CN110063257A - 甜叶菊新品种及高rd、rm含量甜菊糖苷的制备 - Google Patents

甜叶菊新品种及高rd、rm含量甜菊糖苷的制备 Download PDF

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CN110063257A
CN110063257A CN201811345553.5A CN201811345553A CN110063257A CN 110063257 A CN110063257 A CN 110063257A CN 201811345553 A CN201811345553 A CN 201811345553A CN 110063257 A CN110063257 A CN 110063257A
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stevioside
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奥维迪·玛珂善
李善汪
卜宇成
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Abstract

本发明提供了一种甜叶菊变种植物，所述变种植物为807086谱星4号，其愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCC No.9702。本发明同时提供了一种高RM含量的甜菊糖苷产品，其中RM/TSG大于0.11，RD/TSG大于0.17。

Description

甜叶菊新品种及高RD、RM含量甜菊糖苷的制备
本申请是申请号为2015100374358，申请日为2015年1月23日，发明名称为“甜叶菊新品种及高RD、RM含量甜菊糖苷的制备”的发明专利申请的分案申请。
技术领域
本发明涉及一种甜叶菊新品种，所述甜叶菊品种含有高含量的RM和RD，并可由保藏的愈伤组织连续培育产生；涉及从该品种的甜叶菊干叶制备高RD、RM含量甜菊糖苷的方法。
背景技术
甜叶菊(Stevia)，学名为Stevia Rebaudiana Bertoni，是一种多年生菊科草本植物，原产于南美巴拉圭，其叶片中含有甜菊糖苷，其甜度为蔗糖的150～300倍，是一种高甜度、低热能、味质好、安全无毒的新型天然甜味剂，可广泛应用于食品、饮料、医药、日化工业等行业。
甜菊糖苷是多组分糖苷混合物，糖苷组分及含量决定混合物的甜度和口感。甜叶菊中的甜菊糖苷含有13种组分，即甜叶菊苷A(Rebaudioside A，简称RebA或RA)，甜叶菊苷B(Rebaudioside B，简称RebB或RB)，甜叶菊苷C(Rebaudioside C，简称RebC或RC)，甜叶菊苷D(Rebaudioside D，简称RebD或RD)，甜叶菊苷E(Rebaudioside E，简称RebE或RE)，甜叶菊苷F(Rebaudioside F，简称RebF或RF)，甜叶菊苷M(Rebaudioside M，简称RebM或RM)，甜叶菊苷N(Rebaudioside N，简称RebN或RN)，甜叶菊苷O(Rebaudioside O，简称RebO或RO)，甜苷A(Dulcoside A，简称DulA或DA)，甜茶苷(Rubusoside，简称Rub或RU)；甜菊双糖苷(Steviolbioside，简称Sbio或SB)，甜菊糖(Stevioside，简称Stev或ST)；这十三种甜菊糖苷的总和成为总甜菊糖苷(Total Steviol glycosides，TSG)；其中RM和RD相对含量较少，约占总甜菊糖苷的0.15％-0.2％。但是RM的甜度最高约为蔗糖的300倍，RD的甜度最高约为蔗糖的450倍；而且RD、RM味质最好，最接近于蔗糖的口感，不含任何不良余味，被业内人士誉为“甜菊糖苷中的黄金”，售价也是其他糖苷的3～5倍，是一种最为理想的天然甜味剂。因此，制取RD、RM含量高的甜菊糖苷产品具有广阔的市场前景。
