In-vitro determination and classification method for dynamic digestion rate of nutrient components of feed and application of in-vitro determination and classification method

Abstract

The invention provides an in-vitro determination and classification method for the dynamic digestion rate of feed nutrient components and application, and belongs to the technical field of feed science and analysis. According to the technical scheme, the method comprises the following steps: performing simulated in-vitro digestion on a feed sample, wherein the digestion sequentially comprises a simulated stomach digestion stage and a simulated small intestine digestion stage; sampling at a plurality of preset time points in the simulated small intestine digestion stage, and measuring the cumulative digestibility of the target nutrient component in the sample at each time point; the target nutritional component is classified based on the determined cumulative digestibility. Quantitative data of fast, slow and non-digestible components obtained by the method can provide a key scientific basis for designing a precise feed formula and realize optimal matching of nutrition supply and dynamic requirements of animals, so that the utilization efficiency of the animals on feed nutrients is remarkably improved, the growth performance of the animals is improved, the feed cost is reduced, and the economic benefits of the animals are improved. Resource consumption and environmental pollution are reduced, and remarkable economic benefits and application values are achieved.

Description

Technical Field

[0001] This invention relates to the field of feed science and analysis technology, and in particular to an in vitro method and application for determining and classifying the dynamic digestibility rate of feed nutrient components. Background Technology

[0002] In intensive livestock production, the precise and efficient utilization of feed resources is key to controlling costs and improving efficiency. Currently, the industry's evaluation of the nutritional value of feed ingredients largely relies on static indicators, such as effective energy and ileal amino acid digestibility. However, these static indicators only reflect the final result of nutrient digestion and cannot reveal the complete process of dynamic digestion, absorption, and utilization of key nutrients such as starch and protein in the animal's digestive tract.

[0003] The lack of this evaluation dimension makes it difficult to accurately match feed formulation design with the actual nutritional needs of animals at different growth stages, often resulting in poor feed conversion efficiency and resource waste. Although traditional in vivo experiments can obtain dynamic digestion data, their application is greatly limited due to their long experimental cycles, high costs, complex operations, and the unnecessary stress they cause to experimental animals. To overcome the drawbacks of in vivo experiments, those skilled in the art have begun to explore in vitro biomimetic digestion technology. Existing technologies already include some in vitro digestion systems that can simulate the animal gastrointestinal environment, mimicking basic physicochemical conditions such as temperature and pH during digestion.

[0004] However, most of these systems are used to measure endpoint digestibility, that is, the total amount of nutrients digested after a set digestion time. They generally lack a standardized method for dynamically tracking the entire digestion process, and even more so, they fail to provide a scientifically based, widely accepted classification standard to distinguish components in feed with significantly different digestibility rates, such as rapidly digestible, slowly digestible, and indigestible portions. This lack of a method for assessing the dynamic process of digestion limits the ability to refine feed ingredients and achieve precise formulation optimization. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide an in vitro method for determining and classifying the dynamic digestibility rate of feed nutrient components, so as to solve the technical problems faced by the prior art in evaluating the nutritional value of feed, which is that the evaluation indicators are static and cannot reflect the dynamic digestion process, and lacks a standardized evaluation system that can quantitatively classify nutrient components according to digestibility rate.

[0006] The second objective of this invention is to provide an application of an in vitro method for determining and classifying the dynamic digestibility rate of feed nutrient components.

[0007] To achieve one of the above objectives, the present invention provides an in vitro method for determining and classifying the dynamic digestibility of feed nutrient components, comprising the following steps: Step 1: Pre-treat the feed sample to be tested; Step 2: Perform simulated in vitro digestion on the pretreated feed sample, which includes a simulated stomach digestion stage and a simulated small intestine digestion stage. Step 3: Samples are taken at multiple preset time points during the simulated small intestine digestion stage, and the cumulative digestibility of the target nutrient components in the samples at each preset time point is measured; Step 4: Based on the measured cumulative digestibility, classify the target nutrient components, including: The portion digested within the first preset time after the start of the simulated small intestinal digestion stage is defined as the rapidly digested component. The portion that is digested between the first preset time period and the second preset time period is defined as the slow-digesting component; The portion that remains undigested after the second preset time period is defined as an indigestible component.

[0008] Optionally, the target nutrient component is starch or protein.

[0009] Optionally, the first preset duration is 30 minutes, and the second preset duration is 240 minutes.

[0010] Optionally, the simulated gastric digestion stage includes: placing the feed sample in simulated gastric juice containing pepsin, incubating it in an acidic environment with a pH of 3.5, and incubating it at 39°C for 120 minutes.

