CN101007841A - Method for separating and purifying ACE inhibition peptide from rice draff and active peptide obtained therefor - Google Patents

Abstract

The invention discloses a method for separating and purifying ACE-inhibiting active peptides from rice grains. The enzymolyzed rice grains protein enzymolyzate is used as a raw material, comprising the following steps: 1) Sephadex G-15 is used to The enzymatic hydrolyzate of rice grain protein is separated and prepared to obtain isolated components; then the ACE inhibitory activity of each of the obtained isolated components is measured respectively; into lyophilized powder, then the above-mentioned lyophilized powder is dissolved in distilled water to form a solution with a concentration of 5 mg lyophilized powder/mL, and then the above-mentioned solution is separated and analyzed by reversed-phase high performance liquid chromatography, and the obtained chromatographic peak is the largest It is the purified ACE inhibitory active peptide. The ACE-inhibiting active peptide prepared by the method of the invention has a molecular weight of 645.3, and its amino acid primary structure is Phe-Asn-Gly-Phe-Tyr. The method of the invention has the advantages of simple equipment, simple process and good repeatability, and can be widely used.

Description

Method for separating and purifying ACE-inhibiting active peptide from rice grains and the obtained active peptide

technical field

The invention belongs to the fields of bioengineering and protein chemistry, and relates to a method for separating and purifying ACE-inhibiting active peptides from rice grains, and the ACE-inhibiting active peptides prepared by the method.

Background technique

With the improvement of people's living standards and the enhancement of self-protection awareness and safety awareness, non-drug therapy can not only achieve the purpose of curing diseases, but also avoid the damage of synthetic drugs to the human body, so it has been accepted by more and more people. . Hypertension is a common and frequently-occurring disease. The number of people suffering from hypertension worldwide has exceeded 500 million. At the same time, hypertension is the most important factor in coronary heart disease, heart and kidney failure. Therefore, the treatment of hypertension has become a challenge faced by the global medical community. daunting task. At present, there are more than 16 kinds of angiotensin inhibitors officially used clinically at home and abroad, and 80 kinds are under research. Although the angiotensin-converting enzyme (ACE) inhibitory active peptide obtained by enzymatic hydrolysis is not as effective as the chemically synthesized peptide, it has no toxic side effects, high safety, and no side effects on normotensive persons. At present, Japan is the country that studies the most ACE inhibitory active peptides, which are mainly extracted from milk-derived proteins, followed by fish proteins, some of which have been industrialized. However, the research in this area in our country is in its infancy. Our country has a vast land and abundant resources, and there are abundant protein resources and by-products produced in the process of food processing. How to effectively use these resources is the direction of future research.

Peptides commonly used separation and extraction methods include membrane separation technology, gel column chromatography technology, ion exchange chromatography, gel electrophoresis, high performance liquid chromatography, capillary electrophoresis, etc. Since the molecular weight of the ACE-inhibiting active peptide produced after the food protein is hydrolyzed by protease is generally below 1500, high molecular weight protein and unhydrolyzed protein can be removed by filtration or activated carbon, and insoluble substrate and larger molecular weight can be removed by ultrafiltration. For proteins and peptides, short peptides with smaller molecular weights can be obtained. Gel column chromatography separates polypeptides of different molecular weights through a porous gel bed according to the size of the molecules. This technology has a high recovery rate of peptides, no damage to the activity, simple equipment and good repeatability, and is a widely used technology. Kawashima used ODS resin to separate and extract the highly active ACE inhibitory peptide mixture, and then used SP Sephadex C-25 for ion exchange chromatography, and obtained good results; Kawamura Yukio used two gels of Sephadex G-25 and Sephadex G-10, after separating the high-activity ACE-inhibiting active components from soybean protein pepsin hydrolyzate, and then separated by high-performance liquid chromatography twice to obtain high-purity ACE-inhibiting active peptides. Astanwan et al. used Sephadex G-25 gel chromatography and SP Sephadex C-25 ion exchange chromatography to treat Indonesian dried fish enzymatic hydrolyzate, and obtained short peptides with high ACE inhibitory activity.

