White snakehead oligopeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and discloses a white snakehead oligopeptide as well as a preparation method and application thereof. The white snakehead oligopeptide is selected from one of oligopeptides with amino acid sequences as shown in SEQ ID NO.1-8. The invention further discloses a preparation method of the white snakehead oligopeptide. The polypeptide provided by the invention has a relatively high ferrous / zinc absorption rate to a human body, has a certain function of treating iron-deficiency anemia and zinc deficiency, and has a good application prospect in mineral (iron / zinc) supplements, functional foods and health-care products.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to short peptides from white cuttlefish, their preparation methods, and applications. Background Technology

[0002] Iron and zinc are essential trace elements for maintaining human physiological functions; however, their dietary absorption rate is generally low due to the influence of the intestinal environment and antagonistic factors (such as phytic acid and polyphenols). As a core component of hemoglobin and various redox enzymes, iron deficiency directly leads to iron deficiency syndrome. Anemia leads to a decreased oxygen-carrying capacity in the body, manifesting as persistent fatigue, weakened cognitive function, and impaired immunity. It also has a profound negative impact on children's neurodevelopment and pregnancy outcomes in pregnant women. Zinc, as a cofactor for over 300 enzymes, participates in DNA synthesis, cell division, immune function, and wound healing. Zinc deficiency can lead to growth retardation, taste disorders, weakened immune responses, and an increased risk of diarrhea, skin inflammation, and other diseases. Recent studies have found that dietary short peptides are not only easily absorbed by the intestines but can also form stable peptide-metal chelates with iron and zinc ions. These chelate structures effectively shield minerals from binding to intestinal antagonistic factors while promoting transmembrane absorption through peptide transport systems, thereby significantly improving the bioavailability of iron and zinc and minimizing gastrointestinal irritation.

[0003] In view of this, in order to solve the problem of inefficient iron absorption in the human body, this invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a class of bioactive peptides derived from white cuttlefish, which exhibit highly efficient and specific coordination with divalent metal ions through the carboxyl groups of specific acidic amino acids in their sequence, along with their preparation methods and applications. The peptides provided by this invention can enhance the absorption rate of ferrous iron / zinc in the human body and can be applied to mineral (iron) supplements, functional foods, or health products.

[0005] To achieve the above objectives, the first technical solution provided by this invention is as follows: White cuttlefish short peptides are selected from one of the short peptides whose amino acid sequences are shown in SEQ ID NO.1~8.

[0006] The second technical solution provided by this invention is: The peptide-metal chelate comprises the short peptide from white cuttlefish described above, and ferrous or zinc ions that are coordinated with the peptide.

[0007] Preferably, the molar ratio of the polypeptide to the metal ion is 1:(1-3).

[0008] The third technical solution provided by this invention is: The method for preparing short peptides from white cuttlefish includes: homogenizing white cuttlefish meat, simulating in vitro human digestion and enzymatic hydrolysis to obtain an enzymatic hydrolysate; using ultrafiltration to separate and retain ultrafiltration components with a molecular weight cutoff of less than 3000 Da from the enzymatic hydrolysate; performing gel chromatography on the ultrafiltration components; and separating and purifying the components obtained after chromatography by reversed-phase high-performance liquid chromatography.

[0009] The fourth technical solution provided by this invention is: The preparation method of peptide-metal chelate includes the following steps: mixing the above-mentioned white cuttlefish short peptide with ferrous salt or zinc salt in a buffer solution, reacting at a suitable pH and temperature, and separating and purifying the peptide-metal chelate after the reaction is completed.

[0010] Preferably, when preparing the peptide-ferrous chelate, the reaction pH is 7.5-8.0, the temperature is 35-45℃, the mass ratio of the white mullet short peptide to Fe²⁺ is (2-4):1, and ascorbic acid is used as an antioxidant.

[0011] Preferably, when preparing the peptide-zinc chelate, the reaction pH is 2.5-3.5, the temperature is 45-55℃, and the mass ratio of the peptide to Zn²⁺ is (2-4):1.

[0012] The fifth technical solution provided by this invention is: The use of the short peptide from white cuttlefish as described in the first technical solution, or the peptide-metal chelate as described in any of the second technical solutions, in the preparation of a medicine for supplementing minerals.

[0013] Preferably, the mineral is iron or zinc.

[0014] The sixth technical solution provided by this invention is: The application of the short peptide from white cuttlefish as described in the first technical solution, or the peptide-metal chelate as described in any of the second technical solutions, in the preparation of functional foods or health products.

