Application of chicken egg white lysozyme and drinking water additive

Abstract

The invention relates to the field of animal nutrition, and discloses application of chicken egg white lysozyme and a drinking water additive. The invention discloses the defect that the iron supplement treatment cannot completely recover the functions of the intestinal panning cells and the proliferation capacity of the epithelium for the first time, and provides the application of the chicken egg white lysozyme in preparation of the preparation for preventing or treating the secondary intestinal infection after iron supplement, and the chicken egg white lysozyme is mixed with edible-grade water-soluble auxiliary materials to prepare the preparation for preventing or treating the secondary intestinal infection after iron supplement. The preparation is prepared in the form of drinking water, wherein the specific activity of the chicken egg white lysozyme is gt; the purity is greater than or equal to 95%; the final concentration of the preparation is controlled to be 10-50 U / mL, and when the preparation is used, 100-500 U of chicken egg white lysozyme can be ingested every day in the iron supplementing period, so that intestinal infection after iron supplementing is prevented or treated, and the intestinal barrier function is recovered. The method is simple and convenient to operate, is suitable for a preventive strategy of large-scale breeding, and can efficiently promote the recovery of intestinal defense and repair functions, so that the intestinal barrier injury caused by subsequent infection of pathogenic bacteria (such as escherichia coli) is remarkably relieved.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition technology, specifically relating to the use of chicken egg white lysozyme and its use as a drinking water additive, and particularly its application in the prevention or treatment of intestinal infections secondary to iron supplementation. Background Technology

[0002] Iron is an essential trace element for the body, participating in several key physiological processes such as oxygen transport and energy metabolism. Iron deficiency can lead to anemia, growth retardation, and decreased immune function. Therefore, iron supplementation is a routine treatment for iron-deficient individuals in both clinical practice and animal husbandry.

[0003] Lgr5+ intestinal stem cells were first discovered and reported by the Clevers team in 2007. These adult stem cells, located at the base of intestinal crypts and possessing typical markers, were identified as the "origin" cells of the intestinal epithelium. They not only have continuous self-renewal capabilities but also possess the potential for multi-directional proliferation and differentiation, playing a crucial biological role in maintaining the integrity of the intestinal epithelial structure and the homeostasis of the proportions of various functional mature cells. Lgr5+ intestinal stem cells can mainly differentiate into four mature cell types: three secretory mature cells (goblet cells, Paneth cells, and enteroendocrine cells) and one absorptive mature cell (intestinal epithelial absorptive cells), each performing different physiological functions.

[0004] Paneth cells, discovered and named by scientists Schwalbe and Paneth in the late 19th century, are characterized by lysozyme 1 (Lyz1). These cells are located at the base of the small intestinal crypts, adjacent to Lgr5+ stem cells. Their main functions include: ① secreting various growth factors (such as Wnt3 and EGF), providing essential microenvironmental niche signals for Lgr5+ stem cells and directly supporting their maintenance and proliferation; ② synthesizing and releasing a series of antimicrobial peptides (such as α-defensins, lysozyme, and phospholipase A2), constituting the core effector molecules of intestinal innate immunity, enhancing host defense capabilities by directly killing pathogenic microorganisms; and ③ participating in the regulation of intestinal flora balance and inflammatory responses. Therefore, the number and functional status of Paneth cells directly determine the integrity of the intestinal barrier and the capacity for immune response.

[0005] Current research on iron supplementation has clearly established the important regulatory role of iron nutrition in the body's health and intestinal function. Research progress shows that commonly used oral ferrous salt supplements, such as ferrous sulfate, can effectively restore systemic iron metabolism indicators such as erythrocytes and hemoglobin in iron-deficiency anemic animals, correct anemia symptoms, and improve intestinal flora imbalance caused by iron deficiency, increasing the abundance of beneficial bacteria and reducing the proportion of pathogenic bacteria. However, these supplements cannot alleviate intestinal oxidative stress and inflammatory responses caused by iron deficiency, and have no repairing effect on intestinal tissue lesions such as colonic mucosal atrophy and epithelial cell arrangement defects; they may even aggravate intestinal barrier damage. Novel nano-hydrated iron (6-Fh) oral supplements, with their unique absorption mechanism, not only have better iron supplementation efficiency than traditional ferrous salts, but also stabilize the intracellular iron pool, maintain intestinal barrier integrity, and optimize intestinal flora structure, demonstrating good biocompatibility. Furthermore, adequate dietary iron intake can improve mucus layer function by regulating intestinal goblet cell proliferation and mucin expression, enhancing the defense against pathogens. Intramuscular iron injections can alleviate intestinal damage under infectious conditions and repair epithelial tight junctions. However, these traditional studies have mainly focused on the restoration of systemic iron levels, correction of anemia, and macroscopic improvement of gut microbiota / barrier function, without delving into the lasting effects of long-term iron deficiency on the gut's local immune defense system. Summary of the Invention

[0006] In order to overcome the shortcomings of existing technologies that iron supplementation can easily lead to secondary intestinal infections and impaired intestinal function, the present invention aims to provide a use of chicken egg white lysozyme and a drinking water additive to achieve the effects of preventing or treating intestinal infections after iron supplementation and repairing intestinal function.

