| Summary: | In order to obtain a high-protein-content supplement for aquaculture feeds, rich in healthy microorganisms, in this study, <i>Saccharomyces cerevisiae</i> American Type Culture Collection (ATCC) 4126 and <i>Lactobacillus reuteri</i> ATCC 53608 strains were used as starters for fermenting fish waste supplemented with lemon peel as a prebiotic source and filler. Fermentation tests were carried out for 120 h until no further growth of the selected microorganisms was observed and the pH value became stable. All the samples were tested for proteins, crude lipids, and ash determination, and submitted for fatty acid analysis. Moreover, microbiological analyses for coliform bacteria identification were carried out. At the end of the fermentation period, the substrate reached a concentration in protein and in crude lipids of 48.55 ± 1.15% and 15.25 ± 0.80%, respectively, representing adequate levels for the resulting aquafeed, whereas the ash percentage was 0.66 ± 0.03. The main fatty acids detected were palmitic, oleic, and linoleic acids. Saturated fatty acids concentration was not affected by the fermentation process, whereas monounsaturated and polyunsaturated ones showed an opposite trend, increasing and decreasing, respectively, during the process. Coliform bacteria were not detected in the media at the end of the fermentation, whereas the amount of <i>S. cerevisiae</i> and <i>L. reuteri</i> were around 10<sup>11</sup> and 10<sup>12</sup> cells per g, respectively.
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