CN107576655A - The prefabricated reagent of hydroxymethylfurfural quick detection and its preservation, detection method in a kind of honey - Google Patents
The prefabricated reagent of hydroxymethylfurfural 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 hydroxymethylfurfural quick detection in honey and its preservation, detection method, the prefabricated reagent of hydroxymethylfurfural quick detection is made up of the raw material of following parts by weight in the honey：0.5~4 part of 85~95 parts of filler, 0.5~1.5 part of developer, 2.5~7 parts of phosphate, 0.5~4.5 part of hydroxymethylfurfural oxidizing ferment and horseradish peroxidase.The present invention is simple to operate, reagent safety, is easy to carry, and operating process is simple, efficient, and visual result is visible, develops the color in visible region, naked eyes visual color change；Detection sensitivity is high, using spectrophotometer test limit up to 0.15mg/L；The detection method rate of recovery is high, favorable reproducibility, and testing result and national standard high performance liquid chromatography compatibility are high, and for average recovery rate up to 98.5%, relative standard deviation (n=10) is 3.86%, illustrates present invention measure 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 sensitivity, honeybee simple to operate
The prefabricated reagent of hydroxymethylfurfural quick detection and its preservation, detection method in honey.
Background technology
Nectar, secretion or the honeydew of honeybee herborization, after being mixed with itself secretion, the day through fully brewageing
Right 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, have high health-care effect
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, but
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 honeybee
Very big limitation be present in the detection technique of sweet quality.Hydroxymethyl furfural content is one of important indicator of honey quality, when honey is long
Between storage hydroxymethyl furfural content can rise, honey, which mixes syrup (such as sugarcane syrup, corn syrup), can make methylol in product
Furfural content is multiplied, and glucose or fructose is aoxidized generation hydroxymethylfurfural because syrup is heated in preparation process,
Therefore, hydroxymethyl furfural content also can be used to indicate whether product mixes syrup.China《National food safety standard-honey》
(GB14963-2011) provide, hydroxymethyl furfural content≤40mg/kg；U.S. FDA also provides, methylol in common natural honey
Furfural content≤40mg/kg, hydroxymethyl furfural content≤80mg/kg in the natural honey of torrid areas room temperature storage.
Detection method on hydroxymethylfurfural mainly has high performance liquid chromatography, gas chromatography, uv-spectrophotometric
Method and thiobarbituricacidα- colorimetric method etc..High performance liquid chromatography and gas chromatography are uncomfortable due to large-scale instrument to be used
Detected together in field quick detection and laboratories.Ultraviolet spectrophotometry, due to having in honey more than uv-absorbing substance and
Make measure interference more, while be also unsuitable for field quick detection.Thiobarbituricacidα- colorimetric method needs to use strong acid condition, examination
Agent less stable, the reaction time is longer, and other carbohydrates have the shortcomings that certain interference in honey, so as to limit its hydroxyl in honey
Application in the quick detection of methyl furfural.
" quick determination method of hydroxymethylfurfural in a kind of honey " is disclosed in Chinese patent literature, its notification number is
CN104359901A, the invention use mixed proportion as 5:1 toluene amine aqueous solution and the mixed solution of barbiturates solution are examined
Liquid is surveyed, honey sample is added thereto after colour developing by colorimetric card control test.The invention reagent stability is poor, disturbing factor
More, detection operating process is cumbersome, not readily portable dependent on laboratory, is unfavorable for large-scale promotion use.
The content of the invention
The present invention is in order to overcome the problem of hydroxymethylfurfural detection efficiency is low, error is big in traditional honey, there is provided a kind of
High sensitivity, strong antijamming capability honey in hydroxymethylfurfural quick detection prefabricated reagent.
Invention further provides in a kind of simple to operate, good honey of storage effect hydroxymethylfurfural quick detection it is prefabricated
The store method of reagent.
