CN104713990A - Method for identifying Manuka Honey - Google Patents
Method for identifying Manuka Honey Download PDFInfo
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- CN104713990A CN104713990A CN201510071793.0A CN201510071793A CN104713990A CN 104713990 A CN104713990 A CN 104713990A CN 201510071793 A CN201510071793 A CN 201510071793A CN 104713990 A CN104713990 A CN 104713990A
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- honey
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- manuka
- manuka honey
- deionized water
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- 235000012907 honey Nutrition 0.000 title claims abstract description 78
- 240000003553 Leptospermum scoparium Species 0.000 title claims abstract description 50
- 235000016887 Leptospermum scoparium Nutrition 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000012360 testing method Methods 0.000 claims abstract description 31
- 230000005764 inhibitory process Effects 0.000 claims abstract description 15
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 239000008367 deionised water Substances 0.000 claims abstract description 10
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 6
- 102000016938 Catalase Human genes 0.000 claims abstract description 5
- 108010053835 Catalase Proteins 0.000 claims abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 claims description 23
- 229910052739 hydrogen Inorganic materials 0.000 claims description 16
- 239000001257 hydrogen Substances 0.000 claims description 16
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 15
- 235000015097 nutrients Nutrition 0.000 claims description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 239000012086 standard solution Substances 0.000 claims description 7
- 238000009826 distribution Methods 0.000 claims description 6
- 239000006916 nutrient agar Substances 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 102000003992 Peroxidases Human genes 0.000 claims description 4
- 241000295644 Staphylococcaceae Species 0.000 claims description 4
- 230000001419 dependent effect Effects 0.000 claims description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 45
- 239000013641 positive control Substances 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 235000010633 broth Nutrition 0.000 description 3
- 239000007799 cork Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to the field of honey identification, in particular to a method for identifying Manuka Honey. To relatively well identify genuineness of Manuka Honey, the invention discloses the method for identifying Manuka Honey. The method comprises the following steps: respectively mixing a honey sample with a catalase solution and deionized water so as to form a catalase-honey sample to be tested and a deionized water-honey sample to be tested; by taking staphylococcus aureus as a testing bacterium, respectively adding the catalase-honey sample to be tested and the deionized water-honey sample to be tested into sample adding holes; observing change of inhibition zones, and judging that the honey is Manuka Honey if the two samples to be tested both have inhibition zones. Due to the adoption of the method provided by the invention, Manuka Honey can be accurately identified, and the NPA value of Manuka Honey of which the NPA value is greater than or equal to 5 can be quantitatively detected. The method has important significance in honey identification, particularly identification and quality detection on Manuka Honey.
Description
Technical field
The present invention relates to honey qualification field, particularly relate to the authentication method of Manuka honey.
Background technology
Mai Luka (Manuka) honey is that the distinctive a kind of precious honey of New Zealand is planted, and is that honeybee gathers the distinctive a kind of shrub plant of New Zealand---the nectar of Mai Luka (Leptospermum scoparium) is brewageed.Because Manuka honey has unique antibacterial activity, alimentary health-care function is powerful, is more and more subject to the favor of China consumer.
Honey generally all has antibacterial activity, and this is by the high osmosis of honey itself, stronger acidity and general to cause containing superoxide.The antibacterial activity of Manuka honey does not then rely on superoxide, is therefore called as non-peroxidating antibacterial activity (non-peroxide antibacterial activity, NPA).
Due to the singularity of Manuka honey antibacterial activity, the Manuka honey price variance of different brackets is very large.Because honey generally all has antibacterial activity, and the Manuka honey with NPA is few.Therefore, some honey manufacturer often deliberately obscures total antibacterial activity and the NPA of Manuka honey.Such as, Product labelling marks Active or similar wording misguides the consumer.At present, China does not also have coherent detection standard for the NPA of Manuka honey, is badly in need of carrying out correlative study work and supervises import Manuka honey.
