CN105784868A - Method for detecting lactulose in milk - Google Patents
Method for detecting lactulose in milk Download PDFInfo
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- CN105784868A CN105784868A CN201610223749.1A CN201610223749A CN105784868A CN 105784868 A CN105784868 A CN 105784868A CN 201610223749 A CN201610223749 A CN 201610223749A CN 105784868 A CN105784868 A CN 105784868A
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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
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention relates to a method for detecting lactulose in milk.The method comprises the steps that lactose and lactulose in the milk react and are hydrolyzed into galactose, glucose and fructose through beta-D-galactosidase, glucose oxidase is added to oxidize most of the glucose into gluconic acid, finally catalase is added to remove hydrogen peroxide, and a testing solution is obtained; an ion chromatograph with an ampere detector is used for analyzing the testing solution, and the content of the lactulose is obtained.According to the method, the content of the lactulose in the milk can be accurately and sensitively measured by means of easy and convenient operation.
Description
Technical field
The invention belongs to lactulose detection field, particularly to the detection method of lactulose in a kind of milk.
Background technology
Lactulose is lactose isomerization product in milk heat treatment process, is assessment sterilization of milk degree and the one of storage time
Item important indicator, World Milk goods tissue (IDF) has been proposed that the concentration of lactulose is to use pasteuring as distinguishing this dairy products,
Superthermal process and sterilized indicator, therefore in milk, the Accurate Determining of lactulose more seems increasingly important.
Tradition has chromatograph and enzyme to analyze both approaches about the analysis method of lactulose, but owing to milk containing more breast
Sugar, can affect lactulose so that both approaches is difficult to carry out quantitative analysis accurately.Along with ion chromatography in recent years
Constantly ripe, use ion chromatography saccharide to become a kind of new approach measuring lactulose, but lactose and lactulose
Separation is still that a difficult problem, and it is the harshest to the requirement of the conditions such as eluent concentration.
Summary of the invention
The technical problem to be solved is to provide the detection method of lactulose in a kind of milk, and the method can be accurate, clever
Quick, measure the content of lactulose in milk easy and simple to handlely.
The detection method of lactulose in a kind of milk of the present invention, including:
(1) after taking milk sample dilute with water, it is sequentially added into potassium ferrocyanide solution and solution of zinc sulfate, stands, filter, collect filter
Liquid；Add beta-D-galactosidase (β-galactosidase), add a cover after mixing, in 50～60 DEG C of constant temperature culture 1～2h；Then
It is sequentially added into glucoseoxidase (glucose oxidase, GOD), n-octyl alcohol, sodium hydroxide solution, hydrogen peroxide and peroxide
Change hydrogen enzyme, in 45～50 DEG C of constant temperature culture 3～4h；Constant volume after cooling, filters, and purifies, and collects filtrate as liquid to be measured；
(2) use the liquid to be measured in the ion chromatograph analytical procedure (1) with ampere detector, obtain the content of lactulose.
The additive that milk sample is fresh cow milk or milk (such as high calcium milk, student nutrition reinforce-milk etc.) in described step (1).
The concentration of the potassium ferrocyanide solution in described step (1) is 200g/L, is 1:5 with the volume ratio of milk sample；Sulphuric acid
The concentration of zinc solution is 200g/L, is 1:5 with the volume ratio of milk sample.
Buffer [0.34mol/L disodium hydrogen phosphate (Na is added after described step (1) adds solution of zinc sulfate2HPO4)
+ 0.07mol/L sodium dihydrogen phosphate (NaH2PO4), pH=7.5] adjust solution ph to 7.5；Add beta-D-galactosidase constant temperature
Buffer [0.75mol/L triethanolamine hydrochloride (C is added after cultivation6H15NO3HCl)+0.01mol/L magnesium sulfate
(MgSO4·7H2O), pH=7.6] adjust solution ph to 7.6.
Beta-D-galactosidase in described step (1) is 0.1:5 with the volume ratio of filtrate.
The volume of glucoseoxidase, sodium hydroxide solution, hydrogen peroxide, catalase and filtrate in described step (1)
Ratio is 0.1:0.5:0.05:0.1:5.
