Nutritional intervention method, preparation, sustained-release granules and application of egg-laying hen intestinal flora-metabolite regulating eggshell mineralization
By regulating the gut microbiota of laying hens through three-stage precise nutritional intervention, and utilizing bile acid precursors and multi-layer microcapsule technology, the problem of declining eggshell quality in the later stages of egg production was solved, significantly improving eggshell strength and extending the laying cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TONGREN POLYTECHNIC COLLEGE
- Filing Date
- 2025-10-09
- Publication Date
- 2026-05-29
Abstract
Description
Technical Field
[0001] This invention relates to the fields of animal nutrition and microbiology, specifically to nutritional intervention methods, preparations, sustained-release granules, and their applications for regulating eggshell mineralization through intestinal flora and metabolites in laying hens. In particular, it relates to methods and applications for regulating the composition of intestinal flora and its metabolites through multi-stage precise nutritional intervention, thereby promoting the eggshell mineralization process, improving eggshell quality, and extending the laying cycle of laying hens. Background Technology
[0002] With the development of intensive farming techniques, the production performance of laying hens has been significantly improved. However, this high-yield and high-efficiency farming model has also brought about a series of problems, among which the decline in eggshell quality in the later stages of egg production is particularly prominent. Statistics show that the breakage rate of eggs in the later stages of egg production (over 55 weeks of age) can be as high as 6-8%, causing not only direct economic losses but also affecting product quality and safety. Solving this problem and extending the effective production cycle of laying hens is of great significance for improving farming efficiency and reducing resource waste.
[0003] Currently, the main methods for improving eggshell quality include the following:
[0004] 1. Calcium supplementation: This method improves calcium utilization by increasing the calcium and phosphorus content in feed or changing the form of calcium sources (such as calcium carbonate, oyster shell powder, etc.). However, simply increasing the calcium content does not fundamentally solve the problem and may even cause an imbalance in the calcium-to-phosphorus ratio in the intestines, leading to mineral antagonism and decreased utilization.
[0005] 2. Micronutrient and Vitamin Regulation: Adding micronutrients such as zinc, manganese, and copper, as well as vitamin D3, enhances the activity of enzymes related to eggshell formation. While this method has some effect, single-nutrient intervention is insufficient to address the comprehensive decline in the physiological functions of laying hens.
[0006] 3. Compound premix addition method: For example, Chinese patent CN104012804B discloses "A compound premix for improving intestinal health during the laying period of laying hens and its application," which improves intestinal health by adding compound enzyme preparations, mycotoxin adsorbents, and stomachic acids, thereby indirectly improving eggshell quality. Although this technology focuses on the relationship between intestinal health and eggshell formation, it does not reveal the molecular mechanism between the two, and the continuous use of a single formula makes it difficult to adapt to the changing physiological needs of laying hens at different production stages.
[0007] Recent studies have shown that the gut microbiota is closely related to host calcium metabolism and bone health. Chen et al. (published in Metabolites in 2022) found that the gut microbiota can regulate bone metabolism and calcium absorption through the bile acid signaling pathway. Wang et al. (published in IJMS in 2023) further elucidated the role of the bile acid-TGR5 / FXR dual receptor signaling pathway in regulating microbiota composition and metabolite production. However, these studies have mainly focused on human and experimental animal models, with limited research on the gut microbiota-metabolite-shell gland tissue interaction network formed in eggshells. In particular, precise nutritional intervention strategies based on this network have not yet been established.
[0008] Therefore, there is an urgent need to develop a nutritional intervention method based on the latest gut microbiota-metabolite-shell gland mineralization interaction mechanism. By precisely regulating the composition and metabolic activity of the microbiota, the calcium transport and mineralization process of the shell gland tissue can be activated, thereby solving the problem of declining eggshell quality in the later stage of egg production, extending the effective production cycle, and improving breeding efficiency. Summary of the Invention
[0009] The purpose of this invention is to provide a method for mineralizing eggshells based on the interaction network of gut microbiota-metabolites-shell gland tissue. Through a three-stage precise intervention strategy, the composition of gut microbiota and its metabolites are regulated, and the calcium transport and mineralization process of shell gland tissue is activated, thereby improving eggshell quality and extending the laying cycle of laying hens.
[0010] To achieve the above objectives, the present invention adopts the following technical solution:
[0011] Nutritional intervention methods for regulating eggshell mineralization through gut microbiota and metabolites in laying hens include the following steps:
[0012] A. Establishment of gut microbiota homeostasis: Add bile acid precursor complex, microbiota regulation complex and gut environment regulator to the feed of 24-week-old laying hens at a ratio of 0.5% for 14 consecutive days.
[0013] B. Enhanced Metabolite Production Period: During the peak egg production period of laying hens, add metabolite precursor complex and metabolic activity regulator to the laying hen feed at a ratio of 0.8%;
[0014] C. Shell gland mineralization efficiency enhancement period: During the later stage of egg production, calcium transporter activation complex, carbonic anhydrase cofactor, and ion channel regulator are added to the egg-laying hen feed at a ratio of 1.0%.
[0015] The bile acid precursor complex, microbiota regulation complex, intestinal environment regulator, metabolite precursor complex, metabolic activity regulator, calcium transporter activation complex, carbonic anhydrase cofactor, and ion channel regulator work synergistically with Ruminococcus, Lactobacillus, and Enterococcus to activate intestinal microbiota metabolism through the TGR5 and FXR signaling pathways, promote the production of γ-aminobutyric acid, branched-chain amino acids, and 25-hydroxycholesterol, and thereby activate the expression of calbindin-D28k and carbonic anhydrase in the shell gland tissue, thus improving eggshell mineralization efficiency.
[0016] As a preferred embodiment, the bile acid precursor complex is composed of the following components in parts by weight: 30-50 parts of deoxycholic acid, 10-20 parts of chenodeoxycholic acid, and 10-20 parts of taurocholic acid.
[0017] The microbial community regulation complex is composed of the following components in parts by weight: 80-120 parts of fructooligosaccharides, 30-50 parts of inulin, and 20-30 parts of mannan oligosaccharides.
[0018] The intestinal environment regulator is composed of the following components in parts by weight: 20-30 parts citric acid, 10-20 parts malic acid, and 5-10 parts sodium butyrate.
[0019] As a further preferred embodiment, the metabolite precursor complex is composed of the following components in parts by weight: 40-60 parts L-glutamic acid, 20-30 parts leucine, 10-15 parts isoleucine, 10-15 parts valine, 5-10 parts β-sitosterol, and 5-10 parts 7-dehydrocholesterol.
[0020] The metabolic activity regulator is composed of the following components in parts by weight: 10-20 parts taurine, 20-30 parts inositol, and 1-2 parts cobalt-biotin complex.
[0021] As another preferred embodiment, the calcium transporter activation complex is composed of the following components in parts by weight: 1,25-dihydroxyvitamin D3 200-300 IU, vitamin K2 8-12 mg, and zinc-glycine chelate 60-80 mg;
[0022] The carbonic anhydrase cofactor is composed of the following components in parts by weight: 10-15 mg of copper-histidine chelate and 60-80 mg of manganese-protein acid salt.
[0023] The ion channel modulator is composed of the following components in parts by weight: 350-450 mg magnesium citrate and 500-600 mg potassium gluconate.
[0024] The present invention also provides a microbial preparation for implementing the above-described nutritional intervention method, the microbial preparation comprising the following active bacterial strains: Ruminococcus, Lactobacillus, and Enterococcus;
[0025] The rumenococci were identified as Ruminococcus CGMCC 1.23020, with a viable count of 8.0 × 10⁻⁶. 10 CFU / g;
[0026] The lactobacillus mentioned is Lactobacillus CGMCC 1.557, with a viable count of 5.0 × 10⁻⁶. 10 CFU / g;
[0027] The enterococcus was Enterococcus CGMCC 1.2136, with a viable count of 3.0 × 10⁻⁶. 10 CFU / g.
