CN111434339A - Chitosan nano-selenium hydrosol with immunity enhancement effect and preparation, preservation and application thereof - Google Patents
Chitosan nano-selenium hydrosol with immunity enhancement effect and preparation, preservation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of nano-selenium hydrosol, in particular to chitosan nano-selenium hydrosol with immune enhancement effect and preparation, preservation and application thereof, wherein the chitosan nano-selenium hydrosol with immune enhancement effect contains the following components of 0.5 mmol/L-5.0 mmol/L of nano-selenium and 100.0 mg/L-1000.0 mg/L of functionalized chitosan.
Description
Technical Field
The invention relates to the technical field of nano-selenium hydrosol, in particular to chitosan nano-selenium hydrosol with an immunity enhancement effect, and preparation, storage and application thereof.
Background
China is the largest aquatic product producing country in the world, and the economic value directly generated each year is about 9000 million yuan RMB. However, in recent years, frequent outbreaks of aquaculture diseases affect the health condition of aquatic products and cause huge economic losses. In 2012 only, the economic loss of China due to aquaculture diseases reaches 36 million yuan (2013, annual book of Chinese fishery statistics). Aquatic diseases caused by common aquaculture pathogens such as Vibrio anguillarum, Aeromonas hydrophila and Yersinia ruckeri cause enormous economic loss to the aquaculture industry every year. Immunopotentiators have been receiving attention over the years for the prevention and treatment of aquatic diseases and for the enhancement of the immune function of aquatic organisms. Therefore, according to the principle of prevention as the main part and treatment as the auxiliary part, the immunity of aquatic products needs to be improved before the disease outbreak in the prevention and treatment of aquaculture diseases, and the self-generated immunity of aquaculture organisms is used for resisting the invasion of external pathogens.
The immunopotentiator is a substance which can stimulate the enhancement of immune system by using related chemical agents, bacteria and components thereof, polysaccharides, animal or plant extracts, trophic factors, cytokines as raw materials, thereby resisting various breeding germs. In aquaculture application, polysaccharide is a relatively common immunopotentiator, such as Chitosan (Chitosan) extracted from crustacean is applied in various aquaculture enhancement experiments to show relevant immune effects, such as reducing pathogenic infection, improving lysozyme activity,enhancing the alternative complement system, etc. Chitosan, also known as chitosan, is obtained by deacetylating chitin. Chitosan is a cationic polymer, is a natural positively charged high molecular polymer which is rare in nature, and is concerned by the wide application in the fields of food and chemical industry due to rich sources and low price. In addition, chitosan has abundant hydrophilic hydroxyl (-OH) and amino (-NH) groups in its molecular structure2) The group makes the tissue compatible and biodegradable. Therefore, the chitosan is often used for adsorbing and embedding active ingredients to improve the utilization rate of the ingredients, and particularly has good controllability on the synthesis of nano materials.
Selenium is one of fifteen essential trace elements for human body, and can improve immunity of livestock. At present, more than 40 countries or regions around the world have the problem of selenium deficiency, and China also has a plurality of selenium deficiency or low-selenium regions. Selenium is a strong antioxidant ingredient, and can effectively protect the body against oxidative stress, and is helpful for improving the division and differentiation of the immune system and lymphocytes thereof. In recent years, nano-selenium obtained by using nano-technology has become a new research hotspot due to high bioavailability, low toxicity and remarkable biological activity. After the nano selenium is added into the fish feed, the dosage for culturing is reduced, the disease resistance of the fish is enhanced, and the meat quality is improved.
The research on the aquaculture immunopotentiator at home and abroad only focuses on evaluating the efficacy of a single material, and almost no research is carried out on simultaneously using two or more materials. The most ideal immunopotentiator should have nutritive value for water-producing substances, can stimulate a wide range of antibacterial nonspecific immune reactions, has high stability after being mixed with food and water, is compatible with other drugs, has low price, and has nutritive value for human body. In the aquaculture industry, chemicals are generally used for enhancing the immunity of fishes, but the food safety problems such as chemical residue, potential toxicity and the like are caused as a result. On the contrary, selenium is a necessary trace element for animals and humans, chitosan is also a traditional aquatic product immunopotentiator, and the two traditional immunopotentiator materials can improve the immune function of a host, reduce the use and residue of antibiotics and chemicals in aquaculture, and provide nutritional supplement for the problem of selenium deficiency of the national people. At present, the preparation method of nano-selenium is generally a reduction method, specifically, the nano-selenium is prepared by various reduction reactions of oxysalts or oxides of selenium, and a modifier or a regulator is used to modify and regulate the particle size and morphology. However, in the existing preparation methods of various nano-selenium, few application requirements are considered, and the biological activity of nano elemental selenium cannot be effectively improved, especially the immunity of the organism cannot be improved, i.e. the product obtained by the conventional method is not functionalized nano-selenium.
