A method for determining the release rate of a ruminant post-rumen product at various sites

Abstract

This invention relates to a method for determining the release rate at various points of a rumen-exposed product in ruminants, comprising the following steps: water solubility determination: weighing an appropriate amount of rumen-exposed sample and determining its water solubility; acid solubility determination: weighing an appropriate amount of rumen-exposed sample and determining its acid solubility; rumen degradation rate determination: weighing the rumen-exposed sample after water and / or acid solubility determinations, placing it on feed, adding buffer solution and rumen fluid for in vitro fermentation, drying at a constant temperature after fermentation, weighing, and determining the rumen degradation rate; abomasal degradation rate determination: weighing the rumen-exposed sample after rumen degradation rate determination, adding pepsin hydrochloride buffer solution for incubation at a constant temperature, removing and drying at a constant temperature after incubation, weighing, and determining the abomasal degradation rate; small intestinal degradation rate determination: taking the rumen-exposed sample after abomasal degradation rate determination, adding small intestinal buffer solution for shaking incubation, washing, drying at a constant temperature, weighing, and determining the small intestinal degradation rate.

Description

Technical Field

[0001] This invention relates to the field of livestock feed processing technology, and in particular to a method for determining the release rate at various points of a rumen-transparent product for ruminants. Background Technology

[0002] Rumen-passing technology plays a crucial role in ruminant nutrition. Its core objective is to protect specific nutrients (such as proteins, fats, and amino acids) from rumen microbial degradation, enabling their targeted delivery to the small intestine for efficient absorption and thus improving feed utilization. With the widespread application of rumen-passing products, accurately evaluating their stability and targeted release characteristics has become a research hotspot. Traditional evaluation methods mainly include the two-step method proposed by Tilley and Terry (1963) based on "two-stage in vitro digestion," and the improved in vitro three-step method (rumen fluid-pepsin-pancreatic enzyme) by Calsamiglia et al., the latter of which has become the standard method for assessing the rumen-passing properties of proteins. Continuous culture systems (such as Rusitec) can maintain microbial homeostasis by dynamically simulating the rumen environment, making them suitable for long-term microbial fermentation studies. However, it should be noted that the residence time of rumen-passing products in the real gastrointestinal tract is usually significantly shorter than that in vitro, which may lead to evaluation bias. While the semi-in vivo small nylon bag method can directly reflect the rumen environment—by encapsulating samples in 40-50 μm small nylon bags and incubating them in animals with rumen fistulas before measuring residue—it is significantly affected by individual differences among fistula animals, with the coefficient of variation often exceeding 15%. As a commonly used method for determining feed degradation rates in ruminants, the semi-in vivo small nylon bag method has several technical drawbacks in practice: First, the method of fixing the small nylon bags (usually using plastic tubing or metal rods as support structures) is easily affected by rumination movements within the rumen, leading to mechanical entanglement between multiple supports and feed. This physical interaction not only interferes with normal rumen peristalsis rhythms but may also cause mechanical damage to the rumen mucosa, thereby inducing local inflammatory responses. Second, repeated mechanical stimulation may accelerate the fibrosis process of tissues surrounding the fistula, significantly shortening the effective lifespan of experimental animals. Furthermore, this method suffers from low sample processing efficiency; the fistula plug needs to be opened for each sampling, increasing the risk of microbial contamination and the difficulty of maintaining rumen environmental stability. These limitations affect the accuracy and reproducibility of experimental data to some extent, and also pose a potential threat to animal welfare.

[0003] Assessing small intestinal digestibility is equally crucial, but the industry currently suffers from an incomplete evaluation system: many products only label rumen degradation rates but lack small intestinal degradation rate data. This is primarily due to the lack of standardization in vitro small intestinal digestion methods and the low correlation between existing in vitro models and in vivo digestion (r...). 2The concentration of bile salts in the in vitro digestion system is mostly below 0.7. It is particularly noteworthy that the small intestinal digestion assessment of current mainstream fat-based coated products requires strict simulation of physiological conditions, necessitating the consideration of bile salt addition. Bile salts not only emulsify the fat-coated material, promoting its contact with digestive enzymes, but also form mixed micelles—a process crucial for simulating the real intestinal digestive environment. It is recommended to add an appropriate amount of bile salts (such as sodium taurocholate) to the in vitro digestion system to more accurately assess the release characteristics of coated fats in the intestine.

