CN107576651B - The prefabricated reagent and its preservation, detection method that sucrose quickly detects in a kind of honey - Google Patents
The prefabricated reagent and its preservation, detection method that sucrose quickly detects in a kind of honey Download PDFInfo
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Abstract
The prefabricated reagent quickly detected the present invention provides sucrose in a kind of honey and its preservation, detection method, the prefabricated reagent that sucrose quickly detects in the honey is made of the raw material of following parts by weight: 75~83 parts of filler, 0.5~1.5 part of color developing agent, 15~20 parts of phosphate, 0.5~1.2 part of sucrose isomerase, 0.6~1.2 part and 0.5~1.2 part of horseradish peroxidase of glucosyl group oligosaccharides oxidizing ferment.The present invention is easy to carry, and operating process is simple, efficient, and highly-safe, strong antijamming capability does not need large-scale instrument and equipment, and testing result is intuitively visible；Detection sensitivity is high, is limited using spectrophotometer detection up to 1.0mg/L；Detection method rate of recovery height, 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 sensitivities, bee easy to operate
The prefabricated reagent and its preservation, detection method that sucrose quickly detects in honey.
Background technique
Honeybee is nectar, secretion or the honeydew of herborization, after mixing with itself secretion, made of sufficiently making
Natural sweet substance.Contain and the plurality of inorganic salt and vitamin of human serum concentration comparable, iron, calcium, copper, manganese, potassium, phosphorus in honey
Etc. a variety of organic acids and beneficial to the microelement of human health, also contain carbohydrate and a variety of amino acid, makees with high health care
With and nutritive value, the deep favor by consumer.In recent years, domestic and international market constantly expands 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, many kinds of, the complicated component of honey, so that
There is very big limitation in the detection technique of honey quality.Cane sugar content is one of important indicator of honey quality, " food safety country
Standard-honey " (GB14963-2011) regulation, in eucalyptus honey, citrus honey, alfalfa honey, lichee honey and Osmanthus forrestii
Cane sugar content≤10%, other honey≤5%.
Detection method about 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
It is titrated, is calculated with fehling reagent at 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 is that sucrose hydrolysis is recycled glucose oxidase Portugal at glucose and fructose with invertase
Grape sugar, converses cane sugar content.In honey other than sucrose, also contain a large amount of glucose and fructose, to measure hydrolysis front and back
The difference of glucose content in sample, can just be converted into cane sugar content, but due in honey cane sugar content be usually glucose
1/5 to the 1/10 of content, while sample needs extension rate very high, the error of measurement is very big, is not able to satisfy detection needs.This
Outside, acid-hydrolysis method needs titrate under the conditions of boiling, operate comparatively laborious, condition and require more harshness.Therefore, acid-hydrolysis method and
Enzyme hydrolysis method is not suitable for the quick detection of sucrose in honey sample.
It is disclosed in Chinese patent literature " honey quality rapid detection method ", notification number CN101393122A, it should
Invention is measured each component content of honey using the mathematical model between each component content of near-infrared of honey, and this method is former
Reason is complicated, and detection efficiency is low, is unfavorable for large-scale promotion use.
Summary of the invention
The present invention provides one kind in order to which the determination efficiency for overcoming the problems, such as cane sugar content in existing honey is low, error is big
High sensitivity, strong antijamming capability honey in the prefabricated reagent that quickly detects of sucrose.
The prefabricated reagent quickly detected invention further provides sucrose in a kind of honey easy to use, storage effect is good
Store method.
The prefabricated reagent that sucrose quickly detects in a kind of the present invention also provides measurement rate of recovery high, favorable reproducibility honey
Detection method, the method be suitable for laboratories detect and field quick detection.
