CN116426456A - 一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法及其应用 - Google Patents
一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法及其应用 Download PDFInfo
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Abstract
本发明提供一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法及其应用,涉及化妆品技术领域,第一方面,本发明利用植物组织培养技术提供一种植物外泌体的获取方法,包含以下步骤:S1、外植体的选材。选取石斛、人参、雪莲的根段、梗芽、茎尖、叶片、胚、花梗节间的幼嫩部分,切取长0.5‑4cm的切段作为外植体;S2、外植体的消毒。用自来水清洗干净,在饱和漂白粉上清液中浸泡5‑20min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,置于超净工作台上,先用75%酒精消毒10‑60s,无菌水清洗2‑4次,再用0.1‑0.3%升汞浸泡5‑20min。利用植物组织培养技术提供一种植物外泌体,植物来源外泌体对源于肠道菌群长双歧杆菌婴儿亚种(YJXB‑01‑21)的具有促进作用和复合功效作用。
Description
技术领域
本发明涉及化妆品技术领域,尤其涉及一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法及其应用。
背景技术
二裂酵母又称双歧杆菌,普遍定殖于人体肠道,其中长双歧杆菌是双歧杆菌属的成员之一,是公认为安全的宿主有益菌。各个双歧杆菌种在肠道中的丰度呈现个体化特征,在不同饮食习惯、不同年龄以及不同生理状态的宿主个体间差异巨大,其中的长双歧杆菌婴儿亚种主要存在于婴儿的肠道中,更易从婴儿肠道中分离,并具有激活巨噬细胞、刺激免疫因子释放、维持肠道菌群平衡、抗肿瘤、促进微量元素吸收、控制内毒素的产生等重要的生理保健功能,因此长双歧杆菌婴儿亚种的应用价值极高。
植物外泌体是真核细胞分泌的直径为40-100nm的细胞外囊泡,是由多囊体与质膜融合,将多泡内含体释放到细胞外环境中形成的。这些多泡内含体则是外泌体的前体,其包含着RNA(包括mRNA、miRNA和其他非编码RNA)、DNA和脂质。
研究发现,植物来源外泌体中RNA对调节肠道菌群具有重要作用。从生姜外泌体中鉴定出小RNA和miRNA,可调节肠道微生物组成及其代谢产物,其主要作用机制为:(1)富含脂质的ELN生姜外泌体发出一个信号,导致肠道细菌对其进行吸收;(2)外泌体的小RNA介导肠道微生物和宿主免疫系统之间的相互作用,形成免疫和肠道微生物之间的平衡;(3)外泌体RNA调节肠道微生物的组成、代谢产物、生长和定位。具体是生姜外泌体中RNA诱导的肠道益生菌LGGI3A通过激活AHR信号通路促进IL-22表达,在屏障表面诱导抗微生物免疫和组织修复。
此外,其他研究也发现生姜类外泌体纳米粒子可降低炎性细胞因子[肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和IL-1β)]表达,增强结肠炎模型中的抗炎细胞因子(IL-10和IL-22)的表达。研究还发现外泌体可以保护miRNA在超声、高温以及RNase处理下的完整性,保护其免受强酸的腐蚀,使miRNA能够在人体血液中稳定存在,并对人类某些特定基因的表达起到调节作用,这对于核酸治疗这一靶向调控并能有效抑制疾病相关基因表达的治疗方法有着重要作用。
