Maturation of immune and metabolic processes via algal biomass and / or related substances administered to animals

Abstract

Methods and compounds are disclosed for promoting growth and stimulating the immune system in both animals and humans. The disclosed methods utilize compounds derived from gram-negative bacterial lipopolysaccharide, lipid A, and one or more of its fractions, derivatives, and cellular components. The compounds are combined with a conventional feed, such as a corn and soybean feed, for administration to animals, such as poultry. Human applications are also possible. Kinetic analysis of tissues taken from birds fed a mixture of the compounds of the invention and a conventional feed and sacrificed suggests that biomass induces signaling changes in multiple growth-related pathways. These pathways include, but are not limited to, pathways related to vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK or MAP kinase), Ak transforming factor (Akt), and neurotrophic tropomyosin-related kinase (NTRK). Evidence supports the conclusion that the changes represent modulation of various pathways.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. non-provisional application of U.S. Provisional Patent Application No. 63 / 143,444, entitled “Maturation of immune and metabolic processes via algal biomass and / or related substances in animal feed,” filed on January 29, 2021, and is hereby incorporated by reference in its entirety for all purposes.

[0002] The disclosed inventive concepts relate to immune and metabolic processes in animals and humans. More specifically, the disclosed inventive concepts relate to natural feed compounds derived from freshwater algae cultures and methods of using the compounds to cause tissues in animals or human subjects to mature faster, thereby promoting more robust growth, when compared to animals or human subjects not administered the compounds. The disclosed compounds of the present invention further induce immune stimulatory responses that shift certain organs to a more adaptive response, induce lipid metabolism in selected tissues, and induce a broad immune response when the subject is infected with a pathogen. The disclosed inventive concepts have particular application in the poultry industry, but may be applicable beyond poultry to other animals. The disclosed inventive concepts may also be beneficial to humans. [Background technology]

[0003] The commercial animal industry is under constant economic pressure to develop animal rearing methods that maximize healthy animal growth rates while minimizing the costs associated with feeding and caring for the animals. One such industry is the poultry industry, which is facing a dramatic increase in demand. Poultry meat competes with pork for the position of the most consumed meat in the world. Global poultry production is expected to need to meet an increase in demand of more than 120% by 2050.

[0004] In response to this increasing supply demand, poultry producers are constantly looking for ways to increase the efficiency with which birds, such as broilers, convert feed to body mass as a way to increase profit margins by reducing the amount of feed required to produce a bird of a given size (or maximizing the size of the bird produced using a given amount of feed). On farms where diseases such as coccidiosis are a significant problem, the focus is often on improving growth performance, directed toward controlling diseases that negatively impact growth performance. However, this approach does not improve the growth ability of healthy birds.

[0005] Improving growth performance in healthy birds can be achieved by (1) increasing the availability of nutrients in the diet (e.g., by increasing digestibility), (2) altering the bird's physiology to metabolize available nutrients more efficiently, or (3) shifting the bird's energy utilization from non-growth related processes to growth related metabolic pathways. Summary of the Invention [Problem to be solved by the invention]

[0006] It is therefore desirable to develop practical, natural and cost-effective compounds and methods of use that support the overall health of animals by promoting growth and improving immune function, thereby making animals more resilient to disease. [Means for solving the problem]

[0007] The disclosed inventive concept provides natural compounds for use as feed ingredients to promote animal growth. The compounds of the disclosed inventive concept are combined with conventional feed for administration to animals such as poultry. Human applications are also possible. The compounds of the disclosed inventive concept include one or more substances selected from algal biomass / supernatants (including both algae and bacteria), bacterial biomass, and isolated and purified compounds, as well as specific active sites or structures on those compounds. The combination of the disclosed compounds of the invention and oral administration of liquid or dry feed, or both, acts through unique biological pathways, for example in healthy birds, to enhance growth performance while simultaneously stimulating the immune system to promote disease response if disease occurs (disclosed in U.S. Provisional Patent Application No. 63 / 044,841 entitled "Immune Priming to Accelerate / Enhance Immune Response Through Administration of Innate Immune Modulators" and U.S. Provisional Patent Application No. 63 / 056993 entitled "Natural Feed Compositions Derived from Freshwater Algal Cultures for Animal Growth Promotion", both provisional patent applications are incorporated herein by reference). The disclosed compounds of the present invention are natural products and therefore do not adversely affect the environment.

[0008] During the period of use, the compound is administered to the animals via poultry feed, drinking water, or both, along with a nutritionally adequate or standard diet, including but not limited to a corn-soy based diet. Studies based on the use of animal feeds containing the disclosed compounds of the invention or biomass containing algae cultures have demonstrated improved animal growth. It has been found that animals, particularly healthy birds, receive significant benefits in terms of improved growth efficiency when fed the biomass of the disclosed compounds of the invention mixed with conventional feeds, such as corn-soy feeds. Data have shown that healthy chickens, particularly broiler chickens, are fed a corn / soy feed supplemented with the biomass of the compounds of the invention of algae cultures, resulting in improved growth efficiency and concomitant improved immune response, compared to birds fed the same feeds without supplementation with the algae culture biomass. Although reference is made herein to conventional feeds of corn and soy, it is understood that the disclosed compounds can also be advantageously used in combination with other forms of conventional animal feed, such as, but not limited to, wheat.

