CN107576651A - The prefabricated reagent of sucrose quick detection and its preservation, detection method in a kind of honey - Google Patents
The prefabricated reagent of sucrose quick detection and its preservation, detection method in a kind of honey Download PDFInfo
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Abstract
The invention provides a kind of prefabricated reagent of sucrose quick detection in honey and its preservation, detection method, the prefabricated reagent of sucrose quick detection is made up of the raw material of following parts by weight in the honey：0.5~1.2 part of 75~83 parts of filler, 0.5~1.5 part of developer, 15~20 parts of phosphate, 0.5~1.2 part of sucrose isomerase, 0.6~1.2 part of glucosyl group oligosaccharides oxidizing ferment and horseradish peroxidase.The present invention is easy to carry, and operating process is simple, efficient, safe, strong antijamming capability, it is not necessary to which large-scale instrument and equipment, testing result are intuitively visible；Detection sensitivity is high, using spectrophotometer test limit up to 1.0mg/L；The detection method rate of recovery is high, favorable reproducibility.
Description
Technical field
The present invention relates to technical field of rapid detection of food safety, more particularly to a kind of high sensitivity, honeybee simple to operate
The prefabricated reagent of sucrose quick detection and its preservation, detection method in honey.
Background technology
Honeybee is nectar, secretion or the honeydew of herborization, after being mixed with itself secretion, through what is fully brewageed
Natural sweet substance.Contain in honey and the plurality of inorganic salt and vitamin of human serum concentration comparable, iron, calcium, copper, manganese, potassium, phosphorus
Etc. a variety of organic acids and the trace element beneficial to health, also containing carbohydrate and several amino acids, there is high health care to make
With and nutritive value, it is deep to be favored by consumer.In recent years, domestic and international market constantly expands to the demand of honey, so
And the yield of honey is difficult to meet the needs of market, and due to the difference in nectar source, the species of honey is various, complicated component so that
Very big limitation be present in the detection technique of honey quality.Cane sugar content is one of important indicator of honey quality,《Food security country
Standard-honey》(GB14963-2011) provide, in eucalyptus honey, citrus honey, alfalfa honey, lichee honey and Osmanthus forrestii
Cane sugar content≤10%, other honey≤5%.
Detection method on sucrose mainly has high performance liquid chromatography, acid-hydrolysis method and enzyme hydrolysis method.High-efficient liquid phase color
Spectrometry is not suitable for field quick detection and laboratories detection due to large-scale instrument to be used.Acid-hydrolysis method is by sucrose
Titrated, calculated with fehling reagent into glucose and fructose, then under the conditions of boiling in 70 DEG C or so of hydrolyzed under acidic conditions
Cane sugar content；Enzyme hydrolysis method, it is that sucrose hydrolysis is recycled into glucose oxidase Portugal into glucose and fructose with invertase
Grape sugar, converses cane sugar content.In honey in addition to sucrose, also containing substantial amounts of glucose and fructose, before and after determining hydrolysis
The difference of glucose content in sample, can just be converted into cane sugar content, but due in honey cane sugar content be typically glucose
1/5 to the 1/10 of content, while sample needs extension rate very high, the error of measure is very big, it is impossible to meets detection needs.This
Outside, acid-hydrolysis method needs to titrate under the conditions of boiling, and operation is comparatively laborious, condition requirement is more harsh.Therefore, acid-hydrolysis method and
Enzyme hydrolysis method is not suitable for the quick detection of sucrose in honey sample.
" honey quality rapid detection method " is disclosed in Chinese patent literature, its notification number is CN101393122A, should
Invention is measured using the mathematical modeling between each component content of near-infrared of honey to each component content of honey, and this method is former
Reason is complicated, and detection efficiency is low, is unfavorable for large-scale promotion use.
The content of the invention
The present invention is in order to overcome the problem of determination efficiency of cane sugar content in existing honey is low, error is big, there is provided a kind of
High sensitivity, strong antijamming capability honey in sucrose quick detection prefabricated reagent.
