Preparation method of small peptide manganese feed additive for enhancing development of white feather broilers

Abstract

The invention discloses a preparation method of a small peptide manganese feed additive for enhancing development of white feather broilers, and relates to the technical field of feed additives.The method comprises the steps that zein serves as a raw material, and a small peptide solution with the molecular weight concentrated to 500-1200 Daltons is obtained through complex enzyme synergistic enzymolysis; under the protection of nitrogen, directional chelation is carried out according to the molar ratio of small peptide carboxyl to manganese ions being 3: 1; performing ultrafiltration purification to remove free manganese, and performing spray drying to obtain a powder product; the controllable release performance is verified by simulating a poultry digestion environment. The invention aims to solve the technical problems that the existing small peptide chelated manganese feed additive is low in absorption efficiency, limited in growth promoting effect, poor in batch stability and not suitable for physiological characteristics of digestive tracts of poultry when being applied to white feather broilers, and realizes high chelation rate, excellent flowability and storage stability of the feed; the daily gain, the tibia ash content and the feed conversion rate of the white feather broilers are remarkably improved, and the process is green and suitable for industrial continuous production.

Description

[0001] This invention belongs to the field of feed additive technology, and specifically relates to a method for preparing a small peptide manganese feed additive that enhances the development of broiler chickens. Background Technology

[0002] With the increasing demands for functionality and bioavailability of feed additives in the livestock and poultry farming industry, small peptide chelated manganese, as a highly efficient and environmentally friendly organic trace element additive, has shown promising application prospects in promoting animal growth and development. Especially during the rapid growth period of broiler chickens, manganese plays a crucial role in bone development, enzyme system activation, and metabolic regulation. However, existing small peptide chelated manganese feed additives are mostly designed for monogastric animals such as pigs. Their preparation processes, formulations, and release characteristics have not been fully adapted to the physiological characteristics and nutritional needs of broiler chickens, resulting in problems such as insufficient absorption efficiency and limited growth-promoting effects in practical applications.

[0003] The preparation of manganese chelated from small peptides requires consideration of the bioactivity of the small peptide source, the chelation stability of manganese ions, and the release behavior of the product in the poultry digestive tract. Current mainstream technologies include synthesizing small peptides with strong acid resin catalysis followed by manganese chelation, or obtaining small peptides through plant protein hydrolysis followed by metal complexation. However, the former easily introduces difficult-to-remove chemical residues, affecting feed safety; the latter, while using natural raw materials, suffers from a wide pH control range and non-specific enzymatic hydrolysis conditions, making it difficult to ensure the consistency of the small peptide sequence and the targeted exposure of manganese chelation sites, leading to significant batch-to-batch performance fluctuations. More critically, none of the above methods consider the unique physiological characteristics of broiler chickens, such as the high pH value in the foregut, short intestinal transit time, and the concentrated manganese absorption window from brooding to early rearing, resulting in additives that cannot achieve efficient targeted delivery and sustained release of manganese in vivo.

[0004] The aforementioned defects together make it difficult for existing products to simultaneously improve bone mineralization strength, daily weight gain rate and feed conversion rate in broiler chicken farming practices. There is an urgent need for a specialized small peptide manganese feed additive preparation method for this high-growth-performance poultry breed to achieve precise matching between manganese nutrition supply and broiler development rhythm. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a small peptide manganese feed additive to enhance the development of broiler chickens, so as to solve the technical problems of insufficient absorption efficiency, limited growth promotion effect, poor batch stability, and inability to adapt to the physiological characteristics of poultry digestive tract in the application of existing small peptide chelated manganese feed additives in broiler chickens.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A method for preparing a small peptide manganese feed additive to enhance the development of broiler chickens includes the following specific steps:

[0008] Step 1: Select zein as the precursor of small peptides and disperse it in deionized water to form a suspension with a mass concentration of 8%. Adjust the pH of the system to 5.6. Add a compound enzyme preparation composed of alkaline protease and flavor protease at a temperature of 45℃ for synergistic enzymatic hydrolysis. The enzymatic hydrolysis reaction lasts for 3 hours, during which the stirring speed is maintained at 180 rpm. After the reaction is completed, raise the temperature to 95℃ and keep it at 10 minutes to achieve enzyme inactivation, and obtain a small peptide solution with a molecular weight distribution concentrated in 500 to 1200 Daltons.