Studies on the relationship between ACE inhibitory activity and structure suggest that ACE inhibitory activity has an important relationship with the properties of the C-terminal and N-terminal amino acids in the structural amino acid sequence. Cheung et al. believed that the ACE inhibitory activity was higher when the C-terminal amino acid residues were aromatic amino acids (Thr, Tyr, Phe) and proline; in addition, the N-terminal amino acid residues were hydrophobic Val, Leu, Ile or alkali The inhibitory activity of the peptide was higher when the sex amino acid was used. Japanese scholars reported that an ACE inhibitory peptide was obtained by enzymatic hydrolysis of zein, and its primary structural formula was Leu-Pro-Pro. Kunio Suetsuna isolated seven dipeptides from garlic, and their peptide sequences were Ser-Tyr, Gly-Tyr, Phe-Tyr, Asn-Tyr, Ser-Phe, Gly-Phe and Asn-Phe, and these peptides were separated in different After dose synthesis, feeding hypertensive rats with 200mg/kg body weight found that it has obvious blood pressure lowering effect. Among the 11 ACE-inhibiting active peptides obtained by Toshiro et al. using gastrointestinal enzymes to hydrolyze and purify royal jelly, the C-terminal amino acid residues of most peptides are Phe and Tyr, which is also consistent with Cheung et al.

The above research results indicate that the ACE inhibitory activity has an important relationship with the properties of the C-terminal and N-terminal amino acids in the amino acid sequence, but there are also some peptides with strong ACE inhibitory activity that do not meet the above conditions. For example, Matsui et al. used alkaline protease to hydrolyze sardines to obtain ACE inhibitory active peptides, which were mainly composed of acidic amino acids, and the content of hydrophobic amino acids was very low.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for rapidly separating and purifying the ACE-inhibiting active peptide after enzymatic hydrolysis of rice grains, and the active peptide obtained according to the method.

In order to solve the above-mentioned technical problems, the present invention provides a method for separating and purifying ACE-inhibiting active peptides from rice grains, using enzymatic hydrolyzed rice grain protein hydrolyzate as raw material, which is characterized in that it includes the following steps in sequence:

1) Sephadex G-15 was used to separate and prepare rice grain protein hydrolyzate to obtain separated components; the separation conditions were as follows:

Chromatographic column: length 160cm, diameter 30mm,

Filling: Sephadex G-15,

Flow rate: 0.5-5mL/min,

Detection wavelength: 280nm,

Eluent: distilled water,

Sample volume: 25-50mL;

Then carry out the mensuration of ACE inhibitory activity to each isolated fraction of gained;

2), vacuum freeze-dry the isolated component with the highest measured value of the above-mentioned ACE inhibitory activity to make a lyophilized powder, then dissolve the above-mentioned lyophilized powder in distilled water to form a solution with a concentration of 5 mg lyophilized powder/mL, and then Above-mentioned solution adopts reverse-phase high-performance liquid chromatography to carry out separation and analysis, and chromatographic analysis condition is as follows:

Chromatographic column: C18P/N84176, length 300mm, diameter 7.8mm;

Mobile phase: Acetonitrile, water and trifluoroacetic acid are mixed according to the volume ratio of 45/55/0.1 to form the mobile phase;

Detection: UV280nm;

Flow rate: 0.5-1mL/min;

Column temperature: 30°C;

The obtained chromatographic peak with the largest value is the purified ACE inhibitory active peptide.

The present invention also provides the ACE-inhibiting active peptide prepared according to the above method, its molecular weight is 645.3, its amino acid primary structure is Phe-Asn-Gly-Phe-Tyr, named as FNGFY.

Because those with ACE inhibitory activity are generally small peptides with a molecular weight less than 1000, therefore, in the present invention, Sephadex G-15 was selected to separate the rice grain protein hydrolyzate. Sephadex G-15 is generally used to separate small peptides with a molecular weight less than 1500, so it is more suitable for separating ACE inhibitory peptides in rice protein gluten hydrolyzate.

In the course of the invention, the inventors used reverse-phase high-performance liquid chromatography to analyze the separated components F-II, F-III, F-IV, F-V, and F-VI of the ACE inhibitory active peptide Sephadex G-15, and used reverse F-V-IV in component F-V was prepared by phase-high performance liquid chromatography, and its molecular weight and amino acid primary structure were determined by mass spectrometry. The results showed that this component contained FNGFY (Phe-Asn-Gly-Phe -Tyr) pentapeptide. The present invention for the first time obtains a pentapeptide with high ACE inhibitory activity from rice grain protein hydrolyzate by using reversed-phase high-performance liquid chromatography, mass spectrometry and other methods. At home and abroad, no research on this aspect has been reported.