[0028] As a preferred embodiment, the method for preparing the microbial preparation includes the following steps:
[0029] a) Primary seed culture: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557 and Enterococcus CGMCC 1.2136 were inoculated into the corresponding culture media and cultured anaerobically at 37°C for 24 hours.
[0030] b) Secondary fermentation: Inoculate the strain obtained in step a) into the basic culture medium, control the pH to 6.2-6.5, maintain 35°C for the first 24 hours, reduce to 32°C for the next 12 hours, maintain 32°C for the last 12 hours, ferment for 36-48 hours, and control dissolved oxygen to below 10%.
[0031] c) Cell collection and protection: Centrifuge the fermentation broth obtained in step b) at 8000 r / min for 15 minutes at 4℃, collect the cells, and add 10% skim milk powder, 5% maltodextrin and 2% sodium alginate as protectants.
[0032] d) Freeze-drying treatment: The mixture obtained in step c) is first pre-frozen at -45℃ for 4 hours, then dried at -30℃ and vacuum degree less than 20Pa for 24 hours, and finally dried at 20℃ and vacuum degree less than 10Pa for 4 hours to obtain the microbial preparation.
[0033] Furthermore, this invention also provides a bile acid precursor microcapsule formulation for implementing the above-mentioned nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens. The formulation is prepared by the following steps:
[0034] a) Raw material pretreatment: Deoxycholic acid and chenodeoxycholic acid were mixed at a ratio of 7:3 and ground to 100 mesh. At the same time, 10% gum arabic and 15% maltodextrin aqueous solution were prepared as wall material.
[0035] b) Multilayer microencapsulation: Bile acids and soybean oil are mixed in a 1:1 ratio and homogenized by high-speed shearing. Then, a mixture of gum arabic and maltodextrin is added to form an intermediate layer, and finally, a 3% ethyl cellulose solution is used to form an outer layer.
[0036] c) Targeted release treatment: Spray coating with 3% methacrylic acid copolymer solution in a fluidized bed, with the inlet air temperature controlled at 45±2℃ and the material temperature controlled at 30±2℃;
[0037] d) Quality control: Ensure that the final product has a particle size of 3-5 micrometers, an encapsulation rate of not less than 85%, and a release rate of not less than 85% after 2 hours at pH 6.5 and not more than 15% after 2 hours at pH 2.0.
[0038] This invention also provides a metabolite precursor sustained-release granule for implementing the above-mentioned nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens. The sustained-release granule is prepared by the following steps:
[0039] a) Raw material processing: The amino acid salt was physically pulverized to 200 mesh, sieved, and a 3% sodium carboxymethyl cellulose aqueous solution was prepared as a binder;
[0040] b) Granulation and coating: The mixture is granulated in a fluidized bed, the material temperature is controlled at 45±2℃, the atomization pressure is 1.5 bar, the spraying speed is 5 mL / min, and then coated with 5% hydroxypropyl methylcellulose solution, 10% ethyl cellulose-ethanol solution and 10% triethyl citrate by weight of polymer.
[0041] c) Construction of a multilayer sustained-release system: The metabolite precursor complex is used as the core, hydroxypropyl methylcellulose and sodium alginate are mixed in a 7:3 ratio to form the middle layer, and finally ethyl cellulose-succinate copolymer is used to form the outer layer.
[0042] d) Quality control: Ensure that the moisture content of the final product does not exceed 5%, the coefficient of variation of particle size uniformity does not exceed 5%, the compressive strength is not less than 20 Newtons, and the release rate is 25-35% in the first 2 hours and the cumulative release rate is not less than 85% in 12 hours.
[0043] This invention also provides a calcium transporter activation complex microemulsion formulation for implementing the above-mentioned nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens. The formulation is prepared by the following steps:
[0044] a) Microemulsion of core components: Vitamin D3 and K2 are dissolved in medium-chain triglycerides and homogenized under high pressure to form microemulsions with a particle size of less than 200 nanometers;
[0045] b) Carrier composite: Silica and calcium carbonate are mixed in a 3:7 ratio as the main carrier, and 5% hydroxypropyl-β-cyclodextrin is added as an inclusion agent;
[0046] c) Multilayer sustained-release system: Using layer-by-layer self-assembly technology, chitosan with a molecular weight of 50-100kDa and a degree of deacetylation of not less than 90% is alternately deposited with alginate with an M / G ratio of 1.5 and a viscosity of 200-400mPa·s;
[0047] d) Quality control: Ensure that the vitamin D3 content in the final product is 98-102% and the vitamin K2 content is 95-105%, and that the effective content is not less than 90% after being stored at 40℃ and 75% relative humidity for 3 months. The in vitro release rate conforms to zero-order release kinetics, and the release rate is 80% after 6 hours.
[0048] The beneficial effects of this invention are:
[0049] 1. This invention establishes for the first time a complete regulatory network of bile acids-FXR / TGR5-functional flora-metabolites-shell gland mineralization, revealing the molecular mechanism by which bile acids regulate functional flora through a dual receptor pathway, thereby promoting the production of key metabolites and activating shell gland mineralization.
[0050] 2. This invention develops a "three-stage precision intervention" strategy to provide targeted nutritional support for the physiological needs of laying hens at different production stages, thereby achieving stable eggshell quality throughout the entire production cycle.
[0051] 3. This invention screened and verified the synergistic effect of three functional bacteria strains: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557, and Enterococcus CGMCC 1.2136, to achieve precise regulation of the intestinal flora-metabolism network;
[0052] 4. This invention utilizes multilayer microcapsule and sustained-release particle technology to ensure efficient release of active ingredients at specific sites in the intestine, significantly improving bioavailability and intervention efficacy;
[0053] 5. This invention effectively solves the problem of declining eggshell quality in the later stages of egg production, significantly improves eggshell strength by 4.8%, reduces the breakage rate by 4.2%, and extends the egg production cycle by 3.1 weeks, resulting in significant economic benefits. Detailed Implementation
[0054] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0055] Based on recent research findings, this invention establishes a complete regulatory network of "bile acids-FXR / TGR5-functional flora-metabolites-shell gland mineralization". Bile acids, as important signaling molecules, can regulate the composition of the gut microbiota through the G protein-coupled receptor TGR5 and the nuclear receptor FXR. When bile acids activate TGR5, they promote the activation of the cAMP / PKA signaling pathway; simultaneously, they activate the FGF15 / MAPK cascade signaling through FXR. These two signaling pathways jointly regulate the growth and metabolic activities of key flora such as Ruminococcus, Lactobacillus, and Enterococcus. These functional flora further produce metabolites such as γ-aminobutyric acid (GABA), branched-chain amino acids (BCAAs), and 25-hydroxycholesterol. These metabolites reach the shell gland tissue through blood circulation, activating the expression of calcium-binding protein (calbindin-D28k), carbonic anhydrase, and calcium ion channels (such as TRPV6), thereby increasing calcium ion transport and carbonate ion generation, ultimately promoting the eggshell mineralization process.
[0056] Based on the above mechanism, this invention develops a three-stage precision intervention strategy:
[0057] 1. Establishment of gut microbiota homeostasis: By adding bile acid precursor complex, the TGR5 and FXR signaling pathways are activated, and at the same time, microbiota regulation complex and gut environment regulator are provided to provide a suitable growth environment for functional microbiota and establish gut microbiota homeostasis that is conducive to calcium absorption.
[0058] 2. Enhanced metabolite production phase: Provides metabolite precursor complexes and metabolic activity regulators to promote the production of key metabolites such as γ-aminobutyric acid, branched-chain amino acids, and 25-hydroxycholesterol by functional microbiota;
[0059] 3. Shell gland mineralization efficiency enhancement period: By adding calcium transporter activation complex, carbonic anhydrase cofactor and ion channel regulator, it directly acts on the shell gland tissue, synergistically enhancing the effect of metabolites, and maximizing the improvement of eggshell mineralization efficiency.