目前市售二裂酵母发酵产物溶胞物产品因技术要求和成本限制,多数是使用长双歧杆菌进行发酵获得的,未能对长双歧杆菌内包含诸多的亚种进行详细分析,其产品功效无关年龄且较泛,未能在皮肤保湿紧致方面突出效果。因此,从婴儿皮肤角度出发,针对皮肤保湿紧致的方面,利用植物外泌体,结合长双歧杆菌婴儿亚种进行二次发酵,制备出具有皮肤保湿、紧致、抗氧化的二裂酵母发酵产物溶胞物,解决的目前面临的问题。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法及其应用,所述的二裂酵母产物溶胞物具有良好的皮肤保湿紧致抗氧化效果,在化妆品领域具有极大的应用前景。
第一方面,本发明利用植物组织培养技术提供一种植物外泌体的获取方法,包含以下步骤:
S1、外植体的选材。选取石斛、人参、雪莲的根段、梗芽、茎尖、叶片、胚、花梗节间的幼嫩部分,切取长0.5-4cm的切段作为外植体;
S2、外植体的消毒。用自来水清洗干净,在饱和漂白粉上清液中浸泡5-20min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,置于超净工作台上,先用75%酒精消毒10-60s,无菌水清洗2-4次,再用0.1-0.3%升汞浸泡5-20min,无菌水冲洗3-8次;
S3、外植体的接种。将消毒后的外植体接种到经设计含有a-萘乙酸、6-苄氨基腺嘌呤的改良的MS诱导培养基上,诱导丛生芽。接种后的外植体放在培养室培养,培养条件为温度20-35℃、光照强度1000-3000lx、光照时间5-20h/d;
S4、外植体续代培养。将外植体诱导生长出的不定芽的叶片切成5-25mm2大小接种在添加了50-300mL/L椰汁的改良MS培养基上进行原球茎诱导,培养条件为温度20-25℃、光照强度500-2000lx、光照时间10-20h/d,得到类原球茎;
S5、植物外泌体的获取。将类原球茎收集,经组织破碎机破碎、200-500目的滤网过滤获得植物外泌体滤液备用。
作为一种优选方案,所述S1外植体选取石斛茎尖幼嫩部分切取0.5cm。
作为一种优选方案,所述S2外植体在饱和漂白粉上清液中浸泡10-15min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,用75%酒精消毒50-60s,无菌水清洗2次,再用0.1%升汞浸泡10-15min,无菌水冲洗3-4次。
作为一种优选方案,所述S3所述的a-萘乙酸含量为0.5-2.0mg/L、6-苄氨基腺嘌呤5.0-20.0mg/L。
作为一种优选方案,所述S3改良MS培养基按重量百分比包括:硝酸钾1.90g/L,硝酸铵1.65g/L,磷酸二氢钾0.17g/L,硫酸镁0.37g/L,氯化钙0.44g/L,碘化钾0.83g/L,硼酸0.006g/L,硫酸锰0.022g/L,硫酸锌0.009g/L,钼酸钠0.001g/L,硫酸铜0.000025g/L,氯化钴0.000025g/L,硫酸亚铁0.0278g/L,肌醇0.10g/L,盐酸硫胺素0.0001g/L,甘氨酸0.002g/L,盐酸吡哆醇0.0005g/L,烟酸0.0005g/L,乙二胺四乙酸二钠0.0373g/L,pH值5.7±0.1(25℃)。
作为一种优选方案,所述S4所述的不定芽的叶片切成5-10mm2大小,培养条件为温度22-25℃、光照强度800-1000lx、光照时间15-20h/d。
作为一种优选方案,所述S5类原球茎需在低温10-20℃用组织破碎机进行,过400-500目的滤网。
第二方面,本发明提供一株长双歧杆菌婴儿亚种(YJXB-01-21),所述长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)YJXB-01-21于2022年12月23日保藏于中国典型培养物保藏中心,保藏编号为CCTCCM20222021,保藏地址为湖北省武汉市武昌区八一路299号武汉大学校内。