[0011] Optionally, the simulated small intestine digestion stage includes: after the simulated stomach digestion stage is completed, adjusting the digestive environment to neutral, and adding simulated intestinal fluid containing pancreatic enzymes for incubation, wherein the simulated intestinal fluid also contains amylase and invertase.

[0012] Optionally, when the target nutrient component is starch, the cumulative digestibility is determined by measuring the reducing sugar content in the digestive fluid.

[0013] Optionally, when the target nutrient component is protein, the cumulative digestibility is determined by measuring the crude protein content in the digestate using the Kjeldahl method.

[0014] Optionally, the classification is specifically calculated by determining the percentage of each component in the following manner: The percentage of rapidly digestible components is equal to the cumulative digestibility of the target nutrient components measured during the first preset time period; The percentage of slowly digestible components is equal to the difference between the cumulative digestibility of the target nutrient components measured at the second preset time period and the cumulative digestibility of the target nutrient components measured at the first preset time period. The percentage of indigestible components is equal to 100% minus the cumulative digestibility of the target nutrient components measured at the second preset time period.

[0015] To achieve the second objective mentioned above, this invention provides an application of an in vitro method for determining and classifying the dynamic digestibility of feed nutrient components as described above. The application includes the in vitro determination and classification of the proportion of rapidly digestible, slowly digestible, and indigestible starch in 19 common feed ingredients, including corn, sorghum, wheat, barley, puffed corn, peas, bread flour, extruded corn, extruded sorghum, extruded wheat, extruded barley, rice bran, rice bran meal, cassava, wheat middlings, brown rice, broken rice, corn gluten meal, and wheat bran.

[0016] To achieve the second objective mentioned above, this invention provides an application of an in vitro method for determining and classifying the dynamic digestibility of feed nutrient components as described in any of the above-mentioned methods. The application includes the in vitro determination and classification of the proportions of rapidly digestible, slowly digestible, and indigestible proteins in 13 common feed ingredients, including high-protein soybean meal, cottonseed meal, wheat protein powder, peanut meal, corn protein powder, rapeseed meal, potato protein powder, skim milk powder, soybean protein concentrate, soybean meal, sunflower meal, corn, and extruded corn.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a rapid, low-cost, and highly reproducible in vitro assay method. It also proposes for the first time a classification based on two key time points: 30 minutes and 240 minutes of simulated small intestinal digestion. This provides a clear, unified, and operable classification standard for the dynamic digestibility characteristics of starch and protein in feed, solving the core problems of static evaluation indicators and the lack of classification standards in the industry. The quantitative data on rapid, slow, and indigestible components obtained through this method can provide crucial scientific basis for designing precise feed formulations, achieving optimal matching between nutrient supply and the dynamic needs of animals. This significantly improves the efficiency of animal nutrient utilization, enhances animal growth performance, reduces feed costs, and minimizes resource consumption and environmental pollution, demonstrating significant economic benefits and application value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a flowchart of an in vitro method for determining and classifying the dynamic digestibility of feed nutrient components according to the present invention; Figure 2 yes Figure 1 A schematic diagram illustrating the principle of digestibility classification in in vitro methods for determining and classifying the dynamic digestibility of feed nutrient components. in, Figures 1-2 : S40 - Multi-time point sampling; S50 - Target component content determination; S60 - Dynamic digestion rate classification; Zone A - Rapidly digested components; Zone B - Slowly digested components; Zone C - Indigestible components. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] Example 1 This embodiment provides an in vitro method for determining and classifying the dynamic digestibility of feed nutrients. This method simulates the digestive environment within an animal to quantitatively analyze the digestibility of key nutrients (such as starch) at different time points, and based on this, establishes a standardized classification system, thereby providing a scientific basis for the design of precision feed formulations.

[0022] Please see Figure 1 This illustrates the overall process of the method provided in this embodiment. Specifically, the method mainly includes core steps such as sample pretreatment S10, simulated gastric digestion stage S20, simulated small intestinal digestion stage S30, multi-time point sampling S40, target component content determination S50, and dynamic digestion rate classification S60.

[0023] The steps will be explained in detail below.

[0024] Step 1: Sample Pretreatment S10 In this embodiment, to ensure the accuracy and repeatability of the experimental results, the feed samples to be tested need to be standardized and pretreated.

[0025] The purpose of pretreatment is to increase the contact area between the sample and the digestive enzyme and to eliminate experimental errors caused by uneven particle size.

