Compound biological agent for improving phosphorus utilization efficiency of dairy cows, preparation method and application

Abstract

The application relates to a composite biological agent for improving phosphorus utilization efficiency of dairy cows, a preparation method and application in the technical field of phosphorus emission reduction of dairy cows. The composite biological agent comprises butyric acid carboxy bacteria, DNA and phytase connected with nano zinc oxide; the butyric acid carboxy bacteria, the DNA and the phytase connected with nano zinc oxide are sequentially connected. The butyric acid carboxy bacteria in the composite biological agent of the application has strong colonization ability in the intestinal tract, so that the retention time of the phytase connected with the butyric acid carboxy bacteria in the intestinal tract is improved, the role of hydrolyzing phytic acid is effectively played, the nano zinc oxide connected with the phytase can protect the activity of the phytase, the phytase continuously converts phytic acid in feed, the composite biological agent has a long application effect time on the dairy cows, the phosphorus emission reduction effect is good, the components in the composite biological agent are biologically safe, so the composite biological agent can be well applied to reducing phosphorus emission in the process of dairy cow breeding and has a good application prospect.

Description

Technical Field

[0001] This application relates to the field of phosphorus reduction technology in dairy cows, and in particular to a compound biological agent, preparation method and application for improving phosphorus utilization efficiency in dairy cows. Background Technology

[0002] Phosphorus is an important component of dairy cow feed, playing a wide range of roles in metabolic processes such as bone formation and reproductive performance. Phytic acid is a storage form of phosphorus in plant cell walls and seeds, and is widely found in plant foods. Phytic acid phosphorus in feed is difficult for animals to absorb because it combines with minerals such as calcium and magnesium to form insoluble salts and is excreted. However, hydrolyzed phytic acid is converted into inorganic phosphorus, which can be fully absorbed and utilized by animals.

[0003] Studies have found that the main gut microbiota involved in the conversion of phytate and phosphorus in dairy cows' feed are Prevotella, Bifidobacterium, and Shevarl. While these microorganisms can secrete phytase to hydrolyze phytate and improve the digestibility of phosphorus in feed, their proportion and long-term colonization ability in the rumen are relatively poor, making it difficult for them to permanently colonize the rumen epithelium. Furthermore, directly feeding phytase results in its rapid excretion with the digesta, and its activity is easily destroyed by related components in the digestive tract, thus rendering it inactive and unable to sustainably convert phytate in the feed.

[0004] Therefore, constructing a phytase system that can exist in the rumen for a long time can provide important support for reducing phosphorus in dairy cow feed, protecting the environment, and saving costs and increasing efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides a compound biological agent that improves the phosphorus utilization efficiency of dairy cows. This compound biological agent can colonize the rumen for a long period of time and can efficiently degrade phytic acid, providing a solution for phosphorus emission reduction and efficient feed utilization in dairy cows.

[0006] Therefore, the first aspect of this application provides a compound biological agent for improving the phosphorus utilization efficiency of dairy cows, the compound biological agent comprising butyric acid carboxyl bacteria, DNA, and phytase linked with nano-zinc oxide; the butyric acid carboxyl bacteria, DNA, and phytase linked with nano-zinc oxide are sequentially linked together.

[0007] In this application, the composite biological agent comprises butyric acid carboxyl bacteria linked to DNA and phytase linked to nano-zinc oxide. The butyric acid carboxyl bacteria exhibit strong colonization ability and resistance in the intestine (tolerance to high temperatures, acids, alkalis, and bile salts), thus enabling the composite biological agent to colonize the intestine for a long period. The phytase degrades phytic acid, hydrolyzing it into inorganic phosphorus, which can then be fully absorbed and utilized by animals. The nano-zinc oxide acts as a phytase protectant, preserving the activity of the phytase and thus continuously converting phytic acid in the feed. Compared to traditional phytase, this composite biological agent has a longer duration of effect and better phosphorus reduction in dairy cows, making it well-suited for reducing phosphorus emissions during dairy farming.

[0008] In some embodiments, the DNA is a DNA loop formed by a template and primers; the nucleotide sequence of the template is shown in SEQ ID NO:1 (5'-TGTCTTCGCCTTCTTGTTTCCTTTCCTTGAAACTTCTTCCTTTCTTTCTTTCGACTAAGCACC-3'), and the nucleotide sequence of the primers is shown in SEQ ID NO:2 (5'-GGCGAAGACAGGTGCTTAGTC-3').

