A nutritional composition to help enhance immunity and uses thereof

Abstract

This invention belongs to the field of functional substance research, specifically relating to a nutritional composition that helps enhance immunity and its uses. The nutritional composition provided by this invention comprises the essential active ingredients shown in (I) and (II) below: (I) lysophosphatidylethanolamine; (II) lactoferrin; wherein, the essential active ingredient shown in (I) contains at least unsaturated lysophosphatidylethanolamine, and the fatty acid chain length of the unsaturated lysophosphatidylethanolamine is 14-22 carbon atoms, and in the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500). The combination of unsaturated lysophosphatidylethanolamine with a specific fatty acid chain length and lactoferrin can promote the comprehensive construction and functional regulation of adaptive and innate immune systems, with T cell development and activation as the core.

Description

Technical Field

[0001] This invention belongs to the field of functional substance research, specifically relating to a nutritional composition that helps enhance immunity and its uses. Background Technology

[0002] The immune system is the core defense system of the human body, responsible for resisting the invasion of external pathogens, clearing abnormal cells, and maintaining homeostasis. Its proper function directly determines a person's health and disease resistance. Under normal conditions, the immune system effectively identifies and blocks the invasion of pathogenic microorganisms such as bacteria, viruses, and fungi, reducing the risk of infectious diseases. It also participates in the repair of bodily damage, metabolic regulation, and cell apoptosis control, playing a crucial role in preventing chronic inflammation, autoimmune diseases, and malignant lesions. Weakened immunity leads to a comprehensive decline in the body's defense, surveillance, and clearance functions, manifesting as susceptibility to colds, frequent infections with prolonged illness and extended recovery periods. Long-term immune imbalance can also induce various health problems, seriously affecting quality of life and lifespan. Therefore, maintaining stable and appropriate immune function is the core foundation for maintaining human health.

[0003] Infants, children, and the elderly are high-risk groups for weakened immunity due to their physiological development and declining bodily functions, and generally exhibit significant immune deficiencies and health risks. Infants' immune systems are not yet fully developed, and their immune organs and cells are not fully functional. As the passive immunity passed down from the mother gradually fades, their own active immunity is not yet fully established. Infants have extremely weak resistance to external pathogens and are prone to infections of the respiratory, digestive, and other systems, with infections easily worsening and recurring. Children are in a period of rapid growth and development, and their immune systems are still gradually maturing. Factors such as an unbalanced diet, insufficient outdoor activity, and academic pressure often lead to fluctuations in immune function, manifesting as poor disease resistance, an imbalance between nutrient absorption and immune development, and long-term weakened immunity directly hindering normal growth and development in children. As the body ages naturally, the elderly experience atrophy of immune organs, a decrease in the number and activity of immune cells, and a significant decline in immune response. They belong to the primary immune function decline group, which not only greatly increases the risk of infection, but also leads to more complications and poor prognosis after infection. At the same time, the weakened immune surveillance function further increases the probability of the occurrence of chronic diseases and degenerative changes.

[0004] Therefore, immune dysfunction has become a prominent pain point affecting health throughout the entire life cycle. Current interventions for boosting immunity in specific populations such as infants, children, and the elderly suffer from insufficient targeting, limited effectiveness, and poor adaptability, failing to meet the immune nutritional needs of these special physiological stages. Therefore, there is an urgent need to develop nutritional compositions suitable for all life stages, safely and effectively regulating and enhancing immune function. Scientific nutritional interventions can compensate for immune deficiencies and improve immune status, providing crucial technological support for health protection for all life stages, especially infants, children, and the elderly.

[0005] Phospholipids are key bioactive components of breast milk lipids, playing a crucial role in the development of the nervous system and metabolic regulation in infants and young children. Phospholipids can be broadly classified into two categories: glycerophospholipids (PL) and sphingomyelins (SM). PL can be further subdivided into lysophospholipids (LPL) and phosphatidylacetylphospholipids. Lysophospholipids are produced by hydrolyzing one fatty acid in the PL molecule, and the two main types found in breast milk are lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE).

[0006] Lactoferrin (LF) is a multifunctional iron-binding glycosylated globular protein widely found in various exocrine secretions of mammals, such as human milk, cow's milk, saliva, tears, and secretions from the bronchi and intestines. Human colostrum has the highest LF content, approximately 7-9 g / L, which gradually decreases thereafter, reaching 1.5-4.0 g / L in mature human milk. LF possesses functions including bactericidal and bacteriostatic effects, antiviral activity, immunomodulation, antioxidant activity, regulation of intestinal flora, and promotion of bifidobacteria growth. For example, CN117084414B, through the rational combination of lactoferrin, N-acetylneuraminic acid, PQQ, oat β-glucan, and taurine, produces a product with excellent immune-enhancing effects. CN118370386B discloses a lactoferrin composition that regulates macrophage immune activity by compounding lactoferrin, α-lactalbumin, N-acetylneuraminic acid, and yeast β-glucan in specific proportions. The four components work synergistically to significantly enhance macrophage phagocytic activity without altering macrophage activity, regulate macrophage polarization during inflammatory responses, inhibit macrophage polarization towards pro-inflammatory M1 patterns, and promote macrophage polarization towards anti-inflammatory M2 patterns. Simultaneously, it enhances the antigen-presenting capacity of macrophages and the activation of immune T cells, thereby improving the body's immunity. CN103976359B discloses that lactoferrin can regulate the body's immune response, playing a regulatory role in antibody production, T cell production, and the proportion of natural killer cells in lymphocytes. Regarding the antiviral mechanism of LF, it is generally believed that it mainly includes two aspects: on the one hand, LF can inhibit viral replication in cells by inhibiting the binding of the virus to target cells; on the other hand, LF can bind to viral receptors such as glycosaminoglycans, especially heparan sulfate (HS). The binding of LF to HS avoids the first contact between the host cell and the virus, thereby preventing viral infection. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] Addressing the immune enhancement needs of people throughout their entire life cycle, particularly the immune system construction and functional regulation needs of specific populations such as infants, children, and the elderly, this invention has conducted extensive research and aims to develop a nutritional composition based on lysophosphatidylethanolamine and lactoferrin, nutrients naturally found in breast milk and cow's milk. These two components synergistically construct and improve an immune regulatory system centered on T cells. Specifically, this involves building an immune improvement system with T cells at its core, encompassing "whole-chain regulation + cell linkage." This addresses the functional deficiencies of T cells themselves and increases the number of innate immune cells through T cell linkage, ultimately achieving a systematic, synergistic, and lasting improvement in overall immunity. This provides a more precise and effective nutritional intervention plan for immunocompromised individuals and is suitable for long-term use by people throughout their entire life cycle.

[0009] Solution for solving the problem

[0010] [1]. A nutritional composition, wherein the nutritional composition is a nutritional composition having the function of helping to enhance immunity, the nutritional composition comprising the essential active ingredients shown in (I) and (II) below:

[0011] (I) Lysophosphatidylethanolamine;

[0012] (II) Lactoferrin;

[0013] Wherein, the essential active ingredient shown in (I) contains at least unsaturated lysophosphatidylethanolamine, and the fatty acid chain in the unsaturated lysophosphatidylethanolamine has a length of 14-22 carbon atoms, and in the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500), and within the mass ratio range, the essential active ingredient shown in (I) and the essential active ingredient shown in (II) work synergistically to help enhance immunity.

[0014] [2]. According to the nutritional composition of [1], wherein the fatty acid chain in the unsaturated lysophosphatidylethanolamine is linked at the sn-1 position.

[0015] [3]. The nutritional composition according to [1] or [2], wherein the lysophosphatidylethanolamine is provided in the form of dairy products containing it and / or enzymatically hydrolyzed soybean lecithin.

[0016] [4]. The nutritional composition according to any one of [1]-[3], wherein the lactoferrin is provided in the form of whole milk containing lactoferrin, whole milk powder, colostrum powder, whey protein powder and / or lactoferrin powder.

