A casein peptide-zinc chelate TEDELQDKIHP-Zn and a preparation method thereof

Abstract

The application discloses a casein peptide-zinc chelate TEDELQDKIHP-Zn for promoting zinc absorption, and an amino acid sequence of a polypeptide part of the casein peptide-zinc chelate is Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-Ile-His-Pro; and a polypeptide and zinc chelate have a chelate ratio of 1:0.7. The casein peptide-zinc chelate TEDELQDKIHP-Zn has high gastrointestinal digestion stability, good absorption performance, and an effect of promoting zinc absorption is obviously better than that of traditional zinc supplements, such as zinc sulfate and zinc gluconate. The casein peptide-zinc chelate has very important significance for developing food, health products and medicines with a zinc supplement function.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, specifically to a casein peptide-zinc chelate TEDELQDKIHP-Zn that promotes zinc absorption and its preparation method. Background Technology

[0002] As an essential trace element, zinc is particularly important for human growth and development and the proper functioning of the immune system, earning it the title of "element of life." Since the body lacks dedicated zinc storage sites, people need to supplement their diet with sufficient amounts to maintain zinc balance. Currently, approximately 2.2 billion people worldwide are affected by zinc deficiency, with infants, pregnant women, breastfeeding women, and the elderly being the most severely impacted. Therefore, the problem of zinc deficiency urgently needs to be addressed.

[0003] Components in food matrices, such as phytic acid, polyphenols, saponins, and cellulose, can bind with zinc to form insoluble complexes, thus hindering zinc absorption and utilization, leading to zinc deficiency. Furthermore, other minerals in food, such as iron, calcium, and copper ions, can also competitively bind to these carriers, inhibiting zinc absorption. Therefore, to prevent zinc deficiency, consuming zinc-rich foods and zinc supplements are two main ways to improve the body's zinc nutritional status. However, zinc supplementation through zinc-rich foods, due to their low zinc content, cannot fully meet the body's zinc requirements. The development of zinc supplements has mainly gone through three stages: inorganic zinc salts (ZnSO4, ZnO), zinc supplements synthesized using organic weak acids as ligands (zinc gluconate, zinc lactate), and zinc supplements synthesized using amino acids as ligands (zinc glycinate). However, these zinc supplements all have significant drawbacks. For example, inorganic zinc salts not only alter the physicochemical and sensory properties of food but also cause gastrointestinal discomfort; organic weak acid zinc supplements have complex preparation processes and can cause adverse clinical reactions such as nausea and constipation, making them unsuitable for long-term intake; and amino acid zinc supplements have low zinc content, making it difficult to meet the body's needs. Currently, fourth-generation zinc supplements prepared using peptides as ligands (e.g., casein phosphopeptide chelated zinc) are receiving widespread attention in the health supplement and food industries due to their safety, high zinc bioavailability, and certain biological activity.

[0004] Against the above background, the present invention constructs a peptide-zinc chelate based on casein peptides, which can not only avoid the precipitation of zinc ions in the digestive tract, but also reduce the competitive inhibition of other nutrients during the absorption of zinc ions, thereby promoting zinc absorption. Summary of the Invention

[0005] This invention constructs a peptide-zinc chelate based on casein peptide synthesized by solid-phase synthesis, and verifies its zinc absorption-promoting effect through an in vitro simulated digestion and absorption model.

[0006] The technical solution adopted in this invention is a casein peptide-zinc chelate TEDELQDKIHP-Zn, the amino acid sequence of which is: Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-Ile-His-Pro, and the ratio of peptide to zinc chelate is 1:0.7.

[0007] A method for preparing a casein peptide-zinc chelate TEDELQDKIHP-Zn involves preparing a peptide via solid-phase synthesis followed by zinc ion chelation. The specific steps include: dissolving 20-50 mg of casein synthetic peptide TEDELQDKIHP in 10-20 mL of deionized water, then adding food-grade ZnSO4 for reaction. The molar ratio of peptide to zinc sulfate is 1:(3-10). The pH of the reaction system is 5.5-7.5, the reaction temperature is controlled at 50-70℃, and the reaction time is 40-70 min. After the reaction, four times the total volume of anhydrous ethanol is added to the reaction system. After thorough shaking, the mixture is centrifuged at 4℃ and 15000×g for 5 min. The precipitate is collected and freeze-dried to obtain the casein peptide-zinc chelate.

[0008] Application of casein peptide-zinc chelate TEDELQDKIHP-Zn, specifically its use in the preparation of zinc-absorbing foods, zinc-absorbing health products, or pharmaceuticals.

