CN110200287B - Oyster polypeptide-nano selenium particle compound with effects of dispelling effects of alcohol and protecting liver as well as preparation method and application thereof - Google Patents
Oyster polypeptide-nano selenium particle compound with effects of dispelling effects of alcohol and protecting liver as well as preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/65—Addition of, or treatment with, microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses an oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver, and a preparation method and application thereof. The method comprises the following steps: homogenizing oyster meat, adding water and active enzyme, performing enzymolysis, boiling water bath, centrifuging to obtain supernatant, cooling the supernatant to obtain oyster polypeptide powder, adding into water, mixing to obtain solution, adding sodium selenite solution into oyster polypeptide solution, mixing, adding ascorbic acid solution under stirring, performing water bath reaction, dialyzing to obtain functional nano selenium sol, performing ultrasonic treatment, and lyophilizing to obtain oyster polypeptide-nano selenium particle compound with effects of relieving hangover and protecting liver. The oyster polypeptide is used for stabilizing the nano-selenium, so that the stability of the nano-selenium is obviously improved, the nano-selenium can be stably stored for 1 month at 4 ℃, and the nano-selenium particles can reduce the damage of alcohol or hydrogen peroxide to the liver through the MTT method detection. If the product is converted into a health care product with liver protection function and selenium supplementing function, great social and economic benefits can be generated.
Description
Technical Field
The invention belongs to the technical field of nano-selenium nutrition preparation and functional food preparation, and particularly relates to an oyster polypeptide-nano-selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver, and a preparation method and application thereof.
Background
Selenium (Se) is one of essential and indispensable nutrient elements in organisms, is an important constituent of human erythrocyte glutathione peroxidase, and has the main functions of participating in enzyme synthesis, avoiding excessive oxidation of cell membranes, protecting the structure and the function of the cell membranes, and having multiple biological functions of resisting oxidation, resisting tumors, preventing cardiovascular diseases, epilepsy and the like.
The nano selenium belongs to zero-valent selenium, general zero-valent element selenium can not be absorbed and utilized by human body, and the nano selenium can be directly absorbed and utilized by human body due to the special nano structure. Compared with organic selenium, nano-selenium has better bioavailability and higher bioactivity, and lower toxicity, and is the selenium form with the lowest toxicity which has been found at present. In addition, the nano selenium has outstanding biological activity function, has the characteristics of antioxidation, anti-tumor, liver protection, immunoregulation, bacteriostasis and the like, and has good application prospect in the development of selenium supplementing products and the technical field of medicine nano. The conventional synthesis methods of nano-selenium at present include chemical synthesis methods and biological synthesis methods, but the biological synthesis methods have long preparation period, and the formed nano-selenium particles have large application range and are not widely applied. The chemical synthesis method can quickly form red elemental selenium, but the elemental selenium is unstable and easy to aggregate when being singly present, and a regulating agent is generally required to be added to increase the stability of the elemental selenium. At present, researches show that some polysaccharides, proteins, surfactants (polyvinyl alcohol, sodium dodecyl sulfate, sodium carboxymethyl cellulose and the like) and the like can be used as regulators of nano-selenium formation. Compared with other biomacromolecules such as polysaccharide, the protein regulator has more advantages in biocompatibility and physiological activity. However, the nano-selenium particle compound formed by adding the regulating agent is easy to cause poor storage stability of the whole particles due to uneven distribution of the regulating agent on the nano-selenium surface. In addition, the functional activity of the multi-focus selenium is researched, and the research on the synergism of the regulator on the physiological activity of the nano-selenium particles is less.
