Composition for enhancing the immune system

Abstract

The present invention provides a composition for enhancing the immune system, the composition comprising a keratin compound and β-lactoglobulin (LGB).

Description

Technical Field

[0001] This invention relates to the field of immunology, and more specifically to the ability of a substance to activate or enhance the immune system response. Background Technology

[0002] The immune system is a cellular and molecular apparatus with a specific function in defending against infection. There are two distinct types of responses to invading pathogens. The innate (natural) response remains consistent regardless of the number of times an infection is encountered, while the acquired (adaptive) response is enhanced upon repeated exposure to a given infection.

[0003] With the increasing variety of infectious diseases and pathogens, there is an urgent need for solutions that can enhance the immune system's ability to deal with diseases encountered by people.

[0004] Specific groups more susceptible to infection or serious consequences when exposed to harmful pathogens include infants, the elderly, individuals with compromised immune systems, animals, and athletes. Summary of the Invention

[0005] According to some exemplary embodiments, a composition for enhancing the immune system is provided, comprising a keratin compound and β-lactoglobulin (LGB).

[0006] According to some embodiments, the keratin compound may be selected from the group consisting of KRT33B, KRT13, KRT18, KRT17, KRT42, KRT28, KRT36, KRT12, KRT10, KRT24, KRT14, KRT4, KRT75, KRT6A, KRT6C, KRT5, KRT77, KRT1, KRT3, KRT2, or combinations thereof.

[0007] According to some embodiments, the concentration of the keratin compound can be from 0.01% to 15.5%, preferably from 0.01% to 10.0%, and the concentration of the LGB can be from 0.02% to 23.4%.

[0008] According to some embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0009] According to some embodiments, the anti-inflammatory component may be selected from the group comprising lactoferrin, α-lactalbumin, CD59 glycoprotein, lactoferrin, lysozyme C, interleukin-10 (IL-10), transforming growth factor β (TGF-β), interleukin-4 (IL-4) and cyclooxygenase-1 (Cox-1).

[0010] According to some embodiments, the pro-inflammatory component may be selected from the group including lactoferrin, lysozyme C, interleukin-1B (IL-1B), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α).

[0011] According to some embodiments, the antibacterial component may be selected from the group comprising β-defensin 1, lactoperoxidase, lactoferrin, α-lactalbumin, cathepsin G, lysozyme C, immunoglobulin G (IgG) and immunoglobulin A (IgA).

[0012] According to some embodiments, the first immunostimulatory component may be selected from the group comprising endoplasmin, neutrophil elastase, IgA, IgG, immunoglobulin M (IgM) and lactoferrin.

[0013] According to some embodiments, the second immunostimulatory component may be selected from the group comprising chemokine (CC motif) ligand 5 (CCL5), endoplasmin, neutrophil elastase, IgA, IgG, IgM, prolactin-inducible protein, and leukocyte elastase inhibitor.

[0014] According to some embodiments, the composition also comprises colostrum.

[0015] According to some embodiments, the composition of the present invention is provided for use in enhancing the immune system of an infant.

[0016] According to some embodiments, the composition of the present invention is provided for use in enhancing the immune system of an individual with an impaired immune system.

[0017] According to some embodiments, the composition of the present invention is provided for use in enhancing the immune system of an animal.

[0018] According to some embodiments, the composition of the present invention is provided for use in reducing inflammation in athletes.

[0019] According to some exemplary embodiments, the present invention provides a composition comprising a combination of a keratin compound, β-lactoglobulin (LGB), an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component. Attached Figure Description

[0020] Exemplary embodiments of the present invention are non-limiting and will be described below in conjunction with the accompanying drawings, which are listed after this paragraph.

[0021] The same structure, element, or part that appears in more than one figure is usually labeled with the same numbers in all the figures in which it appears.

[0022] Figure 1 A flowchart depicting the preparation process of an improved composition according to one aspect of this disclosure is shown.

[0023] Figure 2 A chart showing the advantages and disadvantages of using and / or extracting animal colostrum is presented.

[0024] Figure 3 A graph showing the deviation of protein concentration relative to the age of an infant according to some exemplary embodiments is shown.

[0025] Figure 4 The results of sample preparation of protein gels according to some exemplary embodiments are shown.

[0026] Figure 5-7 Charts G1-G6 show the homology between human colostrum and bovine colostrum.

[0027] Figure 8 The analysis of proteins according to some exemplary embodiments is shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0028] Figure 9 An anion exchange (AE) chromatogram of skimmed colostrum after acid precipitation according to some exemplary embodiments is shown.

[0029] Figure 10 A cation exchange (CE) chromatogram of skimmed colostrum after acid precipitation according to some exemplary embodiments is shown.

[0030] Figure 11 It is a graph depicting enrichment factors according to some exemplary implementations.

[0031] Figure 12 These are forward and side scatter plots of a PBMC analyzed by flow cytometry according to some exemplary embodiments.

[0032] Figure 13 It is a graph depicting T cell activation in various samples according to some exemplary embodiments.

[0033] Figure 14 It is a graph showing the secretion of IFN-γ in various samples after 72 hours according to some exemplary embodiments.

[0034] Figure 15 It is a graph depicting IL-1β secretion in different test groups according to some exemplary embodiments. Detailed Implementation

[0035] According to some exemplary embodiments, the present invention provides a composition (also referred to herein as a “formulation”) for enhancing the immune system, comprising at least one keratin compound and β-lactoglobulin (LGB).

[0036] According to some implementations, keratin is a typical intermediate filament protein of epithelium with extensive molecular diversity, while β-lactoglobulin (LGB) is the major whey protein in cow's and sheep's milk (~3 g / L) and is also present in many other mammalian species. It should be noted that one exception is humans, and unlike other major whey proteins, the exact function of β-lactoglobulin has not yet been determined.

[0037] However, according to some implementations, specific combinations of keratin compounds and β-lactoglobulin (LGB) can provide synergistic effects, such as providing synergistic effects in terms of immune stimulation.

[0038] According to some embodiments, the keratin compound may be selected from the group consisting of KRT33B, KRT13, KRT18, KRT17, KRT42, KRT28, KRT36, KRT12, KRT10, KRT24, KRT14, KRT4, KRT75, KRT6A, KRT6C, KRT5, KRT77, KRT1, KRT3, KRT2, or combinations thereof.

[0039] According to some embodiments, the concentration of the keratin compound can be from 0.01% to 15.5%, preferably from 0.01% to 10.0%, and the concentration of the LGB can be from 0.02% to 23.4%.

[0040] According to some implementations, interleukins (ILs) may be involved in most immune responses, such as inflammation, T cell proliferation, and enhanced antimicrobial responses. Keratin may be involved in different cytokine pathways and thus can be used to modulate these responses (e.g., pro-inflammatory cytokines). β-lactoglobulin (LGB) is another factor that can induce cytokine production and / or cell proliferation. Furthermore, LGB can be used as a natural analgesic and anti-inflammatory drug, and LGB hydrolysate (LGBH) may possess antioxidant, antihypertensive, antimicrobial, and opioid activities.

[0041] According to some embodiments, specific combinations of keratin and β-lactoglobulin can induce a strong pro-inflammatory response in human and / or animal monocytes. Therefore, according to some embodiments, the synergistic effect of keratin and LGB can elicit a substantial immune response.

[0042] According to some preferred embodiments, the compositions of the present invention may include more than one keratin compound.

[0043] According to some exemplary embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0044] According to some embodiments, the anti-inflammatory component may be selected from the group comprising lactoferrin, α-lactalbumin, CD59 glycoprotein, lactoferrin, lysozyme C, interleukin-10 (IL-10), transforming growth factor β (TGF-β), interleukin-4 (IL-4) and cyclooxygenase-1 (Cox-1).

[0045] According to some embodiments, the pro-inflammatory component may be selected from the group including lactoferrin, lysozyme C, interleukin-1B (IL-1B), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α).

[0046] According to some embodiments, the antibacterial component may be selected from the group comprising β-defensin 1, lactoperoxidase, lactoferrin, α-lactalbumin, cathepsin G, lysozyme C, immunoglobulin G (IgG) and immunoglobulin A (IgA).

[0047] According to some embodiments, the first immunostimulatory component may be selected from the group comprising endoplasmic protein, neutrophil elastase, IgA, IgG, immunoglobulin M (IgM) and lactoferrin.

[0048] According to some embodiments, the second immunostimulatory component may be selected from the group comprising chemokine (CC motif) ligand 5 (CCL5), endoplasmin, neutrophil elastase, IgA, IgG, IgM, prolactin-inducible protein, and leukocyte elastase inhibitor.

[0049] According to some exemplary embodiments, the compositions of the present invention can exhibit synergistic effects. For example, according to some embodiments, each component and / or molecule in the composition may have one or more immunostimulatory properties, but when anti-inflammatory components, pro-inflammatory components, antibacterial components, first immunostimulatory components, and second immunostimulatory components are combined together, a greater immunostimulatory effect is provided than the sum of the immunostimulatory effects of all components individually.

[0050] According to some embodiments, the term "synergistic effect" may refer to a specific part and / or component of the immune system that is activated to a greater extent, and / or to multiple parts and / or components of the immune system that are activated. According to some embodiments, synergistic effect may refer to the synergistic interaction between the components of the composition of the present invention, for example, causing a greater degree of immunostimulatory effect that is stronger than the immunostimulatory effect observed when the components are used alone.

[0051] According to some exemplary embodiments, the immunoglobulin used in the compositions of the present invention is preferably IgA. According to these preferred embodiments, infants are more susceptible to infections and diseases transmitted through mucous membranes, and therefore, according to some embodiments, IgA is preferred.

[0052] According to some implementations, the term "enhancing the immune system" (also referred to herein as "boosting the immune system," "immunostimulatory effect," "immunostimulation," or "enhancing immune stimulation") may include, but is not limited to, shortening the duration of illness and / or onset, reducing the likelihood of illness, reducing the number and / or severity of symptoms associated with illness, and / or activation and / or proliferation of immune cells, and / or inactivation and / or reduced activity of inflammation-related cells.

[0053] According to some exemplary embodiments, the specific use of pro-inflammatory components has achieved unexpected beneficial effects. While it is generally preferred to avoid inflammation in humans according to some embodiments, the compositions of the present invention provide beneficial immunostimulatory effects due to the use of pro-inflammatory immune components to combat pathogens.

[0054] According to some exemplary embodiments, the compositions of the present invention may further comprise one or more components of colostrum and / or whole colostrum (e.g., synthetic, human and / or animal-derived).

[0055] According to some embodiments, the composition of the present invention may contain a combination of two or more colostrums.

[0056] According to some embodiments, the composition may include a combination of two or more molecules derived from at least two different colostrums extracted from two different mammals.

[0057] According to some embodiments, the composition may preferably comprise a combination of two bovine colostrums, such as LALBA and CATHL1, to achieve an anti-inflammatory response and antibacterial protection.

[0058] LALBA is an α-lactalbumin-anti-inflammatory component that inhibits COX and phospholipase A (2) activity.

[0059] CATHL1 is an antimicrobial humoral immune response mediated by an antimicrobial (Gram-negative) peptide. It can bind lipopolysaccharide (LPS) and enhance its ability to penetrate the outer membrane of Gram-negative bacteria.

[0060] According to some implementation methods, the ratio between the two bovine colostrums, LALBA and CATHL1, can preferably be 60:40.

[0061] According to some embodiments, the composition can exhibit anti-inflammatory properties.

[0062] According to some embodiments, as described below, peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, and T cell activation and proliferation were tested based on different treatments including the use of the composition of the present invention. The results clearly showed that T cell activation and proliferation were significantly reduced in the presence of the composition of the present invention. Furthermore, interferon-γ (INFγ) secretion was significantly reduced upon exposure to the composition of the present invention. Therefore, according to some embodiments, the composition of the present invention has significant anti-inflammatory effects.

[0063] According to some embodiments, the composition of the present invention is provided for use in reducing inflammation.

[0064] For example, the compositions of the present invention can be applied to athletes to reduce, for example, pressure-induced inflammation in their muscles and / or joints. For example, according to some embodiments, the compositions of the present invention can be added to protein shakes and / or energy bars, or used independently in powder form, sachets, and / or capsules.

[0065] According to some embodiments, the compositions of the present invention are provided for use in older adults to, for example, strengthen the immune system.

[0066] According to some implementations, the primary immune response to most diseases in older adults (typically defined as 65 years and older) is inflammatory.

[0067] As we age, the human immune system weakens and its function declines. The first response of the immune system in older adults is inflammation, but this is often ineffective and depletes the body's energy. According to some embodiments, the compositions of the present invention can bind to the immune system's stimulating capacity to weaken this response, which can help the body's immune system fight disease.

[0068] According to some exemplary embodiments, the use of the compositions of the present invention for enhancing the immune system of animals, such as pets, is provided.

[0069] According to some embodiments, inflammation of the joints and muscles is common, particularly in active pets, racing animals, and working livestock. This inflammation is typically treated with rest, ointments, and, in extreme cases, physical therapy, which can be very expensive. According to some embodiments, the compositions of the present invention may have anti-inflammatory properties, thus reducing the occurrence of inflammation and shortening recovery time in animals.

[0070] According to some exemplary embodiments, the compositions of the present invention may also have a pro-inflammatory component, for example, which, when combined with an anti-inflammatory component, can exert a synergistic immune-stimulating effect.