The patent of the present invention selects rice waste protein as the research raw material, separates and purifies the enzymatic hydrolyzate obtained by enzymatic hydrolysis to obtain angiotensin-converting enzyme (ACE) inhibitory active peptide, and determines and identifies the primary structure. It can not only promote the comprehensive utilization of rice grain resources, but also find and develop bioactive peptides with specific physiological functions, laying a foundation for the development, synthesis and utilization of new bioactive peptides.

The invention uses a gel column filtration method combined with high performance liquid chromatography and mass spectrometry to quickly separate and purify the enzymatic hydrolyzate and determine its primary structure. The method adopted in the present invention is simple and low in cost, and the obtained ACE inhibitory active peptide does not have too much damage and has high inhibitory activity. In the existing reports, there is no report on the production of ACE-inhibiting active peptides by enzymatic hydrolysis of rice grains, let alone the invention of the structure of such ACE-inhibiting active peptides. Compared with the ACE-inhibiting active peptides of other food protein sources that have been reported so far, the main advantages of the present invention are that the raw materials used are low in price, rice grain protein is a high-quality protein source, the protein content in the raw materials is high, and the produced The sensory quality of the product is excellent.

As a high-quality nutritional protein, rice protein not only has a very reasonable amino acid composition, but also has the characteristics of hypoallergenicity, no peculiar smell, and high digestibility because it does not contain similar allergens. So far, no small peptides with ACE inhibitory activity have been isolated and purified by enzymatic hydrolysis from rice grains. The recovery rate of the peptide prepared by the method of the invention is high, and the activity is not destroyed. The method of the invention has the advantages of simple equipment, simple process and good repeatability, and can be widely used.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is the separation pattern of rice grain protein enzymatic hydrolyzate Sephadex G-15 Sephadex G-15;

Fig. 2 is the IC ₅₀ (mg/mL) comparison chart of rice grain protein hydrolyzate Sephadex G-15 separation fraction;

Fig. 3 is the reversed-phase high-performance liquid chromatographic analysis figure of rice grain protein hydrolyzate Sephadex G-15 separation component F-V;

Fig. 4 is a diagram showing the preparation of F-V-IV fractions separated by Sephadex G-15 from rice waste protein enzymatic hydrolyzate.

Detailed ways

Preparation of rice waste protein hydrolyzate: the production method is described in the applicant's previous invention patent (application number: 200610050845.7, application time: May 2006).

Example 1: Sephadex G-15 was used to separate and prepare rice grain protein hydrolyzate, and the separated components were obtained:

In the separation process, the present invention selects a chromatographic column of 160 cm (length) × 30 mm, the detection wavelength is 280 nm, and the eluent is distilled water. The flow rate of the eluent is 0.5ml/min, the sample volume is 25-30ml, and the sample is collected according to the change of the wavelength value OD ₂₈₀ in the nucleic acid protein detection instrument. According to the above given operating conditions, short peptides with ACE inhibitory activity can be collected and obtained.

It can be seen from Figure 1 that after the hydrolyzate of rice grain protein is separated by Sephadex G-15, it is divided into six relatively obvious polypeptide components, which are named F-I, F-II, F-III, and F-III respectively. -IV, F-V, F-VI. The peak areas of the fractions F-I, F-II, F-III, F-IV, F-V, and F-VI separated by Sephadex G-15 were 36.08%, 45.93%, 7.98%, and 2.13%, respectively , 3.03%, 4.85%. Among them, F-I and F-II accounted for a relatively high proportion, 36.08% and 46.93% respectively. The protein recovery rate of rice waste protein hydrolyzate separated by Sephadex G-15 reached 84.9%.

In this study, in order to further study the ACE inhibitory activity of Sephadex G-15 separation fraction of rice waste protein enzymatic hydrolyzate, F-I, F-II, F-III, F-IV, F-V, The six components of F-VI were tested for their ACE inhibitory activity, and the protein content of the six components was also measured. The specific results are shown in Table 1.