In August, 2013, FSA issues warning of consumption, requires that various places supervision department emphasis checks New Zealand's Manuka honey, because find in some Manuka honeys not containing active antibacterial material through detecting.In addition, according to New Zealand's media report, nearly 1700 ~ 2000 tons of Manuka honey annual production, but global annual turnover exceedes 10000 tons.Therefore, the adulterated problem of Manuka honey becomes urgently to be resolved hurrily.
Summary of the invention
In order to better differentiate the true and false of Manuka honey, we disclosing a kind of authentication method of Manuka honey, comprising the following steps:
(1) honey sample is mixed with Catalase solution and deionized water respectively, form hydrogen peroxidase-honey testing sample and deionized water-honey testing sample;
(2) using gold-coloured staphylococci as test bacterial classification, preparation, to cultivate containing the nutrient agar of bacteria suspension, and pound out the well of Arbitrary distribution on the nutrient culture media formed;
(3) respectively to adding hydrogen peroxidase-honey testing sample and deionized water-honey testing sample in well;
Cultivate more than 18 hours for (4) 37 DEG C, observe the change of inhibition zone, what two kinds of testing samples all presented inhibition zone is Manuka honey.
Wherein using staphylococcus aureus as test bacterial classification, the method of preparation, the nutrient agar of cultivation containing bacteria suspension is specially: inoculation staphylococcus aureus ATCC 9144(Oxoid, Hampshire) in pancreas peptone soybean broth (TSB), 18h is cultivated at 37 DEG C, the light absorption value adjusting 540 nm is 0.5, obtains bacteria suspension; Fresh preparation 150 mL nutrient agar (Becton), 121 DEG C of autoclaving 15 min, put into 50 DEG C of water-baths, after 30 min, 100 uL bacteria suspensions add in agar, after abundant mixing, pour the analysis double dish (Cornig) of 245 x 245 mm into, after to be solidified, be inverted into 4 DEG C of refrigerators, take out after 18-24h, obtain satisfactory nutrient culture media.
In above-mentioned steps, the described method of pounding out the well of Arbitrary distribution is specially: with approximate Latin square (quasi-Latin square) for template, cross over the grid of 25 mm, cut into nutrient culture media with the cork drill of 8 mm of sterilizing, pound out the hole of Arbitrary distribution, be well.
In order to better provide the bacteriostatic activity data of Manuka honey, we still further provides a kind of method that quantitatively can detect its NPA value, further comprising the steps of:
A () prepares the phenol Standard solution of 2%, 3%, 4%, 5%, 6%, 7% respectively;
B the phenol Standard solution of variable concentrations adds in well by ();
C () 37 DEG C was cultivated after 18 hours, with approximate Latin square for template, use vernier caliper measurement antibacterial circle diameter, set up antibacterial circle diameter and phenol solubility dependent equation;
D hydrogen peroxidase-honey sample adds in test media well by ();
E () 37 DEG C was cultivated after 18 hours, with approximate Latin square for template, use vernier caliper measurement antibacterial circle diameter, and by antibacterial circle diameter and phenol solubility dependent equation, obtain corresponding NPA value.
Preferred as one, keep nutrient culture media level without inclination in described (4), (c), (e) step.
When level is set, the horizontal site of culture dish holding can be set by level meter.
Preferred as another, described hydrogen peroxidase is mixed by hybrid heater with honey.
Further, preferred hydrogen peroxidase and honey are under the condition of 37 DEG C, and hybrid heater mixes 30 minutes.
Certainly, the modes such as concussion, stirring also can be adopted to mix, but the preferred hybrid heater mixing of the present invention, and preferably the interior mixing of the hybrid heater of 37 DEG C can obtain optimum efficiency in 30 minutes.
Preferably, staphylococcus aureus used in invention is the bacterial strain within 25 generations.Bacterial activity is kept stable within 25 generations, considers cost, just can detect for bacterial strain without the need to using at every turn.