[0009] Step 2: Dissolve manganese sulfate in deionized water to prepare a manganese ion solution with a molar concentration of 0.4 mol / L. Slowly add the solution to the small peptide under a nitrogen protective atmosphere, control the molar ratio of small peptide carboxyl groups to manganese ions to be 3:1, adjust the pH of the mixture to 6.2, and react at a constant temperature of 50°C for 4 hours under light-protected conditions with shaking, so that manganese ions can directionally chelate with the carboxyl and amino groups of the small peptide side chain to generate a small peptide manganese chelate precursor.

[0010] Step 3: The precursor solution is subjected to ultrafiltration using a hollow fiber membrane module with a molecular weight cutoff of 1000 Daltons. The operating pressure is set to 0.25 MPa, and the dialysate volume is continuously replaced up to 5 times the original volume to remove unchelated free manganese ions and low molecular weight impurities. The retentate is then collected.

[0011] Step 4: The retentate is concentrated to 1 / 3 of its original volume by rotary evaporation, followed by spray drying. The inlet air temperature is controlled at 160℃, the outlet air temperature is controlled at 85℃, and the atomizer speed is controlled at 25000 rpm to obtain a light yellow powdery small peptide manganese feed additive final product with good flowability.

[0012] Step 5: Characterize the quality of the final product. Determine the chelation rate by UV-Vis spectrophotometry, analyze the molecular weight distribution by high-performance gel permeation chromatography, and determine the total manganese content and soluble manganese release curve by inductively coupled plasma mass spectrometry to confirm that the product meets the set standards.

[0013] In step 1, the initial particle size of zein is controlled to be below 80 micrometers after ball milling pretreatment to improve its dispersion uniformity and enzymatic accessibility in the aqueous phase. The mass ratio of alkaline protease to flavor protease used is 2:1, and the total enzyme addition accounts for 1.2% of the substrate mass. This combined enzyme system exhibits a synergistic effect in the pH range of 5.6 to 6.0, significantly improving the yield of specific functional small peptide fragments.

[0014] In step 1, the molecular weight distribution of the small peptides in the enzymatic hydrolysis products meets the following requirements: the proportion of components with a molecular weight less than 700 Daltons is not less than 45%, the proportion of components with a molecular weight between 700 and 1200 Daltons is not more than 55%, and the overall polydispersity index is less than 1.15, ensuring that the small peptide chain length is appropriate and conducive to subsequent transintestinal transport.

[0015] In step 2, the manganese ion solution is added at a rate of 2 ml per minute for 40 minutes to avoid local supersaturation that could lead to precipitation. The dissolved oxygen concentration in the reaction system is maintained below 2 mg / L to prevent divalent manganese from being oxidized to trivalent manganese and thus reducing its biological activity.

[0016] After the chelation reaction in step 2 is completed, the resulting solution is centrifuged at 4000 rpm for 10 minutes to remove trace amounts of insoluble matter. The supernatant is used for subsequent purification steps to ensure product clarity.

[0017] In step 3, the manganese ion concentration of the permeate is measured every 30 minutes during ultrafiltration. The dialysis endpoint is determined when two consecutive measurements are both below 0.01 mg / L, ensuring thorough removal of free manganese. Preferably, in step 4, the moisture content of the powder obtained from spray drying is controlled below 5%, the loose packing density is 0.45 g / mL to 0.55 g / mL, the angle of repose is less than 40°, and it possesses excellent flowability and storage stability. Preferably, in step 5, the chelation rate is calculated using the formula: Chelation rate = (Total manganese content - Extractable free manganese content) / Total manganese content × 100%, where the extractable free manganese content is determined after separation by trichloroacetic acid precipitation, and the final product chelation rate is required to be greater than or equal to 92%.

[0018] In step 5, the determination of soluble manganese release behavior was carried out using a method simulating the foregut digestive environment of broiler chickens. 0.1g of the additive sample was placed in 10mL of simulated digestive fluid, which consisted of 0.2% pepsin, 0.1% trypsin, and 0.3% bile salts. The pH gradient was set to gradually increase from 5.8 to 6.8. Samples were taken every 30 minutes, filtered through a 0.22-micron filter membrane, and the manganese concentration in the filtrate was measured. A cumulative release curve was plotted, requiring a release rate of 60% to 75% within the first 2 hours and complete release within 4 hours.

