CN109601726B - Nano-particle chelated zinc and preparation method and application thereof - Google Patents

Abstract

The invention discloses a nano-particle chelated zinc and a preparation method and application thereof, wherein chitosan, polyacrylic acid and zinc sulfate are used as main materials, the nano-particle chelated zinc is microspherical in appearance, 50-100 nm in diameter and a nano-scale high-molecular composite material, a framework is composed of a copolymer formed by chitosan (outer layer) -polyacrylic acid (inner layer), an inner core is filled with active zinc ions, and the zinc ions are chelated with hydroxyl and amino bonds of chitosan and hydroxyl bonds of polyacrylic acid, so that the zinc ions are stabilized in the inner core of the nano-particle. Research shows that the nano-particle chelated zinc has stronger killing capacity on ETEC, so that NC-Zn has stronger effect of resisting diarrhea caused by ETEC, and can reduce the release of zinc ions in an acidic environment in stomach, so that the zinc ions can be intensively released in an alkaline environment in intestinal tracts, and the sterilization effect of NC-Zn in animal intestinal tracts is improved to a certain extent.

Description

Nano-particle chelated zinc and preparation method and application thereof

Technical Field

The invention relates to the field of animal feed additives, in particular to nano-particle chelated zinc and a preparation method and application thereof.

Background

Zinc is an essential trace element of animal bodies, is an important component of more than 300 proteins and enzymes in animal bodies, and simultaneously, zinc enzymes participate in the synthesis and translation work of DNA and RNA of the bodies and closely influence the metabolic functions of the bodies. In addition, zinc participates in the development of immune organs of the body, influences the specific and nonspecific immunity of the body, and has the function of killing various pathogenic bacteria. Therefore, the zinc source feed additive can be widely applied to animal production practice.

A large number of pathogenic bacteria exist in the complex environment of an animal farm, and after the pathogenic bacteria infect animals, the normal physiological functions of the animals are disturbed, so that the animals generate strong inflammatory reaction, and the production performance of the animals is seriously influenced. Among them, young animals (such as weaned piglets) are more vulnerable to these environmental pathogens because of immature digestion and immune function. Among them, chickens are most affected by salmonella, while pigs are most affected by pathogenic Escherichia coli (ETEC). In order to make these young animals better able to resist pathogenic infestation, antibiotics and high-zinc preparations (mainly zinc sulfate and zinc oxide) are frequently used in animal husbandry. Antibiotics and high zinc preparations are widely used as growth promoters, particularly on weaned piglets. Although the antibiotics and the high-zinc preparation can help the piglets to relieve the weaning stress to a certain extent, the negative effects brought by the antibiotics and the high-zinc preparation are more, wherein the antibiotics can cause the bacteria to be mutated to generate drug resistance and breed super bacteria; the high-dose zinc is added into the feed and seriously exceeds the actual physiological requirement of animals, so that the biological utilization efficiency of the zinc is extremely low, and a large amount of zinc which is not digested and absorbed is discharged into the environment along with excrement, thereby causing serious pollution to the environment; in addition, the long-term use of the high-zinc preparation can also cause toxicity to animals and is not beneficial to the healthy growth of the animals. Therefore, the strategy of 'banning resistance and reducing zinc' in the feed is a great trend of future animal husbandry development in China.

Disclosure of Invention

The invention aims to solve the technical problems of improving the utilization efficiency of zinc and reducing the dosage of a zinc preparation in a feed additive aiming at the defects of the prior art, and provides a novel feed additive in a form of chelated zinc.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the nano-particle chelated zinc is microspherical in appearance, 50-100 nm in diameter and is a nano-scale high-molecular composite material, a framework is composed of a copolymer formed by chitosan-polyacrylic acid, active zinc ions are filled in an inner core, and the zinc ions, hydroxyl and amino bonds of the chitosan and hydroxyl bonds of the polyacrylic acid form chelation, so that the zinc ions are stabilized in the inner core of the nano-particle.

