CN106676155B - Preparation method of mussel polypeptide with antithrombotic activity - Google Patents
Preparation method of mussel polypeptide with antithrombotic activity Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention discloses a preparation method of mussel polypeptide with antithrombotic activity, which reserves anticoagulant peptide in mussel to the maximum extent and concentrates and enriches the anticoagulant peptide. The invention aims to select different types of protease to hydrolyze food-borne mussel protein, namely, a directional enzyme hydrolysis technology is adopted to prepare polypeptide powder with anticoagulant activity, and the active peptide is purified and concentrated. Compared with anticoagulant drugs of chemical synthesis or other sources, the product has strong digestion and absorption, no side effect on human body and strong prevention effect on blood coagulation.
Description
Technical Field
The invention relates to a preparation method of mussel polypeptide with antithrombotic activity.
Background
The marine organisms are rich in resources, various in types, sound in biological chain and strong in regeneration capacity, and account for about half of the total amount of the global organisms. The changeable living environment and the long evolutionary process enable the organisms to have different physiological properties from terrestrial organisms, generate a plurality of bioactive substances with novel structures and special effects, are huge sources of novel bioactive substances, and have great development potential. The bioactive peptide refers to a polypeptide which has the functions of optimizing the metabolic environment of an organism and benefiting the health of the organism. Generally prepared by enzymatic hydrolysis of proteins, usually contain 3-20 amino acid residues, which are not biologically active in the original protein sequence, but are biologically active after release by acid, base or enzymatic hydrolysis, depending on their amino acid composition and their order. Marine organisms are a good resource for the development of bioactive peptides, and in recent years many different kinds of bioactive peptides have been extracted from marine organisms, and byproducts of marine processing. With the increasing awareness of consumers in diet and health, the awareness and demand for functional food ingredients and health care nutritional products is gradually increasing, and bioactive peptides derived from marine organisms and marine processing byproducts have great potential in the development of functional foods because of the advantages of safety, no toxic or side effect, strong price competitiveness, and the like.
Thrombus refers to a semi-clot formed on the surface of a blood vessel or the endocardium of a heart by blood components during the blood flow. Thrombotic diseases include arterial thrombosis, venous thrombosis, microthrombosis, thromboembolic diseases, and the like. Normally, coagulation and anticoagulation are in a dynamic balance in vivo, and when abnormal coagulation forms thrombus, anticoagulation medicine is used for inhibiting coagulation and thrombolytic medicine is used for enhancing fibrinolysis system effect, which are measures for correcting imbalance in vivo after disease occurs. In fact, diseases are caused by abnormal coagulation state in vivo and final thrombosis, and relatively long pathological processes exist in the process. By obtaining the information of the occurrence and the development of the disease in the monitoring of the index change of the blood coagulation state of the patient, the formation of thrombus can be prevented at an early stage, and the guide and the evaluation can be made on the treatment prognosis of the patient who has suffered from the thrombus.
Thrombotic diseases seriously harm the health of middle-aged and elderly people, and the number of patients suffering from thrombotic diseases is obviously increased along with the acceleration of life rhythm in recent years, so people pay more and more attention to the prevention and treatment of thrombus. Blood coagulation is an important physiological defense process of the body, but pathological thrombosis seriously harms human health. Currently, the incidence of cardiovascular and cerebrovascular thrombotic diseases is rising year by year. In order to prevent thrombosis or to arrest the development of established thrombosis, anticoagulant drugs are used. The mechanism of anti-blood coagulation is that the added substances can prevent prothrombin from forming thrombin, thereby inhibiting the conversion of fibrinogen into fibrin; the fibrinolysis mechanism is that plasminogen is activated by adding plasminogen activator to generate plasmin, and the plasmin induces fibrin to be converted into fibrin degradation products; the mechanism of anti-platelet aggregation is to reduce platelets attached to the fibrin skeleton by adding Hangzhou platelet aggregation substances.
