CN115651087A - Preparation method and application of nano selenium enteromorpha polysaccharide - Google Patents
Preparation method and application of nano selenium enteromorpha polysaccharide Download PDFInfo
- Publication number
- CN115651087A CN115651087A CN202210963000.6A CN202210963000A CN115651087A CN 115651087 A CN115651087 A CN 115651087A CN 202210963000 A CN202210963000 A CN 202210963000A CN 115651087 A CN115651087 A CN 115651087A
- Authority
- CN
- China
- Prior art keywords
- selenium
- nano
- enteromorpha polysaccharide
- enteromorpha
- liver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of preparation of nano-selenium enteromorpha polysaccharide, and discloses a preparation method and application of nano-selenium enteromorpha polysaccharide, wherein the preparation method of the nano-selenium enteromorpha polysaccharide comprises the following steps: s1: adding a certain volume of 10mM sodium selenite solution and a certain volume of enteromorpha polysaccharide solution into a beaker, and stirring at a proper temperature by using a constant-temperature magnetic stirrer; s2: adding a certain volume of 10mM ascorbic acid solution into a beaker, and continuously reacting for a period of time to obtain a nano selenium enteromorpha polysaccharide solution; s3: after the reaction is finished, the prepared nano selenium enteromorpha polysaccharide solution is put into a refrigerator for pre-freezing, then a freeze dryer is used for freeze-drying the nano selenium enteromorpha polysaccharide solution into powder, a scanning electron microscope is used for observing the shape and the particle size of the nano selenium, and an energy spectrometer is used for analyzing the element type and the content of the nano selenium enteromorpha polysaccharide. The invention has the following advantages and effects: the composition has obvious relieving effect on liver injury of AFB1 exposed broiler chicken.
Description
Technical Field
The invention relates to the technical field of preparation of nano-selenium enteromorpha polysaccharide, in particular to a preparation method and application of nano-selenium enteromorpha polysaccharide.
Background
The feed hygiene is an important production problem affecting the healthy breeding of the broilers, especially the feed toxin pollution. Statistically, most of the grains contaminated with Mycotoxins (Mycotoxins) are contaminated with Aflatoxins (AF) in the world. According to the investigation of the existence condition of mycotoxin in feed raw materials in China, the AF pollution rate of the corn can reach more than 80 percent. AF is produced by metabolism of aspergillus flavus and aspergillus parasiticus, and the aspergillus is most likely to produce AF in an environment with the temperature of about 30 ℃ and the humidity of about 80%. Under the background of continuous global temperature rise, the high-temperature and high-humidity climate provides a favorable environment for the generation of AF, so that the problem of AF pollution of feed is increasingly severe. Therefore, the development of an economical and effective method for relieving the adverse effect of feed AF pollution on broiler chickens is an important technical requirement of production practice.
Among the discovered AF, AFB1 is the most toxic and widely distributed, and is classified as a class I carcinogen. AFB1 has cytotoxicity, and can induce organism to generate oxidative stress, which causes organ damage. It has been reported that the toxic effects of AFB1 on animals are mainly manifested by liver damage and immunotoxicity, and broiler chickens are highly susceptible to AFB 1. Researches find that the broiler chicken fed with the feed polluted by AFB1 causes acute liver injury and even death, and huge economic loss is brought to poultry industry. The liver is one of the most important detoxification and metabolic organs of animals and is also the main target organ for the toxic effects of AFB 1. Although the broiler chicken has certain detoxification metabolic capability on AFB1, the organs of the broiler chicken are incompletely developed, and the development and normal functions of the liver of the broiler chicken can be influenced by long-term AFB1 exposure. The existing method for detoxifying feed raw material AFB1 mainly comprises the following steps: physical detoxification, chemical detoxification, biological detoxification and combined application types. Biological detoxification refers to a safe and non-side-effect method for improving the detoxification capability of organisms by utilizing bioactive substances and a nutrition regulation technology. Therefore, how to develop a high-efficiency biological antidote to improve the detoxification capability of the broiler chicken exposed by AFB1 becomes a scientific problem to be solved urgently.
Selenium is an indispensable trace mineral element for poultry, and plays multiple biological functions by regulating the Expression of Selenoprotein (SELENOs) of the body, including improving the activity of phase II detoxification enzymes, scavenging free radical ions and promoting the metabolism and discharge of toxins, protecting cells from damage. The main forms of the nutritional selenium supplement include inorganic selenium and organic selenium, the inorganic selenium has high toxicity, and the organic selenium has low toxicity and high nutritional value and is easy to be absorbed by animal organisms. Research shows that the selenium supplement in the two forms can reduce the liver injury of the broiler chickens induced by AFB1 to a certain extent, and the organic form is superior to the inorganic form. The dosage of the nutritional selenium supplement is difficult to control, and poisoning can be caused by slight overdose. Compared with inorganic selenium and organic selenium, the nano selenium is the currently discovered selenium supplement form with the lowest toxicity, and has better bioavailability and higher bioactivity. In addition, the nano-selenium has small particle size, many unsaturated bonds on surface atoms, high activity, high adhesion to gastrointestinal mucosa and easy absorption. However, the stability of the nano-selenium is poor, and the nano-particles are easy to aggregate into precipitates in the preparation process. The preparation method of the nano-selenium comprises a polymer template method, a microemulsion method, a surfactant method, an ultrasonic chemical method and the like. At present, a polymer template method is mostly adopted for preparing the nano selenium, and the method has the advantages of easily obtained raw materials, simple and easy operation, good biocompatibility and the like. The branched structure of the polysaccharide is complex, contains a plurality of active groups, and can adhere to and wrap nanoparticles formed in the initial reduction reaction, thereby preventing the nanoparticles from growing and agglomerating. The polysaccharide is used as the template to prepare the nano-selenium, so that the nutritional function of the selenium element can be fully exerted, the active effect of the polysaccharide can be utilized, the biological effect of the nano-selenium is obviously better than that of the common nano-selenium, and the nano-selenium is an ideal choice for preparing the nano-selenium compound at present. Enteromorpha is low-value algae rich in polysaccharide, and if polysaccharide polymers in the Enteromorpha are developed and applied to animal nutrition, the feed and high-value utilization of marine algae resources can be realized, and the national marine economic strategy is met. Researches show that the enteromorpha polysaccharide shows excellent medicinal value in the aspects of oxidation resistance, virus resistance, bacteria resistance, immunity improvement and the like
Disclosure of Invention
The invention aims to provide a preparation method and application of nano-selenium enteromorpha polysaccharide, which has the effect of remarkably relieving liver injury of the broiler chicken exposed to AFB1, and has better protection effect on liver injury of the broiler chicken induced by AFB1 compared with sodium selenite (Na 2SeO3, inorganic selenium), selenomethionine (SeMet, organic selenium), common nano-selenium (NPSe) and Enteromorpha Polysaccharide (EPS).
