CN111154007A - 一种竹燕窝多糖的提取方法及用途 - Google Patents
一种竹燕窝多糖的提取方法及用途 Download PDFInfo
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- CN111154007A CN111154007A CN202010098071.5A CN202010098071A CN111154007A CN 111154007 A CN111154007 A CN 111154007A CN 202010098071 A CN202010098071 A CN 202010098071A CN 111154007 A CN111154007 A CN 111154007A
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Abstract
本发明涉及一种竹燕窝多糖的提取方法,属于竹燕窝处理领域。本提取方法包括以下步骤:取新鲜竹燕窝,于60℃下恒温烘烤24h,经研磨和过筛,得到竹燕窝干粉;称取竹燕窝干粉,按质量体积比为1g∶40ml加入超纯水,置于60℃恒温水浴中,经旋蒸减压提取5h,得到竹燕窝多糖提取液;将竹燕窝多糖提取液经多次过滤,取出滤液,离心,取上清液,经浓缩,得到浓缩液;向浓缩液中加入无水乙醇,混合均匀后,于4℃下冷藏静置12h,取出后离心,取下层沉淀物,于‑80℃冷冻成块后取出,经真空冷冻干燥2天后,即得到竹燕窝多糖。通过本提取方法能够有效的将竹燕窝多糖提取出来,提取效率高。
Description
技术领域
本发明属于竹燕窝处理领域,具体涉及一种竹燕窝多糖的提取方法及用途。
背景技术
竹燕窝学名海绵胶煤炱菌,又称为竹花菌、竹菌或竹花等。竹燕窝是以竹蚜虫等分泌的蜜露为营养的一种真菌。竹燕窝食用口感弹脆,是一种新型食用真菌,呈现胶质珊瑚状,颜色为浅黄色或黑色。
现阶段研究发现,竹燕窝在生长初期颜色金黄,鲜脆可口,含有较多的营养物质,是一种营养素含量丰富的食用蕈菌。研究发现,每100g竹燕窝中含有总糖38.4g,粗多糖7.4g,粗纤维6.8g,粗蛋白含量12.8g,粗脂肪4.1g,灰分9.3g,水分12.4g,并且总糖和蛋白质含量都明显高于黑木耳、平菇、金针菇、香菇以及榛蘑。每100g竹燕窝中具有人体必须氨基酸苏氨酸480mg,缬氨酸500mg,蛋氨酸100mg,异亮氨酸280mg,亮氨酸460mg,苯丙氨酸230mg,赖氨酸360mg,组氨酸480mg,另外还含有很多其它种类的非必需氨基酸,必须氨基酸与非必需氨基酸的比值为0.71,符合FA0/WH0对理想蛋白质的要求。竹燕窝还富含Ca、K、Mg、Na等大量元素以及B、Cr、Fe、Zn、Se、Cu、Mn等微量元素。潘洁莉等通过实验也发现竹燕窝中含有Cu、Mn、Zn、Fe、Ca等矿质元素,并且其中的重金属Hg、Pb、Cd、As含量符合《食品安全国家标准》。
竹燕窝作为一种新型待开发食用真菌,含有多糖、麦角甾醇等活性物质,具有抑菌、抗氧化、抗肿瘤的作用。目前没有关于竹燕窝多糖的提取分离等方法,不能对竹燕窝多糖加以有效利用,市面上也未见竹燕窝多糖的相关产品的出现,因此对于提成一种竹燕窝多糖的提取方法是非常有必要的。
发明内容
本发明的目的是为了解决现有技术的缺陷,提供一种竹燕窝多糖的提取方法。本发明能够有效的从新鲜竹燕窝中提取出竹燕窝多糖,提取效率高,多糖含量高。
本发明解决上述技术问题的技术方案如下:一种竹燕窝多糖的提取方法,包括以下步骤:
S1、预处理:取新鲜竹燕窝,于60℃下恒温烘烤24h,经研磨和过筛,得到竹燕窝干粉;
S2、多糖提取:称取步骤S1制得的竹燕窝干粉,按质量体积比为1g∶40ml加入超纯水,置于60℃恒温水浴中,经旋蒸减压提取5h,得到竹燕窝多糖提取液;
S3、后置处理:将步骤S2制得的竹燕窝多糖提取液,经多次过滤,取出滤液,离心,取上清液,经浓缩,得到浓缩液;
S4、纯化:向步骤S3制得的浓缩液中加入无水乙醇,混合均匀后,于4℃下冷藏静置12h,取出后离心,取下层沉淀物,于-80℃冷冻成块后取出,经真空冷冻干燥2天后,即得到竹燕窝多糖。
本发明的有益效果是:
(1)本发明首创性的从竹燕窝中提取出竹燕窝多糖,开创了竹燕窝利用的新途径,增加了经济效益,为竹燕窝的开发利用奠定了基础。
(2)本发明的竹燕窝多糖的提取方法,在无需去除蛋白,也无需使用色谱柱等纯化手段的前提下,提取效率高,多糖含量高,节约资源。
(3)本发明提取得到的竹燕窝多糖的抗氧化活性显著,自由基清除率与竹燕窝多糖浓度呈现一定的正相关。在竹燕窝多糖浓度达到2mg/ml时,对DPPH清除率可以达到85%以上;在竹燕窝多糖浓度为2mg/ml时,对羟基自由基清除能力达到70%。因此,本发明提取得到的竹燕窝多糖既可以作为天然抗氧化剂,也可以用于制备其它的抗氧化剂,且对人体无毒副作用。此外,由于竹燕窝在四川省宜宾地区分布较多,来源广泛,可降低抗氧化剂的生产成本,具有广阔的应用前景。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,在步骤S1中,所述过筛采用80目的过滤网。
采用上述进一步方案的有益效果是:能有效过滤掉杂质。
进一步,在步骤S3中,所述多次过滤具体为:先采用多层纱布初步过滤,再采用滤纸二次过滤。
采用上述进一步方案的有益效果是:过滤效果更好。
进一步,在步骤S3中,所述离心的转速为3000r/min,时间为10分钟;所述浓缩是指将上清液置于60℃恒温水浴中,旋蒸浓缩至50ml。
采用上述进一步方案的有益效果是:能够分离出沉淀杂质。
进一步,在步骤S4中,所述无水乙醇的体积量为4倍所述步骤S3制得的浓缩液的体积量。
采用上述进一步方案的有益效果是:能够有效分离出不需要的有机物。
进一步,在步骤S4中,所述离心的转速为3000r/min,时间为10分钟。
采用上述进一步方案的有益效果是:分层效果好。
本发明还提供一种抗氧化剂,包括采用如上述的提取方法提取得到的竹燕窝多糖。
附图说明
图1为本发明的实验例1中,葡萄糖标准曲线图;
图2为本发明的实验例2中,不同浓度的竹燕窝多糖对DPPH的清除率图;
图3为本发明的实验例2中,不同浓度的竹燕窝多糖对·OH的清除率图;
图4为本发明的实验例2中,不同浓度的竹燕窝多糖对ABTS的清除率图。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例
本实施例提供一种竹燕窝多糖的提取方法,包括以下步骤:
S1、预处理:取2kg野生的新鲜竹燕窝,于60℃下恒温烘烤24h,取出经研磨,得到48g粉末,采用80目过滤网过筛,得到30g竹燕窝干粉。
S2、多糖提取:步骤S1制得的30kg竹燕窝干粉,加入1200ml超纯水,置于60℃恒温水浴中,经旋蒸减压提取5h,得到竹燕窝多糖提取液。
S3、后置处理:将步骤S2制得的竹燕窝多糖提取液,先采用多层纱布初步过滤,再采用滤纸二次过滤,取出滤液,采用3000r/min离心10分钟,取出上清液,将上清液置于水浴60℃中旋蒸浓缩至50ml,得到浓缩液。
