ATP生物荧光法快速检测化妆品中菌落总数的研究

本文从快速检测的必要性出发,介绍了ATP生物荧光法及其原理,并针对某款化妆品通过一系列实验建立了一种快速检测菌落总数的方法。将该方法用于CTFA(美国化妆品香料香精协会,Cosmetic Toiletry and Fragrance Association)实验中,并通过与平板计数法结果进行比对,发现该方法可以有效的运用于样品细菌总数的快速检测。     

Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems

AIM: To evaluate the microbial contamination of 91 cosmetics (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use (intact, in-use, ending product) and the protection efficacy of the preservative systems most frequently used in the analysed cosmetic formulations. METHODS AND RESULTS: Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10.6% of tensiolytes (13.5% bath foam, 6.7% shampoo, 10% liquid soaps) were contaminated by Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The efficacy of the preservative systems of two cosmetic products, tested against standard micro-organisms (Staphylococcus aureus ATCC 4338 and Pseudomonas aeruginosa ATCC 9027) and two isolates from cosmetics in this study (S. epidermidis and P. putida), satisfied the Cosmetics, Toiletries, and Fragrance Association and Official Italian Pharmacopeia criteria, while only one tested cosmetic respected the Rapid Challenge Test criterion. CONCLUSIONS: Contaminated cosmetic products are relatively uncommon, but some products, unable to suppress the growth of several micro-organisms, represent a potential health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: The challenge test may be performed not only during the preparation of the preservative system in the intact cosmetics, but also be used to evaluate the protection efficacy during their use.     

<Emphasis Type="Italic">Rubus rosaefolius</Emphasis> Extract as a Natural Preservative Candidate in Topical Formulations

Even though the synthetic preservatives may offer a high antimicrobial efficacy, they are commonly related to adverse reactions and regarded as having potentially harmful effects caused by chronic consumption. The development of natural preservatives provides a way of reducing the amount of synthetic preservatives normally used in pharmaceutical and cosmetic preparations. In addition, these agents have less toxic effects and represent a possible natural and safer alternative of the preservatives. The purpose of this research was to evaluate the Rubus rosaefolius Smith extract efficiency as a natural preservative in base formulations. Of the extract, 0.2% (w/w) was assayed for its effectiveness of antimicrobial protection in two different base formulations (emulsion and gel). The microbial challenge test was performed following the standard procedures proposed by The United States Pharmacopoeia 33nd, European Pharmacopoeia 6th, Japanese Pharmacopoeia 15th, and the Cosmetics, Toiletries, and Fragrance Association using standardized microorganisms. The results demonstrated that R. rosaefolius extract at the studied concentration reduced the bacterial inocula, satisfying the criterion in all formulations, even though it was not able to present an effective preservative behavior against fungi. Thus, the investigation of new natural substances with preservative properties that could be applied in pharmaceutical and cosmetic products is relevant due to the possibility of substituting or decreasing the concentration of synthetic preservatives, providing a way for the development of safer formulas for the use of consumers.     

Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm? for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm? and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.     

Production of environmentally safe biocides from essential oils having antimicrobial activity

Different liquid formulations of anise, coriander and black cumin essential oils were used for preparing some biocides. The prepared formulations were tested for their antimicrobial activity against some post-harvest pathogenic microorganisms. The tested microorganisms were Fusarium oxysporum (Dray rot of potato), Alternaria alternata (Black rot of tomato), Penicillium italicum, P. digitatum, (Blue and green rot of orange, respectively), Botryitus cinerea (Gray rot of strawberry) and Erwinia carotovora (Soft rot of potato).The results revealed that the different formulations showed a complete inhibition effect on the growth of most of the tested microorganisms. Also, the antimicrobial activity of the formulated essential oils did not affect the formulation process compared with the original oil.     