The technical purpose of the invention is realized by the following technical scheme: the preparation method of the nano selenium enteromorpha polysaccharide comprises the following steps:
s1: adding a certain volume of 10mM sodium selenite solution and a certain volume of enteromorpha polysaccharide solution into a beaker, and stirring at a proper temperature by using a constant-temperature magnetic stirrer;
s2: adding a certain volume of 10mM ascorbic acid solution into a beaker, and continuously reacting for a period of time to obtain a nano selenium enteromorpha polysaccharide solution;
s3: after the reaction is finished, the prepared nano selenium enteromorpha polysaccharide solution is placed into a refrigerator for pre-freezing, then a freeze dryer is used for freeze-drying the nano selenium enteromorpha polysaccharide solution into powder, a scanning electron microscope is used for observing the shape and the particle size of the nano selenium, and an energy spectrometer is used for analyzing the element type and the content of the nano selenium enteromorpha polysaccharide.
The invention is further configured as follows: in the S1, the stirring time is set to be 2h.
The invention is further provided with: in S1 and S2, the volume ratio of the ascorbic acid solution to the sodium selenite solution is 3.
The invention is further provided with: and in the S3, the temperature in the refrigerator is set to be-80 ℃.
The invention is further provided with: and in the S3, the pre-freezing time is more than 12 h.
The application of the nano-selenium enteromorpha polysaccharide and the nano-selenium enteromorpha polysaccharide prepared by the preparation method of the nano-selenium enteromorpha polysaccharide are used for healthy breeding of broiler chickens.
The invention has the beneficial effects that:
the nano selenium-enteromorpha polysaccharide (NPSe-EPS) prepared by the invention has an obvious effect of relieving liver injury of the broiler chicken exposed to AFB1, and has a better protection effect on liver injury of the broiler chicken induced by AFB1 compared with sodium selenite (Na 2SeO3, inorganic selenium), selenomethionine (SeMet, organic selenium), common nano selenium (NPSe) and Enteromorpha Polysaccharide (EPS).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be understood that the drawings in the following description are illustrative only, and that the structures, proportions, sizes, and other elements shown in the drawings are incorporated herein by reference in their entirety for all purposes in the present disclosure, which are not intended to limit the scope of the invention, which is to be construed as limiting the invention in any manner, and not necessarily for all purposes, except as technically essential.
Fig. 1 is a schematic diagram of the prepared nano-selenium-enteromorpha polysaccharide solution and the nano-selenium solution after being respectively placed for 24 hours.
Fig. 2 is SEM images of nano-selenium-enteromorpha polysaccharide prepared at different temperatures.
FIG. 3 is a scanning analysis diagram of the energy spectrum plane of nano selenium-Enteromorpha polysaccharide.
Fig. 4 is a histopathological observation of liver.
FIG. 5 is a graph of the effect of different selenium sources on the indicators of oxidative damage to the liver of broiler chickens exposed to AFB 1.
Fig. 6 is a histopathological observation of the liver.
FIG. 7 is a graph of the effect of nano-selenium-Enteromorpha polysaccharide (NPSe-EPS), common nano-selenium (NPSe) and Enteromorpha Polysaccharide (EPS) on liver oxidative damage indicators of broiler chicken exposed to AFB 1.
FIG. 8 is a graph of the effect of nano-selenium-Enteromorpha polysaccharide (NPSe-EPS), common nano-selenium (NPSe) and Enteromorpha Polysaccharide (EPS) on liver tissue structure damage of broiler chicken exposed to AFB 1.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Referring to fig. 1-8, the invention provides a preparation method of nano selenium enteromorpha polysaccharide, which comprises the following steps:
s1: adding a certain volume of 10mM sodium selenite solution and a certain volume of enteromorpha polysaccharide solution into a beaker, and stirring at a proper temperature by using a constant-temperature magnetic stirrer for 2 hours;
s2: adding a certain volume of 10mM ascorbic acid solution into a beaker, wherein the volume ratio of the ascorbic acid solution to the sodium selenite solution is 3;
s3: after the reaction is finished, the prepared nano selenium enteromorpha polysaccharide solution is put into a refrigerator for pre-freezing, the temperature in the refrigerator is set to be 80 ℃ below zero, the pre-freezing time is more than 12 hours, then a freeze dryer is used for freeze-drying the nano selenium polysaccharide solution into powder, a scanning electron microscope is used for observing the shape and the particle size of the nano selenium, and an energy spectrometer is used for analyzing the element type and the content of the nano selenium enteromorpha polysaccharide.
The application of the nano-selenium enteromorpha polysaccharide, the nano-selenium enteromorpha polysaccharide prepared by the preparation method of the nano-selenium enteromorpha polysaccharide, is used for the healthy breeding of broiler chickens.
