Feed supplement or feed additive, feed premix or feed for animals of the family suidae

Abstract

The present invention relates to a probiotic, feed supplement or feed additive, feed premix or feed for animals of the family Suidae, said probiotic or animal feed comprising a strain of Weizmannia faecalis and a strain of Bacillus licheniformis.

Description

[0001] Düsseldorf, 08 July 2024 Our reference: BD42712 ----------------------------------------------------------------------------------------------------------------- Biochem Zusatzstoffe Handels- u. Produktionsgesellschaft mbH ----------------------------------------------------------------------------------------------------------------- Feed supplement or feed additive, feed premix or feed for animals of the family Suidae ----------------------------------------------------------------------------------------------------------------- Field of the invention The present application relates to a probiotic, feed supplement or feed additive, feed premix or feed for animals of the family Suidae. Incorporation by Reference All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there are any inconsistencies between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended. Introduction Worldwide, the swine livestock sector drives more than one-third of produced meat and is a major part of agricultural economics. However, intestinal disease in livestock animals causing huge economic losses in swine industry. Therefore, the support of intestinal health in livestock animals is widely accepted as crucial parameter linked to higher feed efficiency, farm sustainability, and competitiveness of the global swine industry. Traditionally, antibiotics and pharmacological dosages of zinc oxide have been used to prevent gut dysbioses and maintain animal performance (Kirchhelle 2018; Bonetti et al. 2021). However, the concern about antibiotic resistant bacteria, especially in human medicine, and the ban of pharmacological levels of zinc oxide in several regions worldwide, mainly due to environmental concerns, pushes for more environmentally sustainable alternatives (Kirchhelle 2018; Ben et al. 2019; Bonetti et al. 2021). It is therefore an object of the present invention to provide means and methods to support the health of animals of the family Suidae. It is another object of the present invention, to provide value added feed for animals of the family Suidae. It is another object of the present invention, to increase feed efficiency of animals of the family Suidae. The present invention addresses these and other issues. Brief description of the Figures Fig. 1. shows effects of the probiotic according to the present invention on sow performance, litter quality, and suckling piglets Fig. 2 shows effects of the probiotic according to the present invention on growth performance and feed conversion of weaned piglet Fig. 3 shows E. coli inhibition in vitro and in vivo by W. faecalis DSM 32016 and its metabolites. Asteriks indicate significant differences in comparison to negative control (p ≤ 0.05). In vitro: Specific growth rate of pathogenic diarrhea-causing E. coli PS79 and E.coli EcA without (-) and with (+) buffered supernatnat of W. feacalis. E. coli strains were isolated from piglets. In vivo: Data based on mean value of fecal sample analysis after pre-starter, starter I, and starter II feeding phase in weaned piglets. Detailed Description of the Invention Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values. It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done. Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions. According to a first aspect of the invention, a probiotic, feed supplement or feed additive, feed premix or feed for animals of the family Suidae, is provided, said probiotic or animal feed comprising at least a) a strain of Weizmannia faecalis, and b) a strain of Bacillus licheniformis. Members of Weizmannia, previously allocated to Bacillus, are lactic acid producing spore- forming bacteria. The type species, Bacillus coagulans, was first identified as a spoilage agent of milk products. Weizmannia can be used in the production lactic acid from cellulose and hemicellulose acid hydrolysate. Weizmannia also produces other commercially important chemicals like cyclodextrins and amylases. Bacillus licheniformis is a bacterium commonly found in the soil. It is found on bird feathers, especially chest and back plumage, and most often in ground-dwelling birds (like sparrows) and aquatic species (like ducks). High capacity of secretion of the alkaline serine protease has made Bacillus licheniformis one of the most important bacteria in industrial enzyme production. However, Bacillus licheniformis has been found to cause infection in several cases of immunocompromised patients. Generally, probiotics are defined as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (WHO and FAO, 2001). In animal feed, probiotics are typically used to improve gain weight and feed conversion ratio. Due to their beneficial effect on health and stimulation of growth, probiotics are broadly used particularly for pigs and poultry. That type of formulas may contain one or more selected strains of microorganisms, and depending on the species and age of host animals they may be administered as a powder, suspension, capsules, pellet, gel or paste. They are used periodically or constantly, directly as an additive to feed and premixes. Administration of probiotic strains, both individual and combined, has been reported to have a significant effect on absorption and utilisation of feed, daily increase of body weight and total body weight of various animals, including turkeys, chicken, piglets, sheep, goats, cattle, and horses. An addition of probiotic microorganisms to feed results in improved quantity and quality of milk, meat and eggs. As a result, probiotics are well established for being used in commercially relevant livestock, and their use results in improved commercial performance thereof. Weizmannia faecalis DSM32016 has been used as feed additive for fattening piglets as well as birds, including chicken (EFSA Journal 2020;18(6):6158). It has proven effective in weaned piglets and chickens for fattening at 1x109CFU / kg complete feed. This conclusion was extended to suckling piglets, and to other birds for fattening and ornamental birds at the same use level. A specific Weizmannia faecalis DSM32016is approved, inter alia, in Germany, as feed additive for piglets as well as birds, under reference no 4b1900. The inventors of the present application have surprisingly shown that the novel probiotic combination of Weizmannia faecalis and Bacillus licheniformis has beneficial effects on inter alia Food Conversion Ratio in animals of the family Suidae. The inventors’ findings presented herein indicate that the novel probiotic combination of Weizmannia faecalis and Bacillus licheniformis in animals of the family Suidae improves, inter alia, litter quality and growth performance of suckling piglets. Furthermore, the inclusion of this probiotic combination in feed for weaned piglets enhanced their growth performance and feed conversion. Based on these results, the combination of Weizmannia faecalis and Bacillus licheniformis can be