由于RD、RM在甜叶菊中含量低，过去，获得高纯度RD、RM的唯一方法是反复重结晶，反复重结晶的劣势在于产品得率低，不利于成本控制；另外由于在甜叶菊中含量低，无法大规模生产，不能满足客户的需求。因此有必要开发一种高RD、RM含量的甜叶菊新品种，以便降低高RD、RM含量甜菊糖苷产品生产成本并保持其稳定的得率。
发明内容
本发明提供了一种甜叶菊变种植物。在一个实施例中，变种植物为807086谱星4号，其807086谱星4号的愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏(保藏单位地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)，保藏号为CGMCC No.9702，保藏日期为2014年9月22日，其所述807086谱星4号的植物及干叶中RM/TSG大于0.11，RD/TSG大于0.17。在另一个实施例中，变种植物通过以AKH L4为父本，AKH EM1为母本进行杂交，得到F1代，并从所述F1代优选出性状优良且稳定的单株YF003，然后以YF003为父本，PC Star2为母本进行杂交，得到F2代，其中所述F2代表达由上述807086谱星4号的所有生理和形态特征，其807086谱星4号的愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCC No.9702。在另一个实施例中，变种植物或其一部分表达由上述807086谱星4号所有的生理和形态特征，其807086谱星4号的愈伤组织培养由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCCNo.9702。
本发明同时提供了产生甜叶菊变种植物或其一部分的愈伤组织，甜叶菊变种植物的再生细胞及其再生细胞的组织培养。
本发明同时提供了产生甜叶菊变种植物的方法。在一个实施例中，所述方法包括：以AKH L4为父本，AKH EM1为母本进行杂交，得到F1代，并从所述F1代优选出性状优良且稳定的单株YF003，然后以YF003为父本，PC Star2为母本进行杂交，得到F2代，其中所述F2代表达由上述807086谱星4号的所有生理和形态特征，其807086谱星4号的愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCCNo.9702。
本发明同时提供了一种高RD、RM含量甜菊糖苷的制备方法。在一个实施例中，所述制备方法包括：提供用于制备高RD、RM含量甜菊糖苷的原料，其原料为由上述甜叶菊植物或干叶，其植物及干叶中RM/TSG大于0.11，RD/TSG大于0.17；粉碎所述原料；用水或水溶剂提取所述粉碎原料，获得甜菊糖苷产品，其中RM/TSG大于0.11，RD/TSG大于0.17。在另一个实施例中，所述制备方法包括：提供用于制备高RD、RM含量甜菊糖苷的甜叶菊植物或干叶，其植物及干叶中RM/TSG大于0.11，RD/TSG大于0.17；所述甜叶菊植物或干叶通过愈伤组织再生或扦插由上述甜叶菊植物而得到；粉碎所述甜叶菊植物或干叶；用水或水溶剂提取所述粉碎甜叶菊植物或干叶，获得甜菊糖苷产品，其中RM/TSG大于0.11，RD/TSG大于0.17。
本发明同时提供了一种高RD、RM含量的甜菊糖苷产品，其中RM/TSG大于0.11，RD/TSG大于0.17。