[0004] In actual production, rumen-protected products are typically added to total mixed rations (TMRs). Before ruminants consume these products, they may be exposed to moisture or acidic environments (such as silage, pH=4), which could lead to premature release of some of the coating components, affecting their rumen-protecting effect. Therefore, there is an urgent need to establish an in vitro evaluation method that more closely approximates the actual feeding environment to comprehensively assess the water solubility, acid stability, rumen degradation rate, and small intestinal degradation rate of rumen-protected products. Based on this need, this invention provides a more accurate measurement method by simulating the TMR exposure environment (water solubility and acid treatment) and combining it with an improved in vitro culture device, providing a scientific basis for the research and application of rumen-protected products.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for determining the release rate of rumen-passed products at various points in ruminants. The main purpose of this method is to solve the problems in the prior art that mechanical damage is caused to animals when testing rumen-passed products, and that the degradation rate of rumen-passed products in the small intestine after being consumed by ruminants cannot be determined.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides a method for determining the release rate at various points of a rumen-passing product in ruminants, comprising the following steps:

[0009] Water solubility determination: Weigh an appropriate amount of rumen-treated sample and determine its water solubility.

[0010] Acid solubility determination: Weigh an appropriate amount of rumen-treated sample and determine the acid solubility.

[0011] Rumen degradation rate determination: Weigh the rumen-passed sample after water solubility determination and / or acid solubility determination, place it on the feed, add buffer solution and rumen fluid for in vitro fermentation, dry it at constant temperature after fermentation, weigh it, and determine the rumen degradation rate.

[0012] Rumen degradation rate determination: Weigh the rumen-passed sample after the rumen degradation rate determination, add pepsin hydrochloride buffer and incubate at constant temperature. After the incubation is completed, take it out, dry it at constant temperature and weigh it to determine the rumen degradation rate.

[0013] Small intestinal degradation rate determination: Take the rumen-passed sample after the abomasal degradation rate determination, add small intestinal buffer solution for shaking culture, wash, dry at constant temperature and weigh, and then determine the small intestinal degradation rate.

[0014] This invention accurately assesses the small intestinal release rate of nutrients by measuring the small intestinal degradation rate of rumen-treated products using phosphate buffer to simulate the real small intestinal environment, thereby improving the physiological relevance and reliability of in vitro digestion experiments.

[0015] Meanwhile, this invention establishes a complete four-step evaluation system by simulating the feeding environment and digestive process of the digestive tract, including water solubility determination, acid solubility determination, rumen degradation rate determination, and small intestinal degradation rate determination. It fully considers the exposure conditions (moisture contact and acidic environment) of rumen-protected products in actual production. Simultaneously, in vitro culture is conducted to measure the solubility characteristics at different time points, including water solubility, acid solubility, rumen degradation rate, and small intestinal degradation rate. Compared with traditional methods, this invention not only simulates the digestive environment of the rumen and small intestine but also focuses on investigating key factors that may affect the efficacy of rumen-protected products during feed processing and storage (such as moisture in TMR and the acidic environment of silage). Through dynamic monitoring at multiple time points, the targeted release performance of rumen-protected products in the digestive tract of ruminants can be more accurately evaluated, providing a scientific basis for optimizing coating processes and product applications.

[0016] The advantages of this invention over the prior art are:

[0017] 1. This invention proposes a method for determining the release rate of rumen-transfer products at various points in ruminants. It takes into account the loss determination of rumen-transfer products before rumen passage and the laboratory batch culture of rumen fluid through rumen fluid external fermentation. This method not only avoids the mechanical damage to animals caused by the traditional nylon bag method, but also improves experimental efficiency and data reproducibility. It is suitable for feed evaluation and rumen function research.

[0018] 2. This invention measures the small intestinal degradation rate by simulating the real small intestinal environment. It uses pancreatic enzyme digestion combined with bile salt addition to more accurately assess the intestinal release rate of nutrients and improve the physiological relevance and reliability of in vitro digestion experiments.

[0019] 3. This invention achieves precise simulation of the release behavior of rumen-treated products throughout the entire digestive tract of ruminants by constructing a four-in-one evaluation system of "water-soluble pretreatment - acidic environment simulation - dynamic rumen fermentation - targeted intestinal digestion".

[0020] 4. This invention incorporates TMR feeding environment simulation (water solubility and pH=4 acidic pretreatment) into the evaluation system, effectively solving the shortcomings of traditional methods that ignore the impact of feed processing and storage on coating materials, making the evaluation results more consistent with actual production conditions.

[0021] 5. This invention uses fresh rumen fluid from fistula animals as the fermentation medium, which not only preserves the complete microbial community and enzyme system (compared to artificial rumen fluid), but also avoids the mechanical damage to fistula animals caused by the semi-in vivo nylon bag method through the in vitro culture device, thus significantly improving animal welfare while ensuring the authenticity of the data.

[0022] 6. This invention also provides a self-made fermentation bottle. Its innovative closed fermentation device (integrating a one-way gas valve and a gas collection system) not only achieves seamless connection between rumen fermentation and gastrointestinal digestion, but its modular structure (100mL standard fermentation bottle) also increases the experimental throughput by 3-5 times while reducing equipment costs.

