CN105273059B - A kind of octopus calcium chelating protein peptides and preparation method thereof - Google Patents
A kind of octopus calcium chelating protein peptides and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of octopus calcium to chelate protein peptides and preparation method thereof, using octopus albumen as raw material, is digested by alkali protease and flavor protease, and using isolating and purifying the Specific metal Ca chelating peptides purified, overall amino acid sequence is：dfegae.Metal chelating peptide prepared by the present invention can be used for producing novel metal Ca replenishers-protein peptides-calcium, with unique chelating system and transporting mechanism, is easily absorbed, is safe and non-toxic, price is low, can supplement amino acid and calcium simultaneously, being the first choice of calcium complement agent.The present invention provides a new approaches for the application of octopus albumen.
Description
Technical field
The present invention relates to a kind of octopus calcium to chelate protein peptides and preparation method thereof, has related more specifically to a kind of using alkaline
Protease and two step of flavor protease digest the Ca chelating peptides of octopus albumen preparation respectively, belong to biotechnology.
Background technology
In the cultivation and process of octopus, a large amount of octopus residue is accumulated as waste disposal, is rotted smelly,
The problems such as even going bad, and cause economic loss, and causing serious environmental pollution.Octopus as a kind of high-grade marine products,
During being processed, the ratio of discarded leftover bits and pieces can be accounted for up to 45% or more, including internal organ, cartilage, eyeball, neck etc., and this
Containing abundant protein, amino acid and some active constituents such as taurine, carnitine etc. in a little leftover bits and pieces, all do not obtain but fully
It utilizes.Currently, be mostly the research of the vital sign and information substance etc. about octopus both at home and abroad, and about octopus residue
Utilization and its byproduct the report probed into of research and development it is very few.As active peptide studies the rise with product development, we
It was found that octopus protein sources biologically active peptide has the function of high nutritive value, edible safety and physiological hygiene, work(can be used as
In energy property food or food additives or even pharmaceutical applications to people's daily life.
Currently, it is global nutrition problem that calcium constituent, which lacks, our people due to based on vegetable diet, calcium deficiency
Phenomenon is more serious, therefore the important topic in being studied as China's diet nutritional of replenishing the calcium.Traditional inorganic calcium salt, such as carbonic acid
Calcium, calcium phosphate, calcium chloride etc. have certain destruction, biological value relatively low vitamin；Organic calcium salt, as calcium citrate,
Calcium lactate, calisanin etc., although calcium absorptivity increases, the mistake readily soluble greatly of its solubility, and price is high.Studies have shown that
Protein peptides chelate calcium due to its unique chelating system and transporting mechanism, are easily absorbed, is safe and non-toxic, price is low, can mend simultaneously
Polypeptide and calcium are filled, and as first choice of replenishing the calcium.Therefore, how to obtain with the active peptide of metal-chelating calcium, it is just novel as preparing
The urgent research direction of calcium complement agent.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of octopus calcium to chelate protein peptides and preparation method thereof, makes metal
Ca sequestering activities are efficiently realized.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of metal Ca chelating peptides are the hexapeptides being made of 6 amino acid.The amino acid sequence of the polypeptide is：
dfegae。
A kind of preparation method of metal Ca chelating peptides, using octopus albumen as raw material, using alkali protease and flavor albumen
Enzyme compounding digests it, isolates and purifies, is freeze-dried and obtains metal chelating peptide.
The enzymatic hydrolysis condition is：
The peak enzymolysis-ability condition that alkali protease digests octopus residue is solid-liquid ratio 1:6, enzymolysis time 5.5h, temperature 51
℃；On the basis of alkali protease digests, then adds flavor protease and further digest.Alkali protease and flavor protease
Mass ratio be 2:1.
The enzyme is alkali protease and flavor protease, and the optimal conditions of double enzyme fractional hydrolysis is：In alkali protease
After digesting 5.5h, flavor protease is added and continues to digest 3.5h.
