Biological preparation and application thereof in feed

Abstract

The invention belongs to the technical field of biological agents and feed, and discloses a biological agent and application thereof in low-protein feed. The core component of the biological agent is rumen bypass glycine (RPGly), and the biological agent is micro-capsule type particles and is composed of an inner layer coating composed of a glycine capsule core, hydrophobic beewax and hydrogenated castor oil, and an outer layer coating containing chitosan and nano silicon dioxide. The preparation has excellent rumen bypass rate and small intestine release rate, and can be cooperatively used with rumen bypass methionine. When being applied to low-protein feed for ruminants, the low-protein feed additive can regulate and control metabolic pathways such as glyceryl phospholipids and glutathione, so that nitrogen emission is remarkably reduced, the growth performance of animals is maintained, the eye muscle area is remarkably increased, the tenderness and flavor of meat are improved, and quality and efficiency improvement in a low-protein breeding mode is realized.

Description

Technical Field

[0001] This invention belongs to the field of biological agents, specifically relating to rumen-protected amino acid (RPAA) biological agents and their application in feed, especially in ruminants. Technical Background

[0002] Currently, my country's livestock industry heavily relies on imports for protein raw materials, resulting in persistently high breeding costs. More importantly, ruminants have low feed protein utilization rates, with approximately 30-40% of nitrogen being excreted unused through feces and urine. This not only wastes feed resources but also leads to environmental problems such as soil eutrophication and excessive ammonia nitrogen in groundwater. Simply increasing feed protein content to pursue growth performance significantly increases breeding costs and places an additional burden on the ecological environment. The environmental pressure caused by nitrogen emissions from ruminant farming has become a key constraint on the sustainable development of the livestock industry. This technology, through precise nutrient formulation and amino acid balance, reduces the amount of protein raw materials used while ensuring the growth needs of livestock and poultry, alleviating the pressure of protein resource shortages and significantly reducing the environmental impact of nitrogen emissions. In recent years, the application of low-protein feed in goat farming has continued to expand. However, research has found that excessively reducing feed protein levels may inhibit the growth rate of goats and adversely affect their muscle development and meat quality. To address this issue, nutritional compensation by adding functional amino acids has become an effective solution. Because the degradation characteristics of rumen microorganisms in ruminants can degrade crystalline amino acids that have passed through the rumen, reducing the amount of amino acids reaching the small intestine, it is necessary to use coating technology to protect crystalline amino acids.

[0003] However, research on the applicability of achieving the dual goals of improving quality and efficiency and reducing nitrogen emissions without affecting animal production performance in ruminants, especially in the local Chongqing black goat breed, is still lacking. Summary of the Invention

[0004] The purpose of this invention is to provide a biological agent that can improve the growth performance, slaughter performance and meat quality of ruminants, providing a theoretical and practical basis for developing low-cost and high-efficiency animal nutrition regulation technology, and realizing a high-efficiency and environmentally friendly feeding model of "low protein + (conditional) limiting amino acids".

[0005] The objective of this invention is achieved through the following measures: A biological agent comprising rumen-protected glycine (RPGly). The RPGly has a rumen-protected rate of not less than 94.0% and a small intestinal release rate of 88.0% to 93.0%. Preferably, the RPGly has a rumen-protected rate of 95.0% to 96.0% and a small intestinal release rate of 92.0% to 93.0%.

[0006] The above-mentioned RPGly is a microencapsulated granule formulation. The granules consist of three parts: a core, an inner coating, and an outer coating. The core is composed of glycine and a carrier.

[0007] The carrier includes one or more of the following: silica, corn starch, maltodextrin, and sodium carboxymethyl cellulose. The amount of carrier added is 5.0% to 15.0% of the mass of glycine.

[0008] Inner coating: Composed of a composite coating base and antioxidants. The composite coating base is a mixture of hydrophobic beeswax and hydrogenated castor oil in a 3:1 mass ratio. The antioxidants are one or more of the following: ethoxyquinoline (EQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), tert-butylhydroquinone (TBHQ), tea polyphenols (TP), vitamin E, and ascorbyl palmitate (AP).

[0009] The outer coating consists of a composite film-forming material, an organic solvent, and functional additives. The organic solvent is a mixture of ethanol, acetone, and glycerol monoacetate in a volume ratio of 2:1:1. The composite film-forming material is a compound system of hydrogenated palm oil, cellulose acetate, and polyethylene glycol in a mass ratio of 2:1:1. The functional additive is a mixture of chitosan (degree of deacetylation ≥90%) and nano-silica (particle size 50.0–100.0 nm) in a mass ratio of 5:1, with an addition amount of 3.0%–5.0% of the composite film-forming material's mass.

[0010] The total raw material composition of the RPGly particles, by mass percentage, is as follows: glycine 40.0%–60.0%, hydrophobic beeswax 7.5%–22.5%, hydrogenated castor oil 2.5%–7.5%, antioxidant 0.04%–0.2%, composite film-forming material 20.0%–40.0%, functional additives 0.4%–1.6%, organic solvent 1.0%–2.0%, and carrier 4.0%–8.0%, with the sum of the mass percentages of all components being 100%.

[0011] The preparation method of the above RPGly microcapsules includes: (1) Preparation of capsule core microspheres: Glycine and carrier are mixed in proportion to form uniform microspheres with a particle size of 0.5-1.0 mm; then the microspheres are placed in a mixer, the molten composite coating base is added first, and the mixture is stirred at 150-200 r / min for 5 min. Then the antioxidant is slowly added and the mixture is stirred for 5-10 min until the surface of the microspheres is uniformly coated with the inner coating. (2) Preparation of primary coated particles: The microparticles coated with the inner layer are fed into a freeze spray dryer at a drying temperature of 30.0-40.0 ℃ and dried until the moisture content of the particles is ≤ 0.5% to obtain primary coated particles; (3) Coating and preparation of finished product: The composite film-forming material is added to an organic solvent and stirred at 50.0-60.0℃ for 30 min until completely dissolved. Then, functional additives are added and stirring is continued for 20 min to prepare a uniform coating solution. The primary coated particles are fed into a bottom spray fluidized bed coating machine for coating. After completion, the particles are cooled in stages and then screened through a 20-60 mesh sieve to obtain rumen-protected glycine microcapsules.

[0012] The aforementioned biological agents also include rumen-protected methionine (RPMet), with a 1:1 ratio of RPMet to RPGly. The rumen-protected rate of the aforementioned RPMet is 85.0%–90.0%, and the small intestinal release rate is 80.0%–85.0%. These biological agents can synergistically upregulate glycerophospholipid metabolism, glutathione metabolism, and aminoacyl-tRNA biosynthesis, exhibiting synergistic effects in maintaining cell membrane integrity (e.g., glycerophosphocholine accumulation), antioxidant defense (increased γ-glutamylcysteine), and protein translation efficiency (enhanced L-aspartate / L-proline ratio).

[0013] This invention also provides the application of the above-mentioned biological agents in animal feed.