A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Solid Lipid Nanoparticles as Effective Reservoir Systems for Long-Term Preservation of Multidose Formulations

Cosmetic multidose preparations, as well as pharmaceutical ones, are at risk of contamination by microorganisms, due to their high water content. Besides the risk of contamination during manufacturing, multidose cosmetic preparations may be contaminated by consumers during their use. In this paper, the results of the utilization of nanoparticles as reservoir systems of parabens, the most used class of preservatives, were reported. Two different systems, solid lipid nanoparticles (SLN) made of pure precirol and nanostructured lipid carriers (NLC) made of precirol and almond oil, containing three parabens as single molecules or as a mixture, were prepared and tested. All the systems were characterized for size, polydispersion index, zeta potential and encapsulation efficiency. Release experiments, carried out in steady state and sink conditions, allowed to evidence that both SLN and NLC were able to act as reservoir systems. The antimicrobial activity of the systems was tested against Candida albicans ATCC 10231 with repeat insult tests. The results of the release experiments and the antimicrobial tests showed very low water concentration of parabens still maintaining their antimicrobial activity.     

以甘草为原料的食品天然防腐剂的研制

采用不同的溶剂和提取条件,提取甘草中的抗菌有效成分,然后通过抑菌试验筛选出抗菌力最强的制剂,通过测定该制剂对常见食品污染菌的最小抑菌浓度(MIC),证实该制剂对多种常见的食品污染菌均有较强的抑制作用,保存试验证实本制剂能有效地延长食品的保质期。     

Antimicrobial compounds from Coleonema album (Rutaceae)

Coleonema album, a member of the South African fynbos biome, was evaluated for its antimicrobial activity associated with its secondary metabolites. Ethanol- and acetone-based extracts obtained from plants from two different geographical areas were analyzed. A bioassay-guided fractionation methodology was followed for rapid and effective screening for the presence of bioactive compounds. The TLC-bioautographic method, used to screen the plant extracts for antimicrobial activity and localization of the active compounds, indicated the presence of a number of inhibitory compounds with activity against the microorganisms (E. coli, B. subtilis, E. faecalis, P. aeruginosa, S. aureus, M. smegmatis, M. tuberculosis, C. albicans, C. cucumerinum) tested. Evaluation of the inhibitory strength of each extract by the serial microdilution assay indicated that the C. album extracts inhibited effectively all the microorganisms, with the minimum inhibitory concentrations in the low mg ml(-1) range. Identification and structural information of the bioactive components were obtained by a combination of preparative TLC and LC-MS. It revealed the presence of coumarin aglycones which were responsible for the observed antimicrobial activities. The results of this study indicate that C. album possesses strong antimicrobial activity against a wide range of microorganisms that warrants further investigation into the use of the extracts or their active constituents as a potential source for novel drugs.     

贝莱斯芽孢杆菌B6强化驱油机制分析及在高矿化度油藏中的应用

【目的】筛选油藏内源功能微生物并探究其驱油机理,现场试验以确定油藏内源微生物提高原油采收技术应用的工艺和技术可行性。【方法】采集英东油田样品,利用原油平板筛选驱油功能菌,评价其环境适应性并优化培养条件,通过乳化性能、降黏防蜡和烃转化能力等评估其驱油性能并探索其潜在机制,并在油田现场进行微生物强化驱油试验。【结果】从油水样品中分离到的贝莱斯芽孢杆菌(Bacillus velezensis) B6,其乳化活性指数(emulsifying activity index, EI24)值为100.00%,降黏率为97.20%,防蜡率为86.90%,表明菌株B6具有良好的乳化降黏性能,具有提高原油采收的潜力,并且能够减少原油中的重质组分而增加轻质烃类,改善原油物性,提升原油品质。进一步在英东油田、跃进油田和花土沟油田进行微生物单井吞吐和清防蜡作业,共进行62井次现场试验,累计增油1 460.36 t,平均延长洗井周期47 d,经济效益为342.50万元,投入产出比为1:4。【结论】通过室内研究和现场试验,证实内源微生物Bacillus velezensisB6具有显著提高原油采收和油井清防蜡的效果,应用潜力巨大。     

