Methyl heptadecanoate

The fatty acid content of the microalgae was estimated as the amount of fatty acid methyl esters (FAMEs), which was obtained using the direct esterification method. The obtained crude lipid was saponified with KOH-CH₃OH and then FAMEs were prepared by methylene esterification with boron trifluoride in methanol and extracted with n-hexane [31 (link)].
The qualitative and quantitative analyses of the lipid sample were carried out using a GC-2030AF gas chromatograph (Shimadzu, Tokyo, Japan) equipped with a DB-Wax capillary column (30 cm, 0.32 mm ID, 0.5 μm film thickness) and a flame ionization detector (FID). The temperature program was as follows: initially kept 120 °C for 3 min; then increased to 190 °C at 10 °C/min, where the temperature was maintained for 1 min; then increased to 230 °C at 2 °C/min and held there for 15 min with a split ratio of 10:1. Nitrogen was used as a carrier gas at a flow rate of 3 mL/min. Fatty acids were identified by comparing the peak and retention time of the reference standard 21-component fatty acid methyl ester mix (NU-CHEK-PREP, INC). The relative and absolute contents of fatty acids were quantified by the area normalization method using methyl heptadecanoate purchased from Sigma-Aldrich (Merck & Co., Kenilworth, NJ, USA) as an internal standard.

The lipid content was determined by the chloroform-methanol extraction method [15 (link)] and the extracted lipid was subsequently used for fatty acid analysis. The methyl esterification and analysis of the fatty acids were based on the method described by Ma et al. [13 (link)] using a gas chromatography and mass spectrogram (GC-MS; QP2020, Shimadzu, Kyoto, Japan) with Rxi-5 sil MS (30 × 0.25 mm, 0.25 μm). Fatty acids were identified by the external standards (37 Component FAME Mix, Supelco, Bellefonte, PA, USA) and quantified by the internal standard concentration (methyl heptadecanoate, Sigma, Shanghai, China). The atherogenic index (AI) and thrombogenic index (TI) used as evaluating fat quality indices [16 (link)] were calculated as follows: $$\mathrm{AI}=\frac{(\mathrm{C}12:0+4\times \mathrm{C}14:0+\mathrm{C}16:0)}{(\mathsf{\Sigma }\mathrm{MUFAs}+\mathsf{\Sigma }\mathrm{PUFAs})}$$
$$\mathrm{TI}=\frac{(\mathrm{C}14:0+\mathrm{C}16:0+\mathrm{C}18:0)}{(0.5\times \mathsf{\Sigma }\mathrm{MUFAs}+0.5\times \mathsf{\Sigma }\mathrm{n}\text{-}6\mathrm{PUFAs}+3\times \mathsf{\Sigma }\mathrm{n}\text{-}3\mathrm{PUFAs}+\mathsf{\Sigma }\mathrm{n}\text{-}3\mathrm{PUFAs}/\mathsf{\Sigma }\mathrm{n}\text{-}6\mathrm{PUFAs})}$$
where MUFA and PUFA represent monounsaturated fatty acid and polyunsaturated fatty acids, respectively.

Deuterium oxide (D₂O, 99.9% D) and sodium 3-(trimethylsilyl) [2,2,3,3-²H₄] propionate (TSP) were purchased from Cambridge Isotope Laboratories, Inc. (Miami, U.S.A.). Methanol, hexane, K₂CO₃, K₂HPO₄·3H₂O, and NaH₂PO₄·2H₂O (all in analytical grade) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methyl heptadecanoate, methyl tricosanate, and acetyl chloride (99.0%) were bought from Sigma-Aldrich (St. Louis, MO). 3,5-Di-tert-butyl-4-hydroxytoluene (BHT) and a mixed standard methyl esters of 37 fatty acids were obtained from Supelco (Bellefonte, PA). A buffer containing 0.001% TSP and 80% D₂O was prepared with K₂HPO₄ and NaH₂PO₄ (0.15 M, pH 7.43) and used as solvent for NMR analysis of tissue extracts.

Waste fish fat was collected from a local wholesale fish market in Machilipatnam market, Andhra Pradesh. Anhydrous methanol (99.5%) and sodium hydroxide (NaOH) were purchased from M/s Finar, Ahmedabad. Petro-diesel was bought from a nearby BPCL (Bharat Petroleum Corporation Limited) oil filling station. The reference standard solution, methyl heptadecanoate, used to test FAME was purchased from Sigma-Aldrich. The ultrasonic processor was bought from M/s Oscar Ultrasonics Pvt. Ltd., in Mumbai, India. It has a frequency of 20 kHz and a maximum power output of 1 KW.

Diethyl ether (purity ≥ 99.8%), n-hexane (purity ≥ 95%), n-heptane (purity ≥ 99%), and tert-butyl methyl ether (MTBE, purity ≥ 99.8%) were supplied by Sigma-Aldrich, Inc. (St. Louis, MO, USA). Sudan I (1-phenylazo-2-naphthol, CAS number 842-07-9), methyl heptadecanoate (analytical standard, CAS number 1731-92-6), ethyl palmitate (analytical standard, CAS number 628-97-7), ethyl oleate (analytical standard, CAS number 111-62-6), ethyl linoleate (analytical standard, CAS number 544-354), ethyl stearate (analytical standard, CAS number 111-61-5), and silica gel (high-purity grade, Davisil Grade 62, particle size 60-200 mesh) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).