Applied Microbiology

Cheddar cheese ripening creates favorable conditions for desired microbial changes but also allows survival and outgrowth of spores like Clostridium tyrobutyricum and Bacillus licheniformis, leading to late-blowing defects. In the first phase of the study, NSLAB dynamics were evaluated in the presence of spores, where pilot-scale cheeses (110 L) were produced in four treatments: control, T1 (BL), T2 (CT), and T3 (BL+CT), each inoculated at 2.0 Log₁₀ CFU/mL with spores. Results showed that SLAB declined from 8.0 to 0.2 Log₁₀ CFU/g, while NSLAB increased from 2.0 to 8.5 Log₁₀ CFU/g by the third month and remained stable thereafter. Spore counts reached 2.94 ± 0.02 (T2) and 2.48 ± 0.03 (T3) Log₁₀ CFU/g, with visible spoilage signs appearing after five months, indicating that native NSLAB populations were inadequate to control late-blowing defects. In this study, the effect of soluble fiber (inulin) in stimulating NSLAB was evaluated by incorporating 1% inulin into Cheddar cheese across four treatments: T1 (C SF), T2 (BL SF), T3 (CT SF), and T4 (BL+CT SF). Inulin addition resulted in significantly higher NSLAB counts (>10.5 Log₁₀ CFU/g) and suppressed spore levels (<0.91 ± 0.03 Log₁₀ CFU/g), with no spoilage observed. Inulin addition selectively enhanced beneficial NSLAB, suppressing spore-forming bacteria and preventing late-blowing defects without affecting cheese quality. This provides a natural, sustainable strategy to enhance microbial safety during Cheddar cheese ripening. Full article

The genus Tilia (Malvaceae) comprises long-lived broadleaf trees of considerable ecological, cultural, and historical importance in temperate Europe and Asia. Among these, Tilia × europaea L. (common European linden) is a key native species in Central and Northern Europe, with individuals documented to live for several centuries. While the phyllosphere and soil-associated microbiomes of linden have been studied, the internal fungal communities inhabiting ancient trees remain poorly understood. In this study, the complete mycobiome of linden tree wood was analyzed. Wood-inhabiting fungi (the wood mycobiome) include endophytes, saprotrophs, and potential pathogens that can strongly influence host vitality and ecosystem processes. Advances in high-throughput amplicon sequencing (HTAS) now provide unprecedented opportunities to characterize these hidden communities. In this study, we investigated the trunk wood mycobiome of an ancient T. × europaea L. individual using a culture-independent HTAS approach. The results reveal a diverse fungal assemblage, including taxa like Arthinium or Phialemonium not previously reported from living linden wood, and highlight potential implications for tree health and longevity. This work provides a first baseline characterization of the internal mycobiome of the ancient Tilia tree and contributes to broader efforts to conserve its biological and cultural value. Full article

Fructophilic lactic acid bacteria (FLAB), Apilactobacillus kunkeei strains AG8 and AG9 were selected in the current study for in-depth analysis. Cultivation on fructose yeast peptone (FYP) medium with varying fructose concentrations (1%, 10%, and 30%) revealed that higher fructose levels promoted acetate production over lactate, confirming a heterofermentative metabolic profile. Ethanol production was negligible, consistent with the absence of alcohol dehydrogenase (ADH) activity. Enzyme assays showed fructokinase activity doubled at 30% fructose, while acetate kinase activity increased and L-lactate dehydrogenase activity decreased. This shift in enzyme ratios from 1:1 at 1% fructose to 10:1 or 15:1 at higher concentrations explains the metabolic preference for acetate. Apb. kunkeei is an obligate FLAB, growing poorly on glucose unless supplemented with external electron acceptors like pyruvate or oxygen. It lacks ADH, but retains acetaldehyde dehydrogenase (ALDH), enabling acetate production and additional ATP generation, enhancing biomass yield. The absence of the adhE gene contributes to NAD+/NADH imbalance and favors acetate production. Gene expression studies targeting fructose transport enzymes showed elevated expression of ABC transporters and carbohydrate metabolism genes in response to fructose. ADH expression remained low across sugar concentrations. Fructokinase gene expression was shown to be strain specific. Neither strain expressed the ABC transporter ATP-binding protein gene on glucose, nor the bacteriocin ABC transporter gene, correlating with the absence of antibacterial activity. These findings underscore the metabolic specialization of Apb. kunkeei, its reliance on fructose, and the role of ABC transporters in optimizing fermentation. The strain-specific gene expression and metabolic flexibility highlight its potential as a probiotic and feed additive in apiculture and biotechnology. Full article

