CN102628073A - Glycosylated lactalbumin polypeptide with antioxidant activity as well as preparation method and application of glycosylated lactalbumin polypeptide - Google Patents
Glycosylated lactalbumin polypeptide with antioxidant activity as well as preparation method and application of glycosylated lactalbumin polypeptide Download PDFInfo
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Abstract
The invention relates to a glycosylated lactalbumin polypeptide with antioxidant activity as well as a preparation method and an application of the glycosylated lactalbumin polypeptide. According to the invention, the method for preparing the glycosylated lactalbumin polypeptide with antioxidant activity comprises the following steps: after hydrolyzing lactalbumin into protein peptide by using alkaline protease, boiling up the protein peptide and inactivating the enzyme activity of the protein peptide; cooling the protein peptide; mixing the protein peptide with sugar; stirring and heating the mixture at a certain temperature; and after carrying out Maillard reaction on the mixture for a certain time, rapidly cooling and centrifuging the mixture so as to remove precipitates, wherein a supernatant liquor can be directly frozen and dried or frozen and dried after being ultra-filtered. The glycosylated lactalbumin polypeptide obtained by using the method provided by the invention has a stronger anti-oxidation effect and obtains stronger stability than a plurality of natural antioxidants; moreover, because the used raw materials are all edible foods, high safety is obtained.
Description
Technical field
The present invention relates to a kind of whey protein peptide and the sugared method of modifying that carries out Maillard reaction, specifically, the present invention relates to a kind of glycosylation lactalbumin polypeptide with anti-oxidant activity.The invention belongs to the foodstuff additive field.
Background technology
Albumen through with sugar carry out Maillard reaction can improve proteic many functional, as: water-soluble, thermostability, emulsifying property, oxidation-resistance etc. can be applied to fields such as foodstuff additive, healthcare products, pharmaceutical carrier.But it is more rare that the use protein polypeptide carries out the research of Maillard reaction.
Whey-protein itself just has certain antioxygenation; And the whey protein peptide antioxidant effect after the hydrolysis is improved, and is existing at present about lactalbumin hydrolysate Antioxidation Effects in iron catalysis Yelkin TTS lipid oxidation system, copper catalysis lipid oxidation system; Utilize whey-protein, ALA and beta-lact oglobulin to prepare anti-oxidation peptide in addition, and the whey hydrolyzate is applied to reduce in the pork bun of boiling the report of lipid oxidation in the cold storage procedure.
Since proteic glycosylation and proteic hydrolysis reaction all have the lifting antioxidant effect, with the two organic combination, will prepare oxidation resistant product efficiently so.
Summary of the invention
One of the object of the invention is to provide the method for the glycosylation lactalbumin polypeptide that a kind of preparation has anti-oxidant activity;
Two of the object of the invention is to provide a kind of glycosylation lactalbumin polypeptide with anti-oxidant activity that is prepared by the inventive method;
Three of the object of the invention is to provide the application of glycosylation lactalbumin polypeptide in the preparation inhibitor with anti-oxidant activity of the present invention.
In order to achieve the above object, the present invention has adopted following technical scheme:
A kind of method for preparing the glycosylation lactalbumin polypeptide of anti-oxidant activity of the present invention is characterized in that may further comprise the steps:
(1) lactoalbumin soln is heat-treated;
(2) the cooling back of the lactoalbumin soln after the thermal treatment adds the Sumizyme MP reaction that is hydrolyzed;
(3) the hydrolysis reaction product adds sugar heating carrying out glycosylation through the enzyme that goes out;
(4) cooling, the centrifuging and taking supernatant concentrates, and lyophilize promptly gets.
In the present invention, preferred, the concentration of the lactoalbumin soln described in the step (1) is 2-10% (w/w), is preferably 5% (w/w); Described thermal treatment is: lactoalbumin soln was handled 3-8 minute under 90-98 ℃ of temperature; Preferred, lactoalbumin soln was handled 5 minutes under 95 ℃ of temperature, then cooling.
In the present invention; Preferably; The volume of the Sumizyme MP that is added in the step (2) and the weight ratio of whey protein substrate are (1～3): 100, and the volume that just is equivalent to the needed Sumizyme MP of whey protein substrate of every 100g is 1-3mL, is preferably 2mL.
In a specific embodiment of the present invention; The mass concentration of the lactoalbumin soln that is adopted is 5% (w/w); If the gross weight of lactoalbumin soln is 1000g; The whey protein substrate that is then wherein contained is 50g, is calculating in 2: 100 according to the volume of the Sumizyme MP that is added and the weight ratio of whey protein substrate, and the volume that should add Sumizyme MP is 1ml.Wherein, the enzyme activity of basic protein is 1-20 ten thousand U/g.
