The increasing interest in natural preservatives has driven the search for effective microbial agents capable of controlling spoilage molds in cured meat products. In this study, the efficacy of Debaryomyces hansenii strains as biocontrol agents against spoilage molds in dry-cured meat products was evaluated through a dual experimental approach using both synthetic (PDA) and meat-derived media (LBM). While all D. hansenii strains demonstrated strong antifungal activity in nutrient-rich synthetic media, their performance in meat-like conditions was moderate to high, with significant differences depending on the mold species, the yeast strain, and their interaction with the culture medium. Our results highlight that antifungal efficacy is strongly influenced by the growth environment, underscoring the limitations of traditional in vitro assays that depend solely on synthetic media. Incorporating food-mimicking systems early in the screening process proved critical to identify strain–mold–medium combinations with the highest potential. These findings support the potential application of native D. hansenii strains as natural preservatives to enhance the safety and shelf life of dry-cured meats, emphasizing the importance of testing in conditions that closely resemble the target food environment to select the most effective biocontrol solutions.

The rejection of chemical preservatives reflects the growing demand for natural and safe products. This concern has spurred scientific interest in yeasts as biocontrol agents, given their antagonistic activity against undesired fungi, which is one of the main problems associated with preservative reduction. Debaryomyces hansenii is a non-conventional yeast that has shown great potential for inhibiting filamentous fungi in the food industry. This study investigated the role of nutrient competition in the biocontrol activity of D. hansenii against unwanted molds. Potentially pathogenic molds from spoiled food were isolated using different media and identified using Sanger sequencing. The inhibitory effects of different autochthonous D. hansenii strains under varying nutrient conditions were assessed against isolated molds using semipermeable membranes. Inhibition activity was measured by assessing mycelial expansion and spore production using image software analysis and classical cell counting using a Neubauer chamber. The results indicated that D. hansenii effectively inhibited mold growth and sporulation, with the autochthonous strains LR2 and SRF1 showing higher inhibitory activity than the control strain CBS767. The effectiveness of inhibition varied with the yeast–mold combination, highlighting the need for a species-specific analysis. Nutrient competition plays a complementary role in D. hansenii biocontrol but does not directly impact overall inhibition. This suggests that other mechanisms, such as direct cell interactions or metabolite production, may be crucial. These findings enhance our understanding of the potential of D. hansenii as a natural preservative and advance biocontrol methods for food safety.

Increasing consumer demand for natural and safe food products has led to the exploration of biocontrol alternatives to chemical preservatives, especially in the cured meat industry. The yeast Debaryomyces hansenii has emerged as a promising biocontrol candidate due to its antagonistic properties against spoilage fungi. This study assessed the impact of D. hansenii inoculation on the fungal community structure of Iberian cured pork loin using high-throughput sequencing of the ITS1 region. Ion Torrent ITS1 amplicon sequencing, QIIME2/DADA2 pipeline, and ALDEx2 differential abundance analysis were applied to this study. Pork loin samples inoculated with D. hansenii were compared to non-inoculated controls to evaluate changes in the fungal microbiome. Inoculation resulted in a marked decrease in fungal diversity and evenness, indicating strong competition by D. hansenii against native fungal populations. This effect was reflected in a significant reduction in alpha diversity in inoculated samples (Shannon, p = 0.0042; Pielou p = 0.0075; Gini–Simpson, p = 0.0081). Notably, genera associated with spoilage and mycotoxin production, particularly Aspergillus and Penicillium, were significantly reduced in inoculated samples. Simultaneously, D. hansenii became dominant, reducing other yeasts and filamentous fungi. These findings highlight the powerful competitive and biocontrol potential of D. hansenii, demonstrating its ability to improve microbial safety by potentially reducing mycotoxin-associated risks through the suppression of toxigenic genera. This is the first study to characterise the fungal community of Iberian pork loin using metabarcoding under D. hansenii inoculation. The findings confirm that the inoculation of D. hansenii can substantially reduce fungal contamination risks. Overall, the results contribute valuable insights into microbial interactions during meat curing and underscore the practical benefits of targeted starter cultures for enhancing food safety and quality.

The increasing interest in natural preservatives has driven the search for effective mi-crobial agents capable of controlling spoilage molds in cured meat products. In this study, the efficacy of Debaryomyces hansenii strains as biocontrol agents against spoilage molds in dry-cured meat products was evaluated through a dual experimental approach using both synthetic (PDA) and meat-derived media (LBM). While all D. hansenii strains demonstrated strong antifungal activity in nutrient-rich synthetic media, their perfor-mance in meat-like conditions was moderate to high, with significant differences de-pending on the mold species, the yeast strain, and their interaction with the culture medium. Our results highlight that antifungal efficacy is strongly influenced by the growth environment, underscoring the limitations of traditional in vitro assays that rely solely on synthetic media. Incorporating food-mimicking systems early in the screening process proved critical to identify strain–mold–medium combinations with the highest potential. These findings support the potential application of native D. hansenii strains as natural preservatives to enhance the safety and shelf life of dry-cured meats, em-phasizing the importance of testing in conditions that closely resemble the target food environment to select the most effective biocontrol solutions.