[0009] A zinc absorption enhancer product includes casein peptide-zinc chelate TEDELQDKIHP-Zn, wherein the zinc absorption enhancer product includes zinc absorption enhancer foods, zinc absorption enhancer health products, or zinc absorption enhancer drugs.

[0010] The beneficial effects of the present invention are as follows: (1) Based on casein peptide TEDELQDKIHP, it is chelated with food-grade ZnSO4 under mild conditions to form casein peptide-zinc chelate TEDELQDKIHP-Zn, with a peptide-zinc chelation ratio of 1:0.7. (2) In vitro simulated digestion experiments show that casein peptide-zinc chelate TEDELQDKIHP-Zn has excellent gastrointestinal digestion stability. In vitro absorption experiments show that casein peptide-zinc chelate TEDELQDKIHP-Zn can completely pass through the small intestinal epithelial cells and enter the blood circulation, with good absorption (apparent permeability coefficient P). app >1×10 -6 The zinc absorption-promoting effect (cm / s) is 4.67 times that of zinc sulfate and 1.68 times that of zinc gluconate. Attached Figure Description

[0011] Figure 1 Mass spectrum of casein peptide TEDELQDKIHP synthesized by solid-phase method.

[0012] Figure 2 ITC diagram of casein peptide-zinc chelate TEDELQDKIHP-Zn.

[0013] Figure 3 CD diagram of casein peptide-zinc chelate TEDELQDKIHP-Zn.

[0014] Figure 4 Liquid chromatogram of casein peptide-zinc chelate TEDELQDKIHP-Zn gastrointestinal digestive products.

[0015] Figure 5 : Cytotoxicity test of casein peptide-zinc chelate TEDELQDKIHP-Zn against Caco-2 cells.

[0016] Figure 6 : Absorption performance test of casein peptide-zinc chelate TEDELQDKIHP-Zn.

[0017] Figure 7 Casein peptide-zinc chelate TEDELQDKIHP-Zn promotes zinc absorption. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] The purpose of this invention is to provide a casein peptide-zinc chelate that is resistant to digestion and can pass intact through small intestinal epithelial cells, thereby addressing the problem of zinc deficiency in the human body. It is obtained by synthesizing casein peptide TEDELQDKIHP via solid-phase synthesis, followed by chelation reaction with food-grade ZnSO4 under mild conditions.

[0020] (1) Preparation and structural testing of casein peptide-zinc chelate

[0021] 20-50 mg of casein-derived peptide TEDELQDKIHP was dissolved in 10-20 mL of deionized water, and then food-grade ZnSO4 was added for reaction. The molar ratio of peptide to zinc sulfate was 1:(3-10), the pH of the reaction system was 5.5-7.5, the reaction temperature was controlled at 50-70℃, and the reaction time was 40-70 min. After the reaction was completed, a certain amount of ethanol was added for precipitation, followed by low-temperature centrifugation and vacuum freeze-drying to obtain the peptide-zinc chelate TEDELQDKIHP-Zn. The structure of the chelate was then analyzed, mainly including chelation thermodynamics (ITC), chelation site (FTIR), and changes in secondary structure (CD).

[0022] (2) Digestibility and absorption performance of casein peptide-zinc chelate

[0023] The in vitro simulated digestion and absorption performance of the prepared casein peptide-zinc chelate TEDELQDKIHP-Zn was tested. The results showed that the chelate remained intact during digestion, exhibited excellent resistance to digestive enzymes, and could completely pass through the small intestinal epithelial cells into the bloodstream. Its apparent permeability coefficient P app >1×10 -6 It has a speed of cm / s and good absorption performance.

[0024] Implementation Example 1: Preparation and Structural Testing of Casein Peptide-Zinc Chelate

[0025] (1) Solid-phase synthesis of casein peptide TEDELQDKIHP. A certain amount of resin was weighed and placed in a reactor. DCM (dichloromethane) was added to swell the resin for half an hour. Then, the DCM was removed, and the first amino acid in the sequence and DIEA (diisopropylethylamine), an appropriate amount of DMF (dimethylformamide) were added. The reaction was carried out under nitrogen bubbling for 60 minutes. Then, an appropriate amount of methanol was added, and the reaction was carried out for half an hour. The reaction solution was removed, and the mixture was washed with DMF and methanol. The second amino acid in the sequence and HBTU (1-hydroxybenzotrichloroazole tetramethylhexafluorophosphate) and DIEA were added to the reactor. The reaction was carried out under nitrogen bubbling for half an hour, and the liquid was washed off. Ninhydrin was detected, and the ends were sealed with pyridine and acetic anhydride. Finally, after washing, an appropriate amount of decapping solution was added to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, followed by washing and ninhydrin detection. Then, different amino acids from the sequence were added sequentially and various modifications were performed. The resin was dried under nitrogen and removed from the reaction column, poured into a flask, and then a certain amount of cleavage solution (composed of 95% TFA, 2% ethylenedithiol, 2% triisopropylsilane, and 1% water) was added. The mixture was shaken, the resin was filtered off, and a large amount of diethyl ether was added to the filtrate to precipitate the crude product. After centrifugation and washing, the crude product of the sequence was obtained. After high-performance liquid chromatography purification, the product was freeze-dried to obtain the final product. The mass spectrometry results are shown in the appendix. Figure 1 .