Sumizyme MP is to make the 2709 withered grass bar mikrobes that educate through bacterium protoplastis mutafacient system to reach the refining a kind of proteolytic ferment that forms through submerged fermentation, extraction; Belong to the outer high alkaline proteases of a kind of Serine born of the same parents; It can generate polypeptide or amino acid by protein hydrolysate molecule peptide chain, has the ability of stronger decomposing protein.Staple is a subtilisin, is a kind of restriction endonuclease, and catalytic site is a Serine, and molecular weight is about 27300Da.
In the present invention, preferred, the hydrolysis reaction described in the step (2) carries out under following condition: hydrolysising reacting temperature is 50-60 ℃, and the pH value is 8.0-9.0, and hydrolysis time is 4-6 hour; Preferred, in the reaction that is hydrolyzed of following condition: hydrolysising reacting temperature is 65 ℃, and the pH value is 8.5, and hydrolysis time is 5 hours.The present invention finds through test, adopts the reaction that is hydrolyzed of this hydrolysising condition, and degree of hydrolysis (DH) can reach 35.30%.
In the present invention; Preferably, the glycosylation described in the step (3) carries out under following condition: the polypeptide mixed solution that pH value 8.0～9.0, hydrolysis obtain adds sugar; The final concentration that makes the sugar of adding is 6～10% (w/w); Stirring heating is 3～5 hours under 50～90 ℃ of temperature, and wherein, described sugar is preferably glucose.
In the present invention, preferred, the cf-that centrifugation step adopted described in the step (4) is 1000～1200 * g, and the centrifugal time is 10～15min.
In the present invention, preferred, also be included in after glycosylation finishes; Cooling, obtains the polypeptide mixture of molecular weight between 800-5000Da at centrifugal before glycosylated product further the separation; The mixture that obtains is again through overcooling, and the centrifuging and taking supernatant concentrates; Lyophilize promptly gets.
Preferably, described glycosylated product is further separated, employing be the method for ultrafiltration, preferably adopt hollow-fibre membrane to carry out, membrane pore size is respectively 800Da, 5000Da, the temperature of ultrafiltration is 20 ℃, pressure is 0.1MPa.
Adopt hollow fiber ultrafiltration membrane that glycation product is separated, resulting product mainly contains the glycosylation lactalbumin polypeptide that molecular weight is 800-5000Da, has high anti-oxidant activity and the ability of removing radical.
The present invention also provides the glycosylation that is prepared by method of the present invention lactalbumin polypeptide, and preferred, described glycosylation lactalbumin polypeptide mainly is made up of the mixing glycosylation lactalbumin polypeptide of molecular weight between 800-5000Da.
Further, the present invention also provides the application of described glycosylation lactalbumin polypeptide in the preparation inhibitor.
Experiment showed, glycosylation lactalbumin polypeptide of the present invention, its anti-oxidant activity is stable, is that 50-95 ℃, pH value are can both keep highly stable anti-oxidant activity under the effect of 4-8, illumination and sanitas (Sodium Benzoate) in Heating temperature.
Compared with prior art, preparation method of the present invention is simple to operate, compares safe and reliablely with other synthetized oxidation preventive agents, is fit to suitability for industrialized production, can increase substantially the utilization ratio of whey-protein resource, improves the level of whey comprehensive utilization.Prepared glycated milk white protein anti-oxidation peptide anti-oxidant activity is high, and stability is strong.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Experiment material is bought the source:
Sumizyme MP: available from the gloomy Bioisystech Co., Ltd in Wal, Xi'an; PIN: EC0063, enzyme activity 1-20 ten thousand U/g;
Whey-protein: buy from U.S. Hilmar company; PIN: 9410 whey isolate proteins, protein content 92.8%.
Hollow-fibre membrane: be U.S. FS-TFC fluid film
Embodiment 1
Lactoalbumin soln concentration is 5% (w/w), at 95 ℃, after the thermal treatment of 5min; Drop to 60 ℃ of temperature, add Sumizyme MP and be hydrolyzed, the volume of the Sumizyme MP that is added and the weight ratio of whey protein substrate are 2: 100; The volume that just is equivalent to the needed Sumizyme MP of whey protein substrate of every 100g is 2mL; In temperature is that 60 ℃, pH value are under 8.5 the condition, hydrolysis 5h (through detecting, degree of hydrolysis (DH) has reached 35.30%); Hydrolysate through boiling the enzyme that goes out, is added glucose again, make the final concentration of the glucose of adding reach 8% (w/w), at 90 ℃ of following stirring heating 3h, the cooling rapidly of reaction back through the centrifugal 10min of 1200 * g, is removed deposition, supernatant concentration, and lyophilize promptly gets.
