CN116554359A - 一种银耳多糖及其制备方法和在改善肠道环境中的应用 - Google Patents
一种银耳多糖及其制备方法和在改善肠道环境中的应用 Download PDFInfo
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Abstract
本发明属于多糖技术领域,具体涉及一种银耳多糖的制备方法,包括以下步骤:(1)将干制的银耳子实体粉碎后过50‑70目筛,得到银耳干粉;(2)将银耳干粉与蒸馏水按料液比1∶55‑65g/ml的比例搅拌均匀,加热煮沸2‑3h,得到多糖粗提液;(3)待多糖粗提液冷却至室温后,以5000‑7000r/min的转速离心5‑10min,取上清液旋转蒸发至原上清液体积的1/8‑1/10,得到浓缩液;(4)向浓缩液中加入其体积4‑6倍体积浓度为85‑95%的乙醇溶液,搅拌5‑10min后于4‑6℃静置12‑14h,沉淀冻干后得到银耳多糖。本发明还提供制备银耳多糖在制备改善肠道环境药物和/或保健品中的应用。本发明方法制备得到的银耳多糖,具有较高提取率,方法简单,其显著改变样品的微生物群落组成。
Description
技术领域
本发明属于多糖技术领域,具体涉及一种银耳多糖及其制备方法和在改善肠道环境中的应用。
背景技术
近年来,人民的生活水平不断提高,人们对美好生活有了更高的要求,女性对皮肤保养也越发注重,在市场需求的推动下,护肤品行业愈发聚焦于抗衰老、美白等功效。据研究表明多糖因具有良好的抗氧化、美白、吸湿、保湿等生物活性,常作为具有护肤功效物质而被添加在护肤品中。
银耳多糖是一种具有抗氧化、抗衰老、抗肿瘤、降血压、降血脂、增强免疫等功能,且较为安全的天然活性成分。银耳多糖可广泛用于医疗、保健、食品加工、护肤美容、畜牧养殖等多方面领域。而且原材料来源广泛,可以为银耳多糖带来更大的应用空间,对进一步研究银耳多糖的理化性质和生物活性具有重要意义。随着多糖生物活性的开发,越来越多的研究致力于多糖对皮肤保湿、抗老化或美白作用。
但是对于银耳多糖在被摄入口腔后,需要经过包括口腔-胃-肠以及结肠等复杂的消化程序,在这个过程中样品的稳定性和生物利用度会受到消化环境的影响,其功能活性也会受到一定影响,未消化完的部分会进入结肠,被微生物分解利用(发酵),产生一些代谢物摄入口腔后。而现有技术中,银耳多糖进入口腔后,在消化过程中,其总糖、还原糖、蛋白质、糖醛酸等含量、分子量、单糖组成、抗氧化活性和酪氨酸酶、弹性蛋白酶的抑制活性、pH值和短链脂肪酸的含量如何变化,以及肠道微生物结构如何变化,鲜少有研究。
发明内容
本发明旨在解决上述技术问题,提供一种银耳多糖的制备方法,该方法制备出来的银耳多糖能够增加人体肠道微生物种类。
本发明的技术方案为:
一种银耳多糖的制备方法,包括以下步骤:
(1)将干制的银耳子实体粉碎后过50-70目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶55-65g/ml的比例搅拌均匀,加热煮沸2-3h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以5000-7000r/min的转速离心5-10min,取上清液旋转蒸发至原上清液体积的1/8-1/10,得到浓缩液;
(4)向浓缩液中加入其体积4-6倍体积浓度为85-95%的乙醇溶液,搅拌5-10min后于4-6℃静置12-14h,沉淀冻干后得到银耳多糖。
优选地,本发明所述银耳多糖的制备方法,包括以下步骤:
(1)将干制的银耳子实体粉碎后过60目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶60g/ml的比例搅拌均匀,加热煮沸2h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以5000r/min的转速离心5min,取上清液旋转蒸发至原上清液体积的1/10,得到浓缩液;