considered as environmentally friendly feed additive improving piglet performance and feed efficiency as well as farm profitability and sustainability. A probiotic comprising Weizmannia faecalis DSM 32016 (previously allocated to B. coagulans) and Bacillus licheniformis was recently tested in poultry, where it increased production efficacy by enhancing growth performance and feed intake (Elleithy et al. 2023). However, Mammalia and Aves show major differences regarding their physiques, metabolism, immune system, female reproductive system (egg production), as well as gastro-intestinal tract (Scanes 2020). For example, the gut passage time differs tremendously between swine and poultry in livestock production. Whereas the ingesta needs approximately 40 hours to pass the gastrointestinal tract in swine (Wilfart et al., 2007), in poultry the mean transit time is 3 to 5 hours (Hughes, 2008). Furthermore, the general biological strategy to extract energy from ingesta is significantly different between poultry and swine. Poultry is dependent on ceacal fermentation of fine particle and soluble compounds, whereby larger particle are quickly excreted by the cloaca. In marked contrast, swine digestion is mainly based on an extensive microbial fermentation in the colon (up to 9 hours) including only a temporary residence in the cecum (Moran, 1982). These individual characteristics contributing to a selective pressure shaping the intestinal microbial abundance, location, composition and activity. In particular, in poultry the highest microbial load is found in the ceca, whereas in swine the highest microbial load can be detected in the colon (Inoue et al., 2005). Intestinal differences regarding microbial composition are hard to define because the microbial community changes continuous (Torok et al., 2008). However, dominating bacterial families which are known to be representative for their host can be used for a more simple comparison. In this regard, the poultry gut is mainly colonized by Lactobacillaceae, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Enterobacteriaceae, and Peptostreptococaceae (Pourabedin and Zhao 2015), the pig gut shows dominating taxa such as Prevotellaceae, Ruminococcacea, Lactobacillaceae, Veillonellaceae, Paraprevotellaceae, Streptococcaceae, and Cuccinivibrionaceae, (Guevarra et al. 2019). The differences in microbial composition and associated host-microbe interactions between birds and swine animals can be also seen when focusing on the predominant bacterial pathogens. Whereas Clostridium perfringens and Enterococcus cecorum causing severe infections and high mortality rates in poultry (Lee and Lillehoj 2021), in swine especially enterotoxic Escherichia coli leading to severe diarrhea and hight mortality rates (Fairbrother et al. 2005). Based on these assumptions, poultry-based experiences cannot be used, or only partly used, to estimate the probiotic effect in swine. Furthermore, a probiotic combination of W. coagulans, B. subtilis, and life yeast has shown to modulate the gut microbiome and associated metabolic pathways in 45-day-old swine (Li et al. 2023). While effects of feeding this combination on intestinal microbiota and intestinal metabolites were reported, no data on efficacy regarding performance or feed conversion ratio were provided. As used herein the term “probiotic” relates to a composition comprising a live microorganism that is meant to be administered to an animal. As used herein the terms “feed supplement” and “feed additive” relate to products used in animal nutrition for purposes of improving the quality of feed. As used herein, the term “feed premix” relates to a mixture of feed additives or mixtures of one or more feed additives with feed materials or water used as carriers. The premix is meant to be added to regular feed. According to embodiments of the invention, wherein the strain of Weizmannia faecalis is Weizmannia faecalis DSM 32016 and / or the strain of Bacillus licheniformis strain of Bacillus licheniformis DSM 33806. This language explicitly encompasses (1) the combination of the thus specified strains of Weizmannia faecalis and Bacillus licheniformis as well as (2) Weizmannia faecalis DSM 32016 with an unspecified strain of Bacillus licheniformis and (3) Bacillus licheniformis DSM 33806 with an unspecified strain of Weizmannia faecalis. DSM stands for “Deutsche Sammlung von Mikroorganismen und Zellkulturen” (German Collection of Microorganisms and Cell Cultures GmbH). Strains archived at the DSM receive an identifier “DSMXXXX” can be obtained upon demand at the institute’s website https: / / www.dsmz.de / According to another aspect of the invention, a method for producing an animal feed premix or feed is provided. Said method comprises adding a strain of Weizmannia faecalis and a strain of Bacillus licheniformis to an animal feed premix or feed, wherein the animal feed premix or feed is for animals of the family Suidae. According to another aspect of the invention, a method for feeding an animal of the family Suidae is provided. Said method comprises administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis or a probiotic, feed supplement or feed additive, feed premix or feed according any one of claims 1 – 2 to said animal. According to embodiments of the above discussed different aspects of the invention, the animal is Sus scrofa, optionally Sus scrofa domesticus. According to embodiments of the above discussed different aspects of the invention, the animal is at least one of a sow, in particular a pregnant sow or a lactating sow, and / or a piglet, in particular a weaned piglet. According to embodiments of the above discussed different aspects of the invention, Weizmannia faecalis and / or Bacillus licheniformis is provided in the form of endospores. A spore is a dormant, tough, and non-reproductive structure produced by some bacteria associated to the family Bacillaceae. In a bacterium’s natural environment, spore formation is usually triggered by suboptimal environmental conditions such as a lack of nutrients or water. During sporulation, bacteria undergo an intricate sequence of cell differentiation resulting in a single, stress-resistant spore that allows a bacterium to survive unfavourable environmental conditions. The inner part of the spore contains of the bacterial DNA, ribosomes and large amounts of dipicolinic acid. In vitro, starting from vegetative bacterial cells, spore production can be induced according to methods as described for example in Elisashvilj et al (2019). Endospores can withstand harsh environments such as stomach acidity or heat and pressure during pelleting processes. When the environment becomes favorable, like in the animals’ intestine, the spores germinate into active probiotic cells. According to embodiments of the above discussed different aspects of the invention, Weizmannia faecalis and / or Bacillus licheniformis is present on the feed in a final concentration of between ≥ 1.0 x 107and ≤ 1.0 x1011CFU / kg. In embodiments, Weizmannia faecalis is present in the feed in a final concentration of ≥ 1 x 107, ≥ 3 x 107, ≥ 5 x 107, ≥ 7 x 107, ≥ 1 x 108, ≥ 3 x 108, ≥ 5 x 108, ≥ 7 x 108, ≥ 1 x 109, ≥ 1,5 x 109, ≥ 3 x 109, ≥ 5 x 109or ≥ 7 x 109CFU / kg. In embodiments, Weizmannia