Anticoagulants generally block the coagulation process by inhibiting certain key coagulation factors (such as tissue factor, factor X, and thrombin) in the coagulation process. Clinical anticoagulant drugs mostly surround the inhibition of thrombin and other coagulation factors to achieve an anticoagulant effect.
The twenty-first century is the century of oceans, which are the main raw material base for future foods, medicines and health products. The ocean is developed, and the full utilization of ocean resources is an important direction for the technological development at present. Mussels as a marine resource have high protein content, health care and disease prevention, and have high dietotherapy and medicinal effects. Due to the characteristics of strong adaptability, high yield and the like, the artificial propagation is easy to realize in large quantities. Not only has high protein content, but also has amino acid pattern more close to the requirement of human body. The polypeptide with various activities can be released by enzymolysis.
At present, the extraction method of the marine bioactive peptide mainly comprises 3 methods: solvent extraction, microbial fermentation and enzymatic hydrolysis. The enzyme hydrolysis method has become the first choice in the food and pharmaceutical industries because it has no organic solvent and no toxic chemical residue. Enzymatic preparation of biologically active peptides is a process for preparing biologically active peptides by hydrolysis of the protein by a suitable protease. The bioactive peptide with the anticoagulation effect has wide application prospect in preventing thrombosis, thrombolysis treatment and maintenance treatment after interventional therapy. Mainly adopts a synthesis method to prepare the anticoagulant peptide and extracts the anticoagulant peptide from the biological secretion.
At present, the main anticoagulant drugs clinically used at home and abroad are common heparin, low molecular heparin, warfarin and the like, and the drugs have the defects of side effects of thrombocytopenia, hemorrhage and the like or slow effect taking and the like. The polypeptide anticoagulant drug has the outstanding advantages of quick response, low side effect and the like, and is a key development direction of anticoagulant drugs in the future. The Sarmientos is separated from the leech to obtain the anticoagulant polypeptide. Lu et al extract active peptides (SVP) from scorpion venom, and SVP can inhibit platelet aggregation and thrombosis in mice and increase fibrinolytic activity. Barbouche et al isolated two groups of polypeptides Lebetins 1 and Lebetins 2 from venom of Vipera lebeina of the poisonous snake, which inhibit platelet aggregation. However, they have problems in that they are derived from the saliva of water frog or the venom of snake or scorpion, and they are effective components of the saliva of water frog or the venom of snake or scorpion, so that they have side effects of these substances, such as causing massive bleeding. Anticoagulant active peptides derived from food products, which may be digested and absorbed in the human gastrointestinal tract due to their food origin, function in the anticoagulant system of the human body, such as peptide fragments active in inhibiting platelet aggregation and inhibiting fibrinogen binding to platelet surface receptors derived from the C-terminal portion of gastrointestinal digested K-casein isolated from neonatal serum by Hartmann. Therefore, it is expected that the anticoagulant peptide derived from food is highly safe. However, the research of obtaining the bioactive peptide with the anticoagulation effect from food raw materials through enzymolysis is rarely reported at present, the analysis reason is that no proper enzymolysis substrate is found, and scientific researchers engaged in medical research pay more attention to the development of thrombolytic drugs. To a certain extent, the prevention of the occurrence of thrombus is more significant than the thrombolytic therapy after the occurrence of thrombus. The research on the antithrombin activity of the mussel protein hydrolysate develops a functional component with the antithrombin activity to be applied to health-care food and medicines, and has wide application prospect.
Anticoagulant peptide as a medicine for removing thrombus is widely applied to the field of medicines, and anticoagulant drugs in the market at present have low extraction rate, large side effect or high price.
Disclosure of Invention
The invention starts from the mechanism influencing the anticoagulant activity and the existing problems, retains the anticoagulant peptide in the mussel to the maximum extent, and concentrates and enriches the anticoagulant peptide.
