CN113692960A - 一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法 - Google Patents
一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法 Download PDFInfo
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Abstract
本发明公开了一种γ‑氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法,属于农产品加工技术领域。本方法取颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料,经除杂、0.5%NaClO消毒杀菌后,移至25℃恒温培养箱16h光照/8h黑暗条件下发芽,蒸馏水喷施1天后改用0.25~1.00mmol/Lγ‑氨基丁酸联合100mM NaCl水溶液喷施处理,4天后收获西兰花芽苗。本发明使ZEP和VDE基因表达量分别提高0.87~2.41倍和1.14~2.75倍,同时西兰花芽苗中叶黄素含量显著提高,是种子的24.1~29.0倍,芽苗鲜重提高40%以上。本发明所涉及的西兰花种子发芽方法简单便捷、成本低廉,此外西兰花中富含的叶黄素具有较强的抗氧化性活性,有效增强了西兰花芽苗菜功能性产品的功效,具有广阔的应用前景。
Description
技术领域
本发明涉及一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法,属于农产品加工技术领域。
背景技术
叶黄素又称黄体素,是一种无维生素A活性的含氧类胡萝卜素,在蔬果、花卉及一些藻类生物中普遍存在。叶黄素作为抗氧化剂对人眼视网膜中的黄膜有重要保护作用,通过物理或化学猝灭作用能够灭活单线态氧,抵御游离基在人体内造成细胞与器官损伤,从而预防心血管硬化、冠心病等疾病的发生,提高机体的免疫力。除此之外,叶黄素还是人体血液中主要的类胡萝卜素之一,对多种癌症如乳腺癌、胃癌、结肠癌等癌症有抑制作用。因其优越的生理功能,叶黄素在食品、医药行业得到广泛的应用。此前有学者证实叶黄素为西兰花中含量最多的类胡萝卜素,通过类胡萝卜素的代谢途径,叶黄素的合成量受关键酶基因表达的影响显著,在此过程中不仅受到正向调控作用的影响,玉米黄质环氧酶(ZEP)、紫黄质脱环氧酶 (VDE)的逆向调控也起到重要的作用。
西兰花是为十字花科芸薹属一、二年生草本植物,其内部富含蛋白质、VC、VB及矿物质等多种营养物质,如西兰花中的花青素及多酚具有抗氧化能力,能够预防人体慢性疾病的发生。硫苷与黒芥子酶作用的水解产物异硫氯酸酯可通过抑制阶段I酶和激活阶段II酶发挥抗癌功能,预防多种癌症的发生。种子萌发生长的同时伴随着内部物质的相互转化,这使得芽苗菜的营养价值和功能特性有所增强,多项研究表明西兰花芽苗中硫苷含量是成熟的西兰花的10-50倍,目前发现的抗癌作用最强的异硫氰酸酯一萝卜硫素含量是成熟的西兰花的10-100倍,因此近年来西兰花芽苗受到越来越多学者的关注。
γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种四碳天然的非蛋白氨基酸,在动植物种广泛存在。它不仅在哺乳动物中能够作为抑制性神经递质起到分子信号的作用,参与中枢神经系统中多种代谢活动,具有多种生理功能,如促进大脑新陈代谢、改善神经机能、镇定安神、改善呼吸、降血糖、抑制肿瘤、治疗精神疾病等。同时在各种植物的不同组织中具有多种生理效应,但植物组织中γ-氨基丁酸的含量普遍很低,研究表明在逆境胁迫下会促使内源性γ-氨基丁酸得到积累,这受到了学者的广泛关注。申请号为201210464080.7的中国发明专利申请公开了一种即食性高γ-氨基丁酸调味豆类芽菜及其生产工艺,该方法将豆类籽粒在低氧胁迫条件下发芽制得富含γ-氨基丁酸的豆类芽菜,再经柠檬酸和CaCl2混合水溶液烫漂护色,最后加入调味剂灭菌制得即食性食品。申请号为201210464186.7的中国发明专利申请公开了一种富含γ-氨基丁酸低爆腰率发芽糙米及其生产方法,该方法融合酸液加湿调质、低温低氧胁迫发芽及干燥三项工艺生产出GABA含量高、爆腰率低、可碾白的发芽糙米。
