Feed additive composition and method of using same

Abstract

Provided herein are, inter alia, feed or feed additive compositions comprising direct-fed live bacteria (DFM) and other substances, and methods of making and using same for improving performance and maintaining a balanced gut microbiome in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 248,657, filed September 27, 2021, U.S. Provisional Patent Application No. 63 / 248,668, filed September 27, 2021, and U.S. Provisional Patent Application No. 63 / 397,189, filed August 11, 2022, the disclosures of each of which are incorporated by reference in their entireties herein.

[0002] Provided herein are, inter alia, methods and compositions for the treatment and prevention of necrotic enteritis and / or coccidiosis in a subject in need thereof. [Background technology]

[0003] Necrotic enteritis (NE) is estimated to cost the global poultry industry approximately US$6 billion annually (Poultry Federation, 2019). NE affects 40% of the world's broiler population, costing approximately US$0.050-0.063 per bird. NE severity ranges from an acute clinical disease with 10-25% mortality to a more chronic subclinical infection that results in impaired growth performance (weight gain and feed conversion) and low mortality (0.5-2%).

[0004] Antibiotic resistance, especially in relation to large-scale agriculture and meat production, was in the WHO's list of the top 10 threats to global human health in 2019. According to the US Food and Drug Administration, 80% of antibiotics sold are used on livestock. In many countries, bans on antibiotic use in livestock have already been implemented, and in others, consumer pressure has forced the industry to stop using antibiotic growth promoters (AGPs). The sudden cessation of antibiotic growth promoter use has put the livestock industry under great pressure.

[0005] The global trend towards the elimination of antibiotics in poultry systems continues to pose significant challenges for nutritionists and veterinarians. NE and coccidiosis are the top two concerns for reduced antibiotic production. The lack of an antibiotic line of defense has increased the susceptibility of animals to gut health-related diseases (both clinical and subclinical). This translates to reduced animal performance. Despite the absence of AGPs in many parts of the world for over a decade (e.g., European Union), livestock producers continue to be frustrated by the lack of consistent and reliable antibiotic alternatives. Summary of the Invention [Problem to be solved by the invention]

[0006] Current feed additives that are alternatives to AGPs in livestock production (e.g., acidifiers, minerals, prebiotics, direct-fed live bacteria (DFM; also known as probiotics), nucleotides, and plant extracts (Liu et al., 2018, Animal Nutrition, 4:113-125)) all show much lower efficacy (<50%) compared to antibiotics (~95%). Thus, there is a great unmet need to find alternatives to antibiotics that can maintain the health and performance of livestock without the adverse effects associated with the rise of antibiotic resistance. [Means for solving the problem]

[0007] The subject matter disclosed herein addresses this need and provides additional advantages as well.

[0008] Provided herein are, inter alia, feed or feed additive compositions comprising direct-fed live bacteria (DFM) and other substances, and methods of making and using the same for improving performance and maintaining a healthy and / or balanced gut microbiome in a subject.

[0009] Thus, in some aspects, provided herein is a feed additive composition comprising or consisting essentially of direct-fed live bacteria (DFM), including Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus. In some embodiments, the feed additive composition further comprises or consists essentially of an osmolality adjusting agent. In some embodiments of any of the embodiments disclosed herein, the feed additive composition further comprises or consists essentially of at least one essential oil. In some embodiments of any of the embodiments disclosed herein, the feed additive composition further comprises or consists essentially of one or more enzymes selected from the group consisting of proteases, xylanases, beta-glucanases, phytases, and amylases. In some embodiments, the enzymes are encapsulated or in the form of granules or are lyophilized. In some embodiments of any of the embodiments disclosed herein, the feed additive composition comprises or consists essentially of Bifidobacterium animalis subsp. lactis Bl-04 strain. In some embodiments of any of the embodiments disclosed herein, the feed additive composition comprises or consists essentially of Lactobacillus acidophilus NCFM strain. In some embodiments of any of the embodiments disclosed herein, the osmotic modifier comprises betaine. In some embodiments of any of the embodiments disclosed herein, the at least one essential oil comprises cinnamaldehyde, carbachol and / or thymol. In some embodiments of any of the embodiments disclosed herein, the feed additive composition further comprises or consists essentially of at least one additional DFM.In some embodiments of any of the embodiments disclosed herein, the feed additive composition further comprises or consists essentially of one or more of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, and / or vitamin B. In some embodiments of any of the embodiments disclosed herein, the B vitamin is vitamin B1, B6, and / or B. 12 In some embodiments of any of the embodiments disclosed herein, at least one component of the composition is formulated for aqueduct delivery.

[0010] In another aspect, provided herein is an animal feed or premix comprising any of the feed additive compositions disclosed herein.

[0011] In a further aspect, provided herein is a method of treating or preventing necrotic enteritis in a subject in need thereof, comprising administering to the subject an effective amount of any of the feed additive compositions disclosed herein or any of the animal feeds or premixes disclosed herein. In some embodiments, the subject is a poultry or pig. In some embodiments of any of the embodiments disclosed herein, the poultry is a broiler, or a laying chicken, or a turkey. In some embodiments of any of the embodiments disclosed herein, the pig is a piglet, a growing pig, or a sow. In some embodiments of any of the embodiments disclosed herein, the method reduces or prevents the intestinal lesions of necrotic enteritis. In some embodiments of any of the embodiments disclosed herein, the method further reduces feed conversion ratio, increases feed efficiency, reduces mortality, increases final slaughter weight, or increases weight gain in the subject compared to a subject not administered an effective amount of any of the feed additive compositions disclosed herein or any of the animal feeds or premixes disclosed herein. In some embodiments, the subject has clinical or subclinical necrotic enteritis. In some embodiments of any of the embodiments disclosed herein, the method further reduces expression of Clostridium perfringens necrotic enteritis B-like toxin (NetB). In some embodiments of any of the embodiments disclosed herein, the feed additive composition is administered by water supply. In some embodiments of any of the embodiments disclosed herein, the administration is performed without co-administration of an antibiotic to the subject.

[0012] In a further aspect, provided herein is a method of treating or preventing coccidiosis in a subject in need thereof, comprising administering to the subject an effective amount of any of the feed additive compositions disclosed herein or any of the animal feeds or premixes disclosed herein. In some embodiments, the subject is poultry. In some embodiments of any of the embodiments disclosed herein, the poultry is a broiler or egg-laying chicken. In some embodiments of any of the embodiments disclosed herein, the method reduces feed conversion ratio, increases feed efficiency, reduces mortality, increases final slaughter weight, or increases weight gain in the subject. In some embodiments of any of the embodiments disclosed herein, the method reduces one or more intestinal Eimeria species. In some embodiments of any of the embodiments disclosed herein, at least one component of the feed additive composition is administered by water supply.

[0013] In another aspect, provided herein is a method of reducing necrotizing enteritis B-like toxin (NetB) expression in Clostridium perfringens, comprising contacting C. perfringens cells with one or more of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B, and / or the secretome of one or both of B. animalis or L. acidophilus. In some embodiments, the vitamin B is vitamin B1, B6, and / or B7. 12In some embodiments of any of the embodiments disclosed herein, the B. animalis is Bifidobacterium animalis subsp. lactis strain Bl-04, and / or the L. acidophilus is Lactobacillus acidophilus strain NCFM. In some embodiments of any of the embodiments disclosed herein, the C. perfringens cells are in the intestine of a poultry. In some embodiments, the poultry is a chicken, quail, duck, goose, emu, ostrich, pheasant, or turkey. In some embodiments, the chicken is a broiler or egg-laying chicken. In some embodiments of any of the embodiments disclosed herein, the method further comprises contacting the C. perfringens cells with one or more of an osmotic modifier and / or at least one essential oil. In some embodiments, the osmotic modifier comprises betaine. In some embodiments of any of the embodiments disclosed herein, the at least one essential oil comprises cinnamaldehyde, carbachol, and / or thymol.

[0014] In a further aspect, provided herein is a method of reducing necrotic enteritis B-like toxin (NetB) expression in Clostridium perfringens in a subject in need thereof, comprising or consisting essentially of administering an effective amount of any of the feed additive compositions disclosed herein or any of the animal feed or premix compositions disclosed herein, wherein C. perfringens cells are in the intestine of the subject. In some embodiments, the subject is a poultry or pig. In some embodiments of any of the embodiments disclosed herein, the poultry is a broiler, or a laying hen, or a turkey. In some embodiments of any of the embodiments disclosed herein, the pig is a piglet, a growing pig, or a sow. In some embodiments of any of the embodiments disclosed herein, the method reduces or prevents the intestinal lesions of necrotic enteritis. In some embodiments of any of the embodiments disclosed herein, the method further reduces feed conversion ratio, increases feed efficiency, reduces mortality, increases final slaughter weight, or increases weight gain in the subject compared to a subject not administered an effective amount of any of the feed additive compositions disclosed herein or any of the animal feeds or premixes disclosed herein. In some embodiments, the subject has clinical or subclinical necrotizing enterocolitis. In some embodiments of any of the embodiments disclosed herein, the feed additive composition is administered by water supply. In some embodiments of any of the embodiments disclosed herein, the administration is performed without co-administration of an antibiotic to the subject.

[0015] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of that embodiment or aspect.

[0016] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosures of all patents, patent applications and other publications cited herein are incorporated by reference in their entirety for all purposes. [Brief description of the drawings]

[0017] [Figure 1A] Box plots depicting the mean final slaughter weight of birds (D42) per treatment in the study are shown. Each point represents a replicate pen within a treatment. Significance is determined at P<0.05 compared to challenge control (CC). [Figure 1B] Box plots showing the mean feed conversion ratio (FCR) of birds (D42) per treatment in the study are shown. Each point represents a replicate pen within a treatment. Significance is determined at P<0.05 compared to challenge control (CC).

[0018] [Figure 2A] Necrotic enteritis lesion scores for individual birds based on a scoring system of 0 to 4 by treatment on D21 are shown. 24 birds were evaluated per treatment. The treatment with the least NE lesions contained only betaine, EO and DFM. [Figure 2B] Box plots showing the mean weight of birds per treatment at D28 are shown. Each point represents a replicate pen within a treatment. Significance is determined at P<0.05 compared to challenge control (CC). [Figure 2C] Box plots depicting the mean final slaughter weight of birds per treatment (D35) are shown. Each point represents a replicate pen within a treatment. Significance is determined at P<0.05 compared to challenge control (CC). [Figure 2D] Box plots are shown depicting mean serum FITC-dextran levels (μg / mL) across treatments as a method of determining intestinal permeability. One bird from each replicate pen was evaluated, providing eight birds after treatment. Each point represents one bird. Significance is determined at P<0.05 compared to loading control (CC). [Figure 2E]Box plots showing the mean feed conversion ratio (FCR) of birds per treatment (D42) are shown. Each point represents a replicate pen within a treatment. Significance is determined at P<0.05 compared to challenge control (CC).

[0019] [Figure 3A] Figure 1 shows C. perfringens enumeration determined using qPCR and expressed as Log10 CFU / intestinal swab at D21 of the study. [Figure 3B] FIG. 1 shows the expression of the netB gene in intestinal swabs taken on D21 of the study and expressed as log10 gene copy number per ileal swab.

[0020] [Figure 4] FIG. 1 shows a grid illustrating the set-up of the Clostridium perfringens inhibition assay as described in Example 4.

[0021] [Diagram 5] A series of bar graphs showing the inhibition percentage of Clostridium perfringens by Bl-04 and essential oils are shown.

[0022] [Figure 6] A series of bar graphs showing the inhibition rate of Clostridium perfringens by NCFM and essential oils are shown.

[0023] [Figure 7A] Heat map of marker gene expression (z-scores of log-transformed values) from individual C. perfringens cells grown anaerobically in minimal M9 medium organized into 10 clusters. [Figure 7B] 1 shows an outline of the final steps in arginine biosynthesis. [Figure 7C]1 shows a Western blot of NetB toxin secreted into the growth medium by C. perfringens cultures grown in BHI alone or in BHI in the presence of aspartate, ornithine, or arginine. [Figure 7D] 1 shows UMI plots illustrating single cell clustering of C. perfringens cultures grown in either BHI alone (light) or BHI + aspartate (dark). [Figure 7E] Figure 1 shows the cytotoxicity of conditioned medium from C. perfringens cultures grown in human epithelial HT-29 cells in the presence or absence of aspartate, ornithine or arginine.

[0024] [Figure 8] Western blot detecting NetB toxin production when ammonium phosphate, sodium acetate or B vitamins (vitamins B1, B6 and B12) were added to cultures of Clostridium perfringens compared to exotoxins detected when grown in unsupplemented growth medium (BHI).

[0025] [Figure 9A](Top) Single cell clustering of C. perfringens cultures grown in either BHI alone (light), BHI + 2.5% secretome of Bifidobacterium animalis subsp. lactis (medium) or BHI + 5% secretome of Bifidobacterium animalis subsp. lactis (dark); (Bottom) Single cell clustering of C. perfringens cultures grown in either BHI alone (light), BHI + 2.5% secretome of Bifidobacterium animalis subsp. lactis (medium) or BHI + 5% secretome of Bifidobacterium animalis subsp. lactis (dark). 1 shows UMI plots showing complementary C. perfringens single cell expression of the netB gene grown on either C. perfringens or 5% secretomes of C. lactis subsp. [Figure 9B] (Top) Shows single-cell clustering of C. perfringens cultures grown in either BHI alone (light) or BHI + Lactobacillus acidophilus secretome (dark), and (bottom) UMI plots showing complementary C. perfringens single-cell expression of the netB gene grown in BHI or BHI + L. acidophilus secretome. [Figure 9C] FIG. 1 shows a Western blot of NetB toxin secreted into the growth medium by C. perfringens cultures grown in BHI alone or in the presence of Bifidobacterium animalis subsp. lactis secretome or L. acidophilus secretome. [Figure 9D]Figure 1 shows the cytotoxicity of conditioned medium from C. perfringens cultures grown in human epithelial HT-29 cells in the presence or absence of Bifidobacterium animalis subsp. lactis secretome, L. acidophilus secretome or a combination of both secretomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Clostridium perfringens is the primary causative agent of avian necrotic enteritis (NE), an enteric disease of poultry first described in 1961. In chickens, NE manifests as acute or chronic enterotoxemia. The acute disease results in significant mortality due to the development of extensive necrotic lesions in the intestinal wall, while the chronic disease results in significant losses in productivity and welfare. It is estimated that the disease costs the poultry industry billions of US dollars annually.

[0027] C. perfringens is commonly found in the digestive tract of poultry. It is a gram-positive, rod-shaped, spore-forming, oxygen-tolerant anaerobe. C. perfringens is classified into five toxin types (A, B, C, D, and E) based on the production of four suspected major toxins (alpha, beta, epsilon, and iota). Type A is always recovered from the intestine of chickens, while the other types are less common.

