CN108935912B - Fish meat protein peptide with DPP-IV inhibition and anti-fatigue functions and preparation method thereof - Google Patents
Fish meat protein peptide with DPP-IV inhibition and anti-fatigue functions and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of functional food processing, and particularly discloses a fish protein peptide with DPP-IV (dipeptidyl peptidase IV) inhibition and anti-fatigue functions and a preparation method thereof, wherein the preparation method comprises the following steps: carrying out enzymolysis on the fish protein liquid by alkaline protease, neutral protease and papain, carrying out enzymolysis by flavourzyme, collecting protein peptide liquid with the molecular weight of less than 2000 daltons, and carrying out chromatographic separation to obtain the fish protein peptide. The fish protein peptide with DPP-IV inhibition and anti-fatigue functions is developed by using fish as a raw material, performing pretreatment on the fish through ultrahigh pressure, high temperature, high frequency ultrasonic waves and the like, performing stepwise enzymolysis by using a plurality of protein complex enzymes under the condition of not adding any acid and alkali, and performing membrane separation, gel separation and reversed-phase HPLC separation technologies.
Description
Technical Field
The invention relates to the field of functional food processing, in particular to a fish protein peptide with DPP-IV (dipeptidyl peptidase IV) inhibition and anti-fatigue functions and a preparation method thereof.
Background
The blood sugar reducing functional factor refers to a bioactive component capable of reducing blood sugar concentration of diabetic patients and improving symptoms of the diabetic patients. At present, the research on more natural products such as blood sugar reducing factors, mineral blood sugar reducing factors and vitamin blood sugar reducing factors is different in action mechanism. The natural products of hypoglycemic factors can be classified into flavonoids, active polysaccharides, alkaloids, saponins, terpenoids, conjugated linoleic acid, polypeptides and the like according to chemical structures. Since the artificial synthesis of insulin, various oral or injection medicaments such as sulfonylurea medicaments, biguanides, insulin sensitizers, sugar inhibitors and the like come out in succession, however, chemical medicaments often generate certain toxic and side effects, and natural hypoglycemic components have the advantages of mild and lasting action, stable property, almost no toxic reaction, coexistence of various hypoglycemic components, comprehensive action and the like, are favored by patients and the medical field, and also become the main research direction of hypoglycemic functional factors.
In view of the patent application and the authorization situation of the development of the hypoglycemic functional peptide at present, CN201410129977.3 mainly discloses a method for preparing the hypoglycemic peptide by silkworm pupa, CN201610134873.0 discloses a peony seed hypoglycemic peptide, CN201310290233.5 discloses a method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide by hairtail, and CN201410498361.3 discloses DPP-IV inhibitory peptide GPGSPGGPL derived from deer protein, wherein the amino acid sequence of the DPP-IV inhibitory peptide is Gly-Pro-Gly-Ser-Pro-Gly-Gly-Pro-Leu. The polypeptide GPGSPGGPL has DPP-IV inhibitory activity and hypoglycemic activity, and CN201410455009.1 discloses an extraction method of bean dregs protein and a method for preparing DPP-IV inhibitory peptide by using the same. However, at present, no literature report on DPP-IV inhibitory peptides mainly comprising Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro peptides is found at home and abroad.
Disclosure of Invention
In order to solve the problems in the prior art, the invention develops a fish protein peptide with better DPP-IV inhibition and anti-fatigue functions and a preparation method thereof by taking fish as a raw material through a large number of experiments.
In a first aspect, the present invention provides a method for preparing a fish protein peptide with DPP-iv inhibition and anti-fatigue functions, comprising: carrying out enzymolysis on the fish protein liquid by alkaline protease, neutral protease and papain, carrying out enzymolysis by flavourzyme, collecting protein peptide liquid with the molecular weight of less than 2000 daltons, and carrying out chromatographic separation to obtain the fish protein peptide.
Further, the sum of the mass of the alkaline protease, the neutral protease and the papain is 0.3-0.8% of the mass of the protein in the fish protein liquid, and the mass ratio of the alkaline protease, the neutral protease and the papain is 1-2:1: 1; the enzymolysis conditions of the alkaline protease, the neutral protease and the papain are that the enzymolysis reaction is carried out for 1.0 to 2.5 hours at the temperature of between 50 and 60 ℃.
