Natural oyster zinc peptide product and preparation method thereof

Abstract

The application discloses a natural oyster zinc peptide product and a preparation method thereof. The natural oyster zinc peptide crude product is extracted from fresh oyster meat by an ultrasonic crushing method, and is separated and purified by using a Sephadex G-15 gel chromatography column, so that the natural oyster zinc peptide product is finally obtained. It is verified through experiments that the zinc content of the natural oyster zinc peptide product is 377.71±3.85 mg / kg; four peptide segments with better zinc ion combination capacity are contained in the natural oyster zinc peptide product, and the four peptide segments are EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH respectively; and the structure identification result shows that the natural oyster zinc peptide product has the characteristics of a peptide zinc chelate. The extraction of the natural oyster zinc peptide product provides a new idea for high-value utilization of oysters and development of trace element dietary supplements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of natural product chemistry, and particularly relates to a natural oyster zinc peptide product and a preparation method thereof. BACKGROUND

[0002] Oyster is a representative bivalve mollusk, which is widely distributed in the ocean and plays an important role in maintaining the function of the ecological system. Oyster not only has high edible value, but also has good medicinal value. Oyster contains rich nutrients, including protein, active peptide, glycogen, taurine, vitamins, and minerals such as zinc and selenium. Studies have shown that the active ingredients in oyster have functions such as antioxidant, anti-fatigue, anti-tumor, blood pressure reduction, and immune enhancement. With the emphasis on the high-value utilization of marine biological resources, the development of oyster resources has also begun to develop in the direction of increasing its added value, and in the direction of comprehensive utilization. The development trend of comprehensive utilization of oyster resources in the future is to extract and deep process active substances of oyster by using advanced production technology, and to develop functional foods and biological drugs with high added value.

[0003] Zinc is the second most abundant metal in the human body, which plays an indispensable role in the metabolic activity of various metalloenzymes, gene expression, immune system, and human growth. Zinc deficiency can seriously damage human health and cause multiple system damage. At present, zinc deficiency has become a global problem, and the important factor leading to zinc deficiency is the low absorption and bioavailability of zinc. Peptides extracted from food proteins can improve the absorption and utilization of zinc. Polypeptides can chelate divalent metals including zinc to form soluble metal complexes, thereby improving the bioavailability of minerals and alleviating trace element deficiency. Marine biological resources such as oysters contain rich protein, active peptide, and amino acids, which provide a good environment for chelation with zinc ions. Therefore, finding effective and safe ingredients as substitutes for preventing zinc deficiency has gradually become a research focus.

[0004] Oyster zinc peptide (OZP) is the product of oyster peptide combined with zinc. It not only has the characteristics of fast absorption and transport of peptide compounds, but also can improve the absorption rate of zinc. Studies have shown that OZP has multiple functional activities such as antioxidant, protection of alcoholic liver injury, and promotion of cell proliferation. However, current researches are mostly focused on zinc-chelated peptides, and the research on natural zinc peptides is relatively scarce. Therefore, the present application provides a preparation method of a natural oyster zinc peptide product, which provides a new idea for the high-value utilization of oysters and the development of trace element dietary supplements. SUMMARY

[0005] The present application provides a natural oyster zinc peptide product and a preparation method thereof. The extraction raw material of the natural oyster zinc peptide is Crassostrea gigas, which is rich in raw materials, safe, high in nutritional value, and simple in preparation method.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] The present application provides a preparation method of natural oyster zinc peptide product, comprising the following steps:

[0008] (1) Shell oysters, wash oyster meat after adding water and crushing to obtain crushed tissue fluid;

[0009] (2) The tissue crushing liquid of step (1) is subjected to ultrasonic crushing, and the obtained tissue crushing liquid is subjected to centrifugal collection of supernatant;

[0010] (3) The supernatant of step (2) is concentrated and freeze-dried to obtain a crude natural oyster zinc peptide product;

[0011] (4) The crude natural oyster zinc peptide product of step (3) is subjected to separation and purification to obtain a natural oyster zinc peptide product.

[0012] Further, the weight of water added in step (1) is 2-4 times the weight of oyster meat.

[0013] Further, the power of ultrasonic crushing in step (2) is 70W-100W, and the crushing time is 15min-30min.

[0014] Further, in step (3), the supernatant is concentrated by rotary evaporation at 40℃-50℃ using a rotary evaporator.

