CN106153705A - Honeybee Mel and the method for Apis mellifera Mel in differentiating based on major royal jelly proteins composition - Google Patents
Honeybee Mel and the method for Apis mellifera Mel in differentiating based on major royal jelly proteins composition Download PDFInfo
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Abstract
The present invention provides a kind of honeybee Mel and method of Apis mellifera Mel in differentiating based on major royal jelly proteins composition, the present invention is to pass through biological method, the difference on albumen by middle honeybee Mel and Apis mellifera Mel, honey sample carries out protein electrophoresis after treatment, both differential protein bands occur, diversity band is through mass spectral analysis, determine its protein classes, the Mel in two kinds of different honeybee kinds sources is differentiated with this, in solving present on market, honeybee Mel and Apis mellifera Mel are difficult to differentiate, and Apis mellifera Mel pretend to be and mix in this problem of honeybee Mel, can contain and pretend to be the confusion of honeybee Mel in high price with low price Apis mellifera Mel, the discriminating of middle honeybee Mel and Apis mellifera Mel is applied.
Description
Technical field
The invention belongs to honey quality control method, relate to the isolation identification side of major royal jelly proteins composition in a kind of Mel
Method, particularly relates to a kind of method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel, and and is applied
Among the discriminating with Apis mellifera Mel of the middle honeybee Mel.
Background technology
Mel is the nectar of Apis herborization, secretions or honeydew, after mixing with self secretions, through fully brewageing and
The crude sweet material become.The chemical composition of Mel is considerably complicated, has the most identified more than 180 kind materials, Mel in Mel
In main component be glucose and fructose, account for the 65%-80% of Mel total amount, next to that moisture, account for 16%-25%.This
Outward, Mel, possibly together with the required several mineral materials of multivitamin, protein and the mankind and trace element etc., is integration of edible and medicinal herbs
Natural nutriment.
Protein content in Mel is less, and its content is about 0.1%-0.5%, and the protein in Mel is mainly by royal jelly
Main albumen and some digestive enzyme are constituted, and wherein major royal jelly proteins is the main component of Mel protein.The king in different honeybee kind sources
Starch main albumen and have obvious interspecific difference.
Apis mellifera (Apis mellifera ligustica) and apis cerana (Apis cerana cerana) point
Not being the subspecies under Apis mellifera and Eastern bee kind, both differ greatly on body shape and life habit, both kings
Starch main protein ingredient and have obvious interspecific difference, in therefore can being differentiated by the difference of both major royal jelly proteins honeybee Mel and
Apis mellifera Mel.
In the most at home on market, the price of honeybee Mel is 3-5 times of Apis mellifera Mel, and under interests are ordered about, some are illegal
Retailer pretend to be with Apis mellifera Mel or mix in honeybee Mel to earn juice, look forward to Chinese honeybee apiculture person and relevant bee product
Industry causes greater loss, has caused the trust crisis of consumer simultaneously, is unfavorable for the sound development of bee's product market.Therefore,
Need a kind of method badly and differentiate two kinds of Mel, the Mel market that specification is domestic to a certain extent.
Summary of the invention
It is an object of the invention to provide a kind of method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel,
It is achieved by the steps of:
(1) extraction of total protein in Mel: weigh honeybee Mel and Apis mellifera Mel in 10g respectively, be respectively put in 50ml centrifuge tube
In, respectively adding 10ml sterilized water, concussion mixing, under the conditions of 4 DEG C of centrifuge, 8000g/min is centrifuged 10 minutes to remove Cera Flava, pollen
Deng impurity, with in the careful Aspirate supernatant of liquid-transfering gun to new 50ml centrifuge tube, supernatant is placed in 4 DEG C of refrigerator pre-coolings 10 points
Clock, is added in the dehydrated alcohol 20ml of-20 DEG C of refrigerator-freezer pre-coolings, and gentle agitation 15 minutes in ice-water bath, then under the conditions of 4 DEG C
With the centrifugation 10 minutes of 15000g/min, abandoning supernatant, precipitation adds 1.5ml sterilized water, then shakes with Glass rod stirring
Swing mixing, after be dispensed in 1.5ml centrifuge tube that to be placed in-80 DEG C of refrigerators standby, from the Mel different identification mark of different honeybee kinds
Note；
(2) sample protein concentration measures: use determination of protein concentration kit measurement；
(3) process of protein sample: take in 80 μ l honeybee Mel protein sample and Apis mellifera Mel protein sample respectively in 1.5ml
In centrifuge tube, each albumen sample-loading buffer (SDS-PAGE albumen sample-loading buffer (5X)) adding 20 μ l, after concussion mixing 95
DEG C constant-temperature metal bath degeneration 10 minutes；
(4) preparation of polyacrylamide gel: the resolving gel concentration that this experiment uses is 15%, concentrating glue is 5%, adhesive tape
Thickness 1.5mm；
(5) the albuminoid electrophoresis of Mel: using protein electrophoresis piece-rate system, two kinds of albumen applied sample amounts are 200 μ g, initially
Voltage is 90V, and albumen changes 150V into after entering separation gel to be terminated to electrophoresis, and adds albumen Marker with indicator protein molecular weight
Size；
(6) process of adhesive tape: electrophoresis terminates rear adhesive tape and puts into and fix 1 hour in fixative, middle changes a fixative.