[0023] 7. This invention, by optimizing the bile salt-pancreatic enzyme complex system (bile salt addition 2-3g / 100ml), successfully simulated the intestinal mixed micelle formation process in an in vitro model for the first time, demonstrating the correlation (r) between the measured small intestinal release rate of the fat-based coated product and in vivo experiments. 2 Significantly improved;

[0024] 8. The standardized operating procedures and quantitative evaluation indicators provided by this invention provide a technical platform for horizontal comparison of different coating processes (microencapsulation, fat coating, etc.) and various core materials (enzyme preparations, amino acids, etc.).

[0025] 9. The self-made fermentation flask provided by this invention adopts a coupled design of a one-way gas valve and a gas collection bag, realizing in-situ collection and quantitative determination of fermentation gas (it can be directly connected to a syringe or gas bag). At the same time, through the miniaturized reaction system (100mL) and modular culture method, it supports high-throughput parallel sampling at multiple time points (0-24h).

[0026] 10. The self-made fermentation flask provided by this invention innovatively integrates bioactive components (fresh rumen fluid provides a complete microbial community) with a physical simulation system (buffer solution maintains pH 6.5-7.0), which can more accurately reflect the actual rumen fermentation kinetics than traditional artificial rumen fluid (containing only enzyme preparations). This device combines ease of operation (small footprint, low cost) with data reliability, and is particularly suitable for evaluating the dynamic release characteristics of rumen-transfer products with different coating processes.

[0027] It should be understood that the implementation of any embodiment of the present invention does not mean that it will simultaneously possess or achieve multiple or all of the above-mentioned beneficial effects. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0029] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0030] Figure 1 This is a flowchart illustrating the method for determining the release rate at various points of the rumen-exposed product for ruminants, as provided in an embodiment of the present invention.

[0031] Figure 2 This is a plan view of the self-made fermentation bottle provided in an embodiment of the present invention.

[0032] Marked in the image:

[0033] 1-Bottle body; 2-Small nylon bag; 3-One-way valve tube; 4-Valve; 5-Valve connecting tube; 6-Silicone sealing cap.

[0034] The same or corresponding marks in the diagram indicate the same or corresponding parts. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0036] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0037] It should be understood that the terms "comprising / including," "consisting of," or any other variations are intended to cover non-exclusive inclusion, such that a product, apparatus, process, or method that comprises a list of elements includes not only those elements but may also include, where necessary, other elements not expressly listed, or elements inherent to such a product, apparatus, process, or method. Without further limitation, an element defined by the phrase "comprising / including," "consisting of," does not exclude the presence of additional identical elements in the product, apparatus, process, or method that includes said element.

[0038] It should also be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device, component or structure referred to must have a specific orientation, be constructed or operated in a specific orientation, and should not be construed as a limitation of the present invention.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0040] Finally, for any conditions not specified in the examples, follow standard conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] This invention relates to the field of livestock feed processing technology and discloses a method for determining the release rate of rumen-exposed products in ruminants at various points. This method calculates the water solubility, acid solubility, rumen degradation rate, and small intestinal degradation rate of rumen-exposed products, and accurately detects the release characteristics of rumen-exposed products after consumption by ruminants. This solves the problem in the prior art that the digestion of rumen-exposed products in the small intestine is unknown, and it is impossible to understand the extent to which nutrients are absorbed by ruminants.

[0042] The implementation of the present invention will be described in detail below with reference to preferred embodiments.

[0043] Example 1

[0044] This invention provides a self-made fermentation flask for determining the release rates of rumen-transfer products at various points in ruminants, such as... Figure 2As shown, the homemade fermentation bottle includes a bottle body 1, a silicone sealing cap 6, a one-way valve system, and a gas collection bag. The bottle body 1 is a cylindrical bottle with an inner diameter of 29mm at the mouth. The bottle body 1 is a standard fermentation bottle with a width of 60mm, a length of 160mm, and a capacity of 100ml. A small nylon bag 2 is placed inside the bottle body 1. A silicone sealing cap 6 is set at the mouth of the bottle. The one-way valve system passes through the silicone sealing cap 6 and communicates with the inside of the bottle body 1. The gas collection bag is connected to the one-way valve system to collect the gas produced during fermentation.

[0045] The one-way valve system includes a one-way valve tube 3, a valve 4, and a valve connecting tube 5. The one-way valve tube 3 is installed on the silicone sealing cap 6 to connect the bottle body 1 to the outside. The end of the one-way valve tube 3 away from the bottle body 1 is connected to the valve 4. The valve 4 is connected to the gas collection bag through the valve connecting tube 5 to collect the fermentation gas.

[0046] The present invention also provides a method for determining the release rate of rumen-transfer products at various points in ruminants. The method uses the self-made fermentation bottle mentioned above for determination. The sealed self-made fermentation bottle can achieve seamless connection between rumen fermentation and gastrointestinal digestion. At the same time, its modular structure can increase the experimental throughput by 3-5 times and reduce equipment costs.

