A composition for reducing odor pollutant emissions during pig farming from the feed source and its application, pig feed

Abstract

This invention belongs to the field of feed and discloses an additive composition comprising myristic acid, ferulic acid, magnolol, phytosterols, and γ-aminobutyric acid (GABA). The addition amounts of each substance in the feed are as follows: myristic acid 500-1500 mg / kg; ferulic acid 500-1500 mg / kg; magnolol 300-500 mg / kg; phytosterols 100-300 mg / kg; and GABA 100-300 mg / kg. This additive uses myristic acid, ferulic acid, magnolol, phytosterols, and GABA as active ingredients, which can improve the growth performance of pigs, increase the content of total SCFA, acetic acid, propionic acid, and butyric acid in feces, improve immunity, and significantly improve the microbial diversity index. Ultimately, through multiple improvements, it achieves the goal of emission reduction. This invention also provides the application of the additive composition and pig feed.

Description

Technical Field

[0001] This invention relates to the field of feed, specifically to a composition for reducing odorous pollutant emissions during pig farming from the source of feed, its application, and pig feed. Background Technology

[0002] The applicant has conducted long-term research on emissions reduction in pigs, such as:

[0003] The patent application with publication number CN111657394A is for a biological agent composition for adsorbing toxins in pigs and regulating the reduction of intestinal odor substances, and a method for preparing the same.

[0004] Patent application with publication number CN113317230B, subject to a biological composite bedding cake device for poultry farming and a method for treating odor and reducing emissions;

[0005] The patent application with publication number CN117461725B is for a composition, feed and use for reducing odor substances in poultry.

[0006] After long-term research, we believe that the main directions for achieving emission reduction are improving gut health, increasing the content of beneficial gut bacteria, and enhancing animal immunity, among other things. This is a comprehensive research direction.

[0007] The technical problem to be solved in this case is: how to develop a new feed additive with emission reduction function. Summary of the Invention

[0008] One of the objectives of this invention is to provide a composition that reduces the emission of malodorous pollutants during pig farming from the feed source. This additive uses myristic acid, ferulic acid, magnolol, phytosterol, and γ-aminobutyric acid as active ingredients, which can improve the growth performance of pigs, increase the content of total SCFA, acetic acid, propionic acid, and butyric acid in feces, improve immunity, and significantly improve the microbial diversity index. Ultimately, through multiple improvements, the goal of emission reduction is achieved.

[0009] In addition, the present invention also provides the application of the additive composition and pig feed.

[0010] To achieve the above objectives, this invention provides a composition for reducing odorous pollutant emissions during pig farming from the feed source, comprising myristic acid, ferulic acid, magnolol, phytosterols, and γ-aminobutyric acid, wherein the amounts of each substance added to the feed are as follows:

[0011] Myristic acid 500–1500 mg / kg;

[0012] Ferulic acid 500-1500 mg / kg;

[0013] Magnolol 300–500 mg / kg;

[0014] Phytosterols 100–300 mg / kg;

[0015] γ-Aminobutyric acid 100-300 mg / kg.

[0016] The individual effects of the above substances are:

