CN105273059A - Octopus calcium chelating protein peptide and preparation method thereof - Google Patents
Octopus calcium chelating protein peptide and preparation method thereof Download PDFInfo
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Abstract
The invention provides octopus calcium chelating protein peptide and a preparation method thereof. The preparation method is characterized by using the octopus protein as the raw material and obtaining the purified specific metal Ca chelating peptide through enzymolysis with alkaline protease and flavourzyme and then through separation and purification. The amino acid complete sequence of the peptide is dfegae. The prepared metal chelating peptide can be used for producing a novel metal Ca supplement, namely protein peptide-calcium, has a unique chelating mechanism and transport mechanism, is easy to absorb, is safe and nontoxic, is low in price, can be used for simultaneously supplementing amino acids and calcium and is a first choice for the Ca supplement. The invention provides a new idea for application of the octopus protein.
Description
Technical field
The present invention relates to a kind of octopus calcium chelating protein peptide and preparation method thereof, relate more specifically to a kind ofly utilize Sumizyme MP and the flavor protease two step Ca chelating peptide prepared of enzymolysis octopus albumen respectively, belong to biological technical field.
Background technology
In the cultivation and the course of processing of octopus, a large amount of octopus residues is piled up by as waste disposal, rots smelly, even rotten, and causes economic loss, and causes the problems such as serious environmental pollution.Octopus is as the high-grade marine products of one; carrying out in the course of processing; the ratio of its discarded tankage can account for and reach more than 45%; comprise internal organ, cartilage, eyeball, neck etc.; and contain rich in protein, amino acid and some activeconstituentss in these tankage as taurine, carnitine etc., be but all underutilized.At present, mostly be both at home and abroad the research in the vital sign of octopus and information substance etc., and about the exploitation of octopus residue, and the report that the research and development of byproduct are probed into is very few.Along with bioactive peptide research and the rise of product development, we find that octopus protein source biologically active peptides has high nutritive value, edible safety and physiological hygiene function, can be used as functional foodstuff or foodstuff additive and even pharmaceutical applications in people's daily life.
At present, it is global nutrition problem that calcium constituent lacks, and our people is due to based on vegetable diet, and the phenomenon of calcium deficiency is more serious, the important topic become in China's dietary nutrition research of therefore replenishing the calcium.Traditional inorganic calcium salt, as calcium carbonate, calcium phosphate, calcium chloride etc., have certain destruction to supporting one's family, biological value is lower; Organic calcium salt, as citrate of lime, calcium lactate, calisanin etc., although calcium absorptivity increases, the molten mistake of its solubleness great Yi, and price is high.Research shows, protein peptide chelating calcium, due to the chelating system of its uniqueness and transporting mechanism, is easily absorbed, safety non-toxic, price are low, can supplement polypeptide and calcium simultaneously, and become first-selection of replenishing the calcium.Therefore, how to obtain the peptide with metal-chelating calcium activity, just become the research direction that preparation new calcium supplement are urgent.
Summary of the invention
In order to solve the problem, the invention provides a kind of octopus calcium chelating protein peptide and preparation method thereof, metal Ca sequestering activity is realized efficiently.
For achieving the above object, the present invention adopts following technical scheme:
A kind of metal Ca chelating peptide is six peptides be made up of 6 amino acid.The aminoacid sequence of described polypeptide is: dfegae.
A preparation method for metal Ca chelating peptide, with octopus albumen for raw material, adopt Sumizyme MP and flavor protease is composite carries out enzymolysis to it, separation and purification, lyophilize obtain metal chelating peptide.
Described enzymatic hydrolysis condition is:
The peak enzymolysis-ability condition of Sumizyme MP enzymolysis octopus residue is solid-liquid ratio 1:6, enzymolysis time 5.5h, temperature 51 DEG C; On the basis of Sumizyme MP enzymolysis, then add the further enzymolysis of flavor protease.The mass ratio of Sumizyme MP and flavor protease is 2:1.
Described enzyme is Sumizyme MP and flavor protease, and the optimal conditions of two enzyme fractional hydrolysis is: after Sumizyme MP enzymolysis 5.5h, adds flavor protease and continues enzymolysis 3.5h.
The concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARLDEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant to be concentration gradient the be 0.02mol/LpH9.0 containing 0-0.5MNaCl, flow velocity is 0.5mL/min, and elution peak is measured under 214nm; Collect the peak with the highest Ca sequestering activity, then be separated by SephadexG-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3mL/min, and elution peak measures under 214nm; Collect the peak with the highest Ca sequestering activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is with 0-90%(v/v) acetonitrile solution is as elutriant gradient elution, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, elution time is 45min, flow velocity is 2mL/min, collect and there is the peak of the highest metal Ca sequestering activity and retention time is the elution peak of 23.361min, there is the highest metal Ca sequestering activity, obtain described metal chelating peptide.
The concrete steps that the present invention takes are as follows:
(1) optimization of octopus proteolysis condition
The octopus albumen that this technology adopts is from Dongshan Bo Guangtianxing food limited-liability company (Fujian China), and enzyme believes Bioisystech Co., Ltd (Chinese Tianjin) purchased from Novi.Adopt experiment of single factor, respectively four enzymolysis factors are investigated, be 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).Take certain mass octopus protein dissolution in distilled water, then with 2mol/LNaOH by its pH regulator to 8.0.First this solution water-bath is heated to and needs temperature, then add the enzyme of respective amount again by different enzyme-substrate proportionings, start reaction according to the predetermined reaction times.Then go out enzyme 10 minutes again in boiling water bath, centrifugal 10 minutes of 10000rpm again after cooling.After supernatant collection, respectively metal Ca sequestering activity is measured, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition obtaining the enzymolysis solution with maximum metal sequestering activity is: concentration of substrate 5%, and enzymolysis pH is 8.0, peak enzymolysis-ability condition is solid-liquid ratio 1:6, enzymolysis time 5.5h, temperature 51 DEG C.After Sumizyme MP enzymolysis 5.5h, add flavor protease and continue enzymolysis 3.5h, concentration of substrate 5%, enzymolysis pH is 8.0, peak enzymolysis-ability condition is solid-liquid ratio 1:6, temperature 51 DEG C.
(2) separation of enzymolysis product, purifying
First under optimum enzymolysis condition, carry out enzymolysis, the enzymolysis product obtained is first through TOYOPEARLDEAE-650M anion-exchange chromatography (long 100cm, external diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be concentration gradient the be 0.02mol/LpH9.0 of the NaCl of 0-0.5M, flow velocity is 0.5mL/min, and elution peak is measured under 214nm; Collect the peak with the highest Ca sequestering activity, then use SephadexG-25 gel filtration chromatography (long 50cm, external diameter 1.6cm) to be separated, elutriant is deionized water, and flow velocity is 0.3mL/min, and elution peak measures under 214nm; Collect the peak with the highest Ca sequestering activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is with 0-90%(v/v) acetonitrile solution is as elutriant gradient elution, flow velocity is 2mL/min, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, elution time is 45min, collect and there is the peak of the highest metal Ca sequestering activity and retention time is the elution peak of 23.361min, there is the highest metal Ca sequestering activity, obtain described metal chelating peptide.
(3) test of metal chelating activity
The test of calcium sequestering activity is the sequestering action of metal chelating peptide to calcium ion.The present invention is by the CaCl of 1mL5mmol/L
2add in tool plug test tube with the phosphate buffered saline buffer (pH8.0) of 2mL0.2mol/L, then add 1mL white protein peptide solution, be placed in thermostatically heating shaking bath 37 DEG C of incubation 2h, the centrifugal 10min of 10000r/min normal temperature after taking out.Get 1mL supernatant liquor, add o-cresolphthalein nitrite ion 5mL, shake up.Measure light absorption value in spectrophotometer 570nm place after placing 10min, numerical value is substituted in typical curve and calculate solubility calcium binding capacity.
