CN111363768A - 胶红酵母 cicc胞外多糖的制备及其在抗肝癌中的应用 - Google Patents
胶红酵母 cicc胞外多糖的制备及其在抗肝癌中的应用 Download PDFInfo
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Abstract
本发明提供了一种胶红酵母CICC胞外多糖的制备方法及其在抗肝癌中的应用,高压蒸汽灭菌固体培养基,冷却后倒平板,接种菌株,活化,分离纯化,再次培养菌种,得胶红酵母菌种的纯培养菌落;接种于液体培养基扩大培养,分离上清液与胶红酵母细胞,提取胞外多糖;离心分离胶红酵母细胞与培养液,取上清液,依次在不同浓度的乙醇溶液中分别进行醇沉,离心,得胶红酵母胞外多糖粗品;干燥,充分溶解于蒸馏水中,冷冻干燥,分离、纯化,制得胶红酵母R.mucilaginosa CICC 33013胞外多糖。其用于抗肝癌中。该制备方法易与酵母菌体细胞分离、可通过液体培养发酵实现连续生产。
Description
技术领域
本发明属于生物技术领域,涉及一种胶红酵母CICC胞外多糖的制备方法;本发明还涉及一种该胞外多糖在抗肝癌方面的应用。
背景技术
酵母菌胞外多糖(Yeast extracellular polysaccharide, YEPS)作为微生物 多糖的一个大类,具有多种生理活性,可用于生产有机体免疫调节剂应用 在食品与药品中,或作为潜在的天然抗氧化剂,具有广阔的市场前景。
发明内容
本发明的目的是提供一种胶红酵母CICC胞外多糖的制备方法,解决了现有技术中存在的问题。
本发明的另一个目的是提供一种上述制备方法制得的胶红酵母CICC胞外多糖在抗肝癌中的应用。
为实现上述目的,本发明所采用的技术方案是:一种胶红酵母CICC胞外多糖的制备方法,具体按以下步骤进行:
1)配制用于胶红酵母 R.mucilaginosaCICC 33013菌株生长的 YPD 固体培养基(1%酵母膏、 2%葡萄糖、 2%蛋白胨和 2%琼脂粉),取10 个培养皿和1 个100mL 蒸馏水锥形瓶;将YPD 固体培养基在121℃的温度下高压蒸汽灭菌20min;
2)物料高压灭菌后,待 YPD 固体培养基冷却至 50℃左右, 于超净工作台中倒平板备用;
3)将胶红酵母 R.mucilaginosaCICC 33013 菌株保存管从-80℃冰箱中取出,室温下接种于冷却好的平板上,于 28℃培养 2~3d 进行菌种活化;
4)用移液器吸取 40μL 液体悬浮菌液, 稀释涂布法将活化好的胶红酵母R.mucilaginosaCM-1 菌进一步分离纯化;
5)挑选单个菌落,按照 4)所述的方法再次培养菌种,得到胶红酵母菌种的纯培养菌落;
6)挑取胶红酵母 R.mucilaginosa CICC 33013 菌株的单菌落于载玻片上,滴加无菌水,放置于显微镜下进行观察;使胶红酵母 R. mucilaginosa CICC 33013 菌悬液在载玻片上形成均匀薄膜。待载玻片自然干燥后,滴加少量美蓝染液进行染色, 后室温下静置3min,然后镜检;
7)胶红酵母 R.mucilaginosaCICC 33013 细胞液体培养基培养挑取活化好的胶红酵母 R.mucilaginosaCICC 33013 菌落接种于种子液体培养基进行扩大培养,配制 4000mL的酵母菌无机培养液,种子液的接种量为 5%,于 28℃、 80-100r/min条件下培养 6d 后,分离上清液与胶红酵母细胞,提取胶红酵母 R.mucilaginosa CICC 33013菌胞外多糖;
8)胶红酵母 R.mucilaginosaCICC 33013 胞外粗多糖的分离将胶红酵母液体培养基于 9000r/min、4℃、6min 条件下离心分离胶红酵母 R.mucilaginosaCICC 33013细胞与培养液,取上清液,浓缩处理后备用;
9)将浓缩好的含有胶红酵母胞外多糖的上清液依次在浓度为50%、 60%、70%、80%与90%的乙醇溶液中分别醇沉24h后;移入 50mL 离心管中,于4℃、5000r/min 条件下离心10min,得到胶红酵母胞外多糖粗品;置于通风、干燥处,挥发乙醇;量取 30mL 蒸馏水加入到含有胞外多糖的离心管内,使粗胞外多糖样品充分溶解,冷冻干燥后保存备用;
