Postbiotic substance PS/Ent 412 and its beneficial application in Slovak warm-blood horses            

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Because of promising previous results with Enterocin M, we decided to assess the effectiveness of PS/Ent 412 produced by our autochthonous strain Enterococcus faecium EF 412. PS/Ent 412 was applied to 12 horses of various ages and breeds for 21 days in a bolus diet. Sampling was performed at day 0/1, days 21, and 42 (3 weeks of PS cessation). The horses served as their own controls. Following microbiota, when using next-generation sequencing, 15 phyla were detected at higher abundance percentage (%) with the highest % of abundance in the phylum Bacteroidetes (35.38%), followed by Firmicutes (32.25%, Proteobacteria (13.36%), Lentisphaera (10.03%), Spirochaetae (4.01%), and Fibrobacteres (2.01%). For the other phyla, the % of abundance is from 0.05 to 0.65%. After application of PS/Ent 412 (activity 51,200 AU/mL), at day 21 was noted a decrease of the % abundance of Firmicutes, Proteobacteria, Lentisphaera, Spirochetae, Cyanobacteria, Tenericutes, Synergistetes, and Fibrobacteres, while the % of abundance in Euryarcheota, Actinobacteria, Verrumicrobia, Saccharobacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 21, an increasing tendency of PA (69.00 ± 3.91%), was noted compared to day 0/1 (67.59 ± 3.66%). Glutathion-peroxidase was not negatively affected. The values of the enzymatic, hepatic, energy, mineral, and nitrogen profiles were mostly optimized within the physiological range, as were those of the hematological parameters. Although it is necessary to continue testing, so far, postbiotics are a promising new tool to support the healthy breeding of horses. Postbiotic substance Activity Horse Health Selected parameters Figures Figure 1 Introduction Beneficial (probiotic) bacteria are used widely as nutritional supplements and treatment interventions in the management of livestock and companion animals (Cooke et al., 2021). E.g., the immune response of the beneficial non-autochthonous strain Enterococcus faecium AL41 (CCM8558, accession number-AN-MW256492 in GenBank) was noted in chicks experimentally infected with Campylobacter jejuni CCM 6191. The strain AL41 predominantly acted 24 h post- infection with C. jejuni (Revajová et al., 2020 ). It displayed a distinct manner of T and B cell activation. Antimicrobial activity of the strain AL41 and its postbiotic substance PS/Ent M was noticed in faeces and caecum of broiler rabbits against coliforms (p < 0.05), pseudomonads, and S. aureus (Lauková et al., 2012). In spite of the fact that probiotic use in horses is still limited, E. faecium AL41 (CCM 8558) demonstrated, after 14 days of its application, an increasing tendency in phagocytic activity (PA). Also, hydrolytic activity was significantly increased (P < 0.001; Lauková et al., 2020). Moreover, the application of the autochthonous, beneficial strain E. faecium EF 412 (AN MW256494 in GenBank) in horses also demonstrated a tendency to increase PA. Cholesterol and triglycerides in blood sera were decreased at day 21 compared to day 0/1 and day 35 (2 weeks after the strain cessation, Lauková et al., 2023). Those mentioned beneficial strains produce antimicrobially active postbiotic substances of proteinaceous character (bacteriocins) (Franz et al., 2007 ; Nes et al., 2014 ; Lauková et al., 2018, 2023). Although the use of postbiotic substances in horse breeding and/or diet is not frequent, as reported in our previous study, administration of PS/Ent M led to a mathematical reduction of coliforms, campylobacters, and a significant reduction of Clostridium spp. (p < 0.001). An increase in PA values was also noted at day 21 (p < 0.05), and at day 42 p < 0.001), showing the prolonging effect of PS/Ent M (Lauková et al., 2018). The last-mentioned E. faecium EF 412 is susceptible to antibiotics and exhibits sufficient adhesion to human and canine mucus. It was reported to possess genes for the production of enterocins EntA and EntB. The produced antimicrobial proteinaceous substance is thermostable (Lauková et al., 2008 ) and has not yet been purified to homogeneity; according to recent knowledge, it is assigned to the group of postbiotics. They are defined as preparations of inanimate microbiota and/or their components that confer a health benefit to the host (Salminen et al., 2021 ; Chikindas et al., 2025 ). Those antimicrobially effective proteinaceous substances purified to homogeneity are bacteriocins (Chikindas et al., 2025 ). From the perspective of intestinal health, probiotics and postbiotics play an important role, but compared to probiotics, postbiotics have several advantages; e.g., they can effectively act not only in the intestinal tract but also include the oral cavity, skin, etc. (Liang & Xing, 2023). Mosca et al. ( 2022 ) even reported that the postbiotics can improve diarrhea and the immune function of the host. Because of clinical evidence remains limited, we decided to apply the postbiotic substance (PS/Ent 412) produced by the strain E. faecium EF412 in Slovak warm-blood horses to control microbiota, PA, glutathion-peroxidase (GPx), biochemistry, and hematology. Given together, postbiotics can serve as a new tool to support the healthy breeding of horses. Material and Methods All procedures performed in the experiment were approved by the horses‘ owners and by the Ethical Committee of the Institute of Animal Physiology, Center of Biosciences of the Slovak Academy of Sciences in Košice (Slovakia, Apr. no. 339004). All manipulations were in accordance with standard veterinary practices as defined in Slovak legislation (nos. 377/2012, 436/2012), and horse owners agreed to the participation in this study. Preparation of postbiotic substance PS/Ent 412 for application PS/Ent 412 is produced by the autochthonous strain Enterococcus faecium EF 412 deposited in GenBank with accession number MW256494. For application purposes, the substance was prepared according to the protocol for dipeptide Enterocin A/P previously reported by Mareková et al. (2003) with modifications. Briefly, 300 mL of MRS broth (de Man-Rogosa-Sharpe broth, pH 7.1, Merck, Darmstadt, Germany) was inoculated with an overnight 0.1 % pre-culture of the producing strain E. faecium EF 412. The MRS broth inoculated with the strain EF 412 was incubated at 37 ˚C overnight until the absorbance at 600 nm (A 600) reached 1.0. The broth culture was centrifuged at 10,000 x g for 30 minutes. Supernatant (pH 5.0) was precipitated with ammonium sulphate (40% saturation) at 4 °C for 4 h. After centrifugation (10,000 x g for half an hour), the precipitate was removed and re-suspended in a minimal volume of 10 mM phosphate buffer (pH 6.5). Activity of the precipitate was tested using the agar spot test (De Vuyst et al., 1996). Active postbiotic substance PS/Ent 412 reached activity 51,200 AU/mL against the principal indicator strain E. avium EA5 (our strain). Horses and experimental design Twelve clinically healthy horses (six mares and six stallions, Slovak warm-blood breed) were involved in this experiment. They were owned by private clients who agreed with the experimentation. The horses were placed in the stable (CE SK 339004) of the Riding Centre of the Slovak Agricultural University (SAU) in Nitra (Slovakia) under the supervision of Prof. Marko Halo, accepting the Guide for Animal Practice approved by the Ethics Committees of Institutes participating in the experiment (Apr. No. 339004) as previously indicated by Lauková et al. (2023). The horses were placed in separate boxes and ranged of age, as previously reported by Lauková et al. (2023). The animals involved in the experiment were: a 12-years –old stallion (no.1), a 8-year-old mare (no. 2), a 11-years-old mare (no. 3), a 12-years-old mare (no. 4), a 5-years –old horse (no.5); a 7-years-old mare (no. 6), a 5-years old horse (no. 7), a 5-years –old mare (no. 8), a mare aged 10 years (no. 9), a 8 years-old horse (no. 10), a 9-years –old horse (no.11), and horse aged 13 years (no.12). They were fed twice a day with hay and whole oats or grazed with access to water ad libitum (using automatic nipples). The experiment lasted for 42 days. Sampling was performed at the start of the