Invention further provides the prefabricated reagent of sucrose quick detection in a kind of honey easy to use, storage effect is good
Store method.
Present invention also offers it is a kind of determine the rate of recovery is high, in honey of favorable reproducibility sucrose quick detection prefabricated reagent
Detection method, the method be applied to laboratories detect and field quick detection.
To achieve these goals, the present invention uses following technical scheme：
The prefabricated reagent of sucrose quick detection in a kind of honey, the prefabricated reagent of sucrose quick detection is by following heavy in the honey
Measure the raw material composition of part：75~83 parts of filler, 0.5~1.5 part of developer, 15~20 parts of phosphate, sucrose isomerase 0.5~
1.2 parts, 0.5~1.2 part of 0.6~1.2 part of glucosyl group oligosaccharides oxidizing ferment and horseradish peroxidase.
Sucrose isomerase selected in the prefabricated reagent formula system of sucrose quick detection, glucosyl group in honey of the present invention
Oligosaccharides oxidizing ferment and horseradish peroxidase have a selectivity, strong antijamming capability, other carbohydrates in honey, particularly content
Higher glucose and fructose do not influence on measure.Detecting mechanism is specially：Sucrose in honey is in sucrose isomerase
The lower isomery generation isomaltoketose of effect, the latter is in the presence of glucosyl group oligosaccharides oxidizing ferment, oxidation generation 3- ketone-different malt
Ketose, while produce hydrogen peroxide；Hydrogen peroxide horseradish peroxidase effect under be oxidized to water, while make substrate DPD (N,
N- diethyl-p-phenylenediamines) become cationic DPD, pink, the pink depth and caused amount of hydrogen peroxide is presented in the latter
It is linear relevant, it is then linearly related with sucrose concentration initial in sample, therefore, using the pink depth or absorbance
To determine sucrose concentration.
Preferably, the prefabricated reagent of sucrose quick detection is made up of the raw material of following parts by weight in the honey：Filling
80 parts of agent, 1 part of developer, 16 parts of phosphate, 1 part of sucrose isomerase, 1 part of glucosyl group oligosaccharides oxidizing ferment and horseradish peroxidase
1 part of enzyme.
Preferably, the filler is sodium chloride.
Preferably, the developer is N, N- diethyl-p-phenylenediamines, it is colourless liquid, in detection process moderate resistance
Interference performance is strong.
Preferably, the phosphate is the mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, the disodium hydrogen phosphate with
The mol ratio of sodium dihydrogen phosphate is (1:7)~(17:1).
Preferably, on the basis of the prefabricated reagent gross mass of sucrose quick detection in honey, sucrose is fast in the honey
The enzyme activity unit containing sucrose isomerase is 0.3~1.0U/mg in the prefabricated reagent of speed detection, and sucrose is quick in the honey
Enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment in the prefabricated reagent of detection is 0.4~1.0U/mg, sugarcane in the honey
The enzyme activity unit containing horseradish peroxidase is 0.7~2.0U/mg in the prefabricated reagent of sugared quick detection.
In view of the enzymatic reagent directly bought when the present invention prepares prefabricated reagent, enzyme activity unit is certain, present invention
The enzyme activity of three kinds of enzymes in the prefabricated reagent of sucrose quick detection in honey, preferably three kinds of enzymes are controlled using the number of addition
Enzyme activity control within the above range, the accuracy in detection of prefabricated reagent is optimal.
The store method of the prefabricated reagent of sucrose quick detection in a kind of honey, by sucrose quick detection in above-mentioned honey
Prefabricated reagent is packed with detection pipe, and 4~8 DEG C of refrigerations of lucifuge, the detection pipe is that transparent spiral cover glass test tube or height are saturating
Lightness polystyrene plastics bottle.
