Method for measuring content of amino acid in chyme in digestive tract of livestock and poultry

Abstract

The invention belongs to the technical field of detection methods, and provides a method for determining the content of amino acid in chyme in digestive tracts of livestock and poultry, which comprises the following steps: mixing a sample and a peroxyformic acid solution, and reacting to obtain an oxidation system; mixing the oxidation system, a hydrochloric acid solution and a chelating agent solution, and hydrolyzing to obtain a hydrolysis system; mixing the hydrolysis system and alkali liquor, then adjusting the pH value, and then mixing with acid liquor to obtain liquid to be detected; centrifuging the to-be-measured liquid, filtering, injecting a filtering system and the mixed amino acid standard liquid into an automatic amino acid analyzer, and measuring. According to the method, a certain amount of disodium ethylene diamine tetraacetate is added to chelate heavy metals in chyme in digestive tracts of livestock and poultry, and meanwhile, the heavy metals and redundant impurities are removed by adopting the steps of centrifuging, filtering and the like, so that the accuracy of amino acid detection is ensured, and the addicability of digestible amino acid content in daily ration is better. The hydrolysate is treated by adopting simple steps such as pH regulation and centrifugation, so that the operation process is simplified, and the operation error is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of detection methods, and in particular to a method for determining the amino acid content in chyme of livestock and poultry digestive tracts. Background Art

[0002] Amino acids are important nutrients in animals, and are involved in the regulation of various physiological functions of living organisms and the anabolism of body tissues. For farmed animals such as pigs and poultry, amino acids play an indispensable role in maintaining their normal growth and development, improving immunity and production performance (such as meat quality, egg production, etc.). Especially in modern breeding production, accurate quantification of amino acid content is crucial for optimizing feed formula and nutritional management measures, and is the key to improving production efficiency and reducing feeding costs. However, the amino acids in feed cannot be fully absorbed and utilized by pigs and poultry. According to the characteristics of the feed raw materials used, about 10% to 50% of the amino acids will be excreted from the body with feces, causing nutritional waste (see references: Macelline SP, Chrystal PV, Liu SY, Selle PH. The dynamic conversion of dietary protein and amino acids into chicken-meat protein. Animals, 2021, 11 (8): 2288.; Ma Longteng, Wu Cong, Wang Lifen, Zhang Zixi, Song Zehe, He Xi, Ma Xiaokang. Evaluation of effective energy value and amino acid digestibility of feed raw materials from different protein sources for feeding growing pigs. Journal of Animal Nutrition, 2022, 34 (10): 14.). Therefore, accurate determination of the digestible amino acid content in feed can provide data support for the refined formulation of feed, help adjust the amino acid ratio in pig and poultry feed, optimize its nutritional structure, avoid nutritional excess or deficiency, and ensure the accurate supply of amino acid nutritional needs of livestock and poultry.