Research on stability and influence factors of nano selenium-enteromorpha polysaccharide:
fixing the final concentration of sodium selenite in a 10mL reaction system to be 1mM, researching the influence of polysaccharide concentration, time and temperature on the stability of the nano-selenium, and screening the optimal preparation conditions by using the absorbance value ratio (A410/490) of the prepared sample solution at the wavelengths of 410nm and 490nm, wherein the larger the ratio is, the smaller the particle size of the nano-selenium is, and the higher the stability is.
Polysaccharide concentration (A), reaction time (B) and reaction temperature (C) were selected and the design of the experiments was performed according to the L25 (53) orthogonal table, with the levels of the orthogonal test factors as shown in the following table:
the SPSS 23 was used to design an orthogonal experiment, the design and results of which are shown in the following table:
analysis of variance was performed using SPSS 23 software, and the results are shown in the following table:
from the results of the anova, the reaction temperature has a significant effect on the stability of the nano-selenium (P < 0.05). The main and secondary relationship of the three factors is C > B > A, and the optimal preparation temperature is 60 ℃ by a single-factor system meter (shown in the following table).
Effect of polysaccharide concentration
The polysaccharide concentration is set to six gradients of 0, 100, 200, 300, 400, 500mg/L, and other conditions are: the reaction time is 1h, the reaction temperature is 60 ℃, and the following table shows the light absorption values of the nano selenium-enteromorpha polysaccharide solutions prepared from enteromorpha polysaccharides with different concentrations:
as can be seen from the table above, when the reaction temperature is 60 ℃, the reaction time is 1h, and the polysaccharide concentration reaches 400mg/L, the A410/490 reaches the maximum value and tends to be balanced, and in combination with economic benefits, 400mg/L is selected as the optimal polysaccharide concentration for preparing the nano selenium-enteromorpha polysaccharide.
Influence of reaction time
The reaction time is set to five gradients of 0.5, 1, 3, 5 and 7h, and other conditions are as follows: the polysaccharide concentration is 400mg/L, the reaction temperature is 60 ℃, and the following table shows the light absorption values of the nano selenium-enteromorpha polysaccharide solution prepared by different reaction times:
as can be seen from the above table, when the reaction temperature is 60 ℃, the polysaccharide concentration is 400mg/L, and the reaction time is 0.5, 1, 3, 5h, the A410/490 difference is not significant (P > 0.1), and is significantly higher than the A410/490 (P < 0.05) at 7 h.
And (3) integrating the orthogonal design test result and the single-factor investigation result, and selecting the optimal preparation conditions as follows: the polysaccharide concentration is 400mg/L, the reaction temperature is 60 ℃, and the reaction time is 1h.
After the nano-selenium-enteromorpha polysaccharide solution and the nano-selenium solution prepared at the above temperatures are respectively placed for 24 hours, the obvious brick red precipitation of the nano-selenium solution can be seen, and the nano-selenium-enteromorpha polysaccharide solution is still clear. Fig. 1a and 1b are respectively a freshly prepared nano-selenium solution and a nano-selenium-enteromorpha polysaccharide solution, and fig. 1c and 1d are respectively a nano-selenium solution and a nano-selenium-enteromorpha polysaccharide solution after being placed for 24 hours.
Scanning electron microscope and energy spectrum analysis results
And (3) placing the nano-selenium enteromorpha polysaccharide prepared at different temperatures after freeze drying under a scanning electron microscope to observe the morphology and the particle size of the nano-selenium. From the images observed under a scanning electron microscope (fig. 2a, 20 ℃, 2b, 40 ℃, 2c, 60 ℃, 2d. The nano-selenium particles prepared at 20 ℃ and 40 ℃ have different particle sizes, and when the reaction temperature is 60 ℃, the nano-selenium particles are dispersed on the surface of the polysaccharide and have more uniform particle sizes of about 60 nm. At 80 ℃, the product is agglomerated to form a large number of irregular spherical and rod-shaped particles. The energy spectrum surface scanning result (figure 3) shows that the nano selenium enteromorpha polysaccharide prepared by taking the concentration of the enteromorpha polysaccharide as 400mg/L, the reaction time of 60 ℃ as the reaction condition is 18 percent of carbon element (C), 34 percent of oxygen element (O), 23 percent of sodium element (Na), 5 percent of sulfur element (S), 8 percent of chlorine element (Cl) and 12 percent of selenium element (Se).
In summary, the research successfully prepares the nano-selenium enteromorpha polysaccharide by taking the enteromorpha polysaccharide as the template, and the optimal preparation conditions are as follows: the concentration of the enteromorpha polysaccharide is 400mg/L, the reaction temperature is 60 ℃, and the reaction time is 1h; the selenium content of the nano selenium enteromorpha polysaccharide prepared under the optimal conditions obtained in the test is about 12 percent.
Application of nano selenium-enteromorpha polysaccharide
Principal material
The nano selenium-enteromorpha polysaccharide is prepared in a laboratory, and the selenium content is about 10 percent; AFB1 was purchased from Biotechnology, inc. of Kinzau Von, nanjing; total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione S-transferase (GST) and Malondialdehyde (MDA) test boxes are all purchased from Nanjing to build the institute of bioengineering, limited company; the red blood oxygenase 1 (HO-1) and cytochrome P450 (CYP 450) test kits were purchased from Jiangsu enzyme Immunity Co.
Main instrument
A Chemray 800 type full-automatic biochemical analyzer, a KZ-II type high-speed tissue grinder, a D3024R type desk-top high-speed refrigerated centrifuge, a Synergy HTX type multifunctional enzyme labeling instrument and the like.
