Detection of diacetyl and acetaldehyde in culture medium

Schiff's test is a qualitative test for the presence of aldehyde functional groups wherein, a colorless Schiff reagent turns into a characteristic violet color when aldehyde groups are present in the sample added.14 The reaction was performed by adding 500 μL of Schiff's reagent to the 5 mL of medium fermented for 48 h at 30 °C. The media samples were assigned scores between 0 and 5 based on reaction intensity at 5 s after reagent addition, with 0 denoting no acetaldehyde production; 1–3 denoting low production of acetaldehyde, 4 denoting good production of acetaldehyde, and 5 denoting very good production of acetaldehyde. Only samples with high aldehyde content (score 5) were further tested to determine if acetaldehyde was indeed present in high amounts.

2,4-Dinitrophenylhydrazine (DNPH, Brady's reagent) is a chemical compound which can be used to qualitatively detect the presence of ketone or aldehyde functional groups. The test is considered positive if a yellow (aliphatic), orange or red (aromatic) precipitate is present. DNPH does not react with other carbonyl-containing functional groups such as carboxylic acids, amides or esters.15 The test was performed by adding 250 μL of Brady's reagent to 2.5 mL of medium fermented for 48 h at 30 °C. As diacetyl is an aliphatic compound, only those strains that gave a yellow precipitate were selected for further investigation. Similar to Schiff's reaction, the samples were evaluated based on intensity of the yellow precipitate and this was assigned a score between 0 and 5, where 0 denotes no diacetyl production, 1–3 low production of diacetyl, 4 good production of diacetyl and 5 very good production of diacetyl. Further, results from both the Schiff test and the DNPH test were considered together in order to select strains capable of producing both acetaldehyde and diacetyl. For identification of diacetyl and acetaldehyde in the culture medium, the same strain was simultaneously inoculated into both YAD and YPG media.