[0007] Through in-depth research, the applicant has revealed for the first time that in iron-deficient animal models, even after two months of adequate iron supplementation to fully restore serum iron and other systemic iron metabolism indicators to normal, the intestinal immune defense system exhibits significantly asynchronous recovery impairments. Specifically, the number of Paneth cells at the base of the intestinal crypts failed to return to normal levels; and the expression level of the intestinal stem cell marker Lgr5 was significantly lower than that of the control group. This finding contrasts sharply with existing research that focuses solely on the restoration of systemic iron indicators or improvement of macroscopic intestinal function after iron supplementation, further confirming the significant limitations of current iron supplementation strategies—even if systemic iron homeostasis is restored, it cannot repair the deep-seated damage to the core defense units of the intestinal barrier caused by long-term iron deficiency. This finding indicates that iron deficiency may cause irreversible or difficult-to-repair local defects in the intestinal barrier, and also reveals the critical deficiency in current iron supplementation research that neglects the synchronous recovery of the intestinal local immune defense system. There is an urgent need to develop specific intervention strategies targeting the intestinal microenvironment to achieve comprehensive and synchronous repair of the intestinal system and local defense functions after iron deficiency.

[0008] The technical solution of the present invention is as follows: The use of egg white lysozyme in the preparation of a formulation for the prevention or treatment of intestinal infections secondary to iron supplementation is disclosed. The egg white lysozyme is mixed with edible-grade water-soluble excipients to prepare a formulation in the form of drinking water. The specific activity of the egg white lysozyme is >23000 U / mg, and the purity is ≥95%. The final concentration of the formulation is controlled at 10-50 U / mL. During use, it ensures a daily intake of 100-500 U of egg white lysozyme during iron supplementation to prevent or treat intestinal infections after iron supplementation and restore intestinal barrier function.

[0009] The aforementioned uses can enhance the self-renewal capacity of Lgr5+ intestinal stem cells, restore the number of Paneth cells to normal levels, and reduce damage from intestinal infections secondary to iron supplementation.

[0010] The formulation is suitable for animals possessing Paneth cells, including mice, rats, and humans.

[0011] A drinking water additive for improving intestinal function in animals after iron supplementation, comprising chicken egg white lysozyme as an active ingredient, wherein the chicken egg white lysozyme has a purity ≥95% and a specific activity >23000 U / mg, and the additive is prepared at a concentration of 10-50 U / mL, for use in providing animals with 100-500 U of chicken egg white lysozyme daily through drinking water.

[0012] The animals mentioned are those possessing Paneth cells, including mice, rats, and humans.

[0013] The beneficial effects of this invention are: 1. A novel phenomenon of delayed recovery of intestinal immune defense function after iron supplementation was discovered, providing a new explanation and intervention target for infectious complications after clinical iron supplementation.

[0014] 2. This study first proposed and verified that exogenous lysozyme supplementation is an effective means to solve the above problems, with a clear solution and significant results.

[0015] 3. This application has significant clinical and livestock value, as it can reduce the incidence of infection after iron supplementation, reduce antibiotic use, and improve treatment and breeding efficiency.

[0016] 4. Lysozyme, as a natural antimicrobial peptide, has high safety, is easy to produce, and is suitable for development into functional foods, health products, veterinary drugs, or feed additives. This invention administers the active ingredient via drinking water, greatly improving the convenience and feasibility of large-scale application. Animals can receive preventative treatment simply by drinking water freely, eliminating the hassle of gavage or feed mixing.

[0017] 5. The specific dosage, duration, and route of administration are clearly defined, the technical solution is clear, highly reproducible, and easily commercialized. Attached Figure Description

[0018] Figure 1 The results are the serum iron content and total iron-binding capacity of mice after two months of iron-deficient feeding followed by two months of iron supplementation with normal feed. The above results are the average of five independent replicate experiments.

