CN114403428A - 一种仙草多糖凝胶球及其制备方法和应用 - Google Patents
一种仙草多糖凝胶球及其制备方法和应用 Download PDFInfo
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- CN114403428A CN114403428A CN202210308560.8A CN202210308560A CN114403428A CN 114403428 A CN114403428 A CN 114403428A CN 202210308560 A CN202210308560 A CN 202210308560A CN 114403428 A CN114403428 A CN 114403428A
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Abstract
本发明提供一种仙草多糖凝胶球及其制备方法和应用,仙草多糖形成的圆形凝胶球具有良好的耐胃酸能力,有效保护活性成分在胃酸中的稳定性,且在肠道中能实现活性成分的肠靶向释放,有效提高益生菌或功能性成分的利用率,帮助益生菌黏附于肠黏液并在肠道中长期定植,提高其他活性成分的肠道释放和吸收能力。
Description
技术领域
本发明涉及食品生物技术领域。具体地,本发明涉及仙草多糖凝胶球及其制备方法和应用。
背景技术
仙草又名仙人草、凉粉草,学名为MesonachinensisBenth,英文名为MesonaBlume,产于中国台湾地区,福建,浙江,江西,广东,广西等地。它是一种富含仙草多糖(仙草胶)和黄酮类物质的药食两用植物,是生产天然、营养、保健食品的理想原料,具有广泛的加工利用价值。仙草性味涩、甘、寒,具清暑解渴、凉血解暑之功效,可治中暑、高血压、肾脏病和糖尿病等。以仙草为原料加工的食品被报道具有一定的抗氧化特性,其中仙草保健茶具有清除超氧阴离子的功效,仙草蜜具有清除羟自由基和超氧阴离子自由基的作用。仙草多糖是天然益生元,无毒无害且具有多种健康功效,价格低廉,具有良好的抗脂质过氧化作用。目前仙草制品的应用主要有仙草冻、仙草奶茶、凝固型仙草酸奶、保健仙草蜜、仙草发酵酒饮料、仙草复合保健饮料、仙草黑木耳果冻粉等。
传统微胶囊多采用化学聚合物做壁材,摄入后不但对人体有一定毒性而且耐胃酸能力差,不易实现生物活性成分的肠道靶向释放。益生菌、花色苷、姜黄素和溶菌酶等生物活性成分具有多种健康功效,但由于具有耐胃酸能力差、溶解性低、容易变性失活或结构不稳定等缺点,口服后无法有效到达肠道或被人体吸收利用。包埋壁材能有效提高活性成分的水溶性,保护活性成分在胃液中的稳定性。但是目前包埋壁材的种类主要有海藻酸钠、壳聚糖、β-环糊精、改性淀粉等,种类较少且不适用于多种不同特性的生物活性成分的包埋,且化学改性后的壁材原料安全性也有待进一步验证。
发明人实验室研究了氧化魔芋微球(专利文献公开号为CN 110856714 B),介绍了具有巯基修饰的氧化魔芋葡甘露聚糖经反相乳液法与铁离子交联形成益生菌或蛋白包埋微球载体,能提高包埋物的胃液稳定性和在肠道内的粘附性能,使包埋于微球载体上的功能因子具有较高的生物利用率。但其采用化学修饰多糖为原料,在安全性有待提高,不能直接用于食品中。
现有技术中关于仙草多糖的专利文献主要集中宏观凝胶或仙草冻,如公开号为CN110859301A,名称为“一种凉粉草多糖-大豆分离蛋白复合凝胶的制备方法”将仙草多糖和大豆分离蛋白混合,在钠离子或钙离子溶液促进剂的作用下,以及加热过程中蛋白质的变性,聚集,然后交联形成宏观凝胶能作为包埋凝胶使用。公开号为CN 111357817 A,名称为“一种活性养生酸奶仙草冻及其制备方法”将仙草粉与酸奶复配获得具有抗氧化效果的酸奶仙草冻产品,但仙草多糖-蛋白复合凝胶或仙草冻没有实现对益生菌或包埋成分的胃酸稳定性提高和肠靶向释放效果。此外,关于仙草多糖的专利文献还集中在浸提仙草多糖,例如CN 113321750 A一种低钠离子含量的仙草胶及其制备方法介绍了一种超声法提取含低钠的仙草多糖胶的方法,公开号为CN112194734A,名称为“一种仙草胶及其提取方法”的发明专利是采用碱液浸提仙草多糖。
基于此,现需要一种食用安全,且具有良好的耐胃酸能力,能有效保护活性成分在胃酸中的稳定性,且在肠道中能实现活性成分的肠靶向释放;有效提高益生菌存活率,帮助益生菌黏附于肠黏液并在肠道中长期定植;提高其他活性成分的肠道释放和吸收能力的凝胶载体。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题。