The making of typical curve: the accurate Ca working fluid (10ug/mL) 0,0.2,0.4 of label taking respectively, 0.6,0.8,1.0mL is in 10mL test tube, add deionized water 1.0 respectively, 0.8,0.6,0.4,0.2,0mL, adds o-cresolphthalein nitrite ion 5mL, shake up, after placing 10min, measure light absorption value in spectrophotometer 570nm place.With solubility calcium content (ug/mL) for X-coordinate, light absorption value is that ordinate zou is figure, obtains typical curve formula and is: y=0.0973x-0.0401, R2=0.9996.
(4) determined amino acid sequence
The overall amino acid sequence utilizing ESI mass spectrograph (WATERSMALDISYNAPTQ-TOFMS, WatersCo., U.S.A) to measure Specific metal Ca chelating peptide is: dfegae.
The present invention possesses the action site with calcium metal ion chelating based on polypeptide, compound that can be stable with its formation, and polypeptide-metal Ca inner complex has unique chelating system and transporting mechanism, easily absorbed, can be supplemented simultaneously the theoretical basis of amino acid and metal, to come from the octopus albumen of octopus for starting material, controlled by the cutting condition of Sumizyme MP and flavor protease, cutting preparation has the peptide of high metal Ca sequestering activity, and metal Ca sequestering activity is realized efficiently.The present invention is that the application of octopus albumen provides new approaches.
Accompanying drawing explanation
The RP-HPLC-C18 color atlas of Fig. 1 purifying octopus protein source metal chelating peptide.
Embodiment
embodiment 1
preparation method is as follows:
Take 5.0 grams of octopus protein dissolutions in 100ml distilled water, then with 2mol/LNaOH by its pH regulator to 8.0.First this solution water-bath is heated to 51 DEG C, then again according to concentration of substrate 5%, the condition enzymolysis 5.5h of solid-liquid ratio 1:6.After Sumizyme MP enzymolysis 5.5h, add flavor protease and continue enzymolysis 3.5h, concentration of substrate 5%, enzymolysis pH is 8.0, peak enzymolysis-ability condition is solid-liquid ratio 1:6, temperature 51 DEG C.
The mass ratio of Sumizyme MP and flavor protease is 2:1, and enzymolysis time is 7 hours.Then go out enzyme 10 minutes in boiling water bath, and centrifugal 10 minutes of 10000rpm again after cooling, collects supernatant liquor for subsequent use.
By supernatant liquor TOYOPEARLDEAE-650M anion-exchange chromatography (long 50cm, external diameter 1.6cm) be separated, elutriant is the phosphoric acid buffer that concentration gradient contains the 0.02mol/LpH9.0 of the NaCl of 0-0.5M, flow velocity is 0.5mL/min, collects each peak sample and measures metal Ca sequestering activity.
The elution peak with the highest metal Ca sequestering activity of TOYOPEARLDEAE-650M anion-exchange chromatography separation is carried out again to next step separation, with SepadexG-25 gel filtration chromatography (long 100cm, external diameter 2.0cm) collect the peak with the highest metal Ca sequestering activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is with 0-90%(v/v) acetonitrile solution is as elutriant gradient elution, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, elution time is 45min, flow velocity is 2mL/min, collect and there is the peak of the highest metal Ca sequestering activity and retention time is the elution peak of 23.361min, there is the highest metal Ca sequestering activity, obtain described metal chelating peptide.
Adopt o-cresolphthalein colorimetry, measure metal chelating peptide to the sequestering action of calcium ion.By the CaCl of 1mL5mmol/L
2add in tool plug test tube with the phosphate buffered saline buffer (pH8.0) of 2mL0.2mol/L, then add 1mL white protein peptide solution, be placed in thermostatically heating shaking bath 37 DEG C of incubation 2h, the centrifugal 10min of 10000r/min normal temperature after taking out.Get 1mL supernatant liquor, add o-cresolphthalein nitrite ion 5mL, shake up.Measure light absorption value in spectrophotometer 570nm place after placing 10min, numerical value is substituted in typical curve and calculate solubility calcium binding capacity.
Application TOYOPEARLDEAE-650M anion-exchange chromatography, SephadexG-25 molecular sieve, RP-HPLC RPLC etc. are separated means of purification, realize the high efficiency separation purifying of the octopus albumen metal chelating peptide of remarkable activity.