To achieve the goals above, the invention adopts the following technical scheme:
The prefabricated reagent that sucrose quickly detects in a kind of honey, the prefabricated reagent that sucrose quickly detects in the honey by with
The raw material of lower parts by weight forms: 75~83 parts of filler, 0.5~1.5 part of color developing agent, and 15~20 parts of phosphate, sucrose isomerase
0.5~1.2 part, 0.6~1.2 part and 0.5~1.2 part of horseradish peroxidase of glucosyl group oligosaccharides oxidizing ferment.
Selected sucrose isomerase, glucosyl group in the prefabricated reagent formula system that sucrose quickly detects in honey of the present invention
Oligosaccharides oxidizing ferment and horseradish peroxidase have a specificity, strong antijamming capability, other carbohydrates in honey, especially content
Higher glucose and fructose do not influence measurement.Detection mechanism specifically: the sucrose in honey is in sucrose isomerase
It acts on lower isomery and generates isomaltoketose, for the latter under the action of glucosyl group oligosaccharides oxidizing ferment, oxidation generates 3- ketone-different malt
Ketose, while generating hydrogen peroxide；Hydrogen peroxide horseradish peroxidase effect under be oxidized to water, while make substrate DPD (N,
N- diethyl-p-phenylenediamine) become cationic DPD, pink, the amount of hydrogen peroxide of the pink depth and generation is presented in the latter
It is linear related, it is then linearly related with sucrose concentration initial in sample, therefore, using the pink depth or absorbance value
To measure sucrose concentration.
Preferably, the prefabricated reagent that sucrose quickly detects in the honey is made of the raw material of following parts by weight: filling
80 parts of agent, 1 part of color developing agent, 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 color developing agent is N, N- diethyl-p-phenylenediamine is colourless liquid, resists in the detection process
Interference performance is strong.
Preferably, the phosphate be disodium hydrogen phosphate and sodium dihydrogen phosphate mixture, the disodium hydrogen phosphate with
The molar ratio of sodium dihydrogen phosphate is (1:7)~(17:1).
Preferably, sucrose is fast in the honey on the basis of the prefabricated reagent gross mass that sucrose in honey quickly detects
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 that sugar quickly detects.
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 that sucrose in honey quickly detects, preferably three kinds of enzymes are controlled using the number of additional amount
Enzyme activity control within the above range, the accuracy in detection of prefabricated reagent is best.
A kind of store method for the prefabricated reagent that sucrose quickly detects in honey, sucrose in above-mentioned honey is quickly detected
Prefabricated reagent detection pipe sealed package, is protected from light 4~8 DEG C of refrigerations, and the detection pipe is that transparent spiral cover glass test tube or height are saturating
Lightness polystyrene plastics bottle.
The present invention using packing method, by the prefabricated reagent that quickly detects of sucrose in the honey of every batch of preparation with every part
The low dose of individually packaging of 50mg, needs the tedious steps weighed before simplifying the large dosage of packaging detection of tradition, while avoiding 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.Sealed package is protected from light refrigeration
The activity of enzyme in the prefabricated reagent that then sucrose quickly detects in effective guarantee honey.
A kind of detection method for the prefabricated reagent that sucrose quickly detects in honey, comprising the following steps:
(1) after honey sample being diluted 1000~2000 times, 4.0mL sample solution is taken quickly to examine to containing sucrose in honey
In the prefabricated reagent detection pipe of survey, it is uniformly mixed, accurate response 10min at 30~45 DEG C of Yu,；
(2) color of the prefabricated reagent after observation of steps (1) reaction, compares with standard color comparison card, can half-quantitative detection bee
The concentration range of sucrose in sweet sample；
Or the absorbance value of the prefabricated reagent in 540~560nm wave-length coverage after determination step (1) reaction, according to suction
Shading value is searched and is converted from standard curve, can in quantitative detection honey sample sucrose content.