The present invention is using the method dispensed, by the prefabricated reagent of sucrose quick detection in the honey of every batch of preparation with every part
50mg is low dose of individually to be packed, and the tedious steps weighed is needed before the simplified heavy dose of packaging detection of tradition, while avoid repeatedly weighing
Cause reagent contamination.The present invention is easy to use, that is, takes and survey, reagent safety, and accuracy in detection is high.Pack, lucifuge refrigeration
Then in effective guarantee honey in the prefabricated reagent of sucrose quick detection enzyme activity.
The detection method of the prefabricated reagent of sucrose quick detection, comprises the following steps in a kind of honey：
(1) after honey sample being diluted into 1000~2000 times, 4.0mL sample solutions sucrose quick detection in containing honey is taken
In prefabricated reagent detection pipe, it is well mixed, in the accurate response 10min at 30~45 DEG C,；
(2) color of observation of steps (1) reacted prefabricated reagent, is compareed with standard color comparison card, can half-quantitative detection honey sample
The concentration range of sucrose in product；
Or in 540~560nm wave-length coverages determination step (1) reacted prefabricated reagent absorbance, according to absorbance
Value is searched and converted from standard curve, can quantify the content of sucrose in detection honey sample.
In the honey of the present invention in the detection method of the prefabricated reagent of sucrose quick detection, preferably 37 DEG C of reaction temperature, survey
Determine the preferred 553nm of wavelength of absorbance, honey sample is in visible region with the reacted colour developing of prefabricated reagent punishment, visually may be used
See color change, intuitively, conveniently；Also quantitative detection can be carried out with AAS, test limit is up to 1.0mg/L.
Both available standards colorimetric card sxemiquantitative, it is also possible to portable spectrophotometer or common spectrophotometer accurate quantitative analysis
Cane sugar content in sample, suitable for laboratories detection and field quick detection.The present invention has simple to operate, detection quickly
Sensitive, reagent safety, without large-scale instrument, easy to carry, the reduced sugar such as glucose and fructose such as is not influenceed at the spy in by sample
Point.
Therefore, the present invention has the advantages that：
(1) reagent safety, strong antijamming capability, selected enzyme has a selectivity in component, other carbohydrates in honey, especially
It is that the higher glucose of content and fructose do not influence on measure；
(2) it is easy to carry, operating process is simple, efficient, it is not necessary to which large-scale instrument and equipment, testing result is intuitively visible, develops the color
Visible region, naked eyes visual color change；
(3) detection sensitivity is high, using spectrophotometer test limit up to 1.0mg/L；
(4) detection method rate of recovery height, favorable reproducibility, testing result is high with national standard high performance liquid chromatography compatibility, puts down
For the equal rate of recovery up to 97.6%, average relative standard's deviation (n=10) is 3.4%.
Embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, all devices and raw material are commercially available or the industry is conventional, following
Method in embodiment, it is this area conventional method unless otherwise instructed.
Embodiment 1
The prefabricated reagent of sucrose quick detection in a kind of honey, is made up of following raw material：Sodium chloride 8g, N, N- diethyl-to benzene
(mol ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1 by diamines 0.1g, phosphate 1.6g:5), sucrose isomerase 0.1g, grape
Glycosyl oligosaccharides oxidizing ferment 0.1g and horseradish peroxidase 0.1g；With the prefabricated reagent gross mass of sucrose quick detection in honey
On the basis of 10g, the enzyme activity unit containing sucrose isomerase is 0.3U/mg in the prefabricated reagent of sucrose quick detection in honey,
Enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment is 0.7U/mg, and the enzyme activity unit containing horseradish peroxidase is
1.5U/mg；The prefabricated reagent of sucrose quick detection in above-mentioned honey is divided into every part of 50mg, every part with 10mL transparent spiral lid glass
Glass test tube packs, 4 DEG C of refrigerations of lucifuge.
