A 26-week trial was conducted to evaluate the effects of Cyberlindnera jadinii on post-smolt Atlantic salmon (2.310 +/- 0.015 kg). Diets containing 0 (Diet 1, D1), 6 (Diet 2, D2), 12 (Diet 3, D3), and 18% (Diet 4, D4) C. jadinii were fed to duplicate cages. Decreased growth rate relative to D1 (p = 0.0444) was only observed in fish fed D4. The apparent digestibility coefficients of crude protein, crude lipid, and gross energy decreased (p < 0.01) with an increasing level of C. jadinii in the diet. Fish fed D1 or D2 had higher odds of high scores for gaping of the belly and wounds, respectively, relative to fish fed D4. The expression of il-8, tgfb, and il-10 in the distal intestine (DI) was significantly higher in fish fed D4 compared to fish fed D1 or D3 (p <= 0.05). The increased expression of tgfb and il-10 was correlated with il-8 (p <= 0.05). Also, there was a reduction in alpha-diversity in fish fed C. jadinii in digesta- and mucus-associated microbiota, while beta-diversity in digesta-associated microbiota showed a clear separation of fish fed D1 from the fish fed C. jadinii. A greater percentage area of the simple fold with MHCII and CD3 positive cells was reported in the DI of fish fed D4 compared to D1. Overall, this study suggests that an inclusion level of up to 12% C. jadinii is possible in diets for Atlantic salmon reared in seawater cages, supporting growth, health, welfare, and product quality of fish similar to that of a commercial-like control diet.

Engineered synthetic microbial communities (SynComs) forming biofilms with antagonistic activity offer a promising strategy in biotechnology to prevent harmful bacterial settlement and reduce reliance on chemical antimicrobials. However, strain selection criteria and antagonistic mechanisms remain unclear. This study presents a bottom-up approach integrating 3D fluorescence imaging with high-throughput analysis of multistrain biofilms. Our findings reveal that competitive strains against undesirable bacteria may also exclude desirable community members, highlighting the need for compatibility control in SynCom assembly. SynComs composed of Bacillus velezensis and Pediococcus spp. enhanced pathogen exclusion compared to single strains. Temporal analysis of biofilm interactions, supported by mathematical models, showed that pathogen exclusion was primarily driven by nutritional competition (Jameson effect) with additional specific interference dynamics (prey-predator Lotka-Volterra model). Furthermore, pre-established SynComs significantly increased pathogen inhibition, indicating a distinct biofilm-associated exclusion effect. These insights provide a framework for SynCom assembly and refine our understanding of interaction dynamics driving antagonistic biofilm applications.

Weaning is a stressful event that is often accompanied by anorexia, risk of diarrhea, and development of intestinal disorders, making it crucial to provide highly digestible and palatable diets. Novel functional protein sources are being developed to be included in diets fed to weanling pigs. We evaluated in vitro and in vivo the properties of a recently developed hydrolyzed yeast protein source (Yela ProSecure; YPS; Lallemand SAS, Blagnac, France). The objectives were (1) to evaluate in vitro amino acids (AA) digestibility; (2) to assess, in vitro, the impact of the product’s insoluble fraction (YPSi) on the fermentative activity of piglet fecal microbiota; and (3) to test the effects of two inclusion levels (2.5 and 6%) on growth performance and fecal microbiota in weanling piglets. The total AA availability after 3 h of digestion was 76.6%, reaching 89.8% after 48 h. YPSi induced high gas and short-chain fatty acids production. In the in vivo experiment, a significant difference in body weight was observed on day 18 (p < 0.001) post-weaning and on day 40 (p < 0.05), with piglets in both YPS treatments being heavier than control piglets. A higher average daily feed intake was observed between weaning and day 18 (p < 0.01) and overall (p < 0.05) in both YPS treatments, and an improved feed conversion ratio was observed in both YPS treatments between weaning and day 18 (p < 0.001). Moreover, YPS significantly modulated the fecal microbiota composition after 2 days and 16 days of treatment, whereas no lasting effect was evidenced on day 40, namely after 19 days of withdrawal from the diet. Lower relative abundance (RA) of Campylobacterota (p adj. < 0.05) was evidenced in YPS groups compared to the control group. Furthermore, several members of the Lactobacillaceae family, annotated as L. amylovorus, L. mucosae, or L. reuteri, as well as Faecalibacterium prausnitzii, showed higher RA in YPS groups. To conclude, adding YPS to the diet of weanling piglets increased growth performance, probably due to nutrient absorption in the small intestine and its functional role on gut microbiota. Those results suggest complex interconnections between host and microbiota and emphasize the need to consider the holobiont theory when formulating a diet.