Experimental design and feeding management
Selecting 180 AA male broilers with age of 1 day, similar body weight and good health condition, dividing the AA male broilers into 5 groups according to a completely random test design, treating 6 chickens each, and repeating 10 chickens each. The control group (group A) was fed with a basal diet, and the other test groups were fed with 0.1mg/kg of AFB1 (group B), 0.1mg/kg of AFB1+2mg/kg of NPSe-EPS (group C, containing about 0.2mg/kg of Se), 0.1mg/kg of AFB1+4mg/kg of NPSe-EPS (group D, containing about 0.4mg/kg of Se), and 0.1mg/kg of AFB1+6mg/kg of NPSe-EPS (group E, containing about 0.6mg/kg of Se), respectively, based on the basal diet. The experimental basal diet was formulated with reference to NRC (1994), with the diet composition and nutrient levels shown in tables 2-1, with the experimental period of 21 days, and the basal diet composition and nutrient levels shown in the following table:
1 premix feed per kilogram feed: vitamin A5000 IU, vitamin D 3 2500IU, vitamin E1.3 IU, vitamin B 2 2μg。
2 Premix feed provided per kilogram of diet: 75mg of zinc, 100mg of manganese, 80mg of iron, 8mg of copper and 0.35mg of potassium iodide.
3 The other values were measured except for metabolic energy.
Sample collection
Randomly selecting one chicken with the weight similar to that of each repeated cage from each repeated cage when the broiler is 21 days old, performing wing vein blood collection, separating serum, and storing in a refrigerator at-20 ℃. After slaughter, liver tissue was taken, partially fixed with 4% paraformaldehyde for slicing, and partially stored at-80 ℃.
Detection index and method
Biochemical index of serum
Randomly selecting one chicken from each repeated cage, collecting blood by using the infrawing vein for about 3mL, centrifuging at 3000r/min for 15min, and separating serum. Serum liver function-related indices including Total Protein (TP), albumin (ALB), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), serum alkaline phosphatase (ALP), total bilirubin (T-BIL), direct bilirubin (T-BIL), gamma glutamyl transpeptidase (gamma-GT), and Total Bile Acid (TBA) were determined using a fully automated biochemical analyzer.
Liver oxidation resistance and detoxification related indexes
Collecting liver tissue 0.2-1g, rinsing with pre-cooled normal saline, removing blood, placing into a small beaker, adding pre-cooled normal saline (the volume of normal saline is 9 times of tissue weight), and grinding into 10% tissue homogenate with tissue grinder. Centrifuging the prepared tissue homogenate at low temperature of about 3000r/min for 10-15min, and taking supernatant for enzyme activity determination. The protein content of liver tissue, total antioxidant capacity in liver (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione S-transferase (GST), hemoxygenase 1 (HO-1), cytochrome P450 (CYP 450) activity and Malondialdehyde (MDA) were determined according to the kit instructions.
Morphological analysis of liver tissue
The collected liver tissues were fixed in 4% paraformaldehyde for sectioning. hematoxylin-Eosin staining (Haematxylin and Eosin, h.e.) was used and pathological changes were observed under a common light microscope.
Statistical analysis
The SAS 9.4 software is used for analyzing the test data, the Tukey's method is used for analyzing the difference significance, and P <0.05 represents the difference significance.
The influence of the nano-selenium-enteromorpha polysaccharide on the serum biochemical index (liver function) of the broiler chicken exposed to the AFB1 is shown in the following table:
as can be seen from the above table, AFB1 significantly increased ALP, DBIL and γ -GT activities in the broiler serum (P < 0.05) compared to the control group. Compared with the AFB1 group, the addition of 2 and 4mg/kg of NPSe-EPS to the daily ration can reduce the activity of ALP, DBIL and gamma-GT and restore the activity to the level of a control group.
The influence of the nano-selenium-enteromorpha polysaccharide on the oxidation resistance and the detoxification capability of the livers of the broiler chickens exposed to the AFB1 is shown in the table:
as can be seen from the above table, the AFB1 group broiler livers had significantly reduced GST and HO-1 activity (P < 0.05), while the CYP450 activity was significantly increased (P < 0.05) compared to the control group. Compared with the AFB1 group, the addition of 2, 4 and 6mg/kg of NPSe-EPS in the daily ration enables the CYP450 activity in the liver of the broiler chicken to be remarkably reduced (P is less than 0.05). In addition, there was no significant difference in GST activity in the livers of broiler chickens in groups D and E compared to the control group (P > 0.05).
Morphological analysis of liver tissue
From the pathological section of liver (FIG. 4), the liver was red-stained parenchymal tissue by visual observation. Under the low power microscope, compared with the control group, the AFB1 group has disordered lobular structure of the liver, widened liver cords, disordered arrangement and difficult identification, and the liver blood sinuses become narrow or even disappear. Under a high power microscope, the volume of hepatic cells in the AFB1 group is increased, and the hepatic cells contain red-dyed fine particles; the cytoplasm becomes loose and empty, and the local part has circular vacuoles with different sizes and uneven distribution, and partial cell nucleus is extruded to one side and the staining becomes shallow. The liver cells of the broiler chickens in the group D are arranged regularly, the vacuole phenomenon is relieved, and the improvement effect is realized to a certain extent compared with that of the group AFB1, so that the NPSe-EPS has a protection effect on liver tissues of the broiler chickens exposed by the AFB 1.
Comparative study on the detoxifying effect of nano selenium-enteromorpha polysaccharide, inorganic selenium (Na 2SeO 3) and organic selenium (selenomethionine) on the liver of AFB 1-exposed broiler chicken:
principal material
Nano selenium-enteromorpha polysaccharide (NPSe-EPS), which is prepared by laboratories, and has a selenium content of about 10%; feed-grade sodium selenite (Na 2SeO 3) with a selenium content of 1%; L-Selenomethionine (L- (+) -Selenomethionine, seMet) with a selenium content of 2000ppm. AFB1 was purchased from Biotech, inc. of Nanjing Novozam; total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) and Malondialdehyde (MDA) test boxes are purchased from Nanjing institute of bioengineering research, inc.
Main instrument
A KZ-II type high-speed tissue grinder, a D3024R type desk-top high-speed refrigerated centrifuge, a Synergy HTX type multifunctional microplate reader and the like.
