A rumen-protected folic acid preparation, a method for preparing the same and use thereof in improving lactation performance of ruminants

Abstract

The present application belongs to the technical field of veterinary nutritional supplements or feed additives, and particularly relates to a rumen-protected folic acid preparation, a preparation method thereof and application thereof in improving lactation performance of ruminants. The rumen-protected folic acid preparation prepared by the present application is double-coated, and the degradation rate thereof in the rumen can be as low as 12.5% in 12 hours, and the release rate thereof in the small intestine can be as high as 71.7%, effectively solving the technical problem that more than 95% of ordinary folic acid is degraded in the rumen, and significantly improving the absorption and utilization rate of 5-methyltetrahydrofolic acid (5-MTHF) in the small intestine. In addition, ordinary folic acid needs to be reduced by dihydrofolic acid reductase (DHFR) and methylene tetrahydrofolic acid reductase (MTHFR) in two steps to generate active 5-MTHF, and the metabolic efficiency is limited; the present application directly supplements 5-MTHF, bypasses the metabolic bottleneck, and has a more direct and efficient effect, and thus can significantly improve the lactation performance and milk quality of ruminants.

Description

Technical Field

[0001] This invention belongs to the technical field of veterinary nutritional supplements or feed additives, specifically relating to a rumen-exposed folic acid preparation, its preparation method, and its application in improving the lactation performance of ruminants. Background Technology

[0002] Folic acid (vitamin B9) is an important nutrient for regulating the growth and development of ruminants and improving their production performance. It participates in many physiological processes, including cell division, protein metabolism, and DNA and RNA biosynthesis. With the optimization of ruminant breeds and the popularization of large-scale intensive farming models, the production potential of livestock and poultry continues to improve, and the body's nutritional demand for folic acid has also increased significantly.

[0003] Traditional farming practices suggest that rumen microorganisms in ruminants can synthesize sufficient folic acid to meet their physiological needs. However, large-scale, high-yield farming practices have shown that endogenous folic acid synthesis in the rumen is insufficient to meet the nutritional requirements of high-yield livestock and poultry. Meanwhile, modern research confirms that natural folic acid requires two reduction reactions in the body to be converted into its active form, 5-methyltetrahydrofolate (5-MTHF). This active substance is also a core functional substance in animals that participates in one-carbon metabolism and supplies methyl groups.

[0004] However, folic acid is easily decomposed and destroyed by microorganisms in the rumen of ruminants. The vast majority of exogenous folic acid supplemented in the diet is degraded and lost in the rumen, with only a very small portion reaching the small intestine for absorption and utilization. Related research data indicates that over 95% of dietary folic acid is degraded and rendered ineffective by microorganisms in the rumen. Therefore, developing rumen-passing folic acid formulations that can pass smoothly through the rumen and avoid microbial degradation, enabling their targeted transport to the small intestine for efficient absorption, is of significant practical importance for substantially improving folic acid utilization in ruminants and meeting the nutritional needs of high-yield livestock farming. Summary of the Invention

[0005] The purpose of this invention is to provide a rumen-protected folic acid preparation, its preparation method, and its application in improving the lactation performance of ruminants. The rumen-protected folic acid preparation can significantly improve the folic acid utilization rate of ruminants, and at the same time improve the lactation performance and milk quality of ruminants.

[0006] This invention provides a rumen-protected folic acid preparation, wherein the raw materials for preparing the rumen-protected folic acid preparation include a first raw material and a second raw material. The first raw material comprises the following components in parts by weight: 1.8-2.7 parts of 5-methyltetrahydrofolate metal salt, 20.5-30.5 parts of silicon dioxide, 21.7-32.7 parts of palm fat, 22.7-26.7 parts of coconut oil, and 12-18 parts of calcium stearate. The second raw material includes a fatty acid enzyme. The weight ratio of the first raw material to the second raw material is 5000:(4-7).

[0007] Preferably, the 5-methyltetrahydrofolate metal salt includes 5-methyltetrahydrofolate calcium salt with a purity ≥98%; the silica has a particle size of 400 mesh.

[0008] Preferably, the lipase includes triacylglycerol lipase, enzyme classification number EC 3.1.1.3; the enzyme activity of the lipase is 8 × 10⁻⁶ based on the total amount of enzyme activity added. 5 ~1.4×10 6 Raw materials are prepared using U / 100 kg.

[0009] The present invention also provides a method for preparing the rumen-protected folic acid preparation described in the above technical solution, comprising the following steps: A first mixture is prepared by mixing 5-methyltetrahydrofolate metal salt, lipase, and a portion of silica to obtain a first mixture, wherein the portion of silica accounts for 9.5% to 12.5% ​​of the total weight of silica. The first mixture is mixed with the remaining silica to obtain a second mixture; Coconut oil and a portion of palm fat are heated to 55-65°C, then mixed with the second mixture and granulated to obtain a granular semi-finished product. The portion of palm fat is 75%-85% of the total weight of palm fat. The remaining palm fat is heated to 55-65°C and mixed with calcium stearate to coat the outer layer of the granular semi-finished product, thus obtaining a rumen-protected folic acid preparation.

[0010] Preferably, the first and second mixing are carried out using a low-shear mixing device with a rotation speed of 10-30 rpm and a mixing time of 15-30 min.

[0011] The present invention also provides the application of the rumen-protected folic acid preparation described in the above technical solution or the preparation method described in the above technical solution in the preparation of folic acid supplement products.

[0012] Preferably, the folic acid supplement product includes feed additives and / or feed.

[0013] The present invention also provides a feed containing folic acid, comprising a rumen-protected folic acid preparation and a basal diet, wherein the rumen-protected folic acid preparation is the rumen-protected folic acid preparation described in the above technical solution or the rumen-protected folic acid preparation prepared by the preparation method described in the above technical solution.

[0014] The present invention also provides the application of the rumen-protected folic acid preparation described in the above technical solution or the rumen-protected folic acid preparation prepared by the preparation method described in the above technical solution or the folic acid-containing feed described in the above technical solution in improving the folic acid utilization rate of ruminants, improving the lactation performance of ruminants, improving the milk quality of ruminants and regulating mammary gland development.

[0015] Preferably, the application includes supplementing ruminants with the rumen-protected folic acid preparation, with a supplementation amount of 100-300 mg / day per ruminant, calculated based on the dosage of 5-methyltetrahydrofolate, for at least 70 consecutive days.

[0016] Beneficial effects: This invention provides a rumen-protected folic acid preparation (i.e., rumen-protected protective 5-methyltetrahydrofolate, RP5-MTHF) and its preparation method. The RP5-MTHF prepared using the raw materials and method described in this invention has a double-layer coating, exhibiting a degradation rate as low as 12.5% ​​in the rumen after 12 hours and a release rate as high as 71.7% in the small intestine. This effectively solves the technical problem of over 95% degradation of ordinary folic acid in the rumen, significantly improving the absorption and utilization rate of 5-MTHF in the small intestine. Furthermore, ordinary folic acid requires a two-step reduction process involving DHFR and MTHFR to generate active 5-MTHF, limiting its metabolic efficiency. This invention directly supplements 5-MTHF, bypassing the metabolic bottleneck, resulting in a more direct and efficient effect.

