WO2020006940A1 - 一种用酶标仪酶法定量检测液态奶中乳果糖的方法及其试剂盒 - Google Patents

一种用酶标仪酶法定量检测液态奶中乳果糖的方法及其试剂盒 Download PDF

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WO2020006940A1
WO2020006940A1 PCT/CN2018/112496 CN2018112496W WO2020006940A1 WO 2020006940 A1 WO2020006940 A1 WO 2020006940A1 CN 2018112496 W CN2018112496 W CN 2018112496W WO 2020006940 A1 WO2020006940 A1 WO 2020006940A1
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solution
glucose
concentration
fructose
sample
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PCT/CN2018/112496
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French (fr)
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郑楠
文芳
李松励
王加启
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中国农业科学院北京畜牧兽医研究所
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Publication of WO2020006940A1 publication Critical patent/WO2020006940A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

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  • the invention relates to a method for quantitatively determining lactulose in liquid milk by using a enzyme plate reader and an enzyme chemical method, and belongs to the field of enzyme chemical analysis and detection.
  • Lactulose is a product of base isomerization of lactose during heat treatment of milk. It is an isomer with lactose and is an important indicator for evaluating the degree of heat treatment of milk. Both the European Union and IDF propose that the lactulose content of UHT sterilized milk should not exceed 600 mg / L. Therefore, the accurate determination of lactulose is very important.
  • the detection methods of lactulose in liquid milk at home and abroad are relatively complicated. Generally, they are high-performance liquid chromatography and spectrophotometry based on enzyme analysis. Among them, high-performance liquid chromatography has a tens of times higher lactose content than lactulose in the sample, and it is difficult to achieve baseline separation between lactose and lactulose in the separation, which makes it difficult to quantitatively detect lactulose.
  • the spectrophotometric method based on enzyme analysis is a method that uses a standard molar absorption coefficient to quantify (for example, the enzyme analysis spectrophotometry described in standards such as NY / T939-2016 and ISO11285-2004). There is tedious sample processing and the need for measurement.
  • the principle of enzyme chemistry used in the present invention is the same as the principle of enzyme chemistry used in the standard spectrophotometric determination of lactulose in liquid milk, so the standard NY / T 939-2016 is incorporated herein by reference in its entirety.
  • the microplate reader In the method of quantitatively measuring the lactulose content in liquid milk using a microplate reader enzyme method, the microplate reader cannot use the method of quantification of Lambert Beer's law by a spectrophotometer method, and a new quantitative method needs to be established.
  • the inventors of the present invention used fructose as a standard product to establish a standard curve of fructose, which was used to quantitatively detect the content of lactulose in liquid milk by a microplate reader and an enzyme method, which not only solved the problem that can not be quantified in the microplate reader method.
  • the technical problem also avoids the methodological errors caused by the enzymolysis of multiple steps when other substances are used as standards, for example, lactulose is used as a standard.
  • a first aspect of the present invention is to provide a method for quantitatively detecting lactulose in liquid milk by a microplate reader and enzymatic method, which includes the following steps: establishing a standard curve using fructose as a standard.
  • the step of establishing the standard curve includes: adding a fructose standard solution to a microplate for a microplate reader, so that the mass of fructose in each well increases in equal or doubling order, and after adding NADP + and ATP Mix well; then add hexokinase and glucose-6-phosphate dehydrogenase to mix and react, read the OD value of the solution at 340nm on the microplate reader, record it as A 1 , and add phosphoglucose isomerase and mix For the reaction, read the OD value of the solution on a microplate reader at 340 nm and record it as A 2. Take the value of (A 2 -A 1 ) as the net OD value as the ordinate and the fructose mass in each well as the abscissa. Draw the standard Curve or calculated regression equation.
  • a fructose standard solution is added to 4-10 wells in the microplate.
  • the mass of fructose in each well is in ⁇ g.
  • the fructose mass in each well increases in equal order, for example, at a pitch of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ⁇ g / well, or at 0, 2.0, 4.0, 6.0 ⁇ g / well. Pitch increases.
  • a fructose standard solution is added to 7 wells, so that the fructose mass in the wells is 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ⁇ g / well in order.
  • the mass of fructose in each well increases in multiples in sequence, for example, it may increase in multiples of 2 times, 3 times, 4 times and the like.
  • a fructose standard solution is added to 4 wells, so that the fructose mass in the wells is 0, 1.0, 2.0, 4.0 ⁇ g / well in order.
  • NADP + and ATP are added as a mixed solution of NADP + and ATP, in which the concentration of NADP + is 7-12 mg / mL and the concentration of ATP is 40-60 mg / mL.
  • hexokinase and glucose-6-phosphate dehydrogenase are added in the form of a mixed suspension of hexokinase and glucose-6-phosphate dehydrogenase, and the concentration of hexokinase in the suspension is 200-350U / mL, the concentration of glucose-6-phosphate dehydrogenase is 200-350U / mL.
  • hexokinase and glucose-6-phosphate dehydrogenase are added and mixed, and the mixture is allowed to react at 37 ° C for 10 minutes.
  • the concentration of the glucoglucose isomerase solution is 700-900 U / mL.
  • the reaction is carried out at 37 ° C for 15 minutes after adding glucose phosphate isomerase and mixing.
  • the step of establishing the fructose standard curve is: adding a fructose standard solution to a microtiter plate so that the mass of fructose is 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ⁇ g, respectively.
  • buffer B 140.0g / L triethanolamine hydrochloride, 2.5g / L magnesium sulfate, pH 7.5 ⁇ 7.7
  • NADP + and ATP mixed solution and mix well
  • hexokinase and glucose- 6-phosphate dehydrogenase mix and place at 37 ° C for 10 minutes
  • read the OD value of the solution at 340nm on a microplate reader record it as A 1
  • a 2 take the value of (A 2 -A 1 ) as the net OD value as the ordinate and the fructose mass ( ⁇ g / well) as the horizontal Coordinates, draw a standard curve or calculate a regression equation.
  • the detection method further comprises detecting the value of (A 2 -A 1 ) of the milk sample, and converting the content of lactulose in the milk sample according to a fructose standard curve.
  • the conversion formula is:
  • V 1 The volume of milk sample taken at the start of the test, the unit is mL, and the value can be 0.3-1. In one embodiment of the present invention, the value is 0.50, that is, the volume of the milk sample sucked at the start of the test is 0.50 mL.
  • V 2 The volume of the fat and protein in the precipitated sample after centrifugation during sample pretreatment, the unit is mL, and the value can be 1.2-4. In one embodiment of the present invention, the value is 2.00, that is, the volume after sedimentation and centrifugation. 2mL.
  • V 3 the volume of the supernatant after pipetting, the unit is mL, and the value can be 0.3-1, preferably the same as the value of V 1 . In one embodiment of the present invention, the value is 0.50, that is, the volume of the supernatant after suction is 0.50 mL.
  • V 4 the total volume of the sample solution after the glucose oxidation step, the unit is mL, and the value may be 0.606-2.02. In one embodiment of the present invention, the value is 1.01, that is, the sample solution after the glucose oxidation step The total volume is 1.01mL.
  • V 5 The volume of the sample solution added to the microtiter plate when measured by the microplate reader, the unit is mL, and the value can be 0.06-0.2. In one embodiment of the present invention, the value is 0.10, that is, the During the microplate reader measurement, the volume of the sample solution added to the microplate was 0.10 mL.
  • the conversion factor f changes correspondingly according to the volume of the liquid in the corresponding step during the detection process.
  • the conversion factor f 153.52 calculated based on the volume of the liquid in the corresponding step.
  • the detection method of the present invention further includes a sample pretreatment step.
  • the sample pretreatment step includes: precipitating proteins and fats in a liquid milk sample using a zinc sulfate solution and a potassium ferrocyanide solution; after centrifugation, taking a supernatant and adding a ⁇ -galactosidase solution for hydrolysis The lactose and lactulose in the supernatant were then added to glucose oxidase solution and catalase solution to oxidize glucose.
  • centrifugation is performed after the oxidized glucose reaction is completed, and the supernatant is taken for subsequent sample detection.
  • sample liquid the liquid obtained after the sample pre-treatment step, that is, after the glucose oxidation step.
  • sample liquid includes the liquid obtained after the milk sample to be tested has passed the sample pre-treatment step and The supernatant was further centrifuged to distinguish the fructose standard solution.
  • the concentration of the zinc sulfate solution is 168 g / L.
  • the concentration of the potassium ferrocyanide solution is 130 g / L.