Experimental design and feeding management
180 AA male broilers which are 1 day old, similar in weight and good in health condition are selected and divided into 5 groups according to complete random test design, each group is treated for 6 times, and each group is 10 chickens. The control group (CON) was fed with the basic ration, and the other test groups were fed with 0.1mg/kg of AFB1, 0.1mg/kg of AFB1+4mg/kg of NPSe-EPS (containing Se 0.4 mg/kg), 0.1mg/kg of AFB1+40mg/kg of sodium selenite (containing Se 0.4 mg/kg), and 0.1mg/kg of AFB1+200mg/kg of SeMet (containing Se 0.4 mg/kg), respectively, based on the basic ration. The basal diet was formulated with reference to NRC (1994), with the composition and nutritional levels of the diets as in Table 2-1, for a 21 day test period.
Sample collection
Randomly selecting one chicken with the weight similar to that of each repeated cage from each repeated cage when the broiler is 21 days old, performing wing vein blood collection, separating serum, and storing in a refrigerator at-20 ℃. After slaughter, liver tissue was taken, partially fixed with 4% paraformaldehyde for slicing, and partially stored at-80 ℃ for analysis.
Detection index and method
Indexes related to liver oxidative damage
Taking 0.2-1g of liver tissue, rinsing with pre-cooled normal saline, placing into a small beaker after removing blood, adding pre-cooled normal saline (the volume of the normal saline is 9 times of the weight of the tissue), and grinding the liver tissue into 10% tissue homogenate by using a tissue grinder. Centrifuging the prepared tissue homogenate at low temperature of about 3000r/min for 10-15min, and taking the supernatant for enzyme activity determination. Total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) activity and Malondialdehyde (MDA) content in the liver were determined according to the kit instructions.
Morphological analysis of liver tissue
The collected liver tissues were fixed in 4% paraformaldehyde for sectioning. hematoxylin-Eosin staining (Haematxylin and Eosin, h.e.) was used and pathological changes were observed under a common light microscope.
Statistical analysis
And (3) analyzing the test data by using SAS 9.4 software, and analyzing the difference significance by using a Tukey's method, wherein P <0.05 represents that the difference is significant.
Effect of different selenium-derived feed additives on T-AOC of broiler liver exposed to AFB1
As can be seen from FIG. 5A, 100mg/kg AFB1 had no significant effect on T-AOC of broiler liver (P > 0.05). Compared with the AFB1 group, the liver T-AOC (P > 0.05) is not affected significantly by adding the feed additives in the three selenium forms. When 4mg/kg NPSe-EPS was added, the T-AOC of the broiler liver was slightly higher than that of the Na2SeO3 and SeMet groups and higher than that of the AFB1 group.
Effect of different selenium source feed additives on CAT activity of liver of broiler chicken exposed to AFB1
As can be seen from FIG. 5B, AFB1 significantly reduced CAT activity in the liver of broiler chickens (P < 0.05) compared to the control group, while all three forms of selenium supplementation resulted in significantly increased CAT activity in the liver (P < 0.05) compared to the AFB1 group, wherein the effect of NPSe-EPS is slightly better than that of Na2SeO3 and SeMet.
Influence of different selenium source feed additives on T-SOD activity of broiler liver exposed by AFB1
As can be seen from fig. 5C, the addition of AFB1 to the diet significantly reduced T-SOD activity in broiler livers (P < 0.05) compared to the control group, while T-SOD activity in broiler livers of NPSe-EPS group was significantly higher than that of AFB1 group (P < 0.05) and that of Na2SeO3 group and SeMet group (P < 0.05).
Influence of different selenium source feed additives on MDA content of liver of broiler chicken exposed by AFB1
As shown in fig. 5D, the liver of broiler chickens in AFB1 group had a significant increase in MDA content (P < 0.05) compared to the control group. The three forms of selenium supplementation all significantly reduced the MDA content in the liver (P < 0.05), and the NPSe-EPS group had the lowest MDA content.
The results show that the nano selenium-enteromorpha polysaccharide (NPSe-EPS) can effectively relieve AFB 1-induced liver oxidative damage of broiler chickens, and the action effect is superior to that of sodium selenite (Na 2SeO3, inorganic selenium) and selenomethionine (SeMet, organic selenium).
Influence of different selenium source feed additives on liver tissue morphology of broiler chicken exposed by AFB1
Pathological and histological changes of liver of broilers are shown in fig. 6, liver cells of healthy broilers are arranged regularly, and the shapes of cell nuclei are regular. The liver cells in the AFB1 group are disorganized, fuzzy in structure, red in cytoplasm, infiltrated by inflammatory cells mainly accompanied by lymphocytes locally, irregular in shape of cell nucleus and capable of generating vacuolization. The overall lesion of liver tissues of the three selenium supplement groups is obviously reduced, which indicates that NPSe-EPS, na2SeO3 and SeMet can relieve the damage of AFB1 to morphological structures of liver cells to a certain extent, and the nano selenium-enteromorpha polysaccharide (NPSe-EPS) has a better protection effect on the liver tissue structure damage of the broiler induced by AFB1 compared with sodium selenite (Na 2SeO3, inorganic selenium) and selenomethionine (SeMet, organic selenium).
By combining the results of liver oxidative damage and histopathology, the nano selenium-enteromorpha polysaccharide (NPSe-EPS) prepared by the invention has better protection effect on liver damage of the broiler induced by AFB1 compared with inorganic selenium (sodium selenite, na2SeO 3) and organic selenium (selenomethionine, seMet).
Comparative study on the detoxifying effect of the liver of the AFB 1-exposed broiler chicken by using the nano-selenium-enteromorpha polysaccharide, the common nano-selenium and the enteromorpha polysaccharide:
principal material
Nano selenium-enteromorpha polysaccharide (NPSe-EPS) and common nano selenium (NPSe) are prepared by laboratories, and the selenium content is about 10 percent; enteromorpha Polysaccharide (EPS) is purchased from Qingdao sea Biotech limited, and the polysaccharide content is more than 50%. AFB1 was purchased from Biotechnology, inc. of Kinzau Von, nanjing; total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) and Malondialdehyde (MDA) test boxes are purchased from Nanjing institute of bioengineering, inc.