[0017] Based on this, supplementing ruminants with the aforementioned rumen-protected folic acid preparation can significantly improve their lactation performance and comprehensively improve milk quality. Examples have demonstrated that adding RP5-MTHF (200 mg / d) to the diet can increase the actual milk yield of dairy cows by more than 8.3%, the yield of 4% fat-corrected milk by more than 11.6%, the milk fat percentage by more than 6.0%, and the milk protein percentage by more than 6.6%. Simultaneously, it significantly increases the 5-MTHF content in the milk, producing fortified milk rich in active folic acid, achieving a synergistic improvement in lactation performance and milk quality. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The effect of dietary supplementation with a medium dose of rumen-protective 5-methyltetrahydrofolate (5-MTHF) in Example 2 on the expression of proliferation-related proteins in mammary gland tissue of mid-lactation dairy cows was investigated. A represents the protein expression of PCNA, Cyclin D1, BCL2, BAX, caspase-3, and caspase-9 in mammary gland tissue of the control group (0 mg / d 5-MTHF) and the MRP5-MTHF group (200 mg / d 5-MTHF) analyzed by Western blot. B represents the mean ± SEM value of the immunopositive bands for PCNA, Cyclin D1, BCL2, BAX, BCL2 / BAX, caspase-3, and caspase-9. The relative expression levels of each target protein were standardized using the control group as the benchmark. P <0.05 and P <0.01 indicates a significant difference compared to the control group; Figure 2 This study investigated the effect of dietary supplementation with a medium dose of rumen-protective 5-methyltetrahydrofolate (MRP5-MTHF) as described in Example 2 on the Akt-mTOR signaling pathway in mammary gland tissue of mid-lactation dairy cows. A represents the protein expression of SLC19A1, Akt, p-Akt, mTOR, and p-mTOR in mammary gland tissue from the control group (0 mg / d 5-MTHF) and the MRP5-MTHF group (200 mg / d 5-MTHF) analyzed by Western blot. β-actin served as a loading control. B represents the mean ± SEM value of the immunopositive bands for SLC19A1, Akt, p-Akt, mTOR, and p-mTOR. The relative expression levels of each target protein were normalized using the control group as a standard. P <0.05 and P <0.01 indicates a significant difference compared to the control group; Figure 3 To investigate the effects of dietary supplementation with a medium dose of rumen-protective 5-methyltetrahydrofolate (MRP5-MTHF) as described in Example 2 on the expression of fatty acid synthesis-related proteins in mammary gland tissue of mid-lactation dairy cows; A shows the protein expression of AMPK, p-AMPK, PPARγ, SREBP1, ACACA, p-ACACA, FASN, and SCD1 in mammary gland tissue of the control group (0 mg / d 5-MTHF) and the MRP5-MTHF group (200 mg / d 5-MTHF), with β-actin serving as a loading control; B shows the mean ± SEM value of the immunopositive bands for PPARγ, SREBP1, p-ACACA / ACACA, FASN, SCD1, and p-AMPK / AMPK; the relative expression levels of each target protein were standardized using the control group as the standard. P <0.05 and P <0.01 indicates a significant difference compared to the control group.

[0020] Terminology Explanation: 5-Methyltetrahydrofolate (5-MTHF): The main circulating active form of folic acid in the body, which can directly participate in one-carbon metabolism as a methyl donor.

[0021] Rumen protection: A technology that uses physical or chemical methods to reduce the degradation rate of nutrients in the rumen, thereby enabling them to reach the abomasum and small intestine for absorption and utilization.

[0022] SLC19A1: Solute Carrier Family 19 Member 1, also known as proton-coupled folic acid transporter (PCFT), is responsible for transporting 5-MTHF from the extracellular space into the cell. It is a key transporter protein for the uptake of 5-MTHF by mammary epithelial cells.

[0023] RP5-MTHF: Rumen-Protected 5-Methyltetrahydrofolate. Detailed Implementation

[0024] This invention provides a rumen-protected folic acid preparation, wherein the raw materials for preparing the rumen-protected folic acid preparation include a first raw material and a second raw material. The first raw material comprises the following components in parts by weight: 1.8-2.7 parts of 5-methyltetrahydrofolate metal salt, 20.5-30.5 parts of silicon dioxide, 21.7-32.7 parts of palm fat, 22.7-26.7 parts of coconut oil, and 12-18 parts of calcium stearate. The second raw material includes a fatty acid enzyme. The weight ratio of the first raw material to the second raw material is 5000:(4-7).

[0025] In one embodiment, the 5-methyltetrahydrofolate metal salt of the present invention comprises 2.2 to 2.5 parts by weight; the 5-methyltetrahydrofolate metal salt includes calcium 5-methyltetrahydrofolate, sodium 5-methyltetrahydrofolate, potassium 5-methyltetrahydrofolate, or magnesium 5-methyltetrahydrofolate, and may further be calcium 5-methyltetrahydrofolate; in one embodiment, the purity of the 5-methyltetrahydrofolate metal salt is ≥98%.

[0026] In one embodiment, the silica can be in the form of 22.5 to 25.08 parts by weight; the silica has a particle size of 400 mesh; the silica with this particle size has the functional advantages of acting as a dispersing carrier and anti-caking agent, which can ensure the uniform distribution of 5-methyltetrahydrofolate metal salt and lipase in the formulation and improve the mechanical strength of the particles.

[0027] In one embodiment, the palm fat is 24.7 to 30.7 parts by weight; the palm fat may be palm stearin; further, the palm stearin has a melting point range of 44 to 46°C, an iodine value ≤ 5, and a main fatty acid composition (typical value): palmitic acid (C16:0) 50% to 60%, stearic acid (C18:0) 4% to 6%, and oleic acid (C18:1) 25% to 35%. In specific embodiments, Chunjin fat powder or other commercially available products with the above characteristics may be selected.

[0028] In one embodiment, the coconut oil may be present in 23.7 to 24.7 parts by weight. In another embodiment, the calcium stearate may be present in 15 to 17 parts by weight.

[0029] In one embodiment, the lipase includes triacylglycerol lipase, enzyme classification number EC3.1.1.3; further, it is derived from... Thermomyces lanuginosus or Aspergillus niger The feed-grade lipase has an optimal pH of 7.0–8.5, exhibits ≥80% enzyme activity retention after treatment at 60°C for 30 minutes, and demonstrates bile salt tolerance. As one embodiment, the lipase has an enzyme activity of 8 × 10⁻⁶ based on the total amount added. 5 ~1.4×10 6 The raw material can be prepared at a concentration of U / 100 kg, which can further be 1.0 × 10⁻⁶. 6 U / 100 kg of the first preparation raw material, such as Lipolase from Novozymes, can be used. ® 100L of feed-grade lipase from Ningxia Xiasheng Industrial Group Co., Ltd., with an enzyme activity ≥10,000 U / g. This invention introduces lipase into a rumen-protected folic acid preparation, constructing a dual release control system of "physical coating (lipid layer) + enzymatic triggering (lipase)". This design cleverly utilizes the differences in the rumen and small intestine environments (pH, bile salts, enzyme activity) of ruminants to achieve targeted delivery of 5-MTHF to the small intestine, significantly superior to existing technologies that rely solely on physical coating. Furthermore, the addition of lipase is not a simple additive of components, but rather represents a technological leap from "passive release" to "active controlled release". In addition, because the rumen pH is near neutral (about 6.0-7.0) and rich in microbial proteases, exogenous lipases have low activity or are easily degraded in the rumen; however, when the particles enter the small intestine, the pH becomes weakly alkaline (about 7.0-8.0), and there is a bile salt emulsification environment, the lipase activity is significantly activated, and the outer lipid coating is rapidly decomposed, thereby releasing the inner 5-methyltetrahydrofolate for absorption by the small intestine.

[0030] The present invention also provides a method for preparing the rumen-protected folic acid preparation described in the above technical solution, comprising the following steps: A first mixture is prepared by mixing 5-methyltetrahydrofolate metal salt, lipase, and a portion of silica to obtain a first mixture, wherein the portion of silica accounts for 9.5% to 12.5% ​​of the total weight of silica. The first mixture is mixed with the remaining silica to obtain a second mixture; Coconut oil and a portion of palm fat are heated to 55-65°C, then mixed with the second mixture and granulated to obtain a granular semi-finished product. The portion of palm fat is 75%-85% of the total weight of palm fat. The remaining palm fat is heated to 55-65°C and mixed with calcium stearate to coat the outer layer of the granular semi-finished product, thus obtaining a rumen-protected folic acid preparation.