experiment (at day 0/1), at day 21 (3 weeks of PS/Ent 412 application), and at the end of the experiment (at day 42, 3 weeks of PS/Ent 412 cessation). Feces were sampled in the morning after each animal defecation. Blood was sampled from the vena jugularis. Fecal samples were immediately stored in a transporting fridge box (4˚C). After transport to the laboratory, they were analysed, treated, and stored for appropriate analyses. Each horse served as a control animal for each separate sampling, meaning its status at the start of the experiment (day 0/1) was compared with its status after PS/Ent 412 (3 weeks, day 21) application and after its cessation (day 42). After initial sampling, the animals were administered PS/Ent 412 (100 µL) in a small feed ball to ensure that it was eaten by the specified horse. PS/Ent 412 was applied for 3 weeks (21 days). The animals had a standard drinking regimen. After the experiment, they were used by clients and stabled at SAU, Nitra (Slovakia). Microbiota analysis using standard microbiological test Feces (1 g) from each horse were treated using the standard microbiological dilution method (in Ringer solution, ratio 1:9, Merck, pH 7.0), homogenized using a Stomacher IUL (Instruments, Spain) according to the International Organization for Standardization (ISO). Volumes of 100 µL of appropriate dilution were plated on M- Enterococcus agar (ISO 7889, Difco, Detroit, USA) to enumerate the total enterococci. De Man-Rogosa-Sharpe broth supplemented with agar (1.5%, MRS, Merck, Darmstadt, Germany) was used to enumerate lactic acid bacteria (LAB). Baird-Parker agar supplemented with egg yolk tellurite solution (ISO 21527-1, Difco) was used to enumerate coagulase-positive staphylococci (CoPS). Coagulase-negative staphylococci (CoNS) were enumerated on Mannitol salt agar (Difco). Clostridium (Clostridioides) difficile agar with SR0096E supplement and 7% (v/v) defibrinated horse blood (SR0050, ISO 15883, Oxoid Ltd., Basingstoke, Hampshire, England) was used to detect Clostridium spp. Coliforms were detected on MacConkey agar (ISO 7402, Difco). Aeromonas spp. were counted using Aeromonas (Ryan) agar (Oxoid). The plates were cultivated at 37 ºC for 24-48 h. Pseudomonas spp. were enumerated on Pseudomonas agar (Biomark, India) after incubation at 30 ºC for 24 h. Campylobacters were selected on the Campylobacter selective medium-Karmali with supplement CM0935 (Oxoid, USA). In this case, cultivation was provided at 42 °C for 42-48 h. Bacterial counts were expressed in (log 10) colony-forming units per gram (CFU/g) ± SD. Microbiome analysed using next-generation sequencing The protocol used for this analysis, as well as the list of primers, was reported in our previous study (Lauková et al., 2023). There, Enterococcus faecium EF 412, producing PS/Ent412, was tested in horses. Briefly, isolated DNA (according to the manufacturer's protocol) using a PowerLyzerR PowerSoilR isolation kit (Quiagen, Germany) was used as a template in a PCR reaction targeting the hypervariable V3-V4 region (341F-785R) of the bacterial 16S rRNA gene (16S Metagenomic Sequencing Library Preparation protocol, Illumina, USA, Table 1). Sequencing was performed using MiSeq reagent Kits v3 on a MiSeq 2000 sequencer according to manufacturer's instructions (Illumina). Sequences were analyzed using the workflow provided by QIIME 1.9.1. (Aronetsy, 2011). Quality trimming using the Trimmomatic software (Bolger et al., 2014) was included in sequence processing, followed by demultiplexing (a house tool written in Python 3), and joining (Edgar et al., 2010). Operational taxonomic units (OTUs) were constructed by clustering sequences with a 97% sequence similarity threshold and selecting the representative sequences of clusters as OTUs. Clustering was conducted using the UCLUST algorithm (Caporaso et al., 2010). Taxonomy was assigned to each OTU using the USEARCH LCA algorithm, which is based on the SILVA 123 reference database (Quast, 2013) Phagocytic activity analysis , biochemical parameters Blood was sampled into Eppendorf tubes containing microspherical hydrophilic particles (MSHP) and heparin. Direct counting was carried out; ingestion of MSH particles by polymorphonuclear cells (PMN) was determined as follows: 50 mL of SH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100 mL of blood in an Eppendorf-type tube and incubated at 37°C for one h. Blood smears were stained using the May-Grünwald and Giemsa-Romanowski system. Phagocytic activity (PA) was calculated as the number of white cells containing at least three engulfed particles/100 white cells (neutrophils and monocytes). The percentage of phagocytic cells was evaluated using an optical microscope by counting PMN up to 100. Subsequently, the index of PA (IPA) was calculated. Regarding the biochemical parameters in blood/sera of horses, the following profiles were analysed: nitrogen profile (total proteins g/L, albumin g/L, urea g/L, creatinine µmol/L); energetic profile (trigylcerides and cholesterol in mmo/L), enzymatic and hepatic profile (AST, aspartate-aminotransferase,µkat/L; ALT, alanine-aminotransferase, µkat/L, ALP, alkalic phosphatase, µkat/L, and GMT(GGT, gama-glutamyl transferase, µkat/L; total bilirubin, µmmol/L; CK, creatine kinase, µkat/L; GLDH, glutamate dehydrogenase, µkat/L; LDH-L, lactate dehydrogenase, µkat/L.; and mineral profile (Ca-calcium mmol/L, P-phosphorus, Mg-magnesium, and chlorides in mmol/L). Testing was performed using the DiaSys kit (Diagnostic Systems GmbH, Holzheim, Germany) or the commercial tests DIALAB (Czech Republic) using the analyzer Ellipse (AMS, Italy). Minerals were analysed using an EasyLite analyzer via ion-selective electrode (Kolesárová et al., 2008). Hematological analyses were performed using the semi-automatic analyzer Randox RX Monza, as described by Kováčik et al. (2017). Statistical analyses Statistical evaluation of microbiota results was performed using one-way analysis of variance (ANOVA), and a paired t-test; the other parameters were evaluated using one-way ANOVA with the Tukey post hoc test . Data are expressed as the average and standard deviation of the mean (SD). Differences between mean values were considered statistically significant at p<0.05. Results Microbiota determination before PS/Ent 412 application, at days 21 (3 weeks of application) and 42 (3 weeks cessation) Microbiota counting using the standard microbiological method showed that the microbiota were mostly influenced (Table 2a). Regarding the Gram-positive bacteria, after 3 weeks (day 21), enterococci were significantly reduced (p<0.05) with a reincrease at day 42; however, the count remained lower than at the start of the experiment. The count of LAB was also decreased (p<0.01) compared to day 0/1 with day 21 and day 42 (Table 2a). Staphylococci (CoPS, CoNS) were not influenced, although in CoPS a mathematical difference (0.54, 0.74 log cycle) was noted between day 0/1 and days 21 and 42 as well (Table 2a). Clostridiae were influenced; however, their counts were higher at day 21 and also at day 42. In case of the Gram-negative genera, a significant decrease in Campylobacters was noted (Table 2b, p<0.001) when comparing day 0/1 and 42, and also days 21 and 42 (p<0.01). Pseudomonads were not significantly affected; only a slight decrease with differences of 0.67 and 1.01 cycles was observed at day 21 compared to days 0/1 and 42. Aeromonads were significantly reduced (p<0.01) at days 21 and 42 compared to day 0/1. Similarly, coliforms were significantly reduced at day 21 compared to day 0/1 (p<0.001) and also compared day 0/1 and day 42 (p<0.001 ; Table 2b). Following next-generation sequencing at the phylum level, 15 phyla were detected at a higher percentage of abundance (%, Table 3). The highest % of abundance was reached for the phylum Bacteroidetes (35.38%), followed by Firmicutes (32.25%, Table 3, Figure 1), Proteobacteria (13.36%), Lentisphaera (10.03 %), Spirochaetae (4.01%), and Fibrobacteres (2.01%). For the phyla Armatimonadetes, Planctomycetes, Saccharibacteria, Synergistetes, Tenericutes, Verrucomicrobia, Cyanobacteria, Actinobacteria, and Euryarcheota, % of abundance ranged from 0.05 to 0.65% (Table 3). Application of PS/Ent 412 at day 21 led to a decrease of Firmicutes (28.10%), Proteobacteria (8.19%), Lentisphaera (8.83%), Spirochetae (3.34%, Cyanobacteria (0.47%), Tenericutes (0.16%), and Fibrobacteres (1.32%; Table 3); while % of abundance in Euryarcheota, Actinobacteria, Verrumicrobia, Saccharibacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 42 (3 weeks of PS/Ent 412 cessation), Bacteroidetes decreased, and the prolonged effect of PS/Ent 412 was observed for Euryarcheota, Actinobacteria, Verrucomicrobia, Synergistetes, and Saccharibacteria; the other phyla were not influenced; they remained at almost the same levels as at day 21. On the other hand, Fibrobacteres, Proteobacteria, and Lentisphaerae were increased (Table 3). Phagocytic activity analysis, GPx, biochemical parameters The PA and IPA values were well-balanced (Table 4); however, at day 21, an increasing tendency of PA was noted (Table 4), showing a non-significant (p=0.3762) but mathematical increase from 67.59 ± 3.66 % at day 0/1 to 69.00 ± 3.91% at day 21. At day 42, PA values returned to almost the same value as measured at day 0/1 (Table 4). GPx was not negatively affected, with a non-significant effect (p=0.3291) and well-balanced values, only with a slight change at day 21 (Table 4). R eg arding the enzymatic and hepatic profile in blood sera of horses, the value of AST was significantly decreased at day 21 (p<0.05, Table 5) compared to day 0/1 and also day 42. The AST values at day 0/1 and 3 weeks after cessation of PS/Ent412 were nearly identical. However, during the experiment, they reached values close to the upper limit of the reference value for AST (Table 5). PS/Ent412 influenced the effect of the enzyme ALT beneficially because a mathematical decrease (differences 0.82 and 0.95) of the ALT at day 21 was noted (Table 5) compared with day 0/1 and day 42 (Table 5). In this case, values measured during the experiment were in the physiological range. The decrease of the enzyme ALP was also noted, with a prolonged decline (Table 5), although the values were in the physiological range. The values of GMT/GGT were also within the physiological range but decreased slightly after PS/Ent412 administration (Table 5). Significantly reduced activity of the enzyme LDH-L was noted at day 21 (p<0.001) compared to day 0/1 and day 42 (p<0.05; Table 5). The GLDH values were slightly elevated, but remained within the physiological range (Table 5). The CK values were within the physiological range; however, after PS/Ent412 application (day 21), they were slightly decreased (difference 1.22) and then increased to almost the same value as at day 0/1 (Table 5). Moreover, the total bilirubin value was slightly decreased, within the physiological range. Regarding the energetic profile, HDL cholesterol decreased slightly, but LDL cholesterol was significantly lower at day 21 than at day 0/1 (p <0.01), although both were higher than the physiological range. The parameters in the nitrogen profile are well balanced, as are albumin values and urea (Table 7). The values of creatinine were increased, but in the physiological range. The mineral profile was characterized by a decrease of P, a slight decrease of Ca compared to day 0/1 and/or day 42, and a significant decrease of Mg at day 42 (p<0.001), with a slight increase at day 21. Chlorides were also significantly (p<0.01) increased at day 21 with a subsequent decrease to the level noted at day 0/1 (Table 7). Regarding the hematological parameters (Table 8), their values were mostly in the physiological range (Table 8); the average count of erythrocytes (MCV) was slightly increased but still in the physiological range, as thrombocytes were increased (100.0) when at day 21 were under the physiological limit of 74.00. Discussion The advantage of postbiotics lies in their great potential to maintain intestinal homeostasis and health by retarding the growth of intestinal pathogens (Divsalar et al., 2026). They help to decrease the population of bacteria, in general, including non-requested bacteria (Salminen et al., 2021 ). It was reported that the bacterial postbiotic derived from the commercially used probiotic strain Lactobacillus rhamnosus GG could prevent human intestinal smooth muscle cells from the damage caused by intestinal pathogens (Cicenia et al., 2016 ). PS/Ent 412 application in horses influenced microbiota. Enterococci were significantly decreased (p < 0.05), and the count of LAB was decreased (p < 0.05) using the standard microbiological method. The count of staphylococci was not influenced, or it only slightly decreased. Regarding Gram-negative bacteria, campylobacters were significantly decreased (p < 0.001), as were coliforms and aeromonads (p < 0.001). A decrease in pseudomonads was also observed. Similar results were reported in our previous study (Lauková et al., 2018), in which PS/Ent M was applied in horses, and a mathematical reduction of coliforms, a significant reduction of campylobacters (p < 0.05), and clostridia (p<.001) was noted. Although in our application experiment, only semi-purified PS/Ent 412 was used in horses, it showed the characteristics of the II. class of bacteriocins-enterocins, meaning small thermo-stable peptides which are known with a broad antimicrobial spectrum (Franz et al., 2007 ; Nes et al., 2014 ). As previously reported, purified Ent M to homogeneity (but used in semi-purified form in the experiment) showed a similar antimicrobial effect in horses, and it belongs to the same class of bacteriocins. The phyla Bacteroidetes and Firmicutes reached the highest % of abundance in horses after PS/Ent 412 application, which correlates with findings of Dougal et al. ( 2017 ). The % of abundance was almost the same for Firmicutes as in our study (32.25%, 28.10%), respectively. They also detected Spirochaetae, Fibrobacteres, and Actinobacteria at % of abundance of < 4% (Dougal et al., 2017 ). Application of the postbiotic substance means an advantage because PS is safe, and it does not need to declare its safety specifically, as is the case of probiotic/beneficial bacteria (Piskoríková et al., 2010). Up to now, in our case, postbiotics have been administered in healthy horses; however, their application and effects in diseased horses could underscore their antimicrobial efficacy and their stimulation of non-specific immunity parameters, as phagocytic activity. In our study, PA had a tendency to increase after PS/Ent 412 application. The stimulated effect on PA was also previously measured after PS/EntM application (p < 0.05; p < 0.001; Lauková et al., 2018). Similarly to PS/Ent 412 in this study, the application of PS/Ent 412-producing strain E. faecium EF 412 in horses tends to increase PA ( Lauková et al., 2023 ). Application of PS/Ent 412 possessed a beneficial influence on selected parameters of the enzymatic and hepatic profile. Compared to day 0/1, AST was decreased (p < 0.05). Also, significantly lower values of the enzyme LDH (p < 0.001) indicate reparation processes of toxically damaged cells. There were also lower values of the enzymes ALT, GMT/GGT, ALP, creatine kinase, and total bilirubin, as well as a significant decrease of LDL-cholesterol (p < 0.01). In the nitrogen profile, there was no stimulating effect, but in the mineral profile, Ca and Mg were increased. Minerals play a critical role in the health of horses. Higher Ca is a useful parameter, and Mg utilization decreases with aging. So, the age of the horse is important in this parameter (Gálik et al., 2012 ). A decrease of total protein indicates a decrease in protein synthesis. Hisaeda et al. (2024) reported that the season period can influence the concentration of minerals and other parameters in horses' serum. They found that creatinine, total protein, P, and Mg were higher in summer. Ca, chloride, albumin, cholesterol, hemoglobin, and hematocrit were lower in summer. Our sampling was performed in the spring, so we found some values that were similar and others that differed. The results obtained represent a new contribution to the limited research published to date on the efficacy and tolerability of postbiotics in equids (Cooke et al., 2021). Conclusions In 12 horses of various ages and breeds, using next-generation sequencing, 15 phyla in higher percentage (%) of abundance were detected. The others were in