[0060] To verify the effectiveness of the present invention, a series of embodiments and comparative examples will be used to provide a detailed description below.
[0061] Example 1: Screening and preparation of functional bacterial communities
[0062] This embodiment aims to screen and prepare microbial preparations that promote calcium absorption and eggshell mineralization.
[0063] 1.1 Screening of functional microbiota
[0064] Strains that promote calcium absorption and eggshell mineralization were screened through in vitro fermentation experiments and animal experiments. Three functional strains were initially identified: *Ruminococcus rumenans* CGMCC 1.23020, *Lactobacillus rumenans* CGMCC 1.557, and *Enterococcus rumenans* CGMCC 1.2136. These three strains significantly increased the production of γ-aminobutyric acid (GABA), branched-chain amino acids, and 25-hydroxycholesterol under bile acid induction.
[0065] 1.2 Strain Cultivation and Preparation
[0066] 1.2.1 Culture medium formulation
[0067] (1) Lactobacillus culture medium: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, Tween-80 1mL / L, pH 6.2-6.5.
[0068] (2) Culture medium for Ruminococcus and Enterococcus: 15 g / L peptone, 5 g / L yeast extract, 10 g / L glucose, 0.5 g / L L-cysteine, 0.01 g / L vitamin K1, pH 6.8-7.0.
[0069] (3) Fermentation medium: peptone 10g / L, yeast extract 5g / L, glucose 15g / L, sodium acetate 5g / L, magnesium sulfate 0.8g / L, manganese sulfate 0.4g / L, dipotassium hydrogen phosphate 2g / L, L-cysteine 0.5g / L, vitamin K1 0.01g / L, Tween-80 1mL / L, pH 6.2-6.5.
[0070] 1.2.2 Microbial strain preparation process
[0071] (1) Primary seed culture: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557 and Enterococcus CGMCC 1.2136 were inoculated into the corresponding culture media and cultured anaerobically at 37°C for 24 hours.
[0072] (2) Secondary fermentation: The primary seed is inoculated into the fermentation medium at a rate of 5%. The pH is maintained at 6.2-6.5 using an automatic control system. The temperature is maintained at 35°C for the first 24 hours, then reduced to 32°C for the next 12 hours, and then maintained at 32°C for the last 12 hours. Fermentation lasts for 48 hours, and dissolved oxygen is controlled below 5%.
[0073] (3) Collection and protection of bacterial cells: Centrifuge the fermentation broth (8000 r / min, 15 minutes, 4℃), collect the bacterial cells, and add 10% skim milk powder, 5% maltodextrin and 2% sodium alginate as protectants.
[0074] (4) Freeze-drying treatment: The mixture is first pre-frozen at -45℃ for 4 hours, then dried at -30℃ and vacuum degree less than 20Pa for 24 hours, and finally dried at 20℃ and vacuum degree less than 10Pa for 4 hours to obtain the microbial preparation.
[0075] (5) Quality control: The viable count of rumen cocci CGMCC 1.23020 was determined to be 8.5 × 10⁻⁶. 10 The viable count of Lactobacillus CGMCC 1.557 was 5.2 × 10^10 CFU / g, and the viable count of Enterococcus CGMCC 1.2136 was 3.5 × 10^10 CFU / g. 10 CFU / g.
[0076] Example 2: Preparation of bile acid precursor microcapsule formulation
[0077] This embodiment aims to develop a microcapsule formulation of bile acid precursors with targeted release properties, ensuring effective release of bile acid precursors in the small intestine and maximizing activation of the TGR5 and FXR signaling pathways.
[0078] 2.1 Raw material pretreatment
[0079] Deoxycholic acid (purity ≥98%) and chenodeoxycholic acid (purity ≥95%) were mixed in a 7:3 ratio and ground into a uniform powder of 100 mesh. Simultaneously, an aqueous solution of 10% gum arabic (food grade, Shanghai Maclean Biochemical Technology Co., Ltd.) and 15% maltodextrin (DE value 15-20) was prepared as a wall material.
[0080] 2.2 Multilayer microencapsulation
[0081] (1) Inner core preparation: Bile acid mixture and soybean oil (food grade) are mixed at a weight ratio of 1:1 and sheared at 10,000 rpm for 5 minutes in a high-speed shear homogenizer (IKA T25) to form a uniform oil phase.
[0082] (2) Intermediate layer encapsulation: The above oil phase is slowly added to the gum arabic-maltodextrin mixed solution (1:1) and homogenized for 3 cycles at a pressure of 25 MPa in a homogenizer (GEA Niro Soavi NS1001L) to form a primary emulsion.
[0083] (3) Outer coating: The primary emulsion is slowly dripped into a 3% ethyl cellulose (viscosity 45-55 mPa) solution (solvent is ethanol / acetone = 7:3) under stirring conditions, and stirred for 30 minutes to form multilayer microcapsules.
[0084] 2.3 Targeted Release Processing
[0085] Fluidized bed spray coating technology was employed, using a 3% methacrylic acid copolymer (Eudragit L100) solution for coating. The fluidized bed equipment was a Cyrus Mini-Glatt, with the following operating parameters: inlet air temperature 45±2℃, material temperature 30±2℃, atomization pressure 1.8 bar, spray rate 4 mL / min, and coating time approximately 40 minutes.
[0086] 2.4 Quality Control
[0087] (1) Particle size determination: The average particle size was determined to be 4.2 micrometers by a laser particle size analyzer (Mastersizer 3000), and the particle size distribution variation coefficient was 8.5%.
[0088] (2) Encapsulation efficiency determination: The bile acid content was determined by high performance liquid chromatography, and the encapsulation efficiency was 87.3%.
[0089] (3) In vitro release characteristics: The release rate was 12.8% in simulated gastric fluid (pH 2.0) and 88.6% in simulated intestinal fluid (pH 6.5) after 2 hours, which meets the requirements for targeted release.
[0090] Example 3: Preparation of sustained-release particles of metabolite precursors
[0091] This embodiment aims to develop sustained-release particles of metabolite precursors to ensure the slow release of metabolite precursors in the intestine, continuously providing the precursor substances required for metabolite synthesis.
[0092] 3.1 Raw material processing
[0093] L-glutamic acid (food grade), leucine, isoleucine, and valine (all food grade, Shanghai Maclean Biochemical Technology Co., Ltd.), β-sitosterol (purity ≥95%), and 7-dehydrocholesterol (purity ≥90%) were mixed in a weight ratio of 50:25:12:13:7:8, pulverized to 200 mesh using a CSM-V air jet mill, and then passed through a 100-mesh sieve. A 3% sodium carboxymethyl cellulose (medium viscosity, Shanghai Maclean Biochemical Technology Co., Ltd.) aqueous solution was prepared as a binder.
[0094] 3.2 Granulation and Coating
[0095] (1) Fluidized bed mixing and granulation: A fluidized bed equipment (GPCG-1) was used. The pulverized mixture was added to the equipment, and granulation was carried out using a 3% sodium carboxymethyl cellulose solution as a binder. The operating parameters were: material temperature 45±2℃, atomization pressure 1.5 bar, spray rate 5 mL / min, and granulation time approximately 45 minutes.
[0096] (2) Composite Coating: The granulated particles are further coated in a fluidized bed. The coating solution is a mixture of 5% hydroxypropyl methylcellulose (HPMC E15, Shanghai Maclean Biochemical Technology Co., Ltd.) aqueous solution and 10% ethylcellulose (viscosity 45-55 mPa·s) ethanol solution in a 6:4 ratio, with triethyl citrate (10% of the polymer weight) added as a plasticizer. The operating parameters are the same as for granulation, and the coating time is approximately 60 minutes.
[0097] 3.3 Construction of Multilayer Sustained-Release System
[0098] (1) Intermediate layer formation: The coated particles were mixed with a pre-mixed HPMC-sodium alginate complex (7:3) in a high-efficiency mixer (Diosna P1 / 6). Sodium alginate (medium viscosity, Shanghai Maclean Biochemical Technology Co., Ltd.) was used. The mixing parameters were: 200 rpm and 15 minutes.
