CN114469758A - 一种小粒径纳米气泡水及其制备方法以及应用 - Google Patents
一种小粒径纳米气泡水及其制备方法以及应用 Download PDFInfo
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Abstract
本发明提供一种小粒径纳米气泡水及其制备方法以及应用,小粒径纳米气泡水中纳米气泡的总浓度>107个/毫升,其中含有粒径小于50nm的小粒径纳米气泡。该制备方法包括:将需要引入小粒径纳米气泡的水冷却到零摄氏度左右;将冷却后的水放入到加压装置内;向加压装置中通入高压气体或采用机械力加压,以增大加压装置内部压力;维持高压条件,持续一段时间;缓慢释压至常压状态,即得;制备全程保持水温低于10℃,纳米气泡的粒径小于50nm。根据本发明,在不引入外源还原剂的情况下,所制备的小粒径纳米气泡具有能够保护底物免受活性氧氧化的能力,同时本发明还首次提供了这样一种采用冰水混合物加压减压制备小粒径纳米气泡水的方法。
Description
技术领域
本发明涉及界面化学领域,具体涉及一种小粒径纳米气泡水及其制备方法以及应用。
背景技术
活性氧(ROS)是一种细胞代谢的正常产物,伴随着生物体内正常细胞代谢而产生。在适当的浓度下,它们在细胞生理过程中发挥重要作用,但在较高浓度下,它们会对多种重要的细胞成分造成氧化损伤,包括脂质、蛋白质和DNA。在正常的生理功能中,氧化剂和抗氧化剂之间有一个平衡,当氧化剂和抗氧化剂之间的平衡倾向氧化剂时被称为“氧化应激”。氧化应激可导致多种疾病,包括癌症、神经系统疾病、动脉粥样硬化、高血压、缺血/灌注、糖尿病、急性呼吸窘迫综合征、特发性肺纤维化、慢性阻塞性肺病和哮喘。
生物体具有全面的抗氧化系统,包括酶类和非酶类抗氧化剂,通常可以有效阻断ROS的有害作用。酶类抗氧化剂主要包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px);非酶抗氧化剂包括维生素(维生素C和E)、β-胡萝卜素、尿酸和还原性谷胱甘肽(GSH)。
纳米气泡:一种存在于液相中的球状气泡,大小在1000nm以下。纳米气泡(NB)有着许多独特的特性,如长时间稳定性、表面电负性、产生自由基和表面吸附性。近年来,纳米气泡技术在多个领域发现了许多潜在的应用,例如生物工艺、资源回收、水处理和食品生产。其中,纳米气泡的氧化性被广泛的研究和应用,例如破裂能够产生自由基去除污染物和杀菌以用于水处理的各个领域。我们意外发现,当水中含有的纳米气泡粒径足够小,其也具备抗氧化性。该小粒径纳米气泡水可能材料保护、化妆品、食品工业以及饮用水行业有着潜在的应用价值。
目前的体内抗氧化剂主要是食源性抗氧化剂,例如β-胡萝卜素、维生素C和维生素E等抗氧化剂不能在体内合成必须来自膳食。各种膳食抗氧化剂具有特定益处,例如,vonLeeuwen报道大量摄入维生素C、维生素E、锌和β-胡萝卜素与显着降低年龄相关性黄斑变性的风险有关。在动脉粥样硬化预防(ASAP)研究中发现补充维生素C和维生素E与降低高脂血症患者动脉粥样硬化的进展有关。然而,这些抗氧化剂在某些情况下可能是危险的。例如,辛伐他汀和烟酸组合使用能够有益地增加高密度脂蛋白(HDL)浓度和颗粒大小,但该效用被维生素E、维生素C、β-胡萝卜素阻碍。
发明内容
本发明的目的是提供一种小粒径纳米气泡水及其制备方法以及应用,从而解决现有技术中的抗氧化剂依赖于额外添加还原剂,以及存在多种风险的问题。
根据本发明的第一方面,提供一种小粒径纳米气泡水,所述小粒径纳米气泡水中纳米气泡的总浓度>107个/毫升,其中含有粒径小于50nm的小粒径纳米气泡。
优选地,所述小粒径纳米气泡在动态光散射技术(DLS)测量中表现为粒径分布范围在1nm-50nm,更优选为2nm-15nm。
根据本发明提供的小粒径纳米气泡水,所述小粒径纳米气泡在纳米粒子追踪分析系统(NTA)测量中表现为粒径的大小在50nm以下,其中在动态光散射技术(DLS)测量中10nm附近的气泡数占据了总数的绝大部分。优选地,动态光散射技术(DLS)测量中粒径分布范围在8nm-12nm的气泡数占据总数的95%以上。