Embodiment 2
The prefabricated reagent of sucrose quick detection in a kind of honey, is made up of following raw material：Sodium chloride 8.29g, N, N- diethyl-right
(mol ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 17 by phenylenediamine 0.05g, phosphate 1.5g:1), sucrose isomerase 0.05g,
Glucosyl group oligosaccharides oxidizing ferment 0.06g and horseradish peroxidase 0.05g；It is total with the prefabricated reagent of sucrose quick detection in honey
On the basis of quality 10g, the enzyme activity unit containing sucrose isomerase is in the prefabricated reagent of sucrose quick detection in honey
0.65U/mg, the enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment are 0.4U/mg, the enzyme activity containing horseradish peroxidase
Unit of force is 0.7U/mg；The prefabricated reagent of sucrose quick detection in above-mentioned honey is divided into every part of 50mg, it is every part high thoroughly with 10mL
Lightness polystyrene plastics bottle packs, 8 DEG C of refrigerations of lucifuge.
Embodiment 3
The prefabricated reagent of sucrose quick detection in a kind of honey, is made up of following raw material：Sodium chloride 7.49g, N, N- diethyl-right
(mol ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1 by phenylenediamine 0.15g, phosphate 2g:7), sucrose isomerase 0.12g, Portugal
Grape glycosyl oligosaccharides oxidizing ferment 0.12g and horseradish peroxidase 0.12g；With the total matter of the prefabricated reagent of sucrose quick detection in honey
On the basis of measuring 10g, the enzyme activity unit containing sucrose isomerase is 1.0U/ in the prefabricated reagent of sucrose quick detection in honey
Mg, the enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment are 1.0U/mg, the enzyme activity unit containing horseradish peroxidase
For 2.0U/mg；The prefabricated reagent of sucrose quick detection in above-mentioned honey is divided into every part of 50mg, every part with 10mL transparent spiral lids
Teat glass packs, 6 DEG C of refrigerations of lucifuge.
To the prefabricated reagent of sucrose quick detection in embodiment 1-3 honey, the mark of cane sugar content in honey sample is made
Directrix curve, comprise the following steps：
(1) standard working curve is drawn：0,100,200,400,600,1200 and 2000 μ L sucrose standard solution are pipetted respectively
(100mg/L) adds water to supply 5.0mL in seven test tubes；Liquid 4.0mL in this seven test tubes is taken respectively again to being equipped with
In the detection pipe of embodiment 1-3 prefabricated reagent, mix, 10min is accurately incubated under 37 DEG C of water-baths, with addition under 553nm
0mL standard liquids for reference, with 1cm cuvette sequentially determining absorbances.Standard working curve information is obtained, and is recorded in
Table 1；
(2) determination of test limit and quantitative limit：To the least concentration Working Standard Solution in standard working curve, six repetitions are done
Determination test, standard deviation is calculated, be quantitative test limit by detection limit, 10 times of standard deviations of 3 times of standard deviations, as a result record
In table 1.
The testing result of table 1
Note：A is absorbance；C is sucrose concentration (mg/L)
As can be seen from Table 1, the detection sensitivity of the prefabricated reagent of sucrose quick detection is high in honey of the present invention, uses light splitting light
Degree meter test limit is up to 1.0mg/L.
Reappearance and sample recovery of standard addition to detection method and compare analysis with standard method：
The prefabricated reagent of sucrose quick detection in the honey of the embodiment of the present invention 1 is chosen, parallel determination is carried out to certain honey sample
10 times, it is as shown in table 2 to obtain the reappearance result that the present invention determines.In addition, 3 gradients are added in the honey sample respectively
Standard sucrose solution, obtains the mark-on sample of 3 kinds of various concentrations, and measurement result is as shown in table 3.Meanwhile identical mark-on sample state
Family's standard method《Fructose in Honey, glucose, sucrose, the assay method liquid chromatogram differential pulse polarograpll method of maltose content》
(GB/T 18932.22-2003) is determined, as a result as shown in table 3.
