Preparation containing sea cucumber polypeptide and preparation method

Abstract

The present invention relates to the field of marine biological genetic engineering and protein expression technology, in particular to a preparation containing sea cucumber polypeptide and a preparation method thereof, comprising an amino acid sequence as shown in SEQ ID NO: 1, obtained by enzymatic hydrolysis, ultrafiltration, chromatographic separation and purification, and having a good anti-fatigue effect.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering and protein engineering of marine organisms, and relates to obtaining sea cucumber polypeptides with anti-fatigue activity by enzymatic hydrolysis, in particular to a preparation containing sea cucumber polypeptides and a preparation method thereof. Background Art

[0002] The organic components of the dried body wall of sea cucumbers are nearly 90% protein, about 4% lipids, about 6% polysaccharides, and a small amount of nucleic acid. Sea cucumber peptides refer to the protein hydrolyzate obtained by protease hydrolysis and refining of sea cucumbers, which is mainly composed of small molecule peptides and coexists with multiple functional ingredients. It has many physiological functions such as lowering blood pressure, resisting fatigue, improving immunity, and delaying aging. Compared with sea cucumbers, sea cucumber polypeptides have special physical and chemical properties such as good solubility, stability, and low viscosity. Therefore, sea cucumber peptides obtained by enzymatic hydrolysis have the advantages of easy digestion and absorption, high biological efficacy, no antigenicity, and safe consumption, and have higher biological efficacy than ordinary sea cucumber products.

[0003] At present, the domestic consumption of sea cucumbers is still at the stage of rough processing, mainly consuming the whole sea cucumber. However, due to different personal preferences and different absorption efficiencies of sea cucumbers, the active effects of sea cucumbers cannot be fully utilized. In addition, the price of sea cucumbers in China is relatively high, which makes many people give up eating sea cucumbers. Therefore, the small molecule sea cucumber peptides obtained by further refining the sea cucumber enzymatic hydrolysis products through process optimization have the characteristics of easy in vitro preparation and easy promotion and use, which makes the research of sea cucumber peptides have a more far-reaching prospect. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a preparation containing sea cucumber polypeptide and a preparation method.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] A preparation containing a sea cucumber polypeptide comprises a sea cucumber polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

[0007] The nucleotide chain encoding the sea cucumber polypeptide is composed of any one of the following groups:

[0008] (a), a nucleotide chain encoding a polypeptide having an amino acid sequence of SEQ ID NO: 1;

[0009] (b) A nucleotide chain complementary to the nucleotide chain described in (a).

[0010] Furthermore, it also contains preservatives, sweeteners, colorants, thickeners and pure water solvents.

[0011] Furthermore, the sea cucumber polypeptide is used to prepare an anti-fatigue activity promoter for the body.

[0012] A method for preparing a preparation containing sea cucumber polypeptides comprises the following steps:

[0013] S1. A two-step enzymatic hydrolysis method was used to obtain a preliminary enzymatic hydrolysis product from the body wall of the sea cucumber. In the first step, papain was used under the following reaction conditions: pH 6.5, temperature 55°C, enzyme addition amount 6000U / g, and enzymatic hydrolysis time 8h; in the second step, trypsin was used under the following reaction conditions: pH 8, temperature 45°C, enzyme addition amount 7000U / g, and enzymatic hydrolysis time 6h;

[0014] S2. Use ultrafiltration tubes to concentrate and separate the enzymatic hydrolysate. Select millipore ultrafiltration tubes with a volume of 4 mL and a molecular weight cutoff of 50 kDa, 30 kDa, 10 kDa, and 3 kDa to perform step-by-step ultrafiltration and concentration on the enzymatic hydrolysate. Retain 2 mL of each level of concentrated solution, and select the concentrated solution with a molecular weight less than 3 kDa;