本发明自主分离得到的长双歧杆菌婴儿亚种(YJXB-01-21),通过该菌制备得到的二裂酵母发酵产物溶胞物安全无刺激、胞内物质活性高,制备工艺简单,具有更优的使皮肤保湿、紧致、抗氧化等功效。
第三方面,本发明提供利用植物组织培养技术获得的植物外泌体发酵培养长双歧杆菌婴儿亚种(YJXB-01-21)制备二裂酵母发酵产物溶胞物的方法,包含以下步骤
S6、将长双歧杆菌婴儿亚种(YJXB-01-21)斜面挑取一环接种到PDA培养基上,30-40℃下进行活化培养1-3d,得到活化的长双歧杆菌婴儿亚种一级种子液,将活化的长双歧杆菌婴儿亚种一级种子液接种到改良MRS液体培养基中,30-40℃下发酵培养1-3d,得到长双歧杆菌婴儿亚种二级种子液;
S7、配制培养基按重量百分比包括:1.0-5.0%葡萄糖,0.2-3.0%大豆蛋白胨,0.1-0.3%磷酸氢二钾,0.05-0.20%氯化钠,0.10-0.3%酵母粉,115-121℃灭菌20-30min,冷却后将长双歧杆菌婴儿亚种二级种子液接种于灭菌后的培养基中30-40℃培养。
S8、在培养至10-24h时,流加植物外泌体进入发酵液中,流加体积为总体积的0.1-5.0%,流加时间为1-24h,流加后继续培养1-3d。
S9、发酵完成后,将发酵液超声处理后,高压均质,过滤,巴氏灭菌,加入防腐体系,获得二裂酵母发酵产物溶胞物。
本发明利用植物组织培养技术获得的植物外泌体发酵培养长双歧杆菌婴儿亚种(YJXB-01-21)制备二裂酵母发酵产物溶胞物的方法,经试验发现,通过流加植物外泌体,可以促进长双歧杆菌婴儿亚种(YJXB-01-21)的生长,积累胞内产物,提高胞内产物的活性和功效,使二裂酵母发酵产物溶胞物具有更优的使皮肤保湿、紧致、抗氧化等功效。
作为一种优选方案,所述S6的改良MRS液体培养基按重量百分比包括:蛋白胨10g/L,牛肉膏粉2.0g/L,酵母膏粉4g/L,葡萄糖20g/L,吐温80 1.08g/L,磷酸氢二钾(无水)2g/L,乙酸钠(无水)5g/L,柠檬酸三铵(无水)2g/L,硫酸镁(无水)2.0g/L,余量纯净水。
作为一种优选方案,所述S7配制培养基按重量百分比包括:2.0-3.0%葡萄糖,0.5-1.0%大豆蛋白胨,0.10-0.15%磷酸氢二钾,0.05-0.10%氯化钠,0.10-0.2%酵母粉,120-121℃灭菌20-25min,冷却后将长双歧杆菌婴儿亚种二级种子液接种于灭菌后的培养基中35-37℃培养。
作为一种优选方案,所述S8在培养至10-15h时,使用流动泵将植物外泌体流加入发酵液中,流加体积为总体积的0.1-0.3%,流加时间为1-5h,流加后继续培养1-2d。
作为一种优选方案,所述S9发酵液的超声处理时间为2-4h,功率200-800W。
作为一种优选方案,所述S9发酵液的高压均质压力为10-15Mpa,均质时间为10-20min。
作为一种优选方案,所述S9过滤为陶瓷膜过滤,过滤孔径为0.50-2.0um。
作为一种优选方案,所述S9加入防腐体系为1,2-己二醇1-5%,1,3丁二醇1-5%,对羟基苯乙酮0.1-1.0%。
与现有技术相比,本发明的优点和积极效果在于,
1、利用植物组织培养技术提供一种植物外泌体,植物来源外泌体对源于肠道菌群长双歧杆菌婴儿亚种(YJXB-01-21)的具有促进作用和复合功效作用。
2、本发明自主分离得到的长双歧杆菌婴儿亚种(YJXB-01-21),通过该菌制备得到的二裂酵母发酵产物溶胞物安全无刺激、胞内物质活性高,制备工艺简单,具有更优的使皮肤保湿、紧致、抗氧化等功效。
3、本发明利用植物组织培养技术获得的植物外泌体发酵培养长双歧杆菌婴儿亚种(YJXB-01-21)制备二裂酵母发酵产物溶胞物的方法,经试验发现,通过流加植物外泌体,可以促进长双歧杆菌婴儿亚种(YJXB-01-21)的生长,积累胞内产物,提高胞内产物的活性和功效,使二裂酵母发酵产物溶胞物具有更优的使皮肤保湿、紧致、抗氧化等功效。