发芽是生产芽苗菜便捷、成本低廉的重要手段,同时芽苗菜的生产受控因素很少并且可以生食,这在一定程度减少了加工所致的营养成分的损失。申请号为201610886208.7的中国发明专利申请公开了一种富含萝卜硫素的西兰花芽苗及其发芽方法,该方法以电解水为培养液对西兰花种子进行发芽,显著提高了其芽苗中的萝卜硫素含量。申请号为201811607510.X的中国发明专利申请公开了一种绿色富集总黄酮的西兰花芽苗培育方法,该方法将西兰花种子在紫外灯照射、浸泡、避光培养的物理联合化学消毒方法,提高了发芽率和总黄酮的含量。
目前,已知的万寿菊是提取叶黄素的理想原料,近年来,学者们通过研究发现菠菜、柑橘、番茄、南瓜籽等蔬果作物中同样具有丰富的叶黄素。上述专利涉及到植物种子发芽富集γ-氨基丁酸以及以西兰花种子为原料富集萝卜硫素、黄铜类等物质,但利用西兰花芽苗富集叶黄素未涉及专利公布。西兰花富含多种有益人体健康的物质,随着相关研究的深入,芽苗菜将有广阔的应用前景。
在现有技术中,已有相关研究表明适宜浓度的NaCl处理对玉米中叶黄素、发芽大豆中的酚类物质均有促进作用,但是利用NaCl的促进作用联合GABA协同处理西兰花芽苗的发芽方法还尚无研究。另外西兰花中萝卜硫素虽然随芽苗生长有所减少但仍然比成熟西兰花中高很多倍,本发明通过对现有技术进行探索优化,获得一种既可以同时提高西兰花芽苗中叶黄素和萝卜硫素含量又能增大产量富集的培育方法。
发明内容:
本发明的目的在于提供一种富集西兰花芽苗中叶黄素的培育方法。本方法以颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料,经除杂、0.5%NaClO消毒杀菌后,移至25℃恒温培养箱 16h光照/8h黑暗条件下通过γ-氨基丁酸联合NaCl水溶液喷施处理,对种子进行培育,生产出性能优良、根茎壮硕的芽苗,并实现西兰花芽苗中叶黄素的高效富集。
本发明的技术方案:一种富集西兰花芽苗中叶黄素的培育方法。为达到上述目的,所述方法包括以下工艺步骤:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料。
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min。
(3)浸泡处理:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,置于蒸馏水中30℃浸泡4h。
(4)种子催芽:滤干水分,将浸泡后的西兰花种子均匀散落在铺有蛭石的发芽盘上,移至25℃的恒温培养箱,16h光照/8h黑暗条件下用蒸馏水喷施催芽1天。
(5)喷施处理:催芽1天后改用100mM NaCl联合γ-氨基丁酸水溶液喷施处理(浓度为0.25、0.50、 1.00mmol/L),每隔12h喷施20mL(喷施量以芽苗浸润为标准),处理四天后收获西兰花芽苗。
本发明的有益效果:
(1)本发明选用的γ-氨基丁酸广泛存在于动植物及微生物中,作为固有的内源性物质对植物的生长无毒害。
(2)本发明以西兰花种子为原料,100mM NaCl喷施处理西兰花种子4天,显著提高了芽苗中叶黄素的含量,是对照组芽苗的1.4倍,是未萌发的种子的24.1倍。
(3)本发明在NaCl胁迫同时喷施1mMγ-氨基丁酸上调了ZEP基因表达量约0.87~2.41倍,VDE基因表达量约1.14~2.75倍,逆向调控促使西兰花芽苗中的叶黄素含量显著提高,较对照组芽苗高1.7倍,较未萌发的种子高29.0倍。
