Patent Description:
At present, due to an overall consideration on sow reproduction and production efficiency in pig industry, piglets aged <NUM>-<NUM> weeks will be generally weaned, which, however, often leads to diarrhea in piglets weaned at this early stage.

It has been found that the nano zinc oxide with a concentration of <NUM>-<NUM> ppm can reduce the occurrence of diarrhea in piglets. The nano zinc oxide can inhibit the activation and proliferation of intestinal mast cells, improve autoimmune response in piglets, and prevent the reproducing of conditional pathogens, for example, Escherichia coli and Enterococcus, in the intestine. Therefore, at present, the most effective way to prevent and control diarrhea in weaned piglets is to use a high-dose zinc (with a zinc oxide content of about <NUM> ppm).

However, with the increased use of the high-dose zinc, it has been found that, high-dose of zinc leads to a negative influence. That is, excessive zinc, since it cannot be absorbed by piglets, is excreted out of the body of piglets with its feces. The feces containing zinc may enter the soil to pollute the local environment. Long-term use of the high-dose zinc will lead to a negative influence on the piglets, and even lead to tolerance to some drugs in the piglets, which will increase the cost of drugs. Most of the high-dose zinc will be neutralized into Zn<NUM>+ by gastric acid, but a large amount of Zn<NUM>+ will negatively affect the absorption of trace elements such as copper, iron and selenium in piglets.

<CIT> discloses a preparation method of three-dimensional hierarchical porous nano zinc oxide for feed, which includes the following steps: preparing <NUM>-<NUM> mol/l of zinc salt solution, and adding <NUM>-<NUM> of diatomite into each liter of the zinc salt solution to mix into suspension; preparing <NUM>-<NUM> mol/l of ammonia water/carbonate solution, slowly dripping <NUM>-<NUM> times of the zinc salt solution in the step (<NUM>) into the suspension in the step (<NUM>) while stirring until the reaction is complete; and (<NUM>) carrying out centrifugal separation on the solution obtained in the step (<NUM>), washing and drying the obtained precipitate by using deionized water, and calcining the precipitate at the high temperature of <NUM>-<NUM> for <NUM>-<NUM> hours to obtain a product, namely the three-dimensional hierarchical porous nano zinc oxide for the feed. The three-dimensional hierarchical porous nano zinc oxide for the feed has larger specific surface area, more effective sterilization effect and less environmental pollution, and can better prevent the diarrhea of weaned pigs caused by stress. <CIT> discloses a preparation method of a diarrhea-preventing suckling pig feed additive, and the method includes the following steps of (<NUM>) surface modification of palygorskite: gradient heating the palygorskite, and then modifying the palygorskite by using a high-efficiency active dispersant to obtain modified palygorskite; (<NUM>) compounding and cementing zinc oxide and palygorskite: mixing the modified palygorskite powder with the composite zinc oxide, then adding an adhesion promoter solution to perform functional compound cementation on the modified palygorskite and the zinc oxide, magnetically stirring uniformly, drying and grinding into powder to obtain the additive. In a word, the piglet feed additive prepared by the invention has a high-efficiency diarrhea prevention effect, can improve the gastrointestinal environment of piglets, and improves the gastrointestinal adaptability of piglets in the weaning period. <CIT> discloses a feed additive of nanometer size and a process for preparing the same. Said additive consists of a composition of copper modified montmorillonite and zinc modified montmorillonite in a weight proportion of <NUM>:<NUM>-<NUM>. Said additive has such extreme high antibiosis and antiviral properties that it can be used to substitute for the antibiotics and growth promotion feed additives. The present invention also relates to a feed containing the feed additive of nanometer size.

In order to reduce the use amount of zinc and improve the effect of zinc on preventing diarrhea in piglets, promote the growth of piglets and reduce the pollution of residual zinc on the environment, the present application provides a single atomic zinc additive for replacing nano zinc oxide for use in feeds and a preparation method thereof.

In a first aspect, the present application provides a single atomic zinc additive for replacing nano zinc oxide for use in feeds.

A single atomic zinc additive for use in feeds includes a carrier and active metal zinc. The active metal zinc is distributed on the surface of the carrier in single atomic form, without active metal zinc contained inside the carrier.