优选地,小粒径纳米气泡浓度高于动态光散射技术(DLS)检测限,所述纳米气泡的DLS数量强度占比>20%。
根据本发明的第二方面,提供一种小粒径纳米气泡水的制备方法,包括以下步骤:S1:将需要引入小粒径纳米气泡的水冷却到零摄氏度左右;S2:将冷却后的水放入到加压装置内;S3:向所述加压装置中通入高压气体或采用机械力加压,以增大加压装置内部压力;S4:维持高压条件,持续一段时间;S5:缓慢释压至常压状态,即可制备得到一种含有小粒径纳米气泡的水;其中,制备全程保持低于10℃,所述纳米气泡的粒径小于50nm。
优选地,步骤S1包括:将需要引入小粒径纳米气泡的水冷却至冰水混合物。
步骤S1中的所述水可以根据需要任意选择,包含但不限于自来水、蒸馏水、纯净水、矿泉水等等。
优选地,步骤S3包括:向所述加压装置中通入高压气体,根据不同需要选择不同的气体种类。
优选地,步骤S3、S4包括:所述高压条件的压力范围为0.2-1.2MPa,维持时间20min-60min。
进一步优选地,步骤S4中的压力范围为0.4-1.2MPa,维持时间40min-60min。
优选地,步骤S5包括:释压过程采用气体流量计控制释气速率。
优选地,制备全程保持水温低于5℃。
根据本发明的第三方面,提供一种小粒径纳米气泡水在制备抗活性氧氧化试剂中的应用。
根据本发明的研究发现,小粒径(<50nm)纳米气泡具备抑制活性氧氧化的功效,因此含有该小粒径(<50nm)纳米气泡的小粒径纳米气泡水也具有抑制活性氧氧化的功效。在不引入外源还原剂的情况下,小粒径纳米气泡具有能够保护底物免受活性氧氧化的能力。如果粒径超出该范围(≥50nm),可能就不具备抗氧化功效。
截止到目前为止,现有技术从未公开过本发明这样一种采用冰水混合物加压减压制备小粒径纳米气泡水的方法。本发明提供的纳米气泡水的制备方法与最接近的现有技术的不同之处即在于针对冰水混合物进行先加压、后减压。
其次,现有技术也从未公开小粒径(<50nm)纳米气泡具有抗氧化功效的性质,并且也从未将其用于抗氧化,以及用来制备抗活性氧氧化试剂。
本发明通过低温加压减压法来制备小粒径纳米气泡,首次发现低于50nm的纳米气泡具备抗氧化功效。其中,制备小粒径纳米气泡的方法是可替代的。换句话说,通过其他方法制备出的小粒径纳米气泡,应该也具备相同的抗氧化功效。小粒径纳米气泡抗氧化的过程中没有添加特殊的化学还原剂,这意味着小粒径纳米气泡的抗氧化能力可用于安全和可持续地缓解生物体内的氧化应激。
根据本发明提供的一种小粒径纳米气泡水及其制备方法以及应用,其相对现有技术的优越性在于:
1)不添加外源抗氧化剂的情况下,即可制备出一种具有抗氧化功效的小粒径纳米气泡水,其中的绝大多数纳米气泡大小在10纳米附近;
2)通过冰水混合物进行先加压、后减压,提供了一种新的制备小粒径纳米气泡的方法;
3)通过本发明制备的小粒径纳米气泡水的抗氧化功效不依赖于额外添加还原剂,因此能够避免上述化学还原剂的缺陷;
4)本发明提供的这样一种小粒径纳米气泡水的制备方法不仅可用于抗活性氧氧化试剂的制备,还有望应用于材料保护、化妆品、食品工业以及饮用水行业等领域。
附图说明
图1是实施例1中制备得到的小粒径氮气纳米气泡的粒径分布以及抗氧化效果图;其中,a分别示出了超纯水和小粒径纳米气泡水中TMB的氧化曲线,横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;b为纳米粒子追踪分析系统(NTA)测定的小粒径气泡水和超纯水中纳米气泡的粒径分布,其测定范围为50-1000nm;c为使用动态光散射技术(DLS)测定的小粒径气泡水和大粒径纳米气泡水中纳米气泡的粒径分布;d为超纯水、小粒径纳米气泡水、脱气去除部分气泡后水中的TMB氧化曲线,横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;