[0026] The specific procedure is as follows: Nineteen feed ingredients to be tested, such as corn, sorghum, wheat, barley, puffed corn, peas, bread flour, flaked corn, flaked sorghum, flaked wheat, flaked barley, rice bran, rice bran meal, cassava, wheat middlings, brown rice, broken rice, corn gluten meal, and wheat bran, are selected and pulverized using a laboratory grinder. The pulverized samples are then sieved through a standard 40-mesh sieve, and the powder passing through the sieve is collected as the test sample.

[0027] This step ensures that all sample particles entering the digestion system have a relatively consistent maximum particle size, providing uniform substrate conditions for subsequent enzymatic digestion reactions.

[0028] Step 2: Simulating the gastric digestion stage S20 This stage is used to simulate the initial digestion process of food in the gastric environment of a monogastric animal.

[0029] The specific procedure is as follows: Accurately weigh approximately 0.5 grams of the feed sample after pretreatment in step S10 and place it in a clean, dry 50 ml centrifuge tube. To ensure that the sample temperature is consistent with the subsequent reaction temperature, place the centrifuge tube containing the sample in a preheated 39°C water bath shaker for 10 minutes. Setting the temperature to 39°C is intended to simulate the normal physiological body temperature of most livestock. Accordingly, prepare simulated gastric juice.

[0030] In this embodiment, the simulated gastric juice is a buffer solution containing pepsin and a thickener.

[0031] The preparation method is as follows: dissolve pepsin in an appropriate amount of phosphate buffer solution to a final concentration of 5 mg / mL, and dissolve guar gum to a final concentration of 5 mg / mL. Pepsin, as the main proteolytic enzyme in gastric juice, is responsible for the initial degradation of proteins; while the addition of guar gum increases the viscosity of the digestive juice to simulate the viscous state of gastric contents, making the digestive process closer to the actual situation in the body.

[0032] The pH of the pepsin solution was then carefully adjusted to 3.5 using dilute hydrochloric acid to simulate the highly acidic environment of the stomach. Next, 10 ml of the prepared pepsin solution was added to the preheated centrifuge tube.

[0033] In addition, to prevent microbial growth during incubation from interfering with the experimental results, 0.25 ml of chloramphenicol ethanol solution should be added to the centrifuge tube as an antibacterial agent.

[0034] Finally, tighten the caps on the centrifuge tubes and place them in a constant-temperature water bath shaker. Incubate at 39°C and a shaking frequency of 190 rpm for 120 minutes. Continuous shaking ensures that the sample and digestive fluid are thoroughly mixed to simulate gastric peristalsis.

[0035] Step 3: Simulating the small intestine digestion stage S30 After the stomach digestion stage is completed, the digested food (chyme) enters the small intestine for further digestion in a slightly neutral environment. This step is used to simulate this process.

[0036] First, remove the centrifuge tubes from the constant-temperature water bath shaker after 120 minutes of gastric digestion. To simulate the process of chyme being neutralized by bile and pancreatic juice after entering the duodenum from the stomach, quickly add 10 ml of a 0.048 mol / L sodium hydroxide solution preheated to 39°C to the centrifuge tubes. This is intended to neutralize the hydrochloric acid remaining from the gastric digestion stage and adjust the pH of the digestive system to a near-neutral 6.8.

[0037] It should be noted that the moment when the pH value is neutralized is defined as the starting moment (0 minutes) of the simulated small intestinal digestion stage.

[0038] Immediately afterwards, 5 ml of pre-prepared and preheated pancreatic enzyme mixture was added to the centrifuge tube. This pancreatic enzyme mixture is used to simulate the complex enzyme environment in the small intestine. Its specific preparation method is as follows: Pancreatic enzyme was dissolved in a phosphate buffer solution at pH 6.8 to a final concentration of 0.14 g / ml, while amylase (0.01%) and invertase (0.6 mg / ml) were added simultaneously. Pancreatic enzyme contains various proteolytic enzymes and lipases, responsible for the extensive hydrolysis of proteins and fats; amylase and invertase can completely break down starch and oligosaccharides into absorbable monosaccharides (mainly glucose), thus ensuring the complete digestion of starchy substances.

[0039] Finally, the centrifuge tubes containing the trypsin mixture were placed back into a constant temperature water bath shaker at 39°C and 190 rpm for incubation for a total of 240 minutes.

[0040] Step 4: Multi-time point sampling S40 To capture the dynamic digestion process of nutrients, sampling needs to be performed at multiple time points during the simulated small intestinal digestion stage S30. In this embodiment, the preset sampling time points include, but are not limited to: 0, 30, 60, 90, 120, 150, 180, 210, and 240 minutes.