[0015] S3. The concentrate was purified by primary chromatography and secondary circulation chromatography. SP-Sepharose FF cation exchange chromatography was selected for primary chromatography. The column volume was 1.6×20 cm. The equilibrium column was washed with pH 7.5, 20 mmol / L PB buffer, and the concentrate was loaded at a flow rate of 0.5 mL / min; eluted with pH 7.5, 20 mmol / L PB buffer containing 0.1 mol / L NaCl; the components with anti-fatigue activity were collected and further separated with Q-Sepharose FF high-efficiency ion exchange column; after washing with pH 8.0, Tris-HCl equilibrium buffer, step-shaped gradient elution and separation were performed with equilibrium buffer + 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1 mol / L NaCl at a flow rate of 0.5 mL / min; the elution peaks with anti-fatigue activity were combined and concentrated, and Q-Sepharose was performed again according to the above steps. FF high-efficiency ion exchange column is used for step-shaped gradient elution and separation, and the elution peaks with anti-fatigue activity are combined and desalted and concentrated to obtain a high-purity protein solution, which is the sea cucumber polypeptide;

[0016] S4, adding preservatives, sweeteners, colorants and thickeners to the prepared sea cucumber polypeptide and dissolving it in pure water solvent to obtain a preparation containing sea cucumber polypeptide. The preservatives, sweeteners, colorants and thickeners can be selected in type and amount according to conventional methods.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention obtains a small molecule sea cucumber polypeptide by selecting appropriate enzymes and optimizing the enzymatic hydrolysis and refining process, which has been verified by experiments to have a good anti-fatigue effect. The obtained small molecule polypeptide sequence can be synthesized in vitro to reduce the preparation cost, eliminating the limitation of the high price of sea cucumber on market promotion.

[0019] 2. The sea cucumber polypeptide obtained by the present invention has a small molecular weight and is easily absorbed by the human body. It can fully exert its anti-fatigue effect as a component of food or medicine. It has no antigenic components, will not cause human immune response, has no seafood odor, and has good palatability for the public. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is a flow chart of the method of the present invention.

[0021] Figure 2 These are the anti-fatigue activity test results of each level of concentrate.

[0022] Figure 3 This is the spectrum of the elution components of SP-Sepharose FF cation exchange chromatography.

[0023] Figure 4 These are the results of anti-fatigue activity test of the fraction eluted by SP-Sepharose FF cation exchange chromatography.

[0024] Figure 5 This is the first Q-Sepharose FF high-efficiency ion exchange column elution component spectrum.

[0025] Figure 6 This is the result of the first anti-fatigue activity test of the fraction eluted from the Q-Sepharose FF high-efficiency ion exchange column.

[0026] Figure 7 This is the spectrum of components eluted from the second Q-Sepharose FF high-efficiency ion exchange column.

[0027] Figure 8 This is the anti-fatigue activity test result of the second Q-Sepharose FF high-efficiency ion exchange column elution fraction.

[0028] Fig. 9 It is a standard map of amino acid sequence and the map of the first 5 amino acids determined by protein solid phase sequence analyzer.

[0029] Fig.10 This is a map of the 6th to 9th amino acids in the amino acid sequence determined by a protein solid phase sequencer. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0032] Example 1: Isolation and purification of sea cucumber polypeptides

[0033] The sea cucumbers used in this experiment were purchased from Yantai dried sea cucumbers in Shandong. Figure 1 As shown, the following steps are included:

[0034] (1) Extraction and enzymatic hydrolysis of sea cucumber protein

[0035] S1, Experimental preparation stage: 200g of dried sea cucumber was ground into powder, and then passed through a 500-mesh sieve to obtain sea cucumber dry powder. 0.1g of sea cucumber dry powder was poured into 9.9mL of deionized water and shaken to make a 1% sea cucumber powder suspension;

[0036] S2, the sea cucumber powder suspension was crushed by ultrasonic wave, with 6mm horn, 35% power, 3.5s working, 7s rest, and 15min to obtain the crushed sea cucumber powder solution;

[0037] S3, after adjusting the pH of the crushed sea cucumber powder solution with 0.05 mol / L dilute hydrochloric acid and 0.05 mol / L sodium hydroxide solution, papain was added to the sea cucumber powder solution, and the reaction conditions were: pH 6.5, temperature 55°C, enzyme addition amount 6000U / g, and enzymolysis time 8h;

[0038] S4, after inactivating the enzyme in a boiling water bath, keep the temperature at 45°C for 5 minutes, adjust the system pH to 8.0, add 7000U / g of trypsin, and inactivate the enzyme again in a boiling water bath after enzymolysis for 6 hours;

[0039] S5. Cool to room temperature and centrifuge at 5000 rpm for 15 minutes in a centrifuge. The supernatant is the enzymatic hydrolysate.

