Multi-drug resistant bacteria (MDR) pose a major public health challenge. Their ability to exchange resistance genes through Horizontal Gene Transfer (HGT) promotes the appearance of resistant strains, limiting antibiotic treatments for infections caused by these MDR bacteria. Among alternative approaches, phage therapy stands out as a promising strategy that utilizes bacteriophages to specifically target and effectively eliminate bacteria. This narrative review provides an overview of the current knowledge on the use of whole bacteriophages as antimicrobial agents in human and veterinary medicine, as well as in the food industry whether used alone, in cocktails, or combined with antimicrobials. While whole phages offer high specificity and an efficient elimination of bacteria, their application is associated with several limitations, including their contribution to HGT, the emergence of bacterial resistance, their narrow host range, the immune recognition, and the difficulties posed by their regulation. To address these challenges, this review focuses on phage-derived enzymatically active proteins, such as endolysins and depolymerases, as alternative antimicrobial tools, used alone or in combination. These phage components, being smaller and structurally simpler than whole phages, behave more similarly to conventional antimicrobial compounds. They have so far presented a low risk of bacterial resistance appearance and less chance of immune response. In addition, their classification as antimicrobial enzymes or conventional biologics could facilitate regulatory approval by aligning with existing regulatory frameworks. A total of 40 studies were included in this narrative review, highlighting the outcomes of applications involving whole bacteriophages (n = 11) and phage-derived enzymes, including endolysins and depolymerases (n = 27).

This study aimed to develop a compost bedded pack (CBP) model of the active composting layer in dairy housing systems by applying conditions observed on commercial CBP farms along with parameters from published studies. The model was constructed using pack materials collected from 4 commercial CBPs, managed under standardized laboratory conditions to minimize environmental variability. Compost bedded pack material consisting of manure and sawdust obtained from each farm was divided into 6 containers with 3 randomly assigned to control (daily mixing only) and 3 randomly assigned to enriched (daily mixing and manure addition, weekly addition of sawdust). A total of 24 composting containers (n = 24; 6 containers from each of 4 source CBP packs) were managed for 5 weeks. Data were analyzed using a repeated-measures mixed model with treatment, week, and their interaction as fixed effects. The CBP model was assessed by measuring temperature, moisture content, carbon-to-nitrogen ratio, and the levels of cellulose, hemicellulose, and lignin for each container. The enriched treatment supported sustained physicochemical changes consistent with composting processes, as temperature increased from 30.2°C in wk 1 to 40.0°C in wk 5. In contrast, temperatures in the control containers declined from 24.8°C in wk 1 to 22.6°C in wk 5. Moisture in the enriched group remained within optimal composting levels (58.6 - 61.6%), whereas the control showed moisture loss (56.73 - 36.42%). The C:N ratio in the enriched group declined from 28.6 to 16.8 from wk 1 to 5, and overall cellulose content decreased by 26.9% despite weekly sawdust additions. This amount was calculated from data of the ratio of average sawdust addition to manure deposited in summer months on Ontario commercial packs that maintained a temperature above 42°C and between 40% and 65% moisture. The hemicellulose and lignin content did not differ significantly over time even though sawdust was added weekly, suggesting ongoing decomposition. Microbial profiling revealed that Bacillota, Bacteroidota, Pseudomonadota, Actinomycetota, and Verrucomicrobiota were the dominant phyla in both enriched and control containers. Among these, Bacillota, Bacteroidota, and Actinomycetota were detected in 100% of samples at a relative abundance of ≥ 1% and were thus defined as the core phyla. Overall, the laboratory CBP model successfully replicated active composting dynamics and offers a safe and controlled platform for future studies of pathogen survival.

This review aimed to assess the scope of the literature on tracking the microbial community of biofilms, focusing on the dairy farm and processing environments. The majority of studies focused on either production, storage, transport or processing of milk, while 5 combined the investigation of both production and processing facilities. Factors influencing short-term changes in dairy microbiota such as the occurrence of mastitis and season were distinguished from factors revealed through long-term studies, such as feed and weather, rather than the milking equipment. Knowledge gaps were identified in relation to the study design, methods, data analysis and interpretation. The application of DNA sequencing technologies is particularly challenging with respect to samples with low microbial load (milk, swabs). There are few studies on the microbial composition of in situ biofilms, which might require new technologies for detection before sampling. Fundamental studies on the structure of biofilms are needed to identify the on-farm practices impacting the cycle of biofilm development in milking systems.