Microbial biotransformation of Zearalenone (ZEN) is a promising deactivation approach. The residual toxicity and stability of Zearalenone-14-phosphate (ZEN-14-P) and Zearalenone-16-phosphate (ZEN-16-P), two novel microbial phosphorylation products of ZEN, remain unknown. We investigated the cytotoxicity, oxidative stress, proinflammatory, and estrogenic activity of phosphorylated ZENs using porcine intestinal cells, uterine explants, and human endometrial cells and traced their metabolic fate by liquid chromatography-tandem mass spectrometry (LC-MS)/MS analysis. The phosphorylated ZENs significantly decreased the viability of the IPEC-J2 and Ishikawa cells. Similar to ZEN, phosphorylation products induced significant oxidative stress, activated the expression of proinflammatory cytokines, and demonstrated estrogenic activity through upregulation of estrogen-responsive genes, activation of alkaline phosphatase, and proliferation of endometrial glands. LC-MS/MS analysis pointed out that although phosphorylated ZENs are partially hydrolyzed to ZEN, their respective metabolic pathways differ. We conclude that phosphorylation might not be sufficient to detoxify ZEN, leaving its cytotoxic, proinflammatory, and estrogenic properties intact.

Time around parturition is a stressful period for both bitches and their puppies. The use of probiotics has been proposed, e.g., in pigs, to improve health status of sows, their reproductive performances and in turn, the health and performance of their progeny. The objective of the present study was to evaluate the impact, on both dams and puppies, of a supplementation of bitches with the live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB-1079) during the second part of the gestation and the lactation period. A total of 36 bitches of medium and large-sized breeds were enrolled. They were divided into two groups, one of which received 1.3 × 109 colony forming units of live yeast per day. At dam’s level, SB-1079 yeast shaped a different microbiota structure between the two groups just after whelping, impacted alpha diversity and some plasma metabolites related to energy metabolism. Regarding reproductive performances, SB-1079 improved gross energy of the colostrum (1.4 vs. 1.2 kcal of ME/g) as well as the concentration of protein in milk at Day 7 after parturition (10.4 vs. 7.6%). SB-1079 also reduced the odds of having low birth weight in the litter. At puppy’s level, a modulation of immunometabolic phenotype is suggested by the observation of increased growth rates during the early pediatric period (i.e., between 21 and 56 days of life, 225 vs. 190%) and a decrease of the IL-8:IL-10 ratio after vaccination against rabies (4.2 vs. 16.9). Our findings suggest that SB-1079 supplementation during gestation and lactation has the potential to enhance health of bitches and in turn health of puppies through maternal programming.

