2.1.2. Bioprocessing

Bioprocessed wasted bread (bWB) was prepared by mixing ground bread (35%), distilled water (65%), amylase Veron® Mac at the concentration recommended by the manufacturer (3 mg/100 g), and the selected LAB strain at the final cell density of ca. 7 log cfu/g. The biomass was then incubated at 30 ◦C for 24 h, characterized, and used in pot trials. WB was also characterized and used as a control in all the experiments.

### 2.1.3. Characterization of Wasted Bread Biomasses

The pH of the biomasses 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 the AACC method 02–31.01 [13] and expressed as the amount (mL) of 0.1 M NaOH necessary to reach pH of 8.4.

Presumptive LAB were enumerated, before and after fermentation, using MRS agar medium (Oxoid) supplemented with cycloheximide (0.1 g/L). Plates were incubated at 30 ◦C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid). WB and bWB were also characterized for the presence of yeasts, molds, and *Enterobacteriaceae*. Yeasts and molds were cultivated on Yeast Peptone Dextrose Agar medium (Sigma-Merck, Darmstadt, Germany), supplemented with 0.01% chloramphenicol, through pour and spread plate enumeration, respectively, and incubated at 25 ◦C whereas *Enterobacteriaceae* were determined on Violet Red Bile Glucose Agar (Oxoid) at 37 ◦C for 24 h.

Water/salt-soluble extracts (WSE) from wasted bread biomasses were prepared according to the method originally described by Osborne and modified by Weiss et al. [14] using 50 mM Tris–HCl (pH 8.8). After centrifugation, the supernatants were used to determine sugars, organic acids, peptides, and total free amino acid (TFAA) concentration.

Glucose and maltose were measured using the D-Fructose D-Glucose Assay Kit K-FRUGL and the Maltose-Sucrose-D-Glucose Assay Kit K-MASUG (Megazyme International Ireland Limited, Bray, Ireland), respectively, following the manufacturer's instructions.

Organic acids were quantified 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), as described by Rizzello et al. [15].

For the analysis of peptides, WSE were treated with trifluoroacetic acid (0.05% *wt*/*vol*), centrifuged (10.000× *g* for 10 min), and subjected to dialysis (cut-off 500 Da) to remove proteins and free amino acids, respectively. Then, peptide concentration was determined by the *o*-phtaldialdehyde method as described by Church et al. [16], and dialysates were analyzed through Reversed-Phase Fast Performance Liquid Chromatography (RP-FPLC) using a Resource RPC column and ÄKTA FPLC equipment with the UV detector operating at 214 nm (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as described by Rizzello et al. [15]. TFAA was analyzed by a Biochrom 30+ series Automatic Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom), equipped with a Li-cation-exchange column (4.6 × 200 mm internal diameter) [17].

WB and bWB were analyzed for moisture, ash content, pH, and electrical conductivity (EC) according to the methods previously proposed by Trinchera et al. [18]. In detail, the moisture, expressed as a percentage of the initial weight, was determined by drying samples at 105 ◦C overnight; the ash content, expressed as a percentage of the dry matter, was determined by combustion in a Controls 10-D1418/A muffle furnace at 550 ◦C for 12 h. The EC was measured using a Hanna Edge® EC instrument on sample/water extracts (1:10 *w*/*v*) after shaking for 30 min. Total N (TN) content was determined by the Kjeldahl method, while the organic carbon (OC) content was determined by dichromate oxidation and subsequent titration with ferrous sulfate according to Ciavatta et al. [19]. This method is suitable for samples characterized by high OC levels. The total P content was measured spectrophotometrically at 650 nm after incinerating biomass samples at 550 ◦C, suspending ashes in 10% hydrochloric acid solution, and developing the blue color in the filtered solution according to the Olsen method [20].
