*3.1. Substrate Fermentation*

The time course of fermentation by yeast and lactobacilli is shown in Figure 1, as well as the pH trend. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 5 of 11

**Figure 1.** *Lactobacillus reuteri* (circle), *Saccharomyces cerevisiae* (diamond), and coliform (square) concentrations, reported as colony-forming unit (CFU) per g, and pH (triangle) values recorded during the fermentation. **Figure 1.** *Lactobacillus reuteri* (circle), *Saccharomyces cerevisiae* (diamond), and coliform (square) concentrations, reported as colony-forming unit (CFU) per g, and pH (triangle) values recorded during the fermentation.

The growth of *S. cerevisiae* was slow during the first 24 h of fermentation, maintaining a concentration of 108 CFU/g. The amount of *S. cerevisiae* reached a concentration of 1011 CFU/g after 72 h, remaining stable until the end of the process. The growth of *S. cerevisiae* was slow during the first 24 h of fermentation, maintaining a concentration of 10<sup>8</sup> CFU/g. The amount of *S. cerevisiae* reached a concentration of 10<sup>11</sup> CFU/g after 72 h, remaining stable until the end of the process.

*L. reuteri* increased constantly from the beginning of the fermentation until after 96 h, rising from 108 CFU/g up to 1012 CFU/g, reaching a steady state until the end of the process.

The reduction in pH was slow during the first 24 hours of fermentation because of microorganism adaptations at the beginning of the process [19,34]. In the presence of acid lactic bacteria and yeast, after 24 h the pH of the mixture became stable at 3.5 after 96 h. The decrease in pH in the substrate offers evidence of good acidification through lactic fermentation by the starter cultures and represents the most important factor to control in biotransformation. Acidification must be achieved as quickly as possible, in order to inhibit the growth of pathogenic and spoilage microorganisms in the substrate, increasing the shelf life of the resulting fermented substrate [10,35,36]. Moreover, considering that no sterilization procedures were carried out, the quick drop in pH was found to be necessary for maintaining microbial hygiene, along with retaining the quality of the

In fact, the amount of initial substrate total coliforms was 106 CFU/g, whereas no fecal coliforms were detected. The microbiological analysis for total coliform determination showed a net decrease during the fermentation, to reach a complete absence after 96 h

The reduction in coliform numbers could be due to some inhibitory compounds (bacteriocins) formed by the microorganisms employed during lactic acid fermentation and/or to the acidification of the medium [19]. Moreover, the decrease in coliforms may ensure good biopreservation against undesirable and/or hazardous microorganisms.

The final fermented products were low in spoilage microorganisms and rich in healthy microorganisms, representing a healthy final substrate enriched by added value. The starter cultures' capability of growing at low pH can be ascribed to the lemon peel supplementation since polysaccharides, such as pectins, show a protective effect on lactic acid bacteria LAB against low pH [38,39]. Their ability to achieve this on fermenting fish waste supplemented by lemon peel was confirmed by the protein level's increasing

for the resulting aquafeed.

product as an aquaculture feed [37].

(Figure 1).

*L. reuteri* increased constantly from the beginning of the fermentation until after 96 h, rising from 10<sup>8</sup> CFU/g up to 10<sup>12</sup> CFU/g, reaching a steady state until the end of the process. According to Giraffa et al. [17] and Hoseinifar et al. [18], this represents an added value for the resulting aquafeed.

The reduction in pH was slow during the first 24 hours of fermentation because of microorganism adaptations at the beginning of the process [19,34]. In the presence of acid lactic bacteria and yeast, after 24 h the pH of the mixture became stable at 3.5 after 96 h. The decrease in pH in the substrate offers evidence of good acidification through lactic fermentation by the starter cultures and represents the most important factor to control in biotransformation. Acidification must be achieved as quickly as possible, in order to inhibit the growth of pathogenic and spoilage microorganisms in the substrate, increasing the shelf life of the resulting fermented substrate [10,35,36]. Moreover, considering that no sterilization procedures were carried out, the quick drop in pH was found to be necessary for maintaining microbial hygiene, along with retaining the quality of the product as an aquaculture feed [37].

In fact, the amount of initial substrate total coliforms was 10<sup>6</sup> CFU/g, whereas no fecal coliforms were detected. The microbiological analysis for total coliform determination showed a net decrease during the fermentation, to reach a complete absence after 96 h (Figure 1).

The reduction in coliform numbers could be due to some inhibitory compounds (bacteriocins) formed by the microorganisms employed during lactic acid fermentation and/or to the acidification of the medium [19]. Moreover, the decrease in coliforms may ensure good biopreservation against undesirable and/or hazardous microorganisms.

The final fermented products were low in spoilage microorganisms and rich in healthy microorganisms, representing a healthy final substrate enriched by added value.

The starter cultures' capability of growing at low pH can be ascribed to the lemon peel supplementation since polysaccharides, such as pectins, show a protective effect on lactic acid bacteria LAB against low pH [38,39]. Their ability to achieve this on fermenting fish waste supplemented by lemon peel was confirmed by the protein level's increasing during the process, up to 48.55%, making these wastes an excellent raw material for aquafeed production with *Lactobacillus reuteri* and *Saccharomyces cerevisiae*.
