CN1840673A - A method of enzymolyzing wheat bran to prepare feruloyl oligosaccharides - Google Patents
A method of enzymolyzing wheat bran to prepare feruloyl oligosaccharides Download PDFInfo
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技术领域technical field
一种酶解小麦麸皮制备阿魏酰低聚糖的方法，属于农副产品资源开发、功能性食品添加剂和营养保健品技术领域。The invention discloses a method for preparing feruloyl oligosaccharides by enzymatically hydrolyzing wheat bran, which belongs to the technical field of resource development of agricultural by-products, functional food additives and nutritional health products.
背景技术Background technique
小麦是世界上最重要的粮食作物之一，特别是在发达国家，是人类膳食的重要组成部分。我国小麦产量居世界前列。近几年来，我国每年加工的小麦麸皮约在2000万吨左右。小麦麸皮是人类膳食纤维的很好来源，作为饲料组分的经济价值已经明显下降，在国外已有很多文献报道小麦麸皮作为人类膳食纤维对机体健康有益。近年来，对小麦麸皮研究的注意力集中在麦麸的生理活性物质上，如阿拉伯木聚糖、植酸和阿魏酸上，尤其是阿拉伯木聚糖和阿魏酸。小麦麸皮主要由细胞壁多糖组成，富含半纤维素，其中阿拉伯木聚糖占40％，在木聚糖骨架链上木糖残基的O-3、O-2或O-3和O-2位上通常被-L-阿拉伯糖取代。另一个显著特征就是阿魏酸通过酯键连接在阿拉伯糖残基上。在植物学领域，利用多糖水解酶水解植物细胞壁多糖制备阿魏酰低聚糖，主要是为了研究植物细胞壁的结构。到目前为止，从禾本科植物细胞壁上制备的阿魏酰阿拉伯糖基低聚木糖在结构上显示出了很好的一致性，即-L-呋喃型阿拉伯糖残基连接在以-1，4-糖苷键连接的D-木聚糖骨架链上的木糖O-3位上，阿魏酸与阿拉伯糖残基上的O-5位上相连。Wheat is one of the most important food crops in the world, especially in developed countries, and is an important part of human diet. my country's wheat production ranks among the top in the world. In recent years, my country's annual processing of wheat bran is about 20 million tons. Wheat bran is a good source of human dietary fiber, but its economic value as a feed component has declined significantly. There have been many foreign literatures reporting that wheat bran is beneficial to human body health as human dietary fiber. In recent years, research on wheat bran has focused on the physiologically active substances of wheat bran, such as arabinoxylan, phytic acid and ferulic acid, especially arabinoxylan and ferulic acid. Wheat bran is mainly composed of cell wall polysaccharides, rich in hemicellulose, of which arabinoxylan accounts for 40%, and O-3, O-2 or O-3 and O- of xylose residues on the xylan backbone chain The 2-position is usually substituted by -L-arabinose. Another notable feature is that ferulic acid is attached to the arabinose residue through an ester bond. In the field of botany, the use of polysaccharide hydrolase to hydrolyze plant cell wall polysaccharides to prepare feruloyl oligosaccharides is mainly to study the structure of plant cell walls. So far, the feruloylarabinosyl xylooligosaccharides prepared from the cell wall of Poaceae have shown a good structural consistency, that is, the -L-furan-type arabinose residues are linked at -1, On the O-3 position of xylose on the D-xylan backbone chain linked by 4-glycosidic bonds, ferulic acid is connected to the O-5 position on the arabinose residue.
阿魏酰低聚糖的抗氧化活性，阿魏酰低聚糖具有卓越的生物活性，能够有效地抑制低密度脂蛋白的氧化，在防止或减轻动脉硬化进程方面可能具有重要意义，在这一点上，游离阿魏酸是无法与其相比的。The antioxidant activity of feruloyl oligosaccharides, feruloyl oligosaccharides have excellent biological activity, can effectively inhibit the oxidation of low-density lipoproteins, and may be of great significance in preventing or alleviating the process of arteriosclerosis. In terms of nutrition, free ferulic acid cannot be compared with it.