  • the concentration of the ⁇ -galactosidase solution is (1.5-3.0) ⁇ 10 3 U / mL.
  • the reaction condition of the ⁇ -galactosidase solution to hydrolyze lactose and lactulose in the supernatant is 50 ° C. incubator or water bath for 1 h.
  • the concentration of the glucose oxidase solution is (3.0-5.0) ⁇ 10 3 U / mL.
  • the concentration of the catalase solution is (4.0-6.0) ⁇ 10 5 U / mL.
  • the glucose oxidase and catalase are used as a mixed enzyme solution, wherein the concentration of glucose oxidase is (3.0-5.0) ⁇ 10 3 U / mL, and the concentration of catalase is (4.0-6.0) ⁇ 10 5 U / mL.
  • the reaction conditions for oxidizing glucose by the glucose oxidase solution and catalase solution are 40 ° C incubator or water bath for 3h.
  • the operation method of sample pretreatment is: sucking 0.5mL of liquid milk sample, adding 1.3mL of distilled water, 168g / L zinc sulfate solution 0.1mL, 130g / L potassium ferrocyanide solution 0.1 mL, mix and centrifuge at 5000 rpm for 10 minutes. Aspirate the supernatant and divide it into three 500 ⁇ L portions. Add two buffer A (48 g / L disodium hydrogen phosphate, 8.6 g / L sodium dihydrogen phosphate, 1 g / L magnesium sulfate, pH 7.4 to 7.6 respectively).
  • buffer C obtained by 2.5 times dilution of buffer B
  • buffer C obtained by 2.5 times dilution of buffer B
  • 10 ⁇ L of octanol glucose oxidase (4.0 ⁇ 10 3 U / mL)
  • peroxidation 20 ⁇ L of a catalase (5.0 ⁇ 10 5 U / mL) mixed solution and 10 ⁇ L of 30% H 2 O 2 were mixed, cultured in a 40 ° C incubator or a water bath for 3 hours, and centrifuged at 5000 rpm for 10 minutes.
  • the detection method of the present invention further includes a sample measurement step.
  • the sample measurement step includes: adding the treated sample solution (such as a blank control, a measurement sample), NADP + and ATP into the microwells of the microwell plate, adding the hexokinase after mixing. And glucose-6-phosphate dehydrogenase, after mixing and reacting, read the OD value of the solution at 340nm on a microplate reader and record it as A 1 ; then add phosphoglucose isomerase. After mixing and reacting, Read the OD value of the solution at 340 nm on a standard instrument and record it as A 2 .
  • the treated sample solution such as a blank control, a measurement sample
  • NADP + and ATP hexokinase after mixing.
  • glucose-6-phosphate dehydrogenase after mixing and reacting, read the OD value of the solution at 340nm on a microplate reader and record it as A 1 ; then add phosphoglucose isomerase. After mixing and reacting, Read the OD value of the solution at
  • NADP + and ATP are added as a mixed solution of NADP + and ATP, in which the concentration of NADP + is 7-12 mg / mL and the concentration of ATP is 40-60 mg / mL.
  • hexokinase and glucose-6-phosphate dehydrogenase are added in the form of a mixed suspension of hexokinase and glucose-6-phosphate dehydrogenase, and the concentration of hexokinase in the suspension is 200-350U / mL, the concentration of glucose-6-phosphate dehydrogenase is 200-350U / mL.
  • the concentration of the phosphoglucose isomerase solution is 700-900 U / mL.
  • the sample measurement step includes: adding the treated sample solution (such as a blank control, a measurement sample), distilled water, and a buffer solution B (140.0g / L triethanolamine hydrochloride) to the microwells of the microplate. , 2.5g / L magnesium sulfate, pH 7.5 ⁇ 7.7), NADP + and ATP. After mixing, add hexokinase and glucose-6-phosphate dehydrogenase. After mixing, react at 37 ° C for 10min.
  • the treated sample solution such as a blank control, a measurement sample
  • distilled water such as distilled water
  • a buffer solution B 140.0g / L triethanolamine hydrochloride
  • the sample measurement method is: take a 96-well microplate, and add the following substances to each microwell: 100 ⁇ L of sample solution (such as blank control, measurement sample), 100 ⁇ L of distilled water, buffer solution B 20 ⁇ L, NADP + (10 mg / mL) and ATP (50 mg / mL) mixed solution 10 ⁇ L, mix well and let stand for 3 min; then add hexokinase and glucose-6-phosphate dehydrogenase suspension (260U / mL each) 5 ⁇ L, mix in a 37 ° C incubator for 10min, take out the OD value (A1) of the solution at 340nm on a microplate reader; add 5 ⁇ L of phosphoglucose isomerase (800U / mL), mix and place Incubate in a 37 ° C incubator for 15 min. Take out the OD value (A2) of the solution at 340 nm on a microplate reader; (A2-A1) is
  • the detection limit of the detection method of the present invention is that the lactulose content in the liquid milk is 4.6 mg / L.
  • the linear detection range of the detection method of the present invention is 0.03 to 6 ⁇ g of fructose, which is equivalent to the standard detection process, and the range of lactulose content in liquid milk can be determined to be 4.6 to 920 mg / L.
  • a second aspect of the present invention is to provide a detection kit for enzymatically detecting lactulose in liquid milk, the kit comprising: a lyophilized powder of a mixture of glucose oxidase and catalase, NADP + and ATP The mixture was lyophilized.
  • the activity ratio of glucose oxidase and catalase is 1:80 to 1: 600, and preferably 1: 125.
  • a mixture of ATP and NADP + lyophilized powder the mass of ATP and NADP + ratio of 1: 3 to 1: 10, preferably 1: 5.
  • freeze-dried powder of the mixture of glucose oxidase and catalase of the present invention, and the mixture of NADP + and ATP can be prepared by freeze-drying techniques known in the art.
  • a method for preparing a lyophilized powder of a mixture of glucose oxidase and catalase is as follows: take glucose oxidase with an activity of 40KU and catalase with an activity of 5000KU in 10 ml of aseptic In a test tube, 6 mL of sterile water was added to dissolve the whole, and 100 ⁇ L was pipetted into 50 4 mL brown reagent bottles with a dispenser, and placed in a lyophilizer. After freezing at a temperature of 20 ° C to -35 ° C, it is dried for 24 to 72 hours by sublimation. The lyophilized enzyme reagent was sealed and stored in a refrigerator at -20 ° C.
  • a method for preparing a lyophilized powder of a mixture of NADP + and ATP is as follows: Weigh 3000 mg of 5'-adenosine triphosphate disodium salt and 600 mg of nicotinamide adenine dinucleotide phosphate di Sodium salt was placed in a 10 mL sterile test tube, 6 mL of sterile water was added to dissolve the whole, 100 ⁇ L was pipetted into a 50 mL 4 mL brown reagent bottle using a dispenser, and placed in a lyophilizer under a low vacuum environment of 200 Torr , After the solution is frozen at a temperature of -20 ° C to -35 ° C, it is dried for 24-72 hours by sublimation. The lyophilized reagent was sealed and stored in a refrigerator at -20 ° C. When in use, dissolve it in 1mL of distilled water. Use polypropylene tube to separate and store in -20 °
  • the inventors of the present invention have found through research that after mixing glucose oxidase and catalase and lyophilizing them, in the resulting lyophilized powder, the activities of the two enzymes do not affect and interfere with each other.
  • a mixed enzyme solution is made. Will not affect and interfere with the activity of the two enzymes.
  • Making the two into a mixed reagent and adding it to the reaction system in one step can reduce the number of operations for adding reagents. Lyophilized powders of NADP + and ATP mixtures have the same technical advantages.
  • the kit further comprises a suspension of a mixture of hexokinase and glucose-6-phosphate dehydrogenase.
  • concentration of hexokinase is 200-350 U / mL
  • glucose-6 -The concentration of phosphate dehydrogenase is 200-350 U / mL.
  • the kit further includes a ⁇ -galactosidase suspension, and the concentration of ⁇ -galactosidase in the suspension is (1.5-3.0) ⁇ 10 3 U / mL.
  • the kit further comprises: a glucose phosphate isomerase solution, and the concentration of the glucose phosphate isomerase in the solution is 700-900 U / mL.
  • the suspension of the mixture of hexokinase and glucose-6-phosphate dehydrogenase, the ⁇ -galactosidase suspension and the glucoglucose isomerase solution can be in accordance with the enzymatic method-spectrophotometric method in NY / T939-2016 In the method for determining lactulose content, the corresponding solution is prepared.