Adopt authentication method disclosed in this invention, accurately can differentiate Manuka honey, quantitatively can detect the NPA value of the Manuka honey of NPA >=5 simultaneously.Have great importance in the qualification and Quality Detection of honey discriminating, particularly Manuka honey.
Accompanying drawing explanation
Fig. 1 is deionized water-honey testing sample experimental result schematic diagram.
Fig. 2 is hydrogen peroxidase-honey testing sample experimental result schematic diagram.
Fig. 3 is NPA typical curve.
Fig. 4 is the NPA value being placed in the honey sample in 4 wells in non-horizontal placement situation.
Fig. 5 is the NPA value being placed in the honey sample in 4 wells in horizontal positioned situation.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is further elaborated and illustrates.
Unless specifically stated otherwise in the present invention, agents useful for same, instrument are commercially available prod.
Embodiment 1
The preparation of test nutrient culture media
Bacteria suspension: select arbitrarily gold-coloured staphylococci ATCC 9144(Oxoid, the Hampshire within 25 generations) as test bacterial classification, be inoculated in 10 mL pancreas peptone soybean broths, cultivate 18h for 37 DEG C, adjustment absorbance, obtaining light absorption value is the bacteria suspension of 0.5.
Nutrient culture media: fresh preparation 150 mL nutrient agar (Becton), 121 DEG C of autoclaving 15 min, put into 50 DEG C of water-bath 30 min, then 100 μ L bacteria suspensions are added, after abundant mixing, pour the analysis double dish (Cornig) of 245 x 245 mm into, 4 DEG C of refrigerators are put into after culture medium solidifying, take out after 18-24h, with approximate Latin square (quasi-Latin square) for template, cross over the grid of 25 mm, cut into nutrient culture media with the cork drill of 8 mm of sterilizing, pound out the hole of 64 Arbitrary distribution, be well.
The preparation of honey sample solution
In 5g Manuka honey, add the aseptic deionized water of 5 mL, and the mixed solution of honey and deionized water is put into 37 DEG C of hybrid heaters mix 30 min.
Draw 1 mL honey mixed solution respectively to mix with 1 mL Catalase solution (2 mg/mL, Sigma) and 1mL deionized water, obtain hydrogen peroxidase-honey testing sample and deionized water-honey testing sample.
Respectively to adding hydrogen peroxidase-honey testing sample and deionized water-honey testing sample in well.
Double dish is put into the incubator of 37 DEG C, keep double dish horizontal positioned to cultivate 18h.
Take out double dish, observe the inhibition zone of two kinds of samples, as shown in Figure 1, hydrogen peroxidase-honey testing sample experimental result as shown in Figure 2 for deionized water-honey testing sample experimental result.
Sample 11, has obvious inhibition zone in FIG, does not have inhibition zone to occur in fig. 2, can judgement sample 11 not be Manuka honey accordingly.
Sample 4, does not have inhibition zone in FIG, does not have inhibition zone in fig. 2 yet, thus can judgement sample 4 yet non-Manuka honey.
Sample 9, has obvious inhibition zone in FIG, also has obvious inhibition zone in fig. 2, can judgement sample 9 be Manuka honey accordingly.
Embodiment 2
Phenol crystal mixes with aseptic deionized water, preparation 2%, 3%, 4%, 5%, 6%, and the phenol Standard solution of 7%, keeps in Dark Place in-20 DEG C of refrigerators, thaws before using in 4 DEG C.
Add in test media well by 100 ul phenol Standard solution and honey sample solution respectively, with aseptic deionized water as negative control sample, double dish, as positive control sample, is then put into 37 DEG C by Comvita 15+ Manuka honey solution.
After 18h, with approximate Latin square for template, vernier caliper along correct measurement of angle antibacterial circle diameter, the square value its corresponding solubility preparation standard curve of the antibacterial circle diameter of gained phenol Standard solution, as shown in Figure 3.