The mass of the flavourzyme is 0.1-0.2% of the mass of the protein in the fish protein liquid, and the enzymolysis condition of the flavourzyme is that the enzymolysis reaction is carried out for 0.25-0.5h at 50-60 ℃.
Further, after the flavor protease enzymolysis, the obtained enzymolysis liquid is placed at the temperature of 90-95 ℃ for heat preservation for 10-20 minutes, cooled to the room temperature, separated by diatomite, and the separated liquid is collected; and performing ultrafiltration on the separation liquid, and collecting protein peptide liquid with the molecular weight of less than 2000 daltons.
Preferably, the ultrafiltration is a two-step treatment, namely, the ultrafiltration is carried out by utilizing a ceramic membrane with the aperture of 5000 daltons firstly, the protein and the polypeptide with the molecular weight of less than 5000 daltons are separated, and then the ultrafiltration is carried out by utilizing a ceramic membrane with the aperture of 2000 daltons, and the protein peptide with the molecular weight of less than 2000 daltons is separated.
Further, carrying out chromatographic separation on the protein peptide liquid with the molecular weight of less than 2000 daltons, wherein an eluent is deionized water, an elution peak is detected at 280nm, and a 2 nd elution peak is collected; concentrating, and freeze drying to obtain fish protein peptide.
Furthermore, the fish protein liquid is prepared by using fish as a raw material, performing 100-200Mpa ultrahigh pressure treatment on the fish, pulping the fish, adding 1.0-3.0 times of water, homogenizing, centrifuging, degreasing, and performing 90-120kH ultrasonic treatment at 85-90 ℃ for 20-30 minutes.
As a preferable scheme, the preparation method specifically comprises the following steps:
(1) selecting fresh or frozen fish, cleaning with clean water meeting the sanitary standard of drinking water, treating for 1-2 minutes under 200Mpa in a manner of 100-;
(2) carrying out ultrasonic treatment on the fish protein liquid obtained in the step (1) at 85-90 ℃ for 20-30 minutes at 90-120kH to change the tissue structure of the fish protein;
(3) regulating the protein content of fish protein liquid to 9-11%, and performing enzymolysis reaction at 50-60 deg.C for 1.0-2.5 hr with 0.3-0.8% compound protease; secondly, carrying out enzymolysis reaction for 0.25-0.5h at 50-60 ℃ by using 0.1-0.2% of flavourzyme; then preserving the temperature at 90-95 ℃ for 10-20 minutes to inactivate the enzyme, cooling to room temperature, filtering and separating through diatomite, and collecting the separated liquid;
wherein the compound protease consists of alkaline protease, neutral protease and papain in a mass ratio of 1-2:1: 1;
(4) and (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000 daltons, carrying out SephadexG-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, wherein the reversed-phase HPLC separation condition is to use 5-90% acetonitrile solution as eluent, and take 19-20 min collected peptide solution;
(6) and (5) concentrating the peptide solution obtained in the step (5), and freeze-drying to obtain the fish protein peptide powder. The main components of the fish protein peptide powder are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro is 50-59%.
In the embodiment of the present invention, silver carp is taken as an illustrative example.
It should be noted that the fish protein peptide prepared by the method of the present invention also belongs to the protection scope of the present invention.
The application of the fish protein peptide prepared by the invention in preparing medicines or functional foods also belongs to the protection scope of the invention.
Wherein the function of the medicine or functional food depends on the DPP-IV inhibition and anti-fatigue function of the fish protein peptide.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the invention takes fish as raw material, carries out pretreatment on the fish under ultrahigh pressure, high temperature, high frequency ultrasonic waves and the like, utilizes a plurality of protein complex enzymes to carry out stepwise enzymolysis without adding any acid and alkali, and develops the fish protein peptide with specific amino acid polypeptide sequences (Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg, Leu-Leu-Asp-Leu-Gly-Val-Pro) and DPP-IV inhibition and anti-fatigue functions by membrane separation, gel separation and reversed phase HPLC separation technology, thereby establishing a set of simple and efficient preparation method of the multifunctional fish protein peptide.