[0003] The determination of "digestible amino acid content" in feed is mainly carried out by animal digestion and metabolism test, and is calculated by the difference between the total amino acid content in the ingested feed and the content in the excreta (digestive tract chyme or feces). Due to the ineffective contribution of feed amino acids to the animal body in the metabolism of pig and poultry large intestine chyme, the internationally accepted method for determining the digestible amino acid content of pig and poultry feed is mainly to calculate the digestibility before the terminal ileum (see reference: Stein HH, Sève B, Fuller MF, Moughan PJ, De Lange CFM. Invited review: Amino acid bioavailability and digestibility in pig feed ingredients: Terminology and application. J. Anim. Sci., 2007, 85 (1): 172-180.). Therefore, how to accurately determine the amino acid content in feed and digestive tract chyme is crucial for the quantification of digestible amino acid content. At present, the national standard method GB / T18246-2019 "Determination of amino acids in feed" stipulates that this method is only applicable to the determination of amino acid content in feed raw materials, compound feed, concentrated feed, concentrate supplements and additives and mixed feed. In actual production, this method is applied to the determination of amino acid content in the digestive tract of pigs and poultry. It faces a series of limitations, which are specifically manifested in the following aspects: 1) The sample weight is low, and the repeatability and reproducibility of the measurement results are poor. The existing determination method is mainly designed for the determination of amino acids in feed, and its methods and steps fail to fully consider the particularity of digestive tract chyme. The sample weight of the existing method is 50-100 mg. When used for the determination of the amino acid content of digestive tract chyme samples, due to the presence of some unevenly distributed impurities in the digestive tract chyme samples, such as pebbles, mineral elements, etc., the sample weight is difficult to meet the uniformity and representativeness of the sampling. The relative deviation of the results of two independent determinations of the same chyme sample is usually more than 5%, and the absolute difference in the effect on the amino acid digestibility result is more than 1.5% (Reference: Zhou Jinhua, Yuan Jianmin. Research progress and existing problems in the determination of standard ileal amino acid digestibility of broilers. Feed Industry, 2024, 45(12): 59-63.), and the repeatability and reproducibility are poor; 2) Interference of exogenous indicator titanium dioxide. Since the digestive tract end chyme cannot be completely collected, titanium dioxide is often used as an exogenous indicator in the determination of the digestible amino acid content of pig and poultry feed to mark the digestion and absorption of amino acids in the feed. However, titanium dioxide has strong chemical stability and is difficult to completely remove in the digestive tract chyme.Titanium dioxide is easily adsorbed on the cation exchange resin of the amino acid analyzer and is sensitive to interference with the existing amino acid determination method, resulting in large deviations in the determination results, poor repeatability and reproducibility, and serious deviations from the requirements of GB / T 18246-2019 on the precision of amino acid determination; 3) The influence of sample impurities. The digestive chyme of pigs and poultry contains a large amount of impurities, such as undigested feed particles, digestive enzymes, gastrointestinal secretions, etc. These impurities may react with amino acids to form interfering compounds, resulting in incomplete separation of the chromatographic column and affecting the accuracy of the quantitative results; 4) Poor additivity. In feed formulation, the digestible amino acid content in the diet is the weighted sum of the digestible amino acid content provided by the raw materials that make up the formula. This additivity of nutrient content is the core of feed formulation (Ge Kaijing, Wang Yuming, Wang Junli, Jie Jingjing, Sarenna, Liu Huawei, Zhao Feng. Study on the difference in the amino acid content of feed determined by two amino acid analyzers. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3084-3092.). However, the large errors of existing amino acid detection methods in determining digestive tract chyme samples will be transmitted to the calculation of the digestible amino acid content of the raw materials, and affect its additivity in the diet, making it difficult to meet the needs of precise nutrition; 5) The operation is cumbersome. The existing determination methods usually require a long sample pretreatment process. During the treatment process, a rotary evaporator or concentrator is required to evaporate and dry the hydrolyzate and then re-dissolve it. The operation is cumbersome and requires high technical skills of the operator. This complex operation process increases the source of errors in the determination process, and also reduces the convenience and efficiency of the application of this method in production practice; 6) Standardization issues. Due to the lack of a special standardized method for the determination of amino acid content in digestive tract chyme, there are large differences in the analysis results of different laboratories and different batches. This difference makes the accurate determination of amino acid content face great challenges, resulting in the incomparability of the digestible amino acid content determination data of the same feed, and it is impossible to provide accurate and reliable data support for feed formulation, affecting production efficiency and breeding decisions. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a method for determining the amino acid content in chyme of livestock and poultry digestive tract.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for determining the amino acid content in chyme of livestock and poultry digestive tracts, comprising the following steps:

[0007] (1) mixing a sample and a peroxyformic acid solution and reacting them to obtain an oxidation system;

[0008] (2) mixing the oxidation system, the hydrochloric acid solution and the chelating agent solution for hydrolysis to obtain a hydrolysis system;

[0009] (3) mixing the hydrolysis system and the alkali solution, adjusting the pH, and then mixing with the acid solution to obtain a test solution;

[0010] (4) The test solution is centrifuged and then filtered, and the filtration system and the mixed amino acid standard solution are injected into an automatic amino acid analyzer for determination.

[0011] Preferably, the sample in step (1) comprises a digestive tract chyme sample;

[0012] The mass volume ratio of the sample and the peroxyformic acid solution in step (1) is 200-500 mg: 4-6 mL;

[0013] The peroxyformic acid solution in step (1) comprises formic acid and hydrogen peroxide solution;

[0014] The volume ratio of the formic acid to the hydrogen peroxide solution is 80-100:5-15;

[0015] The concentration of the hydrogen peroxide solution is 20-40%.

[0016] Preferably, the mixing temperature in step (1) is 0-1°C and the mixing time is 20-40 minutes;

[0017] The reaction temperature in step (1) is 0-1°C and the reaction time is 14-18h.

[0018] Preferably, the concentration of the hydrochloric acid solution in step (2) is 7 to 9 mol / L;

[0019] The solute of the chelating agent solution in step (2) is disodium ethylenediaminetetraacetate; the concentration of the chelating agent solution is 0.04-0.06 mol / L;

[0020] The mass volume ratio of the sample in step (1) to the hydrochloric acid solution in step (2) is 200-500 mg: 20-30 mL;

[0021] The volume ratio of the hydrochloric acid solution to the chelating agent solution in step (2) is 20-30:1-3.

[0022] Preferably, the hydrolysis in step (2) is a primary hydrolysis and a secondary hydrolysis performed sequentially;

[0023] The temperature of the preliminary hydrolysis is 100-120°C and the time is 0.5-1.5h;

[0024] The secondary hydrolysis is carried out under closed conditions, the temperature of the secondary hydrolysis is 100-120° C., and the time is 20-26 hours.

[0025] Preferably, the alkali solution in step (3) is a sodium hydroxide solution, and the concentration of the alkali solution is 7 to 8 mol / L;

[0026] The volume mass ratio of the alkali solution in step (3) to the sample in step (1) is 20-25 mL: 200-500 mg;

[0027] The solution used to adjust the pH in step (3) is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 1.5 to 2.5 mol / L;

[0028] The target pH value is adjusted to 2-2.5.

