A feed additive for reducing antibiotic-resistant genes in animal feces, a preparation method thereof, and an application thereof

Abstract

This invention belongs to the field of feed additive technology, and particularly relates to a feed additive that reduces antibiotic resistance genes in animal feces, its preparation method, and its application. The feed additive comprises the following components in the indicated mass ratios: rosmarinic acid 10-15%, 5-O-caffeoylshikimic acid 10-15%, Lycopus lucidus flavonoids 8-12%, tea polyphenols 7-10%, DL-α-tocopherol 7-10%, hydroxypropyl-β-cyclodextrin 22-28%, microcapsule wall material 18-22%, EDTA-2Na 0.8-1.2%, and citrate-sodium citrate buffer 1.5-2.2%. This feed additive not only exhibits clear antibacterial activity against Escherichia coli and Salmonella, but also improves the growth performance of weaned piglets, reduces the diarrhea rate in weaned piglets, and decreases the abundance of antibiotic resistance genes in their feces, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and in particular relates to a feed additive that reduces antibiotic resistance genes in animal feces, its preparation method, and its application. Background Technology

[0002] The gut microbiota of livestock and poultry is a complex micro-ecosystem, home to a diverse and abundant community of gut microbes. These microbes are not only the core regulators of the host's gut microecological balance, but also an important reservoir of antibiotic resistance genes (ARGs) between the environment and the host.

[0003] ARGs can spread horizontally across hosts, media, and ecosystems through conjugation, transformation, and transduction. They can also spread to the outside world through the food chain and fecal emissions, polluting the entire ecosystem. Horizontal spread can transfer ARGs to originally drug-resistant symbiotic bacteria or opportunistic pathogens, increasing the drug resistance level of the entire gut microbiota. ARGs in the human and animal gut can transfer to pathogenic strains, leading to the emergence of "superbugs" in clinical practice. This renders the treatment of common infectious diseases ineffective, reduces animal production performance and health, and increases treatment costs and mortality.

[0004] In addition, the spread of ARGs in soil and water can integrate with the inherent resistance gene pool in the environment, giving rise to multidrug-resistant strains and forming a "reservoir of environmental resistance genes" that is difficult to eliminate, posing a long-term potential threat to the ecosystem.

[0005] From the perspective of "One Health," the spread of ARGs not only disrupts livestock and poultry farming ecology and production efficiency but also threatens public health safety and ecological balance. Therefore, it is urgent to reduce the abundance and spread of drug-resistant genes in animal feces at the source. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a feed additive that reduces antibiotic resistance genes in animal feces, its preparation method and application. This feed additive can improve animal growth performance, reduce diarrhea rate and the abundance of antibiotic resistance genes in animal feces.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a feed additive for reducing antibiotic resistance genes in animal feces, comprising the following components in the following mass ratios: rosmarinic acid 10-15%, 5-O-caffeoylshikimic acid 10-15%, Lycopus lucidus flavonoids 8-12%, tea polyphenols 7-10%, DL-α-tocopherol 7-10%, hydroxypropyl-β-cyclodextrin 22-28%, microcapsule wall material 18-22%, EDTA-2Na 0.8-1.2%, and citric acid-sodium citrate buffer 1.5-2.2%.

[0008] Preferably, the microcapsule wall material is made of sodium octenyl succinate starch and gelatin, wherein the mass ratio of sodium octenyl succinate starch to gelatin is 1.5~2.5:1.

[0009] Preferably, the pH of the citric acid-sodium citrate buffer solution is 5.0-5.5, and the total concentration of citric acid and sodium citrate is 10% w / v.

