Quinacrine hydrochloride, feed additive for reducing ammonia emissions from animals and use thereof

Abstract

The application belongs to the technical field of animal ammonia emission, and discloses hydrochloric quinacrine, a feed additive for reducing animal ammonia emission and a use thereof, wherein the hydrochloric quinacrine is used for animal ammonia emission, and the use can be applied to drugs, feeds and other products for reducing animal ammonia emission. It is found that the hydrochloric quinacrine can reduce the gas production, ammonia production and urea nitrogen content of animals, and can effectively reduce the content of free amino acids and amino acid metabolites, thereby effectively reducing the ammonia emission of animals.

Description

Technical Field

[0001] This invention relates to the field of animal ammonia emission technology, specifically to quinacrine hydrochloride, feed additives, and their uses for reducing animal ammonia emissions. Background Technology

[0002] Animals are prone to producing ammonia during the breeding process. The reasons are: (1) Animal feces and urine will release a large amount of ammonia; (2) During the feeding process, the food consumed by animals contains a large amount of protein, and the nitrogen in it will be metabolized into ammonia.

[0003] Ammonia emitted by animals can harm their respiratory system, eyes, immunity, and growth performance: (1) Ammonia can irritate the respiratory mucosa of animals, causing symptoms such as coughing and wheezing; long-term exposure to high concentrations of ammonia can damage the respiratory mucosa of animals, increasing the likelihood of respiratory diseases such as rhinitis, tracheitis, and bronchitis, and in severe cases, it can even cause pulmonary edema and pulmonary hemorrhage; (2) Ammonia has a strong irritant effect on the eyes of animals, causing symptoms such as tearing and redness; (3) long-term exposure to ammonia can reduce the immunity of animals, weaken their resistance to diseases, and make them more susceptible to infection by various bacteria, viruses, and parasites; (4) Ammonia can affect the appetite and feed intake of animals, leading to slower growth and weight loss; it can also affect the feed conversion rate of animals and reduce the breeding efficiency.

[0004] Therefore, how to reduce ammonia emissions from animals during animal husbandry has become an important research topic.

[0005] For example, prior art 1: Chinese patent application 201610115586.5 discloses a feed additive premix for reducing the odor of broiler manure and its application. The premix includes the following components: Bacillus licheniformis, Bacillus subtilis, Clostridium butyricum, Enterococcus faecalis, Lactobacillus reuteri, yucca extract, Lauraceae extract, fructooligosaccharides, and rice protein peptides;

[0006] The premix disclosed in the prior art can effectively reduce the emission of odorous gases such as ammonia and hydrogen sulfide from broilers, improve the growth environment of chickens, and reduce the infection rate and mortality rate of respiratory and intestinal diseases in broilers.

[0007] For example, prior art 2: Chinese patent application 202410524919.4 discloses a feed additive and its use. The feed additive includes yucca saponins, pulsatilla saponins and moringa leaf extract, wherein the contents of yucca saponins, pulsatilla saponins and moringa leaf extract in the feed are 50-300 mg / kg, 10-20 mg / kg and 100-120 mg / kg, respectively.

[0008] The feed additive disclosed in prior art 2 is a compound of yucca saponins, pulsatilla saponins and moringa leaf extract. The three can synergistically stimulate the cecal microorganisms' ability to assimilate nitrogen and synthesize proteins using amino acids, promoting the conversion of unstable ammonium nitrogen and urea nitrogen into more stable protein nitrogen, thereby reducing ammonia emissions.

[0009] Both existing technologies 1 and 2 reduce ammonia emissions through different combinations of raw materials, indicating that current research on reducing ammonia emissions from animals has progressed to the stage of achieving improved effects through existing combinations of components for reducing ammonia emissions from animals.

[0010] However, the applicant of this application believes that there are other substances that can be used to reduce ammonia emissions from animals, and the exploration of raw materials for reducing ammonia emissions from animals remains a good research direction; currently, there are few existing technologies that disclose new substances that can reduce ammonia emissions from animals, and further exploration is needed. Summary of the Invention

[0011] One of the objectives of this invention is to provide the use of quinacrine hydrochloride for reducing ammonia emissions from animals. This invention has found that the use of quinacrine hydrochloride can effectively reduce ammonia emissions from animals, and the effect is significant, thus having high application value.

[0012] Meanwhile, the present invention aims to provide the application of quinacrine hydrochloride in the manufacture of drugs for reducing ammonia emissions from animals. Based on the significant efficacy of quinacrine hydrochloride in reducing ammonia emissions from animals, the use of quinacrine hydrochloride as a raw material to manufacture drugs for reducing ammonia emissions from animals can effectively reduce ammonia emissions from animals and effectively reduce the harm of ammonia to animals.