[0014] An animal feed includes a basal feed and the aforementioned biological agents. The basal feed is a low-protein feed, formulated below the protein nutritional requirements for growing-finishing goats as specified in the "Nutritional Requirements for Meat Sheep" (NY / T 816-2021). In this invention, the crude protein content of the low-protein feed is 11.0%, and the dosage of the aforementioned biological agents is either RPGly 4.0 g / kg or RPMet 2.0 g / kg + RPGly 2.0 g / kg.

[0015] The above feed should be used as follows: Adjust the feeding amount daily based on the previous day's average daily feed intake (ADFI, g / d) to ensure that the remaining amount does not exceed 10.0% of the total feed. Feed once at 08:00 and once at 16:30, feeding roughage first, followed by concentrate 1 hour later: Calculate the concentrate feeding amount for each pen, weigh the corresponding RPAAs, and mix the RPAAs with the concentrate at a total of 4.0 g / kg before feeding. Mix and feed immediately.

[0016] Dosage: Based on feed intake, RPGly alone is 4.0 g / kg per day, and RPMet + RPGly combination is 2.0 g / kg per component per day. For example: If yesterday's concentrate feed intake was 2.0 kg, then today's feed should be 8.0 g of RPAAs.

[0017] Frequency of use: The amount of RPAAs fed is divided into two equal parts, once in the morning and once in the afternoon.

[0018] The aforementioned biological agents and feeds are preferably used for feeding ruminants, and more preferably for black goats, especially during the fattening period.

[0019] The present invention has the following beneficial effects: This invention focuses on the differences in genetic characteristics of local breeds and regional feeding conditions, and systematically develops biological agents and feed programs to study the specific effects of these on the growth, development, carcass quality, and meat flavor of black goats, providing a scientific basis for establishing breed-specific low-protein feeding programs.

[0020] This invention uses the Yudong Black Goat, a genetic resource of Chongqing goats, as the research object to explore the effects of adding specific glycine and methionine to Yudong Black Goats under low-protein conditions on their growth, development, and meat quality and flavor. It also utilizes non-target metabolomics technology to analyze the regulatory mechanisms by which the "low-protein + (conditionally) limiting amino acid" feeding technique affects the meat quality and flavor. Amino acids are not simply supplemented as nutritional substrates, but exert significant physiological regulatory functions through specific metabolic pathways. In particular, the synergistic effect of different RPAAs under the low-protein diet mode showed an unexpected synergistic effect. By comparing and analyzing the growth indicators, serum biochemical indicators, slaughter performance, and meat quality of the experimental and control groups, the study reveals the regulatory pathway of low-protein diet combined with RPAAs technology on the growth and metabolism of Black Goats and evaluates its potential to improve meat flavor and nutritional value. Specifically: This invention uses technologies such as electronic nose, electronic tongue, and metabolomics to study the effects of adding specific RPAAs to low-protein feed on the growth performance, slaughter performance, blood biochemical indicators, and meat quality and flavor of ruminants, proving that low-protein feed can be used in the fattening stage of ruminants.

[0021] This invention shows that reducing feed protein and supplementing with specific RPAAs does not affect the growth performance or slaughter performance of Yudong Black Goats, and the eye muscle area of ​​the MAG group is significantly increased without negatively impacting serum biochemical indicators.

[0022] This invention reduces feed protein and supplements with specific RPAAs, which can significantly improve the quality of Yudong black goat meat and increase the content of umami amino acids and fatty acids in the meat.

[0023] This invention reduces feed protein and supplements specific RPAAs, which can significantly reduce the intensity of unpleasant odors such as nitrogen oxides and sulfides in mutton, enhance the flavor of aromatic benzenes and short-chain alkanes, and show an upward trend in umami, richness and saltiness.

[0024] This invention improves meat quality by reducing feed protein and supplementing with RPAAs, thereby regulating lipid metabolism and energy pathways.

[0025] 6. This invention found that under low-protein conditions, glycine has no different effect on ruminants than methionine, an essential amino acid, and can significantly improve meat quality and flavor, indicating that glycine is also a specific conditionally essential amino acid for ruminants.

[0026] 7. This invention fills the research gap in the precise addition of functional amino acids to low-protein feeds for ruminants, providing new evidence for the theory of amino acid metabolism regulation. By optimizing feed formulation to reduce dependence on soybean meal (responding to the national policy of "reducing and replacing soybean meal"), reducing nitrogen emissions, promoting ecological farming, and improving the quality of Yudong black goat meat, this invention provides a breakthrough direction for cost reduction, quality improvement, and efficiency enhancement in the goat industry, thereby promoting the high-quality development of the local goat industry. Attached Figure Description

[0027] Figure 1 Product image of rumen-protected glycosaminoglycans; Figure 2 Microscopic image of rumen-transmitted glycosides. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0029] Unless otherwise specified, all materials used in the following examples are commercially available products.

[0030] Example 1 Part 1: Effects of RPAAs Addition to Low-Protein Feeds on Growth Performance, Slaughter Performance, and Serum Biochemical Indicators of Yudong Black Goats

[0031] 2.1 Materials and Methods 2.1.1 Test Materials RPMet: Rumen clearance rate 86.9%, small intestinal release rate 82.6%.

[0032] RPGly: Rumen pass-through rate 95.8%, small intestinal release rate 90.2%.

[0033] Methods for testing performance parameters: (1) Method for detecting the rumen pass rate of RPAAs: A.1 Reagents or materials Unless otherwise specified, use only analytical grade reagents.

[0034] A.1.1 Water: GB / T 6682, Grade III.

[0035] A.1.2 Artificial rumen fluid: Take 5.72 g of citric acid monohydrate and 52.1 g of disodium hydrogen phosphate dodecahydrate, add 1000 mL of water, and stir to dissolve.

[0036] A.2 Instruments and Equipment A.2.1 Dissolution tester: temperature accurate to ±0.3 ℃.

[0037] A.2.2 Analytical balance: accuracy 0.1 mg.

[0038] A.3 Test Procedure Perform two parallel tests. Weigh an appropriate amount (approximately equivalent to 0.125 g of RPAAs) of the sample (accurate to 0.1 mg) and place it in the rotating basket of the dissolution apparatus. Add 400 mL of artificial rumen fluid (A.1.2) to the dissolution vessel. Heat to the set temperature of 38°C, completely immerse the rotating basket in the solution in the dissolution vessel, keeping the basket at a consistent height, set the rotation speed to 100 r / min, and rotate for 12 h. Remove the rotating basket, rinse it three times with approximately 20 mL of water, and transfer the residue in the basket to a 250 mL beaker for quantitative analysis.

[0039] A.4 Experimental Data Processing Release rate of the sample (treated with artificial rumen fluid for 12 h) as mass fraction 1. The unit is expressed as a percentage by mass (%), calculated according to formula (A.1):

[0040] In the formula: 2 — The mass concentration of the analyte obtained from the standard curve, in milligrams per milliliter (mg / mL). 2 — Sample dilution volume, in milliliters (mL); 2 — Mass of the sample, in grams (g); 0 — The content of RPAAs in the product, expressed as a percentage (%); 1,000 — Conversion factor.