本发明的一个方面是提供了一种仙草多糖凝胶球及其制备方法,在充分发挥了仙草多糖的健康功效基础上,不但能保护多种不同类型的被包埋活性成分不受胃酸影响,还能起到肠道靶向释放和富集活性成分的作用,该仙草多糖凝胶球具有应用性广、绿色安全等优点。
本发明的仙草多糖原料是天然益生元,无毒无害且具有多种健康功效,价格低廉,能与钙、铁、锌、镁、铝等多种不同价态金属离子交联采用反相乳液法或反相复合乳液法制备凝胶球用于包埋不同特性的生物活性成分;本发明的仙草多糖凝胶球具有提高活性成分耐胃酸能力、肠道靶向释放能力等优点。
具体的,本发明提供的仙草多糖形成的圆形凝胶球,具有良好的耐胃酸能力,有效保护活性成分在胃酸中的稳定性,且在肠道中能实现活性成分的肠靶向释放,有效提高益生菌存活率,帮助益生菌黏附于肠黏液并在肠道中长期定植,提高其他活性成分的肠道释放和吸收能力。
仙草多糖是杂多糖,主要成分包括半乳糖、葡萄糖、鼠李糖、半乳糖醛酸、葡萄糖醛酸、木糖、阿拉伯糖,形成凝胶的主要原因是糖醛酸的羧基与金属离子非共价交联形成凝胶,主要依靠葡萄糖醛酸和半乳糖醛酸参与了交联。
根据本发明的实施例,仙草多糖制备出纳米和微米级凝胶球结构,尺度100 nm~500 μm之间,凝胶球大小可控,大小可通过控制仙草多糖浓度、搅拌转速、乳化工艺、连续相组成等条件来获得。
本发明的一个方面,提供一种仙草多糖凝胶微球,使用金属离子与仙草多糖中的糖醛酸的羧基交联形成凝胶制备获得。
所述糖醛酸为葡萄糖醛酸和半乳糖醛酸,
任选的,所述金属离子Mn2+、Zn2+、Ba2+、Fe3+、Fe2+、Ca2+、Ni2+、Cu2+、Al3+;优选的,金属离子为Fe或Ca。
上述微球呈球状或大颗粒爆珠,
任选地,所述微球载体中铁离子含量为0.2-0.8 mmol/g;
任选地,所述微球载体的粒径为1-1000μm。
本发明的一个方面,提供一种制备仙草多糖凝胶微球的制备方法,包括以下步骤:
将仙草多糖与金属离子分别溶于水中,待多糖完全溶解并与金属离子混合制备水相溶液;
将水相溶液加入油相溶液中搅拌,充分交联;
将油相与水相分离,清洗,将凝胶微球复溶于水中。
上述制备方法中,其中金属离子为Fe或Ca,任选地,仙草多糖溶解于pH为2-7的水中;优选的,pH为2.5-5.5,更优选的,pH为3-5,更优选的,pH为2.5、3、3.5、4、4.5、5;
任选地,仙草多糖与水的质量比为1:(5-50);优选的,质量比为1:(10-40)或1:(20-30);
任选地,金属离子与仙草多糖的质量比为1:(1-100);优选的,质量比为1:(5-50),更优选的,质量比为1:(10-25);
任选地,油相溶液可选用Span80和石蜡;
任选地,搅拌为磁力搅拌,磁力搅拌时间为0.5-2h;
任选地,清洗可选用正己烷和甲醇,清洗次数为2-5次。
仙草多糖凝胶微球中还可加入淀粉,所述淀粉与仙草多糖质量比为(0-1):3;
任选地,淀粉含量不超过仙草多糖总质量的25%。
本发明的另一个方面,提供一种包埋物,包括:
上述仙草多糖凝胶微球;以及功能因子,功能因子包埋于仙草多糖凝胶微球中。
功能因子的包埋率为60-99 %;
任选地,所述功能因子为水溶性小分子;优选的,水溶性小分子为花色苷、维生素C、烟酰胺、维生素B族、儿茶素之一或组合;
任选地,所述功能因子为脂溶性小分子;优选的,脂溶性小分子为姜黄素、辣椒素、脂溶性维生素、β-胡萝卜素之一或组合;
任选地,所述功能因子为蛋白类大分子;优选的,蛋白类大分子为溶菌酶、过氧化物酶、乳铁蛋白和免疫球蛋白;
任选地,所述微生物为益生菌,优选好氧、厌氧或兼性厌氧益生菌;更优选的,包括乳酸乳球菌、保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、干酪乳杆菌、双歧杆菌、酵母菌、枯草芽孢杆菌和酪酸梭菌的至少之一。
本发明的另一方面,提供一种仙草多糖凝胶球,使用金属离子与仙草多糖中的糖醛酸的羧基交联形成凝胶制备获得;任选地,金属离子为Fe或Ca;任选地,凝胶球粒径为1.0-5.0 cm;任选地,凝胶球为爆珠。
本发明的另一方面,提供上述仙草多糖凝胶球或微球在稳定添加到食品体系中的应用,
任选地,食品体系为固体或液体食品;优选的,食品为饮料、酸奶、菌粉、奶酪。
本发明的有益效果包括:
(1)首次利用仙草多糖制备出纳米和微米级凝胶球结构,尺度100 nm~500 μm之间,凝胶球大小可控,大小可通过控制仙草多糖浓度、搅拌转速、乳化工艺、连续相组成等条件来获得。