10)称取100 g 的DEAE-Cellulose 52,置于含200mL蒸馏水的容器中溶胀24h,以0.5mol/LNaOH处理30min, 抽滤,蒸馏水洗至中性后,以0.5mol/L HCI处理30min,抽滤,再用0.5mol/L NaOH漂洗30min,使之转为OH-型,后用0.5mol/L NaCl溶液清洗,使之转为Cl-型,置于缓冲液内进行填料,保证填料柱子完整致密,无小泡;剩余树脂浸泡在75%的乙醇溶液中保留备用;
11)直立固定层析柱,加缓冲溶液,将DEAE-Cellulose 52处理后的浑浊液缓慢的注入到玻璃柱内,自然沉降,当树脂高度到达填料柱高度的三分之一时,使液体均匀地通过柱下方的排液口流出,确保树脂上表面平整且不能暴露在缓冲液外,观察填料柱无小气泡产生;
12)称取2.00g的胶红酵母胞外多糖样品,用50mL蒸馏水溶解,缓慢滴加10%的三氯乙酸, 放置于4℃的冰箱内24h后,于5000r/min、4℃条件下离心10min,去除白色沉淀,将胶红酵母胞外多糖溶液醇沉后进行冻干处理,得到胶红酵母R. mucilaginosa胞外多糖。
从胶红酵母 R.mucilaginosaCICC 33013 液体培养基中分离得到水溶性胞外多糖 RmEPS,其产量为 7.35g/L。多糖 RmEPS 经分离纯化得到单一纯品 RmEPS-11,结果表明, RmEPS-11 是由摩尔比为 1.3:4.4:7.8:86.4 的***糖(Ara),半乳糖(Gal),葡萄糖(Glc)和甘露糖(Man)组成,平均分子量为 2.3×105Da。基于 FT-IR, NMR 和甲基化分析,确定了多糖组分RmEPS-11骨架是由(1→3)-连接的Man组成,分支上是由(1→2)-连接的Glc、(1→2,6)-连接的 Gal 和(1→2,3,4)-连接的 Ara 组成。此外,从胶红酵母R.mucilaginosaCICC 33013 菌株培养液中提取分离出水溶性胞外多糖 REPS,其产量为6.2g/L。多糖 REPS 经分离纯化得到单一多糖组分 REPS2-A,结果表明, REPS2-A 是由摩尔比为 63.1:0.2:18.3:18.3 的半乳糖(Gal)、***糖(Ara)、葡萄糖(Glc)和甘露糖(Man)组成,平均分子量为 7.125×106Da。基于 FT-IR, NMR 和甲基化分析,确定了多糖组分 REPS2-A 是高度分支的多糖,是由(1→3)-连接的 Gal 为骨架组成,分支是由(1→2)-连接的 Glc、(1→4)-连接的 Man、 (1→3)-连接的 Glc、 (1→4,6)-连接的 Man 和(1→2,3,4)-连接的 Ara 组成。
通过对胶红酵母 R.mucilaginosaCICC 33013 菌液体培养基进行浓缩、醇沉、脱蛋白、脱色和透析等方法得到胶红酵母胞外多糖 RmEPS,经苯酚-硫酸法测定胶红酵母胞外多糖量为 7.35g/L。通过 DEAE-52 纤维素阴离子交换层析分离获得 RmEPS-1、RmEPS-2。
本发明的另一个技术方案是:一种上述胶红酵母CICC胞外多糖在抗肝癌方面的应用。
本发明制备方法制得的胞外多糖(EPS)是由酵母细胞产生的,分布于细胞壁之外,或附着到细胞壁上或以粘液的形式分泌到细胞外环境的同多糖或者异多糖,具有易与酵母菌体细胞分离、可通过液体培养发酵实现连续生产等优点。
具体实施方式
下面结合具体实施方式对本发明进行详细说明。
实施例1
配制用于胶红酵母 R.mucilaginosaCICC 33013菌株生长的 YPD 固体培养基(1%酵母膏、 2%葡萄糖、 2%蛋白胨和 2%琼脂粉),取10 个培养皿和1 个100mL 蒸馏水锥形瓶;将YPD 固体培养基在121℃的温度下高压蒸汽灭菌20min;待 YPD 固体培养基冷却至 50℃左右,于超净工作台中倒平板;将胶红酵母 R.mucilaginosaCICC 33013 菌株保存管从-80℃冰箱中取出,室温下接种于冷却好的平板上,于 28℃培养 2~3d 进行菌种活化;用移液器吸取 40μL 液体悬浮菌液,稀释涂布法将活化好的胶红酵母 R.mucilaginosaCICC 33013菌进一步分离纯化;挑选单个菌落,用移液器吸取 40μL 液体悬浮菌液, 稀释涂布法将活化好的胶红酵母 R.mucilaginosaCICC 33013菌进一步分离纯化;得到胶红酵母菌种的纯培养菌落;挑取胶红酵母 R.mucilaginosaCICC 33013 菌株的单菌落于载玻片上,滴加无菌水,放置于显微镜下进行观察;使胶红酵母 R. mucilaginosa CICC 33013菌悬液在载玻片上形成均匀薄膜。