Egg white protein source peptides with improved malnutrition improving immunopotentiation and uses thereof

Abstract

This invention discloses egg white protein-derived peptides that improve malnutrition and enhance immunity, and their applications, belonging to the field of small molecule peptide technology. This invention prepares egg white protein peptides by sequentially hydrolyzing egg white protein with alkaline protease, papain, and neutral protease, among which the peptides contain the immune-enhancing tripeptide LPR. Animal model functional validation shows that egg white protein peptides containing tripeptide LPR can significantly increase serum levels of total protein, albumin, prealbumin, and globulin, as well as the content of short-chain fatty acids in the intestine, achieving the effect of improving malnutrition. The food-derived bioactive peptides have high biosafety. Therefore, tripeptide LPR or egg white protein peptides containing this peptide can be used to prepare drugs or health foods that improve malnutrition and enhance immunity. This invention provides theoretical support for the efficacy of egg white protein peptides in improving malnutrition.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to an egg white protein-derived peptide that improves malnutrition and enhances immunity, and its applications. Background Technology

[0002] Malnutrition is a global problem affecting human health. Its direct cause is that the body's nutrition and energy cannot meet its needs. Typical signs include low body weight and decreased immune function, as well as damage to organs such as the kidneys, liver, pancreas and spleen. In severe cases, it can even lead to death.

[0003] Malnutrition disrupts multiple components of the immune system, such as mucosal barriers and cytokine production, leading to impaired immune system function, reduced resistance to pathogens, and increased risk of infection. Therefore, proper nutrition can improve the body's immune status, enhance resistance to disease, and is of great significance in disease prevention.

[0004] Consuming high-quality protein is one way to supplement nutrition. For example, eggs are rich in high-quality protein. The protein in egg white is mainly ovalbumin and ovoglobulin, which contain the eight essential amino acids for the human body. The composition of human protein is very similar to that of human protein, and the human body can absorb up to 98% of the protein in eggs.

[0005] Bioactive peptides, as a novel research direction in protein studies, release more active peptide components from proteins through natural and green processing methods such as enzymatic hydrolysis. Egg white protein peptides (also known as albumin peptides, because 60% of egg white protein is albumin) are small molecule peptides obtained by hydrolysis of egg white protein and have a variety of biological activities and functional properties. For example, patent document CN118027145A discloses an egg white peptide sequence QVPLW with a skin damage relief effect, which can significantly alleviate mechanical damage to skin fibroblasts (HSF) caused by cell scratching. Ma Sitong et al. analyzed three tetrapeptides (FYCP, YCPI, YLPR) from the enzymatic hydrolysis and digestion products of egg white protein, all of which have good antioxidant activity (differences in antioxidant activity of egg white peptides after in vitro simulated gastrointestinal digestion and alkaline protease treatment and peptide sequence analysis. Food Science, 2020, 41(21): 113-120.).

[0006] Therefore, in-depth analysis of egg white protein peptides, identification of biologically active functional peptides, and development and preparation of egg white protein-derived bioactive peptide products are of great significance for improving the economic benefits of the egg product deep processing industry. Currently, there is no research on the functional components that improve malnutrition. Summary of the Invention

[0007] The purpose of this invention is to provide a natural small molecule bioactive peptide that can improve malnutrition and enhance immunity, and to apply it to the development of products that improve malnutrition.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention utilizes alkaline protease, papain, and neutral protease to sequentially hydrolyze egg white protein, extracting egg white protein peptides from the hydrolysate with a molecular weight less than 1000 Da. The peptide sequences of the egg white protein peptides were analyzed using LC-MS / MS peptide mapping, and the interaction between the peptides and TNF-α receptors was investigated using molecular docking technology. A candidate peptide was screened, and its amino acid sequence was identified as Leu-Pro-Arg (LPR) by mass spectrometry, with a molecular weight of 384.248 Da. Further functional verification through the artificial synthesis of the tripeptide LPR revealed that this peptide has the effect of improving immunodeficiency.

[0009] Therefore, this invention provides the application of tripeptide LPR in the preparation of drugs or health foods that improve immunodeficiency.

[0010] Furthermore, the aforementioned weakened immune function is caused by malnutrition. Malnutrition leads to damage to the body's immune system.