[0015] Further, in step (4), the crude natural oyster zinc peptide product is subjected to separation and purification by using a gel chromatography column.

[0016] Further, the length of the gel chromatography column is 80cm-120cm, the diameter is 2cm-3cm, the eluent condition is pure water, and the flow rate is 0.5mL / min-1mL / min.

[0017] Further, the protein content of the crude natural oyster zinc peptide product in step (3) is 0.32±0.08mg / mL, the polypeptide content is 0.14±0.02mg / mL, and the zinc content is 623.13±4.84mg / kg.

[0018] Further, the oysters are fresh long oysters.

[0019] The present application also provides a natural oyster zinc peptide product prepared by the preparation method.

[0020] Further, the natural oyster zinc peptide product contains four peptide segments, and the amino acid sequences of the peptide segments are respectively EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH.

[0021] Further, the polypeptide content of the natural oyster zinc peptide product is 0.18±0.06 mg / mL, and the zinc content is 377.71±3.85 mg / kg.

[0022] Further, the surface structure of the natural oyster zinc peptide product is relatively rough, obvious wrinkles are formed, and the stretching vibration of the amide bond, amino group and carboxyl group occurs; and there is a relatively wide diffraction peak at 2θ=20°.

[0023] Compared with the prior art, the natural oyster zinc peptide product has the following advantages:

[0024] The extraction raw material of the natural oyster zinc peptide product in the application is abundant, and the natural oyster zinc peptide product obtained has high safety. The natural oyster zinc peptide product is extracted from long oysters, and the polypeptide content of the natural oyster zinc peptide product obtained is 0.18±0.06 mg / mL, the zinc content is 377.71 mg / kg, and the natural oyster zinc peptide product contains four peptide segments EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH which are better combined with zinc ions.

[0025] The structure of the natural oyster zinc peptide product is characterized, and after the natural oyster zinc peptide product is separated and purified, the FTIR shows that the stretching vibration of the amide bond, amino group and carboxyl group occurs; the XRD shows that there is a relatively wide diffraction peak at 2θ=20°, the crystallinity is reduced, and crystallization is not easy during use; and the surface structure is relatively rough, and obvious wrinkles are formed. The development of the natural product will provide a new direction for the high-value utilization of oysters, especially the utilization of minerals, and the natural oyster zinc peptide product also has great potential in the development of trace element dietary supplements. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure is the molecular weight standard curve (A) and the molecular weight distribution graph (B) of the crude natural oyster zinc peptide product.

[0027] Figure 2 The figure is the Sephadex G-15 gel column separation graph (A), the protein polypeptide standard curve (B) and the protein, polypeptide and zinc content graph (C) of each component after separation.

[0028] Figure 3Structure of zinc-binding peptides in natural oyster zinc peptide product; (A) EKSGPGPHCPRC, (B) TVGPITGGGSHK, (C) VIDTNKDRT, (D) SGPSIVH.

[0029] Figure 4 Secondary mass spectrum of zinc-binding peptides in natural oyster zinc peptide product; (A) EKSGPGPHCPRC, (B) TVGPITGGGSHK, (C) VIDTNKDRT, (D) SGPSIVH.

[0030] Figure 5 Infrared spectrum of natural oyster zinc peptide product.

[0031] Figure 6 X-ray diffraction pattern of natural oyster zinc peptide product.

[0032] Figure 7 Scanning electron micrograph of natural oyster zinc peptide product; (A) magnification 500 times, (B) magnification 1000 times. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments, but the scope of protection claimed by the present application is not limited to the scope expressed by the examples.

[0034] Example 1 Extraction of natural oyster zinc peptide product OZP

[0035] The extraction of the natural oyster zinc peptide product in the present application comprises the following steps:

[0036] (1) Shell the long oysters, wash them with deionized water, wash the oyster meat, drain, weigh, add 3 times deionized water (m / m), then pour into a tissue crusher and crush to obtain a tissue crushing liquid;

[0037] (2) The tissue crushing liquid in step (1) is subjected to ultrasonic crushing, the power is 80W, the time is 20min, and the crushing process is carried out in an ice bath to obtain a tissue crushing liquid; centrifuge the crushing liquid at 11000r / min for 20min, and collect the supernatant;

[0038] (3) The supernatant in step (2) is concentrated by rotary evaporation at 40℃ using a rotary evaporator, and dried by freeze-drying method to obtain a crude natural oyster zinc peptide product, and the freeze-dried powder is preserved at -20℃;

[0039] (4) The crude natural oyster zinc peptide product was separated and purified by a Sephadex G-15 gel chromatography column, wherein the length of the Sephadex G-15 gel chromatography column was 100 cm, the diameter was 2.6 cm, the eluent was pure water, and the flow rate was 0.5 mL / min.