The microbiome of howler monkeys is being studied as a potential indicator of forest health. This explorative research aimed to analyze the microbiome, antibiotic resistance genes, and virulence factors of the howler monkey (Alouatta seniculus) in two Colombian Andean forests. A total of six samples were collected from three monkeys in two different forests. The samples were processed and sequenced using 16S rRNA V3-V4 metabarcoding and shotgun metagenomics. No significant differences in microbial diversity were observed between locations. A total of 43 possible resistance genes were identified, 11 of which were associated with plasmids, while 66 virulence genes were detected. The bacterial genera with the highest number of resistance genes were Escherichia and Enterococcus, whereas Escherichia and Citrobacter exhibited the highest number of virulence factors. The bacteria were predominantly resistant to fluoroquinolones, macrolides and beta-lactams, while adherence was the dominant virulence mechanism. This exploratory study suggests that the locations provide similar habitats for howler monkeys and that the presence of resistance genes is primarily due to intrinsic bacterial resistance mechanisms and natural resistance in wild populations despite the environmental presence of bacterial genera with resistance genes and virulence factors. However, acquisition through interaction with domestic animals was not evaluated. Full article

Microorganisms such as methanogenic archaea play a key role in wastewater treatment plants (WWTPs) by breaking down organic matter and pollutants and producing methane, a potential renewable energy source. However, sulphate-reducing bacteria (SRB) compete with archaea for the same substrates under anaerobic conditions, lowering methane production and generating harmful hydrogen sulphide (H₂S). Inhibiting SRB is therefore crucial to enhance methane yield and reduce toxic by-products. By means of manual screening of public databases (KEGG, BRENDA, PDB, PubChem) 12 potential inhibitors of SRB were found. After computational ecotoxicological assessment, four candidates were selected, and one of them experimentally increased methane production, demonstrating that SRB inhibition favours the anaerobic digestion of sludges. In order to further explore new candidates, Quantitative Structure–Activity Relationship (QSAR) models were developed showing reliable predictive performance. These models enabled the virtual screening of COCONUT, a natural product database, identifying 73 potential SRB inhibitors. After an ecotoxicological assessment, five commercially available compounds remained. The identified candidates may reduce competition between SRB and methanogenic archaea, leading to higher methane production and supporting WWTPs in generating their own biogas. This would contribute to a circular economy and help mitigate greenhouse gas emissions. Full article

Bioleaching represents a sustainable and economically viable method for recovering metals from sulfide ores. This study evaluates the bioleaching potential of eight native mesophilic strains isolated from chalcopyrite ore sourced from the Toquepala mine in Peru. The strains were molecularly and phylogenetically identified as Leptospirillum ferriphilum based on 16S rRNA gene sequencing. Bioleaching process was evaluated over 648 h in triplicate flask assays at 30 °C and 120 rpm. All strains demonstrated leaching activity, achieving an average copper recovery above 30%. Notably, strains M1D and M3E demonstrated superior performance, with iron oxidation reaching approximately 50%, copper recoveries of 34.78% and 32.61%, respectively, and peak cell counts of up to 2.45 × 10⁹ cells/mL. Specific growth rates were determined as 0.03469 h⁻¹ for M1D and 0.03651 h⁻¹ for M3E. A positive correlation was observed among microbial growth, iron oxidation, and copper recovery. These results confirm the potential of native mesophilic L. ferriphilum strains as efficient agents for chalcopyrite bioleaching at moderate temperatures, supporting their application in biotechnological metal recovery processes. Full article

This study explored the potential of bacterial consortia to remediate real diesel-contaminated agricultural soils. Two consortia were tested: a native consortium isolated from contaminated soil and an exogenous consortium derived from vermicompost. Bacterial communities (consortia and soils) were characterized through high-throughput sequencing. Within 30 days, total petroleum hydrocarbons (TPH) were removed most efficiently by bioaugmentation with the native consortium (53.32%), followed by the exogenous vermicompost consortium (47.14%) and the indigenous microbiota (42.52%). Gas chromatography confirmed the reduction of polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings; however, terphenyl, chrysene, and pyrene persisted. The highest TPH biodegradation rate was observed in the treatment inoculated with the native consortium (208.5 mg/kg per day), followed by the treatment with indigenous microbiota (181.8 mg/kg per day) and the exogenous consortium (161.9 mg/kg per day). Furthermore, hydrocarbon-degrading bacterial populations increased significantly during the first week but declined after day 21, showing a negative correlation with TPH concentrations across all treatments, indicating that the highest bacterial activity and degradation occurred during the first 14 days. Taxonomic analysis identified Actinobacteria as the most abundant phylum in the initial soil, whereas Proteobacteria dominated both the consortia and the bioremediated soils. Significant differences in community structure and composition were observed between the consortia according to their origin, influencing removal efficiency. Dominant genera shifted from Nocardioides and Streptomyces in untreated soil to Pseudomonas, Sphingobium, and Pseudoxanthomonas following biological treatments, while Nocardia, Rhodococcus, and Bacillus remained nearly constant. These findings underscore the effectiveness of adapted bacterial consortia in restoring real diesel-contaminated agricultural soils and highlight potential microbial succession patterns associated with biodegradation and soil ecological recovery. Full article

Autochthonous enterococci surviving mild thermization of raw milk (RM) before traditional Greek cheese processing may simultaneously comprise safe and virulent thermoduric strains with multiple antibiotic resistances (ARs). Therefore, this study biotyped and then compared the ARs of 60 Enterococcus isolates from two antilisterial sheep milks of native Epirus breeds before (RM) and after (TM) thermization at 65 °C for 30 s; the RM isolates were previously genotyped and evaluated for primary safety traits, namely, hemolytic activity, vanA/vanB, cytolysin, and virulence genes, by molecular methods. Biochemically typical and atypical strains of Enterococcus faecium (six biotypes), E. durans (five biotypes), E. faecalis (two biotypes), and E. hirae (one biotype), which were subdominant to other LAB species in RM (19 isolates), prevailed in TM (41 isolates). E. faecium biotypes 1A, 1D, and 1H included multiple-Ent+ (entA/entB/entP or entA/entB) strains with strong antilisterial CFS activity, whereas E. faecium 1X (entA), E. durans 2A, 2B, 2C, and 2X (entA/entP or entP), E. faecalis 3B, and E. hirae 4A (entA) biotypes displayed direct in vitro antilisterial activity only. Biotypes 1D, 1X, and 2A were selected in TM. All E. faecium/durans isolates were susceptible to vancomycin, but the m-Ent + E. faecium biotype 1A and 1D strains were resistant to penicillin, erythromycin, ciprofloxacin, and ampicillin. In contrast, all biotype 1X isolates were susceptible to all antibiotics tested. All E. faecalis and most E. durans isolates were resistant to penicillin but susceptible to erythromycin and ciprofloxacin. Biotype 2X isolates and one virulent (ace; gelE) E. faecalis isolate from RM were tetracycline-resistant. A sporadic RM isolate of E. hirae that was resistant to penicillin and vancomycin was not retrieved from the counterpart TM, but the inclusion of three vancomycin-resistant isolates from TM in the primary biotype 3B of E. faecalis was a cause for concern. In conclusion, based on the results, antibiotic-susceptible representatives of all strain biotypes of the E. faecium/durans group, as well as antagonistic m-Ent+ E. faecium strains from sheep milk that were susceptible to vancomycin and ampicillin and lacking virulence genes, can be included in safe complex natural starters to be developed for onsite use in traditional Greek hard cheese technologies. Full article

