CN117904233A - 一种黄水低聚糖及其制备方法和应用 - Google Patents
一种黄水低聚糖及其制备方法和应用 Download PDFInfo
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- CN117904233A CN117904233A CN202410108050.5A CN202410108050A CN117904233A CN 117904233 A CN117904233 A CN 117904233A CN 202410108050 A CN202410108050 A CN 202410108050A CN 117904233 A CN117904233 A CN 117904233A
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- oligosaccharide
- yellow water
- yellow
- enzymolysis
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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Abstract
本发明属于食品工程技术领域,公开了一种黄水低聚糖及其制备方法和应用,该制备方法包括原料前处理、酶解、灭酶和除杂步骤,本发明的制备方法简单,原料易得,价格低廉,制得的黄水低聚糖能够显著增加粪便体外发酵液中短链脂肪酸的含量,对维持肠道健康和促进脂肪分解代谢具有显著作用;并且该方法制备的黄水低聚糖得率高,能够满足生产要求。
Description
技术领域
本发明涉及食品工程技术领域,具体来说,涉及一种黄水低聚糖及其制备方法和应用。
背景技术
黄水是在传统固态法酿造白酒过程中,由微生物代谢生成的水与酒醅中的水溶解发酵过程中产生的有机酸、单宁、色素、可溶性淀粉、蛋白质、还原糖及其他香味物质后,沉积到窖池底部而形成的棕黄色粘稠液体。黄水中含有大量残留的可溶性淀粉,经过酶和/或微生物作用能够被进一步降解,产生多糖、寡糖及单糖等物质。因此,对酿酒副产物黄水中糖类物质的深入研究将为合理开发黄水资源提供基础数据,能够极大地提高黄水的利用率,为白酒企业实现“双碳”计划提供重要发展思路。
寡糖又称低聚糖,是一种有特殊生理功能的不消化性糖,具有改善肠道菌群,提高机体免疫力等重要的生理作用,其本质是由2-10个单糖单位通过糖苷键而连接的直链或支链的小聚合体,介于单体单糖与高度聚合的多糖之间。与多糖相比,低聚糖具有粘度低、聚合度低、分子量小、水溶性好、无抗原性、容易吸收、生物利用性好以及在宿主体内具有较弱的积累效应等显著优势,因此,低聚糖在食品、医药、保健品等各个领域的开发和应用已十分广泛。如今,国内外已形成巨大的低聚糖产品市场。
目前,以多糖为原料制备低聚糖的方法主要有化学降解法、物理降解法和酶法降解法,传统的化学降解和物理降解存在反应剧烈、设备维护成本高、环境污染严重、产量较低以及产物聚合度难以控制等问题,而酶解法具有反应条件温和、提取效率高、工艺容易控制且对环境友好等特点,因此酶解法制备低聚糖逐渐成为近年来研究的热点。
孟祥勇、沈赤、毛健等研究了黄酒多糖的分离纯化步骤(黄酒多糖的分离纯化及理化性质研究[J].食品与生物技术学报;2017;036(010):1029-1035),以绍兴黄酒为原料,采用乙醇沉淀,Sevag法脱蛋白得到黄酒粗多糖,经DEAE-Sepharose FF色谱柱和葡聚糖凝胶G75色谱柱对黄酒粗多糖进行分离纯化,并采用高效凝胶渗透色谱法对黄酒多糖的相对分子质量和纯度进行检测,进一步采用紫外光谱,红外光谱以及核磁共振波谱对黄酒多糖组分CRWP1的结构特征进行初步解析。但是该分离纯化方法不满足黄水低聚糖的提取率要求,不适合黄水低聚糖的提取。