The invention aims to select different types of protease to hydrolyze food-borne mussel protein, namely, a directional enzyme hydrolysis technology is adopted to prepare polypeptide powder with anticoagulant activity, and the active peptide is purified and concentrated. Compared with anticoagulant drugs of chemical synthesis or other sources, the product has strong digestion and absorption, no side effect on human body and strong prevention effect on blood coagulation.
The technical scheme of the invention is realized as follows: a method for preparing mussel polypeptide with antithrombotic activity comprises the following steps:
(1) pretreatment: washing fresh commercially available mussels with tap water for three times to remove impurities and salt.
(2) Homogenizing: removing shells of cleaned mussels, cutting mussel meat into pieces, adding deionized water with 3-10 times of volume, mixing uniformly, and homogenizing with a homogenizer (5000-.
(3) Enzymolysis: trypsin and neutral protease are selected to carry out enzymolysis on the mussel homogenate respectively, and the pH value of the system is adjusted and kept stable by 0.5mo1/LNaOH solution in the enzymolysis process. The conditions of the enzymatic hydrolysis were as per Table 1.
Table 1: conditions of enzymolysis
| Class of enzyme | Temperature/. degree.C | pH | Enzymolysis time/min | Enzyme dosage/(u/g) |
| Trypsin | 25-55 | 7.0~9.0 | 20-180 | 1000~5000 |
| Neutral protease | 30-50 | 6.5~8.5 | 20-180 | 1000~5000 |
(4) Enzyme deactivation and centrifugation: heating the enzymolysis liquid in a water bath kettle at 95-100 deg.C for 8-15min to inactivate enzyme, cooling to room temperature, and centrifuging (5000 plus 10000rpm, 10-30 min) to obtain enzymolysis polypeptide supernatant.
(5) And (3) ultrafiltration: and (3) performing membrane interception on the enzymolysis liquid by adopting a filter membrane of 1000-3000Da, and reserving an effluent liquid part to obtain the polypeptide liquid with the molecular weight of less than 1000-3000 Da.
(6) Sequence identification: the molecular weight distribution of the ultrafiltration polypeptide is determined by MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry), and the polypeptide sequence is determined by Q-TOF.
(7) Determination of anticoagulant Activity
The enzyme hydrolysate anticoagulant peptide reacts and mixes with the thrombin standard substance, the OD value under different enzymolysis conditions is measured by an enzyme-labeling instrument, the thrombin inhibition rate of the polypeptide is calculated, and the anticoagulant effect of the polypeptide is analyzed by taking the factor as an index.
(8) Concentrating and drying: the product concentration operation before spray drying is carried out in an evaporation concentration mode, single-effect evaporation or multi-effect evaporation can be adopted, and the solid content is preferably between 20 and 45 percent. Feeding the concentrated liquid into a spray drying tower at a constant speed by a feed pump for spray drying, wherein the air outlet temperature is 75-85 ℃, the air inlet temperature is 138-146 ℃, and the control working voltage of the feed speed is 44-70V; and (3) sending the product after spray drying treatment into a multi-stage cyclone separator for gas-powder separation, and collecting the product after gas-powder separation in a constant-temperature drying chamber provided with a dehumidifier to obtain finished product powder.
Or freeze drying to obtain slurry with thickness of 3-7 mm, and the process conditions are as follows: working pressure is 35-80Pa, freezing temperature is-10 deg.C to-30 deg.C, freezing time is 5h-12h, sublimation temperature is 10-50 deg.C, and resolution temperature is 10-50 deg.C, to obtain dried product.
Crushing the dried substance by using a vacuum freeze drying crusher, sieving by using a 60-80-mesh sieve to obtain powder, namely dried mussel anticoagulant active peptide powder, and drying and storing at low temperature.