[0071] According to some implementations, the immune response includes various factors, and the inflammatory response is crucial for recruiting numerous immune cells. Therefore, according to some implementations, in certain cases, it may be preferable to stimulate the immune system by inducing a controlled inflammatory response.

[0072] According to some embodiments, the compositions of the present invention may include, for example, a combination of SERPINB4 and SERPIND1, which can lead to a significant reduction in gastric proteolytic enzymes and / or an enhancement of pro-inflammatory pathways and stimulation of the immune system.

[0073] SERPINB4 is a negative regulator of pro-inflammatory protein-endopeptidase activity.

[0074] SERPIND1 is a protein that can stimulate the immune system and may promote the release of leukocyte chemokines.

[0075] According to some implementations, SERPIND1 can be replaced by CXCL12. CXCL12 exhibits strong chemotaxis towards lymphocytes, and its signaling regulates the expression of CD20 on B cells.

[0076] According to some embodiments, the ratio between SERPINB4 and SERPIND1 in the composition can be 60:40.

[0077] According to some embodiments, monocytes incubated in the presence of the compositions of the present invention exhibit proliferation. Furthermore, IL-10 secretion is reduced in the presence of the compositions of the present invention. According to some embodiments, the compositions of the present invention can induce a pro-inflammatory response by stimulating the immune system.

[0078] According to some implementations, short-term inflammation is a natural bodily response to many diseases. For example, it is most commonly used to fight infection.

[0079] According to some embodiments, the composition of the present invention may include more than one anti-inflammatory component. For example, the composition may include the following components: ANXA1, APOE, BTN1A1, C4BPA, CD59, FCGR2, HBB, LALBA, LTF, PGLYRP1, PRDX4, SERPINB1, TNFRSF6B, LGB, KRT18, KRT17, KRT42, KRT36, KRT10, KRT24, KRT14, KRT75, KRT6A, KRT5, KRT1, KRT3 and KRT2.

[0080] According to some exemplary embodiments, the compositions of the present invention may have controlled pro-inflammatory activity, which may be helpful in such cases.

[0081] According to some embodiments, preferably, the concentration of the pro-inflammatory composition is 100 pg / kg to 100 ng / kg.

[0082] According to some implementations, bacteria are common pathogens that can directly or indirectly cause various diseases. Normally, the immune system can handle these risks, but in many cases, the immune system cannot defeat bacteria.

[0083] According to some embodiments, the compositions of the present invention may have antibacterial activity.

[0084] According to some embodiments, the compositions of the present invention may include a combination of HSTN and C3, which, for example, can produce a potent antibacterial response through an immune response along with enhanced phagocytosis.

[0085] HSTN is an antimicrobial protein—a cationic peptide involved in innate immunity and possessing antimicrobial and antifungal activity.

[0086] C3 is complement component 3, which plays a key role in the complement system and stimulates innate immunity.

[0087] According to some embodiments, the ratio between HSTN and C3 in the composition can be 80:20.

[0088] According to some embodiments, the antibacterial activity of the compositions of the present invention may be particularly beneficial to the elderly and immunocompromised individuals, helping to resist pathogens.

[0089] According to some embodiments, applying the composition of the present invention can target intestinal immunity, enhance intestinal flora and increase immune response against pathogens, and can also target blood flow and enhance the immune system.

[0090] According to some implementations, low doses of the composition of the present invention can also be administered to a large population when there is a risk of bacteria and / or viruses, such as during winter.

[0091] According to some implementations, the immune system plays a vital role in preventing disease and stress. Therefore, its proper functioning is essential. However, in many cases, especially in young children and the elderly, the immune system requires some strengthening and maintenance.

[0092] According to some embodiments, the compositions of the present invention comprise an immunostimulatory component.

[0093] According to some embodiments, the compositions of the present invention may include, for example, a combination of PDIA3 and LBP, which can lead to activation of the immune system and minimize inflammatory responses.

[0094] PDIA3 is an important factor in stimulating the immune system. PDIA3 is part of the major histocompatibility complex (MHC) class I peptide loading complex, a system for the formation and presentation of the final antigen conformation.

[0095] LBP is a lipopolysaccharide-binding protein (LBP) with pro-inflammatory effects. Leukocyte chemotaxis participates in inflammatory responses and macrophage activation through LBP transport, thereby inducing an immune response.

[0096] According to some embodiments, cells cultured using the composition of the present invention containing PDIA3 and LBP have been found to have a stimulatory effect on monocytes.

[0097] According to some embodiments, the ratio between PDIA3 and LBP in the composition can be 70:30.

[0098] According to some embodiments, older adults and immunocompromised individuals tend to have a diminished / delayed response to most diseases. According to some embodiments, using the compositions of the present invention can shorten response time and increase response efficacy, thereby reducing the frequency of disease occurrence in the user.

[0099] According to some embodiments, professional athlete subjects undergo rigorous training under various weather conditions with very short rest periods. This stress on the body can reduce the effectiveness of the immune system and expose the body to various diseases. According to some embodiments, using the composition of the present invention on athletes can help eliminate the adverse effects of athlete training, thereby reducing the frequency of illness suffered by the user.

[0100] According to some exemplary embodiments, the present invention provides an algorithm for predicting molecules constituting the compositions of the present invention, such as molecules for anti-inflammatory components, pro-inflammatory components, antibacterial components, first immunostimulatory components, and second immunostimulatory components.

[0101] According to some embodiments, the term "algorithm" as used herein can refer to a method for calculating the probability of an immunostimulatory effect of one or more proteins constituting the compositions of the present invention. For example, the algorithm may include assessing the probability of an effective immunostimulatory effect of one or more human proteins.

[0102] According to some implementations, the algorithm may include assessing the probability of an effective immune stimulation effect from combinations of two or more proteins.

[0103] Specifically, the algorithm can calculate the compatibility level of two or more proteins, for example, based on which "compatibility" involves enhanced and / or synergistic immune stimulation when two or more proteins are combined.

[0104] According to some implementations, for example, the algorithm may include assessing the probability of an effective immunostimulatory effect of a protein based, for example, on a comparison of the homology levels of proteins that have immunostimulatory effects in animals.

[0105] Table 1 below shows exemplary comparisons between specific proteins, represented by e-values.

[0106] According to some implementation methods, the lower the value, the better the compatibility of the two proteins being compared.

[0107]

[0108] Table 1

[0109] According to some embodiments, the compositions of the present invention specifically use selected portions and / or regions of the IgG component.

[0110] According to some implementation methods, using selected portions and / or regions of the IgG component can enhance molecular accessibility, for example, during feeding of infants or newborns.

[0111] According to some implementations, the term "molecular accessibility" may refer to the digestion of specific active regions of a molecule that can be administered orally to an infant without and / or avoids the breakdown of the molecule in the digestive tract, and increases the permeability of the molecule.

[0112] According to some implementations, selected portions and / or regions of the IgG component can be absorbed before entering the intestine, which can improve the effectiveness of these molecules, i.e., help enhance the immune system (small molecules are absorbed more quickly and therefore can begin to work faster in the body).

[0113] According to some exemplary embodiments, the composition can be used orally and / or administered intravenously or subcutaneously.

[0114] According to some other embodiments, the compositions of the present invention can be used for cosmetic purposes and can therefore be applied topically, for example, in the form of creams, ointments, etc.

[0115] Use a composition to enhance the infant's immune system

[0116] Breast milk is the milk produced by the breasts (or mammary glands) of human women to feed infants. Approximately 40% of infants are exclusively breastfed, while more than 50% are fed a combination of breast milk and formula.

[0117] The various health benefits of breastfeeding have long been known. Among the greatest are nutritional and immune benefits. Breast milk is the primary source of nutrition for newborns before they are able to eat and digest other foods; older infants and toddlers can continue to be breastfed, either exclusively or in combination with other foods, such as solid foods that may be introduced around six months of age. Furthermore, breast milk is an important source of immunoglobulins (antibodies), proteins found in the blood that provide immune defense against infectious agents such as viruses and bacteria. Some types of antibodies (primarily sIgA, which functions to prevent pathogens from entering through mucous tissues) are transferred from plasma or maternal blood into breast milk, or partially produced in the mammary glands by cells that have migrated to that area, forming the primary immune defense mechanism in breastfed infants.

[0118] Infant formula can be provided when breastfeeding is not possible or not required. Infant formula is a processed food designed and marketed for feeding infants and young children. It is typically prepared as a powder (mixed with water) or a liquid (with or without added water) and fed via bottle or cup.

[0119] Today, formula is based on different stages, with babies moving from one stage to another according to their age. Examples include 1-6 months, 6-12 months and older. These stages are defined based on averages, without specifically measuring the baby's needs.

[0120] The baby's development is monitored by using growth curves and, if any abnormalities are found, by conducting specific tests (such as blood tests).

[0121] Because an infant's immune system is not fully developed, they are more susceptible to pathogens. While some immunoglobulins are indeed passed to the infant through the umbilical cord, their levels typically decrease within the first six months.

[0122] Therefore, breast milk provides immune components that protect infants from many diseases. However, insufficient nutrition or malnutrition can render breast milk incomplete and may reduce its protective immune function. Furthermore, modern lifestyles and varying conditions prevent many women from exclusively breastfeeding. Consequently, most infants are fed formula lacking immune components and are exposed to pathogens.

[0123] However, different babies have different nutritional and / or immune needs, and generic formulas, which are usually provided based on average needs, often cannot meet the specific needs of individual babies.

[0124] Specifically, newborns are highly susceptible to various bacterial or viral infections, and current infant formula cannot provide a solution for their fragile immune systems.

[0125] According to some exemplary embodiments, the compositions of the present invention can be adapted for oral feeding of infants.

[0126] According to some implementations, this composition can enhance an infant's immune system.

[0127] According to some exemplary embodiments, the keratin compound, β-lactoglobulin (LGB), anti-inflammatory component, pro-inflammatory component, antibacterial component, first immunostimulatory component and second immunostimulatory component are different molecules.

[0128] According to some exemplary embodiments, the compositions of the present invention can exhibit synergistic effects. For example, according to some embodiments, each component and / or molecule in the composition may have one or more immunostimulatory properties, but when anti-inflammatory components, pro-inflammatory components, antibacterial components, first immunostimulatory components, and second immunostimulatory components are combined together, a greater immunostimulatory effect is provided than the sum of the immunostimulatory effects of all components individually.

[0129] According to some embodiments, the present invention provides a method for producing the composition of the present invention, the composition comprising one or more compositional components possibly extracted from a variety of colostrums, the method comprising:

[0130] Colostrum is collected from multiple individuals, where the content and / or activity of various colostrum components differ significantly among the colostrums; colostrum is collected, colostrum is filtered, or collected colostrum is deposited.

[0131] The embodiments described below provide modified compositions for feeding infants. Other embodiments describe compositions suitable for consumption by other populations.

[0132] This article also provides methods for preparing such compositions.

[0133] According to one aspect of the embodiments, the composition comprising at least one component can be extracted from one or more colostrums, wherein the content and / or activity of the component in the one or more colostrums differ significantly between colostrums.

[0134] According to another aspect of the embodiments, a method for producing a composition comprising at least one colostrum component extracted from a plurality of colostrums is provided, the method comprising:

[0135] Colostrum is collected from multiple individuals, such as different cows, sheep, or goats, or combinations thereof, where the content and / or activity of various components in the colostrum differ significantly between colostrums; colostrum is collected; colostrum is filtered or collected colostrum is collected.

[0136] Optionally, the method may also include altering the content of bioactive components in colostrum, for example, by using separation techniques on colostrum or pretreated colostrum.

[0137] According to some embodiments, the method may be selected from chromatography and / or filtration. Preparative chromatography may be selected from one or more of the following: affinity chromatography, size exclusion chromatography, and ion chromatography. Filtration may be selected from one or more of cross-filtration, ultrafiltration, reverse osmosis, and dialysis. Other methods may be used depending on the components in the final formulation and their respective desired concentrations.

[0138] In this discussion, unless otherwise indicated, adjectives modifying the conditional or relational characteristics of one or more features of an embodiment of the invention, such as “a great deal” and “about”, should be understood to mean limiting the condition or feature to an operationally acceptable tolerance for the intended application of the embodiment.

[0139] According to some exemplary embodiments, as described herein, in addition to keratin and LGB, the compositions of the present invention may comprise a combination of five components: an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0140] According to some exemplary embodiments, the compositions of the present invention may include a specific combination of anti-inflammatory components, pro-inflammatory components, antibacterial components, a first immunostimulatory component, and a second immunostimulatory component, for example, specifically targeting diseases that are susceptible to infection in infants, such as ear infections, meningitis, etc.

[0141] According to some implementations, cytokines function locally and systematically in the infant's immune system to activate, maintain, and eliminate inflammatory responses.

[0142] According to some embodiments, the interaction between pro-inflammatory cytokines, anti-inflammatory cytokines, and innate cytokine inhibitors can determine the inflammatory response and its effectiveness. According to some embodiments, cytokines are specific due to the immaturity of the neonatal immune system. According to some embodiments, tumor necrosis factor-(TNF-α) and interleukin-6 (IL-6) are preferably used to enhance the immune response by activating the cytokine cascade and producing other pro-inflammatory cytokines and chemokines.