Non-Saccharomyces yeast such as Debaryomyces hansenii or its cell wall-components are emerging candidates for novel functional aquafeeds, as they contain several microbe-associated molecular patterns (MAMPs) that can activate and modulate the host's physiological responses, thereby improving fish health and welfare. In this study, we combined in vitro work using SHK-1 cell line and primary cultures of head kidney leukocytes (HKL) from Atlantic salmon (HKL), with an in vivo trial in which two groups of Atlantic salmon were fed 0.1% hydrolyzed D. hansenii (LAN6) in freshwater (7 weeks) and seawater (6 weeks). In vitro results by ELISA showed that after induction with LAN6, SHK-1 cells increased the levels of TNF alpha and iNOS (at 6 h post-induction), while HKL increased TNF alpha (6 h post-induction) and IL-10 (24 h post-induction), and reduced TNF alpha levels at 24 h postinduction. In addition, during an early natural pathogen challenge in the seawater stage (week 5), an increase in specific plasma immunoglobulins against Moritella viscosa, along with an upregulation in pathways associated with humoral immunity and complement activation in the liver was detected in vaccinated fish fed LAN6 (Group A). These data suggest that LAN6 was able to modulate the immune response of Atlantic salmon making it a promising functional feed additive for aquafeeds to achieve a more resilient farmed salmon.

Whole-plant corn untreated (Control) or treated with an inoculant (Lentilactobacillus buchneri, Lentilactobacillus hilgardii, and Pediococcus pentosaceus, INO) was ensiled for 210 days in mini-silos at constant (MS-C) or variable temperature (MS-V) or for 220 days in bunkers. Bunker samples were collected at 50 (D50) and 150 (D150) cm below silo surface. Samples from MS-V and MS-C had similar pH and concentrations of lactic acid, propionic acid, 1,2-propanediol, and ethanol, but different acetic acid content (P = 0.009). Additionally, MS-V exhibited greater bacterial (P = 0.002) and fungal (P = 0.011) richness than MS-C. Inoculation decreased (P < 0.001) lactic acid levels while increasing (P < 0.05) acetic acid, 1,2-propanediol, and fungal richness in both mini-silos and bunker. In bunkers, samples collected from D50 had a lower aerobic stability (P < 0.05) than D150, but inoculation increased (P < 0.05) aerobic stability compared to Control, regardless of sampling depth. The storage temperature of mini-silos did not markedly impact the fermentation profile or fungal community. Overall, inoculation increased acetic acid production and fungal diversity in mini-silos, regardless of the storage temperature, and in bunkers, irrespective of sampling the depth, improving the aerobic stability of D50 and D150 bunker silages after long-term ensiling.

The use of synthetic microbial communities (SynComs) engineered to form positive biofilms that prevent the settlement of harmful bacteria is emerging as a promising strategy in biotechnology, particularly in reducing reliance on chemical antimicrobials. Despite this potential, the rationale for selecting specific strains in SynComs and the mechanisms underlying their antagonistic effects remains insufficiently understood. In this study, we present a bottom-up approach integrating live-cell imaging with high-throughput analysis of multi-strain biofilms across diverse scenarios. Through this method, we identified beneficial strains based on their superior ability to exclude undesirable bacteria and form mixed biofilms. Notably, our findings revealed that competitive strains against undesirable bacteria could also exclude other beneficial strains, emphasising the need for compatibility control in SynComs design. SynComs composed of B. velezensis and Pediococcus spp. demonstrated enhanced pathogen exclusion compared to single strains. Temporal analysis of biofilm interactions, supported by mathematical models, showed that pathogen exclusion was primarily driven by nutritional competition (Jameson effect) with additional specific interference mechanisms (prey-predator Lotka-Volterra model). Furthermore, pre-establishing SynComs to surfaces significantly increased pathogen inhibition, indicating a distinct biofilm-associated exclusion effect. These insights offer a framework for rational SynCom design and deepen our understanding of the mechanisms underpinning positive biofilm applications. ### Competing Interest Statement The authors have declared no competing interest.