(2)首次发明了采用Mn2+、Zn2+、Ba2+、Fe3+、Fe2+、Ca2+、Ni2+、Cu2+、Al3+等二价或三价金属离子作为交联剂形成多糖与金属离子交联的凝胶结构,不同离子的多糖与交联剂比例范围为(20 ~5):1。
(3)制备的多糖微球具有空心、实心等多种结构,分别具有抵抗胃酸,增加包埋量,能根据需求选用不同结构的微球。
(4)该凝胶球能够包埋好氧、厌氧或兼性厌氧益生菌,包埋率为50%~85%,包埋稳定性高,显著提高益生菌的耐胃酸能力,与未包埋菌相比,包埋益生菌在胃肠道内的存活率能提高50%~90%。
(5)该凝胶球能够包埋水溶性小分子花色苷、维生素C、烟酰胺、维生素B族、儿茶素等,包埋率为85%~99%,包埋稳定性高,包埋后水溶性小分子的耐胃酸能力达60%~80%,同时显著提高小肠吸收能力,帮助其发挥生物功效。
(6)该凝胶球能够包埋脂溶性小分子姜黄素、辣椒素、脂溶性维生素、β-胡萝卜素等,包埋率为80%~95%,包埋稳定性高,显著提高脂溶性小分子的肠道消化吸收能力,帮助其发挥生物功效。
(7)该凝胶球能够包埋蛋白类大分子溶菌酶、过氧化物酶、乳铁蛋白和免疫球蛋白等,包埋率为80%~98%,包埋稳定性高,显著提高蛋白类大分子的耐胃酸能力和小肠吸收能力,帮助其发挥生物功效。
(8)该凝胶球能够包埋水溶性小分子花色苷等成分能显著提高花色苷的抗氧化能力,与游离花色苷相比,凝胶球包埋花色苷的DPPH清除能力和羟自由基清除能力提高15%~50%,仙草多糖凝胶球对功能性成分的功效发挥具有协同促进作用。
(9)仙草凝胶球还能稳定添加到食品体系如饮料、酸奶、菌粉、奶酪等多种固体、液体类型的食品中,作为一种高营养的载体增加食品体系的功能性成分含量和稳定性,促进人体对添加功能性成分的吸收和利用。
(10)仙草是一种天然中药材也是一种天然食材,具有清热解毒、健脾补气、美容养颜和提高机体免疫力等多种功效,且无毒无害,仙草凝胶球还能作为药物载体制备成药物制剂,荷载水溶性、脂溶性或蛋白类药物,提高药物稳定性,增加药物肠道吸收能力,促进被荷载药物的健康功效发挥。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1仙草多糖单糖组成HPLC图
图2仙草多糖微球的能谱扫描
图3仙草多糖和仙草多糖凝胶球的傅里叶红外光谱扫描图
图4仙草多糖微球包埋不同活性成分的光学显微镜图。图4A为仙草多糖凝胶球;图4B为仙草多糖凝胶球包埋益生菌WCFS01;图4C为仙草多糖凝胶球包埋亲水活性成分花色苷;图4D为仙草多糖凝胶球包埋脂溶性活性成分姜黄素;图4E为仙草多糖凝胶球包埋蛋白类活性成分溶菌酶
图5仙草多糖凝胶球包埋自发荧光益生菌NZ9000的共聚焦显微图。图5A为仙草多糖凝胶球包埋自发荧光益生菌NZ9000在荧光通道的结果;图5B 为仙草多糖凝胶球包埋自发荧光益生菌NZ9000在明场的结果;图5C为荧光通道和明场结果的叠加图
图6仙草多糖凝胶球包埋不同活性成分的扫描电子显微结构图。图6A为仙草多糖原料;图6B为仙草多糖凝胶球;图6C为仙草多糖凝胶球包埋益生菌WCFS01;图6D为仙草多糖凝胶球包埋亲水活性成分花色苷;图6E为仙草多糖凝胶球包埋脂溶性活性成分姜黄素;图6F为仙草多糖凝胶球包埋蛋白类活性成分溶菌酶
图7不同结构多糖凝胶球的扫描电子显微结构图。图7A为空心多糖凝胶球;图7B为实心多糖凝胶球
图8模拟人工胃肠液对包埋自发荧光益生菌NZ9000的仙草多糖凝胶球消化的共聚焦显微图。图8A为消化前的包埋益生菌NZ9000的仙草多糖凝胶球;图8B为模拟人工胃液消化60 min的凝胶球;图8C为模拟人工肠液消化2 min的凝胶球
图9 铁离子交联的氧化魔芋多糖凝胶球在人工模拟胃肠液中对益生菌的释放效果
图10 钙离子交联的仙草多糖凝胶球在人工模拟胃肠液中对益生菌的释放效果
图11铁离子交联的添加淀粉的仙草多糖凝胶球在人工模拟胃肠液中对益生菌的释放效果
图12 包埋花色苷的钙离子交联仙草多糖微球在模拟胃肠液中的稳定性
图13 包埋花色苷的铁离子交联淀粉仙草多糖微球在模拟胃肠液中的稳定性
图14铁离子交联添加淀粉的仙草多糖微球制备的宏观爆珠凝胶球。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 高效液相色谱测定仙草多糖组成
具体步骤如下:
(1)待测仙草多糖样品上样溶液配制:
仙草多糖购自武汉远成共创科技有限公司(CAS:542-40-5,标准:企标,级别:食品级,含量:99%),待测仙草多糖样品水解:精密称取仙草多糖样品10 mg至10ml安培瓶中,加入2.0mL 2 mol/L 三氟乙酸于5 mL安瓿中,封管,110℃酸解8h,取出挥干三氟乙酸,加2.0ml水复溶。
样品溶液衍生:精确吸取250uL样品溶液到5mLEP管中,加入250 uL 0.6 mol/LNaOH,500 uL 0.4mol/L1-苯基-3-甲基-5-吡唑啉酮-甲醇,70℃反应1h。冷水中冷却10min;加入500 uL 0.3mol/LHCl中和,再加入1 mL 氯仿漩涡1min,3000r/min离心10min,小心取上清,萃取3次,取上清液即为样品衍生溶液。
(2)单糖标准品对照溶液配制:
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,岩藻糖对照品5 mg,加水溶解稀释至每1ml中各含50 ug的混合对照溶液。
单糖标准品衍生步骤:精确吸取250uL混合对照溶液到5mLEP管中,加入250 uL0.6mol/LNaOH,500 uL 0.4mol/L1-苯基-3-甲基-5-吡唑啉酮-甲醇,70℃反应1h。冷水中冷却10min;加入500 uL 0.3mol/LHCl中和,再加入1 mL 氯仿漩涡1min,3000r/min离心10min,小心取上清,萃取3次。上清用于HPLC。
检测仪器:岛津LC-20AD;色谱柱:Xtimate C18 4.6*200mm 5um;检测条件为:柱温30℃、流速1.0ml/min、检测波长250nm、进样量20ul、流动相0.05M PH 6.70磷酸二氢钾溶液:乙腈=83:17。
表1仙草多糖单糖组成含量表
结果如图1和表1所示,仙草多糖主要由半乳糖、葡萄糖、阿拉伯糖、半乳糖醛酸木糖、鼠李糖、甘露糖、核糖、岩藻糖和葡萄糖醛酸组成,其中半乳糖、葡萄糖、阿拉伯糖含量较高分别为32.58%、20.18%和17.59%。所含的糖醛酸主要为半乳糖醛酸和葡萄糖醛酸,含量分别为11.43%和0.85%。
实施例2 反相乳液法制备仙草微凝胶球
准确称量50 mg仙草多糖,溶于0.5mLpH3的水,涡旋至多糖完全溶解;准确称量5mg氯化铁金属离子。将仙草多糖溶液与金属离子混合。
称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的多糖和铁的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的蒸馏水中。
将仙草微凝胶球用扫描电子显微镜SEM进行能谱扫描,由图2所示,仙草多糖可通过氯化铁中的Fe离子交联形成微球结构。
进一步,采用傅里叶红外光谱FT-IR鉴定了多糖的负电荷基团,图3中,位于1401cm-1和1619 cm-1羧基的两个特征吸收峰(对称和非对称吸收峰)证实了仙草多糖的负电荷基团为羧基。经铁离子交联形成水凝胶后羧基特征峰会红移至1408 cm-1和1627 cm-1。羧基的对称和非对称峰之间的峰间距(Δνas-s=νas(COO−) −νs(COO−))代表了羧基参与的配位类型。Δνas-s≈200 cm-1则代表了桥连配位,而Δνas-s>>200cm-1则代表了单齿配位,微球的FT-IR光谱表明仙草多糖中糖醛酸上的羧基与铁离子的结合为稳固的双齿配位模式。
实施例3包埋益生菌的凝胶微球制备
将0.5 mL6×1010 cfu/mL的益生菌3000 rpm,4℃离心10 min,采用生理盐水洗菌2次,复溶至0.5 mL生理盐水中。将50 mg 仙草多糖,溶于0.5 mLpH3的水中,涡旋至多糖完全溶解;准确称量5 mg氯化铁,将仙草多糖溶液、菌液与金属离子充分混合后加入含有40 g液体石蜡和1 g Span80的50 mL烧杯中,磁力搅拌1 h使多糖与金属离子充分交联,1 h后1000rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将包埋益生菌的微凝胶球复溶至1 mLpH3的蒸馏水中。
实施例4包埋花色苷、姜黄素的凝胶微球制备
将100 mg花色苷、姜黄素分别溶于0.5 mLpH3的水中,将50 mg 仙草多糖溶于0.5mLpH3的水中,涡旋至多糖和活性成分完全溶解;准确称量5 mg氯化铁,将仙草多糖溶液、菌液与金属离子充分混合后加入含有40 g液体石蜡和1 g Span80的50 mL烧杯中,磁力搅拌1h使多糖与金属离子充分交联,1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将包埋花色苷或姜黄素的微凝胶球复溶至1 mLpH3的蒸馏水中。