faecalis is present in the feed in a final concentration of ≤ 7 x 1011, ≤ 5 x 1011, ≤ 3 x 1011, ≤ 1 x 1011, ≤ 7 x 1010, ≤ 5 x 1010, ≤ 3 x 1010, ≤ 1 x 1010, ≤ 7 x 109, ≤ 5 x 109, ≤ 3 x 109, ≤ 1,5 x 109or ≤ 1 x 109CFU / kg. In embodiments, Weizmannia faecalis is present in the feed in a final concentration of 1 x 107CFU / kg, 5 x 107CFU / kg, 1 x 108CFU / kg, 5 x 108CFU / kg, 1 x 109CFU / kg, 1,5 x 109CFU / kg, 5 x 109CFU / kg, 1 x 1010CFU / kg, 5 x 1010CFU / kg, 1 x 1011CFU / kg or 5 x 1011CFU / kg. In one embodiment, Weizmannia faecalis is present in the feed in a final concentration of 1,5 x 109CFU / kg + / - 50 %, preferably + / - 40 %, + / - 30 %, + / - 20 %, or most preferably + / - 10 As used herein, the unit CFU / kg relates to colony-forming units per kg of complete feedstuff. CFU is a unit which estimates the number of microbial cells (bacteria, fungi, viruses etc.) in a sample that are viable, able to multiply via binary fission under the controlled conditions. The following table discloses preferred upper and lower limit combinations: Table 1: preferred combinations of upper and lower limit 9 7 7 7 7 8 8 0 9 0 8 8 919 0 9 1 0 x10 0 0 0 0 0 0x010 CFU / kg x1x 1x 1x 1x 1x 1x 1x 5,1x x 1x1 3≥5≥7≥1≥3 5≥ ≥5≥7≥1≥1≥3≥≥ 7≥ ≤ 7 x 1011X X X X X X X X X X X X X ≤ 5 x 1011X X X X X X X X X X X X X ≤ 3 x 1011X X X X X X X X X X X X X ≤ 1 x 1011X X X X X X X X X X X X X ≤ 7 x 1010X X X X X X X X X X X X X ≤ 5 x 1010X X X X X X X X X X X X X ≤ 3 x 1010X X X X X X X X X X X X X ≤ 1 x 1010X X X X X X X X X X X X X ≤ 7 x 109X X X X X X X X X X X X ≤ 5 x 109X X X X X X X X X X X ≤ 3 x 109X X X X X X X X X X ≤1,5 x 109 X X X X X X X X X≤1 x 109 X X X X X X X XAccording to another aspect of the invention, a method of improving feed conversion ratio and or increasing body weight in an animal of the family Suidae is provided. Said method comprises administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal. According to another aspect of the invention, a method of modulation the gut microbiome of an animal of the family Suidae is provided. Said method comprises administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal. As used herein, the term “modulating the gut microbiome” relates to a change of composition of the bacterial population within the animal’s gut. Preferably, such modulation has a beneficial effect on the animal’s feed conversion ratio, resulting in improved weight gain. According to another aspect of the invention, a method of stimulating the immune system of an animal of the family Suidae is provided. Said method comprises administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal. As used herein, the term “stimulating the immune system” relates to an overall improvement of the animal’s resistance against and / or capacity to fight diseases. In one aspect, such stimulation cam result in an increase of the of IgA antibodies in e,g, the serum or feces. IgA is the antibody subtype that plays a role in the immune function of mucous membranes. Increase of titer is indicative for an increased fitness of the animal’s immune system. According to another aspect of the invention, the use of a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or of a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, for (the manufacture of a medicament for) the treatment of an animal of the family Suidae is provided. This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent). Examples While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. Example 1 Taxonomic reclassification of the probiotic strain DSM 32016 to Weizmannia faecalis is attributed to substantial genetic differences, resulting in unique health-related potentials. Bacterial species are classified taxonomically based on genetic differences, utilizing methods such as comparative genomics. This classification system aligns closely with the unique characteristics linked to certain modes of actions exhibited by each species. For instance, within the realm of probiotics, this genetic variance between different species dictates specific modes of action. Certain strains may possess genes encoding enzymes crucial for feed digestion, enabling them to ferment dietary fibers or proteins and produce short-chain fatty acids. Conversely, other strains may lack this specific capability. Therefore, understanding these genetic disparities not only helps in (a) taxonomic classification but also in (b) gaining insight into the potential modes of action of different probiotic species that contribute to intestinal health and overall well-being. Table 2. ANI values (%) between DSM 32016 and type strains. Analysis conducted by BIOSAFE, Finland. Type Strains Identity to DSM 32016 (%) Weizmannia faecalis Marseille-P9853 98.6 Weizmannia coagulans DSM1 94.6 Weizmannia acidiproducens DSM 23148 76.1 Weizmannia ginsengihumi Gsoil 114 70.1 (a) Taxonomic Reclassification of DSM 32016: from Bacillus coagulans to Weizmannia faecalis The bacterial species “Bacillus coagulans” has always been considered extraordinary due to its special combination of skills. Therefore, previous taxonomic assignments and related designations never appeared to be quite fitting to classify this multifaceted species. Throughout history, designations such as Lactobacillus sporogenes, Bacillus dextrolacticus, Bacillus thermoacidificans, Bacillus thermoacidurans, Bacillus calidolactis, Lactobacillus cereale, and eventually Bacillus coagulans were utilized, with the latter officially recognized as the species' name. Even though the species was officially classified within the genus Bacillus, it was clear that Bacillus coagulans surpassed the typical attributes associated with Bacillus members – suggesting that the similarity with other Bacillus species is lower than expected. To reduce this genetical and physiological heterogeneity in the genus Bacillus a reclassification based on phylogenomic, and comparative genomic analyses was done in 2020. As result, all species not being closely related to Bacillus subtilis were reclassified into new genera, leading to a reassignment of Bacillus coagulans from the genera Bacillus to the new genera Weizmannia (= Weizmannia coagulans). Further analysis through Average Nucleotide Identity (ANI) and core genome alignment revealed that the strain DSM 32016 shares a closer genetic relationship (98.550% identity) with a recently discovered bacterium, Weizmannia faecalis, isolated from the intestine of a healthy individual (Kieu et al., 2022), than with the type species Weizmannia coagulans (94.646% identity). Considering the genetic dissimilarity and the species identification threshold set at 95% (EFSA, 2021), it is evident that DSM 32016 belongs to the novel species W. faecalis rather than W. coagulans. Recently, species within the genus Weizmannia underwent another round of reclassification, now assigned to the genus Heyndrickxia. As a result, the currently scientifically recognized species name for DSM 32016 is Heyndrickxia faecalis, while Weizmannia faecalis is accepted as a synonym. (b) Unique and health-related potentials of W. faecalis DSM 32016, distinguishing it from typical W. coagulans Genetic analysis has revealed several unique genes in the genome of W. faecalis DSM 32016, distinctly setting it apart