附图说明
图1为纯化分离得到的长双歧杆菌婴儿亚种(YJXB-01-21)的菌落形态图;
图2为光学显微镜1600倍下长双歧杆菌婴儿亚种(YJXB-01-21)的染色图。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和实施例对本发明做进一步说明。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开说明书的具体实施例的限制。
实施例一
本实例利用植物组织培养技术提供一种植物外泌体的获取方法,包含以下步骤:
S1、外植体的选材。选取石斛的茎尖的幼嫩部分,切取长0.5cm的切段作为外植体;
S2、外植体的消毒。用自来水清洗干净,在饱和漂白粉上清液中浸泡10min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,置于超净工作台上,先用75%酒精消毒60s,无菌水清洗3次,再用0.1%升汞浸泡10min,无菌水冲洗4次;
S3、外植体的接种。将消毒后的外植体接种到经设计含有1.0mg/La-萘乙酸、5.0mg/L6-苄氨基腺嘌呤的改良的MS诱导培养基上,诱导丛生芽。接种后的外植体放在培养室培养,培养条件为温度25℃、光照强度1500lx、光照时间10h/d;
S4、外植体续代培养。将外植体诱导生长出的不定芽的叶片切成25mm2大小接种在添加了150mL/L椰汁的改良MS培养基上进行原球茎诱导,培养条件为温度20℃、光照强度1000lx、光照时间16h/d,得到类原球茎;
S5、植物外泌体的获取。将类原球茎收集,在低温15℃用组织破碎机进行,过500目的滤网,获得植物外泌体滤液备用。
实施例二
1、长双歧杆菌婴儿亚种(YJXB-01-21)的分离
本实例用TPY固体培养基分离酸奶中开菲尔粒分离菌株。将开菲尔粒用无菌研磨棒研磨,加入无菌水过滤,收集滤液,将收集的滤液涂抹于已灭菌的TPY固体培养基中,充入CO2,28℃下培养3d,选取典型的细菌菌落,并用显微镜观察,接种环划线纯化3代,将分离纯化得到的细菌接种于TPY固体培养基,置于4℃保藏。
2、长双歧杆菌婴儿亚种(YJXB-01-21)的分子生物学鉴定
2.1DNA提取,使用TSINGKE植物DNA提取试剂盒(通用型)。将SpinColumn置于CollectionTube中,加入250μlBufferBL,12000rpm/min离心1min活化硅胶膜;取样本干燥组织(不大于20mg),加入液氮充分研磨;研磨后置于1.5ml离心管中,加入400μlBuffergP1,涡旋振荡1min,65℃水浴10-30min,期间可取出颠倒混匀以充分裂解;加入150μlBuffergP2,涡旋振荡1min,冰浴5min;12000rpm/min离心5min,将上清转移至新的离心管中;加入上清等体积的无水乙醇,立即充分振荡混匀,液体全部转入SpinColumn中,12,000rpm/min离心30s,弃废液;向SpinColumn中加入500μlBufferPw(使用前已加入无水乙醇),12000rpm/min离心30s,弃废液;向SpinColumn中加入500μlWashBuffer(使用前已加入无水乙醇),12000rpm/min离心30s,弃废液;将SpinColumn放回CollectionTube中,12000rpm/min离心2min,开盖晾干1min;取出SpinColumn,放入一个干净的离心管中,在吸附膜的中央处加50-100μLTEBuffer(65℃预热TEBuffer),20-25℃放置2min,12000rpm/min离心2min。
2.2PCR扩增