Probiotics are live yeast or bacterial organisms that have beneficial effects on the host. Several microorganisms exhibit probiotic properties, the most common types being lactic acid bacteria, Bifidobacteria, spore-forming bacteria, and some yeast strains. Saccharomyces cerevisiae var. boulardii is the most important probiotic yeast species. However, another group of foodborne microorganisms, the so-called non-Saccharomyces yeasts (NSYs), has recently been re-evaluated and shown to have enormous potential in various fields of application, ranging from food fermentation to human and animal applications. NSYs are able to produce a range of bioactive compounds such as antimicrobials, mannoproteins, enzymes, polyunsaturated fatty acids, essential amino acids, vitamins, and β-glucans, which increases their potential applications as a new class of probiotics and/or alternatives to antibiotics in animal husbandry. In this review, we aim to highlight the potential and benefits of NSYs as probiotics and natural antimicrobials to improve animal health. Furthermore, the use of NSYs as biological alternatives to antibiotics to control foodborne pathogens in animal production is discussed.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

We describe and discuss the intestinal mycobiota of dairy cows reared in France following variations in dietary regimes and two seasons. Two groups of 21 animals were followed over a summer and winter period, and another group of 28 animals was followed only during the same summer season. The summer diet was based on grazing supplemented with 3–5 kg/d of maize, grass silage and hay, while the winter diet consisted of 30% maize silage, 25% grass silage, 15% hay and 30% concentrate. A total of 69 DNA samples were extracted from the feces of these cows. Amplification and sequencing of the ITS2 region were used to assess mycobiota diversity. Analyses of alpha and beta diversity were performed and compared statistically. The mycobiota changed significantly from summer to winter conditions with a decrease in its diversity, richness and evenness parameters, while beta diversity analysis showed different mycobiota profiles. Of note, the Geotrichum operational taxonomic unit (OTU) was prevalent in the winter group, with a mean relative abundance (RA) of 65% of the total mycobiota. This Geotrichum OTU was also found in the summer group, but to a lesser extent (5%). In conclusion, a summer grazing diet allowed a higher fecal fungal diversity. These data show, for the first time, that a change in diet associated with seasonality plays a central role in shaping hindgut fungal diversity.

A total of 150 21-day-old weaned piglets [(Yorkshire × Landrace) × Duroc] were randomly assigned to 3 groups based on average initial body weight (6.96 ± 0.21 kg) to evaluate the effects of dietary supplementation of probiotic, paraprobiotic, and hydrolyzed yeast mixture (PPY) on growth performance, nutrient digestibility, fecal bacteria counts, fecal calprotectin contents, and diarrhea rate in a 42-day experiment (phase 1: days 1-14; phase 2: days 15-42). There were 10 replicate pens per treatment with 5 pigs per pen (three gilts and two barrows). The experimental diets were a basal diet, without additive (CON), a basal diet supplemented with pharmacological levels of zinc oxide (ZnO; TRT1), and an experimental treatment including PPY (TRT2). Pigs in TRT1 were provided with a basal diet + 3000 mg/kg (as fed) ZnO during phase 1, and a basal diet during phase 2. Pigs in TRT2 were provided with a basal diet + 200 mg/kg (as fed) probiotic for a final concentration of 2 × 109 colony forming units (CFU)/kg diet + 800 mg/kg (as fed) paraprobiotic + 10 g/kg (as fed) hydrolyzed yeast mixture during phase 1, and a basal diet +100 mg/kg (as fed) probiotic + 400 mg/kg (as fed) paraprobiotic mixture during phase 2. Pigs in TRT1 and TRT2 tended to be heavier at day 14 and were significantly heavier at day 42 than CON pigs. Growth rate during days 1-14, 15-42, and 1-42 was similarly affected by treatment while feed efficiency was unaffected by treatment in the first 14 days but was significantly higher for TRT 1 pigs between 15-42 and 1-42 days with TRT2 being intermediate. Apparent nitrogen and energy digestibility were both significantly higher for pigs on TRT1 and TRT2 compared with the CON. There were no significant differences in any parameters measured between TRT1 and TRT2. Therefore, we demonstrated that PPY supplementation had comparable effects as ZnO on nutrient digestibility and the performance of weaned piglets.

