246 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS growth of gram-negatives. With this shift came a corresponding increase in the pH. Our data tends to support their findings that an alkaline pH favors malodor production. Table III shows the change in pH of the incubated system containing the gram-negative organisms before and after 3 hr or 24 hr incubation with L-cysteine as exogenous substrate. The gram-positives, on the other hand, did not cause any appreciable changes in the pH of the incubating medium. Freshly collected whole human saliva has a slightly acid pH and a rather pleasant smell. However, when it is incubated, the saliva becomes more alkaline and takes on a distinctive putrid odor after 8 hr. Instrumental analyses of the headspace of stagnant putrefied saliva show the presence of the VSC similar to those detected in the direct analyses of the breath samples from the mouth (Figure 2). I 2 STERILE SALIVA + GRAM-NEGATIVE ORGANISM + L-CYSTEINE I 2 EARL' MORNING MOUTH AIR SAMPLE WHOLE HUMAN SALIVA + L - CYSTEINE I. H2S 2. CH3SH 3, (CH3} 2 S COLUMN: FEP TEFLON COLUMN PACKED WITH 5% POLYPHENYL ETHER AND 0.05% H3PO 4 ON 30/60 MESH CHROMSORB T ATTENUATION: 1.28 x I0 -IøAFS TEMPERATURE: 60øC CARRIER GAS: AIR Figure 2. GC-FPD chromatogram of headspace of incubated system with gram-negative microorganism compared to chromatograms of mouth air sample and headspace of incubated whole human saliva. Moreover, Schmidt and co-workers (6, 7) showed that volatile sulfur compounds detected instrumentally by GC and a flame photometric detection system from mouth air correlates with the organoleptic ratings of the same subjects. Our results in Table I indicate that the incubated system of sterile saliva and gram-negative microorganism had a malodor and VSC was detected from its headspace. The incubated systems with the gram-positives failed to develop any odor and no VSC was detected instrumentally from analyses of the headspace of these systems. Possibly the volatile sulfur compounds arise from the action of specific gram-negative microorganisms such as Bacteroides melaninogenicus, Fusobacterium nucleatum, Veillonella alcalescens and Kleb- siella pneumoniae, on exogenous and endogenous proteinaceous substrates such as epithelial cells, food debris, saliva and blood. The proteins then undergo proteolysis

INSTRUMENTAL EVALUATION OF ODOR 247 into peptides and constituent amino acids which are further broken down to the volatile and odoriferous sulfur volatiles (8). A comparison of the instrumental evaluation of the headspace of incubated putrefied saliva with L-cysteine after 3 hr of incubation, early morning mouth air samples and the headspace of incubated sterile saliva system containing a gram-negative, Bacte- roides melaninogenicus are shown in Figure 2. It is obvious from the chromatograms and the corresponding retention times, that volatile sulfur compounds, hydrogen sulfide, methyl mercaptan and dimethyl sulfide are present in all these samples and are responsible for the offensive malodor. CONCLUSIONS The results of this study showed that: (1) Oral microorganisms play a role in the production of mouth realodors. (2) Gram-negative anaerobes, but not the gram-positive microorganisms, cause the formation of unpleasant odor in a sterile saliva system with L-cysteine as exogenous substrate in the presence of human blood components. (3) The malodor can be detected organoleptically and can be shown instrumentally to arise from volatile sulfur compounds associated with human mouth odor. (4) The chromatograms of the headspace of sterile saliva incubated with the gram- negative microorganisms are similar to those obtained for human mouth air samples as well as the headspace of stagnant saliva. REFERENCES (1) T. McNamara, J. Alexander and M. Lee, The role of microorganisms in the production of oral malodor, Oral Surg., 34, 41 (1972). (2) J. Tonzetich, Direct gas chromatographic analysis of sulphur compounds in mouth air in man, Arch Oral Biol., 16, 587 (1971). (3) M. C. Solis-Gaffar, H. P. Niles W. C. Rainieri and R. C. Kestenbaum, Instrumental evaluation of mouth odor in a human clinical study,J. Dent. Res., 54, 351 (1975). (4) M. Berg and L. S. Fosdick, Studies in periodontal disease, II. Putrefactive organisms in the mouth. J. Dent. Res., 25, 73 (1946). (5) S. Socransky and S. Manganiello, The oral microbiota of man from birth to senility,J. Periodont., 42, 485 (1971). (6) N. F. Schmidt, S. R. Missan, W.J. Tarbet and A. Cooper, The correlation between organoleptic mouth odor ratings and levels of volatile sulfur compounds, Oral $urg., 45,560 (1978). (7) N. F. Schmidt and W.J. Tarbet, the effect of oral rinses on organoleptic mouth odor ratings and levels of volatile sulfur compounds, Oral $urg., 45,876 (1978). (8) J. Tonzetich, Production and origin of oral malodor. A review of mechanisms and methods of analysis, J. Periodont., 48, 13 (1977).