Disclosure of Invention
Aiming at the problem that the existing aquatic disease prevention and control in the aquaculture industry is lack of an effective immunopotentiator, the invention provides the chitosan nano-selenium hydrosol with the immunopotentiating effect and the preparation method thereof.
On the other hand, the invention also provides a preservation method of the chitosan nano selenium hydrosol with the immune enhancement effect.
And the application of the chitosan nano-selenium hydrosol with the immunity enhancement effect as an aquatic product immunity enhancement agent.
In order to achieve the purpose, the invention adopts the following technical scheme:
a chitosan nano selenium hydrosol with immunity enhancement effect contains the following components:
nano selenium of 0.5 mmol/L-5.0 mmol/L;
the functionalized chitosan is 100.0 mg/L-1000.0 mg/L.
Correspondingly, the preparation method of the chitosan nano-selenium hydrosol with the immunity enhancement effect comprises the following steps:
step S01, adding a solution containing selenium ions and/or selenious ions into the functionalized chitosan aqueous solution to obtain a mixed solution;
step S02, adding a reducing agent solution into the mixed solution obtained in the step S01, and mixing to perform a reduction reaction to obtain a reaction product;
and S03, carrying out constant volume and dialysis treatment on the reaction product obtained in the step S02 to obtain the chitosan nano selenium hydrosol with the immunity enhancing effect.
Correspondingly, the preservation method of the chitosan nano-selenium hydrosol with the immunity enhancement effect is to preserve the chitosan nano-selenium hydrosol in an environment of 0-10 ℃, and the preservation period is 30-60 days.
And, correspondingly, an aquatic product immunopotentiator, which contains the chitosan nano selenium hydrosol with immunopotentiating effect.
The invention has the beneficial effects that:
compared with the prior art, the chitosan nano selenium hydrosol with the immune enhancement effect provided by the invention has the advantages that the polyhydroxy structure in the chitosan can play a strong physical adsorption role on the nano selenium, the nano selenium is prevented from being further aggregated and precipitated, meanwhile, the surface of the nano selenium is effectively modified, the regulation and control on the particle size of the nano selenium are realized, the nano selenium is kept stable in the hydrosol, and meanwhile, the chitosan is used as a functional molecule of the nano selenium and is mutually cooperated with the physiological effect of the nano selenium to promote, so that the immune function enhancement activity is jointly played.
The preparation method of the chitosan nano selenium hydrosol with the immune enhancement effect provided by the invention can successfully prepare the nano selenium aqueous solution with biological activity, particularly immune enhancement activity by combining the functionalized chitosan, the solution containing the selenium ion and/or the selenium ion and the reducing agent at normal temperature and normal pressure. The method does not need to add any other template agent, and avoids the possible adverse effect in practical application. Therefore, the method has the characteristics of simple preparation steps, simple preparation conditions, simple and feasible process and capability of large-scale production.
The preservation method of the chitosan nano-selenium hydrosol provided by the invention can be preserved for 30-60 days at 0-10 ℃ without sedimentation, has small particle size change, and is beneficial to large-scale preparation and preservation.