After having fixed with coomassie brilliant blue R_250 dye liquor dye 3 hours, be subsequently placed on decolorization swinging table decolour clear to band；
(7) identification of differential protein band: the protein electrophoresis result of two kinds of separate sources of record, according to standard protein
Marker indicates, and finds out the protein band of diversity.
(8) Mass Spectrometric Identification of differential protein band: carry out Mass Spectrometer Method after the protein band of diversity is cut glue, mass spectrum is examined
Survey method is Maldi-TOF-TOF, has detected laggard row data search, determines the protein composition of diversity band.
It is a further object to provide described discrimination method should in the discriminating of middle honeybee Mel and Apis mellifera Mel
With.
The present invention proposes a kind of honeybee Mel and method of Apis mellifera Mel in differentiating, in terms of both major royal jelly proteins
Difference differentiate exactly in honeybee Mel and Apis mellifera Mel.The present invention is by biological method, honeybee Mel and Apis mellifera in discovery
Mel difference on albumen, and determine its protein classes, the Mel differentiating two kinds of different honeybee kinds sources with this, solve market
In present on, honeybee Mel and Apis mellifera Mel are difficult to differentiate, and Apis mellifera Mel pretend to be and mix in this problem of honeybee Mel,
Contain to a certain extent and pretend to be the confusion of honeybee Mel in high price with low price Apis mellifera Mel.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE glue figure of middle honeybee Mel and Apis mellifera Mel, and wherein 1-4 is middle honeybee Mel band, and 5-8 is Apis mellifera
Mel band, the differential band for both that arrow marks.
Detailed description of the invention
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment one
Honeybee Mel and the method for Apis mellifera Mel in a kind of discriminating, be achieved by the steps of:
(1) extraction of total protein in Mel: weigh honeybee Mel and Apis mellifera Mel in 10g respectively, be respectively put in 50ml centrifuge tube
In, respectively adding 10ml sterilized water, concussion mixing, under the conditions of 4 DEG C of centrifuge, 8000g/min is centrifuged 10 minutes to remove Cera Flava, pollen
Deng impurity, with in the careful Aspirate supernatant of liquid-transfering gun to new 50ml centrifuge tube, supernatant is placed in 4 DEG C of refrigerator pre-coolings 10 points
Clock, is added in the dehydrated alcohol 20ml of-20 DEG C of refrigerator-freezer pre-coolings, and gentle agitation 15 minutes in ice-water bath, then under the conditions of 4 DEG C
With the centrifugation 10 minutes of 15000g/min, abandoning supernatant, precipitation adds 1.5ml sterilized water, then shakes with Glass rod stirring
Swing mixing, after be dispensed in 1.5ml centrifuge tube that to be placed in-80 DEG C of refrigerators standby, from the Mel different identification mark of different honeybee kinds
Note；
(2) sample protein concentration measures: use determination of protein concentration kit measurement (the green skies, Shanghai biology company limited,
Article No. P0011)；
(3) process of protein sample: take in 80 μ l honeybee Mel protein sample and Apis mellifera Mel protein sample respectively in 1.5ml
In centrifuge tube, each albumen sample-loading buffer [SDS-PAGE albumen sample-loading buffer (5X)] adding 20 μ l, after concussion mixing 95
DEG C constant-temperature metal bath degeneration 10 minutes；
(4) preparation of polyacrylamide gel: the resolving gel concentration that this experiment uses is 15%, concentrating glue is 5%, adhesive tape
Thickness 1.5mm；
(5) the albuminoid electrophoresis of Mel: using protein electrophoresis piece-rate system, two kinds of albumen applied sample amounts are 200 μ g, initially
Voltage is 90V, and albumen changes 150V into after entering separation gel to be terminated to electrophoresis, and adds albumen Marker with indicator protein molecular weight
Size；
(6) process of adhesive tape: electrophoresis terminates rear adhesive tape and puts into and fix 1 hour in fixative, middle changes a fixative.
After having fixed with coomassie brilliant blue R_250 dye liquor dye 3 hours, be subsequently placed on decolorization swinging table decolour clear to band；
(7) identification of differential protein band: the protein electrophoresis result of two kinds of separate sources of record, according to standard protein
Marker indicates, and finds out the protein band of diversity.