[0041] The results are expressed as the arithmetic mean of parallel measurements, and are retained to 3 significant figures.

[0042] A.5 Precision Under repeatability conditions, the absolute difference between two independent measurements should not exceed 5.0%.

[0043] (2) Method for detecting the small intestinal release rate of RPAAs: B.1 Reagents or materials Unless otherwise specified, use only analytical grade reagents.

[0044] B.1.1 Water: GB / T 6682, Grade III.

[0045] B.1.2 Trypsin: Casein conversion power ≥ 25.0, CAS: 8049-47-6.

[0046] B.1.3 1.0 mol / L sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, add 100 mL of water to dissolve, and mix well.

[0047] B.1.4 Phosphate buffer: Weigh 13.6 g of potassium dihydrogen phosphate, dissolve in 1000 mL of water, and use 1.0 mol / L sodium hydroxide solution (B.1.3). Adjust the pH value to 6.8~7.4.

[0048] B.1.5 Artificial intestinal fluid: Weigh 10.0 g of pancreatic enzyme, add 1000 mL of phosphate buffer (B.1.4) to dissolve and mix well.

[0049] B.2 Instruments and Equipment B.2.1 Dissolution tester: temperature accurate to ± 0.3 ℃.

[0050] B.2.2 Analytical balance: accuracy 0.1 mg.

[0051] B.2.3 pH meter: accuracy ±0.01.

[0052] B.3 Test Procedure Perform two parallel experiments. Weigh an appropriate amount of sample (accurate to 0.1 mg) and place it in the rotating basket of the dissolution apparatus. Add 400 mL of artificial rumen fluid (A.1.2) to the dissolution vessel. After heating to the set temperature of 38 °C, completely immerse the rotating basket in the solution in the dissolution vessel, keeping the basket at a consistent height. Set the rotation speed to 100 r / min and rotate for 12 h. Discard the artificial rumen fluid. In a clean dissolution vessel, add 400 mL of artificial intestinal fluid (B.1.5), heat to the set temperature of 38 °C, set the rotation speed to 100 r / min, and rotate for 12 h. Remove the rotating basket. Rinse the rotating basket three times with approximately 20 mL of water. Transfer the residue in the rotating basket to a 250 mL beaker for quantitative analysis.

[0053] B.4 Experimental Data Processing The release rate of the sample (artificial rumen fluid for 12 h + artificial intestinal fluid for 12 h) is expressed as a mass fraction. 2. The unit is expressed as a percentage by mass (%), calculated according to formula (B.1):

[0054] In the formula: 3 — The mass concentration of the analyte obtained from the standard curve, in milligrams per milliliter (mg / mL). 3 — Sample dilution volume, in milliliters (mL); 3 — Mass of the sample, in grams (g); 1 — Release rate of the sample after 12 h of treatment with artificial rumen fluid, expressed as a percentage (%); 0 — The content of RPAAs in the sample, expressed as a percentage (%); 1,000 — Conversion factor.

[0055] The results are expressed as the arithmetic mean of parallel measurements, and are retained to 3 significant figures.

[0056] B.5 Precision Under repeatability conditions, the absolute difference between two independent measurements should not exceed 5.0%.

[0057] 2.1.2 Experimental Animals and Design Sixty 4-month-old Yudong Black Goats with an average weight of 21 kg ± 0.74 kg were randomly divided into a control group and three treatment groups (GLY, MET, and MAG groups), with three replicates per group and five goats per replicate. The experiment lasted for 60 days, including a 10-day pre-feeding period and a 50-day regular feeding period. The control group was fed a conventional crude protein (CP) diet with a CP content of 13.4%. The three experimental groups were fed a low-protein diet with a CP content of 11.0%; the GLY group was supplemented with 4 g / kg RPGly; the MET group was supplemented with 4 g / kg RPGMet; and the MAG group was supplemented with a uniform mixture of 2 g / kg RPGly and 2 g / kg RPGMet. After the experiment, the goats were slaughtered, and samples of the longissimus dorsi muscle and blood were collected for analysis.

[0058] 2.1.3 Experimental Feed The basic feed was formulated based on the national standard NY / T 816-2021 "Nutritional Requirements for Meat Sheep" and combined with the production practice of Yudong Black Goats, with an average daily weight gain of 170 g for goats. The CP level of the control group feed was 13.4%, while the low-protein group was formulated based on the control group feed, with the protein level reduced to 11.0% and RPAAs added.

[0059] Low-protein diets are basic diets formulated below the nutritional requirements for growing and fattening goats as specified in "Nutritional Requirements for Meat Sheep". For example, the daily nutritional requirements for growing goats are shown in Table 1.

[0060]

[0061] The protein content used in this experiment was 11.0%. The dosage of biological agents was 4.0 g / kg.

[0062] The preferred formulation of the basic feed consists of 70.0% peanut vines and 30.0% concentrated supplement, wherein the concentrated supplement comprises corn, wheat, soybean meal, wheat bran, dicalcium phosphate, vegetable oil, limestone powder, baking soda, salt, and premix. See Table 2 for the specific formulation.

[0063]

[0064] Note 1 The premix provides the following per kg of feed: copper 12 mg, zinc 60 mg, selenium 0.3 mg, manganese 40 mg, iron 30 mg, iodine 0.5 mg, vitamin A 8000 IU, vitamin D3 2000 IU, vitamin E 75 IU, and vitamin B1 10 IU; ME is a calculated value, and the rest are measured values.

[0065] Protein content was determined using the Kjeldahl method (GB / T 6432-2018).

[0066] Biological agents: RPGly is a microencapsulated granule formulation. The granules consist of three parts: a core, an inner coating, and an outer coating. The core is composed of glycine and a carrier.

[0067] The carrier consists of corn starch, maltodextrin, and sodium carboxymethyl cellulose (in a mass ratio of 3:1:1). The amount of carrier added is 12.0% of the mass of glycine. This improves the dispersibility and granulation stability of glycine, while preventing agglomeration or uneven particle size during granulation. Sodium carboxymethyl cellulose is used to improve the swelling properties of the capsule core in the small intestine.

[0068] Inner coating: Composed of a composite coating base and antioxidants. The composite coating base is a mixture of hydrophobic beeswax and hydrogenated castor oil in a 3:1 mass ratio. The hydrophobic beeswax has a melting point of 62-67℃, and the hydrogenated castor oil has a melting point of 85-88℃. The two are mixed to form a gradient melting point structure (beeswax is suitable for stable internal temperature, and hydrogenated castor oil enhances mechanical strength), which can form a double protective barrier in the rumen. The antioxidants are a mixture of ethoxyquinoline (EQ), propyl gallate (PG), tea polyphenols (TP), vitamin E, and ascorbyl palmitate (AP).