The aquatic product immunopotentiator provided by the invention contains the chitosan nano selenium hydrosol with the immunopotentiating effect, so that the aquatic product immunopotentiator simultaneously contains chitosan and nano selenium, the physiological functions of the chitosan nano selenium hydrosol and the nano selenium are mutually promoted, and the synergistic immunity is exertedEnhancing function, and can improve immunity of aquatic product and selenium content of aquatic organism by mixing with aquatic feed. The chitosan acts on the non-specific immune system of a host, and the nano selenium regulates specific immunity and the anti-oxidation effect of organisms. In addition, the chitosan molecular chain has hydrophilic hydroxyl (-OH) and amino (-NH) with positive valence bond2) A group. The chitosan is absorbed in fish intestinal tracts through a mucous membrane adhesion mechanism and ion interaction, so that the chitosan with positive valence bonds is combined with sialic acid residues with negative valence bonds in the fish intestinal mucosa, and the fish intestinal mucosa has good histocompatibility and high bioavailability. The affinity of the nano-selenium and immune cells is enhanced, the intake of the immune cells to the nano-selenium is improved, the dosage of the drug is reduced, the overall immune enhancement effect of the nonspecific and specific immune functions of aquatic organisms is improved, and a more effective scheme is provided for the production and development of the aquaculture industry.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a particle size distribution diagram of chitosan nano-selenium hydrosol with immune enhancement effect provided in example 1 of the present invention;
FIG. 2 is a TEM morphology of chitosan nano-selenium hydrosol with immune enhancement effect provided in example 1 of the present invention;
FIG. 3 is a TEM morphology of chitosan nano-selenium hydrosol with immune enhancement effect provided in example 1 of the present invention;
FIG. 4 is a TEM morphology of chitosan nano-selenium hydrosol with immune enhancement effect and the spacing of selenium atomic layers provided in example 1 of the present invention;
FIG. 5 is an EDX elemental analysis chart of chitosan nano-selenium hydrosol with immune enhancement efficacy provided in example 1 of the present invention;
FIG. 6 is a comparison graph of main functional groups of chitosan nano-selenium hydrosol with immune enhancement efficacy provided in example 1 of the present invention;
FIG. 7 is a graph showing the selenium content in muscle tissue after intake of zebrafish fed with chitosan nano-selenium hydrosol with immune enhancement efficacy at different concentrations, which is provided in example 2 of the present invention, mixed with feed;
FIG. 8 is a statistical chart showing the survival rate of zebra fish infected with Aeromonas hydrophila after chitosan nano-selenium hydrosol with immunopotentiation effect provided in example 2 of the present invention is mixed into feed to prepare feed containing chitosan nano-selenium hydrosol with immunopotentiation effect at different concentrations and fed to zebra fish;
FIG. 9 is a statistical chart of a lysozyme activity test (non-specific immunoreaction) of zebra fish serum after chitosan nano selenium hydrosol with immune enhancement provided in example 3 of the present invention is prepared into feeds with different concentrations and fed to zebra fish;
fig. 10 is a statistical chart of a zebra fish lymphocyte proliferation experiment (specific immune response) after the chitosan nano selenium hydrosol with immune enhancement effect provided in example 3 of the present invention is prepared into feeds with different concentrations and fed to zebra fish.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following figures and examples further illustrate the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a chitosan nano-selenium hydrosol with an immunity enhancement effect.
The chitosan nano selenium hydrosol contains the following components in concentration:
nano selenium of 0.5 mmol/L-5.0 mmol/L;
the functionalized chitosan is 100.0 mg/L-1000.0 mg/L.
In any embodiment, the functionalized chitosan refers to chitosan obtained after chitosan is dissolved in organic acid, and the water solubility of chitosan can be improved after the chitosan is dissolved in the organic acid, so that the chitosan exists in the form of aqueous solution before forming hydrosol.
The chitosan nano-selenium hydrosol with the immune enhancement effect provided by the invention takes the functionalized chitosan with the immune regulation effect as the functionalized molecule of the nano-selenium, and the nonspecific immune enhancement effect of the chitosan and the specific cellular immune regulation function of the nano-selenium are mutually coordinated and promoted to jointly exert the strong immune effect, thereby providing a good scheme for the production and disease control of the aquaculture industry.
The chitosan nano-selenium hydrosol has the advantages of 50-150 nm of particle size, uniform particle size distribution and good dispersibility, and is not precipitated after being stored for 30-60 days at 0-10 ℃.
Correspondingly, on the basis of providing the chitosan nano-selenium hydrosol with the immune enhancement effect, the invention further provides a preparation method of the chitosan nano-selenium hydrosol with the immune enhancement effect.
In one embodiment, the preparation method of the chitosan nano selenium hydrosol with the immune enhancement effect comprises the following steps:
step S01, adding a solution containing selenium ions and/or selenious ions into the functionalized chitosan aqueous solution to obtain a mixed solution;
s02, dropwise adding a reducing agent solution into the mixed solution obtained in the step S01, and mixing to perform a reduction reaction to obtain a reaction product;
and S03, carrying out constant volume and dialysis treatment on the reaction product obtained in the step S02 to obtain the chitosan nano selenium hydrosol with the immunity enhancing effect.
In any embodiment, the chitosan is dissolved by the organic acid solution to obtain the functionalized chitosan, and the functionalized chitosan becomes more water-soluble than chitosan and exists in the form of a functionalized chitosan aqueous solution.