[0031] This invention involves a first mixing of 5-methyltetrahydrofolate metal salt, lipase, and a portion of silica to obtain a first mixture. The silica in this invention comprises 9.5% to 12.5% ​​of the total weight of silica, and more specifically, 11.96%. In this invention, the lipase is pre-mixed with 5-methyltetrahydrofolate metal salt and a portion of silica, and embedded in the inner layer of the particles to achieve enzymatically triggered release in the small intestine.

[0032] In one embodiment, the first and second mixing of the present invention are carried out using a low-shear mixing device with a rotation speed of 10-30 rpm and a mixing time of 15-30 min; in another embodiment, the first mixing of the present invention uses a waist drum mixer and the second mixing uses a V-type mixer.

[0033] After obtaining the first mixture, the present invention performs a second mixing with the remaining silica to obtain a second mixture. The present invention achieves "equal incremental addition" (geometric dilution) mixing by mixing silica in steps, ensuring the uniform distribution of trace components. In the first mixing process of the present invention, the amount of silica is equivalent to the total amount of the active ingredient 5-methyltetrahydrofolate, forming a "concentrated premix." Because the two have similar volumes and densities, uniform distribution is easily achieved. In the second mixing process of the present invention, the active ingredient is already uniformly dispersed in the silica. When mixed with the remaining large amount of silica, it is equivalent to "diluting" the concentrated premix, easily achieving overall uniformity. Because the ratio of trace active ingredient (5-methyltetrahydrofolate metal salt) to the main material (silica) is significantly different (1:10 or higher), the mixing efficiency is extremely low, requiring very long mixing times and special mixing equipment to barely achieve uniformity, and electrostatic agglomeration is easily generated. The stepwise mixing method in the present invention avoids the problem of electrostatic agglomeration.

[0034] After obtaining the second mixture, the present invention heats coconut oil and a portion of palm fat to 55-65°C, mixes them with the second mixture, and granulates them to obtain a granular semi-finished product. The portion of palm fat mentioned in the present invention comprises 75%-85% of the total weight of palm fat, more specifically 80.46%. As one embodiment, the present invention heats a portion of palm fat and coconut oil to 60°C, mixes them with the second mixture, and granulates them; the particle size of the granular semi-finished product is 0.8-1.2 mm. In the present invention, heating palm fat and coconut oil to 55-65°C (preferably 60°C) is not a simple conventional process choice, but rather a comprehensive consideration based on balancing lipid fluidity, lipase thermal stability, and granulation formability. Experiments show that when the heating temperature is below 55°C, the viscosity of the molten lipid is too high, resulting in uneven mixing with the second mixture, leading to a decrease in particle coating integrity and an increase in rumen degradation rate; when the temperature is above 65°C, although fluidity is better, the loss of lipase activity increases significantly, and the small intestinal release rate decreases. Therefore, controlling the heating temperature at 55~65℃ (especially 60℃) is one of the key process parameters to achieve the dual effect of "low rumen degradation - high small intestinal release" of this invention.

[0035] To obtain the granular semi-finished product, the present invention heats the remaining palm fat to 55-65°C and mixes it with calcium stearate to coat the outer layer of the granular semi-finished product, thus obtaining a rumen-protected folic acid preparation. As one embodiment, the present invention heats the remaining palm fat to 60°C and mixes it with calcium stearate to coat the outer layer of the granular semi-finished product. As one embodiment, the coating process involves: heating the remaining palm fat to 55-65°C (preferably 60°C) until it is molten, adding calcium stearate and stirring until uniformly dispersed, applying the resulting molten mixture to the surface of the granular semi-finished product by spraying or pouring, ensuring the molten mixture uniformly covers the particle surface under continuous tumbling conditions, and then cooling and solidifying to obtain the rumen-protected folic acid preparation. As one embodiment, the particle size of the rumen-protected folic acid preparation of the present invention is 0.8 mm to 1.6 mm.

[0036] The preparation method of the present invention achieves a double-layer coating of 5-methyltetrahydrofolate. Specifically, the first layer is a drug-containing inner layer composed of a mixture of coconut oil, palm fat, and active ingredients, and the second layer is an outer protective coating composed of palm fat and calcium stearate.

[0037] This invention also provides the application of the rumen-protected folic acid preparation described in the above-described technical solutions or the rumen-protected folic acid preparation prepared by the above-described preparation methods in the preparation of folic acid supplement products. As one embodiment, the folic acid supplement product of this invention includes feed additives and / or feed; as another embodiment, the feed additive can be a nutritional supplement.

[0038] The present invention also provides a feed containing folic acid, comprising a rumen-protected folic acid preparation and a basal diet, wherein the rumen-protected folic acid preparation is the rumen-protected folic acid preparation described in the above technical solution or the rumen-protected folic acid preparation prepared by the preparation method described in the above technical solution.

[0039] The present invention also provides the application of the rumen-protected folic acid preparation described in the above technical solution or the rumen-protected folic acid preparation prepared by the preparation method described in the above technical solution or the folic acid-containing feed described in the above technical solution in improving the folic acid utilization rate of ruminants, improving the lactation performance of ruminants, improving the milk quality of ruminants and regulating mammary gland development.

[0040] In one embodiment, the application of this invention includes supplementing ruminants with the rumen-protected folic acid preparation, calculated at a dosage of 100-300 mg / day per ruminant, for at least 70 consecutive days. In another embodiment, the supplementation dose per ruminant is 200 mg / day. In yet another embodiment, the ruminant can be a cow, more specifically a dairy cow. In yet another embodiment, the supplementation can be administered via feeding, rumen delivery, or abomasal irrigation.

[0041] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the raw materials used in the following embodiments of the present invention can all be purchased from conventional commercial channels in the art.

[0043] Example 1 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 1.8 kg of feed-grade 5-methyltetrahydrofolate calcium salt and lipase (Novozymes' Lipolase). ® 80 g of 100L (same below) and 3 kg of silica (400 mesh) were mixed in a drum mixer at 20 rpm for 20 min until homogeneous. Then, 27.54 kg of silica (400 mesh) was added and mixed in a V-type mixer at 20 rpm for 20 min until homogeneous, yielding a mixed material. 27.7 kg of palm fat and 22.7 kg of coconut oil were heated to 60°C and mixed with the above mixed material until homogeneous. The mixture was passed through a 0.8 mm sieve, and the particles passing through the sieve (undersize) were collected and granulated in a rotary granulator. The resulting particles were placed in the rotary granulator, and 12 kg of calcium stearate was coated onto the outer layer of the particles with 5 kg of palm fat heated to 60°C to obtain the RP5-MTHF product, designated as Formula 1. The finished product particle size range is 0.8~1.5 mm.

[0044] Example 2 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 1.9 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 100 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 24.42 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 27.7 kg of palm fat and 22.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into the rotary pellet mill and coat the outer layer of the granules with 5 kg of palm fat heated to 60°C and 15 kg of calcium stearate. This yields the RP5-MTHF product, designated as Formula 2, with a finished particle size range of 0.8–1.5 mm.

[0045] Example 3 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.0 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 120 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 24.3 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 27.7 kg of palm fat and 22.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into a rotary pellet mill and coat 17 kg of calcium stearate with 5 kg of palm fat heated to 60°C to obtain the RP5-MTHF product, designated as Formula 3. The finished product particle size range is 0.8~1.5 mm.

[0046] Example 4 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.1 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 80 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 27.22 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 24.7 kg of palm fat and 24.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into the rotary pellet mill and coat the outer layer of the granules with 6 kg of palm fat heated to 60°C and 12 kg of calcium stearate. This yields the RP5-MTHF product, designated as Formula 4. The finished product particle size ranges from 0.8 to 1.5 mm.

