Biochrom 30 series amino acid analyzer

L. rossiae DSM 15814^T was propagated for 24 h at 30°C in modified MRS broth (Oxoid, Basingstoke, Hampshire, England) with the addition of fresh yeast extract (5%, vol/vol) and 28 mM maltose to a final pH of approximately 5.6 [34] ; see Text S2). The biosynthesis of GABA from glutamic acid was estimated as described by Siragusa et al. [48] (link) using a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England).

Samples of cheese were analyzed for protein [29 ], fat [30 ], moisture (oven drying at 102°C) [31 ] and salt [32 ]. The pH was measured by Foodtrode electrode (Hamilton, Bonaduz, Switzerland).
Microbiological analyses were carried out as described previously [33 ]. Twenty grams of sample were homogenized with 180 ml of sterile sodium citrate (2%, [wt/vol]) solution. Presumptive mesophilic lactobacilli and lactococci were enumerated onto MRS and M17 agar (Oxoid, Basingstoke, Hampshire, UK), respectively, under anaerobiosis at 30°C for 48 h. Presumptive thermophilic streptococci were enumerated onto M17 agar (Oxoid), under anaerobiosis at 42°C for 48 h. Enterococci were counted onto Slanetz & Barteley Agar (Oxoid) at 37°C for 48 h. Except for enterococci, the media for plating bacteria were supplemented with cycloheximide at 0.1 g/l.
The pH 4.6-insoluble and -soluble nitrogen fractions of the cheeses were analyzed by urea polyacrylamide gel electrophoresis (Urea-PAGE) and reverse phase high pressure liquid chromatography (RP-HPLC), as described by Andrews et al. [34 (link)], and Gobbetti et al. [35 ], respectively. Total and individual free amino acids (FAA) from the pH 4.6-soluble fraction were determined by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK), as described by Di Cagno et al. [36 (link)].

To establish a correlation between the chemical composition of the media and microbial growth, glucose, peptides and amino acids concentrations were determined for each experimental condition. Glucose content was determined by using the D-Fructose/D-Glucose Assay Kit (Megazyme Int., Ireland) following the manufacturer’s instructions. Peptides concentration was estimated by the o-phtaldialdehyde (OPA) method as described by Church et al. (1983) (link). A standard curve prepared using tryptone (0.25 to 1.5 mg/ml) was used as reference. Total free amino acids (TFAA) concentration was analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) with a Na-cation-exchange column as described by Rizzello et al. (2008) (link).

Aiming at selecting the optimal bioprocessing option, total free amino acid (TFAA) concentration and peptide profiles were considered as indexes of the proteolytic degradation and screening parameters. Doughs prior bioprocessing were used as the controls (H, C, and WGB).
Water/salt-soluble extracts (WSEs) from doughs were prepared as reported by Weiss et al. [25 (link)] and used for TFAA and peptides analyses. TFAA were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) with a Na-cation-exchange column (20 by 0.46 cm internal diameter), as described by Rizzello et al. [17 (link)]. For the analysis of peptides, WSE were treated with trifluoroacetic acid (TFA, 0.05% wt/v) and subjected to dialysis (cut-off 500 Da) to remove proteins and FAA, respectively. Then, the peptide concentration was determined by the o-phtaldialdehyde (OPA) method [26 (link)]. Peptide profiles were obtained by Reversed-Phase Fast Performance Liquid Chromatography (RP-FPLC) [26 (link),27 (link)], using an ÄKTA FPLC equipped with a Resource RPC column and a UV detector (214 nm) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Peptide profiles and total peak area were elaborated with Unicorn 4.0 software (GE Life Sciences).

Total free amino acids (FAAs) in water-soluble extracts of fecal media were determined using a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, Waterbeach Cambridge, UK), equipped with a sodium cation-exchange column (20 by 0.46 cm, inner diameter), as previously described [29 (link)]. The analysis was performed after 24 h of probiotic growth in fecal media obtained from pooled and sterilized feces of healthy subjects (HC) or chronic kidney disease patients (CKD).

Kinetics of the growth and acidification of W. cibaria P9 in the different sourdoughs were determined and modeled in agreement with the Gompertz equation, as modified by Zwietering et al. [35 (link)]. The experimental data were modeled by the non-linear regression procedure of the Statistica 12.0 software (Statsoft, Tulsa, OK, USA). The pH of doughs was determined by a M.507 pHmeter (Crison, Milan, Italy) equipped with a food penetration probe. Total Titratable Acidity (TTA) was determined after homogenization of 10 g of dough with 90 mL of distilled water and expressed as the amount (mL) of 0.1 M NaOH required to neutralize the solution to pH 8.4.
Water/salt-soluble extracts (WSE) were used to analyze organic acid, peptides, and free amino acids (FAA). Organic acids were determined by Megazyme kits (Megazyme, Bray, Ireland) K-DLATE and K-ACET (sum of D- and L- lactic and acetic acids, respectively) following the manufacturer’s instructions. The fermentation quotient (FQ) was determined as the molar ratio between lactic and acetic acids. Peptides concentration was determined by the o-phtaldialdehyde (OPA) method as described by Church et al. [36 (link)]. FAA were analyzed by a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a Li-cation-exchange column (20 by 0.46 cm inner diameter).