from typical W. coagulans strains. For instance, the genome of DSM 32016 harbors unique genes related to feed digestibility such as Arabinogalactanase, Alpha- xylosidase, Acetylxylan esterase, Endo-1,4-beta-xylanase, Bifunctional alpha- galactosidase / sucrose kinase, and Oligo-1,6-glucosidase whereas these genes are absent in typical W. coagulans strains like DSM1 (type strain), XZL4, or H-1. This example underscores that genetic disparities exceeding 5% translate into distinct genetic capabilities, potentially influencing health-promoting properties and species-specific host interactions. Table 3. Digestive enzymes unique to DSM 32016 and their function related to feed digestibility. Digestive Enzyme Function Acetylxylan esterase Deacetylation of xylan and xylo- oligosaccharides Alpha-xylosidase Hydrolysis of terminal, non-reducing end of xylan (exo acting) Arabinogalactanase Hydrolysis of arabinogalactan and arabinogalactan-proteins Bifunctional alpha-galactosidase / sucrose Hydrolysis of alpha-galactosides derived kinase from sucrose (e.g., raffinose) Endo-1,4-beta-xylanase Hydrolysis xylan (endo acting) Conclusion: The comprehensive findings underscore distinct genetic disparities between DSM 32016 and the type strains of W. coagulans, warranting its categorization as a novel species, Weizmannia faecalis. As such, W. faecalis DSM 32016 represents the initial exploration of its kind within animals of the order Carnivora (and Suidae), targeting the support of intestinal health, feed conversion, and immune defense (as well as growth performance). Example 2 Materials and Methods Probiotic supplementation in all trials was based on the combination of W. faecalis DSM 32016 and B. licheniformis DSM 33806 and an inclusion rate of 1.5 x 109CFU per kg final feed. The probiotic product (TechnoCare®) was produced by Biochem Zusatzstoffe Handels- und Produktionsges. mbH (Lohne, Germany). Strains were obtained from DSM as vegetative bacterial cells, and spore production was induced according to methods as described for example in Elisashvili et al (2019) Furthermore, diets for lactating sows and weaned piglets were pelleted and formulated to be iso-energetic, iso-nitrogenous, and iso-aminoacidic in control and probiotic diets. Twice a day, all pens were checked for dead animals or animals with clinical symptoms. If necessary, animals were treated. Visually noticed effects, preventive, or therapeutic interventions were recorded. Date and reason (if identifiable) of death of sows or piglets per pen as well as dead body weight was documented. All animals were weekly monitored regarding clinical routine examination by the responsible veterinarian. Animals in this study were raised and treated according to Directive 2010 / 63 / EU of 22 September 2010 and the recommendation of Commission 2007 / 526 / CE for the accommodation and care of animals used for scientific purposes. Procedures and equipment were examined to fit regulations and standard operating procedures. Sow Trial I. Animals, housing, diets, and recorded parameters 7 days before expected farrowing, sows (Danbred x Pietrain) were transferred to the farrowing unit. Under consideration of live weight (Ø 266 kg), body condition score (BCS; Ø 3.1) and parity (Ø 3.3), a total of 160 sows were equally allocated to two experimental groups receiving feed either lacking probiotic (Control group) or including probiotic (Probiotic group). However, at trial start, one sow of the control group died of sudden death. Two sows, one in the control group and one in the probiotic group, were found not being pregnant. Thus, the documentation was based on 157 sows (n=78 for control group and n=79 probiotic group). Sows were kept in individual farrowing crates, each equipped with a heat bulb for piglets until weaning, 25 to 27 days after farrowing. Each crate was equipped with a separate feeder and nipple drinker for sows and piglets. Water was permanently available. Sow diets were mainly based on wheat, cereals bran, and barley (see Table 4 for feed formulation). Total trial duration was approximately 33 days per sow (7 days pre partum plus 25 to 27 days lactation period). Live weights of sows were recorded at trial start and (7 days pre-partum) at weaning of piglets (25 to 27 days post-partum). Individual feed consumption of sows was documented daily. Fecal consistency of sows was scored daily during the first 21 trial days. Score “0” was defined as normally formed, whereas score “1” was defined as obstipation. Number of born piglets (live or dead), mummies, and live weights at birth were documented. To achieve homogenous and healthy litters of 13 piglets per sow, piglets were re-allocated within the first 24 hours after birth between sows of the same group considering individual live weights. Weak and low weight piglets (≤ 0.7 kg) were placed with a nurse sow and excluded from the progressing trial. Prestarter for suckling piglets was provided at day 8 after birth until weaning and was mainly based on corn, soybean meal and skimmed milk powder (see Table 4 for feed formulation). Prestarter consumption was documented per litter on daily basis. Table 4. Lactation feed formulation (sow trial I). Control Probiotic Metabolizable energy (MJ / kg feed) 12.6 12.7 Starch (%) 38.4 37.7 Crude protein (%) 16.3 15.9 Water (%) 12.2 12.1 Crude fiber (%) 5.4 5.4 Crude ash (%) 5.3 5.2 Crude fat (%) 4.0 4.3 Lysine (%) 1.08 1.04 Table 5. Prestarter feed formulation for suckling piglets (sow trial I). Formulation Content Metabolizable energy (MJ / kg feed) 13.9 Crude protein (%) 24.0 Crude fat (%) 3.56 Sodium (%) 2.20 Crude fiber (%) 1.94 Lysine (%) 1.65 Methionine / Cysteine (%) 0.98 Threonine (%) 0.96 Calcium (%) 0.96 Phosphorus (%) 0.75 Digestible phosphorus (%) 0.48 Tryptophan (%) 0.37 Sow Trial II. Animals, housing, diets, and recorded parameters 7 days before expected farrowing, sows (Topigs Norsvin) were transferred to the farrowing unit. Under consideration of live weight (Ø 299 kg), body condition score (BCS; Ø 3.1) and parity (Ø 2.5), a total of 160 sows were equally allocated to two experimental groups receiving feed either lacking probiotic (Control group) or including probiotic (Probiotic group). Sows were kept in individual farrowing crates, each equipped with a heat bulb for piglets until weaning, 25 to 27 days after farrowing. Each crate was equipped with a separate feeder and nipple drinker for sows and piglets. Water was permanently available. Sow diets were mainly based on wheat, cereals bran, and barley (see Table 6 for feed formulation). Total trial duration was approximately 33 days per sow (7 days pre partum plus 25 to 27 days lactation period). Table 6. Lactation feed formulation (sow trial II). Control Probiotic Metabolizable energy (MJ / kg feed) 13.0 13.0 Starch 44.3 44.9 Crude protein 15.8 15.8 Water 13.3 12.5 Crude ash 4.4 4.5 Crude fiber 3.6 3.9 Crude fat 2.4 2.5 Lysine 0.93 0.97 Live weights of sows were recorded at trial start (7 days pre-partum) and at weaning of piglets (25 to 27 days post-partum). Individual feed consumption of sows was documented daily. Fecal consistency of sows was scored daily during the first 21 trial days. Score “0” was defined as normally formed, whereas score “1” was defined as obstipation. Number of born piglets (live or dead), mummies, and live weights at birth were documented. To achieve homogenous and healthy litters of 15 piglets per sow, piglets were re-allocated within the first 24 hours after birth between sows of the same group considering individual live weights. Weak and low weight piglets (≤ 0.7 kg) were placed with a nurse sow and excluded from the progressing trial. Prestarter for suckling piglets (Table 7) was provided at day 8 after birth until weaning and was mainly based on wheat, barley, soybean meal. Prestarter consumption was documented per litter on daily basis. Table 7. Prestarter feed formulation for suckling piglets (sow trial II). Formulation Content Crude protein 17.3 Crude fiber 3.50 Crude fat 2.80 Lysine 1.21 Calcium 0.70 Phosphorus 0.52 Methionine equiv. 