图2是实施例2中小粒径氮气纳米气泡抗氧化能力与抗坏血酸钠的比较图;横坐标为小粒径纳米气泡和10-1000mM抗坏血酸钠,纵坐标代表相对氧化值(相对于超纯水),值越小代表抗氧化能力越强;
图3是实施例3中将不含有小粒径纳米气泡的常规氮气纳米气泡水转化为小粒径氮气纳米气泡水后的粒径分布以及抗氧化效果图;其中,a是在不含有小粒径纳米气泡的常规氮气纳米气泡存在时的TMB的氧化曲线,横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;b和c分别为NTA和DLS测定的大粒径纳米气泡水中纳米气泡的粒径分布,其中NTA显示该水中不含有小于80nm的纳米气泡,DLS图的横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例;d为不含有小粒径纳米气泡的常规氮气纳米气泡通过以公开的冻融法转变为小粒径纳米气泡后的TMB的氧化曲线,横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;e为DLS测定的以公开的冻融法转将常规氮气纳米气泡水转变为小粒径气泡水后水中纳米气泡的粒径分布,横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例;
图4是实施例4制备得到的小粒径氧气纳米气泡的粒径分布以及抗氧化效果图;其中,a是在小粒径氧气纳米气泡存在时TMB的氧化曲线,横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;b和c分别为NTA和DLS测定的气泡水中纳米气泡的粒径分布,其中NTA显示含有小于50nm的纳米气泡,DLS结果图横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例。
具体实施方式
以下结合具体实施例,对本发明做进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围/下面通过实施例来进一步说明本发明,但本发明并不受其限制。
除了特别说明的,实施例中所用原料均为常规原料,所用设备均为常规设备,市购产品。
实施例1小粒径纳米气泡影响自由基对底物的氧化
实验材料和方法:
实验中所用超纯水均来自ELGA LabWater(ELGA Classic-PURELAB)。CuCl2·2H2O、3,3',5,5'-四甲基联苯胺(TMB)(均为分析纯,≥99%);30%H2O2;高纯氮气(≥99.999%);抗坏血酸钠购买自麦克林;二甲基亚砜(DMSO)。
实验步骤:
小粒径纳米气泡的产生和分析:
小粒径纳米气泡是在低温下使用超纯水的冰水混合物通过加压-减压法来制备的。该实验是在一个定制的能够控制压力大小的金属腔内进行的。将超纯水的冰水混合物放入腔体内,通入氮气至气压为0.6MPa持续30分钟,然后在约4小时内缓慢减压至常压。使用纳米粒子跟踪分析(NTA)系统(NS300,Malvern,UK)和动态光散射(DLS)系统(nano-ZS90,Malvern,UK)对制备的纳米气泡水进行粒子数量和尺寸分析测定。
脱气实验:
将制备好的纳米气泡水-20℃冷冻至完全凝固,放入真空干燥箱抽真空(0.01atm)放置24h,循环数次。
TMB(作为底物)氧化曲线的测定:
小粒径纳米气泡对TMB氧化动力学的影响是通过测定TMB随时间被铜离子催化H2O2产生的羟基自由基所氧化的曲线来确定的,反应体系分别在不含纳米气泡的超纯水、大粒径纳米气泡水以及小粒径纳米气泡水中进行。所有样品的H2O2浓度为0.1M。加入H2O2后,将10μL TMB溶液(10mg ml-1于DMSO中)和10μL CuCl2(1mM于水中)加入不同的反应混合物中。通过添加超纯水/常规纳米气泡水/小粒径纳米气泡水使最终反应体积达到1mL。加入底物TMB后,立即将每组200uL(4孔)转移到96孔板中,使用酶标仪(VERSA max microplatereader)监测其在652nm处的光密度变化。将652nm处的吸光度与反应时间作图以获得反应时间曲线。