a slight % of abundance. After PS/Ent 412 application (activity 51,200 AU/mL) for 21 days in a bolus diet, at day 21, a decrease of the % of abundance of Firmicutes, Proteobacteria, Lentisphaera, Spirochetae, Cyanobacteria, Tenericutes, and Fibrobacteres was noted, while the % of abundance in Euryarcheota, Actinobacteria, Verrucomicrobia, Saccharibacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 21 (after 3 weeks of PS/Ent 412 application), an increasing tendency of phagocytic activity was noted compared to day 0/1. Glutathion-peroxidase was not negatively affected. The values of the enzymatic, hepatic, energetic, mineral, and nitrogen profiles were mostly optimized within the physiological range, as were those of the hematological parameters. Although it is necessary to continue testing, postbiotics are a promising new tool to support the healthy breeding of horses. Declarations Funding The r esults were achieved with the support of the joint project SAS-PAS-2024-3 and partially by the Slovak Scientific Agency VEGA no. 2/0009/2025. Declaration of using generative artificial intelligence (AI) and AI-assisted technologies The authors declare they have not used any of the Generative AI tools in creating this article. CRediT authorship contribution statement Andrea Lauková: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing-original draft, Project administration, Funding; Valentína Focková:Methodology, Investigation; Lenka Kosečková Micenková:Methodology, Investigation, Data curation; Soňa Gancarčíková: Investigation, Methodology, Data curation; Iveta Plachá: Methodology; Ľubomíra Grešáková: Methodology, Data curation; Anton Kováčik: Methodology, Data curation; Marko Halo senior: Resources; Branislav Gálik: Resources; Monika Pogány Simonová: Methodology, Funding. Institutional Review Board Statement Sampling from the live animals was performed with the help of veterinarians and/or responsible workers at the Slovak Agricultural University in Nitra and analyzed at both institutions for diagnostic purposes, and approved by the Ethical Committee. All manipulations were performed in accordance with standard veterinary practices as defined by Slovak legislation (nos. 377/2012, 436/2012), and horse owners agreed to participate in this study. Data Availability Statement All data discussed are contained in the article. Acknowledgements The authors would like to thank Mrs. Dana Melišová for her skillful laboratory work. The language was controlled using the program Grammarly. Conflicts of competing interest All authors declare that they have no conflict of interest. References Aronesty E, ea-utils. (2011). Command-line tools for processing biological sequencing data.https://github.com/ExpressionAnalysis/ea-utils. Bolger AM, Lohse Usadel B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinfor 30, 2114-2120. Caparoso JG et al. (2010). QIIME allows analysis of high-throughput community sequencing data. Nat. Meth. 7, 335-336. https://doi.org/10.1038/nmeth.f.303. 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Brno 81, 113-117. doi: 10.2754/avb201281020113 Hisaeda K, Ono T, Shimokawa-Miyma T, Hata A, Iwata E, Hiasa Y, Ohzawa E, Tozaki T, Murase H, Takasu M, Nishii N, Kitagawa H. (2025). Differences in serum iron concentrations between the summer and winter in Noma horses. J. Equine Sci. 36, 1-9. Chikindas ML, Sichel LS, Popov IV, Tagg JR, Lu X, Mitrokhin OV, Todorov SD. (2025). Postbiotics: what are they? Ben. Microbes 1-5. doi: Kováčik A, Arvay J, Tušimová E, Harangozó Ľ, Tvrdá E, Zbynovská K, Cupka P, Andrašovská Š, Tomas J, Massányi P. (2017). Seasonal variations in the blood concentration of selected heavy metals in sheep and their effects on the biochemical and hematological parameters. Chemosphere 168, 365-371. Kolesárová A, Capcarová M, Arpášová H, Kalafová A, Massányi P, Lukáč N, Kováčik J, Schneidgenová M. (2008). Nickel induced blood biochemistry alterations in hens after an experimental per oral administration. Environ. Sci. Health Pt.B 43, 625-632. . Lauková A, Simonová M, Strompfová V, Štyriak I, Ouwehand AC, Várady M. (2008). Potential of enterococci isolated from horses. Anaerobe 14, 234-236. doi:10.1016/j.anaerobe.2008.04.002. Lauková A, Chrastinová Ľ, Pogány Simonová M, Strompfová V, Plachá I, Čobanová K, Formelová Z, Chrenková M, Ondruška Ľ. (2012). Enterococcus faecium AL41: Its Enterocin M and their beneficial use in rabbit husbandry. Prob. Antimicro. Prot . 4, 243-249. doi:10.1007/s12602-012-9118-7. Lauková A, Styková E, Kubašová I, Gancarčíková S, Plachá I, Mudroňová D, Kandričáková A, Miltko R, Belzecki G, Valocký I, Strompfová V. (2018). Enterocin M and its beneficial effects in horses-pilot experiment. Prob. Antimicro. Prot. 10, 420-426. doi:10.1007/s12602-018-9390-2. Lauková A, Styková E, Kubašová I, Strompfová V, Gancarčíková S, Plachá I, Miltko R, Belzecki G, Valocký I, Pogány Simonová M. (2020). Enterocin M-producing Enterococcus faecium CCM 8558 demonstrating probiotic properties in horses. Prob. Antimicro. Prot . 12, 1555 - 1561. https://doi.org/10.1007/s12602- 020-09655-6. Lauková A, Micenková L, Kubašová I, Bino E, Kandričáková A, Plachá I, Štrkolcová G, Gálik B, Kováčik A, Halo M, Pogány Simonová M. (2023). Microbiota, phagocytic activity, biochemical parameters and parasite control in horses with application of autochthonous, bacteriocin-producing, probiotic strain Enterococcus faecium EF 412. Prob. Antimicro. Prot. 15, 139-148. https://doi.org/10.1007/s12602-022- 09918-4 Liang Bing, Xing Dongming. (2023). The current and future perspectives of postbiotics. Prob.Antimicrob. Prot. 15, 1626 - 1643. https://doi.org/10.1007/s12602-023-10045-x. Mareková M, Lauková A, De Vuyst L, Skaugen M, Nes IF (2003). Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13. J. Appl. Microbiol. 94, 523 - 530. https://doi.org/10.1007/S12602. Mosca A, Abreu Y, Abreu AT, Gwee KA, Ianiro G, Tack J, Nguyen TVH et al. (2022). The clinical evidence for postbiotics as microbial therapeutics. Gut Microbes 14, 2117508. Nes IF, Diep DB, Moss MO. (2014). Enterococcal bacteriocins and antimicrobial proteins that contribute to niche control. In Enterococci from commensals to leading of drug resistant infection1-34. Piskoríková, M. Quality and characterization of existing and new probiotics (EFSA QPS). In Proceedings of regulatory Framework Workshop Heath: Claim Approval of Probiotics in the European union. Issues, Barriers, Success Drivers, June 18, 2010; Kosice, Slovakia. Revajová V, Karaffová V, Lavkutová M, Šefcová M, Lauková A, Levkut M. (2020). Reaction of immune cells to Campylobacter jejuni in chicken PBMC treated by different probiotic bacteria in vitro. Appro. Poultry Dairy Vet . 7, 649654. doi.10.31031/APDV.2020.07.000665. Salminen S, Collado MC, Endo A, Hill C, Lebeer S, Quigley EMM et al. (2021). The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 18, 649-667. Vetvička V, Farnousek L, Kopeček J, Kaminková J, Kašpárek L,Vránová M. (1982). Phagocytosis of human blood leucocytes, a simple micromethod. Immunol. Lett. 5, 97-100. Quast C. (2013). SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Tables Tables 1 to 8 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Slovakia","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"pogány","lastName":"Simonová","suffix":""}],"badges":[],"createdAt":"2026-03-03 14:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9021201/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9021201/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104781647,"identity":"f6803f15-e38b-4efe-9358-0dfc8de47df2","added_by":"auto","created_at":"2026-03-17 07:56:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58982,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobiota tested using next-generation sequencing at the phylum level (percentage of abundance)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9021201/v1/6ce43041652055e5d1986150.png"},{"id":106403095,"identity":"d44e05ea-e6df-481d-ac58-715b045fa44f","added_by":"auto","created_at":"2026-04-08 09:13:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":767426,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9021201/v1/c60fa950-d89e-48c1-b4f0-9a524476b231.pdf"},{"id":104780644,"identity":"5c661a43-6817-4392-b3b1-77d0458ba04e","added_by":"auto","created_at":"2026-03-17 