0.38 Sodium 0.20 Piglet Trial I. Animals, diets, housing, and recorded parameters In total 576 weaned male and female 25-to-27-day old piglets (Danbred x Duroc) from 6 weaning groups (group of 96 piglets per week) were randomly allocated to two experimental groups either lacking probiotic (Control group) or including probiotic (Probiotic group) in standard feed. Initial average life weight of both groups was similar (6.85 kg / head). The trial was based on two feeding phases from day 1 to 14 and day 15 to end of trial (day 42). Diets were mainly based on corn, wheat, and barley (see Table 8 for feed formulation). The experimental station consisted of 32 pens. Every pen (1.18 m x 3.35 m) was equipped by a feeder and nipple drinkers. Feeders were filled manually. Water and feed were supplied ad libitum. Weaned piglets were weekly allocated to 8 pens (12 piglets per pen). Table 8. Feed formulation of piglet diet (piglet trial I). Feeding Phase I Feeding Phase II Feed Formulation Control Probiotic Control Probiotic Metabolizable energy (MJ / kg) 13.6 13.8 13.4 13.2 Starch (%) 40.4 41.4 44.5 44.1 Crude protein (%) 19.0 18.5 16.2 15.7 Water (%) 10.3 9.8 11.3 11.7 Crude ash (%) 5.4 5.2 5.5 5.5 Crude fat (%) 3.9 3.8 4.0 4.1 Crude fiber (%) 3.9 3.6 3.9 4.0 Average live weight (at day 1, 14, 42), total and daily weight gain (per feeding phase and entire trial period), feed consumption (per feeding phase and entire period), and incidence of diarrhea (twice per day; from day 1 to 21) was based on single pen evaluation. Diarrhea scoring system included scores from 1 (no diarrhea) to 5 (strong diarrhea). Feed conversion ratio was calculated for both feeding phases and the entire trial period, based on dividing the amount of feed consumed by the amount of live weight gained, and corrected for mortality. All animals were vaccinated against circovirus and mycoplasma. Piglet Trial II. Animals, diets, housing, and recorded parameters In total 360 weaned male and female 26-to-28-day old piglets (Danbred x Duroc) were randomly allocated to two experimental groups either lacking probiotic (Control group) or including probiotic (Probiotic group) in standard feed. Initial average life weight of both groups was similar (6.38 kg / head). The trial was based on two feeding phases from day 1 to 14 and day 15 to end of trial (day 42). Diets were mainly based on barley, extruded grains (cake meal), and wheat (see Table 9 for feed formulation). The experimental station consisted of 40 pens. Every pen (1.45 m x 2.46 m) was equipped by a feeder and nipple drinkers. Feeders were filled manually. Water and feed were supplied ad libitum. Weaned piglets were allocated to 20 pens per group (9 piglets per pen). Table 9. Feed formulation of piglet diet (piglet trial II). Feeding Phase I Feeding Phase II Control Probiotic Control Probiotic Metabolizable energy (MJ / kg feed) 13.6 13.5 13.7 13.8 Starch (%) 38.4 38.3 39.6 40.6 Crude protein (%) 18.7 18.9 18.7 18.4 Water (%) 11.7 11.7 12.5 12.7 Crude ash (%) 5.9 5.6 5.0 4.5 Crude fat (%) 3.5 3.7 4.5 4.3 Crude fiber (%) 3.0 3.4 3.1 2.9 Live weight at day 1, 14, 42, total weight gain as well as daily weight gain (per feeding phase and entire trial period) were measured individually. Feed consumption (per feeding phase and entire period) and incidence of diarrhea (twice per day; from day 1 to 21) was based on single pen evaluation. Diarrhea scoring system included scores from 1 (no diarrhea) to 5 (strong diarrhea). Feed conversion ratio was calculated for both feeding phases and the entire trial period, based on dividing the amount of feed consumed by the amount of live weight gained, and corrected for mortality. All animals were vaccinated against circovirus. Results Probiotic effect on sow performance, litter quality, and suckling piglets No medical treatments of sows or piglets were necessary. No obstipation was observed in sows. All sows had normal fecal consistency during the first 21 days of trial. The lactation duration, initial body weight, weight at weaning, and loss of sows during this time were in both trials similar between groups (Table 9, Line 1-5). Differences between control groups and probiotic groups regarding lactation feed intake of sows was in both trials minor (Table 9, Line 6). The litter weight at birth was slightly enhanced in the probiotic groups compared to the control groups in both trials (Ø 1.5%; Table 10, Line 8 and 19), whereas the differences of individual piglet weights were minor between control and probiotic groups in both trials (Ø -0.4%; Table 10, Line 19). After group-restricted re-allocation, to achieve homogenous litters, weight of litters between control and probiotic groups was more balanced (Table 10, Line 12). At weaning, the average litter weight of the probiotic groups was significantly increased in both trials (Ø 5.1 %; Table 10, Line 16). Consequently, the average daily weight gain of litters in the probiotic group was significantly higher compared to the control group (Ø 6.0%; Table 10, Line 14). An observation partly driven by the increased prestarter intake of piglets in the probiotic groups compared to the control groups of both trials (Ø 3.4%; Table 10, Line 13). Although the difference in percentage of mummies between the control groups and probiotic groups in both trials was minor (Table 10, Line 11), probiotic supplementation of sows increased the percentage of live born piglets (Ø 1.5%; Table 10, Line 9) and reduced the percentages of dead born piglets in both trials (Ø -1.7%, Table 10, Line 10). Additionally, the probiotic groups showed a slightly increased number of weaned piglets per litter (Ø 0.9%, Table 10, Line 15, which was further accompanied by a reduced rate of preweaning mortality (Ø -1.1%, Table 10, Line 17 and 17). The individual evaluation of piglets confirmed the results of average litter evaluation. Accordingly, although average weight after re-allocation were nearly identical between control and probiotic groups in both trials (Table 10, Line 20), weaning weights of piglets in the probiotic groups were significantly increased compared to the piglets in the control groups (Ø 3.7%, Table 10, Line 21). Likewise, the average weight gain of piglets in the probiotic groups was clearly enhanced compared to control groups in both trials (Ø 4.2%, Table 10, Line 22). Exemplary results extracted from Table 10 are shown in Fig. 1. Table 10. Zootechnical evaluation of sows, litter, and suckling piglets.a

