*3.1. Longer Fermentation Periods Are Important for the Highest Lactic Acid Production and the Lowest Sugar Content*

Although lactic acid production is of importance, the major aim of our research was to obtain a disinfecting agent with immediate application to spin-off as an easy, low-cost, technology. While most studies use short fermentation periods such as 24 h [33,39], in the present work we allowed the fermentation to last up to 120 h. The metabolite profile obtained throughout fermentation period is graphed in Figure 1, which shows the amounts of several metabolites: lactose, galactose, acetic and lactic acids, as well as the pH throughout the 120 h. Results show that although the pH dropped from 6.6 to 3.9 during the first 24 h of fermentation, lactic acid production continued to increase through the 120 h, supporting the need to use longer fermentation times, as opposed to the usual procedure in most studies. Lactic acid was the main acid detected, but acetic acid (0.89 g·L<sup>−</sup>1) was produced after 120 h. Starting from 30 g·L−<sup>1</sup> lactose in the unfermented whey, the starter led to a mass conversion rate of

lactose into lactic acid of 0.56, while residual lactose was 2.61 g·L<sup>−</sup>1. The yield of converting lactose into lactic acid was therefore higher in our work when compared to other works, such as Plessas et al. [45] who reported a 0.47 conversion rate. Also, the fact that lactose was reduced to concentrations lower than 3 g·L−<sup>1</sup> makes it suitable to be used in food products as a disinfectant without the problems of lactose intolerance [46].

**Figure 1.** Metabolite profile of whey during fermentation by mesophilic starter bacteria. Results reflect the average of three independent trials ± standard deviation.

Hence, overall results show that our fermentation protocol has the potential to reduce the environmental impact of whey by reducing its organic content.