Oyster (Oyster) is known as "marine milk", is a worldwide shellfish, is one of four well-known economic shellfish for cultivation in China, is widely distributed in Liaoning, hebei, shandong, jiangsu, zhejiang, guangdong, guangxi, hainan and other places, and is also a first medicinal and health-care functional food approved by the Ministry of health in China. The oyster has higher protein content and is a good raw material for preparing bioactive peptide. The active peptide naturally existing in marine organisms has a small content, is difficult to extract and is difficult to mass produce; the chemical synthesis of polypeptides is time-consuming and laborious and costly, so that more and more people use enzymatic methods to extract active peptides from marine proteins. The research on the physiological activity of oyster peptides in the food industry mainly focuses on preventing and treating cardiovascular diseases, anticoagulation, blood fat reduction, antithrombotic, organism immunity improvement and the like. According to the composition characteristics of amino acids and minerals, research on preparing oyster peptides with the functions of dispelling effects of alcohol and protecting liver and regulating and controlling the formation of nano selenium by using a biological enzymolysis technology has not been reported.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a preparation method and application of oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver.
The primary object of the present invention is to provide an oyster polypeptide-nano selenium particle complex. The nano selenium preparation technology and the synergistic incorporation of active molecules have wide application to the research and development of related innovative medicines, and the problems of nano selenium size regulation, stability, active molecule enrichment efficiency and the like mainly exist at present. The invention develops a safe and effective novel oyster polypeptide-nano selenium, identifies and characterizes the form of the oyster polypeptide-nano selenium, and shows better antioxidant activity. A method for preparing nano-selenium by regulating oyster polypeptide and its application in antagonizing alcohol and hydrogen peroxide induced liver injury are disclosed.
Another objective of the invention is to provide a method for preparing nano-selenium by oyster polypeptide regulation. On one hand, oyster polypeptide is taken as a template to stabilize nano selenium, and on the other hand, active factors in the oyster polypeptide are doped into nano selenium particles to realize uniform and stable distribution of the nano particles, and the nano selenium particles have the dual-function nano selenium particles with oyster polypeptide and selenium activity. The prepared red elemental selenium has nano-scale size, spherical particles with average particle diameter smaller than 200 and nm, and selenium content is larger than 40%.
The invention further aims at providing an application of oyster polypeptide to regulation and control of nano-selenium preparation in antagonizing alcohol or hydrogen peroxide induced liver injury.
The object of the invention is achieved by at least one of the following technical solutions.
The invention provides a preparation method of oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver, which comprises the following steps:
(1) Removing shells of oysters, taking meat, cleaning oyster meat, directly homogenizing to obtain homogenate, adding water and active enzyme, and performing enzymolysis reaction to obtain enzymolysis liquid;
(2) Treating the enzymolysis liquid in the step (1) with boiling water bath, centrifuging to obtain supernatant, and freeze-drying the supernatant to obtain oyster polypeptide powder (oyster polypeptide with ADH activating activity);
(3) Adding the Oyster Polypeptide (OPH) powder obtained in the step (2) into water, uniformly mixing to prepare an oyster polypeptide solution, adding a sodium selenite solution into the oyster polypeptide solution, and uniformly stirring to obtain a mixed solution;
(4) Dropwise adding an ascorbic acid solution into the mixed solution obtained in the step (3) under the stirring state, uniformly mixing while dropwise adding, carrying out water bath reaction, and carrying out dialysis treatment to obtain the functionalized nano selenium sol;
(5) Performing ultrasonic treatment on the functionalized nano selenium sol obtained in the step (4), and freeze-drying (the pre-freezing temperature of freeze-drying is-30-40 ℃, the material thickness is 4-7 mm, and the drying time is 24-35 h) to obtain the oyster polypeptide-nano selenium particle compound (OPH-Sepps) with the effects of dispelling the effects of alcohol and protecting the liver.
Further, the mass ratio of the homogenate to the water in the step (1) is 1:1-1:4; the homogenization rate is 10000-15000 r/min; the homogenization time is 30-60s.