It is described isolate and purify the specific steps are：Enzymolysis product is handed over first with TOYOPEARL DEAE-650M anion
Colour changing spectrum is detached, and eluent is that the phosphoric acid that concentration gradient is the 0.02 mol/L pH 9.0 containing 0-0.5 M NaCl is slow
Fliud flushing, flow velocity are 0.5 mL/min, and eluting peak is measured at 214 nm；The peak with highest Ca sequestering activities is collected, then
It is detached with Sephadex G-25 gel filtration chromatographies, eluent is deionized water, and flow velocity is 0.3 mL/min, eluting peak
It is measured at 214 nm；The peak with highest Ca sequestering activities is collected, RP-HPLC-C18 reversed phase high performance liquids are prepared using half
Phase chromatography is further detached again, and separation condition is to use 0-90%（v/v）Acetonitrile solution is as eluent gradient elution, until 90%
Acetonitrile and 10% water（v/v）Mixed liquor terminate, carry out gradient elution, elution time be 45 min, flow velocity be 2 mL/min, receive
Collect the eluting peak that peak, that is, retention time with highest metal Ca sequestering activities is 23.361 min, there is highest metal Ca chelas
Activity is closed, the metal chelating peptide is obtained.
The present invention, which takes, to be as follows：
(1) optimization of octopus proteolysis condition
Octopus albumen used by this technology comes from Dongshan Bo Guangtianxing food limited liability company（Fujian China）, enzyme
Believe Bioisystech Co., Ltd purchased from Novi（Chinese Tianjin）.Using experiment of single factor, four enzymolysis factors are carried out respectively
It investigates, respectively concentration of substrate（1%, 3%, 5% and 7%）, hydrolysis temperature（40,50,55 and 60 DEG C）, solid-liquid ratio（1:2、1:3、1:
4、1:5、1:6、1:7, w/v）And enzymolysis time（1,3,5,7,9 hours）.Weigh certain mass octopus protein dissolution in
In distilled water, its pH is then adjusted to 8.0 with 2mol/L NaOH.It first is heated to the solution water-bath to need temperature, then again
The enzyme of corresponding amount is added by different enzyme-substrate proportionings, starts to react according to the scheduled reaction time.Then again in boiling water bath
Enzyme deactivation 10 minutes, 10000rpm is centrifuged 10 minutes again after cooling.After supernatant collection, metal Ca sequestering activities are surveyed respectively
It is fixed, to determine optimum enzymolysis condition.Obtain that there is the enzymatic hydrolysis condition of the enzymolysis liquid of maximum metal sequestering activity to be：Concentration of substrate
5%, enzymolysis pH is 8.0, peak enzymolysis-ability condition is solid-liquid ratio 1:6, enzymolysis time 5.5h, 51 DEG C of temperature.In alkali protease enzyme
After solving 5.5h, flavor protease is added and continues to digest 3.5h, concentration of substrate 5%, enzymolysis pH is 8.0, peak enzymolysis-ability condition is material
Liquor ratio 1:6,51 DEG C of temperature.
(2) separation, purifying of enzymolysis product
It is first digested under optimum enzymolysis condition, it is cloudy that obtained enzymolysis product first passes through TOYOPEARL DEAE-650M
Ion-exchange chromatography（Long 100cm, outer diameter 2.0cm）It is detached, eluent is the 0.02 of the NaCl that concentration gradient is 0-0.5 M
The phosphate buffer of mol/L pH 9.0, flow velocity are 0.5 mL/min, and eluting peak is measured at 214nm；Collecting has most
The peak of high Ca sequestering activities, then with Sephadex G-25 gel filtration chromatographies（Long 50 cm, 1.6 cm of outer diameter）It is detached, is washed
De- liquid is deionized water, and flow velocity is 0.3 mL/min, and eluting peak is measured at 214 nm；Collecting, there is highest Ca chelatings to live
Property peak, prepare RP-HPLC-C18 reversed-phase high performance liquid chromatography using half is further detached again, and separation condition is to use 0-
90%（v/v）Acetonitrile solution is as eluent gradient elution, and flow velocity is 2 mL/min, until 90% acetonitrile and 10% water（v/v）Mixing
Liquid terminates, and carries out gradient elution, and elution time is 45 min, collects peak, that is, retention time with highest metal Ca sequestering activities
For the eluting peak of 23.361 min, there is highest metal Ca sequestering activities, obtain the metal chelating peptide.
