CN102863548B - 一种从小麦麸皮中提取木聚糖的方法 - Google Patents
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Abstract
本发明公开了一种从小麦麸皮中提取木聚糖的方法，包括：利用吐温-80、中性蛋白酶和中温α-淀粉酶对小麦麸皮进行预处理，然后加酸保温清洗，加NaOH保温浸提，离心去渣，然后加入植酸酶、甘露聚糖酶进一步除去杂质；3次加酒精清洗沉淀，烘干粉碎后即得小麦木聚糖产品。本发明工艺过程简单，条件温和，且制得的木聚糖产品纯度高达95.83%，作为底物测定木聚糖酶的酶活力数据更准确，稳定性更高，具有更广的应用价值。
Description
技术领域
本发明属于植物多糖的生产和应用技术领域，具体涉及一种从小麦麸皮中提取木聚糖的方法。
背景技术
木聚糖是一种多聚五碳糖，是半纤维素中含量最丰富的一种，约占植物细胞干重的35％，是谷物籽实中的主要非淀粉多糖(NSP)之一。木聚糖又具有很高的利用价值，如将其酸水解成单糖可直接生产工业木糖，也可以进一步加工得到木糖醇；水解木聚糖还可以用来生产糠醛，以及进一步加工为糠醇、呋喃、马来酸和糠酸等一系列重要化工产品；另外木聚糖经生物降解得到的低聚木糖是高附加值的功能性生理活性物质；木聚糖也是木聚糖酶酶活测定必须的底物。而在自然界中，植物半纤维素分布广泛，因此，从植物细胞中提取木聚糖具有十分重要的意义。
目前木聚糖的工业化生产一般都是从廉价的玉米芯稻壳提取，在生产过程中为了追求高提取收率而采用高温提取手段。但高温高碱条件容易破坏分解木聚糖大分子，且生产出的木聚糖杂质残留很多，不能满足需要。
另外，作为木聚糖酶酶活测定底物，目前市售木聚糖产品只有燕麦木聚糖、桦木木聚糖和山毛榉木聚糖，而没有作为饲料用酶相关性更高的小麦木聚糖。
发明内容
本发明的目的是提供一种从小麦麸皮中提取木聚糖的方法，可以有效的从小麦麸皮中提取出木聚糖，并能获得更高的纯度，从而弥补了现有技术的不足。
本发明的方法，包括有如下的步骤：
1）将小麦麸皮在吐温-80水溶液中浸泡，再在水溶液中添加蛋白酶和淀粉酶继续浸泡；
2）浸泡结束后，将用水清洗过的小麦麸皮样品加到酸溶液中高温浸泡；浸泡结束后将小麦麸皮用水清洗；
3）再将小麦麸皮放入到碱性水溶液中进行保温浸提，浸提结束后冷却到室温再离心去掉杂质；
4）将离心的上清液调节pH5.0后，再加入植酸酶和/或甘露聚糖酶进行处理；处理结束后将酶失活，在溶液中加入乙醇后进行沉淀，获得的沉淀烘干粉碎后即得小麦木聚糖产品。
上述步骤1）中吐温-80水溶液的体积比浓度为1-1.4%；所述的蛋白酶为中性蛋白酶；所述的淀粉酶为中温α-淀粉酶。
上述步骤2）中的酸溶液为体积分数为0.13%-0.16%的硫酸水溶液，
上述步骤3）中的碱性水溶液为质量体积百分比浓度为3.6%-4.3%的NaOH水溶液。
上述步骤4）中用醋酸调节离心的上清液的pH值至5.0。
上述步骤4）中获得的沉淀用体积分数为95%的乙醇清洗。
本发明利用吐温-80、中性蛋白酶、中温淀粉酶对小麦麸皮进行预处理，吐温-80 的添加加速了麸皮的分散，软化，同时有效促进后续蛋白酶和淀粉酶的降解作用。本发明的反应条件温和，从而减少了对木聚糖大分子的破坏，也大大减少了杂质的产生量。此外，本发明还通过添加植酸酶和甘露聚糖酶进一步去除产物中的杂质，提高木聚糖的纯度。本发明制得的木聚糖产品纯度高达95.83%，以此为底物测定木聚糖酶酶活力数据更准确，稳定性更高。本发明小麦来源的木聚糖比市售的燕麦、桦木、山毛榉木聚糖更适合测定主要作为饲用酶用途的木聚糖酶的酶活，因此具有更广的应用价值。
具体实施方式
本发明所用仪器设备为行业内常用设备所用原料和试剂可选自市售任意一种，例如：中性蛋白酶可选自肇东市日成酶制剂有限公司，酶活50000u/g。
中温α-淀粉酶可选自河南中牟酶制剂厂，酶活2000 u/g。
液体植酸酶可选自康地恩生物集团，酶活10000u/ml。
甘露聚糖酶可选自济南天天香有限公司，酶活50000u/g。
实施例1
1）将小麦麸皮在吐温-80水溶液中浸泡，再在水溶液中添加蛋白酶和淀粉酶后继续浸泡；
