A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products. IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of “good bacteria” to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.

Acidification and nutrient depletion by dairy starter cultures is often sufficient to prevent outgrowth of pathogens during post-processing of cultured dairy products. In the case of cottage cheese, however, the addition of cream dressing to the curd and subsequent cooling procedures can create environments that may be hospitable for the growth of Listeria monocytogenes. We report on a non-bacterio-cinogenic Lacticaseibacillus rhamnosus strain that severely limits the growth potential of L. monocytogenes in creamed cottage cheese. The main mechanism underlying Listeria spp. inhibition was found to be caused by depletion of manganese (Mn), thus through competitive exclusion of a trace element essential for the growth of many microorganisms. Growth of Streptococcus thermophilus and Lactococcus lactis that constitute the starter culture, on the other hand, were not influenced by reduced Mn levels. Addition of L. rhamnosus with Mn-based bioprotective properties during cottage cheese production therefore offers a solution to inhibit undesired bacteria in a bacteriocin-independent fashion.