The testing of disinfectants

Although all disinfectant tests have the same final purpose, namely measuring the antimicrobial activity of a chemical substance or preparation, a large number of testing methods has been described. They are subdivided into suspension tests, carrier and surface disinfection tests and other practice-mimicking tests. The suspension tests comprise qualitative and quantitative suspension tests, and, as derived tests, the determination of the phenol coefficient and capacity tests. There is an essential difference between a carrier test and a surface disinfectant test: in the former case the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in the latter case the disinfectant is applied on the carrier for the application time and thereafter the carrier is drying during the exposure.The basic principle now widely accepted is that the antimicrobial efficiency of a disinfectant is examined at three stages of testing.The first stage concerns laboratory tests in which it is verified whether a chemical compound or a preparation possesses antimicrobial activity. For these preliminary screening tests, suspension tests are considered.In the second stage of tests, disinfection procedures and not disinfectants are examined. It is determined under which conditions and at which use-dilution for a given application the preparation is active: the tests simulate real-life situations; such tests are carrier tests for the disinfection of materials by submersion and surface disinfection tests.The last stage takes place in the field, and comprises the in-loco or in-situ tests with as variants the in-use tests, which examine whether, after a normal period of use, germs are still killed by the disinfectant solution.It is the task of the European and international standardization organisations to develop new standards and to elaborate tests, which predict the effectiveness of a preparation in practice under variable circumstances.     

Hydrophilic Ionic Liquids as Ingredients of Gel-Based Dermal Formulations

Ionic liquids (ILs) have several properties that offer many advantages in dermal drug delivery systems. Depending on the chemical structure, ILs can be used for protection against microorganisms, to enhance skin penetration, and as a solvent. In the present work, SEPINEO? P 600 formulations and hydroxyethylcellulose gels containing the hydrophilic ILs hexylpyridinium chloride, choline dihydrogen phosphate, and 1-ethyl-3-methylimidazolium ethyl sulfate were prepared, and the influence of the ILs on the formulation properties was evaluated. ILs were successfully incorporated into the emulsion structure, resulting in stable formulations. The antimicrobial activity of the ILs was estimated. The minimal inhibitory concentration values for hexylpyridinium chloride are about 2.5 mg/mL. The other two ILs have no antimicrobial activity. Skin penetration enhancement of caffeine, a hydrophilic model substance, was observed in the presence of hexylpyridinium chloride.     

Determination of antimicrobial and antiviral properties of IR3535

Insect repellent is a substance directly applied to skin or clothing in order to repel flies, mosquitoes, ticks etc. IR3535 or Ethyl butylacetylaminopropionate (EBAAP) is a relatively new repellent which is classified as a biopesticide due to exceptional skin tolerance and overall safety. The repellency against various insect and ticks, and the low toxicity of IR3535 are well acknowledged. However, there has been no attempt to investigate the effects on microorganisms or viruses up to now. In the present study, antimicrobial activity was investigated based on disc diffusion and micro-well dilution assays. Disc diffusion assays revealed IR3535 displayed remarkable antimicrobial activity on the microorganisms tested. MIC results showed that the antifungal efficiency of IR3535 is higher with respect to its antibacterial and anticandidal efficiency. Moreover, antiviral test results revealed that IR3535 showed antiviral effects against Poliovirus and Adenovirus. This is the first study that reveals IR3535’s antimicrobial and antiviral properties against a broad range of microorganisms and viruses. In consideration of the antimicrobial and antiviral properties, IR3535 is a promising agent that could be used to develop novel therapeutic approaches, new application areas and formulations in the future.

     

The challenge of testing chemicals for potential carcinogenicity using multiple short-term assays: an analysis of a proposed test battery for hair dyes

Recent reports of the association of hair dyes usage with increased bladder cancer risk in women with the slow NAT-2 acetylator phenotype have resulted both in attempts to identify the putative carcinogen as well as in devising batteries of tests that could be used to screen for such putative carcinogens in hair dye formulations, their intermediates and final products. Analytical studies have reported the presence of traces ( approximately 0.5 ppm) of the carcinogen 4-aminobiphenyl in some hair dye preparations. In parallel, SCCNFP (Scientific Committee on Cosmetic and Non-Food Products Intended for Consumers) has suggested the deployment of a battery of six in vitro assays followed by an in vivo assay. The practicality of deploying and interpreting such a battery is analyzed herein as it is expected to result in 64 and 128 possible test results and SCCNFP does not provide detailed guidance of how the test results are to be interpreted. In this study we have applied a previously described Bayesian approach which takes advantage of the known predictive performances of individual assays, to analyze the possible outcomes of the 6-7 test batteries. While the SCCNFP battery is clearly risk-averse, it is shown that performing all of the assays is not always necessary and moreover it does not necessarily improve predictive performance. Finally, based upon the reported mutagenicity of 4-aminobiphenyl, it is doubtful that this "impurity" would be detected by the test battery.     