[0009] In this application, butyric acid bacteria and phytase linked with nano-zinc oxide can be linked through the DNA. By utilizing the strong colonization of butyric acid bacteria in the intestine, the phytase linked to butyric acid bacteria can be retained in the intestine for a longer time, thereby effectively exerting the function of hydrolyzing phytic acid.

[0010] In some embodiments, the butyric acid carboxyl bacteria are bovine butyric acid clostridium.

[0011] In some specific embodiments, the butyric acid carboxyl bacteria are bovine dung-derived Clostridium butyricum. The butyric acid carboxyl bacteria in this compound biological agent are selected from bovine Clostridium butyricum, thus avoiding biosafety issues. Furthermore, zinc and phytase are both commonly used dairy cow feed additives. Therefore, the compound biological agent of this application can be used as a feed additive for dairy cows.

[0012] The second aspect of this application provides a method for preparing a compound biological agent as described in the first aspect of this application, the method comprising the following steps:

[0013] S1, After the template and primers are circularized, rolling circle amplification is performed to obtain DNA circles;

[0014] S2, the primers are linked to Clostridium butyricum to synthesize a primer-clostridium butyricum linker;

[0015] S3, a ligation reaction is carried out on the reaction system containing the DNA loop, the primer-clostridium butyricum linker, and the phytase linked with nano-zinc oxide to prepare the composite biological agent.

[0016] This application integrates the above methods to form a novel additive-compound biological agent based on bovine Clostridium butyricum-DNA-nano zinc oxide-phytase for efficient utilization of phosphorus in dairy cows. This compound biological agent can greatly improve the role of phytase in reducing excessive phosphorus emissions during dairy farming.

[0017] In some embodiments, step S1 specifically includes: mixing the template, primers, and buffer solution and then annealing the mixture to obtain a template / primer dimer; mixing the template / primer dimer with T4 DNA ligase and T4 DNA ligase buffer to prepare a cross-linking system; performing a cross-linking reaction on the cross-linking system to obtain a circularized template / primer; and performing rolling circle amplification on the circularized template / primer to obtain a DNA circle.

[0018] In this application, the concentration ratio of the template to the primer can be (1-2):4, such as 1.5:4; the buffer can be 1×PBS (phosphate buffer); the annealing temperature of the annealing treatment can be 95-98℃, and the annealing time can be 1-3 minutes.

[0019] In this application, the volume of the cross-linking system can be 20–25 μL, the content of template / primer dimer in the cross-linking system can be 1.5–2 μM, the content of T4 DNA ligase can be 25–30 U / μL, the temperature of the cross-linking reaction can be 15–18 °C, and the time can be 10–15 hours.

[0020] In this application, the rolling circle amplification reaction system can be 60 μL, and the reaction system includes circularized template / primers, dNTPs, DNA polymerase (Phi29 DNA polymerase), and DNA polymerase buffer (10×Phi29 DNA polymerase buffer). The rolling circle amplification program can be: pre-denaturation at 95–98℃ for 3–5 min; denaturation at 90–94℃ for 1–2 min; annealing at 55–60℃ for 35–45 s; extension at 70–72℃ for 40–45 s (denaturation-annealing-extension, 25 cycles); and termination at 70–72℃ for a final extension of 40–45 s.

[0021] This application can effectively prepare DNA loops using the above method for use in subsequent steps.

[0022] In some embodiments, step S2 specifically includes: activating Clostridium butyricum with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to obtain activated Clostridium butyricum; mixing the activated Clostridium butyricum with the primers and performing a ligation reaction to obtain a primer-Clostridium butyricum linker.

[0023] In this application, *Clostridium butyricum* is first activated using activators 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), which enhances the reactivity of the carboxyl groups on the surface of *Clostridium butyricum*, thereby enabling efficient coupling with the amino groups in the primers. This results in the primer-Clostridium butyricum linker. In this application, the ligation reaction can be carried out under anaerobic conditions at a temperature of 20–25°C for 2.5–3 hours.