[0047] In this embodiment, the sample measured by this method is a commercially available rumen-protected amylase product. This product is a rumen-protected product C coated with hydrogenated palm oil as packaging material and core material to packaging material ratio of 5:5 using microcrystalline cellulose excipient granulation. The water solubility, acid solubility, rumen degradation rate, abomasal degradation rate and small intestine degradation rate were measured.

[0048] like Figure 1 As shown, the determination method includes the following steps:

[0049] 1. Water solubility determination:

[0050] Accurately weigh 2g (sample weight before dissolution) of the rumen-protected product into a homemade fermentation flask, add 80mL of distilled water to dissolve, place the homemade fermentation flask on a magnetic stirrer and stir at 10-30rpm at room temperature for 10-30min, keeping the stirring time constant. After stirring, take samples at 0, 2, 4, 8, 12, and 24h, respectively. Then rinse the remaining residue and filter it with quantitative filter paper. Dry it at 45-60℃ to constant weight and weigh it (sample weight after dissolution). Calculate the dissolution rate based on the difference in mass before and after dissolution.

[0051] Water solubility = (weight of sample before dissolution - weight of sample after dissolution) / weight of sample before dissolution × 100%.

[0052] In this embodiment, to achieve better results, the magnetic stirrer speed is 30 rpm, the stirring time is 30 min, and the drying temperature is 55℃.

[0053] 2. Acid solubility determination:

[0054] Accurately weigh 2g (sample weight before dissolution) of the rumen-protected product into a homemade fermentation flask, add 80mL of acidic buffer solution with pH 4 to dissolve, place the homemade fermentation flask on a magnetic stirrer and stir at 10-30rpm at room temperature for 10-30min, keeping the stirring time constant. After stirring, take samples at 0, 2, 4, 8, 12, and 24h, respectively. Then rinse the remaining residue and filter it with quantitative filter paper, dry it at 45-60℃ to constant weight, and weigh it (sample weight after dissolution). Calculate the dissolution rate based on the difference in mass before and after dissolution.

[0055] Acidic solubility = (weight of sample before dissolution - weight of sample after dissolution) / weight of sample before dissolution × 100%.

[0056] In this embodiment, to achieve better results, the magnetic stirrer speed is 30 rpm, the stirring time is 30 min, and the drying temperature is 55℃.

[0057] The acidic buffer solution includes citrate-sodium citrate buffer, acetic acid-sodium acetate buffer, or phosphate-citrate buffer system. Taking citrate-sodium citrate buffer as an example: citrate solution: about 14 mL; sodium citrate solution: about 86 mL; after mixing, calibrate with a pH meter and add water to make up to 100 mL.

[0058] 3. Rumen degradation rate determination:

[0059] a. Preparation of rumen fluid:

[0060] Before morning feeding, the fistula of the ruminant (fistula beef cattle or fistula dairy cattle) is opened, the feed is removed and the crude rumen fluid is squeezed out, and then the feed is put back into the fistula. The crude rumen fluid contains many feed impurities. The crude rumen fluid is filtered through four layers of gauze to obtain the experimental rumen fluid, which is then placed in a thermos for later use.

[0061] b. Prepare the buffer solution:

[0062] Add the following in sequence: 400 mL distilled water, 0.1 mL trace element solution A, 200 mL phosphate buffer B, 200 mL phosphate buffer C, 1 mL 0.1% resazurin indicator D, and 40 mL reducing agent solution E. Mix and incubate at 39°C with continuous CO2 bubbling until the pH reaches 6.8. See the table below for details.

[0063]

[0064] c. Rumen degradation rate determination:

[0065] Accurately weigh 2.0g of the sample (the weight of the sample before rumen digestion) from the sample rinsed in step 1 or step 2 into a pre-weighed small nylon bag (the weight of the small nylon bag itself). The small nylon bag has dimensions of 40um and 8*12cm. In this embodiment, multiple small nylon bags are provided for storing the sample.

[0066] Then, the small nylon bag was placed into a homemade fermentation bottle. 1.0g of pre-weighed TMR (Total Mixed Ration) feed was placed at the bottom of the bottle to provide a fermentation substrate for the microorganisms in the rumen fluid, ensuring their survival. The small nylon bag was placed on top of the feed. 50mL of the buffer solution prepared in step b (preheated at 39℃) and 25mL of filtered rumen fluid were added to the culture bottle, with the buffer solution and rumen fluid mixed at a volume ratio of 2:1.

[0067] Tightly seal the silicone bottle cap and place it in a constant temperature incubator at 35-40℃ for in vitro fermentation with shaking. The shaking speed is 30 rpm. In this embodiment, in order to achieve better results, the internal temperature of the constant temperature incubator is set to 39℃.