This review aimed to assess the scope of the literature on tracking the microbial community of biofilms, focusing on the dairy farm and processing environments. The majority of studies focused on either production, storage, transport, or processing of milk, and 5 studies combined the investigation of both production and processing facilities. Factors influencing short-term changes in dairy microbiota, such as the occurrence of mastitis and season, were distinguished from factors revealed through long-term studies, such as feed and weather, rather than the milking equipment. Knowledge gaps were identified in relation to the study design, methods, data analysis, and interpretation. The application of DNA sequencing technologies is particularly challenging with respect to samples with low microbial load (milk, swabs). There are few studies on the microbial composition of in situ biofilms, which might require new technologies for detection before sampling. Fundamental studies on the structure of biofilms are needed to identify the on-farm practices affecting the cycle of biofilm development in milking systems.

Recent outbreaks of foodborne diseases have highlighted the challenges of maintaining food safety, emphasizing the need for effective strategies to control pathogens and spoilage organisms. Toxins produced by indigenous fungi pose serious economic issues and undermine food security. Mycotoxin spoilage is a ubiquitous hazard that affects all food commodities; however, bakery products, dairy, fruits, vegetables, and meat are particularly vulnerable. The quality of food is perceived through senses such as taste, aroma, and texture. These sensory attributes significantly impact the overall sensation of the product and determine whether it will be accepted or rejected by consumers. Spoilage not only reduces consumer satisfaction but also drastically shortens the shelf life of food. This review highlights the ability of Lactic Acid Bacteria (LABs) to produce diverse antimicrobials, emphasizing antifungal metabolites as effective tools for enhancing food preservation and extending shelf life. As consumer demand for 'clean label' solutions increases, these natural antimicrobials promise safe and effective alternatives for enhancing food safety, reducing fungal spoilage, and extending shelf life of various perishable food commodities and reducing economic losses.

The Simulator of the Human Intestinal Microbial Ecosystem (SHIME) system was provided with baby feed for one week to stabilise the microbial community, followed by a 10-day period with baby feed and another 10-day period with adult feed. The study was conducted using sterilised and standardised feed formulations, which model dietary conditions in vitro. Following the transition from baby to adult feed, a significant reduction in the proportion of butyrate in comparison to total SCFA was found after transitioning to adult feed in both the transverse colon and distal colon bioreactors. Our findings suggest that abrupt early-life dietary changes from simple to complex carbohydrates as well as the exclusion of bovine milk proteins can transiently lower the ability of the microbiota to produce butyrate. The lack of additional microbial input leads to a delay or impairment of the adaptation to the modified feed composition. However, given the short treatment duration and sterilised feed composition, these findings should be interpreted within the limitations of this in vitro model. A reduction in butyrate concentration following the transition to adult feed may reflect a temporary shift in microbial metabolic activity rather than a long-term impact on energy extraction efficiency in vivo.

Biofilms pose major challenges to milk quality and safety, yet their composition in the dairy environment remains under-characterized. This study investigated the prevalence and composition of biofilms on milking system surfaces in commercial dairy farms, focusing on Pseudomonadota, a dominant phylum in raw milk. We sampled bulk tank raw milk (BTRM), tap water, and milking equipment surfaces after cleaning from 20 dairy farms in Québec, Canada, using S1 milk agar, specifically designed to target Pseudomonadota through a culturomics approach. A total of 474 colonies were selected and identified by MALDI-TOF MS. Results demonstrated the presence of multispecies biofilms, within the phylum of Pseudomonadota, including potential spoilage and pathogenic bacteria, such as Pseudomonas aeruginosa. Furthermore, the study revealed a significant presence of yeasts (42% of the isolates), predominantly Candida parapsilosis. Biofilms were predominantly detected in milk pipelines and milking machines (milk pipeline: 5.42 log gene copy number/swab; milking machine: 5.16 log gene copy number/swab), with greater bacterial loads quantified by quantitative PCR in fall than spring or summer. Partial least squares discriminant analysis revealed that the microbial composition of biofilms differed from that of BTRM (classification error rate: 0.23 and area under the curve: 0.95), although several species were shared, such as Candida parapsilosis and P. aeruginosa, and Escherichia coli. Tap water was not identified as a major contamination source of Pseudomonadota for dairy biofilms as only 2 species were shared across water, BTRM, and biofilm samples (Pantoea agglomerans and Serratia liquefaciens). The presence of these biofilms, harboring potentially pathogenic and spoilage microorganisms, poses a challenge to milk quality and safety. These findings provide data on the diversity of culturable Pseudomonadota and yeasts in biofilms on dairy farms and highlight the need for improved sanitation practices to mitigate microbial contamination in milk production.