07:53:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52310,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9021201/v1/0378eb91ce986bb8a1d27e0d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Postbiotic substance PS/Ent 412 and its beneficial application in Slovak warm-blood horses ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBeneficial (probiotic) bacteria are used widely as nutritional supplements and treatment interventions in the management of livestock and companion animals (Cooke et al., 2021). E.g., the immune response of the beneficial non-autochthonous strain \u003cem\u003eEnterococcus faecium\u003c/em\u003e AL41 (CCM8558, accession number-AN-MW256492 in GenBank) was noted in chicks experimentally infected with \u003cem\u003eCampylobacter jejuni\u003c/em\u003e CCM 6191. The strain AL41 predominantly acted 24 h \u003cem\u003epost-\u003c/em\u003einfection with \u003cem\u003eC. jejuni\u003c/em\u003e (Revajov\u0026aacute; et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It displayed a distinct manner of T and B cell activation. Antimicrobial activity of the strain AL41 and its postbiotic substance PS/Ent M was noticed in faeces and caecum of broiler rabbits against coliforms (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), pseudomonads, and \u003cem\u003eS. aureus\u003c/em\u003e (Laukov\u0026aacute; et al., 2012). In spite of the fact that probiotic use in horses is still limited, \u003cem\u003eE. faecium\u003c/em\u003e AL41 (CCM 8558) demonstrated, after 14 days of its application, an increasing tendency in phagocytic activity (PA). Also, hydrolytic activity was significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Laukov\u0026aacute; et al., 2020). Moreover, the application of the autochthonous, beneficial strain \u003cem\u003eE. faecium\u003c/em\u003e EF 412 (AN MW256494 in GenBank) in horses also demonstrated a tendency to increase PA. Cholesterol and triglycerides in blood sera were decreased at day 21 compared to day 0/1 and day 35 (2 weeks after the strain cessation, Laukov\u0026aacute; et al., 2023). Those mentioned beneficial strains produce antimicrobially active postbiotic substances of proteinaceous character (bacteriocins) (Franz et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nes et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Laukov\u0026aacute; et al., 2018, 2023). Although the use of postbiotic substances in horse breeding and/or diet is not frequent, as reported in our previous study, administration of PS/Ent M led to a mathematical reduction of coliforms, campylobacters, and a significant reduction of \u003cem\u003eClostridium\u003c/em\u003e spp. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). An increase in PA values was also noted at day 21 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and at day 42 p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), showing the prolonging effect of PS/Ent M (Laukov\u0026aacute; et al., 2018). The last-mentioned \u003cem\u003eE. faecium\u003c/em\u003e EF 412 is susceptible to antibiotics and exhibits sufficient adhesion to human and canine mucus. It was reported to possess genes for the production of enterocins EntA and EntB. The produced antimicrobial proteinaceous substance is thermostable (Laukov\u0026aacute; et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and has not yet been purified to homogeneity; according to recent knowledge, it is assigned to the group of postbiotics. They are defined as preparations of inanimate microbiota and/or their components that confer a health benefit to the host (Salminen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chikindas et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Those antimicrobially effective proteinaceous substances purified to homogeneity are bacteriocins (Chikindas et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). From the perspective of intestinal health, probiotics and postbiotics play an important role, but compared to probiotics, postbiotics have several advantages; e.g., they can effectively act not only in the intestinal tract but also include the oral cavity, skin, etc. (Liang \u0026amp; Xing, 2023). Mosca et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) even reported that the postbiotics can improve diarrhea and the immune function of the host. Because of clinical evidence remains limited, we decided to apply the postbiotic substance (PS/Ent 412) produced by the strain \u003cem\u003eE. faecium\u003c/em\u003e EF412 in Slovak warm-blood horses to control microbiota, PA, glutathion-peroxidase (GPx), biochemistry, and hematology. Given together, postbiotics can serve as a new tool to support the healthy breeding of horses.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eAll procedures performed in the experiment were approved by the horses\u0026lsquo; owners and by the Ethical Committee of the Institute of Animal Physiology, Center of Biosciences of the Slovak Academy of Sciences in Ko\u0026scaron;ice (Slovakia, Apr. no. 339004).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eAll manipulations were in accordance with standard veterinary practices as defined in Slovak legislation (nos. 377/2012, 436/2012), and horse owners agreed to the participation in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of postbiotic \u0026nbsp;substance PS/Ent 412 for application\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ePS/Ent 412 is produced by the autochthonous strain \u003cem\u003eEnterococcus faecium\u003c/em\u003e EF 412 deposited in GenBank with accession number MW256494. For application purposes, the substance was prepared according to the protocol for dipeptide Enterocin A/P previously reported by Marekov\u0026aacute; et al. (2003) with modifications. Briefly, 300 mL of \u0026nbsp;MRS broth (de Man-Rogosa-Sharpe broth, pH 7.1, \u0026nbsp;Merck, Darmstadt, Germany) was inoculated with an overnight 0.1 % pre-culture of the producing strain \u003cem\u003eE. faecium\u003c/em\u003e EF 412. The MRS broth inoculated with the strain EF 412 was incubated at 37 ˚C overnight until the absorbance at 600 nm (A\u003csub\u003e600) \u0026nbsp;\u003c/sub\u003ereached 1.0. The broth culture was centrifuged at 10,000 \u003cem\u003ex g\u003c/em\u003e for 30 minutes. Supernatant (pH 5.0) was precipitated with ammonium sulphate (40% saturation) at 4 \u0026deg;C for 4 h. After centrifugation (10,000 \u003cem\u003ex g\u003c/em\u003e for half an hour), the precipitate was removed and re-suspended in a minimal volume of 10 mM phosphate buffer (pH 6.5). Activity of the precipitate was tested using the agar spot test (De Vuyst et al., 1996). Active postbiotic substance PS/Ent 412 reached activity 51,200 AU/mL against the principal indicator strain \u003cem\u003eE. avium\u003c/em\u003e EA5 (our strain).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Horses and experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwelve clinically healthy horses (six mares and six stallions, Slovak warm-blood breed) were involved in this experiment. They were owned by private clients who agreed with the experimentation. The horses were placed in the stable (CE SK 339004) of the Riding Centre of the Slovak Agricultural University (SAU) in Nitra (Slovakia) under the supervision of Prof. Marko Halo, accepting the Guide for Animal Practice approved by the Ethics Committees of \u0026nbsp;Institutes participating in the experiment (Apr. No. 339004) as previously indicated by Laukov\u0026aacute; et al. (2023). The horses were placed in separate boxes and ranged of age, as previously reported by Laukov\u0026aacute; et al. (2023). The animals involved in the experiment were: a 12-years \u0026ndash;old stallion (no.1), a 8-year-old mare (no. 2), a 11-years-old mare (no. 3), \u0026nbsp;a 12-years-old mare (no. 4), \u0026nbsp;a 5-years \u0026ndash;old horse (no.5); \u0026nbsp; a 7-years-old mare (no. 6), a 5-years old horse (no. 7), a 5-years \u0026ndash;old mare (no. 8), a mare aged 10 years (no. 9), \u0026nbsp;a 8 years-old horse (no. 10), a 9-years \u0026ndash;old horse (no.11), and horse aged 13 years (no.12). They were fed twice a day with hay and whole oats or grazed with access to water \u003cem\u003ead libitum\u003c/em\u003e (using automatic nipples). The experiment lasted for 42 days. Sampling was performed at the start of the experiment (at day 0/1), at day 21 (3 weeks of \u0026nbsp; PS/Ent 412 application), and at the end of the experiment (at day 42, 3 weeks of \u0026nbsp;PS/Ent 412 cessation). Feces were sampled in the morning after each animal defecation. Blood was sampled from the \u003cem\u003evena jugularis.