Highly immunocompetent chrysalis protein immunomodulating peptide, its preparation method and application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a highly immunomodulatory peptide derived from silkworm pupa protein, its preparation method, and its applications. The invention provides a silkworm pupa protein immunomodulatory peptide, the amino acid sequence of which is shown in SEQ ID NO.2, SEQ ID NO.3, and / or SEQ ID NO.8. This silkworm pupa protein immunomodulatory peptide is prepared by hydrolyzing silkworm pupa protein with alkaline protease, followed by purification of the enzymatic hydrolysis product through ultrafiltration and gel filtration. The most immunomodulatory peptide is screened by determining the immunomodulatory activity index, and identified by LC-MS / MS to obtain a highly immunomodulatory peptide derived from silkworm pupa protein. This immunomodulatory peptide strongly binds to TLR2 and TLR4 / MD-2 receptors through hydrogen bonds and hydrophobic interactions, activating immunity and effectively promoting macrophage proliferation and phagocytosis. This immunomodulatory peptide exhibits good immunomodulatory activity and can be used as a potential functional food ingredient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly immunomodulatory peptide of silkworm pupa protein, its preparation method, and its application. Background Technology

[0002] Silkworm pupae are a byproduct of silk reeling and are rich in vitamins, carbohydrates, fats, and other nutrients. They are high in protein, with a crude protein content of approximately 48-60%, making them a nutritious and high-quality protein source and a natural insect with significant development value. Studies have confirmed that silkworm pupae have multiple uses and values: 1) Relieving fatigue and promoting fat metabolism; 2) Antibacterial and anti-inflammatory properties, inhibiting Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; 3) As a traditional Chinese medicine, it has the effects of quenching thirst, promoting digestion, and regulating qi, and can be used to treat symptoms such as thirst, urinary incontinence, and bloating; 4) Regulating the body's sugar and fat metabolism, with significant effects on treating hypercholesterolemia and improving liver function; 5) Broad-spectrum immune substances extracted from silkworm pupae are effective against cancer, arteriosclerosis, hepatitis, and nephritis; 6) Silkworm pupa oil has certain effects on hypertension, cholesterol, fatty liver, and hyperlipidemia; 7) As a dietary therapy, silkworm pupae are suitable for patients with hypertension, hyperlipidemia, diabetes, as well as those with tuberculosis, emaciation, infantile malnutrition, chronic gastritis, and gastroptosis. In China, due to silk harvesting, more than 300,000 tons of silkworm pupae are produced annually. Most silkworm pupae are used as animal feed or fertilizer, and a small portion is used as medicine or consumed by people, thus their value is not fully utilized. Therefore, it is necessary to explore new ways to utilize silkworm pupae as resources and promote the high-value utilization of silkworm pupae.

[0003] Bioactive peptides are multifunctional compounds derived from proteins, composed of 2-20 amino acids, and released through enzymatic hydrolysis, fermentation, and gastrointestinal digestion. Immunomodulatory peptides are a class of bioactive peptides that regulate the function of the human immune system, playing an important role in host defense mechanisms by regulating immune cell function, antibody synthesis, and cytokine secretion. In recent years, many studies have shown that immunomodulatory peptides extracted from food proteins are easily absorbed, safe, and relatively inexpensive for human health consumption. For example, He et al. isolated and purified immunoactive peptides from duck egg ovalbumin and Litopenaeus vannamei heads, respectively, which significantly enhanced the phagocytic activity of macrophages and promoted the secretion of immune factors such as NO, TNF-α, and IL-6 by macrophages. Lu-Sheng hsiehlu isolated and purified immunoactive peptides from soybean protein hydrolysate, which significantly improved the phagocytic activity of mouse splenic macrophages and neutrophils. Although some studies have reported the immunomodulatory activity of peptides, information on the structure-activity relationship of peptides remains scarce.

[0004] Some researchers have isolated and purified bioactive peptides with various beneficial effects from silkworm pupa proteins. For example, Maria Cerme studied the antioxidant activity of silkworm pupa protein hydrolysates using in vitro antioxidant experiments and in situ reduction of reactive oxygen species (ROS) in HepG2 liver cells. She identified two peptides that exhibited the highest antioxidant activity in HepG2 cells, including ROS reduction, superoxide dismutase expression, and glutathione production activity. Sun Hee Lee, through in vitro mechanism studies, stimulated the differentiation and maturation of 3T3-L1 preadipocytes with insulin and assessed the effects of silkworm pupae and silkworm pupa protein (SPP) on adipogenesis in mature adipocytes. She found that SP and SPP inhibited preadipocyte differentiation and adipogenesis by regulating signal transduction pathways and improved obesity by reducing lipid accumulation and adipocyte size. Li Xiaotong, using flow cytometry, ROS content measurement, and mitochondrial membrane potential detection, found that silkworm pupa protein hydrolysates specifically inhibited the growth of SGC-7901 cells through intrinsic apoptosis pathways, ROS accumulation, and cell cycle arrest. Zhou Yaxi verified the therapeutic effect of SPP on colorectal cancer mice in vivo using a subcutaneous tumor induction method, confirming that the anti-tumor activity of SPP may be achieved by reducing tumor inflammation, inhibiting tumor proliferation and metastasis, and inducing tumor cell apoptosis.

[0005] Currently, research on silkworm pupa protein immunomodulatory peptides in this field is relatively limited, with only a few publications or patents reporting on them. For example, patent CN115232851A discloses a method for preparing silkworm pupa immunopeptides, which uses acidic protease to enzymatically hydrolyze silkworm pupae to obtain silkworm pupa immunopeptides with both antibacterial and antioxidant activities. Therefore, it is necessary to study silkworm pupa protein immunomodulatory peptides and their preparation methods, as this is of great significance for determining the comprehensive effects of active substances in silkworm pupa protein on immune cells. Summary of the Invention

[0006] In view of this, this invention conducts in-depth research on silkworm pupa protein immunomodulatory peptides and their extraction processes. By utilizing different proteases to hydrolyze silkworm pupa protein, combined with NO generation, macrophage proliferation, and phagocytosis experiments, potential immunomodulatory peptides are screened and identified. This invention effectively expands the utilization rate of silkworm pupa protein peptides and provides a theoretical basis for the further processing and utilization of silkworm pupa protein.