(4)向浓缩液中加入其体积4倍体积浓度为95%的乙醇溶液,搅拌5min后于4℃静置12h,沉淀冻干后得到银耳多糖。
本发明还提供所述的方法制备得到的银耳多糖,其提取率为14.27%,总糖含量为82.19%,还原糖含量为1.01%,糖醛酸含量为26.77%,蛋白含量为3.36%,水分含量为6.28%。
本发明还提供所述银耳多糖在制备改善肠道环境药物和/或保健品中的应用,所述改善肠道环境包括改变人体肠道微生物群落组成,所述改变人体肠道微生物群落组成包括提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门中一种或多种的丰度,通过16SrDNA扩增子测序结果表明在微生物的门水平上,本发明的银耳多糖可提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门的丰度。通过OTU指数分析发现,本发明的银耳多糖可以使人体肠道菌群的群落丰富度增加,随着发酵时间延长添加本发明的银耳多糖的发酵液中菌群OTU数值增大,表明本发明的银耳多糖可以提高菌群种类,使人体肠道菌群多样性增多。整体言之,结肠发酵会显著改变样品的微生物群落组成,摄入了本发明的银耳多糖后会增加人体肠道微生物的群落结构。
食物在被摄入口腔后,需要经过包括口腔-胃-肠以及结肠等复杂的消化程序。在这个过程中样品的稳定性和生物利用度会受到消化环境的影响,其功能活性也会受到一定影响。未消化完的部分会进入结肠,被微生物分解利用(发酵),产生一些代谢物。因此,需要对本发明银耳多糖在消化过程中的变化和活性进行进一步研究。而想要准确探究银耳多糖的消化特性,构建活体动物模型是最佳的选择。但是活体动物模型具有高成本、高耗时、以及伦理和不可抗力因素影响。而体外模拟消化模型不仅能反映食物摄入后的消化利用情况,更具有耗时短、成本低、可重复性强且不受道德伦理约束等优点。因此,为了更好的利用银耳多糖,本发明探索银耳多糖的体外模拟消化特性以及结肠发酵过程中,银耳多糖总糖、还原糖、蛋白质、糖醛酸等成分的变化,以及银耳多糖分子量和单糖组成的变化,并对其抗氧化活性和酪氨酸酶、弹性蛋白酶的抑制活性进行了评估。此外,本发明还测定分析了结肠发酵过程中pH的变化和短链脂肪酸的含量,以及肠道微生物的变化情况。本发明银耳多糖经体外模拟消化(口腔、胃、小肠)后总糖含量无明显变化,还原糖在口腔消化阶段无明显变化,胃、小肠消化过程中有少量还原糖生成,分子量无明显变化,单糖种类组成无变化,但各单糖含量发生了改变。经结肠发酵后,我们发现总糖含量显著降低,还原糖含量明显增多;分子量明显变小,单糖组成种类和含量均发生改变。经体外模拟消化后本发明银耳多糖的ABTS、DPPH自由基清除能力有一定的提高,但铁还原能力(FRAP)整体降低,酪氨酸酶抑制活性和弹性蛋白酶抑制活性也略有降低。但经结肠发酵后,添加本发明银耳多糖的实验组,其DPPH自由基清除活性整体高于未添加本发明银耳多糖的空白组。添加本发明银耳多糖的发酵组其FRAP值整体低于空白组,分析原因可能是由于Fe3+与多糖在消化过程中发生反应形成了本发明银耳多糖和铁的配合物。添加本发明银耳多糖的实验组,其酪氨酸酶抑制能力和弹性蛋白酶抑制能力均整体高于空白组。在结肠发酵阶段,发酵液的pH值整体下降,发酵6h时下降最快,发酵12h时pH变化不再明显。同时,我们对每个时间点的发酵液进行了短链脂肪酸的定性、定量分析。结果表明,随着发酵时间增加,发酵液中会生成大量短链脂肪酸,其中乙酸含量最高,丁酸次之。
由于采用上述技术方案,本发明的有益效果为:
本发明方法制备得到的银耳多糖,具有较高提取率,方法简单,其在制备改善肠道环境药物和/或保健品中的应用有一定的成效,特别是显著改变样品的微生物群落组成,摄入了本发明的银耳多糖后会增加人体肠道微生物的群落结构,提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门的丰度。
附图说明
图1为本发明实施例1中银耳多糖样品图及其不同质量浓度溶液。
