Feed additives and related methods for improving animal production performance, digestion and gut health

Abstract

The present invention relates to a feed additive comprising beta-1, 4-endo-xylanase and chitosan oligosaccharide present in an amount that provides a synergistic improvement in monogastric animal gut health or nutrient digestibility. Another aspect of the invention relates to the addition of a composition comprising beta-1, 4-endo-xylanase and chitosan oligosaccharide to an animal ration or water source to improve animal production performance.

Description

Cross-reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 544,329, filed October 16, 2023, entitled “Animal Feed Additives and Related Methods for Promoting Digestion and Gut Health in Animals,” which is incorporated herein by reference in its entirety. Background of the Invention Animal gut health has garnered significant interest from veterinarians, nutritionists, and researchers, and has regained prominence in recent years. This interest stems from the desire to improve animal production—such as growth, survival rates, and milk, meat yields, and egg quality—to meet global demand by enhancing gut health. Researchers have concluded that animal performance, feed efficiency, and overall health depend to a large extent on gut health. It is also recognized that changes in animal production systems and feed regulations, including the phasing out of antibiotic growth promoters, will necessitate the identification of new strategies to optimize gut health in novel and effective ways.

[0003] The gastrointestinal tract has long been considered the habitat of the gut microbiota. The relationship between the gut microbiota and animal health is crucial. Alterations in the gut ecosystem significantly impact animal gut health. Therefore, the animal health industry still urgently needs to identify and characterize novel, potential nutritional products that can provide improved gut health, thereby promoting animal growth and production performance.

[0004] Furthermore, feed additives that promote gut microbiota and feed digestibility are becoming increasingly important in the agri-food industry, especially given rising costs. The animal feed industry has been working to reduce nutritional costs, including rethinking diet composition, due to recent grain price increases and long-term overall price volatility. For example, wheat has become a significant energy source in poultry diets, even in markets that are not traditionally reliant on it, due to corn supply shortages and increased costs. However, poultry do not produce endogenous carbohydrate enzymes capable of breaking down or hydrolyzing non-starch polysaccharides (NSPs) (such as arabinoxylan) present in the sticky grains of such diets. It has even been determined that NSPs reduce the ability of poultry to efficiently utilize the protein and energy in these feed ingredients. Therefore, the use of exogenous enzymes and other feed additives to improve poultry production performance is crucial for poultry diets, and enzyme supplementation has become a common practice in commercial poultry nutrition.

[0005] As background, the applicant has previously demonstrated that the addition of xylanase to poultry diets not only helps reduce feed costs and improve animal production performance, but also positively impacts the cecal microbiota of poultry, which is closely related to poultry gut health. See Van Hoeck et al., Xylanase Impact Beyond Performance: A Microbiome Approach in Laying Hens, PLoS ONE 16(9): e0257681 (2021), which is explicitly incorporated herein by reference in its entirety.

[0006] With overall feed costs and environmental impact becoming areas of significant concern within the feed industry, there is a renewed and urgent need to identify feed additives that ensure optimal digestion and absorption of feed in the animal gut. Therefore, feed additives that promote digestion and gut health are becoming increasingly important for ensuring the profitability and long-term sustainability of the animal feed industry.

[0007] Brief Overview of the Invention This invention relates to feed additives comprising amounts of β-1,4-endo-xylanase enzyme (hereinafter referred to as "xylanase") and chitosan oligosaccharide (hereinafter referred to as "COS"), said amounts providing synergistic improvement in animal gut health. Another aspect of the invention relates to adding a composition comprising β-1,4-endo-xylanase and COS to animal diets or water sources to improve animal production performance.

[0008] Brief description of the attached figures Figure 1-5 The effects of treatment on the production performance of laying hens were described.

[0009] Figure 6-11 The effects of treatment on the digestibility of nutrients in laying hens were described.

[0010] Detailed Overview of the Invention This invention relates to animal feed ingredients or feed additives containing amounts of β-1,4-endoxylanase and COS, said amounts providing a synergistic improvement in animal gut health. Another aspect of the invention relates to animal feed ingredients or feed additives containing amounts of β-1,4-endoxylanase and COS, said amounts providing a synergistic improvement in animal production performance.