[0007] One of the objectives of this invention is to provide a silkworm pupa protein immunomodulatory peptide that has excellent immunomodulatory activity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The silkworm pupa protein immunomodulatory peptide contains the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.8.

[0010] Furthermore, the amino acid sequence of the silkworm pupa protein immunomodulatory peptide is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.8.

[0011] The second objective of this invention is to provide a method for preparing silkworm pupa protein immunomodulatory peptides.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] The preparation method of silkworm pupa protein immunomodulatory peptides includes the following steps:

[0014] (1) Silkworm pupa protein was prepared using defatted silkworm pupa powder as raw material;

[0015] (2) The silkworm pupa protein obtained in step (1) was hydrolyzed with alkaline protease to obtain silkworm pupa protease hydrolysate;

[0016] (3) The silkworm pupa protein hydrolysate obtained in step (2) was separated and purified by ultrafiltration and gel filtration to obtain the separated product;

[0017] (4) The isolated product obtained in step (3) was subjected to LC-MS / MS sequencing to obtain the silkworm pupa protein immunomodulatory peptide.

[0018] Furthermore, step (1) is detailed as follows:

[0019] After dispersing defatted silkworm pupa powder, stir and centrifuge to collect the supernatant; adjust the pH to 4.5 with HCl to precipitate the protein for 1 hour, centrifuge and discard the supernatant; wash the precipitate three times with distilled water, adjust to neutral with NaOH, and freeze-dry under vacuum to obtain silkworm pupa protein;

[0020] Preferably, the dispersant for the defatted silkworm pupa powder is 0.5% NaOH.

[0021] Preferably, defatted silkworm pupa powder and 0.5% NaOH are dispersed at a material-to-liquid ratio of 1:20.

[0022] Preferably, the stirring and centrifugation is performed by stirring at 40°C for 1 hour and then centrifuging at 1000g for 15 minutes.

[0023] Preferably, the concentration of HCl is 1M.

[0024] As a preferred method, the second centrifugation is performed at 3220g for 15 minutes.

[0025] Preferably, the concentration of NaOH used for neutralization is 0.1M.

[0026] Further, in step (2), first, the silkworm pupa protein powder obtained in step (1) is formulated into a suspension of 10 mg / mL, ultrasonically dissolved at 30 °C for 30 min, and then enzymatically hydrolyzed.

[0027] Further, in step (2), the conditions for enzymatic hydrolysis are: pH is 7 - 9, temperature is 40 °C - 60 °C, and time is 2 h - 5 h; more preferably: pH is 8, temperature is 50 °C, and time is 180 min.

[0028] Further, in step (2), the dosage of the alkaline protease is 5000 U / g - 10000 U / g, and more preferably 6000 U / g.

[0029] Further, in step (2), NaOH is continuously added during the hydrolysis process to maintain the pH.

[0030] Further, in step (2), after the enzymatic hydrolysis reaction ends, it is immediately heated to 90 °C for 10 min for inactivation, cooled to room temperature, the pH is adjusted to 4.5, left standing at room temperature for 30 min, and centrifuged at 3220 g for 15 min; the pH of the enzymatic hydrolysate is adjusted to neutral, and after freeze-drying, the silkworm pupa protein hydrolysate is obtained.

[0031] Further, in step (3), the ultrafiltration includes: diluting the silkworm pupa protein hydrolysate with distilled water, and separating it into molecular weight cut-off values of 10 kDa and 3 kDa by an ultrafiltration membrane; the hydrolysis products are divided into three fractions: SPPH-A-I (MW > 10 kDa), SPPH-A-II (10 kDa < MW < 3 kDa), and SPPH-A-III (MW < 3 kDa), and the ultrafiltration product is obtained.

[0032] Further, in step (3), the gel filtration purification is to separate and purify the component with the strongest immunomodulatory activity after ultrafiltration by G-25 gel filtration chromatography; chromatographic purification is carried out using an AKTApuro instrument, the sample concentration is 10 mg / mL, eluted with ultrapure water at 1 mL / min, and the absorbance is measured at 280 nm to obtain the separated product.

[0033] Preferably, the G-25 gel filtration chromatography uses a Sephadex G-25 packed size exclusion chromatography column, and the column size is 1.6 cm × 100 cm.

[0034] Further, for the LC-MS / MS, the liquid phase uses a C18 chromatographic column, with formic acid aqueous solution as mobile phase A and formic acid acetonitrile aqueous solution as mobile phase B, and separation is carried out by gradient elution; after separation, it is analyzed using a mass spectrometer; the gradient elution program is set as follows:

[0035] 0 min - 50 min, a linear gradient of mobile phase B from 4% to 50% and mobile phase A from 96% to 50%;

[0036] A linear gradient of mobile phase B from 50% to 100% and mobile phase A from 50% to 0% was observed from 50 to 54 min.

[0037] Keep solution B at 100% for 54-60 minutes.

[0038] Preferably, the separated product is desalted and then used for mass spectrometry detection.

[0039] Preferably, the chromatographic column used is an RP-C18, manufactured by column Technology Inc., with dimensions of 0.15 mm x 150 mm.

[0040] Preferably, the mobile phase A is a 0.1% formic acid aqueous solution.

[0041] Preferably, the mobile phase B is a 0.1% aqueous solution of formic acid and acetonitrile, wherein the acetonitrile content is 84%.