Determine that the rate of recovery is high, hydroxymethylfurfural quick detection is pre- in honey of favorable reproducibility present invention also offers a kind of
The detection method of reagent processed.
To achieve these goals, the present invention uses following technical scheme：
The prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey, hydroxymethylfurfural quick detection is prefabricated in the honey
Reagent is made up of the raw material of following parts by weight：85~95 parts of filler, 0.5~1.5 part of developer, 2.5~7 parts of phosphate, hydroxyl
0.5~4 part of 0.5~4.5 part of methyl furfural oxidizing ferment and horseradish peroxidase.
The detection mechanism of the prefabricated reagent of hydroxymethylfurfural quick detection in honey of the present invention is：Methylol in honey
Furfural aoxidizes in the case where hydroxymethylfurfural aoxidizes enzyme effect, generates 5-HMFA, while generates hydrogen peroxide, and hydrogen peroxide exists
Water is oxidized under horseradish peroxidase effect, while substrate DPD (N, N- diethyl-p-phenylenediamine) is become cationic
DPD, the latter present pink, the pink depth is linearly relevant with caused amount of hydrogen peroxide, then with sample initially
The concentration of hydroxymethylfurfural is linearly related, therefore, can determine hydroxymethylfurfural concentration using the pink depth or absorbance.
Preferably, in the honey hydroxymethylfurfural quick detection prefabricated reagent by following parts by weight raw material group
Into：1 part of 90 parts of filler, 1 part of developer, 7 parts of phosphate, 1 part of hydroxymethylfurfural oxidizing ferment and horseradish peroxidase.
Preferably, the filler is sodium chloride.
Preferably, the developer is N, N- diethyl-p-phenylenediamines.
Preferably, the phosphate is the mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, its mol ratio is (1:5)~
(12:1)。
Preferably, on the basis of the prefabricated reagent gross mass of hydroxymethylfurfural quick detection in honey, in the honey
Enzyme activity unit containing hydroxymethylfurfural oxidizing ferment in the prefabricated reagent of hydroxymethylfurfural quick detection is 0.5~1.5U/mg,
In the honey in the prefabricated reagent of hydroxymethylfurfural quick detection the enzyme activity unit containing horseradish peroxidase be 1.0~
3.0U/mg。
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 two kinds of enzymes in the prefabricated reagent of hydroxymethylfurfural quick detection in honey is controlled using the number of addition, preferably
Within the above range, the accuracy in detection of prefabricated reagent is optimal for the enzyme activity control of two kinds of enzymes.
The store method of the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey, by methylol chaff in above-mentioned honey
The prefabricated reagent of aldehyde quick detection point is packed with detection pipe, and 4~8 DEG C of refrigerations of lucifuge, the detection pipe is transparent spiral lid
Teat glass or high grade of transparency polystyrene plastics bottle.
The present invention using packing method, by every batch preparation honey in hydroxymethylfurfural quick detection prefabricated reagent with
Every part of 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
Weigh and 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 hydroxymethylfurfural quick detection enzyme activity.
The detection method of the prefabricated reagent of hydroxymethylfurfural quick detection, comprises the following steps in a kind of honey：
(1) after honey sample being diluted into 10~500 times, 4.0mL sample solutions hydroxymethylfurfural in containing honey is taken quickly to examine
In the detection pipe of the prefabricated reagent of survey, 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 hydroxymethylfurfural 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 hydroxymethylfurfural in detection honey sample.
In the honey of the present invention in the detection method of the prefabricated reagent of hydroxymethylfurfural quick detection, reaction temperature preferably 37
DEG C, determine the preferred 553nm of wavelength of absorbance, honey sample and the reacted colour developing of prefabricated reagent punishment be in visible region,
Naked eyes visual color change, intuitively, conveniently；Also quantitative detection can be carried out with AAS, test limit is up to 0.15mg/L.Both
Available standards colorimetric card sxemiquantitative, it is also possible to hydroxyl first in portable spectrophotometer or common spectrophotometer accurate quantitative analysis sample
Base furfural content, suitable for laboratories detection and field quick detection.The present invention has simple to operate, the quick spirit of detection
Quick, reagent safety, without large-scale instrument, it is easy to carry the advantages that.
