CN108578338B - 一种提高中药抗氧化力的发酵方法及其应用 - Google Patents
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Abstract
本发明公开了一种提高中药抗氧化力的发酵方法及其应用,属于发酵技术领域。本发明以总还原力、羟自由基清除率、总酚含量及酪氨酸酶抑制率为指标,筛选出一种新型美白中药五倍子,并通过发酵提高其抗氧化能力、总酚含量及美白能力。本发明发现的新型美白中药五倍子提取物的总还原力为365.94mmol/L,羟自由基清除率为55.74%,总酚含量为269.16mg GAE/g,酪氨酸酶抑制率达到94.85%,并通过酵母菌发酵五倍子,提高了其抗氧化能力、总酚含量进一步提高,具有提高其作为美白成分的效果。
Description
技术领域
本发明涉及一种提高中药抗氧化力的发酵方法及其应用,属于日化和发酵技术领域。
背景技术
随着经济的快速发展及人民生活水平的不断提高,人们对于“美”的关注度也在增加,以肌肤白皙及富有光泽为美的观念越来越受广大女性的推崇。目前,市场中的美白产品主要分为化学类、天然中草药及微生物美白剂,由于化学类产品大多会给人体皮肤细胞带来一定的损伤,但仍是目前国内美白市场的主导产品。我国具有丰富的中草药资源,大力开发具有美白作用的中草药有广阔的应用前景。人体皮肤的颜色主要由肌肤细胞所含黑色素的量决定,它在皮肤基底层的过量异常代谢导致黑色素积累或沉着,使皮肤暗沉、发黑,酪氨酸酶是黑色素产生的关键酶,抑制其活性可以减少黑色素的生成。在衰老的皮肤和组织中检测到的另两类色素物质是脂褐素和腊样质,它们可以使皮肤变黑和粗糙,这两种物质都是由自由基引起,细胞膜分子裂解和过氧化形成的。因此选用具有较强抗氧化力、显著抑制酪氨酸酶活性的中草药成分,应该具有美白的功效。
抗氧化是指抗氧化自由基的简称,人体因为与外界的持续接触,包括呼吸(氧化反应)、外界污染、放射线照射等因素不断的在人体体内产生自由基。抗氧化就是任何以低浓度存在就能有效抑制自由基的氧化反应的物质,其作用机理可以是直接作用在自由基,或是间接消耗掉容易生成自由基的物质,防止发生进一步反应。抗氧化是从根本上减缓皮肤衰老的一个护肤过程。
.酪氨酸酶是一种含铜需氧酶,氧自由基引发和酪氨酸酶的催化作用使酪氨酸逐步氧化,最终形成黑色素。在酪氨酸酶的催化氧化过程中,氧自由基既是引发剂又是反应物,因此对氧自由基的清除作用既阻断了反应的引发,也削弱了酪氨酸酶的供氧作用,从而使酪氨酸氧化反应的强度减弱,即削弱了酪氨酸酶的作用。对氧自由基的清除作用是其对酪氨酸酶的竞争性抑制的一个重要方面,从而阻止黑素的氧化形成。
多酚类物质具有抗炎、抗癌、抗氧化等多种生物活性,植物多酚能有效清除人体代谢过程中产生的自由基,从根源上色斑生成的垃圾毒素代谢干净,对于已经生成的顽固黑色素,在植物多酚的还原作用下黑色素的邻苯二结醌构很易还原成酚型结构,使色素褪色,还原肌肤本色。一些天然中草药中富含多酚类物质,具有较好的抗氧化及美白养颜效果。益生菌发酵中药过程可以改善植物的生物特性,更具体地说,发酵会导致复合物的分解或生物转化,将底物转化为兼容的成分,调节产物特性或改变某种生物活性物质的含量,增强药理特性和治疗作用。发酵作用对天然中草药有效成分进行提取,反应条件温和、能够充分提取有效成分及降低中草药毒性,能够最大程度的保留天然中草药活性成分的功效性。
我国中草药资源得天独厚,很多中草药具有较好的美白功效,因此,寻找高效安全的美白剂,必将受到广大女性的追捧,并产生极大的社会和经济效益。本发明发现五倍子具有很高的抗氧化力、酪氨酸酶抑制率高,其总酚含量、总还原力和酪氨酸酶抑制率均高于目前广泛使用的美白中药-红景天,预示其在美白功效方面要好于红景天。
发明内容
本发明的筛选得到高抗氧化力、酪氨酸酶抑制率高的中药-五倍子,并提供一种进一步提高五倍子抗氧化能力的发酵方法,是以具有抗氧化活性的中药为发酵原料,接种酵母进行发酵。
