Micro-ecological fermented feed suitable for piglets and preparation method of micro-ecological fermented feed

Abstract

The invention discloses a micro-ecological fermented feed suitable for piglets and a preparation method of the micro-ecological fermented feed, and belongs to the technical field of functional feeds. The preparation method comprises the following steps: 1) preparing functional hydrolysate rich in arginine and branched chain amino acid from plant raw materials through composite enzymolysis; (2) inoculating a functional bacterial agent consisting of lactobacillus plantarum with high arginase activity, nitrate reductase positive bacillus subtilis and clostridium butyricum; (3) accurately controlling the fermentation process by taking the molar ratio of arginine to citrulline being 0.8-1.5 as a fermentation end point, directionally enriching nitric oxide precursors such as citrulline in the feed, and constructing an endogenous NO slow-release system; the product can safely and efficiently activate NO metabolism of intestinal tracts of piglets, synergistically improve intestinal barrier, flora balance, digestive function and whole body immunity, significantly increase daily gain, reduce diarrhea rate, replace zinc oxide and antibiotics, and realize green and healthy breeding.

Description

Technical Field

[0001] This invention relates to the field of functional feed technology, and in particular to a microecological fermented feed suitable for piglets and its preparation method. Background Technology

[0002] The weaning period for piglets is a critical bottleneck stage in pig production. The sudden transition from liquid sow's milk to solid feed, accompanied by drastic psychological, environmental, and nutritional changes, triggers typical weaning stress syndrome, mainly manifested as: decreased feed intake, insufficient digestive enzyme secretion, intestinal villus atrophy, crypt hyperplasia, impaired tight junction barrier, intestinal flora imbalance (reduction of beneficial bacteria such as lactobacilli and proliferation of opportunistic pathogens such as Escherichia coli), and systemic inflammatory response. These pathophysiological changes collectively lead to impaired nutrient digestion and absorption, high incidence of diarrhea, and severe growth retardation.

[0003] Traditional animal husbandry has long relied on high doses of zinc oxide (ZnO, often 2000-3000 mg / kg) or antibiotics to prevent post-weaning diarrhea. While zinc oxide has astringent and antibacterial effects, long-term use has many drawbacks, including environmental pollution from zinc emissions, induction of bacterial resistance, interference with the absorption of other trace elements such as copper and iron, and potential damage to gut microbiota diversity. Meanwhile, the use of antibiotics to promote growth is widely restricted globally due to the superbug crisis. Therefore, developing safe and efficient alternatives is an urgent need for the industry.

[0004] Microbial fermented feed is considered a promising alternative. Through fermentation by probiotics, it produces organic acids, digestive enzymes, and vitamins, which can improve palatability and reduce anti-nutritional factors to some extent. However, existing conventional fermented feeds have significant limitations: First, their mode of action is relatively superficial and singular, mainly relying on the colonization competition and acid production to inhibit bacteria, lacking sufficient ability to regulate the host's complex and critical physiological signaling pathways; second, the fermentation process is uncontrollable, easily leading to excessive consumption of valuable functional amino acids (such as arginine and lysine) by microorganisms, resulting in nutrient loss, making it difficult to balance functionality and nutrition.

[0005] In recent years, animal physiological studies have revealed that the gaseous signaling molecule nitric oxide (NO) plays a central role in maintaining gastrointestinal health. Physiological concentrations of NO can dilate intestinal mucosal blood vessels, increase blood flow, promote mucus secretion, maintain epithelial cell integrity, regulate immune responses, and affect intestinal motility. However, NO exhibits a strictly biphasic effect: excessively high concentrations or improperly located NO can produce cytotoxicity. Therefore, how to safely, precisely, and persistently deliver physiological levels of NO locally to the intestines of piglets has become a valuable but challenging scientific problem.

[0006] L-arginine is a classic substrate for NO synthesis in mammals, and certain lactic acid bacteria (such as certain *Lactobacillus plantarum* species) possess an active arginine deiminase (ADI) pathway, capable of converting arginine into citrulline for energy. Citrulline is non-toxic to animals and can be efficiently recycled back to arginine in the intestines and kidneys via enterohepatic circulation, thus providing a stable and controllable substrate supply for the host's own nitric oxide synthase (NOS). This reveals a novel pathway for indirectly and slowly regulating host NO levels through microbial metabolism. However, how to translate this intricate microbial physiological mechanism into a stable and controllable industrial feed production technology, while ensuring the basic nutritional balance of the feed while achieving its function, has not been well-reported both domestically and internationally. Summary of the Invention

[0007] The fundamental objective of this invention is to overcome three major technical shortcomings of existing piglet feeds in addressing weaning stress syndrome: limited options, superficial mechanisms, and a disconnect between nutrition and function. It aims to provide a systematic solution based on the host-microbe co-metabolism theory. Traditional solutions, such as high zinc and antibiotics, are antagonistic treatments that disrupt the ecosystem and are unsustainable; ordinary microecological feeds offer supplementary support with limited effectiveness. This invention aims to achieve regulatory repair, that is, to actively and precisely regulate core physiological signaling pathways in the piglet gut (such as the nitric oxide pathway) through feed as a carrier, while simultaneously providing optimal nutrition, thereby fundamentally enhancing intestinal resilience and growth drive.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A microecological fermented feed suitable for piglets, comprising the following components by weight percentage prepared through a specific process: 20%-35% plant-derived amino acid donors: The plant-derived amino acid donors are composed of arginine donors and branched-chain amino acid donors in a weight ratio of (10-25):(5-15); 60%-75% of the basic energy carrier: The basic energy carrier is selected from one or more of puffed corn, puffed rice, and wheat bran; 2%-5% of compound functional fermentation agent: The compound functional fermentation agent contains Lactobacillus plantarum with arginase activity ≥250U / mg protein, Bacillus subtilis with nitrate reductase positive and Clostridium butyricum. Appropriate amount of post-fermentation nutrient preservation agent: The post-fermentation nutrient preservation agent is a coated amino acid [various amino acid particles coated with polymer membrane materials (such as carrageenan, etc.), such as coated leucine, coated citrulline or coated arginine, etc., used to supplement or adjust the ratio of various amino acids in the raw materials]. If the fermentation products of the raw materials (including plant-derived amino acid donors, basic energy carriers and compound functional fermentation agents) have already met the requirements, then it is not necessary to add the coated amino acid.

[0009] Preferably, the arginine donor is selected from the enzymatic hydrolysis products of pumpkin seed meal, watermelon seed meal or seaweed powder, and its addition amount accounts for 10%-25% of the total weight of the raw materials; the branched-chain amino acid donor is selected from the enzymatic hydrolysis products of hemp seed protein or lupin powder, and its addition amount accounts for 5%-15% of the total weight of the raw materials.

[0010] Furthermore, the enzymatic hydrolysis products of the above-mentioned plant materials are obtained by synergistic enzymatic hydrolysis of the plant materials using neutral protease and cellulase, under the following conditions: temperature 45-55℃, time 3-5 hours, pH 6.5-7.5.

[0011] Preferably, in the compound functional fermentation agent, the ratio of live bacteria of Lactobacillus plantarum, Bacillus subtilis and Clostridium butyricum is (1.5-3.0):(1.0-2.0):(0.5-1.5).

[0012] Preferably, the amount of coating amino acid added is compensated based on the amino acid profile detection results of the fermented product, so that the total arginine content in the final product is ≥1.5%, the effective lysine content is ≥1.3%, and the total branched-chain amino acid content is ≥3.2%; in the microecological fermented feed suitable for piglets, the molar ratio (Arg / CitRatio) of arginine to citrulline is 0.8-1.5.