[0013] Similarly, the purpose of this invention is to provide the use of quinacrine hydrochloride as a feed additive to reduce ammonia emissions from animals. Using quinacrine hydrochloride as a feed additive can also effectively reduce ammonia emissions from animals and effectively reduce the harm of ammonia to animals.

[0014] Furthermore, the present invention aims to provide a feed additive containing quinacrine hydrochloride, thereby significantly reducing ammonia emissions from animals.

[0015] Furthermore, the present invention aims to provide a use for a feed additive used in the preparation of feed for broiler breeders, which can effectively reduce ammonia emissions from broiler breeders, thereby reducing the harm to the respiratory system, eyes, immunity and growth performance of broiler breeders during the rearing process.

[0016] To achieve the above objectives, the present invention provides the use of quinacrine hydrochloride for reducing ammonia emissions from animals.

[0017] The study in this application found that quinacrine hydrochloride can effectively reduce ammonia emissions from animals. By comparing data on gas production, ammonia production, and urea nitrogen, it can be concluded that quinacrine hydrochloride has an excellent effect on reducing ammonia emissions from animals.

[0018] Based on this, this application has discovered a new use for quinacrine hydrochloride, which is used to reduce ammonia emissions from animals; and this use can be applied to products such as drugs and feed that reduce ammonia emissions from animals.

[0019] The present invention also provides the use of quinacrine hydrochloride in the manufacture of a drug for reducing ammonia emissions from animals.

[0020] The present invention also provides the use of quinacrine hydrochloride as a feed additive to reduce ammonia emissions from animals.

[0021] The present invention further provides a feed additive, comprising quinacrine hydrochloride.

[0022] The present invention also provides an application for preparing broiler feed using the feed additives described above.

[0023] Beneficial effects

[0024] Compared with existing technologies, this invention has found that quinacrine hydrochloride can reduce gas production, ammonia production, and urea nitrogen content in animals, and can effectively reduce the content of free amino acids and amino acid metabolites, thereby effectively reducing ammonia emissions from animals. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0026] Figure 1 This is a comparison diagram of the relative gene abundance between the control group and Example 1 group of the present invention;

[0027] Figure 2 This is a comparison diagram of the composition and distribution of cecal flora between the control group and Example 1 group of this invention;

[0028] Figure 3 This is a comparison diagram of the composition and distribution of cecal flora between the control group and Comparative Example 1 of this invention. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0030] To illustrate the technical content of the present invention in detail, the following description is provided in conjunction with the embodiments.

[0031] Example 1

[0032] The quinacrine hydrochloride powder was purchased from Shanghai Lanmu Chemical Co., Ltd. The CAS number for quinacrine hydrochloride is CAS: 69-05-6(HCl).

[0033] Comparative Example 1

[0034] Yucca saponins were purchased from Biosource Biotechnology (Shenzhen) Co., Ltd.

[0035] It should be noted that the technical solution of the present invention only requires testing with quinacrine hydrochloride, and is not limited to the quinacrine hydrochloride powder purchased in Example 1 above. Other commercially available or existingly synthesized quinacrine hydrochloride that meets the CAS number are also within the scope of the technical solution of the present invention.

[0036] Effect test

[0037] I. Biochemical Indicators of Fermentation Broth

[0038] 1. Fermentation broth preparation: Cecal contents of 30-week-old yellow-feathered broiler breeders were used as the fermentation inoculum. After slaughter, the cecal contents of the breeders were collected, mixed thoroughly, and then a buffer solution (60 mL) was added at a ratio of 1:3 (W / V). The mixture was filtered through four layers of gauze and continuously purged with CO2 at 39°C to prepare the in vitro fermentation inoculum. The buffer solution was preheated to 39°C.

[0039] Mix 474 mL of ultrapure water, 237 mL of buffer solution, 237 mL of macro-element solution, 1.22 mL of resazurin solution, and 0.12 mL of trace element solution, continuously purge with CO2 gas for 10 min, add 50 mL of reducing agent solution, and continue purging with CO2 until colorless, to obtain a total of 1000 mL of inoculum.

[0040] The fermentation source and the inoculum were mixed at a ratio of 1:2 to form the in vitro fermentation broth.