[0009] Kinetic analysis of tissues from birds sacrificed on a diet containing the compound of the invention and a conventional diet suggests that biomass induces alterations in signaling in multiple growth-related pathways. These pathways include, but are not limited to, those related to vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK or MAP kinase), Akt, and neurotrophic tropomyosin-related kinase (NTRK). Evidence supports the conclusion that the changes represent modulation (including, but not limited to, activation and deactivation) of various pathways.

[0010] The disclosed inventive concepts have numerous advantageous applications in humans and animals, including, but not limited to: (1) improving growth rate without the use of additional supplements such as antibiotics, enzymes, probiotics, antimicrobials, ionophores and / or other chemicals, (2) providing an entirely natural solution to the need for growth rate improvement, and (3) improving immunity to disease. [Brief description of the drawings]

[0011] For a more complete understanding of the present invention, reference should be made to the accompanying drawings, in which:

[0012] [Figure 1] FIG. 1 shows heat maps of kinotypes of liver and muscle tissue.

[0013] [Diagram 2] FIG. 2 shows the dendrogram of liver and muscle tissue (T2 vs. T1).

[0014] [Diagram 3] FIG. 3 shows heat maps of kinotypes of the small intestine and cecum.

[0015] [Figure 4] FIG. 4 shows the dendrogram of small intestine and cecum tissue (T2 vs. T1).

[0016] [Figure 5A] FIG. 5A shows a pathway diagram depicting novel metabolic riatom pathway members in the small intestine at D14.

[0017] [Figure 5B] FIG. 5B shows a pathway diagram depicting novel metabolic riatom pathway members in the small intestine at D42.

[0018] [Figure 6A] FIG. 6A shows a pathway diagram depicting novel metabolic riatom pathway members in the liver at D14.

[0019] [Figure 6B] FIG. 6B shows a pathway diagram depicting novel metabolic riatom pathway members in liver at D42.

[0020] [Figure 7A] FIG. 7A shows a pathway diagram depicting novel metabolic riatom pathway members in muscle at D14.

[0021] [Figure 7B] FIG. 7B shows a pathway diagram depicting novel metabolic riatom pathway members in muscle at D42.

[0022] [Figure 8] FIG. 8 shows a pathway diagram depicting novel metabolic riatom pathway members in the cecum.

[0023] [Figure 9A] FIG. 9A shows a pathway diagram depicting novel innate immune system riatom pathway members in the small intestine at D14.

[0024] [Figure 9B] FIG. 9B shows a pathway diagram depicting novel innate immune system riatom pathway members in the small intestine at D42.

[0025] [Figure 10A] FIG. 10A shows a pathway diagram depicting novel innate immune system rhatome pathway members in the liver at D14.

[0026] [Figure 10B] FIG. 10B shows a pathway diagram depicting novel innate immune system rhatome pathway members in the liver at D42.

[0027] [Figure 11A] FIG. 11A shows a pathway diagram depicting novel innate immune system rhatome pathway members in muscle at D14.

[0028] [Figure 11B]FIG. 11B shows a pathway diagram depicting novel innate immune system rhatome pathway members in the liver at D42.

[0029] [Figure 12] FIG. 12 shows a pathway diagram depicting novel innate immune system riatom pathway members in the cecum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In the following description, various operating parameters and components are described for different configurations of the embodiment. These specific parameters and components are included as examples and are not intended to be limiting. Unless otherwise noted, all technical and scientific terms used herein are to be given the common meaning as understood by those skilled in the art.

[0031] The disclosed inventive concept method proposes the use of compounds including algal biomass and related substances including, for example, algal supernatant, symbiotic bacteria, bacterial biomass, and bacterial fermentation products, which are combined with conventional feed to produce a feed mixture that is fed to chickens, for example broiler chickens, and other animals, to improve the growth efficiency of the birds.

[0032] Compounds used to promote growth

[0033] The disclosed growth promotion method utilizes active compounds, including algal biomass and related substances, which may be administered early in the life of broilers to achieve optimal growth rates and improved immune responses. The active compounds may be derived from lipopolysaccharide (LPS) of gram-negative bacteria or from sources other than lipopolysaccharide.

[0034] As used herein, the term "inhibitor" refers to a molecule that reduces or attenuates the activity induced by another molecule. As an example, a compound that may block the LPS-dependent regulation of TLR receptors (including but not limited to TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and / or TLR9 receptors) present on the surface of human and animal immune cells would be considered an inhibitor of this particular pathway.

[0035] As used herein, the term "algal culture" is defined as algal organisms and bacteria (one or more species) grown together in a liquid medium. Unless otherwise specified, the term "algal biomass" refers to the algal cells and bacterial cells (with the liquid medium removed). "Algal biomass" can be a wet or dry material.

[0036] Unless otherwise stated, the term "algal supernatant" is defined as the medium in which an algal biomass is grown and includes compounds secreted from the algal biomass. Algal supernatant is obtained by growing an algal biomass in medium for a suitable period of time, followed by removal of algal and bacterial cells by filtration and / or centrifugation.