(3) test of metal chelating activity
The test of calcium sequestering activity is chelation of the metal chelating peptide to calcium ion.The present invention is by 1 mL, 5 mmol/L
CaCl2With the phosphate buffer of 2 mL, 0.2 mol/L（pH 8.0）It is added in tool plug test tube, adds 1 mL albumins
Peptide solution is placed in 37 DEG C of incubation 2h in heated at constant temperature shaking bath, and 10000 r/min room temperature centrifuge 10 min after taking-up.Take 1
ML supernatants are added 5 mL of o-cresol phthalein developing solution, shake up.Suction is measured at 570 nm of spectrophotometer after placing 10 min
Numerical value is substituted into standard curve and calculates solubility calcium binding capacity by light value.
The making of standard curve：Standard Ca working solutions are taken respectively（10 ug/ mL）0,0.2,0.4,0.6,0.8,1.0 mL
In 10 mL test tubes, add 1.0,0.8,0.6,0.4,0.2,0 mL of deionized water respectively, 5 mL of o-cresol phthalein developing solution be added,
It shakes up, light absorption value is measured at 570 nm of spectrophotometer after placing 10 min.With solubility calcium content（ug/ mL）For horizontal seat
Mark, light absorption value are that ordinate does figure, and obtaining calibration curve formula is：Y=0.0973x -0.0401, R2=0.9996.
（4）Determined amino acid sequence
It is measured using ESI mass spectrographs (WATERS MALDI SYNAPT Q-TOF MS, Waters Co., U.S.A) special
The overall amino acid sequence of anisotropic metal Ca chelating peptides is：dfegae.
The present invention has the action site with calcium metal ion chelating based on polypeptide, is capable of the chemical combination of formed stabilization
Object, and polypeptide-metal Ca chelates have unique chelating system and transporting mechanism, are easily absorbed, can supplement amino acid simultaneously
Pass through alkali protease and flavor protease to come from the octopus albumen of octopus as raw material with the theoretical foundation of metal
Cutting condition controls, and cutting prepares the peptide with high metal Ca sequestering activities, and keeps metal Ca sequestering activities efficiently real
It is existing.The present invention provides a new approaches for the application of octopus albumen.
Description of the drawings
Fig. 1 purifies the RP-HPLC-C18 chromatograms of octopus protein sources metal chelating peptide.
Specific implementation mode
Embodiment 1
Preparation method is as follows：
5.0 grams of octopus protein dissolutions are weighed in 100ml distilled water, are then adjusted to its pH with 2mol/L NaOH
8.0.The solution water-bath is first heated to 51 DEG C, then according still further to concentration of substrate 5%, solid-liquid ratio 1:6 condition digests 5.5h.
After alkali protease digests 5.5h, flavor protease is added and continues to digest 3.5h, concentration of substrate 5%, enzymolysis pH is 8.0, most suitable enzyme
Solution condition is solid-liquid ratio 1:6,51 DEG C of temperature.
The mass ratio of alkali protease and flavor protease is 2:1, enzymolysis time is 7 hours.Then it goes out in boiling water bath
Enzyme 10 minutes, 10000rpm is centrifuged 10 minutes again after cooling, and it is spare to collect supernatant.