Therefore, the present invention has following beneficial effect：
(1) very simple is operated, all reagents are mounted in detection pipe in advance, only need sample diluting liquid being added to detection during detection
Guan Zhong, after mixing certain time, you can detection；
(2) reagent safety, it is easy to carry, operating process is simple, efficient, and visual result is visible, develops the color in visible region, visually may be used
See color change；
(3) detection sensitivity is high, using spectrophotometer test limit up to 0.15mg/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 98.5%, relative standard deviation (n=10) is 3.86%, illustrates this method measure rate of recovery height, favorable reproducibility.
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 hydroxymethylfurfural quick detection in a kind of honey, is made up of following raw material：Sodium chloride 9.0g, N, N- diethyl
Base-p-phenylenediamine 0.1g, the phosphate 0.7g (mol ratios 1 of disodium hydrogen phosphate and sodium dihydrogen phosphate:5), hydroxymethylfurfural aoxidizes
Enzyme 0.1g and horseradish peroxidase 0.1g；Using the prefabricated reagent gross mass 10g of hydroxymethylfurfural quick detection in honey as base
Standard, the enzyme activity unit containing hydroxymethylfurfural oxidizing ferment is in the prefabricated reagent of hydroxymethylfurfural quick detection in honey
1.2U/mg, the enzyme activity unit containing horseradish peroxidase are 2.5U/mg；Hydroxymethylfurfural in above-mentioned honey is quickly examined
The prefabricated reagent of survey is divided into every part of 50mg, and every part is packed with 10mL transparent spiral cover glass test tubes, 4 DEG C of refrigerations of lucifuge.
Embodiment 2
The prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey, is made up of following raw material：Sodium chloride 8.5g, N, N- diethyl
Base-p-phenylenediamine 0.05g, the phosphate 0.6g (mol ratios 12 of disodium hydrogen phosphate and sodium dihydrogen phosphate:1), hydroxymethylfurfural oxygen
Change enzyme 0.45g and horseradish peroxidase 0.4g；The prefabricated reagent gross mass 10g of hydroxymethylfurfural quick detection using in honey as
Benchmark, the enzyme activity unit containing hydroxymethylfurfural oxidizing ferment is in the prefabricated reagent of hydroxymethylfurfural quick detection in honey
1.5U/mg, the enzyme activity unit containing horseradish peroxidase are 3.0U/mg；Hydroxymethylfurfural in above-mentioned honey is quickly examined
The prefabricated reagent of survey is divided into every part of 50mg, and every part is packed with 10mL high grade of transparency polystyrene plastics bottles, and 8 DEG C of lucifuge is cold
Hide.
Embodiment 3
The prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey, is made up of following raw material：Sodium chloride 9.5g, N, N- diethyl
Base-p-phenylenediamine 0.15g, the phosphate 0.25g (mol ratios 1 of disodium hydrogen phosphate and sodium dihydrogen phosphate:0.5), hydroxymethylfurfural
Oxidizing ferment 0.05g and horseradish peroxidase 0.05g；With the prefabricated reagent gross mass of hydroxymethylfurfural quick detection in honey
On the basis of 10g, the enzyme activity list containing hydroxymethylfurfural oxidizing ferment in the prefabricated reagent of hydroxymethylfurfural quick detection in honey
Position is 0.5U/mg, and the enzyme activity unit containing horseradish peroxidase is 1.0U/mg；Hydroxymethylfurfural in above-mentioned honey is fast
The prefabricated reagent of speed detection is divided into every part of 50mg, and every part is packed with 10mL transparent spiral cover glass test tubes, and 6 DEG C of lucifuge is cold
Hide.
