CN107602664A - A kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder and its application - Google Patents
A kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder and its application Download PDFInfo
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- CN107602664A CN107602664A CN201711012978.XA CN201711012978A CN107602664A CN 107602664 A CN107602664 A CN 107602664A CN 201711012978 A CN201711012978 A CN 201711012978A CN 107602664 A CN107602664 A CN 107602664A
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Abstract
The invention discloses a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder, the amino acid sequence of this reducing blood lipid pentapeptide is:Gly Ile Glu Trp Ala (GIEWA), ESI MS measure molecular weight are 574.64Da;By brown croaker air bladder slurries through alkali extraction with degreasing brown croaker air bladder is made after alcohol ungrease treatment, air bladder enzymolysis liquid is being digested to obtain through alkali protease, enzymolysis liquid is purified through ultrafiltration, ion exchange chromatography, gel filtration chromatography, RPLC, obtains brown croaker air bladder reducing blood lipid pentapeptide;The present invention also discloses a kind of application for coming from brown croaker air bladder reducing blood lipid pentapeptide in hypolipidemic activity composition, health product and food additives.Brown croaker air bladder reducing blood lipid pentapeptide can be tuned into aliphatic radical down because and can promotes fatty acid beta oxidation, reduces the generation of lipid, has good Lipid-lowering activities, can be developed into reducing blood lipid medicine, health care production, and food additives.
Description
Technical field
The present invention relates to oligopeptides field, more particularly, to a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder and its application.
Technical background
Ocean fish air bladder is one of " ocean eight delicacies ", is known as the reputation of " ocean ginseng ".The main nutrient composition of air bladder is advanced
Collagen isoreactivity composition, wherein protein content is more than 80%, and fat content is relatively low.Therefore, air bladder is preferable height
Albumen low-fat food.
China, which eats air bladder, long history, the Northern Wei Dynasty《Qimin yaoshu》, the Tang Dynasty《New Tang book geography will》And Li Shizhen (1518-1593 A.D.) --
《Compendium of Materia Medica》It is on the books Deng the performance to air bladder.Zhoushan is local, and resident can use brown croaker air bladder to stage of development child
Dried product-fish glue, to improve child's immunity, promote development.The research and utilization to air bladder domestic at present is only limitted to air bladder
Simple working process, the research for ocean fish fish glue is concentrated mainly on eel, vertical fish and large yellow croaker etc..Foreign countries are using air bladder as raw material
Research be also rarely reported, the basic research of air bladder is substantially insufficient.On the other hand, it is existing studies have reported that most of marine sources
Hypolipidemic substance concentrate on polysaccharide, astaxanthin and unrighted acid etc., only less marine active peptide such as seaweed
The reports such as polypeptide have hypolipidemic activity.Therefore, research of the marine organisms polypeptide in terms of reducing blood lipid is also to be strengthened.
The content of the invention
An object of the present invention is to provide a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder, reducing blood lipid pentapeptide can under
Aliphatic radical is tuned into because and can promotes fatty acid beta oxidation, has good Lipid-lowering activities.
The second object of the present invention is to provide a kind of application for the reducing blood lipid pentapeptide for coming from brown croaker air bladder, reducing blood lipid pentapeptide
Hypolipidemic activity composition, health product and food additives can be used as.
The present invention is directed to the problem of being mentioned in background technology, and the technical scheme taken is:A kind of drop for coming from brown croaker air bladder
Blood fat pentapeptide, the amino acid sequence of this reducing blood lipid pentapeptide are:Gly-Ile-Glu-Trp-Ala (GIEWA), ESI-MS determine molecule
Measure as 574.64Da;Reducing blood lipid pentapeptide can reduce oleic acid(OA)The HepG2 lipid within endothelial cells accumulation of induction, to T-CHOL
(TC), triglycerides(TG)In dose dependent decomposition, aliphatic radical can be tuned into down because of SREBP-1c, ACC, SREBP-2, HMGR
With FAS expression, suppress into fat important factor SREBP-1c protein expression level, activate LXR α and LXR β expression.