现有技术中对黄水的研究主要集中在黄水中的有机物质和微生物等方面,尚未有通过酶解黄水多糖制备、提取低聚糖并研究低聚糖生物活性的相关报道。
发明内容
为了解决上述技术问题,本发明提供了一种黄水低聚糖及其制备方法和应用,具体通过黄水的前处理、黄水粗多糖的酶解、灭酶和除杂,制备得到黄水低聚糖,该低聚糖能够显著增加粪便体外发酵液中短链脂肪酸的含量,对维持肠道健康和促进脂肪分解代谢具有显著作用。
为了实现上述目的,本发明采用以下技术方案:
本发明提供了一种黄水低聚糖的制备方法,包括以下步骤:
S1、前处理:黄水醇沉后冻干,得固体黄水粗多糖;
S2、酶解:取固体黄水粗多糖溶于PBS缓冲液得黄水粗多糖溶液,加入复合酶进行酶解,得到酶解液;
S3、灭酶:对酶解液加热灭酶,冷却至室温后离心、过滤得上清液;
S4、除杂:对上清液超滤和透析进行除杂,冻干后得所述的黄水低聚糖。
具体地,步骤S1所述的前处理包括:黄水经离心、过滤去除稻壳、窖泥等杂质,将黄水溶液进行醇沉,之后离心获得沉淀,将沉淀冻干,获得固体黄水粗多糖。
优选地,所述黄水中酒的香型包括清香型、酱香型、浓香型、馥郁香型、芝麻香型、米香型、凤香型中的任意一种或多种。
优选地,步骤S2所述复合酶包括α-淀粉酶和果胶酶;所述复合酶的添加量为5-15μL/10mL黄水粗多糖溶液。
优选地,所述α-淀粉酶和果胶酶的体积比为1-5:1。
进一步优选地,所述α-淀粉酶和果胶酶的体积比为1:1。
优选地,步骤S2所述酶解的条件为:pH 5-7,温度40℃-55℃,时间8h-10h。
进一步优选地,步骤S2所述酶解的条件为:pH 6,温度55℃,时间9h。
优选地,步骤S3所述灭酶的温度为85℃-110℃。
进一步优选地,步骤S3所述灭酶的温度为100℃。
优选地,步骤S3所述离心的条件为:温度20℃-25℃,转速8000rpm-12000rpm,时间8min-12min。
进一步优选地,步骤S3所述离心的条件为:温度20℃,转速10000rpm,时间10min。
优选地,步骤S4所述超滤的滤膜包为2000Da-4000Da。
进一步优选地,步骤S4所述超滤的滤膜包为3000Da。
优选地,步骤S4所述透析的透析袋为100Da-400Da;所述透析的条件为:温度2℃-8℃,时间40h-52h。
进一步优选地,步骤S4所述透析的透析袋为200Da;所述透析的条件为:温度2℃,时间40h。
本发明还提供了一种黄水低聚糖,由上述的制备方法制得。
优选地,所述黄水低聚糖的分子量为1828Da;所述黄水低聚糖组分包括葡萄糖;所述黄水低聚糖为α-构型的吡喃糖。
具体地,纯化后鉴定的黄水低聚糖分子量为1828Da;纯化后检测的黄水低聚糖中的单糖组分包括葡萄糖;纯化后鉴定的黄水低聚糖为α-D构型的吡喃糖。
优选地,所述纯化的步骤为:经DE-52离子交换柱和葡聚糖凝胶G-15纯化。
本发明还提供了一种上述的黄水低聚糖或上述的制备方法制备得到的黄水低聚糖在制备减肥功能食品或保健品或药物中的应用。
优选地,所述的减肥是通过增加肠道发酵过程中短链脂肪酸含量实现的。
进一步优选地,所述的短链脂肪酸包括乙酸、丙酸和丁酸。
本发明的有益效果为:
本发明的制备方法简单,原料易得,价格低廉;本发明制得的黄水低聚糖能够显著增加粪便体外发酵液中短链脂肪酸的含量,对维持肠道健康和促进脂肪分解代谢具有显著作用;本发明的方法得率高,满足生产要求。
附图说明
图1为窄分布聚乙二醇分子量标准曲线图;
图2为本发明实施例1的黄水低聚糖经纯化后的分子量结果图;