[0028] Early studies of NE suggested that a major virulence factor involved in the disease was secreted by the bacteria, and a phospholipase C enzyme called alpha-toxin was proposed to be the primary toxin involved in pathogenesis. However, recent studies have shown that alpha-toxin minus mutant strains could also cause NE, suggesting that alpha-toxin is not an essential virulence factor, thus calling into question the role of alpha-toxin in the overall disease. Subsequent studies have suggested that a novel pore-forming toxin, NetB, plays an important role in the pathogenesis of the disease.

[0029] Coccidiosis remains the number one predisposing factor for NE development. Poultry producers typically use coccidiosis management programs to avoid NE development when possible. Producers continue to be frustrated by the lack of consistency of coccidiosis vaccination, forcing producers to rotate ionophore / chemical programs to avoid resistance development. Therefore, any solutions developed for NE management should be evaluated in models using Eimeria, the causative parasite of coccidiosis, as a predisposing factor for NE, and anti-Eimeria effects should be evaluated in vitro and in vivo (lesion scoring).

[0030] The inventors of the present application have surprisingly discovered that certain active agents, when used in combination, directly interfere with the infection biology of Clostridium perfringens, the pathogen of NE in birds, and / or indirectly interfere with the host and / or its endogenous gut microbiota to modulate the infection biology of C. perfringens. The present invention reduces C. perfringens levels in the gut upon challenge, reduces quorum sensing and therefore reduces toxin expression (specifically, reduces the amount of necrotizing enteritis B-like toxin (NetB) produced by C. perfringens cells), and reduces the occurrence of NE intestinal lesions when birds are susceptible to or challenged with NE (by diet or coccidiosis exposure). Furthermore, the disclosed invention reduces local intestinal inflammation and / or supports / maintains intestinal integrity during NE challenge, resulting in reduced NE mortality and improved bird performance (weight gain and / or feed conversion ratio) in the presence of NE challenge. The invention also supports a positive gut microbiota.

[0031] I. Definition The terms "animal" and "subject" are used interchangeably herein and refer to non-ruminant animals (i.e., monogastric animals). Examples of monogastric animals include, but are not limited to, pigs and swines, such as piglets, growing pigs, sows; and poultry. The term "poultry" as used herein means a domesticated bird kept by humans for eggs, meat, or feathers. The bird is most typically a member of the superorder Galloanserae, and in particular the order Galliformes, which includes, but is not limited to, chickens, quails, ducks, geese, emus, ostriches, pheasants, and turkeys. In further embodiments, the animal is a chicken, such as a broiler or egg-laying chicken. In some embodiments, the subject or animal is not a human.

[0032] As used herein, "prevent," "preventing," "prevention," and grammatical variations thereof refer to a method of partially or completely delaying or preventing the onset or recurrence of a disorder or condition (e.g., necrotizing enterocolitis) and / or one or more of the symptoms associated therewith, or a method of preventing an animal from acquiring or re-acquiring a disorder or condition, or a method of reducing the risk of an animal acquiring or re-acquiring a disorder or condition or one or more of the symptoms associated therewith.

[0033] As used herein, the term "reducing" with respect to a particular feature, characteristic, feature, biological process or phenomenon refers to a decrease in the particular feature, characteristic, feature, biological process or phenomenon. The feature, characteristic, feature, biological process or phenomenon may be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100%.

[0034] As used herein, "administer" or "administering" refers to the act of introducing one or more microbial strains, exogenous feed enzymes, feed additives and / or strains into an animal, such as by feeding or tube feeding. In one embodiment, the composition is administered to the subject via the tap water that the subject drinks.

[0035] As used herein, an "effective amount" refers to an amount of a substance (e.g., betaine, direct-fed live bacteria (DFM), or essential oil (EO)) to improve one or more metrics of an animal. The improvement in one or more metrics of an animal (e.g., but not limited to, improved feed conversion ratio (FCR); improved weight gain; improved feed efficiency; improved gut microbiome status (i.e., more healthy ("good") bacteria and / or less unhealthy ("bad") bacteria; and / or improved carcass quality) may be measured as described herein or by other methods known in the art. An effective amount may be administered to an animal by providing the animal with free access to feed and / or water containing the substance. In addition, the substance (e.g., betaine, DFM, or EO) may also be administered in one or more doses. Similarly, in some embodiments, an effective amount of some substances (e.g., betaine or EO) may be administered in the feed or as a component of the feed, and other substances (e.g., one or more DFMs) may be administered by water supply.

[0036] The term "pathogen" as used herein means any causative agent of disease. Such causative agents may include, but are not limited to, bacterial causative agents, viral causative agents, fungal causative agents, and the like.

[0037] As used herein, "subclinical" means without clinical symptoms; refers to an early stage of an infection or other disease (e.g., necrotizing enterocolitis) or a very mild infection or other disease (e.g., necrotizing enterocolitis) before symptoms and signs become evident or detectable by clinical examination or testing.

[0038] "Feed" and "food" mean any natural or artificial food, meal, or the like, or a component of such a food, intended or suitable to be eaten, ingested, or digested by non-human animals and humans, respectively. As used herein, the term "food" is used broadly to cover food and food products for humans as well as food (i.e., feed) for non-human animals.

[0039] As used herein, the term "feed" is used synonymously herein with "feedstuff." Feed broadly refers to liquid or solid substances used to nourish animals and maintain normal or accelerated growth of animals, such as newborns or young developing animals. The term includes compounds, preparations, mixtures, or compositions suitable for ingestion by animals (e.g., for poultry, such as quail, ducks, turkeys, and chickens). In some embodiments, the feed or feed composition includes a basal food composition and one or more feed additives or feed additive compositions.

[0040] The term "feed additive" as used herein refers to a component included to enhance the nutritional value of a basal feed with additional components to promote feed intake, treat or prevent disease, or alter metabolism. However, in some embodiments, the feed additive is formulated for water supply delivery to animals and is not added directly to the feed. Feed additives include premixes. As used herein, the term "feed additive" also refers to a substance added to the feed or to the water administered with the feed. Feed additives may be added to feed for many reasons, such as to increase the digestibility of the feed, to supplement the nutritional value of the feed, to improve the immune defense of the recipient, and / or to improve the shelf life of the feed. In some embodiments, the feed additive supplements the nutritional value of the feed and / or improves the immune defense of the recipient. In some embodiments, the feed additive is not for administration to humans.

[0041] A "premix," as referred to herein, can be a composition comprised of minor ingredients (such as, but not limited to, one or more of vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients). A premix is ​​typically a composition suitable for blending into a commercially available food product.

[0042] The term "performance" as used herein may be determined by feed efficiency, and / or weight gain of the animal, and / or feed conversion ratio, and / or digestibility of nutrients in the feed (e.g., amino acid digestibility or phosphorus digestibility), and / or digestible or metabolizable energy in the feed, and / or nitrogen retention, and / or the ability of the animal to avoid the adverse effects of disease or the immune response of the subject. Performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; milk production; mineral absorption: mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake ratio (ADFI); average daily gain (ADG) retention and / or secretion of any one or more of copper, sodium, phosphorus, nitrogen and calcium; amino acid retention or absorption; mineralization, bone mineralization, carcass yield and carcass quality.

[0043] As used herein, the term "feed efficiency" refers to the amount of weight gain of an animal that occurs when the animal is fed ad libitum or a specified amount of food over a period of time. "Increased feed efficiency" refers to an increase in weight gain per unit of feed intake due to the use of a feed additive composition according to the invention in the feed or in drinking water by tap delivery, compared to an animal fed without the presence of said feed additive composition.

[0044] As used herein, "feed conversion ratio" refers to a measure of the efficiency of a subject in converting feed mass into a desired increase in production, and is calculated by dividing the mass of feed ingested by the production over a specified period of time. For example, if an animal is raised for meat (e.g., beef), the production may be the mass gained by the animal. If the animal is raised for another intended purpose (e.g., milk production), the production will be different. The term "feed conversion ratio" may be used synonymously with the term "feed conversion ratio" or "feed conversion efficiency." "Lower feed conversion ratio" or "improved feed conversion ratio" means that, due to the use of the feed additive composition in the feed, a lower amount of feed needs to be fed to the animal to increase the animal's weight by a given amount, compared to the amount of feed required to increase the animal's weight by the same amount when the feed does not contain the feed additive composition.

[0045] As used herein, "microorganism" or "microbe" refers to bacteria, fungi (e.g., yeast), viruses, protozoa, and other microorganisms or microscopic organisms.

[0046] The term "direct-fed live bacteria" ("DFM"), as used herein, is a source of live (viable) microorganisms, i.e., prebiotics, that can confer a benefit to the recipient when applied in sufficient numbers. DFM may include one or more of such microorganisms, such as bacterial strains. Categories of DFM include Bacillus, lactic acid bacteria, and yeast. Thus, the term DFM encompasses one or more of direct-fed bacteria, direct-fed yeast, direct-fed yeast, and combinations thereof. Bacillus is a unique Gram-positive rod that forms spores. These spores are extremely stable and can withstand environmental conditions such as heat, moisture, and a range of pH. These spores germinate into active vegetative cells when ingested by the animal and can be used in meal and pelleted feed. Lactic acid bacteria are Gram-positive cocci that produce lactic acid, which are antagonistic to some pathogens. Lactic acid bacteria are not typically used in pelleted feed because they are considered to be somewhat heat-labile. Types of lactic acid bacteria include, but are not limited to, the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus.

[0047] The terms "probiotics", "probiotic cultures" and "DFM" are used interchangeably herein to define live microorganisms (including, for example, bacteria or yeasts) that, when ingested in sufficient numbers or applied topically, beneficially affect the host organism (i.e., by conferring one or more demonstrable benefits, such as digestive and / or performance benefits (e.g., health benefits or benefits of a balanced gut microbiome) to the host organism). Probiotics may improve the microbial balance at one or more mucosal surfaces. For example, the mucosal surface may be the intestine, urinary tract, respiratory tract or skin. The term "probiotics", as used herein, also encompasses live microorganisms that may stimulate beneficial branches of the immune system and simultaneously reduce inflammatory responses at mucosal surfaces (e.g., the intestine). There is no lower or upper limit for probiotic intake, but a daily dose of at least 10 6 ~10 12 , e.g. at least 10 6 ~10 10 , e.g. 10 8 ~10 9 It has been suggested that colony forming units (cfu) are effective in achieving a beneficial effect in a subject.

[0048] The term "CFU" as used herein means "colony forming unit" and is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.

[0049] As used herein, "secretome" refers to the totality of organic molecules and inorganic elements produced and secreted by a cell. When growth conditions are indicated, the secretome is the totality of organic molecules and inorganic elements produced and secreted by the cell under the growth conditions. It will be understood that the secretome may be recovered in the cell supernatant and fractions of that supernatant.

[0050] As used herein, the term "osmotic modifier" refers to a compound that balances the osmotic pressure between the interior and exterior of the cell, thereby promoting cell survival in hyperosmotic environments. In one embodiment, the osmotic modifier is betaine.

[0051] As used herein, the term "betaine" refers to trimethylglycine. This compound is also known as trimethylammonioacetate, 1-carboxy-N,N,N-trimethylmethanaminium, inner salt, and glycine betaine. It has the formula [ka] It is a naturally occurring quaternary ammonium type compound having the formula:

[0052] Betaine has a bipolar structure containing a hydrophilic portion (COO-) and a hydrophobic portion (N+) capable of neutralizing both acidic and alkaline solutions. In its pure form, betaine is a white crystalline compound that is readily soluble in water and lower alcohols. In the present invention, betaine may be used, for example, in anhydrous form or as a hydrate or salt acceptable for animal feed. In one embodiment, betaine, if present, is present as a free zwitterion. In one embodiment, betaine, if present, is present as anhydrous betaine. In one embodiment, betaine, if present, is present as a monohydrate. "Betaine" also includes naturally occurring and synthetic betaines.

[0053] As used herein, "animal feed acceptable salt" refers to any non-toxic salt that can provide, directly or indirectly, a compound or a derivative of a compound described herein upon administration to a recipient. Acids commonly utilized to form acceptable salts include inorganic acids (e.g., hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid) and organic acids (e.g., paratoluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, parabromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid) and related inorganic and organic acids. Thus, salts that are acceptable in such animal feeds include: sulfate, pyrosulfate, disulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-diate, hexaphosphate ... Examples of suitable acid addition salts include benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, [beta]-hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, and similar salts. Preferred animal feed-acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid. Cations suitable for forming feed-acceptable salts include ammonium, sodium, potassium, calcium, magnesium, and aluminum cations, among others.

[0054] As used herein, "essential oil" refers to the set of all compounds that can be distilled or extracted from the plant from which the oil is obtained, or can be synthetically produced, and that contribute to the characteristic aroma of the plant. See, for example, H. McGee, On Food and Cooking, Charles Scribner's Sons, p. 154-157 (1984). Non-limiting examples of essential oils include thymol and cinnamaldehyde.

[0055] The terms "peptide", "protein" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids linked by peptide bonds. A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. One-letter and three-letter codes defined in accordance with the IUPAC-IUB Joint Commission on Biological Nomenclature (JCBN) are used throughout this disclosure for amino acids. The single letter X refers to any of the 20 amino acids. It is also understood that a polypeptide can be encoded by multiple nucleotide sequences due to the degeneracy of the genetic code.

[0056] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in a host organism containing such DNA sequences. In addition, the terms refer to proteins encoded by DNA sequences of synthetic and / or cDNA origin and having the distinguishing characteristics of the protein in question.

[0057] A particular range is expressed herein by a numerical value preceded by the term "about". The term "about" is used herein to provide literal support for the exact number preceding the term as well as a number close to or approximately the number preceding the term. In determining whether a number is close to or approximately a specifically recited number, a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented may be a close or approximately unrecited number. For example, the term "about" in relation to a numerical value refers to a range of -10% to +10% of the numerical value unless the term is clearly defined otherwise in the context.

[0058] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0059] It is further noted that the claims may be drafted to exclude any optional element, in which case this statement is intended to serve as a predicate to the use of exclusive terminology, such as "sole," "only," and the like, in the context of reciting claim elements or using a "negative" limitation.

[0060] It should also be noted that the phrase "consisting essentially of," as used herein, refers to a composition in which the component following this phrase totals less than 30% by weight of the total composition and in the presence of other known components that do not contribute to or interfere with the action or activity of the component.

[0061] Additionally, it should be noted that the term "comprising," as used herein, means including, but not limited to, the component following the term "comprising." The component following the term "comprising" is required or essential, but a composition that includes this component may further include other non-essential or optional components.

[0062] It should also be noted that the term "consisting of," as used herein, means including and limited to the component following the term "consisting of." Thus, the component following the term "consisting of" is necessary or mandatory, and no other component is present in the composition.

[0063] Every individual upper numerical limit given throughout this specification is intended to include every lower individual lower numerical limit, as if such lower individual lower numerical limit was expressly written herein. Every individual lower numerical limit given throughout this specification includes every higher individual upper numerical limit, as if such higher individual upper numerical limit was expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0064] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0065] Throughout this specification, other definitions of terms may appear.