The invention has the beneficial effects that (1) the technology for developing anticoagulant peptide by taking mussels as raw materials does not see related patent documents at present, the enzymolysis preparation method effectively fills the blank of the field, and through identification, the anticoagulant active peptide prepared by the enzymolysis preparation method is different from the like products reported at present. (2) The directional enzymolysis technology is adopted, the cutting site of the peptide segment is more accurately grasped, and the separation and the activity identification of the active peptide are facilitated. The mussel polypeptide liquid after enzymolysis has high anticoagulant activity, and the antithrombin activity unit of the 1mg anticoagulant mussel polypeptide powder is 90 ATU/mg-150 ATU/mg. (3) Mussels are used as raw materials, the source is wide, the price is low, the industrial production in the ground, middle and high scales can be carried out, the processing requirements of factories with various specifications are met, and the method has important guiding significance for deep processing of mussel products and development and utilization of the mussel products in functional foods, health-care foods or medicines. (4) The obtained polypeptide with high anticoagulation activity is enriched, concentrated and dried into powder, the anticoagulation activity is improved, and the product powder is convenient to store and has obvious anticoagulation activity.
Detailed Description
The invention will be further illustrated with reference to specific examples.
1) Pretreatment: washing fresh commercially available mussels with tap water for three times to remove impurities and salt.
2) Homogenizing: removing shells of cleaned mussels, cutting mussel meat into pieces, adding deionized water with 3-10 times of volume, mixing uniformly, and homogenizing with a homogenizer (5000-.
3) Enzymolysis: trypsin and neutral protease are selected to carry out enzymolysis on the mussel homogenate respectively, and the pH value of the system is adjusted and kept stable by 0.5mo1/LNaOH solution in the enzymolysis process. The conditions of the enzymatic hydrolysis were as per Table 1.
Table 1: conditions of enzymolysis
| Class of enzyme | Temperature/. degree.C | pH | Enzymolysis time/min | Enzyme dosage/(u/g) |
| Trypsin | 25-55 | 7.0~9.0 | 20-180 | 1000~5000 |
| Neutral protease | 30-50 | 6.5~8.5 | 20-180 | 1000~5000 |
(4) Enzyme deactivation and centrifugation: heating the enzymolysis liquid in a water bath kettle at 95-100 deg.C for 8-15min to inactivate enzyme, cooling to room temperature, and centrifuging (5000 plus 10000rpm, 10-30 min) to obtain enzymolysis polypeptide supernatant.
(5) And (3) ultrafiltration: and (3) performing membrane interception on the enzymolysis liquid by adopting a filter membrane of 1000-3000Da, and reserving an effluent liquid part to obtain the polypeptide liquid with the molecular weight of less than 1000-3000 Da.
(6) Determination of anticoagulant Activity
The enzyme hydrolysate anticoagulant peptide reacts and mixes with the thrombin standard substance, the OD value under different enzymolysis conditions is measured by an enzyme-labeling instrument, the thrombin inhibition rate of the polypeptide is calculated, and the anticoagulant effect of the polypeptide is analyzed by taking the factor as an index.
(7) Concentrating and drying: the product concentration operation before spray drying is carried out in an evaporation concentration mode, single-effect evaporation or multi-effect evaporation can be adopted, and the solid content is preferably between 20 and 45 percent. Feeding the concentrated liquid into a spray drying tower at a constant speed by a feed pump for spray drying, wherein the air outlet temperature is 75-85 ℃, the air inlet temperature is 138-146 ℃, and the control working voltage of the feed speed is 44-70V; and (3) sending the product after spray drying treatment into a multi-stage cyclone separator for gas-powder separation, and collecting the product after gas-powder separation in a constant-temperature drying chamber provided with a dehumidifier to obtain finished product powder.
The mussel protein anticoagulant active peptide powder obtained by the invention has the characteristic of obvious anticoagulant activity, and the related research methods are as follows:
(1) determination of polypeptide content
The final polypeptide mixture powder was assayed using the BCA method. The specific operation steps are as follows:
A. gradient dilution Bovine Serum Albumin (BSA) standard
B. And mixing the solution A and the solution B in the BCA kit according to the ratio of 50:1 according to the volume of the working solution required by the experiment. Storing at room temperature.
a. 25 μ L of diluted, graded BSA and protein samples to be tested were added to a 96-well plate.
b. Add 200. mu.L of working solution to each tube and mix them well.
c. The 96-well plate was sealed and incubated in a 37 ℃ incubator for 30 min.
d. The 96-well plate was cooled to room temperature. The absorbance of the sample at a wavelength of 562 nm was measured using a microplate reader.