[0015] Compared with the prior art, the present invention has the following advantages: The polypeptide provided by this invention has a high absorption rate of ferrous iron / zinc in the human body and has a certain function in treating iron deficiency anemia and zinc deficiency. It has good application prospects in mineral (iron / zinc) supplements, functional foods and health products. Attached Figure Description

[0016] Figure 1 The chromatograms are those of ultrafiltration fractions separated by Sephadex G-15 gel chromatography. Figure 2 This is a diagram showing the chelation activity of the chromatographically separated components with ferrous iron. Figure 3This is a diagram showing the chelation activity of zinc with the chromatographically separated components; Figure 4 The reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram shows the separation of chromatographic component F-1-2. Figure 5 The reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram shows the separation of chromatographic component F-1-1. Figure 6 The diagram shows the chelation activity of each short peptide with ferrous iron. Figure 7 This is a diagram showing the chelation activity of each short peptide with zinc. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are merely exemplary and not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0018] The first embodiment of the present invention provides a short peptide from white cuttlefish, selected from one of the short peptides shown in SEQ ID NO. 1~8.

[0019] Among them, SEQ ID NO.1 is NDHFVK; SEQ ID NO.2 is DLEKKLN; SEQ ID NO.3 is VGDHREK; SEQ ID NO.4 is RDWPDAR; SEQ ID NO.5 is KDHPYYK; SEQ ID NO.6 is DHPYYK; SEQ ID NO.7 is AKSPDDIKK; SEQ ID NO.8 is DFKSPDDVSRH.

[0020] The short peptide amino acid sequence selected for the white cuttlefish short peptide in the embodiments of the present invention was found in white cuttlefish meat.

[0021] In the following specific embodiments of the present invention, after the ultrafiltration components are chromatographically analyzed using Sephadex G-15 dextran gel chromatography, the components with the best ferrous chelating activity and zinc chelating activity after chromatography are selected, and further separated using reversed-phase high-performance liquid chromatography. The amino acid sequences are identified by liquid chromatography-tandem mass spectrometry, and then the selected amino acid sequences are synthesized into short peptides in solid phase. Each short peptide is then chelated with ferrous and zinc to determine its chelating activity.

[0022] The second embodiment of the present invention provides a peptide-metal chelate, comprising the above-mentioned short peptide from white cuttlefish, and ferrous or zinc ions coordinated with the peptide.

[0023] The preparation method of peptide-metal chelate includes: mixing short peptides of white cuttlefish with ferrous salt or zinc salt in a buffer solution, reacting at a suitable pH and temperature, and separating and purifying after the reaction is completed.

[0024] When preparing peptide-ferrous chelate, the specific method is as follows: dissolve the solid-phase synthesized white cuttlefish short peptide in pure water, use ascorbic acid as an antioxidant, adjust the pH to 7.5-8.0, add white cuttlefish short peptide and FeCl2·4H2O at a mass ratio of (2-4):1, vortex mix, react at 35-45 ℃ for 1 h, let stand and centrifuge, take the supernatant for purification, freeze dry for later use.

[0025] The method for determining ferrous chelating activity is as follows: A 10 mg / mL peptide-ferrous chelate sample was reconstituted with 200 mmol / L pH 5.0 sodium acetate buffer, and 700 μL was added to a 96-well plate. The plate was pre-incubated at 37 ℃ and 200 rpm for 5 min using a constant temperature shaker. Then, 100 μL of 0.2 mmol / L FeCl₂·4H₂O solution was added, and the reaction was carried out at 37 ℃ in the dark for 90 min. The reaction was terminated by adding 200 μL of 5 mmol / L phenanthroline (dissolved in 50 mM HEPES buffer). The absorbance was measured at 562 nm, with the buffer buffer serving as a blank control.

[0026] The formula for calculating ferrous chelation activity is: In the formula, A0 is the absorbance of the buffer solution, A S The absorbance is the value of the sample solution to be tested.

[0027] When preparing peptide-zinc chelates, the specific method is as follows: dissolve white cuttlefish short peptide powder and zinc sulfate heptahydrate in deionized water at a mass ratio of (2-4):1, adjust the pH to 2.5-3.5, incubate in a water bath at 45-55 ℃ for 1 h, purify and freeze dry for later use.

[0028] The specific method for determining zinc chelating activity is as follows: Dissolve the peptide-zinc chelate in 10 mL of ultrapure water, add 10 mL of pH 5.5 acetate-sodium acetate buffer and 2 drops of 0.1% xylenol orange indicator. Titrate with 0.01 mol / L EDTA-Na2 (1 mL / min) until the purple-red color turns bright yellow, and record the volume consumed (accurate to 0.01 mL).