[0040] (2) Concentration and separation of enzymatic hydrolysis products

[0041] Millipore ultrafiltration tubes with a volume of 4 mL and molecular weight cutoffs of 50 kDa, 30 kDa, 10 kDa, and 3 kDa were selected to perform step-by-step ultrafiltration and concentration of the enzymatic hydrolyzate, and 2 mL of each level of concentrate was retained. The centrifugation conditions are shown in Table 1. A concentrate with a molecular weight of 50 kDa or more, a concentrate with a molecular weight of less than 50 kDa and 30 kDa or more, a concentrate with a molecular weight of less than 30 kDa and 10 kDa or more, a concentrate with a molecular weight of less than 10 kDa and 3 kDa or more, and a concentrate with a molecular weight of less than 3 kDa were formed, and the concentrate with the most anti-fatigue activity was retained.

[0042] Table 1 Centrifugation conditions of ultrafiltration tubes of different specifications

[0043] 50kDa 4℃,4500rpm,6min 30kDa 4℃,4500rpm,8min 10kDa 4℃,4500rpm,17min 3kDa 4℃,4500rpm,35min

[0044] (3) Purification of enzymatic hydrolysis products

[0045] SP-Sepharose FF cation exchange chromatography was selected for separation, with a column volume of 1.6×20cm. The equilibrium column was washed with pH7.5, 20mmol / L PB buffer, and the concentrate was loaded at a flow rate of 0.5mL / min. The pH7.5, 20 mmol / LPB buffer was used as elution A, and the elution A containing 0.1 mol / L NaCl was used as elution B. The components with the most anti-fatigue activity were collected and further separated using a Q-Sepharose FF high-efficiency ion exchange column. After washing with equilibrium buffer (pH8.0, Tris-HCl) for 2-3 column volumes, step-shaped gradient elution and separation were performed using equilibrium buffer + 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1mol / L NaCl, with a flow rate of 0.5mL / min. The elution peaks with anti-fatigue activity were combined and concentrated in a concentration / dialysis card. According to the above steps, Q-Sepharose FF high-efficiency ion exchange column was used again for step gradient elution and separation. The elution peaks with anti-fatigue activity were combined and placed in a dialysis bag for desalting. Subsequently, PEG2000 was used to concentrate to 5 mL, and further concentrated to 0.5 mL using a microconcentrator to obtain a high-purity protein solution.

[0046] (4) Determination of amino acid sequence of enzymatic hydrolysis products

[0047] The high-purity protein solution was subjected to a protein solid phase sequence analyzer to determine the polypeptide sequence having anti-fatigue activity.

[0048] Example 2: Detection method of anti-fatigue activity of sea cucumber polypeptide

[0049] (1) Treatment of experimental subjects

[0050] Kunming mice with similar body weight were given 0.5 mL of the concentrate or eluate of each stage in Example 1 by gavage, and the control group was given the same volume of distilled water for 28 consecutive days.

[0051] (2) Mouse weighted swimming experiment

[0052] 30 minutes after the last administration, a lead wire weighing 5% of the mouse's body weight was tied to the mouse's tail, and the mouse was placed in a 30 cm deep pool to swim. The time from the start of swimming to the time when the head was completely submerged in water and could not float to the surface for 8 seconds was recorded as the swimming time.

[0053] (3) Determination of serum urea nitrogen content

[0054] The serum urea nitrogen content increases with the increase of exercise load. The worse the body's ability to adapt to the load, the higher the serum urea nitrogen content. Therefore, the fatigue resistance of mice can be known by observing the generation of serum urea nitrogen. The specific method is: 30 minutes after the last administration, the mice are placed in the swimming pool to swim for 90 minutes. After resting for 60 minutes, 0.5 mL of fundus blood is taken and 150 μL of anticoagulant (trisodium citrate dihydrate solution) is added. Then, the blood urea nitrogen test kit provided by Nanjing Jiancheng Bioengineering Research Institute is used to add each reagent, mix well and boil in water for 15 minutes, and immediately cool with cold water. Finally, the absorbance at a wavelength of 520 nm is measured to calculate the serum urea nitrogen content.