[0040] Example 2 Separation and purification of the natural oyster zinc peptide product

[0041] 1. Polypeptide molecular weight determination of the crude natural oyster zinc peptide product

[0042] The polypeptide molecular weight of the crude natural oyster zinc peptide product obtained in step (3) of Example 1 was determined by high performance size exclusion chromatography (HPSEC).

[0043] (1) Polypeptide molecular weight determination: Cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), glycine-glycine-glycine (189 Da), and Gly-Gly-Tyr-Arg (451 Da) were used as the peptide molecular weight standard solution. The sample and the standard were configured into a 1 mg / mL solution with pure water before injection, and were determined by the machine after passing through a 0.22 μm water-based microporous filter.

[0044] (2) Determination conditions: The chromatographic column used was Shodex Asahi pak GS-320HQ (300 mm x 7.5 mm), and other liquid chromatography system analysis conditions are shown in Table 1. Different molecular weight contents were calculated by GPC software according to the peak area of each.

[0045] Table 1 Liquid chromatography analysis conditions

[0046]

[0047] (3) Standard curve drawing: The standard curve was drawn with the logarithm of molecular weight (Log Mw) as the vertical coordinate and the peak time (t) as the horizontal coordinate, and the standard curve was Log Mw = -0.0525t 2 - 1.7003t + 15.94 (R 2 = 0.9955) (A). Figure 1

[0048] The polypeptide molecular weight distribution results of the crude natural oyster zinc peptide product are shown in Figure 1 B, and the polypeptide molecular weight distribution table is shown in Table 2. The results show that the part with a molecular weight of 189-500 accounts for the largest proportion in the crude product, which is 35.28%; the part with a molecular weight less than 1000 accounts for 42.44% of the crude product.

[0049] Table 2 Polypeptide molecular weight distribution table

[0050] ​

[0051] 2. Isolation and purification of natural oyster zinc peptide product

[0052] The crude natural oyster zinc peptide product obtained in step (3) of Example 1 was separated and purified by Sephadex G-15 gel chromatography column.

[0053] (1) Sample pretreatment: The sample was dissolved in pure water to form a 150 mg / mL solution, centrifuged at 10,000 r / min for 10 min, and the supernatant was taken for sample loading, with a loading volume of 9 mL (about 2% of the column volume) each time;

[0054] (2) Separation: The outlet flow rate was adjusted to 0.5 mL / min, the sample was loaded and flowed along the inner wall of the column, and the liquid level was supplemented with distilled water. The eluate was collected by an automatic collector, and the collected eluate was measured for absorbance at 220 nm. The collected eluate belonging to the same peak was mixed together, concentrated by rotary evaporation at 40°C, freeze-dried, and the freeze-dried components were stored at -20°C for use.

[0055] The results are shown in Figure 2 A. After separation by Sephadex G-15 dextran gel chromatography column, five components OZP-1, OZP-2, OZP-3, OZP-4 and OZP-5 were obtained.

[0056] 3. Component analysis of each component after separation and purification

[0057] (1) Protein and polypeptide content determination

[0058] Folin-phenol standard curve: A 0.5 mg / mL bovine serum albumin (BSA) solution was prepared, and then standard protein solutions with concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL were prepared by dilution. 0.5 mL of protein standard solution with different concentrations was taken, 2.5 mL of Folin-phenol solution A was added, and it was allowed to stand for 10 min; 0.25 mL of Folin-phenol solution B was added, and it was allowed to stand for 30 min. After the reaction was completed, the absorbance of different standard solutions was detected at 500 nm. The standard curve y = 0.3569x + 0.0877 was obtained by calculation, with R 2 = 0.9981, as shown in Figure 2 B.

[0059] Protein content determination: The sample was dissolved in pure water to form a 1 mg / mL solution, and the absorbance of the sample at 500 nm was determined according to the above steps, and the protein concentration of the sample was obtained by calculation.

[0060] Peptide content determination: Prepare a 1 mg / mL solution of the sample with pure water. Add 1 mL of 20% TCA to 1 mL of the sample solution, shake thoroughly to mix, and incubate on ice for 20 min. Centrifuge at 4℃ and 13,500 × g for 20 min. Take 0.5 mL of the supernatant and measure its absorbance. Calculate the peptide concentration of the sample.