[0017] [5]. Use of the nutritional composition according to any one of [1]-[4] in the preparation of products that help enhance immunity.

[0018] [6]. According to the use described in [5], the aid to the construction and functional regulation of the immune system includes at least one of promoting T cell development, promoting T cell activation, balancing the level of inflammatory factors and increasing the number of innate immune cells.

[0019] [7]. According to the use described in [6], wherein the promotion of T cell development includes increasing the expression levels of genes RAG1 and / or RAG2; and / or, the promotion of T cell activation includes increasing the expression levels of genes ZAP70 and / or IFNGR1; and / or, the balancing of inflammatory factor levels includes decreasing the level of TNF-α and / or increasing the level of IL-12α; and / or, the increase in the number of innate immune cells includes increasing the number of neutrophils and / or macrophages.

[0020] [8]. Use of the nutritional composition according to any one of [1]-[4] in the preparation of a product having any one or more of the following (a)-(d):

[0021] (a) Promoting T cell development; preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and / or RAG2; more preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and RAG2; even more preferably, promoting T cell development includes increasing the mRNA levels of genes RAG1 and RAG2.

[0022] (b) Promoting T cell activation; preferably, promoting T cell activation includes increasing the expression levels of genes ZAP70 and / or IFNGR1; more preferably, promoting T cell development includes increasing the expression levels of genes ZAP70 and IFNGR1; even more preferably, promoting T cell development includes increasing the mRNA levels of genes ZAP70 and IFNGR1.

[0023] (c) Balancing inflammatory factor levels; preferably, balancing inflammatory factor levels includes reducing TNF-α levels and / or increasing IL-12α levels; more preferably, balancing inflammatory factor levels includes reducing TNF-α levels and increasing IL-12α levels; even more preferably, balancing inflammatory factor levels includes reducing TNF-α mRNA levels and increasing IL-12α mRNA levels;

[0024] (d) Increasing the number of innate immune cells; preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and / or macrophages; more preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and macrophages.

[0025] [9]. Use according to any one of [5]-[8], wherein the product is solid or liquid.

[0026]

[10] . Use according to any one of [5]-[9], wherein the product contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives and any acceptable excipients.

[0027] The effects of the invention

[0028] This invention proposes for the first time that a combination of unsaturated lysophosphatidylethanolamine and lactoferrin with specific fatty acid chains of a certain length has a synergistic effect on promoting the construction and functional regulation of the body's immune system, with T cell development and activation as the core. When combined in a certain ratio, the two can not only promote the development and activation of T cells, but also regulate the expression of inflammatory factors, improve the disorder of inflammatory factor expression and reduce damage to the body. At the same time, they can also increase the number and improve the activity of innate immune cells such as macrophages and neutrophils, thereby enhancing the body's innate immunity. Through synergistic effects, it helps to comprehensively build and regulate the function of both adaptive and innate immune systems, achieving a comprehensive effect that helps to enhance immunity.

[0029] Furthermore, the nutrients mentioned in this invention, lactoferrin and lysophosphatidylethanolamine, are both natural nutrients found in breast milk or cow's milk, suitable for long-term use, and readily accepted by people throughout their entire life cycle. The nutritional composition can be added to various types of products, including food, nutritional products, nutritional supplements, health foods, and pharmaceuticals. Detailed Implementation

[0030] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0031] <Terminology Definition>

[0032] In this invention, "comprising," "having," "including," or "containing" can mean included or open-ended, and does not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0033] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0034] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0035] In this invention, the numerical ranges represented by "value A ~ value B", "value A - value B", and "value A above / below" refer to the ranges including the endpoint values ​​A and B.

[0036] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0037] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0038] In this invention, all unit names used are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0039] In this invention, "infants and toddlers" refers to the human group under the age of 3 years, including infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0040] In this invention, "children" refers to the human group aged 3-6 years.

[0041] In this invention, "teenagers" refers to the human group aged 7-18.

[0042] In this invention, "pregnant women" includes women who are pregnant and women who are breastfeeding.

[0043] In this invention, "middle-aged and elderly" refers to the human group aged 41 and above, including middle-aged people aged 41-65 and elderly people aged 65 and above.

[0044] In this invention, "animal milk" refers to the liquid obtained from the mammary glands of a mammal in the process of lactation.

[0045] In this invention, "lysophosphatidylethanolamine (LysoPE, LPE)" is a glycerophospholipid composed of a glycerol backbone, a fatty acid chain, a phosphate group, and an ethanolamine head group. Compared to phosphatidylethanolamine, it lacks a fatty acid chain at the sn-1 or sn-2 position of its glycerol backbone.

[0046] In this invention, for ease of description of glycerophospholipids, the following characters are used to refer to different types of fatty acids: straight-chain fatty acids with a chain length of 18 carbon atoms and no carbon-carbon double bonds (18:0), such as stearic acid; straight-chain fatty acids with a chain length of 18 carbon atoms and one carbon-carbon double bond (18:1), such as oleic acid; straight-chain fatty acids with a chain length of 18 carbon atoms and two carbon-carbon double bonds (18:2), such as linoleic acid; and other fatty acid chain lengths are treated similarly. In this invention, for specific lysophosphatidylethanolamines, the format "LPE + the above characters" is used to indicate the type of lysophosphatidylethanolamine linked with different fatty acids, such as LPE 18:0, LPE 18:1, LPE 18:2, etc.

[0047] In this invention, lactoferrin (LF) is a mammalian non-heme cationic protein with a relative molecular mass of about 80,000 Da and composed of about 700 amino acids. It belongs to the transferrin superfamily and has a symmetrical bilobal structure. Its unique structure is mainly used to control the binding and release of iron ions.

[0048] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] This invention is mainly based on the following concept:

[0050] The immune system is the core defense line for the human body against pathogens and maintaining homeostasis. T cells, as the "central regulators" and "executors" of specific immunity, directly determine the effectiveness of the immune system's defenses. However, certain populations commonly exhibit T cell dysfunction. For example, immature T cell development in infants leads to immune vulnerability, while the decline in T cell function in middle-aged and elderly individuals results in immunosenescence. Existing nutritional compositions often focus on single or macroscopic immune manifestations, with few specifically targeting the development or improvement of T cell function to enhance immunity.

[0051] Based on this, the present invention aims to study how to achieve comprehensive immune system construction and functional regulation, especially for special groups such as infants, young children and the elderly, and how to achieve synchronous development or reconstruction and functional regulation of non-specific immunity and specific immunity.

[0052] In response, this invention has discovered through extensive research that when lactoferrin and unsaturated lysophosphatidylethanolamine such as LPE (18:2(9Z,12Z) / 0:0) are combined, they can first promote the development, maturation, and activation of T cells. Subsequently, they can improve the body's inflammatory disorders and reduce damage by regulating inflammatory factors. At the same time, they can regulate the number and activity of macrophages and neutrophils, enhance the body's innate immunity, and memory T cells can generate immune memory that can be rapidly activated upon re-exposure to the same antigen, providing long-term immune protection.

[0053] <Nutritional Combinations>

[0054] The nutritional composition provided by this invention comprises the essential active ingredients shown in (I) and (II) below:

[0055] (I) Lysophosphatidylethanolamine;

[0056] (II) Lactoferrin;

[0057] Furthermore, in the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500).

[0058] This invention does not impose any special requirements on the source of lysophosphatidylethanolamine, as long as its source or form of use meets the requirements of local laws and regulations.

[0059] For example, it can be prepared through chemical synthesis, enzymatic hydrolysis, or physical separation and extraction. For instance, natural phosphatidylethanolamine or natural phospholipids containing phosphatidylethanolamine can be enzymatically hydrolyzed using phosphatase; for example, deacetylation using phosphatase A2 can yield sn-1-acyl-LPE. Furthermore, lysophosphatidylethanolamine with a specific acyl group composition can be obtained using a combination of methods, such as a chemical-enzyme hybridization approach.