[0066] II. Composition Provided herein are compositions that include one or more direct-fed live bacteria (DFM; e.g., a DFM comprising one or more Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus), and / or one or more osmolality modifiers (e.g., betaine), and / or one or more essential oils (e.g., cinnamaldehyde and / or thymol), and / or one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase), and / or one or more of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, and / or B vitamins (e.g., vitamins B1, B6, and / or B12).

[0067] A. Direct-fed live bacteria (DFM) In one embodiment, the DFM may be included in any of the feed additive formulations disclosed herein, optionally formulated as a liquid, dry powder, or granules. In one embodiment, the DFM of any of the feed additive formulations disclosed herein may be formulated as a single mixture. In another embodiment, the DFM of any of the feed additive formulations disclosed herein may be formulated as separate mixtures. In yet another embodiment, separate mixtures of the DFM of any of the feed additive formulations disclosed herein may be administered simultaneously or at different times. In yet another embodiment, separate mixtures of the DFM of any of the feed additive formulations disclosed herein may be administered simultaneously or sequentially. In yet another embodiment, a first mixture comprising the DFM may be administered followed by a second mixture comprising any of the feed additive formulations disclosed herein. In yet another embodiment, a first mixture comprising any of the feed additive formulations disclosed herein may be administered followed by a second mixture comprising the DFM.

[0068] Dry powders or granules can be prepared by means known to those skilled in the art, such as a top spray fluid bed coater, a bottom spray Worcester or drum granulator (eg, high shear granulation), extrusion, pan coating or a microingredient mixer.

[0069] In another embodiment, one or more of the osmolality modifiers (e.g., betaine), essential oils, enzymes, aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6, and / or B12), and / or DFM may be coated, e.g., encapsulated. Suitably, the osmolality modifiers (e.g., betaine), essential oils, enzymes, aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6, and / or B12), and / or DFM may be formulated within the same coating or encapsulated within the same capsule.

[0070] For example, in some embodiments where the DFM is capable of producing endospores, the DFM may be provided without any coating. In such circumstances, the DFM endospores may simply be mixed with an osmotic modifier (e.g., betaine), essential oil, aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6, and / or B12), and / or enzymes. The osmotic modifier (e.g., betaine), essential oil, enzyme, aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6, and / or B12), and / or DFM may be encapsulated as a mixture (i.e., one or more, two or more, three or more, or all), or may be encapsulated separately (e.g., alone).

[0071] At least one DFM may comprise at least one viable microorganism, for example a viable bacterial strain, or a viable yeast, or a viable fungus, or a viable filamentous fungus. In some embodiments, the DFM comprises at least one viable bacterium. The DFM may be a spore-forming bacterial strain, and thus the term DFM may consist of or include spores (e.g., bacterial spores, such as endospores). Thus, the term "viable microorganism" as used herein may include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive composition described herein may not consist of or include microbial spores (e.g., endospores or conidia). The microorganism may be a naturally occurring microorganism or a transformed microorganism.

[0072] The DFM described herein may include microorganisms from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Weissella, Pediococcus, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, and combinations thereof. In some embodiments, the DFM comprises one or more bacterial strains selected from Bacillus spp., including, but not limited to, one or more bacterial strains selected from Bacillus subtilis, Bacillus cereus var. toyoi, Bacillus licheniformis, Bacillus pumilis, Bacillus velezensis, and Bacillus amyloliquefaciens.

[0073] "Bacillus," as used herein, includes all species within the genus "Bacillus" known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. argininos ... ilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. pumilis, and B. thuringiensis. It is recognized that the taxonomic reorganization of the genus Bacillus is ongoing. As such, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus, now called Geobacillus stearothermophilus, or Bacillus polymyxa, now called Paenibacillus polymyxa.The production of resistant endospores under stressful environmental conditions appears to be a defining feature of Bacillus genus, and this feature has been demonstrated in the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, and others. us, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus and Virgibacillus.

[0074] In another embodiment, the DFM may further comprise the following Lactococcus spp: Lactococcus cremoris and Lactococcus lactis, and combinations thereof. The DFM may also contain the following Lactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminisLactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus jensenii, and any combination thereof. "Lactobacillus" as used herein includes all species within the "Lactobacillus" genus known to those of skill in the art. It is recognized that taxonomic reorganization of the Lactobacillus genus continues. For example, as of March 2020, the Lactobacillus genus is comprised of 261 species that are highly diverse phenotypically, ecologically and genotypically. Given recent advances in whole genome sequencing and comparative genomics, the genus Lactobacillus has recently been divided into 25 separate genera, and strains belonging to previously designated Lactobacilli species have been transferred to new species and / or genera (see Zheng et al., 2020, Int. J. Syst. Evol. Microbiol., 70:2782-2858; Pot et al., Trends in Food Science & Technology 94(2019)105-113; and Koutsoumanis et al., 2020, EFSA Journal, 18(7):6174, the disclosures of each of which are incorporated herein by reference). For purposes of this disclosure, the previous classification of Lactobacillus species continues to be adopted. However, in some embodiments, Lactobacillus agilis is also classified as Ligilactobacillus agilis. In other embodiments, Lactobacillus salivarius is also classified as LactobacillusIn a further embodiment, Lactobacillus reuteri is also classified as Limosilactobacillus reuteri.

[0075] In yet another embodiment, the DFM may be further combined with the following Bifidobacterium spp.: Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis and Bifidobacterium angulatum, and any combination thereof.

[0076] The following species of bacteria may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus pumilis, Enterococcus, Enterococcus spp and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococcus acidilactici, acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farciminis, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Clostridium tyrobutyricum, Bifidobacterium animalis ssp. animalis animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp Salivarius, Propionibacterium speciesspp) and combinations thereof.

[0077] The direct-fed microorganisms described herein, including one or more bacterial strains, can be of the same type (genus, species and strain) or can include a mixture of genera, species and / or strains. Alternatively, the DFM may be combined with one or more of the products or microorganisms contained in the products disclosed in WO2012110778 and summarized as follows: Bacillus subtilis strain 2084 accession number NRRLB-50013, Bacillus subtilis strain LSSAO1 accession number NRRL B-50104 and Bacillus subtilis strain 15A-P4 ATCC accession number PTA-6507 (from Enviva® PRO® (formerly known as Avicorr®); Bacillus subtilis strain C3102 (from Calsporin®); Bacillus subtilis strain PB6 (from Clostat®); Bacillus pumilis (8G-134); Enterococcus NCIMB 10415 (SF68) (from Cylactin®); Bacillus subtilis strain C3102 (from Gallipro® & GalliproMax®); Bacillus licheniformis (from Gallipro® Tect®); Enterococcus and Pediococcus (from Poultry star®); Lactobacillus, Bifidobacterium and / or Enterococcus (Protexin®); Bacillus subtilis strain QST713 (Proflora®);Bacillus amyloliquefaciens CECT-5940 (from Ecobiol® & Ecobiol® Plus); Enterococcus faecium SF68 (from Fortiflora®); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B®); Lactobacillus 7 Enterococcus faecium (from Lactiferm®); Bacillus subtilis strain QST 713 (from Baymix® Grobig BS); Lactobacillus strains (from LACTILLUS™); Bacillus strains (from CSI™); Saccharomyces cerevisiae (from Yea-Sacc™); Bacillus subtilis (from Bacillus velezensis NRRL B-67259) (from Correlink™); Enterococcus (from Biomin IMB52™); Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius (from Biomin C5®); Lactobacillus farciminis (from Biacton®); Enterococcus (from Oralin E1707®);Enterococcus (2 strains), Lactococcus lactis DSM 1103 (from Probios-pioneer PDFM®); Lactobacillus rhamnosus and Lactobacillus farciminis (from Sorbiflor®); Bacillus subtilis (from Animavit®); Enterococcus (from Bonvital®); Saccharomyces cerevisiae (from Levucell SB 20®); Saccharomyces cerevisiae (from Levucell SC0 & SC10® from ME); Pediococcus acidilacti (from Bactocell); Saccharomyces cerevisiae (from ActiSaf® (formerly BioSaf®)); Saccharomyces cerevisiae NCYC Sc47 (from Actisaf® SC47); Clostridium butyricum (from Miya-Gold®); Enterococcus (from Fecinor and Fecinor Plus®); Saccharomyces cerevisiae NCYC R-625 (from InteSwine®); Saccharomyces cerevisiae cerevisia (from BioSprint®); Enterococcus and Lactobacillus rhamnosus (from Provita®);Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-C®); Bacillus cereus (from Toyocerin®); Bacillus cereus var. toyoi NCIMB 40112 / CNCMI-1012 (from TOYOCERIN®) or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from BioPlus® YC) and Bacillus subtilis (from GalliPro®);

[0078] The DFM may be combined with Enviva® PRO, commercially available from Danisco A / S. Enviva® PRO is a combination of Bacillus 2084 strain accession number NRRL B-50013, Bacillus LSSAO1 strain accession number NRRL B-50104, and Bacillus 15A-P4 strain ATCC accession number PTA-6507 (as taught in U.S. Pat. No. 7,754,469B, incorporated herein by reference). Preferably, the DFM described herein comprises a microorganism that is generally recognized as safe (GRAS), preferably a GRAS approved microorganism. A person skilled in the art will readily recognize specific species and / or strains of microorganisms within the genera described herein that are used in the food and / or agricultural industry and are generally considered suitable for animal consumption.

[0079] In another embodiment, the DFM may be combined with one or more of the L. reuteri S1 strain, the L. reuteri S2 strain, the L. reuteri S3 strain, the L. reuteri A2 strain, the L. gallinarum H1 strain, the L. salivarius H2 strain, and / or the L. agilis H3 strain, all of which are available from the Westerdijk Fungal Biodiversity Institute (WFDB), Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. The deposits were made in the Netherlands on July 24, 2019 and assigned accession numbers CBS145921, CBS145922, CBS145923, CBS145924, CBS145918, CBS145919 and CBS145920, respectively, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (see also WO2021034660, which is incorporated herein by reference in its entirety).

[0080] In some embodiments, it is important that the DFM be able to withstand heat (i.e., be heat tolerant). This is especially true when the feed is pelleted. Thus, in another embodiment, the DFM can be a heat tolerant microorganism, such as a heat tolerant bacterium, including Bacillus spp.

[0081] In other aspects, it may be desirable for the DFM to include spore-producing bacteria (e.g., Bacillus, e.g., Bacillus spp.). Bacillus can form stable endospores when growth conditions are unfavorable and are highly resistant to heat, pH, humidity and disinfectants.

[0082] The DFM described herein may reduce or prevent the establishment of pathogenic microorganisms (e.g., Clostridium perfringens, and / or E. coli, and / or Salmonella spp., and / or Campylobacter spp.) in the intestine. In other words, the DFM may be anti-pathogenic. The term "anti-pathogenic" as used herein means that the DFM opposes the action (negative effect) of a pathogen.

[0083] As mentioned above, the DFM can be any suitable DFM. For example, the following assay "DFM Assay" can be used to determine the compatibility of a microorganism with a DFM. The DFM Assay used herein is described in more detail in US Patent Publication No. 2009 / 0280090. For the avoidance of doubt, the DFM selected as an inhibitory strain (or anti-pathogenic DFM) according to the "DFM Assay" taught herein is a suitable DFM for use according to the present disclosure, i.e., for use in the feed additive composition according to the present disclosure. Tubes were seeded with representative pathogens (e.g., bacteria) from representative clusters, respectively. The seeded tubes were added with supernatants of potential DFMs grown aerobically or anaerobically (except for controls, where no supernatant was added to the tubes) and incubated. After incubation, the optical density (OD) of the control and supernatant-treated tubes was measured for each pathogen. Colonies of strains (potential DFM) that produce a lower OD compared to the control (no supernatant) may then be classified as inhibitory strains (or anti-pathogenic DFM). The DFM assay used herein is therefore described in more detail in US Patent Application Publication No. 2009 / 0280090. In some embodiments, the representative pathogen used in this DFM assay may be one (or more) of the following: Clostridium, e.g., Clostridium perfringens, and / or Clostridium difficile, and / or E. coli, and / or Salmonella spp., and / or Campylobacter spp.In a preferred embodiment, the assay is performed using one or more of Clostridium perfringens, and / or Clostridium difficile, and / or E. coli, preferably Clostridium perfringens and / or Clostridium difficile, more preferably Clostridium perfringens.

[0084] The DFM may be prepared as cultures and carriers (if used) and mixed in a ribbon or paddle mixer for about 15 minutes, although this timing may be increased or decreased. These components are blended to obtain a homogenous mixture of cultures and carriers. The final product is preferably a dry, free-flowing powder. The DFM containing one or more bacterial strains may then be added (preferably simultaneously with the enzymes described herein) to animal feed or feed premixes, added to water for animals, or administered by other methods known in the art. The content of the individual strains in the DFM mixture may vary in percentage from 1% to 99%, preferably from 25% to 75%. A suitable dosage of DFM in animal feed is about 1×10 3 CFU / g (feed) ~ approx. 1×10 10 CFU / g feed, preferably about 1×10 4 CFU / g (feed) ~ approx. 1×10 8 CFU / g feed, preferably about 7.5×10 4 CFU / g (feed) ~ approx. 1×10 7 In another embodiment, the DMF may range from about 1×10 CFU / g feed. 3 CFU / g feed, preferably about 1×10 4 CFU / g feed, preferably about 5×10 4 CFU / g feed, preferably about 1×10 5CFU / g feed may be administered.

[0085] DFM is present in the feed additive composition at a concentration of about 1 × 10 3 CFU / g (composition) ~ approx. 1×10 13 CFU / g (composition), e.g., 1×10 5 CFU / g (composition) ~ approx. 1×10 13 CFU / g (composition), e.g., about 1×10 6 CFU / g (composition) ~ approx. 1×10 12 CFU / g (composition), e.g., about 3.75 x 10 7 CFU / g (composition) ~ approx. 1×10 11 In another embodiment, the DFM may be administered in a feed additive composition at a concentration of about 1×10 5 CFU / g of composition, e.g., about 1×10 6 CFU / g of composition, e.g., about 3.75×10 7 In one embodiment, the DFM may be administered in a feed additive composition at a concentration of about 2×10 5 CFU / g (composition), e.g., about 2×10 6 CFU / g (composition), preferably about 3.75×10 7 CFU / g of composition.

[0086] In some embodiments, the bacterium is Bifidobacterium animalis subsp. lactis strain Bl-04 and / or Lactobacillus acidophilus strain NCFM. The bacterial strains have been deposited in accordance with the Budapest Treaty at the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig, Germany, by DuPont Nutrition Biosciences ApS, Langebrogade 1, DK-1411 Copenhagen K, Denmark, and are registered under the following accession numbers: strain NCFM (DSM33840), deposited on March 15, 2021; and strain Bl-04 (DSM33525), deposited on May 19, 2020. These bacterial strains are commercially available from DuPont Nutrition Biosciences ApS.