[0069] The outer coating consists of a composite film-forming material, an organic solvent, and functional additives. The organic solvent is a mixture of ethanol, acetone, and glycerol monoacetate in a volume ratio of 2:2:1. This mixed solvent allows for rapid dissolution of the film-forming material with a controllable evaporation rate. The composite film-forming material is a blend of hydrogenated palm oil, cellulose acetate, and polyethylene glycol (PEG) in a mass ratio of 2:1:1. The PEG contains equal masses of PEG 4000 and PEG 2000, creating a molecular weight gradient that enhances the flexibility of the coating film and the gradual swelling in the small intestine. The functional additive is a mixture of chitosan (degree of deacetylation ≥ 90%) and nano-silica (particle size 50.0–100.0 nm) in a mass ratio of 5:1, added at 4.0% of the mass of the composite film-forming material.

[0070] The total raw material composition of the RPGly particles, by mass percentage, is as follows: glycine 50.0%, hydrophobic beeswax 10.2%, hydrogenated castor oil 3.4%, antioxidant 0.2%, composite film-forming material 27.5%, functional additives 1.1%, organic solvent 1.6%, and carrier 6.0%; the sum of the mass percentages of each component is 100%.

[0071] The preparation method of the above RPGly microcapsules includes: 1. Preparation of core microspheres: Glycine and carrier were mixed in proportion and fed into a centrifugal granulation and coating machine. The centrifugal speed was adjusted to 350 r / min and the spray rate was 3 mL / min to prepare uniform microspheres with a particle size of 0.5-1 mm. Then, the microspheres were placed in a twin-helix mixer. Molten composite coating base material (hydrophobic beeswax and hydrogenated castor oil were mixed in proportion, melted at 75-80℃ and kept at that temperature) was added first. After mixing at 200 r / min for 5 min, antioxidant pre-dispersion liquid was slowly added. Mixing continued for 7 min until the surface of the microspheres was uniformly coated with the inner layer. During this period, the temperature inside the mixer was controlled to be maintained at 50.0-55.0℃ to avoid premature solidification of the coating base material. 2. Preparation of primary coated particles: The microcapsules coated with the inner layer are fed into a freeze spray dryer. The feed rate is set to 7 mL / min, the freezing temperature is -35 ℃, the vacuum degree is 0.08~0.1 MPa, and the drying temperature is 38 ℃. The particles are dried until the moisture content is ≤ 0.5% to obtain primary coated particles. 3. Outer Coating and Finished Product Preparation: The composite film-forming material (hydrogenated palm oil, cellulose acetate, and polyethylene glycol mixed in a mass ratio of 2:1:1) is added to an organic solvent (ethanol, acetone, and glycerol monoacetate mixed in a volume ratio of 2:2:1). The mixture is stirred at 55°C for 30 minutes until completely dissolved. Then, a functional additive (chitosan is first dissolved in a 1% acetic acid aqueous solution to prepare a 5% solution, then mixed with nano-silica and sonicated for 20 minutes) is added. Stirring continues for another 20 minutes to prepare a homogeneous coating solution. The coating solution is then filtered through a 1000-mesh screen to remove agglomerated particles. The primary coated particles are fed into a bottom-spray fluidized bed coating machine using a "gradient parameter coating process": In the initial stage (0–10 min), the spray pressure is 0.15 MPa, the inlet air temperature is 55°C, and the spray speed is 5 L / min, forming a dense bottom layer; in the middle stage (10–30 min), the spray pressure is 0.20 MPa, the inlet air temperature is 55°C, and the spray speed is 7 L / min. L / min for rapid film formation; in the later stage (30-40 min), the spray pressure is 0.2 MPa, the inlet air temperature is 50 ℃, and the spray speed is 4 L / min to repair film defects; the outlet air temperature is controlled at 32 ℃ throughout the process, and the coating is carried out until the particle weight gain is 40%-45%; after the coating is completed, segmented cooling is adopted: first air cooling at 35 ℃ for 5 min, then natural cooling to room temperature, and 20-60 mesh sieve to remove agglomerate particles to obtain rumen-protected glycine microcapsules; The RPGly microcapsules obtained in this embodiment have the following properties:

[0072] The RPGly content prepared by the method of the present invention is 45.0%~55.0%, the rumen pass rate is not less than 94.0%, and the small intestine release rate is 88.0%~93.0%.

[0073] 2.1.4 Feeding and Management The experimental sheep were housed in open pens, which were thoroughly cleaned and disinfected before the experiment. Each sheep was ear-tagged and its initial body weight (IBW) was recorded. Vaccinations and deworming were performed according to protocol. Sheep were fed regularly in the morning and evening, ensuring ample feed and water, and clean feed troughs. The health of the flock was monitored. The pens were disinfected every two weeks using a combination of 2.0% sodium hydroxide solution and 0.5% povidone-iodine solution. During the trial period, leftover feed was collected and weighed from each pen every morning. Feeding amounts were adjusted based on the previous day's average daily feed intake, ensuring that the remaining feed did not exceed 10.0% of the total feed intake.

[0074] Feeding method: Feed once at 08:00 and once at 16:30. Feed roughage first, and then feed concentrate 1 hour later. Calculate the amount of concentrate used in each pen, and then weigh the corresponding RPAAs (at 4.0g / Kg). Mix the RPAAs with the concentrate and feed them immediately.

[0075] Dosage of biological agents: Based on feed intake, RPMet or RPGly is 4.0 g / kg, and the mixed amino acid group is (2 g RPMet + 2 g RPGly) / kg. For example: If yesterday's dry matter intake (DMI) was calculated to be 2.0 kg, then 8.0 g of biological agent should be given today.

[0076] RPGly was prepared in Example 1.

[0077] RPMet (Zhejiang Kangdequan Technology Co., Ltd., commercial product "Aimeite"). RPMet has a methionine content of 55.0%–65.0%, a rumen passivity of 85.0%–90.0%, and a small intestinal release rate of 80.0%–85.0%. The rumen-passing DL-methionine used in this example has the following properties: methionine content 61.3%, moisture 1.9%, simulated rumen fluid retention rate of 86.9% after 12 hours, and simulated intestinal fluid release rate of 82.6% after 12 hours.

[0078] Frequency of use: The amount of biological agent fed is divided into two equal parts, once in the morning and once in the afternoon.

[0079] 2.1.5 Measurement Indicators and Methods 2.1.5.1 Determination of growth performance The fasting body weight of the entire flock at the start of the trial period was recorded as IBW, and the fasting body weight on the morning of the last day of the trial was recorded as FBW. The total feed intake of each replicate sheep was recorded daily. Finally, the ADFI, ADG, and FCR of the experimental sheep were calculated.

[0080] 2.1.5.2 Slaughter performance testing After 50 days of the initial feeding period, two Yudong black goats of similar weight from each replicate were selected for slaughter, and their performance indicators were measured. After the experiment, the goats were fasted for 16 hours, and weighed at 7:00 AM the following morning; this was recorded as their pre-slaughter live weight. Slaughter was then carried out according to standard procedures. Immediately afterward, the carcass weight was measured, and the area of ​​the eye muscles was measured. The outline of the vertebral cross-section between the first and second ribs from the bottom was traced on tracing paper with a pencil, and then calculated. The calculation formula is: Eye muscle area (cm²) = Eye muscle width (cm) × Eye muscle thickness (cm) × 0.70.