Further, in the step (1), the active enzyme is one of papain, flavourzyme, alkaline protease, bromelain, pancreatin, composite protease, neutral protease and the like, and the mass of the active enzyme is 0.1-1wt% of that of oyster meat (substrate protein); the temperature of the enzymolysis reaction is 37-60 ℃, the time of the enzymolysis reaction is 0.5-12h, and the pH value of the enzymolysis reaction is 5-11.
Preferably, the active enzyme is preferably pancreatin, and the enzymolysis conditions are as follows: the pH value is 7.0-8.0, the enzyme adding amount is 0.1-1% (w/w) of the substrate protein content, the reaction temperature is 40-60 ℃, and the reaction time is 0.5-12 h.
Further, the boiling water bath treatment time in the step (2) is 10-15min; the temperature of the centrifugation is 4-10 ℃, the rotation speed of the centrifugation is 8000-10000r/min, and the centrifugation time is 10-30min.
Preferably, the vacuum degree of the freeze drying in the step (2) is 1.0-1.5 mbar, the prefreezing temperature is-30-40 ℃, the material thickness is 4-7 mm, and the drying time is 24-35 h.
Further, the oyster polypeptide (abbreviated as OPH) has an activity of activating Alcohol Dehydrogenase (ADH).
Further, the concentration of the oyster polypeptide solution in the step (3) is 0.5-10mg/mL; the concentration of the sodium selenite solution is 0.5-2.0mM; the volume ratio of the sodium selenite solution to the oyster polypeptide solution is 1:1-4:1 (v/v).
Further, the stirring speed in the stirring state in the step (4) is 100-500rpm, preferably, the stirring speed is 100-200rpm; the concentration of the ascorbic acid solution is 1.0-8.0mM; the volume ratio of the mixed solution to the ascorbic acid solution is 1:2-5:16 (v/v).
Further, the temperature of the water bath reaction in the step (4) is 25-45 ℃, and the time of the water bath reaction is 0.5-12h; the dialysis treatment uses a dialysis bag with a specification of 100-1000 Da, preferably a dialysis bag with a specification of 500-1000Da; the dialysis treatment time is 36-48h; the temperature of the dialysis treatment is 4-10 ℃.
Further, the ultrasonic frequency of the ultrasonic treatment in the step (5) is 20-50kHz, the ultrasonic treatment time is 5-15min, and the ultrasonic treatment temperature is less than 25 ℃.
The invention provides oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver, which is prepared by the preparation method, and is called OPH-Sepps for short.
The oyster polypeptide-nano selenium particle compound (OPH-SeNPs) with the effects of dispelling effects of alcohol and protecting liver is spherical particles, the particle size is smaller than 200 and nm, and the selenium content is larger than 40% (w/w).
The oyster polypeptide-nano selenium particle compound (OPH-SeNPs) with the effects of dispelling effects of alcohol and protecting liver can be applied to preparing functional foods or health care products.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The preparation method provided by the invention can not only prepare oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver, but also produce oyster protein source crude peptide for promoting alcohol metabolism, the production method is simple, the oyster protein source crude peptide for promoting alcohol metabolism has obvious effect, and the method has a certain practical significance for large-scale production;
(2) According to the preparation method provided by the invention, the oyster polypeptide with the ADH activating capability is used as a template for regulating and controlling the preparation of nano-selenium, and the prepared oyster polypeptide-nano-selenium particle compound has good effects of dispelling the effects of alcohol and protecting the liver, so that on one hand, the additional value of oyster is improved, on the other hand, the stability of nano-selenium is improved, and the nano-selenium prepared by regulating and controlling the oyster polypeptide is stably preserved for one month at the temperature of 4 ℃, so that the nano-selenium has a certain positive significance for product transportation and storage;
(3) The oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting the liver has stronger liver-protecting activity compared with single oyster polypeptide through MTT method detection, and can prevent the liver injury caused by alcohol and hydrogen peroxide; the effective concentration of the oyster polypeptide-nano selenium particle complex is only one thousandth of Oyster Polypeptide (OPH).