S4、纯化:向步骤S3制得的浓缩液中加入按浓缩液体积量的4倍的无水乙醇,使得混合液的浓度为80%,混合均匀后,放置于4℃下,冷藏静置12h,取出后采用3000r/min,离心10分钟,取下层沉淀物,置于-80℃冷冻成块后取出,经真空冷冻干燥2天后,即得到粉末状的竹燕窝多糖。
实验例1:多糖提取率和多糖含量的测定
对上述实施例提取的竹燕窝多糖的多糖含量进行测定,以确定竹燕窝多糖的提取率,测定具体包括以下步骤:
步骤一、供试品制备:
取上述方法制备得到的竹燕窝多糖,置于100ml烧杯中,加入50ml水,采用磁力搅拌器搅拌使其溶解,得到多糖原液,将其放于4℃冰箱保存。需要测试时,取多糖原液稀释500倍,即得到供试品。
步骤二、葡萄糖标准品制备:
取葡萄糖,在105℃烘箱中干燥至恒重,得到无水葡萄糖。精密称取无水葡萄糖11.0mg,加入蒸馏水定容到100mL,得到0.0110mg/mL的葡萄糖标准溶液,将其放于4℃冰箱保存。
步骤三、标准曲线的绘制:
分别准确称取步骤二中得到的葡萄糖标准溶液0、0.2、0.4、0.6、0.8、1.0mL,再分别置于10mL试管中,各加水至1mL,取1mL蒸馏水做空白对照。在上述各试管中各加入5%苯酚溶液1mL,再各加入5mL浓硫酸,混合均匀,在100℃水浴中加热20min,取出冷却至室温,在490nm处测其吸光度。以标准葡萄糖浓度为横坐标,以吸光度为纵坐标,得到葡萄糖标准曲线,得到回归方程为:y=0.1068x+0.003(R2=0.9964),式中x表示浓度,y表示吸光度,具体如图1所示。
步骤四、竹燕窝多糖含量与多糖提取率的测定
取1ml步骤一制备的供试品,加入5%苯酚溶液1mL,再加5mL浓硫酸,混合均匀,在100℃水浴中加热20min,取出冷却至室温,在490nm处测其吸光度。在图1的葡萄糖标准曲线上读出竹燕窝多糖浓度,根据回归方程换算出多糖浓度。通过如下公式计算得到多糖质量:
多糖质量=多糖浓度*稀释倍数
再通过多糖质量计算出多糖提取率。具体如下公式:
竹燕窝多糖提取率的计算公式为:
多糖提取率(%)=多糖质量/竹燕窝质量×100%。
步骤五、去除误差影响实验
(1)精密度实验:重复5次步骤四,整理吸光度数据,计算吸光度的RSD值。计算得到吸光度RSD值为0.386%,说明精密度良好。
(2)稳定性实验:取1ml步骤一制备的供试品,加入5%苯酚溶液1mL,再加5mL浓硫酸,混合均匀,在100℃水浴中加热20min,取出冷却至室温,在490nm处测其吸光度,每15min,测定一次,共测量4次,在1h内显色稳定,整理吸光度数据,计算吸光度的RSD值。在1h内稳定,计算得到RSD值为1.07%,稳定性好。
(3)重复性实验:重复提取方法分别制备5份竹燕窝多糖,配制成供试品溶液,重复上述步骤一至步骤四,按标准曲线方法测定吸光度,整理吸光度数据,计算多糖含量的RSD值。计算得到RSD值为2.39%,说明该方法重复性良好。
(4)加样回收率实验:取已知含量的竹燕窝多糖5份,精密加入等量的无水葡萄糖制备成测试液,按上述的方法测定吸光度,计算加样回收率。计算得到加样回收率范围1O2.32%,RSD值为2.87%。
通过上述方法计算得到竹燕窝多糖的提取率为13.55%,竹燕窝多糖的含量(g/100g)为73.1g±2.260。
实验例2:抗氧化实验
本实施例通过体外抗氧化实验,验证竹燕窝多糖的抗氧化效果,具体包括如下:
1.DPPH自由基清除能力的测定