In the detection method for the prefabricated reagent that sucrose quickly detects in honey of the invention, preferably 37 DEG C of reaction temperature, survey
Determine the preferred 553nm of wavelength of absorbance, the colour developing after honey sample is reacted with prefabricated reagent punishment visually may be used in visible region
See color change, intuitively, conveniently；Quantitative detection can also be carried out with spectrophotometry, detection is limited 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 is suitable for laboratories detection and field quick detection.The present invention has easy to operate, detection quickly
Sensitive, reagent safety, without large-scale instrument, it is easy to carry, do not influenced etc. by reduced sugars such as glucose in sample and fructose it is special
Point.
Therefore, the invention has the following beneficial effects:
(1) reagent safety, strong antijamming capability, selected enzyme has a specificity in component, other carbohydrates in honey,
Especially the higher glucose of content and fructose do not influence measurement；
(2) easy to carry, operating process is simple, efficient, does not need large-scale instrument and equipment, and testing result is intuitive as it can be seen that aobvious
Color is in visible region, naked eyes visual color change；
(3) detection sensitivity is high, is limited using spectrophotometer detection up to 1.0mg/L；
(4) detection method rate of recovery height, favorable reproducibility, testing result and national standard high performance liquid chromatography compatibility
Height, for average recovery rate up to 97.6%, average relative standard's deviation (n=10) is 3.4%.
Specific embodiment
Below by specific embodiment, the technical solutions of the present invention will be further described.
In the present invention, if not refering in particular to, all devices and raw material is commercially available or the industry is common are following
Method in embodiment is unless otherwise instructed conventional method in that art.
Embodiment 1
The prefabricated reagent that sucrose quickly detects in a kind of honey, is made of following raw material: sodium chloride 8g, N, N- diethyl-
P-phenylenediamine 0.1g, phosphate 1.6g (molar ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1:5), sucrose isomerase 0.1g,
Glucosyl group oligosaccharides oxidizing ferment 0.1g and horseradish peroxidase 0.1g；The total matter of prefabricated reagent quickly detected with sucrose in honey
On the basis of measuring 10g, the enzyme activity unit in the prefabricated reagent that sucrose quickly detects in honey containing sucrose isomerase is 0.3U/
Mg, the enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment are 0.7U/mg, the enzyme activity unit containing horseradish peroxidase
For 1.5U/mg；The prefabricated reagent that sucrose quickly detects in above-mentioned honey is divided into every part of 50mg, every part with 10mL transparent spiral lid
Teat glass sealed package is protected from light 4 DEG C of refrigerations.
Embodiment 2
The prefabricated reagent that sucrose quickly detects in a kind of honey, is made of following raw material: sodium chloride 8.29g, N, N- diethyl
Base-p-phenylenediamine 0.05g, phosphate 1.5g (molar ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 17:1), sucrose isomerase
0.05g, glucosyl group oligosaccharides oxidizing ferment 0.06g and horseradish peroxidase 0.05g；It is quickly detected with sucrose in honey prefabricated
On the basis of reagent gross mass 10g, the enzyme activity unit of sucrose isomerase is contained in the prefabricated reagent that sucrose quickly detects in honey
For 0.65U/mg, the enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment is 0.4U/mg, the enzyme containing horseradish peroxidase
Unit of activity is 0.7U/mg；The prefabricated reagent that sucrose quickly detects in above-mentioned honey is divided into every part of 50mg, every part with 10mL high
Transparency polystyrene plastics bottle sealed package is protected from light 8 DEG C of refrigerations.
Embodiment 3
The prefabricated reagent that sucrose quickly detects in a kind of honey, is made of following raw material: sodium chloride 7.49g, N, N- diethyl
Base-p-phenylenediamine 0.15g, phosphate 2g (molar ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1:7), sucrose isomerase
0.12g, glucosyl group oligosaccharides oxidizing ferment 0.12g and horseradish peroxidase 0.12g；It is quickly detected with sucrose in honey prefabricated
On the basis of reagent gross mass 10g, the enzyme activity unit of sucrose isomerase is contained in the prefabricated reagent that sucrose quickly detects in honey
For 1.0U/mg, the enzyme activity unit containing glucosyl group oligosaccharides oxidizing ferment is 1.0U/mg, the enzyme containing horseradish peroxidase
Unit of activity is 2.0U/mg；The prefabricated reagent that sucrose quickly detects in above-mentioned honey is divided into every part of 50mg, every part saturating with 10mL
Bright screw-cap glass test tube sealed package is protected from light 6 DEG C of refrigerations.