抗氧化剂是21世纪人类健康的物质保障之一。由于人工合成的抗氧化剂，如丁羟基茴香醚(BHA)、丁羟基甲苯(BHT)、叔丁基氢醌(TBHQ)被怀疑会对人体造成负面影响，在国外，上述合成抗氧化剂在食品中应用受到严格的限制。近年来逐渐将注意力转向植物中蕴藏丰富的天然抗氧化剂，特别是那些存在于日常食品、蔬菜、水果中的抗氧化成分，其中植物酚酸化合物就是研究较多的一类，可望成为新型抗氧化剂的重要源泉。阿魏酸是植物界普遍存在的一种酚酸，尤其是在禾本科植物中。谷物中的阿魏酸通过酯键与细胞壁中多糖相连，以结合态的形式存在。Rondini等人报道，以结合态存在的阿魏酸比游离的阿魏酸具有更好的生物利用度。所以对结合态的阿魏酸(包括阿魏酰低聚糖)的研究已引起了极大的兴趣。到目前为止，有关阿魏酰低聚糖方面的研究国内还没有报道。众所周知，低聚木糖是一种性能优越的双歧因子。阿魏酰低聚糖除含有阿魏酰基外，还含有亲水性的低聚木糖体，它是否具有促进双歧杆菌增殖的作用，国内外均没有报道。Antioxidants are one of the material guarantees for human health in the 21st century. Since synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ), are suspected of causing negative effects on the human body, the application of the above-mentioned synthetic antioxidants in food has been criticized abroad. strict restrictions. In recent years, people have gradually turned their attention to the rich natural antioxidants in plants, especially those antioxidant components that exist in daily food, vegetables, and fruits. Among them, plant phenolic acid compounds are a class that has been studied more, and they are expected to become new types of antioxidants. Great source of antioxidants. Ferulic acid is a phenolic acid ubiquitous in the plant kingdom, especially in grasses. Ferulic acid in grains is connected with polysaccharides in the cell wall through ester bonds, and exists in a bound state. Rondini et al. reported that ferulic acid in the bound state had better bioavailability than free ferulic acid. Therefore, the research on ferulic acid (including feruloyl oligosaccharides) in the bound state has attracted great interest. So far, there is no domestic research on feruloyl oligosaccharides. As we all know, xylo-oligosaccharide is a kind of bifidus factor with superior performance. In addition to feruloyl oligosaccharides, feruloyl oligosaccharides also contain hydrophilic xylooligosomes. Whether it has the effect of promoting the proliferation of bifidobacteria has not been reported at home and abroad.
发明内容Contents of the invention
本发明的目的是提供一种酶解小麦麸皮制备阿魏酰低聚糖的方法，实现小麦麸皮的有效增值和利用，所制备的阿魏酰低聚糖具有卓越的生物活性，能够促进双歧杆菌生长和抑制自由基诱导的血红细胞氧化性伤害，对人体健康非常有益，具有很大的经济效益和社会效益。The purpose of the present invention is to provide a method of enzymolyzing wheat bran to prepare feruloyl oligosaccharides to realize the effective value-added and utilization of wheat bran. The prepared feruloyl oligosaccharides have excellent biological activity and can promote The growth of bifidobacteria and the inhibition of free radical-induced oxidative damage to red blood cells are very beneficial to human health and have great economic and social benefits.
本发明的技术方案：本发明是以小麦麸皮为原料，经高温淀粉酶、蛋白酶、糖化酶依次处理，混合物进行离心得沉淀，所得沉淀用热蒸馏水、体积分数95％乙醇和丙酮进行洗涤后得到小麦麸皮不溶性膳食纤维，再用木聚糖酶进行酶解反应，酶解上清液经浓缩、喷雾干燥制备阿魏酰低聚糖粗品。酶解反应所用木聚糖酶为枯草芽孢杆菌(Bacillus subtilis)木聚糖酶。酶解底物为小麦麸皮不溶性膳食纤维，底物质量浓度为90～120g/L，体系中酶质量浓度为2～5g/L，酶解温度为30～50℃，酶解时间为20～40h，酶解体系pH为4.5～5.5。The technical solution of the present invention: the present invention uses wheat bran as raw material, is treated with high-temperature amylase, protease, and glucoamylase in sequence, and the mixture is centrifuged to obtain a precipitate, and the obtained precipitate is washed with hot distilled water, ethanol with a volume fraction of 95% and acetone The insoluble dietary fiber of wheat bran is obtained, and xylanase is used for enzymatic hydrolysis reaction, and the supernatant of enzymatic hydrolysis is concentrated and spray-dried to prepare crude feruloyl oligosaccharides. The xylanase used in the enzymatic hydrolysis reaction is Bacillus subtilis xylanase. The enzymatic hydrolysis substrate is wheat bran insoluble dietary fiber, the substrate mass concentration is 90-120g/L, the enzyme mass concentration in the system is 2-5g/L, the enzymatic hydrolysis temperature is 30-50°C, and the enzymatic hydrolysis time is 20-20～ After 40 hours, the pH of the enzymatic hydrolysis system was 4.5-5.5.