[0037] Embodiments of compounds for use in the growth promoting methods described herein include one or more LPS / Lipid A compounds produced by Gram-negative bacterial strains, including but not limited to fractions, derivatives, and cellular components thereof, for use as selective modulators of one or more TLR signaling pathways. The bacterial strains include one or more of the following: Algoriphaqus aquaticus, Bosea sp., Brevundimonas diminuta, Brevundimonas vesicularis, Desulfovibrio sp., Microbacterium testaceum, Sphingomonas sp., Variovorax paradoxus, and Ochrobactrum pseudogrignonense.

[0038] The disclosed inventive concept involves any combination of three basic steps: (1) Gram-negative bacteria produce LPS / Lipid A compounds; (2) LPS / Lipid A compounds modulate the TLR pathway; and (3) downstream effects result in enhancement of innate and adaptive immune processes, thereby preventing or reversing growth inhibition.

[0039] According to one embodiment, the LPS / Lipid A compounds used as selective modulators of the TLR signaling pathway (including but not limited to TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and / or TLR9 receptors) are produced from one or more strains of Gram-negative bacteria from Argoriphagum aquaticus, Bosea spp., Brevundimonas diminuta, Brevundimonas vesicularis, Desulfovibrio spp., Microbacterium testaceum, Sphingomonas spp., Variovorax paradoxus, and Ochrobactrum pseudogrignonense. The strains may be naturally occurring and may be found in the algal biomass and / or algal supernatant products. For example, the algal biomass may include the green algae species Klebsormidium flaccidum. More specifically, the algal biomass culture may include the algal strain Klebsormidium flaccidum, var. ZIVO.

[0040] According to another embodiment, more specifically, the LPS / Lipid A compound used as a selective modulator of the TLR signaling pathway is produced from a strain of Rhodobacter sphaeroides. Extensive research has been conducted on the structure and function of Rhodobacter sphaeroides in general. More focused studies have investigated the photosynthetic properties of Rhodobacter sphaeroides. Although lipopolysaccharide from Rhodobacter sphaeroides is known to be an effective TLR4 antagonist in human cells and prevents TLR4-mediated inflammation by blocking LPS / TLR4 signaling, the inventors have discovered an LPS compound from Rhodobacter sphaeroides that proved effective in reversing factors that cause growth inhibition. More specifically, although initial data suggested inhibition by LPS-like molecules, it was not until specific tests with Rhodobacter sphaeroides revealed the efficacy of this bacterium in preventing or reversing growth inhibition in poultry. Studies have further demonstrated that combining TLR4 modulators with TLR2 activators (such as LPS from many Gram-negative bacteria) can provide anticoccidiostatic effects.

[0041] In yet another example, LPS / Lipid A compounds may modulate TLR4 through either ligand-dependent or ligand-independent activation, hi another example, LPS / Lipid A compounds may cooperate with other TLR agonists to enhance immune responses while simultaneously reducing metabolic costs to the host.

[0042] Thus, embodiments of the compounds used in accordance with the present disclosure are directed to one or more LPS / Lipid A compounds produced by Gram-negative bacterial strains for use as selective modulators of one or more TLR signaling pathways.

[0043] The LPS / Lipid A compounds used herein can be obtained from Gram-negative bacterial strains by any suitable method, but in certain embodiments, they are prepared by (1) extracting lyophilized bacteria with a solution of phenol / guanidine thiocyanate and collecting the aqueous layer for lyophilization; (2) resolubilizing the lyophilized fraction in water; (3) ultrafiltering the solubilized fraction to remove low molecular weight material and salts; (4) affinity purifying the high molecular weight fraction using a polymyxin B resin column, such as Affi-prep polymyxin matrix material (Bio-Rad), from which active fractions are eluted with 1% deoxycholate; and, optionally, (5) further purification using size exclusion chromatography.

[0044] In some instances, multiple types of LPS extraction protocols are used to obtain LPS compounds from bacteria, and the extraction procedure may be performed multiple times. Once the LPS compounds have been extracted and purified from the bacteria, a lipid A fraction may be prepared by acid hydrolysis or other suitable techniques.

[0045] One or more LPS / Lipid A compounds derived from Gram-negative bacterial strains can selectively inhibit one or more TLR signaling pathways to reduce and / or inhibit inflammatory responses and improve immune health in a variety of applications and uses. According to one embodiment, LPS / Lipid A compounds derived from one or more of the Gram-negative bacterial strains referenced herein can be incorporated into an algae-based feed ingredient to increase growth while simultaneously inducing an improved immune response.

[0046] Growth promoting compounds and methods of use: A non-limiting example of a method for promoting the growth of an animal is described below. Although the following method is aimed at promoting the growth of poultry, it should be understood that the disclosed method can be applied to other animals as well as humans. Thus, the described growth-promoting compounds and methods of use are not intended to be used only in poultry.