Drawings
FIG. 1 is a transmission electron microscope image of oyster polypeptide-nano-selenium particle complexes with anti-hangover and liver protecting effects prepared in embodiment 1 of the present invention;
FIG. 2 is a graph showing the particle size analysis of oyster polypeptide-nano-selenium particle complexes having anti-hangover and liver-protecting effects prepared in embodiment 2 of the present invention;
FIG. 3 is a bar chart showing ADH activation rates of oyster polypeptide-nano-selenium particle complexes having anti-hangover and liver-protecting effects prepared in embodiment 3 of the present invention and oyster polypeptides having the same protein concentration;
FIG. 4 is a bar chart showing the protective effect of oyster polypeptide-nano-selenium particle complexes with anti-hangover and liver-protecting effects and oyster polypeptides with the same protein concentration on alcoholic to liver injury prepared in embodiment 3 of the present invention;
fig. 5 is a bar graph showing the protective effect of oyster polypeptide-nano-selenium particle complex with anti-hangover and liver-protecting effects on hydrogen peroxide to liver injury, prepared in embodiment 4 of the present invention.
Detailed Description
Specific implementations of the invention are further described below with reference to the drawings and examples, but the implementation and protection of the invention are not limited thereto. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art.
In the following examples, the in vitro ADH activation rate was determined using ADH kit (Nanjing, cat. No. A083-2, comprising reagent one, sodium pyrophosphate buffer, reagent two, ethanol, reagent three, NADH.) in combination with ADH enzyme solution, and the specific method was as follows: ADH kit buffer reagent one 0.65 mL (diluted by 1:1 ultrapure water), adding reagent three (powder, diluted by 10 mL ultrapure water before use) of 0.75 mL, then adding reagent two (ethanol) of 0.05 mL, and uniformly mixing to obtain a mixed solution; experimental group A was measured by adding 150. Mu.L of the mixture and 50. Mu.L of 0.5. 0.5 mg/mL of the polypeptide solution (blank group was replaced with water) to a 96-well plate, incubating at 37℃for 5min after mixing, then adding 20. Mu.L of 0.2U/mL of ADH enzyme solution, and counting from the time of adding the sample and mixing 340nm 10 s records one reading and measures continuously for 10 min. The time is plotted on the abscissa, A340nm is plotted on the ordinate, and the slope is the initial reaction rate, which can be used as the relative enzyme activity of ADH. ADH activation rate calculation formula:
ADH activation rate (%) = =. TheV s -V 0 )/V 0 ×100%
Wherein, the liquid crystal display device comprises a liquid crystal display device,V 0 is the initial reaction rate of the negative controlV S Is the initial reaction rate of the test sample.
In the following examples, the particle size was measured as follows: the OPH-Sepps obtained by adjusting and controlling under different conditions of 1.2 and mL are added into a measuring dish, and the particle size distribution is measured by a Nano-particle sizer (Nano-ZS & MPT-2 Nano-particle sizer, malvern Co., UK), the particle absorptivity is 0.001, the dispersing agent is water, the refractive index of the dispersing agent is 1.330, and the measuring temperature is 25 ℃, so that the average particle size (nm) and the particle size volume distribution are obtained.