According to the mode of operation in embodiment 1, obtain sample 9(Manuka honey) inhibition zone, measure the antibacterial circle diameter that honey sample solution 4 repeats, getting its mean value is 17.17, its square value reference standard curve, obtain corresponding phenol solubility 4.65, then be multiplied by extension rate 4, therefore the NPA value of sample 9 is 18.6.
Employ Comvita 15+ Manuka honey as positive control sample in the present embodiment operation, in order to ensure the stability of this positive control sample, the present invention screens its preservation condition.
Concrete mode is: honey sample is placed in respectively 4 DEG C and normal temperature preservation, measures its NPA value after half a year according to aforementioned manner.Result shows, compared with the initial NPA value of honey sample, under 4 DEG C of conditions of storage, the NPA value of sample does not have significant difference, and under normal temperature preservation condition, the NPA value of sample obviously declines.Illustrate that 4 DEG C of conditions of storage are more conducive to keeping the NPA of Manuka honey.
So the Manuka honey as positive control sample should refrigerator cold-storage, and term of life should be less than half a year, selects new Manuka honey as positive control sample afterwards.
Embodiment 3
The preparation of test nutrient culture media
Bacteria suspension: select arbitrarily gold-coloured staphylococci (Staphylococcus aureus) the ATCC 9144(Oxoid within 25 generations, Hampshire) as test bacterial classification, be inoculated in 10mL pancreas peptone soybean broth, cultivate 18h for 37 DEG C, adjustment absorbance, obtaining light absorption value is the bacteria suspension of 0.5.
Nutrient culture media: fresh preparation 150 mL nutrient agar (Becton), 121 DEG C of autoclaving 15 min, put into 50 DEG C of water-bath 30 min, then 100 μ l bacteria suspensions are added, after abundant mixing, pour the analysis double dish (Cornig) of 245 x 245 mm into, 4 DEG C of refrigerators are put into after culture medium solidifying, take out after 18-24h, with approximate Latin square (quasi-Latin square) for template, cross over the grid of 25 mm, cut into nutrient culture media with the cork drill of 8 mm of sterilizing, pound out the hole of 64 Arbitrary distribution, be well.
The preparation of honey sample solution
With three kinds of Manuka honey samples for standard model, its NPA sign value is respectively 16+, 5+, 10+.It is numbered H1, H2, H3 respectively.
In 5g Manuka honey, add the aseptic deionized water of 5 mL, and the mixed solution of honey and deionized water is put into 37 DEG C of hybrid heaters mix 30 min.
Draw 1 mL honey mixed solution and 1 mL Catalase solution (2 mg/mL, Sigma) mixes, obtain hydrogen peroxidase-honey testing sample.
Hydrogen peroxidase-honey testing sample is added in well.
Respectively double dish is done and different puts process:
The first: incubator double dish being put into 37 DEG C, arbitrarily placing and cultivate 18h, there is certain degree of tilt in double dish.
The second: incubator double dish being put into 37 DEG C, keeps double dish horizontal positioned to cultivate 18h.
Sample is put 4 on each double dish and repeats sample.
Under observing two kinds of training methods respectively, the antibacterial circle diameter in the sample hole (Edge) near nutrient culture media edge and the sample hole (Middle) near nutrient culture media center.
As shown in Figure 4, under the first training method, under same dilutability, the antibacterial circle diameter of 2 marginal sample hole Edge1 and Edge2 is less, in addition, media surface is uneven, occur obliquity, the antibacterial circle diameter of featheredge limit sample hole Edge2 is minimum, and another marginal sample hole Edge1 diameter takes second place, maximum with the minimum NPA value of same sample (H1) can differ 4, exceeds error range.
And as shown in Figure 5, under the second training method, under same dilutability, the inhibition zone size there was no significant difference in each sample hole, data are reliable.