[0017] Myristic acid, a multifunctional fatty acid, exhibits important biological functions in multiple fields, including protein modification, signal transduction, and immunomodulation. Myristic acid modifies proteins through N-myristication, a modification that typically occurs at the N-terminal glycine residue of proteins. This modification facilitates protein binding to the cell membrane, thereby affecting its localization and function. While myristication of the catalytic subunit of cAMP-dependent protein kinases does not directly affect their enzymatic activity, it participates in their interactions with other proteins. Myristic acid-treated hepatocytes show increased formation and secretion of VLDL (very low-density lipoprotein) transport vesicles, indicating a regulatory role in lipid metabolism and transport. Furthermore, myristic acid participates in the activation of phospholipase D (PLD), affecting phosphatidic acid (PA) production, thereby regulating cell proliferation and differentiation. Myristic acid-modified lipid A analogs can induce the release of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) from monocytes, demonstrating potential immunomodulatory effects. Myristic acid exhibits good antibacterial activity against bacteria such as Staphylococcus aureus and Streptococcus suis. Its antibacterial mechanism primarily involves interfering with the structure and function of the bacterial cell membrane. Myristic acid interacts with phospholipids in the bacterial cell membrane, disrupting its integrity and causing leakage of cell contents, thereby inhibiting bacterial growth and reproduction. Furthermore, myristic acid can also exert its antibacterial effect by inhibiting toxic proteins secreted by bacteria. For example, studies on Streptococcus suis have shown that myristic acid can significantly inhibit the oligomerization and pore-forming activity of cytolysins, thus reducing their toxicity to host cells. This mechanism allows it to significantly alleviate cytotoxicity induced by Streptococcus suis even without antibacterial activity, providing a potential application direction for the development of novel antibacterial drugs. The antibacterial activity of myristic acid is also closely related to its chemical structure. Studies have shown that functional groups such as the carboxyl group (-COOH) of myristic acid can penetrate the cell wall of microorganisms, thereby interfering with intracellular physiological processes. Furthermore, the antibacterial activity of myristic acid exhibits a concentration-dependent effect, with higher concentrations more effectively inhibiting bacterial growth. Myristic acid derivatives have also been found to possess selective bactericidal activity, achieved by targeting specific glycerophospholipids in bacterial biofilms. Glycosphingolipids containing 2-hydroxymyristic acid can significantly stimulate phagocytosis and phagosome-lysosome fusion in polymorphonuclear leukocytes (PMNs), thereby enhancing the immune response.

[0018] Ferulic acid is a phenolic acid compound widely found in plants with various biological functions, including antioxidant and anti-inflammatory effects. Ferulic acid exhibits significant antioxidant capacity, scavenging free radicals and reducing oxidative stress-induced cell damage. Its antioxidant mechanisms include directly scavenging reactive oxygen species (ROS) and enhancing the activity of endogenous antioxidant enzymes. Furthermore, ferulic acid exerts its anti-inflammatory effect by inhibiting the release of inflammatory mediators (such as the NF-κB signaling pathway), thus reducing inflammatory responses. On the other hand, ferulic acid and its metabolites can promote intestinal mucus secretion and enhance the physical barrier function of the intestinal mucosa. In addition, ferulic acid has a significant regulatory effect on the gut microbiota. Studies have found that ferulic acid can increase the number of beneficial bacteria such as Bifidobacteria and Lactobacillus, while inhibiting the overgrowth of harmful bacteria. In a mouse model of antibiotic stress, ferulic acid improved the gut microbiota structure by upregulating the ratio of Firmicutes to Proteobacteria, promoting the proliferation of beneficial bacteria such as Clostridium, and preventing the overgrowth of opportunistic pathogens such as Enterococcus. Secondly, the protective effect of ferulic acid on intestinal barrier function has also attracted attention. In a mouse model of sepsis, ferulic acid significantly improved intestinal mucosal barrier function, reduced intestinal bacterial translocation, and decreased serum endotoxin levels. Furthermore, ferulic acid can enhance the intestinal mechanical barrier by increasing the expression of tight junction proteins (such as Occludin and ZO-1) and maintaining the integrity of intestinal epithelial cells.