\u003c/em\u003e Fecal samples were immediately stored in a transporting fridge box (4˚C). After transport to the laboratory, they were analysed, treated, and stored for appropriate analyses.\u0026nbsp;Each horse served as a control animal for each separate sampling, meaning its status at the start of the experiment (day 0/1) was compared with its status after PS/Ent 412 \u0026nbsp; (3 weeks, day 21) application and after its cessation (day 42). After initial sampling, the animals were administered PS/Ent 412 (100 \u0026micro;L) in a small feed ball to ensure that it was eaten by the specified horse. PS/Ent 412 was applied for 3 weeks (21 days). The animals had a standard drinking regimen. After the experiment, they were used by clients and stabled at SAU, Nitra (Slovakia).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobiota analysis using standard microbiological test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeces (1 g) from each horse were treated using the standard microbiological dilution method (in Ringer solution, ratio 1:9, Merck, pH 7.0), homogenized using a\u0026nbsp;Stomacher IUL (Instruments, Spain) according to the International Organization for Standardization (ISO). Volumes of 100 \u0026micro;L of appropriate dilution were plated on M-\u003cem\u003eEnterococcus\u003c/em\u003e agar (ISO 7889, Difco, Detroit, USA) to enumerate the total enterococci. De Man-Rogosa-Sharpe broth supplemented with agar (1.5%, MRS, Merck, Darmstadt, Germany) was used to enumerate lactic acid bacteria (LAB). Baird-Parker agar supplemented with egg yolk tellurite solution (ISO 21527-1, Difco) was used to enumerate coagulase-positive staphylococci (CoPS). Coagulase-negative staphylococci (CoNS) were enumerated on Mannitol salt agar \u0026nbsp; (Difco). \u003cem\u003eClostridium (Clostridioides) difficile\u0026nbsp;\u003c/em\u003eagar with SR0096E supplement and 7% (v/v) defibrinated horse blood (SR0050, ISO 15883, Oxoid Ltd., Basingstoke, Hampshire, England) was used to detect \u003cem\u003eClostridium\u003c/em\u003e spp. Coliforms were detected on MacConkey agar (ISO 7402, Difco). \u0026nbsp;\u003cem\u003eAeromonas\u003c/em\u003e spp. were counted using Aeromonas\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Ryan) agar (Oxoid). \u0026nbsp;The plates were cultivated at 37 \u0026ordm;C for 24-48 h. \u003cem\u003ePseudomonas\u0026nbsp;\u003c/em\u003espp. were enumerated on Pseudomonas agar (Biomark, India) after incubation at\u003cem\u003e\u0026nbsp;\u003c/em\u003e30 \u0026ordm;C for 24 h. Campylobacters were selected on the Campylobacter selective medium-Karmali with supplement CM0935 (Oxoid, USA). In this case, cultivation was provided at 42\u0026nbsp;\u0026deg;C for 42-48 h. Bacterial counts were expressed in (log 10) colony-forming units per gram (CFU/g) \u0026plusmn; SD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eMicrobiome analysed using next-generation sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol used for this analysis, as well as the list of primers, was reported in our previous study (Laukov\u0026aacute; et al., 2023). There, \u003cem\u003eEnterococcus faecium\u003c/em\u003e EF 412, producing PS/Ent412, was tested in horses. Briefly, isolated DNA (according to the manufacturer\u0026apos;s protocol) using a PowerLyzerR PowerSoilR isolation kit (Quiagen, Germany) was used as a template in a PCR reaction targeting the hypervariable V3-V4 region (341F-785R) of the bacterial 16S rRNA gene (16S Metagenomic Sequencing Library Preparation protocol, Illumina, USA, Table 1). Sequencing was performed using MiSeq reagent Kits v3 on a MiSeq 2000 sequencer according to manufacturer\u0026apos;s instructions (Illumina). Sequences were analyzed using the workflow provided by QIIME 1.9.1. (Aronetsy, 2011). Quality trimming using the Trimmomatic software (Bolger et al., 2014) was included in sequence processing, followed by demultiplexing (a house tool written in Python 3), and joining (Edgar et al., 2010). Operational taxonomic units (OTUs) were constructed by clustering sequences with a 97% sequence similarity threshold and selecting the representative sequences of clusters as OTUs. Clustering was conducted using the UCLUST algorithm (Caporaso et al., 2010). Taxonomy was assigned to each OTU using the USEARCH LCA algorithm, \u0026nbsp;which is based on the SILVA 123 reference database (Quast, 2013)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhagocytic activity analysis\u003cem\u003e,\u0026nbsp;\u003c/em\u003ebiochemical parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was sampled into Eppendorf tubes containing microspherical hydrophilic particles (MSHP) and heparin. Direct counting was carried out; ingestion of MSH particles by polymorphonuclear cells (PMN) was determined as follows: 50\u0026nbsp;mL of SH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100\u0026nbsp;mL of blood in an Eppendorf-type tube and incubated at 37\u0026deg;C for one h. Blood smears were stained using the May-Gr\u0026uuml;nwald and Giemsa-Romanowski system. Phagocytic activity (PA)\u0026nbsp;was calculated as the number of white cells containing at least three engulfed particles/100 white cells (neutrophils and monocytes).\u0026nbsp;The percentage of phagocytic cells was evaluated using an optical microscope by counting PMN up to 100. Subsequently, the index of PA (IPA) was calculated.\u003c/p\u003e\n\u003cp\u003eRegarding the biochemical parameters in blood/sera of horses, the following profiles were analysed: nitrogen profile (total proteins g/L, albumin g/L, urea g/L, creatinine \u0026micro;mol/L); energetic profile (trigylcerides and cholesterol in mmo/L), enzymatic and hepatic profile (AST, aspartate-aminotransferase,\u0026micro;kat/L; ALT, alanine-aminotransferase, \u0026micro;kat/L, ALP, alkalic phosphatase, \u0026micro;kat/L, and GMT(GGT, gama-glutamyl transferase, \u0026micro;kat/L; total bilirubin, \u0026micro;mmol/L; CK, creatine kinase, \u0026micro;kat/L; GLDH, glutamate dehydrogenase, \u0026micro;kat/L; LDH-L, lactate dehydrogenase, \u0026micro;kat/L.; and mineral profile (Ca-calcium mmol/L, P-phosphorus, Mg-magnesium, and chlorides in mmol/L). Testing was performed using the DiaSys kit (Diagnostic Systems GmbH, Holzheim, Germany) or the commercial tests DIALAB (Czech Republic) using the analyzer Ellipse (AMS, Italy). Minerals were analysed using an EasyLite analyzer via ion-selective electrode (Koles\u0026aacute;rov\u0026aacute; et al., 2008). Hematological analyses were performed using the semi-automatic analyzer Randox RX Monza, as described by Kov\u0026aacute;čik et al. (2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical evaluation of microbiota results was performed using one-way analysis of variance (ANOVA), and a paired t-test; the other parameters were evaluated using one-way ANOVA with the Tukey post hoc test\u003cem\u003e.\u0026nbsp;\u003c/em\u003eData are expressed as the average and standard deviation of the mean (SD). Differences between mean values were considered statistically significant at p\u0026lt;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMicrobiota determination before PS/Ent 412 application, at days 21 (3 weeks of application) and 42 (3 weeks cessation)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobiota counting using the standard microbiological method showed that the microbiota were mostly influenced (Table 2a). Regarding the Gram-positive bacteria, after 3 weeks (day 21), enterococci were significantly reduced (p\u0026lt;0.05) with a reincrease at day 42; however, the count remained lower than at the start of the experiment. \u0026nbsp;The count of LAB was also decreased (p\u0026lt;0.01) compared to day 0/1 with day 21 and day 42 (Table 2a). Staphylococci (CoPS, CoNS) were not influenced, although in CoPS a mathematical difference (0.54, 0.74 log cycle) was noted between day 0/1 and days 21 and 42 as well (Table 2a). Clostridiae were influenced; however, their counts were higher at day 21 and also at day 42. In case of the Gram-negative genera, a significant decrease in Campylobacters was noted (Table 2b, p\u0026lt;0.001) when comparing day 0/1 and 42, and also days 21 and 42 (p\u0026lt;0.01). Pseudomonads were not significantly affected; only a slight decrease with differences of 0.67 and 1.01 cycles was observed at day 21 compared to days 0/1 and 42. \u0026nbsp;Aeromonads were significantly reduced (p\u0026lt;0.01) \u0026nbsp;at days 21 and 42 compared to day 0/1. Similarly, coliforms were significantly reduced at day 21 compared to day 0/1 (p\u0026lt;0.001) and also compared day 0/1 and day 42 (p\u0026lt;0.001\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003eTable 2b).