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 peptide from rice dregs, taking rice dregs protein substrate as raw material. It comprises following steps: (1) separating rice dregs protein substrate with dextran gel Sephadex G-15 and getting separted component; testing ACE inhibiting activity for each got component; (2) vacuum cooling and drying the component with highest test value for ACE inhibiting activity to prepare freeze powder, dissloving freeze powder into distilled water to prepare solution with concentration being 5 mg freeze powder/ ml, separating said solution though inverse high efficiency liquid chromatography, and getting purified ACE inhibiting active peptide with the chromatogram peak value being the largest. The molecular weight of produced product is 645.3, and the primary structure of its amino acid is Phe-Asn-Gly-Phe-Tyr. The invention is characterized by easy device, simple process, good repeatability and wide application.

Description

The method that from rice is poor, separate, purifying ACE suppresses bioactive peptide and active peptide obtained

Technical field

The invention belongs to biotechnology and protein chemistry field, relate to the method for a kind of separation from rice is poor, purifying ACE inhibition bioactive peptide, and the prepared ACE of this method suppresses bioactive peptide.

Background technology

Along with the raising of people's living standard and the enhancing of sense of self-protection and awareness of safety, can reach the purpose of curing the disease, can avoid of the damage of synthetic medicament again by non-drug therapy human body, therefore accepted by increasing people.Essential hypertension is a kind of common frequently-occurring disease, and the whole world is suffered from the hypertension number and surpassed 500,000,000; Essential hypertension is again the main factor of coronary heart disease, heart and renal function depletion simultaneously, and therefore treating hypertension becomes the challenge that global medical circle faces.At present both at home and abroad formally being used for clinical hypertensin inhibitor has more than 16 kinds, just under study for action have 80 kinds.Although suppressing bioactive peptide, the Zinc metallopeptidase Zace1 (ACE) that enzymolysis process obtains do not have the evident in efficacy of chemosynthesis; But it has no side effect, and is safe, and the normotensive is free from side effects.At present, Japan is that research ACE suppresses the maximum country of bioactive peptide, mainly is to extract from milk protein source matter, secondly is exactly the fish albumen, and wherein a part of product has been realized suitability for industrialized production.And China studies in this respect and is in the starting stage, and vast territory and abundant resources in China, and the by product that produces in rich in protein resource and the food processing process is arranged, and is these utilizations of resources from now on research direction effectively how.

Peptide class separating and extracting method commonly used has membrane separation technique, gel filtration chromatography technology, ion exchange chromatography, gel electrophoresis, high performance liquid chromatography, capillary electrophoresis etc.Because food proteins suppresses the bioactive peptide molecular weight generally all below 1500 through the ACE that produces behind the protease hydrolysis, therefore can be with filtering or high molecular weight protein and unhydrolysed protein are gone out in charcoal absorption, remove insoluble substrate, protein and peptide class that molecular weight is bigger with ultrafiltration process, just can obtain the less small peptide of molecular weight.The gel filtration chromatography technology is according to bulk of molecule, isolates the polypeptide of different molecular weight by the porous gel bed.The rate of recovery height of this technology peptide, activity is not destroyed, the simple good reproducibility of equipment, be extensive adopted technology.Chuan Dao carries out ion exchange chromatography with SP Sephadex C-25 after having extracted the ace inhibitory peptide mixture of greater activity with the ODS resin isolation again, has obtained good result; The good fortune of river village is male with two kinds of gels of Sephadex G-25 and Sephadex G-10, be separated to the ACE inhibition active ingredient of greater activity from soybean protein pepsin hydrolysis liquid after, separate having obtained highly purified ACE inhibition bioactive peptide again through twice high performance liquid chromatography.Astanwan etc. handle the dried fish enzymolysis product of Indonesia with Sephadex G-25 gel chromatography and SP Sephadex C-25 ion exchange chromatography, have obtained high ACE and have suppressed bioactive peptide.