The organic acid solution can be any one of citric acid solution and acetic acid solution, and the organic acid is edible organic acid to ensure that the obtained chitosan nano selenium hydrosol can be used as an immune enhancer of aquatic organisms.
In the reduction reaction process, the feeding proportion of the functionalized chitosan to the selenium ions is that the functionalized chitosan (calculated by grams): 1(mol) of selenide (20-2000 (g) by mol), or functionalized chitosan (in grams): 1(mol) of selenite ions (in terms of moles) 20-2000 (g); or functionalized chitosan (in grams): the molar weight of selenium ions and selenite ions mixed is 20-2000 (g) and 1 (mol). Under the feeding proportion, the functionalized chitosan and the selenium ions and/or the selenite ions can form an hydrosol state with uniformly distributed materials.
Preferably, the concentration of the functionalized chitosan aqueous solution is controlled within the range of 120 mg/L-1200 mg/L in the reduction reaction process, under the concentration, the concentration of the functionalized chitosan in the chitosan nano-selenium hydrosol obtained after dialysis treatment can be ensured to reach 100.0 mg/L-1000.0 mg/L, under the concentration, the average particle size of the prepared chitosan nano-selenium hydrosol can be kept about 100nm, the particle size does not change greatly within 30-60 days, the particle size is still kept between 50-150 nm, and good stability is shown.
In any preferred embodiment, the concentration of the solution containing selenium ions, the solution containing selenite ions, or the mixed solution containing both selenium ions and selenite ions is 0.6 mmol/L-6.0 mmol/L, under the concentration, the average particle size of the prepared chitosan nano-selenium hydrosol can be kept about 100nm, the particle size is basically maintained at 50-150 nm within 30-60 days, the particle size is not changed greatly, the uniformity is good, and the stability is good.
The selenium ion-containing solution may be prepared with selenate, and the selenite ion-containing solution may be prepared with selenium dioxide and/or selenite.
Furthermore, sodium selenite in selenite is a component of health food and has high safety, so sodium selenite is adopted as a solute for preparation.
In any embodiment, the concentration of the reducing agent solution is 2.4 mmol/L-42.0 mmol/L.
Preferably, vitamin C is a reducing agent commonly used in the food industry, and is highly safe as well as having high reducing activity, and thus is preferred as a reducing agent.
In the reduction reaction, when the solution containing selenium ions is mixed with a reducing agent to generate the reduction reaction, the solution containing selenium ions and a reducing agent are added according to the molar ratio of the selenium ions to the reducing agent of 1: 4-1: 7 so as to ensure that the reducing agent is slightly excessive and fully reduce the selenium ions into nano selenium; when the added raw materials are a solution containing selenite ions and a reducing agent, the solution containing the selenite ions and the reducing agent are added according to the molar ratio of the selenite ions to the reducing agent of 1: 4-1: 7, so that the reducing agent is slightly excessive, and the selenite ions are fully reduced into nano-selenium; and when the added raw materials are the solution containing both the selenite ions and the selenium ions and the reducing agent, the ratio of the total molar weight of the added selenium ions and the selenite ions to the molar weight of the reducing agent is 1: 4-1: 7, so that the selenium ions and the selenite ions are ensured to be completely reduced into the nano selenium.
After the step S02, the method may further include performing constant volume and dialysis treatment on the obtained reaction product to form the final chitosan nano-selenium hydrosol with immune enhancement effect.
Specifically, after the volume is fixed, the dialysis should be carried out after the red color of the product is not deepened, and the dialysis time is generally 24 hours or more.
In any preferred embodiment, the preparation is carried out in an environment of 15 ℃ to 35 ℃ and 1 standard atmospheric pressure, so as to ensure the stability of the reaction external environment. In addition, the preparation conditions are energy-saving and environment-friendly, the safety is high, and the large-scale production is facilitated.
The preparation method of the chitosan nano-selenium hydrosol with the immune enhancement effect provided by the invention can be successfully prepared by combining the functionalized chitosan, the solution containing selenium ions/sub-selenium ions and the reducing agent at normal temperature and normal pressure, and the obtained chitosan nano-selenium hydrosol with the immune enhancement effect has good biological activity.
The chitosan nano-selenium hydrosol with the immunity enhancement effect successfully prepared by the method has a strong physical adsorption effect on nano-selenium by means of the inherent polyhydroxy structure of functionalized chitosan, well regulates and controls the particle size of the nano-selenium, stabilizes the nano-selenium and avoids the further aggregation and precipitation of the nano-selenium; meanwhile, the method does not need to add any other template agent, thereby avoiding the possible adverse effect in practical application. Therefore, the method has the characteristics of simple preparation steps, simple preparation conditions, simple and feasible process and capability of large-scale production.