\u003c/p\u003e\n\u003cp\u003eFollowing next-generation sequencing at the phylum level, 15 phyla were detected at a higher percentage of abundance (%, Table 3). The highest % of abundance was reached for the phylum Bacteroidetes (35.38%), followed by Firmicutes (32.25%, Table 3, Figure 1), Proteobacteria (13.36%), Lentisphaera (10.03 %), Spirochaetae (4.01%), and Fibrobacteres (2.01%). For the phyla Armatimonadetes, Planctomycetes, Saccharibacteria, Synergistetes, Tenericutes, Verrucomicrobia, Cyanobacteria, Actinobacteria, and Euryarcheota, % of abundance ranged from 0.05 to 0.65% (Table 3). Application of PS/Ent 412 at day 21 led to a decrease of Firmicutes (28.10%), Proteobacteria (8.19%), Lentisphaera (8.83%), Spirochetae (3.34%, Cyanobacteria (0.47%), Tenericutes (0.16%), and Fibrobacteres (1.32%; Table 3); while % of abundance in Euryarcheota, Actinobacteria, Verrumicrobia, Saccharibacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 42 (3 weeks of PS/Ent 412 cessation), Bacteroidetes decreased, and the prolonged effect of PS/Ent 412 was observed for Euryarcheota, Actinobacteria, Verrucomicrobia, Synergistetes, and Saccharibacteria; the other phyla were not influenced; they remained at almost the same levels as at day 21. On the other hand, Fibrobacteres, Proteobacteria, and Lentisphaerae were increased (Table 3).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhagocytic activity analysis, GPx, \u0026nbsp;biochemical parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PA and IPA values were well-balanced (Table 4); however, at day 21, an increasing tendency of PA was noted (Table 4), showing a non-significant \u0026nbsp;(p=0.3762) but mathematical increase from 67.59 \u0026plusmn; 3.66 % \u0026nbsp;at day 0/1 to 69.00 \u0026plusmn; 3.91% at day 21. At day 42, PA values returned to almost the same value as measured at day 0/1 (Table 4). GPx was not negatively affected, with a non-significant effect (p=0.3291) and well-balanced values, only with a slight change at day 21 (Table 4).\u003c/p\u003e\n\u003cp\u003eR\u003cstrong\u003eeg\u003c/strong\u003earding the enzymatic and hepatic profile in blood sera of horses, the value of AST \u0026nbsp;was significantly decreased at day 21 (p\u0026lt;0.05, Table 5) compared to day 0/1 and also day 42. The AST values at day 0/1 and 3 weeks after cessation of PS/Ent412 were nearly identical. However, during the experiment, they reached values close to the upper limit of the reference value for AST (Table 5). PS/Ent412 influenced the effect of the enzyme ALT beneficially because a mathematical decrease (differences 0.82 and 0.95) of the ALT at day 21 was noted (Table 5) compared with day 0/1 and day 42 (Table 5). In this case, values measured during the experiment were in the physiological range. The decrease of the enzyme ALP was also noted, with a prolonged decline (Table 5), although the values were in the physiological range. The values of GMT/GGT were also within the physiological range but decreased slightly after PS/Ent412 administration (Table 5). Significantly reduced activity of the enzyme LDH-L was noted at day 21 (p\u0026lt;0.001) compared to day 0/1 and day 42 (p\u0026lt;0.05; Table 5). The GLDH values were slightly elevated, but remained within the physiological range (Table 5). The CK values were within the physiological range; however, after PS/Ent412 application (day 21), they were slightly decreased (difference 1.22) and then increased to almost the same value as at day 0/1 (Table 5). Moreover, the total bilirubin value was slightly decreased, within the physiological range. Regarding the energetic profile, HDL cholesterol decreased slightly, but LDL cholesterol was significantly lower at day 21 than at day 0/1 (p \u0026lt;0.01), although both were higher than the physiological range. The parameters in the nitrogen profile are well balanced, as are albumin values and urea (Table 7). The values of creatinine were increased, but in the physiological range. The mineral profile was characterized by a decrease of P, a slight decrease of Ca compared to day 0/1 and/or day 42, and a significant decrease of Mg at day 42 (p\u0026lt;0.001), with a slight increase at day 21. Chlorides were also significantly (p\u0026lt;0.01) increased at day 21 with a subsequent decrease to the level noted at day 0/1 (Table 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the hematological parameters (Table 8), their values were mostly in the physiological range (Table 8); the average count of erythrocytes (MCV) was slightly increased but still in the physiological range, as thrombocytes were increased (100.0) when at day 21 were under the physiological limit of 74.00.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe advantage of postbiotics lies in their great potential to maintain intestinal homeostasis and health by retarding the growth of intestinal pathogens (Divsalar et al., 2026). They help to decrease the population of bacteria, in general, including non-requested bacteria (Salminen et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was reported that the bacterial postbiotic derived from the commercially used probiotic strain \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GG could prevent human intestinal smooth muscle cells from the damage caused by intestinal pathogens (Cicenia et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). PS/Ent 412 application in horses influenced microbiota. Enterococci were significantly decreased (p \u0026lt; 0.05), and the count of LAB was decreased (p \u0026lt; 0.05) using the standard microbiological method. The count of staphylococci was not influenced, or it only slightly decreased. Regarding Gram-negative bacteria, campylobacters were significantly decreased (p \u0026lt; 0.001), as were coliforms and aeromonads (p \u0026lt; 0.001). A decrease in pseudomonads was also observed. Similar results were reported in our previous study (Lauková et al., 2018), in which PS/Ent M was applied in horses, and a mathematical reduction of coliforms, a significant reduction of campylobacters (p \u0026lt; 0.05), and clostridia (p\u0026lt;.001) was noted. Although in our application experiment, only semi-purified PS/Ent 412 was used in horses, it showed the characteristics of the II. class of bacteriocins-enterocins, meaning small thermo-stable peptides which are known with a broad antimicrobial spectrum (Franz et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nes et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). As previously reported, purified Ent M to homogeneity (but used in semi-purified form in the experiment) showed a similar antimicrobial effect in horses, and it belongs to the same class of bacteriocins. The phyla Bacteroidetes and Firmicutes reached the highest % of abundance in horses after PS/Ent 412 application, which correlates with findings of Dougal et al. (\u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The % of abundance was almost the same for Firmicutes as in our study (32.25%, 28.10%), respectively. They also detected Spirochaetae, Fibrobacteres, and Actinobacteria at % of abundance of \u0026lt; 4% (Dougal et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Application of the postbiotic substance means an advantage because PS is safe, and it does not need to declare its safety specifically, as is the case of probiotic/beneficial bacteria (Piskoríková et al., 2010). Up to now, in our case, postbiotics have been administered in healthy horses; however, their application and effects in diseased horses could underscore their antimicrobial efficacy and their stimulation of non-specific immunity parameters, as phagocytic activity. In our study, PA had a tendency to increase after PS/Ent 412 application. The stimulated effect on PA was also previously measured after PS/EntM application (p \u0026lt; 0.05; p \u0026lt; 0.001; Lauková et al., 2018). Similarly to PS/Ent 412 in this study, the application of PS/Ent 412-producing strain \u003cem\u003eE. faecium\u003c/em\u003e EF 412 in horses tends to increase PA \u003cb\u003e(\u003c/b\u003eLauková et al., 2023\u003cb\u003e).