Preferably, a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder, its preparation method include:Pre-process, digest, carry
Take, specifically include following steps:
Pretreatment:Brown croaker air bladder is cleaned, after homogenate to pasty state, according to solid-liquid ratio 1g:10-15mL adds 0.1-0.12mol/
LNaOH soaks 12-15 hours in 4-6 DEG C, filtering, neutrality is washed to distillation, according to solid-liquid ratio 1g:5-8mL adds 10-12%
Aqueous isopropanol, degreasing 36-48 hours, filtering, solids rinsed well isopropanol with distilled water, is dried, is obtained degreasing catfish
Fish air bladder;Successively using alkali extraction and alcohol degreasing, the superabundant fats in brown croaker air bladder can be thoroughly removed, are removed in brown croaker air bladder
The impurity such as foreign protein, improve the content of protein, contribute to the progress of further enzyme digestion reaction;
Enzymolysis:Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5-8mL adds distilled water, regulation pH value to 9-10, adds air bladder weight
Measure 2-3% alkali proteases, alkali protease enzyme activity >=2.0 × 105U/g, 50-60 DEG C of enzymolysis 3-4 hours, in 90-100 DEG C
Enzyme deactivation 10-15 minutes, it is cooled to room temperature and centrifuges 15-20 minutes after 12000-12500rmp, collect supernatant, obtain brown croaker air bladder
Enzymolysis liquid;Air bladder polypeptide is obtained using enzymatic isolation method, reaction condition is gentle, and preparation cost is relatively low, and nontoxic secondary organic solvent adds, alkali
Property protease degreasing brown croaker air bladder can be digested to high activity oligopeptides for small molecule, and the composition of active oligopeptide will not be destroyed
With property;
Extraction:Brown croaker air bladder enzymolysis liquid is classified through the milipore filter that molecular cut off is 10kDa and 3kDa, collects retention point
Component of the son amount less than 3kDa, obtains ultrafiltration enzymolysis liquid;By ultrafiltration enzymolysis liquid successively through ion exchange chromatography, gel column layer
Analysis and RPLC(RP-HPLC)Purifying, obtains brown croaker air bladder reducing blood lipid pentapeptide;Ultrafiltration can be with pure with multistage chromatography
Change brown croaker air bladder reducing blood lipid pentapeptide, improve the purity of reducing blood lipid pentapeptide in finished product, be advantageous to the essence of brown croaker air bladder reducing blood lipid pentapeptide
Quasi- application, brown croaker air bladder reducing blood lipid pentapeptide can efficiently reduce the generation of lipid, and its relatively low molecular weight is easier to by body
Absorb, and safe without toxic side effect.
Preferably, the brown croaker in pretreatment isMiichthy smiiuy。
Preferably, the ion exchange chromatography step in extraction step is:Ultrafiltration enzymolysis liquid is made into distilled water
Concentration is 45-55mg/mL solution, 3-4 DEG C, 12000-12500r/min centrifugation 15-20 minutes, removes insoluble impurities;Take
Supernatant is added to DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1mol/L, 0.5mol/L and 1mol/
L NaCl solution elution, collects 0.5mol/LNaCl elution fractions, as ion-exchange chromatography zymolyte;Ion exchange resin
Chromatography has the advantages of easy to operate, inexpensive, Chromatographic purification can be enlarged into large-scale production.
Preferably, the gel filtration chromatography step in extraction step is:Ion-exchange chromatography zymolyte is dissolved in distilled water
The solution that concentration is 45-55mg/mL is made into, 3-4 DEG C, 12000-12500r/min centrifugation 15-20 minutes, is removed insoluble miscellaneous
Matter;Take supernatant to pass through sephadex SephadexG-25 column chromatography for separation, eluted with ultra-pure water, according under 280nm
Absorbance curve collect elution fraction, wherein, the most strong component of hypolipidemic activity is gel chromatography zymolyte;In gel filtration chromatography
Sephadex the molecule of different molecular weight can be made to flow through note bed at different rates so that different size of molecule obtains
With separation.