[0041] The sampling procedure is as follows: at each preset time point, precisely aspirate 0.5 ml of digestion fluid from the vibrating centrifuge tube.

[0042] To immediately stop the activity of all enzymes in the digestive solution and "freeze" the digestion state at that point in time, the aspirated 0.5 ml of digestive solution should be quickly injected into a test tube containing 5 ml of distilled water that has been pre-placed in an ice bath. The low temperature and dilution effect will rapidly inactivate the enzymes.

[0043] After all sampling was completed and the reaction was terminated, the samples were transferred to 50 mL centrifuge tubes and stored at -20°C for subsequent content determination.

[0044] Step 5: Determination of target component content S50 This step is used to quantitatively analyze the degree of digestion of the target nutrient component in the samples at various time points and to calculate the cumulative digestibility. Different determination methods may be used depending on the target nutrient component.

[0045] The target nutrient in this embodiment is starch, and the digestion products of starch are mainly reducing sugars such as glucose. Therefore, the amount of starch digested can be inferred by measuring the content of reducing sugars in the digestive fluid.

[0046] In this embodiment, the starch content in the digestive fluid sample was detected. The sample was placed in a water bath and heated to 60°C. 6 mL of thermostable α-amylase solution was added, and the mixture was kept in a water bath at 75°C-80°C for 1 hour for enzymatic hydrolysis. During this process, the conical flask was gently shaken every 10 minutes to ensure sufficient contact between the sample and the enzyme solution. After hydrolysis, the sample was heated to boiling for 5 minutes to terminate the enzyme reaction. After cooling, the sample was filtered through defatted cotton, and the filtrate was collected and diluted to a 50 mL centrifuge tube for later use. 10 mL of the filtrate was placed in a test tube, 3 mL of 6 mol / L hydrochloric acid solution was added, and a reflux condenser was connected. The sample was heated in a boiling water bath at 100°C for 1 hour for hydrolysis. After hydrolysis, the sample was cooled to room temperature, and an appropriate amount of 200 g / L sodium hydroxide solution was added to adjust the pH to 7.0. The solution was then transferred to a 50 mL centrifuge tube and diluted to volume with distilled water.

[0047] According to the standard method of GB / T 20194-2018, the reducing sugar content is determined using alkaline copper tartrate reagent. The volume of alkaline copper tartrate solution consumed is recorded as follows: The data will be used for subsequent calculations. Based on the measured reducing sugar content, the starch content in the digestive juice will be calculated using the following formula: in, x m1: Starch content in the sample (g / 100g); m2: Mass of reducing sugar (calculated as glucose) equivalent to alkaline copper tartrate solution (mg); m3: Mass of glucose solution consumed in the reagent blank (mg), determined based on the experimental blank measurement value; m: Mass of sample weighed (g). Volume of sample solution consumed (mL) when measuring reducing sugars.

[0048] The final calculation result will be multiplied by the glucose-to-starch conversion factor of 0.9 and the measured reducing sugar content to obtain the actual starch content in the digestive fluid sample.

[0049] Step Six: Dynamic Digestion Rate Classification S60 This step categorizes the target nutrient components based on the cumulative digestibility data obtained in step S50 over a series of time points. Please refer to [link / reference]. Figure 2 This is a schematic diagram illustrating the principle of digestion rate classification.

[0050] The present invention proposes to use two specific time points simulating the small intestine digestion stage as the basis for division: the first preset time T1 = 30 minutes and the second preset time T2 = 240 minutes.

[0051] Understandably, the choice of these two time points is based on the analysis of a large amount of experimental data. 30 minutes usually represents the end of the initial stage where the digestion rate is fastest, while 240 minutes is close to the plateau phase of the digestion curve for most easily digestible components.

[0052] Based on these two time points, the target nutrient components were divided into three parts: 1. Rapidly digestible components (corresponding to) Figure 2 Section A in the diagram refers to the nutrients digested within the first preset time (30 minutes) after the start of simulated small intestinal digestion. This section represents the components that can be rapidly broken down and absorbed by the animal's body to provide energy quickly.

[0053] 2. Slow-digesting components (corresponding to) Figure 2 Zone B in the table refers to the nutrient components that are digested between the first preset time (30 minutes) and the second preset time (240 minutes). This section represents the components that can continuously and steadily release nutrients, providing a long-term supply to the body.

[0054] 3. Indigestible components (corresponding to) Figure 2 Zone C in the text refers to the nutrient components that have not been broken down by the in vitro digestive system after the second preset time (240 minutes). In practical applications, this part is usually considered as a resistant component or an ineffective component that cannot be utilized by animals.