More specifically, the invention has the advantages that (1) acid or alkali is not added in the whole processing process of the developed fish protein peptide to adjust the pH value, the product keeps better functional characteristics, and the ash content of the product is low and is less than 1%. (2) The developed protein peptide has better DPP-IV inhibition function (IC50 value) less than 0.25mg/mL and anti-fatigue function. (3) The fish protein liquid treated by the ultrahigh pressure and the specific frequency ultrasonic technology can obviously improve the sensitivity of the fish protein to enzyme and reduce the using amount of the enzyme. (4) The developed product is safe, the invention adopts a plurality of food-grade compound proteases (alkaline protease, papain, neutral protease and flavourzyme), and fish protein peptide with specific molecular weight and peptide composition is obtained through moderate enzymolysis under mild conditions, and 100 percent of fish protein peptide is obtained without any additive. (5) The protein peptide developed by the invention has good flavor and color by diatomite filtration, and can be widely applied to special foods and nutritional foods. (6) The proportion of the peptide with the molecular weight less than 1000 in the developed fish protein peptide is more than 90 percent, and the fish protein peptide has a clear peptide composition, wherein the content of the peptide of Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg, Leu-Leu-Asp-Leu-Gly-Val-Pro is more than 50 percent.
Detailed Description
The present invention is further illustrated by the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of Fish protein peptide having DPP-IV inhibitory and anti-fatigue Functions
(1) Selecting 100 g of fresh fish, cleaning the fresh fish with clean water meeting the sanitary standard of drinking water, treating the fish for 2 minutes under ultrahigh pressure (100Mpa), pulping the fish into pulp by using a pulping machine, adding water with the weight 1.0 time that of the fish, and homogenizing the pulp for 3 minutes by using a 8000rpm dispersion homogenizer to obtain fish pulp. Centrifuging at 6000g for 10min to remove upper layer fat to obtain defatted fish protein solution.
(2) And (2) adjusting the temperature of the fish protein liquid obtained in the step (1) to 85 ℃, and treating the fish protein liquid for 30 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 100kH) to change the tissue structure of the fish protein.
(3) Adjusting the protein content of the fish protein liquid to 11%, firstly, carrying out enzymolysis reaction for 1.5h at 50 ℃ by using 0.8% of compound protease (composed of alkaline protease, neutral protease and papain in a mass ratio of 1:1: 1); then carrying out enzymolysis reaction for 0.5h at 55 ℃ by using 0.2% flavourzyme in the second step; the mixture was incubated at 95 ℃ for 10 minutes, cooled to room temperature, and then separated by filtration through celite to collect the separated liquid.
(4) And (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for 1 time of separation, and the separation condition of the reversed-phase HPLC is to take 5-90% acetonitrile solution as eluent and take 19-20 minutes of collected peptide solution.
(6) And (5) concentrating the peptide solution obtained in the step (5), and freeze-drying to obtain the fish protein peptide powder. The main components of the fish protein peptide powder are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro is 55 percent.
Example 2 preparation of Fish protein peptide having DPP-IV inhibitory and anti-fatigue Functions
(1) Selecting 500 g of frozen chub meat, cleaning the chub meat with clean water meeting the drinking water sanitary standard, treating the chub meat for 1 minute under ultrahigh pressure (200Mpa), pulping the chub meat into slurry by using a pulping machine, adding water with the weight 2.0 times that of the chub meat, and homogenizing the slurry for 1 minute by using a 10000rpm dispersion homogenizer to obtain fish meat slurry. Centrifuging at 6000g for 10min to remove upper layer fat to obtain defatted fish protein solution.
(2) Adjusting the temperature of the fish protein liquid obtained in the step (1) to 90 ℃, and treating the fish protein liquid for 20 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 120kH) to change the tissue structure of the fish protein.
(3) Regulating the protein content of the fish protein liquid to 9%, firstly, carrying out enzymolysis reaction for 2.5h at 55 ℃ by using 0.5% of compound protease (consisting of alkaline protease, neutral protease and papain in a mass ratio of 2:1: 1); then carrying out enzymolysis reaction for 0.25h at 50 ℃ by using 0.1% flavourzyme in the second step; the mixture was incubated at 90 ℃ for 20 minutes, cooled to room temperature, and then separated by filtration through celite to collect the separated liquid.
(4) And (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for 1 time of separation, and the separation condition of the reversed-phase HPLC is to take 5-90% acetonitrile solution as eluent and take 19-20 minutes of collected peptide solution.
(6) And (5) concentrating the peptide solution obtained in the step (5), and freeze-drying to obtain the fish protein peptide powder. The main components of the fish protein peptide powder are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro is 56%.