图2为本发明实施例1银耳多糖体外模拟消化、结肠发酵后的总糖含量,其中A为体外模拟口腔、胃、小肠消化阶段,空白对照组为未加消化酶组,实验组为添加消化酶组;B为结肠发酵阶段,Blank为未添加TP的空白对照发酵组,FTP为添加TP的发酵实验组。以不同大写字母表示空白组间的显著性差异,以不同小写字母表示实验组间的显著性差异,下同。
图3为本发明实施例1银耳多糖体外模拟消化、结肠发酵后的还原糖含量,其中图2-A为体外模拟口腔、胃、小肠消化阶段,图2-B为结肠发酵阶段。
图4为本发明实施例1银耳多糖体外模拟消化及结肠发酵前后HPGPC色谱图,其中,BLK代表未经消化的TP空白对照组,Oral代表口腔消化后的TP,Gastric代表胃消化后的TP,S-testine代表小肠消化后的TP,FTP24h代表经结肠发酵24h后的TP,下同。
图5为本发明实施例1银耳多糖体外模拟消化及结肠发酵后的单糖组成,其中,1为岩藻糖,2为盐酸氨基半乳糖,3为鼠李糖,4为阿拉伯糖,5为盐酸氨基葡萄糖,6为半乳糖,7为葡萄糖,8为N-乙酰-D氨基葡萄糖,9为木糖,10为甘露糖,11为果糖,12为核糖,13为半乳糖醛酸,14为古罗糖醛酸,15为葡萄糖醛酸,16为甘露糖醛酸。
图6为本发明实施例1银耳多糖口腔、胃、小肠的体外模拟消化抗氧化活性。
图7为本发明实施例1银耳多糖结肠发酵抗氧化活性。
图8为本发明实施例1银耳多糖体外模拟消化、结肠发酵后的酪氨酸酶抑制活性。
图9为本发明实施例1银耳多糖体外模拟消化、结肠发酵后的弹性蛋白酶抑制活性。其中A为体外模拟口腔、胃、小肠消化阶段,B为结肠发酵阶段。
图10为本发明实施例1银耳多糖结肠发酵阶段pH值的变化。
图11为本发明各样品OTU个数分布图。
图12为本发明PCoA分析图。
图13为本发明微生物在门水平的丰度。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种银耳多糖的制备方法,包括以下步骤:
(1)将干制的银耳子实体粉碎后过60目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶60g/ml的比例搅拌均匀,加热煮沸2h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以5000r/min的转速离心5min,取上清液旋转蒸发至原上清液体积的1/10,得到浓缩液;
(4)向浓缩液中加入其体积4倍体积浓度为95%的乙醇溶液,搅拌5min后于4℃静置12h,沉淀冻干后得到银耳多糖,其样品及不同质量浓度溶液样品见图1,如图1所示,银耳多糖呈疏松、絮状的白色片状,对银耳多糖进行水溶性分析发现,其质量浓度为1mg/mL时溶液澄清、透明,可以完全溶解;5mg/mL时溶液呈淡黄色,能完全溶解;10mg/mL时溶液呈均匀胶状,颜色为黄色,能全部溶解。对银耳多糖基本成分进行了测定,结果如表1所示。
表1实施例1银耳多糖提取率及基本成分含量
测定指标 | 数值结果 |
提取率(%) | 14.27±0.46 |
总糖含量(%) | 82.19±0.46 |
还原糖含量(%) | 1.01±0.01 |
糖醛酸含量(%) | 26.77±2.32 |
蛋白质含量(%) | 3.36±0.26 |
水分含量(%) | 6.28±0.02 |
其中,实施例1银耳多糖的提取率按公式(1)计算:
式中:n为TP粗提液稀释倍数,C为粗提液的质量浓度(mg/mL),V为粗提液体积(mL),m为银耳干粉的质量(mg)。
以苯酚-硫酸法、DNS法、考马斯亮蓝法、烘箱干燥法和间羟基联苯法分别测定实施例1银耳多糖样品的总糖、还原糖、蛋白质、水分和糖醛酸的含量。
实施例2
(1)将干制的银耳子实体粉碎后过0目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶55g/ml的比例搅拌均匀,加热煮沸3h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以6000r/min的转速离心10min,取上清液旋转蒸发至原上清液体积的1/10,得到浓缩液;