[0011] Furthermore, the manifestations of the aforementioned immunodeficiency include a significant decrease in the number of macrophages.

[0012] Furthermore, the tripeptide LPR is prepared by solid-phase synthesis or obtained by enzymatic hydrolysis of egg white protein. The enzymatic hydrolysis involves sequentially hydrolyzing egg white protein with alkaline protease, papain, and neutral protease, and then separating the tripeptide LPR from the hydrolysis product.

[0013] Another object of the present invention is to provide an egg white protein peptide that improves malnutrition and enhances immunity, wherein the preparation method of the egg white protein peptide includes the following steps: (1) Mix egg white powder with water at a mass ratio of 1:15-20, and then cut to obtain an egg white solution; (2) Heat the egg white protein powder solution to 50℃, adjust the pH value to 8.0±0.2, add alkaline protease at 0.6%-1.0% of the weight of egg white protein powder, and hydrolyze for 2-2.5 hours; then add papain at 0.4%-0.8% of the weight of egg white protein powder, and continue hydrolyzing for 4-4.5 hours; then add neutral protease at 0.05%-0.1% of the weight of egg white protein powder, and continue hydrolyzing for 2-2.5 hours to obtain the enzymatic hydrolysate; (3) After centrifuging the enzymatic hydrolysate at a speed of 4000-6000 r / min, the supernatant is collected and filtered through a membrane with a molecular weight cutoff of 1000 Da. The filtrate is then concentrated, sterilized, and dried to obtain the egg white protein peptide.

[0014] Further, in step (2), 0.8% of alkaline protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours; then 0.6% of papain by weight of egg white protein powder is added and enzymatically hydrolyzed for 4 hours; then 0.1% of neutral protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours to obtain the enzymatic hydrolysate.

[0015] The egg white protein peptide contains the tripeptide LPR with the amino acid sequence Leu-Pro-Arg. This invention demonstrates that the egg white protein peptide can improve malnutrition and enhance immunity. In a malnutrition model, intervention with the egg white protein peptide significantly increased serum levels of total protein, albumin, prealbumin, and globulin, as well as the content of short-chain fatty acids in the intestine, thus achieving the effect of improving malnutrition.

[0016] Therefore, the present invention provides the application of the egg white protein peptide in the preparation of health foods or medicines that improve malnutrition and enhance immunity.

[0017] Furthermore, the manifestations of malnutrition include: a significant decrease in serum total protein, albumin, prealbumin and globulin, an increase in the albumin / globulin ratio; and a decrease in the content of short-chain fatty acids in the intestine.

[0018] Specifically, the present invention provides a health food for improving malnutrition and enhancing immunity, comprising egg white protein peptides or tripeptides LPR as active ingredients, and food science acceptable excipients.

[0019] The egg white protein peptide or tripeptide LPR in the health food provided by this invention can be used as the sole active ingredient to improve malnutrition, or it can be combined with other active ingredients that improve malnutrition.

[0020] In this invention, the food-grade excipients that are acceptable in food science are those capable of delivering an effective dose of the active substance of this invention without interfering with the bioactivity of the active substance.

[0021] Furthermore, the dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

[0022] This invention provides a drug for improving malnutrition and enhancing immunity, the drug comprising an egg white protein peptide or tripeptide LPR as an active ingredient, and a pharmaceutically acceptable carrier.

[0023] In this invention, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of this invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0024] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0025] The beneficial effects of this invention are as follows: This invention focuses on the efficacy of egg white protein peptides in improving malnutrition, and provides a tripeptide LPR that improves malnutrition and enhances immunity, as well as egg white protein peptides containing this peptide. The tripeptide LPR can be obtained through artificial synthesis or targeted enzymatic hydrolysis of egg white protein powder. Animal model functional validation shows that the tripeptide LPR and the egg white protein peptides containing this peptide can significantly improve malnutrition, increase serum levels of total protein, albumin, prealbumin, and globulin, and decrease the content of short-chain fatty acids in the intestine. The food-derived bioactive peptides exhibit high biosafety. Therefore, the tripeptide LPR or the egg white protein peptides containing this peptide can be used to prepare drugs or health foods that improve malnutrition and enhance immunity. This invention provides theoretical support for the efficacy of egg white protein peptides in improving malnutrition. Attached Figure Description

