Zb wax column

Plasma concentrations of SCFAs were measured as previously described [16 ]. Briefly, 100 μl of plasma were diluted in acidified water spiked with stable isotope-labelled internal standards. Then, SCFA samples were extracted with 2 ml diethyl ether and were measured in an Agilent 6890 gas chromatography (GC) coupled with mass spectrometry (Agilent Technologies, Wilmington, DE, USA). The GC was equipped with a ZB-WAX column (Phenomenex, Cheshire, UK). For the measurement of fecal SCFAs levels, 100 mg fecal contents were mashed in acidic water (pH=2.4) and centrifuged at 12,000 g for 20 min at 4°C. The supernatants were then taken for analysis as previously described [17 ].

Samples of anterior intestine, pyloric caeca and brain from three fish per tank were prepared as pooled homogenates (n = 3 per treatment) and total lipid extracted from 1 g by homogenising in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), and content determined gravimetrically (Folch et al., 1957 (link)). Fatty acid methyl esters (FAMEs) were prepared from total lipid by acid-catalysed transesterification at 50 °C for 16 h (Christie, 2003 ), and FAMEs were extracted and purified as described previously (Tocher and Harvie, 1988 (link)). FAMEs were separated and quantified by gas–liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p.A., Milan, Italy). Individual FAME was identified by comparison to known standards (Supelco™ 37-FAME mix; Sigma-Aldrich Ltd., Poole, UK) and published data (Tocher and Harvie, 1988 (link)).

Triplicates of 2 mL culture were pelleted by centrifugation, washed with ddH₂O and freeze dried at −80 °C. Between 10 and 20 mg were used for the fatty acid analysis. Fatty acid methyl esters (FAMES) were obtained by direct conversion of cell biomass by methanol transesterification [12 (link)].
FAMEs were analyzed on a GC-2025 gas chromatograph from Shimadzu (Nakagyo-ku, Kyōto, Japan) with flame ionisation detector and an AOC-20i auto injector (Shimadzu). 1 μl sample was applied onto a ZB-WAX column (30 m, 0.32 mm ID; 0.25 μm df; Phenomenex (Torrance, CA, USA)) with an initial column temperature of 150 °C (maintained for 1 min). A temperature gradient was applied from 150–240 °C (5 °C/min), followed by 6 min maintenance at 240 °C. Fatty acids were identified according to retention times of authentic standards.

Total lipid was extracted from ~0.5 g of homogenised salmon flesh in 20 volumes of ice-cold chloroform/methanol (2:1 v/v) using an Ultra-Turrax tissue disruptor (Fisher Scientific, Loughborough, UK) and the lipid determined gravimetrically54 (link). Fatty acid methyl esters (FAME) from total lipid were prepared by acid-catalysed transmethylation at 50 °C for 16 h55 . FAME were extracted and purified as described previously56 (link), and separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), ‘on column’ injection and flame ionisation detection. Hydrogen was used as carrier gas with an initial oven thermal gradient from 50 °C to 150 °C at 40 °C.min⁻¹ to a final temperature of 230 °C at 2 °C.min⁻¹. Individual FAME were identified by comparison to known standards (Supelco™ 37-FAME mix; Sigma-Aldrich Ltd., Poole, UK) and published data56 (link). Data were collected and processed using Chromcard for Windows (Version 1.19; Thermoquest Italia S.p.A., Milan, Italy). Fatty acid content per g of tissue was calculated using heptadecanoic acid (17:0) as internal standard.

Fatty acid methyl esters (FAMEs) were obtained by methanol transesterification of lyophilized yeast biomass. The transesterification protocol was originally adopted from Griffiths et al. (2010 (link)) and modified in our lab by Gorner et al. (2016 (link)). FAME profiles were analyzed on a GC-2010 Plus gas chromatograph from Shimadzu (Nakagyo-ku, Kyoto, Japan) with flame ionization detector. One microliter sample was applied by AOC-20i auto injector (Shimadzu) onto a ZB-WAX column [30 m, 0.32 mm ID; 0.25 μm df; phenomenex (Torrance, CA, USA)]. The initial column temperature was set at 150°C (maintained for 1 min). A temperature gradient was applied from 150° to 240°C (5°C.min⁻¹), followed by 6 min maintenance at 240°C. Fatty acids were identified according to retention times of the authentic standard: Marine Oil FAME Mix (Restek, USA). Individual FAME concentrations were based on peak areas relative to Methyl Non-adecaanoate C19 (Sigma, Germany), which was incorporated as an internal standard in all samples.

The products obtained were characterized by gas chromatography-mass spectroscopy (GC-MS) (Agilent Model 7890, United States) and quantified by gas chromatography (Agilent Model 6890, United States) equipped with a flame ionization detector (FID). Compounds were separated by a ZB-Wax column (30 m × 0.25 mm × 0.25 mm) (Phenomenex, United States). The obtained chromatograms were compared with the MS library and chemical standards. Glycerol, glycolic acid and lactic acid were analyzed using the following procedure: the front inlet temperature was controlled at 240°C. Initially, the oven temperature was fixed at 45°C and maintained for 5 min. Later, it was ramped at 10°C/min to reach 240°C at the final temperature and maintained for another 5 min. The sample injection was 1 μL. Glycerol conversion, product selectivity as well as yield were calculated based on the Equations (2–4), respectively.
$\begin{array}{l}Glycerolconversion(\%)=\frac{Gycerolconverted(Cmole)}{Totalglycerolinreactant(Cmole)}\times 100\%\end{array}$
$\begin{array}{l}Productselectivity(\%)=\frac{Product(Cmole)}{Totalofallproductsinliquidphase(Cmole)}\times 100\%\end{array}$
$\begin{array}{l}Productyield(\%)=\frac{Product(Cmole)}{Totalglycerolinreactant(Cmole)}\times 100\%\end{array}$

The extent of lipid contamination in protein pellets, following various purification methods, was measured by accounting for the sum of fatty acid methyl esters (FAMEs), obtained by methanol transesterification. The transesterification protocol was originally adopted from [46 (link)]. and modified in our lab by [47 (link)]. FAME profiles were analyzed on a GC-2025 gas chromatograph from Shimadzu (Nakagyo-ku, Kyōto, Japan) with flame ionization detector. One microliter sample was applied by AOC-20i auto injector (Shimadzu) onto a ZB-WAX column (30 m, 0.32 mm ID; 0.25 μm df; phenomenex (Torrance, CA, USA)). The initial column temperature was 150 °C (maintained for 1 min). A temperature gradient was applied from 150–240 °C (5 °C min⁻¹), followed by 6 min maintenance at 240 °C. Fatty acids were identified according to retention times of the authentic standard: Marine Oil FAME Mix (Restek, USA). Individual FAME concentrations were based on peak areas relative to Methyl Nonadecaanoate C19 (Sigma, Germany), which was incorporated as an internal standard in all samples. Percent lipid was calculated from the sum of individually identified FAMEs with respect to pellet dry weight.