The use of lactose-utilizing microalgae offers a sustainable and cost-effective approach for the bioconversion of dairy industry side-streams and the reduction in microalgae production costs. This work aims to improve the biomass productivity of the lactose-utilizing microalgal strain Graesiella emersonii MSCL 1718 in concentrated cheese whey permeate. It was demonstrated that the mixotrophic growth of the axenic G. emersonii culture resulted in a significantly higher biomass productivity in 20% permeate medium compared to the heterotrophic cultivation. Furthermore, supplementation of the permeate medium with iron, zinc, cobalt, and molybdenum resulted in 12.8%, 12.9%, 9.3%, and 28.9% significant increases (p < 0.05) in biomass synthesis, respectively, compared to the control permeate group. In the subsequent experiment, G. emersonii cultivated in molybdenum-supplemented permeate resulted in 0.34 ± 0.02 g/(L·d) biomass productivity and twofold higher lipid content (30.21 ± 1.29%) compared to the photoautotrophic control in defined synthetic medium. Analysis of the fatty acid composition revealed a twofold increase in saturated fatty acids, reaching 62.16% under mixotrophic cultivation in permeate, compared with the photoautotrophic control. Overall, concentrated cheese permeate proved to be a suitable medium for G. emersonii biomass production, supporting both enhanced growth and increased lipid accumulation. Full article

Soil-borne plant pathogens pose a serious threat to global food security by causing extensive yield losses and compromising crop quality. Conventional chemical-based control methods often prove inadequate, environmentally harmful, and disruptive to beneficial soil microbiota, highlighting the urgent need for sustainable alternatives. Plant growth-promoting fungi (PGPF) have emerged as effective biocontrol agents capable of suppressing diverse soil-borne pathogens while simultaneously enhancing plant growth and resilience. This review synthesizes current knowledge on the tripartite interactions among plants, pathogens, and PGPF within the rhizosphere, with emphasis on their roles in disease suppression, rhizosphere competence, and plant health promotion. The findings highlight that PGPF such as Trichoderma, Penicillium, Aspergillus, non-pathogenic Fusarium, hypovirulent binucleate Rhizoctonia and sterile fungi can significantly reduce diseases caused by fungi, oomycetes, bacteria, nematodes, and protists through mechanisms including antibiosis, hyperparasitism, competition, and induction of systemic resistance. Evidence also indicates that consortium approaches and bioformulations enhance field efficacy compared to single-strain applications. Despite this progress, challenges such as variability in field performance, limited shelf life of inoculants, and gaps in understanding ecological interactions constrain large-scale use. Overall, the review underscores that PGPF-based strategies represent a promising and sustainable alternative to chemical pesticides, with strong potential for integration into holistic crop disease management under changing climatic conditions. Full article

Diabetic foot ulcers (DFUs) and other chronic wounds are major global health challenges, often complicated by infections and delayed healing due to excessive collagen accumulation. Microbial collagenases offer an enzymatic alternative to surgical debridement by selectively degrading collagen and potentially limiting microbial colonization. In this study, an isolated and characterized thermostable collagenase from Streptomyces scabies from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, is investigated. Identification was confirmed via 16S rRNA sequencing, and enzyme production was optimized on gelatin agar. Partial purification was achieved through ammonium sulfate precipitation and dialysis, and molecular weight (~25 kDa) was determined by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Activity was assessed under varying temperatures, pH, substrates, and metal ions, while antibacterial potential was tested against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The collagenase exhibited optimal activity at 80 °C and pH 9, stability under thermophilic and alkaline conditions, activation by Fe²⁺, and notable antibacterial effects at higher concentrations. These results demonstrate that S. scabies collagenase exhibits selective antibacterial activity in vitro, suggesting its potential as an enzymatic tool for further evaluation in diabetic foot debridement and infection control. Full article

This study reports the biosynthesis of selenium nanoparticles (Se-NPs) using four newly isolated strains of lactic acid bacteria, molecularly identified as Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Lactiplantibacillus plantarum, and Lactobacillus acidophilus. The synthesized Se-NPs were characterized using Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and UV-Vis Spectroscopy, and zeta potential analysis. The result revealed that their size ranged from 16 nm to 90 nm with favorable stability and purity. The Se-NPs exhibited significant antimicrobial and antibiofilm activities against certain Gram-positive, Gram-negative bacteria, and Candida albicans, particularly those produced by isolate S4, which showed the lowest MIC values and highest biofilm inhibition. Furthermore, MTT assays revealed selective cytotoxicity against the A549 cancerous lung cell line, with minimal toxicity toward normal Wi38 cells. These findings suggest that biosynthesized Se-NPs are a promising, biocompatible candidate for combating antibiotic-resistant pathogens and biofilm-associated infections. Full article

