Fatty acid compositions were analyzed by gas chromatography (GC) according to the method of Lee et al. [22] . An 6890 Agilent Technologies (Wilmington, DE, USA) gas chromatograph with a fused silica capillary column (length 100 m; internal diameter 0.25 mm; length of film 0.2 μm), Supelco (Bellefonte, PA, USA) was used. Before running the sample in the GC, the sample was prepared methyl ester according to the official method and recommended practices of the AOCS Methods Ce 2-66 (1998). The flow rate of helium was 1.0 ml/min as a carrier gas. The oven temperature was programmed starting at a constant temperature of 130 °C for 3 min, and then it was increased to 240 °C at a rate of 4 °C/min and held at 240 °C for 10 min. 250 °C temperature was fixed for injector and detector. Fatty acid methyl esters were identified by comparison of retention time with standard fatty acid methyl esters mixture (Supelco).
Fused silica capillary column
Manufactured by Merck Group
Sourced in United States
The Fused silica capillary column is a type of laboratory equipment used for chromatographic analysis. It consists of a narrow, flexible tube made of fused silica, which is an amorphous form of silicon dioxide. The column is designed to separate and analyze complex mixtures of chemical compounds.
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Lab products found in correlation
Agilent 6890, by Agilent Technologies (2 mentions)
Poly ethylene glycol phase, by Merck Group (1 mentions)
Autosampler, by Bruker (1 mentions)
Wx vortex, by VELP Scientifica (1 mentions)
R 114 rotary evaporator, by Büchi (1 mentions)
Hp 6890, by Hewlett-Packard (1 mentions)
Agilent 7890b gas chromatograph, by Agilent Technologies (1 mentions)
Openlab chemstation software, by Agilent Technologies (1 mentions)
Sp 2560, by Merck Group (1 mentions)
Agilent 7890b, by Agilent Technologies (1 mentions)
7890b gas chromatograph, by Agilent Technologies (1 mentions)
Rt 6100 microplate reader, by Rayto (1 mentions)
Trace 1310 gas chromatograph, by Thermo Fisher Scientific (1 mentions)
Triplus rsh autosampler, by Thermo Fisher Scientific (1 mentions)
Conflo 4, by Thermo Fisher Scientific (1 mentions)
Mat 253 irms, by Thermo Fisher Scientific (1 mentions)
6 890 model gas chromatograph, by Agilent Technologies (1 mentions)
Sodium methoxide, by Merck Group (1 mentions)
Agilent chemstation software, by Agilent Technologies (1 mentions)
15 protocols using fused silica capillary column
1
Fatty Acid Composition Analysis by GC
2
Determination of Fatty Acid Profiles
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Fatty acid proportions and contents were determined using the method described in detail in Modzelewska-Kapituła et al. [14 (link)]. Briefly, muscle fat was extracted according to Folch et al.’s [18 (link)] method using a mixture of chloroform and methanol (2:1 v/v). Methylation of fatty acids was conducted using a chloroform–methanol–sulphuric acid (100:100:1) mixture [19 (link)]. Chromatographic separation was done using 7890A Agilent Technologies gas chromatograph equipped with a flame-ionization detector (FID) and a 30 m 0.32 mm internal diameter fused silica capillary column (matrix active group: poly(ethylene glycol)phase, Supelco, Bellefonte, PA, USA). Supelco (Bellefonte, PA, USA) standards were used to identify a particular fatty acid by comparing retention times. The results were presented as relative percentage (% total fatty acids) and content (mg/100 g wet tissue) of fatty acids in raw and sous-vide cooked fillets. The content of a particular fatty acid in fish muscle tissue was calculated separately for each sample based on fat content and coefficient for lean fish (0.70) [20 ].
3
Extraction and Analysis of MOH
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To extract MOH from the samples, a WX vortex from Velp Scientifica (Usmate, Italy) was utilized. An R-114 rotary evaporator from Büchi (Flawil, Switzerland) was used for the evaporation of the solvent.
A Scion GC system equipped with an autosampler (Bruker Corporation, Freemont, CA, USA) was used for chromatographic analyses. An ultra-inert liner SPI 0.25/0.32 mm from Agilent was used to simulate on-column injection. A DB-1HT capillary column (15 m × 0.32 mm i.d. × 0.10 µm film thickness) from Agilent (Santa Clara, CA, USA) was utilized for GC separation after an untreated fused silica capillary column used as pre-column (2 m × 0.32 mm) from Supelco (Bellefonte, PA, USA). The two columns were connected with a press-fit column connection from Agilent. Helium was used as carrier gas at a constant flow rate of 3 mL/min (62.2 cm/s linear velocity). Interactive Graphics (Bruker) v8.2.1 software was used for optimization and quantification.
A Scion GC system equipped with an autosampler (Bruker Corporation, Freemont, CA, USA) was used for chromatographic analyses. An ultra-inert liner SPI 0.25/0.32 mm from Agilent was used to simulate on-column injection. A DB-1HT capillary column (15 m × 0.32 mm i.d. × 0.10 µm film thickness) from Agilent (Santa Clara, CA, USA) was utilized for GC separation after an untreated fused silica capillary column used as pre-column (2 m × 0.32 mm) from Supelco (Bellefonte, PA, USA). The two columns were connected with a press-fit column connection from Agilent. Helium was used as carrier gas at a constant flow rate of 3 mL/min (62.2 cm/s linear velocity). Interactive Graphics (Bruker) v8.2.1 software was used for optimization and quantification.