[0060] In some embodiments, the lysophosphatidylethanolamine of the present invention can be separated and extracted from a phospholipid-rich milk source, wherein the phospholipid-rich milk source can be a milk fat globule membrane, and the separation and extraction method can be membrane separation.

[0061] In some embodiments, the lysophosphatidylethanolamine of the present invention is provided in the form of dairy products containing it (e.g., buttermilk products, phospholipid-containing milk proteins, milk phospholipid products, whole milk, whole milk powder, and / or phospholipid-containing cream products, etc.) and / or enzymatically hydrolyzed soybean lecithin. The content of lysophosphatidylethanolamine varies in each source form; for example, in the buttermilk products, the content of lysophosphatidylethanolamine, on a dry weight basis, can be 1-3000 mg / 100g, preferably 1-1000 mg / 100g; wherein the content of LPE (18:2(9Z,12Z) / 0:0) can be 1-1000 mg / 100g, preferably 10-500 mg / 100g; in the phospholipid-containing milk proteins, the content of lysophosphatidylethanolamine, on a dry weight basis, can be 0.1-1000 mg / 100g, preferably 0.5-500 mg / 100g. g; wherein the content of LPE (18:2(9Z,12Z) / 0:0) can be 0.1-300mg / 100g, preferably 0.5-200mg / 100g; in the phospholipid-containing cream product, on a dry weight basis, the content of lysophosphatidylethanolamine can be 0.0001-100mg / 100g liquid, preferably 0.001-50mg / 100g liquid; wherein the content of LPE (18:2(9Z,12Z) / 0:0) can be 0.0001-1mg / 100g liquid, preferably 0.0005-0.5mg / 100g liquid.

[0062] In other embodiments, the lysophosphatidylethanolamine of the present invention is provided in pure form with a purity of 99% or higher.

[0063] This invention does not impose any particular limitation on the source of lactoferrin, as long as its source or form of use meets the requirements of local laws and regulations.

[0064] For example, lactoferrin derived from mammals such as cattle, sheep, and camels can be used, with bovine lactoferrin being preferred. The present invention does not particularly limit the method of obtaining lactoferrin; for example, it can be extracted from fresh milk or dairy products through methods such as protein flocculation, or commercially available lactoferrin products can be used directly.

[0065] In some embodiments, the lactoferrin of the present invention is provided in the form of whole milk containing lactoferrin, whole milk powder, colostrum powder, whey protein powder, and / or lactoferrin powder. For example, the lactoferrin content in each source form can typically be 1%-99% by mass.

[0066] In some embodiments, the essential active ingredient shown in (I) comprises at least unsaturated lysophosphatidylethanolamine, and the fatty acid chain in the unsaturated lysophosphatidylethanolamine has a length of 14-22 carbon atoms, such as LPE 14:1, LPE 17:1, LPE 17:2, LPE 18:1, LPE 18:2, LPE 22:1, LPE 22:2 and / or LPE 22:6, which are present in milk sources; preferably, the essential active ingredient shown in (I) comprises at least unsaturated lysophosphatidylethanolamine with a fatty acid chain length of 18 carbon atoms.

[0067] In some preferred embodiments, the fatty acid chain in the unsaturated lysophosphatidylethanolamine is located at the sn-1 position.

[0068] In some preferred embodiments, the essential active ingredient shown in (I) comprises at least LPE (18:2(9Z,12Z) / 0:0). The present invention has found that the combination of LPE (18:2(9Z,12Z) / 0:0) and lactoferrin can exert a synergistic effect that helps enhance immunity.

[0069] In some embodiments, unsaturated lysophosphatidylethanolamine and lactoferrin, with fatty acid chain lengths of 14-22 carbon atoms, are the main active ingredients of the nutritional composition. That is, the nutritional composition of the present invention mainly relies on the unsaturated lysophosphatidylethanolamine and lactoferrin, with fatty acid chain lengths of 14-22 carbon atoms, to exert specific physiological activities, such as helping to enhance immunity. In other words, in some embodiments, the active ingredients of the nutritional composition (the ingredients that exert specific physiological functions, i.e., the ingredients that help enhance immunity) consist of unsaturated lysophosphatidylethanolamine and lactoferrin, with fatty acid chain lengths of 14-22 carbon atoms.

[0070] In some preferred embodiments, the active ingredients of the nutritional composition comprise, or consist of, unsaturated lysophosphatidylethanolamine and lactoferrin, which have a fatty acid chain length of 18 carbon atoms.

[0071] In some preferred embodiments, the active ingredient of the nutritional composition comprises, or is composed of, unsaturated lysophosphatidylethanolamine and lactoferrin, a fatty acid chain of 18 carbon atoms linked at the sn-1 position.

[0072] In some further preferred embodiments, the active ingredients of the nutritional composition comprise, or consist of, LPE (18:2(9Z,12Z) / 0:0) and lactoferrin.

[0073] In some embodiments, the nutritional composition comprises an active ingredient (a component that performs a specific physiological function, i.e., a component that helps enhance immunity) and an inactive ingredient (a substance that does not help enhance immunity, or at least does not have a synergistic effect with LPE (18:2(9Z,12Z) / 0:0) or lactoferrin). Exemplarily, the inactive ingredient may be other nutrients, any food-acceptable excipient, and / or substances that are generated during the production or acquisition of the active ingredient and cannot be effectively separated from the active ingredient or do not require separation. In some embodiments, the nutritional composition consists of the active ingredient and the inactive ingredient.

[0074] In some embodiments, the nutritional composition further comprises milk proteins other than lactoferrin; for example, other whey proteins (including milk fat globule membrane proteins, α-lactalbumin, β-lactoglobulin, immunoglobulins, etc.), casein, etc. All of the above substances can be present in the nutritional composition as inactive ingredients.

[0075] In some embodiments, in the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500), for example, it can be 1:0.1, 1:0.2, 1:0.4, 1:0.8, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:500, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:500, 1:100, 1:200, 1:300, 1:4 ... The ratios are 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, etc.; preferably, the mass ratio of the two is 1:(0.2-1000); more preferably, the mass ratio of the two is 1:(1-500); even more preferably, the mass ratio of the two is 1:(10-100); preferably, the above ratio is the mass ratio of LPE (18:2(9Z,12Z) / 0:0) to lactoferrin. Within the mass ratio range of (I) and (II), the essential active ingredients (especially LPE (18:2(9Z,12Z) / 0:0)) synergistically exert their effect in enhancing immunity.

[0076] The present invention does not impose any particular limitation on the form of the nutritional composition; typically, it can be a liquid or a solid. From the perspective of ease of production, transportation, storage, and use, the nutritional composition of the present invention is preferably a powdered solid.

[0077] <Uses of Nutritional Combinations>

[0078] This invention provides the use of the above-described nutritional composition to help enhance immunity. In some embodiments, the enhancement of immunity is not intended to prevent or treat disease; that is, this invention provides the use of the above-described nutritional composition for non-therapeutic purposes of enhancing immunity. Based on this, this invention also provides the use of the above-described nutritional composition in the preparation of products that help enhance immunity.

[0079] In some implementations, the product helps to enhance immunity by assisting in the construction and functional regulation of the immune system.

[0080] In some implementations, the ability to enhance immunity includes at least one of promoting T cell development, promoting T cell activation, balancing inflammatory factor levels, and increasing the number of innate immune cells.

[0081] In some specific implementations, the enhancement of immunity includes promoting T cell development; preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and / or RAG2; more preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and RAG2; even more preferably, promoting T cell development includes increasing the mRNA levels of genes RAG1 and RAG2.

[0082] In some specific implementations, the enhancement of immunity includes promoting T cell activation; preferably, promoting T cell activation includes increasing the expression levels of genes ZAP70 and / or IFNGR1; more preferably, promoting T cell development includes increasing the expression levels of genes ZAP70 and IFNGR1; even more preferably, promoting T cell development includes increasing the mRNA levels of genes ZAP70 and IFNGR1.