This study investigates the range of secondary metabolites with antifungal activity produced by bacterial strains that have been previously characterised as antagonistic strains to phytopathogenic fungi. The properties of ten strains were analyzed in silico through functional genomic analysis and in vitro by mass spectrometry. The research has demonstrated that Bacillus and Paenibacillus strains possess diverse gene clusters for biosynthesis of secondary metabolites, including peptides and polyketides. Mass spectrometry analysis confirmed the synthesis of key antimicrobial peptides, such as surfactin, fengycin, macrolactin, and fusaricidin, which are associated with antifungal activity. These strains have been observed to exhibit robust antagonism against phytopathogenic fungi—the diameter of the inhibition zones for the presented strains was at least 20 mm. This feature highlights their potential as biocontrol agents. The presence of numerous uncharacterized gene clusters suggests the possibility of discovering previously undescribed bioactive compounds. Full article

Listeria monocytogenes and Salmonella enterica Typhimurium are leading causes of foodborne illness in the United States and frequently implicated in produce outbreaks. Conventional decontamination methods, such as cold-water washes with chlorine, have limited antibacterial efficacy and environmental sustainability. Power ultrasound has emerged as a promising non-thermal alternative, but the molecular mechanisms remain insufficiently elucidated. This study evaluated transcriptomic responses of L. monocytogenes and S. enterica Typhimurium to (i) ultrasound (20 kHz), (ii) chlorine (50 ppm), and (iii) combined ultrasound + chlorine treatments. RNA-seq analysis identified differentially expressed genes, as well as enriched Gene Ontology and KEGG terms. Results showed that ultrasound and chlorine triggered distinct transcriptomic responses. L. monocytogenes exhibited broad transcriptional shifts under ultrasound, including significant upregulation of phosphotransferase system components and central metabolism. Chlorine alone induced a narrower response, with fewer differentially expressed genes clustering into limited functional categories. In contrast, the combined ultrasound + chlorine treatment elicited the strongest response in S. enterica Typhimurium, with enrichment of multiple energy- and metabolism-related pathways, including the citrate cycle, carbon metabolism, and microbial metabolism in diverse environments. These findings provide new insights into ultrasound-triggered responses in foodborne pathogens and may inform development of optimized ultrasound-based hurdle sanitization strategies for produce safety. Full article

CRISPR-Cas systems are best known as adaptive immune defenses in prokaryotes, but they also function as versatile regulators bridging bacterial immunity with host-related processes. Beyond neutralizing invasive phages and plasmids, these systems influence core aspects of bacterial physiology, such as modulating gene expression, stress responses, biofilm formation, quorum sensing, and virulence. Notably, CRISPR-mediated regulation can facilitate immune evasion at the host-pathogen interface, underscoring these systems as central orchestrators of microbial survival and host interactions. In addition, CRISPR-Cas has rapidly become a cornerstone of synthetic biology and microbiome engineering. Recent strategies repurpose native and engineered CRISPR systems to precisely modulate microbiome composition or deliver sequence-specific antimicrobials, underscoring the expanding translational potential of this system. Collectively, emerging insights highlight both the canonical immune function and non-canonical regulatory roles of CRISPR-Cas, as well as their broad biological and biotechnological relevance. This review provides a critical synthesis of these developments, illustrating how CRISPR-Cas bridges adaptive immunity and microbial physiology, and outlines future directions for harnessing this duality to deepen understanding of microbial physiology and inform new translational applications. Full article

The intestinal microbiota plays a crucial role in maintaining epithelial barrier integrity, while its impairment and the resulting inflammation contribute to numerous human pathologies. To preserve intestinal homeostasis, various probiotics are being developed; however, their selection and validation require accessible yet physiologically relevant models. We recently established a high-throughput Gut-on-Chip model comprising human epithelial (Caco-2) cells and peripheral blood mononuclear cells (PBMCs), demonstrating epithelial barrier disruption and pro-inflammatory cytokine secretion upon inflammation induction. The present study aimed to evaluate the feasibility of co-culturing anaerobic members of the human intestinal microbiota within this model and to assess their effects on inflammation-induced epithelial damage. We successfully co-cultured five intestinal anaerobic bacterial species in direct contact with the epithelial monolayer for two days. As proof of concept, we demonstrate that live Bacteroides thetaiotaomicron and its supernatant preserve epithelial barrier integrity and attenuate CCL2 secretion by Caco-2 cells. In contrast, Clostridium scindens did not prevent epithelial damage but suppressed CCL20 secretion, revealing a promising target for future studies. By recapitulating some of the key aspects of intestinal inflammation, we suggest that the current Gut-on-Chip model has potential as an easy-to-use platform for screening next-generation probiotics and live biotherapeutics with homeostatic and immunomodulatory properties. Full article

