A small molecule peptide derived from lactadherin and its use

Abstract

The present application relates to a kind of small molecule peptides derived from lactadherin and its application.The amino acid sequence of the small molecule peptide is any one shown in SEQ ID NO:1~SEQ ID NO:4.The small molecule peptide of the present application is non-toxic, safe, can be combined with Ca 2+ Specific binding, strong calcium chelating ability, can significantly improve the calcium transport amount of small intestinal epithelial cells, thereby having good activity of improving intestinal calcium absorption, and can be used in food (such as, infant food) and the like.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, and more specifically, to a small molecule peptide derived from lactoferrin and its applications. Background Technology

[0002] Calcium is an essential mineral for the human body, primarily existing in bones as calcium phosphate. Small amounts of calcium exist in ionic form in soft tissues, extracellular fluid, and blood, maintaining a balance with bone calcium. Adequate calcium intake promotes bone health during growth and development and influences bone mass maintenance in later life. Studies have found that a strong adult bone base begins in early childhood. Ensuring sufficient calcium intake during early growth not only reduces the risk of osteoporosis later in life but also prevents stunted growth and fractures.

[0003] For infants and young children, calcium intake and absorption efficiency are crucial factors affecting bone mineralization, nervous system development, and the establishment of immune function. However, the absorption rate of calcium in the infant's intestines is relatively low, especially when inorganic calcium is used as the calcium source in formula milk powder, and there is still significant room for improvement in its bioavailability.

[0004] Previous studies have shown that bioactive peptides from certain sources can form stable chelates with calcium ions, thereby improving their solubility and transmembrane transport efficiency in the intestine. For example, the egg white peptide-calcium chelate prepared by Huang et al. enhanced the calcium transport capacity in the Caco-2 cell model, thereby promoting the absorption of calcium in the intestine.

[0005] Further development of more bioactive peptides that can promote intestinal calcium absorption is needed to meet current calcium supplementation needs in areas such as nutritional supplementation. Summary of the Invention

[0006] The primary objective of this invention is to overcome the aforementioned problem of the lack of existing active peptides that promote intestinal calcium absorption, and to provide a small molecule peptide.

[0007] A further objective of this invention is to provide the application of the above-mentioned small molecule peptides in the preparation of food.

[0008] A further object of the present invention is to provide the application of the above-mentioned small molecule peptides in the preparation of products that can promote calcium absorption.

[0009] A further object of the present invention is to provide the application of the above-mentioned small molecule peptides in the preparation of calcium supplements.

[0010] A further object of the present invention is to provide the application of the above-mentioned small molecule peptides in the preparation of calcium supplements as an adjuvant.

[0011] A further objective of this invention is to provide the application of the above-mentioned small molecule peptides in the preparation of pharmaceuticals.

[0012] A further object of the present invention is to provide the use of the above-described composition of small molecule peptides and calcium agents in the preparation of calcium supplements.

[0013] A further object of the present invention is to provide a composition.

[0014] The above-mentioned objective of the present invention is achieved through the following technical solution: A small peptide derived from lactoferrin, wherein the amino acid sequence of the small peptide is any one of SEQ ID NO:1 to SEQ ID NO:4.

[0015] The amino acid sequence of the small molecule peptide of the present invention is as follows: SEQ ID NO: 1: SELSKELTPK (Ser-Glu-Leu-Ser-Lys-Glu-Leu-Thr-Pro-Lys); SEQ ID NO:2: KLSQEFH (Lys-Leu-Ser-Gln-Glu-Phe-His); SEQ ID NO: 3: HSDESDEVDF (His-Ser-Asp-Glu-Ser-Asp-Glu-Val-Asp-Phe); SEQ ID NO: 4: PTDIPTIA (Pro-Thr-Asp-Ile-Pro-Thr-Ile-Ala).

[0016] The small molecule peptides of the present invention are derived from lactoferrin, which can be obtained either by simulating gastrointestinal digestion (enzymatic hydrolysis) and separation of lactoferrin, or by solid-phase synthesis. Since solid-phase synthesis can be used, large-scale production is possible, and the synthesized small molecule peptides are not only highly pure but also of stable quality, which can meet current market demands.