[0019] The small peptide manganese feed additive prepared by the method is used in the daily diet formula of white-feathered broiler chickens to replace inorganic manganese sulfate. The addition amount is 60 mg of manganese per kilogram of complete feed. It is fed continuously from the first day of chicks to the 42nd day of age. During this period, the daily weight gain is increased by more than 8.5%, the tibia ash content is increased by more than 7.2%, and the feed conversion rate is improved by more than 6.0%.

[0020] It also includes establishing an online quality monitoring system based on near-infrared spectroscopy, collecting spectral data of different batches of products and constructing a partial least squares regression model to achieve rapid prediction of key quality attributes such as chelation rate, moisture content and particle size distribution. The model cross-validation determination coefficient R² is greater than 0.95 and the prediction error is less than 3%, which is used for real-time feedback control of the production process.

[0021] The method is suitable for industrial continuous production, with a single batch processing capacity of over 500 kg. The entire process leaves no strong acid or alkali residues, and the wastewater COD value is below 300 mg / L, meeting the requirements for green feed additive production processes. Preferably, the small peptide manganese additive, when stored in a light-proof, sealed environment with a relative humidity below 60%, has a shelf life of up to 18 months, during which the chelated structure remains stable without significant degradation or precipitation.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0023] This invention utilizes zein as a specific small peptide precursor and combines it with a pH-directed enzymatic hydrolysis process to achieve controllable generation of small peptide sequences, ensuring that the product has a concentrated molecular weight distribution and is rich in functional fragments with high affinity for the avian intestinal transport system.

[0024] This invention establishes a stable directional chelation mechanism by precisely controlling the molar ratio of small peptide carboxyl groups to manganese ions, reaction pH, temperature, and protective atmosphere, significantly improving the chelation rate and product consistency. The designed ultrafiltration purification process effectively removes free manganese and other impurities, ensuring the high purity and safety of the final product. Optimized spray drying parameters give the product good physical properties, facilitating industrial applications.

[0025] The small peptide manganese feed additive prepared in this invention exhibits ideal phased release characteristics under simulated broiler digestive conditions, matching its short-term, high-efficiency nutrient absorption window, thereby significantly improving the bioavailability of manganese. Actual feeding trials show that this additive can simultaneously promote bone mineralization, accelerate daily weight gain, and improve feed conversion efficiency, solving the problems of traditional products having limited functionality and unstable effects on this poultry breed. The entire preparation process does not require the use of strong acid resins or other chemical synthesis pathways, avoiding the risk of harmful residues and aligning with the development direction of modern green farming.

[0026] This invention achieves intelligent monitoring of the production process by introducing near-infrared spectroscopy combined with chemometrics models for quality control, ensuring batch-to-batch stability in large-scale production. This invention has made systematic innovations in raw material selection, reaction mechanism, process parameters and quality evaluation system, filling the technical gap in the customized design of organic manganese additives for the physiological characteristics of broiler chickens, and has significant technological advancement and industrial promotion value. Attached Figure Description

[0027] Figure 1 This is a flowchart of the overall technical solution for the preparation method of a small peptide manganese feed additive that enhances the development of broiler chickens, as proposed in this invention.

[0028] Figure 2 This is a schematic diagram illustrating the core principle framework of the manganese-directed chelation and purification control of small peptides in this invention. Detailed Implementation

[0029] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present invention and not to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely to provide a better understanding of the present invention by illustrating examples of the invention.

[0030] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0031] In the embodiments of the present invention, the same reference numerals denote the same components, and for the sake of brevity, detailed descriptions of the same components are omitted in different embodiments. It should be understood that the thickness, length, width, and other dimensions of various components in the embodiments of the present invention shown in the accompanying drawings, as well as the overall thickness, length, width, and other dimensions of the integrated device, are merely illustrative and should not constitute any limitation on the present invention; the term "multiple" in the present invention refers to two or more (including two).

[0032] Currently, with the increasing demands for functionality and bioavailability of feed additives in the livestock and poultry farming industry, small peptide chelated manganese, as a highly efficient and environmentally friendly organic trace element additive, plays a crucial role in promoting skeletal development, enzyme system activation, and metabolic regulation during the rapid growth period of broiler chickens. However, existing small peptide chelated manganese feed additives are mostly designed for monogastric animals such as pigs. Their preparation processes, formulations, and release characteristics have not been adequately adapted to the unique physiological characteristics of broiler chickens, such as high foregut pH, short intestinal transit time, and a manganese absorption window concentrated in the brooding to early rearing stages. This results in technical problems such as insufficient absorption efficiency, limited growth-promoting effects, poor batch stability, and the inability to achieve efficient targeted delivery and sustained release of manganese in practical applications. To address these technical problems, this invention proposes a systematic preparation method that involves specific raw material selection, directional enzymatic hydrolysis control, precise chelation reaction, efficient purification and separation, and optimization of physical properties. This method is applied to the preparation of a small peptide manganese feed additive that enhances the development of broiler chickens.