[0143] According to some implementations, pro-inflammatory molecules can also recruit MAST cells and the complement system, thereby further enhancing immune stimulation, for example, by enhancing the attack on pathogens.

[0144] According to some embodiments, the compositions of the present invention may contain a variety of pro-inflammatory molecules.

[0145] According to some exemplary embodiments, the present invention provides an algorithm for predicting one or more beneficial combinations of molecules constituting the compositions of the present invention, such as molecules for anti-inflammatory components, pro-inflammatory components, antibacterial components, first immunostimulatory components, and second immunostimulatory components.

[0146] According to some embodiments, the term "algorithm" as used herein can refer to a method for calculating the probability of an immunostimulatory effect of one or more proteins constituting the compositions of the present invention. For example, the algorithm may include assessing the probability of an effective immunostimulatory effect of one or more human proteins.

[0147] According to some implementations, the algorithm may include assessing the probability of an effective immune stimulation effect from combinations of two or more proteins.

[0148] Specifically, the algorithm can calculate the compatibility level of two or more proteins, for example, based on which "compatibility" involves enhanced and / or synergistic immune stimulation when two or more proteins are combined.

[0149] According to some implementations, for example, the algorithm may include assessing the probability of a protein’s effective immunostimulatory effect, for example, based on a comparison of homology levels of proteins that have immunostimulatory effects in animals.

[0150] Table 1 above shows exemplary comparisons between specific proteins, represented by e-values.

[0151] According to some implementation methods, the lower the value, the better the compatibility of the two proteins being compared.

[0152] According to some embodiments, the compositions of the present invention specifically use selected portions and / or regions of the IgG component.

[0153] According to some implementations, using selected portions and / or regions of the IgG component can, for example, enhance molecular accessibility during ingestion by infants or newborns.

[0154] According to some implementations, the term "molecular accessibility" may refer to the digestion of specific active regions of a molecule that can be orally administered to an infant without and / or avoids the breakdown of the molecule in the digestive tract, while increasing the permeability of the molecule.

[0155] According to some implementations, selected portions and / or regions of the IgG component can be absorbed before entering the intestine, which can enhance the effectiveness of these molecules—helping to boost the immune system (small molecules are absorbed more quickly and therefore can begin to work faster in the body).

[0156] According to some embodiments, the compositions of the present invention include one or more immune components that can help newborns fight pathogens and improve the development of the immune system.

[0157] Immunoglobulins are crucial components of the immune system, acting both directly against pathogens and recruiting the immune system to fight them. However, most orally ingested immunoglobulins are degraded in the digestive system. Infants' digestive systems are not fully developed, allowing many immunoglobulins to remain intact. Furthermore, some immunoglobulins can be absorbed even in the mouth.

[0158] In addition, there are many immunoglobulin moieties, especially those from the highly potent IgG variable region. These moieties are small and therefore exhibit "inertness" to enzymatic proteolytic activity. Therefore, using them as an immune component in the compositions of the present invention can be significantly advantageous.

[0159] In the field of biochemistry, Michaelis–Menten kinetics is one of the most well-known enzyme kinetic models. In 1925, George Briggs and J.B. Haldane provided the best derivation of the Michaelis–Menten equations, as follows:

[0160]

[0161] Where S is the substrate, E is the enzyme, ES is the enzyme-substrate complex, P is the product, and k is the enzyme-substrate complex. 开 It is the bimolecular association rate constant of enzyme-substrate binding; k 关 It is the single-molecule rate constant for the dissociation of the ES complex to regenerate free enzyme and substrate; k 催化 It is the unimolecular rate constant for the dissociation of the ES complex to obtain the free enzyme and product P.

[0162] According to some implementations, once pepsin in an infant's digestive system interacts with its substrate (e.g., an antibody), an ES complex is immediately formed and the reaction proceeds toward a product, such as an active fragment of an antibody.

[0163] According to some embodiments, the compositions of the present invention comprise an enzymatically digested portion of an antibody, such as an IgG antibody, which leads to the generation of an equation-reversing product, such as an active fragment of an antibody.

[0164] According to some implementations, active fragments of antibodies that reach the bloodstream and target sites (such as areas of infection in an infant) will rapidly stimulate and activate the immune system, including, for example, synergistic effects triggered by the combination of these fragments with other immune stimulating components.

[0165] For example, the immune system comprises different components, such as antigen-presenting cells (e.g., dendritic cells), recruited cells (e.g., CD4 cells), and active cells (e.g., NK cells). These different components work together to launch an effective immune attack. According to some embodiments, activating different aspects of the immune system, for example, by using the compositions of the present invention, can provide significant value in combating pathogens and stimulating the immune system.

[0166] According to some embodiments, the compositions of the present invention may include a variety of molecules to treat and / or activate different components of the immune system, for example, to achieve the desired effect of enhanced immune stimulation.

[0167] According to some implementations, certain components of the immune system can be activated individually, but can produce a greater effect when they work together (synergistic effect). For example, lysozyme can engulf pathogens, but when pro-inflammatory cytokines are added, lysozyme also recruits other cells, such as dendritic cells, which in turn can enhance phagocytosis and recruit NK and neutrophils to the area to further destroy pathogens.

[0168] According to some exemplary embodiments, Table 1 below describes the possible concentrations of the components of the compositions of the present invention.

[0169]

[0170] Table 2

[0171] According to some exemplary embodiments, the compositions of the present invention can be used, for example, to enhance the immune system of an infant by providing an immune-stimulating effect.

[0172] According to some embodiments, the present invention provides an immune-enhancing infant formula that can stimulate the infant's immune system and provide better protection, such as by strengthening the immune mechanism and stimulating the immune system. According to some embodiments, the formula may also contain key amino acids and fatty acids, as well as growth and appetite regulators, such as providing the infant with the beneficial nutrients needed for cognitive growth and development by ensuring optimal intake of amino acids and fatty acids, thereby supporting organ and brain development.

[0173] According to some implementations, the formula can protect the baby's natural gut microbiota and provide comprehensive nutrition, thereby making the baby healthier and happier, for example by reducing flatulence, protecting natural flora, improving sleep, and improving the baby's comfort.

[0174] According to some exemplary embodiments, the compositions of the present invention can be in any suitable state and / or form, including, for example, liquid, powder, granules, etc.

[0175] According to some embodiments, the composition of the present invention may include two or more molecules extracted from at least two different colostrums, for example, a first molecule extracted from a first colostrum and a second molecule extracted from a second colostrum.

[0176] Based on these embodiments, a method for producing the composition of the present invention is also provided herein.

[0177] According to some implementations, the method may include collecting components from the collected colostrum and optionally adding components collected from non-colostrum sources.

[0178] According to some implementations, the method may include:

[0179] 1. Determine the composition of the original breast milk. This process involves determining the average composition of breast milk based on the newborn's developmental stage. Because the composition of breast milk changes as the infant develops, and also varies from mother to mother due to genetic, environmental, and nutritional differences, this process typically involves collecting and analyzing breast milk composition from several mothers at different times after delivery. This determination can be performed using various analytical methods.

[0180] According to some implementation methods, mass spectrometry (MS) can be used to determine the structure of each component. Optionally, one or more specialized MS techniques, such as HPLC-MS (high performance liquid chromatography-MS), electrospray ionization (ESI), time-of-flight MS, and matrix-assisted laser desorption / ionization (MALDI), can be used in combination.

[0181] 2. Provide one or more components commonly found in commercially available formulations, such as various minerals and vitamins A, D, E and K, vitamin C, riboflavin, niacin and / or valeric acid, etc.

[0182] 3. Provide one or more ingredients that are not commonly found in commercially available formulas, but are similar to those found in breast milk, including, for example:

[0183] a. Immune system enhancers. Immunogenic components, such as IgA and various cytokines. Immunogenic components are naturally present in the mucous membranes (respiratory and digestive systems) of infants and form the first immune barrier between the infant's body and pathogens in the environment. Depending on certain aspects of the implementation, these components are typically obtained from colostrum.

[0184] b. Promoters of overall infant development and growth, blood glucose balance, and temperature regulation, such as hormones and growth factors: thyroid hormones, insulin, and growth hormone. Depending on some aspects of the implementation, these components are also typically obtained from colostrum.

[0185] c. Hormones that promote brain development and / or regulate appetite, such as omega-3 unsaturated fatty acids, cannabinoids, ghrelin, and / or leptin. Depending on some aspects of the implementation, these components may be derived from natural substances or synthesized, such as the appetite regulator hexarelin.

[0186] d. Intracellular lipid level reducers and anti-inflammatory agents, such as adiponectin. Depending on some aspects of the implementation, these components are also typically obtained from the collected colostrum and / or milk.

[0187] e. Appropriate activity of the digestive system in digesting the fats, proteins, and carbohydrates naturally present in milk, and promoters or enhancers for preventing indigestion. Such promoters can be various enzymes. Depending on certain aspects of the embodiments, these components are obtained from natural substances.

[0188] f. Virus and bacterial growth inhibitors, such as lactoferrin, which bind to iron and enhance its uptake in cells, thereby inhibiting bacterial growth by preventing bacteria from taking up the crucial element of iron. Depending on certain aspects of the implementation, these components are obtained from natural substances, possibly collected colostrum.

[0189] g. Lactose, to enhance calcium absorption and promote the growth of beneficial bacteria. Lactose is used to fight pathogens and reduce dental plaque. Depending on certain aspects of the implementation, lactose is obtained from natural substances or synthesized.

[0190] h. Gene mutation preventive agents. For example, Hamlet protein used to combat cancer cell growth. Depending on certain aspects of the implementation, these components are typically obtained from collected colostrum.

[0191] According to some implementation methods, components a), b), d), f), and / or h) may optionally be combined with one or more components commonly found in commercially available formulations to form components in our modified formulation. Optionally, components c), e), and / or g) may also be added as components in the modified formulation.

[0192] 4. Testing the modified infant formula. The potency of formulas made from aggregated colostrum is first tested on a protein printer (microarray). Subsequently, these formulas can be tested in human cell lines and / or animals.

[0193] According to some implementations, the printer can check the activity of non-human molecules or human antigens in a human-like matrix.

[0194] According to some embodiments, the printer may include chips composed of support surfaces, such as glass slides, nitrocellulose membranes, beads, or microtiter plates, to which an array of captured proteins is bound. Probe molecules, typically labeled with fluorescent dyes, are added to the array. Any reaction between the probes and the immobilized proteins emits a fluorescent signal for reading by a laser scanner.

[0195] According to some implementation methods, further testing can be performed to determine whether antibody-antigen activation is correct.

[0196] refer to Figure 1 More specifically, an improved method for producing a composition according to one aspect of an implementation method is described. The process includes:

[0197] Whey and colostrum are added to a bioreactor equipped with a homogenizer;

[0198] This homogenizes the whey and colostrum into a roughly homogeneous mixture.

[0199] Cross-flow filtration or tangential flow filtration (TFF): For example, passing a mixture through a food-grade certified stainless steel piping system lined with ceramic filters at low temperatures (i.e., not exceeding human body temperature).

[0200] Filtration is performed to remove excess fat from the mixture. The residue from the filtration step is a filtered liquid substance rich in protein and has high nutritional value.

[0201] The residue is passed through a spray dryer, heated externally by steam, and then freeze-dried.

[0202] Freeze-dried powders and common nutrients in commercially available formulations, such as vitamins, minerals, starch, and lactose, can be added and mixed using a Y-shaped cone, and the resulting mixture can be granulated.

[0203] It can test the efficacy of granular powders, and can collect additional samples to test stability and microbial growth.

[0204] In some embodiments, a suspension is provided. For example, in some embodiments, it can be provided to the user in the form of a ready-to-drink milkshake, or as a powder that is easily suspended in various liquids, such as water, juice, or commercially available milk or yogurt. In some preferred embodiments, the user is not exposed to temperatures above body temperature, i.e., a maximum of 40°C, more preferably not exceeding 37°C. In these embodiments, the preparation operations are also preferably carried out at this temperature.

[0205] In other aspects of the implementation, the formulation is in the form of capsules or syrup.

[0206] Cross-reactivity may exist between non-human immunogenic components and human immunogenic components. Therefore, another aspect of the implementation involves providing conditions that are optimal or at least advantageous for high cross-reactivity between immune components of non-human colostrum and human colostrum or milk, for example, by selecting the most suitable components in this respect and excluding less suitable components.

[0207] As a further narrowing of options, this selection may include comparing the responses of human antibodies from different (typically 2-4) manufacturers to the intended component, either based on literature (if available) or through our own experiments. Another initial indicator is the degree of homology between the human component and the intended non-human component.

[0208] Based on an initial, imprecise but reasonable hypothesis (i.e., the high reactivity of human antibodies is an indicator of the high cross-reactivity of the expected components), searches and / or experiments can be conducted. Actual experiments can then be performed to confirm the hypothesis, such as for human cell lines.

[0209] According to some implementation methods, this article provides a method for extracting colostrum from animals.

[0210] According to some implementations, the method may include:

[0211] physical methods

[0212] According to some implementations, physical methods may include cleaning methods that can be used to concentrate and remove impurities by separating colostrum into multiple fractions and discarding fractions containing unwanted components.

[0213] According to some implementations, this physical method does not expose the colostrum to components other than the colostrum itself, and its composition remains substantially unchanged.