  • the kit further includes: a buffer solution A and a buffer solution B, wherein the composition of the buffer solution A is: 48 g / L disodium hydrogen phosphate, 8.6 g / L sodium dihydrogen phosphate, and 1 g / L sulfuric acid Magnesium, pH 7.4 ⁇ 7.6.
  • the composition of the buffer B is: 140.0 g / L triethanolamine hydrochloride, 2.5 g / L magnesium sulfate, and pH 7.5 to 7.7.
  • the kit further includes: a zinc sulfate solution.
  • a zinc sulfate solution Preferably, the concentration of zinc sulfate in the zinc sulfate solution is 168 g / L.
  • the kit further comprises: a potassium ferrocyanide solution.
  • a potassium ferrocyanide solution Preferably, the concentration of potassium ferrocyanide in the potassium ferrocyanide solution is 130 g / L.
  • the kit further includes a fructose standard solution, and the concentration of fructose in the fructose standard solution is 50 ⁇ g / mL.
  • the kit further includes: 30% H 2 O 2 .
  • the kit further comprises: a 0.33M NaOH solution.
  • the kit further includes: octanol.
  • the kit further comprises: a microplate suitable for a microplate reader.
  • the micro-well plate may be various types of micro-well plates known in the art, such as a 96-well plate, a 24-well plate, a 48-well plate, a 256-well plate, and the like.
  • a 96-well plate is preferred from the standpoint of the flux of the sample and the suitability of the sample volume.
  • the kit includes the following reagents for a 96-well plate detection amount:
  • Reagents for sample preparation 168g / L zinc sulfate solution 5mL; 130g / L potassium ferrocyanide solution 5mL; buffer A 20mL; ⁇ -galactosidase suspension 1mL; buffer B 12mL; 0.33 M NaOH solution 5mL; octanol 2mL; lyophilized powder of a mixture of glucose oxidase and catalase; 30% H 2 O 2 2mL;
  • Reagents for detection lyophilized powder of NADP + and ATP mixture; 0.6mL suspension of hexokinase and glucose-6-phosphate dehydrogenase mixture; 0.6mL of phosphoglucose isomerase solution; 50 ⁇ g / mL of fructose standard solution 2mL .
  • the sample volume, the reagent volume used in the preferred or specific embodiment, and the amount of the reagent in the detection kit of the second aspect are based on a 96-well plate test sample.
  • the sample volume, reagent volume, and packaging volume in the kit are used in the examples.
  • Those skilled in the art can double or expand the sample volume or reagent used according to the specific number of wells and the volume of the microplate used in the specific test. Volume, kit manufacturers can also expand or reduce the packaging volume of reagents in the kit based on the number of wells and the volume of the microplate in the kit.
  • the present invention solves the technical problem of detecting lactulose content in liquid milk by using a microplate reader method by establishing a standard curve of fructose, and the microplate reader method has strong consistency of results compared with a standard spectrophotometric method. , Can replace the existing standard method.
  • a microtiter plate can be used as a detection carrier, which greatly reduces the amount of reagents and saves costs; moreover, the use of a microtiter plate improves the sample throughput and greatly improves the detection efficiency.
  • the reagent kit of the present invention provides a mixture preparation of several reagents in the reagents used for enzymatic determination, which reduces the number of steps for adding reagents in sample processing and determination, which not only simplifies sample processing, but also makes the determination more simple and convenient. It avoids the problem that the reaction difference caused by the time difference of adding reagents will ultimately affect the OD value.
  • FIG. 1 is a fructose standard curve for the quantitative detection of lactulose according to the present invention.
  • the chemical reagents used in the following examples are all conventional reagents and are commercially available.
  • the lyophilized powder of the mixture was prepared as a suspension with sterile water and used.
  • the lyophilized powder of the mixture was prepared into a solution with sterile water and used.
  • fructose standard substance dissolved in 1000 mL of sterile water, which is a standard fructose stock solution with a concentration of 500 ⁇ g / mL of fructose; pipette 100 mL of standard fructose stock solution into a 1000 mL volumetric flask, add 0.2 g of sodium azide and use Sterile and mix with sterile water, that is, a fructose standard solution with a fructose concentration of 50 ⁇ g / mL, and then aliquot it into a 4 mL brown reagent bottle.
  • lactulose standard substance dissolved in 100 mL of sterile water, which is a standard lactulose solution with a mass concentration of lactulose of 500 ⁇ g / mL, and then aliquot it into a 4 mL brown reagent bottle.
  • buffer solution C Take 6mL of buffer B and dilute to 15mL with distilled water.
  • Suspension of hexokinase and glucose-6-phosphate dehydrogenase A suspension of hexokinase and glucose-6-phosphate dehydrogenase with 3.2mol / L ammonium sulfate solution, hexokinase and glucose The concentration of -6-phosphate dehydrogenase is 260U / mL and 260U / mL, respectively.
  • Glucose phosphate isomerase suspension A 3.2 mol / L ammonium sulfate solution is used to make a glucose phosphate isomerase suspension at a concentration of 800 U / mL.
  • Double-distilled water was autoclaved at 121 ° C for 15 minutes, 30ml per bottle.
  • each kit can contain 3 bottles of sterile water. Used to prepare the above standard solution, enzyme solution, etc.
  • reagents include: 30% H 2 O 2 and octanol, which can be used as the stock solution purchased. When preparing the kit, these reagents can be directly dispensed according to the stock solution purchased.
  • Hydrolyzed lactose and lactulose Take each sample carefully and pipet the supernatant in two 500 ⁇ L each into 2 2 mL polypropylene microcapsules with screw caps. One of the tubes is filled with 200 ⁇ L of buffer A and ⁇ - 20 ⁇ L of galactosidase (2.0 ⁇ 10 3 U / mL) was used as a measurement sample; 200 ⁇ L of buffer A and 20 ⁇ L of distilled water were added to another tube as a blank control. After vortexing, mix in a water bath at 50 ° C for 1 h.
  • Glucose oxidation 200 ⁇ L of buffer C, 50 ⁇ L of 0.33M NaOH solution, 10 ⁇ L of octanol, glucose oxidase (4.0 ⁇ 10 3 U / mL) and catalase (5.0 ⁇ 10 5 U / mL) were added to each tube. ) 20 ⁇ L of the mixed solution and 10 ⁇ L of 30% H 2 O 2. Immediately cover with a tube cap and mix well. Incubate in a 40 ° C incubator or a water bath for 3 hours, and take it out at 5000 rpm for 10 minutes.
  • sample solution 100 ⁇ L of sample solution (blank control or measurement sample), 100 ⁇ L of distilled water, 20 ⁇ L of buffer B, NADP + (10 mg / mL), and ATP (50 mg / mL) mixed solution 10 ⁇ L, mix well and leave for 3min;
  • the OD value (A1) of the solution was read at 340 nm on a microplate reader; glucose glucose isomerase (800 U / mL) After 5 ⁇ L, mix and incubate in a 37 ° C incubator for 15 min, take it out on a microplate reader at 340 nm and read the solution OD value (A2).
  • A2-A1 as the ordinate and fructose mass ( ⁇ g) / well as the abscissa to draw a standard curve or calculate a regression equation.
  • the standard curve diagram is shown in FIG. 1.
  • V 1 volume of sucked milk sample, 0.50mL
  • V 2 volume after precipitation centrifugation, 2.00 mL
  • V 3 suction the volume of the supernatant after centrifugation, 0.50 mL;
  • V 4 volume after glucose oxidation, 1.01 mL
  • V 5 the volume of colorimetric measurement by sucking the sample solution, 0.10mL;
  • the conversion coefficient f 153.52 is calculated.
  • Liquid milk was used as the matrix for the standard addition, and the same detection method as in Example 2 was used to verify the recovery rate of the measurement method of the present invention.
  • the results are shown in Table 2:
  • fructose as the standard curve can eliminate the enzymatic hydrolysis process such as galactosidase, glucose oxidase and catalase, greatly simplifying the operation steps, reducing the use of detection reagents, and saving costs.
  • Example 5 Comparison between the microplate reader method and the spectrophotometric method
  • the microplate reader detection method of the present invention and the standard spectrophotometric method have a good consistency.

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Abstract

本发明提供一种酶标仪酶法定量检测乳果糖的方法及其试剂盒。本发明采用果糖建立标准曲线，解决了酶标仪检测中无法定量的技术问题，使检测方法因采用酶标仪而极大的提高的检测效率，降低了检测试剂用量。另一方面，本发明通过将葡萄糖氧化酶与过氧化氢酶，NADP +与ATP分别按一定比例混合后进行冻干，将己糖激酶和葡萄糖-6-磷酸脱氢酶混合后制成悬浮液的处理方法进行检测试剂的制备，不仅不干扰检测反应及其效果，还减少了试剂添加步骤，简化了操作，提高了检测的准确性。

Description

一种用酶标仪酶法定量检测液态奶中乳果糖的方法及其试剂盒
本申请要求2018年7月3日向中国国家知识产权局提交的专利申请号为201810720180.9，发明名称为“一种用酶标仪酶法定量检测液态奶中乳果糖的方法及其试剂盒”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种采用酶标仪利用酶化学法定量测定液态奶中乳果糖的方法及其试剂盒，属于酶化学分析检测领域。
背景技术
乳果糖是乳糖在牛奶热处理过程中发生碱基异构的产物，与乳糖是同分异构体，是评估牛奶热处理程度的一项重要指标。欧盟和IDF均提议UHT灭菌乳的乳果糖含量不得超过600mg/L。因此，乳果糖的准确测定十分重要。
目前，国内外关于液态奶中乳果糖的检测方法都比较复杂，一般为高效液相色谱法和基于酶分析的分光光度法。其中高效液相色谱法因样品中乳糖含量高于乳果糖的数十倍，在分离中乳糖与乳果糖间很难达到基线分离，从而使定量检测乳果糖存在较大难度。基于酶分析的分光光度法则是采用标准摩尔吸光系数定量的方法(例如在NY/T 939-2016和ISO 11285-2004等标准中记载的酶分析分光光度法)，存在样品处理繁琐、测定时需采用比色皿分别进行测定、检测通量小、耗时长的问题，在大批量样品测定中该问题更为突出。另外，酶分析分光光度法，整个测定过程中使用的酶的种类较多，需分别进行配制，酶试 剂用量大，不但检测成本高，且酶活性难以保障，直接影响测定结果。随着近年来酶标仪测定技术的不断成熟，运用酶标仪提高检测通量，降低检测成本，可能成为测定乳果糖的一种新途径，但是如何去除乳糖的干扰，对乳果糖进行准确定量仍然是一道难题。
发明内容
针对目前使用的酶分析分光光度法定量检测液态奶中乳果糖，测定步骤繁琐、检测效率低、检测成本高，对操作人员技术要求高，须经较长时间的专门培训才可独立操作等缺点，开发了利用酶标仪酶法定量检测液态奶中乳果糖的方法，以及相应的检测试剂盒。
本发明所利用的酶化学原理，与标准的分光光度法测定液态奶中乳果糖含量所用的酶化学原理相同，故标准NY/T 939-2016全文引用在本发明中作为参考。
在采用酶标仪酶法定量检测液态奶中乳果糖含量的方法中，酶标仪无法采用分光光度计法的朗伯比尔定律定量的方法，需要建立新的定量手段。经过研究考察，本发明的发明人采用果糖为标准品，建立果糖的标准曲线，用于酶标仪酶法定量检测液态奶中乳果糖的含量，不仅解决了酶标仪法测量中无法定量的技术问题，还避免了采用其他物质为标准品，例如，以乳果糖为标准品，需经多个步骤酶解带来的方法学误差。
本发明的第一个方面是，提供一种酶标仪酶法定量检测液态奶中乳果糖的方法，其包括如下步骤：以果糖为标准品建立标准曲线。
根据本发明，所述标准曲线的建立步骤包括：在酶标仪用微孔板中加入果糖标准品溶液，使每孔中果糖质量依次呈等差增长或呈倍数增长，加入NADP +和ATP后混匀；再加入己糖激酶和葡萄糖-6-磷酸脱氢酶混匀后反应，在酶标仪上340nm处读取溶液OD值，记为A 1，再加入磷酸葡萄糖异构酶混匀后反应，在酶标仪上340nm读取溶液OD值，记为A 2；以(A 2-A 1)的值为净OD值，作为纵坐标，以每孔中果糖质量为横坐标，绘制标准曲线或计算回归方程。
优选，在微孔板中的4-10个孔中加入果糖标准品溶液。
优选，每孔中的果糖质量以μg为单位。
优选，每孔中的果糖质量依次呈等差增长，例如，以0、1.0、2.0、3.0、4.0、5.0、6.0μg/孔的间距增长，或以0、2.0、4.0、6.0μg/孔的间距增长。在本发明的一个具体实施方式中，在7个孔中加入果糖标准品溶液，使孔中果糖质量依次为0、1.0、2.0、3.0、4.0、5.0、6.0μg/孔。
优选，每孔中的果糖质量依次呈倍数增长，例如，可以是呈2倍、3倍、4倍等倍数增长。在本发明的一个具体实施方式中，在4个孔中加入果糖标准品溶液，使孔中果糖质量依次为0、1.0、2.0、4.0μg/孔。
优选，NADP +和ATP以NADP +和ATP的混合液形式加入，所述混合液中，NADP +的浓度为7-12mg/mL，ATP的浓度为40-60mg/mL。
优选，己糖激酶和葡萄糖-6-磷酸脱氢酶以己糖激酶和葡萄糖-6-磷酸脱氢酶的混合悬浮液的形式加入，所述悬浮液中己糖激酶的浓度为200-350U/mL，葡萄糖-6-磷酸脱氢酶的浓度为200-350U/mL。
优选，加入己糖激酶和葡萄糖-6-磷酸脱氢酶混匀后放于37℃下反应10min。
优选，磷酸葡萄糖异构酶溶液的浓度为700-900U/mL。
优选，加入磷酸葡萄糖异构酶混匀后于37℃下反应15min。
在本发明的一个具体实施方式中，所述果糖标准曲线的建立步骤为：在微孔板中加入果糖标准品溶液，使果糖质量分别为0、1.0、2.0、3.0、4.0、5.0、6.0μg/孔，加入缓冲液B(140.0g/L三乙醇胺盐酸盐、2.5g/L硫酸镁，pH 7.5～7.7)、NADP +和ATP混合液后混匀放置；再加入己糖激酶和葡萄糖-6-磷酸脱氢酶，混匀后放于37℃下反应10min后，在酶标仪上340nm处读取溶液OD值，记为A 1，再加入磷酸葡萄糖异构酶混匀放于37℃下反应15min后，在酶标仪上340nm读取溶液OD值，记为A 2；以(A 2-A 1)的值为净OD值，作为纵坐标，果糖质量(μg/孔)为横坐标，绘制标准曲线或计算回归方程。
根据本发明，所述检测方法还包括，检测奶样的(A 2-A 1)的值，根据果糖 标准曲线换算出奶样中乳果糖的含量。
根据本发明，所述换算公式为：
Figure PCTCN2018112496-appb-000001
式中：
X：样品中乳果糖含量，单位为mg/L；
C：由标准曲线或回归方程计算得到的奶样微孔中果糖含量，单位为μg；
f：转换系数；
V 1：检测开始时吸取的奶样体积，单位为mL，数值可以为0.3-1。在本发明的一个实施方式中，其数值为0.50，即检测开始时吸取的奶样体积为0.50mL。
V 2：样品前处理过程中沉淀样品中脂肪和蛋白质离心后的体积，单位为mL，数值可以为1.2-4，在本发明的一个实施方式中，其数值为2.00，即沉淀离心后的体积为2mL。
V 3：吸取离心后上清液的体积，单位为mL，数值可以为0.3-1，优选和V 1的数值相同。在本发明的一个实施方式中，其数值为0.50，即，吸取离心后上清液的体积为0.50mL。
V 4：葡萄糖氧化步骤后的试样液的总体积，单位为mL，数值可以为0.606-2.02，在本发明的一个实施方式中，其数值为1.01，即，葡萄糖氧化步骤后的试样液的总体积为1.01mL。
V 5：进行酶标仪测定时，加入微孔板中的试样液的体积，单位为mL，数值可以是0.06-0.2，在本发明的一个实施方式中，其数值为0.10，即，进行酶标仪测定时，加入微孔板中的试样液的体积为0.10mL。
M L：乳果糖的摩尔质量，342.3g/mol
M F：果糖的摩尔质量，180.16g/mol
所述转换系数f，根据检测过程中对应步骤液体的体积数，会发生相应变化，在本发明的一个实施方式中，根据对应步骤的液体体积数计算出的转换系数 f＝153.52。
本发明的检测方法，还进一步包括样品前处理步骤。
根据本发明，所述样品前处理步骤包括：采用硫酸锌溶液和亚铁氰化钾溶液沉淀液态奶样品中的蛋白与脂肪，离心后，取上清液，加入β-半乳糖苷酶溶液水解上清液中的乳糖和乳果糖，然后加入葡萄糖氧化酶溶液和过氧化氢酶溶液氧化葡萄糖。
优选，在氧化葡萄糖反应结束后进行离心，取上清液用于后续的样品检测。
在本发明中，为表述需要，经过样品前处理步骤后，即，在氧化葡萄糖步骤后，得到的液体称为“试样液”，包括待测奶样经过样品前处理步骤后得到的液体以及其进一步离心后的上清液，以区别果糖标准品溶液。
优选，所述硫酸锌溶液的浓度为168g/L。
优选，所述亚铁氰化钾溶液的浓度为130g/L。
优选，所述β-半乳糖苷酶溶液的浓度为(1.5-3.0)×10 3U/mL。
优选，β-半乳糖苷酶溶液水解上清液中的乳糖和乳果糖的反应条件为50℃培养箱或水浴1h。
优选，所述葡萄糖氧化酶溶液的浓度为(3.0-5.0)×10 3U/mL。
优选，所述过氧化氢酶溶液的浓度为(4.0-6.0)×10 5U/mL。
优选，所述葡萄糖氧化酶和过氧化氢酶制成混合酶溶液使用，其中葡萄糖氧化酶的浓度为(3.0-5.0)×10 3U/mL，过氧化氢酶的浓度为(4.0-6.0)×10 5U/mL。
优选，葡萄糖氧化酶溶液和过氧化氢酶溶液氧化葡萄糖的反应条件为40℃培养箱或水浴培养3h。
在本发明的一个具体实施方式中，样品前处理的操作方法为：吸取液态奶样品0.5mL，加入蒸馏水1.3mL、168g/L的硫酸锌溶液0.1mL、130g/L的亚铁氰化钾溶液0.1mL，混合，5000rpm离心10分钟。吸取上清液，分为三份，每份500μL，其中两份分别加入缓冲液A(48g/L磷酸氢二钠、8.6g/L磷酸二氢钠、1g/L硫酸镁，pH 7.4～7.6)200μL与β-半乳糖苷酶(2.0×10 3U/mL)20μL作为测定样品； 另一份中加入缓冲液A 200μL与蒸馏水20μL，作为空白对照，涡旋震荡混合后，50℃培养箱或水浴1h后，再分别加入缓冲液C(缓冲液B进行2.5倍稀释得到)200μL、0.33mol/L NaOH溶液50μL、辛醇10μL、葡萄糖氧化酶(4.0×10 3U/mL)和过氧化氢酶(5.0×10 5U/mL)混合液20μL、30％的H 2O 2 10μL，混匀，在40℃培养箱或水浴培养3h，取出于5000rpm离心10分钟。
本发明的检测方法，还进一步包括样品测定步骤。
根据本发明，所述样品测定步骤包括：在微孔板的微孔中，分别加入处理后的试样液(例如空白对照、测定样品)、NADP +和ATP，混匀后，加入己糖激酶和葡萄糖-6-磷酸脱氢酶，混匀后反应后，在酶标仪上340nm处读取溶液OD值，记为A 1；再加入磷酸葡萄糖异构酶，混匀后反应后，在酶标仪上340nm处读取溶液OD值，记为A 2。
优选，NADP +和ATP以NADP +和ATP的混合液形式加入，所述混合液中，NADP +的浓度为7-12mg/mL，ATP的浓度为40-60mg/mL。
优选，己糖激酶和葡萄糖-6-磷酸脱氢酶以己糖激酶和葡萄糖-6-磷酸脱氢酶的混合悬浮液的形式加入，所述悬浮液中己糖激酶的浓度为200-350U/mL，葡萄糖-6-磷酸脱氢酶的浓度为200-350U/mL。
优选，加入己糖激酶和葡萄糖-6-磷酸脱氢酶混匀后，在37℃下反应10min。
优选，所述磷酸葡萄糖异构酶溶液的浓度为700-900U/mL。
优选，加入磷酸葡萄糖异构酶混匀后，在37℃下反应15min。
优选，所述样品测定步骤包括：在微孔板的微孔中，分别加入处理后的试样液(例如空白对照、测定样品)、蒸馏水、缓冲液B(140.0g/L三乙醇胺盐酸盐、2.5g/L硫酸镁，pH 7.5～7.7)、NADP +和ATP，混匀后，加入己糖激酶和葡萄糖-6-磷酸脱氢酶，混匀后37℃下反应10min后，在酶标仪上340nm处读取溶液OD值，记为A 1；再加入磷酸葡萄糖异构酶，混匀后37℃下反应15min后，在酶标仪上340nm处读取溶液OD值，记为A 2。
在本发明的一个具体实施方式中，样品测定方法为：取96孔微孔板，在每 个微孔中分别加入下列物质：样液(例如空白对照、测定样品)100μL、蒸馏水100μL、缓冲液B 20μL、NADP +(10mg/mL)和ATP(50mg/mL)混合液10μL后混匀，放置3min；再加入己糖激酶和葡萄糖-6-磷酸脱氢酶悬浮液(各为260U/mL)5μL，混匀后放于37℃培养箱中培养10min，取出在酶标仪上340nm处读取溶液OD值(A1)；再加入磷酸葡萄糖异构酶(800U/mL)5μL后混匀放于37℃培养箱中培养15min，取出在酶标仪上340nm读取溶液OD值(A2)；(A2-A1)即为净OD值。
本发明检测方法的检测限为液态奶中乳果糖含量为4.6mg/L。
按照酶法检测反应，本发明检测方法的线性检测范围为0.03～6μg果糖，相当于按照标准检测过程，可测定液态奶中乳果糖含量范围为4.6～920mg/L。
由于酶法检测液态奶中乳果糖的方法，所涉及的酶以及试剂很多，为了提高试剂添加操作的便宜性，本发明的发明人通过分析酶法反应过程的原理，研究各试剂之间的配伍适应性和禁忌，提出了用于酶法检测液态奶中乳果糖的检测试剂组合以及相应的试剂盒，所述试剂组合也适用于前述酶标仪法定量检测液态奶中乳果糖的含量，可减少试剂添加的操作次数，提高工作效率，降低样品间因试剂添加时间差异导致的结果差异。
本发明的第二个方面是，提供一种酶法检测液态奶中乳果糖的检测试剂盒，所述试剂盒包括：葡萄糖氧化酶与过氧化氢酶的混合物冻干粉，NADP +与ATP的混合物冻干粉。
根据本发明，葡萄糖氧化酶与过氧化氢酶的混合物冻干粉中，葡萄糖氧化酶和过氧化氢酶的活性比为1:80～1:600，优选为1:125。
根据本发明，NADP +与ATP的混合物冻干粉中，NADP +与ATP的质量比为1:3～1:10，优选为1:5。
可采用本领域已知的冷冻干燥技术制备本发明的葡萄糖氧化酶与过氧化氢酶的混合物冻干粉，以及NADP +与ATP的混合物冻干粉。
在本发明的一个具体实施方式中，葡萄糖氧化酶和过氧化氢酶的混合物冻 干粉的制备方法如下：取活性为40KU的葡萄糖氧化酶与活性为5000KU的过氧化氢酶于10毫升无菌试管中，加入6mL无菌水使其全部溶解，用分液器吸取100μL分装于50个4mL棕色试剂瓶中，放于冻干机中，在200Torr的低真空环境下，使酶溶液在-20℃～-35℃的温度条件下冷冻后再以升华的方式干燥24～72h。将冻干后的酶试剂密封后放于-20℃冰箱中保存。
在本发明的一个具体实施方式中，NADP +和ATP混合物的冻干粉的制备方法如下：分别称取3000mg 5’-腺苷三磷酸二钠盐和600mg烟酰胺腺嘌呤二核苷酸磷酸二钠盐于10mL无菌试管中，加入6mL无菌水使其全部溶解，用分液器吸取100μL分装于50个4mL棕色试剂瓶中，放于冻干机中，在200Torr的低真空环境下，使溶液在-20℃～-35℃的温度条件下冷冻后再以升华的方式干燥24-72h。将冻干后的试剂密封后放于-20℃冰箱中保存。使用时，用1mL蒸馏水溶解，可用聚丙烯管分装后于-20℃冰箱中保存。
本发明的发明人通过研究发现，将葡萄糖氧化酶和过氧化氢酶混合后冻干，所得冻干粉中，两种酶的活性没有相互的影响和干扰，使用时制成混合酶溶液，也不会对两种酶的活性产生影响和干扰。而将两者制成混合试剂，一步加入反应体系中，可减少试剂添加的操作次数。NADP +和ATP混合物的冻干粉也具有同样的技术优点。
根据本发明，所述试剂盒中还包括己糖激酶和葡萄糖-6-磷酸脱氢酶的混合物的悬浮液，所述悬浮液中，己糖激酶的浓度为200-350U/mL，葡萄糖-6-磷酸脱氢酶的浓度为200-350U/mL。
根据本发明，所述试剂盒中还包括：β-半乳糖苷酶悬浮液，所述悬浮液中，β-半乳糖苷酶的浓度为(1.5-3.0)×10 3U/mL。
根据本发明，所述试剂盒中还包括：磷酸葡萄糖异构酶溶液，所述溶液中，磷酸葡萄糖异构酶的浓度为700-900U/mL。
所述己糖激酶和葡萄糖-6-磷酸脱氢酶的混合物的悬浮液，β-半乳糖苷酶悬浮液和磷酸葡萄糖异构酶溶液，可以按照NY/T939-2016中酶法-分光光度法乳果 糖含量测定方法中对应溶液的配制方法进行配制。
根据本发明，所述试剂盒中还包括：缓冲液A和缓冲液B，其中，缓冲液A的组成为：48g/L磷酸氢二钠、8.6g/L磷酸二氢钠、1g/L硫酸镁，pH 7.4～7.6。缓冲液B的组成为：140.0g/L三乙醇胺盐酸盐、2.5g/L硫酸镁，pH 7.5～7.7。
根据本发明，所述试剂盒中还包括：硫酸锌溶液。优选所述硫酸锌溶液中硫酸锌的浓度为168g/L。
根据本发明，所述试剂盒中还包括：亚铁氰化钾溶液。优选所述亚铁氰化钾溶液中亚铁氰化钾的浓度为130g/L。
根据本发明，所述试剂盒中还包括：果糖标准溶液，所述果糖标准溶液中果糖的浓度为50μg/mL。
根据本发明，所述试剂盒中还包括：30％H 2O 2。
根据本发明，所述试剂盒中还包括：0.33M的NaOH溶液。
根据本发明，所述试剂盒中还包括：辛醇。
根据本发明，所述试剂盒中还包括：适用于酶标仪的微孔板。所述微孔板可以是本领域已知的各类微孔板，例如96孔板，24孔板，48孔板，256孔板等。从检测样品的通量以及样品体积的适宜性角度出发，优选96孔板。
在本发明的一个具体实施方式中，所述试剂盒包括：用于一份96孔板检测用量的如下的上述试剂：
1)样品前处理用试剂：168g/L的硫酸锌溶液5mL；130g/L的亚铁氰化钾溶液5mL；缓冲液A 20mL；β-半乳糖苷酶悬浮液1mL；缓冲液B 12mL；0.33M的NaOH溶液5mL；辛醇2mL；葡萄糖氧化酶和过氧化氢酶的混合物冻干粉；30％的H 2O 2 2mL；
2)检测用试剂：NADP +和ATP混合物冻干粉；己糖激酶和葡萄糖-6-磷酸脱氢酶混合物的悬浮液0.6mL；磷酸葡萄糖异构酶溶液0.6mL；果糖标准溶液50μg/mL 2mL。
在本发明第一方面的检测方法中，优选的或者具体的实施方式中所采用的 样品体积、试剂体积，以及第二方面的检测试剂盒中试剂的量，都是以96孔板检测样品为例所使用的样品体积、试剂体积以及试剂盒中的包装体积，本领域技术人员可以根据具体检测时使用的微孔板的具体孔数和孔容积，成倍扩大或者缩小所用的样品体积或者试剂体积，试剂盒厂家也可以根据配套试剂盒中的微孔板的孔数及孔容积，来成倍扩大或缩小试剂盒中试剂的包装体积。
本发明的优点：
1、本发明通过建立果糖标准曲线，解决了利用酶标仪法进行酶法检测液态奶中乳果糖含量的技术问题，并且酶标仪检测法，与标准的分光光度法相比，结果一致性强，可以替代现有的标准方法。
2、由于使用了酶标仪，可采用微孔板作为检测载体，大大降低了试剂用量，节约了成本；并且，使用微孔板提高了检测的样品通量，大大提高了检测效率。
3、本发明的试剂盒中提供了酶法测定所用试剂中的数种试剂的混合物制剂，使得样品处理及测定中添加试剂的步骤减少，不但简化了样品处理，而且其测定更为简单方便，避免了因加入试剂的时间差导致的反应差异，最终影响OD值的问题。
附图说明
图1为本发明所述的用于酶法定量检测乳果糖的检测用果糖标准曲线。
具体实施方式
以下结合实施例对本发明做进一步描述。需要说明的是，实施例不能作为对本发明保护范围的限制，本领域的技术人员理解，任何在本发明基础上所作的改进和变化都在本发明的保护范围之内。
以下实施例所用化学试剂都是常规试剂，均可商购获得。
实施例1检测用试剂的配制
1.葡萄糖氧化酶和过氧化氢酶的混合物冻干粉，NADP +和ATP的混合物冻干粉的制备
(1)葡萄糖氧化酶和过氧化氢酶的混合物冻干粉的制备：取活性为40KU的葡萄糖氧化酶与活性为5000KU的过氧化氢酶于10毫升无菌试管中，加入6mL无菌水使其全部溶解，立即用分液器吸取100μL分装于50个4mL棕色试剂瓶中，放于冻干机中，在200Torr的低真空环境下，使酶溶液在-20℃～-35℃的温度条件下冷冻后再以升华的方式干燥24～72h。将冻干后的酶试剂密封后放于-20℃冰箱中保存。
检测前，将所述混合物冻干粉用无菌水制备成悬浮液使用。
(2)NADP +和ATP混合物冻干粉的制备：分别称取3000mg 5’-腺苷三磷酸二钠盐和600mg烟酰胺腺嘌呤二核苷酸磷酸二钠盐于10mL无菌试管中，加入6mL无菌水使其全部溶解，立即用分液器吸取100μL分装于50个4mL棕色试剂瓶中，放于冻干机中，在200Torr的低真空环境下，使溶液在-20℃～-35℃的温度条件下冷冻后再以升华的方式干燥24-72h。将冻干后的试剂密封后放于-20℃冰箱中保存。
检测前，将所述混合物冻干粉用无菌水制备成溶液使用。
2.果糖标准溶液的制备
准确称取果糖标准物质500mg溶于1000mL无菌水中，即为果糖质量浓度为500μg/mL的标准果糖储备液；吸取标准果糖储备液100mL于1000mL容量瓶中，加入0.2g叠氮化钠后用无菌水定容混匀，即为果糖质量浓度为50μg/mL的果糖标准溶液，然后分装于4mL棕色试剂瓶中。
3.乳果糖标准溶液的制备
准确称取乳果糖标准物质50mg溶于100mL无菌水中，即为乳果糖质量浓度为500μg/mL的标准乳果糖溶液，然后分装于4mL棕色试剂瓶中。
4.硫酸锌溶液(168g/L)、亚铁氰化钾溶液(130g/L)、氢氧化钠溶液(0.33mol/L)的制备
称取168g硫酸锌溶于800mL水中，定容至1L。按5mL/瓶分装于8mL的棕色塑料瓶中。
称取130g亚铁氰化钾溶于800mL水中，定容至1L。按5mL/瓶分装于8mL的棕色塑料瓶中。
称取13.2g氢氧化钠溶于800mL水中，定容至1L。按5mL/瓶分装于8mL的棕色塑料瓶中。
5.缓冲液A(pH为7.5)的制备：
称48g磷酸氢二钠、8.6g磷酸二氢钠和1g硫酸镁溶解于800mL水中，用1mol/L氢氧化钠溶液调整pH到7.5±0.1(20℃)，再定容到1L。按20mL/瓶分装于30mL的玻璃试剂瓶中。
6.缓冲液B(pH为7.6)的制备：
称取140.0g三乙醇胺盐酸盐和2.5g硫酸镁溶解于800mL水中，用1mol/L氢氧化钠溶液调整pH到7.6±0.1(20℃)，再定容到1L。按12mL/瓶分装于30mL的玻璃试剂瓶中。
7.缓冲溶液C的制备：取缓冲液B 6mL，用蒸馏水稀释至15mL。
8.β-半乳糖苷酶悬浮液的制备：用3.2mol/L的硫酸铵溶液将β-半乳糖苷酶制备成浓度为2.0×10 3U/mL的悬浮液。
9.己糖激酶和葡萄糖-6-磷酸脱氢酶的悬浮液：用3.2mol/L的硫酸铵溶液将己糖激酶和葡萄糖-6-磷酸脱氢酶制成悬浮液，己糖激酶和葡萄糖-6-磷酸脱氢酶的浓度分别为260U/mL和260U/mL。
10.磷酸葡萄糖异构酶悬浮液：用3.2mol/L的硫酸铵溶液将磷酸葡萄糖异构酶制成浓度为800U/mL的悬浮液。
11.无菌水制备
将双蒸水在121℃高压灭菌15分钟，每瓶30ml。
在制备试剂盒时，每个试剂盒中可以包含3瓶无菌水。用于配制上述标准溶液、酶溶液等。
12.其它
其他试剂包括：30％H 2O 2、辛醇，可按购买的原液使用。在制备试剂盒时，这些试剂可以按照购买的原液进行直接分装。
实施例2灭菌乳中乳果糖的测定
1)样品处理：
吸取奶样0.5mL于2mL的聚丙烯离心管，分别加入蒸馏水1.3mL、168g/L硫酸锌溶液0.1mL、130g/L亚铁氰化钾溶液0.1mL，扣紧盖后涡旋震荡混合，5000rpm离心10分钟。
水解乳糖和乳果糖：取出每个样品分别小心吸取上清液两份，每份500μL于2个2mL的聚丙烯带螺帽的微管中，其中一个管中分别加入缓冲液A 200μL与β-半乳糖苷酶(2.0×10 3U/mL)20μL作为测定样品；另一管中分别加入缓冲液A200μL与蒸馏水20μL，作为空白对照。涡旋震荡混合后，50℃水浴1h。
葡萄糖氧化：在每个管中分别加入缓冲液C 200μL、0.33M NaOH溶液50μL、辛醇10μL、葡萄糖氧化酶(4.0×10 3U/mL)和过氧化氢酶(5.0×10 5U/mL)混合液20μL、30％H 2O 2 10μL，立刻盖上管帽混匀，在40℃培养箱或水浴中培养3h，取出于5000rpm离心10分钟。
2)样品测定：
取96孔微孔板，在每个微孔中分别加入下列物质：试样液(空白对照或测定样品)100μL、蒸馏水100μL、缓冲液B 20μL、NADP +(10mg/mL)和ATP(50mg/mL)混合液10μL后混匀，放置3min；
加入己糖激酶和葡萄糖-6-磷酸脱氢酶悬浮液(两者的活性浓度均为260U/mL)5μL，混匀后放于37℃培养箱中培养10min，取出在酶标仪上340nm读取溶液OD值(A1)；
加入磷酸葡萄糖异构酶(800U/mL)5μL后混匀放于37℃培养箱中培养15min，取出在酶标仪上340nm读取溶液OD值(A2)；(A2-A1)即为净OD值。 在进行标准曲线对比时，可以以测定样品的净OD值和空白对照的净OD值之差为对象，从标准曲线上对应出相应的果糖质量；也可以分别以测定样品的净OD值和空白对照的净OD值为对象，从标准曲线上对应出相应的果糖质量，再将两者的果糖质量相减。
3)标准曲线绘制：
分别吸取果糖标准溶液0、20、40、60、80、100、120μL于微孔中，用蒸馏水分别补充至200μL，果糖质量分别为0、1.0、2.0、3.0、4.0、5.0、6.0μg/孔。再加入缓冲液B 20μL、NADP +(10mg/mL)和ATP(50mg/mL)混合液10μL后混匀，放置3min；加入己糖激酶和葡萄糖-6-磷酸脱氢酶悬浮液(两者的活性浓度均为260U/mL)5μL，混匀后放于37℃培养箱中培养10min，取出在酶标仪上340nm读取溶液OD值(A1)；加入磷酸葡萄糖异构酶(800U/mL)5μL后混匀放于37℃培养箱中培养15min，取出在酶标仪上340nm读取溶液OD值(A2)。以(A2-A1)为纵坐标，果糖质量(μg)/孔为横坐标绘制标准曲线或计算回归方程。所述标准曲线图见附图1。
对同一灭菌乳样品进行6次同步实验，根据标准曲线和计算公式，获得样品中乳果糖的含量结果如表1。
Figure PCTCN2018112496-appb-000002
式中：
X：样品中乳果糖含量，单位为mg/L
c：由标准曲线或回归方程计算得到的微孔中果糖含量，单位为μg
f：转换系数
V 1：吸取奶样的体积，0.50mL；
V 2：沉淀离心后的体积，2.00mL；
V 3：吸取离心后上清液的体积，0.50mL；
V 4：葡萄糖氧化后的体积，1.01mL；
V 5：吸取试样液进行比色测定的体积，0.10mL；
M L—乳果糖的摩尔质量，342.3g/mol；
M F—果糖的摩尔质量，180.16g/mol；
计算得到转换系数f＝153.52。
表1同一种灭菌乳中乳果糖含量的测定
序号 净OD值 乳果糖含量(mg/L)
1 0.425 502
2 0.431 509
3 0.447 528
4 0.435 514
5 0.419 495
6 0.428 505
由表1结果计算得到的相对标准偏差RSD＝2.24％
由上述可得，相对标准偏差为2.24％，则表明其重现性良好。
实施例3测定方法的回收率
利用液态奶作为基质进行加标，采用实施例2相同的检测方法，检验本发明测定方法的回收率，结果如表2所示：
表2回收率测定结果
Figure PCTCN2018112496-appb-000003
实施例4用乳果糖绘制标准曲线和用果糖绘制标准曲线结果对比
1)果糖标准曲线绘制：分别吸取果糖标准溶液0、20、40、60、80、100、120μL于微孔中，用蒸馏水分别补充至200μL，果糖质量分别为0、1.0、2.0、3.0、 4.0、5.0、6.0μg/孔。再加入缓冲液B 20μL、NADP +(10mg/mL)和ATP(50mg/mL)混合液10μL后混匀，放置3min；加入己糖激酶和葡萄糖-6-磷酸脱氢酶悬浮液(均为260U/mL)5μL，混匀后放于37℃培养箱中培养10min，取出在酶标仪上340nm读取溶液OD值(A1)；加入磷酸葡萄糖异构酶(800U/mL)5μL后混匀放于37℃培养箱中培养15min，取出在酶标仪上340nm读取溶液OD值(A2)。以(A2-A1)为纵坐标，果糖质量(μg)/孔为横坐标绘制标准曲线或计算回归方程。
2)乳果糖标准曲线绘制：分别吸取乳果糖标准溶液0、40、80、120、160、200、240μL于2mL的聚丙烯带螺帽的微管中，用蒸馏水分别补充至500μL，乳果糖质量分别为0、20.0、40.0、60.0、80.0、100.0、120.0μg/管。分别加入缓冲液A 200μL与β-半乳糖苷酶(2.0×10 3U/mL)20μL，涡旋震荡混合后，50℃水浴1h。在每个管中分别加入缓冲液C 200μL、0.33M NaOH溶液100μL、辛醇10μL、葡萄糖氧化酶(4.0×10 3U/mL)和过氧化氢酶(5.0×10 5U/mL)混合液20μL、30％H 2O 210μL，立刻盖上管帽混匀，在40℃培养箱或水浴中培养3h，取出于5000rpm离心10分钟。取96孔微孔板，在每个微孔中分别加入下列物质：上述离心后的标准样液100μL、蒸馏水100μL、缓冲液B 20μL、NADP +(10mg/mL)和ATP(50mg/mL)混合液10μL后混匀，放置3min；加入己糖激酶和葡萄糖-6-磷酸脱氢酶悬浮液(均为260U/mL)5μL，混匀后放于37℃培养箱中培养10min，取出在酶标仪上340nm读取溶液OD值(A1)；加入磷酸葡萄糖异构酶(800U/mL)5μL后混匀放于37℃培养箱中培养15min，取出在酶标仪上340nm读取溶液OD值(A2)；(A2-A1)即为净OD值。以(A2-A1)为纵坐标，乳果糖质量(μg)/孔为横坐标绘制标准曲线或计算回归方程。
3)样品测定：同实施例2。
选取7个液态奶样品，分别采用乳果糖绘制标准曲线和果糖绘制标准曲线进行定量，结果对比如下表3。
表3乳果糖绘制标准曲线和果糖绘制标准曲线定量结果对比
样品编号 乳果糖标准曲线 果糖标准曲线 相对相差(％)
1 807.1 780.6 3.3
2 785.3 743.5 5.5
3 567.7 536.0 5.8
4 605.4 583.7 3.6
5 664.4 687.4 3.4
6 483.8 442.9 8.8
7 387.7 388.3 0.2
由上表可见，本发明用果糖绘制标准曲线和用乳果糖绘制标准曲线的定量结果一致性很好。但是用果糖做标准曲线，可以省去半乳糖苷酶、葡萄糖氧化酶和过氧化氢酶等酶解过程，大大简化了操作步骤，减少了检测试剂使用，节约了成本。
实施例5酶标仪法与分光光度法的对比
选取7个液态奶样品，分别采用实施例2的酶标仪法和NY/T 939-2016的酶法-分光光度法进行检测，结果如下表：
样品编号 酶标仪法 酶法-分光光度计法 相对相差(％)
1 710.1 673.4 5.3
2 654.8 631.6 3.6
3 670.4 655.3 2.3
4 560.8 513.3 8.9
5 438.3 398.7 9.5
6 484.7 446.4 8.2
7 822.6 821.3 0.2
由上表可见，本发明的酶标仪检测法和标准的分光光度法的一致性很好。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

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  1. 一种酶标仪酶法定量检测液态奶中乳果糖的方法，其包括如下步骤：以果糖为标准品建立标准曲线，所述标准曲线的建立步骤包括：在酶标仪用微孔板中加入果糖标准品溶液，使每孔中果糖质量依次呈等差增长或呈倍数增长，加入NADP +和ATP后混匀；再加入己糖激酶和葡萄糖-6-磷酸脱氢酶混匀后反应，在酶标仪上340nm处读取溶液OD值，记为A1，再加入磷酸葡萄糖异构酶混匀后反应，在酶标仪上340nm读取溶液OD值，记为A2；以(A2-A1)的值为净OD值，作为纵坐标，以每孔中果糖质量为横坐标，绘制标准曲线或计算回归方程；
    优选，在微孔板中的4-10个孔中加入果糖标准品溶液；
    优选，每孔中的果糖质量以μg为单位；
    优选，每孔中的果糖质量依次呈等差增长；更优选，孔中果糖质量依次为0、1.0、2.0、3.0、4.0、5.0、6.0μg/孔；
    优选，每孔中的果糖质量依次呈倍数增长。
  2. 如权利要求1所述的方法，其特征在于，还包括检测奶样的(A2-A1)的值，根据果糖标准曲线换算出奶样中乳果糖的含量，所述换算公式为：
    Figure PCTCN2018112496-appb-100001
    式中：
    X：样品中乳果糖含量，单位为mg/L；
    C：由标准曲线或回归方程计算得到的奶样微孔中果糖含量，单位为μg；
    f：转换系数；
    V 1：检测开始时吸取的奶样体积，单位为mL；
    V 2：样品前处理过程中沉淀样品中脂肪和蛋白质离心后的体积，单位为mL；
    V 3：吸取离心后上清液的体积，单位为mL；
    V 4：葡萄糖氧化步骤后的试样液的总体积，单位为mL；
    V 5：进行酶标仪测定时，加入微孔板中的试样液的体积，单位为mL；
    M L：乳果糖的摩尔质量，342.3g/mol
    M F：果糖的摩尔质量，180.16g/mol。
  3. 如权利要求1-2任一项所述的方法，其特征在于，进一步包括样品前处理步骤，所述样品前处理步骤包括：采用硫酸锌溶液和亚铁氰化钾溶液沉淀液态奶样品中的蛋白与脂肪，离心后，取上清液，加入β-半乳糖苷酶溶液水解上清液中的乳糖和乳果糖，然后加入葡萄糖氧化酶溶液和过氧化氢酶溶液氧化葡萄糖；
    优选，所述硫酸锌溶液的浓度为168g/L；
    优选，所述亚铁氰化钾溶液的浓度为130g/L；
    优选，所述β-半乳糖苷酶溶液的浓度为(1.5-3.0)×10 3U/mL；
    优选，β-半乳糖苷酶溶液水解上清液中的乳糖和乳果糖的反应条件为50℃培养箱或水浴1h；
    优选，所述葡萄糖氧化酶溶液的浓度为(3.0-5.0)×10 3U/mL；
    优选，所述过氧化氢酶溶液的浓度为(4.0-6.0)×10 5U/mL；
    优选，所述葡萄糖氧化酶和过氧化氢酶制成混合酶溶液使用，其中葡萄糖氧化酶的浓度为(3.0-5.0)×10 3U/mL，过氧化氢酶的浓度为(4.0-6.0)×10 5U/mL；
    优选，葡萄糖氧化酶溶液和过氧化氢酶溶液氧化葡萄糖的反应条件为40℃培养箱或水浴培养3h。
  4. 如权利要求1-3任一项所述的方法，其特征在于，进一步包括样品测定步骤，所述样品测定步骤包括：在微孔板的微孔中，分别加入处理后的试样液、NADP +和ATP混匀后，加入己糖激酶和葡萄糖-6-磷酸脱氢酶，混匀后反应，在酶标仪上340nm处读取溶液OD值，记为A1；再加入磷酸葡萄糖异构酶，混匀后反应后，在酶标仪上340nm处读取溶液OD值，记为A2。
  5. 如权利要求1-4任一项所述的方法，其特征在于，所述NADP +和ATP以NADP +和ATP的混合液形式加入，所述混合液中，NADP +的浓度为7-12mg/mL，ATP的浓度为40-60mg/mL；
    优选，己糖激酶和葡萄糖-6-磷酸脱氢酶以己糖激酶和葡萄糖-6-磷酸脱氢酶的混合悬浮液的形式加入，所述悬浮液中己糖激酶的浓度为200-350U/mL，葡萄糖-6-磷酸脱氢酶的浓度为200-350U/mL；
    优选，加入己糖激酶和葡萄糖-6-磷酸脱氢酶混匀后，在37℃下反应10min；
    优选，所述磷酸葡萄糖异构酶溶液的浓度为700-900U/mL；
    优选，加入磷酸葡萄糖异构酶混匀后，在37℃下反应15min。
  6. 一种酶法定量检测液态奶中乳果糖含量的检测试剂盒，所述试剂盒包括：葡萄糖氧化酶与过氧化氢酶的混合物冻干粉，NADP +与ATP的混合物冻干粉；
    优选，所述葡萄糖氧化酶与过氧化氢酶的混合物冻干粉中，葡萄糖氧化酶和过氧化氢酶的活性比为1:80～1:600，优选为1:125；
    优选，所述NADP +与ATP的混合物冻干粉中，NADP +与ATP的质量比为1:3～1:10，优选为1:5。
  7. 如权利要求6所述的检测试剂盒，其特征在于，所述试剂盒中还包括己糖激酶和葡萄糖-6-磷酸脱氢酶的混合物的悬浮液，所述悬浮液中，己糖激酶的浓度为200-350U/mL，葡萄糖-6-磷酸脱氢酶的浓度为200-350U/mL；
    优选，所述试剂盒中还包括：β-半乳糖苷酶悬浮液，所述悬浮液中，β-半乳糖苷酶的浓度为1.5×10 3-3.0×10 3U/mL；
    优选，所述试剂盒中还包括：磷酸葡萄糖异构酶溶液，所述溶液中，磷酸葡萄糖异构酶的浓度为700-900U/mL；
    优选，所述试剂盒中还包括：缓冲液A和缓冲液B，其中，缓冲液A的组成为：48g/L磷酸氢二钠、8.6g/L磷酸二氢钠、1g/L硫酸镁，pH7.4～7.6；缓冲液B的组成为：140.0g/L三乙醇胺盐酸盐、2.5g/L硫酸镁，pH7.5～7.7；
    优选，所述试剂盒中还包括：硫酸锌溶液；优选所述硫酸锌溶液中硫酸锌的浓度为168g/L；
    优选，所述试剂盒中还包括：亚铁氰化钾溶液；优选所述亚铁氰化钾溶液中亚铁氰化钾的浓度为130g/L；
    优选，所述试剂盒中还包括：果糖标准溶液，所述果糖标准溶液中果糖的浓度为50μg/mL；
    优选，所述试剂盒中还包括：30％H 2O 2；
    优选，所述试剂盒中还包括：0.33M的NaOH溶液；
    优选，所述试剂盒中还包括：辛醇；
    优选，所述试剂盒中还包括：适用于酶标仪的微孔板。
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CN104360012A (zh) * 2014-12-04 2015-02-18 福建农林大学 一种同时快速测定葡萄糖、果糖和总糖含量试剂盒及应用

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* Cited by examiner, † Cited by third party
Title
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