[0013] This invention also proposes a method for preparing a microecological fermented feed suitable for piglets, comprising the following steps: S1. Preparation of plant-derived amino acid donors: After crushing the plant raw materials, they are subjected to compound enzymatic hydrolysis or pre-fermentation treatment to obtain plant protein hydrolysates rich in small peptides and free amino acids; S2. Mixing and Inoculation: Mix the plant protein hydrolysate and basic energy carrier evenly, then inoculate with the compound functional fermentation agent, mix evenly and adjust the moisture content of the material to 36%-42%; S3. Step-by-step controlled fermentation: The mixture is placed in a fermentation device and fermented sequentially: First stage aerobic fermentation: control the temperature at 32-37℃ and the relative humidity at 80%-90%, for 12-24 hours; The second stage of anaerobic primary fermentation: control the temperature at 36-40℃ and the relative humidity at 75%-85% for 36-72 hours. S4. Termination of Fermentation and Drying: During the second stage of fermentation, monitor the content of arginine and citrulline in the material. When the molar ratio (Arg / Cit Ratio) reaches 0.8-1.5, terminate the fermentation. Dry the fermentation product at a temperature not exceeding 45℃ until the moisture content is ≤12%. S5. Nutritional Compensation and Finished Product Formulation: The amino acid profile of the dried material is detected, the difference is calculated based on the predetermined target amino acid content, and the corresponding amount of coated amino acids is uniformly mixed in for compensation; finally, the material is crushed and packaged to obtain the finished product.

[0014] Preferably, the S1. compound enzymatic hydrolysis prepared from plant-derived amino acid donors includes the following steps: Step 1. Raw material selection and pretreatment: Raw material proportioning and weighing: Accurately weigh the dried plant materials according to the formula requirements. For example, a typical proportion might be: 70 parts pumpkin seed meal and 30 parts hemp seed protein (by weight). This ratio aims to balance the supply of arginine and branched-chain amino acids. All raw materials must be free of mold, have few impurities, and a moisture content of less than 12%.

[0015] Primary grinding: The raw materials are ground using a hammer mill and passed through a 40-60 mesh sieve. Fine particle size significantly increases the contact area between the enzyme and the substrate, improving the efficiency of subsequent enzymatic hydrolysis.

[0016] Step 2. Complex enzymatic hydrolysis treatment: This step employs a phased, multi-enzyme synergistic strategy to maximize hydrolysis efficiency and produce products with an ideal molecular weight distribution. Preparation of enzymatic hydrolysis substrate slurry: Put the pulverized raw materials into an enzymatic hydrolysis reaction tank equipped with stirring and temperature control, add pure water at 50-55℃, and adjust the material-to-water ratio to 1:3 to 1:4 (by weight). This water content provides a good reaction medium for the enzyme and facilitates material flow. The first stage is enzymatic hydrolysis to break down the fiber structure: Cellulase (enzyme activity ≥10,000 U / g) is added at a rate of 0.1%-0.3% of the dry weight of the raw material; the pH is adjusted to 4.5-5.5 (using food-grade citric acid or lactic acid), which is the optimal pH range for this enzyme; the reaction is carried out at 50-55℃ with continuous stirring for 1.5-2.5 hours; the purpose is to destroy the plant cell wall structure, degrade cellulose and hemicellulose, release the encapsulated proteins, and generate some prebiotic oligosaccharides; Second stage enzymatic hydrolysis. Deep protein hydrolysis: No material replacement is required; proceed directly to the second stage. Use a compound protease, preferably a neutral protease (enzyme activity ≥ 50,000 U / g) and a flavor protease (enzyme activity ≥ 20,000 U / g) in a weight ratio of approximately 3:1. The total amount of compound protease added is 0.8%-1.5% of the dry weight of the raw materials. Adjust the pH of the materials to 6.5-7.0 (the optimal range for neutral protease) with dilute alkali (such as NaOH solution), and continuously stir the reaction at 50-55℃ for 3.5-4.5 hours. The purpose is for the neutral protease to cleave the protein molecules from within, rapidly producing a large number of medium-length peptides; and for the flavor protease to cleave the peptide chains from the ends, further generating small peptides (dipeptides, tripeptides) and free amino acids, and effectively hydrolyzing bitter peptides to improve the flavor of the product.

[0017] Enzymatic hydrolysis monitoring: Samples were taken every hour, and the protein conversion rate was determined using the trichloroacetic acid dissolution method. The ideal hydrolysis endpoint was defined as when the degree of hydrolysis reached 25%-35% and the proportion of small peptides (molecular weight less than 1000 Da) in the total soluble protein exceeded 50%.

[0018] Step 3. Enzyme inactivation and product standardization: Enzyme inactivation and sterilization: After the enzymatic hydrolysis reaction reaches its endpoint, immediately raise the material temperature to 85-90℃ and maintain it for 15-20 minutes; this process can completely inactivate enzyme activity, terminate the reaction, prevent excessive hydrolysis, and at the same time kill the raw materials and miscellaneous bacteria introduced during the process, ensuring the biosafety of the product. Solid-liquid separation and concentration: The enzyme-inactivated slurry is separated into solid and liquid components by a plate and frame filter press or centrifuge to remove unhydrolyzed fibers, residues and other insoluble substances; the filtrate rich in small peptides and amino acids is collected and transferred to a vacuum concentration tank; Concentration was carried out at 60-65℃ and a vacuum of -0.08 to -0.09 MPa until the soluble solids content reached 30%-40% (by refractive index). Concentration aimed to reduce volume for easier storage, transportation, and uniform mixing with subsequent fermentation substrates. Quality control indicators for the final product: Sensory characteristics: A brownish-yellow to brown homogeneous slurry with a rich roasted aroma and umami flavor, without bitterness or rancidity; Physicochemical properties: Small peptide (molecular weight < 1000 Da) content: ≥ 50% of total protein; Total free amino acids: accounting for 15%-25% of dry matter; of which, arginine accounts for no less than 10% of free amino acids; the total proportion of branched-chain amino acids (leucine, isocyanate, valine) is no less than 18%; Dry matter content: 30%-40%; pH value: 5.0-6.0.

[0019] Preferably, in step S4, the contents of arginine and citrulline are monitored online or offline using high-performance liquid chromatography.

[0020] Preferably, the finished product obtained in step S5 must simultaneously meet all of the following key indicators before leaving the factory: a) Functional activity index: Total viable count ≥ 1.0 × 10⁻⁶ 9 CFU / g; Citrulline content ≥1.8mg / g; Molar ratio of arginine to citrulline 0.8-1.5; b) Core nutritional indicators: Total arginine content ≥1.5%; Available lysine content ≥1.3%; Total branched-chain amino acid content ≥3.2%; Phytase activity ≥500U / kg; c) Health and safety indicators: aflatoxin B1 ≤ 10 μg / kg, Salmonella not detectable.

[0021] Preferably, the total arginine and total branched-chain amino acid content are determined using an amino acid analyzer; the effective lysine content is determined using a dye-binding method.

[0022] In addition, the present invention also proposes the application of the aforementioned microecological fermented feed suitable for piglets, which is added to the piglet's complete daily dry matter diet at a ratio of 3% to 10% and mixed evenly before use to improve the intestinal health of weaned piglets and promote growth.

[0023] The formulation and process mechanism of this invention: 1. Plant-derived targeted amino acid donor system: 1) Arginine donor: Pumpkin seed meal / watermelon seed meal enzymatic or fermented hydrolysate: includes active peptides. Enzymatic or fermented hydrolysis can release peptides with angiotensin-converting enzyme inhibitory activity and significantly improve protein solubility, emulsification and oil holding capacity. Seaweed powder enzymatic or fermented hydrolysates: seaweed polysaccharides, bioactive peptides, and minerals: Enzymatic hydrolysis enhances the bioactivity of its physiologically active substances and produces beneficial nutrients. Seaweed protein hydrolysates have been proven to have excellent antioxidant capacity, while the seaweed polysaccharides in seaweed powder act as prebiotics and mild immunomodulators, helping to cultivate a healthy gut microbiota and immune environment.

[0024] The above substances can serve as core NO precursor donors: for example, pumpkin seed meal with high arginine content (more than 17% of protein) can be selected. Pumpkin seed meal itself contains a considerable proportion of citrulline and glutamine. Fermentation systems based on it can not only provide arginine substrates but also directly provide some metabolic intermediates and related products, making the microbial metabolic network richer and more efficient. In addition, its rich natural antioxidants (carotenoids, vitamin E) constitute an endogenous antioxidant defense line, which can neutralize the excess reactive oxygen species (ROS) that may be generated during NO metabolism, ensuring the safety of regulation, which is unmatched by synthetic arginine.