小麦麸皮不溶性膳食纤维酶解反应的优化条件经中心组合旋转设计方案的优化模型得出酶解反应优化条件为底物质量浓度为120g/L，体系中酶质量浓度为4.8g/L，酶解温度为42℃，酶解时间为35h，酶解体系pH为5.2。The optimal conditions of the enzymatic hydrolysis reaction of wheat bran insoluble dietary fiber were obtained by the optimization model of the central combination rotation design scheme, and the optimal conditions of the enzymatic hydrolysis reaction were as follows: The hydrolysis temperature was 42°C, the enzymolysis time was 35 hours, and the pH of the enzymolysis system was 5.2.
小麦麸皮主要由细胞壁多糖组成，其中阿拉伯木聚糖占绝大多数，在木聚糖骨架链上木糖残基的O-3、O-2或O-3和O-2位上通常被-L-阿拉伯糖取代，另外，在某些O-2位上阿拉伯糖残基上还连有阿魏酸。要制备阿魏酰低聚糖，在小麦麸皮不溶性膳食纤维的制备过程中，必须不能破坏阿魏酸与细胞壁多糖间的酯键，使得酯化在多糖上的阿魏酸全部保留下来，以有利于内切木聚糖酶的催化水解。在研究过程中，我们一方面采用通常方法利用耐高温α-淀粉酶、碱性内切蛋白酶和精制糖化酶对原料进行脱淀粉、除蛋白质；另一方面，专门对酶处理过的样品再利用乙醇和丙酮等有机溶剂进行洗涤，以除去部分色素、游离的酚酸类化合物等，因而获得了酯化阿魏酸含量较高的小麦麸皮不溶性膳食纤维。Wheat bran is mainly composed of cell wall polysaccharides, of which arabinoxylan accounts for the vast majority, and the O-3, O-2 or O-3 and O-2 positions of xylose residues on the xylan backbone chain are usually -L-arabinose substitution, in addition, in some O-2 positions, there is also ferulic acid attached to the arabinose residue. To prepare feruloyl oligosaccharides, in the preparation process of wheat bran insoluble dietary fiber, the ester bond between ferulic acid and cell wall polysaccharides must not be destroyed, so that the ferulic acid esterified on the polysaccharides is all retained, so that Facilitates catalyzed hydrolysis by endoxylanases. In the research process, on the one hand, we use the usual method to destarch and remove protein from the raw materials by using high temperature resistant α-amylase, alkaline endoprotease and refined glucoamylase; on the other hand, we specially reuse the enzyme-treated samples Washing with organic solvents such as ethanol and acetone to remove some pigments, free phenolic acid compounds, etc., thus obtaining wheat bran insoluble dietary fiber with a higher content of esterified ferulic acid.
小麦麸皮和小麦麸皮不溶性膳食纤维的基本化学组成分析结果(％)如表1所示。The basic chemical composition analysis results (%) of wheat bran and wheat bran insoluble dietary fiber are shown in Table 1.
表1
a未测定。 aNot determined.
将小麦麸皮用高压蒸汽在121℃处理45min，以使其内源性酶失活。将处理过的小麦麸皮悬浮在一定体积的水中，60℃下连续搅拌16h，使其充分溶胀，接着加入75mL/kg麸皮的耐温α-淀粉酶后，混合物在沸水浴中搅拌40min。悬浮液冷却至60℃后，调pH至7.5，再加入30mL/kg麸皮的水解蛋白酶Alcalase，60℃下连续搅拌30min，调pH至4.5，再加入35mL/kg麸皮的精制糖化酶，60℃下连续搅拌30min，混合物离心，弃去上清液，沉淀用热蒸馏水反复洗涤，直至用冷蒸馏水洗涤时悬浮液无浑浊，再用热蒸馏水、体积分数95％乙醇和丙酮依次重复地洗涤两次，离心所得沉淀物在40℃下真空干燥24h，得到小麦麸皮不溶性膳食纤维。耐温α-淀粉酶、水解蛋白酶和精制糖化酶均由Novozymes公司提供。Wheat bran was treated with high-pressure steam at 121 °C for 45 min to inactivate endogenous enzymes. Suspend the treated wheat bran in a certain volume of water, stir continuously at 60°C for 16h to make it fully swell, then add 75mL/kg bran heat-resistant α-amylase, and stir the mixture in a boiling water bath for 40min. After cooling the suspension to 60°C, adjust the pH to 7.5, then add 30mL/kg bran hydrolyzing protease Alcalase, stir continuously at 60°C for 30min, adjust the pH to 4.5, then add 35mL/kg bran refined glucoamylase, 60 Stir continuously at ℃ for 30 min, centrifuge the mixture, discard the supernatant, and wash the precipitate repeatedly with hot distilled water until the suspension is free of turbidity when washed with cold distilled water, then repeatedly wash twice with hot distilled water, 95% ethanol and acetone The second time, the precipitate obtained by centrifugation was vacuum-dried at 40°C for 24 hours to obtain wheat bran insoluble dietary fiber. Thermostable α-amylase, proteolytic enzyme and refined glucoamylase were all provided by Novozymes.