[0042] Preferably, the liquid chromatography column is equilibrated with 95% solution A.

[0043] As a preferred method, capillary high-performance liquid chromatography (HPLC) was used for separation, followed by Q Exactive HF-X mass spectrometry analysis. The analysis time was 60 min, and the detection mode was positive ion.

[0044] The third objective of this invention is to provide an application of the aforementioned silkworm pupa protein immunomodulatory peptide in the preparation of products that enhance the body's immune regulation.

[0045] To achieve the above objectives, the present invention adopts the following technical solution:

[0046] The aforementioned silkworm pupa protein immunomodulatory peptides are used in the preparation of products that enhance the body's immunity.

[0047] Furthermore, the products include one or more of the following: functional foods, dietary supplements, pharmaceuticals, skincare products, and cosmetics.

[0048] The fourth objective of this invention is to provide the application of the aforementioned silkworm pupa protein immunomodulatory peptide in the preparation of a promoter that promotes macrophage proliferation, phagocytosis, and / or NO production.

[0049] The beneficial effects of this invention are as follows:

[0050] 1. The three silkworm pupa protein immunomodulatory peptides provided by this invention can effectively promote the proliferation and phagocytosis of macrophages and have good immune activity.

[0051] 2. This invention provides a method for preparing immunomodulatory peptides from silkworm pupa protein. The invention first compared the effects of different proteases on hydrolyzing silkworm pupa protein, ultimately selecting an alkaline protease with a high degree of hydrolysis. Potential immunomodulatory peptides were screened and identified through NO production, macrophage proliferation, and phagocytosis experiments. Then, three potential immunoactive peptides were screened using LC-MS / MS sequencing combined with molecular docking. Studies confirmed that the bioactive peptides extracted from alkaline protease-hydrolyzed silkworm pupa protein exhibit good immunomodulatory effects on RAW264.7 cells, promoting cell proliferation, NO production, and phagocytosis. They mediate immune activation through hydrogen bonding and hydrophobic interactions with TLR2 and TLR4 / MD-2. The immunomodulatory peptides from silkworm pupa protein and their preparation method provided by this invention offer a strategic basis for the bioactivity identification of silkworm pupa protein peptides and provide new ideas for the application of silkworm pupa protein peptides as functional food ingredients. Attached Figure Description

[0052] Figure 1 The graph shows the results of DH measurement.

[0053] Figure 2 A statistical graph showing the ratio of SPP to the secondary structure content of each isolated and purified product;

[0054] Figure 3 The mass spectrum of Peptide 1, an immunologically active peptide from silkworm pupae.

[0055] Figure 4 The mass spectrum of Peptide 2, an immunologically active peptide from silkworm pupae.

[0056] Figure 5 Mass spectrum of Peptide 3, an immunologically active peptide from silkworm pupae.

[0057] Figure 6 Mass spectrum of Peptide 4, an immunologically active peptide from silkworm pupae.

[0058] Figure 7 Mass spectrum of Peptide 5, an immunologically active peptide from silkworm pupae.

[0059] Figure 8 Mass spectrum of Peptide 6, an immunologically active peptide from silkworm pupae.

[0060] Figure 9 Mass spectrum of Peptide 7, an immunologically active peptide from silkworm pupae.

[0061] Figure 10 The mass spectrum of Peptide 8, an immunologically active peptide from silkworm pupae.

[0062] Figure 11Mass spectrum of Peptide 9, an immunologically active peptide from silkworm pupae.

[0063] Figure 12 A diagram showing the molecular interactions of Peptide 3, Peptide 2, and Peptide 8 with TLR2 and TLR4 / MD-2, where, Figure 12 -A is a diagram showing the molecular interactions between Peptide 3 and TLR2 (PDB:ID:1FYW). Figure 12 -B is a diagram showing the molecular interactions between Peptide 2 and TLR2 (PDB:ID:1FYW). Figure 12 -C is a diagram showing the molecular interactions between Peptide 8 and TLR2 (PDB:ID:1FYW). Figure 12 -D is a diagram showing the molecular interactions between Peptide 3 and TLR4 / MD-2 (PDB:ID:5IJD). Figure 12 -E is a diagram showing the molecular interactions between Peptide 2 and TLR4 / MD-2 (PDB:ID:5IJD). Figure 12 -F is a diagram of the molecular interactions between Peptide 8 and TLR4 / MD-2 (PDB:ID:5IJD);

[0064] Figure 13 The image shows the results of immunological activity assays for three synthetic peptides. Figure 13 -A is a statistical graph showing the proliferation rate of macrophages treated with different peptides. Figure 13 -B is a statistical chart of the phagocytic rate of macrophages treated with different peptides;

[0065] Figure 14 The figure shows the effects of four silkworm pupa protease hydrolysates on macrophage immune activity indicators. Figure 14 -A shows the results of the effects of four enzymatic hydrolysis products on macrophage proliferation; Figure 14 -B is a graph showing the effect of four enzymatic hydrolysis products on NO production in macrophages; Figure 14 -C is a graph showing the effect of four enzymatic hydrolysis products on macrophage phagocytosis;

[0066] Figure 15 The figure shows the effects of three ultrafiltration products on macrophage immune activity indicators. Figure 15 -A shows the results of the effects of three ultrafiltration products on macrophage proliferation; Figure 15 -B is a graph showing the effects of three ultrafiltration products on NO production in macrophages; Figure 15 -C is a graph showing the effects of three ultrafiltration products on macrophage phagocytosis;

[0067] Figure 16The result of the isolation and purification of SPPH-A-III by Sephadex G-25 is shown in the figure.

