CN112708654A - 一种口腔清洁类产品抑菌效果的评价方法 - Google Patents
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Abstract
本发明属于微生物研究领域,为日化产品抑菌效果评价方法,具体涉及一种口腔清洁类产品抑菌效果的评价方法。本发明所述评价方法包括下列步骤:培养试验菌种;调整菌浓度;待测样品制备;试验菌与待测样品作用;细菌培养;菌落计数;效果评价。本发明方法灵敏度高、成本低廉、操作简易、稳定性高,能有效地评价口腔清洁类产品抑菌效果,填补本领域这一技术空白。
Description
技术领域
本发明属于微生物研究领域,为日化产品抑菌效果评价方法,具体涉及一种口腔清洁类产品抑菌效果的评价方法。
背景技术
在人的口腔内,约有400种以上的细菌在繁殖,其菌数约达100亿个。口腔内的细菌混合于人的唾液中,在经过既定时间之后的唾液中,检测到106CFU/ml水平的细菌。在这些细菌中,包含诱发牙齿龋齿、牙龈炎和牙周炎或口腔炎的细菌。
据悉,引起蛀牙是由于各种原因共同作用,但最重要的是齿菌斑。齿菌斑中混合有多种细菌,厌氧型菌如耐酸链球菌和乳酸菌尤其多,其中变形链球菌(Streptococcusmutans)被认为是主要蛀牙菌。现代医学研究认为,变形链球菌生物膜吸附其他细菌在牙釉表面所形成的牙菌斑也是致龋的关键因素之一,减少或抑制生物膜的形成同样可达到防治龋齿的目的。
抗菌口腔清洁类产品可以有效预防该类疾病的发生,现有技术中,日化品抑菌试验主要通过测定该产品某一浓度下和作用时间下对指定菌种抑制率,来评价产品的抑制效果。如生物膜形成量的测定是开展研究工作与评价治疗效果的重要手段,而目前测量生物膜的方法缺点有:灵敏度低、成本高,操作复杂、平行样本的数据重现性差;同时现有的日化品抑菌方法中选用的菌种均为需氧型的细菌/真菌,主要针对于皮肤感染类的菌种,缺少针对于口腔清洁类产品的厌氧型菌种。
因此,寻找一种灵敏度高、成本低廉、操作简易、稳定性高的口腔清洁类产品抑菌效果的评价方法是当务之急。上述这些都是本领域技术人员亟待解决的问题。
发明内容
本发明的目的在于提供一种口腔清洁类产品抑菌效果的评价方法。
本发明公开了一种口腔清洁类产品抑菌效果的评价方法,包括下列步骤:
a.培养试验菌种:接种厌氧型试验菌种于培养基中,36℃培养厌氧培养得到浑浊的增菌液;
b.调整菌浓度:用生理缓冲液调整菌浓度,回收菌数为1*104-9*104cfu/mL。
c.待测样品制备:用生理缓冲液稀释待测样品至最大溶解度后进行检测。
d.试验菌与待测样品作用:取一定体积待测样品稀释液放入灭菌试管中,室温下恒定5-10min;吸取一定体积试验菌液加入到含有待测样品的试管中,迅速混匀,并计时2-5min;
e.细菌培养:取一定体积试验菌与待测样品混合液,加入到一定体积生理缓冲液中,充分混匀,放置5-10min后,选取2-3个稀释度,36℃培养厌氧培养2-3天;同时设置对照组:以生理缓冲液代替试验样品;
f.菌落计数:
重复3次,求其平均值;按照公式计算:抑菌率(%)=(对照组平均菌落数-实验组平均菌落数)/对照组平均菌落数*100%
结果保留至小数点后两位;
g.效果评价:
抑菌率≥50%-90%:产品有抑菌作用;
抑菌率≥90%:产品有较强抑菌作用。
在一优选例中,所述口腔清洁类产品为:牙膏、牙粉、漱口水、口腔清洁剂、口腔喷雾剂、牙用糊剂、膏状制剂、牙齿凝胶或口香糖。
在一优选例中,所述厌氧型试验菌种为变形链球菌(Streptococcus mutans)、牙龈卟啉单胞菌(Porphyromonas gingivalis)、金黄色葡萄球菌(Staphylococcus aureus)、中间普氏菌(Prevotella intermedia)、内氏放线菌(Actinomyces naeslundii)、黏性放线菌(Actinomyces viscosus)、依氏放线菌(Actinomyces israelii)、迈氏放线菌(Actinomyces meyeri)和溶牙放线菌(Actinomyces dentinolyticus)、龋齿罗菌(Rothiadentocariosa)中的一种或者几种;其中优选为变形链球菌(Streptococcus mutans)。