\u003c/b\u003e Application of PS/Ent 412 possessed a beneficial influence on selected parameters of the enzymatic and hepatic profile. Compared to day 0/1, AST was decreased (p \u0026lt; 0.05). Also, significantly lower values of the enzyme LDH (p \u0026lt; 0.001) indicate reparation processes of toxically damaged cells. There were also lower values of the enzymes ALT, GMT/GGT, ALP, creatine kinase, and total bilirubin, as well as a significant decrease of LDL-cholesterol (p \u0026lt; 0.01). In the nitrogen profile, there was no stimulating effect, but in the mineral profile, Ca and Mg were increased. Minerals play a critical role in the health of horses. Higher Ca is a useful parameter, and Mg utilization decreases with aging. So, the age of the horse is important in this parameter (Gálik et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). A decrease of total protein indicates a decrease in protein synthesis. Hisaeda et al. (2024) reported that the season period can influence the concentration of minerals and other parameters in horses' serum. They found that creatinine, total protein, P, and Mg were higher in summer. Ca, chloride, albumin, cholesterol, hemoglobin, and hematocrit were lower in summer. Our sampling was performed in the spring, so we found some values that were similar and others that differed. The results obtained represent a new contribution to the limited research published to date on the efficacy and tolerability of postbiotics in equids (Cooke et al., 2021).\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn 12 horses of various ages and breeds, using next-generation sequencing, 15 phyla in higher percentage (%) of abundance were detected. The others were in a slight % of abundance. After PS/Ent 412 application (activity 51,200 AU/mL) for 21 days in a bolus diet, at day 21, a decrease of the % of abundance of Firmicutes, Proteobacteria, Lentisphaera, Spirochetae, Cyanobacteria, Tenericutes, and Fibrobacteres was noted, while the % of abundance in Euryarcheota, Actinobacteria, Verrucomicrobia, Saccharibacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 21 (after 3 weeks of PS/Ent 412 application), an increasing tendency of phagocytic activity was noted compared to day 0/1. Glutathion-peroxidase was not negatively affected. The values of the enzymatic, hepatic, energetic, mineral, and nitrogen profiles were mostly optimized within the physiological range, as were those of the hematological parameters. Although it is necessary to continue testing, postbiotics are a promising new tool to support the healthy breeding of horses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cstrong\u003e\u0026nbsp;r\u003c/strong\u003eesults were achieved with the support of the joint project SAS-PAS-2024-3 and partially by the Slovak Scientific Agency VEGA no. 2/0009/2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of using generative artificial intelligence (AI) and AI-assisted technologies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have not used any of the Generative AI tools in creating this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAndrea Laukov\u0026aacute;: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing-original draft, Project administration, Funding; Valent\u0026iacute;na Fockov\u0026aacute;:Methodology, Investigation; Lenka Kosečkov\u0026aacute; Micenkov\u0026aacute;:Methodology, Investigation, Data curation; Soňa Gancarč\u0026iacute;kov\u0026aacute;: Investigation, Methodology, Data curation; Iveta Plach\u0026aacute;: Methodology; Ľubom\u0026iacute;ra Gre\u0026scaron;\u0026aacute;kov\u0026aacute;: Methodology, Data curation; Anton Kov\u0026aacute;čik: Methodology, Data curation; Marko Halo senior: Resources; Branislav G\u0026aacute;lik: Resources; Monika Pog\u0026aacute;ny Simonov\u0026aacute;: Methodology, Funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Sampling from the live animals was performed with the help of veterinarians and/or responsible workers at the Slovak Agricultural University in Nitra and analyzed at both institutions for diagnostic purposes, and approved by the Ethical Committee. All manipulations were performed in accordance with standard veterinary practices as defined by Slovak legislation (nos. 377/2012, 436/2012), and horse owners agreed to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All data discussed are contained in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Mrs. Dana Meli\u0026scaron;ov\u0026aacute; for her skillful laboratory work. The language was controlled using the program Grammarly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of \u0026nbsp;competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAronesty E, ea-utils. (2011). 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SILVA ribosomal RNA gene database project: improved data processing and web-based tools. \u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Postbiotic substance, Activity, Horse, Health, Selected parameters","lastPublishedDoi":"10.21203/rs.3.rs-9021201/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9021201/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany beneficial strains can produce antimicrobially active proteinaceous postbiotic substances (PS) and/or bacteriocins. Because of promising previous results with Enterocin M, we decided to assess the effectiveness of PS/Ent 412 produced by our autochthonous strain \u003cem\u003eEnterococcus faecium\u003c/em\u003e EF 412. PS/Ent 412 was applied to 12 horses of various ages and breeds for 21 days in a bolus diet. Sampling was performed at day 0/1, days 21, and 42 (3 weeks of PS cessation). The horses served as their own controls. Following microbiota, when using next-generation sequencing, 15 phyla were detected at higher abundance percentage (%) with the highest % of abundance in the phylum Bacteroidetes (35.38%), followed by Firmicutes (32.25%, Proteobacteria (13.36%), Lentisphaera (10.03%), Spirochaetae (4.01%), and Fibrobacteres (2.01%). For the other phyla, the % of abundance is from 0.05 to 0.65%. After application of PS/Ent 412 (activity 51,200 AU/mL), at day 21 was noted a decrease of the % abundance of Firmicutes, Proteobacteria, Lentisphaera, Spirochetae, Cyanobacteria, Tenericutes, Synergistetes, and Fibrobacteres, while the % of abundance in Euryarcheota, Actinobacteria, Verrumicrobia, Saccharobacteria, Planctomycetes, and Armatimonadetes was slightly increased. At day 21, an increasing tendency of PA (69.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91%), was noted compared to day 0/1 (67.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66%). Glutathion-peroxidase was not negatively affected. The values of the enzymatic, hepatic, energy, mineral, and nitrogen profiles were mostly optimized within the physiological range, as were those of the hematological parameters. Although it is necessary to continue testing, so far, postbiotics are a promising new tool to support the healthy breeding of horses.\u003c/p\u003e","manuscriptTitle":"Postbiotic substance PS/Ent 412 and its beneficial application in Slovak warm-blood horses ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 15:33:49","doi":"10.21203/rs.3.rs-9021201/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32af82ee-0d25-4a74-8800-f62a58c69450","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-05T22:53:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 15:33:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9021201","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9021201","identity":"rs-9021201","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}