The determination of ACE inhibitory activity is carried out by reversed-phase high-performance liquid chromatography (RP-HPLC), which has been recognized and applied at present.

Table 1 Protein content and ACE inhibitory activity of each fraction of rice grain protein hydrolyzate

separate components Protein content (μg/mL) ACE inhibition rate (%) F-I 309.8 23.7 F-II 153.8 47.5 F-III 49.5 74.6 F-IV 24.6 68.4 F-V 16.6 84.7 F-VI 20.9 77.9

It can be seen from the above table 1 that the components F-I, F-II, F-III, F-IV, F-V, and F-VI obtained by separating the enzymatic hydrolyzate of rice grain protein from Sephadex G-15 all have ACE Inhibitory activity, its ACE inhibitory activities were 23.7%, 47.5%, 74.6%, 68.4%, 84.7%, 77.9%, respectively, and the ACE inhibitory activities of F-III, F-IV, F-V, and F-VI were higher.

Embodiment 2: the half-inhibitory concentration (IC50) research and comparison of the ACE inhibitory activity of rice grain protein hydrolyzate Sephadex G-15 separation fraction:

Raw materials: The gel-separated fractions of rice grain protein enzymatic hydrolyzate obtained by the method described in Example 1 were used as research raw materials.

In order to accurately describe the ACE inhibitory activity of the components FI, F-II, F-III, F-IV, FV, and F-VI, the half-inhibitory concentration (IC ₅₀ ) of the ACE inhibitory activity of each component was determined respectively. The measurement results are shown in FIG. 2 . It can be known from Figure 2 that the half-inhibitory concentrations (IC ₅₀ ) of the ACE inhibitory activity of the isolated fractions FI, F-II, F-III, F-IV, FV, and F-VI were 0.502 g/mL and 0.171 mg, respectively. /mL, 0.041mg/mL, 0.019mg/mL, 0.011mg/mL, 0.017mg/mL. These data show that F-III, F-IV, FV, and F-VI have higher ACE inhibitory activity; among them, the component with the highest ACE inhibitory activity is FV, and the half-inhibitory concentration (IC ₅₀ ) of its ACE inhibitory activity is 0.011mg /mL. The results of the study suggested that the component FV may contain components with higher ACE inhibitory activity.

Table 2 lists the half-inhibitory concentrations (IC ₅₀ ) of ACE inhibitory substances from different food sources, and compares them with rice grain protein hydrolyzate. It can be seen from Table 2 that the ACE inhibitory activity of the rice grain protein hydrolyzate is equivalent to the polypeptide IYPNM obtained from Japanese sake, and the rice grain protein hydrolyzate was separated by Sephadex G-15. -II, F-III, F-IV, FV, and F-VI had significantly higher ACE inhibitory activities than egg protein hydrolysates, and among them, F-III, F-IV, FV, and F-VI had higher ACE inhibitory activities than Protein hydrolyzate derived from crab, clove fish, clove fish.

Table 2 The half-inhibitory concentration (IC ₅₀ ) of the ACE inhibitory activity inhibitors from different food sources

ACE Inhibitor IC50(mg/mL) Food Source Hydrolysate 1.22 Eggs Hydrolysate 0.075 Crab Hydrolysate 0.076 Sardines   YVLAGM                                     IYPNM 0.015 Japanese sake Rice grain protein hydrolyzate 3.1 Rice grain Invention/F-I 0.502 Rice grains Invention/F-II 0.171 Rice grains The present invention/F-III 0.041 rice grains The present invention/F-IV 0.019 rice grains The present invention/F-V 0.011 rice grains The present invention/F-VI 0.017 rice grains

Example 3: Chromatographic separation and reversed-phase high-performance liquid chromatography preparation of rice grain protein enzymatic hydrolyzate Sephadex G-15 to separate F-V components:

In order to further prove the component F-V with high angiotensin-converting enzyme (ACE) inhibitory activity, adopt reversed-phase high-performance liquid chromatography to carry out separation and analysis to F-V, specifically as follows:

In this implementation, the proteolyzed dextran gel Sephadex G-15 separation fraction F-V was vacuum freeze-dried to make a freeze-dried powder. The freeze-drying process conditions were: temperature -45°C, vacuum degree 20Pa.