[0017] The inventors of this invention have discovered that the small molecule peptides of this invention are non-toxic, highly safe, and can react with Ca. 2+ It has specific binding and strong calcium chelation ability, which can significantly increase the calcium transport capacity of small intestinal epithelial cells, thus having good activity in enhancing intestinal calcium absorption. It can be used to prepare food (e.g., infant food).

[0018] Preferably, the fourth amino acid at the N-terminus of the sequence shown in SEQ ID NO:1 is further phosphorylated, i.e., SELSpKELTPK, where Sp represents phosphorylated serine.

[0019] Preferably, the third amino acid at the N-terminus of the sequence shown in SEQ ID NO:2 is further phosphorylated, i.e., KLSpQEFH, where Sp represents phosphorylated serine.

[0020] The structural formula of phosphorylated serine is shown below: .

[0021] The application of the above-mentioned small molecule peptides in food preparation is also within the scope of protection of this invention.

[0022] Preferably, the food is at least one of supplementary food, formula food, or health food.

[0023] More preferably, the supplementary food is an infant supplementary food.

[0024] More preferably, the formulated food is at least one of infant formula and special medical purpose formula.

[0025] More preferably, the infant formula is at least one of infant formula, follow-up formula, toddler formula, and infant formula for special medical purposes.

[0026] Preferably, the food is at least one of liquid food, semi-liquid food, solid food, or semi-solid food.

[0027] Preferably, the food is a dairy product.

[0028] More preferably, the dairy product is at least one of fermented milk and modified milk powder.

[0029] The small molecule peptides of the present invention can be obtained by simulating gastrointestinal digestion (enzymatic hydrolysis) and separating lactoferrin. The process of simulating gastrointestinal digestion closely matches the digestive process in the human body, especially in infants and young children. Therefore, the small molecule peptides are used to prepare infant foods (such as infant complementary foods and infant formula foods), which are very beneficial to promoting the absorption of calcium in the intestines of infants and young children.

[0030] The application of the above-mentioned small molecule peptides in the preparation of products that promote calcium absorption is also within the scope of protection of this invention.

[0031] Preferably, the product is one that promotes intestinal calcium absorption.

[0032] The application of the aforementioned small molecule peptides in the preparation of calcium supplements is also within the scope of protection of this invention.

[0033] Preferably, the use of the small molecule peptide in the preparation of calcium supplements is also within the scope of protection of this invention.

[0034] The application of the above-mentioned small molecule peptides in the preparation of pharmaceuticals is also within the scope of protection of this invention.

[0035] Preferably, the medicine is a medicine for the prevention and / or treatment of osteoporosis.

[0036] The use of the above-mentioned combination of small molecule peptides and calcium in the preparation of calcium supplements is also within the scope of protection of this invention.

[0037] Preferably, the calcium agent is a calcium salt.

[0038] More preferably, the calcium salt is at least one of inorganic calcium salt, organic calcium salt, or biological calcium salt.

[0039] More preferably, the inorganic calcium salt is at least one of calcium chloride, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, or tricalcium phosphate.

[0040] More preferably, the organic calcium salt is at least one of calcium gluconate, calcium citrate, calcium lactate, milk mineral salt, L-threonate calcium, calcium glycinate, calcium aspartate, calcium malate, calcium acetate, vitamin E succinate, or calcium glycerophosphate.

[0041] More preferably, the biological calcium salt is at least one of milk calcium, eggshell calcium, seaweed calcium, fish bone calcium, seashell calcium, crustacean calcium, and coral calcium.

[0042] A composition comprising the above-mentioned small molecule peptide and calcium agent.

[0043] Compared with the prior art, the beneficial effects of the present invention are: The small molecule peptides of this invention are non-toxic, highly safe, and can react with Ca. 2+ It has specific binding and strong calcium chelation ability, which can significantly increase the calcium transport capacity of small intestinal epithelial cells, thus having good activity in enhancing intestinal calcium absorption. It can be used to prepare food (e.g., infant food). Attached Figure Description

[0044] Figure 1 This is a chromatogram of the high-performance liquid chromatography analysis of the Mw < 3kDa component in Example 1.