[0099] (2) Outer layer formation: The intermediate layer particles were finally coated in a fluidized bed with an ethyl cellulose-succinate copolymer (prepared by reacting ethyl cellulose with succinic anhydride, self-made) solution (5%, solvent: acetone / ethanol = 3:7). The operating parameters were the same as described above, and the coating time was approximately 40 minutes.
[0100] 3.4 Quality Control
[0101] (1) Moisture content determination: The moisture content was determined to be 4.3% by the Karl Fischer method, which meets the requirements (≤5%).
[0102] (2) Particle size uniformity: The coefficient of variation was 4.2% as determined by a laser particle size analyzer, which meets the requirements (≤5%).
[0103] (3) Compressive strength: The average compressive strength was 24.5 Newtons, which meets the requirements (≥20 Newtons) as determined by the particle compressive strength tester (YPJ-200A).
[0104] (4) In vitro release characteristics: The release rate was 32.6% in the first 2 hours and 88.7% in the cumulative release rate after 12 hours, which met the requirements for sustained release.
[0105] Example 4: Preparation of a microemulsion formulation of a calcium transporter activation complex
[0106] This embodiment aims to develop a microemulsion formulation of a calcium transporter activation complex to improve the bioavailability of vitamins D3 and K2 and maximize the activation of calcium transporter expression in shell gland tissue.
[0107] 4.1 Microemulsion of core components
[0108] Vitamin D3 (400,000 IU / g) and vitamin K2 (MK-7, purity ≥98%) were dissolved in medium-chain triglycerides (caprylic / capric triglycerides) at a weight ratio of 1:3. The mixture was homogenized for 3 cycles at 40 MPa in a high-pressure homogenizer (AH100D) to form a microemulsion with an average particle size of 180 nanometers.
[0109] 4.2 Carrier Composite System
[0110] A 3:7 mixture of silica (fumed silica, A200) and calcium carbonate (precipitated calcium carbonate, purity ≥98%) was used as the main carrier, with 5% hydroxypropyl-β-cyclodextrin (degree of substitution 0.6-0.9) added as an inclusion agent. The microemulsion was slowly added dropwise to the carrier mixture and mixed in a high-efficiency mixer for 30 minutes to form a uniform powder.
[0111] 4.3 Construction of a multi-layered sustained-release system
[0112] A layer-by-layer self-assembly technique was employed to alternately deposit chitosan (molecular weight 70 kDa, degree of deacetylation 95%, Shanghai Maclean Biochemical Technology Co., Ltd.) and alginate (M / G ratio 1.5, viscosity 300 mPa·s) onto the surface of a carrier powder. The specific operation was as follows:
[0113] (1) Disperse the carrier powder in a 0.5% chitosan solution (the solvent is a 1% aqueous acetic acid solution), stir for 30 minutes, collect by centrifugation, and wash once with water.
[0114] (2) Disperse the above materials in a 0.5% sodium alginate solution, stir for 30 minutes, collect by centrifugation, and wash once with water.
[0115] (3) Repeat steps (1) and (2) twice to finally obtain a multilayer coated complex.
[0116] (4) The composite was vacuum dried at 40°C for 24 hours to obtain the final product.
[0117] 4.4 Quality Control
[0118] (1) Content of active ingredients: The content of vitamin D3 was 99.5% and the content of vitamin K2 was 98.2% as determined by high performance liquid chromatography, which met the requirements.
[0119] (2) Stability test: After being stored at 40℃ and 75% relative humidity for 3 months, the vitamin D3 content was 92.3% and the vitamin K2 content was 93.5%, which met the stability requirements (≥90%).
[0120] (3) In vitro release rate: The release rate was determined using a pharmacopoeia dissolution tester. After 6 hours, the release rate was 81.5%, and the release curve conformed to zero-order release kinetics (R0). 2=0.985).
[0121] Example 5: Formulation Development of a Three-Stage Nutritional Intervention Agent
[0122] This embodiment aims to develop specific formulations for a three-stage nutritional intervention preparation to ensure that the nutritional intervention goals at each stage are achieved.
[0123] 5.1 Preparations for establishing gut microbiota homeostasis (Phase 1)
[0124] The first-stage formulation was prepared according to the following formula:
[0125] (1) Bile acid precursor complex (prepared in Example 2): 40 parts deoxycholic acid, 15 parts chenodeoxycholic acid, and 15 parts taurocholic acid;
[0126] (2) Microbial community regulation complex: 100 parts of fructooligosaccharide (purity ≥95%), 40 parts of inulin (purity ≥90%), and 25 parts of mannan oligosaccharide (purity ≥85%);
[0127] (3) Intestinal environment regulators: 25 parts of citric acid (food grade, Shanghai Maclean Biochemical Technology Co., Ltd.), 15 parts of malic acid (food grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), and 7 parts of sodium butyrate (purity ≥98%).
[0128] (4) Functional microbial preparation (prepared in Example 1): 50 portions;
[0129] (5) Carrier (maltodextrin, DE value 15-20): Make up to 1000 parts.
[0130] The above components are mixed in a V-type mixer (VH-14) for 30 minutes to form a uniform powder, which is then packaged and sealed.
[0131] 5.2 Formulations for Enhanced Metabolite Production (Phase Two)
[0132] The Phase II formulation was prepared according to the following formula:
[0133] (1) Metabolite precursor complex (prepared in Example 3): 50 parts L-glutamic acid, 25 parts leucine, 12 parts isoleucine, 13 parts valine, 7 parts β-sitosterol, and 8 parts 7-dehydrocholesterol;
[0134] (2) Metabolic activity regulators: 15 parts of taurine (food grade, Shanghai Maclean Biochemical Technology Co., Ltd.), 25 parts of inositol (food grade), and 1.5 parts of cobalt-biotin complex (self-made, cobalt to biotin molar ratio 1:2);
[0135] (3) Functional microbial preparation (prepared in Example 1): 40 portions;
[0136] (4) Carrier (maltodextrin, DE value 15-20): Make up to 1000 parts.
[0137] The above components are mixed in a V-type mixer for 30 minutes to form a uniform powder, which is then packaged and sealed.
[0138] 5.3 Formulation for improving shell gland mineralization efficiency (Phase III)
[0139] Prepare the three-stage formulation according to the following formula:
[0140] (1) Calcium transporter activation complex (prepared in Example 4): 1,25-dihydroxyvitamin D3 250 IU, vitamin K2 10 mg, zinc-glycine chelate (zinc content 15%, Shanghai Mairui Chemical Technology Co., Ltd.) 70 mg;
[0141] (2) Carbonic anhydrase cofactors: copper-histidine chelate (copper content 12%, self-made) 12mg, manganese-protein acid salt (manganese content 8%, self-made) 70mg;
[0142] (3) Ion channel modulators: magnesium citrate (magnesium content 10%, Shanghai Maclean Biochemical Technology Co., Ltd.) 400mg, potassium gluconate (potassium content 16%, Shanghai Aladdin Biochemical Technology Co., Ltd.) 550mg;
[0143] (4) Functional microbial preparation (prepared in Example 1): 30 portions;
[0144] (5) Carrier (maltodextrin, DE value 15-20): Make up to 1000 parts.
[0145] The above components are mixed in a V-type mixer for 30 minutes to form a uniform powder, which is then packaged and sealed.
[0146] Example 6: Evaluation of the Application Effect of the Three-Stage Nutritional Intervention Method
[0147] This embodiment aims to evaluate the impact of a three-stage nutritional intervention approach on eggshell quality and production performance.
[0148] 6.1 Experimental Design
[0149] (1) Experimental subjects: Jinghong No. 1 laying hens, 32 weeks old, a total of 900 birds, randomly divided into 3 groups, with 10 replicates in each group and 30 birds in each replicate.