[0081] 2.1.5.3 Detection of serum biochemical indicators and hormone levels After the experiment, blood was collected from the jugular vein of goats using a 10 mL anticoagulant-free vacuum blood collection tube. After natural coagulation, the blood was centrifuged at 3000 rpm for 15 min, and the supernatant serum was transferred to a 2 mL cryovial and temporarily stored at -20°C. The serum was then sent to a testing institution to detect biochemical indicators, oxidative stress hormones, and inflammatory factors.

[0082] 2.1.6 Data Processing and Analysis Data were processed, analyzed for variance, and compared using Microsoft Excel 2020 and SPSS 18.0 software. Significant differences (P<0.05) and insignificant differences (P>0.05) were used as the criteria for judgment. Results are expressed as mean ± standard deviation.

[0083] 2.2 Results and Analysis 2.2.1 Effects of RPAAs Added to Low-Protein Feed on Growth Performance of Yudong Black Goats The initial weights of the experimental goats in each group were similar, with no significant difference (P>0.05). The average daily feed intake (ADG) ranged from 170.50 to 177.25 g, achieving the ADG effect expected for meat-type growing-finishing goats. There were no significant differences in dry matter intake and feed conversion ratio among the groups (P>0.05), but the feed conversion ratio (FCR) showed a certain downward trend compared to the control group. The results indicate that adjusting the feed protein level and supplementing with RPAAs can effectively maintain the growth rate of fattening goats, and their production performance is basically the same as the control group.

[0084] 2.2.2 Effects of RPAAs Added to Low-Protein Feed on Slaughter Performance of Yudong Black Goats The dressing percentage of fattening goats in all groups was above 44.0%, and there were no significant differences in carcass weight and dressing percentage (P>0.05). However, compared with the control group, the eye muscle area of ​​the MAG group was significantly increased (P<0.05), and the eye muscle area of ​​the GLY group also increased by 25.35%. This indicates that reducing the protein level in the feed and adding RPAAs does not affect the slaughter performance of fattening goats, and the addition of RPGly can improve the eye muscle area of ​​Yudong Black Goats.

[0085] 2.2.3 Effects of RPAAs Addition to Low-Protein Feed on Serum Biochemical Indicators and Hormone Content in Yudong Black Goats Regarding serum biochemical indicators, the levels of total protein (TP), globulin (GLO), and low-density lipoprotein (LDL-C) in the GLY and MET groups were significantly higher than those in the control group (P < 0.05), while the albumin / globulin ratio (A / G) was significantly lower in the GLY and MET groups (P < 0.05). The level of albumin (ALB) in the MET and MAG groups was significantly lower than that in the control group (P < 0.05), while the level of cholesterol (CHOL) in the GLY group was significantly higher than that in the control group (P < 0.05). Other indicators had no significant effect (P > 0.05).

[0086] 2.3 Summary The initial weight of the sheep in each group was consistent (P>0.05). There was no significant difference in average daily weight gain between the three treatment groups and the control group, but the feed conversion ratio was lower. There were no significant differences in carcass weight, dressing percentage, serum oxidative stress, and inflammatory markers among the groups (P>0.05). Compared with the control group, the MAG group showed a significant increase in ocular muscle area (P<0.05); the GLY and MET groups showed significantly increased TP, GLO, and LDL-C levels (P<0.05), and significantly decreased A / G ratio (P<0.05).

[0087] In summary, the addition of RPGly and RPMet to low-protein diets had no negative impact on the growth performance, slaughter performance, or serum biochemical parameters of Yudong Black Goats. IBW was consistent across groups (P>0.05), ADG was comparable to the control group, and FCR showed a decreasing trend; there were no differences among groups in carcass weight, dressing percentage, serum oxidative stress, and inflammatory markers (P>0.05). Notably, compared with the control group, the MAG group showed a significant increase in ocular muscle area (P<0.05), while the GLY and MET groups showed significant increases in serum total protein, globulin, and low-density lipoprotein (P<0.05), and a decrease in A / G ratio, suggesting that amino acid intervention may improve nutrient utilization efficiency by regulating hepatic protein synthesis and lipid metabolism pathways. In conclusion, the addition of RPAAs to low-protein diets can effectively maintain the production performance, slaughter performance, and health of goats on low-protein diets, providing a theoretical basis for the development of environmentally friendly feeds.

[0088] Example 2 Part Two: Effects of RPAAs Addition to Low-Protein Feeds on Meat Quality, Amino Acid and Fatty Acid Content, and Meat Flavor of Yudong Black Goat 3.1 Materials and Methods 3.1.1 Experimental Materials: Same as Part 1, 2.1.1.

[0089] 3.1.2 Experimental animals and design: Same as Part 1, 2.1.2.

[0090] 3.1.3 Experimental feed: Same as Part 1, 2.1.3.

[0091] 3.1.4 Feeding and management: Same as Part 1, 2.1.4.

[0092] 3.1.5 Measurement Indicators and Methods 3.1.5.1 Determination of routine indicators of meat quality pH: pH was measured in samples from the longest back muscle of the test sheep. 45min and pH 24h The pH of the longissimus dorsi muscle in experimental goats was measured within 45 minutes post-slaughter using a portable pH meter. 45min Then, the longissimus dorsi muscle sample was stored at 4°C for 24 h, and the pH was measured. 24h .

[0093] Flesh color index: The lightness value (L) of the longissimus dorsi muscle cross-section 5 cm from the tailbone was measured using a colorimeter. ✱ ), redness value (a ✱ ) and yellowness value (b ✱ Each sample was measured three times and the average value was taken.

[0094] Moisture content: Measured using samples dried in an oven at 103±2°C.

[0095] Cooking loss: Take the sample after 24 hours of acid removal at 4℃, remove the fascia and fat, accurately weigh and record the weight, put the sample into a self-sealing bag, place it in an automatic temperature-controlled water bath preheated to 80℃, heat for 45 minutes, take out the sample and hang it in a cool and dry place to air dry to room temperature, absorb the surface moisture with filter paper, and weigh the sample after cooking.

[0096] Drip loss: Cut the longest back muscle block with a size of 5×3×2cm, weigh it, hang it in a sealed container, place it at 4℃ for 24 hours, and weigh it again. Calculate the percentage of drip loss by the weight difference.

[0097] Intramuscular fat: As specified in the national food safety standard GB 5009.6 "Determination of fat in food".

[0098] Shear stress: Meat samples were obtained using a cylindrical sampler, and the shear stress value perpendicular to the muscle fiber direction was measured using a texture analyzer. Each sample was measured five times and the average value was taken.

[0099] Muscle texture analysis: The texture properties of meat are evaluated using a texture analyzer, including firmness, elasticity, adhesiveness, cohesion, and chewiness.