[0029] Preferably, the acid solution in step (3) is a hydrochloric acid solution, and the concentration of the acid solution is 0.01 to 0.03 mol / L;

[0030] The volume mass ratio of the test solution in step (3) to the sample in step (1) is 180-220 mL: 200-500 mg.

[0031] Preferably, the centrifugal speed in step (4) is 8000-12000 rpm, and the time is 10-20 min.

[0032] Preferably, the diameter of the filter membrane used in step (4) is 0.2 to 0.25 μm.

[0033] The present invention provides a method for determining the amino acid content in digestive tract chyme of livestock and poultry, comprising the following steps: (1) mixing a sample and a peroxyformic acid solution and reacting the mixture to obtain an oxidation system; (2) mixing the oxidation system, a hydrochloric acid solution and a chelating agent solution and hydrolyzing the mixture to obtain a hydrolysis system; (3) mixing the hydrolysis system and an alkali solution and adjusting the pH value, and then mixing the mixture with an acid solution to obtain a test solution; (4) centrifuging the test solution and filtering the mixture, and injecting the filtered system and a mixed amino acid standard solution into an amino acid automatic analyzer for determination.

[0034] The amino acids in feed and digestive tract chyme of pigs and poultry are all in the form of proteins. Methionine and cystine are oxidized into methionine sulfone and sulfalanine respectively by peroxyformic acid solution. After the reaction, sodium metabisulfite is added to decompose the excess peroxyformic acid. Hydrochloric acid solution and disodium ethylenediaminetetraacetic acid (EDTA-Na) that can chelate heavy metal titanium in digestive tract chyme are added to the oxidized solution to hydrolyze it into free amino acids. After the hydrolysis is completed, the pH value of the hydrolyzate is adjusted with sodium hydroxide, and then impurities such as heavy metal salts that cannot be hydrolyzed are removed by centrifugation, filtration, etc., and then chromatographic separation is performed using an amino acid analyzer. The separated amino acids are mixed with ninhydrin inside the instrument and generate specific color-developing substances in the heated reaction coil. These compounds can be detected by a photometric detector.

[0035] The results of the present invention have good reproducibility and small variation. The uniformity and representativeness of the sample are ensured by increasing the sample weight from 50 to 100 mg (nitrogen content is 1.20 to 4.0 mg) to 200 to 500 mg (nitrogen content is about 10 mg) and simultaneously expanding the hydrolysis system; however, the synchronous expansion of the sample weight and the hydrolysis system in the present application does not present the same proportion as the national standard method. The present application improves the representativeness of the sample on the basis of expanding the volume and increasing the sample weight, avoiding the problem of sample inhomogeneity. During the experiment, the present application found that the expansion of the volume of the solute and the solvent in the hydrolysis system did not increase linearly. The present invention obtained the optimal reaction system and sample weight of the sample through multiple test screening and parameter optimization.

[0036] The invention chelates heavy metals in the digestive tract chyme of livestock and poultry by adding a certain amount of disodium ethylenediaminetetraacetate (EDTA-Na), and simultaneously removes heavy metals and excess impurities by centrifugation, filtration and other steps, thereby ensuring the accuracy of amino acid detection results, thereby improving the additivity of digestible amino acid content in the diet.

[0037] The invention is easy to operate, and the hydrolyzate is processed by adopting simple steps such as adjusting pH and centrifugation, thereby replacing the existing method of using a professional rotary evaporator for vacuum concentration and drying, adding water for repeated evaporation, and sodium citrate buffer solution for re-dissolving, thereby simplifying the operation process and reducing operation errors. DETAILED DESCRIPTION

[0038] The present invention provides a method for determining the amino acid content in chyme of livestock and poultry digestive tracts, comprising the following steps:

[0039] (1) mixing a sample and a peroxyformic acid solution and reacting them to obtain an oxidation system;

[0040] (2) mixing the oxidation system, the hydrochloric acid solution and the chelating agent solution for hydrolysis to obtain a hydrolysis system;

[0041] (3) mixing the hydrolysis system and the alkali solution, adjusting the pH, and then mixing with the acid solution to obtain a test solution;

[0042] (4) The test solution is centrifuged and then filtered, and the filtration system and the mixed amino acid standard solution are injected into an automatic amino acid analyzer for determination.

[0043] In the present invention, the sample in step (1) comprises a digestive tract chyme sample.

[0044] In the present invention, the particle size of the sample in step (1) is preferably ≤0.25 mm, more preferably ≤0.23 mm, and more preferably ≤0.20 mm.

[0045] In the present invention, the mass volume ratio of the sample and the peroxyformic acid solution in step (1) is preferably 200-500 mg: 4-6 mL, more preferably 250-450 mg: 4.5-5.5 mL, and more preferably 300-400 mg: 4.8-5.2 mL.

[0046] In the present invention, the peroxyformic acid solution in step (1) comprises formic acid and hydrogen peroxide solution.