[0010] This invention also discloses a method for preparing the above-mentioned feed additive that reduces antibiotic resistance genes in animal feces, comprising the following steps: (1) Preparation of cyclodextrin solution: Add the prescribed amount of hydroxypropyl-β-cyclodextrin to pure water at 40~45℃ and stir until completely dissolved to obtain cyclodextrin solution; (2) Preparation of inclusion mixture: After mixing the formula amount of rosmarinic acid, 5-O-caffeoyl shikimic acid and zeylan flavonoids evenly, slowly add the cyclodextrin solution from step (1), heat to 40~45℃, and stir at 300~600 rpm for 2~3 h under nitrogen protection to obtain inclusion mixture; (3) Preparation of loose inclusion micro powder: Pour the inclusion mixture from step (2) into a freeze drying tray, place it in a freeze dryer, pre-freeze at -40~-35℃ for 3~5 h, then under vacuum of 5~20 Pa, gradually raise the shelf temperature from -30℃ to 0℃, perform a first drying for 12~18 h, and finally raise the shelf temperature from 0℃ to 25℃, and perform a second drying at this temperature for 4~6 h to obtain loose inclusion micro powder; (4) Preparation of suspension: Add the microcapsule wall material according to the formula to purified water at 50~60℃, stir until completely dissolved, then add the tea polyphenols, DL-α-tocopherol and EDTA-2Na according to the formula, and homogenize at 8000~12000 rpm for 3~8 min to make it evenly dispersed; then slowly add the loosely encapsulated micro powder from step (3), stir and disperse to form a uniform suspension; (5) Preparation of feed additive: The suspension from step (4) is pumped into a granulator and atomized for granulation. During the granulation process, the citric acid-sodium citrate buffer solution of the formula is sprayed through the top atomizing nozzle to adjust the pH of the particle surface and obtain the initial granules. The initial granules are sent to a low-temperature fluidized bed and dried under nitrogen protection to remove surface moisture. Then, 200~300 μm particles are screened through a grading sieve to obtain the feed additive that reduces antibiotic resistance genes in animal feces.

[0011] Preferably, in step (1), the mass ratio of hydroxypropyl-β-cyclodextrin to pure water is 1:15~30.

[0012] In the preferred step (4), the mass ratio of the microcapsule wall material to purified water is 1:4~6.

[0013] Preferably, in step (5), the inlet air temperature of the low-temperature fluidized bed is 35~45℃ and the drying time is 20~40min.

[0014] The present invention also discloses the application of the above-mentioned feed additive in reducing antibiotic resistance genes in animal feces, wherein the feed additive is added to the basal diet of weaned piglets at a dosage of 300 mg / kg.

[0015] Preferably, the antibiotic resistance gene is tet(Q), tet(37), mel, ANT(6)-Ib, erm(F), APH(3)-IIIa, aadS, erm(G), fexA, tet(40), erm(B), lsa(A), efrB, efrA, tet(M), arlR, emeA, dfrE, tetX2, CfxA6, SAT-4, lsa(B), tet(W), mef(En2), aad(6), optrA, sul2, bacA, ANT(6)- Ia and cfr(E) Ia and cfr(E) .

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The feed additive of the present invention has good antibacterial activity against Escherichia coli and Salmonella. The minimum inhibitory concentration experiment shows that the minimum inhibitory concentration of the feed additive of the present invention against Escherichia coli and Salmonella can reach 64~128 μg / mL, and the diameter of the inhibition zone against Escherichia coli and Salmonella is larger than that of colistin sulfate, showing a good antibacterial effect.

[0017] 2. The feed additive of the present invention can improve the growth performance of weaned piglets. Animal experiments show that after feeding a diet containing the product of the present invention, the average daily weight gain of weaned piglets is significantly increased and the feed conversion ratio is reduced.

[0018] 3. The feed additive of the present invention can also reduce the diarrhea rate of weaned piglets and the abundance of drug resistance genes in their feces, and has good application prospects. Attached Figure Description

[0019] Figure 1 The graph shows the storage stability results of the feed additives in Examples 1-3 of this invention.

[0020] Figure 2The graph shows the minimum inhibitory concentration (MIC) results of the feed additives of Examples 1-3 of this invention against Escherichia coli and Salmonella.

[0021] Figure 3 The graph shows the diameter of the inhibition zone of the feed additives in Examples 1-3 of this invention against Escherichia coli and Salmonella.

[0022] Figure 4 The graph shows the effect of the feed additives of Examples 1-3 of this invention on the abundance of drug resistance genes in the feces of weaned piglets. Detailed Implementation

[0023] The present invention will now be described in more detail with reference to the accompanying drawings and specific embodiments.

