CN106706798A - Lactose content detection method - Google Patents
Lactose content detection method Download PDFInfo
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- CN106706798A CN106706798A CN201710073863.5A CN201710073863A CN106706798A CN 106706798 A CN106706798 A CN 106706798A CN 201710073863 A CN201710073863 A CN 201710073863A CN 106706798 A CN106706798 A CN 106706798A
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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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- 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
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a lactose content detection method. The lactose content detection method comprises the following steps: (1) dissolving a to-be-detected sample into deionized water, and dissolving through ultrasonic waves to obtain a mixed solution; (2) transferring the mixed solution obtained in the step (1) to a volumetric flask to meter the volume, centrifugally filtering to obtain a to-be-detected solution; (3) repeating the method of the step (1) and the step (2), replacing the to-be-detected sample with a lactose standard product, and obtaining a standard solution; (4) using liquid chromatogram to detect the standard solution and the to-be-detected solution to obtain the retention time and the peak area of various components in the lactose standard product and the to-be-detected sample, drawing a corresponding standard curve for the peak area of the concentration of the lactose standard product, comparing the retention time and the peak area of the various components in the to-be-detected sample with the standard curve, and calculating to obtain the content of lactose in the to-be-detected sample. According to the lactose content detection method, the detection steps are few, the detection time is short, and the result is accurate.
Description
Technical field
The present invention relates to chemical composition detection technique field, and in particular to a kind of detection method of lactose content, especially
The detection method of the lactose in animal feed and raw material.
Background technology
Lactose be only in mammal milk a kind of naturally occurring disaccharide, by the glucose of a molecule with a molecule
Galactolipin is constituted, and lactose is a kind of Major Nutrient material during it grows, the development to its intelligence for human child
Play a part of particularly significant.
For feed industry, lactose is equally a kind of important feed adding component, is come particularly with wean sucking pig
Say particularly important, weanling sucking pig is faced with many challenges for existence, which includes its not yet full grown Digestive
System will face the transformation from breast milk to solid feed.Substantial amounts of research existing at present shows that addition is suitable in the feed of wean sucking pig
The whey powder raw material of amount can improve the feed feed intake of wean sucking pig, growth rate, survival rate etc..
And the main component in whey powder is exactly lactose, for piglet feed, its raw material and finished product are accurately monitored
Lactose content to improve quality of the fodder, improve forage feed economic benefit for it is particularly important.
The national standard method of special detection lactose is there is no in current feed industry, is using more《NY/T1422-2007 breasts
And in dairy products lactose measure enzyme-colorimetric method》.The method by the way that lactose is digested after determine nicotinoyl amine gland with honourable photometry
The content of purine dinucleotides phosphoric acid carrys out the content of indirect determination lactose, and the method is not only cumbersome, and its enzyme digestion reaction
, it is necessary to the condition of control is more more than step, the risk of detection accident is easily increased in actual applications.
The method for summarizing the above is primarily present some following problem：
1) it is cumbersome, it is necessary to carry out the content of lactose in determination sample after the operation of multi-step；
2) lactose content in itself and indirectly measurement sample, but surveyed indirectly by determining final product after being digested
Amount lactose content, increased the difficulty and repeatability of measure；
3) detection process need to use plurality of reagents, including organic reagent and inorganic reagent, not friendly to operating personnel and environment
It is good.
The content of the invention
The purpose of the present invention be exactly provided for the defect for overcoming above-mentioned prior art to exist a kind of determination step it is few, when
Between the accurate lactose content of short, result detection method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of detection method of lactose content, the method includes following steps：
Step (1), testing sample (feed or other raw materials) is dissolved in deionized water, and it is molten that ultrasonic dissolved solves mixing
Liquid；
Step (2), constant volume in volumetric flask is transferred to by the mixed solution that step (1) is obtained, and centrifugal filtration obtains to be measured molten
Liquid；
The method of step (3), repeat step (1) and step (2), and the testing sample is substituted for lactose standard,
Obtain standard liquid；
Step (4), standard liquid and solution to be measured are detected using liquid chromatogram, obtain lactose standard and to be measured
The retention time and peak area of each component in sample, and it is the corresponding standard curve of peak area to draw the concentration of lactose standard,
The retention time and peak area of testing sample each component are compared with standard curve, be calculated testing sample (feed or
Other raw materials) in lactose content.
Existing method is detected after sample is carried out into enzymolysis processing, is such as detected according to existing national standard method, is grasped
Make very complicated, and poor repeatability.
Ultrasonic wave extraction (i.e. ultrasound on extracting) is the effect by ultrasonic wave to extractant, makes to produce sky in solution
The effects such as cave bubble, explosion concussion, so that the interaction between extractant is more violent such that it is able to quick, complete
Liposoluble constituent is extracted entirely.
Because the high molecular weight protein that be there may exist in sample such as milk in course of dissolution forms turbid liquid, using general
Filter paper cannot leach, therefore selection using centrifugation method process after refilter upper machine.
The step (1), at 50~70 DEG C, ultrasonic time is 10-30min to the temperature of ultrasonic wave extraction.