Correspondingly, on the basis of providing the preparation method of the chitosan nano-selenium hydrosol with the immune enhancement effect, the invention further provides a preservation method of the chitosan nano-selenium hydrosol with the immune enhancement effect. Specifically, the chitosan nano-selenium hydrosol with the immunity enhancement effect is preserved in an environment with the temperature of 0-10 ℃, and the preservation time is 30-60 days.
In any embodiment, the chitosan nano selenium hydrosol with the immunity enhancing effect provided by the invention not only has biological immunity activity, but also can improve the content of selenium in fish muscle tissues and provide reliable and safe edible selenium sources for eaters, so that the invention further provides an aquatic product immunity enhancing agent.
The aquatic product immunopotentiator contains chitosan nano selenium hydrosol with immune enhancement effect, wherein the chitosan nano selenium hydrosol with immune enhancement effect is the chitosan nano selenium hydrosol with immune enhancement effect, and the mass content of the chitosan nano selenium hydrosol in the aquatic product immunopotentiator is 1.0-100%.
When the aquatic product immunopotentiator is used on aquatic organisms, the immunopotentiator can be mixed with aquaculture feed and fed to aquatic organisms after being uniformly mixed. The aquatic organism containing the aquatic product immunopotentiator is ingested, the immunity is improved, the aquatic organism immunopotentiator can be used for improving the survival rate of the aquatic organism such as fish infected by common germs in the aquaculture process, and the selenium content in the body is also improved.
When the aquatic product immunopotentiator of the invention is used in the fields of enhancing the immune function of aquatic organisms and preventing and treating diseases in aquaculture, the functionalized chitosan acts on the nonspecific immune system of a host respectively, and the nano selenium regulates specific immunity and organism antioxidation. The chitosan is functionalized, and then exists in a weak acid aqueous solution in the form of electrolyte, namely protons in organic acid and amino groups on the chitosan react to form ammonium salt, and hydrogen bonds between chitosan molecules are broken to dissolve in water. The nano selenium is physically adsorbed by a polyhydroxy structure in the chitosan, so that aggregation and precipitation are prevented. In the chitosan nano-selenium hydrosol with the immunity enhancement effect, a functional chitosan molecular chain contains hydrophilic hydroxyl (-OH) and protonated amino, so that the chitosan nano-selenium hydrosol has positive charge, can be absorbed by fish intestinal tracts through a mucous membrane adhesion mechanism, and combines chitosan with positive valence bonds with sialic acid residues with negative valence bonds in fish intestinal tracts through ionic interaction, so that the chitosan nano-selenium hydrosol has good histocompatibility and high bioavailability. The nano-selenium has the effects of regulating specific immune system and improving the antioxidation of organisms.
In order to better illustrate the chitosan nano-selenium hydrosol provided by the invention, the preparation method and the application technical scheme, the following examples are further illustrated.
Example 1
A preparation method of chitosan nano-selenium hydrosol with immunity enhancement effect comprises the following steps:
(1) adding 1.0m L, 2.0m L and 10m L mass concentration of 3000 mg/L functionalized chitosan aqueous solution into 25m L volumetric flasks (numbered 1#, 2#, and 3#) filled with 5m L double distilled water respectively at 20 ℃ and 101.325kPa, wherein the functionalized chitosan is chitosan treated by citric acid, and the average molecular weight of the functionalized chitosan is 200000 Da;
(2) adding sodium selenite solution with concentration of 60 mmol/L in the amount of 0.25m L, 1.0m L and 2.5m L into the No. 1#, No. 2# and No. 3# 25m L volumetric flasks, respectively, and shaking up gently to mix them thoroughly to obtain mixed solution;
(3) and (3) dropwise adding 320 mmol/L vitamin C solution 0.19m L, 1.0m L and 3.29m L into the mixed solution in the step (2), slightly shaking while dropwise adding, adding water to a constant volume of 25m L after dropwise adding is finished, standing until the red color is not deepened any more, dialyzing for 24 hours (molecular weight cut-off 8000), and obtaining a product, wherein the selenium content is measured by a nitration ICP method, and the nano-selenium concentration in the obtained product is about 0.5-5.0 mmol/L, and the chitosan concentration is 100.0 mg/L-1000.0 mg/L.