Embodiment 2
Lactoalbumin soln concentration is 2% (w/w), at 90 ℃, after the thermal treatment of 8min; Drop to 60 ℃ of temperature; Add Sumizyme MP and be hydrolyzed, the volume of the Sumizyme MP that is added and the weight ratio of whey protein substrate are 2: 100, and the volume that just is equivalent to the needed Sumizyme MP of whey protein substrate of every 100g is 2mL; In temperature is that 50 ℃, pH value are under 8.0 the condition, hydrolysis 4h; Hydrolysate through boiling the enzyme that goes out, is added glucose again, make the final concentration of the glucose of adding reach 6% (w/w), at 60 ℃ of following stirring heating 4h, the cooling rapidly of reaction back through the centrifugal 15min of 1000 * g, is removed deposition, supernatant concentration, and lyophilize promptly gets.
Embodiment 3
Lactoalbumin soln concentration is 5% (w/w), at 95 ℃, after the thermal treatment of 5min; Drop to 60 ℃ of temperature, add Sumizyme MP and be hydrolyzed, the volume of the Sumizyme MP that is added and the weight ratio of whey protein substrate are 2: 100; The volume that just is equivalent to the needed Sumizyme MP of whey protein substrate of every 100g is 2mL; Under the condition of 60 ℃ of temperature, pH value 8.5, hydrolysis 5h (through detecting, degree of hydrolysis (DH) has reached 35.30%); Hydrolysate through boiling the enzyme that goes out, is added glucose again, make the final concentration of the glucose of adding reach 8% (w/w); 90 ℃ of following stirring heating 3h, the cooling rapidly of reaction back is through the centrifugal 15min of 1200 * g; Remove deposition, supernatant carries out lyophilize again through ultrafiltration and processes, and ultrafiltration adopts hollow-fibre membrane to carry out; Membrane pore size is respectively 800Da, 5000Da, and the temperature of ultrafiltration is 20 ℃, and pressure is 0.1MPa.Mainly being contained molecular weight is the mixture of the glycosylation lactalbumin polypeptide between the 800-5000Da.
Embodiment 4
Lactoalbumin soln concentration is 10% (w/w), at 98 ℃, after the thermal treatment of 8min; Drop to 60 ℃ of temperature; Add Sumizyme MP and be hydrolyzed, the volume of the Sumizyme MP that is added and the weight ratio of whey protein substrate are 3: 100, and the volume that just is equivalent to the needed Sumizyme MP of whey-protein of every 100g is 3mL; Under the condition of 60 ℃ of temperature, pH value 9.0, hydrolysis 6h; Hydrolysate through boiling the enzyme that goes out, is added glucose again, make the final concentration of the glucose of adding reach 10% (w/w); 75 ℃ of following stirring heating 4h, the cooling rapidly of reaction back is through the centrifugal 15min of 1000 * g; Remove deposition, supernatant carries out lyophilize again through ultrafiltration and processes, and ultrafiltration adopts hollow-fibre membrane to carry out; Membrane pore size is respectively 800Da, 5000Da, and the temperature of ultrafiltration is 20 ℃, and pressure is 0.1MPa.Mainly being contained molecular weight is the mixture of the glycosylation lactalbumin polypeptide between the 800-5000Da.
The anti-oxidant activity test of Test Example 1 glycosylation lactalbumin polypeptide of the present invention
1, supplies the prepared glycosylation lactalbumin polypeptide of test agent: embodiment 1 and embodiment 3;
2, TP and result:
Anti-oxidant activity carries out in Yelkin TTS lipid oxidation system (being called for short the Lipsome oxidation system); Lipsome oxidation system compound method is: take by weighing 0.89472g Repone K; 0.0776g Histidine, thin up, are transferred its pH value to 6.8 to 100mL; The soybean lecithin 1g that gets purity 20% again is dissolved in Repone K-Histidine buffered soln, processes the liposome of 2mg/mL.Get the liposome 5mL of 10 times of dilutions when carrying out anti-oxidant the test; Add sample 1mL; And adding 0.1mL 50mm Fecl3 solution and 0.1mL 10mm xitix salts solution, 37 ℃ of water-bath 1h, the TBARS of glycated milk white protein anti-oxidation peptide (thiobarbituricacid value) 2.03mg/L; Compare reduction 40-50% with whey-protein, compare with the whey protein peptide after the hydrolysis and reduce 10-20%; Reducing power (FRAP value) 1873 μ moL/L compare with whey-protein and to have improved 6-8 doubly, compare with the whey protein peptide after the hydrolysis and have improved 2-3 doubly; To the clearance rate 84.23% of ABTS radical, compare raising 7-8 with whey-protein doubly, compare with the whey protein peptide after the hydrolysis and improved 3-4 doubly; Hydroxyl clearance rate 59.31% is compared raising 8-10 doubly with whey-protein, compare with the whey protein peptide after the hydrolysis and improved 4-5 doubly.