[0047] Example 5 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.2 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 100 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 22.08 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 24.7 kg of palm fat and 24.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into a rotary pellet mill and coat the outer layer of the granules with 6 kg of palm fat heated to 60°C and 15 kg of calcium stearate. This yields the RP5-MTHF product, designated as Formula 5, with a finished particle size range of 0.8–1.5 mm.

[0048] Example 6 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.3 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 100 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 24 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 24.7 kg of palm fat and 24.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into a rotary pellet mill and coat 15 kg of calcium stearate with 6 kg of palm fat heated to 60°C to obtain the RP5-MTHF product, designated as Formula 6. The finished product particle size range is 0.8~1.5 mm.

[0049] Example 7 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.4 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 80 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 26.9 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 21.7 kg of palm fat and 26.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into a rotary pellet mill and coat the outer layer of the granules with 7 kg of palm fat heated to 60°C and 12 kg of calcium stearate. This yields the RP5-MTHF product, designated as Formula 7, with a finished particle size range of 0.8–1.5 mm.

[0050] Example 8 Preparation of rumen-protected 5-methyltetrahydrofolate (RP5-MTHF) Weigh 2.6 kg of feed-grade 5-methyltetrahydrofolate calcium salt, 120 g of lipase, and 3 kg of silica (400 mesh). Mix them in a drum mixer at 20 rpm for 20 min until homogeneous. Add 22.66 kg of silica (400 mesh) and mix in a V-type mixer at 20 rpm for 20 min until homogeneous. Heat 21.7 kg of palm fat and 26.7 kg of coconut oil to 60°C and mix them with the above mixture until homogeneous. Pass the mixture through a 0.8 mm sieve and collect the granules (undersize). Perform rotary granulation in a rotary pellet mill. Place the resulting granules into a rotary pellet mill and coat the outer layer of the granules with 7 kg of palm fat heated to 60°C and 16 kg of calcium stearate. This yields the RP5-MTHF product, designated as Formula 8, with a finished particle size range of 0.8–1.5 mm.

[0051] The content of RP5-MTHF products prepared in each embodiment of the present invention was determined. The results showed that the 5-MTHF content in the product had a good linear relationship with the amount of feed, and the dry matter content was all between 96.0% and 97.0%. Among them, the specific determination results of the product in Example 5 are as follows: dry matter content was 96.5%, and 5-MTHF content was 2.2%.

[0052] Application Example 1 Comparative experiments on rumen degradation rate and small intestinal release rate of different formulations of RP5-MTHF in Examples 1-8 The experiment selected 12 dairy bulls (weighing 580 ± 11.2 kg) with permanent rumen and duodenal fistulas, and randomly divided them into 4 groups of 3 bulls each. A 4 × 4 Latin square design was used, and the experiment was conducted for 4 periods. Each period had a 10-day pre-feeding period and a 5-day trial period. The basal diet consisted of: 25% corn silage, 13.2% alfalfa hay, 11.8% oat hay, 25.8% corn, 5.5% wheat bran, 9.8% soybean meal, 2.5% rapeseed meal, 4.6% cottonseed meal, 0.5% calcium carbonate, 0.5% salt, 0.3% dicalcium phosphate, and 0.5% premix (composition as in Application Example 2).

[0053] For each trial, 5 g of RP5-MTHF prepared in Examples 1-8 was accurately weighed and placed into nylon bags (5 cm × 8 cm) of known weight. On the first day of the trial, after morning feeding, the bags were placed 50 cm into the rumen sac. Eight bags were placed in each cow, and four bags were removed at 12 h and 24 h. Two bags were immediately rinsed with water until the water was completely clear, dried at 65°C to constant weight, and the folic acid content after degradation was determined by high-performance liquid chromatography. The other two bags were inserted into the small intestine through a duodenal fistula, and the nylon bags excreted in the feces were collected, rinsed, dried, and the folic acid content in the residue was determined. The rumen degradation rate and small intestinal release rate were calculated. The results are shown in Table 1.

[0054] Table 1. Rumen degradation rate and small intestinal release rate (%) of RP5-MTHF in different embodiments

[0055] As shown in Table 1, the degradation rates of RP5-MTHF prepared in Examples 1-8 in the rumen were 12.5%-20.5% after 12 h and 20.1%-29.3% after 24 h. Among them, Example 5 had the lowest rumen degradation rate (12.5%) and the highest small intestinal release rate (71.7%), indicating that the coating process of Example 5 was optimal and could more effectively protect 5-MTHF from crossing the rumen and entering the small intestine for absorption.

[0056] Application Example 2 Effects of dietary supplementation with RP5-MTHF on lactation performance and mammary gland development in lactating dairy cows 1. Experimental Design: Fifty-six healthy, well-condition Holstein dairy cows in mid-lactation were selected, with an average weight of 671 ± 14.6 kg, an average parity of 2.6 ± 0.12, an average lactation duration of 116 ± 2.8 days, and an average milk yield of 33.4 ± 1.26 kg / d. A randomized block design was used, and the cows were divided into four groups (n=14 / group) according to parity, lactation duration, and milk yield: a control group (basal diet), a low-dose group (100 mg / d 5-MTHF), a medium-dose group (200 mg / d 5-MTHF), and a high-dose group (300 mg / d 5-MTHF). The experiment lasted for 70 days, including a 10-day pre-feeding period and a 60-day formal experimental period. The RP5-MTHF used in the experiment was prepared according to Example 5 of this invention. The composition and nutrient levels of the basal diet are shown in Table 2.

[0057] Table 2. Composition and nutrient levels of the basal diet (dry matter basis, %)

[0058] 1 Each kilogram of premix contains: Fe 20,000 mg, Cu 1,680 mg, Mn 8,200 mg, Zn 7,100 mg, I 118 mg, Se 58 mg, Co 18 mg, VA 825,000 IU, VD 310,000 IU, VE 10,600 IU.

[0059] 2 Non-fiber carbohydrates = 100 - crude protein - neutral detergent fiber - crude fat - crude ash; 3 Net lactation energy was calculated according to NASEM (2021).

[0060] 2. Measurement Indicators 2.1 Feed intake and milk yield of each cow were recorded daily during the experiment. Milk samples were collected every 10 days to determine milk composition. Mixed samples from morning, noon, and evening milkings were collected according to milk yield ratios, and a preservative (2-bromo-2-nitropropane-1,3-diol) was added and stored at 4℃. A MilkoScan FT-120 milk composition analyzer (Foss Electric, Hiller) was used. (d, Denmark) measures milk fat percentage, milk protein percentage, and lactose percentage. 4% fat-corrected milk (FCM) and energy-corrected milk (ECM) are calculated according to the NRC (2001) formula: 4% FCM (kg / d) = 0.4 × milk yield + 15 × milk fat yield; ECM (kg / d) = 0.3246 × milk yield + 12.86 × milk fat yield + 7.04 × milk protein yield; Feed efficiency is expressed as ECM / DMI.

[0061] 2.2 Determination of nutrient digestibility: Cr2O3 was used as an exogenous indicator. During the sampling period of days 50-67, Cr2O3 was administered twice daily, morning and evening, at 55 g / head via capsule (refer to the following literature [Loučka R, Jambor V, Synková H, Homolka P, Kumprechtová D, Koukolová V, et al. Effect of calcareous marinealgae buffer on high-producing dairy cows during peak lactation. Animals2024;14(6):897. https: / / doi.org / 10.3390 / ani14060897.]). During the sampling period of days 63-67, approximately 200 g of rectal fecal samples were collected daily at 07:00, 13:00, 19:00, and 01:00, and 10% tartaric acid was immediately added for nitrogen fixation. The fecal samples from the 5 days were mixed in equal amounts, dried at 65℃, and pulverized through a 1 mm sieve. The Cr content in fecal samples was determined by atomic absorption spectrophotometry (AA-1800H, Shanghai Meixi Instrument Co., Ltd.) (refer to the following literature [Williams CH, David DJ, Iismaa O. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J Agric Sci 1962; 59(3): 381-385. https: / / doi.org / 10.1017 / S002185960001546X.]), and the nutrient digestibility was calculated.