[0024] In some embodiments, the method for preparing the phytase linked to nano-zinc oxide includes the following steps:

[0025] T1, a zinc-polyvinylpyrrolidone solution is obtained by adding a methanol solution containing zinc chloride dropwise to a methanol solution containing polyvinylpyrrolidone and stirring the mixture.

[0026] T2, a methanol solution containing phytase is added dropwise to the zinc-polyvinylpyrrolidone solution and stirred to react. After dialysis of the reaction product, phytase with nano-zinc oxide is obtained.

[0027] This application provides a simple and effective method for linking nano-zinc oxide to phytase. The stirring reaction in step T1 can be carried out at room temperature (20-25°C) with a stirring speed of 300-500 rpm for 10-20 minutes. The stirring reaction in step T2 can also be carried out at room temperature (20-25°C) with a stirring speed of 300-500 rpm for 2.5-3 hours.

[0028] In some embodiments, the mass ratio of phytase, zinc chloride and polyvinylpyrrolidone is 1:(1.5-2.5):(6-7).

[0029] In some preferred embodiments, the mass ratio of phytase, zinc chloride, and polyvinylpyrrolidone is 1:2:6.6.

[0030] By controlling the dosage of phytase, zinc chloride, and polyvinylpyrrolidone within the aforementioned range, this application enables the prepared phytase with nano-zinc oxide to have a better protective effect on phytase, thereby improving the effect of the compound biological agent on the phosphorus utilization efficiency in dairy cow feed.

[0031] In some embodiments, step S3 specifically includes: mixing the DNA loop, primer-clostridium butyricum linker, phytase linked with zinc oxide nanoparticles, DNA polymerase, dNTPs, DNA polymerase buffer, and water to form a reaction system; and subjecting the reaction system to a ligation reaction at 25–30°C for 1.5–2.5 hours to obtain the composite biological agent.

[0032] In this application, the DNA polymerase added to the reaction system can be phi29 DNA polymerase, and the DNA polymerase buffer can be 10×phi29 DNA polymerase buffer.

[0033] In this application, the butyric acid carboxyl bacteria used in the method are bovine butyric acid carboxyl bacteria, specifically bovine manure-derived butyric acid carboxyl bacteria, which can be obtained by separating and culturing dairy cow manure.

[0034] The third aspect of this application provides a feed additive for improving phosphorus utilization efficiency in dairy cow feed, comprising a compound biological agent as described in the first aspect of this application or a compound biological agent prepared by the method described in the second aspect.

[0035] In the compound biological agent of this application, phytase linked to nano-zinc oxide is attached to butyric acid carboxylic acid bacteria via DNA linkage. Utilizing the strong colonization of butyric acid carboxylic acid bacteria in the intestine, the phytase linked to the bacteria can remain in the intestine for a longer period, thus effectively hydrolyzing phytic acid. Furthermore, nano-zinc oxide protects the activity of the phytase, allowing the phytase colonized in the intestine to continuously convert phytic acid in the feed. Compared with traditional phytases, this compound biological agent has a longer duration of effect on dairy cows, better phosphorus reduction, and all components are biosafety-free. Therefore, it can be well applied as a feed additive to improve phosphorus utilization efficiency in dairy cow feed.

[0036] The beneficial technical effects of this application are as follows: The butyric acid carboxyl bacteria in the compound biological agent provided by this application have a strong colonization ability in the intestine, thus increasing the retention time of phytase linked to butyric acid carboxyl bacteria in the intestine, thereby effectively exerting the effect of hydrolyzing phytic acid. Moreover, the nano zinc oxide linked to the phytase can protect the activity of the phytase, enabling the phytase to continuously convert phytic acid in the feed. Therefore, the compound biological agent has a long-lasting effect on dairy cows and a good phosphorus emission reduction effect. Furthermore, all components in the compound biological agent are biosafe, so it can be well applied to reduce phosphorus emissions in the dairy cow breeding process, and its application prospects are good. Detailed Implementation

[0037] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0038] Example 1: Preparation of a compound biological agent to improve phosphorus utilization efficiency in dairy cows

[0039] (1) Cultivation of Clostridium butyricum from cow dung

[0040] Take 5g of cow manure and place it in an Erlenmeyer flask containing 100mL of sterile water. Shake for about 25 minutes to thoroughly mix the manure and water, prepare a diluted solution, and incubate at 85℃ for 15 minutes to kill non-spore-forming bacteria. Then, serially dilute the solution at a 10-fold dilution ratio and inoculate it at a 5% concentration into Clostridium butyricum-enriched medium. Incubate at 37℃ in an anaerobic incubator for 48 hours. Dilute the sample 10-fold and spread it onto tryptophanase-sulfite-neomycin medium, and incubate anaerobicly at 37℃ for 48 hours. Select colonies that conform to the culture characteristics and colony morphology of Clostridium butyricum for subsequent experiments.