[0026] (2) Preparation of the chelate TEDELQDKIHP-Zn. 25 mg of casein synthetic peptide TEDELQDKIHP was dissolved in 10 mL of deionized water and sonicated for 3 min. Then, food-grade ZnSO4 was added to make the molar ratio of peptide to zinc ions 1:6. The pH was adjusted to 6.0 with 0.1 M hydrochloric acid, and the reaction was carried out in a 60℃ water bath for 60 min. Subsequently, 4 times the total volume of anhydrous ethanol was added to the reaction system, and after thorough shaking, the mixture was centrifuged at 4℃ and 15000×g for 5 min. The precipitate was collected and freeze-dried to obtain the casein peptide-zinc chelate.

[0027] (3) Isothermal calorimetric titration (ITC): TEDELQDKIHP (60 μM) and ZnSO4 (600 μM) were dissolved separately in 50 mM 3-morpholinopropanesulfonic acid (MOPS, pH 7.0). Then, ZnSO4 solution was continuously added dropwise to the peptide solution to begin the measurement. The titration data were analyzed using nano-Analyzer software to calculate the chelation coefficient (n), entropy change (ΔS), and enthalpy change (ΔH). The results are attached. Figure 2 As shown, both the enthalpy change and entropy change are negative, indicating that casein peptides can spontaneously react with zinc ions to form peptide-zinc chelates with a chelation ratio of 1:0.7 (peptide:zinc).

[0028] (4) Circular dichroism (CD) assay: TEDELQDKIHP and its chelate TEDELQDKIHP-Zn were dissolved in deionized water (0.5 mg / mL) and subjected to circular dichroism spectroscopy. The spectral recording range was 190-260 nm, the bandwidth was 1.0 nm, and the scan rate was 100 nm / min. The structural changes of the chelate TEDELQDKIHP-Zn were calculated using the obtained chromatographic data. The results are attached. Figure 3 As shown, after chelating zinc ions, the β-sheet structure in the chelate increased significantly (P<0.01), while the β-turn and random coil structure decreased significantly (P<0.05), indicating that the chelate TEDELQDKIHP-Zn structure is more regular and ordered.

[0029] Implementation Example 2: Digestibility and Absorption Performance of Casein Peptide-Zinc Chelate TEDELQDKIHP-Zn

[0030] (1) In vitro digestion stability of the chelate TEDELQDKIHP-Zn. TEDELQDKIHP-Zn was dissolved in 0.01M hydrochloric acid solution at pH 2.0, and pepsin (enzyme:chelate = 1:50, w / w) was added. The mixture was incubated in a 37℃ water bath with shaker for 2 hours to simulate gastric digestion, followed by boiling at 100℃ for 10 minutes to inactivate pepsin. Subsequently, the pH was adjusted to 7.5 with 0.01M NaOH solution, and trypsin (enzyme:chelate = 1:25, w / w) was added. The mixture was then incubated in a 37℃ water bath with shaker for 2 hours to simulate intestinal digestion, followed by boiling at 100℃ for 10 minutes to inactivate trypsin. Samples from the gastric and intestinal digestions were collected and analyzed by high-performance liquid chromatography (HPLC). The results are attached. Figure 4 As shown, the casein peptide-zinc chelate TEDELQDKIHP-Z retained 78.54% of its integrity after gastric digestion and 70.18% after intestinal digestion, demonstrating excellent gastrointestinal digestive stability.

[0031] (2) Construction of the Caco-2 cell uptake model. Caco-2 cells were passaged to 20-30 generations and seeded onto polycarbonate membranes in Transwell culture plates. At 21 days of growth, the luminal side, apical villus side, and intestinal wall side of the differentiated cells were observed using scanning electron microscopy. The expression levels of enzymes related to peptide transport, such as alkaline phosphatase and brush border enzyme, were measured in the cell model using a kit. The transmembrane resistance of the cell model was measured using a Millipore MERS00002 cell resistance meter; a transmembrane resistance >400 Ωcm² was acceptable for the experiment. Simultaneously, the transmembrane flux of fluorescein in the cell model was measured to determine the integrity of the cell monolayer.