[0055] The specific quantitative calculation method is as follows: Percentage of rapidly digested components (%) = Cumulative digestibility measured at 30 minutes.

[0056] Percentage of slowly digested components (%) = (Cumulative digestibility measured at 240 minutes) - (Cumulative digestibility measured at 30 minutes).

[0057] Percentage of indigestible components (%) = 100% - (cumulative digestibility measured at 240 minutes).

[0058] Table 1 shows the changes in starch digestibility at different in vitro biomimetic digestion time points for corn, sorghum, wheat, barley, puffed corn, peas, bread flour, flaked corn, flaked sorghum, flaked wheat, flaked barley, rice bran, rice bran meal, cassava, wheat middlings, brown rice, broken rice, corn gluten meal, and wheat bran. Based on the starch digestibility of these 19 raw materials at different in vitro time points, it was found that the coefficient of variation for starch digestibility at the 30-minute mark was significantly higher than at other time points.

[0059] The above results indicate that the digestibility at 30 minutes of in vitro biomimetic digestion is the most representative indicator for distinguishing the starch digestibility rates of different feed ingredients.

[0060] In addition, flaked corn, flaked sorghum, flaked wheat, and flaked barley have a relatively high proportion of rapidly digestible starch, ranging from 93.44% to 96.00%. Bread flour, rice bran meal, wheat bran, and cassava have a rapidly digestible starch proportion of approximately 57.42% to 68.45%, while slowly digestible starch accounts for approximately 12.93% to 29.74%. Broken rice, wheat middlings, whole rice bran, puffed corn, peas, barley, brown rice, sorghum, wheat, and corn have a higher proportion of slowly digestible starch than rapidly digestible starch, with rapidly digestible starch accounting for approximately 19.38% to 37.87% and slowly digestible starch accounting for approximately 43.27% to 72.50%. Corn, whole rice bran, rice bran meal, peas, corn gluten meal, and wheat bran have a relatively high proportion of indigestible starch, ranging from approximately 10.00% to 37.35%.

[0061] Table 1. Proportion of rapidly digestible, slowly digestible, and indigestible starch in 19 common feed ingredients Coefficient of variation calculation method: Coefficient of variation CV (%) = (Standard deviation SD / Mean) × 100% Through the above six steps, this embodiment can accurately and repeatedly quantify the starch in any feed ingredient into three parts: fast-moving, slow-moving, and indigestible, providing a solid data foundation for the establishment and application of a precision feed formulation database.

[0062] Example 2 This embodiment provides an in vitro method for determining and classifying the dynamic digestibility of feed nutrients. This method simulates the digestive environment within an animal to quantitatively analyze the degree of digestibility of key nutrients (such as protein) at different time points, and based on this, establishes a standardized classification system, thereby providing a scientific basis for the design of precision feed formulations.

[0063] Please see Figure 1This illustrates the overall process of the method provided in this embodiment. Specifically, the method mainly includes core steps such as sample pretreatment S10, simulated gastric digestion stage S20, simulated small intestinal digestion stage S30, multi-time point sampling S40, target component content determination S50, and dynamic digestion rate classification S60.

[0064] The steps will be explained in detail below.

[0065] Step 1: Sample Pretreatment S10 In this embodiment, to ensure the accuracy and repeatability of the experimental results, the feed samples to be tested need to be standardized and pretreated.

[0066] The purpose of pretreatment is to increase the contact area between the sample and the digestive enzyme and to eliminate experimental errors caused by uneven particle size.

[0067] The specific procedure is as follows: Thirteen feed ingredients to be tested, including dehulled soybean meal, cottonseed meal, wheat gluten meal, peanut meal, corn gluten meal, rapeseed meal, potato gluten meal, skim milk powder, soy protein concentrate, soybean meal, sunflower meal, fish meal, and cottonseed protein, are selected and pulverized using a laboratory grinder. The pulverized samples are then sieved through a standard 40-mesh sieve, and the powder passing through the sieve is collected as the test sample.

[0068] This step ensures that all sample particles entering the digestion system have a relatively consistent maximum particle size, providing uniform substrate conditions for subsequent enzymatic digestion reactions.

[0069] Step 2: Simulating the gastric digestion stage S20 This stage is used to simulate the initial digestion process of food in the gastric environment of a monogastric animal.

[0070] The specific procedure is as follows: Accurately weigh approximately 0.5 grams of the feed sample after pretreatment in step S10 and place it in a clean, dry 50 ml centrifuge tube. To ensure that the sample temperature is consistent with the subsequent reaction temperature, place the centrifuge tube containing the sample in a preheated 39°C water bath shaker for 10 minutes. Setting the temperature to 39°C is intended to simulate the normal physiological body temperature of most livestock. Accordingly, prepare simulated gastric juice.

