A method for automatically determining the inulin content in anticoagulated bovine plasma

Abstract

The present application relates to the technical field of plasma polysaccharide detection, and more specifically, it relates to a method for automatically determining the inulin content in anticoagulated bovine plasma. A method for automatically determining the inulin content in anticoagulated bovine plasma is as follows: six inulin anticoagulated bovine plasma standard solutions with different concentration gradients are configured, and the inulin anticoagulated bovine plasma standard solutions contain adenosine triphosphate, magnesium chloride, nicotinamide adenine dinucleotide, hexokinase and 6-phosphate glucose dehydrogenase, and the absorbance of each standard solution at 340nm is measured, and an absorbance-concentration curve is drawn; then the inulin anticoagulated bovine plasma solution to be tested is taken, and after the same operation, the absorbance of the sample solution to be tested at 340nm is measured, and the inulin concentration in the sample is calculated by substituting the curve. The method for the content of inulin in anticoagulated bovine plasma of the present application can reduce the influence of reducing sugars, peroxides, turbidity and pigments on the final data, and the measured data is more accurate.

Description

Technical Field

[0001] The present application relates to the technical field of plasma polysaccharide detection, and more specifically, to a method for automatically determining the inulin content in anticoagulated bovine plasma for performance evaluation of hemodialysis equipment. Background Art

[0002] Inulin is a linear inulin-type fructan. As a natural functional polysaccharide, it has the physiological functions of water-soluble dietary fiber and biological activity. It also has a strong function of proliferating bifidobacteria. It plays a certain role in regulating body balance, restoring gastrointestinal function, improving immunity, promoting the absorption of minerals such as calcium, magnesium, zinc, copper, regulating blood sugar, lowering blood lipids, preventing obesity and anti-cancer.

[0003] The determination of inulin content is an important indicator for the performance evaluation of hemodialysis equipment. At present, the commonly used method for determining inulin is the phenol-sulfuric acid method. However, the result of the phenol-sulfuric acid method is the total sugar content of the sample. The inulin content is equal to the total sugar content minus the reducing sugar content. Therefore, this method cannot identify the reducing sugar content in the sample, that is, the inulin content in the determination result is incorrect. At the same time, when conducting plasma testing, peroxides, turbidity and pigments in plasma will also affect the accuracy of the final inulin content.

[0004] Therefore, there is an urgent need for a method for detecting inulin content in anticoagulated bovine plasma. Summary of the invention

[0005] In order to improve the problem that conventional inulin detection methods cannot identify the reducing sugar content, and that peroxides, turbidity and pigments in plasma also affect the final test data, the present application provides a method for automatically determining the inulin content in anticoagulated bovine plasma.

[0006] The present application provides a method for automatically determining the inulin content in anticoagulated bovine plasma, which adopts the following technical solution:

[0007] A method for automatically determining the inulin content in anticoagulated bovine plasma comprises the following steps:

[0008] Step 1: prepare an aqueous solution containing adenosine triphosphate, magnesium chloride, and nicotinamide adenine dinucleotide, wherein the pH value of the Tris buffer is 7.8, to obtain solution A;

[0009] Step 2: preparing a solution containing hexokinase, 6-phosphoglucose dehydrogenase, bovine serum albumin, disodium hydrogen phosphate, sodium dihydrogen phosphate, p-hydroxybenzoate, and 4-aminoantipyrine to obtain a solution B;

[0010] Step 3, prepare six inulin anticoagulated bovine plasma standard solutions with different concentration gradients, ranging from 0 to 10 mg / L, and dilute 100 times with normal saline;

[0011] Step 4: Take a small amount of the diluted solution in step 3, add acetic acid-sodium acetate solution and hydrochloric acid solution, mix evenly, add inulinase and mix well to obtain solution C;

[0012] Step 5: Keep solution C at 37°C for 2 min to equilibrate to room temperature;

[0013] Step 6: Mix 2.5 μL of solution C and 5 μL of solution A, then add 4.5 μL of solution B and mix well;

[0014] Step 7, measuring the absorbance of each concentration standard solution at 340nm, drawing an absorbance-concentration curve, and removing the 0mg / L inulin anticoagulated bovine plasma standard solution as a blank background;

[0015] Step 8, taking the inulin anticoagulated bovine plasma solution to be tested, and diluting it 100 times with normal saline;

[0016] Step 9: Take a small amount of the diluted solution in step 8, add equal volumes of acetic acid-sodium acetate solution and hydrochloric acid solution, mix evenly, add inulinase and mix well to obtain solution D;

[0017] Step 10: Keep solution D at 37°C for 2 min to equilibrate to room temperature;

[0018] Step 11: Mix 2.5 μL of solution D and 5 μL of solution A, and then add 4.5 μL of solution B and mix well;

[0019] Step 12: Determine the absorbance of the sample solution at 340 nm and substitute it into the curve to calculate the inulin concentration in the sample.

[0020] Inulin, also known as inulin, is a linear fructan with fructose groups connected by β-glycosidic bonds. Most of the glycoside ends have a pyranose glucose residue. Under appropriate pH and temperature, the exonuclease activity of inulinase can specifically enzymatically hydrolyze a pyranose glucose group at the end of the inulin glycoside in anticoagulant bovine plasma into a single molecule of glucose, which can react with adenosine triphosphate and Mg to form a single molecule of glucose. 2+ Together, they are phosphorylated by hexokinase to glucose-6-phosphate.

[0021] Then, glucose 6-phosphate serves as the reaction substrate of glucose dehydrogenase, and the H +Nicotinamide adenine dinucleotide is reduced and colored to obtain NADH. Under 340nm wavelength light, the absorbance is proportional to the increase in NADH, so a mathematical logic operation relationship can be established. By converting the above mathematical logic relationship into analyzer data parameters, the absorbance value obtained by the test can be automatically converted into the concentration value of inulin, thereby reducing the influence of reducing sugar, peroxide, turbidity and pigment in anticoagulated bovine plasma on the final inulin content result.

[0022] Preferably, all steps of the method for automatically determining the inulin content in anticoagulated bovine plasma are implemented on a fully automatic biochemical analyzer, the reaction system and conditions are verified and obtained on a Beckman Coulter AU680 biochemical analysis system, the reaction type is an endpoint method, and the blank rate of the enzyme activity assay reagent is less than 0.002ΔA / min.

[0023] Preferably, in step 1, an aqueous solution containing a final concentration of 2 mmol / L of adenosine triphosphate, a final concentration of 1.32 mmol / L of magnesium chloride, and a final concentration of 1.37 mmol of nicotinamide adenine dinucleotide, 100 mmol / L of Tris buffer, and a pH of 7.8 of Tris buffer is prepared to obtain solution A.

[0024] Preferably, in step 2, a solution containing a final concentration of hexokinase of 900 U / L, a final concentration of 6-phosphoglucose dehydrogenase of 1800 U / L, a final concentration of bovine serum albumin of 20 g / L, disodium hydrogen phosphate of 2.9 g / L, sodium dihydrogen phosphate of 0.3 g / L, p-hydroxybenzoate of 25 mmol / L, and 4-aminoantipyrine of 0.3 mmol / L is prepared to obtain solution B.

[0025] Preferably, in step 3, the gradient standard of the inulin anticoagulated bovine plasma standard solution is as follows:

[0026]

[0027] Among them, the inulin anticoagulant bovine plasma standard solution is a 100-fold diluted solution.

[0028] Preferably, in step 4, 50 μL of the diluted solution in step 3 is taken, and an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution are added, the concentration of the hydrochloric acid solution is 0.1 mol / L, and the pH of the acetic acid-sodium acetate solution is 5.2. After uniform mixing, 30 μL of inulinase is added and mixed, and the inulinase concentration is 600 U / ml to obtain solution C.

[0029] Preferably, in step 4, each concentration solution C is obtained by using a fully automatic preparation machine;

[0030] The fully automatic configuration machine comprises a frame, a raw material conveying assembly for conveying raw materials, a product conveying assembly for conveying products, and a driving member, wherein the raw material conveying assembly mixes inulin anticoagulant bovine plasma standard solutions of different concentrations with other raw materials and transfers the solutions to the product conveying assembly, and the driving member drives the raw material conveying assembly and the product conveying assembly to operate;

[0031] The raw material conveying component includes a driving gear and an inulin anticoagulant bovine plasma standard solution rack, the driving gear is rotatably connected to the frame, the inulin anticoagulant bovine plasma standard solution rack is meshed with the driving gear, and the inulin anticoagulant bovine plasma standard solution rack is slidably connected to the frame; the inulin anticoagulant bovine plasma standard solution rack is provided with six first fixing rings for fixing the inulin anticoagulant bovine plasma standard solution feed pipe, the frame is fixedly connected with a second fixing ring for fixing the inulinase feed pipe and a third fixing ring for fixing other raw material feed pipes, and the driving member drives the six first fixing rings to be transferred to the feed end of the product conveying component in sequence;

[0032] The product conveying assembly includes a support plate slidably connected to the frame and six product barrels placed on the support plate. The nozzles of the inulin anticoagulant bovine plasma standard solution feed pipe, the nozzles of the inulinase feed pipe and the nozzles of other raw material feed pipes are all facing the product barrels. The product barrels are used to mix the inulin anticoagulant bovine plasma standard solution, inulinase and other raw materials.

[0033] Preferably, the driving member includes a driving gear, a driving rack and a driving cylinder, the driving gear is rotatably connected to the frame, the driving gear is coaxially arranged with the driving gear and rotates synchronously, the driving rack is fixedly connected to the support plate, the driving rack and the driving gear are meshed with each other, the driving cylinder is fixedly connected to the frame, and the output end of the driving cylinder is fixedly connected to the support plate.

[0034] When it is necessary to prepare solution C of different concentrations, the staff can first fix the feed tubes of inulin anticoagulant bovine plasma standard solutions of different concentrations at different first fixed rings, fix the inulinase feed tube at the second fixed ring, and fix the other raw material feed tubes at the third fixed ring.

[0035] Afterwards, the staff can start the driving cylinder, drive the cylinder to run and drive the support plate to slide. At this time, the support plate will drive the driving gear to rotate through the driving rack, and the driving gear will drive the active gear to rotate. The rotation of the active gear will drive the rack of the inulin anticoagulant bovine plasma standard solution to slide, and then the first fixed ring of the feed tube of the inulin anticoagulant bovine plasma standard solution with different concentrations will be transferred to the product cylinder, thereby realizing the automatic configuration of solutions C with different concentrations, improving efficiency while reducing labor costs.

[0036] Preferably, in step seven, the formula of the absorbance-concentration curve is as follows:

[0037] C=26.607×(A-A0)+0.4169

[0038] Where:

[0039] C-inulin concentration;

[0040] A-absorbance of the sample solution at 340nm;

[0041] A0-0mg / L inulin anticoagulated bovine plasma standard solution absorbance at 340nm.

[0042] Preferably, in step nine, take 5 μL of the diluted solution in step eight, add an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution, the concentration of the hydrochloric acid solution is 0.1 mol / L, the pH of the acetic acid-sodium acetate solution is 5.2, after uniform mixing, add 30 μL of inulinase and mix well, the inulinase concentration is 600 U / ml, and obtain solution D.

[0043] In summary, this application has the following beneficial effects:

[0044] 1. Inulin, also known as inulin, is a linear fructan with fructose groups connected by β-glycosidic bonds. Most of the glycosides have a pyranose glucose residue at the end. Under appropriate pH and temperature, the exonuclease activity of inulinase can specifically enzymatically hydrolyze a pyranose glucose group at the end of the inulin glycoside in anticoagulant bovine plasma into a single molecule of glucose, which can react with adenosine triphosphate and Mg 2+ Together, they are phosphorylated by hexokinase to glucose-6-phosphate;

[0045] Then, glucose 6-phosphate serves as the reaction substrate of glucose dehydrogenase, and the H + Nicotinamide adenine dinucleotide is reduced and colored to obtain NADH. Under light of 340nm wavelength, the absorbance is proportional to the increase in NADH. Therefore, a mathematical logic operation relationship can be established. By converting the above mathematical logic relationship into analyzer data parameters, the absorbance value obtained by the test can be automatically converted into the concentration value of inulin, thereby reducing the influence of reducing sugar, peroxide, turbidity and pigment in anticoagulated bovine plasma on the final inulin content result.

[0046] 2. When it is necessary to prepare solution C of different concentrations, the staff can first fix the feed tubes of inulin anticoagulant bovine plasma standard solutions of different concentrations at different first fixing rings, fix the inulinase feed tube at the second fixing ring, and fix the other raw material feed tubes at the third fixing ring;

[0047] Afterwards, the staff can start the driving cylinder, drive the cylinder to run and drive the support plate to slide. At this time, the support plate will drive the driving gear to rotate through the driving rack, and the driving gear will drive the active gear to rotate. The rotation of the active gear will drive the rack of the inulin anticoagulant bovine plasma standard solution to slide, and then the first fixed ring of the feed tube of the inulin anticoagulant bovine plasma standard solution with different concentrations will be transferred to the product cylinder, thereby realizing the automatic configuration of solutions C with different concentrations, improving efficiency while reducing labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the structure of the fully automatic configuration machine;

[0049] Figure 2 This is a schematic diagram of the structure of the fully automatic configuration machine in the other direction;

[0050] Figure 3 It is the absorbance-concentration standard curve.

[0051] Description of the drawings: 1. Frame; 2. Raw material conveying assembly; 3. Product conveying assembly; 4. Driving member; 21. Driving gear; 22. Inulin anticoagulant bovine plasma standard solution rack; 23. First fixed ring; 24. Second fixed ring; 25. Third fixed ring; 31. Support plate; 32. Product barrel; 41. Driving gear; 42. Driving rack; 43. Driving cylinder. DETAILED DESCRIPTION

[0052] The following is combined with Figure 1-3 This application is described in further detail.

[0053] raw material

[0054] Inulinase was purchased from SIGMA, CAS: 9001-57-4, I4753-1G;

[0055] Hexokinase was purchased from SIGMA, CAS: 9001-51-8, H6380-1.5KU;

[0056] Glucose-6-phosphate dehydrogenase was purchased from SIGMA, CAS: 9001-40-5, G5885-2KU;

[0057] Bovine serum albumin (powder) was purchased from VETEC, CAS: 9048-46-8, V900933-100g;

[0058] Adenosine triphosphate was purchased from SIGMA (chromatographic grade), CAS: 987-65-5, A6559-25UMO;

[0059] Magnesium chloride was purchased from SIGMA, CAS: 7786-30-3, M8266-100G;

[0060] Nicotinamide adenine dinucleotide was purchased from Millipore, CAS: 53-84-9, 481911-5GM;

[0061] Tris hydrochloric acid was purchased from Millipore, CAS: 1185-53-1, 648313-500GM;

[0062] Sodium hydrogen phosphate was purchased from Sinopharm Reagent, CAS: 7558-79-4, 20040617;

[0063] Sodium dihydrogen phosphate was purchased from Sinopharm Reagent, CAS: 7558-80-7, 20040818;

[0064] Acetic acid-sodium acetate buffer was purchased from SIGMA, CAS: 126-96-5, S7899-500ML;

[0065] Hydrochloric acid was purchased from SIGMA, CAS: 7647-01-0, 2104-50ML;

[0066] Parabens were purchased from SIGMA, CAS: 114-63-6, H3766-25G;

[0067] 4-Aminoantipyrine was purchased from SIGMA, CAS: 83-07-8, A4382-100G;

[0068] Inulin (inulin) was purchased from Aladdin, CAS: 9005-80-5;

[0069] Or, inulin (fructan) was purchased from SIGMA, SKU: 900388-100MG, (from chicory), ≥97atom % 13C, ≥97% (CP);

[0070] This application uses inulin (inulin) as an example;

[0071] Bovine plasma was purchased from Beijing Borsi and anticoagulated with sodium citrate;

[0072] Physiological saline is commonly available in the market.

[0073] Example

[0074] The present application provides a method for automatically determining the inulin content in anticoagulated bovine plasma, comprising the following steps:

[0075] Step 1: prepare an aqueous solution containing a final concentration of 2 mmol / L of adenosine triphosphate, a final concentration of 1.32 mmol / L of magnesium chloride, a final concentration of 1.37 mmol of nicotinamide adenine dinucleotide, 100 mmol / L of Tris buffer, and a pH of 7.8 to obtain solution A;

[0076] Specifically, 200 μmol of adenosine triphosphate, 0.0126 g of magnesium chloride, 0.0909 g of nicotinamide adenine dinucleotide, and 1.576 g of Tris buffer were placed in a 100 mL beaker, 50 mL of purified water was added to fully dissolve, and the mixture was transferred to a 100 mL volumetric flask and made up to 100 mL with purified water to obtain solution A.

[0077] Step 2: preparing a solution containing 900 U / L hexokinase, 1800 U / L 6-phosphogluconate dehydrogenase, 20 g / L bovine serum albumin, 2.9 g / L disodium hydrogen phosphate, 0.3 g / L sodium dihydrogen phosphate, 25 mmol / L p-hydroxybenzoate, and 0.3 mmol / L 4-aminoantipyrine to obtain solution B;

[0078] Specifically, weigh 2.571 g of hexokinase, 3.273 g of 6-phosphoglucose dehydrogenase, and 2 g of bovine serum albumin, put them into a 100 mL beaker, add 70 mL of purified water, mix thoroughly, add 0.29 g of disodium hydrogen phosphate, 0.03 g of sodium dihydrogen phosphate, 0.40 g of p-hydroxybenzoate, and 0.006 g of 4-aminoantipyrine, transfer them into a volumetric flask and dilute to 100 mL to obtain solution B.

[0079] Step 3, prepare six inulin anticoagulated bovine plasma standard solutions with different concentration gradients, ranging from 0 to 10 mg / L, and dilute 100 times with normal saline;

[0080] The gradient standard of inulin anticoagulated bovine plasma standard solution is as follows:

[0081]

[0082] The inulin anticoagulant bovine plasma standard solution refers to a 100-fold diluted solution.

[0083] Step 4: Take 50 μL of the diluted solution in step 3, add an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution, the concentration of the hydrochloric acid solution is 0.1 mol / L, the pH of the acetic acid-sodium acetate solution is 5.2, mix evenly, add 30 μL of inulinase and mix well, the inulinase concentration is 600 U / ml, and obtain solution C.

[0084] In step 4, each concentration solution C is obtained by using a fully automatic preparation machine;

[0085] Reference Figure 1 and Figure 2 The fully automatic configuration machine includes a frame 1, a raw material conveying assembly 2 for conveying raw materials, a product conveying assembly 3 for conveying products, and a driving member 4. The raw material conveying assembly 2 is used to mix the inulin anticoagulated bovine plasma standard solution with different concentrations with other raw materials and transfer them to the product conveying assembly 3, the other raw materials refer to acetic acid-sodium acetate solution and hydrochloric acid solution, and the driving member 4 drives the raw material conveying assembly 2 and the product conveying assembly 3 to operate.

[0086] The raw material conveying assembly 2 includes a driving gear 21 and an inulin anticoagulated bovine plasma standard solution rack 22. The driving gear 21 is rotatably connected to the upper part of the frame 1, and the inulin anticoagulated bovine plasma standard solution rack 22 is slidably connected to the upper part of the frame 1. The driving gear 21 and the inulin anticoagulated bovine plasma standard solution rack 22 are meshed with each other.

[0087] Six first fixing rings 23 for fixing the inulin anticoagulant bovine plasma standard solution feed pipe are fixedly connected to the side wall of the inulin anticoagulant bovine plasma standard solution rack 22, and a second fixing ring 24 for fixing the inulinase feed pipe and a third fixing ring 25 for fixing other raw material feed pipes are fixedly connected to the upper side wall of the frame 1. The driving member 4 drives the six first fixing rings 23 to be transferred to the feed end of the product conveying component 3 in sequence through the driving gear 21 and the inulin anticoagulant bovine plasma standard solution rack 22.

[0088] The product conveying assembly 3 includes a support plate 31 slidably connected to the lower part of the frame 1 and six product barrels 32 placed on the support plate 31. The nozzles of the inulin anticoagulant bovine plasma standard solution feed pipe, the nozzles of the inulinase feed pipe and the nozzles of other raw material feed pipes are all facing the product barrel 32. The product barrel 32 is used to mix the inulin anticoagulant bovine plasma standard solution, inulinase and other raw materials.

[0089] The driving member 4 includes a driving gear 41, a driving rack 42 and a driving cylinder 43. The driving gear 41 is rotatably connected to the lower portion of the frame 1. The driving gear 41 and the driving gear 21 are coaxially arranged and rotate synchronously. The driving rack 42 is fixedly connected to the lower end surface of the support plate 31. The driving rack 42 and the driving gear 41 are meshed with each other. The driving cylinder 43 is fixedly connected to the lower portion of the frame 1, and the output end of the driving cylinder 43 is fixedly connected to the support plate 31.

[0090] When it is necessary to prepare solution C of different concentrations, the staff can first fix the feed tubes of inulin anticoagulant bovine plasma standard solutions of different concentrations to different first fixing rings 23, fix the inulinase feed tube to the second fixing ring 24, and fix the other raw material feed tubes to the third fixing ring 25;

[0091] Afterwards, the staff can start the driving cylinder 43, and the driving cylinder 43 can run and drive the support plate 31 to slide. At this time, the support plate 31 will drive the driving gear 41 to rotate through the driving rack 42, and the driving gear 41 drives the active gear 21 to rotate. The rotation of the active gear 21 drives the rack 22 of the inulin anticoagulant bovine plasma standard solution to slide, and then the first fixed ring 23 of the feed pipe of the inulin anticoagulant bovine plasma standard solution with different concentrations is transferred to the product cylinder 32, thereby realizing the automatic configuration of solutions C with different concentrations.

[0092] Step 5: Keep solution C at 37°C for 2 min and equilibrate to room temperature.

[0093] Step 6: Mix 2.5 μL of solution C and 5 μL of solution A, then add 4.5 μL of solution B and mix well.

[0094] Step 7, measuring the absorbance of each concentration standard solution at 340nm, drawing an absorbance-concentration curve, and removing the 0mg / L inulin anticoagulated bovine plasma standard solution as a blank background;

[0095] The formula for the absorbance-concentration curve is as follows:

[0096] C=26.607×(A-A0)+0.4169

[0097] Where:

[0098] C-inulin concentration;

[0099] A-absorbance of the sample solution at 340nm;

[0100] A0-Absorbance of 0mg / L inulin anticoagulated bovine plasma standard solution at 340nm;

[0101] The absorbance-concentration curve is as follows Figure 3 shown.

[0102] Step 8, taking the inulin anticoagulated bovine plasma solution to be tested, and diluting it 100 times with normal saline;

[0103] Step 9. Take 5 μL of the diluted solution in step 8, add an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution, the concentration of the hydrochloric acid solution is 0.1 mol / L, the pH of the acetic acid-sodium acetate solution is 5.2, mix evenly, add 30 μL of inulinase and mix well, the inulinase concentration is 600 U / ml, and obtain solution D.

[0104] Step 10: Keep solution D at 37°C for 2 min to equilibrate to room temperature;

[0105] Step 11: Mix 2.5 μL of solution D and 5 μL of solution A, and then add 4.5 μL of solution B and mix well;

[0106] Step 12: Determine the absorbance of the sample solution at 340 nm and substitute it into the curve to calculate the inulin concentration in the sample.

[0107] It should be noted that all the above steps were implemented on a fully automatic biochemical analyzer, the reaction system and conditions were verified and obtained on a Beckman Coulter AU680 biochemical analysis system, the reaction type was an endpoint method, and the blank rate of the enzyme activity assay reagent was less than 0.002△A / min.

[0108] In addition, in the embodiments of the present application, the above-mentioned fixed connection can be selected according to actual conditions by conventional fixed connection methods such as welding, bolt connection, and one-piece molding; the above-mentioned rotational connection can be selected according to actual conditions by conventional rotational connection methods such as pin connection; the above-mentioned sliding connection can be selected according to actual conditions by conventional sliding connection methods such as slide rails and slide grooves.

[0109] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for automatically determining the inulin content in anticoagulated bovine plasma, characterized in that: The following steps are involved: Step 1: prepare an aqueous solution containing adenosine triphosphate, magnesium chloride, and nicotinamide adenine dinucleotide, wherein the pH value of the Tris buffer is 7.8, to obtain solution A; Step 2: preparing a solution containing hexokinase, 6-phosphoglucose dehydrogenase, bovine serum albumin, disodium hydrogen phosphate, sodium dihydrogen phosphate, p-hydroxybenzoate, and 4-aminoantipyrine to obtain a solution B; Step 3, prepare six inulin anticoagulated bovine plasma standard solutions with different concentration gradients, ranging from 0 to 10 mg / L, and dilute 100 times with normal saline; Step 4: Take 50 μL of the diluted solution in step 3, add an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution, the concentration of the hydrochloric acid solution is 0.1 mol / L, the pH of the acetic acid-sodium acetate solution is 5.2, mix evenly, add 30 μL of inulinase and mix well, the inulinase concentration is 600 U / ml, and obtain solution C; In step 4, each concentration solution C is obtained by using a fully automatic preparation machine; The fully automatic configuration machine comprises a frame (1), a raw material conveying component (2) for conveying raw materials, a product conveying component (3) for conveying products, and a driving component (4); the raw material conveying component (2) mixes inulin anticoagulant bovine plasma standard solutions of different concentrations with other raw materials and transfers the mixed solutions to the product conveying component (3); and the driving component (4) drives the raw material conveying component (2) and the product conveying component (3) to operate; The raw material conveying component (2) comprises a driving gear (21) and an inulin anticoagulant bovine plasma standard solution rack (22), wherein the driving gear (21) is rotatably connected to the frame (1), the inulin anticoagulant bovine plasma standard solution rack (22) is meshed with the driving gear (21), and the inulin anticoagulant bovine plasma standard solution rack (22) is slidably connected to the frame (1); the inulin anticoagulant bovine plasma standard solution rack (22) is provided with six first fixing rings (23) for fixing an inulin anticoagulant bovine plasma standard solution feed pipe, the frame (1) is fixedly connected with a second fixing ring (24) for fixing an inulinase feed pipe and a third fixing ring (25) for fixing other raw material feed pipes, and the driving member (4) drives the six first fixing rings (23) to be transferred to the feed end of the product conveying component (3) in sequence; The product conveying assembly (3) comprises a support plate (31) slidably connected to the frame (1) and six product barrels (32) placed on the support plate (31), the nozzles of the inulin anticoagulant bovine plasma standard solution feed pipe, the nozzles of the inulinase feed pipe and the nozzles of the other raw material feed pipes all face the product barrel (32), and the product barrel (32) is used to mix the inulin anticoagulant bovine plasma standard solution, inulinase and other raw materials; The driving member (4) comprises a driving gear (41), a driving rack (42) and a driving cylinder (43); the driving gear (41) is rotatably connected to the frame (1); the driving gear (41) and the driving gear (21) are coaxially arranged and rotate synchronously; the driving rack (42) is fixedly connected to the support plate (31); the driving rack (42) and the driving gear (41) are meshed with each other; the driving cylinder (43) is fixedly connected to the frame (1); and an output end of the driving cylinder (43) is fixedly connected to the support plate (31); Step 5: Keep solution C at 37°C for 2 min to equilibrate to room temperature; Step 6: Mix 2.5 μL of solution C and 5 μL of solution A, then add 4.5 μL of solution B and mix well; Step 7, measuring the absorbance of each concentration standard solution at 340nm, drawing an absorbance-concentration curve, and removing the 0mg / L inulin anticoagulated bovine plasma standard solution as a blank background; Step 8, taking the inulin anticoagulated bovine plasma solution to be tested, and diluting it 100 times with normal saline; Step 9: Take a small amount of the diluted solution in step 8, add equal volumes of acetic acid-sodium acetate solution and hydrochloric acid solution, mix evenly, add inulinase and mix well to obtain solution D; Step 10: Keep solution D at 37°C for 2 min to equilibrate to room temperature; Step 11: Mix 2.5 μL of solution D and 5 μL of solution A, and then add 4.5 μL of solution B and mix well; Step 12: Determine the absorbance of the sample solution at 340 nm and substitute it into the curve to calculate the inulin concentration in the sample.

2. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: All steps of the method for automatically determining the inulin content in anticoagulated bovine plasma are implemented on a fully automatic biochemical analyzer, the reaction system and conditions are verified and obtained on a Beckman Coulter AU680 biochemical analysis system, the reaction type is an endpoint method, and the blank rate of the enzyme activity determination reagent is less than 0.002△A / min.

3. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: In step 1, an aqueous solution containing a final concentration of 2 mmol / L of adenosine triphosphate, a final concentration of 1.32 mmol / L of magnesium chloride, a final concentration of 1.37 mmol of nicotinamide adenine dinucleotide, 100 mmol / L of Tris buffer, and a pH of 7.8 of Tris buffer was prepared to obtain solution A.

4. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: In step 2, a solution containing a final concentration of hexokinase of 900 U / L, a final concentration of 6-phosphogluconate dehydrogenase of 1800 U / L, a final concentration of bovine serum albumin of 20 g / L, disodium hydrogen phosphate of 2.9 g / L, sodium dihydrogen phosphate of 0.3 g / L, p-hydroxybenzoate of 25 mmol / L, and 4-aminoantipyrine of 0.3 mmol / L is prepared to obtain solution B.

5. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: In step 3, the gradient standard of inulin anticoagulated bovine plasma standard solution is as follows: ； Among them, the inulin anticoagulant bovine plasma standard solution is a 100-fold diluted solution.

6. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: In step 7, the formula for the absorbance-concentration curve is as follows: C=26.607×(A-A0)+0.4169 Where: C-inulin concentration; A-absorbance of the sample solution at 340nm; A0-0mg / L inulin anticoagulated bovine plasma standard solution absorbance at 340nm.

7. The method for automatically determining the inulin content in anticoagulated bovine plasma according to claim 1, characterized in that: In step nine, take 5 μL of the diluted solution in step eight, add an equal volume of acetic acid-sodium acetate solution and 5 μL of hydrochloric acid solution, the concentration of the hydrochloric acid solution is 0.1 mol / L, the pH of the acetic acid-sodium acetate solution is 5.2, after uniform mixing, add 30 μL of inulinase and mix well, the inulinase concentration is 600 U / ml, and obtain solution D.

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