[0062] The contents of dry matter (AOAC Method 934.01), crude protein (AOAC Method 976.05, N × 6.25), crude fat (AOAC Method 973.18), neutral detergent fiber (refer to the following literature [van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74(10):3583-97. https: / / doi.org / 10.3168 / jds.S0022-0302(91)78551-2.]), acid detergent fiber (AOAC Method 973.18), calcium and phosphorus (AOAC Method 935.13) in fecal and feed samples were determined according to the AOAC (2000) method. Organic matter content = dry matter content - crude ash content.

[0063] 2.3 Serum biochemical index determination On day 70 of the sampling period, 4 hours after morning feeding (10:30), 10 mL of blood was collected from the tail vein into a vacuum blood collection tube (without anticoagulant). The tube was left to stand at room temperature for 30 min, then centrifuged at 3000 g for 15 min to separate the serum, which was then stored at -20℃ for testing.

[0064] Serum glucose (#A154-2-1), total protein (#A045-3-1), albumin (#A028-1-1), triglycerides (#A110-2-1), non-esterified fatty acids (NEFA; #A042-1-1), and β-hydroxybutyrate (BHB; #E030-1-1) levels were determined using an automated biochemical analyzer (HTSH-3000, Qingdao Hantang Technology Co., Ltd.). The kits were purchased from Nanjing Jiancheng Biotechnology Institute. Serum homocysteine ​​(Hcy) levels were determined using an ELISA kit (#H245-1-2, Nanjing Jiancheng Biotechnology Institute).

[0065] Serum concentrations of insulin-like growth factor 1 (IGF-1; #BL-E21687M), estradiol (E2; #BL-E28820M), and prolactin (PRL; #BL-E28845M) were determined using an ELISA kit purchased from Beijing Bio-Lab Biotechnology Co., Ltd., with intra- and inter-assay coefficients of variation both less than 10%.

[0066] The content of vitamin B12 in serum was determined by competitive chemiluminescence immunoassay. The kit was purchased from Siemens Healthineers (#L2KVB2) and the analysis was performed using an Immulite 2000 XPi analyzer.

[0067] The concentration of 5-MTHF in serum was determined using an ELISA kit (#150310, Abbexa Ltd., Cambridge, UK). This kit showed good correlation with the LC-MS / MS method (r = 0.94). P <0.001), detection sensitivity <0.51 ng / mL, intra-batch and inter-batch coefficients of variation less than 10% and 12%, respectively.

[0068] 2.4 Breast tissue sampling and protein expression detection On day 70 of the sampling period, 6 cows were randomly selected from each group (24 cows in total). Mammary tissue samples were collected using a minimally invasive surgical method (refer to the following literature [de Lima LS, Martineau E, De Marchi FE, Palin MF, dos Santos GT, Petit HV. Anew technique for repeated biopsies of the mammary gland in dairy cows, sallotted to Latin-square design studies. Can J Vet Res 2016;80(3):225-9. ISSN0830-9000.]). The cows were fasted for 6 hours before the operation and underwent epidural anesthesia at the tail root (2% lidocaine, 0.2 mg / kg) combined with mammary nerve block (0.5% bupivacaine, 5 mL). After ultrasound localization, a 2 cm incision was made 5 cm perpendicular to the teat on the outer side of the udder, and a 14G biopsy needle was inserted vertically into the mammary parenchyma to remove approximately 1.0 g of mammary tissue. The collected tissues were immediately flash-frozen in liquid nitrogen and stored at -80°C for Western blot analysis.

[0069] The expression of the following proteins in breast tissue was detected using Western blot. The specific experimental method is as follows: Approximately 50 mg of frozen breast tissue was taken and lysed with RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. After homogenization on ice, the tissue was centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was collected. The total protein concentration was determined by BCA method. An equal amount of protein (30-50 μg) was separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane using wet transfer. The membrane was blocked with 5% skim milk powder (dissolved in TBST) at room temperature for 1 h. The corresponding primary antibody (diluted according to the antibody's instructions) was added and incubated overnight at 4°C. The membrane was washed three times with TBST (10 min each time). HRP-labeled secondary antibody (1:5000) was added and incubated at room temperature for 1 h. The membrane was washed three times with TBST, and then developed using ECL chemiluminescence. Grayscale analysis was performed using ImageJ software, and β-actin was used as an internal control for normalization.

[0070] Major antibody: Rabbit anti-proliferating cell nuclear antigen (PCNA): Cell Signaling Technology, USA Rabbit anti-cyclin D1 (Cyclin D1): Proteintech Group, USA Rabbit anti-B-cell lymphoma / leukemia-2 (BCL2): BIOSS, China Rabbit anti-BCL2-associated X protein (BAX): BIOSS, China Rabbit anti-cysteine ​​aspartate protease-3 (Caspase-3): BIOSS, China Rabbit anti-cysteine ​​aspartate protease-9 (Caspase-9): BIOSS, China Rabbit anti-proton-coupled folic acid transporter (SLC19A1 / PCFT): BIOSS, China Rabbit anti-protein kinase B (Akt): Cell Signaling Technology, USA Rabbit antiphosphokinase B (p-Akt, Ser473): BIOSS, China Rabbit anti-mammalian target of rapamycin (mTOR): BIOSS, China Rabbit antiphosphorylated mammalian target of rapamycin (p-mTOR, Ser2448): BIOSS, China Rabbit anti-Janus kinase 2 (JAK2): BIOSS, China Rabbit antiphosphorylated Janus kinase 2 (p-JAK2, Tyr1007 / 1008): BIOSS, China Rabbit anti-signal transduction and transcription activator 5 (STAT5): BIOSS, China Rabbit antiphosphorylation signal transducer and transcription activator 5 (p-STAT5, Tyr694): BIOSS, China Rabbit anti-αs1-casein: BIOSS, China Rabbit anti-β-casein: BIOSS, China Rabbit anti-κ-casein: BIOSS, China Rabbit anti-peroxisome proliferator-activated receptor γ (PPARγ): BIOSS, China Rabbit antisterol regulatory element binding protein 1 (SREBP1): Novus Biologicals, USA Rabbit anti-acetyl-CoA carboxylase α (ACACA): BIOSS, China Rabbit antiphosphorylated acetyl-CoA carboxylase α (p-ACACA, Ser79): BIOSS, China Rabbit anti-fatty acid synthase (FASN): BIOSS, China Rabbit anti-stearoyl-CoA desaturase 1 (SCD1): BIOSS, China Rabbit anti-AMP activated protein kinase (AMPK): BIOSS, China Rabbit antiphosphorylated AMP-activated protein kinase (p-AMPK, Thr172): BIOSS, China Mouse anti-β-actin: Cell Signaling Technology, USA.

[0071] 2.5 Data Analysis All data were statistically analyzed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Residual normality and homogeneity of variance were assessed using the Shapiro-Wilk test and Levene test, respectively. Logarithmic transformations were used when these parametric assumptions needed to be met.