[0071] In this embodiment, the simulated gastric juice is a buffer solution containing pepsin and a thickener.

[0072] The preparation method is as follows: dissolve pepsin in an appropriate amount of phosphate buffer solution to a final concentration of 5 mg / mL, and dissolve guar gum to a final concentration of 5 mg / mL. Pepsin, as the main proteolytic enzyme in gastric juice, is responsible for the initial degradation of proteins; while the addition of guar gum increases the viscosity of the digestive juice to simulate the viscous state of gastric contents, making the digestive process closer to the actual situation in the body.

[0073] The pH of the pepsin solution was then carefully adjusted to 3.5 using dilute hydrochloric acid to simulate the highly acidic environment of the stomach. Next, 10 ml of the prepared pepsin solution was added to the preheated centrifuge tube.

[0074] In addition, to prevent microbial growth during incubation from interfering with the experimental results, 0.25 ml of chloramphenicol ethanol solution should be added to the centrifuge tube as an antibacterial agent.

[0075] Finally, tighten the caps on the centrifuge tubes and place them in a constant-temperature water bath shaker. Incubate at 39°C and a shaking frequency of 190 rpm for 120 minutes. Continuous shaking ensures that the sample and digestive fluid are thoroughly mixed to simulate gastric peristalsis.

[0076] Step 3: Simulating the small intestine digestion stage S30 After the stomach digestion stage is completed, the digested food (chyme) enters the small intestine for further digestion in a slightly neutral environment. This step is used to simulate this process.

[0077] First, remove the centrifuge tubes from the constant-temperature water bath shaker after 120 minutes of gastric digestion. To simulate the process of chyme being neutralized by bile and pancreatic juice after entering the duodenum from the stomach, quickly add 10 ml of a 0.048 mol / L sodium hydroxide solution preheated to 39°C to the centrifuge tubes. This is intended to neutralize the hydrochloric acid remaining from the gastric digestion stage and adjust the pH of the digestive system to a near-neutral 6.8.

[0078] It should be noted that the moment when the pH value is neutralized is defined as the starting moment (0 minutes) of the simulated small intestinal digestion stage.

[0079] Immediately afterwards, 5 ml of pre-prepared and preheated pancreatic enzyme mixture was added to the centrifuge tube. This pancreatic enzyme mixture is used to simulate the complex enzyme environment in the small intestine. Its specific preparation method is as follows: Pancreatic enzyme was dissolved in a phosphate buffer solution at pH 6.8 to a final concentration of 0.14 g / ml, while simultaneously adding proteoglycans at a final concentration of 0.01% and invertase at a final concentration of 0.6 mg / ml. Pancreatic enzymes contain various proteolytic enzymes and lipases, responsible for the extensive hydrolysis of proteins and fats; proteoglycans and invertase can completely break down proteins and oligosaccharides into absorbable monosaccharides (mainly glucose), thus ensuring the complete digestion of protein-like substances.

[0080] Finally, the centrifuge tubes containing the trypsin mixture were placed back into a constant temperature water bath shaker at 39°C and 190 rpm for incubation for a total of 240 minutes.

[0081] Step 4: Multi-time point sampling S40 To capture the dynamic digestion process of nutrients, sampling needs to be performed at multiple time points during the simulated small intestinal digestion stage S30. In this embodiment, the preset sampling time points include, but are not limited to: 0, 30, 60, 90, 120, 150, 180, 210, and 240 minutes.

[0082] The sampling procedure is as follows: at each preset time point, precisely aspirate 0.5 ml of digestion fluid from the vibrating centrifuge tube.

[0083] To immediately stop the activity of all enzymes in the digestive solution and "freeze" the digestion state at that point in time, the aspirated 0.5 ml of digestive solution should be quickly injected into a test tube containing 5 ml of distilled water that has been pre-placed in an ice bath. The low temperature and dilution effect will rapidly inactivate the enzymes.

[0084] After all sampling was completed and the reaction was terminated, the samples were transferred to 50 mL centrifuge tubes and stored at -20°C for subsequent content determination.

[0085] Step 5: Determination of target component content S50 This step is used to quantitatively analyze the degree of digestion of the target nutrient component in the samples at various time points and to calculate the cumulative digestibility. Different determination methods may be used depending on the target nutrient component.

[0086] The target nutrient component in this embodiment is protein, and the digestion products of protein are mainly amino acids.