The obtained product is placed in the environment of 0-10 ℃, and the product exists in the form of hydrosol.
The product obtained in example 1 was characterized by a detection instrument, and the tests mainly included the following:
the average particle size, SD (standard deviation) and particle size distribution of the nanoparticles in the product were determined using a Nanosight NS3000 particle tracking Analyzer (Malvern) and are shown in FIG. 1.
The morphology of the product of example 1 was characterized by a JEM-2010 high-resolution projection electron microscope (JEO L), see FIGS. 2, 3 and 4 for details.
The EDX elemental analysis of the product of example 1 was characterized by a JEM-2010 high resolution projection electron microscope (JEO L) and a light energy source diffusion analyzer (Horiba) model EX-250, the results of which are detailed in FIG. 5.
The products of example 1 were characterized and compared with the main functional groups of chitosan by means of a Fourier transform infrared spectrometer model Equinox55 (Bruker), the results of which are detailed in FIG. 6.
Table 1 average particle size and standard deviation of chitosan nano-selenium particles having immunopotentiating effect obtained in example 1
| |
1# | 2# | 3# |
| Average particle size (nm) of chitosan nano-selenium particles | 112 | 117 | 101 |
| Standard Deviation (SD) | 27 | 32 | 19 |
As can be seen from fig. 1 and table 1, in the product obtained by adding the functionalized chitosan aqueous solution, the Nanosight NS3000 particle tracking analyzer (Malvern) determines that the average particle size is about 100nm, which indicates that the functionalized chitosan has a good wrapping effect on selenium ions, the particle size range of the generated chitosan nano-selenium with the immunity enhancement effect is 85-149nm, the functionalized chitosan polysaccharide has a good regulation and control effect on the particle size of the nano-selenium, the SD value is small, and the nano-selenium particle size distribution is narrow.
As can be seen from FIGS. 2, 3 and 4, the product of example 1 has good dispersibility, the product is spherical nano-selenium, and the distance between the selenium atom layers is
As can be seen from fig. 5, the main element of the obtained product is selenium.
As can be seen from FIG. 6, the stretching vibration of hydroxyl group (-OH) was determined to be 3456.13cm in comparison between Chitosan (Chitosan) and Chitosan nano-selenium (CTS-SeNP)-1Moving to 3417.92cm-1The blue shift of the absorption peak indicates that the functionalized chitosan is bonded to the surface of the nano-selenium through hydroxyl. The combination of the functionalized chitosan and the nano-selenium is beneficial to improving the stability of the nano-selenium; and secondly, the biocompatibility of the nano selenium is improved.
In summary, the chitosan nano selenium hydrosol with the immune enhancement effect obtained by the reaction of the functionalized chitosan, the sodium selenite and the vitamin C is characterized in that the polyhydroxy structure of the polysaccharide part in the functionalized chitosan is utilized to exert strong physical adsorption effect on the nano selenium, further aggregation and precipitation of the nano selenium are avoided, and meanwhile, the surface of the nano selenium is effectively modified to realize the particle size regulation of the nano selenium and keep the stability of the chitosan nano selenium hydrosol.
Example 2
A preparation method of chitosan nano-selenium hydrosol with immune enhancement effect and application of the chitosan nano-selenium hydrosol as an aquatic immune enhancer are disclosed, wherein the preparation method comprises the following steps:
(1) adding 2.0m L mass concentration of 3000 mg/L functionalized chitosan aqueous solution into a 25m L volumetric flask filled with 5m L double distilled water at 20 ℃ and 101.325kPa, wherein the functionalized chitosan is prepared by mixing chitosan and citric acid, so that the water solubility of the chitosan is improved, and the average molecular weight of the functionalized chitosan is 200000 Da;
(2) adding 1.0m L of sodium selenite solution with the concentration of 60 mmol/L into the volumetric flask with the thickness of 25m L, and shaking up gently to mix the solution fully to obtain a mixed solution;
(3) and (3) dropwise adding a vitamin C solution with the concentration of 320 mmol/L into the mixed solution in the step (2) to obtain a vitamin C solution with the concentration of 1.0m L, slightly shaking while dropwise adding, adding water to fix the volume to 25m L after dropwise adding is finished, standing until the red color is not deepened any more, dialyzing (with the molecular weight cut-off of 8000) for 24 hours to obtain a product, and measuring the selenium content by a nitration ICP method to obtain the chitosan nano-selenium hydrosol with the concentration of 2.35 mmol/L and the concentration of functionalized chitosan of 240 mg/L, wherein the product is the chitosan nano-selenium hydrosol with the immunity enhancement effect.