[0026] Figure 1 The effect of the egg white protein peptides prepared in Example 1 on serum total protein is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0027] Figure 2 The effect of the egg white protein peptide prepared in Example 1 on serum albumin is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0028] Figure 3 The effect of the egg white protein peptide prepared in Example 1 on serum globulin is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0029] Figure 4 The effect of the egg white protein peptide prepared in Example 1 on serum prealbumin is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0030] Figure 5The effect of the egg white protein peptide prepared in Example 1 on serum albumin / globulin ratio is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0031] Figure 6 The effect of the egg white protein peptide prepared in Example 1 on intestinal acetic acid is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0032] Figure 7 The effect of the egg white protein peptide prepared in Example 1 on intestinal propionic acid is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0033] Figure 8 The effect of the egg white protein peptide prepared in Example 1 on intestinal butyrate is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0034] Figure 9 The effect of the egg white protein peptide prepared in Example 1 on intestinal isobutyric acid is shown in the figure. The same letter between groups indicates no significant difference, and different letters indicate significant differences. P <0.05).

[0035] Figure 10 The effect of the egg white protein peptide prepared in Example 1 on intestinal valerate is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0036] Figure 11 The effect of the egg white protein peptide prepared in Example 1 on intestinal isovaleric acid is shown in the figure. The same letter between groups indicates no significant difference, and different letters indicate significant differences. P <0.05).

[0037] Figure 12 The effect of the egg white protein peptide prepared in Example 1 on intestinal hexanoic acid is shown in the figure. The same letter between groups indicates no significant difference, while different letters indicate significant differences. P <0.05).

[0038] Figure 13 This is the first-order mass spectrum of the tripeptide LPR.

[0039] Figure 14This is the secondary mass spectrum of the tripeptide LPR. In the figure, pre[1+] represents the charge [1+]; b1 represents the first fragment ion generated by the N-terminal cleavage of the peptide; y1-NH3 represents the first deaminated fragment ion generated by the C-terminal cleavage of the peptide; y1 represents the first fragment ion generated by the C-terminal cleavage of the peptide; y2-NH3 represents the second deaminated fragment ion generated by the C-terminal cleavage of the peptide; and y2 represents the second fragment ion generated by the C-terminal cleavage of the peptide.

[0040] Figure 15 This is a schematic diagram illustrating the binding of the tripeptide LPR to the TNF-α receptor.

[0041] Figure 16 The figure shows the change in macrophage number after intervention with tripeptide LPR in a zebrafish immunodeficiency model. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0044] The egg white protein powder used in the following examples was purchased from Zhejiang Aige Egg Products Co., Ltd.; the alkaline protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Biological Products Co., Ltd.; the papain was purchased from Guangxi Dongheng Huadao Biological Products Co., Ltd.; and the neutral protease (derived from Aspergillus oryzae) was purchased from Nanning Pangbo Biological Products Co., Ltd.

[0045] Example 1: Preparation of egg white protein peptides 1. This embodiment provides a method for sequentially enzymatically hydrolyzing egg white protein powder using alkaline protease, papain, and neutral protease. The specific steps are as follows: (1) Add 1500 kg of water to the reactor, and add 100 kg of egg white protein powder while stirring. Stir and mix evenly, and start shearing for 30 minutes at a shearing condition of 10000 r / min. (2) After shearing, raise the temperature of the liquid to 50°C. Once the temperature is reached, adjust the pH of the material to 8.0 with alkaline solution. Add 800 g of alkaline protease (from Bacillus subtilis) to the egg white protein powder solution and hydrolyze for 2 hours. Then add 600 g of papain and continue hydrolyzing for 4 hours. Finally, add 100 g of neutral protease (from Aspergillus oryzae) and continue hydrolyzing for 2 hours to obtain the hydrolysate. (3) The enzymatic hydrolysate was separated using a disc separator to obtain the supernatant. The supernatant was then passed through a membrane with a molecular weight cutoff of 1000 Da, and the filtrate was collected. (4) Then the liquid is evaporated and concentrated in a vacuum low-temperature thin film evaporator until the sugar content of the liquid is 25° to obtain a concentrated liquid; (5) The concentrated liquid is sterilized at high temperature and spray-dried to obtain egg white protein hydrolysate (egg white protein peptide).