[0100] Crude ash content: As specified in the National Food Safety Standard GB 5009.4-2016 "Determination of Ash Content in Food".

[0101] Centrifugation water loss rate: Weigh an appropriate amount of meat sample and place it in a centrifuge tube. Process it under standard centrifugal force and calculate the water loss rate by the weight change before and after centrifugation.

[0102] Protein and fat content were determined using the Kjeldahl method and Soxhlet extraction method, respectively.

[0103] 3.1.5.2 Determination of amino acids and fatty acids in muscle After slaughtering the experimental goats, the longissimus dorsi muscle was taken and sent to a testing institution for determination of amino acid and fatty acid composition.

[0104] 3.2.5.3 Electronic detection of the longissimus dorsi muscle and nasal tongue Electronic nose analysis: Fresh mutton samples (10 g / sample) were placed in a 100 ml beaker and allowed to stand at room temperature for 30 min before being placed into the machine. The injection needle was directly inserted into the sealed beaker containing the sample, and the electronic nose was used for measurement. The sampling time was 1 s / sample. The sensor self-cleaning time was 80 s, and the zeroing time was 10 s. The sample preparation time was 5 s, and the injection flow rate was 400 mL / min.

[0105] Electronic tongue analysis: The TS-5000Z intelligent taste analysis system (electronic tongue) was used to detect the taste characteristics of fresh mutton samples. This device is equipped with five chemical sensors (CO0 sensor for bitterness and after-bitterness, AE1 sensor for astringency and after-astringency, AAE sensor for umami and richness, CTO sensor for saltiness, and CA0 sensor for sourness) and two reference electrodes. The specific operating procedure is as follows: 30.0 g of minced mutton sample was accurately weighed, 150 mL of distilled water was added, and the mixture was homogenized to extract flavor substances. After centrifugation at 2265×g for 10 min at 4℃, the supernatant was collected for electronic tongue detection to evaluate the taste characteristics of the mutton sample.

[0106] 3.1.6 Data processing and analysis: Same as Part 1, 2.1.6.

[0107] 3.2 Results and Analysis 3.2.1 Effects of RPAAs Added to Low-Protein Feed on Conventional Quality Indicators of Yudong Black Goat Meat The flesh-colored results indicated that, compared to the other three groups, the MAG group had L... ✱ The values ​​(brightness) all decreased significantly. P <0.05), b ✱ The yellowness value (yellowness) also showed a significant difference compared to the control group. P <0.05), the lowest value among the three groups; while the GLY and MET groups, compared with the control group, had b ✱ The values ​​decreased by 23.19% and 21.79%, but the difference was not significant. P >0.05); Shear force results showed that, compared with the control group and the MET group, the MAG group significantly reduced [the stress] by 43.11% and 40.25%, respectively. P <0.05), while the GLY group also showed a 31.21% reduction compared to the control group ( P >0.05); The results of the textural properties showed that, compared with the control group, GLY exhibited significantly improved cohesiveness, elasticity, and adhesiveness by 7.89%, 68.16%, and 27.96%, respectively. P <0.05), the elasticity, adhesiveness, and chewiness of the MET group were significantly improved by 38.54%, 34.39%, and 66.51%, respectively. P <0.05), the cohesion and elasticity of the MAG group were significantly improved by 7.89% and 45.25%, respectively. P <0.05); The crude ash content results showed that the GLY group significantly increased by 7.87% compared to the MET group. P <0.05).

[0108] Other indicators showed that, compared with the control group, the pH, redness, moisture, cooking loss, drip loss, intramuscular fat, hardness, adhesiveness, centrifugal water loss rate, and fat and protein content of the GLY, MET, and MAG groups were not significantly different. P >0.05) 3.2.2 Effects of RPAAs Addition to Low-Protein Feed on Amino Acid and Fatty Acid Content in the Muscle of Yudong Black Goats This experiment measured a total of 17 amino acids, including essential amino acids required to maintain animal life activities and umami amino acids from meat. There were no significant differences in the total amino acid content and the total essential amino acid content. P >0.05), but showed significant differences in total umami amino acids, with the GLY and MAG groups showing significantly higher levels compared to the control group ( P <0.05). Compared with the MET group, the cystine content in the GLY and MAG groups decreased significantly by 33.33% and 23.07%, respectively. P <0.05), and the control group also showed a significant increase of 22.22% compared to the GLY group ( P <0.05); compared with MAG, the isoleucine content in the MET group decreased significantly by 14.28% ( P <0.05); compared with the control group, the tyrosine content in the MAG group was significantly increased by 32.55% ( P <0.05); Regarding fatty acids, compared with the control group, the C20:3n6 of the GLY group, MET group, and MAG group was significantly increased by 43.39%, 54.79%, and 23.51%, respectively ( ). P<0.05), and the C14:0, C18:2n6c, and C20:1 in the MAG group were significantly higher than those in the control group by 20.67%, 24.88%, and 26.25%, respectively. P <0.05).

[0109] 3.2.3 Detection of the flavor of Yudong black goat meat in different experimental groups using electronic nose and electronic tongue 3.2.3.1 Detection of the flavor of Yudong black goat meat in different experimental groups using an electronic nose Electronic noses represent the human sense of smell and can accurately detect various molecules. They are typically equipped with 5 to 100 sensitive sensors, enabling them to identify subtle differences in volatile compounds. The 10 sensors used in this experiment were W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2S, W2W, and W3S, with corresponding sensitivities of aromatic benzenes, nitrogen oxides, aromatic amines, hydrides, short-chain alkanes, methyl compounds, sulfides, alcohols, aldehydes, ketones, organosulfur compounds, and long-chain alkanes, respectively. Compared to the control and MET groups, the response values ​​of W5S, W6S, W1S, W1W, W2S, W2W, and W3S in raw mutton were significantly reduced in the GLY and MAG groups. P <0.05), the response values ​​of W1, W3C and W5C in raw mutton were significantly increased ( P <0.05). This indicates that both the GLY and MAG groups reduced the odor intensity of nitrogen oxides, hydrides, methyl compounds, sulfides, alcohols, aldehydes, ketones, organosulfur compounds, and long-chain alkanes in raw mutton, while enhancing the odor intensity of aromatic benzenes, aromatic amines, and short-chain alkanes.

[0110] Compared with the control group, the W1C, W3C, and W5C response values ​​of cooked mutton in the GLY and MAG groups were significantly increased. P＜ 0.05), W2S and W3S decreased significantly ( P＜ 0.05); W6S in cooked mutton of the MAG group was significantly reduced ( P＜ 0.05). This indicates that both the GLY and MAG groups increased the odor intensity of aromatic benzenes, aromatic amines, and short-chain alkanes in cooked mutton, while decreasing the odor intensity of alcohols, aldehydes, ketones, and long-chain alkanes. The MAG group also decreased the odor intensity of hydrides.

