*3.1. Amendment Characterization*

In bWB, the initial cell density of presumptive LAB corresponded to the targeted inoculum and increased after 24 h of fermentation approximately 2 log cycles, reaching 9.35 ± 0.36 log cfu g<sup>−</sup>1.

The amendments were also characterized for the presence of yeasts, molds, and *Enterobacteriaceae*. Yeasts and molds in WB were 3.8 ± 0.1 and 2.9 ± 0.0 log cfu g<sup>−</sup>1, respectively, whereas *Enterobacteriaceae* were 4.1 ± 0.2 log cfu g<sup>−</sup>1. After bioprocessing, all microbial species investigated were in a notably lower range, compared to WB. Yeasts and molds remained below 2 log cfu g<sup>−</sup>1, whereas *Enterobacteriaceae* were not detected.

Relevant acidification was obtained after fermentation. The pH decreased from 5.92 ± 0.09 of WB to 3.89 ± 0.16 of bWB, with a production of 56 and 2 mmol kg−<sup>1</sup> of lactic and acetic acid, respectively, which were detected in traces in unprocessed WB. As expected, the TTA value was significantly higher in bWB (8.46 ± 0.48 mL) compared to WB (1.54 ± 0.08 mL). Glucose and maltose, present in small amounts in WB (0.95 and 2.79 mg g<sup>−</sup>1, respectively), were also found at higher concentrations in bWB (12.87 and 7.83 mg g<sup>−</sup>1, respectively).

Peptide content in WB was 101.36 ± 4.29 mg g<sup>−</sup><sup>1</sup> and increased by ca. 38% during fermentation. This trend was confirmed by the FPLC chromatograms, where although no differences among peak area/total area ratios were observed at different percentages of acetonitrile, the total area and the number of detected peaks increased by roughly 20% in bWB compared to WB. On the contrary, TFAA slightly but significantly (*p* < 0.05) decreased after fermentation (ca. 700 mg g<sup>−</sup>1). Nevertheless, γ-aminobutyric acid (GABA) content was higher in bWB (149.68 ± 1.36 mg g<sup>−</sup>1) compared to WB (116.39 ± 3.12 mg g<sup>−</sup>1).

WB bioprocessing slightly increased the OC content (7%) compared to unprocessed WB (Table 1). In contrast, WB biomass showed significantly higher EC and TN content than bWB, up to 6% and 9%, respectively. Accordingly, the C/N ratio was significantly higher in bWB than WB. Finally, even though bWB resulted in a numerically higher total P content (2150 ± 15 mg kg−1) compared to WB (1716 ± 246 mg kg−1), they were not significantly different.


**Table 1.** Chemical and physicochemical characteristics of the amendment. WB wasted bread; bWB, bioprocessed wasted bread (treated with amylase and fermented with *Lactiplantibacillus plantarum* H64).

Data are the means of three independent experiments ± standard deviations (*n* = 3). a–b Values in the same column followed by a different letter are significantly different according to the HSD test or Dunn test (¥). \* Significant at *p* ≤ 0.05; \*\* Significant at *p* ≤ 0.01, ns: not significant.