本发明同时提供了一种甜叶菊变种植物或其一部分,源自807086谱星4号的任何一代的后代，其所述807086谱星4号的愈伤组织培养由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCCNo.9702。
通过如下结合附图对优选实施例的详细描述，本发明的目的和优点是显而易见的。
附图说明
本发明的优选实施方案现在将参考附图进行说明，其中类似的附图标记表示相同的元件。
图1、杂交选育甜叶菊植物的示意图。
具体实施方式
1、高RD、RM含量甜叶菊品种育种方法
技术路线如图1所示。
采用三交育苗方法，分别选择AKH EM1、PC Star2为母本，先以AKH L4为父本进行一次杂交，再以优选单株YF003为父本进行二次杂交，采收种子，育苗，移栽大田种植。以单株选择育种方式选择优良单株，经过种植观察其农艺性状和分析检测糖苷含量，性状稳定后，再用组织培养法和无性扦插繁殖方式固定其优良性状，经示范性种植后，成为新品种。
A、选育YF003
通过杂交育种的方法进行选育。首先，将母本AKH EM1和父本AKH L4种植于同一大棚中，通过蜜蜂封闭式授粉的方法进行杂交，收其亲本的种子进行播种、育苗、种植，根据农艺性状及色谱分析，从F1代中优选出性状优良的单株，经无性扦插繁殖固定后的新品系，即YF003号单株。通过父本AKH L4与母本AKH EM1杂交得到的YF003，相对于父本其RA含量明显下降，而RD、RM的含量明显增加。
B、选育807086谱星4号(807086PC Star 4)
以YF003为父本，PC Star 2为母本，同样通过蜜蜂封闭式授粉进行杂交和选择，收其亲本的种子进行播种、育苗、种植，根据农艺性状及色谱分析，从F2代中优选出性状优良的单株，经无性扦插繁殖固定后的新品系，即807086谱星4号(807086PC Star 4)。通过父本YF003和母本PC Star 2杂交得到的807086谱星4号(807086PC Star 4)，其RD、RM含量又有明显的增加。
对杂交种进行检测确认的方法为薄层色谱分析和高效液相分析，分析采自所述植株807086谱星4号(807086PC Star 4)的干燥叶，证实RM/TSG大于0.11，RD/TSG大于0.17。然后对选育出的优良品种807086谱星4号(807086PC Star 4)进行扩繁。扩繁主要采用两种方法：愈伤组织培养和扦插，其中又以愈伤组织培养为主。
甜叶菊新品种807086谱星4号(807086PC Star 4)的愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCC No.9702。本发明的所涉及的植物可由807086谱星4号(807086PCStar 4)品种的保藏愈伤组织再生得到。
2、高RD、RM含量甜菊糖苷产品的制备
(a)预处理：将粉碎的807086谱星4号(807086PC Star 4)甜叶菊干叶于热水中萃取，制成甜菊糖苷萃取液，萃取液经过絮凝沉淀和板框压滤两道工序后，进入下一道工序；
(b)一次吸附及解吸：采用树脂吸附上述萃取液中甜菊糖苷有效成分，到树脂出甜时止；用氢氧化钠稀溶液、盐酸稀溶液、纯水洗树脂、至流出液pH值达到7时止；用乙醇溶液对树脂进行分柱解吸，得RD含量为13.83～15.25％、RM含量为8.92～9.84％、含糖苷量为78～86％的解吸液，解吸液进入下一道工序；
(c)一次离子交换：解析液依次通过一次离子交换除杂后，蒸发浓缩，得到RD含量为14.54～15.61％、RM含量为9.38～10.07％、含苷量在82～88％的浓缩液，浓缩液进入下一道工序；
(d)二次离子交换：将流出液再通过二次离子交换树脂进行深度除杂，浓缩得到RD含量≥14.19％、RM含量≥9.15％、含苷量≥80％的浓缩液，再除菌过滤，浓缩液进入下一道工序；
