*3.2. Antibacterial Activity Is Also Dependent on the Length of Fermentation*

Although several works have already shown that fermented whey can be used as an antibacterial agent in minimally processed salads, being able to reduce overall microorganism growth [8,40], most studies also use fermentation periods around 24 to 72 h [33,39]. Since lactic acid production was higher in longer fermentation periods, we set out to search the best timing along fermentation for higher antibacterial activity. Hence, whey samples were collected daily throughout the 120 h, and firstly tested against reference Gram-positive *Listeria monocytogenes* 4b and Gram-negative *Escherichia coli* O157:H7, and MIC were determined for each day (Table 1). Results show that our fermented whey reduced the growth of both bacteria, from day 1 in *Escherichia coli* O157:H7 and from day 4 in *L. monocytogenes* 4b, concomitantly to the increase in lactic acid produced (Figure 1), respectively, of approximately 5 and 15 g·L<sup>−</sup>1. However, more importantly, activities were significantly higher on the final day of fermentation, where MICs were more significantly reduced. Thus, our results indicate that fermentation periods longer than 72 h should be preferred in this type of study to obtain the best antibacterial activities.

**Table 1.** Minimum inhibitory concentrations (MIC) of fermented whey to control two selected bacterial strains \*.


Note: \* NI = No inhibition observed. Figures are averages of three independent experiments, expressed in % (*v*/*v*) of whey diluted in growth media.
