BACILLUS ISOLATE COMPOSITIONS AND METHODS OF USING AND

PRODUCING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of United States Provisional Patent Application No. 62/865,010, filed June 21, 2019, which is incorporated herein by reference in its entirety.

INTRODUCTION

Feed ingredients used for nutrition of monogastric animals contain anti -nutritional factors that are not only undigestible, but also deleterious to gut health within the animal. The non-starch polysaccharides (NSP) and oligosaccharides present mainly in soybean meal can lead to gut inflammation which results in poor nutrient absorption and bacterial leakage across the mucosal epithelium. The a-galactosides, raffmose and stachyose, are the primary oligosaccharides and make up 5-7% of soybean meal. They are considered anti -nutritional factors because they are poorly digestible and reduce the metabolizable energy (ME) and carbohydrate digestion of the meal. They can also cause wet feces, which can impact litter and air quality in poultry. This problem can be alleviated with the use of a-galactosidase enzymes to pretreat the soybean meal before feeding or by feeding these enzymes directly to the animal. This also reduces the quantity of soybean meal and energy dense feed ingredients ( e.g supplemental lipids) needed due to greater nutritional value and the increased concentration of digestible amino acids.

Recently, numerous biosynthetic enzymes have been marketed to break down non- digestible substrates in the feed. These products contain enzymes that can break down non-starch polysaccharides such as hemicellulose (hemicellulase) and arabinoxylans (xylanases), poorly digestible proteins (proteases), and others. In fact, almost all feed that is commercially produced for pigs and chickens contains exogenous phytase, an enzyme that breaks down phytate to release poorly available phosphorus from plant-based feedstuffs. These feed-grade enzymes have been widely adopted and their use depends on cost, availability, and feed ingredients (i.e., substrate). Enzymes are valuable for animal nutritionists as they allow for increased flexibility of feed ingredient selection based on least cost formulation. It is also thought that enzymes provide a nutritional“safety margin” in the event that the quality of preferred ingredients such as com and soybean meal is less than anticipated due to agronomic or improper storage or processing conditions.

Although the addition of biosynthetic enzymes to animal feed has shown some success, the approach suffers from a number of shortcomings. First, the biosynthetic enzymes may not be stable and thus denature during heat treatments applied during feed processing. Enzyme stability is also a concern in the low pH environment of the animal gut leading to inefficient processing of anti -nutritional factors. Finally, the production and purification of biosynthetic enzymes can be costly leading to higher animal feed costs. Accordingly, there is a need in the art for new cost- effective feed supplements that increase nutrient uptake in animals and which retain stability both during feed processing and within the animal gut.

SUMMARY

In one aspect of the present invention, new isolates of Bacillus are provided. The Bacillus isolates may be in vegetative (i.e., cellular metabolic form) or spore form. A new Bacillus isolate designated as“Isolate 46” or "VWB46" is provided. The isolate has been deposited on June 12, 2020 at the Agricultural Research Culture Collection (NRRL) under the terms of the Budapest treaty as Accession number NRRL B-67957. The isolate is a Bacillus amyloliquefaciens. A new Bacillus isolate designated as“Isolate 40” or "VWB40" is provided. The isolate has been deposited on June 12, 2020 at the Agricultural Research Culture Collection (NRRL) under the terms of the Budapest Treaty as Accession Number NRRL B-67956. The isolate is also a Bacillus amyloliquefaciens.

In another aspect, the present invention relates to probiotic compositions. The probiotic compositions may include any one of or any combination of the Bacillus isolates described herein and a carrier. These compositions may be combined with other bacterial isolates in a probiotic composition.

In a further aspect of the present invention, animal feed compositions are provided. The animal feed compositions may include any of the Bacillus isolates or probiotic compositions described herein and a plant-based food source. Optionally, the animal feed compositions may additionally include a carbohydrate component, a protein component, a fat component, a mineral component, a vitamin component, or any combination thereof.

In a still further aspect, the present invention relates to methods for improving nutritional uptake in a subject. The methods may include administering to the subject an effective amount of any of the compositions {Bacillus isolates, probiotic compositions, or animal feed compositions) described herein.

In another aspect, the present invention relates to methods for producing an animal feed composition. The methods may include introducing any one of the Bacillus isolates or probiotic compositions described herein into the animal feed composition. Suitably, the feed composition is a dry mash or pelleted feed composition and the Bacillus isolate or probiotic composition is added prior to mixing the feed composition and the Bacillus isolates are added as spores.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows isolate growth on ammonia media plates containing 0.0125% glucose with arabinoxylan soft agar overlays.

FIG. 2 shows isolate growth on ammonia media plates containing 0.0125% glucose with cellulose soft agar overlays.

FIG. 3 shows isolate growth on ammonia media plates containing 0.0125% glucose with galactomannan soft agar overlays.

FIG. 4 shows average bird weight at day 21 of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 5 shows the feed conversion ratio (Dl-21) of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 6 shows average bird weight at day 21 of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 7 shows average bird weight at day 21 of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial.

FIG. 8 shows average bird weight at day 14 of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial.

FIG. 9 shows average bird weight at day 21 of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial.

FIG. 10 shows body weight gain (BWG; DO-21) of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial.

FIG. 11 shows feed conversion ratio (FCR; DO-21) of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial. FIG. 12 shows body weight gain (DO-21) of broilers fed a low energy diet with or without dietary inclusion of a Bacillus direct fed microbial.

FIG. 13 shows feed conversion ratio (DO-21) of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 14 shows body weight gain (DO-21) of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 15 shows feed conversion ratio (DO-21) of broilers fed a low energy diet with or without dietary inclusion of & Bacillus direct fed microbial.

FIG. 16 shows body weight gain (DO-21) of pooled data from the previous 6 trials that included isolate 46.

FIG. 17 shows feed conversion ratio (DO-21) of pooled data from the previous 6 trials that included isolate 46.