[0032] (3) Chelate toxicity test (MTT assay). After Caco-2 cells were seeded in 24-well Transwell plates, a certain amount of TEDELQDKIHP-Zn (100-1000 μg / mL) was added. At time points of 0 h, 24 h, 48 h, and 72 h, 20 μL of MTT solution (5 mg / mL) was added to each well for color development. After incubation for another 4 h, 150 μL of DMSO was added to each well to dissolve the crystals. The absorbance at 490 nm was detected using enzyme-linked immunosorbent assay (ELISA), and cell growth was compared with that of a blank control without added chelates. The results are attached. Figure 5 As shown, the cell viability of Caco-2 cells was not significantly different from that of the control group after different concentrations of chelate were added (P>0.05), indicating that casein peptide-zinc chelate is not toxic to cells within a certain range.

[0033] (4) Chelate absorption and transport assay. Caco-2 cells were washed three times with HBSS buffer. 1.5 mL and 3 mL of preheated HBSS buffer (37℃) were added to the upper (AP) and lower (BL) sides of the Caco-2 cell model, respectively, and the cells were equilibrated at 37℃ for 30 min. Then, the HBSS buffer on the AP side was removed, and HBSS buffer solution containing TEDELQDKIHP-Zn (100-1000 μg / mL) was added. The Transwell culture plate was placed in a CO2 cell incubator for absorption and transport for 2 h. The AP and BL solutions were collected and analyzed by high-performance liquid chromatography (HPLC). The apparent permeability coefficient P of the chelate was calculated. app Simultaneously, absorption and transport experiments were conducted on TEDELQDKIHP-Zn, zinc sulfate, and glucose at the same concentration (1000 μg / mL). During the 2-hour absorption and transport process, the BL test solution was collected for zinc ion concentration determination, and the area under the zinc ion absorption curve (AUC) was calculated. 0-120min The apparent permeability coefficients of transmembrane transport of casein peptide-zinc chelate TEDELQDKIHP-Zn at different concentrations are shown in the attached figure. Figure 6 As shown, the apparent permeability coefficient P of the chelate is within the range of 100-1000 ug / mL. app >1×10 -6 The cm / s indicates that this chelate is a substance with good absorption properties. Regarding its effect on promoting zinc absorption, see attached... Figure 7 As shown, compared with commonly used zinc supplements such as zinc sulfate and zinc gluconate, the chelate TEDELQDKIHP-Zn has a higher zinc absorption-promoting effect (AUC). 0-120min =546.06) is superior, being 4.67 times that of zinc sulfate (AUC). 0-120min =117.92), 1.68 times that of zinc gluconate (AUC) 0-120min =325.81).

[0034] The foregoing describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A casein peptide-zinc chelate TEDELQDKIHP-Zn, characterized in that: The amino acid sequence of its polypeptide is: Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-Ile-His-Pro, and the ratio of polypeptide to zinc chelation is 1:0.

7.

2. A method for preparing a casein peptide-zinc chelate TEDELQDKIHP-Zn, characterized in that: After preparing the peptide by solid-phase synthesis, zinc ion chelation is performed. The specific steps include: taking 20-50 mg of casein synthetic peptide TEDELQDKIHP, dissolving it in 10-20 mL of deionized water, and then adding food-grade ZnSO4 for reaction. The molar ratio of peptide to zinc sulfate is 1:(3-10). The pH of the reaction system is 5.5-7.5, the reaction temperature is controlled at 50-70℃, and the reaction time is 40-70 min. After the reaction is completed, 4 times the total volume of anhydrous ethanol is added to the reaction system. After thorough shaking, the mixture is centrifuged at 4℃ and 15000 × g for 5 min. The precipitate is collected and freeze-dried to obtain the casein peptide-zinc chelate.

3. The application of the casein peptide-zinc chelate TEDELQDKIHP-Zn as described in claim 1, characterized in that: The casein peptide-zinc chelate TEDELQDKIHP-Zn is used in the preparation of zinc-absorbing foods or zinc-absorbing health products.

4. A zinc absorption enhancer product, characterized in that: Including the casein peptide-zinc chelate TEDELQDKIHP-Zn as described in claim 1, the zinc absorption promoting product includes zinc absorption promoting foods or zinc absorption promoting health products.

Images