[0068] The small nylon bags were removed at time points of 0, 2, 4, 8, 12, 16 and 24 hours respectively. After cleaning, the small nylon bags were dried in a constant temperature drying oven at 50-60℃ until constant weight, and then weighed (the weight of the small nylon bags after rumen digestion). In this embodiment, in order to achieve better results, the temperature of the constant temperature drying oven was set to 55℃.

[0069] To ensure the accuracy of the experiment, in this embodiment, five small nylon bags were taken at time points 1, 2, 4, 8, 12 and 24 hours, and seven small nylon bags were taken at time point 16 hours, to ensure that there were enough samples for subsequent small intestinal degradation rate determination.

[0070] The rumen degradation rate was calculated based on the difference in mass before and after rumen fermentation.

[0071] Rumen degradation rate = 1 - (weight of small nylon bag after rumen digestion - weight of small nylon bag itself) / weight of sample before rumen digestion.

[0072] 4. Determination of abomasal degradation rate:

[0073] a. Preparation of hydrochloric acid buffer:

[0074] Weigh 1.0 g of pepsin (1:10000) and dissolve it in 1000 mL of 0.1 mol / L hydrochloric acid;

[0075] b. Determination of abomasal degradation rate:

[0076] Take the small nylon bag taken out at the 16h time point in step 3, accurately weigh 0.5g of the sample (the weight of the sample before digestion) into a new small nylon bag (weigh it before use, and the result is the weight of the small nylon bag itself), seal it with a sealing machine, put the small nylon bag into the homemade fermentation bottle, add 80mL of hydrochloric acid buffer solution prepared in step a, and ensure that the liquid can submerge the small nylon bag;

[0077] After sealing the homemade fermentation bottle with the silicone sealant, place it in a constant temperature incubator at 35-40℃ for 1 hour. In this embodiment, to achieve better results, the internal temperature of the constant temperature incubator is set to 39℃.

[0078] After the culture is complete, remove the small nylon bags and dry them at 40-50℃ for 20-30 hours, then weigh them to obtain the weight of the small nylon bags after the abomasal digestion is finished.

[0079] Wherein, the degradation rate of the abomasum = [1 - (weight of the small nylon bag after digestion of the abomasum - weight of the small nylon bag itself) / weight of the sample before digestion].

[0080] In this embodiment, in order to achieve better results, the drying temperature is set to 50°C and the drying time is set to 24 hours.

[0081] 5. Determination of small intestinal degradation rate:

[0082] a. Preparation of small intestinal buffer:

[0083] First, adjust the pH of 80 mL of 0.5 mol / L phosphate buffer to 7.8. Then, add 30 mg / L of thymol, 0.5 g / 100 mL of trypsin and 2 g / 100 mL of bile salts to the phosphate buffer. The trypsin was purchased from Sigma-Aldrich and the bile salts were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0084] The preparation is complete after mixing;

[0085] b. Determination of small intestinal degradation rate:

[0086] The dried nylon bag from step 4b is placed into a homemade fermentation bottle, and the small intestinal buffer prepared in step a is added. After sealing the bottle with a silicone seal, it is placed in a constant temperature incubator at 35-40℃ and shaken at a speed of 10-20 rpm. In this embodiment, in order to achieve better results, the temperature of the constant temperature incubator is set to 39℃ and the shaking speed is set to 20 rpm.

[0087] Five small nylon bags were taken out at time points of 0, 2, 4, 8, 12 and 24 hours respectively. The small nylon bags were washed with ultrapure water until clear and then dried in an oven at 50°C for 24 hours. They were then removed, dried and weighed (the weight of the small nylon bags after small intestinal digestion).

[0088] The small intestinal degradation rate is calculated as follows: [1 - (weight of the small nylon bag after small intestinal digestion - weight of the small nylon bag itself) / weight of the sample before digestion].

[0089] The results of water solubility determination are shown in Table 1, acid solubility determination are shown in Table 2, rumen degradation rate is shown in Table 3, and small intestine degradation rate is shown in Table 4.

[0090] Example 2

[0091] The specific implementation steps are the same as in Example 1. The only difference is that the sample being measured is a commercially available rumen-protected amylase product. This product is a rumen-protected product D coated with hydrogenated palm oil as packaging material and a core material to packaging material ratio of 6:4 using microcrystalline cellulose excipient granulation. The water solubility, acid solubility, rumen degradation rate, abomasal degradation rate, and small intestinal degradation rate were measured.

[0092] The results of water solubility determination are shown in Table 1, acid solubility determination are shown in Table 2, rumen degradation rate is shown in Table 3, abomasal degradation rate is shown in Table 4, and small intestine degradation rate is shown in Table 5.