[0019] Magnolol is a biphenyl compound isolated from the stem bark of the traditional Chinese medicine Magnolia officinalis, possessing physiological functions such as anti-inflammatory, antioxidant, and lipid metabolism-improving effects. In mice with ulcerative colitis, magnolol reduced the activities of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interleukin-12 (IL-12) induced by sodium dextran sulfate by downregulating nuclear factor κB (NF-κB) and upregulating the mRNA expression level of peroxisome proliferator-activated receptor (PPARγ). Furthermore, magnolol significantly increased the mRNA expression levels of ZO-1 and Occludin in mice with sodium dextran sulfate-induced ulcerative colitis. In a renal ischemia-reperfusion injury model, magnolol was found to reduce the levels of TNFα, IL-1β, interleukin-6 (IL-6), and Bax, while inhibiting the phosphorylation of pro-apoptotic mitogen-activated protein kinase (MAPK) and enhancing the mRNA expression levels of interleukin-10 (IL-10) and Bcl-2, thereby inhibiting apoptosis through both intrinsic and extrinsic pathways. Previous studies have shown that magnolol inhibits macrophage inflammation induced by *Porphyromonas gingivalis* lipopolysaccharide by activating the MAPK and Nrf-2 / HO-1 signaling pathways. Similar studies have found that magnolol can effectively eliminate free radicals in animals, enhance the activity of antioxidant enzymes, improve the growth performance and antioxidant capacity of yellow-feathered broilers, and improve meat quality and gut microbiota. Combined use of magnolol with chemotherapy drugs can improve skeletal muscle atrophy and inhibit weight loss in bladder cancer patients. This may be related to magnolol's inhibition of carnosine and activin A formation, increased activity of phosphorylated protein kinase B (p-Akt) and phosphorylated FOXO3 (p-FOXO3), enhanced mRNA expression levels of insulin-like growth factor 1 (IGF-1) and phosphorylated mammalian target of rapamycin (p-mTOR), and reduced activity of pro-inflammatory cytokines. Acrolein is a neurotoxin that can induce neurodegenerative diseases by causing neuroblast apoptosis through inducing oxidative stress and activating MEK / ERK signaling and mitochondrial caspase. Pretreatment of cells with magnolol can reduce ROS production and protect the brain from oxidative damage by inhibiting JNK / mitochondrial caspase, PI3K / MEK / ERK, and PI3K / Akt / FOXO1 signaling pathways. During stress-mediated venous remodeling, magnolol can interfere with venous proliferation, ERK1 / 2 activity, and gelatinase activity in mice without affecting endothelial capillary formation. Studies by Choi et al. have shown that honokiol can exert its protective effect by reducing phospholipid peroxidation in osteoblasts MC3T3-E1 treated with antimycin A.

[0020] In addition to their cholesterol-lowering effects, phytosterols also possess antioxidant and anti-inflammatory properties. Their antioxidant effect primarily protects cell membrane lipids from oxidative damage by scavenging free radicals and interrupting free radical chain reactions. In terms of anti-inflammation, phytosterols may lower cholesterol by reducing the oxidation of low-density lipoprotein, inhibiting the aggregation of inflammatory cells in the arterial intima, and controlling the formation of inflammatory cytokines. Phytosterols can also exert immunomodulatory effects by influencing leukocytes and complement synthesis through cell membrane embedding. The phosphorylation level of NF-κB p65 protein controlled by phytosterols regulates the binding of DNA to the cell nucleus and the nuclear translocation of NF-κB. Phytosterols also induce a natural immune response in cells and tissues by affecting the binding of ligands to nuclear receptors such as the hepatic X receptor.

[0021] Gamma-aminobutyric acid (GABA) can generate inhibitory postsynaptic potentials by binding to GABA receptors on neurons, thereby reducing neuronal excitability. This inhibitory effect helps maintain the stability and balance of the nervous system. GABA plays a crucial role in the central nervous system and has important biological functions, including improving intestinal function, enhancing immune function, increasing antioxidant capacity, and alleviating environmental stress. GABA can mediate somatostatin in the gastrointestinal tract through central neurotransmitters, increasing immunoglobulin levels, inhibiting the production of inflammatory factors, activating B lymphocytes, and inhibiting T lymphocyte cytotoxicity, playing an important role in enhancing the body's non-specific immunity. GABA significantly reduces H2O2-induced ROS formation and inhibits H2O2-induced NF-κB and cysteine ​​aspartate protease 3 (Caspase 3) signaling activation. Simultaneously, under H2O2 stress, GABA can also regulate the Keleh-like epichlorohydrin-associated protein 1 (Keap1)-nuclear factor E2-associated factor 2 (Nrf2) and Notch1 signaling pathways, maintaining redox balance in the body. GABA works by regulating corticotropin-releasing hormone (CRH) neurons in the animal HPA axis, thereby alleviating the animal's stress response.