(4)本发明发现在NaCl胁迫处理时加入γ-氨基丁酸溶液联合作用,能够改善西兰花芽苗在NaCl胁迫下受到的损伤,使其抗性增强,对合成芽苗中的活性物质起到促进作用,使叶黄素和萝卜硫素含量得到提高,同时鲜重增加提高了产量。
(5)本发明的西兰花发芽方法简便易行,安全可靠,收获的西兰花芽苗可直接生食,也可用于做成粉制品及其他功能性食品,对人体抗癌、保健等方面有重要作用。
具体实施方式:
实施例1:
本发明提供一种富集西兰花芽苗中叶黄素的培育方法,包括以下步骤:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料。
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min。
(3)浸泡处理:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,置于蒸馏水中 30℃浸泡4h。
(4)种子催芽:滤干水分,将浸泡后的西兰花种子均匀散落在铺有蛭石的发芽盘上,移至25℃的恒温培养箱,16h光照/8h黑暗条件下用蒸馏水喷施催芽1天。
(5)喷施处理:催芽1天后改用100mM NaCl联合0.25mMγ-氨基丁酸水溶液喷施处理,每隔12h 喷施20mL,处理四天后收获西兰花芽苗,称得单株鲜重约为24.80mg。
(6)冷冻干燥:将芽苗用蒸馏水洗净,用双层滤纸吸干水分,液氮速冻后放置于-80℃冰箱预冻24h,然后转至真空冷冻干燥机进行冻干处理,打粉后待测。
(7)测定分析:利用qRT-PCR分析测得西兰花芽苗中ZEP基因表达量提高1.47倍,VDE基因表达量提高2.06倍,利用高效液相色谱法测定西兰花芽苗中叶黄素的含量为637.74μg/g DW,萝卜硫素含量为 1.72mg/g DW。
实施例2:
本发明提供一种富集西兰花芽苗中叶黄素的培育方法,包括以下步骤:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料。
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min。
(3)浸泡处理:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,置于蒸馏水中 30℃浸泡4h。
(4)种子催芽:滤干水分,将浸泡后的西兰花种子均匀散落在铺有蛭石的发芽盘上,移至25℃的恒温培养箱,16h光照/8h黑暗条件下用蒸馏水喷施催芽1天。
(5)喷施处理:催芽1天后改用100mM NaCl联合0.50mMγ-氨基丁酸水溶液喷施处理,每隔12h 喷施20mL,处理四天后收获西兰花芽苗,称得单株鲜重约为26.98mg。
(6)冷冻干燥:将芽苗用蒸馏水洗净,用双层滤纸吸干水分,液氮速冻后放置于-80℃冰箱预冻24h,然后转至真空冷冻干燥机进行冻干处理,打粉后待测。
(7)测定分析:利用qRT-PCR分析测得西兰花芽苗中ZEP基因表达量提高0.87倍,VDE基因表达量提高1.14倍,利用高效液相色谱法测定西兰花芽苗中叶黄素的含量为569.87μg/g DW,萝卜硫素含量为 1.39mg/g DW。
实施例3:
本发明提供一种富集西兰花芽苗中叶黄素的培育方法,包括以下步骤:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料。
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min。
(3)浸泡处理:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,置于蒸馏水中 30℃浸泡4h。
(4)种子催芽:滤干水分,将浸泡后的西兰花种子均匀散落在铺有蛭石的发芽盘上,移至25℃的恒温培养箱,16h光照/8h黑暗条件下用蒸馏水喷施催芽1天。
(5)喷施处理:催芽1天后改用100mM NaCl联合1.00mMγ-氨基丁酸水溶液喷施处理,每隔12h 喷施20mL,处理四天后收获西兰花芽苗,称得单株鲜重约为26.52mg。