[0061] (2) Determination of zinc content

[0062] Sample pretreatment: Weigh an appropriate amount of sample and add it to a disposable digestion tube. Add 2 mL of nitric acid and 0.5 mL of hydrogen peroxide, and react for 15 min. Then, place the sample digestion tube in a 100℃ water bath for 90 min of continuous digestion. After digestion, remove the digestion tube and allow it to cool naturally to room temperature. Transfer all the digested solution to a 10 mL volumetric flask, add deionized water to make up to the final volume, mix well, and let stand until analysis. Determine the zinc content of the sample by inductively coupled plasma mass spectrometry (ICP-MS).

[0063] The results are as follows Figure 2 As shown in C, the polypeptide contents of OZP-1, OZP-2, OZP-3, OZP-4, and OZP-5 are 0.21±0.03 mg / mL, 0.38±0.08 mg / mL, 0.18±0.06 mg / mL, 0.03±0.05 mg / mL, and 0.08±0.03 mg / mL, respectively; the zinc contents of the five components OZ-1 through OZ-5 are 50.16±3.21 mg / kg, 123.08±4.27 mg / mL, 377.71±3.85 mg / kg, 308.21±4.63 mg / kg, and 338.23±3.29 mg / kg, respectively. Considering the polypeptide content and zinc content of each component, OZP-3 was selected as the target component, thus obtaining the natural oyster zinc peptide product.

[0064] Example 3: Identification of peptide sequences of natural oyster zinc peptide products

[0065] The peptide sequence of the purified natural oyster zinc peptide product from Example 2 was analyzed using an LC-MS / MS system, and the mass spectrometry results were analyzed and matched using relevant supporting software and databases.

[0066] (1) Chromatographic column type: Acclaim PepMap RPLC C18 pre-column (300μm×5mm, 5μm, ); Acclaim PepMap RPLC C18 (150μm×150mm, 1.9μm, ).

[0067] (2) Capillary high performance liquid chromatography detection conditions

[0068] Sample loading amount was 5 μL; mobile phase A was: 0.1% formic acid, 2% acetonitrile; mobile phase B was: 0.1% formic acid, 80% acetonitrile; under the condition of flow rate of 600 nL / min, a total of 66 min of analysis and detection.

[0069] (3) Mass spectrometry conditions

[0070] Mass spectrometry end detection was performed using an electrospray ion source (ESI) in negative ion mode.

[0071] (4) Database search

[0072] The mass spectrometry raw file was searched against the oyster protein database using Byonic.

[0073] The peptide identification results are shown in Figure 3 and Figure 4 Four peptides with better zinc ion binding ability were obtained through secondary mass spectrometry fragment analysis, and the detailed information is shown in Table 3. The four peptides are EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH.

[0074] Table 3 Peptide detailed information

[0075]

[0076] The physicochemical parameters of the polypeptides were calculated using the ProtParam tool. The physicochemical parameter results of the polypeptides are shown in Table 4. Since the three peptides TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH do not contain Trp, Tyr, or Cys, these peptides are invisible under ultraviolet spectrophotometry, i.e., active detection of the sample by ultraviolet method will not interfere with the experimental results. The theoretical isoelectric point (Theoretical pI) of EKSGPGPHCPRC is 8.16; the theoretical pI of TVGPITGGGSHK is 8.44; the theoretical pI of VIDTNKDRT is 5.93; and the theoretical pI of SGPSIVH is 6.46. The prediction results using the instability index show that the four peptides are relatively stable. The grand average of hydropathicity (GRAVY) indicates the strength of polypeptide hydrophobicity, and the results show that the hydrophilicity of peptides EKSGPGPHCRPRC, TVGPITGGGSHK, and VIDTNKDRT is relatively good, while peptide SGPSIVH is a hydrophobic polypeptide. The aliphatic side chain index (Aliphatic index) is mainly used to predict the thermal stability of polypeptides, and the results show that the peptide EKSGPGPHCPRC has poor heat resistance, and the peptides TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH have good heat resistance.