The chitosan nano selenium hydrosol with the immunity enhancement effect is applied as an aquatic product immunity enhancement agent, and is specifically added into aquatic product feed and fed to a water product.
The adding and using method comprises the following steps:
and (3) adding the chitosan nano-selenium hydrosol with the immune enhancement effect prepared in the step (3) into feed according to different concentrations respectively to obtain the feed containing the chitosan nano-selenium hydrosol with the immune enhancement effect, wherein the chitosan nano-selenium hydrosol with the immune enhancement effect contains 0 mu g/g, 2 mu g/g, 5 mu g/g, 10 mu g/g and 20 mu g/g.
ICP-MS (Agilent 7500) inductively coupled plasma mass spectrometry is used for measuring the selenium content change of the muscle tissue of the zebra fish which ingests chitosan nano selenium with different concentrations and the immune enhancing effect, and the detail is shown in figure 7.
The product is subjected to aquaculture disease source challenge experiment, feeds (0 mug/g, 2 mug/g, 5 mug/g, 10 mug/g and 20 mug/g) containing chitosan nano selenium hydrosol with different concentrations and immunity enhancement efficacy are fed to zebra fish for 9 days, then subcutaneous challenge experiment of the zebra fish is carried out by Aeromonas hydrophila (Aeromonas hydrophylla), and the immunity enhancement condition is observed after 96 hours, and the result is detailed as shown in figure 8.
As can be seen from FIG. 7, the chitosan nano-selenium hydrosol with immunity enhancing effect can effectively provide selenium element for fish, and can correspondingly improve the selenium content in fish muscle tissues under the action of different concentrations, and simultaneously provide a reliable and safe edible selenium source for eaters.
As can be seen from FIG. 8, the feed containing the chitosan nano-selenium hydrosol with the immune enhancement effect can well inhibit the infection of the zebra fish by the hydrophilus unicellular bacteria, the survival rate of the zebra fish and the feed concentration of the chitosan nano-selenium added with the immune enhancement effect have a dosage effect, the immunity of the fish can be improved by adding 5-20 mug/g of chitosan nano-selenium in the fish feed as an aquatic product immunity enhancer, and the fish can be helped to resist the germ infection under the condition of effectively controlling the cost.
Example 3
A preparation method of chitosan nano-selenium hydrosol with immune enhancement effect and application of the chitosan nano-selenium hydrosol as an aquatic immune enhancer are disclosed, wherein the preparation method comprises the following steps:
(1) adding 2.0m L of chitosan polysaccharide aqueous solution with the mass concentration of 3000 mg/L into a 25m L volumetric flask filled with 5m L double distilled water at the temperature of 20 ℃ and the pressure of 101.325 kPa;
(2) adding 1.0m L of sodium selenite solution with the concentration of 60 mmol/L into the volumetric flask with the thickness of 25m L, and shaking up gently to mix the solution fully to obtain a mixed solution;
(3) and (3) dropwise adding a vitamin C solution with the concentration of 320 mol/L to the mixed solution in the step (2) to obtain a vitamin C solution with the concentration of 1.0m L, slightly shaking while dropwise adding, adding water to fix the volume to 25m L after dropwise adding is finished, standing until the red color is not deepened any more, dialyzing (with the molecular weight cut-off of 8000) for 24 hours to obtain a product, and determining the selenium content by a nitration ICP method, wherein the concentration of nano selenium in the obtained product is about 2.35 mmol/L, and the concentration of functionalized chitosan is 240 mg/L, so that the product is the chitosan nano selenium hydrosol with the immunity enhancement effect.
The chitosan nano selenium hydrosol with the immunity enhancement effect is applied as an aquatic product immunity enhancement agent, and is specifically added into aquatic product feed and fed to a water product.
The adding and using method comprises the following steps:
and (3) respectively adding the chitosan nano selenium hydrosol with the immune enhancement effect prepared in the step (3) into feed according to different concentrations to obtain the feed containing the chitosan nano selenium hydrosol with the immune enhancement effect, wherein the chitosan nano selenium hydrosol with the immune enhancement effect contains 0 mu g/g, 2 mu g/g, 5 mu g/g, 10 mu g/g and 20 mu g/g.