[0093] Example 3

[0094] The specific implementation steps are the same as in Example 1. The only difference is that the sample being measured is a commercially available rumen-protected amylase product. This product is a rumen-protected product F coated with rumen-protected fat powder granulated with microcrystalline cellulose excipients as packaging material and a core material to packaging material ratio of 6:4. The water solubility, acid solubility, rumen degradation rate, abomasal degradation rate and small intestine degradation rate are measured.

[0095] The results of water solubility determination are shown in Table 1, acid solubility determination are shown in Table 2, rumen degradation rate is shown in Table 3, abomasal degradation rate is shown in Table 4, and small intestine degradation rate is shown in Table 5.

[0096] Example 4

[0097] The specific implementation steps are the same as in Example 1, the only difference being that in the small intestinal release rate determination step, we set different gradients in the amount of pancreatic enzyme added to verify the optimal amount of pancreatic enzyme added. The added amounts were 0g / 100ml, 0.5g / 100ml, 1g / 100ml, 2g / 100ml, and 4g / 100ml, while the added amounts of other substances were the same. The small intestinal degradation rate was measured after 12 hours, and the results are shown in Table 6.

[0098] Example 5

[0099] The specific implementation steps are the same as in Example 1, the only difference being that in the small intestinal release rate determination step, we set different gradients in the amount of bile salts added to verify the optimal amount of bile salts added. The added amounts were 0 g / 100 ml, 0.5 g / 100 ml, 1 g / 100 ml, 2 g / 100 ml, 4 g / 100 ml, and 8 g / 100 ml. The added amounts of other substances were the same, with pancreatic enzyme added at 0.5 g / 100 mL. The small intestinal degradation rate was measured after 12 hours, and the results are shown in Table 7.

[0100] Comparative Example 1

[0101] The specific implementation steps are the same as in Example 1. The only difference is that the sample being measured is a commercially available rumen-protected amylase product. This product is made by granulating microcrystalline cellulose excipients without coating. Since the sample is not coated, it will be completely degraded in the rumen. Therefore, only the water solubility, acid solubility, and rumen degradation rate are measured.

[0102] The results of water solubility determination are shown in Table 1, the results of acid solubility determination are shown in Table 2, and the rumen degradation rate is shown in Table 3.

[0103] Comparative Example 2

[0104] This comparative example uses the traditional semi-in vivo small nylon bag method for determination. The sample tested is the product described in Example 1. The rumen degradation rate was determined using the following steps:

[0105] a. Weighing the sample:

[0106] Small nylon bags with a pore size of 40μm and a size of 12×10cm were numbered in advance, washed, dried at 65℃ for 48h, and weighed. 2g (the weight of the sample in the bag) of rumen amylase product was weighed using an analytical balance and placed into a small nylon bag of known weight and then sealed.

[0107] b. Small nylon bags for placement:

[0108] Small nylon bags containing samples were tied to different chains using nylon ropes, with a maximum of 12 bags tied to each chain. Following the principle of "adding and removing simultaneously," the chains with the bags were inserted into the rumen sac through a rumen fistula. One end of the chain was tied to the fistula cap with a nylon thread. The bags were removed at fermentation times of 0, 2, 4, 8, 16, and 24 hours (the specific times can be selected based on the feed ingredients). To ensure the accuracy of the experiment, three bags were removed at each time point (0, 2, 4, 8, 16, and 24 hours).

[0109] c. Cleaning:

[0110] Rinse the small nylon bag immediately under tap water until the water flowing out of the small nylon bag is clear and bright. After rinsing, gently squeeze out the excess water by hand.

[0111] d. Weighing:

[0112] The small nylon bags were dried at 45℃ for 48 hours, then removed and weighed (after rumen digestion), and transferred to sealed bags. The rumen degradation rate was calculated based on the difference in mass before and after rumen fermentation.

[0113] Rumen degradation rate = (weight of sample in bag – weight of sample after rumen digestion) / weight of sample in bag × 100%.

[0114] The comparative measurement results are shown in Table 8.

[0115] Comparative Example 3

[0116] The specific implementation steps are the same as those in Comparative Example 2, the only difference being that the sample being measured is the same product as in Example 2.

[0117] The comparative measurement results are shown in Table 9.

[0118] Comparative Example 4

[0119] The specific implementation steps are the same as those in Comparative Example 2, the only difference being that the sample being measured is the same product as in Example 3.

[0120] The comparative measurement results are shown in Table 10.

