Sp 2560

A total of 3 mL of fermentation broth was mixed with 4 mL of HCl (12 N) and incubated in a water bath at 65 °C for 50 min. Total lipids from the mixture were extracted four times with 4 mL of n-hexane, and then the collected extracts were mixed and purified by the nitrogen blowing method at room temperature [32 (link)].
Fatty acid methyl esters (FAMEs) were prepared according to the methods of Kueiling et al. and Ren et al. [33 (link), 34 (link)] with modifications: heptadecanoic acid methyl (16 g/L, Sigma, USA) as an internal standard was added to the tubes and mixed by vortexing for 1 min. The upper phase containing FAMEs was applied to a gas chromatograph (Agilent GC 7890, USA) equipped with a 100 m × 0.25 mm capillary column (SP™-2560, USA). Peaks were identified with authentic standards of fatty acid methyl esters (Sigma, USA).

Biomass was determined by gravimetric method. Lipid extraction method was same as Li et al. [5 (link)]. Fatty acid methyl esters (FAMEs) were prepared regarding to previous methods [33 (link)] with some modifications. First, 5 mL 0.5 M KOH–methanol was added to a tube containing total lipids. The tubes were heated in a water bath at 65 °C for 10 min and then 5 mL 30% BF₃-ether was added. The tubes were further heated in a water bath at 65 °C for 30 min and then 5 mL hexane was added when the tubes cooled down to room temperature. Then, 16 g/L methyl heptadecanoic acid (Sigma, San Francisco, USA) served as an internal standard and was added to mixtures. Then, the mixtures settled for separation of two phases after adding 1 mL saturated sodium chloride solution for preventing emulsification. FAMEs were applied to a gas chromatograph (Agilent GC 7890, Sacramento, USA) equipped with a 100 m × 0.25 mm capillary column (SP-2560, Sacramento, USA). The column was increased from 140 to 240 °C at 3 °C/min and then maintained at 240 °C for further 30 min. The temperature of the injector and detector were both set at 260 °C. Nitrogen was used as the carrier gas at 20 cm/s. Peaks were identified using authentic standards of supelco 37 component FAME mix (Sigma, San Francisco, USA). Fatty acids were quantified from the peak areas relative to the peak of the internal standard.

Fatty acid profile was determined by converting the fat into fatty acid methyl esters, briefly, 50 mg fat was taken in a 15 ml test tube, dissolved in 3-ml iso-octane and 2 ml 0.5 N sodium methoxide was added. Test tube was capped and vortex for 3-min at 2200 rpm, after 5 min, supernatant was transferred to GC vials and injected to GC-MS (79890-A Agilent, USA) fitted with fused silica capillary column (SP 2560; 100 m, film thickness 25 μm) and Mass Selective Detector [17 ]. Helium was used as carrier gas at the flow rate of 2 ml/ min; total run time was 35 min. Fatty acid standards, FAME 37 kit (Sigma Aldrich, UK) was used for identification and quantification of fatty acids. Cholesterol in cheese was determined according to the method [18 (link)]. Free fatty acids were determined in terms of oleic acid, for the determination of free fatty acids, absolute ethanol was neutralized to light pink colour, using 3 drops of 1% phenolphthalein indicator, 5 g fat was taken in the same flask, contents of flask were mixed, boiled for 2 min and titrated against standardized 0.1 N NaOH till light pink colour end point and free fatty acids (FFA) were calculated by the following formula [19 ]. $$\%\mathrm{FFA}\left(\text{Oleic Acid}\right)=\frac{\text{Volume\hspace{0.17em}of}0.1\mathrm{N}\text{NaOH}\mathrm{x}\text{Normality\hspace{0.17em}of\hspace{0.17em}NaOH}\mathrm{x}282\mathrm{x}100}{1000\mathrm{x}\text{Weight\hspace{0.17em}of\hspace{0.17em}Sample}}$$