[0072] The following mixed model was used to analyze data on feed intake, milk yield, and milk composition measured repeatedly over multiple weeks: Y ijkl = μ + B i + C j(i) + T k + Wl + TW kl + βX ijkl + ε ijkl Where Y ijkl = Dependent variable, μ = population mean, B i = Random effects of blocks (i = 1 to 14), C j(i) = Random effects of cows within blocks, T k = Fixed effects of treatment (k = CON, L5-MTHF, M5-MTHF, H5-MTHF), W l = Fixed effects of week (l = 1 to 10), TW kl = Treating the fixed interaction effect with Zhou, βX ijkl = Covariate adjustment using covariate period measurements, ε ijkl = Residual error.

[0073] By comparing the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) values ​​for different covariance structures (composite symmetric, first-order autoregressive, and unstructured), the spatial power covariance structure SP (POW) was selected, which provided the best fit (lowest AIC) for most variables.

[0074] For digestibility, serum metabolites, and other single-time-point measurements, the model used is: Y ijk = μ + B i + C j(i) + T k + ε ijk Multiple comparisons between means were performed using the PDIFF option with Tukey-Kramer adjustment. Orthogonal polynomial comparisons were used to assess the dose-response relationship of RP5-MTHF (0, 100, 200, 300 mg / d 5-MTHF) to detect linear and quadratic effects. The significance level was set to [value missing]. P <0.05, 0.05 ≤ PA value <0.10 was considered a trend. For Western blot data, based on our prior assumption that the dose elicits the maximum response, and considering the limited available tissue samples that prevent a complete dose-response analysis, we used the unpaired two-tailed Student's t-test in SigmaPlot 14.0 (Systat Software, San Jose, CA, USA) to compare the CON and M5-MTHF groups. Unless otherwise stated, all data are expressed as least squares means and their corresponding pooled standard errors (SEM).

[0075] 3. Experimental Results and Analysis Lactation performance: The medium-dose group (200 mg / d) showed the best results, with an actual milk yield increase of 8.3%, 4% fat-corrected milk yield increase of 11.6%, milk fat percentage increase of 6.0%, milk protein percentage increase of 6.6%, and 5-MTHF content in milk increase of 29.4% compared to the control group. P <0.05).

[0076] Protein expression in breast tissue: The expression of PCNA and Cyclin D1 in breast tissue was significantly upregulated in the medium-dose group. P <0.01), the BCL2 / BAX ratio increased significantly ( P <0.05), the phosphorylation ratios of p-Akt / Akt and p-mTOR / mTOR were significantly increased ( P <0.05; meanwhile, the expression of PPARγ, SREBP1, FASN and SCD1 proteins was significantly upregulated ( P <0.05), the p-AMPK / AMPK ratio decreased significantly ( P <0.05). The in vivo results were highly consistent with the in vitro cell experiments.

[0077] 3.1 Effects on lactation performance The effects of dietary supplementation with coated 5-MTHF on lactation performance in dairy cows are shown in Table 3. There were no significant differences in dry matter intake among the groups. P >0.05). Actual milk production, 4% fat-corrected milk production, and energy-corrected milk production all exhibit a quadratic curve response ( P <0.05, the medium-dose group (200 mg / d) reached its peak. Compared with the control group, the actual milk production in the medium-dose group increased by 8.3% ( P <0.05), 4% FCM increased by 11.6% ( P <0.05), ECM increased by 12.4% ( P <0.05).

[0078] Milk composition analysis showed that the milk fat percentage and milk protein percentage increased in a quadratic curve.P <0.001, both the medium-dose group and the high-dose group were significantly higher than the control group ( P <0.05%. Compared with the control group, the medium-dose group showed a 6.0% increase in milk fat percentage and a 6.6% increase in milk protein percentage. There was no significant difference in lactose percentage among the groups. P >0.05). Feed efficiency (ECM / DMI) increased linearly ( P = 0.030), all added groups were significantly higher than the control group ( P = 0.019).

[0079] Table 3 Effects of rumen-treated 5-methyltetrahydrofolate supplementation on dry matter intake, lactation performance, and feed efficiency in dairy cows

[0080] CON = Control group; LMTHF = Low-dose RP5-MTHF group; MMTHF = Medium-dose RP5-MTHF group; HMTHF = High-dose RP5-MTHF group.

[0081] a ~ c Different lowercase letters on the same shoulder label indicate significant differences. P <0.05).

[0082] 1 The CON, LMTHF, MMTHF, and HMTHF groups (n=14) were supplemented with 0, 100, 200, and 300 mg / d of 5-MTHF, respectively, derived from 0, 5, 10, and 15 g / d of RP5-MTHF product.

[0083] 2 4% milk fat corrected milk is calculated according to NRC (2001): 4.0% FCM = 0.4 × milk yield (kg / d) + 15 × milk fat content (kg / d).

[0084] 3 Energy-corrected milk is calculated according to NRC (2001): ECM = [0.327 × milk yield (kg / d)] + [12.95 × milk fat (kg / d)] + [7.65 × protein (kg / d)].

[0085] 4 Feed efficiency = Energy-corrected milk production per cow divided by dry matter intake.

[0086] 3.2 Effect on milk fatty acid production Supplementing with RP5-MTHF linearly increased the yield of pre-formed fatty acids. P= 0.001; Table 4), where the levels in the 200 mg / d and 300 mg / d groups were higher than those in the control group ( P = 0.005). Conversely, the yields of de novo synthesized fatty acids and fatty acids from mixed sources were unaffected ( P >0.05). With increasing RP5-MTHF dosage, the daily yields of C18:0, C18:1n9c, C18:2n6c, C18:3, C20:0, C20:3, and C20:4 increased linearly. P <0.05), while the output from C4:0 to C17:0 remained unchanged ( P >0.05). The C18:0, C18:1n9c, C18:2n6c, C20:3, and C20:4 yields in the 200 mg / d and 300 mg / d groups were higher than those in the control group ( P <0.05%. The yield of C18:3 was lowest in the control group, highest in the 200 mg / d group, and intermediate in the 100 mg / d and 300 mg / d groups. P =0.010). C20:0 production was higher in the 100 mg / d and 200 mg / d groups than in the control group ( P = 0.048).

[0087] Table 4. Effects of rumen-protective 5-methyltetrahydrofolate (RP5-MTHF) supplementation on fatty acid production in lactating cows (g / d)

[0088] CON = Control group; LMTHF = Low-dose RP5-MTHF group; MMTHF = Medium-dose RP5-MTHF group; HMTHF = High-dose RP5-MTHF group.

[0089] a~c Different lowercase letters on the same shoulder label indicate significant differences. P <0.05).

[0090] 1 The CON, LMTHF, MMTHF, and HMTHF groups (n=14) were supplemented with 0, 100, 200, and 300 mg / d of 5-MTHF, respectively, derived from 0, 5, 10, and 15 g / d of RP5-MTHF product.

[0091] 2 De novo synthetic fatty acids refer to fatty acids (<16 carbons) derived from de novo synthesis in the mammary glands.

[0092] 3 The mixed source fatty acids are the sum of C16:0 and C16:1.

[0093] 4 Preformed fatty acids are fatty acids (>16 carbons) extracted from the blood.

[0094] 3.3 Effects on nutrient digestibility The effects of dietary supplementation with coated 5-MTHF on the digestibility of nutrients in dairy cows are shown in Table 5. The digestibility of dry matter, organic matter, crude protein, and crude fat increased linearly with increasing 5-MTHF supplementation. P <0.05. The digestibility of dry matter, organic matter, crude protein, and crude fat in the medium-dose and high-dose groups was significantly higher than that in the control group ( P <0.05. There was no significant difference in the digestibility of neutral detergent fiber and acid detergent fiber among the groups. P >0.05).

[0095] Table 5. Effects of rumen-treated 5-methyltetrahydrofolate (RP5-MTHF) supplementation on nutrient digestibility in dairy cows (%)

[0096] CON = Control group; LMTHF = Low-dose RP5-MTHF group; MMTHF = Medium-dose RP5-MTHF group; HMTHF = High-dose RP5-MTHF group.