在一优选例中,所述生理缓冲液可为PBS;
在一优选例中,所述培养基为细菌培养基、放线菌培养基或霉菌培养基中之一,其中细菌培养基优选为BHI肉汤;
本发明方法灵敏度高、成本低廉、操作简易、稳定性高,能有效地评价口腔清洁类产品抑菌效果,填补本领域这一技术空白。
附图说明
图1为本发明方法流程图;
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
操作人员:实验人员甲、乙
所需试剂耗材:
1)菌种:变形链球菌;
2)培养基试剂:磷酸盐缓冲液(PBS)、胰蛋白胨大豆琼脂培养基(TSA);
3)设备耗材:厌氧培养箱(或其他厌氧装置,如厌氧罐、厌氧包等)、生物安全柜、高压灭菌锅、水浴锅、移液枪1mL、5mL。
试验步骤:
1)培养试验菌种:接种试验菌种于BHI肉汤中,36℃培养厌氧培养3天,得到浑浊的增菌液;
2)调整菌浓度:用PBS调整菌浓度,要求取0.1mL滴于5.0mL PBS中,回收菌数为1×104-9×104cfu/mL。
3)样品制备:用PBS稀释牙膏至50%的样品浓度(最大溶解度)。
4)取样品稀释液5.0mL放入灭菌试管中,室温(约20℃)下恒定5min。
5)吸取试验菌液0.1mL加入到含有5.0mL样品的试管中,迅速混匀,并计时2分钟;作用至设定的时间后,取试验菌与样品混合液0.5mL,加入到4.5mL PBS中,充分混匀。
6)放置10分钟后,选取2个稀释度,做菌落计数,36℃培养厌氧培养2-3天。
7)设置对照组:以PBS代替试验样品,按照以上步骤操作。
8)重复3次,求其平均值。
9)按照公式计算:抑菌率(%)=(对照组平均菌落数-实验组平均菌落数)/对照组平均菌落数*100%
结果保留至小数点后两位。
试验数据分析:
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质。
Claims (8)
1.一种口腔清洁类产品抑菌效果的评价方法,包括下列步骤:
a.培养试验菌种:接种厌氧型试验菌种于培养基中,36℃培养厌氧培养得到浑浊的增菌液;
b.调整菌浓度:用生理缓冲液调整菌浓度,回收菌数为1×104-9×104cfu/mL;
c.待测样品制备:用生理缓冲液稀释待测样品至最大溶解度后进行检测;
d.试验菌与待测样品作用:取一定体积待测样品稀释液放入灭菌试管中,室温下恒定5-10min;吸取一定体积试验菌液加入到含有待测样品的试管中,迅速混匀,并计时2-5min;
e.细菌培养:取一定体积试验菌与待测样品混合液,加入到一定体积生理缓冲液中,充分混匀,放置5-10min后,选取2-3个稀释度,36℃培养厌氧培养2-3天;同时设置对照组:以生理缓冲液代替试验样品;
f.菌落计数:
重复3次,求其平均值;按照公式计算:抑菌率(%)=(对照组平均菌落数-实验组平均菌落数)/对照组平均菌落数*100%
结果保留至小数点后两位;
g.效果评价:
抑菌率≥50%-90%:产品有抑菌作用;
抑菌率≥90%:产品有较强抑菌作用。
2.如权利要求1所述的评价方法,其特征在于所述口腔清洁类产品为牙膏、牙粉、漱口水、口腔清洁剂、口腔喷雾剂、牙用糊剂、膏状制剂、牙齿凝胶或口香糖。
3.如权利要求1所述的评价方法,其特征在于所述口腔清洁类产品为抗菌牙膏。
4.如权利要求1所述的评价方法,其特征在于所述厌氧型试验菌种为变形链球菌、牙龈卟啉单胞菌、金黄色葡萄球菌、中间普氏菌、内氏放线菌、黏性放线菌、依氏放线菌、迈氏放线菌、溶牙放线菌、龋齿罗菌中的一种或者几种。
5.如权利要求1所述的评价方法,其特征在于所述厌氧型试验菌种为变形链球菌。
6.如权利要求1所述的评价方法,其特征在于所述生理缓冲液为PBS。
7.如权利要求1所述的评价方法,其特征在于所述培养基为细菌培养基、放线菌培养基或霉菌培养基中之一。
8.如权利要求7所述的检测方法,其特征在于所述细菌培养基为BHI肉汤。
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