提取的DNA样品适量稀释后作为PCR模板,以S×TSE246进行扩增,扩增体系各组分如下:
S×TSE246 | 45ul |
27F(10P) | 2ul |
1492R(10P) | 2ul |
DNA模板 | 1ul |
以上扩增体系按以下扩增程序扩增:
2.3电泳检测
将扩增好的PCR产物进行琼脂糖凝胶电泳(2ul样品+6ul溴酚蓝),300V电压下15分钟,获取鉴定胶图。
将准备好PCR产物进行一代测序并对比
比对结果
(1)用ContigExpress拼接测序结果,并去除两端不准的部分。
(2)将拼接好的序列在NCBI数据库(blast.ncbi.nlm.nih.gov)中进行比对,分离出一株长双歧杆菌婴儿亚种(YJXB-01-21)。
实施例三
本实例提供利用植物组织培养技术获得的植物外泌体发酵培养长双歧杆菌婴儿亚种(YJXB-01-21)制备二裂酵母发酵产物溶胞物的方法,包含以下步骤:
S6、将长双歧杆菌婴儿亚种(YJXB-01-21)斜面挑取一环接种到PDA培养基上,37℃下进行活化培养3d,得到活化的长双歧杆菌婴儿亚种一级种子液,将活化的长双歧杆菌婴儿亚种一级种子液接种到改良MRS液体培养基中,37℃下发酵培养3d,得到长双歧杆菌婴儿亚种二级种子液;
S7、配制培养基按重量百分比包括:2.0%葡萄糖,0.5%大豆蛋白胨,0.1%磷酸氢二钾,0.10%氯化钠,0.10%酵母粉,115℃灭菌30min,冷却后将长双歧杆菌婴儿亚种二级种子液接种于灭菌后的培养基中37℃培养。
S8、在培养至24h时,使用流动泵将植物外泌体流加入发酵液中,流加体积为总体积的0.2%,流加时间为2h,流加后继续培养2d。
S9、发酵完成后,将发酵液超声处理,处理时间为4h,功率800W,然后高压均质压力为10Mpa,均质时间为15min,过滤,过滤孔径为2.0um,巴氏灭菌10min,加入防腐体系1,2-己二醇2.0%,1,3丁二醇5.0%,对羟基苯乙酮1.0%,获得二裂酵母发酵产物溶胞物。
实施例四
一种精华液的制备,其质量百分比为:A组分:去离子水85.12%、EDTA二钠0.03%、尿囊素0.15%、甘油4.00%、羟苯甲酯0.05%、卡波姆0.2%;B组分:上述实例3制备的二裂酵母发酵溶胞物10.00%;C组分为精氨酸0.15%;D组分为苯氧乙醇0.30%。将A组分投入乳化锅升温至85℃搅拌转速500r/min,恒温20min,均质2min,转速1500r/min,B组分降温至50℃加入乳化锅搅拌均匀,C组分降温至45℃加入乳化锅搅拌均匀,再投入D组分,搅拌均匀,出料送检。
实施例五
一种霜的制备,其质量百分比为:A组分:去离子水66.45%、EDTA二钠0.03%、尿囊素0.15%、甘油4.00%、透明质酸0.05%、卡波姆0.25%;B组分为:鲸蜡醇2%,乳木果油2%,水飞蓟籽油3%,霍霍巴籽油3%,狭翅娑罗双籽脂5%,PEG-100硬脂酸酯/甘油硬脂酸酯2%,棕榈酰水解小麦蛋白钾/鲸蜡硬脂醇/甘油硬脂酸酯1.5%,羟苯甲酯0.12%;C组分:实施例3制备的二裂酵母发酵溶泡物;D组分:C组分为精氨酸0.15%;E组分:苯氧乙醇0.3%。将A组分投入水相锅升温至85℃搅拌至溶解完全,B组分投入油相升温至85℃搅拌至溶解完全。C组分真空条件下先抽入A相进乳化锅,再抽入B相进乳化锅搅拌均匀,均质5分钟转速3000r/min。恒温至85℃20min。D组分:降温至45℃相投入乳化锅搅拌均匀。E组分:降温至40℃,D、E相投入乳化锅搅拌均匀,出料送检。
实施例六
一种冻干粉的制备工艺,其质量百分比为:A组分:去离子水84.65%、甘露糖醇5.00%、磷酸氢二钠0.30%、磷酸二氢钠0.05%;B组分:上述实例3制备的二裂酵母发酵溶胞物10.00%。先将A组分散均匀,过滤后在高温度压灭菌冷却后再用,加入B组分搅拌分装,样品预冻至-40℃保温3h,升温至-10℃保温6h,升温至10℃保温6h,升温至30℃保温9h。