[0025] 2) Branched-chain amino acid donors: Hemp seed protein hydrolysate or fermentation hydrolysate: contains branched-chain amino acids and γ-aminobutyric acid (GABA): its hydrolysate is rich in branched-chain amino acids such as leucine, isoleucine and valine, and fermentation can significantly increase the content of GABA. Lupin flour enzymatic hydrolysate or fermentation hydrolysate: contains high-quality protein and bioactive peptides, and can produce peptides with ACE inhibitory activity after enzymatic hydrolysis.

[0026] The above substances can serve as protein synthesis switch donors: for example, hemp seed protein with an extremely high proportion of branched-chain amino acids (BCAAs), especially leucine, is selected. Leucine is the strongest nutrient signal that activates the mammalian target of sirolimus (mTOR) signaling pathway. Weaning stress often leads to the inhibition of the mTOR pathway, causing protein synthesis to stop. By continuously providing a high proportion of leucine in the diet, it is equivalent to continuously tapping the start switch of protein synthesis, effectively counteracting the catabolic state caused by weaning, and together with the improved microcirculation of the NO pathway, it provides the driving force for growth.

[0027] 2. Biological pretreatment process – release and activation: Simple physical pulverization cannot maximize the value of plant materials. This invention employs a compound enzymatic pretreatment (neutral protease synergistically cellulase). This step serves three purposes: Deep release: Target amino acids such as arginine and BCAA, which are enclosed in plant cell walls and complex protein structures, are hydrolyzed to the maximum extent into free or small peptide forms, greatly improving bioavailability.

[0028] Creating prebiotics: Cellulase degrades some insoluble fiber into soluble oligosaccharides, which are a high-quality carbon source for probiotics in subsequent fermentation and can promote their proliferation.

[0029] Reduce anti-nutritional factors: Pretreatment can simultaneously reduce the activity of substances such as trypsin inhibitors and tannins that may be present in the raw materials.

[0030] Through pretreatment, the original plant raw materials are transformed into a functional nutrient concentrate rich in small peptides, free amino acids, and prebiotics, providing a highly readily available and high-quality substrate for subsequent microbial fermentation. The enzymatic hydrolysis mechanism is reported in the following literature: Bioactive Peptides and Hydrolysates from Plant Sources: Nutraceutical Compounds, Applications, and Challenges A Review, Leila Najafian, Fatemeh Khaleghi, Journal of Mazandaran University of Medical Sciences, Jan 2025: 34-241, 159-178.

[0031] 3. Metabolic engineering construction of functional microbial agents: The transformation from probiotics to cell factories: the microbial agents of this invention are not simply a compound of commercially available probiotics, but a specialized engineered microbial community that is screened and combined for specific metabolic tasks.

[0032] 3.1 Screening and Functions of Highly Active Arginase-Promoting Bacteria (Lactobacillus plantarum): Lactic acid bacteria were isolated from the intestinal contents of healthy lactating piglets and initially screened using a restrictive culture medium with L-arginine as the sole nitrogen source. Further screening was conducted by quantitatively measuring the activities of key enzymes in the intracellular arginine deiminase (ADI) pathway (arginine deiminase and ornithine carbamoyltransferase).

[0033] The final product obtained was a complex of *Lactobacillus plantarum* bacteria with an intact ADI pathway and stable arginase activity above 250 U / mg protein (a polymorphic complex indicating the combined action of multiple bacteria). During the anaerobic fermentation stage, *Lactobacillus plantarum* became the dominant bacterium. It utilized the abundant free arginine in the plant hydrolysate, metabolizing it via the ADI pathway: L-arginine → L-citrulline + NH3. This process not only provided energy (ATP) for the bacterial cell's own growth but, more importantly, accumulated a large amount of the target product, L-citrulline, in the feed. This is a crucial step in constructing an in vitro NO precursor pool.

[0034] 3.2 Design and function of multifunctional engineered bacteria (Bacillus subtilis): Screening was conducted on Bacillus subtilis complex cells that exhibited strong nitrate reductase activity and high production of protease, amylase, and non-starch polysaccharide enzymes (such as xylanase). These complex cells were polymorphic, indicating the presence of multiple bacteria working together.

[0035] In the initial stage of fermentation (aerobic stage): the selected Bacillus subtilis multiplies rapidly, consuming a large amount of oxygen in the fermentation system and quickly creating an anaerobic environment conducive to the growth of lactic acid bacteria and Clostridium butyricum. At the same time, the various digestive enzymes secreted by Bacillus subtilis further break down feed macromolecules, working synergistically with enzymatic pretreatment to achieve super-predigestion of the feed.

[0036] Full-process function: Its nitrate reductase can reduce the trace amounts of nitrate naturally present in feed to nitrite, providing another potential pathway for NO generation (non-enzymatic NO generation under acidic conditions). The enzymes it produces can maintain a certain level of activity in the intestines after the feed is ingested, continuously aiding digestion.

[0037] The role of metabolite synergists (Clostridium butyricum): The core purpose of adding *Clostridium butyricum* is to produce butyrate. Butyrate is not only a preferred energy source for intestinal epithelial cells, but its more profound role lies in its epigenetic synergy with NO signaling. Studies have shown that butyrate, as a histone deacetylation inhibitor (HDACi), can open chromatin and promote the expression of multiple genes that maintain intestinal health, including nitric oxide synthase (eNOS). Therefore, the presence of butyrate in this invention not only directly nourishes the intestine but may also upregulate the host's intestinal ability to synthesize NO, producing a powerful synergistic amplification effect with the externally provided citrulline precursor.

[0038] 3.2 Fermentation-Nutrition Dynamic Balance Protection System: Fermentation inevitably involves the consumption of nutrients by microorganisms, and balancing functional transformation with nutrient retention has long been a challenge in the industry. This invention establishes a quantifiable and executable dynamic assurance system.

[0039] Before fermentation: Enhanced feeding model based on metabolic flux analysis: During formulation design, forward-looking calculations are performed. A target increment (ΔCit) of citrulline in the final product is set. Based on the average conversion efficiency (η, determined through preliminary experiments; for example, if 1 molecule of arginine is converted to 0.9 molecules of citrulline, then η = 0.9) of *Lactobacillus plantarum* under specific processes, the amount of dedicated arginine (ΔArg) required for fermentation is calculated backwards. ferment =ΔCit / η). This additional arginine requirement is achieved by increasing the amount of pumpkin seed meal hydrolysate in a moderate proportion in the basic formula, rather than simply adding synthetic amino acids, thus ensuring the consistency and naturalness of the raw material system.

[0040] During fermentation: endpoint determination based on stoichiometry: This invention abandons the traditional fixed fermentation time and innovatively introduces the arginine / citrulline molar ratio (Arg / Cit Ratio) as the core indicator for determining the fermentation endpoint.

[0041] The mechanism is as follows: In the early stages of fermentation, with the increased activity of *Lactobacillus plantarum*, arginine is rapidly consumed, citrulline accumulates rapidly, and the Arg / Cit Ratio decreases rapidly. When this ratio drops below 1.5, it indicates that the directional conversion of arginine is essentially complete. If fermentation continues, the microorganisms will begin to utilize the remaining amino acids as a common nitrogen source for cell proliferation, leading to unnecessary and non-selective amino acid loss.

[0042] Control strategy: Monitor this ratio in real time through online sampling or rapid testing. Fermentation is immediately terminated once it enters the preset 0.8-1.5 range. This range ensures that: ① citrulline production has reached the ideal level (the ratio is low enough); ② an adequate amount of arginine is still retained in the feed to meet nutritional needs as an important essential amino acid (the ratio is not too low). This is the key process control point for achieving functional and nutritional balance.

[0043] Post-fermentation: Targeted compensation mechanism based on precise detection: Even with the above controls, changes in the amino acid profile caused by fermentation still exist. Therefore, this invention adds an amino acid profile diagnosis and compensation step after fermentation and drying.

