CN115960210A - Method for preparing fishy smell-free collagen peptide by electroosmosis-ultrafiltration integrated membrane technology - Google Patents
Method for preparing fishy smell-free collagen peptide by electroosmosis-ultrafiltration integrated membrane technology Download PDFInfo
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- CN115960210A CN115960210A CN202211611472.1A CN202211611472A CN115960210A CN 115960210 A CN115960210 A CN 115960210A CN 202211611472 A CN202211611472 A CN 202211611472A CN 115960210 A CN115960210 A CN 115960210A
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Abstract
The invention relates to a method for preparing fishy smell-free collagen peptide by utilizing an electroosmosis-ultrafiltration integrated membrane technology, which comprises the following steps: placing collagen in an enzyme reactor, adding distilled water, stirring uniformly, performing heat treatment, cooling, adjusting pH, adding papain and alkaline protease for enzymolysis, inactivating enzyme after enzymolysis, cooling, centrifuging, and taking supernatant to obtain collagen hydrolysate; separating collagen hydrolysate with ultrafiltration membrane component, and ultrafiltering to obtain collagen peptide with wide small molecular weight distribution range; selecting an ultrafiltration membrane according to the molecular weight and the charge property of a target product, so that collagen peptides with smaller molecular weight and fishy substances can permeate through the ultrafiltration membrane, and the collagen peptides with larger molecular weight can be intercepted, thereby obtaining the fishy smell-free collagen peptides with narrow molecular weight distribution; nano-filtering, desalting and concentrating; drying to obtain the fishy smell-free collagen peptide. The collagen peptide prepared by the invention has narrow molecular weight distribution and no fishy smell.
Description
Technical Field
The invention relates to the technical field of separation and purification of bioactive peptides, in particular to a method for preparing fishy smell-free collagen peptide by utilizing an electroosmosis-ultrafiltration integrated membrane technology.
Background
Collagen peptide is a product with low molecular weight obtained by hydrolyzing collagen or gelatin, is easily soluble in water, and has a relative molecular weight of several hundred to several thousand daltons. A large number of researches show that the collagen peptide has a plurality of physiological activities of resisting oxidation, regulating immunity, enhancing bone strength and the like. Many studies have shown that the biological activity of collagen peptides is closely related to the range of molecular weights. Collagen peptides with different molecular weights have different biological properties, such as 2-4 amino acid oligopeptides like Ala-Hyp, pro-Hyp-Gly and the like can be directly absorbed, a 1000-2000Da polypeptide fragment has good water retention property, easy digestion and anti-skin aging activity, and the molecular weight range is a key factor influencing the application of the collagen peptides.
At present, the methods for separating and purifying collagen peptide mainly include chromatographic separation technology and membrane separation technology (such as the technologies disclosed in CN103992386A, CN111574585B, etc.), modern chromatographic technology usually adopts step-by-step separation, and although the separation method can finally obtain the target active peptide, the yield obtained by the separation method is often in milligram level, and the chromatographic method is high in economic cost, and is difficult to realize the large-scale separation of the bioactive peptide. On the other hand, although the conventional pressure-driven membrane separation technology can separate collagen peptide on a large scale (such as the technology disclosed in CN 101928744B), due to its low selectivity, it can only separate active peptides with large molecular weight difference, which has good biological activity, but the effective enrichment effect of active peptides with molecular weight being relatively poor compared with that of the close active peptides. And high-flux membrane separation can bring membrane pollution and influence the separation effect.
Fishy smell substances in fish scales, fish skins and fish bones can reduce the quality of the collagen peptide product. In the prior art, the fishy smell is usually removed by adopting methods such as activated carbon adsorption (CN 110105444A), alcohol extraction (CN 108004288A) and oil-water centrifugal separation (CN 101061827), but the problems of high loss rate of liquid nitrogen, reduction of collagen peptide activity, loss of collagen peptide and the like exist.
Disclosure of Invention
In order to solve the problem of poor separation and purification effects of collagen peptides in the prior art, the invention provides a method for preparing fishy smell-free collagen peptides by using an electroosmosis-ultrafiltration integrated membrane technology.