本发明的第一个目的是提供一种提高中药抗氧化力的发酵方法,是以具有抗氧化活性的中药为发酵原料,接种酵母进行发酵。
在本发明的一种实施方式中,所述酵母包括酿酒酵母(Saccharomycescerevisiae)ATCC550和产朊假丝酵母(Candida utilis)ATCC 9950
在本发明的一种实施方式中,所述具有抗氧化活性的中药包括白芷、白芍、红景天、生甘草、五倍子、生首乌、红花、白附子或绿茶叶。
将5g中药粉碎,加100mL水,混匀,调pH为3.5~4.5,添加纤维素酶10U/g中药、果胶酶20U/g中药,在50℃水浴酶解3h,沸水浴煮沸5min将酶灭活,离心,取上清液,定容至100mL,获得美白中药提取物。
在本发明的一种实施方式中,所述五倍子发酵培养基是称取3g五倍子,再加水60mL,加YEPD液体培养基成分:葡萄糖3%,蛋白胨1%,酵母粉0.2%。
在本发明的一种实施方式中,所述酵母菌发酵五倍子是在五倍子发酵培养基中接种按体积计10%的酵母菌种子液,所述酵母菌种子液的菌体浓度为1×109CFU·mL-1,于30℃、180r/min培养48h,然后取适量发酵液,离心,获得五倍子发酵液。
在本发明的一种实施方式中,所述离心是在4℃、8000r/min条件下离心10min。
在本发明的一种实施方式中,所述五倍子在加入至培养基前进行粉碎。
在本发明的一种实施方式中,所述粉碎是粉碎至40~60目。
本发明的第二个目的是提供应用所述方法制备的五倍子发酵液。
本发明还提供所述方法在制备新型美白中药及发酵产品方面的应用。
本发明还提供所述方法在制备化妆品方面的应用。
有益效果:本发明通过对几种常用美白中药的研究,筛选出了抗氧化能力相对较高的中药原材料五倍子,并通过发酵提高了其抗氧化能力,总还原力由231.31mmol/L提高至387.90~393.36mmol/L,羟自由基清除率由25.74%提高至48.81%~49.08%;总酚含量增加16.5~54.19mg GAE/g;酪氨酸酶抑制率达到94.44~95.72%,美白能力进一步提高,为新型美白中药五倍子及其发酵产品在日化工业中的应用提供参考。
具体实施方式
总还原力测定:用紫外分光光度计在700nm下测其吸光度,用半胱氨酸盐酸盐当量对吸光度回归,同样的测量条件下得到标准曲线。标准曲线的制作:以半胱氨酸盐酸盐做标准曲线;以半胱氨酸盐酸盐当量对吸光度进行回归,并拟合线性回归方程:y=1.4656x+0.0178,R2=0.9976。
羟自由基清除能力测定:反应体系:水杨酸,样品溶液,FeSO4溶液,H2O2溶液,水浴反应一定时间,用紫外分光光度计于510nm处测定吸光值,计算羟自由基清除率。
总酚含量测定:用紫外分光光度计在750nm下测其吸光度,标准曲线的制作:以没食子酸做标准曲线;以没食子酸浓度对吸光度进行回归,并拟合线性回归方程:y=0.0268x+0.0141,R2=0.9990。
酪氨酸酶抑制率测定:依次吸取PBS、样品溶液、L-酪氨酸溶液,然后水浴预热,取出后加酪氨酸酶溶液,水浴反应一定时间,在475nm波长下测定吸光度,计算酪氨酸酶抑制率。
实施例1:各美白中药提取物制备
称取5g中药(已粉碎,过筛),加100mL水,混匀,调pH为3.5~4.5,添加纤维素酶10U/g中药、果胶酶20U/g中药,在50℃水浴酶解3h,沸水浴煮沸5min将酶灭活,4℃、8000r/min离心10min,取上清液,定容至100mL,得到浓度为50g/L中药提取物。
实施例2:提取物的总还原力、总酚含量、酪氨酸酶抑制率测定