结果如图1所示,a分别示出了超纯水和小粒径纳米气泡存在时TMB的氧化曲线,其反应原理为铜离子催化过氧化氢产生羟基自由基来氧化四甲基联苯胺(TMB),使TMB在652nm处有吸光值,结果证明小粒径纳米气泡水具有良好的抗活性氧氧化性能。
b为纳米粒子追踪分析系统(NTA)测定的小粒径气泡水和超纯水中纳米气泡的粒径分布,其测定范围为50-1000nm,而我们测定出纳米气泡在50nm处有较多分布,表明其中存在大量的小于50nm的纳米气泡。
c为使用动态光散射技术(DLS)测定的小粒径气泡水和大粒径纳米气泡水中纳米气泡的粒径分布。DLS能够测定0.4nm以上的纳米粒子的大小,听此被用来测定NTA检测不到的纳米气泡。图中横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例,结果显示该小粒径纳米气泡水中含有3.62nm和255nm的纳米气泡,其中3.62nm的纳米气泡的数量占比接近100%。
d为超纯水、小粒径纳米气泡水、脱气去除部分气泡后水中的TMB氧化曲线,其反应原理为铜离子催化过氧化氢产生羟基自由基来氧化TMB,使TMB在652nm处有吸光值。横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化。其中,使用动态光散射技术(DLS)已经无法检测脱气后的小粒径纳米气泡水的纳米气泡粒径,代表着通过脱气方法去除了绝大部分的纳米气泡。结果证明小粒径纳米气泡水相比超纯水、脱气后小粒径纳米气泡水均具有更好的抗活性氧氧化性能。
实施例2对比小粒径纳米气泡和不同浓度的抗坏血酸钠的抗氧化能力
本实施例进行了小粒径纳米气泡与抗坏血酸钠的抗氧化能力的对比。
配置不同浓度的抗坏血酸钠,按上述测定TMB氧化曲线的方法与超纯水一起在96孔板内动态测量TMB的氧化曲线。相对氧化值的计算公式如下:
结果如图2所示,小粒径纳米气泡的相对氧化值为0.4,其抗氧化能力介于100μM和200μM抗坏血酸钠溶液的抗氧化能力之间。
实施例3含大粒径纳米气泡的水不具备抗氧化效果
大粒径纳米气泡的产生和分析:大粒径纳米气泡是室温下通过在超纯水中加压-减压来制备的。该实验是在一个定制的能够控制压力大小的金属腔内进行的。将超纯水放入腔体内,通入氮气至气压为0.6MPa持续30分钟,然后在约4小时内缓慢减压至常压。使用纳米粒子跟踪分析(NTA)系统(NS300,Malvern,UK)和动态光散射(DLS)系统(nano-ZS90,Malvern,UK)对制备的纳米气泡水进行粒子数量和尺寸分析测定。
结果如图1所示,示出了将不含有小粒径纳米气泡的常规氮气纳米气泡水转化为小粒径氮气纳米气泡水后的粒径分布以及抗氧化效果图;其中,a是在不含有小粒径纳米气泡的常规氮气纳米气泡存在时的TMB的氧化曲线,原理为铜离子催化过氧化氢产生羟基自由基来氧化TMB,使TMB在652nm处有吸光值。横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化。b和c分别为NTA和DLS测定的大粒径纳米气泡水中纳米气泡的粒径分布。其中NTA显示该水中不含有小于80nm的纳米气泡,DLS图的横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例,结果显示该小粒径纳米气泡水中含有142nm和396nm的纳米气泡,其中完全不含有小于100nm的纳米气泡。d为不含有小粒径纳米气泡的常规氮气纳米气泡通过以公开的冻融法转变为小粒径纳米气泡后的TMB的氧化曲线。原理为铜离子催化过氧化氢产生羟基自由基来氧化TMB,使TMB在652nm处有吸光值。横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;e为DLS测定的以公开的冻融法转将常规氮气纳米气泡水转变为小粒径气泡水后水中纳米气泡的粒径分布。横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例,结果显示该小粒径纳米气泡水中含有5.61nm和164nm的纳米气泡,其中5.61nm的纳米气泡的数量占比接近100%。