分别配置浓度为0.1mg/mL、0.2mg/mL、0.4mg/mL、0.8mg/mL和1mg/mL的竹燕窝多糖溶液各1mL,各加入0.05mg/mL的DPPH溶液1mL,混匀,避光反应30min,分别以甲醇代替DPPH溶液作样品对照组,以甲醇代替样品做空白组,以不同浓度的抗坏血酸(VC)作为阳性对照组,采用分光光度比色法在517nm处测定吸光度,对DPPH的清除率按下列公式计算:
DPPH自由基清除率(%)=100×[A0-(A1-A2)]/A0,式中,A0为空白吸光度值,A1为样品吸光度值,A2为样品对照组吸光度值。
不同浓度的竹燕窝多糖溶液对DPPH的清除率见图2。DPPH的清除率与竹燕窝多糖的含量成正相关,随着竹燕窝多糖溶液浓度的升高,对DPPH的清除率逐渐增强,当竹燕窝多糖浓度达到2.5mg/ml时,对DPPH清除率为96.05%。随着抗坏血酸浓度的增加,对DPPH的清除率也逐渐增强,当抗坏血酸浓度达到0.01mg/ml时,对DPPH清除率达到89%。由此可见,竹燕窝多糖具有一定的抗氧化能力,与抗坏血酸(VC)相比抗氧化活性稍弱。
2.羟自由基(·OH)清除率的测定
在试管中依次加入9mmol/L的FeSO4溶液1.0mL、9mmol/L的水杨酸-乙醇1.0mL、不同质量浓度的多糖溶液1.0mL,最后加入8.8mmol/L的H2O2溶液1.0mL启动反应。将试管迅速移入37℃水浴锅中,静置30min后,在510nm波长处测定不同质量浓度多糖样品液的吸光度Ax。用蒸馏水代替H2O2,测得对应质量浓度多糖样品液的本底吸光度AxO。用蒸馏水代替样品测得对照吸光度A0,阳性对照为抗坏血酸(VC)。对·OH的清除率按下式计算:
·OH清除率(%)=100×[A0-Ax-Ax0)]/A0,式中,A0为蒸馏水代替样品的对照吸光度,Ax0为蒸馏水代替H2O2的本底吸光度,Ax为样品的吸光度。
为本底吸光度不同浓度的竹燕窝多糖溶液对·OH自由基的清除率结果如图3。由图3可知,不同浓度的竹燕窝多糖溶液对·OH清除率表现出良好的线性关系,随着竹燕窝多糖溶液浓度的增加,对·OH清除率逐渐增加,说明其抗氧化能力逐渐增强,在相同浓度的情况下,抗坏血酸(VC)的抗氧化能力要优于不同浓度的竹燕窝多糖。体外抗氧化研究表明羟基自由基清除IC50为0.923±0.012mg/mL。
3.ABTS自由基清除率的测定
取7mmol的ABTS和2.45mmol过硫化钾等体积混合,室温下置于暗处放置12h,得到ABTS储备液。取5ml的ABTS储备液,使用前用无水乙醇稀释40-45倍,得到ABTS+溶液,使该溶液在734nm下的吸光度值在0.7±0.02(无水乙醇调零)。分别取1mL不同浓度的样品溶液于试管中,依次加入3mL ABTS+溶液,摇匀,室温下避光放置6min后在734nm处测定吸光度。以蒸馏水溶液作为参比,抗坏血酸(Vc)为阳性对照,对ABTS的清除率按下列公式计算:
ABTS自由基清除率%=(A0-Ai+Aj)/A0×100%,式中,Ai为用无水乙醇调零后测定的样品吸光度,A0为蒸馏水代替样品测定对照吸光度,Aj为乙醇代替ABTS溶液测定样品的空白吸光度。
不同浓度的竹燕窝多糖溶液对ABTS自由基清除能力结果如图4。由图4可见,随着竹燕窝多糖溶液浓度的升高,对ABTS自由基的清除率逐渐增强,当竹燕窝多糖溶液浓度达到5mg/ml时,对ABTS自由基清除率为90.31%。
多糖清除ABTS自由基的IC50为1.993±0.026mg/mL。说明竹燕窝多糖具有一定的抗氧化活性,比抗坏血酸(Vc)的抗氧化性稍弱。
