油脂类乳膏药品萃取后微生物在油相与水相中的分布状况研究
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摘要
目的:通过研究萃取后5种不同类型微生物在油相和水相的分布规律,探讨萃取法在非无菌药品微生物限度检查时油脂类供试液制备中的应用。方法:在无菌的十四烷酸异丙酯-pH 7.0氯化钠蛋白胨缓冲液(v/v,1∶1)中分别接种高、低浓度的金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、白色念珠菌和黑曲霉等5种试验菌,混匀静置5 min,照《中华人民共和国药典》2015年版四部1105的方法进行计数检查,计算回收率。应用水分活度测定仪分别测定油相、水相的水分活度(a_w)。对油脂类乳膏剂用萃取法处理,然后薄膜过滤进行计数方法适用性试验。结果:萃取后不同浓度、不同类型的试验菌主要分布在下层水相中,各试验菌回收率均在0.5%～2.0%之间。25℃下油相a_w为0.22,水相a_w为0.99。3种乳膏剂试验菌回收率均在0.5%～2.0%之间。结论:萃取后微生物在油相、水相中非均匀分布,其主要分布特点是微生物趋向于分布在有利于其生存的环境条件水相中。采用十四烷酸异丙酯萃取的方法可用于油脂类乳膏药品微生物限度检查时的供试液制备。
Objective:To study the distribution of 5 different kinds of microorganisms in the oil phase and aqueous phase after extraction,discuss the application of extraction method in the preparation of fatty sample solution for microbiological examination of non-sterile products.Methods:Low and high concentrations of test microorganism which included Staphylococcus aureus,Pseudomonas aeruginosa,Bacillus subtilis,Candida albicans,Aspergillus nigerin were inoculated respectively in sterile isopropyl myristate and pH 7.0 sodium chloride peptone buffer(v/v,1∶1).The samples were mixed and laid for 5 minutes.The recovery rate was calculated according to the method of ChP 2015 edition Volume Ⅳ<1105>counted inspection.Water activity instrument was used to determine water activity of oil phase and aqueous phase respectively.The extraction method was adopted to deal with fatty cream samples,and the method suitability experiments were completed with membrane filter.Results:Different concentrations and different types of microorganisms were mainly distributed in aqueous phase after extraction,the recovery rates of each tested microorganism were 0.5 to 2.0.Water activity(a_w)of oil phase was 0.22 and aqueous phase was 0.99 at 25 ℃.The test bacteria recovery rates of 3 kinds of grease cream drugs were between 0.5 to 2.0.Conclusion:Microorganisms after extraction distributed inhomogeneous in oil and aqueous phase.Its main distribution characteristic is that the microorganisms tend to be distributed in the aqueous phase where is favorable for their survival.The method of extracting with isopropyl myristate could be used to prepare the test solution for microbial limit test for oil and fat samples.
Objective:To study the distribution of 5 different kinds of microorganisms in the oil phase and aqueous phase after extraction,discuss the application of extraction method in the preparation of fatty sample solution for microbiological examination of non-sterile products.Methods:Low and high concentrations of test microorganism which included Staphylococcus aureus,Pseudomonas aeruginosa,Bacillus subtilis,Candida albicans,Aspergillus nigerin were inoculated respectively in sterile isopropyl myristate and pH 7.0 sodium chloride peptone buffer(v/v,1∶1).The samples were mixed and laid for 5 minutes.The recovery rate was calculated according to the method of ChP 2015 edition Volume Ⅳ<1105>counted inspection.Water activity instrument was used to determine water activity of oil phase and aqueous phase respectively.The extraction method was adopted to deal with fatty cream samples,and the method suitability experiments were completed with membrane filter.Results:Different concentrations and different types of microorganisms were mainly distributed in aqueous phase after extraction,the recovery rates of each tested microorganism were 0.5 to 2.0.Water activity(a_w)of oil phase was 0.22 and aqueous phase was 0.99 at 25 ℃.The test bacteria recovery rates of 3 kinds of grease cream drugs were between 0.5 to 2.0.Conclusion:Microorganisms after extraction distributed inhomogeneous in oil and aqueous phase.Its main distribution characteristic is that the microorganisms tend to be distributed in the aqueous phase where is favorable for their survival.The method of extracting with isopropyl myristate could be used to prepare the test solution for microbial limit test for oil and fat samples.
