J. Soc. Cosmet. Chem., 30, 241-247 (July/August 1979) Instrumental evaluation of odor produced by specific oral microorganisms MARIA C. SOLIS-GAFFAR, THOMAS J. FISCHER and ABDUL GAFFAR Colgate-Palmolive Research Center, Piscataway, NJ 08854. Received November 8, 1978. Presented at 56th General Session, International Assodation for Dental Research, March 1978, IVashington, D.C. Synopsis The purpose of this STUDY was to determine the role of SPECIFIC ORAL MICROORGANISMS in the FORMATION of the odoriferous components associated with human mouth ODOR. Eight gram- positive and four gram-negative microorganisms were individually incubated anaerobically for 3 hr or 24 hr in a sterile saliva system with L-cysteine or L-methionine as the exogenous substrate. After the period of incubation, the headspace was analyzed by a sensitive gas chromatography-flame photometric instrumental technique for the presence of the volatile sulfur compounds (VSC) such as hydrogen sulfide, methyl mercaptan and dimethyl sulfide. The results indicated that only the gram-negative organisms, Veillonella alcalescens, Fusobacterium nucleatum, Bacteroides melaninogenicus and Klebsiella pneumoniae produced VSC from the incubated systems. The gram-positive organisms, S. mutans, S. sanguis, S. salivarius, S. faecalis, Lactobacillus acidophilus, Staphylococcus aureus, Candida albicans, and A. naeslundii failed to produce VSC. An unpleasant odor was detected organoleptically when VSC was produced in the incubated systems. Moreover, VSC production by these gram-negative organisms was accompanied by an increase in pH of the reaction mixture. Except for Klebsiella pneumoniae, the other gram-negative organisms tested needed the presence of blood for the production of VSC. However, the gram-positive organisms even with the addition of blood did not form VSC. VSC chromatograms were similar to the data obtained from the analysis of human mouth air samples. INTRODUCTION Microorganisms have a role in producing intrinsic oral realodor. McNamara et al. (1) found that gram-negative anaerobes and not the gram-positive flora could produce odor in vitro. However, they evaluated the odor organoleptically and did not attempt to investigate the compounds responsible for the malodor. Tonzetich (2) showed that volatile sulfur compounds, namely hydrogen sulfide, methyl mercaptan and dimethyl sulfide are major components in human mouth odor as well as in the headspace of putrefied saliva. He used a sensitive gas chromatography-flame photometric instrumental technique to detect these compounds. We have shown this 241

242 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS technique to be useful in conducting human clinical studies to determine the effect of oral agents on intrinsic mouth odor (3). The purpose of this study was to determine the role of specific oral microorganisms in the formation of the odoriferous components associated with mouth odor. EXPERIMENTAL ORAL MICROORGANISMS Eight gram-positive and four gram-negative microorganisms which represent the predominant flora found in the oral cavity were selected (5). Other gram-negatives such as Eikenella corredens and spirochetes which occur in deep gingival pockets or in a rapidly destructive periodontitis were not used because their sampling and cultivation require anaerobic glove box techniques. Each microorganism was cultivated in a media most favorable to its growth, for a period of 24 hr with the exception of Veillonella and Fusobacterium, which are slow growers and were cultivated for 48 hr. Microorganism Broth • Streptococcus mutans ATCC #27352 $treptcoccu$ sanguis ATCC #10556 $treptococcus salivarius ATCC #25975 Streptococcus fa ecalis ATCC//4083 Actinomyces naeslundii ATCC//19039 Veillonella alcalescens ATCC//17745 Lactobacillus acidophilus ATCC//29601 $taphylococcus aureus ATCC ff4012 Klebsiella pneumoniae ATCC//132 Candida albicans ATCC//26555 Bacteroides melaninogenicus ATCC//15930 Fusobacterium nucleatum ATCC//10953 Trypticase Soy w/0.2% glucose Trypticase Soy w/0.2% glucose Trypticase Soy w/0.2% glucose Trypticase Soy w/0.2% glucose Mycoplasma Broth BBL Veillonella Broth LBS (Lactobacillus broth) Mycoplasma Trypticase Soy Trypticase Soy Brain/heart Infusion Brain/heart Infusion After cultivation, the organism was centrifuged at 5000 rpm for 10 min and washed with sterile buffer solution. The buffer was 0.06M phosphate buffer which was sterilized in an autoclave and had a pH of 7.0. The pellet of the organism was then suspended in 10 ml of the sterile buffer and the suspension adjusted to give an optical density of 0.5 at a wavelength of 525 nm 0.5 ml of the suspension was used as inoculum. •BBL, Div. of Beckman & Dickinson, Co., Cockeysville, Md.