(4)向浓缩液中加入其体积5倍体积浓度为85%的乙醇溶液,搅拌10min后于4℃静置14h,沉淀冻干后得到银耳多糖。
实施例3
(1)将干制的银耳子实体粉碎后过50目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶65g/ml的比例搅拌均匀,加热煮沸2.5h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以7000r/min的转速离心5min,取上清液旋转蒸发至原上清液体积的1/8,得到浓缩液;
(4)向浓缩液中加入其体积6倍体积浓度为95%的乙醇溶液,搅拌5min后于6℃静置13h,沉淀冻干后得到银耳多糖。
实施例4:银耳多糖的体外模拟消化特性及其对人体肠道微生物的影响
1、试验方法
(1)体外模拟口腔、胃和小肠消化:
将1g实施例1中的银耳多糖分散在50mL蒸馏水中,然后加入50mL模拟唾液(含3.25mgα-淀粉酶、62.5μL 0.15mol/L六水合氯化镁、20μL 0.5mol/L碳酸铵、1.89mL0.5mol/L氯化钾、0.46mL 0.5mol/L磷酸二氢钾和0.85mL 1mol/L碳酸氢钠)。均匀混合后,调整pH值至6.5,将样品置于37℃的恒温培养箱中,培养15min,进行模拟口腔消化。口腔消化后,取出20mL,置于沸水中10min灭活酶,12000r/min离心5min,取上清液测定活性。
之后,将残留物和50mL模拟胃液(含有125mg胃蛋白酶,50μL 0.15mol/L六水合氯化镁,62.5μL 0.5mol/L碳酸铵,0.86mL 0.5mol/L氯化钾,0.11mL 0.5mol/L磷酸二氢钾,1.56mL 1mol/L碳酸氢钠,和2mol/L 1.48mL氯化钠)均匀混合,pH值调整为2,并放置在37℃恒温孵化器,进行模拟胃消化1h。反应结束后,取20mL的样品溶液进行活性测定,样品处理方法与模拟口腔取样相同。
最后,将剩余部分与50mL模拟肠液(含20mg胰酶、30mg胆盐、0.14mL 0.15mol/L氯化镁、0.85mL 0.5mol/L氯化钾、0.1mL 0.5mol/L磷酸二氢钾、5.31mL 1mol/L碳酸氢钠、1.2mL 2mol/L氯化钠)混合,调整pH值为7,然后放入37C恒温培养箱中消化2h。肠消化过程结束后,取20mL进行分析,其余部分透析、冻干后备用。设置3个平行组,进行口腔、胃、肠内模拟消化,无酶组进行对照分析。
(2)结肠发酵:
人类粪便发酵方案经西南林业大学学术委员会学术道德与科技伦理委员分会审核批准(伦理批准号SWFU-2021015)。首先,从三名健康的人类志愿者身上收集了新鲜的人类粪便(他们至少三个月没有摄入任何药物,也没有同时出现胃肠道疾病或其他可能影响胃肠道的疾病)。然后,将所有新鲜粪便样品混合,后将粪便分散于无菌磷酸盐缓冲液(0.1M,pH 7.2)中,制备粪便浆液(10%,w/v)。然后将得到的粪便浆液以无菌纱布过滤两次,去除大的粪便残留物,获得最终的人粪浆。
其次,将4.5g氯化钠、4.5g氯化钾、2g果胶、4g粘蛋白、0.69g六水合硫酸镁,1g瓜尔胶、0.8g硫酸镁水,1g半胱氨酸、0.5g磷酸二氢钾、0.5g磷酸氢二钾、3g酪蛋白、2g阿拉伯半乳糖、1.5g碳酸氢钠、0.4g胆汁酸、0.005g七水合硫酸亚铁、0.08g氯化钙,1mL吐温80和4mL刃天青(0.025%,w/v)用蒸馏水溶解,并于1L容量瓶中定容。然后,用0.1mol/L盐酸将混合物的pH调整到7.0,最后在121℃下灭菌30min,制成用于发酵实验的基础营养生长培养基。最后,将胃肠道消化后的1.5g TP残留物溶解在100mL基础营养生长培养基中,得到最终浓度为15mg/mL(w/v)。然后,将粪便浆(50mL)与基础营养生长培养基(100mL,含消化1.5g TP残留物)混合作为实验组。此时,反应体系中TP的浓度为10mg/mL。以接种相同粪便浆的培养基作为空白对照组。发酵过程是在37℃的厌氧菌条件下发酵24h。在沸水浴中加热10min后停止反应,并在0、6、12和24h时收集反应混合物。用pH仪测量各溶液在不同时间点的pH值。然后,将样品储存在-80℃冰箱中备用。每个实验设置三个平行组。