[0019] Figure 2 The images show a comparison of Paneth cells in the intestines of mice treated with iron deficiency for 2 months followed by iron supplementation on a normal diet for 2 months. The results are the average of 5 independent replicate experiments.

[0020] Figure 3 The above images show a comparison of Paneth cells in mice after two months of iron deficiency followed by two months of iron supplementation with a normal diet. The results are the average of three independent replicate experiments.

[0021] Figure 4 The above image shows a comparison of intestinal stem cells in mice after treatment with iron-deficient diet for 2 months followed by iron supplementation on normal diet for 2 months. The results are the average of 3 independent replicate experiments.

[0022] Figure 5 The mice were treated with iron deficiency for 2 months, followed by iron supplementation with normal feed for 2 months, and iron supplementation with water and lysozyme for 1 month. The intestinal barrier of the mice was challenged with E. coli for 3 consecutive days. The above results are the average of 3 independent replicate experiments.

[0023] Figure 6 The results are the intestinal cell differentiation marker gene maps of mice after 2 months of iron deficiency, followed by 2 months of iron supplementation with normal feed and simultaneous iron supplementation with water and lysozyme. The results are the average of 3 independent replicate experiments. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] The applicant believes that exogenous supplementation with highly active chicken oocyte lysozyme may work through the following mechanisms: 1) directly replacing the insufficient secretion of endogenous lysozyme by Paneth cells under iron deficiency, thereby enhancing local antibacterial defense; 2) promoting the directed differentiation of Lgr5+ stem cells into Paneth cells through interaction with the Wnt / β-catenin or Notch signaling pathways; 3) alleviating oxidative stress caused by iron supplementation, improving the crypt microenvironment, and thus supporting the recovery of stem cell function.

[0026] Example 1: Verification of delayed recovery of local intestinal function after iron supplementation C57BL / 6 mice were randomly divided into a control group and an iron deficiency group. The iron deficiency group was fed a low-iron diet with less than 5 mg / kg iron for 8 weeks to establish an iron deficiency animal model. Subsequently, the iron deficiency group mice were further randomly divided into an iron supplementation group (Fe) and a low-iron control group. The iron supplementation group was fed a control diet with 165.29 mg / kg iron for 8 weeks, while the low-iron control group continued to be fed a low-iron diet. After the intervention, indicators were measured. All tests were performed independently, with serum iron-related tests performed in 5 independent replicates and intestinal cell tests in 3 independent replicates. Data are expressed as averages to ensure the reliability of the results.

[0027] 1. Systemic iron status detection: Serum iron and total iron binding capacity were detected. The results showed that the systemic iron index of the iron-supplemented group had recovered to the level of the normal iron group (see Figure 1), indicating that feeding mice with normal iron diet for 8 weeks can effectively correct the iron deficiency status of mice and achieve systemic iron nutrition recovery.

[0028] 2. Intestinal Paneth cell function detection: Mouse small intestine tissue was taken, fixed, embedded in paraffin, sectioned, and then subjected to immunofluorescence staining for the Paneth cell marker LYZ1. Figure 2 (5 independent replicates) The results showed that the number of Paneth cells in the iron supplementation group was significantly reduced compared with that in the normal iron group; Figure 3 (3 independent replicates) This conclusion was further verified by quantitative analysis. The results showed that the relative fluorescence intensity of Paneth cells in the iron-supplemented group (reflecting the expression level of LYZ1 protein) was only 49.6% of that in the normal iron group, indicating that even if the systemic iron index returned to normal, the number of Paneth cells in the intestine of the iron-supplemented mice had not been fully restored.

[0029] 3. Intestinal stem cell function testing: Intestinal stem cell marker staining and proliferation activity detection were performed on small intestinal tissue. The results are as follows: Figure 4 As shown in the three independent replicates, the number of Lgr5+ intestinal stem cells in the iron-supplemented group was significantly lower than that in the normal iron group, confirming that the function of intestinal stem cells in mice also did not fully recover after iron supplementation. In summary, Example 1 clearly verifies the phenomenon that the recovery of local intestinal function (Pan's cells, stem cell function) in mice after iron supplementation lags behind the recovery of systemic iron status.

[0030] Example 2: Regulation of differentiation protein levels in mouse duodenal intestinal stem cells by iron The lysozyme used in this experiment was chicken egg white lysozyme (product number: 10837059001) purchased from Roche. It is a solid powder with a purity of ≥95% and a specific activity of >23000 U / mg (Shugar units).