[0041] (2) Synthesis of DNA loops

[0042] The template (SEQ ID NO:1: 5'-TGTCTTCGCCTTCTTGTTTCCTTTCCTTGAAACTTCTTCCTTTCTTTCTTTCGACTAAGCACC-3') and primer (SEQ ID NO:2: 5'-GCGAAGACAGGTGCTTAGTC-3') were mixed at a concentration ratio of 1.5:4. The mixture was then made up to 30 μL with 1×PBS (phosphate buffer). This mixture was added to a 50 μL PCR tube and annealed in a PCR instrument (95°C for 3 minutes) to form a stable template / primer dimer. The tube was then stored at 12°C. Based on this, a 24 μL cross-linking system (final concentration) was prepared with the following components: 2 μM template / primer mixture, 30 U / μL T4 DNA ligase, and 1×T4 DNA ligase buffer. The cross-linking reaction was performed in a 50 μL centrifuge tube to form a circular template / primer with a complete circular structure. The crosslinking reaction steps are as follows: overnight (12 hours) at 16°C, inactivated by heating at 75°C for 10 minutes, and the final product of the reaction is purified by centrifugation in a 10kD ultrafiltration centrifuge tube to remove buffer components in the system and avoid interfering with the subsequent reaction process.

[0043] Take 15 μL of 3 μM circularized template / primers, 6 μL of 10 mM dNTPs, 3 μL of 10 U / μL phi29 DNA polymerase, and 5 μL of 10×phi29 DNA polymerase buffer. Finally, add ultrapure water to make up to 60 μL of rolling circle amplification reaction mixture. The rolling circle amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 55℃ annealing for 45 s, 72℃ extension for 45 s (denaturation-annealing-extension, 25 cycles); 72℃ final extension for 45 s to terminate the reaction. DNA circles are obtained after the reaction and used for subsequent experiments.

[0044] (3) Preparation of primer-Clostridium butyricum linker

[0045] Take 200 μL (2 × 10⁻⁶) of the naturally cultured Clostridium butyricum from step (1). 8 CFUs were centrifuged at 8000 rpm for 5 min, the supernatant was removed and the mixture was resuspended. 2 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution (EDC, 0.11 mg / mL) and 3 μL of N-hydroxysuccinimide solution (NHS, 0.13 mg / mL) were added, mixed and activated for 30 min. After centrifugation at 8000 rpm for 5 min, the supernatant was removed and the mixture was resuspended. 5 μL of 100 mM DNA primers were added, mixed well and incubated under anaerobic conditions for 3 hours (20 °C) to obtain the primer-Clostridium butyricum ligand.

[0046] (4) Preparation of phytase linked with nano-zinc oxide

[0047] Weigh 66 mg of polyvinylpyrrolidone (PVP) and dissolve it in 5 mL of methanol to prepare a PPVP methanol solution, which is then placed in a beaker. Weigh 20 mg of zinc chloride and dissolve it in 1 mL of methanol to prepare a zinc chloride methanol solution. Weigh 10 mg of phytase and dissolve it in 1 mL of methanol to prepare a phytase methanol solution. Add the zinc chloride methanol solution dropwise to the PPVP methanol solution using a 1 mL pipette tip while stirring (300 rpm) at 20°C for 20 minutes to prepare a zinc-PVP solution. Add the phytase methanol solution dropwise to the zinc-PVP solution and stir with a magnetic stirrer (300 rpm) at 20°C for 3 hours. Finally, transfer the reaction product to a 3500 Da dialysis bag and dialyze it in pure water for 18 hours to obtain the final product, phytase with zinc oxide nanoparticles. During the preparation process, the mass ratio of phytase, zinc chloride, and PPVP was 1:2:6.6.