(3)测定银耳多糖体外模拟消化、结肠发酵后的总糖含量、还原糖含量、分子量变化、单糖组成、抗氧化活性、酪氨酸酶抑制活性、弹性蛋白酶抑制活性、pH值与短链脂肪酸。
(4)肠道微生物分析:根据说明书使用DNA粪便提取发酵样品的细菌基因组DNA,并由上海中科新生命生物技术有限公司(中国上海)分析提取的DNA样品。通过正向引物341F(CCTAYGGGRBGCASCAG)和反向引物806R(GGACTACNNGGGTATCTAAT)对V3-V4区域的细菌16SrRNA基因进行PCR扩增,并通过Illumina Miseq平台测序(IIIumina,San Diego,USA),使用Quantitative Insights Into Microbial Ecology(QIIME,v1.8.0)33进行处理。
2、试验结果
银耳多糖体外模拟消化、结肠发酵过程中产物的总糖含量如图2A所示,TP在口腔、胃、小肠模拟消化中无明显变化。而在结肠发酵中实验组的总糖含量变化显著(图2B),发酵24h后,总糖含量从972.28μg/mg降到了617.94μg/mg,发生了显著性的降低(p<0.05),与空白对照组的下降趋势一致。表明在发酵过程中,TP可被肠道微生物分解利用。
经体外模拟消化、结肠发酵后实施例1银耳多糖产物的还原糖含量如图3所示。实施例1银耳多糖在口腔拟消化中无明显变化,在胃、小肠模拟消化阶段,还原糖有少量增加由0.02mg/mL增加至0.23mg/mL,说明实施例1银耳多糖在胃、肠液中有一定程度的降解。而在结肠发酵中还原糖含量变化显著,发酵6h时还原糖含量最高为1.42mg/mL,是发酵0h组(0.41mg/mL)的3.46倍。可能是此时微生物大量繁殖,将大分子实施例1银耳多糖分解为大量小分子糖,从而利用。6h后还原糖含量逐渐减少,24h后还原糖含量变化不明显,可能此时微生物已达k值,达到平衡,还原糖含量变化趋于稳定。与空白对照组的趋势一样,表明在发酵过程中,实施例1银耳多糖可被肠道微生物利用。
从图4中实施例1银耳多糖经体外模拟消化和结肠发酵前后分子量变化可看出,经口腔、胃、肠消化后,实施例1银耳多糖分子量大小未发生明显变化,其分子量分别为1692.03kDa、1691.38kDa、1691.02kDa。而经结肠发酵后,实施例1银耳多糖分子量明显变小,由未经消化的1692.38kDa减小至1164.08kDa。说明TP在结肠发酵阶段可被微生物较好的降解和利用。
实施例1银耳多糖模拟口腔、胃、肠消化以及结肠发酵后其单糖组成分析结果如图5所示,多糖含量变化见表3。通过将银耳单糖组分与单糖标准品进行对比分析,可确定TP消化前后的变化,可以看出口腔、胃、肠消化阶段单糖组成都为岩藻糖,半乳糖,葡萄糖,木糖,甘露糖,葡萄糖醛酸,无其他单糖生成。但在结肠发酵后检测出含有盐酸氨基半乳糖以及盐酸氨基葡萄糖,这可能是由于接种菌液中粪便所含有的。虽然体外模拟口腔、胃、肠消化阶段单糖组成没有发生变化,但单糖的峰面积发生了明显变化。如图中的峰7为葡萄糖,峰面积先增后减,含量从初始的0.011mol上升到0.052mol,发生显著性增加,之后逐渐降低到初始水平。这种情况可解释为,粗多糖先发生降解,暴露较多的片段,使得检测到的葡萄糖含量增加,而后多糖片段被肠道微生物利用,粗多糖含量减少,对应峰面积降低。甘露糖其峰面积变化结果与葡萄糖一致。经结肠发酵后,岩藻糖含量由0.362mol减少至0.31mol,木糖含量由0.423mol减少至0.4mol,半乳糖含量由0.013mol增加至0.082mol,葡萄糖醛酸几乎无明显变化。综上,实施例1银耳多糖经体外模拟消化、结肠发酵后,其单糖组分含量发生了变化,且单糖含量间存在一定程度的转化。