Example 3 preparation of Fish protein peptide having DPP-IV inhibitory and anti-fatigue Functions
(1) Selecting 1000 g of frozen chub meat, cleaning the chub meat with clean water meeting the drinking water sanitary standard, treating the chub meat for 1 minute under ultrahigh pressure (150Mpa), pulping the chub meat by using a pulping machine, adding water with the weight 3.0 times that of the chub meat, and homogenizing the chub meat for 2 minutes by using a 9000rpm dispersion homogenizer to obtain a chub meat slurry. Centrifuging at 6000g for 10min to remove upper layer fat to obtain defatted fish protein solution.
(2) Adjusting the temperature of the fish protein liquid obtained in the step (1) to 90 ℃, and treating the fish protein liquid for 25 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 110kH) to change the tissue structure of the fish protein.
(3) Adjusting the protein content of the fish protein liquid to 10%, firstly, carrying out enzymolysis reaction for 2.0h at 55 ℃ by using 0.6% of compound protease (composed of alkaline protease, neutral protease and papain in a mass ratio of 2:1: 1); then carrying out enzymolysis reaction for 0.5h at 60 ℃ by using 0.2% flavourzyme in the second step; the mixture was incubated at 95 ℃ for 15 minutes, cooled to room temperature, and then separated by filtration through celite to collect the separated liquid.
(4) And (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for 1 time of separation, and the separation condition of the reversed-phase HPLC is to take 5-90% acetonitrile solution as eluent and take 19-20 minutes of collected peptide solution.
(6) And (5) concentrating the peptide solution obtained in the step (5), and freeze-drying to obtain the fish protein peptide powder. The main components of the fish protein peptide powder are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro is 57%.
Comparative example 1 Fish meat protein peptide having hypoglycemic function
1. Selecting 500 g of chub fish, and pulping the fish into pulp by using a pulping machine; then adding water with the weight 1.8 times of that of the fish meat into the beaten fish meat paste, adjusting the temperature to 90 ℃, and stirring for 10 minutes.
2. Adjusting the temperature of the fish pulp obtained in the step 1 to 80 ℃, and then mixing the bromelain and the fish in a mass ratio of 1: 200, adding 2.5 g of bromelain, carrying out enzymolysis for 1.0 hour, cooling to 45 ℃, and mixing the obtained product according to the mass ratio of the flavourzyme to the fish meat of 1: adding flavourzyme 1.25 g into 400, carrying out enzymolysis for 1.5 hours, centrifuging for 5min under the condition of 6000g, carrying out ultrafiltration by utilizing a ceramic membrane with the aperture of 5000 daltons, and taking protein peptide liquid with the molecular weight of less than 5000 daltons. Concentrating, and freeze drying to obtain fish protein peptide powder.
Experimental example 1 measurement test of DPP-IV inhibitory Activity of Fish meat protein peptide
Test samples: samples 1, 2 and 3 were fish protein peptide powders prepared in example 1, example 2 and example 3, respectively; sample 4 was the fish protein peptide powder prepared in comparative example 1; the sample Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and the sample Leu-Leu-Asp-Leu-Gly-Val-Pro are protein peptides synthesized by the amino acid sequences Ala-Ala-Leu-Glu-Gln-Thr-Glu-Arg and Leu-Leu-Asp-Leu-Gly-Val-Pro.
The method comprises the following steps:
the sample was diluted to an appropriate concentration with 100mmol/L Tris-HCl (pH8.0) buffer, 25. mu.L of the sample dilution was aspirated, mixed with 25. mu.L of a substrate (concentration: 1.6mmol/L), and added to a 96-well plate. After incubation for 10min at 37 ℃, adding 50 mu L of DPP-IV enzyme liquid (the enzyme activity is 8U/L), mixing uniformly, then incubating for 60min at 37 ℃, immediately adding 100 mu L of 1mol/L acetic acid-sodium acetate (pH 4.0) buffer solution to terminate the reaction, measuring the absorbance A at 405nm, and calculating the DPP-IV inhibition rate of the sample according to the following formula.