[0044] Diagnosis: The semi-finished product was analyzed using an amino acid analyzer to obtain a full spectrum of its components.

[0045] Compensation: The analysis results were compared with the target amino acid profile set according to the nutritional requirements model for piglets. For deficient essential amino acids, especially lysine and methionine, which may be significantly affected during fermentation, and glutamine, which has special intestinal nutritional functions, precise and quantitative supplementation was carried out using a stabilized coating form. The coating technology ensures that these supplemented amino acids can effectively pass through the acidic environment of the stomach and be absorbed in the intestine.

[0046] This step is for precise calibration, ensuring that the final product, while possessing powerful biological regulatory functions, fully meets or even exceeds the standards of high-quality piglet feed in terms of its basic nutritional indicators (amino acid balance), thus completely resolving the common concerns about nutritional imbalance in fermented feed.

[0047] 3.3 Multi-dimensional product quality control system: To ensure the stability and reliability of each batch of products produced by the aforementioned complex system, this invention establishes a quantitative quality control model consisting of nine key indicators, covering three dimensions of the product: 1) Functional activity dimension (proving its usefulness): Total viable bacteria count: fundamental to ensuring probiotic effects, required to be ≥1.0×10⁻⁶. 9 CFU / g.

[0048] Citrulline content: a quantitative representation of the core functional substance, requiring ≥1.8mg / g.

[0049] Arg / Cit molar ratio: Direct evidence of process control and nutritional balance, requiring a ratio between 0.8 and 1.5.

[0050] 2) Core nutritional dimensions (proving they are harmless and balanced): Total arginine, available lysine, and total branched-chain amino acids: Monitor the levels of key functional amino acids, first limiting amino acids, and anabolic amino acids respectively, and set specific lower limits (e.g., total arginine ≥ 1.5%).

[0051] Phytase activity: an indirect indicator of the ability to degrade anti-nutritional factors and release minerals during fermentation, requiring ≥500 U / kg.

[0052] 3) Hygiene and safety dimension (ensuring its safety): Strict testing for mycotoxins and pathogens is conducted, meeting or exceeding national standards.

[0053] The above indicators are interconnected and mutually verify each other. An abnormality in any indicator may indicate a problem in the upstream raw materials, process, or control links, thereby achieving true full-process traceability and controllable quality management.

[0054] This invention provides a composite functional carrier capable of simultaneously delivering probiotics (live bacteria), regulatory precursors (citrulline), nutrient substrates (small peptides, amino acids), and metabolic synergists (butyric acid) to the piglet intestine. Its mechanism of action can be summarized as follows: After piglets ingest feed, citrulline is absorbed into the enterohepatic circulation, continuously and gradually providing substrates for endogenous NO synthesis in the body (especially intestinal endothelial cells). The resulting physiologically adequate NO dilates intestinal blood vessels, improves mucosal blood flow, and enhances barrier function. Simultaneously, colonized *Lactobacillus plantarum* continuously and moderately metabolizes arginine in the intestine, providing local fine-tuning; butyric acid produced by *Clostridium butyricum* directly provides energy and may epigenetically upregulate host eNOS expression, synergistically enhancing NO; *Bacillus subtilis* and its produced enzymes continuously assist digestion. The entire system, together with optimized amino acid nutrition, creates an internal environment conducive to intestinal villus growth, beneficial bacteria proliferation, pathogen inhibition, and immune balance, thereby systematically overcoming weaning stress and achieving healthy and efficient growth.

[0055] In summary, this invention abandons the traditional approach of using a single additive as the active ingredient and pioneers an integrated design of ecological functional modules. Specifically, this invention treats feed as a programmable bioreactor and drug delivery system. By implanting a designed functional microbiome and a complex plant nutrient source during the feed production stage (in vitro) and controlling specific biochemical reactions, a series of regulatory metabolites (such as citrulline) are pre-synthesized. When piglets consume this feed, these products are released and exert their effects in the intestines (in vivo) according to a designed program, while the introduced probiotics colonize and continuously produce beneficial effects. The entire system constitutes a pre-programmed, slow-release, and regulatory chain from production to consumption, from in vitro to in vivo.

[0056] This invention embodies the application of systems biology principles in the feed industry. It is not a breakthrough in a single technology, but rather a design and implementation of an efficient, stable, and safe overall platform for regulating gut health through interdisciplinary integrated innovation.

[0057] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes screened Lactobacillus plantarum with high arginase activity to directionally convert plant-derived L-arginine into L-citrulline during fermentation, and then enriches it in feed, constructing a safe and efficient endogenous nitric oxide (NO) sustained-release system to improve intestinal health from the signaling source. After piglets consume the feed, citrulline is absorbed into the enterohepatic circulation and can be continuously regenerated into arginine in intestinal and kidney cells, thus providing a persistent and stable substrate for the host's own nitric oxide synthase (NOS). This avoids the toxic side effects of exogenous NO donors and achieves physiological, sustained-release, and localized NO supply.

[0058] 2. The physiological NO produced by the sustained-release system of this invention can effectively dilate blood vessels in the intestinal submucosa, improving villous microcirculation and oxygen supply. Simultaneously, the butyric acid produced during fermentation (reaching 18.7 μmol / g in the cecum of the IP-B group) synergistically interacts with NO signaling: butyric acid is the primary energy source for intestinal epithelial cells and can upregulate tight junction protein expression through epigenetic mechanisms. The synergistic effect of NO and butyric acid jointly promotes epithelial cell proliferation and repair, significantly improving intestinal structure and function, and synergistically enhancing digestion and barrier protection capabilities.

[0059] 3. The fermented feed of this invention is rich in active probiotics (IP-B component has a live bacteria count ≥ 4.8 × 10⁻⁶). 9 (CFU / g). After entering the intestines, it works together with the prebiotic components in the feed (derived from the enzymatic hydrolysis products of plant raw materials), and the organic acids (lactic acid, butyric acid) produced by fermentation rapidly lower the intestinal pH, creating an acidic environment that is unfavorable to pathogens and favorable to the colonization of lactic acid bacteria and Clostridium butyricum; it can optimize the intestinal microecological environment and create a healthy flora dominated by probiotics.

[0060] Unlike traditional zinc oxide, which inhibits gut microbiota, the lactic acid bacteria count in the cecal digesta of piglets fed with the feed of this invention was 9.5 × 10⁻⁶. 7 The CFU / g level was significantly increased, while the number of E. coli was significantly suppressed. The cecal pH level dropped to a healthy 5.8, and the total short-chain fatty acid content increased significantly. This healthy gut microbiota structure is the foundation for maintaining long-term intestinal stability and resisting pathogen colonization, and is also the reason for the sustained and stable effects of this invention.

[0061] 4. Physiological NO has anti-inflammatory and immunomodulatory effects. A healthy intestinal barrier can effectively prevent endotoxin translocation and reduce systemic inflammation. In addition, the plant raw materials selected in this invention (such as pumpkin seed meal) are rich in natural antioxidants (carotenoids and vitamin E), which, together with fermentation metabolites, enhance the body's antioxidant reserves, providing a healthy physiological state of immune balance and low oxidative stress. This lays the foundation for efficient growth, ultimately manifested as a significant increase in average daily weight gain (408g), ensuring the overall health and growth potential of the animals.

[0062] 5. In summary, this invention, through the combined approach of plant nutrient precursor design, functional microbial engineering, precise fermentation control, and dynamic nutritional support, has successfully developed a functional feed that not only provides nutrition but also actively regulates core physiological signals in the piglet gut and reshapes a healthy microecology. Its effects have been rigorously verified through experiments, demonstrating significant superiority over traditional zinc oxide solutions and ordinary fermented feeds in multiple dimensions, including growth performance, gut health, and immune regulation. It possesses broad clinical application prospects and substantial market value. Detailed Implementation

[0063] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0064] Example Group I. Validation Experiment of Plant-Derived Amino Acid Donor Formulation Range: This experiment aims to precisely verify the optimal addition range of the total proportion of plant-derived amino acid donors (20%-35%) and their core components, arginine donors (10%-25%) and branched-chain amino acid (BCAA) donors (5%-15%).