The step (2), the rotating speed of centrifugal filtration is 4000-10000r/min, and the time is 5-15min.
The step (4), with water as mobile phase, liquid chromatogram uses isocratic elution mode to liquid chromatogram.
After using such scheme, compared with prior art, beneficial effects of the present invention are embodied in following several respects：
(1) pretreatment process is simple, and determination step is few, and its testing result high precision, effect is good, realizes in short-term
Interior separation and detection to lactose；
(2) experimentation has only used deionized water for reagent, reduces the usage amount of poisonous and hazardous reagent, more ring
Ensure safety.
Brief description of the drawings
Fig. 1 is the chromatogram of testing sample (concentrate feed)；
Fig. 2 is the chromatogram of various concentrations lactose standard；
Fig. 3 is the standard curve of lactose.
Specific embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed implementation method and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
Embodiment 1
The instrument and chromatographic condition that the present embodiment is used are as follows：
1. instrument and equipment
Electronic balance (is accurate to 0.1mg), laboratory Conventional glass vessel, ultrasonic extractor, 0.22 μm of micro porous filtration
Film, solvent filter, the high performance liquid chromatography with time difference shading detector, centrifuge.
2. chromatographic condition
Chromatographic column：Agilent Hi-Plex H (8 μm, 7.7mm × 300mm)
Column temperature：70℃
Mobile phase：Water
Sample size：20μL
A kind of detection method of lactose content, comprises the following steps：
The accurate testing sample for weighing 0.1-5g, is placed in the beaker of 100mL, adds 60mL deionized waters to stir,
60 DEG C of ultrasonic extraction 10min, solution is transferred in 100mL volumetric flasks, and 100mL is settled to deionized water, takes appropriate upper liquid
Body 8000r/min is centrifuged 15min, takes after the supernatant liquor after centrifugation crosses 0.22 μm of organic miillpore filter of syringe needle to enter by liquid-phase condition
Machine testing on row, mobile phase is water, and using isocratic elution mode, the chromatogram for obtaining is as shown in Figure 1.
Lactose standard liquid：The accurate lactose standard for weighing 0.1g, is placed in the volumetric flask of 100mL, adds deionization
Water is settled to scale, and upper machine testing is carried out by liquid-phase condition after crossing 0.22 μm of organic miillpore filter of syringe needle, and mobile phase is water, uses
Isocratic elution mode.
The vitamin mixed standard solution that will be prepared carries out liquid chromatogram measuring under above-mentioned liquid-phase condition, enters respectively
Sample 0.5mL, 1mL, 2mL, 5mL, 10mL, 20mL record lactose chromatographic peak area, the chromatogram for obtaining is as shown in Fig. 2 and with table 2
In peak area be ordinate, content is abscissa, draw standard curve and calibration curve equation, the standard curve for obtaining as scheme
Shown in 3.
Relative standard deviation (RSD) analysis, the result for obtaining such as table 1 are carried out to 6 retention times of sample introduction in gained Fig. 2
It is shown.
The vitamin A of table 1, vitamine D3, relative standard deviation (RSD) analysis of vitamin E retention time
| Sequence number | Retention time |
| 1 | 8.240 |
| 2 | 8.245 |
| 3 | 8.243 |
| 4 | 8.251 |
| 5 | 8.253 |
| 6 | 8.262 |
| RSD (%) | 0.10 |
The peak area in standard curve and table 2 according to lactose in Fig. 3 is calculated, and the correction of the peak area for obtaining is calculated
As a result, as a result as shown in table 2.As can be known from Table 2 the deviation between correction calculated value and actual content for -1.66%~
2.58%, and the coefficient correlation of its standard curve is R2=0.99999, illustrate that the standard curve is linearly good.
The testing result of the lactose standard liquid of table 2
| Retention time (min) | Content (mg) | Peak area | Correction calculated value | Deviation (%) |
| 8.240 | 0.503 | 7272.3 | 0.490 | 2.58 |
| 8.245 | 1.006 | 14518.5 | 1.000 | 0.56 |
| 8.243 | 2.012 | 28632.7 | 1.995 | 0.87 |
| 8.251 | 5.030 | 72911.5 | 5.113 | -1.66 |
| 8.253 | 10.060 | 143094.6 | 10.057 | 0.03 |
| 8.262 | 20.120 | 285914.1 | 20.116 | 0.02 |
According to table 2, the equation that can obtain the standard curve of above-mentioned lactose standard liquid is：
Calibration curve equation：Y=14197.62077x+315.01961R2=0.99999
Bring the peak area of the lactose of testing sample into calibration curve equation and be calculated on corresponding lactose in machine liquid
X is brought into formula (I) calculate the content for determining lactose in sample again by content X：
In formula：
W --- the content (%) of lactose in sample
X --- by the content (mg) of lactose in the upper machine liquid that sample peak area and standard curve are calculated
V1--- the constant volume (mL) during extraction
V2--- the sample size (mL) on sample during machine
M --- sample mass (g)
The implementation process being set forth below specifically is tested according to above-mentioned detecting step, above-mentioned computational methods meter
Result is calculated, the numerical value such as the sample introduction concentration of test liquid sample mass, peak area and sample are repeated no more, specific calculating is only described
As a result.
