CN116496425A - 一种具益生功能牛蒡根低聚糖的制备方法 - Google Patents
一种具益生功能牛蒡根低聚糖的制备方法 Download PDFInfo
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- CN116496425A CN116496425A CN202310492709.7A CN202310492709A CN116496425A CN 116496425 A CN116496425 A CN 116496425A CN 202310492709 A CN202310492709 A CN 202310492709A CN 116496425 A CN116496425 A CN 116496425A
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- burdock root
- oligosaccharide
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Abstract
本发明涉及一种具益生功能牛蒡根低聚糖的制备方法，属于农产品加工技术领域。本发明包括新鲜牛蒡根原料切片、灭酶、烘干、热水浸提、脱淀粉、浓缩、脱蛋白、醇沉、冻干、复溶、超滤、纯化、冻干，其特征是得到的一种纯度高、均一性好的牛蒡根低聚糖，含糖量为93.45％，峰位分子量Mp为2473Da，重均分子量Mw为2544Da，数均分子量Mn为2120Da，能够促进鼠李糖乳杆菌体外生长，菌落总数是空白对照培养基的1.54～2.38倍，是阳性对照低聚异麦芽糖的1.11～1.71倍，具有显著益生功能，可为牛蒡根低聚葡萄糖在功能性食品中的应用开发提供参考。
Description
技术领域
本发明为一种具益生功能牛蒡根低聚糖的制备方法，属于农产品加工技术领域。
背景技术
牛蒡(Arctium lappa L.)为菊科牛蒡属二年生草本植物，俗称“东洋参”，有“蔬菜之王”的美誉。牛蒡中胡萝卜素的含量比胡萝卜中高150倍，蛋白质和钙的含量为根茎类之首，营养价值极高。牛蒡根作为一种传统的药食同源植物，在《本草纲目》、《名医别录》、《中国药典》等著作中均有详细记载，《名医别录》中记载牛蒡“可疗汗出、中风、面肿、消渴、逐水”；《珍惜名优蔬菜》、《蔬菜栽培学》等著作中介绍：经常食用牛蒡根有促进血液循环、清除肠胃垃圾、防止人体过早衰老、润泽肌肤等功效。牛蒡在我国主要种植在山东的兰陵县、江苏的丰县和沛县等地，是优质的出口创汇型蔬菜，被联合国粮农组织誉为“21世纪人类最佳保健食品之一”，具有极高的营养价值、经济价值和开发应用前景。
牛蒡根富含蛋白质、氨基酸、多种维生素、矿物质以及低聚糖，牛蒡低聚糖具有促使双歧杆菌增殖、防止便秘、保护肝脏、降低血清胆固醇、增强机体免疫力、抗癌等作用，等保健功能，被广泛应用于食品、医药等领域，在国内外市场需求巨大。低聚糖，或称寡糖，是单糖经过糖苷键连接而成的低度聚合糖。目前市场上常用的益生元低聚糖类，主要是聚合度2-9的寡糖，包括低聚异麦芽糖、低聚果糖、低聚半乳糖、低聚木糖、大豆低聚糖、菊粉等。由于人体肠胃道内没有水解这些低聚糖的酶系统，因此它们不被消化吸收而直接进入大肠内，优先被双歧杆菌利用，是双歧杆菌的增殖因子。低聚糖具有预防肿瘤、冠心病、糖尿病、结肠癌、便秘等作用，非消化性低聚寡糖具有双向调节微生态平衡的“整肠”生理功能，它可以被肠道内益生菌利用，促进益生菌的生长繁殖，同时抑制肠道有害菌的生长，促进人体润肠通便，安全排毒，增强机体免疫力，降低血脂和胆固醇，促进矿物质吸收。
发明内容
技术问题
本发明的目的提供一种具益生功能牛蒡根低聚糖的制备方法，基于热水浸提法提取新鲜牛蒡根，利用醇沉法得到牛蒡根水溶性多糖部位，再通过截留分子量为5000Da的超滤装置及阴离子交换柱分离纯化得到高纯度的牛蒡根低聚糖。通过本发明所述方法得到的牛蒡根低聚糖，经验证具有益生功能，促进益生菌生长，可为牛蒡根低聚糖在功能性食药、药品和化妆品中的应用开发提供参考。