[0022] In this invention, by combining the various components, the purpose of odor reduction can be achieved; the odor reduction effect is achieved from different angles, such as the antibacterial and antioxidant properties of each substance, the inhibition of inflammatory factors, and the regulation of intestinal flora.

[0023] In the above composition, the amount of each substance added to the feed is as follows:

[0024] Myristic acid 800–1300 mg / kg;

[0025] Ferulic acid 800-1300 mg / kg;

[0026] Magnolol 350–450 mg / kg;

[0027] Phytosterols 150–250 mg / kg;

[0028] γ-Aminobutyric acid 150-250 mg / kg.

[0029] In addition, the present invention also discloses the use of the composition described above to prepare feed for improving the growth performance of pigs or for reducing pig odor emissions.

[0030] In addition, the present invention also discloses a pig feed containing the following substances:

[0031] Myristic acid 500–1500 mg / kg;

[0032] Ferulic acid 500-1500 mg / kg;

[0033] Magnolol 300–500 mg / kg;

[0034] Phytosterols 100–300 mg / kg;

[0035] γ-Aminobutyric acid 100-300 mg / kg.

[0036] The pig feed mentioned above contains the following substances:

[0037] Myristic acid 800–1300 mg / kg;

[0038] Ferulic acid 800-1300 mg / kg;

[0039] Magnolol 350–450 mg / kg;

[0040] Phytosterols 150–250 mg / kg;

[0041] γ-Aminobutyric acid 150-250 mg / k.

[0042] Beneficial effects

[0043] 1. The composition of the present invention has a positive effect on the growth performance of piglets and can reduce the feed conversion ratio;

[0044] 2. The composition of the present invention can significantly reduce the diarrhea rate;

[0045] 3. The composition of the present invention can reduce the emission of odor components and greenhouse gases;

[0046] 4. The combination of the present invention can increase the content of short-chain fatty acids in feces, improve the immune function of piglets, and increase the abundance of specific microorganisms in feces. Attached Figure Description

[0047] Figure 1This is a comparison chart of the short-chain fatty acid content in the feces of the control group and experimental group 1.

[0048] Figure 2 This is a comparison chart of the immune function of piglets in the control group and experimental group 1.

[0049] Figure 3 This is a comparison chart of the abundance of specific microorganisms in the feces of the control group and experimental group 1. Detailed Implementation

[0050] 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.

[0051] 1. Aquaculture Experiment

[0052] This experiment employed a single-factor experimental design, selecting 36 weaned castrated boar piglets with similar initial weights for each treatment. They were divided into six treatments: a control group and experimental groups 1 through 5. The control group was fed a standard basal diet (see Table 2), while the experimental groups were fed a basal diet plus feed additives (see Table 1). All feeding and management practices were identical for both groups, with frequent small feedings and free access to feed. The experiment lasted for 15 days.

[0053] The following additive compositions were added to each group:

[0054] Table 1. Additive Composition Table

[0055]

[0056]

[0057] Table 2. Basal Diet Composition (%, Feeding Basis)

[0058] corn 55.9 Peeled soybean meal 15.0 Puffed full-fat soybeans 5.0 Soy protein concentrate 4.0 Fish meal 5.0 whey powder 8.0 sucrose 2.0 Soybean oil 1.4 Calcium hydrogen phosphate 1.2 stone powder 0.75 salt 0.2 Lysine 0.45 Methionine 0.2 threonine 0.1 Tryptophan 0.1 Valine 0.2 premix 0.5 total 100

[0059] Performance testing methods:

[0060] Growth performance

[0061] At the start of the experiment, all weaned piglets were weighed, and their initial weight was recorded before the experiment began. At the end of the experiment, they were weighed again, and feed consumption was calculated. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated for the experimental period. Environmental control data and mortality were recorded.