Example 1
A preparation method of oyster polypeptide-nano selenium particle compound with anti-alcohol and liver-protecting effects comprises the following steps:
(1) Taking commercial oyster as a raw material, removing shells, taking meat, cleaning the meat, directly homogenizing (the homogenizing speed is 12000 r/min, the homogenizing time is 60 s), forming homogenate, adding water (the mass ratio of the homogenate to the water is 1:1), and adding Pancreatin for enzymolysis under the following conditions: pH 7.0, enzyme addition amount of 0.1% (w/w) (i.e. enzyme addition amount of 0.1% of oyster meat mass), reaction temperature of 60deg.C, and reaction time of 4. 4 h. After the enzymolysis reaction is finished, inactivating enzyme in boiling water bath for 10 min, centrifuging at 8000 r/min at 4deg.C for 30min, and lyophilizing the obtained supernatant (pre-lyophilization temperature of-40deg.C, material thickness of 7 mm, and drying time of 35 h) to obtain oyster polypeptide powder (OPH);
(2) Adding the oyster polypeptide powder obtained in the step (1) into water, uniformly mixing to prepare an oyster polypeptide solution (the concentration is 10 mg/mL), adding a sodium selenite solution with the concentration of 2 mM into the oyster polypeptide solution with the concentration of 10mg/mL, uniformly stirring, and then dropwise adding an ascorbic acid solution with the concentration of 8.0mM, wherein the ratio of the oyster polypeptide solution to the sodium selenite solution to the ascorbic acid solution is 1:4:4 (v/v/v), uniformly mixing (500 rpm) while dropwise adding, reacting at 25 ℃ under water bath for 0.5 h, and dialyzing at 4 ℃ for 48h by using a dialysis bag with the concentration of 100 Da to obtain the functionalized nano-selenium sol; and carrying out ultrasonic treatment on the functionalized nano selenium sol with ultrasonic output frequency of 20 kHz and ultrasonic time of 5min (controlling the temperature of a sample to be lower than 25 ℃ by using an ice bath in the ultrasonic process), and freeze-drying (pre-freezing temperature of-40 ℃, material thickness of 7 mm and drying time of 35 h) to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver. The morphology was observed by Transmission Electron Microscopy (TEM), and the result is shown in fig. 1, the composite was spherical particles with an average particle size of less than 200 nm. The effects of the other embodiments are similar to those of embodiment 1, and reference is made to fig. 1.
Example 2
A preparation method of oyster polypeptide-nano selenium particle compound with anti-alcohol and liver-protecting effects comprises the following steps:
(1) Taking commercial oyster as a raw material, removing shells, taking meat, cleaning the meat, directly homogenizing (the homogenizing speed is 12000 r/min, the homogenizing time is 60 s), forming homogenate, adding water (the mass ratio of the homogenate to the water is 1:1), and adding pancreatin Papan for enzymolysis under the following conditions: pH 5, enzyme addition amount is 1% (w/w) (i.e. enzyme addition amount is 1% of oyster meat mass), reaction temperature is 37 ℃, and reaction time is 12 h. After the enzymolysis reaction is finished, inactivating enzyme in boiling water bath for 15min, centrifuging at 10deg.C and 10000r/min for 10 min, and lyophilizing the obtained supernatant (pre-lyophilization temperature of-30deg.C, material thickness of 4 mm, and drying time of 24 h) to obtain oyster polypeptide powder (OPH);
(2) Adding the oyster polypeptide powder obtained in the step (1) into water, and uniformly mixing to prepare oyster polypeptide solution (the concentration is 5 mg/mL); adding a sodium selenite solution with the concentration of 0.5 mM into an oyster polypeptide solution with the concentration of 5mg/mL, uniformly stirring, then dropwise adding an ascorbic acid solution with the concentration of 2 mM, wherein the ratio of the oyster polypeptide solution to the sodium selenite solution to the ascorbic acid solution is 1:1:4 (v/v/v), uniformly mixing (100 rpm) while dropwise adding, reacting at the water bath of 45 ℃ for 12h, and dialyzing at the temperature of 10 ℃ for 36 h by using a dialysis bag of 1000Da to obtain the functionalized nano-selenium sol; and carrying out ultrasonic treatment on the functionalized nano selenium sol with ultrasonic output frequency of 50kHz and ultrasonic time of 15min (controlling the temperature of a sample to be lower than 25 ℃ by using an ice bath in the ultrasonic process), and freeze-drying (pre-freezing temperature of-30 ℃, material thickness of 4 mm and drying time of 24 h) to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver. The particle size distribution was measured by Zeta nanosize, and as shown in FIG. 2, the average particle size was less than 200 and nm. The effect of the other embodiments is similar to that of embodiment 2, and reference is made to fig. 2.