技术方案
上述的目的通过以下的技术方案实现：
a)热水浸提：将新鲜牛蒡根切片后立即投入沸水中灭酶防止氧化褐变，捞出牛蒡根片后沥水，而后转移至烘箱中烘至半干。将牛蒡根片按公知方法热水提取，液料比为15～20∶1，温度为60～80℃，时间为1.5～2h，重复两次，合并提取液浓缩至合适体积；
b)脱淀粉：将步骤a所得提取液按公知方法分别调最适pH值，先后加α-淀粉酶、糖化酶脱淀粉，离心去掉酶渣，收集浸提液，于50℃条件下旋蒸浓缩；
c)脱蛋白：将步骤b所得提取液中加入1/5的Sevag试剂，按公知方法除蛋白，至无中间蛋白层；
d)醇沉：向步骤c的提取液中按公知方法加入3倍体积的无水乙醇沉淀多糖，4℃过夜，收集沉淀，并用80％乙醇清洗三次，而后将粗多糖放在通风橱中散去乙醇，用适当体积的蒸馏水复溶多糖，再次醇沉；
e)超滤膜分离：将步骤d得到的多糖配成溶液，采用截留分子量为5000Da的PLC改良纤维素复合膜的超滤系统进行分离，温度30℃，压力差0.1Mpa，收集流出液，冷冻干燥，备用；
f)阴离子交换色谱柱纯化：称取一定量步骤e得到牛蒡根粗多糖充分溶解于去离子水中，采用DEAE-32纤维素层析柱纯化，流速1.0mL/min，使用去离子水、0.1、0.3、0.5mol/L NaCl溶液依次梯度洗脱，采用苯酚-硫酸法检测收集液，在490nm处测吸光值，将洗脱液相同组分进行合并；
g)冻干：将步骤f所得合并洗脱液在50℃下减压浓缩后，冷冻干燥，即得一种牛蒡根低聚糖。
本发明的优点和效果：
(1)本发明的一种具益生功能牛蒡根低聚糖的制备方法，相比于常见的多糖分离方法，本发明采用PLC改良纤维素复合膜的超滤系统进行分离，更易制备小分子量的低聚糖。本发明得到的牛蒡根低聚糖纯度高、均一性好，含糖量超过90％，分子量分布指数接近1。本发明的制备工艺简单，耗能少，经济无毒，方便易行，适用于工业化生产。
(2)本发明得到的牛蒡根低聚糖，具有益生功能，可显著增加鼠李糖乳杆菌菌株的生长，菌落总数是空白对照培养基的1.54～2.38倍，是阳性对照低聚异麦芽糖的1.11～1.71倍。鼠李糖乳杆菌具有平衡肠道菌群，改善胃肠道功能；增强人体自身免疫能力；预防和帮助治疗腹泻；减少受感染的几率，比如呼吸道感染；预防过敏；预防龋齿；增强肠道黏膜屏障的作用。所用的阳性对照低聚异麦芽糖是两个葡萄糖分子以α-1，6糖苷键连接起来的双糖聚合物，低聚异麦芽糖能有效的促进人体内有益细菌-双歧杆菌的生长繁殖，故又称为“双歧杆菌生长促进因子”，简称“双歧因子”，是一种常见的益生元。本发明得到的的牛蒡根低聚糖，根据分子量分析葡萄糖聚合度在13～16之间，其益生功能与葡萄糖双糖聚合物低聚异麦芽糖相当、甚至更优，具有益生活性高的优点。
(3)本发明的原料为传统药食两用牛蒡根，廉价易得，安全无毒，为开发应用牛蒡制作酸奶或其它发酵乳制品、牛奶、新鲜干酪、硬质干酪、婴儿食品、乳饮料或非乳饮料以及医药品，如胶囊、小包药片、牛蒡低聚糖/鼠李糖乳杆菌中药微生态制剂奠定了基础，提高牛蒡根的经济价值，实现牛蒡植物资源的充分利用。
附图说明
图1为牛蒡根低聚糖高效凝胶色谱HPGPC图
图2为牛蒡根低聚糖衍生化水解后单糖分析离子色谱IC图
图3为牛蒡根低聚糖红外分析FT-IR图
图4为牛蒡根低聚糖对鼠李糖乳杆菌的益生功能
具体实施方案
下面结合具体实施例对本发明作进一步说明：
实施例1
牛蒡低聚糖的制备
(1)预处理：样品选取新鲜、二年生开花前的牛蒡根，保证个体均匀相似、茎肉饱满无空心、无虫蛀及虫斑、无霉烂变质；挑选新鲜且品质良好的牛蒡根，新鲜牛蒡根切约5mm厚的片，立即投入沸水中灭酶处理2min，捞出牛蒡片后沥水，而后转移至60℃烘箱中烘2小时，冷却至室温后用粉碎机进行粉碎，粉碎后过60目筛，制得牛蒡根粉。
(2)热水浸提：将牛蒡根粉加水进行热水浸提，所述热水浸提法的提取条件为：料液比1∶20、提取温度80℃、提取时间2h，提取结束后，3000rpm/min离心5min，收集上清液，残渣再重复提取一次，将两次的上清液合并，旋转蒸发浓缩。