[0065] 1.1 Preparation of concentrated plant protein hydrolysate (PHC): raw material: Defatted pumpkin seed meal: crude protein ≥50%, moisture ≤10%, arginine ≥16% of protein.

[0066] Defatted hemp seed protein powder: crude protein ≥45%, moisture ≤10%, total branched-chain amino acids (leucine, isoleucine, valine) account for ≥18% of the protein.

[0067] Enzyme preparations: food-grade cellulase (enzyme activity ≥100,000 U / g), neutral protease (enzyme activity ≥50,000 U / g), flavor protease (enzyme activity ≥20,000 U / g).

[0068] Preparation equipment: hammer mill, 1000L jacketed heated and stirred reactor, plate and frame filter, vacuum concentrator, refractometer, pH meter.

[0069] Detailed preparation steps: Grinding: Weigh the pumpkin seed meal and hemp seed protein powder separately according to the experimental design ratio (see Table 1), mix them, and then grind them using a hammer mill. The mixture is then passed through a 60-mesh standard sieve to obtain the mixed powder.

[0070] Preparation of slurry: Add the mixed powder into the reactor and start stirring. Add purified water at 55±1℃, and control the material-to-water ratio (by weight) to be 1:3.5.

[0071] First stage enzymatic hydrolysis (cell wall disruption): After the temperature stabilizes at 53±1℃, add 0.2% (by dry weight) of cellulase from the mixed powder. Adjust the pH of the system to 5.0±0.1 using a 10% edible citric acid solution. Under these conditions, maintain a constant temperature and stir for 120 minutes.

[0072] Second stage enzymatic hydrolysis (protein hydrolysis): Maintain the temperature and adjust the pH of the system to 6.8±0.1 using 5% sodium hydroxide solution. Add 1.0% (by dry weight) of the mixed powder of a compound protease (neutral protease and flavor protease premixed at a weight ratio of 3:1). Continue the reaction with constant temperature and stirring for 240 minutes. During the reaction, samples were taken at 60, 120, 180, and 240 minutes, and the degree of hydrolysis was determined using the ninhydrin method.

[0073] Enzyme inactivation and solid-liquid separation: When the degree of hydrolysis reaches 30±2%, steam is rapidly introduced to raise the temperature of the feed liquid to 90℃ within 15 minutes, and maintained for 20 minutes for enzyme inactivation and pasteurization. Subsequently, the feed liquid is pumped into a plate and frame filter press to separate the filtrate and residue.

[0074] Concentration: Collect all filtrate and transfer it to a vacuum concentrator. Concentrate at 65±2℃ and a vacuum of -0.085 to -0.090 MPa. Monitor the soluble solids content online using an Abbe refractometer. Stop concentration when the soluble solids content reaches 35±1%, yielding the final product—plant protein hydrolysate concentrate (PHC).

[0075] Quality control: Each batch of PHC samples is taken, and the free amino acid composition is determined by high performance liquid chromatography (HPLC). The content of small peptides (molecular weight <1000 Da) is determined by trichloroacetic acid precipitation-biuret method. Small peptides must account for >50% of total soluble protein, arginine >10% of free amino acids, and the total branched-chain amino acids >18%.

[0076] 1.2 Animal feeding experiment design: Experimental animals and grouping: 216 healthy Duroc × Landrace × Large White crossbred piglets of uniform weight (8.0±0.5 kg) weaned at 28 days of age were selected. They were randomly divided into 6 treatment groups using a completely randomized block design, with 6 replicates (pens) per group and 6 piglets per replicate. The treatment groups were designed based on the total proportion and composition of plant donors in the PHC as follows: Table 1. Group design of plant-derived donor formulation experiments (percentage of dry fermentation substrate) serial number Example Total proportion of plant-derived donors Arginine donor (pumpkin seed meal) ratio Branched-chain amino acid donor (cannabis seed protein) ratio Basic energy carrier ratio E1 Example 1 20% 10% 5% 75% E2 Example 2 25% 15% 8% 70% E3 Example 3 30% 20% 10% 65% C1 Comparative Example 1 15% 8% 2% 80% C2 Comparative Example 2 40% 28% 18% 55% BD Control Group I 0% 0% 0% 95% It should be noted that control group I was fed only with a basal diet for basic nutritional support, and the proportion of basal energy carriers was adjusted to ensure that the diets of each group were equal in nitrogen and energy.

[0077] Diet and Feeding Management: All groups were fed a corn-soybean meal basal diet (meeting the nutritional requirements of NRC, 2012, for piglets weighing 10-20 kg). Each treatment group had 5% of their basal diet replaced with the corresponding formula of PHC (on a dry matter basis), and the diet was remixed. The experiment lasted 21 days. Piglets had free access to pelleted feed and water, and the amount of feed given and leftover feed was recorded daily. Fecal condition was observed daily, and a 0-4 scale was used to score feces (0-1 normal, 2 mild soft stool, 3 diarrhea, 4 watery diarrhea). A score ≥2 was considered diarrhea. Piglets were weighed individually on an empty stomach in the morning of days 1 and 22 of the experiment.

[0078] 1.3 Sample collection and testing methods: At the end of the experiment, 6 piglets with a weight close to the group average were selected from each group. 10 mL of blood was collected from the anterior vena cava, centrifuged at 3000 rpm for 10 minutes after standing, and the serum was separated and stored at -20℃. The piglets were then anesthetized and slaughtered.

[0079] Intestinal morphology: Immediately, approximately 2 cm segments of the middle duodenum, middle jejunum (2 meters from the pylorus), and middle ileum (1 meter from the ileocecal valve) were harvested. The contents were gently rinsed with pre-cooled physiological saline and then fixed in 4% paraformaldehyde phosphate buffer for 24 hours. Routine paraffin embedding, sectioning (5 μm), and hematoxylin-eosin (H&E) staining were performed. Under an optical microscope, image analysis software was used to randomly measure the height of 10 intact villi and the depth of their corresponding crypts on each section, calculating the average value and the villus-crypt ratio (V / C).

[0080] Serum biochemistry: Serum total protein (TP, biuret method) and blood urea nitrogen (BUN, urease-Bohrer colorimetric method) were measured using a fully automated biochemical analyzer.

[0081] Table 2. Effects of different plant donor ratios on growth performance, intestinal morphology, and protein metabolism in weaned piglets. Group Average daily weight gain (ADG, g) Material weight ratio (F / G) Jejunal villus height (μm) Jejunal crypt depth (μm) jejunal villous-to-crystal ratio (V / C) Serum total protein (g / L) Serum urea nitrogen (mmol / L) BD <![CDATA[305±18 d ]]> <![CDATA[1.62±0.05 a ]]> <![CDATA[320±28 d ]]> <![CDATA[250±23 a ]]> <![CDATA[1.28±0.11 d ]]> <![CDATA[47.5±2.4 c ]]> <![CDATA[6.0±0.5 a ]]> C1 <![CDATA[338±20 c ]]> <![CDATA[1.56±0.04 b ]]> <![CDATA[355±30 c ]]> <![CDATA[245±22 a ]]> <![CDATA[1.45±0.12 c ]]> <![CDATA[49.8±2.6b c ]]> <![CDATA[5.6±0.5 ab ]]> E1 <![CDATA[372±22 b ]]> <![CDATA[1.49±0.04 c ]]> <![CDATA[388±32 b ]]> <![CDATA[231±21 ab ]]> <![CDATA[1.68±0.14 b ]]> <![CDATA[52.1±2.8 b ]]> <![CDATA[5.0±0.4 bc ]]> E2 <![CDATA[385±23 ab ]]> <![CDATA[1.47±0.03 c ]]> <![CDATA[402±34 ab ]]> <![CDATA[230±20 ab ]]> <![CDATA[1.75±0.15 ab ]]> <![CDATA[53.8±2.9 ab ]]> <![CDATA[4.7±0.4 c ]]> E3 <![CDATA[390±24 a ]]> <![CDATA[1.46±0.03 c ]]> <![CDATA[410±35 a ]]> <![CDATA[228±20 b ]]> <![CDATA[1.80±0.16 a ]]> <![CDATA[54.5±3.0 a ]]> <![CDATA[4.5±0.4 c ]]> C2 <![CDATA[370±22 b ]]> <![CDATA[1.52±0.04 b ]]> <![CDATA[395±33 ab ]]> <![CDATA[232±21 ab ]]> <![CDATA[1.70±0.15 b ]]> <![CDATA[53.0±2.8 ab ]]> <![CDATA[5.2±0.5 b ]]> It should be noted that in Table 2, different letters in the superscript of the same row indicate significant differences (P < 0.05), while the same letter indicates no significant differences. The same applies to the following tables, and the explanation will not be repeated.