Serum lysozyme activity experiments (non-specific immunoreaction) and lymphocyte proliferation experiments (specific immunoreaction) are carried out on the products, feeds (0 mu g/g, 2 mu g/g, 5 mu g/g, 10 mu g/g and 20 mu g/g) added with chitosan nano selenium hydrosol with different concentrations and with immune enhancement efficacy are respectively used for feeding zebra fish for 9 days, zebra fish serum is taken for lysozyme activity experiments, and pancreatic cells are subjected to lymphocyte proliferation experiments, which are detailed in figures 9 and 10.
As can be seen from FIG. 9, the feed added with the chitosan nano-selenium hydrosol with the immune enhancement effect can well improve the activity of the serum lysozyme of the zebra fish, and the activity of the serum lysozyme and the concentration of the added chitosan nano-selenium hydrosol with the immune enhancement effect have a dosage effect, and according to FIG. 9, the adding concentration of the chitosan nano-selenium hydrosol with the immune enhancement effect is 5-20 μ g/g when the chitosan nano-selenium hydrosol with the immune enhancement effect is used as an aquatic product immune enhancer.
From fig. 10, it can be seen that the feed added with the chitosan nano-selenium hydrosol with the immune enhancement effect can well improve the proliferation capacity of the zebra fish T lymphocytes, and when the concentration of the chitosan nano-selenium hydrosol with the immune enhancement effect is 5-20 μ g/g, the proliferation capacity of the T lymphocytes is better than that of other concentrations.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The chitosan nano selenium hydrosol with the immune enhancement effect is characterized by comprising the following components in concentration:
nano selenium of 0.5 mmol/L-5.0 mmol/L;
the functionalized chitosan is 100.0 mg/L-1000.0 mg/L.
2. The immunopotentiating chitosan nanoseled hydrosol of claim 1, wherein the functionalized chitosan is chitosan treated with organic acid, and the molecular weight of the functionalized chitosan is 50000-375000 Da.
3. The chitosan nano-selenium hydrosol with immune enhancing effect of claim 1, wherein the particle size of the chitosan nano-selenium hydrosol is 50nm to 150 nm.
4. The method for preparing chitosan nano-selenium hydrosol with immune enhancement effect according to any one of claims 1 to 3, which comprises the following steps:
step S01, adding a solution containing selenium ions and/or selenious ions into the functionalized chitosan aqueous solution;
step S02, adding a reducing agent solution into the mixed solution obtained in the step S01, and mixing to perform a reduction reaction to obtain a reaction product;
and S03, carrying out constant volume and dialysis treatment on the reaction product obtained in the step S02 to obtain the chitosan nano selenium hydrosol with the immune enhancement effect.
5. The method for preparing chitosan nano-selenium hydrosol with immune enhancement effect as claimed in claim 4, wherein in the reduction reaction, the material is fed according to the ratio of the mass of the functionalized chitosan to the selenium ions and/or the selenious ions being 20-2000: 1, and the concentration of the functionalized chitosan aqueous solution is controlled to be 120 mg/L-1200 mg/L in the reduction reaction process.
6. The method for preparing chitosan nano-selenium hydrosol with immunity enhancement effect as claimed in claim 4, wherein in the solution containing selenium ions and/or selenious ions, the concentration of selenium ions and/or selenious ions is 0.6 mmol/L-6.0 mmol/L, and the concentration of the reducing agent solution is 2.4 mmol/L-42.0 mmol/L.
7. The method for preparing chitosan nano-selenium hydrosol with immunopotentiating effect according to claim 4, wherein in the reduction reaction, the ratio of the selenium ions and/or the selenite ions to the reducing agent is 1: 4-1: 7, the materials are fed.
8. The method for preparing chitosan nano-selenium hydrosol with immune enhancement efficacy according to claim 4, wherein the solution containing selenite ions is selenite solution; the reducing agent is vitamin C.
9. The method for preserving the chitosan nano-selenium hydrosol with immune enhancement effect according to claim 1 or the method for preparing the chitosan nano-selenium hydrosol with immune enhancement effect according to any one of claims 2 to 8, wherein the chitosan nano-selenium hydrosol with immune enhancement effect is preserved in an environment of 0 to 10 ℃ and has a preservation period of 30 to 60 days.
10. An aquatic product immunopotentiator, which is characterized by comprising an immunopotentiating chitosan nano selenium hydrosol, wherein the immunopotentiating chitosan nano selenium hydrosol is the immunopotentiating chitosan nano selenium hydrosol according to claim 1 or the immunopotentiating chitosan nano selenium hydrosol obtained by the method for preparing the immunopotentiating chitosan nano selenium hydrosol according to any one of claims 2 to 8.
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