[0077] Table 4 Physicochemical parameters of peptides

[0078]

[0079] Basic structural characteristics of natural oyster zinc peptide product of Example 4

[0080] 1. KBr tablet method was used to determine the infrared spectrum of natural oyster zinc peptide product

[0081] The powder OZP of natural oyster zinc peptide product was mixed with dry KBr powder at a ratio of 100:1. After tabletting, infrared spectrum scanning was performed, with a scanning range of 4000 to 400 cm -1 , 32 scans, and a resolution of 4 cm -1 . With blank potassium bromide as the background, after the background was collected, the sample was detected.

[0082] The results are shown in Figure 5 . The characteristic absorption peak at 3214 cm -1 in the infrared spectrum is caused by N-H stretching vibration; the characteristic absorption peak at 1618 cm -1 is the amide I band, which is a typical infrared spectrum characteristic of peptides, and the stretching vibration is mainly attributed to the stretching vibration of C=O and N-H; the absorption peak at 1035 cm -1 is mainly caused by the stretching vibration of -COO- and C-O. In addition, a characteristic absorption peak appears at 1475 cm -1 , which may be the amide II band absorption peak, and several new absorption peaks appear in the range of 605-892 cm -1 , which may be derived from the stretching vibration of C-H and N-H bonds.

[0083] 2. X-ray diffraction method was used to determine the crystal structure of natural oyster zinc peptide product

[0084] The powder OZP of natural oyster zinc peptide product was evenly spread on the sample disc, and the test conditions were set as starting point 5°, ending point 90°, step size 0.02, and the sample was detected.

[0085] The results are shown in Figure 6 . The natural oyster zinc peptide product has a relatively wide diffuse peak near 2θ = 20° and a large peak bottom.

[0086] 3. Scanning electron microscopy was used to observe the morphological characteristics of natural oyster zinc peptide product

[0087] The powder of natural oyster zinc peptide product was evenly coated on the sample disc, and after gold plating film treatment, the ultrastructure of the sample was observed; the magnification was 500-1000 times.

[0088] The results are shown in Figure 7As shown, the natural oyster zinc peptide product surface wrinkles are obvious, structure aggregation, part began to appear fragmentation.

[0089] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, for those skilled in the art, the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A natural oyster zinc peptide product, characterized in that, The natural oyster zinc peptide product contains four peptide segments, and the amino acid sequences of the peptide segments are respectively: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH; the preparation method of the natural oyster zinc peptide product comprises the following steps: (1) The oysters are shelled, and the oyster meat is washed and then crushed by adding water to obtain a crushed tissue liquid; (2) The crushed tissue liquid of step (1) is subjected to ultrasonic crushing, and the obtained tissue crushing liquid is centrifuged to collect the supernatant; the power of the ultrasonic crushing is 70 W-100 W; (3) The supernatant of step (2) is concentrated and freeze-dried to obtain a crude natural oyster zinc peptide product; (4) The crude natural oyster zinc peptide product of step (3) is separated and purified to obtain a natural oyster zinc peptide product; the crude natural oyster zinc peptide product is dissolved in pure water, centrifuged, and the supernatant is loaded; an automatic collector is used to collect the eluate, and the collected eluate is measured for absorbance at 220 nm; after separation by the Sephadex G-15 gel chromatography column, five components OZP-1, OZP-2, OZP-3, OZP-4 and OZP-5 are obtained, and OZP-3 is selected as the target component, i.e. the natural oyster zinc peptide product is obtained; the length of the Sephadex G-15 gel chromatography column is 80 cm-120 cm, the diameter is 2 cm-3 cm, the eluent is pure water, and the flow rate is 0.5 mL / min-1 mL / min.

2. The natural oyster zinc peptide product according to claim 1, characterized in that: The weight of the water added in step (1) is 2-4 times the weight of the oyster meat.

3. The natural oyster zinc peptide product according to claim 1, characterized in that: The ultrasonic crushing time in step (2) is 15 min-30 min.

4. The natural oyster zinc peptide product according to claim 1, characterized in that: In step (3), the supernatant is concentrated by rotary evaporation at 40°C-50°C.

5. The natural oyster zinc peptide product of claim 1, characterized by: The protein content of the crude natural oyster zinc peptide product in step (3) is 0.32±0.08 mg / mL, the polypeptide content is 0.14±0.02 mg / mL, and the zinc content is 623.13±4.84 mg / kg.

6. The natural oyster zinc peptide product of claim 1, characterized by: The polypeptide content of the natural oyster zinc peptide product is 0.18±0.06 mg / mL, and the zinc content is 377.71±3.85 mg / kg.

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