[0033] refer to Figure 1 The schematic diagram of the overall technical solution shows that the method of the present invention starts with the treatment of zein precursor and goes through six core steps in sequence: compound enzymatic hydrolysis, directional chelation, ultrafiltration purification, spray drying and quality characterization, forming a closed-loop, industrially scalable green production process. Figure 2 The core principle framework for the directional chelation and purification control of small peptide manganese was further demonstrated, which clearly indicated the coordination mechanism of carboxyl and amino groups of small peptide side chains to manganese ions, the molecular weight cutoff and impurity removal logic of ultrafiltration membrane, and the staged release behavior regulation strategy under simulated digestion environment.

[0034] The specific steps in the preparation method of the above-mentioned small peptide manganese feed additive that enhances the development of broiler chickens include the following:

[0035] Step 1: Zeatin was selected as the precursor of small peptides and dispersed in deionized water to form a suspension with a mass concentration of 8%. The pH of the system was adjusted to 5.6. A compound enzyme preparation consisting of alkaline protease and flavor protease was added at 45℃ for synergistic enzymatic hydrolysis. The enzymatic hydrolysis reaction lasted for 3 hours, during which the stirring speed was maintained at 180 rpm. After the reaction, the temperature was raised to 95℃ and held for 10 minutes to inactivate the enzyme, obtaining a small peptide solution with a molecular weight distribution concentrated between 500 and 1200 Daltons. Specifically, in Step 1, the initial particle size of zeatin was controlled to below 80 micrometers after ball milling pretreatment. This pretreatment operation used a planetary ball mill with zirconium oxide grinding balls, a ball-to-material ratio of 10:1, a rotation speed of 300 rpm, and a treatment time of 20 minutes to ensure that the surface microcracks of the raw material particles increased and the specific surface area increased, thereby significantly improving its dispersion uniformity and enzymatic accessibility in the aqueous phase. The alkaline protease used was derived from Bacillus subtilis fermentation broth, with an activity of 200,000 U / g, while the flavor protease was derived from Aspergillus oryzae extract, with an activity of 500 U / g. The two were mixed at a mass ratio of 2:1 to form a compound enzyme preparation, with the total enzyme addition accounting for 1.2% of the substrate mass, i.e., 1.2 g of compound enzyme per 100 g of zein. This combined enzyme system exhibited a significant synergistic effect within the pH range of 5.6 to 6.0: the alkaline protease preferentially cleaved peptide bonds between hydrophobic amino acid residues, generating intermediate fragments rich in acidic amino acids such as glutamic acid and aspartic acid; the flavor protease specifically hydrolyzed the N-terminal hydrophobic residues, exposing more free carboxyl groups while retaining the C-terminal hydrophilic structure, thereby jointly promoting the high-yield generation of specific functional small peptide fragments—short-chain peptides with transintestinal wall transport capabilities and sensitivity to avian digestive enzymes. During enzymatic hydrolysis, the reactor is equipped with an automatic pH feedback control system, which maintains the system pH at a constant 5.6±0.1 by adding 0.1mol / L sodium hydroxide solution dropwise. The temperature is precisely controlled by a jacketed circulating water bath, with fluctuations not exceeding ±0.5℃. Anchor-type impellers are used for stirring to ensure that the suspension does not settle or exhibit localized excessive concentrations. The end point of enzymatic hydrolysis is determined by sampling and measuring the degree of hydrolysis (DH value). When the DH value reaches 18% to 22%, the reaction is stopped, and the temperature is immediately raised to 95℃ and held for 10 minutes to cause irreversible denaturation and inactivation of the enzyme protein, thus terminating the hydrolysis process. After cooling to room temperature, the obtained peptide solution exhibited the following molecular weight distribution: the proportion of components with a molecular weight less than 700 Daltons was not less than 45%, the proportion of components with a molecular weight between 700 and 1200 Daltons was not more than 55%, and the overall polydispersity index was less than 1.15. This distribution characteristic was verified by high-performance gel permeation chromatography (HPLC). The chromatographic column packing material was Sephadex G-10 hydroxypropyl dextran gel, the mobile phase was 0.1 mol / L phosphate buffer, pH 6.0, the flow rate was 0.5 mL / min, and the detection wavelength was 220 nm.This narrow distribution characteristic ensures that the small peptide chains are of moderate length, avoiding both excessive length leading to increased transmembrane resistance and excessive shortness resulting in loss of structural stability, thus facilitating efficient transport in the duodenal epithelial cells of broiler chickens.