[0024] Example 1 This embodiment uses the following components in the indicated mass ratios to prepare a feed additive: 12% rosmarinic acid, 12% 5-O-caffeoylshikimic acid, 8% Lycopus lucidus flavonoids, 9% tea polyphenols, 8% DL-α-tocopherol, 26.5% hydroxypropyl-β-cyclodextrin, 22% microcapsule wall material (sodium octenyl succinate starch and gelatin in a mass ratio of 2:1), 1% EDTA-2Na, and 1.5% citric acid-sodium citrate buffer solution (pH 5.2, total concentration of citric acid and sodium citrate 10% w / v).

[0025] The specific preparation method includes the following steps: (1) Preparation of cyclodextrin solution: Hydroxypropyl-β-cyclodextrin and pure water at 42℃ are mixed at a mass ratio of 1:25 and stirred until completely dissolved to obtain cyclodextrin solution; (2) Preparation of inclusion mixture: After mixing rosmarinic acid, 5-O-caffeoylshikimic acid and zeylan flavonoids evenly, slowly add the cyclodextrin solution from step (1), heat to 42°C, and stir at 450 rpm for 2.5 h under nitrogen protection to obtain inclusion mixture; (3) Preparation of loose inclusion micro powder: Pour the inclusion mixture from step (2) into a freeze drying tray, place it in a freeze dryer, pre-freeze at -38℃ for 4 h, then under a vacuum of 10 Pa, gradually raise the shelf temperature from -30℃ to 0℃ and dry for 16 h, and finally raise the shelf temperature from 0℃ to 25℃ and dry for 5 h at this temperature to obtain loose inclusion micro powder; (4) Preparation of suspension: Mix the microcapsule wall material with purified water at 55℃ at a mass ratio of 1:5, stir until completely dissolved, then add tea polyphenols, DL-α-tocopherol and EDTA-2Na in sequence, and homogenize at 9000 rpm for 5 min to make it uniformly dispersed; then slowly add the loosely encapsulated micro powder from step (3), stir and disperse to form a uniform suspension; (5) Preparation of feed additives: The suspension from step (4) is pumped into a pellet mill and atomized into pellets. During the pelleting process, the formula amount of citric acid-sodium citrate buffer solution is sprayed through the top atomizing nozzle to adjust the pH of the pellet surface and obtain the initial pellets. The initial pellets are sent to a low-temperature fluidized bed and dried at 40°C for 30 min under nitrogen protection to remove surface moisture. Then, the pellets are screened through a grading sieve to obtain 200~300 μm particles, which is the product of Example 1.

[0026] Example 2 This embodiment uses the following components in the indicated mass ratios to prepare a feed additive: 10% rosmarinic acid, 15% 5-O-caffeoylshikimic acid, 10% Lycopus lucidus flavonoids, 10% tea polyphenols, 10% DL-α-tocopherol, 22% hydroxypropyl-β-cyclodextrin, 20% microcapsule wall material (sodium octenyl succinate starch and gelatin in a mass ratio of 2.5:1), 0.8% EDTA-2Na, and 2.2% citric acid-sodium citrate buffer solution (pH 5.0, total concentration of citric acid and sodium citrate 10% w / v).

[0027] The specific preparation method includes the following steps: (1) Preparation of cyclodextrin solution: Hydroxypropyl-β-cyclodextrin and pure water at 40℃ are mixed at a mass ratio of 1:15 and stirred until completely dissolved to obtain cyclodextrin solution; (2) Preparation of inclusion mixture: After mixing rosmarinic acid, 5-O-caffeoylshikimic acid and zeylan flavonoids evenly, slowly add the cyclodextrin solution from step (1), heat to 40°C, and stir at 600 rpm for 3 h under nitrogen protection to obtain inclusion mixture; (3) Preparation of loose inclusion micro powder: Pour the inclusion mixture from step (2) into a freeze drying tray, place it in a freeze dryer, pre-freeze at -35℃ for 5 h, then gradually raise the shelf temperature from -30℃ to 0℃ under a vacuum of 5 Pa, perform a first drying for 18 h, and finally raise the shelf temperature from 0℃ to 25℃, and perform a second drying at this temperature for 4 h to obtain loose inclusion micro powder; (4) Preparation of suspension: Mix the microcapsule wall material with purified water at 50℃ at a mass ratio of 1:4, stir until completely dissolved, then add tea polyphenols, DL-α-tocopherol and EDTA-2Na in sequence, and homogenize at 8000 rpm for 8 min to make it uniformly dispersed; then slowly add the loosely encapsulated micro powder from step (3), stir and disperse to form a uniform suspension; (5) Preparation of feed additives: The suspension from step (4) is pumped into a pellet mill and atomized into pellets. During the pelleting process, the formula amount of citric acid-sodium citrate buffer solution is sprayed through the top atomizing nozzle to adjust the pH of the pellet surface and obtain the initial pellets. The initial pellets are sent to a low-temperature fluidized bed and dried at 35°C for 40 min under nitrogen protection to remove surface moisture. Then, the pellets are screened through a grading sieve to obtain 200~300 μm particles, which is the product of Example 2.