[0047] Previous efforts by the applicant have demonstrated some beneficial effects of the inventive algae-based compounds disclosed herein fed to poultry. However, further data was needed regarding the specific mechanism of action, particularly at the protein, post-translational and cell signaling levels. The present invention provides an analysis to characterize the immune and metabolic responses resulting from feeding the inventive algae compounds with a corn-soy-based diet compared to a control corn-soy diet alone. Specific objectives achieved in this study and discussed herein include measuring immune and metabolic changes in the small intestine (particularly adjacent Meckel's diverticulum), liver, cecum, and breast muscle induced by the addition of the inventive algae products to a corn-soy diet of broiler chickens. EXAMPLES

[0048] Working Example

[0049] According to a non-limiting example of the present invention, the compound of the present invention is defined as the algae biomass and related substances including algae supernatant and symbiotic bacteria described above. The compound of the present invention was mixed with conventional feed to form a supplemented "feed mixture" at a certain ratio. This ratio was maintained during the test period. The bird flocks were divided into a control group that received only conventional corn and soybean feed and an experimental group that received the supplemented feed mixture. The compound of the present invention was orally administered via dry or liquid feed to animals at an early stage, defined as some time between the first and second week of life.

[0050] Two growth-promoting treatment regimens were administered: Treatment 1 (T1) = corn and soy diet control Treatment 2 (T2) = corn and soybean diet + algae

[0051] The growth promoting compound according to the inventive concept of the present disclosure was tested in a 42-day broiler pen study at a research university. Overall, the results showed that birds fed a feed composition containing the disclosed compound of the present invention and a corn / soybean mixture from day 14 of age onwards showed improved metabolism and improved immune response compared to control animals fed only a corn / soybean mixture. An ongoing review of a small number of other studies in which healthy birds were fed a composition containing the compound of the present invention for a limited period before a preliminary disease challenge was applied suggests the positive effect of the compound of the present invention in healthy animals when combined with conventional animal feed.

[0052] The treatment compound is a freshwater algal biomass containing one or more of the gram-negative bacteria Algorithm aquaticus, Bosea spp., Brevundimonas diminuta, Brevundimonas vesicularis, Desulfovibrio spp., Microbacterium testaceum, Sphingomonas spp., Variovorax paradoxus, and Ochrobactrum pseudogrignonense, and is provided in drinking water or in combination with a feed additive such as soybean oil, preferably in a ratio of 1 part algal biomass to 2 parts soybean oil, in the animal feed. Once the biomass and feed additive are combined to the desired premix level, the combined batch is poured or dosed evenly into a ribbon mixer containing the finished feed. When including the disclosed inventive concepts as algal biomass in animal feed, the mixed batch is preferably provided in an amount of about 0.5 pounds of composition per ton of finished feed to about 11.0 pounds of composition per ton of finished feed, more preferably about 1.0 pounds of composition per ton of finished feed to about 5.0 pounds of composition per ton of finished feed, and most preferably about 3.0 pounds of composition per ton of finished feed to about 4.0 pounds of composition per ton of finished feed. A recommended, non-limiting ideal ratio is about 3.5 pounds of composition per ton of finished feed that provides good results without waste.

[0053] When incorporating the disclosed inventive concepts as bacterial biomass in animal feed, the combination batch is preferably provided in an amount of from about 20.0 g of composition per ton of finished feed to about 250.0 g of composition per ton of finished feed, more preferably from about 125.0 g of composition per ton of finished feed to about 175.0 g of composition per ton of finished feed, and most preferably from about 100.0 g of composition per ton of finished feed to about 150.0 g of composition per ton of finished feed. A recommended, non-limiting ideal ratio is about 125.0 g of composition per ton of finished feed for maximum effectiveness.

[0054] Research - Treatment

[0055] Mixed sex broiler chicks were obtained from a commercial hatchery on day 0 (day of hatching and placement). Chicks were assessed upon receipt for signs of disease or other complications that may affect the study outcomes. Weak birds were humanely sacrificed. No replacement of birds was performed during the study.

[0056] After testing, chicks were weighed and assigned to pens of the different treatment groups using a randomized block design. Weight distribution across treatment groups was assessed by comparing the standard deviation of the means of the individual test groups with that of the control group before feeding. Weight distribution between groups was considered acceptable in this study if the difference between the control and test groups was within one standard deviation.

[0057] All birds were fed a nutritionally adequate diet. Enclosures were monitored for environmental conditions including temperature, light, water, feed, litter condition and unanticipated housing conditions or events. Pens were inspected daily for mortality. Inspections were performed on all broilers found dead or moribund. Mortality was recorded and investigated.

[0058] After the end of the growth period, muscle, liver, small intestine, and cecum tissues were collected from five birds from each of the two dietary groups at 14 days (D14) and 42 days (D42) after hatching. Tissue samples were removed from the chickens and immediately flash frozen in liquid nitrogen to preserve kinase enzyme activity. Samples were stored on dry ice and at -80°C until the experimental protocol was performed. Tissue samples were thawed and 40 mg sections were collected and placed in a 2.0 mL homogenizer tube containing 1.5 mm zirconium beads and 100 μL of lysis buffer. Samples were homogenized in a Bead Ruptor.

[0059] The homogenized tissue was then incubated on ice for 10 min and centrifuged in a microcentrifuge.The peptide array fabrication was then initiated, generating 771 unique kinase substrate target peptide sequences printed in nine replicates.