To the prefabricated reagent of hydroxymethylfurfural quick detection in embodiment 1-3 honey, methylol in honey sample is made
The standard curve of furfural content, comprises the following steps：
(1) standard working curve is drawn：0,200,500,1000,2000 and 4000 μ L hydroxymethylfurfural standard liquids are pipetted respectively
(10mg/L) adds water to supply 5.0mL in six test tubes；Liquid 4.0mL in this six test tubes is taken respectively again to being equipped with
In the detection pipe of the prefabricated reagent of embodiment 1, mix, accurate insulation 10 minutes under 37 DEG C of water-baths, to add 0mL under 553nm
Standard liquid for reference, with 1cm cuvette sequentially determining absorbances.Standard working curve information is obtained, as shown in table 1.
(2) determination of test limit and quantitative limit：To the least concentration Working Standard Solution in standard working curve, do six times
Replication is tested, and calculates standard deviation, is quantitative test limit by detection limit, 10 times of standard deviations of 3 times of standard deviations, is obtained
As a result it is as shown in table 1.
The testing result of table 1
Note：A is absorbance；C is hydroxymethylfurfural concentration (mg/L)
As can be seen from Table 1, the detection sensitivity of the prefabricated reagent of hydroxymethylfurfural quick detection is high in honey of the present invention, uses
Spectrophotometer test limit is up to 0.1mg/L.
Reappearance and sample recovery of standard addition to detection method and compare analysis with standard method：
The prefabricated reagent of hydroxymethylfurfural quick detection in honey in the embodiment of the present invention 1 is chosen, certain honey sample is put down
Row measure 10 times, it is as shown in table 2 to obtain the reappearance result that the present invention determines.In addition, 3 are added in the honey sample respectively
The standard hydroxymethylfurfural solution of gradient, obtains the mark-on sample of 3 kinds of various concentrations, and measurement result is as shown in table 3.It is meanwhile identical
Mark-on sample national standard method《Assay method liquid chromatogram-uv detection method of hydroxymethyl furfural content in honey》(GB
18932.18-2003) measure, as a result as shown in table 3.
The preparation method of sample solution：Honey 2.0g accurately is weighed into beaker, adds distilled water solution, and all move on to
In 50mL volumetric flasks, and beaker is cleaned 3~4 times with distilled water, cleaning fluid is poured into volumetric flask, constant volume, and this is random sample to be measured
Product solution, it is standby.
Measure：Sample solution 4.0mL accurately is pipetted into the detection pipe equipped with the prefabricated reagent of embodiment 1, is mixed, in 37 DEG C
Accurate insulation 10 minutes, are poured into 1cm cuvettes under water-bath, to add distilled water instead of the other step identicals of sample as ginseng
Than determining absorbance under 553nm.Corresponding concentration value is obtained from the standard working curve of embodiment 2 with absorbance,
And by the extension rate of the present embodiment, the concentration of hydroxymethylfurfural in sample is obtained, as a result as shown in table 3.
Table 2, reappearance result
| Honey sample measure numbering | The content (mg/kg) of hydroxymethylfurfural |
| 1 | 17.8 |
| 2 | 18.6 |
| 3 | 18.9 |
| 4 | 17.3 |
| 5 | 18.4 |
| 6 | 17.9 |
| 7 | 16.9 |
| 8 | 19.1 |
| 9 | 18.4 |
| 10 | 18.6 |
| Average value | 18.2 |
| Relative standard deviation | 3.86% |
As can be seen from Table 2, the detection method reappearance of the prefabricated reagent of hydroxymethylfurfural quick detection is good in honey of the present invention
It is good.
The recovery of standard addition of table 3. and with national standard method comparison result
As can be seen from Table 3, the relative standard deviation of detection method be 3.86%, recovery of standard addition be 96.0~
101.1%, average recovery of standard addition is 98.5%, and compared with national standard high performance liquid chromatography, relative error is in -0.1.46
~2.58%, there is good compatibility.Illustrate the detection of the prefabricated reagent of hydroxymethylfurfural quick detection in honey of the present invention
Method precision is good, the rate of recovery is high.