[0036] Step 2: Dissolve manganese sulfate in deionized water to prepare a manganese ion solution with a molar concentration of 0.4 mol / L. Under a nitrogen atmosphere, slowly add this solution to the small peptide solution, controlling the molar ratio of small peptide carboxyl groups to manganese ions to be 3:1. Adjust the pH of the mixture to 6.2, and react at a constant temperature of 50°C with shaking for 4 hours under light-protected conditions. This allows the manganese ions to directionally chelate with the carboxyl and amino groups of the small peptide side chains, generating a small peptide manganese chelate precursor. Specifically, in Step 2, the manganese sulfate used is an analytical grade reagent, pre-dried at 105°C for 2 hours to remove water of crystallization. Ultrapure water with a resistivity ≥18.2 MΩ·cm is used for preparation. The solution is filtered through a 0.22-micron microporous membrane for sterilization before use. Nitrogen protection is achieved by continuously introducing high-purity nitrogen (≥99.999%) into the top of the reaction vessel at a flow rate of 0.5 L / min. This reduces the oxygen partial pressure in the reaction system to below 50 Pa, corresponding to a dissolved oxygen concentration below 2 mg / L, effectively preventing the oxidation of divalent manganese to trivalent manganese and thus reducing biological activity. The manganese ion solution is added at a rate of 2 mL / min over 40 minutes, with a peristaltic pump precisely controlling the flow rate to avoid the precipitation of manganese carbonate or manganese hydroxide due to excessively high local manganese ion concentrations. The molar number of carboxyl groups in the small peptide is pre-determined using an acid-base titration method: 10 mL of the small peptide solution is titrated with 0.01 mol / L sodium hydroxide standard solution using phenolphthalein as an indicator until a faint pink color appears, the volume consumed is recorded, and the free carboxyl group concentration is calculated. The amount of manganese sulfate added was adjusted accordingly to ensure a strict 3:1 molar ratio of carboxyl groups to manganese ions in the small peptide. This ratio, verified in previous experiments, is the optimal coordination stoichiometry point, ensuring that each manganese ion is coordinated by at least two carboxyl groups to form a stable five-membered ring structure, while avoiding excessive carboxyl groups that could lead to charge repulsion and affect chelation efficiency. The pH of the mixed system was adjusted to 6.2 ± 0.05 using 0.1 mol / L hydrochloric acid or sodium hydroxide solution. This pH value is close to the median of the physiological pH gradient from the crop to the duodenum of broiler chickens (5.8–6.8), which is beneficial for simulating the in vivo chelation environment and improving the stability of the product in the digestive tract. The reaction was carried out in a constant-temperature shaking water bath at a shaking frequency of 120 times / minute to ensure sufficient contact of the reactants. The outer wall of the container was wrapped with aluminum foil to protect it from light and prevent photocatalytic side reactions. After the chelation reaction was completed, the resulting solution was centrifuged at 4000 rpm for 10 minutes to remove trace amounts of insoluble matter. The supernatant was used for subsequent purification steps to ensure product clarity. The centrifugation operation uses a tubular centrifuge with a drum diameter of 100 mm, an effective volume of 5 L, and a separation factor of 8000 G, which can effectively remove aggregated particles with a particle size greater than 1 micrometer.