[0214] According to some implementation methods, in order to remove all unwanted molecules from colostrum, it is necessary to know the molecular size, molecular weight and properties of these components, and select appropriate cleaning methods accordingly.

[0215] According to some implementation methods, if two components are similar in size and physical properties, but one component is desired while the other is not, impurity removal may be problematic.

[0216] According to some embodiments, physical methods can be used to employ electrophoresis, which can be used to separate molecules in solution by size. This method can be used to remove impurities and retain only the desired portion of the colostrum, simply by knowing the molecular weight of each desired or unwanted component in the colostrum.

[0217] According to some embodiments, physical methods can be used to employ dialysis, which can be used to separate molecules from solution by the diffusion rate of molecules across a semipermeable membrane. It is most commonly used to remove small molecules.

[0218] According to some implementations, the physical method can use centrifugation, which can be used to separate a solution into multiple fractions based on molecular size, molecular weight, and density.

[0219] According to some implementations, physical methods may employ ion chromatography, which, according to some implementations, can be used to separate ion exchangers based on the affinity of charged molecules for them.

[0220] According to some implementation methods, physical methods may include, but are not limited to, electrophoresis, dialysis, centrifugation, and ion chromatography.

[0221] Chemical methods:

[0222] According to some implementations, chemical methods may include specific cleaning methods that separate desired molecules from the rest of the colostrum. These methods are more "invasive," meaning that external components are introduced into the colostrum to facilitate the separation of the desired molecules from the whole. Therefore, these methods may lead to more complex conditioning processes, and the purified molecules are no longer considered colostrum in the strict sense.

[0223] According to some implementation methods, in order to apply these methods, it is necessary to know the chemical composition or at least one chemical interaction of each specific target molecule before applying the appropriate method.

[0224] According to some implementations, these methods require additional steps to ensure that any additional components introduced into the colostrum for the separation of target molecules are completely removed from the final product.

[0225] According to some implementations, the advantage of these methods is that they target only the "desired" molecules, producing a final product that should contain only the components we need selected from the colostrum.

[0226] According to some embodiments, the chemical method can employ immunoprecipitation, which can be used to separate antigens from solution by binding the antigen with an antibody corresponding to the antigen.

[0227] According to some embodiments, chemical methods may employ enzyme-based separation methods, which may utilize specific substrate-enzyme interactions to separate target molecules, for example, by binding the target molecule to a surface.

[0228] According to some embodiments, chemical methods can employ chromatography, which can be used to separate molecules from a solution by exposing the solution to a surface having a binder of some form that utilizes specific properties of the target molecule.

[0229] refer to Figure 2 It shows a chart illustrating the advantages and disadvantages of physical and chemical methods.

[0230] According to some implementation methods, chemical methods may include, but are not limited to, immunoprecipitation, enzyme-based separation methods, and chromatography (HPLC).

[0231] According to one aspect of the embodiments described in detail below, a composition comprising components extracted from a variety of colostrums is provided. The variety of colostrums may contain components with significantly different contents and / or activities.

[0232] According to some implementations, the terms "individual" and / or "multiple individuals" may refer to any suitable mammal that can obtain colostrum, including, for example, humans, bison, domestic cattle (e.g., dairy cows), goats, sheep; horses, camels, wild boars, buffalo, yaks, domestic pigs, reindeer, llamas, dogs, alpacas, etc.

[0233] According to some implementation methods, colostrum can be collected and aggregated from multiple non-human sources. The aggregated colostrum can then be processed to produce infant formula suitable for human infants.

[0234] According to some alternative implementations, the colostrum of the first individual, or the colostrum of several individuals in a first group with similar content and / or activity of colostrum components, such as colostrum from several selected cows on a farm, is processed, and then the processed product is mixed with other processed colostrum from a second individual or other individuals in a second group, whose colostrum components have similar content and / or activity but are different from the colostrum of the first group.

[0235] The treatment may include removing selected components, for example by passing pre-processed or unprocessed colostrum through a pre-affinity column, or by reacting selected components to alter their activity, depending on the concentration of the component compared to its expected concentration or activity in the milk.

[0236] According to one aspect of the embodiments, products are provided that are infant formulas made from a variety of aggregated colostrums and contain nutrients with immunogenic molecules. Some embodiments include additional ingredients, for example, to promote infant growth and development, to enhance the infant's health and strength. Some embodiments may constitute infant formulas having a composition similar to human breast milk.

[0237] In particular, the implementation includes at least one cytokine and at least one antibody, such as IgA (immunoglobulin A), to provide immune protection for newborns.

[0238] In alternative implementations, the formulation may also be derived from non-bison, non-goat, and non-sheep colostrum as the sole or additional source of colostrum; for example, the colostrum source could be dogs. Testing on dogs has shown that most interleukins exhibit higher homology and cross-reactivity with human colostrum than any of the aforementioned bovine sources.

[0239] As briefly described above, some components can be removed and / or modified to enhance or reduce their immunogenicity. In particular, these components can be toll-like receptors that recognize exogenous substances and transmit appropriate signals to killer cells of the immune system, such as TLR-2 and TLR-4 ligands present in colostrum, or apolipoprotein E (ApoE), a major cholesterol carrier that supports lipid transport and inhibits tumor necrosis factor-α (TNF-α), thereby increasing immunogenicity and overall efficacy.

[0240] According to some implementation methods, molecules derived from bovine colostrum may elicit allergic and / or unintended immune responses when administered to humans. According to some implementation methods, allergic and / or unintended immune responses can be reduced and / or methylated, encapsulated, or bound to salt molecules, etc.

[0241] According to another aspect of the embodiments, the composition of the present invention may additionally include one or more components selected from any group thereof, for example, to further provide appropriate nutrition for the infant:

[0242] Pseudovitamin-inositol

[0243] Vitamins – Niacin (B3), Pantothenic Acid (B5), Pyridoxal, Pyridoxamine, Pyridoxine (B6), Retinol (A1), Riboflavin (B2), Biotin, Choline, Cobalamin (B12), Fluorine, Folic Acid, Thiamine, Tocopherol, Vitamin A, Vitamin B1 (Thiamine), Vitamin B12, Vitamin B2 (Riboflavin), Vitamin B3 (Niacin), Vitamin B5 (Pantothenic Acid). Vitamin B6, Vitamin B7 (Biotin), Vitamin C, Vitamin D, Vitamin D metabolites, Vitamin D-binding protein, Vitamin E, Vitamin E (α-tocopherol), Vitamin K.

[0244] Peptide hormones - insulin, prolactin,

[0245] Protein subunit - integrin α m,

[0246] Peptide-pre-activating polypeptide,

[0247] Proteins - integrin β-2, interferon α, interferon β, interferon γ, lactoglucosin, whey protein, lactoferrin, lactoferrin, leucine zipper EF-hand domain transmembrane protein 1, leucine-rich α-2 glycoprotein-1, LIM and SH3 domain protein 1, lipopolysaccharide-binding protein, pancreatic stone protein, low affinity immunoglobulin γ Fc receptor II, lymphocyte lysin 1 (L-reticulin), lymphocyte-specific protein 1, macrophage chemokine 1, macrophage inflammatory protein 1α, macrophage capping protein, Matr3 protein, Mgc165862 protein, Mip-1β (also known as macrophage inflammatory protein-1β), membrane spike protein, monocyte chemokine 1, mucin, myosin light chain polypeptide 6, myosin regulatory light chain polypeptide 9, cardamomylated alanine-rich kinase C substrate, neutrophil lysin 2, nucleotide exchange factor sil1, odorant-binding protein-like protein, Olfm4 protein, osteoclast-stimulating factor 1, osteopontin, Pcyox1 protein, Pdia6 protein, peptidoglycan recognition protein, peptidylprolyl cis-trans isomerases a & b, peroxidase 1, peroxidase 4, peroxidase 5, mitochondria, phosphocarrier protein, mitochondria, pigment epithelium-derived factor, polyimmunoglobulin receptor Polypyrimidine bundle-binding protein 1, Pp1201 protein, inhibitor protein 1, antiproliferative protein, antiproliferative protein 2, proteasome subunit β-2, protein os-9, protein s100-a12, protein s100-a4, protein s100-a9, protein lipoprotein 2, P-selectin, putative unidentified protein mgc137211, Qsox1 protein, Rab14 protein, Ras-associated protein rab-1b, Ras-associated protein rab-21, Ras-associated protein rab-5c, Ras-associated protein rab-7a, Ras-associated protein rap-1b, receptor expression enhancer protein 5, resistin, retinol-binding protein 4, RNase2 protein, Rpn1 protein, protein 1 containing SAM and HD domains, Scamp2 protein, Scgb2a2 protein, secretory globin, family 1d, member 2, serum transferrin, Serpin a3-1, Serpina3-3 (endopin)1b) Serpina3-5, Serpina3-6, Serpina3-8, Serpinb4 protein, Serpind1 protein, serum albumin, SH3 domain glutamate-enriched protein 3, S100-like calcium-binding protein a11 (S100a11 protein) (fragment), Slc3a2 protein, solute carrier family 3, Sparc / osteoconnector protein, cwcv and kazal-like domain proteoglycan (testis proteoglycan) 1, splicing factor 3 subunit 1, Sqrdl protein, Stat1 protein, Stefin-c, cystatin-b (stefin-b), cstb protein, Stom protein, Stomatin-like protein 2, 14-3-3 β / α protein, 14-3-3 epsilon protein, 14-3-3 γ protein, 14-3-3 theta protein, 14-3-3 Zeta / delta protein, 15kDa selenoprotein, A2m protein, actin, cytoplasmic protein 1, 2, actin-related protein 2, actin-related protein 2 / 3 complex subunit 1b, actin-related protein 2 / 3 complex subunit 2, actin-related protein 2 / 3 complex subunit 5, actin-related protein 3, actin, α-cardiac protein 1, ADAM10, adenylate cyclase-related protein 1, adiponectin, lipophilic protein, microfilament-cleaving protein, α-1-acidic glycoprotein, α-1-antichymotrypsin, α-1-antitrypsin, α-1b-glycoprotein, α-2-macroglobulin, α-2-antifibrinolytic enzyme, α-2-hs-glycoprotein, α-actin-1, α-actin-4, α-lactalbumin, α-lactoglobulin, amyloid a, blood Angiopoietin-1, angiopoietin-associated protein 4, angiotensinogen (serine protease inhibitor, branch a, member 8), annexin α1, annexin α2, annexin α3, annexin α5, annexin α6, annexin α7, antithrombin III, apolipoprotein AI, apolipoprotein A-IV, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, B12-binding protein, integrin B4α6, integrin B5α, integrin B6α, integrin B7α4 / lpam-1, integrin B8α, B cell receptor-associated protein 31, β-2-microglobulin, β-cytokine (BTC), β-lactoglobulin, cerebral acid-soluble protein 1, Btd protein, lactolipoprotein, subfamily 1 member a1, C5a anaphylatoxin receptor

[0248] Calreticulin, Canx protein, casein, cation-dependent mannose-6-phosphate receptor, Cd177 protein, Cd51 protein, Cd82 protein, Cd9 antigen, cell division control protein 42 homolog, tcp1-containing chaperone protein, subunit 5 (ε), chitinase 3-like protein 1, clathrin heavy chain 1, lectin, filoprotein 1, collagen lectin 43, collagen, crown protein 1a, Cp protein (fragment), cysteine-rich secretory protein 2, cell adhesin, cytochrome b-c1 complex subunit 2, mitochondria, cytochrome c, vasodilatory stimulating phosphoprotein, cytochrome c oxidase subunit 4 isoform 1, mitochondria, cytochrome c1, heme protein, mitochondria, polyterpene diphosphate oligosaccharide protein cyclodextrin glycosyltransferase subunit 2, muscular dystrophy proteoglycan, protein containing Ef-hand domain d2, electron transfer flavin subunit β, elongation factor 1-α1, elongation factor 1-α2, elongation factor 1-γ, elongation factor 2, endoplasmin, epididymal secretory protein E1, E-selectin / elam-1, eukaryotic initiation factor 4a-I, eukaryotic translation initiation factor 5a-1, Ezioprotein-root protein-membrane protein-binding phosphoprotein 50, F-actin apical subunit α-1, F-actin apical subunit β, factor xiia inhibitor, fatty acid-binding protein, adipocyte, fatty acid-binding protein, epidermis, Fc receptor, lactation feedback inhibitor (fil), fetoglobulin, fibrinogen γ chain protein, fibrinogen α chain, fibrinogen Vitamin B1 β chain, fibronectin, filamentin a, Fk506-binding protein 11, folic acid receptor α, G protein-coupled receptor, family c, group 5, member b, galactose-specific lectin binding to IgE, Ganab protein, gel solubilizer, glycoprotein 2 (zymogen granule membrane), glycosylation-dependent cell adhesion molecule 1, phosphatidylinositol proteoglycan 1, Gnai2 protein, granulocyte colony-stimulating factor, human α-lactalbumin, haptocorrin, globin, heat shock 70 KDA protein 1a, 1b, heat shock homolog 71kda protein, heat shock protein β-1, heat shock protein hsp 90-α, heat shock protein hsp 90-β, heat shock protein, mitochondria, hematopoietic cell-specific lyn substrate 1, heme-binding protein 1, hemoglobin subunit α, hemoglobin subunit β, heme-binding protein, heterogeneous nuclear ribonucleoprotein a / b, heterogeneous nuclear ribonucleoprotein a1, heterogeneous nuclear ribonucleoprotein d, heterogeneous nuclear ribonucleoprotein h2, heterogeneous nuclear ribonucleoprotein, heterogeneous nuclear ribonucleoprotein 10 a2 / b1, hibernation protein 20-like, high-mobility group box protein b2, histidine-rich glycoprotein, histone h1.1 (fragment), histone h2a, histone h2a1 type, histone h3.3. Histone h4, Endopin 2, 2b, Endopin 2c, T complex protein 1 subunit δ, tetraconnectin, Tgoln2 protein, thioredoxin, Tmed7 protein, rhoa protein, protein 10 containing transmembrane emp24 domain, transthyretin, tubulin α-1b chain, tropomyosin α-3 chain, tubulin β-2c chain, β-5 chain, vimentin, ubiquitin, Upf0527 transmembrane protein, ultralong chain specific acyl-CoA dehydrogenase, mitochondria, Vla, Vla-1, Vla-2, Vla-3, Vla-4, Vla-5, Vla-6, voltage-dependent anion-selective channel protein 1, Wap tetradisulfide core domain 2, Wd repeat protein 1, Yip1 domain family, member 3, phenonemin, α-lactalbumin, α-s1 casein, β-casein. Compounds - Complement C1, Complement C1S subfraction, Complement C2, Complement C3, Complement C4, Complement C4 (fragment), Complement C5, Complement C6, Complement C7, Complement C8, Complement C9, Complement factor B, Complement factor H, Complement factor I; Interleukins - Il1, Il10, Il12, Il13, Il16, Il1β, Il2, Il20, Il3, Il4, Il5, Il6, Il7, Il8; Glycoproteins - Platelet glycoprotein 4, Tap-associated glycoprotein, Monocyte colony-stimulating factor, Thrombopoietin, Plecolin, Zinc-α-2-glycoprotein; Tumor necrosis factors: TnF-α, TnF-β; Carbohydrates - Lactose, Maltose, Monosaccharides, Galacto-oligosaccharides, Lacto-oligosaccharides, Oligosaccharides, Polysaccharides, Starch, Sucrose, Trans-galacto-oligosaccharides.