[0087] Additionally, in some embodiments, the secretome or fraction thereof of any of the DFMs disclosed herein (e.g., the secretome of Bifidobacterium animalis subsp. lactis strain Bl-04 and / or Lactobacillus acidophilus strain NCFM) may be used in any of the compositions or methods disclosed herein. The secretome of any of the DFMs disclosed herein may be obtained by any standard means known in the art, including, but not limited to, cultured cell supernatants, modified supernatants, supernatant fractions, partially purified secretomes, and secretome fractions of cells.

[0088] B.Essential oil Essential oils are concentrated volatile oils that have the characteristic aroma of the plant from which they are derived. Typically, essential oils are obtained by distillation of plants and contain a mixture of constituent compounds, including anethole, beta-ionone, capsaicin, carvacrol, cinnamaldehyde, citral, cresol, eugenol, guaiacol, limonene, thymol, tannins, and vanillin.

[0089] The animal feed or feed additive composition disclosed herein may comprise at least 1 g of cinnamaldehyde per 1000 kg of animal feed, at least 2 g of cinnamaldehyde per 1000 kg of animal feed, at least 3 g of cinnamaldehyde per 1000 kg of animal feed, at least 4 g of cinnamaldehyde per 1000 kg of animal feed, or at least 5 g of cinnamaldehyde per 1000 kg of animal feed.

[0090] The animal feed or feed additive composition disclosed herein may comprise at least 1 mg of cinnamaldehyde per kg of animal feed, at least 2 mg of cinnamaldehyde per kg of animal feed, at least 3 mg of cinnamaldehyde per kg of animal feed, at least 4 mg of cinnamaldehyde per kg of animal feed, or at least 5 mg of cinnamaldehyde per kg of animal feed.

[0091] For broilers, the animal feed or feed additive composition disclosed herein may contain less than 6g cinnamaldehyde per 1000kg of animal feed, such as less than 5.9g cinnamaldehyde.For pigs, the animal feed may contain less than 18g cinnamaldehyde per 1000kg of animal feed, such as less than 17g cinnamaldehyde per 1000kg of animal feed, less than 16g cinnamaldehyde per 1000kg of animal feed, less than 15g cinnamaldehyde per 1000kg of animal feed, or less than 14g cinnamaldehyde per 1000kg of animal feed.

[0092] The animal feed or feed additive composition disclosed herein comprises thymol, the animal feed comprising at least 1 g thymol per 1000 kg of the animal feed or feed additive composition, at least 2 g thymol per 1000 kg of the animal feed or feed additive composition, at least 3 g thymol per 1000 kg of the animal feed or feed additive composition, at least 4 g thymol per 1000 kg of the animal feed or feed additive composition, at least 5 g thymol per 1000 kg of the animal feed or feed additive composition, at least 6 g thymol per 1000 kg of the animal feed or feed additive composition, at least 7 g thymol per 1000 kg of the animal feed or feed additive composition, at least 8 g thymol per 1000 kg of the animal feed or feed additive composition, at least 9 g thymol per 1000 kg of the animal feed or feed additive composition, at least 10 ... The animal feed or feed additive composition may comprise at least 8g thymol per 1000kg, at least 9g thymol per 1000kg, at least 10g thymol per 1000kg, at least 11g thymol per 1000kg, at least 12g thymol per 1000kg, at least 13g thymol per 1000kg, at least 14g thymol per 1000kg, at least 15g thymol per 1000kg. The animal feed or feed additive composition may comprise less than 50g thymol per 1000kg animal feed or feed additive composition.

[0093] The animal feed or feed additive composition disclosed herein may comprise at least 0.00001% by weight of components (i), (ii) and carvacrol. Suitably, the animal feed or feed additive composition may comprise at least 0.00005% by weight; at least 0.00010% by weight; at least 0.00020% by weight; at least 0.00025% by weight; at least 0.00050% by weight; at least 0.00100% by weight; at least 0.00200% by weight of components (i), (ii) and carvacrol.

[0094] The animal feed or feed additive composition disclosed herein may comprise at least 0.0001% by weight of the feed enzyme. Preferably, the animal feed may comprise at least 0.0005% by weight; at least 0.0010% by weight; at least 0.0020% by weight; at least 0.0025% by weight; at least 0.0050% by weight; at least 0.0100% by weight of the feed enzyme. The feed may comprise at least 0.001% by weight of the animal feed additive. Preferably, the animal feed may comprise at least 0.005% by weight; at least 0.010% by weight; at least 0.020% by weight; at least 0.100% by weight; at least 0.200% by weight; at least 0.250% by weight; at least 0.500% by weight of the animal feed additive or feed additive composition.

[0095] C. Enzyme In one embodiment, the present disclosure relates to a feed or feed additive composition comprising an osmotic modifier (e.g., betaine), an essential oil, aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, B vitamins (e.g., vitamins B1, B6, and / or B12), and / or DFM, and further comprising at least one enzyme (e.g., phytase). Enzymes suitable for use in accordance with the methods disclosed herein include, but are not limited to, glucoamylase, xylanase, amylase, phytase, beta-glucanase, and protease.

[0096] 1. Glucoamylase Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and by ca.

[0097] In one embodiment, provided herein is an osmotic modifier (e.g., betaine), essential oil and / or DFM feed or feed additive composition comprising one or more glucoamylases. The glucoamylase may be any commercially available glucoamylase. A suitable glucoamylase may be 1,4-alpha-D-glucan glucohydrolase (EC 3.2.1.3). All EC enzyme classifications referred to herein relate to the classifications provided in the International Union of Biochemistry and Molecular Biology's Enzyme Nomenclature-Recommendations (1992)-ISBN 0-12-226164-3, which is incorporated herein.

[0098] Glucoamylases have been used successfully in commercial applications for many years. In addition, various mutations have been introduced into fungal glucoamylases (e.g., Trichoderma reesei glucoamylase (TrGA)) to enhance thermostability and specific activity. See, for example, WO 2008 / 045489; WO 2009 / 048487; WO 2009 / 048488 and U.S. Pat. No. 8,058,033. Glucoamylase activity can be assessed using any means known in the art, such as those described in the Examples section below.

[0099] The glucoamylase may be from any suitable source, for example from a microorganism or a plant. The glucoamylase may be of fungal or bacterial origin and may be selected from the group consisting of Aspergillus glucoamylases, such as Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5):1097-1102) or variants thereof, such as those disclosed in WO 92 / 00381, WO 00 / 04136 and WO 01 / 04273 (from Novozymes, Denmark); A. awamori glucoamylase disclosed in WO 84 / 02921 (Hata et al., 1991, Agric. Biol. Chem. 55(4):941-949) or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with increased thermostability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9:499-505); D257E and D293E / Q (Chen et al., 1995, Prot. Eng. 8:575-582); N182 (Chen et al., 1994, Biochem. J. 301:275-281); disulfide bond, A246C (Fierobe et al., 1996, Biochemistry 35:8698-8704; and introduction of Pro residues at positions A435 and S436 (Li et al., 1997, Protein Eng. 10:1199-1204).

[0100] Other glucoamylases include Athelia rolfsii (formerly Corticium rolfsi) glucoamylase (see U.S. Pat. No. 4,727,026 and Nagasaka et al., 1998, Appl. Microbiol. Biotechnol. 50:323-330), Talaromyces glucoamylases, particularly Talaromyces duponti, Talaromyces emersonii (WO 99 / 28448), Talaromyces leycettanus (U.S. Reissue Pat. No. 32,153), and Talaromyces thermophilus. thermophilus) (U.S. Pat. No. 4,587,215).

[0101] Bacterial glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135138) and C. thermohydrosulfuricum (WO 86 / 01831), Trametes cingulata, Pachykytospora papyracea and Leucopaxillus giganteus (all as disclosed in WO 2006 / 069289); or Peniophora ruhomarginata. rufomarginata) (described in WO 2007 / 124285 or PCT / US2007 / 066618); or mixtures thereof. Hybrid glucoamylases may be used in the present invention. Examples of hybrid glucoamylases are disclosed in WO 2005 / 045018. Specific examples include the hybrid glucoamylases disclosed in Tables 1 and 4 in Example 1 (these hybrids are incorporated herein by reference).

[0102] The glucoamylase may have a high degree of sequence identity to any of the glucoamylases described above, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequence described above.

[0103] Commercially available glucoamylase compositions include: AMG 200L; AMG 300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME ULTRA, SPIRIZYME™ EXCEL and AMG™ E (Novozymes A / S, Denmark); OPTIDEX™ 300, GC480™ and GC147™ (Genencor Int., USA); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (Genencor Int.).

[0104] 2. Xylanase Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide β-1,4-xylan to xylose and thus to hemicellulose, one of the major components of plant cell walls. Xylanases (e.g., endo-β-xylanases (EC 3.2.1.8)) hydrolyze the xylan backbone. In one embodiment, provided herein are osmotic modifier (e.g., betaine), essential oil and / or DFM-containing feed or feed additive compositions comprising one or more xylanases.

[0105] In another embodiment, provided herein is a feed or feed additive composition comprising one or more xylanases. In one embodiment, the xylanase can be any commercially available xylanase. Suitably, the xylanase can be endo-1,4-Pd-xylanase (classified as EG3.2.1.8) or 1,4β-xylosidase (classified as EG3.2.1.37). All EC enzyme classifications referred to herein refer to the classifications provided in the Enzyme Nomenclature-Recommendations (1992) -ISBN 0-12-226164-3 of the Commission on Nomenclature of the International Union of Biochemistry and Molecular Biology, incorporated herein.

[0106] In another embodiment, the xylanase can be from the genera Bacillus, Trichoderma, Therinomyces, Aspergillus, and Penicillium. In yet another embodiment, the xylanase can be within Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A / S. In an embodiment, the xylanase can be a mixture of two or more xylanases. In yet another embodiment, the xylanase is an endo-1,4-β-xylanase or a 1,4-β-xylosidase. In yet another embodiment, the xylanase is derived from an organism selected from the group consisting of Bacillus, Trichoderma, Thermomyces, Aspergillus, Penicillium, and Humicola, hi yet another embodiment, the xylanase can be one or more of the xylanases listed in Table 1 or one or more of the commercially available products. [Table 1]

[0107] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanases, in one embodiment the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and more than 750 xylanase units / g of composition.

[0108] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000 and greater than 8000 xylanase units / g of composition.

[0109] One xylanase unit (XU) will be understood to be the amount of enzyme that will liberate 0.5 μmol of reducing sugar equivalents (as xylose by the dinitrosalicylic acid (DNS) assay reducing sugar method) per minute from oat spelt-xylan substrate at pH 5.3 and 50° C. (Bailey, et al., Journal of Biotechnology, Volume 23, (3), May 1992, 257-270).

[0110] 3. Amylase Amylases are a class of enzymes that can hydrolyze starch into shorter chain oligosaccharides such as maltose. Glucose moieties can then be transferred to monoglycerides or glycosyl monoglycerides more readily than from the original starch molecule. The term amylase includes α-amylases (EG3.2.1.1), G4-forming amylases (EG3.2.1.60), β-amylases (EG3.2.1.2) and γ-amylases (EC3.2:1.3). Amylases can be of bacterial or fungal origin, or can be derived from chemically modified or protein engineered mutants. In another embodiment, provided herein are osmoticum (e.g., betaine), essential oil and / or DFM-containing feed or feed additive compositions comprising one or more amylases.

[0111] In one embodiment, the amylase can be a mixture of two or more amylases. In another embodiment, the amylase can be one amylase (e.g., an α-amylase from Bacillus licheniformis) and one amylase (e.g., an α-amylase from Bacillus amyloliquefaciens). In one embodiment, the α-amylase can be an α-amylase within Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A / S. In yet another embodiment, the amylase can be a pepsin-resistant α-amylase, such as a pepsin-resistant Trichoderma (e.g., Trichoderma reesei) α-amylase. Suitable pepsin resistant α-amylases are taught in UK Patent Application No. 1011513.7 (hereby incorporated by reference) and PCT / IB2011 / 053018 (hereby incorporated by reference).

[0112] In one embodiment, the amylase used in the present invention may be one or more of the amylases in one or more of the commercially available products listed in Table 2. [Table 2]

[0113] It will be understood that 1 amylase unit (AU) is the amount of enzyme that will liberate 1 mmol of glycosidic bonds from a water-insoluble crosslinked starch polymer substrate per minute at pH 6.5 and 37° C. (this may be referred to herein as the assay for determining 1 AU).

[0114] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more amylases, in one embodiment the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and more than 750 amylase units / g of composition.

[0115] In one embodiment, the composition is 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7 000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and more than 15000 amylase units / g (composition).

[0116] 4. Proteases The term protease as used herein is synonymous with peptidase or proteinase. The protease may be subtilisin (EG3.4.21.62) or bacillolysin (EG3.4.24.28) or alkaline serine protease (EG3.4.21.x) or keratinase (EG3.4.XX). In one embodiment, the protease is subtilisin. Suitable proteases include proteases of animal, vegetable or microbial origin. Chemically modified or protein engineered variants are also suitable. The protease may be a serine protease or a metalloprotease, such as an alkaline microbial protease or a trypsin-like protease. In another embodiment, provided herein is an osmolyte (e.g., betaine), essential oil and / or DFM-containing feed or feed additive composition comprising one or more proteases.

[0117] Examples of alkaline proteases are subtilisins, particularly those derived from Bacillus sp., such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, for example, U.S. Pat. No. 6,287,841), subtilisin 147 and subtilisin 168 (see, for example, WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and Fusarium proteases (see, for example, WO 89 / 06270 and WO 94 / 25583). Examples of useful proteases also include, but are not limited to, the variants described in WO 92 / 19729 and WO 98 / 20115.

[0118] In another embodiment, the protease can be one or more of the proteases in one or more of the commercially available products listed in Table 3. [Table 3]

[0119] In one embodiment, the protease is selected from the group consisting of subtilisin, bacillolysin, alkaline serine protease, keratinase, and Nocardiopsis protease.

[0120] It will be understood that 1 protease unit (PU) is the amount of enzyme that liberates 1 microgram of phenolic compounds (expressed as tyrosine equivalents) from a substrate (0.6% casein solution) in 1 minute at pH 7.5 (40 mM Na2PO4 / lactate buffer) and about 40° C. This may be referred to as the assay for determining 1 PU.

[0121] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more proteases. In another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanases and a protease. In yet another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more amylases and a protease. In yet another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanases, amylases and proteases.

[0122] In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and greater than 750 protease units / g of composition.

[0123] In one embodiment, the composition is 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 70 00-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 protease units / g (composition).