引文
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[15]ATLAS RM,ATLAS MC.Biodegradation of oil and bioremediation of oil spills[J].Curr Opin Biotechnol,1991,2:440
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[19]李辉,杨晓莉,梁泽梅,等.不同剂型硝酸咪康唑微生物限度检查方法适用性研究与评价[J].中国抗生素杂志,2018,43(1):80LI H,YANG XL,LANG ZM,et al.Study and evaluation on the applicability of different microbiological methods formiconazole nitrate[J].Chin J Antibiot,2018,43(1):80
[20]郑小玲,王征南,李珏.瑞他莫林软膏的微生物限度检查适用性试验[J].中国现代应用药学,2016,33(5):634ZHENG XL,WANG ZN,LI J.Applicability test of microbial limit test for retapamulin ointment[J].Chin J Mod Appl Pharm,2016,33(5):634
[21]钟柳芳,肖文可,孙树周,等.双柏膏微生物限度检查方法适用性试验[J].中国现代药物应用,2017,11(14):195ZHONG LF,XIAO WK,SUN SZ,et al.Applicability test of microbial limit test for Shuangbai ointment[J].Chin J Mod Drug Appl,2017,11(14):195
[22]HERMANSSON M,KJELLEBERG S,KORHONEN TK,et al.Hydrophobic and electrostatic characterization of surface structures of bacteria and its relationship to adhesion to an air-water interface[J].Arch Microbiol,1982,131(4):308
[23]ROSENBERY M,KJELLEBERG S.Hydrophobie Interactions:Role in Bacterial Adhension[M]//MARSHALL KC.Advances in Microbial Ecology.New York:Plenum Press,1986:353
[24]唐啸宇,孙洪林,何冰芳.耐有机溶剂微生物及酶类在生物催化中的应用[J].化学进展,2009,21(12):2726TANG XY,SUN HL,HE BF.Organic solvent tolerant bacteria and enzymes for application in biocatalysis[J].Prog Chem,2009,21(12):2726
[25]SCOTT S.Accuracy of plate counts[J].J valid Technol,2011,17(3):42
[26]USP 40-NF 35.VolⅠ[S].2017:General Information(1227)
[27]李辉,杨晓莉,秦亚红,等.改进培养基制备的平皿法应用于芬太尼透皮贴剂微生物限度检查[J].药学研究,2017,36(9):512LI H,YANG XL,QIN YH,et al.Application of the plate count methods of improved medium preparation on fentanyl transdermal patches microbial limit examination[J].J Pharm Res,2017,36(9):512
[2]雷质文,姜英辉,梁成珠,等.食源微生物检验用样品的抽取和制备手册[M].北京:中国标准出版社,2010:106LEI ZW,JIANG YH,LIANG CZ,et al.Handbook on Sampling and Preparing Foodborne Microbiological Samples[M].Beijing:Standards Press of China,2010:106
[3]中华人民共和国药典2015年版.四部[S].2015:140ChP 2015.VolⅣ[S].2015:140
[4]杨晓莉,李辉,马英英,等.中国药典2015年版非无菌产品微生物限度检查:微生物计数法解读[J].药物分析杂志,2016,36(6):1101YANG XL,LI H,MA YY,et al.Interpretation of the microbiological examination of nonsterile products:microbial enumeration test of Chinese pharmacopoeia 2015edition[J].Chin J Pharm Anal 2016,36(6):1101
[5]周剑,李霞.药品微生物限度检查法中几种样品前处理方法的可行性研究[J].中国药房,2012,23(33):3136ZHOU J,LI X.Pharmaceutical microbial limit test of several sample pretreatment methods and feasibility studies[J].China Pharm,2012,23(33):3136
[6]李趣嫦,曾璞,肖建光,等.50种中成药微生物限度检查法的适用性试验及结果分析[J].药物分析杂志,2017,37(12):2214LI QC,ZENG P,XIAO JG,et al.Microbial limit test for determination of 50 Chinese patent drugs and results analysis[J].Chin J Pharm Anal,2017,37(12):2214
[7]ZHOU L,NIU Y,YANG J,et al.Reaction kinetics for synthesis of isopropyl myristate catalyzed by sulfated titania[J].Korean J Chem Engin,2016,33(8):1
[8]颜红,徐伟娜,刘林,等.气相色谱法测定睾酮凝胶中肉豆蔻酸异丙酯的含量[J].药学研究,2016,35(8):469YAN H,XU WN,LIU L,et al.Determination of isopropyl myristate in testosterone gel by capillary GC[J].Pharm Res,2016,35(8):469
[9]张艳.肉豆蔻酸异丙酯作为油相的微乳液的相行为和增溶能力[D].济南:山东师范大学,2017ZHANG Y.Phase Behavior and Solubility of Isopropyl Myristate as Microemulsion of Oil Phase[D].Jinan:Shandong Normal University,2017
