J. Cosmet. Sci., 69, 35–46 ( January/February 2018) 35 Use of L-Glutamic Acid in a New Enrichment Broth (R-TATP Broth) for Detecting the Presence or Absence of Molds in Raw Ingredients/Personal Care Product Formulations by Using an ATP Bioluminescence Assay YOUJUN YANG and DONALD J. ENGLISH, Avon Products Inc., Suffern, NY 10901. Accepted for publication December 4, 2017. Synopsis The present study reports the effects of adding L-glutamic acid to a new enrichment broth designated as R-TATP broth, to promote the growth of slow-growing mold microorganisms such as Aspergillus brasiliensis and Aspergillus oryzae, without interfering in the growth of other types of microorganisms. This L-glutamic acid containing enrichment broth would be particularly valuable in a rapid microbial detection assay such as an adenosine triphosphate (ATP) bioluminescence assay. By using this new enrichment broth, the amount of ATP (represented as relative light unit ratio after normalized with the negative test control) from mold growth was signifi cantly increased by reducing the time of detection of microbial contamination in a raw ingredient or personal care product formulation from an incubation period of 48–18 h. By using L-glutamic acid in this enrichment broth, the lag phase of the mold growth cycle was shortened. In response to various concentrations of L-glutamic acid in R-TATP broth, there was an increased amount of ATP that had been produced by mold metabolism in an ATP bioluminescence assay. By using L-glutamic acid in R-TATP broth in an ATP bioluminescence assay, the presence of mold could be detected in 18 h as well as other types of microorganisms that may or may not be present in a test sample. By detecting the presence or absence of microbial contamination in 18 h, it is superior in comparison to a 48–96 h incubation period by using either a standard or rapid detection method. INTRODUCTION All living microorganism contain and use adenosine triphosphate (ATP) as a vital part of their energy and metabolic system. Energy is stored within the phosphate bonds of the ATP molecule. By using a luminometer in an ATP bioluminescence assay, the presence of microbial ATP assay is used to detect light energy when ATP is converted to adenosine monophosphate (AMP) by an enzymatic reaction (1,2). This converted energy is detected as light energy and the amount of light is measured by a luminometer that is reported as a value of relative light unit (RLU). A higher RLU value corresponds to a higher level of ATP that is present in a test sample. A positive detection for a microbial contamination Address all correspondence to Donald J. English at don.english@avon.com.

JOURNAL OF COSMETIC SCIENCE 36 in a test sample is represented by a value of an RLU ratio that is greater than 2 in com- parison with an RLU for negative control without ATP. The calculated RLU ratio is based on the RLU of an incubated test sample versus the RLU of a non-incubated test sample (negative control). By using the ATP bioluminescence test kits from Charles River Labo- ratories, Inc. (Charleston, SC), an ATP bioluminescence assay is able to detect the presence or absence of microorganisms in either a nonsterile raw ingredient or a personal care fi nished product formulation that is susceptible to microbial contamination by using an incubation period of 24 h for most applications (1,3,4), 30 h for bacteria and fungi screening (5), or 48 h (6) However, the application of this technology may be limited for most test samples if they are contaminated with a slow-growing microorganism such as mold. The reason for this limitation in detection is that only low levels of ATP are released by growing mold after 24–30 h of incubation which may lead to a false-negative test result. This limitation becomes more signifi cant if a higher nonmicrobial ATP level is also detected to be present in a test sample from a nonmicrobial source. Thus, the increased RLU background signal of the test sample will minimize the RLU ratio, especially when the RLU signal of the microbial ATP level is low from the growth of mold unlike that from bacteria. Based on information from Machlis, it led us to study if the detection of mold microor- ganisms can be enhanced by adding L-glutamic acid to R-TAT broth (7). The effi cacy of L-glutamic acid in shortening the lag phase of the fungal growth cycle was also studied and discussed by Griffi n (8). The application of this information in a rapid microbial detection system has yet been studied or published. In the light of the observed limita- tion in using an ATP bioluminescence assay for detecting the presence or absence of mi- crobial contamination in a test sample, our goal is to increase the detectable level of ATP that is produced by a slow-growing microorganisms, such as mold, by creating a new enrichment broth that can promote the growth of these microorganisms. This new en- richment broth should not affect the growth of bacteria and/or yeasts that may also be part of the microbial bioburden of a test sample. In addition, the new enrichment broth should have either no or very low levels of nonmicrobial ATP present. Before an ATP bioluminescence assay is routinely implemented to screen for the presence or absence of microbial contamination in either a nonsterile raw ingredient or product formulation, the test method needs to be validated by inoculating 1% test suspensions in enrichment broth to demonstrate recovery. This validation testing involves the use of indicator test microorganisms. In our case, we used Staphylococcus aureus ATCC 6538 for demonstrating the recovery of Gram-positive cocci, Bacillus subtilis ATCC 6633 for dem- onstrating the recovery of spore-forming Gram-positive bacilli, Escherichia coli ATCC 8739 for demonstrating the recovery of Gram-negative bacilli, Candida albicans ATCC 10231 for demonstrating the recovery of yeasts, and Aspergillus brasiliensis ATCC 16404 for demonstrating the recovery of mold in a 1% test sample suspension of an ATP biolu- minescence assay by using R-TATP broth as the enrichment broth. MATERIALS AND METHODS TEST MICROORGANISMS The following test microorganisms were used in this study: EZ-colony-forming unit (CFU) cultures of S. aureus ATCC 6538 (Catalog number 0485C), E. coli ATCC 8739