[0060] A glass lifter slip was applied to the microarray to sandwich and distribute the applied lysate. 80 μL of the mixture was applied to the peptide microarray, ensuring that no air bubbles were present in the pipette tip or array slide. The slide was incubated in a humidity chamber for 2 hours. A humidity chamber is a closed container (not in contact with the array) that contains a small amount of water in an incubator. The array was removed from the incubator and humidity chamber and placed in a centrifuge tube containing phosphate-buffered saline. The array was repeatedly immersed in the solution until the lifter slip slid off the array. The array was then immersed and agitated. This process was then repeated with new solution. The array was immersed in ddH2O and agitated. The array slide was removed from ddH2O and immersed in phospho-specific fluorescent stain in a dish and placed on a shaker table. The dish was covered to protect the fluorescent stain from light. The array was then placed in a new dish and immersed in the destaining solution with agitation. The Petri dish was covered to protect the stain from light. This process was repeated twice, with a final wash being with distilled, deionized water.

[0061] The array was then placed in a mL centrifuge tube with a rolled up Kimwipe at the bottom. The tube containing the array was then centrifuged to remove water from the array. The array was scanned at 532–560 nm with a 580 nm filter using a Tecan PowerScanner microarray scanner to detect dye fluorescence.

[0062] Statistics and Data Analysis

[0063] Images were generated and spot intensity signals were collected as the average of pixel intensities using local feature background intensity calculations at default scanner saturation levels.

[0064] Images were gridded and spot intensity signals were collected as the average of pixel intensities using local feature background intensity calculations at default scanner saturation levels. The resulting data were then analyzed with PIIKA2 peptide array analysis software. Briefly, the resulting data points were normalized to eliminate variations due to technical variations, e.g., random variations in staining intensity between arrays or between array blocks within an array. Variance-stabilizing normalization was performed. Note that the arrays were printed in triplicate peptide blocks, so there are three values ​​for each peptide. Using the normalized dataset, comparisons between growth-promoting treated and control groups were performed and fold changes and significance P values ​​were calculated. P values ​​were calculated by performing one-tailed paired t-tests between growth-promoting treated and negative control values ​​for a given peptide. The resulting fold changes and significance values ​​were used to generate any analysis (e.g., heatmaps, hierarchical clustering, principal component analysis, pathway analysis, etc.).

[0065] The four tissues analyzed by chicken-specific kinome peptide arrays (small intestine, liver, muscle, and cecum) were run through the Integrated Intelligent Kinome Analysis 2 (PIIKA2) platform, an analytical pipeline, in two batches: one for liver and muscle, and one for cecum and small intestine (the area surrounding Meckel's diverticulum). Heatmaps of kinome signals for liver and muscle are shown in Figure 1. Each column in the figure represents the total phosphorylation signal of a tissue from the array. This is called the kinome profile or kinotype of the tissue. The connecting lines at the top of the figure represent the clustering, i.e., the relative similarity of the kinotypes between each tissue. There is a strong similarity between samples of the same tissue, regardless of the treatment or age of the bird. This is reflected by the two distinct clusters (liver and muscle) in Figure 1. This similarity is expected, since muscle and liver are very different physiologically and functionally and are expected to show different kinome profiles. Within each tissue cluster, the D14 T1 samples clustered separately from the other groups, indicating that the effect of adding algae to the diet was, overall, to make the D14 tissues more similar to the D42 tissues.

[0066] Figure 1 shows heatmaps of liver and muscle tissue kinotypes after PIIKA2 analysis. Each colored line within each column represents a peptide on the array. Red indicates a relative increase in phosphorylation, and green indicates a relative decrease. General kinotype clustering shows primarily tissue-specific clustering.

[0067] For each time-matched tissue, growth-enhancing treatments were compared to the control (T2 vs. T1) pair to remove signals that were not due to the addition of algae to the diet. This resulted in an increase or decrease in fold change in phosphorylation compared to the control for each peptide (fold change peptide X=T2 peptide X / T1 peptide X). This data was then clustered for relative similarity. Liver and muscle tissues at D14 were found to be the most similar (Figure 2). This indicates that the algal products were exerting similar signaling effects on both tissues at D14, and that the effect was likely strongest at this early time point.

[0068] Figure 2 shows the dendrograms of liver and muscle tissues (T2 vs. T1) showing the clustering of kinos types after calculation of fold changes. By removing the control corn and soybean diet and tissue related responses from the kinos types, it may be seen that the increased diets more closely match the two tissues based on growth promotion treatment and age at D14.

[0069] When considering gut tissues, particularly the small intestine (around Meckel's gut) and cecum (Figure 3), a similar pattern to Figure 1 can be seen. There is a strong similarity between samples of the same tissue, regardless of the treatment or age of the bird. This similarity is expected, as the small intestine and cecum are physiologically and functionally different and would be expected to show different kinome profiles. However, unlike liver and muscle, the T1 D14 small intestine and cecum samples cluster outside of their respective tissue clusters. This indicates that the effect of adding algae to the diet made the D14 tissues more similar to the D42 tissues overall. The clustering pattern is even more pronounced in Figure 3 than in Figure 1, as the T1 D14 tissues are in fact clustered completely away from the other samples, although there were significant differences between the two, as indicated by the length of the lines connecting the two columns.

[0070] Figure 3 shows heatmaps of kinotypes in the small intestine and cecum after PIIKA2 analysis. Each colored line within each column represents a peptide on the array. Red indicates a relative increase in phosphorylation, and green indicates a relative decrease. General kinotype clustering shows tissue-based clustering, except for the D14 T1 group.