待载玻片自然干燥后,滴加少量美蓝染液进行染色, 后室温下静置3min,然后镜检;胶红酵母 R.mucilaginosaCICC 33013 细胞液体培养基培养挑取活化好的胶红酵母 R.mucilaginosaCICC 33013 菌落接种于种子液体培养基进行扩大培养,配制4000mL 的酵母菌无机培养液,种子液的接种量为 5%,于 28℃、80-100r/min条件下培养6d 后,分离上清液与胶红酵母细胞,提取胶红酵母 R.mucilaginosaCICC 33013菌胞外多糖;胶红酵母 R.mucilaginosaCICC 33013 胞外粗多糖的分离将胶红酵母液体培养基于9000r/min、4℃、6min 条件下离心分离胶红酵母 R.mucilaginosa CICC 33013细胞与培养液,取上清液,浓缩处理后备用;将浓缩好的含有胶红酵母胞外多糖的上清液依次在浓度为50%、 60%、 70%、 80%与 90%的乙醇溶液中分别醇沉24h后;移入 50mL 离心管中,于4℃、5000r/min 条件下离心 10min,得到胶红酵母胞外多糖粗品;置于通风、干燥处,挥发乙醇;量取 30mL 蒸馏水加入到含有胞外多糖的离心管内,使粗胞外多糖样品充分溶解,冷冻干燥后保存备用;称取100 g 的DEAE-Cellulose 52,置于含200mL蒸馏水的容器中溶胀24h,以0.5mol/LNaOH处理30min, 抽滤,蒸馏水洗至中性后,以0.5mol/L HCI处理30min,抽滤,再用0.5mol/L NaOH漂洗30min,使之转为OH-型,后用0.5mol/L NaCl溶液清洗,使之转为Cl-型,置于缓冲液内进行填料,保证填料柱子完整致密,无小泡;剩余树脂浸泡在75%的乙醇溶液中保留备用;直立固定层析柱,加缓冲溶液,将DEAE-Cellulose 52处理后的浑浊液缓慢的注入到玻璃柱内,自然沉降,当树脂高度到达填料柱高度的三分之一时,使液体均匀地通过柱下方的排液口流出,确保树脂上表面平整且不能暴露在缓冲液外,观察填料柱无小气泡产生;称取2.00g的胶红酵母胞外多糖样品,用50mL蒸馏水溶解,缓慢滴加10%的三氯乙酸, 放置于4℃的冰箱内24h后,于5000r/min、4℃条件下离心10min,去除白色沉淀,将胶红酵母胞外多糖溶液醇沉后进行冻干处理,得到胶红酵母CICC胞外多糖。
不同胶红酵母(R.mucilaginosa)菌株胞外多糖的抗氧化活性,实验结果显示,多糖组分 RmEPS-11 在浓度为 8mg/mL 时自由基清除活性达到 66.15%、 ABTS 自由基清除活性为 82.8%、还原力(0.753)和氧自由基吸收能力(560.38±6.45μMTE/g)。同时,多糖组分 REPS2-A 在浓度 8mg/mL 时 DPPH 自由基清除活性为 49.1%, ABTS 自由基清除活性为51.2%,还原力为 0.352。 由多糖抗氧化活性实验结果可知, 在相同条件下,胶红酵母胞外多糖的分子量越小,其抗氧化能力越强。
多糖组分 RmEPS-11 对小鼠异烟肼(INH)和利福平(RIF)引起的肝毒性的保护机制。结果显示,与 INH 和 RIF 处理的小鼠相比,多糖 RmEPS-11 给药后小鼠血清 ALT、AST 水平和肝脏 MDA 含量显著降低, 肝脏 SOD 活性和 GSH 含量显著恢复。此外,肝脏的组织病理学损伤和凋 亡肝细胞的数量也通过处理显著改善。 多糖 RmEPS-11 保肝护肝机制研究 表明,肝功能保护作用主要是由于 CYP2E1 mRNA 表达水平的调节,导致 有害自由基和 ROS 的形成量减少,同时伴随着其抗氧化能力的显著诱导, 多糖组分 RmEPS-11 通过抑制肝 CYP2E1 基因抑制脂质过氧化、增加自由 基的清除能力达到护肝的效果。 实验结果也验证了多糖组分 RmEPS-11 相 比 REPS2-A 具有较高的自由基清除能力。