[0082] As shown in Table 2: The optimal performance range: Groups E1, E2, and E3 significantly outperformed comparative groups C1, C2, and BD in terms of average daily weight gain, feed efficiency, jejunal villus development, and protein deposition efficiency (high TP, low BUN) (P < 0.05). Among them, groups E2 and E3 showed the most outstanding effects, indicating that a total plant-derived donor ratio of 25-30%, arginine donor ratio of 15-20%, and branched-chain amino acid donor ratio of 8-10% represents the preferred implementation range of this invention.

[0083] Although the C1 group performed better than the BD group in all indicators, it was significantly worse than the E1 group. This indicates that when the supply of plant-derived functional amino acids is insufficient, it is impossible to provide sufficient precursor substances for subsequent fermentation and host physiology, resulting in limited growth promotion and intestinal repair effects, which proves the necessity of setting a lower limit (20%).

[0084] The ADG and intestinal morphology indicators of group C2 were comparable to or slightly better than those of group E1, but significantly worse than those of group E2 / E3. Crucially, their feed conversion ratio was significantly higher than that of group E2 / E3. Analysis showed that the excessively high proportion of plant protein (40%) led to a relative decrease in dietary energy concentration, disrupting the optimal energy-protein ratio. Although the total amount of functional components increased, the overall feeding efficiency did not improve; in fact, it may have caused a rebound in BUN due to increased metabolic burden. This result clearly demonstrates the rationality of the upper limit of the proportion (35%), namely, that while ensuring functional intake, the overall nutritional balance of the diet must be maintained.

[0085] Example Group II. Validation Experiment on Functional Microbial Agent Ratio and Fermentation Endpoint Control: This experiment aims to verify the scientific validity and key role of the functional microbial agent formulation and the core control parameter of the fermentation endpoint, the arginine / citrulline molar ratio (Arg / Cit Ratio) range (0.8-1.5).

[0086] 2.1 Preparation of Fermented Feed: Basic substrate: PHC (25% total proportion of plant-derived donors) from the E2 formulation in Example Group I was uniformly used as one of the fermentation substrate components.

[0087] bacterial strains: Lactobacillus plantarum (customized by Xi'an Musen Biotechnology Co., Ltd., arginase activity 265U / mg protein).

[0088] Bacillus subtilis (custom-made by Xi'an Musen Bioengineering Co., Ltd., nitrate reductase positive, with strong protease activity).

[0089] Clostridium butyricum (purchased from Jinan Zhiheng Zhiyuan Chemical Technology Co., Ltd., purity > 99%).

[0090] Ordinary commercial compound probiotic powder (Nortrand compound active probiotic powder, as a control, contains multiple unlabeled strains).

[0091] Detailed preparation steps: Activation of bacterial agents and preparation of inoculum: Weigh the freeze-dried bacterial powder required for each experimental group according to the design ratio (see Table 3), activate it with 20 times its weight of 37℃ sterile physiological saline for 30 minutes to prepare inoculum suspension.

[0092] Mixing and conditioning: PHC (25% dry weight), puffed corn (60%), puffed rice (10%), wheat bran (4.5%), and dicalcium phosphate (0.5%) were mixed in a horizontal mixer for 15 minutes. The activated bacterial solution was evenly sprayed into the material, and purified water was added simultaneously. The moisture content of the material was monitored using a rapid moisture analyzer until it was stabilized at 39±1%.

[0093] Step fermentation: Aerobic stage: Spread the material evenly in a constant temperature and humidity fermentation chamber with a thickness of 30cm, control the temperature at 35±1℃ and the ambient humidity at 85±5%, and let it ferment for 20 hours.

[0094] Anaerobic primary fermentation: Transfer the material to a sealed fermenter and compact it appropriately. Control the temperature at 38±1℃ and the ambient humidity at 80±5%. Key control operations: Starting from the 24th hour of fermentation, take approximately 50g samples from different parts of the tank every 6 hours using a sterile sampler. Immediately flash-freeze the samples with liquid nitrogen and send them to the laboratory for quantitative analysis of arginine and citrulline content using high-performance liquid chromatography (HPLC) within 2 hours, and calculate the Arg / Cit Ratio.

[0095] Fermentation termination and drying: When the Arg / Cit Ratio of the sample reaches the preset target value of each group (see Table 3), all the material in the corresponding fermentation tank is immediately discharged and sent to a belt dryer at 45±2℃ for continuous drying until the moisture content of the material is ≤12%.

[0096] Finished product analysis: The dried material was pulverized and passed through a 20-mesh sieve. Sampling and determination: total viable count (GB 4789.35 plate count method), citrulline content (HPLC method), pH value (potential method).

[0097] 2.2 Animal feeding experiment design: The same animal source and feeding management process as in Example Group I were used. Six treatment groups were set up, each fed with 5% fermented feed prepared by the different processes described above as experimental diets. A control group II (FMB) was also set up with 5% ordinary fermented soybean meal added. The experiment lasted for 21 days.

[0098] Table 3. Inoculum formulation and fermentation endpoint test group design serial number Example <![CDATA[Bacterial agent ratio (LP + : BS + : CB)]]> Set the fermentation endpoint Arg / Cit Ratio Actual fermentation value E4 Example 4 1.5：1.0：0.5 1.5 1.48±0.15 E5 Example 5 2.5：1.5：1.0 1.2 1.18±0.12 E6 Example 6 3.0：2.0：1.5 0.8 0.82±0.08 C3 Comparative Example 3 1.0：0.5：0.2 No control (fixed 60h) 2.95±0.30 C4 Comparative Example 4 4.0：3.0：2.0 0.5 0.51±0.05 FMB Control Group II Common commercial fermentation agents No control (fixed 60h) 4.20±0.42 2.3 Detection Method: In addition to growth performance and intestinal morphology, the following tests were added: Intestinal NOS activity: Jejunal mucosal tissue was taken, homogenized, and total NOS activity was measured using a nitric oxide synthase (NOS) assay kit (nitrate reductase method).

[0099] Cecal microorganisms and metabolites: Cecal chyme was aseptically collected, and a portion was plate counted on selective media (MRS medium for lactic acid bacteria, VRBGA medium for Escherichia coli, and reinforced clostridial medium for Clostridium butyricum); the other portion was centrifuged, and the supernatant was used to determine the content of short-chain fatty acids (SCFAs) by gas chromatography.

[0100] Table 4. Effects of different inoculant ratios and fermentation endpoints on product characteristics and piglet physiology.

[0101] Analysis of the results in Table 4 and Table 2 shows that: The core of functional microbial agent formulation: In group C3, insufficient microbial agents led to insufficient power of the conversion engine, low citrulline production, and a low number of viable bacteria in the product, resulting in limited improvement in various animal indicators. Groups E4-E6, with a reasonable formulation, successfully constructed a highly efficient fermentation system, achieving high citrulline conversion rate and high viable bacteria survival rate, proving that a specific range of microbial formulations is fundamental to achieving the expected function.

[0102] The value of fermentation endpoint control (Arg / Cit Ratio): In the FMB and C3 groups, the Arg / Cit Ratio was too high (>2.9) due to the lack of endpoint control, indicating that arginine was not effectively converted and the accumulation of functional precursors was insufficient.