实施例5包埋溶菌酶的凝胶微球制备
将100 mg溶菌酶溶于0.5 mLpH3的水中,将50 mg 仙草多糖溶于0.5 mLpH3的水中,涡旋至多糖和活性成分完全溶解;准确称量5 mg氯化铁,将仙草多糖溶液、菌液与金属离子充分混合后加入含有40 g液体石蜡和1 g Span80的50 mL烧杯中,磁力搅拌1 h使多糖与金属离子充分交联,1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将包埋溶菌酶的微凝胶球复溶至1 mLpH3的蒸馏水中。
实施例6 仙草多糖微球的结构表征
通过光学显微镜、共聚焦显微镜和扫描电镜对仙草多糖凝胶球的结构进行表征。
如图4-7所示,光学显微结果和扫描电镜结果表明采用乳化法能将不规则的仙草多糖原料通过离子交联制备成规则的凝胶球,形成的凝胶球表面光滑,颗粒大小分布均匀,粒径尺寸约20μm,仙草凝胶球能有效将益生菌、水溶性功能性小分子、脂溶性功能性小分子和蛋白类功能性大分子包埋进入凝胶球内部,能作为不同类型的功能性成分的传递载体。
实施例7包埋率测定
将不同浓度的益生菌WCFS01、花色苷、姜黄素、溶菌酶,按前述实施例方法进行包埋,检测其负载量。
测定步骤:将包埋花色苷、姜黄素或溶菌酶的仙草多糖凝胶球冻干后取10 mg复溶至pH7的水溶液中孵育1h破球,采用酶标仪分别在520 nm、425 nm和280 nm波长下测量花色苷、姜黄素或溶菌酶的含量(采用花色苷、姜黄素或溶菌酶标准品做标曲,以等浓度空白微球载体做空白对照)。
将包埋益生菌的仙草多糖凝胶球冻干后取10 mg复溶至pH7的生理盐水溶液中孵育1h破球,采用稀释平板涂布法测定包埋的益生菌数量。
表2仙草多糖凝胶球对不同活性成分的包埋量
经铁离子交联的仙草多糖凝胶球对益生菌的包埋率达50~95%,对水溶功能性小分子花色苷的包埋量为0.90~1.30 mg/mg凝胶球,对脂溶功能性小分子姜黄素的包埋量为0.06~0.10 mg/mg凝胶球,对蛋白类功能性大分子溶菌酶的包埋量为1.20~2.0 mg/mg凝胶球。
结果表明,益生菌、花色苷、姜黄素或溶菌酶等具有不同特性的活性成分能被包埋入仙草多糖凝胶球内。仙草多糖凝胶的包埋量大,稳定性好,适合包埋多种不同功能性成分。
实施例8 凝胶球消化液释放检测
模拟胃肠液释放测定步骤:
1)模拟人工胃肠液液配制:(a)模拟胃液:准确称量3.2 g胃蛋白酶,2 g氯化钠溶于1L超纯水中,用稀盐酸调节胃液pH为1.2。(b)模拟肠液:准确称量10 g胰蛋白酶,6.8 g磷酸二氢钾溶于1L超纯水中,用0.5 M氢氧化钠调节模拟肠液pH为6.8。
2)将包埋益生菌、或花色苷、或姜黄素、或溶菌酶的仙草多糖凝胶球冻干后分别溶于50 mL模拟胃液或模拟肠液中,消化一定时间后取1mL消化液13000 rpm离心5min,采用酶标仪测定上清液中释放的花色苷、或姜黄素、或溶菌酶的含量。采用共聚焦显微镜表征自发荧光的益生菌的释放情况。
表3模拟人工胃液中仙草多糖凝胶球对包埋活性成分的释放率
表4模拟人工肠液中仙草多糖凝胶球对包埋活性成分的释放率
表5模拟人工胃液和肠液消化后仙草多糖凝胶球对益生菌的释放率
铁离子交联仙草多糖凝胶球能实现对益生菌、水溶功能性小分子花色苷、脂溶功能性小分子姜黄素、蛋白类功能性大分子溶菌酶的耐胃酸保护和肠道靶向释放。如图8A-8C和表5,将包埋益生菌在胃肠道内的存活率能提高至68.25±1.78%。包埋益生菌在模拟胃液中仅释放5%~15%,在模拟肠液中释放85%~95%。同时能显著提高肠道黏附定植能力,调控肠道菌群平衡,提高益生菌比例,抑制有害菌存活。表3和表4中,包埋的花色苷、姜黄素和溶菌酶在模拟胃液中仅释放低于30%,超过70%在肠液中释放。
由上述实验证明,仙草多糖微球在模拟胃液中稳定,对活性成分的释放较少,在人工模拟肠液中迅速破裂,能将益生菌、花色苷、姜黄素或溶菌酶等活性成分靶向释放入肠道。
实施例9 包埋花色苷的仙草多糖凝胶球的抗氧化性
1. DPPH清除能力测定
准确称量0.008、0.015、0.03、0.06、0.12、0.24、0.48 mg的游离花色苷分别溶解于pH3的水配成0.008~0.48 mg/mL的游离花色苷溶液,命名为“花色苷组”;
按照前述实施例4的仙草多糖凝胶球制备步骤分别制备出花色苷包埋终浓度为0.008、0.015、0.03、0.06、0.12、0.24、0.48 mg/mL的花色苷仙草多糖凝胶球,命名为“包埋组”;