[0028] Example 3 This embodiment uses the following components in the indicated mass ratios to prepare a feed additive: 15% rosmarinic acid, 10% 5-O-caffeoylshikimic acid, 12% Lycopus lucidus flavonoids, 7% tea polyphenols, 7% DL-α-tocopherol, 28% hydroxypropyl-β-cyclodextrin, 18% microcapsule wall material (sodium octenyl succinate starch and gelatin in a mass ratio of 1.5:1), 1.2% EDTA-2Na, and 1.8% citric acid-sodium citrate buffer solution (pH 5.5, total concentration of citric acid and sodium citrate 10% w / v).

[0029] The specific preparation method includes the following steps: (1) Preparation of cyclodextrin solution: Hydroxypropyl-β-cyclodextrin and pure water at 45℃ are mixed at a mass ratio of 1:30 and stirred until completely dissolved to obtain cyclodextrin solution; (2) Preparation of inclusion mixture: After mixing rosmarinic acid, 5-O-caffeoylshikimic acid and zeylan flavonoids evenly, slowly add the cyclodextrin solution from step (1), heat to 45°C, and stir at 300 rpm for 2 h under nitrogen protection to obtain inclusion mixture; (3) Preparation of loose inclusion micro powder: Pour the inclusion mixture from step (2) into a freeze drying tray, place it in a freeze dryer, pre-freeze at -40℃ for 3 h, then under a vacuum of 20 Pa, gradually raise the shelf temperature from -30℃ to 0℃ and dry for 12 h, and finally raise the shelf temperature from 0℃ to 25℃ and dry for 6 h at this temperature to obtain loose inclusion micro powder; (4) Preparation of suspension: Mix microcapsule wall material with purified water at 60℃ at a mass ratio of 1:6, stir until completely dissolved, then add tea polyphenols, DL-α-tocopherol and EDTA-2Na in sequence, and homogenize at 12000 rpm for 3 min to make it uniformly dispersed; then slowly add the loosely encapsulated micro powder from step (3) to the wall material solution, stir and disperse to form a uniform suspension; (5) Preparation of feed additives: The suspension from step (4) is pumped into a pellet mill and atomized into pellets. During the pelleting process, the formula amount of citric acid-sodium citrate buffer solution is sprayed through the top atomizing nozzle to adjust the pH of the pellet surface and obtain the initial pellets. The initial pellets are sent to a low-temperature fluidized bed and dried at 45°C for 20 min under nitrogen protection to remove surface moisture. Then, the pellets are screened through a grading sieve to obtain 200~300 μm particles, which is the product of Example 3.

[0030] Experimental Example 1 Storage stability of the products from Examples 1, 2, and 3 above at 60°C: like Figure 1 As shown, after three months of sealed storage at 60°C, the products of Examples 1, 2, and 3 exhibited a 95% retention rate of active ingredients; after 12 months of sealed storage at 60°C, over 90% of the active ingredients remained. This demonstrates the good storage stability of the products of this invention.

[0031] Experimental Example 2 Evaluation of the antibacterial effects of the products in Examples 1, 2, and 3 above: Figure 2 and Figure 3 The minimum inhibitory concentrations and inhibition zone diameters of the products from Examples 1, 2, and 3 against Escherichia coli and Salmonella are shown respectively.

