CN1840674A - A method for preparing xylooligosaccharides by enzymatically hydrolyzing wheat bran - Google Patents

Abstract

The preparation method for oligoxylose comprises: with wheat bran as material, processing at high temperature with amylase, proteinase and saccharifying enzyme in turn to obtain the insoluble fiber; taking enzymolysis reaction with bacillus subtilis xylanase; taking enzymolysed supernatant to concentrate, spray dry and obtain the crude product with 12.5% oligoxylose yield and purity more than 60% while xylobiose and xylotriose more than 30%. The product is very benefit to people health. This invention increases value of wheat bran.

Description

A method for preparing xylooligosaccharides by enzymatically hydrolyzing wheat bran

technical field

The invention discloses a method for preparing xylo-oligosaccharides by enzymatically hydrolyzing wheat bran, which belongs to the technical field of resource development of agricultural by-products and functional food additives.

Background technique

Plant hemicellulose is the most abundant polysaccharide in nature except cellulose. Xylan is the main component of plant hemicellulose, its main chain is composed of xylopyranosyl linked by β-D-(1,4)-glycosidic bonds, L-arabinose, D-glucose, D-galactose And methyl, acetyl, etc. constitute a branched chain. The content of xylan and the type and degree of substitution of xylan in different raw materials are different. For example, the hemicellulose content in the grain cortex accounts for 25%-40% of the dry weight, and the main component is arabinoxylan, and arabinose is at the O-2, O-3 position or O-2 and O- 3 positions were substituted.

Xylooligosaccharides are products of xylan degradation. At present, the industrial production of xylooligosaccharides mainly adopts enzymatic method, that is, xylanase is used to catalyze the hydrolysis of xylan. Many microorganisms in nature can produce xylanase, such as: Bacillus subtilis, Streptomyces, Aspergillus, Penicillium, Trichoderma and Chaetomium, etc., mainly bacteria and fungi.

Since the main effective components of xylooligosaccharides are disaccharides and trisaccharides, xylanases with relatively high endoxylanase activity should be selected for the preparation of xylooligosaccharides. At the same time, since xylan is a heterogeneous polysaccharide with complex structure and contains many different substituents, in order to obtain a higher yield of xylo-oligosaccharides, the selected xylanase should have the ability to hydrolyze the heterogeneous polysaccharides in specific substrates. The ability of xylan is strong or the enzyme system contains branch-cutting enzymes (such as arabinosidase) that hydrolyze the branched chain in specific substrates. Different raw materials require the use of different microbial xylanases. Therefore, the key to enzymatic hydrolysis of wheat bran to prepare xylo-oligosaccharides lies in the adaptability of xylanase to wheat bran xylan present in a large amount in the insoluble dietary fiber of wheat bran, that is, to select a suitable xylanase.

Contents of the invention

The object of the present invention is to provide a kind of method that enzymolysis wheat bran prepares xylo-oligosaccharide, realizes the effective value-added and utilization of wheat bran, prepared xylo-oligosaccharide is a kind of outstanding functional oligosaccharide, to It is very beneficial to human health and has great economic and social benefits.

Technical scheme of the present invention: take wheat bran as raw material, adopt common high-temperature amylase, protease, glucoamylase to obtain insoluble dietary fiber after adopting high-temperature amylase, protease, glucoamylase in sequence, the present invention uses xylan again to insoluble dietary fiber of wheat bran The enzyme performs enzymatic hydrolysis reaction, and the supernatant of enzymatic hydrolysis is concentrated and spray-dried to prepare crude xylooligosaccharide. The xylanase used in the enzymatic hydrolysis reaction is Bacillus subtilis xylanase. The enzymatic hydrolysis substrate is wheat bran insoluble dietary fiber, the mass concentration of the substrate is 4%-7%, the mass concentration of the enzyme in the system is 0.4%-0.7%, the enzymolysis temperature is 40-50°C, and the enzymolysis time is 24- After 30 hours, the pH of the enzymatic hydrolysis system was 5.0.

The optimal conditions for the enzymatic hydrolysis of wheat bran insoluble dietary fiber were that the mass concentration of the substrate was 5%-6%, the mass concentration of the enzyme in the system was 0.45%-0.55%, the enzymatic hydrolysis temperature was 45°C, and the enzymatic hydrolysis time was 24h. The pH of the solution system is 5.0.

Because wheat bran contains a large amount of starch and protein, in order to improve the utilization rate of raw materials and reduce the burden of downstream separation and purification, firstly, amylase, protease, and glucoamylase are used to destarch and remove protein in order to obtain wheat bran insoluble meal. fiber.