To the prefabricated reagent that sucrose in the honey of embodiment 1-3 quickly detects, the mark of cane sugar content in honey sample is made
Directrix curve, comprising the following steps:
(1) standard working curve is drawn: it is molten to pipette 0,100,200,400,600,1200 and 2000 μ L sucrose standards respectively
In liquid (100mg/L) Yu Qizhi test tube, and water is added to supply 5.0mL；Take liquid 4.0mL in this seven test tubes to dress respectively again
In the detection pipe for having the prefabricated reagent of embodiment 1-3, mixes, 10min is accurately kept the temperature under 37 DEG C of water-baths, to add at 553nm
Entering 0mL standard solution is reference, with 1cm cuvette sequentially determining absorbance value.Standard working curve information is obtained, and is recorded
In table 1；
(2) it the determination of detection limit and quantitative limit: to the minimum concentration Working Standard Solution in standard working curve, does six times
Replication test, calculates standard deviation, is quantitative detection limit by detection limit, 10 times of standard deviations of 3 times of standard deviations, as a result
It is recorded in table 1.
1 testing result of table
Note: A is absorbance value；C is sucrose concentration (mg/L)
As can be seen from Table 1, the detection sensitivity of the prefabricated reagent that sucrose quickly detects in honey of the present invention is high, using point
The detection of light photometer is limited up to 1.0mg/L.
Reproducibility and sample recovery of standard addition to detection method and analysis is compared with standard method:
The prefabricated reagent that sucrose quickly detects in the honey of the embodiment of the present invention 1 is chosen, certain honey sample is carried out parallel
Measurement 10 times, obtaining the reproducibility that the present invention measures, the results are shown in Table 2.In addition, 3 ladders are added in the honey sample respectively
The standard sucrose solution of degree, 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
With national standard method " the measuring method liquid chromatogram differential refraction inspection of Fructose in Honey, glucose, sucrose, maltose content
Survey method " (GB/T 18932.22-2003) measurement, the results are shown in Table 3.
The preparation method of sample solution: accurately weighing honey 1.0g into beaker, distilled water solution is added, and all move on to
In 100mL volumetric flask, and beaker 3~4 times wash with distilled water, cleaning solution pours 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, spare.
Measurement: sample solution 4.0mL is accurately pipetted to the transparent spiral cover glass test tube that 1 prefabricated reagent of embodiment is housed
In, it mixes, 10 minutes is accurately kept the temperature under 37 DEG C of water-baths, is poured into 1cm cuvette, distilled water is added instead of the other steps of sample
Suddenly identical is reference, measures absorbance value at 553nm.It is obtained from the standard working curve of embodiment 2 with absorbance value
Corresponding concentration value, and the extension rate of the present embodiment is pressed, the concentration of sucrose in sample is obtained, the results are shown in Table 3.
2 reproducibility result of table
| Honey sample measurement number | 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 reproducibility for the prefabricated reagent that sucrose quickly detects in honey of the present invention is good.
3. recovery of standard addition of table and with national standard method comparison result
As can be seen from Table 3, average relative standard's deviation of detection method is 3.4%, and recovery of standard addition is
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 the detection side for the prefabricated reagent that sucrose quickly detects in honey of the present invention
Method precision is good, the rate of recovery is high.
The reproducibility for the prefabricated reagent detection method that sucrose quickly detects 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, step is identical with embodiment 1, repeats no more.