图3为本发明实施例1的黄水低聚糖经纯化后的单糖组成结果图;
图4为本发明实施例1的黄水低聚糖经纯化后的红外分析图;
图5为体外发酵过程中乙酸含量变化图;
图6为体外发酵过程中丙酸含量变化图;
图7为体外发酵过程中为丁酸含量变化图。
具体实施方式
以下实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。对所公开的实施例的下述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例中,而是可以应用于符合与本文所公开的原理和新颖特点相一致的更宽的范围。虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料,本文在此处列举优选的方法和材料。
除非另外定义,本文中使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同意义。
实验材料及仪器设备:黄水取自宜宾五粮液股份有限公司,取样后于-20℃下储存备用;α-淀粉酶(Ban-480L)、果胶酶(Pectinex Ultra SP-L)购于诺维信(中国)生物技术有限公司;单糖标品(甘露糖,核糖,鼠李糖,葡萄糖醛酸,半乳糖醛酸,N-乙酰氨基葡萄糖,葡萄糖,N-乙酰氨基半乳糖,半乳糖,木糖,阿拉伯糖,岩藻糖),均为色谱级,购于Sigma公司;无水乙醇(分析纯),国药集团化学试剂有限公司;高速冷冻离心机(CR22N),日本株式会社日立制作所;高效液相色谱仪(LC20),日本Shimadzu(岛津)公司;TSKgel GMPWXL水相凝胶色谱柱,日本TOSOH(TSK东曹)公司;高效液相色谱仪(U3000),美国赛默飞世尔科技公司;气相色谱仪(7890A),美国安捷伦科技有限公司;红外光谱仪(NicoletTM iSTM10),美国赛默飞世尔科技公司;超滤机(ZJMP-16-078)、超滤膜包(PELLICON 2MINI),美国MILLIPORE(密理博)公司;DE-52离子交换柱,购于上海麦克林生化科技股份有限公司;葡聚糖凝胶G-15,购于上海源叶生物科技有限公司。
实施例1一种黄水低聚糖的制备方法
包括以下步骤:
S1、前处理:黄水经离心、过滤去除稻壳、窖泥等杂质后,加无水乙醇沉淀,于-50℃冻干,得固体黄水粗多糖;
S2、酶解:取固体黄水粗多糖溶于pH为6.0的PBS缓冲液中,制成1mg/mL的黄水粗多糖溶液,加入复合酶(α-淀粉酶和果胶酶的体积比为1:1)进行酶解,复合酶的添加量为10μL/10mL黄水粗多糖溶液,酶解pH为6.0,酶解温度为55℃,酶解时间为9h,得到酶解液;
S3、灭酶:对酶解液100℃加热灭酶,冷却至室温后离心(温度20℃、转速10000rpm、时间10min)去除沉淀,上清液过0.45μm滤膜过滤;
S4、除杂:对上清液进行超滤,滤膜包为3000Da,用来除掉未酶解完全的多糖大分子,将超滤完成的透过液倒入200Da透析袋,于4℃下透析48h,透析的目的是除去小分子糖类、盐和部分色素。透析完成后,于-50℃下冷冻干燥24h得到所述的黄水低聚糖,得率28.24%。
实施例2一种黄水低聚糖的制备方法
包括以下步骤:
S1、前处理:黄水经离心、过滤去除稻壳、窖泥等杂质后,加无水乙醇沉淀,于-50℃冻干,得固体黄水粗多糖;
S2、酶解:取固体黄水粗多糖溶于pH为6.0的PBS缓冲液中,制成1mg/mL的黄水粗多糖溶液,加入复合酶(α-淀粉酶和果胶酶的体积比为5:1)进行酶解,复合酶的添加量为15μL/10mL黄水粗多糖溶液,酶解pH为7.0,酶解温度为50℃,酶解时间为10h,得到酶解液;