确定多糖组分 REPS2-A 对肝癌细胞 Hep-G2 抑制作用最佳的浓度及时效性。当多糖组分 REPS2-A 的浓度为 1mg/mL 时, 肝癌细胞 Hep-G2 的抑制率高于 IC50 时的抑制率,抑制效果明显优于其它浓度。 肝癌细胞Hep-G2 的自发凋亡率是 0.40%,当多糖REPS2-A 浓度为 1mg/mL, 处理 24h、48h 与 72h,肝癌细胞 Hep-G2 的凋亡率分别为77.70%、 88.18%、 97.08%。多糖组分 REPS2-A 使肝癌细胞 Hep-G2 阻滞发生在 G1/S 期。多糖组分REPS2-A 能有效抑制肝癌细胞的增殖,且存在剂量效应。相比于多糖RmEPS-11,多糖组分 REPS2-A 具有显著的抑制肝癌细胞活性, 主要归因于多糖组分 REPS2-A 是由 (1→3)-连接的活性半乳糖(Gal)为骨架组成。
Claims (3)
1.一种胶红酵母CICC胞外多糖的制备方法,其特征在于,具体按以下步骤进行:
1)配制用于胶红酵母 R.mucilaginosaCICC 33013菌株生长的 YPD 固体培养基,取10个培养皿和 1个100mL 蒸馏水锥形瓶;将YPD 固体培养基、培养皿和锥形瓶在121℃的温度下高压蒸汽灭菌20min;待 YPD 固体培养基冷却至 50℃,于超净工作台中倒平板备用;
2)将胶红酵母 R.mucilaginosaCICC 33013 菌株保存管从-80℃冰箱中取出,室温下接种于冷却好的平板上,于 28℃培养 2~3d 进行菌种活化;
3)用移液器吸取 40μL 液体悬浮菌液,稀释涂布法将活化好的胶红酵母R.mucilaginosaCICC 33013菌进一步分离纯化;
4)挑选单个菌落,按照3)所述的方法再次培养菌种,得到胶红酵母菌种的纯培养菌落;
5)胶红酵母 R.mucilaginosaCICC 33013 细胞液体培养基培养挑取活化好的胶红酵母 R.mucilaginosa CICC 33013 菌落接种于种子液体培养基进行扩大培养,配制 4000mL的酵母菌无机培养液,种子液的接种量为 5%,于 28℃、80-100r/min条件下培养 6d 后,分离上清液与胶红酵母细胞,提取胶红酵母 R.mucilaginosaCICC 33013菌胞外多糖;
6)将胶红酵母液体培养基于 9000r/min、 4℃、 6min 条件下离心分离胶红酵母R.mucilaginosaCICC 33013细胞与培养液,取上清液,浓缩处理后备用;
7)将浓缩好的含有胶红酵母胞外多糖的上清液依次在浓度为50%、 60%、70%、80%与90%的乙醇溶液中分别醇沉24h后;移入 50mL 离心管中,于4℃、5000r/min 条件下离心10min,得到胶红酵母胞外多糖粗品;置于通风、干燥处,挥发乙醇;量取 30mL 蒸馏水加入到含有胞外多糖的离心管内,使粗胞外多糖样品充分溶解,冷冻干燥后保存备用;
8)称取100 g 的DEAE-Cellulose 52,置于含200mL蒸馏水的容器中溶胀24h,以0.5mol/LNaOH处理30min, 抽滤,蒸馏水洗至中性后,以0.5mol/L HCI处理30min,抽滤,再用0.5mol/L NaOH漂洗30min,使之转为OH-型,后用0.5mol/L NaCl溶液清洗,使之转为Cl-型,置于缓冲液内进行填料,保证填料柱子完整致密,无小泡;剩余树脂浸泡在75%的乙醇溶液中保留备用;
9)直立固定层析柱,加缓冲溶液,将DEAE-Cellulose 52处理后的浑浊液缓慢的注入到玻璃柱内,自然沉降,当树脂高度到达填料柱高度的三分之一时,使液体均匀地通过柱下方的排液口流出,确保树脂上表面平整且不能暴露在缓冲液外,观察填料柱无小气泡产生;
10)称取2.00g的胶红酵母胞外多糖样品,用50mL蒸馏水溶解,缓慢滴加10%的三氯乙酸, 放置于4℃的冰箱内24h后,于5000r/min、4℃条件下离心10min,去除白色沉淀,将胶红酵母胞外多糖溶液醇沉后进行冻干处理,得到胶红酵母CICC胞外多糖。
2.如权利要求1所述的胶红酵母CICC胞外多糖的制备方法,其特征在于,所述步骤1)中,YPD 固体培养基中含1%酵母膏、2%葡萄糖、2%蛋白胨和 2%琼脂粉。
3.一种权利要求1所述制备方法制得的胶红酵母CICC胞外多糖在抗肝癌中的应用。
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Application publication date: 20200703 |