[0037] Step 3 involves ultrafiltration of the precursor solution using a hollow fiber membrane module with a molecular weight cutoff of 1000 Daltons. The operating pressure is set at 0.25 MPa, and the dialysate volume is continuously replaced up to 5 times the original solution volume to remove unchelated free manganese ions and low-molecular-weight impurities. The retentate is then collected. Specifically, the ultrafiltration system in Step 3 consists of a circulation pump, membrane module, pressure sensor, flow meter, and dialysate storage tank. The membrane module is a hollow fiber membrane made of polyethersulfone, with an inner diameter of 0.8 mm, a wall thickness of 0.2 mm, and an effective membrane area of ​​0.5 square meters. The operating pressure is precisely controlled at 0.25 ± 0.02 MPa via a back pressure valve. Excessive pressure can lead to membrane compaction and fouling, while insufficient pressure results in insufficient flux. The dialysate is deionized water, maintained at a temperature of 25°C, and its flow rate is kept at a 1:1 ratio with the feed flow rate to maintain a stable transmembrane pressure differential. During ultrafiltration, the manganese ion concentration in the permeate was measured every 30 minutes using atomic absorption spectrometry (AAS) at a wavelength of 279.5 nm and a slit width of 0.2 nm. The dialysis endpoint was defined as two consecutive measurements below 0.01 mg / L, ensuring complete removal of free manganese. The total volume of dialysate replaced during the entire dialysis process was five times the original volume; for example, if the original volume was 100 L, the total dialysate volume was 500 L. This multiple was verified through material balance to reduce the residual free manganese to below 0.1% of the initial amount. The main component of the retentate was a small peptide manganese chelate with a molecular weight greater than 1000 Daltons. Its manganese content was determined by inductively coupled plasma mass spectrometry (ICP-MS), confirming a recovery rate of no less than 98%.

[0038] Step 4 involves concentrating the retentate to one-third of its original volume via rotary evaporation, followed by spray drying. The inlet air temperature is controlled at 160°C, the outlet air temperature at 85°C, and the atomizer speed at 25,000 rpm, yielding a free-flowing, light yellow powder of small peptide manganese feed additive. Specifically, in Step 4, rotary evaporation is conducted in a 60°C water bath under a vacuum of -0.095 MPa, with the evaporation rate controlled at a reduction of 10% of the original volume per hour to avoid localized overheating that could damage the chelate structure. The solid content of the concentrate is increased to approximately 24%, and the viscosity is measured at 15 mPa·s at 25°C. Spray drying utilizes a closed-loop nitrogen-protected spray drying tower with a diameter of 1.2 m and a height of 6 m, equipped with two-fluid atomizing nozzles. The inlet air temperature is precisely controlled at 160±2°C by an electric heater, and the outlet air temperature is maintained at 85±1°C by a PID temperature control system. This temperature combination ensures rapid evaporation of moisture while preventing thermal denaturation of the small peptides or breakage of the manganese chelate bonds. The atomizer rotates at 25,000 rpm, producing droplets with an average particle size of 25 micrometers. After instantaneous drying with hot air, these droplets form well-spherical microparticles. The moisture content of the resulting powder is controlled below 5%, as determined using a halogen moisture analyzer; the loose density is 0.45 g / mL to 0.55 g / mL, as determined according to GB / T 16913.3-1997 standard; the angle of repose is less than 40°, measured using the fixed funnel method, indicating that the product possesses excellent flowability and storage stability, facilitating uniform mixing in feed production lines.

[0039] Step 5: Characterize the quality of the final product. The chelation rate is determined by UV-Vis spectrophotometry, the molecular weight distribution is analyzed by high-performance gel permeation chromatography, and the total manganese content and soluble manganese release curve are determined by inductively coupled plasma mass spectrometry to confirm that the product meets the set standards. Specifically, the formula for calculating the chelation rate in step 5 is: ;

[0040] The total manganese content was determined by ICP-MS after the sample was diluted following microwave digestion with nitric acid and hydrogen peroxide. The extractable free manganese content was determined by trichloroacetic acid precipitation: 0.5 g of sample was dissolved in 10 mL of 10% trichloroacetic acid solution, shaken for 30 minutes, and then centrifuged (10,000 rpm for 10 minutes). The supernatant was then used to determine the manganese concentration. This method effectively precipitates macromolecular chelates while retaining free manganese in solution. The final product chelation rate was required to be greater than or equal to 92%. The release behavior of soluble manganese was determined by simulating the foregut digestive environment of broiler chickens. A 0.1g additive sample was placed in 10mL of simulated digestive fluid, which consisted of 0.2% pepsin, 0.1% trypsin, and 0.3% bile salts. The pH gradient was set to gradually increase from 5.8 to 6.8, automatically adjusted every 30 minutes. Digestion was simulated in a 37℃ constant-temperature shaker, with samples taken every 30 minutes. After filtration through a 0.22-micron filter membrane, the manganese concentration in the filtrate was measured, and a cumulative release curve was plotted. The release rate was required to reach 60% to 75% within the first 2 hours and complete release within 4 hours. This release characteristic precisely matches the 2-4 hour nutrient absorption window of the broiler foregut, ensuring efficient release of manganese at the optimal time and location.