DPP-IV inhibition = {1- (sample-a sample blank)/(a negative control-a negative blank) } × 100
Sample A: is the light absorption value A of the sample reaction solution at 405 nm;
sample a blank: Tris-HCL buffer solution is used for replacing DPP-IV enzyme solution as a light absorption value A of a sample blank control;
negative control A: replacing a sample with Tris-HCL buffer solution as a light absorption value of a negative control;
negative blank control a: Tris-HCL buffer solution is used for replacing DPP-IV enzyme solution and sample to be used as light absorption value of negative blank control
IC for DPP-IV inhibition50Value determination:
determining DPP-IV inhibition rate of samples under different concentrations, making a regression curve by logarithmic value of polypeptide concentration and inhibition rate to obtain a regression equation, and calculating IC (integrated Circuit) according to the regression equation50I.e. 50% inhibition of DPP-IV enzyme activity is the concentration of the peptide.
As can be seen from Table 1, the products of the present invention have very good DPP-IV inhibitory activity (IC50 less than 0.25 mg/mL).
TABLE 1 DPP-IV inhibition test results of the fish protein peptide of the present invention
Experimental example 2 determination test of fatigue-delaying function of fish protein peptide of the present invention
Test samples: samples 1, 2 and 3 are the fish protein peptides prepared in examples 1, 2 and 3, respectively, sample 4 is the fish protein peptide powder prepared in comparative example 1, and sample 5 is the fish protein powder obtained by directly freeze-drying the fish protein liquid obtained in step (1) of example 1.
The method comprises the following steps:
1.1 Experimental animals raising and grouping
240 clean-grade Kunming mice are bred adaptively for one week in a clean animal laboratory, one cage is bred in 8-10 cages, the padding is kept dry and sanitary, and the mice are changed and washed once every three days. Cleaning on time every day, performing work such as disinfection and sterilization, and ensuring cleanness and tidiness of a clean animal laboratory. The mouse weight was recorded weekly at 25 + -2 deg.C, relative humidity 50 + -5%, and light illumination dark interval 12h, and fed with mouse quasi-feed and water ad libitum. After a one-week acclimation period, 200 mice were randomly assigned to 6 groups, i.e., blank, sample 1, sample 2, sample 3, sample 4, and sample 5. And (3) gavage the same amount of normal saline into the stomach of the blank group of mice, setting the dosage of other 5 groups to be 140mg/kg/d, gavage the stomach during 9:00-10:30 in the morning every day, and detecting the anti-fatigue effect of the tested sample after the stomach is gavaged for 30 days.
1.1.1 weight bearing swimming test
After 30min from the last sample administration, 10 mice were randomly selected from each group, and the tail roots of the mice were wrapped with 5% weight lead sheet and then placed in a swimming box for swimming. The water depth is not less than 30cm, the water temperature is 25 +/-1 ℃, and the time from swimming to death of the mouse, namely the time of the mouse weight swimming, is recorded.
1.1.2 liver glycogen, Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay
After 30min of the last sample giving, randomly taking 10 mice from each group, drawing eyeballs to collect whole blood, centrifuging the collected blood at 3000r/min for 10min, taking serum for later use, and measuring according to CAT, SOD and GSH-Px test boxes. Immediately dissecting the mouse after blood collection, rinsing the liver with normal saline, sucking the liver with filter paper, accurately weighing 100mg of the liver, and determining according to the instructions of the liver glycogen detection kit.
1.1.3 Whole blood lactate assay
Randomly taking 10 mice in each group, sampling blood after 30min of the last sample feeding, then swimming in water at the temperature of 30 ℃ without load for 10min, stopping, sampling blood from the adjacent eyes by using capillaries, and respectively measuring the lactic acid content of the whole blood before swimming, after 10min of swimming and 20min of rest after swimming.