[0071] As mentioned above, comparing growth-promoting treatments to controls (T2 vs. T1) may remove signals that are not due to the addition of algae to the diet. The data were then clustered for relative similarity. In the small intestine and cecum tissues at D14, it was found that the algal products had similar signaling effects on both tissues at D14, with the effect probably being strongest at earlier time points.

[0072] Figure 4 shows the dendrograms of small intestinal and cecal tissues (T2 vs. T1) showing the clustering of kinotypes after calculation of fold changes. By removing the control corn and soybean diet and tissue-related responses from the kinotypes, it can be seen that the enriched diet more closely matches the two tissues based on growth promotion treatment and age at D14.

[0073] Based on the above data from the four tissues, the strongest impact of dietary algal supplementation appears to occur at D14. This is especially true for intestinal tissues, as the product appears to bring the D14 intestine closer to the D42 intestine, regardless of whether the D42 intestine received the supplement (Figure 3). When comparing treatment and time-matched controls and considering only the effects of algae, the pattern of signaling changes is consistent between tissue pairs: small intestine and cecum, and liver and muscle. Figures 2 and 4 show that the D14 samples cluster separately from the D42 samples. The above data provide evidence that algal products have matured tissues at D14 such that their kinome profiles now appear similar to mature tissues at D42.

[0074] By analyzing what protein changes occurred at D14 compared to D42, we may be able to understand the kinome changes behind this clustering pattern. Proteins that showed statistically significant differences between the algal treatment and control at each tissue and each time point were entered into the STRING database to generate signaling pathways. (As used herein, the "STRING" database refers to the "Search Tool for Interacting Genes / Proteins," a biological database and web resource of known and predictable protein-protein interactions obtained from several sources, including computational prediction methods, public text collections, and experimental data.) This data can be analyzed to determine changes in biological function attributable to the algae.

[0075] In the table below:

[0076] "Proteins" refers to the number of proteins in the pathway that are differentially phosphorylated on the array, "background" is the number of proteins in the pathway, and "FDR" is the false discovery rate significance value of the pathway.

[0077] "Reactome" refers to an open-source, open-access, manually curated, peer-reviewed pathway database used to support basic and clinical research related to genomic analysis and modeling.

[0078] "MYD88" refers to the first known downstream component of TLR4 and TLR2 signaling.

[0079] "TRIF (TICAM1)-mediated TLR4 signaling" or "Toll-like receptor adaptor molecule 1" refers to a gene that encodes a protein. Activated TLR4 signaling pathway is associated with TICAM1.

[0080] "Fc epsilon receptor (FCER1)" refers to the high affinity IgE receptor for the Fc region of immunoglobulin E (IgE). It is also known as FcεRI or FcεRI.

[0081] Tables 1A and B show the top 20 pathways in small intestinal tissue that were altered by addition of algae to the diet on D14 in Table 1A) D14 and Table 1B) D42. Highlighted in bold are pathways specific to that day.

[0082] At D42, algal supplementation increased the adaptive immune response (Table 1B), possibly indicating that the system was more immunologically competent or developed by this time point. It is possible that the supplement stimulated the immune system starting from the innate system. This resulted in a more adaptively biased immune system in supplemented birds by D42. Evidence for this includes substantial changes in adaptive immune system pathways at D42 in supplemented birds, and TLR signaling at D14.

[0083] [Table 1]

[0084] [Table 2]

[0085] Tables 1A and 1B: Small intestine (adjacent to Meckel's) reactome pathways . Statistically significantly differentially phosphorylated proteins from the small intestine at D14 and D42 were entered into the STRING database to generate a list of enriched riatomical pathways. Table 1A) D14 shows the top 20 pathways from D14, and Table 1B) D42 shows the top 20 pathways from D42. Highlighted in bold are unique pathways at each time point. Much of the unique signaling at D14 is related to innate immune signaling, while signaling at D42 is related to growth and the adaptive immune system. "Protein" is the number of proteins in the pathway that are differentially phosphorylated on the array, "Background" is the number of proteins in the pathway, and "FDR" is the false discovery rate significance value for the pathway.

[0086] The same analysis above performed on liver samples shows that there are few unique signaling pathways between D14 and D42 (Tables 2A and B). The unique ones are related to growth, namely “signaling by VEGF” at D14 and “MAPK family signaling cascade” at D42. A particularly interesting difference between the two time points was that there were two innate immune signaling pathways related to TLR signaling in the top 20 list at D14, but not at D42. That does not mean that there was no innate immune signaling or TLR signaling at D42. Instead, this means that there was additional unique TLR signaling at D14. This again indicates a stronger immune stimulation by the algae at the early growth stage, possibly maturing the tissue at this early time point.

[0087] [Table 3]

[0088] [Table 4]

[0089] Tables 2A and 2B: Liver reactome pathways . Statistically significantly differentially phosphorylated proteins from liver at D14 and D42 were entered into the STRING database to generate a list of enriched riatomics pathways. Table 2A) D14 shows the top 20 pathways from D14 and Table 2B) D42 shows the top 20 pathways from D42. Highlighted in bold are unique pathways at each time point. Unique to D14 (Table 2A) D14) is a combination of growth and innate immune signaling. In Table 2B), growth at D42 represents a unique property. This may indicate initial priming and growth followed by supplementation enhancing growth-related signaling.