[0041] The small peptide manganese feed additive prepared by the above method was used in the daily diet of broiler chickens to replace inorganic manganese sulfate. The addition amount was 60 mg of manganese per kilogram of complete feed, and it was fed continuously from day 1 to day 42 of age. The feeding trial was conducted in a standardized chicken house, and the ambient temperature, humidity and light cycle were strictly controlled. The basic feed formula was formulated according to the NRC (1994) broiler nutrition standards. During the period, the daily weight gain increased by ≥8.5%, and the tibia ash content increased by ≥7.2%. Among them, the feed conversion ratio was improved by ≥6.0% after the tibia was calcined in a muffle furnace at 550℃ for 4 hours and then weighed. The feed conversion ratio (FCR) was calculated as feed intake / weight gain. In addition, an online quality monitoring system based on near-infrared spectroscopy was established to collect spectral data of different batches of products, with a wavelength range of 800–2500 nm and a resolution of 8 cm⁻¹. -1 A partial least squares regression model was constructed to rapidly predict key quality attributes such as chelation rate, moisture content, and particle size distribution. The model's cross-validation determination coefficient (R²) was greater than 0.95, and the prediction error was less than 3%, enabling real-time feedback control of the production process. The method is applicable to industrial continuous production, with a single batch processing capacity exceeding 500 kg. No strong acids or alkalis remain throughout the process, and the wastewater COD value is below 300 mg / L, meeting the requirements for green feed additive production processes. When stored in a light-proof, sealed environment with a relative humidity below 60%, the small peptide manganese additive has a shelf life of up to 18 months, during which the chelated structure remains stable without significant degradation or precipitation.

[0042] Example 2

[0043] To further verify the universality and robustness of the technical solution of this invention, some process parameters were adaptively adjusted based on Example 1 to form another feasible implementation scheme. Specifically, in step 1, zein was replaced with an equal mass of corn yellow powder extract, wherein the protein content is ≥60%, the initial particle size is controlled at 75 micrometers, the mass ratio of alkaline protease to flavor protease in the compound enzyme preparation was adjusted to 3:1, the total enzyme addition was increased to 1.5% of the substrate mass, the enzymatic hydrolysis temperature was maintained at 45°C, but the pH was adjusted to 5.8 to adapt to the slightly higher non-protein nitrogen content in the raw material. In the molecular weight distribution of the obtained small peptide solution, the proportion of components less than 700 Daltons was 48%, the proportion of components 700–1200 Daltons was 52%, and the polydispersity index was 1.12. In step 2, the dropping rate of manganese ion solution was adjusted to 2.5 mL per minute, the dropping time was shortened to 32 minutes, the reaction pH was fine-tuned to 6.3, and the other conditions were the same as in Example 1. During the ultrafiltration purification stage, the dialysate volume replacement factor was increased to 6 times to address any trace pigment impurities that might be introduced into the raw materials. The spray drying parameters remained unchanged. The final product achieved a chelation rate of 93.1%, a manganese release rate of 68.5% after 2 hours, and a 42-day feeding trial showed a 9.2% increase in daily weight gain, a 7.8% increase in tibia ash content, and a 6.5% improvement in feed conversion ratio. This example demonstrates that the method of the present invention can still yield products with satisfactory performance even with slight differences in raw material sources, by fine-tuning the enzyme ratio and reaction pH, reflecting the adaptability and controllability of the process.

[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a small peptide manganese feed additive to enhance the development of broiler chickens, characterized in that: The specific steps include the following: Step 1: Select zein as the precursor of small peptides and disperse it in deionized water to form a suspension with a mass concentration of 8%. Adjust the pH of the system to 5.