[0111] 3.2.3.2 Detection of the flavor of Yudong black goat meat in different experimental groups using an electronic tongue Like electronic noses, electronic tongues are widely used, objective, and effective tools commonly used to assess the flavor characteristics of food. Sourness, Bitterness, Astringency, Aftertaste-B, Aftertaste-A, Umami, Richness, and Saltiness represent eight sensors of the electronic tongue, corresponding to sensitivities of sourness, bitterness, astringency, bitter aftertaste, astringent aftertaste, umami, richness, and saltiness, respectively. In this experiment, compared with the control group, there were no significant changes in sourness, bitterness, astringency, bitter aftertaste, astringent aftertaste, umami, richness, and saltiness of lamb in the GLY, MET, and MAG groups. P The concentration of umami (>0.05) was significantly higher than that of the control group, but its umami, richness and saltiness were improved by 20.55%, 13.26% and 34.69% and 16.60%, 13.26% and 40.68% respectively, and 13.55%, 10.37% and 32.65% respectively. The MAG group showed the greatest improvement.

[0112] 3.3 Summary In summary, regarding meat quality, the shear force was significantly reduced in both the GLY and MAG groups. P <0.05), the cohesiveness, elasticity and adhesiveness of the GLY group were significantly improved ( P <0.05), the MET group showed a 66.51% increase in chewing ability, the highest among the four groups. P <0.05); GLY and MAG groups significantly increased umami amino acids in muscle ( P <0.05); Regarding meat flavor, electronic nose testing showed that both the GLY and MAG groups significantly reduced unpleasant odor substances such as nitrogen oxides ( ). P <0.05), while significantly enhancing the strength of aromatic compounds ( P (<0.05), there was no significant difference in taste indicators detected by electronic tongue, but all improved the umami, richness and umami of meat, and reduced sourness.

[0113] The results of this study indicate that supplementing low-protein diets with RPAAs can significantly improve the quality characteristics of mutton. The GLY and MAG groups showed a significant reduction in muscle pH. 45min and pH 24h ( P <0.05), with the GLY group showing a 31.21% decrease in shear force, and the MAG group showing a significantly lower decrease compared to the control group ( P <0.05). Regarding textural properties, the GLY group significantly improved cohesion, elasticity, and adhesiveness, while the MET group had the highest chewiness among the four groups. Flavor analysis showed that the GLY and MAG groups significantly increased umami amino acids (…). P<0.05), electronic nose testing showed that it reduced unpleasant odor substances such as nitrogen oxides, while enhancing the intensity of aromatic compounds ( P <0.05). Principal component analysis effectively distinguished the flavor characteristics of different treatment groups, but electronic tongue detection showed no significant differences in taste indicators. Overall, the addition of RPGly and RPMet shows potential in improving meat tenderness and flavor characteristics.

[0114] Example 3

[0115] Part Three: Metabolomics Analysis of the Effects of RPAAs Addition to Low-Protein Feed on Muscle Quality in Yudong Black Goats 4.1 Materials and Methods 4.1.1 Test materials: Same as Part 2, 2.1.1.

[0116] 4.1.2 Experimental animals and design: Same as Part 2, 2.1.2.

[0117] 4.1.3 Experimental feed: Same as Part 2, 2.1.3.

[0118] 4.1.4 Feeding and management: Same as Part 2, 2.1.4.

[0119] 4.1.5 Measurement Indicators and Methods Non-target metabolites in muscle samples were identified using ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UHPLC-Q-TOF MS). First, an extraction solution of methanol / acetonitrile / water (v / v, 2:2:1) was added to 100 mg of thawed meat sample at 4°C. The mixture was then swirled, sonicated, centrifuged, and subsequently vacuum-dried to obtain the supernatant. Then, a mixture of 50 μL of acetonitrile and water (v / v, 1:1) was added, followed by vortex mixing and centrifugation. The resulting supernatant was then analyzed on a column at 25°C using a 2 μL injection volume. Mobile phase A (ultrapure water containing 25 mmol·L⁻¹ ammonium acetate and 25 mmol·L⁻¹ ammonia) and mobile phase B (mass spectrometry grade acetonitrile) were used at a flow rate of 0.5 mL / min. Mass spectrometry analysis was performed using a triple TOF 6600 mass spectrometer, with detection using electrospray ionization (ESI) in both positive and negative ion modes. After all measurements were completed, data processing was performed using XCMS software, including peak alignment, retention time correction, and peak area extraction. The results were then analyzed using SIMCA18.0 software to determine differences between the two groups. The differentially expressed metabolites were functionally annotated and their corresponding metabolic pathways identified using the Kyoto Encyclopedia of Genetics and Genomes (KEGG, www.genome.jp / kegg).

[0120] 4.1.6 Data processing and analysis: Same as Part 2, 2.1.6.

[0121] 4.2 Summary This study used non-targeted metabolomics analysis to reveal the effects of adding different RPAAs to a low-protein diet on the metabolic pathways and metabolites of the longissimus dorsi muscle in Yudong black goats.

[0122] Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) validated the reliability of the metabolic data (R2>0.5, Q2>0.3), and the addition of amino acids significantly altered the muscle metabolite profile. The number of differentially expressed metabolites screened were 69 in the GLY group, 48 in the MET group, and 70 in the GAM group.

[0123] KEGG enrichment analysis showed that GLY promotes the utilization of unsaturated fatty acids by activating linoleic acid metabolism and the ABC transporter pathway; MET focuses on regulating arginine biosynthesis and the cAMP signaling pathway, enhancing nitric oxide-mediated microcirculation; while the MAG combination synergistically upregulates glycerophospholipid metabolism, glutathione metabolism, and aminoacyl-tRNA biosynthesis, exhibiting an additive effect in maintaining cell membrane integrity (e.g., glycerophosphocholine accumulation), antioxidant defense (increased γ-glutamylcysteine), and protein translation efficiency (enhanced L-aspartate / L-proline). All three experimental groups inhibited apoptosis-related pathways (e.g., reduced sphingosine) and restructured energy metabolism (downregulation of glycolysis and enhancement of lipid metabolism), suggesting that amino acid intervention improves muscle growth and quality by reducing muscle cell loss and optimizing energy distribution.

[0124] Non-target metabolomics analysis further revealed that the low-protein amino acid pattern significantly affected important metabolic pathways and metabolites in the longissimus dorsi muscle of Yudong black goats, clarifying the key metabolic pathways and metabolites that regulate the improvement of meat quality in Yudong black goats under this pattern.

[0125] In summary, this invention demonstrates that adding specific RPAAs to low-protein feed can significantly improve the slaughter performance and meat quality of Yudong Black Goats without affecting their growth and normal physiological metabolism, and has good potential for widespread application.

[0126] RPGly: GLY's main function is to promote the utilization of unsaturated fatty acids by activating linoleic acid metabolism and the ABC transporter pathway.

[0127] RPMet: MET's main function is to regulate arginine biosynthesis and the cAMP signaling pathway, and to enhance nitric oxide-mediated microcirculation.