Further, the invention also provides a preparation method of the nano-particle chelated zinc, which comprises the following steps:

(1) slowly dripping zinc sulfate aqueous solution into polyacrylic acid (PAA) aqueous solution, and stirring for 20-30 min at room temperature to obtain polyacrylic acid-zinc mixed solution (PAA-Zn);

(2) heating acetic acid water solution of Chitosan (CS) to 55-60 ℃, dropwise adding polyacrylic acid-zinc mixed solution (PAA-Zn) prepared in the step (1) under a stirring state, adding a cross-linking agent after dropwise adding, and reacting for 4-5 h after sealing to obtain chitosan-polyacrylic acid-zinc suspension (CS-PAA-Zn);

(3) And (3) centrifuging the chitosan-polyacrylic acid-zinc suspension obtained in the step (2), removing the supernatant, re-centrifuging after re-suspending, washing, drying and crushing to obtain the chitosan-polyacrylic acid-zinc suspension.

In the step (1), the concentration of the zinc sulfate aqueous solution is 0.3-0.6 g/mL; the concentration of the polyacrylic acid aqueous solution is 1.0-2.0 g/100 mL; the volume ratio of the zinc sulfate aqueous solution to the polyacrylic acid aqueous solution is 1-1.5: 5.

Preferably, the zinc sulfate aqueous solution is added into the polyacrylic acid aqueous solution at a dropping speed of 1.5-2.0 ml/min so as to enable Zn to be added²⁺Is wrapped by PAA as much as possible to form Zn²⁺The active core of (1).

In the step (2), the acetic acid aqueous solution of the Chitosan (CS) is prepared by mixing 1.5-2.5 g of chitosan with 100mL of 1 wt% acetic acid aqueous solution.

Preferably, the volume ratio of the acetic acid aqueous solution of Chitosan (CS) to the polyacrylic acid-zinc mixed solution (PAA-Zn) is 25: 6-7.

Preferably, the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) aqueous solution, the concentration is 0.005-0.01 mol/ml, and chitosan aqueous acetic acid solution is added in an amount of 15-20 mu L/ml.

The order of addition is aimed at making CS wrap the periphery of PAA-Zn as much as possible, so that Zn ²⁺Is better encapsulated inside the synthetic material. The purpose of EDC is to crosslink the chemical bonds of CS and PAA, making the composite structure more stable.

Preferably, in the step (3), the rotation speed of the centrifugation is 10000-12000 r/min, and the time is 15-20 min.

Preferably, the resuspension is carried out using an aqueous solution of acetic acid at pH 4.5.

The invention also claims application of the nanoparticle chelated zinc (NC-Zn) as a feed additive, which is used for inhibiting activity of escherichia coli (ETEC) and treating animal intestinal barrier damage and diarrhea caused by escherichia coli infection.

Has the advantages that:

the invention successfully develops Nano-chelating zinc (NC-Zn) by adopting chitosan, polyacrylic acid and zinc sulfate as main materials. The preparation has a number of excellent properties:

(1) nano-particles: the preparation is microspherical, has the diameter of about 50-100 nm, and is a nano-scale feed additive. The nano material has excellent performances of large specific surface area, small size effect and the like, and a large number of researches prove that the nano preparation has higher biological utilization efficiency than a common preparation;

(2) inhibition of ETEC activity: the escherichia coli ETEC infection is one of important factors causing damage and even diarrhea of animal intestinal barriers, and NC-Zn prepared by the research has stronger killing capacity on ETEC, so that the NC-Zn has stronger effect of resisting ETEC-induced diarrhea;

(3) The release of zinc ions in the active core has pH controlled release property: NC-Zn can reduce the release of zinc ions in an acidic environment in the stomach, so that the zinc ions can be released concentratedly in an alkaline environment in the intestinal tract, and the sterilization effect of the NC-Zn in the intestinal tract of the animal is improved to a certain extent;

(4) NC-Zn contains a chitosan component which is difficult to be directly digested and utilized by a host, but can be metabolized by microorganisms and enzymes in intestinal tracts, so that the effect of improving the microbial flora structure of the intestinal tracts is achieved, and in addition, chitosan also has the effect of improving the immune function of the host. This also increases the anti-diarrheal function of NC-Zn. In conclusion, NC-Zn has good application prospect as the anti-diarrhea feed additive.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a TEM observation of the nanoparticle chelated zinc (NC-Zn);

FIG. 2 is a graph showing the bacteriostatic effect of NC-Zn on ETEC;