[0047] In the present invention, the volume ratio of the formic acid to the hydrogen peroxide solution is preferably 80-100:5-15, more preferably 85-95:6-14, and more preferably 88-92:8-12.

[0048] In the present invention, the concentration of the hydrogen peroxide solution is preferably 20 to 40%, more preferably 25 to 35%, and even more preferably 28 to 32%.

[0049] In the present invention, the mixing temperature in step (1) is preferably 0-1°C, more preferably 0.2-0.8°C, more preferably 0.4-0.6°C; the mixing time is preferably 20-40 min, more preferably 25-35 min, more preferably 28-32 min; stirring is maintained during the mixing process.

[0050] In the present invention, after the mixing in step (1) is completed, stirring is stopped and the reaction system is controlled under closed conditions to carry out the reaction.

[0051] In the present invention, the reaction temperature in step (1) is preferably 0-1°C, more preferably 0.2-0.8°C, more preferably 0.4-0.6°C; the reaction time is preferably 14-18h, more preferably 14.5-17.5h, more preferably 15-17h.

[0052] In the present invention, the peroxyformic acid solution in step (1) is prepared by formic acid and hydrogen peroxide solution, and the formic acid and hydrogen peroxide solution are mixed to generate peroxyformic acid. The peroxyformic acid will only oxidize methionine and cystine, and other amino acids will not undergo any changes in the oxidation reaction of step (1). After the step is completed, the peroxyformic acid is decomposed by sodium metabisulfite, and a high-concentration hydrochloric acid solution is added. Under the high-concentration hydrochloric acid, the protein will be decomposed and the peptide chain will be broken, and all the amino acids will be released as single amino acids.

[0053] In the present invention, the reason why oxidative hydrolysis by peroxyformic acid is first performed is that cystine and methionine are sulfur-containing amino acids, which are easily degraded under high concentration hydrochloric acid conditions. Peroxyformic acid is used to convert them into stable sulfalanine and methionine sulfone of equal concentrations, respectively, so that they can withstand subsequent hydrolysis with high concentration acid and high temperature.

[0054] The original amino acids are connected by peptide bonds, disulfide bonds, etc. to form peptide segments containing several amino acid residues, and the peptide segments are then connected by peptide chains to form proteins. The principle of the amino acid pretreatment process in the present invention is to destroy the peptide bonds, disulfide bonds and peptide chains by high-concentration acid solution to free single amino acids, which are then eluted in sequence according to different isoelectric points in an amino acid analyzer through cation exchange resins, and then detected by a detector.

[0055] In the present invention, after the reaction in step (1) is completed, a sodium metabisulfite solution is added to decompose excess peroxyformic acid to terminate the reaction; the concentration of the sodium metabisulfite solution is preferably 0.15-0.20 g / mL, more preferably 0.16-0.19 g / mL, and more preferably 0.17-0.18 g / mL; the volume mass ratio of the sodium metabisulfite solution to the sample in step (1) is preferably 1-10 mL:200-500 mg, more preferably 2-8 mL:250-450 mg, and more preferably 4-6 mL:300-400 mg; stirring is started after the sodium metabisulfite solution is added, and the stirring time is preferably 20-40 min, more preferably 25-35 min, and more preferably 28-32 min.

[0056] In the present invention, the concentration of the hydrochloric acid solution in step (2) is preferably 7 to 9 mol / L, more preferably 7.5 to 8.5 mol / L, and more preferably 7.8 to 8.2 mol / L.

[0057] In the present invention, the solute of the chelating agent solution in step (2) is preferably disodium ethylenediaminetetraacetate; the concentration of the chelating agent solution is preferably 0.04 to 0.06 mol / L, more preferably 0.045 to 0.055 mol / L, and more preferably 0.048 to 0.052 mol / L.

[0058] In the present invention, the mass volume ratio of the sample in step (1) and the hydrochloric acid solution in step (2) is preferably 200-500 mg:20-30 mL, more preferably 250-450 mg:22-28 mL, and more preferably 300-400 mg:24-26 mL.

[0059] In the present invention, the volume ratio of the hydrochloric acid solution to the chelating agent solution in step (2) is preferably 20-30:1-3, more preferably 22-28:1.5-2.5, and more preferably 24-26:1.8-2.2.

[0060] In the present invention, the hydrolysis in step (2) is a primary hydrolysis and a secondary hydrolysis performed sequentially.

[0061] In the present invention, the temperature of the preliminary hydrolysis is preferably 100-120°C, more preferably 105-115°C, more preferably 108-112°C; the time is preferably 0.5-1.5h, more preferably 0.6-1.4h, more preferably 0.8-1.2h.

[0062] In the present invention, the secondary hydrolysis is carried out under closed conditions, and the temperature of the secondary hydrolysis is preferably 100-120°C, more preferably 105-115°C, and more preferably 108-112°C; the time is preferably 20-26h, more preferably 21-25h, and more preferably 22-24h.

[0063] In the present invention, after the secondary hydrolysis is completed, the mixture is cooled to room temperature and then the next operation is performed.

[0064] In the present invention, the alkali solution in step (3) is a sodium hydroxide solution, and the concentration of the alkali solution is preferably 7-8 mol / L, more preferably 7.2-7.8 mol / L, and more preferably 7.4-7.6 mol / L.

[0065] In the present invention, the volume mass ratio of the alkali solution in step (3) to the sample in step (1) is preferably 20-25 mL: 200-500 mg, more preferably 21-24 mL: 250-450 mg, and more preferably 22-23 mL: 300-400 mg.

[0066] In the present invention, the solution used for adjusting the pH in step (3) is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is preferably 1.5 to 2.5 mol / L, more preferably 1.6 to 2.4 mol / L, and more preferably 1.8 to 2.2 mol / L.

[0067] In the present invention, the target value for adjusting pH is preferably 2 to 2.5, more preferably 2.1 to 2.4, and even more preferably 2.2 to 2.3.

[0068] In the present invention, the acid solution in step (3) is a hydrochloric acid solution, and the concentration of the acid solution is preferably 0.01 to 0.03 mol / L, more preferably 0.015 to 0.025 mol / L, and more preferably 0.018 to 0.022 mol / L.

[0069] The volume mass ratio of the test solution in step (3) to the sample in step (1) is preferably 180-220 mL: 200-500 mg, more preferably 185-215 mL: 250-450 mg, and more preferably 190-200 mL: 300-400 mg.

[0070] In the present invention, the centrifugal speed in step (4) is preferably 8000-12000 rpm, more preferably 8500-11500 rpm, more preferably 9000-11000 rpm; the time is preferably 10-20 min, more preferably 12-18 min, more preferably 14-16 min.

[0071] In the present invention, the diameter of the filter membrane used in step (4) is preferably 0.2 to 0.25 μm, more preferably 0.21 to 0.24 μm, and even more preferably 0.22 to 0.23 μm.

[0072] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0073] Example 1

[0074] The sample was passed through a 0.25 mm sieve, and then 5 mL of peroxyformic acid solution (the volume ratio of formic acid and 30% hydrogen peroxide solution was 90:10) was added according to the standard of 300 mg of sample, and stirred at 0°C for 30 min; then the stirring was stopped, the bottle stopper was tightly closed, and the reaction was continued for 16 h at 0°C under closed conditions; after the reaction, 5 mL of sodium metabisulfite solution (concentration of 0.2 g / mL) was added and stirred for 30 min to decompose excess peroxyformic acid to obtain an oxidation system; 25 mL of 8 mol / L hydrochloric acid solution and 2 mL of 0.05 mol / L disodium ethylenediaminetetraacetic acid solution were added to the oxidation system, and hydrolyzed at 110°C for 1 h, and then the bottle stopper was tightly closed, and the hydrolysis was continued for 24 h at 110°C under closed conditions; after the hydrolysis was completed, it was cooled to room temperature to obtain a hydrolysis system. Add 22.5 mL of 7.5 mol / L sodium hydroxide solution to the hydrolysis system, and then adjust the pH to 2.2 with 2 mol / L sodium hydroxide solution; then add 0.02 mol / L hydrochloric acid solution until the volume of the test solution is 200 mL; centrifuge the test solution at 10000 rpm for 15 min, and then filter with a 0.22 μm filter membrane to obtain a filtration system, inject the filtration system and the mixed amino acid standard solution into an amino acid automatic analyzer for determination.

[0075] Configuration of mixed amino acid standard solution:

[0076] First, prepare the amino acid standard solution: containing 16 kinds of amino acids (lysine, methionine, threonine, valine, isoleucine, leucine, phenylalanine, histidine, arginine, cysteine, aspartic acid, serine, glutamic acid, glycine, alanine, proline), the concentration of each is 2.50 μmol / mL.

[0077] Prepare cysteic acid-methionine sulfone standard solution: accurately weigh 42.3 mg of cysteic acid and 45.3 mg of methionine sulfone into a 100-mL volumetric flask containing 50 mL of water. After dissolving, add water to the scale. The concentration of both amino acids is 2.50 μmol / L. It can be stored at 0-4°C for 3 months.

[0078] Accurately pipette 0.5 mL of amino acid standard solution and 0.5 mL of cysteic acid-methionine sulfone standard solution into a 25 mL volumetric flask containing 10 mL of water. After mixing evenly, add water to the mark. The concentration of each component is 50 nmol / mL. This solution is prepared and used immediately.

[0079] The experiment adopted a single-factor completely randomized design, and three portions of terminal ileal digesta from growing pigs fed the same wheat-rapeseed meal diet were taken (from three growing pigs equipped with terminal ileal T-type fistulas).

[0080] Average value (%) = (repeat 1 + repeat 2) / 2

[0081] Relative deviation (%) = |(repeat 1-average value)| / average value*100

[0082] The results of porcine terminal ileum digesta 1 are shown in Table 1, the results of porcine terminal ileum digesta 2 are shown in Table 2, and the results of porcine terminal ileum digesta 3 are shown in Table 3.

[0083] Table 1 Results of determination of amino acid content in pig ileum terminal chyme 1 (air-dried basis, %)

[0084]

[0085]

[0086] Table 2 Results of determination of amino acid content in chyme at the end of pig ileum (air-dried basis, %)

[0087]

[0088]

[0089] Table 3 Results of determination of 3 amino acid contents in pig terminal ileum chyme (air-dried basis, %)

[0090]

[0091] It can be seen from Tables 1 to 3 that the relative deviation of the amino acid content of chyme 1 determined by the method of the present application between two repeated measurement results is 0.55% to 3.74%, chyme 2 is 0.36% to 3.64%, and chyme 3 is 0.04% to 3.61%. The relative deviations are all within 4%, which meets the precision requirements of GB / T18246-2019 "Determination of Amino Acids in Feed".

[0092] The experiment adopted a single-factor completely randomized design, and three portions of terminal ileal digesta of white-feathered broilers fed the same corn-soybean meal diet were taken (from three groups of white-feathered broilers sampled after slaughter).

[0093] The method of the present application is used to determine the amino acid content in the ileal chyme of growing white-feathered broiler chickens.

[0094] Average value (%) = (parallel 1 + parallel 2) / 2

[0095] Relative deviation (%) = |(parallel 1-average value)| / average value*100

[0096] The results of ileum terminal digesta 1 of white-feathered broiler chickens are shown in Table 4, the results of ileum terminal digesta 2 of white-feathered broiler chickens are shown in Table 5, and the results of ileum terminal digesta 3 of white-feathered broiler chickens are shown in Table 6.

[0097] Table 4 Determination results of amino acid content in terminal ileum chyme of white-feathered broiler chickens (air-dried basis, %)

[0098]

[0099] Table 5 Determination results of 2 amino acid contents in terminal ileum chyme of white-feathered broiler chickens (air-dried basis, %)

[0100]

[0101] Table 6 Results of determination of 3 amino acids in terminal ileum chyme of white-feathered broiler chickens (air-dried basis, %)

[0102]

[0103]

[0104] It can be seen from Tables 4 to 6 that under the repeatability measurement conditions, the relative deviation of the amino acid content of chyme 1 determined by the method of the present application between two repeated measurement results is 0.26% to 3.47%, chyme 2 is 0.29% to 2.97%, and chyme 3 is 0.55% to 3.25%. The relative deviations are all within 4%, which all meet the precision requirements of GB / T18246-2019 "Determination of Amino Acids in Feed".

[0105] The experiment adopted a single-factor completely randomized design, and three portions of terminal ileal digesta of Muscovy ducks fed the same corn-sorghum-cottonseed meal diet were taken (from three groups of Muscovy ducks sampled after slaughter).

[0106] The method of the present application is used to determine the amino acid content in the ileal chyme of growing Muscovy ducks.

[0107] Average value (%) = (parallel 1 + parallel 2) / 2

[0108] Relative deviation (%) = |(parallel 1-average value)| / average value*100

[0109] The results of the terminal ileum digesta 1 of the Muscovy duck are shown in Table 7, the results of the terminal ileum digesta 2 of the Muscovy duck are shown in Table 8, and the results of the terminal ileum digesta 3 of the Muscovy duck are shown in Table 9.

[0110] Table 7 Determination of amino acid content in terminal ileum chyme 1 of Muscovy duck (air-dried basis, %)

[0111]

[0112]

[0113] Table 8 Determination of 2 amino acid contents in terminal ileum chyme of Muscovy duck (air-dried basis, %)

[0114]

[0115]

[0116] Table 9 Results of determination of 3 amino acids in terminal ileum chyme of Muscovy duck (air-dried basis, %)

[0117]

[0118] It can be seen from Tables 7 to 9 that under the repeatability measurement conditions, the relative deviation of the amino acid content of chyme 1 determined by the method of the present application between the two repeated measurement results is 0.08% to 3.73%, chyme 2 is 0.41% to 3.59%, and chyme 3 is 0.10% to 3.09%. The relative deviations are all within 4%, which all meet the precision requirements of GB / T18246-2019 "Determination of Amino Acids in Feed".

[0119] Six samples of commonly used feed raw materials for pigs were selected, including three cereal raw materials: corn, wheat and sorghum, two protein raw materials: soybean meal and rapeseed meal, and one fiber raw material: wheat bran. The chemical composition of the raw materials is shown in Table 10.

[0120] Table 10 Nutritional composition of feed raw materials (feeding basis, %)

[0121]

[0122] The animal experiment selected 10 growing pigs equipped with terminal ileal T-type fistulas, and adopted a 10×6 Youden square design, that is, 10 dietary treatments, which were measured in 6 phases. Each pig was fed an experimental diet (numbered 1 to 10) in each phase, forming 6 replicates for each treatment and 1 pig for each replicate. However, the same pig would not be fed the same diet twice or more in different phases.

[0123] Ten Duroc-Changda hybrid castrated boars with an initial body weight of 21.8±2.8kg were selected for the experiment and housed in metabolic cages. After one week of adaptation, a simple T-shaped fistula was installed at the terminal ileum. After the operation, routine care was followed for 2 weeks, and the formal experiment began after the fistula pigs recovered.

[0124] Before each period, the experimental pigs were weighed on an empty stomach. Each experimental period was 7 days, including a 5-day adaptation period and a 2-day digesta collection period. The daily feeding amount was 4% of body weight, and the experimental diet was fed in two meals at 08:00 and 16:00, with free drinking water during the period. Feeding management followed the national regulations on animal welfare, and immunization was carried out according to the normal immunization procedures of the experimental pig farm. The temperature of the metabolic chamber was controlled at 23-26°C, and the basic diet was fed during the experimental interval or the recovery period of the pigs.

[0125] The 6th and 7th days of each test were the chyme collection period. During the chyme collection period, ileal chyme was collected continuously for 12 hours from 08:00 to 20:00 every day, and the collected chyme was immediately stored at -20°C. After each test, all the chyme of each pig was mixed evenly and freeze-dried in a freeze dryer, then pulverized after rehydration at room temperature for 24 hours and stored for later use.

[0126] The experiment included 10 diets, with diets 1-6 consisting of the raw materials to be tested as the only protein source; diets 7-9 consisting of 3-5 of the above 6 raw materials; and diet 10 being a nitrogen-free diet used to determine the basal ileal endogenous amino acid loss. The calcium, phosphorus, vitamins and trace elements in all diets met the nutritional requirements of GB / T39235-2020, and 0.4% titanium dioxide was added as an indicator.

[0127] When testing protein and fiber raw materials, it is necessary to mix an appropriate amount of corn starch, sucrose and soybean oil to prepare a semi-pure diet, adjust the protein level of the diet to about 16%, and refer to GB / T39235-2020 to prepare a semi-pure diet with calcium, phosphorus, sodium chloride, mineral trace elements, vitamin premixes, etc., and the fiber content is preferably 3% to 5%. When testing energy feed raw materials with good palatability, the raw material to be tested is the only protein source, and only calcium, phosphorus, sodium chloride, mineral trace elements, vitamin premixes, etc. need to be added according to GB / T39235-2020. The specific diet composition is shown in Table 11.

[0128] Table 11 Experimental diet composition (feeding basis, %)

[0129]

[0130]

[0131] Tested using the method of this application

[0132] Apparent ileal amino acid digestibility (AID): AID (%) = [1-(AA digesta / AA diet )×(M diet / M digesta )]×100;

[0133] Basal endogenous amino acid loss (BasalIAAend): BasalIAAend = AAdigesta × (M diet / M digesta );

[0134] Standardized ileal amino acid digestibility (SID): SID (%) = AID + (basal IAA end / AA diet )×100;

[0135] Standard ileum digestible amino acid content (SDAA): SDAA (%) = AA ingredient ×SID / 100.

[0136] Standard ileum digestible amino acid content (DSDAA) of the diet: DSDAA = A 1 ×SDAA 1 +……+A n ×SDAA n

[0137] AA digesta Refers to the amino acid content in ileal chyme, basalIAA end Refers to the loss of basic endogenous amino acids, expressed as %DM; M digesta Refers to the indicator content in ileal chyme, M diet Refers to the indicator content in the diet, expressed as %DM; A n Indicates the proportion of raw material n in the diet, SDAA n Indicates the digestible amino acid content in raw material n.

[0138] Average value (%) = (repeat 1 + repeat 2) / 2

[0139] Relative deviation (%) = |(repeat 1-average value)| / average value*100

[0140] Specific test comparison results are shown in Tables 12 to 20.

[0141] Table 12 Comparison of amino acid content in terminal ileum chyme of growing pigs fed corn by two amino acid determination methods

[0142]

[0143]

[0144] Table 13 Comparison of the amino acid content in the terminal ileum chyme of growing pigs fed wheat by two amino acid determination methods

[0145]

[0146]

[0147] Table 14 Comparison of amino acid content in terminal ileum digesta of growing pigs fed sorghum by two amino acid determination methods

[0148]

[0149]

[0150] Table 15 Comparison of the amino acid content in the terminal ileum chyme of growing pigs after feeding on soybean meal by two amino acid determination methods

[0151]

[0152]

[0153] Table 16 Comparison of the amino acid content in the terminal ileum chyme of growing pigs fed rapeseed meal by two amino acid determination methods

[0154]

[0155] Table 17 Comparison of the amino acid content in the terminal ileum digesta of growing pigs fed wheat bran by two amino acid determination methods

[0156]

[0157] Table 18 Comparison of amino acid content in terminal ileum digesta of growing pigs after ingestion of diet 1 by two amino acid determination methods

[0158]

[0159] Table 19 Comparison of amino acid content in terminal ileum digesta of growing pigs after ingestion of diet 2 by two amino acid determination methods

[0160]

[0161]

[0162] Table 20 Comparison of the amino acid content in the terminal ileum digesta of growing pigs after ingestion of diet 3 by two amino acid determination methods

[0163]

[0164]

[0165] Under repeatability conditions, the relative deviations of the amino acid contents in ileal chyme of growing pigs fed with corn, wheat, sorghum, soybean meal, rapeseed meal, wheat bran, diet 1, diet 2 and diet 3 determined by the method of this application between two repeated determination results were 0.34%-3.04%, 0.32%-4.88%, 0.19%-3.88%, 0.16%-3.31%, 0.12%-3.36%, 0.04%-3.17%, 0.02%-3.31%, 0.15%-3.81% and 0.04%-3.55%, respectively, and the average values ​​were 1.37%, 1.87%, 2.08%, 1.58%, 1.81%, 1.47%, 1.52%, 2.23% and 1.92%, respectively, all of which met the requirements of GB / T18246-2019 on the precision of amino acid determination. However, the relative deviations of the amino acid content in the ileal digesta of growing pigs fed the corresponding raw materials and diets determined by GB / T18246-2019 between the two repeated determinations were 1.46%-7.48%, 2.97%-9.27%, 3.65%-10.86%, 2.78%-7.99%, 2.54%-8.05%, 3.91%-8.68%, 2.21%-8.98%, 3.86 ...86%-10.86%, 2.78%-7.99%, 2. .56%~9.49% and 3.42%~10.56%, with average values ​​of 4.92%, 5.57%, 6.28%, 5.59%, 5.52%, 6.55%, 6.24%, 6.38% and 6.03% respectively. It can be seen that the relative deviation between the two repeatability determination results of most amino acids exceeded 4%, which is significantly higher than the requirements of GB / T18246-2019 for the precision of amino acid determination. The above results show that the repeatability of the determination of amino acid content in the chyme of growing pigs by this patent is better, and GB / T18246-2019 is not suitable for the determination of amino acid content in the chyme of the terminal ileum of growing pigs.

[0166] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the amino acid content in digestive tract chyme of livestock and poultry, characterized in that: It includes the following steps: (1) mixing a sample and a peroxyformic acid solution and reacting them to obtain an oxidation system; (2) mixing the oxidation system, the hydrochloric acid solution and the chelating agent solution for hydrolysis to obtain a hydrolysis system; (3) mixing the hydrolysis system and the alkali solution, adjusting the pH, and then mixing with the acid solution to obtain a test solution; (4) The test solution is centrifuged and then filtered, and the filtration system and the mixed amino acid standard solution are injected into an automatic amino acid analyzer for determination.

2. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 1, characterized in that: The sample in step (1) comprises a digestive tract chyme sample; The mass volume ratio of the sample and the peroxyformic acid solution in step (1) is 200-500 mg: 4-6 mL; The peroxyformic acid solution in step (1) comprises formic acid and hydrogen peroxide solution; The volume ratio of the formic acid to the hydrogen peroxide solution is 80-100:5-15; The concentration of the hydrogen peroxide solution is 20-40%.

3. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 1 or 2, characterized in that: The mixing temperature in step (1) is 0-1°C and the mixing time is 20-40 minutes; The reaction temperature in step (1) is 0-1°C and the reaction time is 14-18h.

4. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 3, characterized in that: The concentration of the hydrochloric acid solution in step (2) is 7 to 9 mol / L; The solute of the chelating agent solution in step (2) is disodium ethylenediaminetetraacetate; the concentration of the chelating agent solution is 0.04-0.06 mol / L; The mass volume ratio of the sample in step (1) to the hydrochloric acid solution in step (2) is 200-500 mg: 20-30 mL; The volume ratio of the hydrochloric acid solution to the chelating agent solution in step (2) is 20-30:1-3.

5. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 4, characterized in that: The hydrolysis in step (2) is a primary hydrolysis and a secondary hydrolysis performed sequentially; The temperature of the preliminary hydrolysis is 100-120°C and the time is 0.5-1.5h; The secondary hydrolysis is carried out under closed conditions, the temperature of the secondary hydrolysis is 100-120° C., and the time is 20-26 hours.

6. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 5, characterized in that: The alkali solution in step (3) is a sodium hydroxide solution, and the concentration of the alkali solution is 7-8 mol / L; The volume mass ratio of the alkali solution in step (3) to the sample in step (1) is 20-25 mL: 200-500 mg; The solution used to adjust the pH in step (3) is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 1.5 to 2.5 mol / L; The target pH value is adjusted to 2-2.

5.

7. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 6, characterized in that: The acid solution in step (3) is a hydrochloric acid solution, and the concentration of the acid solution is 0.01 to 0.03 mol / L; The volume mass ratio of the test solution in step (3) to the sample in step (1) is 180-220 mL: 200-500 mg.

8. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 7, characterized in that: The centrifugal speed in step (4) is 8000-12000 rpm, and the time is 10-20 min.

9. The method for determining the amino acid content in the digestive tract of livestock and poultry according to claim 8, characterized in that: The diameter of the filter membrane used in step (4) is 0.2 to 0.25 μm.

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