[0029] The formula for calculating zinc chelation rate is: In the formula, m is the mass of the peptide-zinc chelate (g); M is the relative molecular mass of zinc; c is the concentration of the EDTA-Na2 solution (mol / L); and V is the volume of EDTA-Na2 solution consumed in the titration (mL).

[0030] The third embodiment of the present invention provides the application of the above-mentioned white mullet short peptide, or peptide-metal chelate, in the preparation of a medicine for supplementing minerals.

[0031] Preferably, the mineral is iron or zinc.

[0032] The embodiments of the present invention also provide the application of the above-mentioned short peptides of white cuttlefish, or peptide-metal chelates, in the preparation of functional foods or health products.

[0033] Example 1: Extraction and purification of crude peptides from white cuttlefish After slaughtering, skinning, and deboning the white mullet, weigh 20.0 g of fish pieces and add deionized water at a ratio of 1:4 (meat:water). Homogenize for 180 s using a sterile homogenizer. Adjust the pH of the solution to 8.65 with 1 mol / L NaOH, then add alkaline protease (1%) for enzymatic hydrolysis. After the reaction is complete, heat in a 90 ℃ water bath for 10 min to inactivate the enzyme. After cooling to room temperature, filter to remove residue. Centrifuge the filtrate at 4 ℃ and 10000 r / min for 10 min (centrifugation conditions: 4 ℃, 8000 xg, time: 20 min). Take the supernatant and freeze-dry under vacuum to obtain crude white mullet fish peptide powder, which is stored at -18 ℃.

[0034] The crude peptide powder was subjected to ultrafiltration separation, and ultrafiltration fractions with a molecular weight of less than 3000 Da were retained. The separated fraction F-1 was then freeze-dried and stored at -18 °C.

[0035] The ultrafiltration fraction F-1 was then prepared into a 50 mg / mL solution and filtered through a 0.22 μm aqueous microporous membrane. Sephadex G-15 dextran gel chromatography was performed using ultrapure water as the eluent. The chromatography method was as follows: elution flow rate 2 mL / min, detection wavelength 220 nm, and sample loading volume 4 mL. The chromatographic results are shown below. Figure 1 As shown, the horizontal axis represents the elution time T (min), and the vertical axis represents the absorbance AU. The chromatography process yielded three separate fractions, which were labeled F-1-1, F-1-2, and F-1-3. These three fractions were then collected and freeze-dried under vacuum.

[0036] The ferrous chelating activity and zinc chelating activity of the fractions F-1-1, F-1-2, and F-1-3 obtained after chromatography were determined, and the results are as follows: Figure 2 , 3 As shown. Figure 2The middle component F-1-2 exhibits the best ferrous chelating activity. Figure 3 The middle component F-1-1 exhibits the best zinc chelating activity.

[0037] Fractions F-1-2 and F-1-1 were separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The RP-HPLC conditions were as follows: Sepax BR-C18 column, 4.6*250 mm, 5 μm; injection volume: 10 μL; flow rate: 1 mL / min; temperature: 30 ℃; detection wavelength: UV 214 nm; mobile phase: phase A was pure water, pH 10; phase B was acetonitrile. The gradient elution program was as follows: 0 min, 95% A; 5–10 min, 95% A–92% A; 10–45 min, 92% A–82% A; 45–67 min, 82% A–68% A; 67–69 min, 68% A–5% A; 69–71 min, 5% A; 71–77 min, 5% A–95% A; 77–92 min, 95% A.

[0038] The reversed-phase high-performance liquid chromatography separation results of components F-1-2 and F-1-1 are as follows: Figure 4 , 5 As shown in the figures, the horizontal axis represents elution time (min), and the vertical axis represents absorbance (AU). Figure 4 The middle component F-1-2 was separated into three components by reversed-phase high-performance liquid chromatography, which were designated as F-1-2-1, F-1-2-2, and F-1-2-3 in the order of separation. Figure 5 A component F-1-1 was separated from the middle component by reversed-phase high-performance liquid chromatography and designated as F-1-1-1. All components were collected, lyophilized, and stored at -18 °C for later use.

[0039] Example 2 After identifying all the purified components from Example 1 using liquid chromatography-mass spectrometry, short peptides were synthesized in the solid phase and their chelating activity was determined.