Highly-safe, strong antijamming capability of the invention, selected enzyme has specificity, other sugar in honey in component
The higher glucose of class, especially content and fructose do not influence measurement；Easy to carry, operating process is simple, efficient, no
Large-scale instrument and equipment is needed, testing result is intuitive as it can be seen that colour developing is in visible region, naked eyes visual color change；Detection sensitivity
Height is limited using spectrophotometer detection up to 1.0mg/L；Rate of recovery height, favorable reproducibility are measured, testing result and national standard are high
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%.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, and is not surpassing
There are also other variations and modifications under the premise of technical solution documented by claim out.
Claims (6)
1. the prefabricated reagent that sucrose quickly detects in a kind of honey, which is characterized in that sucrose quickly detects pre- in the honey
Reagent processed is made of the raw material of following parts by weight: 75 ~ 83 parts of filler, 0.5 ~ 1.5 part of color developing agent, and 15 ~ 20 parts of phosphate, sucrose
0.5 ~ 1.2 part of isomerase, 0.6 ~ 1.2 part and 0.5 ~ 1.2 part of horseradish peroxidase of glucosyl group oligosaccharides oxidizing ferment；The filling
Agent is sodium chloride；The color developing agent is N, N- diethyl-p-phenylenediamine；The phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate
Mixture.
2. the prefabricated reagent that sucrose quickly detects in a kind of honey according to claim 1, which is characterized in that the honey
The prefabricated reagent that middle sucrose quickly detects is made of the raw material of following parts by weight: 80 parts of filler, 1 part of color developing agent, and phosphate 16
Part, 1 part of sucrose isomerase, 1 part and 1 part of horseradish peroxidase of glucosyl group oligosaccharides oxidizing ferment.
3. the prefabricated reagent that sucrose quickly detects in a kind of honey according to claim 1, which is characterized in that the phosphoric acid
The molar ratio of disodium hydrogen and sodium dihydrogen phosphate is (1:7) ~ (17:1).
4. the prefabricated reagent that sucrose quickly detects in a kind of honey according to claim 1, which is characterized in that in honey
On the basis of the prefabricated reagent gross mass that sucrose quickly detects, contain sucrose in the prefabricated reagent that sucrose quickly detects in the honey
The enzyme activity unit of isomerase is 0.3 ~ 1.0U/mg, contains glucose in the prefabricated reagent that sucrose quickly detects in the honey
The enzyme activity unit of base oligosaccharides oxidizing ferment is 0.4 ~ 1.0U/mg, is contained in the prefabricated reagent that sucrose quickly detects in the honey
The enzyme activity unit of horseradish peroxidase is 0.7 ~ 2.0U/mg.
5. a kind of store method for the prefabricated reagent that sucrose quickly detects in honey as described in claim 1-4 is any, special
Sign is, by the prefabricated reagent detection pipe sealed package that sucrose quickly detects in above-mentioned honey, is protected from light 4 ~ 8oC refrigeration, it is described
Detection pipe is transparent spiral cover glass test tube or high grade of transparency polystyrene plastics bottle.
6. a kind of detection method for the prefabricated reagent that sucrose quickly detects in honey as described in claim 1-4 is any, special
Sign is, comprising the following steps:
(1) after honey sample being diluted 1000 ~ 2000 times, 4.0mL sample solution sucrose in containing honey is taken quickly to detect
In the detection pipe of prefabricated reagent, it is uniformly mixed, Yu 30 ~ 45oAccurate response 10min under C；
(2) color of the prefabricated reagent after observation of steps (1) reaction, compares with standard color comparison card, can half-quantitative detection honey sample
The concentration range of sucrose in product；
Or the absorbance value of the prefabricated reagent in 540 ~ 560nm wave-length coverage after determination step (1) reaction, according to absorbance value
Search and convert from standard curve, can in quantitative detection honey sample sucrose 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 |
| 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 |
| WO2015052315A3 (en) * | 2013-10-11 | 2016-02-04 | Novozymes A/S | Method for decolorization of sugar solution using enzymes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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