[0002] Probiotic effect on growth performance and feed conversion of weaned piglet In both feeding trials, weaned piglets in control and probiotic groups started with an identical live weight (Table 11, Line 1). However, at the end of both trial, weight of piglets in the probiotic groups was slightly higher compared to control groups (Ø 0.9%, Table 11, Line 2). In both trials, daily weight gain of piglets in probiotic groups was clearly increased (Ø 1.3%, Table 11, Line 3), although daily feed intake was reduced in these groups compared to the control groups (Ø -2.1%, Table 11, Line 4). In both trials, the average feed conversion ratios were significantly improved due to probiotic supplementation (Ø -3.8%, Table 11, Line 5). Fecal scores were similar between probiotic groups and control groups of both trials. Overall, no mild or severe diarrhea was observed, neither in the control groups nor in the probiotic groups. Therefore, no effect of the probiotic on diarrhea incidence was noticeable. In feeding trial I, three deaths in the control group (mortality rate of 1.0%) and no deaths in the probiotic group (mortality rate of 0.0%) were observed. In feeding trial II, two deaths in the control group (mortality rate of 1.1%) and two deaths in the probiotic group (mortality rate of 1.1%) were observed. Exemplary results extracted from Table 11 are shown in Fig. 2 Table 11. Zootechnical evaluation weaned piglets based on entire trial data (day 1- 42).a

