2 nonanone

Aroma compounds were selected based
on their chemical class (aldehydes and ketones), structure (chain
length and carbonyl group position), physicochemical properties (volatility,
hydrophobicity, water solubility), and common use in beverages. Hexanal,
nonanal, and 2-nonanone (Sigma-Aldrich, Zwijndrecht, The Netherlands)
with a purity of ≥95% were chosen, meeting food-grade standards
below their toxicity levels.
Each aroma compound was dissolved
in MiliQ water (pH 7.0) at 10 mg/L in 100 mL amber glasses, following
a modified version of Wang and Arntfield’s protocol.^{30 (link)} The stock solutions were then placed in a bath
at 30 °C for 1 h to ensure optimal mixing.
Table 1 details
the molecular structure and physicochemical properties of the selected
aroma compounds.

Animals starved for 2 hours were tested on food-free NGM plates. Videos of worm behavior were recorded during which a 2 μl drop of 2-nonanone (Sigma, Switzerland) was deposited in one corner of the field of view. Animals that were within 10 mm of the drop but not in direct contact with it were scored for reversal. Results are presented as fractions of animals that were responding with a reversal.

B. subtilis 168- or B. subtilis DG101-fed N2 (10 days-old adults) worms were collected, washed three times with M9 buffer (Cogliati et al., 2020 (link)), and seeded in 10 cm Petri dishes prepared with NGMA without food for 1 h. Then, approximately 75 worms were placed in the center of 6 cm plates prepared with NGMA. After all the animals were transferred to the center of the assay plates, 2 μL of attractant or repellent were seeded 2 cm from the center of the plate, and 2 μL of solvent (control) in which the attractant or repellent was diluted were seeded equidistantly. Both the attractant, repellent, and solvent (control) were added with a 1-μL drop of 1 M azide. The plates were incubated for 1 h at 25°C. Then, worms found at each end of the plates were counted, and the chemotaxis index (CI) was calculated (Bargmann et al., 1993 (link)). The attractant compounds used for the assays were 0.5% diacetyl (DA, Sigma-Aldrich) diluted in ethanol and 1 mM isoamyl alcohol (IAA; Sigma-Aldrich) diluted in water, 1 mM 2,3-pentanedione and 1 mM 2-butanone (Sigma-Aldrich). The repellent compounds used for the assays were 0.3% octanol and 15% 2-nonanone (Sigma-Aldrich). The CI was defined as the number of worms at the attractant or repellent location—the number of worms at the control location divided by the total number of worms on the plate (Bargmann et al., 1993 (link)).

Methanol (≥99.9% purity, HPLC grade) was purchased from Honeywell (Charlotte, NC, USA). α-Pinene, 2-pentyl furan, isoamyl butyrate, 1-hexanol, caryophyllene, methyl salicylate, and caryophyllene oxide were purchased from ChemFaces (Wuhan, Hubei, China). 3-Decanone was obtained from Tokyo Chemical Industry (Tokyo, Japan). α-Guaiene and β-sesquiphellandrene were purchased from Toronto Research Chemicals (North York, NY, USA). β-farnesene and €-2-nonen-1-ol were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-Pinene, 1-undecene, octanal, 2-heptanol, 2-nonanone, 1-octanol, viridiflorol, 2,3-butanediol, 2-methylbutyl 2-methylbutyrate, and a mixture of n-alkanes (C7-C30) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

We sourced 2-nonanone, nonanoic acid and dichloromethane (98–99%) from Sigma-Aldrich, Taufkirchen, Germany. We synthesized ε-nonalactone in the laboratory following the method of Gikonyo et al. [9 (link)] as modified by Wachira et al. [21 (link)]. The ε-nonalactone synthesized consisted of a racemic mixture of equal quantities (50: 50) of (+) ε-nonalactone and (-) ε-nonalactone enantiomers as described by Wachira et al. [21 (link)].
We confirmed the structure of the resultant ε-nonalactone using spectra of the molecule generated from Infra-Red (IR) Spectrometer (Shimadzu, Kyoto, Japan), High Resolution Mass Spectrometer (HR-MS) (Jiangsu Skyray Instrument Co., Ltd., Kunshan, China), Proton (¹H- NMR) and Carbon-13 (¹³C-NMR) Nuclear Magnetic Resonance Spectrometer (Agilent Technologies, Inc., California, USA), as outlined by Skoog et al. [22 ].

The following reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA): C7-C30 saturated alkane mixed standard, (internal standard) 2-methyl-3-heptanone, 2-methylpropyl 2-methylpropanoate, β-myrcene, β-trans-ocimene, ethyl hexanoate, 3-octanone, hexyl acetate, 2-methylbutyl 2-methylbutanoate, (Z)-3-hexenyl acetate, (Z)-hex-3-en-1-ol, 2-nonanone, 3-octanol, ethyl octanoate, (Z)-3-hexenyl butanoate, α-gurgujene, linalool, β-caryophyllene, (Z)-3-hexenyl (E)-2-butenoate, 2-undecanone, methyl benzoate, (Z)-3-hexenyl hexanoate, ethyl benzoate, humulene, α-farnesene, (Z)-3-hexenyl benzoate, and β-ocimene. All chemicals were of analytical grade or higher.