Inoculants combining Lentilactobacillus buchneri and Lentilactobacillus hilgardii have been shown to improve the aerobic stability of high-moisture corn (HMC) and whole-plant corn silage, but the mode of action of this co-inoculation remains to be elucidated. This study used metatranscriptomics to evaluate the effects of inoculation with L. buchneri alone or combined with L. hilgardii on the bacterial community, gene expression, fermentation profile, and starch digestibility in HMC. High-moisture corn not inoculated (Control) or inoculated with L. buchneri NCIMB 40788 (LB) or L. buchneri NCIMB 40788 combined with L. hilgardii CNCM-I-4785 (Combo) was ensiled in mini silo bags for 30, 60, 120, and 180 days. The fermentation profile was evaluated at all time points. Metatranscriptomics was performed on samples collected on day 120. Combo had a greater alpha diversity richness index of contigs than LB and Control, and inoculation with Combo and LB modified the beta-diversity of contigs compared to Control. Out of 69 genes of interest, 20 were differentially expressed in LB compared to Control and 25 in Combo compared to Control. Of those differently expressed genes, 16 (10 of which were associated with carbohydrate metabolism and six with amino acid metabolism) were differently expressed in both LB and Combo compared to Control, and all those genes were upregulated in the inoculated silages. When we compared Combo and LB, we found seven genes expressed differently, four associated with carbohydrate metabolism and downregulated in Combo, and three associated with amino acid metabolism and upregulated in Combo. At day 120, the inoculated silages had more culturable lactic acid bacteria, higher Lactobacillus relative abundance, and lower Leuconostoc relative abundance than Control. The concentration of acetic acid remained low throughout ensiling in Control, but in LB and Combo, it increased up to day 60 and remained stable from day 60 to 180. The 1,2-propanediol was only detected in LB and Combo. Inoculation did not affect the concentration of starch, but starch digestibility was greater in Combo than in Control. Inoculation of HMC with Combo modified the gene expression and fermentation profile compared to Control and LB, improving starch digestibility compared to uninoculated HMC.

Mechanical skin lesions are a persistent issue under aquaculture operations compromising fish health and robustness. Using an established zebrafish model, the study compared the potential skin wound healing benefit of dietary supplementation with a single-strain yeast fraction rich in ll-glucans (ll-glucan diet) or with a multi-strain yeast fraction (MsYF). Adult zebrafish were divided into four triplicated groups (12 tanks; 25 fish/tank) as negative control (unwounded-basal diet); control (wounded-basal diet), ll-glucan (wounded-ll-glucan diet), and MsYF (wounded-MsYF diet) and, following 7-week of nutritional preparation, inflicted with a full-thickness skin wound using a biopsy punch. Wound healing was assessed macroscopically by measuring wound surface area (18 fish individually tracked/group) until full healing within 4 weeks; as well as by histopathological diagnostic, transcriptional and immunoblotting analysis at the wound site at 1, 4 and 9 day post-wound (dpw) over the early healing phase (re-epithelization and inflammation). Following an initial expansion, wound closure was first measured at 4 dpw in the ll-glucan and MsYF groups showing a positive and significantly higher daily wound closure rate compared to the control. Later at 16 dpw, cumulative and daily wound closure were significantly higher in the MsYF compared to the ll-glucan and control groups. Enhanced gross wound healing was confirmed by histopathological diagnosis of mitigated inflammation, earlier re-epithelization and significantly enhanced granulation tissue synthesis in the MsYF group. Targeted transcriptomic and proteomic markers documented an early up-regulation of matrix metalloproteinases and a dampening or quicker resolution of inflammatory and cellular stress markers in the healing tissues of the MsYF compared to the control group while the ll-glucan diet overall elicited intermediary results. Overall, this study demonstrates that dietary supplementation with a yeast-based functional compound can modulate fish immune response in wound healing and promote early wound closure indicating a role for skin health management under aquaculture operations.

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and β-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.