[0068] Figure 17 The figure shows the effects of three Sephadex G-25 isolates on macrophage immune activity indicators. Figure 17 -A shows the results of the effects of three Sephadex G-25 isolates on macrophage proliferation; Figure 17 -B shows the effect of three Sephadex G-25 isolates on NO production in macrophages; Figure 17 -C shows the effect of three Sephadex G-25 isolates on macrophage phagocytosis;

[0069] Figure 18 EdU was incorporated into RAW264.7 cells after treatment with a 200 μg / mL sample solution, and the results are presented as a quantification of total cell count (scale bar = 50 μm). Figure 18 -A is the fluorescence image. Figure 18 -B represents the positive quantitative result graph.

[0070] In the above figures, asterisks indicate statistically significant differences, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no statistical significance. Detailed Implementation

[0071] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0072] The immune system regulates various physiological processes in the body. Therefore, enhancing immunity and immune function are crucial for preventing disease and restoring health. Macrophages are important cells in the body's immune function and serve as the body's first line of defense against infection. Activated macrophages participate directly or indirectly in the immune response, clearing foreign antigens, senescent cells, and cellular debris through phagocytosis, and secreting cytokines such as reactive oxygen species, reactive nitrogen species, TNF-α, and IL-6 to participate in the immune response. Therefore, macrophages are considered an ideal cell model for evaluating the immunomodulatory activity of bioactive compounds. Macrophage activation is mediated by pattern recognition receptors (PRRs), including mannose receptors (MRs), toll-like receptors (TLRs), scavenger receptors, and complement receptors (CRs). Many immunomodulatory substances activate macrophages by interacting with PRRs. For example, maca protein hydrolysate activates RAW 264.7 cells through the NF-κB and MAPK signaling pathways mediated by TLR2 and TLR4 receptors. The TLR family is the most characteristic PRR family in mammals. Compared to other PRRs, TLRs have been reported to interact with a variety of components, including dietary fiber, lipids, proteins, peptides, and nucleic acids. Therefore, in this embodiment of the invention, the immune activity of the peptide is assessed by detecting its effect on the immune activity of macrophages.

[0073] In this embodiment of the invention, the alkaline protease is produced by selected Bacillus licheniformis, and its main active ingredient is subtilisin A. It is an endopeptide that primarily acts on peptide bonds containing hydrophobic carboxyl groups. Under alkaline conditions, it can effectively break down proteins into smaller peptides or amino acids. Because this enzyme exhibits high activity under alkaline conditions, it is classified as an alkaline protease.

[0074] In this embodiment of the invention, defatted silkworm pupa powder was purchased from Nanxun Zhanhong Silkworm Pupa Processing Plant in Huzhou City; trypsin (≥2500U / mg), pepsin (>3000U / mg), flavor enzyme (150U / mg), alkaline protease (2000U / mg), o-phenylenedialdehyde (≥99%), serine (≥99%), DL-dithiothreitol (≥99%), and sodium tetraborate (99.5%) were purchased from Beijing Puxitang Biotechnology Co., Ltd.; lipopolysaccharide (LPS) and CCK8 were obtained from Sigma (USA). Culture medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin antibody, and phosphate-buffered saline (PBS) were purchased from Gibco (USA); NO and neutrophil proliferation kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); ultrafiltration tubes were purchased from Millipore (MA, USA); CCK8 kits were purchased from Beyotime Biotechnology Co., Ltd. (China); RAW 264.7 cells were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA); the BeyClick™ EdU-488 imaging kit was purchased from Sigma, USA; all other chemicals and reagents used were of analytical grade.

[0075] In this embodiment of the invention, the FTIR-650 spectrometer is manufactured by Tianjin Guangdong Technology Co., Ltd. (Tianjin, China); the Zorbax 300SB-C18 peptide trap is manufactured by Agilent Technologies; the Q Exactive HF-X mass spectrometer is manufactured by Thermo Fisher; and the multi-functional microplate reader is manufactured by SpecteaMax iD5, USA.

[0076] In this embodiment of the invention, BIOPEP is a database containing protein sequences, bioactive peptides, and sensitive peptides, developed by the University of Walmia Mazury in Poland. Its webpage is: http: / / www.uwm.edu.pl / biochemia / index.php / en / biopep, accessed.

[0077] In this embodiment of the invention, the preparation method of silkworm pupa protein immunomodulatory peptide is as follows:

[0078] (1) Preparation of silkworm pupa protein: The defatted silkworm pupa powder was dispersed in 0.5% NaOH at a ratio of 1:20 (solid to liquid), stirred at 40 °C for 1 hour, centrifuged at 1000 g for 15 minutes, and the supernatant was taken; the pH was adjusted to 4.5 with 1M HCL to precipitate the protein for 1 hour, and then centrifuged at 3220 g for 15 minutes; the supernatant was discarded, the precipitate was washed three times with distilled water, adjusted to neutral with 0.1M NaOH, and freeze-dried under vacuum to obtain silkworm pupa protein;

[0079] (2) Enzymatic hydrolysis of silkworm pupa protein: The silkworm pupa protein powder obtained in step (1) was formulated into a suspension of 10 mg / mL and ultrasonicated at 30 °C for 30 min; 6000 U / g of alkaline protease was added to the silkworm pupa protein suspension for hydrolysis. The optimal hydrolysis conditions were pH = 8, 50 °C, and 180 min; 1M NaOH was continuously added during the hydrolysis process to maintain the pH; after the reaction, it was immediately heated to 90 °C to inactivate for 10 min, cooled to room temperature, the pH was adjusted to 4.5, and left standing at room temperature for 30 min, then centrifuged at 3220 g for 15 min; the pH of the enzymatic hydrolysate was adjusted to neutral, and freeze-dried to obtain silkworm pupa protein hydrolysate (SPPH-A).