[0041] Add the fermentation substrate and 30 mL of the above in vitro fermentation broth to the fermentation tank, remove the air from the fermentation tank, seal it, and place it in a 39℃ air shaker incubator at a speed of 60 rpm for 12 h; after fermentation, place the fermentation tube on ice to terminate fermentation; separately collect the fermentation broth and store it in a -80℃ freezer for subsequent index detection.

[0042] The preparation method of the macro-element solution is as follows: 5.7g Na2HPO4, 6.2g KH2PO4, 0.6g MgSO4·7H2O, add deionized water to make up to 1000ml;

[0043] Trace element solution: CaCl2·2H2O 13.2g, MnCl2·4H2O 10.0g, CoCl2·6H2O 1.0g, FeCl2·6H2O 0.8g, add deionized water to make up to 1000ml;

[0044] Reducing agent solution: 2.0 ml 1N NaOH, 335 mg Na2S·9H2O, and deionized water to a final volume of 50 ml.

[0045] 2. Biochemical indicator testing methods:

[0046] 1) Gas production: Read the gas production according to the scale on the fermentation tube. Gas production = scale after fermentation - scale before fermentation.

[0047] 2) Ammonia: Nessler's reagent colorimetric method. Inject 1 mL of H₂SO₄ absorption solution into a colorimetric tube, dilute to 25 mL, add 1 mL of potassium sodium tartrate, shake well, then add 0.5 mL of Nessler's reagent, shake well, let stand for 10 min, and measure the absorbance at 420 nm using a spectrophotometer. Plot a standard curve and calculate the regression equation. Use the regression equation to calculate the ammonia content.

[0048] Urea nitrogen: The urea nitrogen content in the fermentation broth of each experimental group was detected by sampling the urea nitrogen test kit from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. For specific operation, please refer to the kit instructions.

[0049] pH value: The pH value of the fermentation broth in each experimental group was measured using a portable pH meter.

[0050] The control group was the fermentation broth; Example 1 was the fermentation broth with added quinacrine hydrochloride, with an addition amount of 50 mg / kg; Comparative Example 1 was the fermentation broth with added yucca saponins, with an addition amount of 500 mg / kg.

[0051] The control group, comparative example 1, and example 1 were tested according to the above test method, and the results are shown in Table 1.

[0052] Table 1. Biochemical indicators of fermentation broth in control group, Example 1, and Comparative Example 1

[0053]

[0054]

[0055] According to Table 1:

[0056] Compared to the control group, yucca saponins in Comparative Example 1 effectively reduced gas production and ammonia production, indicating that yucca saponins can effectively reduce ammonia emissions from animals. At the same time, yucca saponins can also effectively reduce urea nitrogen, indicating that yucca saponins can fully stimulate the cecal microorganisms' ability to assimilate nitrogen and synthesize proteins using amino acids, promoting the conversion of urea nitrogen into more stable protein nitrogen, thereby reducing ammonia emissions.

[0057] According to the data comparison between Example 1 and Comparative Example 1 and the control group, it can be seen that quinacrine hydrochloride can also effectively reduce ammonia emissions in animals, and its stimulating effect on cecal microorganisms is more obvious. Quinacrine hydrochloride is more effective than yucca saponins in reducing ammonia emissions in animals.

[0058] II. Detection of Free Amino Acids and Amino Acid Metabolites

[0059] Detection method: Take 0.2 ml of the control group, Example 1 group, and Comparative Example 1 group respectively, add 1.2 ml of 10% sodium flavonoid homogenate, centrifuge at 12000 rpm / min for 15 min, and filter all the supernatant into a 1.5 ml amino acid sample bottle. Then, use a fully automated amino acid analyzer (L-8900 model, Hitachi, Japan) for determination. The main indicators to be determined include 17 single amino acids, 8 non-essential amino acids, branched-chain amino acids, restriction amino acids, and amino acid metabolites.

[0060] The blank group and the fermentation broths containing Example 1 and Comparative Example 1, respectively, were tested according to the above test method. The results are shown in Table 2.