[0087] The crude protein content in the digestion fluid sample was determined according to the standard method of GB / T 6432-2018: 5 mL of digestion fluid was weighed into a digestion tube, one granule of mixed catalyst was added, and then 10 mL of sulfuric acid was added to the digestion tube. The tube was digested at 420℃ for 90 minutes in a digestion oven. After removal, it was cooled for 30 minutes, and the tube cap was rinsed with a small amount of distilled water. The digestion tube was placed in a semi-automatic Kjeldahl nitrogen analyzer. The end of the condenser of the distillation apparatus was immersed in an Erlenmeyer flask containing 35 mL of boric acid absorption solution and 2 drops of methyl red-bromocresol green mixed indicator. The absorption solution was immediately titrated with 0.1 mol / L hydrochloric acid standard titration solution; the endpoint was reached when the solution changed from blue-green to gray-red.

[0088] : Protein mass fraction in the sample; v : The volume of hydrochloric acid standard solution consumed during sample titration, in mL; c : Concentration of hydrochloric acid standard solution consumed during sample titration, in mol / L.

[0089] Step Six: Dynamic Digestion Rate Classification S60 This step categorizes the target nutrient components based on the cumulative digestibility data obtained in step S50 over a series of time points. Please refer to [link / reference]. Figure 2 This is a schematic diagram illustrating the principle of digestion rate classification.

[0090] The present invention proposes to use two specific time points simulating the small intestine digestion stage as the basis for division: the first preset time T1 = 30 minutes and the second preset time T2 = 240 minutes.

[0091] Understandably, the choice of these two time points is based on the analysis of a large amount of experimental data. 30 minutes usually represents the end of the initial stage where the digestion rate is fastest, while 240 minutes is close to the plateau phase of the digestion curve for most easily digestible components.

[0092] Based on these two time points, the target nutrient components were divided into three parts: 1. Rapidly digestible components (corresponding to) Figure 2 Section A in the diagram refers to the nutrients digested within the first preset time (30 minutes) after the start of simulated small intestinal digestion. This section represents the components that can be rapidly broken down and absorbed by the animal's body to provide energy quickly.

[0093] 2. Slow-digesting components (corresponding to) Figure 2 Zone B in the table refers to the nutrient components that are digested between the first preset time (30 minutes) and the second preset time (240 minutes). This section represents the components that can continuously and steadily release nutrients, providing a long-term supply to the body.

[0094] 3. Indigestible components (corresponding to) Figure 2 Zone C in the text refers to the nutrient components that have not been broken down by the in vitro digestive system after the second preset time (240 minutes). In practical applications, this part is usually considered as a resistant component or an ineffective component that cannot be utilized by animals.

[0095] The specific quantitative calculation method is as follows: Percentage of rapidly digested components (%) = Cumulative digestibility measured at 30 minutes.

[0096] Percentage of slowly digested components (%) = (Cumulative digestibility measured at 240 minutes) - (Cumulative digestibility measured at 30 minutes).

[0097] Percentage of indigestible components (%) = 100% - (cumulative digestibility measured at 240 minutes).

[0098] Table 2 shows the changes in protein digestion rates of dehulled soybean meal, cottonseed meal, wheat protein powder, peanut meal, corn protein powder, rapeseed meal, potato protein powder, skim milk powder, soy protein concentrate, soybean meal, sunflower meal, fish meal, and cottonseed protein at different in vitro biomimetic digestion time points in this embodiment.

[0099] Based on the protein digestibility of 13 raw materials at different time points in vitro, it was found that at the 30th minute of simulated small intestinal digestion in vitro, the digestible protein of most raw materials (except potato protein powder) (calculated by multiplying the digestibility at the 30th minute by the protein content of the raw materials) had exceeded 50% of the digestible protein content at the 240th minute.

[0100] Furthermore, among different time points in various in vitro biomimetic digestion processes, the coefficient of variation for the digestibility of the 13 feed ingredients was highest at the 30-minute mark. These results indicate that the digestibility at the 30-minute mark in in vitro biomimetic digestion is the most representative for distinguishing the digestibility rates of proteins from different feed ingredients.

[0101] In addition, skim milk powder, wheat protein powder, peanut meal, peeled soybean meal, sunflower meal, and rapeseed meal have the highest proportion of rapidly digestible protein, greater than 70%, while the proportion of slowly digestible protein is relatively low, less than 15%. Soybean meal, cottonseed meal, fish meal, and cottonseed protein have a rapidly digestible protein proportion ranging from 45.71% to 68.55%, while the proportion of slowly digestible protein is relatively low, approximately 17.02% to 36.92%. Soy protein concentrate has a relatively high proportion of both rapidly and slowly digestible protein, at 42.81% and 45.08%, respectively. Corn protein powder and potato protein powder have a relatively high proportion of indigestible protein, at 40.76% and 37.99%, respectively.