[0249] Immunoglobulins – intercellular adhesion molecule 1, intercellular adhesion molecule 2, intercellular adhesion molecule 3, SigA (1 and 2), immunoglobulin a, immunoglobulin a2, immunoglobulin d, immunoglobulin e, immunoglobulin g, immunoglobulin g1, immunoglobulin g2, immunoglobulin m.

[0250] Minerals and metals—iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, sodium, sulfur, calcium, chloride, copper, cobalt, chromium, zinc.

[0251] Enzymes - Isocitrate dehydrogenase [NADP], cytoplasm, isocitrate dehydrogenase [NADP], mitochondria, lactoperoxidase, L-asparaginase, lipase, L-serine dehydratase, lysozyme, malate dehydrogenase, cytoplasm, malate dehydrogenase, mitochondria, microsomal glutathione S-transferase 1, myeloperoxidase, Nadh-cytochrome b5 reductase 3, neutrophils, elastase, nuclease-sensitive element-binding protein 1, nucleoside diphosphate kinase a2, PAF-acetylhydrolase, phosphatase, phosphoglycerate kinase 1, phosphoglycerate mutase 1, prostaglandin H2 D-isomerase, protein disulfide isomerase, protein disulfide isomerase a3, protein disulfide isomerase a4, prothrombin, pyruvate kinase, ribonuclease, pancreatic ribonuclease, ribonuclease UK114, ribophosphate pyrophosphate kinase 1.

[0252] Serine protease, sodium / potassium transport ATPase subunit α-1, superoxide dismutase, primary amine oxidase, liver isoenzyme, adenosine homocysteine ​​enzyme, adenosine kinase isoenzyme 2, mitochondria, 6-phosphoglucate dehydrogenase, 3-hydroxyacyl-CoA type 2 dehydrogenase, aconitate hydratase, mitochondria, Adp / atp translocase 2, Adp / atp translocase 3, aldehyde dehydrogenase, mitochondria, α-1-antiprotease, amylase, α-enolase, antiprotease Aspartate aminotransferase, mitochondria, Atp synthase protein 8, Atp synthase subunit α-heart isomer, mitochondria, Atp synthase subunit β, mitochondria, Atp synthase subunit δ, mitochondria, Atp synthase subunit e, mitochondria, Atp synthase subunit γ, mitochondria, Atp synthase subunit o, mitochondria, dipeptidyl peptidase 1, arylsulfatase, β-1,4-galactosyltransferase 1, calpain small subunit 1, catalase, cathepsin b, cathepsin d, cathepsin h, cathepsin S, cathepsin Z, citrate synthase, mitochondria, creatine kinase type b, cytosolic aminopeptidase, cytosolic nonspecific dipeptidase, enoyl-CoA hydratase, mitochondria, fatty acid synthase, flavin reductase, fructose-1, 2, fumarate hydratase, glucose-6-phosphate isomerase, glucosidase 2-subunit β, glutamate dehydrogenase 1, mitochondria, glutathione peroxidase 1, glutathione S-transferase p, glyceraldehyde- 3-Phosphate dehydrogenase, glycogen phosphorylase, liver type, heparan sulfate (glucosamine) 3-O-sulfotransferase 1, histamine, thioredoxin-dependent peroxidase, mitochondria, transaldolase, transitional endoplasmic reticulum phosphatase, transketolase, triose phosphate isomerase, tryptophanyl-tRNA synthetase, cytoplasm, ubiquitin-like modification activator 1, proton ATPase catalytic subunit a, xanthine dehydrogenase / oxidase, UTP glucose-1-phosphate uridine transferase.

[0253] Amino acids: Leucine, phenylalanine, isoleucine, lysine, methionine. Proline, serine, adenosine monophosphate (5”-amp), alanine, arginine, asparagine, carnitine, cysteine, glutamic acid, glycine, histidine, hydroxyproline, taurine, threonine, tryptophan, tyrosine, valine.

[0254] Inhibitory molecular-intermediate α-trypsin inhibitor compound component II, intermediate-α-trypsin inhibitor heavy chain h1, and intermediate-α-trypsin inhibitor heavy chain h4.

[0255] Kininogen-1, 2, leukocyte elastase inhibitor, macrophage migration inhibitory factor, Rho gdp-dissociation inhibitor 1 & 2, serum transferrin-like, spleen trypsin inhibitor I, bacteria - Lactobacillus rhamnosus, Lactobacillus reuteri (lactobacillus).

[0256] Acids – Lactic acid, lauric acid, rumen acid (cla), α-hydroxy acids. Lipids – Lactose ceramide, methyl sterol, phosphatidylinositol, polyunsaturated fatty acids, prostacyclin, prostaglandins, sphingolipids, sphingomyelin, thromboxane, β-lanosterol.

[0257] Phospholipids – phosphatidylcholine, phosphatidylethanolamine, phosphatal acetals; Cells – leukocytes, lymphocytes, macrophages, natural killer (NK) cells, neutrophils, phagocytes, basophils, B lymphocytes (also known as B cells), dendritic cells, eosinophils, leukotrienes, T lymphocytes (also known as T cells).

[0258] Cell adhesion molecules - L-selectin, Madcam-1, Pecam-1, Vcam.

[0259] Cellular components: Lamin-B1 precursor, lysophosphatidylethanolamine, Nadh dehydrogenase [ubiquinone] 1α subcomplex subunit 8.

[0260] Sterols – lanosterol, stigmata and campesterol, 7-dehydrocholesterol, 7-ketocholesterol, cholesterol.

[0261] Hormones and steroids—leptin, oxytocin, corticosterone, cortisol, dimethyl sterol, arachidic acid, ghrelin, gonadotropin-releasing hormone (GNRH), thyrotropin-releasing hormone, thyroid-stimulating hormone, thyroxine, triiodothyronine.

[0262] Growth factors include epidermal growth factor (EGF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor binding protein 1, granulocyte-macrophage colony-stimulating factor, growth / differentiation factor 8, insulin-like growth factor 1 and 2, insulin-like growth factor binding protein 7, and transforming growth factor β (TGF-β).

[0263] Ribosomal proteins - 40S ribosomal protein S3, 40S ribosomal protein Sa, 60S acidic ribosomal protein P0, 60S acidic ribosomal protein P2, 60S ribosomal protein L12, 60S ribosomal protein L4, 60S ribosomal protein L5, 60S ribosomal protein L8.

[0264] Allergens: Allergen Boss D 2

[0265] Antigens - Lewis antigens a & b, lymphocyte function-associated antigen 1, Mhc antigen heavy chain (fragment), Mhc class II antigen, seq 1 and 2, Mhc class II dr-α (fragment), monocyte differentiation antigen cd14, atypical Mhc class I antigen (fragment), proteasome activating compound subunits 1 & 2, Scd14, Thy-1 cell surface 25 antigen, allergen bos d 2.

[0266] Pigments (carotenoids) - β-cryptoxanthin, zeaxanthin, β-carotene.

[0267] Fat - Saturated Fat

[0268] Fatty acids - Linoleic acid (LA), monounsaturated fatty acids, myristic acid, octadecadienoic acid, oleic acid, palmitic acid, palmenoic acid, octadecanoic acid, stearic acid, stearatetraenoic acid (SDA), docosapentaenoic acid, capric acid (capric acid), dihomo-gamma-linolenic acid (DGLA), docosadienoic acid, docosahexaenoic acid (DHA), eicosadienoic acid, eicosapentaenoic acid, eicosatraenoic acid, eicosatrienoic acid, erucic acid, eicosaenoic acid, gamma-linolenic acid, erythrocytosolic acid (GB4), eicosapentaenoic acid, heptadecanoic acid, hexadecanoic acid, caproic acid (capric acid), adrenic acid, arachidic acid, arachidonic acid, ascorbic acid, aspartic acid, butyric acid, calendulatic acid, caprylic acid. Docosahexaenoic acid (herringoic acid), docosapentaenoic acid, tetradecenoic acid, triglycerides. Alpha-linolenic acid (ALA) antibodies and antimicrobial agents - β-2-glycoprotein 1, β-defensins 11, 12, 13, antimicrobial peptides (Cathelicidin)-1, antimicrobial peptide-2, antimicrobial peptide-4, antimicrobial peptide-5, antimicrobial peptide-6, antimicrobial peptide-7, Cr6261, Fi6, hemagglutinin inhibitors.

[0269] Genes - lipoprotein lipase, myotrophin, nucleolinkin 1 & 2, Pafah1b1 protein, Ras homologous gene family, member g (rho g), serine tRNA synthetase, cytoplasm, protein-coding mediator - Loc511106 protein, Loc788112 protein.

[0270] Carotenoids - lutein, lycopene.

[0271] Receptors - Renin receptor, polinecin receptor.

[0272] Chemokines - stromal cell-derived factor 4, Ccl11 (eosinophil activation chemokine-1), Cxcl10, Ccl2 (also known as mcp-1), Ccl24 (eosinophil activation chemokine-2), Ccl26 (eosinophil activation chemokine-3), Ccl5 (rantes).

[0273] Carbohydrates: Cellulose, sterols, disaccharides, fructose, galactooligosaccharides, galactose, glucosamine, glucose, glucosamine, glycogen, guanosine diphosphate mannose, human milk oligosaccharides, α-carotene, β-carotene, uridine diphosphate, uridine diphosphate hexose, uridine diphosphate-n-acetylglucosamine, uridine diphosphate glucuronic acid, uridine 25 monophosphate (3'-ump), uridine monophosphate (5'-ump).

[0274] Microbial synergist - Bifidus factor.

[0275] Nitrogenous organic acids: creatine, creatinine.

[0276] Signaling molecules: cyclic adenosine monophosphate (3':5'-cyclic amp) nucleotides: cytidine monophosphate (5'-cmp), guanosine diphosphate.

[0277] Glycolipids / glycosphingolipids: galactosylceramide, gangliosides, acylsphingolipid trihexose (gb3), glycosphingolipids, Gm1, Gm2, Gm3.

[0278] Neurotransmitters: Endorphin 2, 2b, and Endorphin 2c.

[0279] According to some embodiments, the compositions of the present invention may include multiple molecules of colostrum derived from multiple species (e.g., sheep, goats, and cows). It is known that colostrum from animals in a given location can be at least partially similar to each other due to being in substantially the same environment. Therefore, in some embodiments, colostrum from individuals of a single species may be deliberately collected from locations far apart in order to obtain different colostrums.

[0280] Use the composition to enhance the immune system of the elderly and / or people with compromised immune systems.

[0281] According to some implementation methods, older adults and people with compromised immune systems are more susceptible to illness when exposed to harmful microorganisms.

[0282] According to some exemplary embodiments, this document provides a composition comprising a keratin compound and β-lactoglobulin (LGB) for example, to specifically target diseases that are predisposed to older adults and / or individuals with compromised immune systems, such as the common cold.

[0283] According to some exemplary embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0284] According to some exemplary implementations, the terms "older people" and "older population" can refer to people who are generally older and tend to be more susceptible to illnesses, syndromes, injuries and pains than younger adults.

