Sweat may be the first thing that comes to mind when we think of foot odor, but the skin’s outer keratinized layer can also supply bacteria with material for odor compounds. A 2013 study produced isovaleric acid, a contributor to foot odor, by combining foot callus, a liquid containing enzymes released by callus-degrading bacteria, and a different bacterium.
The aim was to identify the main components of foot odor, investigate the bacteria and metabolism responsible for producing them, and reproduce the process in vitro. Rather than asking only which bacteria are abundant, the study followed what they use as substrates and what they produce.
Analyzing foot samples with instruments and the human nose
The initial donors were three laboratory staff members, two men and one woman, aged 20–35. Bacteria and odor samples were collected from the forefoot skin and by enclosing the feet for six hours in bags containing a small amount of water. This was not a direct measurement inside everyday shoes, but an experiment using moist conditions to facilitate collection of bacteria and compounds.
After six hours, three assessors described the feet and the air inside the bags as cheesy and acidic. The liquid collected in the bags initially had little odor, but acidifying it made the same characteristics more apparent.
Compounds were extracted from the liquid and separated by gas chromatography, with simultaneous mass spectrometry and human assessment of the odor at a sniff port. Two assessors took turns carrying out the olfactory analysis. Butyric acid, isobutyric acid, and isovaleric acid were identified as foot-odor-like compounds, with isovaleric acid judged particularly similar to foot odor.
Depending on the extraction method, indole, skatole, and unidentified volatile sulfur compounds were also associated with foot odor, though to a lesser extent. The study did not reduce foot odor to isovaleric acid alone, but its importance led the subsequent experiments to focus on production of that acid.
Bacteria that produce isovaleric acid from leucine
Seventy-seven strains were isolated from the three donors’ feet. They included the genera Staphylococcus, Corynebacterium, Micrococcus, Kytococcus, and Brevibacterium. Because these were cultured isolates, they did not represent every bacterium present on the feet.
The researchers cultured each isolate with the amino acid leucine and measured the acids produced. Staphylococci were prominent producers of isovaleric acid. Among 15 strains examined, yields varied widely, from approximately 10% to 80% on a molar basis relative to the leucine supplied.
Strains within the same genus can have different functions. Some species appeared to produce more, but only a few strains of each species were examined, and the authors noted that more work would be needed to rank species overall. The Corynebacterium strains tested did not produce isovaleric acid under these conditions.
The study also examined production of isobutyric acid from valine and 2-methylbutyric acid from isoleucine. Yields were lower than for the leucine-to-isovaleric-acid conversion, supporting the focus on isovaleric acid alongside the initial odor analysis. The individual enzymatic pathways between these starting materials and products were proposed using databases; not every step was measured directly in this study.
Some bacteria consume odor compounds rather than only producing them
Bacterial activity did not run only in the direction of producing odor compounds. When representative strains were supplied with amino acids or the acids formed downstream, some Kytococcus, Micrococcus, and Brevibacterium isolates could also use and deplete isovaleric acid and related compounds.
By contrast, the staphylococci studied in detail produced acids from amino acids but did not show an ability to use those acids further. The authors suggested that the amount of odor compounds remaining on feet depends on the balance between producer and consumer populations and their respective reaction rates.
In other words, the presence of bacteria is not itself equivalent to stronger odor. An important finding was the need to consider the combination of bacteria and the metabolic activities they perform.
Reproducing the path from callus to odor in one vessel
Amino acids such as leucine occur in skin proteins as well as in sweat. The researchers therefore used thickened foot callus as a substrate to reproduce isovaleric-acid formation.
The callus-degrading component was concentrated, cell-free supernatant from a culture of Kytococcus sedentarius. This liquid contained enzymes that break down callus keratin, but was not a completely purified single enzyme. Live cells were not used because this bacterium might also consume the isovaleric acid produced.
One gram of callus, the enzyme-containing supernatant, and the strong isovaleric-acid-producing strain Staphylococcus capitis G390 were combined in one vessel and incubated for 48 hours. Isovaleric acid reached 0.62 and 0.58 mg/mL in two experiments. It was not detected without callus, while supernatant plus callus alone and staphylococci plus callus alone each produced smaller amounts.
The result shows that combining the release of substrates through degradation of callus proteins with conversion of those substrates into odor compounds can generate isovaleric acid from callus. It also provides a model for future studies of substances or reactions that inhibit this production.
Applications to foot care are a next step
The human samples came from just three donors, and the work focused on culturable bacteria. The callus model also used in vitro conditions. The study did not measure the relative contributions of sweat-derived and callus-derived substrates on real feet, or the deodorizing effect of removing callus.
Other questions remain, including how butyric acid and unidentified sulfur compounds associated with foot odor are produced. The authors proposed further investigation of foot-bacterial diversity and deodorizing approaches targeting particular bacteria, odor compounds, metabolic pathways, or enzymes.
Summary
Isovaleric acid was identified as one of the main foot-odor-like compounds in collected foot samples, and experiments with isolates showed that staphylococci readily produced it from leucine. Other bacteria could use and reduce the acids produced, suggesting that both production and consumption influence odor.
Combining callus, a liquid containing callus-degrading enzymes, and staphylococci also reproduced the process of generating isovaleric acid from callus. The study helps explain foot odor through connections between skin substrates and bacterial metabolism, rather than sweat quantity alone.
Research funding and affiliations: Unilever funded the research in full. At the time, two of the three authors were affiliated with Unilever and one with the University of Nottingham. No separate conflict-of-interest declaration was identified.
Reference
James AG, Cox D, Worrall K. Microbiological and biochemical origins of human foot malodour. Flavour and Fragrance Journal. 2013;28:231–237. doi:10.1002/ffj.3136 (published online in 2012)