再按照实施例2中的步骤分别制备出不含花色苷但仙草多糖凝胶球浓度与包埋花色苷仙草多糖凝胶球一致的空载仙草多糖凝胶球溶液,命名为“凝胶球载体组”;
如下表,按表6的配比将上述溶液与0.1 mM的DPPH乙醇溶液混合,避光静置30min,在528 nm波长处分别读取样品Am、对照A0、空白An的吸光值,按照下式计算凝胶球载体组、花色苷、包埋组的DPPH清除能力。
表6DPPH清除能力实验不同组所加试剂配比表
表7包埋花色苷的仙草多糖凝胶球对DPPH自由基的清除能力
2. 羟自由基清除能力
准确称量0.06、0.12、0.24、0.47、0.94 mg的游离花色苷分别溶解于1mL pH3的水配成0.06~0948 mg/mL的游离花色苷溶液,命名为“花色苷组”;
按照前述实施例4的步骤分别制备出花色苷包埋终浓度为0.06、0.12、0.24、0.47、0.94 mg/mL的花色苷仙草多糖凝胶球,命名为“包埋组”;
再按照前述实施例2中的步骤分别制备出不含花色苷但仙草多糖凝胶球浓度与包埋花色苷仙草多糖凝胶球一致的空载仙草多糖凝胶球溶液,命名为“包埋组”;
按照表8的配比将上述溶液与9 mM的硫酸亚铁溶液,9 mM的水杨酸-乙醇溶液,8.8mM的过氧化氢溶液混合,避光静置30 min,在510 nm波长处分别读取样品Ax、对照A0、空白Ax0的吸光值,按照下式计算凝胶球载体组、花色苷、包埋组的羟自由基清除率。
表8羟自由基清除能力实验不同组所加试剂配比表
表9包埋花色苷的仙草多糖微球对羟自由基的清除能力
由表7和表9结果可知,仙草多糖凝胶球包埋水溶功能性小分子花色苷后能将花色苷的DPPH清除能力和羟自由基清除能力提高15%~50%。能有效促进被包埋物发挥健康功效。
对比例1铁离子交联氧化魔芋多糖微球对益生菌的包埋制备
1、准确称量50 mg氧化魔芋多糖,溶于0.5mLpH3的水,涡旋至多糖完全溶解;准确称量5 mg氯化铁;将0.5 mL6×1010cfu/mL的益生菌3000 rpm,4℃离心10 min,采用生理盐水洗菌2次,复溶至0.5 mL生理盐水中;将氧化魔芋多糖溶液、铁离子与益生菌混合均匀。
2、称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的氧化魔芋多糖、铁和益生菌的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
3、1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的生理盐水中。
4、同上文实施例7,采用平板涂布法对铁离子交联的氧化魔芋多糖微球包埋益生菌进行表征,说明益生菌能被包埋入铁离子交联的氧化魔芋葡甘露聚糖凝胶球内。
通过共聚焦显微镜对氧化魔芋多糖微球在人工模拟胃肠液中对益生菌的释放进行了表征。发现了铁离子交联氧化魔芋多糖微球在模拟胃液中稳定,对活性成分的释放较少,在人工模拟肠液中前1h微球相对更稳定,极少会出现破裂,1h模拟肠液消化后,氧化魔芋多糖微球会逐渐破裂,能将益生菌缓慢释放入肠道。
将包埋益生菌的氧化魔芋多糖凝胶球冻干后取10 mg复溶至pH7的生理盐水溶液中孵育1h破球,采用稀释平板涂布法测定包埋的益生菌数量。
模拟胃肠液释放测定步骤同前述实施例8
表10氧化魔芋多糖凝胶球对不同活性成分的包埋量
经铁离子交联的添加淀粉的仙草多糖凝胶球对益生菌的包埋率达60.3±7.2%,人工模拟胃肠液消化后益生菌存活率达45.4±2.62%,显著高于未包埋益生菌组,但也显著低于氯化铁交联仙草多糖为微球(见表10和表5)。魔芋多糖凝胶的在胃液中稳定性好,能有效保护益生菌不受胃酸影响,但在肠液中释放益生菌缓慢,释放速度显著低于仙草多糖微球(如图9)。
实施例10钙离子交联仙草微球对益生菌的包埋制备
1、准确称量50 mg仙草多糖,溶于0.5mLpH3的水,涡旋至多糖完全溶解;准确称量5mg氯化钙;将0.5 mL6×1010cfu/mL的益生菌3000 rpm,4℃离心10 min,采用生理盐水洗菌2次,复溶至0.5 mL生理盐水中;将仙草多糖溶液、钙离子与益生菌混合均匀。
2、称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的多糖、钙和益生菌的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
3、1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的生理盐水中。