[0062] Short-chain fatty acids

[0063] Take 1 gram of sample and place it in a 10 mL centrifuge tube. Add 2 mL of 0.1% hydrochloric acid (v / v), place on ice for 25 min, mix well, centrifuge at 15,000 r / min for 15 min, take the supernatant, filter it using a 0.22 μm filter membrane (Millipore, Bedford, OH, USA), and inject it into a gas chromatograph (Agilent HP 6890 series, Santa Clara, CA, USA) to detect the content of volatile fatty acids in the fecal sample.

[0064] Microbial diversity

[0065] Fresh pig fecal samples were collected, and genomic DNA was extracted. Total bacterial DNA was extracted from the samples using a DNA kit (Omega Bio-tek, Norcross, GA, USA), and DNA concentration and purity were determined using NanoDrop 2000. The DNA was extracted using the universal bacterial primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R.

[0066] (5′-CCGTCAATTCMTTTRAGTTT-3′) was used to amplify the 16S rRNA gene of the V4-V5 variable region of bacteria by PCR. The PCR amplification products were recovered and purified using the Axyprep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA), eluted with Tris-HCl, and DNA fragments were detected by 2% agarose gel electrophoresis. Libraries were constructed from the purified amplified fragments and sequenced using the Illumina Miseq platform. The raw sequencing sequences were quality controlled using Trimmomatic (version 3.29) software, assembled using FLASH software, and then clustered into operational taxonomic units (OTUs) based on 97% similarity using UPARSE software; chimeras were removed using UCHIME software. Each sequence was annotated for species classification using RDP classifier and compared with the Silva database (SSU123). The comparison threshold was set to 90%. The 16S rRNA microbial gene sequences were analyzed using PICRUSt software (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States). The resulting OTUs table was used to predict the metabolic function of the microbial community by referencing the KEGG database (Kyoto Encyclopedia of Genes and Genome, KEGG).

[0067] Immune factors

[0068] Serum immunoglobulin A (IgA), IgG, IgM, IL1β, IL-6, and IL-10 were detected according to the enzyme-linked immunosorbent assay (ELISA) instructions.

[0069] Quantitative analysis of fecal microorganisms

[0070] Specific primers were used to amplify specific fragments; the primer sequences are shown in Table 3. A standard plasmid was constructed using pMD 19-TVector (Takara, Dalian, China), and the vector was introduced into competent cells. White single colonies were selected and cultured on LB agar plates containing X-Gal, IPTG, and ampicillin. The cells were then expanded in LB medium. Plasmids were extracted using Omega D6943 (Guangzhou, China), and the target fragment was amplified. Agarose gel electrophoresis was performed, and the fragments of the corresponding sizes were recovered and sequenced to verify the introduction of the target fragment. Subsequent experiments were conducted after successful sequencing. The qualified plasmids were serially diluted by multiples of 10 to construct a linear relationship between different lg (copy number) (x-axis) and quantitative cycle number (y-axis). The absolute copy number of specific microorganisms per gram of feces was calculated. The quantitative reaction system was as follows: SYBR Green Supermix 5.0 μL; fecal bacteria / plasmid DNA 2 μL; upstream primer 0.4 μL; downstream primer 0.4 μL; ddH2O 2.2 μL.

[0071] Table 3 Microbial primer sequences

[0072]

[0073] Fecal polyphenols and indole compounds

[0074] The HPLC method was used to determine the content of phenols and indoles in feces, mainly cresol, phenol, and skatole. The specific operation procedure was referred to in the literature (Jensen, MT, Cox, RP, & Jensen, BB (1995). 3-Methylindole (skatole) and indole production by mixed populations of pig fecalbacteria. Applied and environmental microbiology, 61(8), 3180–3184.).

[0075] Biogenic amine determination

[0076] The contents of methylamine, tryptamine, tyramine, putrescine, cadaverine, spermine, and spermidine in piglet feces were determined by high performance liquid chromatography (HPLC). The total biogenic amine content was then calculated. The specific operation steps were referred to in the previously published literature (Yang YX, Mu CL, Zhang JF, et al. Determination of biogenic amines in digesta by high performance liquid chromatography with precolumn dansylation[J]. Analytical Letters, 2014, 47(8): 1290-1298.).