Based on the method of the invention, the collagen peptide with no fishy smell and narrow molecular weight distribution can be prepared.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a method for preparing fishy smell-free collagen peptide by utilizing an electroosmosis-ultrafiltration integrated membrane technology, which comprises the following steps:
(1) And (3) carrying out compound enzymolysis on the original protein: placing collagen in an enzyme reactor, adding a proper amount of distilled water, stirring and uniformly mixing, carrying out heat treatment, cooling, adjusting pH, adding papain and alkaline protease for enzymolysis, keeping the pH of the system unchanged in the reaction process, carrying out enzyme deactivation after the enzymolysis is finished, cooling, centrifuging and taking supernate to obtain collagen hydrolysate;
(2) Ultrafiltration membrane pretreatment of collagen peptide: separating collagen hydrolysate with ultrafiltration membrane component to remove enzyme, incompletely enzymolyzed collagen and macromolecular collagen peptide, and ultrafiltering to obtain collagen peptide with wide small molecular weight distribution range;
(3) Electro-osmosis-ultrafiltration integrated membrane refining of collagen peptide: selecting a proper ultrafiltration membrane according to the molecular weight and the charge property of a target product, so that collagen peptides with smaller molecular weight and fishy smell substances can permeate through the ultrafiltration membrane, and collagen peptides with larger molecular weight can be intercepted, thereby obtaining the fishy smell-free collagen peptides with narrow molecular weight distribution;
(4) Nano-filtering, desalting and concentrating;
(5) Drying to obtain the fishy smell-free collagen peptide.
In some embodiments of the invention, the collagen is collagen extracted from fish scales, fish skin or fish bones.
In some embodiments of the present invention, in step (1), the ratio of collagen to water (mass ratio) is 1:7 to 1: 7. 1: 8. 1: 9. 1: 10. 1: 11. 1:12.
in some embodiments of the present invention, in step (1), the heat treatment conditions are: heat treating at 60-90 deg.C for 8-15min (preferably 10 min).
In some embodiments of the present invention, in step (1), the heat treatment is preferably performed in a water bath environment.
In some embodiments of the present invention, in step (1), after the heat treatment, the temperature reduction refers to: the temperature is lowered to 30-65 ℃.
In some embodiments of the invention, in step (1), after cooling, the pH is adjusted to 6.5 to 9.5.
In some embodiments of the present invention, in step (1), the ratio of papain to alkaline protease added is 1.
In some embodiments of the present invention, in step (1), the enzymolysis temperature during the enzymolysis process is 30-65 ℃, the enzymolysis pH is 6.5-9.5, and the enzymolysis time is 2-10h.
In some embodiments of the present invention, in step (1), the enzyme deactivation conditions are: inactivating enzyme at 90 ℃ for 10min, wherein the cooling conditions are as follows: cooling to room temperature, and centrifuging under the following conditions: centrifuge at 3000g for 15min.
In some embodiments of the present invention, in the step (2), the ultrafiltration membrane module is one of a spiral wound membrane, a hollow fiber membrane or a plate type membrane, and is made of cellulose acetate, cellulose acetate esters, polyethylene, polysulfone or polyamide, and has a molecular weight cut-off of 2000-10000Da; the operating temperature in the ultrafiltration is 15-45 deg.C, the operating pressure is 0.3-1.5MPa, and the membrane surface flow rate is 1-5m/s.
In some embodiments of the present invention, in step (3), the electrode chamber of the electroosmosis-ultrafiltration integrated membrane device is filled with 15-25g/L NaCl solution to perform ion exchange buffering, and the electric field intensity is 2-20V/cm.
In some embodiments of the present invention, in step (3), the electroosmosis-ultrafiltration integrated membrane device selects an ultrafiltration membrane with a cut-off molecular weight of 5000-50000Da, so that collagen peptides with smaller molecular weight and fishy smell substances can permeate through the ultrafiltration membrane, and collagen peptides with larger molecular weight can be cut off, thereby achieving the purpose of separation.
In some embodiments of the invention, in step (4), nanofiltration desalination concentration with a molecular weight cut-off of 500-1000Da is selected.
In some embodiments of the invention, in step (5), spray drying or freeze drying is selected.
Compared with the prior art, the invention has the following advantages and beneficial effects:
compared with other separation technologies, the electroosmosis-ultrafiltration integrated membrane technology can set proper electric field intensity, feed liquid pH value and feed liquid concentration by selecting a proper ultrafiltration membrane according to isoelectric points and molecular weights of target products, so that separation selectivity is improved, and separation of narrow molecular weight intervals of bioactive peptides is realized.
The method can be used for separating the collagen peptide, and aims at the problems that the traditional pressure type membrane separation selectivity is low and the molecular weight range of the obtained collagen peptide product is wide, the method is driven by electric field force, and the separation is carried out according to the molecular weight and the charge property of the collagen peptide, so that the method has good selectivity and separation effect, and is convenient to operate and beneficial to realizing large-scale production.
The method is used for removing fishy smell of the collagen peptide extracted from fish scales, fish skins and fish bones, and removes fishy smell substances by utilizing the molecular weight and charge difference of the collagen peptide and the fishy smell substances without influencing the activity of the collagen peptide. The collagen peptide prepared by the invention has narrow molecular weight distribution and no fishy smell.
