CN112126222B - 一种提高ε-聚赖氨酸稳定性的方法及其在抑菌产品中的应用 - Google Patents
一种提高ε-聚赖氨酸稳定性的方法及其在抑菌产品中的应用 Download PDFInfo
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Abstract
本发明公开了一种提高ε‑聚赖氨酸稳定性的方法及其在抑菌产品中的应用,在ε‑聚赖氨酸加入产品的同时加入季铵盐类化合物,以提高ε‑聚赖氨酸的稳定性,使其在应用过程中不再变黄和降解,提高了终产品的稳定性和货架期,同时可以减少ε‑聚赖氨酸在终产品中的使用量,本方法还具有简便,易操作,安全环保,重复性好,成本低廉等优点,因此具有优异的应用价值。此外,ε‑聚赖氨酸与季铵盐类化合物的协同还具有很好的抑菌效果,在抑菌产品中也有很好的应用前景。
Description
技术领域
本发明涉及一种提高ε-聚赖氨酸稳定性的方法及其在抑菌产品中的应用,属于ε-聚赖氨酸技术领域。
背景技术
ε-聚赖氨酸(ε-PL)是由白色链球菌(Streptomyces albulus)发酵产生的一种含有25~35个L-赖氨酸残基的同型单体聚合物,分子量在3600-4300,由L-赖氨酸的ε-氨基与另一L-赖氨酸的α-羧基形成ε-酰胺键连接而成,结构式如下:
ε-聚赖氨酸为淡黄色粉末,吸湿性强,略有苦味,易溶于水,微溶于乙醇,是赖氨酸的直链状聚合物。ε-聚赖氨酸是一种天然的生物代谢产品,具有很好的杀菌能力,是具有优良防腐性能和巨大商业潜力的生物防腐剂。1989年,日本厚生劳动省已批准ε-聚赖氨酸作为食品防腐剂,把ε-聚赖氨酸归属于一种天然食品添加剂,并允许使用。2004年,FDA已正式批准ε-聚赖氨酸作为天然食品添加剂。在化妆品中,ε-聚赖氨酸作为防腐剂已被纳入我国2015年版的《已使用化妆品原料名称目录》。鉴于其良好的抑菌性能和安全性,韩国、日本和欧美的化妆品企业已将ε-聚赖氨酸用作防腐剂,在一些化妆水、面霜、乳液和卸妆液类的产品中都有广泛的应用。另外,ε-聚赖氨酸具有很强的吸湿能力,在化妆品中可用作保湿剂,也可与丙烯乙二醇结合形成水凝胶,制备高吸水性聚合物材料,应用于一次性纸尿裤和卫生巾等日化产品中。ε-聚赖氨酸还具有补充皮肤和头发的营养、增加组织活力、减少皮肤皱纹、延缓皮肤老化、促进皮肤健康的作用。同时ε-聚赖氨酸安全无毒、可生物降解,ε-聚赖氨酸是一种多肽,在人体内可分解为赖氨酸,而赖氨酸是人体必须的8种氨基酸之一,因此ε-聚赖氨酸是一种营养型抑菌剂,安全性高于其他化学防腐剂,其急性口服毒性为5g/kg,有研究证明ε-聚赖氨酸对生殖、神经系统、免疫功能、胚胎发育方面均无毒副作用。
此外,ε-聚赖氨酸还不受pH值影响,对热稳定。有人对聚赖氨酸的抑菌性能进行研究,发现ε-聚赖氨酸不仅可抑制耐热性较强的G+的微球菌,而且对其它天然防腐剂(如Nisin)不易抑制的G-的大肠杆菌、沙门氏菌抑菌效果亦非常好,同时还可抑制保加利亚乳杆菌、嗜热链球菌、酵母菌的生长。但是单独使用ε-PL时对枯草芽孢杆菌、黑曲霉抑制不明显,采用ε-PL与醋酸复合处理,对枯草芽孢杆菌抑制作用增强,经高温处理后的ε-聚赖氨酸对微球菌仍有抑菌活性。
但是在使用过程中发现,ε-聚赖氨酸在加入产品中时很容易变黄和降解,严重的影响了ε-聚赖氨酸在化妆品、医药等领域的应用,目前对于该问题还没有很好的解决办法。
发明内容
针对ε-聚赖氨酸加入产品中时容易变黄和降解的问题,本发明提供了一种提高ε-聚赖氨酸稳定性的方法,该方法可以有效的提高ε-聚赖氨酸在产品中的稳定性,使其在应用过程中变黄和降解的情况消失或减少,提高了终产品的稳定性和货架期,同时可以减少ε-聚赖氨酸在终产品的使用量。
本发明具体技术方案如下:
一种提高ε-聚赖氨酸稳定性的方法,该方法是:将ε-聚赖氨酸与季铵盐类化合物同时加入产品中,通过季铵盐类化合物提高ε-聚赖氨酸在产品中的稳定性。
进一步的,所属产品既可以是液体产品,也可以是凝胶产品,还可以是其他类型的产品。
本发明经过研究发现,季铵盐类化合物的存在能很好的避免或缓解ε-聚赖氨酸的降解和变黄问题,起到防止ε-聚赖氨酸在水性液体环境中变黄和降解的问题。优选的,当季铵盐类化合物选择西吡氯铵、苯扎氯铵、苯扎溴铵、苄索氯铵、透明质酸季铵盐、壳聚糖季铵盐、十二烷基三甲基氯化铵和十二烷基二甲基苄基溴化铵中的一种或多种时,具有很好的避免ε-聚赖氨酸的降解和变黄的效果。上述各种季铵盐类化合物都在《已使用化妆品原料名称目录》中,它们的加入不会对最终产品的性能造成不良影响。
更为优选的,所述季铵盐类化合物为苯扎氯铵和苯扎溴铵中的一种或两种。
进一步的,ε-聚赖氨酸与季铵盐类化合物的质量比为50:1-1:2。更为优选的,ε-聚赖氨酸与季铵盐类化合物的质量比为5:1-1:1。
进一步的,ε-聚赖氨酸作为添加剂,在产品中的含量可以根据现有技术中报道的常规含量进行添加,例如添加量为产品质量的0.03-0.3wt%,优选为0.03-0.1wt%。在此含量下,季铵盐类化合物在产品中的含量一般为0.001-0.15wt%,优选为0.006-0.1wt%。如果ε-聚赖氨酸添加量过多,则季铵盐类化合物抑制其降解或变黄的作用降低。
本发明还提供了一种产品,该产品中含有ε-聚赖氨酸与季铵盐类化合物。
进一步的,该产品优选为抑菌产品,因为ε-聚赖氨酸与季铵盐类化合物的协同作用,表现出很好的抑菌效果,同时季铵盐类化合物还能有效提高ε-聚赖氨酸的稳定性,延长产品的保质期。
进一步的,上述产品中,季铵盐类化合物的种类与上述定义一致。
进一步的,上述产品中,ε-聚赖氨酸与季铵盐类化合物的质量比为50:1-1:2,优选为5:1-1:1。
进一步的,上述产品中,ε-聚赖氨酸在产品中的含量为0.03-0.3wt%,优选为0.03-0.1wt%;季铵盐类化合物在产品中的含量为0.001-0.15wt%,优选为0.006-0.1wt%。
本发明在ε-聚赖氨酸加入产品的同时加入季铵盐类化合物,可以有效的提高ε-聚赖氨酸的稳定性,使其在应用过程中变黄和降解的情况得到有效缓解或避免,提高了终产品的稳定性和货架期,同时可以减少ε-聚赖氨酸在终产品中的使用量。本方法具有简便、易操作、安全环保、重复性好、成本低廉等优点,因此具有优异的应用价值。此外,ε-聚赖氨酸与季铵盐类化合物的协同还具有很好的抑菌效果,在抑菌产品中也有很好的应用前景。
具体实施方式
为了更好的理解本发明,下面结合实施例对本发明作详细的说明,但并不只限于以下的实施列。
实施例1-6
按照下表1的配方配制液体产品,制备方法为:分别按表1中的配方称取ε-聚赖氨酸、苯扎氯铵、甘油、尿囊素、依克多因、甜菜碱,将甘油、尿囊素、依克多因、甜菜碱加入适量纯化水中,搅拌溶解完全,然后同时加入ε-聚赖氨酸和苯扎氯铵,用适量氢氧化钠调节pH值至4.0-7.5,即得各实施例液体产品,该液体产品可用于化妆品领域。
表1液体产品配方(wt%)
实施例7-16
按照实施例3的配方和方法制备液体产品,不同的是:所用的季铵盐类化合物不同,具体方法如下:
1、按照ε-聚赖氨酸0.03wt%、季铵盐类化合物0.006wt%、甘油5.0wt%、尿囊素0.1wt%、依克多因0.2wt%、甜菜碱0.5wt%的量称取各原料;
2、将甘油、尿囊素、依克多因、甜菜碱加入适量纯化水中,搅拌溶解完全,然后同时加入ε-聚赖氨酸和季铵盐类化合物,用适量氢氧化钠调节pH值至4.0-7.5,即得各实施例液体产品。
所用季铵盐类化合物如下表2所示:
表2
编号 | 季铵盐类化合物 |
实施例7 | 苯扎溴铵0.006wt% |
