Methyl nonadecanoate

For fatty acid methyl ester (FAME) transformation, 5–10 mg weighed freeze-dried cell powder was used. Cells were suspended in 1 ml NaOH–CH₃OH solution (2 mol/L) and incubated at 75°C for 30 min with shaking. Methyl non-adecanoate (C15:0, 200 μg/ml; Sigma-Aldrich) was applied as the internal standard. After cool-down, 1 ml of HCl–CH₃OH (4 mol/L) was added, and pH was adjusted to below 2.0 using HCl, and the mixture was then incubated at 75°C for 30 min with shaking. The methyl esters of FAs were extracted with 2 ml hexane, then dried by nitrogen blowing, and dissolved in 10 μl of CH₂Cl₂ for GC/MASS analysis. FAMEs were examined by Thermo Trace GC Ultra gas chromatograph coupled to Thermo Polaris Q mass spectrometry (Agilent, Santa Clara, CA, United States) equipped with an HP-5MS column (30 mm × 0.25 mm, film thickness 0.25 μm) as previously described (Wang et al., 2018 (link)).

The Candida antarctica lipase B variant (CalB 1422) immobilized on a methacrylic resin supplied from Korea Research Institute of Bioscience and Biotechnology (KRIBB) was used. It was obtained by directional evolution of wild-type CALB (Candida antartica lipase B), and Saccharomyces cerevisiae, which secretes it, was fermented to produce an enzyme. The enzyme was concentrated by diafiltration and immobilized on Lewatit VP OC 1600 resin. The immobilized concentration was 25–30 mg/g resin. The feedstocks used in this study were soybean oil (SBO), crude waste animal fat (CAF), and refined waste animal fat (RAF). The commercial SBO (CJ CheilJedang, Korea) was purchased from local grocery, and CAF and RAF were supplied by Taegok Oils & Fats. The RWF was the CAF pretreated with filtration and pressing. Liquefied carbon dioxide (99.999%, Dae Myung Specialty Gas, Korea), methanol (99.9%, Sigma-Aldrich, USA), methyl nonadecanoate (98.0%, Sigma-Aldrich, USA) and toluene (99.9%, Sigma-Aldrich, USA) were purchased and used without further purification.

The fatty acid analysis of the animal tissue samples included lipid extraction and transesterification of fatty acids and their further purification, which were described in detail elsewhere [37 ]. The FAME analysis was conducted using a GC-MS (Model 7000 QQQ, Agilent Technologies, Santa Clara, CA, USA). A 30 m (internal diameter is 0.25 mm) capillary HP-FFAP column was used. Detailed descriptions of the chromatographic and mass-spectrometric conditions are given elsewhere [21 (link)]. Peaks of FAME were identified by comparing their mass spectra to those in the integrated database NIST Mass Spectral Search 2.0 (built 22 October 2009) and to mass spectra of FAME in the standard of 37 FAME mixture (U-47885, Supelco, Bellefonte, PA, USA). The FAMEs were quantified according to the peak area of the internal standard—the methyl nonadecanoate (Sigma-Aldrich, St. Louis, MO, USA)—which was added to the samples prior to the lipid extraction.
Lipid dietary indexes, such as the index of thrombogenicity (IT), index of atherogenicity (IA), health-promoting index (HPI), and hypocholesterolemic/hypercholesterolemic index (HH), were calculated using the equations described elsewhere [38 (link),39 (link)].




where UFA is the sum of unsaturated fatty acids, MUFA is the sum of monounsaturated fatty acids, and PUFA is the sum of polyunsaturated fatty acids.