Butyl butyrate

Butyl butyrate, menthol, vanilline, methyl salicylate and eugenol standards were purchased from Sigma-Aldrich (Beijing, China). Ethanol of liquid-chromatography grade was purchased from Merck (Shanghai, China). Chewing gum were obtained from local markets in Qingdao city of Shandong Province. According to Fierens et al.^{26 (link)}, sample contamination can occur in every stage of the analytical procedure because it is ubiquitous in laboratory environment. Therefore, all the glassware was thoroughly washed and rinsed with deionized water and acetone prior to use. Standard stock solutions of Butyl butyrate, menthol, vanilline, methyl salicylate and eugenol were prepared in ethanol. Standard working solutions were prepared by the stock solution with serial dilution. All solutions were stored in amber vials at 4 °C for later use.

Ethyl acetate, ethyl 2-methylpropionate, propyl acetate, ethyl propionate, ethyl butyrate, isobutyl acetate, ethyl 2-methylbutyrate, ethyl 3-methyl butyrate, isoamyl acetate, ethyl valerate, butyl butyrate, ethyl hexanoate, isoamyl butyrate, propyl hexanoate, ethyl heptanoate, ethyl lactate, 3-methylbutyl hexanoate, propyl octanoate, ethyl nonanoate, hexyl hexanoate, ethyl 2-furoate, ethyl caprate, ethyl benzoate, ethyl phenylacetate, ethyl laurate, ethyl 3-phenylpropanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl palmitate, ethyl oleate, and linoleic acid ethyl ester were obtained from Sigma-Aldrich (Shanghai, China). The internal standard (IS) was 2-octanol (Sigma-Aldrich, Shanghai, China). The linear retention index (RI) was determined with a C7–C30 n-alkane mixture (Sigma-Aldrich, Shanghai, China). All of the reagents used were of analytical grade with a purity of at least 97%, and most with a purity exceeding 99%. A Milli-Q purification system provided pure water (Millipore, Bedford, MA, USA). Sodium chloride (analytical grade) and absolute ethanol (analytical grade) were obtained from Sino-pharm Chemical Reagent Co., Ltd (Shanghai, China).

All triglyceride substrates were purchased from Sigma-Aldrich Canada (Oakville, ON) using the highest purity available. Analytical standards for methyl acetate, isoamyl acetate, methyl butyrate, methyl hexanoate, methyl octanoate, ethyl butyrate, propyl butyrate, butyl butyrate and amyl butyrate were also purchased from Sigma-Aldrich. Analytical grade methyl nonanoate was purchased from Chromatographic Specialties (Brockville, ON). All other chemicals were obtained commercially and were of analytical grade.
The strain Escherichia coli DH11S was employed for all subcloning procedures. The strain E. coli BL21 (DE3) was employed for the heterologous expression of the lipase gene. Expression vector pET16b (EMD Millipore) was used for the production of the N-terminal histidine-tagged recombinant protein. Plasmid pIAFD95A was used as template for the amplification of the lipIAF5-2 gene (Genbank EU660533) [22] (link).