实施例8 | 西吡氯铵0.006wt% |
实施例9 | 苄索氯铵0.006wt% |
实施例10 | 透明质酸季铵盐0.006wt% |
实施例11 | 壳聚糖季铵盐0.006wt% |
实施例12 | 十二烷基三甲基氯化铵0.006wt% |
实施例13 | 十二烷基二甲基苄基溴化铵0.006wt% |
实施例14 | 苯扎溴铵0.003wt%、苯扎氯铵0.003wt% |
实施例15 | 透明质酸季铵盐0.003wt%、苄索氯铵0.003wt% |
实施例16 | 十二烷基二甲基苄基溴化铵0.003wt%、西吡氯铵0.003wt% |
对比例1-5
按照下表3的配方配制液体产品,制备方法为:分别按表3中的量称取各原料,将甘油、尿囊素、依克多因、甜菜碱加入适量纯化水中,搅拌溶解完全,然后同时加入ε-聚赖氨酸(如果有的话)和季铵盐类化合物(如果有的话),用适量氢氧化钠调节pH值至4.0-7.5,即得各对比例样品。
表3配方(wt%)
稳定性试验
1、加速试验:
将实施例1-6和对比例1-5的样品置于40℃±2℃/75%RH±5%RH条件下放置6个月,分别于1月、2月、3月、6月取样检验外观、pH及ε-聚赖氨酸含量,结果见表4。
2、长期试验:
将实施例1-6和对比例1-5的样品置于25℃±2℃/60%RH±5%RH条件下放置12个月,分别于3月、6月、9月、12月取样检验外观、pH及ε-聚赖氨酸含量,结果见表5。
3、检验方法
3.1外观:
取试样在室温和非阳光直射下目测观察。
3.2、pH
取各样品,按照《中华人民共和国药典》(2015年版)四部通则0631中规定的方法测定。
3.3、ε-聚赖氨酸含量
3.3.1试剂和材料
硫酸钠溶液:0.30mol/L。称取42.6g硫酸钠溶解并定容至1000mL,用乙酸调pH为4.0。
ε-聚赖氨酸标准贮备溶液:10.0mg/mL。称取1.0gε-聚赖氨酸标准样品,溶解并定容至100mL。
3.3.2仪器和设备
凝胶渗透色谱仪:配有紫外检测器。
色谱条件
凝胶柱:水相凝胶过滤色谱(7.8mm×300mm),检测温度:30℃。
检测波长:210nm,流速:0.5mL/min,进样量:20μL。
3.3.3分析步骤
标准曲线的绘制
分别移取不同量的ε-聚赖氨酸标准溶液至合适的容量瓶中并定容,溶液经0.22μm微孔滤膜过滤。打开色谱仪,并调至工作状态,待基线平稳后,依次将上述ε-聚赖氨酸标准溶液注入凝胶柱中,进样量为20μL,记录峰面积,以标准溶液中ε-聚赖氨酸的浓度为横坐标,峰面积为纵坐标绘制标准曲线。
试样测定
准确称取试样,溶解定容至合适的容量瓶中。配好的试样溶液经0.22μm微孔滤膜过滤,进样量为20μL,进行凝胶渗透色谱检测,记录峰面积,并根据标准曲线查得试样中ε-聚赖氨酸的浓度。
4、结果计算
ε-聚赖氨酸含量的百分含量w,按以下公式计算:
式中:
M1——从标准曲线中查得的ε-聚赖氨酸浓度,单位为mg/mL;
M——标示浓度,即ε-聚赖氨酸的理论原始浓度,单位为mg/mL。
实验结果以平行测定结果的算术平均值为准。在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的2%。
表4加速试验结果
表5长期试验结果
由对比例1和实施例1-6的结果可知,含ε-聚赖氨酸的溶液在常温和40℃时不稳定,易变黄、含量易下降,且pH值也下降,加入苯扎氯铵后可以减轻ε-聚赖氨酸的不稳定。对比例1中,长期试验中储存12个月后ε-聚赖氨酸的含量下降了13.25%,加速试验中储存6个月后ε-聚赖氨酸下降了16.45%;实施例1-6的样品在加速试验和长期试验中的pH值基本未变,外观也未变黄,含量仅下降了4.74-7.03%和2.65-5.03%,稳定性明显高于对比例1。对比例2-5中的ε-聚赖氨酸的稳定性也低于实施例。