[0083] In some specific implementations, the enhancement of immunity includes balancing the levels of inflammatory factors; preferably, balancing the levels of inflammatory factors includes reducing the level of TNF-α and / or increasing the level of IL-12α; more preferably, balancing the levels of inflammatory factors includes reducing the level of TNF-α and increasing the level of IL-12α; even more preferably, balancing the levels of inflammatory factors includes reducing the mRNA level of TNF-α and increasing the mRNA level of IL-12α.

[0084] In some specific implementations, the enhancement of immunity includes increasing the number of innate immune cells; preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and / or macrophages; more preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and macrophages.

[0085] In some preferred embodiments, the enhancement of immunity includes promoting T cell development, promoting T cell activation, balancing inflammatory factor levels, and increasing the number of innate immune cells.

[0086] Based on this, the present invention also provides the use of the above-mentioned nutritional composition in the preparation of products having any one or more of the following (a)-(d):

[0087] (a) Promoting T cell development; preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and / or RAG2; more preferably, promoting T cell development includes increasing the expression levels of genes RAG1 and RAG2; even more preferably, promoting T cell development includes increasing the mRNA levels of genes RAG1 and RAG2.

[0088] (b) Promoting T cell activation; preferably, promoting T cell activation includes increasing the expression levels of genes ZAP70 and / or IFNGR1; more preferably, promoting T cell development includes increasing the expression levels of genes ZAP70 and IFNGR1; even more preferably, promoting T cell development includes increasing the mRNA levels of genes ZAP70 and IFNGR1.

[0089] (c) Balancing inflammatory factor levels; preferably, balancing inflammatory factor levels includes reducing TNF-α levels and / or increasing IL-12α levels; more preferably, balancing inflammatory factor levels includes reducing TNF-α levels and increasing IL-12α levels; even more preferably, balancing inflammatory factor levels includes reducing TNF-α mRNA levels and increasing IL-12α mRNA levels;

[0090] (d) Increasing the number of innate immune cells; preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and / or macrophages; more preferably, increasing the number of innate immune cells includes increasing the number of neutrophils and macrophages.

[0091] The present invention does not specifically limit the products containing or prepared from the above-described nutritional composition, such as food products. When the product is a food product, the components of the nutritional composition are used in a source or form that meets the requirements of local laws and regulations. For example, in some embodiments, the lysophosphatidylethanolamine in the food product is derived from dairy products containing it (e.g., buttermilk products, phospholipid-containing milk proteins, milk phospholipid products, whole milk, whole milk powder, and / or phospholipid-containing cream products, etc.) and / or enzymatically hydrolyzed soybean lecithin, and the lactoferrin in the food product is derived from whole milk containing lactoferrin, whole milk powder, colostrum powder, whey protein powder, and / or lactoferrin powder.

[0092] In some embodiments, the food is infant food, children's food, adolescent food, maternal food, adult food, or food for the middle-aged and elderly. In some embodiments, the food of the present invention is candy, such as hard candy, gel candy, shortbread candy, compressed candy, and aerated candy.

[0093] In some embodiments, the food described in this invention is a beverage, such as carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages. In some embodiments, the food described in this invention is a dairy product, such as milk powder, cheese, yogurt, and liquid milk. In some embodiments, the food described in this invention is a baked product, such as bread, cakes, and biscuits. In some embodiments, the food described in this invention is a dietary supplement, such as hard capsules, soft capsules, tablets, oral liquids, pills, granules, and powders.

[0094] In some embodiments, the food described in this invention is in the form of a liquid or a solid under normal temperature conditions.

[0095] In some embodiments, in the food product of the present invention, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500), for example, it can be 1:0.1, 1:0.2, 1:0.4, 1:0.8, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:500, etc. The ratios are 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, etc.; preferably, the mass ratio of the two is 1:(0.2-1000); more preferably, the mass ratio of the two is 1:(1-500); even more preferably, the mass ratio of the two is 1:(10-100); preferably, the above ratio is the mass ratio of LPE (18:2(9Z,12Z) / 0:0) to lactoferrin.

[0096] This invention does not impose any specific absolute limits on the content of lysophosphatidylethanolamine, LPE (18:2(9Z,12Z) / 0:0), and LF in food, as long as they meet the requirements of local food-related laws and regulations.

[0097] In some embodiments, the content of LPE (18:2(9Z,12Z) / 0:0) in the food may be 1 mg / 100g-100 mg / 100g, and the content of lactoferrin may be 400 mg / 100g-1900 mg / 100g.

[0098] In addition to the components described above in the nutritional composition, the food may also contain other ingredients, such as common food ingredients like proteins / amino acids, carbohydrates, fats, vitamins, and minerals.

[0099] Furthermore, depending on the type of food and the end needs of the target audience, in some embodiments, the food contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0100] Examples of plant-based ingredients include fruits such as figs, pomegranates, kiwis, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, amla, and bilberries, or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, and kidney beans, or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, and ginkgo nuts, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0101] Animal dairy product ingredients can include fresh milk from cows and sheep, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0102] Examples of animal meat product ingredients include pork, beef, mutton, seafood, and poultry.

[0103] Examples of functional additives include vitamin supplements (such as vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, biotin, etc.), mineral supplements (such as iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (e.g.), dietary fiber (e.g., inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.), and functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.).

[0104] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0105] Example

[0106] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0107] Experiment Example 1: Regulation of T cell development by different nutrients and their combinations

[0108] 1. Experimental Methods

[0109] 1.1. Experimental Materials

[0110] LPE 18:2 (LPE(18:2(9Z,12Z) / 0:0), is lysophosphatidylethanolamine with a linoleic acid chain linked at the sn-1 position and lacking a fatty acid chain at the sn-2 position.) was provided by Heilongjiang Feihe Dairy Co., Ltd.; lactoferrin was provided by Heilongjiang Feihe Dairy Co., Ltd.; cyclophosphamide was provided by Shanghai Maclean Biochemical Technology Co., Ltd.; levamisole was provided by Shanghai Maclean Biochemical Technology Co., Ltd.

[0111] E3 medium: 5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate, purchased from Beijing Chemical Reagent Company. All reagents were of analytical grade.

[0112] 1.2. Laboratory Animals

[0113] Wild-type AB lineage zebrafish, bred and maintained by the Precision Nutrition and Active Health Center of Yantai University.

[0114] Zebrafish daily maintenance: Adult zebrafish are housed in an Aisheng zebrafish recirculating aquarium system, with a 14-hour / 10-hour light / dark cycle daily. The water temperature is maintained at 28±0.5℃, pH at 7.0-8.0, and conductivity at around 500 μs. They are fed fresh brine shrimp eggs twice daily.

[0115] Zebrafish spawning: Remove the mating box, insert a separator in the middle to separate the males and females, add circulating water to the system until it is 80% full, and finally place it in the isolation tank to prevent the adult fish from eating the embryos. After the adult zebrafish finish feeding at night, place them on both sides of the separator according to a male:female ratio of 1:2. The next morning, remove the separator. After the male and female fish chase each other, they will spawn and fertilize externally. After 0.5-1 hours, collect the adult fish into the circulating water system, mark the date, and use a filter to collect the embryos in a glass culture dish containing E3 water and culture them at 28±0.5℃.

[0116] Establishment of a zebrafish juvenile immunodeficiency model: Wild-type AB strain zebrafish at 4 dpf were randomly selected and placed in 6-well plates, 30 fish per well. A blank control group, a model group, a positive control group, and a sample treatment group were set up. The blank control group was cultured normally in E3 water. All other groups were treated with 2.5 mM cyclophosphamide, and the positive control group was given 25 μg / mL levamisole. The sample treatment groups received different nutrient compositions.