Drawings
FIG. 1 shows the molecular weight distribution of collagen peptide products before and after refining by electroosmosis-ultrafiltration integrated membrane technology.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
Provides a method for preparing fishy smell-free collagen peptide by using an electroosmosis-ultrafiltration integrated membrane technology, which comprises the following steps:
accurately weighing 2.5kg of decalcified and degreased fish scales, placing the fish scales in an enzyme reactor, adding 28L of distilled water, uniformly stirring, carrying out heat treatment at 90 ℃ for 10min, then cooling to 55 ℃ for stabilization, adjusting the pH to 7.5 by using 0.1M NaOH solution, adding 7000u/g of papain and alkaline protease for hydrolysis for 4h, keeping the pH of the system unchanged in the reaction process, inactivating enzyme at 90 ℃ for 10min after the hydrolysis is finished, cooling to room temperature, centrifuging for 15min under 3000g, taking supernatant, making the obtained collagen hydrolysate pass through an ultrafiltration membrane to be 2000Da, and making the ultrafiltration membrane component be a spiral roll type membrane which is made of polyamide; the operating temperature in the ultrafiltration operation is 25 ℃, the operating pressure is 1.0MPa, and the membrane surface flow rate is 5m/s, so that the collagen peptide with the molecular weight distribution below 1600Da is obtained. In order to further obtain the collagen peptide with narrow molecular weight distribution, an electroosmosis-ultrafiltration integrated membrane technology is adopted for refining, wherein the concentration of NaCl in a polar chamber is 20g/L, the electric field intensity is 9V/cm, and the molecular weight cut-off of an ultrafiltration membrane is 30000Da. Collecting collagen peptide liquid in a feeding chamber, performing nanofiltration, desalting and concentrating by using molecular weight cutoff of 500Da, and freeze-drying to obtain 210.6g of fishy smell-free collagen peptide product, wherein the relative molecular weight range of the fishy smell-free collagen peptide product is 1300-1600Da, as shown in figure 1, A is a molecular weight distribution diagram of the collagen peptide product before refining by the electroosmosis-ultrafiltration integrated membrane technology, and B is a molecular weight distribution diagram of the collagen peptide product after refining by the electroosmosis-ultrafiltration integrated membrane technology.
Example 2
Provides a method for preparing fishy smell-free collagen peptide by using an electroosmosis-ultrafiltration integrated membrane technology, which comprises the following steps:
accurately weighing 3.0kg of degreased and crushed fish skin, placing the fish skin into an enzyme reactor, adding 30L of distilled water, stirring and uniformly mixing, carrying out heat treatment at 90 ℃ for 10min, then cooling to 50 ℃ for stabilization, adjusting the pH to 8.5 by using 0.1M NaOH solution, adding 7000u/g of papain and trypsin for hydrolysis for 3h, keeping the pH of the system unchanged in the reaction process, carrying out enzyme deactivation at 90 ℃ for 10min after the hydrolysis is finished, cooling to room temperature, centrifuging for 15min under the condition of 3000g, taking supernatant, and carrying out ultrafiltration on the obtained collagen hydrolysate by an ultrafiltration membrane to be 5000Da, wherein the membrane component is a hollow fiber membrane and is polysulfone; the operating temperature in the ultrafiltration operation is 30 ℃, the operating pressure is 1.2MPa, and the membrane surface flow rate is 5m/s, so that the collagen peptide with the molecular weight distribution below 2000Da is obtained. In order to further obtain the collagen peptide with narrow molecular weight distribution, an electroosmosis-ultrafiltration integrated membrane technology is adopted for refining, wherein the concentration of NaCl in a polar chamber is 20g/L, the electric field intensity is 10V/cm, and the cut-off molecular weight of an ultrafiltration membrane is 40000Da. Collecting collagen peptide liquid in a feeding chamber, performing nanofiltration desalination concentration by using molecular weight cutoff of 800Da, and freeze-drying to obtain 230.1g of fishy smell-free collagen peptide product, wherein the relative molecular mass range of the fishy smell-free collagen peptide product is mainly 1600-1800 Da.