Example 3
A preparation method of oyster polypeptide-nano selenium particle compound with anti-alcohol and liver-protecting effects comprises the following steps:
(1) Taking commercial oyster as a raw material, removing shells, taking meat, cleaning the meat, directly homogenizing (the homogenizing speed is 12000 r/min, the homogenizing time is 60 s), forming homogenate, adding water (the mass ratio of the homogenate to the water is 1:2), and adding alkaline protease Alcalase 2.4L for enzymolysis, wherein the enzymolysis conditions are as follows: pH 11, enzyme addition amount of 0.4% (w/w) (i.e. enzyme addition amount of 0.4% of oyster meat mass), reaction temperature of 40deg.C, and reaction time of 0.5. 0.5 h. After the enzymolysis reaction is finished, inactivating enzyme in boiling water bath for 10 min, centrifuging at 10000r/min at 4deg.C for 15min, and lyophilizing the obtained supernatant (pre-lyophilization temperature of-35deg.C, material thickness of 5 mm, and drying time of 30 h) to obtain oyster polypeptide powder (OPH);
(2) Adding the oyster polypeptide powder obtained in the step (1) into water, uniformly mixing to prepare an oyster polypeptide solution (the concentration is 0.5 mg/mL), adding the oyster polypeptide solution with the concentration of 1.5 mM sodium selenite solution into the oyster polypeptide with the concentration of 0.5 mg/mL, uniformly stirring, and then dropwise adding an ascorbic acid solution with the concentration of 6 mM, wherein the proportion of the oyster polypeptide solution, the sodium selenite solution and the ascorbic acid solution is 1:1.4 (v/v), uniformly mixing (150 rpm) while dropwise adding, reacting at the water bath of 35 ℃ for 6 h, and dialyzing at the temperature of 4 ℃ for 36 h by using a dialysis bag of 500 Da to obtain the functionalized nano-selenium sol; and (3) carrying out ultrasonic treatment on the functionalized nano selenium sol with ultrasonic output frequency of 20 kHz and ultrasonic time of 10 min (controlling the temperature of a sample to be lower than 25 ℃ by using an ice bath in the ultrasonic process), and freeze-drying (pre-freezing temperature of-35 ℃, material thickness of 5 mm and drying time of 24 h) to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver.
Under the same protein concentration, compared with Oyster Peptide (OPH), the oyster polypeptide-nano selenium particle compound (OPH-SeNPs) with the effects of dispelling effects of alcohol and protecting liver has obviously higher activation rate of ADH by the OPH-SeNPs than by Oyster Peptide (OPH), and the results are shown in figure 3. The protection effect of oyster peptide and oyster polypeptide-nano selenium particle complex on alcoholic liver injury was detected by MTT, and the result is shown in figure 4. Other embodiments have similar effects to embodiment 3, and reference is made to fig. 3 and 4.