[0003]

[0004] Conclusion The collective findings indicate that the novel probiotic combination of Weizmannia faecalis and Bacillus licheniformis in animals of the family Suidae improves, inter alia, litter quality and growth performance of suckling piglets. Furthermore, the inclusion of this probiotic combination in feed for weaned piglets enhanced their growth performance and feed conversion. Based on these results, the combination of Weizmannia faecalis and Bacillus licheniformis can be considered as environmentally friendly feed additive improving piglet performance and feed efficiency as well as farm profitability and sustainability. Example 3 The blend of W. faecalis and B. licheniformis leads to the combination of different health beneficial modes of action, which allows an overreaching support of intestinal health in Suidae. The genetic differences between W. faecalis and B. licheniformis not only lead to taxonomic separation but also result in distinct species-specific as well as strain-specific modes of action. For example, W. faecalis DSM 32016 and B. licheniformis DSM 33806 vary in their (a) capacity to directly inhibit pathogenic growth and (b) production of health-beneficial enzymes. These differences contribute to a more comprehensive activity profile when these specific probiotic strains are combined, particularly in addressing challenges within swine production such as pathogen suppression or improving feed efficiency. (a) Species- and strain-specific pathogen inhibition Scientific evidence confirms that B. licheniformis DSM 33806 effectively inhibits pathogens relevant to Suidae, including various pathogenic strains of Streptococcus suis and Clostridium perfringens A (Table 12). However, it's noteworthy that under these conditions, inhibition of Escherichia coli by B. licheniformis DSM 33806 was not observed. Table 12. Growth inhibition of Suidae-relevant pathogens by B. licheniformis DSM 33806 analyzed via Cross-Streak-Method and quantification of inhibition zone (mm).aPathogen, Type MH-Agar CB-Agar Clostridium perfringens A, 4NE 20 mm 13 mm Clostridium perfringens A, 3 mm 9 mm 64NE Clostridium perfringens A, 6 mm 11 mm 16NE Streptococcus suis, 8 15 mm 13 mm Streptococcus suis, 7 20 mm 14 mm Streptococcus suis, 5 15 mm 7 mmaMH, Mueller–Hinton; CB, Columbia blood In marked contrast, a previous study demonstrated the inhibition of pathogenic, diarrhea- causing E. coli by W. faecalis DSM 32016. Specifically, the metabolites (culture supernatant) of this probiotic directly reduced the growth of this gram-negative pathogen (Figure 3). The effective suppression of Suidae-relevant E. coli by W. faecalis DSM 32016 was further validated in vivo, through examination of fecal samples from weaned piglets. This feeding trial revealed a reduction in E. coli levels, accompanied by significantly improved fecal consistency, enhanced feed conversion (-0.8%), and increased daily weight gain (+2.7%). The capacity to inhibit pathogenic growth not only varies among different bacterial genera but also notably differs among strains within the same genus. This was demonstrated by comparing various Bacillus, including several B. licheniformis strains in addition to DSM 33806 (Table 13). Table 13. Overview of different abilities of Bacillus species / strains to inhibit the growth of pathogenic C. perfringens. Inhibition was analyzed using the agar diffusion assay. Species / Internal ID InhibitionaB. subtilis - a-, No inhibition; +, very small and unclear inhibition; ++, clear, but small inhibition; +++, clear and medium inhibition *, Strain DSM 33806 (b) Species-specific enzyme production Genus-, species-, and strain-related differences extend beyond pathogen-inhibitory abilities and manifest also in the production of beneficial enzymes essential for supporting feed conversion and overall intestinal health. This disparity was demonstrated between B. licheniformis DSM 33806 and W. faecalis DSM 32016 in vitro (Table 14), providing valuable insight into the complex health support that can be achieved in Suidae when these two strains are combined. Table 14. Health-beneficial enzymes produced by W. faecalis DSM 32016 and / or B. licheniformis DSM 33806 and their function. Analysis based on API ZYM Assay. Produced enzymes Function DSM 32016: α-galactosidase Hydrolysis of glycoproteins, glycolipids, and polysaccharides. In human nutrition: Reduction of intestinal gas production, diarrhea, and stomach pain N-acetyl-β-glucosaminidase Hydrolysis of bacterial / fungal cell wall Valin-arylamidase Hydrolysis of peptides DSM 33806: Alkaline phosphatase Hydrolysis of organic phosphate esters including potential detoxification of lipopolysaccharides Arginine dihydrolase Acting on carbon-nitrogen bonds Participates in arginine and proline metabolism Chymotrypsin Hydrolysis of peptides Cystin-arylamidase Hydrolysis of peptides Both: α-glucosidase Hydrolysis of maltose to glucose β-galactosidase Hydrolysis of lactose to galactose und glucose β-glucosidase Hydrolysis of β-D-glucosides (e.g., cellulose) Esterase Hydrolysis of esters Esterase lipase Hydrolysis of lipids Cystin-arylamidase Hydrolysis of peptides Leucin-arylamidase Hydrolysis of peptides Conclusion The genetic differences between W. faecalis and B. licheniformis lead to distinct modes of action, affecting their abilities to inhibit pathogens and produce beneficial enzymes. Therefore, combining these probiotic strains offers comprehensive benefits in swine production. For example, while B. licheniformis DSM 33806 is effective against different Suidae-relevant pathogens, it does not inhibit E. coli. Conversely, W. faecalis DSM 32016 efficiently suppresses different pathogenic diarrhea-causing E. coli. These findings emphasize the importance of species and strain specificity in probiotic selection and combination for swine health and performance. References ^ Li J, Li H, Zhou Y et al. Effects of Compound Probiotics on Cecal Microbiota and Metabolome of Swine. Animals 2023;13:1006. ^ Kirchhelle C. Pharming animals: a global history of antibiotics in food production (1935–2017). Palgrave Commun 2018;4:96. ^ Ben Y, Fu C, Hu M et al. Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: A review. Environ Res 2019;169:483–93. ^ Bonetti A, Tugnoli B, Piva A et al. Towards Zero Zinc Oxide: Feeding Strategies to Manage Post-Weaning Diarrhea in Piglets. Animals 2021;11:642. ^ Elleithy EMM, Bawish BM, Kamel S et al. Influence of dietary Bacillus coagulans and / or Bacillus licheniformis-based probiotics on performance, gut health, gene expression, and litter quality of broiler chickens. Trop Anim Health Prod 2023;55:38. ^ Wilfart A, Montagne L, Simmins H, Noblet J, Milgen JV. Digesta transit in different segments of the gastrointestinal tract of pigs as affected by insoluble fibre supplied by wheat bran. Br J Nutr 2007;98:54e62. ^ Hughes RJ. Relationship between digesta transit time and apparent metabolizable energy value of wheat in chickens. Brit J Poult Sci 2008;49:716e20. ^ Moran Jr ET. Comparative nutrition of fowl and swine. The gastrointestinal systems. ISBN 0-88955-010-7. Ontario, Canada: University of Guelph; 1982. ^ Inoue R, Tsukarahara T,T, Nakanishi N, Ushida K. Development of the intestinal microbiota in the piglet. J Gen Appl Microbiol 2005;51:257e65. ^ Torok VA, Ophel-Keller K, Loo M, Hughes RJ. Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl Environ Microbiol 008;74:783e91. ^ Guevarra RB, Lee JH, Lee SH, Seok M-J, Kim DW, Kang BN, Johnson TJ, Isaacson RE, Kim HB. 2019. Piglet gut microbial shifts early in life: causes and effects. J Anim Sci Biotechnol. 10(1):1. ^ Lee K-W, Lillehoj HS. 2021. Role of Clostridium perfringens Necrotic Enteritis B- like Toxin in Disease Pathogenesis. Vaccines. 10(1):61. ^ Fairbrother JM, Nadeau É, Gyles CL. 2005. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim Health Res Rev. 6(1):17–39. ^ Elisashvili V, Kachlishvili E, Chikindas ML. Recent Advances in the Physiology of Spore Formation for Bacillus Probiotic Production. Probiotics Antimicrob Proteins. 2019 Sep;11(3):731-747. ^ Kieu et al., 2022