Main instrument
A KZ-II type high-speed tissue grinder, a D3024R type desk-top high-speed refrigerated centrifuge, a Synergy HTX type multifunctional enzyme-labeling instrument and the like.
Experimental design and feeding management
Selecting 180 AA male broilers with age of 1 day, similar body weight and good health condition, dividing the AA male broilers into 5 groups according to a completely random test design, treating 6 chickens each, and repeating 10 chickens each. The control group (CON) is fed with basic daily ration, and other test groups are respectively added with 0.1mg/kg of AFB1, 0.1mg/kg of AFB1+4mg/kg of NPSe-EPS (containing Se 0.4 mg/kg), 0.1mg/kg of AFB1+4mg/kg of NPSe (containing Se 0.4 mg/kg) and 0.1mg/kg of AFB1+4mg/kg of EPS on the basis of the basic daily ration. The basal diet was formulated with reference to NRC (1994), with the composition and nutritional levels of the diets as in Table 2-1, for a 21 day test period.
Sample collection
Randomly selecting one chicken with the weight similar to that of each repeated cage from each repeated cage when the broiler is 21 days old, performing wing vein blood collection, separating serum, and storing in a refrigerator at-20 ℃. After slaughter, liver tissue was taken, partially fixed with 4% paraformaldehyde for slicing, and partially stored at-80 ℃ for analysis.
Detection index and method
Indexes related to liver oxidative damage
Collecting liver tissue 0.2-1g, rinsing with pre-cooled normal saline, removing blood, placing into a small beaker, adding pre-cooled normal saline (the volume of normal saline is 9 times of tissue weight), and grinding into 10% tissue homogenate with tissue grinder. Centrifuging the prepared tissue homogenate at low temperature of about 3000r/min for 10-15min, and taking supernatant for enzyme activity determination. Total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) activity and Malondialdehyde (MDA) content in the liver were determined according to the kit instructions.
Morphological analysis of liver tissue
The collected liver tissues were fixed in 4% paraformaldehyde and used for the preparation of sections. hematoxylin-Eosin staining (Haematxylin and Eosin, h.e.) was used and pathological changes were observed under a common light microscope.
Statistical analysis
The SAS 9.4 software is used for analyzing the test data, the Tukey's method is used for analyzing the difference significance, and P <0.05 represents the difference significance.
Influence of nano-selenium-enteromorpha polysaccharide, common nano-selenium and enteromorpha polysaccharide on liver T-AOC of broiler chicken exposed to AFB1
As shown in FIG. 7A, the inclusion of 100mg/kg AFB1 in the diet significantly reduced liver T-AOC levels in broiler chickens (P < 0.05). Compared with the AFB1 injured group, the liver T-AOC (P > 0.05) was not significantly affected by the addition of NPSe and EPS. The addition of NPSe-EPS can obviously improve the AFB1 exposed broiler liver T-AOC level (P < 0.05).
Influence of nano-selenium-enteromorpha polysaccharide, common nano-selenium and enteromorpha polysaccharide on CAT activity of broiler liver exposed by AFB1
As shown in fig. 7B, AFB1 significantly reduced liver CAT activity in broilers (P < 0.05), and the addition of NPSe-EPS and NPSe significantly increased AFB 1-exposed liver CAT activity in broilers (P < 0.05) compared to the control group.
Influence of nano-selenium-enteromorpha polysaccharide, common nano-selenium and enteromorpha polysaccharide on T-SOD activity of broiler liver exposed by AFB1
As shown in FIG. 7C, AFB1 significantly reduced liver T-SOD activity (P < 0.05) in broiler compared to control group, and the addition of NPSe-EPS and NPSe significantly increased liver T-SOD activity (P < 0.05) in broiler exposed by AFB 1.
Influence of nano-selenium-enteromorpha polysaccharide, common nano-selenium and enteromorpha polysaccharide on MDA content of liver of broiler chicken exposed by AFB1
As shown in fig. 7D, the liver of broiler chickens in AFB1 group had a significant increase in MDA content (P < 0.05) compared to the control group. The addition of NPSe-EPS, NPSe and EPS can both significantly reduce the liver MDA content (P < 0.05), and the NPSe-EPS group has the lowest MDA content.
The results show that the effect of NPSe-EPS in relieving AFB 1-induced liver oxidative damage of broiler chicken is better than that of NPSe and EPS.
Influence of nano-selenium-enteromorpha polysaccharide, common nano-selenium and enteromorpha polysaccharide on liver tissue morphology of broiler chicken exposed to AFB1
As shown in FIG. 8, the liver cells of healthy broiler chickens were aligned and the shape of the cell nucleus was regular. The AFB1 group of liver cells are disordered in arrangement, fuzzy in structure, red in cytoplasm, infiltrated by inflammatory cells mainly accompanied by lymphocytes locally, irregular in cell nucleus shape and generated by vacuolization. The overall pathological change of the liver tissues of the NPSe-EPS group is obviously improved, and the overall pathological change of the liver tissues of the NPSe group and the EPS group is improved to a certain degree, so that the nano selenium-enteromorpha polysaccharide (NPSe-EPS) has a better protective effect on AFB 1-induced liver tissue structure damage of broiler chicken compared with common nano selenium (NPSe) and Enteromorpha Polysaccharide (EPS).
By combining the results of liver oxidative damage and histopathology, the nano selenium-enteromorpha polysaccharide (NPSe-EPS) prepared by the invention has a better protection effect on liver damage of the broiler induced by AFB1 compared with the common nano selenium (NPSe) and Enteromorpha Polysaccharide (EPS).