[0055] Example 3: Preparation method of sea cucumber polypeptide

[0056] (1) Extraction and enzymatic hydrolysis of sea cucumber protein

[0057] In the experimental preparation stage, the dried sea cucumber was ground into powder to prepare a sea cucumber powder suspension with a concentration of 1%; ultrasonic crushing was used to obtain a crushed sea cucumber powder solution; at pH 6.5, 55°C, 6000U / g of papain was added and the enzymatic hydrolysis time was 8h; after the enzyme was inactivated in a boiling water bath, the pH was adjusted to 8.0, and 7000U / g of trypsin was added. After enzymatic hydrolysis for 6h, the enzyme was inactivated again; after cooling, the mixture was centrifuged at 5000rpm for 15 minutes, and the supernatant was taken as the enzymatic hydrolyzate.

[0058] (2) Concentration and separation of enzymatic hydrolysis products

[0059] Millipore ultrafiltration tubes were used to carry out step-by-step ultrafiltration and concentration of the enzymatic hydrolysate. 2 mL of each concentrated solution was retained. The results of the anti-fatigue activity test are shown in Table 2 and Figure 2 As shown, it can be seen that the concentrate with a molecular weight less than 3 kDa has the strongest activity.

[0060] Table 2 Anti-fatigue activity test results of each level of concentrate

[0061]

[0062] (3) Purification of enzymatic hydrolysis products

[0063] Select SP-Sepharose FF cation exchange chromatography for separation, column volume 1.6×20cm, use pH 7.5, 20mmol / L PB buffer to wash the equilibrium column, and load the concentrate with molecular weight less than 3kDa at a flow rate of 0.5mL / min. Use pH7.5, 20mmol / L PB buffer containing 0.1 mol / L NaCl for elution. The results are as follows: Figure 3 As shown in Table 3, the anti-fatigue activity of the eluted components was identified. The control group still used experimental mouse samples gavaged with distilled water to collect the components with the most anti-fatigue activity. Figure 4 As shown in the figure, the anti-fatigue activity of peak c3 is the highest. Peak c3 was selected and further separated using a Q-Sepharose FF high-efficiency ion exchange column. After washing with equilibrium buffer (pH 8.0, Tris-HCl), step-shaped gradient elution and separation were performed using equilibrium buffer + 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1 mol / L NaCl at a flow rate of 0.5 mL / min. The elution results are shown in the figure. Figure 5 The four peaks eluted were tested for anti-fatigue activity, and the results are shown in Table 4 and Figure 6 As shown, it can be seen that both peak c1' and peak c2' have anti-fatigue activity. Peak c1' and peak c2' were combined and concentrated, and then separated again by Q-Sepharose FF high-efficiency ion exchange column according to the above steps to obtain Figure 7 The c1" elution peak was detected to detect the anti-fatigue activity of the c1" elution peak. The mice in the distilled water gavage group were used as controls. The results are shown in Tables 5 and Figure 8 As shown, the anti-fatigue activity of the c1" elution peak was significantly higher than that of the control group. The c1" peak was desalted and concentrated to obtain a high-purity protein solution.

[0064] Table 3 Anti-fatigue activity of components eluted by SP-Sepharose FF cation exchange chromatography

[0065] Group Swimming time (s) Blood urea nitrogen content (mmol / L) Control group 538±269 13.31±2.17 c1 549±106 12.86±0.94 c2 554±97 12.15±1.14 c3 1058±214 6.47±0.83 c4 661±84 10.54±1.33 c5 590±96 11.33±1.47 c6 792±88 9.07±1.66 c7 715±132 9.85±1.09 c8 693±61 10.13±1.88

[0066] Table 4 Anti-fatigue activity of each component in the first elution of Q-Sepharose FF high-efficiency ion exchange column

