The pH of doughs was determined by a pH meter (Model 507, Crison, Milan, Italy) with a food penetration probe and total titratable acidity (TTA) was determined according to AACC method 02–31.01 [28 ].
Water/salt-soluble extracts (WSE) from doughs were prepared according to the method originally described by Osborne and modified by Weiss et al. [29 (link)]. Fifteen grams of sample were resuspended in 60 mL of 50 mM Tris–HCl (pH 8.8), held at 4 °C for 1 h, under stirring condition (150 rpm) and centrifuged at 20,000× g for 20 min. The supernatant was used for analyses. WSE were used to determine total free amino acids (TFAA) and organic (lactic and acetic) acids concentration. TFAA were analyzed by a Biochrom 30+ series Automatic Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK), equipped with a Li-cation-exchange column (4.6 mm × 200 mm internal diameter), using lithium citrate buffer eluents following the elution conditions recommended by the manufacturer. A mixture of amino acids at known concentrations (Sigma Chemical Co., Milan, Italy) was added with tryptophan, ornithine and γ-aminobutyric acid (GABA) and used as a standard. Proteins and peptides in the samples were precipitated by addition of 5% (v/v) cold solid sulfosalicylic acid, holding the samples at 4 °C for 1 h and centrifuging them at 15,000× g for 15 min. Organic acids were determined by high performance liquid chromatography (HPLC), using an ÄKTA Purifier system (GE Healthcare, Buckinghamshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad, Richmond, CA, USA), and an UV detector operating at 210 nm. Elution was carried out at 60 °C, with a flow rate of 0.6 mL/min, using H2SO4 10 mM as the mobile phase. The fermentation quotient (FQ) was determined as the molar ratio between lactic and acetic acids. Resistant starch of doughs, prior and after the fermentation, was determined according to the AACC approved methods 32–40.01 [28 ].
Water/salt-soluble extracts (WSE) from doughs were prepared according to the method originally described by Osborne and modified by Weiss et al. [29 (link)]. Fifteen grams of sample were resuspended in 60 mL of 50 mM Tris–HCl (pH 8.8), held at 4 °C for 1 h, under stirring condition (150 rpm) and centrifuged at 20,000× g for 20 min. The supernatant was used for analyses. WSE were used to determine total free amino acids (TFAA) and organic (lactic and acetic) acids concentration. TFAA were analyzed by a Biochrom 30+ series Automatic Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK), equipped with a Li-cation-exchange column (4.6 mm × 200 mm internal diameter), using lithium citrate buffer eluents following the elution conditions recommended by the manufacturer. A mixture of amino acids at known concentrations (Sigma Chemical Co., Milan, Italy) was added with tryptophan, ornithine and γ-aminobutyric acid (GABA) and used as a standard. Proteins and peptides in the samples were precipitated by addition of 5% (v/v) cold solid sulfosalicylic acid, holding the samples at 4 °C for 1 h and centrifuging them at 15,000× g for 15 min. Organic acids were determined by high performance liquid chromatography (HPLC), using an ÄKTA Purifier system (GE Healthcare, Buckinghamshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad, Richmond, CA, USA), and an UV detector operating at 210 nm. Elution was carried out at 60 °C, with a flow rate of 0.6 mL/min, using H2SO4 10 mM as the mobile phase. The fermentation quotient (FQ) was determined as the molar ratio between lactic and acetic acids. Resistant starch of doughs, prior and after the fermentation, was determined according to the AACC approved methods 32–40.01 [28 ].
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