Suppress the research of active and structural relation about ACE and think, the C-terminal in ACE inhibition activity and the structure aminoacid sequence and the character of-terminal amino acid have important relation.Cheung etc. think that ACE suppressed active higher when the C-terminal amino-acid residue was die aromatischen Aminosaeuren (Thr, Tyr, Phe) and proline(Pro); The inhibition activity of peptide was higher when in addition, the n terminal amino acid residue was hydrophobic Val, Leu, Ile or basic aminoacids.The Japan scholar reports that zein obtains a kind of ace inhibitory peptide through enzymolysis, and its primary structure formula is Leu-Pro-Pro.Kunio Suetsuna isolates 7 kinds of dipeptides from garlic, its peptide sequence is respectively Ser-Tyr, Gly-Tyr, Phe-Tyr, Asn-Tyr, Ser-Phe, Gly-Phe and Asn-Phe, and these peptides are found to have obvious hypotensive activity with the synthetic back of various dose with the 200mg/kg body weight Hypertensive Rats of feeding.Toshiro etc. obtain 11 kinds of ACE with gastrointestinal enzyme to royal jelly proteolysis, purifying to be suppressed in the bioactive peptides, and the C-terminal amino-acid residue of most of peptide is Phe, Tyr, and this is identical of views with Cheung etc. also.

Above-mentioned result of study explanation, the C-terminal in ACE inhibition activity and the aminoacid sequence and the character of-terminal amino acid have important relationship, do not meet above-mentioned situation but also there are some ACE to suppress active stronger peptide.Mainly be made up of acidic amino acid as the ACE inhibition bioactive peptide that usefulness Sumizyme MP enzymolysis sardiness such as Matsui obtain, hydrophobic amino acid content is very low.

Summary of the invention

The technical problem to be solved in the present invention provide a kind of can sharp separation, the ACE behind the poor enzymolysis of purifying rice suppresses the method for bioactive peptide, and according to the bioactive peptide of this method gained.

In order to solve the problems of the technologies described above, the invention provides the method for a kind of separation from rice is poor, purifying ACE inhibition bioactive peptide, be 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.

The present invention also provides the ACE that makes according to aforesaid method to suppress bioactive peptide, and its molecular weight is 645.3, and its amino acid primary structure is Phe-Asn-Gly-Phe-Tyr, called after FNGFY.

To suppress active generally all be molecular weight less than 1000 little peptide because have ACE, therefore, in the present invention, selects for use dextrane gel Sephadex G-15 that the white zymolyte of rice Egg preserved in wine is separated.Dextrane gel Sephadex G-15 generally is used for the isolated molecule amount less than 1500 little peptide, therefore, relatively is fit to separate the ace inhibitory peptide in the poor white hydrolyzate of rice egg.

In the invention process, the contriver uses RPLC ACE inhibition bioactive peptide Sephadex G-15 separated portion F-II, F-III, F-IV, F-V, F-VI is analyzed, and utilize the RPLC method to prepare F-V-IV among the component F-V, further adopt mass spectrometry method to determine its molecular weight and amino acid primary structure, the result shows, this component contain molecular weight be 645.3 be the pentapeptide of FNGFY (Phe-Asn-Gly-Phe-Tyr).The present invention uses methods such as RPLC, mass spectrum to obtain to have higher ACE from the white zymolyte of rice Egg preserved in wine to suppress active pentapeptide first, and at home and abroad, relevant research does not in this respect appear in the newspapers as yet.

Patent of the present invention selects for use meter Egg preserved in wine white as the research raw material, will separate by the zymolyte that enzyme solution obtains, purifying obtains Zinc metallopeptidase Zace1 (ACE) and suppress bioactive peptide, and carry out determining and evaluation of primary structure.Can promote the comprehensive utilization of the poor resource of rice, can seek again and develop biologically active peptides, for developing and synthesizing and utilize novel biologically active peptides to lay the foundation with particular physiological function.

The present invention adopts the gel filtration column method in conjunction with high performance liquid chromatography, mass-spectrometric technique zymolyte to be carried out sharp separation, purifying, and determines its primary structure.The method that the present invention adopts is succinct, cost is low, and the ACE of acquisition suppresses not too big infringement of bioactive peptide, suppresses active high.Do not produce the report that ACE suppresses bioactive peptide in the existing report, more do not have this ACE to suppress the structure invention of bioactive peptide about the poor enzymolysis process of rice.Suppress bioactive peptide with other foodstuff protein source ACE that has reported at present and compare, the product sensory that it is protein content height in a kind of fine protein source, the raw material in vain that major advantage of the present invention is embodied in the cheap and rice Egg preserved in wine of the cost of material that is adopted, produce is excellent.

Rice protein is as good nutritional quality albumen, and not only the amino acid composition is very reasonable, and because it does not contain similar sensitizing factor, has the hypoallergenic characteristics, free from extraneous odour, digestibility advantages of higher.Yet there are no rice poor in an enzymolysis separation and purification go out to have ACE and suppress active little peptide.Adopt the peptide rate of recovery height of the inventive method preparation, activity is not destroyed.Method of the present invention has that equipment is simple, technology is succinct, the advantage of good reproducibility, can be widely adopted.

