Dairy propionibacteria are Gram positive Actinomycetota, routinely utilized as starters in Swiss type cheese making and highly appreciated for their probiotic properties and health promoting effects. In this work, within the frame of a circular economy approach, 47 Propionibacterium and Acidipropionibacterium spp. were isolated from goat cheese and milk, and ewe rumen liquor, and characterized in view of their possible utilization for the production of novel pro-bioactive food and feed on scotta, a lactose rich substrate and one of the main by-products of the dairy industry. The evaluation of the Minimum Inhibitory Concentration (MIC) of 13 among the most common antibiotics in clinical practice revealed a general susceptibility to ampicillin, gentamycin, streptomycin, vancomycin, chloramphenicol, and clindamycin while confirming a lower susceptibility to aminoglycosides and ciprofloxacin. Twenty-five isolates, that proved capable of lactose utilization as the sole carbon source, were then characterized for functional and biotechnological properties. Four of them, ascribed to Propionibacterium freudenreichii species, and harboring resistance to bile salts (growth at 0.7–1.56 mM of unconjugated bile salts), acid stress (>80% survival after 1 h at pH 2), osmostress (growth at up to 6.5% NaCl) and lyophilization (survival rate > 80%), were selected and inoculated in scotta. On this substrate the four isolates reached cell densities ranging from 8.11 ± 0.14 to 9.45 ± 0.06 Log CFU mL⁻¹ and proved capable of producing different vitamin B9 vitamers after 72 h incubation at 30°C. In addition, the semi-quantitative analysis following the metabolomics profiling revealed a total production of cobalamin derivatives (vitamin B12) in the range 0.49–1.31 mg L⁻¹, thus suggesting a full activity of the corresponding biosynthetic pathways, likely involving a complex interplay between folate cycle and methylation cycle required in vitamin B12 biosynthesis. These isolates appear interesting candidates for further ad-hoc investigation regarding the production of pro-bioactive scotta.

Mycotoxins are well-known secondary metabolites produced by several fungi that grow and occur in different crops during both pre-harvest and post-harvest conditions. The contamination and occurrence of mycotoxins currently represent some of the major issues in the entire agri-food system. The quantification of mycotoxins in different feeds and foodstuffs is extremely difficult because of the low concentration ranges; therefore, both sample collection and preparation are essential to providing accurate detection and reliable quantification. Currently, several analytical methods are available for the detection of mycotoxins in both feed and food products, and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most reliable instrumental approach. In particular, the fast development of high-throughput methods has made it possible to screen and analyze, in the same analytical run and with high accuracy, multiple mycotoxins, such as those regulated, masked, or modified, and emerging ones. Therefore, the aim of this review is to provide an overview of the state of the art of mycotoxins occurrence, health-related concerns, and analyses, discussing the need to perform multi-screening approaches combined with omics technologies to simultaneously analyze several mycotoxins in different feed and food matrices. This approach is expected to provide more comprehensive information about the profile and distribution of emerging mycotoxins, thus enhancing the understanding of their co-occurrence and impact on the entire production chain.

Corn silage (CS) and high-moisture corn (HMC) represent fundamental ingredients in ruminant diets; however, their chemical complexity and susceptibility to mycotoxin contamination pose challenges for feed safety and quality assessment. This study applied an innovative approach combining untargeted metabolomics and mycotoxin profiling through ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) to characterize the chemical profiles of CS (n = 19) and HMC (n = 13) samples collected from four farms in northern Italy over a period of two years. Fumonisin B1 (FB1) emerged as the most prevalent mycotoxin, with contamination levels significantly higher in HMC than CS, though all the detected levels complied with European Union (EU) guidance limits. Untargeted metabolomics distinguished CS and HMC based on their metabolic signatures: polyamines, amino acids, peptides, and phenolic acids typified CS, while HMC was primarily characterized by flavonoids and mycotoxins. Geographical origin significantly influenced both mycotoxin patterns and metabolite profiles, while the sampling season showed no significant impact. This study highlights the complementary value of metabolomics and mycotoxin screening to assess feed quality, identify biomarkers, and unravel the link between fungal contamination and biochemical composition, offering a robust strategy to support feed safety management in livestock production.