Antimicrobial resistance (AMR) is a concern in the dairy industry. Recent studies have indicated that bedding serves as a reservoir for antimicrobial-resistant bacteria and antimicrobial-resistance genes (ARGs), while silage has been proposed as another possible source. The impact of AMR in dairy farming can be significant, resulting in decreased productivity and economic losses for farmers. Several studies have highlighted the safety implications of AMR bacteria and genes in bedding and silage, emphasizing the need for further research on how housing, bedding, and silage management affect AMR in farm environments. Exposure to sub-lethal concentrations of antibiotics, such as those from contaminated bedding and silage, can prompt bacteria to develop resistance mechanisms. Thus, even if antimicrobial usage is diminished, ARGs may be maintained in the dairy farm environment. By implementing proactive measures to tackle AMR in dairy farming, we can take steps to preserve the health and productivity of dairy cattle while also protecting public health. This involves addressing the prudent use of antibiotics during production and promoting animal welfare, hygiene, and management practices in bedding and farm environments to minimize the risk of AMR development and spread. This narrative review compiles the growing research, positioning the contribution of bedding and silage to the prevalence and dissemination of AMR, which can elicit insights for researchers and policymakers.

Milk residue and the accompanying biofilm accumulation in milking systems can compromise the microbial quality of milk and the downstream processes of cheese production. Over a six-month study, the microbial ecosystems of milk (n = 24), tap water (n = 24) and environmental swabs (n = 384) were cultured by plating decimal dilutions to obtain viable counts of total aerobic mesophilic lactose-utilizing bacteria (lactose-M17), lactic acid bacteria (MRS), yeasts and molds (Yeast, Glucose, Chloramphenicol (YGC) medium). Viable aerobic lactose-M17 plate counts of milk remained well below 4.7 log CFU/ml over five of the months, except for 1 week in November where milk at the facility exceeded 5 log CFU/ml. Swab samples of the farm milking equipment showed consistent viable counts after sanitation, while the bulk tank swabs contained the lowest counts. Viable counts from swabs of the facility were generally below the detection limit in the majority of samples with occasional residual contamination on some food contact surfaces. Extracted DNA was amplified using primers targeting the V3–V4 region of the 16S rRNA gene, and the amplicons were sequenced by MiSeq to determine the shared microbiota between the farm and the processing facility (8 genera). Culture independent analysis of bacterial taxa in milk, water and residual contamination after sanitation with swab samples revealed the shared and distinct microbiota between the sample types of both facilities. Amplicon sequence variants (ASVs) of the V3–V4 region of the 16S rRNA gene revealed that the microbiota of milk samples had lower diversity than water or environmental swabs (279 ASVs compared to 3,444 in water and 8,747 in environmental swabs). Brevibacterium and Yaniella (both Actinomycetota) were observed in all sampling types. Further studies will include whole genome sequencing of Brevibacterium spp. isolates to determine their functionality and diversity within the system.