取经10目过筛的小麦麸皮500g，加入5000 mL水，50mL 吐温-80，40℃浸泡3h，再加入1.8g中性蛋白酶，40℃水浴保温1h；然后加入2g中温α-淀粉酶，80℃水浴保温3h。煮沸灭酶，自来水洗净至清洗麸皮的水清澈。
2）浸泡结束后，将用水清洗过的小麦麸皮样品加到酸溶液中高温浸泡；浸泡结束后将小麦麸皮用水清洗；
将步骤1）预处理的麸皮加入5000ml水，再加入6.5ml浓H2SO4，在60℃水浴保温5h，然后将麸皮用自来水洗净，至清洗麸皮的水pH与自来水pH一致。
3）再将小麦麸皮放入到加碱性水溶液中进行保温浸提，浸提结束后冷却到室温再离心去掉杂质，取上清液待用；
将步骤2）清洗过的麸皮再加入5000ml水，180g NaOH，55℃水浴保温3h，冷却至室温，4500r/min，离心15min，取清液；
4）将离心的上清液调节pH5.0后，再加入植酸酶和/或甘露聚糖酶进行处理；将酶失活后在溶液中加入乙醇后低温沉淀，获得的沉淀烘干粉碎后即得小麦木聚糖产品：
将清液用醋酸调pH至5.0，然后每100ml清夜加液体植酸酶0.1ml，甘露聚糖酶0.001g，37℃水浴保温1h，然后煮沸灭酶；冷却后加体积比为95%的200ml乙醇，混匀，置4℃冰箱过夜，3000r/min，离心10min，去除上清液取沉淀。
将沉淀用95%乙醇清洗3次以上，然后将沉淀捣碎，平铺在平皿上50℃通风烘干7h；粉碎，即得粉状木聚糖产品123.6g。
实施例2
1）将小麦麸皮在吐温-80水溶液中浸泡，再在水溶液中添加蛋白酶和淀粉酶继续浸泡；
取经10目过筛麸皮500g，加入5000 mL水，70mL 吐温-80，40℃浸泡2h。加入2.16g中性蛋白酶，40℃水浴保温1h；然后加入2.4g中温α-淀粉酶，80℃水浴保温3h。煮沸灭酶，自来水洗净至清洗麸皮的水清澈。
2）浸泡结束后，将用水清洗过的小麦麸皮样品加到酸溶液中高温浸泡；浸泡结束后将小麦麸皮用水清洗；
将上述预处理的麸皮加入5000ml水，再加入8.0ml浓H2SO4，60℃水浴保温4h，自来水洗净至清洗麸皮的水pH与自来水pH一致
3）再将小麦麸皮放入到加碱性水溶液中进行保温浸提，浸提结束后冷却到室温再离心去掉杂质，取上清液待用；
在清洗过的麸皮中再加入5000ml水，216g NaOH，55℃水浴保温2h，冷却至室温，4500r/min，离心15min，取清液；
4）将离心的上清液调节pH5.0后，再加入植酸酶和/或甘露聚糖酶进行处理；将酶失活后在溶液中加入乙醇后低温沉淀，获得的沉淀烘干粉碎后即得小麦木聚糖产品：
清液用醋酸调pH值5.0，每100ml清夜加液体植酸酶0.15ml，甘露聚糖酶0.015g，37℃水浴保温1h，然后煮沸灭酶。冷却后加200ml乙醇（体积比95%），混匀，置4℃冰箱过夜，3000r/min，离心10min，去除上清液取沉淀。沉淀用95%乙醇再清洗2-3次后，将沉淀捣碎，平铺在平皿上45℃通风烘干8h。粉碎，得粉状木聚糖产品128.8g。
实施例3
取经10目过筛麸皮500g，加入5000 mL水，55mL 吐温-80，40℃浸泡2.2h。加入2 g中性蛋白酶，40℃水浴保温1h。然后加入2 g中温α-淀粉酶，80℃水浴保温3h。煮沸灭酶，自来水洗净至清洗麸皮的水清澈。
将上述预处理的麸皮加入5000ml水，再加入7ml浓H2SO4，60℃水浴保温3.5h，自来水洗净至清洗麸皮的水pH与自来水pH一致。再加入5000ml水，196g NaOH，55℃水浴保温2.5h，冷却至室温，4500r/min，离心15min，取清液。
清液用醋酸调pH值5.0，每100ml清夜加液体植酸酶0.12ml，甘露聚糖酶0.012g，37℃水浴保温1h，然后煮沸灭酶。冷却后加200ml乙醇（体积比95%），混匀，置4℃冰箱过夜，3000r/min，离心10min，去除上清液取沉淀。将沉淀用95%乙醇再清洗2-3次，每次清洗都置4℃过夜，3000r/min，离心15min去除上清液取沉淀；将沉淀捣碎，平铺在平皿上45℃通风烘干8h后粉碎，得粉状木聚糖产品126.1g。
实施例1-3制得木聚糖产品中木糖含量检测：
D-木糖标准溶液：10mg/100ml
取木糖标准液0.75、1.00、1.25、1.50、1.75、2.00ml分别置于25ml具塞试管中（其中0ml用作水的空白试验），加水补足2ml，加反应液11ml，摇匀，置于沸水浴显色25min后迅速流水冷却，立即于552nm及510nm测定吸光度，结果如下表所示。