The preparation method and the application of the nano-selenium enteromorpha polysaccharide provided by the invention are described in detail above. The principles and embodiments of the present invention have been described herein using specific examples, which are set forth only to help understand the method and its core ideas of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (6)
1. The preparation method of the nano-selenium enteromorpha polysaccharide is characterized by comprising the following steps:
s1: adding a certain volume of 10mM sodium selenite solution and a certain volume of enteromorpha polysaccharide solution into a beaker, and stirring at a proper temperature by using a constant-temperature magnetic stirrer;
s2: adding a certain volume of 10mM ascorbic acid solution into a beaker, and continuously reacting for a period of time to obtain a nano selenium enteromorpha polysaccharide solution;
s3: after the reaction is finished, the prepared nano selenium enteromorpha polysaccharide solution is put into a refrigerator for pre-freezing, then a freeze dryer is used for freeze-drying the nano selenium enteromorpha polysaccharide solution into powder, a scanning electron microscope is used for observing the shape and the particle size of the nano selenium, and an energy spectrometer is used for analyzing the element type and the content of the nano selenium enteromorpha polysaccharide.
2. The method for preparing nano-selenium enteromorpha polysaccharide according to claim 1, wherein in S1, the stirring time is set to be 2h.
3. The method for preparing nano selenium enteromorpha polysaccharide according to claim 1, wherein in S1 and S2, the volume ratio of ascorbic acid solution to sodium selenite solution is 3.
4. The method for preparing nano selenium enteromorpha polysaccharide according to claim 1, wherein in the S3, the temperature in a refrigerator is set to-80 ℃.
5. The method for preparing nano-selenium enteromorpha polysaccharide according to claim 1, wherein in S3, the pre-freezing time is more than 12 h.
6. Application of nano selenium enteromorpha polysaccharide, which is characterized in that the nano selenium enteromorpha polysaccharide prepared by the preparation method of the nano selenium enteromorpha polysaccharide according to the claims 1 to 5 is used for healthy breeding of broiler chickens.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210963000.6A CN115651087A (en) | 2022-08-11 | 2022-08-11 | Preparation method and application of nano selenium enteromorpha polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210963000.6A CN115651087A (en) | 2022-08-11 | 2022-08-11 | Preparation method and application of nano selenium enteromorpha polysaccharide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115651087A true CN115651087A (en) | 2023-01-31 |
Family
ID=85023542
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210963000.6A Pending CN115651087A (en) | 2022-08-11 | 2022-08-11 | Preparation method and application of nano selenium enteromorpha polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115651087A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333267A (en) * | 2013-07-12 | 2013-10-02 | 青岛王牌动物健康产品有限公司 | Enteromorpha se-polysaccharide with biological activity as well as preparation method and application of enteromorpha se-polysaccharide |
| CN103583683A (en) * | 2013-11-26 | 2014-02-19 | 青岛嘉瑞生物技术有限公司 | Fruit preservative based on comprehensive utilization of enteromorpha prolifera and preparation method of fruit preservative |
| CN104671973A (en) * | 2014-12-12 | 2015-06-03 | 青岛中天信达生物技术研发有限公司 | Selenium-rich vegetable compound fertilizer |
| CN109303922A (en) * | 2018-11-19 | 2019-02-05 | 华南理工大学 | A kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and preparation method thereof and the application in hypoglycemic medicine |
| US20190216844A1 (en) * | 2017-12-14 | 2019-07-18 | Morningbell Wellness Llc | Methods to stabilize and reverse atherosclerotic lesions by sulfated polysaccharides |
| CN111406948A (en) * | 2020-02-27 | 2020-07-14 | 广东药科大学 | Preparation method and application of Grateloupia filicina polysaccharide-nano selenium |
| US20200236977A1 (en) * | 2016-03-24 | 2020-07-30 | Terry Yu Tsai Shih | Stabilizing agent for reducing the leaching toxicity of heavy metals contained in foods, foodstuffs, chinese herbs and enhancing food safety and environmental protection and preparation method thereof |
| CN112370465A (en) * | 2020-10-29 | 2021-02-19 | 华南理工大学 | Sargassum fusiforme polysaccharide functionalized nano-selenium and preparation method and application thereof |
| CN112552421A (en) * | 2020-12-10 | 2021-03-26 | 广东省微生物研究所（广东省微生物分析检测中心） | Preparation method of ganoderma lucidum polysaccharide nano-selenium, ganoderma lucidum polysaccharide nano-selenium and application thereof |
| CN114163272A (en) * | 2021-12-08 | 2022-03-11 | 江苏植丰生物科技有限公司 | Selenium-rich microbial agent and preparation method thereof |
-
2022
- 2022-08-11 CN CN202210963000.6A patent/CN115651087A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333267A (en) * | 2013-07-12 | 2013-10-02 | 青岛王牌动物健康产品有限公司 | Enteromorpha se-polysaccharide with biological activity as well as preparation method and application of enteromorpha se-polysaccharide |
| CN103583683A (en) * | 2013-11-26 | 2014-02-19 | 青岛嘉瑞生物技术有限公司 | Fruit preservative based on comprehensive utilization of enteromorpha prolifera and preparation method of fruit preservative |
| CN104671973A (en) * | 2014-12-12 | 2015-06-03 | 青岛中天信达生物技术研发有限公司 | Selenium-rich vegetable compound fertilizer |
| US20200236977A1 (en) * | 2016-03-24 | 2020-07-30 | Terry Yu Tsai Shih | Stabilizing agent for reducing the leaching toxicity of heavy metals contained in foods, foodstuffs, chinese herbs and enhancing food safety and environmental protection and preparation method thereof |