[0032] from Figure 2 It can be seen that: the minimum inhibitory concentration (MIC) of the product in Example 1 against Escherichia coli and Salmonella is 64 μg / mL; the MIC of the product in Example 2 against Escherichia coli and Salmonella is 64 μg / mL and 128 μg / mL, respectively; and the MIC of the product in Example 3 against Salmonella and Escherichia coli is 128 μg / mL and 64 μg / mL, respectively. This indicates that the product of the present invention has a good antibacterial effect. from Figure 3 It can be seen that the inhibition zone diameters of the products in Examples 1, 2 and 3 against Escherichia coli and Salmonella are all larger than those of colistin sulfate (antibiotic), indicating that the antibacterial effect of the products of the present invention is better than that of colistin sulfate.

[0033] Experimental Example 3 The effects of the products from Examples 1, 2, and 3 above on the growth performance of weaned piglets and drug resistance genes in their feces.

[0034] Experimental Design: Twenty-four healthy Duroc × Landrace × Large White weaned piglets weighing approximately 6 kg were randomly divided into four groups based on the principle of no significant difference in body weight, with six replicates per group and one pig per replicate. The basal diet was formulated according to the NRC (2012) "Nutritional Requirements for Swine". The experimental groups were supplemented with 300 mg / kg of the product from Examples 1, 2, and 3, respectively. During the experiment, the ambient temperature (22–25°C) and relative humidity (50–65%) in the livestock housing were controlled. All pigs had free access to feed and water. The experiment lasted 21 days. Feed intake and fecal scores were recorded daily, and pigs were weighed weekly. Production performance (average feed intake, average daily weight gain, feed conversion ratio) and diarrhea rate were statistically analyzed at each stage and throughout the entire experiment, using replicates as the unit. The abundance of drug resistance genes was determined by qPCR.

[0035] Experimental results: A. Impact on production performance: As shown in Table 1, compared with the basal diet group, adding 300 mg / kg of the product from Examples 1, 2 and 3 to the diet of weaned piglets can increase the average daily feed intake and average daily weight gain, and reduce the feed conversion ratio.

[0036] Table 2 shows that adding 300 mg / kg of the products from Examples 1, 2, and 3 to the diets of weaned piglets effectively reduced the diarrhea rates at 1-7 days, 8-14 days, 15-21 days, and 1-21 days. This indicates that the products from Examples 1-3 of this invention can improve the growth performance of weaned piglets and reduce the diarrhea rate.

[0037] Table 1. Effects of the products in Examples 1-3 of this invention on the growth performance of weaned piglets. Table 2. Effects of the products of Examples 1-3 of the present invention on the diarrhea rate (%) in weaned piglets. B. Effect of drug resistance gene abundance in feces like Figure 4 As shown, adding 300 mg / kg of the product from Examples 1, 2, and 3 to the diet of weaned piglets can reduce drug resistance genes in fecal samples. tet(Q), tet(37), mel, ANT(6)-Ib, erm(F), APH(3)-IIIa, aadS, erm (G), fexA, tet(40), erm(B), lsa(A), efrB, efrA, tet(M), arlR, emeA, dfrE, tetX2, CfxA6, SAT-4, lsa(B), tet(W), mef(En2), aad(6), optrA, sul2, bacA, ANT(6)-Ia and cfr(E) The relative abundance of these genes. The above data indicate that the products of Examples 1, 2, and 3 can effectively reduce the abundance of drug resistance genes in fecal samples.

[0038] In summary, the feed additive of this invention can improve the growth performance of weaned piglets, reduce the diarrhea rate of piglets, and decrease the abundance of drug resistance genes in their feces, showing good application prospects.

[0039] The above embodiments are merely illustrative of the present invention and are not intended to limit the invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A feed additive that reduces antibiotic resistance genes in animal feces, characterized in that, The product comprises the following components in the indicated mass ratios: rosmarinic acid 10-15%, 5-O-caffeoylshikimic acid 10-15%, Lycopus lucidus flavonoids 8-12%, tea polyphenols 7-10%, DL-α-tocopherol 7-10%, hydroxypropyl-β-cyclodextrin 22-28%, microcapsule wall material 18-22%, EDTA-2Na 0.8-1.2%, and citrate-sodium citrate buffer 1.5-2.2%.