DETAILED DESCRIPTION

Here, in the non-limiting Examples, the present inventors have developed a serial screening method for the selection of specific spore-forming bacterial isolates (all of the genus Bacillus ) for their ability to robustly form spores of high quality during appropriate incubation. The Bacillus isolate spores were selected to resist treatment with high temperatures, which are commonly applied during commercial feed production, and for their ability to produce enzymes capable of breaking down cellulose, xylans, mannans, and/or raffmose.

The present inventors further demonstrate, using multiple in vivo experiments in poultry, that dietary inclusion of spores from certain Bacillus isolates in chicken feed could improve poultry performance with reduced energy diets, including significant improvement in growth rate and feed efficiency of chickens when fed a low energy diet (LED). Additionally, inclusion of the spores in chicken feed improved carcass characteristics and lowered the incidence of footpad pododermatitis lesions. These results suggest the compositions described herein could be added to feed compositions to lower the cost of raising and caring for animals in meat production and allow the use of food-stuffs to make animal feed that would otherwise be considered too low in quality for such use. This may allow use of what may be considered waste products in animal feeds and could reduce the costs of production.

The present invention is superior to current exogenously produced, biosynthetic enzymes in several ways. First, due to the innate heat stability of Bacillus , the present isolates can be administered in both pelleted and non-pelleted feeds. Most enzyme supplements, on the other hand, are not stable at high temperatures and must be protectively coated or applied post pelleting. Application of products post pelleting however is not preferred, as it is often more expensive and obstructive to the feed milling process. The present Bacillus isolates are heat stable at normal feed milling temperatures and can be applied as a dry powder pre-pelleting.

Another advantage to our Bacillus isolates is that, unlike biosynthetic enzymes which are often produced in GMO (genetically modified organisms), the present isolates are natural producers of these enzymes and are simply selected without genetic modification. Thus, the isolates may help animal feed manufacturers address current consumer preference for non-GMO products.

Finally, biosynthetic enzymes are usually a single enzyme or a combination of a few enzymes. Although the activity of these enzymes is high for their specific substrate, if that substrate is not exposed due to the plant structure then it has no use. The plant packages nutrients in layers and without each specific enzyme to break down each layer, synthetic enzymes are not providing any benefits. The present Bacillus isolates, however, were selected to act on a multitude of substrates in a given feedstuff, not a single, specific substrate. Thus, when selected for the correct target(s), the present isolates produce the necessary enzymes to break down the packaged nutrients and release otherwise indigestible materials.

Compositions

Bacillus Isolates

In one aspect of the present invention, new isolates of Bacillus are provided. The Bacillus isolates may be in vegetative or spore form.

A new Bacillus isolate designated as“Isolate 46” is provided. The isolate has been deposited at the ARS Culture Collection (NRRL Accession No. B-67957).

A new Bacillus isolate designated as“Isolate 40” is provided. The isolate has been deposited at the ARS Culture Collection (NRRL Accession No. B-67956).

Probiotic Compositions

In another aspect, the present invention relates to probiotic compositions. The probiotic compositions may include any one of the Bacillus isolates described herein or a combination thereof and a carrier. Suitable carriers may include substances that aid in the formulation of a Bacillus isolate for administration to a subject. Carriers for water administration may include, without limitation, water; buffers such as phosphate, citrate, or other organic acids; or chelating agents such as EDTA. Carriers for feed administration in powdered form may include, without limitation, calcium carbonate, ground limestone, rice hulls, or other dry material to be added during mixing or on top of existing feed at the farm.

Animal Feed Compositions

In a further aspect of the present invention, animal feed compositions are provided. The animal feed compositions may include a plant-based food source. The animal feed compositions may include any of the Bacillus isolates or probiotic compositions described herein and a plant- or animal-based food source. Optionally, the animal feed compositions may also include a carbohydrate component, a protein component, a fat component, a mineral component, a vitamin component, or any combination thereof.

The animal feed compositions may include key nutrients needed to meet the dietary requirements of a particular subject. These key nutrients may include a protein component, a carbohydrate component, fats and oils, minerals, vitamins, or any combination thereof.

Common protein components found in animal feeds for example may include, without limitation, protein meals or protein derived from plant (vegetable or seed) and animal sources, such as soybean, oilseed, legumes, rice, abattoir, and fish processing by-products. Suitable plant-based food sources include the vegetable or fruit product and the seed, but also derivatives thereof. For example, soybean or other oilseed meal left over from oil or plant-based protein production, such as soybean meal, oilseed meal, wheat meal or bran and rice bran or other derivatives of plant-based food production or processing operations. Suitable oilseed meals may include, without limitation, soybean, rapeseed/canola, sunflower, palm kernel, copra, linseed, peanut and sesame seed meals.

In some embodiments, the animal feed compositions comprise a fermented plant-based meal, such as fermented soybean meal. Soybean meal contains a variety of antinutritional factors (ANFs), such as trypsin inhibitor, phytate, lectins, and soya globulins, which limits its utilization as a plant-derived protein source for animal feed, especially for young animals. Fermentation using bacteria or fungi has been shown to improve the nutritional value of soybean meal, resulting in the degradation of various anti -nutritional factors and increasing the availability of small peptides and amino acids. Feeding non-ruminants with fermented soybean meal (as compared to unfermented soybean meal) offers several benefits, including increased average daily gain, improved growth performance, better protein digestibility, decreased immunological reactivity, and reduced undesirable morphological changes (Asian Australas. J. Anim. Sci. (2016) 29: 1523-1529). Several studies have demonstrated that fermented soybean meal can be used to improve performance in broiler chickens. For example, feeding chicks soybean meal fermented with Aspergillus oryzae has been shown to significantly increase the average daily weight gain and average daily feed intake of the chicks as compared to standard soybean meal (Anim. Feed Sci Tech (2007) 134:235-242), and dietary supplementation with soybean meal fermented with Aspergillus niger has been shown to increase body weight, improve feed conversion ratio, and reduce feed consumption in broiler chickens (Int. J. Poult. Sci. (2006) 5(9):868-872). Further, fermentation with Aspergillus usamii almost completely degrades phytate phosphorus in soybean meal, and has been shown to improve phosphorus bioavailability in chicken feed (Poult Sci (1998) 77(4):552-6). The soybean meal may be fermented as discussed in the references above or alternatively may be fermented with the Bacillus strains provided herein prior to pelleting to produce a Bacillus fermented soybean meal based food. The Bacillus strains would sporulate as fermentation is complete and become part of the fermented soybean meal food product.