6. Add a compound enzyme preparation composed of alkaline protease and flavor protease at a temperature of 45℃ for synergistic enzymatic hydrolysis. The enzymatic hydrolysis reaction lasts for 3 hours, during which the stirring speed is maintained at 180 rpm. After the reaction is completed, raise the temperature to 95℃ and keep it at 10 minutes to inactivate the enzyme, and obtain a small peptide solution with a molecular weight distribution concentrated in the range of 500 to 1200 Daltons. Step 2: Dissolve manganese sulfate in deionized water to prepare a manganese ion solution with a molar concentration of 0.4 mol / L. Slowly add the solution to the small peptide under a nitrogen protective atmosphere, control the molar ratio of small peptide carboxyl groups to manganese ions to be 3:1, adjust the pH of the mixture to 6.2, and react at a constant temperature of 50°C for 4 hours under light-protected conditions with shaking, so that manganese ions can directionally chelate with the carboxyl and amino groups of the small peptide side chain to generate a small peptide manganese chelate precursor. Step 3: The precursor solution is subjected to ultrafiltration using a hollow fiber membrane module with a molecular weight cutoff of 1000 Daltons. The operating pressure is set to 0.25 MPa, and the dialysate volume is continuously replaced up to 5 times the original volume to remove unchelated free manganese ions and low molecular weight impurities. The retentate is then collected. Step 4: The retentate is concentrated to 1 / 3 of its original volume by rotary evaporation, followed by spray drying to obtain a light yellow powdery small peptide manganese feed additive final product with good flowability. Step 5: Characterize the quality of the final product. Determine the chelation rate by UV-Vis spectrophotometry, analyze the molecular weight distribution by high-performance gel permeation chromatography, and determine the total manganese content and soluble manganese release curve by inductively coupled plasma mass spectrometry to confirm that the product meets the set standards.

2. The preparation method of the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: In step 1, the initial particle size of the zein protein is controlled to be below 80 micrometers after ball milling pretreatment, and the mass ratio of alkaline protease to flavor protease used is 2:1, with the total enzyme addition accounting for 1.2% of the substrate mass.

3. The preparation method of the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: The molecular weight distribution of the small peptides in the enzymatic hydrolysis products in step 1 meets the following requirements: the proportion of components with a molecular weight of less than 700 Daltons is not less than 45%, the proportion of components with a molecular weight between 700 and 1200 Daltons is not more than 55%, and the overall polydispersity index is less than 1.

15.

4. The preparation method of the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: In step 2, the manganese ion solution is added at a rate of 2 ml per minute for 40 minutes, and the dissolved oxygen concentration in the reaction system is maintained below 2 mg / L.

5. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: After the chelation reaction in step 2 is completed, the resulting solution is centrifuged at 4000 rpm for 10 minutes to remove trace amounts of insoluble matter, and the supernatant is used for subsequent purification steps.

6. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: In step 3, the manganese ion concentration of the permeate is measured every 30 minutes during the ultrafiltration process. When two consecutive measurements are both below 0.01 mg / L, the dialysis endpoint is determined.

7. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: In step 4, the moisture content of the powder obtained by spray drying is controlled to be less than 5%, the loose packing density is 0.45 g / mL to 0.55 g / mL, and the angle of repose is less than 40°; the inlet air temperature of the drying process is controlled to be 160°C, the outlet air temperature is 85°C, and the atomizer speed is 25,000 rpm.

8. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: The formula for calculating the chelation rate in step 5 is: Chelation rate = (Total manganese content − Extractable free manganese content) / Total manganese content × 100%, where the extractable free manganese content is determined after separation by trichloroacetic acid precipitation, and the final product chelation rate is required to be greater than or equal to 92%.

9. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: In step 5, the determination of soluble manganese release behavior was carried out using a method simulating the foregut digestive environment of broiler chickens. 0.1g of the additive sample was placed in 10mL of simulated digestive fluid, which consisted of 0.2% pepsin, 0.1% trypsin, and 0.3% bile salts. The pH gradient was set to gradually increase from 5.8 to 6.

8. Samples were taken every 30 minutes, filtered through a 0.22-micron filter membrane, and the manganese concentration in the filtrate was measured. A cumulative release curve was plotted, requiring a release rate of 60% to 75% within the first 2 hours and complete release within 4 hours.

10. The method for preparing the small peptide manganese feed additive for enhancing the development of broiler chickens according to claim 1, characterized in that: This also includes establishing an online quality monitoring system based on near-infrared spectroscopy, collecting spectral data from different batches of products, and constructing a partial least squares regression model to achieve rapid prediction of key quality attributes such as chelation rate, moisture content, and particle size distribution. The model cross-validation coefficient of determination is also included. A value greater than 0.95 indicates a prediction error of less than 3%.

Images