[0045] Figure 2 The graph shows the test results of calcium transport capacity testing in Example 3.

[0046] Figure 3 Figure A shows the results of intracellular calcium retention testing in Example 4. Figure 3 B is a graph showing the intracellular calcium fluorescence intensity results of the calcium retention test in Example 4.

[0047] Figure 4 A is an intracellular calcium fluorescence imaging image of the Control group in Example 4, used for the calcium retention test. Figure 4 B is an intracellular calcium fluorescence imaging image of the SELSpKELTPK group in Example 4, used for calcium retention testing. Figure 4C is an intracellular calcium fluorescence imaging image of the KLSpQEFH group in Example 4, used for calcium retention testing. Figure 4 D is an intracellular calcium fluorescence imaging image of the HSDESDEVDF group in Example 4, used for calcium retention testing. Figure 4 E is an intracellular calcium fluorescence imaging image of the PTDIPTIA group in Example 4, used for calcium retention testing. Detailed Implementation

[0048] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0049] The materials and reagents used in various embodiments of this invention are described below: Lactopontin (L-OPN) was provided by Biostime (Changsha) Nutritional Foods Co., Ltd.; trypsin (P7545, enzyme activity 6.4 U / mg), pepsin (P7000, enzyme activity ≥250 U / mg), and bile salts were purchased from Sigma-Aldrich, USA; DA201-C macroporous adsorption resin was purchased from Tianjin Yunkai Resin Technology Co., Ltd.; BCA protein detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; Caco-2 cells (catalog number: TCHU146) were purchased from the Cell Bank of the Chinese Academy of Sciences; DMEM basal medium, penicillin, streptomycin solution, fetal bovine serum, 0.25% trypsin, and TRIzol lysis buffer were purchased from Gibco; 96-well cell culture plates and 12-well Transwell cell culture plates were purchased from Corning; other reagents were of analytical grade; Dynamic In Vitro Human Gastrointestinal System (DHSI-IV) was purchased from Xiaodong Yijian Instrument Equipment Co., Ltd.

[0050] Cell Culture: Caco-2 cells (catalog number: THu146) were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells (passage number 10-20) were seeded into culture flasks and placed in a cell culture incubator at 37℃ and 5% CO2. The medium was changed every two days. When the cells covered approximately 80%-90% of the culture flask, they were digested with 0.25% trypsin for 3 minutes and then passaged. The complete culture medium consisted of 79% DMEM, 1% penicillin-streptomycin solution, and 20% fetal bovine serum.

[0051] Example 1: Preparation of small molecule peptides 1.1 Dynamic simulation of gastrointestinal digestion Lactoadenosine solution was added to DHSI-IV via a sample funnel to initiate the digestion program. The sample underwent digestion and peristalsis sequentially through the esophagus, stomach, duodenum, and small intestine at 37°C for 180 minutes. After digestion, the digestion products were collected and placed in a 95°C water bath for 10 minutes to inactivate the enzymes. After cooling, the digestion products were centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. The supernatant was then desalted using DA201-C macroporous adsorption resin to obtain lactoadenosine peptides.

[0052] 1.2 Purification of Small Molecule Peptides 1.2.1 Ultrafiltration The lactoferrin peptides were placed in a 50 mL ultrafiltration tube with a 3 kDa molecular weight cutoff ultrafiltration membrane (Merck Millipore, Billerica, MA, USA). During ultrafiltration, the ultrafiltration tube was centrifuged at 4000 rpm for 25 min, and the fraction with Mw > 3 kDa in the inner tube and the fraction with Mw < 3 kDa in the filtrate in the outer tube were collected.