[0150] (2) Experimental grouping:
[0151] Control group: basal diet, without any added nutritional interventions;
[0152] Standard calcium supplementation group: basal diet + 3.8% calcium carbonate;
[0153] Three-stage intervention group: The three-stage nutritional intervention was carried out according to the method of the present invention.
[0154] (3) Basic feed composition: corn 60%, soybean meal 28%, limestone powder 8%, dicalcium phosphate 1.5%, salt 0.3%, laying hen premix 2.2%.
[0155] (4) Feeding conditions: temperature 21±2℃, relative humidity 65±5%, light 16h / d.
[0156] (5) Experimental period: 20 weeks (including the late egg production period and the recovery period after forced molting).
[0157] 6.2 Implementation of the Three-Stage Intervention Approach
[0158] (1) Establishment period of gut microbiota homeostasis (weeks 1-2): Add the Phase 1 formulation prepared in Example 5.1 to the feed of 24-week-old laying hens at a ratio of 0.5% (by weight) for 14 consecutive days;
[0159] (2) Enhanced metabolite production period (weeks 3-12): Add the Phase 2 formulation prepared in Example 5.2 to the basal diet at a ratio of 0.8% (by weight).
[0160] (3) Period of improved shell gland mineralization efficiency (weeks 13-20): Add the Phase 3 formulation prepared in Example 5.3 to the basal diet at a ratio of 1.0% (by weight).
[0161] 6.3 Measurement Indicators and Methods
[0162] (1) Eggshell quality parameters:
[0163] Eggshell strength: 50 whole eggs were randomly selected from each group each week and measured using an Instron tester, unit: kg / cm²;
[0164] Eggshell thickness: Measured using a digital micrometer, with 4 measurements taken for each egg and the average value taken, unit: mm;
[0165] Broken egg rate: The percentage of broken eggs out of the total number of eggs laid each day;
[0166] Eggshell microstructure: Crystal growth and arrangement were assessed using scanning electron microscopy.
[0167] (2) Microbiome and metabolome analysis:
[0168] On days 0, 7, 14, 60 and 120, 10 laying hens were randomly selected from each group to collect fresh fecal samples.
[0169] 16S rRNA sequencing analysis of changes in gut microbiota composition;
[0170] Targeted metabolomics analysis of GABA, BCAAs, and 25-hydroxycholesterol levels;
[0171] Bile acid spectroscopy analysis: LC-MS / MS method;
[0172] Gene expression: RT-PCR was used to detect the expression levels of TGR5 and FXR in intestinal tissue.
[0173] (3) Analysis of the shell gland tissue:
[0174] At the end of the experiment, six laying hens were randomly selected from each group, and shell gland tissue samples were collected.
[0175] Expression of calcium-binding protein (calbindin-D28k): Western blot analysis;
[0176] Carbonic anhydrase activity: determined by colorimetric method;
[0177] Calcium ion transporter expression: Immunohistochemical and RT-PCR analysis;
[0178] Morphological assessment of the shell gland tissue: observation with H&E staining.
[0179] (4) Production performance indicators:
[0180] Egg production rate: Record the number of eggs produced daily and calculate the egg production rate;
[0181] Feed consumption: Record feed consumption weekly;
[0182] Feed conversion ratio: Calculates the amount of feed required to produce one unit of eggs;
[0183] Egg production cycle: Record the duration of continuous egg production by laying hens.
[0184] 6.4 Test Results
[0185] 6.4.1 Eggshell quality parameters
[0186] Table 1. Effects of different treatments on eggshell quality
[0187] index control group Regular calcium supplementation group Three-stage intervention group Eggshell strength (kg / cm²) <![CDATA[3.85±0.19 * ]]> <![CDATA[3.92±0.21 ** ]]> <![CDATA[4.21±0.23 *** ]]> Eggshell thickness (mm) <![CDATA[0.341±0.017 * ]]> <![CDATA[0.352±0.014 ** ]]> <![CDATA[0.372±0.015 *** ]]> Broken egg rate (%) <![CDATA[6.5±0.8 *** ]]> <![CDATA[5.1±0.6 ** ]]> <![CDATA[2.3±0.4 * ]]>
[0188] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0189] As shown in Table 1, the eggshell strength and thickness of the three-stage intervention group were significantly higher than those of the control group and the conventional calcium supplementation group (P<0.05), while the egg breakage rate was significantly lower than that of the control group and the conventional calcium supplementation group (P<0.05). This indicates that the three-stage nutritional intervention method of the present invention can effectively improve eggshell quality and significantly reduce the egg breakage rate.
[0190] 6.4.2 Microbiome and Metabolome Analysis
[0191] Table 2. Effects of different treatments on gut microbiota composition and metabolite content
[0192] index control group Regular calcium supplementation group Three-stage intervention group Relative abundance of R. gauvreauii (%) 0.82±0.12* 0.91±0.15** 2.35±0.28*** relative abundance of L. plantarum (%) 1.25±0.22* 1.42±0.25** 3.04±0.36*** E. faecium relative abundance (%) 0.63±0.09* 0.71±0.11** 1.67±0.20*** GABA content (μg / g) 3.42±0.45* 3.68±0.52** 6.02±0.75*** BCAAs content (μg / g) 52.6±6.8* 58.3±7.5** 85.2±9.3*** 25-Hydroxycholesterol (ng / g) 18.3±2.4* 20.5±2.8** 28.9±3.5*** relative expression level of TGR5 in the gut 1.00±0.15* 1.23±0.19** 2.47±0.32*** Relative expression level of intestinal FXR 1.00±0.14* 1.18±0.17** 2.35±0.29***
[0193] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0194] As shown in Table 2, the relative abundance of functional flora in the three-stage intervention group was significantly higher than that in the control group and the conventional calcium supplementation group (P<0.05), and the content of key metabolites was also significantly higher in the three-stage intervention group than in the control group and the conventional calcium supplementation group (P<0.05). Simultaneously, the expression levels of intestinal TGR5 and FXR in the three-stage intervention group were significantly higher than those in the control group and the conventional calcium supplementation group (P<0.05). This indicates that the three-stage nutritional intervention method of the present invention can effectively activate the bile acid-TGR5 / FXR signaling pathway, increase the abundance of functional flora, and promote the production of key metabolites.
[0195] 6.4.3 Analysis of shell gland tissue
[0196] Table 3 Effects of different treatments on shell gland tissue parameters
[0197] index control group Regular calcium supplementation group Three-stage intervention group Calbindin-D28k relative expression level 1.00±0.16* 1.32±0.21** 2.70±0.35*** Carbonic anhydrase activity (U / mg protein) 0.58±0.09* 0.74±0.12** 1.80±0.25*** TRPV6 relative expression level 1.00±0.14* 1.35±0.20** 2.40±0.31*** Density of shell glands (number per mm²) 42.5±5.8* 48.3±6.5** 58.7±7.6***
[0198] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0199] As shown in Table 3, the expression levels of Calbindin-D28k, carbonic anhydrase activity, TRPV6 expression, and shell gland density in the shell gland tissue of the three-stage intervention group were significantly higher than those in the control group and the conventional calcium supplementation group (P<0.05). This indicates that the three-stage nutritional intervention method of the present invention can effectively activate the calcium transport and carbonate ion generation-related factors in the shell gland tissue, thereby improving the eggshell mineralization efficiency.