[0124] 5. Phytase In another embodiment, provided herein is an osmotic modifier (e.g., betaine), essential oil and / or DFM-containing feed or feed additive composition comprising one or more phytases. The phytase used in the present invention may be classified as a 6-phytase (classified as EC 3.1.3.26) or a 3-phytase (classified as EC 3.1.3.8). In one embodiment, the phytase used in the present invention may be one or more of the phytases in one or more of the commercially available products in Table 4 below. [Table 4]

[0125] In one embodiment, the phytase is a Citrobacter phytase, e.g., from Citrobacter freundii, e.g., C. freundii NCIMB 41247 and variants thereof, as disclosed in WO 2006 / 038062 (hereby incorporated by reference) and WO 2006 / 038128 (hereby incorporated by reference), Citrobacter braakii YH-15, as disclosed in WO 2004 / 085638, Citrobacter braakii ATCC 41161, as disclosed in WO 2006 / 037328 (hereby incorporated by reference), Citrobacter freundii ... 51113 and variants thereof, e.g., Citrobacter amalonaticus, as disclosed in WO 2007 / 112739 (herein incorporated by reference) and WO 2011 / 117396 (herein incorporated by reference). In some embodiments, Citrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC 25407, Citrobacter gillenii, as disclosed in WO 2006037327 (herein incorporated by reference).In some embodiments, Citrobacter gillenii DSM 13694 or Citrobacter intermedius, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Citrobacter species polypeptides or variants thereof disclosed in WO2006037327, incorporated herein by reference.

[0126] In some embodiments, the phytase is an E. coli phytase sold under the name Phyzyme XP™ by Danisco A / S. Alternatively, the phytase is a Buttiauxella phytase, such as a Buttiauxella agrestis phytase, such as the phytase enzymes taught in WO 2006 / 043178, WO 2008 / 097619, WO 2009 / 129489, WO 2008 / 092901, PCT / US2009 / 41011, or PCT / IB2010 / 051804, all of which are incorporated herein by reference. Alternatively, the phytase may be an engineered robust high Tm clade phytase polypeptide, such as a phytase disclosed in WO 2020 / 106796, which is incorporated herein by reference.

[0127] In one embodiment, the phytase can be a phytase from the genus Hafnia, such as a phytase from Hafnia alvei, such as a phytase enzyme taught in US Patent Publication No. 2008263688, which is incorporated herein by reference. In one embodiment, the phytase can be a phytase from the genus Aspergillus, such as a phytase from Aspergillus oryzae. In one embodiment, the phytase can be a phytase from the genus Penicillium, such as a phytase from Penicillium funiculosum.

[0128] In some embodiments, the phytase is present in the feed or feed additive composition in the range of about 200 FTU / kg feed to about 1000 FTU / kg feed. In some embodiments, about 300 FTU / kg feed to about 750 FTU / kg feed. In some embodiments, about 400 FTU / kg feed to about 500 FTU / kg feed. In one embodiment, the phytase is present in the feed at more than about 200 FTU / kg feed, preferably more than about 300 FTU / kg feed, preferably more than about 400 FTU / kg feed. In one embodiment, the phytase is present in the feed at less than about 1000 FTU / kg feed, preferably less than about 750 FTU / kg feed. In some embodiments, the phytase is present in the feed additive composition in the range of about 40 FTU / g to about 40,000 FTU / g composition; about 80 FTU / g to about 20,000 FTU / g composition; about 100 FTU / g to about 10,000 FTU / g composition; and about 200 FTU / g to about 10,000 FTU / g composition. In one embodiment, the phytase is present in the feed additive composition in greater than about 40 FTU / g composition, preferably greater than about 60 FTU / g composition, preferably greater than about 100 FTU / g composition, preferably greater than about 150 FTU / g composition, preferably greater than about 200 FTU / g composition. In one embodiment, phytase is present in the feed additive composition at less than about 40,000 FTU / g composition, preferably less than about 20,000 FTU / g composition, preferably less than about 15,000 FTU / g composition, preferably less than about 10,000 FTU / g composition.

[0129] As used herein, 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from a substrate in 1 minute under reaction conditions defined in the ISO 2009 Phytase Assay - a standard assay for determining phytase activity - and it will be understood that 1 FTU can be found in International Standard ISO / DIS 30024:1-17, 2009. In one embodiment, the enzyme is classified using the EC classification above, which designates an enzyme that has this activity when tested in the assay taught herein to determine 1 FTU.

[0130] D. Feed and feed additives The enzymes may be encapsulated for use in animal feed or premixes, either alone or in combination with at least one direct-fed probiotic, an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, B vitamins (e.g., vitamins B1, B6 and / or B12), and / or at least one other enzyme. Additionally, the enzymes may be in the form of granules, whether encapsulated or not, alone or in combination with at least one direct-fed probiotic, an osmotic regulator (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate salts), ornithine, arginine, phosphate, acetate, B vitamins (e.g., vitamins B1, B6 and / or B12), and / or in combination with at least one protease, amylase, xylanase, beta-glucosidase and / or phytase.

[0131] Animal feed may include plant materials such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of these plant materials or plant protein sources for poultry, swine, ruminants, aquaculture, and pets. The terms "animal feed", "feed" and "livestock feed" are used interchangeably and may include one or more feed ingredients selected from the group including: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and / or large grains (e.g., corn or sorghum); b) cereal by-products, such as corn gluten meal, distillers dried grains with solubles (DDGS) (especially corn-based distillers dried grains with solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel and citrus pulp; c) proteins obtained from sources such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, rapeseed, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame, etc.; d) fats and oils obtained from plant and animal sources; and / or e) minerals and vitamins.

[0132] When used as a feed, such as a functional feed, or in the preparation of such a feed, the enzyme or feed additive composition of the present invention may be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, and a nutritionally active ingredient. For example, at least one component selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben, and propylparaben.

[0133] In a preferred embodiment, the enzyme or feed additive composition of the present invention is mixed with a feed mix ingredient to form a feed. The term "feed ingredient" as used herein means all or a part of a feed. A part of a feed can mean one component of the feed or multiple, for example two, or three, or four or more components of the feed. In one embodiment, the term "feed ingredient" encompasses a premix or premix ingredients.

[0134] Preferably, the feed may be a livestock feed or a premix thereof, a compound feed or a premix thereof. The feed additive composition according to the present invention may be mixed with a compound feed, a compound feed component, or a premix of a compound feed, or a livestock feed, a livestock feed component, or a premix of a livestock feed.

[0135] The term "livestock feed" as used herein means any food that is provided to animals (rather than the animals having to forage for it themselves). Livestock feed includes cut plants. In addition, livestock feed includes silage, pressed and pelleted feed, oil and mixed feed, and also germ grains and legumes.

[0136] Livestock feed can be obtained from one or more of the plants selected from the following: corn (maize), alfalfa (lucerne), barley, lotus grass, oilseed rape, Chau moellier, kale, rapeseed (canola), rutabaga (sweet turnip), turnip, clover, astragalus pratense, red clover, subterranean clover, white clover, fescue, brome, millet, oats, sorghum, soybeans, trees (tree shoots clipped short for woody pasture), wheat and legumes.

[0137] The term "formulated feed" refers to commercially available feed in the form of meal, pellets, nuts, cakes or grinds. Formulated feed may be blended from a variety of raw materials and additives.

[0138] The blend is formulated according to the specific requirements of the target animal.

[0139] Formulated feeds may be complete feeds providing all of the daily required nutrients, concentrates providing a portion of the feed (protein, energy), or supplements providing only additional micronutrients such as minerals and vitamins. The main ingredients used in formula feeds are feed grains including corn, wheat, canola meal, rapeseed meal, lupin, soybean, sorghum, oats and barley.

[0140] Suitably, a "premix" as referred to herein may be a composition comprised of minor ingredients (e.g., vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients). A premix is ​​usually a composition suitable for blending into a commercially available food product.

[0141] As used herein, the term "contacting" refers to the indirect or direct application of the enzyme to a product (e.g., feed), alone or in combination with at least one direct-fed live bacteria, an osmotic regulator (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6 and / or B12), and / or at least one other enzyme. Examples of application methods that may be used include, but are not limited to, treating the product in a substance containing the feed additive composition, directly applying the feed additive composition by mixing it with the product, spraying the feed additive composition on the surface of the product, or immersing the product in a preparation of the feed additive composition. In one embodiment, the feed additive composition of the present invention is preferably mixed with the product (e.g., feed). Alternatively, the feed additive composition may be included in an emulsion or ingredient of the feed.

[0142] The enzymes can be homogenized alone to produce a powder or in combination with at least one direct-fed probiotic, an osmotic regulator (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, B vitamins (e.g., vitamins B1, B6 and / or B12), and / or at least one other enzyme to produce a powder. In alternative embodiments, the enzymes may be formulated into granules alone as described in WO 1997 / 016076 or WO 1992 / 012645, herein incorporated by reference (referred to as TPT granules), or in combination with at least one direct-fed live bacteria, an osmotic agent (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, B vitamins (e.g., vitamins B1, B6 and / or B12), and / or at least one other enzyme. "TPT" stands for Thermal Protection Technology.

[0143] In another aspect, when the feed additive composition is formulated into a granule, the granule comprises a hydration barrier salt coated onto the protein core. The benefits of such a salt coating are improved heat resistance, improved storage stability, and protection from other feed additives that would otherwise have a detrimental effect on the enzyme. Preferably, the salt used in the salt coating has a water activity of greater than 0.25 or a moisture content of greater than 60% at 20°C. In some embodiments, the salt coating comprises Na2SO4.

[0144] The method of preparing the enzyme alone or in combination with at least one direct-fed probiotic, an osmotic regulator (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B (e.g., vitamin B1, B6 and / or B12) and / or at least one other enzyme may also include the further step of pelleting the powder. This powder may be mixed with other components known in the art. The powder or a mixture containing this powder may be extruded through a die and the resulting strands cut into suitable pellets of variable length.

[0145] Optionally, the pelletizing process may include a steaming or conditioning step prior to the formation of pellets. The mixture containing the powder may be placed in a conditioner (e.g., a mixer with steam injection). The mixture is heated in the conditioner to a specific temperature, such as 60-100°C, with typical temperatures being 70°C, 80°C, 85°C, 90°C, or 95°C. Residence times may vary from a few seconds to a few minutes, or even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour.

[0146] It will be understood by those skilled in the art that different animals have different feed requirements, and that the same animal may have different feed requirements depending on the purpose for which the animal is being raised. Optionally, the feed may also include additional minerals, such as calcium, and / or additional vitamins. In some embodiments, the feed is corn soybean meal, wheat, or a mixed grain mix.

[0147] Feed is typically produced in a feed mill, where the raw material is first ground to a suitable particle size and then mixed with appropriate additives. The feed can then be produced as a mash or pellets; the latter typically involves raising the temperature to a target level and then passing the feed through a die to produce pellets of a specific size. The pellets are cooled. Liquid additives such as fats and enzymes can then be added. Feed production can also include further steps including extrusion or expansion prior to pelleting, in particular by suitable techniques that may include at least the use of steam.

[0148] The feed may be for monogastric animals such as poultry (e.g., broilers, egg-laying chickens, broiler breeders, turkeys, ducks, geese, waterfowl) and pigs (all age categories), ruminants such as cattle (e.g., dairy cows or bulls (including calves)), horses, sheep, pets (e.g., dogs, cats) or fish (e.g., stomachless fish, stomached fish, freshwater fish such as salmon, cod, trout and carp, e.g. koi carp, sea bass, and crustaceans such as shrimp, mussels and scallops).

[0149] The feed additive composition and / or the feed containing it may be used in any suitable form. The feed additive composition may be used in the form of a solid or liquid preparation or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts and granules, which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous solutions, organic solutions or aqueous-organic solutions, suspensions and emulsions.

[0150] In some applications, the feed additive composition may be mixed with the feed or administered in drinking water (e.g., drinking water from wells, fountains, shallow wells, semi-dug and dug wells, municipal water, lakes or streams). In other embodiments, one or more components of the feed additive composition (e.g., one or more of osmotic modifiers, essential oils, DFM or feed enzymes) are administered in drinking water, and one or more components of the feed additive composition (e.g., one or more of osmotic modifiers, essential oils, DFM or feed enzymes) are administered in the feed at the same time. When water supply delivery is intended, administration of the feed additive composition may include, but is not limited to, one or more of mixing the composition with water, rehydrating the components of the composition (e.g., DFM), placing the hydrated composition in a medicator and administering it to the water supply by a dosatron or other pumping means.

[0151] A feed additive composition comprising a DFM as taught herein (e.g., a DFM comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) and / or one or more osmolality adjusting agents (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and / or one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase and amylase), aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, and / or B vitamins (e.g., vitamins B1, B6 and / or B12) in combination with a feed acceptable carrier, diluent or excipient, and (optionally) packaging.

[0152] The feed and / or feed additive composition may be combined with at least one mineral and / or at least one vitamin. The composition thus obtained may be referred to herein as a premix. The feed may comprise at least 0.0001% by weight of the feed additive. Suitably, the feed may comprise at least 0.0005% by weight; at least 0.0010% by weight; at least 0.0020% by weight; at least 0.0025% by weight; at least 0.0050% by weight; at least 0.0100% by weight; at least 0.020% by weight; at least 0.100% by weight; at least 0.200% by weight; at least 0.250% by weight; at least 0.500% by weight of the feed additive.

[0153] Preferably, the food or feed additive composition may further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na2S04, talc, PVA, and mixtures thereof. In a further embodiment, the food or feed additive may further comprise a metal ion chelating agent. The metal ion chelating agent may be selected from EDTA or citric acid.

[0154] In some embodiments, the food or feed additive composition comprises one or more enzymes (e.g., a protease, phytase, xylanase, glucoamylase, or amylase) at a level of at least 0.0001 g / kg, 0.001 g / kg, at least 0.01 g / kg, at least 0.1 g / kg, at least 1 g / kg, at least 5 g / kg, at least 7.5 g / kg, at least 10.0 g / kg, at least 15.0 g / kg, at least 20.0 g / kg, or at least 25.0 g / kg.

[0155] In some embodiments, the food or feed additive comprises one or more enzymes (e.g. protease, phytase, xylanase, glucoamylase or amylase) at a level such that when added to a food or feed material, the feed material comprises 1-500 mg / kg, 1-100 mg / kg, 2-50 mg / kg or 2-10 mg / kg of the one or more enzymes. In some embodiments of the invention, the food or feed material comprises at least 100, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000, 50000, 100000, 500000, 1000000 or 2000000 Units of enzyme per kg of feed or food material. In some embodiments, one unit of a-1,2-fucosidase activity can be defined as the amount of enzyme that can catalyze the release of one molecule of substrate per minute under standard assay conditions.

[0156] Formulations containing any of the enzymes described herein may be made in any suitable manner to ensure that the formulation contains active enzyme. Such formulations may be liquid, dry powder or granules. Preferably, the feed additive composition is in a solid form suitable for addition onto or into feed pellets.

[0157] Dry powders or granules may be prepared by means known to those skilled in the art, such as high shear granulation, drum granulation, extrusion, spheronization, fluid bed agglomeration, fluid bed spray drying, and the like.

[0158] The feed additive compositions described herein may be formulated into dry powders or granules as described in WO 2007 / 044968 (referred to as TPT granules), or WO 1997 / 016076, or WO 1992 / 012645, each of which is incorporated herein by reference.