[0090] The differential response in muscle was functionally quite distinct between the D14 and D42 pathways (Tables 3A and B). In muscle at D42, there was a mix of growth-promoting signaling (MAPK, AKT) and innate immune signaling (TLR4 and 9) (Table 3B). At D14, innate immune signaling via TRAF6 was present, while the other two unique pathways were related to NTRK signaling (Table 3A). NTRK is a neuron-associated signaling receptor that leads to cell differentiation and MAPK-associated proliferation. This function has been shown to be related to muscle-bone formation, cell growth, and immune response to Clostridium perfringens alpha-toxin. These effects of algae on muscle have many intriguing possibilities, such as improved muscle / bone development, further muscle growth, or immune priming, all of which are seen in early muscle compared to later growth stages.

[0091] [Table 5]

[0092] [Table 6]

[0093] Tables 3A and 3B: Muscle reactome pathways . Proteins from muscle that were statistically significantly differentially phosphorylated at D14 and D42 were entered into the STRING database to generate a list of enriched riatomical pathways. Table 3A) shows the top 20 pathways from D14 and Table 3B) shows the top 20 pathways from D42. Highlighted in bold are pathways unique to each time point. NTRK signaling is shown in Table 3A) muscle at D14 and Table 3B) muscle at D42, with a mix of immune signaling and growth-related signaling. "Protein" is the number of proteins in the pathway that are differentially phosphorylated on the array, "Background" is the number of proteins in the pathway, and "FDR" is the false discovery rate significance value for the pathway.

[0094] In the cecum, there are a limited number of unique signaling pathways between D14 and D42. Signaling by NTRK was unique to D14, whereas signaling by NTRK1 appeared at both D14 and D42, and this pathway is likely related to immune-related simple cell proliferation responses. Signaling by VEGF is also unique to D14, whereas signaling by the VEGFA-VEGFR2 pathway is present at D42, a subset of this larger group of pathways, also related to cell proliferation signals. At D42, Akt signaling and TLR signaling via MyD88 are unique, representing a mix of proliferation and immune signaling that is absent at D42. Overall, however, algae do not appear to result in significant changes between D14 and D42 in the cecum compared to other tissues.

[0095] [Table 7]

[0096] [Table 8]

[0097] Tables 4A and 4B: Cecal reactome pathways . Statistically significantly differentially phosphorylated proteins from D14 and D42 cecum were entered into the STRING database to generate a list of enriched riatomical pathways. Table 4A) D14 shows the top 20 pathways from D14 and Table 4B) D42 shows the top 20 pathways from D42. Highlighted in bold are unique pathways at each time point. "Protein" is the number of proteins in the pathway that are differentially phosphorylated on the array, "Background" is the number of proteins in the pathway, and "FDR" is the false discovery rate significance value for the pathway.

[0098] Although not included in the top 20 riatom pathways, there was a widespread pathway called metabolism that showed significant changes between algae supplementation and control across all tissues and time points. Proteins that were significantly differentially phosphorylated within metabolic pathways were compared between D14 and D42 per tissue. Proteins unique to each time point were uploaded into STRING. Within the resulting protein interaction groups (Figures 5-8), there were specific macronutrient metabolic processes that were enriched. The fact that unique proteins generated overwhelmingly contiguous protein-protein interaction networks indicates that these proteins are part of interacting metabolic processes and not simply heterogeneous phosphorylation changes.

[0099] In the small intestine, the unique protein interaction group for “lipid metabolism” in the metabolic pathway was enriched at both D14 and D42 (Fig. 5). Thus, despite the difference in the response to algae at two different ages in birds, the main effect was to alter lipid metabolism in the intestine.

[0100] Figures 5A and 5B show novel metabolic riatom pathway members in the small intestine (adjacent to Meckel's duct) at D14 and D42, respectively. Proteins unique to each time point within the metabolic riatom pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "metabolism of lipids" pathway at both D14 (A) and D42 (B).

[0101] In the liver, the unique protein interaction groups for "metabolism of lipids" and "metabolism of fatty acids" in the metabolic pathways were enriched at both D14 and D42 (Figure 6A and B). Thus, despite the difference in the response to algae at two different ages, the main effect was to alter the metabolism of fats in the intestine. Since the liver is the main processing device for lipids and fats, lipid metabolism is altered in the intestine. This result is consistent with the data above.

[0102] Figures 6A and 6B show novel metabolic riatome pathway members in liver at D14 and D42, respectively. Proteins unique to each time point within the metabolic riatome pathway were entered into STRING to generate protein-protein interaction groups. For both D14 (A) and D42 (B), proteins in red are members of the "metabolism of lipids" pathway and proteins in purple are members of the "fatty acid metabolism" pathway.

[0103] In muscle, the unique protein interaction groups for "pyruvate metabolism and TCA cycle" and "carbohydrate metabolism" were enriched at both D14 and D42 within the metabolic pathways, respectively (Figure 7). Thus, the main effect of algae supplementation was to shift the TCA cycle to earlier and carbohydrate metabolism to later. As muscle is the main consumer of glucose, this is consistent with increased muscle deposition, and thus growth. This may even indicate a higher potential feed conversion ratio.