Filtration in described step (1) uses 0.45 μm aqueous phase membrane filtration；Purify and use the little column purification of C18.
The condition of the ion chromatograph analysis in described step (2) is:
Detector: ED40 electrochemical detector, reference electrode is Ag/AgCl, Au electrode；
Chromatographic column: CarboPac PA10 chromatography column；
Flowing phase: ultra-pure water；
Flow velocity: 1.0mL/min；
Leacheate: 10mmol/L KOH；
Sampling volume: 25 μ L.
The specification of described chromatographic column is: length 250mm × internal diameter 4mm.
The content of the lactulose in described step (2) uses quantified by external standard method.
The present invention use carbohydrate chromatography post CarboPac PA10 (4mm*250mm) separate each different component
Sugar.
The present invention analyzes the ultimate principle of lactulose by having the ion chromatograph of ampere detector: the lactose in sample and breast
After fructose is hydrolyzed into galactose, glucose and fructose, separated by carbohydrate chromatographic column.Based on ligand exchange reaction, sugar
On molecule, each hydroxyl is with a very weak negative charge, and the hydroxyl carried on anomer carbon can by deprotonation,
Thus bring a negative charge the strongest.These negative charges on glycan molecule and between the positive charge of the metal ion on resin surface
Interaction make sugar retained, thus reach to separate, therefore each component retention time in the chromatography column is the most different, Jing Guoyi
After fixed column length, the most separated from one another, leave chromatographic column in order and enter detector, reach efficiently separating of several sugar, logical
Cross its retention time on a column qualitative, peak area quantification.By the fructose content recorded, fructose in deducting at the bottom of sample copy
Content, the fructose content that available lactulose enzymolysis produces, thus calculate the content of lactulose in milk sample.
Present invention potassium ferrocyanide and solution of zinc sulfate, as precipitant, can effectively get rid of albumen and fat to lactulose analysis
Interference.
The present invention uses enzymatic isolation method by lactose substantial amounts of in milk and least a portion of lactulose complete hydrolysis, and whole pretreatment process is adopted
Lactose and lactulose cannot be kept completely separate one produced due to lactose too high levels is well solved by the method
Individual problem.
In the present invention, use carbohydrate chromatography post to separate, divide by having the ion chromatograph of ampere detector
Analysis lactulose content.The hydrolyzate of lactulose and lactose is glucose, galactose and fructose, and these three sugar is at CarboPac PA10
On carbohydrate chromatography post, separating degree is preferable.Milk blank sample adds the lactulose standard substance of 1.0mg/L, warp
Sample pre-treatments, after Instrument measuring, the signal to noise ratio (S/N) of measured object is 8.9 more than 3, shows the detection limit of the inventive method
Can be to 1.0mg/L.
Beneficial effect
(1) present invention beta-D-galactosidase, glucoseoxidase and catalase carry out enzymolysis to sample, can effectively by
In sample, lactose and galactose extract completely, can guarantee that again their enzymatic hydrolysate is kept completely separate and do not interfere with each other；
(2) present invention carries out the detection of sugar with the ion chromatograph with ampere detector, and the method is the most ripe, and sugar is each
Separating degree and the response value of individual component are relatively good；
(3) during the present invention is milk, the detection of lactulose content provides a kind of highly sensitive detection means.
Accompanying drawing explanation
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 (the most enzyme-added) in embodiment 1；
Fig. 3 is the chromatography of ions figure of background in embodiment 1 (after enzyme-added hydrolysis process)；
Fig. 4 is that in embodiment 1, sample adds target chromatography of ions figure (mark-on level is 5.0mg/L)；
Fig. 5 is the chromatography of ions figure of embodiment 2 empty sample；
Fig. 6 is that in embodiment 2, sample adds target chromatography of ions figure (mark-on level is 1.0mg/L).
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
Shake up after adding lactulose standard solution in the milk blank sample weighed, prepare the lactulose mark-on sample of the present embodiment.