[0103] Although group C4 (Ratio=0.5) had the highest citrulline production, it experienced a significant rebound in diarrhea rates. Analysis suggests that excessive arginine consumption may have affected its direct nutritional effect on the intestinal mucosa; simultaneously, excessive microbial metabolism may have altered the gut microbiota balance (its cecal lactobacilli and butyrate levels were lower than in the E5 / E6 groups) or produced certain irritating metabolites. This result is crucial, clearly defining the safe boundaries of fermentation and demonstrating the necessity of maintaining the Arg / Cit Ratio above 0.8 to avoid the side effects of over-conversion.

[0104] The E4-E6 group, by controlling the endpoint within the range of 0.8-1.5, perfectly balanced functional conversion (high citrulline production) and nutritional safety (low diarrhea rate), and optimally activated the host gut NOS activity, demonstrating the practicality of using the Arg / Cit Ratio as the core parameter for judging fermentation endpoints.

[0105] Preferred embodiment: The E5 group (ratio 2.5:1.5:1.0, endpoint ratio=1.2) showed the most balanced and excellent performance in promoting growth, maintaining intestinal health, activating endogenous NO pathways and shaping beneficial flora, and is the preferred embodiment of the present invention.

[0106] Example Group III. Comprehensive Comparative Verification Experiment: This experiment is the final integration verification of the present invention. It aims to comprehensively compare the complete product of the present invention with current mainstream alternatives and products lacking key elements, to fully evaluate its comprehensive benefits and to deeply explain its mechanism of action.

[0107] 3.1 Experimental Design: Six treatment groups were set up for a 28-day feeding trial. The basal diet was the same as before.

[0108] NC group: basal diet (negative control).

[0109] PC group: basal diet + 2250 mg / kg zinc oxide (pharmacological dose, industry positive control).

[0110] MF group: basal diet + 5% commercially available mainstream brand microbial fermented feed (control).

[0111] IP-A group: basal diet + 5% of the core fermentation product of this invention (prepared using the E5 group process, but without precise amino acid compensation after fermentation).

[0112] IP-B group: Basal diet + 5% of the complete final product of this invention (prepared using the E5 group process, and after fermentation, based on amino acid profile detection, it is precisely supplemented with coated L-arginine, L-glutamine and L-lysine, so that the total arginine reaches 1.60% and the effective lysine reaches 1.40%).

[0113] Arg group: The basal diet was supplemented with synthetic crystalline L-arginine and L-lysine to make the total arginine and lysine levels in the diet exactly the same as those in the IP-B group (to compare the differences between different supply routes).

[0114] 3.2 Detection method: In addition to all the aforementioned indicators, the following is added: Serum markers: total antioxidant capacity (T-AOC, FRAP method), immunoglobulin G (IgG, immunoturbidimetric method), tumor necrosis factor-α (TNF-α, ELISA method).

[0115] Intestinal barrier: Jejunal tissue was collected, and total RNA and protein were extracted. The relative expression levels of the tight junction protein ZO-1 gene and protein were detected by real-time quantitative PCR (qPCR) and Western blotting.

[0116] Nitric oxide metabolism: Measurement of serum nitrite / nitrate (NO) x The content of nitric oxide (Griess method) serves as a stable metabolic marker for nitric oxide production in the body.

[0117] Table 5. Results of the comprehensive comparative test index NC Group PC group (ZnO) MF group IP-A group IP-B group Arg group ADG(g) <![CDATA[308±19 e ]]> <![CDATA[370±22 c ]]> <![CDATA[362±21 c ]]> <![CDATA[392±23 b ]]> <![CDATA[408±24 a ]]> <![CDATA[315±19 d ]]> Diarrhea rate (%) <![CDATA[18.2 a ]]> <![CDATA[6.0 c ]]> <![CDATA[8.8 b ]]> <![CDATA[4.2 d ]]> <![CDATA[2.5 e ]]> <![CDATA[17.0 a ]]> Jejunal vitamin C <![CDATA[1.30±0.12 e ]]> <![CDATA[1.80±0.16 c ]]> <![CDATA[1.72±0.15 c ]]> <![CDATA[2.02±0.18 b ]]> <![CDATA[2.20±0.20 a ]]> <![CDATA[1.38±0.13 e ]]> <![CDATA[Serum NO x (μM)]]> <![CDATA[16.5±1.7 e ]]> <![CDATA[18.8±1.9 d ]]> <![CDATA[19.5±2.0 d ]]> <![CDATA[26.8±2.7 b ]]> <![CDATA[30.2±3.0 a ]]> <![CDATA[18.2±1.8 d ]]> Serum T-AOC (U / mL) <![CDATA[7.8±0.8 e ]]> <![CDATA[9.5±1.0 c ]]> <![CDATA[9.8±1.0 c ]]> <![CDATA[12.0±1.2 b ]]> <![CDATA[13.5±1.4 a ]]> <![CDATA[8.2±0.8 d ]]> Serum TNF-α (pg / mL) <![CDATA[132±14 a ]]> <![CDATA[98±10 b ]]> <![CDATA[105±11 b ]]> <![CDATA[78±8 c ]]> <![CDATA[65±7 d ]]> <![CDATA[128±13 a ]]> <![CDATA[Lactobacillus caecorum (10 7 CFU / g)]]> <![CDATA[2.0±0.3 e ]]> <![CDATA[1.8±0.3 e ]]> <![CDATA[4.8±0.6 c ]]> <![CDATA[8.0±0.9 b ]]> <![CDATA[9.5±1.0 a ]]> <![CDATA[2.2±0.3 e ]]> Cecal pH <![CDATA[6.9±0.2 a ]]> <![CDATA[7.0±0.3 a ]]> <![CDATA[6.4±0.2 b ]]> <![CDATA[6.0±0.2 c ]]> <![CDATA[5.8±0.2 c ]]> <![CDATA[6.8±0.2 a ]]> ZO-1 protein expression <![CDATA[1.00±0.11 d ]]> <![CDATA[1.35±0.14 c ]]> <![CDATA[1.28±0.13 c ]]> <![CDATA[1.78±0.18 b ]]> <![CDATA[2.05±0.21 a ]]> <![CDATA[1.05±0.11 d ]]> As shown in Table 5, the IP-B group demonstrated significant and consistent superiority across all core evaluation indicators. It not only exhibited the best growth performance but also comprehensively outperformed in improving intestinal morphology, enhancing barrier function (high ZO-1), boosting systemic antioxidant capacity (high T-AOC), reducing systemic inflammation (low TNF-α), and optimizing the intestinal microenvironment (high lactic acid bacteria, low pH). This demonstrates the multi-target, systemic health-promoting effects of the present invention's technical system.

[0118] Comparison of IP-A and IP-B: The IP-A group without nutrient compensation was significantly superior to all traditional control groups (PC, MF), highlighting the efficacy of the plant donor + functional bacteria targeted fermentation combination. The IP-B group, through precise nutrient compensation after fermentation, showed improvements in ADG and serum NO levels. x Significant improvements have been achieved in key indicators, demonstrating that a dynamic nutrition guarantee system is indispensable for fully releasing product potential and achieving optimal amino acid balance.

[0119] IP-B and MF / PC Comparison: The product of this invention is significantly superior to zinc oxide (PC) in promoting growth and regulating gut microbiota, and overcomes the drawback of zinc oxide potentially inhibiting beneficial bacteria. Compared with conventional fermented feed (MF), this invention significantly activates endogenous physiological pathways in the host (such as significantly increasing serum NO). xIt has made a qualitative leap in terms of T-AOC, revealing its deeper mechanism of action.

[0120] Activation of the NO metabolic axis: Extremely high serum NO levels in the IP-B group x The level of V / C and ZO-1 expression in the jejunum, along with the lowest diarrhea rate, forms a perfect logical loop, clearly revealing the core pathway of providing microbial-transformed citrulline → enhancing host endogenous NO production → improving intestinal blood flow and barrier integrity → promoting health and growth.