Preferably, the RP-HPLC purifying in extraction step:Gel chromatography zymolyte is made into 80-100 μ with distilled water
G/mL solution, is purified using RP-HPLC, and RP-HPLC purification conditions are:Sample size is 10-15 μ L;Chromatographic column
KromasilC18(250mm × 4.6mm, 5 μm);Mobile phase is 40% acetonitrile;Elution speed is 0.8-1.0mL/min;Ultraviolet detection
Wavelength is 280nm;Screened according to each component hypolipidemic activity and obtain reducing blood lipid pentapeptide Gly-Ile-Glu-Trp-Ala (GIEWA);
RPLC(RP-HPLC)With non-polar stationary phase and the material of polarity mobile phase separating-purifying opposed polarity, tool
There is the advantages of separating rate is fast, purification efficiency is high.
Compared with prior art, the advantage of the invention is that:Brown croaker air bladder reducing blood lipid pentapeptide Gly- provided by the present invention
Ile-Glu-Trp-Ala (GIEWA) can reduce oleic acid(OA)The HepG2 lipid within endothelial cells accumulation of induction;Reducing blood lipid pentapeptide is in 1-
To T-CHOL under 10 μm of ol/L concentration(TC), triglycerides(TG)In dose dependent decomposition;Gly-Ile-Glu-
Trp-Ala (GIEWA) can be tuned into expression of the aliphatic radical because of SREBP-1c, ACC, SREBP-2, HMGR and FAS down, suppress into fat
Important factor SREBP-1c protein expression level, activation LXR α and LXR β expression.Therefore, Gly-Ile-Glu-Trp-Ala
(GIEWA) aliphatic radical can be tuned into down because and can promotes fatty acid beta oxidation, reduces the generation of lipid, and there is good lipid-loweringing to live
Property, it can be developed into reducing blood lipid medicine, health care production, and food additives.
Brief description of the drawings
Fig. 1 is the DEAE-52 anion exchange resin tomographic maps of the present invention;
Fig. 2 is the sephadex SephadexG-25 tomographic maps of the present invention;
Fig. 3 sephadexes SephadexG-25 prepares the RP-HPLC analyses of zymolyte;
Fig. 4 Gly-Ile-Glu-Trp-Ala (GIEWA) mass spectrogram;
The influence that Fig. 5 Gly-Ile-Glu-Trp-Ala (GIEWA) breed to HepG2 cells;
The influence that Fig. 6 Gly-Ile-Glu-Trp-Ala (GIEWA) accumulate to HepG2 lipid within endothelial cells;
The Microscopic observation result that Fig. 7 Gly-Ile-Glu-Trp-Ala (GIEWA) accumulate to HepG2 lipid within endothelial cells;
Fig. 8 Gly-Ile-Glu-Trp-Ala (GIEWA) are to HepG2 intracellular triglycerides(TG)The influence of content;
Fig. 9 Gly-Ile-Glu-Trp-Ala (GIEWA) are to the intracellular T-CHOLs of HepG2(TC)The influence of content;
The influence that Figure 10 Gly-Ile-Glu-Trp-Ala (GIEWA) are expressed HepG2 lipid within endothelial cells synthesis related gene;
Figure 11 Gly-Ile-Glu-Trp-Ala (GIEWA) aoxidize the influence of related gene expression to HepG2 lipid within endothelial cells;
Figure 12 Gly-Ile-Glu-Trp-Ala (GIEWA) SREBP-1c and ACC genes intracellular to HepG2 are in protein level
Expression influences;
The influence that Figure 13 Gly-Ile-Glu-Trp-Ala (GIEWA) are expressed the intracellular liver receptor-inducibles of HepG2.
Embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
A kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder, the amino acid sequence of this reducing blood lipid pentapeptide are:Gly-Ile-Glu-Trp-
Ala (GIEWA), ESI-MS measure molecular weight are 574.64Da, and reducing blood lipid pentapeptide can be used as hypolipidemic activity composition, health product
And food additives;The preparation method of reducing blood lipid pentapeptide includes:Pretreatment, enzymolysis, extraction, specifically include following steps:
Pretreatment:Brown croaker(Miichthysmiiuy)Air bladder is cleaned, after homogenate to pasty state, according to solid-liquid ratio 1g:10mL is added
0.1mol/LNaOH soaks 12 hours in 4 DEG C, filtering, neutrality is washed to distillation, according to solid-liquid ratio 1g:5mL add 10% it is different
Propanol solution, degreasing 36 hours, filtering, solids are rinsed well isopropanol with distilled water, are dried, are obtained degreasing brown croaker air bladder;
Successively using alkali extraction and alcohol degreasing, the impurity in brown croaker air bladder is removed;
Enzymolysis:Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5mL adds distilled water, regulation pH value to 9, adds the alkali of air bladder weight 2%
Property protease, alkali protease enzyme activity be 2.0 × 105U/g, 50 DEG C digest 3 hours, in 90 DEG C of enzyme deactivations 10 minutes, are cooled to
Room temperature centrifuges 15 minutes after 12000rmp, collects supernatant, obtains brown croaker air bladder enzymolysis liquid;Alkali protease can be by degreasing catfish
Fish air bladder enzymolysis is the oligopeptides of small molecule, is prepared for further extraction reducing blood lipid pentapeptide;
Extraction:Brown croaker air bladder enzymolysis liquid is classified through the milipore filter that molecular cut off is 10kDa and 3kDa, collects retention point
Component of the son amount less than 3kDa, obtains ultrafiltration enzymolysis liquid;By ultrafiltration enzymolysis liquid successively through ion exchange chromatography, gel column layer
Analysis and RPLC(RP-HPLC)Purifying, obtains brown croaker air bladder reducing blood lipid pentapeptide;Ultrafiltration can be with pure with multistage chromatography
Change brown croaker air bladder reducing blood lipid pentapeptide, improve the purity of reducing blood lipid pentapeptide in finished product, be advantageous to the essence of brown croaker air bladder reducing blood lipid pentapeptide
Quasi- application.
Implement 2:
The preparation technology flow of brown croaker air bladder reducing blood lipid pentapeptide is as follows:Brown croaker air bladder " degreasing " enzymolysis " zymolyte " ultrafiltration " ion
Displacement chromatography " gel permeation chromatography " high performance liquid chromatography prepares " reducing blood lipid pentapeptide " functional evaluation.
Concretely comprise the following steps:
1)Brown croaker air bladder pre-processes:Brown croaker(Miichthysmiiuy)Air bladder is after over cleaning, homogenate to pasty state, according to 1g:
10mL adds 0.1mol/LNaOH and 15h is soaked in 4 DEG C, filtering, neutrality is washed to distillation, according to 1g:5mL add 10% it is different
Propanol solution, degreasing 48h, filtering, solids are rinsed well isopropanol with distilled water, are dried, are obtained degreasing brown croaker air bladder;
2)The enzymolysis of brown croaker air bladder:Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5mL adds distilled water.PH value is adjusted to 10, is added
Enter the alkali protease of air bladder weight 2.5%, alkali protease enzyme activity is 2.5 × 105U/g, 55 DEG C enzymolysis 3.5h, in 95 DEG C,
10min enzyme deactivations, room temperature is cooled to, 12000rmp centrifugation 20min, collects supernatant, i.e. brown croaker air bladder enzymolysis liquid;
3)The preparation of brown croaker air bladder reducing blood lipid oligopeptides:Through molecular cut off it is the super of 3kDa and 10kDa by brown croaker air bladder enzymolysis liquid
Filter membrane is classified, and is collected molecular cut off and is less than 3kDa components, obtains ultrafiltration enzymolysis liquid.Ultrafiltration enzymolysis liquid is handed over through ion successively
Change resin chromatography, gel filtration chromatography and RPLC(RP-HPLC)Purifying, obtains brown croaker air bladder reducing blood lipid pentapeptide.