(1) sample pre-treatments
Pipette 10.00mL milk sample to be measured in 150mL triangular flask, be sequentially added into the ferrous cyanogen that 2mL concentration is 200g/L
Change potassium solution, 2mL concentration are solution of zinc sulfate and 6.5mL buffer [the 0.34mol/L disodium hydrogen phosphate of 200g/L
(Na2HPO4)+0.07mol/L sodium dihydrogen phosphate (NaH2PO4), pH=7.5], every reagent adding is intended to fully shake up, and all adds
Rear standing 30min, filters, and discards just filtrate, collects filtrate.
Take filtrate 5mL in 10mL volumetric flask, add the beta-D-galactosidase suspension of 100 μ L, shake up, add a cover,
Cultivate 1 hour at 50 DEG C.2.0mL buffer [0.75mol/L triethanolamine hydrochloride it is sequentially added in cultured sample
(C6H15NO3HCl)+0.01mol/L magnesium sulfate (MgSO4·7H2O), pH=7.6], 100 μ L glucose oxidase suspensions,
1 n-octyl alcohol, 0.5mL0.33mol/L sodium hydroxide solution, 50 μ L hydrogen peroxide (30%) and 100 μ L catalases hang
Supernatant liquid.Often add a kind of reagent to be intended to shake up, in 40 DEG C of water-baths or baking oven, after all adding, cultivate 3h.Determine with distilled water after cooling
Holding to 10mL, after passing sequentially through 0.45 μm aqueous phase filter membrane and the little column purification of C18, collecting filtrate is liquid to be measured.
(2) ion chromatograph with ampere detector measures
Chromatography of ions condition is: detector: ED40 electrochemical detector, and reference electrode is Ag/AgCl, Au electrode.Chromatograph
Post: CarboPac PA10 chromatography column (4mm*250mm).Flowing phase: ultra-pure water；Flow velocity: 1.0mL/min；Drip washing
Liquid: 10mmol/L KOH；Sampling volume: 25 μ L；
By above-mentioned analysis method, sample is carried out mark-on recovery test, analyze every time and can obtain collection of illustrative plates and quantitatively letter accurately
Breath, reference standard curve, calculating response rate result such as table 1:
Lactulose mark-on recovery test result in table 1 milk
Embodiment 2
Shaking up after adding lactulose standard solution in the student drinking milk blank sample weighed, the lactulose preparing the present embodiment adds
Standard specimen product.
(1) sample pre-treatments
Pipette 50.00mL School Milk to be measured sample in 250mL triangular flask, be sequentially added into the ferrous iron that 5mL concentration is 200g/L
Potassium cyanide solution, 5mL concentration are solution of zinc sulfate and 10mL buffer [the 0.34mol/L disodium hydrogen phosphate of 200g/L
(Na2HPO4)+0.07mol/L sodium dihydrogen phosphate (NaH2PO4), pH=7.5], every reagent adding is intended to fully shake up, and all adds
Rear standing 30min, filters, and discards just filtrate, collects filtrate.
Take filtrate 5mL in 10mL volumetric flask, add the beta-D-galactosidase suspension of 100 μ L, shake up, add a cover,
Cultivate 1 hour at 50 DEG C.2.0mL buffer [0.75mol/L triethanolamine hydrochloride it is sequentially added in cultured sample
(C6H15NO3HCl)+0.01mol/L magnesium sulfate (MgSO4·7H2O), pH=7.6], 100 μ L glucose oxidase suspensions,
1 n-octyl alcohol, 0.5mL0.33mol/L sodium hydroxide solution, 50 μ L hydrogen peroxide (30%) and 100 μ L catalases hang
Supernatant liquid.Often add a kind of reagent to be intended to shake up, in 40 DEG C of water-baths or baking oven, after all adding, cultivate 3h.Determine with distilled water after cooling
Holding to 10mL, after passing sequentially through 0.45 μm aqueous phase filter membrane and the little column purification of C18, collecting filtrate is liquid to be measured.