[0046] 2. Detection of egg white protein peptides The physicochemical test results of egg white protein peptides are shown in Table 1, and the relative molecular weight distribution of egg white protein peptides is shown in Table 2. All results are presented as mass percentages. The moisture content detection method refers to the national standard GB 5009.3-2016 "Determination of Moisture in Food", the ash content detection method refers to the national standard GB 5009.4-2016 "Determination of Ash in Food", the protein content detection method refers to the national standard GB 5009.5-2025 "Determination of Protein in Food", the hydrolysis degree detection method refers to the o-phthalaldehyde method of Nielsen et al., and the relative molecular weight distribution detection method refers to Appendix A of the national standard GB / T 22729-2008 "Marine Fish Oligopeptide Powder".

[0047] Table 1. Physicochemical detection results of egg white protein peptides Moisture 4.19 Ash 6.24 Protein content 78.8 Degree of hydrolysis 40.92 Table 2. Relative molecular weight distribution of egg white protein peptides ＞10000 0.13 5000-10000 0.23 3000-5000 0.69 2000-3000 1.80 1000-2000 6.43 500-1000 19.96 180-500 62.16 ＜180 8.61 The results in Tables 1 and 2 show that egg white protein peptides have high protein content and degree of hydrolysis, and their relative molecular weights are mainly distributed below 1000 Da. This indicates that the enzymatic hydrolysis method of this embodiment can effectively hydrolyze egg white protein powder into small molecule peptides, especially peptides with relatively small molecular weights, which is beneficial for the human body to absorb and utilize these peptides.

[0048] Example 2: Efficacy of egg white protein peptides in improving a malnourished mouse model In this embodiment, the egg white protein peptide prepared in Example 1 was applied to a dexamethasone-induced malnourished mouse model to study its effect on improving malnutrition.

[0049] 1. Experimental Principle Malnutrition stems from a negative energy balance, meaning that energy intake cannot meet the body's needs. High-dose glucocorticoid treatment can affect weight and body composition. Studies have also found that this treatment can induce an excessive catabolic state, leading to muscle loss, inhibiting protein synthesis, causing a temporary increase in protein degradation, and a negative nitrogen balance.

[0050] Dexamethasone is a type of glucocorticoid that can induce malnutrition models by inhibiting protein synthesis and promoting catabolism.

[0051] Serum total protein (TP), which can be divided into albumin and globulin, plays an important physiological role in the body. The determination of serum total protein is one of the important items in clinical biochemical testing. Serum proteins have multiple functions, including maintaining normal blood colloid osmotic pressure and pH, transporting various metabolites, regulating the physiological functions of transported substances and detoxifying them, immune function, and nutritional function. Serum total protein can be used not only for monitoring the body's nutritional status but also for the diagnosis and differential diagnosis of diseases.

[0052] Serum albumin is the most abundant protein in mammalian blood. It is produced through the modification of prealbumin in the liver endoplasmic reticulum and is crucial for maintaining tissue osmotic pressure. A decrease in serum albumin levels lowers blood osmotic pressure, causing fluid to flow into tissues and resulting in edema. Malnourished patients often present with hypoalbuminemia; therefore, albumin is frequently used as a marker of protein-energy malnutrition.

[0053] Serum prealbumin is the gold standard for evaluating and monitoring a patient's nutritional status. Prealbumin is produced in liver and gastrointestinal mucosal cells and can represent the protein status of organs.

[0054] Serum globulins are a mixture of various proteins, including immunoglobulins and complement, which play a defensive role and are present in large quantities, as well as various glycoproteins. Globulins are produced by the body's immune organs, with most being generated outside liver cells. They are related to the body's immunity. Elevated globulin levels are usually due to the body being infected by a foreign virus. The immune system will fight against the foreign virus, leading to an increase in globulin levels.

[0055] Gut fatty acids are key metabolites for maintaining gut immune homeostasis. A healthy gut immune system is the core organ for ensuring nutrient absorption. Conversely, good nutrition is the material basis for producing beneficial fatty acids and maintaining immune function. An imbalance in any of these areas can trigger a chain reaction, leading to a vicious cycle. Therefore, in clinical and public health interventions (such as treating malnutrition and autoimmune diseases), it is essential to address all three levels simultaneously to effectively break the vicious cycle and restore health.