[0200] 6.4.4 Production Performance Indicators
[0201] Table 4. Effects of different treatments on laying hen production performance
[0202] index control group Regular calcium supplementation group Three-stage intervention group Late-stage egg production rate (%) 67.3±2.8* 71.2±3.1** 78.6±3.5*** Egg ratio 2.18±0.06*** 2.12±0.05** 2.03±0.04* The egg-laying cycle has been prolonged (in weeks). 0* 1.2±0.3** 3.1±0.4*** Increased economic benefits (yuan / unit) 0* 4.5±0.7** 12.3±1.5***
[0203] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0204] As shown in Table 4, the late-stage egg production rate of the three-stage intervention group was significantly higher than that of the control group and the conventional calcium supplementation group (P<0.05), the feed conversion ratio was significantly lower than that of the control group and the conventional calcium supplementation group (P<0.05), the laying cycle was significantly longer than that of the control group and the conventional calcium supplementation group (P<0.05), and the economic benefits were significantly increased than those of the control group and the conventional calcium supplementation group (P<0.05). This indicates that the three-stage nutritional intervention method of the present invention can not only improve eggshell quality, but also improve the laying performance of laying hens, prolong the laying cycle, and bring significant economic benefits.
[0205] Comparative Example 1: Single-Stage Nutritional Intervention
[0206] This comparative study aims to assess the difference in effectiveness between a single-stage nutritional intervention and a three-stage nutritional intervention.
[0207] The experimental subjects, basal diet, and feeding conditions were the same as in Example 6, and the experimental groups were as follows:
[0208] Control group: basal diet, without any added nutritional interventions;
[0209] Single intervention group: The Stage 3 formulation prepared in Example 5.3 was added to the basal diet at a ratio of 1.0% and used throughout the entire course of treatment;
[0210] Three-stage intervention group: The three-stage nutritional intervention was carried out according to the method in Example 6.
[0211] Table 5 Comparison of the effects of single-stage and three-stage nutritional interventions
[0212] index control group Single intervention group Three-stage intervention group Eggshell strength (kg / cm²) 3.85±0.19* 4.02±0.22** 4.21±0.23*** Broken egg rate (%) 6.5±0.8*** 3.8±0.5** 2.3±0.4* Relative abundance of R. gauvreauii (%) 0.82±0.12* 1.05±0.18** 2.35±0.28*** GABA content (μg / g) 3.42±0.45* 4.12±0.58** 6.02±0.75*** Calbindin-D28k relative expression level 1.00±0.16* 1.85±0.26** 2.70±0.35*** The egg-laying cycle has been prolonged (in weeks). 0* 1.8±0.3** 3.1±0.4***
[0213] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0214] As shown in Table 5, although the indicators in the single intervention group were improved compared with the control group, they were significantly lower than those in the three-stage intervention group (P<0.05). This indicates that the three-stage precision nutrition intervention method has a better effect than the single-stage intervention, and can more effectively regulate the gut microbiota-metabolite-shell gland mineralization network, improve eggshell quality, and prolong the laying cycle.
[0215] Comparative Example 2: Three-stage nutritional intervention without functional gut microbiota
[0216] This comparative study aims to evaluate the role of functional microbiota in a three-stage nutritional intervention.
[0217] The experimental subjects, basal diet, and feeding conditions were the same as in Example 6, and the experimental groups were as follows:
[0218] Control group: basal diet, without any added nutritional interventions;
[0219] Aseptic intervention group: Three-stage nutritional intervention was carried out according to the method in Example 6, but no functional microbial preparations were added to any of the preparations;
[0220] Complete intervention group: The three-stage nutritional intervention was carried out according to the method in Example 6.
[0221] Table 6 Comparison of the effects of aseptic intervention and complete intervention
[0222] index control group Aseptic intervention group Complete intervention group Eggshell strength (kg / cm²) 3.85±0.19* 3.98±0.21** 4.21±0.23*** Broken egg rate (%) 6.5±0.8*** 4.2±0.6** 2.3±0.4* GABA content (μg / g) 3.42±0.45* 4.05±0.55** 6.02±0.75*** 25-Hydroxycholesterol (ng / g) 18.3±2.4* 21.2±2.9** 28.9±3.5*** Calbindin-D28k relative expression level 1.00±0.16* 1.65±0.24** 2.70±0.35*** The egg-laying cycle has been prolonged (in weeks). 0* 1.5±0.3** 3.1±0.4***
[0223] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0224] As shown in Table 6, the indicators in the aseptic intervention group were improved compared with the control group, but significantly lower than those in the complete intervention group (P<0.05). This indicates that functional flora plays a key role in the three-stage nutritional intervention and is an indispensable link in the "bile acid-functional flora-metabolites-shell gland mineralization" network.
[0225] Comparative Example 3: Three-stage nutritional intervention without bile acid precursors
[0226] This comparative study aims to evaluate the role of bile acid precursors in a three-stage nutritional intervention.
[0227] The experimental subjects, basal diet, and feeding conditions were the same as in Example 6, and the experimental groups were as follows:
[0228] Control group: basal diet, without any added nutritional interventions;
[0229] Bile acid-free intervention group: Three-stage nutritional intervention was carried out according to the method of Example 6, but bile acid precursor complex was not added to the formulation of the first stage;
[0230] Complete intervention group: The three-stage nutritional intervention was carried out according to the method in Example 6.
[0231] Table 7 Comparison of effects between bile acid-free intervention and complete intervention
[0232] index control group Bile acid-free intervention group Complete intervention group Eggshell strength (kg / cm²) 3.85±0.19* 3.95±0.20** 4.21±0.23*** Broken egg rate (%) 6.5±0.8*** 4.5±0.6** 2.3±0.4* Relative abundance of R. gauvreauii (%) 0.82±0.12* 1.35±0.20** 2.35±0.28*** relative expression level of TGR5 in the gut 1.00±0.15* 1.32±0.18** 2.47±0.32*** Relative expression level of intestinal FXR 1.00±0.14* 1.28±0.17** 2.35±0.29*** The egg-laying cycle has been prolonged (in weeks). 0* 1.4±0.3** 3.1±0.4***
[0233] Note: The number of different asterisks in the same row (*, **, ***) indicates a significant difference (P<0.05).
[0234] Table 7 shows that the indicators in the bile acid-free intervention group were improved compared with the control group, but significantly lower than those in the complete intervention group (P<0.05). In particular, the expression levels of intestinal TGR5 and FXR were significantly lower in the group than in the complete intervention group, resulting in lower abundance of functional flora, which in turn affected the subsequent metabolite production and shell gland mineralization process. This indicates that bile acid precursors play a crucial role in initiating the three-stage nutritional intervention and are key triggering factors for activating the "bile acid-TGR5 / FXR-functional flora-metabolite-shell gland mineralization" network.
[0235] This invention, based on a complete regulatory network of "bile acids-TGR5 / FXR-functional microbiota-metabolites-shell gland mineralization," developed a three-stage precision nutritional intervention method, achieving significant improvements in eggshell quality and extending the egg-laying cycle. The mechanism of action is analyzed in detail below:
[0236] Bile acids, as important signaling molecules, can regulate intestinal function through two main pathways:
[0237] (1) TGR5 pathway: Bile acids activate the G protein-coupled receptor TGR5 on the intestinal epithelial cell membrane, leading to increased cAMP levels and activation of the PKA signaling pathway. PKA further phosphorylates CREB (cAMP response element-binding protein), enhancing downstream gene transcription and promoting intestinal barrier function and anti-inflammatory response. The experimental results of this invention show that the intestinal TGR5 expression level in the three-stage intervention group was significantly higher than that in the control group and the single intervention group, indicating that the bile acid precursor complex successfully activated the TGR5 signaling pathway.
[0238] (2) FXR pathway: After bile acids enter intestinal epithelial cells, they bind to the nuclear receptor FXR, activating the FXR-RXR heterodimer and regulating the transcription of downstream genes. After FXR activation, it inhibits hepatic bile acid synthesis by inducing FGF15 / 19 expression; and it suppresses intestinal inflammation and improves the intestinal microenvironment by downregulating the ASBT / NF-κB pathway. The experimental results of this invention show that the intestinal FXR expression level in the three-stage intervention group was significantly higher than that in the control group and the single intervention group, indicating that the bile acid precursor complex simultaneously activates the FXR signaling pathway.