[0011] In at least one embodiment, animal gut health can be measured histologically by examining intestinal samples, including villus height and crypt depth. In another embodiment, the efficacy of the invention can be demonstrated by measuring blood cytokines, which may be pro-inflammatory or anti-inflammatory biomarkers depending on interactions within the intestinal epithelium.

[0012] In at least one embodiment, the addition of the composition of the present invention results in positive changes in the cecal microbiota of poultry, which can be measured by conventional agar plate culture or more generally by modern microbiota 16S ribosomal DNA sequencing technology (e.g., Illumina (Novogene) or Nanopore (Pathosense) based technology).

[0013] In at least one embodiment, animal production performance is measured by milk and meat production, egg quality, and / or egg production. For example, in at least one embodiment, poultry production performance can be measured by feed conversion ratio (FCR), egg quality, egg production, or egg characteristics (including egg weight, yolk color, Haugh unit, and the proportion of dirty or broken eggs).

[0014] In at least one embodiment, the composition of the present invention comprises sufficient amounts of xylanase and COS to have a positive effect on the microbial community, including by the measurement methods described herein.

[0015] In another embodiment, the feed additive of the present invention improves the digestion and absorption of nutrients, resulting in increased weight gain, for example, by at least about 1 gram of weight gain per egg or an average weight gain of about 1.6% per egg.

[0016] In another embodiment, the feed additive of the present invention can trigger macrophages to express anti-inflammatory and immune-stimulating pathways.

[0017] In some embodiments, the composition may optionally include any compound commonly used in animal nutrition, including β-glucanase, cellulase, protease, α-amylase, probiotics, prebiotics, organic acids or their salts, antioxidants, vitamins, minerals, lipids, lecithin and lysophosphatidylcholine, clay, carrier material, sugars, polyols, flavorings or aromas.

[0018] In at least one embodiment of the present invention, the COS is an oligomer of N-acetylglucosamine (NACOS) with an average degree of polymerization of 2 to 6, a degree of acetylation of more than 90%, and an average molecular weight of less than 2 kDa.

[0019] In at least one embodiment of the invention, the average molecular weight of the COS ranges from about 0.2 kDa to 4 kDa. For example, in at least one embodiment, the average molecular weight of the COS is less than 3.9 kDa, and each polymer chain contains fewer than 20 monomer units.

[0020] In some embodiments, the composition comprising xylanase and COS is fed to monogastric animals at doses of about 10 g / ton of feed to about 100 g / ton of feed, for example about 20 g / ton of feed to about 90 g / ton of feed, for example about 30 g / ton of feed to about 80 g / ton of feed. In at least one embodiment, the composition is present at an amount of at least 10 g / ton of feed. In at least one embodiment, the composition is present at an amount of at least 20 g / ton of feed. In yet another embodiment, the composition is present at an amount of at least 30 g / ton of feed. In yet another embodiment, the composition is present at an amount of at least 40 g / ton of feed.

[0021] In some embodiments, the ratio of xylanase to COS falls within the range of 1:5 to 1:1, for example, the components are present in a ratio of about 1:5, about 2:5, about 3:5 or 4:5.

[0022] In some embodiments, the composition comprises about 15% to about 50% xylanase and about 50% to about 85% COS by weight.

[0023] According to at least one embodiment, the composition is soluble in water.

[0024] According to at least one embodiment, the composition of the present invention is administered by adding the composition to animal feed or drinking water.

[0025] In some implementations, "animal" refers to monogastric animals, including but not limited to poultry, pigs, horses, rabbits, dogs, and cats, as well as aquatic animals, or pre-ruminant animals, such as calves.

[0026] According to at least one embodiment, the compositions of the present invention are suitable for animal feed and can be combined with known animal feed ingredients, including but not limited to corn flour, soybean meal, fish meal, sunflower seed meal, wheat, barley, sorghum, sunflower seed meal, rapeseed cake, soybean oil cake, dried distillers grains with solubles (DDGS), etc.

[0027] For the purposes of this disclosure, "animal feed" means animal rations, and "water" means drinking water or water supply for livestock. "Animal feed" may be provided to animals by any conventional means known to those skilled in the art, including but not limited to top-dressing, hand mixing, pelleting, mixing with crushed grains, etc.