[0097] a~d Different lowercase letters on the same shoulder label indicate significant differences. P <0.05).

[0098] 1 The CON, LMTHF, MMTHF, and HMTHF groups (n=14) were supplemented with 0, 100, 200, and 300 mg / d of 5-MTHF, respectively, derived from 0, 5, 10, and 15 g / d of RP5-MTHF product.

[0099] 3.4 Effects on serum biochemical indicators The effects of dietary supplementation with coated 5-MTHF on serum biochemical parameters in dairy cows are shown in Table 6. Serum glucose, total protein, albumin, 5-MTHF, and vitamin B12 levels increased linearly with increasing 5-MTHF supplementation. P <0.05. Serum glucose and total protein levels in the medium-dose and high-dose groups were significantly higher than those in the control group ( P <0.05%, 5-MTHF and vitamin B12 levels increased in a gradient with increasing dosage ( P <0.001). Serum homocysteine ​​levels decreased linearly ( P <0.001, the medium-dose group and the high-dose group were significantly lower than the control group ( P<0.001). Serum BHB levels decreased linearly ( P = 0.004), the medium-dose and high-dose groups were significantly lower than the control and low-dose groups (P = 0.028). There was no significant difference in NEFA content among the groups (P = 0.004). P >0.05).

[0100] Serum IGF-1 and prolactin levels increased linearly. P <0.001. The IGF-1 levels in the medium-dose and high-dose groups were significantly higher than those in the control group ( P = 0.001). The prolactin levels in the medium-dose and high-dose groups were significantly higher than those in the control and low-dose groups. P <0.001). Estradiol content increased linearly with increasing 5-MTHF addition ( P <0.01, the medium-dose group and the high-dose group were significantly higher than the control group ( P <0.05).

[0101] Table 6 Effects of rumen-treated 5-methyltetrahydrofolate (RP5-MTHF) supplementation on serum biochemical parameters in dairy cows

[0102] CON = Control group; LMTHF = Low-dose RP5-MTHF group; MMTHF = Medium-dose RP5-MTHF group; HMTHF = High-dose RP5-MTHF group.

[0103] a~d Different lowercase letters on the same shoulder label indicate significant differences. P <0.05).

[0104] 1 The CON, LMTHF, MMTHF, and HMTHF groups (n=14) were supplemented with 0, 100, 200, and 300 mg / d of 5-MTHF, respectively, derived from 0, 5, 10, and 15 g / d of RP5-MTHF product.

[0105] 3.5 Effects on the expression of cell proliferation-related proteins in breast tissue Compared with the control group, supplementation with 200 mg / d RP5-MTHF-derived 5-MTHF increased Cyclin D1 ( P <0.01), PCNA ( P <0.01) and BCL2 ( P Protein expression levels <0.05, and the BCL2 / BAX ratio ( P <0.05, while reducing BAX ( P<0.05), caspase-3 ( P <0.05) and caspase-9 ( P The level of <0.01 ( Figure 1 (A and B). Furthermore, supplementation with 200 mg / d 5-MTHF also increased the protein expression level of SLC19A1 ( ). P <0.01) and p-Akt / Akt ( P <0.05) and p-mTOR / mTOR ( P Phosphorylation ratio <0.01 ( Figure 2 (A and B in the middle).

[0106] 3.6 Effects on the expression of fatty acid synthesis-related proteins in breast tissue Compared with the control group, supplementation with 200 mg / d RP5-MTHF significantly increased PPARγ, SREBP1, and FASN ( P <0.01) and SCD1 ( P Protein expression <0.05 ( Figure 3 (A and B). Furthermore, ACACA phosphorylation levels are increased ( P <0.05), while AMPK phosphorylation levels decreased, manifested as changes in the p-ACACA / ACACA and p-AMPK / AMPK ratios, respectively. P <0.05).

[0107] This invention is the first to discover and confirm that, compared to traditional folic acid, its active form, 5-methyltetrahydrofolate (5-MTHF), is a key factor determining differences in mammary gland development in dairy cows. This invention utilizes the SLC19A1 transporter-mediated "Akt-mTOR" and "AMPK-PPARγ-SREBP1" dual signaling axes to simultaneously regulate mammary cell proliferation and milk fat synthesis, achieving a synergistic enhancement of lactation performance with significantly better results than ordinary folic acid.

[0108] Comparative Example 1 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, with the only difference being that lipase was not added, while the other components and amounts were exactly the same.

[0109] Application Example 3 Using the method described in Application Example 1, the degradation rate and small intestinal release rate of the RP5-MTHF products in Example 5 and Comparative Example 1 were determined in the rumen over 12 h, and the results are shown in Table 7.

[0110] Table 7. Rumen degradation rate and small intestinal release rate (%) of RP5-MTHF prepared in Example 5 and Comparative Example 1.

[0111] The results in Table 7 show that the addition of lipase reduced the rumen degradation rate from 16.8% to 12.5% ​​(a decrease of 25.6%) over 12 hours, while increasing the small intestinal release rate from 58.4% to 71.7% (an increase of 22.8%). This indicates that lipase not only did not disrupt the rumen protective effect, but also significantly improved the release rate and bioavailability of 5-MTHF through enzymatic hydrolysis in the small intestine.

[0112] Application Example 4 Comparison with regular folic acid (non-rumen-protected) 1. Experimental Design: Forty Holstein dairy cows in mid-lactation were selected and randomly divided into four groups (n=10): Control group: basal diet (composition shown in Table 2); Regular folic acid group: Regular folic acid (non-rumen-protected) was added to the basal diet, so that each dairy cow could ingest 200 mg of folic acid per day; Low-dose RP5-MTHF group: The RP5-MTHF product prepared in Example 5 was added to each cow to provide 100 mg of 5-MTHF per day; Medium-dose RP5-MTHF group: The RP5-MTHF product prepared in Example 5 was added to each cow to provide 200 mg of 5-MTHF per day.

[0113] The experiment lasted 70 days. The serum 5-MTHF content, milk yield and milk fat percentage of Holstein dairy cows were measured. The results are shown in Table 8.

[0114] Table 8 Comparison of RP5-MTHF and regular folic acid (non-rumen-crossed)

[0115] Different letters in the same column indicate significant differences. P <0.05).

[0116] Table 8 shows that the serum 5-MTHF level in the regular folic acid group (200 mg / d) was only 15.2% higher than that in the control group, with no significant increase in milk yield and milk fat percentage. This indicates that regular folic acid is largely degraded in the rumen and requires two steps of reduction to be converted into its active form, resulting in extremely low utilization efficiency. In contrast, the present invention directly supplements rumen-protected 5-MTHF. Even at only 100 mg / d (50% of the regular folic acid dose), the serum 5-MTHF level, milk yield, and milk fat percentage are significantly better than those in the regular folic acid group; the 200 mg / d group shows even better results. This demonstrates that the present invention achieves a breakthrough in "low dosage, high efficacy."

[0117] Comparative Example 2 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, with the only difference being that the amount of palm fat was increased from 24.7 kg to 40 kg, while the other components and amounts were exactly the same.

[0118] Comparative Example 3 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, with the only difference being that the amount of calcium stearate was increased from 15 kg to 8 kg, while the other components and amounts were exactly the same.

[0119] Comparative Example 4 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, except that the amount of coconut oil was increased from 24.7 kg to 35 kg and the amount of palm fat was reduced from 24.7 kg to 15 kg, while the other components and amounts were exactly the same.

[0120] Application Example 5 Using the method described in Application Example 1, the degradation rate and small intestinal release rate of the RP5-MTHF products in Example 5 and Comparative Examples 2-4 were determined in the rumen over 12 h. The results are shown in Table 9.