鲑鱼鱼精蛋白对食品防腐特性的研究

测定了鲑鱼鱼精蛋白食品常见污染菌的抗菌范围和抗菌力,探讨ＰＨ值、温度、食品中的我机成分、有机成分对鱼精蛋白菌活性的影响,阐明了鱼精帽白的抗菌特点和作为食品天然防腐剂的开发价值。     

A Practical Approach to Evaluation of the Germicidal Efficiency of a General Purpose Military Disinfectant

The bactericidal activity of a general purpose disinfectant consisting of 25% sodium-o-phenylphenolate and 75% sodium-4- and 6-chloro-2-phenylphenolate was evaluated by a simulated in-use, surface-square dilution method. Common floor (asphalt, rubber, and unglazed tiles) and wall (stainless steel tile, ceramic tile, and painted wood) surfaces of various porosities and compositions were selected to simulate actual-use conditions.

The method used consisted of inoculating the surfaces of 1-in. square sections of floor and wall covering with a test organism, air-drying the inoculated surface, applying the disinfectant, allowing it to act for 10 min, and recovering the survivors by plating. Confirmatory results of the standard phenol coefficient and use-dilution tests indicated 700 ppm of the disinfectant to be a safe use concentration. The in-use surface-square dilution studies have shown that this is a more than adequate safe concentration for stainless steel, both glazed and unglazed ceramic tile, and nonwaxed surface of asphalt tile. However, concentrations ranging between 2,500 and 6,000 ppm for plastic-fortified rubber tile, 1,500 and 2,000 ppm for waxed asphalt tile, and 2,000 ppm for painted wood were required to achieve 99.9% reduction of either Salmonella choleraesuis or Salmonella schottmuelleri. These results indicate that a disinfectant concentration derived from the Association of Official Agricultural Chemists use-dilution test cannot always be relied upon to provide a dependable index to actual safe use-dilution when a disinfectant is supplied to certain wall or floor surfaces.

     

A Comparison of the British Pharmacopoeia (1973) and United States Pharmacopeia (1975) Methods for Detecting Pseudomonads

The British Pharmacopoeia (Anon. 1973) test for contamination by pseudomonads and the United States Pharmacopeia (Anon. 1975) test for the detection of Pseudomonas aeruginosa have been evaluated using type cultures representative of a wide spectrum of Pseudomonas spp. Media containing cetrimide are recommended by both Pharmacopoeias for the detection of pseudomonads. Of the 28 strains tested only five were detected by the BP method and 11 by the USP method. However, only one strain of P. aeruginosa failed to be detected using the USP method. A correlation between m.i.c. values for cetrimide and detection of the organisms was noted. The BP method failed to detect organisms with m.i.c. values of ± 0.03% whilst the USP method did not detect strains with m.i.c. values ± 0.015%.     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。     

一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性

【目的】为发掘和利用蜂粮中拮抗菌资源,对分离获得的拮抗细菌菌株PC2进行分类鉴定,并测定其发酵液抑菌物质基本特性。【方法】采用改良牛津杯双层平板法测定菌株发酵液抑菌谱及温度、p H、紫外线和蛋白酶对其抑菌活性稳定性的影响,菌株鉴定结合形态学、生理生化特征和16S r RNA基因序列分析,硫酸铵沉淀法和盐酸沉淀有机溶剂提取法进行抑菌活性物质的初步分离。【结果】从3种蜂粮中分离筛选得到17株拮抗菌株,其中1株细菌PC2以马铃薯葡萄糖液体培养基发酵制备的无菌发酵液对7种供试菌株具有较强抑制作用,经形态、生理生化特征及16S r RNA基因序列分析,将其初步鉴定为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。菌株发酵液抑菌活性对温度、酸和紫外线具有较强的稳定性,对蛋白酶K、胃蛋白酶、碱性蛋白酶处理敏感。菌株发酵液存在抑菌蛋白和脂肽类物质。【结论】菌株PC2在食品保鲜和农业生防中具有潜在的开发应用价值。     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.