Profiling Volatile Compounds from Culture Supernatants of Periodontal Bacteria Using “MonoTrap” and GC/MS/

Significance of the Topic

Halitosis, commonly known as bad breath, is primarily driven by volatile compounds produced by anaerobic periodontal bacteria in the oral cavity. Understanding the full range of these volatile substances is essential for developing noninvasive diagnostic methods and identifying novel biomarkers for oral health assessment.

Study Objectives and Overview

This study aimed to perform a comprehensive analysis of volatile compounds generated by Fusobacterium nucleatum and Porphyromonas gingivalis. By employing a silica monolithic adsorbent (MonoTrap RGPS TD) coupled with gas chromatography-mass spectrometry-olfactometry (GC/MS/O), the researchers sought to identify and characterize odor-active molecules that contribute to halitosis.

Methodology and Instrumentation

The bacterial strains F. nucleatum ATCC 25586 and P. gingivalis ATCC 33277 were cultivated under strict anaerobic conditions at 37 °C. Following plate and liquid pre-cultivation phases, culture supernatants were diluted to OD600 0.1 and incubated for 24 hours. Volatile compounds in the headspace were adsorbed onto MonoTrap at 40 °C for two hours, thermally desorbed, and introduced into the GC/MS/O system. Data processing involved peak detection and alignment with MS-DIAL, compound annotation via GC-Analyzer using the NIST20 library, and olfactory evaluation with a human sniffing port.

Instrumentation Used

• Gas chromatograph-mass spectrometer GCMS-TQ8050 NX
• Multimode inlet OPTIC-4 with cryotrap
• Autosampler AOC-6000 Plus with CDC station
• Olfactometry port OP275 Pro II
• MonoTrap RGPS TD silica monolithic adsorbent
• Column InertCap FFAP (60 m × 0.32 mm, df 0.50 µm)
• Software: GCMSsolution Ver. 4.52, MS-DIAL Ver. 4.70, Evolution Workstation Ver. 4.6.3, Olfactory Voicegram V2.3.0

Main Results and Discussion

Comparative analysis against blank media identified 110 volatiles from F. nucleatum and 100 from P. gingivalis, including sulfur compounds, short-chain fatty acids, ketones, alcohols, pyrazines and indole derivatives. Key findings included:

• Detection of 17 malodorous compounds from F. nucleatum, 9 of which were volatile sulfur compounds (VSCs).
• Detection of 20 malodorous compounds from P. gingivalis, including 6 VSCs.
• Significant increases in indole and butyric acid, both known contributors to halitosis.
• Identification of S-methyl thiobutyrate as a novel bacterial volatile potentially linked to bad breath.
• Observed biological variability in methanethiol production (RSD ≥ 70 %), indicating caution for quantitative biomarker use.

Benefits and Practical Applications

This profiling approach allows sensitive detection and odor correlation of trace volatile compounds. The data support the development of targeted, noninvasive breath tests for halitosis diagnosis and may guide the discovery of new biomarkers for periodontal disease monitoring.

Future Trends and Potential Applications

• Extension of volatile profiling to clinical breath samples and direct oral cavity analyses.
• Integration with metagenomic and microbiome data to link specific bacteria to odor signatures.
• Advances in portable or real-time GC/MS technologies for point-of-care diagnostics.
• Application of machine learning to classify halitosis severity based on volatile fingerprints.

Conclusion

The combined use of MonoTrap sampling and GC/MS/O provided a comprehensive volatile profile of key periodontal pathogens, revealing both established and novel malodorous compounds. This method holds promise for advancing noninvasive halitosis diagnostics and expanding our understanding of oral microbial metabolism.

References

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3. Monedeiro F, Milanowski M, Ratiu IA, Zmysłowski H, Ligor T, Buszewski B. VOC profiles of saliva in assessment of halitosis and submandibular abscesses using HS-SPME-GC/MS technique. Molecules. 2019;24:2977.