Other common plant-based components found in animal feeds for example may include, without limitation, cereal grains such as com, wheat, sorghum, barley, rye, triticale, or oats. These plant-based components may include one or more anti -nutritional factors. As used herein, an“anti-nutritional factor” is a poorly digestible carbohydrate or protein that may reduce the metabolizable energy (ME) or phosphorus or amino acid availability within a feed composition. Suitable anti-nutritional factors may include, without limitation, a-galactosides (e.g., raffmose, stachyose, and verbascose), cellulose, hemicellulose, galactomannan, arabinoxylans, xylose, phytate, lectins, trypsin inhibitors and other enzyme-inhibiting or antigenic proteins (e.g., b- conglycinin).

In some embodiments, the animal feed composition may be a poultry feed composition. As used herein, a“poultry feed composition” is poultry feed commonly used to raise poultry. Exemplary poultry feed compositions are well-described in the art and may be found, for example, in Scott’s Nutrition of the Chicken 4th Edition, written by Steven Leeson and John D. Summers, University Books Guelph, Ontario, Canada 2001.

In some embodiments, the animal feed composition may be a low energy diet (LED) feed composition. A LED feed would contain lower inclusion levels of concentrated energy sources, such as supplemental lipids, compared with standard poultry feeds. Additionally, LED feeds could contain higher concentrations of dietary fiber. In some embodiments, the LED feed has 1- 10% lower, 2-8% lower, or preferably 3-5% lower energy content (e.g, measured as kcal/kg ² ) as compared to a standard poultry feed. In particular embodiments, the LED feed has about 4% lower energy content as compared to a standard poultry feed.

A standard poultry diet may vary greatly based on several factors, such as the age, breed, and intended use of the poultry (e.g, egg laying vs. meat production). For example, different methods are used to feed chickens that are raised for the production of eggs for human consumption (e.g, Leghorn-type), which have a small body size and are prolific layers, as opposed to chickens used as broilers or broiler breeders (meat-type), which have rapid growth rates and a large body size. Poultry are commonly fed on a three to five phase diets. For example, a three phase feeding program might include starter, grower, and finisher feeds, with the starter diet containing the highest level of protein and fed soon after hatching (from roughly 0-2 weeks). Then the chickens are switched to a“grower” diet (2-4 weeks), and finally to a “finisher” diet (4-6 weeks), in which the protein content is lower than starter because as the birds grow, their protein requirements decrease while their energy requirements increase. Thus, as used herein, the term "standard poultry diet" refers to a diet that would be considered appropriate for the particular bird and purpose at hand. The Bacillus isolate may be within the animal feed composition at a concentration ranging from between about 10 ⁴ -10 ¹² cells/spores of the Bacillus isolate per gram of total animal feed composition, or any range therein. Suitably, between 10 ⁴ and 10 ⁹ , or between 10 ⁵ and 10 ⁸ , or between 10 ⁶ and 10 ⁷ spores per gram are used in the feed. Methods

Methods for Improving Nutritional Uptake

In a still further aspect, the present invention relates to methods for improving nutritional uptake in a subject. The methods may include administering to the subject an effective amount of any of the compositions (Bacillus isolates, probiotic compositions, or animal feed compositions) described herein.

Improved nutritional uptake may be demonstrated by, for example, improved body weight gain in a subject, improved cumulative feed conversion ratio, or improved carcass characteristics in a subject as compared to a control subject. The Feed Conversion Ratio (FCR) is the metric for conversion of feed to body weight gain (feed intake/body weight gain). Improvements in feed conversion are attributed to improved absorption of nutrients by the animal. This can mean that the animal is better able to absorb nutrients because of a change in digestion and absorptive capacity resulting in more available nutrients in the gastrointestinal tract of the animal. With this measurement, a lower number/ratio is better than a higher one. Body weight/body weight gain (BW or BWG) is a measure of the growth rate and is increased when the overall health of the animal is improved (less resources spent on stress or disease), the gastrointestinal tract of the animal is working more optimally, more nutrients are made available to the animal, and/or feed intake increases. Carcass characteristics include both the weight and yield percentage (i.e., percent of live animal weight) of the hot carcass, hot fat pad, and chilled carcass. "Hot carcass" weights and yields are based on defeathered carcasses, with the neck, head, feet, and viscera removed, immediately after processing and before chilling. "Hot fat pad" weight and yield reflect abdominal fat tissue which is a reliable indicator of total body fat content. "Chilled carcass" weights and yields are based on carcasses after being placed in ice water for a four-hour chill. Carcass weights and yields may also be determined for specific body parts. For example, breast weight and yield are determined after cutting the pectoralis major from the chilled carcass, while tender weights and yield are determined from cutting the pectoralis minor from the chilled carcass. Total white meat is the sum of the pectoralis major and pectoralis minor weights, as white meat is the most expensive and desirable cut of chicken in the U.S. market, and an increase in yield is profitable to the industry. These measurements are typically used in part to determine the quality of a feed product. Feed intake (FI) is a measure of the amount of feed eaten by the animal. Thus, at equal BW, a lower FI number is better and indicates that the animal is more efficiently converting feed to body weight, resulting in a lower FCR.

As used herein, “administering” may be carried out through any of the variety of procedures used to apply compositions to a subject that will be apparent to the skilled artisan. Suitable application methods may include incorporating composition into the feed or water source for the subject. Accordingly, the compositions used in accordance with the present methods may be any one of compositions disclosed herein. In some embodiments, the composition of the present methods may be a liquid including water such as drinking water that may be given to the subject. In other embodiments, the composition is a dry food composition or a pelleted feed composition. In these embodiments Bacillus spores may be added as a dry powder to the feed either before or after pelleting of the feed. As used herein, a“subject” may include any animal species. Suitable subjects may include, without limitation, a poultry species, a cow species, a pig species, a sheep species, a goat species, a fish species, or a mammalian species such as human and non-human mammalian species. Preferably, the subject is a poultry species such as a chicken or turkey species.