In an animal, only zinc atoms in direct contact with the animals can work. However, in a conventional process, most of the zinc in nano zinc oxide is wrapped inside, having a low use rate of zinc atoms. In the present applicant, the active metal zincs are distributed on the surface of the carrier in single atomic form, and there is no active metal zinc contained inside the carriers, so that the utilization rate of the zinc atom can be improved. In the bodies of animals, zinc oxide will be neutralized into Zn<NUM>+ by gastric acid, but a large amount of Zn<NUM>+ will affect the absorption of trace elements such as copper, iron and selenium in piglets. In the present applicant, single atomic zincs are loaded on the surface of carriers, there will be no residual Zn<NUM>+ in the present application since zinc will not be changed into Zn<NUM>+ due to the carrying effect of the carriers. Therefore, zinc can achieve the effect of treating diarrhea, promote the growth of piglets and cause no pollution on the environment in the present application.

Further, the carrier is nano silica, and a mass ratio of metal zinc to nano silica is <NUM>:(<NUM>-<NUM>).

By controlling the mass ratio of the active metallic and the carrier to control the amount of metal loaded on the carrier, the active metal loaded on each carrier can play the best effect.

In a second aspect, the present applicant provides a preparation method of single atomic zinc additive for use in feeds, including the following steps:.

By ultrasonic treatment of the carrier and the single atomic precursors of active metals, the active metal can be more evenly distributed on the surface of the carrier, thereby improving the effect of treating piglet diarrhea and promoting the growth of piglets by the present method.

Further, the preparation method of nano silica carrier includes the following steps:.

Dripping the sodium silicate solution into aqueous ammonium chloride solution can control the reaction process. Washing the mixed liquid prepared by mixing and stirring can further improve the dispersion degree of silica, reduce the possibility of silica agglomeration, improve the bonding strength between the carrier and zinc atoms, improve the effect of the product prepared in the present application on diarrhea, and promote the growth of piglets, causing no pollution to the environmental.

Further, in S11, the sodium silicate solution is prepared by the following steps: preparing an aqueous ethanol solution by a volume ratio of ethanol to water of <NUM>: (<NUM>-<NUM>), adding a sodium silicate solution to the aqueous ethanol solution to obtain a mixed solution of <NUM>-<NUM> mol/L, and adding cationic surfactant cetyltrimethylammonium bromide (CTAB) to the mixed solution to obtain the sodium silicate solution.

Cationic surfactant cetyltrimethylammonium bromide is a cationic surfactant with excellent emulsifying effect, which can provide a well-mixed aqueous ethanol solution and sodium silicate solution. Cetyltrimethylammonium bromide can reduce the agglomeration of nano silica and improve the uniformity and stability of the obtained sodium silicate solution. Cetyltrimethylammonium bromide has biodegradability and sterilizing function itself, so that the sterilizing effect of the present application can be further improved.

Further, <NUM>-<NUM> cationic surfactant cetyltrimethylammonium bromide is added per <NUM> of the sodium silicate solution.

By controlling the use amount of cetyltrimethylammonium bromide, the possibility of precipitation due to a high concentration can be reduced, so that cetyltrimethylammonium bromide can improve the stability of sodium silicate solution while giving full play to its efficacy.

Further, the washing solution in S13 is obtained by mixing cetyltrimethylammonium bromide with the aqueous ethanol solution, in which the volume ratio of ethanol to water in aqueous ethanol solution is <NUM>: (<NUM>-<NUM>), and <NUM>-<NUM> mol of cetyltrimethylammonium bromide is contained per <NUM> of the aqueous ethanol solution.

Cetyltrimethylammonium bromide is easily soluble in ethanol. Adding cetyltrimethylammonium bromide to the ethanol solution can further reduce the agglomeration of nano silica, so that more zinc atoms can be loaded on the surface of the carrier to improve the utilization rate of the carrier.

Further, in S2, the single atomic precursor of active metal zinc is prepared by the following steps: adding the sodium carbonate solution to zinc nitrate solution by <NUM>-<NUM>µL/s, stirring and mixing at <NUM>-<NUM> rpm for <NUM>-<NUM>, heating to <NUM>-<NUM> within <NUM>-<NUM>, performing mixing and stirring at the same speed for another <NUM>-<NUM>, and then cooling to room temperature to obtain the single atomic precursor of active metal zinc.