| 木糖浓度 | 75ug | 100ug | 125ug | 150ug | 175ug | 200ug |
| 552nm比色 | 0.239 | 0.300 | 0.373 | 0.429 | 0.502 | 0.556 |
| 510nm比色 | 0.059 | 0.076 | 0.096 | 0.109 | 0.130 | 0.144 |
| 吸光差 | 0.180 | 0.224 | 0.278 | 0.320 | 0.372 | 0.412 |
以木糖浓度为y轴，吸光差为x轴绘制标准曲线，为Y=530.89x-20.53
称取实施例1-3制得的小麦木聚糖1.000g，加入0.32g氢氧化钠，再加入90ml水加热，直至完全溶解，冷却，加冰乙酸调pH 至5.5，然后用pH5.5乙酸-乙酸钠缓冲溶液定容至100ml，使用前摇匀，取1ml木聚糖溶液用水稀释一定倍数，取2ml稀释液，置于25ml具塞试管中（其中0ml用作水的空白试验加水补足2ml）,加11ml反应液，摇匀，置于沸水浴显色25min后迅速流水冷却，立即于552nm及510nm测定吸光度，结果如下表所示。
结果显示，本发明制备得到的木聚糖产品木糖含量高，其中实施例2所述木聚糖产品的木糖含量最高，达95.83%。
本发明的方法由于添加吐温-80，从而加速了麸皮的分散和软化，有效促进了后续蛋白酶和淀粉酶的降解作用，减少了后续沉淀步骤中杂质的量，使提取的木聚糖的纯度相比于现有的方法大大的提高了。
以实施例1-3制得的小麦木聚糖产品为底物测定木聚糖酶酶活，并以市售燕麦木聚糖、桦木木聚糖和山毛榉木聚糖做对照。
取2 ml浓度为1%的木聚糖底物（pH5.5乙酸-乙酸钠缓冲液配制），加入到比色管中，37℃平衡10 min，再加入2 ml经pH5.5乙酸-乙酸钠缓冲液适当稀释并经37℃平衡好的酸性木聚糖酶酶液，混匀于37℃精确保温反应30 min。反应结束后，加入5 ml DNS试剂，混匀以终止反应。然后沸水浴煮沸5 min，用自来水冷却至室温，加蒸馏水定容至25 ml，混匀后，以标准空白样为空白对照，在540 nm处测定吸光值AE。
酶活单位定义：在37℃、pH值为5.5的条件下，每分钟从浓度为5 mg/ml的木聚糖溶液中释放1 μmol还原糖所需要的酶量即为一个酶活力单位U。
酶活计算公式：
式中：XD为稀释酶液中木聚糖酶的活力，U/ml；AE为酶反应液的吸光度；AB为酶空白液的吸光度；K为标准曲线的斜率；C0为标准曲线的截距；M为木糖的摩尔质量，150.2 g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1 mmol=1000 μmol。
经测定，木聚糖酶酶活力如下表所示：
从表中可以看出，以小麦木聚糖作为底物测得的木聚糖酶酶活力远高于以市售燕麦木聚糖、桦木木聚糖和山毛榉木聚糖为底物测定的酶活力，说明本发明制备得到的小麦木聚糖用于测定木聚糖酶酶活的数据更准确，稳定性更高。
Claims (4)
1.一种从小麦麸皮中提取木聚糖的方法，其特征在于，所述的方法包括有如下的步骤：
1)将小麦麸皮在吐温-80水溶液中浸泡，再在水溶液中添加中性蛋白酶和中温α-淀粉酶继续浸泡；其中吐温-80水溶液的浓度为1-1.4％；
2)浸泡结束后，将用水清洗过的小麦麸皮样品加到酸溶液中在60℃浸泡；浸泡结束后将小麦麸皮用水清洗；
3)再将小麦麸皮放入到碱性水溶液中进行保温浸提，浸提结束后冷却到室温再离心去掉杂质；
4)将离心的上清液调节pH5.0后，再加入植酸酶和甘露聚糖酶进行处理；处理结束后将酶失活，在溶液中加入乙醇后进行沉淀，获得的沉淀即为小麦木聚糖。
2.如权利要求1所述的方法，其特征在于所述的步骤2)中的酸溶液为0.13-0.16％的硫酸水溶液。
3.如权利要求1所述的方法，其特征在于所述的步骤3)中的碱性水溶液为3.6-4.3％的NaOH溶液。
4.如权利要求1所述的方法，其特征在于所述的步骤4)中获得的沉淀用体积分数为95％的乙醇清洗。
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