[0121] Table 1. Results of water solubility determination in Examples 1-3 and Comparative Example 1

[0122] 0h 2h 4h 8h 12h 24h Example 1 0.21％ 2.61％ 3.24％ 3.30％ 4.36％ 7.49％ Example 2 0.25％ 3.02％ 3.38％ 5.23％ 10.23％ 12.05％ Example 3 0.79％ 8.45％ 11.61％ 13.86％ 22.51％ 25.32％ Comparative Example 1 10.12％ 24.21％ 38.16％ 59.13％ 77.12％ 85.57％

[0123] Table 2. Results of acid solubility determination in Examples 1-3 and Comparative Example 1

[0124] 0h 2h 4h 8h 12h 24h Example 1 0.21％ 2.61％ 3.24％ 3.30％ 4.36％ 7.49％ Example 2 0.25％ 3.02％ 3.38％ 5.23％ 10.23％ 12.05％ Example 3 0.93％ 13.36％ 19.70％ 20.73％ 23.27％ 27.97％ Comparative Example 1 12.34％ 30.76％ 44.19％ 66.09％ 83.27％ 92.15％

[0125] Table 3. Results of rumen degradation rate determination in Examples 1-3 and Comparative Example 1

[0126] 0h 2h 4h 8h 12h 24h Example 1 0.67％ 2.55％ 2.68％ 3.18％ 4.69％ 6.32％ Example 2 0.68％ 3.18％ 5.34％ 7.72％ 8.40％ 10.69％ Example 3 0.30％ 5.83％ 10.59％ 15.70％ 17.68％ 19.58％ Comparative Example 1 14.55％ 36.76％ 57.68％ 89.90％ 100％ 100％

[0127] Table 4. Results of 1-hour degradation rate determination in abomasum for Examples 1-3

[0128] Example 1 Example 2 Example 3 Abdominal gastric degradation for 1 hour 0.59％ 1.00％ 6.23％

[0129] Table 5 Results of small intestinal degradation rate determination in Examples 1-3

[0130] 0h 2h 4h 8h 12h 24h Example 1 0.47％ 1.55％ 2.70％ 15.74％ 33.04％ 42.22％ Example 2 0.69％ 1.65％ 5.00％ 27.43％ 41.22％ 52.03％ Example 3 0.36％ 2.77％ 22.65％ 23.32％ 31.77％ 37.99％

[0131] Table 6 Results of small intestinal degradation rate determination in Example 4

[0132] Project (enzyme concentration) 0g / 100ml 0.5g / 100ml 1g / 100ml 2g / 100ml 4g / 100ml Small intestinal degradation rate 0.20％ 7.25％ 7.33％ 7.46％ 7.47％

[0133] Table 7 Results of small intestinal degradation rate determination in Example 5

[0134] Project (bile salt concentration) 0g / 100ml 0.5g / 100ml 1g / 100ml 2g / 100ml 4g / 100ml 8g / 100ml Small intestinal degradation rate 0.48％ 1.69％ 3.14％ 40.01％ 40.50％ 40.78％

[0135] Table 8 Results of Rumen Degradation Rate Measurement in Example 1 and Comparative Example 2

[0136] 0h 2h 4h 8h 12h 24h Example 1 0.67％ 2.55％ 2.68％ 3.18％ 4.69％ 6.32％ Comparative Example 2 1.23％ 3.09％ 3.77％ 4.11％ 5.40％ 7.09％

[0137] Table 9 Results of rumen degradation rate determination in Example 2 and Comparative Example 3

[0138] 0h 2h 4h 8h 12h 24h Example 2 0.68％ 3.18％ 5.34％ 7.72％ 8.40％ 10.69％ Comparative Example 3 0.98％ 4.89％ 7.17％ 8.90％ 10.76％ 11.87％

[0139] Table 10 Results of rumen degradation rate determination in Example 3 and Comparative Example 4

[0140] 0h 2h 4h 8h 12h 24h Example 3 0.30％ 5.83％ 10.59％ 15.70％ 17.68％ 19.58％ Comparative Example 4 0.69％ 6.21％ 11.21％ 17.90％ 18.65％ 21.09％

[0141] The data in Tables 1-5 show that the uncoated granulated product degrades rapidly at all sites, reaching almost complete degradation in the rumen. The coated product, however, is coated with fat, resulting in poor water and acid solubility, yet it degrades more significantly in the rumen. This indicates that microorganisms also participate in the degradation process in our in vitro rumen system, as lipases are not added to the rumen and can only be produced by microorganisms. Furthermore, comparing the data from Example 1 and Example 2 shows that Example 1, with its thicker coating, exhibits lower rumen and small intestinal degradation rates compared to Example 2. This demonstrates that the degradation intensity increases with decreasing coating thickness, consistent with the degradation characteristics of our product and the rumen. In the small intestinal degradation, we can conclude that the addition of bile salts and pancreatic enzymes effectively degrades the rumen-crossed product and achieves a good release rate.

[0142] As can be seen from the data in Table 6, when different concentration gradients of pancreatic enzyme are added, the degradation rate in the small intestine increases with the increase of the added amount. When the added amount is 0.5g / 100ml, the degradation rate changes very little with the increase of the added amount. Therefore, the appropriate added amount of pancreatic enzyme is 0.5g / 100ml.