The Specific metal chelating peptide that purifying obtains has very high metal Ca sequestering activity, and as can be seen from Table 1, compared with enzymolysis solution, the metal chelating at C peak makes a concerted effort there has been large increase.
The metal chelating of the Specific metal chelating peptide of table 1 purifying is made a concerted effort
((WATERSMALDISYNAPTQ-TOFMS, WatersCo., U.S.A) measures the aminoacid sequence of Specific metal chelating peptide to utilize ESI mass spectrograph to the Specific metal chelating peptide of purifying.The aminoacid sequence of described metal chelating peptide is: dfegae.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCELISTING
<110> University of Fuzhou
<120> octopus calcium chelating protein peptide and preparation method thereof
<130>1
<160>1
<170>PatentInversion3.3
<210>1
<211>6
<212>PRT
<213> protein peptide sequence
<400>1
AspPheGluGlyAlaGlu
15
Claims (6)
1. an octopus calcium chelating protein peptide, is characterized in that: the aminoacid sequence of described peptide is: dfegae.
2. octopus calcium chelating protein peptide according to claim 1, is characterized in that: described metal is Ca.
3. the preparation method of an octopus calcium chelating protein peptide as claimed in claim 1, it is characterized in that: with octopus albumen for raw material, adopt Sumizyme MP and flavor protease two step to carry out enzymolysis to it respectively, separation and purification, lyophilize obtain metal chelating peptide.
4. the preparation method of octopus calcium chelating protein peptide according to claim 3, is characterized in that: the peak enzymolysis-ability condition of described Sumizyme MP enzymolysis octopus residue is solid-liquid ratio 1:6, enzymolysis time 5.5h, temperature 51 DEG C; On the basis of Sumizyme MP enzymolysis, then add the further enzymolysis of flavor protease, the mass ratio of Sumizyme MP and flavor protease is 2:1.
5. the preparation method of octopus calcium chelating protein peptide according to claim 3, it is characterized in that: described enzyme is Sumizyme MP and flavor protease, the optimal conditions of two enzyme fractional hydrolysis is: after Sumizyme MP enzymolysis 5.5h, adds flavor protease and continues enzymolysis 3.5h.
6. the preparation method of octopus calcium chelating protein peptide according to claim 3, it is characterized in that: the concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARLDEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant to be concentration gradient the be 0.02mol/LpH9.0 containing 0-0.5MNaCl, flow velocity is 0.5mL/min, and elution peak is measured under 214nm; Collect the peak with the highest metal chelating activity, then be separated by SephadexG-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3mL/min, and elution peak measures under 214nm; Collect the peak with the highest metal chelating activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is with 0-90%(v/v) acetonitrile solution is as elutriant gradient elution, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, elution time is 45min, flow velocity is 2mL/min, collect and there is the peak of the highest metal Ca sequestering activity and retention time is the elution peak of 23.361min, there is the highest metal Ca sequestering activity, obtain described octopus calcium chelating protein peptide.
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| CN106632597A (en) * | 2017-02-08 | 2017-05-10 | 福州大学 | Marine-sourced calcium chelating peptide and preparation method thereof |
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| ATE139572T1 (en) * | 1990-04-09 | 1996-07-15 | Upjohn Co | METHOD FOR PURIFYING RECOMBINANT PROTEINS AND USEFUL PRODUCTS THEREFOR |
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| CN106632597A (en) * | 2017-02-08 | 2017-05-10 | 福州大学 | Marine-sourced calcium chelating peptide and preparation method thereof |
| CN106632597B (en) * | 2017-02-08 | 2020-09-01 | 福州大学 | Marine source calcium chelating peptide and preparation method thereof |
| CN106866785A (en) * | 2017-04-15 | 2017-06-20 | 福州大学 | A kind of 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 |
| 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 |
| CN110627896A (en) * | 2019-09-03 | 2019-12-31 | 华南农业大学 | Calcium chelating peptide and preparation method and application thereof |
| 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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