The basic composition of wheat bran after analysis is shown in Table 1. The basic composition of wheat bran insoluble dietary fiber obtained after destarching and protein removal is shown in Table 2.

Table 1 Basic composition of wheat bran Element moisture Ash starch protein Pentosan other content(%) 10.98 6.98 14.21 13.97 21.40 Untested

Table 2 Basic composition of wheat bran insoluble dietary fiber Element Ash starch protein Pentosan other content(%) 1.46 2.20 3.97 54.70 Untested

Usually, suspend wheat bran in hot water, add 75mL/kg bran heat-resistant α-amylase, stir at 95°C for 1 hour, cool to 60°C and adjust pH to 7.5, then add 30mL/kg bran hydrolyzing protease Alcalase, stirred at 60°C for 30 minutes, adjusted the pH to 4.5, then added 35mL/kg bran glucoamylase, stirred at 60°C for 30 minutes, centrifuged to collect the precipitate, and washed the precipitate repeatedly with hot distilled water to obtain wheat bran insoluble dietary fiber . Thermostable α-amylase, proteolytic enzyme and refined glucoamylase were all provided by Novozymes.

After screening, it was confirmed that Bacillus subtilis (Bacillus subtilis) xylanase was used to enzymatically hydrolyze wheat bran insoluble dietary fiber, because of its high enzymolysis efficiency for wheat bran insoluble dietary fiber, the enzyme was developed by Wuhan Xinhua Yang Supplied by Bio Ltd.

The substrate concentration has a great influence on the enzymatic reaction. When the substrate concentration is low, increasing the substrate concentration can increase the reaction rate, but when the substrate concentration is high, the further increase of the substrate concentration will reduce the reaction rate. The increase becomes slow or even stops. Moreover, an increase in substrate concentration often leads to a decrease in product yield. Increasing the enzyme concentration can increase the reaction speed, but at the same time it will increase the cost. The appropriate enzyme concentration should be selected so that the enzyme reaction can be completed within an acceptable time.

In the preliminarily determined enzymatic hydrolysis process, the five factors (substrate concentration, enzyme concentration, temperature, time, pH) that affect the enzymatic hydrolysis reaction were carried out in an orthogonal design, and their levels are shown in Table 3.

Table 3 Experimental factors and levels level Substrate concentration [S] Enzyme concentration [E] Temperature T(°C) time t(h) pH 123 4% 6% 8% 0.3% 0.5% 0.7% 604050 243018 456

According to the characteristics of factors and levels, we use the orthogonal table L ₁₈ (3 ⁷ ) to conduct experiments.

The experimental results are shown in Table 4.

Table 4 Orthogonal result table Test No. [S] [E] T t pH Reducing sugar yield (%) 123456789101112131415161718 1(4%)112(6%)223(8%)33111222333 1(0.3%)2(0.5%)3(0.7%)123123123123123 1(60)2(40)3(50)123231312231312 1(24)2(30)3(18)231123312312231 1(4)2(5)3(6)231312231123312 17.0826.0416.4821.1819.6723.8517.8022.7421.9223.1319.8522.2617.9723.8818.7617.0714.8923.28

From the reducing sugar yields of 18 experiments, it can be seen that the yield of experiment 2 is 26.04%, and the corresponding experimental conditions are substrate concentration 4%, enzyme concentration 0.5%, temperature 40°C, time 30h, pH 5.0.

Statistical analysis showed that pH value was an important factor, reaction time, temperature and enzyme concentration were also important factors, while substrate concentration was a less influential factor. The pH of the reaction system is 5.0, the temperature is 50°C, the enzyme concentration is 0.5%, and the substrate concentration is 6%. From the perspective of production, it is more appropriate to determine the reaction time as 24h.

Except for temperature and substrate concentration, the optimal level combination of statistical analysis and the optimal level combination obtained intuitively from the orthogonal table had the same enzyme concentration and pH in the experimental condition No. 2. In order to further compare the two conditions, we carried out enzymatic hydrolysis of wheat bran insoluble dietary fiber for 24 hours under these two conditions respectively. The result of the best level combination is better than that of No. 2 experiment, and the yield of reducing sugar to wheat bran insoluble dietary fiber is 25.80% and 24.71% respectively.

In summary, we determined the optimal process conditions for Bacillus subtilis xylanase to act on wheat bran insoluble dietary fiber: pH 5, reaction time 24h, temperature 45°C, enzyme concentration 0.45%-0.55%, substrate concentration 5%-6%. The enzymolysis solution obtained under this condition was freeze-dried and weighed, and the yield of xylo-oligosaccharides was 12.50% to wheat bran.

Beneficial effects of the present invention: domestically, there have been researches on enzymolysis of wheat bran to produce oligosaccharides; existing compound enzyme preparations using phytase, cellulase, protease and oligosaccharase have been used to crush and deionize oligosaccharides after drying. Wheat bran after soaking in water carries out enzymatic hydrolysis, adjusts the research of pH value preparation xylo-oligosaccharide condition (seeing Chinese patent CN1438319A); Existing adopts α-amylase, alkaline protease to process wheat bran, then adopts decolorization, separation Refining methods such as handover and filtration remove insoluble matter to prepare oligosaccharides, and the content reaches 80% in open literature reports (see Chinese patent CN1438319A). There have been published literature reports on the use of wheat bran as a substrate to study the action characteristics of xylanases from different sources. One (1→4)-β-endoxylanase, two (1→4) -β-Endoxylanase (xylanase of family 11 and family 10), two pure enzymes (thermostable xylanase XP1 and XP2) enzymatically hydrolyze wheat bran and study the conditions of enzymatic hydrolysis .

There is no public literature on the yield, purity and xylobiose and xylotriose content of xylooligosaccharides prepared by hydrolyzing wheat bran insoluble dietary fiber with Bacillus subtilis xylanase Reports; foreign countries have not used α-amylase, alkaline endoprotease and refined glucoamylase to treat wheat bran, and used Bacillus subtilis xylanase to catalyze the hydrolysis of wheat bran insoluble dietary fiber to prepare xylooligosaccharides. Public literature reports on the yield, purity and xylobiose and xylotriose content of xylan.

The yield of xylooligosaccharide prepared by the invention is 12.5% (calculated by wheat bran), the purity of xylooligosaccharide exceeds 60%, and the content of xylobiose and xylotriose is more than 30%. The invention realizes effective value-added and utilization of wheat bran, and the prepared xylooligosaccharide is an excellent functional oligosaccharide, which is very beneficial to human health and has great economic and social benefits.

Detailed ways

Suspend wheat bran in hot water, add 75mL/kg of bran-resistant α-amylase, stir at 95°C for 1 hour, cool to 60°C and adjust the pH to 7.5, then add 30mL/kg of bran-hydrolyzing protease Alcalase , stirring at 60°C for 30 minutes, adjusting the pH to 4.5, adding 35m/Lkg bran glucoamylase, stirring at 60°C for 30 minutes, centrifuging to collect the precipitate, and washing the precipitate repeatedly with hot distilled water to obtain wheat bran insoluble dietary fiber. Yield rate is 35%, promptly every kilogram of wheat bran gets 350g wheat bran insoluble dietary fiber, and control substrate concentration is 4%-7%, adds 35g bacillus subtilis xylanase (xylanase) by every kilogram of bran then The carbohydrase was prepared with 6.6L pH5.0 sodium acetate buffer), and the enzymolysis was carried out at 50°C for 24h. The reaction mixture was centrifuged, and the supernatant was taken, concentrated and spray-dried to obtain a crude xylo-oligosaccharide with a yield of 12.5% based on wheat bran.

Claims (2)

1. the method for a preparing xylo-oligosaccharide by enzymolysis of wheat bran, comprise that Testa Tritici obtains the Testa Tritici insoluble dietary fibre after alpha-amylase, proteolytic enzyme, saccharifying enzyme are handled successively, it is characterized in that the Testa Tritici insoluble dietary fibre carries out enzyme digestion reaction with zytase again, the enzymolysis supernatant liquor prepares the xylo-oligosaccharide crude product through concentrated, spraying drying; The used zytase of enzyme digestion reaction is subtilis (Bacillus subtilis) zytase; The enzymolysis substrate is the Testa Tritici insoluble dietary fibre, and the substrate mass concentration is 4%-7%, and the enzyme mass concentration is 0.4%-0.7% in the system, and hydrolysis temperature is 40-50 ℃, and enzymolysis time is 24-30h, and enzymatic hydrolysis system pH is 5.0.

2. method according to claim 1, the optimal conditions that it is characterized in that Testa Tritici insoluble dietary fibre enzyme digestion reaction is 5%-6% for the substrate mass concentration, and the enzyme mass concentration is 0.45%-0.55% in the system, and hydrolysis temperature is 45 ℃, enzymolysis time is 24h, and enzymatic hydrolysis system pH is 5.0.