[0056] 2. Experimental Methods Six- to eight-week-old male C57BL / 6J mice (weighing 20-25 g) were selected and acclimatized for one week. Dexamethasone was used to establish the model. After 35 days of intervention, the mice were sacrificed, and serum and intestinal contents samples were collected. Serum albumin, total serum protein, prealbumin, and serum globulin levels were measured using a kit. The fatty acid content in the intestinal contents was determined by gas chromatography. This experiment was approved by the Animal Ethics Center of Zhejiang University of Traditional Chinese Medicine, ethics approval number IACUC-20250331-15.

[0057] Administration: Mice were administered the appropriate dose of the test substance daily according to their body weight, at a gavage dose of 0.01 mL / g bw, once daily, with the following specific groupings: Control group (CON): Distilled water, administered by gavage at a rate of 0.01 mL / g bw; Model group (DEX): DEX (solvent is water), injection dose is 20 mg / kg / d.ih; Low-dose group of egg white protein peptides (OP-L+DEX): Egg white protein peptides prepared in Example 1 (dose: 0.25 g / kg / d.bw) + DEX (dose: 20 mg / kg / d.ih); High-dose egg white protein peptide group (OP-H+DEX): Egg white protein peptide prepared in Example 1 (dose: 0.5 g / kg / d.bw) + DEX (dose: 20 mg / kg / d.ih).

[0058] 3. Experimental Results like Figures 1-5 As shown, compared with the blank group, the model group showed a significant decrease in total protein, albumin, prealbumin and globulin, while the albumin / globulin ratio increased, indicating that the modeling was successful.

[0059] Compared with the model group, intervention with egg white protein peptides significantly increased the levels of total protein, albumin, prealbumin, and globulin. Specifically, low-dose (equivalent to 1.5 g / 60 kg / day for adults) egg white protein peptides increased the levels of total protein, albumin, prealbumin, and globulin by 14.6%, 6.9%, 177.5%, and 28.1%, respectively; while high-dose (equivalent to 3.0 g / 60 kg / day for adults) egg white protein peptides increased the levels of total protein, albumin, prealbumin, and globulin by 11.2%, 6.3%, 183.7%, and 19.7%, respectively.

[0060] like Figures 6-12 As shown, compared with the blank group, the short-chain fatty acids of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid in the model group decreased significantly, indicating that the modeling was successful.

[0061] Compared with the model group, the intervention of egg white protein peptides, specifically the high-dose group (equivalent to 3.0 g / 60 kg / day for adults), increased the content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid by 281.31%, 370.30%, 375.79%, 468.86%, 366.92%, 315.65%, and 353.68%, respectively.

[0062] 4. Experimental Conclusions Egg white protein peptides significantly improved serum protein levels in a dexamethasone-induced malnourished mouse model, with both high- and low-dose groups showing positive regulatory effects, indicating that the effective dose of egg white protein peptides in improving malnutrition is low. Further analysis revealed that the low-dose group had a particularly significant effect on increasing prealbumin, suggesting that it may play a key role by promoting liver synthetic function or regulating nutritional metabolic pathways. While the high-dose group showed a slightly lower increase in globulin than the low-dose group, it still maintained a stable immunomodulatory effect, indicating that different doses may improve nutritional status through multi-target synergistic effects. Experimental data also showed that after intervention with egg white protein peptides, the albumin / globulin ratio recovered to levels close to the control group, further validating its comprehensive effect of improving malnutrition by balancing serum protein composition.

[0063] Dexamethasone-induced malnutrition in mice leads to a decrease in beneficial bacteria that produce short-chain fatty acids in the gut, while also reducing the levels of major intestinal energy-providing fatty acids such as butyrate and propionic acid, damaging the intestinal barrier, and causing imbalances in immune and metabolic regulation. Egg white protein peptides can significantly improve the intestinal short-chain fatty acid indicators in a dexamethasone-induced malnutrition mouse model. High doses show a positive and significant regulatory effect, essentially restoring the mice to a normal state, and the levels of acetic acid, butyrate, and caproic acid are higher than those in the control group.

[0064] The significant positive effects of egg white protein peptides on serum proteins and intestinal short-chain fatty acids indicate that egg white protein peptides can improve malnutrition through synergistic effects across multiple targets and pathways.