(2) ion chromatograph with ampere detector measures
Chromatography of ions condition is: detector: ED40 electrochemical detector, and reference electrode is Ag/AgCl, Au electrode.Chromatograph
Post: CarboPac PA10 chromatography column (4mm*250mm).Flowing phase: ultra-pure water；Flow velocity: 1.0mL/min；Drip washing
Liquid: 10mmol/L KOH；Sampling volume: 25 μ L；
By above-mentioned analysis method, sample is carried out mark-on recovery test, analyze every time and can obtain collection of illustrative plates and quantitatively letter accurately
Breath, reference standard curve, calculating response rate result such as table 2:
Lactulose mark-on recovery test result in table 2 student drinking milk
Claims (10)
1. a detection method for lactulose in milk, including:
(1) after taking milk sample dilute with water, it is sequentially added into potassium ferrocyanide solution and solution of zinc sulfate, stands, filter, collect filter
Liquid；Add beta-D-galactosidase, add a cover after mixing, in 50～60 DEG C of constant temperature culture 1～2h；Then Fructus Vitis viniferae glycosyloxy it is sequentially added into
Change enzyme, n-octyl alcohol, sodium hydroxide solution, hydrogen peroxide and catalase, in 45～50 DEG C of constant temperature culture 3～4h；After cooling
Constant volume, filters, and purifies, and collects filtrate as liquid to be measured；
(2) use the liquid to be measured in the ion chromatograph analytical procedure (1) with ampere detector, obtain the content of lactulose.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (1)
Milk sample is the additive of fresh cow milk or milk.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (1)
The concentration of potassium ferrocyanide solution is 200g/L, is 1:5 with the volume ratio of milk sample；The concentration of solution of zinc sulfate is 200g/L,
It is 1:5 with the volume ratio of milk sample.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: described step adds in (1)
Add buffer after entering solution of zinc sulfate and adjust solution ph to 7.5；Buffering is added after adding beta-D-galactosidase constant temperature culture
Liquid adjusts solution ph to 7.6.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: the β-D-in described step (1)
Tilactase is 0.1:5 with the volume ratio of filtrate.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (1)
Glucoseoxidase, sodium hydroxide solution, hydrogen peroxide, catalase are 0.1:0.5:0.05:0.1:5 with the volume ratio of filtrate.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (1)
Filter and use 0.45 μm aqueous phase membrane filtration；Purify and use the little column purification of C18.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (2)
The condition of ion chromatograph analysis is:
Detector: ED40 electrochemical detector, reference electrode is Ag/AgCl, Au electrode；
Chromatographic column: CarboPac PA10 chromatography column；
Flowing phase: ultra-pure water；
Flow velocity: 1.0mL/min；
Leacheate: 10mmol/L KOH；
Sampling volume: 25 μ L.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: the specification of described chromatographic column is:
Length 250mm × internal diameter 4mm.
The detection method of lactulose in a kind of milk the most according to claim 1, it is characterised in that: in described step (2)
The content of lactulose uses quantified by external standard method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107462642A (en) * | 2017-07-21 | 2017-12-12 | 华南农业大学 | The rapid assay methods of lactulose in a kind of dairy products |
| CN108982829A (en) * | 2018-07-03 | 2018-12-11 | 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 |
| CN111855829A (en) * | 2020-03-23 | 2020-10-30 | 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 |
| WO2021165343A1 (en) | 2020-02-20 | 2021-08-26 | Triviumvet Designated Activity Company | Bienzymatic biosensor for the detection of lactulose in blood |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107462642A (en) * | 2017-07-21 | 2017-12-12 | 华南农业大学 | The rapid assay methods of lactulose in a kind of dairy products |
| CN107462642B (en) * | 2017-07-21 | 2020-04-14 | 华南农业大学 | Method for rapidly determining lactulose in dairy product |
| CN108982829A (en) * | 2018-07-03 | 2018-12-11 | 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 |
| 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 |
| WO2021165343A1 (en) | 2020-02-20 | 2021-08-26 | Triviumvet Designated Activity Company | Bienzymatic biosensor for the detection of lactulose in blood |
| CN111855829A (en) * | 2020-03-23 | 2020-10-30 | 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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