Francisco Javier Ruiz-Castilla

Exponential population growth continues to intensify pressure on global food systems, reinforcing the need for sustainable protein-rich alternatives. Among the most promising emerging alternatives are edible insects, lab-grown meat, and single-cell protein, the latter often facilitated by fungi. In this context, filamentous fungi (FF), which have long been central to enzyme production and food fermentation, are now leveraged in increasingly diverse and innovative ways. Therefore, the objective of this review is to (i) assess the current status and primary applications of major FF genera within the European context by analyzing safety evaluations conducted by the European Food Safety Authority (EFSA), and (ii) provide an updated overview of novel FF-based uses in the agri-food sector. EFSA data show that Aspergillus and Trichoderma account for around 70% of FF-related safety notifications, mainly linked to food enzyme production and zootechnical additives (digestibility and performance enhancers in animal nutrition), respectively. Evaluations from 2020 to 2025 highlight hydrolases (EC 3), particularly glycosylases (EC 3.2) and esterases (EC 3.1), as key enzyme classes produced by Aspergillus spp. mainly associated with established food processing applications. Beyond these long-established applications, FF are increasingly explored in novel agri-food uses, including mycoprotein-based meat alternatives, functional ingredients for bakery products, and nutrient-rich fermented beverages. Moreover, species such as A. niger , T. reesei , Penicillium spp., and Fusarium spp. are increasingly employed in the biosynthesis of high-value aromatic compounds. The economic significance of FF-based innovations is reflected in an estimated market impact exceeding 2,140 billion USD, positioning FF as scalable and multifunctional agents that integrate sustainability, nutrition, and technological advancements.

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.

Debaryomyces hansenii es una levadura halotolerante y xerotolerante perteneciente al filo Ascomycota (familia Debaryomycetaceae). Es, de hecho, considerada como modelo eucariota para el estudio de la adaptación a ambientes hipersalinos. Su extraordinaria capacidad para crecer en condiciones de estrés osmótico, oxidativo y de pH, junto a su carácter no patógenico y su estatus GRAS/QPS (Generally Recognized As Safe/Qualified Presumption of Safety), la han situado como un microorganismo de referencia en biotecnología alimentaria, ambiental y agrícola. En la última década, la aparición y mejora de las tecnologías “ómicas” y bioprocesos controlados ha renovado el interés por esta levadura no convencional, revelando mecanismos moleculares de adaptación únicos y un potencial aún no explotado en el contexto de la biotecnología.

Iron (Fe) is an essential micronutrient for crop productivity, but its low availability in alkaline and calcareous soils limits the growth of rice (Oryza sativa L.), which employs a combined strategy for its acquisition based on the release of phytosiderophores (PS) and the use of specific transporters. In this study, the effect of the rhizospheric bacterium Pseudomonas simiae WCS417 and the halotolerant yeast Debaryomyces hansenii CBS767 as inducers of responses to Fe deficiency in rice grown under hydroponic conditions was evaluated. Plants were inoculated in nutrient solutions with and without Fe, and PS production and the expression of genes associated with biosynthesis and transport were determined by qRT-PCR. The results showed that both microorganisms significantly increased PS production compared to controls, especially under Fe-deficient conditions, although P. simiae also exerted an effect under Fe sufficiency. Furthermore, induction of key genes (OsNAAT, OsIRO2, OsTOM1, OsYSL15, and OsIRT1), as well as genes related to the ethylene pathway (OsEIN2, OsACS2, and OsACO3), was observed, pointing to a regulatory role for this hormone in the response. In conclusion, P. simiae and D. hansenii act as inducers of Fe acquisition mechanisms in rice, offering a sustainable biotechnological approach to improve iron nutrition in limiting environments.

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 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.

Calcareous soils, characterized by high pH and calcium carbonate content, often limit the availability of essential nutrients for crops such as rice (Oryza sativa L.), reducing yield and nutritional quality. In this study, we evaluated the effect of the halotolerant yeast Debaryomyces hansenii on the growth, nutrient uptake, and phosphorus acquisition mechanisms of rice plants cultivated in calcareous soil under controlled greenhouse conditions. Plants inoculated with D. hansenii, particularly via root immersion, exhibited significantly higher SPAD chlorophyll index, plant height, and grain yield compared to controls. A modest increase (~4%) in dry matter content was also observed under sterilized soil conditions. Foliar concentrations of Fe, Zn, and Mn significantly increased in plants inoculated with D. hansenii via root immersion in non-sterilized calcareous soil, indicating improved micronutrient acquisition under these specific conditions. Although leaf phosphorus levels were not significantly increased, D. hansenii stimulated acid phosphatase activity, as visually observed through BCIP staining, and upregulated genes involved in phosphorus acquisition under both P-sufficient and P-deficient conditions. At the molecular level, D. hansenii upregulated the expression of acid phosphatase genes (OsPAP3, OsPAP9) and a phosphate transporter gene (OsPTH1;6), confirming its influence on P-related physiological responses. These findings demonstrate that D. hansenii functions as a plant growth-promoting yeast (PGPY) and may serve as a promising biofertilizer for improving rice productivity and nutrient efficiency in calcareous soils, contributing to sustainable agricultural practices in calcareous soils and other nutrient-limiting environments.

Calcareous soils, characterized by high pH and calcium carbonate content, often limit the availability of essential nutrients for crops such as rice (Oryza sativa L.), reducing yield and nutritional quality. In this study, we evaluated the effect of the halotolerant yeast Debaryomyces hansenii (Dh) on the growth, nutrient uptake, and phosphorus acquisition mechanisms of rice plants cultivated in calcareous soil under controlled greenhouse conditions. Plants inoculated with Dh, particularly via root immersion, exhibited significantly higher SPAD chlorophyll index, plant height, dry matter content, and grain yield compared to controls. Foliar concentrations of Cu, Fe, Zn, and Mn also increased under certain conditions, suggesting improved micronutrient acquisition. Although phosphorus levels in leaves were not significantly enhanced, Dh stimulated acid phosphatase activity in both P-sufficient and P-deficient treatments. At the molecular level, Dh upregulated the expression of acid phosphatase genes (OsPAP3, OsPAP9) and a phosphate transporter gene (OsPTH1;6), confirming its influence on P-related physiological responses. These findings demonstrate that D. hansenii functions as a plant growth-promoting yeast (PGPY) and may serve as a promising biofertilizer for improving rice productivity and nutrient efficiency in calcareous soils, contributing to sustainable agricultural practices in challenging edaphic environments.

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.

Iron (Fe) deficiency is among the most important agronomical concerns under alkaline conditions. Bicarbonate is considered an important factor causing Fe deficiency in dicot plants, mainly on calcareous soils. Current production systems are based on the use of high-yielding varieties and the application of large quantities of agrochemicals, which can cause major environmental problems. The use of beneficial rhizosphere microorganisms is considered a relevant sustainable alternative to synthetic fertilizers. The main purpose of this work has been to analyze the impact of the inoculation of tomato (Solanum lycopersicum L.) seedlings with the WCS417 strain of Pseudomonas simiae, in the presence or absence of bicarbonate, on plant growth and other physiological parameters. To conduct this research, three different inoculation methods were implemented: root immersion, foliar application, and substrate inoculation by irrigation. The results obtained show the ability of the P. simiae WCS417 strain to induce medium acidification in the presence of bicarbonate to increase the SPAD index and to improve the growth and development of the tomato plants in calcareous conditions provoked by the presence of bicarbonate, which indicates that this bacteria strain could have a great potential as an Fe biofertilizer.