[0239] The synergistic activation of these two pathways not only improved the gut microenvironment but also regulated the composition of the gut microbiota, creating favorable conditions for the colonization and growth of functional flora. Comparative Example 3 also demonstrated that the bile acid precursor complex is a key initiating factor in the three-stage nutritional intervention; the intervention effect was significantly reduced without bile acid precursors.
[0240] This invention screened three functional strains with synergistic effects: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557, and Enterococcus CGMCC 1.2136. These three strains formed a functionally complementary micro-ecosystem in the gut.
[0241] (1) Ruminococcus CGMCC 1.23020: Belonging to the Firmicutes phylum, it is mainly distributed in the colon of the intestine. It can decompose complex polysaccharides and produce short-chain fatty acids such as butyric acid. Butyric acid is the main energy source for intestinal epithelial cells, which can enhance the intestinal barrier function and affect host metabolism by regulating gene expression. In this experiment, the increase of rumenococcus was significantly correlated with the increase of GABA content.
[0242] (2) Lactobacillus CGMCC 1.557: Belonging to the Firmicutes phylum, it is widely distributed in the intestine and can produce lactic acid, lowering the intestinal pH and inhibiting the growth of harmful bacteria. Simultaneously, Lactobacillus can synthesize various vitamins and amino acids, especially branched-chain amino acids. In this experiment, the increase in Lactobacillus was significantly correlated with the increase in BCAAs content.
[0243] (3) Enterococcus CGMCC 1.2136: Belonging to the Firmicutes phylum, it is mainly distributed at the junction of the small intestine and colon in the intestine. It can convert cholesterol precursors and produce active metabolites such as 25-hydroxycholesterol. In this experiment, the increase of Enterococcus was significantly correlated with the increase of 25-hydroxycholesterol content.
[0244] The synergistic effect of these three bacterial strains forms a complete metabolic network, jointly producing a variety of key metabolites related to eggshell mineralization. The results of Comparative Example 2 also demonstrate that functional microbiota is an indispensable component of the three-stage nutritional intervention; the intervention effect is significantly reduced without functional microbiota.
[0245] This invention focuses on the association between three key metabolites and crust gland mineralization:
[0246] (1) γ-Aminobutyric acid (GABA): GABA is a non-protein amino acid that mainly functions as an inhibitory neurotransmitter in animals. Recent studies have found that GABA also participates in calcium signal transduction and cellular metabolic regulation. In shell gland tissue, GABA enhances calcium transport capacity by activating calcium-sensitive receptors (CaSRs), promoting the release of intracellular calcium stores and the influx of extracellular calcium. In this experiment, the GABA content in the three-stage intervention group was significantly higher than that in the control group and the single intervention group, and was significantly positively correlated with the expression level of Calbindin-D28k in shell gland tissue.
[0247] (2) Branched-chain amino acids (BCAAs): including leucine, isoleucine, and valine, are important raw materials for protein synthesis and also participate in energy metabolism and signal transduction. In shell gland tissue, BCAAs promote protein synthesis, especially the synthesis of calcium-binding proteins and transport proteins, by activating the mTORC1 signaling pathway. In this experiment, the BCAA content in the three-stage intervention group was significantly higher than that in the control group and the single intervention group, and was significantly positively correlated with the expression level of TRPV6 in shell gland tissue.
[0248] (3) 25-Hydroxycholesterol: It is an important intermediate product of cholesterol metabolism and plays an important role in vitamin D metabolism and calcium absorption. In the shell gland tissue, 25-hydroxycholesterol can directly activate the vitamin D receptor (VDR) and promote the expression of calcium transporter and carbonic anhydrase. In this experiment, the 25-hydroxycholesterol content in the three-stage intervention group was significantly higher than that in the control group and the single intervention group, and was significantly positively correlated with the carbonic anhydrase activity in the shell gland tissue.
[0249] These three types of metabolites work synergistically through different mechanisms to enhance the calcium ion transport and carbonate ion generation capacity, ultimately promoting the eggshell mineralization process.
[0250] The three-stage precision nutrition intervention strategy developed in this invention provides targeted nutritional support for the physiological needs of laying hens at different production stages:
[0251] (1) Establishment of gut microbiota homeostasis: The TGR5 / FXR signaling pathway is activated by bile acid precursor complex, while providing microbiota regulation complex and gut environment regulators to create favorable conditions for the colonization and growth of functional microbiota and establish gut microbiota homeostasis that is conducive to calcium absorption.
[0252] (2) Enhanced metabolite production period: Based on the stable colonization of functional microbiota, metabolite precursor complexes and metabolic activity regulators are provided to provide sufficient substrates and cofactors for functional microbiota, thereby maximizing the production of key metabolites.
[0253] (3) Shell gland mineralization efficiency enhancement period: On the basis of stable improvement of metabolite levels, calcium transporter activation complex, carbonic anhydrase cofactor and ion channel regulator are directly added to work synergistically with metabolites on shell gland tissue to maximize the improvement of eggshell mineralization efficiency.
[0254] These three stages are interconnected, forming a complete intervention chain. Each stage lays the foundation for the next, ultimately achieving comprehensive improvement in eggshell quality and effective extension of the egg-laying cycle. The results of Comparative Example 1 also demonstrate that the three-stage precise intervention is more effective than single-stage intervention, and can more effectively regulate the gut microbiota-metabolite-shell gland mineralization network.
[0255] In summary, this invention elucidates the complete regulatory network of "bile acids-TGR5 / FXR-functional microbiota-metabolites-shell gland mineralization," and develops a three-stage precision nutrition intervention method. This method successfully solves the problem of declining eggshell quality in the later stages of egg production, extends the effective production cycle, improves breeding efficiency, and provides a new paradigm for precision nutrition regulation in the egg-laying hen industry.
[0256] This invention provides a method for mineralizing and nutrient-intervening eggshells based on the gut microbiota-metabolite-shell gland tissue interaction network. Through a three-stage precise intervention strategy, the following objectives were successfully achieved:
[0257] 1. The complete regulatory network of "bile acids-TGR5 / FXR-functional microbiota-metabolites-shell gland mineralization" was elucidated, providing a new theoretical basis for the regulation of eggshell quality;
[0258] 2. The synergistic effect of three functional bacteria strains, namely Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557 and Enterococcus CGMCC 1.2136, was screened and verified to achieve precise regulation of the intestinal flora-metabolism network;
[0259] 3. A "three-stage precision intervention" strategy was developed to provide targeted nutritional support for the physiological needs of laying hens at different production stages, thereby achieving stable eggshell quality throughout the entire production cycle.
[0260] 4. We prepared bile acid precursor microcapsule formulations, metabolite precursor sustained-release particles, and calcium transporter activation complex microemulsion formulations with targeted release properties to ensure efficient release of active ingredients at specific sites in the intestine.
[0261] 5. Significantly improved eggshell quality, increased eggshell strength by 4.8%, reduced egg breakage rate by 4.2%, extended egg production cycle by 3.1 weeks, resulting in significant economic benefits.
[0262] This invention solves the industry pain point of declining eggshell quality in the later stages of egg production, and provides a new paradigm for precise nutritional regulation in the egg-laying hen industry, which has important theoretical value and application prospects.
[0263] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.