[0143] As shown in Table 7, when different concentration gradients of bile salts were added, the small intestinal degradation rate increased with the increase of the added amount. The degradation rate changed slightly at 0.3 g / 100 ml and 1 g / 100 ml of bile salts. However, the change was very large when the added amount was 2 g / 100 ml, and the degradation rate increased significantly. This indicates that the concentration of bile salts was sufficient to form micelles of fat on the surface of the rumen-protected product coating, increasing the contact area with fat and pancreatic enzymes, thereby improving digestibility. Further increases in concentration did not change the degradation rate much, proving that it was an appropriate addition concentration.

[0144] As can be seen from the data in Table 8-10, both the traditional semi-in vivo nylon bag method and the method for determining the release rate of rumen-passing products in ruminants provided in this application can measure the rumen degradation rate. Both can accurately reflect the rumen degradation rate of rumen-passing products. However, this application involves in vitro culture, which can greatly reduce mechanical damage to animals and significantly improve animal welfare while ensuring the authenticity of the data.

[0145] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for determining the release rate at various points of a rumen-transfer product in ruminants, characterized in that, Includes the following steps: Water solubility determination: Weigh an appropriate amount of rumen-passed sample, add distilled water and incubate at a constant temperature. After incubation, rinse and filter, dry to constant weight and then determine the water solubility. Acid solubility determination: Weigh an appropriate amount of rumen-passed sample, add an acidic buffer solution with pH 4, and incubate at a constant temperature. After incubation, rinse and filter, dry to constant weight, and then determine the acid solubility. Rumen degradation rate determination: Weigh the rumen-passed sample after water solubility and / or acid solubility determination, place it on the feed, place the feed at the bottom of the bottle of the self-made fermentation device, add buffer solution and rumen fluid for in vitro fermentation, dry at constant temperature after fermentation, weigh it, and determine the rumen degradation rate. The homemade fermentation device includes: a bottle body, which is a standard fermentation bottle with a capacity of 100ml, with a small nylon bag placed inside the bottle body; a silicone sealing cap, which is set at the mouth of the bottle body; a one-way gas valve system, which is connected to the bottle body through the silicone sealing cap; and a gas collection bag, which is connected to the one-way gas valve system to collect the gas produced during fermentation. Rumen degradation rate determination: Weigh the rumen-passed sample after the rumen degradation rate determination, add pepsin hydrochloride buffer and incubate at constant temperature. After the incubation is completed, take it out, dry it at constant temperature and weigh it to determine the rumen degradation rate. Small intestinal degradation rate determination: Rumen-passed samples after abomasal degradation rate determination were taken, and small intestinal buffer was added for shaking culture. After washing, the samples were dried at constant temperature and weighed for small intestinal degradation rate determination. The small intestinal buffer included phosphate, thymol, trypsin, and bile salts. The amount of phosphate in the small intestinal buffer was 0.5 mol / L, the amount of thymol was 30-50 mg / L, the amount of trypsin was 0.5-4 g / 100 mL, and the amount of bile salts was 2-4 g / 100 mL.

2. The determination method according to claim 1, characterized in that, In the determination of small intestinal degradation rate, the temperature of the shaking culture is 35-40℃, the shaking speed is 10-30rpm, and the shaking time is 0-24h; the temperature of the drying and weighing is 40-50℃, and the time is 24-48h.

3. The determination method according to claim 1, characterized in that, In the determination of rumen degradation rate, the in vitro fermentation temperature is 35-39℃ and the time is 0-24h, and the constant temperature drying temperature is 50-60℃.

4. The determination method according to claim 1, characterized in that, In the determination of water solubility, the rumen-passed sample is added to distilled water and then incubated at a constant temperature, which includes the following steps: Incubate at a constant temperature with shaking speed of 10-30 rpm for 10-30 min. The drying temperature is 45-60℃.

5. The determination method according to claim 1, characterized in that, In the acid solubility determination, the process of adding the rumen-passed sample to an acidic buffer solution with a pH of 4 and then incubating it at an isothermal temperature includes the following steps: Incubate at a constant temperature with a rotation speed of 10-30 rpm for 10-30 minutes; The drying temperature is 45-60℃.

6. The determination method according to any one of claims 1-5, characterized in that, The bottle body is a cylindrical bottle with an inner diameter of 29mm at the bottle mouth.

7. The determination method according to claim 1, characterized in that, The one-way valve system includes a one-way valve pipe, a valve, and a valve connecting pipe. The one-way valve tube is located at the silicone sealing cap and extends out of the silicone sealing cap at both ends. The end of the one-way valve tube away from the bottle body is connected to the valve. The valve is connected to a valve connecting tube, which is connected to the gas collection bag.

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