(2) Method for measuring anticoagulant activity (thrombin inhibition rate)
The microplate reader method is adopted, the temperature of the microplate reader is set to be 37 ℃, and the measurement wavelength is 405 nm. Both fibrinogen and thrombin solutions were formulated in Tris-HCl buffer (0.05 mo1/L, pH = 7.4). Add 140. mu.L of 1mg/ml (W/V) fibrinogen solution and 40. mu.L of sample solution to the microplate, mix well and read (A)SB). The reaction was started by adding 10. mu.L of thrombin solution (12U/mL), and the reading (A) was taken after preheating at 37 ℃ for 10minS). The absorbance (A) was measured by using 40. mu.L of Tris-HCl buffer solution instead of the sample solution and the same tube as the aboveCB) And AC。
Definition of thrombin inhibitory activity is the antithrombin activity per mg mussel protein, units: ATU/mg.
Thrombin inhibitory activity = Ce*Y*Ve/(Ch*Vh*m)
Ve-standard thrombin loading volume/microliter
CeStandard thrombin Activity U/ml
Y-inhibition%
ChSample concentration, mg/ml
VhSample application volume/microliter
m-mussel protein content, mg
Y=[(AC-ACB)-(AS-ASB)]/ (AC-ACB) *100%
(3) Method for measuring degree of hydrolysis
Measuring the hydrolysis degree of the mussel protein by a pH-stat method, timing after adding protease, adding 0.5 mol/L NaOH during hydrolysis to maintain the pH constant, and recording the alkali consumption every 20min until the hydrolysis is finished. The degree of hydrolysis was calculated by the following formula.
DH(%) =VNaOH*NNaOH/(α*Mp*Htot)*100
In the formula:
a=10pH-pK/(1+10pH-pK)
pK=7.8+2400*(298-t)/298T
T=273.15+t
VNaOH: titration of the volume of base consumed (mL)
C: titration of the consumed base equivalent concentration (mol/L)
And Mp: mass of protein (g)
α: average degree of dissociation of alpha-amino group
t: temperature of reaction environment
(4) Moisture measuring method
The measurement was carried out according to the moisture measurement method in GB 50093-2010:
firstly, drying an aluminum weighing bottle and a centrifuge tube in a 105 ℃ oven overnight before the experiment, cooling the aluminum weighing bottle and the centrifuge tube in a drier for 1-2 hours before weighing, and recording the weight of the weighing bottle as m1。
② adding 100mL of deionized water preheated to 30 ℃ in advance into a homogenizer, then adding 3 drops of defoamer, then adding 4g (accurate to 0.001 g) of sample, slowly adjusting the rotating speed of a stirrer to 1500rpm, and stirring for 10 min.
Thirdly, 5mL of mixed liquid is immediately taken out of three aluminum weighing bottles, and the weight is recorded as m2. Placing the mixture in an oven at 105 ℃ until the weight is constant, and weighing the mixture and recording the weight as m3。
The following formula is used for calculation:
moisture content M% = (M)3-m1）/（m2-m1）×100%
(5) Protein assay method
The measurement is carried out by adopting an automatic Kjeldahl apparatus method. Weigh 0.2g mussel to 0.001 g. The test was performed as required by the instrument instructions. The coefficient of nitrogen converted to protein at the time of calculation was 6.25. The results retain three significant figures when the protein content is greater than or equal to 1 g/100 g, as represented by the arithmetic mean of the results of two independent determinations made under repetitive conditions.