[0065] Example 3: Screening of active ingredients in egg white protein peptides In this embodiment, LC-MS / MS analysis was used to perform peptide mapping on the egg white protein peptides prepared in Example 1, and then molecular docking technology was used to screen for functional peptides. Details are as follows: 1. LC-MS / MS analysis

[0066] 2. Molecular docking Nutritional imbalance poses a serious challenge to the homeostasis and maintenance of physiological functions in all organisms. Mammalians have evolved the ability to automatically regulate the utilization and storage of nutrients. When nutrients are in excess, the body stores energy in adipose tissue, liver, and muscles; when nutrients are insufficient, the stored energy is mobilized to maintain physiological functions. During this process, the volume of adipose tissue changes (increases or decreases) depending on nutrient excess or deficiency, thereby affecting the secretion of adipose tissue hormones and adipokines such as leptin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and adiponectin. Based on literature reports, TNF-α receptors are used as docking targets for molecular docking when screening for bioactive peptides.

[0067] The PeptdieRanker score (≥0.6) and relative peak area (≥10) were used to determine the performance of the peaks. 6 The peptide fragment of [a specific peptide] was molecularly docked with the TNF-α receptor. The docking score (≤-7.0), number of hydrogen bonds (≥3), frequency of amino acid residue docking, and amino acid residues associated with regulating immune responses and improving nutritional status were used for sequence screening and identification. Finally, the tripeptide LPR was determined to have the potential to improve malnutrition, as shown in Table 3.

[0068] Table 3. Potentially bioactive peptides that bind to TNF-α receptors LPR 0.69 -6 385.256 1 <![CDATA[9.46×10 7 ]]> 4 The primary and secondary structures of the tripeptide LPR were analyzed by mass spectrometry. Figure 13 , Figure 14 It can be seen that the [M+H]+ ion signal of the tripeptide LPR in the primary structure is 385.256 m / z, which is basically consistent with the molecular weight of tripeptide LPR of 384.248 Da, and the amino acid sequence of tripeptide LPR in the secondary structure is Leu-Pro-Arg.

[0069] Molecular docking analysis results ( Figure 15 The results indicate that the interaction between LPR and TNF-α involves amino acid residues GLU125, TYR87, and ARG82, and the binding is achieved through four hydrogen bonds. This multi-point hydrogen bond interaction mode enhances the binding stability of LPR to the TNF-α receptor, suggesting that it may exert an immunomodulatory effect by blocking the TNF-α-mediated inflammatory signaling pathway, thereby improving malnutrition.

[0070] Example 4: Synthesis of active peptides In this embodiment, the LPR peptide was synthesized artificially. Specifically, it was synthesized by Shenzhen Borunsida Biotechnology Co., Ltd., with a purity of ≥98%.

[0071] S1. Weigh Fmoc-Arg-Wang Resin and place it in a glass reaction column. Add DCM to swell the solution for 30 min, then remove the DCM under reduced pressure. S2. Wash the resin three times with DMF, add 20% piperidine / DMF solution and react for 20 min to remove the protecting group fmoc, remove the solution under reduced pressure, and wash with DMF six times. S3. Weigh out the second amino acid Fmoc-Pro-OH and TBTU respectively and add them to the resin. Dissolve them in DMF and add DIEA. React for 30 minutes. Take the resin to perform a color reaction and observe the color of the solution and the color of the resin. If the solution is bright yellow and the resin is yellow, it means that the reaction is complete. Remove the solvent under reduced pressure. S4. Repeat steps S2 and S3 until the last amino acid Fmoc-Leu(otbu)-oh is precipitated. Then wash three times each with DMF, DCM and methanol, and dry the resin. S5. Add lysis buffer to remove resin and amino acid side chain protecting groups, filter with sand core, add diethyl ether to the filtrate to precipitate, centrifuge and wash the solid 3 times, dry and detect by MS to obtain tripeptide LPR.

[0072] Example 5: Efficacy Verification of Active Peptides In this embodiment, the efficacy of the active peptides was verified using a malnourished and immunocompromised zebrafish model induced by cyclophosphamide.

[0073] 1. Experimental Principle Malnutrition can severely interfere with a person's health development, making them more susceptible to infection. The immune response triggered by infection involves multiple mechanisms, in which macrophages play an important role.