对实施例1中制备的各美白中药提取物进行抗氧化能力、总酚含量及酪氨酸酶抑制率的研究,不同美白中药各指标测定结果(表1)。中药的抗氧化能力用总还原力与羟自由基清除率进行评价,美白能力用体外酪氨酸酶活性抑制实验进行评价。在几种常用的美白中药中,五倍子提取物的总还原力显著高于其他中药提取物,为365.94mmol/L,其次红景天的总还原力也较高,为47.27mmol/L;对于羟自由基清除率,红景天、五倍子及绿茶叶提取物的羟自由基清除率较高,均高于50%;对于总酚含量,五倍子中总酚含量显著高于其他中药,为269.16mg GAE/g,红景天中总酚含量也较高,为101.76mg GAE/g,多酚类物质具有抗氧化、抗癌、抗炎等多种生物活性,这与它们的抗氧化活性、酪氨酸酶活性抑制能力有关;五倍子提取物的酪氨酸酶抑制率显著高于其他中药,为94.85%,其次为红景天提取物,其酪氨酸酶抑制率为67.70%,其余中药也表现出一定的酪氨酸酶抑制能力。
表1不同美白中药各指标测定结果
注:总酚含量是每克提取物中所含没食子酸毫克数,单位是mg GAE/g。
实施例3:五倍子发酵方法
称取3g五倍子,粉碎至40~60目,再加水60mL,混匀,加入至40mL YEPD液体培养基中,于30℃度下发酵2天。YEPD液体培养基成分为:葡萄糖3%,蛋白胨1%,酵母粉0.2%。
向含有五倍子的YEPD培养基中接种按体积计10%酵母菌种子液(菌体浓度为1×109CFU·mL-1),于30℃、180r/min培养48h,研究不同酵母菌发酵五倍子对其抗氧化能力、总酚含量及美白能力的影响。以在相同条件下接种相同浓度市售活性干酵母发酵的培养液作为对照。
对于总还原力,酿酒酵母ATCC550与假丝酵母ATCC9950分别发酵五倍子后,总还原力均增加,明显高于对照,增加量分别为162.05mmol/L、156.59mmol/L,表明两株酵母菌发酵五倍子均对其抗氧化能力有较大影响(表2)。
表2不同酵母菌发酵五倍子对其总还原力的影响
对于羟自由基清除率,酿酒酵母ATCC550与假丝酵母ATCC9950分别发酵五倍子后,羟自由基清除率分别达到49.08%、48.81%,均明显高于对照,表明五倍子经发酵后捕捉羟自由基能力均增强,抗氧化能力增强(表3)。
表3不同酵母菌发酵五倍子对其羟自由基清除率的影响
对于总酚含量,酿酒酵母ATCC550与假丝酵母ATCC9950分别发酵五倍子后,总酚含量均增加,增加量分别为16.5mg GAE/g、54.19mg GAE/g,表明酵母菌假丝酵母ATCC9950发酵五倍子对其总酚含量影响显著(表4)。
表4不同酵母菌发酵五倍子对其总酚含量的影响
对于酪氨酸酶抑制率,酿酒酵母ATCC550与假丝酵母ATCC9950分别发酵五倍子后,酪氨酸酶抑制率分别为94.44%、95.72%,较对照均有所增加,表明五倍子经发酵后其酪氨酸酶活性抑制能力变强,美白能力变强(表5)。
表5不同酵母菌发酵五倍子对其酪氨酸酶抑制率的影响
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (6)
1.一种提高中药抗氧化力的发酵方法,其特征在于,以具有抗氧化活性的中药为发酵原料,接种酵母进行发酵;所述酵母为酿酒酵母(Saccharomyces cerevisiae)ATCC 550或产朊假丝酵母(Candida utilis)ATCC 9950;所述具有抗氧化活性的中药为五倍子;所述发酵培养基按重量计,含有葡萄糖3~5%,蛋白胨0.5~1%,酵母粉0.2~0.5%,五倍子3~10%。
2.根据权利要求1所述的方法,其特征在于,所述五倍子经过粉碎;所述粉碎是粉碎至40~60 目。
3. 根据权利要求1所述的方法,其特征在于,在五倍子发酵培养基中接种按体积计10%的酵母菌种子液,所述酵母菌种子液的菌体浓度为1×109 CFU·mL-1 ~9×109 CFU·mL-1,于28~30℃培养24~48 h。
4.根据权利要求1或3所述的方法,其特征在于,对发酵结束后的发酵液进行纯化。
5.应用权利要求1~4任一所述的方法制备的五倍子发酵液。
6.权利要求5所述的五倍子发酵液在制备美白食品或化妆品方面的应用。
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