对比例1
本对比例提供一种发酵培养长双歧杆菌婴儿亚种(YJXB-01-21)制备二裂酵母发酵产物溶胞物的方法,与实施例3的区别仅在于培养至24h时,不流加植物外泌体于发酵液中,直接发酵3d,参照实例3所述步骤S6、S7、S9制备二裂酵母发酵产物溶胞物。
对比例2
本对比例提供一种利用植物组织培养技术获得植物外泌体发酵培养长双歧杆菌婴儿亚种制备二裂酵母发酵产物溶胞物的方法,与实施例3的区别仅在于保藏编号为CCTCCM20222021的长双歧杆菌婴儿亚种更换为编号BNCC185971的长双歧杆菌婴儿亚种(购于商城北纳创联生物科技有限公司),参照实例3所述步骤制备二裂酵母发酵产物溶胞物。
对比例3
本对比例为参照专利申请号为CN202010763305.3的专利文件中实施例5记载的二裂酵母发酵溶胞物的制备方法,制备得到二裂酵母发酵溶胞物。
测试例1人体功效测试-皮肤经皮水分流失
皮肤水分流失TEWL对评估皮肤水分保护层的功能是非常重要的参数,在国际上已经得到了广泛的认可。皮肤保护层越完好,水份的含量就会越高,皮肤水分流失TEWL的数值就越低,TEWL的单位为:g/㎡h。在化妆品的研制过程中,通过测试皮肤水分流失TEWL的数值可评价保湿化妆品的功效,
为了进一步证明本发明的效果,提供了以下测试方法,在使用实施例3、对比例1、对比例2、对比例3中的制备的二裂酵母发酵溶胞物,添加10%于精华液后,涂抹20位受试者的手臂内侧皮肤,用皮肤水分散失测试仪AquaFlux对皮肤的皮肤经皮水分流失进行测试:
表1测试结果
表1经皮水分散失TEWL值统计分析结果(n=20)
测试仪器显示,使用实例3制备的二裂酵母发酵溶胞物添加10%于精华液中,使用2周和4周,皮肤的经皮水分散失TEWL值远小于对比例1、对比例2和对比3,说明采用实例3制备的二裂酵母发酵溶胞物,在使用同等添加量的情况下,其皮肤保湿效果比对比例1、对比例2和对比3的效果显著。
测试例2细胞紧致功效测试-对I型胶原蛋白的合成能力
使用细胞ELISA法:
(1)细胞培养:试验前36h制备细胞悬液,将细胞悬液接种于96孔细胞培养板,每孔0.1mL,每孔细胞数为5000个,培养30h。
(2)测试:弃细胞培养板中培养液,每孔加入0.1mL实施例3、对比例1、对比例2、对比例3中制备的二裂酵母发酵溶胞物的测试样品,分别加入依次为5%,阳性对照组Vc浓度为100μg/mL,培养箱孵育24h。
(3)收集样品:离心取上清液或储存于-20℃备用。
(4)ELISA测试:在酶标仪450nm波长处测定吸收值。
每次测试根据标准样品的标曲计算样品中I型胶原蛋白的含量,对测试结果采用GraphPad6.0进行统计学分析。
具体结果见表2
表2I型胶原蛋白的含量
组别 | OD450 | I型胶原蛋白(ng/mL) | 相对含量(%) |
空白组 | 0.921 | 11.3245 | 100.00 |
Vc(100μg/mL) | 1.675 | 26.3643 | 249.74 |
实例3 | 1.452 | 22.3543 | 232.34 |
对比1 | 1.123 | 15.2435 | 157.53 |
对比2 | 1.345 | 20.4352 | 164.24 |
对比3 | 1.043 | 15.0245 | 149.35 |
测试结果:
阳性对照Vc能促进I型胶原蛋白的含量。实例3较对比例1、对比例2、对比例3具有更能够促进I型胶原蛋白的含量的效果,实例3制备的二裂酵母发酵产物溶胞物具有皮肤紧致的功效。
测试例3生化试剂抗氧化功效测试-ABTS清除自由基实验