Resulting glycated milk white protein anti-oxidation peptide is divided into different three sections of range of molecular weight distributions through hollow fiber ultrafiltration membrane: below the 0.8KDa, 0.8-5KDa is more than the 5KDa.Wherein the 0.8-5KDa effect is best, TBARS (thiobarbituricacid value) 1.86 (mg/L); Reducing power (FRAP value) 2118 μ moL/L; Clearance rate 99.08% to the ABTS radical; Hydroxyl clearance rate 66.23%, Cu
2+Sequestering power reaches 88.28%, Fe
2+Sequestering power reaches 41.47%.
The anti-oxidant activity stability test of Test Example 2 glycosylation lactalbumin polypeptides of the present invention
Supply test agent: the glycosylation lactalbumin polypeptide that embodiment 1-4 is prepared;
In Heating temperature is that 50-95 ℃, pH value are to measure anti-oxidant activity (reducing power FRAP value is to the clearance rate of ABTS radical) under the effect of 4-8, illumination and sanitas (Sodium Benzoate).
The result shows the glycosylation lactalbumin polypeptide under above condition changes, and anti-oxidant activity changes minimum, still has stronger anti-oxidant activity.
Claims (10)
1. method for preparing the glycosylation lactalbumin polypeptide with anti-oxidant activity is characterized in that may further comprise the steps:
(1) lactoalbumin soln is heat-treated;
(2) the cooling back of the lactoalbumin soln after the thermal treatment adds the Sumizyme MP reaction that is hydrolyzed;
(3) the hydrolysis reaction product adds sugar behind the enzyme that goes out, and glycosylation is carried out in heating;
(4) cooling, the centrifuging and taking supernatant concentrates, and lyophilize promptly gets.
2. according to the described method of claim 1, it is characterized in that: the concentration of the lactoalbumin soln described in the step (1) is 2-10% (w/w), is preferably 5% (w/w); Described thermal treatment is: lactoalbumin soln was handled 3-8 minute under 90-98 ℃ of temperature; Preferably, lactoalbumin soln was handled 5 minutes under 95 ℃ of temperature, then cooling.
3. according to the described method of claim 1; It is characterized in that: the volume of the Sumizyme MP that is added in the step (2) and the weight ratio of whey protein substrate are (1～3): 100; The volume that is the needed Sumizyme MP of whey protein substrate of every 100g is 1-3ml, is preferably 2ml.
4. according to the described method of claim 1, it is characterized in that: the hydrolysis reaction described in the step (2) carries out under following condition: hydrolysising reacting temperature is 50-60 ℃, and the pH value is 8.0-9.0, and hydrolysis time is 4-6 hour; Preferably, in the reaction that is hydrolyzed of following condition: hydrolysising reacting temperature is 65 ℃, and the pH value is 8.5, and hydrolysis time is 5 hours.
5. according to the described method of claim 1; It is characterized in that: the glycosylation described in the step (3) carries out under following condition: pH value 8.0～9.0; The polypeptide mixed solution that hydrolysis obtains adds sugar, and the final concentration that makes the sugar of adding is 6～10% (w/w), and stirring heating is 3～5 hours under 50～90 ℃ of temperature; Wherein, described sugar is glucose.
6. according to the described method of claim 1, it is characterized in that: the cf-that centrifugation step adopted described in the step (4) is 1000～1200 * g, and the centrifugal time is 10～15min.
7. according to the described method of claim 1, after it is characterized in that also being included in glycosylation and finishing, glycosylated product is further separated, obtain the polypeptide mixture of molecular weight between 800-5000Da.
8. according to the described method of claim 7, it is characterized in that: described glycosylated product is further separated, employing be the method for ultrafiltration; The preferred hollow-fibre membrane that adopts carries out; Membrane pore size is respectively 800Da, 5000Da, and the temperature of ultrafiltration is 20 ℃, and pressure is 0.1MPa.
9. by the prepared glycosylation lactalbumin polypeptide that obtains of each described method of claim 1-8, preferred, described glycosylation lactalbumin polypeptide mainly is made up of the mixing glycosylation lactalbumin polypeptide of molecular weight between 800-5000Da.
10. the application of the described glycosylation lactalbumin polypeptide of claim 9 in the preparation inhibitor.
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| CN103805664A (en) * | 2014-03-06 | 2014-05-21 | 渤海大学 | Low-sensitivity glycosylation whey protein hydrolysate and preparation method thereof |
| CN103805664B (en) * | 2014-03-06 | 2017-04-05 | 渤海大学 | A kind of subsensitivety glycated milk albumin hydrolysate and preparation method thereof |
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| CN113061170B (en) * | 2019-12-16 | 2023-04-07 | 青海师范大学 | Alfalfa antioxidant glycoprotein and preparation method and application thereof |
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