The detection results show that the mussel anticoagulant active polypeptide powder comprises the following basic components: the total protein content is more than or equal to 60 percent, wherein the polypeptide content of less than 1000-3000Da is more than 50 percent, the hydrolysis degrees of 2 hours of the enzymolysis liquid of trypsin and neutral protease are respectively 15.67 percent and 12.76 percent, the ash content is 0.1 to 0.5 percent, and the water content is less than or equal to 4 percent. The antithrombin activity units of 1mg of mussel trypsin and neutral protease enzymolysis solution dry powder are respectively 135.63 ATU/mg and 91.44 ATU/mg.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (1)
1. A method for preparing mussel polypeptide with antithrombotic activity is characterized in that fresh mussels are washed to remove impurities and salt; removing shells and homogenizing to obtain homogenate; carrying out enzymolysis on the mussel homogenate by using trypsin and neutral protease respectively; heating the enzymolysis liquid to inactivate enzyme, cooling, centrifuging to obtain enzymolysis polypeptide supernatant, performing membrane interception on the enzymolysis liquid by adopting a 1000-and 3000Da filter membrane, and retaining an effluent liquid part; spray drying or freeze drying to obtain polypeptide powder; wherein,
the homogenate is: cutting mussel meat after the shells of the mussels are removed, adding 3-10 times of deionized water by volume, uniformly mixing, and then performing homogenization treatment by using a homogenizer, wherein the rotation speed of the homogenizer is 5000-;
the enzymolysis conditions are as follows: the enzymolysis temperature of the trypsin is 25-55 ℃, the pH value is 7.0-9.0, the enzymolysis time is 20-180min, the enzyme dosage is 5000 mu/g, the enzymolysis temperature of the neutral protease is 30-50 ℃, the pH value is 6.5-8.5, the enzymolysis time is 20-180min, and the enzyme dosage is 1000-;
in the enzymolysis process, the pH value of the system is adjusted by 0.5mo1/LNaOH solution; heating the enzymolysis liquid in a water bath kettle at 95-100 ℃ for 8-15min to inactivate enzyme, cooling to room temperature, centrifuging, wherein the rotation speed of a centrifuge is 5000-;
before the spray drying, evaporation concentration is carried out, single-effect evaporation or multi-effect evaporation is adopted for the evaporation concentration, so that the solid content is 20% -45%, and then the spray drying is carried out; spray drying at inlet air temperature of 138-146 deg.C and outlet air temperature of 75-85 deg.C, separating gas and powder in a multi-stage cyclone separator, and collecting polypeptide powder in a constant temperature drying chamber;
the freeze drying is carried out, the thickness of the slurry is 3mm-7mm, and the process conditions are as follows: working pressure is 35-80Pa, freezing temperature is-10 deg.C to-30 deg.C, freezing time is 5h to 12h, sublimation temperature is 10-50 deg.C, and resolution temperature is 10-50 deg.C to obtain dried substance, pulverizing the dried substance with vacuum freeze drying pulverizer, and sieving with 60-80 mesh sieve to obtain polypeptide powder.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558296A (en) * | 2010-12-16 | 2012-07-11 | 浙江海洋学院 | Mytilus edulis enzymolysis polypeptide and preparation method and application thereof |
| WO2016153363A1 (en) * | 2015-03-24 | 2016-09-29 | The New Zealand Institute For Plant And Food Research Limited | Water-soluble mussel extract |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558296A (en) * | 2010-12-16 | 2012-07-11 | 浙江海洋学院 | Mytilus edulis enzymolysis polypeptide and preparation method and application thereof |
| WO2016153363A1 (en) * | 2015-03-24 | 2016-09-29 | The New Zealand Institute For Plant And Food Research Limited | Water-soluble mussel extract |
Non-Patent Citations (3)
| Title |
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| 我国海洋贝类资源的利用现状和发展趋势;刘媛;《现代食品科技》;20130315;第29卷(第3期);第673-677页 * |
| 海湾扇贝酶解物的制备、鉴定及其体内外抗肿瘤作用研究;刘媛;《中国博士学位论文全文数据库 工程科技Ⅰ辑》;20160215(第2期);第3页第3段 * |
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