The peak area of testing sample is brought into calibration curve equation the content X that is calculated lactose in upper machine liquid and by its
In substitution formula (I), the content of lactose in testing sample can be obtained, its result is as shown in table 3.
Lactose content testing result in the testing sample of table 3
| Measured value (%) | Mark-on amount (%) | The rate of recovery (%) | National standard method (%) | Relative deviation (%) | |
| Whey powder | 133.1 | 50 | 99 | 86.1 | 3.0 |
| Concentrated feed | 19.2 | 10 | 102 | 9.2 | 7.0 |
| Mixed feed | 0.19 | 0.1 | 95 | 0.11 | 10.0 |
Therefrom it is known that adopting the lactose content being obtained by the present invention, breast in sample can be more accurately obtained
The content of sugar, and relative to GB detection, simple to operate, the used time is few.Using national standard method, due to there is other carbohydrates to be hydrolyzed into
Itself also has a certain amount of glucose in glucose, or sample, causes the result of detection generally higher.
Embodiment 2
Using the feed of another brand, using with the identical operating procedure of embodiment 1, the final result of gained is such as
Shown in table 4.
Lactose content testing result in the testing sample of table 4
| Measured value (%) | Mark-on amount (%) | The rate of recovery (%) | National standard method (%) | Relative deviation (%) | |
| Mixed feed | 0.21 | 0.1 | 91 | 0.10 | 8.2 |
The present invention can effectively reduce the time of pre-treatment operation, and the input for using manpower and material resources sparingly saves toxic reagent
Use.And can accurately detect lactose content.
Embodiment 3
Using the feed of another brand, using with the identical operating procedure of embodiment 1, the final result of gained is such as
Shown in table 5.
Lactose content testing result in the testing sample of table 5
| Measured value (%) | Mark-on amount (%) | The rate of recovery (%) | National standard method (%) | Relative deviation (%) | |
| Concentrated feed | 21.9 | 10 | 106 | 11.9 | 5.3 |
The present invention can effectively reduce the time of pre-treatment operation, and the input for using manpower and material resources sparingly saves toxic reagent
Use.Lactose content can accurately be detected.
Claims (4)
1. a kind of detection method of lactose content, it is characterised in that including following steps：
Step (1), testing sample is dissolved in deionized water, and ultrasonic dissolved solves mixed solution；
Step (2), constant volume in volumetric flask is transferred to by the mixed solution that step (1) is obtained, and centrifugal filtration obtains solution to be measured；
The method of step (3), repeat step (1) and step (2), and the testing sample is substituted for lactose standard, obtain
Standard liquid；
Step (4), standard liquid and solution to be measured are detected using liquid chromatogram, obtain lactose standard and testing sample
The retention time and peak area of middle each component, and it is the corresponding standard curve of peak area to draw the concentration of lactose standard, will be treated
The retention time and peak area of test sample product each component are compared with standard curve, are calculated containing for lactose in testing sample
Amount.
2. a kind of detection method of lactose content as claimed in claim 1, it is characterised in that the step (1), ultrasonic wave is carried
At 50~70 DEG C, ultrasonic time is 10-30min to the temperature for taking.
3. a kind of detection method of lactose content as claimed in claim 1, it is characterised in that the step (2), centrifugal filtration
Rotating speed be 4000-10000r/min, the time is 5-15min.
4. a kind of detection method of lactose content as claimed in claim 1, it is characterised in that the step (4), liquid chromatogram
With water as mobile phase, liquid chromatogram uses isocratic elution mode.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315053A (en) * | 2017-05-31 | 2017-11-03 | 南京威尔药业股份有限公司 | About the EFI fog detector liquid phase chromatography analytical method of material in a kind of lactose |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105424858A (en) * | 2015-12-31 | 2016-03-23 | 广州甘蔗糖业研究所 | Efficient liquid chromatography tandem mass spectrometry detection method for galactooligosaccharides in milk powder |
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2017
- 2017-02-10 CN CN201710073863.5A patent/CN106706798A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424858A (en) * | 2015-12-31 | 2016-03-23 | 广州甘蔗糖业研究所 | Efficient liquid chromatography tandem mass spectrometry detection method for galactooligosaccharides in milk powder |
Non-Patent Citations (3)
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| UPENDRA SHARMA 等: "Simultaneous Determination of Ten Sugars in Tinospora cordifolia by Ultrasonic Assisted Extraction and LC-ELSD", 《CHROMATOGRAPHIA》 * |
| 梁龙 等: "高效液相色谱法测定混菌发酵乳中乳糖、葡萄糖及半乳糖含量的变化", 《食品工业科技》 * |
| 王浩 等: "液相色谱-质谱联用技术测定无糖食品中果糖、葡萄糖、蔗糖、麦芽糖和乳糖", 《分析化学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315053A (en) * | 2017-05-31 | 2017-11-03 | 南京威尔药业股份有限公司 | About the EFI fog detector liquid phase chromatography analytical method of material in a kind of lactose |
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Application publication date: 20170524 |