Summary: Diet can affect ruminal microbial protein production. Ruminal microbial protein production could be estimated by noninvasive methods, such as analysis of milk fatty acids or milk metabolome, but how do milk odd-chain fatty acids, a proxy for microbial protein production, correlate with milk compounds from pyrimidine and purine metabolism? In a crossover trial using different carbohydrate types, milk from dairy cows was analyzed for metabolomic profile and fatty acid concentration. The milk metabolome and fatty acid concentration could differentiate the 4 diets used in the trial, whereas the milk metabolome alone could only differentiate the 2 silage types. Some milk odd-chain fatty acids were correlated with pyrimidine and purine derivatives. Our results indicate that the milk metabolome could provide an indication of the protein efficiency of the diet; however, validation of the metabolomic findings with actual ruminal microbial protein synthesis data is necessary.

To protect ruminants from the harmful effects of mycotoxins, anti-mycotoxin agents can be added to the dietary ration, thus guaranteeing animal health and production. Therefore, the objective of this study was to evaluate the in vitro ruminal initial sequestration (weak binding) and subsequent desorption (strong binding) of an anti-mycotoxin agent based on a mixture of adsorbing material, turmeric and milk thistle extracts and yeast-based components to adsorb or bio-convert aflatoxins (AF), fumonisins B1 and B2 (FB), trichothecene deoxynivalenol (DON), T-2 and HT-2 toxins, and zearalenone (ZEN). Two doses were tested: Dose 1 simulated 30 mg/cow/d, while Dose 2 simulated 90 mg/cow/d of the anti-mycotoxin agent. Each treatment involved three analytical replicates at each of three incubation times (1, 4, and 24 h post-incubation), with two independent experimental runs providing experimental replicates. Analytical methods, including UHPLC-HRMS and multivariate analyses, were used to both quantify mycotoxin concentrations and reveal dose-dependent reductions, with statistical validations indicating significant changes in mycotoxin levels across both dose and time. The results indicated that the anti-mycotoxin agent was able to highly bind AFB1, T2, and HT-2 toxins since its concentration was always under the limit of detection (<1 ppb). Regarding ZEN (weak binding mean: 94.6%; strong binding mean: 62.4%) and FBs (weak binding mean: 58.7%; strong binding mean: 32.3%), orthogonal contrasts indicated that the anti-mycotoxin agent was able to effectively bind these toxins using Dose 1 (p < 0.05). This finding suggests that Dose 1 may be sufficient to achieve the targeted effect and that a further increase does not significantly improve the outcome. Regarding DON, a strong linear relationship was observed between dose and adsorption. However, the complex interactions between the mycotoxin, the ruminal environment, and the anti-mycotoxin agent made it difficult to establish a clear dose–effect relationship (p > 0.10). UHPLC-HRMS analysis identified over 1500 mass features in rumen samples, which were further analyzed to assess the effects of the anti-mycotoxin agent. Hierarchical clustering analysis (HCA) revealed significant changes in the untargeted metabolomic profiles of samples treated with mycotoxins compared to control samples, particularly after 24 h with the anti-mycotoxin treatments. Clear differences were noted between strong binding and weak binding samples. Further analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) highlighted distinct metabolomic profiles, with stronger predictive ability in the strong binding group (Q² cumulative value of 0.57) compared to the weak binding group (0.30). The analysis identified 44 discriminant compounds in the strong binding model and 16 in the weak binding model. Seven compounds were common to both groups, while silibinin, known for its antioxidant and anti-inflammatory properties, was found among the unique compounds in the weak binding group. Overall, the findings suggest that both doses of the anti-mycotoxin agent significantly influenced the chemical profiles in the rumen, particularly enhancing the binding of mycotoxins, thereby supporting the role of phytogenic extracts in mitigating mycotoxin effects.

Summary: In this study, 977 noncorn forage samples from dairy farms located mainly in Northern Italy were analyzed over a 7-year period; samples included 642 silages and 335 hays or straws. Deoxynivalenol (DON), zearalenone (ZEN), and total fumonisins (FUM) were quantified using competitive enzyme-linked immunosorbent assay (ELISA) methods. Deoxynivalenol was the most frequently detected mycotoxin (48% of samples), with particularly high concentrations in straw and mixed-cereal silages. Zearalenone showed lower overall prevalence but reached higher levels in sorghum silages and straw. Total fumonisins were less common across all forage types, with the highest occurrence in sorghum silages and negligible contamination in legume hays. Climatic variability across years, especially the wetter conditions recorded in 2018 through 2019, contributed to the observed differences in mycotoxin occurrence. Overall, the findings confirm that noncorn forages are susceptible to Fusarium mycotoxins, with contamination patterns influenced by botanical composition, environmental conditions during growth and harvest, and farm-level conservation practices. Image generated using the design program Mind the Graph (https://mindthegraph.com/; accessed Oct. 8, 2025).