In accordance with the present methods, the body weight gain of the agricultural animal species may be improved by increasing by at least 5%, 10%, 20%, or 30% the body weight of the agricultural animal as compared to a control animal.

As used herein, a“control subject” or "control animal" refers to an animal species that is given a composition that excludes the Bacillus isolates described herein. Suitably, the control animal is fed a similar food product without any bacterial component added or without the specific isolates described herein. For example, an appropriate control animal for a chicken species that is subjected to the present methods would be the same chicken species fed a composition that excludes the Bacillus isolate.

In accordance with the present methods, the cumulative feed conversion ratio of the subject may be improved by decreasing the cumulative feed conversion ratio by at least 5%, 10%, 20%, 30%, 40%, or 50% as compared to a control subject.

Methods for Producing an Animal Feed Composition

In another aspect, the present invention relates to methods for producing an animal feed composition. The methods may include introducing any one of the Bacillus isolates or probiotic compositions described herein into any of the animal feed compositions described herein.

Optionally, the methods may further include pelleting the animal feed composition. In such embodiments, the Bacillus isolate or probiotic composition is introduced into the animal feed composition prior to, concurrently, or after the pelleting of the animal feed composition. For use in these processes, the Bacillus may be used in spore form.

Methods for Preventing Footpad Dermatitis

In another aspect, the present invention provides methods for reducing the incidence of footpad dermatitis (FPD) in poultry. The methods involve administering to the poultry an effective amount of any of the compositions {Bacillus isolates, probiotic compositions, or animal feed compositions) described herein.

FPD, also known as pododermatitis, is a condition characterized by lesions on the feet of poultry caused by ammonia bums. Ammonia is produced by a combination of excess litter moisture and nitrogen, both of which are increased in birds fed diets with elevated levels of galactooligosaccharides and non-starch polysaccharides ( e.g ., high soybean meal or alternative grain diets). Chicken paws are a major export for U.S. poultry integrators, and a reduction in paw condemnations at the processing plant increases profitability. Importantly, FPD is a threat to the health and welfare of the birds, so decreasing its incidence improves well-being.

FPD is commonly assessed using a subjective scoring system. For instance, the inventors used a 0-2 rating scale to compare the incidence of FPD across groups of chickens, and found that there was a reduced incidence of FPD in chickens that were fed at least one Bacillus isolate (isolate 46 or a combination of isolate 46 and isolate 40) as compared to chicken that were not fed a Bacillus isolate (see Table 11).

The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms“including,”“comprising,” or“having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as“including,”“comprising,” or “having” certain elements are also contemplated as“consisting essentially of’ and“consisting of’ those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 0.01% to 5%, it is intended that values such as 0.025% to 0.50%, 0.10% to 1.0%, or 0.025% to 0.075%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word“about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference in their entirety, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

Unless otherwise specified or indicated by context, the terms“a”,“an”, and“the” mean “one or more.” For example,“a protein” or“an RNA” should be interpreted to mean“one or more proteins” or“one or more RNAs,” respectively.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

EXAMPLES

Selection of Direct Fed Microbials (DFMs) for in situ diminution of anti-nutritional factors associated with non-starch polysaccharides (NSP) and free sugars in the diet of monogastric animals

Example 1 - In vitro Isolate Screening

Bacillus isolates were obtained from fecal samples gathered from various poultry flocks. The various poultry flocks included five separate flocks. The age of the chicken flocks ranged from 29 to 65 weeks old and the turkey flocks ranged from 16 to 18 weeks old. We hypothesized that using fecal samples would increase the chances of finding host-adapted Bacillus isolates that have a niche within the chicken gastrointestinal tract. Candidate isolates were grown in solid state fermentation (SSF) media to ensure robust sporulation of 10 ^L 10 or 10 ^L 11 spores/gram to confirm their ability to be mass-produced commercially. The spores were also tested under extreme heat conditions to ensure heat stability for the pelleting process of commercial feed production. The isolates were exposed to 90 °C for 10 minutes, which is the maximum temperature during the pelleting process and for a much longer duration than the typical pelleting process. Isolates were also selected based on biofilm production, which is important for survival and replication within the bird.

Following isolation, the Bacillus isolates were screened for galactosidase, cellulase, xylanase, and beta-mannase activities. Galactosidase activity was evaluated using a simple plate media with raffmose as the sole source of energy to support growth of the organism. 1 Omΐ of each isolate grown up in broth culture and drop plated onto the center of an ammonia media agar plate containing 0.2% raffmose as the only source of nutrition. As shown in Table 1, several of the Bacillus isolates exhibited galactosidase activity in this assay including isolates 26, 30, 31, 34, 36, 40, 46, 65, 86, 94, 96, 101, 102, and 105.

Table 1: Bacillus isolate growth on ammonia media plates containing 0.2% raffmose

Cellulase, xylanase, and beta-mannase activities of candidate isolates were evaluated using insoluble dyed substrates in agar plates. These insoluble substrates rapidly hydrate to form gel particles which are readily and rapidly hydrolyzed by specific enzymes, releasing soluble dye-labelled fragments.

To test for xylanase activity, we used soft agar overlays consisting of ammonia media containing only high purity dyed, crosslinked and finely milled insoluble AZCL-Arabinoxylan (Wheat) for identification of enzyme activity. A change in coloration when compared to the test plate indicates that the isolate is capable of enzymatically hydrolyzing the carbohydrate included in the overlay as its only means of nutrition. As shown in FIG. 1 and Table 2, isolates 26, 30, 31, 34, 36, 40, 46, 65, 86, 94, 96, 101, 102, and 105 exhibited xylanase activity.