[0159] In one embodiment, the animal feed may be formulated as a granule for a feed composition comprising a core; an active agent; and at least one coating, wherein the active agent of the granule maintains at least 50% activity, at least 60% activity, at least 70% activity, at least 80% activity after being subjected to conditions selected from one or more of: a) a feed pelleting process, b) a steam heating feed pretreatment process, c) storage, d) storage as an ingredient in a non-pelleted mixture, and e) storage as an ingredient in a feed base mix or feed premix comprising at least one compound selected from trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that result in an acidic or basic feed base mix or feed premix.

[0160] For granules, the at least one coating may comprise a moisture hydrating material that accounts for at least 55% w / w of the granule; and / or the at least one coating may comprise two coatings. The two coatings may be a moisture hydrating coating and a moisture barrier coating. In some embodiments, the moisture hydrating coating may be 25% to 60% w / w of the granule and the moisture barrier coating may be 2% to 15% w / w of the granule. The moisture hydrating coating may be selected from inorganic salts, sucrose, starch and maltodextrin, and the moisture barrier coating may be selected from polymers, gums, whey and starch.

[0161] The feed additive composition may be formulated into a granule for animal feed comprising a core; an active agent, the active agent of the granule retaining at least 80% activity after storage and after the steam heated pelleting process of which the granule is a component; a moisture barrier coating; and a moisture hydrating coating that is at least 25% w / w of the granule, the granule having a water activity of less than 0.5 prior to the steam heated pelleting process.

[0162] The granules may have a moisture barrier coating selected from polymers and gums, and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may be 25% to 45% w / w of the granule, and the moisture barrier coating may be 2% to 10% w / w of the granule.

[0163] Granules may be produced using a steam heated pelletizing process which may be carried out at 85°C to 95°C for up to several minutes.

[0164] Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumables include one or more of a buffer, a salt, sorbitol and / or glycerol.

[0165] Similarly, the feed additive composition may be formulated by applying (e.g., spraying) the enzyme onto a carrier substrate, such as ground wheat. In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, the premix may include one or more feed components, such as one or more minerals and / or one or more vitamins.

[0166] In one embodiment, the at least one DFM and / or enzyme (e.g., protease, amylase, xylanase, beta-glucosidase, and / or phytase) is formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

[0167] Pharmaceutically acceptable salts (e.g., mineral acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or salts of organic acids such as acetate, propionate, malonate and benzoate) may be used. The pharma-ceutically acceptable carrier in the feed additive composition and / or the water supply composition may further comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in such compositions. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient. Once formulated, the compositions of the present invention may be administered directly to the subject. The subject to be treated may be an animal. However, in one or more embodiments, the compositions are suitable for administration to human subjects.

[0168] III. Method A. Methods of Treating or Preventing Necrotizing Enterocolitis The present disclosure relates to a method of treating or preventing necrotizing enteritis in a subject, comprising administering to the animal an effective amount of a feed, feed additive composition, or premix comprising a direct-fed bacteria (DFM) comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus. However, in some embodiments, the DFM comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus may be delivered to the subject in drinking water via the water supply. In another embodiment, the method relates to treating or preventing necrotizing enteritis in a subject comprising administering to the animal an effective amount of a feed, feed additive composition or premix containing water (e.g., water delivered by water mains) containing direct-fed bacteria (DFM) including Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus. The feed, feed additive composition or premix may further comprise one or more of an osmolality modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase).

[0169] In some embodiments, the subject is administered an effective amount of a feed or feed additive composition comprising a DFM comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or a secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus), thereby improving the survival and quality of the subject's diet by increasing the survival and quality of the subject's diet. In one embodiment, the incidence of necrotizing enterocolitis is reduced (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and all values ​​between these percentages) compared to the incidence of necrotizing enterocolitis present in subjects not administered an effective amount of a feed or feed additive composition comprising DFM comprising a secretome of Lactobacillus acidophilus (Lactobacillus subsp. lactis) and / or a secretome of Lactobacillus acidophilus. The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0170] In one embodiment, treating or preventing necrotizing enterocolitis comprises preventing or alleviating intestinal pathology in a subject. Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). Intestinal lesions are reduced (i.e., decreased in number and / or less severe) compared to the incidence of necrotizing enterocolitis present in subjects not administered an effective amount of a feed or feed additive composition comprising DFM containing a secretome of Lactobacillus subsp. lactis and / or Lactobacillus acidophilus (e.g., reduced by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and all values ​​between these percentages). The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0171] In another embodiment, treating or preventing necrotic enteritis comprises reducing the feed conversion ratio (FCR) of the subject. Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). In one embodiment, the FCR is reduced by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, and all values ​​between these percentages, compared to the FCR of a subject not administered an effective amount of a feed or feed additive composition comprising a DFM comprising a secretome of Lactobacillus subsp. lactis and / or Lactobacillus acidophilus. The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0172] In another embodiment, treating or preventing necrotizing enterocolitis comprises reducing mortality in a subject or a group of subjects. Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). lactis and / or Lactobacillus acidophilus secretome), the mortality rate is reduced (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, and all values ​​between these percentages) compared to the mortality rate of a subject or group of subjects not administered an effective amount of a feed or feed additive composition comprising a DFM comprising a secretome of Lactobacillus acidophilus and / or Lactobacillus subsp. lactis. The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0173] In another embodiment, treating or preventing necrotizing enterocolitis comprises increasing feed efficiency in a subject. Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). lactis and / or Lactobacillus acidophilus secretome), as compared to the feed efficiency of a subject not administered an effective amount of a feed or feed additive composition comprising DFM (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 700%, 700%, 710%, 720%, 730%, 740%, 750%, 76 (Increases of 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500% and all values ​​in between these percentages).The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0174] In another embodiment, treating or preventing necrotizing enterocolitis comprises increasing the final slaughter weight of the subject (i.e., the body weight of the subject entering the final stage of feeding).Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). a final slaughter weight is increased (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 555%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 850%, 800%, %, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500% and all values ​​between these percentages).The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0175] In another embodiment, treating or preventing necrotizing enterocolitis comprises increasing weight gain in the subject. Specifically, the present invention relates to a method for the production and / or propagation of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) by administering to a subject an effective amount of a feed or feed additive composition containing a DFM containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). lactis and / or Lactobacillus acidophilus secretome) as compared to the weight gain of a subject not administered an effective amount of a feed or feed additive composition comprising DFM (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, (Increases of 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500% and all values ​​in between these percentages).The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0176] In another embodiment, treating or preventing necrotizing enterocolitis comprises decreasing the expression of Clostridium perfringens necrotizing enterocolitis B-like toxin (NetB) in a subject. NetB is produced by C. perfringens toxin type A strains and, to a lesser extent, by type C strains (Kaldhusdal et al. (1999) FEMS Immunol Med Microbiol vol 24:337-343). The protein is 322 amino acids long in its active form with a predicted molecular weight of 36.5 kDa. Although the molecular basis of toxicity remains largely unknown, several studies have suggested that NetB is a novel member of the small β-pore-forming toxins (β-PFTs) because it is capable of forming pores in membranes and shares amino acid sequence similarity with several other related members of the pore-forming toxin family (38% identity with beta-toxin from C. perfringens, 40% identity with C. perfringens delta-toxin, and 31% identity with alpha-toxin from S. aureus) (Keyburn et al. (2008) PLoS Pathog vol 4:e26; Manich et al. (2008) PLoS One vol 3:e3764). Initially, alpha-toxin produced by the same bacteria was thought to be the main virulence factor causing NE, but experiments with alpha-toxin mutants showed that the strains were still virulent and could cause disease (Keyburn et al. (2006) Infect Immun vol 74:6496-6500). In contrast, netB mutants could not cause NE, whereas wild-type and complemented mutants could (Keyburn et al. (2008) PLoS Pathog vol 4:e26; Manich et al. (2008) PLoS One vol 3:e3764).However, it remains unclear whether NetB is a critical virulence factor in causing NE, since it has been reported that in some cases, C. perfringens strains lacking the NetB gene still have virulence capabilities (Cooper & Songer (2009) Vet Microbiol vol 142:323-328). Additionally, immunization studies using alpha toxin and other antigens (e.g., hypothetical zinc metalloprotease and pyruvate-ferredoxin oxidoreductase) have been shown to provide modest protection in chickens from developing NE (Cooper et al. (2009) Vet Microbiol vol 133:92-97; Zekarias et al. (2008) Clin Vaccine Immunol vol 15:805-816; Kulkarni et al. (2010) Clin Vaccine Immunol vol 17:205-214; Kulkarni et al. (2007) Clin Vaccine Immunol vol 14:1070-1077).

[0177] In some embodiments, the subject is administered an effective amount of a feed or feed additive composition comprising a DFM comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or a secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus), thereby improving the survival and quality of the subject's diet by increasing the survival and quality of the subject's diet. In some embodiments, the expression of NetB is reduced (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% and all values ​​between these percentages) compared to expression of NetB in a subject not administered an effective amount of a feed or feed additive composition comprising a DFM comprising a secretome of Lactobacillus acidophilus (Lactobacillus subsp. lactis) and / or a secretome of Lactobacillus acidophilus. The feed or feed additive composition may further include one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., a laying chicken, or a broiler, or a turkey) or a pig (e.g., a piglet, a growing pig, or a sow).

[0178] B. Methods of Treating or Preventing Coccidiosis Coccidiosis is an intestinal disease of poultry caused by infection with intracellular protozoan parasites of the genus Eimeria. Coccidiosis is the most economically devastating parasitic disease of poultry. Anticoccidiopathic drugs and losses due to coccidiosis are estimated to cost the poultry industry hundreds of millions of US dollars annually. The disease is spread from one animal to another by contact with infected feces or by ingesting infected tissue. Diarrhea, which may be bloody in severe cases, is the main symptom. Most animals infected with coccidiosis are asymptomatic, but young or immunocompromised animals suffer from severe symptoms and may die.

[0179] The present disclosure relates to a method of treating or preventing coccidiosis in a subject, comprising administering to the animal an effective amount of a feed, feed additive composition, or premix comprising directly fed live bacteria (DFM) comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). The feed, feed additive composition or premix may further comprise one or more of an osmotic regulator (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol) and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase and amylase).

[0180] In some embodiments, the efficacy and safety of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus) is improved by administering to a subject or group of subjects an effective amount of a feed or feed additive composition comprising a DFM comprising Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus). In one embodiment, the incidence or incidence of coccidiosis is reduced (e.g., by about any of the following percentages) relative to the incidence of coccidiosis present in a subject or group of subjects not administered an effective amount of a feed or feed additive composition comprising a DFM comprising a secretome of Lactobacillus subsp. lactis and / or Lactobacillus acidophilus. The feed or feed additive composition may further comprise one or more of an osmotic modifier (e.g., betaine), one or more essential oils (e.g., cinnamaldehyde and / or thymol), and one or more enzymes (e.g., protease, xylanase, beta-glucanase, phytase, and amylase). The subject may be a poultry (e.g., layer chicken or broiler) or a pig (e.g., piglet, growing pig, or sow).

[0181] C. Methods for reducing NetB toxin expression in Clostridium perfringens Also provided herein is a method of reducing necrotizing enteritis B-like toxin (NetB) expression in Clostridium perfringens by contacting C. perfringens cells with one or more of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, vitamin B, and / or the secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus.

[0182] Contacting C. perfringens cells with aspartic acid or its ionic form, aspartate, reduces C. perfringens NetB toxin expression. Aspartic acid is an α-amino acid used in protein biosynthesis. In microorganisms, aspartate is the precursor of several amino acids, such as methionine, threonine, isoleucine, lysine, asparagine, and arginine. The conversion of aspartate to these other amino acids begins with the reduction of aspartic acid to its "semialdehyde" O2CCH(NH2)CH2CHO. Asparagine arises from aspartate via transamidation. The role of aspartate in arginine biosynthesis is shown in Figure 7B. Thus, in some embodiments, contacting C. perfringens cells with aspartic acid or its ionic form, aspartate, reduces C. perfringens NetB toxin expression (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with aspartic acid. In some embodiments, the C. perfringens cells are further contacted with an osmotic modifier (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In further embodiments, the C. perfringens cells are in the intestine of poultry (e.g., chickens, such as broilers or egg-laying hens).

[0183] Exposing C. perfringens cells to ornithine reduces C. perfringens NetB toxin expression. Ornithine is a non-proteinogenic amino acid that plays a role in arginine biosynthesis (see FIG. 7B). Thus, in some embodiments, contacting C. perfringens cells with ornithine reduces C. perfringens NetB toxin expression (e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% and any values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with ornithine. In some embodiments, the C. perfringens cells are further contacted with an osmotic agent (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In a further embodiment, the C. perfringens cells are in the intestine of poultry (eg, chickens such as broilers or egg-laying chickens).

[0184] Exposing C. perfringens cells to arginine reduces C. perfringens NetB toxin expression. Arginine is an α-amino acid used in protein biosynthesis. Thus, in some embodiments, contacting C. perfringens cells with arginine reduces C. perfringens NetB toxin expression (e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with arginine. In some embodiments, the C. perfringens cells are further contacted with an osmotic agent (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In a further embodiment, the C. perfringens cells are in the intestine of poultry (eg, chickens such as broilers or egg-laying chickens).

[0185] C. perfringens NetB toxin expression is reduced by contacting C. perfringens cells with a source of phosphate, or orthophosphate [PO4]. 3- has three protons H + It is derived from phosphoric acid by the removal of one or two protons to give the dihydrogen phosphate ion [H2PO4], respectively. - and hydrogen phosphate ion [HPO4] 2-These names are also used for salts of these anions, such as, but not limited to, ammonium dihydrogen phosphate and trisodium phosphate. Thus, in some embodiments, contacting C. perfringens cells with a phosphate source reduces C. perfringens NetB toxin expression (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with a phosphate source. In some embodiments, the C. perfringens cells are further contacted with an osmotic modifier (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In further embodiments, the C. perfringens cells are in the intestine of poultry (e.g., chickens, such as broilers or egg-laying hens).

[0186] Exposing C. perfringens cells to acetate reduces C. perfringens NetB toxin expression. Acetate is a salt formed by the combination of acetic acid with a base (e.g., an alkali base, an earth base, a metal base, a nonmetallic base, or a radical base). In some microorganisms, pyruvate is converted to acetyl-coenzyme A (acetyl-CoA) by the enzyme pyruvate dehydrogenase. This acetyl-CoA is then converted to acetate with the concomitant production of ATP by substrate-level phosphorylation. Two enzymes are required for acetate formation: phosphate acetyltransferase and acetate kinase. Thus, in some embodiments, contacting C. perfringens cells with acetate reduces C. perfringens NetB toxin expression (e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with acetate. In some embodiments, the C. perfringens cells are further contacted with an osmotic agent (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In a further embodiment, the C. perfringens cells are in the intestine of poultry (eg, chickens such as broilers or egg-laying chickens).

[0187] Contacting C. perfringens cells with one or more B vitamins reduces C. perfringens NetB toxin expression. B vitamins are a type of water-soluble vitamin that play an important role in cellular metabolism. Non-limiting examples of B vitamins include vitamins B1, B6 and / or B 12Thus, in some embodiments, contacting C. perfringens cells with one or more B vitamins reduces C. perfringens NetB toxin expression (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with one or more B vitamins. In some embodiments, the C. perfringens cells are further contacted with an osmotic modifier (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In further embodiments, the C. perfringens cells are in the intestine of poultry (e.g., chickens, such as broilers or egg-laying hens).