(e)喷雾干燥：将除菌过滤后的浓缩液喷雾干燥，得到RM/TSG大于0.11，RD/TSG大于0.17，TSG≥80％的甜菊糖苷产品。
如需要制备更高纯度的产品，则在步骤(c)后按以下步骤进行：
(d)二次吸附提纯：用二次吸附树脂三柱串联过饱和吸附上述浓缩液除杂，得到含苷量≥95％的甜菊糖苷提纯液，提纯液进入下一道工序；
(e)二次离子交换：将提纯液通过二次离子交换树脂进行深度除杂，浓缩得到含苷量≥95％的浓缩液，再经过除菌滤膜除菌过滤，浓缩液进入下一道工序；
(f)喷雾干燥：将除菌过滤后的浓缩液喷雾干燥，得到RM/TSG大于0.11，RD/TSG大于0.17，TSG≥95％的甜菊糖苷产品。
实施例
下面结合实施例对本发明做做进一步详细描述。
实施例1
新品系807086谱星4号(807086PC Star 4)的育种
用于杂交育种的父母本性状：
母本一AKH EM1：2010年从巴拉圭引进的RebA含量中等(以下简称RA)的甜菊新品种。株高30～50cm，叶子成广披针状，叶子边缘具有明显锯齿，颜色为绿色。干叶中RebA含量为9.20％，St含量为2.10％。
父本一AKH L4：2010年从巴拉圭引进的高RebA含量甜菊新品种。该品种为为早熟品种，株高51～70cm，叶子成广披针状，叶子边缘锯齿不明显，颜色为深绿色。干叶中RebA含量为11.50％，St含量为1.30％。
母本二谱星2号(PCstar2)：谱赛科(江西)生物技术有限公司选育的高RA甜菊新品种。该品种属于晚熟品种，株高80～120cm，株型直立，生长整齐，茎粗且近地面气根发达，抗性好，叶片大且厚实，一级分枝5～8个，二级分枝少而短，利于脱叶。大田种植前期生长慢，中后期旺盛。一茬大田生长期125～135天。总苷含量13％以上，其中Reb A含量10％以上。亩产干叶220～300公斤。
父本二YF003：第一次杂交后代中性状优良且稳定的优良单株，经无性扦插繁殖固定后的新品系。其RebA含量中等，RD、RM含量较高，分枝能力强，抗性一般，干叶产量高。
杂交育种过程：
2011年8月中旬开始，将所选中等RebA含量的AKH EM1(母本)和RebA糖苷含量高的AKH L4(父本)移入育种大棚内，进行杂交育种，10月初，亲本现蕾之前放入蜂箱，利用蜜蜂进行封闭式授粉杂交；11月中旬开始采收杂交种子，一直采收至12月中旬。2012年初将所得种子播种于育苗大棚内，待其长到3叶1心(约10厘米)时，将种苗移植至6×8cm的育苗袋中，并放于大棚内培养，3月初浇施一次可溶性复合肥500倍液及尿素1000倍液，3月中旬气温回暖后，将种苗移栽至试验田，并对其进行田间管理等措施。
2012年7月份采收植株鲜叶，并进行干燥，采用薄层色谱及高效液相色谱法分析干叶糖苷含量，选择RA糖苷含量中等且有RD、RM糖苷含量的单株，即YF003。
YF003及其亲本以及亲本含苷量检测结果见表1：
表1：YF003及其亲本检测结果
ND–Not Detected(未检出)。
然后在2012年8月中旬移入大棚内进行杂交育种。2013年1月初将所得种子播种在谱赛科公司塑料大棚内进行育种，种子萌发后，秧苗长至3叶1心时，移植至6×8cm的育苗袋中，并放于大棚内培养，3月初浇施一次可溶性复合肥500倍液及尿素1000倍液，3月中旬气温回暖后，将种苗移栽至试验田，并对其进行田间管理等措施。
2013年7月份采收植株鲜叶，并进行干燥，采用薄层色谱及高效液相色谱法分析干叶糖苷含量，选育出所含RM占总甜菊糖苷量比例超过11％，所含RD占总甜菊糖苷量比例超过17％，所含RA占总甜菊糖苷量比例超过45％的单株，即807086谱星4号(807086PC Star4)。
807086谱星4号及其亲本含苷量检测结果见表2：
表2：807086谱星4号(807086PC Star 4)及其亲本含苷量检测结果
ND–未检出。