[0117] 1.3. Experimental Apparatus

[0118] Zebrafish recirculating aquaculture system (ESEN-AW-S1, Beijing Aisheng Technology Development Co., Ltd.); Electronic balance (AR-2140, Shanghai Ohaus Instruments Co., Ltd.); Ultrapure water system (TC-RO-100, Shanghai Likang Instruments Co., Ltd.); pH meter (pH9500, Shanghai Peirui Instruments Co., Ltd.); Conductivity meter (HM-100TDS, HM Digital Co., Ltd., South Korea); Thermometer (high precision, Odasys Technology Co., Ltd.); Ultra-low temperature freezer (HYCD-205, Qingdao Haier Co., Ltd.); High-speed refrigerated centrifuge (Micro 21R, Thermo Fisher Scientific, USA); Mini centrifuge (LX-500, Anhui Zhongke Co., Ltd.); Real-time quantitative PCR system (7500 Fast, Thermo Fisher Scientific, USA); Ultra-micro spectrophotometer (ND5000, Wuxi Baitek Biotechnology Co., Ltd.).

[0119] 1.4. Experimental Intervention Design

[0120] In this study, LPE 18:2 and lactoferrin were combined in different single doses or ratios to investigate their synergistic effect in improving immunodeficiency. The experimental design is shown in Table 1. The control group consisted of normal zebrafish without any treatment or intervention. The model group consisted of zebrafish treated with 2.5 mM cyclophosphamide. Specifically, 2.5 mM cyclophosphamide was dissolved in E3 water, and 3 ml of the solution was placed in the wells of a 6-well plate and fed to the zebrafish. The positive control group consisted of zebrafish treated with 2.5 mM cyclophosphamide + 25 μg / mL levamisole. Specifically, 2.5 mM cyclophosphamide was dissolved in E3 water, and then 25 μg / mL levamisole was dissolved in the same solution. 3 ml of this solution was placed in the wells of a 6-well plate and fed to the zebrafish to form the positive control group. The nutrient intervention group consisted of zebrafish treated with 2.5 mM cyclophosphamide and other nutrients. Specifically, cyclophosphamide was dissolved in E3 water, and the test sample was then dissolved in the solution. 3 ml of the solution was then placed in the wells of a 6-well plate containing zebrafish to feed them, forming the nutrient intervention group. Comparative Examples 1-3 were intervention groups treated with 2.5 mM cyclophosphamide + 0.06 μg / mL, 0.5 μg / mL, and 1 μg / mL LPE 18:2; Comparative Examples 4-6 were intervention groups treated with 2.5 mM cyclophosphamide + 0.2 μg / mL, 30 μg / mL, and 60 μg / mL lactoferrin; Examples 1-8 were intervention groups treated with compositions formed by combining 2.5 mM cyclophosphamide + LPE 18:2 and lactoferrin at different concentration ratios (1:0.2-1000).

[0121] Table 1. Experimental Intervention Design

[0122]

[0123] 1.5. qPCR method for analyzing immune-related genes

[0124] Wild-type AB strain zebrafish with a 4 dpf growth rate were randomly selected and randomly assigned to 6-well plates, with 30 zebrafish treated in each well (experimental group). After treatment at 28℃ for 24 h, all 30 zebrafish from each well were collected into 2 mL RNase-free centrifuge tubes, the water was aspirated, and 350 μL of pre-chilled lysis buffer (Ciscotech, catalog number: AC0202-B) and 2 sterilized 3 mm steel beads were added. The cells were then lysed in a cell disruptor at 4℃.

[0125] Total RNA was extracted using the Cisco SPARKeasy RNA Rapid Extraction Kit. The simplified steps included: lysis followed by mixing with anhydrous ethanol, transferring to an adsorption column, centrifuging, and discarding the eluent; washing the adsorption column sequentially with washing buffer; and finally eluting RNA with RNase-free water. RNA concentration and purity were determined using a micro-spectrophotometer. An A260 / A280 ratio of 1.8–2.0 was considered acceptable. Acceptable samples were stored at -80°C for later use.

[0126] An equal volume of total RNA was used to synthesize cDNA using a Cisco reverse transcription kit. Genomic DNA was first removed by treatment with a gDNA Eraser at 42°C for 2 min. Then, SPARKscript II RT Plus Master Mix was added, and the reaction was terminated by heating at 85°C for 5 min. The cDNA product was stored at -20°C.

[0127] The mRNA expression level of the gene was detected using the Cisco Real-Time Quantitative PCR Kit.

[0128] The reaction mixture (20 μL) contained: 10 μL of 2×SYBR qPCR Mix, 0.4 μL each of forward and reverse primers, 1 μL of cDNA template, and 8.2 μL of RNase-free H2O. Each sample was tested in triplicate, normalized to an internal control gene, and subjected to 2... ΔΔCt The relative gene expression levels were calculated using a method. Primer sequences are shown in Table 2.

[0129] Experimental data are expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism software, and Dunnett's test was used for comparisons between groups. p < 0.05 was considered statistically significant.

[0130] Table 2 Primer Sequences

[0131]

[0132] 2. Experimental Results

[0133] 2.1 Toxicity test results

[0134] The immune system plays a crucial role in maintaining homeostasis, defending against pathogen invasion, and responding to adverse external stimuli. Cyclophosphamide (CTX) is a classic alkylating immunosuppressant that exhibits significant toxicity to rapidly dividing immune cells by interfering with DNA synthesis, inhibiting cell proliferation, and inducing apoptosis. CTX also significantly inhibits lymphocyte production and differentiation, disrupting the normal development and function of the immune system, thereby leading to a decreased immune response. Therefore, CTX is often used to construct stable and reproducible animal models of immune dysfunction or immunodeficiency.

[0135] In this experiment, the effects of different concentrations of cyclophosphamide on zebrafish were first investigated. The results showed that the survival rate of zebrafish was still 100% when the maximum concentration of cyclophosphamide was 2.5 mM. When the concentration was further increased to 5 mM, the survival rate dropped to 82%. When the concentration was further increased to 10 mM, the survival rate of zebrafish was 0. Therefore, in subsequent experiments, the cyclophosphamide treatment concentration of 2.5 mM was uniformly selected.

[0136] Before investigating the immune-enhancing function, the toxicity and lethality of the two nutrients, both monomers and their combinations, in zebrafish were examined. This study found that LPE18:2 at three concentrations (0.06 μg / mL, 0.5 μg / mL, and 1 μg / mL), LF at three concentrations (0.2 μg / mL, 30 μg / mL, and 60 μg / mL), and in eight different combinations formed by combining the two in varying proportions, all exhibited 100% survival rates without any other toxicity. This indicates that the monomer concentrations of the two nutrients used in this experiment, and their synergistic combinations, do not pose a toxic threat to zebrafish and do not affect their survival rate.

[0137] 2.2 Results of T cell development-related gene expression detection

[0138] Nutrients can significantly upregulate the mRNA expression and protein activity of RAG1 and RAG2 by activating T cell development signaling pathways (such as the Notch signaling pathway) within the thymus. RAG1, as the core catalytic subunit for T cell receptor (TCR) gene rearrangement, forms a recombination complex with RAG2, which can specifically recognize and cleave TCR gene fragments, promoting V(D)J rearrangement efficiency. The synergistic effect of both can increase the number of mature T cells (CD4+). + / CD8 + The amount of T cells produced.