[0040] Liquid chromatography conditions: 100 μm id × 180 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm analytical column; mobile phase: phase A 0.1% formic acid, phase B 0.1% formic acid, 80% acetonitrile; flow rate: 600 nL / min, analysis time per fraction 66 min. Analytical program: 0–2 min, 4% B–8% B; 2–35 min, 8%–28% B; 35–55 min, 28% B–40% B; 55–56 min, 40% B–95% B; 56–66 min, 95% B. Mass spectrometry conditions: mass spectrometry scan range 300–1800 m / z, first-order resolution 70,000 (AGC 3e6, IT 100 ms). Precipitating ions with an intensity ≥20 were selected for fragmentation using a high-energy collisional fragmentation method. The second-order resolution was 17,500 (AGC 1e5, IT 50 ms), and the collision energy was 28%. Raw mass spectrometry data were generated.

[0041] By searching and comparing data in the Byonic database, 361 peptides were identified in fraction F-1-2-1, 101 peptide molecules in fraction F-1-2-2, 52 peptides in fraction F-1-2-3, and 305 peptides in fraction F-1-1-1. Peptides with scores greater than 300 were selected, and after screening, 8 peptides were finally chosen. These 8 peptides were then used to synthesize short peptides using solid-phase chromatography, and their purity and chelation activity with ferrous / zinc iron were determined. The sequence information of each short peptide is shown in Table 1 below.

[0042] surface Information on each short peptide sequence .

[0043] The chelation activities of each short peptide with ferrous iron and zinc are as follows: Figure 6 , 7 As shown. Figure 6 The chelation results of the short peptides and ferrous iron showed that the short peptide with sequence number SEQ ID NO.4 had the highest chelation activity with ferrous iron, reaching 30.36%, proving that the short peptide with sequence number SEQ ID NO.4 was the most suitable for chelation with ferrous iron among the eight short peptides. Figure 7 The chelation results of each short peptide and zinc showed that the short peptide with sequence number SEQ ID NO.2 had the highest chelation activity with zinc, reaching 21.55%. Therefore, the short peptide with sequence number SEQ ID NO.2 is the most suitable for chelation with zinc among the eight short peptides.

[0044] Finally, it should be noted that although the description of the present invention is quite detailed and the embodiments described have been described, it is not intended to be limited to any of these details or embodiments. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, combinations, simplifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Short peptides from white cuttlefish, characterized in that, It is selected from one of the short peptides whose amino acid sequences are shown in SEQ ID NO.1~8.

2. A peptide-metal chelate, characterized in that, It contains the short peptide from white cuttlefish as described in claim 1, and ferrous or zinc ions that are coordinated with the polypeptide.

3. The peptide-metal chelate as described in claim 2, characterized in that, The molar ratio of the white cuttlefish short peptide to the metal ion is 1:(1-3).

4. The method for preparing the white cuttlefish short peptide as described in claim 1, characterized in that, include: The white cuttlefish meat was homogenized and then subjected to enzymatic hydrolysis in a manner that simulates in vitro human digestion to obtain an enzymatic hydrolysate. Ultrafiltration was used to separate ultrafiltration fractions with a molecular weight cutoff of less than 3000 Da from the enzymatic hydrolysate. The ultrafiltration components were subjected to gel chromatography; and the components obtained after chromatography were separated and purified by reversed-phase high-performance liquid chromatography.

5. The method for preparing the peptide-metal chelate as described in claim 2 or 3, characterized in that, The process includes the following steps: mixing the short peptide from the white cuttlefish described in claim 1 with ferrous salt or zinc salt in a buffer solution, reacting at a suitable pH and temperature, and then separating and purifying the peptide-metal chelate after the reaction is complete.

6. The preparation method according to claim 5, characterized in that, When preparing the peptide-ferrous chelate, the reaction pH is 7.5-8.0, the temperature is 35-45℃, the mass ratio of the white cuttlefish short peptide to Fe²⁺ is (2-4):1, and ascorbic acid is used as an antioxidant.

7. The preparation method according to claim 5, characterized in that, When preparing the peptide-zinc chelate, the reaction pH is 2.5-3.5, the temperature is 45-55℃, and the mass ratio of the white cuttlefish short peptide to Zn²⁺ is (2-4):

1.

8. The use of the white mullet short peptide as described in claim 1, or the peptide-metal chelate as described in any one of claims 2-3, in the preparation of a medicine for supplementing minerals.

9. The application as described in claim 8, characterized in that, The mineral is iron or zinc.

10. The use of the short peptide from white cuttlefish as described in claim 1, or the peptide-metal chelate as described in any one of claims 2-3, in the preparation of functional foods or health products.

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