Claims
1. A nutritional intervention method for regulating eggshell mineralization by gut microbiota and metabolites in laying hens, characterized in that, Includes the following steps: A. Establishment of gut microbiota homeostasis: Add bile acid precursor complex, microbiota regulation complex, gut environment regulator and functional microbiota preparation to the feed of 24-week-old laying hens at a ratio of 0.5% for 14 consecutive days. B. Enhanced Metabolite Production Period: During the peak egg production period of laying hens, add metabolite precursor complexes, metabolic activity regulators, and functional microbial preparations to the laying hen feed at a ratio of 0.8%. C. Shell gland mineralization efficiency enhancement period: During the later stage of egg production in laying hens, calcium transporter activation complex, carbonic anhydrase cofactor, ion channel regulator, and functional microbial preparation are added to the laying hen feed at a ratio of 1.0%. in: The bile acid precursor complex is composed of the following components in parts by weight: 30-50 parts deoxycholic acid, 10-20 parts chenodeoxycholic acid, and 10-20 parts taurocholic acid. The microbial community regulation complex is composed of the following components in parts by weight: 80-120 parts of fructooligosaccharides, 30-50 parts of inulin, and 20-30 parts of mannan oligosaccharides. The intestinal environment regulator is composed of the following components in parts by weight: 20-30 parts citric acid, 10-20 parts malic acid, and 5-10 parts sodium butyrate. The metabolite precursor complex is composed of the following components in parts by weight: 40-60 parts L-glutamic acid, 20-30 parts leucine, 10-15 parts isoleucine, 10-15 parts valine, 5-10 parts β-sitosterol, and 5-10 parts 7-dehydrocholesterol. The metabolic activity regulator is composed of the following components in parts by weight: 10-20 parts taurine, 20-30 parts inositol, and 1-2 parts cobalt-biotin complex; The calcium transporter activation complex is composed of the following components in parts by weight: 1,25-dihydroxyvitamin D3 200-300 IU, vitamin K2 8-12 mg, and zinc-glycine chelate 60-80 mg; The carbonic anhydrase cofactor is composed of the following components in parts by weight: 10-15 mg of copper-histidine chelate and 60-80 mg of manganese-protein acid salt. The ion channel modulator is composed of the following components in parts by weight: magnesium citrate 350-450 mg, potassium gluconate 500-600 mg; The functional microbial preparation comprises the following active strains: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557, and Enterococcus CGMCC 1.2136, wherein the viable count of Ruminococcus CGMCC 1.23020 is 8.0 × 10⁻⁶. 10 The viable count of the Lactobacillus CGMCC 1.557 was 5.0 × 10⁻⁶ CFU / g. 10 The viable count of the Enterococcus CGMCC 1.2136 was 3.0 × 10⁻⁶ CFU / g. 10 CFU / g; The bile acid precursor complex, microbiota regulation complex, intestinal environment regulator, metabolite precursor complex, metabolic activity regulator, calcium transporter activation complex, carbonic anhydrase cofactor, and ion channel regulator work synergistically with the rumen cocci, lactobacilli, and enterococci to activate intestinal microbiota metabolism through the TGR5 and FXR dual receptor signaling pathways, promote the production of γ-aminobutyric acid, branched-chain amino acids, and 25-hydroxycholesterol, and thereby activate the expression of calbindin-D28k and carbonic anhydrase in the shell gland tissue, thus improving eggshell mineralization efficiency.
2. The nutritional intervention method for regulating eggshell mineralization by gut microbiota and metabolites in laying hens according to claim 1, characterized in that, The preparation method of the functional microbial community preparation includes the following steps: a) Primary seed culture: Ruminococcus CGMCC 1.23020, Lactobacillus CGMCC 1.557 and Enterococcus CGMCC 1.2136 were inoculated into the corresponding culture media and cultured anaerobically at 37°C for 24 hours. b) Secondary fermentation: Inoculate the strain obtained in step a) into the basic culture medium, control the pH to 6.2-6.5, maintain 35°C for the first 24 hours, reduce to 32°C for the next 12 hours, maintain 32°C for the last 12 hours, ferment for 36-48 hours, and control dissolved oxygen to below 10%. c) Cell collection and protection: Centrifuge the fermentation broth obtained in step b) at 8000 r / min for 15 minutes at 4℃, collect the cells, and add 10% skim milk powder, 5% maltodextrin and 2% sodium alginate as protectants. d) Freeze-drying treatment: The mixture obtained in step c) is first pre-frozen at -45°C for 4 hours, then dried at -30°C under a vacuum of less than 20 Pa for 24 hours, and finally dried at 20°C under a vacuum of less than 10 Pa for 4 hours to obtain the functional microbial preparation.
3. A bile acid precursor microcapsule formulation, used for the delivery of the bile acid precursor complex in step A of the nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens as described in claim 1 or 2, characterized in that, The microcapsule formulation is prepared by the following steps: a) Raw material pretreatment: Deoxycholic acid and chenodeoxycholic acid were mixed at a ratio of 7:3 and ground to 100 mesh. At the same time, 10% gum arabic and 15% maltodextrin aqueous solution were prepared as wall material. b) Multilayer microencapsulation: Bile acids and soybean oil are mixed in a 1:1 ratio and homogenized by high-speed shearing. Then, a mixture of gum arabic and maltodextrin is added to form an intermediate layer, and finally, a 3% ethyl cellulose solution is used to form an outer layer. c) Targeted release treatment: Spray coating with 3% methacrylic acid copolymer solution in a fluidized bed, with the inlet air temperature controlled at 45±2℃ and the material temperature controlled at 30±2℃; d) Quality control: Ensure that the final product has a particle size of 3-5 micrometers, an encapsulation rate of not less than 85%, and a release rate of not less than 85% after 2 hours at pH 6.5 and not more than 15% after 2 hours at pH 2.
0.
4. Metabolite precursor sustained-release particles, used for the delivery of the metabolite precursor complex in step B of the nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens as described in claim 1 or 2, characterized in that, The sustained-release granules are prepared by the following steps: a) Raw material processing: The amino acid salt was physically pulverized to 200 mesh, sieved, and a 3% sodium carboxymethyl cellulose aqueous solution was prepared as a binder; b) Granulation and coating: Mix and granulate in a fluidized bed, with the material temperature controlled at 45±2℃, the atomization pressure at 1.5 bar, and the spraying speed at 5 mL / min. Then, coat the polymer with 5% hydroxypropyl methylcellulose solution, 10% ethyl cellulose-ethanol solution, and 10% triethyl citrate by weight of the polymer. c) Construction of a multilayer sustained-release system: The metabolite precursor complex is used as the core, hydroxypropyl methylcellulose and sodium alginate are mixed in a 7:3 ratio to form the middle layer, and finally ethyl cellulose-succinate copolymer is used to form the outer layer. d) Quality control: Ensure that the moisture content of the final product does not exceed 5%, the coefficient of variation of particle size uniformity does not exceed 5%, the compressive strength is not less than 20 Newtons, and the release rate is 25-35% in the first 2 hours and the cumulative release rate is not less than 85% in 12 hours.
5. A calcium transporter activation complex microemulsion formulation, used for the delivery of the calcium transporter activation complex in step C of the nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens as described in claim 1 or 2, characterized in that, The microemulsion formulation is prepared by the following steps: a) Microemulsion of core components: Vitamin D3 and K2 are dissolved in medium-chain triglycerides and homogenized under high pressure to form microemulsions with a particle size of less than 200 nanometers; b) Carrier composite: Silica and calcium carbonate are mixed in a 3:7 ratio as the main carrier, and 5% hydroxypropyl-β-cyclodextrin is added as an inclusion agent; c) Multilayer sustained-release system: Using layer-by-layer self-assembly technology, chitosan with a molecular weight of 50-100kDa and a degree of deacetylation of not less than 90% is alternately deposited with alginate with an M / G ratio of 1.5 and a viscosity of 200-400mPa·s. d) Quality control: Ensure that the vitamin D3 content in the final product is 98-102% and the vitamin K2 content is 95-105%, and that the effective content is not less than 90% after being stored at 40℃ and 75% relative humidity for 3 months. The in vitro release rate conforms to zero-order release kinetics, and the release rate is 80% after 6 hours.
6. The nutritional intervention method for regulating eggshell mineralization by gut microbiota-metabolites in laying hens as described in claim 1 or 2, in improving eggshell quality and extending the laying cycle of laying hens, is characterized in that... The application increased eggshell strength by 4.8%, reduced egg breakage rate by 4.2%, and extended the egg-laying cycle by 3.1 weeks; it also increased calbindin-D28k expression by 2.7 times, carbonic anhydrase activity by 3.1 times, and calcium channel TRPV6 expression by 2.4 times in shell gland tissue.