[0139] This invention found that the expression levels of RAG1 and RAG2 genes in zebrafish induced by cyclophosphamide were significantly lower than those in the control group. However, after intervention with a positive control drug, the expression levels of both key genes were upregulated. The experimental results are shown in Table 3, and the significance analysis of the differences between groups is shown in Table 4. From Table 3, we can see that in comparative examples 1-3, after intervention with LPE 18:2 monoclonal antibody, the expression levels of both RAG1 and RAG2 genes were increased to varying degrees. Comparative example 3 showed the highest increase in the expression levels of these two genes. Furthermore, the significance analysis revealed that the expression levels of both RAG1 and RAG2 genes in all three comparative examples were significantly higher than those in the model group (p-values ​​are shown in Table 4). This indicates that LPE... 18:2 LF intervention alone, at a certain dose, can increase the expression levels of RAG1 and RAG2 genes in zebrafish. Comparative Examples 4-6 show that LF intervention alone also increases the expression levels of RAG1 and RAG2 genes to some extent. However, after significance analysis, it was found that there was no significant difference between the low-dose intervention (Comparative Example 4) and the model group (p>0.05), while the expression levels of both RAG1 and RAG2 genes were significantly higher than those in the model group under medium and high-dose interventions (Comparative Examples 5 and 6). This indicates that LF intervention alone, at a certain dose, can effectively increase the expression levels of RAG1 and RAG2 genes in zebrafish. The expression levels of RAG1 and RAG2 genes were effectively reduced. Examples 1-8 showed that combined intervention with the two nutrients in different proportions significantly increased the expression levels of RAG1 and RAG2 genes in zebrafish, with both genes exceeding 1. As shown in Table 3, the expression level of RAG1 gene was the lowest in Example 1, averaging about 1.015, while it was the highest in Example 8, at about 1.66. Similarly, the expression level of RAG2 gene was the lowest in Example 5, at about 1.06, while it was the highest in Example 8, at about 1.92. Further analysis revealed that all eight examples were significantly higher than the model group (p < 0.0001), and all eight examples were significantly higher than the six comparative examples (p values ​​are shown in Table 4). This indicates that the combined intervention of LPE 18:2 and LF is more effective than the intervention of either of the individual nutrients alone. Further analysis revealed that the effects of the eight examples on the expression levels of RAG1 and RAG2 genes were significantly higher than the direct sum of the effects of the interventions at the same individual doses compared to the model group. This indicates that the two nutrients, LPE 18:2 and LF, played a synergistic role.

[0140] This part of the study shows that the combination of LPE 18:2 and LF in a ratio of 1:0.2-1000 can promote T cell development and maturation, increase the "functional T cell pool that can recognize pathogens", and lay the foundation for immune cell reserves.

[0141] Table 3. Effects of different monomers and nutrient compositions on the expression of T cell development-related genes.

[0142]

[0143] Table 4. Analysis of significant differences in the expression of T cell development-related genes in zebrafish from different groups.

[0144]

[0145]

[0146] Experiment Example 2: Effects of different nutrients and combinations on T cell activation

[0147] 1. Experimental Methods

[0148] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0149] 1.2 The experimental animals were the same as in Experiment 1.

[0150] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0151] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0152] 1.5 qPCR method was used to analyze immune-related genes, as in Experiment 1.

[0153] 2. Experimental Results

[0154] T cell activation is a crucial turning point in the transition of T cells from a "resting state" to a "functional state." It is a core link connecting "T cell development and maturation" with "immune effector output," and its role directly determines the efficiency, intensity, and direction of the immune response. This invention uses two core indicators, ZAP70 and IFNGR1, to examine the effects of different nutrient interventions on T cell activation.

[0155] ZAP70 is a core kinase in the T cell antigen receptor (TCR) signaling pathway. After TCR recognizes an antigen, it can be recruited by the CD3ζ chain and undergo tyrosine phosphorylation. Enhanced binding efficiency of ZAP70 to the CD3ζ chain promotes the activation of downstream LAT and PLC-γ1 molecules, ensuring the transformation of T cells from a "resting state" to an "activated state." In this invention, ZAP70 was used as a standard for T cell activation. As shown in Table 5, the ZAP70 gene expression level in zebrafish after cyclophosphamide modeling was significantly lower than that in the control group (p < 0.0001, as shown in Table 6), while the ZAP70 gene expression level significantly increased back to the control group level after intervention with positive control drugs. Similarly, as shown in Table 5, in Comparative Examples 1-3, after intervention with LPE 18:2 as a single agent, the gene expression level of ZAP70 increased continuously with the increase of intervention concentration. After significant difference analysis, it was found that, except for Comparative Example 1, the other two comparative examples were significantly higher than the model group (p values ​​are shown in Table 6). This indicates that LPE 18:2 alone at a certain dose can increase the gene expression level of ZAP70 in zebrafish. Comparative Examples 4-6 showed that after intervention with LF alone, the gene expression level of ZAP70 also increased to varying degrees, but compared with LPE... The increase in ZAP70 expression was slightly lower with the 18:2 ratio, and significant difference analysis revealed that, except for Comparative Example 4 which showed no significant difference from the model group (p > 0.05), Comparative Examples 5 and 6 were significantly higher than the model group (p values ​​are shown in Table 6). This indicates that LF alone at a certain dose can also increase the expression level of ZAP70 in zebrafish. Examples 1-8 showed that when the two substances were combined in different ratios, the combined intervention further increased the expression level of ZAP70 in zebrafish compared to the comparative examples. In particular, the expression level in Example 8 reached a maximum of 1.720, while the expression level in Example 4 was the lowest at 1.099, but still higher than the control group. Significant difference analysis revealed that all 8 examples were significantly higher than the model group (p values ​​are shown in Table 6), and all 8 examples were significantly higher than the 6 comparative examples (p values ​​are shown in Table 6). This indicates that the combined intervention of LPE 18:2 and LF is more effective than the intervention of the two individual nutrients alone. Further analysis revealed that the effects of the eight examples on ZAP70 gene expression levels were significantly higher than the direct sum of the effects of the interventions at the same single dose compared to the model group. This indicates that the two nutrients, LPE 18:2 and LF, have a synergistic effect. This suggests that the combination of LPE 18:2 and LF can significantly increase ZAP70 gene expression levels, implying that the synergistic effect of the two substances can fully open the T cell receptor (TCR) signaling pathway, allowing mature T cells to respond rapidly to antigens and avoiding "immune sluggishness."

[0156] IFNGR1, as the α-chain receptor for interferon-γ (IFN-γ), directly determines the T cell's ability to receive IFN-γ signals. Nutrients can activate the JAK-STAT pathway, promoting the localized expression of IFNGR1 on the T cell membrane, enhancing the T cell's sensitivity to IFN-γ, and further amplifying activation signals (such as promoting T cell proliferation). This invention found that after cyclophosphamide modeling, the IFNGR1 gene expression level in zebrafish was significantly lower than that in the control group (p < 0.0001, as shown in Table 6), while after intervention with positive control drugs, the IFNGR1 gene expression level significantly increased back to the control group level. Similarly, as shown in Table 5, in Comparative Examples 1-3, after intervention with LPE 18:2 alone, the gene expression level of IFNGR1 increased continuously with the increase of intervention concentration. Furthermore, after significance analysis, all three comparative examples were significantly higher than the model group (p values ​​are shown in Table 6). This indicates that LPE 18:2 alone can increase the gene expression level of IFNGR1 in zebrafish. Comparative Examples 4-6 show that IFNGR1 gene expression also increased to varying degrees after intervention with LF alone, but compared to LPE... The increase in IFNGR1 expression was slightly lower with the 18:2 ratio, and statistical analysis revealed no significant difference between the low-dose group (Comparative Example 4) and the model group. However, Comparative Examples 5 and 6 were significantly higher than the model group (p values ​​are shown in Table 6). This indicates that LF alone at a certain dose can also increase the IFNGR1 gene expression level in zebrafish. Examples 1-8 showed that the combined intervention of the two substances in different ratios further increased the IFNGR1 gene expression level in zebrafish compared to the comparative examples, especially in Example 8 where the expression level reached a maximum of 1.077. Statistical analysis revealed that all eight examples were significantly higher than the model group (p values ​​are shown in Table 6), and all eight examples were significantly higher than the six comparative examples (p values ​​are shown in Table 6). This indicates that the combined intervention of LPE 18:2 and LF is more effective than the intervention of either single nutrient alone. Further analysis revealed that the effects of the eight examples on IFNGR1 gene expression were significantly higher than the direct sum of the effects of interventions at the same individual doses compared to the model group. This indicates that the two nutrients, LPE 18:2 and LF, played a synergistic role, which means that the synergistic effect of the two substances enhanced the activation efficiency and functional orientation of T cells.

[0157] Table 5. Effects of different monomers and nutrient compositions on the expression levels of T cell activation-related genes.

