Experimental diets and excreta samples were analysed in triplicate for dry matter (DM, method 930.15), crude protein (CP, Method 990.03) and calcium, and phosphorus (method 985.01) according to AOAC (2007) . Gross energy (GE) of diets and excreta were measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Molin, IL), and chromium concentrations was determined with an automated spectrophotometer (Jasco V-650, Jasco Corp., Tokyo, Japan) according to the procedure of Fenton and Fenton (1979) . The content of β-mannan in feed ingredients, diets and excreta samples was estimated from the total mannose contents according to the method of Englyst and Cummings (1984) . In brief, after removing fat, a stock buffer solution was made by adjusting the pH to 5.2 by acetate buffer solution using of a-amylase at 45°C for 16–18 h to hydrolysis of starch. The mannose separated and identified on Rtx-2330 using gas liquid chromatography (HP 6890 Plus; Hewlett Packard, Houston, TX, USA). Commercial kits (Fujifilm Corp., Saitama, Japan) were used for analysis of serum metabolites (total cholesterol, triacylglycerides, glucose, total protein, and blood urea nitrogen) using an automated chemistry analyzer (Fuji Dri-chem 3500i, Fujifilm Corp.).
Hp 6890 plus
Manufactured by Hewlett-Packard
Sourced in United States
The HP 6890 Plus is a gas chromatograph designed for versatile, high-performance analysis. It features advanced electronics and software control to deliver consistent, reliable results.
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8 protocols using hp 6890 plus
1
Nutritional Analysis of Diets and Excreta
2
GC/MS Analysis of E. cava
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E. cava GC/MS analysis was performed at Kyungpook National University by the “Center of Scientific Instruments” using an HP 6890 Plus GC gas chromatograph with an HP 5973 mass selective detector (Hewlett-Packard, Palo Alto, CA, USA). The dilution of the sample was prepared by v:v ratio (1:1000) with DCM (HPLC grade). For this, 1 µL of the sample was injected into the HP-5 column. The GC oven temperature was set at 50 ℃ for 4 min, increased to 280 ℃ at a rate of 4 ℃ per minute, and held at the final temperature for 2 min. The carrier gas He (99.99%) velocity was 0.7 mL/min. Quantitative analysis was performed by using the area normalization method.
3
GC-MS Analysis of Organic Compounds
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GC-MS analysis of DFNTE, EFNTE, and BFNTE was performed by “Center for Scientific Instruments” of Kyungpook National University and carried out using a HP 6890 Plus GC gas chromatograph with a (MSD)—HP 5973 MSD mass selective detector (Hewlett-Packard). Samples were diluted 1 : 1000 (v : v) with HPLC grade dichloromethane. Aliquots of the sample (1 μL) were injected into an HP-5 column. The GC oven temperature was set at 50°C for 4 min, increased to 280°C at a rate of 4°C/min, and held at the final temperature for 2 min. Velocity of the He carrier gas (99.99%) was 0.7 mL/min. Quantitative analysis was performed using the area normalization method.
4
Quantification of Individual Sugars
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Individual sugars were assessed according to Mrabet et al. [17 (link)]. Prior to determining the neutral sugars, the sugar fraction was hydrolyzed with trifluoro acetic acid (TFA) for 1 h at 121 °C. The released sugars were reduced and then acetylated and, finally, identified and quantified as alditol acetates using gas chromatography (HP 6890 Plus, Hewlett-Packard, Palo Alto, CA, USA) equipped with a capillary column (30 m × 250 μm × 0.20 mm, SP-2330, Supelco, Bellefonte, PA, USA). The flow of the vector gas (Helium) was 2.2 mL/min at a pressure of 148.24 kPa. The run time was 40.7 min, during which the injector and flame ionization detector (FID) temperatures were 250 and 300 °C, respectively. After 2 min of the injection (splitless mode), the oven temperature was 50 °C; it was then held progressively at 180 °C and finally increased to 220 °C for 22 min. The internal standard was Myo-inositol. The results were expressed as milligrams per kg of dry weight (mg/kg DW).