表2实施例1银耳多糖体外模拟消化及结肠发酵后的单糖含量(摩尔比)
注:BLK代表未经消化的TP空白对照组,Oral代表口腔消化后的TP,Gastric代表胃消化后的TP,S-testine代表小肠消化后的TP,FTP24h代表经结肠发酵24h后的TP,下同。
研究表明,银耳的生物活性主要依赖于其多糖类物质,具有较强抗氧化活性的TP与多种慢性疾病的预防有关。通过ABTS、DPPH自由基清除活性和铁还原能力三个指标来评估实施例1银耳多糖在体外消化过程中的抗氧化活性。发现实施例1银耳多糖经口腔、胃、小肠模拟消化以及结肠发酵后其抗氧化活性有明显变化。如图6所示,经消化后实施例1银耳多糖的ABTS、DPPH自由基清除活性高于未经消化的组,而FRAP值则相反。经小肠消化后实施例1银耳多糖的ABTS、DPPH自由基清除活性最高,其当量值分别为62.89、13.50μg/10mg,其ABTS、DPPH当量值分别是口腔空白组(54.26、3.29μg/mg)的1.16倍,4.11倍。而实施例1银耳多糖的FRAP值最高是未经胃消化的组,其FRAP值为55.99μg/mg,是经小肠消化后组(14.69μg/mg)的3.81倍。总的来说,实施例1银耳多糖在体外消化过程中仍表现出较强的抗氧化活性,但实施例1银耳多糖的FRAP值消化组低于未消化组,这可能是由于Fe3+与多糖在消化过程中发生反应形成了TP和铁的配合物。
如图7所示,经发酵后实施例1银耳多糖的ABTS、DPPH自由基清除活性以及铁还原能力都有显著改变。随着发酵时间的变化,发现TP的ABTS、DPPH当量值最高都为发酵6h组,分别为84.03、22.17μg/mg,是发酵24h组(78.80,7.11μg/mg)的1.06倍,3.11倍。TP的FRAP值最高为未发酵组(189.74μg/mg),其是发酵24h组(73.82μg/mg)的2.57倍。总的来说,实施例1银耳多糖经发酵后其ABTS、DPPH自由基清除活性显著高于未经发酵的体外消化组,但其活性随发酵时间而变化,发酵6h其抗氧化活性最好。
经体外模拟消化以及结肠发酵后,我们对实施例1银耳多糖的酪氨酸酶抑制活性测定结果如图8所示。实验结果表明,经口腔、胃、小肠消化后,TP对酪氨酸酶活性的抑制率略有降低(图8A)。在胃消化阶段,未消化的实施例1银耳多糖展示出最强的酪氨酸酶抑制能力,抑制率达75.26%。这可能是胃消化液过酸(pH为2.0)导致酪氨酸酶失活而产生的结果。通过结肠发酵,实施例1银耳多糖整体展现出更强的酪氨酸酶抑制活性。尤其是经过发酵6h后,实施例1银耳多糖对酪氨酸酶活性的抑制率达74.45%,而对照组无明显变化,排除了pH变化带来的影响。随着发酵时间延长,实施例1银耳多糖对酪氨酸酶酶活性的抑制率有所降低(发酵12h、24h,TP对酪氨酸酶活性抑制率分别为70.05%,65.03%),但仍高于未发酵组(54.73%)。
如图9所示,经体外模拟消化以及结肠发酵后,TP对弹性蛋白酶活性的抑制率变化趋势与酪氨酸酶类似。即经口腔、胃、小肠消化后,TP对弹性蛋白酶活性抑制能力有所降低。其中,经小肠消化后,TP对弹性蛋白酶活性抑制率降低至27.97%。而小肠发酵阶段,未经消化的TP依然显示出最高的抑制活性(84.29%)。同样地,经结肠发酵后添加TP的发酵液对弹性蛋白酶的抑制能力显著提升。含TP的发酵液发酵6h后,显示出最强的弹性蛋白酶抑制率(92.72%)。但随着发酵时间的延长,发酵液对弹性蛋白酶的抑制能力略有降低。含TP的发酵液达12h时,其对弹性蛋白酶的抑制率降为89.27%,24h时,其对弹性蛋白酶的抑制率降为84.29%。