Claims (6)
1. an authentication method for Manuka honey, is characterized in that, comprises the following steps:
(1) honey sample is mixed with Catalase solution and deionized water respectively, form hydrogen peroxidase-honey testing sample and deionized water-honey testing sample;
(2) using gold-coloured staphylococci as test bacterial classification, preparation, to cultivate containing the nutrient agar of bacteria suspension, and with approximate Latin square for template, the nutrient culture media formed pounded out the well of Arbitrary distribution;
(3) respectively to adding hydrogen peroxidase-honey testing sample and deionized water-honey testing sample in well;
Cultivate more than 18 hours for (4) 37 DEG C, observe the change of inhibition zone, what two kinds of testing samples all presented inhibition zone is Manuka honey.
2. the authentication method of Manuka honey according to claim 1, is characterized in that, further comprising the steps of:
A () prepares the phenol Standard solution of 2%, 3%, 4%, 5%, 6%, 7% respectively;
B the phenol Standard solution of variable concentrations adds in well by ();
C () 37 DEG C was cultivated after 18 hours, with approximate Latin square for template, use vernier caliper measurement antibacterial circle diameter, set up antibacterial circle diameter and phenol solubility dependent equation;
D hydrogen peroxidase-honey sample adds in test media well by ();
E () 37 DEG C was cultivated after 18 hours, with approximate Latin square for template, use vernier caliper measurement antibacterial circle diameter, and by antibacterial circle diameter and phenol solubility dependent equation, obtain corresponding UMF value.
3. the authentication method of Manuka honey according to claim 1 and 2, is characterized in that, keeps nutrient culture media level without inclination in described (4), (c), (e) step.
4. the authentication method of Manuka honey according to claim 1 and 2, is characterized in that, described hydrogen peroxidase is mixed by hybrid heater with honey.
5. the authentication method of Manuka honey according to claim 4, is characterized in that, hydrogen peroxidase and honey mix 30 minutes in the hybrid heater of 37 DEG C.
6. the authentication method of Manuka honey according to claim 1, is characterized in that, staphylococcus aureus is the bacterial strain within 25 generations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510071793.0A CN104713990A (en) | 2015-02-11 | 2015-02-11 | Method for identifying Manuka Honey |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510071793.0A CN104713990A (en) | 2015-02-11 | 2015-02-11 | Method for identifying Manuka Honey |
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| CN201510071793.0A Pending CN104713990A (en) | 2015-02-11 | 2015-02-11 | Method for identifying Manuka Honey |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075187A (en) * | 2021-03-30 | 2021-07-06 | 南京海关动植物与食品检测中心 | Method for rapidly characterizing Manuka honey |
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|---|---|---|---|---|
| CN102636526A (en) * | 2012-04-05 | 2012-08-15 | 上海交通大学 | Electrochemical method for rapidly detecting oxidation resistance of bee product |
| CN103642850A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Determination method of ferulic acid antioxidant activity in vitro |
| CN103743724A (en) * | 2013-11-30 | 2014-04-23 | 万洪转 | New method for determination of catalase activity in honey |
-
2015
- 2015-02-11 CN CN201510071793.0A patent/CN104713990A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636526A (en) * | 2012-04-05 | 2012-08-15 | 上海交通大学 | Electrochemical method for rapidly detecting oxidation resistance of bee product |
| CN103743724A (en) * | 2013-11-30 | 2014-04-23 | 万洪转 | New method for determination of catalase activity in honey |
| CN103642850A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Determination method of ferulic acid antioxidant activity in vitro |
Non-Patent Citations (5)
| Title |
|---|
| CHRISTOPHER. J. ADAMS等: "Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka(Leptospermum scoparium) honey", 《CARBOHYDRATE RESEARCH》 * |
| K.L.ALLEN等: "A survey of the antibacterial activity of some New Zealand honeys", 《J. PHARM.PHARMACOL.》 * |
| 朱威等: "蜂蜜的抗菌机理及其抗菌效果的影响因素", 《天然产物研究与开发》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075187A (en) * | 2021-03-30 | 2021-07-06 | 南京海关动植物与食品检测中心 | Method for rapidly characterizing Manuka honey |
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Application publication date: 20150617 |