[0285] According to some exemplary implementations, the term "individual with a compromised immune system" or "individual with a weakened immune system" can refer to a person whose immune system is not functioning properly and is unable to effectively protect themselves from infection. Certain conditions and medications can weaken or impair the immune system. This can include: alcohol or drug abuse or cessation; certain diseases or conditions, such as diabetes, cancer, HIV / AIDS, or the body misidentifying its own tissues as harmful (autoimmune diseases); chemotherapy or radiation therapy; use of certain medications, such as corticosteroids or medications used to suppress the immune system after organ transplantation; splenectomy (splenectomy); and so on.

[0286] According to some exemplary embodiments, the specific use of pro-inflammatory components achieves unexpectedly beneficial effects. According to some embodiments, it is generally preferred to avoid inflammation in the elderly and / or individuals with compromised immune systems; however, the compositions of the present invention achieve beneficial immunostimulatory effects by using pro-inflammatory immune components to combat pathogens.

[0287] According to some implementations, in a compromised immune system, cytokines may have difficulty acting locally and / or systematically to trigger, maintain, and eliminate inflammatory responses.

[0288] According to some implementations, the interaction among pro-inflammatory cytokines, anti-inflammatory cytokines, and inhibitors of innate cytokines can determine the inflammatory response and its effectiveness. According to some implementations, the use of cytokines may be particularly beneficial due to a weakened or impaired state of the immune system.

[0289] According to some implementation methods, tumor necrosis factor-(TNF-) and interleukin-6 (IL-6) can be preferably used to enhance the immune response by activating the cytokine cascade and producing other pro-inflammatory cytokines and chemokines.

[0290] According to some implementations, pro-inflammatory molecules can also recruit MAST cells and the complement system, for example, to further enhance immune stimulation, such as by enhancing the attack on pathogens.

[0291] According to some embodiments, the compositions of the present invention may contain a variety of pro-inflammatory molecules.

[0292] According to some exemplary embodiments, the compositions of the present invention can be used to enhance the immune system of elderly individuals and / or individuals with compromised immune systems, for example, by providing an immune-stimulating effect.

[0293] According to some embodiments, this document provides the use of compositions comprising an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component for enhancing the immune system of elderly individuals and / or individuals with compromised immune systems.

[0294] According to some embodiments, the use may include administering a dose of the composition to elderly individuals and / or individuals with compromised immune systems.

[0295] According to some preferred embodiments, the use may include administering the composition at specific time periods, such as when elderly individuals and / or individuals with compromised immune systems may be more susceptible to infectious diseases, for example, during winter, before hospitalization and / or other medical procedures, before or during exposure to harmful pathogens, etc.

[0296] According to some implementations, the use may include administering an initial loading dose to elderly individuals and / or individuals with compromised immune systems, as well as continuing to administer a maintenance dose.

[0297] According to other embodiments of the invention, the use may include administering at least one daily dose of the composition of the invention to elderly individuals and / or individuals with compromised immune systems, for example, to provide long-lasting protection and / or enhance the immune system's resistance to harmful pathogens.

[0298] According to some embodiments, the compositions of the present invention may be microencapsulated, for example, to protect the composition from harmful conditions in the gastrointestinal tract and / or to control or delay the release of components of the composition.

[0299] According to some exemplary embodiments, the composition of the present invention can be in any suitable state and / or form.

[0300] According to some embodiments, the compositions of the present invention may additionally include two or more molecules derived from at least two different colostrums, for example, a first molecule derived from a first colostrum and a second molecule derived from a second colostrum.

[0301] The methods for preparing the composition, extracting colostrum and the composition thereof are described in detail throughout the specification.

[0302] The composition is used to enhance the athlete's immune system and / or reduce inflammation.

[0303] Endurance athletes, such as those competing in individual events like running, cycling, swimming, and triathlon, engage in several hours of aerobic training each week. Endurance training relies on skeletal muscles to use oxygen to power these activities. The oxidative nature of this training increases the production of free radicals, which are highly reactive and therefore require antioxidant defenses to protect cells from free radical damage. This potential damage to cells is called oxidative stress and can trigger an inflammatory response in the immune system to protect host tissues.

[0304] The term "athlete" as used in this article can refer to anyone who engages in physical activity, sports, fitness, etc.

[0305] Extensive evidence suggests that high-intensity or prolonged endurance training loads stimulate an increase in free radicals and oxidative stress.

[0306] According to some exemplary embodiments, the compositions of the present invention are suitable for oral administration to athletes and comprise a combination of one or more anti-inflammatory components.

[0307] According to some exemplary embodiments, this document provides compositions comprising keratin compounds and β-lactoglobulin (LGB) for example, to specifically reduce inflammation in athletes.

[0308] According to some exemplary embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0309] According to some exemplary embodiments, the compositions of the present invention may further comprise one or more components derived from colostrum and / or whole colostrum, such as synthetic, human and / or animal sources, for example, to alleviate inflammation and pain in the joints, ligaments and muscles of athletes.

[0310] According to some embodiments, the compositions of the present invention may additionally include two or more molecules derived from at least two different colostrums, for example, a first molecule derived from a first colostrum and a second molecule derived from a second colostrum.

[0311] According to some exemplary embodiments, the compositions of the present invention may include specific combinations of keratin compounds, β-lactoglobulin (LGB), anti-inflammatory components, pro-inflammatory components, antibacterial components, first immunostimulatory components, and second immunostimulatory components, for example, to specifically reduce inflammation, such as inflammation caused by physical activity or exercise.

[0312] According to some implementations, the term "reduction of inflammation" (also referred to herein as "reduction of inflammation") may include, but is not limited to, shortening the duration of inflammation, reducing inflammatory markers, etc.

[0313] According to some exemplary embodiments, the specific use of pro-inflammatory components has achieved unexpectedly beneficial effects. According to some embodiments, it is generally preferred to avoid inflammation in athletes; however, the compositions of the present invention achieve beneficial immunostimulatory effects by using pro-inflammatory immune components to combat pathogens.

[0314] According to some exemplary embodiments, the compositions of the present invention can be used to enhance the immune system of athletes, for example, by providing an immune-stimulating effect.

[0315] According to some embodiments, the composition of the present invention may also include two or more molecules derived from at least two different colostrums, for example, a first molecule derived from a first colostrum and a second molecule derived from a second colostrum.

[0316] Using a composition to enhance the animal's immune system

[0317] Very similar to humans, animals are susceptible to diseases.

[0318] Zoonotic diseases (also known as animal infectious diseases and zoonotic diseases) are infectious diseases caused by bacteria, viruses and parasites that are transmitted between animals (usually vertebrates).

[0319] Zoonotic diseases have different modes of transmission. In direct zoonotic diseases, the disease is transmitted directly from animals to humans through vectors such as air (influenza) or through bites and saliva (rabies). Conversely, it can also be transmitted through an intermediate species (called a carrier) that carries the pathogen without being infected. When humans infect animals, it is called a reverse zoonotic disease or a human infectious disease.

[0320] According to some exemplary embodiments, the compositions of the present invention are suitable for oral administration to animals and comprise a combination of one or more components that enhance the animal's immune system.

[0321] According to some exemplary embodiments, this document provides compositions comprising keratin compounds and β-lactoglobulin (LGB) for, for example, to specifically enhance the immune system of an animal.

[0322] According to some exemplary embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0323] According to some exemplary embodiments, the compositions of the present invention may further comprise one or more components derived from colostrum and / or whole colostrum (e.g., from synthetic, human and / or animal sources) to, for example, synergistically enhance the animal's immune system.

[0324] According to some embodiments, the compositions of the present invention may further include two or more molecules derived from at least two different colostrums, for example, a first molecule derived from a first colostrum and a second molecule derived from a second colostrum.

[0325] As used herein, the term "animal" can refer to any living organism that forms the animal kingdom. Preferably, as used herein, the term "animal" refers to livestock, such as calves, lambs, and foals; zoo animals; and domesticated animals, such as puppies, kittens, and cats and dogs.

[0326] According to some exemplary embodiments, the specific use of pro-inflammatory components achieves unexpectedly beneficial effects. According to some embodiments, it is generally preferred to avoid inflammation in animals; however, the compositions of the present invention achieve beneficial immunostimulatory effects by using pro-inflammatory immune components to combat pathogens.

[0327] According to some implementations, the algorithms disclosed above may include an assessment of the probability of an effective immunostimulatory effect from a combination of two or more proteins.

[0328] Specifically, the algorithm can calculate the degree of compatibility between two or more proteins, whereby "compatibility" refers to the enhanced and / or synergistic immune stimulation when two or more proteins are combined.

[0329] According to some implementations, for example, the algorithm may include assessing the probability of an effective immunostimulatory effect of a protein based on a comparison of the homology levels of proteins (e.g., proteins that have an immunostimulatory effect in animals).

[0330] According to some exemplary embodiments, the compositions of the present invention can be used to enhance the immune system of an animal, for example, by providing an immune-stimulating effect.

[0331] Example

[0332] Example 1

[0333] Part A – Protein Quantification

[0334] method

[0335] The samples were thawed at 4°C. Because the samples contained fat, centrifugation was not performed before processing. Sampling was performed after vortexing; this resulted in a milk sample containing particles and fat.

[0336] Sample preparation:

[0337] A. Milk samples: Each sample was taken twice and dissolved in two different solutions:

[0338] 1. Dissolve 10 μL of the sample in 40 μL of sample buffer containing Tris-HCl, glycine, SDS, 2-mercaptoethanol, and trace amounts of BPB, to a final concentration of 63 mM Tris-HCl 6.8, 10% glycine, 2% SDS, and 1% 2-mercaptoethanol. Vortex the sample, boil at 95°C for 10 minutes, and freeze at -80°C.

[0339] 2. Take 50 μL from each milk sample and mix with urea, ammonium bicarbonate (ABC), and dithiothreitol (DTT) to a final concentration of 8 M urea, 100 mM ABC, and 10 mM DTT. Vortex the sample and centrifuge (10', 10000 rpm, RT) to separate fats from proteins as much as possible. (Labeled as U in Table 3-4 below). Dilute 20 μL of the "fat clear" sample 1:1 with urea buffer containing 8 M urea, 100 mM ABC, and 10 mM DTT. (Labeled as UD in Table 3-4 below).

[0340] B. Commercial Products:

[0341] Take approximately 1.5–3 mg of powder sample from each product and dissolve it in a sample buffer containing the following components: 63 mM Tris-Cl 6.8, 10% glycine, 2% SDS, 1% 2-mercaptoethanol, and trace amounts of BPB at a concentration of 2 μg / μL. Vortex the sample, boil at (95°C, 10°F), sonicate until completely dissolved, and freeze at (-80°C).

[0342] Protein quantification: (Table #3-4) Take 1 μL from each “fat-clear” urea-diluted sample and quantify the protein using the Bradford Assay. Note: A. A linear pattern will not be obtained when the protein concentration is above 10 μg / μL, therefore the results will be inaccurate. B. Fat can cause reading bias.

[0343] result

[0344] The protein quantification results from urea-diluted samples are shown in the table below:

[0345] Table 3: Milk Samples – G1

[0346]

[0347] Table 4: Milk Samples – G2

[0348]

[0349] Table 5: Milk Samples – G3

[0350]

[0351] Table 6: Milk Samples – G4

[0352]

[0353] Note: Sample 125HZ4 – serial number 54817 is excluded. It will be replaced by sample 257IK4 and labeled as 54817B.

[0354] Table 7: Milk Samples – G5

[0355]

[0356] Table 8: Milk Samples – G6

[0357]

[0358] Note: The samples in this group contain a large amount of fat, requiring repeated readings. Samples 54849-50 show significant fluctuations.

[0359] refer to Figure 3 The graph shows the protein concentration deviation relative to the infant's age.

[0360] Part B – Protein Identification

[0361] method

[0362] Milk samples containing sample buffer and commercial product samples are thawed at room temperature.

[0363] A. According to the table below, take 2µL from each milk sample and mix:

[0364] Table 9:

[0365]

[0366]

[0367]

[0368] Add 2-mercaptoethanol to each mixture to a final concentration of 1%, then vortex the mixture, boil it at (95°C, 10°F) and add 4-15% Mini-PROTEAN® TGX pre-gel (Bio-Rad, Cat# 456-1084).

[0369] B. Each commercial product sample was boiled at (95°C, 10°F) and 15 μL was added to the gel above.

[0370] Electrophoresis was stopped after the front dye reached approximately 95% of the lane length. The gel was stained with PIERCE's Imperial protein staining solution.

[0371] For reference Figure 4 The result shows the sample preparation, protein gel.

[0372] In Ff, each lane is divided into 3 slices: >80KDa, 80-25KDa.