[0188] Contacting C. perfringens cells with a secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus (e.g., Bifidobacterium animalis subsp. lactis strain Bl-04 and / or Lactobacillus acidophilus strain NCFM) reduces C. perfringens NetB toxin expression. Thus, in some embodiments, the production of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus is achieved by contacting C. perfringens cells with a secretome of Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus. In some embodiments, C. perfringens NetB toxin expression is reduced (e.g., by about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and all values ​​between these percentages) compared to C. perfringens NetB toxin expression in cells not contacted with the secretome of C. acidophilus. In some embodiments, the C. perfringens cells are further contacted with an osmotic agent (e.g., betaine) or an essential oil (e.g., thymol or cinnamaldehyde). In a further embodiment, the C. perfringens cells are in the intestine of poultry (eg, chickens such as broilers or egg-laying chickens).

[0189] The present invention can be further understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting. EXAMPLES

[0190] Example 1: Use of betaine, essential oils and DFM to reduce the adverse effects of necrotizing enterocolitis This example demonstrates that betaine, essential oils and DFM, when used in specific combinations, reduce the deleterious effects of necrotic enteritis (NE) as evidenced by improved lesion scores and bird performance.

[0191] Materials and Methods Briefly, 40-day-old male Cobb 500 broilers were housed in floor pens with nine replicate pens per treatment and seven total treatments. All birds were fed a commercial representative corn / soybean diet for three periods and had free access to water. Treatment groups were as follows: unloaded control (UC), loaded control (CC), CC+BE (betaine; contents were betaine (1 kg / ptt), Enviva® EO (cinnamaldehyde and thymol essential oil; 100 g / ptt)); CC+BE+DUAL; CC+BE+NCFM; CC+50% BE+DUAL; CC+betaine (1 kg / ptt)+DUAL. DUAL refers to the DFM combination of L. acidophilus NCFM and B. animalis subsp. lactis Bl-04. From D1 to D28 of this study, BE was administered via the feed and DFM was administered via the water supply every other day, with the birds receiving 10 9 All diets contained 750 FTU Axtra® PHY. Birds were administered approximately 1 x 10 CFU on days 18-20. 8NE was induced by oral gavage of 1.0 ml of liquid thioglycollate (FTG) broth containing CFU / ml Clostridium perfringens (Cp; netB+ strain). Control birds were inoculated with sterile FTG once daily from 18–20 days of age. BWG, FI and FCR were recorded on days 0, 14, 21, 28 and 42 of the study. At D28, NE lesion scores were performed (0–4).

[0192] result Briefly, the results demonstrate that BE alone was insufficient at the levels tested to mitigate the effects of NE, but BE+NCFM significantly (p<0.05) reduced mortality at D42, as shown in Figure 1A and Figure 1B. The most favorable impact on bird lesion scores was seen with the BE+NCFM or BE+DUAL combinations, with no birds scoring a lesion score above 1, whereas the BE-only treatment had three birds scoring a lesion score of 2. Furthermore, when the BE dose was reduced by 50% (500g and 50g betaine and EO, respectively), there was an increase in birds scoring a lesion score above 1.0, and removal of EO resulted in a dose-response increase in more moderate lesions.

[0193] Such findings were translated to final BW (D42), which was significantly (p<0.05) improved for BE+NCFM or DUAL and 50% BE+DUAL versus CC, whereas the BW for the combination of betaine+DUAL was only numerically (p>0.05). Thus, this example demonstrates that it is the correct combination of active agents that results in the desired outcome of reducing the adverse effects of NE. Without being bound by theory, it is believed that these observed improvements are likely mediated by reduced Cp levels in vivo, prevention of Clostridium overgrowth, quorum sensing and subsequent reduction in NetB toxin production, as measured in the tests according to Example 3 below.

[0194] Example 2: Not all active agents can reduce the adverse effects of necrotizing enterocolitis Example 2 demonstrates that, surprisingly, not all active agents with promising antimicrobial activity demonstrate the ability to reduce NE incidence when used as part of a combination. Glucose oxidase (GOX) has been reported in the literature to have anti-Cp activity and is commonly used as a food enzyme due to its antimicrobial activity through the production of hydrogen peroxide.

[0195] Materials and Methods In this example, 2160 day old male Cobb 500 broilers were assigned to one of six treatment pens with eight replicate pens / treatment (45 birds per pen). All birds were fed a commercial representative corn / soybean diet for three periods and had free access to water. All diets contained 750 FTU Axtra PHY. Treatment groups were as follows: Unloaded Control (UC), Loaded Control (CC), CC+BEG (Betaine (1 kg / ptt), Enviva® EO (100 g / ptt, GOX (100 g / ptt))+NCFM and Bl-04 (DUAL); CC+BEG+NCFM; CC+BE+DUAL; CC+BE+NCFM. From D1-28 of the study, BEG active was administered via the feed and DFM was administered via the water daily to birds for 10-28 days. 9 Birds were administered coccidiosis vaccine on day 0 and 10 CFU were delivered daily by oral gavage on days 19, 20 and 21 for all birds except the UC group. 8~9 CFU field Cp strain (Cp4) was administered. Such challenge resulted in the development of mild NE. Three birds per pen were sacrificed on days 21 and 28 for scoring of NE lesions (0–4) and evaluation of intestinal permeability (FITC-dextran assay and tight junction protein gene expression). Feed intake, weight gain and FCR were calculated on days D14, 21, 28 and 35. Mortality was recorded daily.

[0196] result Lesion scores at D21 demonstrated that all combinations of active agents reduced NE lesion scores compared to CC (Figure 2A), while the greatest reduction in NE lesion scores was observed in birds receiving only the combination of betaine, EO and DFM, regardless of whether the combination contained one or two DFM strains. The same results were seen in the assessment of intestinal permeability using the FITC-DEXTRAN assay at D28 (Figure 2D), demonstrating an improvement in intestinal integrity. These results were translated to the weight of the birds at D28 (Figure 2B) and D35 (Figure 2C). For example, birds receiving BEG+DUAL gained 69 grams BW compared to CC, while the same combination, excluding GOX, gained 185 grams BW at D35. Thus, birds receiving only BE+DFM had the highest BW, with a significant difference (P<0.05) compared to CC. Such performance improvement was observed in FCR at D35 (Figure 2E); BEG+NCFM improved FCR by 5.18 points versus 8.15 points for the same combination excluding GOX.

[0197] Therefore, this example demonstrates that not all administered antimicrobial active agents have an equally positive impact on NE development.

[0198] Example 3: Reduction of C. perfringens and netB Expression in Treated Animals This example shows that certain active agents can reduce the levels of C. perfringens and netB expression upon NE challenge.

[0199] Materials and Methods Briefly, 1680 male Cobb 500-day-old chickens were assigned to one of seven treatments with eight replicate pens per treatment (30 birds / pen). Treatments were as follows: unloaded control (UC), loaded control (CC), CC+BE (contents were betaine (1 kg / ptt), Enviva® EO (100 g / ptt)); CC+BE+3 strains DFM; CC+BE+DUAL; CC+BE+NCFM; CC+BEG (betaine (1 kg / ptt), Enviva® EO (100 g / ptt, GOX (100 g / ptt))+NCFM. DUAL refers to the DFM combination of L. acidophilus NCFM and B. animalis subsp. lactis Bl-04. From D1 to 28 of the study, BE and BEG actives were administered via the feed and DFM was administered daily via the water supply to the birds for 10-28 days. 9 CFU were delivered daily. All diets contained 750 FTU Axtra® PHY.

[0200] On day 7 of the study, birds in all treatments except UC were administered 10 times the recommended dose of Advent coccidiosis vaccine to make them susceptible to NE. At D17, these birds received approximately 10 8 CFU C. perfringens field isolate was administered. Bird performance parameters were measured on days 0, 14, 21, 28 and 42. Birds were sacrificed on D21 to score for NE lesions and ileal swabs were collected for quantification of C. perfringens by Q-PCR and netB expression.

[0201] result Bird performance was numerically (p>0.05) or significantly (p<0.05) improved by additive supplementation. Microbial analysis demonstrated that C. perfringens levels and netB expression were significantly reduced by the BE+DUAL or NCFM combination (Figures 3A, 3B, 4A, and 4B).

[0202] In summary, this example demonstrates that by lowering C. perfringens levels and reducing the amount of microbial toxins produced, there is less damage to the intestinal tract, thereby maintaining bird performance up to UC-like levels.

[0203] Example 4: Synergistic improvement of Clostridium perfringens when combined with probiotic-derived essential oil CFS The aim of this study was to evaluate the potential synergistic effect of Enviva® EO (essential oil) and cell-free supernatant (CFS) from a proposed blended combination of Bifidobacterium animalis subsp. lactis strain Bl-04 and Lactobacillus acidophilus strain NCFM, a probiotic against Clostridium perfringens (CP).

[0204] Materials and Methods Increasing amounts of CFS and Enviva® EO essential oil of each probiotic were added together in a checkerboard pattern and CP was inoculated and grown overnight. The inhibition percentage was then calculated relative to the untreated control (no CFS and no EO). Briefly, Bl-04 and NCFM strains were grown anaerobically at 37°C in De Man, Rogosa and Sharpe medium (MRS) for approximately 48 hours.

[0205] The cells were pelleted by centrifugation at 8000×g for 10 min, and CFS was harvested by filtering the supernatant through a 0.2 um aPES membrane. For inhibition assays, wells of a 96-well microtiter plate (Corning Costar #3370, Corning, NY) were filled with 130 μL of Brain Heart Infusion medium (BHI).

[0206] A 17.6 g / L solution of Enviva® EO was made in sterile water and diluted 50:50 in BHI to a final concentration of 8.8 g / L. Two-fold dilutions were then made in BHI to give 10x stock concentrations of 4.4, 2.2, and 1.1 g / L. The Bl-04 CFS was then added to rows of a microtiter plate containing 130 μL of BHI, as shown in FIG. 4. To each well in row A, 50 μL of CFS was added. To row B, 45 μL was added. This process continued in decreasing amounts of 5 μL down the plate until 20 μL was added to row G. Row H contained no CFS. 20 microliters of 10× EO stock was then added to each column. To column 2, 20 μL of 1.1 g / L (10×) EO stock was added to each well. To column 3, 20 μL of 2.2 g / L stock was added. To column 4, 20 μL of 4.4 g / L stock was added. To column 5, 20 μL of 8.8 g / L stock was added to each well. To column 1 (no EO), 20 μL of BHI medium was added. The final volume of each well was brought to 200 μL with MRS. This set up was the same as for the NCFM strains, except the CFS ranged from 5 μL to 35 μL in 5 μL increments.

[0207] A single colony of Clostridium perfringens (CP) from a BHI agar plate was inoculated into 200ul of BHI and grown for 3.5 hours to an OD600 of 0.5-0.8. Two microliters of this was added to each well. The plates were covered with a breathable membrane and incubated anaerobically at 37°C for approximately 16-18 hours. Optical density was then read at 600nm on a BioTek Synergy MX microplate reader (BioTek Instruments, Winooski, VT). Inhibition of CP growth was then calculated as the ratio of zero CFS / zero EO control wells.

[0208] result The results show that synergy between the two DFM candidates and Enviva® EO was present, especially at higher EO concentrations (Figures 5 and 6). For example, in the case of NCFM, no EO and 15 μL CFS did not inhibit CP. In addition, 0.44 g / L EO and no CFS did not inhibit CP. However, 0.44 g / L EO and 15 μL CFS resulted in 91% inhibition. Similar synergistic effects could be found at other concentrations of CFS and EO with both Bl-04 and NCFM.

[0209] Example 5: Modulation of Clostridium perfringens netB toxin expression by addition of metabolites In this example, a novel technique for assessing gene expression in single bacterial cells is used to identify single cell populations of Clostridium perfringens characterized by high or low netB expression, respectively. Genes encoding components of a metabolic pathway that are reduced in expression in high netB expressing cells are identified, and the high netB expressing cells are then supplemented with products of this metabolic pathway to determine the effect on netB expression.

[0210] Materials and Methods Probe design and library creation: To take advantage of existing microfluidic single-cell sequencing platforms, we devised a method to tag individual transcripts with DNA probes. This approach required the creation of a large oligonucleotide library complementary to all protein-coding sequences in the genome. Multiple DNA regions of 50 bp were selected from each ORF based on uniqueness determined by UPS2 software or based on already published oligonucleotide arrays. These sequences then served as hybridization regions for ssDNA probes designed to target mRNAs by sequence complementarity. The probes contained a 5' PCR handle for library creation, a Unique Molecular Identifier (UMI) and a 3' polyadenosine tail (A) for incorporation of prokaryotic transcripts into the 10X Genomics Chromium Single Cell 3' system. 30 ) were also included. Multiple probes (complementary to various regions) were designed for each gene to enhance the efficiency of transcript capture and reduce noise caused by poor hybridization and / or underamplification of any given probe. The full species library included 11,723 probes for Clostridium perfringens, targeting 3189 C. perfringens genes.

[0211] Libraries were ordered from Twist Biosciences in sub-femtomole quantities and amplified by rolling circle amplification to obtain sufficient concentration for scRNA-seq experiments (0.25mg = 10.25nM / library or approximately 0.35pM of each probe). Probe libraries were completed by addition of randomized 12bp UMI sequences and polyA tails and purified by PAGE. The completed libraries were homogenous in probe coverage.

[0212] Bacteria were fixed and permeabilized with 1% paraformaldehyde prior to microfluidic encapsulation. Permeabilized bacteria were incubated with the corresponding DNA probe library. Unhybridized probes were washed away. Bacteria were then passed 10x through the controller to capture and barcode the DNA probes. The resulting libraries were sequenced, preprocessed with custom scripts, and analyzed with the standard CellRanger pipeline and the Seurat analysis package.

[0213] Clostridium perfringens: Clostridium perfringens strain 25037-CP01 was grown anaerobically at 37°C in BHI medium supplemented with 0.05% cysteine-HCL and, where indicated, ornithine, aspartate or arginine at a final concentration of 0.625 mg / ml. Anaerobic conditions were maintained using gas packs and anaerobic incubation boxes. Oxygen indicators in all experimental replicates showed no oxygen contamination in the chamber.

[0214] HT-29 human colorectal adenocarcinoma cell line: HT-29 cells were obtained from ATCC and cultured in McCoy's 5A medium (1.5 mM L-glutamine; 2200 mg / L sodium bicarbonate) supplemented with 10% certified FBS (complete medium). Cells were cultured in T75 flasks in a 37°C incubator with 5% CO2 and 95% humidity until the cells were 80-90% confluent. Cells were subcultured by detaching the cells using 0.05% trypsin, centrifuging, and resuspending in 10 mL complete medium. New flasks were seeded 1:10 and incubated under the same conditions as above or cells were seeded in 96-well plates for the experiments described.