By adding sodium carbonate into the zinc nitrate solution, the temperature of zinc nitrate solution is improved, and in turn the stability of the obtained mixture is improved.

Further, the sodium carbonate solution has a mass concentration of <NUM>%, the zinc nitrate solution has a concentration of <NUM>-<NUM>/L, and a volume ratio of the sodium carbonate solution to the zinc nitrate solution is (<NUM>-<NUM>): <NUM>.

By mixing sodium carbonate with zinc nitrate in proportion, the stability of the prepared mixed solution is further improved.

Further, in S4, the activation at a high temperature includes: activating the single atomic zinc additive precursor powder obtained in step S3 at a high temperature of <NUM>-<NUM> for <NUM>-<NUM> in <NUM>% hydrogen-argon mixture atmosphere, cooling, and grinding to <NUM> to obtain the single atomic zinc additive.

The hydrogen-argon mixture can reduce the metal single atomic precursor, avoid oxidation thereof at high temperature, and improve the ability of the atomic zinc additive prepared in the present application to activate oxygen, thereby improving the effect of the single atomic zinc additive prepared in the present application on the prevention of piglet diarrhea.

In summary, the present application achieves the following beneficial effects:.

The present application will be further described in details below in connection with the accompanying drawings.

Preparation of washing solution: the washing solution was obtained by mixing cationic surfactant cetyltrimethylammonium bromide with an aqueous ethanol solution. The volume ratio of ethanol to water in the aqueous ethanol solution was <NUM>:<NUM>, and <NUM> mol cetyltrimethylammonium bromide was contained per <NUM> of the aqueous ethanol solution.

A preparation method of single atomic zinc additive for replacing nano zinc oxide for use in feeds was performed by the following steps:.

A preparation method of single atomic zinc additive for replacing nano zinc oxide for use in feeds included the following steps:.

Referring to <FIG>, in the single atomic zinc additive for replacing nano zinc oxide for use in feeds in the present applicant, zinc atom <NUM> is ankled on the surface of the nano-silica carrier <NUM>, and there is no zinc atom <NUM> contained inside the nano-silica carrier <NUM>.

Comparative Example <NUM> differs from Example <NUM> in that: for preparing single atomic zinc additive precursor in S3, in Comparative Example <NUM>, the mass ratio of active metal zinc and nano silica carrier in S3 is <NUM>:<NUM>.

Example <NUM> differs from Example <NUM> in the high-temperature for activating in S4. In particular, in S4 of Example <NUM>, the single atomic zinc additive precursor powder prepared in S3 was activated at a high temperature of <NUM> in a <NUM>% air atmosphere for <NUM>, cooled and ground to <NUM> on a planetary mill (zirconia ball mill) to obtain the single atomic zinc additive.

Example <NUM> differs from Example <NUM> in S13. In particular, in S13 of Example <NUM>, the mixed solution obtained in S12 was continuously mixed and stirred under <NUM> rpm at <NUM> for <NUM> to obtain a precipitate, which was centrifugally washed with water.

Example <NUM> differs from Example <NUM> in the sodium silicate solution in S11. In particular, in S11 of Example <NUM>, sodium silicate and cationic surfactant cetyltrimethylammonium bromide were added to the aqueous ethanol solution with a volume ratio of ethanol to water of <NUM>:<NUM> to obtain <NUM> mol/L sodium silicate solution, in which <NUM> CTAB was contained per <NUM> solution.

Example <NUM> differs from Example <NUM> in the sodium silicate solution in S11. In particular, in S11 of Example <NUM>, sodium silicate and cationic surfactant cetyltrimethylammonium bromide were added to the aqueous ethanol solution with a volume ratio of ethanol to water of <NUM>:<NUM> to obtain <NUM> mol/L sodium silicate solution, in which <NUM> cetyltrimethylammonium bromide was contained per <NUM> solution.