[0053] Samples (lactoadenosine peptide, Mw<3kDa fraction, and Mw>3kDa fraction) were chelated at 37℃ and pH=7 with a sample:CaCl2 mass ratio of 1:1. The calcium content was measured, and the calcium chelating activity was analyzed. The results showed that the Mw<3kDa fraction exhibited the most significant calcium chelating activity, reaching 89.67 μg / mg; the lactoadenosine peptide showed the second highest activity; while the Mw>3kDa fraction had the lowest activity, significantly lower than the Mw<3kDa fraction (P<0.05).

[0054] 1.2.2 High Performance Liquid Chromatography Separation The fraction with Mw < 3 kDa was filtered through a 0.22 μm filter membrane and then further separated using a preparative liquid chromatography system (chromatographic system: Prep150 preparative liquid chromatography system; reversed-phase column: SunFirePrepC18OBDT™ 19 mm × 250 mm, 5 μm, Waters). The separation conditions were as follows: mobile phase A: deionized water + 0.1% TFA; mobile phase B: methanol + 0.1% TFA; injection volume 8 mL; flow rate 5 mL / min; elution gradient: 0–20 min, 10%–25% mobile phase B; 20–65 min, 25%–45% mobile phase B; 65–80 min, 45%–75% mobile phase B; 80–110 min, 75%–90% mobile phase B; 110–135 min, 90% mobile phase B.

[0055] The results of high performance liquid chromatography separation are as follows Figure 1As shown, after collecting and concentrating each absorption peak by rotary evaporation and freeze-drying, the calcium chelating activity of each fraction (F1, F2, F3, F4, F5, F6, F7, F8, and F9) was measured. The calcium chelating activity of the liquid fractions was determined using the o-cresphthalenephthalein colorimetric method: 1 mL of 1 mg / mL sample solution was placed in a 10 mL centrifuge tube, and 2 mL of 0.02 mol / L sodium phosphate buffer was added. The mixture was then reacted in a shaker at 37°C for 60 min. The reaction solution was centrifuged at 4000 rpm for 20 min to precipitate insoluble calcium phosphate salts, and the calcium content in the supernatant was then determined using the o-cresphthalenephthalein colorimetric method.

[0056] The results showed that fraction F9 exhibited the most outstanding calcium chelating activity, reaching 84.49 μg / mL, significantly higher than the other eight fractions (P < 0.05). This result indicates that fraction F9 is enriched with the core active substances capable of calcium chelation. Fraction F9 was identified as the target fraction with the strongest calcium chelating activity, and its peptide sequences were subsequently identified using liquid chromatography-tandem mass spectrometry to screen for peptides with strong calcium chelating capabilities.

[0057] 1.2.3 Structural Identification of Peptides One mg of lyophilized F9 fraction was desalted using a C18 desalting column and then analyzed by LC-MS / MS equipped with an online nanospray ionization source. A total of 5 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm × 25 cm). The sample was separated using a gradient ionization technique with a 60-min time. The column flow rate was controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray ionization voltage at 2 kV. The gradient started at 4% B phase (80% ACN, 0.1% FA), increased non-linearly to 50% within 53 min 40 sec, increased to 95% within 40 sec, and was maintained for 5 min 40 sec. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 100-1500; resolution: 120,000; normalized AGC target: 200%; maximum injection time: 100ms; (2) HCD-MS / MS: resolution: 50,000; normalized AGC target: 200%; maximum injection time: 86ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30s.

[0058] Tandem mass spectra were analyzed using PEAKS Studio version 10.6. The database was uniprot-Bostaurus_2024, with enzymatic digestion set to None. Search parameters: fragment ion mass tolerance: 0.02 Da; parent ion mass tolerance: 10 ppm.

[0059] After identification, the F9 component consisted of 705 peptides with molecular weights ranging from 299 to 3103 kDa.

[0060] 1.2.4 Molecular docking The 705 peptides obtained were compared with Ca using AutodockVina. 2+ Molecular docking was performed, with the screening criteria set as "-10lgP ≥ 60 (high confidence) and peptide chain length ≤ 10 amino acids", ultimately yielding 60 peptides. Of the 44 peptides obtained through molecular docking screening, all could bind to Ca. 2+ Spontaneous binding occurred. Based on the binding energy ranking, four small peptides with the strongest calcium binding capacity were screened out as SELSpKELTPK, KLSpQEFH, HSDESDEVDF, and PTDIPTIA, as shown in Table 1.