[0028] Example Example 1 Materials and methods Chitosan oligosaccharide (COS) was used to screen for potential biological effects in the gastrointestinal tract. Intestinal CaCo2 cells were used as the intestinal cell model in this study. Cells were exposed for 24 hours. A total of 8 replicates were generated for each treatment. Cells were recovered, flash-frozen, RNA was extracted, and RNA sequencing was performed using a NovaSeq PE150 platform (Illumina, San Diego, USA).

[0029] result Gene expression data showed that COS had a significant impact on the transcriptome of CaCo2 cells. Treatment with 2 mg / ml compared to the control resulted in differential expression of 3161 genes, of which 1683 were upregulated and 1478 were downregulated. Treatment with 4 mg / ml compared to the control resulted in differential expression of 5815 genes, of which 2971 were upregulated and 2844 were downregulated. Therefore, the effect was dose-dependent, with the highest dose leading to more differentially expressed genes than the lowest dose. Furthermore, very consistently, pathways enriched in all COS-treated groups were related to ribosome biosynthesis and activity compared to the control. This may indicate that the treated cells had a higher protein biosynthetic capacity compared to untreated cells. Genes related to antioxidant capacity were transcribed and enriched. In conclusion, COS has a positive effect on the animal gut.

[0030] Example 2 Materials and methods A polarized cell culture system was established to mimic the gut, in which CaCo2 cells were seeded on the apical side and RAW 264.7 macrophages were cultured in the basal lateral compartment.

[0031] result Gene expression data showed that COS had a significant impact on the macrophage transcriptome. This effect was dose-dependent, with the highest dose resulting in more differentially expressed genes than the lowest dose. For macrophages, 3345 genes were differentially expressed when compared to the control at 2 mg / ml, and 4246 genes were differentially expressed when compared to the control at 4 mg / ml. Here, compared to the control group, all pathways enriched in all COS-treated groups were associated with cell differentiation. The data from this study support the conclusion that COS is a biologically active compound that has a direct impact on both intestinal cells and immune regulatory cells.

[0032] Example 3 Researchers conducted an in vivo trial using laying hens as a model to assess whether and to what extent COS can have an effect on animals.

[0033] Materials and methods The effects of dietary supplementation with COS were evaluated in a 60-day laying hen trial. Twenty HiSex laying hens were used in this study, employing two diets with ten replicates for each diet. Dosage was determined based on extrapolation from in vitro cell culture data. Hens were assigned to two distinct groups: a (T1) control group and a (T2) COS group (prepared via partial hydrolysis of shrimp chitin; 50 mg / kg feed). The diets were based on wheat (~55%), soybean, and sunflower seed meal.

[0034] result: Throughout the experiment, COS treatment showed beneficial effects on laying hen performance, nutrient digestibility (with particular attention to the fiber portion), and gut health (as shown in Table 1). Furthermore, based on LDA analysis, 16S sequencing data of the cecal microbiota revealed that Veillonellales-Selenomonadales (FC=1.7 enriched; P=0.01) and Prevotellaceae (FC=1.5 downregulated; P=0.001) were biomarkers in animals supplemented with COS.

[0035] Table 1. Average effects of COS on laying hen performance, egg quality, nutrient digestibility, and intestinal morphology. Average values ​​with different superscript letters (a, b) exist within the same row. P A significant difference of <0.05.

[0036] While previous studies have investigated COS, these studies have included poorly characterized heterogeneous mixtures, and COS is conventionally derived from highly deacetylated chitosan rather than chitin. For example, Piao et al. (2008) described a chitosan-derived COS and its effects on the poultry microbiota (DOI: 10.2527 / jas.2007-0668 · Source: PubMed). In contrast, the COS used in this example is well-defined in terms of molecular weight (1008 g / mol), average degree of polymerization (4), and high degree of acetylation (>90%). These properties are crucial because the results indicate that they strongly influence the bioactivity of COS, which was unknown prior to this work. Furthermore, the researchers determined that the product is water-soluble, which provides interesting application opportunities. The laying hen data in this example suggest that COS may provide health benefits, particularly when combined with other ingredients, providing a stimbiotic approach for animal gut health.

[0037] Example 4 Researchers combined xylanase and COS in a 2x2 factorial design study to investigate the effects of the combined xylanase and COS on animal gut health, particularly on the microbiome.