[0158]

[0159] Table 6. Analysis of significant differences in the expression levels of T cell activation-related genes among groups.

[0160]

[0161]

[0162] Experiment Example 3: Effects of different nutrients and their combinations on the expression of inflammatory factors in the body

[0163] 1. Experimental Methods

[0164] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0165] 1.2 The experimental animals were the same as in Experiment 1.

[0166] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0167] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0168] 1.5 qPCR method was used to analyze immune-related genes, as in Experiment 1.

[0169] Inflammatory factors are a core effector mechanism in the immune function of T cells. After promoting T cell development, maturation, and activation, inflammatory factors are the manifestation of T cells converting intracellular activation signals into a systemic immune response, reflecting their ability to fight pathogens or regulate other immune cells. This invention investigated the changes in two pro-inflammatory factors, tumor necrosis factor-α (TNF-α) and interleukin-12α subunit (IL-12α). The gene expression levels of these two inflammatory factors are shown in Table 7, and the results of the significance analysis of differences between groups are shown in Table 8.

[0170] Tumor necrosis factor-α (TNF-α) is mainly secreted by activated macrophages, T cells, NK cells, mast cells, and certain tissue cells (such as adipocytes). TNF-α is a core initiator of the inflammatory cascade, potently pro-inflammatory and inducing apoptosis, playing a crucial role in autoimmune diseases, sepsis, and metabolic diseases. This invention first investigated the effects of LPE 18:2 and LF monomers and compositions on the expression level of the inflammatory cytokine TNF-α gene in immunodeficient zebrafish. Table 7 shows that the TNF-α gene expression level in the immunodeficient model group increased to 1.94. One-way ANOVA analysis revealed a significant difference between the model group and the control group (p < 0.0001). After intervention with the positive control drug levamisole, the TNF-α gene expression level decreased to 0.472, indicating that the positive control drug intervention inhibited the initiation and outbreak of the inflammatory cytokine TNF-α. Furthermore, we investigated the effects of different doses of LPE 18:2 and LF as monomers and the complex formed by combining the two in different proportions on the expression level of TNF-α gene in zebrafish after modeling. Table 7 shows that LPE18:2 as a single agent significantly reduced TNF-α gene expression in zebrafish (Comparative Examples 1-3). Statistical analysis revealed that all three comparative examples were significantly lower than the model group (p < 0.0001), indicating that LPE18:2 alone can significantly reduce the expression of the pro-inflammatory factor TNF-α gene in zebrafish. Comparative Examples 4-6 showed that LF alone also significantly reduced TNF-α expression (p < 0.0001), but the reduction was slightly less than that of LPE18:2 alone. Examples 1-8 showed that combined intervention with different proportions of the two substances further enhanced the effect, with the highest TNF-α gene expression level at 0.766 (Example 4) and the lowest at 0.381 (Example 8). Statistical analysis revealed that all eight examples were significantly lower than the model group (p < 0.0001), and all eight examples were significantly lower than the six comparative examples (p values ​​are shown in Table 8). This indicates that LPE18:2 alone can significantly reduce the expression of the pro-inflammatory factor TNF-α gene in zebrafish. The combined intervention of LPE 18:2 and LF was more effective than the intervention of either nutrient alone. Further analysis revealed that the effect of the model group on the pro-inflammatory factor TNF-α in the eight examples was significantly higher than the direct sum of the effects of the interventions at the same individual doses, indicating that the two nutrients, LPE 18:2 and LF, had a synergistic effect. This suggests that the combination of LPE 18:2 and LF can reduce the occurrence of chronic inflammation, tissue damage, immune depletion, and metabolic disorders caused by persistently high TNF-α expression.

[0171] Interleukin-12 (IL-12) is a key cytokine primarily secreted by antigen-presenting cells (APCs), such as monocytes, macrophages, and dendritic cells, in response to infection or inflammation. IL-12 activates NK cells and T cells, promotes Th1 cell differentiation, bridges innate and adaptive immunity, has anti-tumor and anti-infection effects, and enhances memory T cell function. In the later stages of infection, it can improve the immune system's ability to prevent subsequent infections by promoting the generation and maintenance of memory T cells. IL-12α and IL-12β together form IL-12, a key guiding factor for regulating T cell function. Insufficient expression of IL-12α leads to impaired Th1 cell differentiation, resulting in activated T cells with a deviated functional direction. Table 7 shows that the expression level of IL-12α gene in zebrafish in the immunocompromised model group was significantly lower than that in the control group (p < 0.0001). However, after intervention with the positive control drug levamisole, the expression level of TNF-α gene increased to 1, indicating that the positive control drug intervention promoted the expression of IL-12α. Further, we investigated the effects of different doses of LPE 18:2 and LF as monomers and complexes formed by their different ratios on the expression level of IL-12α gene in zebrafish after modeling. Table 7 shows that after intervention with LPE 18:2 as a monomer (Comparative Examples 1-3), the expression level of IL-12α gene was increased to varying degrees, and it increased continuously with the increase of LPE 18:2 concentration. However, after significance analysis, except for Comparative Example 1 which showed no significant difference from the model group (p > 0.05), Comparative Examples 2 and 3 were significantly higher than the model group (p < 0.0001). This indicates that LPE 18:2 and LF 18:2 as monomers had a significant effect on the expression level of IL-12α gene in zebrafish after modeling. When 18:2 was used alone, it significantly increased the expression level of the IL-12α gene in zebrafish at a certain dose. Comparative Examples 4-6 showed that LF alone also increased the expression level of the inflammatory factor IL-12α to varying degrees. However, at the same low concentration (Comparative Example 4), there was no significant difference between the two groups (p>0.05), while at medium and high concentrations (Comparative Examples 5 and 6), the expression levels were significantly higher than the model group (p<0.0001). This indicates that LF alone can also increase the expression level of IL-12α in zebrafish at a certain concentration. Examples 1-8 showed that the combined intervention of the two substances in different proportions further enhanced the effect. The highest IL-12α gene expression level was 0.961 (Example 8), and the lowest was 0.746 (Example 4). After significance analysis, all 8 examples were significantly higher than the model group (p<0.0001), and the 8 examples were significantly higher than the 6 comparative examples (p values ​​are shown in Table 8). This indicates that LPE The combined intervention of 18:2 and LF was more effective than the intervention of either single nutrient alone.Further analysis revealed that the effects of the eight embodiments on the inflammatory factor IL-12α were significantly higher than the direct sum of the effects of the interventions at the same single dose compared to the model group. This indicates that the two nutrients, LPE 18:2 and LF, have a synergistic effect. This suggests that the combination of LPE 18:2 and LF can promote the expression of the important positive immune regulatory factor IL-12α, indicating enhanced immune defense function, improving the body's immune function without causing excessive inflammatory damage.

[0172] Table 7. Effects of different monomers and nutritional compositions on the expression levels of inflammatory factor genes.

[0173]

[0174] Table 8. Analysis of significant differences in the expression levels of inflammatory factor genes in zebrafish from different groups.

[0175]

[0176]

[0177] Experiment Example 4: Effects of different nutrients and combinations on the body's innate immune cells

[0178] 1. Experimental Methods

[0179] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0180] 1.2 The experimental animals were the same as in Experiment 1.

[0181] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0182] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0183] 1.5 Neutrophil detection (Sudan Blick B staining method)

[0184] Juvenile zebrafish (7 dpf) were washed 2-3 times with PBS and then fixed overnight in freshly prepared 4% paraformaldehyde (PFA) solution at 4°C in the dark using a shaker. After fixation, the samples were washed 3 times with PBS (5 min each time). Subsequently, the samples were stained for 1.5-2 h at room temperature in the dark using Sudan Blick B working solution with gentle shaking. After staining, the samples were washed 3 times with 70% ethanol (5-10 min each time) to remove unbound dye, and then rehydrated twice with PBS-Tween (1× PBS + 0.1% Tween-20). The samples were then transferred to KOH / H2O2 destaining solution and treated at room temperature for 5-10 min. The degree of melanin removal was monitored under a microscope, and the samples were then washed twice with PBS-Tween to stabilize the signal. After decolorization, the juvenile fish were preserved in PBS and single-plane horizontal scanning imaging (xy plane) was performed using a laser confocal microscope with a water immersion objective (10×). Z-stick scanning was not performed, and only the distribution of neutrophils within the same focal plane was observed and analyzed.