[0061] Table 1 Small molecule peptides and Ca 2+ Molecular docking results

[0062] 1.2.5 Synthesis of Small Peptides The small molecule peptides SELSpKELTPK, KLSpQEFH, HSDESDEEVDF, and PTDIPTIA were synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase synthesis method and are used for subsequent testing.

[0063] Example 2: Cytotoxicity test of small molecule peptides After digesting Caco-2 cells in the logarithmic growth phase with trypsin, they were then subjected to a reaction at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in 96-well plates and incubated for 24 h. Then, the complete culture medium was removed, and 100 μL of complete culture medium solution containing different concentrations (0.05, 0.1, 0.2, 0.25, 0.5, 1 mg / mL) of Mw < 3 kDa components and small peptides was added to the sample groups, while the blank control group only received complete culture medium. The plates were then incubated. After 24 h of incubation, the complete culture medium was discarded, and 100 μL of 0.5 mg / mL MTT solution was added for 4 h. Finally, the MTT solution was removed, and 100 μL of LDMSO was added to dissolve the purple crystalline product, and the absorbance at 490 nm was measured. The cell viability was calculated using the formula shown in (1): (1).

[0064] The results showed that within the concentration range of 0.05–1 mg / mL, the survival rate of Caco-2 cells in the Mw < 3 kDa fraction, the SELSpKELTPK, KLSpQEFH, HSDESDEVDF, and PTDIPTIA treatment groups all exceeded 94%, indicating that none of the four small peptides had cytotoxic effects within this concentration range. Therefore, subsequent calcium transport experiments can be conducted within this concentration range.

[0065] Example 3: Calcium transport capacity test of small molecule peptides 3.1 Establishment of a Caco-2 cell monolayer model With 1×10 5 Cells were seeded at a density of cells / well into 12-well Transwell nested plates (PET Corning, 3460) and cultured for 21 days, with medium changes every other day. Transepithelial resistance (TEER) values ​​were measured on days 8, 15, and 21 to detect the integrity of the Caco-2 monolayer cell membrane.

[0066] During cell monolayer culture, the TEER (transepithelial resistance) value gradually increased with increasing culture time, indicating continuous cell proliferation and the formation of a dense cell barrier. After inoculation, Caco-2 cells rapidly proliferated and firmly attached to the culture membrane, exhibiting a flat and irregular morphology. With prolonged culture time, the cells gradually formed clusters. By day 7, Caco-2 cells had covered the entire polyester Transwell membrane, forming a monolayer with uniform cell distribution. With further increases in cell culture time (a total of 21 days), the Caco-2 cell monolayers on the polyester Transwell membrane became more tightly connected and structurally more complete, with a TEER value reaching 807 Ω × cm², indicating dense cell monolayer growth, complete differentiation of the apical-basal side membrane, and stable barrier function, meeting the model requirements for calcium transport experiments. Under the culture conditions, they spontaneously formed a polarized intestinal model resembling the villi of small intestinal epithelial cells.

[0067] 3.2 Calcium transport capacity test Before the transfer experiment, prepare a new 12-well plate and add 1.5 mL of HBSS buffer to the lower chamber. Wash the 21-day cultured cell monolayer three times with HBSS buffer and then transfer it to the new 12-well plate. Premix the pre-prepared CaCl2 solution with the small molecule peptides and incubate at 37°C for 1 hour (CaCl2 + Ca ... 2+ The concentration was 300 μg / mL; the ratio of small molecule peptide to CaCl2 was 1:1 (m:m). The control group (Control group) only had CaCl2 added. 2+(Concentration 300 μg / mL) After transport for 30, 60, 120, and 180 min, 1 mL of the transport solution was taken from the lower chamber to determine the calcium transport amount, and 1 mL of HBSS buffer was quickly added. The calcium concentration in the transport solution taken at each time point was determined by flame atomic absorption spectrometry.