[0038] Materials and methods This study evaluated the effects of dietary supplementation with a xylanase / COS combination (prototype or "CXNRGY") in a 60-day laying hen trial. Forty HiSex laying hens were used in the trial, employing four different diets, with ten replicates for each diet. Dosage was determined based on extrapolation from in vitro cell culture data. Hens were assigned to four distinct groups: (T1) control group; (T2) COS-only group (prepared via partial hydrolysis of shrimp chitin; 50 mg / kg feed); (T3) xylanase-only group using Xygest HT (Kemin Industries, Inc.); and (T4) xylanase and COS combination group. The diets were based on wheat (~55%), soybean, and sunflower meal.

[0039] result Data confirms that the combination of these two compounds has a synergistic positive effect on gut health and thus on animal production performance, such as... Figure 1-5 As shown in more detail in Table 2. More specifically, the egg weight of hens supplemented with COS was significantly increased ( P The difference was most pronounced in the CXNRGY group (P < 0.0001), showing a significantly higher egg weight than the other three groups. Furthermore, researchers reported a significant increase in egg production rate in both the Xygest HT and CXNRGY groups compared to the control group. Finally, and very importantly, the FCR (fetal rate) of the CXNRGY-treated animals was significantly lower than that of the control animals (P < 0.0001). Interestingly, the CXNRGY group showed a trend towards achieving a better FCR compared to the Xygest HT and COS-treated groups. This suggests that the CXNRGY treatment had a more significant impact on production performance than either molecule (COS or Xygest HT) alone.

[0040] Table 2. Synergistic effect of COS and xylanase combination in prototype (COS and xylanase combination) compared with control treatment.

[0041] This study shows that individual compounds can beneficially affect gut health (measured by egg weight and feed conversion ratio (FCR)) and thus affect animal production performance, but going a step further, the data surprisingly show a synergistic effect when a combination containing both xylanase and COS is added to animal diets.

[0042] Digestibility data (e.g.) Figure 6-11As shown (as illustrated), the original compound had a significant effect compared to the control group, the xylanase-only group, or the COS-only group. More specifically, when examining the effect of COS alone, significant improvements were observed in almost all digestibility parameters studied. Even better effects were observed in the Xygest HT treatment group. The results indicate that the CXNRGY treatment achieved the best nutrient digestibility compared to the other three treatments. The CXNRGY treatment consistently demonstrated numerically better efficiency in nutrient digestibility compared to each individual compound.

[0043] In addition, researchers observed that the egg quality of the laying hens receiving the prototype was significantly improved compared with the control group (T1), the COS-only group (T2), the xylanase-only group (T3), or the COS and xylanase combination group (T4), as summarized in Table 3.

[0044] Table 3. Summary of Egg Characteristics.

[0045] In summary, the group receiving prototypes containing both xylanase and COS had a positive impact on laying hen production performance, egg quality, and nutrient digestibility.

[0046] Example 5 Researchers conducted a study in growing poultry, combining xylanase and COS in a dose-response study to investigate the effects of the combination of xylanase and COS on poultry growth.

[0047] Materials and methods This study evaluated the effects of dietary supplementation with xylanase / COS in a 35-day broiler trial. A total of 1200 Ross broilers were used in this experiment, employing five different diets, with 12 replicates for each diet. As shown in Table 4, the broilers were assigned to five different groups.

[0048] Table 4. Summary of processing.

[0049] resultThe results of the breeding performance are summarized in Table 5, showing that the heaviest body weight (BW) was observed in the T2 and T4 treatment groups, while the lightest broilers were in the T3 group. No significant differences in production performance were observed between the treatment groups during the brooding period (0–14 days). During the growth period (15–35 days), broilers in the T4 and T5 treatment groups grew more and exhibited lower feed conversion ratios than those in the T3 and T1 groups. Looking at overall production performance, broilers in the T2, T4, and T5 treatment groups grew significantly more and exhibited higher EPEFs than those in the T3 group. Furthermore, broilers in the T4 and T5 groups showed significantly lower feed conversion ratios than those in the T1 and T3 groups.

[0050] Table 5. Aquaculture production performance at each research stage and overall stage.

[0051] Based on the experimental conditions, the researchers were able to conclude that a combination of COS at a dose of 25 g / ton and xylanase (XygestHT) at a dose of 10 g / ton showed improved overall broiler production performance, particularly improved broiler growth and reduced FCR.