2. The feed additive for reducing antibiotic resistance genes in animal feces as described in claim 1, characterized in that, The microcapsule wall material is made of sodium octenyl succinate starch and gelatin, wherein the mass ratio of sodium octenyl succinate starch to gelatin is 1.5~2.5:

1.

3. The feed additive for reducing antibiotic resistance genes in animal feces as described in claim 1, characterized in that, The pH of the citric acid-sodium citrate buffer solution is 5.0-5.5, and the total concentration of citric acid and sodium citrate is 10% w / v.

4. A method for preparing a feed additive that reduces antibiotic resistance genes in animal feces as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of cyclodextrin solution: Add the prescribed amount of hydroxypropyl-β-cyclodextrin to pure water at 40~45℃ and stir until completely dissolved to obtain cyclodextrin solution; (2) Preparation of inclusion mixture: After mixing the formula amount of rosmarinic acid, 5-O-caffeoyl shikimic acid and zeylan flavonoids evenly, slowly add the cyclodextrin solution from step (1), heat to 40~45℃, and stir at 300~600 rpm for 2~3h under nitrogen protection to obtain inclusion mixture; (3) Preparation of loose inclusion micro powder: Pour the inclusion mixture from step (2) into a freeze drying tray, place it in a freeze dryer, pre-freeze at -40~-35℃ for 3~5 h, then under vacuum of 5~20 Pa, gradually raise the shelf temperature from -30℃ to 0℃, perform a first drying for 12~18 h, and finally raise the shelf temperature from 0℃ to 25℃, and perform a second drying at this temperature for 4~6 h to obtain loose inclusion micro powder; (4) Preparation of suspension: Add the microcapsule wall material according to the formula to purified water at 50~60℃, stir until completely dissolved, then add the tea polyphenols, DL-α-tocopherol and EDTA-2Na according to the formula, and homogenize at 8000~12000 rpm for 3~8 min to make it evenly dispersed; then slowly add the loosely encapsulated micro powder from step (3), stir and disperse to form a uniform suspension; (5) Preparation of feed additives: The suspension from step (4) is pumped into a pellet mill and atomized into pellets. During the pelleting process, the formula amount of citric acid-sodium citrate buffer solution is sprayed through the top atomizing nozzle to adjust the pH of the pellet surface and obtain the initial pellets. The pre-processed granules are fed into a low-temperature fluidized bed and dried under nitrogen protection to remove surface moisture; then, they are screened through a grading sieve to obtain 200-300 μm particles, thus obtaining a feed additive that reduces antibiotic resistance genes in animal feces.

5. The method for preparing the feed additive that reduces antibiotic resistance genes in animal feces as described in claim 4, characterized in that, In step (1), the mass ratio of hydroxypropyl-β-cyclodextrin to pure water is 1:15~30.

6. The method for preparing the feed additive that reduces antibiotic resistance genes in animal feces as described in claim 4, characterized in that, In step (4), the mass ratio of the microcapsule wall material to purified water is 1:4~6.

7. The method for preparing the feed additive that reduces antibiotic resistance genes in animal feces as described in claim 4, characterized in that, In step (5), the inlet air temperature of the low-temperature fluidized bed is 35~45℃, and the drying time is 20~40 min.

8. The application of the feed additives as described in claims 1 to 3 in reducing antibiotic resistance genes in animal feces.

9. The application of the feed additive as described in claim 8 in reducing antibiotic resistance genes in animal feces, characterized in that, The feed additive was added to the basal diet of weaned piglets at a rate of 300 mg / kg.

10. The application of the feed additive as described in claim 8 or 9 in reducing antibiotic resistance genes in animal feces, characterized in that, The antibiotic resistance gene is tet(Q), tet(37), mel, ANT(6)-Ib, erm(F), APH(3)- IIIa, aadS, erm(G), fexA, tet(40), erm(B), lsa(A), efrB, efrA, tet(M), arlR, emeA, dfrE, tetX2, CfxA6, SAT-4, lsa(B), tet(W), mef(En2), aad(6), optrA, sul2, bacA, ANT(6)-Ia and cfr(E) .

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