[0121] Table 9. Rumen degradation rate and small intestinal release rate (%) of RP5-MTHF prepared in Example 5 and Comparative Examples 2-4

[0122] Table 9 shows that when the amount of palm fat is too high (Comparative Example 2), the outer coating is too thick, making it difficult for lipases in the small intestine to hydrolyze the particles quickly, resulting in a decrease in the release rate. When the amount of calcium stearate is too low (Comparative Example 3), the mechanical strength of the particles is insufficient, leading to premature disintegration in the rumen and an increased degradation rate. When the proportion of coconut oil is unbalanced (Comparative Example 4), the hydrophilicity of the coating layer changes, which also leads to a poorer rumen-crossing effect. The above results demonstrate that the range of amounts of palm fat (21.7%~32.7%), coconut oil (22.7%~26.7%), and calcium stearate (12%~18%) specified in this invention has critical significance; exceeding this range makes it impossible to simultaneously achieve low rumen degradation and high small intestinal release.

[0123] Comparative Example 5 The RP5-MTHF product was prepared according to the raw material dosage and preparation method in Example 5. Unlike the preparation method in Example 5 where silica was mixed in steps, 2.2 kg of 5-methyltetrahydrofolate calcium salt, 100 g of lipase and all 25.08 kg of silica were added into a V-type mixer at once and mixed evenly.

[0124] Application Example 6 During the preparation of the RP5-MTHF product, 10 samples (approximately 1 g each) were randomly selected from the final mixtures in Example 5 and Comparative Example 5, and the 5-MTHF content was determined by HPLC. The coefficient of variation (CV%) was calculated. The results are shown in Table 10.

[0125] Table 10. Content and coefficient of variation of 5-MTHF in the mixture of Example 5 and Comparative Example 5

[0126] The results in Table 10 show that the coefficient of variation (CV) of the one-step mixing group in Comparative Example 5 was as high as 8.6%, exceeding the requirements for feed additive uniformity (typically CV ≤ 5%), resulting in a low product qualification rate. In contrast, the stepwise, equal-volume incremental mixing reduced the CV to 1.8%, significantly improving product uniformity. This demonstrates that the stepwise mixing process employed in this invention (first equal-volume premixing, then serial dilution) is necessary and superior for ensuring the uniform distribution of trace active ingredients in a large number of carriers.

[0127] Comparative Example 6 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, with the only difference being that the heating temperature of coconut oil and palm fat was adjusted to 50°C (below 55°C), while the other components and amounts were exactly the same.

[0128] Comparative Example 7 The RP5-MTHF product was prepared according to the formulation and preparation method of Example 5, with the only difference being that the heating temperature of coconut oil and palm fat was adjusted to 70°C (above 65°C), while the other components and amounts were exactly the same.

[0129] Application Example 7 Using the method described in Application Example 1, the 12-h rumen degradation rate and small intestinal release rate of the RP5-MTHF products prepared in Example 5 (60℃), Comparative Example 6 (50℃), and Comparative Example 7 (70℃) were determined, and the mixing uniformity and particle appearance during the granulation process were observed. The results are shown in Table 11.

[0130] Table 11 Effect of different heating temperatures on key performance characteristics of RP5-MTHF (%)

[0131] As shown in Table 11, when the heating temperature drops to 50℃, the excessively high lipid viscosity leads to uneven mixing and impaired coating integrity, causing the rumen degradation rate to increase from 12.5% ​​to 18.6%. When the heating temperature rises to 70℃, although the mixing effect is good, the thermal inactivation of lipase is significant, and the small intestinal release rate decreases from 71.7% to 62.4%. These results fully demonstrate that only by strictly controlling the heating temperature within 55~65℃ (especially 60℃) can the dual objectives of low rumen degradation rate (≤12.5%) and high small intestinal release rate (≥71.7%) be simultaneously achieved. This temperature range is critical; exceeding this range will prevent the achievement of the intended technical effects of this invention.

[0132] The results from Examples 3-7 demonstrate that the addition of lipase is a core technical feature for achieving targeted release of the formulation into the small intestine and improving bioavailability. Compared to existing conventional physical coating methods, it produces unexpectedly outstanding technical results. This invention directly selects 5-methyltetrahydrofolate as the raw material and implements rumen protection, resulting in a comprehensive effect far superior to traditional folic acid. Furthermore, experimental verification shows that only by limiting the amount of each component of the coating material within the range set by this invention can the optimal rumen protection effect and targeted release performance be achieved; the stepwise equal-volume incremental mixing process is an important technical method to ensure uniform mixing and stable product quality.

[0133] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A rumen-exposed folic acid preparation, characterized in that, The raw materials for preparing the rumen-protected folic acid preparation include a first raw material and a second raw material. The first raw material includes the following components in parts by weight: 1.8-2.7 parts of 5-methyltetrahydrofolate metal salt, 20.5-30.5 parts of silicon dioxide, 21.7-32.7 parts of palm fat, 22.7-26.7 parts of coconut oil, and 12-18 parts of calcium stearate. The second raw material includes a fatty acid enzyme. The weight ratio of the first raw material to the second raw material is 5000:(4-7).

2. The rumen-protected folic acid preparation according to claim 1, characterized in that, The 5-methyltetrahydrofolate metal salt includes 5-methyltetrahydrofolate calcium salt with a purity ≥98%; the silica has a particle size of 400 mesh.

3. The rumen-protected folic acid preparation according to claim 1, characterized in that, The lipase includes triacylglycerol lipase, enzyme classification number EC 3.1.1.3; the enzyme activity of the lipase is 8 × 10⁻⁶ based on the total amount of enzyme activity added. 5 ~1.4×10 6 Raw materials are prepared using U / 100 kg.

4. The method for preparing the rumen-exposed folic acid preparation according to any one of claims 1 to 3, characterized in that, Includes the following steps: A first mixture is prepared by mixing 5-methyltetrahydrofolate metal salt, lipase, and a portion of silica to obtain a first mixture, wherein the portion of silica accounts for 9.5% to 12.5% ​​of the total weight of silica. The first mixture is mixed with the remaining silica to obtain a second mixture; Coconut oil and a portion of palm fat are heated to 55-65°C, then mixed with the second mixture and granulated to obtain a granular semi-finished product. The portion of palm fat is 75%-85% of the total weight of palm fat. The remaining palm fat is heated to 55-65°C and mixed with calcium stearate to coat the outer layer of the granular semi-finished product, thus obtaining a rumen-protected folic acid preparation.

5. The preparation method according to claim 4, characterized in that, The first and second mixing are carried out using low-shear mixing devices with a rotation speed of 10-30 rpm and a mixing time of 15-30 min.

6. The use of the rumen-protected folic acid preparation according to any one of claims 1 to 3 or the rumen-protected folic acid preparation prepared by the preparation method according to claim 4 or 5 in the preparation of folic acid supplement products.

7. The application according to claim 6, characterized in that, The folic acid supplement products include feed additives and / or feeds.

8. A feed containing folic acid, characterized in that, The product includes a rumen-protected folic acid preparation and a basal diet, wherein the rumen-protected folic acid preparation is the rumen-protected folic acid preparation according to any one of claims 1 to 3 or the rumen-protected folic acid preparation prepared by the preparation method according to claim 4 or 5.

9. The use of the rumen-protected folic acid preparation according to any one of claims 1 to 3, or the rumen-protected folic acid preparation prepared by the preparation method according to claim 4 or 5, or the folic acid-containing feed according to claim 8, in at least one of improving folic acid utilization in ruminants, improving lactation performance in ruminants, improving milk quality in ruminants, and regulating mammary gland development.

10. The application according to claim 9, characterized in that, The application includes supplementing ruminants with the rumen-protected folic acid preparation, calculated based on the dosage of 5-methyltetrahydrofolate, at a supplementation amount of 100-300 mg / day per ruminant, for at least 70 consecutive days.

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