[0048] (5) Preparation of compound biological agents

[0049] Take 200 μL (2 × 10⁻⁶) of primer-Clostridium butyricum ligand. 8CFUs were centrifuged at 25°C (8000 rpm) for 5 minutes, then the supernatant was removed and the mixture was washed once with phosphate buffer. 30 μL of the DNA loop prepared in step (2), 15 μL of 10 U / μL phi29 DNA polymerase, 9 μL of 10 mM dNTPs, 10 μL of phi29 10× DNA polymerase buffer, 2.64 μL of phytase solution with zinc oxide nanoparticles (the concentration of phytase with zinc oxide nanoparticles in the solution was 1.5 mol / L), and 138.36 μL of purified water were added to form a reaction system. The reaction system was reacted in an anaerobic environment at 30°C for 2 hours to obtain the composite biological agent (Clostridium butyricum-DNA-zinc oxide nanoparticles-phytase).

[0050] Example 2: Preparation of a compound biological agent to improve phosphorus utilization efficiency in dairy cows

[0051] The preparation process is basically the same as in Example 1, except that step (4) is as follows:

[0052] Weigh 66 mg of polyvinylpyrrolidone (PVP) and dissolve it in 5 mL of methanol to prepare a PPVP methanol solution, which is then placed in a beaker. Weigh 10 mg of zinc chloride and dissolve it in 1 mL of methanol to prepare a zinc chloride methanol solution. Weigh 10 mg of phytase and dissolve it in 1 mL of methanol to prepare a phytase methanol solution. Add the zinc chloride methanol solution dropwise to the PPVP methanol solution using a 1 mL pipette tip while stirring (300 rpm) at 20°C for 20 minutes to prepare a zinc-PVP solution. Add the phytase methanol solution dropwise to the zinc-PVP solution and stir with a magnetic stirrer (300 rpm) at 20°C for 3 hours. Finally, transfer the reaction product to a 3500 Da dialysis bag and dialyze it in pure water for 18 hours to obtain the final product, phytase with zinc oxide nanoparticles. During the preparation process, the mass ratio of phytase, zinc chloride, and PPVP was 1:1:6.6.

[0053] Example 3: Preparation of a compound biological agent to improve phosphorus utilization efficiency in dairy cows

[0054] (1) Cultivation of Clostridium butyricum from cow dung

[0055] Take 5g of cow manure and place it in an Erlenmeyer flask containing 100mL of sterile water. Shake for about 25 minutes to thoroughly mix the manure and water, prepare a diluted solution, and incubate at 85℃ for 15 minutes to kill non-spore-forming bacteria. Then, serially dilute the solution at a 10-fold dilution ratio and inoculate it at a 5% concentration into Clostridium butyricum-enriched medium. Incubate at 37℃ in an anaerobic incubator for 48 hours. Dilute the sample 10-fold and spread it onto tryptophanase-sulfite-neomycin medium, and incubate anaerobicly at 37℃ for 48 hours. Select colonies that conform to the culture characteristics and colony morphology of Clostridium butyricum for subsequent experiments.

[0056] (2) Synthesis of DNA loops

[0057] The template (SEQ ID NO:1: 5'-TGTCTTCGCCTTCTTGTTTCCTTTCCTTGAAACTTCTTCCTTTCTTTCTTTCGACTAAGCACC-3') and primer (SEQ ID NO:2: 5'-GCGAAGACAGGTGCTTAGTC-3') were mixed at a concentration ratio of 1.5:4. The mixture was then made up to 30 μL with 1×PBS (phosphate buffer). This mixture was added to a 50 μL PCR tube and annealed in a PCR instrument (95°C for 3 minutes) to form a stable template / primer dimer. The tube was then stored at 12°C. Based on this, a 24 μL cross-linking system (final concentration) was prepared with the following components: 2 μM template / primer mixture, 30 U / μL T4 DNA ligase, and 1×T4 DNA ligase buffer. The cross-linking reaction was performed in a 50 μL centrifuge tube to form a circular template / primer with a complete circular structure. The crosslinking reaction steps are as follows: overnight (12 hours) at 16°C, inactivated by heating at 75°C for 10 minutes, and the final product of the reaction is purified by centrifugation in a 10kD ultrafiltration centrifuge tube to remove buffer components in the system and avoid interfering with the subsequent reaction process.