Example <NUM> differs from Example <NUM> in the preparation method of the active metal single atomic precursor of S2. In particular, in S2 of Example <NUM>, <NUM> <NUM>% aqueous sodium carbonate solution was directly added into <NUM> of <NUM>/L aqueous zinc nitrate solution, and then stirred under <NUM> rpm for <NUM>. The mixed solution was heated to <NUM> within <NUM>, stirred at a constant speed for <NUM>, and then cooled to room temperature to obtain the mixed solution.

Example <NUM> differs from Example <NUM> in the preparation method of the active metal single atomic precursor of S2. In particular, in S2 of Example <NUM>, <NUM> <NUM>% aqueous sodium carbonate solution was directly added into <NUM> of <NUM>/L aqueous zinc nitrate solution at <NUM>µL/s, and then stirred at <NUM> rpm, <NUM> for <NUM>, and then cooled to room temperature to obtain the mixed solution.

Example <NUM> differs from Example <NUM> in the preparation method of single atomic zinc additive precursor in S3. In particular, in S2 of Example <NUM>, the carrier prepared in S1 was added to the mixed solution obtained in S2 at <NUM>/min. The mass ratio of active metal zinc to nano silica carrier was <NUM>:<NUM>. The mixed solution was stirred and mixed at <NUM> rpm for <NUM>, thoroughly washed and filtered with water to neutral, dried and ground to <NUM> on a planetary mill (zirconia ball mill) to obtain single atomic zinc additive precursor powder.

According to the use amount in Examples <NUM>-<NUM>, two dose groups of <NUM>/kg and <NUM>/kg and a blank control group were established for each type. <NUM>-<NUM> white mice were divided into groups, <NUM> mice in each group, half male and half female. Two dose groups were used for Test A and Test B, respectively. The mice in the blank control group were not given drugs, and the mice in Test group A and Test group B were continuously given drugs with preset doses for <NUM> days. The spirit, appetite, water drinking, activity and poisoning of the mice were observed every day, and the death number of mice was recorded.

<NUM>-<NUM> white mice were divided into groups, <NUM> mice in each group, half male and half female. For the single atomic zinc additives prepared in Example <NUM>-<NUM>, each was tested with two dose groups and one control group. The comparison was repeated and divided into Test group A and Test group B. The mice in the blank control group were not given drugs, and the mice in test groups were intragastrically administered by two doses, that is, <NUM>/kg and <NUM>/kg, for <NUM> days. During the administration period, the spirit, appetite, death and abnormal reactions of the mice were observed. After the administration was stopped, the death and weight changes of mice were observed within <NUM> days.

Test scheme: weaned piglets were fed with zinc oxide feeds and single atomic zinc additives prepared in Examples <NUM>-<NUM>, and the effects of single atomic zinc additive and zinc oxide on diarrhea and production performance of piglets were studied. A batch of <NUM> weaned piglets with diarrhea were observed. After weaned piglets were fed with normal pig food in the first two days, they were randomly divided into <NUM> groups with <NUM> pigs in each group. After <NUM> days, the piglets in the <NUM> groups were fed with <NUM> ppm single atomic zinc additive and zinc oxide in addition to normal pig food. Piglets were kept indoors under the same temperature, humidity and ventilation conditions.

Index determination and method: the test period was <NUM> days, during which the feeding amount in each feeding was accurately recorded. The piglets and the remaining feed were weighed every week, and the average daily feed intake, average daily gain and feed conversion rate of piglets were measured accordingly. The mortality and the content of zinc in piglet feces were recorded every day (the content of zinc was measured by slurry method using a dissolution instrument).

The single atom zinc additive and nano zinc oxide for use in feeds prepared in examples <NUM>-<NUM>, and Comparative Examples <NUM>-<NUM> were tested for drug resistance antibacterial activity:.

Step <NUM>: bacteria (drug-resistant Escherichia coli, drug-resistant Clostridium welchii and Methicillin-resistant Staphylococcus aureus) freshly cultured for <NUM>-<NUM> were prepared. The bacteria were washed to remove fungus moss and prepared into a bacterial suspension with <NUM> PBS solution (<NUM> mol/L). Then the bacterial suspension was diluted with PBS solution to desired concentrations (<NUM>µL bacterial suspension was dropped on a control sample tablet, and the number of recovered bacteria was <NUM> × <NUM><NUM>-<NUM> × <NUM><NUM> cfu/tablet);.

