Over billions of years, organisms have evolved chemical defenses to combat pathogenic microbial growth. With a parallel goal in mind, home and personal care products are formulated with chemical preservatives that prevent contamination by microbes.^1,2 Unfortunately, many conventional, broad-spectrum preservatives have been associated with adverse human and environmental health outcomes and negative consumer perception.² These concerns have led to interest in developing a broader collection of safe and effective chemical preservatives that are inspired by, or derived from, natural sources.


Despite this interest, safer natural preservatives are often less effective than conventional ones. While preservation can be easily achieved at acidic (e.g., pH < 4) and basic (e.g., pH > 10) ranges, many preservatives do not function optimally at neutral pH ranges, the latter of which are more likely to allow the growth of pathogenic bacteria. Preservatives may also be deactivated by other components (e.g. emulsifiers) in the formula.³ Due to these complex factors, the financial and technical barriers to testing prospective preservatives present a significant hurdle to development and adoption of sustainable preservatives in home and personal care products. This challenge is especially significant for screening potential alternatives.


To reduce these hurdles, we developed a rapid and inexpensive protocol to evaluate the efficacy of promising chemical preservatives (Fig. 1). Protocol reliability and reproducibility were independently confirmed by third party testing, completed by Antimicrobial Test Laboratories (Table 1). Notable attributes of this approach are low and fixed costs, minimal equipment requirements, and short testing durations, which enable rapid screening of potentially large chemical libraries. While this protocol is not a replacement for full preservative challenge testing, it may lower costs of preservative testing by reducing the number of considered preservatives, thereby facilitating judicious use of third-party testing expertise.


The equipment used to conduct these tests included an incubator, plastic Falcon culture tubes, disposable inoculation loops, Mueller-Hinton broth and agar, Petri dishes, disposable hemocytomers, Bunsen burner, autoclave or pressure cooker, refrigerator, and microscope. The total cost of these tools is affordable ($1,000-5,000, depending on sourcing) making this screening accessible even for small research organizations. However, it should be noted that work with potentially pathogenic species (e.g. the biosafety level two (BSL 2) organism Pseudomonas aeruginosa), warrants additional safety measures and expenses.


Triaging can be accomplished by initially testing a hardy problem organism at a challenging pH range, which enables only the most promising preservatives to be identified early in the testing. For this reason, initial tests evaluated the antimicrobial susceptibility of the mold Aspergillus brasiliensis (ATCC 16404), which grows readily at neutral pH. We observed that of the 109 test samples examined in our screen, about half (59) effectively inhibited A. brasiliensis at approximately 1% by mass. Of these 59, slightly more than half (33) also inhibited the growth of the Gram-negative bacterium P. aeruginosa (ATCC 9027). However, it should be noted that the susceptibilities of different organisms to chemical preservatives often are not correlated. Rather, we would argue that the selection of compounds for screening is a more important factor.


This process is not a substitute for full preservative challenge testing, but rather a complement whereby chemical libraries can be narrowed to a few promising candidates. Adoption of this simplified procedure facilitates access to in-house preservative testing, which could be used to identify promising alternatives, evaluate synergistic interactions between multiple ingredients, and derive estimates of minimum inhibitory concentrations for new preservatives. Such information can assist formulators in optimizing the concentration required for effective product preservation prior to confirmation by an external testing agency and is amenable to broad variations in testing protocol.^2,4 We envision that the adoption of simplified and inexpensive preservative testing protocols such as the one described herein will enable efficient use of third party testing expertise and represents a first step toward accelerated development of preservatives with improved human and environmental health properties.


References

1. Reisch, A. S. (2014). Close Scrutiny of Cosmetic Preservatives Continues. C&E News, 92, 22. October 9, 2015, http://cen.acs.org/articles/92/i23/Close-Scrutiny-Cosmetic-Preservatives-Continues.html
2. Anath, V., Rook, T., Shaw, D.A., Ganguly-Mink, S., Vitolo, P., Brutofsky, M., Detwiler, C. (2015). Preservative Efficacy Testing. Household and Personal Products Industry. Retrieved October 9, 2015, http://nitesh_happi.rodpub.com/issues/2015-04-01/view_features/preservative-efficacy-testing/
3. Browne, B. A., Geis, P., Rook, T. (2012). Conventional vs. natural preservatives. Household and Personal Products Industry. Retrieved October 9, 2015, http://nitesh_happi.rodpub.com/issues/2012-05/view_features/conventional-vs-natural-preservatives/
4. Shaw, D. A., Browne, B. A., Rook, T. Geis, P., Anath, V. (2014) Critical Elements of Household Product Preservation. Household and Personal Products Industry. Retrieved October 9, 2015, http://nitesh_happi.rodpub.com/issues/2014-05-01/view_features/critical-elements-of-household-product-preservation/