[0058] Take 15 μL of 3 μM circularized template / primers, 6 μL of 10 mM dNTPs, 3 μL of 10 U / μL phi29 DNA polymerase, and 5 μL of 10×phi29 DNA polymerase buffer. Finally, add ultrapure water to make up to 60 μL of rolling circle amplification reaction mixture. The rolling circle amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 55℃ annealing for 45 s, 72℃ extension for 45 s (denaturation-annealing-extension, 25 cycles); 72℃ final extension for 45 s to terminate the reaction. DNA circles are obtained after the reaction and used for subsequent experiments.

[0059] (3) Preparation of primer-Clostridium butyricum linker

[0060] Take 200 μL (2 × 10⁻⁶) of the naturally cultured Clostridium butyricum from step (1). 8CFUs were centrifuged at 8000 rpm for 5 min, the supernatant was removed and the mixture was resuspended. 2 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution (EDC, 0.11 mg / mL) and 3 μL of N-hydroxysuccinimide solution (NHS, 0.13 mg / mL) were added, mixed and activated for 30 min. After centrifugation at 8000 rpm for 5 min, the supernatant was removed and the mixture was resuspended. 5 μL of 100 mM DNA primers were added, mixed well and incubated under anaerobic conditions for 3 hours (20 °C) to obtain the primer-Clostridium butyricum ligand.

[0061] (4) Preparation of compound biological agents

[0062] Take 200 μL (2 × 10⁻⁶) of primer-Clostridium butyricum ligand. 8 CFUs were centrifuged at 25°C (8000 rpm) for 5 minutes, then the supernatant was removed and the mixture was washed once with phosphate buffer. 30 μL of the DNA loop prepared in step (2), 15 μL of 10 U / μL phi29 DNA polymerase, 9 μL of 10 mM dNTPs, 10 μL of phi29 10× DNA polymerase buffer, 2.64 μL of phytase solution (the concentration of phytase in the solution is 1.5 mol / L) and 138.36 μL of purified water were added to form a reaction system. The reaction system was reacted in an anaerobic environment at 30°C for 2 hours to obtain the composite biological agent (Clostridium butyricum-DNA-phytase).

[0063] Test Example 1: Phytase Activity Assay

[0064] A 5.0 mmol / L sodium phytate solution was prepared at 37°C and adjusted to pH 5.5 with hydrochloric acid. The solution was divided into four equal portions. 1 g of the compound biological agent prepared in Example 1 (containing 700 mg of phytase), 1 g of the compound biological agent prepared in Example 2 (containing 700 mg of phytase), 1 g of the compound biological agent prepared in Example 3 (containing 700 mg of phytase), and 700 mg of phytase were added to each of the four portions. The inorganic phosphorus released per minute from the sodium phytate solution was measured using the method described in GB6730.19-86 (Bismuth Phosphomolybdenum Blue Spectrophotometric Method for Determination of Phosphorus Content). The results showed that the compound biological agent group prepared in Example 1 released 1024 μmol of inorganic phosphorus, the compound biological agent group prepared in Example 2 released 1017 μmol of inorganic phosphorus, the compound biological agent group prepared in Example 3 released 985 μmol of inorganic phosphorus, and the phytase group released 988 μmol of inorganic phosphorus. Therefore, it can be seen that the composite biological agents prepared in Examples 1-3 of this application did not reduce the activity of phytase. In the composite biological agents prepared in Examples 1 and 2, the activity of phytase was higher than that of phytase, while in the composite biological agent prepared in Example 3, the activity of phytase was comparable to that of phytase.

[0065] Test Example 2: Determining the effect of feeding compound biological agents or phytase on phosphorus conversion efficiency in dairy cow feed.

[0066] ① Select 50 lactating dairy cows with the same milk production, lactation days, and parity, and divide them into 5 groups of 10 cows each for the experiment.