[0074] 2. Experimental Methods (1) Zebrafish rearing and acquisition of molted embryos Wild-type zebrafish (AB) were bred at the zebrafish platform of Zhejiang University School of Medicine, and this experiment was approved by the Zhejiang University Center for Experimental Animal Ethics (ethics number ZJU20250649).

[0075] AB wild-type zebrafish were reared separately by sex in zebrafish breeding units, with a light / dark cycle of 14 h / 10 h, constant temperature (28℃) and humidity, and fed brine shrimp twice daily at fixed times and in fixed quantities. Healthy adult zebrafish were placed in a breeding tank the evening before, at a 1:1 female-to-male ratio, with the water level at approximately 2 / 3 full, and separated by a partition. The partition was removed at 8:00 AM the following morning to allow free fertilization, at which point the fertilization rate was 0 hpf. Fertilized eggs were collected at 1 hpf, impurities were removed, and approximately 50-100 eggs were placed in petri dishes containing methylene blue system water (60 μg / mL sea salt solution, methylene blue concentration approximately 0.1%), and cultured in a constant temperature incubator at 28.5℃.

[0076] Unfertilized white oocytes were removed at 24 hpf, the water in the methylene blue system was replaced with fresh water, phenylthiourea (PTU) was added, and the cells were cultured in a constant temperature incubator at 28.5℃.

[0077] Embryos at 48 hpf were placed in a 10 mg / mL streptomycin solution, and the digestion was observed in real time under a dissecting microscope. Once an embryo with ruptured membrane appeared (digestion at 28.5℃ for about 10 min), a large amount of systemic water was immediately added to dilute the enzyme solution, and the embryo was gently blown away with a dropper until the membrane was removed. The ruptured embryos were repeatedly washed with systemic water to remove residual protease.

[0078] (2) Effects of peptides on macrophages Wild-caught AB line zebrafish embryos (48 hpf) that have undergone demembranous embryo transfer were selected and placed in six-well plates, with two parallel wells per group and 10-15 embryos per well. The groups are as follows: Blank control group (CON): 0.5% dimethyl sulfoxide (DMSO) + system water + PTU; Model group (MOD): 200 μg / mL cyclophosphamide (dissolved in DMSO) + system water + PTU, with a final DMSO volume fraction of 0.5%; Test group: 0.5, 1, 5, 10, 25, 50 μg / mL of tripeptide LPR + 200 μg / mL cyclophosphamide (dissolved in DMSO) + system water + PTU, with a final volume fraction of 0.5% for DMSO.

[0079] AB zebrafish embryos (treated with PTU at 24 hpf) were shed at 48 hpf. After 48 h of treatment with PTU, each well was stained with 2.5 μg / mL neutral red dye and PTU for 6 h in the dark. The embryos were washed with systemic water in the dark, anesthetized, and fixed with 6% methylcellulose. Images were taken under a stereomicroscope, and the number of macrophages in the head was counted. Data were analyzed using GraphPad Prism software, and ANOVA and T-tests were used to analyze differences.

[0080] 3. Results Analysis from Figure 16 As can be seen, compared with the blank control group (CON), the number of macrophages in the head of zebrafish in the model group (MOD) was significantly reduced (p<0.0001), indicating that the model was successfully established.

[0081] Compared with the model group (MOD), 0.5 μg / mL of tripeptide significantly improved the effect of cyclophosphamide on the reduction of macrophages in zebrafish, and significantly increased the number of macrophages in the head of zebrafish by 34.84%, indicating that tripeptide LPR can significantly improve the immune decline caused by cyclophosphamide, thereby improving malnutrition.

[0082] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of tripeptide LPR in the preparation of drugs for improving immunodeficiency or health foods for enhancing immunity, characterized in that, The amino acid sequence of the tripeptide LPR is Leu-Pro-Arg.

2. The application as described in claim 1, characterized in that, The aforementioned weakened immune function is caused by malnutrition.

3. The application as described in claim 1, characterized in that, The manifestations of the weakened immune function include a significant decrease in the number of macrophages.

4. The application as described in claim 1, characterized in that, The tripeptide LPR is prepared by solid-phase synthesis or by enzymatic hydrolysis of egg white protein.

5. The application as described in claim 1, characterized in that, The dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

6. The application as described in claim 1, characterized in that, The drug comprises an egg white protein peptide as the active ingredient and a pharmaceutically acceptable carrier.

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