4
Quantification of SCFAs and Lactate in Fermented Samples
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The SCFAs and lactate contents in the PM and fermented PM supernatants were determined using GC (HP 6890, Hewlett-Packard Co., CA, USA) using a flame ionization detector (FID). The used column was a fused silica capillary column (30 m × 0.32 mm × 0.25 µm; Cat No. 2-4131, SUPELCO, PA, USA). The supernatants of the PM and fermented PM (1 mL) were mixed to 50 µL of 2% pivalic acid, and then, they were transferred to a vial, respectively. Pivalic acid was used as an internal standard. A standard solution was produced by adding acetate, propionate, butyrate, lactate, and pivalic acid into deionized water. One microliter of the supernatant sample was injected into the inlet of gas chromatography (split 1:70). The oven temperature program was as follows: the oven temperature initially set at 100 °C for 1 min, it was increased by 10 °C/min to 150 °C, it was held at 150 °C for 7 min, it was increased by 20 °C/min to 180 °C, and then it was held at 180 °C for 1 min. The flow rate of the carrier gas (helium) was maintained at 1.5 mL/min.
5
Quantification of Cecal Short-Chain Fatty Acids
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Approximately 35 mg of cecal content was weighed, snap-frozen and stored at −80°C until analysis. The samples were extracted with water and proteins precipitated with phosphotungstic acid. 2-Ethylbutyrate was added to supernatants at a ratio of 1: 4 as an internal standard. The SCFA content was determined from a 0.3 µL volume of supernatant by gas chromatography (Agilent 7890B gas chromatograph, Agilent Technologies, Les Ulis, France) equipped with a split-splitless injector, a flame-ionization detector and a fused silica capillary column (15 m × 0.53 mm × 0.5 µm; Supelco, Saint-Quentin-Fallavier, France). The carrier gas (H2) flow rate was 10 ml/min. The oven temperature was initially set at 100°C for 10 min, then increased from 100 to 180°C at a rate of 20 C/min and held for 2 min. The detector temperature was 240°C. Samples were analyzed in duplicate. The peaks obtained were integrated using OpenLAB Chemstation software (Agilent Technologies, Les Ulis, France).
6
Fatty Acid Profiling of Omega-3 PUFA Concentrates
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Fatty acid composition of SFBO and prepared ω-3 PUFAs concentrates were determined using a 6890 Agilent (Agilent Technologies, Wilmington, USA) gas chromatograph facilitated with a fused silica capillary column (100 m length × 0.25 mm internal diameter, 0.2 μm of film) (Supelco, Bellefonte, USA). Fatty acid methyl esters for SFBO, 2-MAG, PUFA enriched acylglycerols, and ω-3 PUFFAs concentrate were prepared according to the methods of American Oil Chemists’ Society (AOCS), Ce 2-66 (2) and Ce 2-66 (3) [34 ], respectively. Oven temperature was programmed to start with a constant temperature of 130 °C for 3 min, then increased to 240 °C at a rate of 4 °C/min, and then held at 240 °C for 10 min. The injector and detector temperature were maintained at 250 °C. Standard fatty acid methyl esters were used for identifying the fatty acid methyl esters and quantification was done by obtained peak area (%).
7
Fatty Acid Profiling of SRM 2378
8
Fatty Acid Composition Analysis of C. racemosa and U. lactuca
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The fatty acid composition of C. racemosa and U. lactuca were determined using a Fatty Acid Composition Analysis (Agilent Technologies, Wilmington, NC, USA) gas chromatograph with a fused silica capillary column (Supelco, Bellefonte, PA, USA). Methylation of fatty acids (fatty acid methyl esters; FAMEs Supelco, Bellefonte, PA, USA) were prepared according to The American Oil Chemists’ Society’s protocols. The oven temperature was turned on at 130 °C and run for 180 s, and then the temperatures were increased up to 240 °C at a rate of 4 °C/min and then maintained at 240 °C for 600 s. Both the injector and the detector were set to 250 °C. The FAMEs were identified by comparison of retention time with a standard fatty acid methyl ester mixture (Supelco, Bellefonte, PA, USA).
9
Hydrogen Analysis in Headspace Gas
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The relative abundance of hydrogen in the head-space gas in the bottle was measured by a GC system (Agilent 7890B, Agilent, California, United States). The gas sample was injected into a GC, and the gases were separated on packed GC columns (Fused Silica Capillary Column, Supelco, United States) at 80°C column temperature, 200°C injection temperature, 200°C TCD temperature, and air pressure and carrier gas (nitrogen) pressure of 0.05 MPa. The volume of hydrogen was calculated according to the method of Li et al. (2020) (link).
10
Fecal Fermentation Characteristics Analysis
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Faecal samples were mixed with distilled water at a ratio of 1:4 (w/v) in tubes. The pH values of the mixture were measured using a portable pH meter immediately after mixing. Then, faecal mixtures were centrifuged at 3,000 g for 15 min to obtain the liquid supernatant for analysing fermentation characteristics. Ammonia-N concentration was analysed using a colourimetry method according to Broderick and Kang [16] (link). The concentration of microbial crude protein was determined using the method of Makkar et al [17] (link) with Coomassie blue solution and an RT-6100 microplate reader (Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China). Concentrations of volatile fatty acids were determined using a 7890B gas chromatograph (Agilent Technologies, USA) equipped with a fused silica capillary column (Supelco, Bellefonte, PA, USA) as described by Jin et al [18] (link). The contents of ammonia-N, microbial crude protein, and volatile fatty acids in faecal samples were obtained by their concentrations in mixtures of faeces and water multiplied by the dissolution ratio (1:4, w/v).
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