| Summary: | Cheese whey has been described as an environmental hazard due to its high organic content. Although it has been suggested that whey can be used as food disinfectant, it continues to pose an environmental problem because it still contains a high organic load. Here, we aimed to develop a low-cost, scalable fermentation protocol to produce a disinfectant from dairy waste that has very little organic content and high levels of lactic acid. Fermentation was achieved with industrial whey from ewe, goat, and cow’s milk, using a specific mesophilic-lactic acid bacteria starter mix over 120 h, which yielded the highest lactic acid production and the lowest lactose content. Antibacterial activity was observed against <i>Listeria monocytogenes</i>, <i>Salmonella</i> enterica, and <i>Escherichia coli</i> O157:H7, plus a total of thirteen other food pathogenic and spoilage strains, and antibacterial activities were determined to be highest after 120 h. We further validated this whey’s application as a disinfectant in shredded lettuce and compared its efficacy to that of chlorine, evaluating microbial quality, texture, color, and sensory perception, pH, and O<sub>2</sub> and CO<sub>2</sub> determinations. Results showed that not only was microbial quality better when using our whey solution (<i>p</i> < 0.05), but also the quality indicators for whey were statistically similar to those treated with chlorine. Hence, our work validates the use of an industrial waste whey as a low-cost, efficient, and environmentally safe disinfectant, with potential applications for minimally processed foodstuffs as an alternative to chlorine.
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