[0215] Cytotoxicity assay: Cytotoxicity was examined on conditioned medium from C. perfringens cultures used in Western blot analysis. 4HT-29 cells were seeded into each well and incubated for 2 days in a 37°C incubator with 5% CO2 and 95% humidity. The medium was changed, 5 μL of conditioned C. perfringens supernatant was added to each well, and the cells were incubated overnight in a 37°C incubator with 5% CO2 and 95% humidity. After incubation, images were acquired using an EVOS FLoid microscope and cytotoxicity was measured using a Cell Proliferation Assay Kit (BioVision) according to the manufacturer's protocol. Briefly, 20 μL of a 1:50 (final 1:500) dilution of nuclear dye was added. The plate was incubated for 15 min at room temperature on a plate shaker (100 rpm). Cells were lysed and 480 / 538 nm fluorescence was measured using a plate reader (Tecan).

[0216] result We sought to determine whether heterogeneous expression of virulence genes in true pathogens could be identified using single-cell transcriptional analysis. To this end, we investigated toxin production in Clostridium perfringens, the causative agent of necrotic enteritis. NetB, the major toxin associated with necrotic enteritis, is a secreted β-barrel pore-forming toxin that has been shown to be a virulence factor directly involved in pathogenesis in chickens (REFS). Single-cell analysis was performed on C. perfringens grown in rich medium (BHI, Materials and Methods) until late exponential phase, when the toxin is expressed and accumulates in the growth medium. Although the NetB toxin was expressed to some basal extent from all clusters, differential overexpression of netB was a defining feature of one cluster of cells (Figure 7A, cluster 0), and NetB was selected as a marker gene with a P value of at least 0.005 in individual independent analyses of four biological replicates taken across several different ODs around the time of the exponential to stationary phase transition (data not shown). Interestingly, cells in the cluster with reduced expression levels of NetB differentially overexpressed genes associated with distinct physiological states, including arginine synthesis genes (Figure 7A, cluster 2), putative phage genes (Figure 7A, cluster 4), and purine and pyrimidine synthesis (Figure 7A, cluster 3).

[0217] In cluster 2, which expressed low levels of toxin genes, cells overexpressing arginine synthesis genes and genes for the arginine / ornithine exchanger were present, and the reduced expression levels of toxin genes prompted us to speculate whether toxin production and the size of the toxin-producing population could be controlled by supplying specific metabolites involved in arginine biosynthesis. To test this hypothesis, we perturbed the culture medium by adding ornithine, arginine, and aspartate, which are metabolites related to arginine biosynthesis (Figure 7B), and found that the addition of aspartate, and to a lesser extent ornithine and arginine, reduced the levels of the 33 kDa NetB toxin secreted into the medium (Figure 7C). To confirm the reduction in the levels of extracellular toxins, we performed single-cell analysis in cultures grown with the addition of the three metabolites. The addition of aspartate caused a dramatic change in cell clustering, and in single-cell data from this culture, we observed a significant reduction in the main toxin-producing population (Figure 7D). Addition of ornithine and arginine caused smaller but still significant changes in the cells, slightly decreasing NetB expression in the cells. Without being bound by theory, the larger changes caused by addition of aspartate led us to speculate that aspartate may have effects beyond its contribution to arginine biosynthesis, which may be related to its role in other differentially expressed pathways in the original single-cell data, such as pyrimidine synthesis and aspartate catabolism (Figure 7A).

[0218] Following the hypothesis that virulence could be controlled by adding metabolites that would reduce NetB toxins and downregulate the proportion of virulence-expressing cells in the total population, human HT29 mammalian epithelial cells, a cell line commonly used for in vitro tissue culture cytotoxicity studies, were then used to test the cytotoxicity of conditioned media from cells grown in the presence of ornithine or aspartate (Figure 7E). It was found that the addition of 5 μl of bacterial culture supernatant grown in standard BHI medium to cultures of HT29 cells (195 ul) resulted in approximately 58% cell death. Consistent with the reduced NetB levels and the smaller proportion of cells in a virulent state, the addition of 5 μl of bacterial culture medium administered aspartate resulted in low toxicity to HT29 cells, with less than 10% cell death. Addition of ornithine also resulted in a slight reduction in toxicity, with approximately 32% cell death. Overall, the results with C. perfringens demonstrate that toxin production can be differentially expressed by specific cells and can reduce virulence by providing favorable growth conditions for other cellular states, thereby decreasing the proportion of pathogenic cells in a clonal bacterial population.

[0219] Example 6: Addition of ammonium phosphate, sodium acetate, or B vitamins (vitamins B1, B6, and B12) reduces the levels of extracellular NetB produced by Clostridium perfringens In this example, data obtained from the single-cell transcriptional analysis described in Example 5 was used to determine that Clostridium perfringens cells characterized by high netB gene expression were also characterized by reduced expression associated with phosphate, acetate, or vitamin B metabolism.

[0220] Materials and Methods Growth of Clostridium perfringens: Clostridium perfringens strain 25037-CP01 was grown anaerobically at 37°C in BHI medium supplemented with 0.05% cysteine-HCL and, where indicated, ornithine, aspartic acid or other additives at a final concentration of 0.625 mg / ml. Anaerobic conditions were maintained using gas packs and anaerobic incubation boxes. Oxygen indicators in all experimental replicates showed no oxygen contamination in the chamber.

[0221] Western Blot Analysis: To harvest conditioned medium from C. perfringens cultures, cell cultures in late exponential growth (OD of approximately 0.7-0.8) were pelleted by centrifugation at 4,200 x G for 4 min. The supernatant was then filtered through a 0.2 uM filter. Filtered conditioned medium was diluted 1:10 in dH2O and 5 ul of sample was incubated with MES SDS running buffer for 10 min at 95°C. Samples were loaded in equal amounts and run on a 4-12% Bis Tris polyacrylamide gel. PAGE gels were transferred onto invitrolon 45 uM PVDF membranes presoaked in methanol using an Xcell-II blot apparatus (invitrogen) according to the manufacturer's instructions. The toxin NetB (33 Kd) was detected using a custom polyclonal rabbit antibody and the WesternBreeze rabbit chromogenic Western Blot kit (Invitrogen). All experiments were performed on multiple independent days using at least biological duplicates and technical triplicates, and representative images were selected.

[0222] result Western blots detecting NetB toxin demonstrated that the levels of extracellular NetB toxin were reduced when the metabolites ammonium phosphate, sodium acetate, or vitamin B (vitamins B1, B6, and B12) were added to Clostridium perfringens cultures compared to the extracellular toxin detected when cultured in unsupplemented growth medium (BHI). As shown in Figure 8, the ammonium phosphate band is fainter than the band from unsupplemented BHI medium. Similarly, as shown in Figure 8, the BHI band is darker than the band from medium supplemented with sodium acetate, vitamin B1, vitamin B6, or vitamin B12. Therefore, based on these results, it appears that the addition of any of the metabolites ammonium phosphate, sodium acetate, vitamin B1, vitamin B6, or vitamin B12 to Clostridium perfringens may reduce the extracellular NetB toxin, which is the primary pathogen of necrotic enteritis.

[0223] Example 7: Addition of direct-fed live bacteria reduces the levels of extracellular NetB produced by Clostridium perfringens Necrotic enteritis (NE) caused by C. perfringens is a re-emerging threat to the poultry industry following increasing pressure to reduce antibiotic use. Alternatives to antibiotics are known to be significantly inconsistent in their effectiveness. There is a need to fully understand the etiology and characteristics of the intestinal health problem as a means to improve consistency in order to develop the next generation of solutions.

[0224] In this example, the effect of the secretome of two direct-feeding bacteria (DFM), L. acidophilus strain NCFM and Bifidobacterium animalis subsp. lactis strain Bl-04, on the production of NetB (a pore-forming toxin that is considered the main virulence factor among various different toxins) and cytotoxicity of pathogenic (or NE-induced) C. perfringens strains, was analyzed and deciphered using scRNAseq technology as described in Example 5. NetB expression analysis was performed as in Examples 5 and 6.

[0225] result The combined secretome of both strains reduced NetB production and overall cytotoxicity of C. perfringens. Treatment of HT-29 cells with supernatants of C. perfringens grown in the presence of both DFM secretomes reduced C. perfringens cytotoxicity by 60% (FIG. 9D; p-value <0.05). To better understand the effect of the DFM secretome on controlling C. perfringens biology and virulence, the transcriptomes of single cells after growth in and without the presence of DFM secretome were examined using a novel scRNAseq methodology disclosed in Example 5.

[0226] Heterogeneous gene expression in the C. perfringens population was demonstrated with the netB toxin gene being expressed primarily by a fraction of cells. Furthermore, it was shown that the B. animalis secretome altered the overall gene expression and biological structure of the C. perfringens population, resulting in an 8-fold decrease in netB expression (Figure 9A; p-value < 6 × 10 -7 ).

[0227] Heterogeneous gene expression in C. perfringens populations was demonstrated with the netB toxin gene being expressed primarily by a fraction of cells.Furthermore, it was shown that the L. acidophilus secretome altered the global gene expression and biological structure of the C. perfringens population and was associated with a decrease in netB expression (Figure 9B).

[0228] Western blots detecting NetB toxins demonstrate that levels of extracellular NetB toxins were reduced when B. animalis and L. acidophilus secretomes were added to cultures of Clostridium perfringens compared to the extracellular toxins detected when cultured in unsupplemented growth medium (BHI). As shown in Figure 9C, the bands corresponding to NetB toxins are fainter with increasing amounts of B. animalis secretome compared to the bands in unsupplemented BHI medium. As also shown in Figure 9C, the BHI bands are darker compared to the bands in medium supplemented with increasing amounts of L. acidophilus secretome. Therefore, based on these results, it appears that the addition of either B. animalis or L. acidophilus secretome to Clostridium perfringens may reduce extracellular NetB toxin, the primary pathogenic agent of necrotizing enterocolitis.

[0229] In accordance with the hypothesis that virulence may be controlled by adding a bacterial secretome that reduces NetB toxins and downregulates the proportion of virulence-expressing cells in the total population, human HT29 mammalian epithelial cells, a cell line commonly used for in vitro tissue culture cytotoxicity studies, were used to test the cytotoxicity of conditioned media from cells grown in the presence of B. animalis or L. acidophilus secretome (Figure 9D). Addition of 5 μl of bacterial culture supernatant grown in standard BHI medium to a culture of HT29 cells (195 μl) was found to result in approximately 65% ​​cell death. Consistent with the reduced NetB levels and a smaller proportion of cells in a virulent state, addition of 5 μl of bacterial culture medium administered B. animalis or L. acidophilus secretome was less toxic to HT29 cells, resulting in less than 30% cell death. The combination of B. animalis and L. acidophilus was also slightly less toxic, with cell death of approximately 44%.

[0230] Overall, the results with C. perfringens demonstrate that toxin production can be differentially expressed by specific cells and can reduce virulence by providing favorable growth conditions for other cellular states, thereby decreasing the proportion of pathogenic cells in a clonal bacterial population.

Claims

1. A feed additive composition comprising or consisting essentially of direct-fed live bacteria (DFM) including Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus.

2. 10. The feed additive composition of claim 1, further comprising or consisting essentially of at least one of: (a) an osmotic agent; (b) at least one essential oil; (c) one or more enzymes selected from the group consisting of proteases, xylanases, beta-glucanases, phytases, and amylases; or (d) at least one additional DFM.

3. 3. The feed additive composition of claim 2, wherein the enzyme is encapsulated or in the form of granules or freeze-dried.

4. 2. The feed additive composition according to claim 1, comprising or consisting essentially of Bifidobacterium animalis subsp. lactis strain Bl-04.

5. 2. The feed additive composition of claim 1, comprising or consisting essentially of Lactobacillus acidophilus strain NCFM.

6. 3. The feed additive composition of claim 2, wherein the osmolality adjusting agent comprises betaine.

7. 2. The feed additive composition of claim 1, further comprising or consisting essentially of at least one of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate and / or vitamin B.

8. 3. The feed additive composition of claim 2, wherein at least one component of the composition is formulated for water line delivery.

9. An animal feed or premix comprising the feed additive composition according to any one of claims 1 to 8.

10. 10. A method for treating or preventing necrotizing enterocolitis in a non-human subject in need thereof, the method comprising administering to the subject an effective amount of the feed additive composition according to any one of claims 1 to 8, or an animal feed or premix comprising the feed additive composition according to any one of claims 1 to 8.

11. The method of claim 10, wherein the subject is a poultry or pig.

12. 11. The method of claim 10, which (a) reduces or prevents the intestinal lesions of necrotic enterocolitis, (b) further reduces the feed conversion ratio in said subject, (c) increases feed efficiency, (d) reduces mortality, (e) increases final slaughter weight, (f) increases body weight gain, or (g) further reduces the expression of Clostridium perfringens necrotic enterocolitis B-like toxin (NetB), compared to a non-human subject not administered an effective amount of the feed additive composition of any one of claims 1 to 8 or an animal feed or premix comprising the feed additive composition of any one of claims 1 to 8.

13. 11. The method of claim 10, wherein the administering is performed without co-administration of an antibiotic to the subject.

14. 10. A method for treating or preventing coccidiosis in a non-human subject in need thereof, comprising administering to the subject an effective amount of a feed additive composition according to any one of claims 1 to 8, or an animal feed or premix comprising the feed additive composition according to any one of claims 1 to 8.

15. The method of claim 14, wherein the subject is poultry.

16. 15. The method of claim 14, wherein one or more intestinal Eimeria species are reduced.

17. 15. The method of claim 14, wherein at least one component of the feed additive composition is administered by water supply.

18. 1. A method of reducing necrotizing enteritis B-like toxin (NetB) expression in Clostridium perfringens, comprising contacting C. perfringens cells with one or more of aspartic acid (aspartate), ornithine, arginine, phosphate, acetate, B vitamins, and / or the secretome of one or both of B. animalis or L. acidophilus.

19. 19. The method of claim 18, wherein the B. animalis is Bifidobacterium animalis subsp. lactis strain Bl-04 and / or the L. acidophilus is Lactobacillus acidophilus strain NCFM.

20. 19. The method of claim 18, wherein the C. perfringens cells are in the intestine of a poultry.

21. 21. The method of any one of claims 18-20, further comprising contacting the C. perfringens cells with one or more of an osmotic agent and / or at least one essential oil.

22. 10. A method for reducing necrotizing enteritis B-like toxin (NetB) expression in Clostridium perfringens in a non-human subject in need thereof, comprising or consisting essentially of administering to the subject an effective amount of an animal feed or premix comprising the feed additive composition of any one of claims 1 to 8, wherein C. perfringens cells are in the intestine of the subject.

23. 23. The method of claim 22, wherein the subject is a poultry or pig.

24. 23. The method of claim 22, wherein the intestinal lesions of necrotizing enterocolitis are reduced or prevented.

25. 23. The method of claim 22, wherein the feed additive composition is administered by tap water.

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