[0031] Experimental Design: A mouse iron deficiency model was constructed according to the method described in Example 1 (fed on a low-iron diet for 8 weeks). Mice with successfully established iron deficiency models were then fed a normal iron diet containing 165 mg / kg of iron and randomly divided into two groups. A normal iron control group (Control group) was also established. Mice in each group had free access to food and water. The intervention lasted for 4 weeks. The iron supplementation group (Fe-Rescue group) drank ordinary sterilized water, while the iron supplementation + lysozyme group (Fe-Rescue + Lyz group) drank a sterilized lysozyme solution with a final concentration of 40 U / mL (this solution was prepared fresh and changed every two days; based on an average daily water intake of 5 mL for each mouse, the average daily lysozyme intake per mouse was approximately 200 U).

[0032] Pathogen challenge and detection: After the intervention, mice in each group were challenged with 10^9 CFU of Escherichia coli. After three consecutive days of gavage infection, the mice were sacrificed and duodenal tissue was taken for subsequent index detection and evaluation. All detection experiments were set up in 3 independent replicates, and the data are expressed as average values.

[0033] 1. Intestinal barrier integrity detection: Intestinal barrier integrity was detected by Western blotting using intestinal tight junction proteins (such as ZO-1 and Occludin). The results are as follows: Figure 5 As shown in the image analysis, the continuity and integrity of the tight junction proteins in the intestines of mice in the Fe-Rescue group were severely disrupted, and the protein expression level was significantly reduced; while the damage to the tight junction proteins in the Fe-Rescue+Lyz group was significantly alleviated, approaching the damage level in the Control group, indicating that lysozyme supplementation can effectively improve the integrity of the intestinal barrier in mice after iron supplementation (expression level was 78.6% of that in the control group).

[0034] 2. Detection of intestinal cell differentiation marker gene expression: The mRNA expression levels of intestinal cell differentiation marker genes were detected using Western blotting (WB). The results are as follows: Figure 3 and Figure 6 As shown in the data, the expression levels of Lgr5 (a stem cell marker gene) and LYZ1 (a Paneth cell differentiation marker gene) in the Fe-Rescue group mice were 8% and 12% of those in the Control group, respectively, both significantly reduced; while the expression levels of the above genes in the Fe+Lyz group increased to 50% and 65% of those in the Control group, respectively, significantly higher than those in the Fe-Rescue group. Combined with the detection results of Paneth cells and stem cells in Example 1, this indicates that lysozyme can improve the differentiation function of intestinal cells after iron supplementation by promoting the directed differentiation of intestinal stem cells into mature intestinal epithelial cells and Paneth cells.

[0035] 3. Verification of Paneth cell count: (The sentence is incomplete and requires further context to be translated accurately.) Figure 3Immunofluorescence staining analysis of LYZ1 showed that the number of Paneth cells in the intestine of Fe+Lyz group mice was significantly higher than that in Fe group, further confirming the promoting effect of lysozyme on Paneth cell differentiation.

[0036] In conclusion, supplementing mice with 200 U / mouse of chicken egg white lysozyme daily via drinking water for one month during iron supplementation intervention can effectively improve the incomplete recovery of intestinal defense function after iron supplementation and significantly enhance the mice's resistance to Escherichia coli infection.

[0037] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. The use of a chicken egg white lysozyme in the preparation of a formulation for the prevention or treatment of intestinal infections secondary to iron supplementation, characterized in that: Chicken egg white lysozyme is mixed with edible-grade water-soluble excipients to prepare a preparation in the form of drinking water. The specific activity of the chicken egg white lysozyme is >23000 U / mg, and the purity is ≥95%. The final concentration of the preparation is controlled at 10-50 U / mL. When used, it can ensure that 100-500 U of chicken egg white lysozyme is ingested daily during iron supplementation to prevent or treat intestinal infections after iron supplementation and restore intestinal barrier function.

2. The use according to claim 1, characterized in that: It can enhance the self-renewal capacity of Lgr5+ intestinal stem cells, restore the number of Paneth cells to normal levels, and reduce the damage caused by intestinal infections following iron supplementation.

3. The use according to claim 1, characterized in that: The formulation is suitable for animals possessing Paneth cells, including mice, rats, and humans.

4. A drinking water additive for improving intestinal function in animals after iron supplementation, characterized in that, The additive contains chicken egg white lysozyme as an active ingredient, wherein the chicken egg white lysozyme has a purity ≥95% and a specific activity >23000 U / mg, and the concentration of the additive is 10-50 U / mL, and is used to allow animals to ingest 100-500 U of chicken egg white lysozyme daily through drinking water.

5. The drinking water additive according to claim 4, characterized in that: The animals mentioned are those possessing Paneth cells, including mice, rats, and humans.

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