[0061] Table 2 Results of free amino acids and amino acid metabolites

[0062] Group control group Comparative Example 1 Example 1 SEM p-value Glycine <![CDATA[0.047 b ]]> <![CDATA[0.052 b ]]> <![CDATA[0.242 a ]]> 0.020 ＜0.001 Isoleucine Ile <![CDATA[0.081 b ]]> <![CDATA[0.078 b ]]> <![CDATA[0.137 a ]]> 0.007 ＜0.001 Leucine Leu <![CDATA[0.052 b ]]> <![CDATA[0.051 b ]]> <![CDATA[0.092 a ]]> 0.007 ＜0.001 Tyrosine Tyr <![CDATA[0.023 b ]]> <![CDATA[0.019 b ]]> <![CDATA[0.031 a ]]> 0.003 0.008 Phenylalanine (Phe) <![CDATA[0.040 ab ]]> <![CDATA[0.033 b ]]> <![CDATA[0.053 a ]]> 0.003 0.033 <![CDATA[Ammonia NH3]]> <![CDATA[6.652 a ]]> <![CDATA[2.816 b ]]> <![CDATA[2.811 b ]]> 0.40 ＜0.001 ethanolamine <![CDATA[0.27 a ]]> <![CDATA[0.26 ab ]]> <![CDATA[0.26 ab ]]> 0.006 0.045

[0063] According to the data in Table 2:

[0064] According to the comparison of data from the control group and Comparative Example 1, although yucca saponins can effectively reduce ammonia content, they do not affect the content of free amino acids and amino acid metabolites.

[0065] However, according to the data in Example 1, quinacrine hydrochloride can effectively reduce the content of free amino acids and amino acid metabolites in the fermentation broth.

[0066] III. Results of 16s Measurement of Cecal Contents

[0067] Test methods: Fermentation broth samples from the control group, Example 1 group, and Comparative Example 1 group were sequenced using the Illumina HiSeq2500 sequencing platform for 16S rDNA gene sequencing. The original 16S rDNA sequences were screened and assembled using QIIME V1.9.0 and FLASH software packages. High-quality sequences were compared with the Silva reference database (https: / / www.arb-silva.de / ). The UCLUST algorithm was used to cluster the samples into operational taxonomic units (OTUs) at a 97% similarity level. Species annotation was performed based on the RDP database. α-diversity (ACE index, Chao index, Shannon index, Simpson index) was analyzed using the QIIME2 platform (version 2021.08). The Bray-Curtis algorithm was used to perform hierarchical cluster analysis on the cecal flora structure of quail, comparing the composition and distribution of the two groups of cecal flora at the phylum and genus levels. The PICRUST v1.1.327 software was used to predict the function of the cecal flora and obtain the corresponding orthologous clusters. The information on orthologous groups (COG) and the functional information and relative abundance of genes in pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0068] Gene relative abundance results as follows Figure 1 As shown in the figure, R1 is the control group and R3 is Example 1;

[0069] according to Figure 1 It can be seen that, comparing the control group with Example 1: the control group contained Bacteroides,

[0070] The abundance of Clostridium sensu stricto and Bifidobacterium was significantly higher than in Example 1.

[0071] The abundance of Fusobacterium, Escherichia-Shigella, and Sutterella was significantly lower than that in Example 1; indicating that the addition of quinacrine hydrochloride can alleviate the fermentation of amino acids in the body and reduce the production of ammonia.

[0072] A comparison of the composition and distribution of cecal flora between the control group and Example 1 is as follows: Figure 2 As shown in the figure, R1 is the control group and R3 is Example 1;

[0073] Comparison between the control group and Example 1: As shown in the figure, the abundance of Clostridium sensu stricto 1 (genus), Clostriales, Clostriaceae, Bacteroidetes, class, order, family, genus, and species in the control group was significantly higher than that in Example 1; at the same time, the abundance of Clostridium, class, order, family, genus, Trichophyceae and Trichophyceales, Proteobacteria, Gammaproteobacteria, Enterobacteriaceae, and Escherichia coli-Shigella genus in Example 1 was significantly higher than that in the control group; indicating that the addition of quinacrine hydrochloride can alleviate the fermentation of amino acids in the body and reduce the production of ammonia.

[0074] Comparison of cecal microbiota composition and distribution between the control group and Comparative Example 1: Figure 3 As shown in the figure, R1 is the control group. Since the abundance of Clostridium_sensu_stricto_1 (genus), Clostridiumles, Clostridiumceae, Bacteroidetes, class, order, family, genus and species in Comparative Example 1 is lower than that in the control group, it is completely covered and will not be displayed.

[0075] Compared with the results of Comparative Example 1, the abundance of Clostridium sensu stricto 1 (genus), Clostridiumlesales, Clostridiumceae, Bacteroidetes, class, order, family, genus, and species in the control group was significantly higher than that in Comparative Example 1.

[0076] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. The use of quinacrine hydrochloride in the manufacture of drugs for reducing ammonia emissions from animals.

2. Quinacrine hydrochloride is used in the manufacture of feed additives for reducing ammonia emissions from animals.

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