[0080] (3) Ultrafiltration: The silkworm pupa protein hydrolysate obtained in step (2) was diluted with distilled water and separated by an ultrafiltration membrane into molecular weight cut-off values of 10 kDa and 3 kDa; the hydrolysis products were divided into three fractions: SPPH-A-I (MW > 10 kDa), SPPH-A-II (10 kDa < MW < 3 kDa), and SPPH-A-III (MW < 3 kDa); all fractions were collected and freeze-dried to obtain the ultrafiltration product;

[0081] (4) Gel chromatography purification: The fraction with the strongest immunomodulatory activity (SPPH-A-III) after ultrafiltration was separated and purified by G-25 gel chromatography. The column size was 1.6 cm × 100 cm; AKTA puro instrument was used for chromatographic purification. The sample concentration was 10 mg / mL, eluted with ultrapure water at 1 mL / min, the absorbance was measured at 280 nm, each elution peak was collected, and freeze-dried to obtain the separated products (F1, F2, and F3);

[0082] LC-MS / MS sequencing: The separated products were desalted for mass spectrometry detection; the chromatographic conditions in the liquid phase included: a C18 chromatographic column (0.15 mm * 150 mm, RP-C18, column Technology Inc.) was used, 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile aqueous solution was used as mobile phase B. The acetonitrile content in mobile phase B was 84%; the liquid chromatographic column was equilibrated with 95% of solution A; the sample was sent from the auto sampler to a Zorbax 300SB-C18 peptide trap and separated on the chromatographic column; the liquid phase gradient was set as follows:

[0083] A linear gradient of mobile phase B from 4% to 50% and mobile phase A from 96% to 50% was observed from 0 min to 50 min.

[0084] A linear gradient of mobile phase B from 50% to 100% and mobile phase A from 50% to 0% was observed from 50 to 54 minutes.

[0085] Keep solution B at 100% for 54-60 minutes.

[0086] Separation was performed by capillary high-performance liquid chromatography (HPLC), and analysis was conducted by Q Exactive HF-X mass spectrometry (MS-X). The analysis time was 60 min, and the detection mode was positive ion. The mass-charge ratios of peptides and peptide fragments were collected as follows: 10 fragment images were collected after each full scan (MS2 scan).

[0087] Example 1. Preparation method of silkworm pupa protein immunomodulatory peptides

[0088] 1. Preparation of silkworm pupa protein

[0089] Defatted silkworm pupa powder was dispersed in 0.5% NaOH at a ratio of 1:20 to liquid, stirred at 40℃ for 1 hour, and then centrifuged at 1000g for 15 minutes, collecting the supernatant. The pH was then adjusted to 4.5 with 1M HCl to precipitate the protein for 1 hour, followed by centrifugation at 3220g for 15 minutes again. The supernatant was discarded, the precipitate was washed three times with distilled water, adjusted to neutral with 0.1M NaOH, and then freeze-dried under vacuum to obtain silkworm pupa protein, which was stored at -20℃ for later use.

[0090] 2. Enzymatic hydrolysis of silkworm pupa protein

[0091] The silkworm pupa protein powder obtained in step 1 was prepared into a 10 mg / mL suspension and sonicated at 30 °C for 30 min. Hydrolysis was then performed using four different proteases, with the optimal hydrolysis conditions being alcalase (pH = 8, 50 °C), flavorenzyme (pH = 6.5, 50 °C), pepsin (pH = 1.8, 37 °C), and trypsin (pH = 7, 37 °C). 6000 U / g of protease was added to the silkworm pupa protein suspension for hydrolysis for 180 min, with 1 M NaOH continuously added to maintain the pH during the hydrolysis process. Immediately after the reaction, the protein was heated to 90 °C for 10 min to inactivate the protein. After cooling to room temperature, the pH was adjusted to the isoelectric point of the silkworm pupa protein (pH = 4.5), allowed to stand at room temperature for 30 min, and then centrifuged at 3220 g for 15 min. The pH of the hydrolysate was adjusted to neutral, and the protein was freeze-dried to obtain the silkworm pupa protein hydrolysate, which was stored at -20 °C. The four hydrolysates were named SPPH-A, SPPH-F, SPPH-P, and SPPH-T, respectively.

[0092] 3. Separation and purification of hydrolysis products

[0093] (1) Ultrafiltration

[0094] The hydrolyzate was diluted with distilled water and separated by an ultrafiltration (UF) membrane into molecular weight cut-off (MWCO) values of 10 kDa and 3 kDa. The hydrolyzate was divided into three fractions: MW > 10 kDa, 10 kDa < MW < 3 kDa, and MW < 3 kDa. All fractions were collected and freeze-dried for activity verification.

[0095] (2) Gel chromatography purification

[0096] The fraction with the strongest immunomodulatory activity after ultrafiltration was separated and purified by G-25 gel chromatography. The column size was 1.6 cm × 100 cm. Chromatographic purification was performed using an AKTA puro instrument. The sample concentration was 10 mg / mL, and it was eluted with ultrapure water at 1 mL / min. The absorbance was measured at 280 nm, and each elution peak was collected and freeze-dried for storage.

[0097] Example 2. Effects of different proteases on DH

[0098] During protein hydrolysis, the percentage of hydrolyzed peptide bonds in the total protein is called the degree of hydrolysis (DH). DH is a measure of the decomposition of proteins by enzymes and can be used to evaluate the properties of proteins or peptides. Research has shown that the degree of enzymatic hydrolysis affects the biological activity of polypeptides, and different proteases produce enzymolysis products with different structures. In this study, four common proteases, alkaline protease, flavor protease, pepsin, and trypsin, were selected to hydrolyze silkworm pupa protein, and the proteases with high enzymatic hydrolysis efficiency were screened by measuring DH. The OPA method was used in this study to determine the degree of hydrolysis of silkworm pupa protein. 400 μL of the sample solution was accurately reacted with 3 mL of the OPA solution for 2 min, and the absorbance was measured at 340 nm using a multifunctional microplate reader.