Step <NUM>: a certain amount of single atomic zinc additive and zinc oxide were weighed and dispersed in PBS solution to form a sample solution (the concentration of single atomic zinc additive and zinc oxide was <NUM> ppm), which was put into a <NUM> conical flask;.

Step <NUM>: the conical bottle was fixed on a shaking table and shaken at <NUM> r/min for <NUM>; and.

Step <NUM>: <NUM> of the sample solution (or sample solution properly diluted with PBS) were taken at <NUM> and after shaking for <NUM> respectively, inoculated in a flat dish with agar pouring method, and, incubated in <NUM>-<NUM> incubator for <NUM>-<NUM>, followed by bacterial colonies counting.

The test was repeated for <NUM> times, and the antibacterial rate was calculated according to the formula: <MAT> where,.

In the acute toxicity test, the mice in Test group A, Test group B and blank control group in Examples <NUM>-<NUM> showed no abnormal performance compared with that in the control group after the acute toxicity test <NUM> days, and the mice in the two test groups showed normal appetite, mental state, water drinking and food intake after the acute toxicity test. After administration of <NUM>/kg single atomic feed additive in group A and group B respectively, no poisoning death occurred in the two groups of mice after acute toxicity test. According to the evaluation standard of toxicology, there was no need to determine the median lethal dose, that was, LD > <NUM>/kg. The results of acute toxicity test showed that the single atomic zinc additive was non-toxic.

After the cumulative toxicity test, the mice in Test group A and Test group B in Examples <NUM>-<NUM> showed normal appetite, spirit and drinking water after administration <NUM> days, and there was no death. After stopping the Administration for <NUM> days, the mice showed normal performance. It can be seen from table <NUM> that after administration <NUM> days, there was no significant difference in initial weight, end weight and average weight gain between the mice in the two test groups using the single atomic zinc additive prepared in Examples <NUM>-<NUM> and the blank control group.

It can be seen from Examples <NUM>-<NUM> and Table <NUM> that, the diarrhea of piglets is significantly improved after feeding piglets with the single atomic zinc additive of the present applicant. Moreover, the average daily feed intake, average daily gain and feed conversion rate of piglets with single atomic zinc additive with zinc content of <NUM> ppm are the same as or even better than those for zinc oxide. There is no zinc in the feces of piglets after feeding single atomic zinc additive, so that zinc can be repeatedly absorbed by piglets and will not cause environmental pollution. Therefore, it shows that the present application can replace zinc oxide in preventing piglet diarrhea and promoting growth while achieving a more excellent effect.

It can be seen from Examples <NUM>-<NUM>, Comparative Example <NUM> and Table <NUM> that, when the addition amount of single atomic zinc additive is <NUM> ppm, the killing rates of single atomic zinc additive to drug-resistant Escherichia coli, drug-resistant Clostridium welchii,.

Methicillin-resistant Staphylococcus aureus are better than <NUM> ppm zinc oxide. The results show that single atomic zinc additive has better ability of kill piglet diarrhea bacteria than zinc oxide for feeds, without producing drug resistance. When the mass ratio of active metal zinc to nano silica carrier is <NUM>: (<NUM>-<NUM>), it has excellent ability of killing piglet diarrhea bacteria, without producing drug resistance. In addition, the single atomic zinc additive prepared in Example <NUM> achieves a killing rate of <NUM>%, indicating that, when the mass ratio of active metal zinc to nano silica carrier is <NUM>:<NUM>, it has the best effect of killing piglet diarrhea bacteria.

It can be seen from Example <NUM>, Comparative Example <NUM>, Example <NUM> and Table <NUM> that, the single atomic zinc additives prepared therein all achieve a killing rate <NUM>%, but the raw materials used in Example <NUM> are less than those in Comparative Example <NUM> and Example <NUM>. Therefore, Example <NUM> has better economic benefits.

Claim 1:
A single atomic zinc additive for use in feeds, characterized in that, the single atomic zinc additive comprises a carrier and active metal zinc, wherein the active metal zinc is distributed on a surface of the carrier in single atomic form, without active metal zinc contained inside the carriers; wherein the carrier is nano silica, and a mass ratio of metal zinc to nano silica is <NUM>:(<NUM>-<NUM>).