本实施例展示了含大粒径纳米气泡的水不具备抑制自由基氧化的能力,而通过一定的方法将大粒径纳米气泡转变为小粒径纳米气泡后其重新表现出抗氧化能力。
实施例4含不同气体的小粒径纳米气泡的水的抗氧化能力
实验材料
高纯氧气(≥99.999%)。
实验步骤:
纳米气泡的产生和分析:使用氧气气源替代氮气气源按照上述方法制备小粒径纳米气泡水。
TMB氧化曲线的测定:使用小粒径氧气纳米气泡水按照上述方法测定TMB的氧化曲线。
结果如图4所示,示出了实施例4制备得到的小粒径氧气纳米气泡的粒径分布以及抗氧化效果图;其中,a是在小粒径氧气纳米气泡存在时TMB的氧化曲线。原理为铜离子催化过氧化氢产生羟基自由基来氧化TMB,使TMB在652nm处有吸光值。横坐标代表反应时间,纵坐标的吸收值越高代表越多的TMB被氧化;b和c分别为NTA和DLS测定的气泡水中纳米气泡的粒径分布。其中NTA显示含有小于50nm的纳米气泡,DLS结果图横坐标代表纳米气泡的粒径,黑色实线为不同粒径纳米气泡对光的散射强度所占的比例,灰色实线为不同粒径的纳米气泡所占数量的比例,结果显示该小粒径纳米气泡水中含有13.5nnm、106nm和295nm的纳米气泡,其中13.5nm的纳米气泡的数量占比接近100%。
本实施例利用氧气气源来制备小粒径纳米气泡,研究发现其也具备抗氧化能力,证明小粒径纳米气泡的抗氧化能力与内部气体的种类无关。
以上所述的,仅为本发明的较佳实施例,并非用以限定本发明的范围,本发明的上述实施例还可以做出各种变化。凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰,皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。
Claims (10)
1.一种小粒径纳米气泡水,其特征在于,所述小粒径纳米气泡水中纳米气泡的总浓度>107个/毫升,其中含有粒径小于50nm的小粒径纳米气泡。
2.根据权利要求1所述的小粒径纳米气泡水,其特征在于,所述小粒径纳米气泡的粒径分布范围为2nm-15nm。
3.根据权利要求1所述的小粒径纳米气泡水,其特征在于,所述小粒径纳米气泡的DLS数量强度占比>20%。
4.一种小粒径纳米气泡水的制备方法,其特征在于,包括以下步骤:
S1:将需要引入小粒径纳米气泡的水冷却到零摄氏度左右;
S2:将冷却后的水放入到加压装置内;
S3:向所述加压装置中通入高压气体或采用机械力加压,以增大加压装置内部压力;
S4:维持高压条件,持续一段时间;
S5:缓慢释压至常压状态,即可制备得到一种含有小粒径纳米气泡的水;
其中,制备全程保持水温低于10℃,所述纳米气泡中部分气泡的粒径小于50nm。
5.根据权利要求4所述的制备方法,其特征在于,步骤S1包括:将需要引入小粒径纳米气泡的水冷却至冰水混合物。
6.根据权利要求4所述的制备方法,其特征在于,步骤S3包括:向所述加压装置中通入高压气体,根据不同需要选择不同的气体种类。
7.根据权利要求4所述的制备方法,其特征在于,步骤S3、S4包括:所述高压条件的压力范围为0.2-1.2MPa,维持时间20min-60min。
8.根据权利要求4所述的制备方法,其特征在于,步骤S5包括:释压过程采用气体流量计控制释气速率。
9.根据权利要求4所述的制备方法,其特征在于,制备全程保持水温低于5℃。
10.一种小粒径纳米气泡水在制备抗活性氧氧化试剂中的应用,其特征在于,所述小粒径纳米气泡水中含有粒径小于50nm的纳米气泡。
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