Example 4
A preparation method of oyster polypeptide-nano selenium particle compound with anti-alcohol and liver-protecting effects comprises the following steps:
(1) Taking commercial oyster as a raw material, removing shells, taking meat, cleaning the meat, directly homogenizing (the homogenizing speed is 12000 r/min, the homogenizing time is 60 s), forming homogenate, adding water (the mass ratio of the homogenate to the water is 1:4), and adding Pancreatin for enzymolysis under the following conditions: pH 7.0, enzyme addition amount of 1% (w/w) (representing that enzyme addition amount is 1% of oyster meat mass), reaction temperature of 55deg.C, and reaction time of 2 h. After the enzymolysis reaction is finished, inactivating enzyme in boiling water bath for 15min, centrifuging at 10deg.C for 30min at 10000r/min, and lyophilizing the obtained supernatant (pre-lyophilization temperature of-35deg.C, material thickness of 5 mm, and drying time of 30 h) to obtain oyster polypeptide powder (OPH);
(2) Adding the oyster polypeptide powder obtained in the step (1) into water, uniformly mixing to prepare an oyster polypeptide solution (the concentration is 5 mg/mL), adding the oyster polypeptide solution with the concentration of 1.5 mM sodium selenite solution into the oyster polypeptide with the concentration of 5mg/mL, uniformly stirring, then dropwise adding an ascorbic acid solution with the concentration of 6 mM, wherein the ratio of the oyster polypeptide solution to the sodium selenite solution to the ascorbic acid solution is 1:4:4 (v/v/v), uniformly mixing (150 rpm) while dropwise adding, reacting at 25 ℃ under water bath for 6 h, and dialyzing at 4 ℃ for 36 h by using a dialysis bag of 500 Da to obtain the functionalized nano-selenium sol; and carrying out ultrasonic treatment on the functionalized nano selenium sol with ultrasonic output frequency of 50kHz and ultrasonic time of 10 min (controlling the temperature of a sample to be lower than 25 ℃ by using an ice bath in the ultrasonic process), and freeze-drying (pre-freezing temperature of-35 ℃, material thickness of 5 mm and drying time of 30 h) to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver.
The protection effect of oyster polypeptide-nano selenium particle complex (OPH-SeNPs) with anti-hangover and liver protecting effects on hydrogen peroxide-induced liver injury is detected by MTT method, and the result is shown in figure 5. Other embodiments have similar effects to those of embodiment 4, and reference is made to fig. 5.
As can be seen from the above examples, the oyster peptide obtained by enzymolysis with Pancreatin, 4 h is used as a template, and after being stirred uniformly with 2 mM sodium selenite solution at a concentration of 10mg/mL, 8 mM ascorbic acid is added dropwise for reacting for a period of time (wherein the volume ratio of oyster polypeptide, sodium selenite and ascorbic acid is 1:4:4), and ultrasound is carried out for 5min, so as to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver, which is approximately spherical particles, and the average particle size of the oyster polypeptide-nano selenium particle compound is less than 200nm (fig. 1). And (3) performing enzymolysis by using Papan, taking oyster peptide obtained by 12h as a template, uniformly stirring with 0.5 mM sodium selenite solution at the concentration of 5mg/mL, dropwise adding 2 mM ascorbic acid to react for a period of time (wherein the volume ratio of oyster polypeptide to sodium selenite to ascorbic acid is 1:1:4), and performing ultrasonic treatment for 15min to obtain the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver, wherein the average particle size is smaller than 200nm (figure 2). The oyster peptide obtained by carrying out enzymolysis on Alcalase 2.4L and taking 0.5 h as a template is uniformly stirred with 1.5 mM sodium selenite solution at the concentration of 0.5 mg/mL, after the reaction of dropwise adding 6 mM ascorbic acid for a period of time (wherein the volume ratio of oyster polypeptide to sodium selenite to ascorbic acid is 1:1:6.4), the oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting liver is obtained after ultrasonic treatment for 10 min, the ADH activation rate activity of the oyster polypeptide-nano selenium particle compound is better than that of the oyster peptide at the same protein concentration (figure 3), and the protective effect of the oyster polypeptide-nano selenium particle compound on alcohol-induced liver injury is detected by an MTT method, and the result is shown in figure 4. The oyster peptide obtained by taking Pancreatin pancratin as a template and 2h is uniformly stirred with 1.5 mM sodium selenite solution at the concentration of 5mg/mL, after being dropwise added with 6 mM ascorbic acid to react for a period of time (wherein the volume ratio of oyster polypeptide to sodium selenite to ascorbic acid is 1:4:4), ultrasonic treatment is carried out for 10 min, and the oyster polypeptide-nano selenium particle compound with the effects of dispelling effects of alcohol and protecting liver is obtained, and the effect of the oyster polypeptide-nano selenium particle compound on protecting liver induced by hydrogen peroxide is detected by MTT (methyl thiazolyl tetrazolium sulfonate), and the result is shown in figure 5.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.