[0067] ② Five groups of dairy cows were fed a basal diet supplemented with the following additives: 0 additive (control group), 2000 FTU / kg phytase (phytase group), 2000 FTU / kg of the compound biological agent prepared in Example 1 (Example 1 group), 2000 FTU / kg of the compound biological agent prepared in Example 2 (Example 2 group), and 2000 FTU / kg of the compound biological agent prepared in Example 3 (Example 3 group), for a total of 8 weeks. After feeding was stopped, the milk yield, phosphorus deposition in milk, feces, and urine of the three groups of dairy cows were observed to determine the phosphorus conversion efficiency of the feed. Simultaneously, the lactation performance, feed intake, and fecal and urinary phosphorus excretion of different groups of dairy cows were measured during the 8-week experimental period. The results are shown in Table 1.

[0068] Table 1: Effects of adding compound biological agents or phytase on phosphorus conversion efficiency in dairy cow feed

[0069]

[0070]

[0071] As shown in Table 1, compared with the control group and phytase, the addition of the compound biological agent to the diet effectively increased the phosphorus content in dairy cow milk and reduced the phosphorus content in feces and urine. The compound biological agent prepared in Example 1 showed the best effect. Therefore, the compound biological agent provided in this application improves the absorption efficiency of feed phosphorus, reducing phosphorus pollution while maintaining dairy cow production performance.

[0072] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A compound biological agent for improving phosphorus utilization efficiency in dairy cows, characterized in that, The compound biological agent includes butyric acid carboxyl bacteria, DNA, and phytase linked with nano-zinc oxide; the butyric acid carboxyl bacteria, DNA, and phytase linked with nano-zinc oxide are sequentially linked together. The DNA is a DNA loop formed by a template and primers; the nucleotide sequence of the template is shown in SEQ ID NO:1, and the nucleotide sequence of the primers is shown in SEQ ID NO:

2.

2. The compound biological agent according to claim 1, characterized in that, The butyric acid carboxyl bacteria are bovine butyric acid clostridium.

3. A method for preparing the composite biological agent as described in claim 1 or 2, characterized in that, The method includes the following steps: S1, After the template and primers are circularized, rolling circle amplification is performed to obtain DNA circles; S2, the primers are linked to Clostridium butyricum to synthesize a primer-clostridium butyricum linker; S3, a ligation reaction is carried out on the reaction system containing the DNA loop, the primer-clostridium butyricum linker, and the phytase linked with nano-zinc oxide to prepare the composite biological agent.

4. The method according to claim 3, characterized in that, Step S1 specifically includes: annealing the template, primers, and buffer to obtain a template / primer dimer; preparing a cross-linking system by mixing the template / primer dimer with T4 DNA ligase and T4 DNA ligase buffer; and obtaining a circularized template / primer by performing a cross-linking reaction on the cross-linking system; and performing rolling circle amplification on the circularized template / primer to obtain a DNA circle.

5. The method according to claim 3 or 4, characterized in that, Step S2 specifically includes: activating Clostridium butyricum with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to obtain activated Clostridium butyricum; mixing the activated Clostridium butyricum with the primers and performing a ligation reaction to obtain a primer-clostridium butyricum linker.

6. The method according to claim 3 or 4, characterized in that, The method for preparing phytase linked with zinc oxide nanoparticles Includes the following steps: T1, a zinc-polyvinylpyrrolidone solution is obtained by adding a methanol solution containing zinc chloride dropwise to a methanol solution containing polyvinylpyrrolidone and stirring the mixture. T2, a methanol solution containing phytase is added dropwise to the zinc-polyvinylpyrrolidone solution and stirred to react. After dialysis of the reaction product, phytase with nano-zinc oxide is obtained.

7. The method according to claim 6, characterized in that, The mass ratio of phytase, zinc chloride and polyvinylpyrrolidone is 1:(1.5~2.5):(6~7).

8. The method according to claim 3 or 4, characterized in that, Step S3 specifically includes: mixing the DNA loop, primer-clostridium butyricum linker, phytase linked with zinc oxide nanoparticles, DNA polymerase, dNTPs, DNA polymerase buffer, and water to form a reaction system; and carrying out the ligation reaction of the reaction system in an anaerobic environment at 25-30°C for 1.5-2.5 hours to obtain the composite biological agent.

9. A feed additive for improving phosphorus utilization efficiency in dairy cow feed, comprising the compound biological agent as described in claim 1 or 2, or the compound biological agent prepared by the method described in any one of claims 3-8.

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