[0104] Figures 7A and 7B show novel metabolic riatome pathway members in muscle at D14 and D42, respectively. Proteins unique to each time point within metabolic riatome pathways were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "pyruvate metabolism and TCA cycle" pathway at D14 (A) and "carbohydrate metabolism" at D42 (B).

[0105] In the cecum at D14, unique protein interaction groups related to the metabolic pathway "metabolism of lipids" were enriched (Figure 7). Again, the main effect of algae supplementation is observed to alter lipid metabolism early. There were no significant enrichments in protein interaction groups or functions in the cecum at D42. Thus, most of the unique changes occurred at an early stage of metabolism. Consistent with the metabolic data of the small intestine tissue, lipid metabolism in the gut was altered by algae.

[0106] Figure 8 shows novel metabolic riatom pathway members in the cecum. Proteins unique to each time point in the metabolic riatom pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "metabolism of lipids" pathway in D14 (A). D42 did not show significant protein interaction groups and is therefore not shown.

[0107] In all tissues and time points, the innate immune system was present in the top 20 significant riatom pathways, indicating that this immune response was significantly altered between algae supplementation and control (Tables 1-4). Proteins that were significantly differentially phosphorylated within the innate immune system pathway were compared between D14 and D42 for each tissue. Proteins unique to the time point were uploaded into STRING. Within the resulting protein interaction groups (Figures 9-12), Toll-like signaling was enriched in all cases except for the cecum and liver at D42. The fact that this pathway was not present in the unique responses of these two tissues at D42 is further evidence that the main effect of algae supplementation occurs early in growth.

[0108] Figures 9A and 9B show novel innate immune riatom pathway members in the small intestine (adjacent to Meckel's duct) at D14 and D42, respectively. Proteins unique to each time point within the innate immune riatom pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "Toll-like receptor cascade" pathway at both D14 (A) and D42 (B).

[0109] Figure 10A and 10B show novel innate immune rhatome pathway members in the liver at D14 and D42, respectively. Proteins unique to each time point within the innate immune rhatome pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "Toll-like receptor cascade" pathway at D14 (A). No "Toll-like receptor cascade" pathway was found at D42 (B).

[0110] Figures 11A and 11B show novel innate immune rhatome pathway members in muscle at D14 and D42, respectively. Proteins unique to each time point within the innate immune rhatome pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "Toll-like receptor cascade" pathway at D14 (A). No "Toll-like receptor cascade" pathway was found at D42 (B).

[0111] Figure 12 shows novel innate immune riatom pathway members in the cecum. Proteins unique to each time point in the innate immune riatom pathway were entered into STRING to generate protein-protein interaction groups. Red proteins are members of the "Toll-like receptor cascade" pathway at D14 (A). No protein interaction groups could be generated at D42.

[0112] result

[0113] In general, the analysis of the results supports the conclusion that the inclusion of the innovative compound as part of the conventional diet demonstrated a significant increase in metabolism and an increase in innate immune response. Furthermore, after administration of the disclosed compound to selected birds, samples from both the composition-fed and control-fed birds were examined by gross dissection, including internal examination. Kinetic analysis of tissues taken from birds fed a mixed diet of the compound of the present invention and conventional feed and then sacrificed confirmed that biomass alters multiple growth-related pathways as proposed, thus initiating pathway regulation.

[0114] The results are summarized as follows:

[0115] Tissue kinotypes were primarily clustered by tissue type, and within those clusters the D14 control diets were differentiated.

[0116] When tissue samples were compared to time-matched controls (peptide FC=T2 / T1) and the resulting data were clustered, D14 tissues from birds supplemented with algae were clustered, regardless of tissue type.

[0117] Overall, algal products significantly alter the immunometabolism of all tissues (see pathway table above).

[0118] When considering the addition of algae, the differences between D14 and D42 can be explained by: A more innate response occurs early and an adaptive response occurs later in the small intestine. More TLR signaling in early compared with later stages of the liver Increased NTRK signaling in early muscle stages, Increased AKT- and MyD88-dependent TLR signaling at the late cecal stage.

[0119] Considering the metabolic response of specific proteins at each time point, we find that: Lipid metabolism in the small intestine -Lipid and fatty acid metabolism in the liver, Pyruvate metabolism and the TCA cycle early, carbohydrate metabolism late in muscle, · In the early stage, lipids in the cecum are metabolized, and in the later stage, no significant specific changes are observed.

[0120] Considering the innate immune response of unique proteins at each time point, at D42, the Toll-like receptor cascade appears in all tissues except the liver and cecum.

[0121] Overall, algae supplementation appears to cause tissues to mature more quickly than controls, induce an immune stimulatory response that shifts the small intestine towards a more adaptive response, induce lipid metabolism in all tissues except muscle, and induce an immune response in all tissues.

Claims

[Claim 1] 1. A method for altering signal transduction in multiple biological pathways in an animal, comprising orally administering to the animal an effective amount of a composition derived from the group consisting of a biomass or supernatant from an algae culture, a biomass consisting of gram-negative bacteria, and one or more of lipopolysaccharides, lipid A, and fractions, derivatives, and cellular components thereof from gram-negative bacteria, the method also comprising a step of initially administering an effective amount of said composition to the animal from an early stage in the animal's life.

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