5
Fatty Acid Composition Analysis
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The fatty acid composition was determined by the conversion of oil to fatty acid methyl esters (FAMEs) according to the modified method by Zahran and Tawfeuk (2019) . The FAMEs were separated with an HP 6890 plus gas chromatography (Hewlett Packard, USA), using a capillary column Supelco TM SP-2380 (60 m  0.25 mm  0.20 mm), (Sigma-Aldrich, USA), Detector (FID) and the injector and detector temperature was 250 °C. The column temperature was 140 °C (hold for 5 min) and rises to 240 °C, at a rate of 4 °C/min, and holds at 240 °C for 10 min. The carrier gas was helium at a flow rate of 1.2 mL min À1 . FAMEs were identified by comparing their relative and absolute retention times to those authentic standards of FAMEs (Supelco TM 37 component FAME mix). The fatty acid composition was reported as a relative percentage of the total peak area.
6
Determination of Fatty Acid Composition
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The fatty acids composition was determined by the conversion of oil to fatty acid methyl esters (FAMEs) according to the modified method described by Zahran and Tawfeuk (2019) . FAMEs were separated with an HP 6890 plus gas chromatography (Hewlett Packard, USA), using a capillary column Supelco™ SP-2380 (60 m×0.25 mm×0.20 μm), (Sigma-Aldrich, USA), Detector (FID) and the injector and detector temperatures were 250°C. The column temperature was 140°C (hold for 5 min) and raised to 240°C, at rate of 4°C/min, and holds at 240°C for 10 min. The carrier gas was helium at flow rate 1.2 mL min -1 . FAMEs were identified by comparing their relative and absolute retention times to those authentic standards of FAMEs (SupelcoTM 37component FAME mix). The fatty acid composition was reported as a relative percentage of the total peak area.
7
Fatty Acid Composition Analysis by GC
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The fatty acid composition was determined by the conversion of oil to Fatty Acid Methyl Esters (FAMEs) according to the modified method of Zahran and Tawfeuk [19] . FAMEs were analyzed using an HP 6890 plus Gas chromatography (GC) (Hewlett Packard, USA), a capillary column Supelco™ SP-2380 (60 m×0.25 mm×0.20 μm), (Sigma-Aldrich, USA), Detector (FID) while the injector and detector temperature were 250°C. The column temperature was 140°C (hold for 5 min) and rise to 240°C at 4°C/min., and hold at 240°C for 10 min. The carrier gas was helium at a flow rate of 1.2 mL min -1 . FAMEs were identified by comparing their relative and absolute retention times to those authentic standards of FAMEs (Supelco TM 37component FAME mix). The fatty acid composition was reported as a relative percentage of the total peak area.
8
Saeman Hydrolysis for Carbohydrate Analysis
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Saeman hydrolysis was applied to solid samples. Sulphuric acid 72% (0.5 mL) was added to 10 mg of sample (in triplicate) in screw-cap tubes. The tubes were placed in a heating block at 40º C for 2 hours. Afterwards, 5.5 mL water was added to each tube and the hydrolysis continued at 100º C for 2 hours. 0.5 mL of the hydrolysate was neutralized with NH 4 OH and 50 µg myo-inositol were added as internal standard. After reduction and peracetilation, the released sugars were quantified as alditol acetates by gas chromatography (Dos-Santos et al., 2011) (link). A HP 6890 Plus+ gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) fitted with a 30 m × 250 μm× 0.20 mm capillary column (SP-2330, Supelco, Bellefonte, PA, USA) was used. The carrier gas was helium with a constant flow of 2.2 mL/min and pressure of 21.5 psi (148.24 kPa).
The injection was performed in splitless mode. The oven temperature was held at 50° C for 2 min after injection, then programmed to 180° C at 35° C/min, held at 180° C for 5 min, and then immediately increased to 220° C at 5° C/min and held at 220° C for 22 min. The total run time was 40.7 min. The injector temperature was 250° C and the flame ionization detector was set at 300° C.
The injection was performed in splitless mode. The oven temperature was held at 50° C for 2 min after injection, then programmed to 180° C at 35° C/min, held at 180° C for 5 min, and then immediately increased to 220° C at 5° C/min and held at 220° C for 22 min. The total run time was 40.7 min. The injector temperature was 250° C and the flame ionization detector was set at 300° C.
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