The mutualistic relationship between Streptococcus thermophilus (S. thermophilus) and L. delbrueckii subsp. bulgaricus (L. bulgaricus) is responsible for milk coagulation, gel formation, and the flavour of yogurt. Under set-style yogurt processing conditions, the performance of a mixed culture composed of these species depends on key technological parameters such as the capacity for acidification and proteolytic activity. This study aimed to determine the extent of phenotypic diversity by comparing the key traits of acidification and proteolytic activity among isolates found in yogurt starter cultures. Seventy-two isolates from three industrial starter cultures were ranked by either their fast or slow acidification activity (time to reach pH 4.6, 16 h), proteolytic activity, cell envelope proteinase (CEP) activity, redox potential and titratable acidity. The integration of multiple phenotype measures by hierarchical clustering and non-metric dimensional scaling (NMDS) clustered groups of isolates by multifactor similarity. A significant difference (p-value < 0.05) was observed between the clusters regarding redox potential and the proteolytic activity of both S. thermophilus and L. bulgaricus. The integration of multiple phenotypes points to the diversification that may have occurred over repeated culturing of yogurt starter bacteria. The phenotypic diversity may explain the divergence in starter performance and be used to refine the formulation of new starter cultures. Future work will investigate the correlation between the activity of specific enzymes based on the phenotype to explain the separation between the fast and slow acidification of isolates.

The effects of farm management practices and seasonal variation on the microbial community and chemical composition of corn and grass-legume silage are largely understudied due to the advantages of controlled mini-silo experiments. This study aims to investigate the effects that some key farm factors (use of an inoculant, farm region, and bunker or tower silo) and seasonal variations have on corn and grass-legume silage from farms across Ontario, Quebec, and New York. The silage was either treated with a commercial inoculant (Lallemand Biotal Buchneri 500® or Chr Hansen SiloSolve FC®) or left untreated. The bacterial communities of silage were compared to those of raw bulk tank milk from the same farm to determine if they were similarly affected by management practices or seasonal variations. Family level analysis of the 16S rRNA V3-V4 gene amplicon bacterial community, the ITS1 amplicon fungal community, NMR water soluble metabolome, and mycotoxin LC–MS were performed on silage over a two-year period. Chemical compounds associated with the use of inoculants in corn and grass-legume silage were higher in inoculated corn (acetate, propane-1,2-diol, γ-aminobutyrate; p < 0.001) and grass-legume (propionate; p = 0.011). However, there was no significant difference in the relative abundance (RA) of Lactobacillaceae in either silage type. Leuconostocaceae was higher in non-inoculated corn (p < 0.001) and grass-legume (p < 0.001) silage than in inoculated silage. Tower silos had higher RA of Leuconostocaceae (p < 0.001) and higher pH (p < 0.001) in corn and grass-legume silage. The one farm that used liquid manure with no other fertilizer type had higher RA of Clostridiaceae (p = 0.045) and other rumen/fecal (p < 0.006) bacteria in grass-legume silage than all other farms. Seasonal variation affected most of the key silage microbial families, however the trends were rarely visible across both years. Few trends in microbial variation could be observed in both silage and bulk tank milk: two farms had higher Moraxellaceae (p < 0.001) in milk and either corn or grass-legume silage. In farms using an inoculant, lower Staphylococcaceae was observed in the raw bulk tank milk.

In this study, we evaluated the impact of Lactiplantibacillus plantarum (L. plantarum) with ropy and non-ropy phenotypes on gel structure and protein conformation of fermented milk. Ropy L. plantarum (T1 & CL80) secreted EPS with high molecular weight (1.41 × 106, 1.19 × 106 Da) and intrinsic viscosity (486.46, 316.32 mL/g), effectively enhances fermented milk viscosity and water holding capacity (WHC) (65.4%, 84.6%) by forming a dense gel structure. Non-ropy L. plantarum (CSK & S-1A) fermented milk gel's high surface hydrophobicity and free sulfhydryl content caused high hardness and low WHC. Raman spectroscopy combined with circular dichroism analysis showed that high levels of α-helix (29.32-30.31%) and random roil (23.06-25.36%) protein structures are the intrinsic factors that contribute to the difference among fermented milk gels of ropy and non-ropy strains. This study provides a basis for understanding the structural variability of fermented milk gels using ropy or non-ropy lactic acid bacteria.