[0052] The invention has been described with reference to specific compositions, effectiveness theories, etc., and those skilled in the art will understand that the invention is not intended to be limited to these illustrative embodiments or mechanisms, and modifications can be made without departing from the scope or spirit of the invention as defined in the appended claims. All such obvious modifications and variations are intended to be included within the scope of the invention as defined in the appended claims. The claims are intended to cover any sequence of claimed components and steps that effectively achieve their intended objectives, unless the context specifically indicates otherwise.

[0053] It should also be understood that minor dosage and formulation modifications, as well as modifications to the scope set forth herein, may be made to the compositions, and these shall still fall within the scope and spirit of the invention.

[0054] The foregoing description is for illustrative purposes only. It is not intended to be an exhaustive list or to limit the invention to the precise forms disclosed. It is anticipated that other alternative processes and methods, obvious to those skilled in the art, will be included in the invention. This description is merely an example of embodiments. It should be understood that any other modifications, substitutions, and / or additions may be made, all within the spirit and scope contemplated by this disclosure. As can be seen from the foregoing, the exemplary aspects of this disclosure achieve at least all the intended objectives.

Claims

1. A method for improving animal production performance, comprising the following steps: Adding a composition containing β-1,4-endoxylanase and chitosan oligosaccharide (COS) to animal diets or water in an amount sufficient to improve animal production performance.

2. The method according to claim 1, wherein the composition is water-soluble.

3. The method according to claim 1, wherein the animal is a monogastric animal.

4. The method of claim 1, wherein the animal production performance is measured by increased weight gain or decreased feed conversion ratio.

5. The method of claim 1, wherein the animal production performance is measured by increased milk production, improved egg quality score, increased weight gain per egg, increased egg production, or improved egg characteristics, said egg characteristics including egg weight, yolk color, Haugh units, and / or the proportion of dirty or broken eggs.

6. The method of claim 1, wherein the animal production performance is measured by reduced serum TNF-α, IL-1α and IL-10 concentrations.

7. The method according to claim 1, wherein the COS is an oligomer of N-acetylglucosamine having an average degree of polymerization of 2 to 6 monomers and a degree of acetylation greater than 90%.

8. The method according to claim 1, wherein the average molecular weight of the COS is less than 4 kDa.

9. The method according to claim 8, wherein the average molecular weight of the COS is less than 2 kDa.

10. The method of claim 1, wherein the animal diet comprises the composition in an amount of about 10 g / ton of feed to about 100 g / ton of feed.

11. Feed additives or supplements containing amounts of β-1,4-endoxylanase and chitosan oligosaccharide (COS) sufficient to improve intestinal health and / or nutrient digestion and absorption in monogastric animals.

12. The feed additive or supplement according to claim 11, wherein the composition is water-soluble.

13. The feed additive or supplement of claim 11, wherein the animal gut health is measured by demonstrating a positive effect on the animal's microbiome.

14. The feed additive or supplement of claim 11, wherein the digestion and absorption of the nutrient is measured by increased weight gain or decreased feed conversion ratio.

15. The feed additive or supplement according to claim 11, wherein the digestion and absorption of the nutrients are measured by increased milk production, improved egg quality score, increased egg production, or improved egg characteristics, said egg characteristics including egg weight, yolk color, Haugh units, and / or the proportion of dirty or broken eggs.

16. The feed additive or supplement according to claim 11, wherein the COS is an oligomer of N-acetylglucosamine having an average degree of polymerization of 2 to 6 monomers and a degree of acetylation greater than 90%.

17. The feed additive or supplement according to claim 11, wherein the average molecular weight of said COS is less than 4 kDa.

18. A method for improving the production performance of monogastric animals, comprising the following steps: Compared to applying xylanase alone or chitosan oligosaccharide (COS) alone, adding a composition containing an effective amount of the xylanase and the COS to animal diets or water.

19. The method of claim 18, wherein the animal production performance is measured by increased milk production, improved egg quality score, increased egg production, or improved egg characteristics, said egg characteristics including egg weight, yolk color, Haugh units, and / or the proportion of dirty or broken eggs.

20. The method of claim 18, wherein the COS is an oligomer of N-acetylglucosamine having an average degree of polymerization of 2 to 6 monomers and a degree of acetylation greater than 90%.

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