[0099] DH was calculated using the following formula:

[0100]

[0101] where h tot —— The total number of peptide bonds per protein equivalent obtained by chemical hydrolysis (h tot depends on the type of raw material and is 8.6 mmol / g in this study);

[0102] X —— The weight of the sample;

[0103] P —— The protein content of the sample;

[0104] β and α —— Depend on the type of protein;

[0105] h0 —— The degree of hydrolysis of the sample relative to the original substrate before the hydrolysis reaction;

[0106] h1 — The degree of hydrolysis of the sample relative to the original substrate after the hydrolysis reaction.

[0107] Results: DH levels of different proteases, such as Figure 1 As shown, alkaline protease has the highest degree of hydrolysis, followed by trypsin, flavor enzymes, and pepsin.

[0108] Example 3. Identification of silkworm pupa protein hydrolysates

[0109] 1. Secondary Structure Analysis

[0110] This invention utilizes FTIR spectroscopy to analyze the secondary structure of four silkworm pupa protein hydrolysates. The samples were freeze-dried, mixed with KBr at a ratio of 1:150, and then ground into powder using a mortar. After being pressed into thin tablets using a tablet press, infrared spectroscopy was performed. The FTIR-650 spectrometer was set to a wavelength range of 4000 cm⁻¹. -1 ~400cm -1 The number of scans was 32, and the resolution was 4cm. -1 Each sample was tested three times under the same conditions. Curve fitting analysis was performed using Omnic 9.2 and Peak Fit v4.12.

[0111] Amide I segment (1600-1700cm) -1 The β-sheet primarily exhibits C=O stretching vibrations and is commonly used to reflect protein secondary structure. Secondary structure is obtained through Gaussian curve fitting, combined with Fourier autoreflexive convolution and the second derivative. The characteristic absorption peak of the β-sheet is generally considered to be between 1600-1640 cm⁻¹. -1 The characteristic absorption peak of the irregularly curled shape is 1640-1650 cm⁻¹. -1 The characteristic absorption peak of the α-helix is ​​1650-1660 cm⁻¹. -1 The characteristic absorption peak of the β-turn is 1660-1700 cm⁻¹. -1 Generally, the secondary structure of proteins is linked by various types of hydrogen bonds, among which α-helices and β-sheets are relatively ordered structures. The content of SPP in the secondary structure of various isolated and purified products is compared. Figure 2 As shown, from Figure 2 It can be seen that the relative content of the secondary structure of SPPH-A changed compared to SPP, with a decrease in β-sheet content and an increase in β-turn content. This may be due to the action of proteolytic enzymes, which led to the breaking of intermolecular hydrogen bonds, the unfolding of the protein structure, and the disruption of the ordered and stable β-sheet structure. It was transformed into a relatively flexible and stretched β-turn structure.

[0112] 2. Amino acid sequencing

[0113] The purified F3 was sequenced using LC-MS / MS. The peptide was desalted for mass spectrometry detection. The HPLC conditions included: a C18 column (0.15 mm * 150 mm, RP-C18, column Technology Inc.), with 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile aqueous solution as mobile phase B, wherein the acetonitrile content in mobile phase B was 84%; the column was equilibrated with 95% solution A; the sample was fed from an autosampler to a Zorbax 300SB-C18 peptide capture device for separation on the column; the HPLC gradient was set as follows:

[0114] A linear gradient of mobile phase B from 4% to 50% and mobile phase A from 96% to 50% was observed from 0 min to 50 min.

[0115] A linear gradient of mobile phase B from 50% to 100% and mobile phase A from 50% to 0% was observed from 50 to 54 minutes.

[0116] Keep solution B at 100% for 54-60 minutes.

[0117] This invention employs capillary high-performance liquid chromatography (HPLC) for separation and Q Exactive HF-X mass spectrometry (MS) for analysis. The analysis time is 60 min, and the detection mode is positive ion. The mass-charge ratio of peptides and peptide fragments is collected as follows: 10 fragment images are collected after each full scan (MS2 scan).

[0118] Results: A total of 609 peptides were identified. Peptide toxicity was initially predicted using Toxinpred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php) and [other methods](https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php), and all specific peptides were found to be non-toxic. Peptides were then scored and screened using PeptideRanker to predict the probability of biological activity; higher probability values ​​indicated a greater likelihood of biological activity, with a probability value >0.8 generally used for screening. Allergen sensitization screening was then performed on the AllerTOP Server Page, and finally, the BIOPEP database was searched to screen for peptide novelty. Nine peptides were screened; see [link to relevant documentation]. Figures 3-11 See Table 1.

[0119] Table 1. Peptide information and molecular docking obtained from F3.

[0120]

[0121]

[0122] Example 4. Molecular docking

[0123] Molecular docking contributes to a better understanding of the molecular mechanisms of immunomodulatory peptides. In this invention, toll-like receptor proteins TLR2 (ID: 1FYW) and TLR4 / MD-2 (ID: 5IJD) were retrieved from the protein database (http: / / www.rcsb.org / pdb) and downloaded. The resulting structures were then imported into PyMOL-2.5.2 to remove solvents and organic matter. Hydrogen was added using AutoDockTools-1.5.7 software, and the structures were exported in "pdbqt" format as docking receptors. Furthermore, two-dimensional structures of various peptides were collected from ChemDraw-20.0 and imported into Chem3D-20.0 software to create three-dimensional structures with minimized free energy. These structures were then exported in "mol2" format as docking ligands. Finally, AutoDock Vina 1.1.2 software was used to dock the receptors and ligands, the binding affinity was scored, and the molecular docking results were visualized using PyMOL-2.5.2.