按照上述同样的方法,对实施例7-16的样品进行加速试验和长期试验,实验期间各样品pH值基本不变,外观也未变黄,ε-聚赖氨酸含量变化如下表6和7所示。
表6实施例7-16加速试验结果
表7实施例7-16长期试验结果
抑菌试验
试验步骤如下:
a)用PBS液将试验菌悬液进行适当稀释,要求浓度为:取0.1ml滴于5.0ml对照液(PBS)内,回收菌数为1×104~9×104cfu/ml;
b)将试验样品用无菌标准硬水稀释至规定的浓度;
c)分别吸取实施例3、对比例1和对比例3的样品原液5.0ml放入灭菌试管中,20℃恒温5min;
d)吸取试验菌液0.1ml,加入到各含5.0ml样品的试管中,迅速混匀,并立即计时;
e)作用至设定时间后,取试验菌与样品混合液0.5ml,加入到含4.5ml经灭菌的PBS试管中,充分混匀;
f)放置10min后,吸取样液1ml,置于灭菌平皿内,每个样液接种于两个灭菌平皿。用凉至40℃~45℃的营养琼脂培养基(大肠杆菌、金黄色葡萄球菌)或沙氏琼脂培养基(白色念珠菌)15ml作倾注,转动平皿,使其充分均匀,琼脂凝固后翻转平皿,(35±2)℃培养48h(大肠杆菌、金黄色葡萄球菌)或72h(白色念珠菌)后,做活菌菌数计数;
g)以PBS代替试验样品,同时按以上步骤操作,作为对照样品;
实验重复三次,求其平均值。
抑菌率按以下公式计算:
Ⅰ—对照样品平均菌落数;
Ⅱ—试验样品平均菌落数。
表8抑菌试验结果
由表8可知,仅含0.006%苯扎氯铵溶液的抑菌率不足50%,不具有抑菌作用,仅含有0.03%ε-聚赖氨酸的溶液的抑菌率为50%多,有抑菌作用,但是同时含有0.03%ε-聚赖氨酸和0.006%苯扎氯铵的溶液对大肠杆菌、金黄色葡萄球菌、白色念珠菌三种菌的抑菌率均>96%,说明ε-聚赖氨酸和苯扎氯铵在抑菌上有协同增效的作用。
实施例17
按照以下质量百分比称取原料:
ε-聚赖氨酸0.1%、苯扎氯铵0.1%、甘油5.0%、尿囊素0.1%、依克多因0.2%、甜菜碱0.5%,HEC30000 1.5%,余量为水。
按照以下方法制备抑菌凝胶:
称取配方量的HEC30000加入合适的容器中,再加适量的90℃以上的热水搅拌(热水浴保温)至上述原料溶解完全。然后降温至60℃以下再加入配方量的甘油、尿囊素、依克多因、甜菜碱、ε-聚赖氨酸、苯扎氯铵,补足水至100%,搅拌溶解,溶解完全后,用适量的5%氢氧化钠或盐酸调节pH至4.5,即得抑菌凝胶。
所制备的抑菌凝胶产品分别进行6个月加速稳定性试验和抑菌试验。结果表明,6个月加速试验结果与0月比外观、PH均未有变化,ε-聚赖氨酸含量仅下降了4.8%。该凝胶在20min对大肠杆菌、金黄色葡萄球菌、白色念珠菌三种菌的抑菌率均>95%。
虽然,上文中,已用一般性说明及实施例对本发明做了详尽的说明,但在本发明的基础上,可以做一些修改或改进,这对本领域技术人员是显而易见的。因此,在不偏离本发明精神的基础上所做的修改或改进,均属于本发明要求保护的范围。
Claims (3)
1.一种提高ε-聚赖氨酸稳定性的方法,其特征是:将ε-聚赖氨酸与季铵盐类化合物同时加入产品中,通过季铵盐类化合物提高ε-聚赖氨酸在产品中的稳定性;所述季铵盐类化合物为苯扎氯铵和苯扎溴铵中的一种或两种,ε-聚赖氨酸与季铵盐类化合物的质量比为5:1-1:1,ε-聚赖氨酸在产品中的含量为0.03-0.1wt%,季铵盐类化合物在产品中的含量为0.006-0.1wt%。
2.根据权利要求1所述的方法,其特征是:所述提高ε-聚赖氨酸稳定性指的是使ε-聚赖氨酸在水性液体环境中变黄和降解的情况消失或减少。
3.根据权利要求1或2所述的方法,其特征是:所述产品为液体产品或凝胶产品。
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