新品系807086谱星4号(807086PC Star 4)的扩繁及栽培管理：
2013年10月至2014年2月，采用组织培养法将807086单株扩繁到50000株。2014年2月下旬将组培苗移植至育苗穴盘中进行炼苗；2014年3月中下旬，将练好的种苗移栽到大田，并进行相应的田间管理。2014年4月初(移栽后10天左右)进行查苗补缺工作，2014年4月中下旬(苗高15cm左右)开始进行打顶摘心，2014年4月中下旬开始进行第一次追肥，追施的肥料为可溶性复合肥和尿素，用量为可溶性复合肥500倍液，尿素1000倍液，2014年5月份开始进行第二次追肥管理，追施的肥料为可溶性复合肥和尿素，用量为可溶性复合肥500倍液，尿素1000倍液，后期(6月份)补施磷酸二氢钾叶面肥。2014年7月初开始进行收获，收割采用人工采收，打谷机脱叶方式，然后进行晒干，并装袋保存。
分析采自所述植株的干燥叶，通过高效液相色谱测定其成分。
807086 PC Star 4与相似品种(PC Star)中甜菊糖苷含量、各组分占总甜菊糖苷比例以及性状的比较见表3、表4以及表5。
表3：807086PC Star 4与相似品种(PC Star)中甜菊糖苷含量比较(％wt/wt,干基)
ND–未检出。
表4：807086PC Star 4与相似品种(PC Star)中各组分占总甜菊糖苷比例比较(％)
ND–未检出。
表5：807086PC Star 4与相似品种(PC Star)性状比较
实施例2
高RD、RM含量甜菊糖苷产品的制备
(a)预处理：将甜叶菊叶子与50℃软水按重量比1:15混合，制成甜菊糖苷提取液，经过絮凝沉淀和板框压滤两道工序，板框过滤后的滤液进入下一道工序；
(b)一次吸附及解吸：用一次吸附树脂吸附上述萃取液中甜菊糖苷有效成分，用氢氧化钠溶液、盐酸溶液处理吸附了甜菊糖苷有效成分的树脂，纯水淋洗树脂，至流出液PH值达7时止，再用乙醇溶液进行解吸，得到RD含量为14.10％、RM含量为9.10％、含苷量79.5％的解吸液，解吸液去醇进入下一道工序；
(c)一次离子交换：去醇解吸液依次通过一次离子交换除杂，蒸发浓缩得到RD含量为14.52％、RM含量为9.37％、含苷量为81.86％的一次浓缩液，浓缩液进入下一道工序；
(d)二次离子交换：将浓缩液通过二次离子交换树脂进行深度除杂，经过浓缩得到RD含量为14.60％、RM含量为9.42％、含苷量为82.30％的二次离子交换液，再进行除菌滤芯除菌过滤，无菌液进入下一道工序；
(e)喷雾干燥：将无菌液进行喷雾干燥，得到甜菊糖苷产品，RD含量为14.60％、RM含量为9.42％、TSG为82.30％，其中RD/TSG为0.1774，RM/TSG为0.1145，经高效液相色谱分析测定其成分：
表6产品高效液相检测结果
实施例3
(a)预处理：将甜叶菊叶子与70℃软水按重量比1:20混合，制成甜菊糖苷提取液，经过絮凝沉淀和板框压滤两道工序，板框过滤后的滤液进入下一道工序；
(b)一次吸附及解吸：用一次吸附树脂吸附上述脱盐滤液中甜菊糖苷有效成分，用氢氧化钠溶液、盐酸溶液处理吸附了甜菊糖苷有效成分的树脂，再用乙醇溶液进行解吸，得到RD含量为14.54％、RM含量为9.38％、含苷量82.0％的解吸液，解吸液去醇进入下一道工序；
(c)一次离子交换：去醇解吸液依次通过一次离子交换除杂，蒸发浓缩得到RD含量为14.63％、RM含量为9.44％、含苷量为82.5％的一次浓缩液，浓缩液进入下一道工序；
(d)二次吸附提纯：用二次大孔吸附树脂三柱串联吸附上述离子交换液除杂，得到RD含量为16.88％、RM含量为10.89％、含苷量为95.17％甜菊糖苷提纯液，提纯液进入下一道工序；