FIG. 3 is an SEM observation picture of morphological destruction of ETEC cells by NC-Zn (left picture is a control group, right picture is an NC-Zn treated group);

FIG. 4 is a graph of the effect of different pH on the release of zinc ions from NC-Zn;

FIG. 5 shows the body weight change before and after challenge (left graph is the body weight change before challenge, right graph is the body weight change after challenge);

FIG. 6 is a graph showing the effect of NC-Zn on feed intake of ETEC infected weaned rats (the left graph is a feed intake change graph before challenge, and the right graph is a feed intake change graph after challenge);

FIG. 7 is a graph of the effect of NC-Zn on the diarrhea rate of ETEC infected weaned rats;

FIG. 8 is a graph showing the effect of NC-Zn on the number of E.coli in feces of ETEC infected weaned rats;

FIG. 9 is a graph of the effect of NC-Zn on the small intestine weight of ETEC infected weaned rats;

FIG. 10 is a graph showing the effect of NC-Zn on the morphology of the jejunum of ETEC-infected weaned rats (A: HE staining of morphology of rat jejunum; B: villus height; C: crypt depth; D: crypt ratio);

FIG. 11 is a graph showing the effect of NC-Zn on serum antioxidant performance of ETEC-infected weaned rats.

Detailed Description

The invention will be better understood from the following examples.

First, material synthesis process

1 preparation of main reagent

1) 1.5% PAA (m/V): 1.5g of Polyacrylic acid (PAA; MW 240000-250000, BR grade) is added with 100mL of deionized water and mixed for standby.

2) 2% CS (m/V): 2g of chitosan (chitosan, CS; MW 80000-90000; CS content > 90%; Deacetylation Degree > 95%; particle size 80 mesh; viscosity: 50mpa. s) was added to 100mlL 1% acetic acid solution and mixed for use.

3)0.5g/mL ZnSO₄.7H₂O (m/V): 50g of zinc sulfate heptahydrate (ZnSO)₄.7H₂O) constant volume to 100mL, and mixing uniformly for later use.

4) EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride): EDC stock solution was prepared by adding 0.666g (0.0035mol) of EDC crosslinker to 5mL of water.

2 synthetic method

1) Preparing a PAA-Zn mixed solution: 1.2mL of 0.5g/mL zinc sulfate was slowly added dropwise to 5mL of 1.5% PAA solution, and the mixture was stirred at room temperature for 20 min. So that Zn is present²⁺Is wrapped by PAA as much as possible to form Zn²⁺The active core of (1).

2) Heating 25mL of 2% CS solution to 60 ℃, stirring with a magnetic stirrer (the rotating speed is not lower than 500r/min), and slowly dropwise adding the prepared PAA-Zn mixed solution into the heated CS solution. After the dropwise addition, 375 mu L of EDC solution is added, the preservative film is sealed, and the reaction is continued for 4 hours under the temperature. The order of addition is aimed at making CS wrap the periphery of PAA-Zn as much as possible, so that Zn²⁺More preferably the interior of the composite material is encapsulated. The purpose of EDC is to crosslink the chemical bonds of CS and PAA, making the composite structure more stable.

3) Centrifuging the prepared CS-PAA-Zn liquid at 12000r/min for 15min, discarding the supernatant, using acetic acid water with pH of 4.5, resuspending, centrifuging again, washing for 2 times, freeze-drying, and pulverizing to obtain the Nano CS-PAA-Zn feed additive, wherein the synthesized CS-PAA-Zn is a Nano-particle chelated zinc (NC-Zn) preparation.

Material characteristics of synthetic NC-Zn

1) Zinc content

The zinc content in NC-Zn was 7.77% by ICP measurement.

2) Form of the composition

The NC-Zn is microspherical and has a diameter of about 50 to 100nm, as observed by TEM (FIG. 1).

3) Bacteriostatic ability

In vitro Oxford cup test (figure 2) is adopted to prove that NC-Zn has stronger killing capacity to enterotoxigenic Escherichia coli ETEC; the minimum inhibitory concentration of NC-Zn to ETEC is 1250ug/mL by MIC method test; and NC-Zn is found to have strong destructive effect on the cell morphology of ETEC through SEM observation (FIG. 3). In vivo experiments, it was also observed that the synthetic material was able to significantly reduce the amount of fecal escherichia coli in weaned rats (fig. 8).