[0185] 1.6 Macrophage detection (Neutral Red staining)

[0186] Wild-type zebrafish juveniles (7 dpf) were used. Neutral Red was dissolved in E3 culture medium to prepare a 2.5 μg / mL working solution under light-protected conditions. The juveniles were transferred to the dye-containing E3 solution and incubated at 28.5℃ in the dark for 6-8 h. After staining, the fish were washed 2-3 times with fresh E3 to remove excess dye, and then anesthetized for 1-2 min with 0.2 mg / mL Tricaine (MS-222, buffered with NaHCO3 to pH 7.0-7.4). Subsequently, observation and imaging were performed using a stereomicroscope. The red spots appearing in the juvenile fish represented the accumulation signal of Neutral Red in macrophages within lysosomes, used to assess the number and distribution of macrophages.

[0187] 2. Experimental Results

[0188] After activation, T cells do not function in isolation. Instead, they secrete pro-inflammatory factors to provide "activation signals" to innate immune cells, such as macrophages and neutrophils, thereby achieving synergistic enhancement of innate immune cells.

[0189] Neutrophils are the main force in acute infections, reacting rapidly but with a short lifespan, primarily responsible for phagocytizing and eliminating pathogens. Table 9 shows that the average fluorescence intensity of neutrophils in the control group of zebrafish was 111, while after cyclophosphamide modeling, the number of neutrophils significantly decreased, with fluorescence intensity dropping to 35.33. One-way ANOVA analysis revealed a significant difference (p < 0.0001). However, after intervention with the positive control drug levamisole, the average fluorescence intensity of neutrophils recovered to approximately 107. Furthermore, we investigated the effects of different doses of LPE 18:2 and LF monomers and complexes formed by their different ratios on the fluorescence intensity of neutrophils in zebrafish after modeling. Table 9 shows that after LPE 18:2 monomeric intervention (comparative examples 1-3), the mean fluorescence intensity of neutrophils continuously increased with increasing concentration. Furthermore, after significant difference analysis, all three comparative examples were significantly higher than the model group (p < 0.01 or p < 0.0001). This indicates that LPE... 18:2 alone can increase the number of neutrophils. Comparative examples 4-6 showed that the average fluorescence intensity of neutrophils was also increased after LF alone. However, after significance analysis, it was found that there was no significant difference between comparative example 4 and the model group (p>0.05), while comparative examples 5 and 6 were significantly higher than the model group (p<0.0001). This indicates that LF alone can also increase the number of neutrophils when a certain dose is reached. Examples 1-8 showed that the combined intervention of the two substances in different proportions further enhanced the effect. The lowest fluorescence intensity of neutrophils was 73 (Example 1) and the highest was 97 (Example 8). After significance analysis, it was found that all 8 examples were significantly higher than the model group (p<0.0001) and significantly higher than the 6 comparative examples (p values ​​are shown in Table 10). This indicates that the combined intervention of LPE 18:2 and LF is better than the intervention of the two individual nutrients alone. Further analysis revealed that the improvement in neutrophil fluorescence intensity in the eight examples was significantly higher than the direct sum of the intervention effects at the same single dose compared to the model group, indicating that the two nutrients, LPE 18:2 and LF, played a synergistic role.

[0190] Macrophages have a more persistent effect, participating not only in phagocytosis and degradation of cellular debris and pathogens, but also in antigen presentation and tissue repair, and regulating immune responses by releasing cytokines. Table 9 shows that the average fluorescence intensity of reactive macrophages in the control group zebrafish was 100.33±3.21, while after cyclophosphamide modeling, macrophage numbers significantly decreased, with fluorescence intensity dropping to 14.67±1.15. One-way ANOVA analysis revealed a significant difference between the two (p<0.0001). However, after intervention with the positive control drug levamisole, macrophage fluorescence intensity recovered to 92.00±3.00. Furthermore, we investigated the effects of different doses of LPE 18:2 and LF monomers and complexes formed by their different ratios on the fluorescence intensity of macrophages in zebrafish after modeling. Table 9 shows that after LPE 18:2 monomeric intervention (comparative examples 1-3), the average fluorescence intensity of macrophages increased to the range of 23.33-43. Significant differences were observed in all three comparative examples, which were significantly higher than the model group (p < 0.01 or p < 0.0001). This indicates that LPE... 18:2 alone can increase the number of macrophages; Comparative Examples 4-6 show that the average fluorescence intensity of macrophages increased to 23.33-36.67 after intervention with LF alone. Similarly, the significance analysis showed that all three comparative examples were significantly higher than the model group (p<0.01 or p<0.0001), indicating that intervention with LF alone can increase the number of macrophages; Examples 1-8 show that the combined intervention of the two substances in different proportions further enhances the effect. The lowest macrophage fluorescence intensity was 51.00 (Example 1) and the highest was 82.67 (Example 8). The significance analysis showed that all 8 examples were significantly higher than the model group (p<0.0001), and the 8 examples were significantly higher than the 6 comparative examples (p values ​​are shown in Table 10). This shows that the combined intervention of LPE 18:2 and LF is better than the intervention of the two individual nutrients alone. Further analysis revealed that the improvement in macrophage fluorescence intensity in the eight examples was significantly higher than the direct sum of the intervention effects at the same single dose compared to the model group, indicating that the two nutrients, LPE 18:2 and LF, played a synergistic role.

[0191] Table 9. Effects of different monomers and nutritional compositions on immune cells.

[0192]

[0193] Table 10. Significant differences in immune cells among different groups of zebrafish.

[0194]

[0195]

Claims

1. A nutritional composition, characterized in that, The nutritional composition is a nutritional composition that helps enhance immunity, and the nutritional composition contains the necessary active ingredients shown in (I) and (II) below: (I) Lysophosphatidylethanolamine; (II) Lactoferrin; Wherein, the essential active ingredient shown in (I) contains at least unsaturated lysophosphatidylethanolamine, and the fatty acid chain in the unsaturated lysophosphatidylethanolamine has a length of 18 carbon atoms, the fatty acid chain in the unsaturated lysophosphatidylethanolamine is attached at the sn-1 position, and in the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(0.1-1500), and within the mass ratio range, the essential active ingredient shown in (I) and the essential active ingredient shown in (II) synergistically exert an effect that helps to enhance immunity.

2. The nutritional composition according to claim 1, characterized in that, The lysophosphatidylethanolamine is provided in the form of dairy products containing it and / or enzymatically hydrolyzed soybean lecithin.

3. The nutritional composition according to claim 1, characterized in that, The lactoferrin is provided in the form of whole milk containing lactoferrin, whole milk powder, colostrum powder, whey protein powder and / or lactoferrin powder.

4. Use of the nutritional composition according to any one of claims 1-3 in the preparation of products that help enhance immunity.

5. The use according to claim 4, characterized in that, The benefits of enhancing immunity include at least one of the following: promoting T cell development, promoting T cell activation, balancing inflammatory factor levels, and increasing the number of innate immune cells.

6. The use according to claim 5, characterized in that, The promotion of T cell development includes increasing the expression levels of genes RAG1 and / or RAG2; and / or, the promotion of T cell activation includes increasing the expression levels of genes ZAP70 and / or IFNGR1; and / or, the balancing of inflammatory factor levels includes decreasing the level of TNF-α and / or increasing the level of IL-12α; and / or, the increase in the number of innate immune cells includes increasing the number of neutrophils and / or macrophages.

7. The use according to any one of claims 4-6, characterized in that, The product is either solid or liquid.

8. The use according to any one of claims 4-6, characterized in that, The product contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, and functional additives.

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