The preparation method of sample solution：Honey 1.0g accurately is weighed into beaker, adds distilled water solution, and all move on to
In 100mL volumetric flasks, and beaker is cleaned 3~4 times with distilled water, cleaning fluid is poured into volumetric flask, constant volume, takes solution 5.0mL
Into another 100mL volumetric flask, and with distilled water constant volume, this is sample solution to be determined, standby.
Measure：The accurate sample solution 4.0mL that pipettes is to the transparent spiral cover glass test tube equipped with the prefabricated reagent of embodiment 1
In, mix, accurate insulation 10 minutes, pour into 1cm cuvettes, the other steps of sample are replaced to add distilled water under 37 DEG C of water-baths
Rapid identical is reference, and absorbance is determined under 553nm.Obtained with absorbance from the standard working curve of embodiment 2
Corresponding concentration value, and by the extension rate of the present embodiment, the concentration of sucrose in sample is obtained, as a result as shown in table 3.
The reappearance result of table 2
| Honey sample measure numbering | The content (g/100g) of sucrose |
| 1 | 2.78 |
| 2 | 2.84 |
| 3 | 2.87 |
| 4 | 3.03 |
| 5 | 3.01 |
| 6 | 2.76 |
| 7 | 2.75 |
| 8 | 2.83 |
| 9 | 2.79 |
| 10 | 2.91 |
| Average value | 2.86 |
| Average relative standard's deviation | 3.4% |
As can be seen from Table 2, the detection method reappearance of the prefabricated reagent of sucrose quick detection is good in honey of the present invention.
The recovery of standard addition of table 3. and with national standard method comparison result
As can be seen from Table 3, average relative standard's deviation of detection method be 3.4%, recovery of standard addition be 92.0~
102%, average recovery of standard addition is 97.6%, compared with national standard high performance liquid chromatography, relative error -0.79~
2.51%, there is good compatibility.Illustrate that the detection method of the prefabricated reagent of sucrose quick detection in honey of the present invention is accurate
Spend, rate of recovery height.
The reappearance of the prefabricated reagent detection method of sucrose quick detection in the honey of the embodiment of the present invention 2,3 is chosen respectively
Analysis is compared with sample recovery of standard addition and with standard method, its step is identical with embodiment 1, repeats no more.
Safe, strong antijamming capability of the invention, selected enzyme has a selectivity in component, other sugar in honey
The higher glucose of class, particularly content and fructose do not influence on measure；It is easy to carry, operating process is simple, efficient, no
Large-scale instrument and equipment is needed, testing result is intuitively visible, develops the color in visible region, naked eyes visual color change；Detection sensitivity
Height, using spectrophotometer test limit up to 1.0mg/L；Rate of recovery height, favorable reproducibility are determined, testing result is high with national standard
Effect liquid phase chromatogram method compatibility is high, and for average recovery rate up to 97.6%, average relative standard's deviation (n=10) is 3.4%.
Presently preferred embodiments of the present invention is the foregoing is only, any formal limitation not is made to the present invention, not surpassed
There are other variants and remodeling on the premise of going out the technical scheme described in claim.
Claims (8)
1. the prefabricated reagent of sucrose quick detection in a kind of honey, it is characterised in that sucrose quick detection is pre- in the honey
Reagent processed is made up of the raw material of following parts by weight：75~83 parts of filler, 0.5~1.5 part of developer, 15~20 parts of phosphate,
0.5~1.2 part of 0.5~1.2 part of sucrose isomerase, 0.6~1.2 part of glucosyl group oligosaccharides oxidizing ferment and horseradish peroxidase.
2. the prefabricated reagent of sucrose quick detection in a kind of honey according to claim 1, it is characterised in that the honey
The prefabricated reagent of middle sucrose quick detection is made up of the raw material of following parts by weight：80 parts of filler, 1 part of developer, phosphate 16
1 part of part, 1 part of sucrose isomerase, 1 part of glucosyl group oligosaccharides oxidizing ferment and horseradish peroxidase.
3. the prefabricated reagent of sucrose quick detection in a kind of honey according to claim 1, it is characterised in that the filling
Agent is sodium chloride.