(e)二次离子交换：通过二次离子交换树脂进行深度除杂，经过浓缩得到RD含量为16.88％、RM含量为10.89％、含苷量为95.19％的二次离子交换液，再进行除菌滤芯除菌过滤，无菌液进入下一道工序；
(f)喷雾干燥：将无菌液进行喷雾干燥，得到甜菊糖苷产品。经高效液相色谱分析，产品RD含量为16.80％、RM含量为10.90％、含苷量为95.14％，其中RD/TSG为0.1766，RM/TSG为0.1146。
表7产品高效液相检测结果
本发明中所有样品，在下列条件下，通过高效液相色谱测定其成分。高效液相色谱检测：
一种新的高效液相色谱方法检测甜菊糖苷。每个样品需用2种方法综合检测。
方法1是用于分析RebE、RebD、RebM、RebN、RebO；方法2是用于分析RebA、Stev、RebF、RebC、DulA、Rub、RebB、Sbio。
所有糖苷的标准品,包括RebE、RebD、RebM、RebN、RebO均购于美国ChromaDex公司。
检测仪器：配备二元泵、自动进样器的安捷伦1200高效液相色谱仪，DAD检测器配合化学工作站软件使用。
方法1仪器条件：
色谱柱：安捷伦Poroshell 120SB-C18 2.7μm,4.6x 150mm
柱温：40℃
流动相A：10mM磷酸二氢钠pH2.6:乙腈，75％:25％(v/v)
流动相B：水：乙腈，50％:50％(v/v)
流量：0.5mL/min
进样量：5μL
检测：UV 210nm
运行时间：25分钟
过度时间：10分钟
进样器温度：常温
梯度程序，(％，v/v:)
时间(min) A(％) B(％)
0.0 100 0
14.0 100 0
14.5 0 100
25.0 0 100
方法2仪器条件：
色谱柱：安捷伦Poroshell 120SB-C18 2.7μm,4.6x 150mm
柱温：40℃
流动相：10mM磷酸二氢钠pH2.6:乙腈，68％:32％(v/v)
流量：1.0mL/min
进样量：5μL
检测：UV 210nm
运行时间：20分钟
进样器温度：常温

Claims (5)

1.一种含有高甜叶菊苷D、甜叶菊苷M含量的甜菊糖苷产品的制备方法，其特征在于，所述含有高甜叶菊苷D、甜叶菊苷M含量的甜菊糖苷产品由甜叶菊变种植物807086谱星4号的叶提取得到，所述甜叶菊变种植物807086谱星4号的叶通过再生甜叶菊变种植物807086谱星4号的愈伤组织而得到，其中，所述甜叶菊变种植物807086谱星4号的愈伤组织由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，保藏号为CGMCC No.9702。
2.根据权利要求1所述的方法，其中，所述提取包括以下步骤：
(a)预处理：将所述甜叶菊变种植物807086谱星4号的叶子与50～70℃软水按重量比1:15～20混合，制成甜菊糖苷提取液，经过絮凝沉淀和过滤，得到滤液；
(b)吸附及解吸：用吸附树脂吸附上述滤液中的甜菊糖苷，用氢氧化钠溶液、盐酸溶液处理吸附了甜菊糖苷的树脂，再用乙醇溶液进行解吸，得到解吸液并对解吸液去醇；
(c)一次离子交换：将步骤(b)的去醇解吸液通过一次离子交换树脂除杂，蒸发浓缩得到一次浓缩液；
(d)二次离子交换：将步骤(c)的一次浓缩液通过二次离子交换树脂进行深度除杂，浓缩、除菌和过滤，得到除菌液；
(e)喷雾干燥：将上述无菌液进行喷雾干燥，得到所述含有高甜叶菊苷D、甜叶菊苷M含量的甜菊糖苷产品。
3.根据权利要求2所述的方法，其中，
在步骤(c)后及步骤(d)之前，还包括用大孔吸附树脂三柱串联对步骤(c)得到的一次浓缩液进行除杂。
4.一种根据权利要求1-3任一项所述的方法制备的含有高甜叶菊苷D、甜叶菊苷M含量的甜菊糖苷产品。
5.根据权利要求4所述的产品，其为甜味剂。
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