Example 3
Provides a method for preparing fishy smell-free collagen peptide by using an electroosmosis-ultrafiltration integrated membrane technology, which comprises the following steps:
accurately weighing 2.5kg of degreased and crushed fishbone, placing the degreased and crushed fishbone into an enzyme reactor, adding 25L of distilled water, uniformly stirring, carrying out heat treatment at 90 ℃ for 10min, then cooling to 50 ℃ for stabilization, adjusting the pH to 8.5 by using 0.1M NaOH solution, adding 7000u/g of papain and alkaline protease for hydrolysis for 3h, keeping the pH of the system unchanged in the reaction process, inactivating enzyme at 90 ℃ for 10min after the hydrolysis is finished, cooling to room temperature, centrifuging for 15min under the condition of 3000g, taking supernatant, and obtaining collagen hydrolysate with the ultrafiltration membrane of 3000Da, wherein the ultrafiltration membrane component is a plate-type membrane and is made of cellulose acetate; the operating temperature in the ultrafiltration operation is 25 ℃, the operating pressure is 1.0MPa, and the membrane surface flow rate is 4m/s, so that the collagen peptide with the molecular weight distribution below 1600Da is obtained. In order to further obtain the collagen peptide with narrow molecular weight distribution, an electroosmosis-ultrafiltration integrated membrane technology is adopted for refining, wherein the concentration of NaCl in a polar chamber is 15g/L, the electric field intensity is 8V/cm, and the molecular weight cutoff of an ultrafiltration membrane is 20000Da. Collecting collagen peptide liquid in a feeding chamber, performing nanofiltration desalination by using molecular weight cutoff of 1000Da, concentrating, freezing and drying to obtain a fishy smell-free collagen peptide product of 150.8g, wherein the relative molecular mass range of the fishy smell-free collagen peptide product is mainly 1400-1600 Da.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.
Claims (10)
1. A method for preparing fishy smell-free collagen peptide by using electroosmosis-ultrafiltration integrated membrane technology is characterized by comprising the following steps:
(1) And (3) carrying out compound enzymolysis on the original protein: placing collagen in an enzyme reactor, adding distilled water, stirring uniformly, performing heat treatment, cooling, adjusting pH, adding papain and alkaline protease for enzymolysis, keeping the pH of the system unchanged during the reaction process, inactivating enzyme after enzymolysis is finished, cooling, centrifuging, and taking supernate to obtain collagen hydrolysate;
(2) Ultrafiltration membrane pretreatment of collagen peptide: separating collagen hydrolysate with ultrafiltration membrane component to remove enzyme, incompletely enzymolyzed collagen and macromolecular collagen peptide, and ultrafiltering to obtain collagen peptide with wide small molecular weight distribution range;
(3) Electro-osmosis-ultrafiltration integrated membrane refining of collagen peptide: selecting an ultrafiltration membrane according to the molecular weight and the charge property of a target product, so that collagen peptides with smaller molecular weight and fishy substances can permeate through the ultrafiltration membrane, and the collagen peptides with larger molecular weight can be intercepted, thereby obtaining the fishy smell-free collagen peptides with narrow molecular weight distribution;
(4) Nano-filtering, desalting and concentrating;
(5) Drying to obtain the fishy smell-free collagen peptide.
2. The method for preparing fishy smell-free collagen peptide by using the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein the collagen is collagen extracted from fish scales, fish skin or fish bones.
3. The method for preparing fishy smell-free collagen peptide by using electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (1), the mass ratio of the collagen to the water is 1: 7-1.
4. The method for preparing fishy smell-free collagen peptide by using electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (1), the heat treatment conditions are as follows: heat treating at 60-90 deg.C for 8-15min.
5. The method for preparing fishy smell-free collagen peptide by using the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein the step (1) of cooling after the heat treatment is performed is that: reducing the temperature to 30-65 ℃; in the step (1), after cooling, the pH is adjusted to 6.5-9.5.
6. The method for preparing the fishy smell-free collagen peptide by utilizing the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (1), the adding proportion of the papain and the alkaline protease is 1.
7. The method for preparing fishy smell-free collagen peptide by using the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (1), the enzymolysis temperature in the enzymolysis process is 30-65 ℃, the enzymolysis pH is 6.5-9.5, and the enzymolysis time is 2-10h.
8. The method for preparing the fishy smell-free collagen peptide by utilizing the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (2), the ultrafiltration membrane component is one of a spiral wound membrane, a hollow fiber membrane or a plate-type membrane, the material is cellulose acetate, cellulose acetate esters, polyethylene, polysulfone or polyamide, and the cut-off molecular weight is 2000-10000Da; the operating temperature in the ultrafiltration is 15-45 deg.C, the operating pressure is 0.3-1.5MPa, and the membrane surface flow rate is 1-5m/s.
9. The method for preparing fishy smell-free collagen peptide by using the electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (3), the polar chamber of the electroosmosis-ultrafiltration integrated membrane device is filled with 15-25g/L NaCl solution, and the electric field intensity is 2-20V/cm.
10. The method for preparing fishy smell-free collagen peptide by using an electroosmosis-ultrafiltration integrated membrane technology according to claim 1, wherein in the step (3), an electroosmosis-ultrafiltration integrated membrane device selects an ultrafiltration membrane with the molecular weight cutoff of 5000-50000Da, so that collagen peptide with smaller molecular weight and fishy smell substances can permeate through the ultrafiltration membrane, and collagen peptide with larger molecular weight can be cut off, thereby achieving the purpose of separation;
in the step (4), nanofiltration, desalination and concentration are carried out, wherein the molecular weight cut-off is 500-1000 Da.
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