S3、灭酶:对酶解液110℃加热灭酶,冷却至室温后离心(温度25℃、转速12000rpm、时间8min)去除沉淀,上清液过0.45μm滤膜过滤;
S4、除杂:对上清液进行超滤,滤膜包为4000Da,用来除掉未酶解完全的多糖大分子,将超滤完成的透过液倒入400Da透析袋,于8℃下透析52h,透析的目的是除去小分子糖类、盐和部分色素。透析完成后,于-50℃下冷冻干燥24h得到所述的黄水低聚糖,得率26.52%。
实施例3一种黄水低聚糖的制备方法
包括以下步骤:
S1、前处理:黄水经离心、过滤去除稻壳、窖泥等杂质后,加无水乙醇沉淀,于-50℃冻干,得固体黄水粗多糖;
S2、酶解:取固体黄水粗多糖溶于pH为6.0的PBS缓冲液中,制成1mg/mL的黄水粗多糖溶液,加入复合酶(α-淀粉酶和果胶酶的体积比为3:1)进行酶解,复合酶的添加量为5μL/10mL黄水粗多糖溶液,酶解pH为5.0,酶解温度为40℃,酶解时间为8h,得到酶解液;
S3、灭酶:对酶解液90℃加热灭酶,冷却至室温后离心(温度22℃、转速8000rpm、时间12min)去除沉淀,上清液过0.45μm滤膜过滤;
S4、除杂:对上清液进行超滤,滤膜包为2000Da,用来除掉未酶解完全的多糖大分子,将超滤完成的透过液倒入100Da透析袋,于2℃下透析40h,透析的目的是除去小分子糖类、盐和部分色素。透析完成后,于-50℃下冷冻干燥24h得到所述的黄水低聚糖,得率25.27%。
对比例1一种黄水低聚糖的制备方法
与实施例1相比,区别在于:步骤S2中α-淀粉酶和果胶酶的体积比为1:3,其它步骤同实施例1,得率18.77%。
对比例2一种黄水低聚糖的制备方法
与实施例1相比,区别在于:步骤S2中用α-淀粉酶替代果胶酶,其它步骤同实施例1,得率19.93%。
对比例3一种黄水低聚糖的制备方法
与实施例1相比,区别在于:步骤S2中用果胶酶替代α-淀粉酶,其它步骤同实施例1,得率11.24%。
效果例1黄水低聚糖的结构分析
(1)取实施例1得到的黄水低聚糖经DE-52离子交换柱和葡聚糖凝胶G-15纯化后,使用高效凝胶渗透色谱测定分子量,条件为:色谱柱为TSKgel GMPWXL水相凝胶色谱柱,流动相为0.1M NaNO3和0.06%NaN3水溶液,流速为0.6mL/min,柱温为35℃,以不同分子量的窄分布聚乙二醇(Mw 903000、580000、146000、44200、1000、600)为标准品,做出校正曲线,如图1所示;根据校正曲线求出样品的分子量为1828Da,如图2所示。
(2)取实施例1得到的黄水低聚糖经DE-52离子交换柱和葡聚糖凝胶G-15纯化后,使用高效液相色谱测定黄水低聚糖的单糖组成:精密称取3mg黄水低聚糖于10mL安瓿瓶中,加入3.0mL 2mol/L三氟乙酸(TFA),充氮,封管,120℃酸解4h。取出加入甲醇,氮吹挥干TFA,加3.0ml水复溶。精确吸取250μL样品溶液到5mL离心管中,加入250μL0.6 mol/L的NaOH,500μL 0.4mol/L的PMP-甲醇,70℃反应1h。冷水中冷却10min;加入500μL 0.3mol/L HCl中和,再加入1mL氯仿涡旋1min,3000r/min离心10min,小心取上清,萃取3次。取上清液,备用。精密称取甘露糖,核糖,鼠李糖,葡萄糖醛酸,半乳糖醛酸,N-乙酰氨基葡萄糖,葡萄糖,N-乙酰氨基半乳糖,半乳糖,木糖,阿拉伯糖和岩藻糖对照品适量,加水溶解稀释至每1ml中各含50μg的混合对照溶液,按照上述方法进行衍生。采用高效液相色谱(HPLC)测定单糖组成。HPLC条件:Xtimate C18 4.6*200mm 5μm色谱柱,柱温30℃,流速1.0mL/min,流动相为0.05M磷酸二氢钾溶液(用NaOH溶液调pH为6.70):乙腈=83:17,检测波长为250nm,进样量为20μL。