[0067] Group Swimming time (s) Blood urea nitrogen content (mmol / L) Control group 501±187 11.98±3.02 c1＇ 1069±105 7.04±0.37 c2＇ 1144±67 6.78±1.14 c3＇ 598±87 10.62±1.96 c4＇ 534±103 11.91±0.87

[0068] Table 5 Anti-fatigue activity of each component in the second elution of Q-Sepharose FF high-efficiency ion exchange column

[0069] Group Swimming time (s) Blood urea nitrogen content (mmol / L) Control group 574±241 12.77±2.92 c1＂ 1128±75 6.54±0.49

[0070] (4) Determination of amino acid sequence of enzymatic hydrolysis products

[0071] The high-purity protein solution was used to determine the polypeptide sequence with anti-fatigue activity using a protein solid phase sequence analyzer. The principle is to use the Edman degradation method to sequentially release amino acids from the amino end of the sequence and capture and identify them. The measured results are as follows: Fig. 9 and Fig.10 Wherein, (a) is the standard amino acid spectrum, (b)-(j) are the measured amino acids, and the amino acid sequence of the polypeptide obtained by the present invention is shown in SEQ ID NO: 1: Asp-Arg-leu-Ile-Phe-Phe-Gly-Tyr-Val (DRLIFFGYV).

[0072] It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

Claims

1. A preparation containing sea cucumber polypeptide, characterized in that: The invention comprises a sea cucumber polypeptide having an amino acid sequence as shown in SEQ ID NO:

1.

2. The preparation containing sea cucumber polypeptide according to claim 1, characterized in that: It also contains preservatives, sweeteners, colorants, thickeners and pure water solvent.

3. The preparation containing sea cucumber polypeptide according to claim 1, characterized in that: The sea cucumber polypeptide is used for preparing an anti-fatigue preparation for the body.

4. A method for preparing a preparation containing sea cucumber polypeptides as claimed in claim 1, characterized in that: The following steps are involved: S1. A two-step enzymatic hydrolysis method was used to obtain a preliminary enzymatic hydrolysis product from the body wall of the sea cucumber. In the first step, papain was used under the following reaction conditions: pH 6.5, temperature 55°C, enzyme addition amount 6000U / g, and enzymatic hydrolysis time 8h; in the second step, trypsin was used under the following reaction conditions: pH 8, temperature 45°C, enzyme addition amount 7000U / g, and enzymatic hydrolysis time 6h; S2. Use ultrafiltration tubes to concentrate and separate the enzymatic hydrolysate. Select millipore ultrafiltration tubes with a volume of 4 mL and a molecular weight cutoff of 50 kDa, 30 kDa, 10 kDa, and 3 kDa to perform step-by-step ultrafiltration and concentration on the enzymatic hydrolysate. Retain 2 mL of each level of concentrated solution, and select the concentrated solution with a molecular weight less than 3 kDa; S3. The concentrate was purified by primary chromatography and secondary circulation chromatography. SP-Sepharose FF cation exchange chromatography was selected for primary chromatography. The column volume was 1.6×20 cm. The equilibrium column was washed with pH 7.5, 20 mmol / L PB buffer, and the concentrate was loaded at a flow rate of 0.5 mL / min. The column was eluted with pH 7.5, 20 mmol / L PB buffer containing 0.1 mol / L NaCl. The components with anti-fatigue activity were collected and further separated with Q-Sepharose FF high-efficiency ion exchange column. After washing with pH 8.0, Tris-HCl equilibrium buffer, step-shaped gradient elution and separation were performed with equilibrium buffer + 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1 mol / L NaCl at a flow rate of 0.5 mL / min. The elution peaks with anti-fatigue activity were combined and concentrated, and Q-Sepharose was performed again according to the above steps. FF high-efficiency ion exchange column is used for step-shaped gradient elution and separation, and the elution peaks with anti-fatigue activity are combined and desalted and concentrated to obtain a high-purity protein solution, which is the sea cucumber polypeptide; S4. Add preservatives, sweeteners, colorants and thickeners to the prepared sea cucumber polypeptide and dissolve it in pure water solvent to obtain a preparation containing the sea cucumber polypeptide.

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