The prefabricated reagent detection method of hydroxymethylfurfural quick detection in the honey of the embodiment of the present invention 2,3 is chosen respectively
Reappearance and sample recovery of standard addition and analysis is compared with standard method, its step is identical with embodiment 1, repeats no more.
Therefore, present invention operation very simple, all reagents are mounted in detection pipe in advance, only need to dilute in sample during detection
Liquid is added in detection pipe, after mixing certain time, you can detection；Reagent safety, it is easy to carry, operating process is simple, efficient,
Visual result is visible, develops the color in visible region, naked eyes visual color change；Detection sensitivity is high, is detected using spectrophotometer
Limit is up to 0.15mg/L；The detection method rate of recovery is high, favorable reproducibility, and testing result is consistent with national standard high performance liquid chromatography
Property it is high, average recovery rate is 3.86% up to 98.5%, relative standard deviation (n=10), illustrate present invention measure rate of recovery height, again
Existing property is good.
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)
- A kind of 1. prefabricated reagent of hydroxymethylfurfural quick detection in honey, it is characterised in that hydroxymethylfurfural in the honey The prefabricated reagent of quick detection is made up of the raw material of following parts by weight：85~95 parts of filler, 0.5~1.5 part of developer, phosphoric acid 0.5~4 part of 2.5~7 parts of salt, 0.5~4.5 part of hydroxymethylfurfural oxidizing ferment and horseradish peroxidase.
- 2. the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey according to claim 1, it is characterised in that institute The prefabricated reagent for stating hydroxymethylfurfural quick detection in honey is made up of the raw material of following parts by weight：90 parts of filler, developer 1 1 part of part, 7 parts of phosphate, 1 part of hydroxymethylfurfural oxidizing ferment and horseradish peroxidase.
- 3. the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey according to claim 1, it is characterised in that institute It is sodium chloride to state filler.
- 4. the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey according to claim 1, it is characterised in that institute It is N to state developer, N- diethyl-p-phenylenediamines.
- 5. the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey according to claim 1, it is characterised in that institute Phosphate is stated as disodium hydrogen phosphate and the mixture of sodium dihydrogen phosphate, its mol ratio is (1:5)~(12:1).
- 6. the prefabricated reagent of hydroxymethylfurfural quick detection in a kind of honey according to claim 1, it is characterised in that with In honey on the basis of the prefabricated reagent gross mass of hydroxymethylfurfural quick detection, hydroxymethylfurfural quick detection in the honey Enzyme activity unit containing hydroxymethylfurfural oxidizing ferment in prefabricated reagent is 0.5~1.5U/mg, hydroxymethylfurfural in the honey The enzyme activity unit containing horseradish peroxidase is 1.0~3.0U/mg in the prefabricated reagent of quick detection.
- A kind of 7. preservation side of the prefabricated reagent of hydroxymethylfurfural quick detection in honey as described in claim 1-6 is any Method, it is characterised in that pack the prefabricated reagent of hydroxymethylfurfural quick detection in above-mentioned honey with detection pipe, lucifuge 4 ~8 DEG C of refrigerations, the detection pipe is transparent spiral cover glass test tube or high grade of transparency polystyrene plastics bottle.
- A kind of 8. detection side of the prefabricated reagent of hydroxymethylfurfural quick detection in honey as described in claim 1-6 is any Method, it is characterised in that comprise the following steps：(1) after honey sample being diluted into 10~500 times, 4.0mL sample solutions hydroxymethylfurfural in containing honey is taken quickly to examine In the detection pipe of the prefabricated reagent of survey, 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 hydroxymethylfurfural 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 hydroxymethylfurfural in detection honey sample.
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