[0124] Results: All nine peptides successfully docked with TLR2 and TLR4 / MD-2. Binding energy is generally considered a predictive reference value; the lower the binding energy, the lower the binding energy of the ligand-receptor interaction, and the easier it is to form a more stable molecular binding conformation. Generally, a binding energy ≤7 kcal / mol is considered a strong binding. The docking results are shown in Table 2 above. From the binding energy perspective, all peptides showed strong binding to TLR4 / MD-2, and TLR4 / MD-2 has a relatively large cavity, indicating it is primarily hydrophobic. These peptides can stably bind to the hydrophobic cavity and are occupied by complementary structures. Peptide 2, Peptide 3, and Peptide 8 showed strong binding energies to both TLR2 and TLR4 / MD-2. The binding energies of Peptide 2, Peptide 3, and Peptide 8 to TLR2 were -9.3 kcal / mol, -7.2 kcal / mol, and -7 kcal / mol, respectively. The binding energies of Peptide 2, Peptide 3, and Peptide 8 to TLR4 are -13.5 kcal / mol, -8.9 kcal / mol, and -9.3 kcal / mol, respectively. Therefore, we selected these three peptides for ligand-receptor interaction site mapping analysis to elucidate the molecular mechanism of their immunomodulatory activity.

[0125] Reports indicate that hydrogen bonding interactions have a greater influence than charge interactions and π-π bond interactions, playing a crucial role in stabilizing the crystal structure of ligand-acceptor complexes. For example... Figure 12 As shown, the peptide binds to TLR2 and TLR4 / MD-2 mainly through hydrogen bonds and hydrophobic interactions. Figure 12In the image, the protein is pink, in a cartoon style; the peptide is cyan and rod-shaped, and the bound amino acid residues are yellow and rod-shaped. Peptide 3 forms with HIS 697, SER-696, ASP-730, and SER-636 residues of TLR2. and Four hydrogen bonds, see details Figure 12 -A. Peptide 3 forms an interaction with the ILE-46 residue of TLR4 / MD-2. Long hydrogen bonds, see details Figure 12 -D. Peptide 2 forms a complex with ARG-650, TRP-764, and ARG-771 residues of TLR2. and Six hydrogen bonds, see details Figure 12 -B. Peptide 2 forms with asn-359 and LYS-360 residues of TLR4 / MD-2. and Two hydrogen bonds, see details Figure 12 -E. Peptide 8 forms with ASP-678, GLU-664, ASN-688, LYS-695, and SER-692 residues of TLR2. and Eleven hydrogen bonds, see details Figure 12 -C. Peptide 8 forms with LEU-94, HIS-96, ASP-101, and LYS-263 residues of TLR4 / MD-2. and Four hydrogen bonds, see details Figure 12 -F. In addition to hydrogen bonds, these three peptides also bind to TLR2 and TLR4 / MD-2 via hydrophobic forces.

[0126] Example 5. Synthesis, identification, and immunological activity verification of polypeptides.

[0127] 1. Synthesis and Identification of Polypeptides

[0128] To determine whether Peptide 2, Peptide 3, and Peptide 8 were novel sequences to be studied, we searched for these sequences on BIOPEP on July 15, 2024, but they did not appear in any publications. The three peptides were then synthesized using a solid-phase method, and their immunomodulatory activities were investigated. The peptides were synthesized by Shanghai Hongtai Biotechnology Co., Ltd. (Shanghai, China), and their purity and molecular weight were identified by HPLC and MS. Peptide 3 had a purity of 98.85% and an actual molecular weight of 984.13 Da. Peptide 2 had a purity of 99.07% and an actual molecular weight of 782.95 Da. Peptide 8 had a purity of 98.54% and an actual molecular weight of 1365.57 Da. The actual molecular weights of the three peptides were less than 0.1% of their theoretical molecular weights.

[0129] 2. Verification of the immunomodulatory activity of synthetic peptides

[0130] Cells were treated with different concentrations of synthetic peptides. CCK8 results showed that all three synthetic peptides promoted cell growth without exhibiting cytotoxicity. Figure 13 -A and Figure 13 As shown in Figure B, compared with the control group, all three synthetic peptides significantly promoted the proliferation and phagocytic activity of macrophages, and this increase was dose-dependent. These findings suggest that peptides extracted from silkworm pupa proteins have the potential for immunomodulatory enhancement.

[0131] Example 6. Effects of enzymatic hydrolysis products, ultrafiltration products, and separated products on macrophage immune activity indicators

[0132] Immunomodulatory peptides do not interact directly with pathogens, but rather promote host defense responses by binding to receptors on the surface of immune cells. Studies have reported that most amino acids with immunomodulatory effects are hydrophobic, the most common being glycine (Gly), valine (Val), leucine (Leu), proline (Pro), phenylalanine (Phe), the negatively charged amino acid glutamate (Glu), and the aromatic amino acid tyrosine (Tyr). Other studies have shown that peptides containing isoleucine (Ile) and alanine (Ala) can promote immune cell proliferation and stimulate the production of immune-related immune factors. Therefore, Peptide 2, Peptide 3, and Peptide 8 bind to the active sites of TLR4 / MD-2 and TLR2 through hydrogen bonding and hydrophobic interactions, potentially activating signal transduction pathways, stimulating macrophage activation, and regulating the body's immunity.

[0133] This invention verifies the above hypothesis through a cell immunomodulatory activity detection experiment. The specific method and results are as follows:

[0134] 1. Detection of cellular immune activity

[0135] (1) Cell Culture

[0136] RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum and 0.5% penicillin-streptomycin, and then incubated at 37°C in an incubator containing 5% CO2.

[0137] (2) Cell proliferation assay

[0138] The CCK-8 assay was used to detect the proliferative activity of the samples against RAW264.7 cells. 5 × 10⁶ cells were seeded in 96-well plates. 4 RAW264.7 cells were suspended at 100 μL per well. After 12 h, 100 μL of different sample solutions were added and incubated for 24 h, with six replicates for each sample solution. After 24 h, the culture medium was removed, and the wells were washed with PBS. Alternatively, all viability assays were performed using a CCK8 microplate reader. Absorbance was detected using a multi-functional microplate reader.