枯草芽孢杆菌(Bacillus subtilis)木聚糖酶(简称木聚糖酶)由武汉新华扬生物有限公司提供。Bacillus subtilis (Bacillus subtilis) xylanase (xylanase for short) was provided by Wuhan New Huayang Biological Co., Ltd.
温度对微生物酶的影响情况很复杂，它不仅影响酶蛋白质的天然构象、参与酶促反应功能性基团的解离状态，而且还影响到酶与底物的亲和力、酶-底物络合物的分解，甚至还影响酶与激活剂、抑制剂的亲和力等。pH也是决定酶催化活性的一个重要参数，除影响酶的构象、酶活性部位催化基团和结合基团的解离状态外，还影响底物的带电状态。因此，温度和pH对酶的催化反应速度影响非常明显。The influence of temperature on microbial enzymes is very complicated. It not only affects the natural conformation of enzyme proteins and the dissociation state of functional groups participating in enzymatic reactions, but also affects the affinity of enzymes and substrates, and the enzyme-substrate complexes. The decomposition of the enzyme even affects the affinity of the enzyme with the activator and inhibitor. pH is also an important parameter that determines the catalytic activity of enzymes. In addition to affecting the conformation of the enzyme, the dissociation state of the catalytic group and the binding group at the active site of the enzyme, it also affects the charged state of the substrate. Therefore, the influence of temperature and pH on the catalytic reaction rate of enzymes is very obvious.
在较低温度范围内(30℃～50℃)，木聚糖酶酶活力随温度升高而增大；在较高温度范围内(60℃～80℃)，木聚糖酶酶活力随温度升高而迅速降低，因为温度升高，酶蛋白的热变性失活速度加快；该酶反应的最适温度为50℃，60℃时该酶具有97％的活力，而在80℃时酶的活力仅有6％。适当稀释的酶液在不同温度下保温1h，取出后立即冷却，按常规方法测定相对酶活。结果表明该酶在50℃以下最稳定，60℃时能维持38％的酶活，80℃时酶基本完全失活。In the lower temperature range (30℃～50℃), the enzyme activity of xylanase increases with the increase of temperature; in the higher temperature range (60℃～80℃), the activity of xylanase increases with the temperature As the temperature increases, the thermal denaturation and inactivation speed of the enzyme protein is accelerated; the optimum temperature of the enzyme reaction is 50°C, and the enzyme has 97% activity at 60°C, and the enzyme's activity at 80°C Vitality is only 6%. Appropriately diluted enzyme solution was incubated at different temperatures for 1 h, cooled immediately after taking out, and the relative enzyme activity was measured by conventional methods. The results showed that the enzyme was most stable below 50°C, 38% of the enzyme activity could be maintained at 60°C, and almost completely inactivated at 80°C.
木聚糖酶在pH3.0时，相对活力为37％；在pH6.0时，相对活力为91％；在pH9.0时，相对活力仅为26％；该酶反应时最适pH值为5.0。将酶用不同pH缓冲液适当稀释，在室温下保温1h，按常规方法测定相对酶活，该酶的pH稳定范围为4.0～6.0。At pH 3.0, the relative activity of xylanase is 37%; at pH 6.0, the relative activity is 91%; at pH 9.0, the relative activity is only 26%; the optimal pH value of the enzyme reaction is 5.0. Properly dilute the enzyme with different pH buffers, incubate at room temperature for 1 h, and measure the relative enzyme activity according to conventional methods. The stable pH range of the enzyme is 4.0-6.0.