Using amino acid sequence analysis instrument and mass spectroscopy its structure, detailed process is:
1. ion exchange chromatography:Above-mentioned ultrafiltration enzymolysis liquid is made into the solution that concentration is 50mg/mL with distilled water, 4 DEG C,
12000r/min centrifuges 20min, removes insoluble impurities, and supernatant is added to DEAE-52 anion exchange resin chromatographic columns, according to
It is secondary with distilled water, 0.1,0.5 and 1mol/L NaCl solution elute, collect 0.5mol/L components(DMS-Ⅲ), as ion friendship
Change chromatography zymolyte(Fig. 1);
2. gel filtration chromatography:DMS- III is dissolved in distilled water and is made into the solution that concentration is 50mg/mL, 4 DEG C, 12000r/min centrifugations
20min, removes insoluble impurities, and supernatant passes through sephadex SephadexG-25 column chromatography for separation, carried out with ultra-pure water
Elution, according to the absorbance curve under 280nm(Fig. 2)Collect elution fraction GS-III, as gel chromatography zymolyte;
3. RP-HPLC is purified and structure detection:GS-III is made into 100 μ g/mL solution with distilled water, utilizes RP-HPLC(Enter
The μ L of sample amount 15;Chromatographic column KromasilC18(250mm × 4.6mm, 5 μm);Mobile phase:40% acetonitrile;Elution speed 1.0mL/min;
Ultraviolet detection wavelength 280nm)Purified, hypolipidemic activity oligopeptides is prepared according to hypolipidemic activity(Fig. 3), examined through RP-HPLC
Survey as simple spike, it is Gly-Ile-Glu-Trp-Ala (GIEWA) to determine amino acid sequence using protein/polypeptide sequenator,
ESI-MS measure molecular weight is 574.64Da(Fig. 4).
Obtained brown croaker air bladder reducing blood lipid oligopeptides Gly-Ile-Glu-Trp-Ala (GIEWA) is subjected to activity rating, as a result
Show:Gly-Ile-Glu-Trp-Ala (GIEWA) can reduce oleic acid(OA)The HepG2 lipid within endothelial cells accumulation of induction(Fig. 5, figure
6th, Fig. 7);To T-CHOL under 1-10 μM of concentration(TC), triglycerides(TG)In dose dependent decomposition(Fig. 8, figure
9);Gly-Ile-Glu-Trp-Ala (GIEWA) can be tuned into aliphatic radical down because of SREBP-1c, SREBP-2, FAS, ACC and HMGR
Expression(Figure 10, Figure 11);Westernblot experiments show that Gly-Ile-Glu-Trp-Ala (GIEWA) can suppress into fat weight
Want the protein expression level of factor S REBP-1c, ACC(Figure 12), activation LXR α and LXR β expression(Figure 13).Therefore, Gly-
Ile-Glu-Trp-Ala (GIEWA) can be tuned into aliphatic radical down because and can promotes fatty acid beta oxidation, reduces in terms of lipid-loweringing mechanism
The generation of lipid, lipid lowering level are more than lipid generative capacity, have good Lipid-lowering activities.
Routine operation in operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
Sequence table
<110>Zhejiang Ocean university
<120>A kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 5
<212> PRT
<213>Brown croaker (Miichthys miiuy)
<400> 2
Gly Ile Glu Trp Ala
1 5
Claims (8)
- A kind of 1. reducing blood lipid pentapeptide for coming from brown croaker air bladder, it is characterised in that:The reducing blood lipid pentapeptide for coming from brown croaker air bladder Amino acid sequence is:Gly-Ile-Glu-Trp-Ala (GIEWA), molecular weight 574.64Da.