4) Has pH controlled release property for releasing zinc ions

In vitro bionic test results show that the NC-Zn has certain pH controlled release property for releasing zinc ions, as can be seen from figure 4, the NC-Zn has low zinc ion release rate and is relatively stable under low pH, and has strong zinc ion release capacity under alkaline pH, so that the characteristic can be beneficial to reducing the zinc release amount of the NC-Zn in the stomach and increasing the zinc release of the NC-Zn in intestinal tracts, further improving the sterilization effect of the NC-Zn in animal intestinal tracts and improving the biological efficiency of zinc.

Protection effect of NC-Zn on ETEC (ethylene-tetra-ethyl-carbonate) toxicity attack

1 test grouping

The animal experiment is carried out in the animal experiment center of Nanjing university of agriculture for 9-10 months in 2018. In the experiment, 60 weaned rats with initial weight of 55.63 +/-2.01 g are selected and randomly divided into A, B, C, D and E groups, and during the experiment, two groups of A (Control group, CON) and B (Negative Control group, N-CON) are fed with basic ration; feeding group C with basal diet +50mg/kg Aureomycin (ANTIB); feeding group D with basal diet and High-dose zinc oxide (High-ZnO group, H-ZnO), with the addition amount of 300mg/kg (calculated by zinc content); group E was fed with basal diet + Nano-chelated Zinc (NC-Zn) at an additive amount of 50mg/kg (in terms of Zinc content). The whole course of the formal test is 14 days, and at 9 am on the 10 th day of the test, B, C, D, E groups of bacterium liquid containing ETEC are respectively subjected to intragastric administration, and the intragastric administration dosage is 10¹⁰CFU/(day, only), group A was perfused with equal volume of LB medium for 3 consecutive days and all sacrificed 48 hours after the last lavage and samples were taken.

2 index determination

2.1 weight and feed intake

During the test period, food intake was recorded daily and weighed at 8 am on test days 1, 4, 7, 10, 11, 12, 13 and 14, respectively.

2.2 diarrhea Rate

Diarrhea status of each weaned rat was observed at 8 pm on test days 9, 10, 11, 12, 13, respectively, and diarrhea index and diarrhea rate were recorded based on fecal status.

2.3 fecal Escherichia coli

At 8 pm on the 13 th day of the experiment, the feces of each group of mice were collected and counted by the dilution spread plate method using meccankey agar medium.

2.4 Small intestine weight and jejunum morphology identification

After the rats are slaughtered, the small intestines (including duodenum, jejunum, ileum and chyme thereof) are weighed, then the middle sections of all the jejunum groups are taken, HE staining is carried out, the height of villus and the depth of crypt are measured, and the ratio of the height of the villus to the depth of the crypt is calculated and used for evaluating the protection effect of NC-Zn on the intestinal morphology after ETEC is attacked.

2.5 serum antioxidant index

The kit is adopted to measure the contents of SOD, MDA, T-AOC and GSH-px in the serum of each group of rats so as to evaluate the protection effect of NC-Zn on an organism oxidation system after ETEC infection.

3 results

3.1 Effect of NC-Zn on weight and feed intake of weaned rats after ETEC challenge

As can be seen from fig. 5 and 6, before toxicity attack, the nano-chelated zinc has a tendency of significantly increasing the body weight of weaned rats (P ═ 0.09); after ETEC is attacked, the weight and the feed intake of N-CON weaned rats are obviously reduced, and the feed intake of the weaned rats can be improved by adding NC-Zn into the feed, and the weight loss of the rats caused by the ETEC attack is reduced.

3.2 Effect of Nano chelated Zinc on diarrhea Rate after ETEC challenge

As can be seen from figure 7, after ETEC is attacked, the diarrhea rate of N-CON weaned rats is obviously improved, and the diarrhea incidence rate can be obviously reduced by feeding NC-Zn.

3.3 Effect of Nano-chelated Zinc on fecal Escherichia coli count after ETEC challenge

As can be seen from FIG. 8, the number of E.coli in N-CON group rats was significantly increased after the ETEC challenge, and the feeding of NC-Zn decreased the number of E.coli in feces, which may be one of the important reasons for the alleviation of the diarrhea rate.