枯草芽孢杆菌(Bacillus subtilis)木聚糖酶能够水解小麦麸皮不溶性膳食纤维，随意断开膳食纤维中阿拉伯木聚糖主链上的糖苷键，释放阿魏酰低聚糖。随着酶浓度的增大，阿魏酰低聚糖的浓度也不断增加。当酶量达到4.0g/L时，阿魏酰低聚糖的浓度增加比较平缓。Bacillus subtilis xylanase can hydrolyze wheat bran insoluble dietary fiber, randomly break the glycosidic bonds on the main chain of arabinoxylan in dietary fiber, and release feruloyl oligosaccharides. As the enzyme concentration increased, the concentration of feruloyl oligosaccharides also increased. When the enzyme amount reached 4.0g/L, the concentration of feruloyl oligosaccharides increased relatively gently.
在低底物浓度10g/L时，阿魏酰低聚糖浓度的最大值相对较小，仅有0.090mmol/L。然而，当底物浓度超过50g/L时，阿魏酰低聚糖的最大浓度增加相对较快，在底物浓度70g/L时，阿魏酰低聚糖浓度的最大值达到1.217mmol/L。At a low substrate concentration of 10g/L, the maximum concentration of feruloyl oligosaccharides was relatively small, only 0.090mmol/L. However, when the substrate concentration exceeds 50 g/L, the maximum concentration of feruloyl oligosaccharides increases relatively quickly, and when the substrate concentration is 70 g/L, the maximum concentration of feruloyl oligosaccharides reaches 1.217 mmol/L .
B.subtilis木聚糖酶最适反应温度在50℃；最适反应pH值为5.0；该酶在50℃下保温酶活基本稳定，其稳定pH范围为pH4.0～pH6.0。The optimal reaction temperature of B. subtilis xylanase is 50°C; the optimum reaction pH value is 5.0; the enzyme activity is basically stable at 50°C, and the stable pH range is pH4.0～pH6.0.
选择反应温度45℃，pH5.5，反应时间36h，酶量3.0g/L，底物浓度50g/L作为中心组合旋转设计的中心条件。Select reaction temperature 45℃, pH 5.5, reaction time 36h, enzyme amount 3.0g/L, substrate concentration 50g/L as the central conditions of the central combination rotation design.
各因子的中心组合旋转设计方案与实验结果如表2。Table 2 shows the central combination rotation design scheme and experimental results of each factor.
表2 5水平，5因子的中心组合旋转设计方案与实验结果a Table 2 5-level, 5-factor central combination rotation design scheme and experimental results a
a平均绝对相对偏差为5.75％，**FOs表示阿魏酰低聚糖。 a The mean absolute relative deviation is 5.75%, ** FOs represent feruloyl oligosaccharides.
综上分析，使用优化模型可得出阿魏酰低聚糖浓度最大时木聚糖酶水解小麦麸皮不溶性膳食纤维的条件，酶解反应温度42℃，pH5.2，酶解反应时间35h，酶量4.8g/L，底物浓度120g/L，在优化条件下，模型预测阿魏酰低聚糖的最大浓度为1.497mmol/L。在优化条件下获得的阿魏酰低聚糖由酯化的阿魏酸、阿拉伯糖和木糖所组成。Based on the above analysis, using the optimization model, the conditions for xylanase to hydrolyze wheat bran insoluble dietary fiber can be obtained when the concentration of feruloyl oligosaccharides is the highest. The enzyme amount is 4.8g/L, the substrate concentration is 120g/L, under the optimal conditions, the model predicts the maximum concentration of feruloyl oligosaccharides to be 1.497mmol/L. Feruloyl oligosaccharides obtained under optimized conditions consisted of esterified ferulic acid, arabinose and xylose.
本发明的有益效果：以小麦麸皮为原料，利用酶法脱淀粉、除蛋白质制备小麦麸皮不溶性膳食纤维，再利用枯草芽孢杆菌木聚糖酶水解小麦麸皮不溶性膳食纤维制备阿魏酰低聚糖，在反应温度42℃，pH5.2，反应时间35h，酶量4.8g/L，底物浓度120g/L的条件下，阿魏酰低聚糖浓度达到1.497mmol/L。Beneficial effects of the present invention: use wheat bran as raw material, use enzymatic method to destarch and remove protein to prepare wheat bran insoluble dietary fiber, and then use Bacillus subtilis xylanase to hydrolyze wheat bran insoluble dietary fiber to prepare feruloyl low For polysaccharides, under the conditions of reaction temperature 42°C, pH 5.2, reaction time 35h, enzyme amount 4.8g/L, and substrate concentration 120g/L, the concentration of feruloyl oligosaccharides reached 1.497mmol/L.