Table 2: Bacillus Isolate Growth on Ammonia Media Plates with 0.0125% glucose with an

Arabinoxylan soft agar overlay

To test for cellulase activity, we used soft agar overlays consisting of ammonia media containing only high purity dyed, crosslinked and finely milled insoluble AZCL-HE-Cellulose for identification of enzyme activity. A change in coloration when compared to the test plate indicates that the isolate is capable of enzymatically hydrolyzing the carbohydrate included in the overlay as its only means of nutrition. As shown in FIG. 2 and Table 3, isolates 26, 30, 31, 34, 36, 40, 46, 65, 86, 94, 96, 101, 102, and 105 exhibited cellulase activity. Table 3: Bacillus Isolate Growth on Ammonia Media Plates with 0.0125% glucose with a

Cellulose soft agar overlay

To test for beta-mannase activity, soft agar overlays consisted of ammonia media containing only high purity dyed, crosslinked and finely milled insoluble galactomannan. RedCL-Galactomannan (Carob) was used for identification of enzyme activity. A change in coloration when compared to the test plate indicates that the isolate is capable of enzymatically hydrolyzing the carbohydrate included in the overlay as its only means of nutrition. As shown in FIG. 3 and Table 4, isolates 26, 30, 31, 34, 36, 40, 46, 65, 86, 94, 96, 101, 102, and 105 exhibited beta-mannase activity.

Table 4: Bacillus Isolate Growth on Ammonia Media Plates with 0.0125% glucose with a

Galactomannan Soft Agar Overlay

Example 2 - In vivo Testing of Bacillus Isolates

In vivo experiments using the Bacillus isolates (i.e, isolates 31, 40, 46, and 65) identified above were performed to determine the effectiveness of the isolate in a low energy corn and soybean meal based diet fed to broiler chickens. The Bacillus isolates were added to the feed premix prior to the mixing of the individual treatments to ensure proper distribution of the spores throughout the feed. Spores were added to the feed at a quantity that would ensure a final spore count of approximately 10 ⁶ in the finished feed. Birds were provided feed ad libitum for the life of the trial with the respective spore treatment groups receiving spores for the duration of the trial. Birds were reared in floor pens or battery cages and under standard commercial management practices. Several parameters were measured in the in vivo trials at days 7, 14, and 21. There was a 125 kcal/kg measured energy (ME) difference between the control, high energy diet (HED) and low energy diet (LED) used in these trials (Table 5). Table 5. Ingredient and calculated nutrient composition (% as-fed) of the starter basal diets (high energy diet (HED) and low energy diet (LED)) for 21d trials.

Ingredients (%)

Corn 53.34 55.71

Soybean Meal 40.00 40.00

Soy Oil 2.88 0.53

Dicalcium Phosphate 1.67 1.66

Limestone 0.99 1.00

DL-methionine 0.27 0.27

L-lysine HCL 0.07 0.06

L-threonine 0.05 0.04

Salt 0.69 0.69

Vitamin premix 0.10 0.10

Mineral premix 0.10 0.10

Choline Chloride (60%) 0.05 0.05

Se Premix (0.06%) 0.02 0.02

Santoquin 0.02 0.02

Inert Filler ¹ 0.02 0.02

Calculated analysis

AMEn, kcal/kg ² 3,050 2,925

CP (%) 23.62 23.79

_ dLys (%) ³ 1.20 1.22

Spores were added at the expense of sand

2 AMEn = Nitrogen corrected apparent metabolizable energy

3dLys = Digestible lysine

A brief explanation of the significance of each measure is included here

Feed Conversion Ratio (FCR): This is the metric for conversion of feed to body weight (Feed intake/body weight). Improvements in feed conversion are attributed to improved absorption of nutrients by the animal. This can mean that the animal is better able to absorb nutrients because of a change in absorptive capacity or more available nutrients in the gastrointestinal tract (GIT) of the animal. With this measurement, a lower number/ratio is better than a higher one.

Body weight / body weight gain (BW or BWG): growth rate is increased when the overall health of the animal is improved (less resources spent on stress or disease), the GIT is working more optimally, more nutrients are made available to the animal, and/or feed intake increases.

Feed intake (FI) is a measure of the amount of feed eaten by the animal. At equal BW, a lower number is better and indicates that the animal is more efficiently converting feed to body weight, resulting in a lower FCR.

Hot carcass weights and yields are based on defeathered carcasses, with the neck, head, feet, and viscera removed, immediately after processing and before chilling. Hot fat pad weight and yield reflect abdominal fat tissue which is a reliable indicator of total body fat content. Chilled carcass weights and yields are based on carcasses after being placed in ice water for a four-hour chill. Breast weight and yield are determined after cutting the pectoralis major from the chilled carcass. Tender weights and yield are determined from cutting the pectoralis minor from the chilled carcass. Total white meat is the sum of the pectoralis major and pectoralis minor weights, as white meat is the most expensive and desirable cut of chicken in the U.S. market, and an increase in yield is profitable to the industry.

Footpad dermatitis (FPD) is a condition characterized by lesions on the feet of poultry caused by a combination of excess litter moisture and nitrogen, leading to ammonia production. Moisture and nitrogen excretion are increased in birds fed diets with elevated levels of galactooligosaccharides and non-starch polysaccharides as seen with high soybean meal or alternative grain diets. The size of the associated lesions increase with severity and are subjectively scored on a scale from 0-2, in which a score of 0 indicates no lesions or very small superficial lesions, a score of 1 indicates mild lesions and discoloration of the footpad, and a score of 2 indicates severe lesions with ulcers or scabs and swollen foot pads. Chicken paws are a major export for U.S. poultry integrators, and a reduction in paw condemnations at the processing plant increases profitability. Importantly, FPD is a threat to the health and welfare of the birds, so decreasing its incidence improves well-being.