[0068] The results are as follows Figure 2 As shown, the calcium transport capacity of the Mw<3kDa fraction and the small molecule peptide treatment groups were significantly higher than that of the Control group in each time period (P<0.05), confirming that the addition of small molecule peptides can significantly promote calcium transmembrane transport and absorption. At 30 min of incubation, the calcium transport capacity of all sample groups showed a rapid upward trend, with the calcium transport capacity of the KLSpQEFH group being significantly higher than that of other sample groups (P<0.05). At 180 min of incubation, the calcium transport capacity of the Mw<3kDa group, SELSpKELTPK group, KLSpQEFH group, HSDESDEEVDF group, and PTDIPTIA group were 57.06 μg / well, 58.04 μg / well, 61.80 μg / well, 54.24 μg / well, and 57.43 μg / well, respectively, which were 83.8%, 83.4%, 95.4%, 71.5%, and 81.6% higher than that of the Control group, respectively. Among them, the calcium transport-promoting effect of the KLSpQEFH group was the most significant. These results indicate that small molecule peptides can significantly increase the calcium transport capacity of small intestinal epithelial cells, thereby having a good activity in enhancing intestinal calcium absorption.

[0069] Example 4: Calcium Retention Test of Small Molecule Peptides After 180 minutes of calcium transport, the transport medium was removed from the 12-well plate, and the cell monolayer was washed with HBSS buffer. 400 μL of RIPA lysis buffer was added to each well to lyse the cell monolayer. The calcium content in the cells was determined using flame atomic absorption spectrometry, and the calcium retention was calculated. Simultaneously, calcium fluorescence analysis was performed on the Caco-2 cell monolayer model after 180 minutes of transport. The procedure was followed according to the Elabscience® Fluo-4 calcium ion fluorescence assay kit instructions. The bright-field cells were located under an inverted fluorescence microscope, the fluorescence field was switched, and fluorescence images of the cells were captured. The fluorescence intensity was analyzed using ImageJ software.

[0070] The results are as follows Figure 3As shown in Figure A, the cellular calcium retention levels in the Control group (CaCl2 group), SELSpKELTPK group, KLSpQEFH group, HSDESDEEVDF group, and PTDIPTIA group were 9.73 μg / mL, 7.22 μg / mL, 6.15 μg / mL, 5.97 μg / mL, and 4.20 μg / mL, respectively. The cellular calcium retention levels in each small peptide treatment group were lower than those in the Control group. Intracellular calcium fluorescence imaging analysis further validated these results. Figure 3 B and Figure 4 A~4E ( Figure 4 A, Figure 4 B. Figure 4 C Figure 4 D、 Figure 4 E shows intracellular calcium fluorescence imaging images of the Control group, SELSpKELTPK group, KLSpQEFH group, HSDESDEEVDF group, and PTDIPTIA group (representing the control, SELSP, KELTPK, KLSpQEFH, HSDESDEEVDF, and PTDIPTIA groups, respectively). The intracellular calcium fluorescence intensity of each small peptide treatment group was lower than that of the Control group, directly reflecting the lower intracellular calcium content, consistent with quantitative detection results. This further indicates that small peptides can significantly promote calcium ion translocation across the cell monolayer through an active transport mechanism. This manifests as rapid transport of intracellular calcium ions to the basal side, resulting in reduced intracellular calcium retention and high calcium accumulation in the transport medium on the basal side.

[0071] Example 5: Transmembrane transport assay of small molecule peptides A Caco-2 cell monolayer model was constructed, and a small peptide HBSS transport solution was prepared. After 180 min of transport, the solutions from the upper and lower chambers were collected for mass spectrometry analysis to observe the degradation and absorption of the small peptides.