By supernatant TOYOPEARL DEAE-650M anion-exchange chromatographies（Long 50 cm, outer diameter 1.6cm）Divided
From eluent is the phosphate buffer of 0.02 mol/L pH 9.0 for the NaCl that concentration gradient contains 0-0.5M, flow velocity 0.5
ML/min collects each peak sample and measures metal Ca sequestering activities.
The elution with highest metal Ca sequestering activities to the separation of TOYOPEARL DEAE-650M anion-exchange chromatographies
Peak carries out the separation of next step again, with Sepadex G-25 gel filtration chromatographies（Long 100 cm, 2.0 cm of outer diameter）Collection has
The peak of highest metal Ca sequestering activities prepares RP-HPLC-C18 reversed-phase high performance liquid chromatography using half and is further detached again,
Separation condition is to use 0-90%（v/v）Acetonitrile solution is as eluent gradient elution, until 90% acetonitrile and 10% water（v/v）Mixing
Liquid terminates, and carries out gradient elution, and elution time is 45 min, and flow velocity is 2 mL/min, and collecting, there is highest metal Ca chelatings to live
Property peak, that is, retention time be 23.361 min eluting peak, there is highest metal Ca sequestering activities, obtain the metal chelating
Close peptide.
Using o-cresol phthalein colorimetric method, chelation of the metal chelating peptide to calcium ion is measured.By 1 mL, 5 mmol/L
CaCl2With the phosphate buffer of 2 mL, 0.2 mol/L（pH 8.0）It is added in tool plug test tube, adds 1 mL albumins
Peptide solution is placed in 37 DEG C of incubation 2h in heated at constant temperature shaking bath, and 10000 r/min room temperature centrifuge 10 min after taking-up.Take 1
ML supernatants are added 5 mL of o-cresol phthalein developing solution, shake up.Suction is measured at 570 nm of spectrophotometer after placing 10 min
Numerical value is substituted into standard curve and calculates solubility calcium binding capacity by light value.
Using TOYOPEARL DEAE-650M anion-exchange chromatographies, Sephadex G-25 molecular sieves, RP-HPLC reverse phases
High performance liquid chromatography etc. detaches means of purification, and that realizes the octopus albumen metal chelating peptide of remarkable activity efficiently separates purifying.
Purifying obtained Specific metal chelating peptide has very high metal Ca sequestering activities, as can be seen from Table 1, with enzyme
Liquid phase ratio is solved, the metal chelating resultant force at the peaks C has large increase.
The metal chelating resultant force for the Specific metal chelating peptide that table 1 purifies
To the Specific metal chelating peptide of purifying using ESI mass spectrographs ((WATERS MALDI SYNAPT Q-TOF MS,
Waters Co., U.S.A) measure Specific metal chelating peptide amino acid sequence.The amino acid sequence of the metal chelating peptide
For：dfegae.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of octopus calcium chelating protein peptides and preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
<213>Albumen peptide sequence
<400> 1
Asp Phe Glu Gly Ala Glu
1 5
Claims (1)
1. a kind of octopus calcium chelates protein peptides, it is characterised in that：The amino acid sequence of the peptide is：dfegae.
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| CN106632597B (en) * | 2017-02-08 | 2020-09-01 | 福州大学 | Marine source calcium chelating peptide and preparation method thereof |
| CN107056885A (en) * | 2017-04-15 | 2017-08-18 | 福州大学 | The method that two enzymes method prepares calcium chelating peptide |
| CN106866785A (en) * | 2017-04-15 | 2017-06-20 | 福州大学 | A kind of calcium chelating peptide and preparation method thereof |
| CN107788429A (en) * | 2017-11-19 | 2018-03-13 | 荣成海锐芯生物科技有限公司 | A kind of method that oral agents of replenishing the calcium are prepared using squid |
| CN110627896B (en) * | 2019-09-03 | 2022-02-18 | 华南农业大学 | Calcium chelating peptide and preparation method and application thereof |
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