In FIG. 4, the growth rate of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 46 and 65. LED + Isolate 46 and HED had a significant increase in average body weight at D21 when compared to the LED without Bacillus supplementation. Each P-value was generated from a T-test against the LED. In FIG. 5, the feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 46 and 65. LED + Isolate 46 and HED had a significant decrease in feed conversion ratio from Dl-21 when compared to the low energy diet without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 6, the growth rate of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 40 and 46. LED + isolate 40 and HED had a significant increase in average body weight at D21 when compared to the LED without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 7, the growth rate of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). The isolate used in this experiment consisted of isolate 40. HED had a significant increase in average body weight at D21 when compared to the LED without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 8, the growth rate of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 31, 40, and 46. LED + isolate 46 had a significant increase in average body weight at D14 when compared to the low energy diet without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 9, the growth rate of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 31, 40, and 46. LED + isolate 46 and HED had a significant increase in average body weight at D21 when compared to the LED without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 10, the body weight gain of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 31, 40, and 46. LED + isolate 46 and HED had a significant increase in body weight gain from DO-21 when compared to the LED without Bacillus supplementation. Each P-value was generated from a T-test against the LED.

In FIG. 11, the feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or a high energy diet (HED). Isolates used in this experiment consisted of isolates 31, 40, and 46. HED had a significant decrease in feed conversion ratio from DO-21 when compared to the low energy diet without Bacillus supplementation. Each P-value was generated from a T-test against the LED. The group fed a LED diet and isolate 46 was intermediate between the HED and the LED groups which were statistically different.

In FIG. 12, the body weight gain of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed low (LED) or high energy diets (HED). Isolates used in this experiment consisted of isolate 40 and a low (46L) and high (46H) dose of isolate 46. Those doses were used to confirm there would be no detrimental effects if spores were added at a lower or higher dose than recommended. Birds in the LED + 40 and HED groups had increased body weight gain from DO-21 when compared to those fed LED without Bacillus supplementation. The LED + 46H treatment numerically increased body weight compared to LED and was not statistically different from birds fed HED. The overall ANOVA /’-value is shown with letters above bars denoting significant differences among groups according to a Tukey’s honest significance difference (HSD) test.

In FIG. 13, the feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed low (LED) or high energy diets (HED). Isolates used in this experiment consisted of isolate 40 and low (46L) and high (46H) dose of isolate 46. The LED group had increased FCR when compared to the birds fed HED. The LED + 40 group was intermediate between the HED and LED groups, while both doses of isolate 46 significantly reduced FCR compared to LED and were not statistically different from the HED group. The overall ANOVA /’-value is shown with letters above bars denoting significant differences among groups according to a Tukey’s honest significance difference (HSD) test.

In FIG. 14, the body weight gain of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed low (LED) or high energy diets (HED). Isolates used in this experiment consisted of isolates 40, 46, and the combination of the two. In this experiment there were no statistical differences across all groups, though inclusion of isolate 40, 46, and the combination tended to increase body weight gain compared to the LED and HED. The overall ANOVA C- value is shown.

In FIG. 15, the feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed low (LED) or high energy diets (HED). Isolates used in this experiment consisted of isolates 40, 46, and the combination of the two. Birds fed the HED had increased FCR compared to birds fed the LED. There was a significant reduction in FCR in birds fed the LED + 46 compared to birds fed the HED. The overall ANOVA P- value is shown with letters above bars denoting significant differences among groups according to a Tukey’s honest significance difference (HSD) test.

In FIG. 16, data were pooled from the previous six experiments to further evaluate the responses across all trials in which isolate 46 was used due to the consistent positive responses to this isolate. Across all trials, birds fed the LED had a reduction in body weight gain of 10% compared to birds fed the HED, but birds fed the LED + 46 were not statistically different from the birds fed the HED. The overall ANOVA C- value is shown with letters above bars denoting significant differences among groups according to a Tukey’s honest significance difference (HSD) test.

In Fig. 17, data were pooled from the previous six experiments to further evaluate the responses across all trials in which isolate 46 was used due to the consistent positive responses to this isolate. Across all trials, FCR was significantly increased by 13 points for birds fed LED compared to those fed HED. The LED + 46 group had a lower FCR than the LED group and was not statistically different from the HED group. The overall ANOVA C- value is shown with letters above bars denoting significant differences among groups according to a Tukey’s honest significance difference (HSD) test. Thus isolate 46 seems to counteract the effect of feeding a LED.

In the following experiment, birds were reared in floor pens from 0-35 d poast-hatch and fed starter (0-14d), grower (15-28d), and finisher (29-35d) diets. A control diet was formulated to industry relevant nutrient specifications and based on com soybean meal (HED), whereas a low energy diet (LED) was formulated to be similar, except for 125 kcal/kg reduction in dietary energy (Table 6). All experimental diets were fed as both mash and crumble or pelleted diets to confirm efficacy of Bacillus isolates in pelleted diets. Bacillus isolates were added to the feed premix prior to the mixing of the individual treatments to ensure proper distribution of the spores throughout the feed. Spores were added to the feed at a quantity that would ensure a final spore count of approximately 10 ⁶ in the finished feed. Birds were provided feed ad libitum for the duration of the trial with the spores provided in their respective treatment throughout all feeding phases. Growth performance parameters were measured at the end of each feeding phase on days 14, 28, and 35. Birds were processed on D36 to determine foot pad dermatitis lesions and carcass characteristics.

Table 6. Ingredient and calculated nutrient composition (% as-fed) of starter, grower, and finisher diets (high energy diet (HED) and low energy diet (LED)), supplemented without or with Bacillus isolates from 0 to 35 d Post-hatch.