(6)冷冻干燥:将芽苗用蒸馏水洗净,用双层滤纸吸干水分,液氮速冻后放置于-80℃冰箱预冻24h,然后转至真空冷冻干燥机进行冻干处理,打粉后待测。
(7)测定分析:利用qRT-PCR分析测得西兰花芽苗中ZEP基因表达量提高2.41倍,VDE基因表达量提高2.75倍,利用高效液相色谱法测定西兰花芽苗中叶黄素的含量为681.90μg/g DW,萝卜硫素含量为 1.64mg/g DW。
对比例1:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料。
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min。
(3)冷冻干燥:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,用双层滤纸吸干水分,液氮速冻后放置于-80℃冰箱预冻24h,然后转至真空冷冻干燥机进行冻干处理,打粉后待测。
(4)液相分析:利用高效液相色谱法测定西兰花种子中叶黄素的含量为23.50μg/gDW,萝卜硫素的含量为5.53mg/g DW。
对比例2:
与实施例1相比,步骤(4)后继续蒸馏水喷施处理,每隔12h喷施20mL,处理四天后收获西兰花芽苗,称得单株鲜重约为17.73mg。将芽苗用蒸馏水洗净,用双层滤纸吸干水分,液氮速冻后放置于-80℃冰箱预冻24h,然后转至真空冷冻干燥机进行冻干处理,打粉后利用高效液相色谱法测定西兰花芽苗中叶黄素的含量为397.17μg/g DW,萝卜硫素的含量为0.17mg/g DW。
以上所述实施例仅为本发明的优选的实施方式,不应被视为对本发明范围与构思的限制。对于本领域的技术人员,可以在不脱离本发明的保护范围的情况下,作出同等的若干替换与改进,这些均应属于本发明的保护范围。本发明请求保护的技术内容已记入权利要求书中,说明书中的具体实施方式等可用于解释权利要求书的内容。
Claims (3)
1.一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法,其特征在于,筛选颗粒饱满、具有发芽能力的“青峰”品种西兰花种子为原料,除去杂质后浸泡于次氯酸钠中杀菌消毒,喷施蒸馏水催芽1天后,改用氯化钠联合γ-氨基丁酸水溶液喷施处理,收获富含高含量叶黄素的西兰花芽苗。
2.根据权利要求1所述的一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素的方法,其特征在于,包括以下步骤:
(1)原料的筛选:选择颗粒饱满、大小均匀、具有发芽能力的“青峰”品种西兰花种子为原料;
(2)杀菌消毒:将种子中掺杂的杂质去除,用蒸馏水冲洗1-2次,浸泡于0.5%NaClO溶液15-20min;
(3)浸泡处理:滤干NaClO溶液后,用蒸馏水冲洗5-8次至西兰花种子pH呈中性,置于蒸馏水中30℃浸泡4h;
(4)种子催芽:滤干水分,将浸泡后的西兰花种子均匀散落在铺有蛭石的发芽盘上,移至25℃的恒温培养箱,16h光照/8h黑暗条件下用蒸馏水喷施催芽1天;
(5)喷施处理:催芽1天后改用γ-氨基丁酸(γ-氨基丁酸浓度为0.25~1.00mmol/L)联合100mM NaCl水溶液喷施处理,每隔12h喷施20mL(喷施量以芽苗浸润为标准),处理四天后收获西兰花芽苗。
3.根据权利要求2所述的一种γ-氨基丁酸联合氯化钠胁迫高效富集西兰花芽苗中叶黄素方法,其特征在于,γ-氨基丁酸联合NaCl处理后叶黄素合成关键酶-玉米黄质环氧酶基因(ZEP)和紫黄质脱环氧酶基因(VDE)的表达量分别提高0.87~2.41倍和1.14~2.75倍,芽苗中叶黄素含量是种子的24.1~29.0倍。而且GABA能显著缓解NaCl对西兰花芽苗生长的抑制,与不加盐组相比,芽苗鲜重提高40%以上。
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