[0102] Table 2. Composition of rapidly digestible, slowly digestible, and indigestible proteins in 13 feed ingredients Coefficient of variation calculation method: Coefficient of variation CV (%) = (Standard deviation SD / Mean) × 100% Through the above six steps, this embodiment can accurately and repeatedly quantify the protein in any feed ingredient into three parts: fast-producing, slow-producing, and indigestible, providing a solid data foundation for the establishment and application of a precision feed formulation database.

[0103] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An in vitro method for determining and classifying the dynamic digestibility rate of feed nutrient components, characterized in that, Includes the following steps: Step 1: Pre-treat the feed sample to be tested; Step 2: Perform simulated in vitro digestion on the pretreated feed sample, which includes a simulated stomach digestion stage and a simulated small intestine digestion stage. Step 3: Samples are taken at multiple preset time points during the simulated small intestine digestion stage, and the cumulative digestibility of the target nutrient components in the samples at each preset time point is measured; Step 4: Based on the measured cumulative digestibility, classify the target nutrient components, including: The portion digested within the first preset time after the start of the simulated small intestinal digestion stage is defined as the rapidly digested component. The portion that is digested between the first preset time period and the second preset time period is defined as the slow-digesting component; The portion that remains undigested after the second preset time period is defined as an indigestible component.

2. The method for in vitro determination and classification of dynamic digestibility of feed nutrient components according to claim 1, characterized in that, The target nutrient component is starch or protein.

3. The method for in vitro determination and classification of dynamic digestibility of feed nutrient components according to claim 1, characterized in that, The first preset duration is 30 minutes, and the second preset duration is 240 minutes.

4. The in vitro determination and classification method for dynamic digestibility of feed nutrient components according to claim 1, characterized in that, The simulated gastric digestion stage includes: placing the feed sample in simulated gastric juice containing pepsin, incubating it in an acidic environment with a pH of 3.5, and incubating it at 39°C for 120 minutes.

5. The method for in vitro determination and classification of dynamic digestibility of feed nutrient components according to claim 1, characterized in that, The simulated small intestine digestion stage includes: after the simulated stomach digestion stage is completed, adjusting the digestive environment to neutral, and adding simulated intestinal fluid containing pancreatic enzymes for incubation. The simulated intestinal fluid also contains amylase and invertase.

6. The in vitro determination and classification method for dynamic digestibility of feed nutrient components according to claim 2, characterized in that, When the target nutrient component is starch, the cumulative digestibility is determined by measuring the reducing sugar content in the digestive fluid.

7. The in vitro determination and classification method for dynamic digestibility of feed nutrient components according to claim 2, characterized in that, When the target nutrient component is protein, the cumulative digestibility is determined by measuring the crude protein content in the digestate using the Kjeldahl method.

8. The method for in vitro determination and classification of dynamic digestibility of feed nutrient components according to claim 1, characterized in that, The classification is specifically calculated by determining the percentage of each component in the following way: The percentage of rapidly digestible components is equal to the cumulative digestibility of the target nutrient components measured during the first preset time period; The percentage of slowly digestible components is equal to the difference between the cumulative digestibility of the target nutrient components measured at the second preset time period and the cumulative digestibility of the target nutrient components measured at the first preset time period. The percentage of indigestible components is equal to 100% minus the cumulative digestibility of the target nutrient components measured at the second preset time period.

9. The application of an in vitro method for determining and classifying the dynamic digestibility rate of feed nutrient components as described in any one of claims 1-8, characterized in that, The application includes in vitro determination and classification of the proportion of rapidly digestible, slowly digestible, and indigestible starch in 19 common feed ingredients, including corn, sorghum, wheat, barley, puffed corn, peas, bread flour, extruded corn, extruded sorghum, extruded wheat, extruded barley, rice bran, rice bran meal, cassava, wheat middlings, brown rice, broken rice, corn gluten meal, and wheat bran.

10. The application of an in vitro method for determining and classifying the dynamic digestibility rate of feed nutrient components as described in any one of claims 1-8, characterized in that, The application includes in vitro determination and classification of the proportion of rapidly digestible, slowly digestible, and indigestible protein in 13 common feed ingredients, including high-protein soybean meal, cottonseed meal, wheat protein powder, peanut meal, corn protein powder, rapeseed meal, potato protein powder, skim milk powder, soybean protein concentrate, soybean meal, sunflower meal, corn, and extruded corn.

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