[10]GB 5009.238-2016食品安全国家标准食品水分活度的测定[S].2016GB 5009.238-2016National food safety standards of food and water activity determination[S].2016
[11]郑西强,蔡昌凤,李娜.萃取-比浊法测定焦化废水中微生物疏水性的研究[J].安徽工程科技学院学报,2010,25(1):11ZHENG XQ,CAI CF,LI N.Extraction-turbidimetric measuring hydrophobic microorganism in coking wastewater[J].J Anhui Univ Technol Sci,2010,25(1):11
[12]LIANG B,ZHANG K,WANG LY,et al.Different diversity and distribution of archaeal community in the aqueous and oil phases of production fluid from high-temperature petroleum reservoirs[J].Front Microbiol,2018,9:841
[13]虞志强,陈杏娟,赵刚,等.两相系统在疏水性有机污染物高效降解菌富集培养中的应用研究进展[J].微生物学通报,2016,43(7):1605YU ZQ,CHEN XJ,ZHAO G,et al.Research progress on the application of two-phase systems to enrich highly efficient bacteria for hydrophobic organic pollutants degradation[J].Microbiol China,2016,43(7):1605
[14]LI XX,MBADINGA SM,LIU JF,et al.Microbiota and their affiliation with physiochemical characteristics of different subsurface petroleum reservoirs[J].Intern Biodeter Biodegr,2017,120:170
[15]ATLAS RM,ATLAS MC.Biodegradation of oil and bioremediation of oil spills[J].Curr Opin Biotechnol,1991,2:440
[16]WANG LY,KE WJ,SUN XB,et al.Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches[J].Appl Microbiol Biotechnol,2014,98(9):4209
[17]MADIGAN MT,MARTINKO JM,STAHL DA,et al.Brock Biology of Microorganisms[M].13th EdNew York:Benjamin Cummings,2012
[18]SCOTT WJ.Water relations of food spoilage microorganisms[J].Adv Food Res,1957,7:83
[19]李辉,杨晓莉,梁泽梅,等.不同剂型硝酸咪康唑微生物限度检查方法适用性研究与评价[J].中国抗生素杂志,2018,43(1):80LI H,YANG XL,LANG ZM,et al.Study and evaluation on the applicability of different microbiological methods formiconazole nitrate[J].Chin J Antibiot,2018,43(1):80
[20]郑小玲,王征南,李珏.瑞他莫林软膏的微生物限度检查适用性试验[J].中国现代应用药学,2016,33(5):634ZHENG XL,WANG ZN,LI J.Applicability test of microbial limit test for retapamulin ointment[J].Chin J Mod Appl Pharm,2016,33(5):634
[21]钟柳芳,肖文可,孙树周,等.双柏膏微生物限度检查方法适用性试验[J].中国现代药物应用,2017,11(14):195ZHONG LF,XIAO WK,SUN SZ,et al.Applicability test of microbial limit test for Shuangbai ointment[J].Chin J Mod Drug Appl,2017,11(14):195
[22]HERMANSSON M,KJELLEBERG S,KORHONEN TK,et al.Hydrophobic and electrostatic characterization of surface structures of bacteria and its relationship to adhesion to an air-water interface[J].Arch Microbiol,1982,131(4):308
[23]ROSENBERY M,KJELLEBERG S.Hydrophobie Interactions:Role in Bacterial Adhension[M]//MARSHALL KC.Advances in Microbial Ecology.New York:Plenum Press,1986:353
[24]唐啸宇,孙洪林,何冰芳.耐有机溶剂微生物及酶类在生物催化中的应用[J].化学进展,2009,21(12):2726TANG XY,SUN HL,HE BF.Organic solvent tolerant bacteria and enzymes for application in biocatalysis[J].Prog Chem,2009,21(12):2726
[25]SCOTT S.Accuracy of plate counts[J].J valid Technol,2011,17(3):42
[26]USP 40-NF 35.VolⅠ[S].2017:General Information(1227)
[27]李辉,杨晓莉,秦亚红,等.改进培养基制备的平皿法应用于芬太尼透皮贴剂微生物限度检查[J].药学研究,2017,36(9):512LI H,YANG XL,QIN YH,et al.Application of the plate count methods of improved medium preparation on fentanyl transdermal patches microbial limit examination[J].J Pharm Res,2017,36(9):512
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