4. the prefabricated reagent of sucrose quick detection in a kind of honey according to claim 1, it is characterised in that the colour developing
Agent is N, N- diethyl-p-phenylenediamines.
5. the prefabricated reagent of sucrose quick detection in a kind of honey according to claim 1, it is characterised in that the phosphoric acid
Salt is the mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, and the mol ratio of the disodium hydrogen phosphate and sodium dihydrogen phosphate is (1:7)
~(17:1).
6. the prefabricated reagent of sucrose quick detection in a kind of honey according to claim 1, it is characterised in that with honey
On the basis of the prefabricated reagent gross mass of sucrose quick detection, contain sucrose in the prefabricated reagent of sucrose quick detection in the honey
The enzyme activity unit of isomerase is 0.3~1.0U/mg, contains glucose in the prefabricated reagent of sucrose quick detection in the honey
The enzyme activity unit of base oligosaccharides oxidizing ferment is 0.4~1.0U/mg, is contained in the honey in the prefabricated reagent of sucrose quick detection
The enzyme activity unit of horseradish peroxidase is 0.7~2.0U/mg.
7. the store method of the prefabricated reagent of sucrose quick detection in a kind of honey as described in claim 1-6 is any, it is special
Sign is, the prefabricated reagent of sucrose quick detection in above-mentioned honey is packed with detection pipe, 4~8 DEG C of refrigerations of lucifuge, described
Detection pipe is transparent spiral cover glass test tube or high grade of transparency polystyrene plastics bottle.
8. the detection method of the prefabricated reagent of sucrose quick detection in a kind of honey as described in claim 1-6 is any, it is special
Sign is, comprises the following steps：
(1) after honey sample being diluted into 1000~2000 times, 4.0mL sample solutions sucrose quick detection in containing honey is taken
In the detection pipe of prefabricated reagent, it is well mixed, in the accurate response 10min at 30~45 DEG C,；
(2) color of observation of steps (1) reacted prefabricated reagent, is compareed with standard color comparison card, can half-quantitative detection honey sample
The concentration range of sucrose in product；
Or in 540~560nm wave-length coverages determination step (1) reacted prefabricated reagent absorbance, according to absorbance
Value is searched and converted from standard curve, can quantify the content of sucrose in detection honey sample.
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Cited By (1)
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|---|---|---|---|---|
| CN108226151A (en) * | 2018-01-31 | 2018-06-29 | 安徽泓顺源生物科技有限公司 | A kind of assay method of Fructose in Honey and glucose content |
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| CN101393122A (en) * | 2008-10-31 | 2009-03-25 | 中国农业大学 | Honey quality rapid detection method |
| CN101846617A (en) * | 2009-12-29 | 2010-09-29 | 中国科学院地球化学研究所 | Sterile detection method of cane sugar content in culture media based on spectrum analysis |
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| WO2015052315A3 (en) * | 2013-10-11 | 2016-02-04 | Novozymes A/S | Method for decolorization of sugar solution using enzymes |
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2017
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|---|---|---|---|---|
| CN101393122A (en) * | 2008-10-31 | 2009-03-25 | 中国农业大学 | Honey quality rapid detection method |
| CN101846617A (en) * | 2009-12-29 | 2010-09-29 | 中国科学院地球化学研究所 | Sterile detection method of cane sugar content in culture media based on spectrum analysis |
| WO2015052315A3 (en) * | 2013-10-11 | 2016-02-04 | Novozymes A/S | Method for decolorization of sugar solution using enzymes |
| CN104730185A (en) * | 2013-12-20 | 2015-06-24 | 江苏中谱检测有限公司 | Quick determination method of beet syrup characteristic substances in adulterated honey |
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| CN108226151A (en) * | 2018-01-31 | 2018-06-29 | 安徽泓顺源生物科技有限公司 | A kind of assay method of Fructose in Honey and glucose content |
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