4、按照实施例7的方法,采用平板涂布法对钙离子交联仙草微球包埋益生菌进行表征,说明益生菌能被包埋入钙离子交联的仙草多糖凝胶球内。
通过共聚焦显微镜对仙草多糖微球在人工模拟胃肠液中对益生菌的释放进行了表征。发现了钙离子交联仙草多糖微球在模拟胃液中稳定,对活性成分的释放较少,在人工模拟肠液中迅速破裂,能将益生菌靶向释放入肠道。
将包埋益生菌的仙草多糖凝胶球冻干后取10 mg复溶至pH7的生理盐水溶液中孵育1h破球,采用稀释平板涂布法测定包埋的益生菌数量。
模拟胃肠液释放测定步骤同实施例8。
表11仙草多糖凝胶球对不同活性成分的包埋量
经钙离子交联的仙草多糖凝胶球对益生菌的包埋率达86.88±2.31%,人工模拟胃肠液消化后益生菌存活率达66.44± 2.25%,显著高于未包埋益生菌组(见表11)。仙草多糖凝胶的包埋量大,在胃液中稳定性好,能有效保护益生菌不受胃酸影响,在肠液中能迅速释放益生菌(如图10)。
实施例11 钙离子交联仙草多糖凝胶球对花色苷的包埋
1、准确称量50 mg仙草多糖和100mg花色苷,溶于0.5 mLpH3的水,涡旋至多糖完全溶解;准确称量5 mg氯化钙;将仙草多糖溶液、花色苷和钙离子混合均匀。
2、称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的多糖和钙离子的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
3、1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的水中。
4、通过酶标仪检测(520 nm)和激光共聚焦显微镜对钙交联仙草多糖微球的包埋率和模拟人工胃肠液释放率进行表征。
表12模拟胃液消化时钙离子交联仙草多糖微球对花色苷的释放率
表13模拟肠液消化时钙离子交联仙草多糖微球对花色苷的释放率
如图12和表12、13所示,通过激光共聚焦显微镜和酶标仪检测对钙交联仙草多糖微球的包埋效果和模拟人工胃肠液释放效果进行表征,发现了钙交联仙草多糖微球在模拟胃液中稳定,对花色苷的释放较少,在人工模拟肠液中迅速破裂,能将花色苷靶向释放入肠道。
实施例12铁离子交联添加淀粉的仙草微球对益生菌的包埋制备
1、准确称量12.5mg淀粉、37.5 mg仙草多糖,溶于0.5mLpH3的水,涡旋至多糖完全溶解;准确称量5 mg氯化铁;将0.5 mL6×1010cfu/mL的益生菌3000 rpm,4℃离心10 min,采用生理盐水洗菌2次,复溶至0.5 mL生理盐水中;将仙草多糖溶液、淀粉溶液、铁离子与益生菌混合均匀。
2、称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的多糖、钙和益生菌的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
3、1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的生理盐水中。
4、按照实施例7,采用平板涂布法对铁离子交联的淀粉仙草微球包埋益生菌进行表征,说明益生菌能被包埋入铁离子交联的淀粉仙草多糖凝胶球内。
通过共聚焦显微镜对淀粉仙草多糖微球在人工模拟胃肠液中对益生菌的释放进行了表征。发现了铁离子交联淀粉仙草多糖微球在模拟胃液中稳定,对活性成分的释放较少,在人工模拟肠液中迅速破裂,能将益生菌靶向释放入肠道。
将包埋益生菌的淀粉仙草多糖凝胶球冻干后取10 mg复溶至pH7的生理盐水溶液中孵育1h破球,采用稀释平板涂布法测定包埋的益生菌数量。
模拟胃肠液释放测定步骤同实施例8。
表14掺淀粉的仙草多糖凝胶球对不同活性成分的包埋量
经铁离子交联的添加淀粉的仙草多糖凝胶球对益生菌的包埋率达90.43±1.7%,人工模拟胃肠液消化后益生菌存活率达66.58± 2.69%,显著高于未包埋益生菌组(见表14)。仙草多糖凝胶的包埋量大,在胃液中稳定性好,能有效保护益生菌不受胃酸影响,在肠液中能迅速释放益生菌(如图11)。
实施例13铁离子交联添加淀粉的仙草多糖凝胶球对花色苷的包埋
1、准确称量12.5mg淀粉、37.5 mg仙草多糖,溶于0.5mLpH3的水,涡旋至多糖完全溶解;准确称量5 mg氯化铁;准确称量100mg花色苷;将仙草多糖溶液、淀粉、花色苷和铁离子混合均匀。
2、称量40 g液体石蜡和1 g Span80于50 mL烧杯中,加入完全混合均匀的多糖和钙离子的混合液,磁力搅拌1 h使多糖与金属离子充分交联。