Starter Grower Finisher

Item HED LED HED LED HED LED

Ingredients (%)

Corn 54.19 57.07 62.19 65.08 64.87 67.76 Soybean Meal 38.91 38.45 31.14 30.68 28.27 27.81

Soy Oil 3.19 0.77 2.94 0.51 3.53 1.11 Dicalcium- 1.63 1.62 1.51 1.50 1.31 1.29 Phosphate

Limestone 0.91 0.92 0.90 0.91 0.84 0.86 DL-methionine 0.26 0.26 0.28 0.28 0.25 0.25

L-lysine HCL 0.03 0.04 0.15 0.16 0.12 0.12

L-threonine 0.07 0.07 0.08 0.08 0.06 0.06

NB 3000 0.25 0.25 0.25 0.25 0.25 0.25

Salt 0.40 0.40 0.40 0.40 0.40 0.40 Choline 0.06 0.06 0.04 0.04 0.04 0.03 Chloride (60%)

BioCox 60 0.05 0.05 0.05 0.05

Inert Filler ¹ 0.02 0.02 0.02 0.02 0.02 0.02

Calculated

analysis

AMEn, kcal/kg ² 3,025 2,900 3,095 2,970 3,165 3,040

CP (%) 23.00 23.00 20.00 20.00 18.75 18.75 dLys (%) ³ 1.22 1.22 1.12 1.12 1.02 1.02

Spores were added at the expense of sand

2 AMEn = Nitrogen corrected apparent metabolizable energy

3dLys = Digestible lysine Table 7. Live performance of broilers fed mash or pelleted high soybean meal control (HED) or low energy diets (LED), with or without Bacillus-based DFM supplementation, during starter (0-14 d) and grower (15-28 d) phases. ¹

Starter (0-14 d) Grower (15-28 d)

Item 2 BWG, kg FI, kg FCR BWG, kg FI, kg FCR

Main effect of FF

Mash 0.280 ^b 0.41 l ^b 1.490 ^a 1.010 ^b 1.50 l ^b 1.493

Crumble/Pellet 0.404 ^a 0.530 ^a 1.320 ^b 1.275 ^a 1.894 ^a 1.487

SEM 0.010 0.010 0.018 0.021 0.031 0.005

Main effect of Diet

HED 0.350 0.478 1 394 ^ab 1.160 1.677 1.450 ^b

LED 0.334 0.471 1.444 ^a 1.129 1.704 1.507 ^a

LED + 46 0.344 0.460 1.361 ^b 1.139 1.709 1.503 ^a LED + combo 0.339 0.472 1 42i ^ab 1.141 1.699 1.50 l ^a SEM 0.013 0.014 0.025 0.030 0.044 0.007

P-Values ³

FF <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.35

Diet 0.233 0.373 0.016 0.364 0.644 <0.0001

FF x Diet 0.459 0.727 0.76 6 0.673 0.984 0.261 ^ab Means within a column that do not share a common superscript are different (P < 0.05)

Values are LSMeans of 24 replicate pens for FF and 12 replicate pens for Diet

Abbreviations: DFM = direct fed microbial; LED = low energy diet with 125 kcal/kg reduction in apparent ME _n ; LED + 46 = LED with Bacillus isolate 46; LED + Combo = LED with Bacillus isolates 40 + 46; FF = feed form

³ Overall ANOVA /< values

The body weight gain, feed intake, and feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or high energy diet (HED) during the starter and grower phases (Table 7). Isolates used in this experiment consisted of isolate 46 and combination of 40 and 46 (combo). There were no statistical differences in BWG or FI during the starter phase, however the LED + 46 treatment numerically increased BWG compared to the LED and lowered FI compared to all other groups. Birds fed LED + 46 had lower FCR than birds fed the LED and a similar FCR to birds fed the HED. Similarly, in the grower phase, there was no statistical differences among groups in BWG or FI, but the birds fed LED + 46 and LED + combo had numerically higher BWG compared to those fed the LED. Birds fed the HED had significantly lower FCR than all other groups.

Table 8. Live performance of broilers fed mash or pelleted high soybean meal control (HED) or low energy diets (LED) supplemented without or with Bacillus-based DFM candidates _ during finisher (29-35 d) and overall (0-35 d) phases. ¹ _

Finisher (29-35 d) Overall (0-35 d) Item ² BWG, kg FI, kg FCR BWG, kg FI, kg FCR

Main effect of FF

Mash 0.748 2.408 ^b 1.637 ^b 2.038 ^b 4.321 ^b 1.548 ^a

Crumble/Pellet 0.775 2.603 ^a 1.678 ^a 2.454 ^a 5.027 ^a 1.528 ^b

SEM 0.013 0.022 0.010 0.034 0.059 0.005

Main effect of

Diet

HED 0.791 2.426 ^b 1.639 2.302 4.583 1.50 l ^b

LED 0.770 2.542 ^a 1.664 2.234 4.719 1.554 ^a

LED + 46 0.753 2.536 ^a 1.650 2.237 4.705 1.546 ^a LED + combo 0.730 2.517 ^a 1.675 2.211 4.689 1.552 ^a SEM 0.018 0.031 0.014 0.049 0.083 0.008

P- Values ³

FF 0.156 <0.0001 0.003 <0.0001 <0.0001 0.005

Diet 0.125 0.001 0.238 0.064 0.104 <0.0001

FF x Diet 0.825 0.644 0.018 ⁴ 0.982 0.986 0.843 a ^b Means within a column that do not share a common superscript are different (P < 0.05)

Values are LSMeans of 24 replicate pens for FF and 12 replicate pens for Diet

Abbreviations: DFM = direct fed microbial; LED = low energy diet with 125 kcal/kg reduction in apparent ME _n ; LED + 46 = LED with Bacillus isolate 46; LED + Combo = LED with Bacillus isolates 40 + 46; FF = feed form

³ Overall ANOVA /< values

interaction means of FF x Diet for Finisher FCR: Mash, HED = 1.586 ^b ; Mash, LED = 1.662 ^ab ; Mash, LED + 46 = 1.651 ^ab ; Mash, LED + combo = 1.650 ^ab ; Pellet, HED = 1.694 ^a ; Pellet, LED = 1.668 ^ab ; Pellet, LED + 46 = 1.649 ^ab ; Pellet, LED + combo = 1.702 ^a

The body weight gain, feed intake, and feed conversion ratio of birds fed a low energy diet supplemented with a Bacillus direct fed microbial were compared to birds fed only a low energy diet (LED) or high energy diet (HED) during the finisher and overall phases (Table 8). Isolates used in this experiment consist of isolate 46 and combination of 40 and 46. There were no differences in body weight gain or FCR during the finisher phase, however birds fed HED had a lower feed intake (FI) than all other groups. For the overall period, there were no differences in BWG or FI, but birds fed HED had significantly lower FCR compared to other groups.