Then the above lyophilized powder was dissolved in distilled water to form a solution with a concentration of 5 mg lyophilized powder/mL, which was separated and analyzed by _C18 reverse-phase high performance liquid chromatography. Its chromatographic conditions are as follows:

Chromatographic column: C18P/N84176 (300mm×7.8mm)

Mobile phase: acetonitrile/water/trifluoroacetic acid, 45/55/0.1(V/V)

Detection: UV280nm

Flow rate: 0.5mL/min

Column temperature: 30°C.

According to the six chromatographic peaks obtained by the above method (see Figure 3), the activity of F-V-IV is the largest, and F-V-IV was prepared by reversed-phase high performance liquid chromatography. Its chromatographic conditions are as follows:

Chromatographic column: YWG-C18 (300mm×7.8mm)

Mobile phase: A: 0.1% TFA/water; B: 0.1% TFA/ACN

Detection: UV220nm

Flow rate: 1.0mL/min

Column temperature: 30°C

The parts shown in Figure 3 were collected repeatedly, then freeze-dried, and stored for the next step of mass spectrometry analysis.

Example 4: Mass spectrometry analysis of F-V-IV fractions separated by Sephadex G-15 of Sephadex G-15 enzymatic hydrolyzate of rice grain protein:

Mass spectrometry plays an important role in the determination of the primary structure of proteins. In order to determine the amino acid sequence of the polypeptide in fractions F-V-IV of Sephadex G-15 Sephadex G-15 separation fraction of rice grain protein hydrolyzate, the LC MS.M mass spectrum of the polypeptide was determined. Liquid chromatography-mass spectrometry analysis of Sephadex G-15 fractions F-V-IV of rice waste protein enzymatic hydrolyzate. Analyzed with Bruker DataAnalysis 3.0 software, a component with a molecular weight of 645.3 can be clearly seen in the mass spectrum of component F-V-IV.

A fraction with a molecular weight of 645.3 in Sephadex G-15 Sephadex G-15 separation fraction F-V-IV was analyzed by MS/MS by MS/MS. According to the simple fragmentation rules in mass spectrometry, peptides can produce amino acid mass fragments and one or several amino acid residues + CO + NH, residue + CO, residue + NH, residue -NH, residue - CO or mass fragments of the residue -CO-NH. By comparing these mass fragments with amino acids, the amino acid sequence in the original peptide can be known. The results of amino acid alignment showed that the primary amino acid structure of the peptide with a molecular weight of 645.3 was FNGFY (Phe-Asn-Gly-Phe-Tyr).

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (2)

1, a kind of separation from rice is poor, purifying ACE suppress the method for bioactive peptide, are raw material with the white zymolyte of rice Egg preserved in wine behind the enzymolysis, it is characterized in that may further comprise the steps successively:

1), adopt dextrane gel Sephadex G-15 that the white zymolyte of rice Egg preserved in wine is separated preparation, must separate component; Separation condition is as follows:

Chromatography column: long 160cm, diameter 30mm,

Weighting material: sephadex G-15,

Flow velocity: 0.5-5mL/min,

Detect wavelength: 280nm,

Elutriant: distilled water,

Applied sample amount: 25-50mL;

Then every kind of separated portion of gained is carried out ACE respectively and suppress active mensuration;

2), above-mentioned ACE is suppressed make lyophilized powder after the highest separated portion vacuum lyophilization of determination of activity value, again above-mentioned lyophilized powder is dissolved in and is made into the solution that concentration is 5mg lyophilized powder/mL in the distilled water, adopt reversed-phased high performace liquid chromatographic to carry out compartment analysis to above-mentioned solution then, chromatographiccondition is as follows:

Chromatographic column: C18P/N84176, long 300mm, diameter 7.8mm;

Moving phase: the volume ratio according to 45/55/0.1 is mixed the back with acetonitrile, water and trifluoroacetic acid and is formed moving phase;

Detect: UV280nm;

Flow velocity: 0.5-1mL/min;

Column temperature: 30 ℃;

The ACE that is behind the purifying of the chromatogram peak value maximum that obtains suppresses bioactive peptide.

2, a kind of ACE that makes according to the described method of claim 1 suppresses bioactive peptide, and it is characterized in that: the molecular weight that described ACE suppresses bioactive peptide is 645.3, and its amino acid primary structure is Phe-Asn-Gly-Phe-Tyr.

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