3、1 h后1000 rpm离心2 min,将油相和水相分离,弃去上层油相,下层水相用正己烷和甲醇各洗微球3次,最后将微凝胶球复溶至1 mLpH3的水中。
4、通过酶标仪检测(520 nm)和激光共聚焦显微镜对铁交联的添加淀粉的仙草多糖微球的包埋率和模拟人工胃肠液释放率进行表征。
表15模拟胃液消化时铁离子交联淀粉仙草多糖微球对花色苷的释放率
表16模拟肠液消化时铁离子交联淀粉仙草多糖微球对花色苷的释放率
如图13和表15、16所示,通过激光共聚焦显微镜和酶标仪检测铁交联淀粉仙草多糖微球对花色苷的包埋效果和模拟人工胃肠液释放效果进行表征,发现了铁交联添加淀粉的仙草多糖微球在模拟胃液中稳定,对花色苷的释放较少,在人工模拟肠液中迅速破裂,能将花色苷靶向释放入肠道。
实施例14钙离子/铁离子交联添加淀粉的仙草多糖凝胶宏观爆珠制备
准确称量1.25 g淀粉、3.75 g仙草多糖,溶于5 mLpH3的水,涡旋至多糖完全溶解,准确称量10 g三氯化铁金属离子溶于100 mL蒸馏水;将淀粉仙草多糖溶液逐滴低加入三氯化铁溶液中,50 rpm磁力搅拌10 min使多糖与金属离子充分交联形成宏观爆珠凝胶球,将宏观爆珠凝胶球捞出后用pH3的水冲洗后拍照。
钙离子交联的仙草多糖凝胶球与上述制备步骤相同,仅将三氯化铁金属离子替换为氯化钙金属离子。
如图14所示,铁离子交联添加淀粉的仙草多糖微球能制备成直径0.5±0.32cm的宏观爆珠凝胶球,爆珠凝胶球颗粒大小均一,具有凝胶弹性,能应用于饮料、载体等食品或药品领域。钙离子交联的仙草多糖凝胶球与铁离子交联微球大小相近,性状相似。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种仙草多糖凝胶微球,其特征在于,使用金属离子与仙草多糖中的糖醛酸的羧基交联形成凝胶制备获得。
2.根据权利要求1所述的仙草多糖凝胶微球,其特征在于,所述糖醛酸为葡萄糖醛酸和半乳糖醛酸,金属离子为Fe或Ca。
3.根据权利要求1或2所述的仙草多糖凝胶微球,其特征在于,所述微球呈球状或大颗粒爆珠,微球载体中铁离子含量为0.2-0.8 mmol/g;微球载体的粒径为1-1000μm。
4.一种制备如权利要求1-3任一项所述仙草多糖凝胶微球的制备方法,其特征在于,包括以下步骤:
将仙草多糖与金属离子分别溶于水中,待多糖完全溶解并与金属离子混合制备水相溶液;
将水相溶液加入油相溶液中搅拌,充分交联;
将油相与水相分离,清洗,将凝胶微球复溶于水中。
5.根据权利要求4所述的制备方法,其特征在于,所述金属离子为Fe或Ca,pH为2.5-5.5;仙草多糖与水的质量比为1:(5-50);金属离子与仙草多糖的质量比为1:(1-100);油相溶液可选用Span80和石蜡;搅拌为磁力搅拌,磁力搅拌时间为0.5-2h;清洗可选用正己烷和甲醇,清洗次数为2-5次。
6.根据权利要求4或5所述的制备方法,其特征在于,所述仙草多糖凝胶微球中还可加入淀粉,所述淀粉与仙草多糖质量比为(0-1):3;淀粉含量不超过仙草多糖总质量的25%。
7.一种包埋物,其特征在于,包括:
如权利要求1-3任一所述仙草多糖凝胶微球或如权利要求4-6任一制备方法制备的仙草多糖凝胶微球;以及功能因子,所述功能因子包埋于仙草多糖凝胶微球中。
8.根据权利要求7所述的包埋物,其特征在于,所述功能因子的包埋率为60%-99%;
所述功能因子为水溶性小分子;水溶性小分子为花色苷、维生素C、烟酰胺、维生素B族、儿茶素之一或组合;
所述功能因子为脂溶性小分子;脂溶性小分子为姜黄素、辣椒素、脂溶性维生素、β-胡萝卜素之一或组合;
所述功能因子为蛋白类大分子;蛋白类大分子为溶菌酶、过氧化物酶、乳铁蛋白和免疫球蛋白;
所述功能因子为微生物;微生物为益生菌,包括乳酸乳球菌、保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、干酪乳杆菌、双歧杆菌、酵母菌、枯草芽孢杆菌和酪酸梭菌的至少之一。
9.一种仙草多糖凝胶球,其特征在于,使用金属离子与仙草多糖中的糖醛酸的羧基交联形成凝胶制备获得;金属离子为Fe或Ca;凝胶球粒径为1.0-5.0 cm;凝胶球为爆珠。
10.如权利要求1-3任一所述仙草多糖凝胶微球或如权利要求4-6任一制备方法制备的仙草多糖凝胶微球、如权利要求7或8的包埋物以及权利要求9的仙草多糖凝胶球在稳定添加到食品体系中的应用,食品体系为固体或液体食品。
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