bran to the NaOH solution is 1g:15ml, and the NaOH solution contains 0.5% of H by volume fraction₂O₂② adjusting pH of the supernatant obtained in step ① to 4.5 with HCl, centrifuging to obtain supernatant, ③ concentrating the supernatant obtained in step ② under reduced pressure to 1/4 of the original volume, precipitating with ethanol, dissolving the precipitate in water, centrifuging, and repeating the above steps.

Preferably, the deproteinization in the step (3) adopts a Sevage method to remove proteins, and specifically comprises the following steps: adding Sevage reagent into the polysaccharide solution, oscillating vigorously for 1h, centrifuging, collecting water layer, adding Sevage reagent again, repeating for 5-6 times until free protein is completely removed, concentrating the supernatant under reduced pressure, and freeze drying; the ratio of the volume of Sevage reagent added to the volume of polysaccharide solution added was 1: 4.

Preferably, the enzyme treatment of step (4) comprises the steps of: preparing a polysaccharide solution with the mass fraction of 4%, adding high-temperature-resistant alpha-amylase into the polysaccharide solution, reacting for 2 hours at the temperature of 95 ℃, boiling and cooling to room temperature, wherein the enzyme activity of the high-temperature-resistant alpha-amylase is 100 u/g; adding amyloglucosidase into the solution obtained in the step I, reacting for 4 hours at 55 ℃, boiling for 30min after reaction, centrifuging, and dialyzing the obtained supernatant with deionized water.

Preferably, the enzymolysis process in step (5) includes the following steps: dispersing a polysaccharide solution in a NaAc buffer solution, preparing a suspension with the mass fraction of 2%, adding pentosanase, reacting for 2 hours at the temperature of 50 ℃ and the pH value of 5.0, and centrifuging to obtain a supernatant; the enzyme activity of the pentosanase is 5000 u/g.

Preferably, the alcohol precipitation fractionation of the step (6) comprises the following steps:

and A, carrying out alcohol precipitation grading on the polysaccharide solution subjected to enzymolysis in the step (5) to obtain a polysaccharide solution:

and B, carrying out alcohol precipitation grading on the polysaccharide solution which is not subjected to enzymolysis in the step (4) to obtain a polysaccharide solution:

and C, respectively standing the polysaccharide solutions obtained by the alcohol precipitation and classification in the step A, B at 4 ℃ overnight, centrifuging to obtain precipitates, dissolving the precipitates in water, and freeze-drying and storing the obtained crude polysaccharide.

Preferably, the secondary enzyme treatment of step (7) comprises the steps of: preparing the crude polysaccharide freeze-dried and stored in the step (6) into a polysaccharide solution with the mass fraction of 0.5%, adding lichenase, reacting for 1h at the temperature of 40 ℃, cooling, centrifuging, dialyzing the obtained supernatant with deionized water, and freeze-drying and storing; the enzyme activity of the lichenase is 20 u/g.

Preferably, the membrane tube in the dialysis process of the step (4) and the step (7) is 3500Da, and the dialysis time is 36 h.

Preparation method of arabinoxylan.

Compared with the prior art, the invention has the beneficial effects that:

1. the preparation method is simple and feasible, can improve the utilization rate of the raw material wheat bran, improves the added value of grain processing products, increases economic benefits, and simultaneously obtains the polysaccharide with higher purity, thereby having good application value and market potential;

2. the enzyme used in the invention is simple and easy to obtain, and because of the structure of AX in the bran and physical or chemical combination between AX molecules and between AX and other cell wall substances such as lignin and cellulose molecules, the combination can be broken under alkaline conditions, so that the original insoluble AX is changed into soluble AX; meanwhile, when a single NaOH solution is used as an extracting agent, polysaccharide and cell wall substances can only be subjected to rough hydrolysis without selectivity, and the arabinoxylan cannot be subjected to high-selectivity specific hydrolysis, so that the yield is high but the purity is low, the release of the polysaccharide is aggravated by adding hydrogen peroxide, the dissolution degree of the polysaccharide is improved, and the extraction rate and the yield of an obtained sample are correspondingly improved;

3. AX obtained by alkali extraction has a certain amount of ferulic acid, and the change of extraction conditions has certain influence on the content of ferulic acid, and in order to reduce the deficiency of the alkali extraction method, the reaction conditions such as alkali solution concentration and H need to be strictly controlled₂O₂Content, reaction temperature and time, etc.;

4. the polysaccharide component extracted from wheat bran is mostly crude polysaccharide, and relatively pure AX can be obtained only by removing impurities such as starch, protein and glucan, etc., while the protein can be removed by sevage method, and the starch and the glucan can be separated by adding related specific enzyme for enzymolysis reaction to prevent the polysaccharide structure from being damaged; meanwhile, the enzyme treatment is also a purification process of the alkali-extracted polysaccharide, the first enzymolysis is to remove the soluble starch with small molecular weight in the polysaccharide, the lichenase used in the second enzymolysis has specificity to the beta-glucan, the beta-glucan in the polysaccharide can be removed by dialysis after the reaction, and the AX with higher purity can be obtained after the second enzymolysis;

5. the fractional precipitation method is to add another solvent (such as ethanol, acetone, etc.) into the polysaccharide water solution to change the polarity of the mixed solvent, so as to precipitate part of the substances, thereby realizing separation, achieving the purpose of primarily purifying the polysaccharide by gradually increasing the volume concentration of the ethanol, maintaining higher yield, and the polysaccharides precipitated according to different ethanol concentrations have different molecular weights and biological activities, generally speaking, the larger the molecular weight of the polysaccharide is, the lower the polarity is, and the lower the concentration of the ethanol can precipitate the polysaccharide;

6. the method can effectively remove protein and starch in polysaccharide solution, can ensure the existence of polysaccharide, and can remove the residue of organic matters in the polysaccharide solution through dialysis.

Drawings

FIG. 1 is a GPC chart of a water-soluble arabinoxylan enzymatic hydrolyzed sample;

FIG. 2 GPC chart of water-soluble arabinoxylan unlysed sample;

FIG. 3 is a UV scanning spectrum of water-soluble arabinoxylan;

FIG. 4 is a schematic diagram of the preparation method of arabinoxylan.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.