[0077] Greenhouse gas detection

[0078] A representative gas sample of 100 mL was collected using a gas bag, and 1 mL of the gas sample was injected into a gas chromatograph (Agilent 8890, USA) to determine the contents of CH4, CO2 and N2O in the gas sample.

[0079] Test results

[0080] The results of the growth performance test are shown in Table 4;

[0081] Table 4. Results of Growth Performance Test

[0082] Initial weight, kg 9.80 9.80 9.87 9.83 9.83 9.87 Final weight at the end of the test, kg 16.26 16.80 16.47 16.73 16.67 16.50 Daily weight gain, g 431 467 440 460 456 442 Daily feed intake, g 756 763 758 774 790 756 Material weight ratio 1.76 1.64 1.72 1.68 1.74 1.71 Diarrhea rate, % 6.32 3.26 3.98 3.35 3.63 3.38

[0083] The test results for the emissions of indole, skatole, total biogenic amines, and greenhouse gases in feces are shown in Table 5.

[0084] Table 5. Emission statistics of indole, skatole, total biogenic amines, and greenhouse gases in feces.

[0085] Methane, ppm / kg feces 63.53 54.16 61.00 58.00 63.00 60.00 Carbon dioxide, ppm / kg feces 1592.47 1163.96 1476.18 1334.67 1388.88 1566.85 Nitrous oxide, ppm / kg feces 2.47 2.30 2.42 2.34 2.46 2.43 Indole, μg / g 24.00 13.70 22.97 14.30 22.00 14.10 Skatine, μg / g 45.00 28.00 42.33 31.67 38.77 29.33 Total biogenic amines, μg / g 308.00 226.00 287.00 257.00 274.00 260.00

[0086] In addition, this invention also detected the content of short-chain fatty acids in feces, microbial diversity index, immune function and inflammatory factors, and abundance of specific microorganisms in feces in the control group and experimental group 1. The detection results are as follows: Figure 1 , Figure 2 , Figure 3 And Table 6;

[0087] Table 6. Statistical Results of Microbial Biodiversity Index

[0088] Ace 348.53 343.59 Chao1 365.66 350.44 Sobs 333.02 331.76 Shannon 3.56 4.28 Simpson 0.09 0.05

[0089] Results analysis:

[0090] 1. As shown in Table 3, experimental group 1 showed the best growth effect, followed by experimental group 3, indicating that ferulic acid had a slightly smaller impact on growth performance than myristic acid. Based on the data from experimental groups 2, 3, 4, 5 and the control group, we can basically conclude that magnolol and myristic acid have a relatively important impact on improving growth performance.

[0091] 2. In terms of improving the diarrhea rate, magnolol, myristic acid, and ferulic acid are almost equally important;

[0092] 3. From the perspective of reducing greenhouse gas emissions, myristic acid plays a crucial role. In the presence of magnolol, myristic acid and ferulic acid show obvious synergy.

[0093] 4. In terms of odor emission, myristic acid performed almost identically to the experimental group with the combination of myristic acid and ferulic acid, indicating that myristic acid is also important in reducing odor emission.

[0094] 5. The results of detecting the content of short-chain fatty acids in feces, microbial diversity index, immune function and inflammatory factors, and abundance of specific microorganisms in feces of the control group and the experimental group demonstrate that the formulation of the present invention achieves the above-mentioned performance optimization based on its effects on multiple aspects such as immune function, biodiversity, anti-inflammatory and antioxidant properties, and intestinal microbial balance.

Claims

1. The application of a composition in the preparation of feed that reduces odorous pollutant emissions during pig farming from the feed source, characterized in that, The composition includes myristic acid, ferulic acid, magnolol, phytosterols, and γ-aminobutyric acid, and the amount of each substance added to the feed is as follows: Myristic acid 1000 mg / kg; Ferulic acid 1000 mg / kg; honokiol 400 mg / kg; Phytosterols 200 mg / kg; γ-aminobutyric acid 200 mg / kg.

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