Description of drawings

Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.

Fig. 1 is the white zymolyte dextrane gel of a meter Egg preserved in wine Sephadex G-15 separating spectrum;

Fig. 2 is the IC of the white zymolyte Sephadex of meter Egg preserved in wine G-15 separated portion ₅₀(mg/mL) comparison diagram;

Fig. 3 is the rp-hplc analysis figure of the white zymolyte Sephadex of meter Egg preserved in wine G-15 separated portion F-V;

Fig. 4 is that the white zymolyte dextrane gel of meter Egg preserved in wine Sephadex G-15 separates F-V-IV component preparation figure.

Embodiment

The preparation of rice Egg preserved in wine white zymolyte: production method see the previous patent of invention of the applicant described (application number: 200610050845.7, the application time: in May, 2006).

Embodiment 1: adopt dextrane gel Sephadex G-15 that the white zymolyte of rice Egg preserved in wine is separated preparation, must separate component:

In sepn process, the present invention selects 160cm (length) * 30mm chromatography column for use, and the detection wavelength is 280nm, and elutriant is a distilled water.Eluent flow rate is 0.5ml/min, and applied sample amount is 25-30ml, according to the wavelength numerical value OD in the nucleic acid-protein detecting instrument ₂₈₀Change and carry out the collection of sample.According to above-mentioned given operational condition, can collect and obtain to have ACE and suppress active small peptide.

As can be seen from Figure 1, the white zymolyte of rice Egg preserved in wine is divided into six apparent in view polypeptide fraction after dextrane gel Sephadex G-15 separates, respectively called after F-I, F-II, F-III, F-IV, F-V, F-VI.The isolated component F-I of dextrane gel SephadexG-15, F-II, F-III, F-IV, F-V, the shared peak area of F-VI are respectively 36.08%, 45.93%, 7.98%, 2.13%, 3.03%, 4.85%.Wherein F-I and F-II proportion are higher, are respectively 36.08% and 46.93%.The protein recovery of the white zymolyte of rice Egg preserved in wine after dextrane gel Sephadex G-15 separates reached 84.9%.

In this research, for the ACE that further studies the white zymolyte dextrane gel of rice Egg preserved in wine Sephadex G-15 separated portion suppresses active, separating obtained F-I, F-II, F-III, F-IV, six components of F-V, F-VI have been carried out ACE and suppressed active mensuration, also measured the protein content of six components simultaneously, concrete outcome sees Table 1.

ACE suppresses active mensuration and measures with reversed-phased high performace liquid chromatographic (RP-HPLC), and this method gets the nod at present and uses.

It is active that the protein content of white each separated portion of zymolyte of table 1 meter Egg preserved in wine and ACE suppress

Separated portion	Protein content (μ g/mL)	ACE inhibiting 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

From last table 1 as can be seen, rice Egg preserved in wine white zymolyte dextrane gel Sephadex G-15 separating obtained each component F-I, F-II, F-III, F-IV, F-V, F-VI all have ACE and suppress active, its ACE suppresses activity and is respectively 23.7%, 47.5%, 74.6%, 68.4%, 84.7%, 77.9%, and wherein the ACE of F-III, F-IV, F-V, F-VI suppresses active higher.

Embodiment 2: the ACE of the white zymolyte Sephadex of rice Egg preserved in wine G-15 separated portion suppresses active 503nhibiting concentration (IC50) research and compares:

Raw material: adopt embodiment 1 described method to obtain the gel separation component of the white zymolyte of rice Egg preserved in wine respectively as the research raw material.

Active for the ACE inhibition of accurate description component F-I, F-II, F-III, F-IV, F-V, F-VI, the ACE that has measured each component respectively suppresses active 503nhibiting concentration (IC ₅₀).Measurement result as shown in Figure 2.Can learn that by Fig. 2 the ACE of separated portion F-I, F-II, F-III, F-IV, F-V, F-VI suppresses active 503nhibiting concentration (IC ₅₀) be respectively 0.502g/mL, 0.171mg/mL, 0.041mg/mL, 0.019mg/mL, 0.011mg/mL, 0.017mg/mL.These data show that the ACE of F-III, F-IV, F-V, F-VI suppresses active higher; Wherein, it is F-V that ACE suppresses the highest active component, and its ACE suppresses active 503nhibiting concentration (IC ₅₀) be 0.011mg/mL.May contain ACE among the result of study prompting component F-V and suppress active higher component.