(8) Mass Spectrometric Identification of differential protein band: carry out Mass Spectrometer Method after the protein band of diversity is cut glue, mass spectrum is examined
Survey method is Maldi-TOF-TOF, has detected laggard row data search, determines the protein composition of diversity band.
Embodiment two
Honey sample information such as table 1 below in the present embodiment, it is standby that sample puts 4 DEG C of refrigerators.Method according to embodiment one
Step is tested, wherein the 1-4 duct in 4 corresponding glue figures in 1-in sample number into spectrum, in the sample number into spectrum corresponding glue figures of meaning 1-meaning 4
5-8 duct, slice result is shown in that Fig. 1, mass spectral results are shown in Table 2.
Table 1 honey sample information table
The mass spectral results of differential protein band is as follows:
Table 2 differential protein band Mass Spectrometric Identification result
| Band is numbered | Honeybee kind is originated | Mass Spectrometric Identification result |
| C1 | Apis cerana | Middle honeybee MRJP1,2,3 |
| C2 | Apis cerana | Middle honeybee MRJP1,2,3 |
| C3 | Apis cerana | Middle honeybee MRJP1-7 |
| M1 | Apis mellifera | Apis mellifera MRJP1-9 |
| M2 | Apis mellifera | Apis mellifera MRJP1,2 |
| M3 | Apis mellifera | Apis mellifera MRJP1-4 |
| M4 | Apis mellifera | Apis mellifera MRJP1,2 |
| M5 | Apis mellifera | Apis mellifera MRJP1,2 |
| M6 | Apis mellifera | Apis mellifera MRJP1,2,3 |
From glue figure and mass spectral results it can be seen that two kinds of Mel have obvious difference on albumen composition, can be according to it
The difference of major royal jelly proteins differentiates both.
Although the present invention has been made detailed description by above-described embodiment, but protection scope of the present invention is not limited to
This, also can make its amendment without departing from center to the solution of the present invention, and they broadly fall into protection scope of the present invention.
Claims (5)
1. a method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel, it is characterised in that by following
Step realizes:
(1) extraction of total protein in Mel: weigh middle honeybee Mel and Apis mellifera Mel respectively, be respectively put in centrifuge tube, respectively add nothing
Bacterium water, concussion mixing, centrifugal 10 minutes, in Aspirate supernatant to new centrifuge tube, supernatant is placed in 4 DEG C of refrigerator pre-coolings 10 points
Clock, adds dehydrated alcohol, and gentle agitation 15 minutes in ice-water bath, then recentrifuge 10 minutes, abandoning supernatant, precipitation adds
Sterilized water, then stirring shake mixing, after be dispensed in centrifuge tube that to be placed in-80 DEG C of refrigerators standby, from the Mel of different honeybee kinds
Use different identification labelling；
(2) sample protein concentration measures: use determination of protein concentration kit measurement；
(3) process of protein sample: take middle honeybee Mel protein sample and Apis mellifera Mel protein sample respectively in centrifuge tube, respectively add
Enter albumen sample-loading buffer, constant-temperature metal bath degeneration 10 minutes after concussion mixing；
(4) preparation of polyacrylamide gel: using resolving gel concentration is 15%, concentrating glue is 5%, adhesive tape thickness 1.5mm；
(5) the albuminoid electrophoresis of Mel: using protein electrophoresis piece-rate system, two kinds of albumen applied sample amounts are 200 μ g, initial voltage
For 90V, albumen changes 150V into after entering separation gel to be terminated to electrophoresis, and it is big with indicator protein molecular weight to add albumen Marker
Little；
(6) process of adhesive tape: electrophoresis terminates rear adhesive tape and puts into and fix 1 hour in fixative, middle changes a fixative, fixing
After completing with coomassie brilliant blue R_250 dye liquor dye 3 hours, be subsequently placed on decolorization swinging table decolour clear to band；
(7) identification of differential protein band: the protein electrophoresis result of two kinds of separate sources of record, refers to according to standard protein Marker
Show, find out the protein band of diversity；
(8) Mass Spectrometric Identification of differential protein band: carry out Mass Spectrometer Method, Mass Spectrometer Method side after the protein band of diversity is cut glue
Method is Maldi-TOF-TOF, has detected laggard row data search, determines the protein composition of diversity band.
A kind of method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel the most according to claim 1,
It is characterized in that, the dehydrated alcohol wherein added is the dehydrated alcohol-20 DEG C of refrigerator-freezer pre-coolings.