本实验应用ABTS测定自由基清除率。配制浓度为7mmol/L的ABTS和2.45mmol/L的过硫酸钾混合溶液,室温避光静置12h,用水稀释,使其在734nm下的吸光值为0.7±0.02(记为A0),在ABTS工作液试管中分别加入实施例3、对比例1、对比例2、对比例3中制备的二裂酵母发酵溶胞物1mL,ABTS工作液1.5mL。以上各组室温避光放置1h,在734nm下测定其吸光值(记为A),清除率(%)=(A0-A)/A0×100%,具体结果见表3。
表3测试结果
表3阳性维生素C对照结果
组别 | 吸光度值(试样组)A | 样品本底液A | 清除率(%) |
空白样 | 1.038 | 0.001 | |
0.0001mg/mL维C溶液 | 0.982 | -0.002 | 6.33 |
0.001mg/mL维C溶液 | 0.894 | 0.003 | 32.22 |
0.01mg/mL维C溶液 | 0.675 | -0.001 | 40.86 |
0.1mg/mL维C溶液 | 0.002 | -0.002 | 97.94 |
0.5mg/mL维C溶液 | 0.001 | 0.004 | 99.54 |
测试结果:
阳性对照Vc清除自由基,实例3较对比例1、对比例2、对比例3更能有效的清除自由基,清除率达到34.16%,结果表明实例3制备的二裂酵母发酵产物溶胞物具有皮抗氧化的功效。
以上,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域,但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
Claims (10)
1.一种利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于,包含以下步骤:
S1、外植体的选材,选取石斛、人参、雪莲的根段、梗芽、茎尖、叶片、胚、花梗节间的幼嫩部分,切取长0.5-4cm的切段作为外植体;
S2、外植体的消毒,用自来水清洗干净,在饱和漂白粉上清液中浸泡5-20min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,置于超净工作台上,先用75%酒精消毒10-60s,无菌水清洗2-4次,再用0.1-0.3%升汞浸泡5-20min,无菌水冲洗3-8次;
S3、外植体的接种,将消毒后的外植体接种到经设计含有a-萘乙酸、6-苄氨基腺嘌呤的改良的MS诱导培养基上,诱导丛生芽,接种后的外植体放在培养室培养,培养条件为温度20-35℃、光照强度1000-3000lx、光照时间5-20h/d;
S4、外植体续代培养,将外植体诱导生长出的不定芽的叶片切成5-25mm2大小接种在添加了50-300mL/L椰汁的改良MS培养基上进行原球茎诱导,培养条件为温度20-25℃、光照强度500-2000lx、光照时间10-20h/d,得到类原球茎;
S5、植物外泌体的获取,将类原球茎收集,经组织破碎机破碎、200-500目的滤网过滤获得植物外泌体滤液备用;
S6、将长双歧杆菌婴儿亚种(YJXB-01-21)斜面挑取一环接种到PDA培养基上,30-40℃下进行活化培养1-3d,得到活化的长双歧杆菌婴儿亚种一级种子液,将活化的长双歧杆菌婴儿亚种一级种子液接种到改良MRS液体培养基中,30-40℃下发酵培养1-3d,得到长双歧杆菌婴儿亚种二级种子液;
S7、配制培养基按重量百分比包括:1.0-5.0%葡萄糖,0.2-3.0%大豆蛋白胨,0.1-0.3%磷酸氢二钾,0.05-0.20%氯化钠,0.10-0.3%酵母粉,115-121℃灭菌20-30min,冷却后将长双歧杆菌婴儿亚种二级种子液接种于灭菌后的培养基中30-40℃培养,
S8、在培养至10-24h时,流加植物外泌体进入发酵液中,流加体积为总体积的0.1-5.0%,流加时间为1-24h,流加后继续培养1-3d,