- A kind of 2. reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 1, it is characterised in that:Described comes from catfish The preparation method of the reducing blood lipid pentapeptide of fish air bladder is:Pretreatment:Brown croaker air bladder is cleaned, after homogenate to pasty state, according to solid-liquid ratio 1g:10-15mL adds NaOH solution immersion, Filtering, is washed to neutrality, according to solid-liquid ratio 1g with distillation:5-8mL adds aqueous isopropanol degreasing, filtering, solids distillation Water rinses isopropanol well, dries, obtains degreasing brown croaker air bladder;Enzymolysis:Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5-8mL adds distilled water, adjusts pH value, adds alkali protease, enzyme Solution, enzymolysis terminated after 90-100 DEG C of enzyme deactivation 10-15 minute, were cooled to room temperature after 15-20 points of 12000-12500rmp centrifugations Clock, supernatant is collected, obtains brown croaker air bladder enzymolysis liquid;Extraction:Brown croaker air bladder enzymolysis liquid is classified through the milipore filter that molecular cut off is 10kDa and 3kDa, collects retention point Component of the son amount less than 3kDa, obtains ultrafiltration enzymolysis liquid;By ultrafiltration enzymolysis liquid successively through ion exchange chromatography, gel column layer Analysis and RPLC(RP-HPLC)Purifying, obtains brown croaker air bladder reducing blood lipid pentapeptide.
- A kind of 3. preparation method of reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 2, it is characterised in that:Institute The concentration of NaOH solution is 0.1-0.12mol/L in the pre-treatment step stated, and soaking temperature is 4-6 DEG C, soak time 12-15 Hour, the mass fraction of aqueous isopropanol is 10-12%, and degreasing time is 36-48 hours.
- 4. a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 2 and its application, it is characterised in that:It is described The addition of alkali protease in enzymolysis step be air bladder weight 2-3%, enzyme activity >=2.0 × 10 of alkali protease5U/ G, hydrolysis temperature are 50-60 DEG C, pH 9-10, and enzymolysis time is 3-4 hours.
- 5. a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 2 and its application, it is characterised in that:It is described Extraction step in ion exchange chromatography be:It is the molten of 45-55mg/mL that ultrafiltration enzymolysis liquid is made into concentration with distilled water Liquid, 3-4 DEG C, 12000-12500r/min centrifugation 15-20 minutes, remove insoluble impurities;Supernatant is taken to be added to DEAE-52 the moon Ion exchange chromatography post, eluted, collected with distilled water, 0.1mol/L, 0.5mol/L and 1mol/L NaCl solution successively 0.5mol/LNaCl elution fractions, as ion-exchange chromatography zymolyte.
- 6. a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 2 and its application, it is characterised in that:It is described Gel filtration chromatography in extraction step is:It is 45-55mg/mL's that ion-exchange chromatography zymolyte, which is dissolved in distilled water to be made into concentration, Solution, 3-4 DEG C, 12000-12500r/min centrifugation 15-20 minutes, remove insoluble impurities;Supernatant is taken to be coagulated by glucan Glue SephadexG-25 column chromatography for separation, is eluted with ultra-pure water, and elution group is collected according to the absorbance curve under 280nm Point, wherein, the most strong component of hypolipidemic activity is gel chromatography zymolyte.
- 7. a kind of reducing blood lipid pentapeptide for coming from brown croaker air bladder according to claim 1 and its application, it is characterised in that:Institute Stating the purifying of the RP-HPLC in extraction step is:Gel chromatography zymolyte is made into 80-100 μ g/mL solution with distilled water, profit Purified with RP-HPLC, RP-HPLC purification conditions are:Sample size is 10-15 μ L;Chromatographic column KromasilC18(250mm× 4.6mm, 5 μm);Mobile phase is 40% acetonitrile;Elution speed is 0.8-1.0mL/min;Ultraviolet detection wavelength is 280nm;According to each The screening of component hypolipidemic activity obtains reducing blood lipid pentapeptide Gly-Ile-Glu-Trp-Ala (GIEWA).
- A kind of 8. application for the reducing blood lipid pentapeptide for coming from brown croaker air bladder, it is characterised in that:Described reducing blood lipid pentapeptide is as drop blood The application of fat active component, health product and food additives.
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