如图10所示在结肠发酵阶段pH由6.83降至4.73,其与空白对照组的趋势一样,表明在发酵过程中,TP可被肠道微生物利用,且代谢产生了大量脂肪酸、乳酸,从而使得pH降低。结肠发酵过程中,短链脂肪酸含量如表4所示,由表可知,添加实施例1银耳多糖的发酵组其短链脂肪酸总量从197.31增加到1937.33μg/mL,发生显著性增加;其中乙酸和丁酸量上升趋势最显著,6h含量分别是0h的8.50倍、6.82倍;在6h丙酸、异丁酸、戊酸、异戊酸含量均略微下降,结合总糖、还原糖以及pH的变化,推测可能是该时段微生物大量繁殖,被微生物利用所致。与0h对比24h时7种短链脂肪酸均显著增多。空白组发酵过程中短链脂肪酸变化趋势与添加实施例1银耳多糖的实验组基本一致,但空白组所含总的短链脂肪酸以及各类别短链脂肪酸含量均低于添加TP组。在6h时添加TP组的乙酸和丁酸含量比空白组分别多238.00和32.66μg/mL,同样24h时乙酸和丁酸的含量也显著高于空白组。有报道称低酸性环境有利于乙酸和丁酸的生成。从图10可知多糖组pH呈弱酸性,低于空白组,所以有利于乙酸和丁酸的生成。乙酸和丁酸是有益的短链脂肪酸,能抑制去乙酰化酶活性,可以调节宿主的基因表达,能预防和治疗肠炎、肠癌等肠道疾病。总体而言,多糖组结肠发酵短链脂肪酸含量髙于空白组,含量有显著增加。
表3实施例1银耳多糖结肠发酵产物短链脂肪酸含量
注:BL为未加TP的发酵组,FTP为添加TP的发酵组。BL0h=未加TP未发酵组;BL6h=未加TP发酵6h组;BL24h=未加TP发酵24h组;FTP6h=添加TP发酵6h组;FTP24h=添加TP发酵24h组,下同。
根据OTUs(Operational Taxonomic Units)聚类分析,可以探明各样品中微生物的物种组成多样性。不同样品间的共有和特有OTU如图11所示,发酵24h后样品OTU数目明显增多,发酵6h后样品OTU数目次之,未发酵的样品OUT数目最少。此外,添加TP的样品OTU数目高于未添加TP的样品。研究结果表明,结肠发酵能改变人体肠道微生物的物种组成多样性,且添加TP后,可在一定程度上增加人体肠道微生物的OTU数目。
利用主坐标分析(Principal Co-ordinates Analysis,PCoA),能有效地找出数据中最“主要”的元素、结构和影响因素,从而区分各个样本,根据样本远近展示样本间的相似性和差异性[135]。如图12所示,BL0h、BL6h和BL24h各组间分隔较远,表明这三个样品组之间的微生物组成差异较大。FTP24h和BL24h组也彼此分离,这两组间的微生物组成差异也较大。而FTP6h和FTP24h组相对聚集,说明该两组的微生物组成相似度较高。结果表明,人体肠道微生物群落结构会随发酵时间而改变,TP也可明显改变微生物群落组成。
各样品组在门水平的相对丰度结果如图13所示,样品中微生物主要以厚壁菌门(Firmicutes),放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),变形菌门(Proteobacteria),疣微菌门(Verrucomicrobia)和蓝细菌门(Cyanobacteria)为主。BL0h组主要微生物为厚壁菌门和放线菌门,含少量变形菌门、拟杆菌门、疣微菌门以及软壁菌门(Tenericutes)。经发酵6h后,BL6h组微生物组成以蓝细菌门和厚壁菌门为主。发酵达24h时,BL24h组微生物主要含厚壁菌门和拟杆菌门。添加TP并发酵6h的组(FTP6h),与BL0h组相比,厚壁菌门、蓝细菌门、拟杆菌门丰度有所增加,变形菌门丰度减少。添加TP的发酵液,发酵达24h时(FTP24h)其放线菌门、变形菌门丰度增大,但其主要微生物为厚壁菌门和放线菌门。据研究表明,放线菌门可以产生、提炼抗菌素(如:链霉素、土霉素、庆大霉素等),还可以提炼部分维生素。变形菌门可以同植物共生(如:有固氮作用的根瘤菌),还具有降解化合物的作用等。厚壁菌门具有较强的化能合成作用,可有效吸收食物中的热量,帮助消化。还有研究发现,厚壁菌门丰度与皮肤痤疮有一定联系,当皮肤出现痤疮时厚壁菌门丰度会明显下降。研究结果表明,随着发酵时间的改变,肠道微生物在门水平上的丰度也会发生改变。添加TP的发酵液在6h时使得厚壁菌门、蓝细菌门和拟杆菌门的丰度增加,在24h时主要影响并提高放线菌门和变形菌门丰度。
综上可知:
(1)TP经体外模拟消化(口腔、胃、小肠)后总糖含量无明显变化,还原糖在口腔消化阶段无明显变化,胃、小肠消化过程中有少量还原糖生成,分子量无明显变化,单糖种类组成无变化,但各单糖含量发生了改变。经结肠发酵后,我们发现总糖含量显著降低,还原糖含量明显增多;分子量明显变小,单糖组成种类和含量均发生改变。
(2)经体外模拟消化后TP的ABTS、DPPH自由基清除能力有一定的提高,但铁还原能力(FRAP)整体降低,酪氨酸酶抑制活性和弹性蛋白酶抑制活性也略有降低。但经结肠发酵后,添加TP的实验组,其DPPH自由基清除活性整体高于未添加TP的空白组。添加TP的发酵组其FRAP值整体低于空白组,分析原因可能是由于Fe3+与多糖在消化过程中发生反应形成了TP和铁的配合物。添加TP的实验组,其酪氨酸酶抑制能力和弹性蛋白酶抑制能力均整体高于空白组。
(3)在结肠发酵阶段,发酵液的pH值整体下降,发酵6h时下降最快,发酵12h时pH变化不再明显。同时,我们对每个时间点的发酵液进行了短链脂肪酸的定性、定量分析。结果表明,随着发酵时间增加,发酵液中会生成大量短链脂肪酸,其中乙酸含量最高,丁酸次之。
(4)通过16S rDNA扩增子测序结果表明在微生物的门水平上,TP可提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门的丰度。通过OTU指数分析发现,TP可以使人体肠道菌群的群落丰富度增加,随着发酵时间延长添加TP的发酵液中菌群OTU数值增大,表明TP可以提高菌群种类,使人体肠道菌群多样性增多。总而言之,结肠发酵会显著改变样品的微生物群落组成,摄入了TP后会增加人体肠道微生物的群落结构。
综上可知,本发明的银耳多糖可在制备改善肠道环境药物和/或保健品中的应用,所述改善肠道环境包括改变人体肠道微生物群落组成,所述改变人体肠道微生物群落组成包括提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门中一种或多种的丰度。
需要说明的是,本发明提及的TP为本发明实施例1的银耳多糖。
上述说明是针对本发明较佳可行实施例的详细说明,但实施例并非用以限定本发明的专利申请范围,凡本发明所提示的技术精神下所完成的同等变化或修饰变更,均应属于本发明所涵盖专利范围。
Claims (6)
1.一种银耳多糖的制备方法,其特征在于,包括以下步骤:
(1)将干制的银耳子实体粉碎后过50-70目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶55-65g/ml的比例搅拌均匀,加热煮沸2-3h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以5000-7000r/min的转速离心5-10min,取上清液旋转蒸发至原上清液体积的1/8-1/10,得到浓缩液;
(4)向浓缩液中加入其体积4-6倍体积浓度为85-95%的乙醇溶液,搅拌5-10min后于4-6℃静置12-14h,沉淀冻干后得到银耳多糖。
2.如权利要求1所述银耳多糖的制备方法,其特征在于,包括以下步骤:
(1)将干制的银耳子实体粉碎后过60目筛,得到银耳干粉;
(2)将银耳干粉与蒸馏水按料液比1∶60g/ml的比例搅拌均匀,加热煮沸2h,得到多糖粗提液;
(3)待多糖粗提液冷却至室温后,以5000r/min的转速离心5min,取上清液旋转蒸发至原上清液体积的1/10,得到浓缩液;
(4)向浓缩液中加入其体积4倍体积浓度为95%的乙醇溶液,搅拌5min后于4℃静置12h,沉淀冻干后得到银耳多糖。
3.如权利要求1或2所述的方法制备得到的银耳多糖。
4.如权利要求3所述的银耳多糖在制备改善肠道环境药物和/或保健品中的应用。
5.如权利要求4所述的应用,其特征在于:所述改善肠道环境包括改变人体肠道微生物群落组成。
6.如权利要求5所述的应用,其特征在于:所述改变人体肠道微生物群落组成包括提高厚壁菌门、蓝细菌门、放线菌门、变形菌门和拟杆菌门中一种或多种的丰度。
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