[0072] To systematically elucidate the transmembrane transport mechanism, structural stability, and potential absorption mechanism of the small peptide KLSpQEFH, LC-MS / MS was used to analyze the peptide composition and degradation characteristics in the upper and lower chamber transport solutions after 180 min of transport incubation in a Caco-2 cell monolayer model. The results are shown in Tables 2 and 3. Four peptides were detected in the upper chamber transport solution (simulated intestinal lumen side) (Table 2), while seven peptides were detected in the lower chamber transport solution (simulated blood side) (Table 3). The lower chamber peptides contained all four peptides in the upper chamber. Combined with the total ion chromatogram and secondary mass spectrometry, it can be seen that KLSpQEFH may be partially degraded by peptidases on the surface of intestinal epithelial cells in the intestinal simulated environment. However, the small molecular weight peptide fragments generated by its degradation did not accumulate in the intestinal lumen but could be effectively recognized by Caco-2 intestinal epithelial cells and transported across the membrane. Based on the physiological characteristics of intestinal absorption, low-molecular-weight peptides (especially fragments with a molecular weight <1 kDa) can be rapidly absorbed through paracellular pathways or carrier-mediated pathways, consistent with previous research findings, confirming that KLSpQEFH and its degradation products have good intestinal absorption potential. The intact KLSpQEFH peptide was detected in both the upper and lower compartments of a Transwell plate, and notably, its abundance was highest in both compartments. Observations revealed that the Area value of KLSpQEFH in the lower compartment was significantly higher than other peptides, indicating a relatively higher content. This data fully demonstrates its structural stability from two dimensions: firstly, in the simulated intestinal lumen environment of the upper compartment, KLSpQEFH was not completely degraded by peptidases, and most of it remained intact, indicating that its amino acid sequence arrangement or spatial conformation has anti-degradation properties; secondly, the intact peptide could cross the Caco-2 cell monolayer barrier to enter the lower compartment, confirming that it was not completely hydrolyzed by intracellular peptidases during transmembrane transport, thus maintaining its structural integrity.

[0073] Table 2. Peptide composition in the transwell plate chamber after 180 min of transport.

[0074] Table 3. Peptide composition in the lower compartment of the Transwell plate after 180 min of transport.

[0075] LC-MS / MS analysis also revealed the degradation site characteristics of KLSpQEFH, with degradation mainly occurring at the two ends of the peptide, while the core region in the middle of the peptide showed no significant breakage. This conclusion is the core logical link connecting "transmembrane transport" and "promoting calcium absorption": whether KLSpQEFH crosses the membrane intact or retains its core chelating region after partial degradation, it can maintain its affinity for Ca²⁺. + Its chelating ability allows it to bind Ca²⁺ in the form of "peptide-calcium chelates". +It is transported to the intestinal epithelial cells or directly enters the bloodstream, avoiding Ca²⁺. + It precipitates and is lost in the intestinal lumen, ultimately significantly improving the bioavailability of calcium.

[0076] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A small molecule peptide derived from lactoferrin, characterized in that, The amino acid sequence of the small molecule peptide is any one of SEQ ID NO:1 to SEQ ID NO:

4.

2. The small molecule peptide according to claim 1, characterized in that, The fourth amino acid at the N-terminus of the sequence shown in SEQ ID NO:1 is also phosphorylated.

3. The small molecule peptide according to claim 1, characterized in that, The third amino acid at the N-terminus of the sequence shown in SEQ ID NO:2 is also phosphorylated.

4. The use of the small molecule peptide according to any one of claims 1 to 3 in the preparation of food.

5. The application according to claim 4, characterized in that, The food is at least one of supplementary food, formula food, or health food.

6. The application according to claim 5, characterized in that, The supplementary food is an infant supplementary food; the formula food is at least one of infant formula food and special medical purpose formula food.

7. The application according to claim 4, characterized in that, The food is at least one of liquid food, semi-liquid food, solid food, or semi-solid food.

8. The use of the small molecule peptide according to any one of claims 1 to 3 in the preparation of products that promote calcium absorption.

9. The use of the small molecule peptide according to any one of claims 1 to 3 in the preparation of calcium supplements.

10. A composition, characterized in that, Includes the small molecule peptides and calcium agents described in any one of claims 1 to 3.

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