S9、发酵完成后,将发酵液超声处理后,高压均质,过滤,巴氏灭菌,加入防腐体系,获得二裂酵母发酵产物溶胞物。
2.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S1外植体选取石斛茎尖幼嫩部分切取0.5cm。
3.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S2外植体在饱和漂白粉上清液中浸泡10-15min,浸泡时不断搅动,浸泡后的外植体用流水冲洗干净,用75%酒精消毒50-60s,无菌水清洗2次,再用0.1%升汞浸泡10-15min,无菌水冲洗3-4次。
4.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S3所述的a-萘乙酸含量为0.5-2.0mg/L、6-苄氨基腺嘌呤5.0-20.0mg/L。
5.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S3改良MS培养基按重量百分比包括:硝酸钾1.90g/L,硝酸铵1.65g/L,磷酸二氢钾0.17g/L,硫酸镁0.37g/L,氯化钙0.44g/L,碘化钾0.83g/L,硼酸0.006g/L,硫酸锰0.022g/L,硫酸锌0.009g/L,钼酸钠0.001g/L,硫酸铜0.000025g/L,氯化钴0.000025g/L,硫酸亚铁0.0278g/L,肌醇0.10g/L,盐酸硫胺素0.0001g/L,甘氨酸0.002g/L,盐酸吡哆醇0.0005g/L,烟酸0.0005g/L,乙二胺四乙酸二钠0.0373g/L,pH值5.7±0.1(25℃)。
6.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S4所述的不定芽的叶片切成5-10mm2大小,培养条件为温度22-25℃、光照强度800-1000lx、光照时间15-20h/d。
7.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S5类原球茎需在低温10-20℃用组织破碎机进行,过400-500目的滤网。
8.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S6的改良MRS液体培养基按重量百分比包括:蛋白胨10g/L,牛肉膏粉2.0g/L,酵母膏粉4g/L,葡萄糖20g/L,吐温80 1.08g/L,磷酸氢二钾(无水)2g/L,乙酸钠(无水)5g/L,柠檬酸三铵(无水)2g/L,硫酸镁(无水)2.0g/L,余量纯净水;所述S7配制培养基按重量百分比包括:2.0-3.0%葡萄糖,0.5-1.0%大豆蛋白胨,0.10-0.15%磷酸氢二钾,0.05-0.10%氯化钠,0.10-0.2%酵母粉,120-121℃灭菌20-25min,冷却后将长双歧杆菌婴儿亚种二级种子液接种于灭菌后的培养基中35-37℃培养。
9.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S8在培养至10-15h时,使用流动泵将植物外泌体流加入发酵液中,流加体积为总体积的0.1-0.3%,流加时间为1-5h,流加后继续培养1-2d。
10.根据权利要求1所述的利用植物外泌体发酵制备二裂酵母产物溶胞物的方法,其特征在于:所述S9发酵液的超声处理时间为2-4h,功率200-800W,所述S9发酵液的高压均质压力为10-15Mpa,均质时间为10-20min,所述S9过滤为陶瓷膜过滤,过滤孔径为0.50-2.0um,所述S9加入防腐体系为1,2-己二醇1-5%,1,3丁二醇1-5%,对羟基苯乙酮0.1-1.0%。
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