Spore-forming bacteria in silage may reduce dry matter intake or affect dairy product quality when transferred to milk. The present study investigated the effects of three facultative anaerobes (Bacillus cereus, Bacillus subtilis, and Bacillus licheniformis) and two strict anaerobes (Clostridium tyrobutyricum and Clostridium beijerinckii) commonly found in low-quality silage, milk, and cheese. Maize silage was intentionally contaminated with these spore formers in separate mini silos at 1 × 105 CFU spore former per g and treated with commercial silage inoculant Lentilactobacillus buchneri NCIMB 40788 at 4 × 105 CFU per g or left untreated. The microbial and chemical profiles of maize silage, which were determined using Nuclear Magnetic Resonance (NMR), were measured after fermentation for 100 days, and they were also measured for silage exposed to air for 72 h after opening at 100 days. The effect of the inoculant strain L. buchneri NCIMB 40788 on these contaminated silages was investigated to determine if the inoculant could prevent/limit the negative impacts caused by the spore former contaminants. Overall, inoculation improved silage quality and aerobic stability. Acetic acid content was higher in the INOC samples than in the NIS (p < 0.001), while lactic acid content was lower in INOC than in NIS (p < 0.001). Both lactic and acetic acid levels were higher in the silage samples contaminated with B. cereus. Contamination with the spore formers increased the aerobic and anaerobic spore counts of the samples contaminated with B. subtilis and B. licheniformis compared to the control silage after opening. After 3 days of aerobic exposure, the samples contaminated with B. cereus, B. subtilis, and B. licheniformis showed higher aerobic spore counts than the control. The dominant bacterial population was significantly modified by inoculation. Neither inoculation nor contamination types impacted fungal populations upon opening, but a dominance of Wickerhamomyces was observed after aerobic exposure in the B. cereus silages. The γ-aminobutyrate (GABA) content after aerobic exposure was higher than the uncontaminated control for the silage contaminated with B. licheniformis. The samples contaminated with Clostridium species remained largely unchanged compared to the control samples. Physicochemical and bacterial profiles were mainly affected by inoculation, and a limited impact of spore contaminant was noted. Our results show that when L. buchneri inoculation was carried out, higher aerobic and anaerobic spore counts following contamination with bacterial spore formers were mitigated after reaching aerobic stability.

Ultra-processed, plant-based burgers (PB) and traditional comminuted-beef burgers (BB) share similar organo-leptic characteristics, yet a knowledge gap exists in understanding how consumption of these divergent physical structures alters the lipemic response and gut microbiota. PB, comprised of highly refined ingredients, is formulated with no intact whole food structure, while BB entraps lipids throughout the myofibrillar protein network. PB presented significantly higher free fatty acid (FFA) bioaccessibility (28.2 +/- 4.80 %) compared to BB (8.73 +/- 0.52 %), as obtained from their FFA release profiles over digestion time after characterizing them with a modified logistic model (SLM), using the simulated TIM Gastro-Intestinal Model (TIM-1). Additionally, the rate of lipolysis, k, obtained from the SLM for PB (90% CI [0.0175, 0.0277] min-1) was higher than for BB (90% CI [0.0113, 0.0171] min-1). Using the Simulated Human Intestinal Microbial Ecosystem (SHIME (R)), the Firmicutes to Bacteroidetes ratio (F/B ratio) was significantly higher for PB than BB; and linear discriminant analysis effect size (LEfSe) showed Clostridium and Citrobacter were more highly represented in the microbial community for the PB feed, whereas BB feed differentially enriched Megasphaera, Bacteroides, Alistipes, and Blautia at the genus level. Additionally, short-chain fatty acid (SCFA) production was altered (p < 0.05) site-specifically in each colon vessel, which could be attributed to the available substrates and changes in microbial composition. Total SCFAs were significantly higher for PB in the ascending colon (AC) and descending colon (DC) but higher for BB only in the transverse colon (TC). This research illustrates the crucial role of meat analog physical structure in modu-lating nutritional aspects beyond food composition alone.