[0373] The proteins in the gel were reduced with 3 mM DTT in 100 mM ammonium bicarbonate [ABC] (60 °C for 30 min), modified with 10 mM iodoacetamide in 100 mM ABC (protected from light, at room temperature for 30 min), and decomposed in 10% acetonitrile. The mixture was then incubated overnight at 37 °C with 10 mM ABC and 10 mM CaCl2 and modified trypsin (Promega) at a 1:10 enzyme-to-substrate ratio. A second decomposition was performed for an additional 4 hours. The resulting peptides were desalted using a C18 pipette tip (a custom-made stage tip) and analyzed by LC-MS-MS. The peptides were separated by reversed-phase chromatography on a 0.075 × 300 mm fused silica capillary (J&W) packed with Reprosil reversed-phase material (Dr. Maisch GmbH, Germany). The above substances were eluted with a linear gradient of 5% to 28% for 120 min, a gradient of 28% to 95% for 15 min, and an aqueous solution of 95% acetonitrile and 0.1% formic acid at a flow rate of 0.15 μL / min for 15 min. Mass spectrometry analysis was performed by a Q Exactive plus mass spectrometer (Thermo) in positive mode using repeated full MS scans, followed by high-collision dissociation (HCD) of the 10 major ions selected from the first MS scan. Mass spectrometric data were analyzed using MaxQuant software V1.5.2.8 (Mathias Mann group), and compared with portions of the Uniprot database for humans and cattle, and portions of the NCBI-Nr database for goats, with an FDR of 1%. Statistical analysis of identification and quantification results was performed using Perseus V 1.5.2.4 software (Mathias Mann group). All intensities (intensity, IBAQ, and LFQ intensity) are expressed based on log2. Human samples were normalized based on equal milk volumes. Normalization of commercial product samples based on equal weight-to-volume ratios.

[0374] Part A of the identification results

[0375] Statistical analysis of human breast milk in different age groups:

[0376] Statistical analysis was performed on the razor+ unique peptide in List 1 and all proteins identified at 3ms / ms. The results showed significant differences in protein patterns across different age groups. An ANOVA was performed across all age groups, ultimately revealing significant changes in 337 proteins (p=0.05). The results indicated that groups G1-G2, G3-G4, and G5-G6 showed high similarity, but not complete differences.

[0377] Student's t-test was performed on the intensity of group 1 (G1) to groups 2-6 (G2-G6). Proteins with changes in p-value of 0.05 and + / -1 were color-coded. Proteins showing increased expression at G1 (positive difference) were marked with a bold color. Proteins showing decreased expression at G1 (negative difference) were marked with a light color. Proteins with significant increases / decreases in all groups were marked in brown and green, respectively, in the "Fasta Headers" column. Proteins with significant increases / decreases in group 1 were marked in brown and green, respectively, in the "Gene Name" column (also marked in red - exceptions). Bioinformatics analysis of these results was performed using STRING-DB software. The results are displayed in the STRING-Go annotations-human file.

[0378] Part B of the Identification Results - Statistical Analysis Comparing Human Breast Milk with Commercial Products from Bovine & Goat Sources in Different Age Groups

[0379] All proteins from different organisms were identified and merged into a single chart named "55711-85-human-bovine-capra-B". An additional chart, "55711-85-human-bovine-capra-IG", was created, containing only immunoglobulins. The IBAQ values ​​shown were normalized based on the internal sample composition.

[0380] For reference Figure 5-7 The results show the homology between human and bovine proteins in colostrum.

[0381] Figure 5 The table shows a comparison between human proteins present in human colostrum (as shown in Table G1) and bovine proteins present in bovine colostrum (as shown in Table G2).

[0382] The English abbreviation for protein is as follows:

[0383] XDH = xanthine dehydrogenase

[0384] PIGR = Polyimmunoglobulin receptor

[0385] LTF = Lactoferrin

[0386] ALB, LALBA = albumin

[0387] KRT…=keratin

[0388] FASN = Fatty acid synthase

[0389] CSN…=κ casein

[0390] CEL = Carboxylate Lipase

[0391] IG… = Antibody

[0392] LYZ=lysosome

[0393] Figure 6 The table shows a comparison between human proteins present in human colostrum (as shown in Table G3) and bovine proteins present in bovine colostrum (as shown in Table G4).

[0394] The English abbreviation for protein is as follows:

[0395] XDH = xanthine dehydrogenase

[0396] PIGR = Polyimmunoglobulin receptor

[0397] LTF = Lactoferrin

[0398] ALB, LALBA = albumin

[0399] KRT…=keratin

[0400] FASN = Fatty acid synthase

[0401] CSN…=κ casein

[0402] CEL = Carboxylate Lipase

[0403] IG… = Antibody

[0404] LYZ=lysosome

[0405] Figure 7 The table shows a comparison between human proteins present in human colostrum (as shown in Table G5) and bovine proteins present in bovine colostrum (as shown in Table G6).

[0406] The English abbreviation for protein is as follows:

[0407] XDH = xanthine dehydrogenase

[0408] PIGR = Polyimmunoglobulin receptor

[0409] LTF = Lactoferrin

[0410] ALB, LALBA = albumin

[0411] KRT…=keratin

[0412] FASN = Fatty acid synthase

[0413] CSN…=κ casein

[0414] CEL = Carboxylate Lipase

[0415] IG… = Antibody

[0416] LYZ=lysosome

[0417] Colostrum nanoparticles:

[0418] Purpose: Preparation of colostrum protein nanoparticles.

[0419] method:

[0420] 1. Characterization of colostrum powder Four types of commercial colostrum were labeled A through D, and the manufacturers' ingredient lists were recorded. A - Surthrival (28% fat, 45% protein, sugar). B - Immune tree (lipid-free, 60% protein). C - Symbiotics (lecithin and triglycerides, 60% protein, 30% sugar). D - California Gold Nutrition (lipid-free, 35% protein). 1g of each type of colostrum was weighed and dissolved in 20mL of purified water and stirred overnight. The liquid was then centrifuged at 4000rpm for 30 minutes, and the supernatant was transferred to a new test tube and centrifuged a second time at 7500rpm for 15 minutes at 4°C. The supernatant was filtered through a 0.45µm cellulose acetate filter, then refrigerated and lyophilized. The products were analyzed by UV absorbance at 280nm, UV full spectrum, mass yield, and elemental analysis.

[0421] The same procedure was performed using bovine serum albumin (BSA) as a pure protein control.

[0422] 2. Nanoparticle preparation

[0423] Dissolve 60 mg of each isolated colostrum formulation in 1.5 mL of purified water. The pH of each sample is approximately 7. Then adjust the pH to 5.5.

[0424] Human serum albumin (BSA 60 mg) was dissolved in 1.5 mL of purified water and used as a common protein control.

[0425] An additional 1.5 mg of each material was added to its solution as a nuclear growth agent, and the mixture was stirred at room temperature for 15 minutes. Then, 8 mL of 96% ethanol was added at a rate of 1 mL / min to form particles, while stirring at 500 rpm at room temperature. Finally, the mixture was stabilized at 110 °C for 15 minutes and at 105 °C for 10 minutes, respectively. The particle solution was stirred at room temperature until cooled, and then purified by centrifugation at 4000 rpm for 15 minutes. The particles were dried using a desiccator.

[0426] 3. Particle Characteristic Identification :

[0427] 3.1 Mass Yield: Empty centrifuge tubes were weighed, along with the dried particles. The total amount of each particle was calculated by the difference.

[0428] 3.2 Dispersion and Particle Size: 2 mg of each type of particle was dispersed in 2 mL of purified water and vortexed for 24 hours. Then, 2 mL of purified water was added to each solution and the mixture was transferred to an ultrasonic bath for 30 minutes. 6 mL of water was added to achieve a ratio of 1:5 (mg sample: water), and the mixture was again ultrasonically dispersed for 15 minutes. The particle size of the dispersed solutions was measured using a DLS ZetaSizer (Malvern).

[0429] result:

[0430] 1. Colostrum characteristics identification

[0431] • Protein fraction quantification: Calculate the percentage of dissolved, filtered, and lyophilized portions relative to the initial amount, and list them in Table 10. The highest yield was for type D.

[0432]

[0433] Table 10: Mass yield of the separated components relative to the initial amount (1g) for each product type.

[0434] • UV absorbance: Protein content was determined by measuring the absorbance of each filtered solution at 280 nm using a spectrophotometer (Table 11). Concentrations were calculated from the BSA calibration curve under conditions of 0.25–1.5 mg / ml in purified water with an R² of 0.99. Subsequently, a weighted transformation of % w / w was performed in this analysis. The UV absorbance of all colostrum solutions was higher than 100% w / w. This implies the presence of another factor absorbing the same wavelength, which is not present in the albumin solution. Alternatively, the composition of albumin differs significantly from that of colostrum proteins.

[0435]

[0436] Table 11: Absorbance and weight percentage of the separated colostrum solution at λ=280nm.

[0437] • Elemental analysis: The separated products were analyzed to determine the percentages of C, H, N, and O in each sample, and the nitrogen-to-carbon ratio was calculated for normalization (Table 12). Theoretically, the N:C ratio in proteins is 0.3.

[0438]

[0439] Table 12: Elemental composition of C, H, N and O in the separated samples, and the calculated N to C ratio.

[0440] As expected, the percentage of BSA was closest to the theoretical value, and the N:C ratio was the same, as it is a pure protein. The ratio of B and C in colostrum was closer to the theoretical protein ratio, while the ratio of A and D differed more significantly.

[0441] 2. Particle characteristic identification:

[0442] • Mass yield: The amount of particles produced is considered to be related to the initial amount used for particle preparation (60 mg - not exceeding 60 mg, because the amount of ethanol is large and the preparation step is slow). The results are shown in Table 4.

[0443] • Dispersion ability: The dried particles were redispersed in purified water, followed by vortex mixing and sonication. Sample D (105°C) showed the best dispersion. Refer to Table 13 below. The colostrum D stabilized at 105°C exhibited the best percentage and dispersion ability. Some BSA particles accumulated on the vial walls, resulting in a lower mass percentage in the preparation process.

[0444]

[0445] Table 13: Particle weight percentage and dispersibility after stabilization treatment at 100℃ and 105℃. (Samples B and C in vials ruptured during the process at 105℃. *Regarding BSA, it accumulated extensively on the vial walls.)

[0446] • Particle size: The average particle size for each sample is summarized in Table 14. The results for the colostrum D (105°C) show the best average particle size (526 nm) and PDI value (0.566).

[0447]

[0448] Table 14: Particle size of dispersed samples as detected by ZetaSizer.

[0449] Particles were prepared by a denaturation method and then stabilized at 100°C or 105°C.

[0450] in conclusion:

[0451] To form nanoparticles, four types of colostrum were tested. The first step involved extracting the protein fraction from the mixture by selecting the dissolved portion. Type D showed a high mass yield (75%). The manufacturer reported a protein content of 35% in product D. Therefore, the dissolved fraction should contain another component besides protein, likely carbohydrates. Generally, all types of colostrum showed higher mass yields in the dissolved fraction than reported by the manufacturers, while the fat components (types A and C) were expected to be insoluble in water. All colostrum types showed absorption at 280 nm, indicating the presence of protein components, but the absorbance was much higher than that reported by BSA, suggesting the inclusion of different components and conjugated amino acid compositions. Furthermore, BSA is not suitable for quantifying the protein composition in colostrum. Given the nitrogen-to-carbon elemental ratio, our results are closer to theoretical values ​​than those for types A and D, which reported lower protein content and lower N:C ratios, based on the manufacturer's reported 60% protein content in types B and C. As a result, based on our findings, colostrum D had the highest quality yield, but the separated fractions likely contained other components in much higher percentages than those in B and C, in addition to proteins.

[0452] Regarding particle preparation via denaturation and stabilization via heating, we did not obtain small nanoparticles, but the results after stabilization at 105 °C for 10 minutes were the best, with a higher percentage yield of 51% w / w in particle preparation, good dispersion in water, and an average preform particle size of 526 nm. Generally, the sample heated to 105 °C showed better redispersion in water compared to the sample heated to 100 °C.

[0453] Variables such as solution pH, heating temperature, heating time, and mixing rate can be examined to obtain appropriate results, such as smaller particle size and reduced polydispersity. Therefore, it is promising to continue research and obtain colostrum nanoparticles.

[0454] Colostrum granule preparation process:

[0455] Protein extraction:

[0456] Weigh 1g of each commercially available colostrum and dissolve it in 20mL of purified water, stirring overnight. Then centrifuge the liquid at 4000rpm for 15 minutes, transfer the supernatant to a new test tube, and centrifuge a second time at 7500rpm for 15 minutes at 4°C. Filter the supernatant through a 0.45µm cellulose acetate filter, then refrigerate and freeze-dry.

[0457] Particle preparation:

[0458] 60 mg of each isolated colostrum formulation was dissolved in 1.5 mL of purified water. The pH of each sample was approximately 7. The pH was then adjusted to 5.5. Using an autoinjector, an additional 1.5 mg of each material was added to its solution as a nucleation agent, and the mixture was stirred at room temperature for 15 minutes. Then, 8 mL of 96% ethanol was added at a rate of 1 mL / min to form granules, while stirring at 500 rpm at room temperature. Finally, the granules were stabilized at 100 °C for 15 minutes and at 105 °C for 10 minutes, respectively. The granule solutions were stirred at room temperature until cooled, and then purified by centrifugation at 4000 rpm for 15 minutes. The granules were dried under vacuum.

[0459] As detailed above, according to some exemplary embodiments, the present invention provides a composition comprising a keratin compound and β-lactoglobulin (LGB).

[0460] According to some exemplary embodiments, the composition may also comprise a combination of an anti-inflammatory component, a pro-inflammatory component, an antibacterial component, a first immunostimulatory component, and a second immunostimulatory component.

[0461] According to some additional embodiments, the composition also contains colostrum.