3.4 Effect on Small intestine weight and jejunum morphology

As can be seen in FIG. 9, after ETEC challenge, the small intestine weight of rats in the N-CON group is significantly reduced, and NC-Zn feeding can improve the loss condition of the small intestine weight. Meanwhile, as can be seen from fig. 10A to 10D, the villi of the N-CON group rats become shorter, thinner and the crypt is deeper after the ETEC attack, and these results show that the morphological structure of the jejunum of the weaned rats is seriously damaged after the ETEC attack. And the damage degree of jejunum villi can be obviously improved by feeding NC-Zn.

3.5 Effect on body Oxidation resistance

As can be seen from FIG. 11, after ETEC attack, the activity of GSH-PX, SOD and T-AOC in the serum of N-CON weaned rats is significantly reduced, and MDA is significantly increased, which means that the balance state of the antioxidant system of the organism of the weaned rats after ETEC attack is destroyed. And the NC-Zn feeding can obviously improve the antioxidant balance condition of the rat body.

The present invention provides a method and a system for preparing nanoparticle chelated zinc, and a method and a system for applying the same, and a plurality of methods and ways for implementing the technical scheme are provided, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims (9)

1. A nano-particle chelated zinc is characterized in that the nano-particle chelated zinc is microspherical in appearance, 50-100 nm in diameter and is a nano-scale high-molecular composite material, a framework is composed of a copolymer formed by chitosan-polyacrylic acid, an inner core is filled with active zinc ions, and the zinc ions are chelated with hydroxyl and amino bonds of the chitosan and hydroxyl bonds of the polyacrylic acid so as to be stabilized in the inner core of the nano-particle;

the preparation method of the nano-particle chelated zinc comprises the following steps:

(1) slowly dropwise adding a zinc sulfate aqueous solution into a polyacrylic acid aqueous solution, and stirring at room temperature for 20-30 min to obtain a polyacrylic acid-zinc mixed solution;

(2) Heating acetic acid water solution of chitosan to 55-60 ℃, dropwise adding the polyacrylic acid-zinc mixed solution prepared in the step (1) under a stirring state, adding a cross-linking agent after dropwise adding, and reacting for 4-5 hours after sealing to obtain chitosan-polyacrylic acid-zinc suspension;

(3) and (3) centrifuging the chitosan-polyacrylic acid-zinc suspension obtained in the step (2), removing the supernatant, re-centrifuging after re-suspending, washing, drying and crushing to obtain the chitosan-polyacrylic acid-zinc suspension.

2. The nanoparticle chelated zinc as claimed in claim 1, wherein in step (1), the concentration of the aqueous solution of zinc sulfate is 0.3-0.6 g/mL; the concentration of the polyacrylic acid aqueous solution is 1.0-2.0 g/100 mL; the volume ratio of the zinc sulfate aqueous solution to the polyacrylic acid aqueous solution is 1-1.5: 5.

3. The nanoparticle chelated zinc according to claim 1 or 2, wherein the aqueous zinc sulfate solution is added to the aqueous polyacrylic acid solution at a dropping rate of 1.5-2.0 ml/min.

4. The nanoparticle chelated zinc as claimed in claim 1, wherein in step (2), the acetic acid aqueous solution of chitosan is prepared by mixing 1.5-2.5 g chitosan with 100mL 1 wt% acetic acid aqueous solution.

5. The nanoparticle chelated zinc as claimed in claim 4, wherein the volume ratio of the acetic acid aqueous solution of chitosan to the polyacrylic acid-zinc mixed solution is 25: 6-7.

6. The nanoparticle chelated zinc as claimed in claim 1, wherein in step (2), the cross-linking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in water solution with concentration of 0.005-0.01 mol/ml, and chitosan is added in an amount of 15-20 μ L/ml in acetic acid water solution.

7. The nano-particle chelated zinc as claimed in claim 2, wherein in step (3), the rotation speed of the centrifugation is 10000-12000 r/min for 15-20 min.

8. The nanoparticle chelated zinc as claimed in claim 1, wherein the preparation method of the nanoparticle chelated zinc is that in step (3), the resuspension is carried out using acetic acid water solution with pH 4.5.

9. Use of the nanoparticulate chelated zinc according to claim 1 as a feed additive.

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