Fats, oils, and grease (FOG) deposits are hardened, sticky, insoluble solids that accumulate in sewage systems globally. These deposits contribute to pipe blockages and sanitary sewer overflows, releasing pathogens and pollutants into the environment, posing significant environmental and public health risks. Current removal methods are labor-intensive and costly, emphasizing the need for alternatives. While biological strategies offer a viable alternative, the microbial breakdown of FOG is poorly understood. In this study, we evaluated the potential of individual microbial strains and synthetic microbial communities to biodegrade wastewater-derived FOG deposit samples. These biological agents were applied to a range of FOG samples, and biodegradation was assessed through visual observations such as color change or gas bubbles, particle size, cell counts, pH, weight loss, and changes in fatty acid profile. Results demonstrate that microbial augmentation can enhance FOG degradation, offering an alternative or complementary approach for reducing maintenance burdens and preventing sewer blockages. Full article

The role of petting zoo animals in the dissemination of disease has been widely studied, yet understanding the potential reservoir of antimicrobial resistance (AMR) in these centres has not been explored in the United Kingdom (UK). To understand the carriage of AMR pathogens within petting zoos, this study aimed to identify AMR in E. coli and Staphylococcus intermedius group (SIG) isolated from faeces and skin, respectively, including selective cultures for ESBL-E. coli and methicillin-resistant staphylococci. Faecal samples and skin swabs were collected from 166 petted mammals across eight UK centres to recover E. coli and coagulase-positive staphylococci (CoPS), respectively, through enrichment culture methods, plating onto non-selective (tryptone bile-x agar, mannitol salt agar) and selective media (ESBL ChromID, mannitol salt agar with 6 mg/L oxacillin). Antimicrobial susceptibility was assessed using Kirby-Bauer disc diffusion, covering eight classes of antimicrobials. Antimicrobial usage records from the past 12-months were obtained from 7/8 centres. Overall, 145/166 faecal samples yielded 223 E. coli isolates, with an overall AMR prevalence of 42.6%. Thirteen E. coli isolates (from 8.5% of animals) were classified as multidrug-resistant. ESBL-producing E. coli were detected in 5/166 faecal samples. From 166 skin swabs, 84 yielded CoPS isolates, with S. aureus (n = 70), SIG (n = 13) and S. hyicus (n = 1) identified. Overall, 25.3% of SIG isolates exhibited resistance to at least one antimicrobial. Antimicrobial usage correlated positively with AMR prevalence for E. coli (p < 0.001), though was not associated with multidrug-resistance. This study demonstrates for the first time the presence of AMR within bacteria isolated from UK petting zoo animals, highlighting this reservoir of AMR bacteria. Full article

In efforts to enhance the sensory profile of coffee, fermentation variations have been implemented, including extending the process for prolonged periods. Such practices create imbalances among the microbial groups involved and increase populations of filamentous fungi, compromising product safety. To identify the filamentous fungi present in fermentations for up to 192 h, coffee samples were collected from fermentations conducted under semi-anaerobic (SA) and self-induced anaerobic fermentation (SIAF) conditions. Microscopic, metabolic, and rDNA sequencing techniques were applied to identify the filamentous fungi. Relative abundance and taxonomic classification were obtained through High-Throughput Sequencing of the ITS region. In addition, the presence of Ochratoxin A (OTA) was evaluated through HPLC/FLD. The most abundant genera identified was Aspergillus in SA fermentations, and Fusarium in SIAFs both at 192 h. 3438 OTUs of filamentous fungi were obtained, distributed across 11 orders, 20 families, and 17 genera. The results suggest a greater presence of mycotoxin-producing genera in fermentations with longer processing times, particularly under semi-anaerobic conditions. However, OTA levels remained below 0.8 ppb (µg/kg). These findings provide essential information for microbiological control of fermentation, supporting the maintenance of product safety in subsequent stages and ensuring the safety of the final product. Full article