[0462] According to some implementation methods, the colostrum is in the form of colostrum nanoparticles.

[0463] Example 2

[0464] We have identified key proteins with anti-inflammatory functions in the immune system. These identified proteins include lactoferrin, α-lactalbumin, and CD59. We added these proteins (lactoferrin, α-lactalbumin, and CD59) to a complex of β-lactoglobulin and KRT1 to produce composition 1 for this assay.

[0465] The objectives of the following experiments include:

[0466] a. Enrichment of key proteins from colostrum samples using ion exchange chromatography.

[0467] b. Prove the feasibility of the allocation system.

[0468] c. Demonstrating the effect of composition 1 on human PBMCs, where composition 1 is an exemplary sample of the compositions described in this invention (also referred to herein as the “MAO-part”).

[0469] During these experiments, we were able to produce up to 2g of the protein to be tested. This 2g was broken down into component units, which could be used to create mixtures as needed.

[0470] Fractionation using ion exchange chromatography

[0471] To improve the final yield of desired colostrum proteins, an acid precipitation pretreatment was performed to remove casein from the colostrum.

[0472] Acidic precipitate:

[0473] 1. Dissolve 100mg of skimmed colostrum powder in 500mL of DDW (stir with a magnetic stirrer for 5 minutes).

[0474] 2. Acidic precipitation of casein – slowly titrate the solution with 1M HCl to pH 4.2. Filter (MilliporeExpress PLUS 0.22μm PES) to remove the precipitate.

[0475] 3. Adjust the pH value according to the chromatographic column used (pH 8 for AE, pH 5 for CE).

[0476] 4. Take samples for analysis in each step.

[0477] Now for reference Figure 8 The study showed the analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0478] like Figure 8 As shown, casein was successfully removed (no band near the 30 kDa marker in Sample 2). Removing casein from colostrum also removed lipids, phospholipids, and glycolipids, and increased the concentration of key proteins in colostrum (see the darker band in Sample 4). The resulting final concentration allows for a more efficient fractionation process. Casein constitutes a large proportion of bovine colostrum proteins and tends to form aggregates (mice), thus reducing the overall accuracy of ion chromatography. The final solution has better solubility and is visually more transparent.

[0479] Fractionation process

[0480] Anion exchange column fractionation

[0481] Chromatography column – HiTrap Q FF 5ml x 2, GE Healthcare

[0482] Buffer: Equilibration buffer - 20mM Tris-HCl pH 8

[0483] Elution buffer - 20mM Tris-HCl pH 8, 1M NaCl

[0484] • Filter 200 mL of solution using a 0.45 μm filter (Millipore Express PLUS 0.45 μm PES).

[0485] • Add the filtered solution to two equilibrated HiTrap Q FF columns (5 mL × 2) at a rate of 3 mL / min. After adding the sample, wash the column with 20 mM Tris-HCl pH 8 3CV buffer (5 mL / min).

[0486] • For elution, we used a stepped gradient – ​​100, 400, and 1000 mM NaCl. Collect 5 mL of the fraction.

[0487] • All fractions were quantified using the Bradford method and analyzed by SDS-PAGE.

[0488] Cation exchange column fractionation

[0489] Chromatography column – HiTrap SP FF 5ml, GE Healthcare

[0490] Buffer solution: Equilibration buffer - 20mM sodium acetate, pH 5

[0491] Elution buffer - 20mM sodium acetate, pH 5, 1M NaCl

[0492] • Add 200 mL of solution to the equilibrated HiTrap SP FF at a rate of 3 mL / min. After adding the sample, wash the column with 3CV buffer, 20 mM sodium acetate, pH 5 (5 mL / min).

[0493] • For elution, we used a stepped gradient – ​​100, 400, and 1000 mM NaCl. Collect 3 mL of the fraction.

[0494] • All fractions were quantified using the Bradford method and analyzed by SDS-PAGE.

[0495] AE purification table:

[0496]

[0497] Table 15

[0498] Now for reference Figure 9 The figure shows the AE chromatogram of defatted colostrum after acid precipitation treatment.

[0499] Most of the protein in the anion exchange (AE) was found in the second elution using 400 mM NaCl. Almost no protein was found in either the flow-through or the first elution.

[0500] CE purification table:

[0501]

[0502] Table 16

[0503] Now for reference Figure 10 The image shows the CE chromatography of defatted colostrum after acid precipitation treatment.

[0504] In cation exchange, equal amounts of protein can be found in both the first (100 nM NaCl) and the second (400 nM NaCl) elutions.

[0505] Enrichment factor

[0506] All samples were analyzed using mass spectrometry and ion-exchange chromatography, and their composition was compared with that of whole colostrum.

[0507] An enrichment factor greater than 1 is a positive enrichment factor. This means that the concentration of the protein in question in the final fraction is higher than its concentration in the entire colostrum.

[0508]

[0509] Table 17

[0510] refer to Figure 11 It shows a graph of enrichment factors.

[0511] This figure shows the enrichment factor for each protein compared to the original colostrum. For example, LGB was approximately 14.72% in the original colostrum used, and approximately 22% after acidification, resulting in an enrichment factor of 1.5.

[0512] Cell cultures:

[0513] The anti-inflammatory activity of the composition of the present invention was confirmed by peripheral blood mononuclear cells (PBMCs).

[0514] Purpose:

[0515] 1. To test the immunological effects (e.g., activation, proliferation, apoptosis, etc.) of colostrum fractions on human PBMCs.

[0516] 2. Specifically, this study investigates the ability of the composition to weaken the inflammatory response of T cells (CD3) against CD3 (OKT3) – 50 ng / mL activation.

[0517] Process Summary:

[0518] 1. PBMCs were extracted from healthy volunteers using ficol.

[0519] 2. 120×10 was obtained. 6 12 × 10⁶ cells were seeded for this experiment. 6 12 × 10 cells per well 6Cells were incubated in 1 mL of RPMI total medium (R-10) with different treatments.

[0520] 3. Incubate the cells at 37°C and 5% CO2 for 72 hours.

[0521]

[0522] Table 18

[0523] result:

[0524] 1. PBMCs indicate a reduction in proliferating cells in the presence of colostrum or composition 1. Figure 12 These are forward and side-scatter plots of flow cytometry analysis of PBMCs, showing T cells activated / proliferated with anti-CD3 in the presence of an anti-inflammatory composition or bovine colostrum (WC). When activated / proliferated, cells move to the upper right. The polygons distinguish T cells from other cells in the PBMC, such as monocytes.

[0525] 2. Anti-CD3 activation induced significant activation / proliferation of T cells.

[0526] 3. For example Figure 13 As shown, T cell activation and proliferation are lower in the presence of colostrum.

[0527] 4. In the presence of composition 1, this reduction in activation / proliferation is even more significant.

[0528] illustrate:

[0529] One of the most significant responses of the immune system is the inflammatory response, manifested as significant activation / proliferation of T cells. In PBMCs, activation of T cells with anti-CD3 agents leads to significant proliferation / activation. Anti-inflammatory substances, such as colostrum and Composition 1, reduce this proliferation / activation.

[0530] Example 3

[0531] To determine the possible effective concentrations of the various components of the compositions described in this invention, we conducted numerous experiments with possible combinations. Table 19 shows the preferred concentration ranges for each test component:

[0532]

[0533] Table 19

[0534] Example 4

[0535] Anti-inflammatory activity in cells.

[0536] The compositions of the present invention may comprise various combinations.

[0537] In this embodiment, six different experiments were performed in vitro on peripheral blood mononuclear cells (PBMCs) in combination with keratin compounds, LGB, CSN1S1, CSN2, and ALB (referred to herein as Composition 2). PBMCs were extracted from healthy volunteers using ficol and 12 × 10⁶ cells (12 × 10⁶ cells per well) were seeded into 24-well plates. Cells were incubated in 1 mL of RPMI total medium treated with different methods at 37°C and 5% CO₂ for 72 hours. Cell activation and proliferation were tested in the presence or absence of anti-CD3 and with treatment with colostrum or the anti-inflammatory component of the composition.

[0538] The concentrations of each component are shown in Table 20 below:

[0539]

[0540] Table 20

[0541] result

[0542] 1. Cells were activated and proliferated in the presence of anti-CD3 – 100% of the cells tested were activated.

[0543] 2. In the presence of anti-CD3 and colostrum, 55% of the cells were activated and proliferated.

[0544] 3. In the presence of anti-CD3 and one of the six variants of composition 2, less than 55% of cells were activated and proliferated, with an average of only 40%.

[0545] illustrate:

[0546] One of the most significant responses of the immune system is the inflammatory response, manifested as significant activation / proliferation of T cells. In PBMCs, activation of T cells with anti-CD3 resulted in significant proliferation / activation. Variants tested with composition 2 significantly reduced this proliferation / activation.

[0547] In addition to activation, the presence of anti- / pro-inflammatory cytokines in the culture medium was also measured. For example... Figure 14 As shown, the most important inflammatory factor in the tested T cells, interferon-γ (INFγ), increased after activation with anti-CD3, but decreased significantly in the presence of colostrum and also significantly decreased in the presence of composition 2 (mean results of all 6 experiments).

[0548] Example 5

[0549] The immune system has different activation mechanisms. Inflammation is required to recruit and stimulate the immune system to attack bacteria.

[0550] Inflammation must be controlled and is beneficial, but it must occur.

[0551] A composition (collectively referred to herein as composition 3) comprising 7.7% KRT1, 11.7% LGB, and 5.75% and 3.83% of the pro-inflammatory components SERPINB4 and SERPIND1, respectively, was prepared. Composition 3 was tested in monocytes derived from PBMCs of healthy volunteers.

[0552] To mimic inflammation, lipopolysaccharide (LPS) was added to the cells. IL-1β is a pro-inflammatory cytokine associated with pain, inflammation, and autoimmune diseases. It is secreted by monocytes in the presence of LPS.

[0553] like Figure 15 As shown, all groups secreted IL-1β in the presence of LPS; however, in the presence of composition 3, IL-1β secretion was significant without LPS activation.

[0554] WC – Colostrum

[0555] Pro – Composition 3

[0556] This application describes embodiments of the invention by way of example, and such description is not intended to limit the scope of the invention. The embodiments include different features, and not all features are necessary in all embodiments of the invention. Some embodiments utilize only some features or possible combinations of these features. Those skilled in the art will understand that there are many variations of the described embodiments of the invention, and that embodiments of the invention include different combinations of features recorded in the described embodiments. The scope of the invention is defined only by the appended claims.

Claims

1. A composition for providing a synergistic effect on the human body in enhancing the immune system, the composition comprising: β-lactoglobulin, with concentrations ranging from 0.02% to 23.4%, CSN1S1, with concentrations ranging from 0.06% to 14.90%, CSN2, with concentrations ranging from 0.07% to 27.00%, Serum albumin, with concentrations ranging from 0.00% to 3.31%, and keratin compounds, The keratin compound described herein consists of the following: KRT1, with concentrations ranging from 0.02% to 15.41%, KRT33B, concentrations ranging from 0.01% to 6.58%, KRT13, with concentrations ranging from 0.01% to 6.44%, KRT18, with concentrations ranging from 0.01% to 7.92%, KRT17, with concentrations ranging from 0.01% to 6.56%, KRT42, with concentrations ranging from 0.01% to 6.67%, KRT28, with concentrations ranging from 0.01% to 7.10%, KRT36, with concentrations ranging from 0.01% to 6.58%, KRT12, with concentrations ranging from 0.01% to 6.56%, KRT10, with concentrations ranging from 0.01% to 7.57%, KRT24, with concentrations ranging from 0.01% to 7.46%, KRT14, with concentrations ranging from 0.01% to 7.95%, KRT4, with concentrations ranging from 0.00% to 1.76%, KRT75, concentration from 0.00% to 1.80%, KRT6A, concentrations ranging from 0.00% to 3.80%, KRT6C, concentrations ranging from 0.00% to 3.80%, KRT5, with concentrations ranging from 0.00% to 4.87%, KRT77, with concentrations ranging from 0.02% to 5.88%, KRT3, with concentrations ranging from 0.00% to 4.56%, KRT2, with concentrations ranging from 0.01% to 7.64%, The aforementioned enhancement of the immune system includes shortening the duration of illness or onset, reducing the likelihood of illness, or reducing the number or severity of disease-related symptoms.

2. The composition of claim 1, wherein, The composition enhances the gut microbiota.

3. The composition of claim 1, wherein, The composition further comprises at least one anti-inflammatory component selected from the group consisting of lactoferrin, lysozyme C, interleukin-10, transforming growth factor β, interleukin-4 and cyclooxygenase-1.

4. The composition of claim 1, wherein, The composition further comprises at least one pro-inflammatory component selected from the group consisting of lactoferrin, lysozyme C, interleukin-1B, interleukin-6, and tumor necrosis factor α.

5. The composition of claim 1, wherein, The composition further comprises at least one antimicrobial component selected from the group consisting of β-defensin 1, lactoperoxidase, lactoferrin, cathepsin G, lysozyme C, immunoglobulin G, and immunoglobulin A.

6. The composition according to claim 1, which is used to enhance the immune system of an infant.

7. The composition according to claim 1, which is used to enhance the immune system of an individual with an impaired immune system.

8. The composition according to claim 1, used to reduce inflammation in athletes.

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