Claims

What is claimed is:

1. A probiotic, feed supplement or feed additive, feed premix or feed for animals of the family Suidae, said probiotic or animal feed comprising at least c) a strain of Weizmannia faecalis, and d) a strain of Bacillus licheniformis 2. The probiotic, feed supplement or feed additive, feed premix or feed according to claim 1, wherein the strain of Weizmannia faecalis is Weizmannia faecalis DSM 32016 and / or the strain of Bacillus licheniformis strain of Bacillus licheniformis DSM 33806 3. A method for producing an animal feed premix or feed, said method comprising adding a strain of Weizmannia faecalis and a strain of Bacillus licheniformis to an animal feed premix or feed, wherein the animal feed premix or feed is for animals of the family Suidae.

4. A method for feeding an animal of the family Suidae, said method comprising administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis or a probiotic, feed supplement or feed additive, feed premix or feed according any one of claims 1 – 2 to said animal.

5. The probiotic, feed supplement or feed additive, feed premix or feed according to any one of the aforementioned claims wherein the animal is Sus scrofa, optionally Sus scrofa domesticus.

6. The probiotic, feed supplement or feed additive, feed premix or feed according to any one of the aforementioned claims wherein the animal is at least one of a sow, in particular a pregnant sow or a lactating sow, and / or a piglet, in particular a weaned piglet.

7. The probiotic, feed supplement or feed additive, feed premix or feed according to any one of the aforementioned claims, wherein Weizmannia faecalis and / or Bacillus licheniformis is provided in the form of endospores.

8. The feed or method according to any one of claims, in which Weizmannia faecalis and / or Bacillus licheniformis is present on the feed in a final concentration of between ≥ 1.0 x 107and ≤ 1.0 x1011CFU / kg.

9. A method of improving feed conversion ratio and or increasing body weight in an animal of the family Suidae, said method comprising administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal.

10. A method of modulation the gut microbiome of an animal of the family Suidae, said method comprising administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal.

11. A method of stimulating the immune system of an animal of the family Suidae, said method comprising administering a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, to said animal.

12. Use of a strain of Weizmannia faecalis and a strain of Bacillus licheniformis, or of a probiotic, feed supplement or feed additive, feed premix or feed comprising such two strains, for (the manufacture of a medicament for) the treatment of an animal of the family Suidae.

Images