The ACE that has enumerated the different food sources of part in the table 2 suppresses the 503nhibiting concentration (IC of active inhibitory substance ₅₀), and compare with the white zymolyte of rice Egg preserved in wine.From table 2 as seen, the ACE of the white zymolyte of rice Egg preserved in wine suppresses the active polypeptide IYPNM that is equivalent to obtain from Japanese sake, and the white zymolyte of rice Egg preserved in wine suppresses activity all apparently higher than the egg protein zymolyte through the ACE of dextrane gel Sephadex G-15 separating obtained each component F-I, F-II, F-III, F-IV, F-V, F-VI, wherein, the ACE of F-III, F-IV, F-V, F-VI suppresses the active protein zymolyte that is higher than crab, cloves fish, cloves fish source.

The ACE of the different food sources of table 2 part suppresses the 503nhibiting concentration (IC of active inhibitory substance ₅₀)

ACE inhibition IC50 (mg/mL) food source
	Hydrolysate 1.22 eggs
Hydrolysate 0.075 crab
	Hydrolysate 0.076 sardines
YVLAGM 0.7 cloves fish
	IYPNM 0.015 Japanese sake
3.1 meters of the white zymolytes of rice Egg preserved in wine are poor
	0.502 meter of the present invention/F-I is poor
0.171 meter of the present invention/F-II is poor
	0.041 meter of the present invention/F-III is poor
0.019 meter of the present invention/F-IV is poor
	0.011 meter of the present invention/F-V is poor
0.017 meter of the present invention/F-VI is poor

Embodiment 3: the white zymolyte dextrane gel of rice Egg preserved in wine Sephadex G-15 separates the chromatographic separation and the RPLC preparation of F-V component:

Have the active component F-V of high Zinc metallopeptidase Zace1 (ACE) inhibition for further verifying, adopt reversed-phased high performace liquid chromatographic that F-V is carried out compartment analysis, specific as follows:

In this enforcement, will make lyophilized powder after the protein zymolyte dextrane gel Sephadex G-15 separated portion F-V vacuum lyophilization, cryodesiccated processing condition are: temperature-45 ℃, vacuum tightness 20Pa.

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, use C ₁₈RPLC has carried out compartment analysis.Its chromatographic condition is as follows:

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

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

Detect: UV280nm

Flow velocity: 0.5mL/min

Column temperature: 30 ℃.

Research obtains six chromatographic peak (see figure 3)s according to the method described above, and wherein F-V-IV's is active maximum, with RPLC F-V-IV is prepared.Its chromatographic condition is as follows:

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

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

Detect: UV220nm

Flow velocity: 1.0mL/min

Column temperature: 30 ℃

After collecting part shown in Figure 3 repeatedly, carry out lyophilize, preserve and carry out next step mass spectroscopy.

Embodiment 4: the white zymolyte dextrane gel of rice Egg preserved in wine Sephadex G-15 separated portion F-V-IV mass spectroscopy:

Mass spectrum plays a significant role in proteinic primary structure is determined.For determining meter amino-acid sequence of the middle polypeptide of the white zymolyte dextrane gel of Egg preserved in wine Sephadex G-15 separated portion F-V-IV, measured the LC MS.M mass spectrum of polypeptide.F-V-IV carries out liquid phase-mass spectroscopy to the white zymolyte dextrane gel of rice Egg preserved in wine Sephadex G-15 separated portion.Analyze with Bruker DataAnalysis 3.0 softwares, can find out obviously in the mass spectrum of component F-V-IV that a molecular weight is 645.3 component.

To molecular weight among the white zymolyte dextrane gel of the rice Egg preserved in wine Sephadex G-15 separated portion F-V-IV is that 645.3 component is carried out second order ms (MS/MS) analysis.According to the fracture,simple rule in the mass spectroscopy, the peptide class can produce the mass fragments of amino acid masses fragment and certain or certain several amino-acid residue+CO+NH, residue+CO, residue+NH, residue-NH, residue-CO or residue-CO-NH.These mass fragments and amino acid compared just can learn aminoacid sequence in the original peptide.Show that through the amino acid comparison result molecular weight is that the amino acid primary structure of 645.3 peptide is FNGFY (Phe-Asn-Gly-Phe-Tyr).

At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought 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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