2 3 pentanedione

The chemicals for the synthesis of the complexes were purchased from Sigma-Aldrich (Darmstadt, Germany) (copper(II) perchlorate hexahydrate (≥99.99% trace metals basis), 2,2′-biyridine (bpy, 99%), 1,10-phenantroline (phen, 99%), 2,3-pentanedione (97%) and 3-amino-4H-1,2,4-triazole (96%)) and Merck (Darmstadt, Germany; dibenzo-18-crown-6-ether, potassium superoxide) at reagent grade and were used as received, without further purification. The 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) was synthesized by [1+1} condensation of 3-amino-4H-1,2,4-triazole and 2,3-pentanedione.

The protocol for olfactory assays was adopted from Bargmann et al. (1993) (link). The volatile odorants were diluted in ethanol, which was used as the neutral control. Chemotaxis assays were conducted with indicated concentrations of butanone, benzaldehyde, IAA, 2,3-pentanedione, TMT, diacetyl, pyrazine or 1-octanol (Sigma-Aldrich). One microliter of 1 m sodium azide (Sigma-Aldrich) was dropped in advance to both odorant and control spots to paralyze animals reaching them. Assay plates with 150–200 animals were incubated for 120 min at 20°C, after which plates were moved to 4°C to stop animal movements. Chemotaxis indices (CI = O − C/O + C) were calculated as the number of animals that moved toward the attractive odorant (O) minus the number of animals toward the control (C), divided by the total number of animals. Avoidance indices were calculated similarly, but taken into account the number of animals that moved away from the repellent.

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)).

Standards of most volatile compounds were obtained from Sigma-Aldrich (Shanghai, China): 1-octen-3-ol (98%), (E)-2-octen-1-ol (97%), 1-heptanol (98%), propanal (97%), pentanal (98%), hexanal (98%), heptanal (97%), octanal (99%), (E)-2-octenal (97%), nonanal (99.5%), (E)-2-nonenal (97%), benzaldehyde (99.5%), 2-pentylfuran (98%), and 2,3-pentanedione (97%). The 3-methylbutanal (98%) was supplied by Aladdin (Shanghai, China). The n-alkanes (C₇-C₄₀, ≥97%, external standard) was obtained from o2si Smart Solutions (Shanghai, China). The 2-methyl-3-heptanone (99%) was supplied by Dr. Ehrenstorfer (Beijing, China) as an internal standard.

The commercial cheeses used in this study were a regular salt cheese (RSC) and a low-salt cheese (LSC, –33% salt) from Orval Abbey (Villers-devant-Orval, Belgium). These are semihard cheeses, pertaining to the “Trappist” cheese category. For RSC as for LSC, dry matter was 54%, fat in dry matter: 58%, moisture on fat-free cheese: 61%, fat content: 32%, pH: 5.24. Salt content was 1.78% for RSC and 1.19% for LSC. RSC and LSC were made with pasteurized cow milk, rennet, lactic ferment, and salts. Salts were incorporated in the cheese curd by brining during different times. LSC and RSC came each one from the same brining batch. In addition, after grating, the pieces of cheese were mixed to obtain portions whose homogeneity in salt content was controlled.
A salt-congruent complex aroma, reminiscent of the odor of smoked sardine (Givaudan, Argenteuil, France) and further called the “sardine aroma,” and a fat-congruent aroma compound, 2,3-pentanedione (Sigma–Aldrich, Saint Quentin Fallavier, France), reminiscent of the odor of butter and further called the “butter aroma,” were used to flavor the low-salt cheeses. These aromas were dissolved in propylene glycol (Sigma–Aldrich). All materials were food-grade quality.