[0128] RPGly and RPMet: The main function of the glycine and methionine combination is to synergistically upregulate glycerophospholipid metabolism, glutathione metabolism, and aminoacyl-tRNA biosynthesis, exhibiting an additive effect in maintaining cell membrane integrity (e.g., glycerophosphocholine accumulation), antioxidant defense (increased γ-glutamylcysteine), and protein translation efficiency (enhanced L-aspartate / L-proline). Amino acid intervention improves muscle growth and quality by reducing muscle cell loss and optimizing energy distribution.

[0129] In summary, this invention, through the combination of "low-protein diet + specific rumen-protected biological agents," has demonstrated through a series of verified experimental results that it improves nitrogen utilization efficiency and achieves the goal of improving quality and efficiency by reducing nitrogen input, maintaining the same growth performance, and producing higher quality meat products (more eye muscle and better flavor).

[0130] Nitrogen reduction at the input end: By adopting a low-protein diet and adjusting the feed formula (such as reducing the amount of soybean meal), the crude protein (CP) level of the basic feed was significantly reduced from 13.4% in the control group to 11.0%. This directly reduced the total nitrogen intake of animals at the source, providing a necessary prerequisite for achieving "nitrogen reduction in feces and urine".

[0131] Achieving efficiency and quality improvement at the output end: (1) Achieving substantial effects of nitrogen reduction and emission reduction: Although the total nitrogen intake in the feed is reduced, the present invention, by adding specific RPGly and / or RPGet biological agents, makes the average daily weight gain (ADG) of the experimental group not significantly different from that of the control group, and the feed conversion ratio (FCR) shows a decreasing trend. More importantly, the eye muscle area (i.e., body protein deposition) of the MAG group (RPGly+RPMet) is significantly increased. This strongly proves that the present scheme significantly improves the efficiency of animal utilization of feed nitrogen by precisely supplementing (conditional) limiting amino acids. Animals synthesize the same or even more body protein (muscle) with less nitrogen intake, which inevitably leads to a corresponding reduction in the excretion of "ineffective nitrogen" as metabolic waste through feces and urine. (2) This scheme not only maintained the yield, but also brought about a significant improvement in quality: the tenderness of the mutton was significantly improved (the shear force of the MAG group was reduced), the texture of the meat was improved (such as the elasticity and adhesiveness of the GLY group were improved), especially in terms of flavor: the content of umami amino acids in the GLY and MAG groups was significantly increased, while the intensity of unpleasant odors such as nitrogen oxides and sulfides was significantly reduced, and the aroma of aromatic benzenes was improved.

Claims

1. A biological agent comprising rumen-passing glycine RPGly; wherein the rumen-passing rate of said RPGly is not less than 94.0% to 97.0%, and the small intestinal release rate is 88.0% to 93.0%.

2. The biological agent according to claim 1, wherein RPGly is a microcapsule formulation, and the microcapsule comprises three parts: a core, an inner coating, and an outer coating, wherein: The outer coating comprises a composite film-forming material and functional additives, including chitosan and nano-silica.

3. The biological agent as described in claim 1, wherein RPGly is a microencapsulated granule formulation, and the granule comprises three parts: a core, an inner coating, and an outer coating, wherein: The capsule core is composed of glycine and a carrier; The carrier includes one or more of the following: silica, corn starch, maltodextrin, and sodium carboxymethyl cellulose. The amount of carrier added is 5.0%–15.0% of the mass of glycine. Inner coating: Composed of a composite coating base material and antioxidants. The composite coating base material is a compound of hydrophobic beeswax and hydrogenated castor oil. The antioxidants are one or more of the following: ethoxyquinoline, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tert-butylhydroquinone, tea polyphenols, vitamin E, and ascorbyl palmitate. Outer coating: composed of composite film-forming material, organic solvent and functional additive; wherein the organic solvent is a mixture of ethanol, acetone and glycerol monoacetate; the composite film-forming material is a compound system of hydrogenated palm oil, cellulose acetate and polyethylene glycol; the functional additive is a mixture of chitosan and nano silica, and the amount added is 3% to 5% of the mass of the composite film-forming material.

4. The biological agent as described in claim 1, wherein the RPGly microcapsules comprise the following components by mass percentage: glycine 40%–60%, hydrophobic beeswax 7.5%–22.5%, hydrogenated castor oil 2.5%–7.5%, antioxidant 0.04%–0.2%, composite film-forming material 20%–40%, functional additives 0.4%–1.6%, and organic solvent 1.0%–2.0%. Alternatively, glycine 40.0%–60.0%, hydrophobic beeswax 7.5%–22.5%, hydrogenated castor oil 2.5%–7.5%, antioxidant 0.04%–0.2%, composite film-forming material 20.0%–40.0%, functional additives 0.4%–1.6%, and organic solvent 1.0%–2.0%; carrier 4.0%–8.0%, and the sum of the mass percentages of all components is 100%.

5. The biological agent according to claim 1, wherein the RPGly microcapsules are prepared by the following steps: (1) Preparation of capsule core microspheres: Glycine and carrier are mixed in proportion to form uniform microspheres with a particle size of 0.5-1.0 mm; then the microspheres are placed in a mixer, the molten composite coating base is added first, and the mixture is stirred at 150-200 r / min for 5 min. Then the antioxidant is slowly added and the mixture is stirred for 5-10 min until the surface of the microspheres is uniformly coated with the inner coating. (2) Preparation of primary coated particles: The micro-particles coated with the inner layer are fed into a freeze spray dryer at a drying temperature of 30.0-40.0 ℃ and dried until the moisture content of the particles is ≤ 0.5% to obtain primary coated particles; (3) Coating and preparation of finished product: The composite film-forming material is added to an organic solvent and stirred at 50.0-60.0℃ for 30 min until completely dissolved. Then, functional additives are added and stirring is continued for 20 min to prepare a uniform coating solution. The primary coated particles are fed into a bottom spray fluidized bed coating machine for coating. After completion, the particles are cooled in stages and then screened through a 20-60 mesh sieve to obtain rumen-protected glycine microcapsules.

6. The biological agent as described in claim 1 further comprises rumen-exposed methionine RPMet, wherein the ratio of RPMet to RPGly is 0.7:1 to 1.2:1; wherein the rumen-exposed rate of RPMet is 85.0% to 90.0%, and the small intestinal release rate is 80.0% to 85.0%.

7. The biological agent as described in any one of claims 1-6, wherein the biological agent is used in ruminant feed; preferably in black goats, especially during the fattening period.

8. An animal feed comprising a basal feed and any one of the biological agents of claims 1-6, wherein the basal feed is a low-protein feed.

9. The animal feed as described in claim 8, wherein the protein content of the low-protein feed is not more than 11.0%, and the dosage of the biological agent is 3.0~5.0g of biological agent per kg of concentrate feed.

10. The method of using animal feed as described in claim 9, firstly calculate the amount of biological agent based on the previous day's feed intake, mix the biological agent with the concentrate and feed it, mix and feed immediately; feed once in the morning and once in the afternoon, with the same amount in the morning and afternoon; feed hay first each time, and feed concentrate 1 hour later.

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