Table 9. Carcass characteristics of broilers fed high soybean meal (HED) or low energy diets (LED) supplemented without or with Bacillus-based DFM candidates from 0 to 35 d and processed at 36 d post-hatch ¹

Hot Carcass Hot Fat Pad Chilled Carcass

Weight, kg Yield, % Weight, kg Yield, % Weight, kg Yield, % 1.808 73.03 ^b 0.027 1.15 1.833 74 07 ^b 1.797 73.28 ^ab 0.028 1.19 1.820 74.24 ^ab 1.825 73.86 ^a 0.029 1.19 1.854 75.04 ^a LED + combo 1.803 73.65 ^ab 0.027 1.29 1.827 74.62 ^ab

SEM 0.015 0.22 0.0008 0.040 0.016 0.252 f ^> - value ³ 0.630 0.033 0.517 0.116 0.540 0.030 a ^b Means within a column that do not share a common superscript are different (P < 0.05)

Values are LSMeans of 8 replicate pens

² Abbreviations: DFM = direct fed microbial; LED = low energy diet with 125 kcal/kg reduction in apparent ME _n ; LED + 46 = LED with Bacillus isolate 46; LED + Combo = LED with Bacillus isolates 40 + 46

3Overall ANOVA C- values

On day 36, 8 pens per group of only the birds fed pelleted diets were randomly selected for processing and carcass characteristics were compared across groups (Table 9). Birds fed the LED + 46 had numerically higher hot carcass weights than all other groups. This group also had a significantly higher hot carcass yield percentage compared to the HED group. There was no difference in hot fat pad weight or yield among groups. Similarly, the LED + 46 treatment numerically increased chilled carcass weights of birds over all other groups and increased yield when compared to the HED group.

Table 10. Parts weights and yields of broilers fed high soybean meal (HED) or low energy diets (LED) supplemented without or with Bacillus-based DFM candidates from 0 to 35 d and processed at 36 d post-hatch. ¹

Breast Tenders Total White Meat

Item ² Weight, kg Yield, % Weight, kg Yield, % Weight, kg Yield, %

HED 0.473 19.1 l ^ab 0.093 3.74 ^b 0.566 22.86 ^ab

LED 0.464 18.90 ^b 0.093 3.80 ^ab 0.557 22.65 ^b

LED + 46 0.484 19.56 ^a 0.096 3.92 ^a 0.581 23.48 ^a

LED + 0.473 19.28 ^ab 0.094 3.84 ^ab 0.567 23.13 ^ab combo

SEM 0.006 0.16 0.001 0.041 0.006 0.19

f*- value ³ 0.132 0.020 0.162 0.014 0.125 0.007

a ^b Means within a column that do not share a common superscript are different (P < 0.05)

Values are LSMeans of 8 replicate pens

² Abbreviations: DFM = direct fed microbial; LED = low energy diet with 125 kcal/kg reduction in apparent ME _n ; LED + 46 = LED with Bacillus isolate 46; LED + Combo = LED with Bacillus isolates 40 + 46

³ Overall ANOVA C- values Weight and yield were also compared across parts (i.e., breast, tenders, and total white meat; Table 10). The breast yield percentage for LED + 46 was significantly higher than that of the LED group. The same effect was observed for tender yield as LED +46 which was significantly increased in the LED + 46 group compared to the HED group. Correspondingly, total white meat weight was numerically increased in the LED + 46 group compared to all other groups and the yield percentage for the LED + 46 group was significantly higher than that of the LED group.

Table 11. Percentage distribution of footpad dermatitis scores of

broilers fed high soybean meal (HED) or low energy diets (LED)

supplemented without or with Bacillus- based DFM candidates from 0 to 35

HED 11.65 ^b 83.14 ^a 5.21

LED 10.2 l ^b 78.24 ^a 11.55

LED + 34.47 ^a 59.00 ^b 6.53

46

LED + 33.52 ^a 57.95 ^b 8.52

combo

SEM 5.88 5.44 3.21

F*- value ³ 0.0002 0.0002 0.625

a ^b Means within a column that do not share a common superscript from arc

sine transformed data are different ( P < 0.05)

Values are LSMeans of 8 replicate pens

Abbreviations: DFM = direct fed microbial; LED = low energy diet with

125kcal/kg reduction in apparent ME _n ; LED + 46 = LED with Bacillus

isolate 46; LED + Combo = LED with Bacillus isolates 40 + 46

³ Overall ANOVA / ^J - values from arc sine transformed data

Footpad dermatitis was subjectively scored across the treatment groups on a scale from 0- 2, in which a score of 0 indicates no lesions or very small superficial lesions, a score of 1 indicates mild lesions and discoloration of the footpad, and a score of 2 indicates severe lesions with ulcers or scabs and swollen foot pads. There was a significant increase in the proportion of “0” scores for the LED + 46 and LED + combo groups compared to the HED and LED groups

(Table 11). Additionally, there was a significant reduction in scores of“1” in the LED + 46 and LED + combo groups compared to HED and LED groups. There were no differences observed in scores of“2” among all groups.

In conclusion, feeding isolate 46 in a low energy diet provided similar performance to higher energy, more expensive diet, and also improved carcass characteristics and lowered the incidence of footpad pododermatitis lesions.

DEPOSIT INFORMATION

A deposit of the Bacillus amyloliquefaciens isolate designated as“Isolate 46” disclosed above and recited in the appended claims has been made with the ARS Culture Collection (NRRL), 1815 N. University Street, Peoria, IL 61604. The date of deposit was June 12, 2020. All restrictions will be irrevocably removed upon granting of a patent, and the deposit is intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. The NRRL Accession Number is B- 67957. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

A deposit of the Bacillus amyloliquefaciens isolate designated as“Isolate 40” disclosed above and recited in the appended claims has been made with the ARS Culture Collection (NRRL), 1815 N. University Street, Peoria, IL 61604. The date of deposit was June 12, 2020. All restrictions will be irrevocably removed upon granting of a patent, and the deposit is intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. The NRRL Accession Number is B- 67956. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.