IntroductionThe microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk.MethodsWe identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells.Results and discussionThree community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Shotgun metagenomic sequencing was used to investigate the diversity of the microbial community of Cheddar cheese ripened over 32 months. The changes in taxa abundance were compared from assembly-based, non-assembly-based, and mOTUs2 sequencing pipelines to delineate the community profile for each age group. Metagenomic assembled genomes (MAGs) passing the quality threshold were obtained for 11 species from 58 samples. Although Lactococcus cremoris and Lacticaseibacillus paracasei were dominant across the shotgun samples, other species were identified using MG-RAST. NMDS analysis of the beta diversity of the microbial community revealed the similarity of the cheeses in older age groups (7 months to 32 months). As expected, the abundance of Lactococcus cremoris consistently decreased over ripening, while the proportion of permeable cells increased. Over the ripening period, the relative abundance of viable Lacticaseibacillus paracasei progressively increased, but at a variable rate among trials. Reads attributed to Siphoviridae and Ascomycota remained below 1% relative abundance. The functional profiles of PMA-treated cheeses differed from those of non-PMA-treated cheeses. Starter rotation was reflected in the single nucleotide variant profiles of Lactococcus cremoris (SNVs of this species using mOTUs2), while the incoming milk was the leading factor in discriminating Lacticaseibacillus paracasei/casei SNV profiles. The relative abundance estimates from Kraken2, non-assembly-based (MG-RAST) and marker gene clusters (mOTUs2) were consistent across age groups for the two dominant taxa. Metagenomics enabled sequence variant analysis below the bacterial species level and functional profiling that may affect the metabolic interactions between subpopulations in cheese during ripening, which could help explain the overall flavour development of cheese. Future work will integrate microbial variants with volatile profiles to associate the development of compounds related to cheese flavour at each ripening stage.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

The microbiota of silage is a key determinant of its quality. Although commercial inoculants are often used to improve silage quality, studies to analyze their impact on the microbiota of preserved forage at farm-scale facilities are scarce. We assessed the diversity of viable bacterial communities of hay (unfermented dry forage) and grass or legume (GL) and corn (C) silage to deepen our knowledge of how inoculant addition drives microbial occurrence patterns on dairy farms. Forage samples were collected from 24 dairy farms over two sampling periods. Samples were analyzed by high-throughput sequencing and quantitative PCR after being treated with propidium monoazide to account for viable cells. We found consistent significant differences between hay and silage community structures across sampling periods. Silage was generally dominated by lactic acid bacteria (LAB), while Pantoea and Sphingomonas were the main co-dominant genera in hay. The GL silage dominated by Pediococcus, Weissella, and Bacillus was phylogenetically different from C silage enriched in Acetobacter. The use of inoculants including Lentilactobacillus buchneri either alone or in combination with Lactiplantibacillus plantarum, Lacticaseibacillus casei, Pediococcus pentosaceus, or Enterococcus faecium did not systematically prevent the occurrence of undesirable bacteria, especially when corn-based, probably because of factors that can mitigate the effect of inoculation on the microbiota. The core Lactobacillales constituted the dominant LAB in silage with up to 96% relative abundance, indicating either the ubiquity of inoculants or the high competitiveness of epiphytes. Silage chemical profiles varied inconsistently with sampling periods and the use of inoculants. Multivariate multi-table analyses allowed the identification of bacterial clusters mainly driven by moisture and magnesium content in hay, while pH, lactic, and fatty acids were the main drivers for silage. Bacterial network analyses showed considerable variations in the topological roles with the use of inoculants. These results may help evaluate the effectiveness of forage management practices implemented on dairy farms and, therefore, are useful for fine-tuning the search for new additives. Such knowledge can be used by forage makers to adjust processing routines to improve the hygienic quality, nutritional potential, and aerobic stability of preserved forage.

The diversity of microorganisms in raw milk come from the cow, the milking equipment, transportation, storage, and the environment. Microorganisms in milk can be either detrimental (pathogenic, spoilage) or beneficial (positive effects on processing or on animal and human health). Some microorganisms, such as lactic acid bacteria, can be put to work to produce fermented dairy foods when they are allowed to grow under controlled conditions. Milk is generally a good growth medium for many microorganisms, so controls over storage temperature and hygiene during production and processing are critical for maintaining an acceptable product.