(3)纯化：将(2)中得到的提取液pH调至α-淀粉酶最适pH 6.0，按酶的添加量加到提取液中，60℃处理1h，用碘化钾溶液检验直到碘液不变色，冷却，pH调糖化酶最适pH 4.5，按酶添加量添加至提取液中，60℃处理1h，冷却后pH调至7，离心去掉酶渣，收集浸提液，于50℃条件下旋蒸浓缩至1/5后，加Sevage试剂(三氯甲烷∶正丁醇＝4∶1)混合，搅拌20min，7000rpm/min离心10min，将有机溶剂层和变性蛋白与水层分离，保留水层，继续用Sevage法除蛋白，至无中间蛋白层除去蛋白；向脱完蛋白的提取液中加入四倍体积的无水乙醇使多糖沉淀下来，4℃过夜，收集沉淀，用去离子水复溶，再用等体积的乙醇溶液漂洗，最后用三倍乙醇醇沉，而后将粗多糖放在通风橱中散去乙醇，用适当体积的蒸馏水复溶多糖，将多糖溶液倒入平皿中，放入-18℃冰箱中冷冻6h以上，而后冻干；得到的牛蒡粗提物，用去离子水配成溶液，采用截留分子量为5000Da的再生纤维素平板膜进一步分离，温度30℃，压力差0.1Mpa，收集滤液，利用旋转蒸发仪浓缩，用超纯水配成浓度10mg/mL溶液，将配制的溶液上样于DEAE-32阴离子交换柱以超纯水、0.1、0.3、0.5mol/L NaCl溶液梯度洗脱，用苯酚-硫酸法检测各收集管490nm处的吸光值，收集超纯水洗脱的中性部位，冷冻干燥，既得一种牛蒡根低聚糖。
实施例2
(1)糖含量测定：按公知方法采用苯酚-硫酸法测定总糖含量，计算得出总糖含量为93.45％。
(2)分子量测定：采用高效凝胶渗透色谱(HPGPC)对实施例1纯化出来的牛蒡根低聚糖进行分子量测定。精密称取样品和标准品，样品配制成5mg/mL溶液，12000rpm离心10min，上清液用0.22μm的微孔滤膜过滤，然后将样品转置于1.8mL进样小瓶中。标准品为分子量1152、5000、11600、23800、48600、80900、148000、273000、409800、667800的Dextran。高效液相色谱仪：岛津Shimadzu LC-10A；色谱柱：BoRui Saccharide BRT105-104-102串联凝胶柱(8×300mm)；流动相：0.05M NaCl溶液；流速：0.6mL/min，柱温：40℃；进样量：20μL；检测器：示差检测器RI-10A。所得结果如图1所示，牛蒡根低聚糖主峰的保留时间为45.535min，带入线性回归方程得到牛蒡根低聚糖的峰位分子量Mp为2473Da，重均分子量Mw为2544Da，数均分子量Mn为2120Da(表1)，分子量分布指数1.2，接近1，均一性较好。
表1.分子量信息
(3)单糖组成测定：使用离子色谱仪测定实施例3得到的纯化牛蒡根低聚糖中单糖组成。精密称量10mg样品置于安瓿瓶中，加入3M TFA 10mL，120℃水解3h。准确吸取酸水解溶液转移至管中氮吹吹干，加入10mL水涡旋混匀，吸取100μL加入900μL去离子水，12000rpm离心5min。取上清进IC分析。取16种单糖标准品(岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸、氨基半乳糖盐酸盐、盐酸氨基葡萄糖、N-乙酰-D氨基葡萄糖、古罗糖醛酸、甘露糖醛酸)配成标准母液溶液。根据绝对定量方法，测定不同单糖质量，根据单糖摩尔质量计算出摩尔比。离子色谱仪：ThermoFisherICS5000；色谱柱：DionexCarbopacTMPA20(3*150)；流动相：A：H2O；B：15mM NaOH；C：15mMNaOH&100mM NaOAC；流速：0.3mL/min；进样量：5μL；柱温：30℃；检测器：电化学检测器。得到的牛蒡根低聚糖中单糖组成分析结果见图2和表2。
表2.单糖组成分析
(4)红外图谱分析：通过FT-IR分析实施例3得到的纯化牛蒡根低聚糖的官能团。精密称取样品2mg和溴化钾200mg，压制成片，空白对照采用溴化钾粉末压片而成。置于傅里叶变换红外光谱仪FT-IR650进行扫描记录，如图3所示，吸收带在3600-3200cm-1是-OH的伸缩振动吸收峰，这个区域的吸收峰是糖类的特征峰。具体如下：3367cm-1是O-H的伸缩振动吸收峰，是糖类的特征峰。在2929cm-1处有一个吸收峰，可能归属于C-H伸缩振动。在1635cm-1处有一个吸收峰，可能归属于结晶水。在1417cm-1处、1133cm-1处有吸收峰，可能归属于C-O伸缩振动。在1218cm-1处、1029cm-1处有吸收峰，可能归属于O-H变角振动。在927cm-1处有吸收峰，可能归属于吡喃环的非对称环伸缩振动。在867cm-1处有吸收峰，可能归属于吡喃环的环上次甲基的横摇振动。
实施例3
益生活性检测：实施例1得到的纯化牛蒡根低聚糖进行益生功能检测。将鼠李糖乳杆菌接种至MRS培养基中，添加0.1g/mL的牛蒡根低聚葡萄糖样品于基础培养基中，培养16-18h后，用细胞计数仪检测细菌数，每个样品重复三次，阳性对照组采用相同浓度的低聚异麦芽糖，空白对照组为MRS培养基。经牛蒡根低聚葡萄糖处理的实验组鼠李糖乳杆菌的活菌数、菌株存活率均高于空白对照组(MRS培养基)，如图4所示，空白对照组、阳性对照组、样品组的鼠李糖乳杆菌的菌落总数分别为(1.93±0.285)、(2.687±0.211)、(3.753±0.663)×108CFU/mL，牛蒡根低聚糖除了组的菌落总数是空白对照组的1.54～2.38倍，牛蒡根低聚糖对鼠李糖乳杆菌增殖的作用是阳性对照品低聚异麦芽糖的1.11～1.71倍，说明牛蒡根低聚葡萄糖为促生因子。
以上详细说明了本发明的实施方式，但这只是为了便于理解而举的实例，不应被视为是对本发明范围的限制。同样，任何所属技术领域的技术人员均可根据本发明的技术方案及其较佳实施例的描述，做出各种可能的等同改变或替换，但所有这些改变或替换都应属于本发明的权利要求的保护范围。
Claims (3)
1.一种具益生功能牛蒡根低聚糖的制备方法，其特征在于，包括如下步骤：
a)热水浸提：将新鲜牛蒡根切片后立即投入沸水中灭酶防止氧化褐变，捞出牛蒡根片后沥水，而后转移至烘箱中烘至半干。将牛蒡根片按公知方法热水提取，液料比为15～20∶1，温度为60～80℃，时间为1.5～2h，重复操作，合并提取液浓缩至合适体积；
b)脱淀粉：将步骤a所得提取液按公知方法分别调最适pH值，先后加α-淀粉酶、糖化酶脱淀粉，离心去掉酶渣，收集浸提液，于50℃条件下旋蒸浓缩；
c)脱蛋白：将步骤b所得提取液中加入1/5的Sevag试剂，按公知方法除蛋白，至无中间蛋白层；
d)醇沉：向步骤c的提取液中按公知方法加入3倍体积的无水乙醇沉淀多糖，4℃过夜，收集沉淀，并用80％乙醇清洗三次，而后将粗多糖放在通风橱中散去乙醇，用适当体积的蒸馏水复溶多糖，再次醇沉；
e)超滤膜分离：将步骤d得到的多糖配成溶液，采用截留分子量为5000Da的PLC改良纤维素复合膜的超滤系统进行分离，温度30℃，压力差0.1Mpa，收集流出液，冷冻干燥，备用；
f)阴离子交换色谱柱纯化：称取一定量步骤e得到牛蒡根粗多糖充分溶解于去离子水中，采用DEAE-32纤维素层析柱纯化，流速1.0mL/min，使用去离子水、0.1、0.3、0.5mol/LNaCl溶液依次梯度洗脱，采用苯酚-硫酸法检测收集液，在490nm处测吸光值，将洗脱液相同组分进行合并；
g)冻干：将步骤f所得合并洗脱液在50℃下减压浓缩后，冷冻干燥，即得一种牛蒡根低聚糖。
2.根据权利要求1所述制得的牛蒡根低聚糖，其特征在于：多糖提取原料为新鲜牛蒡根，纯化部位为PLC改良纤维素复合膜的超滤系统分离得到的截留分子量小于5000Da部分，纯化低聚糖的含糖量为93.45％，峰位分子量Mp为2473Da，重均分子量Mw为2544Da，数均分子量Mn为2120Da。
3.根据权利要求1所述制得的牛蒡根低聚糖，其特征在于：能够促进鼠李糖乳杆菌生长，菌落数是空白对照的1.54～2.38倍。
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