结果如图3所示,结果表明黄水低聚糖主要成分为葡萄糖(95.41%),另外含有少量葡萄糖醛酸(1.25%)、甘露糖(1.04%)、阿拉伯糖(0.87%)、半乳糖(0.57%)、木糖(0.52%)和核糖(0.34%)。
(3)黄水低聚糖的红外光谱分析:经DE-52离子交换柱和葡聚糖凝胶G-15纯化后的黄水低聚糖的傅里叶变换红外光谱,由Nicolet iS10 FT-IR光谱仪记录,扫描范围为4000至400cm-1,扫描32次。光谱仪分辨率为4cm-1,信噪比(S/N)为50000:1。测量前,用KBr粉末研磨3mg实施例1得到的黄水低聚糖,并根据KBr圆盘法压制成薄片之后上样。
结果如图4所示,3337cm-1附近的峰是多糖分子间或分子内部O-H的伸缩振动峰;在2932cm-1附近的峰是烷基的C-H伸缩振动吸收峰;在1638cm-1处是羰基的伸缩振动吸收峰,这三个峰是糖类物质的特征吸收峰。1363cm-1和1417cm-1是C-H的弯曲振动峰。在1151cm-1、1082cm-1和1024cm-1附近的3个吸收峰是吡喃环的伸缩振动峰,表明黄水低聚糖属于吡喃糖。848cm-1和762cm-1的吸收峰表明糖链中存在α-糖苷键。
效果例2黄水低聚糖对短链脂肪酸含量的影响
(1)粪便接种物的体外发酵:将四名成年人(两男两女,3个月内没有服用抗生素,无肠胃疾病)的粪便样本混匀后,均匀分散到无菌PBS缓冲液(pH为7.0)中,以得到10%(w/v)的粪便混悬液。将粪便混悬液在500×g离心5min,放到超净台中备用。培养基采用BHI培养基,胰蛋白胨6.0g,无水磷酸氢二钠0.6g,牛心浸出粉3g,氯化钠3g,葡萄糖1.2g。实验前将配置好的培养基加入不同底物后高温高压灭菌(121℃,20min),放到超净台冷却至室温,向已灭菌的50mL血清瓶中加入29mL培养基和1mL粪便混悬液,阴性对照无额外碳源,阳性对照额外碳源为100mg菊糖(购自上海麦克林生化科技有限公司,货号I811905)。各实验组培养基组成详见表1。吹扫恒定N2流至血清瓶中以排除氧气,然后迅速旋螺旋口。所有处理组均在37℃摇床中与粪便接种物一起孵育,使用厌氧产气袋使整个过程保持厌氧环境。发酵第0,6,12,24小时后取出样品进行分析。
表1各实验组发酵液配比表
(2)发酵液中短链脂肪酸含量的测定:使用气相色谱法对发酵液进行短链脂肪酸的测定。分别取不同时间的1.5mL发酵液于10000×g、4℃下离心5min,取400μL发酵液过0.45μm滤膜,加入20μL 2-乙基丁酸混匀,加入100μL 50%H2SO4酸化,涡旋(2000rpm/min)振荡后加入1000μL无水乙醚,再次涡旋(2000rpm/min)振荡10-15s后静置2min萃取。3000×g4℃冷冻离心5min,转移700μL上层有机相至1.5mL气相小瓶中。使用配备火焰离子化检测器(FID)的气相色谱仪检测生成的短链脂肪酸,使用DB-WAX检测柱。检测条件:初始柱温100℃,持续1min,以5℃/min的速度升温至180℃,保持2min,再以20℃/min速度升温至230℃,保持2min。FID检测器温度为250℃,进样口(注射器)温度250℃。上样量为1μL,分流比为5:1,载气为N2,,流速为2.5mL/min,尾吹气为N2,吹起流速为30mL/min;H2流量为40mL/min,空气流量为400mL/min。