A kind of method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel the most according to claim 1,
It is characterized in that, centrifugal condition is 8000g/min under the conditions of 4 DEG C for the first time, second time is centrifugal be under the conditions of 4 DEG C with
The centrifugation of 15000g/min 10 minutes.
A kind of method based on honeybee Mel in the discriminating of major royal jelly proteins composition with Apis mellifera Mel the most according to claim 1,
It is characterized in that, step (3) albumen sample-loading buffer is SDS-PAGE albumen sample-loading buffer, 5X.
Method the most according to claim 1 is applied in the discriminating of middle honeybee Mel and Apis mellifera Mel.
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Cited By (8)
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| CN107344961A (en) * | 2017-07-03 | 2017-11-14 | 中国农业科学院烟草研究所 | A kind of method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium and the otherness peptide fragment of identification extraction |
| CN108802163A (en) * | 2018-06-12 | 2018-11-13 | 福建出入境检验检疫局检验检疫技术中心 | A method of different honey categories of quickly reflecting |
| CN109142596A (en) * | 2018-08-27 | 2019-01-04 | 福建出入境检验检疫局检验检疫技术中心 | A kind of recognition methods of honey or royal jelly authenticity |
| CN109470785A (en) * | 2018-10-23 | 2019-03-15 | 广东省生物资源应用研究所 | The construction method and identification application of a kind of natural middle bee honey of lungan flowers and natural Apis mellifera honey of lungan flowers finger-print |
| CN109470779A (en) * | 2018-09-28 | 2019-03-15 | 广东省生物资源应用研究所 | The construction method and identification application of a kind of natural middle bee honey of lychee flowers and natural Apis mellifera honey of lychee flowers finger-print |
| CN109541060A (en) * | 2018-11-28 | 2019-03-29 | 杭州谱胜检测科技有限责任公司 | A method of honey adulteration is identified by protein detection |
| CN110376384A (en) * | 2019-06-24 | 2019-10-25 | 浙江大学 | The ELISA detection kit of bee honey and Apis mellifera honey in detection |
| CN111398498A (en) * | 2020-03-19 | 2020-07-10 | 中国农业科学院蜜蜂研究所 | Application of indole-3-methyl acetate in identifying apis cerana honey and apis mellifera honey |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107344961A (en) * | 2017-07-03 | 2017-11-14 | 中国农业科学院烟草研究所 | A kind of method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium and the otherness peptide fragment of identification extraction |
| CN108802163A (en) * | 2018-06-12 | 2018-11-13 | 福建出入境检验检疫局检验检疫技术中心 | A method of different honey categories of quickly reflecting |
| CN109142596A (en) * | 2018-08-27 | 2019-01-04 | 福建出入境检验检疫局检验检疫技术中心 | A kind of recognition methods of honey or royal jelly authenticity |
| CN109470779A (en) * | 2018-09-28 | 2019-03-15 | 广东省生物资源应用研究所 | The construction method and identification application of a kind of natural middle bee honey of lychee flowers and natural Apis mellifera honey of lychee flowers finger-print |
| CN109470779B (en) * | 2018-09-28 | 2021-11-23 | 广东省科学院动物研究所 | Construction method and identification application of natural apis cerana litchi honey and natural apis mellifera litchi honey fingerprint |
| CN109470785A (en) * | 2018-10-23 | 2019-03-15 | 广东省生物资源应用研究所 | The construction method and identification application of a kind of natural middle bee honey of lungan flowers and natural Apis mellifera honey of lungan flowers finger-print |
| CN109470785B (en) * | 2018-10-23 | 2021-11-23 | 广东省科学院动物研究所 | Construction method and identification application of natural Chinese honeybee longan honey and natural Italian honeybee longan honey finger prints |
| CN109541060B (en) * | 2018-11-28 | 2021-06-29 | 杭州谱胜检测科技有限责任公司 | Method for identifying adulteration of honey through protein detection |
| CN109541060A (en) * | 2018-11-28 | 2019-03-29 | 杭州谱胜检测科技有限责任公司 | A method of honey adulteration is identified by protein detection |
| CN110376384B (en) * | 2019-06-24 | 2020-06-16 | 浙江大学 | ELISA detection kit for detecting Chinese bee honey and Italian bee honey |
| CN110376384A (en) * | 2019-06-24 | 2019-10-25 | 浙江大学 | The ELISA detection kit of bee honey and Apis mellifera honey in detection |
| CN111398498A (en) * | 2020-03-19 | 2020-07-10 | 中国农业科学院蜜蜂研究所 | Application of indole-3-methyl acetate in identifying apis cerana honey and apis mellifera honey |
| CN111398498B (en) * | 2020-03-19 | 2023-01-03 | 中国农业科学院蜜蜂研究所 | Application of indole-3-methyl acetate in identifying apis cerana honey and apis mellifera honey |
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