| US20190216844A1 (en) * | 2017-12-14 | 2019-07-18 | Morningbell Wellness Llc | Methods to stabilize and reverse atherosclerotic lesions by sulfated polysaccharides |
| CN109303922A (en) * | 2018-11-19 | 2019-02-05 | 华南理工大学 | A kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and preparation method thereof and the application in hypoglycemic medicine |
| CN111406948A (en) * | 2020-02-27 | 2020-07-14 | 广东药科大学 | Preparation method and application of Grateloupia filicina polysaccharide-nano selenium |
| CN112370465A (en) * | 2020-10-29 | 2021-02-19 | 华南理工大学 | Sargassum fusiforme polysaccharide functionalized nano-selenium and preparation method and application thereof |
| CN112552421A (en) * | 2020-12-10 | 2021-03-26 | 广东省微生物研究所（广东省微生物分析检测中心） | Preparation method of ganoderma lucidum polysaccharide nano-selenium, ganoderma lucidum polysaccharide nano-selenium and application thereof |
| CN114163272A (en) * | 2021-12-08 | 2022-03-11 | 江苏植丰生物科技有限公司 | Selenium-rich microbial agent and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| HAITAO LÜ 等: "Preparation and antibacterial activity studies of degraded polysaccharide selenide from Enteromorpha prolifera", 《CARBOHYDRATE POLYMERS》, vol. 107, pages 98 - 102, XP028841900, DOI: 10.1016/j.carbpol.2014.02.045 * |
| KHAZRAEI SEYED KAVEH 等: "Selenium nanoparticles are more efficient than sodium selenite in reducing the toxicity of aflatoxin B1 in Japanese quail", 《VETERINARY MEDICINE AND SCIENCE》, vol. 08, no. 01, pages 254 - 266 * |
| 杨玉澜 等: "不同硒源对黄曲霉毒素B1致鸡肝损伤的缓解作用", 《畜牧与兽医》, vol. 47, no. 10, pages 23 - 28 * |
| 郭艳: "纳米硒-浒苔多糖制备及对AFB1诱导肉仔鸡肝脏损伤的保护作用研究", 《万方数据知识服务平台》 * |
| 高玉杰 等: "浒苔多糖和硒化浒苔多糖抑菌作用研究", 《食品科技》, vol. 38, no. 01, pages 195 - 198 * |
| 高玉杰 等: "浒苔硒多糖的制备及其活性研究", 《化学与生物工程》, vol. 29, no. 05, pages 40 - 45 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Abd El-Naby et al. | Dietary chitosan nanoparticles enhance the growth, production performance, and immunity in Oreochromis niloticus | |
| Karthikeyeni et al. | Biosynthesis of Iron oxide nanoparticles and its haematological effects on fresh water fish Oreochromis mossambicus | |
| Shen et al. | Effects of nano-selenium on antioxidant capacity in Se-deprived Tibetan gazelle (Procapra picticaudata) in the Qinghai–Tibet Plateau | |
| Song et al. | Effects of nano-copper poisoning on immune and antioxidant function in the Wumeng semi-fine wool sheep | |
| Kaoud et al. | Bioremediation the toxic effect of mercury on liver histopathology, some hematological parameters and enzymatic activity in Nile tilapia, Oreochromis niloticus | |
| Suparmi et al. | Anti-anemia effect of chlorophyll from katuk (Sauropus androgynus) leaves on female mice induced sodium nitrite | |
| WO2019242044A1 (en) | Use of composite nanoparticle of carbon and copper | |
| Delavari et al. | Modulatory effect of dietary copper nanoparticles and vitamin C supplementations on growth performance, hematological and immune parameters, oxidative status, histology, and disease resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss) | |
| Song et al. | Dietary copper requirements of postlarval coho salmon (Oncorhynchus kisutch) | |
| Hu et al. | Toxicity effects of different dietary selenium forms on sea cucumber, Apostichopus japonicus | |
| Ishwarya et al. | Facile synthesis of haemocyanin-capped zinc oxide nanoparticles: Effect on growth performance, digestive-enzyme activity, and immune responses of Penaeus semisulcatus | |
| Fatahian-Dehkordi et al. | Serum biochemical status and morphological changes in mice ovary associated with copper oxide nanoparticles after thiamine therapy | |
| CN115651087A (en) | Preparation method and application of nano selenium enteromorpha polysaccharide | |
| Al-Salmany et al. | Effect of Cinnamon and Turmeric Nanoparticles Extract In Quality Characteristics Of Ground Beef During Freeze Storage | |
| Veisi et al. | Dietary beet molasses enhanced immunity system of common carp (Cyprinus carpio) exposed to copper nano oxide, CuO-NP | |
| Kausar et al. | Characterization, dose dependent assessment of hepatorenal oxidative stress, hematological parameters and histopathological divulging of the hepatic damages induced by Zinc oxide nanoparticles (ZnO-NPs) in adult male Sprague Dawley rats | |
| Reddy et al. | Tender Coconut Water Uses, Health Benefits, Good Nutritive Value and Antioxidant Capacity. | |
| Vlizlo et al. | Disturbance of antioxidant protection and natural resistance factors in rats with different availabilities of trivalent chromium | |
| Rajan et al. | Impact of different quantity of green synthesized iron oxide nanoparticles on growth, enzymatic, biochemical changes and hematology of zebrafish Danio rerio | |
| Tussupbekova et al. | The study of hematological parameters of animals in the application of enterosorbent food fiber | |
| KR100708984B1 (en) | Effect of sprout blended powderbroccoli sprout, alfalfa sprout, red clover sprout on heavy metal detoxification and the Saengshik composition containing theses powder | |
| Robin et al. | Bio-elimination of lead (Pb) from the organs of red tilapia (Oreochromis sp.) using Gliricidia sepium compost as a feed additive. | |
| Rajan et al. | Different quantities of copper oxide nanoparticles incorporated feed on growth and haematological parameters Tilapia Oreochromis mossambicus | |
| Ali et al. | The physiological response of broilers chickens (Ross 308) to adding nano-boron to the diet | |
| AlNoori et al. | Effects of Dietary Addition of Natural and Nano Zinc Oxide to the Diet on the Physiological State of Common Carp |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |