CN105784868B - The detection method of lactulose in a kind of milk - Google Patents
The detection method of lactulose in a kind of milk Download PDFInfo
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- CN105784868B CN105784868B CN201610223749.1A CN201610223749A CN105784868B CN 105784868 B CN105784868 B CN 105784868B CN 201610223749 A CN201610223749 A CN 201610223749A CN 105784868 B CN105784868 B CN 105784868B
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to a kind of detection method of lactulose in milk, including：Lactose in milk and lactulose are hydrolyzed into galactolipin, glucose and fructose by the reaction of β D galactosidases, add glucose oxidase and most of glucose is oxidized to gluconic acid, it is eventually adding catalase and removes hydrogen peroxide, obtains test fluid；Test fluid is analyzed using the ion chromatograph with ampere detector, obtains the content of lactulose.The method of the present invention can be accurate, sensitive, determines the content of lactulose in milk easy to operately.
Description
Technical field
The invention belongs to lactulose detection field, the detection method of lactulose in more particularly to a kind of milk.
Background technology
Lactulose is isomerization product of the lactose in milk heat treatment process, is when assessing sterilization of milk degree and storage
Between an important indicator, international dairy produce tissue (IDF) have been proposed that the concentration of lactulose as distinguish the dairy products be with bar
Family name's method is sterilized, superthermal processing and sterilized indicator, therefore the Accurate Determining of lactulose more seems increasingly important in milk.
Tradition on lactulose analysis method have chromatogram and enzyme analysis both approaches, but due in milk contain compared with
More lactose, lactulose can be influenceed so that both approaches are difficult to carry out accurate quantitative analysis.With chromatography of ions in recent years
The continuous maturation of technology, with ion chromatography carbohydrate become measure lactulose a kind of new approach, but lactose and
The separation of lactulose is still a difficult problem, and its requirement to conditions such as eluent concentrations is quite harsh.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of detection method of lactulose in milk, and this method can be accurate
Really, it is sensitive, the content of lactulose in milk is determined easy to operately.
The detection method of lactulose in a kind of milk of the present invention, including：
(1) after taking milk sample to be diluted with water, potassium ferrocyanide solution and solution of zinc sulfate are sequentially added, is stood, filtering,
Collect filtrate；Add beta-D-galactosidase (β-galactosidase), be capped after mixing, in 50~60 DEG C incubated 1~
2h；Then glucose oxidase (glucose oxidase, GOD), n-octyl alcohol, sodium hydroxide solution, hydrogen peroxide are sequentially added
And catalase, in 45~50 DEG C of incubated 3~4h；Constant volume after cooling, filter, purification, collect filtrate as prepare liquid；
(2) using the prepare liquid in the ion chromatograph analytical procedure (1) with ampere detector, containing for lactulose is obtained
Amount.
Milk sample in the step (1) for the additive of fresh cow milk or milk, (such as strengthen by high calcium milk, student nutrition
Milk etc.).
The concentration of potassium ferrocyanide solution in the step (1) is 200g/L, and the volume ratio with milk sample is 1:5；
The concentration of solution of zinc sulfate is 200g/L, and the volume ratio with milk sample is 1:5.
Buffer solution [0.34mol/L disodium hydrogen phosphates (Na is added after solution of zinc sulfate is added in the step (1)2HPO4)+
0.07mol/L sodium dihydrogen phosphates (NaH2PO4), pH=7.5] solution ph is adjusted to 7.5；Add beta-D-galactosidase constant temperature
Buffer solution [0.75mol/L triethanolamine hydrochlorides (C is added after culture6H15NO3HCl)+0.01mol/L magnesium sulfate (MgSO4·
7H2O), pH=7.6] solution ph is adjusted to 7.6.
The volume ratio of beta-D-galactosidase and filtrate in the step (1) is 0.1:5.
Glucose oxidase, sodium hydroxide solution, hydrogen peroxide, catalase and filtrate in the step (1)
Volume ratio is 0.1:0.5:0.05:0.1:5.
Filtering in the step (1) uses 0.45 μm of aqueous phase membrane filtration；Purification uses the small column purifications of C18.
In the step (2) ion chromatograph analysis condition be：
Detector：ED40 electrochemical detectors, reference electrode Ag/AgCl, Au electrode；
Chromatographic column：CarboPac PA10 chromatography columns；
Mobile phase：Ultra-pure water；
Flow velocity：1.0mL/min；
Leacheate：10mmol/L KOH；
Sampling volume：25μL.
The specification of the chromatographic column is：Length 250mm × internal diameter 4mm.
The content of lactulose in the step (2) uses quantified by external standard method.
Each difference is separated using carbohydrate chromatographic column CarboPac PA10 (4mm*250mm) in the present invention
The sugar of component.
The general principle that the present invention analyzes lactulose by ion chromatograph with ampere detector is：Breast in sample
After sugar and lactulose are hydrolyzed into galactolipin, glucose and fructose, pass through carbohydrate chromatogram post separation.It is anti-based on ligand exchange
Should, each hydroxyl carries a very weak negative electrical charge on glycan molecule, and the hydroxyl of institute's band can be gone on anomer carbon
Protonation, so as to take a very strong negative electrical charge.These negative electrical charges on glycan molecule and the metal ion on resin surface
Interaction between positive charge is retained sugar, and so as to reach separation, therefore the retention time of each component in the chromatography column is just
Difference, after certain column length, is just separated each other, is left chromatographic column in order and is entered detector, has reached several sugars
Efficiently separate, peak area quantification qualitative by its retention time on a column.By the fructose content measured, sample is deducted
The content of fructose in product background, fructose content caused by lactulose enzymolysis is can obtain, so as to which lactulose in milk sample be calculated
Content.
The present invention can effectively exclude albumen and fat to lactulose by the use of potassium ferrocyanide and solution of zinc sulfate as precipitating reagent
The interference of analysis.
The present invention uses enzymatic isolation method by substantial amounts of lactose and least a portion of lactulose complete hydrolysis, whole pre-treatment in milk
Process solved well due to lactose too high levels using the method and it is caused can not be complete by lactose and lactulose
A fully separating problem.
In the present invention, separated using carbohydrate chromatographic column, pass through the ion color with ampere detector
Spectrometer analyzes lactulose content.The hydrolysate of lactulose and lactose is glucose, galactolipin and fructose, and these three sugar exist
Separating degree is preferable in CarboPac PA10 carbohydrate chromatographic columns.1.0mg/L newborn fruit is added in milk blank sample
Standard for Sugars material, through sample pre-treatments, after Instrument measuring, the signal to noise ratio (S/N) of measured object is more than 3 for 8.9, shows present invention side
The detection limit of method can be to 1.0mg/L.
Beneficial effect
(1) present invention is digested with beta-D-galactosidase, glucose oxidase and catalase to sample, can
Effectively lactose in sample and galactolipin are extracted completely, and can ensures that their enzymolysis product is kept completely separate and do not done mutually
Disturb；
(2) present invention carries out sugared detection with the ion chromatograph with ampere detector, and this method is at present more
Maturation, the separating degree and response of each component of sugar are all relatively good；
(3) present invention provides a kind of highly sensitive detection means for the detection of lactulose content in milk.
Brief description of the drawings
Fig. 1 is the chromatography of ions figure of Plays material of the present invention (lactulose, glucose, fructose, sucrose, lactose)；
Fig. 2 is the chromatography of ions figure of background (not enzyme-added) in embodiment 1；
Fig. 3 is the chromatography of ions figure of background (after enzyme-added hydrolysis process) in embodiment 1；
Fig. 4 is the chromatography of ions figure of sample mark-on in embodiment 1 (mark-on level is 5.0mg/L)；
Fig. 5 is the chromatography of ions figure of blank sample in embodiment 2；
Fig. 6 is the chromatography of ions figure of sample mark-on in embodiment 2 (mark-on level is 1.0mg/L).
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
Shaken up after adding lactulose standard liquid in the milk blank sample of weighing, the lactulose that the present embodiment is made adds
Standard specimen product.
(1) sample pre-treatments
10.00mL milk samples to be measured are pipetted in 150mL triangular flasks, sequentially add the ferrous iron that 2mL concentration is 200g/L
Potassium cyanide solution, the solution of zinc sulfate and 6.5mL buffer solutions [0.34mol/L disodium hydrogen phosphates that 2mL concentration is 200g/L
(Na2HPO4)+0.07mol/L sodium dihydrogen phosphates (NaH2PO4), pH=7.5], it is intended to fully shake up per reagent adding, all adds
After stand 30min, filter, discard primary filtrate, collect filtrate.
Filtrate 5mL is taken in 10mL volumetric flasks, 100 μ L beta-D-galactosidase suspension is added, shakes up, is capped,
Cultivated 1 hour at 50 DEG C.2.0mL buffer solutions [0.75mol/L triethanolamine hydrochlorides are sequentially added in cultured sample
(C6H15NO3HCl)+0.01mol/L magnesium sulfate (MgSO4·7H2O), pH=7.6], 100 μ L glucose oxidizing ferment suspension, 1
N-octyl alcohol, 0.5mL0.33mol/L sodium hydroxide solutions, 50 μ L hydrogen peroxide (30%) and 100 μ L catalases are dripped to suspend
Liquid.Often plus a kind of reagent is intended to shake up, and 3h is cultivated in 40 DEG C of water-baths or baking oven after all adding.Distilled water constant volume is used after cooling
To 10mL, after passing sequentially through 0.45 μm of aqueous phase filter membrane and the small column purifications of C18, collection filtrate is prepare liquid.
(2) ion chromatograph with ampere detector determines
Chromatography of ions condition is：Detector：ED40 electrochemical detectors, reference electrode Ag/AgCl, Au electrode.Chromatogram
Post：CarboPac PA10 chromatography columns (4mm*250mm).Mobile phase：Ultra-pure water；Flow velocity：1.0mL/min；Leacheate：
10mmol/L KOH；Sampling volume：25μL；
By above-mentioned analysis method to sample carry out mark-on reclaims experiment, every time analysis can obtain accurate collection of illustrative plates and
Quantitative information, reference standard curve, calculate rate of recovery result such as table 1：
Lactulose mark-on reclaims result of the test in the milk of table 1
Embodiment 2
Shaken up after adding lactulose standard liquid in the student drinking milk blank sample of weighing, the breast of the present embodiment is made
Fructose mark-on sample.
(1) sample pre-treatments
50.00mL student's milk samples to be measured are pipetted in 250mL triangular flasks, sequentially add the Asia that 5mL concentration is 200g/L
Potassium ferricyanide solution, the solution of zinc sulfate and 10mL buffer solutions [0.34mol/L disodium hydrogen phosphates that 5mL concentration is 200g/L
(Na2HPO4)+0.07mol/L sodium dihydrogen phosphates (NaH2PO4), pH=7.5], it is intended to fully shake up per reagent adding, all adds
After stand 30min, filter, discard primary filtrate, collect filtrate.
Filtrate 5mL is taken in 10mL volumetric flasks, 100 μ L beta-D-galactosidase suspension is added, shakes up, is capped,
Cultivated 1 hour at 50 DEG C.2.0mL buffer solutions [0.75mol/L triethanolamine hydrochlorides are sequentially added in cultured sample
(C6H15NO3HCl)+0.01mol/L magnesium sulfate (MgSO4·7H2O), pH=7.6], 100 μ L glucose oxidizing ferment suspension, 1
N-octyl alcohol, 0.5mL0.33mol/L sodium hydroxide solutions, 50 μ L hydrogen peroxide (30%) and 100 μ L catalases are dripped to suspend
Liquid.Often plus a kind of reagent is intended to shake up, and 3h is cultivated in 40 DEG C of water-baths or baking oven after all adding.Distilled water constant volume is used after cooling
To 10mL, after passing sequentially through 0.45 μm of aqueous phase filter membrane and the small column purifications of C18, collection filtrate is prepare liquid.
(2) ion chromatograph with ampere detector determines
Chromatography of ions condition is：Detector：ED40 electrochemical detectors, reference electrode Ag/AgCl, Au electrode.Chromatogram
Post：CarboPac PA10 chromatography columns (4mm*250mm).Mobile phase：Ultra-pure water；Flow velocity：1.0mL/min；Leacheate：
10mmol/L KOH；Sampling volume：25μL；
By above-mentioned analysis method to sample carry out mark-on reclaims experiment, every time analysis can obtain accurate collection of illustrative plates and
Quantitative information, reference standard curve, calculate rate of recovery result such as table 2：
Lactulose mark-on reclaims result of the test in the student drinking milk of table 2
Claims (7)
1. the detection method of lactulose in a kind of milk, including：
(1) after taking milk sample to be diluted with water, potassium ferrocyanide solution and solution of zinc sulfate are sequentially added, is stood, is filtered, is collected
Filtrate；Beta-D-galactosidase is added, is capped after mixing, in 50~60 DEG C of incubated 1~2h；Then glucose is sequentially added
Oxidizing ferment, n-octyl alcohol, sodium hydroxide solution, hydrogen peroxide and catalase, in 45~50 DEG C of incubated 3~4h；Cooling
Constant volume afterwards, filter, purification, collect filtrate as prepare liquid；Wherein beta-D-galactosidase, glucose oxidase, sodium hydroxide
Solution, hydrogen peroxide, catalase and the volume ratio of filtrate are 0.1:0.1:0.5:0.05:0.1:5；
(2) using the prepare liquid in the ion chromatograph analytical procedure (1) with ampere detector, the content of lactulose is obtained；
Wherein ion chromatograph analysis condition be：
Detector：ED40 electrochemical detectors, reference electrode Ag/AgCl, Au electrode；
Chromatographic column：CarboPac PA10 chromatography columns；
Mobile phase：Ultra-pure water；
Flow velocity：1.0mL/min；
Leacheate：10mmol/L KOH；
Sampling volume：25μL.
2. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：In the step (1)
Milk sample for fresh cow milk or milk additive.
3. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：In the step (1)
The concentration of potassium ferrocyanide solution be 200g/L, the volume ratio with milk sample is 1:5；The concentration of solution of zinc sulfate is
200g/L, the volume ratio with milk sample are 1:5.
4. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：In the step (1)
Buffer solution adjustment solution ph is added to 7.5 after adding solution of zinc sulfate；Add the incubated rear addition of beta-D-galactosidase
Buffer solution adjusts solution ph to 7.6.
5. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：In the step (1)
Filtering use 0.45 μm of aqueous phase membrane filtration；Purification uses the small column purifications of C18.
6. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：The rule of the chromatographic column
Lattice are：Length 250mm × internal diameter 4mm.
7. the detection method of lactulose in a kind of milk according to claim 1, it is characterised in that：In the step (2)
The content of lactulose use quantified by external standard method.
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| CN107462642B (en) * | 2017-07-21 | 2020-04-14 | 华南农业大学 | Method for rapidly determining lactulose in dairy product |
| CN108982829B (en) * | 2018-07-03 | 2019-10-25 | 中国农业科学院北京畜牧兽医研究所 | A kind of method and its kit with lactulose in microplate reader enzyme process quantitative detection liquid milk |
| AU2021223585A1 (en) | 2020-02-20 | 2022-07-28 | Triviumvet Designated Activity Company | Bienzymatic biosensor for the detection of lactulose in blood |
| CN111855829B (en) * | 2020-03-23 | 2022-07-29 | 唐山市食品药品综合检验检测中心(唐山市畜牧水产品质量监测中心) | Method for detecting lactulose in dairy product |
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| CN201072414Y (en) * | 2006-12-30 | 2008-06-11 | 上海特敏生物医药科技有限公司 | Reagent kit for measuring content of lactulose, mannitol and lactose |
| CN101526510B (en) * | 2009-04-22 | 2012-07-04 | 南昌大学 | Method for rapidly determining content of lactosucrose in food |
| CN102166216A (en) * | 2011-01-31 | 2011-08-31 | 四川健能制药有限公司 | Lactulose oral solution and quality control method thereof |
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