上述实验结果表明,本发明制得的竹燕窝多糖具有抗氧化性能,能够作为抗氧化功能食品或制备抗氧化剂。
因此,采用如上述的提取方法提取的竹燕窝多糖能够在食品添加剂或功能食品领域中应用。
采用如上述的提取方法提取的竹燕窝多糖制备的抗氧化剂能够在化妆品领域中应用。
采用如上述的提取方法提取的竹燕窝多糖制备的抗氧化剂能够在医药药品领域中应用。
本发明中起到抗氧化性能的是竹燕窝多糖,所以竹燕窝多糖或含有竹燕窝多糖的其他提取物均具有抗氧化性能,抗氧化能力的强弱与多糖的浓度呈正相关,一般竹燕窝多糖浓度越高,抗氧化能力越强,浓度过高时,其抗氧化能力趋于平稳。
其中,1,1-二苯基-2-三硝基苯肼(DPPH)为上海源叶生物科技有限公司生产试剂,无水乙醇、苯酚、浓硫酸、无水葡萄糖、丙酮、乙醚、甲醇均为分析纯,为成都科隆化学品有限公司生产试剂。
实验所用设备如下:
G-12A歌能超声波清洗机:歌能清洗设备有限公司;
RSD-V17小型研磨机:合肥荣事达小家电有限公司;
TDZ5-WS台式低速离心机:长沙湘仪离心机仪器有限公司;
Spectramax 190美国MD全波长酶标仪:美谷分子仪器(上海)有限公司;
JD200-4千分之一电子天平:沈阳龙腾电子有限公;
DHG-9053A电热恒温鼓风干燥箱:上海精宏实验设备有限公司;
DZKW-4电子恒温水浴锅:北京中兴伟业仪器有限公司;
T6可见光分光光度计:北京普析通用仪器有限责任公司。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种竹燕窝多糖的提取方法,其特征在于,包括以下步骤:
S1、预处理:取新鲜竹燕窝,于60℃下恒温烘烤24h,经研磨和过筛,得到竹燕窝干粉;
S2、多糖提取:称取步骤S1制得的竹燕窝干粉,按质量体积比为1g:40ml加入超纯水,置于60℃恒温水浴中,经旋蒸减压提取5h,得到竹燕窝多糖提取液;
S3、后置处理:将步骤S2制得的竹燕窝多糖提取液经多次过滤,取出滤液,离心,取上清液,经浓缩,得到浓缩液;
S4、纯化:向步骤S3制得的浓缩液中加入无水乙醇,混合均匀后,于4℃冷藏静置12h,取出后离心,取下层沉淀物,于-80℃冷冻成块后取出,经真空冷冻干燥2天后,即得到竹燕窝多糖。
2.根据权利要求1所述的竹燕窝多糖的提取方法,其特征在于,在步骤S1中,所述过筛采用80目的过滤网。
3.根据权利要求1所述的竹燕窝多糖的提取方法,其特征在于,在步骤S3中,所述多次过滤具体为:先采用多层纱布初步过滤,再采用滤纸二次过滤。
4.根据权利要求1所述的竹燕窝多糖的提取方法,其特征在于,在步骤S3中,所述离心的转速为3000r/min,时间为10分钟;所述浓缩是指将上清液置于60℃恒温水浴中,旋蒸浓缩至50ml。
5.根据权利要求1所述的竹燕窝多糖的提取方法,其特征在于,在步骤S4中,所述无水乙醇的体积量为4倍所述步骤S3制得的浓缩液的体积量。
6.根据权利要求1所述的竹燕窝多糖的提取方法,其特征在于,在步骤S4中,所述离心的转速为3000r/min,时间为10分钟。
7.一种采用如权利要求1-6任一项所述的提取方法提取的竹燕窝多糖在食品添加剂或功能食品领域中的应用。
8.一种抗氧化剂,其特征在于,包括采用如权利要求1-6任一项所述的提取方法提取得到的竹燕窝多糖。
9.一种如权利要求8所述的抗氧化剂在化妆品领域中的应用。
10.一种如权利要求8所述的抗氧化剂在医药药品领域中的应用。
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