TABLE 2 Effect of the Fish protein peptide of the present invention on the anti-fatigue ability of mice
As can be seen from Table 2, the fish protein peptide can prolong the swimming time of the mouse, improve the energy metabolic system of the mouse, and promote the synthesis of liver glycogen, thereby storing more energy substances, improving the activities of CAT, SOD and GSH-Px enzymes, and delaying physical fatigue; the composition has obvious inhibition effect on the increase of the lactic acid level after short-time movement, and can help organisms delay the accumulation of lactic acid to a certain extent; after resting for 20min, the lactic acid content is remarkably reduced, and the composition can help the organism to accelerate the removal of the accumulation of lactic acid. The products developed by the present invention, sample 1, sample 2, sample 3, were significantly better than sample 4 and sample 5.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. A preparation method of a fish protein peptide with DPP-IV inhibition and anti-fatigue functions is characterized by comprising the following steps:
(1) cleaning fresh or frozen chub fish meat, treating the chub fish meat for 1-2 minutes by 100-;
(2) treating the fish protein liquid obtained in the step (1) by 90-120kH ultrasonic waves at the temperature of 85-90 ℃ for 20-30 minutes;
(3) regulating the protein content of fish protein liquid to 9-11%, and performing enzymolysis reaction at 50-60 deg.C for 1.0-2.5 hr with 0.3-0.8% compound protease; secondly, carrying out enzymolysis reaction for 0.25-0.5h at 50-60 ℃ by using 0.1-0.2% of flavourzyme; then preserving the heat at 90-95 ℃ for 10-20 minutes, cooling to room temperature, filtering and separating through diatomite, and collecting a separation solution;
wherein the compound protease consists of alkaline protease, neutral protease and papain in a mass ratio of 1-2:1: 1;
(4) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons;
(5) taking protein peptide liquid with molecular weight less than 2000 daltons, carrying out Sephadex G-25 gel separation, wherein an eluent is deionized water, an elution peak is detected at 280nm, and a 2 nd elution peak is collected; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, wherein the reversed-phase HPLC separation condition is to use 5-90% acetonitrile solution as eluent, and take 19-20 min collected peptide solution; concentrating, and freeze drying to obtain fish protein peptide powder.
2. A fish protein peptide produced by the production method according to claim 1.
3. Use of the fish protein peptide of claim 2 for the preparation of a medicament or a functional food.
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| CN110437309A (en) * | 2019-09-04 | 2019-11-12 | 安徽国肽生物科技有限公司 | A kind of algae protein peptides and preparation method thereof inhibiting function with DPP-IV |
| CN114711324A (en) * | 2021-02-04 | 2022-07-08 | 云南海王水产有限公司 | Fish scale collagen peptide with probiotic growth promoting and antioxidant effects and preparation method thereof |
| CN113151388B (en) * | 2021-04-19 | 2022-09-09 | 国肽生物工程（常德）有限公司 | Sea cucumber peptide with antioxidant and DPP-IV (dipeptidyl peptidase IV) inhibiting functions and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333940A (en) * | 2013-07-10 | 2013-10-02 | 浙江大学宁波理工学院 | Method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide through using hairtail |
| CN106434809A (en) * | 2016-11-07 | 2017-02-22 | 中国农业大学 | Fish protein peptide with ACE inhibitory function and preparation method thereof |
| CN107164445A (en) * | 2017-06-08 | 2017-09-15 | 中国农业大学 | Suppress fish-skin protein peptides of function and preparation method and application with DPP IV |
| CN107821726A (en) * | 2017-10-25 | 2018-03-23 | 浦江县欧立生物技术有限公司 | The purposes of grass carp anti-fatigue active peptide |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE542896T1 (en) * | 2004-12-23 | 2012-02-15 | Campina Nederland Holding Bv | PROTEIN HYDROLYZATE ENRICHED WITH DPP-IV INHIBITING PEPTIDES AND USE THEREOF |
| JP5887630B2 (en) * | 2011-08-11 | 2016-03-16 | 岡山県 | Method for producing dipeptidyl peptidase-IV inhibitor |
-
2018
- 2018-06-21 CN CN201810644982.6A patent/CN108935912B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333940A (en) * | 2013-07-10 | 2013-10-02 | 浙江大学宁波理工学院 | Method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide through using hairtail |
| CN106434809A (en) * | 2016-11-07 | 2017-02-22 | 中国农业大学 | Fish protein peptide with ACE inhibitory function and preparation method thereof |
| CN107164445A (en) * | 2017-06-08 | 2017-09-15 | 中国农业大学 | Suppress fish-skin protein peptides of function and preparation method and application with DPP IV |
| CN107821726A (en) * | 2017-10-25 | 2018-03-23 | 浦江县欧立生物技术有限公司 | The purposes of grass carp anti-fatigue active peptide |
Non-Patent Citations (1)
| Title |
|---|
| 鱼蛋白肽的制备及其作为功能食品基料的应用;王思远等;《广州城市职业学院学报》;20170630;第11卷(第2期);54-57 * |
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