体外发酵所产生的短链脂肪酸如图5-7所示,其中图5显示,黄水低聚糖组在发酵6h时的乙酸含量与除菊糖外的其他组无显著差异;而发酵12h时后,实施例1、2、3组的乙酸含量显著高于对比例1、2、3组和阴性对照组;尤其显著的是,实施例1中24h后的乙酸产量为10.47mM,与实施例2、3组无显著性差异,显著高于阴性对照组的6.36mM、菊糖组的9.70mM,以及对比例1、2、3组。
图6显示丙酸含量的变化,在发酵6h前各组丙酸含量无显著性差异;在发酵12h时后实施例1、2、3和发酵菊糖所产生的丙酸含量无显著性差异,但均显著高于对比例1、2、3组和阴性对照组;值得注意的是,发酵24h后,实施例1、2、3组丙酸含量显著高于对比例1、2、3组、阴性对照组和菊糖组,其中实施例1的含量最高,为10.91Mm。
如图7所示,发酵0-6h,各实验组中丁酸的含量并无明显差异,均低于菊糖组;12h后,实施例1、2、3组和菊糖组的丁酸含量开始显著增加,且实施例1、2、3组的丁酸含量显著高于对比例1、2、3组和阴性对照组;发酵24h后,实施例1组的的丁酸含量最高,达到了7.51mM,与实施例2、3组无显著性差异,但均显著高于阴性对照组、菊糖组和对比例1、2、3组。
短链脂肪酸是由肠道菌群发酵低聚糖、非淀粉性多糖、抗性淀粉或其他膳食纤维等产生的代谢产物,主要包括乙酸、丙酸和丁酸等,能够对肠道健康产生重要的影响。研究表明,短链脂肪酸在维持肠道健康、调节免疫系统、促进脂肪分解代谢和调节能量代谢等方面具有一定作用。本发明实施例制备的黄水低聚糖在发酵后24h后能够显著增加乙酸、丙酸和丁酸短链脂肪酸的产量,对维持肠道健康、促进脂肪分解代谢、制备减肥功能食品或保健品或药物具有优异效果。
以上是结合具体实施例对本发明进一步的描述,但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
Claims (10)
1.一种黄水低聚糖的制备方法,其特征在于,包括以下步骤:
S1、前处理:黄水醇沉后冻干,得固体黄水粗多糖;
S2、酶解:取固体黄水粗多糖溶于PBS缓冲液得黄水粗多糖溶液,加入复合酶进行酶解,得到酶解液;
S3、灭酶:对酶解液加热灭酶,冷却至室温后离心、过滤得上清液;
S4、除杂:对上清液超滤和透析进行除杂,冻干后得所述的黄水低聚糖。
2.根据权利要求1所述的制备方法,其特征在于,步骤S2所述复合酶包括α-淀粉酶和果胶酶;所述复合酶的添加量为5-15μL/10mL黄水粗多糖溶液。
3.根据权利要求2所述的制备方法,其特征在于,所述α-淀粉酶和果胶酶的体积比为1-5:1。
4.根据权利要求1所述的制备方法,其特征在于,步骤S2所述酶解的条件为:pH 5-7,温度40℃-55℃,时间8h-10h。
5.根据权利要求1所述的制备方法,其特征在于,步骤S3所述灭酶的温度为85℃-110℃。
6.根据权利要求1所述的制备方法,其特征在于,步骤S3所述离心的条件为:温度20℃-25℃,转速8000rpm-12000rpm,时间8min-12min。
7.根据权利要求1所述的制备方法,其特征在于,步骤S4所述超滤的滤膜包为2000Da-4000 Da;步骤S4所述透析的透析袋为100Da-400 Da;所述透析的条件为:温度2℃-8℃,时间40h-52h。
8.一种黄水低聚糖,其特征在于,由权利要求1-7任一项所述的制备方法制得。
9.根据权利要求8所述的黄水低聚糖,其特征在于,所述黄水低聚糖的分子量为1828Da;所述黄水低聚糖的组分包括葡萄糖;所述黄水低聚糖为α-构型的吡喃糖。
10.权利要求8-9任一项所述的黄水低聚糖或权利要求1-7任一项所述的制备方法制备得到的黄水低聚糖在制备减肥功能食品或保健品或药物中的应用。
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