Claims (5)
1. An oyster polypeptide-nano selenium particle compound with the effects of dispelling the effects of alcohol and protecting the liver is prepared by the following method, wherein the oyster polypeptide-nano selenium particle compound prepared by the following method is spherical particles with the particle size of less than 200nm, and the selenium content is more than 40%; the specific preparation method comprises the following steps:
(1) Removing shells of oysters, taking meat, washing oyster meat, homogenizing to obtain homogenate, adding water and active enzyme, and performing enzymolysis reaction to obtain enzymolysis liquid; the active enzyme is one of papain, flavourzyme, alkaline protease, bromelain, pancreatin, composite protease and neutral protease, and the mass of the active enzyme is 0.1-1wt% of that of oyster meat; the temperature of the enzymolysis reaction is 37-60 ℃, the time of the enzymolysis reaction is 0.5-12h, and the pH value of the enzymolysis reaction is 5-11; the mass ratio of the homogenate to the water is 1:1-1:4;
(2) Treating the enzymolysis liquid in the step (1) in boiling water bath, centrifuging to obtain supernatant, and freeze-drying the supernatant to obtain oyster polypeptide powder;
(3) Adding the oyster polypeptide powder obtained in the step (2) into water, uniformly mixing to prepare an oyster polypeptide solution, adding a sodium selenite solution into the oyster polypeptide solution, and uniformly stirring to obtain a mixed solution; the volume ratio of the sodium selenite solution to the oyster polypeptide solution is 1:1-4:1; the concentration of the oyster polypeptide solution is 0.5-10mg/mL; the concentration of the sodium selenite solution is 0.5-2.0mM;
(4) Dropwise adding an ascorbic acid solution into the mixed solution obtained in the step (3) under the stirring state, uniformly mixing, carrying out water bath reaction, and carrying out dialysis treatment to obtain a functionalized nano selenium sol; the temperature of the water bath reaction is 25-45 ℃, and the time of the water bath reaction is 0.5-12h; the specification of a dialysis bag used for the dialysis treatment is 500-1000Da; the dialysis treatment time is 36-48 hours; the temperature of the dialysis treatment is 4-10 ℃; the concentration of the ascorbic acid solution is 1.0-8.0mM; the volume ratio of the mixed solution to the ascorbic acid solution is 1:2-5:16;
(5) Performing ultrasonic treatment on the functionalized nano-selenium sol obtained in the step (4), and freeze-drying to obtain the oyster polypeptide-nano-selenium particle compound with the effects of dispelling the effects of alcohol and protecting the liver; the ultrasonic frequency of the ultrasonic treatment is 20-50kHz, the ultrasonic treatment time is 5-15min, and the ultrasonic treatment temperature is less than 25 ℃.
2. The oyster polypeptide-nano-selenium particle complex with anti-hangover and liver-protecting effects according to claim 1, wherein the homogenization rate in step (1) is 10000-15000r/min and the homogenization time is 30-60s.
3. The oyster polypeptide-nano-selenium particle complex with anti-hangover and liver-protecting effects according to claim 1, wherein the boiling water bath treatment time of step (2) is 10-15min; the temperature of the centrifugation is 4-10 ℃, the rotating speed of the centrifugation is 8000-10000r/min, and the time of the centrifugation is 10-30min.
4. The oyster polypeptide-nano-selenium particle complex with anti-hangover and hepatoprotective effects according to claim 1, wherein the stirring rate in the stirring state of step (4) is 100-500rpm.
5. The use of oyster polypeptide-nano selenium particle complex with anti-hangover and liver protecting effects in preparing functional food or health care product according to claim 1.
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