[0121] Decisive evidence of pathway specificity: There were no significant differences in any indicators between the Arg group (simply supplemented with synthetic amino acids) and the NC group; their serum NO levels were significantly lower. x The levels were not effectively improved. This result is decisive: it strongly demonstrates that the superior effect of this invention does not stem from simply increasing the concentration of arginine or lysine in the diet, but rather relies entirely on the unique biotransformation pathway of converting plant-derived arginine into citrulline through specific microbial fermentation, and the resulting comprehensive ecological effects of probiotics, organic acids, etc. This fundamentally distinguishes this invention from conventional nutritional supplementation strategies, highlighting its inventiveness.

[0122] Summary of feed products and processes: Based on the above three sets of detailed, systematic, and logically rigorous implementation examples, the following conclusions can be drawn: 1) The ranges for the proportion of plant-derived donors, the ratio of functional microbial agents, and the control parameters for the fermentation endpoint are all scientifically defined ranges determined through extensive experiments, ensuring optimal technical results while avoiding side effects. The comparative data clearly reveal the decrease in effectiveness or problems caused by deviations from these ranges.

[0123] 2) The superior effects of this invention stem from the synergistic effect of a complete technical system: optimized plant donors, engineered functional microbial communities, precise fermentation control, and dynamic nutrient compensation. The absence or deviation of any link in this chain (as shown in the comparative examples) will lead to a significant reduction in the final effect.

[0124] 3) This invention utilizes a series of traditional and reliable physiological, biochemical, and microbiological detection methods to fully demonstrate its multi-target mechanism of action, which systematically promotes animal health and growth by activating the host's endogenous NO metabolic axis and synergistically optimizing gut microbiota and barrier function. Its comprehensive effect significantly surpasses current mainstream zinc oxide and conventional fermented feed solutions, providing an innovative, efficient, and safe piglet feed solution. All embodiments are detailed, with complete data and strong reproducibility.

[0125] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A microecological fermented feed suitable for young pigs, characterized in that, The following components are prepared by a specific process: 20%-35% plant-derived amino acid donors: the plant-derived amino acid donors are composed of arginine donors and branched-chain amino acid donors in a weight ratio of (10-25):(5-15); 60%-75% base energy carriers: the base energy carriers are selected from one or more of puffed corn, puffed rice, and bran; 2%-5% complex functional fermentation agents: the complex functional fermentation agents include Lactobacillus plantarum with arginine enzyme activity ≥250 U / mg protein, Bacillus subtilis positive for nitrate reductase, and Clostridium butyricum; an appropriate amount of post-fermentation nutritional guarantee agent: the post-fermentation nutritional guarantee agent is a coated amino acid, and the addition amount of the coated amino acid is compensated based on the detection results of the amino acid profile of the post-fermentation product, so that the total arginine content in the micro-ecological fermented feed is ≥1.5%, the effective lysine content is ≥1.3%, and the total branched-chain amino acid content is ≥3.2%; in the micro-ecological fermented feed, the molar ratio of arginine to citrulline is 0.8-1.5:

1.

2. The microecological fermented feed for piglets according to claim 1, characterized in that, The arginine donors are selected from the enzymatic products of pumpkin seed meal, watermelon seed meal, or seaweed powder, and the addition amount accounts for 10%-25% of the total weight of the raw materials; the branched-chain amino acid donors are selected from the enzymatic products of hemp seed protein or lupin powder, and the addition amount accounts for 5%-15% of the total weight of the raw materials.

3. The microecological fermented feed for piglets according to claim 1, characterized in that, The enzymatic products are obtained by synergistic enzymatic hydrolysis of plant raw materials with neutral protease and cellulase, and the enzymatic hydrolysis conditions are as follows: temperature 45-55℃, time 3-5 hours, and pH 6.5-7.

5.

4. The microecological fermented feed for piglets according to claim 1, characterized in that, In the complex functional fermentation agent, the viable cell count ratio of Lactobacillus plantarum, Bacillus subtilis, and Clostridium butyricum is (1.5-3.0):(1.0-2.0):(0.5-1.5).

5. A method for preparing a microecological fermented feed for young pigs according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1. Preparation of plant-derived amino acid donors: after the plant raw materials are crushed, they are subjected to complex enzymatic hydrolysis or pre-fermentation treatment to obtain plant protein hydrolysate rich in small peptides and free amino acids; S2. Mixing and inoculation: the plant protein hydrolysate and the base energy carrier are uniformly mixed, and then the complex functional fermentation agent is inoculated, mixed uniformly, and the moisture content of the material is adjusted to 36%-42%; S3. Stepwise controllable fermentation: the mixed material is placed in a fermentation device, and the following steps are performed in sequence: First-stage aerobic fermentation: control the temperature at 32-37℃ and the relative humidity at 80%-90% for 12-24 hours; Second-stage anaerobic main fermentation: control the temperature at 36-40℃ and the relative humidity at 75%-85% for 36-72 hours; S4. Termination of fermentation and drying: when the molar ratio of arginine to citrulline in the material reaches 0.8-1.5 during the second-stage fermentation, the fermentation is terminated; the fermentation product is dried at a temperature not higher than 45℃ until the moisture content is ≤12%; S5. Nutritional compensation and productization: detect the amino acid profile of the dried material, calculate the difference according to the predetermined target amino acid content, uniformly mix the corresponding amount of coated amino acid for compensation, and finally crush and package to obtain the finished product.

6. The method of claim 5, wherein the microecological fermented feed for piglets is prepared by the steps of: a) mixing the probiotic microorganism with the feed; b) fermenting the feed with the probiotic microorganism; c) drying the fermented feed; and d) packaging the dried fermented feed. The S1. plant source amino acid donor prepared composite enzymolysis includes the following steps: First step. Raw material selection and pretreatment: According to the formula requirements, the dry plant raw materials are weighed, and the raw materials are crushed using a hammer crusher and passed through a 40-60 mesh screen; Second step. Composite enzymolysis treatment: Prepare the enzymolysis substrate slurry: put the crushed raw materials into an enzymolysis reaction tank with stirring and temperature control, add 50-55℃ pure water, and adjust the solid-liquid ratio to 1:3 to 1:4; First stage enzymolysis: use cellulase with enzyme activity ≥10,000 U / g, the addition amount is 0.1%-0.3% of the dry weight of the raw materials, adjust the pH to 4.5-5.5, and continuously stir at 50-55℃ for 1.5-2.5 hours; Second stage enzymolysis: Use neutral protease with activity ≥50,000 U / g and flavor protease with enzyme activity ≥20,000 U / g to prepare a composite protease with a weight ratio of about 3:1, and the total addition amount of the composite protease is 0.8%-1.5% of the dry weight of the raw materials; adjust the pH of the material to 6.5-7.0, and continuously stir at 50-55℃ for 3.5-4.5 hours; Third step. Enzyme inactivation and product standardization: Enzyme inactivation and sterilization: immediately raise the temperature of the material to 85-90℃ after the enzymolysis reaction reaches the end point, and maintain for 15-20 minutes; Solid-liquid separation and concentration: after enzyme inactivation, the slurry is subjected to solid-liquid separation by plate and frame filter press or centrifuge to remove insoluble substances, and the filtrate rich in small peptides and amino acids is collected and transferred to a vacuum concentration tank, and concentrated to a soluble solid content of 30%-40%.

7. The method for preparing a microecological fermented feed suitable for piglets according to claim 5, characterized in that, The S5 finished product must meet all the following key indicators: a) Functional activity indicators: total viable count > 1.0 x 10 9 CFU / g; citrulline content > 1.8 mg / g; molar ratio of arginine to citrulline 0.8-1.5; b) Core nutritional indicators: total arginine content ≥1.5%; effective lysine content ≥1.3%; total branched chain amino acid content ≥3.2%; phytase activity ≥500 U / kg; c) Health and safety indicators: aflatoxin B1 ≤10 μg / kg, Salmonella must not be detected.

8. Use of a microecological fermented feed for young pigs according to any one of claims 1 to 4, characterized in that, Add the micro-ecological fermented feed at a proportion of 3% to 10% of the total dry matter weight of the whole pig diet, mix evenly, and use.

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