阿魏酰低聚糖不仅能够体外促进双歧杆菌生长，而且还具有独特的抗氧化活性，能够有效地保护人红细胞免受AAPH引发的氧化性应激伤害，与红细胞内源性抗氧化剂协同抵抗AAPH所诱导的伤害。Feruloyl oligosaccharides can not only promote the growth of bifidobacteria in vitro, but also have unique antioxidant activity, which can effectively protect human erythrocytes from oxidative stress induced by AAPH, and cooperate with endogenous antioxidants in erythrocytes to resist AAPH-induced injury.
本发明实现了小麦麸皮的有效增值和利用，所制备的阿魏酰低聚糖具有卓越的生物活性，能够促进双歧杆菌生长和抑制自由基诱导的血红细胞氧化性伤害，对人体健康非常有益，具有很大的经济效益和社会效益。The invention realizes the effective value-added and utilization of wheat bran, and the prepared feruloyl oligosaccharide has excellent biological activity, can promote the growth of bifidobacteria and inhibit the oxidative damage of red blood cells induced by free radicals, which is very harmful to human health It is beneficial and has great economic and social benefits.
经查新未见相关公开文献报道。No relevant public literature reports have been found after searching.
具体实施方式Detailed ways
将小麦麸皮用高压蒸汽在121℃处理45min，以使其内源性酶失活。将处理过的小麦麸皮悬浮在一定体积的水中，60℃下连续搅拌16h，使其充分溶胀，接着加入75mL/kg麸皮的耐温α-淀粉酶后，混合物在沸水浴中搅拌40min。悬浮液冷却至60℃后，调pH至7.5，再加入30mL/kg麸皮的水解蛋白酶Alcalase，60℃下连续搅拌30min，调pH至4.5，再加入35mL/kg麸皮的精制糖化酶，60℃下连续搅拌30min，混合物离心，弃去上清液，得沉淀，即为通常的脱淀粉、除蛋白质制备小麦麸皮不溶性膳食纤维的常用方法，本发明专门采取将沉淀用热蒸馏水反复洗涤，直至用冷蒸馏水洗涤时悬浮液无浑浊，再用热蒸馏水、体积分数95％乙醇和丙酮依次重复洗涤两次以除去部分色素、游离的酚酸类化合物等，离心所得沉淀物在40℃下真空干燥24h，得到小麦麸皮不溶性膳食纤维。小麦麸皮不溶性膳食纤维再用枯草芽孢杆菌木聚糖酶进行酶解，酶解条件为反应温度42℃、pH5.2、反应时间35h、酶量4.8g/L、底物浓度120g/L时，阿魏酰低聚糖的浓度为1.546±0.037mmol/L(实验重复3次)。Wheat bran was treated with high-pressure steam at 121 °C for 45 min to inactivate endogenous enzymes. Suspend the treated wheat bran in a certain volume of water, stir continuously at 60°C for 16h to make it fully swell, then add 75mL/kg bran heat-resistant α-amylase, and stir the mixture in a boiling water bath for 40min. After cooling the suspension to 60°C, adjust the pH to 7.5, then add 30mL/kg bran hydrolyzing protease Alcalase, stir continuously at 60°C for 30min, adjust the pH to 4.5, then add 35mL/kg bran refined glucoamylase, 60 Stir continuously at ℃ for 30min, centrifuge the mixture, discard the supernatant, and obtain a precipitate, which is a common method for preparing wheat bran insoluble dietary fiber by destarching and removing protein. The present invention specially adopts repeated washing of the precipitate with hot distilled water, Until the suspension is not turbid when washed with cold distilled water, it is washed twice with hot distilled water, ethanol with a volume fraction of 95% and acetone to remove part of the pigment, free phenolic acid compounds, etc., and the precipitate obtained by centrifugation is placed in a vacuum at 40°C. Dry for 24 hours to obtain wheat bran insoluble dietary fiber. Wheat bran insoluble dietary fiber is then enzymatically hydrolyzed with Bacillus subtilis xylanase. The enzymolysis conditions are reaction temperature 42°C, pH 5.2, reaction time 35h, enzyme amount 4.8g/L, substrate concentration 120g/L , the concentration of feruloyl oligosaccharides was 1.546±0.037mmol/L (the experiment was repeated 3 times).
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