[0139] (3) EdU staining

[0140] To investigate the effect of the peptide on cell proliferation, cell proliferation capacity was measured using the BeyClick™ EdU-488 imaging kit according to the manufacturer's instructions and operating manual. 1 mL of logarithmic growth phase RAW264.7 cell suspension (5 × 10⁶ cells / well) was seeded into 24-well plates. 4 Cells were incubated at 1 mL / well for 12 hours, followed by incubation for 24 hours with 1 mL of different sample solutions added. EdU staining was performed according to the manufacturer's instructions for the BeyClick™ EdU-488 imaging kit. EdU fluorescence images were obtained using an inverted fluorescence microscope, and the percentage of EdU-positive cells was randomly counted from three microscopic fields of view.

[0141] (4) NO production determination

[0142] NO production in cell culture supernatant was detected using the Griess reaction kit. Standards were diluted according to the manufacturer's instructions to establish a standard curve. Logarithmic growth phase macrophages (5 × 10⁶ cells / year) were used. 4 (Nuclear samples per well) were added to 96-well plates containing 100 μL of culture medium and incubated at 37°C with 5% CO2. After 12 h, 100 μL of different samples were added and incubated for 24 h. Each sample was repeated 6 times. After 24 h, 50 μL of supernatant was collected per well, and 50 μL of Griess Reagent I and Griess Reagent II were added to each well sequentially. The absorbance was measured at 540 nm, and the NO yield was calculated using a standard curve.

[0143] (5) Phagocytic capacity of macrophages

[0144] Macrophages in logarithmic growth phase grow at a rate of 5 × 10⁻⁶. 4Cells / well were seeded into 96-well plates containing 100 μL of culture medium and incubated at 37°C with 5% CO2. After 12 h, 100 μL of different samples were added and incubated for 24 h. Each sample was repeated 6 times. After 24 h, the culture medium was removed, washed 1-2 times with PBS, and 200 μL of cell culture medium and 20 μL of neutral red staining solution were added. The cells were incubated in a cell culture incubator for 2 h. After 2 h, the culture medium was removed, washed 1-2 times with PBS, and 200 μL of neutral red lysis buffer was added. The cells were lysed on a shaker at room temperature for 10 min, and the absorbance was measured at 540 nm.

[0145] 2. Effects of silkworm pupa protease hydrolysates on macrophage immune activity indicators

[0146] The relative growth rate of macrophages is one of the most commonly used indicators for evaluating immune activity. In this invention, RAW264.7 macrophages were treated with different enzymatic hydrolysates obtained in Example 1. The results showed that none of the four enzymatic hydrolysates were cytotoxic to macrophages. The alkaline protease hydrolysate (SPPH-A) significantly promoted macrophage activation and proliferation. (See details...) Figure 14 -A.

[0147] NO is an active mediator that can exert immunomodulatory effects by activating macrophages. The degree of macrophage activation can be evaluated by measuring NO production. Figure 14 As shown in Figure -B, SPPH-A produced significantly more NO than other components, indicating that SPPH-A can upregulate intracellular NO production. Macrophage phagocytosis is an important indicator of macrophage immune regulation; macrophages can protect the body from infections caused by both biological and non-biological attacks. The results of the neutral red phagocytosis experiment are shown below. Figure 14 As shown in Figure -C, there was no significant difference in the phagocytic rate of macrophages among the various enzymatic hydrolysis products. These findings suggest that SPPH-A may be a potential immunomodulator, therefore we focused on SPPH-A to identify key functional peptides.

[0148] 3. Effects of ultrafiltration products on macrophage immune activity indicators

[0149] To identify the key functional peptides of SPPH-A, this invention further purified the hydrolysate of silkworm pupa protease. SPPH-A was dissolved in ultrapure water at a concentration of 1 mg / mL and separated into three fractions using different ultrafiltration membranes, named SPPH-A-Ⅰ (>10 kDa), SPPH-A-Ⅱ (10⁻³ kDa), and SPPH-A-Ⅲ (<3 kDa). The immunomodulatory activity of each fraction was measured by macrophage proliferation, phagocytosis, and NO production, and the strongest fraction was screened. The results showed that, compared with the control group, SPPH-A-Ⅲ significantly stimulated macrophage activation, proliferation, and phagocytosis. (See details below.) Figure 15This indicates that SPPH-A-III contains an active peptide that activates the immune activity of RAW 264.7 cells. Consistent with previous studies, lower molecular weight peptides generally exhibit better activity.

[0150] 4. Effects of the isolated products on macrophage immune activity indicators

[0151] To identify the fraction with strong immunoreactivity, SPPH-A-III was separated into three fractions using a Sephadex G-25 packed size exclusion column, such as... Figure 16 As shown, the effects of three components on macrophage proliferation, NO production, and phagocytosis were measured. The results showed that, compared with the control group, F3 significantly stimulated macrophage activation and proliferation, NO production, and phagocytosis. (See details in [link to study]). Figure 17 In addition, cell proliferation was assessed using EdU staining. The results showed that, compared to the control group, the percentage of edu-positive cells in cells incubated with F3 was significantly increased (see details). Figure 18 This indicates that F3 has higher immune activity and contains more active peptides that activate the immune activity of RAW 264.7 cells.

Claims

1. Silkworm pupa protein immunomodulatory peptide, characterized in that, The amino acid sequence of the silkworm pupa protein immunomodulatory peptide is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

8.

2. The application of the silkworm pupa protein immunomodulatory peptide according to claim 1 in the preparation of products that enhance the body's immunity, characterized in that, The product is either a functional food or a dietary supplement.

3. The application according to claim 2, characterized in that, The silkworm pupa protein immunomodulatory peptide promotes macrophage proliferation, phagocytosis, and NO production.

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